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cancer is a serious life - threatening disease . despite the improvements in cancer medicine that are transforming cancer into a chronic and even curable disease patient - perceived severity can be considered a proxy for beliefs about the objective controllability of disease , and is related to health behavior under certain conditions , such as perceived vulnerability.1 health psychology research has demonstrated the beneficial nature of secondary control beliefs ( perceived control is sustained by primary control , an attempt to change the environment , or secondary control , to adjust psychologically to one s environment)2 and identified numerous coping strategies for individuals faced with cancer.35 according to the health belief model,6 perceived vulnerability and disease severity combine to form a threat , which in turn motivates action and is likely to change patient perceptions of control . in the extended parallel process model,7 there is a balance between threat and efficacy beliefs , ie , if efficacy beliefs exceed levels of threat , then healthy behavior is adopted ( danger control ) , whereas if threat beliefs exceed levels of efficacy , then efforts are focused on managing fear ( fear control).8 because the potential consequences of a health threat can be diverse , it is not surprising that severity appears to be a multidimensional concept.9 therefore , in addition to the characteristics of the illness and the efficacy of medical therapy , patient risk perception and efficacy beliefs could affect their health behavior and psychological well - being . evidence of an association between perceived control and adaptation to illness has been reported in women with breast cancer.1013 several studies have shown that perceived control decreases with age1417 but increases with educational level.18,19 the aims of this study were to measure patient - perceived severity and curability of the disease , and to assess the relationship between patient - perceived degree of severity and curability , psychological well - being , and quality of life in patients with cancer . a sample of 135 cancer patients ( 2481 years of age ) participated in this cross - sectional investigation , which was approved by our local medical ethics committee . the research participants were patients affected by cancer and recruited from the division of oncology , istituto dermopatico dellimmacolata- istituto di ricerca e cura a carattere scientifico , a hospital located in rome , italy . participants were required to be over the age of 18 years and fluent in italian . subjects regularly attending the division were consecutively enrolled and asked to complete the study questionnaires during a hospital visit . written informed consent , following thorough information provision about the purpose of the study , was obtained . one hundred and thirty - five of 137 eligible patients agreed to participate ( 98.5% ) . participants were required to rate the perceived severity and curability of their disease on two ten - point scales ranging from 1 ( not severe , easy to cure ) to 10 ( high severity , difficult to cure ) . disease severity was used as a proxy for objective controllability of the disease20 and curability was used as the opportunity to exert control . usually , disease severity is measured using a general item , and to improve its predictive utility , we weighted it according to the likelihood of disease curability . the functional assessment of cancer therapy - general ( fact - g)21 is a 27-item scale of general questions divided into four primary quality of life domains of well - being , ie , physical , social / family , emotional , and functional . higher scores indicate better quality of life.21,22 in this study , internal consistency was good to excellent for all the fact - g domains , with cronbach s alpha coefficients ranging from 0.73 to 0.88 . the hospital anxiety and depression scale ( hads)23 is a questionnaire consisting of 14 items , ie , seven for anxiety ( hads - a ) and seven for depression ( hads - d ) , with each being scored from 0 ( not present ) to 3 ( highly present ) . scores > 10 were chosen to identify cases of anxiety or depression.23 for descriptive purposes , the study variables were categorized whereby subjects were subdivided into three groups with respect to years of education ( < 8 , 813 , > 13 ) , and time elapsed since diagnosis ( < 1 , 13 , > 3 years ) . marital status ( no / yes ) , religious beliefs ( no / yes ) , belonging to a religious community ( no / yes ) , and severity and ( in)curability were dichotomized into two groups ( 0 , 15 ; 1 , 610 ) . four categories based on patient perception of severity and curability of their disease were defined as follows : not severe / easy to cure ; not severe / difficult to cure ; severe / easy to cure ; and severe / difficult to cure . fact - g domain and total scores were computed on a 0100 point scale ( t values ) characterized by a distribution with a mean of 50 and a standard deviation of 10 . the descriptive statistics included percentages or mean values , depending on the nature of each variable , as well as standard deviations , whenever applicable . multivariable logistic regression models were used to assess the relationship between control perception or quality of life ( independent variables ) , and hads - defined cases of depression or anxiety ( dependent variables ) , adjusting for sociodemographic and medical factors . quality of life levels were categorized as fact - g - specific domain scores either below or above the median value of 50 . medical factors considered were presence of metastasis and treatment - related side effects ( no / yes ) . covariates significantly associated ( p < 0.10 ) with the outcome were entered into a multiple regression model and subjected to backward selection until all remaining covariates had a p value < 0.05 adjusted for the other remaining covariates . patient age , sex , site of cancer , and time elapsed since diagnosis were considered potentially confounding variables . all the statistical analyses were carried out using stata version 11.0 ( stata corporation , college station , tx ) . for descriptive purposes , the study variables were categorized whereby subjects were subdivided into three groups with respect to years of education ( < 8 , 813 , > 13 ) , and time elapsed since diagnosis ( < 1 , 13 , > 3 years ) . marital status ( no / yes ) , religious beliefs ( no / yes ) , belonging to a religious community ( no / yes ) , and severity and ( in)curability were dichotomized into two groups ( 0 , 15 ; 1 , 610 ) . four categories based on patient perception of severity and curability of their disease were defined as follows : not severe / easy to cure ; not severe / difficult to cure ; severe / easy to cure ; and severe / difficult to cure . fact - g domain and total scores were computed on a 0100 point scale ( t values ) characterized by a distribution with a mean of 50 and a standard deviation of 10 . the descriptive statistics included percentages or mean values , depending on the nature of each variable , as well as standard deviations , whenever applicable . multivariable logistic regression models were used to assess the relationship between control perception or quality of life ( independent variables ) , and hads - defined cases of depression or anxiety ( dependent variables ) , adjusting for sociodemographic and medical factors . quality of life levels were categorized as fact - g - specific domain scores either below or above the median value of 50 . medical factors considered were presence of metastasis and treatment - related side effects ( no / yes ) . covariates significantly associated ( p < 0.10 ) with the outcome were entered into a multiple regression model and subjected to backward selection until all remaining covariates had a p value < 0.05 adjusted for the other remaining covariates . patient age , sex , site of cancer , and time elapsed since diagnosis were considered potentially confounding variables . all the statistical analyses were carried out using stata version 11.0 ( stata corporation , college station , tx ) . close to one third of the patients ( n = 40 , 32.3% ) scored positive for anxiety , and almost one quarter ( n = 26 , 21.0% ) scored positive for depression . the mean patient - perceived severity score was 6.04 2.60 and patient - perceived curability was 5.96 2.42 . visual inspection of the distributions of raw scores revealed that both measures were negatively skewed , and probability density curves , produced by kernel density estimation , were asymmetrical ( figures 1 and 2 ) . patients who perceived their disease as severe ( scores ranging from 6 to 10 , n = 78 , 57.8% ) as opposed to not severe ( scores ranging from 1 to 5 , n = 57 , 42.2% ) were younger ( 55 versus 65 years ; p = 0.010 ) , had more than 8 years of education ( p = 0.010 ) , were affected by lung cancer ( p = 0.043 ) , and had metastasis ( p = 0.010 ) . patients who perceived their disease as difficult to cure or incurable ( 5 , n = 59 ; 43.7% ) as opposed to those who perceived their disease as curable ( > 5 , n = 76 ; 56.3% ) were affected by lung cancer ( p = 0.017 ) , had metastasis ( p 0.001 ) , a shorter duration of disease ( 5.3 versus 21.0 months , p = 0.031 ) , and were younger ( 54 versus 60.5 years , p = 0.019 ) . disease was judged by patients as not severe / easy to cure in 51 cases ( 37.8% ) , not severe / difficult to cure in six ( 4.4% ) , severe / easy to cure in 25 ( 18.5% ) , and severe / difficult to cure in 53 ( 39.8% ) . as shown in table 2 , patients who assessed their disease as severe / easy to cure had a lower physical and emotional fact - g well - being score compared with the other two groups , while the prevalence of hads - defined cases of anxiety varied significantly throughout . no group differences emerged for social and functional well - being fact - g domains or prevalence of hads - defined cases of depression . patient perception of their disease as severe / difficult to cure was strongly associated with hads - defined cases of depression ( odds ratio [ or ] 6.93 ; 95% confidence interval [ ci ] 1.0247.23 ; p = 0.048 ) when adjusted for sex , age , site of cancer , and time from diagnosis . factors independently associated with hads - defined cases of anxiety were perception of the disease being severe / difficult to cure ( or 15.73 ; p = 0.018 ) , having religious beliefs ( or 49.74 ; p = 0.013 ) , and metastasis ( or 18.42 ; p = 0.015 ) , when adjusted for sex , marital status , site of cancer , and time from diagnosis . this study shows that health - related quality of life , as reported by an unselected hospital - based cohort of cancer patients , was generally high , except in the area of functional well - being . however , more than 20% of patients had hads - defined depression and more than 30% had hads - defined anxiety . in the entire sample , 58% of patients perceived their disease as severe , and 44% perceived it as difficult to cure . among 75 nonmetastatic patients , 71% considered their cancer to be curable ; however , among 60 metastatic patients , 70% judged their disease as severe , and 62% judged it as difficult to cure . these findings suggest a marked increase in awareness of disease severity when compared with data collected for an italian sample more than 10 years earlier.24 the main findings of this study clarify the relationship between disease curability and depression or anxiety in cancer patients . among those patients who perceived their disease as severe and difficult to cure , there was an increased risk of hads - defined cases of depression or anxiety , regardless of cancer site and time from diagnosis . high levels of anxiety were independently associated with metastasis and major involvement in religious activity , compared with scores for anxiety below the cut - off . despite the cross - sectional design of our study , we can suppose that metastatic patients were more anxious than those who were nonmetastatic , and that anxious patients try to find comfort in connection with their religious traditions ( illness and death raise profound spiritual questions and have a tremendous impact on patients ) . while some studies have shown that patients who attend religious services have better health care and mental health outcomes,25,26 not all do so.2729 recently , we have found that the absence of anxiety disorder , and coping strategies characterized by acceptance and positive reinterpretation of the stressor , independently increased the likelihood of existential well - being ( or 4.5 and or 7.7 , respectively ) , whereas religious well - being was not significantly associated with these variables.30 the diagnosis of cancer is generally regarded as a low - control situation . in our sample , more severe cancer was not considered a threatening situation by all patients , and it is the perception of curability ( easy or difficult ) that determines controllability . in a recent prospective study,31 a decline in perception of control was noted both before and after diagnosis , which resulted in perception being maladaptive because it accounted for psychological distress at 6 and 12 months after diagnosis , regardless of the prognosis . the authors assumed that changes in control perception could be the result of the diagnosis itself , and that they can be caused by being confronted with an event over which no control could be exerted ( ie , a diagnosis of cancer ) , by the limited possibility to exert control over the consequences of the event ( ie , treatment ) , or by a lack of opportunity to control the course of the disease . in our sample , we noted tremendous variability in time elapsed since diagnosis , making it difficult to analyze the impact of diagnosis and treatment . it should be noted that this study has some limitations . although we used standardized validated questionnaires to measure quality of life and psychological distress , limitations inherent in patient self - assessment and choice of cut - off points should be considered . a cross - sectional study can not generally identify the direction of detected associations , and we can not state with certainty that the low - control disease perception ( severe and difficult to cure ) is a cause rather than a consequence of psychological distress . longitudinal prospective studies are needed to measure the incidence of psychological distress and its time of occurrence . the findings of this study have implications for both screening policies and practical management of patients with cancer . our sample is characterized by high quality of life scores . however , of great concern is the high prevalence of hads - defined depression and anxiety in patients with a perception of low - control disease . the assistance of trained counselors is needed for patients to learn how to manage internal fear and uncertainty ( a common emotional response to health threats ) and to improve or gain a sense of control even in situations where there are few opportunities to do so.32,33 psychological interventions emphasizing a sense of control over illness could be effective in enhancing well - being . the use of validated screening questionnaires such as hads , facilitating recognition of psychological problems in the nonpsychiatric setting , and use of easy tools such as ten - point scales to measure severity and curability are useful in identifying individuals who may benefit from professional assessment and treatment , and should become part of the routine management of cancer patients . as noted by brez et al,11 the evolution of distress can be predicted from the initial value and rate of change of control perception .

spontaneous basal ganglia hemorrhage ( bgh ) is the most common hemorrhagic stroke , imposing significant socio - economic burden on the victims.18 ) anatomically , the basal ganglia generally refer to a set of subcortical nuclei , such as striatum , composed of the caudate nucleus and putamen , globus pallidus , substantia nigra , and subthalamic nucleus . the basal ganglia are supplied by various deep perforators.7)11 ) functionally , the basal ganglia play a crucial role in neurologic pathways , including motor , sensory , and cognition.14 ) therefore , bgh may often present with a broad spectrum of clinical manifestations , radiologic features , and outcomes according to its regional location . in this study , we attempted to classify bghs into regional subgroups according to anatomic location , and to extrapolate the distinct characteristics of each group of bgh , which may affect clinical presentation , course , and prognosis . a total of 247 patients diagnosed with spontaneous intracerebral hemorrhage from january 2008 to december 2012 at the author 's institution were reviewed retrospectively . patients with an underlying cause such as trauma , vascular disease , tumor , etc . were excluded from this study . as a result , 103 patients met the criteria of the spontaneous bgh group . functional status was assessed by modified rankin scale ( mrs ) at the time of admission , discharge and six months after discharge , and was categorized according to good ( 0 - 3 ) or poor ( 4 - 6 ) group . motor weakness was estimated according to manual muscle strength scale and the worst affected site was recorded . motor weakness was dichotomized according to good ( 4 - 5 ) or poor ( 0 - 3 ) . bghs were classified into four subgroups according to anatomical region based on computed tomography ( ct ) scans ; the anterior bgh mainly involves the caudate head , and possibly extends to the anterior limb of the internal capsule and the rostral part of the putamen ; the posterior bgh refers to hemorrhage mainly involving putamen , globus pallidus , and posterior limb of the internal capsule ; the lateral bgh is located below the insular cortex and outside the putamen ; the massive bgh occupies all three other regions . hematoma volume was measured using the abc/2 method on non - contrast brain ct scan.10 ) hematoma expansion was defined as a minimum volume increment of 30% documented on follow - up ct scan within 48 hours compared to initial ct scan.9 ) chi - square test , fisher exact test , and kruskal - wallis test were used for analysis of data . risk factors contributing to poor outcome ( mrs 4 - 6 at six months ) were determined by single and multiple logistic regression analysis . ibm spss statistics software version 20.0.0 ( ibm , armonk , ny , united states ) was used for all statistical analyses . correlation between double layer on initial ct and hematoma expansion was calculated using pearson 's correlation equation . no significant differences with regard to age , sex , hematoma expansion , anti - platelet medication and history of diabetes , hypertension , smoking and alcohol consumption were observed between the four subgroups . the largest mean amount of hematoma was observed in massive type , and the smallest volume was observed in anterior type ( p = 0.005 ) , while posterior and lateral bghs were similarly intermediate in amount . initial gcs score was the highest in anterior type , followed by posterior , lateral , and lowest in massive type . layered density on initial ct was noted in 45 bghs , of which 13 bghs ( 28.9% ) had expanded on short - term interval follow - up ct . on the correlation test , double layer showed strong correlation with further hematoma expansion ( p = 0.016 , coefficient = 0.237 ) . on follow up check the best functional outcome was observed in anterior type ( mean mrs = 0.67 ) , followed by lateral ( mean mrs = 2.3 ) , posterior ( mean mrs = 2.7 ) , and worst in massive bghs ( mean mrs = 4.83 ) . in agreement with these results , the mean delta mrs ( mrs = admission mean mrs - six - month mean mrs ) showed the highest recovery potentials in anterior bghs ( mrs = 2.33 ) , followed by lateral ( mrs = 1.61 ) , posterior ( mrs = 0.94 ) , and the least in massive bghs ( mrs = 0.09 ) . among borderline amount of bgh groups of lateral and posterior type , the mean mrs of posterior type was initially better than that of lateral type at the time of admission ( 3.64 vs. 3.92 , respectively ) , but these results were reversed at six months after discharge ( 2.31 vs. 2.7 , respectively ) ( fig . age , amount of hematoma , ivh , initial gcs , and massive type bgh were considered possible risk factors for poor outcome ( mrs 4 - 6 at six months ) , while , after multiple logistic regression analysis , only age and initial gcs remained statistically significant risk factors ( table 3 ) . this type mainly involves the caudate head and possibly extends to the anterior limb of the internal capsule and rostral part of the putamen ( fig . hematoma was generally round in shape and the smallest mean volume was observed among subgroups of bgh , measured as 9.7 8.6 ml . three patients ( 33.3% ) had accompanying intraventricular hemorrhage ( ivh ) , however , development of hydrocephalus did not occur in any case ( fig . none of the patients was assessed as below 8 points on the glasgow coma scale ( gcs ) and eight patients ( 88.9% ) were over gcs 13 on initial presentation . two patients presented with poor motor weakness at admission , but all had recovered at discharge ( table 2 ) . only one patient underwent navigator assisted intracerebral hemorrhage ( ich ) catheter insertion . the posterior type was most common ( 56/103 , 54.4% ) and mainly involved putamen , globus pallidus , and posterior limb of the internal capsule ( fig . hematoma tended to be irregular in shape and the mean volume was 26.3 19.9 ml . thirty one patients ( 55.4% ) underwent navigator assisted ich catheter insertion and one patient underwent craniotomy and hematoma removal . poor motor grade was demonstrated in 45 patients ( 80.4% ) at admission , 41 patients ( 73.2% ) at discharge , and 35 patients ( 62.5% ) at six months after discharge ( table 2 ) . the lateral bgh is located below the insular cortex and outside the putamen ( fig . hematoma was generally ovoid in shape and the mean volume was 27.7 18.3 ml ( fig . hematoma expansion was observed in seven patients ( 26.9% ) , most commonly among the four subgroups . four patients had ivh , but no further development of hydrocephalus was observed in any case . most patients ( 17 , 65.4% ) showed a relatively good level of consciousness as over 13 gcs , and only one patient scored below 8 on gcs . twenty patients ( 76.9% ) had poor limb motor grade at the time of admission , which remained in 14 patients ( 53.8% ) at follow - up check ( table 2 ) . eighteen patients ( 69.2% ) underwent navigator assisted ich catheter insertion and subsequent fibrinolytic treatment . hematoma was generally irregular in shape and the largest mean volume was 115.8 38.4 ml . accompanying ivh was observed in all cases and development of acute hydrocephalus was observed in two patients . all patients in this type presented with poor motor grade , which did not recovered until six months after discharge . ten patients underwent surgical treatment ; craniotomy and hematoma removal in four patients and navigator assisted ich catheter insertion followed by fibrinolytic treatment in six patients ( table 2 ) . this type mainly involves the caudate head and possibly extends to the anterior limb of the internal capsule and rostral part of the putamen ( fig . hematoma was generally round in shape and the smallest mean volume was observed among subgroups of bgh , measured as 9.7 8.6 ml . three patients ( 33.3% ) had accompanying intraventricular hemorrhage ( ivh ) , however , development of hydrocephalus did not occur in any case ( fig . none of the patients was assessed as below 8 points on the glasgow coma scale ( gcs ) and eight patients ( 88.9% ) were over gcs 13 on initial presentation . two patients presented with poor motor weakness at admission , but all had recovered at discharge ( table 2 ) . the posterior type was most common ( 56/103 , 54.4% ) and mainly involved putamen , globus pallidus , and posterior limb of the internal capsule ( fig . hematoma tended to be irregular in shape and the mean volume was 26.3 19.9 ml . thirty one patients ( 55.4% ) underwent navigator assisted ich catheter insertion and one patient underwent craniotomy and hematoma removal . poor motor grade was demonstrated in 45 patients ( 80.4% ) at admission , 41 patients ( 73.2% ) at discharge , and 35 patients ( 62.5% ) at six months after discharge ( table 2 ) . the lateral bgh is located below the insular cortex and outside the putamen ( fig . hematoma was generally ovoid in shape and the mean volume was 27.7 18.3 ml ( fig . hematoma expansion was observed in seven patients ( 26.9% ) , most commonly among the four subgroups . four patients had ivh , but no further development of hydrocephalus was observed in any case . most patients ( 17 , 65.4% ) showed a relatively good level of consciousness as over 13 gcs , and only one patient scored below 8 on gcs . twenty patients ( 76.9% ) had poor limb motor grade at the time of admission , which remained in 14 patients ( 53.8% ) at follow - up check ( table 2 ) . eighteen patients ( 69.2% ) underwent navigator assisted ich catheter insertion and subsequent fibrinolytic treatment . 4 ) . hematoma was generally irregular in shape and the largest mean volume was 115.8 38.4 ml . accompanying ivh was observed in all cases and development of acute hydrocephalus was observed in two patients . all patients in this type presented with poor motor grade , which did not recovered until six months after discharge . ten patients underwent surgical treatment ; craniotomy and hematoma removal in four patients and navigator assisted ich catheter insertion followed by fibrinolytic treatment in six patients ( table 2 ) . bghs differ variously in size , shape , and location , which may affect clinic - radiologic manifestations . we assumed that bgh could show some standardized pattern because the consistency of each nucleus of striatum , capsular fibers , and peri - ventricular matter is differently discrete , and , as a resultant , may act as a boundary of the hemorrhagic cavity . in the previous report by chung et al.,4 ) bghs were categorized according to six types based on vascular territory , including anterior , middle , posteromedial , posterolateral , lateral , and massive type . each type presented with its own unique location , morphological characteristics , and clinical presentations and outcomes , similar to ours . however , in clinical application , it often seemed confusing and slightly cumbersome to differentiate one type of bgh from another type , particularly between middle , posteromedial , and posterolateral type . therefore , we tried to categorize bghs more simply into four subgroups mainly based on anatomical structures and could assure that this modified subgroup of bgh still reflect well enough their clinico - radiological characteristics . the amount of hemorrhagic volume is the critical factor related to initial neurologic status and subsequent long term prognosis.5)8)15)16 ) therefore , it was notable that the volume of hematoma showed a distinct trend according to its location . the largest mean amount of hematoma was observed in massive type , as intuitively expected , and anterior type showed the smallest volume . in addition , initial gcs score was the highest in anterior type , meaning that most patients with anterior bghs are less neurologically stunned . in general , initial gcs score was slightly higher in posterior type than in lateral type , but posterior type showed a broad spectrum in gcs . because the lateral and posterior type tended to show a moderate amount of hemorrhage ( 20~30ml ) , more sophisticated decision making regarding surgical intervention may be mandatory . according to our observation , the potentials for recovery from motor weakness were better in lateral type than in posterior type . in lateral type , motor weakness showed rapid recovery during hospitalization before discharge , however , in the posterior type , recovery of motor grade was gradual during long term rehabilitation after discharge . it appeared that the posterior type damaged basal ganglia and adjacent structures directly , but lateral bgh did not directly affect the neural tracts , but simply compressed them . for this reason , patients in lateral type bgh may require early surgical intervention for rescue of a salvageable neural tract . the well circumscribed , ovoid shape of lateral bgh is also best fit to stereotactic catheter aspiration and subsequent fibrinolytic treatment , whereas posterior bghs are often shaped in an irregular staghorn . previous reports have advocated that larger ichs were significantly more irregular in shape , heterogeneous in density , and had greater growth , and density heterogeneity independently predicted ich growth.1)9 ) in this study , a layered density of hematoma on initial ct scan showed correlation with hematoma expansion , as in a previous study . a layered density of hematoma on initial computed tomography was seen mainly in massive type . however , hematoma expansion among bghs showing layered density was most frequently identified in the lateral type of bgh . this may be because emergent surgical intervention was more readily adopted in posterior and massive bghs so that the time interval between initial and follow - up ct was too short to recognize identifiable hematoma expansion . actually , hematoma expansion was observed in two patients who did not undergo surgical treatment . at least , we could assure that the layered density within the hematoma cavity is a strong predictor of hematoma growth . because the medial side of the caudate nucleus is composed of the lateral wall of the frontal horn of the ventricle , anterior bghs can spread into the ventricle more frequently than posterior and lateral bghs do . many reports advocate that the combined ivh is a negative factor for good recovery,6)8)11 ) however , the existence of ivh appeared not to affect the final outcome if combined with anterior bgh . on the contrary , if ivh is combined with another type of bgh , it may implicate the explosive nature of hemorrhage , resulting in a relatively poor clinical course . regarding functional outcome on follow up check , the highest recovery potentials were observed in anterior bghs , followed by lateral , posterior , and massive bghs were most ominous . among borderline amount of bgh groups of lateral and posterior type , it was interesting that the mean mrs of posterior type was initially better than the lateral type at the time of admission , but these results were reversed at six months after discharge . as previously described regarding recovery potentials in motor weakness , the initial insult by the larger volume of hematoma might reflect slightly poorer neurological status in lateral bghs , however , simply stunned neurophysiological function in lateral bghs could better retain their ability soon after alleviation of mechanical and chemical stresses than destructed neural pathway in posterior bghs could do . the corticospinal tract , a major neural tract in the human brain for motor function , runs along the posterior limb of the internal capsule . therefore , this neural pathway may be more preserved in anterior and lateral type bgh than posterior type bgh , which can affect functional outcome . 5 , the mean mrs of lateral bghs nearly catches up with that of the posterior bghs at the time of discharge , and finally surpassed at six months after discharge . among these two deep seated , medium volumes of ich groups , the final prognosis might differ as to the primary locations of hematoma . one patient with massive type who underwent direct surgical evacuation of hematoma recovered and scored mrs 2 at six months after discharge . although there is significant criticism regarding performance of surgical intervention in deep seated ichs,2)3)12)13)17 ) we experienced and believed that a certain patient may still possess recovery potential . over 75% of patients with massive bghs were unconscious , uncommunicable in devastating status , thus , selection of patients who might benefit from surgical intervention seemed difficult . therefore , an aggressive strategy to include all possible surgical candidates can be the final opportunity for recovery for life threatening ich victims , once providing that the surgical intervention can meet the condition of ' at least do no harm ' . this is a retrospective study , harboring possible bias in patient selection , assessment , and follow - up plan . furthermore , we did not assess additional clinical manifestations , such as language impairment , cognitive function , and other non - motor deficits . in addition , because variables between hematoma volume , initial gcs , ivh , and type of bgh may be co - related , and the number of data in each group seems relatively small , the statistical calculation failed to show significance of regional location of bgh in assessing risk factors for poor outcome . all of these are possible limitations of this report , and subject to future investigation . we observed that bghs can be grouped according to regional location and each group may have distinct characteristics . thus , more sophisticated clinical strategy tailored to each group of bghs can be implemented .

the symmetry of biomolecules originating from gene duplication and consolidated by evolution , while often only approximate , is intimately linked to functionality . for transmembrane proteins ( tmps ) in particular , symmetry is one of the common properties shared in their functional states , and it has been related to dynamics , fast folding kinetics , high stability , and allosteric regulation . in addition , engineering of proteins with internal symmetry has become an emerging field with a growing body of reported success . tmps , such as g protein - coupled receptors and ion channels , are crucial targets in drug discovery due to their physiological roles as direct rectors for drug - like solutes ; it has been suggested that for receptors of neurotransmitters , for instance , an indirect and less specific mechanism whereby solutes absorbed into the lipid bilayer can affect the receptor . with around half of current drug targets being these tmps and most of those drugs targeting only a few members , it is hardly surprising that the study of tmps has become an active field of research with an increasing amount of experimental and computational efforts for potential pharmaceutical applications . despite advances in both hardware and software for atomistic molecular simulations , there is still a large gap between the duration of all - atom molecular dynamics ( md ) trajectories produced on a routine basis ( typically , microseconds ) and time scales of biologically relevant events observed in experiments involving tmps ( usually milliseconds to seconds ) . one fruitful strategy to overcome this gap and better bridge experiments and simulations is to apply a coarse - graining approach . the structure - based coarse - grained ( cg ) representation encompasses a reduced level of detail of the system , as atoms are grouped into effective particles also termed cg sites , and many of the biofunctionally irrelevant degrees of freedom are integrated out . high resolution level in which each amino acid is represented by several cg sites or beads . highly cg level , where each cg site or bead represents some number of amino acids ( e.g. , tens or more ) . a variety of modern coarse - graining approaches have been developed to define highly cg protein models , including essential dynamics coarse - graining , topology representing network , and rigid unit recognition . at the highly coarse - grained level , constructing cg models that satisfy the correct underlying physics is by no means a trivial task and often the resulting models are neither unique nor transferable . to simulate tmps at very large spatial and temporal scales relevant for most biological processes , it is both useful and necessary to resort to models of the lowest , such as ultracoarse - grained ( ucg ) models , where one cg site represents many amino acid residues and may also have internal states to represent the various conformations , chemical forms , etc . the ucg methodology , often motivated in the context of modeling of the actin filament , has only recently been applied to other families of proteins but not yet to tmps . this work therefore describes our most recent efforts to construct highly cg models for tmps based on the essential dynamics coarse - graining ( ed - cg ) method . the ed - cg method ( or a similar approach ) is a systematic variational way of creating cg models that capture the most essential functional motions of biomolecules by a direct mapping of their atomistic motions . in this context , the essential dynamics from the atomistic simulations is used as a proxy for the functionally relevant motions . the ed - cg method determines the assignment of atoms to cg sites ( the cg mapping ) such that the essential dynamics subspace is best preserved at a given resolution . the ed - cg method has been applied to a variety of globular proteins and protein complexes , including a ribosome , actin filaments , and a hydrogenase . however , two limitations of the ed - cg method should be taken into consideration . first , the ed - cg approach does not by itself automatically determine the optimal resolution level of a cg model . the total number of cg sites is an externally set parameter by the user . ( we note that this issue has been partially resolved in our previous work where we developed a set of criteria to choose optimal numbers of cg sites in different parts of a large biomolecular complex in a self - consistent way . ) second , there is no guarantee that the ed - cg technique will create the same cg model for a protein in two or more discrete functional states . a previous study from our group shows that the ed - cg models of globular proteins like g - actin only share 6080% of similarity between the atp- and adp - bound states . this creates a difficulty in using a cg representation , especially when it is desirable to study effects of transitions between distinct topological conformations . in this work , we have focused on addressing these issues for an important class of proteins , namely , tmps . as pointed out in prior work , it is important to understand the biomolecular features and peculiarities of the systems in order to construct meaningful cg models . it is generally appreciated in the field of coarse - graining that even small inadequacies in the cg mapping can manifest as damage beyond repair when the usual pairwise interaction potentials are used ; two - site methanol is a classic example of a problematic cg mapping for a molecular liquid . for tmps , the membrane environment imposes particular constraints onto the structure and dynamics of the transmembrane domains inserted into the lipid bilayer and differentiates them from extra- or intracellular domains of tmps or their soluble counterparts . such constraints give rise to many intriguing structural and dynamic properties of tmps to account for their functions , such as symmetry . although tmps often exist in multimeric symmetric complexes of several repeating subunits with similar tertiary structures ( even though the primary sequences of these subunits may be diverse ) , they also frequently possess approximate internal symmetry . this work is primarily focused on tmps with approximate internal symmetry , but the findings have the potential to be extended to cases with generalized symmetry ; a comparison is made between cg models built using ed - cg methods and ones built on a simple and intuitive heuristic that exploits the molecular symmetry . it is shown that , by exploiting symmetry , we are able to construct cg mappings of tmps for highly cg simulations consistent with the mappings resulting from the systematic bottom - up ed - cg method without the need for fine - grained md trajectories and complex numerical optimization schemes . in principle , the ed - cg method could be applied to all the atoms in a given protein structure . however , as a matter of practice , we use a residue - based strategy instead , wherein the position of each residue is represented solely by its c atom . given a protein of nres amino acid residues and ncg cg sites to assign ( nres ncg ) , we can calculate the ed - cg variational residual and use it as a measure of the accuracy of a cg mapping to an underlying atomistic md trajectory with nt frames . as defined in prior work , the residual is given by1where ried(t ) is the fluctuation of the c atom of residue i in the essential subspace at time t , calculated from principal component analysis of the atomistic md simulation . if the c atom of another residue j exhibits motion ( in the essential subspace ) similar to that of the c atom of residue i , then it is reasonable to assign residues i and j to the same cg site i. this idea is mirrored in the definition of the ( a cost function ) by summing terms of fluctuation differences |ried(t ) rjed(t)| over pairs of atoms belonging to the same cg site . in this scheme , the ed - cg method samples a variety of possible ways to group atoms / residues and selects the one with the minimum residual as the optimal cg model . the ed - cg approach comes in two main variations , namely , sequence - based and space - based ed - cg . both methods group the atoms into cg sites based on minimizing intrasite correlated fluctuations , but the different variants of the method applies different rules in sampling to locate the global minimum of . the sequence - based ed - cg method divides the primary sequence of the protein into contiguous cg domains , while the spaced - based ed - cg method favors cg site definitions with atoms / residues close in the three - dimensional space . provided the contiguous sequence constraint , the sequence - based ed - cg method is less demanding in sampling , but it does not permit nonadjacent domains in the same cg site , even if they are correlated in fluctuation but separated in the sequence ( for example , in the case of a disulfide bond ) . because of the much greater number of cg mappings allowed by space - based ed - cg , a brute - force search for the global minimum of would require looking through an exponentially greater number of combinations in comparison to sequence - based ed - cg . the use of simulated annealing and steepest descent techniques significantly decreases the number of combinations to be considered . nevertheless , the amount of computations required to achieve a reasonably low value of is still greater in the case of the space - based ed - cg , and this gap increases with the number of atoms or residues in the biomolecule under investigation . in our prior work , it was demonstrated that the ed - cg residual for the optimal cg map with a given number of cg sites can be approximated by a simple function of the protein size and the number of cg sites,2where the anomalous dimension is a protein - specific parameter , is a protein - independent coefficient , and c(t ) is a temperature - dependent prefactor . for a wide class of proteins , the value of was found to range from 0.00 to 0.91 ( however , tmps were not included into the studied set of proteins ) , while 0.35 . internal symmetry will provide additional restraints in the coarse - graining of tmps . in the context of biomolecules , we use the term internal symmetry for symmetry operations obeyed by the three - dimensional structure of the primary polypeptide chain sequence . on the basis of normal - mode analysis of md simulations and group theory , matsunaga and co - workers revealed that structural symmetry of homooligomers is a principal determinant of the entire protein complex s symmetric fluctuation . in the same way , tmps with internal symmetry should also have symmetric thermal fluctuation , which can be captured by ed - cg methods . mapped onto the cg model , the symmetric domains of a tmp should result in identical cg domains . directly , this suggests that the cg model should better describe symmetric fluctuation of the target tmp if it is consistent with the protein symmetry . in the simplest case of building a two - site cg model for a protein with perfect s - fold symmetry , we can always obtain the lowest ed - cg residual when either cg site contains half of the residues . our direct method ( without ed - cg ) of systematically constructing directly comparable cg mappings ( of adjustable resolution ) that satisfy the three - dimensional structural symmetry of the molecule that it represents is as follows . the contiguous protein sequence is evenly divided into ncg domains , which gives rise to a cg model that has an identical number of residues in each cg site ( setting aside rounding errors ) ; we shall refer to this construction as a symmetric model in this present work because these mappings satisfy a modular symmetry in the sense that each cg site is of equal size and separation in sequence space ( n.b . only a subset of these mappings will be consistent with the structural symmetry of the molecule ) . we have collected a representative benchmark data set of eight important tmps from protein data bank ( pdb ) ( table 1 ) that all exhibit approximate internal symmetry in order to compare the cg models built by the ed - cg method to these symmetric cg models and thereby elaborate on the necessity of preserving symmetries , exact or approximate , when constructing highly cg mappings for biomolecular systems . we selected a set of test cases by choosing tmps with internal symmetry and no missing residues in the sequence . our set of eight proteins represents tmps of different size , structure , symmetry , function , and complexity and includes structures of either -helical bundles or -barrels ( see table 1 and figure 1 ) . we note that all of these proteins are folded and fluctuate around the stable equilibrium structure with no large - scale conformational rearrangements . these protein models were set up in a membrane - bound environment before performing the atomistic md simulations . with maestro ( schrdinger , inc . ) , each pdb structure was prepared using protein preparation wizard and embedded in a 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) bilayer by the system builder . the tmp - membrane assemblies were placed in the simulation boxes , which were filled with explicit water ( tip3p water model ) and physiological salt ( 0.15 m nacl ) on both sides of the membrane . the distance between protein atoms and the box boundaries was at least 12 in all directions . charmm22/cmap protein and charmm36 lipid force fields were used to assign parameters with the tool viparr . after a 9-step standard relaxation protocol , which has been successfully applied in previous studies , each atomistic md simulation was run for 30 ns in the isothermal isobaric ensemble with constant temperature , t = 310 k , and constant pressure , p = 1 atm , using the martyna - tobias - klein coupling scheme . van der waals and short - range electrostatics were cut off at 9 . all md simulations were performed in the desmond 3.0 simulation package with an integration time step of 2 fs . hereafter , we applied the ed - cg method to build the cg models from the simulated all - atom md trajectories . plots were prepared using grace ( xmgrace ; http://plasma-gate.weizmann.ac.il/grace ) and numpy / matplotlib . for the different sets of cg models for each tmp with the same cg resolution level , we computed and analyzed the nave model similarity defined as the fraction of residues assigned to the same cg sites in the two compared models , where nres is the number of residues and m(i),n(i ) is the kronecker delta function adding to the similarity whenever residue i is mapped to the same cg site by the two mappings of equal resolution , m and n. initial tests were performed to compare the cg models built with space- and sequence - based ed - cg methods . we decided to proceed with the sequence - based variant as a suitable representative approach ; results were almost identical for the systems and resolutions studied in this paper ( in general , though , they will not be ) , as the space - based method exhibited much slower convergence rates . to compare the highly cg models built with the ed - cg method to the symmetric cg models , we calculated the value of the residual ( figure 2 ) over a range of different numbers of cg sites ( i.e. , the cg resolution ) , corresponding to the highly cg mapping regime where multiple amino acid residues are represented by a single cg site . it is seen that the residuals for the symmetric models tend to exhibit an oscillatory behavior compared to ed - cg models and that the period of this oscillation depends on the cg resolution . these oscillatory footprints indicate that the collective dynamics , which encompasses symmetric modes for structurally symmetric molecules , is better captured by cg mappings that preserve the dominant symmetries . since the calculated ed - cg residuals are a good proxy for the lower bound of the residual at a certain mapping resolution , we can identify a subset of the symmetric models that is optimal in the sense that the symmetric residual is almost identical to its lower bound for these models . the error of the symmetric model can be estimated by comparing its residual to the ed - cg -residual - minimized mapping . while this error tends to be small , it increases systematically whenever the cg mapping does not preserve the structural symmetry of the tmps , giving rise to what we shall call a symmetry mismatch that appears as an oscillatory difference in between the symmetric model and the ed - cg model ( figure 2 ) . it can therefore be eliminated to the point where the residual sym2 edcg2 by appropriately choosing the symmetric model that optimally aligns with the topological features of the tmp . the penalty for a symmetry mismatch follows the same power law relation as the ed - cg residual , and the relative error is therefore strongly dampened as the resolution of the mapping is increased . plots of the residuals for the symmetric mappings ( squares , green ) and the ed - cg method resulting mappings ( circles , red ) for the eight transmembrane proteins plotted against numbers of cg sites ( ncg ) . the panel with blue dots below each major plot shows the difference in between the symmetric model and the ed - cg model . for all the test cases ( table 1 and figure 1 ) , it is observed that , for low values of ncg ( highly cg models ) , the optimal subset of symmetric models always contains models for which the number of cg sites complies with the symmetry point group in the sense that for the tmp with approximate s - fold internal symmetry . when this rule is not obeyed , there will in general be a penalty in the residual . our results also show a number of differences between small and large tmps . for small proteins , such as tmem14a ( 75 residues ) , we observe excellent agreement between the two cg models when the ncg is a multiple of 3 , which can be visually understood looking at the cg map with 6 sites ( figure 3 ) . the relatively large symmetry - mismatch penalty observed in the residual for tmem14a is attributed to two factors : ( 1 ) the small size of the protein and ( 2 ) the fact that the protein has three modular repeats ( -helices in this case ) , which coincides with the approximate 3-fold axis of symmetry ( c3 ) . for the larger tmps in our set of test cases , this effect is weaker ( figure 2 ) . model similarity between the ed - cg mapping and the symmetric mapping at this level of resolution was very high ( 8090% ) in all tested cases . the backbone of the atomistic x - ray crystal structure is shown as translucent ribbons . the geometric planes that flank the molecule in the long ( transmembrane ) dimension are indicated by dashed - line triangles . the symmetry mismatch penalty for the higher resolution models is negligible . while model similarity between the ed - cg mapping and the symmetric mapping at this level of resolution for the tested cases varied somewhat ( 4575% ) , the absolute difference in the values of the residual is subtle ( figure 2 , lower panels ) . this makes physical sense because , at a certain threshold resolution ( here , 10 amino acids per cg site ) , there will be enough cg sites in the asymmetric subunit to adequately represent the dynamics of the unit in the essential subspace . these oscillations can be interpreted as mismatches ( albeit numerically very small compared with the previously described symmetry mismatches ) to the modular repeats in the secondary structure of the tmp . for -helical bundles ( -barrels ) , the modular repeats are the individual helix - loop ( strand - loop ) motifs . on the basis of the data plotted in figure 2 , we calculated the values of the anomalous dimension and the temperature - dependent prefactor c(t , nres ) , as defined by eq 2 . as shown in table 2 , falls in a small range around 1.0 for -helical bundles and in another small range around 1.5 for -barrels . these values are generally higher than the values previously reported for globular proteins like ubiquitin ( = 0.50 ) or g - actin ( = 0.33 ) , implying that decreases faster for tmps than for other proteins when the resolution of the cg mapping is increased . our results also show that the anomalous dimension falls within a very well - defined range for specific tmps with similar topology . in addition , the similar values between the sequence - based ed - cg models and the symmetric cg models indicate good agreement with respect to scaling behavior through the whole range of mapping resolutions . standard deviations of our estimates of are shown in parentheses . to explain why the values of in the case of tmps are typically higher than in the case of globular proteins studied earlier , we studied two simplified models : one of a solid ball and the other of a straight rod . the anomalous dimensions for these two extreme case model systems are demonstrated to be 0 and 1 , respectively ( see appendix a for details ) . most proteins considered in the previous work are globular ; hence , it is reasonable that their anomalous dimensions are typically closer to 0 . on the other hand , a set of straight rods , in the approximation of weak interactions between the rods , has the same anomalous dimension as a single rod does ( for details , see appendix b ) . this analytical result explains why the anomalous dimensions of tmps are closer to 1 and , therefore , greater than those for globular proteins . the difference in the anomalous dimensions of the two groups of proteins ( or , in general , any biomolecules ) leads to an interesting consequence for a multimolecular complex formed by weakly interacting nrod components ( such as -helices embedded into a lipid bilayer ) and nball ball - shaped molecules ( such as extra- or intracellular parts of membrane - associated proteins ) . in this case , an increase in the average resolution level of the cg model of the complex leads to a higher resolution representation of the ball - shaped parts in comparison to the rod - shaped parts or , in other words , the new cg sites added to the complex upon increasing resolution mainly end up in ball - shaped ( e.g. , extra- or intracellular ) components of the complex . in mathematical terms , if the total number of cg sites in the multimolecular complex is denoted ncgtotal , then the ratio of the optimal number of cg sites per each rod - shaped component ncg per rod to the optimal number of cg sites per each ball - shaped component ncg per ball has the following asymptotic behavior as ncgtotal :3or , equivalently , the fraction of cg sites within rod - shaped components decreases with the increase of the resolution level of a cg model of the complex4inversely , in coarser cg models , for example , ucg models , the optimal distribution of the cg sites implies a more detailed description of rod - shaped components ( e.g. , filamentous proteins or -helices in a protein ) . the oscillatory behavior of the (ncg ) curves for tmps with nsymm - fold rotational or rotoreflection symmetry can be explained on the basis of the universal scaling behavior for provided by eq 2 and the fact that the anomalous dimension for straight rods equals 1 ( see details in appendix b ) . the dependence of on ncg predicted by this simple model is shown in figure 4 in black solid lines . the behavior of these (ncg ) curves is qualitatively similar to those in figure 2 ( especially , tmem14a , amt1 , and cox1- ) despite the fact that the model of weakly interacting rods provides a simplified representation of dynamical behavior of tmps . a model of nsymm = 3 ( left panels ) and nsymm = 7 ( right panels ) weakly interacting straight rods demonstrates an oscillatory behavior of the (ncg ) curves ( shown with solid lines and circles ; the corresponding lower2(ncg ) curves are shown with dashed lines ) . therefore , the damped oscillatory behavior of the (ncg ) curves for tmps analyzed in this article ( see figure 2 ) is qualitatively captured by the simple model approximating tmps by several interaction rods . very recently , foley et al . investigated the connection between the entropic component of the potential of mean force ( pmf ) and the cg representation both in general terms and for concrete models , notably the gaussian linear chain model where an exact explicit pmf could be derived . their analysis suggests that there are bounds on the resolution range wherein information - efficient cg mappings can found . our results presented herein add a new perspective by emphasizing that careful consideration of structural symmetries and local modularities in approximately symmetric transmembrane proteins may help to choose between cg mappings that preserve a comparable fraction of nontrivial information . in this work , we have demonstrated that accurate and precise cg mappings can be generated for a diverse class of tmps without the use of computationally expensive md simulations and subsequent global residual minimization . to investigate the design principle in a general sense , we have studied cg mappings that partition transmembrane domains into contiguous segments of equal length along the primary sequence . the relative error in resulting from the use of the proposed heuristic rule is oscillatory and strongly damped , which has two practical consequences . second , it is possible for the heuristic to produce cg mappings with negligible relative difference in values to ed - cg methods even in the ucg regime , as long as the number of cg sites agrees with the overall symmetry group of the system ( most important for low - resolution cg models ) and conforms with the modular repeats ( most important for medium - resolution cg models ) . it is likely that this heuristic will be especially useful when used in conjugation with other procedures to select optimal cg mappings on a case - by - case basis . moreover , from the analysis of simple models , we predict that low resolution ucg models generated with the ed - cg approach should contain more cg sites in rod - shaped parts of proteins and protein complexes , such as -helices immersed into a lipid bilayer , while higher - resolution cg models with more cg sites contain a larger fraction of cg sites in ball - shaped parts of the system , such as extra- or intracellular parts . in summary , our study provides new insight into highly cg modeling of tmps and facilitates cg simulations by demonstrating that simple symmetry - preserving cg mappings are fast and reliable constructions , which have potential applications to future highly cg ( or ucg ) simulations of large tmps and tmp assemblies on long time scales .

vascular complications after renal transplantation occur frequently and can lead to the deterioration of renal function , graft loss , and even death . iliac vein stenosis is a rare complication after renal transplantation , with only a few cases reported to date , , . here , we describe a case of iliac vein stenosis that developed 16 years after transplantation in a 56-year - old renal transplant recipient . written informed consent was obtained from the study participant for publication of this case report and any accompanying images . a 56-year - old male with a 16-year history of renal transplantation was admitted for a painless right lower limb edema that had developed 1 month earlier . the leg swelling waxed and waned over the course of each day without continuous leg pain , chest discomfort , or dyspnea . the clinical workup revealed pitting edema and a loss of arterial pulsation in the right leg without discoloration or focal tenderness . laboratory examinations showed normal prothrombin time and activated partial thromboplastin time , with a d - dimer concentration of 0.52 g / ml fibrinogen - equivalent units ( feu ; reference concentration < 0.5 g / ml feu ) . his serum creatinine concentration was measured at 1.34 mg / dl , and his estimated glomerular filtration rate ( gfr ) according to the modification of diet in renal disease ( mdrd ) formula was 55 ml / min/1.73 m , which were slightly changed from his baseline serum creatinine concentration ( 1.1 mg / dl ) and estimated gfr ( 69 ml / min/1.73 m ) . radionuclide venography demonstrated a delay in upward drainage at the level of the right common iliac vein , and collateral flow into the left iliac vein . computed tomography ( ct ) angiography revealed severe stenosis in the right common iliac vein , with kinking at the bifurcation of the right common iliac artery . venous flow from the transplanted renal vein drained through the right external iliac vein , subsequently collecting in the regional collateral and left iliac veins . neither extrinsic compression due to perivascular fibrosis nor peritransplant fluid collection , such as hematoma and lymphocele , was detected . pelvic venography after puncture of the right groin with 7f sheaths confirmed near - complete occlusion of the right common iliac vein and collateral venous flow ( fig . a self - expanding 14 mm6 cm zilver vena stent ( cook medical , bloomington , in , usa ) was deployed without using angioplasty balloons , across the area of stenosis . follow - up venograms demonstrated a brisk flow through the right common iliac vein without collateral filling and a patent renal transplant vein ( fig . the right lower limb edema rapidly resolved after the intervention , and the patient s serum creatinine concentration stabilized at 0.91 mg / dl 3 days after venous stenting . the patient was free of procedure - related complications and was discharged in a stable condition with only aspirin therapy . after 6 months of follow - up , the patient remained asymptomatic with a functioning graft and the absence of lower limb edema . according to the patient s medical records , he had received a living related donor kidney transplant 16 years previously for end - stage renal disease secondary to hypertensive nephropathy . prior to transplantation , the patient had undergone hemodialysis for 14 years via a left - arm arteriovenous fistula , with no prior insertion of femoral dialysis catheters . the transplant kidney had two arteries and two veins , which were terminolaterally anastomosed onto the corresponding right external iliac vasculature . his postoperative course was uneventful , with neither surgical complications nor graft dysfunction requiring dialysis . the patient was discharged with a well - functioning graft and a serum creatinine concentration of 1.1 mg / dl . five years after renal transplantation , the patient was admitted to hospital because of an abrupt onset of graft tenderness , decreased urine output , and elevated level of serum creatinine ( 5.1 mg / dl ) . prerenal causes of acute kidney injury were less likely because the fractional excretion of filtered sodium was 1.2% . a doppler ultrasonography ( us ) examination of the graft showed that echogenicity and flow in the renal vasculature were within normal limits , with a resistive index of 0.74 . us also revealed no signs of abnormal fluid collection or hydronephrosis . when evaluating the renal biopsy to differentiate causes of graft dysfunction , azotemia was rapidly resolved without specific treatment . his serum creatinine concentration was measured as 2.4 mg / dl 2 days after admission and as 1.4 mg / dl at the time of discharge . the exact cause of graft dysfunction remained unknown , but no further diagnostic evaluation was attempted owing to obvious clinical improvement . this episode of acute kidney injury may have been due to chronic venous stenosis and transient thrombosis in the right common iliac vein , although lower extremity edema was not apparent . his serum creatinine level returned to baseline ( 1.1 mg / dl ) 2 weeks after discharge , and remained around the baseline concentration until the development of iliac vein stenosis . once considered a rare complication of renal transplantation , iliac vein stenosis has been consistently reported since 1972 and shown to be related to may thurner syndrome in left - side transplant recipients , . the risk factors for development of iliac vein stenosis include the prior insertion of femoral dialysis catheters , postoperative fluid collection , hematoma , urinoma , and lymphocele . renal transplant vein stenosis has also coincided with acute graft rejection , local infection , kinking of the renal vein , and external pressure / compression by the crossing iliac artery . none of these postoperative complications was observed in our patient . it has been suggested that chronic trauma may lead to intimal proliferation with scarring and septal formation in may similarly , in renal transplant recipients , a local inflammatory reaction due to chronic extrinsic pressure may induce repetitive endothelial injury and subsequent vessel stenosis . surgical clamp injury , intimal dissection , and faulty suturing may also damage the vein directly , resulting in intimal proliferation and fibrosis . our patient may have experienced gradual fibrosis or scarring of the vessel wall caused by chronic compression from the bifurcation of the right common iliac artery . however , the abrupt narrowing and dilatation of the downstream vessel in our patient were not typical findings . iliac vein stenosis can be complicated by thrombosis of the iliofemoral and/or transplanted renal vein , . most patients present with distinctive clinical features , including lower limb edema ipsilateral to the transplant kidney and varying degrees of allograft dysfunction ( table 1 ) . occasionally , acquired renal transplant vein stenosis may present with similar clinical findings , although unilateral leg swelling may not occur , . although the clinical symptoms and signs are nonspecific , the co - occurrence of unexplained graft dysfunction combined with ipsilateral leg swelling in renal transplant recipients should be regarded as indicative of iliac vein stenosis . other causes of renal allograft dysfunction , including ureteral obstruction , acute graft rejection , and bk virus nephropathy , should be excluded . the differential diagnosis should also include deep vein thrombosis of the lower extremity . because slowly developing venous stenosis can progressively worsen renal allograft function but remain undiagnosed for several years after transplantation , as in our current case , a high index of suspicion should be maintained until diagnosing this rare disease . the absence of venous flow and/or reversed arterial diastolic flow is an indirect indication of acute venous complications in renal transplant recipients . without thrombosis and acute pressure overload in the renal vasculature , however , chronic venous stenosis may only moderately impair venous flow , making it difficult to detect on us examination . when us evaluation shows no reasons for allograft dysfunction , additional imaging methods , such as ct angiography , should be performed . further imaging can reveal several obvious causes , including peritransplant fluid collections and deep vein thrombosis . only by thorough evaluation , interventional radiological management , including catheter - directed thrombolysis and angioplasty with or without endovascular stenting , has been found to improve allograft function in some patients with iliac vein stenosis , , . balloon angioplasty alone without stent implantation is of little use owing to unsatisfactory long - term results , including early restenosis and resistance to ballooning . recently , iliofemoral venous stents that were purposefully designed for the iliofemoral venous segment were introduced , demonstrating better flexibility , larger diameters , longer lengths , and higher radial force . to our knowledge , no previous studies have described the use of these newly developed stents to manage complications of renal transplantation . a successful endovascular stenting at the right common iliac vein of our patient restored venous patency and immediately relieved his lower extremity edema . in summary , patients with a swollen leg and renal allograft dysfunction , even months or years after transplantation , should be suspected of having iliac vein stenosis , a rare , but potentially treatable , complication of renal transplantation . percutaneous angioplasty with endovascular stenting is a safe and effective approach and may be the treatment of choice for managing iliofemoral venous stenosis . the authors have no commercial conflict of interest to report , and nothing to disclose .

there are a few qol questionnaires dedicated for patients with ais : srs-22 , scoliosis quality of life index ( sqli ) and sf-36 seem to be the most diffused [ 2 , 3 ] . two questionnaires for monitoring the level of stress in patients currently being managed conservatively for progressive scoliosis have been constructed : they are designated to assess the stress induced by the deformity ( bad sobernheim stress questionnaire deformity , bssq - deformity ) , as well as the stress induced by the treatment with a brace ( bad sobernheim stress questionnaire these questionnaires do not assess the overall qol . in 2006 vasiliadis et al . proposed the brace questionnaire ( brq ) , an instrument for measuring the qol of scoliotic adolescents who are being treated conservatively with wearing a corrective brace . being familiar with its english version ( not validated ) , we have found this questionnaire to be potentially helpful for monitoring the qol of our patients . the aim of the study was to carry on the process of cultural adaptation of the brq into polish . brace questionnaire is a 34 likert scale items questionnaire , and consists of eight specific domains ; they are : ( a ) general health perception , ( b ) physical functioning , ( c ) emotional functioning , ( d ) self - esteem and aesthetics , ( e ) vitality , ( f ) school activity , ( g ) bodily pain and ( h ) social functioning . the questionnaire was designed to be self - administrated and developmentally appropriate for ages 918 years . scoring of the brq was planned as follows : for items 4 , 5 , 6 , 12 , 14 , 15 , 16 , 17 always receives a score of 5 , most of the time receives a score of 4 , sometimes receives a score of 3 , almost never receives a score 2 and never receives a score of 1 . for items 1 , 2 , 3 , 7 , 8 , 9 , 10 , 13 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 always receives a score of 1 , most of the time receives a score of 2 , sometimes receives a score of 3 , almost never receives a score of 4 and never receives a score of 5 . next , each item score is multiplied by 20 and the total score is divided by 34 . thus , the minimum score of the questionnaire is 20 and the maximum is 100 . higher scores are better quality of life . a subscale score can be calculated for each of the eight domains by dividing the total score of each dimension by the number of its items . the process of cross - cultural adaptation of the brq was performed in accordance with the guidelines set up by international quality of life assessment ( iqola ) . in the first stage one of the translators , who had a medical background , was instructed on the whole process of adaptation . second stage consisted of comparison of the original and two translated versions . during that stage , the two translators and the authors identified differences in translations and produced a combined version . in the third stage the so - called reversed translation two independent translators , who were native in greek , translated the polish version into the language of the original document ( greek ) . the objective of this stage was to assure equivalence of the two versions and to identify possible mistranslations . at the last fourth stage , a commission composed of a specialist in orthopaedics , translators , a statistician and a psychologist reviewed the translations . as a result of consensus , the so - called pre - final version was drafted . thirty - five patients with idiopathic scoliosis were enrolled for the assessment using the polish version of brq , twice within one - week interval . all patients were treated with the same kind of brace ( chneau orthosis ) and by the same specialist in orthopaedics ( second author ) . all patients and parents gave their informed consent prior to their inclusion in the study . the following inclusion criteria were applied : ( 1 ) patients at the age of 918 years , ( 2 ) who have been wearing the brace for at least 3 months for at least 12 h per day , ( 3 ) with cobb angle between 20 and 45 , ( 4 ) having thoracic , thoracolumbar or lumbar scoliosis . mean age of the patients at the time of completing the questionnaire was 14.0 years ( 1.5 years ) , for details see table 1 . patients have been wearing the brace for an average duration of 17.9 months ( 11.7 months ) . patients have been wearing the brace for 17.0 h per day ( 5.2 h ) . among the patients , 25.7% of patients had left curve pattern and 74.3% of patients had right curve pattern.table 1description of the study subjectsmean ( sd)age ( years)14.0 ( 1.5)body weight ( kg)48.0 ( 8.9)height ( cm)162.5 ( 8.4)cobb angle ( degrees)35.1 ( 10.6)angle of trunk rotation ( degrees)7.0 ( 2.9)angle of trunk rotation as measured with bunnell scoliometer description of the study subjects angle of trunk rotation as measured with bunnell scoliometer statistical analysis was performed using statistica 9.1 software . shapiro wilk test for normality did not identify the data to be normally distributed ; therefore , non - parametric tests were used . firstly , descriptive statistics was used to calculate mean scores and standard deviations for a given question and a domain . retest design was used to measure temporal stability of the questionnaire with kendall s tau ( r ) coefficient . to reduce the memory effect , there was a 7-day period between tests . the brq was assessed for item convergent validity ( item - scale correlation should be 0.4 ) , floor and ceiling effects . the distribution of results indicates the number ( percentage ) of patients with minimum score ( floor effect ) and the number ( percentage ) of patients with maximum score ( ceiling effect ) . polish adaptation of brq as a research project has been approved by the bioethical commission at the university ( decision number 541/11 ) . average , lowest , highest scores and 95% confidence interval obtained using the brq are presented in table 2 . the mean score for the brq was 77.1 points ( 12.2 points ) in the first test and 76.5 points ( 12.1 points ) in the second test . the mean duration of completing the questionnaire was 7.9 min ( 1.36 min).table 2distribution of mean , minimal and maximal scores , 95% confidence interval of polish brqquestionnairenminmaxmean95% confidence intervalstandard deviationfromtobrq first trial35529277.170.779.112.2brq second trial35529176.571.379.612.1 distribution of mean , minimal and maximal scores , 95% confidence interval of polish brq value of cronbach s alpha and kendall s tau ( r ) coefficient of the polish version of the brq assessed with the use of test retest method in comparison with greek results are presented in table 3.table 3the value of cronbach s alpha and kendall s tau coefficientsquestionnairepolish versiongreek version ( original)cronbach alphakendall tau ( r)cronbach alphakendall tau ( r)brq0.940.820.82 the value of cronbach s alpha and kendall s tau coefficients mean , standard deviation , floor and ceiling effects for each brq question are presented in table 4 , while the values for each brq domain are presented in table 5 . mean values for individual brq questions ranged from 1.0 ( question 34 ) to 5.0 ( questions 5 , 6 , 20 , 22 , 28 and 31).table 4mean , standard deviation , floor and ceiling effects for each brq questionbrqmeanstandard deviationnumber ( % ) floor effectceiling effectquestion 14.00.80 ( 0.0)11 ( 31.4)question 23.51.00 ( 0.0)7 ( 20.0)question 33.51.21 ( 2.9)6 ( 17.1)question 43.01.24 ( 11.4)4 ( 11.4)question 55.00.50 ( 0.0)32 ( 91.4)question 65.00.10 ( 0.0)33 ( 94.3)question 74.01.10 ( 0.0)9 ( 25.7)question 84.01.22 ( 5.7)8 ( 22.9)question 94.01.01 ( 2.9)7 ( 20.0)question 104.00.90 ( 0.0)13 ( 37.1)question 114.01.00 ( 0.0)12 ( 34.3)question 123.01.26 ( 17.4)0 ( 0.0)question 132.51.45 ( 14.3)5 ( 14.3)question 144.01.00 ( 0.0)11 ( 31.4)question 153.51.00 ( 0.0)6 ( 17.4)question 163.01.02 ( 5.7)1 ( 2.9)question 173.50.91 ( 2.9)2 ( 5.7)question 183.51.23 ( 8.6)3 ( 8.6)question 194.51.00 ( 0.0)16 ( 45.7)question 205.00.80 ( 0.0)31 ( 88.6)question 214.51.00 ( 0.0)17 ( 48.6)question 225.00.60 ( 0.0)26 ( 74.3)question 234.50.90 ( 0.0)16 ( 45.7)question 244.01.00 ( 0.0)9 ( 25.7)question 254.01.00 ( 0.0)10 ( 28.6)question 264.50.90 ( 0.0)15 ( 42.9)question 273.51.43 ( 8.6)11 ( 31.4)question 285.01.12 ( 5.7)18 ( 51.4)question 293.01.13 ( 8.6)2 ( 5.7)question 303.51.42 ( 5.7)9 ( 25.7)question 315.00.70 ( 0.0)24 ( 68.6)question 324.51.22 ( 5.7)15 ( 42.9)question 334.01.11 ( 2.9)14 ( 40.0)question 341.01.022 ( 62.9)1 ( 2.9)table 5mean and standard deviation for each brq domainbrq domainnumber of itemsmedianstandard deviationgeneral health perception27.51.5physical functioning727.54.3emotional functioning517.54.2self esteem and aesthetics27.02.0vitality27.01.7school activity314.02.2bodily pain625.54.4social functioning725.55.0 mean , standard deviation , floor and ceiling effects for each brq question mean and standard deviation for each brq domain item convergent validity , cronbach s alpha , floor and ceiling effects for each brq domain are presented in table 6 . there were no floor or ceiling effects when completing the questionnaire for the first and the second time.table 6item convergent validity , cronbach s alpha and floor and ceiling effects for each brq domainbrq domainnumber of itemsitem convergent validity ( % ) cronbach s alphafloor effect ( % ) ceiling effect ( % ) general health perception21000.510 ( 0)3 ( 8)physical functioning7300.740 ( 0)0 ( 0)emotional functioning51000.820 ( 0)0 ( 0)self esteem and aesthetics21000.910 ( 0)1 ( 3)vitality200.520 ( 0)0 ( 0)school activity31000.710 ( 0)0 ( 0)bodily pain6700.820 ( 0)4 ( 11)social functioning7400.770 ( 0)0 ( 0 ) item convergent validity , cronbach s alpha and floor and ceiling effects for each brq domain cronbach s alpha is considered to be a proper method for estimating reliability of multi - item scales , it provides an estimate of internal consistency that expresses both the number of items and their average correlation . even though cronbach s alpha disregards other possible material sources of measurement error ( e.g. temporal instability ) , these sources of measurement error usually have a minimal impact on the measure of reliability . the polish version of the brq had a high value of cronbach s alpha coefficient ( 0.94 ) , exceeding the minimum recommended value of 0.80 and indicating satisfactory internal consistency as a factor of satisfactory reliability of the brq . preliminary validation of the italian version of the brq questionnaire had the value of cronbach s alpha of 0.86 , indicating satisfactory internal consistency . kendall s tau ( r ) coefficient of the polish version of the brq assessed with the use of test retest method was 0.82 . the criterion for item convergent validity ( item - scale correlations 0.40 ) was fulfilled by the items related to general health perception , emotional functioning , self - esteem and aesthetics , school activity , bodily pain and social functioning . in the present study , the criterion for item convergent validity was not fulfilled by the items related to physical functioning and vitality . in the italian validation , the test retest reliability showed a good temporal stability ( r = 0.88 , p < 0.001 ) . for the brq overall score , in the present study 0% of patients scored at floor and 0% scored at ceiling . therefore , there were no floor or ceiling effects for the brq overall score . conservative treatment of scoliosis with a rigid brace can have a significant impact on patients wellbeing and negatively affect their qol . ais can lead to multiple impairments , non - only of physical but also of psychosocial character [ 7 , 9 ] . the effectiveness of the conservative scoliosis treatment has been demonstrated to be dependent on the patients treatment compliance [ 10 , 11 ] . the level of stress during therapy is one of the factors determining compliance and can be assessed using the bssq questionnaire . weiss reported brace treatment to be associated with higher level of stress and poor quality of life . noticed that the bssq is helpful for determining the level of stress during scoliosis therapy . however , the bssq is not able to measure the influence of family , school environment or physical activity on patient s qol . the brq is the first questionnaire specially developed and validated to measure the quality of life of adolescent currently being under conservative scoliosis treatment with a corrective brace . according to vasiliadis and grivas , when assessing the effectiveness of conservative treatment of ais , the health - related quality of life ( hrqol ) variables are more important than radiographic results or pulmonary function tests . emphasize growing interest in demonstrating the effect of treatments on the health - related quality of life of patients with idiopathic scoliosis . according to vasiliadis et al . , a specific instrument , such as the brq , has evident strengths by virtue of its increased sensitivity to the problems related to the brace itself . emphasize that qol monitoring should be routinely implemented during brace treatment , with type of bracing , gender , curve pattern and cobb angle taken into account , to provide professional psychological support if needed . our results indicate that the brq is an effective tool for evaluating qol of patients with ais being treated with a corrective brace . cronbach s alpha is considered to be a proper method for estimating reliability of multi - item scales , it provides an estimate of internal consistency that expresses both the number of items and their average correlation . even though cronbach s alpha disregards other possible material sources of measurement error ( e.g. temporal instability ) , these sources of measurement error usually have a minimal impact on the measure of reliability . the polish version of the brq had a high value of cronbach s alpha coefficient ( 0.94 ) , exceeding the minimum recommended value of 0.80 and indicating satisfactory internal consistency as a factor of satisfactory reliability of the brq . preliminary validation of the italian version of the brq questionnaire had the value of cronbach s alpha of 0.86 , indicating satisfactory internal consistency . kendall s tau ( r ) coefficient of the polish version of the brq assessed with the use of test retest method was 0.82 . the criterion for item convergent validity ( item - scale correlations 0.40 ) was fulfilled by the items related to general health perception , emotional functioning , self - esteem and aesthetics , school activity , bodily pain and social functioning . in the present study , the criterion for item convergent validity was not fulfilled by the items related to physical functioning and vitality . in the italian validation , the test retest reliability showed a good temporal stability ( r = 0.88 , p < 0.001 ) . for the brq overall score , in the present study 0% of patients scored at floor and 0% scored at ceiling . therefore , there were no floor or ceiling effects for the brq overall score . conservative treatment of scoliosis with a rigid brace can have a significant impact on patients wellbeing and negatively affect their qol . ais can lead to multiple impairments , non - only of physical but also of psychosocial character [ 7 , 9 ] . the effectiveness of the conservative scoliosis treatment has been demonstrated to be dependent on the patients treatment compliance [ 10 , 11 ] . the level of stress during therapy is one of the factors determining compliance and can be assessed using the bssq questionnaire . weiss reported brace treatment to be associated with higher level of stress and poor quality of life . noticed that the bssq is helpful for determining the level of stress during scoliosis therapy . however , the bssq is not able to measure the influence of family , school environment or physical activity on patient s qol . the brq is the first questionnaire specially developed and validated to measure the quality of life of adolescent currently being under conservative scoliosis treatment with a corrective brace . according to vasiliadis and grivas , when assessing the effectiveness of conservative treatment of ais , the health - related quality of life ( hrqol ) variables are more important than radiographic results or pulmonary function tests . emphasize growing interest in demonstrating the effect of treatments on the health - related quality of life of patients with idiopathic scoliosis . according to vasiliadis et al . , a specific instrument , such as the brq , has evident strengths by virtue of its increased sensitivity to the problems related to the brace itself . emphasize that qol monitoring should be routinely implemented during brace treatment , with type of bracing , gender , curve pattern and cobb angle taken into account , to provide professional psychological support if needed . our results indicate that the brq is an effective tool for evaluating qol of patients with ais being treated with a corrective brace . the brq takes less than 10 min to be completed and covers most of the aspects of life affected by the brace . polish version of the brq is reliable and can be used in adolescents with idiopathic scoliosis wearing the brace to assess their quality of life . 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 . gorset ortopedyczny ponisza ankieta zawiera pytania dotyczcego tego , co mylisz o stanie swojego zdrowia i jak si czujesz . nie jest to aden test , w ktrym istniej poprawne i bdne odpowiedzi.przeczytaj uwanie kade pytanie.wybierz odpowied , ktr uwaasz za waciw i postaw x w odpowiednim kwadracie . prosimy , o podanie nam informacji dotyczcych ciebie : jeste : dziewczyn chopcem wiek : lat data . w ostatnich trzech miesicach nigdyrzadkoczasamiczstozawsze1 uwaasz , e terapia z zastosowaniem gorsetu bya dla ciebie korzystnaw ubiegym miesicu nigdyrzadkoczasamiczstozawsze15 . bya/-e z siebie dumny/-a16 . bya/-e z siebie zadowolona/-y17 z powodu noszenia gorsetu miaa/-e trudnoci w odrabianiu lekcji i z nauk20 . z powodu noszenia gorsetu opuszczaa/-e zajcia szkolne21 . uwaasz , e twoje relacje z rodzin i koleankami / kolegami byyby lepsze , gdyby nie nosia/- gorsetu33 .

the robert wood johnson foundation funded hablamos juntos ( hj ) , a $ 10-million multiyear demonstration to improve access to health care for latinos with limited english proficiency and to explore cost - effective ways for health care organizations to provide language access services . in this manuscript , the authors draw on their experiences in evaluating hj , provide brief descriptions of innovative interventions , estimate operating costs , and synthesize lessons learned about implementation . , it highlights the need for health care organizations to involve physicians in the design and adoption of language services . in 2002 , rwjf launched the hj national demonstration ( http://www.hablamosjuntos.org/ ) to develop affordable models for health care organizations to offer language access services to latino patients with lep . the demonstration provided grants of up to $ 1 million to 10 organizations in communities with new and rapidly growing latino populations . there were 3 main areas of required activities pertaining to language services as set out by the national program office ( npo ) for the hj demonstration . first , sites were required to plan and carry out activities related to the recruitment , assessment , training , and placement of medical interpreters at key access points in their health care systems . second , sites were required to develop activities to create an environment and culture supportive of interpretation , such as developing institutional leadership , recruiting physician champions , and providing payment for medical interpretation services . third , sites were asked to beta test an assessment of language proficiency and develop comprehensive training programs for interpreters . in addition , some sites also put resources into translation and development of new spanish language materials , and supported the npo s efforts to develop universal way - finding symbols for hospitals and clinics . the foundation and the npo purposefully selected a diverse group of organizations for this demonstration , each with a distinct approach to addressing language access in their communities . this seemed prudent at the time ( 2002 ) given the lack of an evidence base to suggest that any one approach or model would be superior to another . rwjf also provided grants to the rand corporation to conduct a cross - site evaluation of the hj demonstration . briefly , the goals of the evaluation were to provide sufficiently detailed descriptions of the interventions , their implementation , and operating costs ; to enhance the likelihood of replication at other sites ; and to show the impact of the interventions on patient - centered outcomes of care . the methods and detailed findings from the evaluation are reported elsewhere.18 below we briefly describe the interventions and discuss lessons learned from the evaluation , focusing on the costs , barriers , and facilitators of implementation . these observations are drawn from a multisite case study in which we collected and analyzed descriptive information from key informant interviews with a broad range of hj project stakeholders in each of the 10 hj sites , and from a cost study in which we collected cost information from project directors in 8 hj sites . the grantee organizations included 4 hospital systems , 2 medicaid health maintenance organizations ( hmo ) , 1 area health education center ( ahec ) , 1 school of public health , and 2 community organizations . all but 2 of the hj demonstration projects provided language access services in clinical health care settings , most commonly in emergency departments ( eds ) and obstetric / gynecology units , which are critical access points for latino patients . table 1project features of the participating sites and estimated annual operating costs in 2004 dollars ( arranged in descending order of costs)site idgrantee organizationlocation in census region ( urban / rural)demonstration unitsdemo . unit patient volumelatino ( % ) lep ( % ) among latinoprimary interventionstraining interventionpersons in projectinterpreter fteestimated annual costs*site_1hospital systemsouth ( mixed)a language services agency that is partnering with 13 clinical units in three hospitalsn / an / an / adeveloped a new language services agency providing on - site and on - call interpreters to local hospitalscollege certificate ( no credit)4515.5$1,181,000site_2medicaid hmonortheast ( urban)health plan partnering with 4 hospitals involving 6 clinical units ( ed , ob / gyn , urgent care)16,700/mo.2,500/mo . ( 28%)provided reimbursement for medical interpretation and supported development of interpreter programs in partner hospitalsno educational partner3512.5$989,000site_3medicaid hmowest ( urban)a call center to serve spanish - speaking members of health plan81,00046,500 ( 57%)34,000 ( 73%)provided 24/7 bilingual nurse advice line offering nurse advice and medical interpretationmodule for medical school standardized lep patient exam and use of interpreters346$848,000site_4hospital systemnortheast ( urban)two hospital eds and one hospital ob / gyn unit8,200/mo.3,600/mo . ( 50%)provided dedicated , dual role and contracted interpretersgraduate - level interdisciplinary seminar series ( no credit)94$561,000site_5area health education centermidwest ( rural)ahec with a video interpreter service partnering with five hospitals and two higher education institutionsn / an / an / aprovided video medical interpreters via high speed communication links among partnering hospitalscommunity college certificate ( credit)133.25$534,000site_6hospital systemsouth ( mixed)the entire hospital but focus on ob / gyn inpatients1,400/mo.183 ( 13%)165 ( 90%)improved availability of interpreters to match patient demandcollege course ( credit)124$492,000site_7hospital systemsouth ( urban)two hospital eds and one hospital ob / gyn unit20,000/mo.1,900/mo . ( 85%)improved availability and accessibility of dedicated medical interpreters / patient navigatorsin development185.4$402,000site_8community organizationwest ( rural)the community organizationn / an / an / aworked to affect state and service provider policy on language access services , assisted partner organizations , and provided interpretation for clientscommunity college course ( no credit)13n / a$324,000site_9community organizationsouth ( mixed)a full - service language services agency for hire by clinical institutionsn / an / an / acreated a language services agency for interpreter training and supplyin developmentdid not participate in cost studysite_10school of public healthwest ( urban)a school of public health partnering with six provider sites for internship rotationn / an / an / aestablished a health interpreting and health applied linguistics graduate - level program with internship ( credit ) and developed spanish health education materialsdid not participate in cost studyn / a = not applicable , ed = emergency departments , ob / gyn = obstetric / gynecology , lep = limited english proficiency , fte = full - time equivalent*the costs were operating costs in providing the primary interventions from october 2004 to september 2005 , valued by 2004 dollars.number of medicaid hmo members in the service area.these are bilingual registered nurses providing nurse advice and medical interpretation . there were additional 3.75 fte bilingual medical assistant receptionists transferring latino members calls to a bilingual nurse , as well as providing membership services to these members ( e.g. , verifying membership , scheduling appointment).because of conflicts that developed within the program , the site was dropped before the end of the national demonstration . project features of the participating sites and estimated annual operating costs in 2004 dollars ( arranged in descending order of costs ) n / a = not applicable , ed = emergency departments , ob / gyn = obstetric / gynecology , lep = limited english proficiency , fte = full - time equivalent * the costs were operating costs in providing the primary interventions from october 2004 to september 2005 , valued by 2004 dollars . there were additional 3.75 fte bilingual medical assistant receptionists transferring latino members calls to a bilingual nurse , as well as providing membership services to these members ( e.g. , verifying membership , scheduling appointment ) . because of conflicts that developed within the program , the site was dropped before the end of the national demonstration . in the majority of the study sites three hospitals hired , trained , and certified additional professional interpreters and redeployed interpreters in response to system and patient care demands ( sites 4 , 6 , and 7 ) . two of these hospitals also offered financial incentives ( salary increase or bonus ) to bilingual staff to encourage them to be assessed for language proficiency and to receive interpreter training . one of the hospital systems developed an innovative model , called the patient navigator , to allow interpreters to accompany patients between clinical departments ( e.g. , ed and radiology ) , rather than restricting the interpreters to a single unit , to enhance continuity within an episode of care ( e.g. , an ed visit ) . two grantees collaborated with local partners to establish language service agencies ( sites 1 and 9 ) . these agencies recruited , trained , and deployed their interpreters to the participating health care facilities . one of the participating medicaid hmo grantees encouraged the use of interpreters by reimbursing interpreter services ( site 2 ) . the other medicaid hmo enhanced its existing member call center with a bilingual nurse advice line providing interpretation , patient referral , and support 24 hours a day ( site 3 ) . the ahec grantee , which was located in a rural area with widely dispersed patients and providers , purchased equipment , wiring , and routers for its partnering hospitals and provided video medical interpretation via high - speed telecommunication links with the hospitals ( site 5 ) . the school of public health grantee developed a masters degree program in medical interpreting and health applied linguistics that included internship rotations in health care facilities ( site 10 ) . the final site , another community organization serving a mostly rural area , focused on advocacy for policy change at the state and local levels . they provided assistance to local health care providers as well as direct language assistance to clients applying for public benefits ( site 8) . most sites fulfilled the hj interpreter training requirement by collaborating with local educational institutions to develop college - level interpreter training programs . we defined operating costs of the hj interventions as the dollar value of the resources ( including labor , contracts , equipment , materials , and supplies ) used by the grantee and all major partners . costs were assessed for 1 year from october 2004 to september 2005 , which corresponds to the second , and last , year of the hj demonstration . by that point all sites had implemented their interventions . our cost analysis methodology is described elsewhere.18 on average , the annual costs of operating the hj interventions were $ 666,000 per site , with a range of $ 324,000 to $ 1,181,000 ( see table 1 ) . the hj sites that provided interpreter services in the greatest number of clinical units incurred the highest costs ( sites 1 and 2 ) . another high - cost site operated a bilingual nurse advice line within a call center serving a large population of medicaid hmo members ( site 3 ) . these three sites had many more personnel and full - time equivalent interpreters involved in the hj intervention than the other sites . the three hospital systems that focused on improving the availability of interpreters in a few high - volume clinical units ( sites 4 , 6 , and 7 ) had fewer personnel and incurred lower costs . these cost findings can serve as a rough guideline for other similar health care organizations interested in implementing similar interventions in their communities . the case studies of the hj sites were organized according to a framework suggested by a recent systematic review of the literature on adoption and diffusion of innovation in service organizations.19 we observed that there were a number of barriers and facilitators that , in part , explained some of the strengths and weaknesses we observed in the implementation of the hj interventions in the various sites . a variety of motivations facilitate change administrators , staff , and stakeholders at each of the hj sites were asked to reflect on the primary motivations of the grantee and their partners , including internal and external forces for change . most reported some combination of a growing latino population with lep in their catchment area , a sense of altruism that most health care organizations share , and concerns about the legal ramifications of ignoring either the office of civil rights guidance on title vi16 or their own concerns about malpractice risks associated with language barriers . in only a few cases were sites responding to pressure from latino advocacy organizations as many of their constituency were immigrants who did not feel empowered to demand better services . providers need convincing about the quality rationale whereas some projects spent much time cultivating and persuading administrators , the assumption was that the usefulness of the interpreters would be obvious to physicians and other health care providers . most hj project leaders assumed they would not need to convince physicians of the need to hire , support , and utilize qualified medical interpreters in their practice settings . many believed that once they were able to assess , train , and certify interpreters as qualified , hospitals , clinics , physicians and nurses would immediately recognize the difference between a professionalized workforce and the ad hoc interpreters they had previously used ( e.g. , family , bilingual housekeeping staff ) . whereas reports from administrators were mixed , front - line interpreters routinely told us that they faced an uphill struggle to integrate themselves into health care teams . unfortunately , most of the project leaders realized too late in the intervention period that they had not sufficiently communicated to physicians the rationale for adoption of interpreter programs . implementation strategies to ensure program adoption are critical in many ways , at both the hj national demonstration level and the local project level , there was a lack of attention to handling implementation within busy health care institutions . many physicians and nurses had become used to improvising and in busy and often chaotic settings ( e.g. , eds ) did not feel they could wait for an interpreter to be paged or to finish interpreting for another patient . because coverage was not 24/7 in any of the sites , there were different procedures for accessing language services at different times of day . and in academic medical settings residents and other trainees work in rotations , meaning that new staff are constantly being introduced . unfortunately , to the extent that hj projects attempted to orient physicians and nurses to the new interpreter programs , they did so through in - service trainings at busy staff meetings rather than through the methods that the literature suggests are effective , such as directly involving physicians in the planning for the program design and implementation , and cultivating physician champions to foster behavior change . interpreter proficiency standards are lacking the hj national demonstration put a spotlight on the need to develop comprehensive training programs for medical interpretation , and most hj sites found educational partners and worked with them to develop credit or noncredit courses or certificates . however , many of the sites also sponsored 40-hour training using the bridging the gap curriculum.20 there was no agreement across sites as to how much training is appropriate and there are no universally accepted benchmarks by which to judge the proficiency of interpreters . several hj sites suggested that different levels of training may be required for interpreters depending on the nature of the communication being interpreted . for example , ad hoc interpreters may be adequate for helping patients navigate a health care facility whereas highly proficient interpreters are needed to interpret in clinical encounters . sites participated in beta testing of a language proficiency examination but it received only mixed reviews . the field still lacks benchmarks and tools to measure proficiency . uncertain job market for trained interpreters threatens sustainability hj projects spent time and resources attempting to professionalize the role of interpreter and create educational programs that will produce a steady stream of individuals qualified to provide medical interpretation services . the fact is , however , these are low - wage jobs with little in the way of a career ladder . interpreters , especially the least experienced ones , struggled to gain respect from the health professionals with whom they worked . some reported that their motivation to become an interpreter was intrinsic but whether such motivations will be enough to prevent high rates of turnover is questionable . whether the educational programs initiated under the hj grants will be able to sustain themselves depends on whether individuals are willing to pay for college ( or graduate level training ) to get a low - wage job . in a few instances , employers were willing to subsidize the costs of training it remains an open question whether there will be sufficient demand for medical interpreters in local markets to sustain college - level training programs . physicians tend to avoid tele - technology given the costs of in - person interpretation , some have suggested that technology is the only reasonable approach to providing language access services for large numbers of patients and/or across large institutions . however , physicians and nurses in the hj sites preferred on - site interpreters and tended to avoid telephonic language lines and remote video - conferencing , even when attempts were made to make the technologies readily available and user - friendly . the business case for medical interpretation is difficult to make in spite of the foundation s urging at the inception of the demonstration , most hj sites had not developed a business case to sustain their interventions by the close of the demonstration . a few sites attempted but did not succeed in producing data to suggest that there were cost offsets related to medical interpretation . most health care institutions that retained their interpreter programs at the end of the demonstration simply absorbed the ongoing operating costs . in the end , as suggested by health care ceo and cfos with whom we spoke , a quality of care or risk management argument may be more feasible and successful in spurring health care providers and purchasers to provide language access services than an argument based on cost offsets.although hj projects faced a number of barriers , their experience also suggests that there are a number of facilitators that can enhance the success of future efforts . using the words of the project staff , administrators , and community stakeholders with whom we spoke , some of the lessons learned include the following : health care organizations are hierarchical , therefore it is important to have active , vocal support at the highest levels of medical and administrative leadership in the organization;if there is little motivation for the issue of language access , it is important to connect it to one of the driving issues of the organization ( e.g. , patient safety , quality of care ) , to help gain momentum for the project;projects that succeed involve good planning taking time to consider all of the options rather than adopting a one size fits all model;assuming that problems will arise in implementation of any innovation , projects that adopt a systematic approach to problem identification and trouble shooting are able to keep the project from being derailed;prioritizing staff time devoted to efforts related to critical success factors is associated with smoother implementation;the local latino community could become either a facilitator or a barrier to implementation ; it is important that health care organizations collaborate with the local community rather than implement their own solutions without consultation;the most successful projects have leaders on the ground who have a vision for what they want to accomplish along with substantial business and interpersonal skills;programs that are sustainable are those that are integrated into the normal operations and workflow of an organization from the outset not grafted on as special projects ; andtraining was critical to implementation of language access services ; if training is not a part of the mission of the organization , partnering with an experienced training organization is important . health care organizations are hierarchical , therefore it is important to have active , vocal support at the highest levels of medical and administrative leadership in the organization ; if there is little motivation for the issue of language access , it is important to connect it to one of the driving issues of the organization ( e.g. , patient safety , quality of care ) , to help gain momentum for the project ; projects that succeed involve good planning taking time to consider all of the options rather than adopting a one size fits all model ; assuming that problems will arise in implementation of any innovation , projects that adopt a systematic approach to problem identification and trouble shooting are able to keep the project from being derailed ; prioritizing staff time devoted to efforts related to critical success factors is associated with smoother implementation ; the local latino community could become either a facilitator or a barrier to implementation ; it is important that health care organizations collaborate with the local community rather than implement their own solutions without consultation ; the most successful projects have leaders on the ground who have a vision for what they want to accomplish along with substantial business and interpersonal skills ; programs that are sustainable are those that are integrated into the normal operations and workflow of an organization from the outset not grafted on as special projects ; and training was critical to implementation of language access services ; if training is not a part of the mission of the organization , partnering with an experienced training organization is important . the hj national demonstration provides health care organizations many valuable lessons regarding the provision or enhancement of language access services . first , there are a variety of creative models that can be employed to develop , enhance , and sustain language access services . second , there are considerable operating costs to consider and the scale of implementation is the main cost driver . the ultimate costs and benefits of these language service programs depend on how they are implemented . the most common lesson drawn from the evaluation was the extent to which the hj sites underestimated the difficulty of changing the culture of health care organizations , and the lack of attention they paid to the role that physicians play ( or could have played ) in the adoption of innovative language services . other challenges to be resolved are the need for benchmarks and tools to measure the proficiency of interpreters , and the creation of a sustainable market environment for professional , college - level interpreter educational programs . implementing innovative programs in health care organizations takes resources , leadership , and implementation strategies . we hope the lessons learned from the hj evaluation will be useful to policymakers and health care providers across the country as they take steps to overcome language barriers to serve a growing and dispersed immigrant population .

the objective of this report is to document the combination of intussusception and malrotation in an adult and to discuss clinical features and the treatment used in this case . a 25-years - old female patient presented in surgical emergency with complaints of diffuse pain abdomen , associated with vomiting , inability to pass feces and flatus , and distension of abdomen of 4 days duration . general physical examination revealed tachycardia with a pulse rate of 110/min , bp of 106/68 mmhg , dry , coated tongue and temperature of 101 f. abdomen examination showed distension and features of frank peritonitis and absent bowel sounds . on rectal examination after resuscitation with crystalloids , x - ray abdomen erect was done which showed multiple air fluid levels , and ultrasonography showed target like mass , dilated fluid filled a peristaltic bowel loops . in view of features of peritonitis , after resuscitation patient was taken up for emergency laparotomy with a probable diagnosis of acute intestinal obstruction secondary to intussusceptions . the involved intestine was discolored from congestion , edema , and haemorrhage into the wall . duodeno - jejunal flexure was found in an abnormal position , it was lying onto the right of midline . ileocaecal junction , ascending colon and hepatic flexure were lying in the midline with unfixed caecum and ascending colon ( figures 13 ) . in view of gangrenous changes in distal small bowel , resection of distal ileum , caecum and proximal half of ascending colon with end - to - end anastomosis was done . intussusception in adults originates in distal small bowel near ileocaecal valve secondary a variety of pathologies , which serve as a lead point , but in some cases there may not be a lead point . in waugh 's syndrome , there is no lead point and it is the presence of an abnormally placed gut loops along with unfixed and mobile caecum and ascending colon which might be a precursor for intussusception . brereton et al . who named this association of intussusceptions and malrotation as waugh 's syndrome , after george e. waugh , suggested that malrotation by its nature is associated with mobile right hemicolon which may be a predisposing factor for intussusceptions . patients with intussusceptions are optimally managed by air insuffulation or hydrostatic enema in paediatric age group but recommendation in adults is operation . surgical intervention becomes necessary when a patient presents with unequivocal peritonitis when the intussusceptions can not be reduced manually , this generally implies necrosis and a resection and anastomosis is performed . in our case a manual reduction by compression of the colon was not possible because the intussusception was irreducible and resection and end - to - end anastomosis was performed . it is concluded that we should remember that lack of normal rotation and fixation of the intestine and its mesentery is an important factor in the etiology of idiopathic intussusceptions , and failure and recurrence chances are more with non - operative methods .

chronic obstructive pulmonary disease ( copd ) is predicted and reported as the third cause of death in 2030 by world health organization ( who ) and is receiving gaining attention due to the increase in elderly population , in addition to the pollution worsening all over the world . besides , it is known that occurrence rate and death rate increases every subsequent year this way , managing respiratory diseases is very important including copd1 . patients with copd have anatomophysiological changes such as a limitation in the shape of the diaphragm and thoracic cage2 . these make patients more seriously impaired , leading to complains about dyspnea , a symptoms caused by airway obstruction while exercising . as a result , it causes changes of muscle mass due to decrease of momentum and the stamina and becomes the factor affecting respiratory muscles3 . when patients with copd breathe , the actual respiration volume is decreasing despite of the effort for respiration . this occurs because the demand for respiration is not consistent with the energy ratio supplied by respiratory muscles4 . and as the disease gets worse , respiratory failure appears and other body functions are negatively influenced due to deformation of the ventilation mechanism by overuse of respiratory muscles synergists5 . if copd occurs once , it is impossible to cure it completely . because it causes not only damage of the respiratory machinery , but also several physical damages and affects overall life pattern and normal body function6 . thus , fragmentary studies on characteristics of respiratory muscles are being done to increase respiratory capacity by classifying exercises into voluntary respiratory exercises which improves symptoms and prevents copd and exercise using breathing exercise equipment7 . but when the studies analyzing the respiratory muscles activity changes are being done , it is necessary to pay attention to respiratory muscles activity changes before and after the respiratory exercise . this research intends to examine the effect of agonist and synergist muscles by comparing respiratory muscles activity after the patients were submitted to respiratory exercises , as well as to find out the effective intervention method by comparing the respiratory muscles pattern and its effects on body functions . this study was approved by sehan university center s bioethics committee ( irb ) ( approval number : 2014 - 11 ) in october 1 , 2014 and included 30 male patients with mild copd aged between 55 and 70 , who were receiving medical treatment in a medical institution located in jeollanamdo , south korea from october of 2014 to december of 2014 . this experiment was composed of those who did nt have major medical diseases such as ischemic heart diseases and intermittent claudications , complications which would make participation on this program impossible ; those who did nt have accompanied damages such as rib fracture and congenital thoracic cage deformations ; those who did nt have rapid copd symptoms . all subjects agreed to participate in this research and understood the objective of the research ( table 1table 1.characteristic of subjectsexperimental group i ( n=15)experimental groupii ( n=15)age ( years)65.14.164.74.4hight ( cm)166.53.4164.93.9weight ( kg)66.23.964.94.0bmi ( kg / m)23.63.822.84.3fev1 ( % ) 60.44.859.83.0fev1/fvc ( % ) 50.63.753.23.4mean sd . experimental group ii : feedback respiration exercise group as experiment designs , we selected 15 subjects for experiment group i that performed diaphragm respiration exercise and 15 subjects for experiment group ii that performed feedback respiration exercise . the mediation program was done for 5 weeks , three times a week , once a day , and 30 minutes a session . before doing the exercise , respiratory muscles activity was measured with surface electromyogramand body function was measured with bode index using pulmonary function , 6-minute walking test , dyspnea criteria , bmi and then the result was analyzed through comparison of the data obtained before and after the exercise within the group and between groups . method of feedback respiration exercise consisted on the patient sitting straight and holding a handle with respiration pocket , having a mouth piece in the mouth and staring at the connector . the therapists pushed the start button of the connector . and when the patients see the orange gradation of the body reaching the in mark , they inhale and when it goes to out mark , they exhale . if there is a beeping sound with a green light when orange mark is on in or out , that means normal feedback respiration exercise . enough explanation was given to them before doing exercise so that patients understand the method of this exercise . it was conducted within the range not to happen hypocarbia during inhaling and exhaling at maximum level8 . method of diaphragm respiration exercise consists on the therapists hands placing on rectus abdominis located just below front costal cartilages and then they induce patients to inhale slowly and deeply through their nose . at this time , therapists apply proper resistance to rectus abdominis of patients when rectus abdominis rises and induce them to inhale deeply , and patients relax their shoulder and maintain it realaxed during inhaling deeply . upper chest is fixed not to be moved and only rise of abdomen is allowed9 . surface emg mp 100 system ( biopac , usa ) 4 channels were used to measure muscle activity of respiratory muscle and sampling rate for emg signal collection was set 1000 and frequency band filter was set 20450 hz . to minimize skin resistance of emg signal , subjects hair was removed from their skin and dead skin cells were removed using thin sandpaper . and then their skin was rubbed with alcohol cotton and was kept in clean conditions . two surface electrodes of ag / agcl were attached to belly of each muscle in a direction parallel to muscular fiber at 2 cm interval . standard electrodes were attached to spine of 12th backbone and scapula spine that are protruding parts . emg signals of right sternocleidomastoid muscle , scalene muscle , external intercostal muscle and diaphragm were collected10 , 11 . signals of measured muscle were converted to root mean square ( rms ) and emg signal analysis was done using acknowledge 3.9.1 software program ( biopac , usa ) . besides , root mean square was measured for 10 times by breathing naturally after making patients sit comfortably to normalize emg signal of subjects . average value of rms of eight respirations was set as rms when doing standard motion except rms of first and final respiration . in addition , subjects breathed at the level 30% of maximum inhalation pressure using threshold inspiratory muscle trainer ( new jersey , usa ) which is an inhalation respiration exercise equipment and rms value was quantified10 . chestgraph hi-701 ( chest , japan ) was used to measure pulmonary function and it was measured in the way that patients hold the respiration machine in their mouth in a sitting position and inhale at maximum level and then exhale with all their strength . pulmonary capacity was measured over 3 times at least and measured figure within 200 ml or 5% in the difference between the biggest figure and the second biggest figure . it marked the track at 1 m interval in indoor space and measurers turned on the stopwatch designed to be rung 6 minutes later when subjects started to walk , and when bell rang 6 minutes later , he measured the distance calculating the intervals marked by 1 m. bode index is composed of 4 items which are forced expiration capacity for 1 second ( fev1%pred ) , 6-minute walking distance ( m ) , mmrc dyspnea score , bmi ( bmi , kg / m ) . each item was evaluated with 03 score and then the total score was added for measurement6 . spss 18.0 for window was used as data processing method and shapiro - wilk was used to test the normality of general characteristics of subjects , activity of respiratory muscle and body function of groups . paired t - test was used for comparison of change within the group and analysis of covariance ( ancova ) was used in comparison of change between groups , significance level was set as =0.05 . this study was approved by sehan university center s bioethics committee ( irb ) ( approval number : 2014 - 11 ) in october 1 , 2014 and included 30 male patients with mild copd aged between 55 and 70 , who were receiving medical treatment in a medical institution located in jeollanamdo , south korea from october of 2014 to december of 2014 . this experiment was composed of those who did nt have major medical diseases such as ischemic heart diseases and intermittent claudications , complications which would make participation on this program impossible ; those who did nt have accompanied damages such as rib fracture and congenital thoracic cage deformations ; those who did nt have rapid copd symptoms . all subjects agreed to participate in this research and understood the objective of the research ( table 1table 1.characteristic of subjectsexperimental group i ( n=15)experimental groupii ( n=15)age ( years)65.14.164.74.4hight ( cm)166.53.4164.93.9weight ( kg)66.23.964.94.0bmi ( kg / m)23.63.822.84.3fev1 ( % ) 60.44.859.83.0fev1/fvc ( % ) 50.63.753.23.4mean sd . as experiment designs , we selected 15 subjects for experiment group i that performed diaphragm respiration exercise and 15 subjects for experiment group ii that performed feedback respiration exercise . the mediation program was done for 5 weeks , three times a week , once a day , and 30 minutes a session . before doing the exercise , respiratory muscles activity was measured with surface electromyogramand body function was measured with bode index using pulmonary function , 6-minute walking test , dyspnea criteria , bmi and then the result was analyzed through comparison of the data obtained before and after the exercise within the group and between groups . method of feedback respiration exercise consisted on the patient sitting straight and holding a handle with respiration pocket , having a mouth piece in the mouth and staring at the connector . the therapists pushed the start button of the connector . and when the patients see the orange gradation of the body reaching the in mark , they inhale and when it goes to out mark , they exhale . if there is a beeping sound with a green light when orange mark is on in or out , that means normal feedback respiration exercise . enough explanation was given to them before doing exercise so that patients understand the method of this exercise . it was conducted within the range not to happen hypocarbia during inhaling and exhaling at maximum level8 . method of diaphragm respiration exercise consists on the therapists hands placing on rectus abdominis located just below front costal cartilages and then they induce patients to inhale slowly and deeply through their nose . at this time , therapists apply proper resistance to rectus abdominis of patients when rectus abdominis rises and induce them to inhale deeply , and patients relax their shoulder and maintain it realaxed during inhaling deeply . upper chest is fixed not to be moved and only rise of abdomen is allowed9 . surface emg mp 100 system ( biopac , usa ) 4 channels were used to measure muscle activity of respiratory muscle and sampling rate for emg signal collection was set 1000 and frequency band filter was set 20450 hz . to minimize skin resistance of emg signal , subjects hair was removed from their skin and dead skin cells were removed using thin sandpaper . and then their skin was rubbed with alcohol cotton and was kept in clean conditions . two surface electrodes of ag / agcl were attached to belly of each muscle in a direction parallel to muscular fiber at 2 cm interval . standard electrodes were attached to spine of 12th backbone and scapula spine that are protruding parts . emg signals of right sternocleidomastoid muscle , scalene muscle , external intercostal muscle and diaphragm were collected10 , 11 . signals of measured muscle were converted to root mean square ( rms ) and emg signal analysis was done using acknowledge 3.9.1 software program ( biopac , usa ) . besides , root mean square was measured for 10 times by breathing naturally after making patients sit comfortably to normalize emg signal of subjects . average value of rms of eight respirations was set as rms when doing standard motion except rms of first and final respiration . in addition , subjects breathed at the level 30% of maximum inhalation pressure using threshold inspiratory muscle trainer ( new jersey , usa ) which is an inhalation respiration exercise equipment and rms value was quantified10 . chestgraph hi-701 ( chest , japan ) was used to measure pulmonary function and it was measured in the way that patients hold the respiration machine in their mouth in a sitting position and inhale at maximum level and then exhale with all their strength . pulmonary capacity was measured over 3 times at least and measured figure within 200 ml or 5% in the difference between the biggest figure and the second biggest figure . it marked the track at 1 m interval in indoor space and measurers turned on the stopwatch designed to be rung 6 minutes later when subjects started to walk , and when bell rang 6 minutes later , he measured the distance calculating the intervals marked by 1 m. bode index is composed of 4 items which are forced expiration capacity for 1 second ( fev1%pred ) , 6-minute walking distance ( m ) , mmrc dyspnea score , bmi ( bmi , kg / m ) . each item was evaluated with 03 score and then the total score was added for measurement6 . spss 18.0 for window was used as data processing method and shapiro - wilk was used to test the normality of general characteristics of subjects , activity of respiratory muscle and body function of groups . paired t - test was used for comparison of change within the group and analysis of covariance ( ancova ) was used in comparison of change between groups , significance level was set as =0.05 . as a result of comparing changes of experimental group i , only diaphragm showed meaningful difference in activity of respiratory muscles ( p<0.05 ) . and there was a meaningful difference in fev1 and 6-minute walking in bodyfunction ( p<0.05 ) ( table 2table 2.comparison of change in respiratory muscle activity and physical function within experimental group iexperimental group i ( n=15)pre - testpost - test%rmsscm43.23.443.02.3scalenius56.04.755.05.0diaphragm5.92.37.51.9external intercostalis15.91.416.11.7pysical functionfev160.44.861.34.26mwt360.417.3369.412.3bode index2.50.52.50.5p<0.05 , p<0.001 . experimental group ii : feedback respiration exercise group as a result of changes of experimental group ii , there was a meaningful difference in sternocleidomastoid muscle , scalene muscle and diaphragm regarding activity of respiratory muscles . and there was a meaningful difference in fev1 , 6-minute walking and bode index regarding bodyfunction ( p<0.05 ) ( table 3table 3.comparison of change in respiratory muscle activity and physical function within experimental group iiexperimental group ii ( n=15)pre - testpost - test%rmsscm43.85.740.63.7scalenius56.73.452.83.5diaphragm5.92.37.72.0external intercostalis15.81.716.42.5physical functionfev159.83.062.02.56mwt368.318.1379.513.4bode index2.90.12.270.5p<0.05 , p<0.001 . experimental group ii : feedback respiration exercise group there was a meaningful difference in sternocleidomastoid muscle and scalene muscle regarding activity of respiratory muscles in change comparison between groups . and there was a meaningful difference in 6-minute walking and bode index in bodyfunction ( p<0.05 ) ( table 4table 4.comparison of change in respiratory muscle activity and physical function between experimental group i and experimental group iiexperimental group i ( n=15)experimental group ii ( n=15)pre - testpost - testpre - testpost - test%rmsscm43.23.443.02.343.85.740.63.7scalenius56.04.755.05.056.73.452.83.5diaphragm5.92.37.51.95.92.37.72.0external intercostalis15.91.516.11.715.81.716.42.5physical functionfev160.44.861.34.259.83.062.02.56mwt360.417.3369.412.3368.318.1379.513.4bode index2.50.52.50.52.90.12.30.5p<0.05 , p<0.01 . when respiratory function of patients with copd becomes insufficient , activity of respiratory muscle synergists increases and the patients also take a more comfortable position to breathe . to set useful method for ventilation , they change their posture , which causes changes of the functions of involuntary muscles12 . legrand et al.13 says muscles around the neck bone are used for mechanical advantage in case of abnormal respiration pattern of copd patients . especially , sternocleidomastoid and scalene muscles are more activated for high mechanical advantage when breathing and it can be interpreted as role of supporting respiratory muscles caused by mechanical problems of worsening respiration patterns . duiverman et al.11 found that activity of sternocleidomastoid and scalene muscles in patients with copd increases a lot when compared to normal adults while exercising . this finding was a result of comparing muscle activity and dyspnea after a particular set of exercises performedfor 10 minutes targeting patients with copd . as tomich et al.14 observed , the air - centered activity of sternocleidomastoid muscle in the exercises performed is a result of its activity . the movements of chest and abdomen observed while conducting respiratory exercises with diaphragm respiration , air - center and capacity - center have identified 17 normal people . in this research , we could see that activities of sternocleidomastoid and scalene muscles decrease in the group performing feedback respiration exercise when compared to the group performing diaphragm respiratory exercise . deep and slow inhalation , as done in feedback respiratory exercise decreases activity of sternocleidomastoid muscle . chiti et al.15 found that activity of scalene muscle was increased in dyspnea , inhalation induction , inhalation effort and it would be useful to reduce synergist muscle activity such as scalene muscle during mechanical ventilation . loring et al.2 conducted a research in which 12 healthy people performed 2 sets of 30 repetitions each with 40% of initial maximum inspiratory pressure and as a result , muscle activity of diaphragm that is agonist of the respiratory muscles was greatly increased when inhaling . changes in muscle activity of diaphragm were shown to improve respiratory ability because of its synergy effect while these two muscles are doing inhalation . these findings and supported this research , so he can see that increase of activity by feedback respiration exercise is effective to reduce mechanical respiratory load . copd makes respiration hard due to decrease of lung capacity caused by deformation of thoracic cage by mechanical dynamics . thus , the respiratory synergists play an important role in the maintenance of pulmonary function in these patients16 . to copd patients , body function s state forms very close relation in conducting daily routines and 6-minute walk and bode index are meaningful in comparing the effect that indicates functional activity by mediation of lung6 . alexandra et al.17 found that level of 6-minute walking and functional activity have increased in the group that has performed mobile feedback every day for 2 weeks targeting copd patients . and mei18 mediated respiration pursing up lips that can heighten the activity of respiratory synergist in daily routine movements and exercise training by session targeting 101 copd and after this mediation , bode index and daily routine function were improved . this research could also see that 6-minute walk and bode index have increased significantly in group for feedback respiratory exercise in comparison to the group for diaphragm respiratory exercise . yamaguti et al.9 mediated diaphragm respiratory exercise for a total of 45 minutes repeating 3 sets a session of 10 repetitions each , three times a week for 4 weeks targeting 30 patients with copd and there was a meaningful difference in 6-minutes walking test and motility of diaphragm 4 weeks later . but there was no meaningful difference in bode index and this fact supported this research . the reason is that respiratory muscle exercise can obtain inhalation flow or inhalation pressure set as aims using resistant respiration during training for improvements of respiratory function19 . especially feedback respiratory exercise helps to improve endurance and strength of muscle by applying load to inhalation synergist and diaphragm20 . so , feedback respiratory exercise mobilizing respiratory synergist when compared to respiratory exercise of diaphragm is considered to help to improve body function . watz et al.21 say that another problem of copd which is not researched is the difference between different physical activities at severe stage of copd . although symptoms are weak , it is difficult to recover after there was a lung damage and the progress of symptoms can change the functional pattern of life with structural problems , it implies importance of breathing exercise before reaching severe levels of damage . this research found that of respiratory muscles synergists activities in patients with mild copd decreased because deformed thoracic cage and contraction of peripheral muscles were solved by mechanical advantage and it worked as a necessary factor for maintenance of respiratory function . it was possible to see the significant improvement in activity of synergists of respiratory muscles when breathing before doing breathing exercises , but it is valuable to reduce too much mobilization respiratory muscles synergists through the proper intervention . it is considered that it is necessary to study body function for the improvement of respiratory function for patients with copd .

all participants signed informed consent forms , and the study adhered to the tenets of the declaration of helsinki . the study initially included 24 eyes of 24 patients who presented to the retina clinic at the hospital , were diagnosed with unilateral exudative amd , and underwent anti - vegf treatment with intravitreal ranibizumab . all subjects received three intravitreal injections of ranibizumab at monthly intervals from the diagnosis date . additional injections were administered if necessary , depending on the presence of macular edema or hemorrhage . subjects were excluded if they had an eye disorder that could affect rnfl thickness , such as glaucoma or retinal vessel obstruction , a history of photodynamic treatment for macular degeneration , bilateral exudative amd , or a history of intraocular surgery such as vitrectomy . in addition , four of the initial patients were excluded because of poor data quality , resulting in a total of 20 eyes of 20 patients included for final analysis . all subjects underwent intraocular pressure measurement , slit - lamp examination , and fundus examination . the average and four - quadrant rnfl thicknesses were measured in the affected and normal counterpart eyes using a cirrus spectral domain oct ( carl zeiss meditec , dublin , ca , usa ) . rnfl thickness was measured before and 6 and 12 months after intravitreal injection with the optic disc cube mode of cirrus spectral domain oct . poor - quality images with a signal strength less than 5 and any scans with visible eye movements , blinking artifacts , or poor centration were excluded . also , images with missing parts , misplacement of boundaries between retinal layers , or images showing seemingly distorted anatomy that resulted in readings of zero or otherwise abnormally low values were discarded to prevent algorithm segmentation failure . all eyes were scanned twice to evaluate reproducibility , and good reproducibility was achieved based on intraclass correlation coefficients of 0.941 to 0.989 . the optic disc cube 200 200 scanning program ( cirrus hd - oct , carl zeiss meditec ) obtains 200 a - scans from 200 linear b - scans evenly distributed in a 6 mm 6 mm area centered over the optic nerve . this scanning mode was used to measure the average and four - quadrant rnfl thicknesses . rnfl thicknesses were measured in affected eyes before and 6 and 12 months after intravitreal injection for sequential comparison . rnfl thickness was divided into 12 segments of 30 degrees for 12 clock - hour rnfl thickness measurements . under the assumption that macular lesions affect rnfl thickness , changes in average thickness were evaluated after dividing the 12 clock - hour rnfl thickness measurements into pathologic area , including the lesion , and non - pathologic area of the affected eye and the corresponding area of the normal fellow eye ( fig . intraocular pressure measurements were obtained using a goldmann applanation tonometer before and 6 and 12 months after intravitreal injection for sequential comparison . sequential time - related changes in rnfl thickness in affected and normal eyes were analyzed using wilcox signed - ranks tests . the comparison of rnfl thickness between affected and normal eyes at different time points was conducted using a mann - whitney u - test . all participants signed informed consent forms , and the study adhered to the tenets of the declaration of helsinki . the study initially included 24 eyes of 24 patients who presented to the retina clinic at the hospital , were diagnosed with unilateral exudative amd , and underwent anti - vegf treatment with intravitreal ranibizumab . all subjects received three intravitreal injections of ranibizumab at monthly intervals from the diagnosis date . additional injections were administered if necessary , depending on the presence of macular edema or hemorrhage . subjects were excluded if they had an eye disorder that could affect rnfl thickness , such as glaucoma or retinal vessel obstruction , a history of photodynamic treatment for macular degeneration , bilateral exudative amd , or a history of intraocular surgery such as vitrectomy . in addition , four of the initial patients were excluded because of poor data quality , resulting in a total of 20 eyes of 20 patients included for final analysis . all subjects underwent intraocular pressure measurement , slit - lamp examination , and fundus examination . the average and four - quadrant rnfl thicknesses were measured in the affected and normal counterpart eyes using a cirrus spectral domain oct ( carl zeiss meditec , dublin , ca , usa ) . rnfl thickness was measured before and 6 and 12 months after intravitreal injection with the optic disc cube mode of cirrus spectral domain oct . poor - quality images with a signal strength less than 5 and any scans with visible eye movements , blinking artifacts , or poor centration were excluded . also , images with missing parts , misplacement of boundaries between retinal layers , or images showing seemingly distorted anatomy that resulted in readings of zero or otherwise abnormally low values were discarded to prevent algorithm segmentation failure . all eyes were scanned twice to evaluate reproducibility , and good reproducibility was achieved based on intraclass correlation coefficients of 0.941 to 0.989 . the optic disc cube 200 200 scanning program ( cirrus hd - oct , carl zeiss meditec ) obtains 200 a - scans from 200 linear b - scans evenly distributed in a 6 mm 6 mm area centered over the optic nerve . this scanning mode was used to measure the average and four - quadrant rnfl thicknesses . rnfl thicknesses were measured in affected eyes before and 6 and 12 months after intravitreal injection for sequential comparison . rnfl thickness was divided into 12 segments of 30 degrees for 12 clock - hour rnfl thickness measurements . under the assumption that macular lesions affect rnfl thickness , changes in average thickness were evaluated after dividing the 12 clock - hour rnfl thickness measurements into pathologic area , including the lesion , and non - pathologic area of the affected eye and the corresponding area of the normal fellow eye ( fig . intraocular pressure measurements were obtained using a goldmann applanation tonometer before and 6 and 12 months after intravitreal injection for sequential comparison . intraocular pressure in affected and normal eyes was also compared . sequential time - related changes in rnfl thickness in affected and normal eyes were analyzed using wilcox signed - ranks tests . the comparison of rnfl thickness between affected and normal eyes at different time points was conducted using a mann - whitney u - test . twenty eyes of 20 patients were enrolled , of whom 13 were male and seven were female with a median age of 67.1 8.9 years . the average best - corrected visual acuity was 0.5 0.6 ( logarithm of the minimum angle of resolution ) , and the mean intraocular pressure was 15.3 2.6 mmhg . the mean number of anti - vegf injections was 5.0 1.0 , and the mean clock - hour segment in the pathologic area was 4.8 0.5 in the 12 clock - hour rnfl thickness analysis ( table 1 ) . the overall average rnfl thickness at baseline was 98.0 6.8 m in affected eyes and 94.5 7.3 m in normal eyes ( p = 0.146 ) . postinjection differences in rnfl thickness between the two groups were not significant at 6 or 12 months ( p = 0.892 and 0.776 , respectively ) . the average rnfl thickness in affected eyes was 96.3 4.2 m at 6 months and 95.5 4.3 m at 12 months after intravitreal injection , showing no significant difference from baseline ( p = 0.153 and 0.070 , respectively ) . segmental rnfl thickness in the superior , inferior , and nasal quadrants did not exhibit significant changes in the affected or normal eyes at 6 and 12 months postinjection compared to baseline rnfl thickness ( p > 0.05 ) . postinjection changes in rnfl thickness were not significant in affected eyes over time ( p > 0.05 ) . temporal rnfl thickness at baseline was 84.4 20.9 m in affected eyes , which was significantly thicker than the 73.0 9.7 m observed in normal eyes ( p = 0.043 ) . however , postinjection differences in temporal rnfl thickness between the two groups were not significant at 6 or 12 months ( p = 0.464 and 0.787 , respectively ) . time - related changes in temporal rnfl thickness were significant in affected eyes 6 and 12 months after injection compared to baseline ( p = 0.012 and 0.006 , respectively ) ( table 2 and fig . the overall average rnfl thickness at baseline was 85.8 21.2 m in the pathologic area of affected eyes , which was significantly greater than the 74.0 10.0 m measured in normal eyes ( p = 0.048 ) . however , postinjection differences in rfnl thickness were not significant between affected and normal eyes at 6 or 12 months ( p = 0.607 and 0.665 , respectively ) . longitudinal changes in rnfl thickness in the pathologic areas were significant in affected eyes at 6 and 12 months postinjection compared to baseline ( p = 0.011 and 0.005 , respectively ) . the average rnfl thickness in the non - pathologic area was not significantly different between affected and normal eyes ( p > 0.05 ) . the average postinjection rnfl thickness did not change significantly in affected eyes over time ( table 3 and fig . the average intraocular pressure at baseline was 15.3 2.6 mmhg in affected eyes and 16.0 2.9 mmhg in normal eyes , showing no significant difference ( p = 0.473 ) . postinjection differences in intraocular pressure between the two groups were not significant at 6 or 12 months ( p = 1.000 and 0.534 , respectively ) ( table 4 ) . the overall average rnfl thickness at baseline was 98.0 6.8 m in affected eyes and 94.5 7.3 m in normal eyes ( p = 0.146 ) . postinjection differences in rnfl thickness between the two groups were not significant at 6 or 12 months ( p = 0.892 and 0.776 , respectively ) . the average rnfl thickness in affected eyes was 96.3 4.2 m at 6 months and 95.5 4.3 m at 12 months after intravitreal injection , showing no significant difference from baseline ( p = 0.153 and 0.070 , respectively ) . segmental rnfl thickness in the superior , inferior , and nasal quadrants did not exhibit significant changes in the affected or normal eyes at 6 and 12 months postinjection compared to baseline rnfl thickness ( p > 0.05 ) . postinjection changes in rnfl thickness were not significant in affected eyes over time ( p > 0.05 ) . temporal rnfl thickness at baseline was 84.4 20.9 m in affected eyes , which was significantly thicker than the 73.0 9.7 m observed in normal eyes ( p = 0.043 ) . however , postinjection differences in temporal rnfl thickness between the two groups were not significant at 6 or 12 months ( p = 0.464 and 0.787 , respectively ) . time - related changes in temporal rnfl thickness were significant in affected eyes 6 and 12 months after injection compared to baseline ( p = 0.012 and 0.006 , respectively ) ( table 2 and fig . the overall average rnfl thickness at baseline was 85.8 21.2 m in the pathologic area of affected eyes , which was significantly greater than the 74.0 10.0 m measured in normal eyes ( p = 0.048 ) . however , postinjection differences in rfnl thickness were not significant between affected and normal eyes at 6 or 12 months ( p = 0.607 and 0.665 , respectively ) . longitudinal changes in rnfl thickness in the pathologic areas were significant in affected eyes at 6 and 12 months postinjection compared to baseline ( p = 0.011 and 0.005 , respectively ) . the average rnfl thickness in the non - pathologic area was not significantly different between affected and normal eyes ( p > 0.05 ) . the average postinjection rnfl thickness did not change significantly in affected eyes over time ( table 3 and fig . the average intraocular pressure at baseline was 15.3 2.6 mmhg in affected eyes and 16.0 2.9 mmhg in normal eyes , showing no significant difference ( p = 0.473 ) . postinjection differences in intraocular pressure between the two groups were not significant at 6 or 12 months ( p = 1.000 and 0.534 , respectively ) ( table 4 ) . intravitreal anti - vegf injection is commonly used in the treatment of a wide variety of retinal diseases , including exudative amd . however , intravitreal injection can result in complications , including endophthalmitis , intraocular hemorrhage , and ocular hypertension . temporary ocular hypertension following intravitreal anti - vegf injection is caused by increased intraocular volume . reported that the average intraocular pressure soared to 36.3 mmhg from a baseline of 15.2 mmhg 3 minutes after intravitreal injection of 0.05 ml bevacizumab in 70 patients . however , the mean intraocular pressure decreased to 24.6 mmhg at 10 minutes , and intraocular pressure in all eyes decreased to less than 30 mmhg after 15 minutes . also reported that intraocular pressure increased to more than 40 mmhg immediately after intravitreal injection of 0.05 ml ranibizumab in 71.1% of 45 patients but decreased to 21 mmhg at 10 minutes . on the other hand , reported six cases of chronic ocular hypertension after one - time or repeat injection of bevacizumab and cited aqueous outflow induced by an accumulated trabecular meshwork and inflammatory responses as the cause of sustained ocular hypertension . in our study , intraocular pressure measurements of affected and normal eyes were obtained 6 and 12 months after intravitreal injection for comparison . the difference in mean intraocular pressure was not significantly different between the affected and normal eyes . in addition , anti - vegf drugs can have adverse effects on neuronal cells due to the blockage of vegf . considering that vegf is involved in the survival of neuronal cells and has neuroprotective and neurotrophic actions in addition to its angiogenesis role , anti - vegf treatment can affect the neurophysiologic role of vegf and cause damage to optic nerves . because the effects of anti - vegf agents are of limited duration , repeated intravitreal injections are required in many cases . as a result , brief ocular hypertension , increased intraocular pressure fluctuations , changes in ocular blood flow , and adverse effects on the optic nerve can ensue . seth et al . analyzed changes in the cup - to - disc ratio in 23 eyes over 9 months following repeat intravitreal injection of pegaptanib . horsley et al . retrospectively assessed changes in rnfl thickness in patients with amd following repeat intravitreal anti - vegf injection using stratus oct and reported no significant postinjection changes in rnfl thickness throughout the follow - up period . another study assessed rnfl thickness in 49 eyes of 49 patients with amd using stratus oct for 12 months after an average of 4.8 intravitreal injections of an anti - vegf agent . they found that average rnfl was significantly thinner at 12 months compared to baseline values . they also found that rnfl thickness significantly decreased in the superior , inferior , and temporal quadrants at 12 months compared to baseline data . however , there was no significant change in nasal rnfl thickness . in the present study , rnfl thickness at baseline was significantly greater in the temporal and pathologic areas of affected eyes compared to normal eyes . rnfl thickness in the temporal and pathologic areas of affected eyes did , however , display significant changes at sequential time points after injection . longitudinal changes in rnfl thickness were not significant in quadrants other than the temporal quadrant in non - pathologic areas of affected eyes . therefore , the reduction in rnfl thickness in the pathologic area is more likely to be due to a change in the macular lesion rather than a result of increased pressure due to anti - vegf administration . that is , rnfl thickness was increased at baseline due to an exudative lesion , edema , or hemorrhage in the macular area , and rnfl thickness was reduced 6 and 12 months after injection because the exudative lesion improved after repeat injection . this conclusion is supported by the findings of greater rnfl thickness in the temporal quadrant in affected eyes at baseline compared to that of normal eyes , without a significant difference in postinjection rnfl thickness between the two groups . this area overlapped that of the temporal quadrant , leading to similar changes in rnfl thickness . hwang et al . described two reasons for increased rnfl thickness in diabetic macular edema ( dme ) patients . first , an increase in temporal sector rnfl thickness in patients with acutely worsening dme might be related to a change in macular tomography due to macular edema . second , the breakdown of the inner blood retinal barrier in rnfl causes rnfl edema , which then results in increased rnfl thickness in all sectors in patients with chronic dme . thus , a similar increase in temporal sector rnfl thickness at baseline in patients with amd might be related to changes in macular tomography due to macular edema . spectral domain - oct was used , which offers better reproducibility than time - domain oct . four - quadrant rnfl thickness measures were conducted in addition to an overall assessment of average rnfl thickness . additionally , pathologic and non - pathologic areas close to and distant from the macular lesions were separately evaluated . in conclusion , repeat intravitreal anti - vegf treatment did not have a significant effect on overall rnfl thickness . however , rnfl thickness significantly decreased with time in the temporal and pathologic areas . it can be concluded that the reduction in rnfl thickness was more associated with changes in the macular lesion rather than with anti - vegf injection . long - term prospective studies with more subjects are needed to investigate the long - term effects of anti - vegf treatment in various disease states .

neuropathic pain is a devastating condition , and our lack of knowledge regarding its pathogenesis has hampered efforts to find an effective treatment . increasing evidence has shown that mitogen - activated protein kinase ( mapk ) plays important roles in the induction and maintenance of chronic pain . the mapk family consists of three primary members : extracellular signal - regulated kinases ( erk ) , p38 mapk , and c - jun n - terminal kinase ( jnk ) ; these three kinases correspond to three distinct signaling pathways . experiments were designed to elucidate the signaling cascades that induce allodynia in a neuropathic pain model . previously published studies have described the role of mapks in the regulation of allodynia and hyperalgesia . mapks have , therefore , been suggested as a specific target in neuropathic pain responses . to investigate whether the inhibition of neuropathic pain by najanalgesin is regulated by a mapk pathway , we examined the effect of najanalgesin on neuropathic pain by measuring the phosphorylation of erk , jnk , and p38 mapk . najanalgesin is purified from cobra venom and consists of a single polypeptide with a molecular mass of 6714 da with an ld50 of 2.69 mg / kg . in our laboratory , we found that najanalgesin had an analgesic effect on pain resulting from heat and chemical burns . additionally , we evaluated the antinociceptive effect of najanalgesin in a rat model of neuropathic pain , which was induced by l5 spinal nerve ligation ( snl ) . we observed that najanalgesin had antinociceptive effects on the central and peripheral system in a rat neuropathic pain model . however , the effect of najanalgesin on the mapk pathways after nerve injury remains unclear . in the present study , we investigated the effect of najanalgesin in rats using a neuropathic pain model , focusing on the mapk family members jnk , erk , and p38 mapk the mapk kinase ( mek ) 1/2 inhibitor ( pd98059 ; 0.1 g/l ; emd biosciences , la jolla , ca , usa ) , the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl ) imidazole ( sb203580 ; 0.5 g/l ; calbiochem , usa ) , and the jnk inhibitor anthrax 1,9-cd ] pyrazol-6 ( 2h)-one ( sp600125 ; 2.5 g/l ; calbiochem ) were prepared in 50% dimethyl sulfoxide ( dmso ) . both dmso ( 50% ) and normal saline were used as vehicle controls . the doses used for intrathecal najanalgesin ( 40 ng / kg ) were selected according to our pilot study . jnk , phosphor - jnk ( thr183/tyr185 ) , phosphor - p38 , phosphor - erk , and phosphor - c - jun antibodies were purchased from cell signaling technology inc . , usa . male sprague - dawley rats ( 200220 g ) were purchased from the laboratory animal center at sun yat - sen university . all procedures were conducted in accordance with the national institutes of health guidelines on animal care . the experimental design resulted in four groups of rats ( n = 8 per group ) : ( 1 ) control group of the sham - operated rats ; ( 2 ) snl group of rats with l5 spinal nerve ligation ; ( 3 ) najanalgesin group of treatment injury rats with 40 ng / kg najanalgesin ; ( 4 ) inhibitor group of treatment rats with pd98059 or sb203580 or sp600125 . all rats were housed in separate cages with free access to food and water and maintained on a 12-h light / dark cycle . a sterile polyethylene-10 catheter was inserted into the rat 's lumbar subarachnoid space , and drugs were administered in a volume of 10 l . the l5 spinal nerve was tightly ligated with silk suture . in sham - operated rats , the nerve was exposed without ligature . , the rats were brought into the behavioral room at least 30 min prior to the test session . mechanical sensitivity was assessed using the up - down method as previously described using a set of von frey hairs ( ugo basile , italy ) . equal amounts of the samples were run on a 12% dodecyl sulfate - polyacrylamide gel and separated using sds - page . antibodies against jnk ( 1:1000 ; cell signaling technology ) , erk ( 1:1000 ; cell signaling technology ) , and p38 ( 1:1000 ; cell signaling technology ) , as well as a monoclonal primary antibody against -actin ( mouse , 1:500 ; boshide ) , were diluted in tris - buffered saline containing 0.1% triton x-100 and 5% bovine serum albumin and used as primary antibodies . immunohistochemistry was performed on 25-m free - floating l5 spinal cord sections as previously described . spinal cord sections were incubated overnight at 4c with jnk primary antibody ( 1:1000 ) and subsequently incubated with a cyc3-conjugated secondary antibody ( 1:6000 , boshide , china ) . to assess nonspecific staining , control sections were incubated in the absence of primary antibodies . the mean value of jnk - ir was obtained within the spinal dorsal horn laminae i all data were expressed as the mean standard error ( se ) of the mean . one - way analysis of variance ( anova ) followed by tukey 's test was used for immunohistochemistry and western blotting data . a p < 0.05 was considered as statistically significant . the mapk kinase ( mek ) 1/2 inhibitor ( pd98059 ; 0.1 g/l ; emd biosciences , la jolla , ca , usa ) , the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl ) imidazole ( sb203580 ; 0.5 g/l ; calbiochem , usa ) , and the jnk inhibitor anthrax 1,9-cd ] pyrazol-6 ( 2h)-one ( sp600125 ; 2.5 g/l ; calbiochem ) were prepared in 50% dimethyl sulfoxide ( dmso ) . both dmso ( 50% ) and normal saline were used as vehicle controls . the doses used for intrathecal najanalgesin ( 40 ng / kg ) were selected according to our pilot study . jnk , phosphor - jnk ( thr183/tyr185 ) , phosphor - p38 , phosphor - erk , and phosphor - c - jun antibodies were purchased from cell signaling technology inc . , usa . male sprague - dawley rats ( 200220 g ) were purchased from the laboratory animal center at sun yat - sen university . all procedures were conducted in accordance with the national institutes of health guidelines on animal care . the experimental design resulted in four groups of rats ( n = 8 per group ) : ( 1 ) control group of the sham - operated rats ; ( 2 ) snl group of rats with l5 spinal nerve ligation ; ( 3 ) najanalgesin group of treatment injury rats with 40 ng / kg najanalgesin ; ( 4 ) inhibitor group of treatment rats with pd98059 or sb203580 or sp600125 . all rats were housed in separate cages with free access to food and water and maintained on a 12-h light / dark cycle . a sterile polyethylene-10 catheter was inserted into the rat 's lumbar subarachnoid space , and drugs were administered in a volume of 10 l . the l5 spinal nerve was tightly ligated with silk suture . in sham - operated rats , the nerve was exposed without ligature . on each testing day , the rats were brought into the behavioral room at least 30 min prior to the test session . mechanical sensitivity was assessed using the up - down method as previously described using a set of von frey hairs ( ugo basile , italy ) . equal amounts of the samples were run on a 12% dodecyl sulfate - polyacrylamide gel and separated using sds - page . antibodies against jnk ( 1:1000 ; cell signaling technology ) , erk ( 1:1000 ; cell signaling technology ) , and p38 ( 1:1000 ; cell signaling technology ) , as well as a monoclonal primary antibody against -actin ( mouse , 1:500 ; boshide ) , were diluted in tris - buffered saline containing 0.1% triton x-100 and 5% bovine serum albumin and used as primary antibodies . immunohistochemistry was performed on 25-m free - floating l5 spinal cord sections as previously described . spinal cord sections were incubated overnight at 4c with jnk primary antibody ( 1:1000 ) and subsequently incubated with a cyc3-conjugated secondary antibody ( 1:6000 , boshide , china ) . to assess nonspecific staining the mean value of jnk - ir was obtained within the spinal dorsal horn laminae i all data were expressed as the mean standard error ( se ) of the mean . one - way analysis of variance ( anova ) followed by tukey 's test was used for immunohistochemistry and western blotting data . the rats subjected to the neuropathic pain model developed robust behavioral allodynia after snl , which persisted for at least 2 weeks . sham - operated rats showed no apparent response to mechanical stimuli at any of the investigated time points . in the mechanical withdrawal threshold testing , groups of rats were injected intrathecally with saline , najanalgesin , or sp600125 ; the paw withdrawal threshold was measured for 24 h after injection . the injection of najanalgesin resulted in the significantly high threshold compared with saline treatment ( p < 0.05 ) started 1 h and lasted past 24 h. sp600125 produced antinociceptive action than those observed with saline treatment at 1 , 3 , and 9 h after injection ( p < 0.05 ) , [ figure 1 ] . our study suggested that najanalgesin has a significant antinociceptive effect in a neuropathic pain model in rats . to determine whether the inhibition of neuropathic pain by najanalgesin is regulated by the mapk pathways , we examined the effect of najanalgesin on the phosphorylation levels of erk , jnk , and p38 mapk by western blot analysis . effects of najanalgesin on mechanical allodynia in the rat spinal nerve ligation model up to 24 h after drug administration . after administration of najanalgesin , saline , or sp600125 , the paw withdrawal threshold in response to mechanical stimuli was measured at different time points after the injection ( n = 8) . * p < 0.05 , p < 0.01 compared with the mean values of saline group . as shown in figure 2 , p - p38 mapk was consistently increased in the spinal dorsal cord of injured rats . however , there was no significant change after treatment with najanalgesin . when we investigated the possible role of erk , we observed a significant increase in erk phosphorylation levels after nerve injury . interestingly , najanalgesin treatment had no effect on this increase in phosphorylated erk ( p - erk ) [ figure 3 ] . changes in p38 expression in the spinal dorsal horn after l5 spinal nerve ligation and najanalgesin treatment . ( a ) immunoblots against p38 in the spinal dorsal horn of the control , spinal nerve ligation , and najanalgesin treatment groups . ( b ) images of protein bands corresponding to p - p38 after intrathecal sb203580 administration . the data are represented as the mean standard error of the mean ( n = 4 ) . * p < 0.05 versus the control group . changes in extracellular - regulated kinase expression in the spinal dorsal horn after l5 spinal nerve ligation and najanalgesin treatment . ( a ) immunoblots against extracellular - regulated kinase in the spinal dorsal horn of the control , spinal nerve ligation , and najanalgesin treatment groups . the extracellular - regulated kinase expression at all of the investigated time points was quantified and graphed . ( b ) images of protein bands corresponding to p - extracellular - regulated kinase after intrathecal pd98059 administration . the data are represented as the mean standard error of the mean ( n = 4 ) . * p < 0.05 versus the control group . peripheral nerve injury significantly induced the activation of jnk without causing any significant changes in total jnk protein levels . jnk activation was increased by eight - fold in the snl group compared with the sham group ( p < 0.01 ) . interestingly , we observed a significant decrease in the phosphorylation of jnk at 3 and 9 h after treatment with 40 ng / kg najanalgesin [ figure 4 ] . the phosphorylation of c - jun followed a similar pattern to jnk after treatment with najanalgesin . the levels of phosphorylated c - jun decreased progressively from 3 h to 9 h [ figure 5 ] . based on these results , we used three known pharmacological inhibitors of various meks to confirm the specificity of najanalgesin activity . we used inhibitors against erk ( pd98059 ) , p38 ( sb203580 ) , and jnk ( sp600125 ) . all of these inhibitors decreased the phosphorylation of p38 [ figure 1 ] , erk [ figure 2 ] , and jnk [ figure 3 ] . western blotting revealed that rats subjected to nerve injury had increased levels of p - jnk , p38 , and p - erk . treatment with najanalgesin inhibited the injury - induced increase in p - jnk , whereas p - erk and p38 were unchanged . this result suggests that jnk may have a key role in the antinociceptive effect of najanalgesin on neuropathic pain . changes in c - jun n - terminal kinase expression in the spinal dorsal horn after l5 spinal nerve ligation and najanalgesin treatment . ( a ) immunoblots against c - jun n - terminal kinase in the spinal dorsal horn of the control , spinal nerve ligation , and najanalgesin treatment groups . the c - jun n - terminal kinase expression at all of the investigated time points was quantified and graphed . ( b ) images of the protein bands corresponding to p - jnk after intrathecal sp600125 administration . the data are represented as the mean standard error of the mean ( n = 4 ) . * p < 0.05 versus the control group ; p < 0.05 versus the spinal nerve ligation group . changes in c - jun expression in the spinal dorsal horn after l5 spinal nerve ligation and najanalgesin treatment . ( a ) immunoblots against p - c - jun in the spinal dorsal horn of the control , spinal nerve ligation , and najanalgesin treatment groups . ( b ) images of the protein bands corresponding to p - c - jun after intrathecal sp600125 administration . the c - jun expression at all of the investigated time points was quantified and graphed . * p < 0.05 versus the control group ; p < 0.05 versus the spinal nerve ligation group . the changes in jnk expression were confirmed by immunohistochemistry analysis . the robust increase of jnk expression in jnk - ir cells was observed in the superficial region ( laminae i iii ) of the dorsal horn . consistent with the results obtained by western blot analysis , najanalgesin treatment of rats subjected to l5 snl led to a decrease in ipsilateral jnk immunoreactivity at 3 , 6 , and 9 h compared with the control group [ figure 6 ] . these findings suggest that the antinociceptive effect of najanalgesin inhibits jnk in a rat model of neuropathic pain . changes in c - jun n - terminal kinase protein expression in the spinal dorsal horn after l5 spinal nerve ligation , transection - induced neuropathic pain , and najanalgesin treatment . images show c - jun n - terminal kinase immunostaining in the spinal dorsal horn in the control , spinal nerve ligation , najanalgesin treatment , and sp600125 treatment groups . c - jun n - terminal kinase - ir activation was not observed in the control group ( a , d , g ) . prominent astrocyte activation was observed in rats treated with intrathecal saline after spinal nerve ligation ( b , e , h ) . these responses were markedly inhibited in rats treated with either intrathecal najanalgesin ( c , f , i ) or intrathecal sp600125 ( j , k , l ) for 3 , 6 , and 9 h. the c - jun n - terminal kinase protein expression at all of the investigated time points was quantified and graphed . * p < 0.05 versus the control group ; p < 0.05 versus the spinal nerve ligation group . increasing evidence has shown that mapks play important roles in the induction and maintenance of chronic pain . erk , p38 , and jnk represent three different signal transduction pathways that contribute to pain sensitization after tissue and nerve injury . erk , p38 , and jnk are downstream of many kinases and are activated in primary sensory neurons , dorsal horn neurons , and spinal glial cells , and contribute to neuropathic pain via transcriptional , translational , and posttranslational regulation . the inhibition of all three mapk pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models . in the present study , we observed that peripheral nerve injury resulted in a marked increase in the activation of p38 , erk , and jnk ( p < 0.01 ) . the increased activation of both erk and p38 mapk was not attenuated by najanalgesin administration . however , the increase in jnk was attenuated with najanalgesin . the transcription c - jun is the best - known substrate of jnk , and it is activated mainly through double phosphorylation by the jnk and also has a phosphorylation - independent function . our study showed that the levels of phosphorylated c - jun were markedly increased in the snl group and that this increase was prevented by najanalgesin treatment , which implies the involvement of jnk in the antinociceptive effect of najanalgesin . consistent with these results , we also demonstrated by immunohistochemistry that the activation of jnk is involved in the antinociceptive effect of najanalgesin . the mapk pathways are complex and not yet fully understood , as different cell types respond to the same nerve injury by activating different signaling pathways . it has been suggested that the erk pathway , which is primarily triggered in response to growth factors and mitogen stimulation , is involved in cellular proliferation and differentiation . specifically , erk activation in spinal cord dorsal horn neurons occurs by nociceptive activity by regulating the activity of glutamate receptors and potassium channels , as well as inducing gene transcription . based on our results , erk may be not involved in the analgesic effect of najanalgesin treatment , suggesting that najanalgesin may act through another signaling pathway . p38 was shown to be rapidly and robustly activated in the superficial spinal dorsal horn after nerve injury and localized primarily to microglia . peripheral nerve injury leads to the activation of both microglia and astrocytes in the spinal cord but with different time courses . microglia are involved in the early response to neuropathic pain , but the persistent expression of astrocyte markers correlates with the persistence of neuropathic pain symptoms . activated glial cells release proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and il-6 , as well as other substances that enhance neuronal central sensitization and nerve injury - induced persistent pain . in this study , najanalgesin treatment was administered after the initial pain was inflicted and during the maintenance of the pain . therefore , p38 may be not involved in antinociception of najanalgesin on the maintenance of neuropathic pain . whether p38 would be involved in the antinociception with najanalgesin on the early response to neuropathic pain , we need to investigate it further . the previous study showed that jnk is persistently activated in the spinal dorsal horn after nerve injury . jnk3 is primarily expressed in the brain and has different roles than jnk1 and jnk2 . however , jnk1 and jnk2 are heavily expressed in the spinal cord and are the predominant isoforms after nerve injury . pjnk1/2 is persistently increased in astrocytes on 1 , 3 , 10 , and 21 days in the spinal cord after partial sciatic nerve injury . additionally , c - jun , the major transcription factor downstream of jnk , is also activated in the spinal cord after nerve injury . the jnk / c - jun pathway is activated in the spinal astrocytes , which is also critical for the maintenance of neuropathic pain . in this study , there were marked increases in the jnk and c - jun levels after snl , and both of these protein levels decreased after najanalgesin treatment . in addition , we have observed that astrocytes were involved in the antinociception of najanalgesin during the maintenance of pain in the previous study . therefore , the effect of najanalgesin may act through the jnk signaling pathway in the spinal cord on the maintenance of neuropathic pain . ctx is the primary neurotoxic component of crotalus durissus terrificus snake venom , which modulates inflammatory responses . this toxin inhibits cytokine release ( e.g. , il-2 , il-4 , and il-10 ) in human serum albumin - immunized mice . our previous study showed that najanalgesin attenuated the activation of spinal astrocytes and reduced the release of proinflammatory cytokines ( tnf- , il-1 ) in the spinal cord . based on the results of our previous and current studies , najanalgesin produces an antinociceptive effect on the maintenance of neuropathic pain , by attenuating the activation of jnk , and reducing the release of proinflammatory cytokines . and then , we should investigate the mrna of jnk and give jnk inhibitor to observe the expression of proinflammatory cytokines , in order to make the molecule mechanism of najanalgesin clear in the future . the present study evaluates the intracellular signaling pathways implicated in the antinociceptive effect of najanalgesin , a toxin isolated from naja naja atra . this inhibition could be one of the mechanisms of najanalgesin that protects against peripheral nerve injury . the results suggest that the antinociceptive effect of najanalgesin is due to inhibition of jnk based on the neuropathic pain model . this study was supported by the grants from the national natural science foundation of china ( no . this study was supported by the grants from the national natural science foundation of china ( no .

controlling tb in india is a tremendous challenge and the burden is still staggering.(2 ) the revised national tuberculosis control programme ( rntcp ) , based on the dots strategy , was implemented in the country since 1993 . a dots observer watches and helps the patient to swallow the drug and ensures the treatment for the entire course . dots is the only reliable and proven strategy of ensuring adherence to the treatment that has proven effective in controlling of tb on a mass basis.(3 ) the adoption of dots has given impressive results with higher treatment success being reported from developing and industrialized countries.(4 ) in ahmedabad corporation area , treatment for tb is supervised by tuberculosis health visitors ( tbhvs ) who are appointed on a contractual basis as dots providers and also by non - tbhvs such as anganwadi workers , community volunteers , teachers , private practitioners , etc . it is accepted that the most effective dots provider will be the one who is accessible , acceptable to the patient , and accountable to the health system . thus the present study was carried out to assess the effectiveness of different dots providers working under rntcp . patients who were diagnosed and treated during the last quarter of year 2004 were selected for the study . there were seven tus in ahmedabad corporation area . the number of beneficiaries selected from each tu was based on the number of new cases treated in a year ( out of which 10% of the cases were selected ) and sample size came out to be a total of 200 . from the register maintained in the tu , patients were selected randomly supervised by tbhvs and non - tbhvs . maximum effort was made to include only category i patients , however , in some of the centers new cases were not supervised by non - tbhvs , in such cases , categories ii and iii were also included in the study . the study was carried out by the medical social worker ( msws ) with predesigned and pretested proforma . investigator followed these selected patients till the completion of the treatment to assess the outcome and other variables such as treatment taken during intensive and continuation phase , regularity of the treatment , health education , sputum examination , collection of empty blister packs , defaulter action , etc . they visited each patient three times , one in the beginning of the treatment , second completing the 2 months , and third at the end of the full course of the treatment . definitions for cured , treatment completed , defaulter , died , and failure were used as per the rntcp guideline.(5 ) to calculate the cured rate only patients treated under categories i and ii were included and patients treated under all the three categories were included to calculate the treatment success rate . ethical clearance for the study was obtained from the institutional ethical committee and verbal consent taken from all the patients . out of a total of 200 patients , 57.0% were males and 43% were females . both males and females showed similar age distribution with the mean age 33.9 14.9 years and 33.85 14.03 years , respectively . tbhvs supervised total 105 patients of which 61.83% were of category i and treatment of 95 patients was supervised by non - tbhvs . different providers included under non - tbhvs were private practitioners , ngos , cured tb patients , link workers , and anganwadi workers [ table 1 ] . category - wise patients supervised by different dots provider around 95% of the patients who were under the supervision of tbhvs and non - tbhvs took the medicine under the direct observation of the provider . a total of 4 out of 10 patients who had taken treatment at home without direct observation were supervised by private practitioners . the average distance to a dots center was 0.82 km 1.07 and to a dots provider place in the case of a non - tbhv was 0.55 km 0.706 . a total of 97% of the patients had been informed about the importance of the regularity of the treatment . this observation was slightly higher in tbhvs ( 98.09% ) than non - tbhvs ( 95.98% ) but the difference was statistically insignificant ( z = 0.88 , p > 0.05 ) . more than 90% of the patients had completed the intensive phase ( ip ) therapy in both the groups . all the patients ( 35 ) who were supervised by a link worker or an anganwadi worker had completed the intensive phase . no significant difference was observed in the outcome of the intensive phase between tbhvs and non - tbhvs ( z = 1.50 , p0 > 0.05 ) [ table 2 ] . provider versus result of intensive phase therapy a total of 184 patients out of 200 patients had continued the treatment after the intensive phase . it was found that 2 patients died within first 2 months and 14 were defaulters . at the end of the continuation phase , total 182 patients were analyzed as 2 more patients died during the continuation phase ( cp ) . overall 159 ( 87.36% ) were reported to have taken the first dose of treatment in a week under supervision . but compliance to this norm was significantly high ( z = 3.14 , p0 < 0.001 ) in the case of tbhvs ( 94.74% ) compared to non - tbhvs ( 79.31% ) . however , both the groups collected empty blister packets while collecting another weeks treatment in 95% of the patients . regarding the treatment outcomes with the different models of dots providers , there was a significant difference ( risk ratio 1.53 , ci 1.15 - 2.03 ) in the cured rate which was 71.74% ( 66/92 ) with tbhvs and 60% ( 39/65 ) with non - tbhvs . however , no significant difference was observed in the treatment success rate ( risk ratio 0.87 , ci 0.76 - 0.99 ) between tbhvs ( 80/105 ) and non - tbhvs ( 83/95 ) . a 100% cure or completed rate was observed among patients supervised by link workers and anganwadi workers . no significant difference was observed in the defaulter rate between tbhvs ( 14.28% ) and non - tbhvs ( 9.47% ) [ table 3 ] . in dots , each and every dose of the drugs during the intensive phase should be taken in front of dots providers . lack of supervision was found to be more in non - tbhvs as compared to tbhvs ; however , there was no significant difference in the outcome of intensive phase therapy between tbhvs and non - tbhvs . regular supervision is required to ensure that the patients actually take all the drugs even during continuation phase but compliance was significantly better in tbhvs than non - tbhvs . distance traveled to the provider did not affect the outcome though the average distance to the dots center was significantly more compared to the distance to the providers place in the case of non - tbhvs . all the patients should be made aware about the risks of irregular or incomplete treatments . in the present study , more than 95% of the patients though the overall supervision both during intensive and continuation phase was better with the tbhvs , there was no significant difference in the outcome of treatment in terms of completed rate and defaulter rate ( except for the cure rate ) . among non - tbhvs , private practitioners of allopathy , ayurveda and other alternative systems are the people , more than 50% of the tuberculosis patients consult for advice and service initially . being a scientific group with deep - rooted ideas , this group requires a different approach for adopting the dots strategy.(6 ) though the compliance during intensive and continuation phase therapy was better in the case of patients supervised by tbhvs , however there was no significant difference in the defaulter rates among tbhvs and non - tbhvs . it is universally accepted that no public health program can succeed without the total involvement and participation of the community.(6 ) available community workforce could be involved in supervising the intermittent short - course chemotherapy as there was no difference in the outcome between tbhvs and non - tbhvs . providers other than tbhvs especially private practitioners need to be trained and motivated to monitor the treatment for better supervision .

the life - style of women in developing countries is changing due to a shift in age at marriage and child bearing . working mothers show a low inclination toward breast feeding and an increased trend in early weaning . fine - needle aspiration cytology ( fnac ) of palpable breast lesions has become a boon in developing countries since it is a quick , easy and cost - effective technique . in many centers , fnac is the only pre - operative investigating procedure to analyze the breast tumors . now , however , it has become a tool to differentiate between various benign and malignant lesions of the breast . masood developed a cytological grading system to delineate benign and malignant lesions into four groups based on the cellular arrangement , the degrees of cellular pleomorphism and anisonucleosis , the presence of myoepithelial cells and nucleoli and the status of the chromatin pattern . this has been recognized as the masood cytologic index ( mci ) [ table 1 ] . grading system for interpretation of fnac by masood cytologic index similar to the nottingham prognostic index that provides a useful guideline for deciding the systemic adjuvant therapy , studies have revealed that neoadjuvant therapy can minimize the morbidity and enhance the prognostication in breast carcinoma . the overall aim of the present study was to propose an expansion of malignant category of mci to target the neoadjuvant therapy to appropriate patients . specific objectives were ( a ) to construct an expansion of malignant category of mci based on scoring of selected cytological features , ( b ) to validate the same by comparing with the gold standard histological grading system ( i.e. , modified bloom richardson [ mbr ] grading system ) and ( c ) to assess the role of various cytological features in determining the grading scores . this was a prospective study of 50 cases of breast carcinoma over a period of 3 years . women diagnosed with breast carcinoma on fnac and subsequently subjected to surgical excision were selected for the study . the aspirate was expressed and thinly spread on 4 - 5 clean dry glass slides . hematoxylin - eosin and papanicolaou stains were used for the slides , which were fixed in 95% ethyl alcohol . scores ranging from 1 to 4 were assigned to each of the 6 features under consideration as described in table 1 by two independent blindfolded observers . these 6 scores were added to arrive at the final score for grading of the carcinoma . thus , the final grading score for each of the individual cases ranged from a minimum of 6 to a maximum of 24 . score range : tumor grade 6 - 10 : non - proliferative breast disease 11 - 14 : proliferative breast disease without atypia 15 - 18 : proliferative breast disease with atypia 19 - 20 : carcinoma , grade i 21 - 22 : carcinoma , grade ii 23 - 24 : carcinoma , grade iii wherever the scores differed between the two observers , the slides were reviewed for final score . since the cases under study also underwent excision surgery and specimens were histopathologically examined , these carcinomas were graded according to mbr grading system . this grading system was taken as the gold standard to validate the proposed expansion of mci grading . we estimated concordance rates for the same , for individual grades as well as for the overall grade . multiple regression analysis was performed to assess the role of different cytological features in the grading scores of a carcinoma . the regression coefficients were tested for the significance applying student 's t - test at 5% level . scores ranging from 1 to 4 were assigned to each of the 6 features under consideration as described in table 1 by two independent blindfolded observers . these 6 scores were added to arrive at the final score for grading of the carcinoma . thus , the final grading score for each of the individual cases ranged from a minimum of 6 to a maximum of 24 . score range : tumor grade 6 - 10 : non - proliferative breast disease 11 - 14 : proliferative breast disease without atypia 15 - 18 : proliferative breast disease with atypia 19 - 20 : carcinoma , grade i 21 - 22 : carcinoma , grade ii 23 - 24 : carcinoma , grade iii wherever the scores differed between the two observers , the slides were reviewed for final score . since the cases under study also underwent excision surgery and specimens were histopathologically examined , these carcinomas were graded according to mbr grading system . this grading system was taken as the gold standard to validate the proposed expansion of mci grading . we estimated concordance rates for the same , for individual grades as well as for the overall grade . multiple regression analysis was performed to assess the role of different cytological features in the grading scores of a carcinoma . the regression coefficients were tested for the significance applying student 's t - test at 5% level . proposed expansion of malignant section of mci has been presented in the previous section . for validation of the proposed method , we cross - tabulated 50 cases under study according to the two grading systems : the proposed one and the mbr . mbr classified 14% of the cases under study as grade i carcinomas , 54% grade ii and the remaining 32% grade iii . all the 7 grade i carcinomas were classified as grade i according to the proposed method also giving a concordance rate for grade i carcinomas as 100% . similarly for grade ii and grade iii carcinomas [ figure 1 ] , the concordance rates were 85% and 81% respectively . 43 of 50 cases graded by the proposed method were in consistency with the mbr giving an overall concordance rate of 86% . thus , the performance of the proposed grading system appears to be almost perfect for early carcinomas and is useful for advance carcinomas . the left panels showing expanded masood 's cytological grade i , ii and iii respectively ( pap , 400 ) ( d - f ) the right panels showing corresponding histopathology with grade i , ii and iii ( h and e , 200 ) the results of multiple regression analysis of grading scores on the 6 cytological features under study have been presented in table 2 . the analysis revealed a r2 value of 60% indicating that 60% of the variation in grading scores could be explained by the 6 parameters under study . table 2 shows that anisonucleosis , nucleoli and chromatin pattern had a significant role in the prediction of overall grading scores . quantitatively , chromatin pattern was the highest contributor to the total grading score , followed by anisonucleosis . a unit change in the score of chromatin pattern was associated with a change of 0.63 in the total grading score . the breast is an easily palpable organ and so , more importance is being given to early diagnosis of breast carcinoma than the various treatment modalities . fnac has been practiced for more than 160 years . even though , breast fnac has been known for such a long time , its utility was underestimated . diagnosis of palpable breast lesions in developing countries by fnac is the initial method of pathological assessment as a component of triple test . with increasing use of fnac in the diagnosis of palpable and non - palpable breast lesions , the scope of cytology has been gradually shifting from the mere identification of malignancy to accurate cytologic categorization of various benign and malignant breast lesions . the evaluation of malignant breast aspirates , should provide not only the diagnosis of malignancy and type of carcinoma , but also information on the cytological grade of ductal carcinomas . patients with grade i carcinomas have a significantly better survival than those with grade ii and iii carcinomas . a grading system based on cytology would be helpful in the selection of patients for appropriate therapy . recently , neoadjuvant therapy is gaining more attention as primary medical treatment for breast carcinoma , resulting in more importance for cytological grading . administering neoadjuvant therapy to low - grade carcinomas leads to overtreatment and increased morbidity that can be avoided . the need to assess the biological aggressiveness of breast carcinoma by cytological grading before any surgical intervention is thus mounting . therefore , neoadjuvant therapy with tamoxifen gives better result by more rapidly shrinking the carcinoma when given to high - grade ( grade iii ) than low - grade carcinomas ( grade i ) . the nottingham prognostic index is a useful and sensitive guide for selecting adjuvant systemic therapy and there is no reason why such an index should not be developed for neoadjuvant treatment . with the advent of neoadjuvant therapy , with all this , the importance of breast fnac has been upgraded as a diagnostic , therapeutic and prognostic procedure . was 86% and in our study it is 86% , which is consistently high in all three studies . even though , mci has been helpful in delineating all categories of breast carcinomas , there were no studies to further grade the carcinoma category . here , an attempt has been made to grade them without altering the scoring and grouping methods described by masood . on multiple regression analysis , the nuclear features such as anisonucleosis , chromatin clumping and nucleoli were significant , showing a p < 0.05 , while the cellular arrangement , presence or absence of myoepithelial cells and cellular pleomorphism were not significant . in a manner similar to that of the mbr system , where emphasis is laid on the nuclear features , it seems that the same features may also help in grading malignancies through the mci system . since the nuclear grade provides such important prognostic information and is a fundamental cytologic parameter , the nuclear grade should appear in fine - needle aspiration biopsy reports on breast carcinomas . discriminant analysis by other authors showed that the features with the closest correlation with histological grade were nuclear diameter , nuclear pleomorphism and the presence of nucleoli . a scoring system based on these three parameters enabled the classification of carcinomas into high and low cytological grades , which showed a close correlation with histological grade . similar to the standardization of the histopathological grading system , there is a need to standardize cytological grading systems . the present study , with the expansion of carcinoma category of mci into three grades similar to mbr , will help the surgeon to plan the management accordingly . however , the expanded mci will help the cytologist to classify benign breast lesions into different categories as well as grade malignant breast lesions . hence , this novel cytological grading system will help the cytologist to categorize any breast lump accordingly . the results obtained in this study need to be subjected to multicentric study with a large number of cases .

twenty - two sites in five european countries ( france , germany , norway , romania , and spain ) participated in this phase 2 , open - label , randomized , controlled 16-week trial . the trial protocol was approved by local institutional review boards , and all subjects gave written informed consent . the study was conducted in accordance with the declaration of helsinki ( 14 ) and good clinical practice guidelines ( 15 ) . adults with type 2 diabetes were enrolled if they were 1875 years of age , had an a1c of 711% , and had a bmi of 2537 kg / m . subjects had to be insulin - nave ( no previous insulin treatment or insulin treatment for 14 days in the 3 months prior to trial ) , and had to be treated with up to two oads in the 2 months prior to trial at stable maximum doses or at least half maximum allowed doses . subjects were excluded if they had been treated with thiazolidinediones in the 3 months preceding the trial . other exclusion criteria included cardiac disease ( heart failure : new york heart association class iii or iv , unstable angina pectoris , or a myocardial infarction ) within 12 months of the trial , severe hypertension ( systolic blood pressure 180 mmhg or sitting diastolic blood pressure 100 mmhg ) , recurrent severe hypoglycemia or hypoglycemia unawareness , use of drugs likely to affect glycemia , impaired hepatic function ( alanine aminotransferase > 2.5-fold the upper local reference limit ) , pregnancy , and breast - feeding . prior to randomization , eligible subjects discontinued their pretrial oad treatment and underwent a 2-week forced metformin titration period ( dose increased up to 2,000 mg / day : 1,000 mg each at breakfast and the evening meal ) followed by a 1-week metformin maintenance period . subjects taking metformin at enrollment could undergo a modified titration period or advance directly to the metformin maintenance period . metformin could be decreased to a minimum of 1,500 mg / day in the case of unacceptable hypoglycemia or other adverse events . subjects were eligible for randomization provided the maximum daily metformin dose ( 2,000 mg ) or maximum tolerated dose ( 1,500 mg ) remained unchanged in the maintenance period and the median prebreakfast self - measured plasma glucose ( smpg ) value ( measured on the 3 days prior to randomization ) was 7.5 randomization was carried out using a telephone- or web - based randomization system , with subjects stratified according to pretrial oad treatment ( table 1 ) . eligible subjects were randomized ( 1:1:1 ) to receive once - daily subcutaneous injections of either idegasp ( 70% ideg and 30% iasp ; novo nordisk a / s , bagsvrd , denmark ; 100 u / ml ) , af ( 55% ideg and 45% iasp ; novo nordisk a / s , bagsvrd , denmark ; 100 u / ml ) or iglar ( lantus ; sanofi - aventis , paris , france ; 100 u / ml ) for 16 weeks , all in combination with metformin . characteristics of randomized population and subject disposition data are means sd or n ( % ) unless otherwise indicated . serious adverse event of transient ischemic attack considered by the investigator to be unlikely related to trial product . the insulin starting dose was 10 units administered in the abdomen ( idegasp , af ) or thigh ( iglar ) before the evening meal . idegasp and af were administered using a 3 ml flexpen device ( novo nordisk a / s , bagsvrd , denmark ) ; iglar was administered using a 3 ml optiset device ( sanofi - aventis , paris , france ) . based on smpg levels before breakfast ( lowest fpg value from 3 consecutive days ) , insulin doses were individually titrated once a week throughout the trial ( by clinic or telephone contacts ) aiming at an fpg level of 4.06.0 mmol / l ( 72108 mg / dl ) . mmol / l ( 109144 mg / dl ) , by 4 units if fpg was 8.19.0 mmol / l ( 145162 mg / dl ) , or by 6 units if fpg was > 9.0 mmol / l ( > 162 mg / dl ) ; doses were decreased by 2 units ( or 5% reduction if dose > 45 units ) if fpg was 3.13.9 mmol / l ( 5671 mg / dl ) , and by 4 units ( or 10% reduction if dose > 45 units ) if fpg was < 3.1 mmol / l ( < 56 mg / dl ) . if the fpg target was reached , idegasp and af doses could be further adjusted ( in suggested increments of 2 units ) to achieve a dinner postprandial plasma glucose target value of < 8.0 mmol / l ( < 145 mg / dl ) , provided that no hypoglycemia had occurred . other efficacy end points included changes in laboratory - measured fpg and 9-point smpg profile . the proportion of subjects achieving a1c < 7.0% and 6.5% at end of trial , and the proportion reaching these a1c targets without confirmed hypoglycemia ( confirmed by a plasma glucose measurement of < 3.1 mmol / l [ 56 mg / dl ] or if classified as severe ) in the last 4 weeks of treatment ( subjects treated for 8 weeks ) were also determined . safety variables included adverse events , hypoglycemic episodes , vital signs , physical examination , fundoscopy , electrocardiogram , standard biochemical and hematology measures , and insulin antibodies ( ideg - specific , iasp - specific and antibodies cross - reacting between ideg and iasp and between ideg and human insulin ) . mmol / l [ 56 mg / dl ] irrespective of symptoms , or if classified as severe ) . hypoglycemia was considered nocturnal if the time of onset was between 2300 and 0559 h. laboratory analyses were performed by quintiles central laboratories ( edinburgh , scotland ) . a1c was assayed using a validated high - performance liquid chromatography method certified by the national glycohemoglobin standardization program . fpg was measured using the gluco - quant system ( roche , mannheim , germany ) . insulin antibodies were analyzed by celerion ( fehraltorf , switzerland ) using a subtraction radioimmunoassay method ( 16 ) , validated according to standard procedures ( 17 ) . subjects determined smpg using glucose meters ( abbott diabetes care , alameda , ca ) and recorded values in diaries . the statistical evaluation of a1c , fpg , and 2-h postprandial plasma glucose increment was based on all randomized subjects following the intention - to - treat principle . treatment differences in a1c and fpg values after 16 weeks of treatment were estimated by a linear model , in which the estimates were adjusted by country , sex , oad therapy at screening , age , and baseline values . mean 2-h postprandial plasma glucose increments ( plasma glucose concentration measured 2 h after a meal minus the plasma glucose concentration measured immediately prior to the meal ) were analyzed in an identical fashion to the primary end point except for the inclusion of the premeal plasma glucose value as an additional covariate . testing for superiority or noninferiority was not the aim of this exploratory trial ; no confirmatory hypotheses were prespecified , and no formal statistical testing was undertaken . instead , the aim was to estimate a treatment difference in a1c with sufficient precision : a 95% ci for the treatment difference with a total width of 0.8% ( absolute ) was considered sufficient for this proof - of - concept trial and would be obtained with 50 completed subjects per treatment arm . on the basis of the chosen precision for a1c and an expected dropout rate of 15% , 60 subjects were to be randomized to each treatment arm . values are presented as mean sd for descriptive statistics and as estimated mean ( 95% ci ) for inferential statistics from the linear model . adults with type 2 diabetes were enrolled if they were 1875 years of age , had an a1c of 711% , and had a bmi of 2537 kg / m . subjects had to be insulin - nave ( no previous insulin treatment or insulin treatment for 14 days in the 3 months prior to trial ) , and had to be treated with up to two oads in the 2 months prior to trial at stable maximum doses or at least half maximum allowed doses . subjects were excluded if they had been treated with thiazolidinediones in the 3 months preceding the trial . other exclusion criteria included cardiac disease ( heart failure : new york heart association class iii or iv , unstable angina pectoris , or a myocardial infarction ) within 12 months of the trial , severe hypertension ( systolic blood pressure 180 mmhg or sitting diastolic blood pressure 100 mmhg ) , recurrent severe hypoglycemia or hypoglycemia unawareness , use of drugs likely to affect glycemia , impaired hepatic function ( alanine aminotransferase > 2.5-fold the upper local reference limit ) , pregnancy , and breast - feeding . prior to randomization , eligible subjects discontinued their pretrial oad treatment and underwent a 2-week forced metformin titration period ( dose increased up to 2,000 mg / day : 1,000 mg each at breakfast and the evening meal ) followed by a 1-week metformin maintenance period . subjects taking metformin at enrollment could undergo a modified titration period or advance directly to the metformin maintenance period . metformin could be decreased to a minimum of 1,500 mg / day in the case of unacceptable hypoglycemia or other adverse events . subjects were eligible for randomization provided the maximum daily metformin dose ( 2,000 mg ) or maximum tolerated dose ( 1,500 mg ) remained unchanged in the maintenance period and the median prebreakfast self - measured plasma glucose ( smpg ) value ( measured on the 3 days prior to randomization ) was 7.5 randomization was carried out using a telephone- or web - based randomization system , with subjects stratified according to pretrial oad treatment ( table 1 ) . eligible subjects were randomized ( 1:1:1 ) to receive once - daily subcutaneous injections of either idegasp ( 70% ideg and 30% iasp ; novo nordisk a / s , bagsvrd , denmark ; 100 u / ml ) , af ( 55% ideg and 45% iasp ; novo nordisk a / s , bagsvrd , denmark ; 100 u / ml ) or iglar ( lantus ; sanofi - aventis , paris , france ; 100 u / ml ) for 16 weeks , all in combination with metformin . characteristics of randomized population and subject disposition data are means sd or n ( % ) unless otherwise indicated . serious adverse event of transient ischemic attack considered by the investigator to be unlikely related to trial product . the insulin starting dose was 10 units administered in the abdomen ( idegasp , af ) or thigh ( iglar ) before the evening meal . idegasp and af were administered using a 3 ml flexpen device ( novo nordisk a / s , bagsvrd , denmark ) ; iglar was administered using a 3 ml optiset device ( sanofi - aventis , paris , france ) . based on smpg levels before breakfast ( lowest fpg value from 3 consecutive days ) , insulin doses were individually titrated once a week throughout the trial ( by clinic or telephone contacts ) aiming at an fpg level of 4.06.0 mmol / l ( 72108 mg / dl ) . mmol / l ( 109144 mg / dl ) , by 4 units if fpg was 8.19.0 mmol / l ( 145162 mg / dl ) , or by 6 units if fpg was > 9.0 mmol / l ( > 162 mg / dl ) ; doses were decreased by 2 units ( or 5% reduction if dose > 45 units ) if fpg was 3.13.9 mmol / l ( 5671 mg / dl ) , and by 4 units ( or 10% reduction if dose > 45 units ) if fpg was < 3.1 mmol / l ( < 56 mg / dl ) . if the fpg target was reached , idegasp and af doses could be further adjusted ( in suggested increments of 2 units ) to achieve a dinner postprandial plasma glucose target value of < 8.0 mmol / l ( < 145 mg / dl ) , provided that no hypoglycemia had occurred . other efficacy end points included changes in laboratory - measured fpg and 9-point smpg profile . the proportion of subjects achieving a1c < 7.0% and 6.5% at end of trial , and the proportion reaching these a1c targets without confirmed hypoglycemia ( confirmed by a plasma glucose measurement of < 3.1 mmol / l [ 56 mg / dl ] or if classified as severe ) in the last 4 weeks of treatment ( subjects treated for 8 weeks ) were also determined . safety variables included adverse events , hypoglycemic episodes , vital signs , physical examination , fundoscopy , electrocardiogram , standard biochemical and hematology measures , and insulin antibodies ( ideg - specific , iasp - specific and antibodies cross - reacting between ideg and iasp and between ideg and human insulin ) . mmol / l [ 56 mg / dl ] irrespective of symptoms , or if classified as severe ) . hypoglycemia was considered nocturnal if the time of onset was between 2300 and 0559 h. laboratory analyses were performed by quintiles central laboratories ( edinburgh , scotland ) . a1c was assayed using a validated high - performance liquid chromatography method certified by the national glycohemoglobin standardization program . fpg was measured using the gluco - quant system ( roche , mannheim , germany ) . insulin antibodies were analyzed by celerion ( fehraltorf , switzerland ) using a subtraction radioimmunoassay method ( 16 ) , validated according to standard procedures ( 17 ) . subjects determined smpg using glucose meters ( abbott diabetes care , alameda , ca ) and recorded values in diaries . the statistical evaluation of a1c , fpg , and 2-h postprandial plasma glucose increment was based on all randomized subjects following the intention - to - treat principle . missing values for a1c and fpg treatment differences in a1c and fpg values after 16 weeks of treatment were estimated by a linear model , in which the estimates were adjusted by country , sex , oad therapy at screening , age , and baseline values . mean 2-h postprandial plasma glucose increments ( plasma glucose concentration measured 2 h after a meal minus the plasma glucose concentration measured immediately prior to the meal ) were analyzed in an identical fashion to the primary end point except for the inclusion of the premeal plasma glucose value as an additional covariate . testing for superiority or noninferiority was not the aim of this exploratory trial ; no confirmatory hypotheses were prespecified , and no formal statistical testing was undertaken . instead , the aim was to estimate a treatment difference in a1c with sufficient precision : a 95% ci for the treatment difference with a total width of 0.8% ( absolute ) was considered sufficient for this proof - of - concept trial and would be obtained with 50 completed subjects per treatment arm . on the basis of the chosen precision for a1c and an expected dropout rate of 15% , values are presented as mean sd for descriptive statistics and as estimated mean ( 95% ci ) for inferential statistics from the linear model . a total of 226 people were screened for the trial , of which 46 failed screening criteria , and 2 were run - in failures . the remaining 178 subjects were randomized and exposed to treatment following the metformin run - in period ( table 1 ) . baseline characteristics at randomization were comparable across treatment groups , with the exception of a higher percentage of males in the iglar group ( table 1 ) . a similar proportion ( 710% ) of subjects withdrew from each treatment group during the trial ( table 1 ) . 1a ) ; after 16 weeks , a1c had decreased by 1.3 1.0 % -point ( idegasp ) , 1.5 1.4 % -point ( af ) , and 1.3 1.1 % -point ( iglar ) to similar end - of - trial values ( 7.0 1.0 , 7.2 1.0 , and 7.1 1.3% , respectively ) . idegasp was associated with a 0.11 % -point greater reduction in a1c compared with iglar ( estimated mean treatment difference [ idegasp - iglar ] : 0.11 [ 95% ci 0.41 to 0.19 ] ) and a 0.08 % -point greater reduction in a1c compared with af ( estimated mean treatment difference [ af - idegasp ] : 0.08 [ 95% ci 0.22 to 0.38 ] ) . mean a1c over time ( a ) , percentage of subjects achieving a1c targets of < 7.0% and 6.5% at end of study ( b ) , and percentage of subjects treated for at least 8 weeks achieving a1c targets of < 7.0% and 6.5% at end of study in the absence of confirmed hypoglycemia ( hypo . ) in the last 4 weeks of treatment ( c ) . a similar proportion of subjects ( 50% ) reached an a1c of < 7.0% by trial end , whereas a higher proportion of subjects in the idegasp and iglar groups achieved an a1c value of 6.5% compared with af ( fig . treatments were comparable in terms of the percentage of subjects reaching an a1c of < 7.0% in the absence of confirmed hypoglycemia in the last 4 weeks of treatment ( fig . however , compared with af , a greater percentage of subjects in the idegasp and iglar groups reached an a1c of 6.5% in the absence of confirmed hypoglycemia in the last 4 weeks of treatment ( fig . 2 ) . mean 2-h postprandial plasma glucose increments ( plasma glucose concentration measured 2 h after a meal minus the plasma glucose concentration measured immediately prior to the meal ) were similar for all treatments after breakfast and lunch . however , the increase in mean 2-h postdinner plasma glucose was substantially lower for idegasp ( 0.13 3.43 mmol / l ) and af ( 0.24 3.48 mmol / l ) compared with iglar ( 1.63 3.18 mmol / l ) . mmol / l [ 95% ci 2.45 to 0.23 ] for idegasp - iglar and 0.01 mmol / l [ 95% ci 1.09 to 1.12 ] for af - idegasp . mmol / l in the af group , and by 5.1 3.9 mmol / l in the iglar group to comparable end - of - trial values ( 6.8 2.5 , 7.4 2.8 , and 7.0 2.5 mmol / l , respectively ) . idegasp was associated with a 0.13 mmol / l greater reduction in fpg compared with iglar ( estimated mean treatment difference [ idegasp - iglar ] : 0.13 mmol / l [ 95% ci 1.03 to 0.77 ] ) and a 0.64 mmol / l greater reduction in fpg compared with af ( estimated mean treatment difference [ af - idegasp ] : 0.64 mmol / l [ 95% ci 0.25 to 1.53 ] ) . the self - monitored fpg titration target of 4.06.0 mmol / l was reached by 43 , 54 , and 51% of participants on iglar , idegasp , and af , respectively , at trial end ; the postdinner plasma glucose target ( < 8.0 mmol / l ) was reached by 42 , 76 , and 76% , respectively . the median time to reach the fpg target for the first time was similar for all treatments ( 5 weeks ) . there was no obvious difference in baseline characteristics between subjects who did or did not meet fpg targets other than a slightly higher ( 0.40.9 mmol / l ) baseline fpg in the latter group ( supplementary table 1 ) . baseline ( starting ) insulin doses were comparable across treatment arms ( 0.12 units / kg ) , with doses increasing for all groups during the trial . at end - of - trial , mean daily insulin doses were 20% lower for idegasp and af than iglar ( 0.38 0.16 , 0.36 0.16 , and 0.45 0.20 units / kg , respectively ) . only small changes in mean body weight were observed from baseline to week 16 for idegasp ( 0.4 2.3 kg ) , af ( 0.3 2.2 kg ) , and iglar ( 0.1 3.2 kg ) . mmol / l ) was infrequent and reported for 22% ( 13 subjects ; 20 events ) , 31% ( 18 subjects ; 41 events ) and 15% ( 9 subjects ; 12 events ) of subjects in the idegasp , af , and iglar groups , respectively ; rates of confirmed hypoglycemia were lower for idegasp and iglar than af ( 1.2 , 0.7 , and 2.4 events / patient year ) . nocturnal hypoglycemia occurred rarely for idegasp ( 1 subject ; 1 event ) and iglar ( 3 subjects ; 3 events ) compared with af ( 10 subjects ; 27 events ) . three serious adverse events were reported : two for idegasp ( 2 subjects ; 2 events : depression , transient ischemic attack ; the latter leading to the subject s discontinuation ) and one for af ( epistaxis ) , none of which was considered to be related to trial product by the investigator . adverse events judged to have possible or probable relation to insulin were only reported for af ( 5 subjects ; 5 events : diarrhea , nausea , diabetic retinopathy , ecchymosis , and hematoma ) . overall , levels of antibodies specific to iasp and ideg remained low or undetectable during the trial . small increases were observed from baseline to end - of - trial ( to a median level of 0.1% b / t at end - of - trial ) in antibodies cross - reacting between ideg and iasp ( for both idegasp and af groups ) and between ideg and human insulin ( idegasp group only ) . there was no apparent association between these minor changes in antibodies and a1c , hypoglycemia , insulin dose , or body weight ( results not shown ) . no clinically relevant differences were observed between treatments in physical examination findings , vital signs , standard laboratory analyses ( hematology and biochemistry ) , fundoscopy , or electrocardiogram . 1a ) ; after 16 weeks , a1c had decreased by 1.3 1.0 % -point ( idegasp ) , 1.5 1.4 % -point ( af ) , and 1.3 1.1 % -point ( iglar ) to similar end - of - trial values ( 7.0 1.0 , 7.2 1.0 , and 7.1 1.3% , respectively ) . idegasp was associated with a 0.11 % -point greater reduction in a1c compared with iglar ( estimated mean treatment difference [ idegasp - iglar ] : 0.11 [ 95% ci 0.41 to 0.19 ] ) and a 0.08 % -point greater reduction in a1c compared with af ( estimated mean treatment difference [ af - idegasp ] : 0.08 [ 95% ci 0.22 to 0.38 ] ) . mean a1c over time ( a ) , percentage of subjects achieving a1c targets of < 7.0% and 6.5% at end of study ( b ) , and percentage of subjects treated for at least 8 weeks achieving a1c targets of < 7.0% and 6.5% at end of study in the absence of confirmed hypoglycemia ( hypo . ) in the last 4 weeks of treatment ( c ) . a similar proportion of subjects ( 50% ) reached an a1c of < 7.0% by trial end , whereas a higher proportion of subjects in the idegasp and iglar groups achieved an a1c value of 6.5% compared with af ( fig . treatments were comparable in terms of the percentage of subjects reaching an a1c of < 7.0% in the absence of confirmed hypoglycemia in the last 4 weeks of treatment ( fig . however , compared with af , a greater percentage of subjects in the idegasp and iglar groups reached an a1c of 6.5% in the absence of confirmed hypoglycemia in the last 4 weeks of treatment ( fig . 2 ) . mean 2-h postprandial plasma glucose increments ( plasma glucose concentration measured 2 h after a meal minus the plasma glucose concentration measured immediately prior to the meal ) were similar for all treatments after breakfast and lunch . however , the increase in mean 2-h postdinner plasma glucose was substantially lower for idegasp ( 0.13 3.43 mmol / l ) and af ( 0.24 3.48 mmol / l ) compared with iglar ( 1.63 3.18 mmol / l [ 95% ci 2.45 to 0.23 ] for idegasp - iglar and 0.01 mmol / l [ 95% ci 1.09 to 1.12 ] for af - idegasp . mmol / l in the iglar group to comparable end - of - trial values ( 6.8 2.5 , 7.4 2.8 , and 7.0 2.5 mmol / l , respectively ) . idegasp was associated with a 0.13 mmol / l greater reduction in fpg compared with iglar ( estimated mean treatment difference [ idegasp - iglar ] : 0.13 mmol / l [ 95% ci 1.03 to 0.77 ] ) and a 0.64 mmol / l greater reduction in fpg compared with af ( estimated mean treatment difference [ af - idegasp ] : 0.64 mmol / l [ 95% ci 0.25 to 1.53 ] ) . the self - monitored fpg titration target of 4.06.0 mmol / l was reached by 43 , 54 , and 51% of participants on iglar , idegasp , and af , respectively , at trial end ; the postdinner plasma glucose target ( < 8.0 mmol / l ) was reached by 42 , 76 , and 76% , respectively . the median time to reach the fpg target for the first time was similar for all treatments ( 5 weeks ) . there was no obvious difference in baseline characteristics between subjects who did or did not meet fpg targets other than a slightly higher ( 0.40.9 mmol / l ) baseline fpg in the latter group ( supplementary table 1 ) . baseline ( starting ) insulin doses were comparable across treatment arms ( 0.12 units / kg ) , with doses increasing for all groups during the trial . at end - of - trial , mean daily insulin doses were 20% lower for idegasp and af than iglar ( 0.38 0.16 , 0.36 0.16 , and 0.45 0.20 units / kg , respectively ) . only small changes in mean body weight were observed from baseline to week 16 for idegasp ( 0.4 2.3 kg ) , af ( 0.3 2.2 kg ) , and iglar ( 0.1 3.2 kg ) . mmol / l ) was infrequent and reported for 22% ( 13 subjects ; 20 events ) , 31% ( 18 subjects ; 41 events ) and 15% ( 9 subjects ; 12 events ) of subjects in the idegasp , af , and iglar groups , respectively ; rates of confirmed hypoglycemia were lower for idegasp and iglar than af ( 1.2 , 0.7 , and 2.4 events / patient year ) . nocturnal hypoglycemia occurred rarely for idegasp ( 1 subject ; 1 event ) and iglar ( 3 subjects ; 3 events ) compared with af ( 10 subjects ; 27 events ) . three serious adverse events were reported : two for idegasp ( 2 subjects ; 2 events : depression , transient ischemic attack ; the latter leading to the subject s discontinuation ) and one for af ( epistaxis ) , none of which was considered to be related to trial product by the investigator . adverse events judged to have possible or probable relation to insulin were only reported for af ( 5 subjects ; 5 events : diarrhea , nausea , diabetic retinopathy , ecchymosis , and hematoma ) . overall , levels of antibodies specific to iasp and ideg remained low or undetectable during the trial . small increases were observed from baseline to end - of - trial ( to a median level of 0.1% b / t at end - of - trial ) in antibodies cross - reacting between ideg and iasp ( for both idegasp and af groups ) and between ideg and human insulin ( idegasp group only ) . there was no apparent association between these minor changes in antibodies and a1c , hypoglycemia , insulin dose , or body weight ( results not shown ) . no clinically relevant differences were observed between treatments in physical examination findings , vital signs , standard laboratory analyses ( hematology and biochemistry ) , fundoscopy , or electrocardiogram . the main objective of this exploratory , clinical proof - of - concept trial was to assess the feasibility of insulin initiation with once - daily administration of idegasp , the first available soluble coformulation of distinct rapid - acting and basal insulin analogs , as add - on therapy to metformin in patients with type 2 diabetes insufficiently controlled with oads . idegasp achieved clinically meaningful improvements in a1c of 1.4 % -point that were comparable to those seen with iglar . the reduction in a1c was slightly ( nonsignificantly ) greater with idegasp ( estimated mean treatment difference [ idegasp - iglar ] : 0.11 [ 95% ci 0.41 to 0.19 ] ) but confirmatory trials are needed to establish potential treatment differences . nevertheless , the observation that 50% of subjects achieved an a1c target of < 7.0% with idegasp , and that rates of hypoglycemia were similar to iglar despite greater reductions in 2-h postdinner plasma glucose increment , is promising and warrants further investigation in larger , longer - term trials . in principle , initiation of insulin therapy with a coformulation of two distinct insulin analogs , such as idegasp , has the potential to combine the simplicity of one insulin injection with the advantages of the more physiological profiles of both prandial and basal insulin analogs . insulin regimens with prandial components have been shown to achieve lower a1c levels than basal insulin alone , but at the expense of higher rates of hypoglycemia and greater weight gain ( 13,18,19 ) . until now , it has not been feasible to combine rapid - acting prandial insulin analogs with a long - acting basal analog in one injection . this might be convenient not only for insulin initiation but also for intensification of insulin therapy because many patients will require prandial insulin on top of basal insulin therapy ( 13,20,21 ) . indeed , by addressing both prandial and basal insulin needs from the outset , glycemic control is expected to be achieved in more subjects and sustained for longer , thereby delaying the need to intensify treatment with additional injections . therefore , it should be tested in adequately powered studies whether or not the benefits previously seen with basal and rapid - acting insulin analogs ( e.g. , better postprandial glucose control , less [ nocturnal ] hypoglycemia ) can be achieved through the early introduction of and long - term treatment with a coformulation such as idegasp . it is important , though , to choose the right proportions of rapid - acting and basal insulin ; in this trial we also tested a formulation of idegasp containing a higher proportion of iasp ( 45% ) . this af was associated with a twofold higher rate of confirmed , mostly nocturnal , hypoglycemia and , consequently , fewer subjects achieved a1c targets without hypoglycemia in the last 4 weeks of treatment . in view of these findings , the clinical development of the af has been discontinued . in contrast to af , the overall safety profile of idegasp was comparable to iglar : no relevant changes in safety assessments occurred , hypoglycemia rates were low ( only one nocturnal hypoglycemic event was reported for idegasp ) , and no increases in mean body weight were observed . with respect to efficacy , idegasp provided markedly better postdinner plasma glucose control than iglar ( at a similarly low level of nocturnal hypoglycemia ) . however , this did not translate into improved overall glycemic control ; at end of trial , no apparent difference in a1c was found between treatments . it can not be ruled out that the period of time when postdinner plasma glucose levels differed between treatments was of too short a duration to translate into a significant reduction in a1c , but it should be noted that the lack of an a1c difference is in accordance with early trials with other novel insulin preparations . in fact , initial studies with short - acting insulin analogs did not show a superior effect on a1c lowering compared with human insulin despite lower postprandial glucose levels , and it was not before basal insulin therapy was optimized that beneficial effects on a1c and/or hypoglycemia were observed ( 22 ) . because this was the first trial of idegasp in a clinical setting , a relatively conservative titration algorithm was used , which resulted in 50% of subjects in each treatment arm achieving the fpg target after 16 weeks of treatment . it therefore seems conceivable that a more intense titration algorithm , over a longer time period , may lead to greater glucose and a1c improvements with idegasp . in addition , it is possible that glycemic control could have been further optimized by tailoring the dosing of idegasp to coincide with the meal inducing the largest postprandial glucose excursion ( in the present trial , mean postmeal glucose excursions at baseline were greatest after breakfast ) . it is noteworthy that similar glycemic control was achieved with lower doses of idegasp , although this needs to be confirmed in larger studies . the design of ongoing phase 3 trials with idegasp will take many of the limitations of the current trial into account : more intense treatment algorithms will be applied so that more subjects are anticipated to reach titration targets . this is not expected to lead to higher rates of hypoglycemia than in this study because dosing of idegasp will be targeted to the most appropriate meal of the day rather than fixed to a certain meal . despite the limitations of this proof - of - concept study with a small sample size , a relatively short treatment duration and an open study design , it did show idegasp to be a promising treatment option for initiating ( and potentially intensifying ) insulin therapy in subjects with type 2 diabetes inadequately controlled with oads . idegasp was safe and well tolerated , providing comparable overall glycemic control to iglar with lower doses and with the additional benefit of postdinner plasma glucose control that did not result in an increased risk of nocturnal hypoglycemia .

portal vein thrombosis ( pvt ) after liver transplantation ( lt ) is one of serious complications and reportedly ranges from 2% to 13% . a 60-year - old female underwent living - donor lt with the left liver graft for end - stage liver disease related to chronic hepatitis c. after reperfusion , indocyanine green ( icg)-fluorescence imaging was performed to confirm the graft perfusion , which pointed out an insufficient perfusion on the surface of segment 4 . following intraoperative ultrasonography revealed thrombus in the portal vein of segment 4 , which was successfully removed by heparinized saline flush . this enhances the importance of the surveillance for pvt in the postoperative period as well as the intraoperative period . however , the modality to identify pvt during surgery is limited mainly to intraoperative ultrasound . icg - fluorescence imaging can visualize regions with impaired hepatic perfusion due to pvt in real time during lt in addition to visualization of hepatic flows of reconstructed vessels and evaluation of regions with venous occlusion . icg - fluorescence imaging can be simply performed with single icg injection and is expected to have potential roles to enhance the safety of lt . this work was supported by a grant - in - aid for scientific research from the ministry of education , culture , sports , science , and technology of japan and grants - in - aid for research on hiv / aids , and research on measures for intractable diseases from the ministry of health , labor , and welfare of japan .

geneexpression profiling approaches divided all into several subcategories , in which prognoses and frequencies according to age differ significantly.12 herein , we introduce one of the new all categories , phlike all , which is related to highrisk all . philadelphia chromosome positivity signifies the result of a translocation that gives rise to the bcrabl1 oncogene and is one of the most detrimental hallmarks observed in all patients . two groups independently proposed that multiple patients with phnegative blineage all had geneexpression profiles similar to those of patients with phpositive all.13 , 14 such phnegative all cases were categorized as philadelphia chromosomelike all ( phlike all ) . philadelphia chromosomelike all comprises 10% and 13% of standard and highrisk childhood blineage all , respectively.15 the frequency of phlike all increases with age , accounting for > 25% of young adult cases , whose eventfree and overall survival expectation is extremely poor , similar to that of phpositive cases.15 given that the definition of phlike all was based only on the similarity of geneexpression profiles to phpositive all , the genetic abnormalities associated with phlike all cases were unlikely to be homogeneous . therefore , transcriptome and wholegenome sequencing was carried out to understand genetic alterations underlying phlike all.15 , 16 among 1725 blineage all cases , 154 patients were determined as phlike all and underwent genomic analyses . these approaches subcategorized phlike all into seven groups : type i , ablclass fusions ( abl1 , abl2 , csf1r , pdgrb ) ; type ii , erythropoietinreceptor ( epor ) or jak2 rearrangements ; type iii , cytokine receptorlike factor 2 ( crlf2 ) rearrangements ( often accompanied by jak2 mutations and jakstat signal activation ) ; type iv , other mutations activating jakstat signaling ( il7r , flt3 , sh2b3 , tyk2 , il2rb ) ; type v , uncommon miscellaneous kinase mutations ( ntrk3 , dgkh ) ; type vi , raspathway mutations ( kras , nras , ptpn11 , nf1 ) ; and type vii , no mutations in kinase genes ( fig . 2 ) . categorization of philadelphia chromosomelike acute lymphoblastic leukemia ( phlike all ) according to genetic abnormalities . genomic analyses have shown that phlike all is heterogeneous , but involves a high frequency of kinase gene alterations . the data summarized in this figure are from roberts et al.15 the type i category with ablclass fusions accounted for 22% of phlike all cases and was independent from the abnormalities associated with the jakstat signaling pathway . the type ii category accounted for 18% of phlike all , while type iii cases showed the most frequently observed genetic abnormality ( approximately 20% ) in phlike all cases . interestingly , more than half of the crlf2 rearrangements associated with type iii cases also harbored missense or multiple mutations in jak2 , which activate jakstat signaling . the type iv category was related with other jakstatactivating abnormalities , in which il7r and/or flt3 mutations were common . cases involving the type v and type vii categories were infrequent and difficult to characterize , and type vi occurred in a minority of phlike all cases , exhibiting genomic abnormalities activating ras signaling . importantly , the high frequency of such kinaseactivating mutations in phlike all suggested that the treatment outcomes of phlike all might improve with specific inhibitors ( fig . , the mullighan group reported that the addition of imatinib induced remission in a refractory phlike all patient who had an activating rearrangement of pdgfb.17 kinase gene alterations and their inhibitors in philadelphia chromosomelike acute lymphoblastic leukemia ( phlike all ) . the tyrosine kinase inhibitors for each kinase mutation in phlike all are indicated as a possible therapy . deletions , amplifications , mutations , and structural rearrangements in key transcription factors promoting early lymphoid differentiation ( e.g. , tcf3/e2a , ebf , lef1 , ikzf1 , ikzf3 , pax5 , and blnk ) were detected in 40% of blineage all cases.18 among these , mutations in the ikzf1 gene encoding the ikaros transcription factor were frequently observed and more highly correlated with poor prognosis associated with all than were mutations in genes encoding other transcription factors . notably , many phlike all cases , regardless of the subcategories mentioned above , revealed mutations in the ikzf1 gene , which is also a common finding in phpositive all . ikzf1 encodes the transcription factor ikaros , which is indispensable for the induction of blineage differentiation in hscs.19 , 20 its mutations are also strongly associated with lymphoid blast crisis of cml.21 therefore , here , we introduce accumulating data pertaining to ikzf1 mutations associated with highrisk all ( table 1 ) . features of ikzf1mutated acute lymphoblastic leukemia ( all ) aberrant ikzf1 mutations are likely some of the most detrimental driver mutations , accounting for > 80% of phpositive all . ikzf1 deletions were not detectable in chronicphase cml , but emerged simultaneously when cml transformed to lymphoid blast crisis.21 ikzf1 alterations were also common in phlike all cases , regardless of the type of kinase gene mutation described above,15 and suggested significantly lower 5year eventfree survival rates of phlike all patients compared to those without an ikzf1 alteration . with respect to tlineage all , ikzf1 mutations were also observed more frequently in etpall , the phenotype of which is characterized as tlineage markernegative and hsc / myeloid markerpositive , than in other tlineage all cases.22 homozygous germline ikzf1null mice lacked t , b , and nk lymphocytes and their early progenitors , and heterozygous dominantnegative ikzf1mutated mice rapidly acquired tlineage all.19 , 23 , 24 dominantnegative ikzf1 mutations are more deleterious and oncogenic , likely due to crossinterference with other ikaros family members . most ikzf1 deletions identified in phpositive all were monoallelic and lacked exons 36 of the ikzf1 gene , which encode the nterminal zinc finger dnabinding domain.21 this deletion results in dominantnegative isoforms that inhibit both wildtype ikaros and other family members.25 interestingly , genomic breakpoints in the ikzf1 gene are located in the vicinity of cryptic heptamerrecombinationsignal sequences , which are recognized by the rag enzyme complex.21 these observations suggested that ikzf1 deletions likely occurred with rag expression , which marked the specification of hematopoietic stem / progenitor cells toward the lymphoid lineage.26 , 27 , 28 recent studies have indicated that histone h3 , trimethylated on lysine at position 4 , in proximity to cryptic recombinationsignal sequences , might contribute to misleading the rag complex into producing aberrant recombination and promoting oncogenes.29 thus , epigenetic instability in phpositive hscs or their proximate progenitors could underlie the incidence of ikzf1 mutations . although ikzf1 alterations strongly correlate with refractory all , the underlying mechanism of which remains unknown , recent efforts to investigate the biological features of ikzf1mutated cells offered clues to overcome this intractable disease . the georgopoulos group showed that induction of dominantnegative ikaros isoforms in early preb cells arrested their differentiation at the proliferative large preb cell stage and culminated in oligoclonal expansion with the occurrence of blineage all in transplanted recipients.30 , 31 these ikarosdeleted preleukemic and leukemic cells expressed higher levels of integrins than those in normal counterpart cells , and were more dependent on bm stromal cells through integrinmediated adhesion for their growth and survival . notably , inhibitors for focal adhesion kinase , which transduces integrin signaling into cells , significantly abrogated adhesion and induced apoptosis in ikarosdeleted leukemic cells.30 the mullighan group also reported that ikzf1 alterations induced adhesive potential and hscrelated characteristics in phpositive all cells , while reducing their responsiveness to tyrosine kinase inhibitors.32 ikzf1altered all cells infiltrated bm and interacted with perivascular mesenchymal cells and arterial endothelial cells , which are thought to comprise the hsc niche.33 it is noteworthy that treatment with retinoidreceptor agonists enhanced ikzf1 expression , reversed hsclike features of ikzf1altered phpositive all cells , and recovered their sensitivity to tyrosine kinase inhibitors.32 while retinoids have long been known to affect the integrity of hscs and the differentiation of lymphohematopoietic progenitors by directly or indirectly regulating a number of transcription factors,34 , 35 , 36 numerous nuclear receptors also potentially regulate transcription factors in hscs.37 thus , it is worth examining the types of nuclearreceptor signals that upregulate ikzf1 expression . intragenic deletions of erg , which encodes an ets family member transcription factor , were recently identified in a subset of childhood blineage all.38 intriguingly , although dominantnegative ikzf1 deletions were frequently associated with ergdeleted all cases , the response to treatment and survival of these patients were unexpectedly positive . in fact , within blineage all cases involving ikzf1 deletion , 8year eventfree survival improved to > 85% in cases involving erg deletions , whereas the rate was only 51% without the deletions.38 an independent group also reported similar results , suggesting that erg deletions might mitigate the negative impact of ikzf1 deletion in all prognoses.39 future studies on the molecular mechanisms underlying such exceptional cases might discover more sophisticated strategies to overcome ikzf1 alterations in all cells . historically , germline variations inherited from ancestors were rarely considered to influence all incidence or features . until a decade ago , all pathogenesis was thought to be mostly attributed to acquired mutations in hscs or lymphoid progenitors . however , recent studies have identified multiple genomic variants that increase all susceptibility and affect prognosis . genomewide association studies for childhood all cases identified multiple germline polymorphisms showing high association with all incidence and characteristics.11 some of the inheritable variants were found in lymphoid neoplasmrelated genes , including ikzf1 , arid5b , and cdkn2.40 , 41 , 42 , 43 , 44 ikzf1 encodes the lymphoidlineage transcription factor ikaros mutation , which is intimately associated with all as previously discussed . notably , this protein is also an integral component combining transcription factors with the chromatinremodeling network.45 arid5b is a member of the atrich dnainteraction domain family,46 and although the arid5b function in lymphohematopoiesis has not been well studied , it may be involved in epigenetic regulation of gene expression in hscs and early lymphoid progenitors , similar to other atrich dnabinding proteins.47 , 48 , 49 , 50 interestingly , arid5b polymorphisms are associated with blineage hyperdiploid all and racial differences associated with all incidence.40 , 51 cdkn2 encodes ink4a / arf , which regulates hsc selfrenewal and differentiation under the control of the transcriptional repressor and polycomb group protein bmi1.52 , 53 therefore , hereditary predisposition to all may be related to the epigenetic instability of hscs and lymphoid progenitors . a hyperdiploid karyotype is more common in childhood all compared to adult all ; however , the associated mechanisms remain unknown . moriyama and colleagues studied familial all and identified a nonsense variant of etv6 , a member of the erythroblast transformationspecific family , which is involved in hematopoiesis and oncogenesis , with high prevalence among familial all cases.54 , 55 , 56 they undertook a broad survey for etv6 mutations in > 4405 all children and identified 31 somatic etv6 variants potentially related to all susceptibility . interestingly , all children with etv6 variants were significantly older than those without such variants when diagnosed , and more often had a hyperdiploid karyotype . holmfeldt and colleagues used genomic profiling on 124 childhood all cases with a hypodiploid karyotype57 and identified inherited alterations in tp53 , as well as ras , dnarepair , and receptor tyrosine kinase signalingrelated genes . the higher frequency of all in children relative to adults suggests that the influence of genetic predisposition to the disease might be more profound at younger ages . however , a recent study showed that inherited gata3 variants strongly enhanced susceptibility to all in adolescents and young adults.58 the detected gata3 polymorphisms were also detectable in childhood phlike all,59 and the frequency of all patients with those gata3 variations was positively correlated with patient age at diagnosis . furthermore , gata3 variations were also associated with poor treatment response and high risk of relapse . these findings suggested that agerelated differences in all biology might reflect , at least in part , the genetic variations resident in hscs . in the last decade , advances in geneexpression profiling and genomewide sequencing have revolutionized our understanding of all pathogenesis . as described in this short review , many pathogenic all mutations have been identified in hscrelated genes , which are often associated with all treatment failure and early relapse . as some of the mutations are also associated with activation of certain kinase pathways , the invention of simple , convenient , and costeffective sequencing technologies would enable earlier and more sophisticated therapeutic intervention with specific inhibitors . on the other hand , we must stress that leukemiainitiating mutations are still undetectable in > 10% of childhood all patients , and that the genetic information of older adult all patients has not been catalogued at this stage . nonetheless , we believe that studies in the coming decade will completely describe the genomic landscape of all across all generations and refine the therapeutic algorithm to be more targeted and individualized . the authors have no conflicts of interest relating to the topic of this article . abbreviationsallacute lymphoblastic leukemiaamlacute myeloid leukemiabmbone marrowcmlchronic myeloid leukemiaetpearly tlineage progenitorflt3fmslike tyrosine kinase3hschematopoietic stem cellil7rinterleukin7 receptornknatural killerphlikephiladelphia chromosomelikeragrecombinationactivating genestatsignal transducer and activator of transcription acute lymphoblastic leukemia acute myeloid leukemia chronic myeloid leukemia early tlineage progenitor fmslike tyrosine kinase3 hematopoietic stem cell interleukin7 receptor philadelphia chromosomelike recombinationactivating gene signal transducer and activator of transcription

reversible posterior leukoencephalopathy syndrome ( rpls ) is radiologically characterized by symmetrical subcortical areas of vasogenic edema that are preferentially parieto - occipital , and it typically resolves after appropriate treatment . we present a patient with strikingly unilateral rpls that developed 21 days after coiling of an anterior communicating artery aneurysm and several days of triple - h therapy . cortical and subcortical vasogenic edema and enhancement developed only in the left hemisphere , with a pattern suggesting rpls . after 7 months the pathophysiological mechanism underlying rpls is still not well understood , which makes it difficult to explain the unilateral appearance in this case . since the imaging findings may be confused with other conditions such as ischemia , recognition of rpls after coiling reversible posterior leukoencephalopathy syndrome ( rpls ) , also referred to as posterior reversible encephalopathy syndrome , typically presents with headache , altered mental status , visual disturbances , and seizures in the setting of acute blood pressure alterations.1 however , it has also been associated with sepsis , shock , posttransplantation , postchemotherapy , and medications such as cyclosporine and tacrolimus in the absence of blood pressure changes.2 radiologically , rpls is characterized by symmetrical subcortical areas of vasogenic edema that are preferentially parieto - occipital , and it typically resolves within a few weeks after appropriate treatment.1,3,4 the term rpls may not cover the complete spectrum of the condition , since nonreversible lesions are also reported , as well as lesions in other parts of the brain including the gray matter.3,5 as another example of nontypical rpls we present a patient with unilateral rpls that developed 21 days after coiling of an intracranial aneurysm . she had also experienced an epileptic seizure and had a slightly lowered consciousness at admission . nonenhanced computed tomography ( ct ) showed subtle signs of subarachnoid hemorrhage ( sah ) with some frontobasal subarachnoid blood ( fig . 1 ) . ct angiography revealed a 4-mm aneurysm of the left anterior cerebral artery ( aca ) . at admission on the first day of admission her mean arterial pressure ( map ) varied between 81 and 99 mm hg , with a mean of 88 mm hg . she had no history of hypertension , was no taking any medication , and exhibited a good neurological condition ( hunt and hess grade of 1 ) . the procedure took longer than usual ( 2 hours ) because of the smallness of the aneurysm , which made it difficult to enter it safely with the microcatheter . during the procedure , both internal carotid arteries were catheterized to optimize depiction of the local vascular anatomy ( fig . 2 ) . however , a microcatheter was only advanced in the left aca to reach the aneurysm . according to the local protocol , 5000 ie of heparin was given during the procedure ; no other medication was given during the procedure . the aneurysm was successfully packed with two small coils ( 3 cm long ; 2-mm ultrasoft , boston scientific , natick , ma , usa ) using a left approach . no complications were noted and the patient experienced no focal neurological symptoms . after the procedure the patient developed periods of bradycardia with sinus arrest in the intensive care unit ( icu ) but without clinical symptoms . there were assumed to be secondary to the sah , and were treated conservatively . transcranial doppler ( tcd ) performed during the following week showed mildly increased flow velocities in the middle cerebral artery ( mca ) that were just within the normal range ( 111 and 82 cm / s in the left and right mcas , respectively ) , suggesting mild vasospasm . oral nimodipine at 30 mg taken 12 times daily and triple - h therapy were started according to the local protocol . on day 4 of the triple - h therapy tcd performed 5 days later showed normal mca velocities , although the velocities in the mca and aca continued to be higher on the left than on the right , albeit still within the normal range . the patient was discharged in good clinical condition after 12 days with a prescription for oral nimodipine ( 60 mg taken six times daily for 20 days ) . the evening after taking the last prescription of nimodipine , which was 21 days after coiling and 15 days after the cessation of triple - h therapy , the patient presented at the emergency room with progressive headache , right - sided homonymous hemianopsia , right - sided tactile extinction , and a slight paresis of the right arm . her map during the first day of re - admission was 81 - 120 mm hg ( mean 108 mm hg ) . renal function was normal with serum creatinine , sodium , and potassium levels of 47 mol / l , 140 mmol / l , and 4.4 mmol / l , respectively . nonenhanced ct performed 21 days after the first onset of the sah revealed subcortical hypodense areas in the left hemisphere occipitally and around the insula . magnetic resonance imaging ( mri ) showed vasogenic edema mostly subcortically in the entire left hemisphere , with a preference for the watershed areas ( fig . the combination of headache , confusion , visual disturbances , and vasogenic edema on mri was suggestive of rpls . further clinical analysis revealed no signs of infection , and dural sinus thrombosis was ruled out with ct venography . because late - onset secondary brain ischemia was initially suspected clinically , this improved the clinical status of the patient , with headache being the most prominent symptom ; this was reduced by prescription of dexamethasone at 2 mg twice daily . slow tapering of dexamethasone led to the return of slight headaches , but they could now be controlled with acetaminophen . because the initial clinical suspicion of ischemia was ruled out by the imaging workup , a diagnosis of rpls was eventually suggested on the fifth day of re - admission based on clinical signs and mri findings . by the sixth day of re - admission the map had decreased to 82 - 100 mm hg ( mean 89 mm hg ) , possibly due to the nimodipine treatment , and so no antihypertensive medication was administered in our patient . the mri abnormalities had diminished but were not completely resolved . however , mri performed 7 months after the first presentation showed further reduction of the lesions , with a single small area of signal enhancement remaining in the left frontal lobe ( fig . rpls is generally thought to be associated with a sudden increase in blood pressure , which may exceed the cerebral autoregulation capacity and thus cause vasogenic edema in the brain.1 a second theory has also emerged after reports of rpls combined with regions of vasospasm,5 in which hypoperfusion is assumed to cause injury and subsequent edema . in general , etiological causes reported in cases of rpls include eclampsia , chemotherapy , and systemic diseases such as systemic lupus erythematosus and wegener 's disease . rpls has also been reported in patients after carotid endarterectomy ( cea).6,7 because the brain perfusion is usually chronically low prior to a cea procedure in patients with carotid stenosis , the restoration of ' normal ' blood pressure may result in the autoregulation capacity being exceeded , thus provoking rpls . unilateral rpls similar to our case has been reported8 in a patient after sah and aneurysm clipping . in that case the unilateral distribution might have been due to treatment of combined carotid stenosis on the same side as the rpls . another report of unilateral rpls9 has described a relation with cyclosporine . however , what distinguishes our patient from both of these previous cases is that she had no history of chronic hypoperfusion in the brain and was not on any long - term medication , which makes the unilateral distribution difficult to explain . bilateral rpls has been described after coiling or clipping.10 - 13 giraldo et al.10 suspected that this phenomenon was associated with hemodynamic augmentation ( triple - h therapy ) for symptomatic cerebral vasospasm after sah . some of the reported patients received hemodynamic augmentation therapy , despite having normal tcd results , which may have been the cause of their rpls . a case report was recently published concerning a patient developing asymmetrical rpls after sah , clipping , two angiography procedures , and triple - h therapy.14 the distribution of rpls was asymmetrical rather than being totally unilateral , and mri showed characteristic bilateral rpls lesions in the posterior lobes of the brain . consistent with giraldo et al.,10 hemodynamic augmentation therapy to treat vasospasm was suggested as the underlying cause . our patient also received triple - h therapy for a few days because of suspected vasospasm . however , her rpls symptoms started 15 days after the cessation of triple - h therapy . analysis of the time course of our patient revealed that only the cessation of nimodipine coincided with the onset of rpls . based on the vasoconstriction theory , it is possible that the acute cessation of nimodipine caused vasoconstriction . the left hemisphere previously showed slight vasospasm immediately after sah and might have been more sensitive to changes in perfusion than the right hemisphere , which could explain the unilateral distribution . the restarting of nimodipine was associated with a reduction of headaches and neurological symptoms , and no symptoms returned when nimodipine was slowly tapered thereafter . however , we found no reports in the literature suggesting a causative relationship between the cessation of oral nimodipine and rpls . an alternative explanation is that the elevated map at re - admission combined with the new clinical symptoms was associated with the brain abnormalities . such blood pressure elevations may be treated with intravenous labetalol until they decrease by 25% or a diastolic pressure of 110.120 mm hg is achieved . however , this was unnecessary in our patient since the map decreased within a few days of re - admission , possibly because of nimodipine treatment . in summary , we have presented a case of unilateral rpls related to sah , endovascular aneurysm treatment , and vasospasm treatment . although we can only speculate about the underlying cause in our patient , the presented results emphasize the importance of distinguishing rpls radiologically from other conditions such as ischemia in order to avoid inadequate treatment .

the science of outcome prediction is particularly useful in the setting of the emergency room the entry point of many to acute care . in this issue of critical care jaimes and coworkers evaluate the usefulness of artificial neural networks ( anns ) in predicting hospital mortality in patients presenting to the emergency room with suspected sepsis . the construction of a prediction tool is a difficult undertaking ; it requires careful methodological consideration and validation before the predictions can be deemed valid and reliable in nave patients . these tools identify the presence of associations between the outcome of interest and empiric risk factors that contribute to this outcome . a well designed tool will typically possess three qualities : discrimination ( the ability to identify accurately those patients who will reach the outcome from those who will not ) , goodness of fit ( the ability to match accurately predicted and actual outcomes , such as mortality rate , in subgroups of patients ) , and the ability to achieve these predictions in cohorts of patients similar to those in which the tool was developed . most predictive tool use logistic regression a well vetted statistical technique that is applicable to situations in which the outcome is binary ( e.g. survival / death ) , measured at a predetermined time in the future . the technique can precisely quantify the relative contribution of each risk factor to outcome , typically crystallized as the odds ratio ( i.e. the relative odds that a patient with the risk factor has of reaching the outcome as compared with a patient without the risk factor ) . the key difference between the two techniques is that the contribution of each risk factor is not as rigidly dictated with anns as it is in a logistic regression model . arguably , if a modeler had foresight of the important interactions present , then they could design an accurate standard logistic regression model without the heavy price associated with the use of an ann . is readily available , but there is little guidance as to how ann parameters , such as number of intermediate ( hidden ) layers of neurons , number of neurons in those layers , learning rate , activation functions , and several other tuning parameters , should be chosen and tuned for optimal performance by a novice user . anns , by virtue of the presence of numerous weights linking the neurons , can accommodate nonlinearity but they also include very large numbers of parameters . consequently , the relative contribution of each input risk factor to the outcome is difficult to quantify . because of the large number of parameters , it is very easy for an ann to overfit the development dataset . in other words , the predictions offered by the ann will be overly optimistic in the original population of patients , and this good performance will not generalize well to populations to which the ann is nave . jaimes and coworkers do not offer a prediction in a nave population , nor do those investigators clearly indicate whether techniques were applied to minimize the risk of overfitting . thus , their conclusion may be overstated . limited measures guarding against overfitting can be applied within a development set , without specific recourse to an independent validation set . finally , preparing input data for an ann requires some amount of a priori knowledge . for example , white blood cell count or fever typically have a ' u'-shaped relationship to outcome . both low and high values portend a bad outcome , whereas intermediate values are normal . the predictions of an ann will be significantly improved if this unintuitive relationship between risk factor and outcome is already ' known ' to the modeler ahead of time . anns have been used by many investigators to predict outcomes in the critically ill [ 7 - 11 ] . clearly , anns as prediction tools require significant expertise to build , have significant shortcomings , and should be developed and validated as rigorously as logistic regression models . similarly , anns are easily scalable to ordinal or categorical outcomes , and to survival analysis , which is much less familiar territory for the critical care physician . however , anns can easily be misused , typically because their limitations are not well understood . therefore , in settings in which familiar tools such as logistic regression can be applied , anns should be reserved for those situations where standard models do not perform well ; where one suspects the presence of intense but poorly characterized interactions between risk factors ; where one does not particularly care about quantifying the relative contributions of risk factors ; where appropriate validation is possible ; and , most importantly , where proper expertise is readily available . the science of developing prediction models is best left to the expert , but the clinician can contribute invaluable content knowledge to the process . dr clermont is a paid consultant to amgen , altana , and immunetrics , llc . the current manuscript was realized without any financial or other material support from commercial entities .

some examples include clostridium difficile , methicillin - resistant staphylococcus aureus ( mrsa ) , extended spectrum beta - lactamase producing gram - negative bacilli and acinetobacter baumanii . there is overwhelming evidence that the use of antibiotics is a driving factor for the emergence of resistance . this problem is particularly severe in intensive care areas where antibiotic use is high . does the current practice of prescribing of erythromycin for prokinesis constitute prudent use of antimicrobials ? the mechanisms by which antibiotic use can increase antibiotic resistance have been reviewed by lipsitch and samore and include : selecting in favour of resistant strains ; creating colonization opportunities for resistant strains ( assuming competition between resistant and susceptible strains ) ; and encouraging an increased colonisation ' load ' . in addition , antibiotic use can more rarely select in favour of the emergence of de novo resistance . we recently conducted an observational carriage study that supported the view that mrsa and methicillin - susceptible s. aureus strains compete for colonisation space in the anterior nares . thus , exposure to macrolides has the potential to alter the composition of the resident bacterial microbiota in the anterior nares , leading to selection of mrsa . in support of this , treatment with slow release clarithromycin has been shown to eliminate nasal carriage of s. aureus . this would leave patients more susceptible to colonisation and infection with mrsa . berg and co - workers also showed that treatment with a macrolide increased macrolide resistance in the oropharyngeal flora . erythromycin as a prokinetic agent is used at sub - therapeutic doses , which particularly promotes selection of mutational resistance . hospitals in north america and europe are experiencing a rise in c. difficile infections in the inpatient population caused by a strain ( nap1/027 ) that is characterized by increased toxin production . c. difficile strains isolated from uk hospitals are resistant to several antibiotics , including erythromycin . in addition , the transfer of erythromycin resistance to sensitive c. difficile strains has been linked to simultaneous acquisition of a gene homologous to c. difficile toxin a in nontoxigenic strains of c. difficile . early enteral feeding improves outcome in critically ill patients by increasing gut blood flow and gut function , improving wound healing and reducing septic complications . early nutrient intake ( within 24 to 48 hours of admission ) inappropriate gastrointestinal motility may cause macro- or micro - aspiration of the gastric contents into the lower respiratory tract , which may act as a risk factor for ventilator - associated pneumonia . motility agents are routinely prescribed in patients with high gastric content , identified by high volume of fluids aspirated from the nasogastric tube . the prokinetic agents available have been reviewed , although a subsequent commentary highlighted the lack of any large methodological studies on which to base treatment recommendations . in addition , there is little evidence from clinical trials that erythromycin used as a prokinetic agent improves the outcome of patients in intensive care . although benefits of early enteral nutrition are evident , there is lack of evidence to support the use of erythromycin . prudent use of antibiotics is an essential component of any strategy aimed at reducing the spread of antimicrobial resistance and health care associated infections . the use of sub - inhibitory concentrations of erythromycin as a prokinetic agent contributes to the antibiotic burden , is likely to lead to the spread of antimicrobial resistance in the intensive care unit and may increase the likelihood of c. difficile - associated disease . this practice is incompatible with the principle of ' prudent antimicrobial prescribing ' and should be reserved only for patients in whom alternative agents are contraindicated .

the chromosome 11q13 deletion syndrome , the otodental syndrome or oculo - oto - dental syndrome , is a rare but severe autosomal dominant craniofacial anomaly1 ) . they described that the syndrome is characterized by multiple dental abnormalities that included large , globe - shaped molars , absent or small premolars , delayed eruption of teeth , and high - frequency hearing loss3456789 ) . this syndrome presented a variable expressivity and penetrance with an autosomal dominant inheritance pattern in familial cases , although some sporadic cases have been also reported2345 ) . common clinical manifestations include grossly enlarged canines and fused molars ( globodontia ) , high - frequency sensorineural hearing loss , and ocular coloboma in some cases23 ) . we report a 1-year - old girl who presented with delayed dentition and other craniofacial dysmorphic features . the array - comparative genomic hybridization ( cgh ) analysis revealed a deletion in chromosome 11q13.2-q13.3 . a 1-year - old girl was referred to our neurodevelopmental clinic because of her developmental delay . she was born at 38 weeks of gestational age by vaginal delivery and her weight was 2,090 g ( < 3rd percentile ) . the mother was 27 years old at the time of delivery , and the father was 28 years old . she could hold her head up at 4 months and roll over at 7 months after birth . at the age of 12 months , her body weight was 6.6 kg ( < 5th percentile ) , her height was 71.5 cm ( 10th25th percentile ) , and her head circumference was 42 cm ( < 5th percentile ) . she had dysmorphic features including ptosis of the left eye , right auricular deformity , microcephaly , high - arched palate , and simian line on the right hand . she presented right microtia ( small ear ) with shortened upper parts of the auricles . however , the results of the otoscopic examination and auditory brainstem response measurement were normal . during the ophthalmic examination , no visual or structural abnormalities were observed , except the congenital ptosis of the left upper eyelid . dentition was delayed , but normal teeth were observed upon mandible x - ray examination . the korean bayley scales of infant development test ii had been used to assess her development . according to the test , she had a moderate developmental delay ; a mental development index score of 50 ( 8 months ) and a psychomotor development index score of 50 ( 6 months ) at 12 months of age . however , the array - cgh ( nimbelgen 135k cgx-3 array , roche nimblegen , inc . , madison , wi , usa ) analysis revealed a deletion of a 2.75-mb segment in the region spanning from 67,524,823 to 70,272,728 in chromosome 11q13.2-q13.3 ( fig . she was reexamined at 17 months of age , when her body weight was 8 kg ( < 3rd percentile ) . by this time her left upper eyelid drooped more than it did before , and she underwent ophthalmic surgical treatment . the typical tooth morphology in patients with the otodental syndrome was first described in 1969 by denes and csiba from hungary in a mother and her son2410 ) . the dental abnormalities were classified as those in eruption , number , size , shape , and structure2 ) . globodontia ( large , globe - shaped molars ) occurs in both the primary ( deciduous ) and the permanent dentition . other , less frequent , dental findings include hypoplastic yellow areas in the enamel of the canines and molars , microdontic premolars , numerous supernumerary conoid teeth , deformed maxillary arch with a deep - narrow palate , and odontoma24567 ) . in general , sensorineural hearing loss ( hearing threshold greater than 25 db in one or both ears ) is more pronounced at frequencies of about 1,000 hz . the age of onset of hearing loss varies from early childhood to middle age ; however , the first symptoms usually occur before the patients are in their 20s . regardless of the age of onset , hearing loss is bilateral and progressive , but it may plateau by 40 years of age2456 ) . the otodental syndrome is associated with a congenital coloboma of the eye in some cases , and such a syndrome was termed as an oculo - oto - dental syndrome12511 ) . some authors have reported dysmorphic facial features including a long face , full cheek , anteverted nostrils , a long philtrum , and a cleft lip in some patients with this syndrome23 ) . dental abnormalities and hearing loss usually coexisted in the patients . however , it was found that some patients developed only sensorineural hearing loss , while others showed dental abnormalities without the hearing loss345 ) . in our case , we observed a delayed eruption of the teeth ; however , sensorineural hearing loss was not detected . because the onset of hearing loss may vary from early childhood to middle age 1 ) . in 2007 , gregory - evans et al.1 ) found that the underlying genetic defect causing the otodental syndrome was the hemizygous microdeletion at chromosome 11q13.3 . this finding suggested that the haploinsufficiency of fgf3 is likely to be the cause of the dental and hearing defects . in addition , the hemizygosity of the fadd gene appears to be responsible for the ocular coloboma formation . the otodental syndrome ( omim 166750 ) was named as the chromosome 11q13 deletion syndrome . recently , recessive mutations in the fgf3 gene have been reported in a new form of severe syndromic deafness , characterized by microtia and microdontia ( small teeth ) without eye abnormalities ( omim 610706 : lamm ) . some of the affected individuals were reported to have delayed gross motor development1213 ) . it is possible that the microdeletions may have unmasked a recessive allele of the fgf3 gene on the normal chromosome in individuals with the otodental syndrome1 ) . four of the previously described cases ( omim 166750 , decipher 2389 , 251808 and isca nssv 577346 ) had overlapping symptoms with our patient . these cases were reported in association with otodental syndrome , ocular coloboma , global developmental delay , and mental retardation ( fig . 1 ) . the database of chromosomal imbalances and phenotypes using ensemble resources ( decipher ) reported 2 patients , both showing overlapping 11q deletions . patient 2389 carried a smaller , completely overlapping deletion spanning from 68.072 to 70.307 mb and had congenital deafness . patient 251808 had a much smaller deletion between 67.644 and 68.044 mb and presented clinical features including low - set ears , down - slanting palpebral fissures , speech delay , and mental retardation14 ) . wischmeijer et al.14 ) reported the identification of a cryptic interstitial deletion ( 3.4 mb ) in chromosome 11q13.2-q13.4 that spans from 67.525 to 70.964 mb in a child . the child was diagnosed with developmental delay , severe speech delay , moderate / severe mental retardation , and some dysmorphic features . the patient presented a preauricular tag , small low - set ears , ptosis of the upper eyelid , broad nasal bridge , and a short philtrum . the deleted region in the patient 's chromosome in this study included a region of the shank2 gene . shank2 is a member of a family of genes ( comprising of shank1 , shank2 , and shank3 ) encoding scaffold proteins . these genes are localized in the postsynaptic density of the excitatory synapses in the brain1415 ) . berkel et al.15 ) identified shank2 loss - of function mutations in patients with autism spectrum disorder and mental retardation and suggested a potential role of this gene in the described pathogenesis . in conclusion , the chromosome 11q13 deletion syndrome ( omim 166750 : otodental syndrome ) shows variable clinical manifestations that involve craniofacial dysmorphology . we recommend performing an array - cgh analysis whenever clinical features of patients may lead to uncertain diagnosis of the otodental syndrome and requiring a functional workup for hearing , ophthalmic examination , and long - term dental care .

primary central nervous system lymphoma ( pcnsl ) is a rare variant of extranodal non - hodgkins lymphoma ( nhl ) . cure is achieved only in a minority of patients using high - dose chemotherapy with or without whole brain irradiation . however , combined chemoradiotherapy often is associated with severe neurotoxicity leading to dementia . therefore , new treatment targets are urgently needed . increased expression of nuclear factor kappa - b ( nf - kb)-regulated genes has been detected suggesting that this signaling pathway might be of particular importance . b - cell activating factor belonging to the tumor necrosis factor family ( baff ) and a proliferating inducing ligand ( april ) physiologically stimulate survival of b - cells through activation of the nf - kb - pathway . baff is usually expressed by macrophages , monocytes , and t- , but not b - cells . it binds to three receptors : baff receptor ( baff - r ) , b - cell maturation antigen ( bcma ) and transmembrane activator and calcium modulator cyclophilin ligand interactor ( taci ) . the baff / april system might play an important role in the pathogenesis of systemic b - cell malignancies . aim of this study was to assess the expression profile of the baff / april system in pcnsl . formalin - fixed , paraffin - embedded tissue sections from biopsy specimens of five newly diagnosed pcnsl were assessed . antigen retrieval was done with target retrieval solution ( s1699 , dako , germany ) for 10 min at 98.5c . we used the following primary antibodies : rabbit anti - human baff ( ab117256 , dilution 1:12.5 ) , goat anti - human april ( ab110848 , dilution 1:100 ) , rabbit anti - human baff - r ( ab5965 , dilution 1:100 ) , rabbit anti - human bcma ( ab115315 , dilution 1:50 ) , rabbit anti - human taci ( ab79023 , dilution 1:50 ) , mouse anti - human cd20 [ ab9475 ( l26 ) , dilution 1:35 ] , rabbit anti- human cd3 ( ab5690 , dilution 1:100 ) ( all abcam , cambridge , ma , usa ) , mouse anti - human cd68 ( pg - m1 , m 0876 , dilution 1:100 , dako , glostrup , denmark ) and rabbit anti - human baff ( abin740175 , dilution 1:50 , antibodies - online inc . , the following secondary antibodies were used : horseradish peroxidase conjugated polyclonal goat anti - rabbit ( dilution 1:100 ) , horseradish peroxidase conjugated polyclonal goat anti - mouse ( dilution 1:200 , both dako , glostrup , denmark ) , and horseradish peroxidase conjugated polyclonal donkey anti - goat ( dilution 1:125 , dianova , germany ) . all tumors were histologically classified as diffuse large b - cell lymphoma ( dlbcl ) . immunohistochemistry revealed extensive nuclear expression of baff in all specimens ( table 1 , figure 1 ) . expression of baff and april was not restricted to stromal or inflammatory cells , but was found mainly in cd20 positive tumor cells . furthermore , all tumors showed at least weak expression of all three receptors ( table 1 , figure 1 ) . two tumors showed extensive expression of taci and weak to moderate expression of bcma and baff - r . as for the ligands , expression of baff - r , bcma and taci was found mainly within cd20 positive cells . all in all , most cd20 positive cells expressed not only baff / april , but also at least one corresponding receptor . table 1semi - quantitative assessment of expression of the baff / april system in the different tumor specimens.pcnslbaffaprilbaff-rbcmatacie123+++++++++++e236+++++++++++e370++++++++++e582++++++e753++++++++pcnsl , primary central nervous system lymphoma ; baff , b - cell activating factor belonging to the tumor necrosis factor family ; april , a proliferating inducing ligand ; baff - r , baff receptor ; bcma , b - cell maturation antigen ; taci , transmembrane activator and calcium modulator cyclophilin ligand interactor.negative;+<30% positive cells;++3060% positive cells;+++>60% positive cells . pcnsl , primary central nervous system lymphoma ; baff , b - cell activating factor belonging to the tumor necrosis factor family ; april , a proliferating inducing ligand ; baff - r , baff receptor ; bcma , b - cell maturation antigen ; taci , transmembrane activator and calcium modulator cyclophilin ligand interactor . 3060% positive cells ; figure 1immunohistochemical stainings of specimen e582 : haematoxylin & eosin staining ( a ) shows densely packed lymphoid tumor cells , which are positive for cd20 ( b ) . reactive cd3 positive t cells ( c ) and cd68 positive microglial cells ( d ) are found within the tumor mass . cd20 positive tumor cells show extensive expression of baff ( e ) and weak to moderate expression of baff - r ( g ) , bcma ( h ) and taci ( i ) . validation of anti - baff staining using a second antibody in tumor specimen e236 ( k ) . cerebellar tissue was used as positive control of anti - april staining ( l ) . original magnification x200 ( k , l ) , x400 ( a j ) . immunohistochemical stainings of specimen e582 : haematoxylin & eosin staining ( a ) shows densely packed lymphoid tumor cells , which are positive for cd20 ( b ) . reactive cd3 positive t cells ( c ) and cd68 positive microglial cells ( d ) are found within the tumor mass . cd20 positive tumor cells show extensive expression of baff ( e ) and weak to moderate expression of baff - r ( g ) , bcma ( h ) and taci ( i ) . validation of anti - baff staining using a second antibody in tumor specimen e236 ( k ) . cerebellar tissue was used as positive control of anti - april staining ( l ) . original magnification x200 ( k , l ) , x400 ( a j ) . expression of components of the baff / april signaling pathway has been shown in various systemic lymphomas . overexpression of april has been correlated with higher aggressiveness of b - cell lymphomas and expression of taci has been associated with poor prognosis in burkitt lymphoma . another study suggested that baff is able to decrease apoptosis and to enhance survival of malignant b - cells . furthermore , patients with increased serum baff levels had a worse prognosis and baff levels correlated with transformation to a more aggressive tumor phenotype . so far , only one report deals with this signaling pathway in pcnsl : krumbholz et al evaluation of expression of the corresponding proteins , however , was done only for baff - r by immunohistochemistry . the authors assumed that baff might be expressed by astrocytes since they had found baff - expressing astrocytes in the context of multiple sclerosis lesions . in contrast , our results show for the first time , that cd20 positive tumor cells themselves extensively produce baff . therefore , rather autocrine than paracrine stimulation of this signaling pathway might promote proliferation of lymphoma cells . these results indicate a pathogenic role of the baff / april system not only in systemic b - cell lymphomas , but also in pcnsl . therefore , this signaling pathway might represent a new treatment target . indeed , lyu et al . found a significant antitumor activity of a baff fusion toxin in dlbcl cell lines , which expressed baff recep - tors . other groups recently achieved promising results using baff - mediated targeting of liposomal vincristine to malignant b - cells or monoclonal antibodies directed against april . further in vitro and in vivo studies should address the role of the baff / april system in pcnsl and analyze whether blocking this signaling pathway might be beneficial in the treatment of this disease .

atrial septal defect ( asd ) is a tissue defect which allows blood passing between both atria , and accounts for approximately 5 - 10% of all congenital heart defects.1 ) under clinical situations such as high proportions of left - to - right shunts , right ventricular volume overloads and paradoxical embolisms , the closure of asd may be required . for the treatment of asd , depending on the type and anatomical characteristics , percutaneous closure or surgical repair techniques are applied . the surgical approach is based on closing the defects with a direct suture or patch.2 ) embolic complications can occur after surgical interventions,3 ) but cases reported after primary sutures are uncommon.4)5 ) herein , we report two cases of large right atrial thrombus developed in the late stage after repairs of asds which were treated surgically . a 24-year - old female patient with a history of primary repair for asd performed 8 years ago admitted to our clinic for her annual checkups . in her previous primary repair surgery , the defect was closed directly with an atriotomy incision followed by 5/0 polypropylene and 4/0 polypropylene sutures . the patient had attended her ensuing controls every three months for a period of one year , and no pathological findings were identified during this period . physical examination was normal , and routine hematological and biochemical laboratory analysis were within normal levels . transthoracic echocardiography ( tte ) revealed normal left ventricular systolic functions , mildly dilated right chambers , minimal mitral regurgitation , mild tricuspid regurgitation , systolic pulmonary artery pressure of 30 mm hg , and an irregular - shaped mass in the right atrium . on her transesophageal echocardiography ( tee ) examination , a 3.73.5 cm sized giant pedunculated mobile mass was observed being attached to the septum in the right atrium ( fig . chest computed tomography ( ct ) showed no evidence of pulmonary embolism , and ventilation / perfusion scans indicated no problems . protein c , protein s , and antithrombin iii levels were within the normal range . the right atrium was being opened , and a large mass filling the entire atrium and obstructing the tricuspid valve was observed . the mass was intimately attached to the free atrial wall , it was irregularly - shaped , and 2.24.1 cm in dimension . the patient was weaned off with cardiopulmonary bypass without any difficulty . the mass consistent with thrombus formation originating from the suture line a 42-year - old female , without cardiac complaints , was admitted to our clinic presented with chest discomforts , one month history of exertional dyspnea and persistent dry coughs . she had a history of primary asd repair 3 years ago , and quitted routine follow - ups . in her previous primary repair surgery , the defect was closed directly with an atriotomy incision followed by 5/0 polypropylene and 4/0 polypropylene sutures . the patient had attended her ensuing controls every two months for a period of one year , and no pathological findings were identified during this period . tte revealed normal left ventricular systolic functions , heart chambers within normal size , and a mass in the right atrium . on tee examination , a 3.22.4 cm mobile and irregularly - shaped mass was observed in the right atrium . the patient 's bilateral doppler of lower extremity , abdominal ultrasonography , and hypercoagulability screening panel was negative . excision of the mass with redo sternotomy was decided for treatment . during the surgery , exploration of the right atrium revealed a 3.22.3 cm globular mass with a tanned , gelatinous appearance and multifocal areas of calcification . the mass was attached to the free wall of the right atrium by a 1-cm stalk . this mass originating from the free wall of the right atrium was then excised and its histological examination later revealed it to be an organized thrombus . both patients had uneventful postoperative courses , and were discharged with warfarin ( international normalized ratio : 2 - 3 ) combined with acetylsalicylic acid therapy on postoperative seventh and sixth day , respectively . the tte and tee showed no thrombus after 6 months of follow - up for both patients . a 24-year - old female patient with a history of primary repair for asd performed 8 years ago admitted to our clinic for her annual checkups . in her previous primary repair surgery , the defect was closed directly with an atriotomy incision followed by 5/0 polypropylene and 4/0 polypropylene sutures . the patient had attended her ensuing controls every three months for a period of one year , and no pathological findings were identified during this period . physical examination was normal , and routine hematological and biochemical laboratory analysis were within normal levels . transthoracic echocardiography ( tte ) revealed normal left ventricular systolic functions , mildly dilated right chambers , minimal mitral regurgitation , mild tricuspid regurgitation , systolic pulmonary artery pressure of 30 mm hg , and an irregular - shaped mass in the right atrium . on her transesophageal echocardiography ( tee ) examination , a 3.73.5 cm sized giant pedunculated mobile mass was observed being attached to the septum in the right atrium ( fig . chest computed tomography ( ct ) showed no evidence of pulmonary embolism , and ventilation / perfusion scans indicated no problems . protein c , protein s , and antithrombin iii levels were within the normal range . the right atrium was being opened , and a large mass filling the entire atrium and obstructing the tricuspid valve was observed . the mass was intimately attached to the free atrial wall , it was irregularly - shaped , and 2.24.1 cm in dimension . the patient was weaned off with cardiopulmonary bypass without any difficulty . the mass consistent with thrombus formation originating from the suture line a 42-year - old female , without cardiac complaints , was admitted to our clinic presented with chest discomforts , one month history of exertional dyspnea and persistent dry coughs . she had a history of primary asd repair 3 years ago , and quitted routine follow - ups . in her previous primary repair surgery , the defect was closed directly with an atriotomy incision followed by 5/0 polypropylene and 4/0 polypropylene sutures . the patient had attended her ensuing controls every two months for a period of one year , and no pathological findings were identified during this period . tte revealed normal left ventricular systolic functions , heart chambers within normal size , and a mass in the right atrium . on tee examination , a 3.22.4 cm mobile and irregularly - shaped mass was observed in the right atrium . the patient 's bilateral doppler of lower extremity , abdominal ultrasonography , and hypercoagulability screening panel was negative . excision of the mass with redo sternotomy was decided for treatment . during the surgery , exploration of the right atrium revealed a 3.22.3 cm globular mass with a tanned , gelatinous appearance and multifocal areas of calcification . the mass was attached to the free wall of the right atrium by a 1-cm stalk . this mass originating from the free wall of the right atrium was then excised and its histological examination later revealed it to be an organized thrombus . both patients had uneventful postoperative courses , and were discharged with warfarin ( international normalized ratio : 2 - 3 ) combined with acetylsalicylic acid therapy on postoperative seventh and sixth day , respectively . the tte and tee showed no thrombus after 6 months of follow - up for both patients . the right atrium contains crista terminalis , eustachian valve and chiari network , and these anatomical structures often cause incorrect interpretations for the evaluation of masses.6 ) thrombi , myxomas , and vegetations should be kept in mind for the differential diagnosis of right atrial masses . fifteen percent of the myxomas derived from the right atrium and are usually linked to the interatrial septum with a broad base and a narrow stalk.7 ) myxomas are usually slow - growing , but sometimes may show rapid progressions and may occur after repairs of asd.8 ) we offered possible diagnoses among our cases , the right atrial myxoma and thrombus were definitely diagnosed by histopathological examination after surgery . after the asd closure , possible complications include pericardial effusion , arrhythmias and thrombus formation . embolization complications have been reported after asd closure with transcatheter devices,9 ) prosthetic patches10 ) and pericardial patches.11 ) however , cases of embolization or intracardiac thrombus developed after primary closure of asd are less common . right atrial thrombi appearing in the postoperative period is a rare finding and possibly originated from the site of interatrial trauma by suction and devices or suture line . previously published cases of atrial thrombus detected in the free wall of the right atrium were also detected in the postoperative third month and third year.4)5 ) in addition , in the report of thrombus on the primary sutured patent foramen ovale site and complicated with systemic embolization by rodriguez et al.,12 ) the atrial thrombus was detected six months later after the operation . in the cases which we presented , thrombus were detected at longer periods such as 8 years and 3 years after surgery , and they were treated with surgical interventions . right atrial thrombus , which are almost always accompanied by pulmonary embolism , carry a higher mortality rate in the preoperative and perioperative period . treatment of right chamber thrombi is controversial ; medical , surgical procedures and percutaneous interventional techniques can be applied.13 ) when using thrombolytic agents for the treatment of immobile intra - cardiac thrombus , there is a risk that the dissolution of the peduncule might lead to the migration of the thrombus , which in return may result in embolization.14)15 ) in addition , one of our patients were complicated with pulmonary embolism . these could have easily migrated into both pulmonary arteries that were nearly completely occluded , therefore , we preferred surgical treatments . anticoagulation treatment for 3 months was previously proposed and implemented.4)16 ) as seen in our cases , for the detection of thrombus formations over the years after operation , further studies on long - term antiplatelet and anticoagulant therapy are necessary . right atrial incisions are used in many other intracardiac procedures , such as congenital repair . therefore , it is important to bear in mind and apply the above mentioned precautions following the completion of these procedures . the long - term detection of atrial thrombus after operation shows the importance of routine echocardiography . echocardiographic follow - ups after the procedure are recommended , usually at 24 hours , 1 month , 6 months , 1 year and then , at regular intervals.17 ) as a result , thrombosis may occur after closure of asd in the right atrium . caution should be exercised in the long - term follow - up period , especially in terms of intra - atrial thrombus .

a 42-year - old man , who had complained of seizure and headache , was admitted to another hospital , where angiography and ct revealed an avm in the left occipital lobe , with hematoma ( fig . preembolic cerebral angiography revealed a left - sided occipital avm fed by the left posterior parietal artery and branches of the left posterior cerebral artery ( fig . after local anesthesia , a 6-fr guiding catheter was positioned in the high cervical internal carotid artery using the femoral approach and the seldinger method . to prevent systemic coagulation , 5000 units of heparin was used , and to ensure that the anticoagulative effect was adequate , an activated clotting time of greater than 300 seconds was employed . throughout the procedure , all catheters were continuously flushed with saline . using a flow - directed 1.8-fr magic catheter ( balt , montmorency , france ) and a hydrophilic terumo 0.010 wire ( terumo , tokyo , japan ) , selective catheterization was performed . the tip of the magic catheter was j - shaped , with steaming , and initial embolization was attempted via the left posterior parietal artery ; for assessment of this vessel , a 1.8-fr magic catheter combined with a terumo 0.010 wire was employed . using a mixture of 1 ml of histoacryl and 3 ml of lipiodol , embolization was successfully performed . guided by a terumo 0.010 wire , a 1.8-fr magic microcatheter was advanced by flow control through the left vertebral artery into a branch of the posterior cerebral artery . the wire did not extend beyond the tip of the catheter , as commonly occurs in the standard microcatheter / wire system , and the microcatheter was subsequently advanced into a turn approximately 1.5 cm from the nidus , at which point it failed to progress . at this juncture , a lateral injection demonstrated no evidence of extravasation , but an anteroposterior injection revealed the extravasation of contrast medium at the point at which the parieto - occipital branch changed direction , at the a site of the microcatheter tip , approximately 1.5 cm from the nidus ( fig . the position of the magic microcatheter was not changed and angiography was again performed ; this time neither occlusion of the parieto - occipital branch nor contrast medium extravasation at the rupture site was seen . we believe that arterial rupture had occurred at the point at which the parieto - occipital branch turned sharply , approximately 1.5 cm from the nidus . because no changes had been made to the microcatheter or position of the wire , the reason for perforation was thought to be over - injection rather than catheter wedging . the tip of the magic catheter was located extravascularly , so in order to occlude the site of arterial rupture , the distal portion of the microcatheter was positioned at the perforation site . the extravascular compartment were sealed with a small amount of a 40% mixture of histoacryl and lipiodol , injected through the same magic catheter ( fig . control vertebral angiography revealed occlusion of the perforation site , with subtle narrowing at the point at which the parieto - occipital branch changed direction . this vessel was reselected and the feeder , including the sealed perforation site , was occluded . the remaining feeders of the posterior cerebral artery were occluded with a 25% mixture of histoacryl and lipiodol . control angiography of the vertebral artery after embolization revealed complete anatomic cure of the avm . after embolization and surgery , two days later , the patient no longer complained of neurologic symptoms or headache . although several reports have dealt with the vascular perforation that can occur during neurointerventional procedures , none has -to our knowledge- described vessel perforation by a flow - directed microcatheter itself and management of the perforation using this same microcatheter . the outcome of vascular perforation has ranged from asymptomatic to massive hemorrhage and death ( 2 , 3 ) . rupture of a vessel is usually related to guidewire manipulation and the use of steerable microcatheters such as the tracker catheter ( 2 , 3 ) . for the embolization of brain avms , the variable , stiff , guidewire - guided microcatheter has thus been gradually replaced by the very floppy , flow - directed catheter ( 4 ) . since january 1996 , we have used the hydrophilic terumo 0.010 wire in conjunction with the 1.8-fr magic microcatheter for superselective catheterization of the nidus of avms . the wire does not extend beyond the catheter tip , as is commonly seen in standard microcatheter / wire systems . endovascular embolization using flow - directed microcatheters has undergone considerable technical evolution in recent years , and during avm - related procedures , periembolic complications are now less frequent than before . even so , during endovascular embolization using flow - directed microcatheters , perforation of a vessel may occur at vessel angulations or in associated aneurysms -whether congenital , flow - related feeder , or intranidal -due to the fact that the wire is stiffer than the microcatheter ( 5 ) . the mechanisms involved in arterial perforation during neurointerventional procedures have been described in several reports ( 2 , 6 ) and fall into three groups : mechanical perforation of a normal vessel , mechanical disruption of a dysplastic vessel or aneurysm , and fluid overinjection ( 2 ) . we consider that in our case , perforation occurred due to the overinjection of contrast medium at a sharp angulation . the management of arterial rupture occurring during embolization has included surgical closure of the perforation , closure with various embolic materials , or conservative management(2 ) . we believe that the most expeditious treatment is occlusion of the perforation by embolization via the offending catheter . despite new developments in the field of flow - directed microcatheters and associated techniques , prompt recognition and closure of the perforation site by embolization via the offending catheter are essential .

numerous syndromes of a suspected infectious origin remain of unknown microbial aetiology even after investigation by a thorough microbial diagnostic assessment that includes the use of broad - range bacterial pcr targeting the 16s rrna encoding gene on normally sterile samples . however , this technique fails to detect bacteria from the chlamydiales order because of the highly different chlamydiales 16s rrna sequences compared to bacteria from other orders . we thus wondered whether members of the chlamydiales order may be implicated in the occurrence of cryptogenic infections . in this study , we performed a pan - chlamydiales pcr on all samples received in our laboratory between 11 may 2012 and 11 march 2013 and all samples that tested negative by 16s rrna pcr analysis . a total of 107 samples from 69 patients were investigated by our homemade pan - chlamydiales pcr in duplicate , as previously described . dna sequencing was not successful in any of these samples , probably because of the low amount of targeted dna . ( dna was detected between a range of 36.2 to 44.7 cycle threshold ( ct ) , corresponding to 3160 and 8 copies of dna / ml respectively ) . the first patient , a 60-year - old woman who had undergone several knee joint replacements and who had metabolic syndrome , was admitted to the emergency ward with fever , dyspnoea and an increasing systemic inflammatory response syndrome . joint infection was documented by a knee arthrocentesis revealing a high amount of polymorphonuclear cells . concomitantly , the patient received a treatment with intravenous amoxicillin / clavulanic acid , which was switched to an oral therapy of rifampicin and doxycycline for 3 months because of the high suspicion of prosthesis intracellular bacterial infection due to the negative cultures and broad - range bacterial 16s rrna pcrs . interestingly , the pan - chlamydiales pcr was positive for two samples from this patient ; one sample was taken from the prosthesis , and the other was a sample of synovial fluid obtained 3 weeks after spacer implantation . the infection could have been of nosocomial origin because the last prosthesis replacement was performed 7 months before the patient 's presentation to the hospital . chlamydia spp . could have been the cause of the infection even though these bacteria are more likely to cause reactive arthritis , . the second patient , a 41-year - old man with no relevant medical history , experienced flulike symptoms while he was in south africa . two months later , back in switzerland , he developed two episodes of bronchopneumonia with fever , cough , dyspnoea , chest pain and a radiologic infiltrate that was located at the left superior lobe . a bronchoscopy was performed after the second episode of bronchopneumonia , revealing a stenosis of a segmental bronchi . all the cultures remained sterile , whereas both the 16s rrna pcr and the broad - range mycobacterial pcrs were negative on all tested lower respiratory tract samples . the bronchial biopsy sample showed signs of chronic inflammation and fibrosis without any signs of neoplasia . the patient had a good clinical outcome with a treatment of amoxicillin / clavulanate ( 16 days ) and clarithromycin ( 17 days ) and after stenting the bronchi with a metallic stent . our work suggests a role for chlamydiales in orthopaedic prosthesis infections and in the pathogenesis of chronic bronchial stenosis or bronchial stenosis superinfection . this relies on the absence of any alternative identified pathogen despite multiple in - depth investigations , a favourable outcome after beginning empirical therapy with antibiotics with intrinsic activity against most pathogens of the chlamydiales order ( i.e. doxycycline , rifampicin and clarithromycin ) and the plausibility that the infectious syndromes were caused by intracellular bacteria . in that regard , several chlamydiaceae and chlamydia - related bacteria have already been associated with pulmonary infections , , . the value of this work is limited by the absence of identification of the aetiological agent at the species level . however , this work shows the added value of pan - chlamydiales pcr in such cases of infections of unknown aetiology and suggests a role of some member of the chlamydiales order as an agent of prosthetic infection and in the pathogenesis of bronchial stenosis or bronchial stenosis superinfection . further studies should be done in order to precisely identify the chlamydiales species involved in these syndromes .

heart failure is one of the most common causes of hospitalization and death , with an increasing trend in the number of patients living with heart failure.1 it has been acknowledged that several comorbid conditions and biomarkers are associated with disease progression and outcome in heart failure.2 indeed , because the thyroid hormones increase heart rate and cardiac contractility and decrease systemic vascular resistance , both overt hyper and hypothyroidism can be a cause of heart failure . therefore , the american college of cardiology / american heart association guidelines for the diagnosis and management of heart failure in adults recommend measurement of thyroid function.3 a low triiodothyronine ( t3 ) concentration in nonthyroidal illness , socalled lowt3 syndrome , is considered to be an adaptive compensatory mechanism and a beneficial response to preserve energy consumption , and the magnitude of the change in t3 concentration varies according to the severity of the illness.4 it has been reported that lowt3 syndrome was also observed in patients with heart failure , and that impairment of thyroxine ( t4 ) to t3 conversion was found to be proportional to the clinical severity of heart failure.5 , 6 furthermore , it has been reported that the impaired concentration recovers to the baseline after adequate therapy for heart failure.7 several studies indicated that lowt3 syndrome was an independent prognostic factor in patients with heart failure.8 , 9 , 10 meanwhile , subclinical thyroid dysfunction is defined biochemically as altered thyroid stimulating hormone ( tsh ) but normal thyroid hormones levels . although thyroid hormones show normal levels , the cardiovascular alterations , such as left ventricular ( lv ) diastolic dysfunction and endothelial dysfunction , which have been reported in overt hypothyroidism have also been observed in patients with subclinical hypothyroidism.11 additionally , the prognostic impact of subclinical hypothyroidism has been reported in patients with heart failure,10 , 12 although the results were inconsistent . as described above , it should be noted that lower thyroid hormones concentrations are observed in patients with more severe heart failure , and that lowered thyroid hormones concentrations return to the baseline when the patient recovers from a decompensated state of heart failure , and that the magnitude of the change is proportional to the severity of heart failure . accordingly , its prognostic impact may also differ with the phase of heart failure . in previous studies of lowt3 syndrome and subclinical thyroid dysfunction in patients with heart failure , thyroid hormones concentrations however , the prevalence and prognostic impact of abnormal thyroid function in an acute decompensated phase of heart failure have not been investigated . we , therefore , examined the prevalence and prognostic impact of lowt3 syndrome and subclinical thyroid dysfunction in patients with acute decompensated heart failure ( adhf ) . from october 2007 to july 2012 , 401 patients were hospitalized with adhf in the national cerebral and cardiovascular center . diagnoses of heart failure were retrieved from the medical records and validated using the modified framingham criteria.13 among the 401 patients , 74 patients ( 18% ) who did not receive thyroid function tests ( tsh , free t4 , and free t3 levels ) on admission were excluded . = 2 ) or thyroxine replacement ( n = 31 ) ] or amiodarone ( n = 28 ) were excluded . this study was approved by the ethics committee of the national cerebral and cardiovascular center , and patients gave informed consent . electrochemiluminescence immunoassay ( eclia ) ( cobas 8000 < e602 > ; roche diagnostics , japan ) was used for the analysis of tsh , free t3 , and free t4 in our institution 's laboratory . the normal reference intervals in our laboratory are : free t3 , 4.0 to 7.1 pmol / l ( 2.6 to 4.6 pg / ml ) , and free t4 , 14.2 to 23.2 pmol / l ( 1.1 to 1.8 ng / dl ) . thyroid function was analyzed as continuous variables using the data of free t3 , free t4 , and tsh , and as categorical variables . for categorical analysis , patients were assigned to the following groups using free t4 and tsh levels , using a common definition of subclinical thyroid dysfunction based on expert reviews and prior reports:14 , 15 euthyroidism defined as tsh of 0.45 to 4.49 miu / l ; subclinical hypothyroidism defined as tsh of 4.5 to 19.9 miu / l with normal free t4 ; subclinical hyperthyroidism defined as tsh < 0.45 miu / l with normal free t4 ; overt hypothyroidism defined as tsh > 4.49 miu / l with decreased free t4 ; overt hyperthyroidism defined as tsh < 0.45 miu / l with elevated free t4 . undetermined. additionally , using free t3 level , lowt3 syndrome was defined as tsh of 0.45 to 4.49 miu / l with decreased free t3 . for free t3 and free t4 , we used our institution 's cutoffs because free t3 and free t4 measurements show greater intermethod variation than do tsh assays.16 , 17 patients underwent standardized evaluation including detailed medical history ( comorbid conditions and medication ) , physical examination , blood chemistry , 12lead electrocardiogram , and chest xray on admission . estimated glomerular filtration rate ( egfr ) was calculated using the modification of diet in renal disease equation : egfr ( ml / min/1.73m2 ) = 186 [ plasma creatinine ] [ age ] [ 0.742 if female ] [ 1.212 if black].18 lv ejection fraction and lv dimensions were estimated by echocardiography in a stable state of heart failure after therapy for heart failure during hospitalization . cardiac death and rehospitalization for heart failure were assessed as cardiac events after admission for adhf . the primary endpoint of this study was composite cardiovascular events , defined as cardiac death and rehospitalization for heart failure . statistical analyses were performed using jmp10 ( sas institute , cary , nc , usa ) . the results were presented as mean sd , median values with their respective 25th and 75th percentiles , or number ( % ) . continuous variables were compared using unpaired student 's ttest or anova followed by tukey 's posttest as appropriate . btype natriuretic peptide ( bnp ) , free t3 , free t4 , and tsh levels were analyzed as continuous variables after natural logarithmic transformation to normalize their distribution . categorical variables were compared among subgroups using chisquared test or fisher 's exact test as appropriate . longterm survival was estimated by kaplan meier analysis , and differences in survival were assessed using the logrank test . univariate and multivariate cox proportional hazards regression models were constructed to investigate baseline data as predictors of composite cardiovascular events . receiveroperating characteristic ( roc ) curves were used to examine the performance characteristics of the indices of thyroid function . area under the curve ( auc ) and 95% confidence of the roc curves were calculated to provide a measure of the accuracy of the indices of thyroid function to predict composite cardiovascular events . from october 2007 to july 2012 , 401 patients were hospitalized with adhf in the national cerebral and cardiovascular center . diagnoses of heart failure were retrieved from the medical records and validated using the modified framingham criteria.13 among the 401 patients , 74 patients ( 18% ) who did not receive thyroid function tests ( tsh , free t4 , and free t3 levels ) on admission were excluded . = 2 ) or thyroxine replacement ( n = 31 ) ] or amiodarone ( n = 28 ) were excluded . this study was approved by the ethics committee of the national cerebral and cardiovascular center , and patients gave informed consent . electrochemiluminescence immunoassay ( eclia ) ( cobas 8000 < e602 > ; roche diagnostics , japan ) was used for the analysis of tsh , free t3 , and free t4 in our institution 's laboratory . the normal reference intervals in our laboratory are : free t3 , 4.0 to 7.1 pmol / l ( 2.6 to 4.6 pg / ml ) , and free t4 , 14.2 to 23.2 pmol / l ( 1.1 to 1.8 ng / dl ) . thyroid function was analyzed as continuous variables using the data of free t3 , free t4 , and tsh , and as categorical variables . for categorical analysis , patients were assigned to the following groups using free t4 and tsh levels , using a common definition of subclinical thyroid dysfunction based on expert reviews and prior reports:14 , 15 euthyroidism defined as tsh of 0.45 to 4.49 miu / l ; subclinical hypothyroidism defined as tsh of 4.5 to 19.9 miu / l with normal free t4 ; subclinical hyperthyroidism defined as tsh < 0.45 miu / l with normal free t4 ; overt hypothyroidism defined as tsh > 4.49 miu / l with decreased free t4 ; overt hyperthyroidism defined as tsh < 0.45 miu / l with elevated free t4 . undetermined. additionally , using free t3 level , lowt3 syndrome was defined as tsh of 0.45 to 4.49 miu / l with decreased free t3 . for free t3 and free t4 , we used our institution 's cutoffs because free t3 and free t4 measurements show greater intermethod variation than do tsh assays.16 , 17 patients underwent standardized evaluation including detailed medical history ( comorbid conditions and medication ) , physical examination , blood chemistry , 12lead electrocardiogram , and chest xray on admission . estimated glomerular filtration rate ( egfr ) was calculated using the modification of diet in renal disease equation : egfr ( ml / min/1.73m2 ) = 186 [ plasma creatinine ] [ age ] [ 0.742 if female ] [ 1.212 if black].18 lv ejection fraction and lv dimensions were estimated by echocardiography in a stable state of heart failure after therapy for heart failure during hospitalization . cardiac death and rehospitalization for heart failure were assessed as cardiac events after admission for adhf . the primary endpoint of this study was composite cardiovascular events , defined as cardiac death and rehospitalization for heart failure . statistical analyses were performed using jmp10 ( sas institute , cary , nc , usa ) . a p value less than 0.05 was considered statistically significant . the results were presented as mean sd , median values with their respective 25th and 75th percentiles , or number ( % ) . continuous variables were compared using unpaired student 's ttest or anova followed by tukey 's posttest as appropriate . btype natriuretic peptide ( bnp ) , free t3 , free t4 , and tsh levels were analyzed as continuous variables after natural logarithmic transformation to normalize their distribution . categorical variables were compared among subgroups using chisquared test or fisher 's exact test as appropriate . longterm survival was estimated by kaplan meier analysis , and differences in survival were assessed using the logrank test . univariate and multivariate cox proportional hazards regression models were constructed to investigate baseline data as predictors of composite cardiovascular events . receiveroperating characteristic ( roc ) curves were used to examine the performance characteristics of the indices of thyroid function . area under the curve ( auc ) and 95% confidence of the roc curves were calculated to provide a measure of the accuracy of the indices of thyroid function to predict composite cardiovascular events . baseline characteristics of patients divided by free t4 and tsh levels are presented in table 1 . among 274 patients , 188 patients ( 69% ) showed euthyroidism and 58 ( 21% ) showed subclinical hypothyroidism . patients included in other criteria of thyroid function were not common ( figure 1 ) . patient characteristics according to thyroid function status on admission binary data are presented as number ( % ) ; continuous variables are presented as mean ( sd ) or median ( interquartile range ) . acei , angiotensin converting enzyme inhibitor ; bnp , btype natriuretic peptide ; arb , angiotensin ii receptor blocker ; egfr , estimated glomerular filtration rate ; hdl , high density lipoprotein ; ldl , low density lipoprotein ; lv , left ventricular ; tsh , thyroid stimulating hormone ; t3 , triiodothyronine ; t4 , thyroxine . * p value < 0.05 vs. euthyroidism . no differences were found regarding age , sex , systolic blood pressure , lv ejection fraction , and plasma bnp among the groups . compared with euthyroidism patients , subclinical hypothyroidism patients had a lower prevalence of ischemic heart disease and hypertension , higher prevalence of atrial fibrillation and diuretic ( loop and/or thiazide ) use , lower heart rate , and higher total bilirubin level . thyroid hormones concentrations , free t3 and free t4 levels , were similar between patients with subclinical hypothyroidism and euthyroidism . among 188 euthyroidism patients , 95 patients ( 35% of the whole population ) showed decreased free t3 ( < 4.0 pmol / l ) and 93 patients showed normal free t3 ( 4.07.1 pmol / l ) ( figure 1 ) . baseline characteristics of patients with lowt3 syndrome on admission are shown in table 2 . compared with patients with normal free t3 , lowt3 syndrome patients were older and had lower levels of egfr , hemoglobin , and triglycerides , but a higher level of creactive protein . baseline characteristics in subjects with normal free t3 and lowt3 syndrome binary data are presented as number ( % ) ; continuous variables are presented as mean ( sd ) or median ( interquartile range ) . the mean duration of followup was 31.8 21.7 months . during the followup period , 27 ( 10% ) patients died of heart disorders , including 6 inhospital deaths and 21 deaths after discharge . among the patients , 20 patients died of heart failure and 7 patients died of ventricular arrhythmia . additionally , 86 ( 32% of discharged patients ) patients were rehospitalized for heart failure . in univariate cox regression analysis , a high tsh level was significantly associated with an elevated risk of composite cardiovascular events ( hazard ratio : 1.34 per 1sd increment in logtransformed tsh value ; 95% confidence interval : 1.09 to 1.64 ; p = 0.006 ) , whereas free t3 and free t4 levels were not . in multiple cox regression analysis adjusting for age , heart rate , egfr , and hemoglobin , the prognostic impact of tsh level persisted ( table 3 ) . in the present patient population , roc curve analysis indicated that tsh on admission had modest ability to predict composite cardiovascular events ( auc = 0.651 ; 95% confidence interval : 0.579 to 0.718 ) . a cutoff level of tsh of 4.39 miu / l best predicted composite cardiovascular events , with sensitivity of 47% and specificity of 81% . univariate and multivariate cox proportional hazards models for composite cardiovascular events age reflects risk with increase of 10 years . with increase of 10 mmhg . with increase of 5 bpm . with increase of 10 ml / min/1.73 m . with increase of 5 mmol / l . with increase of 1 g / dl . with increase of 1 mg / dl . with increase of 1sd in log bnp . with increase of 1sd in log free t3 . with increase of 1sd in log bnp , btype natriuretic peptide ; ci , confidence interval ; egfr , estimated glomerular filtration rate ; hr , hazard ratio ; lv , left ventricular ; t3 , triiodothyronine : t4 , thyroxine : tsh , thyroid stimulating hormone . fifty ( 27% ) euthyroidism patients , 34 ( 59% ) subclinical hypothyroidism , 8 ( 47% ) overt hypothyroidism , and 1 ( 50% ) overt hyperthyroidism had adverse cardiovascular events . no adverse cardiovascular events were observed in patients with subclinical hyperthyroidism . in the present study , we examined the prognostic impact of subclinical hypothyroidism , compared with euthyroidism the survival rate of patients with subclinical hypothyroidism was lower than that of patients with euthyroidism ( logrank : 2 = 15.99 ; p < 0.001 ) . in multivariate cox proportional hazards regression among variables including age , heart rate , egfr , and hemoglobin , patients with subclinical hypothyroidism showed a 2.31fold higher risk of composite cardiovascular events when compared with euthyroidism patients ( table 3 , hazard ratio : 2.31 ; 95% confidence interval:1.44 to 3.67 ; p < 0.001 ) . as demonstrated in the kaplan meier plots of groups with normal free t3 and lowt3 syndrome , the survival rate was comparable between patients with normal free t3 and lowt3 syndrome ( figure 3 ) . meier survival curves for composite cardiovascular events for patients with normal free t3 and lowt3 syndrome . baseline characteristics of patients divided by free t4 and tsh levels are presented in table 1 . among 274 patients , 188 patients ( 69% ) showed euthyroidism and 58 ( 21% ) showed subclinical hypothyroidism . patients included in other criteria of thyroid function were not common ( figure 1 ) . patient characteristics according to thyroid function status on admission binary data are presented as number ( % ) ; continuous variables are presented as mean ( sd ) or median ( interquartile range ) . acei , angiotensin converting enzyme inhibitor ; bnp , btype natriuretic peptide ; arb , angiotensin ii receptor blocker ; egfr , estimated glomerular filtration rate ; hdl , high density lipoprotein ; ldl , low density lipoprotein ; lv , left ventricular ; tsh , thyroid stimulating hormone ; t3 , triiodothyronine ; t4 , thyroxine . * p value < 0.05 vs. euthyroidism . no differences were found regarding age , sex , systolic blood pressure , lv ejection fraction , and plasma bnp among the groups . compared with euthyroidism patients , subclinical hypothyroidism patients had a lower prevalence of ischemic heart disease and hypertension , higher prevalence of atrial fibrillation and diuretic ( loop and/or thiazide ) use , lower heart rate , and higher total bilirubin level . thyroid hormones concentrations , free t3 and free t4 levels , were similar between patients with subclinical hypothyroidism and euthyroidism . among 188 euthyroidism patients , 95 patients ( 35% of the whole population ) showed decreased free t3 ( < 4.0 pmol / l ) and 93 patients showed normal free t3 ( 4.07.1 pmol / l ) ( figure 1 ) . baseline characteristics of patients with lowt3 syndrome on admission are shown in table 2 . compared with patients with normal free t3 , lowt3 syndrome patients were older and had lower levels of egfr , hemoglobin , and triglycerides , but a higher level of creactive protein . baseline characteristics in subjects with normal free t3 and lowt3 syndrome binary data are presented as number ( % ) ; continuous variables are presented as mean ( sd ) or median ( interquartile range ) . the mean duration of followup was 31.8 21.7 months . during the followup period , 27 ( 10% ) patients died of heart disorders , including 6 inhospital deaths and 21 deaths after discharge . among the patients , 20 patients died of heart failure and 7 patients died of ventricular arrhythmia . additionally , 86 ( 32% of discharged patients ) patients were rehospitalized for heart failure . in univariate cox regression analysis , a high tsh level was significantly associated with an elevated risk of composite cardiovascular events ( hazard ratio : 1.34 per 1sd increment in logtransformed tsh value ; 95% confidence interval : 1.09 to 1.64 ; p = 0.006 ) , whereas free t3 and free t4 levels were not . in multiple cox regression analysis adjusting for age , heart rate , egfr , and hemoglobin , the prognostic impact of tsh level persisted ( table 3 ) . in the present patient population , roc curve analysis indicated that tsh on admission had modest ability to predict composite cardiovascular events ( auc = 0.651 ; 95% confidence interval : 0.579 to 0.718 ) . a cutoff level of tsh of 4.39 miu / l best predicted composite cardiovascular events , with sensitivity of 47% and specificity of 81% . univariate and multivariate cox proportional hazards models for composite cardiovascular events age reflects risk with increase of 10 years . with increase of 10 mmhg . with increase of 5 bpm . with increase of 10 ml / min/1.73 m . with increase of 5 mmol / l . with increase of 1 g / dl . with increase of 1 mg / dl . with increase of 1sd in log bnp . with increase of 1sd in log free t3 . with increase of 1sd in log free t4 . with increase of 1sd in log tsh . bnp , btype natriuretic peptide ; ci , confidence interval ; egfr , estimated glomerular filtration rate ; hr , hazard ratio ; lv , left ventricular ; t3 , triiodothyronine : t4 , thyroxine : tsh , thyroid stimulating hormone . fifty ( 27% ) euthyroidism patients , 34 ( 59% ) subclinical hypothyroidism , 8 ( 47% ) overt hypothyroidism , and 1 ( 50% ) overt hyperthyroidism had adverse cardiovascular events . no adverse cardiovascular events were observed in patients with subclinical hyperthyroidism . in the present study the survival rate of patients with subclinical hypothyroidism was lower than that of patients with euthyroidism ( logrank : 2 = 15.99 ; p < 0.001 ) . in multivariate cox proportional hazards regression among variables including age , heart rate , egfr , and hemoglobin , patients with subclinical hypothyroidism showed a 2.31fold higher risk of composite cardiovascular events when compared with euthyroidism patients ( table 3 , hazard ratio : 2.31 ; 95% confidence interval:1.44 to 3.67 ; p < 0.001 ) . kaplan meier survival curves for composite cardiovascular events for patients with euthyroidism and subclinical hypothyroidism . meier plots of groups with normal free t3 and lowt3 syndrome , the survival rate was comparable between patients with normal free t3 and lowt3 syndrome ( figure 3 ) . meier survival curves for composite cardiovascular events for patients with normal free t3 and lowt3 syndrome . we demonstrated that the presence of subclinical hypothyroidism on admission for adhf was an independent predictor of longterm adverse cardiovascular events including cardiac death and rehospitalization for heart failure , although the presence of low t3 concentration on admission was not . these findings suggest that measurements of thyroid hormones concentrations could be useful for early risk stratification of patients with adhf . subclinical hypothyroidism is characterized by a high serum tsh concentration with normal levels of serum t4 ( and t3).15 thyroid hormones are regulated via a feedback loop of tsh and thyroid releasing hormone . as long as the regulation system is preserved , an increase in tsh level indicates deficiency of circulating thyroid hormones . the prevalence of subclinical hypothyroidism has been reported to be between 4 and 20% of communitybased populations,19 , 20 and subclinical hypothyroidism is an independent risk factor for coronary heart disease,21 heart failure,15 , 22 and cardiac death23 in communitybased populations , whereas a large population study of danish national patient registry has demonstrated that risk of major adverse cardiovascular events and developing heart failure was not elevated in patients with subclinical hypothyroidism.24 a few previous studies examining the prognostic impact of subclinical hypothyroidism in patients with heart failure have been reported . although , they have apparently shown inconsistent results , it is noteworthy that the study endpoints differed among the studies . for example , iacoviello et al . demonstrated that a higher tsh level was independently associated with a higher incidence of composite outcomes including death , urgent heart transplantation , or hospitalization because of worsening heart failure.12 on the other hand , frey et al . reported that subclinical hypothyroidism was not associated with allcause death , 42% of which comprised noncardiac deaths.10 in the present study , we demonstrated that a higher tsh level and the presence of subclinical hypothyroidism were independent predictors of composite cardiovascular events including cardiac death and hospitalization for worsening heart failure . considering these results , higher tsh level or the presence of subclinical hypothyroidism might be associated with higher incidence of cardiac events , rather than noncardiac death , suggesting that a close relationship between the thyroid and cardiovascular system could exist in patients with heart failure . during severe systemic illness , the alteration of thyroid hormones levels is also affected by the factors other than the hypothalamicpituitarythyroid axis . more than 80% of t3 , which is considered to be the biologically active form of thyroid hormones , derives from peripheral conversion of the prohormone t4 via a reaction catalyzed by 5monodeiodinases in organs such as the liver , kidney , and skeletal muscle.25 , 26 it has been reported that a decrease in 5monodeiodination leads to low serum t3 concentration in patients with any severe systemic illness , including acute myocardial infarction,27 and chronic heart failure.8 besides the deiodination of t4 , the alterations in thyroid hormones binding to serum proteins and t4 transport to t3producing tissues are involved in such transient changes in plasma concentrations of free thyroid hormones.28 , 29 , 30 proinflammatory cytokines such as interleukin6 ( il6 ) and tumor necrosis factor ( tnf ) , inducing the release of il6 , are considered to contribute to the transient change in the thyroid hormones levels . administration of il6 and tnf into healthy subjects reproduces alterations in both peripheral and central thyroid hormones similar to lowt3 syndrome.31 , 32 actually , the increase of plasma il6 and tnf is observed in patients with heart failure and associated with poor prognosis.33 , 34 therefore , the transient increase in proinflammatory cytokines in adhf patients gives rise to temporary changes in peripheral , and central thyroid hormones not only via the hypothalamicpituitarythyroid axis but possibly also because of a direct negative effect of the cytokines on both hypothalamic and/or pituitary levels.35 t3 level alters with the severity and stage of the illness via the mechanisms described above ; t3 becomes lower as the illness becomes more severe , probably in order to suppress systemic energy consumption , and returns to the baseline as the illness resolves.7 , 36 actually , in a previous report , subclinical thyroid dysfunction at discharge had normalized spontaneously six months after discharge in four fifths of patients hospitalized for heart failure,10 suggesting the presence of the transient subclinical thyroid dysfunction in the course of heart failure . in the extremely severe stage of heart failure , peripheral t4 to t3 conversion would be more strongly suppressed , and subsequently plasma tsh level might increase via a feedback loop of the hypothalamicpituitarythyroid axis . therefore , elevated tsh level on admission of adhf patients might indicate that the patients were in the very severe or lifethreatening systemic state in the adhf phase . frey et al . reported that lowt3 syndrome is an independent prognostic factor in patients with adhf.10 however , we demonstrated that lowt3 syndrome was not associated with composite cardiovascular events in adhf patients . the difference in the timing of evaluating thyroid function may account for the inconsistent results . as described above , the temporal alteration of thyroid hormones levels exists in the clinical course of heart failure.10 in the stable phase of heart failure , lowered t3 level may indicate the intrinsic severity of heart failure , so that lowt3 syndrome might have a poor prognosis . on the other hand , lowered t3 level in the acute decompensated phase may reflect temporal hemodynamic deterioration that could be restored by adequate therapy . we demonstrated that tsh level on admission was an independent prognostic marker in adhf patients , and had modest ability to predict adverse outcomes . although some indicators , including sodium and bnp levels , are considered to be useful to predict cardiac events , it has been reported that few indicators evaluated at the decompensated stage are independent prognostic markers . for example , a high bnp level at discharge is generally considered to be a strong independent marker of death or readmission for heart failure.37 , 38 however , the prognostic impact of bnp level on admission on adhf appears to be weak and is less than that of bnp level at discharge after appropriate treatment for adhf . indeed , bnp level on admission was not a prognostic marker of cardiovascular events in our study . in contrast , the present study indicated that higher tsh level on admission for adhf was independently associated with adverse cardiovascular events after discharge . thus , tsh level could be a practical indicator to stratify the risk of future adverse cardiovascular events , even in the early stage of treatment for adhf . second , in view of the nature of the study design , we can not infer causeeffect relationship between thyroid hormones and heart failure , suggesting that this study can not be used to support the clinical benefit of replacement therapy with thyroid hormones . third , although we demonstrated that the presence of subclinical hypothyroidism at admission was a predictor of worse prognosis in the present study , we can not determine whether a raise of tsh level on admission for adhf had been preexisting before developing adhf , or arose in this adhf phase . in other words , we can not mention the main pathophysiology of worse prognosis : preexisting subclinical thyroid dysfunction and/or the acute exacerbation of heart failure which causes severe general condition and instability of the hypothalamicpituitarythyroid axis . tsh level before hospitalization or after discharge , as well as antithyroid peroxidase antibodies , may be useful to refer to the point , although we do not have sufficient information on them . we demonstrated that the presence of subclinical hypothyroidism on admission for adhf was an independent predictor of adverse cardiovascular events . elevated tsh level could serve as a promising marker to predict cardiovascular events in adhf patients .

non - small cell lung cancer ( nsclc ) is the most common diagnosed cancer and the leading cause of cancer death worldwide ( 1 , 2 ) . in china , new nsclc cases are approximately 569 , 000 annually ( 3 ) . platinum - based chemotherapy remains the first - line treatment for advanced nsclc , especially in patients without mutations in certain genes such as epidermal growth factor receptor ( egfr ) and anaplastic lymphoma kinase ( alk ) . however , the efficacy of different platinum - based regimens appears to have reached a plateau , with the median survival ranges from 8 to 12 months ( 4 , 5 ) , even with the addition of targeted agents , such as bevacizumab and cetuximab ( 6 , 7 ) . novel systemic therapy strategies are in great need to improve the survival in advanced nsclc . chidamide ( cs055 ) is a novel member of the benzamide class of histone deacetylase ( hdac ) inhibitor . it was rationally designed to block the catalytic pocket of class i hdacs with enhanced metabolic stability , durable induction of histone acetylation , and distinct gene expression profiling relative to existing benzamide and hydroxamic acid class inhibitors ( 8 - 14 ) . previous studies have demonstrated that chidamide , functioning as an epigenetic modulator , selectively inhibits the activity of hdac1 , 2 , 3 and 10 , and performs its antitumor action via multiple mechanisms , including induction of growth arrest and apoptosis in blood and lymphoid - derived tumor cells ( 9 ) , activation of natural killer cell- and antigen - specific cd8 + cytotoxic t lymphocyte - mediated cellular antitumor immunity ( 10 , 11 ) , and reversion of epithelial - mesenchymal transitions and drug - resistance of tumor cells ( 12 ) . chidamide has also been shown to synergistically enhance platinum - induced dna damage responses and apoptosis in nsclc cells ( 13 ) . chidamide has shown good tolerability , antitumor activity , and favorable pharmacokinetics and pharmaco - dynamics profiles in phase i and phase ii studies in patients with advanced solid tumors or lymphomas ( 8 , 14 ) . based on the results from a multicenter pivotal phase ii study ( 14 ) , chidamide has been approved for the treatment of relapsed or refractory peripheral t - cell lymphoma ( ptcl ) in china . on the basis of the preclinical and clinical findings , we designed this phase i study to evaluate the safety and pharmacokinetics of chidamide in combination with paclitaxel and carboplatin as first - line treatment in patients with advanced nsclc . patients ( age 18 years ) with cytologically or histologically confirmed nsclc , stage iiib or iv disease , eastern cooperative oncology group ( ecog ) performance status of 0 or 1 , treatment - naive or recurrent after surgery without systematic therapy were eligible . presence of measurable diseases , life expectancy of 3 months and body weight 47 - 87 kg for men and 35 - 75 kg for women were required . qualifying laboratory criteria were as follows : leukocytes 410/l , platelets 10010/l , hemoglobin 11 g / dl , total bilirubin 1.5 upper limits of normal ( uln ) , alanine transaminase ( alt)/aspartate aminotransferase ( ast ) 1.5uln , and creatinine 1.5uln . pregnant women and men with fertility desires were excluded , and women with reproductive potential were required to use contraception . other exclusion criteria were : 1 ) history of qtc prolongation , ventricular tachycardia ( vt ) , auricular fibrillation ( af ) , heart block ( hb ) , myocardial infarction ( mi ) onset within one year , congestive heart failure ( chf ) , or clinically significant coronary artery disease which needs drug treatment ; 2 ) patients who had undergone organ transplantation ; 3 ) patients with active bleeding or newly diagnosed thromboembolic diseases ; 4 ) patients with active ulcer or bleeding in gastrointestinal tract ; 5 ) patients with active infections ; 6 ) patients with symptomatic brain metastases ; or 7 ) patients with mental disorders . the study was approved by china food and drug administration ( cfda ) and the ethics committee of cancer institute & hospital , chinese academy of medical science & peking union medical college . the study was registered on world health organization international clinical trials registry platform ( registration no . chictr - onc-12002283 ) , and was conducted in accordance with the declaration of helsinki and good clinical practice guidelines . the primary purpose of this open - label study was to determine the recommended phase ii dose of chidamide combined with fixed dose of paclitaxel and carboplatin in a 3-week cycle in patients with advanced nsclc . paclitaxel and carboplatin were commercially available from bristol - myers squibb ( new york , usa ) . the starting dose of chidamide was 20 mg and escalated by 5 mg with a 3 + 3 ( 3 to 6 patients per cohort ) " up and down " design . a dose - limiting toxicity ( dlt ) was defined as the occurrence of any of the following events in treatment cycle 1 : 1 ) grade 4 neutropenia lasting > 7 d ; 2 ) grade 3 febrile neutropenia with fever > 38.5 ; 3 ) grade 4 thrombocytopenia ; 4 ) grade 3 nausea or vomiting lasting > 48 h despite maximal symptomatic treatment ; 5 ) grade 4 nausea or vomiting ; 6 ) grade 3 or higher non - hematologic toxicity excluding nausea , vomiting , or alopecia ; 7 ) cycle 2 treatment delay > 2 weeks due to intolerable events ; and 8) drug - related and clinically significant grade 3 or higher non - hematologic laboratory abnormity . the grade of toxicities and the relationship of adverse events ( ae ) with the study drug / combination treatment regimen were evaluated and determined by investigators . chidamide tablets were administered orally 30 min after breakfast twice weekly ( biw ) on d 1 and d 5 every 3 weeks of each cycle . on d 5 of each cycle , along with oral chidamide , paclitaxel ( 175 mg / m ) was infused intravenously over 3 h , immediately followed by a 30 min infusion of carboplatin [ area under the curve ( auc)=5 mg / ml / min ] . patients with stable disease ( sd ) or better after four cycles of combination treatment were administered with single chidamide with the same dose as maintenance , until disease progression or unacceptable toxicity . paclitaxel - treated patients received dexamethasone ( 20 mg oral doses 12 and 6 h before paclitaxel ) , diphenhydramine [ 50 mg intravenous injection ( i.v . ) ] , and a histamine receptor 2 antagonist ( 50 mg i.v dose reductions were required in patients who had no dlt in cycle 1 and experienced grade 3 or higher non - hematologic toxicity , or grade 4 hematologic toxicity , or grade 3 neutropenia lasting > 7 d or febrile neutropenia with fever > 38.5 in the subsequent period . the principle of dose reduction was as follows : 1 ) if toxicity was chidamide - related , the dose of chidamide was reduced to the next lower level for the subsequent treatment cycles ; 2 ) if toxicity was paclitaxel - related , the dose of paclitaxel was reduced by 25 mg / m , and if carboplatin - related , the dose of carboplatin was reduced by 1 mg / ml / min ; and 3 ) patients with 2 or more dose reductions should be withdrew from the study . blood samples ( 3 ml ) for chidamide when given alone ( d 1 , cycle 1 ) , and with paclitaxel and carboplatin ( d 5 , cycle 1 ) , were collected from all patients before and 1 , 2 , 3 , 6 , 12 , 24 , 48 and 72 h after administration of chidamide , and after the onset of paclitaxel and carboplatin infusion , respectively . plasma concentrations of chidamide and paclitaxel were determined by a validated , sensitive , and specific high - performance liquid chromatography method with tandem mass spectrometric detection ( lc - ms / ms ) ( 15 ) . the assay was linear over a range of 1 - 1 , 000 ng / ml with a lower limit of quantification ( lloq ) of 1 ng / ml for chidamide , and 20 - 10 , 000 ng / ml with a lloq of 20 ng / ml for paclitaxel , respectively . baseline evaluations included physical examination , performance status , complete blood count ( cbc ) , biochemistry , urinalysis , electrocardiograph ( ecg ) , cardiac ultrasound and computed tomography ( ct ) scan . cbc tests were performed biw in combination treatment period , and once weekly in single chidamide treatment period . responses were assessed every 6 weeks by ct scan using response evaluation criteria in solid tumors ( recist , version 1.0 ) , and toxicity was graded using national cancer institute the common toxicity criteria ( nci - ctc , version 3.0 ) for aes . median of patient age was calculated based on the baseline data from 10 patients enrolled . all descriptive statistical analyses were carried out by using the sas software ( version 9.2 , sas institute inc . , pharmacokinetic parameters of chidamide and paclitaxel were analyzed using non - compartmental methods by phoenix 6.0 winnonlin , version 5.2 ( pharsight corporation , mountain view , ca , usa ) . the maximum plasma drug concentration ( cmax ) and time to reach maximum concentration ( tmax ) were obtained from experimental observation . the paired two - tailed t test was employed using ibm spss statistics ( version 19.0 , ibm corp . , new york , usa ) to assess mean pharmacokinetic variables for chidamide ( before and after combination with paclitaxel / carboplatin ) and paclitaxel ( among each chidamide combination dose group ) in cycle 1 for all the patients , and p<0.05 ( two - tailed ) was considered statistically significant . patients ( age 18 years ) with cytologically or histologically confirmed nsclc , stage iiib or iv disease , eastern cooperative oncology group ( ecog ) performance status of 0 or 1 , treatment - naive or recurrent after surgery without systematic therapy were eligible . presence of measurable diseases , life expectancy of 3 months and body weight 47 - 87 kg for men and 35 - 75 kg for women were required . qualifying laboratory criteria were as follows : leukocytes 410/l , platelets 10010/l , hemoglobin 11 g / dl , total bilirubin 1.5 upper limits of normal ( uln ) , alanine transaminase ( alt)/aspartate aminotransferase ( ast ) 1.5uln , and creatinine 1.5uln . pregnant women and men with fertility desires were excluded , and women with reproductive potential were required to use contraception . other exclusion criteria were : 1 ) history of qtc prolongation , ventricular tachycardia ( vt ) , auricular fibrillation ( af ) , heart block ( hb ) , myocardial infarction ( mi ) onset within one year , congestive heart failure ( chf ) , or clinically significant coronary artery disease which needs drug treatment ; 2 ) patients who had undergone organ transplantation ; 3 ) patients with active bleeding or newly diagnosed thromboembolic diseases ; 4 ) patients with active ulcer or bleeding in gastrointestinal tract ; 5 ) patients with active infections ; 6 ) patients with symptomatic brain metastases ; or 7 ) patients with mental disorders . the study was approved by china food and drug administration ( cfda ) and the ethics committee of cancer institute & hospital , chinese academy of medical science & peking union medical college . the study was registered on world health organization international clinical trials registry platform ( registration no . chictr - onc-12002283 ) , and was conducted in accordance with the declaration of helsinki and good clinical practice guidelines . the primary purpose of this open - label study was to determine the recommended phase ii dose of chidamide combined with fixed dose of paclitaxel and carboplatin in a 3-week cycle in patients with advanced nsclc . paclitaxel and carboplatin were commercially available from bristol - myers squibb ( new york , usa ) . the starting dose of chidamide was 20 mg and escalated by 5 mg with a 3 + 3 ( 3 to 6 patients per cohort ) " up and down " design . a dose - limiting toxicity ( dlt ) was defined as the occurrence of any of the following events in treatment cycle 1 : 1 ) grade 4 neutropenia lasting > 7 d ; 2 ) grade 3 febrile neutropenia with fever > 38.5 ; 3 ) grade 4 thrombocytopenia ; 4 ) grade 3 nausea or vomiting lasting > 48 h despite maximal symptomatic treatment ; 5 ) grade 4 nausea or vomiting ; 6 ) grade 3 or higher non - hematologic toxicity excluding nausea , vomiting , or alopecia ; 7 ) cycle 2 treatment delay > 2 weeks due to intolerable events ; and 8) drug - related and clinically significant grade 3 or higher non - hematologic laboratory abnormity . the grade of toxicities and the relationship of adverse events ( ae ) with the study drug / combination treatment regimen were evaluated and determined by investigators . chidamide tablets were administered orally 30 min after breakfast twice weekly ( biw ) on d 1 and d 5 every 3 weeks of each cycle . on d 5 of each cycle , along with oral chidamide , paclitaxel ( 175 mg / m ) was infused intravenously over 3 h , immediately followed by a 30 min infusion of carboplatin [ area under the curve ( auc)=5 mg / ml / min ] . patients with stable disease ( sd ) or better after four cycles of combination treatment were administered with single chidamide with the same dose as maintenance , until disease progression or unacceptable toxicity . paclitaxel - treated patients received dexamethasone ( 20 mg oral doses 12 and 6 h before paclitaxel ) , diphenhydramine [ 50 mg intravenous injection ( i.v . ) ] , and a histamine receptor 2 antagonist ( 50 mg i.v . dose reductions were required in patients who had no dlt in cycle 1 and experienced grade 3 or higher non - hematologic toxicity , or grade 4 hematologic toxicity , or grade 3 neutropenia lasting > 7 d or febrile neutropenia with fever > 38.5 in the subsequent period . the principle of dose reduction was as follows : 1 ) if toxicity was chidamide - related , the dose of chidamide was reduced to the next lower level for the subsequent treatment cycles ; 2 ) if toxicity was paclitaxel - related , the dose of paclitaxel was reduced by 25 mg / m , and if carboplatin - related , the dose of carboplatin was reduced by 1 mg / ml / min ; and 3 ) patients with 2 or more dose reductions should be withdrew from the study . blood samples ( 3 ml ) for chidamide when given alone ( d 1 , cycle 1 ) , and with paclitaxel and carboplatin ( d 5 , cycle 1 ) , were collected from all patients before and 1 , 2 , 3 , 6 , 12 , 24 , 48 and 72 h after administration of chidamide , and after the onset of paclitaxel and carboplatin infusion , respectively . plasma concentrations of chidamide and paclitaxel were determined by a validated , sensitive , and specific high - performance liquid chromatography method with tandem mass spectrometric detection ( lc - ms / ms ) ( 15 ) . the assay was linear over a range of 1 - 1 , 000 ng / ml with a lower limit of quantification ( lloq ) of 1 ng / ml for chidamide , and 20 - 10 , 000 ng / ml with a lloq of 20 ng / ml for paclitaxel , respectively . baseline evaluations included physical examination , performance status , complete blood count ( cbc ) , biochemistry , urinalysis , electrocardiograph ( ecg ) , cardiac ultrasound and computed tomography ( ct ) scan . cbc tests were performed biw in combination treatment period , and once weekly in single chidamide treatment period . responses were assessed every 6 weeks by ct scan using response evaluation criteria in solid tumors ( recist , version 1.0 ) , and toxicity was graded using national cancer institute the common toxicity criteria ( nci - ctc , version 3.0 ) for aes . median of patient age was calculated based on the baseline data from 10 patients enrolled . all descriptive statistical analyses were carried out by using the sas software ( version 9.2 , sas institute inc . , pharmacokinetic parameters of chidamide and paclitaxel were analyzed using non - compartmental methods by phoenix 6.0 winnonlin , version 5.2 ( pharsight corporation , mountain view , ca , usa ) . the maximum plasma drug concentration ( cmax ) and time to reach maximum concentration ( tmax ) were obtained from experimental observation . the paired two - tailed t test was employed using ibm spss statistics ( version 19.0 , ibm corp . , new york , usa ) to assess mean pharmacokinetic variables for chidamide ( before and after combination with paclitaxel / carboplatin ) and paclitaxel ( among each chidamide combination dose group ) in cycle 1 for all the patients , and p<0.05 ( two - tailed ) was considered statistically significant . this phase i study was conducted at cancer institute & hospital , chinese academy of medical sciences . a total of 10 patients were enrolled from march 2011 to april 2012 to receive protocol treatment . as listed in table 1 , the median age was 55.6 ( range 35.6 - 63.1 ) years old , with 7 patients having an ecog performance status equal to 1 , 3 patients with ecog performance status equal to 0 . nine patients ( 90% ) were treatment - naive , and 1 patient received radiotherapy for brain metastasis . baseline patient characteristics ( n=10 ) all 10 patients received at least one cycle of combination therapy and were evaluable for dlt and safety . as shown in table 2 , 4 patients completed the 4 scheduled cycles of combination therapy , and 2 patients went on to receive chidamide maintenance monotherapy . no patients in this cohort experienced dlt and all completed the planned 4-cycle combination therapy , in which 2 patients continued to receive the maintenance chidamide . in the 25 mg cohort , two patients discontinued treatment due to disease progression , and one due to lack of treatment - benefit based on investigator 's discretion . one patient completed the 4-cycle combination therapy , 2 patients discontinued treatment in cycle 1 due to dlts , and 1 patient experienced serious adverse event ( sae ) and withdrew from the study . two out of 4 patients in the 30 mg cohort experienced dlts defined by the protocol : 1 patient had grade 4 thrombocytopenia , and the other had treatment delay over 14 d for the subsequent treatment because of grade 3 neutropenia and grade 2 thrombocytopenia . myelo - suppression , particularly neutropenia , was the principal hematologic toxicity of chidamide / pactlitaxel / carboplatin regimen ( table 3 ) . grade 3/4 neutropenia was observed in all patients , but not associated with significant complications . nausea , alopecia and fatigue were relatively common ( > 50% ) non - hematologic toxicities , but all were grade 1 . other non - hematologic toxicities were most grade 1 or grade 2 , and only 1 case of grade 3 hypocalcemia was documented in the 20 mg cohort . one patient at the 20 mg dose level and 2 patients at the 30 mg dose level had dose reduction of paclitaxel or carboplatin per the study protocol . one patient at the 30 mg dose level experienced sae . on d 15 of the third cycle , the patient had numbness , weakness and limitation of motion on the left limbs . acute cerebral infarction was diagnosed , and the patient was hospitalized for treatment and withdrawn from the study . plasma samples for pharmacokinetic studies were obtained from all the patients to detect potential relevant drug - drug interactions . figure 1 shows the mean plasma concentrations of chidamide at different doses when administered alone and with paclitaxel and carboplatin . in general , no significant difference in chidamide concentration - time curve furthermore , similar chidamide clearance was observed between single dose vs. combination with paclitaxel / carboplatin among all three doses in average ( 55.327.9 vs. 51.626.1 and there is no significant difference of chidamide values in cmax ( p=0.08 ) and auc0 - 72 ( p=0.97 ) by a paired t test between single and combination dosage . mean plasma concentrations of chidamide before and after co - administration of paclitaxel and carboplatin ( pc ) . ( a ) 20 mg ; ( b ) 25 mg ; ( c ) 30 mg . major pharmacokinetic parameters of paclitaxel when combined with chidamide at the dose of 20 , 25 and 30 mg are presented in table 4 . similar paclitaxel pharmacokinetic estimates were seen when combined with chidamide at different doses ( p>0.05 ) , and these values are in agreement with reported values for comparable dose of single - agent paclitaxel administered as a 3-h i.v . main pharmacokinetic parameters of paclitaxel ( 175 mg / m ) combined with oral chidamide 20 mg , 25 mg or 30 mg there are 8 patients were evaluable for tumor response since 2 patients in the 30 mg cohort withdrew from the study due to dlts before response assessment . confirmed partial response ( pr ) was observed in 1 patient in the 20 mg cohort , with a duration of 166 d. sd was seen in 4 patients : 1 in 20 mg cohort , 1 in 25 mg cohort , 2 in 30 mg cohort , and the duration of sd was 1 + , 28 + , 47 and 105 d , respectively ( " + " denotes a censored value ) . progressive disease ( pd ) five patients ( 3 in 20 mg cohort , 1 in 25 mg cohort and 1 in 30 mg cohort ) had brain metastases prior to study entry . two patients in the 20 mg cohort with brain lesions of 6 - 9 mm achieved intracranial complete response ( cr ) after 6-week treatment , which lasted for 12 weeks ( data not shown ) . this phase i study was conducted at cancer institute & hospital , chinese academy of medical sciences . a total of 10 patients were enrolled from march 2011 to april 2012 to receive protocol treatment . as listed in table 1 , the median age was 55.6 ( range 35.6 - 63.1 ) years old , with 7 patients having an ecog performance status equal to 1 , 3 patients with ecog performance status equal to 0 . nine patients ( 90% ) were treatment - naive , and 1 patient received radiotherapy for brain metastasis . all 10 patients received at least one cycle of combination therapy and were evaluable for dlt and safety . as shown in table 2 , 4 patients completed the 4 scheduled cycles of combination therapy , and 2 patients went on to receive chidamide maintenance monotherapy . no patients in this cohort experienced dlt and all completed the planned 4-cycle combination therapy , in which 2 patients continued to receive the maintenance chidamide . in the 25 mg cohort , 3 patients were enrolled and all treated for 2 cycles without dose reduction . two patients discontinued treatment due to disease progression , and one due to lack of treatment - benefit based on investigator 's discretion . one patient completed the 4-cycle combination therapy , 2 patients discontinued treatment in cycle 1 due to dlts , and 1 patient experienced serious adverse event ( sae ) and withdrew from the study . two out of 4 patients in the 30 mg cohort experienced dlts defined by the protocol : 1 patient had grade 4 thrombocytopenia , and the other had treatment delay over 14 d for the subsequent treatment because of grade 3 neutropenia and grade 2 thrombocytopenia . myelo - suppression , particularly neutropenia , was the principal hematologic toxicity of chidamide / pactlitaxel / carboplatin regimen ( table 3 ) . grade 3/4 neutropenia was observed in all patients , but not associated with significant complications . nausea , alopecia and fatigue were relatively common ( > 50% ) non - hematologic toxicities , but all were grade 1 . other non - hematologic toxicities were most grade 1 or grade 2 , and only 1 case of grade 3 hypocalcemia was documented in the 20 mg cohort one patient at the 20 mg dose level and 2 patients at the 30 mg dose level had dose reduction of paclitaxel or carboplatin per the study protocol . one patient at the 30 mg dose level experienced sae . on d 15 of the third cycle , the patient had numbness , weakness and limitation of motion on the left limbs . acute cerebral infarction was diagnosed , and the patient was hospitalized for treatment and withdrawn from the study . plasma samples for pharmacokinetic studies were obtained from all the patients to detect potential relevant drug - drug interactions . figure 1 shows the mean plasma concentrations of chidamide at different doses when administered alone and with paclitaxel and carboplatin . in general , no significant difference in chidamide concentration - time curve furthermore , similar chidamide clearance was observed between single dose vs. combination with paclitaxel / carboplatin among all three doses in average ( 55.327.9 vs. 51.626.1 and there is no significant difference of chidamide values in cmax ( p=0.08 ) and auc0 - 72 ( p=0.97 ) by a paired t test between single and combination dosage . mean plasma concentrations of chidamide before and after co - administration of paclitaxel and carboplatin ( pc ) . ( a ) 20 mg ; ( b ) 25 mg ; ( c ) 30 mg . major pharmacokinetic parameters of paclitaxel when combined with chidamide at the dose of 20 , 25 and 30 mg are presented in table 4 . similar paclitaxel pharmacokinetic estimates were seen when combined with chidamide at different doses ( p>0.05 ) , and these values are in agreement with reported values for comparable dose of single - agent paclitaxel administered as a 3-h i.v . main pharmacokinetic parameters of paclitaxel ( 175 mg / m ) combined with oral chidamide 20 mg , 25 mg or 30 mg there are 8 patients were evaluable for tumor response since 2 patients in the 30 mg cohort withdrew from the study due to dlts before response assessment . confirmed partial response ( pr ) was observed in 1 patient in the 20 mg cohort , with a duration of 166 d. sd was seen in 4 patients : 1 in 20 mg cohort , 1 in 25 mg cohort , 2 in 30 mg cohort , and the duration of sd was 1 + , 28 + , 47 and 105 d , respectively ( " + " denotes a censored value ) . progressive disease ( pd ) was seen in 3 patients . five patients ( 3 in 20 mg cohort , 1 in 25 mg cohort and 1 in 30 mg cohort ) had brain metastases prior to study entry . two patients in the 20 mg cohort with brain lesions of 6 - 9 mm achieved intracranial complete response ( cr ) after 6-week treatment , which lasted for 12 weeks ( data not shown ) . chidamide represents a novel benzamide class hdac inhibitor with unique mechanism of actions and significant orally single - agent activity against relapsed or refractory ptcl as single agent ( 14 ) . this dose - escalation phase i study was designed and conducted to determine the safety and maximum tolerated dose ( mtd ) of chidamide in combination with paclitaxel and carboplatin to identify a regimen suitable for further phase ii / iii evaluation . in this study , the most frequently reported aes were myelosuppression , which were principally neutropenia , thrombocytopenia , leucopenia and anemia . all patients ( 100% ) and 3 patients ( 30% ) respectively experienced grade 3/4 neutropenia and thromcytopenia , which were more frequent than those in previous studies evaluating paclitaxel / carboplatin alone ( 17 - 19 ) . it was probably due to the overlapping hematological toxicity of chidamide and paclitaxel / carboplatin . consistent with the side effect profiles for paclitaxel - carboplatin or single chidamide , the combination treatment showed mild to moderate non - hematological toxicities without grade 3/4 aes , which suggested little additive non - hematological toxicities in those patients to the regimen . dlts were documented in 2 patients from the 30 mg chidamide cohort with a total 4 patients enrolled , including 1 grade 4 thrombocytopenia , and 1 treatment delay due to grade 3 neutropenia and grade 2 thrombocytopenia . therefore , dlt for chidamide combined with paclitaxel / carboplatin was identified as 30 mg , and no further patients were enrolled in this cohort . although similar frequency and severity of aes were represented between the 25 mg and 20 mg cohorts , all 3 patients in the 25 mg cohort had treatment discontinuation after cycle 2 due to the disease progression and investigator 's discretion , which prevented this cohort from adequate evaluation for repeated administration . based on these considerations and potential efficacy observed in the 20 mg cohort , 20 mg chidamide in combination with fixed dose of paclitaxel ( 175 mg / m ) and carboplatin ( auc=5 mg / ml / min ) was recommended as phase ii regimen for further evaluation . chidamide in combination with paclitaxel / carboplatin displayed similar pharmacokinetic properties to those from phase i study of single chidamide ( 8) . regardless of dose levels , no significant difference was seen in the plasma clearance ( cl ) of chidamide before and after combination with paclitaxel and carboplatin . we also investigated the potential effects of chidamide on the pharmacokinetics of paclitaxel : similar paclitaxel pharmacokinetic estimates were seen in co - administration with chidamide at different doses . paclitaxel had a half life ( t1/2 ) of 11.23.2 h and a cl of 8.31.9 l / h / m when administered at 175 mg / m in our study . it was favorably comparable with the pharmacokinetic parameters of paclitaxel ( bristol - myers squibb , usa ) in the fda database with a t1/2 of 9.04.9 h , and a cl of 10.56.4 l / h / m at the dose of 170 - 175 mg / m , as well as with reported values for comparable dose of single - agent paclitaxel administered ( 16 ) . these results suggested lack of significant drug - drug interaction between chidamide and paclitaxel during co - administration . of 5 patients with brain metastases , 2 from the 20 mg cohort had complete remission of brain tumors after 4-cycle treatment . poor permeability across the blood brain barrier was reported for paclitaxel and carboplatin ( 20 , 21 ) , and intracranial control was rarely seen in patients treated with either of the agents ( 22 ) . however , whether the observed antitumor activity for brain metastasis in this trial could be contributed to the addition of chidamide to the chemotherapy regimen needs further study to elucidate . the results of this study on 10 patients with advanced nsclc showed that the combination regimen of chidamide plus paclitaxel / carboplatin is generally tolerable , with the preliminary evidence of tumor control and no apparent chidamide - paclitaxel pharmacokinetic interactions . a phase ii study has been performed to further evaluate the efficacy and safety of chidamide 20 mg biw plus paclitaxel / carboplatin for the treatment of advanced nsclc patients with wild - type egfr genotype .

ventricular septal defect ( vsd ) complicating acute myocardial infarction ( ami ) is reported in 0.2 % the patients in this thrombolytic era . in the first week emergent surgical closure of post infarction vsd with or without appropriate revascularization irrespective of the clinical status had been the standard treatment . many surgeons prefer surgical closure after a 3 4 week delay to allow scarring of the surrounding tissue , which allows better anchoring of sutures . an interventional approach using a device is a less invasive option , and allows immediate complete closure after initial hemodynamic stabilization . immediate reduction of the left - to - right shunt , even if the vsd is not completely closed , may stabilize the patient enough to function as a bridge to surgery . current interventional reports are mainly restricted to vsd closure in the sub acute or chronic setting , or for residual shunts after initial surgical closure.[79 ] we report a case of anterior myocardial infarction ( mi ) which was subjected to per - cutaneous transluminal coronary angioplasty ( ptca ) of the culprit lesion onfourth day of myocardial infarction , and closure of vsd with a device 3 weeks later , to allow reasonable stabilization of patient and the rims of the defect . a 60 year old gentleman without previous history of hypertension , diabetes mellitus and smoking , sustained an acute anterior wall myocardial infarction , and was thrombolysed with streptokinase within a window period of six hours at a local hospital . he was shifted to our hospital on detection of a ventricular septal defect , ongoing angina and breathlessness at rest next day . on admission , he was in congestive heart failure of killip ii with a pulse rate of 109/minute , blood pressure of 100/70 mm hg and a grade iv pan systolic murmur at left lower parasternal area . his 12 lead surface electrocardiogram ( ecg ) was consistent with evolved acute anterior wall myocardial infarction [ figure 1 ] . two - dimensional and 3-dimensional echocardiogram showed akinetic mid and distal interventricular septum and apex , moderate left ventricular dysfunction and a muscular ventricular septal defect of 10 mm size in the anterior - middle part of the septum [ figure 2 ] . cardiac catheterization done on the same day revealed a single muscular ventricular septal defect of size 10 mm [ figure 3 ] , with a significant step up of oxygen saturation at right ventricular level by 23% andthe ratio of pulmonary blood flow ( qp ) to the systemic blood flow ( qs ) depicted as qp / qs equal to 1.8:1 . pulmonary artery pressure ( 40/12/15 mm hg ) and pulmonary wedge pressure ( mean-17 mmhg ) was mildly elevated . left anterior descending artery was the only affected vessel with proximal 90% stenosis ( type b ; society of coronary angiography and interventions ( scai ) [ figures 4 and 5 ] . coronary angioplasty using a drug eluting ( endeavour resolute 3 mm 15 mm ) stent was done with good angiographic result [ figure 6 ] . the device closure was planned after 4 weeks ; however had to be done earlier as patient was unwilling to stay longer than 3 weeks in hospital due to some domestic problem . 12 lead electrocardiography shows persistent st segment elevation in v2-v4 even after thrombolysis as an evidence of septal aneurysm echocardiography in apical four chamber view single and discrete muscular septal defect left ventricular angiogram in left anterior oblique view shows single discrete muscular vsd of size 10 mm selective coronary hooking of right coronary artery reveals normal right coronary artery selective left coronary injection shows type b proximal significant stenosis in left anterior descending artery successful angioplasty and stenting of left anterior descending artery with very good angiographic result on day-23 , the right femoral artery and right internal jugular vein were cannulated under local anesthesia . after a left ventricular angiogram ventricular septal defect was crossed using 5 french right judkin 's ( jr ) catheter and a 0.035 260 cm angled tip terumo wire ( terumo , tokyo , japan ) using a retrograde arterial approach . it was passed into right ventricle , to main pulmonary artery and then into the left pulmonary artery . the right judkin 's catheter was advanced a little more into right ventricular outflow tract ( rvot ) and the terumo wire was exchanged with 0.035 260 cm teflon exchange length wire ( medtronic ) . the distal end of that wire was then caught with a 10 mm amplatzer goose - neck snare ( microvena , mn ) in the left pulmonary artery [ figure 7 ] , and extracted outside via the right internal jugular sheath . a 12 fr 180 degree curve device delivery sheath with its dilator ( star way medical technology , inc ) was advanced from the jugular vein into the left ventricle ( lv ) across the vsd . then dilator and the guide wire a 14 mm cardio - o - fix septal occluder was screwed onto the delivery cable , and introduced into the sheath . the distal disc ( left ventricle ) was initially opened and pulled back onto the lv side of the septum under trans - thoracic echocardiography ( tte ) and fluoroscopic guidance . after confirming the septal alignment , the proximal disc ( right ventricle ) was also opened . once adequate placement was ascertained by tte and left ventricular angiogram , the device was released [ figure 8 ] . tte and left ventricular angiography showed proper positioning of device with only minimal shunting through the device . tte on the next day showed only a trivial residual shunt [ figure 9 ] . the patient was discharged on day 4 in a stable condition . at 4 month follow - up successful snaring of regular guide wire from left pulmonary artery from right internal jugular venous approach appropriate placement of cardio - fix septal occluder across ventricular septal defect with tiny residual shunt is evident from left ventricular angiogram in left anterior oblique view two dimensional color doppler showing septal occluder exactly across the ventricular septal defect after four days of procedure ventricular septal defect ( vsd ) complicates acute myocardial infarction in about 1 - 2% cases in the first week . surgical closure had been the gold standard of this fatal complication . as the mortality rates of surgical closure remains high at 20 87% in acute stage , surgeons recommend a 3 4 week delay to allow scarring of the surrounding tissue to occur , which allows for better anchoring of sutures . device closure of post infarction vsd is more studied in sub - acute and chronic stages and for residual shunts after surgical closure . till today there are no guidelines available for device closure of ventricular septal defect after myocardial infarction . the small series and anecdotal reports highlighted that device closure in acute settings has a high mortality and more complications like device embolization , major shunting , left ventricular rupture and malignant arrhythmias.[710 ] the reported number of interventional post infarction vsd procedures till nov 2009 were less than 200 in literature . in a recent study , demkow and colleagues described their 5 year experience with amplatzer device with satisfactory results . for very severely ill patients , surgery may be a better option.[1214 ] the possible difficulties faced by intervention cardiologist are i ) lack of expertise due to the rarity of the procedure , ii ) the rigid delivery sheath that has to cross the ventricular septal defect ( vsd ) and needs to be advanced into the direction of the left ventricular outflow tract , might tear the borders of the vsd , resulting in an increased vsd size or , in the worst case , left ventricular rupture iii ) the requirement of guide wire removal after insertion of the delivery sheath may result in kinking of sheath or jumping of device into right ventricle and iv ) the currently available device sizes of the muscular vsd occluder not sufficient to close the large and complex vsd . furthermore , healing of the infarcted myocardium over time may increase the size of the vsd leading to device malposition and embolization . this requires the use of devices that are larger than the measured vsd size at the time of implantation . the higher transventricular pressure for a vsd leads to persistent shunting until thrombus formation and endothelialization of the device has occurred . in contrast to surgical vsd closure , inferior and basal vsds are more easily closed with an interventional technique due to the more favorable access from the upper jugular vein . we critically analyzed all four variables : the hemodynamic status , coronary artery disease burden , vsd profile and financial aspects before adopting this strategy for ventricular defect closure after myocardial infarction . the timing of the procedure was critical . as the patient had angina and we found a significant critical lesion , we did angioplasty of the coronary artery lesion on fourth day . as the acute setting has potential for more complications , we staged the vsd closure to a later date . we had to take up the device closure a few days earlier than a desirable gap of 4 four weeks as the patient gave only conditional consent . literature review showed only few anecdotal reports for device closure with cardio - o - fix vsd occluder which is used by us . the use of this device in a case of post - operative residual vsd was recently presented in . the starwaycardi - o - fix vsd occluder is a self - expandable , double disc implant device made from a nitinol wire mesh . the two discs are linked together by a short connecting waist corresponding to the size of the vsd . in order to increase its closing ability , the discs and the waist the polyester fabric is securely sewn to each disc by surgical sutures . in our experience in reasonably hemodynamically stable patient , with a favorable coronary anatomy and ventricular septal defect anatomy , an initial percutaneous revascularization of culprit lesion and elective device closure of vsd at a later date may make a safer strategy as alternative to surgery.this strategy also helps using local anesthesia instead of general anesthesia , tte in place of trans - esophageal echocardiogram and cardio - o - fix device as an used alternative to amplatzer vsd occluder , hence adding to the safety and reduced cost of the whole procedure .

there are two conventional wisdoms about weather and electoral participation ( see knack 1994 ; gomez et al . 2007 ; and references therein ) . one is that inclement weather on election day reduces voter turnout , whereas pleasant weather encourages people to vote in greater numbers . the common belief is that poor weather drives down turnout as voters would be less willing to venture out of doors to the polling places if they have to deal with rain , low temperatures and other inclement weather . hence weather makes going to the polls more or less costly depending on the outside conditions . some u.s . studies ( merrifield 1993 ; shachar and nalebuff 1999 ; gatrell and bierly 2002 ; gomez et al . 2007 ) as well as our recent dutch research ( eisinga et al . 2011 ) support the notion that rainfall diminishes turnout . the weather effects we found are modest in size , however , with a maximum downturn of approximately 1.5% in dutch parliamentary turnout . the other long - held belief is that depressed voter turnout attributable to poor weather benefits some political parties at the expense of others . in the two - party u.s . system , where republicans and democrats dominate presidential voting , rainy weather is taken to lower the democratic party s relative vote share rather than republican voting ( knack 1994 ; gomez et al . the argument goes that more democrats live in urban areas and therefore rely more strongly on transportation that is susceptible to weather , such as walking and public transit , to get to the polls . a rainy election day may discourage many democrat voters from waiting for busses and from standing in long lines at busy urban polling places . also , the popular wisdom is that republicans are favored by lower turnout due to precipitation since supposedly some key democratic voting blocks , such as the elderly , are more weather sensitive than the rest of the voting population . in the same vein , one of the enduring myths of multi - party dutch politics is that inclement weather on election day hurts the left - wing social democratic ( labor ) and socialist parties more than other political parties , such as the christian democratic party . unpleasant weather , so the logic goes , prevents traditional labor and socialist supporters such as the poor , unemployed and elderly citizens from casting their ballot . fine weather , on the other hand , sees labor and socialist supporters turning out to vote in greater numbers , stopping off at the polling station on their way to or from work . little empirical work has been done , however , either to justify or to refute statements about weather s contribution to voting with comprehensive and reliable data . gomez et al . ( 2007 ) examined the effect of weather conditions in 14 u.s . presidential elections and in their study rainfall is shown to benefit the republican party s vote share . but apart from this exhaustive work the academic literature provides little evidence on the role weather plays in electoral politics . this study examines the effects of rainfall and temperature on share of the votes of eight political parties and party groupings that participated in 13 national parliament elections , held in the netherlands from 1971 to 2010 . it matches for each election the voting results of over 400 dutch municipalities with election - day weather observations drawn from the weather station closest to the polls . the local election - day rainfall amount in millimeters and daily mean temperature in degrees celsius were obtained from all available weather stations of the royal netherlands meteorological institute . during the time period in question the netherlands had 17 to 35 meteorological stations and 222 to 235 precipitation stations , with the number of stations increasing over time . the weather station closest to the local municipality was selected using the haversine equation , giving the shortest distance between station and municipality from their longitudes and latitudes . the mean distance between municipality and meteorological station was 17.7 km ( sd 9.8 ) . the daily mean outside temperature ranged from 1.7 to 19.9c , with a mean of 12c ( sd 4.4 ) . the amount of rainfall was obtained from the closest precipitation station , the mean distance between municipality and precipitation station being 4.4 km ( sd 2.3 ) . local rainfall on election day ranged from 0 to 43.9 mm ( mean 2.6 , sd 4.0 ) . the municipality voting figures were obtained from the dutch electoral council . they include , for each municipality , the number of voters eligible to vote , the number of voters who casted ( valid , invalid and blank ) votes and the number of valid votes for the participating political parties . the municipalities growing in number over time from 412 to 425 by creating new land from sea were classified according to the 2010 codes issued by statistics netherlands . municipality turnout is the percentage of eligible voters in a municipality who voted in the election , whereas party vote share is the percentage of the total valid votes obtained by a party in a municipality . turnout varied from 56.5 to 100% , with a mean of 83.3% ( sd 5.8 ) . the party vote shares were obtained for the parties that participated in ( almost ) all of the elections surveyed . votes for parties that contested in one or a few elections were collapsed into the category called other parties. table 1 offers a description of the dutch political parties in terms of vote share and the left - right ideological position and church membership of their voters . the parties are sorted from top to bottom according to the voters left - right political orientation ( eisinga and franses 1996).table 1dutch political partiesabbreviationdutch nameenglish equivalentvote shareleft - rightchurchglgroenlinksgreenleft5.82.50.39spsocialistische partijsocialist party4.32.80.42pvdapartij van de arbeidsocial democratic ( labor ) party26.43.00.49d66democraten 66democrats 666.73.60.49cdachristen democratisch appelchristian democratic appeal27.94.80.93vvdpartij voor vrijheid en democratiepeople s party for freedom and democracy17.74.90.56cu / sgpchristen unie / staatskundig gereformeerde partijchristian union / reformed political party4.35.70.98other parties7.24.40.69vote share is the party s observed percentage of the votes aggregated over the 13 national parliament elections , 19712010 . left - right indicates the mean ideological position of the party supporters on a 7-point scale running from left ( 1 ) to right ( 7 ) . the column labeled church offers the proportion of the party supporters who consider themselves ( catholic or protestant ) church members . the figures in the two right - most columns were taken from sample survey data ( n = 871,844 ) collected in the 19762000 period ( source : eisinga 2005 ) dutch political parties vote share is the party s observed percentage of the votes aggregated over the 13 national parliament elections , 19712010 . left - right indicates the mean ideological position of the party supporters on a 7-point scale running from left ( 1 ) to right ( 7 ) . the column labeled church offers the proportion of the party supporters who consider themselves ( catholic or protestant ) church members . the figures in the two right - most columns were taken from sample survey data ( n = 871,844 ) collected in the 19762000 period ( source : eisinga 2005 ) the political parties include three major and four minor dutch parties in terms of vote shares . the major parties are the cda : a right - of - the - center christian democratic party with a mean share of the vote aggregated over elections of 27.9% ( sd 4.5 ) ; the pvda : a left - wing social democratic ( labor ) party ( mean 26.4% , sd 4.4 ) ; and the vvd : a right - wing liberal party ( mean 17.7% , sd 3.8 ) . the minor parties include gl : a left - wing ( green ) environmentalist party ( mean 5.8% , sd 2.3 ) ; the sp : a left - wing socialist party ( mean 4.3% , sd 2.0 ) ; d66 : a center - liberal party ( mean 6.7% , sd 2.5 ) ; and cu and sgp : two right - wing orthodox religious parties collapsed into a single category ( mean 4.3% , sd 2.0 ) . the mean share of the vote for other parties is 7.2% ( sd 2.6 ) . the 5,507 observation counts ( i.e. , number of municipalities times number of elections ) for each party s vote share represent 113 million valid votes and 28 million abstentions ( including half million invalid and blank votes ) . the model we used examines the effect of rainfall and temperature on share of the votes for each contesting party separately . in addition to their main effects we also investigated the potential joint effect of the two weather variables , as the interplay of a cold and rainy day may be the major weather issue that keeps voters from going to the polls . in order to mitigate confounding influences , the weather effects were controlled for the weather - related variable electoral turnout and other variables . party vote shares at the municipality level are strongly related across elections as a result of party loyalty . to account for this temporal continuity the model incorporates the share of the vote for the party in question in the two preceding elections by including their moving average . the vote shares for the elections prior to 1971 were additionally coded to obtain complete observations for the first two elections . because contemporary issues and changing domestic and international affairs all contribute to the uniqueness of a particular national election , election dummy variables were entered in the model that allow the election intercepts to vary . some party strongholds are to a lesser or greater extent geographically concentrated in particular dutch regions . the southern part of the netherlands is a traditional christian democratic stronghold , for example , and there is a strip of land called the dutch bible belt which is inhabited mainly by conservative protestants overwhelmingly voting for cu or sgp . to account for this regional concentration , the municipality 's longitude and latitude ( in degrees , decimal degrees ) were included in the model . the variable included in the analysis is the natural logarithm of the number of eligible voters per municipality square kilometer . the data were analyzed party - by - party using two different three - level hierarchical models linear and logistic with voters for a particular party at level one , nested within municipality - by - election at level two and municipality at level three . as the parameter estimates of the linear and logistic models obtained identical effect signs and near equivalent p - values , we opted for the presentation of the results of the linear models as they are easier to communicate . the effects of the weather parameters on each of the party s share of the vote are presented in table 2 . we restrict our discussion to the most important results.table 2maximum - likelihood hierarchical linear models of municipality - level political party s share of the votes in dutch national parliament elections , 19712010independent variables ( iv)glsppvdad66cdavvdcu / sgpother partiesfixed effectsintercept ( iv mean centered)5.36 ( 0.14)2.83 ( 0.10)22.87 ( 0.23)7.78 ( 0.13)29.83 ( 0.27)14.97 ( 0.19)6.45 ( 0.10)9.37 ( 0.17)rainfall ( mm)0.02 ( 0.01)0.07 ( 0.01)0.07 ( 0.01)0.02 ( 0.01)0.13 ( 0.02)0.01 ( 0.01)0.02 ( 0.01)0.05 ( 0.01)temperature ( c)0.02 ( 0.02)0.04 ( 0.01)0.00 ( 0.03)0.04 ( 0.01)0.04 ( 0.03)0.00 ( 0.02)0.05 ( 0.01)0.06 ( 0.02)rainfall temperature0.00 ( 0.00)0.02 ( 0.00)0.02 ( 0.00)0.00 ( 0.00)0.01 ( 0.00)0.01 ( 0.00)0.00 ( 0.00)0.02 ( 0.00)moving average vote share previous two elections0.29 ( 0.01)1.11 ( 0.01)0.82 ( 0.01)0.40 ( 0.02)0.81 ( 0.00)0.75 ( 0.01)1.01 ( 0.00)0.48 ( 0.01)electoral turnout0.02 ( 0.01)0.06 ( 0.00)0.03 ( 0.01)0.01 ( 0.01)0.09 ( 0.01)0.11 ( 0.01)0.01 ( 0.00)0.08 ( 0.01)municipality latitude0.61 ( 0.11)0.06 ( 0.03)0.11 ( 0.10)0.27 ( 0.08)0.35 ( 0.11)0.66 ( 0.13)0.11 ( 0.03)0.48 ( 0.06)municipality longitude0.23 ( 0.09)0.14 ( 0.03)0.72 ( 0.07)0.34 ( 0.06)0.39 ( 0.08)1.19 ( 0.11)0.11 ( 0.02)0.37 ( 0.04)log voting - age population density0.68 ( 0.06)0.06 ( 0.02)0.25 ( 0.05)0.41 ( 0.04)0.56 ( 0.06)0.12 ( 0.07)0.01 ( 0.02)0.11 ( 0.03)random effectsfitted modelmunicipality election1.65 ( 0.03)1.37 ( 0.03)5.94 ( 0.16)1.34 ( 0.03)7.82 ( 0.17)2.72 ( 0.06)1.20 ( 0.02)3.56 ( 0.07)municipality1.43 ( 0.12)0.00 ( 0.00)0.53 ( 0.21)0.61 ( 0.07)0.51 ( 0.12)2.00 ( 0.32)0.00 ( 0.00)0.07 ( 0.03)null modelmunicipality election4.18 ( 0.08)26.70 ( 0.55)35.81 ( 0.71)12.52 ( 0.25)76.34 ( 1.52)20.19 ( 0.40)1.55 ( 0.03)38.93 ( 0.74)municipality3.41 ( 0.26)0.00 ( 0.00)55.16 ( 3.98)1.98 ( 0.20)100.87 ( 7.33)31.71 ( 2.28)58.68 ( 4.03)0.00 ( 0.00)proportional reduction in error0.590.950.930.870.950.910.980.91standard error in parenthesis . the parameter estimates are significant at the 0.05 level , except those reported in italic . the parameters of the sp vote share were estimated for the 11 elections held in the 19772010 period . the proportional reduction in prediction error is obtained as the ratio of explained variation to total variation and may be interpreted as indicating the proportion of variation explained . descriptions of the abbreviations can be found in table 1 maximum - likelihood hierarchical linear models of municipality - level political party s share of the votes in dutch national parliament elections , 19712010 standard error in parenthesis . the parameter estimates are significant at the 0.05 level , except those reported in italic . the parameters of the sp vote share were estimated for the 11 elections held in the 19772010 period . the proportional reduction in prediction error is obtained as the ratio of explained variation to total variation and may be interpreted as indicating the proportion of variation explained . descriptions of the abbreviations can be found in table 1 rainfall was seen to have a significant negative effect on the vote shares of the socialist party sp , the social democratic party pvda , and the other parties , and a positive effect on the vote shares of the christian democratic party cda , the center - liberal party d66 , the left - wing environmentalist party gl , and the orthodox religious parties cu / sgp . the outside temperature was found to have a significant positive effect on the shares of the vote for d66 and cu / sgp , and a negative effect on the sp s vote share and those of the other parties . equally or even more important , temperature is observed to modify the effect of rainfall , most notably in the case of the social democratic party pvda and the socialist party sp . basically , the detrimental effect of rain on the social democratic and the socialist party vote shares diminishes as the outside temperature increases . with respect to the non - weather variables we note that a large turnout benefits the left - wing environmentalist party gl and the christian democratic party cda and not , as frequently claimed , the social democratic party pvda . the estimates for municipality latitude confirm that much of the christian democratic electorate is concentrated in the southern part of the country . to examine the political significance of the weather parameters , table 3 displays the predicted deviations in terms of number of seats in parliament from the party 's estimated mean seat count for three settings of rainfall ( 0 , 5 , 10 mm ) as well as temperature ( 5 , 10 , 15c).table 3predicted mean deviations in number of parliamentary seats from party s estimated mean seat count by rainfall ( mm ) and temperature ( c ) , 19712010temperature ( c)rainfall ( mm)0510gl ( mean 7.94 , se 0.11 ) 50.06 ( 0.20)0.40 ( 0.23)0.74 ( 0.33 ) 100.04 ( 0.13)0.16 ( 0.13)0.36 ( 0.18 ) 150.14 ( 0.13)0.08 ( 0.13)0.01 ( 0.15)sp ( mean 6.72 , se 0.04 ) 51.17 ( 0.18)0.15 ( 0.21)1.48 ( 0.30 ) 100.56 ( 0.08)0.19 ( 0.09)0.94 ( 0.14 ) 150.06 ( 0.06)0.23 ( 0.06)0.41 ( 0.09)pvda ( mean 39.15 , se 0.09 ) 50.88 ( 0.33)0.74 ( 0.39)2.36 ( 0.59 ) 100.48 ( 0.14)0.37 ( 0.16)1.23 ( 0.28 ) 150.08 ( 0.16)0.13 ( 0.16)0.11 ( 0.22)d66 ( mean 10.04 , se 0.08 ) 50.57 ( 0.17)0.37 ( 0.20)0.16 ( 0.29 ) 100.24 ( 0.09)0.06 ( 0.10)0.12 ( 0.15 ) 150.09 ( 0.10)0.25 ( 0.10)0.41 ( 0.12)cda ( mean 41.97 , se 0.10 ) 51.20 ( 0.37)0.17 ( 0.44)1.53 ( 0.67 ) 100.74 ( 0.16)0.32 ( 0.18)1.38 ( 0.31 ) 150.28 ( 0.18)0.47 ( 0.18)1.22 ( 0.24)vvd ( mean 27.18 , se 0.13 ) 50.26 ( 0.25)0.21 ( 0.29)0.69 ( 0.42 ) 100.12 ( 0.15)0.09 ( 0.16)0.30 ( 0.22 ) 150.03 ( 0.16)0.03 ( 0.16)0.08 ( 0.19)cu / sgp ( mean 6.48 , se 0.03 ) 50.58 ( 0.14)0.45 ( 0.17)0.32 ( 0.26 ) 100.23 ( 0.06)0.09 ( 0.07)0.05 ( 0.12 ) 150.11 ( 0.07)0.26 ( 0.07)0.42 ( 0.09)other parties ( mean 11.00 , se 0.06 ) 50.21 ( 0.25)1.12 ( 0.29)2.03 ( 0.45 ) 100.23 ( 0.10)0.22 ( 0.12)0.20 ( 0.21 ) 150.26 ( 0.12)0.68 ( 0.12)1.62 ( 0.16)standard error in parenthesis . descriptions of the abbreviations can be found in table 1 predicted mean deviations in number of parliamentary seats from party s estimated mean seat count by rainfall ( mm ) and temperature ( c ) , 19712010 standard error in parenthesis . descriptions of the abbreviations can be found in table 1 the dutch national parliament has 150 seats that , once the election results are known , are allocated to the contesting parties . the number of votes cast is divided by the 150 seats available , to render a threshold for each seat . each party 's number of votes is divided by this threshold to give an initial number of seats . any party that received fewer votes than the threshold fails to gain representation in parliament . after the initial seats are allocated if we disregard both the method of allocating remainder seats and electoral alliances of parties to gain remainder seats , we may equate a single seat to represent ( 100/150 ) 0.667% of the votes . with this in mind , table 3 reveals that the weather parameters have a politically significant effect on the seat gains and losses for some political parties . most notable are the consequences for the social democratic party pvda and the socialist party sp on the one hand and the christian democratic party cda on the other . the two left - wing parties seem to profit from cold ( 5c ) and dry ( 0 mm rainfall ) conditions . however , if the weather is both cold and wet ( 10 mm ) , the social democratic party pvda is seen to lose 1 ( 10c ) to 2 seats ( 5c ) in parliament . about the christian democratic party , on the contrary , is predicted to benefit from an extremely rainy ( 10 mm ) election day , irrespective of temperature , and to gain one extra seat in parliament under such conditions . cold ( 5c ) and dry ( 0 mm ) election day weather cause this party to lose a seat in parliament however . the electoral gains and losses experienced by these three parties either impose costs on or create benefits for the other parties . the remaining political parties are shown neither to win nor to lose in an electorally significant manner as a result of inclement weather . this paper validates the long - held belief in dutch politics that inclement weather on election day measured by local rainfall and low temperatures diminishes the share of the vote received by the left - wing social democratic ( labor ) party and the socialist party . however , the impact is not as strong as many people , politicians and journalist alike , believe it to be . cold and rainy election day weather causes these parties to lose one or two seats in the 150-seat dutch national parliament . their major opponent , the christian democratic party , is shown to profit electorally from an extremely rainy election day as it offers the party one additional seat in parliament . one explanation for the weather - related seat gain of the christian democratic party is that the party is exclusively supported by religious people . it may be that the religious part of the electorate has a somewhat higher level of civic duty which prevents them from letting the rain stop their voice from being heard . an additional explanation may be that the christian democratic party is traditionally sought to represent the interests of farmers and fishermen , who are used to work in all kinds of weather , and their families . however this may be , our results indicate that the left - wing social democratic and socialist parties should cross their fingers for dry and cold temperatures on dutch election day , whereas christian democrat supporters should pray for large amounts of rain .

a 32-year - old male from nepal presented to the oculoplasty clinic with complaints of redness and swelling of both the upper eyelids followed by progressive drooping for the last 5 years . there was no history of diurnal variation , double vision , trauma or any previous surgery . the patient also gave history of recurrent episodes of painful , swollen joints ( hip , knee and ankle ) . similar ocular as well as orthopaedic complaints were seen in one of his paternal uncles and grandfather . on examination meibography revealed severe dropout of meibomian glands with marked distortion in the architecture [ fig . 2 ] . ( b ) external photograph showing pachydermia of forehead , face , and eye lids with mechanical ptosis and deep forehead furrows . ( e ) elongated upper tarsal plate meibography showing distorted architecture of meibomian glands with severe dropout bone scan revealed osteopenia of the hip joint . patient was advised to start oral residronate and osteocalcium by orthopaedist , and his joints showed marked symptomatic improvement . the patient also underwent bilateral upper lid wedge excision with lateral tarsal strip with external levator advancement . per operatively the tarsal plates were found to be hypertrophied with increased width and height [ fig . a good correction of ptosis and lid laxity was noted in the immediate postoperative period [ fig . 3 ] . histopathological examination of the resected tarsal tissue showed fibrosis , sebaceous gland hyperplasia , mucin deposition and nonspecific chronic inflammation , which are typically seen in pdp [ fig . postoperative ( day 1 ) external photograph showing good correction of ptosis and floppy eye lids microphotograph ( h and e ; 20 ) showing sebaceous gland hyperplasia , fibrosis , and nonspecific chronic inflammation patient was started on aggressive lubricants in view of meibomian gland dysfunction and was asked to review after 6 weeks . pdp was first described by nikolaus friedrich in 1868 as a form of acromegaly . later on in 1935 touraine , solente , and gole identified the condition to be quite distinct and described it as syndrome of pulmonary hypertrophic osteoarthropathy . hypertrophic osteoarthropathy can be either primary or secondary with similar clinical features including pachydermia , cutis verticus gyrata , digital clubbing , and thickening of long bones ( periostosis ) . the secondary form is associated with an underlying pathology , is almost always painful and rapidly progressive . it is seven times more common in males , inherited in autosomal dominant fashion with variable expression . the onset of pdp is usually in the adolescence , wherein there is clubbing and broadening of limbs resulting from diffuse periostial ossifications . the changes in pdp progress over the next 520 years and then stabilize to remain unchanged throughout life . ptosis , caused by thickening of the eyelids , has been associated with pdp ; but floppy eyelids have rarely been described . we also documented the presence of severe distortion in meibomian gland architecture , a feature not previously described , by meibography . pdp needs to be differentiated from similar conditions like acromegaly , leprosy , syphilis , and paget 's disease . development of eyelid thickening in pdp has been attributed to sebaceous gland hyperplasia and mucin deposition . medical management includes nonsteroidal anti - inflammatory drugs , corticosteroids , tricyclic antidepressants , resedronate , pamidronate , and tamoxifen citrate to control arthritis . surgical management of pdp is challenging and mostly combines horizontal lid shortening with ptosis correction , frontal rhytidectomy , and brow lift . however , we feel that the lid laxity and the blepharoptosis can be addressed simultaneously and gratifying results can be obtained . as these patients can present initially with ocular symptoms , early diagnosis and referral to appropriate specialties ( dermatology , rheumatology ) is crucial for management and prevention of disabilities like deafness , kyphosis , and osteonecrosis of the femoral head . it can be associated with mechanical ptosis , floppy eye lids , and meibomian gland dysfunction . characteristic morphological appearance and body habitus along with typical radiological features help in clinching the diagnosis .

amyotrophic lateral sclerosis ( als ) is a fatal neurodegenerative disease that causes progressive muscular atrophy and death within 35 years after its onset . most patients have the so - called sporadic als , with unknown etiology ; and approximately 10% of the patients have the familial form of the disease , when more than one individual from the same family is affected . in recent years knowledge of the disease physiopathology has increased greatly , and different genes have been associated with the familial form . currently , the gene most commonly associated with als is the c9orf72 gene , which affects approximately 40% of cases of familial als , followed by cu / zn - superoxide dismutase1 ( sod1 ) , tar dna - binding protein 43 ( tardbp ) , fused in sarcoma ( fus ) and other less - frequent mutations ( su et al . , 2014 ) . despite the significant advances in knowledge of als pathology , at the moment the only available treatment is riluzole , which extends the survival time by only three months , with no improvement in the quality of life . therefore , it is imperative to search for new alternatives to treat als , and cell therapy appears to be a promising therapy . in 1994 , the first als mouse model was described , leading to enormous breakthroughs in this field ( gurney , 1994 ) . in this mouse model , the genome carries multiple copies of the mutant sod1 human gene , which contains a substitution of glycine for alanine at position 93 in the amino acid chain ( sod1g93a ) . although this mouse model represents only a small percentage ( 2% ) of als patients , it shows a progressive degeneration , with muscle atrophy and loss of motorneurons in the ventral horn of the spinal cord , culminating in the animal 's death , which is similar to the human pathology . subsequently , other mutations have been used to produce different animal models of als , but most of the disease mechanisms were elucidated in the g93a model . researchers have formed a consensus that als is a multifactorial disease , in which other cells beyond the motor neurons are involved , such as astrocytes and microglia ( mcgoldrick et al . , 2013 ) . the multifactorial feature of the disease leads researchers to believe that in order to develop an effective treatment it is necessary to address multiple compromised pathways . in this respect , unlike a canonical pharmacological approach , cell therapy , for instance , could produce multiple clinical effects regulating these disturbed pathways . initially , the main goal of cell therapy was to replace lost or damaged tissue . the discovery of stem cells in different niches , including the central nervous system , and the isolation of pluripotent embryonic stem cells contributed to this prospect . although cell replacement is possible in some damaged tissues such as bone marrow , in the central nervous system this goal is still far from being achieved , especially concerning lost motor neurons , the affected cells in als patients . considering the multifactorial nature of als , the limited knowledge of the mechanisms that initiate neuronal death , and the necessity for some kind of axonal guidance to direct long - distance and specific axonal innervation , it appears to be a difficult matter to use cell replacement for this purpose . a second possibility may be to replace cells other than the motorneurons , such as astrocytes . sod1 mutated astrocytes show a harmful profile , being toxic to motor neurons . in this respect , the injection of glial restricted precursors ( grp ) into the spinal cord of sod1 mice has been tested , and resulted in the engraftment of normal astrocytes , the protection of surrounding motor neurons , and an increase in the animals lifespan ( rizzo et al . , 2014 ) . in addition , cell therapy , especially using bone marrow cells , also proved to be very useful to protect the neural tissue . this approach has been tested in different types of brain disease or traumatic lesions with promising results . it has been demonstrated , for instance , that bone marrow transplantation , either intravenously or locally , stimulates angiogenesis , neurogenesis and axonal outgrowth , and decreases inflammation and neuronal death ( mesentier - louro et al . , 2016 ) . another advantage of the use of bone marrow cells is the possibility to perform autologous transplantation , reducing the risk of adverse effects triggered by immune responses . however , it is necessary to determine whether the autologous transplanted cells maintain the same beneficial potential , especially in genetic als cases . bone marrow cell therapy includes the use of the mononuclear fraction of bone marrow cells ( bmmc bone marrow mononuclear cells ) or mesenchymal stromal cells ( msc ) resident in this region . bmmc comprise both hematopoietic stem cells and msc in a small percentage , several hematopoietic progenitors , and differentiated bone marrow cells such as lymphocytes and monocytes . both msc and bmmc have been used in cell therapy . in a rat model of focal ischemia , these cell populations showed similar beneficial effects on the animals functional recovery , although only one - tenth the number of msc was needed to achieve the same effect as bmmc ( de vasconcelos dos santos et al . , 2010 ) . ( 2012 ) obtained a better outcome using whole bone marrow cells , compared to msc transplant , in the mdf / ocd mouse model of motoneuron degeneration . nevertheless , for clinical use , bmmc therapy is safer than msc , since the latter cells require extensive laboratory manipulation and weeks of culturing procedures , increasing the possibilities of cell contamination or even chromosomal alterations . on the other hand , bmmc could be injected in the same day of their isolation . in sod1 mice models of als , initially , the strategy aimed toward bone marrow replacement , using high doses of irradiation . although some groups observed an increase in animal survival , the use of irradiation in humans could be a dangerous procedure ( corti et al . , 2004 ) . different routes of injection , either intraspinally or systemically , and different doses have been tested . most published results show beneficial effects of the use of msc , demonstrating an increase in animal survival , protection of motorneurons , and decreases in inflammation and in the number of microglia and astrocytes ( lewis and suzuki , 2014 ) . low doses of msc ( < 500,000 cells ) , especially when administered intrathecally , showed no beneficial effects . another important issue that should be addressed in pre - clinical studies is the timing of the treatment . currently , pre - clinical studies have shown a beneficial effect of treating animals in the presymptomatic phase or at the onset of the disease . it is known that by the time the symptoms become apparent , a significant number of motor neurons have already died , supporting the importance of this approach . however , most patients have sporadic als , and even those with the familial form of the disease are not necessarily diagnosed early . added to this , the diagnosis is usually difficult and may take months to become conclusive . for these reasons it is important to be certain that therapies are beneficial even when the disease is advanced . in our work , we demonstrated , for the first time , different outcomes when we treated als mice with intraspinal injection of bmmc . as mentioned previously , it has been shown that therapy with these cells is capable of reducing cell death , microglial activation and to enhance the axonal regeneration . bone marrow cells were showed to protect neurons from apoptosis through growth factors releasing , such as gdnf ( pastor et al . , 2012 ) . others growth factors and cytokines also seems important to keep a protective and regenerative environment , such as fibroblast growth factor 2 ( fgf-2 ) ( mesentier - louro et al . , 2016 ) . in addition , clinical trials with bmmc demonstrated that they are safe and feasible for human use as well as being easy to isolate requiring little manipulation , a clear advantage comparing to msc . although we did not observe an increase in the animals survival using this approach , animals treated in the presymptomatic phase showed delays in the disease progression , analyzed by their functional motor performances , using rotarod and hanging wire tests . when we treated symptomatic animals , no modification of the progression of the disease was observed , demonstrating that bmmc could not reverse the established damage ( gubert et al . , 2016 ) . it is important to point out that , only in bmmc - treated animals in the symptomatic phase , we observed a couple of animals that survive for a longer period , more than 160 days , while non - treated animals did not exceeds 150 days . therefore , it would be necessary to test if increasing the number of cells would result in a positive effect in lifespan . local injections have the advantage of ensuring that the injected cells are near the region of interest . although spinal - cord injections involve some risk , intra - parenchymal transplants are feasible . msc injected into the spinal cord seem to survive in the injection site for at least 10 weeks in als animal models ( vercelli et al . , 2008 ) . however , testing different strategies to track bmmc , we demonstrated that these cells did not remain for a long time in this region ( gubert et al . , 2016 ) . this characteristic could explain the transitory effects of these cells in als mice observed by our group . the hazardous environment of the spinal cord could impair the transplanted cells and be responsible for damage / disappearance of the injected . in this respect , it has been shown that msc have both antioxidant and immunomodulatory potentials that could protect them from the harmful environment . intravenous treatment using msc or human umbilical - cord cells also showed positive outcomes ( lewis and suzuki , 2014 ) . these results are very promising , since this approach is less invasive , allowing multiple injections in a potential clinical trial . concerning this aspect , ongoing studies in our laboratory it is known that bone marrow cells migrate to the damaged area , even in neurodegenerative diseases such as als ( corti et al . , 2004 ) . however , the main contribution of these cells in slowing the progression of the disease may be through modulating inflammation . the contribution of immune cells , especially t lymphocytes and microglia , has been demonstrated , and the differentiation of these cells to an anti - inflammatory profile could delay the disease progression ( figure 1 ) . intramuscular and intracerebroventricular transplants are other potential routes of injection that have already been tested ( lewis and suzuki , 2014 ) . cell therapy is a promising treatment for amyotrophic lateral sclerosis ( als ) , but it is still necessary to find out the best protocol and best type of cell to use . some other important features should also be carefully tested , such as the number of cells and the route of administration . the best time of injection should also be investigated , especially considering that , probably , the therapy would occur in symptomatic patients . bone marrow derived cells showed promising results in animal models of als , and these cells seem to act not only on the motor neurons , but also on non - neuronal cells , such as astrocytes , microglia and lymphocytes , reducing the inflammatory toxic environment into the spinal cord . bdnf : brain - derived neurotrophic factor ; fgf2 : fibroblast growth factor 2 ; igf-1 : insulin - like growth factor 1 ; il-10 : interleukin 10 ; il-13 : interleukin 13 ; mcp-1 : monocyte chemoattractant protein-1 ; tgf beta : transforming growth factor beta . as mentioned above , modulation of inflammation is a possible strategy for bone marrow therapy . in recent years , many groups have demonstrated a relationship between an inflammatory pathway and the progression of the disease , in both human patients and animal models . it has been demonstrated in the animal model that the disease progresses more slowly during its onset than in a more - advanced phase ( beers et al . , 2011 ) . this initial stable phase was associated with the presence of regulatory t cells and with an anti - inflammatory profile of the microglia . in contrast , the rapid - progression phase was associated witha decrease of regulatory t cells , an increase of t cytotoxiclymphocytes , and with a pro - inflammatory microglia . in human patients , the infiltration of lymphocytes in the spinal cord was also observed ( rizzo et al . , 2014 ) . in this respect , bmmc could contribute to regulate the inflammatory profile , especially considering the presence of lymphocytes and monocytes in this population . msc has also been shown to regulate the immune system , e.g. , by reducing the proliferation of cd8 t cells through transforming growth factor beta ( tgf beta ) secretion and downregulating the production of ifn of natural killer cells ( chen et al . , the promising results using bone marrow cells have encouraged clinical trials for als patients . at the moment , most trials seek to determine if bone marrow therapy is safe and feasible , although the clinical outcome may also be analyzed . a retrospective analysis found that bmmc therapy had a significant effect on patient survival ( sharma et al . , 2015 ) . however , a randomized study is still necessary , analyzing a larger cohort to prove the efficacy of bmmc transplants . msc are also being tested , and were found to be safe when injected intrathecally , intraspinally and intravenously . bone marrow cell therapy for als is in its infancy , and much has to be done before we can determine the true potential of this approach . many groups , including ours , are working intensively to find the best protocol that could be translated to the clinic . however , until now , the best outcome extends an animal 's lifespan by only a few weeks . it is essential to continue to investigate the mechanisms that initiate and spread the degenerative process , in order to fight them and further delay the progression of the disease ( figure 1 ) . multiple injections , especially intravenously , could sustain the effect longer , but it is important to think in broad terms to deal with als . a combination of cell therapy with gene therapy could potentiate the results , and pharmacological approaches should be added to treat specific targets . finally , inflammation seems to be a potential issue in als and should also be considered a potential target in the investigation of als disease treatment .

emmons - type olefinations . incorporating -substitution in a stereodefined ,-unsaturated ester has been a significant limitation to date for this method . there are several valuable natural product cores that can be elaborated using the described -arylated phosphonoacetates , especially the cinnamic acid core , shown in scheme 2 . the 2-arylcinnamic acid derivatives have been studied for their antimitotic activity , as well as their activity as endothelin a receptor antagonists . unfortunately , derivatization has been limited due to an inability to broadly functionalize the -arene . despite their clear utility , there are few reported methods to synthesize any variety of -arylated phosphonoacetates . arbuzov reaction , which requires high temperatures and has limited tolerance for sterically hindered substrates ( scheme 3a ) . this method is also limited by the availability of the -halo--aryl acetate starting materials , and the electrophilic functional group tolerance is particularly limited . becker reaction , which uses the corresponding phosphonic acids , proceeds in poor yield , especially for sterically hindered tertiary phosphonoacetates . in addition , strong bases are required to deprotonate the phosphonic acids , which are incompatible with many desirable functional groups . the starting phosphonic acids are also not readily available , which further limits the utility of the method . an alternative bond disconnection to this structural class utilizes an aryl halide and phosphonoacetate ( scheme 3b , c ) . there is extensive literature precedent for the -arylation of acidic substrates to form tertiary centers , using activating functional groups such as esters , ketones , nitro groups , and amides . however , in the literature to date , only the -arylation of phosphonoacetates using aryl iodides has been reported , and the substrate scope was not thoroughly explored ( scheme 3b ) . iodobenzene works well in this transformation , but aryl bromides do not couple effectively under the reaction conditions . since fewer aryl iodides are available relative to the bromo and chloro arenes , we targeted this transformation for study . notably , walsh and co - workers recently published the -arylation of benzyl phosphonates , but we have found that the addition of an acetate coordinating group greatly alters the optimal reaction conditions ; such acidic substrates readily form stable chelated adducts with the metal catalyst which are not productive reaction intermediates . in this report , we describe the first intermolecular -arylation of phosphonoacetates with readily available aryl bromides and chlorides ( scheme 3c ) . an initial survey of cross - coupling conditions from related acidic substrates failed to cause -arylation of phosphonoacetates . using bromobenzene , 12 ligands and eight solvents were evaluated using pd2(dba)3 as a palladium source and 1.2 equiv of k3po4 . as shown in table 1 , cyclopentyl methyl ether ( cpme ) was quickly identified as the best solvent for this arylation , and both brettphos and sphos afforded the product in good isolated yield upon 0.2 mmol scale validation of the microscale leads . reactions were conducted at 100 c and 0.1 m in solvent , with 5 mol % of pd2(dba)3 , 20 mol % of ligand , 1.2 equiv f base , and 1.1 equiv of bromobenzene . most reactions still had trace starting material remaining at the end of the reaction time . with cpme as a solvent , 12 different bases and the top two ligands were again assessed via parallel microscale experimentation . the top results of that screen were validated on a 0.2 mmol scale and are shown in table 2 . reactions were conducted at 100 c and 0.2 m in cpme , with 2.5 mol % of pd2(dba)3 , 10 mol % of ligand , 1.2 equiv of base , and 1.1 equiv of bromobenzene . overall , reactions using brettphos as the ligand had a cleaner reaction profile , and cs2co3 was the most effective base for the transformation . a further concentration study showed that moving from 0.1 to 0.2 m caused an 11% improvement in the isolated yield of the arylated product 3a . proceeding at a 0.2 m reaction concentration , the reactivity of aryl bromides was compared to aryl chlorides . as shown in table 3 , both aryl bromides and aryl chlorides perform well at 2.5 mol % of pd2(dba)3 . the reactivity of chlorobenzene dropped off at 1.25 mol % of pd2(dba)3 , but that of bromobenzene was well maintained . reactions were conducted at 100 c in cpme , in a 2:1 ligand : metal ratio , with 1.2 equiv cs2co3 , and 1.1 equiv aryl halide . with these conditions in hand , the substrate scope of the reaction was investigated , as shown in schemes 4 and 5 . both electron - poor and electron - rich substrates are well - tolerated , as are several heterocyclic substrates . r ) generally did not perform well in the reaction unless the nitrogen basicity was moderated , as with substrates 3n and 3o . aryl halides with ortho - substituents did not perform well in the coupling ( 3e ) ; presumably , the steric bulk of the ortho - group upon coordination to the palladium center hinders transmetalation or reductive elimination . notably , substrates with electrophilic functional groups ( 3 g ) are coupled in high yield . additionally , only 3a , 3k , and 3l in schemes 4 and 5 can be synthesized via the arbuzov reaction from commercially available starting materials . both aryl chlorides and aryl bromides are well - tolerated for a diverse array of functional groups . reactions were conducted at 100 c in cpme with 1.25 mol % of pd2(dba)3 , 5 mol % of brettphos , 1.2 equiv of cs2co3 , and 1.1 equiv of aryl bromide . reactions were conducted at 100 c in cpme with 2.5 mol % of pd2(dba)3 , 10 mol % of brettphos , 1.2 equiv of cs2co3 , and 1.1 equiv of aryl bromide . reactions were conducted at 100 c in cpme , with 1.25 mol % of pd2(dba)3 , 5 mol % of brettphos , 1.2 equiv of cs2co3 , and 1.1 equiv of aryl chloride . reactions were conducted at 100 c in cpme , with 2.5 mol % of pd2(dba)3 , 10 mol % of brettphos , 1.2 equiv of cs2co3 , and 1.1 equiv of aryl chloride . as shown in scheme 6 , on a 5.0 mmol scale , this previously unreport -arylated phosphonoacetate was able to be isolated in 80% yield . we propose a mechanism for this transformation similar to those proposed for the -arylations of other enolic substrates . in this case , however , the palladium can chelate to the phosphonate or the ester , as shown in scheme 7 . the different chelation modes are shown in structures c , d , and e. as proposed by culkin and hartwig , reductive elimination likely occurs from the -c - bound structure ( c ) , which is accessible only in the presence of bulky ligands on the transition metal . the increased stability of the chelated form d presents the key challenge to this method , causing the reductive elimination step to be comparatively slow . we have also begun to investigate whether this reaction can be applied to quaternary centers , a much more challenging c c bond construction . as shown in scheme 8 , a quaternary -arylated product , 5 , could be achieved in an unoptimized 50% yield . in conclusion , the method provides an efficient route to complex arylated products that are not otherwise accessible . this process can be utilized for a variety of functionalized aryl bromides and aryl chlorides to afford these highly useful compounds in good to excellent yields . unless otherwise noted , all reagents were reagent grade and used without further purification . cyclopentyl methyl ether ( cpme ) and toluene were distilled over cah2 and stored under argon . analytical thin - layer chromatography ( tlc ) was performed using 0.25 mm silica gel 254-f plates . h nmr and c nmr spectra were recorded at 25 c on 300 , 360 , or 500 mhz spectrometers . p nmr spectra were recorded at 25 c on 300 or 360 mhz spectrometers and are proton decoupled . chemical shifts are reported relative to the solvent resonance peak 7.27 ( cdcl3 ) for h and 77.23 ( cdcl3 ) for c. for f spectra , chemical shifts are reported relative to a capillary internal standard of 76.55 ( trifluoroacetic acid ) . for p spectra , chemical shifts are reported relative to a capillary internal standard 0 ( h3po4 ) . peaks are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , bs = broad singlet , m = multiplet ) , coupling constants , and number of protons . high - resolution mass spectra were obtained using a tof mass analyzer in esi ionization mode . all yields refer to isolated yields , and product purity was determined by h nmr spectroscopy . in a glovebox , a flame - dried microwave vial containing a magnetic stir bar was charged with cs2co3 ( 78 mg , 0.24 mmol ) , pd2(dba)3 ( 2.3 mg , 0.0025 mmol ) , brettphos ( 5.8 mg , 0.01 mmol ) , and the aryl halide ( 2 ) ( if a solid ) ( 0.22 mmol ) . cpme was added via syringe , followed by the aryl halide ( 2 ) ( if a liquid ) ( 0.22 mmol ) and triethyl phosphonoacetate ( 1 ) ( 40 l , 0.20 mmol ) . the vial was sparged with dry argon and then heated to 100 c in an oil bath with vigorous stirring . upon consumption of the triethyl phosphonoacetate , as monitored by tlc , h , or p nmr , the reaction mixture was allowed to cool to room temperature and then quenched with 1.0 ml of 1.0 m hcl . this mixture was diluted with h2o and extracted with etoac ( 3 15 ml ) . the resulting residue was purified by flash column chromatography to afford the pure -arylated phosphonoacetates . the general method was followed with a reaction time of 19 h. purification by chromatography ( 50% etoac / hexanes ) provided the title compound as a pale yellow oil ( 47 mg , 78% ) . the general method was followed with a reaction time of 17 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a yellow solid ( 62 mg , 88% ) : mp 6567 c ; h nmr ( 360 mhz , cdcl3 ) 7.99 ( m , 1h ) , 7.867.82 ( m , 3h ) , 7.68 ( ddd , j = 8.6 hz , 1.6 hz , 1.4 hz , 1h ) , 7.507.47 ( m , 2h ) , 4.43 ( d , jh p = 23.4 hz , 1h ) , 4.313.97 ( m , 6h ) , 1.29 ( t , j = 6.8 hz , 3h ) , 1.28 ( t , j = 6.7 hz , 3h ) , 1.20 ( t , j = 7.2 hz , 3h ) ; c{h } nmr ( 125.7 mhz , cdcl3 ) 167.7 ( d , j = 1.5 hz ) , 133.2 ( d , j = 1.5 hz ) , 133.8 , 128.81 , 128.75 , 128.5 ( d , j = 5.3 hz ) , 128.1 ( d , j = 0.9 hz ) , 128.0 , 127.6 , 127.3 ( d , j = 5.0 hz ) , 126.2 ( d , j = 3.8 hz ) , 63.4 ( d , j = 6.3 hz ) , 63.1 ( d , j = 7.5 hz ) , 61.8 , 52.4 ( d , j = 134.6 hz ) , 16.3 ( d , j = 6.3 hz ) , 16.2 ( d , j = 6.3 hz ) , 14.1 ; p{h } nmr ( 145.8 mhz , cdcl3 ) 19.10 ( s ) ; ir ( neat ) 3058 , 2988 , 2940 , 1733 , 1300 , 1253 , 1050 , 1026 cm ; hrms ( esi ) calcd for c18h23o5pna [ m + na]m / z = 373.1181 , found 373.1189 . the general method was followed with a reaction time of 18 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a yellow oil ( 46 mg , 70% ) . the general method was followed with a reaction time of 17 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a yellow oil ( 49 mg , 72% ) : h nmr ( 300.1 mhz , cdcl3 ) 7.81 ( m , 1h ) , 7.62 ( d , j = 2.2 hz , 1h ) , 7.497.42 ( m , 2h ) , 6.776.76 ( m , 1h ) , 4.34 ( d , jh p = 23.5 hz , 1h ) , 4.323.89 ( m , 6h ) , 1.283 ( t , j = 7.1 hz , 3h ) , 1.276 ( t , j = 7.1 hz , 3h ) , 1.19 ( t , j = 7.1 hz , 3h ) ; c{h } nmr ( 90.6 mhz , cdcl3 ) 168.2 , 154.8 , 145.7 , 127.9 , 126.1 ( d , j = 6.3 hz ) , 125.6 ( d , j = 8.1 hz ) , 122.6 ( d , j = 6.7 hz ) , 111.5 , 106.9 , 63.5 ( d , j = 6.8 hz ) , 63.3 ( d , j = 7.1 hz ) , 62.0 , 52.2 ( d , j = 135.8 hz ) , 16.5 ( d , j = 5.8 hz ) , 16.4 ( d , j = 5.8 hz ) , 14.2 ; p{h } nmr ( 121.5 mhz , cdcl3 ) 18.04 ( s ) ; ir ( neat ) 2985 , 2930 , 1733 , 1468 , 1446 , 1256 , 1050 , 1026 cm ; hrms ( esi ) calcd for c16h21o6pna [ m + na]m / z = 363.0973 , found 363.0975 . the general method was followed with a reaction time of 23 h. purification by chromatography ( 40% etoac / hexanes ) provided the title compound as a pale oil ( 56.3 mg , 86% ) : h nmr ( 360 mhz , cdcl3 ) 7.12 ( s , 2h ) , 6.94 ( s , 1h ) , 4.303.97 ( m , 7h ) , 2.31 ( s , 6h ) , 1.28 ( t , j = 7.2 hz , 6h ) , 1.22 ( t , j = 7.0 hz , 3h ) ; c{h } nmr ( 90.6 mhz , cdcl3 ) 167.8 , 138.0 ( d , j = 1.8 hz ) , 130.6 ( d , j = 8.1 hz ) , 129.6 ( d , j = 3.6 hz ) , 127.3 ( d , j = 6.3 hz ) , 63.3 ( d , j = 7.2 hz ) , 63.1 ( d , j = 7.2 hz ) , 61.7 , 52.1 ( d , j = 134.9 hz ) , 21.3 , 16.3 ( d , j = 5.9 hz ) , 16.2 ( d , j = 5.9 hz ) , 14.0 ; p{h } nmr ( 145.8 mhz , cdcl3 ) 19.47 ( s ) ; ir ( neat ) 2985 , 2928 , 1735 , 1602 , 1256 , 1051 , 1024 , 733 cm ; hrms ( esi ) calcd for c16h26o5p [ m + h]m / z = 329.1518 , found 329.1516 . the general method was followed with a reaction time of 21 h. purification by chromatography ( 50% etoac / hexanes ) provided the title compound as a pale yellow oil ( 41.6 mg , 58% ) : h nmr ( 360 mhz , cdcl3 ) 8.01 ( d , j = 8.3 hz , 2h ) , 7.60 ( dd , j = 8.6 hz , 2.2 hz , 2h ) , 4.31 ( d , jh p = 23.8 hz , 1h ) , 4.283.96 ( m , 6h ) , 3.91 ( s , 3h ) , 1.27 ( t , j = 7.0 hz , 3h ) , 1.26 ( t , j = 7.0 hz , 3h ) , 1.20 ( t , j = 7.0 hz , 3h ) ; c{h } nmr ( 90.6 mhz , cdcl3 ) 167.1 ( d , j = 3.6 hz ) , 166.7 , 136.2 , 136.1 , 129.73 , 129.66 , 63.5 ( d , j = 7.2 hz ) , 63.3 ( d , j = 7.2 hz ) , 62.0 , 52.4 ( d , j = 133.1 hz ) , 52.1 , 16.3 ( d , j = 4.8 hz ) , 16.2 ( d , j = 4.8 hz ) , 14.0 ; p{h } nmr ( 145.8 mhz , cdcl3 ) 18.44 ( s ) ; ir ( neat ) 2984 , 2910 , 1724 , 1279 , 1257 , 1021 , 734 cm ; hrms ( esi ) calcd for c16h23o7p [ m + na]m / z = 381.1079 , found 381.1085 the general method was followed with a reaction time of 19 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a pale oil ( 42.3 mg , 61% ) . all reported spectra were in agreement with published literature values : ir ( neat ) 2984 , 2940 , 1735 , 1532 , 1025 , 735 cm . the general method was followed with a reaction time of 19 h. purification by chromatography ( 40% etoac / hexanes ) provided the title compound as a pale oil ( 48.5 mg , 66% ) : h nmr ( 360 mhz , cdcl3 ) 7.78 ( m , 1h ) , 7.757.73 ( m , 1h ) , 7.597.57 ( m , 1h ) , 7.47 ( dd , j = 7.8 hz , 7.8 hz , 1h ) , 4.324.00 ( m , 6h ) , 4.30 ( d , jh p = 24.1 hz , 1h ) , 1.29 ( t , j = 7.2 hz , 3h ) , 1.27 ( t , j = 7.2 hz , 3h ) , 1.21 ( t , j = 7.0 hz , 3h ) ; c{h } nmr ( 125.8 mhz , cdcl3 ) 167.3 ( d , j = 5.0 hz ) , 133.3 ( d , j = 6.3 hz ) , 132.4 ( d , j = 8.8 hz ) , 131.0 ( dq , j = 32.1 hz , 1.9 hz ) , 129.1 ( d , j = 2.5 hz ) , 126.8126.6 ( m ) , 125.0124.9 ( m ) , 124.1 ( q , jc f = 272.1 hz ) , 63.8 ( d , j = 6.3 hz ) , 63.5 ( d , j = 7.5 hz ) , 62.3 , 52.2 ( d , j = 134.6 hz ) , 16.4 ( d , j = 6.3 hz ) , 16.3 ( d , j = 6.3 hz ) , 14.2 ; p{h } nmr ( 145.8 mhz , cdcl3 ) 18.30 ( s ) ; ir ( neat ) 2987 , 2938 , 1736 , 1330 , 1026 , 736 cm ; hrms ( esi ) calcd for c15h20f3o5p [ m + h]m / z = 369.1079 , found 369.1080 . in a glovebox , a flame - dried microwave vial containing a magnetic stir bar was charged with cs2co3 ( 78 mg , 0.24 mmol ) , pd2(dba)3 ( 4.6 mg , 0.005 mmol ) , and brettphos ( 11.6 mg , 0.02 mmol ) . the vial was capped and brought out of the glovebox . cpme was added via syringe followed by chlorobenzene ( 2k ) ( 22 l , 0.22 mmol ) and triethyl phosphonoacetate ( 1 ) ( 40 l , 0.20 mmol ) . the vial was sparged with dry argon and then heated to 100 c in an oil bath with vigorous stirring . upon consumption of the triethyl phosphonoacetate , as monitored by tlc , h , or p nmr , the reaction mixture was allowed to cool to room temperature and then quenched with 1.0 ml of 1.0 m hcl . the resultant mixture was diluted with h2o and extracted with etoac ( 3 15 ml ) . purification by chromatography ( 50% etoac / hexanes ) provided the title compound as a pale yellow oil ( 49.8 mg , 83% ) . the general method was followed with a reaction time of 18 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a pale yellow oil ( 50.1 mg , 79% ) . all reported spectra were in agreement with the published literature values : p{h } nmr ( 145.8 mhz , cdcl3 ) 18.85 ( d , j = 12.6 hz ) ; ir ( neat ) 2985 , 2936 , 1735 , 1509 , 1050 , 1026 , 735 cm ; hrms ( esi ) calcd for c14h20fo5pna [ m + na]m / z = 341.0930 , found 341.0923 . the general method was followed with a reaction time of 17 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a pale yellow oil ( 61.1 mg , 83% ) : h nmr ( 300 mhz , cdcl3 ) 7.687.60 ( m , 4h ) , 4.333.96 ( m , 6h ) , 4.31 ( d , jh p = 23.7 hz , 1h ) , 1.29 ( t , j = 7.1 hz , 3h ) , 1.28 ( t , j = 7.1 hz , 3h ) , 1.22 ( t , j = 7.1 hz , 3h ) ; c{h } nmr ( 125.8 mhz , cdcl3 ) 167.2 ( d , j = 3.8 hz ) , 135.4 ( d , j = 8.8 hz ) , 130.4 ( dq , j = 32.7 hz , jc f = 2.5 hz ) , 130.2 ( d , j = 6.3 hz ) , 125.6125.5 ( m ) , 124.2 ( q , jc f = 272.5 hz ) , 63.8 ( d , j = 7.5 hz ) , 63.5 ( d , j = 6.3 hz ) , 62.3 , 52.3 ( d , j = 133.3 hz ) , 16.5 ( d , j = 6.9 hz ) , 16.4 ( d , j = 6.9 hz ) , 14.2 ; p{h } nmr ( 145.8 mhz , cdcl3 ) 18.03 ( s ) ; ir ( neat ) 2985 , 2936 , 1736 , 1325 , 1020 , 736 cm ; hrms ( esi ) calcd for c15h21f3o5p [ m + h]m / z = 369.1079 , found 369.1071 . the general method was followed with a reaction time of 19 h. purification by chromatography ( 60% etoac / hexanes ) provided the title compound as a pale yellow oil ( 65.6 mg , 90% ) : h nmr ( 300.1 mhz , cdcl3 ) 7.59 ( dd , j = 8.7 hz , 2.3 hz , 2h ) , 7.39 ( d , j = 8.3 hz , 2h ) , 7.10 ( dd , j = 2.2 hz , 2.2 hz , 2h ) , 6.35 ( dd , j = 2.1 hz , 2.1 hz , 2h ) , 4.343.96 ( m , 6h ) , 4.27 ( d , jh f = 23.8 hz , 1h ) , 1.304 ( t , j = 7.1 hz , 3h ) , 1.295 ( t , j = 7.1 hz , 3h ) , 1.23 ( t , j = 7.1 hz , 3h ) ; c{h } nmr ( 125.8 mhz , cdcl3 ) 167.5 ( d , j = 3.8 hz ) , 140.4 , 130.8 ( d , j = 6.3 hz ) , 128.2 ( d , j = 7.5 hz ) , 120.3 ( d , j = 2.5 hz ) , 119.1 , 110.6 , 63.4 ( d , j = 6.3 hz ) , 63.2 ( d , j = 6.3 hz ) , 51.6 ( d , j = 133.3 hz ) , 16.31 ( d , j = 6.3 hz ) , 16.26 ( d , j = 6.3 hz ) , 14.03 ; p{h } nmr ( 121.5 mhz , cdcl3 ) 18.79 ( s ) ; ir ( neat ) 3054 , 2984 , 2930 , 1733 , 1521 , 1265 , 735 cm ; hrms ( esi ) calcd for c18h25no5p [ m + h]m / z = 366.1470 , found 366.1472 . the general method was followed with a reaction time of 21 h. purification by chromatography ( 40% etoac / hexanes ) provided the title compound as a pale yellow oil ( 85.6 mg , 97% ) : h nmr ( 500 mhz , cdcl3 ) 8.10 ( d , j = 8.0 hz , 1h ) , 7.75 ( dd , j = 2.0 hz , 2.0 hz , 1h ) , 7.59 ( d , j = 3.5 hz , 1h ) , 7.44 ( ddd , j = 8.6 hz , 1.9 hz , 1.9 hz , 1h ) , 6.56 ( d , j = 3.5 hz , 1h ) , 4.33 ( d , jh p = 23.0 hz , 1h ) , 4.283.91 ( m , 6h ) , 1.67 ( s , 9h ) , 1.270 ( t , j = 7.0 hz , 3h ) , 1.268 ( t , j = 7.0 hz , 3h ) , 1.19 ( t , j = 7.0 hz , 3h ) ; c{h } nmr ( 125.8 mhz , cdcl3 ) 168.2 ( d , j = 1.8 hz ) , 149.8 , 135.0 , 130.9 , 126.6 , 125.9 ( d , j = 4.3 hz ) , 125.3 ( d , j = 6.1 hz ) , 122.3 ( d , j = 5.0 hz ) , 115.3 , 107.5 , 84.0 , 63.6 ( d , j = 4.5 hz ) , 63.2 ( d , j = 4.5 hz ) , 61.9 , 52.2 ( d , j = 97.8 hz ) , 28.3 , 16.52 ( d , j = 5.7 hz ) , 16.48 ( d , j = 5.7 hz ) , 14.2 ; p{h } nmr ( 145.8 mhz , cdcl3 ) 19.58 ( s ) ; ir ( neat ) 2981 , 2934 , 1733 , 1024 , 731 cm ; hrms ( esi ) calcd for c21h31no7p [ m + h]m / z = 440.1838 , found 440.1823 . in a glovebox , a flame - dried schlenk flask containing a magnetic stir bar was charged with cs2co3 ( 1.95 g , 6.0 mmol ) , pd2(dba)3 ( 57.2 mg , 0.0625 mmol ) , and brettphos ( 134.2 mg , 0.25 mmol . the flask was sealed and brought out of the glovebox . cpme ( 25 ml ) was added , followed by 3,5-dimethylbromobenzene ( 657 l , 5.5 mmol ) and triethyl phosphonoacetate ( 1 ) ( 992 l , 5.0 mmol ) . the flask was heated to 85 c in an oil bath with vigorous stirring . upon consumption of the triethyl phosphonoacetate , as monitored by tlc , the reaction mixture was allowed to cool to room temperature and then quenched with 25 ml of 1.0 m hcl . this mixture was diluted with h2o and extracted with etoac ( 3 15 ml ) . the resulting residue was purified by flash column chromatography ( 40% etoac / hexanes ) to provide the title compound as a pale oil ( 1.31 g , 80% ) . under an argon atmosphere , a round - bottom flask containing a magnetic stir bar and equipped with a reflux condenser was charged with ethoxydiphenylphosphane . the resulting residue was purified by flash column chromatography ( 50% etoac / hexanes ) to afford the title compound as a white powder ( 10.28 g , 38% , unoptimized ) : mp 146147 c ; h nmr ( 499.7 mhz , cdcl3 ) 7.927.84 ( m , 4h ) , 7.657.60 ( m , 2h ) , 7.567.52 ( m , 4h ) , 5.70 ( dd , jh p = 8.0 hz , jh f = 47.0 hz , 1h ) , 4.91 ( q , j = 7.2 hz , 2h ) , 1.11 ( t , j = 7.0 hz , 3h ) ; c{h } nmr ( 125.7 mhz , cdcl3 ) 165.2 ( d , j = 22.0 hz ) , 133.3 ( d , j = 2.8 hz ) , 133.1 ( d , j = 2.9 hz ) , 132.1 ( d , j = 2.0 hz ) , 132.04 ( d , j = 2.9 hz ) , 131.96 ( d , j = 2.9 hz ) , 131.88 ( d , j = 1.8 hz ) , 129.0 ( d , j = 14.8 hz ) , 128.9 ( d , j = 14.8 hz ) , 88.5 ( dd , jc p = 70.5 hz , jc f = 203.6 hz ) , 62.6 , 14.1 ; f{h } nmr ( 282.4 mhz , cdcl3 ) 202.2 ( d , jf p = 59.3 hz ) ; p{h } nmr ( 145.8 mhz , cdcl3 ) 26.3 ( d , jp f = 57.7 mhz ) ; ir ( neat ) 3058 , 2924 , 1756 , 1246 , 1190 , 1071 , 698 cm ; hrms ( esi ) calcd for c16h17o3fp [ m + h]m / z = 307.0899 , found 307.0902 . in a glovebox , a flame - dried microwave vial containing a magnetic stir bar was charged with khmds ( 47.9 mg , 0.24 mmol ) , [ pd(allyl)cl]2 ( 3.7 mg , 0.01 mmol ) , xantphos ( 5.8 mg , 0.01 mmol ) , and ethyl 2-(diphenylphosphoryl)-2-fluoroacetate ( 4 ) ( 61.3 mg , 0.2 mmol ) . the vial was capped and brought out of the glovebox . the vial was then heated to 110 c in an oil bath with vigorous stirring . after 16 h , the reaction mixture was allowed to cool to room temperature and then quenched with 1.0 ml of ph = 7 phosphate buffer . this mixture was diluted with h2o and extracted with ch2cl2 ( 3 15 ml ) . the resulting residue was purified by flash column chromatography ( 50% etoac / hexanes ) to afford the title compound as a pale yellow oil ( 38.3 mg , 50% ) : h nmr ( 500 mhz , cdcl3 ) 8.148.1 ( m , 2h ) , 7.77.63 ( m , 3h ) , 7.597.56 ( m , 2h ) , 7.507.46 ( m , 3h ) , 7.377.3 ( m , 5h ) , 4.1 ( q , j = 7.0 hz , 2h ) , 1.0 ( t , j = 7.0 hz , 3h ) ; c{h } nmr ( 125.8 mhz , cdcl3 ) 166.5 ( dd , j = 4.6 hz , 23.8 hz ) , 133.0 ( d , j = 2.8 hz ) , 132.7 ( d , j = 1.9 hz ) , 132.6 ( d , j = 2.1 hz ) , 132.4 ( dd , j = 3.3 hz , 8.8 hz ) , 131.7 ( d , j = 20.7 hz ) , 131.1 , 129.1 , 129.0 , 128.9 ( d , j = 12.1 hz ) , 128.3 , 128.2 , 128.1 , 125.8 ( dd , j = 2.9 hz , 10.2 hz ) , 62.9 , 13.9 ; f{h } nmr ( 338.9 mhz , cdcl3 ) 169.4 ( d , jf p = 74.5 hz ) ; p{h } nmr ( 145.8 mhz , cdcl3 ) 27.5 ( d , jp f = 74.4 mhz ) ; ir ( neat ) 3057 , 1750 , 1591 , 1265 , 1246 , 1206 , 1116 cm ; hrms ( esi ) calcd for c22h20o3fpna [ m + na]m / z = 405.1032 , found 405.1050 .

they are present in the eye in high concentrations . the retina and lens in general and the macular region in the center of the retina in particular there is increasing evidence that lutein and zeaxanthin may play an important role in protecting against several eye diseases , such as age - related macular degeneration ( amd ) [ 38 ] . therefore , lutein and zeaxanthin are widely used as nutrient supplements for the prevention and treatment of amd and other eye diseases . the protective effects of lutein and zeaxanthin may be related to their short wave light - screening effect and antioxidant properties [ 19 ] . recent studies indicated that they also influence cell function through various signal pathways or transcription factors and have anti - inflammatory effect [ 9 , 10 ] . it has been reported that lutein can suppress the development of uveitis caused by injection of lipopolysaccharide ( lps ) in rats and mice [ 10 , 11 ] . injection of lps into mice or rats can generate endotoxin - induced uveitis ( eiu ) , which is an important experimental uveitis model in animals [ 1016 ] . interleukin-8 ( il-8)/cxcl8 , a proinflammatory chemokine , plays an important role in the pathogenesis of lps - induced uveitis . animal studies indicated that lutein reduces the secretion of proinflammatory cytokines and chemokines and this may be the mechanism of inhibition of lps - induced uveitis by lutein . however , the cell type and the signal pathways involved in this process remain to be studied . uveal melanocytes ( um ) are the predominant cell type in the uvea . in the past , very little was known about the function of um and the role of um in the pathogenesis of various eye diseases . in the past decades , after the development of methods for the culture of um and the establishment of in vitro models for studying the function of um , it has been reported that um produce various growth factors , cytokines , and chemokines , in addition to their functions related to melanin [ 1719 ] . reported that um produce il-8 constitutively and the secretion of il-8 could be increased significantly by the stimulation of lps . this suggested that um might play a role in the pathogenesis of ocular inflammatory diseases and may be involved in the inhibitory effects of lutein on lps - induced uveitis . however , the effects of lutein and zeaxanthin on the secretion of chemokines by um have not been reported previously . the purposes of this study were to investigate the effects of lutein and zeaxanthin on lps - induced secretion of il-8 in human um in vitro and to study the signal pathways involved in this process . cell culture medium , fetal bovine serum , and trypsin were obtained from gibco ( grand island , ny , usa ) . lps , dimethyl sulfoxide ( dmso ) , collagenase , lutein , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt ) , phosphate buffered saline ( pbf ) , and protease inhibitor cocktail were obtained from sigma ( st . louis , mo , usa ) . quantikine il-8 elisa kit was obtained from r&d system ( minneapolis , mn , usa ) . p38 mitogen - activated protein kinase ( mapk ) , extracellular signal - regulated kinases 1/2 ( erk1/2 ) , and c - jun n - terminal kinase 1/2 ( jnk1/2 ) elisa kits and cell extraction buffer , pmsf , hypotonic buffer , and nuclear factor - kappa b ( nf-b ) elisa kits were obtained from invitrogen ( carlsbad , ca , usa ) . human um were isolated from donor eyes by trypsin - collagenase sequential method and cultured in fic medium as previously reported [ 1719 ] . cells from a primary culture of melanocytes isolated from choroid at passage levels 35 were used in this study . mtt assay was used to determine the effects of lps , lutein , and zeaxanthin on cell viability of cultured um as previously described . for each experiment , 5 10 cells were seeded into each well in 96-well plates . after incubation for 24 h , lps , lutein , or zeaxanthin at different levels was added . twenty - four hours later , 50 l of 1 mg / ml mtt was added and culture plates were incubated at 37c for 4 h. the medium was removed and 100 l of dmso was added . the optical density as the parameter of cell viability was measured at 540 nm with a microplate reader ( multiskan ex , thermo , ventana , finland ) . human um ( 1 10 cells / well ) were seeded into the 24-well plates and cultured . when cells reached 8090% confluence , they were rinsed with pbs and incubated in serum - free culture medium with or without lps . after 24 h , human um ( 1 10 cells / well ) were seeded into the 24-well plates and cultured . when cells reached 8090% confluence , they were rinsed with pbs and incubated in serum - free culture medium with or without lutein or zeaxanthin . human um ( 1 10 cells / well ) were seeded into the 24-well plates and cultured , and culture medium was replaced as described above . two hours later , lps at the final levels of 0.1 g / ml was added to the cultures . enzyme - linked immunosorbent assay ( elisa ) was used for the measurement of il-8 protein levels in the supernatant of cultured cells . a commercially available quantikine il-8 elisa kit was used to determine il-8 protein levels according to the protocol provided by the manufacturer . the optical density of the elisa samples was measured at 450 and 540 nm using a microplate reader . for the study of phosphorylated- ( p- ) p38 mapk , p - erk1/2 , and p - jnk1/2 levels , human cultured um ( 1 10 cells / well ) were seeded into 6-well plates and cultured for 24 h and culture medium was replaced as described above . two hours later , lps at the final levels of 0.1 g / ml was added into cultures and cultured for 60 min . cell extraction buffer with protease inhibitor cocktail and pmsf were added to the pellets and cultured for 30 min and centrifuged . the supernatants were collected and stored at 80c . for the study of nf-b , cells were cultured , treated with or without lutein , zeaxanthin , and lps , and collected as described above . collected cells were treated with hypotonic buffer and centrifuged . the pellets that contain nuclear fraction were collected , treated with cell extraction buffer , vortexed , and centrifuged . p38 mapk , erk1/2 , and jnk1/2 elisa kits were used to determine p - p38 mapk , p - erk1/2 , and p - jnk1/2 levels in cell extracts , respectively . the sensitivity of these kits was 0.8 u / ml . nf-b elisa kits were used to determine nf-b levels in cell nuclear extracts according to the protocol provided by the manufacturer . each experiment was replicated 3 times and the data were presented as mean standard deviation ( sd ) . a one - way analysis of variance ( anova ) test was performed to assess the significance . all data analysis was performed using specific software ( spss 19.0 , spss inc . , mtt assay showed that lps at the final levels of 0.01 , 0.1 , and 1 g / ml did not influence the cell viability of cultured human um ( p > 0.05 , compared with cells not treated with lps ) ( figure 1(a ) ) . lutein and zeaxanthin at the final levels of 1 , 3 , and 10 m also had no effects on the cell viability of cultured um ( p > 0.05 , compared with cells not treated with lps ) ( figures 1(b ) and 1(c ) ) . therefore , level ranges of 0.011 g / ml of lps and 110 m of lutein and zeaxanthin were chosen for subsequent experiments . elisa analysis of cell supernatants of um cultured with serum - free culture medium detected a low level of il-8 protein ( 9.20 0.90 pg / ml ) , indicating a low level of constitutive secretion of cultured um . lps at 0.011.0 g / ml caused a dose - dependent significant increase of il-8 levels ( p < 0.05 at all levels of lps as compared with cells not treated with lps , figure 2 ) . il-8 protein levels in cell supernatants from cells treated with lutein or zeaxanthin ( 1 , 3 , and 10 m ) did not significantly differ from those from cells not treated with lutein and zeaxanthin ( p > 0.05 , figure 3 ) , suggesting that lutein and zeaxanthin do not affect the constitutive secretion of il-8 from um . lutein ( 1 , 3 , and 10 m ) dose - dependently decreased lps - induced secretion of il-8 in cultured um ( p < 0.05 as compared with cells treated by 0.1 g / ml lps only , figure 4(a ) ) . however , il-8 levels in cells treated with lutein and lps were still greater than those from cells not treated with lps ( p < 0.05 ) , suggesting that lutein has a partial inhibitory effect on lps - induced secretion of il-8 . zeaxanthin ( 1 , 3 , and 10 m ) dose - dependently decreased lps - induced secretion of il-8 in cultured um ( p > 0.05 , zeaxanthin 1 m versus the controls and p < 0.05 , zeaxanthin 3 and 10 m versus the controls , figure 4(b ) ) . il-8 levels in cells treated with zeaxanthin and lps were still greater than those from cells not treated with lps ( p < 0.05 ) , suggesting that zeaxanthin also has a partial inhibitory effect on lps - induced secretion of il-8 . lps at 0.1 g / ml level significantly increased p - jnk levels ( p < 0.05 as compared with cells not treated with lps ; figure 5(a ) ) but not p - p38 mapk and p - erg1/2 levels in cultured um ( p > 0.05 , figures 5(b ) and 5(c ) ) . addition of lutein or zeaxanthin significantly reduced lps - induced increase of p - jnk levels ( both p < 0.05 , figure 5(a ) ) but did not affect p - p38 mapk and p - erg1/2 levels ( both p > 0.05 , figures 5(b ) and 5(c ) ) in cultured um . nf-b levels in cell nuclear extracts from cells treated with lps were significantly greater than those from cells not treated with lps ( p < 0.05 , figure 5(d ) ) . lutein or zeaxanthin significantly reduced lps - induced increase of nf-b levels in cell nuclear extracts ( both p < 0.05 , figure 5(d ) ) . these results suggested that jnk1/2 and nf-b ( but not p38 mapk and erk1/2 ) play an important role in lps - induced increased secretion of il-8 and in the inhibitory effects of lutein and zeaxanthin on lps - induced increased secretion of il-8 . in the present study , we demonstrated that lutein and zeaxanthin inhibited lps - induced secretion of il-8 in cultured human um and this effect was mediated by jnk1/2 and nf-b signaling pathways . uveitis is a common eye disease and a major cause of visual impairment throughout the world [ 10 , 11 , 20 ] . intraocular or systemic injection of lps can induce uveitis in experimental animals ( eiu ) . eiu is a well - known model of experimental uveitis used for the study of human uveitis [ 1016 , 19 ] . lps is an endotoxin and is the major component of the outer membrane of gram - negative bacteria . lpe - induced uveitis is thought to be the result of a cytokine - chemokine cascade [ 10 , 14 ] . il-8 levels increased significantly in lps - induced uveitis [ 15 , 16 ] . intraocular injection of anti - il-8 antibody inhibits leukocyte accumulation and decreases the clinical and histological grades of inflammation in lpe - induce uveitis . in vitro studies suggested that lps induces expression of il-8 in various cell types [ 2123 ] . chemokines could be classified into four subfamilies based on the number and location of the cysteine residues at the n - terminus of the molecule and are named cc ( with two adjacent cysteines near the n - terminus of the molecule ) , cxc ( the two cysteines being separated by an amino acid ) , cx3c ( having three amino acids between the two cysteines ) , and c ( having a specific amino acid sequence of glutamic acid - leucine - arginine immediately before the first cysteine ) , in agreement with the systematic nomenclature . in the two main subfamilies ( cc and cxc ) , cxc chemokines are important in the attraction of neutrophils and cc chemokines have powerful chemoattractants and activators for monocytes and lymphocytes [ 19 , 21 , 22 ] . il-8 is a prototype of cxc chemokine family and is a potent stimulus for neutrophils recruitment and activation . it also triggers the migration and adhesion of t cells , monocytes , and basophils to vascular endothelium and leads to extravasation of these cells into the tissues [ 19 , 21 ] . biological activities of il-8 are mediated by two cell surface g - protein - coupled receptors , cxcr1 and cxcr2 [ 21 , 22 ] . il-8 acts as a proinflammatory chemokine and plays an important role in the pathogenesis of the inflammatory process [ 21 , 22 ] . in the eye , il-8 levels are significantly increased in the aqueous humor or vitreous from patients with various types of uveitis [ 19 , 20 , 25 , 26 ] . it has been reported that intravenous injection or oral administration of lutein suppresses the development of lpe - induced uveitis in rats and mice , respectively [ 10 , 11 ] . the anti - inflammatory effect of 100 mg / kg lutein was as strong as that of 1 mg / kg dexamethasone . during the development of eiu the mechanism of the anti - inflammatory effect of lutein may be related to the suppression of proinflammation signal pathways . in addition to the anti - inflammatory effects , lutein has neuroprotective effects on retinal neurons during experimental uveitis and retinitis caused by lps . in the present study , lps significantly induced the secretion of il-8 by um in a dose - dependent manner , which is consistent with previous reports . lutein and zeaxanthin dose - dependently inhibit lps - induced increased secretion of il-8 in cultured um ; this is consistent with the animal studies , which showed that lutein suppresses the occurrence of lpe - induced uveitis . to the best of our knowledge , this is the first report showing that lutein and zeaxanthin inhibit lps - induced expression of il-8 in um . the mechanism of lutein and zeaxanthin inhibition of lps - induced expression of chemokines in um has not been previously reported . the present study demonstrated that lutein and zeaxanthin inhibited the secretion of il-8 induced by lps through the activation of jnk1/2 and nf-b signal pathway , but not p38 and erk pathway ; this is consistent with the animal study , which showed that lutein inhibited the activation of nf-b in the iris - ciliary body in lps - induced uveitis and in cultured macrophages [ 9 , 10 ] . in conclusion , this study demonstrated that lutein and zeaxanthin inhibited lps - induced secretion of il-8 in cultured human um and this effect was mediated by jnk1/2 and nf-b signaling pathways . inhibition of secretion of il-8 by lutein and zeaxanthin might be explored as a therapeutic approach in the management of uveitis and other inflammatory diseases of the eye .

it is well - established that obesity is associated with a substantial burden of illness and health care costs . obesity is associated with an increased risk of hypertension , diabetes mellitus , hyperlipidemia , coronary artery disease and many other noncontiguous diseases . the economic costs associated with the management of obesity and obesity - related diseases are remarkable . overweight and obesity are determined by combination of factors such as ; genetic , nutritional , metabolic , behavioral , environmental , cultural and socioeconomic influences . however , overweight and obesity in most individuals result from excessive energy consumption and/or inadequate physical activity . previous studies in this area showed that one in four children aged 614 years is presently overweight in developed and developing countries , which ranges from 11% to 39% . the prevalence of overweight and obesity was found similar in boys and girls , or significantly different between genders , with difference found in studies that reported higher prevalence of overweight and obesity in either girls or boys . the level of prevalence in european school children is reported to be as high as 27.7% and 28% for boys and girls of the eastern region , respectively . based on published data in this regards prevalence rates of overweight and obesity for iranian school children is 13.8% . physical factors such as an increase in sedentary lifestyles and the availability and marketing of foods , an increase in the use of computers and television viewing , greater dependence on vehicles for transportation , and decreases in physical activity in schools are considered major determinants of obesity . some international surveys suggest that the proportion of students who did not participate in at least 20 min of regular physical activity on 3 or more of the past 7 days and did not do at least 30 min of moderate physical activity on 5 or more of the past 7 days was 33.4% . the percentage of students who participated in no vigorous or moderate physical activity during the past 7 days was 11.5% , the percentage of students who attended physical education class daily was only 28.4% , the percentage of students who watched 3 or more hours of tv / day on an average school day was 38.2% , the percentage of students who ate five or more servings of fruits and vegetables per day during the past 7 days was 22% , the percentage of students who were at risk for becoming overweight was 15.4% , while the percentage of students who were overweight was 13.5% . to the best of our knowledge , only a few data are available about the effects of school - based physical activity programs on overweight and obese students in the city of isfahan and even in our country , iran . therefore , this study was carried out to determine the effects of a short - term physical activity intervention on obesity and cardiovascular fitness of middle school boy students in the city of isfahan . to examine the efficacy of a selected short - term exercise program for improving aerobic fitness and body composition of children , this semi - practical study has been conducted . a number of 127 middle school boy students , with written consent of their parents or guardians , volunteered to take part in this study and selected as subjects . subjects selected from a different area of the city of isfahan , who based on preliminary assessments during february to may 2011 , have been determined as overweight or obese . for the entry conditions , participants were in good health , free from musculoskeletal dysfunctions , and metabolic and heart diseases . all of the subjects were informed that they could withdraw from the study at any time . participants were in good health , free from musculoskeletal dysfunctions , and metabolic and heart diseases . subjects completed a physical activity and medical history questionnaire as each subject underwent 1-day testing session . during this session , skin folds at three sites ( triceps , sub - scapular , and medial calf ) were obtained using a caliper . the landmarks were identified and measured according to lohman and timothy procedure , and the mean of three measurements was used for representing skinfold thickness . body - fat percentage was determined according to gender - specific equations as following : if triceps and sub - scapular skin folds > 35 mm ; % fat = 0.546 sf + 9.7 females ; and if triceps and sub - scapular < 35 mm ; % fat = 1.33 [ sf ] 0.013 [ sf ] + 2.5 females . standard calibrated scales and stadiometers were used to determine the height , weight , and body mass index ( bmi ) of students . heights were measured by a stadiometer ( novin , iran ) to the nearest 0.5 cm , while body mass were obtained to the nearest 0.2 kg using a calibrated balance beam scale ( novin , iran ) . children 's waist - circumference was measured using a flexible tape at the level of the narrowest point between the lower costal border and the iliac crest , and hip circumference was measured at the widest region . cardiovascular or aerobic fitness of subjects was determined using pacer 20 m shuttle - run test . subjects were required to run back and forth on a 20 m course and be on the 20 m line at the same time a beep is emitted from tape . the frequency of sound - signals increased in such a way that running speed started at 8.5 km / h and was increased by 0.5 km / h each minute . when the subjects could no longer follow the pace , the stage the subjects were able to run for was recorded and used for calculating the aerobic power or vo2 max in children . the selected 6 weeks intervention program consisted of adding two 90 min sessions of physical education courses for selected students in their leisure time . with collaboration of school principals , teachers , school health services and parental committees , our physical activity intervention was programmed to increase participants physical activity during both school hours and their leisure time , for 6 weeks intervention period . in addition , we prepared physical activity and nutritional fact sheets for parents ( including student - parent tasks ) , and hence that they were informed of the program they had to follow in holidays . furthermore , the intervention schools received some exercising equipment ( such as balls , jump ropes , etc . ) to promote physical activity during exercise intervention time period . the physical activity intervention program included 10 min of warm up , performing push - ups , curl - ups , 20 m shuttle - run tests in groups , practicing some basic sport skills like soccer , volleyball , basketball and badminton , playing one of the mentioned sports for 2030 min and finally 5 min cool down activities . the intervention strategies were aimed to increase the total physical activity level of all participants in general and specifically overweight or obese boy students . we used paired t - test to determine differences between results of pre and posttests in measured variables , with p < 0.05 considered as statistically significant . data were analyzed using the spss - pc software ( version 20.0 , ibm spss inc . to examine the efficacy of a selected short - term exercise program for improving aerobic fitness and body composition of children , this semi - practical study has been conducted . a number of 127 middle school boy students , with written consent of their parents or guardians , volunteered to take part in this study and selected as subjects . subjects selected from a different area of the city of isfahan , who based on preliminary assessments during february to may 2011 , have been determined as overweight or obese . for the entry conditions , participants were in good health , free from musculoskeletal dysfunctions , and metabolic and heart diseases . all of the subjects were informed that they could withdraw from the study at any time . participants were in good health , free from musculoskeletal dysfunctions , and metabolic and heart diseases . subjects completed a physical activity and medical history questionnaire as each subject underwent 1-day testing session . during this session , skin folds at three sites ( triceps , sub - scapular , and medial calf ) were obtained using a caliper . the landmarks were identified and measured according to lohman and timothy procedure , and the mean of three measurements was used for representing skinfold thickness . body - fat percentage was determined according to gender - specific equations as following : if triceps and sub - scapular skin folds > 35 mm ; % fat = 0.546 sf + 9.7 females ; and if triceps and sub - scapular < 35 mm ; % fat = 1.33 [ sf ] 0.013 [ sf ] + 2.5 females . standard calibrated scales and stadiometers were used to determine the height , weight , and body mass index ( bmi ) of students . heights were measured by a stadiometer ( novin , iran ) to the nearest 0.5 cm , while body mass were obtained to the nearest 0.2 kg using a calibrated balance beam scale ( novin , iran ) . children 's waist - circumference was measured using a flexible tape at the level of the narrowest point between the lower costal border and the iliac crest , and hip circumference was measured at the widest region . cardiovascular or aerobic fitness of subjects was determined using pacer 20 m shuttle - run test . subjects were required to run back and forth on a 20 m course and be on the 20 m line at the same time a beep is emitted from tape . the frequency of sound - signals increased in such a way that running speed started at 8.5 km / h and was increased by 0.5 km / h each minute . when the subjects could no longer follow the pace , the stage the subjects were able to run for was recorded and used for calculating the aerobic power or vo2 max in children . all intervention subjects participated in a twice - weekly training program . the selected 6 weeks intervention program consisted of adding two 90 min sessions of physical education courses for selected students in their leisure time . with collaboration of school principals , teachers , school health services and parental committees , our physical activity intervention was programmed to increase participants physical activity during both school hours and their leisure time , for 6 weeks intervention period . in addition , we prepared physical activity and nutritional fact sheets for parents ( including student - parent tasks ) , and hence that they were informed of the program they had to follow in holidays . furthermore , the intervention schools received some exercising equipment ( such as balls , jump ropes , etc . ) to promote physical activity during exercise intervention time period . the physical activity intervention program included 10 min of warm up , performing push - ups , curl - ups , 20 m shuttle - run tests in groups , practicing some basic sport skills like soccer , volleyball , basketball and badminton , playing one of the mentioned sports for 2030 min and finally 5 min cool down activities . the intervention strategies were aimed to increase the total physical activity level of all participants in general and specifically overweight or obese boy students . descriptive statistics was run on all the variables . we used paired t - test to determine differences between results of pre and posttests in measured variables , with p < 0.05 considered as statistically significant . data were analyzed using the spss - pc software ( version 20.0 , ibm spss inc . subject characteristics including height , weight , waist - hip ratio ( whr ) , bmi , body - fat percentage and cardiovascular fitness are summarized in table 1 . the results of statistical analysis and comparison of measured variables are shown in table 2 and figures 1 - 3 . this study showed that subjects body - fat percentage changed of 17.84% ( 42.25% pretest vs. 34.71% posttest ) , whr levels changed 0.44%,(0.915 pretest vs. 0.911 posttest ) , vo2 max changed 8.54% ( 27.84 pretest vs. 30.22 posttest ) , while bmi was changed 2.61% ( 26.81 pretest vs. 26.03 posttest ) . results also revealed that there was a greater decrease in fat percent and increase in cardiovascular fitness levels among the intervention participants , and this difference between fat percent , ( p = 0.001 ) and vo2 max of subjects were significant ( p = 0.001 ) . on the other hand , although bmi levels of students decreased from pre and posttests , but this difference did not reach statistical significance ( p = 0.452 ) . descriptive data related to measured variables in girl students ( n=129 ) dependent t - test and comparison of variables in subjects ( n=129 ) comparison of pre posttests cardiovascular fitness of boy students comparison of pre posttests fat percent of boy students comparison of pre posttests body mass index of boy students the interventional strategies in this study were aimed to increase the total physical activity level of all participants in general and specifically obese or overweight boys . our intervention was associated with significant weight loss , reduced bmi , reduced body - fat percentage , and increased cardiovascular fitness , however we believed that duration of intervention was too short and many factors we have to control in this regards . preventive or treatment programs for childhood obesity require a multidisciplinary approach and should include dietary modifications , nutritional education , changes in life style , behavioral modification , and parental involvement . in a school - based intervention that was held in britain and implemented in 10 primary schools with 634 children , it was found that the program resulted in remarkable improvements to achieve aims of the study . furthermore , in an intervention in britain , with the aim of reducing consumption of carbonated drinks to prevent excessive weight gain in children , consumption of carbonated drinks was decreased ( 0.6 glasses with average glass size of 250 ml . ) in the intervention group , whereas it increased in the control group ; which in 12 months resulted in a decrease in the percentage of overweight and obese children in the intervention group , while it increased in the control group . in a study carried out on american - indian students aged 811 years old , the purpose of the study was to evaluate the effectiveness of a school - based multi - component intervention for reducing body - fat percentage in different states , it consisted of four components : classroom curriculum , food service modifications , physical activity and family involvement . it has been reported that , the body - fat percentage levels of subjects decreased in terms of physical activity implementation but , significant differences were not found . in some studies , it has been reported that a multi - component school - based intervention can affect physical activity patterns in adolescents by increasing overall physical activity . effects of this intervention seemed to be more profound in girls than boys , low - active adolescents compared with high - active adolescents , participants with normal weight compared with overweight , and for participants with parents having middle education level as opposed to high and low education levels , respectively . the findings above mentioned researches revealed that implementation of interventional strategies in the school system may have a beneficial effect on public health by increasing overall physical activity among adolescents and possibly among children , and low - active adolescents in particular . however , as we mentioned in some literatures in this regard , comparison studies of different kinds of school - based interventions are necessary to distinguish the most effective strategies to prevent obesity or improvements of health status of children . we believe that more accurate measurement methods of physical activity and multidisciplinary approaches are required to determine the effects of school - based physical activity interventions in children . moreover , it is better to have control groups to modify and control other covariates . finally , it can be said that unfortunately duration of intervention in this study was short due to inadequate facilities and other problems . we believe that more accurate measurement methods of physical activity and multidisciplinary approaches are required to determine the effects of school - based physical activity interventions in children . moreover , it is better to have control groups to modify and control other covariates . finally , it can be said that unfortunately duration of intervention in this study was short due to inadequate facilities and other problems . the data of this study demonstrated that although duration of school - based program of this study was short , but intervention resulted in significant positive effects on body - fat percentage , bmi , whr and aerobic fitness of the participants . our results suggest that aerobic fitness as an indicator of physical activity is linked to decreased level of adiposity in children . according to the results of the present study and the fact that iranian academic curriculum includes only 90 min physical activity per week , initiatives should be put forward to promote physical activity in iranian children in both school and out - of - school environments .

the migration of peripheral circulating monocytes into tissues is considered an important process in the immune response , including inflammatory reactions . during this process , monocytes migrate from the circulation , across the endothelium and the basement membrane , and into the affected tissue . when monocytes enter tissues , they eventually mature into macrophages to carry out their main functions . as reported concerning other leukocytes , the monocyte migratory response occurs through a multistep adhesion mechanism . interactions established between leukocytes and extracellular matrix ( ecm ) components are crucial to the cell migration through the perivascular tissue . lectins may mediate many of the adhesive interactions during leukocyte extravasation and directly influence the migratory response of these cells [ 2 , 3 ] . galectin-3 , a -galactoside - binding lectin belonging to the galectin family , is involved in many processes during the inflammatory response . this lectin is expressed and secreted by various inflammatory cells and induces migration of monocytes / macrophages . the mechanisms by which galectin-3 induces monocyte / macrophage migration are not fully elucidated , and those involving interactions with the ecm have not been addressed to date . galectin-3 is composed of a carboxyl - terminal carbohydrate recognition domain ( crd ) and aminoterminal tandem repeats . upon binding to its glycan ligands at the cell surface , galectin-3 oligomerizes by self - assembly of its n - terminal regulatory domain , a characteristic that makes it suited for a cell adhesion function , including cell - to - cell and cell - to - extracellular matrix protein interactions . indeed , a role in the traversing of neutrophils through the basement membrane at inflammation sites has been suggested for this lectin . although a great deal of effort has led to the characterization of the initial steps of the leukocyte migration process , considerably less is known concerning the movement of leukocytes across the endothelium and through the extravascular tissue . in the present study , we sought to investigate whether interactions established between galectin-3 and ecm glycoproteins might be involved in monocyte migration , given that little is known about the mechanisms by which monocytes move through a complex meshwork of proteins and carbohydrates such as the ecm . we show that monocytes migrate through distinct mechanisms that involve galectin-3 combinations with laminin and fibronectin . this study was approved by the national research ethics committee ( conep ; document number 10461/2006 ) , and a written informed consent was obtained from all the volunteers before their inclusion in the study . hrgal-3 and the carboxyl - terminal domain fragment of gal-3 ( truncated galectin-3 ( tgal-3 ) ) were prepared as described previously . recombinant gal-3 was further purified by affinity chromatography on detoxi - gel beads to eliminate lipopolysaccharide ( lps ) contamination , according to the manufacturer 's recommendations ( pierce chemical co. , usa ) . the lectin purity was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) , and protein concentration was determined using a commercial kit ( bca protein assay kit , pierce ) . biotinylation of hrgal-3 and tgal-3 with sulfo - nhs - lc - biotin ( pierce ) was performed according to the manufacturer 's recommendations . human monocytes ( 1 10 ) were incubated or not with biotinylated - hrgal-3 ( 25 g / ml ) for 1 h at 4c . in some assays , biotinylated - hrgal-3 samples were previously incubated with 10 mm lactose or sucrose ( sigma ) for 16 h at 4c . cells were washed and then incubated , for 45 min at 4c in the dark , with fitc - conjugated streptavidin ( 1 : 1000 ; pierce ) , pe - conjugated mouse anti - human cd14 monoclonal ab ( caltag laboratories , usa ) , or igg isotype control ( 10 g / ml ; caltag laboratories , usa ) . cells were acquired using a facsort system ( becton dickinson immunocytometry systems , usa ) , and data were analyzed using the flowjo v7.2.5 software . monocytes ( 1 10 ) were added into a 24-well culture plate ( corning incorporated costa , usa ) , containing glass slides , where they were maintained for 2 h at 5% co2 and 37c . cells were incubated for 1 h at 4c with biotinylated hrgal-3 ( 25 g / ml ) or in rpmi only . in some assays , biotinylated hrgal-3 was previously incubated with 10 mm lactose or sucrose for 16 h at 4c . cells were washed twice with pbs and incubated , for 40 min at 4c in the dark , with alexa fluor 594-conjugated streptavidin ( molecular probes eugene , usa ) and fitc - conjugated mouse anti - human cd14 monoclonal ab ( caltag laboratories , usa ) or igg isotype control ( 10 g / ml ; caltag laboratories , usa ) . after fifty washes using pbs , cells were fixed with 2% paraformaldehyde at room temperature for 20 min . coverslips were mounted with fluoromount - g , and images were acquired using a leica tcs sp5 confocal microscope . monocyte migration was evaluated in a transwell system ( 6.5 mm - diameter polycarbonate membranes with 5.0 m - diameter pores ; corning ) . cells ( 1 10 ) were added into inserts and chemoattractants into the lower wells . monocyte chemoattractant protein-1 ( mcp-1 ) ( 100 ng / ml ; prepotech , usa ) was used as positive control and rpmi as negative . after 40 min at 37c , nonmigrating monocytes were wiped off the insert , and the migrating cells present on the lower side of the membrane were fixed with 70% methanol and stained with hema3 ( biochemical sciences , usa ) . the number of migrating cells was counted in five fields for each condition , assayed in triplicate . , filters were previously treated with different concentrations of the lectin for 90 min at 37c in a humidified atmosphere . after being washed with pbs , filters were dried and transferred to the transwell system . the basis for migration assays using immobilized molecules were established previously . to evaluate the role of matrix glycoproteins in monocyte- or macrophage - induced migration by galectin-3 , laminin , fibronectin , or vitronectin ( sigma ) previous incubations of hrgal-3 samples with 10 mm lactose ( or sucrose ) for 16 h at 4c were performed to test the involvement of the lectin carbohydrate recognition domain in its attractant activity . a 96-well microplate was coated overnight at 4c with 1 g / well of laminin or fibronectin diluted in 100 l of carbonate buffer . wells were washed three times with pbs with 0.05% tween 20 ( pbs - t ) and 3% gelatin ( w / v ) was added to the wells to block nonspecific binding . the plate was incubated for 2 h at 37c washed and incubated for additional 2 h with biotinylated hrgal-3 or truncated gal-3 ( 25 g / ml ) . in some assays , biotinylated - hrgal-3 was previously incubated with 10 mm lactose or sucrose for 16 h at 4c . the plate was washed three times with pbs - t and incubated with streptavidin - peroxidase ( 1 : 500 ) for 1 h at 37c . after washing three times with pbs - t , 100 l of 3,3,5,5-tetramethylbenzidine ( tmb ) substrate solution ( bd biosciences , usa ) was added to each well for development of peroxidase reaction , and the optical density was measured at 450 nm . data are summarized as the mean sem results were statistically analysed using a paired t - test . to study the binding of galectin-3 to ligands on the surface of human monocytes , cells were incubated with biotinylated lectin and analyzed by flow cytometry and confocal microscopy . we also assayed the inhibition of hrgal-3 binding to the cell surface by the specific sugar , lactose . the flow cytometry results showed that 95.5% of cd14 gated monocytes incubated with hrgal-3 were labeled , and in the presence of lactose , hrgal-3 binding to monocytes was significantly inhibited , as only 17.7% of cells were labeled ( figure 1(a ) ) . on the other hand , sucrose had no significant effect on hrgal-3 binding to monocytes , as 89.6% of cells were labeled in its presence ( figure 1(a ) ) . for confocal microscopy analysis , we labeled monocytes with anti - cd14 antibody stained with fitc , and hrgal-3 stained with alexa594 . as shown in figure 1(b ) , hrgal-3 was evenly distributed on the surface of cd14-labeled monocytes . binding of the lectin was inhibited in the presence of lactose , whereas sucrose had no effect . these results indicate that galectin-3 binds to ligands expressed on the surface of human monocytes and that such an interaction is dependent on gal-3 lectin binding activity . we used transwell systems to test the attractant activity of soluble hrgal-3 ( sgal-3 ) toward human monocytes . soluble hrgal-3 ( 0.0030.9 m ) induced monocyte migration in a dose - dependent manner , yielding a typical bell - shaped dose - response curve . maximum attractant activity was observed at 0.1 m , similar to that induced by mcp-1 , a potent attractant for monocytes used as positive control ( figure 2(a ) ) . to evaluate the ability of galectin-3 to induce monocyte haptotaxis , polycarbonate filters containing different concentrations of immobilized hrgal-3 ( igal-3 ; 0.0010.9 m ) immobilized hrgal-3 , as soluble hrgal-3 , induced monocyte migration in a dose - dependent manner . however , when compared to chemotaxis , a ten - times lower concentration of hrgal-3 ( 0.01 m ) was sufficient to induce the maximum response , similar to that determined by mcp-1 ( figure 2(b ) ) . these results indicate that a substrate of galectin-3 , formed through its immobilization on polycarbonate filters , improves the migratory response of monocytes induced by the lectin . to verify the involvement of the extracellular matrix glycoproteins laminin , fibronectin , or vitronectin in the attractant activity of galectin-3 , we evaluated the monocyte migration in response to hrgal-3 stimulus in the presence of gradients of these molecules . to this end , 0.01 m hrgal-3 was used given that this concentration determined the maximum response of monocytes in our previous assay , as described above . as shown in figure 3(a ) when soluble hrgal-3 was assayed in the presence of immobilized laminin or fibronectin , but not vitronectin , we observed a monocyte migration significantly higher than that obtained in response to soluble hrgal-3 alone ( figure 3(a ) ) . soluble or immobilized laminin , fibronectin , and vitronectin , in the absence of galectin-3 , were not able to induce monocyte migration ( data not shown ) . to investigate if the carbohydrate recognition domain ( crd ) of galectin-3 is involved in the monocyte migration in response to the lectin in combination with laminin or fibronectin , the assays were performed using soluble hrgal-3 previously incubated with sugars . in agreement with the results described above ( figure 3(a ) ) , soluble hrgal-3 induced monocyte migration in the presence of immobilized laminin or fibronectin , showing similar responses in both conditions . lactose significantly inhibited monocyte migration induced by soluble hrgal-3 alone ( not shown ) or in combination with laminin or fibronectin . a nonspecific sugar , sucrose , did not affect the cell migration induced by hrgal-3 in combination with both glycoproteins ( figure 3(b ) ) . these results indicate that monocyte migration induced by galectin-3 is potentiated in the presence of laminin and fibronectin , but not vitronectin . the crd of the lectin is implicated in the monocyte migration induced by galectin-3 in combination with laminin or fibronectin . to investigate if galectin-3 binds to laminin and fibronectin , we performed galectin-3 binding assays to laminin- or fibronectin - coated microplates . both hrgal-3 and its truncated form , which lacks the n - terminal domain , bound to laminin . on the other hand , hrgal-3 did not bind to fibronectin . confirming our previous results , lactose inhibited hrgal-3 binding to laminin , whereas sucrose had no effect on this binding ( figure 4 ) . these results indicate that galectin-3 binds to laminin but not fibronectin , and that such an interaction is dependent on the carbohydrate recognition domain of the lectin . upon leaving the vasculature , if the mechanisms by which galectin-3 induces the migration of monocytes are also involved in the migratory response of macrophages to this lectin , the migration assays were performed using human monocyte - derived macrophages ( figure 5 ) . as observed for monocytes , both soluble and immobilized hrgal-3 induced the migration of macrophages , and the response was higher toward immobilized lectin . when compared to the macrophage migration toward immobilized hrgal-3 alone , the presence of fibronectin potentiated the response induced by the lectin , since it was 60% higher , whereas the association of hrgal-3 with laminin did not . this behavior differed from that observed for monocytes , as their migration was improved when soluble hrgal-3 was combined with both glycoproteins , laminin , or fibronectin . these results show that macrophage migration induced by galectin-3 is potentiated by fibronectin but not by laminin . recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to an attractant gradient . we attempted to gain further insights into the mechanisms involved in monocyte migration and found that interactions between galectin-3 and components of the extracellular matrix are differentially involved in the process . thus , while monocytes migrate in response to associations / combinations of galectin-3 and laminin or fibronectin , monocyte - derived macrophages have a distinct behavior and migrate only in response to galectin-3 combined with fibronectin . monocyte movement , as reported for other leukocytes , is driven by a gradient of an attractant molecule . previous studies have shown that both monocytes and macrophages migrate toward galectin-3 , but the molecular interactions between these leukocytes and the vessel wall or the perivascular tissue , essential to the migratory process , were not investigated . first , we studied the binding of galectin-3 to the surface of monocytes , and we showed that binding of galectin-3 to monocytes is dependent on the carbohydrate recognition domain of the lectin , in agreement with previous work that demonstrated the involvement of the crd in monocyte migration . furthermore , we verified that galectin-3 ligands are evenly distributed on the monocyte surface . by virtue of its potential multivalence , galectin-3 can establish concomitant interactions with glycans on different substrates , such as the extracellular matrix and monocyte surface . therefore , it is conceivable that monocyte migration induced by galectin-3 occurs through haptotaxis , a mechanism in which cell migration is driven by an immobilized gradient of the attractant molecule , which binds to substrates such as the ecm components on the surface of endothelial cells , in the basement membrane , and in the connective tissue [ 9 , 10 ] . the glycoprotein fibronectin is present in the interstitial space and plays a major role in cell adherence to the tissue [ 1214 ] . this molecule is also found in the basement membrane along with laminin , which is the most abundant glycoprotein in this layer [ 15 , 16 ] . in our study , using transwell systems , we showed that monocytes migrated in response to both soluble and immobilized galectin-3 . however , when immobilized , lower concentrations of the lectin determined higher responses . we then analyzed the involvement of extracellular matrix glycoproteins in monocyte migration in response to galectin-3 and observed significantly higher migratory responses in the presence of laminin and fibronectin when compared to those determined by both soluble and immobilized galectin-3 alone . vitronectin , on the other hand , did not affect the monocyte migration toward galectin-3 . migration induced by galectin-3 in combination with laminin or fibronectin was dependent on the lectin crd . these findings suggest that galectin-3 interacts selectively with specific molecules present in the extracellular matrix . as reported previously , the interactions of galectin-3 with extracellular matrix components are involved in cell adhesion . the lectin promotes adhesion of neutrophils to laminin and fibronectin and enhances the adhesion of a tumor cell line to these molecules as well as to vitronectin . although the findings obtained by these groups demonstrate the adhesive role of galectin-3 , they can not be directly extrapolated to cell migration , as after adhesion to the vascular endothelium , cells need to breach this vascular barrier to reach the underlying tissue , and this is achieved after an appropriate activation of intracellular signaling . we showed that although both laminin and fibronectin potentiated the monocyte migration induced by galectin-3 , the lectin was able to bind to laminin but not to fibronectin . galectin-3 laminin associations are dependent on the lectin 's carbohydrate - binding activity , as they were selectively inhibited by lactose . furthermore , the nh2-terminally truncated form of galectin-3 generated by collagenase digestion of the lectin , which retains carbohydrate binding ability , also bound to laminin . therefore , it is plausible that in vivo galectin-3 , by binding to carbohydrate residues of laminin , can form a substrate to drive the transmigration of monocytes across the endothelium and their movement through the extravascular tissue . by cross - linking appropriately glycosylated receptors on opposing surfaces , namely , the monocyte surface and laminin molecules on the ecm , the lectin could bridge monocytes to laminin . on the other hand , a mechanism independent of physical associations between galectin-3 and fibronectin may be involved in monocyte movement along a fibronectin - rich substrate . as previously reported for neutrophils , monocytes may bind to fibronectin as a consequence of a cell activation process induced by galectin-3 . indeed the lectin , upon binding to extracellular domains of transmembrane proteins , can either upregulate their expression or modulate the binding affinity and clustering of these proteins to their extracellular ligands [ 17 , 19 ] . we did not detect any difference in the surface levels of the integrins cd11b and cd11c and of l - selectin in galectin-3-stimulated monocytes . likewise , the levels of icam , vcam , vla-4 , lfa-1 , psgl-1 , and mac-1 mrna transcripts did not increase upon monocyte stimulation with galectin-3 ( data not shown ) . therefore , monocyte migration on a fibronectin substrate is most likely due to the redistribution of adhesion molecules on the cell surface and/or to increased ligand binding affinity or avidity to fibronectin induced by galectin-3 . considering that both laminin and fibronectin are recognized by a number of cell surface receptors of the integrin family [ 3 , 2022 ] and that integrin molecules are included among surface receptors for galectin-3 on different cell types , including monocytes / macrophages [ 23 , 24 ] , these adhesion molecules are potentially involved in monocyte movement induced by galectin-3 along laminin and fibronectin substrates . indeed , galectin-3-induced modulation of integrin clustering / redistribution has been previously demonstrated to regulate cell adhesion or motility on the ecm [ 2527 ] . interestingly , a functional synergistic loop between 1-integrins and galectin-3 has been recently demonstrated . while galectin-3 expression was induced by the introduction of the integrin 1 in ge11 epithelial cells , galectin-3 expression , in turn , enhanced 1 integrin - mediated cell adhesion to fibronectin and laminin , as well as cell migration . on their recruitment to sites of injury , monocytes leave the blood stream and after crossing the vessel wall , reach the extravascular space , and eventually mature into macrophages . in the connective tissue , macrophages contact the extracellular matrix , which modulates their maturation . present in the ecm , fibronectin contributes to the monocyte / macrophage differentiation process and has been described as an important function modulator of macrophages . in the present study , we demonstrated that the migratory response of monocyte - derived macrophages toward galectin-3 differs from monocytes , as macrophage migration is potentiated when the lectin is combined with fibronectin but not laminin . in general , haematopoietic cell differentiation is determined by finely regulated signals acting on gene expression and leading to the achievement of terminally differentiated phenotypes within the proper cell lineages . cellular modifications occur as a consequence of specific leukocyte maturation , and several proteins are differentially expressed on the cell surface . noteworthy is the fact that when monocytes differentiate into macrophages , galectin-3 expressed on the cell surface is upregulated . in addition , monocyte - derived macrophages differentiated into different phenotypes have been shown to exhibit specific patterns of galectin-3 expression . based on the distinct migratory response presented by monocytes and macrophages in our study , we cogitate that the modulation of cell surface molecules might affect how migrating cells perceive and discriminate their extracellular matrix ligands . it is conceivable that galectin-3 highly expressed on the macrophage surface would indirectly mediate their contact with fibronectin , contributing to their interstitial migration . in summary , these data show that monocytes migrate through selective interactions with the extracellular matrix driven by galectin-3 . both ecm glycoproteins , laminin and fibronectin , would be involved in monocyte migration , whereas only fibronectin would be implicated in macrophage migration . we speculate that during inflammatory response , mononuclear phagocytes may migrate towards injured sites through discriminating molecular interactions mediated by different attractants , including now galectin-3 . a better understanding of the mechanisms underlying galectin-3-mediated cell migration and cell matrix interactions is critical to examine the role of the lectin as a potential therapeutic target in inflammatory disorders .

oral cavity cancer involves the lesions occurred in the tongue , lip mucosa and floor of the mouth . one of the standard methods for control of oral cavity tumors is the external radiation therapy in which the linac or cobalt 60 source ( 60co ) is used for irradiation . beside the positive effect of radiation in oral cavity tumors , the radiation may cause damages to the normal tissue such as dental caries , loss of taste , xerostomia , mucositis , trismus , and osteoradionecrosis , with significant impairment of the patient 's quality of life . therefore many techniques are considered to protect the sensitive organs of oral cavity during irradiation . there are few studies about the stents and shields , which can be placed inside the oral cavity . investigated a stent including thermal plastic molding patient jaws and two bars of acrylic resin for keeping the mouth open . because this stent keeps the tongue below the radiation therefore it prevents the tongue from extra dose . this stent makes an extra distance between upper and lower jaws and makes it possible to irradiate the tumor without irradiation of upper jaw . two columns of acrylic resin keep the distances constant during treatment course and improve the reproducibility of the treatment setups . an important restriction of this stent is that it is not applicable to the patients with severe bone resorption with missing teeth . verrone et al . designed a stent from acrylic resin that made a 1.5 cm opening between upper and lower jaws during treatment . in this method , a protective layer from acrylic resin on the lower jaw was used and patient was treated with imrt ( intensity - modulated radiation therapy ) technique . that this work carried out by dosimetric analysis of treatment planning system . the results of this study showed that with the use of this stent the average dose to normal tissue in oral cavity , parotids and salivary glands are reduced compare to the case without the stent . the dryness of the mouth and the ulcers were also reduced with the use of stent . kaanders et al . built a stent from lipowitz alloy with an acrylic resin cover . the lipowitz alloy is a good absorbent of radiation , and the acrylic resin cover reduces the backscatter radiation . they concluded that with this kind of alloy the radiation to normal tissue around the tumor is reduced . in this study , the use of lead suggested , however , the 0.5 cm cover is needed to reduce backscattered radiation , which was high for the lead shield . in this study , for optimization of the material and design of the oral cavity shield many combinations of various materials are investigated . the measurement of backscatter radiation is also performed with film dosimetry , which is very important aspect of any shielding since it might cause severe burn of the normal tissue . the shields which are used in treatment of the cancers in one side of the oral cavity is placed inside the cavity to protect the other side of the cavity , which is normal tissue . the purpose of the design of the shield is to keep the mouth open during treatment and reduce the absorbed radiation on the opposite side of tumor . the type of the radiation is photon beam with relatively low energy in radiation therapy . the shield ( or stent ) could be uniform material or layers of different materials the combined materials may decrease the absorb dose to the opposite side and reduce the backscatter radiation at the same time . to avoid the toxicity of the metals in direct contact with the mouth and tongue metal shields are covered with thin layer of plastic . in this study , a special phantom considering the geometry of the open oral cavity is built . all the details , dimensions and thicknesses of this phantom are designed similar to a real oral cavity of a patient undergoing a lateral radiation field . the phantom is designed to be irradiated form left lateral or right lateral direction the material of the phantom is plexiglass with a density of 1.19 g / cm which is close to water and soft tissue density . inside the phantom , a long cubic piece with dimensions of 4 1 1 cm is attached , which simulates the gum of the patient . the material of this part is teflon ( polytetrafluoroethylene or ptfe ) with a density of 2.12.3 g / cm close to the density of the bone . the shields that are built and used in this study were slabs with dimensions of 1 4 4 cm . the maximum height of the shield should not be uncomfortable for the patient for opening the mouth . the length of the shield should not be too much that causes the nausea in the patient . the thickness of the shield also should be limited to make sure that it does not make a problem in patients breathing . the shields were made of five materials : lead , plexiglass , acrylic resin , silicon and plaster . as it is illustrated in figure 2 , for each material we have three similar layers with 1 cm thickness . each slab has 1 cm thickness and for each material there are three slabs for film dosimetry in this study , gafchromic ebt2 films were used . ebt2 is a radiochromic film which needs no cover and there is also no need for processing of the film . because of these characteristics radiochromic films are very useful for this kind of dosimetry since it is very hard task to use a typical radiographic film in small parts since this kind of film is sensitive to visible light and cutting and covering of these films in small pieces is very sensitive and time consuming . however using radiochromic film one can easily cut the film to small sheets in a room with an ordinary day light . the instantaneous response of the ebt2 films is also very convenient for initial visual evaluation of the film dosimetry results . in this way with a simple visual check of the films one can evaluate the major problems and possible errors in experiment . the analysis of the results of film dosimetry is performed with matlab software . for experiments and irradiation the co-60 beam and 6 mv photon beam of nepton 10 pc linac in seyed - o - shohada hospital , isfahan , iran , these energies are selected according to the realistic cases in a typical oral cavity cancer in radiation therapy . the setup of the radiation for this study is illustrated in figures 3 and 4 . the surface of the phantom is placed in 100 source to surface distance ( ssd ) for . the gantry of the machine field is placed in lateral position according to routine treatment plans . in each time of irradiation , two pieces of the films are placed in two points of the phantom as it is illustrated in figures 3 and 4 . film 1 is placed in the opposite side of the tumor after the shield so it measures the absorbed dose in normal tissue of the mouth . film 2 is placed right before the shield in contact with the shield to evaluate the amount of the backscattering radiation . the film is placed in opposite side of the incident radiation the setup of the film dosimetry and location of film 2 in phantom . the film is in direct contact with the shield the first set of experiments was performed using the shields with uniform material and 3 cm thickness . for each of five materials , the field size was 6 6 cm to cover one side of the phantom which is supposed to be treated . in the second step , the shield in this case has two layers of the lead , and the third layer is one of four other materials such as acrylic , resin , and silicon . there is also a set of experiments for just two layers with the same material such as 2 cm lead . in this setting , the entire shield is thinner than 3 cm . the comparison of absorbed dose in normal tissue after the shield for uniform shield of five material : lead ( l ) , silicon ( s ) , plexi glass ( pg ) , acrylic resin ( ar ) and plaster ( p ) . the energy of photos is 6 mv after the experiment , the irradiation films are scanned . it is recommended that the films are scanned at least 24 h after irradiation for self - development of film emulsion . in this study , then , the films should be scanned with a special scanner ( mikroteck - scanmaker-9800 plus ) which has the capability to scan the films in transitional mode . for dose measurement in film dosimetry it is needed to obtain the calibration curve of the ebt2 films . for calibration of the film each strip with 2 10 cm dimensions was irradiated , which had a known dose and the doses are increased from 5 cgy to 2000 cgy . the dose steps were 10 , 20 , and 100 cgy from low dose to high dose . it should be noted that for calibration of the film with 6 mv photon beams , a 1.5 cm rw3 slab should be placed on the film to provide sufficient build up on the surface of the film . below the film all the films should also be scanned five times before the irradiation to avoid the error due to no - uniform distribution of the background on the film . the average of the background in each point is then considered for obtaining the dose for that point . after obtaining the calibration curve of the films , all the irradiated films in above experiments were converted to absorbed dose using an in - house matlab routine the format of the images was tiff to have a better quality since in jpeg format one loses the details of the image . the resolution of the scanner was set to 300 dot per inch ( dpi ) . it should be noted that higher resolution produces very large images which is then hard to process in matlab . the original image of the film contains three channels of colors , red , green , and blue . each color is related to one channel of the scanner . for better results in all stages of film dosimetry such as calibration and the dosimetry of each point the red channel of the image the setup of the radiation for this study is illustrated in figures 3 and 4 . the surface of the phantom is placed in 100 source to surface distance ( ssd ) for . the gantry of the machine field is placed in lateral position according to routine treatment plans . in each time of irradiation , two pieces of the films are placed in two points of the phantom as it is illustrated in figures 3 and 4 . film 1 is placed in the opposite side of the tumor after the shield so it measures the absorbed dose in normal tissue of the mouth . film 2 is placed right before the shield in contact with the shield to evaluate the amount of the backscattering radiation . the film is placed in opposite side of the incident radiation the setup of the film dosimetry and location of film 2 in phantom . the film is in direct contact with the shield the first set of experiments was performed using the shields with uniform material and 3 cm thickness . for each of five materials , the field size was 6 6 cm to cover one side of the phantom which is supposed to be treated . in the second step , the shield in this case has two layers of the lead , and the third layer is one of four other materials such as acrylic , resin , and silicon . there is also a set of experiments for just two layers with the same material such as 2 cm lead . in this setting , the comparison of absorbed dose in normal tissue after the shield for uniform shield of five material : lead ( l ) , silicon ( s ) , plexi glass ( pg ) , acrylic resin ( ar ) and plaster ( p ) . the energy of photos is 6 mv after the experiment , the irradiation films are scanned . it is recommended that the films are scanned at least 24 h after irradiation for self - development of film emulsion . in this study , then , the films should be scanned with a special scanner ( mikroteck - scanmaker-9800 plus ) which has the capability to scan the films in transitional mode . for dose measurement in film dosimetry it is needed to obtain the calibration curve of the ebt2 films . for calibration of the film each strip with 2 10 cm dimensions was irradiated , which had a known dose and the doses are increased from 5 cgy to 2000 cgy . the dose steps were 10 , 20 , and 100 cgy from low dose to high dose . it should be noted that for calibration of the film with 6 mv photon beams , a 1.5 cm rw3 slab should be placed on the film to provide sufficient build up on the surface of the film . below the film all the films should also be scanned five times before the irradiation to avoid the error due to no - uniform distribution of the background on the film . the average of the background in each point is then considered for obtaining the dose for that point . after obtaining the calibration curve of the films , all the irradiated films in above experiments were converted to absorbed dose using an in - house matlab routine the format of the images was tiff to have a better quality since in jpeg format one loses the details of the image . the resolution of the scanner was set to 300 dot per inch ( dpi ) . it should be noted that higher resolution produces very large images which is then hard to process in matlab . the original image of the film contains three channels of colors , red , green , and blue . each color is related to one channel of the scanner . for better results in all stages of film dosimetry such as calibration and the dosimetry of each point the red channel of the image the results of the absorbed dose in film 1 which is related to the opposite side of the oral cavity and normal tissue is illustrated in figures 5 - 8 . figure 6 is for experiments with two layers of lead and third layer of different materials . figure 7 illustrates the various thickness of lead shield from 1 cm to 3 cm for 6 mv . in all figures , the abbreviation of materials is as follows : lead ( l ) , silicon ( s ) , plexiglas ( pg ) , acrylic resin ( ar ) and plaster ( p ) . the comparison of the dose for shields with two layers of lead and third layer of different materials the absorbed dose in film 1 for various thickness of lead shield from 1 cm to 3 cm for 6 mv the dose in film1 for cobalt 60 and uniform shields with 3 cm thickness as it is expected the maximum absorption of the dose to prevent the dose to the normal tissue on the opposite side is for 3 cm lead shield . for 150 cgy prescribed dose to the tumor side , the amount of the dose in film1 is 21 cgy for 6 mv and 32 cgy in 60co photons . the minimum absorption of the dose was for silicon shield in which the absorbed dose in films 1 was 116 cgy and 147 cgy for two energies . although for both energies three layers of lead provide the best shielding for normal tissue in the opposite side of the oral cavity . the amount of the dose for film 2 was 116 and 147 cgy for 6 mv and 60co respectively . however it also has an effect on the jaw and other tissues around the tumor . as it is illustrated in figure 6 , the absorbed doses in these samples are very close together and the difference between these cases is around 2 cgy . therefore , two layers of lead and one layer of other materials has no major difference in terms of the dose to the opposite side of the oral cavity . one can compare the backscattering of these cases and choose the proper shielding . in figure 7 as it is expected with increasing the thickness of the shield from 1 cm to 3 cm there is a significant change in the dose of the normal tissue . the measured dose in film 2 which is placed right before the shield consists of the primary beam which is constant and backscattering radiation from the shield . the results of the absorbed dose in film 2 are illustrated in figures 9 - 13 for 6 mv and 60co . figures 9 and 11 are for the cases in which all three layers are identical . figures 10 and 12 are for two layers of lead and third layer of different materials . for the lead shield , the absorbed dose is 177 cgy and 219 cgy for 6 mv and 60co respectively which is quite considerable . the minimum amount of the backscattering is related to resin acrylic shield for which the absorbed dose in film 2 is 102 and 118 cgy for two energies . the comparison of absorbed dose in film 2 illustrating the backscattered radiation for various materials and 6 mv photons the backscattered dose for film 2 in two layers of lead and third layers of different material and 6 mv photon beams the comparison of absorbed dose in film 2 illustrating the backscattered radiation for various materials and cobalt 60 photons the absorbed dose in film 2 , backscattered , in combination of one layer and two layer of lead the absorbed dose in film1 and film 2 various composition of shield in cobalt 60 with increasing the atomic number of shielding material ( z ) the backscattering radiation is increase . as it is seen in the above numbers , although for 6 mv the amount of the monitor unit is the same , however the difference between the amount of the absorbed dose of lead and acrylic resin ( 177 and 102 ) is 75 cgy , which is a significant difference . this magnitude of difference for 60co is 101 cgy , which is larger compare to75 cgy for 6 mv photon beams . this is also reasonable since the magnitude of backscattering is reduced as the energy of the photon beam is increased . on the other hand if there in only the primary incident beam one expect to have the same amount of the dose for all cases in film 2 for each energy , since there is only a constant layer of the phantom is in the path of the film , which is assumed to be the tumor . the differences of the doses in the film 2 are only due to difference of backscattering radiation of various materials . according to results of composite layers , figures 10 and 12 , two layers of lead and an acrylic resin together produced the minimum backscattering radiation . as it is illustrated a layer acrylic resin absorbers a considerable amount of the back scattered radiation . the absorbed dose for two layers of lead alone is 147 cgy and it is reduced to about 103 cgy with an additional acrylic resin layer . the measured dose in film 2 which is placed right before the shield consists of the primary beam which is constant and backscattering radiation from the shield . the results of the absorbed dose in film 2 are illustrated in figures 9 - 13 for 6 mv and 60co . figures 9 and 11 are for the cases in which all three layers are identical . figures 10 and 12 are for two layers of lead and third layer of different materials . for the lead shield , the absorbed dose is 177 cgy and 219 cgy for 6 mv and 60co respectively which is quite considerable . the minimum amount of the backscattering is related to resin acrylic shield for which the absorbed dose in film 2 is 102 and 118 cgy for two energies . the comparison of absorbed dose in film 2 illustrating the backscattered radiation for various materials and 6 mv photons the backscattered dose for film 2 in two layers of lead and third layers of different material and 6 mv photon beams the comparison of absorbed dose in film 2 illustrating the backscattered radiation for various materials and cobalt 60 photons the absorbed dose in film 2 , backscattered , in combination of one layer and two layer of lead the absorbed dose in film1 and film 2 various composition of shield in cobalt 60 with increasing the atomic number of shielding material ( z ) the backscattering radiation is increase . as it is seen in the above numbers , although for 6 mv the amount of the monitor unit is the same , however the difference between the amount of the absorbed dose of lead and acrylic resin ( 177 and 102 ) is 75 cgy , which is a significant difference . this magnitude of difference for 60co is 101 cgy , which is larger compare to75 cgy for 6 mv photon beams . this is also reasonable since the magnitude of backscattering is reduced as the energy of the photon beam is increased . on the other hand if there in only the primary incident beam one expect to have the same amount of the dose for all cases in film 2 for each energy , since there is only a constant layer of the phantom is in the path of the film , which is assumed to be the tumor . the differences of the doses in the film 2 are only due to difference of backscattering radiation of various materials . according to results of composite layers , figures 10 and 12 , two layers of lead and an acrylic resin together produced the minimum backscattering radiation . as it is illustrated a layer acrylic resin absorbers a considerable amount of the back scattered radiation . the absorbed dose for two layers of lead alone is 147 cgy and it is reduced to about 103 cgy with an additional acrylic resin layer . in this study , various shields for cancer of oral cavity is designed , and a film dosimetry is performed to evaluate the amount of absorption and backscattering for each shield . the shield is used to keep the mouth open in irradiation of oral cavity and to prevent the dose to the opposite side of the cavity . the typical field for oral cavity cancer is a lateral field with low energy photons . in terms of the protection of the opposite side of the oral cavity however , the magnitude of back scattering is significant and it may cause damage to the normal tissue and lower gum before the shield . the relative amount of the backscattering was larger for low energy photons as it is expected . the amount of the backscattering for 60co increased the dose to almost double of prescribed dose for lead shielding . the silicon and other low z materials have a negligible backscattering , however they can not be used alone to protect the opposite side of the oral cavity . they have to be used as a combined shield with a layer of lead and acrylic resin to absorb the radiation in the opposite side of the oral cavity in lateral fields of radiation therapy . in our study , two layers of lead and one layer of acrylic resin had a good performance to reduce the backscattering radiation and absorb the dose in the normal tissue on the opposite side of oral cavity . keyvan jabbari received b.s . degree in physics from isfahan university of technology , isfahan , iran in 1999 , m.s . degree in medical radiation engineering from amir kabir university , tehran in 2002 , m.s . degree in medical physics from university of manitoba , winnipeg , canada , in 2004 . since then he is assistant professor in department of medical physics and medical engineering , school of medicine , isfahan university of medical sciences . he is also clinical medical physicist in isfahan milad hospital and seyed - o - shohada cancer center . his research interest is fast monte carlo in treatment planning , dose calculation and plan optimization . degree in physics from alzahra university science & research of tehran , iran in 2002 . degree in medical physics from isfahan university of medicalscience , isfahan , iran in 2015 . her research interest is designing and dosimetry of a shield for photon fields of radiation therapy . e - mail : [email protected] mahnaz roayaei received specialist m.d . degree from isfahan university of medical sciences , isfahan , iran in 2004 . she is currently assistant professor in school of medicine , isfahan university of medical sciences . e - mail : [email protected] masoumeh rostampour rostampour received b.sc degree in physics from razi university , kermanshah , iran ( 2010 ) , m.sc degree in medical physics from isfahan university of medical science , isfahan , iran ( 2013 ) . currently she is doing phd in tehran university of medical sciences , tehran , iran .

we present an atypical presentation of this disease with rare pathological finding in the setting of ulcerative colitis ( uc ) . this patient came to our institution as a referral for management of worsening colitis and difficulty passing stool . initial colonoscopic and radiographic studies revealed a large mass in the right ischiorectal fossa that appeared entirely extraluminal without pathological evidence of mucosal dysplasia . concern was raised for a soft tissue sarcoma until percutaneous biopsy of the mass demonstrated a cylindroma - like basaloid carcinoma of the anal region . a 51-year - old caucasian male with 20-year history of uc presents with decreased stool caliber , inability to empty completely , as well as bloody stools . his uc was well controlled until the previous year , at which point he required the addition of oral mesalamine and hydrocortisone suppositories . recent colonoscopy visualized left - sided disease , and biopsies did not reveal any dysplasia . magnetic resonance imaging ( mri ) and computed tomography ( ct ) showed a large soft tissue mass in the right ischiorectal fossa with mass effect on the pelvic diaphragm muscles , prostate and rectum ( fig . 1 ) . however , ct - guided percutaneous biopsy of the mass revealed a cylindroma - like basaloid carcinoma of the anal region . it was at stage ii , t3n0m0 , according to american joint committee on cancer guidelines . he received 5 weeks of radiation ( 54 gy ) , plus chemotherapy with 5-fluorouracil ( 5fu ) and mitomycin c during weeks 1 and 5 of radiation with only a modest response ( 20% ) . given his history of uc , we performed total colectomy and end ileostomy in conjunction with wide abdominoperineal resection of the tumor . we reconstructed his perineum with a rectus abdominis myocutaneous flap from the left abdomen , and the omentum was used to obliterate pelvic space . ( b ) this is a ct image of the same mass after neoadjuvant therapy , marked by dashed line . ( b ) this is a ct image of the same mass after neoadjuvant therapy , marked by dashed line . microscopically , the tumor showed a proliferation of basaloid cells arranged in a trabecular and nested growth pattern . the neoplastic cells had hyperchromatic nuclei with minimal cytoplasm and mild pleomorphism ( fig . there was no evidence of dysplasia in the overlying rectal and anal epithelia . in some areas , the lesion also exhibited a jigsaw puzzle ( mosaic - like ) arrangement of basaloid cells surrounded by bright eosinophilic hyaline basement membrane material , simulating cylindroma of the skin or adenoid cystic carcinoma . by immunohistochemistry , the lesional cells were positive for ck7 and partially positive for p16 ( fig . 3b ) . areas of patchy high - molecular - weight keratin immunoreactivity were also observed . ck20 , monoclonal cea , cd117 , calponin , s-100 and p40 stains were completely negative . finally , all 15 regional lymph nodes were negative for metastatic carcinoma . figure 3:basaloid carcinoma of the anal canal . ( a ) relatively small basaloid neoplastic cells arranging in a trabecular pattern , resembling cylindroma of the skin or adenoid cystic carcinoma . ( a ) relatively small basaloid neoplastic cells arranging in a trabecular pattern , resembling cylindroma of the skin or adenoid cystic carcinoma . he was recently seen at his 6-month postoperative follow - up with no evidence of recurrent disease on imaging , although he reported loss of sexual function and mild urinary incontinence . this includes cancers of the anorectal canal and anal margin . in 2014 , the american cancer society estimates 7210 new cases of anal cancer and 950 deaths . nearly half of all anal cancers are squamous cell carcinomas . about a third are basaloid carcinomas ( otherwise known as cloacogenic or transition cell tumors ) . human papilloma virus ( hpv ) infection is postulated to be a prerequisite for anal cancer development , and markers for hpv infection are found in the vast majority of tumor specimens [ 3 , 4 ] . in the rectum and anal canal , only a few examples of basaloid carcinomas with cylindromatous microcystic spaces or spiradenocylindroma - like pattern have been reported [ 57 ] . the clinical significance and associated pathologic variables of this unusual morphologic pattern in basaloid anal carcinomas are uncertain at this point . notably , kacerovska and colleagues previously detected the presence of hpv-16 in the lesional tissue , thus indicating hpv as a potential causative factor . in our case , the tumor cells were also partially highlighted by p16 immunohistochemistry , a surrogate marker for transcriptionally active hpv . however , strong and diffuse p16 expression can be observed in the absence of hpv e6/e7 mrna expression . therefore , additional molecular confirmation will be necessary in establishing the presence of high - risk hpv in our case . this patient 's tumor was entirely extraluminal , which allowed it to grow to a large size before detection . lower gastrointestinal bleeding , although present at the time of diagnosis , was likely a result of his colitis and unrelated to his tumor . in terms of risk factors , aside from his hpv positivity , inflammatory bowel disease ( ibd ) is also known to be associated with tumorigenesis . the chronic inflammatory state and long - term use of immunosuppressive therapy are thought to be causative factors . although there is no clear evidence to suggest that incidence of anal cancer is higher in the ibd population , an association is likely . in this patient , colectomy was indicated because uc significantly increases his risk for developing colon cancer , and chemoradiation may exacerbate symptoms of colitis . fortunately , his cancer was discovered at an early stage , and because of the case complexity , surgical resection was planned in advance involving surgical oncology , colorectal surgery , urology , orthopedic surgery , and plastic and reconstructive surgery . although he had a modest response to neoadjuvant therapy , a r0 resection was accomplished while preserving his genitourinary and skeletal anatomy . flap reconstruction was planned in advance in anticipation of poor primary tissue healing as a result of radiation and the large defect in his perineum after extirpation . likewise , the colectomy and ileostomy portions of the operation were planned to ensure the integrity of the rectus flap . postoperatively , he had mild perineal pain that had resolved at 3 months postoperatively and sustained some functional deficit of the genitourinary system .

a number of surgical options are available for treating medial unicompartmental osteoarthritis ( oa ) of the knee , such as high tibial osteotomy ( hto ) , unicompartmental knee arthroplasty , and total knee arthroplasty . however , the activities of patients after arthroplasty may be limited and revision surgery is invariably required after 10 or 20 years . accordingly , valgus hto is becoming more popular for relatively active young patients with medial unicompartmental oa and varus deformity1234567 ) . opening wedge technique is becoming more popular because it has some advantages over closing wedge osteotomy . it has been shown that adequate stable fixation is mandatory for safe healing of the osteotomy to minimize the risk of nonunion and loss of correction after medial opening wedge hto8 ) . some special implants have been developed in an attempt to make medial opening wedge hto procedure stable . , naples , fl , usa ) is the most widely used fixation device for the medial opening hto . however , several studies have reported higher complication rates of nonunion , fixation failure , and correction loss , due to unstable fixation in medial opening hto using the puddu plate910 ) . hence , internal fixators such as tomofix plate ( synthes , oberdorf , switzerland ) and aescular plate ( b. braun korea , seoul , korea ) were developed to achieve optimal stability and maintain the attained correction . stoffel et al.11 ) suggested that the tomofix plate would provide superior stability under both compression and torsion compared to the puddu plate in a situation where the lateral cortex is damaged . jung et al.8 ) insisted that loss of correction after opening - wedge hto using the aescular plate was significant compared with the tomofix plate . this study was undertaken to compare the mechanical stability of the three fixation plate systems for opening wedge hto based on the mechanical test results obtained using a porcine tibia model . our hypothesis was that the aescular plate would show no difference compared to other fixation plate systems with respect to mechanical stability . fifty - four tibias from 1-year - old domestic pigs weighting 90 - 100 kg were used . specimens were stored at -70 until required and thawed at room temperature for 24 hrs before experiments . the soft tissues were completely removed , but fibular and tibiofibular joints ( anterior , posterior tibiofibular ligament and interosseous membrane ) were spared . a medial opening wedge osteotomy was performed at 20 mm below the tibial plateau targeting the tibiofibular joint using an oscillating saw . the osteotomy was opened carefully using a small osteotome and an osteotomy wedge was formed gradually so as not to break the lateral cortex of the tibia . after 10 mm opening of the osteotomy site , fixation was carried out on the osteotomy site using either aescular plate ( b. braun korea ) , a puddu plate ( arthrex inc . ) , or a tomofix plate ( synthes ) without a bone graft . in the aescular group , two plate sizes were used , that is , the anterior plate was smaller than the posterior plate to prevent subsequent increases in posterior slope . two 6.5 mm cancellous screws were used for each plate fixation . in the puddu group , two 6.5 mm cannulated screws were used for proximal fixation and two 4.5 mm cortical screws for distal fixation . in the tomofix group , three 4.5 mm locking screws were used for proximal fixation and four 4.5 mm locking screws for distal fixation . the ends of both plates were then molded with bone cement for mechanical testing ( fig . this process was performed using a jig that aligned the long axis of the tibia with the axis of the instron 5569 series unit ( instron , norwood , ma , usa ) ( fig . six of the original 54 specimens were excluded due to lateral cortical breakage during osteotomy or the opening procedure . thus , 48 fresh - frozen specimens were assigned to the three study groups , that is , aescualr group ( 16 specimens ) fixed with aescular plates ; puddu group ( 16 specimens ) with a puddu plate , and tomofix group ( 16 specimens ) with a tomofix plate . axial compression load testing and compression - extension cyclic load testing were both conducted on 8 tibias per group . initially , a preload of 50 n was applied for 3 minutes before axial compression . the width of the ventral and dorsal parts of the osteotomy space was measured by micrometer ( tolerance 0.1 mm ) during this time interval . the point for measurement was marked with a water proof fine liner on the ventral and dorsal tibial edges . axial compression load testing involved a gradual axial compression load test and a maximal compression axial load test . axial displacement was measured by increasing the axial compression force in steps from 200 to 2,000 n ( 200 , 500 , 1,000 , and 2,000 n)12 ) . specimens were then loaded to failure to determine maximal failure loads ; testing was stopped when loads fell by 30% of maximum loads ( fig . the testing machine was operated at a constant speed of 20 mm / min for all tests . compression - extension cyclic load testing involved the cyclic application of compressive force and an extension force from 0 to 2,000 n. extension means distraction of proximal tibia . loading was applied to the tibial shaft for 100 cycles at 1 hz , and the loading condition was from 0 to 2,000 n13 ) . extension length ( mm ) and strain ( % ) were recorded during the last complete test cycle . extension length was recorded as the difference of length between the initial cycle and the last cycle . so , strain was calculated as the extension length divided by the initial length . a compressive load of 2,000 n corresponds to the load exerted on the knee by a person weighing 70 kg during normal walking . analysis of variance was used to compare the three groups with respect to displacement and loads under compression and cyclic loads . spss ver . 17.0 ( spss inc . , chicago , il , usa ) was used to compare results , and statistical significance was accepted for p - values of < 0.05 . fifty - four tibias from 1-year - old domestic pigs weighting 90 - 100 kg were used . specimens were stored at -70 until required and thawed at room temperature for 24 hrs before experiments . the soft tissues were completely removed , but fibular and tibiofibular joints ( anterior , posterior tibiofibular ligament and interosseous membrane ) were spared . a medial opening wedge osteotomy was performed at 20 mm below the tibial plateau targeting the tibiofibular joint using an oscillating saw . the osteotomy was opened carefully using a small osteotome and an osteotomy wedge was formed gradually so as not to break the lateral cortex of the tibia . after 10 mm opening of the osteotomy site , fixation was carried out on the osteotomy site using either aescular plate ( b. braun korea ) , a puddu plate ( arthrex inc . ) , or a tomofix plate ( synthes ) without a bone graft . in the aescular group , two plate sizes were used , that is , the anterior plate was smaller than the posterior plate to prevent subsequent increases in posterior slope . two 6.5 mm cancellous screws were used for each plate fixation . in the puddu group , two 6.5 mm cannulated screws were used for proximal fixation and two 4.5 mm cortical screws for distal fixation . in the tomofix group , three 4.5 mm locking screws were used for proximal fixation and four 4.5 mm locking screws for distal fixation . the ends of both plates were then molded with bone cement for mechanical testing ( fig . this process was performed using a jig that aligned the long axis of the tibia with the axis of the instron 5569 series unit ( instron , norwood , ma , usa ) ( fig . six of the original 54 specimens were excluded due to lateral cortical breakage during osteotomy or the opening procedure . thus , 48 fresh - frozen specimens were assigned to the three study groups , that is , aescualr group ( 16 specimens ) fixed with aescular plates ; puddu group ( 16 specimens ) with a puddu plate , and tomofix group ( 16 specimens ) with a tomofix plate . axial compression load testing and compression - extension cyclic load testing were both conducted on 8 tibias per group . initially , a preload of 50 n was applied for 3 minutes before axial compression . the width of the ventral and dorsal parts of the osteotomy space was measured by micrometer ( tolerance 0.1 mm ) during this time interval . the point for measurement was marked with a water proof fine liner on the ventral and dorsal tibial edges . axial compression load testing involved a gradual axial compression load test and a maximal compression axial load test . axial displacement was measured by increasing the axial compression force in steps from 200 to 2,000 n ( 200 , 500 , 1,000 , and 2,000 n)12 ) . specimens were then loaded to failure to determine maximal failure loads ; testing was stopped when loads fell by 30% of maximum loads ( fig . the testing machine was operated at a constant speed of 20 mm / min for all tests . compression - extension cyclic load testing involved the cyclic application of compressive force and an extension force from 0 to 2,000 n. extension means distraction of proximal tibia . loading was applied to the tibial shaft for 100 cycles at 1 hz , and the loading condition was from 0 to 2,000 n13 ) . extension length ( mm ) and strain ( % ) were recorded during the last complete test cycle . extension length was recorded as the difference of length between the initial cycle and the last cycle . so , strain was calculated as the extension length divided by the initial length . a compressive load of 2,000 n corresponds to the load exerted on the knee by a person weighing 70 kg during normal walking . analysis of variance was used to compare the three groups with respect to displacement and loads under compression and cyclic loads . spss ver . 17.0 ( spss inc . , chicago , il , usa ) was used to compare results , and statistical significance was accepted for p - values of < 0.05 . as axial compressive load was gradually applied , plate displacements increased in all three groups . in the aescular group , the osteotomy model showed a mean displacement of 0.270.16 mm at 200 n , but this increased to 2.701.38 mm at 2,000 n. in the puddu group , the osteotomy model showed a mean displacement of 0.170.14 mm at 200 n , which increased to 2.081.16 mm at 2,000 n. the tomofix group showed a displacement of 0.150.09 mm at 200 n and 1.811.06 mm at 2,000 n. all three groups showed similar increases in displacement with the increase of axial compressive loads ( table 1 ) , but no significant intergroup difference in mean values was observed under different amount of loading conditions ( p>0.05 ) . the maximum load at the time of failure was 6,055.11,184.7 n in the aescular group , 6,798.2998.7 n in the puddu group , and 6,973.8499.7 n in the tomofix group . at the time of failure , the aescular , pudda , and tomofix groups showed a displacement of 7.542.32 mm , 7.312.05 mm , and 6.413.31 mm , respectively ( fig . however , no significant difference was found in the mean maximal loads or displacements at the time of failure among three groups ( p=0.41 , p=0.41 ) . in the aescular , puddu , and tomofix groups , the mean extension under cyclic loading test was 2.910.39 mm , 2.560.38 mm , and 2.020.27 mm , respectively , and corresponding mean strain ( % ) was 9.201.23 , 8.081.20 , and 6.380.86 , respectively ( table 2 ) . the mean extension and strain were slightly smaller in the tomofix group than the other two groups . however , no significant differences were observed among groups in terms of extension ( p=0.21 ) and strain ( p=0.43 ) as axial compressive load was gradually applied , plate displacements increased in all three groups . in the aescular group , the osteotomy model showed a mean displacement of 0.270.16 mm at 200 n , but this increased to 2.701.38 mm at 2,000 n. in the puddu group , the osteotomy model showed a mean displacement of 0.170.14 mm at 200 n , which increased to 2.081.16 mm at 2,000 n. the tomofix group showed a displacement of 0.150.09 mm at 200 n and 1.811.06 mm at 2,000 n. all three groups showed similar increases in displacement with the increase of axial compressive loads ( table 1 ) , but no significant intergroup difference in mean values was observed under different amount of loading conditions ( p>0.05 ) . the maximum load at the time of failure was 6,055.11,184.7 n in the aescular group , 6,798.2998.7 n in the puddu group , and 6,973.8499.7 n in the tomofix group . at the time of failure , the aescular , pudda , and tomofix groups showed a displacement of 7.542.32 mm , 7.312.05 mm , and 6.413.31 mm , respectively ( fig . however , no significant difference was found in the mean maximal loads or displacements at the time of failure among three groups ( p=0.41 , p=0.41 ) . in the aescular , puddu , and tomofix groups , the mean extension under cyclic loading test was 2.910.39 mm , 2.560.38 mm , and 2.020.27 mm , respectively , and corresponding mean strain ( % ) was 9.201.23 , 8.081.20 , and 6.380.86 , respectively ( table 2 ) . the mean extension and strain were slightly smaller in the tomofix group than the other two groups . however , no significant differences were observed among groups in terms of extension ( p=0.21 ) and strain ( p=0.43 ) opening wedge osteotomy has higher complication rates than closing wedge osteotomy with respect to fixation failure , loss of correction , nonunion , and delayed union1014 ) . in particular , the rate of nonunion has been reported to be higher for opening wedge hto than closing wedge hto , ranging between 10% and 35% . furthermore , after medial opening wedge osteotomy , the gap created in the proximal tibia facilitates instability and loss of correction , the latter of which is mainly caused by a lack of primary stability15 ) . al.16 ) reported a 5.4% nonunion rate ( 10 of 182 patients ) for medial opening wedge hto , and concluded that lateral cortex preservation and secure fixation were most important in terms of preventing complications after opening wedge hto . in another study , inadequate fixation stability of opening wedge plates was found to be a reason for opening wedge hto failure17 ) . of the implants specifically designed for medial opening wedge hto this plate is fixed to the medial side of the tibial plateau , and a spacer is inserted into the osteotomy gap , thus preventing gap closure . however , some authors have reported high failure rates of this implant and technique in hto due to unstable fixation1118 ) . for these reasons , a new implant with a long , rigid titanium plate ( tomofix ) with the curvature of the medial tibial metaphysis was designed for opening wedge hto . stoffel et al.11 ) examined the construct stiffness and fixation strengths of the puddu and tomofix plates , and found that both were able to maintain correction at a load of more than 2,500 n , which concurs with our results . this finding suggests that both plates can provide sufficient stability to maintain an axial compressive load in the knee of an adult in a single limb stance or during walking19 ) . however , tomofix plates were found to provide better stability in both compression and torsion than puddu plates when the lateral cortex was fractured . in the present study , mean displacements of 2.08 and 2.13 mm were observed for puddu and tomofix plates , respectively , at a compressive load of 2,000 n , indicating no significant difference between the plates . however , tomofix plates showed better stability during cyclic load testing ( 2.5 mm vs. 2.0 mm displacement for puddu and tomofix plates , respectively ) . although tomofix plates provide sufficient initial stability after opening wedge osteotomy , they are too large if a small incision is required and the posterior tibial slope is not easily controlled . to resolve these problems , an opening wedge plate system consisting of two plates ( aescular plate ; b. braun korea ) was developed . this system consists of anterior and posterior plates ; the posterior plate is placed as posteriorly as possible at the corner of the proximal tibia , and the anterior plate is placed behind the tibial tuberosity . thus , surgeons can control the tibial slope and prevent an unintended increase in posterior tibial slope . however , the fixation stability of this fixation device has not been fully established . in this study , primary fixation stability of this plate the amount of displacement of the aescular plate during axial compression load testing was not significantly different from the other two plates . 6,973.8499.8 n for the aescular , puddu , and tomofix plates , respectively , which were also not significantly different . all three plates were able to maintain correction at loads of greater than 2,500 n , which approximates to the axial compression load in the knee of an adult during walking19 ) . during cyclic load testing using 100 cycles of a 2,000 n compressive force , the mean displacement of the aescular system was comparable to that of the puddu system ( 2.91 mm vs. 2.56 mm ) . however , the tomofix system exhibited greater stability ( 2.02 mm ) than the aescular and puddu systems , although the intergroup differences in the mean displacement were not significant and less than 1 mm during the cyclic load test . these results showed that the primary stability of three systems were similar each other under cyclic loading . first of all , we fully acknowledge that the porcine bone could have different mechanical properties to a human bone even though we performed a bone quality test using quantitative computed tomography . second , the osteotomy site of hto is broad ; therefore , there can be a little difference of displacement in the medial and lateral osteotomy sites . we are aware that we did not consider the difference of displacement in the medial and lateral osteotomy sites so that the result of difference in medial and lateral side displacement was not taken into consideration . third , the study was aimed at evaluating the stability of the plates during walking ; however , we used instron instead of the femur for the upper loading , and were unable to accurately simulate the walking condition . forth , this study only dealt with an axial load without any other load components . based on the comparison of stability of three types of fixation plate systems , the aescular , puddu , and tomofix plate systems were found to provide fixation sufficient stability to bear axial compression and cyclic loads during walking .

transplantation of mobilized peripheral blood stem cells ( pbscs ) is used to facilitate hematologic recovery after high - dose chemotherapy.1,2 although several methods for mobilization of pbscs have been described,35 leukapheresis is a commonly used collection procedure . pbsc yield is enhanced if collection is performed at the time of recovery from chemotherapy , and while patients are receiving treatment with granulocyte colony - stimulating factors ( g - csfs).5,6 recently , biosimilar g - csf products have been introduced to the market . some trials have revealed their noninferiority for treatment of neutropenia during chemotherapy , compared with the originator.710 the us food and drug administration describes biosimilars as highly similar to a previously approved biological product , known as the biological reference product , and as having been shown to have no clinically meaningful differences from the reference product . furthermore , published data from prospective randomized and retrospective studies on the use of biosimilar filgrastim for pbsc mobilization in an autologous setting suggest a substantially similar efficacy and safety in comparison with the originator.1121 however , in asia , particularly in japan , there is currently a lack of data for pbsc mobilization by biosimilar g - csf . therefore , evaluation in a japanese context is needed to assess the clinical efficacy and safety of bio - similar g - csf for hematopoietic stem cell transplantation , including autologous stem cell transplantation ( asct ) . filgrastim bs - nk ( fbnk ) ( nippon kayaku co. , ltd . , tokyo , japan ) is a genetically modified biosimilar g - csf drug and is the same as tevagrastim ( xm02).11 the production and sale of fbnk was approved in japan in february , 2013 . in this study , we evaluated the efficacy of fbnk compared with the originators for peripheral blood cell mobilization and transplantation . 1 ) biosimilar group : from july 2014 to october 2015 , 14 consecutive patients affected by malignant lymphoma ( ml ) and multiple myeloma ( mm ) were scheduled for collection of pbscs for asct , after induction chemotherapy . 2 ) originator group : 57 consecutive patients who underwent the same procedure between december 2006 and july 2013 were enrolled . the originator g - csf was filgrastim for 34 patients and lenograstim for 23 patients . the study protocol was approved by institutional review board in kansai medical university and written informed consent was obtained from each patient . for mobilization of pbscs , all patients received chemotherapy priming consisting of various regimens ( table 1 ) . pbscs from patients with ml were either harvested after cyclophosphamide and g - csf treatment , or following a cycle of chemotherapy including rituximab , ifosfamide , etoposide , or cytarabin . all patients were treated with chemotherapy followed by the administration of fbnk , filgrastim , or lenograstim ( 5 g / kg ) . leukapheresis was performed with a cell separator ( cobe spectra , gam - bro k.k . , the procedure was commenced when the white blood cell count recovered to 110/l , and continued until the number of harvested mononuclear cells exceeded 310/kg body weight . the apheresis product was enriched for mononuclear cells , cryopreserved in 6% hydroxyethyl starch , 5% dimethylsulfoxide , and 4% human albumin , and then stored at 80c . a sample of each leukapheresis product was analyzed according to the method in our previous reports.22,23 all samples were washed to remove platelets and were stained with an anti - cd34 monoclonal antibody ( qbend-10 ; immunotech , marseille , france ) . after staining , the erythrocytes were lysed with ammonium chloride . flow cytometric analysis was performed using an epics xlmcl ( beckman coulter , brea , ca , usa ) . it is very difficult to predict the quantity of cd34 cells collected from patients or donors , because the collection efficiency of cd34 cells differs between individuals . therefore , we counted the numbers of cd34 + cells used , with 65,500 cells being analyzed per sample , according to previous reports.24,25 after gating for forward and right scatter , a second gate was drawn around the cd34 lower right scatter population to determine the percentage of hematopoietic progenitors corrected for the control . the target dose of pbscs to collect was > 210 cd34 cells / kg for patients affected by ml , and > 410 cd34 cells / kg for patients affected by mm . 1 ) biosimilar group : from july 2014 to october 2015 , 14 consecutive patients affected by malignant lymphoma ( ml ) and multiple myeloma ( mm ) were scheduled for collection of pbscs for asct , after induction chemotherapy . 2 ) originator group : 57 consecutive patients who underwent the same procedure between december 2006 and july 2013 were enrolled . the originator g - csf was filgrastim for 34 patients and lenograstim for 23 patients . the study protocol was approved by institutional review board in kansai medical university and written informed consent was obtained from each patient . for mobilization of pbscs , all patients received chemotherapy priming consisting of various regimens ( table 1 ) . pbscs from patients with ml were either harvested after cyclophosphamide and g - csf treatment , or following a cycle of chemotherapy including rituximab , ifosfamide , etoposide , or cytarabin . all patients were treated with chemotherapy followed by the administration of fbnk , filgrastim , or lenograstim ( 5 g / kg ) . leukapheresis was performed with a cell separator ( cobe spectra , gam - bro k.k . , the procedure was commenced when the white blood cell count recovered to 110/l , and continued until the number of harvested mononuclear cells exceeded 310/kg body weight . the apheresis product was enriched for mononuclear cells , cryopreserved in 6% hydroxyethyl starch , 5% dimethylsulfoxide , and 4% human albumin , and then stored at 80c . a sample of each leukapheresis product was analyzed according to the method in our previous reports.22,23 all samples were washed to remove platelets and were stained with an anti - cd34 monoclonal antibody ( qbend-10 ; immunotech , marseille , france ) . after staining , the erythrocytes were lysed with ammonium chloride . flow cytometric analysis was performed using an epics xlmcl ( beckman coulter , brea , ca , usa ) . it is very difficult to predict the quantity of cd34 cells collected from patients or donors , because the collection efficiency of cd34 cells differs between individuals . therefore , we counted the numbers of cd34 + cells used , with 65,500 cells being analyzed per sample , according to previous reports.24,25 after gating for forward and right scatter , a second gate was drawn around the cd34 lower right scatter population to determine the percentage of hematopoietic progenitors corrected for the control . the target dose of pbscs to collect was > 210 cd34 cells / kg for patients affected by ml , and > 410 cd34 cells / kg for patients affected by mm . mononuclear cells ( 210 ) were cultured in triplicate in semisolid medium ( 0.8% methylcellulose in medium ) supplemented with 10 ng / ml recombinant g - csf , 10 ng / ml recombinant human granulocyte / macrophage - csf , 100 iu / ml recombinant human interleukin-3 , 2 u / ml recombinant human erythropoietin , and 10% placental conditioned medium as a source of csf . culture plates were incubated for 14 days at 37c in a humidified atmosphere ( 5% co2 in air ) and granulocyte / macrophage colony forming units ( cfu - gm ) were counted under an inverted microscope . the choice of conditioning regimen was based on the patient s diagnosis ( table 1 ) . on day 0 administration of recombinant human g - csf ( biosimilar or originator ) was started on day 5 after the pbsc infusion , at a daily dose of 5 g / kg administered subcutaneously , except for some patients for whom support therapy was anticipated because of their indications . to evaluate the impact of g - csf on the asct , we calculated the median times to leucocyte engraftment , defined as an absolute neutrophil count of > 0.510/l on the first 3 consecutive days , and to platelet recovery , defined as a platelet count of > 2010/l for 2 consecutive days without platelet transfusions . ml and mm patients were evaluated both as separate cohorts , and combined into a single cohort . welch s t - test was used for the comparison of data for each case before and after g - csf treatment . all analyses were performed using the statflex program , version 6 ( artec inc . , the choice of conditioning regimen was based on the patient s diagnosis ( table 1 ) . on day 0 administration of recombinant human g - csf ( biosimilar or originator ) was started on day 5 after the pbsc infusion , at a daily dose of 5 g / kg administered subcutaneously , except for some patients for whom support therapy was anticipated because of their indications . to evaluate the impact of g - csf on the asct , we calculated the median times to leucocyte engraftment , defined as an absolute neutrophil count of > 0.510/l on the first 3 consecutive days , and to platelet recovery , defined as a platelet count of > 2010/l for 2 consecutive days without platelet transfusions . ml and mm patients were evaluated both as separate cohorts , and combined into a single cohort . welch s t - test was used for the comparison of data for each case before and after g - csf treatment . all analyses were performed using the statflex program , version 6 ( artec inc . , the numbers of all leukocytes , neutrophils , and monocytes peaked on day 5 after g - csf treatment . different parameters to characterize the yield and composition of the grafted cells are displayed in figure 2 as dot plots . the upper three graphs show the total cd34 cell numbers , the cd34 cells / kg and the cfu - gm scores for all patients . the lower graphs show the cd34 cells / kg and the cfu - gm scores for ml versus mm patients . there were no significant differences in cd34 cell numbers and cfu - gm scores among the fbnk , filgrastim , and lenograstim treatments , not only for the combined patient cohort , but also for ml and mm patients evaluated separately . in addition , there were no significant differences in the frequencies of side effects induced by the three g - csf drugs ( table 2 ) . the numbers of all leukocytes , neutrophils , and monocytes peaked on days 810 after transplantation . figure 4 shows the times to engraftment for the combined , ml , and mm patient cohorts . there were no significant differences in the time to engraftment among the fbnk , filgrastim , and lenograstim treatments , not only for the combined patient cohort , but also for ml and mm patients evaluated separately . in addition , there were no significant differences in complications after asct among the three drugs ( table 2 ) . g - csf has become widely used to treat therapy - induced neutropenia , for mobilization of pbscs , and to accelerate engraftment after hematopoietic stem cell transplantation.26,27 in addition , several forms of biosimilar nonglycosylated recombinant human g - csf have recently been clinically developed . these have been approved by the european medicines agency.2831 in japan , applications to approve the production of biosimilars are progressed according to guidelines for efficacy and security notified by the ministry of health , labour , and welfare . fsk0808 is a domestic product , and the others , tkn732 and ep2006 , are imported from foreign countries . the fbnk used in the present study corresponds to tkn732 , an imported filgrastim biosimilar product sold in europe as xm02 . therefore , the safety data of 541 overseas phase iii randomized controlled trials can be extrapolated to this fbnk product.2831 there are several publications regarding the use of bio - similar g - csf for pbsc mobilization.1121,32 however , the world marrow donor association does not recommend the use of biosimilars for stem cell mobilization in healthy adult donors , because the long - term safety is considered poor.33 in the present study , there were no significant differences in cd34 cell numbers and cfu - gm scores among fbnk , filgrastim , and lenograstim treatments for the total patient , ml and mm patient groups . similarly , there were no significant differences in the time to engraftment after asct among the fbnk , filgrastim , and lenograstim treatments . lefrre et al12 reported that no significant difference was found between the biosimilar g - csf zarzio and neupogen for stem cell mobilization in 40 patients , compared with an historical control . andreola et al11 also showed successful mobilization and engraftment using plerixafor and xm02 in a study of 14 patients . furthermore , publicover et al14 and bassi et al19 reported the same results using other biosimilars . the chief physician should monitor drug dosage and patient response , and strict safety monitoring of pharmaceutical products should be performed after marketing.28 previous phase iii randomized controlled trials conducted internationally in many facilities reported that the adverse effects and tolerability were similar between the originator and g - csf biosimilars.2830 in the present study , there were no significant differences in the frequencies of side effects such as bone pain and fever among the three g - csf drugs ( fbnk , filgrastim , and lenograstim ) , and there were no significant differences in complications after asct . a study of the effect on medical expenses reported that biosimilar g - csf is advantageous in this regard in europe.11 these results appear to be compatible with japan . however , an original evaluation is necessary , because the japanese medical system is different from the european medical system . the use of biosimilars may offer significant cost savings and help reduce health care costs . therefore , further observation will be very important to establish the clinical use of fbnk . in comparison with the originator g - csfs , biosimilar fbnk shows the same efficacy and safety for facilitating bone marrow recovery in japanese patients with ml and mm who undergo stem cell transplantation . in addition , it reduced the patients hospitalization costs because it is less expensive than the originators .

surfactant proteins ( sps ) , originally known as human lung surfactants , are essential to proper respiratory structure and function . they are unique in composition , consisting of about 90% lipids , mostly phospholipids , and 810% surfactant - associated proteins . sps line the alveolar surface , and reduce surface tension at the air fluid interface . pulmonary surfactant is stored mainly in type ii alveolar epithelial cells in the form of densely packed bilayers called lamellar bodies that are secreted and efficiently transferred to the interface . there are 4 subtypes , sp - a , sp - b , sp - c and sp - d , the characteristics of which are summarized in table 1table 1 . characteristics of sp - a , sp - b , sp - c and sp - d ; the table contents were obtained from the literature and modified for use here . there are many reports regarding the role of sps in lung inflammation or chronic obstructive pulmonary disease ( copd ) . however , the expression of sp - a , sp - b , sp - c or sp d in lung tumors has received only limited attention . previously , in order to establish an appropriate bioassay for detection of promoting potential for lung tumor development after intratracheal instillation ( it ) of fine particles in rats , sequential histopathological changes up to 30 weeks were examined after initiation of lung tumorigenesis with 0.1% n - bis ( 2-hydroxypropyl ) nitrosamine ( dhpn ) in drinking water for 2 weeks in male f344 rats . it has been reported that activating mutations of the kras gene at codon 12 are detected in almost 50% of rat lung neoplastic lesions induced by a carcinogen . furthermore , we previously examined the lung toxicity of fine particles of various materials in an in vivo bioassay using an it approach . in humans , coal miners and building construction workers who are exposed to quartz dust demonstrate obstructive and restrictive loss of lung capacity , as well as copd this is associated with an inflammatory cell responses characterized by alveolitis with recruitment of inflammatory cells , particularly neutrophils , and may result in pulmonary fibrosis and impaired lung function . it of quartz into rats produces inflammatory reactions followed by histological changes characteristic of lung fibrosis , similar to human conditions . with a dose of 2 mg / rat it , fine particles of quartz caused toxicity with severe inflammatory changes characterized by neutrophil infiltration and edema after 28 days . in the present study , the expressions of sp - a , sp - b , sp - c and sp - d in lung neoplastic lesions induced by dhpn and in inflammatory lesions caused by quartz were examined and compared immunohistochemically . experimental animals were maintained in the division of animal experiments , life science research center , kagawa university , according to the institutional regulations for animal experiments . all were housed in polycarbonate cages with white wood chips for bedding and given free access to drinking water and a basal diet , ce-2 ( clea japan inc . , tokyo , japan ) , under controlled conditions of humidity ( 60 10% ) , lighting ( 12-h light / dark cycle ) and temperature ( 24 2c ) . formalin fixed paraffin embedded ( ffpe ) lung samples featuring inflammation were obtained with the rat quartz instillation model on day 28 and ffpe lung samples including neoplastic lesions , hyperplasias and adenomas , were obtained with the rat dhpn - induced lung carcinogenesis model . briefly , in the rat quartz instillation model , male 10-week old f344/ducrlcrj rats ( charles river laboratories japan , inc . , kanagawa , japan ) were exposed by it to quartz with a particle diameter of less than 7 m ( dq-12 , dmt gmbh & co. kg , germany ) at a dose of 2 mg / rat suspended in saline ( 0.2 ml ) ( isotonic sodium chloride solution , otsuka pharmaceutical factory , inc . , tokushima , japan ) on day 0 and sacrificed on day 28 . for the rat dhpn induced lung carcinogenesis model , kyoto , japan ) in drinking water for 2 weeks and sacrificed in week 30 . at autopsy in both experiments , the lungs were removed , including the trachea and heart , infused through the trachea with 10% phosphate buffered formalin , rinsed in the same fixative and then immersed in the fixative for approximately 48 h. slices were then routinely processed for embedding in paraffin for histopathological examination . lung lesions from the rat dhpn - induced lung carcinogenesis model were categorized as bronchioloalveolar hyperplasia ( hyperplasia ) and bronchioloalveolar adenoma ( adenoma ) in accordance with the established criteria given in the international harmonization of nomenclature and diagnostic criteria ( inhand ) . in detail , bronchioloalveolar hyperplasia was diagnosed from the finding of solitary or multiple , segmental ( cone - shaped ) foci of increased cellularity , a lack of strongly convex or spherical borders , the bronchioloalveolar architecture still being detectable and epithelial cells , usually a single layer , still being dominant and the cause of hypercellularity . bronchioloalveolar adenomas were frequently located at the lung periphery and usually small in size ( less than 34 mm in diameter ) , featuring well - circumscribed areas of high epithelial cell density , usually with a strongly convex border , an underlying alveolar architecture obscured to various degrees , sharp demarcation from the surrounding tissue and relatively uniform neoplastic epithelial cells with mitotic figures rare or absent . small foci of mild atypia were sometimes present , and occasionally extension into adjacent bronchioles was evident . for sp - a , sp - b and sp - c , all immunohistochemical staining processes from deparaffinization to counterstaining with hematoxylin were performed automatically using the ventana discovery staining system ( ventana medical systems , tucson , az , usa ) . for sp - d , immunostaining was performed by hand with an envision kit ( dako , tokyo , japan ) . d02647 , ventana medical systems , tucson , az , usa ) for sp - a and sp - c , cc1 solution ( lot . d06855 , ventana medical systems , tucson , az , usa ) for sp - b and target retrieval solution ( dako , tokyo , japan ) for sp - d . the details of the sp - a , sp - b , sp - c and sp d antibodies used in the experiments are summarized in table 2table 2 . the expressions for each marker , sp - a , sp - b , sp - c and sp - d , were evaluated as none ( ) , weak ( + ) and strong ( + + ) compared with the histopathological normal area without inflammation or a proliferative lesion in the same specimens . experimental animals were maintained in the division of animal experiments , life science research center , kagawa university , according to the institutional regulations for animal experiments . all were housed in polycarbonate cages with white wood chips for bedding and given free access to drinking water and a basal diet , ce-2 ( clea japan inc . , tokyo , japan ) , under controlled conditions of humidity ( 60 10% ) , lighting ( 12-h light / dark cycle ) and temperature ( 24 2c ) . formalin fixed paraffin embedded ( ffpe ) lung samples featuring inflammation were obtained with the rat quartz instillation model on day 28 and ffpe lung samples including neoplastic lesions , hyperplasias and adenomas , were obtained with the rat dhpn - induced lung carcinogenesis model . briefly , in the rat quartz instillation model , male 10-week old f344/ducrlcrj rats ( charles river laboratories japan , inc . , kanagawa , japan ) were exposed by it to quartz with a particle diameter of less than 7 m ( dq-12 , dmt gmbh & co. kg , germany ) at a dose of 2 mg / rat suspended in saline ( 0.2 ml ) ( isotonic sodium chloride solution , otsuka pharmaceutical factory , inc . , tokushima , japan ) on day 0 and sacrificed on day 28 . for the rat dhpn induced lung carcinogenesis model , kyoto , japan ) in drinking water for 2 weeks and sacrificed in week 30 . at autopsy in both experiments , the lungs were removed , including the trachea and heart , infused through the trachea with 10% phosphate buffered formalin , rinsed in the same fixative and then immersed in the fixative for approximately 48 h. slices were then routinely processed for embedding in paraffin for histopathological examination . lung lesions from the rat dhpn - induced lung carcinogenesis model were categorized as bronchioloalveolar hyperplasia ( hyperplasia ) and bronchioloalveolar adenoma ( adenoma ) in accordance with the established criteria given in the international harmonization of nomenclature and diagnostic criteria ( inhand ) . in detail , bronchioloalveolar hyperplasia was diagnosed from the finding of solitary or multiple , segmental ( cone - shaped ) foci of increased cellularity , a lack of strongly convex or spherical borders , the bronchioloalveolar architecture still being detectable and epithelial cells , usually a single layer , still being dominant and the cause of hypercellularity . bronchioloalveolar adenomas were frequently located at the lung periphery and usually small in size ( less than 34 mm in diameter ) , featuring well - circumscribed areas of high epithelial cell density , usually with a strongly convex border , an underlying alveolar architecture obscured to various degrees , sharp demarcation from the surrounding tissue and relatively uniform neoplastic epithelial cells with mitotic figures rare or absent . small foci of mild atypia were sometimes present , and occasionally extension into adjacent bronchioles was evident . for sp - a , sp - b and sp - c , all immunohistochemical staining processes from deparaffinization to counterstaining with hematoxylin were performed automatically using the ventana discovery staining system ( ventana medical systems , tucson , az , usa ) . for sp - d , immunostaining was performed by hand with an envision kit ( dako , tokyo , japan ) . d02647 , ventana medical systems , tucson , az , usa ) for sp - a and sp - c , cc1 solution ( lot . d06855 , ventana medical systems , tucson , az , usa ) for sp - b and target retrieval solution ( dako , tokyo , japan ) for sp - d . the details of the sp - a , sp - b , sp - c and sp d antibodies used in the experiments are summarized in table 2table 2 . the expressions for each marker , sp - a , sp - b , sp - c and sp - d , were evaluated as none ( ) , weak ( + ) and strong ( + + ) compared with the histopathological normal area without inflammation or a proliferative lesion in the same specimens . inflammatory lesions were observed in the lungs with the rat quartz instillation models on day 28 . the main findings were neutrophil infiltration in the walls and spaces of the alveoli , pulmonary edema , pulmonary fibrosis , alveolar macrophage infiltration and restructuring of the alveolar walls and granulation - like changes with giant cells and macrophages in the alveoli . for evaluation of the expressions of the sps , the histopathological normal area of the lungs treated with quartz was used as a control ( see figure 1-d , e , f , i and jfig . histopathological and immunohistochemical findings for inflammatory lesions induced by quartz in the f344 rat lung ( 200 ) . b and e , sp - a ; c and f , sp - b ; g and i , sp - c ; h and j , sp - d . a , b , c , g and h , inflammatory lesions ; d , e , f , i and j , noninflammatory lesions ( control ) . 1-b , strongly positive staining of alveolar epithelial cells and bronchial epithelial cells in fig . 1-c , alveolar epithelial cells in fig . the lesions were observed to be strongly positive for sp - a in the mucus in alveoli ( see figure 1-b ) , weakly positive for sp - b in the mucus and strongly positive for sp - b in the alveolar epithelial cells and bronchial epithelial cells ( see figure 1-c ) , weakly positive for sp - c in the mucus and strongly positive for sp - c in the alveolar epithelial cells ( see figure 1-g ) , and strongly positive for sp - d in the mucus and partially positive for sp - d in the alveolar epithelial cells ( see figure 1-h ) . the bronchiolar epithelial cells after treatment with quartz were strongly positive ( + + ) for sp - b and weakly positive ( + ) for sp - a , sp - c and sp - d ( see figure 2fig . 2 . immunohistochemical findings for bronchiolar epithelial cells in the f344 rat lung after quartz exposure ( 200 ) . a , weak positivity ( + ) for sp - a ; b , strong positivity ( + + ) for sp - b ; c , weak positivity ( + ) for sp - c ; d , negative for sp - d . ) . dhpn - induced proliferative lesions , hyperplasias and adenomas , in f344 rat lungs were weakly positive ( + ) for sp - a ( see figure 3-bfig . 3 . histopathological and immunohistochemical findings for dhpn - induced proliferative lesions , hyperplasias and adenomas , in the f344 rat lung ( 200 ) . a and d , h.e . ; b and e , sp - a ; c and f , sp - b ; g and i , sp - c ; h and j , sp - d . a , b , c , g and h , proliferative lesions ; d , e , f , i and j , nonproliferative lesions ( control ) . note the weak positivity ( + ) for sp - a in fig . 1-b , strong positivity ( + + ) for sp - b in fig . 1-c , strong positivity ( + + ) for sp - d in fig . 1-g and almost negative ( ) staining for sp - d in fig . 1-h . ) , strongly ( + + ) positive for sp - b ( see figure 3-c ) , strongly positive ( + + ) for sp - d ( see figure 3-g ) , and almost negative ( ) for sp - d , although the normal alveolar epithelium was also positive ( see figure 3-h ) . for evaluation of the expressions the sps , the histopathological normal area of the lungs treated with dhpn was used as a control ( see figure 3-d , e , f , i and j ) . the findings for the expressions in the control areas were similar ( table 3table 3 . summary of the expressions of sp - a , sp - b , sp - c and sp - d ) . histopathological and immunohistochemical findings for inflammatory lesions induced by quartz in the f344 rat lung ( 200 ) . a and d , h.e . ; b and e , sp - a ; c and f , sp - b ; g and i , sp - c ; h and j , sp - d . a , b , c , g and h , inflammatory lesions ; d , e , f , i and j , noninflammatory lesions ( control ) . note the strong positive reactions in the mucus in the alveoli in fig . 1-b , strongly positive staining of alveolar epithelial cells and bronchial epithelial cells in fig . immunohistochemical findings for bronchiolar epithelial cells in the f344 rat lung after quartz exposure ( 200 ) . a , weak positivity ( + ) for sp - a ; b , strong positivity ( + + ) for sp - b ; c , weak positivity ( + ) for sp - c ; d , negative for sp - d . histopathological and immunohistochemical findings for dhpn - induced proliferative lesions , hyperplasias and adenomas , in the f344 rat lung ( 200 ) . a and d , h.e . ; b and e , sp - a ; c and f , sp - b ; g and i , sp - c ; h and j , sp - d . a , b , c , g and h , proliferative lesions ; d , e , f , i and j , nonproliferative lesions ( control ) . note the weak positivity ( + ) for sp - a in fig . 1-b , strong positivity ( + + ) for sp - b in fig . the expressions of sp - a and sp - d were strongly positive in the mucus in the alveoli with inflammatory changes . sp - b and sp - c showed strong positivity in the proliferative lesions , hyperplasias and adenomas , whereas the expressions of sp - a and sp - d were weak and lacking , respectively . in the present experiment , the expressions of sp - a and sp - d appeared to be similar , with strong positivity in the mucus in alveoli of inflammatory lesions induced by quartz . sp - a and sp - d both play significant roles in surfactant homeostasis and pulmonary immunity and are known to bind to a range of microbial pathogens that invade the lungs and target them for phagocytic clearance by resident alveolar macrophages . they are also involved in the removal of apoptotic and necrotic cells and subsequent resolution of pulmonary inflammationin vivo studies have shown that sp - a regulates responses involved in initiation and potentiation of inflammation by regulating the production of proinflammatory cytokines , such as tumor necrosis factor ( tnf- ) , in response to lipopolysaccharide ( lps ) or by accelerating the clearance of a variety of pathogens . alveolar macrophages are thought to play a critical role in host defense in the lung . in vitro , both sp - a and sp - d can stimulate alveolar macrophages to generate oxygen radicals , as measured by chemiluminescence . as hydrophilic surfactant proteins , they participate in the innate immune response by binding to bacterial and viral pathogens and activating alveolar macrophages . the strong expression of sp - a and sp - d in the present experiment suggests activation of macrophages to deal with instilled quartz particles . expression of sp - a was weak in the proliferative lesions in the present experiment , and this was noteworthy because sp - a has been used as a serous marker for human lung cancer in clinical examinations in japan . lung cancer is the leading cause of cancer - related deaths worldwide in both developing and developed regions . in one study , sp - a some studies have suggested that sp - a might have a role in suppressing lung cancer progression , and sp - a expression by tumor cells leads to recruitment and activation of nk cells and tumor - associated macrophages . in another study , activating mutations of the kras gene at codon 12 were reported in 47% of lesions induced by dhpn in the f344 rat . overexpression of oncogenic kras leads to activation of the nf-b pathway , which appears to be required for the development of tumors in a mouse model of lung adenocarcinoma . sp - a has been shown to inhibit lps - induced nf-b , tnf- production , inducible no synthase protein expression or activity and , in the presence of diverse stimuli , nadph oxidase in immunocompetent cells . in the present experiment , only hyperplasias and adenomas , not adenocarcinomas , were observed as neoplastic lesions , and this suggests that kras mutations and activation of nf-b might be associated with later stages of malignancy . sp - c , which is a specific marker of type ii epithelial cells in the lung , showed the strong positivity in proliferative lesions in the present experiment . sp - b and sp - c have physicochemical properties that reduce the surface tension of biological interfaces . in the lung tumorigenesis model induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and benzo[a]pyrene ( bap ) in a / j mice , pulmonary sp - a , sp - b , and sp - c were identified in the mouse lung , but only the sp - c levels increased in carcinogen - treated versus untreated mice . in the present experiment , sp - b was also positive in neoplastic lesions . the main function of sp - b is to accelerate the formation of a surface - active film composed of phospholipids at the air - water interface by increasing the adsorption rate . sp - b has anti - inflammatory properties and may be involved in protecting the lung against oxidative stress and circulating pro sp - b is reported to be a biomarker for early detection of non small - cell lung cancer ( nsclc ) . on synthesis , pro - sp - b quickly undergoes proteolytic cleavage by cysteine proteases in the endoplasmic reticulum , resulting in the production and secretion of a 9-kd noncollagenous hydrophobic lung tumor cells , especially adenocarcinomas , have dysregulated sp - b synthesis , leading to the overexpression of pro - sp - b with a muted ability to posttranslationally modify the precursor into the mature hydrophobic form . as described in the introduction page , sps are stored in the lung type ii epithelial cells , and in the present experiment , the lung proliferative lesions , hyperplasias and adenomas , were also positive for sps . this result suggests the lung hyperplasias and adenomas would have their origin in type ii alveolar cells . in our previous experiment , napsin a was determined to be a good marker for detection of hyperplastic lesions linked to actual neoplasia . napsin a is an aspartic proteinase involved in the maturation of sp - b and is expressed in the cytoplasm of type ii pneumocytes and clara cells in the lung and also in the proximal tubular renal epithelium and exocrine pancreas . human clinical studies suggest that napsin a may be a highly specific marker for adenocarcinoma in the lung . however , when there is a need to rule out lung metastasis from other organs , for example , from renal , thyroid or endometrial carcinomas , implementation of other biologically specific markers should be considered . in the present experiment , the positive area of sp - b seemed not to correspond completely with that of napsin a. in conclusion , sp - b and sp - c showed strong expression in lung hyperplasias and adenomas and expression of sp - a and sp - d was observed in mucus or exudates in inflammatory alveoli . these results suggest the possibility that sp - b and sp - c are linked to lung tumorigenesis .

sh is defined as a serum tsh concentration below the reference range , with normal free t4 ( ft4 ) and free t3 ( ft3 ) levels . it can be divided into two groups ; patients with a mildly reduced tsh ( 0.10.4 mu / liter ) can be classified as having grade i sh , whereas those with a serum tsh of less than 0.1 mu / liter , including a completely suppressed tsh level ( < 0.01 mu / liter ) are classified as having grade ii sh ( 1 ) . the prevalence of sh has been reported to range from 0.63 to 2.1% in epidemiological studies ( 24 ) . most people with sh have no symptoms of hyperthyroidism , and relatively few develop specific complications attributable to sh : atrial fibrillation or osteoporosis ( 5 ) . prognostically , many people with sh have only a transient abnormality of thyroid function , with resolution of biochemical abnormalities found in 2575% of those with grade i sh on repeat testing ( 6 , 7 ) . thus , the majority of individuals with grade i sh do not have intrinsic thyroid disease , and the low tsh reflects nonthyroidal illness or drug effects . nevertheless , approximately 12% of patients over the age of 60 with sh progress to overt hyperthyroidism , with progression most likely in those with grade ii sh . this reflects the indolent nature of multinodular goiter as the dominant cause in this age group , with mild thyroid autonomy developing slowly . a small number of individuals with sh have early graves ' disease , and progression to overt hyperthyroidism is more probable in these cases ( 7 ) . conversely , 12% of elderly individuals have a persistent and stably low serum tsh that remains unexplained by any primary thyroid pathology . thus , these alterations in thyroid function may reflect either altered physiology or pathology associated with advanced age . the study of the physiological changes in the thyroid axis during aging is complicated by the presence of confounders such as medication , chronic illness , and increased risk of thyroid disease with age . nevertheless , many observational studies in healthy older individuals , which took into account common confounders , have revealed an age - dependent decline in serum tsh and ft3 and an age - dependent increase in rt3 with maintenance of stable serum ft4 levels ( 8 , 9 ) . a further study demonstrated that thyroid function was well preserved until the eighth decade of life , with a decreased level of serum ft3 only observed in centenarians ( 10 ) . however , the upper limit of serum tsh has also been shown to increase with age ( 11 ) , leading to a wider spread of the tsh distribution with age , expanding both above and below the reference intervals for younger individuals . the mechanisms responsible for this age - related decline in serum tsh have not been fully examined . one hypothesis suggests an age - related reduction in the secretion of pituitary tsh and/or hypothalamic trh , due to an increased pituitary thyrotrope sensitivity to peripheral t3/t4 negative feedback ( 12 ) . interestingly , the pattern of tsh pulsatility is also changed in the healthy elderly , with a blunted nocturnal tsh peak being observed , followed by a 1- to 1.5-h backward shift in the circadian rhythm of tsh secretion , leading to an earlier peak ( 13 , 14 ) . equally compelling , a primary defect in thyroid hormone inactivation and disposal might explain the observation of unchanged serum t4 levels despite reduced tropic drive from lower pituitary tsh secretion . therefore , reduced t4 and t3 degradation and clearance may lead to reduced throughput of hormone and a lower tone for the axis ( 15 ) . the population prevalence of dementia is about 7% , and this rises to more than 30% in those aged 85 yr and above ( 16 , 17 ) . alzheimer 's disease ( ad ) , frontal temporal lobe dementia , and lewy body dementia are caused by primary degeneration of the brain , comprising about 70% of cases of primary dementia ( 18 ) . the cholinergic hypothesis of ad pathogenesis is well accepted , with characteristic depletion of acetylcholine and presynaptic cholinergic markers in ad cortex and hippocampus ( 20 , 21 ) . studies using single photon emission computed tomography or magnetic resonance spectroscopy have found similar characteristics in ad and in cognitively impaired patients with hyperthyroidism ( 2224 ) . correspondingly , reduced levels of choline - related compounds in the brain have been demonstrated in hyperthyroidism , with a gradual normalization after antithyroid medication ( 22 ) . paradoxically , t4 administration led to an enhancement of rats ' learning ability , along with increased cholinergic activity in the frontal cortex and hippocampus ( 25 ) . interestingly , trh has also been shown to exert strong stimulant action on the cholinergic pathways of the cortex and hippocampus ( 26 ) . intraseptal injection and intracerebroventricular administration of trh increased the turnover of acetylcholine in rat hippocampus and parietal cortex , respectively ( 27 , 28 ) . hence , reduced trh secretion could contribute to acetylcholine depletion , which is associated with the cognitive changes in ad . the amyloid plaque is the pathological hallmark of ad , and the potential pathways of amyloid - mediated neurotoxicity include inhibition of acetylcholine activity in the cortex and hippocampus , oxidative stress from free radical damage , mitochondrial dysfunction , and ultimately apoptotic cell death ( 2933 ) . at the same time experimental and clinical studies have demonstrated increased reactive oxygen species and lipid peroxidases in the hyperthyroid state , resulting in diminishing antioxidative enzymes ( 34 , 35 ) . furthermore , t3 has been shown to affect splicing of certain -amyloid precursor protein isoforms , which are preferentially expressed in the ad brain ( 36 ) . therefore , thyroid hormones could also have a role in modulating the intracellular and extracellular contents of -amyloid precursor protein isoforms and directly influence the pathogenesis of ad ( 36 , 37 ) . we performed a systematic search through the pubmed , embase ( 1996 to 2012 wk 4 ) , cochrane library , and medline ( 1996 to january wk 4 , 2012 ) electronic databases . the key search terms were subclinical hyperthyroidism , subclinical thyroid disorders or dysfunction , cognitive decline or * impairment , dementia , and alzheimer 's disease . we included all cross - sectional or longitudinal studies published or translated into the english language . meta - analysis has not been performed in light of the heterogeneity in study design , statistical analysis methods , and outcome measures among the studies . we performed a systematic search through the pubmed , embase ( 1996 to 2012 wk 4 ) , cochrane library , and medline ( 1996 to january wk 4 , 2012 ) electronic databases . the key search terms were subclinical hyperthyroidism , subclinical thyroid disorders or dysfunction , cognitive decline or * impairment , dementia , and alzheimer 's disease . we included all cross - sectional or longitudinal studies published or translated into the english language . meta - analysis has not been performed in light of the heterogeneity in study design , statistical analysis methods , and outcome measures among the studies . eighty - four studies were identified that investigated the relationship between thyroid dysfunction or serum thyroid parameters and cognitive impairment . we excluded investigations that involved participants with overt thyroid disorders and only included published papers that examined the relationship between sh , or variations of thyroid function within reference intervals , with cognitive function . twenty - three studies published from 1996 to january 2012 , evaluating a total of 31,482 patients , fulfilled the inclusion criteria . fifteen studies indicated the correlation between thyroid function and dementia as the primary study objective . eight studies included other entities such as cardiovascular or osteoporosis risk or imaging changes as the primary outcome , with the correlation of thyroid function and cognition as a secondary objective . of the 23 studies , 13 were case - control or cross - sectional single - phase trials , and the remaining 10 studies had a prospective longitudinal population - based design . to date , there have been no randomized interventional studies examining the effects of antithyroid treatment on cognition in sh . six studies made a categorical analysis , either of sh vs. euthyroidism or of quantiles of serum tsh and thyroid hormone concentration in relation to cognitive function ( table 1 ) . the sao paulo ageing and health study , a large cross - sectional study comprising 1119 community - dwelling participants aged 65 yr and over , found an association between sh and dementia after multivariate adjustment ( 38 ) . the effect was strongest for vascular dementia [ odds ratio ( or ) for all types of dementia , 4.9 ; 95% confidence interval ( ci ) , 1.515.7 ; vascular dementia or , 5.8 ; 95% ci , 1.433.1 ] . this was confirmed using an analysis of quintiles of tsh ; those with the lowest serum tsh quintile showed more than a 3-fold increase in risk for all types of dementia [ age - adjusted or for dementia , 3.6 ( 95% ci , 1.48.9 ) ; vascular dementia , 9.3 ( 95% ci , 1.175.5 ) ] . similarly , the inchianti study , a population - based study of 916 italians aged 65 yr and older , demonstrated a significantly lower mini - mental state examination ( mmse ) score among those with sh compared with the euthyroid group ( 22.61 6.88 vs. 24.72 4.52 ; p < 0.03 ) ( 39 ) . furthermore , sh was associated with more than a 2-fold risk of scoring less than 24 out of 30 on the mmse ; a standard threshold for significant dementia [ hazard ratio ( hr ) = 2.26 ( 95% ci , 1.323.91 ) ; p = 0.003 ] . these two large studies were well designed with more than a 90% participant response rate , and both carefully excluded patients with overt thyroid dysfunction as well as those taking thyroid medication . ( 41 ) ] , involving 469 and 119 participants , respectively , supported these findings . van osch et al . ( 40 ) investigated a cognitively intact group and an ad cohort aged greater than 52 yr . interestingly , the euthyroid participants with a tsh level in the lowest tertile ( tsh , 0.51.3 mu / liter ) had more than a 2-fold increase in ad prevalence compared with those with serum tsh in the highest tertile ( 2.16.0 mu / liter ) [ or , 2.04 ( 95% ci , 1.183.53 ) ; p = 0.01 ] . ( 41 ) demonstrated a significantly lower tsh level in participants with both ad and vascular dementia ( p < 0.01 ) compared with controls without cognitive impairment . cross - sectional and case control studies on the relationship between sh and cognitive decline ( 19962011 ) ns , nonsignificant ; mmse , mini - mental state examination ; cerad , consortium to establish a registry for alzheimer 's disease ; bmi , body mass index ; dm , diabetes mellitus ; cvd , cardiovascular disease ; ra , rheumatoid arthritis ; pvd , peripheral vascular disease ; ldl , low - density lipoprotein ; hdl , high - density lipoprotein ; tft , thyroid function test ; apoe4 , apolipoprotein e4 ; bp , blood pressure ; pd , parkinson 's disease ; mi , myocardial infarction . in contrast , the largest cross - sectional population - based study , involving 5868 participants , showed no association between sh and cognition ( 42 ) . however , this study invited participants by mail , and the response rate was poor at 38% . although the responders were representative of the regional population with respect to demographic characteristics , the average mmse score was above 27 in all subgroups , suggesting that the responder population was skewed toward a cognitively intact group . another cross - sectional observational study , involving 829 consecutive unselected referrals to a hospital geriatric clinic , did not demonstrate any difference in tsh level between the group with ad and the cohort without dementia ( 43 ) . however , comorbidities and medication use were not described , so the results may be confounded by nonthyroidal illness , thyroid medication , or overt thyroid dysfunction . an additional seven studies performed multivariate analyses to explore the relationship between cognition and thyroid function . 44 ) were the first to suggest an association between poorer cognitive performance and lower serum tsh concentrations within the reference range . a significant positive correlation was found between a lower episodic memory score and serum tsh concentration ( p < 0.01 ) in 200 healthy , euthyroid , community - dwelling participants aged over 75 yr . in keeping with these findings , stuerenburg ( 45 ) demonstrated an inverse correlation ( p = 0.04 ) between serum ft4 level and mmse score in 227 patients aged 4991 yr with mild to moderate ad . in contrast , the remaining five studies of this design failed to demonstrate any association between cognition and low serum tsh or high serum ft4 level ( 4650 ) . in a cross - sectional study involving 1219 community - dwelling participants , global cognitive impairment was not increased in those with sh ( n = 34 ) ( 46 ) . in a hospital - based study involving 409 euthyroid patients aged 5294 yr diagnosed with probable ad , two smaller studies involving 120 healthy volunteers and 69 hospital - based participants , respectively , showed similar findings ( 48 , 49 ) . paradoxically , a study of 44 healthy , community - dwelling male volunteers with a mean age of 72 yr observed a significant association between higher total t4 ( tt4 ) levels and better cognition [ verbal performance in wechsler adult intelligence scale ( p < 0.05 ) and global cognitive performance ( p < 0.01 ) ] but failed to reproduce this trend with tt3 and ft4 ( 50 ) . it is worth noting that almost all of these studies have relatively small sample sizes , that each made a single measurement of thyroid function , and they used different cognitive performance tests , which limits the generalizability of these results . six studies performed categorical analyses , either of sh vs. euthyroidism or quantiles of serum thyroid hormone concentration in relation to cognitive function ( table 2 ) . the rotterdam study was the first longitudinal study to examine the relationship between sh and dementia in 1893 population - based subjects ( mean age , 69 yr ) ( 51 ) . over a 2-yr follow - up , a 3-fold increased risk of dementia from all causes [ relative risk ( rr ) , 3.5 ; 95% ci , 1.210 ] and ad ( rr , 3.5 ; 95% ci , 1.111 ) was found among those with a low baseline tsh level ( < 0.4 mu / liter ) . this study was limited by the small number of the sh group with dementia ( n = 25 ) and a short follow - up period . nevertheless , the positive findings in this study are supported by two well - conducted , large population - based studies with a longer follow - up period . the first study , a community - based observational study , was carried out over a period of 12 yr by tan et al . ( 52 ) in 1864 patients who were free of dementia for 3 yr at recruitment . the patients were then divided into three tertiles according to baseline tsh : t1 , less than 1.0 mu / liter ; t2 , 1.02.1 mu / liter ; and t3 , more than 2.1 mu / liter . a positive correlation in women with a serum tsh level in the lowest ( t1 ) tertile ( hr , 2.26 ; 95% ci , 1.363.77 ; p = 0.002 ) and the highest ( t3 ) tertile ( hr , 1.84 ; 95% ci , 1.103.08 ; p = 0.003 ) with ad risk was found . this relationship was not found in men , but other indices of thyroid function were not studied . however , the two aforementioned large prospective studies involved only a single measurement of thyroid function . this limitation has recently been addressed by the largest observational study involving 12,115 participants ( 2,004 with sh ; 10,111 euthyroid individuals ) with a mean age of 66.5 yr and a median follow - up period of 5.6 yr ( 53 ) . this well - designed , population - based study had a carefully selected sh cohort , including only individuals with two confirmatory measurements of serum tsh at least 4 months apart . patients whose tsh normalized or who developed overt thyroid disease during the course of follow- up were excluded . a positive association between sh and dementia was observed ( adjusted hr , 1.64 ; 95% ci , 1.202.25 ) . interestingly , when patients were divided into two groups according to the tsh level ( 0.10.4 vs. < 0.1 mu / liter ) , no relationship could be established between suppressed tsh ( < 0.1 mu / liter ) and dementia , but a significant association remained for the group with tsh at 0.10.4 mu / liter . longitudinal populational - based studies on the relationship between sh and cognitive decline ( 19962011 ) gms - a , geriatric mental state schedule ; camdex , cambridge examination for disorders of the elderly ; bmi , body mass index ; apoe4 , apolipoprotein e4 ; dm , diabetes mellitus ; pvd , peripheral vascular disease ; rai , radioiodine therapy ; tft , thyroid function test . in addition to these three large population - based studies , de jong et al . ( 54 ) carried out a smaller prospective study among 615 japanese - american men in the honolulu heart program . the cohort had a mean age of 77.5 yr , and the mean duration of follow - up was 5 yr . this study observed a 20 and 30% increased risk for dementia and ad , respectively , for each sd increase in serum ft4 ( hr for dementia , 1.21 ; 95% ci , 1.041.40 ; and hr for ad , 1.31 ; 95% ci , 1.141.51 ) . in addition , neuropathological examinations from the autopsy program instituted as part of this study demonstrated a higher neocortical neurofibrillary tangle count per sd increase in tt4 ( 0.25 ; 95% ci , 0.050.46 ) . in contrast with the studies discussed so far , the rotterdam scan study , another prospective community - based study involving 1025 subjects aged 6090 yr with a mean follow - up interval of 5.5 yr , showed no association between dementia and serum tsh or thyroid hormone levels ( 55 ) . nevertheless , they found a positive association between a higher ft4 and rt3 and greater atrophy at the hippocampus and amygdala on magnetic resonance imaging ( mri ) , in keeping with the finding in the honolulu aging study . although the rotterdam scan study had greater power than the original rotterdam study , due to a larger - sized dementia cohort ( n = 60 vs. 25 ) and a longer follow - up period , it is limited by single measurements of thyroid function and a small number of dementia cases with low tsh ( n = 7 ) . finally , a community - based study involving 464 euthyroid women aged 65 yr or older found an association between lower ft4 concentrations within the reference range and cognitive impairment over a 3-yr period ( rr , 1.97 ; 95% ci , 1.103.5 ) ( 56 ) however , the same pattern was not exhibited in those with a frankly low tsh or an elevated ft4 level . there was a 14% dropout rate during follow - up , largely due to missing mmse scores , which might have introduced a significant bias . four studies made multivariate analyses using thyroid function parameters or cognitive function ( mmse ) as continuous variables . ( 57 ) conducted a large prospective study involving 1047 community - dwelling patients aged 6494 yr with mmse scores of at least 25 in the united kingdom and wales . higher serum ft4 concentrations within the reference range were associated with lower baseline mmse scores and accelerated cognitive decline after 2 yr [ or , 1.13 ( 95% ci , 1.031.23 ) ; p = 0.006 ] . prospective follow - up of 558 individuals aged 85 living in leiden , the netherlands , demonstrated an association between higher serum tsh and better memory function ( , 0.13 ; p = 0.03 ) ( 58 ) . nevertheless , this study had a small sample size and a 41.5% loss of follow - up due to death or refusal to continue participation . after the cross - sectional study in 1998 ( 44 ) , wahlin et al . ( 59 ) conducted a follow - up survey on the same cohort and found an association between low serum tsh levels within the reference range and decreased verbal memory or episodic recall deficits at the 6-yr follow - up ( , 0.290 ; p < 0.05 ) . ( 60 ) demonstrated that for each unit decrease in serum tsh , the or of developing dementia increased by 3.5 . twenty - three studies that met our criteria have examined the association between sh and cognition . fourteen of these studies , including several well - designed and well - powered cross - sectional and longitudinal analyses , have shown a consistent finding of an association of sh , or low serum tsh within the reference interval , with cognitive impairment or dementia . in particular , this association was seen in more than three fourths of the prospective longitudinal studies , providing reliable evidence from robust studies . several of the studies that did not confirm this result are potentially compromised by small samples sizes , recruitment from hospital environments , or the challenging nature of working with participants with cognitive problems ( e.g. failure to return questionnaires ) . eighty - four studies were identified that investigated the relationship between thyroid dysfunction or serum thyroid parameters and cognitive impairment . we excluded investigations that involved participants with overt thyroid disorders and only included published papers that examined the relationship between sh , or variations of thyroid function within reference intervals , with cognitive function . twenty - three studies published from 1996 to january 2012 , evaluating a total of 31,482 patients , fulfilled the inclusion criteria . fifteen studies indicated the correlation between thyroid function and dementia as the primary study objective . eight studies included other entities such as cardiovascular or osteoporosis risk or imaging changes as the primary outcome , with the correlation of thyroid function and cognition as a secondary objective . of the 23 studies , 13 were case - control or cross - sectional single - phase trials , and the remaining 10 studies had a prospective longitudinal population - based design . to date , there have been no randomized interventional studies examining the effects of antithyroid treatment on cognition in sh . six studies made a categorical analysis , either of sh vs. euthyroidism or of quantiles of serum tsh and thyroid hormone concentration in relation to cognitive function ( table 1 ) . the sao paulo ageing and health study , a large cross - sectional study comprising 1119 community - dwelling participants aged 65 yr and over , found an association between sh and dementia after multivariate adjustment ( 38 ) . the effect was strongest for vascular dementia [ odds ratio ( or ) for all types of dementia , 4.9 ; 95% confidence interval ( ci ) , 1.515.7 ; vascular dementia or , 5.8 ; 95% ci , 1.433.1 ] . this was confirmed using an analysis of quintiles of tsh ; those with the lowest serum tsh quintile showed more than a 3-fold increase in risk for all types of dementia [ age - adjusted or for dementia , 3.6 ( 95% ci , 1.48.9 ) ; vascular dementia , 9.3 ( 95% ci , 1.175.5 ) ] . similarly , the inchianti study , a population - based study of 916 italians aged 65 yr and older , demonstrated a significantly lower mini - mental state examination ( mmse ) score among those with sh compared with the euthyroid group ( 22.61 6.88 vs. 24.72 4.52 ; p < 0.03 ) ( 39 ) . furthermore , sh was associated with more than a 2-fold risk of scoring less than 24 out of 30 on the mmse ; a standard threshold for significant dementia [ hazard ratio ( hr ) = 2.26 ( 95% ci , 1.323.91 ) ; p = 0.003 ] . these two large studies were well designed with more than a 90% participant response rate , and both carefully excluded patients with overt thyroid dysfunction as well as those taking thyroid medication . ( 41 ) ] , involving 469 and 119 participants , respectively , supported these findings . van osch et al . ( 40 ) investigated a cognitively intact group and an ad cohort aged greater than 52 yr . interestingly , the euthyroid participants with a tsh level in the lowest tertile ( tsh , 0.51.3 mu / liter ) had more than a 2-fold increase in ad prevalence compared with those with serum tsh in the highest tertile ( 2.16.0 mu / liter ) [ or , 2.04 ( 95% ci , 1.183.53 ) ; p = 0.01 ] . similarly , dobert et al . ( 41 ) demonstrated a significantly lower tsh level in participants with both ad and vascular dementia ( p < 0.01 ) compared with controls without cognitive impairment . cross - sectional and case control studies on the relationship between sh and cognitive decline ( 19962011 ) ns , nonsignificant ; mmse , mini - mental state examination ; cerad , consortium to establish a registry for alzheimer 's disease ; bmi , body mass index ; dm , diabetes mellitus ; cvd , cardiovascular disease ; ra , rheumatoid arthritis ; pvd , peripheral vascular disease ; ldl , low - density lipoprotein ; hdl , high - density lipoprotein ; tft , thyroid function test ; apoe4 , apolipoprotein e4 ; bp , blood pressure ; pd , parkinson 's disease ; mi , myocardial infarction . in contrast , the largest cross - sectional population - based study , involving 5868 participants , showed no association between sh and cognition ( 42 ) . however , this study invited participants by mail , and the response rate was poor at 38% . although the responders were representative of the regional population with respect to demographic characteristics , the average mmse score was above 27 in all subgroups , suggesting that the responder population was skewed toward a cognitively intact group . another cross - sectional observational study , involving 829 consecutive unselected referrals to a hospital geriatric clinic , did not demonstrate any difference in tsh level between the group with ad and the cohort without dementia ( 43 ) . however , comorbidities and medication use were not described , so the results may be confounded by nonthyroidal illness , thyroid medication , or overt thyroid dysfunction . an additional seven studies performed multivariate analyses to explore the relationship between cognition and thyroid function . wahlin et al . ( 44 ) were the first to suggest an association between poorer cognitive performance and lower serum tsh concentrations within the reference range . a significant positive correlation was found between a lower episodic memory score and serum tsh concentration ( p < 0.01 ) in 200 healthy , euthyroid , community - dwelling participants aged over 75 yr . in keeping with these findings , stuerenburg et al . ( 45 ) demonstrated an inverse correlation ( p = 0.04 ) between serum ft4 level and mmse score in 227 patients aged 4991 yr with mild to moderate ad . in contrast , the remaining five studies of this design failed to demonstrate any association between cognition and low serum tsh or high serum ft4 level ( 4650 ) . in a cross - sectional study involving 1219 community - dwelling participants , global cognitive impairment was not increased in those with sh ( n = 34 ) ( 46 ) . in a hospital - based study involving 409 euthyroid patients aged 5294 yr diagnosed with probable ad , no association was found between serum tsh or ft4 and dementia ( 47 ) . two smaller studies involving 120 healthy volunteers and 69 hospital - based participants , respectively , showed similar findings ( 48 , 49 ) . paradoxically , a study of 44 healthy , community - dwelling male volunteers with a mean age of 72 yr observed a significant association between higher total t4 ( tt4 ) levels and better cognition [ verbal performance in wechsler adult intelligence scale ( p < 0.05 ) and global cognitive performance ( p < 0.01 ) ] but failed to reproduce this trend with tt3 and ft4 ( 50 ) . it is worth noting that almost all of these studies have relatively small sample sizes , that each made a single measurement of thyroid function , and they used different cognitive performance tests , which limits the generalizability of these results . six studies made a categorical analysis , either of sh vs. euthyroidism or of quantiles of serum tsh and thyroid hormone concentration in relation to cognitive function ( table 1 ) . the sao paulo ageing and health study , a large cross - sectional study comprising 1119 community - dwelling participants aged 65 yr and over , found an association between sh and dementia after multivariate adjustment ( 38 ) . the effect was strongest for vascular dementia [ odds ratio ( or ) for all types of dementia , 4.9 ; 95% confidence interval ( ci ) , 1.515.7 ; vascular dementia or , 5.8 ; 95% ci , 1.433.1 ] . this was confirmed using an analysis of quintiles of tsh ; those with the lowest serum tsh quintile showed more than a 3-fold increase in risk for all types of dementia [ age - adjusted or for dementia , 3.6 ( 95% ci , 1.48.9 ) ; vascular dementia , 9.3 ( 95% ci , 1.175.5 ) ] . similarly , the inchianti study , a population - based study of 916 italians aged 65 yr and older , demonstrated a significantly lower mini - mental state examination ( mmse ) score among those with sh compared with the euthyroid group ( 22.61 6.88 vs. 24.72 4.52 ; p < 0.03 ) ( 39 ) . furthermore , sh was associated with more than a 2-fold risk of scoring less than 24 out of 30 on the mmse ; a standard threshold for significant dementia [ hazard ratio ( hr ) = 2.26 ( 95% ci , 1.323.91 ) ; p = 0.003 ] . these two large studies were well designed with more than a 90% participant response rate , and both carefully excluded patients with overt thyroid dysfunction as well as those taking thyroid medication . ( 41 ) ] , involving 469 and 119 participants , respectively , supported these findings . van osch et al . ( 40 ) investigated a cognitively intact group and an ad cohort aged greater than 52 yr . interestingly , the euthyroid participants with a tsh level in the lowest tertile ( tsh , 0.51.3 mu / liter ) had more than a 2-fold increase in ad prevalence compared with those with serum tsh in the highest tertile ( 2.16.0 mu / liter ) [ or , 2.04 ( 95% ci , 1.183.53 ) ; p = 0.01 ] . similarly , dobert et al . ( 41 ) demonstrated a significantly lower tsh level in participants with both ad and vascular dementia ( p < 0.01 ) compared with controls without cognitive impairment . cross - sectional and case control studies on the relationship between sh and cognitive decline ( 19962011 ) ns , nonsignificant ; mmse , mini - mental state examination ; cerad , consortium to establish a registry for alzheimer 's disease ; bmi , body mass index ; dm , diabetes mellitus ; cvd , cardiovascular disease ; ra , rheumatoid arthritis ; pvd , peripheral vascular disease ; ldl , low - density lipoprotein ; hdl , high - density lipoprotein ; tft , thyroid function test ; apoe4 , apolipoprotein e4 ; bp , blood pressure ; pd , parkinson 's disease ; mi , myocardial infarction . in contrast , the largest cross - sectional population - based study , involving 5868 participants , showed no association between sh and cognition ( 42 ) . however , this study invited participants by mail , and the response rate was poor at 38% . although the responders were representative of the regional population with respect to demographic characteristics , the average mmse score was above 27 in all subgroups , suggesting that the responder population was skewed toward a cognitively intact group . another cross - sectional observational study , involving 829 consecutive unselected referrals to a hospital geriatric clinic , did not demonstrate any difference in tsh level between the group with ad and the cohort without dementia ( 43 ) . however , comorbidities and medication use were not described , so the results may be confounded by nonthyroidal illness , thyroid medication , or overt thyroid dysfunction . an additional seven studies performed multivariate analyses to explore the relationship between cognition and thyroid function . wahlin et al . ( 44 ) were the first to suggest an association between poorer cognitive performance and lower serum tsh concentrations within the reference range . a significant positive correlation was found between a lower episodic memory score and serum tsh concentration ( p < 0.01 ) in 200 healthy , euthyroid , community - dwelling participants aged over 75 yr . in keeping with these findings , stuerenburg et al . ( 45 ) demonstrated an inverse correlation ( p = 0.04 ) between serum ft4 level and mmse score in 227 patients aged 4991 yr with mild to moderate ad . in contrast , the remaining five studies of this design failed to demonstrate any association between cognition and low serum tsh or high serum ft4 level ( 4650 ) . in a cross - sectional study involving 1219 community - dwelling participants , global cognitive impairment was not increased in those with sh ( n = 34 ) ( 46 ) . in a hospital - based study involving 409 euthyroid patients aged 5294 yr diagnosed with probable ad , two smaller studies involving 120 healthy volunteers and 69 hospital - based participants , respectively , showed similar findings ( 48 , 49 ) . paradoxically , a study of 44 healthy , community - dwelling male volunteers with a mean age of 72 yr observed a significant association between higher total t4 ( tt4 ) levels and better cognition [ verbal performance in wechsler adult intelligence scale ( p < 0.05 ) and global cognitive performance ( p < 0.01 ) ] but failed to reproduce this trend with tt3 and ft4 ( 50 ) . it is worth noting that almost all of these studies have relatively small sample sizes , that each made a single measurement of thyroid function , and they used different cognitive performance tests , which limits the generalizability of these results . six studies performed categorical analyses , either of sh vs. euthyroidism or quantiles of serum thyroid hormone concentration in relation to cognitive function ( table 2 ) . the rotterdam study was the first longitudinal study to examine the relationship between sh and dementia in 1893 population - based subjects ( mean age , 69 yr ) ( 51 ) . over a 2-yr follow - up , a 3-fold increased risk of dementia from all causes [ relative risk ( rr ) , 3.5 ; 95% ci , 1.210 ] and ad ( rr , 3.5 ; 95% ci , 1.111 ) was found among those with a low baseline tsh level ( < 0.4 mu / liter ) . this study was limited by the small number of the sh group with dementia ( n = 25 ) and a short follow - up period . nevertheless , the positive findings in this study are supported by two well - conducted , large population - based studies with a longer follow - up period . the first study , a community - based observational study , was carried out over a period of 12 yr by tan et al . ( 52 ) in 1864 patients who were free of dementia for 3 yr at recruitment . the patients were then divided into three tertiles according to baseline tsh : t1 , less than 1.0 mu / liter ; t2 , 1.02.1 mu / liter ; and t3 , more than 2.1 mu / liter . a positive correlation in women with a serum tsh level in the lowest ( t1 ) tertile ( hr , 2.26 ; 95% ci , 1.363.77 ; p = 0.002 ) and the highest ( t3 ) tertile ( hr , 1.84 ; 95% ci , 1.103.08 ; p = 0.003 ) with ad risk was found . this relationship was not found in men , but other indices of thyroid function were not studied . however , the two aforementioned large prospective studies involved only a single measurement of thyroid function . this limitation has recently been addressed by the largest observational study involving 12,115 participants ( 2,004 with sh ; 10,111 euthyroid individuals ) with a mean age of 66.5 yr and a median follow - up period of 5.6 yr ( 53 ) . this well - designed , population - based study had a carefully selected sh cohort , including only individuals with two confirmatory measurements of serum tsh at least 4 months apart . patients whose tsh normalized or who developed overt thyroid disease during the course of follow- up were excluded . a positive association between sh and dementia was observed ( adjusted hr , 1.64 ; 95% ci , 1.202.25 ) . interestingly , when patients were divided into two groups according to the tsh level ( 0.10.4 vs. < 0.1 mu / liter ) , no relationship could be established between suppressed tsh ( < 0.1 mu / liter ) and dementia , but a significant association remained for the group with tsh at 0.10.4 mu / liter . longitudinal populational - based studies on the relationship between sh and cognitive decline ( 19962011 ) gms - a , geriatric mental state schedule ; camdex , cambridge examination for disorders of the elderly ; bmi , body mass index ; apoe4 , apolipoprotein e4 ; dm , diabetes mellitus ; pvd , peripheral vascular disease ; rai , radioiodine therapy ; tft , thyroid function test . in addition to these three large population - based studies , de jong et al . ( 54 ) carried out a smaller prospective study among 615 japanese - american men in the honolulu heart program . the cohort had a mean age of 77.5 yr , and the mean duration of follow - up was 5 yr . this study observed a 20 and 30% increased risk for dementia and ad , respectively , for each sd increase in serum ft4 ( hr for dementia , 1.21 ; 95% ci , 1.041.40 ; and hr for ad , 1.31 ; 95% ci , 1.141.51 ) . in addition , neuropathological examinations from the autopsy program instituted as part of this study demonstrated a higher neocortical neurofibrillary tangle count per sd increase in tt4 ( 0.25 ; 95% ci , 0.050.46 ) . in contrast with the studies discussed so far , the rotterdam scan study , another prospective community - based study involving 1025 subjects aged 6090 yr with a mean follow - up interval of 5.5 yr , showed no association between dementia and serum tsh or thyroid hormone levels ( 55 ) . nevertheless , they found a positive association between a higher ft4 and rt3 and greater atrophy at the hippocampus and amygdala on magnetic resonance imaging ( mri ) , in keeping with the finding in the honolulu aging study . although the rotterdam scan study had greater power than the original rotterdam study , due to a larger - sized dementia cohort ( n = 60 vs. 25 ) and a longer follow - up period , it is limited by single measurements of thyroid function and a small number of dementia cases with low tsh ( n = 7 ) . finally , a community - based study involving 464 euthyroid women aged 65 yr or older found an association between lower ft4 concentrations within the reference range and cognitive impairment over a 3-yr period ( rr , 1.97 ; 95% ci , 1.103.5 ) ( 56 ) . however , the same pattern was not exhibited in those with a frankly low tsh or an elevated ft4 level . there was a 14% dropout rate during follow - up , largely due to missing mmse scores , which might have introduced a significant bias . four studies made multivariate analyses using thyroid function parameters or cognitive function ( mmse ) as continuous variables . ( 57 ) conducted a large prospective study involving 1047 community - dwelling patients aged 6494 yr with mmse scores of at least 25 in the united kingdom and wales . higher serum ft4 concentrations within the reference range were associated with lower baseline mmse scores and accelerated cognitive decline after 2 yr [ or , 1.13 ( 95% ci , 1.031.23 ) ; p = 0.006 ] . prospective follow - up of 558 individuals aged 85 living in leiden , the netherlands , demonstrated an association between higher serum tsh and better memory function ( , 0.13 ; p = 0.03 ) ( 58 ) . nevertheless , this study had a small sample size and a 41.5% loss of follow - up due to death or refusal to continue participation . after the cross - sectional study in 1998 ( 44 ) , wahlin et al . ( 59 ) conducted a follow - up survey on the same cohort and found an association between low serum tsh levels within the reference range and decreased verbal memory or episodic recall deficits at the 6-yr follow - up ( , 0.290 ; p < 0.05 ) . similarly , in a small hospital - based study , annerbo et al . ( 60 ) demonstrated that for each unit decrease in serum tsh , the or of developing dementia increased by 3.5 . six studies performed categorical analyses , either of sh vs. euthyroidism or quantiles of serum thyroid hormone concentration in relation to cognitive function ( table 2 ) . the rotterdam study was the first longitudinal study to examine the relationship between sh and dementia in 1893 population - based subjects ( mean age , 69 yr ) ( 51 ) . over a 2-yr follow - up , a 3-fold increased risk of dementia from all causes [ relative risk ( rr ) , 3.5 ; 95% ci , 1.210 ] and ad ( rr , 3.5 ; 95% ci , 1.111 ) was found among those with a low baseline tsh level ( < 0.4 mu / liter ) . this study was limited by the small number of the sh group with dementia ( n = 25 ) and a short follow - up period . nevertheless , the positive findings in this study are supported by two well - conducted , large population - based studies with a longer follow - up period . the first study , a community - based observational study , was carried out over a period of 12 yr by tan et al . ( 52 ) in 1864 patients who were free of dementia for 3 yr at recruitment . the patients were then divided into three tertiles according to baseline tsh : t1 , less than 1.0 mu / liter ; t2 , 1.02.1 mu / liter ; and t3 , more than 2.1 mu / liter . a positive correlation in women with a serum tsh level in the lowest ( t1 ) tertile ( hr , 2.26 ; 95% ci , 1.363.77 ; p = 0.002 ) and the highest ( t3 ) tertile ( hr , 1.84 ; 95% ci , 1.103.08 ; p = 0.003 ) with ad risk was found . this relationship was not found in men , but other indices of thyroid function were not studied . however , the two aforementioned large prospective studies involved only a single measurement of thyroid function . this limitation has recently been addressed by the largest observational study involving 12,115 participants ( 2,004 with sh ; 10,111 euthyroid individuals ) with a mean age of 66.5 yr and a median follow - up period of 5.6 yr ( 53 ) . this well - designed , population - based study had a carefully selected sh cohort , including only individuals with two confirmatory measurements of serum tsh at least 4 months apart . patients whose tsh normalized or who developed overt thyroid disease during the course of follow- up were excluded . a positive association between sh and dementia was observed ( adjusted hr , 1.64 ; 95% ci , 1.202.25 ) . interestingly , when patients were divided into two groups according to the tsh level ( 0.10.4 vs. < 0.1 mu / liter ) , no relationship could be established between suppressed tsh ( < 0.1 mu / liter ) and dementia , but a significant association remained for the group with tsh at 0.10.4 mu / liter . longitudinal populational - based studies on the relationship between sh and cognitive decline ( 19962011 ) gms - a , geriatric mental state schedule ; camdex , cambridge examination for disorders of the elderly ; bmi , body mass index ; apoe4 , apolipoprotein e4 ; dm , diabetes mellitus ; pvd , peripheral vascular disease ; rai , radioiodine therapy ; tft , thyroid function test . in addition to these three large population - based studies , de jong et al . ( 54 ) carried out a smaller prospective study among 615 japanese - american men in the honolulu heart program . the cohort had a mean age of 77.5 yr , and the mean duration of follow - up was 5 yr . this study observed a 20 and 30% increased risk for dementia and ad , respectively , for each sd increase in serum ft4 ( hr for dementia , 1.21 ; 95% ci , 1.041.40 ; and hr for ad , 1.31 ; 95% ci , 1.141.51 ) . in addition , neuropathological examinations from the autopsy program instituted as part of this study demonstrated a higher neocortical neurofibrillary tangle count per sd increase in tt4 ( 0.25 ; 95% ci , 0.050.46 ) . in contrast with the studies discussed so far , the rotterdam scan study , another prospective community - based study involving 1025 subjects aged 6090 yr with a mean follow - up interval of 5.5 yr , showed no association between dementia and serum tsh or thyroid hormone levels ( 55 ) . nevertheless , they found a positive association between a higher ft4 and rt3 and greater atrophy at the hippocampus and amygdala on magnetic resonance imaging ( mri ) , in keeping with the finding in the honolulu aging study . although the rotterdam scan study had greater power than the original rotterdam study , due to a larger - sized dementia cohort ( n = 60 vs. 25 ) and a longer follow - up period , it is limited by single measurements of thyroid function and a small number of dementia cases with low tsh ( n = 7 ) . finally , a community - based study involving 464 euthyroid women aged 65 yr or older found an association between lower ft4 concentrations within the reference range and cognitive impairment over a 3-yr period ( rr , 1.97 ; 95% ci , 1.103.5 ) ( 56 ) . however , the same pattern was not exhibited in those with a frankly low tsh or an elevated ft4 level . there was a 14% dropout rate during follow - up , largely due to missing mmse scores , which might have introduced a significant bias . four studies made multivariate analyses using thyroid function parameters or cognitive function ( mmse ) as continuous variables . ( 57 ) conducted a large prospective study involving 1047 community - dwelling patients aged 6494 yr with mmse scores of at least 25 in the united kingdom and wales . higher serum ft4 concentrations within the reference range were associated with lower baseline mmse scores and accelerated cognitive decline after 2 yr [ or , 1.13 ( 95% ci , 1.031.23 ) ; p = 0.006 ] . prospective follow - up of 558 individuals aged 85 living in leiden , the netherlands , demonstrated an association between higher serum tsh and better memory function ( , 0.13 ; p = 0.03 ) ( 58 ) . nevertheless , this study had a small sample size and a 41.5% loss of follow - up due to death or refusal to continue participation . after the cross - sectional study in 1998 ( 44 ) , wahlin et al . ( 59 ) conducted a follow - up survey on the same cohort and found an association between low serum tsh levels within the reference range and decreased verbal memory or episodic recall deficits at the 6-yr follow - up ( , 0.290 ; p ( 60 ) demonstrated that for each unit decrease in serum tsh , the or of developing dementia increased by 3.5 . twenty - three studies that met our criteria have examined the association between sh and cognition . fourteen of these studies , including several well - designed and well - powered cross - sectional and longitudinal analyses , have shown a consistent finding of an association of sh , or low serum tsh within the reference interval , with cognitive impairment or dementia . in particular , this association was seen in more than three fourths of the prospective longitudinal studies , providing reliable evidence from robust studies . several of the studies that did not confirm this result are potentially compromised by small samples sizes , recruitment from hospital environments , or the challenging nature of working with participants with cognitive problems ( e.g. failure to return questionnaires ) . given the weight of information suggesting an association of sh and lower serum tsh concentrations with cognitive impairment summarized above , we need to consider several explanations that could explain these findings ( table 3 ) . a conventional explanation ( explanation a ) might be that mild endogenous sh reflects true thyroid overactivity causing excessive thyroid hormone action on the central nervous system . toxic effects of thyroid hormones on the brain could be mediated by increased brain oxidative stress caused by the mild hyperthyroidism , which promotes reactive oxygen species production ( 35 ) . alternatively , thyroid hormone effects on the heart could mediate vascular dementia via thromboembolism from a combination of atrial fibrillation , myocardial and endothelial dysfunction , and hypercoagulability ( 5 ) . first , only about 20% of individuals with sh have a suppressed tsh ( grade ii sh ) , and so most do n't have true endogenous hyperthyroidism . clearly , neither do those people with serum tsh concentrations in the lower centiles of the healthy reference range . indeed , the low but not suppressed tsh in the 0.10.4 mu / liter range is most commonly seen as a manifestation of nonthyroidal illness and is associated with a lowering of circulating ft3 , rather than thyroid hormone excess . second , for this explanation to be true , we would expect to see a biological gradient with cognitive defects being worse in individuals with the lowest tsh . ( 53 ) where , if anything , the cognitive effects were most marked in the grade i sh group . possible mechanism for association of cognitive impairment with sh or low serum tsh an alternative explanation ( explanation b ) is that the organic brain diseases causing cognitive impairment also reduce trh secretion from the hypothalamus and other brain areas . knock - on effect leading to lower tsh secretion and hence reduced thyroid hormone turnover ( production and excretion)in effect , a state of mild central hypothyroidism . it is known that there are projections from many areas of the brain to the paraventricular nucleus containing the trh - secreting neurons , such as the c13 adrenergic neurons of the brainstem , the hypothalamic arcuate nucleus , and the neurons of the hypothalamic dorsomedial nucleus , which exert different effects on the hypophysiotropic trh neurons ( 6166 ) . furthermore , trh is not only released from the paraventricular nucleus of hypothalamus but is also localized in neurons of the septal nuclei , preoptic area , raphe nuclei of the medulla oblongata , and spinal cord ( 6769 ) . thus , trh has a more generalized role as a central nervous system ( cns ) neurotransmitter , and the brain involution of the dementia process may lead to a widespread perturbation of neurotransmitters , including trh . if we accept this explanation as being plausible , then paradoxically , a study of thyroid hormone supplementation in dementia might be warranted . a third explanation refers back to our understanding of thyroid hormone metabolism in older age . with this mechanism , one has to consider that low serum tsh ( and indeed higher ft4 ) may be a marker of biological age , reflecting reduced hepatic clearance of thyroid hormones ( including reduced type 1 deiodinase activity ) and a consequent reduction in thyroid axis turnover . the well - established observation that older people with hypothyroidism require less levothyroxine replacement is the clinical correlate of this phenomenon ( 15 , 70 ) . in effect , the epidemiological studies reviewed above identify a group of individuals within a cohort who have more advanced biological age , using tsh as the biomarker ( 1 ) . these individuals therefore have an excess of the degenerative disorders of advanced age , which includes cognitive impairment and dementia . compellingly , sh is also associated with excess rates of atrial fibrillation , vascular events , low bone mineral density , fracture , and reduced muscle strength ( 7174 ) . thus , dementia can be viewed as another noncausal association of low tsh / sh ( 1 ) . individuals with cognitive impairment and dementias have a high burden of comorbidity and associated medication use . this may include many episodic intercurrent illnesses , such as transient infection , as well as more chronic degenerative conditions . all of these comorbidities can lead to reduced serum tsh , consequent to the well - described changes in serum thyroid hormones associated with nonthyroidal illness , known as similarly , a wide variety of medications for conditions other than thyroid diseases including glucocorticoids , opiates , l - dopa , amiodarone , and metformin are known to reduce serum tsh concentrations ( 1 , 75 ) . thus , the combination of excess comorbidity and the consequent additional medications that may be prescribed for this may also explain a noncausal association of sh / low tsh with cognitive impairment . the fact that most of the studies performed in this field have relied upon a single tsh measurement makes these data particularly vulnerable to this interpretation . nevertheless , although it is possible that this explanation contributes to some of the association between sh and dementia , it is unlikely to be the dominant factor . ( 53 ) involving 12,115 patients showed that this association persists after stringent ascertainment of a cohort categorized as having sh after repeated serum tsh measurement . at the current time , we believe that there are insufficient data to allow discrimination between the various mechanistic possibilities outlined above ( table 3 ) . indeed , it is likely that there may be contributions from several of the above factors to the observed association between sh or low serum tsh and cognitive impairment . until such time we have performed the first detailed review of a large and heterogeneous literature relating to the association between low serum tsh and cognitive impairment in older people . overall , taking into account the largest and most robustly designed studies , there is a strong body of evidence to support the association between sh and cognitive impairment . however , there is no clear mechanistic explanation for these associations , nor is there any evidence to support the use of antithyroid measures to prevent or improve cognitive decline in this patient group . larger and more detailed prospective longitudinal or randomized controlled trials are required to inform these important questions .

the most currently used adhesive system classification is based on the number of steps necessary for clinical application and their interaction with dental hard tissues . total etch adhesive systems , which remove the smear layer with phosphoric acid and combine the functions of primer and adhesive in one bottle , have been widely used . although long term clinical success has been achieved with total - etch systems , the demand for simplified application has increased , resulting in the development of self - etching adhesive systems . the bonding mechanism of self - etching adhesive systems is based on the simultaneous etching and priming of enamel and dentin without rinsing forming a continuum in the substrate and incorporating smear plugs into the resin tags . effective adhesives are expected to seal the etched dentin surfaces by intertubular and peritubular hybridization and by resin tag formation in the opened dentinal tubules . this seal prevents fluid shift across the tubules occurring in response to mechanical , thermal , or osmotic stimuli . however , abiding by the hydrodynamic theory , if the tags formed within the dentinal tubules were too long , it could cause post - operative sensitivity . but combining a resin adhesive with a previous application of desensitizing agent seems to be contradictory at first sight , considering its effect on resin tag formation and the bond strength of composite resin to the tooth structure . even though the use of a desensitizing agent prior to application of dentin bonding resin seems to be promising , we are not aware of its effect on the degree of conversion of the dentin bonding resin . the degree of conversion ( dc ) of the resin material is related to many properties , including mechanical properties , solubility , dimensional stability , colour change and biocompatibility . hence , the aim of this in vitro study was to determine the degree of conversion of an etch - and - rinse dentin bonding resin and a self - etching adhesive resin used with and without a desensitizing agent using fourier transform infrared ( ftir ) spectroscopy . adper single bond ( 3 m espe ) , an etch - and - rinse bonding resin and adhse one ( ivoclar vivadent ) , a self - etching adhesive resin were chosen for the study . the four study groups are as follows : group i : adper single bond ( n=10 ) group ii : adper single bond + vivasens ( n=10 ) group iii : adhese one ( n=10 ) group iv : adhese one + vivasens ( n=10 ) materials used , their manufacturer , and composition group i and group iii specimens were prepared by curing 1 ml of the bonding resin in a vial for 20 seconds . group ii and group iv specimens were prepared by mixing 1 ml each of the respective bonding agent and vivasens in a vial , which was then immersed in an ultrasonic bath and mixed for 1 minute . the specimens were then sent for ftir spectroscopy ( spectrum one , perkin elmer , usa ) analysis . first , the spectrum of the unpolymerized resin was measured and then the infrared spectrum was recorded . during spectra recording , the resin was continuously in contact with the sensor . dc% was calculated from the aliphatic c = c peak at 1638 cm and normalized against the aromatic c = c peak at 1608 cm according to the following formula dc% = { 1- [ caliphatic/ caromatic ] / [ ualiphatic/ uaromatic ] } 100 where caliphatic = absorption peak at 1638 cm of the polymerized specimen , caromatic = absorption peak at 1608 cm of the polymerized specimen ualiphatic = absorption peak at 1638 cm of the unpolymerized specimen and uaromatic = absorption peak at 1608 cm of the unpolymerized specimen statistical analysis was based on anova and post - hoc tests with a level of significance of p<0.05 first , the spectrum of the unpolymerized resin was measured and then the infrared spectrum was recorded . during spectra recording , dc% was calculated from the aliphatic c = c peak at 1638 cm and normalized against the aromatic c = c peak at 1608 cm according to the following formula dc% = { 1- [ caliphatic/ caromatic ] / [ ualiphatic/ uaromatic ] } 100 where caliphatic = absorption peak at 1638 cm of the polymerized specimen , caromatic = absorption peak at 1608 cm of the polymerized specimen ualiphatic = absorption peak at 1638 cm of the unpolymerized specimen and uaromatic = absorption peak at 1608 cm of the unpolymerized specimen statistical analysis was based on anova and post - hoc tests with a level of significance of p<0.05 table 2 shows the mean degree of conversion ( dc% ) values and standard deviations of the study groups . mean degree of conversion ( dc% ) values and standard deviations of the study groups ftir spectra of group i ( adper single bond ) ftir spectra of group ii ( adper single bond + desensitizer ) ftir spectra of group iii ( adhese one ) ftir spectra of group iv ( adhese one + desensitizer ) statistical analysis showed a significantly higher dc% values for group ii ( adper single bond + desensitizer-47.492.79 ) compared to group i ( adper single bond - 37.613.03 ) . comparing groups iii and iv , group iv ( adhese one+desensitizer - 40.933.65 ) showed a significantly higher dc% values compared to group iii ( adhese one-36.712.77 ) . comparing the groups without desensitizer , no statistically significant difference was found between group i ( 37.613.03 ) and group iii ( 36.712.77 ) . comparing the groups with desensitizer , group ii ( 47.492.79 ) showed a significantly higher dc% compared to group iv ( 40.933.65 ) . the most commonly used esthetic restorative materials are the resin composites that are bonded to the tooth structure via an adhesive material . adhesion of resins to tooth structure has been a goal for dental researchers for many years . the interest of adhesion focussed on the factors and techniques for enhancement of monomer penetration . dentin - bonding agents have evolved over the years , with the introduction of a new system of bonding in every generation . etch - and - rinse adhesives combine the primer and the bonding agent into a single solution . as this system requires several time consuming steps , manufacturers attempted to reduce the number of steps and corresponding application time , making more user - friendly adhesive systems . in recent years , self etching resins containing acidic methacrylates have been introduced in an attempt to eliminate the steps of etching and rinsing . the application of dentin adhesive can result in the formation of long resin tags within the dentinal tubules , which may lead to postoperative sensitivity . a method of reducing postoperative sensitivity is by using a desensitizer along with a dentin bonding agent . studies have been done to check the effect of application of a layer of desensitizing agent on the bond strength of the bonding agent , but no studies have been done to check the degree of conversion of the bonding agent when a desensitizing agent is incorporated into it . hence this study was done to assess the degree of conversion of dentin bonding agents on addition of a desensitizing agent using ftir spectroscopy . the extent of polymerization of a material is expressed as degree of conversion of monomeric c = c bonds into polymeric c - c bond , which affects both physical and mechanical properties . incomplete conversion results in residual unreacted monomer which may cause a reduction in material properties , a decrease in the long term stability of the restoration and pulpal irritation.[1315 ] ftir was used in this study as it provides a direct measure of unreacted methacrylate groups . it is a reliable and fast technique used for detecting c = c bond stretching vibrations directly before and after curing of resins . according to this study , the combination of a dentin adhesive with a desensitizer showed an increase in the degree of conversion ( group iii and group iv ) compared to when dentin adhesive was used alone ( group i and group ii ) . dentin adhesive / desensitizer combination can be used to mechanically block patent dentinal tubules or physiologically decrease the excitability of the intradental nerves to relieve hypersensitivity . vivasens , the desensitizing agent contains organic acids such as phosphoric acid methacrylate and solvents such as ethanol which induce the precipitation of proteins in the dentin liquid . moreover , the sealing effect is enhanced by co - precipitating polyethylene glycol dimethacrylate , which is present in vivasens . polyethylene glycol dimethacrylate is a condensation polymer , which acts as an organic filler and cross links molecular chains of polymers . therefore an increase in the degree of conversion in group ii and group iv can probably be attributed to the presence of polyethylene glycol dimethacrylate . within the limitations of this in - vitro study , it can be concluded that the use of a desensitizing agent along with a dentin bonding resin does not affect the polymerization of the bonding resin and can be recommended to effectively prevent postoperative sensitivity associated with composite restorations .

its frequency increases during childhood but it usually resolves by the age of 30 years . acetylation is a metabolic pathway in a number of drugs , which exhibits a genetically controlled bimodal distribution within any given population which is phenotyped as slow or rapid acetylators . studies had been done to determine acetylation phenotype in middle east population,[58 ] which showed slow acetylator predominance . therefore , we examined ad in this population to find any possible association with the disease severity . thirty - six pediatric patients , 21 males and 15 females , aged from 8 to 11 years ( mean 9.660.9 sd ) besides , 42 healthy children , 27 males and 15 females , aged from 7 to 11 years ( mean 8.931.1 sd ) as a control group , participated in this prospective and open study . approval to conduct this study , was granted by the appropriate ethical committee in baghdad college of medicine . the participants were recruited from the outpatient clinic of department of dermatology of baghdad teaching hospital from october 2007 to december 2008 . the nature of the trial was explained to the children 's parents and their consent was obtained . after an overnight fast , each individual ( patient and control ) received a single oral dose of dapsone 1.54 mg / kg body weight . a blood sample ( 5 ml ) was taken in a test tube containing heparin , 3 hours after the drug intake . plasma was separated within 1 hour of collection and then high performance liquid chromatography ( hplc ) was used to estimate plasma concentrations of dapsone and its metabolite ( monoacetyldapsone ) . individuals were considered slow acetylators if their acetylation ratio ( ratio of monoacetyldapsone to dapsone ) was less than 0.30 and rapid acetylators if their acetylation ratio was greater than 0.30 . twenty - six of the 36 ad pediatric patients ( 72.2% ) were slow acetylators compared with 29 of 42 control individuals ( 69.4% ) , and this was found to be a statistically non - significant result [ table 1 ] . in ad patients , a significant relationship appeared between familial history of allergy and slow acetylators who represented 73% ( 19 out of 26 slow acetylator patients ) [ table 2 ] . application of hanifin - rajka scoring system revealed that 30.5% ( 11 of 36 ad patients ) were severe cases and all were slow acetylators [ table 3 ] . in the ad patients , eczematous lesions which presented in the limbs ( elbows , knees , hands and feet ) were found mostly in slow acetylators who represented 84.6% ( 22 out of 26 eczematous patients ) , whereas lesions that presented in the face and neck were found mainly in rapid acetylators who represented 60.0% ( 6 out of 10 rapid phenotypes ) . frequency distribution of acetylation phenotype in ad patients and control the acetylation phenotype and history of allergy in ad patients the acetylation phenotype and hanifin - rajka scoring system in ad patients the acetylation phenotype and distribution of eczematous lesions in ad patients this study demonstrated that there is a predominance of slow acetylators in ad patients as compared with the control group . the results are in accordance with those of studies that showed a predominance of ad patients carrying the allele for slow acetylation.[58 ] however , our study succeeded to show a relation between acetylator status and different aspects of the disease course . a significant association was found between the severity of ad and slow acetylator status in which hanifin - rajka scoring system determined that a large number of slow acetylator ad patients had scores ( 50 points ) which means severe cases , while no one is a rapid acetylator . this indicates that n - acetylation phenotype variation in human skin could account for variation in the disease severity . this result agreed with that of a study on acetylator phenotype among children with allergic contact dermatitis in which an association was found between the site of allergic lesions and acetylator status . in a population of slow acetylators , it appears that the slow acetylatation phenotype can be considered as a genetic predisposing state for ad and is a clue for severity of the disease .

cervical pedicle screw fixation can achieve three - column stability and rigid fixation , and is especially suitable for patients with cervical spine instability caused by osteoporosis , cervical spine injuries , bone degeneration , and tumors [ 68 ] . however , cervical pedicle screw placement involves higher risks than pedicle screw placement in the thoracolumbar spine , and demands much more surgical skill . the cervical pedicles are small and have very thin lateral walls , with great variations in their directions . thus , cervical pedicle screw insertion has a certain risk of perforation , which may cause complications such as nerve root compression , vertebral artery injury , and even spinal cord injury [ 1015 ] . a cervical pedicle screw can be inserted using one of the three methods : manual insertion , computer - assisted image - guiding insertion , and placement with cervical pedicle three - dimensional locator guidance . manual placement varies greatly in accuracy depending on the specific insertion method and surgeon s experience , and therefore has variable safety . computer - assisted insertion has good accuracy , stability , and safety , and one of its advantages is percutaneous placement of screws . conflicting results have been obtained on whether computer - assisted placement can lower the incidence of pedicle perforation [ 11,12,17,2328 ] . cervical pedicle locator guidance is cost - effective and can work with all imaging systems . this study aimed to compare the accuracy of screw placement between the three - dimensional locator guidance and manual manipulation . eighteen formalin - embalmed adult cadavers were randomly divided into group a ( n=9 ) and group b ( n=9 ) . the c3c7 vertebrae were scanned using computed tomography ( ct ) ( a 256-slice brilliance ict scanner , philips medical systems , nederland ) in the axial and sagittal planes . the slice thickness was 1 mm . the ct images were measured for the reference of screw insertion . paravertebral muscles were dissected to expose the bilateral masses and inferior borders of the articular process . in group data from the ct image of the c3c7 vertebrae were input into the locator to regulate the processus articularis feet , lateral mass feet , columella , angular arm , and gunsight . the hooklets attached to the processus articularis feet were inserted into the joint space and pulled tight to maintain the processus , with articularis feet tightly touching the inferior borders of inferior articular process . a hole was made using a tailor - made long drill and a screw of 2-mm diameter was inserted . in group b , the screw was inserted using manual manipulation . the entering point of the screw was determined using the abumi technique and a caliper . the abduct angle of the pedicle was measured using an angle measuring instrument . in the sagittal plane , after insertion of screws in the two groups , each cadaver underwent sagittal and axial ct scans . the parallel offset and angular offset were measured between the screw and the pedicle axis in ct images with a caliper and an angle - measuring instrument . the offsets were graded according to the following criteria : i : parallel offset 1 mm and angulate offset 5. ii : parallel offset > 1 mm . parallel offset 1 mm and angulate offset 5. fair : the screw is completely within the pedicle . all statistical analyses were performed using spss 17.0 software ( spss , chicago , il , usa ) . eighteen formalin - embalmed adult cadavers were randomly divided into group a ( n=9 ) and group b ( n=9 ) . the c3c7 vertebrae were scanned using computed tomography ( ct ) ( a 256-slice brilliance ict scanner , philips medical systems , nederland ) in the axial and sagittal planes . the slice thickness was 1 mm . the ct images were measured for the reference of screw insertion . paravertebral muscles were dissected to expose the bilateral masses and inferior borders of the articular process . data from the ct image of the c3c7 vertebrae were input into the locator to regulate the processus articularis feet , lateral mass feet , columella , angular arm , and gunsight . the hooklets attached to the processus articularis feet were inserted into the joint space and pulled tight to maintain the processus , with articularis feet tightly touching the inferior borders of inferior articular process . a hole was made using a tailor - made long drill and a screw of 2-mm diameter was inserted . in group b , the screw was inserted using manual manipulation . the entering point of the screw was determined using the abumi technique and a caliper . the abduct angle of the pedicle was measured using an angle measuring instrument . in the sagittal plane , after insertion of screws in the two groups , each cadaver underwent sagittal and axial ct scans . the parallel offset and angular offset were measured between the screw and the pedicle axis in ct images with a caliper and an angle - measuring instrument . the offsets were graded according to the following criteria : i : parallel offset 1 mm and angulate offset 5. ii : parallel offset > 1 mm . parallel offset 1 mm and angulate offset 5. fair : the screw is completely within the pedicle . all statistical analyses were performed using spss 17.0 software ( spss , chicago , il , usa ) . a total 90 screws were inserted in the c3c7 vertebrae in group a. among them , 81 screws ( 90% ) were entirely within the pedicle and 9 screws ( 10% ) breached the pedicle cortex . two screws injured the nerve root and one screw injured the vertebral artery ( table 1 ) . in the axial ct image , 63 screws ( 70% ) had a parallel offset 1 mm and an angular offset 5 from the pedicle axis , 19 screws ( 21.1% ) had a parallel offset > 1 mm , and 8 screws ( 8.9% ) had an angular offset > 5 ( table 2 ) . parallel offsets in the axial and sagittal ct image were 0.560.70 mm and 1.040.99 mm , respectively ; angular offsets in the axial and sagittal ct image were 1.692.41c and 6.547.08c , respectively ( figures 1 , 2 ) . a total 90 screws were inserted in the c3c7 vertebrae in group b. among them , 50 screws ( 55.6% ) were entirely within the pedicle and 40 screws ( 44.4% ) breached the pedicle cortex breach . nineteen screws injured the nerve root and four screws injured the vertebral artery ( table 3 ) . in the axial ct image , 25 screws ( 27.8% ) had a parallel offset 1 mm and an angular offset 5 from the pedicle axis , 38 screws ( 42.2% ) had a parallel offset > 1 mm , and 54 screws ( 60% ) had an angular offset > 5 ( table 4 ) . parallel offsets in the axial and sagittal ct images were 1.191.02 mm and 1.350.99 mm , respectively ; angular offsets in the axial and sagittal ct images were 11.279.34c and 9.848.22c , respectively ( figures 1 , 2 ) . the parallel offset in the axial ct images was significantly lower in group a than group b ( p<0.01 ) . the parallel offset in the sagittal ct images in group was significantly higher than the axial images ( p<0.01 ) ( figure 1 ) . however , there was no significant difference in the parallel offset between group a and group b in the sagittal ct image ( p=0.05 ) . the parallel offset of groups also did not differ significantly between the axial and sagittal ct images ( p>0.05 ) ( figure 1 ) . the angular offset in the axial and sagittal ct images of group a was significantly higher than that of group b ( p<0.01 ) . the angular offset of group a differed significantly between the axial and sagittal ct images ( p<0.01 ) ( figure 2 ) . there was no significant difference in angular offset of group between the axial and sagittal ct images ( p>0.05 ) ( figure 2 ) . a total 90 screws were inserted in the c3c7 vertebrae in group a. among them , 81 screws ( 90% ) were entirely within the pedicle and 9 screws ( 10% ) breached the pedicle cortex . two screws injured the nerve root and one screw injured the vertebral artery ( table 1 ) . in the axial ct image , 63 screws ( 70% ) had a parallel offset 1 mm and an angular offset 5 from the pedicle axis , 19 screws ( 21.1% ) had a parallel offset > 1 mm , and 8 screws ( 8.9% ) had an angular offset > 5 ( table 2 ) . parallel offsets in the axial and sagittal ct image were 0.560.70 mm and 1.040.99 mm , respectively ; angular offsets in the axial and sagittal ct image were 1.692.41c and 6.547.08c , respectively ( figures 1 , 2 ) . a total 90 screws were inserted in the c3c7 vertebrae in group b. among them , 50 screws ( 55.6% ) were entirely within the pedicle and 40 screws ( 44.4% ) breached the pedicle cortex breach . nineteen screws injured the nerve root and four screws injured the vertebral artery ( table 3 ) . in the axial ct image , 25 screws ( 27.8% ) had a parallel offset 1 mm and an angular offset 5 from the pedicle axis , 38 screws ( 42.2% ) had a parallel offset > 1 mm , and 54 screws ( 60% ) had an angular offset > 5 ( table 4 ) . parallel offsets in the axial and sagittal ct images were 1.191.02 mm and 1.350.99 mm , respectively ; angular offsets in the axial and sagittal ct images were 11.279.34c and 9.848.22c , respectively ( figures 1 , 2 ) . the parallel offset in the axial ct images was significantly lower in group a than group b ( p<0.01 ) . the parallel offset in the sagittal ct images in group was significantly higher than the axial images ( p<0.01 ) ( figure 1 ) . however , there was no significant difference in the parallel offset between group a and group b in the sagittal ct image ( p=0.05 ) . the parallel offset of groups also did not differ significantly between the axial and sagittal ct images ( p>0.05 ) ( figure 1 ) . the angular offset in the axial and sagittal ct images of group a was significantly higher than that of group b ( p<0.01 ) . the angular offset of group a differed significantly between the axial and sagittal ct images ( p<0.01 ) ( figure 2 ) . there was no significant difference in angular offset of group between the axial and sagittal ct images ( p>0.05 ) ( figure 2 ) . our study showed that locator guidance has better accuracy than manual manipulation in cervical pedicle screw placement and lower risks of injuries of the pedicle , nerve root , and vertebral artery . parallel offset of screws from the pedicle axis in axial ct images can help to evaluate the offset of insertion points . angular offset of screws from the pedicle axis can help to evaluate the abduct angle offset of screw trajectory . in our study , c - arm x - ray guidance was not used to assist screw placement ; therefore , the accuracy of locator guidance and manual manipulation was compared in the same condition . according to the abumi procedure , the insertion point was 5 mm medial to the lateral border of the lateral mass and slightly inferior to the inferior border of the articular process of the superior vertebra . however , due to the arched shape of the lateral border of the lateral mass , it was difficult to precisely determine the distance between the insertion point and the lateral border of the lateral mass , regardless of ct image , or during the operation , even when aided by a caliper . therefore , the parallel offsets between axes were so large that 38 ( 42.2% ) screws had a parallel offset > 1 mm ( table 4ii ) . in contrast , the three - dimensional locator determined the horizontal insertion point by locating the posterior tangent line of the lateral masses and the inferior point of the inferior articular process and measuring the posterior tangent line dd of the lateral masses in ct image . this can decrease the number of screws with a parallel offset > 1 mm to 19 ( 21.1% ) screws ( table 2ii ) . the abduct angle in manual manipulation was determined by measuring the ct image ( range from 25 to 45 ) . an angle - measuring instrument was used to accurately measure the abduct angle of the pedicle and to guide the screw insertion . however , 54 screws ( 60% ) had an angular offset > 5 ( table 4 v ) because the surface of the lateral mass was uneven and the spinous process was not absolutely upright . the orientation of the locator depends on its parts ( angular arm and gunsight ) and results from measurement of the ct image referring to the surface of the lateral masses rather than the spinous process . therefore , only 8 ( 8.9% ) screws had an angular offset > 5 ( table 2v ) . in the axial ct images , both incidences of parallel offset ( 1 mm ) and angulate offset ( 5 ) were less , and the screw axis was close to the pedicle axis . there was great contrast between locator guidance ( 63 screws , 70% ) and manual manipulation ( 25 screws , 27.8% ) ( tables 2 , 4i ) . the difference in both parallel offset ( p<0.01 ) and angular offset ( p<0.01 ) was statistically significant between group a and group b in the axial ct image ( figures 1 , 2 ) . these results indicate that the locator can enhance the breadthwise accuracy of screw placement compared with manual manipulation . also , the parallel offset ( p<0.01 ) and angular offset ( p<0.01 ) of the locator were significantly different between the axial and sagittal ct images ( figures 1 , 2 ) . these results suggest that the breadthwise accuracy was higher than that of the lengthwise accuracy . there were no significant difference in the parallel offset ( p>0.05 ) and angular offset ( p>0.05 ) of the manual manipulation between the axial and sagittal ct images ( figures 1 , 2 ) . breach of the pedicle cortex is currently the most common standard for evaluation in both cadaveric and clinical studies . our study , however , further graded the screw placement accuracy to excellent ( grade i : 1 mm and 5 ) and fair ( grade ii : > 1 mm and > 5 ) . the failure rate of screw insertion manipulated by abumi himself was 6.7% . during screw placement , abumi ground the cortex at the point for insertion , explored the pedicle marrow cavity using a probe , and used x - ray guidance . the first two steps were indispensable for correcting the offset of insertion point and angle . therefore , abumi s procedure was based on experience rather than quantitative parameters , despite the reference of ct image . on the contrary , the locator guidance can be easily manipulated and is a precise method with minimal experience required . for locator guidance , the two factors of point location and screw trajectory in abumi s procedure were reduced to only one location on the posterior surface of the lateral masses for placing the locator . for the placement of cervical pedicle screws , locator guidance has better accuracy than manual manipulation . we speculate that locator guidance might provide better safety than manual manipulation in placing cervical screws .

ruptured rudimentary horn is a life threatening obstetrical emergency encountered frequently in the emergency department where the diagnosis is either missed or delayed . unicornuate uterus results from abnormal development and fusion of the mullerian ducts usually associated with various degrees of rudimentary horn which may be communicating or non - communicating with the uterine cavity . there is no communication between the two cavities in 75% to 90% of the cases and the incidence of pregnancy in non - communicating horn is high as 83% with incidence of uterine rupture observed in 90% of cases mostly in second trimester as was observed in our case.12 the thin muscular wall of the pregnant uterus ruptures early because of under development and poor distensibility of the myometrium . an unusual case of a primigravida married four months back with pregnancy 22 weeks reported to nizwa regional referral hospital with acute abdominal pain of two hours . on admission she was in shock with pale , cold , clammy extremities , feeble thready pulse 120 beats / minute , blood pressure 78/45 mmhg and respiratory rate 18/min . abdomen was enlarged to 28 weeks size , tense with generalized acute tenderness all over . abdominopelvic ultrasound showed a fetus of 22 weeks with increased free intraperitoneal fluid collection and absent fetal cardiac activity . uterus with cervix separate from the gestational sac was seen clearly lower down in the pelvis . she was taken for emergency laparotomy with the provisional diagnosis of abdominal pregnancy with fetal death . during laparotomy haemoperitoneum of around there was complete rupture of left rudimentary horn of the uterus with the dead fetus lying in the intact amniotic sac covered with 1000 gms of clots [ figure 1 ] . no evidence of placental adherence to the rudimentary horn was observed . the left fallopian tube and left ovary appeared normal and both were attached to the left rudimentary horn [ figure 2 ] . the fetus within the amniotic sac [ figure 3 ] along with the placenta and membranes was removed from the abdominal cavity . there was no communication between the rudimentary horn and the main unicornuate uterine cavity which was confirmed with a probe . the uterus lying separate in the pelvis was soft in consistency , globular and enlarged to 8 weeks size . the right fallopian tube and ovary found healthy were attached normally to the unicornuate uterus . excision of the rudimentary horn and left fallopian tube with conservation of the left ovary was done . the specimen was sent for histopathological examination which was reported as sections from the uterine horn show areas of haemorrhage and necrosis . follow up appointment was arranged for her with a plan for intravenous urogram to rule out any associated renal anomalies . she reported after three weeks with complaints of pain abdomen which subsided with mild analgesics . a repeat abdominopelvic ultrasonography followed by ct abdomen and pelvis was found to be absolutely normal . ( a ) left ruptured rudimentary horn ( b ) fetus in intact amniotic sac ( a ) right unicornuate uterus ( b ) left ruptured rudimentary horn ( c ) left fallopian tube ( d ) left ovary fetus in intact amniotic sac obstructive genitourinary malformations may be diagnosed in prepregnancy workup for complaints of dysmenorrhoea , endometriosis , infertility , and for various pregnancy complications like recurrent miscarriages , preterm labor and malpresentations . however if the rudimentary horn is underdeveloped with non - functional endometrium dysmenorrhoea may be absent . the use of ultrasonography , ct scan , magnetic resonance imaging , 3d ultrasound and laparoscopy may be helpful for diagnosing such abnormalities . buntugu3 used placement of a foley 's catheter into the uterine cavity prior to performing a transabdominal ultrasound for diagnosing an extrauterine pregnancy although not accepted as a preferred method . the associated urologic anomalies are reported to be as high as 50%-80% due to close approximity of the two systems and must be diagnosed either at laparotomy by palpation or postnatally by magnetic resonance imaging or intravenous urogram.4 pregnancy in a non - communicating horn of uterus is possible by intraperitoneal sperm and ovum transmigration or contralateral tubal pick up of the fertilized ovum within the peritoneal cavity . the reported incidence of pregnancy in the rudimentary is 100,000 to 140,000 being a rare form of ectopic pregnancy.5 rupture of pregnancy in rudimentary horn by second trimester is the most common outcome but silent rupture with continuation of pregnancy as secondary abdominal pregnancy was reported in some studies . cases of pregnancy progressing to third trimester and resulting in a live birth after caesarean section have also been documented . pregnancy continued till term as abdominal after ruptured rudimentary horn of a unicornuate uterus and the placenta was attached in part to the myometrium of the horn deriving the blood supply for the live fetus.68 a very unusual case of twin pregnancy in a unicornuate uterus with one fetus in the non - communicating rudimentary horn has been reported where the outcome was successful birth of twins by caesarean section.4 chances of placental adherence are increased due to poorly developed musculature , scant decidualization and small size of the horn . magnetic resonance imaging has been a useful preoperative tool for both diagnosing pregnancy in rudimentary horn and any abnormal placentation . ultrasound has a sensitivity of 33.3% for diagnosing this anomaly and sensitivity reduces with advancing pregnancy adding to the diagnostic dilemma.1 sonographic diagnostic criteria suggested by tsafri9 are presence of pseudo - pattern of an asymmetrical bicornuate uterus , absent visual continuity between the cervical canal and the lumen of the pregnant horn and the presence of myometrial tissue surrounding the gestational sac . immediate surgery is recommended whenever rudimentary horn pregnancy is diagnosed but conservative management until viability is achieved , has been advocated in very select cases with larger myometrial mass where facilities for emergency surgery may be possible any time . a rudimentary horn pregnancy , can never be delivered vaginally and mode of delivery is always a laparotomy both with eventuality of ruptured horn or if pregnancy continues as abdominal post rupture . surgical removal of the rudimentary horn is mandatory to avoid risk of recurrence of rupture with increased maternal morbidity . however , laparoscopic excision of unruptured rudimentary horn pregnancy has been increasingly carried out with safe and favorable outcome in many expert centers now.10 a careful examination of the uterus by experienced obstetrician in every case suspected as mullerian anomaly may help to avoid misdiagnosis and catastrophic haemorrhage . high clinical suspicion , early diagnosis and timely laparotomy can reduce maternal and perinatal mortality and morbidity .

bochdalek hernia is a congenital posterior lateral diaphragmatic defect , which allows abdominal viscera to herniate into the thorax . intrathoracic ectopic kidney accounts for 5% of all renal ectopias and its association with congenital diaphragmatic hernia has been reported to have an incidence of only 0.25% . we report a case of a female who had a left thoracic kidney associated with left bochdalek hernia . a 20 year - old female presented with complaints of dry cough for 15 days and intermittent fever of 4 days duration . the patient does not have any siblings and her mother denied any history of infections or drug abuse during pregnancy . as part of routine investigation chest x - ray was done which showed a left retro cardiac homogenous opacity [ figure 1 ] . abdominal ultrasound ( us ) showed the right kidney to be normal ; left kidney was not visualized . postcontrast computer tomography ( ct ) with 3d reconstruction showed posterior diaphragmatic hernia with herniation of the left kidney into the thorax which was posteriorly located , suggestive of left - sided bochdalek hernia with the upper pole of left kidney in the thorax [ figures 2 and 3 ] . there were no other associated congenital anomalies of renal vessels or any other abdominal organ . an intra venous pyelography done confirmed the diagnosis of a normally located right kidney and properly functioning kidney partially located in the left side of thorax . x - ray of chest showing intrathoracic mass ct image showing intrathoracic kidney ct scan of thorax , axial cuts showing intrathoracic kidney patient was treated symptomatically for her fever and cough . as there were no direct complications due to the hernia or ectopic kidney no definitive treatment was initiated for it . the posterolateral defect in the diaphragm through which abdominal organs might herniate into the thorax was first described by vincent alexander bochdalek , a czech anatomist in 1848 , hence bears his name bochdalek 's hernia . this is presumably due to the fact that the pleuroperitoneal canal closes earlier on the right side , or due to the narrowing of the right pleuroperitoneal canal by the caudate lobe of the liver . the ectopic kidney is usually pelvic , iliac , abdominal , thoracic , contra lateral , or crossed . ectopic kidney is slightly common on the left side as was the case in our patient . the intrathoracic location of an ectopic kidney is the rarest with a reported incidence of 5% of all ectopic kidneys . the association of a bochdalek hernia and intrathoracic renal ectopia is even rarer at 0.25% . during embryogenesis the developing kidney which is located in the pelvis moves upwards to fuse with the adrenals ; and thereby this ascent is arrested . sometimes , this upward progression of the developing kidney does not stop at its designated point and thus the kidney reaches into the thorax . one of the reasons ascribed to this disorderly ascent is the delay in closure of the developing diaphragm . the ectopic kidney has been known to be associated with many other defects ranging from acromelic frontonasal dysplasia to williams syndrome . it is most often detected incidentally on chest imaging or at the time of thoracotomy for a suspected mediastinal tumor . in such patients the symptoms and x - ray picture might be easily confused for pleuritis , pulmonary tuberculosis , or pneumothorax especially in the pediatric age group . chest radiographs are not appropriate , ct scan and mri would be better for diagnosing diaphragm defects . as such an incidentally detected intrathoracic kidney which is asymptomatic does not require any active intervention . its clinical significance lies in its potential to be confused as a pathological thoracic mass leading to its erroneous excision . diagnosis of both these conditions thoracic ectopic kidney and bochdalek hernia can be reached if and only if there is a high degree of suspicion on the part of the treating physician . usually bochdalek hernia is detected incidentally , but very rarely patients might present as an acute emergency due to the strangulation of herniated abdominal contents .

at present , while the trend of cigarette smoking is either stable or decreasing , the trend of other forms of tobacco use , most notably the waterpipe is increasing . waterpipe has a history of as long as four centuries and originates from india and ancient persia . during the past decade , waterpipe smoking ( also known as hookah , goza , shisha , narghile , ghelyan , and hubble - bubble ) has been witnessing a surge in popularity not only in the eastern mediterranean region ( emr ) countries but also in western countries . prevalence studies indicate that the prevalence of current waterpipe smoking is 5 - 17% among american adolescents and 6 - 34% among emr adolescents . although these differences are because of many reasons including sociocultural differences , smoking norms , and societal or legal situations , the consumption has increased in emr and western countries . some waterpipe and cigarette smokers may like some specific features of this form of tobacco use such as its smell or taste and stress relief . maassel , a form of waterpipe tobacco with a variety of flavors ( fruit , coffee , etc . ) , was introduced in the 1990s and tends to be more attractive to youths than the unflavored traditional tobacco used in the waterpipe . waterpipe smoking often occurs during social activities among friends and family members in and out of home . while a large body of studies has examined the role of parent and sibling smoking or the lack of parent - adolescent closeness on smoking , a few studies have investigated parent reactions toward smoking in relation to adolescents smoking . these studies indicate that when parents set rules not to smoke , discuss about smoke - related topics , and punish their children when they smoke , adolescents smoking decreases even when the parents are smokers . moreover , waterpipe smoking is perceived by parents as an acceptable social activity in some cultures such as the arab american community . smoking every tobacco product has adverse health effects and the severity of those effects vary substantially among products . however , people believe that the waterpipe is a safe alternative to cigarette smoking as it causes its smoke to pass through water as a filter . this misconception may be one of the possible reasons for the waterpipe 's popularity . therefore , the present study aims at gaining more insight on waterpipe and cigarette smoking based on sociodemographic factors , perceived parental reaction , and the appeal and repelling factor of smoking among adolescents . the result can improve the understanding of the role of these factors on waterpipe smoking and may also help to take action to prevent this health hazard . an ongoing cross - sectional survey entitled isfahan tobacco use prevention program ( itupp ) was accomplished collaboratively by the isfahan health centre and cardiovascular research center in iran in 2010 . five thousand five hundred students in middle and high schools ( grades 6 - 12 ) were asked to fulfill a self - administered questionnaire in the classroom . schools were then selected randomly from among each cluster , and finally , students were taken from among the selected schools using a random numbers table . within each cluster , stratified sampling was performed based on the school level ( high / middle school ) , gender , and area of residence ( rural or urban area ) . all participants were required to sign a consent form after receiving knowledge on the study goal and design . the students answered the questionnaires in a 30-min period during class time . trained staff members collected the data and provided help to the students in completing the questionnaires . five thousand four hundred and eight questionnaires were completed and returned , corresponding to a 98.3% response rate . trained staff had the responsibility of explaining the study objectives and helping the students in case they needed more explanation . participation was voluntary and filling the questionnaire indicated the student 's consent to take part in the research . the study was approved by the ethics committee of isfahan university of medical science ( 87139 ) . the student 's age , gender ( boy / girl ) , residential area ( urban / rural ) , parental smoking ( yes / no ) , and sibling smoking ( yes / no ) were collected using a self - administered questionnaire . the strata consisted of those who had never been users and never tried , not even one puff , those who tried at least one puff or more , those who tried at least once a month but less than once a week , those who tried at least once a week but less than once a day , andthose who tried at least once a day . those who had never been users and never tried , not even one puff , those who tried at least one puff or more , those who tried at least once a month but less than once a week , those who tried at least once a week but less than once a day , and those who tried at least once a day . the first strata was considered as those who had never smoked , strata 2 and 3 were considered as triers , and 4 and 5 were considered as smokers . students perceptions of the harmful effects of waterpipe were gauged by question how do you judge the waterpipe in comparison to the cigarette ? the answer options were : less harmful , the waterpipe is as harmful as cigarette , andmore harmful . the waterpipe is as harmful as cigarette , and students were asked what they liked most about the waterpipe and cigarette , separately . participants were asked to indicate how their parents would react if they would smoke cigarettes orwould smoke the waterpipe . would smoke cigarettes or would smoke the waterpipe . they punish me , they get angry , they discuss with me , andthey have no reaction . students were asked about the main reasons that were responsible for the waterpipe 's increased popularity among students by using nine items , namely , stressful life and need for relaxation , trend and habit , increased availability , media , peer imitation , freedom , ignorance , belief that it was less harmful than the cigarette , self - expression , and appearance . we computed absolute and relative frequencies for categorical variables and mean and standard deviation for continious one . we used chi - square statistics to determine the relation between waterpipe and cigarette smoking status and the main reasons for the recent increase in waterpipe smoking . we used univariate logistic regression to determine the association between perceived factors related to waterpipe and cigarette smoking separately ( the triers data omitted in this part of the analysis ) . in multiple logistic regression , we controlled age , gender , residency , and parent and sibling smoking . chi - square test compared waterpipe smokers and never - smokers with regard to the most important reasons of increase in waterpipe smoking . for all analyses , we defined statistical significance a priori with a two - tailed alpha of 0.05 . statistical analyses were conducted using the statistical package for the social sciences ( spss ) version 15 ( spss inc . , chicago , illinois , usa ) . an ongoing cross - sectional survey entitled isfahan tobacco use prevention program ( itupp ) was accomplished collaboratively by the isfahan health centre and cardiovascular research center in iran in 2010 . five thousand five hundred students in middle and high schools ( grades 6 - 12 ) were asked to fulfill a self - administered questionnaire in the classroom . schools were then selected randomly from among each cluster , and finally , students were taken from among the selected schools using a random numbers table . within each cluster , stratified sampling was performed based on the school level ( high / middle school ) , gender , and area of residence ( rural or urban area ) . all participants were required to sign a consent form after receiving knowledge on the study goal and design . the students answered the questionnaires in a 30-min period during class time . trained staff members collected the data and provided help to the students in completing the questionnaires . five thousand four hundred and eight questionnaires were completed and returned , corresponding to a 98.3% response rate . trained staff had the responsibility of explaining the study objectives and helping the students in case they needed more explanation . participation was voluntary and filling the questionnaire indicated the student 's consent to take part in the research . the study was approved by the ethics committee of isfahan university of medical science ( 87139 ) . the student 's age , gender ( boy / girl ) , residential area ( urban / rural ) , parental smoking ( yes / no ) , and sibling smoking ( yes / no ) were collected using a self - administered questionnaire . the strata consisted of those who had never been users and never tried , not even one puff , those who tried at least one puff or more , those who tried at least once a month but less than once a week , those who tried at least once a week but less than once a day , andthose who tried at least once a day . those who had never been users and never tried , not even one puff , those who tried at least one puff or more , those who tried at least once a month but less than once a week , those who tried at least once a week but less than once a day , and those who tried at least once a day . the first strata was considered as those who had never smoked , strata 2 and 3 were considered as triers , and 4 and 5 were considered as smokers . students perceptions of the harmful effects of waterpipe were gauged by question how do you judge the waterpipe in comparison to the cigarette ? the answer options were : less harmful , the waterpipe is as harmful as cigarette , andmore harmful . the waterpipe is as harmful as cigarette , and students were asked what they liked most about the waterpipe and cigarette , separately . participants were asked to indicate how their parents would react if they would smoke cigarettes orwould smoke the waterpipe . would smoke cigarettes or would smoke the waterpipe . they punish me , they get angry , they discuss with me , andthey have no reaction . students were asked about the main reasons that were responsible for the waterpipe 's increased popularity among students by using nine items , namely , stressful life and need for relaxation , trend and habit , increased availability , media , peer imitation , freedom , ignorance , belief that it was less harmful than the cigarette , self - expression , and appearance . the strata consisted of those who had never been users and never tried , not even one puff , those who tried at least one puff or more , those who tried at least once a month but less than once a week , those who tried at least once a week but less than once a day , andthose who tried at least once a day . those who had never been users and never tried , not even one puff , those who tried at least one puff or more , those who tried at least once a month but less than once a week , those who tried at least once a week but less than once a day , and those who tried at least once a day . the first strata was considered as those who had never smoked , strata 2 and 3 were considered as triers , and 4 and 5 were considered as smokers . students perceptions of the harmful effects of waterpipe were gauged by question how do you judge the waterpipe in comparison to the cigarette ? the answer options were : less harmful , the waterpipe is as harmful as cigarette , andmore harmful . the waterpipe is as harmful as cigarette , and students were asked what they liked most about the waterpipe and cigarette , separately . participants were asked to indicate how their parents would react if they would smoke cigarettes orwould smoke the waterpipe . would smoke cigarettes or would smoke the waterpipe . they punish me , they get angry , they discuss with me , andthey have no reaction . students were asked about the main reasons that were responsible for the waterpipe 's increased popularity among students by using nine items , namely , stressful life and need for relaxation , trend and habit , increased availability , media , peer imitation , freedom , ignorance , belief that it was less harmful than the cigarette , self - expression , and appearance . we computed absolute and relative frequencies for categorical variables and mean and standard deviation for continious one . we used chi - square statistics to determine the relation between waterpipe and cigarette smoking status and the main reasons for the recent increase in waterpipe smoking . we used univariate logistic regression to determine the association between perceived factors related to waterpipe and cigarette smoking separately ( the triers data omitted in this part of the analysis ) . in multiple logistic regression , we controlled age , gender , residency , and parent and sibling smoking . chi - square test compared waterpipe smokers and never - smokers with regard to the most important reasons of increase in waterpipe smoking . for all analyses , we defined statistical significance a priori with a two - tailed alpha of 0.05 . statistical analyses were conducted using the statistical package for the social sciences ( spss ) version 15 ( spss inc . , chicago , illinois , usa ) . of the 5,500 students selected for participation , we received completed questionnaires from 5,408 ( 98.32% ) individuals , all of whom were included in the final sample of the study . there was a significant relation between cigarette and waterpipe smoking status ( ( df = 4 ) = 172.88 , p < .001 ) . the prevalence of waterpipe smoking was higher than cigarette smoking ( 11.5% versus 5.8% ) . most of the nonwaterpipe smokers ( 96.4% ) were also noncigarette smokers ; however , 79.1% of noncigarette smokers were nonwaterpipe smokers . cigarette smokers were more common ( 70.9% ) waterpipe smokers , and only 35.7% of waterpipe smokers puffed the cigarette as well [ table 1 ] . cross - tabulations of cigarette smoking and waterpipe smoking the boys had almost six times more probability of smoking either the waterpipe ( or = 6 . 06 ; 95% ci : 4.87 - 7.46 ) or the cigarette ( or = 6 . students in urban areas had also a higher chance of smoking ( 2.15-fold for cigarette smoking ( 95% ci 1.31 - 3.37 ) and 1.87 folds for waterpipe smoking ( 95% ci 1.35 - 2.58 ) ) . students whom owning smoker sibling more probably smoked cigarettes ( or : 3.53 , 95% ci 2.68 - 4.64 ) or waterpipe ( or : 5.06 , 95% ci 4.07 - 6.29 ) . although with lower or than sibling smoking , the adolescent was impressionable to parental smoking too . parental smoking increases the odds of cigarette smoking by 1.59-fold ( 95% ci : 1.26 - 2.01 ) and the odds of waterpipe smoking by 2.41-fold ( 95% ci : 2.02 - 2.87 ) . the student perception that parents do react to their child 's smoking behavior was a protective factor for cigarette and waterpipe smoking . the belief that the parents would punish , get angry , or discuss against smoking decreased student cigarette smoking probability by 33% ( or = 0.63 ; 95% ci : 0.50 - 0.79 ) , 56% ( or = 0.44 ; 95% ci : 0.35 - 0.56 ) , and 55% ( or = 0.45 ; 95% ci : 0.35 - 0.58 ) , respectively , when compared to those without these belief . the equivalent decrease for waterpipe smoking was 66% ( or = 0.34 ; 95% ci : 0.28 - 0.41 ) , 71% ( or = 0.29 ; 95% ci : 0.24 - 0.34 ) , and 60% ( or = 0.40 ; 95% ci : 0.33 - 0.48 ) . however , students who thought that their parents showed no reaction had higher odds of 2.48 ( 95% ci : 1.88 - 3.27 ) for cigarette smoking and 3.89 ( 95% ci : 3.22 - 4.71 ) for waterpipe smoking . among appealing characteristics of smoking , the taste ( or = 14.37 ; 95% ci : 11.10 - 18.60 ) and stress relief ( or = 11.03 ; 95% ci : 8.52 - 14.27 ) were more dominant among cigarette smokers , and taste ( or = 18.07 ; 95% ci : 14.80 - 22.07 ) and smell ( or = 13.40 ; 95% ci : 11.00 - 16.69 ) more influential for waterpipe smokers . the repellent characteristics of smoking were perceived strongly by nonsmokers than smokers [ table 2 ] . univariate analysis of influential perceptional factors on cigarette smoking and waterpipe smoking in table 3 , after adjustment , the odds of smoking for students who believed in the equal harm of both the products was 1.33 ( 95% ci : 1.01 , 1.77 ) for cigarette and 1.84 ( 95%ci : 1.4 - 2.31 ) for waterpipe smoker . but those who did not smoke the cigarette and waterpipe thought the cigarette to be more dangerous than the waterpipe . even after adjustment , the approach of the parent 's reaction responses being punish me , get angry , and discuss with me were higher in nonsmokers . smoking in those who expected less extensive parent reactions ( parents who do nothing at all ) increase among cigarette smokers with or = 1.89 ( 95% ci : 1.35 - 2.63 ) and waterpipe smokers with or = 2.75 ( 95% ci : 2.16 - 3.50 ) . taste was rated the most attractive feature by waterpipe and cigarette smokers with or = 2.83 ( 95% ci : 2.06 - 3.90 ) and or = 4.14 ( 95% ci : 2.79 - 6.12 ) , respectively . on the other hand , the social environment had no significant association with waterpipe and cigarette smoking . yellowish teeth with odds of 0.67 and adverse health effect with odds of 0.79 were the most repellent factors of smoking . adjusted multiple logestic regression of influential perceptional factors on cigarette and waterpipe smoking table 4 summarized the main reasons for the current increase in waterpipe smoking among students . 62.7 percent of nonsmokers and 58.4% of smokers expressed stressful life as a reason for the current increase of waterpipe smoking . compared with no waterpipe smokers , higher proportions of waterpipe smokers expressed the trend and habit as the most common reason of increased use of smoking ( 73.8 of the smokers vs 64.6% of nonsmokers , p < 0.001 ) . higher percentage of nonsmokers expressed media ( 62% ) , freedom ( 70.8% ) , ignorance ( 61.6% ) , wrong belief about the absence of harmful effect of the waterpipe ( 63.3% ) , and self - expression ( 66.9% ) as the reasons for increasing the consumption of smoking . there was no difference between the two groups with regard to increased availability and peer imitation . in this study , approximately 35% of waterpipe smokers were cigarette smokers and 70% of cigarette smokers were waterpipe smokers . the present study revealed that parental reaction to adolescent smoking is a protector of smoking . in addition , waterpipe smokers considered taste and smell as the favorable factors of smoking and malodor and yellowish teeth as the unfavorable ones . on the other hand , cigarette smokers considered taste , stress relief , and the social environment as favorable factors and adverse health effect as an unfavorable one . at present , the prevalence of waterpipe smoking has been witnessing a surge among youth in the world , especially eastern countries . in our sample , the result is consistent with the global youth tobacco survey ( gyts ) and new jersey ( nj ) youth tobacco survey . according to gyts and nj youth tobacco survey , current waterpipe smoking had a range of 6 - 34% among 13 - 15-year - olds and 9.7% among high school students . additionally , in the study conducted in london , england , the prevalence of waterpipe smoking was more than double of cigarette smoking , which was similar to our findings . the present findings reveal that although there was a predominance of cigarette smokers who smoked the waterpipe too , only a small number of waterpipe smokers smoked cigarette . these data are consistent with previous studies , which found a strong association between these forms of tobacco use . for example , a study among college students in the united states found that cigarette smokers were twice as likely as nonsmokers to report lifetime waterpipe smoking . additionally , the large proportion of both waterpipe and cigarette smokers perceived the waterpipe to be as harmful as or less harmful than regular cigarettes . this might have been because they considered the waterpipe to be a safer substitution than the cigarette or this might point to the belief that waterpipe is more socially acceptable than cigarette smoking , especially in eastern countries such as iran . for example , some families let their children smoke at home and therefore , are the initiators and supporters of waterpipe smoking among youth , especially in females . also , waterpipe cafes are considered as the most prevalent social places for waterpipe smoking . we also examined the role of familial factors on adolescent smoking by perceived parental reaction toward smoking and the influence of parents and sibling smoking . parent reaction is thought to be the primary source of social influence on smoking and other unhealthy behaviors among adolescents . in our sample , adolescents who perceived that their parents would punish them or discuss with them if they smoked were less likely to be smokers . the finding is in keeping with the finding of the research in 2010 , which reported that the adolescent 's perception of parental punishment was related to reduction in adolescent smoking . the present study also shows that parent and sibling smoking have both been linked to increase adolescent smoking in accordance with a finding reported by several studies . the explanations for this might be that smoking parents are involved in antismoking socialization practices less frequently and less constructively than nonsmoking parents and also some smoking parents believe that smoking in the presence of their children is inevitable . furthermore , we assessed the attitude toward smoking in waterpipe smokers opposed to cigarette smokers . while taste and stress relief are considered as the main positive attributes in the large prevalence of cigarette smokers , most waterpipe smokers like this type of smoking because of its smell and preparation and the minority of waterpipe smokers mention stress relief as a favorable factor . this result is consistent with the outcome of the research by maziac et al . except the stress relief , which is considered as the second reason in both types of smoking in the study . according to several studies conducted in iran , it seems that most iranian people perceived the cigarette as a stress relief and the waterpipe as a hobby . in addition , the majority of cigarette smokers dislike ther cigarette due to its health hazard , which is not an important factor in waterpipe smokers . according to the study conducted by aljarah et al . , higher percentages of waterpipe smoking were associated with the belief that it was less harmful than the cigarette and this belief can play a role in increasing the percentage of regular waterpipe smokers . this study and other studies conducted in the same field reflect that increasing waterpipe smoking , which might have been due to the belief of its less harmfulness . in addition , most studies considered hobby as the main motive behind the increase in waterpipe popularity . for example , in a study conducted in iran among youth aged 12 - 20 years , most respondents considered the waterpipe as a means of entertainment , hospitability , and as a symbol of fashion . but we found that a large proportion of waterpipe smokers believed that habit and dependency were the main factors behind the maintenance of waterpipe smoking . the study 's main limitation was the cross - sectional design of the present study , which only measured the association than causation . second , the data was self - reported and might be subject to social desirability and underreporting . third , we selected a sample as representative of iranian students ; however , sampling from only one province might affect the generalizability of the study . despite these limitations , the strengths of the current study include a large sample with a high response rate ( 98.3% ) and pioneer information about the attitudes and beliefs and the role of parent reaction among iranian adolescents , which is a clue for policymaker in planning the first step of prevention strategies . the study 's main limitation was the cross - sectional design of the present study , which only measured the association than causation . second , the data was self - reported and might be subject to social desirability and underreporting . third , we selected a sample as representative of iranian students ; however , sampling from only one province might affect the generalizability of the study . despite these limitations , the strengths of the current study include a large sample with a high response rate ( 98.3% ) and pioneer information about the attitudes and beliefs and the role of parent reaction among iranian adolescents , which is a clue for policymaker in planning the first step of prevention strategies . in summary , our study shows that the perception of no reaction from the parents and lack of students knowledge about waterpipes health hazards and addictive features of the waterpipe might be associated with an increase in waterpipe smoking among students . additionally , we found that the attitudes toward smoking were different among waterpipe smokers in contrast to the cigarette smokers . hr contributed in the conception and design of the work , drafting and revising the draft , analyzing and interpreting of data , approval of the final version of the manuscript , and agreed to all aspects of the work . zk contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed to all aspects of the work . kh contributed in the conception and design of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed to all aspects of the work . ro contributed in the conception and design of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed to all aspects of the work . ta contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed to all aspects of the work . mn contributed in the conception of the work , data acquisition , approval of the final version of the manuscript , and agreed to all aspects of the work . mj contributed in the conception of the work , data acquisition , approval of the final version of the manuscript , and agreed to all aspects of the work . ms contributed in the conception of the work , analyzing and interpreting of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

rett syndrome ( rtt ) , an x - linked neurodevelopment disorder affecting almost exclusively females , is associated with a single monogenic mutation ( methyl - cpg binding protein 2 , mecp2 ) in up to 95% of cases , more rarely by mutations in cyclin - dependent kinase - like 5 ( cdkl5 ) , and forkhead box protein g1 ( foxg1 ) gene . rtt syndrome , formerly placed in the heterogeneous group of autism spectrum disorders ( asd ) , is now regarded as a distinct pathological entity . the recently issued clinical criteria for rtt diagnosis have clearly defined the core features of the disease . approximately 80% of rtt patients show the typical clinical features : early neurological regression , followed by loss of acquired cognitive , social , and motor skills in a typical four - stage neurological regression , together with development of autistic behavior . rtt patients with atypical clinical presentation usually harbor cdkl5 ( early seizure variant , esv ) or foxg1 ( congenital variant , cv ) mutations , while the preserved speech variant ( psv ) is usually linked to mecp2 mutations . despite almost two decades of research into the functions and role of mecp2 , research , focused largely on in vivo functions of mecp2 in transgenic mice lacking the mecp2 gene , has provided evidence for a repressive action of the transcription factor on neuronal genes associated with dendrite arborization as well as other neuronal functions . in contrast , evidences indicate that , in central nervous systems ( cns ) cells other than neurons , as astrocytes and microglia , mecp2 ablation impairs upregulation of genes , which support dendritic arborization , and the inherent phagocytic activity of microglia [ 7 , 8 ] . in light of demonstrated involvement of microglia in disease progression in mecp2-null mice , we hypothesize the coexistence of a perturbation of the immune system in rtt patients . in particular , a derangement of microglia immune responsiveness might be likely to occur in these patients , as neuroinflammation is a powerful modulator of the cns immune system . in pathological cns conditions , such as rtt and others ( e.g. , multiple sclerosis , ms ) in which neither the antibodies nor the antigens involved in the disease are known , the detection of putative antibodies in biological fluids may be achieved by employing a library of unnatural peptides , which , by virtue of their conformation and posttranslational modifications ( e.g. , n - glucosylation ) , recognize and bind to the relevant antibodies [ 9 , 10 ] . we have previously used this approach to characterize antibodies present in multiple sclerosis patients ' serum by applying a further refinement , that is , the n - glucosylation , to the molecule of the surrogate peptide antigen [ 11 , 12 ] . the rationale for this chemical modification stems from the recognition that n - glucosylation is a pervasive feature of biomolecules occurring in cns pathologies characterized by neuronal disruption . in this work , we took advantage of prior experience gained on ms antibodies using the csf114(glc ) peptide antigenic probe , with the aim of investigating the presence of antibodies in serum of rtt patients , and by comparison to healthy controls and non - rtt pervasive developmental disorders patients . this population consisted of three clearly distinguishable groups : the rtt syndrome group versus non - rtt pervasive developmental disorders ( non - rtt pdd ) group on the basis of the clinical features and the presence of mutated rtt - related genes and healthy , age - matched controls . the rtt group consisted of 53 patients ( mean age 12.8 10.7 years ) who were referred to our clinical unit ( a referral center for a vast region in central italy ) subdivided into n = 41 ( 77.4% ) with classical clinical presentation with proven mecp2 gene mutation and n = 12 ( 12.6% ) atypical presentation ( of whom n = 9 , n = 2 , and n = 1 linked to cdkl5 , foxg1 , and mecp2 mutations , resp . ) . these patients were hospitalized for 1 week every 6 months , in the child neuropsychiatric unit , university hospital azienda ospedaliera universitaria senese of siena ( italy ) , during the course of the study . criteria for inclusion in the study were clinical diagnosis of rtt syndrome coupled with positive identification for the presence / absence of mutated mecp2 , cdkl5 , or foxg1 genes . the age - matched non - rtt pdd group consisted of 82 patients ( mean age 13.0 9.5 years ) , as diagnosed on the basis of well - established criteria . blood samplings in the patients ' group were performed during the routine follow - up study at hospital admission , while the samples from the control group were carried out during routine health checks , sports , or blood donations , obtained during the periodic clinical checks . parents , tutors , or guardians of all the participants provided their written informed consent for the minors to participate in this study . the study design , methods , and consent procedure were approved by the institutional review board of azienda ospedaliera universitaria senese . determination of total plasma igg and igm was performed using the cobas 6000 system ( roche diagnostics ) . briefly , the roche test is based on the principle of agglutination immunoassay where anti - ig antibodies react with the antigen in the sample , forming an antigen - antibody complex , which , after agglutination , is measured turbidimetrically . it is important to note that this method is standard in the clinical chemistry laboratories and is independent from the recognition of the synthetic n - glucosylated peptide epitope . ninety - six well activated polystyrene elisa plates ( nunc maxisorp sigma ) were coated with 1 g per 100 l of glycopeptide in carbonate buffer 0.05 m ( ph 9.6 ) and incubated at + 4c overnight . after five washes with saline containing 0.05% tween 20 , nonspecific binding sites were blocked by fetal bovine serum ( fbs ) , 10% in saline tween 20 ( 100 l per well ) at room temperature for 60 min . sera diluted from 1 : 100 to 1 : 10,000 were applied at + 4c overnight in saline / tween 20/10% fbs . after five washes , we added to each well 100 l of alkaline phosphatase conjugated antihuman igm ( diluted 1 : 200 in saline / tween 20/fbs ; sigma ) or igg ( diluted 1 : 8000 in saline / tween 20/fbs ; sigma ) . after 3 h at room temperature incubation and five washes , 100 l of substrate solution consisting of 1 mg ml p - nitrophenyl phosphate ( sigma ) in 10% diethanolamine buffer was applied . after 30 min , the reaction was stopped with 1 m naoh ( 50 l ) , and the absorbance was read in a multichannel elisa reader ( tecan sunrise ) at 405 nm . elisa plates , coating conditions , reagent dilutions , buffers , and incubation times were tested in preliminary experiments . antibody levels are expressed as absorbance in arbitrary units at 405 nm ( sample dilution 1 : 100 ) . all variables were tested for normal distribution ( d'agostino - pearson test ) and data were presented as either means standard deviation ( sd ) for normally distributed variables or medians and interquartile range values for non - normally distributed data . the efficiency of anti - csf114(glc ) igm in discriminating rtt from either non - rtt pdd patients or healthy control subjects was evaluated using a receiver operating characteristic ( roc ) curve analysis . the medcalc version 12.1.4 statistical software package ( medcalc software , mariakerke , belgium ) was used . the presence of putative serum antibodies ( abs ) in the selected patients ' population and healthy subjects was evaluated by both a conventional agglutination procedure and solid - phase elisa , using an n - glucosylated -turn peptide probe , termed csf114(glc ) . the igm antibody titers clearly segregate the two patient groups ( figure 1 ) . in fact , the individual igm antibody titers found in rtt patients were significantly higher than those in the control groups , that is , both healthy subjects and non - rtt pdd patients . these changes of igm antibodies were detected either by assaying antibodies through an agglutination assay routinely used in the hospital laboratory ( roche cobas 6000 ) or by the n - glucopeptide - based immunoenzymatic assay based on the novel csf114(glc ) antigen probe ( figures 1(a ) and 1(b ) , p < 0.05 ) . although the two assays are not directly comparable , since they provide different absolute values of antibody titers , nonetheless they both indicate a consistent relative increase of serum igm in the rtt group . moreover , we observed a linear correlation between anti - csf114(glc ) serum igm antibody titers and total plasma igm levels in rtt patients ( figure 2 ) . whereas the agglutination assay detects the total serum antibody population , the csf114(glc ) identifies a small fraction of this population . these observed differences are , in our view , evidence that csf114(glc ) captures specific antibodies associated with the disease . receiver operating characteristic ( roc ) curves indicated that anti - csf114(glc ) titers are able to discriminate rtt syndrome from either non - rtt pdd patients or control subjects ( figure 3 ) . at variance , while the csf114(glc ) antigen failed to detect significant differences in auto igg auto - antibody titers among the three groups ( rtt vs. non - rtt pdd vs. healthy controls ) , the total plasma igg titer in the rtt population was significantly decreased . this report describes the changes observed in serum immunoglobulins igm and igg , in rtt patients in comparison to healthy subjects and patients afflicted by diffused autistic traits ( i.e. , non - rtt pdd ) . the two patients groups were defined on the basis of the presence of known gene mutations ( mainly mecp2 ) for the rtt group and of well - established diagnostic criteria for the non - rtt pdd group . although a number of studies have addressed the relationship between rtt and immune system dysfunction , these have mainly focused on their influence on peripheral immune cells , although two early reports investigated humoral immunity in rtt patients , observing evidences of immune system activation [ 13 , 14 ] . here , we demonstrate that rtt patients show a consistent and highly significant increase of the serum igm fraction relative to both healthy controls and non - rtt pdd patients group . we assayed immunoglobulin by a conventional , widely used , hospital procedure based on agglutination and as a complementary assay , an antibody recognition procedure based on a surrogate antigen , termed csf114(glc ) , a peptide bearing n - glucosylation on its moiety . in our view , the igm fraction recognized by csf114(glc ) , tiny in comparison to the large one detected by the agglutination procedure , represents an antibody pool of pathophysiological relevance for this disease . supporting this view is the remarkable specificity of interaction between csf114(glc ) and igm antibodies demonstrated by the receiver operating curve , widely considered a test of accuracy for discriminating between diseased patients and normal subjects . thus , it appears that the rtt patients group can be distinguished with confidence on the basis of serum igm titer detected by the csf114(glc)-based assay . it is of interest that the large serum igm increases , noted here , were also observed by us and others , in a subset of multiple sclerosis patients as well as in meningitis and encephalitis patients ( and references therein ) . it is plausible that the igm changes , noted in these cns pathologies , reflect a common underlying mechanism , most probably a pervasive neuroinflammation . we suggest that the association between neuroinflammation and the observed serum igm rise might be causal to the rtt disease initiation and/or progression . the question arises whether the neuronal derangement elicits the immune response , or conversely , the immune dysfunction might trigger the neuroinflammation . chicken or egg question , there are indications that igm overexpression represents primarily a line of defense against noxious stimuli . in contrast to igm fraction , we found small changes in the serum igg of rtt patients when assayed with the agglutination assay , while the csf114(glc ) assay performed poorly in detecting igg antibodies . further considerations concern the growing research area of epigenetics , which reportedly appears to be involved in autoimmune diseases . these are generally considered to be caused by a combination of epigenetic modification , deregulated immunomodulation , and environmental factors . several single - nucleotide polymorphisms ( snps ) at an x chromosome locus harboring the mecp2 gene have previously been shown to be associated with susceptibility to systemic lupus erythematosus , as well as with a variety of systemic autoimmune diseases . recent reports on experimental models in mice have placed microglia at the center stage of the rtt progression , as the mecp2-deficient mice reproduce most of the human rtt phenotype features [ 7 , 16 ] . because microglia exert a prominent immune surveillance role in the cns , it is likely that the large serum igm increase reflects and signals a severe disturbance of immune regulation in brain . in conclusion , the results of this work contribute to elucidate that the two disease conditions such as rett syndrome and the diffuse autism , seemingly contiguous as they share some behavioral traits , are in fact dramatically different for their severity , life - span expectancy , and , as we now learn , immune system derangement .

leishmaniasis , also known as kala - azar ( literally meaning black fever or black disease ) is a zoonosis caused by protozoan of the genus leishmania , which is transmitted by bite of insect vector phlebotomus sandfly . still it is a rare infection in postrenal transplant setting with atypical presentation leading to delayed diagnosis and considerable morbidity and mortality . high index of suspicion , particularly in patients from endemic areas , is required in early diagnosis and improve the disease outcome . we report a case of 50-year - old male renal allograft recipient who presented to us with fever , sore throat , hoarseness of voice , lymphadenopathy , and splenomegaly and was diagnosed to have visceral and laryngeal leishmaniasis . a 50-year - old male , resident of nepal underwent live - related renal transplant in 2003 for a basic renal disease no induction was used and he was on maintenance immunosuppression with cyclosporine , azathioprine , and prednisolone . he presented after 7 years with the history of sore throat of 3 months duration and fever of 1-month duration . he consulted a local ent surgeon 3 months before presenting to us and was told to have vocal cord polyp . he was also seen by a local practitioner for fever and treated empirically with ceftriaxone 2 g daily for 7 days , however fever persisted . on examination he was febrile , had bilateral cervical lymphadenopathy measuring up to 3 cm . blood investigations revealed serum creatinine 1.1 mg / dl , total bilirubin 1.5 mg / dl , direct bilirubin 0.5 mg / dl , ast 108 u / l , alt 91 u / l , total protein 5.0 g / dl , serum albumin 2.3 g / dl , and serum globulin 2.7 g / dl . he had pancytopenia ( hb , 9.3 g / dl ; total leukocyte count , 1,560 cells / mm , platelet count , 85 10/l ) . in addition , there were intra- and extracellular leishman donovan bodies ( amastigote form ) [ figure 1 ] . ultrasound abdomen showed splenomegaly ( spleen size , 14 cm ) and multiple enlarged retroperitoneal lymph nodes . bone marrow trephine biopsy show intra- and extracellular amastigote forms of leishmaniasis ( h and e stain , 100 ) excisional biopsy of cervical lymph node revealed complete replacement of nodal architecture by infectious pathology and was studded with intra - and extracellular amastigote form of protozoa ( leishman donovan bodies ) accompanied with plasma cell response . he was also evaluated for hoarseness of voice and sore throat by ent surgeon . microlaryngoscopy showed irregular lesion on the left vocal cord , that was diffuse and involving full length . donovan bodies ( amastigote form ) and accompanying acute inflammatory cells [ figure 2 ] . giemsa , pas , and silver stain in both lymph node and vocal cord biopsy confirmed the organisms as leishmaniasis . vocal cord biopsy shows numerous extracellular amastigote forms of leishmaniasis ( pasm stain , 40 ) he was started on treatment with liposomal amphotericin b. he completed total cumulative dose of 2 g iv amphotericin b. the fever subsided and pancytopenia improved . after 8 months of follow - up , patient has remained asymptomatic without any further recurrence of the disease . leishmaniasis is endemic in india , bangladesh , nepal , parts of africa , southwest asia , spain , as well as other mediterranean coast . in indian subcontinent , transmission is anthroponotic and humans are the reservoir . cell - mediated immunity determines the outcome of infection , and hence only minority of infected individuals develop clinically apparent disease . the immunological response , particularly interleukin-12 driven th1 response has been widely accepted as promoting protective immunity against all species of leishmania . immunosuppressive drugs used in transplant patients hamper t - cell activation and proliferation , thereby altering defense mechanisms against intracellular microorganisms . with increasing frequency of renal transplantation as the preferred renal replacement treatment modality , several cases of visceral leishmaniasis are being detected in renal transplant recipients . among the organ transplant leishmaniasis disease , that is most commonly seen with disease is : cyclosporine plus azathioprine and steroids . the total number of cases reported in renal transplant is less than 100 . hernandez - perez et al . reported five cases of leishmaniasis in solid organ transplant recipient out of which three were renal transplant recipients . oliveira et al . reported eight cases ( 1% ) of leishmaniasis in renal transplant recipients . reported five renal transplant recipients presenting in early post - transplant period and eventually leading to graft loss in two . it presents with fever ( 94% ) , hepatomegaly ( 42% ) , splenomegaly ( 75% ) , and pancytopenia ( 85% ) . our patient had classical presentation of fever , splenomegaly , pancytopenia suggesting bone marrow suppression and in addition , lymphadenopathy . he had the deranged liver function test in form of mild hyperbilirubinemia , hypoalbuminemia , and elevated ast and alt . histopathological diagnosis rests on visualization of amastigotes , which are spherical , or ovoid bodies measuring 1 to 5 m . the diagnosis of vl and ml in our case was done on the basis of biopsy of the lymph node , bone marrow , and vocal cord lesion , which provided unequivocal evidence of leishman donovan bodies . laryngeal involvement in leishmaniasis is a rare presentation even in general population.[611 ] it can either present with other systemic manifestations or isolated laryngeal involvement . mucosal involvement of the nose , oral cavity , pharynx , or larynx occurs in 25% of persons infected with leishmania braziliensis . described three cases of post kala - azar dermal leishmaniasis presenting with hoarseness of voice and biopsy of nodular lesions on vocal cord confirmed leishmaniasis in nontransplant patients . in separate reports some of the patients have presented like mass lesions simulating neoplasia . despite extensive pubmed literature search , we could not find a report of vocal cord leishmaniasis in a renal allograft recipient . agents used for treatment of visceral leishmaniasis include amphotericin b , pentavalent antimonial drugs , paromomycin , and miltefosine . liposomal amphotericin b has highest therapeutic efficacy and it is safest among all these , although conventional amphotericin b has considerable renal toxicity . in india and nepal , high level of resistance to antimonial drugs has been observed and hence amphotericin b is being used as first line agents.[1416 ] although leishmaniasis is a rare disease in a post - transplant setting even in endemic areas , it has got high morbidity and mortality in untreated cases . clinical presentation may be atypical and high index of suspicion is a prerequisite for timely evaluation and appropriate treatment which can be life saving .

the drosophila slowpoke channel ( dslo ) is a large conductance voltage - gated , calcium - dependent potassium channel ( atkinson et al . , 1991 ; dslo has many tissue - specific splice variants with different properties ( becker et al . , 1995 ; atkinson et al . , 1998 ) , and plays important roles in neuronal excitability , neurotransmitter release , and muscle contraction ( warbington et al . , 1996 ; atkinson et al . , 2000 unlike most other potassium channels , dslo channels have a large carboxy - terminal tail region . 2001 ) and is the target of phosphorylation and binding of other regulatory proteins that modulate channel activity ( xia et al . , 1998 ; schopperle et al . , 1998 ; wang et al . , 1999 ) . the dslo binding protein slob from drosophila melanogaster was discovered by a yeast two - hybrid screen using the carboxy - terminal tail region of dslo as bait ( schopperle et al . , 1998 ) . it binds to and modulates the dslo channel ( schopperle et al . , 1998 ; zhou et al . , 1999 , 2003 ) slob was originally described as a 511amino acid protein that contains a protein kinase like domain , and in vitro assays indicated that slob exhibits weak but regulatable protein kinase activity ( zeng et al . , 2004 ) . slob mrna ( mcdonald and rosbash , 2001 ; claridge - chang et al . , 2001 ueda et al . , 2002 ) and protein ( jaramillo et al . , 2004 ) cycle in a circadian manner in vivo , and may participate directly or indirectly in circadian pathways ( jaramillo et al . , 2004 ) . previous studies of the modulation of dslo by slob used an epitope - tagged slob construct , ha - slob57 , in which an ha tag was fused to the amino - terminal region of slob . we reported that coexpression of dslo with this construct shifts the voltage dependence of dslo activation to less depolarized voltages ( zhou et al . , 1999 ) . recently , we have found that there are several slob proteins , resulting from alternative splicing and multiple translational start sites ( unpublished data ) , and have named them based on their molecular weights ( in kd ) . the results indicate that slob57 ( the originally characterized variant ) and slob51 shift the voltage dependence of dslo activation to more depolarized voltages , cause an apparent inactivation of dslo , and make the channel close faster . this is in contrast to the hyperpolarizing shift we originally reported with an amino - terminal ha - tagged version of slob57 ( zhou et al . , 1999 ) . all other slobs shift the conductance voltage relationship of dslo to less depolarized voltages and have no significant effect on dslo kinetics . the difference between slob57/51 and other slobs ( including ha - slob57 ) is in their amino termini . thus the amino - terminal region of slob57/51 appears to play a critical role in modulating the voltage dependence and deactivation kinetics of dslo . all slob cdnas were subcloned into the pires2-egfp vector ( clontech laboratories , inc . ) for mammalian expression and electrophysiological studies . site - directed mutagenesis was performed using the quikchange site - directed mutagenesis kit , following the manufacturer 's instructions ( stratagene ) . the slowpoke channel used in this study is the alternative splice variant a1c2e1g3 ( genbank / embl / ddbj accession no . m96840 ) , which was subcloned into the prc / cmv vector ( invitrogen ) . chinese hamster ovary ( cho ) cells were maintained in 75-cm culture dishes with ham 's f-12 nutrient mixture ( invitrogen ) , supplemented with 10% fbs ( invitrogen ) and 100 u / ml penicillin and streptomycin ( invitrogen ) . plasmids were transfected into cho cells using lipofectamine 2000 reagent , according to the manufacturer 's specifications ( invitrogen ) . cells were harvested the second day after transfection . in brief , after harvest and wash with pbs , cells were resuspended in 500 l lysis buffer ( 25 mm tris - hcl , ph 7.5 , 150 mm nacl , 50 mm naf , 50 mm kcl , 1% chaps , 1 mm phenylmethylsulfonyl fluoride , 2 mm dtt , 1 mg / ml each aprotinin , leupeptin , and pepstatin a ) with rotation at 4c for 30 min . lysates were centrifuged at 16,000 g for 10 min , and the supernatants were used for western blot analysis of expression . cell lysates were loaded onto 415% gradient sds polyacrylamide gels ( bio - rad laboratories , inc . ) . membranes were blocked in tris - hcl buffered saline with 0.1% tween 20 ( tbst ) containing 5% nonfat milk . membranes were incubated with primary antibody ( schopperle et al . , 1998 ) for 1 h at room temperature . following washes with tbst , membranes were incubated with an appropriate secondary antibody ( horseradish peroxidase conjugated donkey anti - rabbit antibody ) at room temperature for 1 h. labeled proteins were detected using an enhanced chemiluminescence reagent according to the manufacturer 's specifications ( amersham biosciences ) . cho cells were transiently transfected with dslo and different slob cdnas in a 1:1 molar ratio using lipofectamine as described above . 1 d after transfection , whole cell currents were recorded from cells exhibiting green fluorescence , using an axiovert 25 inverted fluorescence microscope ( carl zeiss microimaging , inc . ) and an axopatch 200a amplifier ( axon ) . glass pipettes with 24 m resistance were pulled from thinwall glass ( wpi ) using a two - stage vertical puller ( narishige ) . both the bath and pipette solutions contained ( in mm ) 100 kcl , 0.5 hedta , 2 mgcl2 , and 10 hepes , ph 7.2 ( symmetrical conditions ) . the pipette solution also contained different amounts of free ca ( 4 m , 20 m , 40 m , or 110 m ) . total cacl2 required to generate the desired free ca was calculated ( webmaxc ; http://www.stanford.edu/~cpatton/webmaxcs.htm ) , and the appropriate amount of cacl2 from a 100 mm cacl2 solution ( orion ) was added to the pipette solution . the final concentration of free ca was measured with a fisher accumet calcium - sensitive electrode , and determined against a standard curve of free ca generated with the calcium - sensitive electrode and calcium standards ( wpi ) . currents were elicited by depolarizing test pulses to different voltages from a holding potential of 80 mv , followed by a hyperpolarizing step to 120 mv to measure tail currents . currents were filtered at 2 khz , digitized at 10 khz with a digidata 1322a digitizer ( axon ) , and acquired with pclamp9 software ( axon ) . peak tail currents were normalized to the maximum peak tail current of the patch and fitted with a boltzmann function to generate the conductance voltage curves . all slob cdnas were subcloned into the pires2-egfp vector ( clontech laboratories , inc . ) for mammalian expression and electrophysiological studies . site - directed mutagenesis was performed using the quikchange site - directed mutagenesis kit , following the manufacturer 's instructions ( stratagene ) . the slowpoke channel used in this study is the alternative splice variant a1c2e1g3 ( genbank / embl / ddbj accession no . m96840 ) , which was subcloned into the prc / cmv vector ( invitrogen ) . chinese hamster ovary ( cho ) cells were maintained in 75-cm culture dishes with ham 's f-12 nutrient mixture ( invitrogen ) , supplemented with 10% fbs ( invitrogen ) and 100 u / ml penicillin and streptomycin ( invitrogen ) . plasmids were transfected into cho cells using lipofectamine 2000 reagent , according to the manufacturer 's specifications ( invitrogen ) . cells were harvested the second day after transfection . in brief , after harvest and wash with pbs , cells were resuspended in 500 l lysis buffer ( 25 mm tris - hcl , ph 7.5 , 150 mm nacl , 50 mm naf , 50 mm kcl , 1% chaps , 1 mm phenylmethylsulfonyl fluoride , 2 mm dtt , 1 mg / ml each aprotinin , leupeptin , and pepstatin a ) with rotation at 4c for 30 min . lysates were centrifuged at 16,000 g for 10 min , and the supernatants were used for western blot analysis of expression . cell lysates were loaded onto 415% gradient sds polyacrylamide gels ( bio - rad laboratories , inc . ) . membranes were blocked in tris - hcl buffered saline with 0.1% tween 20 ( tbst ) containing 5% nonfat milk . membranes were incubated with primary antibody ( schopperle et al . , 1998 ) for 1 h at room temperature . following washes with tbst , membranes were incubated with an appropriate secondary antibody ( horseradish peroxidase conjugated donkey anti - rabbit antibody ) at room temperature for 1 h. labeled proteins were detected using an enhanced chemiluminescence reagent according to the manufacturer 's specifications ( amersham biosciences ) . cho cells were transiently transfected with dslo and different slob cdnas in a 1:1 molar ratio using lipofectamine as described above . 1 d after transfection , whole cell currents were recorded from cells exhibiting green fluorescence , using an axiovert 25 inverted fluorescence microscope ( carl zeiss microimaging , inc . ) and an axopatch 200a amplifier ( axon ) . glass pipettes with 24 m resistance were pulled from thinwall glass ( wpi ) using a two - stage vertical puller ( narishige ) . both the bath and pipette solutions contained ( in mm ) 100 kcl , 0.5 hedta , 2 mgcl2 , and 10 hepes , ph 7.2 ( symmetrical conditions ) . the pipette solution also contained different amounts of free ca ( 4 m , 20 m , 40 m , or 110 m ) . total cacl2 required to generate the desired free ca was calculated ( webmaxc ; http://www.stanford.edu/~cpatton/webmaxcs.htm ) , and the appropriate amount of cacl2 from a 100 mm cacl2 solution ( orion ) was added to the pipette solution . the final concentration of free ca was measured with a fisher accumet calcium - sensitive electrode , and determined against a standard curve of free ca generated with the calcium - sensitive electrode and calcium standards ( wpi ) . currents were elicited by depolarizing test pulses to different voltages from a holding potential of 80 mv , followed by a hyperpolarizing step to 120 mv to measure tail currents . currents were filtered at 2 khz , digitized at 10 khz with a digidata 1322a digitizer ( axon ) , and acquired with pclamp9 software ( axon ) . peak tail currents were normalized to the maximum peak tail current of the patch and fitted with a boltzmann function to generate the conductance voltage curves . slob protein was discovered by a yeast two - hybrid screen using the carboxy - terminal tail region of dslo as bait ( schopperle et al . , 1998 ) . it binds to dslo and modulates the channel activity ( schopperle et al . , 1998 ; zhou et al . , 1999 recently we found that at least four different slob transcripts are present in the fly ( unpublished data ) . these transcripts can initiate translations at either met1 or met124 and contain or exclude a spliceable exon , exon 7 of the slob gene . slob71 , the largest variant , starts at met1 and contains 634 amino acid residues , including exon 7 ( fig . 1 , green ) , which encodes the region from ser197 to thr252 ( all numbering throughout the text is relative to met1 , the first amino acid residue in slob71 ; fig . the slob that also starts at met1 , but excludes exon 7 , is a 65-kd protein called slob65 ( fig . 1 ) . the smaller slob , which initiates at met124 and contains exon 7 , is the slob variant isolated in the yeast two - hybrid screen ; it is now renamed slob57 . initially , an ha - tagged version of slob57 ( ha - slob57 ) was used in our electrophysiological analysis ( zhou et al . , 1999 ) . the ha epitope was appended to an arginine residue ( arg151 ) near the amino terminus of slob57 ( fig . 1 ) . six slob proteins from a single slob gene . there are at least four different transcripts of the slob gene in vivo . these transcripts can initiate translations at either met1 ( m1 ) or met124 ( m124 ) , and contain or exclude the spliceable exon 7 ( green ) . we named them slob71 , slob65 , slob57 , and slob51 based on their molecular weights . a third met ( m163 ) can be recognized by the ribosome to translate two more slobs : slob53 and slob47 . the epitope - tagged slob used in our previous study was slob57 , with an amino - terminal ha epitope appended to arg151 ( r151 ) . all amino acid numbering is relative to the first met ( m1 ) in slob71 . to facilitate in vitro electrophysiological analysis of slobs , we subcloned the cdnas of different slobs into the pires2-egfp vector , which expresses gfp , in order to express slobs in cho cells . to prevent multiple initiations of translation , the atg ( encoding met124 ) in the slob71 and slob65 constructs was mutated to ctg ( encoding leu ) . we first tested the heterologous expression of these slob constructs in cho cells using western blot with anti - slob antibody that recognizes all slobs . to our surprise , we found multiple bands in these western blots ( fig . 2 a ) . for each slob , in addition to the band with the expected molecular weight ( the top bands in lanes 14 of fig . 2 a ) , there is another major band with a lower molecular weight . ( a ) different slobs were transiently transfected into cho cells . on the second day , proteins in cell lysates were separated by gel electrophoresis , transferred onto nitrocellulose membranes , and blotted with anti - slob antibody . lane 1 , slob71 construct with m124l mutation ; lane 2 , slob65 construct with m124l mutation ; lane 3 , slob57 construct ; lane 4 , slob51 construct ; lane 5 , pires2-egfp vector only . ( b ) individual slob variants can be expressed by mutating one or more translational start sites ( see fig . lane 1 , slob71 construct with m124l and m163l mutations ; lane 2 , slob65 construct with m124l and m163l mutations ; lane 3 , slob57 construct with m163l mutation ; lane 4 , slob57 construct with m124l mutation , which expresses only slob53 ; lane 5 , slob51 construct with m163l mutation ; lane 6 , slob51 construct with m124l mutation , which expresses only slob47 ; lane 7 , pires2-egfp vector only . a closer examination of the cdna sequence of slob reveals that around met163 , the cdna sequence is attatgaag . there is an adenine ( bold ) at the 3 position of this atg ( encoding met163 ) , which is critical for the ribosome to recognize that particular atg and initiate translation ( kozak , 1991 , 2002 ) . therefore , we suspected that the lower bands are two smaller slobs initiated at met163 ( fig . 1 ) . to test this hypothesis , we mutated the atg ( encoding met163 ) to ctg ( encoding leu ) in our original constructs of slob71 , slob65 , slob57 , and slob51 . when these constructs are expressed , only the top bands with the expected molecular weights remain ( compare lanes 13 and 5 in fig . 2 a ) . furthermore , when we mutated the atg ( encoding met124 ) in the original constructs of slob57 and slob51 to ctg ( encoding leu ) , but kept the atg of met163 intact , we saw two new bands on the western blot ( fig . 2 b , lanes 4 and 6 ) , corresponding to the lower bands in lanes 14 of fig . 2 a. therefore two additional slobs , slob53 and slob47 , can be expressed in this heterologous expression system . these two slobs lack the first 39 amino acid residues at the amino - terminal region of slob57 and slob51 ( fig . there is also an unidentified band observed in all slob - expressing cells that migrates at an apparent molecular weight slightly smaller than slob47 ( fig . previous studies indicated that slob57 can coimmunoprecipitate with dslo in a heterologous expression system and in vivo ( schopperle et al . the dslo binding region in slob is from asn314 to glu356 ( zhou et al . , 2003 ) . as expected , anti - slob antibody was able to coimmunoprecipitate dslo when it was coexpressed with any of the slobs ( unpublished data ) . we examined the effects of all the slobs on dslo channel function by cotransfecting each with dslo in cho cells , which exhibit very low levels of endogenous potassium current ( fig . , we found that a high concentration of free ca is required to achieve full dslo activation and tail current saturation in the presence of slob57 . therefore we chose to study the modulatory effects of the different slobs in the presence of 110 m free ca . whole cell outward currents were elicited by a 350-ms test pulse to different voltages from a holding potential of 80 mv , followed by hyperpolarization to 120 mv to measure tail currents ( fig . 3 , c f ) of various slobs ( slob71 , slob65 , slob53 , and slob47 ) indicates that there is no change in activation or deactivation kinetics . such shifts are reminiscent of ( although smaller than ) those reported previously with ha - tagged slob57 ( zhou et al . , 1999 ; unpublished data ) . whole cell currents evoked by depolarizing voltage steps ( g ) with 110 m free ca , in cells transfected with vector alone ( a ) , dslo alone ( b ) , or together with different slobs as indicated ( c f ) . currents were elicited by a 350-ms test pulse to different voltages from a holding potential of 80 mv , followed by hyperpolarization to 120 mv to measure tail currents . voltage relationships for dslo transfected alone or together with slob71 , slob65 , slob53 , or slob47 were measured from tail currents with 110 m free ca , as shown in fig . voltage relationship by slob71 , 65 , 53 , and 47 the v1/2 for dslo activation and the slope of the conductance the pattern of modulation is very different when slob57 or slob51 is cotransfected with dslo , as shown in fig . coexpression with these slobs causes an apparent inactivation of the dslo current during the 350-ms depolarizing pulse . because this apparent inactivation compromises our ability to measure the conductance voltage relationship from tail currents , we repeated this experiment using a shorter depolarizing pulse , during which the channel does not have time to inactivate . as shown in fig . 5 ( e and f ) , slob57 and slob51 cause a decrease in current compared with dslo cotransfected with control vector ( fig . 5 d ) . the channel also appears to close faster in the presence of these two slobs , as indicated by the more rapid deactivation of the tail currents . as shown in fig . 6 and table ii , slob57 and slob51 shift the conductance in addition , there is a significant decrease in the slope of the conductance voltage curve . voltage relationships are summarized for slobs71 , 65 , 53 , and 47 in table i , and for slobs57 and 51 in table ii . whole cell currents , with 110 m free ca , were evoked by either 350 ms ( a c ) or 100 ms ( d f ) depolarizing pulses , followed by hyperpolarization to 120 mv to measure tail currents . dslo was expressed either alone ( a and d ) or together with either slob57 ( b and e ) or slob51 ( c and f ) . 4 , except the effects of slob57 and slob51 were measured using the shorter pulse protocol in fig . 5 ( d f ) . modulation of the dslo conductance voltage relationship by slob57 and 51 the v1/2 for dslo activation and the slope of the conductance 6 ( short pulse protocol ) . significantly different from dslo + vector ( p < 0.001 ; student 's t test ) . we fitted the current traces with standard exponential functions to obtain the activation and deactivation time constants of dslo when it is expressed with vector alone or with one of the slob variants . we found that the time course of dslo deactivation was very different in the presence of slob57 or slob51 ( fig . there were no differences in deactivation time constants in the presence of the other slobs , and there were no differences in activation time constants of dslo in the presence of any slob ( unpublished data ) . when dslo is expressed alone , the time constant of dslo deactivation can be well fitted using a single exponential function with a mean value of 8.4 ms . in contrast , when dslo is coexpressed with slob57 , two exponential functions are required to fit the deactivation time course of dslo . these time constants are voltage independent . the mean fast time constant f is 3.3 ms with a weight of 72% , while the mean slow time constant s is 26 ms and contributes 28% . both the f and the s are significantly different ( p < 0.001 ) from the single of dslo when it is expressed alone . deactivating tail currents following a 100-ms test pulse to + 40 mv , with 110 m free ca , in the absence or presence of slob57 or slob51 , were normalized and superimposed . the deactivation time constants for dslo expressed alone , or together with slob57 or slob51 , were obtained by fitting the tail currents with exponential functions . both the current traces themselves and the exponential fits to the data ( smooth lines ) are shown in the figure . when dslo is expressed without slobs , in the voltage range from 20 to + 40 mv , the tail currents can be fitted with a single exponential function with a mean , voltage - independent of 8.4 0.5 ms . in the presence of slob57 or slob51 , the tail currents can only be fitted , in the voltage range from + 20 to + 180 mv , with two voltage - independent exponential functions . both the f and the s of dslo in the presence of slob57 or slob51 are significantly different ( p < 0.001 ) from the of dslo expressed alone . to determine whether the modulation by various slobs is influenced by the intracellular free ca concentration , we examined the effect of slob53 and slob71 in the presence of 40 or 20 m free ca ( the latter is the lowest ca concentration at which we could achieve tail current saturation in the voltage range of our amplifier ) . 8 , under these conditions , neither slob53 nor slob71 produces the small leftward shift in the conductance voltage relationship that is seen at 110 m free ca ( fig . 4 and table i ) , suggesting that this modulatory response is calcium dependent . although the lack of tail current saturation prevented us from measuring the effects of slob57 and slob51 on the conductance voltage relationship at these lower free ca concentrations , we did examine whether the apparent inactivation of dslo caused by slob57 is ca dependent . 9 , slob57 causes a similar inactivation of the dslo current during a 350-ms depolarizing pulse , over a wide range of free ca concentrations . 4 , except the effects of slob53 and slob71 were measured with 20 m ( filled symbols ) or 40 m ( open symbols ) free ca . whole cell currents , with ( a ) ca - free solution ( no added ca ) , ( b ) 4 m free ca , ( c ) 20 m free ca , ( d ) 40 m free ca , were evoked by the same pulse protocol as shown in fig . 3 g. dslo was expressed either alone or together with slob57 as indicated . we have found a total of six slob proteins , resulting from alternative splicing and multiple translational start sites from a single slob gene . at least four of these slobs are expressed in adult fly heads in vivo ( unpublished data ) , and it is conceivable that the other two may be expressed in other tissues and/or at other developmental stages . all of these slobs bind dslo and modulate the voltage dependence of the slowpoke channel . slob57 and slob51 cause an apparent inactivation of dslo , significantly shift the voltage dependence of dslo activation to more depolarized voltages , greatly decrease the slope of the conductance voltage relationship , and increase the deactivation rate of the channel . in contrast , all other slobs , including the amino - terminal ha - tagged ha - slob57 , do not cause this apparent inactivation , do not change the deactivation rate of dslo , and shift the conductance although we have found independent mrna transcripts in fly heads coding for slob71 , slob65 , slob57 , and slob51 using rt - pcr ( unpublished data ) , we do not yet have direct evidence for the existence of slob53 and slob47 in vivo . however , ribosomes can recognize the atg of met163 and initiate translation there , and the efficiency for the ribosome to recognize this start site appears to be high , as indicated by the following : ( a ) an adenine at the 3 position of this atg site makes this region a kozak sequence - like region , which is efficiently recognized by the ribosome to start translation ( kozak , 1991 , 2002 ) ; ( b ) in the slob57 construct , when the atg for met163 is intact , the yield of slob57 and slob53 is almost equal ( fig . 2 a ) ; ( c ) in the same slob57 construct , when the start site ( met124 ) for slob57 is mutated , slob53 can still be well expressed ( fig therefore , it seems likely that the atg of met163 is an independent start site of translation of slob mrna , and slob53 and slob47 are likely to exist in vivo . the fact that the ribosome can translate both slob57 and slob53 from a single slob mrna is not an isolated example , as many studies have shown that ribosomes can initiate translation at different start sites in a single message ( stacey et al . , 2000 ; xiong et al . , 2001 ; takeda et al . , 2004 ; wang and rothnagel , 2004 ; yang et al . , 2004 ) . since both slob53 and slob47 contain the dslo binding region ( zhou et al . , 2003 ) , it is not a surprise that they both bind dslo ( unpublished data ) . when slob71 is coexpressed with dslo , it slightly but significantly shifts the voltage dependence of dslo activation to less depolarized voltages at a high concentration of free ca . however , at lower concentrations of free ca , slob71 has little effect on dslo , suggesting that slob71 activates dslo in a ca - dependent manner . we have not yet explored the mechanism of the ca dependence of this modulatory phenomenon . slob65 , slob53 , and slob47 have a similar effect on the voltage dependence of dslo activation . ha - slob57 , the slob construct used in our earlier studies , has a similar but larger effect on dslo ( zhou et al . , 1999 ) . in contrast , the untagged slob57 and slob51 modulate dslo in an entirely different way . under our experimental conditions , they cause an apparent inactivation of dslo that is ca independent ( fig . 9 ) , while dslo expressed alone or coexpressed with other slobs does not inactivate during a 350-ms depolarizing pulse . to circumvent this apparent inactivation , a shorter test pulse was used to study the effect of slob57 and slob51 on the voltage dependence of dslo activation ; a similar procedure has been used in other studies that involve channel inactivation ( jerng et al . , 1999 ; xia et al . , 2000 ) . the conductance voltage relationship of dslo , when expressed alone , is the same whether the short or long pulse is used ( tables i and ii ) . studies using the short pulse show that slob57 and slob51 significantly shift the conductance voltage relationship of dslo to more depolarized voltages , and greatly decrease the slope of the conductance voltage curve . besides right shifting the conductance voltage relationship , slob57 and slob51 also cause dslo to close faster , as indicated from an examination of the deactivating tail currents ( fig . 7 ) . the fitting of the deactivation time constant requires two exponential functions , indicating that in the presence of slob57 or slob51 , dslo needs two steps , a dominant fast one and a minor slow one , to close . dslo expressed alone in cho cells can close in a single step under our experimental conditions , although dslo expressed in xenopus oocytes needs two steps to close ( dichiara and reinhart , 1995 ) . since slob57 and slob51 produce very similar effects , it seems that the spliceable region ( from ser197 to thr252 ) of slob plays little role in this modulation of dslo . the modulatory effects of slob57 and slob51 on dslo are totally different than those of other slobs . the difference in amino acid sequence between slob57/slob51 and the other slobs is at the amino - terminal region . slob71/slob65 have a 123-residue extension in front of the amino terminus of slob57/slob51 , whereas slob53/slob47 lack the amino - terminal 39 amino acid residues of slob57/slob51 . in the case of ha - slob57 , the 10-residue ha epitope was appended to arg151 , the 28th amino acid residue of slob57 ( fig . the correlation between the different amino termini and the different modulatory effects indicates that the amino - terminal 39-residue region of slob57/slob51 plays a critical role in the modulation of dslo activity . it must form part or all of the functional domain that causes the apparent inactivation of dslo , shifts the voltage dependence of dslo to more depolarized voltages , and makes dslo close faster . when this 39-residue region is removed , the strong inhibitory effect of slob on dslo is eliminated , and the remaining protein ( i.e. , slob53 and slob47 ) slightly activates dslo . in the larger slobs , slob71 and slob65 , the extra 123-residue region that precedes this critical region may mask its effects , such that the larger slobs have the same minimal effects on dslo as slob53 and slob47 . dslo belongs to the large conductance , voltage - gated , and calcium - dependent potassium ( bk ) channel family . although dslo inactivation has not been reported previously , some mammalian bk channels do exhibit inactivation ( solaro and lingle , 1992 ; li et al . , 1999 ; pyott et al . , this trypsin - sensitive ( solaro and lingle , 1992 ) and ca - dependent ( hicks and marrion , 1998 ; ding and lingle , 2002 ) inactivation is caused by auxiliary 2 or 3 subunits ( wallner et al . , 1999 ; xia et al . , 1999 , 2000 ; uebele et al . , 2000 ) . the amino - terminal residues 24 of the 2 subunit may directly block the bk channel to cause inactivation ( xia et al . , 2003 ) , and the amino - terminal 33 residues of the 3 subunit also play a critical role in causing bk inactivation ( xia et al . , 1999 ) . therefore , it seems possible that slob57 and slob51 may function like these subunits to cause dslo inactivation , and this inactivation depends on the amino - terminal 39-residue region of these slobs . interestingly , the slowpoke channel from periplaneta americana , which is 90% identical to dslo , does not inactivate when it is heterologously expressed , but does so in native cells ( derst et al . , 2003 ) . it will be interesting to determine whether the various slobs bind to and modulate dslo in a dynamic way , particularly in view of the finding that slob57 protein cycles in a circadian manner in vivo ( jaramillo et al . , 2004 ) . for example , if the amount of slob57 cycles , and different slobs compete for dslo within a cell , then the activity of the dslo channel in the cell may change as a function of time of day . we show here that it strongly inhibits dslo activity . studies of the downstream , physiological in vivo effects of this inhibition are ongoing . the amino - terminal 39-residue region of slob57/slob51 plays a critical role in this regulation of dslo . our data reveal that simply by masking or removing this amino - terminal region , slob can modulate dslo in very different ways . thus alternative splicing and/or choice of different translational

ankylosing spondylitis ( as ) is a chronic inflammatory rheumatic disease that affects especially males in the second and third decades of life , with a prevalence of 0.50.9% . the main clinical symptom is inflammatory back pain typically occurring at night and morning stiffness improving after exercise . the pain , stiffness , and limited mobility of the spine can cause severe limitations in daily life activities . the last decade there is an increasing interest in the spinal involvement in as visible on magnetic resonance imaging ( mri ) [ 25 ] . there is a wide range of abnormalities described in the spine of as patients . apart from syndesmophytes and ankylosis of the spine resulting in rigidity , in longstanding as , also focal destructive discovertebral lesions ( andersson lesion ( al ) ) can occur , also known as als [ 6 , 7 ] . in 1996 , rasker et al . recently , we described a review on als , which show disk space narrowing or widening , vertebral bone destruction , a surrounding zone of sclerosis , and local kyphosis at radiographs of the spine ( fig . 1 ) . one of the causes of an al is the local inflammation in the spine in combination with a minor trauma . this lesion can be differentiated from the signs of inflammation at the mri of the spine of active disease in as , as is often observed in patients who are candidates for tumor necrosis factor ( tnf ) blocking treatment . an al might be difficult to detect on clinical symptoms alone because most as patients suffer from back pain . more important is the fact that an al requires a different treatment , for instance immobilization , in contrast with the physical therapy normally prescribed in as patients . 1a lateral radiograph of the lumbar spine shows an andersson lesion with extensive bony destruction of the l1l2 disk with irregular endplates and increased sclerosis of adjacent vertebral bodies ( from van royen et al . with permission ) a lateral radiograph of the lumbar spine shows an andersson lesion with extensive bony destruction of the l1l2 disk with irregular endplates and increased sclerosis of adjacent vertebral bodies ( from van royen et al . with permission ) the aim of this study was to investigate whether in as patients , who are candidates for treatment with anti - tnf , als could be detected . as patients were derived from the outpatient clinics of the vu university medical center ( vumc ) and the jan van breemen institute ( jbi ) in amsterdam . all as patients who fulfilled the modified new york criteria and met the criteria for starting treatment with tnf - blocking agents according to the international asas consensus statement [ 9 , 10 ] were included . following this asas statement , all patients had an active disease as defined by a bath ankylosing spondylitis disease activity index ( basdai ) of 4 ( scale 010 ) , had failed to previous therapy with at least two nonsteroidal anti - inflammatory drugs and sulfasalazine in case of peripheral arthritis , and should be treated with tnf - blocking agents according to an expert . patients were enrolled consecutively , but mainly because of limited availability of the mr scanner , several patients could not be included . furthermore , one patient did not fit into the scanner due to severe kyphosis , and several patients suffered from claustrophobia . at baseline , data that conform to daily clinical practice during treatment with tnf - blocking agents were collected in all patients who gave their written informed consent . standing anteroposterior and lateral full - length plain films and an mri of the whole spine were performed before the start of anti - tnf therapy . mri were obtained at baseline and after 624 months of anti - tnf therapy , using t1-weighted spin - echo sequences ( t1 ) before and after administration of the contrast medium gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) , t2-weighted sequences ( t2 ) , and fat - suppression sequences ( short tau inversion recovery ( stir ) ) . in patients included in the jbi , mri of the spine was performed in the amstelland hospital amstelveen with a field strength of 0.5 tesla , starting from the third cervical vertebra ( c3 ) and downwards . in patients included in the vumc , mri of the whole spine pre- and post-(gd ) t1 tse and stir mris were acquired . in the amstelland hospital amstelveen , the conventional radiographs and mri were read by two observers , one orthopedic surgeon ( bvr ) and a radiologist ( rm ) , who were blinded to the status of the subjects . the extent of spinal ankylosis in the total study cohort was estimated by counting extensive syndesmophyte formation or bamboo spine features on conventional radiographs counted by segment . the extent of abnormalities in the discovertebral unit was compared with the same discovertebral unit in different sagittal mr sequences and the other way around . an al was defined as an abnormality in the discovertebral junction that shows on mri diffuse endplate destruction , with associated bone marrow edema and fat replacement or sclerosis . the fracture cleft through the disk space or vertebra itself is depicted as decreased signal on t1 and increased signal on t2 , stir , and t1 imaging after gd - dtpa administration , reflecting reactive edema of the fracture . conventional radiography of the al must show a fracture line , irregular endplates , and increased sclerosis of adjacent vertebral bodies . continuous variables were reported as the mean sd or , if skewed , as the median ( range ) . as patients were derived from the outpatient clinics of the vu university medical center ( vumc ) and the jan van breemen institute ( jbi ) in amsterdam . all as patients who fulfilled the modified new york criteria and met the criteria for starting treatment with tnf - blocking agents according to the international asas consensus statement [ 9 , 10 ] were included . following this asas statement , all patients had an active disease as defined by a bath ankylosing spondylitis disease activity index ( basdai ) of 4 ( scale 010 ) , had failed to previous therapy with at least two nonsteroidal anti - inflammatory drugs and sulfasalazine in case of peripheral arthritis , and should be treated with tnf - blocking agents according to an expert . patients were enrolled consecutively , but mainly because of limited availability of the mr scanner , several patients could not be included . furthermore , one patient did not fit into the scanner due to severe kyphosis , and several patients suffered from claustrophobia . at baseline , data that conform to daily clinical practice during treatment with tnf - blocking agents were collected in all patients who gave their written informed consent . standing anteroposterior and lateral full - length plain films and an mri of the whole spine were performed before the start of anti - tnf therapy . mri were obtained at baseline and after 624 months of anti - tnf therapy , using t1-weighted spin - echo sequences ( t1 ) before and after administration of the contrast medium gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) , t2-weighted sequences ( t2 ) , and fat - suppression sequences ( short tau inversion recovery ( stir ) ) . in patients included in the jbi , mri of the spine was performed in the amstelland hospital amstelveen with a field strength of 0.5 tesla , starting from the third cervical vertebra ( c3 ) and downwards . in patients included in the vumc , mri of the whole spine was performed with a field strength of 1.5 tesla . pre- and post-(gd ) t1 tse and stir mris were acquired . in the amstelland hospital amstelveen , the conventional radiographs and mri were read by two observers , one orthopedic surgeon ( bvr ) and a radiologist ( rm ) , who were blinded to the status of the subjects . the extent of spinal ankylosis in the total study cohort was estimated by counting extensive syndesmophyte formation or bamboo spine features on conventional radiographs counted by segment . the extent of abnormalities in the discovertebral unit was compared with the same discovertebral unit in different sagittal mr sequences and the other way around . an al was defined as an abnormality in the discovertebral junction that shows on mri diffuse endplate destruction , with associated bone marrow edema and fat replacement or sclerosis . the fracture cleft through the disk space or vertebra itself is depicted as decreased signal on t1 and increased signal on t2 , stir , and t1 imaging after gd - dtpa administration , reflecting reactive edema of the fracture . conventional radiography of the al must show a fracture line , irregular endplates , and increased sclerosis of adjacent vertebral bodies . continuous variables were reported as the mean sd or , if skewed , as the median ( range ) . in total , 56 as patients were included , 36 in the vumc and 20 in the jbi , and the baseline characteristics of all patients are summarized in table 1 . patients from the two centers . table 1baseline characteristicsmean sdrangenumber56age ( in years)43 10.82273male gender ( % ) 3868disease duration ( in years)11 8.7141symptom duration ( in years)21 11.3149basdai ( 010)6.4 1.44.09.7tragus - to - wall distance ( cm ; normal , < 15 cm)16 6.01144lumbar flexion index ( cm ; normal , > 5 cm)2.5 1.20.35lumbar side flexion ( cm ; normal , > 10 cm)10 4.93.819chest expansion ( cm ; normal , > 5 cm)3.4 1.50.57disease duration : mean time between the diagnosis and baselinesymptom duration : mean time between the first symptoms and baselinebasdai : bath ankylosing spondylitis disease activity index ; mean value , 11 baseline characteristics disease duration : mean time between the diagnosis and baseline symptom duration : mean time between the first symptoms and baseline basdai : bath ankylosing spondylitis disease activity index ; mean value , 11 systematic examination of conventional radiographs of the cervical , thoracic , and lumbar spine did not reveal any lesions that met the definition of al . distinct ankylosis with extensive syndesmophyte formation was seen in 13 of 55 ( 24% ) of the patients determined with conventional radiography of the spine . spinal ankylosis or bridging syndesmophytes over multiple vertebrae were seen at the cervical , thoracic , and lumbar segment in 16% , 6% , and 7% of patients , respectively . conventional radiographs and mris of the whole spine showed no al according to our definition . this abnormality showed bone marrow edema of more than 50% of the vertebral bodies adjacent to the intervertebral disk of t7/t8 and t9/t10 ( fig . t1 after administration of gd - dtpa demonstrated high signal intensity around the disk margins of t7/t8 and t9/t10 . however , no fracture line was visible on conventional radiology . 2a t1-weighted gadolinium diethylenetriamine pentaacetic acid postcontrast image demonstrated high - signal intensity around the disk margins of t7t8 and t9t10 . b t1-weighted image shows low - signal intensity bone marrow edema in the adjacent vertebral endplates of the t7/t8 and t9/t10 disk spaces . c t1-weighted image , obtained 24 months after treatment with anti - tumor necrosis factor agents a t1-weighted gadolinium diethylenetriamine pentaacetic acid postcontrast image demonstrated high - signal intensity around the disk margins of t7t8 and t9t10 . b t1-weighted image shows low - signal intensity bone marrow edema in the adjacent vertebral endplates of the t7/t8 and t9/t10 disk spaces . c t1-weighted image , obtained 24 months after treatment with anti - tumor necrosis factor agents the clinical characteristics of this patient did not differ from the other patients . he was a male , aged 65 years , with a basdai and crp of 5 and 35 mg / l , respectively , indicative of an active disease . he did not report any specific localizing complaints or a preceding ( minor ) trauma . he had suffered from complaints of low back pain for 40 years and was diagnosed with as at the age of 51 . after 24 months treatment with infliximab , t1-weighted images showed a decrease of signs of acute inflammation such as bone marrow edema and maybe even an increase of post - inflammatory fatty bone marrow degeneration around the disk margins . the t1-weighted post - contrast images demonstrated no high signal intensity around the disk margins of t7/t8 and t9/t10 anymore . there were signs of progression of the ankylosis and kyphosis in the thoracic segment after 2 years of follow - up despite anti - tnf treatment . apart from the focus on als , other abnormalities involving the discovertebral unit as described in literature on mri in as were encountered . romanus lesions , which are acute inflammatory lesions at the corners of the endplates of the vertebra , were often detected . these lesions show bone marrow edema in the corners of vertebral endplates and are depicted as hypodense signal areas in t1- and high signal in t2-weighted images . systematic examination of conventional radiographs of the cervical , thoracic , and lumbar spine did not reveal any lesions that met the definition of al . distinct ankylosis with extensive syndesmophyte formation was seen in 13 of 55 ( 24% ) of the patients determined with conventional radiography of the spine . spinal ankylosis or bridging syndesmophytes over multiple vertebrae were seen at the cervical , thoracic , and lumbar segment in 16% , 6% , and 7% of patients , respectively . conventional radiographs and mris of the whole spine showed no al according to our definition . this abnormality showed bone marrow edema of more than 50% of the vertebral bodies adjacent to the intervertebral disk of t7/t8 and t9/t10 ( fig . t1 after administration of gd - dtpa demonstrated high signal intensity around the disk margins of t7/t8 and t9/t10 . however , no fracture line was visible on conventional radiology . 2a t1-weighted gadolinium diethylenetriamine pentaacetic acid postcontrast image demonstrated high - signal intensity around the disk margins of t7t8 and t9t10 . b t1-weighted image shows low - signal intensity bone marrow edema in the adjacent vertebral endplates of the t7/t8 and t9/t10 disk spaces . c t1-weighted image , obtained 24 months after treatment with anti - tumor necrosis factor agents a t1-weighted gadolinium diethylenetriamine pentaacetic acid postcontrast image demonstrated high - signal intensity around the disk margins of t7t8 and t9t10 . b t1-weighted image shows low - signal intensity bone marrow edema in the adjacent vertebral endplates of the t7/t8 and t9/t10 disk spaces . c t1-weighted image , obtained 24 months after treatment with anti - tumor necrosis factor agents the clinical characteristics of this patient did not differ from the other patients . he was a male , aged 65 years , with a basdai and crp of 5 and 35 mg / l , respectively , indicative of an active disease . he did not report any specific localizing complaints or a preceding ( minor ) trauma . he had suffered from complaints of low back pain for 40 years and was diagnosed with as at the age of 51 . after 24 months treatment with infliximab , t1-weighted images showed a decrease of signs of acute inflammation such as bone marrow edema and maybe even an increase of post - inflammatory fatty bone marrow degeneration around the disk margins . the t1-weighted post - contrast images demonstrated no high signal intensity around the disk margins of t7/t8 and t9/t10 anymore . there were signs of progression of the ankylosis and kyphosis in the thoracic segment after 2 years of follow - up despite anti - tnf treatment . apart from the focus on als , other abnormalities involving the discovertebral unit as described in literature on mri in as were encountered . romanus lesions , which are acute inflammatory lesions at the corners of the endplates of the vertebra , were often detected . these lesions show bone marrow edema in the corners of vertebral endplates and are depicted as hypodense signal areas in t1- and high signal in t2-weighted images . in 56 as patients , who fulfilled the criteria for treatment with tnf - blocking agents , only one lesion resembling an al of the thoracic spine was detected with mri , but this lesion lacked a fracture line on the conventional radiograph . the low prevalence of als was unexpected because this group of as patients had a relatively high rate of ankylosis of the spine ( 24% ) and signs of active inflammation . this was in concordance with the moderate impairment of the mobility of the spine in these patients . the mean tragus - to - wall distance was 16 cm ( normal , < 15 cm ) , mean lumbar flexion index was 2.5 cm ( normal , > 5 cm ) , the mean lumbar side flexion was 10 cm ( normal , > 10 cm ) , and the mean chest expansion was 3.4 ( normal , > 5 cm ) . the percentage of als as described in literature in this subset of severe as patients varies between 1% and 28% [ 1521 ] . many studies report that the presence of an al is associated with long standing disease in as and with a rigid , ankylosed spine [ 2224 ] , concluding that an al is a late complication of as . the low prevalence of als in our study was probably caused by the relatively short disease duration , with a mean of 11 years and a mean age of 43 years . this could be due to a selection bias , for mostly young patients with an active disease were referred for treatment with tnf - blocking agents , whereas the efficacy of tnf blockers in older and more severe cases of as with complete ankylosis of the spine was still doubtful at the time of the study . a severe kyphotic deformity and an osteoporotic spine put a patient at risk of development of a stress fracture in the ankylosed spine [ 25 , 26 ] . however , we did not find any features of stress fractures in this susceptible subgroup of patients , probably due to the small group size . another cause of the low prevalence of als in this study might be due to the radiographic techniques used to detect them , although the combination of conventional radiographs of the spine and mri with gadolinium should be sensitive enough . hermann et al . compared mri and conventional radiography of spinal changes in patients with spondyloarthritis . they concluded that the best way to detect syndesmophytes was through radiography ; ankylosis was detected equally well by both radiography and mri , but for all other lesions , mri was the preferred method . a disadvantage of conventional radiographs of the spine is the difficult analysis of the vertebrae of the thoracic segment because of the overimposed lung tissue , while most disorders of the discovertebral junction occur in the lower part of the thoracic and upper parts of the lumbar spine [ 28 , 29 ] . mri is important for early detection of discovertebral lesions and described as a sensitive method to detect als , in particular , after administration of gadolinium . some mris in our study showed features of severe degenerative disk disease ( ddd ) , specifically lumbar - sacral . the mri features of these degenerative abnormalities were sometimes difficult to distinguish from destructive discovertebral lesions as was described by jevtic et al . , who showed that the appearance of an al can resemble modic type iii degenerative lesion . one point to differentiate between al and ddd in as is the fact that an al occurs as a result of inflammation in combination with mechanical stress of the ankylosed spine . another point is that al differs from ddd in the localization , because most al lesions occur at the level of the cervical and thoracolumbar spine , whereas the ddd predominate at the lumbosacral level . the debate about the prevalence of als is hampered by the lack of uniformity in the nomenclature of these lesions . for example , the term al has been employed for severe destructive discovertebral lesions , but also for minor abnormalities such as degenerative changes and local inflammation around in the discovertebral junction , as described in our case [ 7 , 12 ] . to conclude , no als was detected by using conventional radiographs and mri in this study in 56 severe as patients , whereas a higher prevalence was described in literature . the discrepancy might be due to the relatively small group of patient with a short disease duration and young age despite the severe ankylosed spine in our study cohort . despite the absence of als in our study , we would like to increase the awareness of this complication in as . in case of a minor spine trauma , an mri should be combined with the conventional spine radiographs in order to detect this lesion in the stiff and vulnerable spine , which is often osteoporotic as well . the treatment of an al lesion consists of immobilization instead of the routinely advised exercise program or even surgical decompression in case of neurological deficits . in previous studies , there has been no sequential mri study of the spine in patients with as that visualizes the evolution from an early discovertebral lesion , as described in our patient , into a severe destructive discovertebral lesion , by some authors called an al . thus , it remains unclear whether and how often an abnormality in the discovertebral junction develops into an al . more research with a long - term follow - up of discovertebral lesions is necessary to clarify the evolution of these lesions .

episiotomy is performed with a pudendal block during second stage of labor to expand the opening of the vagina which improves maternal and neonatal outcomes.it is one of the most common medical procedures performed on women in iran , which have been reported 88.3% at ommolbanin hospital of mashhad , in 2006 . also khajavi et al . , reported that the prevalence of episiotomy is 97.3% in primiparous women in tehran , iran which is higher than other countries . although its routine use in childbirth has steadily declined in recent decades , it is still widely practiced in asian countries . it could be explained because of the short perineal and sturdy tissue of asian women . , reported that medio - lateral episiotomy is widely used in iran despite its disadvantages . , showed that perineal pain in the first , tenth , fortieth and ninetieth day after birth was 96.4% , 63% , 25% and 12% , respectively . episiotomy pain is always difficult and stressful for primiparous women , which has several negative aspects such as negative effect on the first experience of motherhood , mothers ' inability to care the infant , delay in mother - infant communication , insomnia , fatigue , confusion , anxiety , and ignoring health educations in relation to maternal- infant care . thus , we have to recover women more quickly after childbirth by removing their perineal pain and discomfort . non - drug methods include using hot and cold compression , and salt water or diluted savlone in the bathroom . drug methods such as acetaminophen , aspirin , and ibuprofen and diclofenac sodium . despite empirical evidence of drug treatments , some issues should be considered in selecting them such as : pain intensity , constipation property , digestive problems , risks and possible complications of baby through the breast milk . drug treatments make high costs for consumer and the health care system as well as it has more side effects . world health organization ( who ) has a great emphasis on using herbal medicine because of complications of chemical drugs . currently , finding the most effective method with minimal complications , availability and easily acceptance by women has always been considered by researchers . chamomile is one of the plants that are currently used in iranian traditional medicine as an analgesic , which contains 120 chemical compositions including -bisabolol , bisabolol oxides , spiro - ethers , chamazulene and flavonoids . episiotomy pain often caused by inflammation and edema . due to the strong anti - inflammatory effect of chamomile which has been mentioned in several studies , , it seems that chamomile could be used as a pain killer by reducing edema and inflammation in the first days after delivery . in addition to the mechanisms in inhibiting the prostaglandin synthesis in reducing pain , other painless mechanisms are also possible . based on the importance of reducing episiotomy pain , the complications of chemical drugs , the general trend towards herbal medicine , the researchers decided to conduct a study on the effect of chamomile cream on episiotomy pain and provide more favorable services by using its findings to ensure maternal health. this triple blind clinical trial was done on 114 eligible women admitted to ommolbanin hospital in mashhad , iran . to determine the sample size based on a preliminary study , the mean and deviation of the 10 day were obtained in experimental group 0.87 ( 0.90 ) and in control group 0.56 ( 1.10 ) . after the approval of the ethics committee of mashhad university of medical sciences , the study was registered in the iranian registry center of clinical trials with irct2013051413336n1 code . dried chamomile flowers ( matricaria chamomile ) was purchased from zardband company of tehran and its identity was confirmed by herbarium lab of mashhad pharmacy school . for making chamomile extract , 60 gr of crushed chamomile which made by mill was poured in to each thimble and extracted with 70% alcohol by soxhlet extractor and then after removing the solvent by rotary evaporator , concentrated extract was prepared in the laboratory of pharmacology , school of medicine , mashhad . then concentrated extract was weighed and its efficiency was obtained and according to charousaei and dabirian , chamomile cream 1.3% was made of the combination of chamomile extract with cold cream and placebo cream containing simple foundation of cream ( cold cream ) by the pharmacist and were coded ( code a and b(.tubes of chamomile and placebo creams were similar in appearance . it should be mentioned that participants , statistical analysts , researcher and research assistant were unaware of codes until the end of the study . the inclusion criteria of this study were as following : primiparous women aged 18 - 35 years , vaginal delivery with episiotomy , living with her husband , body mass index in the range of 19.8 - 30 , absence of disease impaired wound healing , not using drugs affecting wound healing , singleton pregnancy with cephalic presentation , lack of symptomatic infection of the vagina and vulva ( infectious discharge , itching , and irritant ) , no revealed abnormalities in the baby , no severe rectocele or cystocele ( grade 2 or higher ) , no septum or mass in vagina , no obstetric problem history , not using sedative for 4 hours before delivery and no reconstructive surgery history of the vagina and the perineum . the exclusion criteria were as following : the presence of disorder in the progress of labor , duration of second stage of labor for more than 2 hours , extension of the incision area and turning into grade 3 or 4 tear or rupture except episiotomy tear , abnormal vaginal bleeding , shoulder dystocia ( when maneuvers used to relieve shoulder dystocia except mac robert maneuver ) , expulsion the placenta by hand , hematoma formation , having sexual intercourse until end of the study ( 15 days postpartum ) , curettage or resection within the first 24 hours after delivery , undesired events during the first 14 days after delivery , using cream regularly , having puerperal fever , infection of the episiotomy area , need to suturing again and complications arising from the use of chamomile cream . in this study , data were gathered by questionnaires including base information and examination results ( selection form of the subjects , forms of delivery stages and episiotomy , daily form of sedative , antibiotics consumption , health and nutrition care , etc . ) , constipation assessment scale in pregnancy and short - form mcgill pain questionnaire. the validity of the base information and examination and the constipation assessment form was confirmed by content validity . the validity of short - form mcgill pain questionnaire was determined by criterion - related validity on 10 participants . thus , the ability of the instrument to measure episiotomy pain compared to the pain visual tool ( correlation coefficient r=0.85 ) . the reliability of the selection form and daily data entry form were confirmed . the reliability of labor stages forms and episiotomy information were confirmed with the assessor agreement ( r= 0.89 , r=0.94 ) . the reliability of short - form mcgill pain questionnaire was approved by internal reliability ( cronbach 's alpha ) ( r = 0.91 ) . in addition , the reliability of constipation assessment scale has been confirmed by brusard and brend ( 1998 , r = 0.92 ) and tabatabai chehr ( 2001 , r = 0.82 ) in pregnant women . the procedure were as following : after the approval of the ethics committee of mashhad university of medical sciences ( code 911324 ) , the purposes of the study was explained to eligible women referred to ommolbanin hospital . they were selected by simple sampling method and then they were randomly assigned into two groups a or b using random blocks after obtaining informed consent . delivery stages care , delivery , repair of episiotomy was done by the researcher assistant . after expulsion the placenta and repair of episiotomy in lithotomy position , information about the baby , episiotomy incision size , type of yarn used and the number of stitches were recorded . after repair of episiotomy , the researcher gave the necessary training about stitch and perineum care , personal hygiene , how to complete daily information form ( such as sedative and antibiotics consumption , using cream for the stitches , hygiene , nutrition and physical activity ) to the mothers . an encoded package cream was given to the mothers 2 hours after episiotomy recovery and they were taught how to use it . they had to rub 0.5gr of the cream ( in the size of knuckle ) on the stitches after washing their hands and perineum so that it completely covers the surface of the wound and repeat it twice a day ( every 12 hours ) to ten days . pain in the perineal area were evaluated immediately before the intervention ( 2 hours after perineal repair ) , 12 hours after the complete repair of episiotomy and on 1 , 7 , 10 days after delivery and also 14 day ( if there was any moderate to severe pain on tenth day after birth ) . the questionnaire consisted of three parts : sensory - emotional - verbal descriptions ( in the range of painless to severe pain ) , visual pain scale ( 0 - 10 scores ) and pain intensity ( in the range of painless to severe pain ) . the total score of pain is equal to the total score obtained in almost all categories in different aspects of pain , which is in the rage of 0 - 60 scores . omitting variables of the study smirnov test . to compare the two groups in terms of quantitative variables with normal distribution , independent t - test in with non - normal distribution mann - whitney test was used . to compare the groups in terms of qualitative ordinal variables , the kruskal - wallis test was used and for qualitative nominal variables , chi - square test was applied . to compare the quantitative variables changes in the two groups , repeated measures during the study ( may- november 2013 ) , 16 patients were excluded due to the elimination criteria ( 7 patients in the control group and 9 in the intervention group due to not using the cream regularly , not referring and access to the researcher , infection and the need for suturing again ) . the analysis was conducted on the 48 patients in the control group and 50 patients in the intervention group ( per protocol or on treatment analysis ) . the difference between two groups was not statistically significant in terms of characteristics such as age , education , socioeconomic status , characteristics of pregnancy , doing strengthening exercises , pelvic floor muscle and other intervening variables during labor such as fatigue and hunger in the second stage of labor , disturbance in progress of the first stage of labor , duration of second and third stage of labor , the number of vaginal examinations for first and second stage of labor , fetal head position at birth , the interval time between injection of lidocaine and episiotomy incision , information about the baby weight , sex and height , pain during urinating and defecating , having constipation , antibiotics consumption , hygiene care , nutrition , physical activity in the first and 10 days after delivery and other variables related to episiotomy ( p>0.05 ) ( table 1 ) . flow chart of the study comparison of scores from 17 variables of mcgill pain questionnaire before intervention ( p= 0.16 ) , 12 hours after episiotomy recovery ( p=0.38 ) and the first day after delivery ( p=0.11 ) using t - test showed that there was no significant difference between two groups , but episiotomy pain on the 7 day ( p=0.03 ) , the 10 day ( p=0.02 ) and 14 days after delivery ( p=0.03 ) were significant between two groups ( table 2 ) . comparison of mean scores of episiotomy pain between two groups using anova with repeated measures indicated that the episiotomy pain intensity in patients treated with chamomile cream was decreased on the seventh day , tenth and fourteenth days after delivery compared to patients treated with placebo ( p=0.05 ) . mann - whitney test showed that none of the sensory and affective components of pain before the intervention , 12 hours after episiotomy recovery , the first and fourteen days after delivery were significantly different in two groups ( p>0.05 ) , except irritant pain on the first day ( p=0.04 ) and fourteenth day ( p=0.03 ) and the sensitivity on the first day ( p=0.03 ) and fourteenth day ( p=0.04 ) had significant differences between the two groups. considering sensory components of episiotomy pain based on the mann - whitney test showed that irritant pain , being sensitive on the seventh and tenth day , heaviness feeling on seventh day and cramp pain on tenth day after delivery were significantly different in the two groups ( p<0.05 ) . among emotional components , tormentor pain on the tenth day after delivery was significantly different between two groups ( p<0.05 ) . in this study , the rate of using gelofen tablet , diclofenac , indomethacin and acetaminophen suppository was not significantly different between the two groups . but the amount of acetaminophen tablet was significantly lower in patients treated with chamomile cream than the placebo group ( p=0.001 ) . it appears that chamomile cream decreases the need for sedative by reducing pain. in present study , there was no report on side effects of using chamomile cream and placebo cream among mothers in both experimental and control groups . mothers satisfaction of using the cream on the seventh day after birth ( p=0.05 ) was not significantly different between the two groups , however on the tenth day after the birth , mothers had more satisfaction about chamomile cream than placebo cream ( p=0.03 ) . the results of this study indicated that the using chamomile cream has not reduced pain12 hours after episiotomy recovery ( p=0.38 ) and also on the first day after birth . qnais showed that chamomile extract at a dose of 100 , 200 and 300 mg / kg has dose - dependent analgesic effect.the prevalence and the rate of pain reported more in the first few days after birth . khajavi et al . , showed that perineal pain were 96.4% , 63% , 25% and 12% in the first , tenth and fortieth and ninetieth day after birth , respectively . also , macarthur et al . , in the university of toronto in canada reported perineal pain 97% during 24 hours after delivery , which is similar to khajavi et al . in the present study , 98% of patients complained perineal pain in the first 24 hours after delivery ranging from 5 - 49 by mcgill pain questionnaire which is similar to the other studies . thus , it seems that patients require more analgesic drugs within the first day after birth . regarding to the dose - dependent analgesic effect of chamomile , it seems that the dose used in this study for analgesic in the first 12 hours and the first day after birth was less than the needed dose . , study these authors , reported that chamomile essence has not reduced episiotomy pain in the first 12 hours after delivery . the findings of this study showed that the use of chamomile cream was effective for pain relief on seventh ( p = 0.03 ) , tenth ( p = 0.02 ) and fourteen days ( p = 0.03 ) after delivery .the mean pain score in seventh and tenth days after delivery in the placebo group versus chamomile group was 1.3 ( the pain score on the seventh day in chamomile group was 11.36 ( 5.04 ) versus 14.88 ( 7.34 ) in control group and on the tenth day after delivery in chamomile group was 7.10 ( 4.10 ) versus 9.96 ( 4.81 ) . the mean pain score on the fourteen day in placebo group versus chamomile group was 1.7 . in present study , chamomile may be effective through several mechanisms ( opioidergic , serotonergic , anti - inflammatory ) in analgesia . as a result , several studies have raised the possibility of analgesic effect of chamomile through opioidergic . in this , showed that chamomile in both phases of the formalin test has analgesic effect on mice . in the first phase , the second phase of the formalin test is partly due to the inflammation process.there are some evidences that shows extract effect is somewhat similar to receptor antagonists of serotonergic in reducing pain ; of course , the extent of this effect and its similarity depends on the amount of germacranolide and parthenolide in the extract . therefore , it is possible that part of the nociceptive is applied through serotonergic system . the anti - inflammatory effect of chamomile essence is due to inhibitory effect of chamomile essences especially bisabolol and chamazulene on cyclooxygenase and lipooxygenase enzymes . according to the reports , acquired lakton from chamomile contains epoxyartemurin and partenolid which have preventing effects on creating components resulting in arachidonic acid that is thromboxane b2 and leukotriene.epigenin in the extract has analgesic effect and also has interference with the body 's histamine system and cause reversible inhibition and dose - dependent inflammation of the skin . pain , warmth , redness , swelling and loss of function are some local signs of inflammation . in the present study , it seems that chamomile , because of its strong anti - inflammatory properties , has relieved episiotomy pain by reducing pain mediators in the inflammatory part and reducing inflammatory prostaglandins and due to its anti - inflationary property resulting from azulene , chamazulene , alpha bisabolol essence by reducing swelling and inflammation in the first days after birth . , showed that the severity of menstrual pain was significantly lower in the group taking chamomile capsules than the group taking mefenamic acid capsules two days after using drugs in the first month of treatment ( p<0.001 ) and the second month of treatment(p<0.001 ) . these results confirmed that analgesic effect of chamomile is aligned . in charousaei s & dabirian study stoma symptoms(pain , itching ) was removed in group , which used chamomile compresses twice a day , more than the group using hydrocortisone 1% cream . confirming the analgesic effect of aromatherapy with chamomile , lim & lee showed that inhalation aromatherapy for ten minutes after tonsillectomy , with a mixture of lavender and chamomile oil in the ratio1 : 2 , reduces the amount of perceived pain , systolic and diastolic blood pressure and the heart beating . pazandeh et al . , used chamomile essence produced by steam distillation method to reduce episiotomy pain ; participants poured 3 drops of chamomile essence in two liters of warm water and used it 2 times a day for 20 minutes during 2 weeks as aromatherapy while sitting in bath . the result showed that chamomile essence has not lessened episiotomy pain in the first 12 hours after birth and in 7 and 14 days after birth , which is not consistent with the results of the present study . this difference could be related to the differences in the type , quality and quantity of the prescribed herbal medication. mcgill pain tools describe pain in 3 dimentions : sensory , emotional and evaluative . based on our results , irritant pain and sensitivity 24 hours after labor and on the seventh , tenth and fourteenth days after delivery , feeling of heaviness on the seventh day and cramp pain on the tenth day was significantly lower in mothers using chamomile cream than ones using the placebo cream . in the present study , chamomile may lead to reduction in sensitivity and cramp pain and heaviness feeling through softening and moisturizing wound skin , as well as its anti - inflammatory effects resulting from -bisabolol and chamazulene essence and its anti - spasmodic effect resulting from flavonoids and comarin . sahba and mohammadalipour found that chamomile mouth wash reduces the needed time for controlling pain and irritant feeling , that is in line with the results of the present study . in traditional books , consuming chamomile products as external use has been also recommended for softening necrosis and granulated tissue ( rigid tissue ) , swelling , inflammation , ulcer guard and skin rash . chamomile is also a key ingredient in creams used to treat eczema and used as an emollient for hairs . the limitations of this study were as follows : individual and genetic differences , impossibility of monitoring the use of creams and observing personal hygiene . although it was attempted to control them by providing similar training and giving pamphlets to mothers. it is recommended to evaluate the effect of chamomile with more doses on reduction of episiotomy pain on the first day after delivery as well as the effect of chamomile cream prepared by the soxhlet method comparing to the percolated method . this study confirms analgesic and anti - inflammatory effect of this plant which has been reported in traditional medicine text books. since chamomile cream reduces episiotomy pain and no significant side effect has been reported by mothers , and also because in recent years the tendency of people to use herbal medicine has been increased , chamomile cream could be used as a pain killer of episiotomy in primiparous women to improve the quality of obstetric services. we thanks to the deputy of research and ethics committee of the university , staff of ommolbanin hospital ( midwives and physicians ) and also the mothers.

baseline samples from two studies of depression and distress among adult type 2 diabetic patients were included . study 1 , our primary sample , used baseline data from the distress and depression in diabetes study ( 3d study ) , a noninterventional , three - wave , 18-month study of the prevalence and persistence of dd and depressive symptoms among 506 adult type 2 diabetic patients ( 2 ) . study 2 used baseline , preintervention data from the reducing distress and enhancing effective management ( redeem ) study , a randomized controlled trial designed to reduce dd and enhance management among 392 type 2 diabetic adults ( 12 ) . patients in both studies were recruited using the diabetes registries of several bay area community - based medical groups and diabetes education centers . inclusion criteria for both studies were patients with type 2 diabetes for 12 months or more , age 21 years or older , read and speak english fluently , no severe diabetes complications , and no active psychosis , substance use , or dementia . additional , more restrictive criteria for redeem study patients included displaying a mean item score of 1.5 or higher on the dds2 to indicate elevated dd , displaying a score of 15 or higher on the patient health questionnaire 8 to exclude patients with clinical depression , and displaying a deficit in at least one of three areas of diabetes self - management ( diet , physical activity , medication use ) . a modification of the summary of diabetes self - care activities ( sdsca ) ( 13 ) was used to define a deficit as not following their diet or physical activity plan 3 or more days during the last week or not taking prescribed diabetes medications 2 or more days during the last week . for both studies , letters were sent to each patient from their health care facility , cosigned by facility and project representatives , informing them of the project and that they would receive a phone call from the project office if one of two opt - out procedures was not initiated : patient returned an enclosed postcard or called an 800 phone number . a screening phone call followed and , for eligible patients , an appointment was made to explain the project in detail , collect informed consent , and begin the baseline assessment . baseline assessment in both studies included a 1.5-h home visit for completion of questionnaires , physical measurements , and interviews , as well as a visit to a community laboratory for collection of blood and urine specimens . both studies were approved by the institutional review boards at the university of california , san francisco , and at each participating facility . control variables for both studies included patient age , sex ( female = 1 , male = 0 ) , education , ethnicity ( white = 1 , nonwhite = 0 ) , years with type 2 diabetes , use of insulin ( yes or no ) , and bmi . the dds is a 17-item measure that uses a likert scale to score each item from 1 ( no problem ) to 6 ( a serious problem ) during the last month ( = 0.93 ) ( 6 ) . previous analyses identified that the dds2 had good sensitivity and specificity with the dds17 ( 7 ) and was considered a reliable screening composite for use in clinical practice . the dds17 and dds2 were associated with four diabetes - related variables . hba1c was collected at a community facility and processed at the same community laboratory in both studies . also included were measures of diabetes self - efficacy , diet , and physical activity , although different measures were used in each of the two studies . diabetes self - efficacy is a 10-item scale ( = 0.88 ) , adapted from coyne and smith ( 14 ) , that assesses the patient s perceived confidence about taking care of diabetes ( 15 ) . items are rated on a 4-point scale from not at all sure to very sure . diet and physical activity were assessed by the summary of diabetes self - care activities ( 13 ) . respondents indicated the number of days during the past week that they completed their diet and physical activity regimens . diabetes self - efficacy was assessed by a 15-item scale developed by lorig et al . diet was assessed by five items from the starting the conversation survey ( 17 ) that asks respondents to indicate the number of times per week during the past 4 months that they ate certain types of unhealthy foods ( e.g. , fast foods , sodas , sweet tea ) . physical activity was assessed by the community healthy activities model program for seniors ( 18 ) . nineteen items regarding frequency of specific physical activities ( e.g. , walk fast or briskly , swim , ride a bicycle ) assessed caloric expenditures per week in moderate - intensity physical activity activities , capped at a value of 6,000 calories ( 19 ) . comparisons were undertaken between 3d and redeem on key patient demographics using and t tests . separate step - wise multiple regression equations were conducted for the dds17 and dds2 for each of the four dependent variables for each sample : step 1 , demographics and disease status ; step 2 , dds score ( linear term ) ; step 3 , dds score ( quadratic term ) . quadratic effects were interpreted only if the related t test , with one degree of freedom at step 3 , was significant . the goal in these analyses was to determine if the shape of the relationships yielded a common pattern that would define a meaningful cut point for the dds17 and dds2 . a minimum change of 0.5 sd units in dependent variables was used to help establish cut points ( 20 ) . the results of each significant effect were placed on scatterplots , with fitted linear or quadratic lines , to examine the shape and start - point of the relationship between each dds score and each of the dependent variables . mean - item dds values up to 4.0 were plotted because these encompassed more than 90% of the sample and dds values note that the direction of the curves varies as a function of the direction of the regression coefficient and the direction of the scale . baseline samples from two studies of depression and distress among adult type 2 diabetic patients were included . study 1 , our primary sample , used baseline data from the distress and depression in diabetes study ( 3d study ) , a noninterventional , three - wave , 18-month study of the prevalence and persistence of dd and depressive symptoms among 506 adult type 2 diabetic patients ( 2 ) . study 2 used baseline , preintervention data from the reducing distress and enhancing effective management ( redeem ) study , a randomized controlled trial designed to reduce dd and enhance management among 392 type 2 diabetic adults ( 12 ) . patients in both studies were recruited using the diabetes registries of several bay area community - based medical groups and diabetes education centers . inclusion criteria for both studies were patients with type 2 diabetes for 12 months or more , age 21 years or older , read and speak english fluently , no severe diabetes complications , and no active psychosis , substance use , or dementia . additional , more restrictive criteria for redeem study patients included displaying a mean item score of 1.5 or higher on the dds2 to indicate elevated dd , displaying a score of 15 or higher on the patient health questionnaire 8 to exclude patients with clinical depression , and displaying a deficit in at least one of three areas of diabetes self - management ( diet , physical activity , medication use ) . a modification of the summary of diabetes self - care activities ( sdsca ) ( 13 ) was used to define a deficit as not following their diet or physical activity plan 3 or more days during the last week or not taking prescribed diabetes medications 2 or more days during the last week . for both studies , letters were sent to each patient from their health care facility , cosigned by facility and project representatives , informing them of the project and that they would receive a phone call from the project office if one of two opt - out procedures was not initiated : patient returned an enclosed postcard or called an 800 phone number . a screening phone call followed and , for eligible patients , an appointment was made to explain the project in detail , collect informed consent , and begin the baseline assessment . baseline assessment in both studies included a 1.5-h home visit for completion of questionnaires , physical measurements , and interviews , as well as a visit to a community laboratory for collection of blood and urine specimens . both studies were approved by the institutional review boards at the university of california , san francisco , and at each participating facility . control variables for both studies included patient age , sex ( female = 1 , male = 0 ) , education , ethnicity ( white = 1 , nonwhite = 0 ) , years with type 2 diabetes , use of insulin ( yes or no ) , and bmi . the dds is a 17-item measure that uses a likert scale to score each item from 1 ( no problem ) to 6 ( a serious problem ) during the last month ( = 0.93 ) ( 6 ) . previous analyses identified that the dds2 had good sensitivity and specificity with the dds17 ( 7 ) and was considered a reliable screening composite for use in clinical practice . the dds17 and dds2 were associated with four diabetes - related variables . hba1c was collected at a community facility and processed at the same community laboratory in both studies . also included were measures of diabetes self - efficacy , diet , and physical activity , although different measures were used in each of the two studies . diabetes self - efficacy is a 10-item scale ( = 0.88 ) , adapted from coyne and smith ( 14 ) , that assesses the patient s perceived confidence about taking care of diabetes ( 15 ) . items are rated on a 4-point scale from not at all sure to very sure . diet and physical activity were assessed by the summary of diabetes self - care activities ( 13 ) . respondents indicated the number of days during the past week that they completed their diet and physical activity regimens . diabetes self - efficacy was assessed by a 15-item scale developed by lorig et al . diet was assessed by five items from the starting the conversation survey ( 17 ) that asks respondents to indicate the number of times per week during the past 4 months that they ate certain types of unhealthy foods ( e.g. , fast foods , sodas , sweet tea ) . physical activity was assessed by the community healthy activities model program for seniors ( 18 ) . nineteen items regarding frequency of specific physical activities ( e.g. , walk fast or briskly , swim , ride a bicycle ) assessed caloric expenditures per week in moderate - intensity physical activity activities , capped at a value of 6,000 calories ( 19 ) . diabetes self - efficacy is a 10-item scale ( = 0.88 ) , adapted from coyne and smith ( 14 ) , that assesses the patient s perceived confidence about taking care of diabetes ( 15 ) . items are rated on a 4-point scale from not at all sure to very sure . diet and physical activity were assessed by the summary of diabetes self - care activities ( 13 ) . respondents indicated the number of days during the past week that they completed their diet and physical activity regimens . diabetes self - efficacy was assessed by a 15-item scale developed by lorig et al . diet was assessed by five items from the starting the conversation survey ( 17 ) that asks respondents to indicate the number of times per week during the past 4 months that they ate certain types of unhealthy foods ( e.g. , fast foods , sodas , sweet tea ) . physical activity was assessed by the community healthy activities model program for seniors ( 18 ) . nineteen items regarding frequency of specific physical activities ( e.g. , walk fast or briskly , swim , ride a bicycle ) assessed caloric expenditures per week in moderate - intensity physical activity activities , capped at a value of 6,000 calories ( 19 ) . comparisons were undertaken between 3d and redeem on key patient demographics using and t tests . separate step - wise multiple regression equations were conducted for the dds17 and dds2 for each of the four dependent variables for each sample : step 1 , demographics and disease status ; step 2 , dds score ( linear term ) ; step 3 , dds score ( quadratic term ) . quadratic effects were interpreted only if the related t test , with one degree of freedom at step 3 , was significant . the goal in these analyses was to determine if the shape of the relationships yielded a common pattern that would define a meaningful cut point for the dds17 and dds2 . a minimum change of 0.5 sd units in dependent variables was used to help establish cut points ( 20 ) . the results of each significant effect were placed on scatterplots , with fitted linear or quadratic lines , to examine the shape and start - point of the relationship between each dds score and each of the dependent variables . mean - item dds values up to 4.0 were plotted because these encompassed more than 90% of the sample and dds values > 4 were based on small numbers with greater error . note that the direction of the curves varies as a function of the direction of the regression coefficient and the direction of the scale . screening identified 640 eligible patients and 506 participated ( 79% ) , with no demographic or diabetes status differences between those who did and did not participate . the final sample was 56.9% women , average age was 57.79 ( sd , 9.84 ) years , and average years since diagnosis was 8.14 ( 7.49 ) ( table 1 ) . about 37% self - identified as white , 87.7% completed high school , and 15% of patients were taking insulin . sample descriptions of 3d and redeem studies after step1 control variables were entered into the equations , multiple regression analyses indicated significant linear and quadratic effects between the dds17 and each of the four dependent variables ( table 2 ) , with increases in dd associated with poorer glycemic control ( p < 0.02 ) , self - efficacy ( p < 0.001 ) , diet ( p figure 1 indicates that substantive associations between dds17 and diabetes variables were evident at unexpectedly low levels of dds17 . relationships between the dds17 and the dependent variables began linearly between dds17 scores of 1 and 2 , continued but were somewhat muted between dds17 scores of 2 and 3 , and reached a maximum between dds17 scores of 3 and 4 . the significant increases in dds17 mean - item scores across the four diabetes variables increased by 0.5 sd units for each dependent variable for each of three dds17 score groups : below a dds mean - item score of 2 ( n = 276 , 54.6% of patients ) , between a score of 2 and 3 ( n = 139 , 27.4% ) , and above 3 ( n = 91 , 18.0% ) . standardized coefficients for dds17 and dds2 linear and curvilinear effects associations between 3d study dds17 scores and the key diabetes variables of hba1c ( a ) , self - efficacy ( b ) , healthy diet ( c ) , and physical activity ( d ) using fitted quadratic lines . findings from the dds2 analyses approximated those from the dds17 : after control variables were entered into the equations , significant linear and quadratic effects were found for hba1c ( p = 0.02 ) and self - efficacy ( p < 0.001 ) , but only significant linear effects were found for diet ( p < 0.001 ) and physical activity ( p = 0.005 ; table 1 ) . furthermore , where they occurred , the shape of the curvilinear relationships for the dds2 with the diabetes variables was virtually identical to those found for the full dds17 scale ( data not shown ) : substantive linear relationships began between a mean item score of 1 and 2 , continued but were muted between scores of 2 and 3 , and reached a maximum at a mean item score of 4 . likewise , about a half - sd unit of change in each dependent variable occurred up to a mean - item score of 2 , between 2 and 3 , and over 3 . of 588 subjects who were screened as eligible , 392 ( 67% ) completed the baseline assessment ( table 1 ) . there were no significant differences between participating and nonparticipating eligible patients on demographics or disease status . average age was 56.12 ( sd , 9.5 ) years , 56.5% were women , and diabetes duration was 7.9 ( 6.33 ) years . almost all patients completed high school ( 97.4% ) , 40% identified as white , and 18% were taking insulin . although redeem patients had to meet more restrictive inclusion criteria than 3d patients , between - group differences were minimal : redeem study patients were slightly younger , had higher education and initial diabetes distress , but displayed no hba1c differences ( table 1 ) . after the control variables were entered into the equations , significant quadratic effects occurred for the dds17 on hba1c ( p = 0.03 ) , self - efficacy ( p < 0.004 ) , and diet ( p = 0.04 ) , but not on physical activity ( p = ns ) : high dds17 was associated with poor glycemic control , low self - efficacy , and poor diet . as in the 3d study , notable linear associations between dds17 and the diabetes variables occurred between dds17 scores of 1 and 2 ( n = 135 , 34.5% of patients ) , continued between dds17 scores of 2 and 3 ( n = 162 , 41.3% ) , and a maximum was observed between a mean - item score of 3 and 4 ( n = 95 , 24.2% ) , with approximate changes in half - sd units of the dependent variables for each of the 3 dds score intervals . where they occurred , the degree and shape of the quadratic effects were virtually identical to those in the 3d study ( fig . 1 ) . for the dds2 , significant curvilinear effects occurred for hba1c ( p = 0.02 ) , for self - efficacy ( p = 0.05 ) , and for physical activity ( p = 0.03 ; table 2 ) . where curvilinear effects were found , substantive linear relationships occurred between a mean - item score of 1 and 2 , with more muted linear increases between scores of 2 and 3 , and a maximum was reached between mean - item scores of 3 and 4 . approximate half - sd increases in the dependent variables occurred for each dds2 score interval and the degree and shape of the curves were identical to those in the 3d study ( fig . only significant linear effects occurred for the dds2 on diet ( = 0.21 , p < 0.001 ) , with high distress associated with poor diet . screening identified 640 eligible patients and 506 participated ( 79% ) , with no demographic or diabetes status differences between those who did and did not participate . the final sample was 56.9% women , average age was 57.79 ( sd , 9.84 ) years , and average years since diagnosis was 8.14 ( 7.49 ) ( table 1 ) . about 37% self - identified as white , 87.7% completed high school , and 15% of patients were taking insulin . sample descriptions of 3d and redeem studies after step1 control variables were entered into the equations , multiple regression analyses indicated significant linear and quadratic effects between the dds17 and each of the four dependent variables ( table 2 ) , with increases in dd associated with poorer glycemic control ( p < 0.02 ) , self - efficacy ( p < 0.001 ) , diet ( p figure 1 indicates that substantive associations between dds17 and diabetes variables were evident at unexpectedly low levels of dds17 . relationships between the dds17 and the dependent variables began linearly between dds17 scores of 1 and 2 , continued but were somewhat muted between dds17 scores of 2 and 3 , and reached a maximum between dds17 scores of 3 and 4 . the significant increases in dds17 mean - item scores across the four diabetes variables increased by 0.5 sd units for each dependent variable for each of three dds17 score groups : below a dds mean - item score of 2 ( n = 276 , 54.6% of patients ) , between a score of 2 and 3 ( n = 139 , 27.4% ) , and above 3 ( n = 91 , 18.0% ) . standardized coefficients for dds17 and dds2 linear and curvilinear effects associations between 3d study dds17 scores and the key diabetes variables of hba1c ( a ) , self - efficacy ( b ) , healthy diet ( c ) , and physical activity ( d ) using fitted quadratic lines . findings from the dds2 analyses approximated those from the dds17 : after control variables were entered into the equations , significant linear and quadratic effects were found for hba1c ( p = 0.02 ) and self - efficacy ( p < 0.001 ) , but only significant linear effects were found for diet ( p < 0.001 ) and physical activity ( p = 0.005 ; table 1 ) . furthermore , where they occurred , the shape of the curvilinear relationships for the dds2 with the diabetes variables was virtually identical to those found for the full dds17 scale ( data not shown ) : substantive linear relationships began between a mean item score of 1 and 2 , continued but were muted between scores of 2 and 3 , and reached a maximum at a mean item score of 4 . likewise , about a half - sd unit of change in each dependent variable occurred up to a mean - item score of 2 , between 2 and 3 , and over 3 . of 588 subjects who were screened as eligible , 392 ( 67% ) completed the baseline assessment ( table 1 ) . there were no significant differences between participating and nonparticipating eligible patients on demographics or disease status . average age was 56.12 ( sd , 9.5 ) years , 56.5% were women , and diabetes duration was 7.9 ( 6.33 ) years . almost all patients completed high school ( 97.4% ) , 40% identified as white , and 18% were taking insulin . although redeem patients had to meet more restrictive inclusion criteria than 3d patients , between - group differences were minimal : redeem study patients were slightly younger , had higher education and initial diabetes distress , but displayed no hba1c differences ( table 1 ) . results from the multiple regression analyses for redeem paralleled 3d findings . after the control variables were entered into the equations , significant quadratic effects occurred for the dds17 on hba1c ( p = 0.03 ) , self - efficacy ( p < 0.004 ) , and diet ( p = 0.04 ) , but not on physical activity ( p = ns ) : high dds17 was associated with poor glycemic control , low self - efficacy , and poor diet . as in the 3d study , notable linear associations between dds17 and the diabetes variables occurred between dds17 scores of 1 and 2 ( n = 135 , 34.5% of patients ) , continued between dds17 scores of 2 and 3 ( n = 162 , 41.3% ) , and a maximum was observed between a mean - item score of 3 and 4 ( n = 95 , 24.2% ) , with approximate changes in half - sd units of the dependent variables for each of the 3 dds score intervals . where they occurred , the degree and shape of the quadratic effects were virtually identical to those in the 3d study ( fig . 1 ) . for the dds2 , significant curvilinear effects occurred for hba1c ( p = 0.02 ) , for self - efficacy ( p = 0.05 ) , and for physical activity ( p = 0.03 ; table 2 ) . where curvilinear effects were found , substantive linear relationships occurred between a mean - item score of 1 and 2 , with more muted linear increases between scores of 2 and 3 , and a maximum was reached between mean - item scores of 3 and 4 . approximate half - sd increases in the dependent variables occurred for each dds2 score interval and the degree and shape of the curves were identical to those in the 3d study ( fig . only significant linear effects occurred for the dds2 on diet ( = 0.21 , p < 0.001 ) , with high distress associated with poor diet . significant curvilinear relationships occur between the dds17 and measures of glycemic control , diabetes - specific self - efficacy , diet , and physical activity in two community - based samples of type 2 diabetic adults , with the exception of physical activity in redeem . the degree and shape of these relationships are almost identical for two patient samples of type 2 diabetic adults : a highly diverse community sample ( 3d study ) and a more restricted community sample that displayed high initial levels of distress , no clinical depression , and poor self - management ( redeem study ) . the similar degree and shape of relationships across studies , across dependent variables , and across the dds17 and dds2 highlight two important findings with respect to establishing a clinically meaningful cut point for high distress . first , the significant relationship of diabetes - specific distress with each of the four dependent variables begins at unexpectedly low levels of distress far less dd is required to demonstrate a relationship with diabetes - specific biologic and behavioral indicators than was previously considered . a previous report suggested that a mean item score of 3.0 provided a valid cut point for high dd ( 7 ) . our new findings confirm and expand these findings : even relatively low levels of dd are associated with diabetes - related indicators . second , to help determine where an appropriate dds cut point should be , we focus on the notable consistency in the shape of the significant curvilinear relationships between the dds17 across the dependent variables in both 3d and redeem : the curve rises linearly from a dds17 mean item score of 1 to 2 , continues linearly but in a more muted fashion between scores of 2 and 3 , and reaches a maximum between scores 3 and 4 . we note also that , in general , there is an 0.5 sd increase in each of the four dependent variables corresponding to increases in dds17 mean item scores from 1 to 2 , 2 to 3 , and above 3 . findings for the dds2 approximate those for the dds17 , even with the many fewer items and far less information contained in dds2 . the similar pattern of significant linear and curvilinear effects across the four diabetes variables , the similar degree of association , and the similar shape of the respective curves of association all suggest that the dds2 behaves relatively similarly to the dds17 with respect to the four diabetes variables we studied . these findings suggest that dichotomizing the distribution of dds17 scores to denote a high distress group and a low distress group does not accurately reflect the shape of the relationships between the dds17 and the four diabetes indicators . it therefore may be more helpful to define three dds categories : little or no dd ( dds < 2.0 ) , moderate dd ( dds = 2.02.9 ) , and high dd ( dds 3.0 ) . creating defined categories of moderate and high dd provides a better reflection of the shape of these relationships across diabetes indicators than a single cut point and allows greater flexibility for use in clinical and research settings . 3d study results , based on a diverse community sample , indicate that 45.4% of patients with type 2 diabetes display at least moderate dd . ( percentages for redeem are considerably higher65.5%because this study required high dd as an inclusion criterion . ) these findings suggest that significant levels of dd occur among almost half of patients with type 2 diabetes and that the possible reciprocal influence of dd on diabetes indicators indicates increased risk for poor treatment outcomes . because 70% of these patients are not clinically depressed ( 2 ) , interventions for dd may best be focused on ongoing conversations about dd and diabetes management in the clinical setting . highly rated dds17 items can be used to identify areas of specific patient concern , and patients are often relieved when clinicians initiate a discussion that labels feelings overtly , links them with diabetes - related difficulties , and normalizes them in ways that provide both reassurance and perspective ( 1 ) . even the verbalization of emotional experiences surrounding diabetes can be therapeutic and can lead to action planning for behavioral change . the dds17 may display different levels and patterns of association with other diabetes - related indicators . second , we did not explore how patient demographic and diabetes - related factors qualify these relationships ; instead , we controlled for many of them in equations . although our goal was to report findings with the greatest generalizability , differences in results may occur for subgroups of patients . last , our findings are cross - sectional and associational , such that causation between dd and diabetes indicators can not be inferred . a previous study suggested that these associations are bidirectional ( 2 ) , with one affecting the other sequentially over time . we used a data - oriented approach to establish empirically dds cut points for significant diabetes distress among adult patients with type 2 diabetes . in two community samples we show a consistent pattern of curvilinear relationships between the dds and hba1c , diabetes self - efficacy , diet , and physical activity . the degree and shape of these relationships suggest cut points on the dds17 and the dds2 for three patient subgroups : those with little or no distress , moderate distress , and high distress .

this is a prospective cohort study of patients admitted to a specialized geriatric icu at the liuhuaqiao hospital in guangzhou , china . founded in 1933 , it grew out of the general hospital of guangzhou military command . since 2009 , it has been open to the general public , and named liuhuaqiao , meaning flowing flowers bridge . it treats all eligible patients , from the army or the community , following the same standards . the 16-bed specialized geriatric icu , established in 2007 , provides critical care for older patients with serious conditions . it addresses the challenges of providing care , especially to senior military officials , in a cultural expectation that filial piety is often best demonstrated by doing everything without regard to a patient s age , prior level of function , or potential outcomes ( 13 ) . over 500 patients the specialized geriatric icu has significantly improved survival of older adults , for example recording a 50% mortality reduction compared to when they were cared for in the general icu . here , all icu patients aged 65 + years admitted between july 2011 and december 2011 were evaluated ( n = 155 ; including 114 retired military officials ) . mortality data were collected up to november 1 , 2012 through death certificates , decedent records , and medical records , or contact with the next of kin or other caregivers . several scores commonly used in icu and/or geriatric care settings were recorded , including the glasgow coma scale ( 33 ) , karnofsky performance scale ( 34 ) , palliative performance scale ( 35 ) , acute physiology score ( 36 ) , and acute physiology and chronic health evaluation an fi was constructed for each patient , using variables drawn from icu admission records of acute medical conditions , chronic diseases , symptoms , signs , premorbid function ( from an informant ) , lifestyle , health attitude , psychological health , and laboratory measures . following a standard procedure ( 39 ) , all items which met the criteria for being counted as a deficit in the fi were selected . to be counted as a deficit , a variable should : be health related ; be present in the general population in at least 1% of people , but in fewer than 80% ; increase in prevalence with age in the general population ; be related to death or other adverse health outcomes ( eg , institutionalization ) , and ; contain < 5% missing data . any fi should have > 30 variables , covering several organ systems ( 39 ) . premorbid status ( eg , mobility and dependence scores ) was defined as the average performance 1 month prior to admission . of the 13 variables with any missing values , most were missing in just 1 or 2 patients ; two variables were missing a maximum of 9 cases ( 4.3% ) . missing values were replaced using the variable s nonmissing mean . to better understand which items most influenced fi behavior , we also subdivided it into a chronic fi ( n = 23 items ) and an acute fi ( n = 31 items ) ( supplementary appendix 1 ) . the icu risk scores typically did not contain neuropsychiatric items , as these are not addressed in icu guidelines ( supplementary appendix 2 ) . among the 52 variables , 15 had 2 levels ( absent / present ) , 6 had 3 levels , 2 had 5 levels , 1 had 7 levels , and the remaining 31 were continuous . each variable was recoded to a value between 0 and 1 , that is , 1 indicating the highest level of a problem and 0 its absence . cut - point selection for recoding was based on established criteria and the diagnostic recommendations used in the hospital ( supplementary appendix 1 ) . an fi value was calculated for each subject as the proportion of deficits present ( ie , all deficits summed and divided by 52 ) . theoretically , fi values can range from 0 ( no deficits present ) to 1 ( all deficits present ) . even so , an fi > 0.7 is seldom ( < 1% ) observed ; this appears to represent a natural limit to frailty , beyond which survival is not possible ( 1,22,23 ) . higher fi values indicate a greater level of frailty , and thus worse health and greater vulnerability to adverse outcomes , and vice versa . variable selection , grouping , and recoding were performance prior to fi construction and further statistical analyses . data are presented as means and standard deviations for continuous variables and percentages for discrete variables . analysis of variance and chi - square ( ) tests were used respectively to compare group differences in the means or percentages . to evaluate the relationship between each measure and survival , logistic regression was used to calculate the likelihood and 95% confidence intervals ( cis ) of early death ( less than 30 days ) versus survival to the end of the study period ( 300 days ) . the akaike information criterion was calculated for each score based on maximum likelihood from logistic regression to evaluate the relative quality of the model fitting . cox proportional hazard models were employed to estimate the age / sex adjusted survival probability . fi values were graded to 0100 integers by rounding the fi after multiplying it by 100 , so as to evaluate the change in risk seen with each percent increment of the fi and other measures . to evaluate the value added by the fi , the fi and each score was tested in separate models , as well as in the same model . the all factor fi and the acute or chronic fi were also included in the cox proportional hazard model to test which index explained more variability when adjusting for each other . performance of the scores in classifying patients who died and who survived was assessed by the area under receiver operating characteristic curves ( supplementary appendix 3 details the sensitivity and specificity at various cut points ) . kaplan meier survival probabilities were evaluated using each score , stratified to assay clinically useful cut - points ( ie , 30% subsample with the best score , 10% with the worst , and two levels in between ) . data analyses were conducted using spss version 19.0 and codes developed using matlab v8 . statistical significance level was set at p = 0.05 . the data analysis protocol was approved by the research ethics board at the capital district health authority , where the analyses took place . this is a prospective cohort study of patients admitted to a specialized geriatric icu at the liuhuaqiao hospital in guangzhou , china . founded in 1933 , it grew out of the general hospital of guangzhou military command . since 2009 , it has been open to the general public , and named liuhuaqiao , meaning flowing flowers bridge . it treats all eligible patients , from the army or the community , following the same standards . the 16-bed specialized geriatric icu , established in 2007 , provides critical care for older patients with serious conditions . it addresses the challenges of providing care , especially to senior military officials , in a cultural expectation that filial piety is often best demonstrated by doing everything without regard to a patient s age , prior level of function , or potential outcomes ( 13 ) . over 500 patients the specialized geriatric icu has significantly improved survival of older adults , for example recording a 50% mortality reduction compared to when they were cared for in the general icu . here , all icu patients aged 65 + years admitted between july 2011 and december 2011 were evaluated ( n = 155 ; including 114 retired military officials ) . mortality data were collected up to november 1 , 2012 through death certificates , decedent records , and medical records , or contact with the next of kin or other caregivers . several scores commonly used in icu and/or geriatric care settings were recorded , including the glasgow coma scale ( 33 ) , karnofsky performance scale ( 34 ) , palliative performance scale ( 35 ) , acute physiology score ( 36 ) , and acute physiology and chronic health evaluation an fi was constructed for each patient , using variables drawn from icu admission records of acute medical conditions , chronic diseases , symptoms , signs , premorbid function ( from an informant ) , lifestyle , health attitude , psychological health , and laboratory measures . following a standard procedure ( 39 ) , all items which met the criteria for being counted as a deficit in the fi were selected . to be counted as a deficit , a variable should : be health related ; be present in the general population in at least 1% of people , but in fewer than 80% ; increase in prevalence with age in the general population ; be related to death or other adverse health outcomes ( eg , institutionalization ) , and ; contain < 5% missing data . any fi should have > 30 variables , covering several organ systems ( 39 ) . premorbid status ( eg , mobility and dependence scores ) was defined as the average performance 1 month prior to admission . of the 13 variables with any missing values , most were missing in just 1 or 2 patients ; two variables were missing a maximum of 9 cases ( 4.3% ) . missing values were replaced using the variable s nonmissing mean . to better understand which items most influenced fi behavior , we also subdivided it into a chronic fi ( n = 23 items ) and an acute fi ( n = 31 items ) ( supplementary appendix 1 ) . the icu risk scores typically did not contain neuropsychiatric items , as these are not addressed in icu guidelines ( supplementary appendix 2 ) . among the 52 variables , 15 had 2 levels ( absent / present ) , 6 had 3 levels , 2 had 5 levels , 1 had 7 levels , and the remaining 31 were continuous . each variable was recoded to a value between 0 and 1 , that is , 1 indicating the highest level of a problem and 0 its absence . cut - point selection for recoding was based on established criteria and the diagnostic recommendations used in the hospital ( supplementary appendix 1 ) . an fi value was calculated for each subject as the proportion of deficits present ( ie , all deficits summed and divided by 52 ) . theoretically , fi values can range from 0 ( no deficits present ) to 1 ( all deficits present ) . even so , an fi > 0.7 is seldom ( < 1% ) observed ; this appears to represent a natural limit to frailty , beyond which survival is not possible ( 1,22,23 ) . higher fi values indicate a greater level of frailty , and thus worse health and greater vulnerability to adverse outcomes , and vice versa . variable selection , grouping , and recoding were performance prior to fi construction and further statistical analyses . data are presented as means and standard deviations for continuous variables and percentages for discrete variables . analysis of variance and chi - square ( ) tests were used respectively to compare group differences in the means or percentages . to evaluate the relationship between each measure and survival , logistic regression was used to calculate the likelihood and 95% confidence intervals ( cis ) of early death ( less than 30 days ) versus survival to the end of the study period ( 300 days ) . the akaike information criterion was calculated for each score based on maximum likelihood from logistic regression to evaluate the relative quality of the model fitting . cox proportional hazard models were employed to estimate the age / sex adjusted survival probability . fi values were graded to 0100 integers by rounding the fi after multiplying it by 100 , so as to evaluate the change in risk seen with each percent increment of the fi and other measures . to evaluate the value added by the fi , the fi and each score was tested in separate models , as well as in the same model . the all factor fi and the acute or chronic fi were also included in the cox proportional hazard model to test which index explained more variability when adjusting for each other . performance of the scores in classifying patients who died and who survived was assessed by the area under receiver operating characteristic curves ( supplementary appendix 3 details the sensitivity and specificity at various cut points ) . kaplan meier survival probabilities were evaluated using each score , stratified to assay clinically useful cut - points ( ie , 30% subsample with the best score , 10% with the worst , and two levels in between ) . the data analysis protocol was approved by the research ethics board at the capital district health authority , where the analyses took place . most patients were married ( 91.6% ) men ( 87.1% ) who lived with family members ( 99.4% ) . the mean age was 82.77.1 years ( median : 84 ; range : 65103 ) . during the 300-day follow - up , 60 patients died ( 38.7% ) , among whom 27 ( 45.0% ) died within the first 30 days . most ( 53 ) of the 60 deaths occurred in hospital , two in long - term care institutions , and five at home . notably , on average people who died were not older than those who survived ; instead , they had higher fis ( poorer health ) and worse prognostic scores ( table 1 ) . most patients ( n = 139 , 89.7% ) had multiple deficits , with sepsis , hypertension , coronary heart disease , diabetes , dementia , and renal dysfunction being most common . when considered individually , 29 of the 52 deficits used in the fi were significantly associated with death ( p < 0.05 ) . of the remaining 23 deficits , 16 were more often present in those who died ( supplementary appendix 1 ) . characteristics of the sample at admission , by icu survival outcome notes : data are presented as mean ( standard deviation ) , unless as stated otherwise . ( apache = acute physiology and chronic health evaluation ) * a lower score is associated with poorer outcomes . the fi score was correlated with each prognostic score , that is , glasgow coma scale , karnofsky performance scale , palliative performance scale , acute physiology score , and acute physiology and chronic health evaluation apache - ii and apache - iv ( r ranged between 0.52 and 0.72 , ps < 0.001 ) , and varied significantly by survival status ( figure 1 ) : higher in patients who died within 30 days ( fi = 0.410.11 ) than in the survivors ( fi = 0.220.14 , f = 38.91 , p < 0.001 ; table 1 ) . all patients with fi scores < 0.22 ( n = 62 or 40% ) survived 30 days , whereas all patients with fi scores > 0.46 ( n = 15 or 10% ) died within 90 days ( figure 1 ; maximum survival = 66 days ) . the auc was 0.89 ( 95% ci = 0.830.95 ) for the fi in classifying individuals who died within 30 days against those who survived , comparable with the prognostic scores ( table 2 ) . including the fi in the same model with a prognostic score often led to an improved akaike information criterion or auc , or both ( table 2 ) . proportional distribution of the frailty index ( fi ) by survival status of patients admitted to the specialized geriatric icu . symbols ( dots ) represent observational data ; lines represent curves fit to the data . blue : patients who survived 300 days ; red : died between 31 and 300 days ; black : died within 30 days . the fi ranged from 0.06 to 0.63 . here , all patients who died had fi > 0.46 ( n = 15 ) , whereas all patients who survived 30 days had fi < 0.22 ( n = 63 ) . akaike information criterion ( aic ) and area under the curve ( auc ) for the risk of death using each score with ( upper lines ) or without ( lower lines ) including the all - factor frailty index in the model notes : aic = akaike information criterion ; apache = acute physiology and chronic health evaluation ; aps = acute physiology score ; auc = area under the roc curve ; ci = confidence interval ; fi = the 52-item all - factor frailty index ; gcs = glasgow coma scale ; ks = karnofsky scale ; pps = palliative performance scale ; p = level of significance . in an age - sex adjusted model , the fi was associated with an increased risk of death ( with each 1% increase in the fi from the previous level , for example , from 0.18 to 0.19 , the relative risk ratio for 30-day death = 1.11 , 95% ci = 1.071.15 ) . similarly , the fi was also associated with the risk of 300-day mortality ( supplementary appendix 4 ) . as a worked example , people of the same age and sex would be nine times more likely to die within 30 days if the fi at admission was 0.38 than if it was 0.18 ( ie , e = 9.02 ; table 3 , model 1 ) . the risk ratio for the fi remained significant when the icu scores were also adjusted for in the models ( p < 0.05 ) , whereas age was not , nor the glasgow coma scale or acute physiology and chronic health evaluation ( table 3 , model 2 ) . the acute and chronic fis showed lower predictive values than did the all - factor fi ( table 3 ) . cox proportional hazard ratio for the risk using the frailty index and the prognostic scores notes : apache = acute physiology and chronic health evaluation ; aps = acute physiology score ; ci = confidence interval ; fi = the 52-item all - factor frailty index ; gcs = glasgow coma scale ; ks = karnofsky scale ; pps = palliative performance scale ; p = level of significance . a robust dose - responsive survival probability was observed with the fi , as with most of the prognostic scores ( figure 2a , b ) . the 15 patients with fi > 0.46 and 100% mortality were not so identified using other scores ( figure 2a , b ) . five of these 15 patients did not also have the worst apache - iv scores , that is , the two best - performing scores identified different subsets of patients with the highest risks of death . kaplan meier survival probability ( km ) of patients admitted to the specialized geriatric icu as a function of time ( days ) . panel a : black frailty index ; red apache - iv ( acute physiology and chronic health evaluation iv ) . panel b : yellow gcs ( glasgow coma scale ) ; blue kps ( karnofsky performance scale ) ; purple pps ( palliative performance scale ) ; cyan aps ( acute physiology score ) ; green apache - ii ( acute physiology and chronic health evaluation ) . for each score , survival data were stratified into four clinically useful groups ; ie , 30% subsample with the best score ( dashed lines ) , 10% with the worst score ( solid lines ) , and two levels in between representing 30% subsamples each ( dotted lines and dot - dashed lines ) . we evaluated whether frailty was related to survival in older people admitted to a specialized geriatric icu , and compared an fi , with several commonly used icu prognostic scores . all were strongly associated with survival , and the performance of the fi was at least as good as any icu score . in addition , the fi identified a group of people who were least frail ( with fi < 0.22 ; corresponding to 40% of the sample ) all of whom survived 30 days , and a least fit group ( fi > 0.46 ; 10% of the sample ) , none of whom no one survived beyond 66 days . in short , the degree of frailty , as estimated by both acute deficits and information gathered on function , mobility , and health attitude 1 month prior to admission , was closely related to critical care survival in older adults . our data contribute to understanding whether knowledge about the extent of premorbid deficit accumulation adds prognostic value in acutely ill patients ( 2832 ) . an important part of understanding prognosis is in needing to advise people about near - term outcomes of hospital admission for frail older adults who are acutely ill ( 2830,40 ) . for this reason , examining older patients who died within days to months is of particular interest . here , the fi showed that the subset patients with the worst fi scores all died , whereas the subset with the best scores all survived . clinically , it is often the prediction of the best and worst outcomes that is most valuable in knowing how to gauge expectations . by contrast , considerable uncertainty typically accords any intermediate score , so impacts less on care options . that the high - risk subsets from the fi and apache - iv ( the best performing prognostic scores ) were not identical , suggests that older adults who died quickly could have heterogeneous profiles , not always identified now . for example , here , an apache - iv score as low as 60 ( indicating low risk ) was observed in two people whose fi > 0.46 ( indicating high risk ) and who did not survive . this brings to attention the potential value in taking frailty into account when assessing critically ill older patients . by using the fi and the risk scores in the same model , we were able to address whether the fi added value to the icu scores . the fi allows many pieces of data to contribute information , even when some deficits are not individually significant ( 1 ) . further , the all - item fi includes 33 items that were not considered by apache iv , and 35 items not considered by the acute physiology score . although the standard icu scores shared certain items with the fi , they notably omitted essential information about premorbid function . here , the auc changed variably with use of the fi , to a high level between 0.89 and 0.93 . while an increase of auc was observed in most cases , the kaplan meier auc remained 0.90 even though akaike information criterion was lower . this likely suggests the following : ( a ) fi helps improve performance , especially when it was not high originally ; ( b ) to better understand the data , different analysis approaches may allow examinations from different angles ; and ( c ) information imbedded in the data can determine the accuracy limit of a model , reflected by the < 100% accuracy by using multiple models . the redundancy of the human body makes it unlikely that two summary scores would assess risk and be completely independent , even with no overlap in their items . indeed , this redundancy allows for different versions of the fi using differing items , differing numbers of items , and items of differing natures ( eg , self - reported vs clinical assessments)to give similar results ( 41 ) . likewise , the items that make up an fi can be sampled at random without loss of explanatory power ( 42 ) . the results suggest that the all - item fi explained more variability compared to the partial ones ; by taking into account the accumulation of a large number of deficits , each can reflect an aspect of the system and thus add information to understanding the data . how to best incorporate frailty in the icu setting is challenging , given that older patients needing critical care typically have complex health conditions , and that multiple , interacting problems can vary between individuals . short - term mortality in older survivors arises in relation not just to their acute illness , but to their overall state of health , which is what the fi quantifies ( 1,3,43,44 ) . multiple deficits in the same individuals challenge modern healthcare systems , which have experienced enormous progress ( and train people for , are reimbursed and evaluated ) using a paradigm of single system illness and most responsible diagnoses ( 3,45 ) . surrogate measures of physiologic age offer the ability to stratify the risk for rapid deterioration related to a major event . here , the fi combined the effects of multiple problems in a single , graded variable that was easily operationalized from routine icu assessments and combined them with clinically relevant information that often is known by informants . using such instruments is part of a shift in how prognostic factors for icu outcomes are evaluated ( 9 ) . similarly , some measures now commonly employed in icus might inform acute care of frail older adults in geriatric medicine ( 43 ) . such interplay is part of the rationale for specialized geriatric intensive care , which offers a range of approaches for example early mobilization ( 15 ) and playing attention to sleep ( 16)that can make routine care less hazardous . our data also contribute to understanding frailty . here , despite the high prevalence of critical illness , the fi remained closely related to the risk of death , consistent with other observations in seriously ill patients ( 1,2830 ) . the limit to frailty ( ie , fi < 0.7 ) notably held . in short , this study joins virtually all others , including from community - dwelling older chinese adults ( 22,24 ) , in demonstrating a quantifiable extent to how many things an individual can have wrong with them and survive . such a submaximal limit is shared with many other frailty measures , suggesting that locally adapted scales can capture this important feature . first , this is a single centre study ; whether the conclusion is generalizable warrants multicentre investigations . second , reflecting historical and social circumstances , the sample consists mostly of men , and many from the military . previous studies have shown close relationships between age and the fi and between fi and mortality in both men and women ( 1 ) . third , our analysis has centred on 30-day mortality , and even though the 300-day data demonstrated similar trends , these might not hold for other follow - up periods . furthermore , as is common even with research studies , there were missing values for some variables , which can affect generalizability . here , our data showed consistent features in other icu samples ; for example , the high proportion with comorbidities was also seen in a large study where 79.4% of icu patients had comorbidities ( 46 ) . even though our study suggests that the fi can help improve the performance of the various common icu scores , further research will be needed to address which scores are most valid for mortality prediction in icu . here , the karnovsky performance scale was compared with the fi because , unlike the other scores , in many parts of china it used routinely . we focused on understanding 30-day death chiefly because short - term survival is most critical for icu outcome evaluation . for survival outcome analysis , the most updated health status has been shown to be more important than prior health states , making 30-day mortality a way to understand overall outcomes in this group , where there is net decline : that is , on average , decline outweighs recovery ( 21 ) . recognizing that any net positive score is likely to be early , we have also examined mortality over 300 days in relation to the risk scores and the trend of the results held true ( supplementary appendix 4 ) . note too , although death was readily verifiable , survival might be less so , given that some families have a financial interest in not reporting death immediately , as older adults receive social benefits . this is a less serious concern for short - term mortality as most patients ( n = 138 , 89.0% ) remained in hospital / institution , and deaths were recorded immediately as they occurred . with its very large population of older adults , understanding frailty in china is important in its own right . health deficits , even when including acute illness , are only one part of what puts people at risk : the social and physical environment is also important , something not directly measured here . previous research has suggested that the fi is robust to exactly which variables are used in its construction ( 1 ) . even so , in the critical care setting , this must be established formally through larger studies . older patients needing critical care are typically both critically ill and also often frail , which collectively determine their outcomes . our study examined the value of the health deficit accumulation - based fi in a specialized geriatric icu . the data showed that the greater the degree of frailty , the greater the risk of death . here , the frailest group of patients ( with the highest fi scores ) showed 100% mortality , some of whom were not identified by the other commonly used measures , which do not focus on older adults . our study also verified that the empirical limit to deficit accumulation ( fi ~ 0.7 ) also applies to seriously ill older adults : when the limit of frailty is reached , death occurs within days to months . in short , our data showed a close association between frailty and icu survival , and suggest that the fi can add value to commonly used icu prognostic scores . paying attention to premorbid health and function can help care providers better estimate the likelihood of benefit from icu admission . in particular , since it is often difficult to make people better than they were a month before they became ill , that level of function might serve as a benchmark for individual care planning . this research was partially supported by the china - canada joint health research initiative grant from the canadian institutes of health research ( cihr cci-92216 ) , and operating grants from the national natural science foundation of china ( e0603 - 59976016 ; 61300107 ) , chinese military medicine and health foundation ( 119 - 10bjz09 ) , natural science foundation of guangdong ( s2012010010212 ) , science and technology planning project of guangdong , china ( 2012a061400010 ) , science and technology planning project of guangzhou , china ( 201505031501397 ; 201504301341059 ) , and guangdong key laboratory of geriatric infection and organ function . additional funding for analysis came from the fountain innovation fund of the queen elizabeth ii health sciences research foundation , halifax , canada .

chronic renal failure is a progressive deterioration of renal function impairment which leads to interference with the metabolism of body water and electrolytes . two - three percent of the current world 's population is suffering from chronic renal failure and every year the rate increases twice . the standard recommended treatments are renal transplantation , hemodialysis ( hd ) , and peritoneal dialysis for patients with renal failure . the patients underwent hd for a long time , suffer from physical and mental stresses , and experience serious changes in lifestyle and personality . in the last 3 decades , psychological factors have attracted more attention in end stage like patients with chronic renal failure . previous researches have reported some psychological problems such as suicide ideation , depression , anxiety , sexual disorders , interpersonal problems , paranoia , physical complain , compulsive disorders , psychoses , aggression , and phobia ; although researchers are not unanimous in terms of prevalence and intensity of these problems among hd patients . navidian et al . , reported that 10% of hd patients had a history of psychiatric disorders while it was only 2.5% in the general population . stress refers to the consequence of the failure of a person to respond appropriately to emotional or physical threats . inappropriate coping decreases quality of life and leads to physical , mental , economic , social , and emotional problems . regarding pharmacological stress control methods relaxation is one of most useful nonpharmacological technique which reduces stress through impact on mental and physical conditions , depression , mood , anxiety , and self - steam . among relaxation methods , benson 's relaxation method ( 1970 ) is one of the easiest to learn and administration . despite the importance of reduction of stress and anxiety among hd patients , there is no evidence to evaluate the impact of nonpharmacological methods especially relaxation in hd patients in iran . therefore , this study aimed to evaluate the impact of benson 's relaxation method of stress reduction in hd patients in iran . the study population included 80 hd patients from imam khomeini and fatemeh zahra hospitals in the mazandaran city during 2011 , who met the inclusion criteria . eighty ( n = 40 in experimental group and n = 40 in the control group ) patients were selected randomly based on a random number table . sampling was parallel , the first eligible patients assigned in the intervention group and the next one in the control group . all samples who met the inclusion criteria were selected randomly and had the same chance of participation . the inclusion criteria were : age range between 18 and 65 years ; at least 2 months dialysis history ; active file recordation in mentioned hospitals ; no comorbidity with other chronic disease like cancer , heart disease , etc . ( diabetes and hypertension are included , because usually this comorbidities are a part of renal failure ; excluding them leads to limitation in generalization ) . exclusion criteria were : the unwillingness of a patient , physical disability , regular tranquillizer or sedative drugs ; had a previous history of psychiatric disorders , suffered from stress other than dialysis in the past 6 months , incomplete training ( below 55 sessions ) , and peritoneal dialysis or a kidney transplant . sample size was calculated based on the pilot study with : considering mean and standard deviation ( sd ) before ( 16.5 6.9 ) and after ( 13.13 2.27 ) with 95% confidence level , sample size was calculated as n = 37 , however prediction of a 10% drop during intervention n = 40 was the final sample size . based on research aims demographic characters included : age , gender , marital status , income level , education level , dialysis time and duration , shelter status , medicine usage , other disease , etc . depression , stress , and anxiety was assessed by depression anxiety stress scale ( dass21 ) , as these questionnaires assessed the three variables contemporarily with limited questions in comparison to other standardized questionnaires . the dass is a quantitative measure of distress along the three axes of depression , anxiety , and stress . this questionnaire had been presented for the first time by lovibond in 1995 which included three subscales and every subscale included seven questions . in translated version , each item has choices of never , little , moderate , and many . the lowest score is related to every zero question and the highest score is 3 . in this questionnaire the questions 2 , 4 , 7 , 9 , 15 , 19 , and 20 are related to anxiety ; 3 , 5 , 10 , 13 , 16 , 17 , and 21 are related to depression and the questions 1 , 6 , 8 , 11 , 12 , 14 , and 18 assess the stress . defined cutoff points are as follows : stress : scores from 0 - 7 is normal , 8 - 9 mild , 10 - 12 average , 13 - 16 severe , and higher than 17 is very severe.anxiety : 0 - 3 is normal , 4 - 5 mild , 6 - 7 average , 8 - 9 severe , and higher than 10 is very severe.depression : score 0 - 4 is considered normal , 5 - 6 mild , 7 - 10 average , 11 - 13 severe , and higher than 14 is very severe . stress : scores from 0 - 7 is normal , 8 - 9 mild , 10 - 12 average , 13 - 16 severe , and higher than 17 is very severe . anxiety : 0 - 3 is normal , 4 - 5 mild , 6 - 7 average , 8 - 9 severe , and higher than 10 is very severe . depression : score 0 - 4 is considered normal , 5 - 6 mild , 7 - 10 average , 11 - 13 severe , and higher than 14 is very severe . , on 970 students and armies , the authors reported the translated questionnaire is comparable with the original one and there is high internal correlation 0.77 , 0.79 and 0.78 for depression , anxiety and stress respectively , according to this questionnaire . the study by moradi panah in iran has also reported 0.94 cronbach 's alpha for depression 0.92 for anxiety 0.82 for stress . dass21 questionnaire was administrated by nurses blindly before and after intervention by self - reporting method or nurses recorded data in case they were illiterate or had visual disorders . after patients signed consent to participate , we used video exhibition to train the benson 's relaxation method to patients and their care givers in dialysis center . in first step , the patients and their care givers learned relaxation through exhibition in hd center , then the cd was provided and asked care giver to observe and guide patients to practice correctly twice a day in morning and evening for 15 min during 4 weeks . relaxation was practiced whenever they came to the center or contacted them by phone daily to remind on time practice . the instruction of the benson 's relaxation technique included the following steps : stay in confidence position.close your eyes.calm down and relax your body , relax from your toes to top of your head.take a breath from your nose and keep your awareness . exhale from the mouth whenever exhaling , repeat one word or number , inhale , and exhale with comfort and confidence.do this for 15 min . try to keep your body and muscles relaxed . then open your eyes slowly and do not move for few minutes.do not worry , it is not important to which level of relaxation you have reached , calm down and relax your body , relax from your toes to top of your head . exhale from the mouth whenever exhaling , repeat one word or number , inhale , and exhale with comfort and confidence . do this for 15 min . try to keep your body and muscles relaxed . then open your eyes slowly and do not move for few minutes . do not worry , it is not important to which level of relaxation you have reached , leave your body and let it happen itself . do not care about interfering thoughts and let them go . data were analyzed using the spss software ( statistical package for the social sciences , version 16.0 , spss inc , chicago , ill , usa ) . descriptive statistics such as central and dispersion indexes were first run and then regarding normal distribution the independent sample t - test was applied to compare the two groups . according to the terms of the scattering data regarding correlation was not significant , there was not a meaningful significant linear relationship ( data distribution is not significant ) . therefore , the simplest parametric method was used for analysis ( independent and paired t - test ) . demographic data categorized and quantitative data converted to qualitative data and k - square was used to analyze the obtained data . pre- and post - intervention scores in each group were compared using the paired t - test . the study population included 80 hd patients from imam khomeini and fatemeh zahra hospitals in the mazandaran city during 2011 , who met the inclusion criteria . eighty ( n = 40 in experimental group and n = 40 in the control group ) patients were selected randomly based on a random number table . sampling was parallel , the first eligible patients assigned in the intervention group and the next one in the control group . all samples who met the inclusion criteria were selected randomly and had the same chance of participation . the inclusion criteria were : age range between 18 and 65 years ; at least 2 months dialysis history ; active file recordation in mentioned hospitals ; no comorbidity with other chronic disease like cancer , heart disease , etc . ( diabetes and hypertension are included , because usually this comorbidities are a part of renal failure ; excluding them leads to limitation in generalization ) . exclusion criteria were : the unwillingness of a patient , physical disability , regular tranquillizer or sedative drugs ; had a previous history of psychiatric disorders , suffered from stress other than dialysis in the past 6 months , incomplete training ( below 55 sessions ) , and peritoneal dialysis or a kidney transplant . sample size was calculated based on the pilot study with : considering mean and standard deviation ( sd ) before ( 16.5 6.9 ) and after ( 13.13 2.27 ) with 95% confidence level , sample size was calculated as n = 37 , however prediction of a 10% drop during intervention n = 40 was the final sample size . based on research aims demographic characters included : age , gender , marital status , income level , education level , dialysis time and duration , shelter status , medicine usage , other disease , etc . depression , stress , and anxiety was assessed by depression anxiety stress scale ( dass21 ) , as these questionnaires assessed the three variables contemporarily with limited questions in comparison to other standardized questionnaires . the dass is a quantitative measure of distress along the three axes of depression , anxiety , and stress . this questionnaire had been presented for the first time by lovibond in 1995 which included three subscales and every subscale included seven questions . in translated version , each item has choices of never , little , moderate , and many . the lowest score is related to every zero question and the highest score is 3 . in this questionnaire the questions 2 , 4 , 7 , 9 , 15 , 19 , and 20 are related to anxiety ; 3 , 5 , 10 , 13 , 16 , 17 , and 21 are related to depression and the questions 1 , 6 , 8 , 11 , 12 , 14 , and 18 assess the stress . defined cutoff points are as follows : stress : scores from 0 - 7 is normal , 8 - 9 mild , 10 - 12 average , 13 - 16 severe , and higher than 17 is very severe.anxiety : 0 - 3 is normal , 4 - 5 mild , 6 - 7 average , 8 - 9 severe , and higher than 10 is very severe.depression : score 0 - 4 is considered normal , 5 - 6 mild , 7 - 10 average , 11 - 13 severe , and higher than 14 is very severe . stress : scores from 0 - 7 is normal , 8 - 9 mild , 10 - 12 average , 13 - 16 severe , and higher than 17 is very severe . anxiety : 0 - 3 is normal , 4 - 5 mild , 6 - 7 average , 8 - 9 severe , and higher than 10 is very severe . depression : score 0 - 4 is considered normal , 5 - 6 mild , 7 - 10 average , 11 - 13 severe , and higher than 14 is very severe . for instance , in a study done by sahebi et al . , on 970 students and armies , the authors reported the translated questionnaire is comparable with the original one and there is high internal correlation 0.77 , 0.79 and 0.78 for depression , anxiety and stress respectively , according to this questionnaire . the study by moradi panah in iran has also reported 0.94 cronbach 's alpha for depression 0.92 for anxiety 0.82 for stress . dass21 questionnaire was administrated by nurses blindly before and after intervention by self - reporting method or nurses recorded data in case they were illiterate or had visual disorders . after patients signed consent to participate , we used video exhibition to train the benson 's relaxation method to patients and their care givers in dialysis center . in first step , the patients and their care givers learned relaxation through exhibition in hd center , then the cd was provided and asked care giver to observe and guide patients to practice correctly twice a day in morning and evening for 15 min during 4 weeks . relaxation was practiced whenever they came to the center or contacted them by phone daily to remind on time practice . the instruction of the benson 's relaxation technique included the following steps : stay in confidence position.close your eyes.calm down and relax your body , relax from your toes to top of your head.take a breath from your nose and keep your awareness . exhale from the mouth whenever exhaling , repeat one word or number , inhale , and exhale with comfort and confidence.do this for 15 min . try to keep your body and muscles relaxed . then open your eyes slowly and do not move for few minutes.do not worry , it is not important to which level of relaxation you have reached , leave your body and let it happen itself . calm down and relax your body , relax from your toes to top of your head . exhale from the mouth whenever exhaling , repeat one word or number , inhale , and exhale with comfort and confidence . do this for 15 min . try to keep your body and muscles relaxed . then open your eyes slowly and do not move for few minutes . do not worry , it is not important to which level of relaxation you have reached , leave your body and let it happen itself . data were analyzed using the spss software ( statistical package for the social sciences , version 16.0 , spss inc , chicago , ill , usa ) . descriptive statistics such as central and dispersion indexes were first run and then regarding normal distribution the independent sample t - test was applied to compare the two groups . according to the terms of the scattering data regarding correlation was not significant , there was not a meaningful significant linear relationship ( data distribution is not significant ) . therefore , the simplest parametric method was used for analysis ( independent and paired t - test ) . demographic data categorized and quantitative data converted to qualitative data and k - square was used to analyze the obtained data . pre- and post - intervention scores in each group were compared using the paired t - test . patient who practiced 55 times and above was considered as a participant who completed the study . there was no drop in experimental and control groups because of daily follow - up and patients good cooperation . the samples comprised 44 male ( 55% ) and 36 females ( 45% ) and the mean age was 47.98 12.53 . seventy - one people were married ( 88.8% ) , eight were single ( 10% ) , and one was ( 1.2% ) table 1 . two groups were compared with k - square and no significant differences were seen in demographic characters between the two groups . the comparison of two mean scores before and after intervention in terms of stress and anxiety showed a significant difference between two time frames ( p > 0.001 ) . however in the control group , there was no significant difference in two time frame scores ( p < 0.11 , p < 0.18 ) . distribution of patients according to demographic characters there were no significant difference between depression levels in the experimental and control groups , just in experimental group there was an insignificant difference after intervention ( p < 0.22 ) [ table 2 ] . the mean comparison between two groups stress , anxiety , and depression there were significant relationships between anxiety , stress , and depression with demographic characters such as age , gender , marital status , economic level , education , dialysis duration , dialysis frequency in year and week , shelter status , content disease , and using other medicine ( exception renal failure ) ( p < 0.05 ) . based on the finding in present study ; means of stress , anxiety , and depression is high among the patients with hemodialysis ( hd ) . as it is obvious from [ table 2 ] , there are significant differences in mean and standard deviation of control and intervention groups in case of stress and anxiety scores . , mollahadi et al . , shafipour et al . , eghbali et al . , and rajamanickam and kumar . they also suggested that stress reduction influences quality of life in the patients , but they need regular psychotherapy also . this study has shown that there is a difference between the mean and standard deviation of the group who received benson 's relaxation and who did not receive . there is growing evidence to evaluation of the influence of benson 's relaxation as a cost effective and safe technique on different chronic disease . hosseini et al . , confirmed that psychological training based on the standard protocols and the use of medicine resulted in comparable decreases in the severity of depressive and anxiety symptoms of hd patients . in another study rahimi et al . , which evaluated palliative care effects during 4 - 6 months on stress , anxiety , and depression ; the results showed positive effects of care on hd patients . , also explored the effect of music therapy on stress , anxiety , and depression in hd patients and confirmed significant stress reduction in hd patients after music therapy . according to evidence practice of the benson 's relaxation technique , results have shown significant gains in tons if practiced regularly for long time period . hence , in present study insignificancy of intervention and control group in depression is explainable by limited practiced time period . the results of this study and other related studies in this context showed that using cost effective , low risk , and easy methods by patients could help the patients to reduce the stress and lead to several benefits if practiced daily ; beside the palliative care and alternative treatments in hd patients . the level of stress was high among hd patients , although it decreased after the intervention , but still it was at a higher level . regarding some limitations of this study , researchers could not observe all sessions and there can be some fake report from patients or care givers or it could be due to limited practice time ( 1 month ) . on the other hand , dass21 is not a categorical measure of clinical diagnoses . generally we have mentioned in benson 's relaxation method in text , our findings confirmed the effect of relaxation on stress and anxiety with several studies and the recommendations are mostly emphasized on long - term , regular practice . the most effects of relaxation works through the decrease of metabolism and strengthening of heart contractions , respiration , and blood pressure ; and release of epinephrine on the sympathetic system of a patient 's physiological condition . thus , by teaching the nurses can benefit the patients can by lower cost and prevent from extra problems as it is easy to use and teach to all levels of patients .

cholesterol , l - a - pa , stearylamine ( sa ) , lactoperoxidase , monensin , 2,5-diphenyl oxazole , 1,4-bis[2-(5-phenyl oxazole ) ] benzene , guar gum , penicillin - g ( potassium salt ) , streptomycin sulphate , 2.5% trypsin in hbss ( hank s balanced salt solution ) and hepes ( ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) were purchased from sigma chemical co. , st . dspe - mpeg ( 1000 , 2000 , 3000 and 5000 ) ( distearoyl phosphatidylethanolamine polyethylene glycol ) was purchased from avanti polar lipids , inc . , alabama , usa . sepharose cl 6b , sephacryl s-300 and sephadex g-25 were obtained from pharmacia fine chemicals , uppsala , sweden . powdered roswell park memorial institute ( rpmi ) 1640 ( deficient ) medium was obtained from sigma chemical co. , st . louis , usa . dulbecco s modified eagle s medium ( dmem ) purchased from invitrogen corporation , new york , usa . [ h ] leucine and na i were bought from american radiochemicals inc . , st . louis , usa . human kb cells were procured from national centre for cell science , pune , india and these cells were maintained in dmem . the culture media were supplemented with 10% fcs , hepes , nahco3 , penicillin ( 100 units / ml ) and streptomycin ( 100 g / ml ) . cells were then grown in a humidified incubator at 37 , 5% co2 and 95% air atmosphere . ricin was purified from the seeds of ricinus communis and radiolabelled with na i by lactoperoxidase according to the method reported earlier . liposomes composed of spc , cholesterol , pa and dspe - mpeg-2000 in a molar ratio of 40:45:10:5 were prepared by hand shaken method as described earlier . liposome size and zeta potential were determined using a zeatasizer nano zs ( zen 3600 , malvern instruments , worcestershire , uk ) as presented in table 1 . size , zeta potential and intercalation efficiency of monensin liposomes liposomes were prepared using the spc and cholesterol in a molar ratio of 70:30 by hand shaken method . briefly the lipids ( total 50 mol ) were dissolved in chloroform and ethanol solution of monensin ( 10 mol% of the lipid mixture ) was added to it in 100 ml round bottom flask . the organic solvent was evaporated to dryness in a rotary evaporator ( wheaton ) at 37 under reduced pressure to obtain a homogeneous lipid film on the walls of the round bottom flask . the lipid film so obtained was desiccated for 1 h , followed by hydration with 1 ml psb ( 20 mm , ph 7.4 ) . the round bottom flask containing liposomes suspension was stored overnight ( under n2 atmosphere to avoid lipid oxidation ) at 4 for complete hydration . the following day , liposomes were sonicated in a bath type sonicator ( branson ) at 25 for 30 min in 10 min intervals to avoid the heat generation . the liposomes after sonication were extruded 10 times through 100 nm polycarbonate membrane . to prepare the negatively and positively charged monensin liposomes , 21 mol% either phosphatidic acid ( pa ) or sterylamine ( sa ) were added during the preparation of lipid film , respectively . sterically stabilised liposomes intercalated with monensin were prepared exactly as described above only by adding various ( 1 to 7.5 ) mol% of dspe - mpeg of different chain lengths ( 1000 , 2000 , 3000 and 5000 ) during the preparation of lipid film . liposomal monensin was separated from the free monensin by gel permeation chromatography using the sepharose cl 6b column preequilibrated with phosphate buffer saline ( pbs ) ( 20 mm , ph 7.4 ) at 25. briefly , 1 ml liposomal suspension ( 50 mol total lipids in 1 ml ) was loaded on the sepharose cl 6b column ( 143 cm ) . the column was eluted with pbs ( 20 mm , ph 7.4 ) at a flow rate of 15 ml / h . liposome size and zeta potential were determined and are presented in table 1 . in vitro cytotoxicity was assessed by [ h ] leucine incorporation assay by established procedure in our laboratory . briefly , human nasopharyngeal carcinoma ( kb ) cells were plated in 24 well plates at a cell density of 810 cells / well in 1ml dmem containing 10% fcs , penicillin ( 100 units / ml ) and streptomycin ( 100 g / ml ) , 24 h prior to the experiment . the monolayer cultures were washed twice with 1 ml dbss and incubated in 0.9 ml serum free dmem medium containing penicillin and streptomycin for 1 h at 37. cells were then incubated with different concentrations of ricin in free and different liposomal form for 4 h at 37 , followed by washing with dbss twice and incubated with 0.9 ml leucine free medium for 1 h at 37. after 1 h of pre - incubation with leucine free medium , 100 l [ h ] leucine ( 0.5 ci / ml ) in rpmi-1640 was added to the cells and the monolayer cultures were further incubated for 1 h at 37. the monolayers were then fixed with 3% ( w / v ) perchloric acid and 0.5% ( w / v ) phosphotungstic acid twice , washed with dbss twice and dissolved in 0.5 ml of 0.5 n naoh . a 50 l aliquot of the solubilised cell extract was transferred to a scintillation vial containing 5 ml scintillation cocktail , neutralized with 25 l 1 n hcl and counted in liquid scintillation analyser ( tri - carb 2900tr , perkin elmer , usa ) . for the determination of the effect of monensin , cells were preincubated with monensin ( 50 nm ) for 1 h , followed by incubation with ricin . cellular uptake of neutral conventional and various sterically stabilized liposomal formulations of monensin was assessed using nbd - pc ( 1-palmitoyl -2-{12-[(7-nitro-2 - 1,3-benzoxadiazol-4-yl)amino ] lauroyl}-sn - glycero-3-phosphocholine ) as fluorescent surface marker . briefly , cells at a density of 810 cells / well/1 ml were plated in 24 well plates , 24 h before the experiment . the monolayer cultures were washed twice with dbss and incubated with or without 50 nm monensin intercalated in liposomes containing 0 - 7.5 mol% dspe - mpeg of different chain length ( 1000 , 2000 , 3000 and 5000 ) at a final lipid concentration of 20 - 25 nm / ml for 1 h at 37. then , varying concentrations of liposomal ricin in 100 l dbss was added and cells were incubated at 37 for 4 h. cells were then washed thrice with ice cold dbss to remove unbound liposomes . the cells were fixed with 2% paraformaldehyde , detached by a brief trypsin - edta ( ethylenediaminetetraacetic acid ) treatment and the fluorescence intensity in cells was analysed by fluorescence activated cell sorter ( facs canto ii , bd bioscience , usa ) . cellular uptake of the liposomal formulations was quantified on the basis of fluorescence intensity associated with the cells . ricin was purified from the seeds of ricinus communis and radiolabelled with na i by lactoperoxidase according to the method reported earlier . liposomes composed of spc , cholesterol , pa and dspe - mpeg-2000 in a molar ratio of 40:45:10:5 were prepared by hand shaken method as described earlier . liposome size and zeta potential were determined using a zeatasizer nano zs ( zen 3600 , malvern instruments , worcestershire , uk ) as presented in table 1 . liposomes were prepared using the spc and cholesterol in a molar ratio of 70:30 by hand shaken method . briefly the lipids ( total 50 mol ) were dissolved in chloroform and ethanol solution of monensin ( 10 mol% of the lipid mixture ) was added to it in 100 ml round bottom flask . the organic solvent was evaporated to dryness in a rotary evaporator ( wheaton ) at 37 under reduced pressure to obtain a homogeneous lipid film on the walls of the round bottom flask . the lipid film so obtained was desiccated for 1 h , followed by hydration with 1 ml psb ( 20 mm , ph 7.4 ) . the round bottom flask containing liposomes suspension was stored overnight ( under n2 atmosphere to avoid lipid oxidation ) at 4 for complete hydration . the following day , liposomes were sonicated in a bath type sonicator ( branson ) at 25 for 30 min in 10 min intervals to avoid the heat generation . the liposomes after sonication were extruded 10 times through 100 nm polycarbonate membrane . to prepare the negatively and positively charged monensin liposomes , 21 mol% either phosphatidic acid ( pa ) or sterylamine ( sa ) were added during the preparation of lipid film , respectively . sterically stabilised liposomes intercalated with monensin were prepared exactly as described above only by adding various ( 1 to 7.5 ) mol% of dspe - mpeg of different chain lengths ( 1000 , 2000 , 3000 and 5000 ) during the preparation of lipid film . liposomal monensin was separated from the free monensin by gel permeation chromatography using the sepharose cl 6b column preequilibrated with phosphate buffer saline ( pbs ) ( 20 mm , ph 7.4 ) at 25. briefly , 1 ml liposomal suspension ( 50 mol total lipids in 1 ml ) was loaded on the sepharose cl 6b column ( 143 cm ) . the column was eluted with pbs ( 20 mm , ph 7.4 ) at a flow rate of 15 ml / h . in vitro cytotoxicity was assessed by [ h ] leucine incorporation assay by established procedure in our laboratory . briefly , human nasopharyngeal carcinoma ( kb ) cells were plated in 24 well plates at a cell density of 810 cells / well in 1ml dmem containing 10% fcs , penicillin ( 100 units / ml ) and streptomycin ( 100 g / ml ) , 24 h prior to the experiment . the monolayer cultures were washed twice with 1 ml dbss and incubated in 0.9 ml serum free dmem medium containing penicillin and streptomycin for 1 h at 37. cells were then incubated with different concentrations of ricin in free and different liposomal form for 4 h at 37 , followed by washing with dbss twice and incubated with 0.9 ml leucine free medium for 1 h at 37. after 1 h of pre - incubation with leucine free medium , 100 l [ h ] leucine ( 0.5 ci / ml ) in rpmi-1640 was added to the cells and the monolayer cultures were further incubated for 1 h at 37. the monolayers were then fixed with 3% ( w / v ) perchloric acid and 0.5% ( w / v ) phosphotungstic acid twice , washed with dbss twice and dissolved in 0.5 ml of 0.5 n naoh . a 50 l aliquot of the solubilised cell extract was transferred to a scintillation vial containing 5 ml scintillation cocktail , neutralized with 25 l 1 n hcl and counted in liquid scintillation analyser ( tri - carb 2900tr , perkin elmer , usa ) . for the determination of the effect of monensin , cells were preincubated with monensin ( 50 nm ) for 1 h , followed by incubation with ricin . cellular uptake of neutral conventional and various sterically stabilized liposomal formulations of monensin was assessed using nbd - pc ( 1-palmitoyl -2-{12-[(7-nitro-2 - 1,3-benzoxadiazol-4-yl)amino ] lauroyl}-sn - glycero-3-phosphocholine ) as fluorescent surface marker . briefly , cells at a density of 810 cells / well/1 ml were plated in 24 well plates , 24 h before the experiment . the monolayer cultures were washed twice with dbss and incubated with or without 50 nm monensin intercalated in liposomes containing 0 - 7.5 mol% dspe - mpeg of different chain length ( 1000 , 2000 , 3000 and 5000 ) at a final lipid concentration of 20 - 25 nm / ml for 1 h at 37. then , varying concentrations of liposomal ricin in 100 l dbss was added and cells were incubated at 37 for 4 h. cells were then washed thrice with ice cold dbss to remove unbound liposomes . the cells were fixed with 2% paraformaldehyde , detached by a brief trypsin - edta ( ethylenediaminetetraacetic acid ) treatment and the fluorescence intensity in cells was analysed by fluorescence activated cell sorter ( facs canto ii , bd bioscience , usa ) . cellular uptake of the liposomal formulations was quantified on the basis of fluorescence intensity associated with the cells . in the present investigation , monensin was intercalated in neutral , negatively and positively charged liposomes and its ability to potentiate the cytotoxicity of ricin entrapped in negatively charged sterically stabilised liposomal form was evaluated in kb cells . these results of liposomal monensin were compared with free monensin to develop an optimal delivery system for liposomal ricin in combination with monensin for selective elimination of tumour cells . the extent of enhancement of cytotoxicity of ricin by monensin intercalated in various charged liposomes is found to be dependent on the lipid composition of monensin liposomes and significantly higher as compared to free monensin . monensin exhibit maximum enhancing ability ( 106.5 fold ) when delivered through intercalated into neutral liposomes followed by negatively ( 94.2 fold ) and positively charged ( 90 fold ) liposomes as compared to free monensin ( 88.6 fold ) ( fig . the cellular uptake studies of monensin liposomes clearly indicate that there is a direct correlation between the enhancements of the cytotoxicity of ricin encapsulated into sterically stabilised liposomes by monensin intercalated into various charged liposomes with their amount of cell association ( fig . these results are highly comparable with earlier reports that interaction of liposomes with mammalian cells is highly dependent on several factors like lipid composition , size , charge , bilayer fluidity , hydrophilicity of the liposomes as well as cell types . as our results clearly showed that liposomes can be used as delivery vehicle for monensin to enhance the cytotoxicity of liposomal ricin and as neutral monensin liposome has the maximum ability to enhance the cytotoxicity of liposomal ricin , all subsequent work was therefore carried out using monensin intercalated into neutral liposomal formulations . effect of monensin on potentiation of the cytotoxicity of liposomal ricin and cellular uptake effect of monensin in free and liposomal forms on potentiation of the cytotoxicity of liposomal ricin ( a ) and cellular uptake of monensin liposomes ( b ) each bar represents meansd ( n=3 ) studies on the effect of various densities of dspe - mpeg-2000 on the surface of neutral monensin liposomes revealed that monensin exhibits maximum enhancement ( 117.6 fold ) of liposomal ricin cytotoxicity at 2.5 mol% dspe - mpeg-2000 on the surface ( fig . 2 ) . in order to evaluate whether the differential efficacy exhibited by liposomal monensin on the enhancement of the cytotoxicity of liposomal ricin is due to the difference in cellular uptake , 3 shows the cellular uptake of monensin liposomes having different densities of dspe - mpeg-2000 as expressed in the form of percentage of fluorescence of kb cells studied by facs . cellular uptake studies and measurement of zeta potential of monensin liposomes having various densities of dspe - mpeg-2000 on the surface revealed that there is high correlation between the enhancement of the cytotoxicity of ricin with their amount of cell association and zeta potential on the surface of monensin liposomes . the maximum enhancement of the cytotoxicity of liposomal ricin was observed when the cellular uptake of liposome was maximum ( 49.4% ) and the zeta potential value on the surface of liposomes was 0.645 at 2.5 mol% dspe - mpeg-2000 on the surface of liposomes . keeping 2.5 mol% dspe - mpeg as constant , we varied the chain length of peg on the surface of monensin liposomes and examined the efficacy of monensin in these liposomes on the enhancement on the cytotoxicity of liposomal ricin in kb cells ( fig . it was observed that the peg chain length of 2000 has maximum influence on the ability of the monensin in neutral peg - liposomes to enhance the cytotoxicity of liposome entrapped ricin . the liposomal ricin cytotoxicity enhancing potency of monensin in peg - liposomes is reduced from 117.6 fold to 82.4 and 63.0 fold when chain length of peg was increased from 2000 to 3000 and 5000 . it was also observed that the fold enhancement of the cytotoxicity of liposomal ricin was again reduced ( from 117.6 to 82.4 fold ) on reducing the length of peg from 2000 to 1000 . the maximum enhancement of the cytotoxicity of liposomal ricin by peg - liposomal monensin was observed when the cellular uptake of liposome was maximum ( 42.0% ) and the zeta potential value on the surface of liposomes was 0.645 at 2.5 mol% dspe - mpeg of chain length 2000 on the surface of liposomes ( fig . 5 ) . these results clearly showed that the value of zeta potential on the surface of peg liposomes is crucial for expression of the biological activity of monensin in peg - liposomes and the optimum value is around 0.645 . these results are in agreement with earlier reports that the interaction of liposomes with cancer cells not only depends on the surface charge , but also on density , chain length and lipid anchor of peg - lipid . this zeta potential on the surface of liposomes can be modulated by two ways , either by changing the density of dspe - mpeg-2000 or by changing the chain length of peg in dspe - mpeg on the surface of liposomes . this report is the first of its kind to show that liposomal monensin is very effective in enhancing the cytotoxicity of liposomal ricin in human cancer cells and liposome can be used as in vivo delivery vehicle to eliminate tumour cells . fold potentiation of the cytotoxicity of liposomal ricin by monensin liposomes having different densities of peg on their surface fold potentiation = id50 of ricin without monensin / id50 with liposomal monensin ( 50 nm ) having different densities of peg . cell - associated fluorescence intensity measured by facs canto ii was expressed as cell - associated liposomal monensin . potentiation of cytotoxicity of liposomal ricin by monensin liposomes fold potentiation of the cytotoxicity of liposomal ricin by monensin liposomes having different chain lengths of peg on their surface . cellular uptake of monensin liposomes having different chain lengths of dspe - mpeg on their surface . cell - associated fluorescence intensity measured by facs canto ii was expressed as cell - associated liposomal monensin .

systemic lupus erythematosus ( sle ) is an autoimmune , chronic inflammatory disorder affecting the connective tissue of multiple organ systems . it occurs on the background of disturbances of environmental , hormonal , and genetic factors . no single clinical , laboratory , neuropsychological , and imaging test can be used to differentiate neuropsychiatric systemic lupus erythematosus ( npsle ) from non - npsle patients with similar neuropsychiatric manifestations . the most common neuropsychiatric manifestations in sle are cognitive deficit ( 49.33% ) , lupus headache ( 23.11% ; in 57.69% of these patients , tension - type ) , psychoses ( 12.00% ) , seizures ( 10.67% ) , and cerebrovascular events ( 9.78% ) . typical central nervous system ( cns ) changes for npsle during magnetic resonance image examination are the multiple lacunar infarctions with temporal localization as well as a parenchymatous cerebral atrophy . skin vasculitis , serositis , and pulmonary involvement are the most common extracerebral manifestations in patients with sle and data for neuropsychiatric lesions ( seizures , psychoses , and cerebrovascular events ) being signs of general vascular pathology disease activity . the american college of rheumatology established case definitions for 19 central and peripheral nervous system syndromes listed in table 1 . a 20-year - old unmarried woman from middle socioeconomic status with urban bac kground presented to the department of psychiatry . she was a known case of sle with predominant skin manifestations since 5 years , and she was treated by a dermatologist . the patient discontinued medication after 2 years on her own , as there was an improvement of symptoms after attaining the menarche . again , for 3 months the patient noticed frequent hair fall , itching over the scalp , and intermittent low - grade fever . the patient also had continuous withdrawn / dull behavior , suspiciousness , referential ideas , hearing of voices , decreased self - care , and occasional crying episodes in the last 15 days . the dermatologist started 30 mg / day of prednisolone ( tablet ) after the onset of psychiatric symptoms . there was dark pigmentation all over her body and sparse and thin hair over her scalp , but there were no neurological deficits or signs of meningeal irritation . on mental status examination , the patient was found to be poorly kempt , restless , irritable , had poverty of speech , third person auditory hallucination , delusion of persecution , delusion of reference , and blunt affect but she was conscious and oriented . baseline biochemical and hematological investigations were within normal limits ; antinuclear antibody ( ana ) test was positive ( index ratio was 3.5 on ana detect ) ; and rheumatoid antibody test was negative . the patient was diagnosed to have organic delusional ( schizophrenia - like ) disorder ( international classification of diseases ( icd)-f06.2 ) secondary to sle . eventually the patient was started on 10mg / day of olanzepine ( tablet ) . she remained free from psychiatric symptoms for the last 2 months . a 17-year - old unmarried woman from rural background with lower socioeconomic status reported to the general medicine department . she had a history of 2 months of fever and joint pain and a history of 5 days of cough . she was diagnosed to have cutaneous lupus erythematosus for a year , and she was under 20 mg/ day of prednisolone ( tablet ) in divided doses . general physical examination revealed pallor , icterus , hyperpigmentation all over her body , including face , erythematosus skin lesions , diffuse hair loss , depigmented patches over the scalp , and erosions in the buccal mucosa . chest radiograph was suggestive of miliary tuberculosis , and an electromyographic study was suggestive of mixed neurogenic and myogenic pathology . most of the baseline biochemical and hematological investigations ( except hb% 8 g% and total bilirubin 4 mg / dl ) were within normal limits . immunological investigations showed positive anti - ui / sm ribonucleoprotein antibody ( rnpab ) , anti - sm ab , anti - ds deoxyribonucleic acid ( dna ) ab , anti - histone ab , anti - nucleosomal ab , and anti - ribosomal ab . the human immunodeficiency virus ( hiv ) test was nonreactive . after the review by the immunologist , the patient was diagnosed to have sle with myositis with military tuberculosis . she was treated with 200 mg of ciprofloxacin bid ( injection ) , 100 mg of amikacin iv tid ( injection ) , 100 mg of hydrocortisone iv bid ( injection ) , 1 g of ceftriaxone iv od ( injection ) , and 400 mg / day of ethambutol ( tablet ) . the patient was free from psychiatric complaints for initial 1.5 months . for 15 days , the patient presented with muttering , smiles to self , occasional crying spells , irrelevant speech , anger outbursts , and decreased sleep . there was no alteration in medication , changes in dosages , or onset of any fresh physical complaints in these 15 days . there was no history of psychosis , depression , or mania , but she was premorbidly well adjusted . mental status examination revealed poorly kempt , irrelevant speech , second person auditory hallucination , reduced range of mood , and absence of insight . the patient was diagnosed as organic delusional [ schizophrenia - like ] disorder ( icd - f06.2 ) . the patient was started on 10 mg / day of olanzepine ( tablet ) and increased to 20 mg / day . the patient showed 50% improvement in the first week , and she was discharged later . a 35-year - old woman was diagnosed with sle since 2 years , and she was admitted in dermatology department for acute cutaneous exacerbation of sle . she was on 20 mg / day of predinisolone ( tablet ) and local skin applications for skin lesions . the patient was referred to psychiatric evaluation for the complaints of fearfulness and decreased sleep for 2 days . there were no significant family history , history of major psychiatric illness , and no similar or significant psychiatric illness . mental status examination revealed that the patient had delusion of persecution , delusion of reference , irritability , and absence of insight . the patient was diagnosed as organic delusional ( schizophrenia - like ) disorder ( icd - f06.2 ) and started on 10 mg/ day of olanzepine ( tablet ) . the patient remitted from psychiatric symptoms in 1 week , and she was symptom - free even after follow - up of 2 months . a 20-year - old unmarried woman from middle socioeconomic status with urban bac kground presented to the department of psychiatry . she was a known case of sle with predominant skin manifestations since 5 years , and she was treated by a dermatologist . the patient discontinued medication after 2 years on her own , as there was an improvement of symptoms after attaining the menarche . again , for 3 months the patient noticed frequent hair fall , itching over the scalp , and intermittent low - grade fever . the patient also had continuous withdrawn / dull behavior , suspiciousness , referential ideas , hearing of voices , decreased self - care , and occasional crying episodes in the last 15 days . the dermatologist started 30 mg / day of prednisolone ( tablet ) after the onset of psychiatric symptoms . there was dark pigmentation all over her body and sparse and thin hair over her scalp , but there were no neurological deficits or signs of meningeal irritation . on mental status examination , the patient was found to be poorly kempt , restless , irritable , had poverty of speech , third person auditory hallucination , delusion of persecution , delusion of reference , and blunt affect but she was conscious and oriented . baseline biochemical and hematological investigations were within normal limits ; antinuclear antibody ( ana ) test was positive ( index ratio was 3.5 on ana detect ) ; and rheumatoid antibody test was negative . the patient was diagnosed to have organic delusional ( schizophrenia - like ) disorder ( international classification of diseases ( icd)-f06.2 ) secondary to sle . eventually the patient was started on 10mg / day of olanzepine ( tablet ) . a 17-year - old unmarried woman from rural background with lower socioeconomic status reported to the general medicine department . she had a history of 2 months of fever and joint pain and a history of 5 days of cough . she was diagnosed to have cutaneous lupus erythematosus for a year , and she was under 20 mg/ day of prednisolone ( tablet ) in divided doses . general physical examination revealed pallor , icterus , hyperpigmentation all over her body , including face , erythematosus skin lesions , diffuse hair loss , depigmented patches over the scalp , and erosions in the buccal mucosa . chest radiograph was suggestive of miliary tuberculosis , and an electromyographic study was suggestive of mixed neurogenic and myogenic pathology . most of the baseline biochemical and hematological investigations ( except hb% 8 g% and total bilirubin 4 mg / dl ) were within normal limits . immunological investigations showed positive anti - ui / sm ribonucleoprotein antibody ( rnpab ) , anti - sm ab , anti - ds deoxyribonucleic acid ( dna ) ab , anti - histone ab , anti - nucleosomal ab , and anti - ribosomal ab . the human immunodeficiency virus ( hiv ) after the review by the immunologist , the patient was diagnosed to have sle with myositis with military tuberculosis . she was treated with 200 mg of ciprofloxacin bid ( injection ) , 100 mg of amikacin iv tid ( injection ) , 100 mg of hydrocortisone iv bid ( injection ) , 1 g of ceftriaxone iv od ( injection ) , and 400 mg / day of ethambutol ( tablet ) . the patient was free from psychiatric complaints for initial 1.5 months . for 15 days , the patient presented with muttering , smiles to self , occasional crying spells , irrelevant speech , anger outbursts , and decreased sleep . there was no alteration in medication , changes in dosages , or onset of any fresh physical complaints in these 15 days . there was no history of psychosis , depression , or mania , but she was premorbidly well adjusted . mental status examination revealed poorly kempt , irrelevant speech , second person auditory hallucination , reduced range of mood , and absence of insight . the patient was diagnosed as organic delusional [ schizophrenia - like ] disorder ( icd - f06.2 ) . the patient was started on 10 mg / day of olanzepine ( tablet ) and increased to 20 mg / day . the patient showed 50% improvement in the first week , and she was discharged later . a 35-year - old woman was diagnosed with sle since 2 years , and she was admitted in dermatology department for acute cutaneous exacerbation of sle . she was on 20 mg / day of predinisolone ( tablet ) and local skin applications for skin lesions . the patient was referred to psychiatric evaluation for the complaints of fearfulness and decreased sleep for 2 days . there were no significant family history , history of major psychiatric illness , and no similar or significant psychiatric illness . mental status examination revealed that the patient had delusion of persecution , delusion of reference , irritability , and absence of insight . the patient was diagnosed as organic delusional ( schizophrenia - like ) disorder ( icd - f06.2 ) and started on 10 mg/ day of olanzepine ( tablet ) . the patient remitted from psychiatric symptoms in 1 week , and she was symptom - free even after follow - up of 2 months . all the three cases noted above fulfilled the criteria for npsle . from the same institution , a series of four cases of sle were reported in 1982 , of which two had depressive episodes and one had acute psychosis during the exacerbation of sle . in the present series , all of them had acute psychosis ( organic delusional ( schizophrenia - like ) disorder or organic psychosis according to icd-10 ) . according to icd-10 , diagnostic features of organic psychosis are as follows : evidence of cerebral disease , damage , or dysfunction or of a systemic physical disease , known to be associated with one of the listed syndromes;a temporal relationship ( weeks or a few months ) between the development of the underlying disease and the onset of the mental syndrome;recovery from the mental disorder following removal or improvement of the underlying presumed cause ; andabsence of evidence to suggest an alternative cause of the mental syndrome ( such as a strong family history or precipitating stress ) . evidence of cerebral disease , damage , or dysfunction or of a systemic physical disease , known to be associated with one of the listed syndromes ; a temporal relationship ( weeks or a few months ) between the development of the underlying disease and the onset of the mental syndrome ; recovery from the mental disorder following removal or improvement of the underlying presumed cause ; and absence of evidence to suggest an alternative cause of the mental syndrome ( such as a strong family history or precipitating stress ) . recently in 2008 , monov and monova had developed an approach for making the diagnosis of npsle . common differential diagnosis that has to be considered are functional psychosis , delirium , steroid psychosis , and other drug - induced psychosis . as there were no apparent significant stressors apart from exacerbation of sle and absence of family history of psychosis , functional acute psychosis is ruled out . on detailed assessment of above cases , it was found that they did not have symptoms of delirium ( icd-10 ) , such as impairment of consciousness and attention , global disturbance of cognition , and disturbance of sleep - wake cycle . lastly , none of the drugs used ( apart from steroids ) to treat above cases are known to exacerbate or cause psychosis . steroids may cause psychosis , but psychiatric side effects with corticosteroids appear to be dose dependent . the boston collaborative drug surveillance program ( n=676 ) examined psychiatric symptoms in patients free of psychiatric disease prior to steroid treatment . severe psychiatric illness was uncommon ( 1.3% ) with doses less than 40 mg / day of prednisolone but increased to 18.4% at doses above 80 mg / day of prednisolone . two of the above three cases were on less than 40 mg / day of prednisolone and one was on 200 mg / day of hydrocortisone ( injection ) , which is equivalent to 50 mg of prednisolone ( 5 mg of prednisolone is equivalent to 20 mg of hydrocortisone ) . the most commonly reported corticosteroid - induced psychiatric disturbances are affective , including mania , depression , or mixed states . most often , patients receiving short - term corticosteroid therapy present with euphoria or hypomania whereas long - term therapy tends to engender depressive symptoms . most of the psychiatric symptoms secondary to steroids occur in first 12 weeks . in the above case series , thus , the psychiatric symptoms in the above cases described are unlikely to be due to steroids . in any case of suspected organic psychiatric illness , psychotropics have to be started at lower dose and gradually built up because they are prone for more cns side effects , such as extrapyramidal side effects ( eps ) . in the above cases , the patients were started on olanzepine 2.5 mg initially and gradually built up to 1020 mg / day . we chose olanzepine because it has good efficacy and tolerability and is less likely to have eps . the pattern of psychotic spectrum disorders in sle is not systemically studied , but most common form appears to be acute psychosis , which was true in the above case series . prognosis is good for acute psychosis ; even present cases had good prognosis on short - term follow - up . findings of npsle on magnetic resonance imaging ( mri ) of the brain are frequently unremarkable , and abnormalities are nonspecific . a quantitative mri technique , concluded that in sle patients with a history of neuropsychiatric symptoms , the gray matter is particularly affected . sle is a chronic autoimmune disorder with occasional cns manifestations . in a few situations , psychosis in patients with sle has to be differentiated from functional psychosis , delirium , steroid psychosis , and other drug - induced psychosis . most of these patients are prone to cns side effects because of already underlying cns damage and so antipsychotics with least eps , such as quetiapine or olanzepine , can be used .

moreover , in chemical ionization mass spectrometry ( cims ) , humidity is involved in the complex processes taking place at and after chemical ionization . from a practical point of view , humidity in cims is relevant for quantification . several general concepts are understood , such as the influence of protonated higher water clusters and dependence on proton affinity in cims . however , the underlying reaction mechanisms of how humidity influences fragmentation are often unknown and are probably reaction specific . solvation has been proposed as one possible reaction principle , e.g. , for isoprene ; however , it has yielded contradictory experimental results . in support of our humidity dependent calibration efforts and in continuation of our studies on sulfur compounds it is found in foods such as garlic , onion , leek , and cooked beef and is also attributed to garlic breath . proton transfer reaction - mass spectrometry ( ptr - ms ) is a mass spectrometric technique deploying soft chemical ionization with hydronium ions . it allows for direct injection analysis without sample preparation and online detection of gas phase samples . even elusive ( organo)-sulfur compounds can be detected with ptr - ms , down to the particularly low ppt(vol ) concentration range required for direct or real time ( in vivo ) analysis , e.g. , of human breath . for the protonation reaction in the drift tube of the ptr - ms , a volatile organic compound ( voc ) reacts with the hydronium ion:1 on the basis of theoretical considerations , the concentration of voc in the sample can be calculated via the ion count rates of voch and h3o in the mass spectrometer and the reaction rate kh3o+ and the ( known ) reaction time :2 hence , in principle , ptr - ms allows for quantification without an external calibration standard . however , eq 2 is a simplification , in particular because of protonated water clusters ( h2o)nh3o , which can act as reagent ions . . measurements with ptr - ms allow for a variation of the collisional energy in the chemical reaction chamber , a flow drift tube . for instrumental intercomparison , the collisional energy is expressed as the ratio of the electric field e to the number density of the gas n in the drift tube of the ptr - ms , hence e / n , which is measured in townsend ( 1 td = 10 vcm ) . fragmentation is strongly influenced by the applied collisional energy , usually thought to occur directly through the elevated energy conditions . at a higher e / humidity is known to influence the protonation reactions of certain compounds in the ptr - ms , e.g. , for isoprene , toluene , benzene , formaldehyde ( sifdt ) , alpha pinene , terpenes , sesquiterpenes , isocyanates , hcn , h2s ( sift ) , and the mix of breath gas . the choice of the collisional energy is usually a compromise between minimizing water cluster abundance , which otherwise might obscure spectra , and minimizing the amount of fragmentation at high energies . a major influence from humidity is probably the change of composition of reagent ions:3 hence , higher humidity results in the higher abundance of protonated higher water clusters h2oh3o and ( h2o)nh3o . the protonated higher water clusters have a smaller proton affinity ( pa ) , and hence the protonation reaction is less voluntary , or might not take place at all . pa of the protonation reaction ( compare eq 1 ) is approximately equivalent to the negative change in gibbs energy ( g ) . for efficient reactions , a minimum pa 35 kj / mol is required . moreover , proton affinity has an influence on fragmentation : reaction products from direct protonation reactions with protonated higher water clusters , as well as products from ligand switching reactions , are less likely to undergo subsequent fragmentation . again , the reason lies in the lower difference in proton affinities compared to direct hydronium ion protonation . there is less excess energy for breaking bonds in the protonated voc ; protonation is softer . increased levels of larger protonated water clusters are found either at increased humidity in the drift tube ( e.g. , via humid samples ) or through lower e / n . subsequent changed fragmentation patterns due to softer reactions ( via direct protonation from protonated higher water clusters or alternatively from ligand switching ) have been proposed . in another work , we investigated the collisional energy dependence of fragmentation of ams and of other sulfides . ams displayed a markedly different fragmentation and a rather abundant fragmentation pattern with about six major fragments ( with > 2% of the total ions from ams ) compared to the other small saturated organosulfur compounds , probably due to its unsaturated allyl group . we measured the collisional energy - dependent fragmentation pattern and calculated possible reaction pathways with quantum chemical ( qc ) methods . comparison with literature data showed differences in the found fragments and fragment abundance , which we attributed to differences in sample humidity between literature and our measurements . it indicated that humidity considerably influences fragmentation of ams , which warranted further investigation . hence , in the present study , we deepen our understanding of ams fragmentation but also use ams as a model compound for humidity dependence . we study the influence of water vapor pressure ( wvp ) on the fragmentation of ams , in combination with the effect of varying collisional energies . we use logarithmic dilution approaches ( inert gas stripping and desorption from inlet lines ) to distinguish true fragments from the background . furthermore , we deploy extensive qc calculations for further studies on fragmentation reaction pathways . the study of the humidity dependence yields a better understanding of the influence of humidity on the fragmentation reaction , which is of importance for calibrations , both , from theory and with gas standards . the following compounds were used : allyl methyl sulfide ( ams ) cas 10152 - 76 - 8 from aldrich ( 98% purity ) , deionized water ( conductivity 0.1 siemenscm ) , and high purity ( pa ) n2 from rivoira ( italy ) . measurements were performed with a commercially available ptr - ms from ionicon analytik gmbh ( innsbruck , austria ) in the hs ( high sensitivity ) version equipped with a quadrupole mass analyzer as described previously ( see supporting information s1 ) . the fragmentation spectra were averaged typically over a minimum of 5 cycles per e / n value . measurements from dry n2 were performed with typically 3 cycles per e / n value . fragment ion signals were averaged by directly calculating the percentages in relation to the protonated primary ion at each cycle and then averaging the percentages from several cycles . isotopologue patterns , literature , and software were used as additional aids for fragment identification . percent fragments are usually given as % of the measured signal of m / z 89 , protonated ams , not considering isotopologues , unless stated otherwise . for details on humidity , see supporting information s2 ; for information on fragmentation in the figures , compare supporting information s3 . in order to achieve ( pseudo)logarithmic declining concentrations of ams in the gas phase , inert gas stripping was used for humid samples , and desorption from inlet lines was used for the dry n2 experiment . for the latter , after removing the ams feed sample , desorption was induced from the inlet lines by purging with pure nitrogen ( flux of 100 sccm ( standard cubic centimeter per minute ) ) . gas stripping and desorption effects both allow for the distinction between parent ions and derived fragments from other ions , which display a different time - dependence . this is particularly important for isotopologues of h2oh3o ( m / z 39 ) and ( h2o)2h3o ( m / z 55 ) . four settings of humidity were used ( dry , least humid , less humid , and mid humid ) with a humidity range of 0140% rh @ 25 c . further details on the ptr - ms settings and conditions for all measurements can be found in the supporting information s1 . because ptr - ms is also frequently used in breath gas analysis and exhaled breath is typically saturated with water vapor at 37 c , we prefer to call our highest humidity setting mid humid . given for the samples , as well as corresponding calculated sample water pressure and relative humidity , an estimated value for humidity from the ion source leaking into the drift tube . an example of a reagent ion distribution is given ( see supporting information s1 for full list ) in % of h3o ( m / z 19 monitored via its isotopologue at m / z 21 ) . least humid ion source water vapor amount is a rough estimate , hence the question mark ( see supporting information s2 for details ) . ew and : data with italics indicate recalculated values from ion abundance data ( m / z 37 and 55 ) and italics with error range indicate estimated values . sv : switching valve of ptr - ms for sri ( switchable reagent ion ) ; vp . , estimated ; recalculated from m / z 21 ( isotopologue of h3o at m / z 19 ) ; rh , relative humidity . at t = 25 c and ambient pressure . the water vapor content was varied as follows : during the gas stripping process , nitrogen gas takes up not only the desired vocs but also water vapor , and thus , measurements are performed under humidified sample conditions in inert gas stripping . the water vapor content was varied by a change in the temperature of the water ( 2531 c ) and the flux of the nitrogen ( 20200 sccm ) ( standard cubic centimeter ) . settings for least humid consisted of a low flux with slightly elevated amounts of parasitic reagent ions o2 ( m / z 32 ) and no ( m / z 30 ) , compared to dry n2 . however , the concentrations are still low and , in the present context , are considered not to be relevant ; compare also supporting information s1 . samples measured with saturated water vapor n2 are considered to have 100% relative humidity at the given temperature in the humidifier ( gas stripper ) , see table 1 . according to buck , the temperature dependence of water vapor saturation pressure can be estimated for total pressures > 800 mbar and the relevant temperature range via4where ew is the saturated vapor pressure expressed in hectopascal ( hpa ) , t is the so - called dry bulb temperature in c , and p is the absolute pressure in hpa ( assumed to be 1013.25 hpa ) . the relative humidity rh ( or ) is calculated via5with ew indicating the actual water vapor pressure in the sample and inserting ew from eq 4 as reference points , in particular for the temperature . by applying eqs 4 and 5 , the relative humidity rh and ew * from t = 30.6 c were recalculated to relative humidities and ew at 25 c , see table 1 . reported a linear relationship between sample water vapor pressure ( wvp ) and ion signal of mass 37 ( h2oh3o ) , which we used similarly for calculating the values for least humid settings and estimating wvp from the ion source . the tabulated results for ew and rh can be found in table 1 ; for more details on the calculations , see supporting information s2 . the pathways for the fragmentation of allyl methyl sulfide were calculated with the mp2 method and the 6 - 31g(d , p ) basis set . for improved interaction energies , mp2/6 - 311+g(3df,2p ) single - point calculations were carried out at the stationary points . they were checked to possess one imaginary frequency corresponding to the reaction coordinate by frequency calculations at the corresponding level of theory ( cf . supporting information s4 ) . because of the different approximations inherent in the functional and the effects of basis set size on thermochemistry and geometry , it is difficult to give precise error bars for the calculations . the influence of humidity was investigated with both the polarizable continuum model ( pcm ) and by explicitly including microhydration with one , two , or three water molecules along the reaction coordinate . for the calculation of proton affinities , the gibbs free energies ( g ) of the reactions were obtained from g4mp2 calculations . figure 1 demonstrates the overall humidity - dependent fragmentation of ams in ptr - ms ( panel a ) . therein experimentally , humidity quantitatively reduces the overall fragmentation of ams ( amsh ) in cims . minor deviations can be explained as e / n variations and different modes of analysis of fragmentation ( background correction , selection of fragments , overlap with other fragments , e.g. , in the case of m / z 55 and 39 ) . compare panel b for changes in relative fragmentation . the relationship between humidity and the abundance at m / z 37 of the protonated first water cluster is displayed in panel c. humidity suppresses fragmentation in the entire e / n range from 90 to 140 td . e / n has a stronger effect on fragmentation than humidity ( panel a ) . total fragmentation for the 9 most abundant signals from ams as dependent on humidity ( four settings ) and for an e / n range of 90140 td . open symbols indicate values for which some low abundant fragments could not be determined quantitatively and hence were set to zero ; e.g. , at low e / n , fragment m / z 55 could not be determined , and similarly , in mid humid settings for lower e / n , no m / z 61 and 55 were determined . panel a : fragmentation is given as % of total ions from ams from the 9 most abundant signals ( m / z 89 , 90 , 91 , 61 , 55 , 47 , 41 , 39 , and 49 with background corrections ) , with m / z 89 , 90 , and 91 treated as nonfragments ( isotopologues of amsh ) and the others as fragments . considering the isotopologue abundances for amsh , the maximum possible fragmentation here is 91.0% . panel b : change in percent of total fragmentation relative to 0% rh ( relative humidity ) . panel c : abundance of h2oh3o , expressed as percent of total reagent ions ( m / z 19 + 37 + 55 ) for the data from panel a. the results for single ions for the humidity - dependent fragmentation of ams in ptr - ms for e / n 90140 td can be seen in figure 2 . the strongest humidity - dependent fragmentation can be seen at an e / n 140 td ( figure 2e ) . the energy dependence of single fragments can be seen in detail in figure 3 . all major fragments of ams reactions in ptr - ms follow the same basic pattern of increased fragmentation at higher e / n ( as expected ) and decreased fragmentation at higher humidity . humidity- and energy - dependent fragmentation patterns of ams . e ) , as % of the most abundant ions ( protonated ams at m / z 89 ) . for comparison , the literature values ( lit . ) from schwarz et al . are given in panel d , which had been measured in dry nitrogen at e / n 126 td collisional energy dependence of the main single ion fragments from ams in ptr - ms plotted for four humidity ranges . fragment abundance is given in % of m / z 89 , interpreted as amsh , but not considering isotopologues . we study dehydrogenation reactions and minor fragmentation pathways following protonation of ams with chemical ionization in ptr - ms ( figure 4 ) and investigate the humidity dependence of single fragmentation reactions ( see figures 5 and 6 ) . the calculations complement and therein , we investigated with ptr - ms the fragmentation behavior of mono- and disulfides under constant humidity conditions but under varying internal energy conditions . in addition , for ams , we deployed quantum chemical calculations in order to explain the observed major fragmentation pathways without water . throughout this work , we assume kinetic control in the drift tube , due to the short reactions times ; compare ref ( 8) . e ( using the labels from our other study ) and the labeling of the studied reactions r1r5 . the chemical reaction equations for the studied reactions are given in table 2 , including the short names used for the reactions . additional results for the effect of humidity on the reaction r5 can be found in supporting information s5 . in addition , the effect of humidity on the major reactions r2 ( figure 5 ) and r1 ( figure 6 ) was studied in detail with both the pcm ( polarizable continuum method ) ( panels a in figures 5 and 6 ) and with a microhydration approach where h2o is explicitly taken into account ( panels b and c in figures 5 and 6 ) . further details therein can be found in supporting information s6 , including microhydration results for three water molecules . results on the reaction temperature dependence of calculations on hydration ( both , pcm and microhydration ) can be found in supporting information s7 . calculations for the proton affinities were performed , and the results are given in table 3 , details can be found in supporting information s8 . reactions were calculated at the mp2/6 - 311+g(3df,2p)//mp2/6 - 31g(d , p ) level of theory . the temperature for the gibbs free energy g is 298.15 k. panel a : overview of the reactions r1r5 . panel b : conversion of amsh to c3h5 ( m / z 41 ) and hsch3 ( m 48 ) ( r1 ) in vacuum . panel c : subsequent fragmentation ( dehydrogenation ) of m / z 41 to m / z 39 ( r2 ) in vacuum . panel d : direct fragmentation of amsh to m / z 47 ( r3 ) . panel f : possible ( but unlikely ) fragmentation ( dehydrogenation ) of m / z 49 to m / z 47 ( r5 ) . the reaction r5 in panel d is given in parentheses because according to the quantum chemical results r3 ( direct fragmentation to m / z 47 ) is the preferred reaction pathway . humidity effect on energy profiles of the fragmentation from c3h5 ( m / z 41 ) to c3h3 ( m / z 39 ) ( r2 ) . panel a : reaction energy profiles in vacuum without water ( solid line ) and with the pcm model ( dashed line ) . panel c : reaction profiles comparing microhydration with one ( dashed line ) , two ( dashed - dotted line ) , and three ( dotted line ) water molecules . panel d : comparison of the activation energies of the rate determining steps calculated for microhydration , vacuum , and pcm . reactions were calculated at the mp2/6 - 311+g(3df,2p)//mp2/6 - 31g(d , p ) level of theory . the temperature used was 298.15 k. humidity effect on energy profiles for the fragmentation from amsh ( m / z 89 ) to c3h5 ( m / z 41 ) ( r1 ) . panel a : reaction energy profiles in vacuum without water ( solid line ) and with water ( dashed line ) ( pcm ) . panel c : reaction profiles comparing microhydration with one ( dashed line ) , two ( dashed - dotted line ) , and three ( dotted line ) water molecules . panel d : activation energies of the rate determining steps from 4 qc calculations . reactions were calculated at the mp2/6 - 311+g(3df,2p)//mp2/6 - 31g(d , p ) level of theory . the temperature used was 298.15 k. the gibbs free energies ( g ) of the reactions were obtained from g4mp2 calculations at a temperature of 298.15 k. camsh is cyclic protonated ams . moreover , we calculated all results in addition to the mp2 method also with the m06 - 2x method ( see supporting information s9 ) . according to the experimental results with ptr - ms , collisional energy increases fragmentation of amsh , both in total fragmentation ( figure 1 ) and for the single fragments ( figure 2 ) . humidity reduces fragmentation , again , both overall ( figure 1 ) as well as for single fragments ( figures 2 and 3 ) . e / n ( 90145 td ) studied cover in the most extreme cases quasi zero fragmentation at e / n 90 td and rh 141 [ % @ 25 c ] , on the one side and , on the other side , 92% of the maximum possible fragmentation . interestingly , humidity suppresses fragmentation by 2025% of the overall fragmentation ( figure 1 ) , very uniformly ( parallel graphs in figure 1a , but see also figure 1b ) in the entire studied e / n range . we attribute this to a uniform reaction mechanism , of which the energy diagrams ( activation energies ) do not change ( much ) , i.e. , uniform reaction mechanisms in the fragmentation suppression of the dominating ions . hence , presumably , with a change in water concentration , the fragmentation changes the same way for all energy ( e / n ) conditions . the fragmentation of amsh to ch2sh ( m / z 47 ) could in principle proceed via two different pathways , either via the formation of ch3sh2 ( m / z 49 ) and subsequent dehydrogenation to ch2sh ( m / z 47 ) ( r5 ) or via the direct formation of ch2sh ( m / z 47 ) ( r3 ) ( compare figure 4a ) . both products , m / z 47 and 49 , are found experimentally , and previously , we had contemplated a two - step mechanism . in this study , we go into the details of minor fragmentation pathways , such as this one , and perform quantum chemical calculations thereon . experimentally , the qualitative energy and humidity dependent behavior of fragment m / z 49 is similar to that of m / z 47 ( figure 3e , b ) , which is in contrast to the behavior of m / z 39/41 ( figure 3a , b ) . this different behavior might indicate that the fragment m / z 47 is rather derived directly from amsh . the calculated qc reaction pathways ( in vacuum ) suggest a rather high activation energy for the rate determining step for the dehydrogenation reaction to m / z 47 ( r5 ) ( figure 4f ) of g 105.9 kcal / mol , compared to g = 30.3 kcal / mol for direct fragmentation ( r3 ) ( figure 4d ) . the qc results therefore indicate that direct fragmentation from amsh to ch2sh ( m / z 47 ) takes place ( r3 ) , rather than dehydrogenation from ch3sh2 ( m / z 49 ) ( r5 ) , and hence , the reaction r5 is set in parentheses in figure 4a . interestingly , reaction r3 to m / z 47 ( figure 4d ) from amsh proceeds via a complex transition state . in the transition state , the breaking of the s c3 bond and a proton transfer ( h2 ) from c4 to c1 occurs , and this leads to the products ch2sh ( m / z 47 ) and ch2chch3 . this is highly reminiscent of the concerted intramolecular -hydrogen shift and propene elimination in the retro - ene reaction of ams ( unprotonated ) , calculated for pyrolysis reactions . hence , we suggest that a similar retro - ene reaction is taking place for amsh fragmentation to ch2sh ( m / z 47 ) ( r3 ) . notably , its fragmentation reaction ( r4 , compare figure 4e ) has a high activation energy for the rate limiting first step ( 89.2 kcal / mol ) , yet a comparatively small endothermic total gibbs energy of the reaction of 5.9 kcal / mol for the associated product . the threshold for efficient hydronium protonation reactions in the gas phase is considered to be a difference in proton affinities of 3540 kj / mol ( corresponding to about 10 kcal / mol ) . amsh has two isomers and the protonation reaction with h3o to both surpass the threshold ( table 3 ) . hence , the protonation reactions are expected to be efficient and are expected to proceed at a collisional rate , here k 2.28 10 cm s ( e / n = 140 td ; tdrift = 90 c ) . for the direct protonation reaction with the first protonated water cluster h2oh3o , the cyclic , but not the linear form , of protonated ams provides enough energy for an efficient reaction , the linear form being borderline ( compare table 3 ) . hence , in theory , humidity - dependent changes in fragmentation patterns could be due to less available energy when proton transfer takes place from higher water clusters . however , the uniform experimental results for all e / n ranges for the change in overall humidity dependence ( mostly caused by fragments m / z 41 and 39 ) of about 2025% less fragmentation ( compare figure 1 ) with increased humidity , indicate the contrary : e / n has a uniform influence on humidity - dependent fragmentation of amsh . ( in the literature , for ethanol a constant influence of humidity on fragmentation is seen , whereas for isoprene it is not ( compare figure 5 in ref ( 2 ) ) . ) however , the amount of protonated water clusters present decreases with increasing e / n ( figure 1c ) . this influence has been described by a roughly quadratic function ; compare refs ( 27 ) and ( 47 ) . hence , a decreasing influence of protonated water clusters with increasing e / n , not an approximately constant influence of a reduction of ca . note that a small systematic change in fragmentation with e / n can be seen ( compare figure 1b ) , particularly for e / n 90 td . as interpretation , we suggest that the strong uniform behavior of the humidity dependence of ams fragmentation is not solely due to lower internal energy ( caused by higher water cluster proton affinities , as reported for other compounds such as some terpenes ) . from this , we conclude a stabilizing effect of water directly on amsh and the fragment at m / z 41 c3h5 , which consequently reduces fragmentation . concerning the reaction r2 , the resulting fragment at m / z 39 shows the strongest energy- and humidity - dependent change , and , importantly , also the largest changes ( 1st derivation ) ( figure 3a ) . for calibrations , this means that this fragment is prone to introduce the largest errors in calibrations , in particular at high e / n and under dry conditions . a second , important fragment is m / z 41 , by its shear abundance in the e / n range 125145 td . together , these two fragments are responsible for the major part of the absolute humidity dependence of ams fragmentation . for modeling hydration , continuum solvent methods like the reaction field or explicit consideration of water molecules ( classical or quantum chemically described ) or mixtures of them can be used . pcm ( polarizable continuum model ) is a qc method that simulates water as a polarizable continuum . all species have been reoptimized to account for solvation - induced geometrical changes . for r2 , according to pcm ( figure 5a ) , all species become more stable with hydration . this holds true for both , the first step , the hydride shift , which is the linearization of c3h5 ( m / z 41 ) , and , to a greater extent , for the second step , the dehydrogenation . however , these qc results from pcm contradict the experimental results : according to the pcm results , the presence of water would facilitate dehydrogenation of c3h5 ( m / z 41 ) , be it as a kinetically determined reaction ( which would be rather expected ) , but just as well for a thermodynamically controlled reaction . thus , the experimental ptr - ms gas - phase conditions in the reaction chamber ( drift tube ) are not well modeled by an infinite solvent . the counterpart to solvation in an infinite solvent is an atomistic microhydration approach , which simply means the addition of water molecules to the reacting species ( compare figure 5b ) . of course , this is computationally much more challenging due to the larger size of the quantum system and the need to optimize floppy structures with multiple local minima . with single molecules of water added , a new 2-step reaction mechanism appears ( see figure 5b ) , in which the water molecule inhibits fragmentation : at first , water forms a covalent bond with c3h5 and donates a hydrogen atom for the formation of h2 . water is regenerated from the ch2ch2coh intermediate , while the intermediate is converted to the c3h3 ( m / z 39 ) product in the rate - determining step of the reaction . a comparison of the energy profiles for adding one , two , or three water molecules as microhydration can be seen in figure 5c . the detailed steps for one , two , and three water molecules can be seen in supporting information s6 , s6_figure 1 . when one , two , or three water molecules are added , the activation energies for the c3h5 ( m / z 41 ) dehydrogenation to c3h3 ( m / z 39 ) ( r2 ) increase for both reaction steps ( see figure 5c ) . the gibbs activation energies of the rate - determining step in microhydration ( 71.2 , 75.2 , and 76.7 kcal / mol for one , two , and three water molecules , respectively ) are higher than the activation energy of c3h5 dehydrogenation in vacuum ( 49.4 kcal / mol ) ( figure 5d ; compare also figure 5c to figure 5a for vacuum without water ) . according to the microhydration results , the dehydrogenation reaction of c3h5 ( m / z 41 ) to c3h3 ( m / z 39 ) is actually inhibited by the water molecule , and this inhibition increases from one to three water molecules . hence , in this unusual case , limited quantities of water actually act as an anticatalyst , an inhibitor : water is used and regenerated during a reaction cycle . the reaction without water has a lower activation barrier ( compare summary in figure 5d ) . contrary to the continuum model , the microhydration results are in line with the experimental results and allow for the conclusion that water indeed acts as an inhibitor . as the conditions in the drift tube , the reaction chamber of the chemical ionization , are probably proceeding at temperatures above thermal energies , also calculations for higher energies were performed ; see supporting information s7 , which basically yield the same results : microhydration is inhibited compared to vacuum . for the reaction of m / z 89 to 41 , the situation of the humidity dependence is similar to that for m / z 39 ( figure 6 ) . experimental results show a stabilization with humidity against fragmentation ( figure 3d and figure 2e ) . qc results for solvation in comparison to vacuum ( pcm ) ( figure 6a ) show a tiny ( g = 0.1 and 2.4 kcal / mol for the first and the second steps , respectively ) kinetic inhibition of fragmentation with solvation . in the microhydration scenario ( cf . the reaction of amsh to c3h5 ( m / z 41 ) proceeds also favorably in two steps ( energy profiles in figure 6c ) and involves intramolecular rearrangements leading to bond breakage . the second , rate - determining step with one h2o ( 70.1 kcal / mol ) is similar to the vacuum system ( 70.1 kcal / mol ) . when more water is added ( see figure 6c , d for a comparison ) , the activation energy increases to 70.5 and 72.1 kcal / mol for two and three h2o , respectively . this is mainly due to the stronger binding ( association ) of the water molecules in the intermediate complexes and is similar to the effect of h2o in r2 , the dehydrogenation of c3h5 ( m / z 41 ) to c3h3 ( m / z 39 ) ( see figure 5c ) . here again , calculations for higher ion temperatures were performed , yielding comparable results as those with thermal calculations ; see supporting information s8 . schwarz et al . performed energy - dependent fragmentation pattern measurements for ams with ptr - ms from dry n2 . for comparison with our results we can now discount our previous theory that humidity is responsible for the differences in fragmentation patterns between schwarz et al . and our results . however , similar contradictory results on fragmentation pathways have been reported for isoprene , which has a similar ( same ) fragment(s ) as ams at m / z 41 and 39 . stabilization for a similar ( same ) fragment m / z 41 from isoprene by humidity was also proposed by schwarz et al . our qc calculations with microhydration confirm the theory of stabilization ( figure 5 ) . this could also explain the strong changes in sensitivity , in particular , when moving from completely dry samples to slightly humid samples , as reported in literature for other compounds . changes in relative abundance ( fragmentation ) due to humidity changes are known and , in part , have been implemented as humidity - dependent calibration factors , e.g. , here , and the use of the ratio abundance of m / z 37/19 has been proposed for better consideration of fragmentation . however , ptr - ms literature is still scarce on the theory and implementation thereof for calibration considerations ; compare ref ( 47 ) . currently , the focus concentrates rather on direct application of humidity calibrations to measured samples . recently , comparison measurements of calculated data to data measured with ptr - tof - ms demonstrated that reaction rates can be well measured , even for humid conditions , as well as predicted . however , therein , e / n has to be kept above 120 td , mainly because then the influence of higher water clusters can be neglected . our results from ams re - emphasize that quantification of ptr - ms data still requires knowledge of the fragmentation patterns and the compound - specific humidity dependence thereof . we present a combined experimental and theoretical study on the humidity- and energy - dependent fragmentation of allyl methyl sulfide ( ams ) in chemical ionization mass spectrometry ( cims ) . at low humidity concentrations , as typically found in ptr - ms conditions , humidity reduces fragmentation of amsh . at high concentrations of water , water would favor fragmentation of amsh according to qc calculations as the pcm results become valid at high humidity concentrations . the reason for this amphoteric behavior of water is a change in the reaction mechanism . at high humidity concentrations , water acts as a solvent in contrast , at low water concentrations , a different reaction mechanism takes place , in which water participates directly : the activation energy of the new rate determining step is higher because water stabilizes the complex . consequently at low humidity , fragmentation is suppressed , and water acts as an inhibitor to fragmentation . this represents a new reaction mechanism of humidity in fragmentation , an inhibition against fragmentation via the direct influence of water . furthermore , we conclude that the specific reactions of h2o with amsh rather than changes in proton affinities or solvation are the reason for the observed humidity - dependent fragmentation patterns for ams . for cims and ptr - ms , the presented results reemphasize the importance of considering humidity- and energy - dependent fragmentation in calibrations for quantitative measurements and present a further step toward absolute calibration procedures from theory . as humidity often influences the reaction profile of gas phase ion molecule reactions , the newly found mechanism may be relevant in a number of fields , including upper atmospheric ion chemistry , prebiotic atmosphere of planets , ion chemistry of plasma , and plasma sources .

medical records were retrospectively reviewed for cats with nasal lymphoma treated with radiation therapy at the rakuno gakuen university veterinary teaching hospital . the 30 cats included in this study were treated between april 2004 and december 2011 . all cats had biopsies taken under general anesthesia and were diagnosed with lymphoma before any treatments . pretreatment evaluation included complete blood counts , serum chemistry , three - view thoracic radiographs , abdominal ultrasounds , ct scans of head , chest and abdomen , and aspiration cytology of lymph nodes or organs . follow - up information was obtained from the medical records or by contacting the referring veterinarians . radiation therapy was performed by use of an orthovoltage x - ray machine ( titan-450s , ge , tokyo , japan ) with a half - value layer of 4.8 mm of cu at 450 kv and 10 ma . the exposure rate was 1.68 gy / min with a filter of 1.0 mm of al , 0.3 mm of cu and 0.5 mm of sn . the distance from the x - ray source to the skin was 60 cm . radiation was delivered via single dorsal portal directed to the tip line of the nose to the top of the head . the response was assessed based on ct images at one month after finishing the radiation therapy . complete response ( cr ) was determined , if the tumor disappeared from ct images . partial response ( pr ) was defined as a decrease of 30% or greater in the sum of the longest diameters . stable disease ( sd ) was defined as between a decrease of 30% and an increase of 20% in the sum of the longest diameter . progressive disease ( pd ) was defined as an increase of 20% or greater in the sum of the longest diameters . apoptotic and proliferative activities in pretreatment biopsy samples in feline nasal lymphoma were detected . a terminal deoxynucleotidyl transferase dutp nick end - labeling ( tunel ) assay was carried out using an apoptag in situ apoptosis detection kit ( s7100 , chemicon merck millipore , billerica , ma , u.s.a . ) to detect apoptotic cells . all 3-m sections from paraffin - embedded tissues that were taken before radiation therapy were cut . the samples were pretreated with proteinase k ( 20 g / ml , dako , glostrup , denmark ) . working - strength tdt solution was then added to the slides at 37c for 1 hr . the reaction was stopped by addition of working - strength wash / stop buffer for 10 min . anti - digoxigenin peroxidase was applied , and the tissue sections were then incubated for 30 min at room temperature . 3 - 3-diaminobenzidine ( kanto chemical co. , tokyo , japan ) solution was used for color developing , and then , mayer s hematoxylin was used for counterstaining . . the antigen was retrieved by autoclaving at 121c for 15 min with 0.1 m citrate buffer ( ph 6.0 ) . all sections were blocked for endogenous peroxidase activity and non - specific binding with 3% hydrogen peroxide and 1% horse serum in phosphate - buffered saline ( pbs ) . sections were then incubated with a mouse anti - ki-67 monoclonal antibody ( clone mib-1 , dako ) at a 1:100 dilution in pbs for 60 min at room temperature . the secondary antibody dilution and avidin - biotin complex solution ( vectastain abc kit , vector , burling , ca , u.s.a . ) were applied to the sections and incubated for 30 min each at room temperature . 3 - 3-diaminobenzidine solution and mayer s hematoxylin were used as chromogen and counterstain , respectively . the apoptotic index ( ai ) was determined as the percentage of apoptotic positive tumor cells that stained brown in pretreatment biopsy samples . the ki-67 labeling index , defined as the percentage of ki-67-positive tumor cells , was counted in five random fields . the ki-67 index was divided into two groups ( high and low ki-67 ) with the level of 40% as the cut point . for statistical analysis , 30 samples were divided into two groups based on the clinical findings ( age , sex , presence of cribriform plate destruction and response to radiation ) . we compared the two indices ( apoptosis and ki-67 ) and analyzed the differences between these groups using t - test for independent samples . the correlation between the ai and ki-67 indices was analyzed according to spearman s correlation method . the cats that had a stage i disease ( single organ affected ) and treated with radiation alone were studied with survival time . survival time of the patient was defined from the starting date of radiation therapy to the time of death . the correlation between the msts and the indices was estimated using the kaplan meier method , and statistically significant differences between survival curves were analyzed using a log - rank method . a p value of < 0.05 ( two - sides ) was considered statistically significant . thirteen of the enrolled cats were neutered males , thirteen were spayed females , two were intact males , and two were intact females . the age of the cats that started treatment with radiation therapy ranged from 4 to 17 years with mean and median of 10.7 and 11 years , respectively . mean weight was 4.2 kg ( range of 2.5 to 8.7 kg , median of 4.7 kg ) . only one pure breed of persian cat was represented in this study , and the others were mixed - breed cats ( n=29 ) . the most common clinical sign at the time of diagnosis was purulent or mucous nasal discharge ( 73.3% ) followed by facial deformity , nose bleeding , sneezing and exophthalmos . fourteen cats ( 46.7% ) were found to have cribriform plate destruction due to the tumor on pre - treatment ct images . the mean of the total skin surface radiation dose was 34.4 gy ( median of 36 gy ; range of 28 to 42 gy ) . thirteen cats also received chemotherapy after radiation therapy , because of having remaining nasal mass or evidence of systemic disease . fourteen cats died owing to distant disease ( kidney , n=12 ; spleen , n=1 ; and brain , n=1 ) , four cats died from local recurrence and brain involvement , three cats died from renal failure but no evidence of renal lymphoma , two cats died of causes other than tumor , three cats died of unknown causes , and four cats were still alive at the time of data analysis . the ai and ki-67-positive rates are shown in table 1table 1.comparison of the apoptotic and ki-67 indices according to clinical findingsnumber of samplesindices ( mean sd ) ( % ) aiki-67age11 years160.92 0.7240.61 13.22>11 years140.98 0.6136.39 15.46genderfemale150.87 0.7035.58 15.39male151.02 0.6341.37 12.42destruction of cribriform plateyes141.05 0.7039.84 15.15no160.87 0.6337.54 13.89response to treatmentcr161.22 0.7744.43 10.76pr90.77 0.2439.64 12.68sd50.44 0.4316.30 9.41significant differences were calculated with the t - test . a ) p<0.05 .. the ai of all pre - treatment biopsy samples in feline nasal lymphoma ranged from 0 to 2.4% , with a median of 0.85% and mean of 0.94% . the mean ai for cr samples was 1.22% , and the mean ais for the pr and sd samples were 0.77% and 0.46% , respectively . a statistically significant difference was observed between the samples from cats with cr and from cats with sd ( p=0.045 ) . the percentage of ki-67-positive tumor cells in all samples ranged from 7.5 to 57.1% ( median of 41.6% and mean of 38.9% ) . the samples from the cats with cr and pr had mean ki-67 index of 44.4% and 39.6% . the ki-67 index in both the cr and pr samples was significantly higher than in the sd samples ( p<0.001 and p=0.008 ) . however , a significant difference was not observed between the samples from cats with cr and pr ( p=0.334 ) . in all samples of feline nasal lymphoma , a significant positive correlation was found between ai and ki-67 index ( r=0.509 ; p=0.005 ) ( fig . 1fig . 1.correlation between apoptotic and ki-67 indices in cats with nasal lymphoma ( r=0.509 ; correlation between apoptotic and ki-67 indices in cats with nasal lymphoma ( r=0.509 ; p=0.005 ) . seventeen cats diagnosed with stage i disease at first diagnosis day and only treated with radiation therapy were estimated with overall survival times . the cut - off levels of the apoptotic and ki-67 indices were 0.9% and 40% , respectively . the respective msts for cats with high ai and low ai were 402.5 days and 135 days , respectively , but there was no statistical difference between the cats with high and low ai ( p=0.202 ; fig . 2.kaplanmeier curves for overall survival time in 17 cats comparing with different levels of apoptotic and ki-67 indices . ( a ) the mst in cats with high ai and low ai was 402.5 days and 135 days , respectively ( p=0.202 ) . ( b ) the mst in cats with high ki-67 and low ki-67 was 582 days and 77 days , respectively ( p=0.015 ) . ) . on the other hand , the cats with a high ki-67 index had significantly longer survival time compared with those with a low ki-67 tumor ( 582 days vs. 77 days ; p=0.015 ; fig . meier curves for overall survival time in 17 cats comparing with different levels of apoptotic and ki-67 indices . ( a ) the mst in cats with high ai and low ai was 402.5 days and 135 days , respectively ( p=0.202 ) . ( b ) the mst in cats with high ki-67 and low ki-67 was 582 days and 77 days , respectively ( p=0.015 ) . nasal lymphoma is the most common nasal tumor in cats [ 4 , 13 , 20 , 23 ] . chemotherapy should be considered as a top priority of treatment in feline nasal lymphoma , because it is generally a systemic disease . previous larger studies of cats with nasal lymphoma that were treated with chemotherapy alone reported to have msts ranged from 98 to 456 days [ 12 , 13 , 30 , 31 , 33 ] . owing to the nature of lymphomas being sensitive to radiation , radiation therapy is also thought to be treatment choice for localized form of lymphomas [ 12 , 28 , 37 ] . a large retrospective study by haney et al . of 97 cats with nasal lymphoma showed that the mst for radiation therapy alone was 456 days . additionally , the nasal lymphoma cats treated with a combination of local radiation therapy and systemic chemotherapy had a mst from 174 to 955 days [ 12 , 28 ] . a small number of studies showed prognostic indicators for feline nasal lymphomas , including anemia , irradiation dose , response and destruction of the cribriform plate [ 12 , 28 ] . to our knowledge , this study is the first report to correlate the rate of apoptosis and cell proliferation in pretreatment biopsy samples , and the outcome of radiation therapy in feline nasal lymphoma . in veterinary medicine , immunohistochemistry has not been widely used to assess the response to irradiation at the cellular level and has not been used to indicate outcome to radiation [ 11 , 21 ] . ionizing radiation may cause dna damage . the cells occur death when the damage of dna is irreparable , which triggers the cellular pathways . mitotic death is the cells failing to mitosis completely and then death , whereas interphase cell death is the irradiated cells undergoing cell death through apoptosis before they enter mitosis , particularly soon after treatment . previous studies suggested that radiation - induced apoptosis closely correlates with radiosensitivity [ 1 , 9 , 22 ] . the tumor with a high spontaneous apoptotic rate would occur apoptosis easily after irradiation . the relationship between apoptosis and outcome in radiation has been studied in several human cancers . a high apoptotic rate is strongly associated with good prognosis in cancer patients treated with radiation therapy [ 1 , 29 , 35 ] . however , there were few studies focused on spontaneous apoptotic rate for a prognostic predictor in lymphomas . apoptosis - induced death is considered major reason in lymphoid cells compared to other type cells after radiation . although it was unsignificant , the spontaneous apoptosis trended to be predictive to response to in vivo irradiation in a study of human non - hodgkin s lymphoma . moreover , one study of feline lymphoma cell lines also suggested that the more radioresistent cell line was prone to a lower ratio of spontaneous apoptosis . the tunel method is a common technique used for detecting dna fragmentation associated with apoptosis . tunel was used in this study , and the ai was considered as a response predictor for feline nasal lymphomas . the tumor achieving cr showed significantly higher rate of apoptosis than which with sd ( mean of 1.22% vs. 0.46% ; p=0.045 ) . although no significant difference was observed , the cats with a high spontaneous apoptosis rate in the pretreated biopsy sample trended to a longer survival time ( mst of 402.5 vs. 135 days ) . some proteins also have been linked to radiosensitivity , such as p53 , bcl-2 and survivin . p53 gene contributed to the delay of induction of apoptosis in the more radioresistant human lymphoid cell lines . cell nuclei were more likely to appear swelling and multi - nucleated after irradiation in the feline lymphocyte cell line that appeared to be resistant to radiation - induced apoptosis . it is possible that some lymphoma cells may die via mitotic death after radiation . some human cancers have been shown to have higher ki-67 tumors and were linked to better outcomes with radiation therapy [ 2 , 7 , 10 , 18 ] . a study on feline squamous cell carcinoma also found that cats with higher ki-67 had markedly favorable prognosis than cats with lower ki-67 . nevertheless , no significant difference was observed in canine nasal carcinomas . cell cycle of g0 phase is also considered to be a radioresistant period in cells . thus , a low level of ki-67 in tumor cells is believed to reflect hypoxia and radioresistance [ 7 , 16 ] . ki-67 expression detects the proliferating cells , which has also been reported in feline and canine lymphomas [ 8 , 17 ] . the rapidly proliferating tissues may have more radiosensitivity than those growing slowly . in our study , cats with a good response had a higher ki-67 index than cats with a poor response , by a statistically significant margin ( cr and pr vs. sd ) . moreover , cats with a higher level of ki-67 treated with radiation therapy had significantly longer mst than cats with a low ki-67 ( 582 vs. 77 days , respectively ) . despite the relation between proliferation and prognosis being suggested , the reason of this relationship is necessary to be clarified by a further study in lymphomas . in our study , a significant positive correlation was observed between the rate of spontaneous apoptosis and ki-67-positive cells in feline nasal lymphomas ( r=0.509 ; p=0.005 ) . high apoptotic factors have been associated with high proliferation indices in canine lymphomas and human non - hodgkin s lymphomas [ 19 , 24 ] . many of the genes that control cell death also regulate proteins involved in cell division , such as p53 . therefore , cell apoptosis and proliferation control appear to be closely linked with each other [ 15 , 24 , 34 ] . faster proliferating tumors usually indicate better response to irradiation , and higher apoptosis levels may also reflect rapidly proliferating tumors . our results show that the spontaneous apoptotic and ki-67 indices are both strong predictors of response to radiation therapy in feline nasal lymphomas . in conclusion , although our study was retrospective and the study was limited in population size , the response predictive power of measuring apoptosis and proliferation was strong . assessment of apoptosis and proliferation before radiation therapy could be helpful for decision making to decide an appropriate protocol of treatment . because there are many regulators of apoptosis and proliferation in tumor cells , and because radiosensitivity of cancer treatment is not clearly known , additional larger studies would be necessary in order to increase the benefit of treatment .

cytokines are an intriguing group of soluble protein mediators with a large number of described actions but short biological range . these molecules are produced by a variety of cells , including monocytes , macrophages , lymphocytes , and endothelial cells . such proteins include the pro - inflammatory cytokines interleukin ( il)-6 and soluble tumour necrosis factor receptor-1 ( stnfr-1 ) , as measured by jimenez and colleagues in their study . cytokines have been shown to be important in all biological processes , including inflammation in which the major pro - inflammatory cytokines are considered to be tumour necrosis factor ( tnf ) , il-1 , and interferon - gamma in addition to il-6 . they also have anti - inflammatory roles ( il-10 , transforming growth factor - beta , and il-4 ) . the control of this balance between pro- and anti - inflammatory cytokines is vital to the understanding of inflammation and the inflammatory response in human disease processes . cytokines are normally subject to tight homeostatic control and are produced in response to a variety of physiologic and pathologic stimuli . pro - inflammatory cytokines play a pivotal role in initiating and amplifying the inflammatory process . furthermore , the plasma concentrations of certain cytokines , such as il-1 and il-6 , have been shown to be predictive of outcome in specific subgroups of critically ill patients . in particular , tnf and il-1 are elevated early following cardiac surgery , with il-6 and il-8 peaking later . this damaging pro - inflammatory cytokine response is well documented in patients who develop systemic inflammatory response syndrome ( sirs ) after cardiac surgery . in this sirs patient group , il-8 and il-18 the association of elevated levels of pro - inflammatory cytokines with poor outcomes following cardiac surgery has been demonstrated by many studies , but a direct cause - and - effect relationship has not been demonstrated . the pro - inflammatory cytokine response to injury , including cardiac surgery , is countered by the release of anti - inflammatory cytokines . this anti - inflammatory response is not limited to cytokine proteins alone ( such as il-10 ) but also includes the release of soluble cytokine receptors ( stnfr-1 and -2 ) and cytokine receptor antagonists ( il-1 receptor antagonist ) . this complex balance between pro- and anti - inflammatory molecules is likely to influence outcome following many pathophysiological insults , including cardiac surgery . while in some respects the coagulation cascade and the inflammatory response are separate processes , they are closely interconnected in acute disease . the activation of coagulation is a key component of the acute inflammatory response and vice versa , with the endothelium intricately involved in both processes . pro - inflammatory cytokines are released at sites of local inflammation , leading to activation of the endothelium and initiation of the coagulation cascade . cardiac surgery ( in particular , cardiopulmonary bypass ) induces activation of the immune system by factors that include ( but are not limited to ) contact activation of immunological cells , ischaemia - reperfusion injury , and endotoxaemia . once activated , the immune system amplifies its response with activation of complement , cytokine production , coagulation / fibrinolysis , endothelium , and the cellular immune system . all of these processes , if unchecked , may lead to the development of sirs and a poorer outcome in this patient group . the use of tranexamic acid in the study by jimenez and colleagues to attenuate inflammation reveals a glimpse of how the fibrinolytic and pro - inflammatory responses are interlinked . the interplay of other major pro- and anti - inflammatory mediators is worthy of further study in this patient group ( in particular , tnf and il-10 ) to further elucidate the pathways involved and highlight the biological targets involved in this attenuation of the inflammatory response . their study also , perhaps more importantly , yields a practical , clinically applicable method of influencing these often catastrophic cascades that , once initiated , can lead to significant mortality and morbidity in subgroups of patients following cardiac surgery . in the last two decades , much scientific effort has led us closer to a definition of the molecular basis of the immunological response to biological insults and to the development of specific ' targeted therapies ' such as anti - tnf treatment for rheumatoid arthritis . each additional insight into the control of inflammation and its interaction with other biological responses , such as coagulation and fibrinolysis , has the potential to lead to more effective interventions for the clinician perioperatively and in the management of critically ill patients . il = interleukin ; sirs = systemic inflammatory response syndrome ; stnfr-1 = soluble tumour necrosis factor-1 ; tnf = tumour necrosis factor .

a 47-year - old man was admitted with the chief complaint of persistent high fever and transient loss of consciousness . although his general condition improved after intravenous acyclovir administration , the patient presented with visual loss in both eyes 4 days after admission . visual acuity in his right eye was 20/200 and his left eye had light perception alone . progression of retinal detachment was prevented in both eyes ; however , visual acuity of the left eye was totally lost because of neovascular glaucoma . although cases of bilateral acute retinal necrosis have been reported after herpetic encephalitis , this condition is rare after herpetic meningitis . prophylactic acyclovir therapy and early panretinal photocoagulation may prevent retinal detachment and improve the prognosis . neurologists and ophthalmologists should be aware that not only herpetic encephalitis but also herpetic meningitis can lead to acute retinal necrosis within a very short interval . acute retinal necrosis ( arn ) was first described as a rapidly progressive unilateral necrotizing retinitis in 1971 by urayama et al.1 arn usually presents unilaterally and has a poor prognosis . bilateral cases have also been reported.2 to prevent a poor outcome , arn requires aggressive management.3 although several case reports have described the occurrence of arn after herpetic encephalitis,48 arn after herpetic meningitis is rare.9 here , we report the case of a healthy immunocompetent man with bilateral arn that occurred in combination with herpetic meningitis during the administration of intravenous acyclovir . a 47-year - old man was admitted with the chief complaint of persistent high fever and transient loss of consciousness . the patient was somnolent and complained of posterior cervical pain but nuchal rigidity was unclear . a painless erythematous macule with blistering was observed on both upper eyelids and several painless erythematous macules with blisters were observed on the left side of the chest . glucose was maintained at a normal level and cerebrospinal fluid was negative for herpes simplex virus ( hsv ) dna and varicella zoster virus ( vzv ) dna . brain t2-weighted magnetic resonance imaging revealed no findings of herpetic encephalitis , such as hyperintensity corresponding to edematous changes in the temporal lobes ( figure 1 ) . therefore , viral meningitis was suspected and administration of intravenous acyclovir was initiated at 750 mg every 8 hours ( 10 mg / kg every 8 hours ; the patient s body weight was 75 kg ) . three days after admission , the patient s consciousness status slightly improved but binocular diplopia and blurred vision suddenly occurred . in addition , several new sharply circumscribed erythematous macules appeared on the right and left upper and lower extremities . four days after admission , the visual acuity was 20/200 oculus dexter ( od ) and light perception oculus sinister ( os ) . the right and left corneas were clear , but flare and many cells were observed in both anterior chambers and vitreous bodies . optic disc swelling , macular edema , exudation along blood vessels , narrowing of arterioles , dilatation of venules , and superficial retinal hemorrhages were observed in both retinas ( figure 2a ) . oral administration of prednisolone and ocular instillation of betamethasone sodium phosphate and tropicamide were initiated and subsequently tapered over several weeks . five days after admission , panretinal photocoagulation was performed for both eyes . although the patient s body temperature had returned to normal 5 days after admission , medical treatment was continued . inflammation , optic disc swelling , macular edema , and exudation along blood vessels were alleviated in the right eye and the visual acuity of this eye had improved to 20/20 4 months later . however , massive retinal hemorrhage derived from central vein occlusion appeared in the left eye 19 days after panretinal photocoagulation and the hemorrhage extended to vitreous 26 days after photocoagulation . all sight was lost due to neovascular glaucoma approximately 1 month after the occurrence of the hemorrhage ; however , rhegmatogenous retinal detachment was prevented by photocoagulation in both eyes ( figure 2b ) . by 25 days after admission , the patient s general condition and ophthalmoscopic findings had improved , and he was treated as an outpatient . the onset of arn occurred a few days after the administration of intravenous acyclovir , although the patient was not immunocompromised . watanabe et al11 reported a case in which bilateral arn occurred after hsv type 1 meningitis . in their case , the retinal findings deteriorated and retinal detachment occurred in both eyes . in our case , we prevented rhegmatogenous retinal detachment in both eyes . this may be partly due to the administration of intravenous acyclovir before the onset of retinal symptoms as well as early panretinal photocoagulation . serological test results were negative for vzv antibodies and the results of cerebrospinal examination were negative for hsv dna and vzv dna . administration of acyclovir improved the patient s general condition , suggesting that herpes viruses were the causal agent . ganatra et al12 reported six out of seven patients diagnosed with arn in whom hsv type 1 dna was detected had a history of hsv encephalitis and three out of six patients with hsv type 2 dna had a likely history of hsv meningitis . therefore , they concluded that herpes simplex virus is likely to be the viral cause of central nervous system infection in a patient with acute retinal necrosis syndrome . it is possible that isolated meningitis can be accidentally coincident with arn ; however , in our case , we believe this was a part of the disseminated herpetic virus infection . vandercam et al13 reported an overall interval of 5 weeks ( 14 days to 2 months ) between vzv encephalitis and arn . however , as far as the authors of the present report are aware , a bilateral arn occurring in an immunocompetent patient following vzv encephalitis has not been previously described . in this patient , arn occurred in both eyes simultaneously and the occurrence of arn after encephalitis was very short . our patient showed a very rapid and rare clinical course of arn occurring immediately after herpetic meningitis . neurologists and ophthalmologists should be aware that not only herpetic encephalitis but also herpetic meningitis can lead to arn within a very short interval .

sarcoidosis is a systemic disorder of unknown etiologycharacterized by widespread development of noncaseating epithelioid cell granulomas in more than one organ system . a review of the literature suggests that the relationship between sarcoidosis and malignancy is controversial ; however , new onset sarcoidosis has been observed repeatedly and reported in oncology patients [ 2 - 4 ] . we report on a case of sarcoidosis mimicking cancer recurrence , which developed after chemo - therapy for ovarian cancer . a 52-year - old female was admitted to our hospital with a one - month history of abdominal pain on the left side . her abdominal computed tomography ( ct ) scan showed ascites , omental caking , and peritoneal thickening . she received combination chemotherapy comprising paclitaxel 175 mg / m+carboplatin 400 mg / m for eight cycles for seven months after surgery . however , four months later , she presented with a dry cough , and multiple painless subcutaneous nodules that had developed on her right arm and leg . a plain chest radiograph showed bilateral hilar enlargement compared with a previous film ( fig . computed tomography ( pet / ct ) scan for evaluation of cancer recurrence showed no uptake in the subcutaneous nodules , however , multiple areas of high uptake were observed in the enlarged paratracheal , left supraclavicular , retrocrural , and diaphragmatic lymph nodes ( the maximum standardized uptake value [ suvmax ] of the lymph nodes ranged from 12.3 to 21.5 ) ( fig . the same features were observed on both chest ct scan and pet / ct ( fig . 3a ) . however , her serum levels of carbohydrate antigen 125 ( ca125 ) and carbohydrate antigen 19 - 9 ( ca19 - 9 ) were unremarkable ; ca125 level was 9.42 u / ml ( reference range , 0 to 35.0 u / ml ) and ca19 - 9 level was 3.12 u / ml ( referencerange , 0 to 37 u / ml ) . to be certain of the diagnosis of cancer recurrence , biopsy of the mediastinal lymph nodes using endobronchial ultrasound - guided transbronchial needle aspiration and biopsy of the subcutaneous nodules ziehl - neelsen and polymerase chain reaction detection of mycobacterium tuberculosis in tissues were negative for mycobacterial infection , and a serum interferon - gamma release assay test was also negative . she had mild symptoms , including intermittent dry cough , and no specific treatment was required . she was observed closely at our clinic for three months , and the multiple painless subcutaneous nodules resolved spontaneously . her follow - up chest ct scan showed an interval decrease in the size of multiple mediastinal lymph nodes ( fig . however , newly developed perilymphatic and centrilobular nodules were detected in both lungs on the same ct scan ( fig . lymphangitic metastasis of ovarian cancer was suspected radiologically , therefore , she underwent video - assisted thoracoscopic wedge resection of the nodules in the right upper lobe . however , the pathological findings of the lung nodules showed noncaseating granuloma , which was similar to that of the mediastinal lymph nodes and skin ( fig . to the best of our knowledge , this is the first report of sarcoidosis that occurred following surgery and chemotherapy for ovarian cancer in south korea . in addition , this case demonstrates that the clinical presentation and radiological examinations , including a pet / ct scan , may not be able to discriminate between cancer recurrence and sarcoidosis . according to the literature , 4 - 14% of all patients with malignancy can exhibit histopathological evidence of sarcoidosis . in oncology patients , noncaseating granulomas have been observed most frequently in the lymph nodes draining the cancer . in the current case , development of noncaseating granulomas occurred not only in the mediastinal lymph nodes , but also in the subcutaneous tissue and lung parenchyma in the absence of recurrence of ovarian cancer , which led to diagnosis of sarcoidosis . it is difficult to explain why the reaction occurred only in distant lymph nodes and organs and not in regional lymph nodes . understanding of the pathogenesis of sarcoidosis associated with cancer remains incomplete . according to one hypothesis , localized sarcoidosis may be related to degenerative and necrotic changes within the tumor itself and that systemic sarcoidosis at sites not involved with the tumor may be mediated by humoral and t cell - mediated factors , and subsequent recruitment and activation of macrophages . development of sarcoidosis during or immediately after chemotherapy is rare and has been reported only in a limited number of cases of malignant lymphoma , osteosarcoma , and solid tumors [ 7,9 - 11 ] . the pathophysiological mechanisms of sarcoidosis during or after high - dose chemotherapy are unknown because chemotherapy inhibits the local immunological responses there by preventing granuloma formation and development of associated disease symptoms . it has been postulated that sarcoidosis can be caused by a microorganism because immunosuppression induced by antineoplastic therapy might spread this agent , leading to an immune response resulting ingranuloma formation . this phenomenon has been associated with several different chemotherapeutic agents ; the most common causative agent is the biologic modifier alpha - interferon . there is only one previous report on development of sarcoidosis associated with combination chemotherapy paclitaxel and carboplatin . reported a case of sarcoidosis that developed during induction chemotherapy comprising paclitaxel and carboplatin for lung cancer . in the current case , development of sarcoidosis occurred four months after chemotherapy ; however , the patient described by umezu et al . was diagnosed with sarcoidosis during chemotherapy . paclitaxel and carboplatin may have induced the reaction , however , conduct of further studies will be required in order to explain the pathogenesis of sarcoidosis during or after chemotherapy . as in the current case , in addition , the current case showed higher uptakes in the pet / ct scan ( e.g. , suvmax of lymph nodes , 12.3 to 21.5 ) than in previous reports , suggesting that the suvmax in the pet / ct scan is not accurate for distinguishing between sarcoidosis and malignancy . therefore , evaluation of other clinical parameters or the serum ace level might be necessary in order to discriminate between sarcoidosis and metastasis . in conclusion , when cancer recurrence is suspected despite successful chemotherapy for a neoplastic disease , the clinician should consider the possibility of sarcoidosis even if it occurs during or immediately after treatment . histopathological examinations can be useful for making a precise diagnosis and for appropriate decision making .

starting in 2000 , pegylated interferon- ( peg - ifn ) has been the treatment of choice for chronic hepatitis c virus ( hcv ) infections . , we report for the first time a patient who experienced a relapse of vogt - koyanagi - harada ( vkh ) disease during treatment with peg - ifn and ribavirin therapy for hcv infection . a 60-year - old woman was referred to our department for fundus examination on june 25 , 2008 , and was scheduled to begin peg - ifn and ribavirin therapy for chronic hcv infection . she had been diagnosed with vkh disease in 1991 and had been treated for 15 months with both injection and oral steroid therapies ( fig . 1a , b ) . after the initial treatment , no further symptoms or evidence of the disease were observed over the next 17 years . at the time of the examination , she had no symptoms , and her visual acuity was 1.2 in her right eye and 1.0 in her left eye . the fundus of both eyes showed a sunset appearance , but slit lamp and fundus examinations showed no active inflammation . based on our findings , treatment with peg - ifn and ribavirin was started . at 3 weeks after the start of the therapeutic intervention , the patient complained of visual blurring , eye pain , and an increased hearing loss . her visual acuity was reduced to 0.2 in her right eye and remained at 1.0 in her left . fluorescein angiography showed multiple leakage of fluorescein dye from the choroid into the subretinal space ( fig . 2c , auditory acuity was mildly diminished in both ears . as we diagnosed a relapse of the patient 's vkh disease , we began administration of intravenous pulses of methylprednisolone ( 1,000 mg / day for 3 days ) , followed by daily oral corticosteroids , which were then tapered off over time . two months after the symptoms first appeared , her visual acuity returned to 1.0 in both eyes without ocular inflammation and remained stable thereafter for more than 15 months . over the last decade , peg - ifn and ribavirin therapy has become the standard treatment for hcv . ifn- belongs to the type 1 interferon family , which is involved in the host 's natural defense against viruses , bacteria , and neoplasia . recent publications have emphasized the effects of ifn- on refractory uveitis , including vkh disease . although the mechanisms of its efficacy are believed to be multifactorial , they have not yet been well elucidated . in contrast , several autoimmune disorders , such as sarcoidosis , mooren 's ulcer , graeve 's disease , systemic lupus erythematosus , type i diabetes or rheumatoid arthritis , have been observed during ifn- and ribavirin therapy . these cases have been attributed to immunomodulatory actions related to the therapy [ 3 , 4 ] . there have been some reports stating that vkh disease occurs after ifn therapy for hcv . in these reports , the disease was indicated to have emerged 3 months to 2 years after the start of ifn therapy [ 5 , 6 , 7 ] . in the current case , however , we identified the relapse of vkh disease at only 3 weeks after starting ifn therapy , even though the disease had been dormant for 17 years . the immunological activities of ifn , including enhanced lymphocyte cytotoxity , increased expression of major histocompatibility class i antigens , production of proinflammatory cytokines , and differentiation of antigen - presenting cells , may predispose susceptible individuals to the development of an autoimmune disorder [ 3 , 4 , 5 ] . moreover , ifn- can cause an immune response shift to t - helper-1-type predominance and alter the expression of histocompatibility class i and ii antigens , possibly leading to the development of vkh disease [ 5 , 8 ] . in addition , administration of ifn- may lead to the development of vkh disease via an increase in the production of endogenous ifn- , and it has been reported that serum endogenous ifn- titers are significantly elevated in vkh disease patients [ 9 , 10 ] . these possible mechanisms may explain how ifn exacerbates preexisting autoimmune conditions and leads quickly to the relapse of vkh disease , as shown in this case . in conclusion , when ifn therapy is administered to hcv patients , especially those who have a history of vkh disease must be closely monitored for ophthalmologic complications due to immediate relapse of vkh disease .

an enormous change has taken place in ocular health issues , however , there are various problems facing ophthalmic public health worldwide . in the previous years , the world health organization and the international agency for the prevention of blindness in partnership launched the global initiative to eradicate avoidable blindness by the year 2020 , vision 2020 the right to sight . it has concentrated on the prevention of blindness disability and recognized a health issue - sight as a human right . in view of challenges ahead of visual sciences , close collaboration between international agencies at the global level to implement new strategies and monitor the progress will be mandatory . in these circumstances non - governmental organizations should not be neglected . world sight day 2012 would be a great opportunity to be a focus on importance of visual impairment as an important public health issue and discovering new challenges ahead . world sight day is an international day of awareness , held annually on the second thursday of october to focus attention on the global issue of avoidable blindness and visual impairment . this year , there is no global theme for world sight day 2012 however this would be a great opportunity to focus on importance of visual impairment as an important public health issue . global initiatives are becoming more and more common , as well as more feasible in light of these changes . ophthalmology and visual sciences have been progressing quickly in recent decades through globalization of science . an enormous change has taken place in ophthalmic public health issues and has faced new challenges worldwide . in the meantime , ophthalmology and visual sciences might well be a discipline with the highest number of advancements ( 1 ) . our ability to learn so soon about advances made by ophthalmologist and visual scientists in other parts of the world is a relatively recent phenomenon . today it is much easier to share information developed with other ophthalmologists around the world ( 2 ) . in addition , an astonishing array of organizations and societies are now working together to focus on correcting global delinquent issues so that the vision loss can be avoided ( 3 ) . of that number , 39 million are blind and the remaining 246 million have severe to moderate vision impairment . the tragedy is that up to 80% of that number was preventable and/or treatable ( 4 ) . on an ongoing basis , over 2 million people per year go blind , with cataracts being three - fourths of the cause ( 5 ) and the leading cause of blindness . sixty - five percent of the world s population is visually impaired , and 82% that are blind are over the age of 50 , which represents 20% of the world s population . clearly , some action has to be taken but one of the many challenges is that 90% of the untreated population lives in the developing countries where there is a low ratio of healthcare resources particularly when compared to the developed countries ( 4 ) . substantial impact is now possible on account of a combination of advances in pharmacology and technology . medications for onchocerciasis , trachoma , and vitamin a deficiency are now widely accessible . on the other hand novel technologies , sutures , and intraocular lenses in cost affordable components ( 5 ) . despite these advancements , as well as various effort in globalization of ophthalmology by creating multilingual education material ( 6 ) global treatment for the visual impairments was seen to be woefully inadequate . for that reason , various organizations combined their forces to create international ophthalmology strategic plan to preserve and restore vision its mandate is to both encourage and enhance collaborative activities of the 150,000 ophthalmologists and other thousands of health professionals to create a sustainable program designed to dramatically reduce of the world s population the blind and visually impaired ( 7 ) . a free textbook with hundreds topics has been posted on the internet in english as a complimentary resource . the plan is to translate that text so that all countries can benefit from a global , current ophthalmological knowledge repository . other areas of focus in the vision for the future are the development of clinical guidelines for the provision of eye care within the existing infrastructure , however limited . at a more macro level , vision for the future calls for the establishment of a basic international standard for the eye care , and the last but not the least , for ophthalmologists to continue to advocate for the preservation and restoration of vision ( 5 ) . concurrently , there is another key program : global initiative for the elimination of avoidable blindness that is vision 2020 : the right to sight . its objective is literally to improve eyesight globally , with a special focus on under - developed countries where surgically remedial cataracts are rampant in the aging population . its main area of focus is the control of major causes of blindness : cataracts , trachoma , onchocerciasis , childhood blindness , vitamin a deficiency , surgically treatable disorders and the statistically largest group , refractive errors and low vision . other areas of focus include human research development ( secondary and tertiary workers ) , and infrastructure and technology development ( 5 ) . currently , the program is underway ( and it has been since 1999 ) but faces challenges in terms of funding and required functionality . the ophthalmologic community is creating curriculum for both current and ongoing education , which is tasked with continuing ophthalmologic research , all with a view to the betterment of eye care everywhere by the year 2020 ( 7 ) . nevertheless , while the statistics are disheartening , i.e. , there is so much visual impairment issues that could have been prevented and/or treated , the fact that so many disparate yet common organizations ( both governmental and other ) would collaborate to this degree , is heart - warming . their goals , despite the challenges faced by the under - developed countries , seem achievable . clearly the ophthalmologic communities must grow and band together to make these targets realizable to be successful in security funds necessary to build the infrastructure needed perhaps under a global department . in these circumstances non - governmental organizations and we live in an age of information that is increasingly global in scope . medical breakthroughs abound and our population is aging and living longer . global initiatives are becoming more and more common , as well as more feasible in light of these changes . ophthalmology and visual sciences have been progressing quickly in recent decades through globalization of science . an enormous change has taken place in ophthalmic public health issues and has faced new challenges worldwide . in the meantime , ophthalmology and visual sciences might well be a discipline with the highest number of advancements ( 1 ) . our ability to learn so soon about advances made by ophthalmologist and visual scientists in other parts of the world is a relatively recent phenomenon . today it is much easier to share information developed with other ophthalmologists around the world ( 2 ) . in addition , an astonishing array of organizations and societies are now working together to focus on correcting global delinquent issues so that the vision loss can be avoided ( 3 ) . the statistics are staggering : 285 million people are visually impaired world - wide . of that number , 39 million are blind and the remaining 246 million have severe to moderate vision impairment . the tragedy is that up to 80% of that number was preventable and/or treatable ( 4 ) . on an ongoing basis , over 2 million people per year go blind , with cataracts being three - fourths of the cause ( 5 ) and the leading cause of blindness . sixty - five percent of the world s population is visually impaired , and 82% that are blind are over the age of 50 , which represents 20% of the world s population . clearly , some action has to be taken but one of the many challenges is that 90% of the untreated population lives in the developing countries where there is a low ratio of healthcare resources particularly when compared to the developed countries ( 4 ) . substantial impact is now possible on account of a combination of advances in pharmacology and technology . medications for onchocerciasis , trachoma , and vitamin a deficiency are now widely accessible . on the other hand novel technologies , sutures , and intraocular lenses in cost affordable components ( 5 ) . despite these advancements , as well as various effort in globalization of ophthalmology by creating multilingual education material ( 6 ) global treatment for the visual impairments for that reason , various organizations combined their forces to create international ophthalmology strategic plan to preserve and restore vision its mandate is to both encourage and enhance collaborative activities of the 150,000 ophthalmologists and other thousands of health professionals to create a sustainable program designed to dramatically reduce of the world s population the blind and visually impaired ( 7 ) . a free textbook with hundreds topics has been posted on the internet in english as a complimentary resource . the plan is to translate that text so that all countries can benefit from a global , current ophthalmological knowledge repository . other areas of focus in the vision for the future are the development of clinical guidelines for the provision of eye care within the existing infrastructure , however limited . at a more macro level , vision for the future calls for the establishment of a basic international standard for the eye care , and the last but not the least , for ophthalmologists to continue to advocate for the preservation and restoration of vision ( 5 ) . concurrently , there is another key program : global initiative for the elimination of avoidable blindness that is vision 2020 : the right to sight . its objective is literally to improve eyesight globally , with a special focus on under - developed countries where surgically remedial cataracts are rampant in the aging population . its main area of focus is the control of major causes of blindness : cataracts , trachoma , onchocerciasis , childhood blindness , vitamin a deficiency , surgically treatable disorders and the statistically largest group , refractive errors and low vision . other areas of focus include human research development ( secondary and tertiary workers ) , and infrastructure and technology development ( 5 ) . vision 2020 is to be organized and developed by each country . currently , the program is underway ( and it has been since 1999 ) but faces challenges in terms of funding and required functionality . the ophthalmologic community is creating curriculum for both current and ongoing education , which is tasked with continuing ophthalmologic research , all with a view to the betterment of eye care everywhere by the year 2020 ( 7 ) . nevertheless , while the statistics are disheartening , i.e. , there is so much visual impairment issues that could have been prevented and/or treated , the fact that so many disparate yet common organizations ( both governmental and other ) would collaborate to this degree , is heart - warming . their goals , despite the challenges faced by the under - developed countries , seem achievable . clearly the ophthalmologic communities must grow and band together to make these targets realizable to be successful in security funds necessary to build the infrastructure needed perhaps under a global department . in these circumstances non - governmental organizations and the closer collaboration between international agencies especially international council of ophthalmology , world health organization , and international agency for the prevention of blindness at the global level is required . more care , more research and , most of all , more education are required if we are willing to decrease rates of blindness and other visual impairments globally . in summary , with the world health organization , international agency for the prevention of blindness , international non - governmental organizations , and the international ophthalmic community , we can all promote to up - keep to prevent and treat visual impairments worldwide ( 5 ) . as long as those ends are met , and there is continuity of effort , there seems to be no reason why the visual impairment can not be dramatically reduced within the next decade . world sight day 2012 would be a great opportunity to elaborate these issues and locating appropriate solutions worldwide .

the extracellular matrix ( ecm ) is an insoluble supra - structure comprised of a variety of matrix components including fibronectin , glycosaminoglycans , chrondronectin , osteonectin , collagens , laminin , proteoglycans , and growth factors [ 16 ] . the ecm provides the scaffold for cell attachment which is necessary for several diverse cellular activities , including cytoskeletal remodeling , polarization , differentiation , migration , and invasion [ 79 ] . binding to the ecm is regulated by various signaling pathways that control the assembly and disassembly of three distinct , but functionally related actin and integrin - containing adhesion structures known as focal adhesions , podosomes , and invadopodia . in this review , we will discuss our current understanding of the similarities and differences between focal adhesions , podosomes , and invadopodia . we also will highlight several important tyrosine kinases and other signaling proteins that are known to control the formation and function of these adhesion structures , and we will discuss their role in pathophysiology . focal adhesion formation and turnover has been used as a model system for understanding the mechanisms of cellular adhesion . although focal adhesions , podosomes , and invadopodia share common signaling proteins , they are distinct in cellular architecture and function ( summarized in table 1 ) . focal adhesions , also known as focal contacts , were identified over 30 years ago by electron microscopy and described as electron - dense plaques associated with actin filament bundles . focal adhesions can be considered to be large protein assembly complexes that spread mechanical forces from sites of cell adhesion to the cell body . in addition , focal adhesions regulate intracellular signaling pathways necessary for cell migration , growth , proliferation , embryogenesis , wound healing , and tissue repair [ 1114 ] . focal adhesions are comprised of a wide range of signaling proteins , such as the tyrosine kinases pyk2 [ 16 , 17 ] , fak [ 18 , 19 ] , src [ 20 , 21 ] , abl , and integrin - linked kinase ; the phosphatases ptp - pest and ptp1b ; the actin - binding proteins paxillin [ 26 , 27 ] , talin [ 23 , 2830 ] , vinculin [ 23 , 2830 ] and tensin , the gtpases dynamin and cdc42/rho [ 33 , 34 ] , as well as scaffolding proteins p130cas and crk . many of these proteins have been shown to play predominantly a structural role or are involved in signal transduction . these protein platforms recruit adaptor proteins and lead to the activation of complex network of signaling cascades that regulate basic cellular functions [ 16 , 36 ] . an important tyrosine kinase found in focal adhesions is the focal adhesion kinase ( fak ) . fak is a 125 kda cytoplasmic tyrosine kinase that is activated upon integrin engagement and controls signaling pathways crucial for cell proliferation , migration , and survival . the c - terminal domain of fak is known as the focal adhesion targeting domain ( fat ) . as its name implies , the fat domain is involved in directing fak to focal adhesion complexes in a variety of cells . in contrast , the n - terminal domain of fak is known as the ferm domain ( f for the 4.1 protein , ezrin , radixin , and moesin ) . the central kinase domain of fak , which itself is activated by phosphorylation , directs the phosphorylation of several signaling protein such as paxillin , grb2 and p130cas . in vitro studies have shown that the ferm binds directly to the intracellular domain of the 1-integrin subunit and regulates fak kinase activity . it was also discovered that blocking 1-integrin function leads to fak dephosphorylation , which in turn increases the sensitivity of malignant tumors to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines . fak and the tyrosine kinase src play a central regulatory role in focal adhesion turnover , and deletion of either of these kinases increases focal adhesion stability . in addition , it has been shown that fak and src work in concert with the gtpase dynamin to regulate microtubule - induced focal adhesion disassembly . in studies by ezratty and colleagues , fak/ fibroblasts exhibited reduced dynamin accumulation around focal adhesions compared to controls , suggesting that fak regulates dynamin localization and recruitment to focal adhesions . in addition , wang and others demonstrated that src phosphorylates dynamin at tyrosine residues , which promotes the translocation of dynamin to focal adhesions by fak . disruption of the src - fak - dynamin complex blocked focal adhesion disassembly and fibroblast migration . using bone - forming osteoblasts as our model system , we also found that dynamin is expressed in osteoblasts and inhibition of its gtpase activity with the chemical inhibitor dynasore , increased the number of vinculin and paxillin - positive focal adhesions in osteoblasts , compared to control cells ( figure 1 ) . interestingly , we found that dynamin is also localized to actin - rich podosomes , in bone - resorbing osteoclasts [ 44 , 45 ] . moreover , dynamin knockdown with shrna or overexpression of a gtpase - inactive dynamin mutant increased podosome stability and the thickness of the podosome belt and decreased osteoclast bone resorbing activity . together , these studies reveal that dynamin 's gtpase activity is necessary for both focal adhesion turnover in osteoblasts as well as podosome turnover in osteoclasts . furthermore , these findings suggest potential similarities in the mechanism of turnover of focal adhesions and podosomes , which is likely to be dependent on the complement of signaling and scaffolding proteins present in different cell types . podosomes are highly dynamic adhesion structures found in a wide variety of migratory cells including macrophages , osteoclasts , endothelial cells [ 4650 ] , transformed fibroblasts , and carcinoma cell lines . they were first identified in the 1980s in v - src - transformed fibroblasts [ 53 , 54 ] . podosomes and focal adhesions are both cell - matrix adhesion sites , but they differ in their structural design and turnover rates [ 5559 ] ( table 1 ) despite sharing a large number of common signaling proteins , such as fak , dynamin , talin , paxillin , wasp , and vinculin . podosomes turnover occurs very rapidly with an apparent half - life of 212 min and involves the polymerization and depolymerization of the central f - actin core [ 50 , 61 ] . podosomes first appear as small actin dots which are then reorganized into small rings or rosettes with a diameter of 0.51 m and a depth of 0.20.4 m [ 62 , 63 ] ( figure 2 ) . the assembly of podosomes in macrophages and osteoclasts is dependent on an intact microtubule system [ 49 , 64 ] . the central core of f - actin is surrounded by a ring of molecules that are involved in adhesion , matrix degradation , or migration . these proteins include the tyrosine kinases pyk2 and src , actin - associated proteins [ 51 , 65 ] , integrins , and their associated proteins , intermediate filaments , motor proteins and metalloproteases [ 50 , 68 ] . in vitro studies demonstrated that rhoa , rac1 , and cdc42 are also involved in the regulation of podosomes turnover [ 69 , 70 ] and perhaps in recruiting podosomes to the leading edge of cells following microtubule - dependent cell polarization [ 64 , 69 , 7173 ] . in contrast to focal adhesions , podosomes are found at sites of ecm degradation [ 51 , 74 ] . the metalloproteases mt1-mmp and mmp-9 have been localized to podosomes , strongly supporting a role for podosomes in ecm degradation [ 56 , 68 , 75 ] in addition to adhesion . this is well illustrated in osteoclasts , the primary bone - resorbing cells found in the body . in mature osteoclasts , podosomes are organized into a ring or belt - like structure at the cell periphery ( referred to as the sealing zone ) [ 50 , 77 ] . this unique actin- and integrin - rich structure functions to dock osteoclasts to ecm proteins in bone and seals off the bone - resorbing compartment . this allows for the localized secretion of acidifying protons , chloride ions , and bone matrix - degrading metalloproteases . in response to integrin engagement , and in the presence of intracellular calcium , pyk2 is autophosphorylated at tyrosine residue y402 , which is essential for its catalytic activity [ 37 , 78 , 79 ] and for downstream signaling via p130cas , src , cbl , integrins , gelsolin , and paxillin and the tyrosine phosphatase ptp - pest [ 24 , 8082 ] . pyk2 is expressed at high levels in the nervous system and in various hematopoietic cells [ 57 , 83 ] . pyk2 is expressed in osteoblasts [ 84 , 85 ] and osteoclasts [ 45 , 64 ] . deletion of pyk2 in osteoblasts affects differentiation , migration , and actin remodeling [ 84 , 85 ] . in osteoclasts , pyk2 is localized to the podosome belt and deletion of pyk2 leads to a decrease in osteoclast bone resorption , which contributes to the osteopetrotic phenotype observed in pyk2-deficient mice [ 64 , 84 ] . whereas deletion of pyk2 in osteoblasts affects focal adhesion turnover ( our unpublished findings ) , osteoclasts lacking pyk2 exhibit structurally disorganized podosomes . src has also been shown to be indispensable for osteoclast function and is necessary for podosome assembly / disassembly [ 86 , 87 ] . leupaxin is a member of the focal adhesion - associated adaptor proteins and has been found to be associated with the podosome - belt ( sealing zone ) in osteoclasts [ 89 , 90 ] . it was also demonstrated that leupaxin forms a signaling complex with pyk2 , c - src , and ptp - pest which regulates the migration of prostate cancer cells . finally , as discussed above , the gtpase dynamin regulates podosome assembly and dynamics in osteoclasts [ 44 , 63 , 92 ] in a process that involves src . these studies and others demonstrate that distinct signaling proteins work in concert to regulate podosome organization and turnover in osteoclasts , and perhaps in podosome - containing migratory cells . the tyrosine kinase pyk2 is a homolog of fak and shares 45% overall sequence identity and 60% amino acid identity within the catalytic domain . structurally , pyk2 also contains an n - terminal ferm domain , a central catalytic core , several proline rich domains ( prds ) , and a c - terminal fat domain [ 64 , 79 , 93 ] . the ferm domain is involved in localizing pyk2 to the plasma membrane and facilitates pyk2 binding to phosphatidylinositol bisphosphate ( pip2 ) [ 94 , 95 ] . although structural similarities exist between fak and pyk2 , these proteins appear to exhibit unique effects on adhesion structures in different cells . recently , it was reported that deletion of fak in osteoclasts leads to the formation of peripheral podosome belt , whereas deletion of pyk2 resulted in small podosome rings . in addition , deletion of fak but not pyk2 , in lung carcinoma cl1 - 5 cells resulted in decreased formation of podosomes rosettes . these findings suggest that fak and pyk2 may regulate different patterning of podosome organization in osteoclasts . although the mechanism is unknown , the recruitment of downstream effector proteins is likely to be important in the differential roles of these kinases . invadopodia appear as dynamic protrusions of the plasma membrane , containing a central actin core surrounded by adhesion proteins , signaling molecules , and scaffolding proteins . in addition , invadopodia are sites of ecm degradation and are often observed in highly migratory metastatic cancer cells . invadopodia share , overlapping features with podosomes , especially with regards to their intracellular localization , composition of proteins , and cell types in which they are found [ 55 , 59 , 62 , 9799 ] ( see table 1 ) . in particular , podosomes are short lived ( minutes ) and found in phagocytic cells such as osteoclasts , whereas invadopdia persist for hours and found primarily in cancer cells [ 97 , 100 ] . like podosomes , invadopodia are regulated by a multitude of signaling proteins such as the src - family kinases , protein kinase c [ 55 , 101104 ] , cdc42 , n - wasp , and arp2/3 [ 105 , 106 ] . dynamin has also been shown to participate in focal extracellular matrix degradation by invasive cells . although integrin signaling in the initiation of podosome formation is well established , the role of integrins in invadopodia is not yet clear . the life cycle of invadopodia involves initiation , extension , ecm degradation , and disassembly . each of these steps involves f - actin remodeling and the activation / deactivation of signaling proteins around the central actin core . the initiation of invadopodia is known to be stimulated by egf , pdgf and reactive oxygen species ( ros ) . following initiation by egf receptor activation , src and the tyrosine kinase abl ( abelson ) are recruited and activated [ 102 , 105 , 109 ] . this results in an increase in actin polymerization and cortactin phosphorylation within the elongating invadopodium [ 102 , 105 , 109 ] . microscopic imaging has shown that cortactin accumulates in invadopodia prior to f - actin nucleation , matrix metalloprotease accumulation , and matrix degradation , suggesting that cortactin isan early player in this process . in addition to the filamentous actin network , microtubules and intermediate filaments also participate in the elongation and extension of invadopodia , with the resulting structure resembling the arrangement of actin filaments in podosomes . the growing protrusive membrane is supplied by vesicular trafficking to sites of invadopodia extension and is controlled by the golgi apparatus and by f - bar proteins such as cip4 ( cdc42 interacting protein ) [ 107 , 111 ] and the ena / vasp family protein , mena [ 112114 ] . membrane fusion and actin remodeling by dynamin have also been shown to be involved in invadopodia formation [ 115 , 116 ] . like podosomes , the formation and stabilization of invadopodia involves microtubule - dependent transport [ 108 , 110 ] . the third step in the life cycle of invadopodia , and the major function of these structures , is ecm degradation . ecm degradation is facilitated by secretion of a variety of matrix metalloproteases and serine proteases [ 56 , 112 , 117 , 118 ] and is thought to be regulated by cortactin , an actin regulating protein . the secreted proteases act to degrade components of the ecm , thereby facilitating cellular migration and invasion [ 119 , 120 ] . finally , the disassembly of invadopodia involves depolymerization of the actin core and has been shown to be regulated by erk , paxillin , and the calcium - dependent cysteine protease , calpain , which degrades cortactin [ 121 , 122 ] . the src family kinases have been demonstrated to be critical for invadopodia formation and maturation . however , several lines of evidence support a role for src in focal adhesion and podosome stability [ 123125 ] . similarly , as discussed above , fak is important for focal adhesion turnover but deletion of fak has been also shown to increase invadopodia formation [ 6 , 18 ] and suppress podosomes rosettes formation n fibroblasts . moreover , fak has been shown to regulate a switch from phosphotyrosine - containing proteins at focal adhesions to invadopodia through the temporal regulation of active src . in the same study , it was shown that fak - src signaling also plays a significant role in cancer cell invasion [ 6 , 116 ] . the apparent overlapping role of fak and src in different adhesion structures can be explained by the formation of dynamic proteins complexes between these molecules . for example , the major autophosphorylation site in fak is y397 ( y402 in pyk2 ) which serves as an sh2-binding site , allowing src to bind fak ( or pyk2 ) . the binding of src leads to release of its own autoinhibitory catalytic domain , leading to the full activation of src and to the activation of distinct downstream signaling cascades . in the following section , and summarized in table 2 , we provide an overview of the role of key focal adhesion proteins and their potential link to human diseases . bones provide structural rigidity to the skeleton and are constantly remodeled to maintain calcium and mineral homeostasis and repair skeletal damage . bone architectural integrity relies in part on the rate of apoptosis of bone - forming osteoblasts . the activity of pyk2 is also linked with a variety of metabolic conditions , including the regulation of bone mass . deletion of pyk2 leads to increased bone mass in mice [ 64 , 84 ] due in part to defects in focal adhesion signaling in osteoblasts ( our unpublished findings ) as well as changes in podosome dynamics in osteoclasts [ 14 , 87 ] . deletion of src impairs osteoclast bone resorbing activity and mice lacking src exhibit severe osteopetrosis and exhibit defects in tooth eruption . studies have shown that disruption of the interaction of -actinin with integrins at focal adhesions increases osteoblast apoptosis , which shifts the balance in favor of osteoclast activity , resulting in bone loss . as discussed above , invadopodia formation is tightly linked with cancer metastasis . for example , recently , it was demonstrated that the transcription factor twist1 , a central regulator of the epithelial mesenchymal transition , promotes invadopodia formation through upregulation of platelet - derived growth factor receptor expression and activity , which play significant role in human breast cancer metastasis . invadopodia formation and therefore cancer invasion also involves the adaptor proteins tks4 and tks5 ( tyrosine kinase substrate 4 and 5 ) . it was also found that tks5 colocalizes to invadopodia in different human cancer cells and that decreased tks5 expression leads to decreased podosome formation and to reduced tumor metastasis . thus , tks4 and tks5 could potentially be used as therapeutic targets for the treatment of certain types of cancer . other studies have demonstrated that loss of function of the fgd1 gene , which encodes a gtp - exchange factor , was associated with a rare inherited human developmental disease called faciogenital dysplasia . however , fgd1 was also shown to be involved in invadopodia biogenesis and ecm degradation , and to be expressed in human prostate and breast cancer cells , suggesting it may also be critical for cancer progression and tumorigenesis . several independent studies have demonstrated a critical role for fak in tumor progression and invasion . elevated fak phosphorylation has been observed in several cancers , including breast , colon , thyroid , prostate , oral , neck , and ovarian cancer [ 134 , 135 ] . deletion of fak from tumor cells or breast cancer cells resulted in decreased tumor progression [ 136 , 137 ] , while in endothelial - specific tamoxifen - inducible fak knockout mice , tumor growth and angiogenesis were reduced , indicating that fak may be important for tumorigenesis . in addition , quantitative real - time pcr has shown an elevation of fak expression in malignant gastrointestinal stromal tumors . increased fak expression was also detected in esophageal squamous cell carcinomas and was associated with cell differentiation , tumor invasiveness , and lymph node metastasis . also , it was found that fak was overexpressed in esophageal squamous cell carcinoma which have led to cell differentiation , tumor invasiveness , and lymph node metastasis . in vitro evidence also demonstrates that src - fak signaling is associated with elevated tumor cell metastases , invadopodia formation , and promotes cell invasion [ 141 , 142 ] . the src family of tyrosine kinases are important for embryonic stem cell self - renewal and are key regulators of signal transduction in various cells , including cancer cells [ 143 , 144 ] . collectively , these findings provide strong evidence that overexpression of fak and other proteins localized to invadopodia are important for invadopodia formation and tumor metastasis . although there is a strong correlation between the expression of fak and src in invadopodia and the potential link of these kinases in cancer progression and invasion , it is not yet clear if src - fak signaling specifically in invadopodia is critical role for tumor growth . nevertheless , fak may be a useful biomarker for cancer cell metastasis and inhibitors to fak or src may be useful to limit disease progression . to this end , the fak inhibitor pnd-1186 was found to dramatically decrease fak activity in breast carcinoma cells , resulting in tumor cell apoptosis . lung cancer is considered to be one of the leading causes of mortality among the malignant tumors worldwide . it has been reported that small cell lung cancers ( sclcs ) constitute 1525% of all newly diagnosed primary lung cancers . in the same study , it was shown that inhibition of pyk2 by lentiviral rnai or src using a chemical inhibitor ( pp2 ) reduced sclc survival and proliferation in liquid culture and in soft agar . in addition , it was demonstrated that pyk2 also plays an important role in human non - small cell lung cancer ( nsclc ) . this was based on the detection of higher levels of pyk2 , as determined by western blotting and immunohistochemistry , in nsclc biopsies compared to nontumors . in other studies , fak signaling was shown to be important in the early stages of mammary adenocarcinoma lung metastasis . it was further demonstrated that the dominant - negative fak inhibitor , frnk , blocked lung metastasis if added one day before tumor cell injection , but had no effect if given several days after tumor cell injection . furthermore , it was demonstrated that depletion of fak , but not pyk2 , in lung carcinoma cl1 - 5 cells , decreased the formation of podosome rosette structures and decreased cell invasion . nevertheless , despite strong in vitro and ex vivo evidence linking fak , pyk2 , and src to various cancers , a direct link between kinase activity , effects on podosome / invadopodia formation , and cancer cell metastasis / function is currently lacking . several studies also suggest a link between the adhesion kinases and prostate cancer . for example , it has been shown that metastatic prostate cancer cells express elevated fak mrna levels and protein phosphorylation . more recent studies also suggest that inhibition of pyk2 and fak may be an important therapeutic strategy to decrease prostate cancer progression . sun et al . used a mouse xenograft model injected with a chemical inhibitor of fak and pyk2 ( pf-562,271 ) . after two weeks of treatment with pf-562,271 ( 25 mg / kg ) , the mouse xenograft model showed a 62% decrease in tumor growth , compared to control mice . leupaxin was found to associate with pyk2 , c - src , and ptp - pest . in vitro studies also suggest that the migration of prostate cancer cells ( pc-3 ) may be regulated by protein complexes involving leupaxin , pyk2 , and the tyrosine phosphatase ptp - pest , which dephosphorylates pyk2 [ 91 , 152 ] . furthermore , it was shown that invasion of pc3 cells in a gelatin matrix is controlled by invadopodia and ecm degradation . astrocytomas represent the most common intracranial neoplasms accounting for 60% of all primary brain tumors . in separate studies , fak and pyk2 expressions in addition , a novel kinase inhibitor of fak ( tae226 ) has been shown to increase tumor cell apoptosis in brain tumors . finally , others have demonstrated that administration of src family kinase inhibitors , pp1 and dasatinib , results in a dramatic increase in apoptosis of several pediatric brain tumor cell lines , compared to control cell lines was observed . collectively , the above findings suggest that inhibition of pyk2 and fak and other signaling molecules impair tumor migration by blocking the biogenesis of invadopodia which are important for ecm degradation . pyk2 was identified as a central regulator for angiogenesis of pulmonary vascular endothelial cells . additional studies show that pyk2 is essential in regulating airway inflammation , th2 cytokine secretion , and airway hyper - responsiveness in ovalbumin - sensitized mice during antigen challenge in vivo . inhibition of pyk2 blocked broncho - alveolar lavage , eosinophilia , mucous gland hyperplasia , and airway hyper - responsiveness , conditions that are also characteristic of the asthmatic state in humans . in addition , deletion of pyk2 leads to developmental defects , abnormal macrophage activity , obesity , and insulin resistance under a high - fat diet [ 161 , 162 ] . although the mechanism by which pyk2 regulates these physiological processes is still unknown , therapeutic strategies that target pyk2 might be a novel approach for the treatment of a variety of metabolic and pathological diseases . finally , it has been shown that dynamin mutations are associated with human centronuclear myopathy and charcot - marie - tooth neuropathy [ 164166 ] . however , it is interest to note that dynamin plays an important role in actin remodeling , which is linked to its function in membrane endocytosis [ 115 , 116 ] . therefore , it is possible that dynamin 's role in actin remodeling and adhesion structure turnover [ 43 , 44 , 63 , 92 , 107 ] may also be involved in these pathologies , although this remains to be determined . in summary , focal adhesions , podosomes , and invadopodia facilitate adhesion to the matrix and cellular migration . in addition to adhesion , podosomes and invadopodia have evolved the unique function of ecm degradation . the focal adhesion kinases , fak and pyk2 , exhibit overlapping and unique roles in the biogenesis , stability , and disassembly of these different adhesion structures . there is currently a growing body of evidence linking these and other kinases to the biogenesis of different adhesion structures . in addition , a great deal of studies suggests a link between the expression levels of these kinases and several human diseases , especially cancer ( see table 2 ) . finally , emerging evidence suggests that disrupting the activity of the adhesion kinases not only disrupts the formation of the adhesion structures , but it may also be useful in the treatment of serious medical conditions such as cancer and osteoporosis . a greater understanding of the function of adhesion kinases and the adhesion structures they control will offer future avenues for therapeutic interventions against several human diseases .

in this study , we analyzed 12 species of aquatic heteroptera belonging to five families : belostomatidae ( belostoma anurum and belostoma micantulum ) , gelastocoridae ( gelastocoris angulatus and gelastocoris flavus flavus ) , gerridae ( rheumatobates crassifemur crassifemur ) , notonectidae ( buenoa amnigenus , buenoa unguis , martarega brasiliensis , martarega membranacea , and martarega uruguayensis ) , and veliidae ( rhagovelia tenuipes and rhagovelia zela ) . we analyzed 20 adult males of each species collected in the region of so jos do rio preto city ( 20 4732 s , 49 2137 w ) , sp , brazil . after collection , samples were fixed in methanol : acetic acid ( 3:1 ) and kept at 4c . with the aid of a stereomicroscope leica , germany 16 , the testicles were extracted and analyzed with respect to their morphology , the peritoneal sheath , and the number of lobes . thereafter , the testes were placed on slides , lacerated , and stained with orcena lacto - actica . images were acquired using a microscope and the image analysis program axio vision ( zeiss , germany ) , version 4.8 . for the morphometric comparison of cells in early meiotic prophase , we analyzed a representative of each family : b. micantulum ( belostomatidae ) , g. f. flavus ( gelastocoridae ) , r. c. crassifemur ( gerridae ) , m. brasiliensis ( notonectidae ) , and rha . zela ( veliidae ) . for each species , we analyzed 50 cells using the measure tool in axio vision and 4.8 in m scale . an analysis of variance ( anova ) test was used for statistical analysis using the program minitab 16.1.0 . the degree of reliability was considered to be highly significant at a value of p 0.05 . the 12 species analyzed have testes surrounded by a transparent peritoneal sheath with variable morphology ( round , round / spiral , or elongated / spiral ) . species of the same family were found to be similar to one another , with the exception of the notonectidae family in which the buenoa genera have testes with round / spiral morphology , whereas the martarega genera have elongated / spiral morphology . the testes of the species of the belostomatidae family ( b. anurum and b. micantulum ) are coiled in the distal region of the ejaculatory duct , forming a rounded structure ( fig . 1a ) with five testicular lobes that are individualized in the proximal region of the ejaculatory duct ( fig . in gelastocoridae ( g. angulatus and g. f. flavus ) , the testicles are formed by two elongated lobes that are closely matched in the proximal region of the ejaculatory duct ; in the distal region , they are separated and spiral with approximately five turns ( fig . 1.testes of b. anurum ( a ) and b. micantulum ( b ) ; the distal region ( dr ) is rounded and the proximal region ( pr ) elongated . note the five testicular lobes in ( b ) ; in ( c ) , testes of g. f. flavus consisting of two lobes that are elongated with a spiral distal extremity ; in ( d ) , testes of bu . unguis characterized as rounded and highly spiral ; in ( e ) , testes of m. membranacea formed by two elongated and spiraling lobes ; in ( f ) , testes of r. c. crassifemur ; in ( g ) , testes of rha . tenuipes ; in ( h ) , testes of rha . zela rounded and formed by two ( f ) or one lobe ( g , h ) . testes of b. anurum ( a ) and b. micantulum ( b ) ; the distal region ( dr ) is rounded and the proximal region ( pr ) elongated . note the five testicular lobes in ( b ) ; in ( c ) , testes of g. f. flavus consisting of two lobes that are elongated with a spiral distal extremity ; in ( d ) , testes of bu . unguis characterized as rounded and highly spiral ; in ( e ) , testes of m. membranacea formed by two elongated and spiraling lobes ; in ( f ) , testes of r. c. crassifemur ; in ( g ) , testes of rha . tenuipes ; in ( h ) , testes of rha . zela rounded and formed by two ( f ) or one lobe ( g , h ) . unguis of the notonectidae family also have a rounded morphology , but they are highly spiral ( fig . the testes are elongated and spiral , even though these species are in the notonectidae family ( fig . the number of turns is approximately three , and the testicles are formed of two lobes ( fig . the species of the gerridae family present two testicular lobes , both rounded and separated ( fig . 1f ) , and the species of the veliidae family have a single lobe ( fig table 1.characteristics of the testes and chromosome complement from the 12 species analyzedfamilyspeciestestis morphologylobe morphologyno . of lobes2nbelostomatidaeb . anurumrounded in the distal region and elongated in the proximal regioncoiled in distal region and elongated in proximal region529 ( 26a + x1x2y)b . micantulumrounded in the distal region and elongated in the proximal regioncoiled in distal region and elongated in proximal region516 ( 14a + xy)gelastocoridaeg . angulatuselongated and spiral at the distal extremityelongated and with lobes spiraling216 ( 14 + xy)g . ( 30a + x1x2x3x4y)gerridaer . c. crassifemurroundround221 ( 20a + x0)notonectidaebu . ( 22a + x0 ) characteristics of the testes and chromosome complement from the 12 species analyzed the behavior of cells during meiotic prophase i varies according to species . the cells of r. c. crassifemur do not exhibit heteropycnotic corpuscles ; in some cells , a small and elongated heteropycnotic region near the nucleolar corpuscle can be observed ( fig . in contrast , the cells of b. micantulum have a single large heteropycnotic corpuscle that is heavily stained and rounded ( fig . 2c ) , and the cells of m. brasiliensis have a corpuscle with no defined morphology ( fig . 2d ) . fig . 2.cells of the seminiferous tubules of r. c. crassifemur ( a ) , g. f. flavus ( b , e ) , b. micantulum ( c ) , m. brasiliensis ( d ) , b. anurum ( f ) , and m. uruguayensis ( g ) stained with lacto - acetic orcein . ( a ) prophase i with a small heteropycnotic corpuscle near the nucleolus ( arrow ) ; ( b ) prophase i with several small heteropycnotic corpuscles ; ( c ) prophase i with heteropycnotic corpuscle ( arrow ) ; ( d ) prophase i with a large corpuscle heteropycnotic , rounded with no defined morphology ( arrow ) ; ( e , f ) diplotene / diakinesis with interstitial chiasmata that give the chromosome a cross - shaped morphology ( arrow ) or double terminals chiasmata that give the chromosomes a rounded morphology ( arrowhead ) ; ( g ) diplotene / diakinesis with telomeric associations between the autosomes ( arrow ) . cells of the seminiferous tubules of r. c. crassifemur ( a ) , g. f. flavus ( b , e ) , b. micantulum ( c ) , m. brasiliensis ( d ) , b. anurum ( f ) , and m. uruguayensis ( g ) stained with lacto - acetic orcein . ( a ) prophase i with a small heteropycnotic corpuscle near the nucleolus ( arrow ) ; ( b ) prophase i with several small heteropycnotic corpuscles ; ( c ) prophase i with heteropycnotic corpuscle ( arrow ) ; ( d ) prophase i with a large corpuscle heteropycnotic , rounded with no defined morphology ( arrow ) ; ( e , f ) diplotene / diakinesis with interstitial chiasmata that give the chromosome a cross - shaped morphology ( arrow ) or double terminals chiasmata that give the chromosomes a rounded morphology ( arrowhead ) ; ( g ) diplotene / diakinesis with telomeric associations between the autosomes ( arrow ) . i , it is possible to observe the interstitial chiasmata , which make the chromosomes cross - shaped , or terminals or double terminals chiasmata , which give the chromosomes a rounded morphology , as observed in g. f. flavus and b. anurum ( fig . some species , such as m. uruguayensis , also exhibit telomeric associations between the autosomes ( fig . the diploid chromosome complement was verified with the presence of 2n = 16 ( 14a+xy , b. micantulum and g. angulatus ) , 21 ( 20a+x0 , r. c. crassifemur ) , 23 ( 22a+x0 , rha . tenuipes ) , 25 ( 24a+x0 , bu . amnigenus and bu . unguis ; 22a+2m+x0 , m. membranacea ) , 27 ( 24a+2m+x0 , m. brasiliensis and m. uruguayensis ) , 29 ( 26a+x1x2y , b. anurum ) and 35 ( 30a+x1x2x3x4y , g. f. flavus ) ( fig . 3a 3.cells of the seminiferous tubules in metaphase i stained with lacto - acetic orcein , showing chromosome complements of ( a ) b. anurum , 2n = 29 ( 26a+x1x2y ) ; ( b ) b. micantulum , 2n = 16 ( 14a+xy ) ; ( c ) g. angulatus , 2n = 16 ( 14a+xy ) ; ( d ) g. f. flavus , 2n = 35 ( 30a+x1x2x3x4y ) ; ( e ) r. c. crassifemur , 21 ( 20a+x0 ) ; ( f ) bu . amnigenus , 2n = 25 ( 24a+x0 ) ; ( g ) bu . unguis , 2n = 25 ( 24a+x0 ) ; ( h ) m. brasiliensis , 2n = 27 ( 24a+2m+x0 ) ; ( i ) m. membranacea , 2n = 25 ( 22a+2m+x0 ) ; ( j ) m. uruguayensis , 2n = 27 ( 24a+2m+x0 ) ; ( k ) rha . tenuipes , 2n = 23 ( 22a+x0 ) ; ( l ) rha . zela , 2n = 23 ( 22a+x0 ) . arrows , x chromosomes ; arrowhead , y chromosomes ; and asterisk , m - chromosomes . bars : 10 m . cells of the seminiferous tubules in metaphase i stained with lacto - acetic orcein , showing chromosome complements of ( a ) b. anurum , 2n = 29 ( 26a+x1x2y ) ; ( b ) b. micantulum , 2n = 16 ( 14a+xy ) ; ( c ) g. angulatus , 2n = 16 ( 14a+xy ) ; ( d ) g. f. flavus , 2n = 35 ( 30a+x1x2x3x4y ) ; ( e ) r. c. crassifemur , 21 ( 20a+x0 ) ; ( f ) bu . amnigenus , 2n = 25 ( 24a+x0 ) ; ( g ) bu . unguis , 2n = 25 ( 24a+x0 ) ; ( h ) m. brasiliensis , 2n = 27 ( 24a+2m+x0 ) ; ( i ) m. membranacea , 2n = 25 ( 22a+2m+x0 ) ; ( j ) m. uruguayensis , 2n = 27 ( 24a+2m+x0 ) ; ( k ) rha . arrows , x chromosomes ; arrowhead , y chromosomes ; and asterisk , m - chromosomes . bars : 10 m . at metaphase i , in the polar view , the arrangement of chromosomes is variable , particularly with respect to the sex chromosomes . the location of the sex chromosomes depends on the particular system . with x0 , the x chromosome if the system is x0 with m - chromosomes , in most cases , the autosomes are arranged circularly with the m - chromosomes in the center and the x outside of the ring ( fig . the x is inside the ring formed by autosomes , and the y is located with the autosomes in the ring ( fig . 3b ) . however , with x1x2y , the x and the y chromosomes are inside of the ring ( fig . the x chromosomes are in the center of the ring formed by the autosomes , and the y is included in the ring with the autosomes ( fig . , we observed that the species exhibit regular migration of the chromosomes , with the exception of m. brasiliensis , m. membranacea , and m. uruguayensis . in these species , we observed chromosomes with late migration , both in the first and second division ( fig . i , we could see the m - chromosomes in the center of the ring formed by the autosomes , and the x was outside and migrated late ( fig . 4.cells of the seminiferous tubules of m. membranacea ( a , c ) , m. brasiliensis ( b , d ) , b. anurum ( e ) , b. micantulum ( f j ) , g. f. flavus ( k , n , o ) , and g. angulatus ( l , m , p , q ) stained with lacto - acetic orcein . ( a ) anaphase i / telophase i chromosome with late migration of the x sex chromosome ( arrow ) and the m - chromosomes in the center of the ring ( asterisk ) ; ( b , c ) anaphase ii / telophase ii with late migration of the x chromosome ( arrows ) ; ( d ) telophase ii with the x chromosome only in one of the cells ( arrow ) ; ( e , f ) round spermatids with the heteropycnotic material on one side of the spermatid ( arrows ) ; ( g , h ) spermatids in a teardrop shape ; ( i ) elliptical spermatids with small vesicles ( arrowheads ) ; ( j ) spermatid in final elongation ; ( k m ) round spermatids with heteropycnotic material evident ( arrows ) and a large vesicle ( arrowheads ) ; ( n , o ) elongating spermatids with a large vesicle in the anterior region ( arrowheads ) ; ( p , q ) elliptical spermatids . bars : 10 m . cells of the seminiferous tubules of m. membranacea ( a , c ) , m. brasiliensis ( b , d ) , b. anurum ( e ) , b. micantulum ( f j ) , g. f. flavus ( k , n , o ) , and g. angulatus ( l , m , p , q ) stained with lacto - acetic orcein . ( a ) anaphase i / telophase i chromosome with late migration of the x sex chromosome ( arrow ) and the m - chromosomes in the center of the ring ( asterisk ) ; ( b , c ) anaphase ii / telophase ii with late migration of the x chromosome ( arrows ) ; ( d ) telophase ii with the x chromosome only in one of the cells ( arrow ) ; ( e , f ) round spermatids with the heteropycnotic material on one side of the spermatid ( arrows ) ; ( g , h ) spermatids in a teardrop shape ; ( i ) elliptical spermatids with small vesicles ( arrowheads ) ; ( j ) spermatid in final elongation ; ( k m ) round spermatids with heteropycnotic material evident ( arrows ) and a large vesicle ( arrowheads ) ; ( n , o ) elongating spermatids with a large vesicle in the anterior region ( arrowheads ) ; ( p , q ) elliptical spermatids . bars : 10 m . the behavior of cells during spermiogenesis differs between families ; however , some features are similar , for example , the morphology of early spermatids . h , it is only on one side of the cell , but in gelastocoridae ( fig . m ) , the heteropycnotic material is rounded and heavily stained . in the other families , there are also round spermatids , but the chromatin material is homogeneously distributed and more strongly stained . 5.cells of the seminiferous tubules of r. c. crassifemur ( a g ) , m. uruguayensis ( h j ) , bu . zela ( n , o , p , q , t , v ) , and rha . tenuipes ( r , s , u ) stained with lacto - acetic orcein . ( a ) round spermatids ; ( b ) elongating spermatids with the heteropycnotic material in the posterior region ( arrow ) and a large vesicle in the anterior region ( arrowhead ) ; ( c e ) elongating spermatids with the rod - shaped heteropycnotic material ( arrows ) ; ( f , g ) elongating spermatids ; ( h ) round spermatids ; ( i ) elongating spermatids with the rod - shaped heteropycnotic material ( arrow ) ; ( j ) elongating spermatids with the posterior region of the tail v - shaped ( arrowhead ) ; ( k ) spermatids with an irregular morphology and heteropycnotic material distributed along the periphery ; ( l , m ) elongating spermatids with a small vesicle inside ( arrowheads ) ; ( n ) round spermatids ; ( o q ) round spermatids with sickle - shaped heteropycnotic material ( arrows ) and a small vesicle ( arrowheads ) ; ( r , s ) elongating spermatids with the heteropycnotic material in the anterior region ( arrows ) and a vesicle in the posterior region ( arrowheads ) ; ( t v ) elongating spermatids with rod - shaped heteropycnotic material ( arrows ) . cells of the seminiferous tubules of r. c. crassifemur ( a g ) , m. uruguayensis ( h j ) , bu . zela ( n , o , p , q , t , v ) , and rha . tenuipes ( r , s , u ) stained with lacto - acetic orcein . ( a ) round spermatids ; ( b ) elongating spermatids with the heteropycnotic material in the posterior region ( arrow ) and a large vesicle in the anterior region ( arrowhead ) ; ( c e ) elongating spermatids with the rod - shaped heteropycnotic material ( arrows ) ; ( f , g ) elongating spermatids ; ( h ) round spermatids ; ( i ) elongating spermatids with the rod - shaped heteropycnotic material ( arrow ) ; ( j ) elongating spermatids with the posterior region of the tail v - shaped ( arrowhead ) ; ( k ) spermatids with an irregular morphology and heteropycnotic material distributed along the periphery ; ( l , m ) elongating spermatids with a small vesicle inside ( arrowheads ) ; ( n ) round spermatids ; ( o q ) round spermatids with sickle - shaped heteropycnotic material ( arrows ) and a small vesicle ( arrowheads ) ; ( r , s ) elongating spermatids with the heteropycnotic material in the anterior region ( arrows ) and a vesicle in the posterior region ( arrowheads ) ; ( t v ) elongating spermatids with rod - shaped heteropycnotic material ( arrows ) . i ) and gelastocoridae ( fig . 4p and q ) or a rod shape , as in gerridae ( fig . 5c v ) . between the rounded phases and the rod - shaped or elliptical phases , some families have large vesicles , as in gelastocoridae ( fig . 5b ) , whereas others have small vesicles , as in veliidae ( fig . 5o the heteropycnotic material is located in the posterior region of the cell ( figs . 4 m and 5b ) , whereas in cells with small vesicles , the heteropycnotic material is located in the anterior region . v ) . in species that do not have vesicles ( belostomatidae and notonectidae ) , spermatids are first rounded ( fig . 4e and f ) and later resemble a teardrop ( fig . the spermatids are first rounded ( fig . 5h and k ) posteriorly and then assume a rod shape ( fig . although belostomatidae and notonectidae do not have vesicles at this stage , the elliptical spermatids have two small vesicles ( belostomatidae ) ( fig . 4i ) or a single large vesicle ( notonectidae ) ( fig . 5l and m ) . during the final elongation 4j , p , and q and 5f , g , and v ) , except the species m. uruguayensis , which is v - shaped in the posterior region of the tail ( fig . we analyzed 50 cells in early meiotic prophase of b. micantulum ( belostomatidae ) , g. f. flavus ( gelastocoridae ) , r. c. crassifemur ( gerridae ) , m. brasiliensis ( notonectidae ) and rha . zela have the smallest cells with an average area of 396 and 596 m , respectively ( table 2 ) . g. f. flavus has the highest cell area and also the greatest variability in size ( fig . we observed cells of 2,154.3727,154.04 m with an average of 10,641 m ( table 2 ) . r. c. crassifemur and m. brasiliensis have intermediate cell sizes compared with b. micantulum , g. f. flavus and r. zeal , with an average of 3,800 and 3,651 m , respectively ( table 2 ) . r. c. crassifemur and g. f. flavus also presented very different cell sizes , varying from 1,165.64 to 10,500.48 m . the statistical analysis ( anova test ) performed using the program minitab 16.1.0 showed that b. micantulum and r. zeal have similar cell sizes , as well as r. c. crassifemur and m. brasiliensis no significant differences between these species and between the two groups were observed . g. f. flavus it has the highest area was placed separately the other species , showing a significant difference in the area of cells in prophase i of this species , however , and the differences between the four other species also were significant by statistical analysis ( fig . 6.distribution of cell area ( m ) in prophase i of the five species of heteroptera analyzed . table 2.averages and standard deviations obtained from the morphometric analysis of 50 cells in prophase i of the five species of aquatic heteropteraspeciesnaverage ( m)standard deviationb . zela50596275 distribution of cell area ( m ) in prophase i of the five species of heteroptera analyzed . averages and standard deviations obtained from the morphometric analysis of 50 cells in prophase i of the five species of aquatic heteroptera the 12 species analyzed have testes surrounded by a transparent peritoneal sheath with variable morphology ( round , round / spiral , or elongated / spiral ) . species of the same family were found to be similar to one another , with the exception of the notonectidae family in which the buenoa genera have testes with round / spiral morphology , whereas the martarega genera have elongated / spiral morphology . the testes of the species of the belostomatidae family ( b. anurum and b. micantulum ) are coiled in the distal region of the ejaculatory duct , forming a rounded structure ( fig . 1a ) with five testicular lobes that are individualized in the proximal region of the ejaculatory duct ( fig . in gelastocoridae ( g. angulatus and g. f. flavus ) , the testicles are formed by two elongated lobes that are closely matched in the proximal region of the ejaculatory duct ; in the distal region , they are separated and spiral with approximately five turns ( fig . 1.testes of b. anurum ( a ) and b. micantulum ( b ) ; the distal region ( dr ) is rounded and the proximal region ( pr ) elongated . note the five testicular lobes in ( b ) ; in ( c ) , testes of g. f. flavus consisting of two lobes that are elongated with a spiral distal extremity ; in ( d ) , testes of bu . unguis characterized as rounded and highly spiral ; in ( e ) , testes of m. membranacea formed by two elongated and spiraling lobes ; in ( f ) , testes of r. c. crassifemur ; in ( g ) , testes of rha . tenuipes ; in ( h ) , testes of rha . zela rounded and formed by two ( f ) or one lobe ( g , h ) . testes of b. anurum ( a ) and b. micantulum ( b ) ; the distal region ( dr ) is rounded and the proximal region ( pr ) elongated . note the five testicular lobes in ( b ) ; in ( c ) , testes of g. f. flavus consisting of two lobes that are elongated with a spiral distal extremity ; in ( d ) , testes of bu . unguis characterized as rounded and highly spiral ; in ( e ) , testes of m. membranacea formed by two elongated and spiraling lobes ; in ( f ) , testes of r. c. crassifemur ; in ( g ) , testes of rha . tenuipes ; in ( h ) , testes of rha . zela rounded and formed by two ( f ) or one lobe ( g , h ) . unguis of the notonectidae family also have a rounded morphology , but they are highly spiral ( fig . the testes are elongated and spiral , even though these species are in the notonectidae family ( fig . the number of turns is approximately three , and the testicles are formed of two lobes ( fig . the species of the gerridae family present two testicular lobes , both rounded and separated ( fig . 1f ) , and the species of the veliidae family have a single lobe ( fig table 1.characteristics of the testes and chromosome complement from the 12 species analyzedfamilyspeciestestis morphologylobe morphologyno . of lobes2nbelostomatidaeb . anurumrounded in the distal region and elongated in the proximal regioncoiled in distal region and elongated in proximal region529 ( 26a + x1x2y)b . micantulumrounded in the distal region and elongated in the proximal regioncoiled in distal region and elongated in proximal region516 ( 14a + xy)gelastocoridaeg . angulatuselongated and spiral at the distal extremityelongated and with lobes spiraling216 ( 14 + xy)g . ( 30a + x1x2x3x4y)gerridaer . c. crassifemurroundround221 ( 20a + x0)notonectidaebu . ( 22a + x0 ) characteristics of the testes and chromosome complement from the 12 species analyzed the behavior of cells during meiotic prophase i varies according to species . the cells of r. c. crassifemur do not exhibit heteropycnotic corpuscles ; in some cells , a small and elongated heteropycnotic region near the nucleolar corpuscle can be observed ( fig . in contrast , the cells of b. micantulum have a single large heteropycnotic corpuscle that is heavily stained and rounded ( fig . 2c ) , and the cells of m. brasiliensis have a corpuscle with no defined morphology ( fig . 2d ) . fig . 2.cells of the seminiferous tubules of r. c. crassifemur ( a ) , g. f. flavus ( b , e ) , b. micantulum ( c ) , m. brasiliensis ( d ) , b. anurum ( f ) , and m. uruguayensis ( g ) stained with lacto - acetic orcein . ( a ) prophase i with a small heteropycnotic corpuscle near the nucleolus ( arrow ) ; ( b ) prophase i with several small heteropycnotic corpuscles ; ( c ) prophase i with heteropycnotic corpuscle ( arrow ) ; ( d ) prophase i with a large corpuscle heteropycnotic , rounded with no defined morphology ( arrow ) ; ( e , f ) diplotene / diakinesis with interstitial chiasmata that give the chromosome a cross - shaped morphology ( arrow ) or double terminals chiasmata that give the chromosomes a rounded morphology ( arrowhead ) ; ( g ) diplotene / diakinesis with telomeric associations between the autosomes ( arrow ) . cells of the seminiferous tubules of r. c. crassifemur ( a ) , g. f. flavus ( b , e ) , b. micantulum ( c ) , m. brasiliensis ( d ) , b. anurum ( f ) , and m. uruguayensis ( g ) stained with lacto - acetic orcein . ( a ) prophase i with a small heteropycnotic corpuscle near the nucleolus ( arrow ) ; ( b ) prophase i with several small heteropycnotic corpuscles ; ( c ) prophase i with heteropycnotic corpuscle ( arrow ) ; ( d ) prophase i with a large corpuscle heteropycnotic , rounded with no defined morphology ( arrow ) ; ( e , f ) diplotene / diakinesis with interstitial chiasmata that give the chromosome a cross - shaped morphology ( arrow ) or double terminals chiasmata that give the chromosomes a rounded morphology ( arrowhead ) ; ( g ) diplotene / diakinesis with telomeric associations between the autosomes ( arrow ) . i , it is possible to observe the interstitial chiasmata , which make the chromosomes cross - shaped , or terminals or double terminals chiasmata , which give the chromosomes a rounded morphology , as observed in g. f. flavus and b. anurum ( fig . some species , such as m. uruguayensis , also exhibit telomeric associations between the autosomes ( fig . the diploid chromosome complement was verified with the presence of 2n = 16 ( 14a+xy , b. micantulum and g. angulatus ) , 21 ( 20a+x0 , r. c. crassifemur ) , 23 ( 22a+x0 , rha . tenuipes ) , 25 ( 24a+x0 , bu . amnigenus and bu . unguis ; 22a+2m+x0 , m. membranacea ) , 27 ( 24a+2m+x0 , m. brasiliensis and m. uruguayensis ) , 29 ( 26a+x1x2y , b. anurum ) and 35 ( 30a+x1x2x3x4y , g. f. flavus ) ( fig . 3a 3.cells of the seminiferous tubules in metaphase i stained with lacto - acetic orcein , showing chromosome complements of ( a ) b. anurum , 2n = 29 ( 26a+x1x2y ) ; ( b ) b. micantulum , 2n = 16 ( 14a+xy ) ; ( c ) g. angulatus , 2n = 16 ( 14a+xy ) ; ( d ) g. f. flavus , 2n = 35 ( 30a+x1x2x3x4y ) ; ( e ) r. c. crassifemur , 21 ( 20a+x0 ) ; ( f ) bu . amnigenus , 2n = 25 ( 24a+x0 ) ; ( g ) bu . unguis , 2n = 25 ( 24a+x0 ) ; ( h ) m. brasiliensis , 2n = 27 ( 24a+2m+x0 ) ; ( i ) m. membranacea , 2n = 25 ( 22a+2m+x0 ) ; ( j ) m. uruguayensis , 2n = 27 ( 24a+2m+x0 ) ; ( k ) rha . tenuipes , 2n = 23 ( 22a+x0 ) ; ( l ) rha . zela , 2n = 23 ( 22a+x0 ) . arrows , x chromosomes ; arrowhead , y chromosomes ; and asterisk , m - chromosomes . bars : 10 m . cells of the seminiferous tubules in metaphase i stained with lacto - acetic orcein , showing chromosome complements of ( a ) b. anurum , 2n = 29 ( 26a+x1x2y ) ; ( b ) b. micantulum , 2n = 16 ( 14a+xy ) ; ( c ) g. angulatus , 2n = 16 ( 14a+xy ) ; ( d ) g. f. flavus , 2n = 35 ( 30a+x1x2x3x4y ) ; ( e ) r. c. crassifemur , 21 ( 20a+x0 ) ; ( f ) bu . amnigenus , 2n = 25 ( 24a+x0 ) ; ( g ) bu . unguis , 2n = 25 ( 24a+x0 ) ; ( h ) m. brasiliensis , 2n = 27 ( 24a+2m+x0 ) ; ( i ) m. membranacea , 2n = 25 ( 22a+2m+x0 ) ; ( j ) m. uruguayensis , 2n = 27 ( 24a+2m+x0 ) ; ( k ) rha . arrows , x chromosomes ; arrowhead , y chromosomes ; and asterisk , m - chromosomes . bars : 10 m . at metaphase i , in the polar view , the arrangement of chromosomes is variable , particularly with respect to the sex chromosomes . the location of the sex chromosomes depends on the particular system . with x0 , the x chromosome if the system is x0 with m - chromosomes , in most cases , the autosomes are arranged circularly with the m - chromosomes in the center and the x outside of the ring ( fig . the x is inside the ring formed by autosomes , and the y is located with the autosomes in the ring ( fig . 3b ) . however , with x1x2y , the x and the y chromosomes are inside of the ring ( fig . the x chromosomes are in the center of the ring formed by the autosomes , and the y is included in the ring with the autosomes ( fig . , we observed that the species exhibit regular migration of the chromosomes , with the exception of m. brasiliensis , m. membranacea , and m. uruguayensis . in these species , we observed chromosomes with late migration , both in the first and second division ( fig . i , we could see the m - chromosomes in the center of the ring formed by the autosomes , and the x was outside and migrated late ( fig . 4.cells of the seminiferous tubules of m. membranacea ( a , c ) , m. brasiliensis ( b , d ) , b. anurum ( e ) , b. micantulum ( f j ) , g. f. flavus ( k , n , o ) , and g. angulatus ( l , m , p , q ) stained with lacto - acetic orcein . ( a ) anaphase i / telophase i chromosome with late migration of the x sex chromosome ( arrow ) and the m - chromosomes in the center of the ring ( asterisk ) ; ( b , c ) anaphase ii / telophase ii with late migration of the x chromosome ( arrows ) ; ( d ) telophase ii with the x chromosome only in one of the cells ( arrow ) ; ( e , f ) round spermatids with the heteropycnotic material on one side of the spermatid ( arrows ) ; ( g , h ) spermatids in a teardrop shape ; ( i ) elliptical spermatids with small vesicles ( arrowheads ) ; ( j ) spermatid in final elongation ; ( k m ) round spermatids with heteropycnotic material evident ( arrows ) and a large vesicle ( arrowheads ) ; ( n , o ) elongating spermatids with a large vesicle in the anterior region ( arrowheads ) ; ( p , q ) elliptical spermatids . bars : 10 m . cells of the seminiferous tubules of m. membranacea ( a , c ) , m. brasiliensis ( b , d ) , b. anurum ( e ) , b. micantulum ( f j ) , g. f. flavus ( k , n , o ) , and g. angulatus ( l , m , p , q ) stained with lacto - acetic orcein . ( a ) anaphase i / telophase i chromosome with late migration of the x sex chromosome ( arrow ) and the m - chromosomes in the center of the ring ( asterisk ) ; ( b , c ) anaphase ii / telophase ii with late migration of the x chromosome ( arrows ) ; ( d ) telophase ii with the x chromosome only in one of the cells ( arrow ) ; ( e , f ) round spermatids with the heteropycnotic material on one side of the spermatid ( arrows ) ; ( g , h ) spermatids in a teardrop shape ; ( i ) elliptical spermatids with small vesicles ( arrowheads ) ; ( j ) spermatid in final elongation ; ( k m ) round spermatids with heteropycnotic material evident ( arrows ) and a large vesicle ( arrowheads ) ; ( n , o ) elongating spermatids with a large vesicle in the anterior region ( arrowheads ) ; ( p , q ) elliptical spermatids . bars : 10 m . the behavior of cells during spermiogenesis differs between families ; however , some features are similar , for example , the morphology of early spermatids . h , it is only on one side of the cell , but in gelastocoridae ( fig . m ) , the heteropycnotic material is rounded and heavily stained . in the other families , there are also round spermatids , but the chromatin material is homogeneously distributed and more strongly stained . 5.cells of the seminiferous tubules of r. c. crassifemur ( a g ) , m. uruguayensis ( h j ) , bu . zela ( n , o , p , q , t , v ) , and rha . tenuipes ( r , s , u ) stained with lacto - acetic orcein . ( a ) round spermatids ; ( b ) elongating spermatids with the heteropycnotic material in the posterior region ( arrow ) and a large vesicle in the anterior region ( arrowhead ) ; ( c e ) elongating spermatids with the rod - shaped heteropycnotic material ( arrows ) ; ( f , g ) elongating spermatids ; ( h ) round spermatids ; ( i ) elongating spermatids with the rod - shaped heteropycnotic material ( arrow ) ; ( j ) elongating spermatids with the posterior region of the tail v - shaped ( arrowhead ) ; ( k ) spermatids with an irregular morphology and heteropycnotic material distributed along the periphery ; ( l , m ) elongating spermatids with a small vesicle inside ( arrowheads ) ; ( n ) round spermatids ; ( o q ) round spermatids with sickle - shaped heteropycnotic material ( arrows ) and a small vesicle ( arrowheads ) ; ( r , s ) elongating spermatids with the heteropycnotic material in the anterior region ( arrows ) and a vesicle in the posterior region ( arrowheads ) ; ( t v ) elongating spermatids with rod - shaped heteropycnotic material ( arrows ) . cells of the seminiferous tubules of r. c. crassifemur ( a g ) , m. uruguayensis ( h j ) , bu . zela ( n , o , p , q , t , v ) , and rha . tenuipes ( r , s , u ) stained with lacto - acetic orcein . ( a ) round spermatids ; ( b ) elongating spermatids with the heteropycnotic material in the posterior region ( arrow ) and a large vesicle in the anterior region ( arrowhead ) ; ( c e ) elongating spermatids with the rod - shaped heteropycnotic material ( arrows ) ; ( f , g ) elongating spermatids ; ( h ) round spermatids ; ( i ) elongating spermatids with the rod - shaped heteropycnotic material ( arrow ) ; ( j ) elongating spermatids with the posterior region of the tail v - shaped ( arrowhead ) ; ( k ) spermatids with an irregular morphology and heteropycnotic material distributed along the periphery ; ( l , m ) elongating spermatids with a small vesicle inside ( arrowheads ) ; ( n ) round spermatids ; ( o q ) round spermatids with sickle - shaped heteropycnotic material ( arrows ) and a small vesicle ( arrowheads ) ; ( r , s ) elongating spermatids with the heteropycnotic material in the anterior region ( arrows ) and a vesicle in the posterior region ( arrowheads ) ; ( t v ) elongating spermatids with rod - shaped heteropycnotic material ( arrows ) . i ) and gelastocoridae ( fig . 4p and q ) or a rod shape , as in gerridae ( fig . 5c v ) . between the rounded phases and the rod - shaped or elliptical phases , some families have large vesicles , as in gelastocoridae ( fig . 5b ) , whereas others have small vesicles , as in veliidae ( fig . 5o the heteropycnotic material is located in the posterior region of the cell ( figs . 4 m and 5b ) , whereas in cells with small vesicles , the heteropycnotic material is located in the anterior region . v ) . in species that do not have vesicles ( belostomatidae and notonectidae ) , spermatids are first rounded ( fig . 4e and f ) and later resemble a teardrop ( fig . the spermatids are first rounded ( fig . 5h and k ) posteriorly and then assume a rod shape ( fig . although belostomatidae and notonectidae do not have vesicles at this stage , the elliptical spermatids have two small vesicles ( belostomatidae ) ( fig . 4i ) or a single large vesicle ( notonectidae ) ( fig . 5l and m ) . during the final elongation 4j , p , and q and 5f , g , and v ) , except the species m. uruguayensis , which is v - shaped in the posterior region of the tail ( fig . we analyzed 50 cells in early meiotic prophase of b. micantulum ( belostomatidae ) , g. f. flavus ( gelastocoridae ) , r. c. crassifemur ( gerridae ) , m. brasiliensis ( notonectidae ) and rha . zela have the smallest cells with an average area of 396 and 596 m , respectively ( table 2 ) . g. f. flavus has the highest cell area and also the greatest variability in size ( fig . we observed cells of 2,154.3727,154.04 m with an average of 10,641 m ( table 2 ) . r. c. crassifemur and m. brasiliensis have intermediate cell sizes compared with b. micantulum , g. f. flavus and r. zeal , with an average of 3,800 and 3,651 m , respectively ( table 2 ) . r. c. crassifemur and g. f. flavus also presented very different cell sizes , varying from 1,165.64 to 10,500.48 m . the statistical analysis ( anova test ) performed using the program minitab 16.1.0 showed that b. micantulum and r. zeal have similar cell sizes , as well as r. c. crassifemur and m. brasiliensis no significant differences between these species and between the two groups were observed . g. f. flavus it has the highest area was placed separately the other species , showing a significant difference in the area of cells in prophase i of this species , however , and the differences between the four other species also were significant by statistical analysis ( fig . 6.distribution of cell area ( m ) in prophase i of the five species of heteroptera analyzed . table 2.averages and standard deviations obtained from the morphometric analysis of 50 cells in prophase i of the five species of aquatic heteropteraspeciesnaverage ( m)standard deviationb . zela50596275 distribution of cell area ( m ) in prophase i of the five species of heteroptera analyzed . averages and standard deviations obtained from the morphometric analysis of 50 cells in prophase i of the five species of aquatic heteroptera the 12 species analyzed have testes surrounded by a transparent peritoneal sheath with variable morphology ( round , round / spiral , or elongated / spiral ) . species of the same family were found to be similar to one another , with the exception of the notonectidae family in which the buenoa genera have testes with round / spiral morphology , whereas the martarega genera have elongated / spiral morphology . the testes of the species of the belostomatidae family ( b. anurum and b. micantulum ) are coiled in the distal region of the ejaculatory duct , forming a rounded structure ( fig . 1a ) with five testicular lobes that are individualized in the proximal region of the ejaculatory duct ( fig . in gelastocoridae ( g. angulatus and g. f. flavus ) , the testicles are formed by two elongated lobes that are closely matched in the proximal region of the ejaculatory duct ; in the distal region , they are separated and spiral with approximately five turns ( fig . 1.testes of b. anurum ( a ) and b. micantulum ( b ) ; the distal region ( dr ) is rounded and the proximal region ( pr ) elongated . note the five testicular lobes in ( b ) ; in ( c ) , testes of g. f. flavus consisting of two lobes that are elongated with a spiral distal extremity ; in ( d ) , testes of bu . unguis characterized as rounded and highly spiral ; in ( e ) , testes of m. membranacea formed by two elongated and spiraling lobes ; in ( f ) , testes of r. c. crassifemur ; in ( g ) , testes of rha . tenuipes ; in ( h ) , testes of rha . zela rounded and formed by two ( f ) or one lobe ( g , h ) . testes of b. anurum ( a ) and b. micantulum ( b ) ; the distal region ( dr ) is rounded and the proximal region ( pr ) elongated . note the five testicular lobes in ( b ) ; in ( c ) , testes of g. f. flavus consisting of two lobes that are elongated with a spiral distal extremity ; in ( d ) , testes of bu . unguis characterized as rounded and highly spiral ; in ( e ) , testes of m. membranacea formed by two elongated and spiraling lobes ; in ( f ) , testes of r. c. crassifemur ; in ( g ) , testes of rha . tenuipes ; in ( h ) , testes of rha . zela rounded and formed by two ( f ) or one lobe ( g , h ) . unguis of the notonectidae family also have a rounded morphology , but they are highly spiral ( fig . the testes are elongated and spiral , even though these species are in the notonectidae family ( fig . the number of turns is approximately three , and the testicles are formed of two lobes ( fig . the species of the gerridae family present two testicular lobes , both rounded and separated ( fig . 1f ) , and the species of the veliidae family have a single lobe ( fig table 1.characteristics of the testes and chromosome complement from the 12 species analyzedfamilyspeciestestis morphologylobe morphologyno . of lobes2nbelostomatidaeb . anurumrounded in the distal region and elongated in the proximal regioncoiled in distal region and elongated in proximal region529 ( 26a + x1x2y)b . micantulumrounded in the distal region and elongated in the proximal regioncoiled in distal region and elongated in proximal region516 ( 14a + xy)gelastocoridaeg . angulatuselongated and spiral at the distal extremityelongated and with lobes spiraling216 ( 14 + xy)g . ( 30a + x1x2x3x4y)gerridaer . c. crassifemurroundround221 ( 20a + x0)notonectidaebu . ( 22a + x0 ) characteristics of the testes and chromosome complement from the 12 species analyzed the cells of r. c. crassifemur do not exhibit heteropycnotic corpuscles ; in some cells , a small and elongated heteropycnotic region near the nucleolar corpuscle can be observed ( fig . in contrast , the cells of b. micantulum have a single large heteropycnotic corpuscle that is heavily stained and rounded ( fig . 2c ) , and the cells of m. brasiliensis have a corpuscle with no defined morphology ( fig . 2d ) . 2.cells of the seminiferous tubules of r. c. crassifemur ( a ) , g. f. flavus ( b , e ) , b. micantulum ( c ) , m. brasiliensis ( d ) , b. anurum ( f ) , and m. uruguayensis ( g ) stained with lacto - acetic orcein . ( a ) prophase i with a small heteropycnotic corpuscle near the nucleolus ( arrow ) ; ( b ) prophase i with several small heteropycnotic corpuscles ; ( c ) prophase i with heteropycnotic corpuscle ( arrow ) ; ( d ) prophase i with a large corpuscle heteropycnotic , rounded with no defined morphology ( arrow ) ; ( e , f ) diplotene / diakinesis with interstitial chiasmata that give the chromosome a cross - shaped morphology ( arrow ) or double terminals chiasmata that give the chromosomes a rounded morphology ( arrowhead ) ; ( g ) diplotene / diakinesis with telomeric associations between the autosomes ( arrow ) cells of the seminiferous tubules of r. c. crassifemur ( a ) , g. f. flavus ( b , e ) , b. micantulum ( c ) , m. brasiliensis ( d ) , b. anurum ( f ) , and m. uruguayensis ( g ) stained with lacto - acetic orcein . ( a ) prophase i with a small heteropycnotic corpuscle near the nucleolus ( arrow ) ; ( b ) prophase i with several small heteropycnotic corpuscles ; ( c ) prophase i with heteropycnotic corpuscle ( arrow ) ; ( d ) prophase i with a large corpuscle heteropycnotic , rounded with no defined morphology ( arrow ) ; ( e , f ) diplotene / diakinesis with interstitial chiasmata that give the chromosome a cross - shaped morphology ( arrow ) or double terminals chiasmata that give the chromosomes a rounded morphology ( arrowhead ) ; ( g ) diplotene / diakinesis with telomeric associations between the autosomes ( arrow ) . i , it is possible to observe the interstitial chiasmata , which make the chromosomes cross - shaped , or terminals or double terminals chiasmata , which give the chromosomes a rounded morphology , as observed in g. f. flavus and b. anurum ( fig . some species , such as m. uruguayensis , also exhibit telomeric associations between the autosomes ( fig . the diploid chromosome complement was verified with the presence of 2n = 16 ( 14a+xy , b. micantulum and g. angulatus ) , 21 ( 20a+x0 , r. c. crassifemur ) , 23 ( 22a+x0 , rha . tenuipes ) , 25 ( 24a+x0 , bu . amnigenus and bu . unguis ; 22a+2m+x0 , m. membranacea ) , 27 ( 24a+2m+x0 , m. brasiliensis and m. uruguayensis ) , 29 ( 26a+x1x2y , b. anurum ) and 35 ( 30a+x1x2x3x4y , g. f. flavus ) ( fig . 3.cells of the seminiferous tubules in metaphase i stained with lacto - acetic orcein , showing chromosome complements of ( a ) b. anurum , 2n = 29 ( 26a+x1x2y ) ; ( b ) b. micantulum , 2n = 16 ( 14a+xy ) ; ( c ) g. angulatus , 2n = 16 ( 14a+xy ) ; ( d ) g. f. flavus , 2n = 35 ( 30a+x1x2x3x4y ) ; ( e ) r. c. crassifemur , 21 ( 20a+x0 ) ; ( f ) bu . amnigenus , 2n = 25 ( 24a+x0 ) ; ( g ) bu . unguis , 2n = 25 ( 24a+x0 ) ; ( h ) m. brasiliensis , 2n = 27 ( 24a+2m+x0 ) ; ( i ) m. membranacea , 2n = 25 ( 22a+2m+x0 ) ; ( j ) m. uruguayensis , tenuipes , 2n = 23 ( 22a+x0 ) ; ( l ) rha . zela , 2n = 23 ( 22a+x0 ) . arrows , x chromosomes ; arrowhead , y chromosomes ; and asterisk , m - chromosomes . bars : 10 m . cells of the seminiferous tubules in metaphase i stained with lacto - acetic orcein , showing chromosome complements of ( a ) b. anurum , 2n = 29 ( 26a+x1x2y ) ; ( b ) b. micantulum , 2n = 16 ( 14a+xy ) ; ( c ) g. angulatus , 2n = 16 ( 14a+xy ) ; ( d ) g. f. flavus , 2n = 35 ( 30a+x1x2x3x4y ) ; ( e ) r. c. crassifemur , 21 ( 20a+x0 ) ; ( f ) bu . amnigenus , 2n = 25 ( 24a+x0 ) ; ( g ) bu . unguis , 2n = 25 ( 24a+x0 ) ; ( h ) m. brasiliensis , 2n = 27 ( 24a+2m+x0 ) ; ( i ) m. membranacea , 2n = 25 ( 22a+2m+x0 ) ; ( j ) m. uruguayensis , 2n = 27 ( 24a+2m+x0 ) ; ( k ) rha . zela , 2n = 23 ( 22a+x0 ) . arrows , x chromosomes ; arrowhead , y chromosomes ; and asterisk , m - chromosomes . bars : 10 m . at metaphase i , in the polar view , the arrangement of chromosomes is variable , particularly with respect to the sex chromosomes . the x chromosome is located outside of the ring formed by the autosomes ( fig . 3 g ) . if the system is x0 with m - chromosomes , in most cases , the autosomes are arranged circularly with the m - chromosomes in the center and the x outside of the ring ( fig . the x is inside the ring formed by autosomes , and the y is located with the autosomes in the ring ( fig . 3b ) . however , with x1x2y , the x and the y chromosomes are inside of the ring ( fig . the x chromosomes are in the center of the ring formed by the autosomes , and the y is included in the ring with the autosomes ( fig . , we observed that the species exhibit regular migration of the chromosomes , with the exception of m. brasiliensis , m. membranacea , and m. uruguayensis . in these species , we observed chromosomes with late migration , both in the first and second division ( fig . 4a i , we could see the m - chromosomes in the center of the ring formed by the autosomes , and the x was outside and migrated late ( fig . 4.cells of the seminiferous tubules of m. membranacea ( a , c ) , m. brasiliensis ( b , d ) , b. anurum ( e ) , b. micantulum ( f j ) , g. f. flavus ( k , n , o ) , and g. angulatus ( l , m , p , q ) stained with lacto - acetic orcein . ( a ) anaphase i / telophase i chromosome with late migration of the x sex chromosome ( arrow ) and the m - chromosomes in the center of the ring ( asterisk ) ; ( b , c ) anaphase ii / telophase ii with late migration of the x chromosome ( arrows ) ; ( d ) telophase ii with the x chromosome only in one of the cells ( arrow ) ; ( e , f ) round spermatids with the heteropycnotic material on one side of the spermatid ( arrows ) ; ( g , h ) spermatids in a teardrop shape ; ( i ) elliptical spermatids with small vesicles ( arrowheads ) ; ( j ) spermatid in final elongation ; ( k m ) round spermatids with heteropycnotic material evident ( arrows ) and a large vesicle ( arrowheads ) ; ( n , o ) elongating spermatids with a large vesicle in the anterior region ( arrowheads ) ; ( p , q ) elliptical spermatids . bars : 10 m . cells of the seminiferous tubules of m. membranacea ( a , c ) , m. brasiliensis ( b , d ) , b. anurum ( e ) , b. micantulum ( f j ) , g. f. flavus ( k , n , o ) , and g. angulatus ( l , m , p , q ) stained with lacto - acetic orcein . ( a ) anaphase i / telophase i chromosome with late migration of the x sex chromosome ( arrow ) and the m - chromosomes in the center of the ring ( asterisk ) ; ( b , c ) anaphase ii / telophase ii with late migration of the x chromosome ( arrows ) ; ( d ) telophase ii with the x chromosome only in one of the cells ( arrow ) ; ( e , f ) round spermatids with the heteropycnotic material on one side of the spermatid ( arrows ) ; ( g , h ) spermatids in a teardrop shape ; ( i ) elliptical spermatids with small vesicles ( arrowheads ) ; ( j ) spermatid in final elongation ; ( k m ) round spermatids with heteropycnotic material evident ( arrows ) and a large vesicle ( arrowheads ) ; ( n , o ) elongating spermatids with a large vesicle in the anterior region ( arrowheads ) ; ( p , q ) elliptical spermatids . bars : 10 m . the behavior of cells during spermiogenesis differs between families ; however , some features are similar , for example , the morphology of early spermatids . h , it is only on one side of the cell , but in gelastocoridae ( fig . m ) , the heteropycnotic material is rounded and heavily stained . in the other families , there are also round spermatids , but the chromatin material is homogeneously distributed and more strongly stained . 5.cells of the seminiferous tubules of r. c. crassifemur ( a g ) , m. uruguayensis ( h j ) , bu . zela ( n , o , p , q , t , v ) , and rha . tenuipes ( r , s , u ) stained with lacto - acetic orcein . ( a ) round spermatids ; ( b ) elongating spermatids with the heteropycnotic material in the posterior region ( arrow ) and a large vesicle in the anterior region ( arrowhead ) ; ( c e ) elongating spermatids with the rod - shaped heteropycnotic material ( arrows ) ; ( f , g ) elongating spermatids ; ( h ) round spermatids ; ( i ) elongating spermatids with the rod - shaped heteropycnotic material ( arrow ) ; ( j ) elongating spermatids with the posterior region of the tail v - shaped ( arrowhead ) ; ( k ) spermatids with an irregular morphology and heteropycnotic material distributed along the periphery ; ( l , m ) elongating spermatids with a small vesicle inside ( arrowheads ) ; ( n ) round spermatids ; ( o q ) round spermatids with sickle - shaped heteropycnotic material ( arrows ) and a small vesicle ( arrowheads ) ; ( r , s ) elongating spermatids with the heteropycnotic material in the anterior region ( arrows ) and a vesicle in the posterior region ( arrowheads ) ; ( t v ) elongating spermatids with rod - shaped heteropycnotic material ( arrows ) . bar : 10 m . cells of the seminiferous tubules of r. c. crassifemur ( a g ) , m. uruguayensis ( h j ) , bu . zela ( n , o , p , q , t , v ) , and rha . tenuipes ( r , s , u ) stained with lacto - acetic orcein . ( a ) round spermatids ; ( b ) elongating spermatids with the heteropycnotic material in the posterior region ( arrow ) and a large vesicle in the anterior region ( arrowhead ) ; ( c e ) elongating spermatids with the rod - shaped heteropycnotic material ( arrows ) ; ( f , g ) elongating spermatids ; ( h ) round spermatids ; ( i ) elongating spermatids with the rod - shaped heteropycnotic material ( arrow ) ; ( j ) elongating spermatids with the posterior region of the tail v - shaped ( arrowhead ) ; ( k ) spermatids with an irregular morphology and heteropycnotic material distributed along the periphery ; ( l , m ) elongating spermatids with a small vesicle inside ( arrowheads ) ; ( n ) round spermatids ; ( o q ) round spermatids with sickle - shaped heteropycnotic material ( arrows ) and a small vesicle ( arrowheads ) ; ( r , s ) elongating spermatids with the heteropycnotic material in the anterior region ( arrows ) and a vesicle in the posterior region ( arrowheads ) ; ( t v ) elongating spermatids with rod - shaped heteropycnotic material ( arrows ) . i ) and gelastocoridae ( fig . 4p and q ) or a rod shape , as in gerridae ( fig . 5c v ) . between the rounded phases and the rod - shaped or elliptical phases , some families have large vesicles , as in gelastocoridae ( fig . 4l 5b ) , whereas others have small vesicles , as in veliidae ( fig . 5o s ) . in species with large vesicles , the heteropycnotic material is located in the posterior region of the cell ( figs . 4 m and 5b ) , whereas in cells with small vesicles , the heteropycnotic material is located in the anterior region . v ) . in species that do not have vesicles ( belostomatidae and notonectidae ) , the spermatids are first rounded ( fig . 5h and k ) posteriorly and then assume a rod shape ( fig . although belostomatidae and notonectidae do not have vesicles at this stage , the elliptical spermatids have two small vesicles ( belostomatidae ) ( fig . 4i ) or a single large vesicle ( notonectidae ) ( fig . 5l and m ) . during the final elongation , 4j , p , and q and 5f , g , and v ) , except the species m. uruguayensis , which is v - shaped in the posterior region of the tail ( fig . we analyzed 50 cells in early meiotic prophase of b. micantulum ( belostomatidae ) , g. f. flavus ( gelastocoridae ) , r. c. crassifemur ( gerridae ) , m. brasiliensis ( notonectidae ) and rha . zela have the smallest cells with an average area of 396 and 596 m , respectively ( table 2 ) . g. f. flavus has the highest cell area and also the greatest variability in size ( fig . we observed cells of 2,154.3727,154.04 m with an average of 10,641 m ( table 2 ) . r. c. crassifemur and m. brasiliensis have intermediate cell sizes compared with b. micantulum , g. f. flavus and r. zeal , with an average of 3,800 and 3,651 m , respectively ( table 2 ) . r. c. crassifemur and g. f. flavus also presented very different cell sizes , varying from 1,165.64 to 10,500.48 m . the statistical analysis ( anova test ) performed using the program minitab 16.1.0 showed that b. micantulum and r. zeal have similar cell sizes , as well as r. c. crassifemur and m. brasiliensis no significant differences between these species and between the two groups were observed . g. f. flavus it has the highest area was placed separately the other species , showing a significant difference in the area of cells in prophase i of this species , however , and the differences between the four other species also were significant by statistical analysis ( fig . 6.distribution of cell area ( m ) in prophase i of the five species of heteroptera analyzed . table 2.averages and standard deviations obtained from the morphometric analysis of 50 cells in prophase i of the five species of aquatic heteropteraspeciesnaverage ( m)standard deviationb . zela50596275 distribution of cell area ( m ) in prophase i of the five species of heteroptera analyzed . averages and standard deviations obtained from the morphometric analysis of 50 cells in prophase i of the five species of aquatic heteroptera the pigmentation of the peritoneal sheath that covers the testicles and testicular lobes has not been explored extensively in heteroptera . other species described in the literature have a reddish sheath , as in hyalymenus sp . and neomegalotomus pallescens ( alydidae ) ( souza et al . 2009 ) ; a yellowish sheath , as in zicca annulata ( souza et al . 2007a ) ; or colorless sheath , as in limnogonus aduncus ( gerridae ) ( castanhole et al . considering our results and the few studies in the literature , there appears to be no pattern associated with sheath coloring . within the coreidae family , for example , there are reddish , yellowish , or colorless sheaths . nonetheless , all aquatic heteroptera described in the literature so far have transparent peritoneal sheaths ; thus , it may be common within aquatic heteroptera . common to all heteroptera is that their testicles are formed of lobes that are elongated and always side by side . the number of lobes varies , with some species having two , four , six , or seven lobes . testicles with seven lobes are considered to be the ancestral morphology ( grozeva and kuznetsova 1992 ) . 2008 ) has two testicular lobes ; mormidae v - luteum ( pentatomidae , souza et al . 2008 ) three lobes ; oebalus poecilus , oebalus ypisilongriseus ( pentatomidae , souza et al . 2008 ) , z. annulata , and chariesterus armatus ( coreidae , souza et al . 2007a ) have four lobes ; antitheuchus tripterus ( pentatomidae , souza et al . 2007c ) , anasa bellator , athaumastus haematicus , dallacoris obscura , dallacoris pictus , leptoglossus gonagra , leptoglossus zonatus , and sphictyrtus fasciatus ( coreidae , souza et al . the aquatic species analyzed in this study showed different testis morphology than the land species , that is , rounded in the distal region and elongated in the proximal for the belostomatidae family , elongated and spiral at the distal extremity for gelastocoridae , elongated and spiral or rounded for notonectidae and rounded for veliidae and gerridae and the number of lobes was one , two , or five . in aquatic species , it was more common to observe testis morphology with rounded lobes arranged side by side . these data are important because when related to the phylogeny of the heteroptera , it will be possible to propose an evolutionary hypothesis to explain the differences between terrestrial , aquatic , and semiaquatic species . the heteroptera have holocentric chromosomes ( ueshima 1979 , souza et al . 2007a , costa et al . zygotene , the x chromosome is heteropycnotic and located on the periphery of the nucleus ; in zygotene and pachytene , the chromosomes are tangled . at the end of diakinesis , the species analyzed in this study also exhibited these characteristics , indicating that these behaviors are common for heteroptera . according to the literature , however , the sex chromosome is not always heteropycnotic ; in belostoma dentatum , bivalent autosomes are continually condensed , whereas both sex chromosomes are decondensed and negatively heteropycnotic during diakinesis ( papeschi and bidau 1985 ) . during prophase i , the species analyzed in this study showed interstitial or terminal chiasmata , single or double . this behavior has also been observed in coreidae ( souza et al . the placement of the other chromosomes ( x , y , and m - chromosomes ) varies : if the species has the sex chromosome system x0 , the x is out of the ring , similar to other species of coreidae ( souza et al . 2007a ) ; when the system is x0 with m - chromosomes , most of the time , the x is out of the ring and the m - chromosomes are in the center of the ring , as in species of coreidae ( souza et al . when the system is xy or x1x2x3x4y , the x chromosome is inside the ring and the y is together with the autosomes forming the ring . in contrast , other species such as pentatomidae place the two sex chromosomes inside the ring formed by autosomes ( souza et al . 2008 ) , and in the x1x2y system , the sex chromosomes are also inside the ring . the number of autosomes varies in heteroptera from 4 ( belostomatidae ) to 80 ( miridae ) ; these numbers are not typical for the suborder ( ueshima 1979 ) . it is difficult to say what the ideal chromosome number is for the whole suborder because all eight of the largest infraordens within heteroptera have not been studied in depth cytologically ( grozeva and nokkala 1996 ) . a larger complement of chromosomes is thought to arise from a smaller complement through the duplication or fragmentation of a pair of autosomes ( ueshima and ashlock 1980 ) . according to jacobs ( 2004 ) , because heteroptera have holocentric chromosomes , fragmentation is the most likely origin for an increase in chromosome number as it generates segments that can regularly migrate to the poles during anaphase and persist for many cell generations . chromosome fusion is also theoretically easier in organisms with holocentric chromosomes . in the belostomatidae family , b. micantulum has 2n = 16 and b. anurum has 2n = 29 ; in the gelastocoridae family , g. angulatus is 2n = 16 and g. f. flavus 2n = 35 ; in the notonectidae family , bu . unguis , and m. membranacea have 2n = 25 ; and m. brasiliensis and m. uruguayensis , 2n = 27 . the species with more chromosomes must have originated from species with less through fragmentation of the autosomes and/or sex chromosomes according to the logic mentioned above . because of the lack of a morphologically differentiated centromere and longitudinal chromosome differentiation , it is difficult to detect structural variation . thus , chromosomal rearrangements , such as inversions and reciprocal translocations , are rarely reported in these organisms . a greater number of species should be evaluated with numerous techniques to better understand this group that is so understudied . during the analysis , we observed that the cells in prophase i of aquatic species were much larger than those of terrestrial species described in the literature , thus , we analyzed cells of one representative of each family in prophase i. the species g. f. flavus , which is a semiaquatic species that lives in aquatic or terrestrial environments , showed the greatest difference in cell area . b. micantulum , which is also semiaquatic , presented a cell area in prophase i that was similar to rha . zela , which lives on the water surface ; thus , we can not conclude that the environment determines cell area . r. c. crassifemur , which also lives on the surface , was similar to m. brasiliensis , which lives underwater . the environment probably does not affect cell area , but more research will be necessary to confirm this finding . the underlying reasons for the large differences in cell area between species would be interesting for understanding the evolution of these species and should be the subject of future work .

a 6-year - old girl 102 cm tall and weighing 17 kg , with chronic intestinal pseudo - obstruction was scheduled for multivisceral organ transplantation . four - years earlier , she visited a hospital for abdominal distention and was diagnosed with gastric volvulus and congenital megacolon , for which she underwent gastropexy and segmental resection of the transverse colon . since her symptoms did not improve over the next year , she was referred to our center and underwent a transverse colostomy . after that , she was repeatedly hospitalized and maintained on total parenteral nutrition due to continuously recurring mechanical ileus , malnutrition , and electrolyte imbalance . two months before surgery , she was admitted due to abdominal distension and a malfunctioning colostomy . the colostomy function was normalized 2 days later , and she received conservative medical care , including total parenteral nutrition . the patient was scheduled for multivisceral organ transplantation , including seven abdominal organs , the liver , spleen , stomach , duodenum , small bowel , colon and pancreas . cadaveric donor was 5-year - old female 110 cm tall and weighed 21 kg , who had brain death due to increased intracranial pressure caused by medulloblastoma . histidine - tryptophan - ketoglutarate solution was used as preservation solution and ischemic time was 170 min . her blood pressure was 120/64 mmhg , her heart rate was 120 beats / min , and her oxygen saturation on pulse oxymetry was 100% before induction of anesthesia . pre - operative blood laboratory data were within normal ranges , including hemoglobin ( 9.4 g / dl ) , na ( 142 mmol / l ) , k ( 3.7 mmol / l ) , creatinine ( 0.34 mg / dl ) , aspartate transaminase ( ast ; 43 iu / l ) , total bilirubin ( 0.5 mg / dl ) , albumin ( 4.4 g / dl ) , and prothrombin time ( 1.00 inr ) . anesthesia was induced with 75 mg of pentothal sodium , 15 mg of rocuronium , and 50 g of fentanyl . after tracheal intubation , anesthesia was maintained with 1% sevoflurane in 50% oxygen in air and continuous infusion of fentanyl ( 100 g / h ) and vecuronium ( 2 mg / h ) . a central venous catheter was inserted into her right internal jugular vein and a second into her subclavian vein to monitor central venous pressure and venous oxygen saturation . ten minutes before graft reperfusion , her vital signs and arterial gas analysis were as follows ; mean arterial blood pressure ( map ) was 86 mmhg , heart rate was 90 bpm , central venous pressure was 4 mmhg , femoral venous pressure was 16 mmhg , and body temperature was 36.7 , ph was 7.32 with a base excess of -9.5 meq / l , arterial lactate concentration was 5.6 mmol / l , na was 142 mmol / l , k was 2.5 mmol / l , ca was 0.83 mmol / l , hemoglobin was 10.8 g / dl , and blood glucose was 98 mg / dl . femoral venous pressure decreased after ivc unclamping from 16 mmhg to 4 mmhg and central venous pressure was maintained around 4 mmhg . because the decrease of arterial pressure was too severe to expect spontaneous recovery and met the definition of prs ( 30% decrease in map occurring within 5 minutes after reperfusion and lasting for at least 1 minute ) . she was immediately administered 5 g of epinephrine , resulting in an map of 44 mmhg . however , repeated injections of 10 g epinephrine and volume replacement were required to increase her map due to prolonged hypotension . hypotension was sustained over 10 minutes , during which her averaged arterial pressure was 38 mmhg despite intermittent surge of map > 60 mmhg ( fig . arterial blood gas analysis 5 minutes after reperfusion showed severe metabolic acidosis , with a arterial blood ph of 7.09 with a base excess of -17.9 meq / l , and the arterial lactate concentration was 8.5 mmol / l , k was 4.8 mmol / l , ca was 0.84 mmol / l , glucose was 114 mg / dl , and hemoglobin was 8.5 g / dl . to correct metabolic acidosis and hypotension , she was immediately and repeatedly injected with 30 meq of nahco3 and 10 g of epinephrine . her body temperature had decreased to 33.4 just after reperfusion , with hypothermia sustained for 30 min ( nadir body temperature : 33.0 ) despite active warming with an air warmer . subsequently , continuous infusion of norepinephrine ( 0.1 g / kg / min ) until the end of the operation was required to maintain her map within an acceptable range . at the end of surgery , her body temperature increased to 35.7 , but metabolic acidosis was sustained despite frequent administration of nahco3 ( arterial ph : 7.23 ; base excess : -10.0 the patient was infused with a total of 2,900 ml of fluid , including 1,500 ml of balanced crystalloid solution , 100 ml of half - normal saline , 300 ml of 5% dextrose water , and 1,000 ml of 5% albumin , as well as 2 units of packed red blood cells . after surgery , the ast and alt concentrations of the patient increased rapidly ( immediate postoperative ast 2,108 iu / l and alt 2,351 iu / l ) , reaching 7,300 iu / l and 6,400 iu / l , respectively , on the second day after the operation . she required continuous infusion of fresh frozen plasma due to prolongation of prothrombin time ( 3.07 - 5.69 inr ) . her total bilirubin concentration , which was 0.5 mg / dl preoperatively , increased to 3.4 mg / dl postoperatively . the patient was diagnosed with primary hepatic graft failure and scheduled for re - transplantation of the liver . an adult to child living donor liver transplantation was performed on the third day after multivisceral transplantation . during this operation , the patient 's vital signs remained stable until reperfusion , including a map of 70 - 80 mmhg . ten minutes before reperfusion , her arterial blood ph was 7.49 with a base excess of -0.1 meq / l , arterial lactate concentration was 3.8 mmol / l , hemoglobin was 8.2 g / dl , and blood glucose was 170 mg / dl . after reperfusion she received three injections each of 4 g epinephrine and 20 g of phenylephrine to maintain map within an acceptable level . after transfusion of one unit of packed red blood cells , her vital signs remained stable ( map : 70 - 76 mmhg ) until the end of the operation . at that time , plasma hemoglobin concentration was 11.9 g / dl , ast was 453 iu / l , alt was 695 iu / l , total bilirubin was 3.6 mg / dl , creatinine was 0.6 mg / dl , albumin was 2.9 g / dl , and prothrombin time was 1.74 inr . her serum ast and alt concentrations decreased gradually . on the seventh day after liver transplantation iu / l , respectively , her total bilirubin was 1.4 mg / dl , prothrombin time was 1.07 inr , and feeding through gastrostomy tube was started . the patient was managed conservatively for 3 months including 50 day stay in the intensive care unit and discharged in an improved condition 139 days after multivisceral transplantation . we describe a pediatric patient who experienced severe prs with metabolic acidosis and hypothermia during multivisceral organ transplantation . the hypotensive period lasted for about 10 minutes after the reperfusion . following multivisceral transplantation , this patient experienced hepatic graft failure , requiring re - transplantation of the liver alone on the third postoperative day . since the first descriptions of small bowel and multivisceral abdominal transplantation in 1960 , this procedure has become the standard treatment for children with intestinal failure who experience difficulties with total parenteral nutrition . graft survival rates have increased yearly due to advanced immunosuppression protocols and surgical techniques , with 1-year and 5-year graft survival rates 77.7% and 55.0% , respectively . small bowel transplantation has been recommended for children who can not receive at least half of their expected caloric requirements enterally and who also suffering from growth failure , deterioration of liver function , loss of venous access , and/or recurrent sepsis . diagnoses suggesting the need for small bowel transplantation include volvulus , intestinal atresia , gastroschisis , pseudo - obstruction , hirschsprung 's disease , and trauma . pretransplant work - up , including gastrointestinal contrast studies and liver biopsy , is required to determine whether to transplant small bowel alone or with other organs . graft reperfusion during small bowel transplantation may cause prs , resulting in severe hemodynamic instability and metabolic disturbances . analysis of 27 patients who underwent small bowel transplantation showed decreases in map from 74.6 5.0 mmhg during preenterectomy to 65.8 8.2 mmhg 5 min after reperfusion ( 12% decreased ) ; decreases in systemic vascular resistance index from 1,303 228 dynem / cm during preenterectomy and 980 242 dynem / cm 5 min after reperfusion ( 25% decreased ) ; and decreases in ph from 7.41 0.06 during preenterectomy and 7.38 0.05 5 min after reperfusion ( 0.4% decreased ) . thus , ischemia - reperfusion injury to the small intestine may result in metabolic changes during the post - reperfusion period . in our case , map of the patient decreased from 86 mmhg before reperfusion to 39 mmhg after reperfusion ( 55% decreased ) , whereas ph decreased from 7.32 to 7.09 ( 3% decreased ) . thus , prs in our case was more severe than previously reported cases , although systemic vascular resistance was not measured in our case . unlike in small bowel transplantation and liver transplantation , prs has not been well - documented in multivisceral transplantation . additionally , the definition of prs after simultaneous reperfusion of multi - organ is unclear , thus prevention strategy of prs is also not established in multivisceral transplantation . it is likely , however , that the degree of prs would be similar or greater following multivisceral transplantation because en - bloc grafts contain multiple organs , including the small bowel and/or liver . the patient described here experienced prolonged hypotension , severe metabolic acidosis , hypothermia , and mild hypocalcemia . the period of prs was sustained for over 10 minutes , until severe metabolic acidosis was improved and norepinephrine was continuously infused . however , mild metabolic acidosis still remained , even after repeat injections of nahco3 , and hypothermia was not corrected for over 30 min after reperfusion , despite active warming . several hypotheses can be advanced to explain the prolonged prs and severe metabolic acidosis in this patient . first , the large size of the en - bloc graft , which included several organs , may have caused large amounts of acidic loading and hypothermia . our patient , who weighing only 17 kg , underwent en - bloc transplantation of the liver , spleen , stomach , duodenum , small bowel , colon and pancreas , suggesting that the graft volume was relatively high and may have caused severe acid loading resulting in severe prs . second , poor graft status may worsen the severity of the metabolic acidosis after graft reperfusion . ui / l and clotting factor support for over 2 days ; and primary graft non - function as a requirement for re - transplantation within 7 days . in our patient , iu / l and 6,000 iu / l , respectively , on the second postoperative day . in addition , since prothrombin time extended over 3.0 inr , the patient required continuous transfusion with fresh frozen plasma . although we can not assure that the most important cause of primary non - functioning of hepatic graft after multivisceral transplantation in our case was due to the prolonged severe prs or poor initial graft dysfunction from the cadaveric donor , it is possible to consider that poor initial hepatic graft dysfunction and subsequent severely prolonged prs may further aggravate hepatic graft dysfunction , because the significant associations between low map and negative surgical outcomes , including initial poor graft function , primary graft non - function , or death has been reported . as shown in the present case , severe prs , including hemodynamic instability , hypothermia , and metabolic disturbance , were encountered at the reperfusion of organ grafts in a pediatric patient undergoing multivisceral organ transplantation . meticulous surveillance and immediate management of aggravating factors for prs , such as hypotension , acid - base imbalance , electrolyte imbalance , and hypothermia , may be indicated for managing patients undergoing multivisceral organ transplantation .

myofibroblasts were first described in healing skin wounds , where it was hypothesized that they were responsible for the phenomenon of wound contraction.1 since then , cells morphologically similar to myofibroblasts have been described in many tissues , predominantly in pathological states where their sustained presence is generally a marker of fibrosis and scarring.2 early studies identified myofibroblasts on the basis of their ultrastructural morphology , with prominent microfilament bundles in their cytoplasm distinguishing them from myofibroblasts also possessed fibronexus junctions between cells and the surrounding extracellular matrix ( ecm ) , thus in some ways appearing to share some of the morphological characteristics of smooth muscle ( sm ) cells.3 many tissues and pathologies have been described in which myofibroblasts have been identified , including hypertrophic and keloid scars in the skin , fibrotic liver as seen in liver cirrhosis and other liver pathologies , renal fibrosis , and idiopathic pulmonary fibrosis.4 more recently , cells with phenotypic features of myofibroblasts have also been found in and around a number of epithelial tumors , where they have been termed cancer - associated fibroblasts or stromal myofibroblasts.57 the role of myofibroblasts in driving fibrotic diseases , and the recent finding that cancer - associated myofibroblasts likely influence tumor growth and correlate with poor clinical prognosis , has increased our interest in their cellular origins , their regulation , and their role in repair and disease.8 after early studies that defined myofibroblasts on the basis of their ultrastructural morphology , later research using antibodies and immunohistochemistry resulted in myofibroblasts and their microenvironment being more clearly defined.9 it is now accepted that myofibroblasts go through a precursor stage of expressing large stress fibers that are not seen in quiescent fibroblasts , prominent bundles of microfilaments that permit some contraction and pre - stressing and remodeling of the surrounding ecm.10 later , fully differentiated myofibroblasts show expression of the usually sm - specific cytoskeletal protein , -sm actin , which is now often used to define the myofibroblast phenotype.9,11 the presence of a splice variant form of fibronectin ( ed - a fibronectin ) in the microenvironment adjacent to the myofibroblast is also a defining feature and appears to be required for their differentiation.12 de novo expression of osteoblast ( ob ) cadherin has also been reported to be found on the surface of differentiated myofibroblasts , and is not seen on -sm actin - negative fibroblasts.13 the other defining feature of myofibroblasts is that they fail to express the full repertoire of sm cell markers , allowing myofibroblasts to be distinguished from sm cells . specifically , myofibroblasts in most cases are negative for sm cell markers such as sm myosin heavy chain,14 n - caldesmon,15 and smoothelin.16 desmin has also been used as a negative marker of myofibroblasts . generally , sm cells express desmin and vimentin as well as sm myosin , while myofibroblasts express only vimentin . however , some situations have been reported in the literature where myofibroblasts in some pathologies have been found to be desmin positive.17 distinguishing myofibroblasts from pericytes is perhaps more problematic since pericytes can closely resemble myofibroblasts in being -sm actin positive , vimentin and desmin positive , but sm myosin negative.15,18 indeed , pericytes may in some cases be a source of myofibroblasts in some conditions , including wound repair , where myofibroblasts may represent a pericyte that has lost some phenotypic features such as desmin expression.19 similarly , sm cells from the media of an injured blood vessel may lose late differentiation markers such as desmin , smoothelin , and sm myosin and acquire a myofibroblast phenotype.20 lastly , myofibroblasts show both fibronexus junctions with other cells and specialized junctional complexes with the ecm ; these large mature focal adhesions allow them to make strong attachments , contract and remodel the ecm , and provide a means of transducing mechanical force in the tissue.21,22 the contractile nature of myofibroblasts has some similarities to sm cells , despite the differences in expression of cytoskeletal features . for example , the calcium signaling in myofibroblasts appears to be similar to that in sm cells , and the arrangement of cells into something resembling that of sm cells in tissues with single - unit sm is also similar , that is , with cells having junctional complexes and connections ( including gap junctions ) that allow the spread of contraction signals through the tissue . contraction of myofibroblasts seems to be possible through ca - dependent mechanisms that are similar to those present in sm cells , with increased free ca regulating phosphorylation of myosin light chain . this may explain the rapid contractile responses of myofibroblasts in vitro to agonists such as angiotensin ii or endothelin . slower and more sustained contraction , which is perhaps more typical of what occurs during slow retractile contraction of connective tissue in granulation tissue , involves activity of the guanosine triphosphate ( gtp)-ase rhoa and activation of its downstream target rho - associated kinase ( rock ) . immediately after wounding , the healing process commences , leading to ( partial ) restoration of injured tissue . wound healing proceeds in three interrelated dynamic phases that temporally overlap ( figure 1 ) . based on morphological changes over the course of the healing process , these phases are defined as the inflammatory phase , the proliferative phase ( the development of granulation tissue ) , and the regeneration phase , including maturation , scar formation , and re - epithelialization.24 the inflammatory phase begins with damage of capillaries , triggering the formation of a blood clot consisting of fibrin and fibronectin . this provisional matrix fills in the lesion and allows a variety of recruited cells to migrate into the lesion . platelets present in the blood clot release multiple chemokines , which participate in the recruitment of inflammatory cells , neutrophils , and macrophages , but also in chemotaxis and recruitment of fibroblasts and endothelial cells . angiogenesis , which is critical for the wound healing process , allows new capillaries to deliver nutrients to the wound , and contributes to the proliferation of fibroblasts . initially the wound is hypoxic due to the loss of vascular perfusion , but with the development of a new capillary network , vascular perfusion is restored . regulating wound angiogenesis in itself may represent a means for improving healing in some cases , particularly where delayed or defective angiogenesis is implicated in healing impairment.25 in the granulation tissue , fibroblasts are activated and acquire -sm actin expression and become myofibroblasts . these myofibroblastic cells synthesize and deposit the ecm components that eventually replace the provisional matrix ( figure 2 ) . these cells exhibit contractile properties , due to the expression of -sm actin in microfilament bundles or stress fibers , playing a major role in the contraction and maturation of the granulation tissue.26 presently , it is accepted that the myofibroblastic modulation of fibroblastic cells begins with the appearance of the protomyofibroblast , whose stress fibers contain only - and -cytoplasmic actins . protomyofibroblasts generally evolve into differentiated myofibroblasts , the most common variant of this cell , with stress fibers containing -sm actin ( for review , see tomasek et al27 ) . myofibroblasts can , depending on the experimental or clinical situation , express other sm - related contractile proteins , such as sm myosin heavy chains or desmin ; however , the presence of -sm actin represents the most reliable marker of the myofibroblastic phenotype.27 the third phase of healing , scar formation , involves a progressive remodeling of the granulation tissue . during this remodeling process , proteolytic enzymes , essentially matrix metalloproteinases ( mmps ) and their inhibitors ( tissue inhibitor of metalloproteinases [ timps ] ) play a major role.28 the synthesis of ecm is not totally stopped , but considerably reduced , and the synthesized components are modified as the matrix is remodeled . progressively , collagen type iii , the major component of the granulation tissue , is replaced by collagen type i , which is the main structural component of the dermis . lastly , elastin , which contributes to skin elasticity and is absent in the granulation tissue , also reappears . in the resolution phase of healing , the cell number is dramatically reduced by apoptosis of both vascular cells and myofibroblasts.29 to date , it is not known whether myofibroblasts can reacquire a quiescent phenotype , that is , return to a normal dermal fibroblast phenotype with no expression of -sm actin ( figure 2 ) . it is generally accepted that the major source of myofibroblasts are local connective tissue fibroblasts that are recruited into the wound.30 dermal fibroblasts located at the edges of the wound can acquire a myofibroblastic phenotype and participate in tissue repair.31 however , important heterogeneity in fibroblastic cell subpopulations has also been observed . these subpopulations reside in different locations within the skin and have specific activation and deactivation properties . at least three subpopulations have been identified in the dermis : superficial ( or papillary ) fibroblasts ( papillary dermis is around 300400 m deep and is arranged as a ridge - like structure ) , reticular fibroblasts , which reside in the deep dermis ( made of thick collagen and elastin fibers arranged parallel to the surface of the skin ) , and fibroblasts associated with hair follicles . these cell subpopulations can be isolated and exhibit , depending of the nature and age of the original skin , distinct phenotypic differences when cultured separately.32,33 recently , the involvement in tissue repair of local mesenchymal stem cells has been increasingly raised . these progenitor cells have been described in the dermal sheath that surrounds the outside of the hair follicle facing the epithelial stem cells . they are involved in the regeneration of the dermal papilla and can also became wound healing ( myo)fibroblasts after a lesion or injury.34,35 recent data have also implicated circulating cells , dubbed fibrocytes , in the tissue repair process . fibrocytes enter injured skin together with inflammatory cells and may then acquire a myofibroblastic phenotype.36 in postburn scars , fibrocytes are recruited to the site of the lesion where they stimulate the local inflammatory response and produce ecm proteins , thus contributing to hypertrophic scar formation.37 another type of circulating cell originating from bone marrow has also been suggested to play a role in tissue repair . mesenchymal stem cells are bone marrow - derived non - hematopoietic precursor cells38 that contribute to the maintenance and regeneration of connective tissues through engraftment.39 indeed , they have the capacity to seed into several organs and to differentiate into wound - healing myofibroblasts . this engraftment in injured organs is regulated by the severity of the damage.40 finally , differentiated ( or malignant ) epithelial and endothelial cells can undergo a phenotypic conversion that gives rise to matrix - producing fibroblasts and myofibroblasts ( through epithelial- and endothelial - to - mesenchymal transition processes).41 this mechanism is increasingly recognized as an integral part of tissue fibrogenesis after injury , but seems to play a limited role during normal tissue repair . overall , mesenchymal stem cells , fibrocytes , bone marrow - derived cells , and cells derived from epithelial - and endothelial - to - mesenchymal transition processes may represent alternative sources of myofibroblasts when local fibroblasts are not sufficient to carry out tissue repair and remodeling . myofibroblasts are implicated in many fibrotic and scarring diseases , where they carry out the important process after initial injury of providing mechanical support and integrity to the tissue . in normal physiological conditions , they are then lost via apoptosis , generally when the tissue integrity has been sufficiently restored to be mechanically coherent.9,29 thus , in normal physiological situations like skin wound healing , myofibroblasts disappear in a prominent wave of apoptosis , leaving a markedly less cellular scar . however , it is now assumed that , in many fibrotic and scarring conditions , as well as in the stromal response to tumors , myofibroblasts fail to undergo cell death , persist , and thus in turn lead to ongoing pathology and scarring ( figure 3 ) . an example of reduced or inhibited apoptosis leading to scarring is in a model of hypertrophic scarring , where mechanical loading increases survival of myofibroblasts and was found to lead to greater scar formation.42,43 a better understanding of the control and signaling that governs apoptosis ( or autophagy ) in myofibroblasts may lead to more targeted approaches to combatting fibrosis and scarring . it has been demonstrated that elevated nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase 4-derived hydrogen peroxide , supported by concomitant decreases in nitric oxide signaling and reactive oxygen species scavengers , are central factors in the molecular pathogenesis of fibrosis.44 redox signaling could therefore represent an interesting target to restore the physiological fibroblast myofibroblast ratio . apoptosis in myofibroblasts is thought to be regulated by a reduction in the local growth factors that drive and sustain myofibroblast differentiation . in particular , local concentrations of transforming growth factor ( tgf)-1 and endothelin-1 play a role in stimulating myofibroblast survival via protein kinase b ( akt ) activation.45 however , changes in mechanical signaling such as unloading of mechanical force likely also plays a role ( discussed below ) . the importance of myofibroblasts in causing fibrosis in internal organs and the skin ( hypertrophic scars ) , and the role that persistence of stromal myofibroblasts appear to play in tumor growth and spread , makes the ( down)regulation of myofibroblasts and the potential regulation of myofibroblast disappearance through apoptosis of increasing interest ( figure 4 ) ( for review , see hinz and gabbiani46 ) . mechanical signals have been shown to play a role in myofibroblast differentiation as the ecm that surrounds the fibroblasts in damaged tissue changes its composition and its stiffness as tissue repair proceeds.47 the early ecm present in damaged tissue , or provisional matrix , is rich in fibrin and has been estimated to be very compliant . fibroblasts cultured in compliant ecm such as soft three - dimensional ( 3d ) collagen gels , show little development of stress fibers . these fibroblasts then form only small adhesions with the ecm.48,49 fibroblasts grown in stiffer collagen matrices have been shown to form stress fibers and mature focal adhesions , though they are still negative for the myofibroblast marker -sm actin . lastly , the stiff matrix found either in 3d cultures using stiff ( higher concentration ) collagen matrix or in vivo in granulation tissue and fibrotic tissues is able to induce full myofibroblast differentiation in concert with growth factor stimulation from tgf-1.22 the contractile nature of myofibroblasts itself leads to an increase in stiffness and mechanical stress of the ecm as healing progresses , leading to a positive - feedback loop where increased stress signals myofibroblast differentiation and also increases myofibroblast survival.42 for this reason , mechanical feedback is considered to be important in driving pathological conditions such as contractures post - injury . the role of mechanical stress in stimulating myofibroblast activity has also been shown in experiments where dermal wounds in mice are mechanically stressed by stretching or splinting the wound , where increased myofibroblast activity results in increased scar formation , to some extent mimicking hypertrophic scarring that is seen in humans.42 in cancer biology , matrix stiffness can be used as a diagnostic indicator of the risk of malignancy and appears to correlate with increased invasiveness of tumors , for example in breast cancer where density of tissue on mammography correlates strongly with the risk of cancer formation . recent studies have suggested this may be due to increased cell proliferation of epithelial and mesenchymal cells on stiffer matrices . conversely , releasing mechanical stress or reducing stiffness has been shown to induce both apoptosis and a reduction in -sm actin expression and contractility in myofibroblasts.50,51 mechanical signaling and stress modulate myofibroblast differentiation via a number of pathways and mechanisms . stress may directly activate transcription of the -sm actin gene , since application of force across integrin binding sites has been shown to up - regulate -sm actin promoter activity.52 as mentioned above , mechanical force alone is not generally sufficient to induce myofibroblast differentiation and other factors are needed to act in concert , specifically tgf-1 . mechanical signaling and tgf-1 stimulation also increase collagen gene expression by fibroblasts , emphasizing the role these factors play in stimulating a pro - fibrotic phenotype as is shown by activated myofibroblasts . tgf-1 also favors deposition of ecm proteins over degradation by up - regulating timps while decreasing expression of the mmps themselves.53 stimulation of myofibroblasts by tgf-1 itself is affected by mechanical forces within the damaged or fibrotic tissue . tgf-1 released from a variety of inflammatory cells and platelets in the microenvironment of damaged or fibrotic tissue is in a latent form . indeed , myofibroblasts themselves release latent tgf-1 complexed with latency - associated peptide ( lap ) . together with a binding protein , tgf-1 is bound to ecm proteins , providing a reservoir of latent tgf-1 that can be activated as healing and scar formation progress.54,55 myofibroblasts express integrins that can bind to the lap , and mechanical stress applied to the integrins , either by mechanical stress on the matrix and/or via myofibroblast contraction , can effectively activate tgf-1 without cleaving the lap and allow its binding to cell membrane receptors.51 thus , both increased mechanical stress and contraction can further increase myofibroblast contractile and matrix synthetic activity . this mode of activation provides another possible pathway for regulating myofibroblast activity by blocking integrin binding to latent tgf-1 , for example by blocking the integrin involved in latent tgf-1 activation , v5.56 inhibition of other integrin - binding sites has also been shown to inhibit myofibroblast development , including blocking of integrins 31,57 111,58 v5,59 or 1.60 tissue oxygenation or hypoxia may play a role in both normal healing and pathological situations . in normal wound healing , staining for the hypoxia - induced transcription factor , hypoxia inducible factor ( hif)-1 shows both areas of the early granulation tissue and the overlying migrating keratinocytes to be hypoxic . during normal healing , this hypoxia hypoxia signaling can induce a number of growth factors that are beneficial to the healing process , prominent amongst them being vascular endothelial growth factor ( vegf ) and , thus , acute hypoxia likely plays a beneficial role in healing . however , the same may not be true for chronic hypoxia and chronic hypoxia signaling . hypoxia has been reported to reduce myofibroblast activation and reduce collagen synthesis and -sm actin expression.61 in vivo studies using hif-1-deficient mice showed that reducing hif-1 availability during healing resulted in reduced collagen synthesis and delayed myofibroblast differentiation , suggesting that , overall , in vivo acute hypoxia during healing was normally compensated by induction of genes that allow tissue to adapt to transient hypoxia , such as vegf.62 in fact , fibroblasts that show reduced hif-1 expression during hypoxia show inhibited migration and collagen synthesis in vitro . it is possible that , in some organs where there is pathological fibrosis and scarring , hypoxia and hif signaling , possibly during more chronic hypoxic states , actually drives fibrosis , which has been suggested in the case of renal fibrosis . in some cells at least , there is cross talk between hypoxia signaling and tgf- signaling that may exacerbate matrix synthesis and thus fibrosis.63,64 anti - fibrotic and anti - scarring therapies have proven to be a difficult and elusive area for research , with relatively few advances until quite recently . as the growth factor tgf- is central to many of the mechanisms of pathological scarring and fibrosis , it has been the target of some therapeutic strategies . some positive results have been reported with the drug pirfenidone , particularly in lung fibrosis , where the drug lowered tgf- expression and both tissue and lavage fluid levels of the protein.65 interfering with activation of latent tgf- is another potential target for anti - scarring therapies , and the role of integrin binding in tgf- activation makes integrin - blocking antibodies a potential therapy for lowering tgf- activation and thus downstream signaling.66,67 specific inhibitors of tgf- signaling have also been suggested as possible treatments for scarring and fibrosis . tgf- exerts its pro - fibrotic effects through transcription factor signaling smad3 , and selective inhibition of smad3 phosphorylation and inhibition of smad3 interaction with smad4 has been shown to reduce fibroblast activation to the myofibroblast phenotype and also reduce ecm synthetic activity of the cells.68 other molecular targets include tyrosine kinases , and the drug imatinib mesylate has been reported to be anti - fibrotic through inhibiting downstream molecules that are required for the tgf- response while having an additional anti - fibrotic role by also inhibiting platelet - derived growth factor signaling.69 interestingly , hmg - coa reductase inhibitors such as statins have been shown to have anti - fibrotic effects , likely through inhibition of rock.70 the widespread use of , and low rate of side effects associated with , these drugs may make them promising as anti - fibrotic therapies in the future . since their first description in the early 1970s , our knowledge about myofibroblast biology has increased greatly , and our interest in the biology of myofibroblasts has also increased , as this cell has been implicated in many pathological situations in addition to their role in normal wound repair . despite major advances in our understanding of the origins of myofibroblasts and the factors that regulate their differentiation and activity , it remains a challenge and a major goal of researchers to find ways in which to regulate their activity to improve healing and scarring in the clinic . it is densely innervated by different sensory nerve fiber subtypes that react to tissue injury , temperature variation , and tactile stimuli ( figure 5 ) . cutaneous sensory nerve fibers are endings of dorsal root ganglia ( or spinal ganglia ) neurons that carry signals from sensory organs toward the appropriate integration center of the brain or of the spinal cord . several clinical observations indicate that damage to the peripheral nervous system influences wound healing , sometimes resulting in chronic wounds within the affected area . patients with cutaneous sensory defects due to spinal cord injury or diabetic neuropathy have an increased risk of developing ulcers that fail to heal . in addition , in aged patients , cutaneous repair processes are less efficient and this could be partly due to a deterioration of the peripheral nervous system at the skin level . interestingly , factors that are required for sustaining peripheral nerves , the neurotrophin network , have also been shown to have direct effects on dermal fibroblasts in regulating ecm secretion , fibroblast differentiation , and tensile strength via effects on myofibroblasts.71 understanding the role of innervation during wound healing and myofibroblastic differentiation therefore represents an interesting domain . in addition , cutaneous innervation is certainly necessary to provide good hemostasis and to maintain the mechanical and cosmetic properties of the skin.72 in conclusion , the recent advances made in understanding control of differentiation , proliferation , and survival of myofibroblasts will hopefully lead to new therapeutic approaches to limit scarring and improve healing in the not - too - distant future.73

fibrosing colonopathy ( fc ) is a rare entity associated with cystic fibrosis ( cf ) . until now , case report on an adult with cf who developed persistent abdominal pain due to a non - passable stricture in the right transverse colon . initially we treated the patient with prednisolone pulse therapy and additive antibiotic therapy . for maintenance therapy we administered budesonide . the patient underwent clinical , laboratory and endoscopic follow - up over a three - year period . the stricture healed and was easy to pass . a relapse in the cecum at the ileocecal valve again improved under steroid and antibiotic therapy . we present a novel therapeutic approach for advanced stricturing fc in an adult patient which successfully avoided surgery ( right hemicolectomy ) over a three year follow up . a 25-year - old man suffering from pre - existing cystic fibrosis was admitted with persistent pain in the right lower - quadrant . he reported a history of meconium ileus at birth , appendectomy and recurrent distal intestinal obstruction syndrome ( dios ) . he was substituted with high - strength pancreatic enzymes ( pancreatin 150.000 units / d ) . during adolescence he took pancreatin 10.000 units for fat - rich nutrition only . on his first presentation in july 2007 , the patient reported about a 20-month history of recurrent pain in the right lower abdominal quadrant which now had increased dramatically . on admission he appeared in good physical condition with a stable body weight ( bml 22.2 kg / m ) and physical examination revealed focal tenderness in the right - lower quadrant and normal bowel sounds . laboratory values showed an elevated c - reactive protein ( 10.7 mg / dl ) and a slight anemia ( hemoglobin : 10.6 g / dl ) . transabdominal ultrasound demonstrated hypoechoic bowel wall thickness of the transverse colon ( 10 mm ) and a 30 mm long stricture in the right transverse colon ( figure 1 ) . the bowel wall of the ascending colon and cecum was mildly thickened , while no abnormalities could be observed in the terminal ileum . colonoscopy was incomplete because of a nonpassable circular stricture in the right transverse colon ( figure 1 ) . distal of the stricture the colonic mucosa appeared hyperemic , edematous and ulcerated for about 20 cm . the histopathologic examination of the endoscopic biopsies showed ulcerative inflammation , cryptitis , irregular hyperplasia of goblet cells , submucosal fibrosis and disruption of the muscularis mucosa consistent with the diagnosis of fibrosing colonopathy ( figure 1 ) . after the patient had given his informed consent with regard to the experimental character of the therapy we initiated intravenous prednisolone pulse therapy ( 75 mg / d ) for 10 days followed by a slow dose tapering ( 10 mg / week to 25 mg , than 5 mg / week ) over 10 weeks . in addition , we administered intravenous antibiotic therapy ( mezlocillin 6 g / d , metronidazole 1 g / d ) . furthermore , the stricture had healed with some minor scars remaining and could now be passed allowing a view on the ascending colon and cecum . here we found multiple pseudopolyps and a lack of haustration while the terminal ileum appeared normal . half a year later the patient presented without any abdominal pain and increasing body weight . he had further decreased the dosage of budesonide to 6 mg / d by himself . however , in january 2009 , 18 months after his first admission , the patient once more presented with crampy pain in the right lower abdominal quadrant . while the region of the former stricture in the right transverse colon was unremarkable , we now found areas of ulcerative inflammation in the cecum and at the ileocecal valve which could not be passed due to inflammatory stenosis . as during the first episode we treated the patient with prednisolone pulse therapy and antibiotics with again good clinical response and administered budesonide 9 mg / d for maintenance therapy . in july 2009 , colonoscopy demonstrated that the stricture in the right transverse colon was now healed and easy to pass . in the cecum multiple the ileocecal valve was constricted over a very short distance and nearly passable with the colonoscope . finally after more than three years , in march 2011 the patient presented in an excellent physical condition with stable weight under maintenance therapy . it has generally been reported in young children approximately 12 months after exposure to high dose pancreas enzyme supplementation ( hdpe ) . predisposing factors are young age ( 2 - 13 years ) , history of gastrointestinal complications ( dios , meconium ileus ) , previous intestinal surgery , hdpe and use of histamine h2-receptor blockers , corticosteroids or recombinant human deoxyribonuclease ( dnase ) . to our knowledge , only five cases of fc in adults were reported [ 3 - 7 ] : three patients with cf , one developed fc after hdpe in pancreatic insufficiency and one suspicious of crohn 's disease and treated with mesalazine . interestingly , development of fc in these five adults was not clearly associated with hdpe : two patients developed fc under treatment with hdpe , one patient after discontinuation of hdpe and two without hdpe at any time . all of these patients were symptomatic due to strictures and the diagnosis of fc was discovered during surgery . clinical symptoms are usually those of intestinal obstruction with abdominal pain , constipation or diarrhea and hematochezia . abnormal bowel wall thickness is a common finding in cf , but it is usually not associated with clinical features and is no predisposing factor for fc . endoscopy may demonstrate a friable , hyperemic , edematous , stiff or ulcerated mucosa often with strictures , colonic shortening and loss of haustration . fc usually spares the rectum and may be either segmental , mostly in the right colon , or involves the complete colon . characteristic histologic features include submucosal fibrosis , disruption of the muscularis mucosa , ulcerative inflammation , eosinophilia and cryptitis [ 10 - 13 ] . in contrast , in ulcerative colitis common features are an acute and chronic inflammatory cell infiltrate , distortion of crypts , crypt abscesses and goblet cell depletion . the differentiation to stricturing crohn 's disease can be challenging in the absence of characteristical epitheloid granuloma . to our knowledge this is the first report of a nonpassable colonic stricture in fc which was successfully treated without surgical intervention . additional antibiotic therapy aimed at treating bacterial translocation due to barrier dysfunction of the intestinal mucosa . in order to avoid side effects of longterm systemic steroids treatment was continued with topical active budesonide . however , maintenance therapy with budesonide did not protect the patient from a second stenosis at the cecum . if clinical symptoms from this stenosis should accelerate in the future , balloon dilatation of the ileocecal valve or a limited surgical intervention might be the treatment of choice . however , until now we avoided the risk of long - term immunosuppression due to the fear of bronchopulmonary infections in cf . body weight ( bmi 25 kg / m ) and physical condition improved under therapy with budesonide . interestingly , lung function improved remarkably under therapy demonstrating an increase of forced expiratory volume in one second ( fev1 ) from 67% to 93% which may point to a relationship between the inflammatory status of the bowel and pulmonary affection in cf . according to the current guidelines we recommended a pancreatic enzyme supplementation on demand which should not exceed 10.000 units of lipase per kg per day . in conclusion , this case demonstrates a non - passable colonic stricture in fc which was discovered endoscopically and was successfully treated conservatively over a three - year period . this suggests a new therapeutic approach in cf patients with stricturing fc .

neuroferritinopathy is an autosomal dominant neurodegenerative disorder characterized by the deposition of iron and ferritin in the brain and a decreased level of serum ferritin . seven different pathogenic mutations of the ferritin light chain gene have been identified [ 17 ] . these mutations are predicted to affect the tertiary structure and stability of the ferritin light chain polypeptide and may cause inappropriate iron release from ferritin polymers [ 8 , 9 ] . it is supposed that the excess iron induces free toxic radical production , which leads to tissue oxidative stress and neuronal cell death [ 1012 ] . the clinical features of neuroferritinopathy are characterized by the adult onset of extrapyramidal motor symptoms : dystonia , chorea , choreoathetosis , parkinsonism , and tremor . some patients may present cerebellar ataxia , cognitive decline , and pyramidal signs [ 2 , 3 , 57 ] . the phenotypic signs of the disease are variable , even among members of the same family [ 1 , 3 ] . generally , there are no nonneurological symptoms , different from in other neurodegenerative brain iron accumulation diseases . the clinical features of neuroferritinopathy are not specific , and they overlap with those of common extrapyramidal disorders . brain mr imaging in the disease is quite characteristic and it may facilitate differential diagnosis of neuroferritinopathy from other extrapyramidal disorders . we will review the findings in neuroferritinopathy with conventional mri methods , t1-weighted imaging , t2-weighted imaging , and t2 * -weighted imaging . on t1wi , there is a sharp contrast between the parenchyma and ventricles , and it is adequate for evaluating brain atrophy and cystic changes . t2wi is suitable for detecting the pathological processes with an increase in water content , such as gliosis , edema and axonal / neuronal loss , as hyperintense signals . on t2*wi with a gradient echo sequence , signal abnormalities on brain mr imaging were observed in all affected individuals previously reported except for one case [ 1315 ] . despite the clinical differences , the findings are usually bilateral and symmetric but sometimes asymmetric [ 3 , 17 ] . radiological findings in patients with neuroferritinopathy have been shown to correlate with the observed pathology . the abnormalities observed on mri reflect four pathological changes : iron deposition , edema and gliosis , cystic changes , and cortical atrophy [ 13 ] . iron is essential for normal neuronal metabolism , but excessive iron may be harmful [ 19 , 20 ] . it is known that iron overload can cause free - radical formation and neuronal damage . physiologically , brain iron appears to be found predominantly in the extrapyramidal system , in particular the globus pallidus , substantia nigra , red nucleus , and putamen . it has been shown that moderate levels of iron occur in the striatum , thalamus , cerebral cortex , cerebellar cortex , and deep white matter . the brain histopathology of affected individuals with neuroferritinopathy involves excess iron and ferritin deposits throughout the forebrain and cerebellum , notably in the basal ganglia [ 13 ] . however , these regions still exhibit the general distribution pattern for iron in the normal aging brain . on fast spin echo t2wi , iron deposits are demonstrated as low - intensity areas and as signal loss on gradient echo t2*wi [ 13 , 22 ] . comparison of t2wi and t2*wi sequences suggests that the t2 * one is more sensitive for the detection of iron , while the t2 fast spin echo t2wi sequence is more frequently used in routine clinical practice . in particular , the cortical iron deposition in neuroferritinopathy is hardly detectable on t2wi but is easily observed on t2*wi . t2 hyperintense abnormalities are seen in the pallidum , putamen , caudate nucleus [ 1 , 3 ] , thalamus and dentate nucleus , and sometimes in the red nucleus and substantia nigra [ 16 , 24 ] in patients with neuroferritinopathy . the border of a lesion has a tendency to be unclear and the signal is unequal . because of the increased water content , the lesions are detected as hyperintense signals on t2wi . around these hyperintense areas , cavities are demonstrated as low - intensity signals on t1wi and high - intensity signals on t2wi , compared with the csf signal . in the region adjacent to a cystic lesion , severe loss of nerve cells and neuropil is observed pathologically . in one case , vidal et al . reported that microcavities measuring up to 1.5 mm in diameter were seen in the putamen anatomically and that these cavities were consistent with small hypointense areas on t1wi and to hyperintense ones on t2wi on mri . mcneill et al . analyzed the mri findings in 21 patients with neuroferritinopathy . in 52% ( 11/21 patients ) , they found that the globus pallidus and/or putamen coincided with a confluent area of hyperintensity and that this hyperintense area was likely to be due to fluid within an area of cystic degeneration . the presence of large cysts is thought to be a finding observed at an advanced stage . on brain mri in neuroferritinopathy , atrophy is sometimes noted in the cerebellar cortices and cerebral cortices , notably in the frontal lobe . regarding on clinicoradiologic correlation , patients having cerebellar atrophy present ataxia [ 2 , 3 , 26 ] , and ones having cerebral atrophy present cognitive decline [ 23 , 26 ] . the first mri change is loss of the t2 * signal due to iron deposits . in an early symptomatic stage , and even in an asymptomatic carrier , there is obvious signal loss on t2 * imaging in the basal ganglia , especially in the globus pallidus , at considerable frequency . in conventional spin echo mr sequences , . there has only been one report of that brain mr t2wi was normal without evidence of iron deposition ; however , it was obtained six years after the onset of neuroferritinopathy symptoms . in this case , the t2 * sequence was not examined at that time . the follow - up mri performed 16 years after the onset , however , showed typical abnormalities . with disease progression , the t2 hypointense signal and t2 * signal loss become more pronounced . the changes eventually extend to the thalamus , dentate nucleus , substantia nigra , red nucleus , and cerebral cortex . in the middle stage of the disorder , t2 hyperintense abnormalities reflecting tissue edema and gliosis are observed . in the basal ganglia , this change is thought to represent precystic degeneration . the combination of hyperintense and hypointense abnormalities is found in the pallidum , putamen , thalamus , and dentate nucleus frequently and sometimes in the red nucleus and substantia nigra [ 17 , 27 ] . the characteristic finding on brain mri at the advanced stage is symmetrical cystic degeneration of the basal ganglia [ 16 , 28 ] . pathologically , many microcavities due to the loss of neurophils and neurons are observed , which are consistent with hypointense areas on t1wi and with hyperintense ones on t2wi on mri . it is supposed that small cavities merge to form larger cavities with progression of the disease . brain mr images of our case are presented in figures 1 , 2 , 3 , and 4 . our patient is a 42-year - old japanese man who first developed hand tremor in his middle teens . he noticed his right foot dragging at age 35 , and generalized hypotonia , hyperextensibility , aphonia , micrographia , hyperreflexia , dystonia of his face , and cognitive impairment at age 42 . he was tested by means of the molecular technique and diagnosed as having neuroferritinopathy because a mutation of the ferritin light chain gene was detected . all images presented here were taken with a 1.5 tesla mr system . t1-weighted , t2-weighted , and t2 * -weighted sequences were collected in the transverse plane . a t1-weighted image is useful for evaluating the atrophy and size of a cyst . as compared with the image at 35 years ( figure 1 ) , that at 42 years demonstrates progression of the cystic formation and deterioration of the cortical atrophy in the frontal lobes ( figures 2(c ) and 3(c ) ) . a t2-weighted image is valuable for detecting the combination of degenerative change and iron accumulation . a clear hyperintense lesion with a hypointense signal was found in the center of the dentate nucleus ( figure 3(a ) ) . foggy high signal changes were found in the inner part of the thalamus bilaterally ( figure 3(c ) ) . iron deposits were indicated as signal loss in the dentate nuclei ( figure 4(a ) ) , red nuclei ( figure 4(b ) ) , thalamus ( figure 4(c ) ) , at the periphery of the cysts ( figure 4(c ) ) , and in the cerebral cortex ( figure 4(e ) ) in t2 * -weighted images . in this section , we provide an overview of the mri findings in three other subtypes of neurodegeneration with brain iron accumulation ( nbia ) : pantothenate kinase-2 associated neurodegeneration ( pkan , formerly known as hallervorden - spatz syndrome ) , aceruloplasminemia , and infantile neuroaxonal dystrophy ( inad ) for the differential diagnosis of iron deposition in the basal ganglia . over the last decade , iron deposition in the adult brain is being increasingly recognized as an indicator of neurodegenerative processes in many chronic neurologic disorders including parkinson disease and alzheimer disease . pkan is a childhood - onset extrapyramidal disorder with aberrant iron metabolism caused by a mutation of the pantothenate kinase-2 ( pank2 ) gene . brian mri findings in patients with the pank2 mutation include hypointensity with an area of central hyperintensity in the globus pallidi on t2- and t2 * -imaging , this characteristic sign being called the eye - of - the - tiger sign [ 14 , 3032 ] . mcneill et al . reported that two of 21 cases of neuroferritinopathy presented the eye - of - the - tiger he emphasized the importance of repeat imaging for a more accurate clinical diagnosis . in the majority of pkan cases , abnormalities are restricted to the globus pallidus and substantia nigra . in neuroferritinopathy , lesions in the globus pallidus , putamen , and dentate nuclei are consistently accompanied by ones in the caudate nuclei or thalami in a subset . the widespread location of lesions throughout the central nervous system is one of the characteristic mr findings in neuroferritinopathy patients . aceruloplasminemia is an adult onset extrapyramidal disorder with iron deposition in the brain , liver , and reticuloendothelial system . the iron deposition in the central nervous system in aceruloplasminemia exhibits a distribution comparable to that in neuroferritinopathy , but in aceruloplasminemia , all basal ganglia nuclei and thalami are simultaneously involved as seen in t2-weighted and t2 * -weighted images . a further distinguishing feature is the lack of the combination of hyperintense and hypointense abnormalities that is often observed in neuroferritinopathy . the cystic changes of the basal ganglia observed in neuroferritinopathy are rarely seen in aceruloplasminemia [ 13 , 14 , 33 ] . inad is an autosomal recessive disorder with motor and mental deterioration , appearing within the first two years of life . the characteristic mri finding in inad patients is cerebeller atrophy , which is often accompanied by signal hyperintensity in the diffuse cerebellar cortex . in inad , abnormal iron accumulation , detected as hypointense lesions on t2wi and t2*wi , is mainly observed in the globus pallidus , sometimes in the substantia nigra , and occasionally in the dentate nuclei . even in advanced cases of inad , there has been no report of iron accumulation in other structures . it is different from the frequent involvement of the putamen , caudate and thalami in neuroferritinopathy . it has been observed in two inad families with pla2g6 mutations that no iron accumulation was detectable on mri despite severe clinical symptoms . patients with parkinson disease ( pd ) may show t2 hypointensity in many anatomic areas compared to normal controls including the substantia nigra pars compacta , dentate nucleus , subthalamic nucleus , and basal ganglia , probably reflecting an excess iron content . quantitative studies have shown a 25% to 100% increase in substantia nigra iron levels in patients with pd compared to in normal controls . the correlation between t2 hypointensity of the substantia nigra and clinical severity has been demonstrated . in patients with alzheimer disease ( ad ) , iron deposition in neurons , neurofibrillary tangles , and plaques has been reported pathologically . to detect brain iron accumulation using mri in ad , investigators tried the high - solution 4.7 t mri or field - dependent - rate - increase ( fdri ) technique . in these studies , increased iron levels were found in the basal ganglia . the signal change on mri reflecting an excess iron content observed in patients with chronic neurologic disorders such as pd and ad is usually slight as compared with that in patients with nbia including neuroferritinopathy . for exact measurement of brain iron or mineralization as a major risk factor for neurodegenerative diseases , multimodal and advanced mri techniques the variety of mri findings including cystic degeneration of the basal ganglia , the combination of hyperintense and hypointense abnormalities , t2 hypointense lesions reflecting iron deposits , and cortical atrophy are specific to neuroferritinopathy . in cases of suspected neuroferritinopathy , mri may be useful for the detection and confirmation of such findings . at an early stage , since the abnormal iron deposits might be not detectable on t2-weighted imaging , t2 * -weighted imaging is recommended . in most cases , there are clear distinguishing features for neuroferritinopathy and other iron accumulative disorders , including pkan , inad , and aceruloplasminemia . however , there is a degree of radiological overlap between neuroferritinopathy and these other iron accumulative disorders . the multimodal and advanced mri techniques being developed to more sensitively and specifically quantify brain iron will be important for correct diagnosis and better understanding of the neurodegenerative processes in the pathological brain .

the mouse hypothalamic cell line n29/4 was cultured as previously described ( 24 ) . min6 cells ( obtained with permission from jun - ichi miyazaki , osaka university ) were used between passages 24 and 30 and grown in dulbecco 's modified eagle 's medium containing 25 mm glucose and supplemented with 12.5% heat - inactivated fetal calf serum , 4 mm l - glutamine , 100 m 2-mercaptoethanol , and 1% penicillin and streptomycin in a humidified atmosphere of 95% air and 5% co2 . wild - type c57bl/6 mice ( 2025 g ) were used for islet isolation and killed by cervical dislocation immediately before the islet isolation procedure . all animal procedures conformed to the uk animals ( scientific procedures ) act 1986 and were approved by our institutional ethical review committee . pancreatic islets were isolated from mice under sterile conditions , and isolated cells were cultured as described previously ( 12 ) . n29/4 cells were plated in 6-well plates at 40% confluence 24 h before transfection . 5 l of lipofectamine ( invitrogen ) and 2 g of pten dna were mixed with 100 l of serum - free opti medium , incubated at room temperature for 20 min to form the transfection complexes , and diluted with 500 l of culture medium . the mixture was applied to the cells , left overnight at 37 c , replaced with culture medium , and incubated for 48 h before use . in experiments where wild - type and mutant pten constructs were overexpressed , f - actin or ptdins(3,4,5)p3 levels were determined from 60 cells for control and each treatment from a total of 4 experiments ( comparing transfected cells , as determined by pten antibody staining , with control cells ) . similarly , 72 cells were examined from 4 experiments to measure f - actin in control and leptin with or without ct99021 and/or 2-dimethyl - amino-4,5,6,7-tetrabromo-1h - benzimidazole ( dmat ) . cells were treated with dmat ( calbiochem ) and/or ct99021 ( 25 ) in normal saline ( 135 mm nacl , 5 mm kcl , 1 mm mgcl2 , 1 mm cacl2 , 10 mm hepes , and 0.2% bsa , ph 7.4 ) for 20 min before challenge with leptin or saline in the continuous presence of inhibitor for 60 min before fixing as described previously ( 13 ) . for staining , cells were washed in phosphate - buffered saline ( pbs ) , permeabilized in pbs , 0.5% triton x-100 for 10 min , rinsed , blocked with 20% goat serum ( sigma ) , and incubated with rhodamine - conjugated phalloidin or 2 units ml alexa 488-phalloidin ( molecular probes ) for 90120 min . immunocytochemical staining was performed as described previously ( 13 ) using a monoclonal antibody against pten ( chemicon ) or ptdins(3,4,5)p3 ( molecular probes ) . the secondary antibodies were cy3 or fluorescein isothiocyanate - conjugated anti - mouse igg ( jackson immunoresearch ) . fluorescence - labeled proteins or lipids in cells were observed with a 63 oil objective , and images were acquired by confocal microscopy ( zeiss lsm 510 ) . for quantitative analysis of f - actin and ptdins(3,4,5)p3 levels , aida / two - dimensional densitometry was used to measure pixel intensity of defined areas of transfected , treated , and control cells . after subtraction of background , intensity values for treated cells were expressed as a ratio of control cell intensity . n29/4 and min6 cells , grown in 6-well plates , were exposed to normal saline for 30 min before treatment with leptin , insulin , or saline in the presence and absence of ly294002 ( sigma ) , dmat , or ct99021 for the required time ( inhibitors added 2030 min before challenge ) . protein isolation , content , immunoblotting , and analysis procedures were as previously described ( 13 ) . pten and phospho - pten antibodies , ser-380/thr-382/thr-383 were from cell signaling ( 1:5000 ) , ser-385 was from biosource ( 1:500 ) , and ser-370 and thr-366 were from nick leslie ( 1:500 ) . phosphorylated ( p)-pkb ( ser-473 ) and p - gsk3 ( ser-9/ser-21 ) were obtained from cell signaling and used at 1:1000 . p - gsk3 ( tyr-216/tyr-279 ) , pkb , and gsk3 antibodies ( all polyclonal ) were obtained from bd transduction laboratories and used at 1:2500 ( p - gsk3 ) and 1:1000 ( pkb and gsk3 ) . protein bands on gels were quantified by densitometry , where total density was determined with respect to constant area , background was subtracted , and average relative band density was calculated . immunoprecipitation and assay of gsk3cell extracts ( 0.5 mg ) were incubated with protein g - sepharose conjugated to 2.5 mg of anti - gsk3/ antibody , upstate biotechnology , inc . ( lake placid , ny ) for 1 h at 4 c on a shaking platform . the immunocomplexes were pelleted and washed with 1 ml of lysis buffer containing 0.5 m nacl and twice with 1 ml of assay buffer ( 50 mm tris - hcl , ph 7.5 , 150 mm nacl , 0.03% ( v / v ) brij-35 , and 0.1% ( v / v ) -mercaptoethanol ) . the immunoprecipitated gsk3 was incubated at 30 c for 20 min in a total volume of 50 ml containing 50 mm tris - hcl , ph 7.5 , 100 mm nacl , 0.03% ( v / v ) brij-35 , 0.1% ( v / v ) -mercaptoethanol , 10 mm mgcl2 , 0.1 mm [ -p]atp ( 0.5 10 cpm / nmol ) , and 30 mm phosphoglycogen synthase 2 peptide ( available from upstate ) . radiolabeled peptide produced was separated from unreacted atp by binding to p81 paper and washing in 75 mm h3po4 before counting in 2 ml of scintillant . one unit of kinase activity is defined as the amount that catalyzes the phosphorylation of 1 nmol of substrate in 1 h. figure 1.inhibitors of ck2 and gsk3 reduce leptin - mediated phosphorylation of pten in n29/4 cells . the sequence of amino acids for part of the c - terminal tail domain is shown , with the main phosphorylation sites denoted in bold , and the putative phosphorylating kinase shown below . b , phosphorylated pten levels in n29/4 cells using the phospho - specific pten antibodies indicated under non - stimulated conditions ( c ) , in response to 10 nm leptin alone ( l ) , and in the presence of 10 m dmat ( l+d ) , 2 m ct99021 ( l+ct ) , and 10 m ly294002 ( l+ly ) . c , the bar graphs show mean normalized levels of phosphorylated pten at ser-385 , ser-370 , and thr-366 under non - stimulated conditions ( c ) and for leptin in the absence ( l ) and presence of dmat ( l+d ) and ct99021 ( l+ct ; n = 6 for each ) . d , bar graphs showing mean normalized levels of phosphorylated pten at thr-366 and ser-370 under non - stimulated conditions ( c ) and after stimulation of n29/4 cells for 5 or 30 min with 10 nm insulin . inhibitors of ck2 and gsk3 reduce leptin - mediated phosphorylation of pten in n29/4 cells . a , schematic representation of pten . the sequence of amino acids for part of the c - terminal tail domain is shown , with the main phosphorylation sites denoted in bold , and the putative phosphorylating kinase shown below . b , phosphorylated pten levels in n29/4 cells using the phospho - specific pten antibodies indicated under non - stimulated conditions ( c ) , in response to 10 nm leptin alone ( l ) , and in the presence of 10 m dmat ( l+d ) , 2 m ct99021 ( l+ct ) , and 10 m ly294002 ( l+ly ) . c , the bar graphs show mean normalized levels of phosphorylated pten at ser-385 , ser-370 , and thr-366 under non - stimulated conditions ( c ) and for leptin in the absence ( l ) and presence of dmat ( l+d ) and ct99021 ( l+ct ; n = 6 for each ) . d , bar graphs showing mean normalized levels of phosphorylated pten at thr-366 and ser-370 under non - stimulated conditions ( c ) and after stimulation of n29/4 cells for 5 or 30 min with 10 nm insulin . mouse cultured -cells were superfused at room temperature ( 2225 c ) with normal saline . wholecell current and voltage clamp recordings were used to monitor membrane potential and macroscopic current , respectively , using an axopatch 200b amplifier . whole - cell experiments were maintained in current - clamp mode to monitor the cell resting membrane potential , with short excursions into voltage clamp mode to examine macroscopic current - voltage relations . in voltage - clamp recordings the membrane potential was held at -70- and 20-mv steps of 2 s duration , with 20 ms between pulses applied ( range of voltages , -160 to -40 mv ) . whole cell voltage clamp data were analyzed using pclamp 8.2 software ( molecular devices ) . current clamp data were recorded and stored onto digital audio tapes and replayed for analysis and illustration on a gould ta240 chart recorder . recording electrodes were pulled from borosilicate glass and had resistances of 510 megaohms when filled with pipette solution , which comprised 140 mm kcl , 5 mm mgcl2 , 3.8 mm cacl2 , 3 mm mgatp , 10 mm egta , 10 mm hepes , ph 7.2 ( free [ ca ] of 100 nm ) . application of drugs was made via the bath perfusion system except for dmat and the gsk3 inhibitors , which were present in the pipette solution . statistical analysis all statistical analyses were performed using analysis of variance , one sample t test , student 's paired or unpaired t tests . we used the leptin- and insulin - sensitive hypothalamic cell line n29/4 ( 12 , 24 ) to examine pten phosphorylation at thr-366 , ser-370 , and ser-385 ( fig . 1a ) on stimulation with leptin ( 10 nm ) or insulin ( 10 nm ) . leptin rapidly ( < 5 min ) increased the level of p - pten at all three sites ( fig . 1b ) , an action sustained for at least 60 min for each site ( data not shown ) . this leptin - mediated increase in p - pten was unaffected by the presence of the pi3k inhibitor , ly294002 ( 10 m ; n = 6 for each ; fig . previous studies indicated that the cluster site , ser-385 and ser-370 , may be phosphorylated by protein kinase ck2 ( 17 , 18 , 23 ) , and thr-366 may be phosphorylated by gsk3 ( 23 ) . consequently , we examined leptin phosphorylation of thr-366 , ser-370 , and ser-385 pten residues in the absence and presence of dmat ( a ck2 inhibitor ( 26 , 27 ) and ct99021 ( a specific gsk3 inhibitor ( 28 , 29 ) ) . dmat ( 10 m ) prevented leptin from phosphorylating ser-370 and ser-385 but not thr-366 in n29/4 cells ( fig . 1 , b and c ) . conversely , leptin phosphorylation of thr-366 was prevented by ct99021 ( 2 m ) , whereas phosphorylation of ser-370 and ser-385 was unaffected by this inhibitor ( fig . 1 , b and c ) . 1d ) or 50 nm ( data not shown ) insulin had no effect on p - pten levels in n29/4 cells at thr-366 or ser-370 residues , with the ser-385 site not tested . leptin re - organization of the actin cytoskeleton is dependent on pi3k activity and inhibition of the lipid and protein phosphatase activity of pten . overexpression of cluster site p - pten mutants demonstrated that phosphorylation at this site was required to limit pten phosphatase activity and prevent leptin - induced f - actin depolymerization ( 12 ) . accordingly , we have examined whether phosphorylation of pten at ser-370 and/or thr-366 is also required for transmission of the leptin signal to f - actin . first , we sought to determine whether phosphorylation of thr-366 or ser-370 altered the activity of pten in cells by overexpression of pten mutants with alanine or aspartic acid substitutions . ptdins(3,4,5)p3 and f - actin levels were assessed by staining with a monoclonal ptdins(3,4,5)p3 antibody and alexa 488-conjugated phalloidin respectively , after transfection of pten mutants into n29/4 cells . in control experiments overexpression of wild - type pten had no effect per se but prevented leptin from decreasing f - actin ( fig . overexpression of t366a pten or s370a pten reduced ptdins(3,4,5)p3 levels ( data not shown ) , indicative of enhanced ptdins(3,4,5)p3 phosphatase activity , did not affect f - actin in the absence of leptin and inhibited leptin - induced f - actin reduction ( fig . , overexpression of t366d pten or s370d pten increased ptdins(3,4,5)p3 levels ( fig . 2c ) . the presence of the 366d pten mutant in n29/4 cells decreased f - actin levels in the absence of leptin in a pi3k - dependent manner ( fig . surprisingly , overexpression of s370d pten , although increasing ptdins(3,4,5)p3 , did not per se alter f - actin staining in the absence of leptin nor occlude the effect of leptin on f - actin ( fig . this action of s370d pten indicates , as observed previously ( 12 ) , that increasing cellular ptdins(3,4,5)p3 levels alone is not a predictor or indeed sufficient to initiate pten - dependent downstream signaling events such as f - actin depolymerization . figure 2.effect of pten phosphorylation mutants on ptdins(3,4,5)p3 and f - actin levels in n29/4 cells . wild - type pten and t366a pten ( a ) and s370a pten ( b ) were overexpressed in n29/4 cells ( upper panels , cy3 secondary antibody on cells treated with monoclonal pten antibody ) and f - actin visualized with alexa 488-phalloidin ( lower panels ) with and without stimulation by 10 nm leptin . note that untransfected cells in the leptin ( l)-treated group are not easily visible in the right lower panels due to leptin - mediated f - actin depolymerization . bar graphs showing mean levels of f - actin after treatment of n29/4 cells overexpression of pten or t366a pten ( a ) or s370a pten ( b ) with 10 nm leptin . c , ptdins(3,4,5)p3 levels in n29/4 cells transfected with t366d pten or s370d pten were investigated using a ptdins(3,4,5)p3 antibody . the bar graph shows mean levels of ptdins(3,4,5)p3 ( as a ratio of control ) in n29/4 cells after treatment with these pten phosphorylation mutants . d and e , f - actin levels were visualized ( lower panels ) in n29/4 cells transfected ( upper panels ) with t366d pten ( d ) or s370d pten ( e ) in the absence ( left ) and presence ( right ) of 10 m ly294002 ( ly , d ) or 10 nm leptin ( e ) . the bar graphs show mean levels of f - actin after treatment of n29/4 cells overexpressing t366d pten ( d ) or s370d pten ( e ) . f , cellular f - actin levels in the absence or presence of 10 nm leptin and in cells pretreated with 2 m ct99021 alone ( ct ) or in combination with 10 m dmat ( d ) before leptin challenge . note that f - actin is visualized in this series of experiments with rhodamine - conjugated phalloidin . values represent the mean s.e . for 34 experiments per group . , p < 0.05 compared with the appropriate labeled control for each treatment . effect of pten phosphorylation mutants on ptdins(3,4,5)p3 and f - actin levels in n29/4 cells . wild - type pten and t366a pten ( a ) and s370a pten ( b ) were overexpressed in n29/4 cells ( upper panels , cy3 secondary antibody on cells treated with monoclonal pten antibody ) and f - actin visualized with alexa 488-phalloidin ( lower panels ) with and without stimulation by 10 nm leptin . note that untransfected cells in the leptin ( l)-treated group are not easily visible in the right lower panels due to leptin - mediated f - actin depolymerization . bar graphs showing mean levels of f - actin after treatment of n29/4 cells overexpression of pten or t366a pten ( a ) or s370a pten ( b ) with 10 nm leptin . c , ptdins(3,4,5)p3 levels in n29/4 cells transfected with t366d pten or s370d pten were investigated using a ptdins(3,4,5)p3 antibody . the bar graph shows mean levels of ptdins(3,4,5)p3 ( as a ratio of control ) in n29/4 cells after treatment with these pten phosphorylation mutants . d and e , f - actin levels were visualized ( lower panels ) in n29/4 cells transfected ( upper panels ) with t366d pten ( d ) or s370d pten ( e ) in the absence ( left ) and presence ( right ) of 10 m ly294002 ( ly , d ) or 10 nm leptin ( e ) . the bar graphs show mean levels of f - actin after treatment of n29/4 cells overexpressing t366d pten ( d ) or s370d pten ( e ) . f , cellular f - actin levels in the absence or presence of 10 nm leptin and in cells pretreated with 2 m ct99021 alone ( ct ) or in combination with 10 m dmat ( d ) before leptin challenge . note that f - actin is visualized in this series of experiments with rhodamine - conjugated phalloidin . pip3 , ptdins(3,4,5)p3 . the dependence of f - actin depolymerization by leptin on the phosphorylation status of the cluster ( 12 ) and thr-366 sites led us to explore whether this action of leptin was sensitive to ck2 and/or gsk3 inhibition . the presence of the gsk3 inhibitor , ct99021 ( 2 m ) or the ck2 inhibitor dmat ( 10 m ) alone had no effect on f - actin ( data not shown ) . leptin decreased the mean f - actin level , and in the presence of ct99021 this was significantly reduced ( fig . the combined presence of ct99021 and dmat inhibited f - actin depolymerization by leptin significantly more than ct99021 alone but did not completely occlude leptin from affecting f - actin levels ( fig . nevertheless , the presence of the gsk3 and ck2 inhibitors at concentrations that significantly reduced pten phosphorylation also significantly reduced the ability of leptin to depolymerize f - actin . phosphorylation of pten in -cells and katp activation it was previously demonstrated that katp channel opening and hyperpolarization of mouse pancreatic -cells by leptin could be inhibited by the lipid phosphatase dead g129e pten mutant and mimicked by the dual lipid and protein phosphatase dead c124s pten mutant ( 12 ) . this is consistent with inhibition of pten lipid and protein phosphatase activity for the successful transmission of the leptin signal to katp channels in -cells . as leptin has not been shown to alter p - pten status in -cells , we used min6 -cells to explore this action of leptin . although the effects of leptin on p - pten were not as robust as observed for n29/4 cells , leptin ( 10 nm ) rapidly ( 1 min ) increased phosphorylation of the cluster site , ser-370 and thr-366 , actions sustained for at least 30 min in each case ( fig . 3 , a and b ) . insulin ( 10 nm ) had no effect on pten phosphorylation levels at any of these sites over this time period ( fig . 3 , a c ) . the ck2 inhibitor dmat ( 2 m ) inhibited leptin - mediated pten phosphorylation at ser-370 and thr-366 , whereas the gsk3 inhibitor ct99021 ( 10 m ) inhibited phosphorylation at thr-366 only ( fig . consequently , we predicted that the f - actin reduction and opening of katp channels by leptin ( 12 , 13 ) would be susceptible to inhibition of ck2 and gsk3 . therefore , we examined the ability of leptin to hyperpolarize the membrane potential and activate katp currents in whole - cell current and voltage clamp recordings , respectively , from mouse primary cultured pancreatic -cells in the absence and presence of dmat and various gsk3 inhibitors . in control experiments -cells were dialyzed with an electrode solution containing 3 mm atp , which resulted in a mean membrane potential of -41.8 1.5 mv application of leptin ( 10 nm ) resulted in a slowly developing hyperpolarization ( fig . 4a ) to a mean value of -69.4 1.6 mv ( n = 22 , p < 0.05 ) , which was reversibly inhibited ( to -34.5 1.9 mv ; n = 17 , p < 0.05 ) by the sulfonylurea tolbutamide ( 100 m ) . voltage clamp analysis ( fig . 4a ) showed that leptin increased the mean cell slope conductance from 0.73 0.11 to 1.62 0.22 nanosiemens ( n = 19 ; p < 0.05 ) , an action reduced by tolbutamide to 0.42 0.04 nanosiemens ( n = 14 ; p < 0.05 ) , indicating that the increase in current responsible for the augmented slope conductance and -cell hyperpolarization by leptin is due to katp channel opening ( 14 , 15 , 30 ) . the presence of dmat ( 10 m ) in the electrode solution ( and , therefore , cell interior ) resulted in a mean resting membrane potential of -44.8 3.4 mv ( p > 0.05 compared with control ) . subsequent addition of leptin caused a small , insignificant ( p > 0.05 ) hyperpolarization to -52.4 8.4 mv 4b ) , which was significantly different ( p < 0.05 ) from the control leptin response . voltage clamp analysis showed that dmat also prevented leptin from increasing the -cell mean slope conductance ( fig . the katp channels were operational in the presence of dmat as -cells were readily responsive ( fig . 4b ) to the direct - acting katp activator , diazoxide ( 200 m ) , which caused hyperpolarization to -75.2 1.7 mv ( p < 0.05 ) and increased slope conductance from 0.82 0.07 to 3.1 0.8 nanosiemens ( n = 5 ; p < 0.05 ) . ct99021 ( 2 m ) , in the electrode solution , resulted in a mean -cell membrane potential of -41.5 3.3 mv . 4c ) caused hyperpolarization to -51.5 4.1 mv , which although less than the control leptin response , was significantly different ( n = 5 , p < 0.05 ) from the mean resting potential before leptin challenge . however , voltage clamp analysis showed that in the presence of ct99021 leptin did not significantly increase the -cell slope conductance ( fig . was elicited by leptin in the presence of ct99021 , we decided to confirm that gsk3 inhibition does occlude leptin - mediated -cell hyperpolarization and increase in katp current . consequently , we examined the effects of the gsk3 inhibitors , ar - a0144 - 18 ( 28 , 31 ) and kenpaullone ( 29 ) on leptin - induced opening of katp . the presence of ar - a0144 - 18 ( 2 m ) or kenpaullone ( 10 m ) in the electrode solution did not alter ( p < 0.05 ) -cell membrane potential ( -40.4 1.6 mv and -35.2 2.1 mv , respectively ) compared with control recordings . leptin ( 10 nm ) caused a small hyperpolarization of the -cells for both ar - a014418 ( to -51.8 6.4 ; n = 5 ) and kenpaullone ( to -45.5 4.6 mv ; n = 6 ) , which was not significant ( p > 0.05 ) for either inhibitor ( fig . voltage clamp analysis confirmed that the presence of either gsk3 inhibitor prevented an increase in slope conductance by leptin ( fig . 4 , d and e ) . diazoxide ( 200 m ) , in contrast , reversibly hyperpolarized -cells in the presence of each gsk3 inhibitor ( fig . ( n = 5 ; p < 0.05 ) , ar - a014418 to -72.4 4.1 mv ( n = 5 , p < 0.05 ) , and kenpaullone , to -72.3 2.4 mv ( n = 6 , p < 0.05 ) and significantly increased the mean slope conductance to values comparable for control -cells ( p < 0.05 ) . a , phosphorylated pten levels in min6 cells using the phospho - specific pten antibodies indicated ( cluster site , ser-370 and thr-366 ) , in response to 10 nm leptin or 10 nm insulin for the indicated times ( minutes ) . b , the bar graph shows mean normalized levels of phosphorylated pten at the cluster phosphorylation site ( 380/2/3 ) for untreated cells and for cells stimulated with leptin ( filled bars ; n = 6 ) and insulin ( open bars ; n = 8) up to 30 min ( , p < 0.05 , versus control ( c ) or insulin ) . c , bar graph shows the mean normalized levels of phosphorylated thr-366 pten after stimulation with 10 nm insulin for 5 and 30 min . d , bar graphs showing mean normalized levels of phosphorylated thr-366 pten and ser-370 pten under non - stimulated conditions ( c ) and after stimulation with 10 nm leptin in the absence ( l ) and presence of dmat ( l+d ) and ct99021 ( l+ct ; n = 7 for each ) . , a , phosphorylated pten levels in min6 cells using the phospho - specific pten antibodies indicated ( cluster site , ser-370 and thr-366 ) , in response to 10 nm leptin or 10 nm insulin for the indicated times ( minutes ) . b , the bar graph shows mean normalized levels of phosphorylated pten at the cluster phosphorylation site ( 380/2/3 ) for untreated cells and for cells stimulated with leptin ( filled bars ; n = 6 ) and insulin ( open bars ; n = 8) up to 30 min ( , p < 0.05 , versus control ( c ) or insulin ) . c , bar graph shows the mean normalized levels of phosphorylated thr-366 pten after stimulation with 10 nm insulin for 5 and 30 min . d , bar graphs showing mean normalized levels of phosphorylated thr-366 pten and ser-370 pten under non - stimulated conditions ( c ) and after stimulation with 10 nm leptin in the absence ( l ) and presence of dmat ( l+d ) and ct99021 ( l+ct ; n = 7 for each ) . , figure 4.activation of katp channels in mouse pancreatic -cells by leptin is occluded by the presence of ck2 or gsk3 inhibitors . representative whole - cell current clamp recordings from a mouse cultured -cell dialyzed with an electrode solution containing 3 mm atp . in this and leptin ( 10 nm ) , tolbutamide ( tolb , 100 m ) , or diazoxide ( dzx , 200 m ) were applied for the time indicated on each trace ( a e ) . the action of leptin on -cell membrane potential was determined in the absence ( a ) and presence of 10 m dmat ( b ) , 2 m ct99021 ( c ) , 10 m kenpaullone ( d ) , and 2 m ar - a014418 ( e ) added to the electrode solution and , hence , cell interior . the bar graphs to the right of each trace denote the mean slope conductance , determined from voltage clamp current - voltage relations in the absence and presence of leptin . in control cells ( a ) the hyperpolarization and increased slope conductance induced by leptin are inhibited by the presence of tolbutamide . for drug - treated cells leptin has no effect on mean cell conductance , although in each case application of 200 m diazoxide hyperpolarizes the cells , which is associated with a significant increase in slope conductance . , p < 0.05 , compared with control . activation of katp channels in mouse pancreatic -cells by leptin is occluded by the presence of ck2 or gsk3 inhibitors . representative whole - cell current clamp recordings from a mouse cultured -cell dialyzed with an electrode solution containing 3 mm atp . in this and subsequent panels the trace begins 2 min after obtaining the recording configuration . leptin ( 10 nm ) , tolbutamide ( tolb , 100 m ) , or diazoxide ( dzx , 200 m ) were applied for the time indicated on each trace ( a e ) . the action of leptin on -cell membrane potential was determined in the absence ( a ) and presence of 10 m dmat ( b ) , 2 m ct99021 ( c ) , 10 m kenpaullone ( d ) , and 2 m ar - a014418 ( e ) added to the electrode solution and , hence , cell interior . the bar graphs to the right of each trace denote the mean slope conductance , determined from voltage clamp current - voltage relations in the absence and presence of leptin . in control cells ( a ) the hyperpolarization and increased slope conductance induced by leptin are inhibited by the presence of tolbutamide . for drug - treated cells leptin has no effect on mean cell conductance , although in each case application of 200 m diazoxide hyperpolarizes the cells , which is associated with a significant increase in slope conductance . , p < 0.05 , compared with control . phosphorylation and kinase activity of gsk3these results taken together with those of ning et al . ( 12 ) indicate that leptin inhibits pten phosphatase activity via phosphorylation at multiple sites on the c terminus of pten , which results in increased ptdins(3,4,5)p3 levels , actin restructuring , and katp activation . however , the dependence on pten thr-366 phosphorylation on gsk3 activity and consequent influence on downstream signaling events such as katp channel regulation gives rise to a quandary . leptin stimulation of rat arcuate increases pkb phosphorylation with subsequent gsk3 phosphorylation at ser-9/ser-21 ( 13 ) , actions also reported for leptin on mouse skeletal muscle cells ( 32 , 33 ) , mouse cortical neurons ( 34 ) , and mcf-7 breast cancer cells ( 35 ) . these latter actions of leptin replicate those of insulin , which increases pi3k activity and raises ptdins(3,4,5)p3 levels with subsequent increased pkb and gsk3 phosphorylation ( 5 ) . importantly , the phosphorylation of gsk3 at these serine residues by pkb is associated with reduced kinase activity ( 36 , 37 ) . this outcome is inconsistent with the requirement for active gsk3 causing phosphorylation of pten thr-366 allowing subsequent downstream signaling . indeed , leptin ( 10 nm ) increases the phosphorylation of pkb ( ser-473 ) and gsk3 ( ser-9/ser-21 ) in n29/4 hypothalamic cells ( fig . 5a ) , although this is not well sustained in the hypothalamic cells , as previously reported for rat arcuate ( 13 ) and mouse muscle ( 32 , 33 ) . insulin ( 10 nm ) induced a sustained rise in pkb ( ser-473 ) and gsk3 ( ser-9/ser-21 ) phosphorylation in n29/4 cells ( fig . 5b ) . similar results were obtained for min6 -cells , although in this case both leptin ( 10 nm ) and insulin ( 10 nm ) produced sustained increases ( fig . 5 , c and d ) in the levels of phosphorylated pkb ( ser-473 ) and gsk3 ( ser-9/ser-21 ) . these results are ostensibly inconsistent with the view that leptin signaling to actin and katp channels requires gsk3-dependent phosphorylation and inhibition of pten activity . however , gsk3 also has a tyrosine residue in the activation loop of the enzyme ( tyr-216 in gsk3 and tyr-279 in gsk3 ) , the phosphorylation of which is important for gsk3 activity ( 38 , 39 ) . most studies conclude that this site is not physiologically regulated by growth factors or cytokines ( 40 ) . insulin ( 10 nm ) did not affect gsk3 tyr-216/tyr-279 phosphorylation levels in either n29/4 cells ( fig . leptin ( 10 nm ) rapidly increased gsk3 phosphorylation at tyr-216/tyr-279 in n29/4 ( fig . 6d ) cells , which was sustained for at least 1 h. thus , in contrast to insulin , which increases phosphorylation of gsk3 serine sites only ( consistent with kinase inhibition ) , leptin increases the phosphorylation level of both serine and tyrosine sites on gsk3 . to define the outcome of this dual phosphorylation of gsk3 , we examined the effects of leptin ( 10 nm ) and insulin ( 50 nm ) on gsk3 activity in n29/4 cells . stimulation of n29/4 cells by insulin for 30 min results in a substantial inhibition of gsk3 activity , as expected from increased pkb activity causing gsk3 ser-9/ser-21 phosphorylation . in contrast , stimulation of n29/4 cells with leptin ( 10 nm ) for 30 min , a time when pten phosphorylation is increased , ptdins(3,4,5)p3 levels are high , f - actin is depolymerized , and -cells are hyperpolarized , results in maintained gsk3 activity ( fig . this outcome is consistent with the finding that , although both hormones increase gsk3 ser-9/ser-21 phosphorylation levels , leptin , but not insulin , also increases gsk3 tyr-216/tyr-279 phosphorylation . leptin phosphorylation of pten at the cluster site is ck2-dependent and leads to inhibition of pten phosphatase activity with a consequent increase in ptdins(3,4,5)p3 levels in n29/4 cells ( 12 ) . here , we show that leptin increases pten phosphorylation at three further sites , thr-366 , ser-370 , and ser-385 in n29/4 cells . we also show that leptin increases p - pten at the cluster site , thr-366 and ser-370 , in min6 pancreatic -cells . as demonstrated for the cluster site ( 12 ) , phosphorylation of pten in n29/4 cells at thr-366 and ser-370 is important for transfer of the leptin signal downstream to cause f - actin depolymerization . leptin - mediated reduction in f - actin levels was previously shown to be dependent on basal pi3k activity and inhibition of both lipid and protein phosphatase activity of pten ( 12 ) . correspondingly , overexpression of the pten phosphorylation site mutants , s370a pten and t366a pten , in n29/4 cells occluded leptin - driven reduction in f - actin . the thr-366 and ser-370 alanine and aspartic acid pten mutants , when overexpressed in n29/4 cells , alter ptdins(3,4,5)p3 levels exactly as reported for the cluster site mutants . this outcome does not agree with in vitro assays , which show no alteration in enzyme activity associated with these mutants ( 41 ) , unlike the corresponding mutations of the cluster site residues ( 12 ) . thus , s370d and t366d both raised ptdins(3,4,5)p3 levels , indicative of reduced lipid phosphatase activity per se and dominant negative behavior with respect to native pten activity . interestingly , these mutants produced different outcomes on f - actin levels and leptin signal transduction . overexpression of t366d pten per se mimicked the effects of leptin by increasing ptdins(3,4,5)p3 and inducing f - actin depolymerization in a pi3k - dependent manner . this replicates the effect of overexpression of c124s pten and d3pten ( cluster site mutant ) in these cells ( 12 ) and is consistent with phosphorylation of the thr-366 site reducing pten lipid and protein phosphatase activity . in contrast , overexpression of s370d pten was unable to elicit f - actin depolymerization per se even in the face of increased cellular ptdins(3,4,5)p3 levels and did not occlude leptin - mediated f - actin depolymerization . consequently , mimicking phosphorylation by aspartate substitution at ser-370 does not exactly replicate the effect of aspartate substitution at thr-366 or the cluster phosphorylation site ( 12 ) , with all three mutant forms of pten increasing ptdins(3,4,5)p3 levels but only the latter two reducing f - actin levels . the s370d pten mutant also does not duplicate the action of the lipid phosphatase dead , protein phosphatase active , mutant g129e pten . overexpression of g129e pten in n29/4 cells increases ptdins(3,4,5)p3 levels with no reduction in f - actin and prevents leptin - mediated f - actin depolymerization ( 12 ) , whereas the presence of s370d pten allows leptin depolymerization of actin . our conclusion from these data is that s370d is lipid phosphatase dead but protein phosphatase active and that leptin inhibits the protein phosphatase activity by phosphorylation at the thr-366 and cluster sites , whereas the protein phosphatase activity of g129e pten can not be overcome by phosphorylation at these sites . thus , phosphorylation of pten at ser-370 is adequate to inhibit pten lipid phosphatase activity and increase ptdins(3,4,5)p3 in n29/4 cells but is not sufficient per se for efficacious transmission of the leptin signal to f - actin . figure 5.leptin and insulin increase pkb and gsk3 serine phosphorylation in n29/4 and min6 cells . pkb ( ser-473 ( s ) ) and gsk3 ( ser-9/ser-21 ) phosphorylation was determined in n29/4 ( a and b ) and min6 ( c and d ) cells , stimulated with 10 nm leptin ( a and c ) , or 10 nm insulin ( b and d ) for the indicated times ( minutes ) . the bar graphs below each set of blots show mean normalized p - pkb and p - gsk3 levels for untreated cells and cells stimulated with leptin ( n29/4 ( a , n = 9 ) , min6 ( c , n = 6 ) ) or with insulin ( n29/4 ( b , n = 9 ) , min6 ( d , n = 6 ) ) . , p < 0.05 , compared with control level for each treatment . leptin and insulin increase pkb and gsk3 serine phosphorylation in n29/4 and min6 cells . pkb ( ser-473 ( s ) ) and gsk3 ( ser-9/ser-21 ) phosphorylation was determined in n29/4 ( a and b ) and min6 ( c and d ) cells , stimulated with 10 nm leptin ( a and c ) , or 10 nm insulin ( b and d ) for the indicated times ( minutes ) . the bar graphs below each set of blots show mean normalized p - pkb and p - gsk3 levels for untreated cells and cells stimulated with leptin ( n29/4 ( a , n = 9 ) , min6 ( c , n = 6 ) ) or with insulin ( n29/4 ( b , n = 9 ) , min6 ( d , n = 6 ) ) . , figure 6.leptin , but not insulin , increases gsk3 tyrosine phosphorylation in n29/4 and min6 cells . gsk3 ( tyr-216/tyr-279 ( y ) ) phosphorylation was determined in n29/4 ( a and b ) and min6 cells ( c and d ) stimulated with 10 nm insulin ( a and c ) or 10 nm leptin ( b and d ) for the indicated times ( minutes ) . the bar graphs below each set of blots show mean normalized p - gsk3 for untreated cells and cells stimulated with insulin ( n29/4 ( a , n = 9 ) , min6 ( c , n = 6 ) ) or with leptin ( n29/4 ( b , n = 9 ) , min6 ( d , n = 6 ) . , p < 0.05 , compared with control level for each treatment . e , n29/4 cells were stimulated with either 10 nm leptin or 50 nm insulin for 30 min . the bar graph denotes the mean normalized gsk3 activity and shows that insulin significantly inhibited gsk3 activity , whereas leptin had no effect on gsk3 activity ( n = 3 for each ) . leptin , but not insulin , increases gsk3 tyrosine phosphorylation in n29/4 and min6 cells . gsk3 ( tyr-216/tyr-279 ( y ) ) phosphorylation was determined in n29/4 ( a and b ) and min6 cells ( c and d ) stimulated with 10 nm insulin ( a and c ) or 10 nm leptin ( b and d ) for the indicated times ( minutes ) . the bar graphs below each set of blots show mean normalized p - gsk3 for untreated cells and cells stimulated with insulin ( n29/4 ( a , n = 9 ) , min6 ( c , n = 6 ) ) or with leptin ( n29/4 ( b , n = 9 ) , min6 ( d , n = 6 ) . , p < 0.05 , compared with control level for each treatment . e , n29/4 cells were stimulated with either 10 nm leptin or 50 nm insulin for 30 min . the bar graph denotes the mean normalized gsk3 activity and shows that insulin significantly inhibited gsk3 activity , whereas leptin had no effect on gsk3 activity ( n = 3 for each ) . a number of phosphorylation sites have been identified within the pten c - terminal tail , with the protein kinase , ck2 , purported to phosphorylate multiple residues , including the cluster site , ser-385 and ser-370 , but not thr-366 ( 1719 , 23 , 41 ) . pten protein stability and activity are considered to be dependent on phosphorylation status of the c - terminal tail ( 17 , 18 ) , with phosphorylation argued to reduce association of pten with the plasma membrane ( 20 , 42 , 43 ) . it is presently unclear what effect leptin - induced phosphorylation will have on pten protein stability , as it has been reported that t366a and s370a are more stable than wild - type pten and gsk3 inhibition increases the stability of wild - type pten ( 41 ) , whereas alanine mutation of the cluster site results in reduced protein stability ( 17 , 19 ) . the inhibition of leptin - induced phosphorylation of the cluster site ( 12 ) , ser-370 and ser-385 , in n29/4 cells and of the cluster site and ser-370 in min6 cells by dmat supports the view that ck2 is the kinase responsible for phosphorylation at these sites by this hormone . inhibition of pi3k had no effect on leptin - mediated phosphorylation at ser-370 , ser-385 , or thr-366 in n29/4 cells , indicating no evidence for pi3k - dependent feedback phosphorylation ( 44 ) consistent with leptin - induced phosphorylation of the cluster site . for gsk3 to phosphorylate its substrate , the substrate has to be primed by phosphorylation of a site four or five residues c - terminal of the target ser / thr of gsk3 ( 45 , 46 ) . thus , phosphorylation of ser-370 by ck2 is considered a priming event for subsequent gsk3 phosphorylation of thr-366 ( 23 ) , and inhibition of gsk3 blocks phosphorylation of pten on thr-366 ( 41 ) . occlusion of leptin driven - increased p - pten on thr-366 by the gsk3 inhibitor ct99021 in n29/4 and min6 cells agrees with these findings . consequently , we expected dmat to reduce or inhibit leptin - mediated phosphorylation of thr-366 in these cells . this was observed for min6 pancreatic -cells but not for the hypothalamic cells , where dmat prevented leptin - induced phosphorylation of ser-370 but not thr-366 . thus , either an additional unidentified priming kinase is responsible or there is sufficient phosphorylation of ser-370 remaining in dmat - treated n29/4 cells to allow gsk3 priming . interestingly , an in vitro study of pten phosphorylation has shown that phosphorylation of ser-370 by ck2 is less efficient than that of the cluster site and that gsk3 is capable of phosphorylating pten at thr-366 without priming , although at low efficiency ( 41 ) . thus , we predicted that inhibition of ck2 and gsk3 would prevent or reduce transmission of the leptin signal via pten to actin filament restructuring and katp channel opening . our results show that the gsk3 inhibitor ct99021 per se or in combination with dmat significantly reduces leptin - mediated f - actin depolymerization . furthermore , leptin - driven hyperpolarization of pancreatic -cells by katp activation was prevented by the presence of dmat or any one of three structurally dissimilar gsk3 inhibitors . this is consistent with the view that leptin - mediated phosphorylation of pten and subsequent inhibition of its phosphatase activity is a necessary step for signal transmission to katp channels . in contrast , insulin does not cause f - actin depolymerization in n29/4 cells nor does it hyperpolarize pancreatic -cells by opening katp channels , and this differential output may be mediated via pten ( 12 ) . the results presented here indicate a key role for pten thr-366 phosphorylation via gsk3 activity for successful transmission of the leptin signal . for this to occur , gsk3 activity has to be maintained or increased by leptin . however , leptin , like insulin , increases pkb phosphorylation with the resultant phosphorylation of gsk3 at ser-9/ser-21 consistent with inhibition of gsk3 activity . indeed , both insulin and leptin increased the phosphorylation of gsk3 at ser-9/ser-21 in n29/4 and min6 cells . in contrast , leptin , but not insulin , caused a rapid and sustained increase in gsk3 phosphorylation at tyr-216/tyr-279 in both cell types . phosphorylation of gsk3 at this site is considered to be activating and necessary for functional activity ( 38 ) . indeed , direct gsk3 kinase assay showed that insulin stimulation of n29/4 cells substantially reduced gsk3 activity , whereas leptin had no effect . taken together , the semiquantitative western blot data show that insulin significantly alters n - terminal serine phosphorylation of gsk3 , which would reduce its activity toward primed substrates . however , phosphorylation of pten at thr-366 in response to leptin does not appear to require priming ( phosphorylation of this site is unaffected by dmat ) , and this may explain why insulin does not reduce thr-366 phosphorylation . in contrast , gsk3 in leptin - treated cells increased phosphate in tyr-216/tyr-279 compared with control or insulin - treated cells . this is a possible explanation why leptin and not insulin induces thr-366 phosphorylation . however , there are other ways to regulate gsk3 , including protein - protein interaction ( e.g. wnt signaling ) and thr-380 phosphorylation ( e.g. cytokine signaling ) that also require further investigation . identification of the kinase ( or phosphatase ) responsible for the increase in tyrosine phosphorylation of gsk3 and development of a quantitative multiple reaction monitoring liquid chromatographymass spectrometric approach for each of the phosphorylation sites on gsk3 would be required to elucidate the exact mechanism of control by leptin . these subtle signaling differences downstream of insulin and leptin receptors are likely to contribute to the ability of each to perform their distinct physiological actions through modulation of the same signaling pathways / molecules . in conclusion , this study has demonstrated that leptin increases the phosphorylation of pten at multiple residues in a ck2- and gsk3-dependent manner . this action of leptin is required for successful transmission of the leptin signal to cause cytoskeletal remodeling , the opening of katp channels , and subsequent hyperpolarization of membrane potential . interestingly , complete loss of pten activity in mouse hypothalamic proopiomelanocortin neurons permanently activates katp channels and hyperpolarizes the neurons resulting in hyperphagia , leptin resistance , and diet - induced obesity ( 10 ) . insulin signaling inhibits gsk3 activity , and this may act via an alteration in pten thr-366 phosphorylation as a negative feedback loop for leptin signaling in cells . in contrast , leptin , regardless of whether it stimulates pi3k activity in all cells or not , by maintaining gsk3 activity may result in a positive feedback to pi3k signaling in cells . such interplay between leptin and insulin signaling in cells may have important implications for downstream signaling events . for example , inhibition of pten in peripheral tissues such as liver , muscle , and fat demonstrate improved insulin sensitivity and systemic glucose tolerance under normal and high fat diets ( 48 ) . dysregulation of pten ( lipid and protein phosphatase ) activity has also been implicated in certain cancers ( 49 ) and neurodegenerative disease ( e.g. parkinson disease ( 47 ) ) . thus , modulation of pten function plays multiple and important roles in numerous physiological and pathological phenomena .

alzheimer s disease ( ad ) is a progressive neurodegeneration and a complex multifactorial disorder among the elderly . the pathological features of ad are the loss of neurons in conjunction with the presence of oxidative stress , axonal dystrophy , senile plaques , and neurofibrillary tangles . because of its polygenic nature , ad is thought to be caused not by defects in a single gene , but instead by variations in many genes , proteins , and their complex interactions . thus , it is challenging to develop novel effective medications targeting multiple proteins in order to stop or reverse the progression of the disease . great efforts have been devoted to carrying out bioscience research with rapid accumulation of a large volume of scientific data relevant to ad . in particular , studies involved in ad neurosignaling pathways and ad - targeted new chemical ligands have been steadily proliferating . the quantity and the quality of the ad special class of molecules are expected to grow at a much faster rate in the future , thanks to rapid technology advancement in biochemistry , biophysics , medicinal chemistry , and pharmacology . unfortunately , the venues to publicize ad - specific research have been limited to archival journals and periodicals . although scifinder and other databases have archived most of the documentation , the reported works are scattered . thus , it is difficult to find , associate , and validate reported ad - related active chemical molecules and reuse the reported research results for ad target research . several ad - related databases have been reported to explore the molecular mechanisms , such as alzgene and alzpathway . the alzgene database has been developed for investigating genetic association in the field of ad . it contains almost all genes related to ad and focuses on systematic meta - analyses of information on ad genetic association . alzpathway , a comprehensive map of signaling pathways of ad , was constructed for exploring the ad pathogenesis . however , these databases were mainly designed to investigate the pathogenic mechanisms of ad . to our knowledge , there is still no publicly available ad specific chemical genomics ( or chemogenomics ) database focusing on small molecules that target proteins related to ad for drug research . herein , an integrated cloud computing server , alzplatform , has been developed in response to the needs . the platform assembles a large repertoire of ad related chemogenomics data , including genes , protein targets , and small chemical molecules with their bioactivity records , bioassays , and references , as well as approved drugs or those in clinical trial for ad treatments . alzplatform enables cloud computing and sourcing services and provides powerful computational algorithms and implemented online computing programs / tools , including our established targethunter , htdocking , and bbb predictor for target identification , drug repurposing , and polypharmacology analysis associated with ad ( figure 1 ) . therefore , alzplatform is a valuable platform for investigating and sharing ad targets and small chemical drug molecules at chemogenomics scale for better understanding the mechanisms of system polypharmacology in aid of new anti - ad drug discovery . overview of alzplatform database featured with integrated computing and data - mining functions ( www.cbligand.org/ad ) . alzplatform was constructed based on the established molecular database prototype cbid ( www.cbligand.org/cbid ) , with a mysql ( http://www.mysql.com ) database and an apache ( http://www.apache.org/ ) web server . the information of protein targets and chemicals associated with ad was gathered according to the approved drugs , clinical trial drugs , and literatures from various databases , including the drugbank , clinicaltrials.gov , bindingdb , alzgene , pubchem , chembl , and scifinder database . the corresponding information on signaling pathway of these targets was compiled from the kegg database . all the chemical structures , affinity values , and additional data including pathways , bioassays , and references were archived in relational database structure formats at the backend of the alzplatform database . alzplatform provides a user - friendly interface with a powerful search engine for the detailed information on ad chemicals and targets . this includes gene / protein symbol , compound name / id , and basic pharmacological properties . jme is used as the input interface , and openbabel is the search engine at the backend . in the structure search window , users can either sketch the structure in the jme interface or upload a file containing a small chemical molecule . after submission , the search is performed by openbabel at the server side and the results will be returned to the client side by loading a new page , including i d , structure of compound , target name , and the corresponding links to the literature . target identification and drug design with desired properties are top priorities for medicinal chemists . the data on chemogenomics and cheminformatics collected in alzplatform provide a valuable opportunity to explore the underlying targets ; absorption , distribution , metabolism , and excretion ( adme ) and toxicity prediction ; and also calculation of molecular properties and drug - likeness . as such , state - of - art machine learning algorithms and chemoinformatics tools have been deployed on the platforms for facilitating ad drug design and target identification as briefed below . targethunter was implemented in alzplatform to provide online computing algorithm to predict the possible targets or off - targets of compounds . the basic principle of the targethunter program is based on a known medicinal chemistry concept : structurally similar compounds have similar physical properties that may result in similar biological profiles . this predicts the targets of a query compound by use of the powerful data - mining algorithm ( tamosic ) , which assigns the targets associated with the most similar compounds of a query chemical as the predicted targets . targethunter is a powerful cloud computing tool with attractive features : ( i ) ease of use ; ( ii ) query data retrieval function ; ( iii ) user choices of desired fingerprints and databases ; ( iv ) high accuracy ; and ( v ) bioassay finder implemented bioassaygeomap function to find the authors who have published a bioassay for validation . such a tool will assist researchers to develop bioactive compounds for research on ad target . in addition to the ligand - based targethunter tool online , we have also established a high - throughput docking ( htdocking , http://www.cbligand.org/ad/docking_search.php ) program . it is a web - based computing tool that automates docking procedure to search for protein targets and to explore interactions between compound and protein . in the current version of alzplatform database , crystal structures of proteins related to ad have been collected from the protein data bank ( pdb ) to build an ad domain - specific subset . water molecules and ligands were removed , hydrogen atoms were added , and the active sites of each protein were defined by the residues around the cocrystallized ligands or generated using the autodock utility scripts . autodock vina can provide 35 predicted binding affinity values ( g values ) from different docking poses for each compound in a binding pocket of a protein . in our htdocking program , we only consider the best binding affinity value which is further transformed as docking score . the docking score is calculated as pki , where pki = log(predicted ki ) and the predicted ki = exp . the docking score of a queried compound from each protein structure is used to assess and rank the potential protein partners or targets . the blood brain barrier ( bbb ) is the bottleneck in ad drug development and is the single most important factor limiting the future growth of neurotherapeutics . considering this , a bbb predictor was specially designed to classify whether a compound can cross the blood this predictor was built by applying the support vector machine ( svm ) and licabeds algorithms on four types of fingerprints of 1593 reported compounds . the bbb predictor is available at http://www.cbligand.org / bbb/. in addition , alzplatform provides toxicity prediction with the toxtree package ( http://cbligand.org/tox ) , an online service for removal of false positive results ( http://cbligand.org/pains ) , and property calculator for the calculation of molecular properties , such as molecular weight , formula , number of rotatable bonds , hydrogen bond donors and acceptors , polar surface area , xlogp , and lipinski s rule of five . the properties calculator was implemented with the cdk package and is available at http://www.cbligand.org/cbid/property_explorer.php . furthermore , links are provided for quickly accessing chemoinformatics resources , such as actelion s property explore for adme prediction and calculation of molecular properties and drug - likeness with molinspiration . thus , alzplatform acts as a chemoinformatics hub to other tools and databases , which can facilitate researchers in ad drug development and target identification . methyl sandaracopimarate ( ms ) , a known diterpenoid compound , was isolated and identified from the extract of seeds of platycladus orientalis . the caenorhabditis elegans ( c. elegans ) strains cl4176 , also the escherichia coli op50 strain , were obtained from the caenorhabditis genetics center ( cgc ; university of minnesota , minneapolis , mn ) . the transgenic nematode cl4176 strain , as an ad model , is a temperature - sensitive mutant strain that expresses human a142 when it reaches nonpermissive temperatures . the nematodes were maintained and assayed on nematode growth medium ( ngm ) agar plates with escherichia coli op50 at 16 c . all worms used were raised from eggs obtained after sodium hypochlorite treatment of hermaphrodites . the paralysis assay was measured according to the method described previously , with slight modifications . the strain cl4176 maintained at 16 c was egg - synchronized onto the 35 mm culture plates with or without methyl sandaracopimarate ( 300 m ) . induction occurred 36 h after egg laying and lasted until the last worm became paralyzed . for the paralysis assay , the survival of worms was determined by touch - provoked movement . worms were scored as paralyzed when they failed to respond to repeated touching with a platinum wire . hepg2 cells were plated at a density of 2 10 cells on a 48 well plate 24 h before transfection . plasmids were transfected using trans - it lt ( mirus , madison , wi ) according to the manufacturer s protocol . to evaluate the binding of methyl sandaracopimarate to ppar , triplicate transfections were performed using the following plasmids : pcmx - tk - ppre - luc ( 400 ng ) , pcmx - ppar ( 200 ng ) , pcmx ( 200 ng ) , and ( beta galactosidase ) -gal ( 50 ng ) . at 24 h after transfection , the cells were treated with rosiglizone ( 10 m ) , vehicle , or the methyl sandaracopimarate at increasing concentrations ( 0.1 , 1 , 10 , 50 , 100 , and 500 m ) . at 24 h after the compound treatment , the cells were lysed and the luciferase signal was quantified using a standard luminometer ( perkin - elmer).the luciferase signal was normalized to -gal signal . alzplatform ( www.cbligand.org/ad/ ) archived 928 genes , 320 ad related proteins , 194 ad drugs approved and in clinical trials , and 405 188 chemicals associated with 1 023 137 records of reported ad bioactivities from 38 284 ad corresponding bioassays and 10 050 references . figure 1 shows an overview of the web - interfaced molecular information database for ad with implemented chemoinformatics computing tools and programs ( www.cbligand.org/ad ) . the current version of alzplatform consists of varieties of ad related proteins ( a total of 320 ) : ( i ) 172 enzymes such as acetylcholinesterase ( aces ) , monoamine oxidase b , angiotensin - converting enzyme , and cyclooxygenase-2 ; ( ii ) 38 membrane receptors , such as serotonin receptors , c c chemokine receptors , and beta adrenergic receptors ; ( iii ) 14 ion channels , such as glutamate n - methyl - d - aspartate ( nmda ) receptors and neuronal acetylcholine receptors ; and ( iv ) 96 other proteins ( figure 2a ) . for most of these targets for example , at least 283 of them are expressed in the corpus striatum and 281 are expressed in the cerebral cortex ( figure 2b ) . besides the 194 drugs for ad clinical treatments , alzplatform also contains 225 additional drugs that are reported to interact with ad - related proteins but are used for treatment of other diseases . some of these drugs may have the potential to be repurposed for ad research or treatment . moreover , small molecules and their bioactivities against these targets could be used for systematic in silico screening of anti - ad lead compounds ( figure 2c ) . the statistics on these ad drugs , in different development phases , were plotted according to their interacting targets . as shown in figure 2d , ad drugs approved by the fda interact with hands of targets , including aces and nmda receptors . among these proteins , muscarinic acetylcholine receptor m1 ( acm1 ) probably is not an ideal drug target for ad treatment because 7 of 8 drugs targeting acm1 are discontinued or withdrawn due to undesirable adverse effects , and only one is in phase ii of clinical trial . in addition , it is well - known that cleavage of amyloid precursor protein ( app ) by -secretase ( bace1 ) is the rate - limiting step in beta - amyloid production , which suggests a potential target for drug development of ad . currently , most of bace1 inhibitors are still in phase i and ii clinical trials . mk-8931 developed by merck is the only bace1 inhibitor that is currently in phase iii clinical trial . however , the research and development pace on bace1 as the major ad therapeutic target has been slow . several concerns have been raised about the potential side effects of bace1-targeted inhibitors , because bace1 also has important roles in myelination , retinal homeostasis , brain circuitry , and synaptic function . pen2 could be a good target because it has a gradient number of drugs ( 9 , 2 , and 1 , respectively ) in phases i , ii , and iii . ( a ) summary of ad - related targets . ( b ) tissue distribution of targets associated with ad . the yellow lines denote that these tissues are located in the central nervous system . these approved and clinical trials ad drugs were classified by different phases with distinct colors . the yellow and black lines indicate the approved and discontinued ad drugs , respectively . the blue , pink , and green lines denote clinical trial drugs in phases i , ii , and iii , respectively . ( e ) ad targets and their drugs were plotted according to the pathways the targets involved . the statistics of ad targets and their drugs were plotted according to the pathways the targets involved ( figure 2e ) . it is not a surprise that alzheimer s disease pathway ( keggid : hsa05010 ) is among the top of the pathway list . we also notice that pathways related with drug addiction , such as amphetamine addiction ( keggid : hsa05031 ) , cocaine addiction ( keggid : hsa05030 ) , and alcoholism ( keggid : hsa05034 ) , are also enriched on the top list , which could imply that ad shares some common pathways with drug addictions . furthermore , our data show that cannabinoid receptors , the key drug abuse related proteins , are among the top list , which is congruent with the reports that cannabinoid receptors are important in the pathology of ad , and cannabinoids succeed in preventing the neurodegenerative process in ad . alzplatform also features an integrated cloud computing service with intrinsic scalability and convenient features for further expansion . it provides powerful computing and sourcing services for both computational queried data storage and reretrieval , which can facilitate ad drug research and development . in the next section , three case studies demonstrate the usage of these computational tools on data - mining of the chemogenomics database for ad drug research . target identification of small chemicals is essential for unraveling the underlying mechanisms of their bioactivities . often , natural products exhibit significant efficacy , yet their molecular mechanisms remain elusive . we used a natural product to illustrate how the alzplatform and high - throughput docking ( htdocking , https://www.cbligand.org/ad/ ) can be used to identify potential targets and explore the mechanism of action for natural products in which there are often multiple chemical components . in our previous study , the extract of seeds of platycladus orientalis can significantly extend lifespan of c. elegans and protect against a toxicity in transgenic c. elegans expressing human a. in addition , methyl sandaracopimarate ( ms ) , a known diterpenoid compound , was isolated and identified from the active extract . in order to evaluate the protective effect of the compound against a-induced toxicity , the paralysis assay was conducted in the transgenic c. elegans.cl4176 strain , which expresses human a142 . as shown in figure 3a , ms significantly protected against a-induced rapid paralysis at 300 m in comparison with the untreated control ( p < 0.05 ) . moreover , ( a ) time course of a-induced paralysis in the transgenic c. elegans.cl4176 treated with standard nematode growth medium ( ngm ) and methyl sandaracopimarate ( ms ) . huperzine a was used as a positive control . ( b ) the chemical structure query window for ad targets prediction of ms by htdocking server . ( c ) molecular docking study of ms in the active site of ppar ( pdb : 2om9 ) . ( d ) the predicted target was further validated by in vitro ppar responsive luciferase assay . ms activates ppar in a concentration dependent manner with ec50 value of 15 m . the significance of differences from normal control group is at * p < 0.05 . to explore further the underlying mechanisms of neuroprotection for ms , the result shows that the compound is predicted to interact with the peroxisome proliferator - activated receptor gamma ( ppar ) , acetylcholinesterase and cgmp - specific 3 , 5-cyclic phosphodiesterase ( pde5a ) ( figure 3b ) . among them , ppar is listed as one of the top targets with docking score 6.5 , suggesting that ppar is likely to be a key target for ms . furthermore , the predicted interactions between the compound and the ligand binding domain of ppar were shown in figure 3c . ser342 , ile341 , phe287 , cys285 , arg288 , gly284 , and his266 form a hydrophobic interaction cleft around ms , and the carbonyl group of the compound exhibits the hydrogen - bonding interactions with the backbone of his266 and lys265 , which are vital residues modulating the activation of the receptor , suggesting that ms might be a potential ppar agonist . the prediction was further validated by in vitro ppar responsive luciferase assay that allows the quantification of the ligand activated ppar on the basis of its specific binding to ppre sequences . the result indicated that the ms activates ppar in a concentration dependent manner with ec50 value of 15 m ( figure 4d ) . according to accumulating evidence , ppar overexpression decreases -secretase1 gene transcription and reduces the intracellular and plaque a generation in vivo . moreover , neurotoxic activities under inflammatory conditions of microglia and astrocytes are reduced by ppar agonists . on the basis of our study , enhancing ppar expression may be one of the mechanism by which ms protected against a-induced toxicity . overview of the application of the targethunter program for ad target prediction of small molecules . ( a ) input interface ; ( b ) backend server ; ( c ) output predicted results ; and ( d ) the bioassay geomap function can be used to find potential collaborators for targets validation experimentally . in addition to the protein - based htdocking program , we have established the ligand - based targethunter tool ( http://cbligand.org/targethunter ) which is an online program for targets identification and drug repurposing . we also used natural product to illustrate the application of targethunter for target prediction . acteoside , isolated from orobanche minor , can significantly inhibit the aggregation of amyloid- with ic50 value of 8.9 m and can attenuate the a induced toxicity . however , the neuroprotective mechanisms are still not exactly known . to identify further the underlying targets , the structure of acteoside was submitted as a query to the targethunter program . as shown in figure 4a c , two related compounds ( chembl510539 and 455827 , with scores of 0.82 and 0.75 , respectively ) were retrieved . the first compound , chembl510539 , targets the endoplasmic reticulum - associated amyloid beta - peptide - binding protein ( erab , tested in the pubchem bioassay aid : 886 with potency value of 0.1 m ) . the protein is an intracellular a-binding protein that contributes to the pathogenesis of ad . the toxic effect of a on neuroblastoma cells is prevented by blocking erab and is enhanced by overexpression of erab . therefore , the inhibition of erab is likely to the cause of the protection against a induced toxicity by acteoside . in addition , the cyclooxygenase-2 ( cox-2 ) targeted by another compound ( chembl455827 ) is an important target associated with inflammatory regulation in the pathogenesis of ad . to facilitate the further target validation , bioassaygeomap ( http://cbligand.org/targethunter/bioassaygeomap.php ) is implemented in the alzplatform to locate the nearby potential collaborators who reported their established bioassay . as show in figure 4d , two research laboratories near the university of pittsburgh were found by the program , and they could be potential collaborators for further experimental validation of the predicted target cox-2 . prediction of polypharmacology of known drugs is highly useful for finding new system polypharmacotherapy . there has been increasing interest in identifying additional targets for known drugs and predicting drug accordingly , we used established chemoinformatics tools to predict potential target and polypharmacology for five fda - approved ad drugs . among them , four ad drugs ( tacrine , rivastigmine , galantamine , and donepezil ) are acetylcholinesterase ( ache ) inhibitors and the other one ( memantine ) is an n - methyl - d - aspartate ( nmda ) receptor antagonist . the protein targets identified by htdocking program for each known ad drug were tabulated in an output window and ranked by docking scores . the five drugs and their top candidate targets ( docking score higher than 6.0 , green and pink nodes ) were compiled to build a polypharmacological interacting network with cytoscape 2.8 ( figure 5 ) . unsurprisingly , the result shows that the known acetylcholinesterase and nmda receptors ( green nodes ) were targeted by four ache inhibitors and memantine , respectively . moreover , the comparison of the predicted and experimental pki values for known ad drugs was visualized in table 1 . the result illustrates that the predicted targets and the binding affinities are correlated with reported experimental data . indeed , the additional predicted associations or drug / protein networks ( green nodes and edges ) , such as beta - secretase1 ( bace1 ) , glycogen synthase kinase-3 beta ( gsk3b ) , and monoamine oxidase type b ( mao - b ) , have already been reported in the literature ( table 2 ) , indicating the reliability of the htdocking program . also , the remaining predicted targets ( pink nodes ) could be the new targets for the known drugs that merit further validation by experiments . illustration of htdocking server ( https://www.cbligand.org/ad/ ) for polypharmacology analysis of 5 approved ad drugs . the large circles ( cyan ) represent fda - approved ad drugs ( tacrine , donepezil , rivastigmine , galantamine , and memantine ) . each drug is linked to its predicted targets . among them , the green nodes and edges denote the known targets of drugs . others pink nodes represent new potential off - targets and their interactions are linked by cyan dotted edges . experimental data from ref ( 66 ) . experimental data from ref ( 67 ) . experimental data from ref ( 68 ) . experimental data from ref ( 69 ) . experimental data from ref ( 70 ) experimental data from ref ( 71 ) . experimental data from ref ( 72 ) . experimental data from ref ( 73 ) . another finding in the network is the polypharmacological effects for two known drugs , galantamine and memantine ( figure 5 ) . the network shows that besides binding to ache , galantamine is predicted to interact with bace1 , mitogen - activated protein kinase 14 ( mapk14 ) , and adenosine a2a receptor ( aa2ar ) . inhibitions of these proteins have effects on decreasing the a production and a-induced toxicity and increasing the expression of nicotinic receptors . similarly , memantine is predicted to interact with gsk3b , bace1 , mao - b , and nitric oxide synthase 1 ( nos1 ) besides binding to nmda receptors . inhibition of these proteins can prevent the accumulation of the misfolded proteins ( tau and a ) and enhance neuronal function . such in silico analysis of polypharmacological effects may explain why the combined use of memantine and galantamine can produce greater memory improvement than either treatment alone in clinical trials , which will guide to design and discover new drug - like leads with the multitarget synergetic therapeutics for ad . alzheimer s disease is a complex multifactorial disorder . with the extensive accumulation of molecular biological elucidations of ad signaling pathway at genes and proteins levels , these databases together with other disease specific databases , such as hlungdb ( human lung cancer database ) and cvdhd ( cardiovascular disease herbal database ) , provide alternative avenues to explore the molecular mechanisms and signaling pathways of diseases . however , there is no comprehensive ad specific chemical genomics knowledgebase available for polypharmacology targets identification to facilitate novel ad drug discovery . comparing with other general in silico docking platforms , such as dock blaster , our alzplatform also offers an ad domain - specific chemogenomics database with user - friendly query functions and polytarget identification algorithms implemented with ligand - based targethunter and protein structure - based htdocking . alzplatform provides a promising alternative to bridge the knowledge gap between biology and chemistry related to ad , enhancing ad target research , polypharmacology analysis , and new drug discovery . our pilot studies demonstrated that the protein - based htdocking program has been successfully used to identify the ad - related targets for small molecules , such as drugs , lead compounds , and natural product . htdocking program provides a list of predicted targets and corresponding computational docking - based binding affinity ( scores ) . the reliability of the htdocking program has been confirmed by comparison of the predicted with the experimental pki values reported for known ad drugs , also by our in vitro experimental validation for an active natural product . of course , htdocking has certain limitations on availability of high - quality protein structures . as a complementary partner , the ligand - based targethunter tool is designed to predict the potential targets and off - targets of chemicals using our established chemogenomics database . our established programs also can be useful in the application of drug repurposing , and in the investigation of potential side effects related to ad drugs . targethunter is a powerful cloud - computing tool with attractive features : usability , flexibility , and veracity . furthermore , it embeds an important query function , i.e. , the geographical bioassay locator can assist users to find nearby collaborators who have reported suitable bioassays in order to validate the target prediction , which will enhance the productivity of collaborative researchers and facilitate the chemogenomics data sharing and information communications . target associations can benefit the discovery of novel therapeutic applications and also reveal the possible side effects of drugs . it will transform the one - target - one - drug development process to a new multitarget multidrug paradigm , thereby expanding the opportunity for system multitarget drug discovery . by assembling many ad related drugs and small molecules with target annotations , alzplatform provides specific data and tools to help researchers conduct in - depth analysis for ad related targets and drugs and will also enable the chemists to design multitarget small molecules and to perform bioactivity test with the collaborators , which will boost the more effective system pharmacotherapy and drug design discovery . alzplatform , a one - stop integrated cloud computing server , has been specifically developed as a public repository http://www.cbligand.org/ad/ for ad drug and targets research . the cloud computing server will augment our capacity to benefit the ad research community and will help break to the knowledge barrier , enhance the productivity of chemogenomics researchers , and accelerate advances in system biology computer - aided drug design by consolidating existing data and computational technology .

high blood pressure is estimated to affect more than one in three adults aged 25 and over , or about 1 billion people worldwide.1 this means that nearly 30% of our patients are suffering from hypertension . various drugs are currently used to treat hypertension , but the effects of these drugs on other age - related problems have not been well studied . drugs that are the primary choice for the treatment of hypertension include calcium channel blockers ( ccbs).2 dihydropyridine derived ccbs , such as amlodipine ( aml ) , lacidipine , and nicardipine , are widely used to treat hypertension . large clinical trials have confirmed their usefulness for preventing cardiovascular events by lowering blood pressure and having an antioxidant effect on vessels in vitro.3,4 ccbs primarily inhibit calcium influx through the l - type voltage - dependent calcium channels at the level of vascular smooth muscle , thereby disrupting the excitation contraction process.5,6 however , as these ccbs interfere in the transportation of calcium through cell membranes , they may affect many metabolic processes , including bone metabolism.7,8 a number of clinical and experimental studies have reported that the effects of ccbs are associated with skeletal calcium metabolism due to the presence of l - type calcium channels in osteoblastic cells.9 regulators of bone formation modulate the channel s expression , suggesting a functional involvement of l - type calcium channels in bone remodeling.10 aml is a third generation dihydropyridine l - type ccb used in the treatment of angina pectoris , arterial hypertension , and cardiac arrhythmias.11 chronic use of ccbs in hypertensive patients may affect the repair process of osteogenesis after surgical and clinical procedures and may be important in the healing period , especially in guided bone regeneration procedures . halici et al12 suggested that aml has a beneficial effect on bone metabolism and an antihypertensive effect . moraes et al13 stated that chronic use of aml compromised bone formation in the early stage of the repair process of surgical defects in the ramus of rat mandible . ushijima et al14 showed that aml prevents reduction in bone density during repeated dosing in stroke - prone , spontaneously hypertensive rats . however , shimizu et al9 proposed that aml showed no action on bone metabolism in ovariectomized hypertensive rats . despite the convincing evidence given above , there are insufficient data about the effects of ccb use on bone physiology in humans . during wound healing , platelets are among the first cells to respond at a wound site , as they are critical to the initiation of this process.1517 recently , platelet rich plasma ( prp ) has become a valuable adjunct used to promote healing in many procedures such as oral surgery , periodontology , otolaryngology , head neck surgery , and neurosurgery.18,19 prp is a product derived from autologous blood , and its preparation is intended to obtain a high platelet concentration in a small volume of plasma . one of the potential advantages of prp is that it is possible for it to be obtained from the patient s own blood after a simple centrifugation procedure.20 both , the plasma and its preparates contain growth factors that play a role during the initial phase of healing and bone regeneration.21,22 various authors have described prp as an effective means of improving the healing of both hard and soft tissues , resulting in reductions in pain , inflammation , and trismus , as well as an acceleration of the bone regeneration process . however , there are some controversial results in the literature , and there have been low numbers of systematic studies carried out to date.23,24 the rapid recovery after surgical procedures is crucial in order to regain normal functioning of the bone and its surrounding structures . there have been an increasing number of studies on this subject.25 the purpose of this study was to make a biochemical and histological evaluation of the effects of aml , prp , and a combination of both materials on the repair process of a surgical defect in rat tibia , as well as to verify possible alterations in alkaline phosphatase ( alp ) and serum bone - specific alkaline phosphatase ( s - bone alp ) levels . the study included 56 male wistar albino rats ( mean age 12 weeks ; weight 290350 g ) and was conducted at the health institution of research centre , afyon kocatepe university , afyonkarahisar , turkey . the animals were housed in groups of seven per plastic cage in a controlled environment ( 22c ; 12-hour light dark cycle ) with free access to drinking water and a diet of standard laboratory rat food pellets . the experimental protocol of the study was approved by the animal experimentation ethics committee of kocatepe university ( 2013 - 49533702/361 ) . the animals were maintained and used in accordance with the animal welfare act and guidance for the care and use of laboratory animals . the rats were randomly divided into four groups : group a ( control group ) , tibia defect model with no treatment ( n=14);group b ( aml group ) , tibia defect model treated with aml , 0.04 mg / rat / day by oral gavage ( n=14);group c ( prp group ) , tibia defect model treated with local prp ( n=14);group d ( aml + prp group ) , tibia defect model treated with 0.2 ml local prp and with aml 0.04 mg / rat / day by oral gavage ( n=14 ) . group a ( control group ) , tibia defect model with no treatment ( n=14 ) ; group b ( aml group ) , tibia defect model treated with aml , 0.04 mg / rat / day by oral gavage ( n=14 ) ; group c ( prp group ) , tibia defect model treated with local prp ( n=14 ) ; group d ( aml + prp group ) , tibia defect model treated with 0.2 ml local prp and with aml 0.04 mg / rat / day by oral gavage ( n=14 ) . groups a and c received daily oral doses of sterile physiological saline ( 0.9% nacl ) in order to simulate any possible influence of physiological and physical stress on bone healing caused by gavage . amlodis ( 5 mg tablet ) and cefamezin ( 1 g vial ) were purchased from zentiva ( istanbul , turkey ) . ketalar 10 ml was purchased from pfizer ( istanbul , turkey ) and xylazine 25 ml was purchased from bayer ( istanbul , turkey ) . aml was dissolved in sterile physiological saline ( 0.9% nacl ) and diluted to the given concentration . blood was obtained from two rats after anesthesia ; a total of 20 ml of whole blood from two rats was collected through intracardiac aspiration and mixed with 2 ml of the anticoagulant , citrate dextrose a. after centrifugation at 3,500 rpm for 12 minutes , red blood cells , prp , and platelet - poor plasma were separated . the prp was divided into 0.2 ml portions for each rat in these two groups . the total platelet count in 1 ml of prepared prp was determined and confirmed with a phase - contrast microscopic platelet count . surgical procedures were performed under general anesthesia with a combination of ketamine chlorhydrate ( ketalar ; pfizer 0.08 ml/100 g body weight ) and xylazine 2% ( rompun ; bayer ; 0.04 ml/100 g body weight ) . a monocortical bone defect on the left tibia taking the right and left legs into a flexion position , the overlying skin of the left tibia was shaved and disinfected with iodated alcohol . a 1.5-cm skin and muscle incision was then made to expose the bone surface of the middle third of the tibia where , on the wider region , a monocortical defect was created . a 1 mm diameter rod and a size 12 stainless steel dental burr ( meisinger gmbh , neuss , germany ) were used on a low - speed hand piece , under constant sterile saline irrigation , to create an oval bone defect measuring approximately 2 mm in width and 3 mm in length , with the cortical thickness . the muscular layer was sutured with resorbable 5.0 catgut suture ( dogsan , trabzon , turkey ) , and the skin was sutured with interrupted 3.0 silk suture ( dogsan ) . upon completion of the surgical procedure , each animal received a single dose of cefazolin sodium , 50 mg / kg ( cefamezin ; zentiva ) by intramuscular injection . following surgery , seven rats per study group were killed on days 21 and 30 , using high dose combination of ketamine and xylazine . histological samples were fixed in 10% formalin for 72 hours and decalcified in 10% ethylenediamine tetraacetic acid ( edta ) solution ( for decalcification ) for approximately 2 months . after complete decalcification , dehydration was carried out in a graded alcohol series , and the samples were embedded in paraffin blocks . findings of fibrous tissue , cartilage tissue , immature ossification , and mature bone formation were seen in the groups . according to these findings , semiquantitative classification of the bone healing was made as follows : good healing = three ( 3 ) points = trabecular bone with lamellar bone cells and compact bone ( mature bone).fair healing = two ( 2 ) points = trabecular bone with wowen bone cells ( immature bone).poor healing = one ( 1 ) point . good healing = three ( 3 ) points = trabecular bone with lamellar bone cells and compact bone ( mature bone ) . fair healing = two ( 2 ) points = trabecular bone with wowen bone cells ( immature bone ) . blood samples were collected at the time of sacrifice by cardiac puncture and centrifuged for plasma separation . for biochemical examination , alp levels were measured using an autoanalyzer ( cobas c501 ; roche diagnostics , in , usa ) in the biochemistry laboratories of afyon kocatepe university . the statistical calculations were carried out with the spss software package , version 15.0 ( spss inc . , the study included 56 male wistar albino rats ( mean age 12 weeks ; weight 290350 g ) and was conducted at the health institution of research centre , afyon kocatepe university , afyonkarahisar , turkey . the animals were housed in groups of seven per plastic cage in a controlled environment ( 22c ; 12-hour light dark cycle ) with free access to drinking water and a diet of standard laboratory rat food pellets . the experimental protocol of the study was approved by the animal experimentation ethics committee of kocatepe university ( 2013 - 49533702/361 ) . the animals were maintained and used in accordance with the animal welfare act and guidance for the care and use of laboratory animals . the rats were randomly divided into four groups : group a ( control group ) , tibia defect model with no treatment ( n=14);group b ( aml group ) , tibia defect model treated with aml , 0.04 mg / rat / day by oral gavage ( n=14);group c ( prp group ) , tibia defect model treated with local prp ( n=14);group d ( aml + prp group ) , tibia defect model treated with 0.2 ml local prp and with aml 0.04 mg / rat / day by oral gavage ( n=14 ) . group a ( control group ) , tibia defect model with no treatment ( n=14 ) ; group b ( aml group ) , tibia defect model treated with aml , 0.04 mg / rat / day by oral gavage ( n=14 ) ; group c ( prp group ) , tibia defect model treated with local prp ( n=14 ) ; group d ( aml + prp group ) , tibia defect model treated with 0.2 ml local prp and with aml 0.04 mg / rat / day by oral gavage ( n=14 ) . groups a and c received daily oral doses of sterile physiological saline ( 0.9% nacl ) in order to simulate any possible influence of physiological and physical stress on bone healing caused by gavage . amlodis ( 5 mg tablet ) and cefamezin ( 1 g vial ) were purchased from zentiva ( istanbul , turkey ) . ketalar 10 ml was purchased from pfizer ( istanbul , turkey ) and xylazine 25 ml was purchased from bayer ( istanbul , turkey ) . aml was dissolved in sterile physiological saline ( 0.9% nacl ) and diluted to the given concentration . blood was obtained from two rats after anesthesia ; a total of 20 ml of whole blood from two rats was collected through intracardiac aspiration and mixed with 2 ml of the anticoagulant , citrate dextrose a. after centrifugation at 3,500 rpm for 12 minutes , red blood cells , prp , and platelet - poor plasma were separated . the prp was divided into 0.2 ml portions for each rat in these two groups . the total platelet count in 1 ml of prepared prp was determined and confirmed with a phase - contrast microscopic platelet count . surgical procedures were performed under general anesthesia with a combination of ketamine chlorhydrate ( ketalar ; pfizer 0.08 ml/100 g body weight ) and xylazine 2% ( rompun ; bayer ; 0.04 ml/100 g body weight ) . a monocortical bone defect on the left tibia taking the right and left legs into a flexion position , the overlying skin of the left tibia was shaved and disinfected with iodated alcohol . a 1.5-cm skin and muscle incision was then made to expose the bone surface of the middle third of the tibia where , on the wider region , a monocortical defect was created . a 1 mm diameter rod and a size 12 stainless steel dental burr ( meisinger gmbh , neuss , germany ) were used on a low - speed hand piece , under constant sterile saline irrigation , to create an oval bone defect measuring approximately 2 mm in width and 3 mm in length , with the cortical thickness . the muscular layer was sutured with resorbable 5.0 catgut suture ( dogsan , trabzon , turkey ) , and the skin was sutured with interrupted 3.0 silk suture ( dogsan ) . upon completion of the surgical procedure , each animal received a single dose of cefazolin sodium , 50 mg / kg ( cefamezin ; zentiva ) by intramuscular injection . following surgery , seven rats per study group were killed on days 21 and 30 , using high dose combination of ketamine and xylazine . histological samples were fixed in 10% formalin for 72 hours and decalcified in 10% ethylenediamine tetraacetic acid ( edta ) solution ( for decalcification ) for approximately 2 months . after complete decalcification , dehydration was carried out in a graded alcohol series , and the samples were embedded in paraffin blocks . findings of fibrous tissue , cartilage tissue , immature ossification , and mature bone formation were seen in the groups . according to these findings , semiquantitative classification of the bone healing was made as follows : good healing = three ( 3 ) points = trabecular bone with lamellar bone cells and compact bone ( mature bone).fair healing = two ( 2 ) points = trabecular bone with wowen bone cells ( immature bone).poor healing = one ( 1 ) point . good healing = three ( 3 ) points = trabecular bone with lamellar bone cells and compact bone ( mature bone ) . fair healing = two ( 2 ) points = trabecular bone with wowen bone cells ( immature bone ) . blood samples were collected at the time of sacrifice by cardiac puncture and centrifuged for plasma separation . for biochemical examination , alp levels were measured using an autoanalyzer ( cobas c501 ; roche diagnostics , in , usa ) in the biochemistry laboratories of afyon kocatepe university . the statistical calculations were carried out with the spss software package , version 15.0 ( spss inc . , chicago , il , usa ) for windows . no unwanted condition developed in any rat , and none of the rats were excluded from the study . all of the animals tolerated the procedure well and demonstrated good homeostasis and rapid recovery from anesthesia . according to multiple comparisons with kruskal wallis test , there were significant differences among all study groups at day 21 ( p<0.05 ) , but no significant differences at day 30 ( p>0.05 ) . whitney u - test , there was no statistically significant difference between days 21 and 30 in group b ( p>0.05 ) . the level of bone healing in groups c ( prp ) and d ( aml + prp ) was significantly higher than that of group a ( control ) at day 21 ( p<0.05 ) . there was also significantly better healing in group d ( aml + prp ) than group a ( control ) at day 30 ( p<0.05 ) ( table 1 ) ( figures 18 ) . the serum levels of alp and s - bone alp showed statistically significant mean values for the experimental groups when compared with the control group . changes in alp levels as a bone remodeling marker after treatments are given in tables 2 and 3 . according to multiple comparisons with kruskal wallis test , there were significant differences among all the study groups at day 21 ( p<0.05 ) , but no significant differences at day 30 ( p>0.05 ) . statistically ( with mann whitney u - test ) , aml did not affect alp and s - bone alp activity at 21 and 30 days ( p>0.05 ) , but prp and aml + prp increased alp and s - bone alp activity statistically ( p<0.05 ) . wallis test , there were significant differences among all study groups at day 21 ( p<0.05 ) , but no significant differences at day 30 ( p>0.05 ) . whitney u - test , there was no statistically significant difference between days 21 and 30 in group b ( p>0.05 ) . the level of bone healing in groups c ( prp ) and d ( aml + prp ) was significantly higher than that of group a ( control ) at day 21 ( p<0.05 ) . there was also significantly better healing in group d ( aml + prp ) than group a ( control ) at day 30 ( p<0.05 ) ( table 1 ) ( figures 18 ) . the serum levels of alp and s - bone alp showed statistically significant mean values for the experimental groups when compared with the control group . changes in alp levels as a bone remodeling marker after treatments are given in tables 2 and 3 . according to multiple comparisons with kruskal wallis test , there were significant differences among all the study groups at day 21 ( p<0.05 ) , but no significant differences at day 30 ( p>0.05 ) . statistically ( with mann whitney u - test ) , aml did not affect alp and s - bone alp activity at 21 and 30 days ( p>0.05 ) , but prp and aml + prp increased alp and s - bone alp activity statistically ( p<0.05 ) . this study aimed at assessing the possible effects of antihypertensive drugs and prp on bone healing . the effects of aml and prp on the healing stage of a tibial bone defect in rats were evaluated by histopathological analyses , and levels of alp were also examined . appropriate hypertension treatment with antihypertensive drugs seems to provide a better quality of life with low morbidity and mortality rates . ccbs , angiotensin - converting enzyme inhibitors , angiotensin ii receptor type 1 antagonists , diuretics , -blockers , and -blockers may be used as a primary choice for the treatment of hypertension . as ccbs are widely used in the treatment of hypertension and angina,12 aml was selected as the working material for this study . the daily dose used was 0.04 mg of aml , consistent with the prescription for humans of 510 mg / day , the lower dose being suggested for elderly patients and those with hepatic insufficiency.24 the outcome of various clinical and surgical procedures in the treatment of bone defects is affected by several factors such as patient , defect , and surgical variables . an awareness of systemic conditions and drugs that could affect alveolar bone may be important to identify patients at increased risk of poor clinical and postoperative results . thus , the present study focused on the impact of aml on defect healing . the physiological , cellular , and molecular mechanisms by which antihypertensive drugs may affect bone tissues are not yet fully understood , and to the best of our knowledge the results of the current study showed that aml had neither a positive nor a negative effect on bone defect healing , although the combination of aml and prp could be beneficial to the defect healing process . to the best of the authors knowledge , this is the first study that used an aml and prp combination for the treatment of a bone defect . these results may have arisen due to the synergistic effect of aml and prp on bone healing . a study by tefilo et al,8 demonstrated the alveolar bone repair process in rats under conditions of chronic aml , used at a dose identical to that of the current study ; a decrease from 20% to 30% in the volume of newly formed bone was reported . in another study by moraes et al13 a period of 14 and 30 days was evaluated for both the aml and control groups , and newly formed bone was reduced when compared with the control group in 14 days , but aml did not affect newly formed bone in 30 days , with the same dose as used as in the current study . gradoov et al26 assumed that aml did not significantly influence bone mineral density for 8 weeks . however , 1 year before this study , gradoov et al27 suggested that aml could prevent osteoporosis for 12 weeks . a study by ushijima et al14 reported that aml prevented the reduction of bone density of the femur . accordingly , amlodipine and lacidipine prevented ovariectomy - induced bone loss in osteopenic rat femur.12 despite the numerous studies mentioned above , there is insufficient knowledge about the effects of ccb use on bone healing metabolism . during the last 20 years , prp has been used to enhance bone regeneration and soft tissue maturation for maxillofacial surgery as well as periodontology , ranging from filling postextraction alveolar sockets to more complex surgery.2831 prp can be used alone or in conjunction with bone grafts in the surgical site.24 it is prepared with the intention of influencing soft and hard tissue repair and/or regeneration.32 it is thought that the contribution of prp to the bone healing process is based on the growth factors present in it which promote proliferation , chemotaxis , and the differentiation of cells which are essential to osteogenesis . the concentration of growth factors is seven times higher in prp than in normal blood clots.33 prp contains growth factors such as platelet - derived growth factor , epithelial growth factor , transforming growth factor- , vascular endothelial growth factor , insulin growth factor-1 , and basic fibroblast growth factor.24,34 therefore , prp may influence bone healing through a variety of pathways . in addition , prp has been reported to have benefits such as the improved handling of particulate grafts , facilitation of graft placement and stability , improved rate and quality of new bone formed , hemostasis when used in conjunction with the cell - poor serum layer containing factor xa , shorter implant exposure time , enhanced vascular ingrowth , mitogenic effects , natural biological sealant , no risk of disease transmission due to its autologous nature , and it is not expensive.35 the vast majority of published clinical studies suggests that prp accelerated bone healing,18,23,3641 while other authors have reported different results.4248 the scientific evidence regarding the efficacy and efficiency of prp is still controversial . however , in a recent study , daif 49 investigated the effect of autologous prp on bone regeneration in mandibular fractures . the authors concluded that direct application of prp along the fracture lines may enhance bone regeneration . similarly , prp improved bone repair alone , not in 30 days , but in 21 days . these effects are mainly noticeable during the early stages of wound healing.50 controversial results regarding the effectiveness of prp may partly be due to wide variations in manufacturing methods.51 it has been reported that differences in proportion and levels of growth factors may be found between currently available commercial prp systems because of the concentration of platelet that , in turn , may affect results.52 differences in the results of the studies might also be related to the study design . alp activity is a marker for the differentiation of cells with osteogenic potential into mature osteoblasts.53 alp is generally considered to play a key role in calcification in vitro . calcification does not occur in the absence of alp activity.54 alp was evaluated as a bone - remodeling marker by biochemical analysis in the current study . in this study , there was no statistically significant difference in the serum levels of alp and s - bone alp between the control group and the aml groups for the periods after 21 and 30 days . similar to the histopathological results , the combination of aml and prp increased the alp levels . tefilo et al8 demonstrated that alp levels of the aml groups were of nearly the same concentration as in the control group at day 30 , but there was a significant decrease at day 14 . gradoov et al26 suggested that the serum concentration of s - bone alp greatly decreased to 12% in the aml group in comparison with the control group . on the other hand , kosaka and uchii55 found that benidipine , a ccb , increased alp activity of osteoblastic cells isolated from neonatal mouse calvaria . the findings of the current study revealed the positive effects of prp on alp activities . kasten et al57 showed that addition of prp reduced the alp activity in mesenchymal stem cells in -tricalcium phosphate ( msc / beta - tcp ) composites significantly at explantation . in accordance with our study and incompatible with the above mentioned studies , chen et al58 showed significantly increased alp concentration in blood at 4 weeks in the prp group . the mechanisms of effects of ccbs and prp have not been fully understood yet , but they are mostly attributed to changes in bone remodeling . . however , the variability in the histopathological and biochemical results of the studies can be related to the differences in methodological designs . a single dose of aml ( 0.04 mg / rat / day ) was applied to the rats , and it can be speculated about the effect of different dosing regimens on bone defect healing . however , there are some ethical restrictions due to the increase in the number of animals for different dosing regimens . another limitation was that in the current study , the immunohistochemical expression of the markers , osteopontin , osteonectin , and osteocalcin , that play an important role in the mineralization and healing of the bone could not be examined . besides , histopathologic and serum findings could not be supported with radiographic measures to determine the healing outcome of the study groups . the current study has other limitations , such as the brief monitoring period as well as the fact that there was no hemodynamic monitoring of the animals during our experiment . it can be concluded that aml had neither a positive nor a negative effect on bone healing at the repair stage . however prp is beneficial in the tibial healing process both at 21 days . in light of these results , prp , especially , could be a viable alternative to accelerate the healing of bone in the early stages . but in the daily clinical practice , many clinicians are still skeptical about the current levels of evidence about the impact of ccbs on bone mineral metabolism and efficacy of prp on soft / hard tissue healing . high quality , robust , randomized controlled studies are needed to investigate the effects of different dose regimens of aml and prp in the promotion of bone healing . such information from in vivo / in vitro studies may clarify the potent action of aml and prp on bone defect healing and may lead to finding a novel therapeutic target for surgical procedures , and systemic and bone diseases .

in a modern lifestyle people are chronically exposed to elevated amounts of lipids and nutrients , potentially leading to tissue dysfunction and disease . the adipose tissue is highly involved in the development of metabolic disorders such as cardiovascular disease ( cvd ) and type 2 diabetes mellitus ( t2 dm ) . it is the body 's largest storage site for triglycerides and plays an important role as an endocrine organ in energy homeostasis . the adipose tissue is divided into specific regional depots with differences in structural organization , cellular size , and biological function . the distribution of fat between these depots seems to be more important than the total adipose tissue mass for the risk of developing obesity - associated diseases ( figure 1 ) . normally , fat storage in the form of neutral triglycerides takes place during food intake to be replaced by fat mobilization processes during fasting or situations with elevated energy demand ( figure 2(a ) ) . adipocytes produce a number of endocrine hormones that contribute to the regulation of this mechanism , such as adiponectin and leptin . as adipocytes play a key metabolic role as a source of fuel , they are required to respond acutely to changes in the nutritional levels . as a result they are tightly regulated by both hormonal ( e.g. , insulin ) and sympathetic ( e.g. , adrenergic ) stimulation . subjects will display elevated levels of glucose and nonesterified fatty acids , also called free fatty acids ( ffas ) , and show symptoms of one or more afflictions amongst which hypertension , hypertriglyceridemia , diabetes , and obesity are the most usual . lipotoxitcy and the accumulation of saturated fat in peripheral tissues are an important step in the development of metabolic syndrome . fatty liver ( liver steatosis ) is found in a large proportion of individuals with body mass index ( bmi ) above 30 . liver steatosis contributes to insulin resistance and may lead to the development of nonalcoholic fatty liver diseases ( naflds ) , including nonalcoholic steatohepatitis ( nash ) and cirrhosis . t2 dm is usually linked to obesity and lipid redistribution to nonadipose organs and tissues and is caused by a reduced sensitivity to insulin in muscle , liver , and adipose tissue . during insulin resistance , an increase in insulin does not lead to a corresponding increased uptake of glucose in muscle and liver . as a compensatory mechanism insulin production t2 dm occurs when the increased insulin production is no longer sufficient , or when lipid - induced beta cell malfunction takes place , particularly in genetically predisposed individuals . it is believed that high levels of circulating ffa and lipotoxic mechanisms can cause damage on -cell and nonislet tissues . several of the high - risk factors for cvd result directly or indirectly from obesity and a malfunctioning adipose tissue : dyslipidemia , chronic inflammation , diabetes , and hypertension . mitochondrial dysfunction could also contribute to cvd due to their role as targets and sources of reactive oxygen species ( ros ) , both in the formation of vascular lesions and in their involvement in the development of insulin resistance and t2 dm . a well - functioning adipose tissue with capacity to neutralize and store nutritional overload is therefore necessary to protect the body from peripheral insulin resistance . two closely linked processes in adipose tissue are important for the development of metabolic disease : adipose tissue inflammation and adipose tissue hypertrophy . adipose tissue inflammation due to the recruitment of t - cells and macrophages has been shown to contribute to insulin resistance in obese individuals . this inflammation leads to a disturbed adipokine - balance and an uncontrolled release of free fatty acids and inflammatory cytokines . as a result , lipoprotein metabolism and insulin sensitivity in other organs will be affected . during obesity , the adipocyte size increases ( hypertrophy ) , and they are eventually unable to store excess lipids even with enhanced adipocyte proliferation ( hyperplasia ) . this redirects fatty acids to the liver promoting dyslipidemia , characterized by elevated plasma ffa , triglycerides ( tgs ) , and small dense low - density lipoprotein ( ldl ) , and the reduction of high - density lipoproteins ( hdli ) . in addition large adipocytes are characterized by an increased lipolysis , the breakdown of stored tg by lipases . this results in ffa and glycerol , which further increases plasma ffa - levels and upregulates tg synthesis in the liver . a recent publication demonstrated that enlarged adipocytes in obese men had a reduced rate of ffa - delivery to the blood stream , resulting in normal systemic ffa concentrations . however , after meals the adipose tissue storage capacity was severely impaired , thus potentially causing ectopic lipid deposition . how well the adipose tissue functions as a storage organ for excess energy intake and the distribution of fat and mitochondria between the different adipose depots may determine the large differences in health observed in obese individuals . here , we will first focus on the importance of mitochondrial function in adipose tissue and then describe the different adipose tissue depots , in rodent animal models as well as humans ( figure 1 ) , and their impact on the development of metabolic diseases . pharmacological treatments that specifically target mitochondria have great potential in the treatment of obesity - related disorders , and their differential effect on adipose depots will be discussed . an elevated metabolism during obesity results in increased production of reactive oxygen species ( ros ) . ros production is an early event in glucose intolerance , contributing to pancreatic -cell dysfunction as well as liver steatosis . -cells may be specially susceptible to ros - damage due to their relatively low expression of free - radical - detoxifying enzymes compared to most other cell types . there are indications that mitochondrial content and function in adipose tissue might be disrupted in metabolic disorders . it has been suggested that insulin resistance is linked to mitochondrial dysfunction [ 10 , 11 ] . furthermore , it is increasingly evident that oxidative capacity and thus mitochondria are fundamental to avoid the development of toxicity during excess energy intake . for instance , increase in mitochondrial ros is observed in adipose tissue during obesity [ 1214 ] , and in skeletal muscle , the mitochondrial h2o2 emission is higher using fatty acids as substrates versus carbohydrate - based substrates [ 15 , 16 ] . two models of insulin resistance , induced by either tumor necrosis factor ( tnf ) or glucocorticoid treatment , were both shown to involve increased ros levels . also , antioxidants attenuating mitochondrial h2o2 emission completely restored insulin sensitivity in skeletal muscle . these findings show that mitochondria play a central role in the physiology leading to insulin resistance . in addition , the nox family of nadph oxidases , located to the plasma membrane , cytosol and cytosolic membranes , is a major source of ros generation . nox2 is an energy transporter that catalyses the reduction of oxygen to o2 in an nadph - dependent reaction . the involvement of nox enzymes in the development of metabolic syndrome has been reviewed by krause and coauthors [ 18 , 19 ] . in low concentrations , ros participates in the cell signaling as secondary messengers . in response to insulin , nox will release ros that activates the distal insulin - signaling cascade , mobilizing glucose transporters to the surface of adipocytes . however in higher amounts it will have serious harmful effects on the cells , including decreased glucose uptake in adipocytes . several mouse obesity models have found an increase in oxidative stress in adipose tissue associated with overexpression of nadph oxidase and repression of antioxidative enzymes such as catalase . also , mitochondrial proliferation is inhibited by increased ros production [ 22 , 23 ] , indicating that ros can modulate adipocyte generation . mature adipocytes require large amounts of atp to maintain their activities such as lipolysis , fatty acid -oxidation , and fatty acid synthesis . in the organism , mitochondria produce energy and are central in the fatty acid metabolism since more than 98% of fatty acids are oxidized in mitochondria . recent evidence indicates that a reduced mitochondrial function and altered biogenesis play important roles in the etiology of obesity , insulin resistance , and t2 dm . an impaired metabolic flexibility is the inability of an organism to adapt fuel oxidation to fuel availability . the ability to switch from fat to carbohydrate oxidation is usually impaired in subjects prone to obesity and in subjects with a family history of diabetes [ 2629 ] . indeed , there is strong evidence that defects in substrate switching cluster together with disturbances in mitochondrial content and/or function and might be a manifestation of an underlying mitochondrial disorder . in this respect , it has been demonstrated that insulin - resistant offspring of patients with t2 dm have reduced mitochondrial function , reduced atp synthesis and accumulation of fat in skeletal muscle , the liver , and other cells . the major adipose depots in the body consist of white adipose tissue ( wat ) . mature white adipocytes require large amounts of atp to maintain their diverse functions , and during adipogenesis mitochondrial biogenesis is an important process . one of the main tasks of adipose tissue is to generate glycerol 3-phosphate and acetyl - coa for esterification into tgs ( lipogenesis ) , processes localized to the mitochondrial matrix as well as the cytosol , that require an abundant mitochondrial population [ 32 , 33 ] . mitochondria in wat are also believed to be important for the regulation of lipolysis , the process of degrading tg into ffa and glycerol ( figure 2(a ) ) . when inhibiting the mitochondrial respiratory chain or decreasing intracellular atp by the use of mitochondrial uncouplers , lipolysis is inhibited . inhibition of the amp - activated protein kinase ( ampk ) , which is an essential molecular sensor regulated by the atp / amp level in the cells , will decrease lipolysis stimulated by agonists of -adrenoreceptors . the ffas produced by lipolysis can subsequently be utilized in the adipocytes as a source of energy , through -oxidation in the mitochondria . thus , mitochondria in white fat cells are involved in both de novo lipogenesis and lipolysis . depending on the anatomical position in the body for example , rat epididymal ( vat ) adipocytes have more mitochondria than inguinal ( sat ) adipocytes . the higher number of mitochondria per mg tissue in vat than sat is also observed in obese human individuals . although mitochondrial respiratory flux per cell and per mitochondrial content was lower in vat compared to sat due to smaller cells , the visceral fat was bioenergetically more active and responsive to substrates of the electron transport chain . in general , some studies show that obesity and t2 dm in humans reduce mitochondrial number in white adipocytes , while others show a connection between mitochondrial number and lipogenesis , but not body mass index ( bmi ) or overall insulin sensitivity . the link between mitochondrial dysfunction and t2 dm is indicated by the reduced oxidative phosphorylation ( oxphos ) capacity in elderly subjects , and in obese subjects , where a reduced fa -oxidation in several tissues including adipocytes will increase blood ffa levels and alter glucose uptake [ 10 , 11 ] . nonalcoholic steatohepatitis , a disease caused by lipid accumulation in the liver , is associated with mitochondrial dysfunction due to increased lipid peroxidation , alterations in mitochondrial ultrastructure , depletion in mtdna , and low oxphos activity . these patients also commonly display abdominal obesity , diabetes , and hypertriglyceridemia with insulin resistance . in rodent genetic or high - fat fed obesity models , the expression of genes involved in mitochondrial atp production , energy uncoupling and other genes important for mitochondrial function was downregulated compared to lean control animals . although brown adipose tissue ( bat ) is mainly found in newborns , recent studies have demonstrated its presence in adults [ 44 , 45 ] . bat originates from a different cellular lineage than wat , but they share many features . however , mature bat contains a higher number of mitochondria and lower numbers of lipid droplets . during cold temperatures and activation of the sympathetic nervous system , brown adipocyte lipolysis occurs ( figure 2(b ) ) . instead of being released for subsequent oxidation in organs such as liver and skeletal muscle , the resulting ffas are oxidized in the bat mitochondria and used to generate heat ( see review in ) . this process involves transport of ffa complexed to fatty acid binding proteins ( fabps ) into the mitochondria by the carnitine shuttle system , where they are oxidized and used to generate heat by uncoupling protein 1 ( ucp1 ) , a process called nonshivering thermogenesis . this process is important in newborn humans and in rodents , but appears to be almost lost in adult humans . although ucp1 is the hallmark of bat , increased ucp1 expression and energy expenditure can also be seen in wat under some conditions and may be a desirable effect during the treatment of obesity . the increased expression of the ucp1 gene in both bat and wat by a fat - specific ap2 promoter generated mice partially resistant to age - related obesity , both genetically and high - fat diet induced [ 50 , 51 ] . peroxisome proliferator - activated receptors ( ppars ) are important regulators of fatty acid metabolism . they serve as lipid sensors since they are activated by metabolic derivates of fatty acids in the body . there are three ppar superfamily members ppar , ppar , and ppar ( also called ppar ) that act upon ligand activation by controlling networks of target genes . it exceeds the scope of this paper to describe their function in detail , but the relevant ppars will be mentioned briefly here . for a comprehensive review on the role of ppars in metabolic syndrome , see guri et al . . ppar is the most important ppar in the liver and has many important target genes involved in fatty acid -oxidation . during fasting , the increase in lipolysis provides ffas that are used as energy source in other tissues . the subsequent ppar-regulated metabolism of fatty acids mainly takes place in the liver , through mitochondrial and peroxisomal -oxidation , but the heart , adipose tissue , and skeletal muscle can also utilize ffa for energy . the decrease in lipogenesis is on the other hand due to downregulation of the two main lipogenic transcription factors , ppar and steroyl regulatory element binding protein 1c ( srebp1c ) [ 54 , 55 ] . two isoforms of ppar exist , of which ppar1 is expressed in a wide variety of tissues , while ppar2 is mainly expressed in adipose tissue . ppar activation stimulates preadipocyte differentiation , promotes the storage of fatty acids in mature adipocytes , and can activate glut4 , facilitating increased fatty acid synthesis from glucose . its activation has been shown to create ligands for ppar , as well as regulate ppar expression in cultured adipocytes . lipogenesis mainly takes place in the cytosol , but the activation of ppar using rosiglitazone has been shown to alter both mitochondrial density and morphology in adipocytes suggesting that ppar also controls mitochondrial functions . in obese db / db mice , the adiponectin expression and mitochondrial content of white adipocytes are reduced , and these effects are reverted by the activation of ppar by rosiglitazone . it is interesting to note that rosiglitazone can increase expression of mitochondrial genes / mitochondrial biogenesis in rodent obesity models . although the total adipose tissue is important for the development of insulin resistance , it is believed that some fat depots are more linked to risk factors for disease than others . the main adipose depots of interest are found in the abdomen and can be divided into the sat and vat , and the visceral tissue can again be divided into omental and mesenteric , the latter being the more deeply buried depot surrounding the intestine ( figure 1 ) . the distribution of sat and vat shows person - to - person variations and is dependent on several factors such as age , nutrition , sex , and the energy homeostasis of the individual adipose tissues . although several similarities exist , the differences in rodent and human adipose tissues entail caution when choosing which depots to study and when extrapolating information between species . particularly , visceral fat depots , including omental and mesenteric adipose tissue , represent a risk factor for the development of cvd and t2 dm . visceral adipose tissue mass correlates with development of insulin resistance , while total or subcutaneous tissue mass does not [ 62 , 64 , 65 ] . it has been thoroughly confirmed that the adipocytes of visceral fat tissue are more lipolytically active than subcutaneous adipocytes and thus contribute more to the plasma free fatty acid levels [ 62 , 66 ] . this was found in particular in diabetic obese individuals , where it was linked to a significant upregulation of leptin and downregulation of adiponectin gene expression in mesenteric vat compared to sat and omental vat . the metabolic activity of a cell is dependent on its mitochondrial content , and it has been shown that , in rats , epididymal ( vat ) adipocytes have more mitochondria than inguinal ( sat ) adipocytes . in adipose tissue from obese individuals undergoing bariatric surgery the relative oxphos activity was found to be higher in omental vat than sat . individuals with a polymorphism in the ucp1 promotor reducing ucp1 gene expression are prone to have a high bmi , in particular due to abdominal obesity . thus , the level of mitochondrial uncoupling and energy efficiency may have an effect on obesity in wat as well as bat . the higher expression level of beta - adrenergic receptors in vat could contribute to the higher lipolytic activity . in addition , insulin - stimulated glucose uptake was found to be higher in vat compared to sat . as a result , excess visceral fat will enhance the level of free fatty acid delivered to the liver , thus increasing hepatic glucose and very low - density lipoprotein particles ( vldls ) output , and impair the hepatic insulin response . the expression of ppar mrna is increased in the adipose tissue of obese subjects , and it has been shown that while there was no difference in omental vat and sat , ppar was significantly higher expressed in mesenteric vat and remarkably so in obese diabetic subjects . lipid synthesis is shown to be higher in internal adipose tissues compared to sat . a study comparing the gene expression pattern in rat retroperitoneal ( rvat ) , mesenteric vat ( mvat ) , and inguinal sat ( isat ) showed that the larger cells in rvat expressed high amounts of the lipogenic transcription factors ppar and srebp1c compared to mvat and isat that showed a high expression of lipogenesis - related genes and a low expression of fatty acid oxidation - related genes . they also showed that the genes involved in lipid metabolism changed more rapidly as a result of fasting in the internal rvat depot compared to sat . as sat is less metabolically active than vat , it may have better short - term and long - term storage capacity . thus , this depot is important to accumulate tg in periods of excess energy intake and supply the organism with ffas in periods of fasting , starvation , or exercise . another suggested role of sat is to be a buffer during intake of dietary lipids , thus protecting other tissues from lipotoxic effects . in humans , sat is anatomically divided by a stromal fascia ( fascia superficialis ) into superficial ( ssat ) and deep subcutaneous adipose tissue ( dsat ) , with distinct histological features . while ssat has not been linked to risk for t2 dm , the size of dsat depots is significantly linked to the fasting insulin level and insulin - stimulated glucose utilization , as is total fat and vat [ 76 , 77 ] . the association between dsat and insulin resistance is particularly seen in male obese patients [ 77 , 78 ] . when studying the expression and secretion of hormones and cytokines in lean subjects , interestingly , sat seem to have an exclusive role in leptin secretion , since it correlates with plasma leptin levels ( in contrast to plasma insulin levels which correlate with inter abdominal fat ) . rat studies have shown that ppar2 expression is higher in male than in female sat , indicating a gender - linked variation in this adipose depot . the subcutaneous gluteofemoral fat tissue is measured by hip or thigh circumference , or leg adipose tissue mass . accumulation of fat in this depot is believed to have a protective role against diabetes and cardiovascular disease . indeed , low amount of this tissue has been associated with an unfavorable lipid and glucose profile . likewise , an increase in gluteofemoral tissue size has been connected to increase in hdl - cholesterol and decrease in total- and ldl - cholesterol levels in several studies [ 8385 ] . tracing of specific lipid fraction in blood samples from veins draining femoral or abdominal fat showed that there was a lower metabolic flux from femoral fat and that femoral fat had a preference for uptake of ffa and vldl - tg compared to chylomicron - tg , thus accumulating recycled fat rather than dietary fat . while the abdominal subcutaneous adipose tissue has a role as buffer during daily fatty acid intake , the gluteofemoral fat tissue may have an important role in tg storage . since it shows less metabolic activity and is more lipolytically inert than upper - body adipose depots , it seems to be involved in the long - term sequestering of fatty acids . few studies have focused on intermuscular adipose tissue ( imat ) , and little is known about its specific metabolic activities . however , due to its increased level in t2 dm patients , imat has been suggested be a risk factor along with vat for the development of obesity - related diseases . the amount of imat seems to be associated with age and lack of activity , and increases the likelihood of metabolic abnormalities in elderly subjects with normal body weight . in one study , only one third of men and less than half of women with t2 dm were obese , but in the normal weight subjects , high amounts of imat was associated with higher fasting insulin levels . also , imat correlates negatively with glucose infusion rate , and fasting glucose and total cholesterol level in caucasians , indicating its involvement in the development of insulin resistance . the amount of imat seem to be hereditary since african - americans , who are also prone to develop t2 dm , have more of this fat type . epicardial adipose tissue ( eat ) is the visceral fat layer located around the heart and is believed to be important for the buffering of the coronary arteries , and in providing fatty acids as a source of energy for the cardiac muscle . the release of adiponectin and adrenomedullin could have a protective effect on the heart during metabolic or mechanical insults . on the other hand , it has recently been shown that eat will locally influence heart and vasculature through the secretion of pro - inflammatory cytokines and will contribute to coronary atherosclerosis [ 9597 ] . studies indicate that the amount of eat can be related to carotid artery stiffness in obese patients with hypertension , while waist circumference shows no statistically significant link . age seem to determine the risk associated with epicardial fat , since there is no relationship between eat thickness and insulin resistance and metabolic syndrome in obese children . a study of patients undergoing coronary artery bypass surgery showed that the fat layer closest to the heart expressed 5-fold higher ucp1 mrna than the more distal substernal fat , while ucp1 expression was undetectable in subcutaneous thoracic fat . the ucp1 expression was influenced by age and body mass index , but showed no relationship to epicardial fat volume , waist circumference , metabolic syndrome or t2 dm . this , along with the increased expression of brown adipocyte differentiation transcription factors prdm16 and pgc-1 , indicate that epicardial fat may have a function similar to brown fat . this could possibly give an additional protection of the myocardium and coronary vessels from hypothermia , but its effect on obesity - linked disease is unknown . gonadal vat is one of the largest adipose depots in rodents and is found around the testis of males ( epididymal ) and around the ovaries of females ( periovarian ) . gonadal vat has been shown to express more of ppar and srebp1c as well as another key adipogenic transcription factor , ccaat enhancer - binding protein alpha ( c / ebp - alpha ) compared to sat . interestingly , ppar2 protein was found at a significantly higher level in female than in male rat perigonadal adipose tissue , indicating that sex hormones can affect the regulation of ppar2 in wat . it is possible that this could contribute to the gender differences observed with different ppar agonists . the largest region of brown adipose tissue is found in the upper back region of rodents ( interscapular bat ) . in humans , small areas are found in the thorax region ( supraclavicular ) , and in the chest and abdomen . there are indications that the nonshivering heat generation is important for the development of obesity . mice lacking ucp1 demonstrates increased obesity with age when fed high - fat diets , and selective destruction of bat leads to obesity and reduced energy expenditure and insulin resistance . interestingly , overnurished rat pups displayed excess weight gain , reduced thermogenic capacity , and lower levels of ucp1 as adults . it seems that reduced amount and thermogenic capacity of bat may contribute to a life - long predisposition for obesity . although adult humans possess only a small percentage of bat compared to wat , the possibility to pharmacologically increase bat amount or activity would be a potential treatment for obesity . recent studies have demonstrated that the forced expression of transcription factors prdm16 and c / ebp - beta is sufficient to generate brown fat cells from myoblastic precursors . the activation of ppar by different agonists has been shown to induce the expression of brown adipose genes in white adipocytes , the differentiation of brown adipose progenitor cells , and the proliferation of bat in rats and monkeys . however , long - term activation of the sympathetic nervous system by drugs such as ephedrine and bat proliferation with the ppar agonist the thiazolidinedione darglitazone have been linked to numerous side effects [ 108 , 109 ] . adipose tissue plays a major role in the inflammation , insulin resistance , and dyslipidemia associated with obesity . it is therefore beneficial to use compounds that therapeutically target adipose tissue to avoid high - risk complications of obesity such as t2 dm and cvd . dietary lipids may influence the energy balance and are shown to have a differential effect on different fat depots . studies in rats have shown that fish oil or n-3 polyunsaturated fatty acids ( n-3 pufa ) intervention reduces epididymal and perirenal adipose tissues although the body weight stays constant [ 110 , 111 ] . it is believed that fish oil can affect adipose tissue by the activation of ppar and , leading to decreased lipolysis , improved lipid storage capacity in subcutaneous adipose tissue , as well as anti - inflammatory effects possibly due to inhibitory action on nf-b . it has been shown that fish oil at 15% ( w / w ) level of substitution in a 35% high - fat diet prevented fat accumulation in c57bl/6 mice , preferentially in abdominal fat depots [ 113115 ] . this dose of fish oil also induced mitochondrial biogenesis in white fat , with a stronger effect in epididymal than in subcutaneous adipose tissue . ampk could be involved in this metabolic switch that increases adipocyte fatty acid metabolism , decreases lipolysis , and upregulates mitochondrial biogenesis . the anti - inflammatory effect of n-3 pufa in mice has been associated with increased adiponectin secretion [ 116 , 117 ] , possibly resulting from ampk activation . this induction of adiponectin is probably mediated by ppar and is higher in epididymal fat than in sat . the artificially made 3-thia sulphur fatty acid tetradecylthioacetic acid ( tta ) has a targeted effect on mitochondria and exhibit high potency compared to fish oil , both with regard to increased fatty acid -oxidation and anti - inflammatory effects . tta thus shows great promise in the treatment of metabolic syndrome ( figure 3 ) . it is a 16-carbon saturated fatty acid where a sulfur atom is inserted between the second and the third carbon in the -position , thus making it unavailable for fatty acid -oxidation . the mechanism of action of tta is based mainly on the activation of ppars , which induce mitochondrial biogenesis and enhance fatty acid oxidation in the liver [ 119121 ] . this has been suggested to remodel wat tissues due to drainage of fatty acids towards the liver . tta was shown to specifically reduce epididymal fat in young obese zucker ( fa - fa ) rats and epididymal and retroperitoneal fat in male wistar rats fed a high - fat diet for 3 weeks . to further study the effect of tta - induced adipose tissue remodelling , wistar rats were fed a high - fat lard diet ( 40% energy from fat ) for 7 weeks , and the distribution of fat was studied by magnetic resonance imaging ( mri ) . the adipose tissue mass was significantly reduced in the tta - fed rats , particularly in the perirenal and epididymal depots . interestingly , while most of the genes involved in lipolysis , fatty acid -oxidation , mitochondrial biosynthesis , and immune response were unchanged by tta in all studied adipose tissues , ucp1 was highly increased at the mrna level in epididymal and mesenteric depots . also , a de novo production of ucp3 in the liver was detected both at the mrna and protein level along with high induction of genes involved in -oxidation . the possible increased energy expenditure due to uncoupling of mitochondria combined with the enhanced hepatic fatty acid -oxidation supports the hypothesis of hepatic lipid drainage . as a result , tta treatment will channel tgs to the liver for -oxidation and the higher metabolic activity of the vat tissues could explain the specific reduction of these risk - linked depots , perhaps also influenced by their increase in bat - like features ( figure 2 ) . although the effect of tta is mostly due to activation of hepatic mitochondrial genes , activation of some adipose genes involved in -oxidation was observed . the lipogenic genes acetyl - coa carboxylase alpha ( acaca ) and fatty acid synthase ( fasn ) were induced by tta in perirenal and epididymal vat . in a directly comparable long - term rat study , the 3-n pufa eicosapentaenoic acid ( epa ) was found to increase acaca and fasn in vat while cpt2 mrna - level was increased in mesenteric vat , indicating an increase in genes involved in fatty acid degradation also with marine oils . tta and epa were able to significantly increase the gene expression of ampk , cpt2 , ucp1 , and acyl - coa oxidase 1 ( acox1 ) , but tta induced a higher level of ampk and ucp1 . importantly , ampk is believed to function as a metabolic switch resulting in upregulation of fa oxidation in parallel with reduced maloyl - coa level and downregulated lipogenesis and tg biosynthesis . the mitochondrial gene pyruvate kinase ( pklr ) was upregulated by tta specifically in perirenal vat , indicating mitochondrial biogenesis in this fat depot . this is important as the generation of new mitochondria is severely compromised in several obesity - models [ 36 , 43 ] . in accordance , a coactivator central for mitochondrial biogenesis , ppar coactivator 1 ( pgc-1 ) was reduced in a rodent obesity model and in morbidly obese humans . although tta reduces dyslipidemia in diabetic patients , a direct effect on glucose metabolism and insulin sensitivity is more disputed [ 121 , 123 , 127 ] . a reduced amount of risk - related adipose tissue in obese individuals would however be important to reduce the likelihood of developing disease . the efficient reduction of plasma ffa and tg by tta , seen in both human and animal studies , may also prevent ectopic lipid deposition and lipotoxic effects . tta induces mitochondrial biogenesis in liver , but the effect on mitochondrial respiration has not been fully elucidated . although the number of mitochondria increases in the liver during tta - treatment , the hepatocytes are enlarged , and the respiration per mg tissue stays constant ( not published results ) . little work has been done to measure oxygen consumption / oxphos in adipose tissue , probably due to the difficulties caused by a low mitochondrial density in adipocytes compared to skeletal muscle . some studies have successfully determined mitochondrial oxphos in cells or organelles isolated from both brown and white adipose tissues , and vat and sat have been compared using similar techniques . information on mitochondrial oxphos activity will be of importance when testing for improved adipose tissue function during treatment with mitochondrial - targeted compounds . thiazolidinediones ( tzds ) , such as pioglitazone and rosiglitazone , activate ppar and are associated with increased body weight due to the ability to stimulate preadipocyte differentiation [ 131 , 132 ] . in particular the retroperitoneal vat [ 133 , 134 ] and studies show that the activation of ppar by fenofibrates or oleoyletanolamide reduces adiposity and increases lipolysis , while a pan - ppar ligand , lm 4156 , had no effect on adipose tissue size . another study in accordance with this showed that a ppar agonist reduced feed intake and body weight gain but had less effect on glucose intolerance ( gi ) , a ppar agonist improved gi and adiponectin release but enhanced feed intake and body weight gain , while an agent with dual ppar - activation demonstrated a combined , more beneficial effect . tta activates both ppar and , the former with higher efficiency [ 136138 ] , and this may partly explain the effect on lipid degradation in the liver , as well as the reduced weight gain and hepatic induction of ucp3 primarily associated with ppar activation [ 132 , 139 ] . however , we have shown the existence of ppar dependent and independent induction of ucp2 by tta . therefore , mechanisms not involving ppar - activation may also be important for the effects seen with tta . there is mounting evidence that the increased release of h2o2 , mainly originating from mitochondria , may be an important step in the development of insulin resistance in different tissues during high - fat intake [ 15 , 16 ] . mitochondrial damage is linked to insulin resistance and could be a secondary effect of the increase in ros . a number of studies have shown that treatment with antioxidants will both improve oxidative stress and restore insulin sensitivity [ 141143 ] . while antioxidants studied so far have had little effect on human atherogenesis , it could be worthwhile to investigate mitochondrial - targeted antioxidants in the treatment of cvd . tta mainly affects mitochondrial functions and is a very potent antioxidant [ 144 , 145 ] and anti - inflammatory agent [ 120 , 146 ] , both of which may be important for its reduction of dyslipedemia in diabetic patients . tta inhibits the lipoprotein oxidation in rats , indicating that its effects as an antioxidant may influence the development of atherosclerosis . it also reduces the stenosis development after balloon angioplasty injury of rabbit iliac arteries . recently we have shown downregulated expression of ppar target genes and reduced mitochondrial fatty acid oxidation in the liver of mice transgenic for htnf . the different adipose depots have specific roles based on their level of lipolysis and rate of tg storage . it is becoming clear that while abdominal visceral adipose tissue increases the risk of obesity - related disease , subcutaneous adipose tissue , especially located to the lower - body parts , protects from lipotoxic effects through short - term and long - term storage of tgs . in rodent studies , intake of bioactive lipids gives specific reduction of risk - related adipose tissues , and this targeted effect may be due to the higher metabolic activity of these depots . treatments that increase energy expenditure through mild uncoupling of mitochondria and fatty acid -oxidation show great promise in the treatment of obesity . enhanced expression of uncoupling proteins together with an induction of hepatic -oxidation suggests that tta may increase energy consumption via increased uncoupling in liver and/or wat . in addition its activity as an antioxidant and anti - inflammatory agent will have a direct positive effect on the diseases linked to obesity , such as t2 dm and cvd .

ever since its origins in 1988 in the usa and france , laparoscopic cholecystectomy has fast become the gold standard for operative gall bladder removal . studies have shown that laparoscopic cholecystectomy , as compared to the previous method of open cholecystectomy , has advantages in terms of lower mortality , lesser complications and shorter length of total hospital stay . this , along with better cosmesis and faster postoperative recovery allowing patients an earlier return to their daily activities made laparoscopic cholecystectomy a clear choice for gall bladder disease requiring surgical intervention . in recent years , surgeons have further been advancing the technique of standard laparoscopic cholecystectomy ( slc ) . the goals for newer techniques included better cosmesis , postoperative recovery times and postoperative pain by reducing the size of the instruments used during slc . the newer instruments being developed with a < 10 mm size showed initial studies claiming better postoperative pain control and cosmesis . in this study , we have evaluated and presented the benefits and drawbacks of mini - laparoscopic cholecystectomy ( mlc ) in comparison with slc in terms of operative time , postoperative pain management using visual analogue scale ( vas ) , intraoperative blood loss , time taken for postoperative ambulation , length of patient 's hospital stay , return to work and overall cost of surgery . the latter , we believe will play a large factor in a lower socioeconomic country such as pakistan with low per capita income . using these variables , we assess if mlc may , in the future , become universally accepted as the standard of care . over the time period between february - april 2014 , a prospective non - randomised study was conducted on a total of 72 patients presenting to ziauddin university hospital , karachi , pakistan with symptomatic gall bladder disease . patients were informed regarding both procedures and consent was obtained before being assigned to either mlc or slc groups ; patients were informed beforehand regarding the operative technique that would be used for their operation . the surgeon was notified at the time of scheduling the procedure regarding the method to be used . the researcher collecting the data and performing the analysis was blinded regarding the procedure performed . preoperative percutaneous abdominal ultrasound , complete blood count and liver function tests were ordered for all patients . for the purposes of this study , mlc was performed using three 3 mm ports and one 10 mm port , whereas slc was performed using two 5 mm and two 10 mm ports . following general anaesthesia , patients undergoing mlc were placed in reverse trendelenburg position with the surgeon on the left . a veress needle was introduced to create pneumoperitoneum , after which a 10 mm transumbilical port and laparoscope were introduced . a specially designed stab knife of 3 mm width was used for subxiphoid , right midclavicular and right anterior axillary line incisions , followed by ports . fundus of the gall bladder retracted using gall bladder grasper ; and calot 's triangle was identified and dissected using sharp dissection , keeping the use of diathermy to a minimum . , a 2.7 mm camera was introduced through the epigastric port and a 10 mm camera was retrieved ; a 10 mm clip applicator was introduced through the 10 mm umbilical port and clips were placed under direct visualization . after clip application , we switched back to visualization with a 10 mm laparoscope for dividing these structures and after dissection from the liver bed . all miniports were closed with steri - strips and no sutures were applied while the umbilical port was closed with subcuticular absorbable sutures . approval from the ethics committee and review board was obtained prior to the start of the study . the patients ' informed consent was obtained regarding both procedures and assigned to either mlc or slc groups . patients were aware beforehand regarding the operative technique that would be used for their operation . the surgeon knew in advance , at the time of scheduling surgery , which operative technique was to be used . data was collected prospectively using predesigned questionnaire and processed retrospectively using statistical package for the social sciences ( spss ) v21 ( ibm , new york , usa ) ; categorical data was calculated using chi - squared test and continuous variables using the student 's t - test . patients in neither groups experienced any significant postoperative complication and there was no loss to follow - up among the study groups . the patients were divided into two groups , one undergoing mlc and the other slc with none of our patients required to be converted from mlc to slc . mean age in the mlc group was 36.4 years ( 19 - 49 years ) and for the slc group was 39.1 years ( 20 - 55 years ) ( p < 0.852 ) . gender ratio for mlc was 2:30 and for slc was 2:38 ( p = 0.645 ) . body mass index ( bmi ) again being comparable for both the groups ; mlc = 24.2 ( 21.4 - 28.5 ) and slc= 25.3 ( 20.2 - 30.1 ) ( mean , min - max ) . the outcome measures included mean operative times slc : 37.5 min versus mlc : 38.2 min ( p = 0.576 ) . other parameters compared between both techniques included : mean blood loss mlc : 18 ml versus slc : 21.4 ml ( p = 0.258 ) . postoperative pain was assessed using the vas and was found to be mlc : 1.6 versus slc : 1.9 ( p = 0.327 ) . postoperative time till patients were fully mobile were assessed and found to be mlc 2.1 h versus slc 2.7 h ( p = 0.231 ) . furthermore , we additionally took into account the total time of the patient 's hospital stay ; mlc : 14.2 h versus slc 26.4 h ( p < 0.001 ) reaching statistical significance [ figure 1 ] . comparison between the lengths of hospital stay on follow - up , we evaluated the average number of days patients in both groups took to return to work and found the results to be : mlc : 2.2 days versus slc : 3.9 days ( p < 0.001 ) [ figure 2 ] . comparison of mean days required to return to routine work comparison between the cost of procedure between mlc and slc ( all figures have been converted from pakistani rupees to usd , according to sep 2014 foreign exchange rates ) mlc was introduced in 1997 as a further advancement to slc with the goal of enhancing the benefits of the latter . ever since then , there have been studies that support its implementation based on results pointing toward better cosmetic outcomes , less postoperative pain due to smaller instruments resulting in lesser tissue damage . while at the same time , no significant increase in complications has been reported . apart from cholecystectomy , global studies have reported significant improvement in postoperative surgical pain and cosmesis using needlescopic instruments including , but not limited to , heller myotomy , splenectomy , adrenalectomy and sigmoid resection in selected patients . this is the first study to be reported from pakistan to the best of our knowledge ; we have compared mlc and slc to evaluate if mlc can become the standard of care by comparing results to slc . in terms of total intraoperative times , the results of our mlc patients compared favourably against those reported by lee et al . 68.8 31.9 min ; however , this may be due to surgeon to surgeon operative time variation in that study . our operative times of slc : 37.5 min versus mlc : 38.2 min are closest and comparable to sarli et al . of 45 ( 20 - 120 ) and 50 ( 20 - 170 ) min ( lc and mlc respectively ) . however , difference in our operative times between slc and mlc did not reach statistical significance ( p = 0.576 ) . although , our intraoperative time for our first mlc patient was the longest for the entire series ( 61 min ) ; this improved with time with our final results for both operative techniques being comparable . out of the earlier reported studies , it has been indicated that difference in intraoperative blood loss between slc and mlc is negligible with huang et al . comparing the differences between three groups undergoing slc , mlc and 5 mm lc and finding no statistical significance between the three groups . our study group , when evaluated for the same parameter , reflected a similar finding and did not find a statistically significant difference between the two groups with mlc 18 ml versus slc : 21.4 ml ( p = 0.258 ) of intraoperative blood loss . the variation in postoperative pain is reported differently across multiple studies according to the size of instruments used . some series we used as comparisons include ecs by lai et al . , which reports that vas scores for trocars 3 mm or less were below 3 by 76% of patients , whereas patients in their comparison group with 5 mm trocars by 21% of patients reported significant postoperative pain ( p < 0.01 ) . some studies suggest that despite there being a significant decrease in postoperative pain in the immediate 24 h after surgery , over the longer term there will be no significant difference in vas in the patients who underwent slc . compared postoperative pain using vas at time intervals of : postoperative , postoperative day 1 , postoperative day 3 , postoperative day 7 and postoperative day 28 and coming to a similar conclusion with only statistical significance at postoperative day 1 ( p < 0.04 ) . in our study , postoperative pain was assessed using vas and results have shown that there was not a significant difference between the mlc and slc groups at 24 h ( p = 0.327 ) . the direct link between postoperative pain and incision size is not well established , with many other factors playing a role in postoperative pain including age , pressure of pneumoperitoneum and preoperative neuroticism being part of the multivariate etiology . and , therefore , even though incision size is one of the factors to reduce postoperative pain , our study shows that it is not the only factor to consider as evidenced by difference in vas scores in both study groups not reaching statistical significance . a secondary variable evaluated was the immediate time taken for the patient to mobilize following surgery . with studies suggesting that due to decreased postoperative pain with mlc techniques patients were able to mobilize in a lesser amount of time than those who underwent slc . in our experience , both mlc and slc groups were able to mobilize in under 3 h with the difference between the groups not reaching statistical significance ( p = 0.231 ) . one of the most important benefits to the patient in terms of overall health of laparoscopic cholecystectomy has been a reduced length of hospital stay and an earlier resumption of the day - to - day activities for the patient . it would be logical to assume that if there is a significant reduction in postoperative pain , there would be a directly correlated shorter hospital stay . however , studies conducted for a comparison between scl and mlc have thus far provided conflicting results regarding mean hospital stay . with prospective studies by gupta et al . and look et al . reporting that despite a significantly decreased postoperative pain in the mlc group , there was no significant difference in mean times for hospital stay with a total study population of 40 and 64 respectively . however , lai et al . reported during a prospective study of 150 patients that mlc group not only had statistically significant decrease in postoperative pain but also had a significantly reduced mean hospital stay . our study showed that patients in the mlc group were able to get an earlier discharge to return home compared with the slc group who stayed for a longer period of time ( p < 0.001 ) . the complications reported by lai et al . included : subcutaneous emphysema , intraperitoneal bile spillage , perforation of the gall bladder upon grasper application and slipped cystic artery duct clip . reported minor incidents including conversion of mlc to slc in 2.8% of patients . these , along with minor rates of 1.9% minor umbilical site infections and 1% umbilical herniation , however , were not significant to recommend against mlc . another concern regarding the use of finer instruments for operative procedures is durability in the intraoperative setting . reported that the major reason for suboptimal performance of mlc instruments was in the setting of inflamed and thickened gall bladder . mlc instruments were found to be more susceptible to damage and smaller diameter laparoscopes gave a lower quality resolution . however , it was concluded that due to similar operative times for both techniques and comparative postoperative morbidity this was not viewed as a major drawback against mlc . a meta - analysis of 18 studies done by thakur et al . reported that there was a decreased trend of slc to conversion to open technique versus mlc , with a pooled odds ratio of 1.05 ( 0.52 - 2.12 ) . a study done by chekan et al . to compare cost burden clinical implications of different cholecystectomy procedures in the us hospitals found a minimal but statistically significant difference in total costs between mlc versus slc . however , in comparing costs in a lower socioeconomic country such as pakistan , we must take into account that most patients do not subscribe to health insurance plans . our findings conclude that there is a markedly increased cost of mlc versus slc $ 766 versus $ 529 with most of the cost difference being attributed to more expensive mlc instruments $ 147 - 244 versus slc $ 49 - 98 . in conclusion , mlc is found to have similar surgical risks , postoperative pain and mobilization time compared to slc . it is found that the mlc technique has a minimal learning curve , reflected by the fact that the first mlc intraoperative time took 61 min , however , by the end of the study , both groups had comparable intraoperative times . mlc , additionally , has favourable outcomes , allowing patients to request faster postoperative hospital discharge and earlier return to work . however , due to the significantly increased cost burdens associated with mlc and with no significant improvements in other variables including operative time , intraoperative blood loss and postoperative pain , this approach should not be routinely recommended to patients and can not be recommended as the gold standard approach in a country of a lower socioeconomic status such as pakistan .

afferent loop obstruction is a potentially life - threatening complication of billroth ii gastrectomy or roux - en - y hepaticojejunostomy ( ryhj ) . afferent loop syndrome caused by an enterolith is rare.1 formation of the enterolith is presumed to be related to hypomotility or stasis of the bowel that is altered anatomically after billroth ii subtotal gastrectomy . when the enterolith obstructs the lumen of the afferent limb , the intraluminal pressure of the intrahepatic duct ( ihd ) and proximal jejunal limb may be elevated , and bacterial overgrowth in the lumen of an obstructed bowel may cause ascending bacterial infection of the biliary tree . most cases are treated with surgery for removal of the enterolith because the afferent loop of the jejunum is long and tortuous and endoscopic access to the enterolith is difficult.2 - 7 here , we report a case of afferent loop syndrome with acute cholangitis caused by an enterolith that was treated with a percutaneous transhepatic cholangioscopic procedure . a 74-year - old woman was admitted with a 2-day history of fever and acute abdominal pain . the patient had undergone choledochal cyst excision , left hepatectomy , and ryhj for ihd stones and a choledochal cyst 12 years previously . on admission , blood pressure was 90/57 mm hg , pulse rate was 116 beats per minute , temperature was 38.5 , and tenderness in the right upper quadrant of the abdomen was detected . laboratory findings were as follows : white blood cell count , 14,900/mm ( normal range , 4,000 to 10,800 ) ; platelet cell count , 81,000/mm ( normal range , 150,000 to 400,000 ) ; c - reactive protein , 11 mg / dl ( normal range , 0 to 0.3 ) ; aspartate aminotransferase , 129 iu / l ( normal range , 7 to 38 ) ; alanine aminotransferase , 187 iu / l ( normal range , 4 to 43 ) ; total bilirubin , 6.2 mg / dl ( normal range , 0.1 to 1.3 ) ; and alkaline phosphatase , 432 iu / l ( normal range , 103 to 335 ) . an abdominal computed tomography ( ct ) scan showed a single stone in the sixth branch of the right ihd and a stone measuring 3 cm in the afferent loop with diffuse dilatation of the upstream small bowel loop and ihd ( fig . afferent loop syndrome caused by an enterolith was diagnosed on the basis of the clinical features and imaging studies . because the patient was septic and her condition was unstable , we performed urgent percutaneous transhepatic biliary drainage ( ptbd ) . a 10 fr pigtail catheter for ptbd was passed over the guide wire and placed in the jejunal limb through b6 after b6 of the ihd was punctured using a 21-gauge hollow needle under ultrasound guidance , and a guide wire was inserted through the needle into the bile duct . because the enterolith was located in the jejunal limb near the hepaticojejunostomy site on abdominal ct and cholangioenterogram , peroral endoscopic access to the enterolith was difficult , and surgical treatment was refused by the patient , we decided to perform percutaneous transhepatic cholangioscopy ( ptcs ) for removal of the ihd stone and the enterolith . first , the ptbd tract was dilated to 18 fr using a dilator to allow insertion of a standard choledochoscope ( chfp20q ; olympus co. , tokyo , japan ) into the bile duct 7 days after ptbd . the ihd stone was a black - pigmented stone and the jejunal loop stone was grayish and hard ( fig . 2 ) . because the hepaticojejunostomy site was not stenotic and an enterolith was located in the jejunal limb near the hepaticojejunostomy site , the cholangioscope could access the enterolith . an electrohydraulic shock wave generator ( lithotron el 25 ; walz - electronic inc . , rohrdorf , germany ) set at an output of 2,000 v was used for the generation of shock waves of increasing frequency ( intensity of 500 mj ) , which were applied with a continuous sequence of discharge . after stone fragmentation , the enterolith was pushed out through the jejunojejunal anastomosis to the efferent loop using the cholangioscope and a saline flush ( fig . the cholangiogram showed that the ihd and the afferent loop were clear , without any remnant stone . the ptbd catheter was removed , and the patient began oral intake 1 day after the procedure . the patient made a full recovery without any complications , and was discharged 2 days after the procedure . afferent loop obstruction is an unusual but potentially life - threatening complication of billroth ii gastrectomy or ryhj . possible causes of the obstruction include mechanical obstruction of the afferent limb caused by adhesions , internal hernias , volvulus , intussusception , intestinal kinks / strictures , malignancy , and rarely , an incarcerated enterolith.1 the etiology of enterolith formation in the afferent limb includes limb dysmotility and bacterial overgrowth , and the cause of limb dysmotility is the vagal denervation or proximal disconnection of the afferent limb from the main pacemaker in the duodenum.2 in such cases , surgery has been the treatment of choice for enterolith removal because nonsurgical removal through the peroral or percutaneous route is difficult technically.3 - 7 successful endoscopic removal of the enterolith in the afferent loop is rare.7 however , theoretically , endoscopic treatment through the percutaneous route should be possible for removal of an enterolith from the afferent loop in special cases such as an enterolith located proximal to the hepaticojejunostomy site or through the use of balloon enteroscopy . ptcs with ehl is a low - risk procedure for removal of the enterolith after ptbd . in the absence of complications 1 day after the procedure , the ptbd catheter can be removed and patients can resume oral nutrition . in the present case , we first created a ptbd tract for retrieval of the ihd stone . after removal of the ihd stone , we attempted to access the enterolith in the afferent loop through the ptbd tract and hepaticojejunostomy using the cholangioscope . we could approach the enterolith in the afferent loop because it was located in the jejunal limb adjacent to the hepaticojejunostomy site . because of the risk of afferent loop syndrome caused by enterolith recurrence in the present case , regular clinical follow - up examinations are recommended for such cases in the future . in addition , the use of prokinetic agents or restrictions in the administration of proton - pump inhibitors should be considered in such cases . in conclusion , ptcs with ehl may be a treatment option for afferent loop syndrome caused by an enterolith in patients who underwent ryhj .

sarcomas arising in abdomen and pelvis constitute 2530% of all soft tissue sarcomas ( sts ) , of which retroperitoneal sarcomas account for 15% , and intra - abdominal sarcomas account for 1015% ; < 5% of sarcomas appear as primary abdominal wall tumors . the most commonly encountered histologic subtypes of retroperitoneal sarcomas are liposarcoma , leiomyosarcoma , malignant fibrous histiocytoma , fibrosarcoma , and malignant peripheral nerve sheath tumor . the most common gynecological sarcomas occur in the uterus and comprise the histological subtypes : leiomyosarcoma and endometrial stromal sarcoma . sts of the anterior abdominal wall include liposarcoma , synovial sarcoma , fibrosarcoma , dermatofibrosarcoma protuberans ( dfsp ) , and others . complete margin - negative resections are difficult to achieve in retroperitoneal sarcomas in view of large size and complex anatomy . limited data exist on treatment outcome and prognostic factors in patients with abdominopelvic sarcomas . to fill these lacunae in literature , we aimed to review our experience with 88 patients of abdominopelvic sarcomas treated at our center over a 10-year period . this study involves data review of all patients with proven diagnosis of abdominopelvic nonrhabdomyosarcomatous sarcomas treated at our department from june 2003 to december 2012 . exclusion criteria were patients in whom complete information on the clinical profile was not available , patients who received 2 cycles of chemotherapy at our center and were lost to follow - up without progression or event and patients in whom response was not evaluated . the tumors were categorized on the basis of site of origin into ( a ) retroperitoneal tumors ( those arising from retroperitoneum and pelvis ) , ( b ) tumors of intra - abdominal and pelvic organs . since there were no intra - abdominal tumors in our study , those arising from uterus and vagina baseline demographic profile , clinical characteristics , treatment given , outcome , and treatment - related toxicities ( according to national cancer institute common terminology criteria for adverse events , version 3.0 ) were collected for all patients . evaluation of primary disease was done by computed tomography ( ct ) scan or magnetic resonance imaging . metastatic work - up included chest radiograph and/or chest ct , abdominal ultrasound / ct abdomen , and bone scan wherever required . treatment protocol consisted of surgery , radiotherapy , and systemic chemotherapy . for patients with localized disease , treatment consisted of surgical resection followed by adjuvant therapy . for patients with unresectable and metastatic disease , response to chemotherapy was assessed radiologically after 2 cycles and at the completion of therapy . complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , and progressive disease ( pd ) were defined according to revised response evaluation criteria in solid tumors version 1.1 . the following variables were analyzed for their potential prognostic value : age , gender , baseline presentation ( localized or metastatic ) , duration of symptoms , baseline eastern cooperative oncology group performance status , baseline serum albumin , tumor size , tumor site , histological subtype , grade , and the type of treatment received . survival was estimated by the kaplan meier method and compared using the log - rank test . event - free survival ( efs ) was calculated from the date of diagnosis to the date of disease relapse or progression or death from any cause . overall survival ( os ) was calculated from the date of initial diagnosis to the date of death ( whatever the cause ) or last follow - up . a univariate cox proportional hazard model followed by stepwise multivariate cox regression analysis factors with significance ( p 0.05 ) in univariate analysis were taken into multivariate analysis . stata / se 11.0 ( statacorp lp , college station , tx , usa ) was used for statistical analysis . evaluation of primary disease was done by computed tomography ( ct ) scan or magnetic resonance imaging . metastatic work - up included chest radiograph and/or chest ct , abdominal ultrasound / ct abdomen , and bone scan wherever required . treatment protocol consisted of surgery , radiotherapy , and systemic chemotherapy . for patients with localized disease , treatment consisted of surgical resection followed by adjuvant therapy . for patients with unresectable and metastatic disease , response to chemotherapy was assessed radiologically after 2 cycles and at the completion of therapy . complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , and progressive disease ( pd ) were defined according to revised response evaluation criteria in solid tumors version 1.1 . the following variables were analyzed for their potential prognostic value : age , gender , baseline presentation ( localized or metastatic ) , duration of symptoms , baseline eastern cooperative oncology group performance status , baseline serum albumin , tumor size , tumor site , histological subtype , grade , and the type of treatment received . survival was estimated by the kaplan meier method and compared using the log - rank test . event - free survival ( efs ) was calculated from the date of diagnosis to the date of disease relapse or progression or death from any cause . overall survival ( os ) was calculated from the date of initial diagnosis to the date of death ( whatever the cause ) or last follow - up . a univariate cox proportional hazard model followed by stepwise multivariate cox regression analysis was done to identify the predictors of outcome . factors with significance ( p 0.05 ) in univariate analysis were taken into multivariate analysis . stata / se 11.0 ( statacorp lp , college station , tx , usa ) was used for statistical analysis . of 696 patients with nonrhabdomyosarcomatous sts , 112 ( 16% ) patients had sarcomas arising from abdomen and pelvis . twenty - four patients were excluded ( 14 patients who did not take treatment and 10 patients who took 2 cycles of chemotherapy ) . hence , here , we have analyzed 88 patients , and their clinicopathologic characteristics are summarized in table 1 . clinicopathologic characteristics of 88 patients median age was 40 years ( range : 178 years ) with a median duration of symptoms of 8 months ( range : 1360 months ) . the most common histological subtypes were leiomyosarcoma ( 36% ) and liposarcoma ( 17% ) . forty - six ( 52% ) patients underwent surgical resection out of which wide excision was performed on 43 patients and marginal resection in 3 patients . negative margins were achieved in 34 patients . of 46 patients who underwent surgical resections , 23 patients had primary tumors located in the retroperitoneum , 15 had abdominal wall primary , and 8 patients had uterine tumors . thirty - three ( 72% ) patients received adjuvant therapy ( radiotherapy alone in 14 patients , chemotherapy alone in 7 patients , and chemotherapy plus radiotherapy in 12 patients ) . nineteen patients received adjuvant chemotherapy and 42 patients received systemic chemotherapy as a primary therapy with palliative intent . of 42 patients who received chemotherapy with palliative intent , 21 patients had unresectable disease at baseline , and 21 patients were metastatic . thirty - one patients received ifosfamide - doxorubicin combination , 23 patients received single agent doxorubicin , and 7 patients received gemcitabine - docetaxel combination . the response evaluation in these 42 patients was as follows : cr in 5 ( 12% ) patients and pr in 6 ( 14% ) patients . sd was seen in 17 ( 40% ) patients and 14 ( 33% ) patients developed pd . there was one death due to febrile neutropenia . at a median follow - up of 43 months ( range : 294 months ) , the 5-year efs and os of whole cohort were 35% and 42% , with a median of 22 months and 43 months , respectively . for efs , univariate analysis identified male gender ( p - 0.01 ) , baseline metastatic disease ( p < 0.001 ) , serum albumin 4 g / dl ( p - 0.04 ) , grade iii tumors ( p - 0.001 ) , and unresectability ( p < 0.001 ) as factors predictive of poor survival . for os , male sex ( p - 0.01 ) , baseline metastatic disease ( p < 0.001 ) , retroperitoneal disease ( p - 0.03 ) , grade iii tumors ( p - 0.001 ) , and unresectability ( p < factors affecting event - free survival and overall survival in univariate analysis for efs , male gender ( p - 0.03 ) , baseline metastatic disease ( p - 0.01 ) , and grade iii tumors ( p - 0.02 ) were identified as poor prognostic factors [ figure 1a c ] , whereas for os , baseline metastatic disease ( p < 0.001 ) , and unresectability ( p - 0.01 ) emerged as poor prognostic factors [ figure 2a and b ] [ table 3 ] . ( b ) based on surgery multivariate analysis of factors affecting event - free survival and overall survival of 696 patients with nonrhabdomyosarcomatous sts , 112 ( 16% ) patients had sarcomas arising from abdomen and pelvis . twenty - four patients were excluded ( 14 patients who did not take treatment and 10 patients who took 2 cycles of chemotherapy ) . hence , here , we have analyzed 88 patients , and their clinicopathologic characteristics are summarized in table 1 . clinicopathologic characteristics of 88 patients median age was 40 years ( range : 178 years ) with a median duration of symptoms of 8 months ( range : 1360 months ) . the most common histological subtypes were leiomyosarcoma ( 36% ) and liposarcoma ( 17% ) . forty - six ( 52% ) patients underwent surgical resection out of which wide excision was performed on 43 patients and marginal resection in 3 patients . of 46 patients who underwent surgical resections , 23 patients had primary tumors located in the retroperitoneum , 15 had abdominal wall primary , and 8 patients had uterine tumors . thirty - three ( 72% ) patients received adjuvant therapy ( radiotherapy alone in 14 patients , chemotherapy alone in 7 patients , and chemotherapy plus radiotherapy in 12 patients ) . nineteen patients received adjuvant chemotherapy and 42 patients received systemic chemotherapy as a primary therapy with palliative intent . of 42 patients who received chemotherapy with palliative intent , 21 patients had unresectable disease at baseline , and 21 patients were metastatic . thirty - one patients received ifosfamide - doxorubicin combination , 23 patients received single agent doxorubicin , and 7 patients received gemcitabine - docetaxel combination . the response evaluation in these 42 patients was as follows : cr in 5 ( 12% ) patients and pr in 6 ( 14% ) patients . sd was seen in 17 ( 40% ) patients and 14 ( 33% ) patients developed pd . forty - six ( 52% ) patients underwent surgical resection out of which wide excision was performed on 43 patients and marginal resection in 3 patients . of 46 patients who underwent surgical resections , 23 patients had primary tumors located in the retroperitoneum , 15 had abdominal wall primary , and 8 patients had uterine tumors . thirty - three ( 72% ) patients received adjuvant therapy ( radiotherapy alone in 14 patients , chemotherapy alone in 7 patients , and chemotherapy plus radiotherapy in 12 patients ) . nineteen patients received adjuvant chemotherapy and 42 patients received systemic chemotherapy as a primary therapy with palliative intent . of 42 patients who received chemotherapy with palliative intent , 21 patients had unresectable disease at baseline , and 21 patients were metastatic . thirty - one patients received ifosfamide - doxorubicin combination , 23 patients received single agent doxorubicin , and 7 patients received gemcitabine - docetaxel combination . the response evaluation in these 42 patients was as follows : cr in 5 ( 12% ) patients and pr in 6 ( 14% ) patients . sd was seen in 17 ( 40% ) patients and 14 ( 33% ) patients developed pd . at a median follow - up of 43 months ( range : 294 months ) , the 5-year efs and os of whole cohort were 35% and 42% , with a median of 22 months and 43 months , respectively . for efs , univariate analysis identified male gender ( p - 0.01 ) , baseline metastatic disease ( p < 0.001 ) , serum albumin 4 g / dl ( p - 0.04 ) , grade iii tumors ( p - 0.001 ) , and unresectability ( p < 0.001 ) as factors predictive of poor survival . for os , male sex ( p - 0.01 ) , baseline metastatic disease ( p < 0.001 ) , retroperitoneal disease ( p - 0.03 ) , grade iii tumors ( p - 0.001 ) , and unresectability ( p < factors affecting event - free survival and overall survival in univariate analysis for efs , male gender ( p - 0.03 ) , baseline metastatic disease ( p - 0.01 ) , and grade iii tumors ( p - 0.02 ) were identified as poor prognostic factors [ figure 1a c ] , whereas for os , baseline metastatic disease ( p < 0.001 ) , and unresectability ( p - 0.01 ) emerged as poor prognostic factors [ figure 2a and b ] [ table 3 ] . ( b ) based on surgery multivariate analysis of factors affecting event - free survival and overall survival for efs , univariate analysis identified male gender ( p - 0.01 ) , baseline metastatic disease ( p < 0.001 ) , serum albumin 4 g / dl ( p - 0.04 ) , grade iii tumors ( p - 0.001 ) , and unresectability ( p < p - 0.01 ) , baseline metastatic disease ( p < 0.001 ) , retroperitoneal disease ( p - 0.03 ) , grade iii tumors ( p - 0.001 ) , and unresectability ( p < 0.001 ) were associated with poor survival [ table 2 ] . for efs , male gender ( p - 0.03 ) , baseline metastatic disease ( p - 0.01 ) , and grade iii tumors ( p - 0.02 ) were identified as poor prognostic factors [ figure 1a c ] , whereas for os , baseline metastatic disease ( p < 0.001 ) , and unresectability ( p - 0.01 ) emerged as poor prognostic factors [ figure 2a and b ] [ table 3 ] . ( b ) based on surgery multivariate analysis of factors affecting event - free survival and overall survival a unique feature of many retroperitoneal and pelvic sarcomas concerns their large size at the time of diagnosis which was reflected in our study also as 65% patients had tumors measuring > 10 cm in size . we observed leiomyosarcoma as the most common histologic subtype arising in retroperitoneum and uterus , whereas dfsp and synovial sarcoma were the most common histologic subtypes in the abdominal wall . the most significant factors which affected survival in our study were baseline disease presentation , histologic grade , gender , and surgical resection . it is well - known that the long - term survival from sarcomas is most dependent on the completeness of resection and that complete resection remains the only chance for long - term survival . this was also reflected in our study as patients who underwent surgical resection had a 5-year survival of 61% as compared to 26% for those who were treated with systemic chemotherapy alone . the identification of prognostic factors other than the adequacy of resection has been inconsistent across studies . tumor size > 5 cm is a poor prognostic factor for abdominal wall sarcomas but has not been identified as a predictor of survival in retroperitoneal sarcomas since virtually all retroperitoneal sarcomas are larger than 5 cm at presentation . tumor grade has been reported as a significant factor in some studies , with the weight of evidence supporting shorter recurrence - free and os for patients with high - grade tumors . our study clearly demonstrates the impact of grade on survival as patients having grade iii tumors did worse compared to grade i / ii tumors ( 5-year os ; grade i - 76% vs. grade ii - 70% vs. grade iii - 28% , p - 0.001 ) . interestingly , gender was found to impact survival in our study as efs was worse in males compared to females . the reason for this gender difference is not known as this has not been observed in any other published study . however , some studies focusing on molecular expression profiling have seen gender - specific differences in survival in sts . studied the prognostic significance of expression of s - phase kinase - associated protein 2 related to gender , estrogen receptor , and progesterone receptor in sts . in men , but not women , estrogen receptor positive / progesterone receptor negative co - expression profile was an independent negative prognostic factor for disease - specific survival . another study by valkov et al . showed that estrogen receptor positivity was a significant favorable indicator for disease - specific survival in women ( p = 0.017 ) , while progesterone receptor positivity had inverse impact in men ( p = 0.001 ) . the impact of gender on survival in sts is unclear and needs to be confirmed by other studies . sarcomas of abdomen and pelvis constitute a diverse group of tumors where baseline metastatic disease , high - grade tumors , male gender , and unresectability have a negative impact on survival . more studies are required to derive any meaningful conclusion on the prognostic impact of gender so that the treatment can be optimized depending on high - risk characteristics of patients .

total daily energy expenditure is the sum of the thermic effect of feeding , the energy expended during physical activity , and resting energy expenditure ( ree ) . ree represents the largest ( i.e. , 6075% ) component of total daily energy expenditure . subsequently , increases in ree can influence daily energy utilization and affect overall energy balance when energy intake is stable . under normal circumstances there is little day - to - day variability in ree ( coefficient of variation ( cv ) = 1.54.0% ) [ 3 , 4 ] . however , an acute bout of heavy resistance exercise has a powerful influence on energy expenditure as it has been shown to elevate ree for 14 to 72 hours after exercise [ 5 , 6 ] . one explanation for the increase in ree in upwards of 24 hours after an acute bout of resistance exercise is increased muscle protein synthesis . the synthesis of muscle proteins is energetically expensive and occurs as a result of the cellular and molecular mechanisms governing mrna translation . this interaction is one of the most complex activities in the cell as it requires a precise coordination between charged trna , amino acids , ribosomes , mrna , numerous proteins , and energy . translation elongation appears to require the most energy as four adenosine triphosphate ( atp ) equivalent molecules are required for every amino acid added to the peptide chain . several studies have investigated the acute ( <3 hours ) changes in energy expenditure during interventions that stimulate muscle protein synthesis . for example , amino acids infused at rates that were known to increase muscle protein synthesis were positively correlated with an increase in ree ( r = 0.79 ) . pre- and/or postresistance exercise intake of amino acids or protein has also been known to increase the fractional rate of muscle protein synthesis and resulted in an elevation of excess postexercise oxygen consumption . overall , it is estimated that the energy contribution from increased muscle protein synthesis in a highly trained male can be as high as 2030 kj day and may account for 20% of ree . the type of macronutrient metabolized for energy has also been shown to change following an acute bout of resistance exercise . previous studies have shown that the nonprotein respiratory exchange ratio ( rer ) , an indirect assessment of substrate utilization , declines 10 and 24 hours after an acute resistance exercise bout . over a longitudinal time frame when muscle protein synthesis is elevated from resistance exercise training and nutritional intake , the increase in lean body mass may be accompanied by a decrease in fat mass . this was demonstrated in a recent study where essential amino acid - carbohydrate supplements were ingested in conjunction with 12 weeks of resistance training in young men . in this study , those who consumed the essential amino acid - carbohydrate after resistance exercise showed greater increases in fat free mass and larger declines in fat mass compared to other subjects that consumed essential amino acids only , carbohydrate only , or a placebo after resistance exercise . these adaptations may be related to the acute energy expenditure and substrate utilization responses in the postexercise period . understanding these changes could be significant when considering that resistance training on consecutive days reflects the training habits of competitive athletes and recreational exercisers . therefore , the purpose of this investigation was to examine how multiple bouts of resistance exercise with and without the strategically timed intake of amino acids affect ree and rer . it was hypothesized that intake of amino acids with each resistance exercise session would lead to greater perturbations of ree and rer . ten male participants ( mean sd , 23.4 2.5 yr , 175.3 9.7 cm , 77.73 13.95 kg , and 14.4 6.2% body fat ) were recruited for the study . all participants were recreationally trained , which was defined as having participated in general resistance exercise for a minimum of 3 days per week for at least 6 months prior to initial testing . prior to participation , all participants read and signed a written informed consent form that was approved by the institutional review board at syracuse university . body mass was measured to the nearest 0.01 kg using the electric scale within the bod pod system ( life measurement inc . , concord , ca , usa ) . body density was estimated from the measurement of body volume using air displacement plethysmography via the bod pod . one repetition maximum ( 1rm ) strength was determined on three free weight exercises ( squats , bench press , and weighted dumbbell lunges ) and eight machine exercises ( lat pulldown , shoulder press , leg extension , leg curl , biceps curl , triceps extension , seated calf raise , and weighted abdominal twists ) in the same order . a research assistant determined the success or failure of each attempt and recorded the final weight that was successfully lifted as the 1rm . because a 1rm test could not be performed for sit - ups , a sit - up endurance test was performed by counting the maximum number of sit - ups a participant could properly perform in one minute . the results of the 1rm and abdominal endurance tests were used to set exercise prescription during two trials where participants performed four acute bouts of upper and lower body resistance exercise with a seven - day period . all participants completed two seven - day trials where ree and rer were obtained each morning , and four resistance exercise sessions were completed ( figure 1 ) . therefore , participants served as their own control because they participated in both trials . on the first day of each trial resistance exercise was implemented after metabolic measurements on days 2 , 3 , 4 , and 5 . on the second and fourth days the exercises were performed in the following order : squats , dumbbell lunge , hamstring curl , leg extension , calf raise , and sit - ups . on the third and fifth days the exercise was performed in the following order : bench press , lat pulldown , shoulder press , biceps curls , triceps extension , and weighted abdominal twists . exercise prescription for sit - ups was determined by calculating a target repetition goal of ~75% of the maximum number of sit - ups performed on the abdominal endurance test . one hundred and twenty seconds of rest was allowed between sets for bench press and squats , while 90 seconds was allowed for all other exercises . on days six and seven only metabolic measures were obtained . during each trial , nutritional intake with resistance exercise was manipulated by providing ( 1 ) amino acids and carbohydrate ( aa - rt ) or ( 2 ) nonnitrogenous , isoenergetic carbohydrate only ( cho - rt ) using a double - blind protocol . assignment of aa - rt or cho - rt was randomly assigned and counterbalanced , such that an equal number of participants performed aa - rt in their first trial and an equal number performed cho - rt in their first trial . the amino acid beverage ( twinlab : amino fuel ; 22 g protein ( 6 g essential amino acids : l - phenylalanine : 633 mg ; l - valine : 781 mg ; l - tryptophan : 133 mg ; l - threonine : 679 mg ; l - isoleucine : 565 mg ; l - methionine : 292 mg , l - histidine : 282 mg ; l - leucine : 1350 mg ; l - lysine : 1449 mg ) ) dosage was chosen because 6 g essential amino acids had previously shown to increase muscle protein synthesis in the postresistance exercise period . the amino acids beverage was mixed with a sports recovery drink ( 36 g carbohydrate , 0 g fat ) to increase palatability and minimize any potential gastrointestinal discomfort . cho - rt was a sports recovery beverage that contained 58.5 g carbohydrate , 0 g fat , and 0 g protein . both aa - rt and cho - rt ingested beverages were mixed with 800 ml of cold water by a research assistant that was not involved in supervising the resistance exercise sessions or performing metabolic data collection or analyses . of this mixture , 400 ml was ingested immediately prior ( < 5 min ) to the resistance training session , while the remaining was consumed during the rest periods between sets during the resistance exercise workout at the leisure of the participant . there was a slight color difference between the nutritional beverages ; therefore , aluminum foil was placed over each beverage container , and participants were instructed not to discuss the taste of the beverages with the researcher during exercise sessions . nutritional intake outside of resistance exercise sessions during each training period was recorded by having participants document their food and beverage consumption during each trial using a dietary journal . prior to the first training period , participants were educated on dietary intake recording including the proper documentation of serving sizes of foods and beverages by a member of the research team . additionally , participants were encouraged to maintain their normal dietary habits over the course of the first training period ( except for experimentally manipulated intake ) . after the first training period , the journals were collected by the research team and then returned to each participant prior to starting the second training period . in the second training period total energy and macronutrient intake were analyzed using nutritional software ( the food processor sql nutritional analysis software from esha research , salem , or , usa ) . resting metabolism was measured via indirect calorimetry during each seven - day trial ( 0630 hr ) using recommendations described previously . briefly , co2 and o2 were measured using a ventilated hood method connected to a sensormedics ( vmax series 2900 ) metabolic system . prior to testing , participants adhered to the following conditions : ( 1 ) refrained from alcohol and caffeine for 24 hours and 12 hours , respectfive , and ( 2 ) avoided eating or drinking anything but water for 8 hours prior to testing . immediately before each test , the metabolic system was calibrated in a temperature stable room ( 20c24c ) for gas and flow using known gas concentrations and a 3l syringe . participants rested supine for 1015 minutes prior to having the canopy placed over their head . during metabolic testing , participants remained supine and minimized movement for 1520 minutes . a polar heart rate monitor was used to monitor heart rate during each minute of the testing period . the first 5 minutes of data was removed via a laboratory standard procedure with metabolic testing . fat and carbohydrate oxidation were determined indirectly by monitoring the nonprotein rer ( vco2 vo2 ) . nonprotein rer was determined by averaging data from the same 15-minute period used to determine ree . all metabolic analyses were performed by one investigator that was blinded to the nutritional intervention . exercise volume ( kg lifted number of sets repetitions completed ) on each day of exercise was compared between aa - rt and cho - rt using paired student 's t - tests . all metabolic and resting heart rate measures obtained on the first two mornings ( before experimental manipulation ) were averaged and served as baseline preintervention variables ( no significant differences were observed in all variables ( p > 0.05 ) ) . separate 2 6 ( condition by time ) anovas with repeated measures were performed for each variable ( ree , rer , heart rate , nutritional intake ) . on average , one month separated each seven - day training period for each participant ( 30 9 days ) . there were no differences in resistance exercise volume ( indicator of performance ) between aa - rt and cho - rt on any training day ( figure 2 , p > 0.05 ) . total energy intake was not different between aa - rt ( 132.09 15 kj kg day ) and cho - rt ( 136.26 14 kj kg day ) during each training period ( p = 0.443 ) . however , there were differences in macronutrient composition due to the nutritional manipulation with resistance exercise ( table 1 ) . for ree , there were significant condition ( p = 0.028 ) and time ( p < 0.001 ) effects . the significant condition effect which determined average ree during the 7-day trial was greater in aa - rt compared to cho - rt ( figure 3 ) . the significant time effect showed that ree was increased significantly from baseline on each morning after multiple resistance exercise sessions were initiated regardless of condition ( figure 4 ) . a significant time effect was also observed for rer ( p < 0.001 ) , which was decreased significantly from baseline on each morning after resistance exercise sessions were initiated regardless of condition ( figure 5 ) . there were no differences in resting heart rate between ( p = 0.411 ) or within ( p = 0.08 ) conditions ( table 2 ) . the main finding of this investigation was over the course of a seven - day period ree was 3.61% higher when amino acids were consumed with resistance exercise . ree represents the largest component of total daily energy expenditure ( 6085% ) and has been implicated as a major contributor to overall body mass management . for example , average ree in the aa - rt training period was equivalent to 7897 kj per day ( 1886 kcals d ) compared to 7622 kj per day ( 1820 kcals d ) in cho - rt . the net difference represented an additional 275 kj ( 66 kcals ) of energy expended each day at rest in the aa - rt . if this is extrapolated across the seven - day training period , the aa - rt would have utilized 1934 kj ( 462 kcals ) more energy than cho - rt . these data suggest that one benefit of consuming amino acids with resistance exercise , in addition to muscle hypertrophy with consistent training , is the acute elevation in energy expenditure which is likely related to the process of muscle growth . although the net change in ree on a given day ( 66 kcals ) is small , over time , subtle elevations in ree may help with body composition management . although the current study was not designed to understand the precise mechanisms responsible for the observed changes in ree , we can speculate into how these alterations may occur . within the first few hours after ingesting amino acids and performing resistance exercise , the fractional rate of skeletal muscle protein synthesis is enhanced by increasing the delivery and uptake of amino acids into skeletal muscle , thereby activating cell - signaling cascades that facilitate increased rates of mrna translation . the synthesis of muscle proteins is energetically expensive as four atp molecules are utilized for every amino acid incorporated into a growing peptide chain . however , the anabolic response from timing amino acid intake with resistance exercise is not limited to the first few hours of recovery . insulin - like growth factor i ( igf-1 ) , a known activator of muscle protein synthesis via the pi3k - akt - mtor signaling pathway , has a delayed secretion where peak values may not be observed until 1628 hours after resistance exercise . in a recent study , igf-1 concentrations were elevated on days 2 and 3 of a three - day paradigm when protein and carbohydrate were ingested before and after each resistance exercise session . therefore , it is plausible that a basal hormonal environment in favor of muscle protein synthesis may provide a mechanism for how ree may be elevated 24 hours after amino acid intake is coupled with heavy resistance exercise . alternatively , the present study does not indicate that there is any additional benefit from the amino acid intake with resistance exercise on total body fat oxidation . previous studies have shown that rer ( indicator of lipid utilization ) is reduced for as long as 24 hours following an acute bout of resistance training [ 2 , 14 ] . it was hypothesized that aa - rt would provide more of an anabolic response compared to cho - rt , which would result in a corresponding increase in energy expenditure and lipid utilization ( because lipid is the primary fuel source at rest ) . our data clearly demonstrated that rer decreased the morning following each resistance exercise session in both conditions . however , we could not detect any differences between aa - rt and cho - rt conditions . therefore , it appears that acute resistance exercise performed on multiple days facilitates increased fat utilization , although ingestion of amino acids does not enhance the response . in summary , all participants completed four acute bouts of resistance exercise ( alternating lower and upper body ) within a seven - day period during two separate trials . during one trial , acute resistance exercise was combined with amino - acid and carbohydrate intake . in the second trial , indicators of resting metabolism ( ree and rer ) were measured before the resistance exercise and nutritional interventions began and on each morning after the resistance exercise and nutritional interventions were initiated . we conclude the additional provision of amino acids with multiple bouts of acute resistance exercise enhances energy expenditure at rest without altering the utilization of lipid .

srus manifests as rectal bleeding , mucoid discharge , straining , altered bowel habits , incontinence , tenesmus , rectal prolapse , and a sensation of incomplete defecation.1,2 the endoscopic findings of srus show variable patterns , including hyperemic mucosa , ulceration , and polypoid lesions.3 because of variable clinical and endoscopic manifestations , srus is often misdiagnosed as malignancy , ibd , chronic vascular insufficiency , and colitis cystica profunda.4 when srus is difficult to distinguish from malignancy , unnecessary surgery is performed . in addition , if srus is accompanied by ibd , an accurate diagnosis is more difficult to achieve . to date , we report an additional case of a 61-year - old man with coexistent srus and ulcerative colitis ( uc ) , and review the literature pertaining to this condition . a 61-year - old man was admitted to chonnam national university hwasun hospital ( jeonnam , korea ) with a 1-day history of rectal bleeding . he denied prior gastrointestinal bleeding , peptic ulcer diseases , use of ulcerogenic medications , or alcohol ingestion during the past year . however , the patient had experienced excessive straining with defecation , tenesmus , and frequent lower abdominal pain for 6 months . on admission , the patient was afebrile , his blood pressure and pulse rate were normal , and he appeared well nourished . the abdomen was nontender without ascites , the spleen tip was not palpable , and bowel sounds were normoactive . all laboratory examination results , including complete peripheral blood cell counts , blood biochemistry , and tumor markers , were within normal range . colonoscopy showed a well - demarcated , shallow ulcerative lesion with polypoidal growth involving the entire circumference of the lower rectal lumen . loss of vascular pattern , edema , and mucopus coating in the distal sigmoid colon and the upper rectum were also seen ( fig . there was no evidence of malignancy . however , abdominal ct revealed thickening of the concentric wall with enhancement in the rectum and enlargement of the regional lymph nodes ( fig . 2 ) . fluorine-18-fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)/ct revealed increased fdg uptake in the lower rectum and regional lymph nodes ( fig . the imaging findings , including those from abdominal ct and fdg - pet / ct , resembled those of rectal cancer . therefore , mucosal biopsy was repeated ; the histopathological findings were similar to those of the previous biopsy . however , clinical symptoms , including hematochezia , persisted despite medical treatment , and occult rectal cancer could not be ruled out . histopathological examination of the resected specimen revealed fibromuscular obliteration of the lamina propria , thickening of the muscularis mucosa , crypt abscesses , focal gland destruction , and increased inflammatory cells in the lamina propria ( fig . the patient received medical treatment for uc , and he was asymptomatic at the follow - up examination . srus is a poorly understood syndrome that affects men and women equally and often occurs in young adults;5 the condition has also been described in the geriatric population.6 its pathogenesis is probably multifactorial , and most accepted causes implicate direct trauma by repetitive self - digitation and ischemic injury to the rectal mucosa.7,8 srus is diagnosed by a combination of clinical aspects , endoscopic presentations , and histologic findings . srus usually presents with rectal bleeding and passage of mucus on defecation.1 other symptoms are tenesmus , straining , constipation , diarrhea , incontinence , and a sensation of incomplete evacuation . the endoscopic features of srus vary greatly , from hyperemic mucosa to ulcerated or polypoid lesions in different numbers and sizes.9 the histologic findings of srus are characterized by fibromuscular obliteration of the lamina propria , thickened muscularis mucosa , and distortion of crypt architecture.10 srus is an uncommon benign disorder of defecation and is difficult to distinguish from malignancy and ibd because of variable clinical and endoscopic manifestations.11 misdiagnosis of srus as malignancy can lead to unnecessary surgery . colonoscopic biopsy showed an ulcerative rectal mucosa and granulation tissue with no evidence of malignancy and ibd . however , the findings of abdominal ct and fdg - pet / ct resembled those of rectal cancer . therefore , repeat mucosal biopsy was performed and histopathologic findings were similar to those of the previous biopsy . srus accompanied by ibd is extremely rare , and an accurate differential diagnosis is difficult to achieve . in our case , the patient frequently experienced tenesmus and lower abdominal pain , although typical symptoms of uc such as chronic diarrhea or long - term hematochezia had not occurred . colonoscopy showed loss of vascular pattern and presence of mucopus coating , which are symptoms different from those associated with srus . symptomatic improvement following initial treatment with biofeedback therapy and laxatives was not satisfactory , which made diagnosis more difficult . to date , only three cases of srus associated with uc ( including the present case ) have been reported in the medical literature ( table 1).12,13 the patients were men aged 23 - 76 years ( mean age , 53.3 years ) . , we diagnosed the patient with srus and uc simultaneously by surgery . in two cases , excluding ours , 5-asa was used to treat uc . the disease extent of uc varied from proctitis to pancolitis . the most common symptom was passage of blood from the rectum . in two cases , including ours , excessive straining with defecation was a possible cause of srus . in the endoscopic features of srus , all three cases showed well - demarcated , single ulcer with or without polypoid lesions in the lower rectum . previously , endoscopic findings of srus revealed shallow ulcers in the majority of cases , and polypoid / nodular lesions or hyperemic mucosa in the remaining cases . all three patients received medical treatment consisting of 5-asa , laxatives , and biofeedback therapy . at the follow - up examination , the symptoms and lesion remained unchanged or slightly improved . furthermore , our patient was refractory to medical treatment , and occult malignancy could not be excluded with imaging studies . the characteristic histological findings in all three cases were fibromuscular obliteration of the lamina propria , thickening of the muscularis mucosa , and elongation and distortion of the crypt . no known mechanism can link uc to the development of srus . in our case , it is unclear whether uc developed earlier than srus because atypical symptoms such as tenesmus and abdominal pain persisted for 6 months . however , the development of the solitary rectal ulcer near the uc lesion indicates a possible sequential relationship between srus and uc . in conclusion , although coexistence of srus and uc is extremely rare , we suggest that the association between srus and uc may not be fortuitous . additional studies are warranted to clarify the relationship between srus and uc , including underlying mechanisms responsible for their development and predisposing factors .

vascular dementia ( vad ) is the impairment of memory and cognitive functioning resulting from cerebrovascular disease ( cvd ) such as infarcts and leukoaraiosis . although vad is considered to be the second most common cause of cognitive impairment after alzheimer 's disease ( ad ) in the elderly , controversy remains concerning the terminology , classification , and diagnostic criteria of vad . cognitive status after a stroke is unstable and wide variability in the rates of cognitive impairment and dementia after stroke is reported , regardless of study duration or methods.1 it is estimated that 6% to 10% of people over the age of 65 have dementia and up to 60% of patients with ad likely have an overlap with vad . vad alone is presumed to account for nearly 20% to 40% of dementia cases.2 seventy - five percent of all strokes occur in individuals older than 65 years.3 in poststroke patients , age is the greatest risk factor for the development of vad.4 although the incidence rate of vad varies considerably depending on the study methodology and criteria used , in one study , the risk of dementia with stroke was higher for those older than 80 years of age ( odds ratio [ or]=12.7 ) or aged 70 - 79 years ( or=3.0 ) than for those aged 60 - 69 years , for those with 8 or fewer years of education versus those with 13 or more years ( or=4.1 ) , for major hemispheric stroke versus posterior fossa ( or=3.0 ) , for those with diabetes mellitus ( or=1.8 ) , for anterior / posterior cerebral artery distribution versus other vascular territories ( or=1.7 ) , and for those with prior stroke ( or=1.7).5 research has established a strong relationship between age , low education level , and poststroke dementia as well as vascular risk factors ( e.g. , hypertension , diabetes , hyperlipidemia , smoking).3,5 ethnic comparisons in the cardiovascular health study showed nearly twofold higher incidence rates among african americans than in whites.6 men , those of asian descent , and older people have a tendency for a higher risk of vad.5,6 vad is considered to result from interactions between vascular etiologies , changes in the brain , host factors , and cognition.7 some studies have found that infarcts may precipitate dementia through an additive or synergistic relationship with ad neuropathology.8 - 10 post - stroke dementia , which includes multi - infarct dementia ( mid ) , strategic infarct dementia , subcortical vascular dementia ( svd ) , and hypoperfusion dementia , is defined as dementia occurring in close temporal relation to thromboembolic or hemodynamic events.11 large - artery infarctions are responsible for most cases of poststroke dementia . mid is characterized by transient ischemic attack ( tia ) and stroke episodes in close temporal relation to the onset or development of dementia . neuroimaging ( brain computed tomography [ ct ] or magnetic resonance imaging [ mri ] ) shows multiple cortico - subcortical infarcts ( fig . mid is related to atherothrombotic strokes , cardiac embolic strokes , and major hemodynamic events . typical clinical features of mid are focal neurological signs such as hemiparesis or sensory deficits and stepwise progression with cognitive impairment . the presence of ' patchy ' or unequal cognitive deficits are only to be expected in mid where there are only very few ( two or three ) cortical infarcts . strategic infarct dementia is caused by isolated infarcts in regions that are important for cognition of the brain , such as the thalamus , hippocampus , caudate , or genu of the internal capsule ( fig . it is characterized by the abrupt onset of cognitive or behavioral changes , which vary depending on the infarct location . most vad results from multi - infarcts where cortical damage is responsible for cognitive impairment . however , this is now known to be a relatively rare pattern of disease in vad and in the far more common subcortical form of vad ( svd ) , a history of stroke may be absent in up to 40%.12 the primary types of brain lesions in svd are lacunar infarcts and ischemic white matter lesions ( wmls ) ( fig . , accumulation of small infarcts in the deep white matter and gray matter may lead to svd . lacunar infarcts represent approximately 25% of symptomatic ischemic strokes.12 wmls have been associated with age , hypertension , diabetes , metabolic syndrome , microvascular retinopathy , elevated homocysteine levels , and ischemic heart diseases.13 the onset of svd is insidious in over half of patients and the course is usually continuous and slowly progressive.13,14 neurological symptoms and signs related to svd include cognitive deterioration , gait disturbance ( imbalance , fall , short - stepped gait ) , urinary dysfunction , and dysarthria . behavioral and cognitive changes include mental slowness ( bradyphrenia ) , emotional lability , personality changes , and depression.15 depression is common in vad , occurring in up to 20% of cases , and is disproportionately prominent in those cases with small amounts of infarction . it may be particularly related to frontal deep white matter lesions.16 cognitive and behavioral changes in svd are due to disruption of the frontal cortico - subcortical circuit , resulting in ' frontal - subcortical syndrome ' or ' dysexecutive syndrome.'17 the primary pathophysiological mechanism underlying svd seems to be vessel wall damage in the long penetrating arterioles that drain into the white matter , with subsequent white matter degeneration and occurrence of lacune.18 orthostatic hypotension and blood pressure variability may play a role in hypoperfusion dementia or wmls.19 proposed mechanisms for this role include nocturnal hypotension superimposed upon limited perfusion reserve and impaired vasoreactivity producing partial ischemia leading to incomplete infarction.20 neuropsychological tests used in the assessment of ad such as the mini - mental state examination ( mmse ) are commonly used to evaluate cognition in vad but are not appropriate tools in patients with vad because neuropsychological characteristics in both subtypes of dementia are not similar . vad predominantly involves deficits in executive functioning , whereas ad involves earlier and more significant memory impairment.12,15 - 17 instruments that include assessment of frontal , executive , and subcortical function are required for the diagnosis of vad . because the pathogenesis , risk factors , and comorbidities for vad are similar to those for ad , it is sometimes difficult to differentiate the two.13 the major clinical differences between vad and ad are summarized in table 1 . the diagnosis of vad is developed by considering clinical , imaging , and pathological findings . given the heterogeneity of the vad phenotype , large , three basic elements for a diagnosis of vad are 1 ) evidence of dementia or cognitive impairment , 2 ) evidence of cvd , and 3 ) evidence of a casual relationship between cognitive impairment and cvd . although there are no uniform diagnostic criteria for vad , guidelines for interpreting neuroimaging findings have been incorporated into recent criteria for the clinical diagnosis of vad . the main set of criteria for the clinical diagnosis of vad are the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv)21 ; the international classification of diseases 10th version ( icd-10)22 ; the state of california alzheimer 's disease diagnostic and treatment centers ( addtc ) criteria23 ; and the national institute of neurological disorders and stroke and association internationale pour la recherch et l'enseignement en neurosciences ( ninds - airen ) criteria.24 all the currently used clinical criteria are ' consensus criteria ' derived from expert opinion based on prevailing knowledge and pathogenetic hypotheses of dementia . all are based on the ischemic infarct concept , with the ninds - airen , dsm - iv , and icd-10 also including hemorrhages . the dsm - iv criteria for vad require focal neurological signs and symptoms or laboratory evidence of focal neurological damage clinically judged to be etiologically related to the disturbance , but the criteria lack neuroimaging guidelines . the icd-10 criteria require unequal , patchy distribution of cognitive deficits , focal neurological signs , and significant cvd judged to be etiologically related to the dementia , but the shortcomings of these criteria include a lack of detailed etiological and neuroimaging guidelines . the addtc criteria are exclusive for ischemic vad and require two or more ischemic strokes by history , neurological signs , or neuroimaging . the ninds - airen criteria include definitions for the dementia syndrome , cvd , and their relationship . a relationship between dementia and cvd is inferred from the onset of dementia within 3 months following recognized stroke , or abrupt deterioration in cognitive functions , or fluctuating , stepwise progression of cognitive deficits . the ninds - airen criteria had moderate to substantial inter - rater reliability and were more specific ( 80% ) , although less sensitive ( 58%).25 the various diagnostic criteria are neither perfect nor interchangeable and none of these criteria have the required sensitivity and specificity to identify patients with mixed vad reliably.26,27 this accounts for the variations in published prevalence and incidence rates and in treatment outcomes . the heterogeneity in the clinical presentation of vad is compounded by the lack of consensus regarding the pathologic diagnosis of vad . the main features of the most commonly used clinical criteria for vad are summarized in table 2 . although neuroimaging is not required for diagnosis by all clinical references , it is recommended in patients with suspected vad owing to a stroke history , vascular risk factors , or abnormal neurological examination . if ct shows significant evidence of vascular pathology , mri is not necessary . however , mri is more sensitive than ct for ischemic damage and vascular lesions . the absence of cerebral vascular lesions on ct or mri excludes the diagnosis of vad . features on ct or mri that are suggestive of vad include cortical or subcortical infarctions , multiple lacunar strokes , and wmls.28 because stroke is common and its risk factors are well defined and modifiable , it is reasonable to intensify efforts to control cardiovascular risk factors to reduce the population of stroke and vad . rigorous control of vascular risk factors is important in the primary and secondary prevention of vad . in terms of specific treatment , the use of cholinesterase inhibitors and n - methyl - d - aspartate receptor antagonists for vad has been evaluated.29 donepezil ( aricept ) 5 mg to 10 mg per day showed improvement in ad assessment scale - cognitive ( adas - cog ) subscale scores.30 - 32 galantamine ( reminyl ) has been studied for the treatment of vad in two randomized , double - blind , placebo - controlled trials of 592 patients and 788 patients , respectively . adas - cog score was a primary efficacy measure . at 26 weeks , there were improvements in galantamine - treated patients at doses of 16 mg to 24 mg per day . however , a significant percentage of patients dropped out owing to adverse events , particularly gastrointestinal effects.33,34 rivastigmine ( exelon ) was studied in a randomized , placebo - controlled trial of 710 patients and it was superior to placebo on the adas - cog score as a primary efficacy measure , except in younger ( age 50 - 75 years ) patients.35 memantine ( ebixa ) was studied for the treatment of vad in two large trials and both trials concluded that memantine 20 mg per day improved adas - cog scores.36,37 the therapeutic approach to vad is summarized as 1 ) primary prevention with cardiovascular risk factor control , 2 ) correct diagnosis of vad , 3 ) secondary stroke prevention with anti - platelet therapy , and 4 ) cholinesterase inhibitors or nmda receptor antagonists . the principles of anti - dementia drugs for vad are summarized below38 : make an accurate diagnosis of the dementia syndromecharacterize the severity of the dementia ( mild , moderate , or severe)inform the patient and caregiver of reasonable treatment expectations ( small symptomatic improvements and delay in decline are typical responses)optimize the dose of each medicationslow the titration of the medication if side effects emerge during the titration periodmonitor multiple domains for beneficial responses ( e.g. , cognition , behavior , function)monitor side effects ( especially gastrointestinal side effects with cholinesterase inhibitors)switch from one cholinesterase inhibitor to another if the patient is intolerant or shows no response to the current treatmentcontinue therapy until the severe phases of dementia are reachedmonitor for rapid emergence of new cognitive , behavioral , or functional symptoms if the medications are discontinuedcombine a cholinesterase inhibitor and memantine for optimal therapeutic response make an accurate diagnosis of the dementia syndrome characterize the severity of the dementia ( mild , moderate , or severe ) inform the patient and caregiver of reasonable treatment expectations ( small symptomatic improvements and delay in decline are typical responses ) optimize the dose of each medication slow the titration of the medication if side effects emerge during the titration period monitor multiple domains for beneficial responses ( e.g. , cognition , behavior , function ) monitor side effects ( especially gastrointestinal side effects with cholinesterase inhibitors ) switch from one cholinesterase inhibitor to another if the patient is intolerant or shows no response to the current treatment continue therapy until the severe phases of dementia are reached monitor for rapid emergence of new cognitive , behavioral , or functional symptoms if the medications are discontinued combine a cholinesterase inhibitor and memantine for optimal therapeutic response the prognosis of vad varies considerably according to the criteria used to make the diagnosis . mid shortens life expectancy to about 50% of normal at 4 years from the initial evaluation.39 about one - third of the most severely affected elderly die from complications of the dementia itself , one - third from cerebrovascular disease , 8% from other cardiovascular disease , and the rest from miscellaneous causes.40 overall , the effect of vad on mortality is similar to or mildly worse than that of ad.5 vad is characterized most commonly by subcortical frontal and executive dysfunction of cognitive impairment as opposed to the concept of amnesia - predominant ad . it may begin with very subtle deficits arising on the basis of leukoaraiosis or wmls and proceeds through infarction to a more advanced state . early identification of vascular cognitive impairment due to vascular brain damage as an early stage of vad and proper management through modification of vascular risk factors especially in midlife are important for the prevention of vad .

extrahepatic biliary duct duplication , also commonly called duplicated common bile duct ( dcbd ) , is exceptionally rare and mostly turn to be unexpected finding during surgery exposing surgeons to unusual surprises leading to iatrogenic bile duct injuries ( bdi ) . this anomaly has been reported to be associated with multiple disorders including cholelithiasis , choledocholithiasis , choledochal cyst , pancreatobiliary maljunction ( pbm ) and malignancy . recognition of the existence of this anomaly is important to prevent iatrogenic biliary injury and most importantly help in making the right surgical decision . after the first case of dcbd reported by vesalius in 1543 , numerous cases of different variants have been reported in the english literature across the globe [ 25 ] . only seven out of nine variants currently described in the literature can be classified by available classification systems . presented here - in , is a new variant of dcbd discovered after iatrogenic bile duct injury during laparoscopic cholecystectomy . the importance of recognition of its existence to avoidance bdi during surgery coupled with the need for a comprehensive classification system to encompass newly discovered variants are highlighted . a 64-year - old female presented to the surgical emergency unit with a day 's history of right upper quadrant pain . her physical examination revealed right upper quadrant tenderness with no rebound tenderness or muscular guarding . laboratory test showed leukocytosis of 17 10/l and elevated c - reactive protein of 51 mg / l ( 05 ml abdominal ultrasound revealed thickened gallbladder ( gb ) wall ( 4.7 mm ) with a 25 mm gallstone . a diagnosis of acute calculous cholecystitis was made and intravenous fluid and antibiotic treatment were initiated . gb wall was found to be inflamed with omental adhesions . using the critical view of safety approach , after careful dissection of the gb from the liver bed small amount of bile was observed oozing from a spot proximal to the ligated cystic duct stump . irrigation of the spot revealed a small tear at what was thought to be the common hepatic duct . a 5-fr drainage catheter ( 50 cm long , 1.4 mm in diameter ) was placed into the opened duct and exteriorized through the mid - clavicular 5 mm port site for post - operative drainage . a tube cholangiography done on post - operative day 7 showed duplication of the common hepatic duct proximal to the cystic duct stump with the injury occurring on the right - sided duct ( fig . was taken which confirmed the presence of the duplicated common hepatic duct with the cystic duct stump distal to the duplication ( fig . her post - operative course was uneventful with an average biliary drainage output of about 300 ml . drainage tube was clamped on post - operative day 9 and finally removed 4 weeks after surgery . figure 1:cholangiogram showing the double duct originating from the hepatic hilus with injury on the right duct . figure 2:mrcp showing the double common bile duct , cystic duct stump and pancreatic duct . cholangiogram showing the double duct originating from the hepatic hilus with injury on the right duct . the wide variation in morphology of reported cases of dcbd has posed a great challenge in its precise anatomical definition and classification . proposed modified classification system describes five groups , with subtypes , involving seven variants as depicted in fig . 3 . in the case presented here - in , the anomaly was discovered post - operatively by tube cholangiography following iatrogenic bile duct injury during laparoscopic cholecystectomy . the double ducts were noted to originate from the hepatic duct confluence separately and rejoined distally with the cystic duct noted to open into the unified duct ( figs 1 and 2 ) . this variant appears to be different from previously reported type v in which the cystic duct opens into the right duct before joining the left duct . literature review showed similar reported cases of inadvertent bdi associated with dbcd during surgery [ 6 , 7 ] and other configurations that could not be classified with existing classification systems [ 3 , 5 , 8 ] [ fig . 4 ] . these reports reaffirm and underscore the importance of prior knowledge of the existence of this anomaly and careful dissection within the hepato - gastroduodenal ligament to avoid bdi , particularly during laparoscopic cholecystectomy . the clinical importance of this anomaly lies with its association with conditions such as cholecysto - choledocholithiasis and choledochal cyst , the risk of malignant transformation and the risk of exposing patients to biliary injury during surgery , if not detected pre - operatively . the type of malignant transformation seems to be directly related to the drainage site of the ducts ( gastric , duodenal or pancreatic ) and the presence of pbm . in a review of 47 cases in the japanese literature , yamashita et al . reported an incidence of cholelithiasis in 28% , choledochal cyst in 11% , pbm in 30% and malignancy in 26% of the cases . review of 24 cases in the chinese literature also revealed cholelithiasis in 37% , choledocholithiasis in 79% , choledochal cyst in 33% and pbm in 8% of the reported cases . the high incidence of gallstone formation associated with this anomaly is thought be due to bile stasis and repetitive cholangitis resulting from reflux of gastrointestinal content into the anomalous duct . figure 3:modified double common bile duct classification proposed by choi et al . type i : cbd separated by septum ; type ii : cbd that bifurcates to drain separately ; type iii : double biliary drainage without extrahepatic communication channels ( without [ iiia ] or with [ iiib ] intrahepatic connecting channels ) ; type iv : double biliary drainage with one or more extrahepatic communication channels ; type v : single biliary drainage of double extrahepatic bile ducts without ( va ) or with ( vb ) communication channels . sahu et al.(b ) variant described by kosar et al.(c ) variant described in current report . type i : cbd separated by septum ; type ii : cbd that bifurcates to drain separately ; type iii : double biliary drainage without extrahepatic communication channels ( without [ iiia ] or with [ iiib ] intrahepatic connecting channels ) ; type iv : double biliary drainage with one or more extrahepatic communication channels ; type v : single biliary drainage of double extrahepatic bile ducts without ( va ) or with ( vb ) communication channels . sahu et al.(b ) variant described by kosar et al.(c ) variant described in current report . extrahepatic bile duct duplication could be a recipe for disaster during surgery and a prior knowledge of its existence is important to avoid inadvertent bdi . a thorough review of all reported variants is needed for a comprehensive classification system of clinical significance .

firstly , a child 's eye continues to grow during the initial years of life and early childhood . refractive elements of the eye undergo radical changes ; axial elongation and changes in corneal curvature are major factors influencing refractive changes in early childhood . for instance average corneal curvature flattens from 52d at birth , to 43.5d at the age of 18 months.1,2 in addition , axial length ( al ) increases from an average of 16.8 mm at birth to 23.6 mm in adulthood.3,4 the second issue is that the immature visual system in young children puts them at risk of amblyopia if visual input is defocused or unequal between the two eyes . third , certain complications , which may be acceptable in adults , are unacceptable in children . during the past decade , the optical correction of aphakia in children has improved dramatically ; however for obvious reasons accurate optical rehabilitation and postoperative supervision in these cases is more difficult than adults.5 optical rehabilitation in pediatric aphakic patients remains a challenge for ophthalmologists . the aim of this review is to cover issues regarding optical correction of pediatric aphakia , types of optical correction , indications , timing of intraocular lens ( iol ) implantation , types of iol , site of implantation , iol power calculations and selection , complications of iol implantation in pediatric patients , and finally to discuss the preferred choice of optical correction . an aphakic eye , especially in children , has optical properties which are different from normal phakic eyes . nowadays optical correction of aphakia in children includes aphakic glasses , aphakic contact lenses ( cls ) and primary or secondary iol implantation each having specific advantages and disadvantages . restrictions on using aphakic glasses are poor optics ; these include visual field narrowing to about 30 , an increase in nystagmus amplitude , and marked disparity in retinal image size of about 30%.6,7 furthermore , anisometropia exceeding 3d in spherical error or 1.5d in cylindrical error make glasses an improper choice . anisometropia produces confusion which may lead to permanent suppression , amblyopia or anomalous retinal correspondence and development of concomitant strabismus . another disadvantage of wearing glasses in newborns and infants is its extra weight and size . correction of aphakia with aphakic glasses is justified in rare cases or in the absence of parents co - operation . aphakic contact lenses contacts lenses may be fitted on eyes in all age groups and are a highly effective device in the visual rehabilitation of pediatric aphakia . the most common grounds for pediatric contact lens fitting are unilateral or bilateral aphakia ; in unilateral cases they can be applied as primary treatment in association with obturation of the normal eye . many bilateral aphakic subjects apply contact lenses with additional plus power for near vision correction in infancy , shifting to bifocals as toddlers . extended - wear soft or rigid lenses are generally well tolerated , though frequent power changes and lost lenses are significant financial barriers for many families . three types of contact lenses are utilized for pediatric aphakia : rigid gas permeable ( rgp ) , silicone elastomer and hydrogel lenses . silicone elastomer lenses are highly permeable to oxygen , even more than rgp lenses . due to the physical properties of silicone elastomer , lipid - mucin deposits easily accumulate on the surface of such lenses leading to corneal and conjunctival complications , therefore such lenses should be worn only during waking hours . hydrogel lenses , in principle , should be used in children over 4 years of age . these lenses are manufactured commercially in selected parameters , which are considered a disadvantage with the small eyeballs and steep corneas of newborns and infants . in pediatric aphakia , high plus power lenses have a thick central portion which inherently decreases oxygen permeability resulting in several corneal and conjunctival complications such as conjunctivitis , giant papillary conjunctivitis , neovascularization , corneal edema , abrasions , infective keratitis , endothelial polymegathism , and acute red eye reactions . such lenses can be damaging to the small developing globe . however , their sole advantage is low cost . nowadays , the majority of clinicians apply this type of contact lenses.8 - 12 special fitting considerations are required in case of microphthalmic eyes following congenital cataract surgery which have steep corneas and medium post - operative astigmatism . due to small corneal diameter , a narrow lid fissure with tense lids , rgp lenses are a good option . in comparison with other types of contact lenses , . however the principal problems of wearing contact lenses are poor compliance with long term use , loss of lenses , and ocular irritation and infection.13 iol implantation in children provides the benefit of reducing dependency on compliance in comparison with other external optical devices ( aphakic glasses and contact lenses ) providing at least partial correction . however , concerns about primary iol implantation are technical difficulties of implanting an iol in eyes of children , selecting an appropriate iol power , and the risk of visual axis opacification ( vao ) or posterior capsular opacification ( pco ) after implantation . despite primary posterior capsulectomy and vitrectomy , the rate of vao is higher in pseudophakic infantile eyes as compared to aphakic infantile eyes.14 on the other hand , although it is possible for an eye with unilateral infantile cataract to achieve good vision following contact lens correction , such an outcome is an exception rather than the rule . both iols and aphakic contact lenses may provide similar visual acuity ( va ) after surgery for unilateral cataract in the presence of good compliance with contact lens wear . however , iols provide better va when compliance with contact lens wear is moderate or poor.15 for bilateral aphakia , glasses and/or contact lenses may be a reasonable option . regarding unilateral cataracts in infancy , the issue of when to implant an iol is unresolved . the results of ongoing multicenter clinical trials are likely to guide us in near future . for children beyond infancy , iol implantation is less controversial . parental counseling cataract surgery in children is only one step in the long way toward visual rehabilitation , not the end of it . the parents/ caregivers play a crucial role in the postoperative care of the eye and treatment of amblyopia following aphakia . they should be made aware that a successful visual outcome depends on more than the surgical procedure ; it also depends to a great extent on their ability to maintain adequate aphakic correction and follow through with amblyopia therapy . before moving forward with iol implantation , it is of high significance to discuss the major pros and cons of available options with parents / legal guardian . a child operated on for cataract requires regular scheduled care in the first decade of life , and then every 1 - 2 years throughout life . so , to achieve the best visual outcome for the child , long - term commitment from the parents is required . the parents should also be informed about treatment of vao , strabismus , glaucoma , and rarely , decentered iol , synechiolysis or removal of a loose stitch . for eyes operated during early infancy , parents should be made aware that follow - up in the first six months is crucial . despite performing primary posterior capsulectomy and vitrectomy , many infant eyes develop vao , mostly within the first six postoperative months . for eyes operated on with an intact posterior capsule , parents should be made aware of the requirement of a secondary procedure for pco . parents of children with lens implants are also made aware that glasses will likely still be necessary postoperatively even with iol implantation . in addition , spectacle correction may need to be changed frequently after surgery , due to changing refraction . site of iol implantation placement of the iol in the capsular bag is preferred when capsular support is adequate . when stability of the capsular bag is compromised , such as with traumatic cataracts and zonular damage which is a common cause of unilateral aphakia in children , a capsular tension ring ( ctr ) can be used . ciliary sulcus fixation of the iol can also be performed in the absence of adequate capsular support for in - the - bag implantation . however , the incidence of uveitis and pupillary capture is higher with sulcus fixation.16 the younger the child , the greater the challenge for in - the - bag implantation of the iol especially due to difficulties of capsulorhexis in this age group . pediatric capsulorhexis performing a complete manual anterior continuous curvilinear capsulorhexis ( ccc ) and posterior ccc is a critical step to ensure safe placement of the iol inside the capsular bag . anterior and posterior ccc can be hard to perform because of high capsule elasticity and tension in children.17,18 capsulorhexis of the anterior capsule in young patients is more difficult than posterior ccc.19 capsular dye - assisted cataract surgery has been used to improve visibility and increase the rate of complete anterior and posterior ccc . saini et al conducted a prospective randomized trial to create anterior and posterior ccc in pediatric cataract surgery with and without applying trypan blue dye . the majority ( 91.3% ) of the eyes had complete anterior ccc and 82.6% had complete posterior ccc when trypan blue was used to stain the capsule , in comparison with 73.6% and 52.6% of anterior and posterior ccc , respectively , in eyes without trypan blue . sharma et al evaluated the efficacy of trypan blue in posterior capsulorhexis with optic capture in pediatric cataracts in a prospective randomized study . optic capture was possible in 17 of 18 eyes in trypan blue assisted surgery and 11 of 17 eyes in which no dye was used ( p=0.04).20 secondary iol implantation the vast majority of children undergoing secondary iol implantation have had primary posterior capsulotomy and anterior vitrectomy . if adequate peripheral capsular support is present , the iol is placed into the reopened capsular bag or in the ciliary sulcus . an all - polymethylmethacrylate ( pmma ) iol is ideal for sulcus placement and should be considered , especially when capsular remnants are insufficient . the most commonly used iol for secondary implantation is the three piece acrysof intraocular lenses ( alcon laboratories , inc . , texas , usa , model ma60ac ) . however , the haptics are soft and decentration may occur , particularly in eyes with large anterior segments and axial length greater than 23 mm . when inadequate capsular support is present for sulcus fixation in a child , implantation of an iol anterior chamber iols and scleral or iris - fixated posterior chamber iols are used in children when other viable options are lacking , although the long - term consequences of these placements are unknown . anterior chamber iols should be of an open - loop flexible design and sized appropriately for the anterior chamber . scleral - sutured iols are usually fixed with 10 - 0 prolene suture but concerns over biodegradation have been raised because late iol decentrations ( 5 - 15 years after surgery ) have been documented . iris fixation is also an alternative in children when adequate capsular support for sulcus or bag fixation is lacking . iris fixation as in the lobster - claw style lenses ( verisyse ) are utilized for phakic iol implantation in some high myopic children . the aphakic version of this iol is available for compassionate use but must be requested through the fda on a case - by - case basis . timing for iol implantation despite advances in adult iol implantation , transition to primary iol implantation in children , specifically those less than 1 year of age , has been gradual . reasons for reluctance to use iols in young children include ocular growth , the higher incidence of pco , and the eye s greater reactivity.13 there are several reports of iol implantation in children older than one year and , to a lesser extent , in younger children.21,22 the trend toward iol implantation in monocular patients aged 6 months and older is likely to continue , with some surgeons using iols in even younger children . bilateral iol implantation has been reported less frequently than unilateral cases , and most series describe children older than 2 to 3 years . for children younger than 2 years , little data exists on bilateral iol implantation , possibly because many children who are bilaterally aphakic can be managed well with aphakic spectacles or contact lenses.23 challenges of pediatric iol power calculation and selection calculating and selecting an optimum the requirement to implant a fixed power lens into a growing eye makes it difficult to choose an optimum iol power providing the most benefits for the child s eye . the younger the child at the time of surgery , the more difficult is the issue . this is a challenging task for ophthalmologists of industrialized countries , and probably even more complicated for ophthalmologists in developing nations.4 the lack of operating room instrumentation in many parts of the developing world , such as the hand - held keratometer and the a - scan ultrasound , increases the difficulty of calculating iol power for pediatric cataract surgery . even with the availability of the a - scan and automated keratometers in the operating room , iol power calculation for small eyes of children is challenging . we also use formulas originally designed for adult eyes . to accurately predict optimal iol power , formulas require measurement of al , corneal power and anterior chamber depth ( acd ) . when ultrasonic echo - impulse techniques are used for biometry , errors in predicted refraction after iol implantation are attributed to faulty al measurement ( 54% ) , keratometric errors ( 38% ) and errors in estimation of post - operative acd ( 8% ) . improving the accuracy of al determination has been suggested to have the greatest impact on improving iol power prediction . this is because an al measurement error of 0.5 mm for example , is capable of inducing a postoperative refractive error of up to 1.4d.24,25 two devices using low coherence reflectometry , which is a similar technique to partial coherence interferometery ( pci ) have been developed , namely lenstar ls900 ( haag - streit , koeniz , switzerland ) and allegro biograph ( wavelight , erlangen , germany).26 these devices have been shown to be as accurate and repeatable as the iolmaster and also advantageous to capturing all measurements needless of realignment and measurement of additional components of the anterior chamber such as corneal thickness for use in probably new biometry algorithms in future . a - scan ultrasound and keratometry measurements on children can be difficult or unattainable in the office . inaccurate al measurement is the most significant source of error in iol power calculation , nearly equating to 2.5 d / mm . in very short eyes ( 20 mm ) , this error rises dramatically to 3.75 d / mm . important details to keep in mind include the velocity required for use in any given eye ( phakic , aphakic , or pseudophakic ) and the a - constant for a specific iol . it has been reported that al measurements made with a contact technique were , on average , 0.24 to 0.32 mm smaller than measurements made using an immersion technique.27,28 reliable autokeratometer devices should be used for accurate measurement of corneal curvature in pediatric eyes . to avoid inaccuracy when taking repeated measurements , it is recommended to take the mean value for iol power calculation.4 iol power calculation formulas in adult patients several generations of iol power formulas have evolved , resulting in vast improvements in the accuracy of post - operative refractive prediction . srk - t , holladay 1 & 2 , hoffer q and haigis are commonly used formulas . although in eyes with average axial length , they only differ slightly in predicting optimal iol power , some are more accurate than others for axial lengths outside the mean . the following guidelines have been recommended for the choice of formulas:29 for al < 22 mm , hoffer q or srk / t ; for al from 22 to 24.5 mm , srk / t , holladay 1 or hoffer q ; for al > 24.6 , srk / t . the haigis and holladay 2 are newer formulas and hence have not been featured in the above guidelines . in one large series , it has been shown to be more accurate than hoffer q in extreme hyperopia.30 it was also found to be the most accurate for long eyes ( al>25.0mm).31 the holladay 2 formula uses seven variables namely axial length , lens thickness , corneal power ( average k ) , horizontal white - to - white corneal diameter , acd , preoperative refraction and patient age . one study investigating the accuracy of iol power prediction using the hoffer q , holladay 1 and 2 and srk / t formulas found no statistically significant difference between them in all subsets of axial lengths32 . now the question is which iol formula should be used in children ? because of the relatively large iol formula errors demonstrated in pediatric studies , no single formula can be considered to be accurate for all children . andreo and coworkers reported that all formulas were slightly less accurate in eyes with shorter al . in this group , the hoffer q formula had the lowest error ( 1.4 d ) and the srk - ii had the highest error ( 1.8 d ) . although no formula has been proven to have an advantage , it is preferable to use theoretical formulas ( e.g. srk - t , holladay i and hollday ii , hoffer i and ii , hoffer q and haigis ) because they are generally more accurate for small eyes , and in pediatric studies they appear to be slightly more accurate overall.4,33 individual surgeons continue to use their favored formulae to give them iol calculations but newer formulas should help reduce residual refractive errors , especially in more extreme cases of biometrics . the eyes of normal children grow from an average al of 16.8 mm at birth to 23.6 mm in adult life . most of the axial growth occurs during the first two years of life , but there is no sharp cut - off date ; instead the rate of change gradually decreases throughout childhood . as eye size increases , the power of the optical component decreases proportionately . the natural lens power decreases from 34.4 to 18.8 d.2 after the crystalline lens is removed surgically ; every millimeter of axial growth of the globe changes the refractive error of the eye by more than 2.5 d. in contrast to -0.9 d refractive change in normal phakic eyes , aphakic eyes have an average myopic shift of 10 d from infancy to adulthood . historically , three major approaches have been used for iol power selection in children : initial high hypermetropia , initial emmetropia , or initial low hypermetropia . regardless the chosen approach , refraction is changing and not stable probably until 20 years of age . thus , regular follow - up visits , and regular change of correction for residual refraction is required . initial high hypermetropia offers the advantage that with axial growth of an eye , hyperopia will improve , and adult refraction would probably be at or near plano , therefore low myopia or low hypermetropia may be achieved . however , this advantage must be balanced by the fact that the uncorrected hyperopic refractive error in children may cause or deteriorate amblyopia . since initial emmetropia reduces the risk of amblyopia , some surgeons prefer to aim for it to help treat amlyopia . however , significant late myopia will be more apparent as years pass since young children s eyes continue to grow . most physicians who have been implanting iols in young children have chosen an intermediate power between what the formulas would predict for the eye at the time of implantation and what the expected adult power would be for the specific eye . most physicians implanting an iol consider age at the time of surgery ( tables 1 , 2 and 3 ) , status of the fellow eye , the probability of compliance with amblyopia therapy , etc . when an iol is implanted in infancy , marked axial growth must be expected over the first 1 to 2 years after surgery . therefore , iols implanted in infancy are usually selected to produce 20% or more undercorrection . the closer to birth , the more marked this under - correction will need to be.4 status of the fellow eye it is important to consider refractive status in the fellow eye . more hyperopia may be acceptable when surgery is performed bilaterally since noncompliance with glasses is less amblyogenic in such children , or in an eye with monocular cataract , if the fellow eye is pseudophakic . attempts should be made to minimize aniseikonia in these eye.4 dense amblyopia may prompt a decision to leave less hyperopia ( or even achieve emmetropia ) in an effort to help recover vision by minimizing the need for glasses and emphasizing occlusion therapy . in this instance , late myopia is acceptable if it helps recover vision during years of amblyopia treatment . furthermore , myopia can probably be more easily handled with refractive surgery.4 parents refractive error it has been noted that if both parents are myopic , 30% to 40% of their children will become myopic , whereas if only one of the parents is myopic , 20% to 25% of their offspring will become myopic . if neither of the parents is myopic , fewer than 10% of their children will become myopic . anticipating more eye growth , these children may be left with more initial hypermetropia.4 amount of undercorrection in general , the higher the iol power , the more undercorrection is needed . for example , at age 1 month , if a child has an emmetropic power of 50 d and another child at the same age has an emmetropic power of 40 d , the first child will require higher residual hyperopic refraction . in other words , one may consider an approximate expected refraction of + 12 d in the first child , while in second child + 10 d may be suitable.4 effective iol power by site of implantation if the site of iol fixation needs to be changed during surgery , an appropriate adjustment may need to be made . a plus - power iol that is shifted more anteriorly in the eye will have a greater refractive effect in comparison with relatively posterior location . intraocular positioning of the iol will affect the predicted error , with sulcus fixation producing relative myopic shift from bag fixation . the iol power intended for capsular bag placement should be decreased by 0.75 to 1.00d ( depending on iol power ) when placed in the ciliary sulcus.4 power calculation for secondary iol implantation for secondary iols , power can be calculated without al or k - values simply by using the aphakic refraction.34 the pediatric iol calculator can also be used for iol power calculation in secondary implantation . add the child s age , the a - constant of the iol and an approximate k and al value . put in a power of 0 for iol power , and the program will tell you the predicted resulting refraction . next , adjust the value of al until the resulting refraction equals the measured refraction for that eye . finally , put in your goal refraction ; the resulting iol to use output should be accurate.4,35 all known factors affecting axial growth should be taken into account . besides these , several other factors ( e.g. , gender , race , etc . ) have been reported to affect growth of the normal eye , and may also influence eye growth after cataract surgery . surgeons who implant iols in young children must be prepared for wide variability in long - term myopic shift . both the magnitude of the myopic shift and the variance in this shift are likely to be greatest in children having surgery in the first few years of life . anticipation of this myopic shift , and its appropriate correction or compensation , will help achieve better anatomical and functional outcomes in young eyes undergoing cataract surgery.4 management of refractive surprises in spite of best efforts , refractive surprises do happen . this may be due to errors in biometry and the use of inappropriate power calculation formulas . sometimes as a result of human error , a wrong lens can be implanted . in every case of unexpected refractive outcome hospital critical incident procedures should be invoked for a multi - disciplinary approach with a view to learning from mistakes and minimizing risk in future . unexpected astigmatism may result from poor wound construction ( high surgically induced astigmatism ) , unplanned intraoperative conversion to a large incision to express lens fragments or due to high pre - existing corneal astigmatism which had been masked by lenticular compensation . complications of iol implantation visual axis opacification secondary vao is one of the most common complications of pediatric cataract surgery , especially when the posterior capsule is left intact . pco is generally delayed in eyes with hydrophobic acrylic iols as compared to pmma iols . vao after acrylic iol implantation with an intact posterior capsule is more proliferative as compared to the fibrous reaction commonly observed in conjunction with pmma iols . after a primary posterior capsulectomy and anterior vitrectomy when infant eyes are implanted with an iol , vao is common despite performing posterior capsulectomy and vitrectomy . using hydrophobic acrylic iols , various articles have reported an average of 44.0% rate for vao , ranging from 8.1% ( reviewing children under 2 years of age ) to 80% ( including operated children below 6 months of age).36 - 40 secondary vao in eyes implanted in infancy tends to occur within the first 6 months after cataract surgery.37 thus , longer follow - up will not likely change the incidence of vao in infantile eyes . eyes with ocular anomalies ( e.g. , anterior segment dysgenesis , iris hypoplasia , or persistent fetal vasculature ) are at 9 times higher risk of developing vao as compared to eyes without such anomalies.37 in children older than 2 years of age at the time of cataract surgery , the rate of secondary vao after primary posterior capsulectomy and vitrectomy varies from 0% to 20.6% with an average of 5.1% . in older children , some authors prefer to perform only posterior capsulorhexis ( without vitrectomy ) . the average rate of secondary intervention in these eyes is 13.8% ( range 0 - 68% ) . with an intact posterior capsule , various articles have reported pco ranging from 14.7% to 100% ( average 25.1% , excluding eyes with 100% pco in children younger than 4 years of age ) . whereas originally the lens biomaterial was thought to be a major determinant , it is now largely recognized that the design of the iol , principally a square edge of the optic , acts as a barrier to the migration of these cells.41 enhanced square edge designs are now available providing a raised edge and consequently greater barrier function . the optical properties of newer lenses would be seriously degraded following capsular opacification and its removal . posterior optic buttonholing is a technique whereby a 4 mm or smaller opening is made in the posterior capsule and the optic is prolapsed into the opening . this technique was adopted from pediatric cataract surgery where the pco rate following cataract surgery is extremely high . in a consecutive series of 1,000 patients , in addition to their direct effects on vision , aphakia and other lens disorders in children may also cause visual loss due to amblyopia particularly in young infants with unilateral aphakia . the onset of amblyopia in this setting is rapid and profound , and early intervention is necessary to maximize the chance for a good visual outcome . a good outcome in optical correction of pediatric aphakia depends on 3 issues : successful surgical removal of the lens opacity . the risk of amblyopia is greatly increased in younger children , and the younger the child , the greater the risk . unilateral cataracts and aphakia in particular are highly amblyogenic in newborns . they need to be removed within 4 to 6 weeks of age to maximize the potential for good vision . bilateral cataracts may also cause amblyopia in newborns , but the time frame for optimal removal in this setting extends to 2 to 3 months . the risk of amblyopia in patients with acquired cataracts decreases as children get older , but persists until 5 years of age or more . if an older child presents with a unilateral cataract , the prognosis for improvement depends on the age at which the opacity developed . if it has been present since early infancy , the prognosis is poor , because of amblyopia , even if the surgery is successful in itself . ongoing treatment for amblyopia is critical in children with lens disorders , particularly infants with unilateral opacities . patching is often necessary during the initial years of life to achieve the best possible vision . children do not well cooperate for eye examination and iop measurement , so glaucoma diagnosis can be easily missed . most cases of pediatric aphakic glaucoma are of the open - angle type and the prognosis is guarded . frequent , lifelong follow - up is required in order to screen for glaucoma since it may manifest many years after congenital cataract surgery.43 - 45 the primary function of the crystalline lens is to focus light on the retina . in aphakic patients , intraocular lenses are usually the best option in older children , because they most closely restore the eye to its natural state . many patients require glasses in addition to the intraocular lenses to fine - tune the focusing . because in aphakic patients the intraocular lens has only one power , a bifocal is necessary in order to focus at near . first , the incidence of complications related to iols , such as glaucoma , vao , lens displacement , and inammation is much higher in the first few months of life . second , the eye grows rapidly during the first 1 to 2 years of life , and this growth affects refraction . intraocular lenses are not adjustable , and a lens that focuses correctly in a 1-month - old child will be substantially overpowered by the time the child is 2 . therefore , most infants with unilateral aphakia are treated with contact lenses for the first few years of life , after which an iol can be implanted as a secondary procedure . aphakic glasses are also an option for replacing focusing power , but are very thick , causing distortion . the parents / caregivers play a critical role in the postoperative care of the eye and treatment of amblyopia following aphakia . frequent and lifelong follow - up is also necessary to screen for glaucoma since glaucoma may become manifest several years after congenital cataract surgery .

insects exhibit symbiotic relationships with bacteria and they provide several benefits to their host . essential insect symbionts offer a desirable target for the control of insect pests that rely upon them . the complete elimination of endosymbionts using antibiotics reduces the lifespan of the insect and suppresses the population within a few days or weeks . in the insect pest complex in agricultural ecosystem , mealy bugs ( homoptera : pseudococcidae ) have been rated as a major pest , since they play a dual role as pest and vector in field crops [ 4 , 5 ] . mealy bugs suck sap from their host and the nutrient deficient plant becomes stunted and distorted and shows reduced vigor [ 6 , 7 ] . the infestation of the mealy bug , rhizoecus amorphophalli betrem , on stored tubers of aroids and yams is a major concern among the tuber crop farmers . tuber crops including aroids and yams play a vital role in food security [ 8 , 9 ] and are the important staple or subsidiary food for one - fifth of the world population . mealy bugs suck cell sap from the tubers and the severely infested deformed tubers find no place in market , nor are they accepted for cooking . mealy bugs multiplication is rapid during the season with high temperature and less humidity and they spread all over the tubers with white powdery mealy substance and disfigure the tubers . since rapid and effective methods to control insect pests represent one of the many scientific achievements of the twentieth century , use of antibiotics for pest management is a viable option . elimination of gut bacteria of southern green stink bug , nezara viridula l. , using antibiotics reduced the weight of newly emerging adults . since endosymbionts play a vital role in the physiology of their host , revealing the types of bacteria associated with mealy bug will give basic information , which may throw light on the management of this pest . the present study deals with the isolation and identification of endosymbionts by classical and molecular approach and their antimicrobial activity . adult females of r. amorphophalli collected from the stock culturewere surface sterilized with absolute ethanol and chlorine bleach , were homogenized in sterile 0.9% saline , and were plated directly on nutrient agar media and kept for aerobic overnight incubation at 30c . pure culture of each bacterium for biochemical identification was obtained by streaking the individual colony on a fresh nutrient agar plate and incubated for 24 h at 35c . the gram staining was performed using hi - media kit ( hi - media laboratories pvt . ltd . , motility of the isolates was tested using hanging drop method ( hi - media ) . place 2 drops of culture in the middle of the slide and place a clean cover slip over the drop and examined under microscope . monosaccharide ( fructose ) , disaccharide ( sucrose , lactose , and maltose ) , polyhydric alcohol ( mannitol ) , and hexose ( dextrose ) were tested . triple sugar - iron agar test ( tsi ) was also done according to the manufacture 's protocol ( hi - media , mumbai , india ) . the production of tryptophanase , sufficient amount of alcohol , acetyl methyl carbinol , and citrate utilisation were noted using the indole test , methyl red ( mr ) test , voges - proskauer ( vp ) test , and citrate utilization test , respectively , according to the protocol of mackie mccartney . exoenzymes produced were noted using urease test , o - nitrophenyl - beta - d - galactoside ( onpg ) ( -galactosidase ) test , and nitrate reduction test according to the protocol of mackie mccartney . pure cultures were maintained by subsequent subculture / transfer in nutrient agar medium every 15 days . total genomic dna was extracted from bacterial cultures and treated with 100 l of rnase ( banglore genei , india ) for eliminating the rna contamination . the bacterial isolates were identified using 16s rdna gene specific primers ( 16sp0 , 5-gaagagtttgatcctggctcag-3 , and 16sp6 , 5-ctacggctaccttgttacga-3 ) . pcr amplification was carried out in 25 l reaction volume which included the following components : 510 l of genomic dna ( 500 ng ) , 20 picomoles of each primer , 10 mm tris hcl ( ph-8.3 ) , 50 mm kcl , 2.5 mm mgcl2 , 0.25 mm of each dntp , and 0.5 u of taq dna polymerase ( fermentas life sciences , eu ) . amplification was carried out in a thermal cycler ( applied biosystems , veriti , usa ) with cycling regime94c for 4 minutes as initial denaturation followed by 35 cycles of 94c for 30 seconds , 55c for 45 seconds , 72c for 45 seconds , and 72c for 10 minutes as final extension . the amplified products resolved on 1.2% agarose gel were stained with ethidium bromide ( 10 g / ml ) and visualized in a gel documentation system ( uvp , analytika jena , germany ) . pcr products were gel - purified and ligated into t / a cloning vector ptz57r / t ( instaclone pcr cloning kit , fermentas , eu ) and transformed into competent dh5 cells according to the manufacturer 's protocol . the transformed cells were spread on lb agar plates supplemented with x - gal ( 300 g / ml ) , iptg ( 120 g / ml ) , and ampicillin ( 100 g / ml ) and incubated at 37c overnight . blue / white selection was carried out and the plasmids were isolated from the positive clones using gene jet plasmid miniprep kit ( fermentas , eu ) according to the manufacturer 's protocol . sequencing was carried out in an automated sequencer ( abi prism 310 ; applied biosystems , usa ) using m13 universal primers in both directions . the nucleotide sequence obtained was processed to remove low quality reads and transformed into consensus sequences with geneious pro software version 5.6 . the resulted high quality sequences were analyzed with blastn ( ncbi ; http://www.ncbi.nlm.nih.gov ) to confirm the authenticity of the isolate . the sequences of related species and genus were downloaded from the genbank database and a phylogenetic study was carried out with the program mega version 5 . the sequences were aligned using the computer package clustalw and were analyzed to determine the relationships between isolates by the neighbor - joining method using the maximum composite likelihood model . bootstrap values were generated using 2000 replicates to infer the robustness of the tree topology . the bacterial cultures maintained in nutrient agar slant at 4c were subcultured in nutrient broth to obtain the working cultures approximately containing 1 10 cfu / ml . mueller hinton agar plates ( hi - media , mumbai , india ) were swabbed with each bacterial strain and the antibiotic disks were placed on the plates . disks of cephalexin ( 30 g / disc ) ( cp ) , ciproflax ( 5 g / disc ) ( cfx ) , endrofloxaxin ( 10 g / disc ) ( ex ) , and cefixime ( 5 g / disc ) ( cfix ) were used . the sensitivity and resistance of each isolate were determined by the criteria of the national committee for clinical laboratory standards ( 1997 ) . a total of five bacterial strains were successfully isolated from the r. amorphophalli and were assigned code numbers as isolates rhizo 1 , rhizo 2 , rhizo 3 , rhizo 4 , and rhizo 5 for laboratory purposes . isolates rhizo 1 , rhizo 2 , and rhizo 3 showed similar biochemical characters ( table 1 ) . acid was produced from dextrose and lactose and no acid was produced from sucrose , mannitol , maltose , fructose , and starch . in imvic test , citrate was positive and indole , methyl red and vp was negative . nutrient agar colonies were irregular , lobate , and creamy yellow in colour ( table 1 ) . the isolate rhizo 5 differed from other isolates with small , irregular , and white colonies . they were gram positive and cocci shaped and even though all other isolates were motile , this bacterium was nonmotile . the biochemical characters of all isolates were verified using bergey 's manual of systematic bacteriology volume 2 ( 2005 ) and bergey 's manual of systematic bacteriology volume 3 ( 2009 ) . based on morphological and biochemical characteristics ( table 1 ) , although biochemical tests revealed the preliminary identity of the isolates , for definitive identification 16s rdna sequencing was performed . blast analysis of isolates rhizo 1 , rhizo 2 , and rhizo 3 showed 100% similarity to bacillus subtilis available in the genbank database and thus the bacterium was identified as bacillus subtilis . similarly , isolate rhizo 4 showed 100% similarity to staphylococcus gallinarum sequence and was identified as staphylococcus gallinarum . the isolate rhizo 5 showed 100% similarity to staphylococcus saprophyticus sequence and was identified as staphylococcus saprophyticus . the sequence data generated from the study was deposited in the genbank nucleotide database ( ncbi ) and the accession numbers assigned are as follows : isolate 1 : kf015514 ; isolate 2 : kf015515 ; isolate 3 : kf015516 ; isolate 4 : kf015518 ; isolate 5 : kf015517 . the bacterial isolates were successfully grouped to their respective reference strains obtained from the genbank database confirming the authenticity of the isolate ( figures 1 and 2 ) . the antibiotic activity against the endosymbiotic bacteria isolated from r. amorphophalli is presented in table 2 . loss of these microorganisms often results in abnormal development and reduces survival of the insect host . previously , many reports described the isolation of bacteria from mealy bugs and other sap sucking insects . however , the present study is the first to identify bacteria associated with mealy bug , rhizoecus amorphophalli , employing phenotypic and 16s rdna sequence analysis . three culturable bacteria , namely , bacillus subtilis , staphylococcus gallinarum , and staphylococcus saprophyticus , were isolated from r. amorphophalli . blast search for these isolates showed the highest match with the respective species and all the sequences were deposited with ncbi - genbank . when compared with bergey 's manual of systematic bacteriology ( 2009 ) it was seen that the biochemical tests of three isolates showed similarity with bacillus sp . but , there were few exceptions as for bacillus acid will be produced all sugars . however , in our study acid was produced only from dextrose and lactose and all other sugars tested showed negative reaction . similarly , isolates rhizo 4 and rhizo 5 showed biochemical characters of staphylococcus when compared with agreement with bergey 's manual of systematic bacteriology volume 2 ( 2005 ) . but present results showed dissimilarity in sugar test and nitrate reduction test when compared with bergey 's manual . a combination of biochemical and molecular methods may prove to be more helpful in obtaining a precise and credible identification for the isolates . therefore one should associate the results of biochemical test with the molecular methods to confirm the identification of the isolate under study . in the current study , bacteria of genus bacillus and staphylococcus were previously reported from many insect species but , among the three isolates , s. saprophyticus was less reported as endosymbiont from insects . bacillus subtilis was previously reported as an endosymbiont of whitefly , b. tabaci . from the intestine of silk worm , bombyx mori l. , nine bacterial strains belonging to genera bacillus , brevibacterium , corynebacterium , staphylococcus , klebsiella , and stenotrophomonas were identified . from the gut of aedes aegypti l. , b. subtilis and serratia sp . were found . the phenotypic characteristics of 15 probable species of bacillus wereidentified and it was found that they are gram positive rods which were capable of hydrolysing starch and casein . in this study they found that the most dominating species was b. subtilis when compared with the other species found ( b. pumilus and b. mycoides ) . b. pumilus , one of the most dominant bacteria of all the populations in insects , is a gram positive , aerobic , rod - shaped , soil - dwelling bacterium . like other bacillus species , the spores produced by b. pumilus are more resistant than vegetative cells to heat , desiccation , uv radiation , -radiation , h2o2 , and starvation . the presence of this has been previously reported from gut flora of adult phlebotomus papatasi . but b. pumilus and b. mycoides are found in mealy bug . one important power possessed by bacillus strains isolated is their ability to produce amylase enzyme [ 30 , 31 ] , and these amylases are involved in the initial breakdown of cassava starch into simple sugars . similarly , the endosymbiont b. subtilis may be helping the r. amorphophalli , by production of amylase enzyme required for conversion of starch to simple sugars . eleven isolates from the silk worm were identified which included the gram positive bacillus circulans that degrade cellulose , pectin , and starch and have impact on digestion . staphylococcus gallinarum and s. saprophyticus were isolated from b and q types of b. tabaci , respectively . several gram positive bacteria including lactococcus garvieae and s. saprophyticus were isolated from the gut of red imported fire ant , solenopsis invicta buren , using 16s rdna sequences . staphylococcus saprophyticus was less reported as endosymbionts of insects but was recorded from many living systems . bacillus and staphylococcus from the whitefly were reported for their potential to produce medium length sugars from sucrose and contribute to the stickiness of the honeydew secreted by the host insect and these bacteria were recorded from r. amorphophalli also . the bacterial association with arthropods has been used to develop novel control strategies of agricultural , medical , and veterinary important pests . based on microecology theory , insects lack a complete enzyme system and thus need gut microorganisms to provide different kinds of enzymes for food digestion , nutrient absorption , and biological metabolism . antibiotic susceptibility tests showed that all the isolates were resistant to the antibiotics except cephalexin . antibiotic sensitivity pattern of bacillus sp . was studied and results showed that b. subtilis was resistant to gentamicin , ampiclox , and zinnacef and susceptible to ciprofloxacin . staphylococcus saprophyticus was susceptible to ampicillin , cephalexin , and trimethoprim - sulfamethoxazole and resistant to nalidixic acid and novobiocin . endosymbionts could be used as effective biocontrol agents ( bcas ) and the present study will stand as a connecting link in identification and effective utilization of these endosymbionts as bcas and also in formulating newer ipm strategy for r. amorphophalli by employing these three bacteria .

asthma is a chronic inflammatory airway disorder characterized by airway hyper - responsiveness , reversible airway obstruction , and airway remodeling . it is commonly thought that asthma is a multi - factorial disease resulting from complex interactions between genetic predisposition and environmental factors . studies have shown that genetic influences on asthma are substantial , with heritability estimates ranging between 35% and 95% . notably , a large number of polymorphisms in genes positioned throughout the genome have been implicated in asthma causation , including the gene encoding toll - like receptor 4 ( tlr4 ) . toll - like receptors ( tlrs ) are a class of evolutionarily conserved membrane - bound pattern recognition receptors ( prrs ) presented on the cell surface of innate immune cells . tlrs recognize pathogen - associated molecular patterns ( pamps ) exclusively expressed by microbial pathogens , and activate cellular signaling pathways to induce immune - response genes , including inflammatory cytokines . tlr4 , the best - studied tlr , is expressed on macrophages , dendritic cells , and other cell types , and mainly recognizes lipopolysaccharide ( lps ) of gram - negative bacteria . interaction of lps with tlr4 can activate the nuclear factor kappa b ( nf-b ) signaling pathway , increase expression of inflammatory cytokines and disturb the th1/th2 balance in asthma . tlr4 also regulates innate immune responses to respiratory syncytial virus infection , which is a risk factor for the development of asthma . two single - nucleotide polymorphisms ( snps ) , tlr4 + 896a / g ( rs4986790 , also known as asp299gly ) and tlr4 + 1196c / t ( rs4986791 , also known as thr399ile ) , have been demonstrated to modify the receptor s response to endotoxin , which is an important trigger of asthma . this genetically determined alteration in endotoxin responsiveness several studies have evaluated the association of tlr4 + 896a / g and + 1196c / t polymorphisms with asthma [ 1331 ] . however , due to the limitation of subjects , the results were inconsistent and controversial . a previous meta - analysis has shown a marginal association of tlr4 + 896a / g with asthma , and no association between tlr4 + 1196c / t polymorphism and asthma , but it did not cover all eligible studies . the exact correlation between tlr4 + 896a / g and + 1196c / t polymorphisms and asthma has not been entirely established . therefore , we performed a meta - analysis including all eligible case - control studies to clarify and quantify the authentic effect of tlr4 + 896a / g and + 1196c / t polymorphisms on the risk of asthma . this study was conducted according to the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines . a literature search was performed using pubmed , embase , web of science , chinese national knowledge infrastructure ( cnki ) , and wanfang data from inception to july 2015 . the search strings was : ( asthma or asthmatic ) and ( toll - like receptor 4 or no restrictions were applied for language , population , sample size , publication date , or type of report . studies were included in the meta - analysis that met the following criteria : 1 ) case - control study design , 2 ) evaluation of the association between the tlr4 + 896a / g ( asp299gly ) and + 1196c / t ( thr399ile ) polymorphisms and risk of asthma , 3 ) genotype frequency was available or sufficient data could be extracted to calculate odds ratios ( ors ) and 95% confidence intervals ( cis ) , and 4 ) not animal studies . for overlapping studies , the following data were extracted : year of publication , first author , country , ethnicity , age , atopic status , detection methods , sample size , and genotype frequencies in cases and controls . all the statistical analyses were conducted using stata software version 12.0 ( stata corporation , college station , tx , usa ) . we first evaluated hardy - weinberg equilibrium ( hwe ) in the control group for each study using the chi - square test , and it was considered statistically significant when p<0.05 . the strength of association between tlr4 + 896a / g and + 1196c / t polymorphisms and the risk of asthma was assessed by calculating ors with 95% cis . potential publication bias was assessed by begg s rank correlation test and egger s linear regression test , and p<0.05 was considered significant publication bias . a literature search was performed using pubmed , embase , web of science , chinese national knowledge infrastructure ( cnki ) , and wanfang data from inception to july 2015 . the search strings was : ( asthma or asthmatic ) and ( toll - like receptor 4 or no restrictions were applied for language , population , sample size , publication date , or type of report . studies were included in the meta - analysis that met the following criteria : 1 ) case - control study design , 2 ) evaluation of the association between the tlr4 + 896a / g ( asp299gly ) and + 1196c / t ( thr399ile ) polymorphisms and risk of asthma , 3 ) genotype frequency was available or sufficient data could be extracted to calculate odds ratios ( ors ) and 95% confidence intervals ( cis ) , and 4 ) not animal studies . for overlapping studies , the most recent article or the one with the largest sample size was selected . the following data were extracted : year of publication , first author , country , ethnicity , age , atopic status , detection methods , sample size , and genotype frequencies in cases and controls . all the statistical analyses were conducted using stata software version 12.0 ( stata corporation , college station , tx , usa ) . we first evaluated hardy - weinberg equilibrium ( hwe ) in the control group for each study using the chi - square test , and it was considered statistically significant when p<0.05 . the strength of association between tlr4 + 896a / g and + 1196c / t polymorphisms and the risk of asthma was assessed by calculating ors with 95% cis . potential publication bias was assessed by begg s rank correlation test and egger s linear regression test , and p<0.05 was considered significant publication bias . the study selection process was shown in figure 1 . in total , 72 english articles and 119 chinese articles met search criteria after we initially searched pubmed , embase , web of science , cnki , and wanfang data . after reading titles and abstracts , 131 articles were excluded because they did not refer to tlr4 gene polymorphisms and asthma risk . the remaining 60 articles were identified for full - text view ; 22 were excluded because they investigated other gene polymorphisms , 16 were excluded because they were reviews , 2 were not case - control studies , and 1 was repeated . a total of 19 relevant studies investigating the tlr4 + 896a / g and + 1196c / t polymorphisms and asthma for meta - analysis were identified . however , 5 studies were monomorphic at site and were excluded from this meta - analysis . finally , 14 case - control studies were included in the present meta - analysis . the characteristics of the selected studies are listed in table 1 , with a total of 13 studies of 2649 asthma cases and 2542 controls for investigating + 896a / g polymorphism and 7 studies of 1443 asthma cases and 1693 controls for + 1196c / t polymorphism . as for ethnicity , 11 studies investigated white populations [ 13,17,19,20,2328,31 ] , 2 studies investigated asian populations , and 1 study investigated the u.s . the genotype frequencies of tlr4 + 896a / g and + 1196c / t polymorphisms in each study are presented in table 2 . the main results of the relationship between tlr4 + 896a / g and + 1196c / t polymorphisms and asthma risk are listed in table 3 . overall , no significant association between tlr4 + 896a / g polymorphism and asthma risk was found in the allele model ( g vs. a : or=1.05 , 95% ci=0.901.23 , p=0.51 ) , the dominant model ( gg + ag vs. aa : or=1.05 , 95% ci=0.891.24 , p=0.56 ) , or the codominant models ( ag vs. aa : or=1.04 , 95% ci=0.881.23 , p=0.64 ) . after categorizing subjects into different subgroups on the basis of ethnicity the recessive model ( gg vs. ag+aa ) and codominant model ( gg vs. aa ) were not performed due to the low frequency of the gg genotype in cases and controls . as for tlr4 + 1196c / t polymorphism , a protective association was found between tlr4 + 1196c / t polymorphism and asthma in the allele model ( t vs. c : or=0.79 , 95%ci=0.630.99 , p=0.04 ) , the dominant model ( tt+ct vs. cc : or=0.76 , 95%ci=0.590.96 , p=0.03 ) , and the codominant model ( ct vs. cc : or=0.74 , 95%ci=0.580.95 , p=0.02 ) . this association was not examined via the recessive model ( tt vs. ct+cc ) or codominant model ( tt vs. cc ) due to the low frequency of the tt genotype in cases and controls . the subgroup analysis by ethnicity showed that the t allele of tlr4 + 1196c / t polymorphism was a significant protective gene for the development of asthma in asians ( t vs. c : or=0.73 , 95%ci=0.540.98 , p=0.04 ; tt+ct vs. cc : or=0.70 , 95%ci=0.510.96 , p=0.03 ; ct vs. cc : or=0.69 , 95%ci=0.500.96 , p=0.03 ) , but there was no statistically significant difference in whites ( t vs. c : or=0.87 , 95%ci=0.601.26 , p=0.46 ; tt+ct vs. cc : or=0.84 , 95%ci=0.571.24 , p=0.39 ; ct vs. cc : or=0.82 , 95%ci=0.551.22 , p=0.33 ) ( figures 35 ) . there was no significant heterogeneity between any studies when analyzing the association of tlr4 + 896a / g and + 1196c / t polymorphisms and asthma risk in all genetic models , so we used fixed - effects models . exclusion of the hwe - deviated studies did not meaningfully change the pooled estimates ( data not shown ) . the funnel plot was used to evaluate publication bias , and there was no obvious asymmetry . furthermore , no significant publication bias was detected by begg s test and egger s test ( all p>0.05 ) . the study selection process was shown in figure 1 . in total , 72 english articles and 119 chinese articles met search criteria after we initially searched pubmed , embase , web of science , cnki , and wanfang data . after reading titles and abstracts , 131 articles were excluded because they did not refer to tlr4 gene polymorphisms and asthma risk . the remaining 60 articles were identified for full - text view ; 22 were excluded because they investigated other gene polymorphisms , 16 were excluded because they were reviews , 2 were not case - control studies , and 1 was repeated . a total of 19 relevant studies investigating the tlr4 + 896a / g and + 1196c / t polymorphisms and asthma for meta - analysis were identified . however , 5 studies were monomorphic at site and were excluded from this meta - analysis . finally , 14 case - control studies were included in the present meta - analysis . the characteristics of the selected studies are listed in table 1 , with a total of 13 studies of 2649 asthma cases and 2542 controls for investigating + 896a / g polymorphism and 7 studies of 1443 asthma cases and 1693 controls for + 1196c / t polymorphism . as for ethnicity , 11 studies investigated white populations [ 13,17,19,20,2328,31 ] , 2 studies investigated asian populations , and 1 study investigated the u.s . the genotype frequencies of tlr4 + 896a / g and + 1196c / t polymorphisms in each study are presented in table 2 . the main results of the relationship between tlr4 + 896a / g and + 1196c / t polymorphisms and asthma risk are listed in table 3 . overall , no significant association between tlr4 + 896a / g polymorphism and asthma risk was found in the allele model ( g vs. a : or=1.05 , 95% ci=0.901.23 , p=0.51 ) , the dominant model ( gg + ag vs. aa : or=1.05 , 95% ci=0.891.24 , p=0.56 ) , or the codominant models ( ag vs. aa : or=1.04 , 95% ci=0.881.23 , p=0.64 ) . after categorizing subjects into different subgroups on the basis of ethnicity the recessive model ( gg vs. ag+aa ) and codominant model ( gg vs. aa ) were not performed due to the low frequency of the gg genotype in cases and controls . as for tlr4 + 1196c / t polymorphism , a protective association was found between tlr4 + 1196c / t polymorphism and asthma in the allele model ( t vs. c : or=0.79 , 95%ci=0.630.99 , p=0.04 ) , the dominant model ( tt+ct vs. cc : or=0.76 , 95%ci=0.590.96 , p=0.03 ) , and the codominant model ( ct vs. cc : or=0.74 , 95%ci=0.580.95 , p=0.02 ) . this association was not examined via the recessive model ( tt vs. ct+cc ) or codominant model ( tt vs. cc ) due to the low frequency of the tt genotype in cases and controls . the subgroup analysis by ethnicity showed that the t allele of tlr4 + 1196c / t polymorphism was a significant protective gene for the development of asthma in asians ( t vs. c : or=0.73 , 95%ci=0.540.98 , p=0.04 ; tt+ct vs. cc : or=0.70 , 95%ci=0.510.96 , p=0.03 ; ct vs. cc : or=0.69 , 95%ci=0.500.96 , p=0.03 ) , but there was no statistically significant difference in whites ( t vs. c : or=0.87 , 95%ci=0.601.26 , p=0.46 ; tt+ct vs. cc : or=0.84 , 95%ci=0.571.24 , p=0.39 ; ct vs. cc : or=0.82 , 95%ci=0.551.22 , p=0.33 ) ( figures 35 ) . there was no significant heterogeneity between any studies when analyzing the association of tlr4 + 896a / g and + 1196c / t polymorphisms and asthma risk in all genetic models , so we used fixed - effects models . exclusion of the hwe - deviated studies did not meaningfully change the pooled estimates ( data not shown ) . the funnel plot was used to evaluate publication bias , and there was no obvious asymmetry . furthermore , no significant publication bias was detected by begg s test and egger s test ( all p>0.05 ) . asthma is a chronic inflammatory airway disorder with complex etiologies involving both genetic and environmental contributions . several candidate genes , such as tlr4 , cd14 , stat6 , adam33 , and il-13 , have been reported to be associated with asthma susceptibility [ 18,3336 ] . recognition of lps by tlr4 plays a crucial role in the activation of subsequent immune and inflammatory responses against invaders . numerous studies have indicated the role of tlr4 in the pathogenesis of asthma [ 3740 ] . demonstrated that lps impair the development of th2 immunity , signaling via tlr4 and myd88 molecules and via the il-12/ifn- axis , and the synthetic tlr4 agonists protect against allergic asthma development . genetic polymorphisms of tlr4 gene have been demonstrated to be associated with diminished airway responsiveness to inhaled lps , and to be closely involved in the susceptibility to many diseases , including asthma , juvenile spondyloarthritis , inflammatory bowel disease , and systemic lupus erythematosus [ 18,4345 ] . two co - segregating single - nucleotide polymorphisms , + 896 a / g and + 1196c / t , in human tlr4 gene that result in amino acid changes in the extracellular domain of the tlr4 protein have been widely studied . these 2 variants have been reported to be associated with a blunted response to inhaled endotoxin on bronchial challenge testing and a reduced systemic inflammatory response to low - dose inhaled endotoxin . non - carriers of these polymorphisms have been found to be more frequently affected by asthma . several case - control studies have evaluated the association of tlr4 + 896a / g and + 1196c / t polymorphisms with asthma susceptibility . a previous meta - analysis has shown a marginal association of tlr4 + 896a / g with asthma , and no association between tlr4 + 1196c / t polymorphism and asthma . however , the previous meta - analysis did not cover all eligible studies related to asthma . therefore , to obtain a more precise conclusion we conducted this meta - analysis including all eligible case - control studies . on the basis of 7 case - control studies including 1443 asthma cases and 1693 controls , the present meta - analysis found that tlr4 + 1196c / t polymorphism might be a protective factor against the development of asthma . the discrepancy stemmed from the fact that tizaoui and coworkers only included 4 studies with 541 asthma cases and 486 controls . next , we conducted the stratified analysis by ethnicity . a significant protective association between tlr4 + 1196c / t polymorphism and asthma was detected in asians . however , we found no significant relationship between tlr4 + 1196c / t polymorphism and asthma in whites . these results suggest that the effect of tlr4 + 1196c / t polymorphism on asthma risk might be influenced by ethnicity . / g polymorphism , no significant correlation was observed between tlr4 + 896a / g polymorphism and asthma risk . our results are consistent with previous meta - analyses . therefore , tlr4 + 896a / g polymorphism seemed not to be associated with the risk of asthma development . however , a study conducted in turkish children with asthma observed that both tlr4 + 896a / g and + 1196c / t polymorphisms were statistically more frequent in the mild asthma group . a strong association between tlr4 + 896a / g and asthma course has been found in a russian study , which reported that the minor g allele was associated with moderate / severe asthma . in addition , the g allele has been suggested to be significantly associated with moderate - severe asthma compared to mild asthma in an egyptian population study . these findings indicate that tlr4 + 896a / g polymorphism might be associated with the severity of asthma , but not susceptibility to asthma . firstly , the number of studies and subjects included in the present meta - analysis were relatively small . secondly , only published studies with sufficient data were included , so the possibility of publication bias can not be completely ruled out , even though funnel plot and egger s test did not detect publication bias . thirdly , the frequencies of gg genotype and tt genotype were low , which may undermine the findings . moreover , the potential interactions between gene - gene and gene - environment during development of asthma were not conducted due to a lack of original data . considering these limitations , well - designed case - control studies with larger sample sizes and different population characteristics are needed to confirm these results . in summary , this meta - analysis suggests that the t allele of the tlr4 + 1196c / t might act as a protective factor against the development of asthma , but there was no significant association between tlr4 + 896a / g polymorphism and risk of asthma . larger well - designed studies based on different ethnic groups should be performed to confirm our findings .

one of the adverse events associated with serotonergic agents is serotonin syndrome ( ss ) . the development of ss can be caused not only by an overdose of serotonergic agents but also through an interaction with other drugs ( 1,2 ) . it is has been reported that more than 85% of physicians are not aware of ss ( 3 ) . we encountered a patient who was treated with noradrenergic and specific serotonergic antidepressants ( nassas ) , who developed ss after receiving an intramuscular injection of metoclopramide , which was administered to alleviate nausea . a 40-year - old woman with bipolar disorder who was taking mirtazapine , lamotrigine , aripiprazole , and lorazepam presented to the clinic with a chief complaint of nausea , and was treated with an intramuscular injection of metoclopramide . following this treatment , the patient was brought to the emergency department by her family who described her condition as being unusual , lethargic and unresponsive . on physical examination , a neurological examination revealed that her glasgow coma scale ( gcs ) was e4v5m6 , conversation was established without any issue , slight mydriasis of the pupils was noted ( 5/5 ) ; the other cranial nerve , motor and sensory findings were all normal . overall increased tendon reflexes , negative pathological reflex , and occasional myoclonus in the legs were observed . the results of blood tests revealed no significant abnormalities other than a creatine phosphokinase level of 362 iu / l . the intake of serotonergic agents followed by myoclonus symptoms and muscle rigidity were consistent with a diagnosis of ss , according to the hunter criteria . the symptoms improved after 3 days of treatment with an increased dose of lorazepam and a decreased dose of mirtazapine , along with the administration of cyproheptadine ( 24 mg , per day ) . the patient in the present case was a 40-year - old woman who took both serotonergic agents and antipsychotics and who developed acute symptoms that were related to autonomic hyperactivity , such as mydriasis and abnormal diaphoresis , as well as myoclonus and muscle rigidity . it is always necessary to keep ss in mind when patients taking serotonergic agents demonstrate acute changes in their mental status , autonomic hyperactivity , neuromuscular abnormalities or other relevant conditions . the symptoms of ss are divided into the following three categories : 1 ) changes in mental condition ( anxiety , agitated delirium , restlessness , and disorientation ) ; 2 ) autonomic hyperactivity ( diaphoresis , tachycardia , hyperthermia , hypertension , vomiting , and diarrhea ) ; and 3 ) neuromuscular abnormalities ( tremor , muscle rigidity , myoclonus , hyperreflexia , and bilateral babinski sign ) ( 1 ) . the neuromuscular abnormalities are more likely to be observed within the lower extremities ( 4 ) . the hunter criteria , which display 84% sensitivity and 97% specificity in the diagnosis of ss , are the preferred diagnostic criteria ( 5 ) . the hunter criteria are satisfied in a patient who meets one of the following conditions ( in the setting of administration of serotonergic agents ) : spontaneous clonus , inducible clonus and agitation or diaphoresis , ocular clonus and agitation or diaphoresis , tremor and hyperreflexia , hypertonia , a body temperature of > 38c and ocular clonus or inducible clonus . the treatment of ss includes the discontinuation of serotonergic agents , supportive care with the stabilization of body temperature , and the administration of benzodiazepines to control agitation and autonomic instability as well as the administration of cyproheptadine ( a histamine-1 receptor antagonist with nonspecific 5-ht1a and 5-ht2a antagonistic properties ) . patients with a temperature of > 41.1c require immediate sedation with paralysis with nondepolarizing agents such as vecuronium , as well as endotracheal intubation ; the use of antipyretic agents such as acetaminophen should be avoided ( 1 ) . since the patient 's medical history included the use of antipsychotics in addition to serotonergic agents , neuroleptic malignant syndrome ( nms ) was a potential differential diagnosis . both nms and ss are associated with fever , consciousness disturbance , autonomic symptoms , muscular rigidity , and increased creatine phosphokinase levels , which sometimes makes it difficult to arrive at a differential diagnosis . ss is associated with the administration of serotogenic agents , while nms is associated with antipsychotics . the second point is the course of the onset of symptoms . the majority of patients with ss present within 24 hours , with most presenting within six hours , after a change in dose or the initiation of a drug . on the other hand , nms develops within a few days to a few weeks after the initiation of a drug . symptoms such as myoclonus and increased tendon reflexes are observed in patients with ss , but not in patients with nms ( 6 ) . the distinctions between serotonin syndrome ( ss ) and neuroleptic malignant syndrome ( nms ) ( 1 - 2 ) . ss : serotonin syndrome , nms : neuroleptic malignant syndrome in addition to nms , anticholinergic poisoning , sympathetic nervous system poisoning , encephalitis , meningitis , malignant hyperthermia , intoxication from sympathomimetic agents and sedative - hypnotic withdrawal are the main differential diagnoses of ss ; these conditions have recently been termed cluster diseases ( 7 ) . frequently used drugs , including metoclopramide , can also cause ss . it is important to avoid prescribing drugs that have the potential to cause the development of ss to patients receiving serotonergic agents . in previous reports on ss caused by metoclopramide , two cases were caused by the additional administration of a single dose of metoclopramide ( 10 mg ) to patients who were taking serotonergic agents . there are no reported cases in which ss was caused by metoclopramide alone ( 8,9 ) . nassas inhibit the alpha 2 , 5-ht2 and 5-ht3 receptors , thereby reinforcing the effects of noradrenaline and serotonin . accordingly , nassas enhance the release of norepinephrine and 5-ht1a - mediated serotonergic transmission . with regard to the mechanism , it has been reported that metoclopramide has a 5-ht3 receptor blocking effect ; although this effect is weak , it may influence the development of ss ( 10 ) . drugs that are frequently used in the daily medical care setting can also cause ss . tramadol , triptan , fentanyl , pentazocine , valproate , carbamazepine , sumatriptan , linezolide , dextromethorphan , lithium , monoamine oxidase inhibitors , ondansetron , levodopa , carbidopa - levodopa and various drugs are listed as having the potential to cause ss ( 1 ) . although ss is a rare disease that can lead to serious consequences , it is preventable . clinicians should recognize this potentially fatal medical condition and be careful of avoiding drugs that may interact to cause ss .

wegener s granulomatosis ( wg ) , first described by friedrich wegener in stuttgart in 1936 , is multi - system disease characterized by a necrotizing granulomatous arteritis of the upper and lower respiratory tract and a necrotizing crescentic glomerulonephritis . vasculitis may also affect other organs such as the eyes , skin , joints , heart and the nervous system . upper respiratory tract manifestations particularly sinusitis , ulcerations of the nasal mucosa and epistaxis are common and debilitating presentations . subglottic stenosis ( sgs ) and tracheal stenosis ( ts ) , however , are potentially life threatening presentations of wg . sgs has been found to occur in approximately 1623% in patients carrying a diagnosis of wg [ 2 , 3 ] . this narrowing of the upper airway at the level of cricoid cartilage and/or upper tracheal rings presents a management dilemma . dilatation tracheoscopy ( dt ) is one of the possible options for treatment of sgs and ts . the long - term effects of dt in patients with wg have never been reported . it can be used for elective and emergency intervention in patients with subglottic and tracheal stenosis . dikkers , dilatation tracheoscopy in treatment of subglottic and tracheal stenosis , submitted for publication ) . patients suffering from wg present differences ( age , sex , response to treatment ) compared to other aetiologies , which justifies separate publication . the objective of this study is therefore to evaluate the effect of the treatment of sgs and ts by dt in patients with wg . we retrospectively identified all patients from our institution that underwent a dt between february 2001 and september 2005 . the total cohort of benign , grade ii ( myer - cotton ) subglottic or tracheal stenosis consisted of patients with a history of posttraumatic tracheal stenosis , thermal tracheal stenosis , posttracheotomy cicatricial stenosis , tracheal rupture , postintubation stenosis , and wg , amongst others . as stated before , the cohort is described elsewhere . from the cohort we identified a total of nine wg patients . these nine patients had been diagnosed with wg between may 1990 and august 2003 . during this period a total of 195 patients in our institution were diagnosed as having wg ( including the nine patients who underwent dt ) . from this cohort , three other patients were diagnosed with relatively mild sgs or ts that did not necessitate dt . the reference group for the comparison of demographic and serological data consisted of the total cohort , minus the patients who underwent dt and minus the three patients with mild tracheal stenosis who did not undergo dt . differences in age between the dt group and the reference group were tested with the mann whitney u test ; differences in the male / female ratio and antineutrophil cytoplasmatic autoantibodies ( anca ) specificity were tested with chi - square test . although this was a retrospective study , the clinical , serological and histopathological data of both the dt group and the control group had been prospectively collected by a standardised protocol starting at the time of the diagnosis of wegener s granulomatosis . sera from all patients were assayed for the presence of antibodies against proteinase 3 ( pr3anca ) , myeloperoxidase ( mpo anca ) and elastase as described previously . dt was performed when patients complained of progressive dyspnoea in combination with decrease of peak flows . an intubation laryngoscope , a groningen optical dilatation tracheoscope ( karl storz 1033r ) ( fig . 1 ) , telescope and suction tubes are required for this procedure . the proximal end of the tracheoscope is designed in such a way that customary ventilation tubes and a 30 cm hopkins straight forward telescope ( karl storz 27005aa ) can be connected . the distal end of the instrument contains numerous lateral tiny openings , which enable air to come through in the centre of the stenosis . the appropriate size of the instrument is determined by the size of the patient s larynx and the healthy portion of the trachea . 1groningen dilatation tracheoscope groningen dilatation tracheoscope the intervention is carried out under general anaesthesia . following the administration of the anaesthesia , with ventilation taking place via an anaesthesia mask , the dilatation tracheoscope is introduced under endoscopic control . the stenosis is then visible through the vocal cords ( fig . 2 ) . the bevelled design of the tip , which can be advanced forward through the stenosis , ensures that the ventilation is maintained during the process . the conical construction of the tip enables the instrument to be advanced up to the wider section of the tracheoscope ( fig . 3 ) , after which the tracheoscope remains in place for 5 to 10 min . parts of the tissue protrude through the tiny distal openings of the tracheoscope patient nr 9 pre dilatation . during laryngotracheoscopy parts of the tissue protrude through the tiny distal openings of the tracheoscope the constellation of the tracheoscope is suitable for most grade ii ( myer - cotton ) subglottic or tracheal stenosis . the intervention can be repeated after any time interval . typically , no antibiotics or corticosteroids are used . the use of mitomycin - c was considered in each case , but it was never used , because mitomycin - c should be used in fresh wounds . peakflows were measured three times ( respironics healthscan inc . , cedar groove , nj , usa ) in an upright position . the total cohort of patients who underwent dt because of sgs and ts was 25 . the causes in these 16 patients were posttraumatic , postintubation , idiopathic , post tracheotomy and post thermal injury . the mean age of the patient group at first presentation of wg was 41.6 11.2 years ( table 1 ) . the interval between the presentation of wg and the first symptoms of laryngeal and tracheal stenosis varied between 0 and 122 months ( mean 48.9 44.1 ) . the average age at of the time of presentation of the stenosis was 45.6 11.9 years ( table 1 ) . table 1patient characteristics of nine patients with wegener s granulomatosis ( wg ) and subglottic stenosis ( sgs ) or tracheal stenosis ( ts ) . clinical and laboratory findings . anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidasepatient no.sexage at presentation of wgorgans involved at first presentation of wganca specificitynumber of relapses of wgorgans involved at first relapse of wgfollow - up after diagnosis of wg ( years)age at first presentation of sgs or tsinterval between diagnosis of wg and first treatment of ts or sgs ( months)1m44jointspr312joints , skin1751922f26lungs , tracheapr31lungs , kidney142603f43tracheampo1lungs124304f57jointspr32ear , eye , kidney , joints12671225f26tracheapr31kidney , eye1131606f51mastoidpr32nose ( concha inferior ) , mastoid1058817f42ear drumatypical0lung , ear drum845358f52nosepr31nose ( septum and concha ) , joints , eye756489f33nasal vesselspr30vessels4332 patient characteristics of nine patients with wegener s granulomatosis ( wg ) and subglottic stenosis ( sgs ) or tracheal stenosis ( ts ) . anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidase at diagnosis , all wg patients had additional organ involvement outside the otorhinolaryngologic area . the skin was affected once ( table 1 ) . as shown in table 1 , the number of relapses of wg varied between 0 and 12 . there was no relation between the number of relapses and the occurrence of ts or sgs . in only one of the dt procedures there was endoscopic and/or histopathological evidence of vasculitis activity . the ts or sgs occurred in 95% in periods where the disease appeared to be less active . in table 2 the patient characteristics of the 9 patients who underwent dt for sgs or ts are compared with those patients who were diagnosed with wg in the same time frame but who did not have evidence of sgs or ts . in the dt group , 89% were female whereas 43% of patients in the reference group were female ( p < 0.01 ) . in the dt group the age at diagnosis of wg was significantly lower in comparison with the reference group ( p < 0.05 ) . table 2patient characteristics of nine patients with wegener s granulomatosis who underwent dt for sgs or ts compared with the reference group of 182 patients who were diagnosed with wg in the same period without evidence of sgs or tswg patients who underwent dt ( n = 9)wg patients without evidence of sgs or ts ( n = 182)p valuemale/ female number ( % ) 1/8 ( 11%/89%)104/78 ( 57%/43% ) < 0.01age at diagnosis of wg ( years , mean sd)41.6 11.253.3 17.0<0.05anca specificitypr3-anca : n = 8 ( 89%)mpo - anca : n = 1 ( 11%)pr3-anca : n = 160 ( 88%)mpo - anca : n = 13 ( 7.1%)hne - anca : n = 1 ( 0.5%)anca - negative : n = 8 ( 4.4%)not significantwg wegener 's granulomatosis , dt dilatation tracheoscopy , sgs subglottic stenosis , ts tracheal stenosis , anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidase , hne human neutrophil elastase patient characteristics of nine patients with wegener s granulomatosis who underwent dt for sgs or ts compared with the reference group of 182 patients who were diagnosed with wg in the same period without evidence of sgs or ts wg wegener 's granulomatosis , dt dilatation tracheoscopy , sgs subglottic stenosis , ts tracheal stenosis , anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidase , hne human neutrophil elastase in the nine patients identified with sgs or ts due to wg , a total of 22 dt s were performed ( table 3 ) . the mean follow - up after treatment was 25.4 14.1 months ( table 3 ) . two patients ( numbers 7 and 8) did not experience a recurrence of significant stenosis . six patients ( numbers 1 , 2 , 3 , 4 , 6 , 9 ) required a second dt without recurrence of local disease activity after the second dt . one of these patients ( patient number 6 ) had acute wg in the trachea , proven by biopsy ( fig . she was treated with high dose prednisolone ( intravenous methylprednisolone 1000 mg on three consecutive days ) and intubation for a week . one patient ( number 5 ) developed a cricoid stenosis 5 years after the diagnosis of wg . initially the stenosis was treated with two dt s . during a pregnancy after pregnancy she was treated with a co2 laser and three dt s . unfortunately , however , she required a definitive tracheostomy . one patient ( number 2 ) became respiratory insufficient at diagnosis of wg and had a tracheotomy for 3 months . she subsequently developed a cicatricial sgs , but she has enough lumen to live without a tracheostomy ( table 3 ) . table 3follow - up after last dtpatient numberlocation of stenosisnumber of dtsfollow - up after last treatment ( months)tracheotomy required1subglottic219no2subglottic221yes , temporary3trachea243no4trachea214no5cricoid812yes6subglottic216no7subglottic154no8subglottic126no9subglottic224nofig . the picture was taken one week after fig . 4 follow - up after last dt patient nr 6 with acute wg during biopsy of a subglottic stenosis patient nr 6 after pulse therapy and intubation . 4 the effect of dt on peakflows was evaluated in five patients . in these patients the mean ( sd ) peakflow increased from 164 45.7 l / min before dt to 226 69.6 l / min after dilatation . an example of the effect of dt on the peakflow is shown in fig . two months after this patient had been diagnosed with wg she developed complaints of dyspnoea . after treatment with high - dose corticosteroids she had a regression of the wg activity . fourteen and 28 months after first diagnosis of sgs a dilatation tracheoscopy was performed successfully . the arrows indicate the dt procedures peak flow measurements after dilatation of patient number 9 . the total cohort of patients who underwent dt because of sgs and ts was 25 . the causes in these 16 patients were posttraumatic , postintubation , idiopathic , post tracheotomy and post thermal injury . the mean age of the patient group at first presentation of wg was 41.6 11.2 years ( table 1 ) . the interval between the presentation of wg and the first symptoms of laryngeal and tracheal stenosis varied between 0 and 122 months ( mean 48.9 44.1 ) . the average age at of the time of presentation of the stenosis was 45.6 11.9 years ( table 1 ) . table 1patient characteristics of nine patients with wegener s granulomatosis ( wg ) and subglottic stenosis ( sgs ) or tracheal stenosis ( ts ) . clinical and laboratory findings . anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidasepatient no.sexage at presentation of wgorgans involved at first presentation of wganca specificitynumber of relapses of wgorgans involved at first relapse of wgfollow - up after diagnosis of wg ( years)age at first presentation of sgs or tsinterval between diagnosis of wg and first treatment of ts or sgs ( months)1m44jointspr312joints , skin1751922f26lungs , tracheapr31lungs , kidney142603f43tracheampo1lungs124304f57jointspr32ear , eye , kidney , joints12671225f26tracheapr31kidney , eye1131606f51mastoidpr32nose ( concha inferior ) , mastoid1058817f42ear drumatypical0lung , ear drum845358f52nosepr31nose ( septum and concha ) , joints , eye756489f33nasal vesselspr30vessels4332 patient characteristics of nine patients with wegener s granulomatosis ( wg ) and subglottic stenosis ( sgs ) or tracheal stenosis ( ts ) . anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidase at diagnosis , all wg patients had additional organ involvement outside the otorhinolaryngologic area . the skin was affected once ( table 1 ) . as shown in table 1 , the number of relapses of wg varied between 0 and 12 . there was no relation between the number of relapses and the occurrence of ts or sgs . in only one of the dt procedures there was endoscopic and/or histopathological evidence of vasculitis activity . the ts or sgs occurred in 95% in periods where the disease appeared to be less active . in table 2 the patient characteristics of the 9 patients who underwent dt for sgs or ts are compared with those patients who were diagnosed with wg in the same time frame but who did not have evidence of sgs or ts . in the dt group , 89% were female whereas 43% of patients in the reference group were female ( p < 0.01 ) . in the dt group the age at diagnosis of wg was significantly lower in comparison with the reference group ( p < 0.05 ) . table 2patient characteristics of nine patients with wegener s granulomatosis who underwent dt for sgs or ts compared with the reference group of 182 patients who were diagnosed with wg in the same period without evidence of sgs or tswg patients who underwent dt ( n = 9)wg patients without evidence of sgs or ts ( n = 182)p valuemale/ female number ( % ) 1/8 ( 11%/89%)104/78 ( 57%/43% ) < 0.01age at diagnosis of wg ( years , mean sd)41.6 11.253.3 17.0<0.05anca specificitypr3-anca : n = 8 ( 89%)mpo - anca : n = 1 ( 11%)pr3-anca : n = 160 ( 88%)mpo - anca : n = 13 ( 7.1%)hne - anca : n = 1 ( 0.5%)anca - negative : n = 8 ( 4.4%)not significantwg wegener 's granulomatosis , dt dilatation tracheoscopy , sgs subglottic stenosis , ts tracheal stenosis , anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidase , hne human neutrophil elastase patient characteristics of nine patients with wegener s granulomatosis who underwent dt for sgs or ts compared with the reference group of 182 patients who were diagnosed with wg in the same period without evidence of sgs or ts wg wegener 's granulomatosis , dt dilatation tracheoscopy , sgs subglottic stenosis , ts tracheal stenosis , anca antineutrophil cytoplasmatic autoantibodies , pr3 proteinase 3 , mpo myeloperoxidase , hne human neutrophil elastase in the nine patients identified with sgs or ts due to wg , a total of 22 dt s were performed ( table 3 ) . the mean follow - up after treatment was 25.4 14.1 months ( table 3 ) . two patients ( numbers 7 and 8) did not experience a recurrence of significant stenosis . six patients ( numbers 1 , 2 , 3 , 4 , 6 , 9 ) required a second dt without recurrence of local disease activity after the second dt . one of these patients ( patient number 6 ) had acute wg in the trachea , proven by biopsy ( fig . she was treated with high dose prednisolone ( intravenous methylprednisolone 1000 mg on three consecutive days ) and intubation for a week . one patient ( number 5 ) developed a cricoid stenosis 5 years after the diagnosis of wg . initially the stenosis was treated with two dt s . during a pregnancy the patient required an emergency tracheotomy because of a threatened airway . after pregnancy she was treated with a co2 laser and three dt s . unfortunately , however , she required a definitive tracheostomy . one patient ( number 2 ) became respiratory insufficient at diagnosis of wg and had a tracheotomy for 3 months . she subsequently developed a cicatricial sgs , but she has enough lumen to live without a tracheostomy ( table 3 ) . table 3follow - up after last dtpatient numberlocation of stenosisnumber of dtsfollow - up after last treatment ( months)tracheotomy required1subglottic219no2subglottic221yes , temporary3trachea243no4trachea214no5cricoid812yes6subglottic216no7subglottic154no8subglottic126no9subglottic224nofig . the picture was taken one week after fig . 4 follow - up after last dt patient nr 6 with acute wg during biopsy of a subglottic stenosis patient nr 6 after pulse therapy and intubation . 4 the effect of dt on peakflows was evaluated in five patients . in these patients the mean ( sd ) peakflow increased from 164 45.7 l / min before dt to 226 69.6 l / min after dilatation . an example of the effect of dt on the peakflow is shown in fig . two months after this patient had been diagnosed with wg she developed complaints of dyspnoea . after treatment with high - dose corticosteroids she had a regression of the wg activity . fourteen and 28 months after first diagnosis of sgs a dilatation tracheoscopy was performed successfully . the arrows indicate the dt procedures peak flow measurements after dilatation of patient number 9 . there are multiple causes of benign laryngeal or tracheal stenosis , the most common being traumatic . however , if there is no prior history of tracheal trauma , the aetiology of the stenosis may be obscure and difficult to determine , necessitating a systematic approach to make the diagnosis . excluding trauma , the differential diagnosis of ts can be subdivided into four categories : congenital , neoplastic , infectious , and inflammatory . congenital ts is really quite rare and is often the result of posterior fusion of the tracheal rings , thereby forming complete rings . other causes of congenital stenosis include vascular rings and other congenital cardiovascular anomalies such as an anomalous subclavian artery primary benign tumours of the trachea such as chondromas , fibromas , squamous papillomas , hemangiomas , and granular cell tumours are also unusual causes of stenosis . in addition , extrinsic compression of the trachea can occur by thyroid neoplasms and goiters . fungal infections such as histoplasmosis and blastomycosis should always be considered when the aetiology of the stenosis is unclear . non - infectious , inflammatory causes of ts include sclerosing mediastinitis , primary amyloidosis , and sarcoidosis . wg and relapsing polychondritis can also cause ts , but they are almost always seen in combination with other , more classic hallmarks of these diseases . a laryngeal or tracheal stenosis is optimally diagnosed via tracheal visualization , which is generally performed by an otorhinolaryngologist . indirect and fiberoptic laryngoscopy are non - invasive examination techniques that can be performed in the office , but usually do not show the entire trachea . a subglottic stenosis is not always visible , therefore direct rigid tracheoscopy in general anaesthesia is indicated in cases suspect of wg . patients with wg who develop laryngotracheal disease have usually already been diagnosed as such because of the presence of disease in other organs at the same time or at a previous occasion . isolated involvement of the subglottic larynx is conspicuously rare only a few cases have been reported in the literature [ 2 , 7 , 8 ] . in our series , however , three patients had sgs or ts as presenting symptom of their wg . this contrasts significantly with the male / female ratio in the reference group that consisted of wg patients that were diagnosed in the same period as the dt group but who did not have evidence of sgs or ts . anatomically the female airway is narrower than the male , making it more prone for post - intubation stenosis . more research in larger series has to be performed to explain this phenomenon . in cases where the diagnosis of wg is suspected , anca testing should be part of the routine laboratory evaluation . c - anca has been shown to be a specific marker for wg with rare false - positive results ; p - anca testing is much less specific . only the particular laboratory findings of c - anca reacting with pr3 and p - anca reacting with myeloperoxidase ( mpo ) are specific for the autoimmune vasculitides [ 9 , 10 ] . notably , seven of our nine patients that underwent dt were pr3-anca positive and one patient was mpo - anca positive . the disease presented as a stenosis , and laryngotracheoscopy revealed the size and site of the lesion . organs involved showed no pattern in which there should be additional suspicion for the development of sgs or ts . there was no relation between the number of relapses and the development of ts or sgs . the ts or sgs occurred more often in periods where the disease appeared to be less active . remarkably , ts or sgs predominantly became manifest in periods in which wg appeared to be inactive . interestingly , seven out of our nine patients who developed ts or sgs had not been diagnosed previously with tracheal involvement of wg , neither at first presentation nor at relapse(s ) . we speculate that during active disease a subclinical tracheal involvement occurs which may subsequently heal with scar formation . to examine this hypothesis in patients , elective tracheoscopy should be performed in all patients with wg . the time interval between presentation of clinical stenosis in these seven patients ( mean 63 months , median 60 , range 2122 ) dt with the groningen dilatation tracheoscope is a safe , minimally invasive procedure for the treatment of cotton - myer grade ii subglottic or tracheal stenosis of various origin . it is an elegant , self - standing surgical intervention , where no additional interventions are needed . for an overview of the effect of dt the reader dikkers , dilatation tracheoscopy in treatment of subglottic and tracheal stenosis , submitted for publication ) . nowadays , mitomycin - c , an alkylating agent that inhibits cell division , protein synthesis , and fibroblast proliferation , is increasingly used as adjuvant treatment in the management of selected cases of laryngeal and tracheal stenosis , for example luminal obstruction in fresh circular sutured wounds . however , the laryngeal and tracheal stenoses of wg patients are almost always of older age and display advanced scarring at the time of diagnosis . however , this damage is considerably different from granulomatous scarification in sutured lumina and , therefore , we do not expect an additional favourable response to mitomycin . therefore , we have not applied mitomycin - c in our wg patient cohort . in cases where ts or sgs develops , there is always the question whether or not to intervene , and the question of timing of the intervention . one might see this as a two - by - two table , with complaints on the x - axis , and peakflow values on the y - axis . progressive complaints and progressive decline of peakflow values ( + /+ ) indicate intervention by dt . absence of progressive complaints with decline of peakflow values ( /+ ) need to be addressed when the values reach a critical level , in which it can be expected that a common cold might lead to severe stenosis of the airways . finally , progressive complaints with unchanged peakflow values ( + / ) indicate that pulmonary function tests should be performed , and , if negative , indicate that the patient has an incorrect perception of his physical potentials . unfortunately , in only five of the nine patients in this retrospective study , we have peak flow measurements . a variety of surgical techniques has been used to treat sgs or ts of other aetiologies [ 12 , 13 ] . the success of these surgical techniques in upper airway stenosis related to wg has been variable [ 3 , 14 ] . in our series two out of nine patients were treated once for upper airway stenosis with dilatation without any re - stenosis because of wg . one was temporary , but one patient needed a permanent tracheotomy ( 11% ) : this was the pregnant woman needing an emergency procedure . wg as such is a rare disease , and sgs and ts are rare symptoms in patients with wg . patient complaints and monitoring of peakflow values offer simple tools for the decision whether or not to intervene .

oral cancer is the eleventh most prevalent cancer worldwide . oral cancers in australia account for approximately 2 - 3% of all cancers and approximately 1% of all cancer deaths , with an increasing incidence over the past decades . the most common oral cancer is oral squamous cell carcinoma ( oscc ) , which makes up 90% of all oral cancers , and if diagnosed early has a five - year survival rate of around 85% . however , the early phase of oral cancer is often asymptomatic . mortality for oral cancer is high because most patients seek care only when they experience late - stage symptoms ( pain , persistent ulceration , unexplained bleeding , or an oral or neck mass ) , at which stage the disease is advanced and the survival rate decreases as low as 1550% . early detection of oral cancer is therefore paramount for improving survival rates and prognosis for patients with the disease . current diagnostic techniques focus on detection of malignant and potentially premalignant lesions in the oral cavity . early lesions may present as unhealing lesions , mucosal colour changes , pain , tenderness or numbness , protuberances , or rough , thickened , crusted , or eroded areas . typically , premalignant and malignant lesions begin as a subtle red or white patch ( erythroplakia or leukoplakia ) that eventually ulcerates and progresses to an exophytic mass . regular comprehensive examinations of the oral cavity form the backbone of oral cancer screening and are especially critical in patients with identified risk habits and factors such as tobacco smoking , excessive alcohol consumption , and human papilloma virus infection . the advantage of the standard visual and tactile examination is that it is simple to perform and requires no added equipment . however , subtle lesions may pass undetected , and it is difficult to make a visual distinction between benign , premalignant , and malignant lesions . adjunctive techniques have been developed in recent years to facilitate making this distinction and enhance the effectiveness of oral examinations . techniques such as vital staining ( toluidine blue ) and visualisation adjuncts ( velscope and vizilite ) highlight abnormal mucosa by targeting tissues undergoing rapid cell division and areas of high metabolic turnover . another adjunctive technique employs transepithelial sampling of the oral mucosa for cytologic analysis ( oralcdx brush test system ) . while promising , these emergent technologies have yet to reproduce the sensitivity and specificity of examination via tissue biopsy and histopathological examination , which remains the gold standard for oral cancer diagnosis . a new focus of research is the use of salivary diagnostics for early detection of oscc , which have the advantage of being noninvasive and nontoxic . proteins , mrna , enzymes , and chemicals extracted from saliva have been found at sufficiently distinct levels between oscc and control samples to be considered as potential biomarkers . these biomarkers could be important indicators of physiological or pathological states and provide information for the detection of early and differential markers for disease . salivary biomarkers offer an easy , inexpensive , safe , and noninvasive approach for disease detection . they have the potential to serve as a widely available screening tool that does not rely on the localization of a lesion for diagnosis . this advantage over other detection methods gives salivary biomarker screening the potential to identify patients with malignant and potentially malignant lesions . recent studies have assessed variation in biomarkers in patients with oral cancer . using an array of biomarkers from oral rinses from 40 hnscc patients and 39 controls assessed by elisa assays , it has been shown that it is possible to distinguish hnscc cases from controls , particularly when the patients demographics were also considered . further , extensive analyses of the plasma levels of 48 proteins ( 26 cytokines , 10 chemokines , and 12 growth factors ) in 111 untreated oscc patients , 112 healthy individuals , and 107 individuals with potentially malignant oral mucosal lesions showed that the levels of 12 proteins were significantly dysregulated in oscc patients serum . furthermore , a recent extensive study had been undertaken to substantiate the development of salivary biomarkers . this study assessed a panel of putative oscc markers in 395 subjects in 5 independent validation cohorts and found them to be independently validated , reproducible , and robust for use in a reference laboratory . thus , such studies indicate the potential of specific deregulated proteins as predictive biomarkers in oral cancer . this review will discuss the potential of cytokines and chemokines as salivary biomarkers for the early diagnosis of oral squamous cell carcinoma . cytokines are a group of small , mainly secreted proteins that affect the behaviour of cells in a diverse number of ways . the binding of cytokines to specific cell membrane cytokine receptors can induce a number of activities within the cell , such as growth , differentiation , or death . most cytokines have pleiotropic effects ; however , some are generally considered as proinflammatory , such as interferon - gamma ( ifn- ) , tumour necrosis factor - alpha ( tnf- ) , and interleukin-1beta ( il-1 ) [ 1619 ] , whereas others are associated with anti - inflammatory effects , such as transforming growth factor - beta-1 ( tgf-1 ) . over the last 5 years a large number of nonredundant proteins have been recognised in saliva , with one study reporting over 1400 and another almost 2,000 , reflecting the diversity of salivary biomarker profiles that may identify and potentially aid in the management of a range of diseases . of particular interest has been the use of salivary cytokine levels as markers of both cell proliferation and oral cancer . the most studied cytokines include epidermal growth factor ( egf ) , interleukins-6 and -8 , vascular endothelial growth factor ( vegf ) , interleukins-4 and -10 , tumour necrosis factor ( tnf ) and endothelin [ 24 , 25 ] . several studies have assessed interleukin-6 ( il-6 ) , a multifunctional cytokine that participates in the inflammatory and immune responses and has been shown to promote the growth of cancer cells as well as associated with an increased rate of metastasis and an altered immune status [ 2632 ] . interestingly , il-6 would appear to have different effects on different cell populations , stimulatory for some cell types while inhibitory for others . il-6 can promote tumor cell proliferation in several tumor cell lines , including human cervical carcinomas mediated cachexia . in contrast , another study indicated that expression of il-6 and its receptor can be inhibitory for cell proliferation and is correlated with good prognoses for patients with breast cancer . il-6 also has a demonstrable direct effect on cancer cells via inactivation of the p53 tumour suppressor gene as seen in human multiple myeloma cell line kas 6/1 . irrespective of the role of il-6 , there is increasing evidence to support higher levels of il-6 in the saliva of patients with oral cancer , as well as oral potentially malignant lesions , than in normal controls . in a recent trial of 29 consecutive patients being treated for oral cancer , it was shown that patients had much higher salivary concentration of il-6 than controls and that this concentration increased during the treatment period returning to baseline levels at discharge . other studies however have assessed a panel of pro - inflammatory cytokines as markers of malignancy [ 27 , 37 ] . a recent study assessing the levels of il-1 , il-6 , il-8 , vegf- , and tnf- in saliva , measured using quantitative elisa , these salivary biomarkers were demonstrated to be increased in patients with oral cancer ; significantly increased in a subgroup of patients with endophytic tongue cancer and il-8 levels ; particularly shown to correlate with poor prognosis ; and intriguingly also found to be higher in control individuals who both smoked and consumed alcohol daily . however , it should be noted that elevated levels of il-6 and il-8 have also been detected in other studies in the saliva of patients with periodontitis [ 39 , 40 ] . the main limitation of this study is the relatively small sample size ( n = 10 ) ; nevertheless , although il-8 was found to be higher in patients with periodontitis than in healthy controls , it is detected at significantly much greater levels in patients with oscc . yet if this is true , it should be possible to differentiate between an inflammatory process and a neoplastic process by the amount of il-6 and il-8 . the study in 2008 by arellano - garcia et al . has multiple important innovative aspects , including the fact that it showed that multiplex bead based assays were as effective as elisa assays for quantification of proteins in saliva and that il-8 and il-1beta were expressed at significantly higher levels in oscc patients . although there were only 20 cancer patients , with 20 age and gender matched controls , this study nevertheless clearly indicated the potential of constituents of saliva as biomarkers for oral cancer . a further study assessing salivary levels of tnf- , il-1 , il-6 , and il-8 in a group of nine patients with oscc with matched healthy controls attempted to assess the relative influence of periodontal inflammation by using a modified gingival index to matched patients and control samples . interestingly though , several patients were edentulous , and thus neither they nor their matched controls would help discriminate the role of periodontal inflammation in relative salivary chemokine level , a fact compounded by the small sample size ( 9 patients with oscc ) . thus far then , the results of a number of studies would indicate that salivary cytokine levels are very likely to provide useful information of the presence of disease , epithelial behaviour , the local inflammatory response , and carcinogenesis . however , larger sample sized studies are required to investigate salivary cytokines and their role in the diagnosis of pml and oscc while at the same time being able to deal with the obvious local confounding factor of inflammation and in particular periodontal disease . further studies of the potential of a panel of salivary cytokines as a screening tool for oral cancer are apparently ongoing , the results of which are eagerly awaited as this is likely to have a profound impact on the early detection of oral cancer and thus morbidity and mortality . the complexity of undertaking such a study , that would require a large number of patients who have oral cancer , patients with potentially malignant mucosal disease , and sufficient health controls as well as patients with non - neoplastic mucosal disease . this comprehensive study , thoroughly analysing the diversity of these salivary biomarkers present in health and disease , is at the same time both daunting and necessary . chemokines are a superfamily of structurally related cytokines , which share an ability to chemotactically attract their target cells along a concentration gradient . it is through this ability that these molecules play an integral role in the migration of immune cells to areas of pathogen challenge . chemokines also mediate the movement of specific cells involved in inflammatory responses that subsequently result in cellular interactions critical for mounting immune responses . all chemokines are small proteins , ranging in weight from 6 to 14 kda . chemokines and chemokine receptors can be classified into 4 main structural families , dependent upon the position of the cysteine residues near the n - terminus . these families are the cc , cxc , c , and cx3c , with the x denoting the number of amino acids between the cysteine residues [ 45 , 46 ] . chemokines are secreted in response to signals such as proinflammatory cytokines such as interleukin ( il)-1 , tumor necrosis factor ( tnf ) , and interferon - c ( ifn - c ) and thus they play an important role in selectively recruiting monocytes , neutrophils , and lymphocytes . once induced , the directed migration of cells expressing the appropriate chemokine receptors occurs along a chemical ligand gradient known as the chemokine gradient . chemokines induce chemotaxis through the activation of g - protein - coupled receptors ( gpcrs ) , subsequently involving adhesion molecules and glycosaminoglycans ( gags ) . chemokines bind to specific cell surface transmembrane receptors coupled with heterotrimeric g proteins , whose activation leads to the activation of intracellular signaling cascades that prompt migration toward the chemokine source ( chemotaxis ) . it has been shown that a single ligand can bind to multiple receptors and in turn a chemokine receptor may bind multiple ligands . also , many of the inflammatory chemokines have wide target cell selectivity , with some acting both on the cells of the innate and adaptive immunity . the function of chemokines can be subdivided into two main families : those that are induced after inflammatory stimuli , the inflammatory chemokines , and those produced constitutively in tissues , the homing chemokines . in addition to their roles in the immune system , chemokines and chemokine receptors are also involved in the pathology of a number of diseases , such as infections ( e.g. , hiv-1/aids ) , autoimmune disorders ( e.g. , psoriasis , rheumatoid arthritis , and multiple sclerosis ) , pulmonary diseases ( asthma and chronic obstructive pulmonary disease ) , transplant rejection , cancer , and vascular disease . furthermore , there would appear to be significant overlap between chemokines as some of the inflammatory chemokines appear to be produced constitutively in some areas of the body and some of the chemokines designated as homing chemokines can be upregulated by inflammatory stimuli . for example , larc / mip-3 plays a role in both homeostatic trafficking of leukocytes , as well acting as an inflammatory chemokine during host defense . although the detection of chemokine levels by enzyme - linked immunosorbent assay ( elisa ) has become a sensitive and specific method to determine the chemokine profile in patient fluids , this is not able to fully represent the actual inflammatory conditions in vivo . indeed , many chemokines are posttranslationally modified by proteolytic cleavage , which can render an agonist more active or inactive or even convert the active chemokine into a receptor antagonist of the intact molecule . nevertheless , using elisa , a recent study assessed the saliva of patients with oral cancer for the presence of both inflammatory chemokines ( cxcl8 , cxcl10 , and ccl2 ) , homeostatic chemokines ( cxcl4 , ccl14 , and ccl18 ) . further , individuals with and without periodontitis were used as controls and it was found that h&n carcinomas give rise to a change in the chemokine composition of the oral fluid with a significant increase in cxcl8 , cxcl10 , and ccl14 before therapy , a finding that was not reproduced after therapy . however , the levels detectable by elisa were very low and it is likely that more refined methods could indicate not only intact chemokines , but also those modified posttranslationally . these authors conclude that it can be expected that specific truncated chemokines , as well as the proteases involved in this truncation , will be linked to particular disease states . these authors postulate that proteomic analysis of biological fluids will further our understanding of the pathogenesis of specific diseases and provide solutions for new diagnostic and treatment options . since chemokines in disease can be occasionally involved in excessive recruitment of inflammatory cells , prevention of this recruitment may be an effective anti - inflammatory strategy . furthermore , chemokine receptors are intimately involved in cellular recruitment and , along with cd4 , have been shown to be an essential cofactor enabling hiv-1 viruses to infect cells . thus , in diseases that have a profound effect on the immune system , such as infection with hiv , there are various points of potential intervention that could provide anti - inflammatory and anti - hiv infectivity therapeutics , including prevention of the receptor - ligand interaction , prevention of the chemokine - glycosaminoglycan interaction , interfering with the signaling pathways that are induced upon receptor activation , and modification of receptor trafficking pathways . however , these postulated potential interventions need considerable further study as the apparent redundancy in the expression of chemokines , and the overlap between homeostatic and inflammatory chemokines pathways , makes them difficult targets for diagnosis of diseases and therapeutics . in addition , it has been shown that the enhancement of the inflammatory response is aided by synergistic activity of chemokines for leukocyte migration . hence , blockage of a single chemokine may downregulate other immune responses , because of the inhibitory effect on its synergy with other chemokines . thus , there is a need to refine our ability to assess the presence of chemokines in disease states , the presence and specificity of chemokine receptors , and the specificity of functional active chemokines in specific disease states , prior to being able to define selective and specific targets for treatment . significant change in our understanding of the role of chemokines in oscc has occurred in a fairly short time . a relatively early study assessed the presence of a particular chemokine ( cxcl12 ) and its specific receptor ( cxcr4 ) , revealing that the receptor was more prevalent in oral cancers that metastasized , suggesting that this chemokine / receptor may be important in the regulation of tumour growth and organ - specific lymphatic spread . a further , extensive study of 85 patients with oral sccc utilized immunohistochemistry , rt - pcr , and western blot to assess the expression of a different chemokine , ccr7 and its ligand ccl21 , in 85 patients with oral squamous cell carcinoma . it was shown that ccr7 expression was positively correlated with lymph node metastasis , tumour size , and clinical stage , and these authors postulated that the interaction between this chemokine and its receptor may be significant for the induction of lymphatic spread . the mechanism by which chemokines and chemokine receptors are involved in oral carcinogenesis has been extensively studied [ 19 , 6264 ] . , normal t cell expressed and secreted ) has been shown to play a crucial role in migration and metastasis in human cancer cell lines and further showed that that ccl5/ccr5 axis enhanced migration of oral cancer cells , probably via mmp-9 . an extensive investigation of 253 oral cancer patients , matched with 347 controls , the presence of mutations ( single nucleotide polymorphisms ) in the genes of specific chemokine ligands and receptors ( ccl5 and ccr5 ) revealed an interesting dichotomy of the presence of mutations increasing risk for oral cancer while at the same time raising the potential that oral cancers with a specific chemokine profile may well have enhanced protection from metastases . in a bid to rectify the dysregulated cc chemokine receptor ( ccr5)/ligand , a recent study used interferon-2b ( ifn-2b ) , known to upregulate ccr5 expression , in a small cohort of 12 oral cancer patients . these investigators showed that enhanced t - cell - mediated tumor cell killing upon ifn-2b treatment and they postulate that this immunotherapy treatment may be combined with standard chemotherapy for better clinical outcome . the sdf-1/cxcr4 ( stromal cell derived factor 1/chemokine ( c - x - c motif ) receptor 4 ) pathway has been suggested to play a role in the metastatic dissemination of neoplasms with migration toward sdf-1 by tumor cells bearing cxcr4 . mutation in the gene of a specific chemokine receptor ( cxcr4 ) has been noted to have an increased likelihood of more advanced oral cancer ( stage iii and iv by 2.66-fold ) . a study of 71 patients with hnscc assessing the tissue expression levels of sdf-1 and cxcr4 found that patients with low sdf-1 had poorer metastasis - free survival ( p = 0.026 ) , disease - free survival ( p = 0.006 ) and overall survival rates ( p = 0.002 ) . a recent immunohistochemical study has confirmed that this relationship showing a significant relationship between cxcl12 and cxcr4 was found both in potentially malignant lesions and oral cancer . an in vitro experiment has recently shown that , with synthetic biology approaches , signalling selective inhibition of the cxcr4 prevented the metastatic spread of neoplastic cells . previously , investigations based on the known association between the chemokine ligand cxcl13 and prognosis of oral cancer found that the chemokine ligand / receptor axis of cxcl13/cxcr5 is not only important for cancer bone invasion and metastasis but may also be a potential therapeutic target to prevent oscc bone invasion / osteolysis . current research has identified deregulated cytokines in oscc as well as oral potentially malignant lesions , robust and reproducible methods for the assessment of these cytokines in saliva , and the possibility of a rapid salivary test as an indicator of disease and risk of malignancy . ongoing development of such a method will have profound impact on oral cancer screening and the early diagnosis of oral cancer , potentially resulting in early treatment and a decrease in the high levels of morbidity and mortality associated with oscc . furthermore , there are indications that it may be possible to utilize our enhanced understanding of the chemokines associated with disease progression , metastases , and bone invasion to develop novel methods for the treatment of oral cancer .

the who regional committee of the western pacific about a new bmi for asians defined 23 kg / m or higher bmi as overweight , and 25 kg / m or higher as obesity1 . it is associated with increased body fat , bmi , and waist measurements , which adversely affect the activities in daily living and physical activities2 . also the incidence of degenerative foot diseases increases with increasing life expectancy , and diabetic foot disease also increases with the growing number of diabetes patients3 . plantar pressure is frequently measured to solve foot problems in patients with pain , diabetes , or rheumatism4 . when foot bones are not properly distributed , the body weight is unevenly applied to the sole , thereby resulting in various symptoms5 . rheumatoid arthritis patients experience foot symptoms in the early stage of their disease ( 20% ) as well as in the latter stage ( 90% ) ; then , after 10 years , most of them experience foot deformity5 , 6 . the effects of the pressure applied to the foot can be investigated through measurements that can be used to solve the foot problems of diabetes or rheumatoid arthritis patients7 . tests on body weight support of the foot include the sole pattern test and the foot - scan test , which is used to observe the sole contact features and to automatically calculate plantar pressure values that are used to evaluate balance and movement8 , 9 . it is necessary to understand the nature of maintaining balance to differentiate the cause of balance disorders from other factors . studies on balance and the maintenance of balance have been conducted10,11,12 . as skeletomuscular factors , muscular strengths and their stability have been studied . posture and balance control through the improvement of the spontaneity index have been reported . studies on neurologic factors , including somesthesia , visual senses , and vestibular sense have also been conducted13 , 14 . thus , the purpose of this study is to suggest physiotherapy programs and to determine foot stability according to the results of plantar pressure and spontaneity balance in the normal group and in the obesity group based on bmi . the subjects were divided into two groups : a normal bmi groups ( thin body type ) and obesity groups ( plump body type ) . the subjects without those who had gait disorders , foot pain , foot deformity , and foot diseases were excluded . general characteristics of the subjects are shown in table 1table 1.general features of the subjectsweightheightbminormal bmi group ( n=10)47.1 2.5159.6 3.518.5 1.1obesity bmi group ( n=10)71.4 6.2162.4 5.827.4 1.4mean sd . all the subjects signed an informed - consent form , and the study was approved by k women s university . , korea ) after the subjects age , height , and body weight was measured . , the subjects comfortably stood on the foot scan on bare feet with even body weight on both legs . they stood facing forward or looking at a red spot in the front without moving their feet and without holding anything . the balance tester , tetrax interactive balance system ( sunlight inc . , israel ) , had four ground reaction force sensors at each right and left foot and forefoot and hindfoot area to measure the pressure changes . spontaneity index was calculated after two of the a , b , c , and d , ground reaction force sensors were selected . the subjects stepped on the force platform on bare feet without holding anything , and stood still , facing forward . the tests were conducted in a standing position facing forward with their eyes open , and in the same position on an unstable spring plate ( 31 cm 12 cm ) . when the subjects were on the spring plate , the tester was there to stand by them to prevent any accidents . the statistical analyses were performed using windows spss version 12.0 . the mann - whitney u test was used to compare pressure distribution according to bmi . the two - way anova test with repeated measurement was also used to compare balance according to bmi . both groups did not show a significant difference in forefoot peak pressure in the static condition according to bmi ( table 2table 2.comparison of forefoot peak pressure ( kg / cm ) between the groups in the static conditionstatic forefootpeak pressurenormal bmi group ( n=10)139.7 23.8obesity bmi group ( n=10)138.1 21.4mean sd ) ; and no significant difference in hindfoot peak pressure either ( table 3table 3.comparison of hindfoot peak pressure ( kg / cm ) between the groups in the static conditionstatic hindfootpeak pressurenormal bmi group ( n=10)164.1 11.9obesity bmi group ( n=10)152.3 22.8mean sd ) . both groups showed significant differences in the spontaneity index on a stable surface according to bmi between the areas , but showed no significant difference between the groups . moreover , no significant difference was observed between the groups and areas ( table 4table 4.comparison of spontaneity index ( % ) in both groups on a stable surface ( % ) abcdacbdnormal bmi group ( n=10)783.6 99.2795.4 118.1515.4 215.9692.9 139.0obesity bmi group ( n=10)538.3 326.2608.4 282.9109.6 379.4594.3 431.4mean sd . ab : left fore / hindfoot ; cd : right fore / hindfoot ; ac : left / right hindfoot ; bd : left / right forefoot ) . on an unstable surface , both groups showed significant difference between the areas , but showed no significant difference between the groups , and there was no significant difference between the groups and areas either ( table 5table 5.comparison of spontaneity index ( % ) in both groups on an unstable surface ( % ) abcdacbdnormal bmi group ( n=10)799.5 116.1746.3 189.6561.1 278.2718.1 120.7obesity bmi group ( n=10)523.5 345.2701.1 209.143.7 626.1665.6 93.3mean sd . ab : left fore / hindfoot ; cd : right fore / hindfoot ; ac : left / right hindfoot ; bd : left / right forefoot ) . ab : left fore / hindfoot ; cd : right fore / hindfoot ; ac : left / right hindfoot ; bd : left / right forefoot mean sd . ab : left fore / hindfoot ; cd : right fore / hindfoot ; ac : left / right hindfoot ; bd : left / right forefoot in this study , both plantar pressure and spontaneity balance were investigated according to bmi to obtain data for future use in the fields of physiotherapy intervention , assistive devices , and shoes that address foot diseases . according to the kim et al.16 study on peak pressure changes in the forefoot area according to the metatarsal pad size and shape , the peak forefoot plantar pressure decreased with the use of a shoe pad . according to the lee et al.17 study on effects of the metatarsal pad on the sole pressure in normal adults , the pressure was significantly low in the areas of hindfoot , midfoot , and the 3rd , 4th , and 5th metatarsal bones . this meant that the use of a metatarsal pad had resulted in no significant changes in foot pressure . shoes and metatarsal pads were not used in this study , but no significant changes were observed in the forefoot and hindfoot areas . according to the barbosa et al.18 study on the ability to maintain balance in the daily activities of low and high bmi groups , the high bmi group showed decreased quickness and ability to maintain balance . lee et al.19 on the gait of mentally retarded children , the contact surface decreased in all areas , and the contact ratio decreased in the hindfoot and midfoot areas , while the ratio increased in the forefoot area . similar to the above - mentioned study , a decreased hindfoot contact ratio and spontaneity balance with swaying posture were expected in this research . the outcomes correspond with those of this study , which confirmed a decrease in the spontaneity balance index in the hindfoot area of both the normal and obese bmi groups . this is thought as a mechanism of compensating for the decreased hindfoot balance in the forefoot area , and forefoot pressure will increase to compensate for balance . many studies on plantar pressure and balance have been conducted , but the effects of bmi on plantar pressure have not been sufficient . moreover , many comparative studies on plantar pressure and balance in patients suffering from specific diseases have been conducted , but not many of them were on foot changes among the general population . among the limitations of this study is its small sample size . further studies in the future involving more samples of various ages may enhance reliability with a variety of outcomes . the results of this study may be used as basic data for exercise programs to enhance hindfoot spontaneity balance in the obesity group by increasing forefoot balance , or for studies on reducing foot swaying .

diabetic nephropathy ( dn ) is one of the most serious microvascular complications of diabetes , which is the leading cause of end - stage renal disease . previous studies have reported that podocytes are highly differentiated , insulin - dependent glomerular epithelial cells contributing to glomerular filtration barrier . there are several proteins on podocyte membrane that are important in the regulation of the glomerular filtration such as nephrin , podocin , and podocalyxin [ 3 , 4 ] . podocalyxin is a sialoprotein contributing to the negative charge of glomerular membrane and maintaining integrity of filtration function of the glomerulus . recently , some researches reported that metformin ( 1,1-dimethylbiguanide ) , as the first - line hypoglycemic drugs for the treatment of t2 dm , can prevent and delay the occurrence and progress of diabetic nephropathy through some mechanisms , which include improving glucolipid metabolism , improving insulin resistance , and some other nonhypoglycemic action mechanisms . in this study , we aim to investigate the protective effect and possible mechanisms of metformin on podocyte damage in type 2 diabetic rats so as to provide an experimental basic for the use of metformin in the clinical prevention and treatment of dn . forty - five male sprague - dawley ( sd ) rats ( 180200 g ) aged two months were purchased from the experimental animal center of anhui medical university . each three rats were fed in one cage with enough food and water under condition of relative humidity of 40%60% and temperature of 2030c . animals were treated strictly according to the rules in the ethics committee of anhui provincial hospital affiliated to anhui medical university . the following reagents were used : stz ( sigma , usa ) , metformin ( shanghai shiguibao medicine co. , ltd . , china ) , tc , tg , and ldl - c kits ( beijing bhkt clinical reagent co. , ltd . ) , urine creatinine ( ucr ) and urea nitrogen ( bun ) kits ( jiancheng technology co. , nanjing , china ) , urinary albumin ( ualb ) kit ( tianjing , xiehe medicine co. , ltd . , china ) , urinary pcx elisa test kit ( shanghai dr . dean biotechnology co. , china ) , ihc kits ( abcam , france ) , and trizol , real - time pcr kit , and primers of pcx ( takara , china ) . apparatuses ultra 2 full - automatic glycosylated hemoglobin analyzer ( primus , usa ) , sn-695b -counter ( china ) , ht1 enzyme - linked immune detector ( biocell , austria ) , uv-2600 uv - vis spectrophotometer ( youni ke instrument co. , ltd . , shanghai ) , jem-1230 transmission electron microscopy ( jeol , japan ) , and an abi7500 apparatus ( bio - rad , usa ) were used in this study . t2 dm was induced by high - fat diet and low - dose stz as mentioned previously [ 79 ] . briefly , sd rats were fed a high - fat diet ( a basal diet + 10% lard compound + 2% cholesterol , 50% calories from fat ) for 4 weeks followed by single intraperitoneal ( i.p . ) injection of a low dose of streptozotocin ( stz , 30 mg / kg , in citrate buffer , ph 4.5 ) . modeling was considered to be successful if blood glucose level was higher than 16.7 mmol / l accompanied with dieresis and polydipsia . methods : 8 sd rats served as normal control group ( group nc , n = 8) and sterile citrate buffer ( i.p . ) was administered to them . t2 dm rats were divided randomly into group dm ( n = 9 ) treated with vehicle alone or group m1 ( n = 8) , group m2 ( n = 8) , or group m3 ( n = 7 ) treated with metformin ( 150 , 300 , and 500 mg / kg per day , resp . ) for 8 weeks . the dose of metformin was based on studies with long - term metformin treatment in stz - diabetic rats . 24 h urinary samples were collected at the end of 8 weeks and then centrifuged at 2,000 g for 10 min for the determination of urinary albumin ( ualb ) , urinary sediment pcx ( upcx ) , and urinary creatinine ( ucr ) . jaffe 's assay was used to measure ucr . to eliminate the impact of urine volume , urinary albumin and urinary sediment pcx were expressed as urinary ualb / ucr ( uacr ) and upcx / ucr ( upcr ) , respectively . these were calculated using the following equations : uacr ( mg / g ) = ualb ( mg / l ) [ ucr ( mol / l ) the molecular weight of cr ( 113.12 g / mol ) 10 ] . upcr ( ng / g ) = upcx ( ng / l ) [ ucr ( mol / l ) the molecular weight of cr ( 113.12 g / mol ) 10 ] . uacr ( mg / g ) = ualb ( mg / l ) [ ucr ( mol / l ) the molecular weight of cr ( 113.12 g / mol ) 10 ] . / l ) [ ucr ( mol / l ) the molecular weight of cr ( 113.12 g / mol ) 10 ] . then all rats were anesthetized and blood was withdrawn from abdominal aorta for the detection of hba1c , insulin ( ins ) , serum bun , tg , tc , and ldl - c . bun was tested by the urease method , tg and tc were tested by the enzyme reaction , and ldl - c was tested by selective precipitation method . all rats were sacrificed ; then the pathological changes of the kidney tissue were observed by electron microscope . 1 mm renal cortex average harmonic mean of gbm in each group was calculated as gbmt of the corresponding group [ 11 , 12 ] . from each photo , the gbm and the part of fused foot process were traced and measured in an image processing and analysis program ( image pro plus 6.0 ) . foot process fusion rate ( frfp ) = the total length of gbm / the sum length of fused foot process covered in the corresponding gbm . for each photo , frfp was calculated and used to finally calculate a mean frfp for each group [ 13 , 14 ] . small blocks of kidney tissue were embedded in paraffin , following dewaxing and adding antigen for microwave repair . kidney sections were stained for pcx ( 1 : 300 ) at 37c for 3 h. then polymerization - hrp anti - rabbit igg was used for incubating sections at 37c for 30 min . the renal pcx protein expression was employed by integrated optical density ( iod ) detection . image pro plus 6.0 was used to calculate the average iod of the positive reaction of 10 images of each sample . the sections were examined with light microscopy by an experienced pathologist , and 10 images of each sample were acquired by continuous manual acquisition with magnification of 400 . total rna was isolated using trizol and reverse - transcribed using a reverse transcription kit . duplicate pcr reactions were tested using the following amplification protocol : 95c for 10 seconds followed by 40 cycles at 95c for 5 seconds and at 60c for 34 seconds . upstream and downstream primers of pcx gene sequence were cca cag ccc tac caa cca and gtg tgt gaa ttc ttc ggt cgt . upstream and downstream primers of -actin gene sequence were gcc t ta gcc tgg acc ca t agt and gac cac caa tcc aca caga . results were normalized to the -actin mrna levels and represented using the comparative threshold cycle method . the cycle threshold ( ct ) value of each sample was analyzed by the 7500 software . the relative changes in gene expression were calculated using the relative quantitative method ( 2 ) . the results were presented as means standard deviation ( sd ) . for the data conforming to homogeneity of variances , one - way analysis of variance ( anova ) was used to determine significant differences among groups , and tukey 's b test was used for comparison between individual groups . for the data not conforming to homogeneity of variances , t2 dm rats induced by high - fat diet / streptozotocin showed a profound elevation in the level of blood glucose , hba1c , and ins ( approximately 4-fold ) while a marked decrease in the body weight as compared to nc group ( figures 1(a ) and 1(c ) ) was noticed . in addition , induction of t2 dm was evidenced by significant increases in tg , tc , ldl - c , and bun after 8 weeks ( figures 1(b ) and 1(d ) ) . treatment of t2 dm rats with low dose of metformin ( 150 mg / kg ) for 8 weeks only significantly decreased the levels of ldl - c , tc , and bun ( p < 0.05 ) but not bg , hba1c , and ins , whereas higher doses of metformin ( 300 , 500 mg / kg ) obviously restored all measured biochemical parameters after 8 weeks ( p < 0.05 ) as compared with group dm ( figure 1 ) . urinary sample collected from dm rats after 8 weeks showed a marked increase in ualb , upcx , uacr , and upcr levels accompanied with a significant decrease in ucr ( figure 2 ) . low dose of metformin treatment ( 150 mg / kg ) caused a significant decrease in ualb and uacr levels ( p < 0.05 ) ; in addition , the highest dose of metformin ( 500 mg / kg ) more noticeably caused decrease in ualb and uacr as compared to group m1 ( p < 0.05 ) ( figures 2(b ) and 2(d ) ) , while figures 2(c ) and 2(e ) indicated only highest dose of metformin ( 500 mg / kg ) significantly restored the increase in upcx and upcr ( p < 0.05 ) . on the other hand , metformin treatment obviously increased the level of ucr as compared to group dm ( p < 0.05 ) without difference between different met groups ( figure 2(a ) ) . histopathological examination of kidney tissue obtained from nc rats showed normal gbm without foot process fusion . however , both the foot process fusion ( nearly 80% ) and gbm and gbmt ( 266.50 10.23 nm ) were found increased significantly in t2 dm rats ( p < 0.05 ) ; meanwhile , some foot processes were completely ruined and even vanished and the architecture of gbm became ambiguous ( figure 3 ) . both frfp and gbmt were alleviated by met treatment compared to those of group dm ( p < 0.05 ) . compared to low dose of metformin ( 150 mg / kg ) treatment , gbmt decreased much more ( 200.53 20.36 versus 150.98 17.25 ; 145.94 16.10 nm ) in higher doses of metformin ( groups m2 , m3 ) ( p < 0.05 ) and frfp was restored to 15% in m3 group ( p < 0.05 ) , as shown in figure 3 . to investigate the role of metformin regulating the expression of pcx in renal tissue , the renal tissue pcx protein expression was detected within the visceral surface of bowman 's capsule in the podocyte with a membranous pattern of staining in normal control samples ( figure 4(a ) ) . pcx protein expression decreased significantly in four diabetic groups which was accompanied with a significant decrease in pcx mrna expression , especially in group dm ( p < 0.05 ) when compared to the group nc ( figure 4 ) . treatment rats with different doses of metformin significantly restored the decreased protein and mrna expression of pcx , which was more prominent in higher dose of metformin group ( 300 , 500 mg / kg ) than low dose of metformin group ( 150 mg / kg ) ( p < 0.05 ) . diabetic nephropathy is one of the most serious complications of both type 1 and type 2 diabetes mellitus and also is one of the most common causes of end - stage renal disease . metformin is recommended , in conjunction with lifestyle modification , as a first - line oral therapy in the recent guidelines of the ada ( american diabetes association ) and easd ( european association of the study of diabetes ) [ 16 , 17 ] . the main effect of this drug is to acutely decrease hepatic glucose production , mostly through mild and transient inhibition of the mitochondrial respiratory chain complex i . recently some experimental and clinical observations suggest that metformin has a specific protective effect on diabetic nephropathy ( dn ) independently of its hypoglycemic effect [ 10 , 19 ] . in this study , the levels of serum bun , uacr , and gbmt in t2 dm rats were higher than those in group nc . these indexes were significantly decreased after met treatment , the value of bg and hba1c in m1 group had no significant changes compared with the group nc , while there was a significant decrease in serum bun , uacr , and gbmt , which indicated that met has a remarkable protective effect on the kidney independently of the hypoglycemic manner . the mechanisms are not clear completely , which may be related to its effects in activating amp , activated protein kinase , improving oxidative stress , inhibiting inflammation and hyperfiltration of the kidney , reducing the formation of advanced glycation end products , lightening lipid deposition , and so forth [ 10 , 19 ] . the filtration barrier of the kidney glomerulus that prevents plasma proteins from leaking into primary urine comprises endothelial cells , a glomerular basement membrane , and visceral epithelial cells ( podocytes ) . podocyte injury has been considered as one of the most important factors in diabetic nephropathy . several medicines that protect from podocyte injury have a role in prevention of the development of diabetic nephropathy [ 20 , 21 ] . in the present study , the foot cell fusion was gradually relieved by metformin treatment , especially in group m2 and group m3 , which confirmed that met can ameliorate podocyte damage in t2 dm in a dose - dependent manner . podocalyxin , located on the surface of podocytes that faces the urinary space , is one of the major negatively charged glomerular proteins in the glomerular basement membrane and plays an important role in the maintenance of the intricate glomerular basement membrane and plays an important role in the maintenance of the intricate glomerular podocyte for optimal filtration . the increased urinary pcx ( upcx ) excretion is a particularly sensitive index for indicating podocyte injury in patients with diabetes and can be used as an early specific indicator of glomerular podocyte dysfunction [ 23 , 24 ] . in fact , decreased mrna expression and protein components of slit diaphragm are related to the degree of proteinuria in several models of kidney diseases . in our research , the upcx was significantly increased in t2 dm model rats compared to normal rats ; moreover high dose of metformin ( m3 ) remarkedly decreased upcx excretion . pcx protein in normal rats was present along with glomerular peripheral capillary loops , which was reduced significantly in t2 dm model rats . treatment with high dose of metformin ( m3 ) obviously inhibited the downregulation of pcx protein expression . compared with group nc , the mrna expression of pcx in group dm was obviously decreased , which was upregulated by met treatment , and that was greater in groups m2 and m3 than in group m1 while the value of bg , hba1c , and ins in m1 group had not been significantly restored compared with the group nc . from all the above , the effect of metformin adjusting renal tissue pcx expression is in a dose - dependent manner while it is independent of the hypoglycemic manner . some researches elucidated possible underlying mechanisms of podocyte protection of metformin . some researches elucidated possible underlying mechanisms : advanced glycation end products suppress pcx expression in high glucose - treated human podocytes through extracellular regulated protein kinases ( erk1/2 ) signal transduction pathway . hypoglycemic effect of metformin which was presented in this study can alleviate this suppression effect . cholesterol is an important regulator in the development of proteinuric kidney disease and cholesterol homeostasis in podocytes is important to the formation of the slit diaphragm . in the experiment , metformin decreased the level of tg , tc , and ldl - c , reduced the excretion of ualb and upcx , and relieved the frfp . therefore , we may infer that metformin develops its podocytes protection effect partly through ameliorating the level of lipid in dm rats . in addition to the contents discussed above , an experiment in cultured podocytes provided evidence that metformin can decrease ros production through inhibiting nad(p)h oxidase . kim et al . reported that metformin can suppress the diabetes - induced loss of podocytes through the repression of oxidative injury in a rat model of type 2 diabetes . similar mechanisms were proposed in which metformin can inhibit ecto - atpase activity leading to an increased extracellular atp concentration , subsequent activation of p2 receptors , and eventual reduction of nad(p)h oxidase activity and enhancement of ampk activity ; these changes may have some beneficial effects for podocytes during metformin therapy . the present study also presents the fact that a certain dose of metformin not only ameliorates hyperglycemia and hyperlipidemia , but also improves insulin level . from all what is mentioned above , met can reduce urinary pcx excretion as long as it adjusts the expression of pcx , so as to infer some podocyte protection in type 2 diabetic rats beyond its hypoglycemic effects .

critical illness may occur at any time of life , having permanent effects not only on one?s health but on other aspects of life , including spiritual , social , and familiar issues . despite this , mortality is still the most frequently assessed outcome in critically ill patients and is routinely used as the primary end - point in intervention trials , cohort studies , and benchmarking analysis . in recent years , the number of studies assessing survival and the overall medical condition of patients after icu and hospital discharge has markedly increased . however , there is still a need for additional sound data on reliable predictors of relevant , early and late , patient - centered outcomes . in a recent issue of critical care , cuthbertson and colleagues , using icu admission as the starting point , reported an ongoing long - term mortality rate of 61% in patients with severe sepsis . also using icu admission as the starting point , nesseler and colleagues recently showed a long - term mortality of 45% among patients with septic shock . in contrast , brinkman and colleagues conducted a systematic review of long - term mortality of critically ill patients , whose data were drawn from 24 studies . this number counterpoints the high mortality observed in the cited studies and highlights the importance of using the same starting point when comparing studies , as discussed by the authors . to illustrate the importance of defining the follow - up starting point , we recovered data from recent trials and cohort studies of patients with sepsis and compared the reported mortality if starting point was changed . as depicted in table?1 , the difference of long - term mortality could reach 40.6% with data from the catecholamine study . the 90-day mortality was 51.2% if we chose icu admission as the starting point ; however , if we chose icu discharge ( that is , excluding patients who died in the icu ) , the long - term mortality was 10.6% . from the studies in table?1 , if the starting point follow - up is changed , the mean difference is 25.0% and the mean reduction in reported long - term mortality is 54.0% . this analysis has the important limitation that , when the follow - up starting point is changed to icu discharge , the total observation period will be less than 90 days ; that is , it will be equal to 90 days minus the icu length of stay . effect on long - term mortality rate of patients with sepsis if the starting point of follow - up is changed cats , catecholamine study ; prowess , protein c worldwide evaluation in severe sepsis ; vasst , vasopressin versus norepinephrine infusion in patients with septic shock . we believe that , despite important advances in describing the natural history of icu patients , it is crucial to better define our research questions . indeed , recommendations provided directions to improve the research in this topic , and standardization of some definitions and outcomes would result in studies that are more homogenous . first of all , what is the best starting point for the follow - up ? should follow - up start at icu admission , after icu discharge , or even after hospital discharge ? this definition , though practical , will include different strata of patients in regard to illness severity and reason for icu admission , including a range of patients with scheduled icu admission , which form a very distinct group . in fact , the first days of icu admission are associated with higher risk of death and greatest vulnerability for the patient . the first phase is followed by the recovery phase , which can be further complicated by secondary insults , especially hospital - acquired infections . patients who had a secondary insult in the icu are probably different than those who had a smooth course after admission and may not reflect the prognosis of all icu survivors . icu discharge can also be used as a starting point . at icu discharge , residual organ failure and need for organ support if this starting point is used , the risk of death is better balanced among critically ill patients , and this starting point is interesting for studying the role of important topics in long - term mortality , such as icu readmission and critical illness rehabilitation . finally , using hospital discharge as the starting point will include patients who have the minimal requirements to sustain an adequate condition in a non - monitored environment and who therefore are more similar to patients who had no recent critical illness . on the other hand , icu survivors may represent patients with better previous health status ( that is , patients who survived critical illness because of good previous health condition and status performance ? in other words ? long - term mortality ? for outcomes that are not limited to post - discharge mortality , resulting in a myriad of possible interpretations . the heterogeneity of case mix and other factors usually make for an intricate analysis , but different starting points play a central role in the differences between studies . for intervention trials and for benchmarking , the starting point should be either the icu admission or syndrome / disease diagnosis . for attributable mortality , icu admission instead of time of diagnosis would be better , as recently reported for ventilator - associated pneumonia . icu discharge is a remarkable starting point to identify high - risk patients who could receive beneficial close attention in the hospital and in outpatient clinics . therefore , to achieve better answers , we must know where , in the natural course of critical illness , our question lies .

the bacterial plaque has been implicated as the primary etiologic factor in inflammatory periodontal disease , but recently , several studies have focused on the role of the immune system in the evolution of the periodontal disease , indicating that bacterial antigens trigger an immunopathologic reaction and that the susceptibility of the patients determines the ultimate outcome of the disease process . unicellular endocrine glands due to their ability to release a wide variety of chemical mediators that have a potent biological action on target tissues . in inflammatory lesions of the oral cavity substantial evidence has implicated certain immune and inflammatory responses as destructive mechanisms in the periodontal disease process . the aim of this study was to evaluate and correlate the relationship between mast cell count in different stages of human periodontal diseases . the american academy of periodontology ( aap ) guidelines were followed for classification of the periodontal disease and conditions . based on aap guidelines samples were divided into three groups : group 1 - control group comprising of normal oral mucosa probing depth ( pd ) <3 mm with no bleeding on probinggroup 2 - dental plaque induced gingivitis [ dpig ] pd <3 mm and clinical attachment loss ( cal ) < 1 mm with bleeding on probinggroup 3 - localized chronic periodontitis [ lcp ] pd and cal > 4 mm with bleeding on probing . group 1 - control group comprising of normal oral mucosa probing depth ( pd ) <3 mm with no bleeding on probing group 2 - dental plaque induced gingivitis [ dpig ] pd <3 mm and clinical attachment loss ( cal ) < 1 mm with bleeding on probing group 3 - localized chronic periodontitis [ lcp ] pd and cal > 4 mm with bleeding on probing . the study sample comprised 50 patients , which were divided into three groups , consisting of 10 cases of clinically healthy gingival tissues ( control group ) [ figure 1 ] , 20 cases of dpig with no attachment loss [ figure 2 ] and 20 cases of lcp characterized by the loss of periodontal support [ figure 3 ] . clinical image of a case in periodontally healthy group clinical image of a case in dental plaque - induced gingivitis group clinical image of a case in localised chronic periodontitis group the study was undertaken after approval from the institutional ethical committee . the samples of groups dpig and lcp were obtained from patients undergoing periodontal surgery ( modified widman flap surgery and gingivectomy ) . clinical parameters such as plaque index , gingival index , pd and cal were recorded for each of the cases . the biopsied specimens were immediately fixed in 10% neutral buffered formalin for further processing and then dehydrated , cleared and embedded in paraffin wax . one section was stained with hematoxylin and eosin and categorized to the particular groups and the second section was stained with special stain , i.e. , 1% toluidine blue used specifically to demonstrate mast cells . hematoxylin and eosin stained tissue sections from the surgical specimens fixed in 10% formalin and embedded in paraffin wax were reviewed to confirm the histological diagnosis and representative sites on tissue sections blocks were selected . examination was carried out on olympus bx 51 trinocular light microscope with provision for photomicrograph with olympus e331 slr digital camera . mast cells were analyzed quantitatively by counting the total number of mast cells as well as qualitatively by counting the number of intact and degranulated mast cells in toluidine blue stained sections . mast cells were counted using an oculometer grid under a magnification of 40 [ figures 46 ] . the area of the microscopic field was calibrated - with an ocular grid fitted inside the eyepiece with an area of 0.04 mm . the mast cells were counted throughout each of the tissue sections in 10 representative and consecutive grid fields ( 40 ) . the mean of ten values was calculated and expressed as mean standard deviation ( sd ) per mm . the field was analyzed in a step - ladder fashion and care was taken to prevent the overlapping of fields . the mast cell counts of each field were re - assessed after 4 weeks by same observer and other observer in random order to observe intra- and inter - observer variability . histological section showing mast cells in a case of periodontally healthy group ( toluidine blue stain , 400 ) histological section showing mast cells in dental plaque - induced gingivitis ( toluidine blue stain , 400 ) histological section showing mast cells in localized chronic periodontitis with occulometer grid ( toluidine blue stain , 400 ) the continuous data were summarized as mean sd while discrete ( categorical ) in number and percentage continuous groups were compared using analysis of variance and the significance of the mean difference between the groups was done by tukey 's post hoc test . the intra- and inter - observer variability were analyzed using intraclass correlation coefficient and pearson correlation coefficient analyses , respectively . a two - tailed ( = 2 ) p < 0.05 was considered statistically significant . number of positively stained mast cells in the periodontal tissues was determined in 10 consecutive microscopic high power ( 800 ; objective 64 ; eyepiece 12.5 ; tube factor 1 ) fields ( each field has an area of 0.017 gg 25 mm , obtained from the mathematical expression a = [ d]/4 , where p = 3.14 , d = 0.15 ) in a representative section of each specimen . the continuous data were summarized as mean sd while discrete ( categorical ) in number and percentage continuous groups were compared using analysis of variance and the significance of the mean difference between the groups was done by tukey 's post hoc test . the intra- and inter - observer variability were analyzed using intraclass correlation coefficient and pearson correlation coefficient analyses , respectively . a two - tailed ( = 2 ) p < 0.05 was considered statistically significant . number of positively stained mast cells in the periodontal tissues was determined in 10 consecutive microscopic high power ( 800 ; objective 64 ; eyepiece 12.5 ; tube factor 1 ) fields ( each field has an area of 0.017 gg 25 mm , obtained from the mathematical expression a = [ d]/4 , where p = 3.14 , d = 0.15 ) in a representative section of each specimen . table 1 shows that the mean mast cell counts of lcp group was the highest followed by dpig group and control group the least ( control < dpig < lcp ) . on statistical analysis using spss software ( windows version 18.0 ) , we found significantly ( p < 0.001 ) high intra- and inter - observer reliability / reproducibility of mast cell counts . the mast cell counts ( meanstandard deviation ) of three groups the observed mean pd and cal of three groups are summarized in table 2 . the mean probing depth and clinical attachment loss of three groups the correlation between pd and mast cell counts of all three groups are summarized graphically in [ figure 7 ] . pearson correlation analysis revealed a significant and positive ( direct ) correlation between pd and mast cells counts ( r = 0.90 , p < 0.001 ) . correlation between probing depth and mast cell counts the correlation between cal and mast cell counts of lcp group are summarized in [ figure 8 ] . pearson correlation analysis revealed a significant and positive ( direct ) correlation between cal and mast cells counts ( r = 0.90 , p < 0.001 ) . mast cells originate from pluripotential hematopoietic cells in the bone marrow , undergo part of differentiation in this site , then enter the circulation and complete their differentiation in peripheral mucosal or connective tissue microenvironments . mast cells are scattered throughout gingival connective tissue , often in close association with endothelial cells , but are also found sub- and intra - epithelially . in inflamed and in healing gingiva , number of mast cells mast cells are characterized by oval to round nuclei with the cytoplasm densely packed with bright red granules . mast cells may be round , oval , or spindle with abundant cytoplasm or thin and elongated resembling fibroblast . when activated , mast cells may either undergo explosive degranulation and then resynthesize their granules or they may release solitary granules into their environment on an on - going basis , a process termed piecemeal degranulation that has been observed in both the oral mucosa and skin . following degranulation , mast cell mediators are deposited in large quantities in the extracellular environment , where they exert effects on endothelial cells and other cell types . mast cells may subsequently synthesize and secrete additional mediators that are not preformed in their granules . key mediators that are preformed in mast cells are serine proteases tryptase , chymase cathepsin g , histamine , heparin , serotonin , acid hydrolases and the cytokines tumor necrosis factor- - and interleukin- 16 ( il-16 ) . following activation , mast cells can synthesize a range of mediators , including the ils il-1 , il-3 , il-4 , il-5 , il-6 , il-8 , il-10 , il-13 , and il-16 , together with granulocyte macrophage colony - stimulating factor , platelet activating factor , rantes , macrophage inflammatory protein-1a and the arachidonic acid metabolites prostaglandin 2 and leukotriene c4 . the results of our study indicates that mast cells have higher counts in chronic periodontitis compared to healthy / gingivitis cases , which is consistent with the results of studies carried out by batista et al . using the same immunohistochemistry marker , observed that the numbers of tryptase - positive mast cells were decreased in chronic periodontitis tissue sections compared with healthy / gingivitis samples . one possible explanation for this discrepancy could be that the clinically healthy and marginal gingivitis samples were grouped together ( healthy / gingivitis ) in gemmell 's study , while we used separate samples of dpig and clinically healthy gingival tissues ( health ) . the results of this study suggest that mast cell counts may be associated with periodontitis . one of the biological and biochemical factors is histamine , which breaks down the tissue barrier , causes edema and helps cellular infiltration . in addition , mast cells are believed to contain most of the body 's histamine . another reason is that the expression of matrix metalloproteinases ( mmps ) 1 , 2 and 8 are strongest in mast cells . mmps are crucial in the degradation of the main components in extracellular matrices . furthermore , tryptase can cleave the third component of collagen and activate latent collagenase that can participate in tissue destruction in periodontitis . furthermore a change from gingivitis to periodontitis involves a shift from predominantly t -cell lesion to a b - cell / plasma cell lesion . mast cells seem to be able to present antigens to t - cells . the resultant t - cell activation would activate mast cells , leading to both degranulation and cytokine release . in our study , we found an increase in number of mast cells in inflamed site as compared to periodontally healthy site , suggesting important dynamic alterations in the migration and localization of mast cells in the evolution of periodontal disease , which need to be more precisely speculated . based on our results , the increase of mast cells speculates the possible participation of mast cells in the defense mechanism and destructive events both as effector and responsive cells in chronic inflammation , as well as the possible functional relationship between mast cells and immunocompetent cell populations in periodontal lesions . based on the concept that mast cells play a critical role in the induction of inflammation , it is logical to use therapeutic agents to alter mast cell function and secretion , to prevent inflammation in its earliest phase . we put forward a concept where in mast cell stabilizers as a group of drugs , can be used in the prevention of periodontal disease . mast cell stabilizers can be used to advantage for host modulation therapy in periodontitis : mast cell stabilizers are currently being used successfully in prophylactic treatment of bronchial asthma . some of the commonly used mast cell stabilizers are sodium cromoglycate and nedocromil sodium ( ns ) . ns not only reduces allergic inflammation by inhibiting mediator release from mast cells but also exhibits a wide range of additional anti - inflammatory activities , including inhibition of increased vascular permeability induced by individual mediators such as histamine . for longer bioavailability of mast cell stabilizers in the periodontal pocket , they can be incorporated into a biodegradable matrix of cross - linked hydrolyzed gelatine and made into a chip . after insertion into the pocket , the chip will come into contact with the gingival crevicular fluid and swell , which prevents the chip from falling out of the pocket . in the first 24 h , there will be a burst effect and a portion of drug will be released into the pocket . thereafter , the release profile of the drug will be in zero order ( constant release rate ) , up to 10 days . within 710 days , it provides optimal levels of the drug in the periodontal pocket . at the same time , the chip is biodegraded within 710 days due to enzymatic degradation . placement of the mast cell stabilizer incorporated in a bioadhesive local drug delivery mode , will result in higher bioavailability of the drug for a longer period of time . hence , the best option of delivery of the drug into the narrow periodontal pocket environment is through local drug delivery systems . placement of the drug in the periodontal pocket environment followed by sealing the periodontal pocket environment with cyanoacrylate glue or a periodontal dressing would be advised . the possible relationships between mast cells and pathogenesis of periodontal diseases is putforth in this study . further research is needed to elucidate the cellular interactions and immunological and dynamic aspects of the disease so that the pathogenesis of periodontitis might be elucidated more clearly and effective treatment approaches can be suggested . the therapeutic implications of the findings and suggestions here in presented include strategies directed toward the possible use of drugs to influence mast cell secretion and there by prevent inflammation and maintenance of chronicity or even with the aim of improving periodontal regeneration . further studies on mast cells can prove to be beneficial in determining the efficacy and effectiveness of mast cell stabilizers in limiting the progression of periodontal diseases .

blindness is one of the most prevalent disabilities worldwide which is known as one of the most severe health conditions affecting individuals , families and communities . according to the latest world health organization ( who ) estimates in 2010 , approximately 39 million people in the world were blind . as revealed in the literature , parents of blind children experience parenting stress due to some reasons such as decreased parent - child emotional interactions and communication performance , and socially and physically developmental delays . such reaction results from the parents negative perceptions toward themselves and their children in association with their parenting responsibilities . parents with blind children take on additional parenting responsibilities such as making attempts to enhance the children s learning about social activities , self - care , and independence . evidently , the parents who experience stress and psychological pressure can not fulfill their parenting roles and , eventually , may even harm the children s social , psychological and physical growth . parenting stress in combination with other factors such as anger expression can lead to more serious parenting problems such as child abuse which , in its turn , would be followed by more serious consequences . accordingly , psychological status of the parents with a blind child is found to have a profound effect on the child s life . according to surveys conducted , hence , conducting interventions to investigate and control parenting stress seems necessary in order to decrease such negative consequences . furthermore , it is necessary to provide consultations and psychological training to parents to reduce parenting stress . life skills are psychological abilities for positive and adaptive behaviors which enable the people to deal effectively with the challenges and demands of daily life . the review of the literature has shown that the child s disability is not the only factor which harms the normal child - parent interactions . the parents psychological issues such as stress and depression play a major role in this regard . teaching stress - coping skills has also been reported to have positive effects on parenting stress of the mothers with children suffering from attention deficit hyperactivity disorder . likewise , life skills training has had positive effects on parenting skills and promoted developmental skills of school - aged children . some factors such as the levels of the parents knowledge about proper interaction skills and the amount of social support received by families have more effects on parenting stress than do aspects of child functioning . therefore , preventing and controlling parenting stress seems crucial . since parenting stress can negatively affect the parenting quality of the parents with blind children and the formation of social behaviors in such children , this study aimed to examine the effect of an lst program , as a strategy for coping with stress , on reducing parenting stress in the parents with blind children . therefore , the present study was designed to evaluate the efficacy of lst in parenting stress of the mothers with blind children aged 7 to 12 years . the results obtained from the current research are of great importance to consultants and health care professionals . this study was approved by the ethics committee of shiraz university of medical sciences ( ethics committee approval number : ct93767006 ) . in this non - blinded randomized controlled trial , we enrolled 52 mothers with blind children aged 7 to 12 years , who were studying at shoorideh shirazi educational complex in the school year of 2013 - 14 . inclusion criteria were willingness to participate in the study , ability to attend educational sessions and completing the questionnaire as well as residence in the city in which the study was done ; while exclusion criteria were the parents unwillingness to continue their participation and not following the interventions , experience of participation in other lst program , consuming psychiatric medications , and absence of more than two sessions . since there is only one primary school for the blind and partially sighted children in shiraz , all samples were collected using census sampling method from shoorideh shirazi boarding school during one month . we enrolled 92 mothers with blind children , of whom 35 who were not available and lived outside shiraz , were excluded . out of the 57 participants who were selected using census sampling method , 4 were excluded as they were consuming psychiatric medications under the supervision of a psychiatrist ( n=5 ) or had an experience of participation in life skills training programs ( n=1 ) . the final 52 participants were randomly assigned into the intervention ( n=26 ) and control ( n=26 ) groups , using balanced block randomization method ( block size=4 ) ( figure 1 ) . data were collected from the mothers using a demographic questionnaire and the parenting stress index - full form ( psi - ff ) by the researcher . design and protocol of the study the psi - ff , which is a self - report questionnaire , takes 30 minutes to be filled out . psi was firstly developed by abidin ( 1990 ) to measure the level of stress in the parent - child relationship in parents of children aged 1 month to 12 years . the normal scores range from 175 to 245 with the score of 260 and above reflecting a high level of parenting stress where professional consultation for the stress is required . abidin ( 1990 ) reported internal consistency reliability coefficients ( cronbach s alpha ) of 0.89 for the total score and 0.90 and 0.93 for child and parent domains respectively based on a study on 2633 mothers of children aged 1 month to 12 years of age in the united states . the scale reliability and validity was also estimated and confirmed in iran by dadsetan et al . the authors reported the chronbach s alpha of 0.88 for validity and test - retest reliability coefficient of 0.94 with a 10-day interval . demographic questionnaire was designed by the researcher including two separate sections enquiring about the parent s characteristics and the child s characteristics . the researcher explained the aims and method of the research to the participants in an orientation session and their anonymity and confidentiality of the data were guaranteed . after obtaining written informed consent , those who were willing to participate in the study filled out the demographic questionnaire . after excluding those who did not meet the inclusion criteria , the participants of both intervention and control groups afterwards , the mothers in the intervention group attended a training program consisting of 5 two - hour sessions per week for 5 consecutive weeks . the presented topics and the purpose of the sessions , which were designed based on the guidelines of who and the united nations children s fund , were as follows : session one : teaching self - awareness skills including contents concentrating on self - awareness and its importance , self - esteem , different types of self - awareness , emotional intelligence and characteristics of self - aware people . session two : teaching the techniques of dealing / coping with emotions including contents concentrating on concepts of emotions , strategies for coping with anger and techniques of assertiveness along with decisiveness . session three : teaching the skills of coping with stress , the definition of stress , stress and individuals characteristics , the adverse effects of psychological stress , the symptoms of stress and its effects , techniques of coping with stress and stress management skills . session four : teaching problem - solving and decision - making skills , creative thinking , critical thinking , the importance of problem - solving ability , defining the problem , solving the problem and making decision , consultation , having positive and negative thoughts facing the problem as well as critical and creative thinking . session five : teaching empathy and interpersonal relationship skills including contents concentrating on empathy and its importance , techniques of empathy and obstacles to empathy , the results of empathic behaviors , effective communication skills and principles of correct communication with a blind child . the psi questionnaire was filled out again by the participants of both the intervention and control groups once immediately after and once one month after the intervention to ensure the durability of the training effect . statistical descriptive tests such as mean , standard deviation ( sd ) and frequency were used to describe the features of the data . chi - square , independent t - test and repeated measures analysis of variances ( anova ) tests were also used as appropriate . ( k - s ) test was also applied to determine the normality of the distributions of quantitative variables . this study was approved by the ethics committee of shiraz university of medical sciences ( ethics committee approval number : ct93767006 ) . the researcher explained the aims and method of the research to the participants in an orientation session and their anonymity and confidentiality were guaranteed after obtaining written informed consent . the control group subjects were also trained as those mothers in the intervention group at the end of the study through one lecturing session and giving educational booklets and cds . quantitative variables were distributed normally . independent t - test and c - square test were used to compare quantitative and qualitative demographic variables respectively in the intervention and control groups . the results showed no significant difference between the two groups with respect to qualitative demographic variables including the mother s marital , educational and occupational status , number of children in the family , number of blind children in the family , sex of the blind child , birth order of the blind child , causes of blindness and severity of the blindness ( p>0.05 ) . also , the meansd age of the mothers in the intervention and control groups were 34.856.20 and 35.696.25 , respectively and the results of independent t - test showed no significant difference between the two groups in this regard ( p=0.86 ) . the results of analysis of repeated measures showed no significant difference between the two groups with respect to the scores of all parenting stress domains ( including parent and child domain as well as life stress ) before the intervention ( table 1 ) . frequency distribution of demographic variables in the intervention and control groups * statistically significant at 0.05 ; moreover , the mean total scores of parenting scores were very similar in both groups and no significant difference was observed between the groups in this regard ( p=0.76 ) . the results showed a significant decrease in the mean scores of parenting stress in the intervention group compared to the control group before , immediately after and one month after the intervention ( p<0.001 ) ( table 2 ) . comparison of the mean change of the parent and child domains scores and total parenting stress score in the participants of the intervention and control groups before , immediately after and one month after the intervention analysis of repeated measures furthermore , the changes in the mean - scores of parenting stress were observed in the subscales of acceptability and reinforcement ( child domain ) and sense of competence , attachment , role restriction , depression , and social isolation ( parent domain ) ( p<0.001 ) . it is noteworthy to mention that95% confidence interval of the difference was applied for all continuous variables in this study . studying the effects of lst program on parenting stress , we found that the meansd total parenting stress score decreased significantly in the intervention group immediately after the intervention , reflecting the positive effect of trainings on decreasing the stress levels of the mothers with blind child / children . our results were similar to that of other researchers who reported that lst could reduce psychological tension in mothers with normal and mentally retarded children . likewise , in another study , the analysis of covariance test results proved the effectiveness of behavior management training on reducing parenting stress of autistic children s mothers and enhancing their ability to cope with such stress . howe , also , concluded that parental psychological status is one of the most important factors which contribute to interactive problems between the parents and their disabled children . on the other hand , training parents to develop parenting skills increased their parenting satisfaction and competence and decreased the child - parent conflicts as well . analysis of the changes occurring in the child domain signifies the fact that the training programs could improve the compatibility mechanisms in parents through providing adequate information on the children s condition and appropriate behaviors with them . accordingly , the program helps the parents better accept the children conditions and play more effective roles in their children progress so that it consequently can reduce their stress . kazdin concluded that providing cognitive problem solving skills and parenting management trainings to the parents of the children with antisocial behaviors , as one of the ten elements of life skill , had decreased such behaviors in their children . the changes of stress score in the adaptability and demandingness subscales from child domain were not statistically significant in the intervention group . for instance , yen et al . concluded that psychological interventions on children can reduce the child s anxiety and parenting stress in their mothers in all child domains subscales including adaptability and demandingness . so , it seems that it would be better to concentrate on the problematic children to reach stress score reduction in such subscales of parenting stress . in the current study , the parent domain has also undergone significant changes probably due to the fact that lst program played a supportive role to the mothers than their children . a study on the efficacy of stress coping skills training on parenting stress of mothers with children suffering from attention deficit hyperactivity disorder reported that the training program could significantly decrease the total parenting stress scores in the participants of the intervention group in the subscales of demandingness , restrictions of role , depression and social isolation . their findings were consistent with ours regarding the changes occurring in the parent domain . lst program could nt diminish stress score in the parent domain about mothers relationship with their spouses in this study while mohammadi et al . showed that lst program could qualify the relationship between parents of children with epileptic problems . however , we did nt find a significant reduction in stress score in parental health subscale from parent domain of the questionnaire . it seems that if the program had taken longer , it would have been more effective in this area . training the parents with blind children on life skills is recommended to the authorities of consultation , supportive and medical centers as well as department of education of exceptional children . the present study only addressed the mothers of the blind children while large scale studies are also required to examine the effects of the training programs on the fathers of such children . considering our small sample size , as another limitation of the study , our findings can not be generalized to other communities in other parts of the world since it was conducted at only one center and one region in iran . therefore , the researchers suggest further studies with a larger sample size to be carried out on the parents with deaf children or those with autism , mental retardation and other disabilities . differences in individual and psychological characteristics of the participants especially in learning and completing the questionnaire were also other limitations that could not be denied . it is concluded that lst program could reduce parenting stress in the mothers with blind children . since parenting stress can also have negative personal and social consequences on physical and mental health of the blind children and their parents , lst is highly recommended to consultation , educational and health care centers as an efficient , cost - effective and simple technique for preventing and managing parenting stress of the parents with exceptional children . for future studies , we suggest the researchers to investigate on the effectiveness of life skills training on parenting stress of fathers of blind children and compare it to those of mothers , or on the qol of their children .

a ti : sapphire laser at 830 nm ( 80 mhz , ~140 fs pulse width , chameleon , coherent , ca ) pumped an optical parametric oscillator ( opo , ape , coherent , ca ) , providing a 1064 nm output with ~260 fs pulse width . the 1064 nm beam the pump beam was modulated by an acousto - optic modulator ( aom , gooch & housego , ca ) at ~7 mhz . the saturation beam was directed to a phase - only spatial light modulator ( pluto , holoeye , germany ) and a doughnut shape was produced by inputting a helical phase ramp from 0 to 2 ( fig . relay optics were applied to project the doughnut shape beam to the back aperture of the objective . the 830 nm beam was used as the probe and was collinearly combined with the pump and saturation beams . the three beams were sent to a laser scanning microscope ( ix71 , olympus , ca ) , and focused into a sample by a water immersion objective ( na=1.2 , olympus , ca ) . the transmitted probe beam was collected by another water immersion objective , filtered by two bandpass filters ( 825/150 chroma , vt ) , and detected by a photodiode ( s3994 - 01 , hamamatsu , japan ) followed by a resonant amplifier . a lock - in amplifier ( hf2li , zurich instrument , zurich ) was used to extract the probe signal at the modulation frequency . the pixel dwell time was ~4 s . for imaging , the delay between the pump and saturation pulses was 0.4 ps . a 1:6 diluted s-1805 photoresist film was spun - coated on the quartz wafer at 10,000 rpm , and the thickness of the coated film was less than 100 nm . the coated quartz wafer was covered by another piece of quartz wafer , and then mounted on a sample stage in a vacuum chamber . before growing graphite nano - platelets , the chamber was pumped and purged by high - purity n2 gas , and maintained at a pressure below 0.1 torr . a continuous wave ( cw ) nd : yag laser with a wavelength of 532 nm was focused on the s-1805 film through the transparent quartz substrate using a lens of 150 mm focal length . with this optical setup , the graphite nano - platelets sample was produced with a laser power of 2.8 w , irradiated for 35 minutes . the size and the coverage of the graphene platelets are determined by the growth parameters . afm ( veeco dimension 3100 ) images were taken with the tapping mode under ambient conditions . graphene was grown by thermal chemical vapor deposition on a cu foil , at a temperature of 1000 c and under 1 atm pressure with methane as the precursor gas . a ti : sapphire laser at 830 nm ( 80 mhz , ~140 fs pulse width , chameleon , coherent , ca ) pumped an optical parametric oscillator ( opo , ape , coherent , ca ) , providing a 1064 nm output with ~260 fs pulse width . the 1064 nm beam the pump beam was modulated by an acousto - optic modulator ( aom , gooch & housego , ca ) at ~7 mhz . the saturation beam was directed to a phase - only spatial light modulator ( pluto , holoeye , germany ) and a doughnut shape was produced by inputting a helical phase ramp from 0 to 2 ( fig . relay optics were applied to project the doughnut shape beam to the back aperture of the objective . the 830 nm beam was used as the probe and was collinearly combined with the pump and saturation beams . the three beams were sent to a laser scanning microscope ( ix71 , olympus , ca ) , and focused into a sample by a water immersion objective ( na=1.2 , olympus , ca ) . the transmitted probe beam was collected by another water immersion objective , filtered by two bandpass filters ( 825/150 chroma , vt ) , and detected by a photodiode ( s3994 - 01 , hamamatsu , japan ) followed by a resonant amplifier . a lock - in amplifier ( hf2li , zurich instrument , zurich ) was used to extract the probe signal at the modulation frequency . the pixel dwell time was ~4 s . for imaging , the delay between the pump and saturation pulses was 0.4 ps . a 1:6 diluted s-1805 photoresist film was spun - coated on the quartz wafer at 10,000 rpm , and the thickness of the coated film was less than 100 nm . the coated quartz wafer was covered by another piece of quartz wafer , and then mounted on a sample stage in a vacuum chamber . before growing graphite nano - platelets , the chamber was pumped and purged by high - purity n2 gas , and maintained at a pressure below 0.1 torr . a continuous wave ( cw ) nd : yag laser with a wavelength of 532 nm was focused on the s-1805 film through the transparent quartz substrate using a lens of 150 mm focal length . with this optical setup , the graphite nano - platelets sample was produced with a laser power of 2.8 w , irradiated for 35 minutes . the size and the coverage of the graphene platelets are determined by the growth parameters . afm ( veeco dimension 3100 ) images were taken with the tapping mode under ambient conditions . graphene was grown by thermal chemical vapor deposition on a cu foil , at a temperature of 1000 c and under 1 atm pressure with methane as the precursor gas .

to describe the work - up and treatment of rare ectopic acromegaly caused by a biopsy - proven somatotroph pituitary adenoma located within the bony intersphenoid septum of a patient with empty sella syndrome ( ess ) . we report the presentation , clinical course , diagnostic work - up , and lesion localization and treatment challenges encountered in a 55-year - old patient , with a brief review of relevant literature . a 55-year - old african - american man presented with acromegaly and ess . attempts to definitively localize the causative tumor were unsuccessful , though petrosal sinus sampling supported central growth hormone production and imaging suggested bone - enclosed subsellar pituitary tissue . endoscopic endonasal transphenoidal exploration was undertaken with resection of a somatotroph pituitary microadenoma , and subsequent clinical improvement and biochemical remission . retrospective review revealed the patient 's pituitary to have been located ectopically within a unique bony intersphenoid septum . this report describes the first known case of an ectopic pituitary adenoma located within the midline bony intersphenoid septum , which we postulate to have resulted from anomalous embryological pituitary migration . intra - intersphenoid septal tumors should be considered in cases of apparent central acromegaly with ess or absence of tumor tissue within the paranasal sinuses or other peripheral locations . empty sella syndrome ( ess ) describes a cerebrospinal - fluid - filled sella turcica associated with a shrunken and/or flattened pituitary gland . pituitary function is generally preserved , though hyperprolactinemia or partial - to - total hypopituitarism occurs in approximately 50% of patients.1 2 3 the most prevalent hormone deficiency in this syndrome is growth hormone ( gh ) deficiency , affecting 35 to 61% of adult patients with ess.4 5 6 pituitary hyperfunction only rarely occurs in ess , and acromegaly is perhaps the least common manifestation in this setting.7 8 9 while autoremission of acromegaly after pituitary infarction or hemorrhage has been described , it is uncommon for an acromegaly - causative pituitary adenoma to evade visualization on pituitary magnetic resonance imaging ( mri ) , much less for an empty sella ( es ) alone to be present.10 11 in cases where only an es is visualized , ectopic acromegaly should be considered , though it is responsible for less than 1% of cases . the vast majority of ectopic acromegaly results from neuroendocrine gh releasing hormone ( ghrh)-secreting tumors , but isolated reports have described ectopic acromegaly from gh - secreting pancreatic islet - cell or lymphoma neoplasms,12 13 and 14 cases have been ascribed to abnormally located pituitary tissue.14 this case describes the work - up and treatment of a phenomenon not previously documented rare ectopic acromegaly caused by a biopsy - proven somatotroph pituitary adenoma located within the bony intersphenoid septum of a patient with baseline ess . a 55-year - old african - american man presented with a history of having grown bigger in his late thirties , with particular hand , foot , and hat size enlargement of the prior 3 years . physical examination was notable for a blood pressure of 151/83 mm hg , rate - controlled atrial fibrillation , weight = 147 kg , height = 67 , and body mass index = 37 . the patient appeared to be acromegalic , with frontal bossing , coarse facial features , enlarged nose , macrognathia , macroglossia , and widened interdental spaces . laboratory tests confirmed acromegaly , with a serum insulinlike growth factor 1 ( igf-1 ) level of 560 ng / ml ( 81225 ng / ml ) and gh level of 16.7 ng / ml ( 0.053.00 ng / ml ) . pituitary mri showed an enlarged es with subtle left anterolateral pituitary tissue but no distinct intrasellar adenoma ; however , a sphenoid sinus mass extending from the infundibulum was also visualized , projecting inferiorly from the sellar floor ( fig . this seemed suggestive of a bone - enclosed ectopic pituitary tumor ; however , further confirmatory work - up was felt indicated as such a tumor had not previously been described . sagittal enhanced spin echo t1 thin section through the sella shows a mass in the sphenoid sinus ( solid arrow ) in continuity with the infundibulum , and an empty sella ( dotted arrow ) with tissue along its floor . pan - computed tomography ( ct ) with contrast demonstrated gross cardiomegaly but was otherwise negative for concerning masses.1 11 in - labeled octreotide scintigraphy was also unremarkable , and further work - up to rule out possible gh hypersecretion was not immediately pursued as per the patient 's request . the patient was treated briefly with octreotide , but self - discontinued this due to visual hallucinations and declined further medical or surgical therapy . repeat laboratory tests showed persistent acromegaly ( gh : 6.37 ng / ml ; igf-1 : 771 ng / ml ) , and a 75-g oral glucose tolerance test failed to suppress gh . other pituitary - axis hormones including prolactin ( 10.4 ng / ml ; reference : 2.117.7 ng / ml ) remained normal . brain ct again did not recognize distinct tumor , but demonstrated stable es configuration and unknown subsellar tissue within the bony intersphenoid septum ( fig . 2 ) . a low - normal ghrh level of 7 pg / ml ( 518 pg / ml ) ruled out an ectopic neuroendocrine tumor , and repeated.1 11 in - labeled octreotide scintigraphy was negative . the patient continued to decline surgery and therefore was resumed on octreotide , but he again discontinued this due to side effects . coronal and sagittal computed tomography ( ct ) head . left : coronal enhanced ct scanning through the midsella using a bone window demonstrates the widened intersphenoid septum containing ectopic pituitary tissue ( solid arrow ) . right : sagittal enhanced ct shows a sellar floor defect , enhancing pituitary tissue within the intersphenoid septum ( dotted arrow ) and marginally lining the sellar floor . igf-1 level was unexpectedly normal at 92 ng / ml ( confirmed on recheck 1 week later with igf-1 level of 174 ng / ml ) ; igf - binding protein-3 was elevated at 6,430 ng / ml ( 2,5924,770 ng / ml ) , ruling out elevated free igf-1 . pituitary mri again revealed an es without evidence of hemorrhage or infarction , and stable tissue - filled bony projection from the sellar floor extending into the medial aspect of the sphenoid sinus ( fig . 3 ) . given the patient 's normalized igf-1 and absence of distinct intrasellar pituitary adenoma on imaging , spontaneous remission of acromegaly was considered ; however , 75-g oral glucose tolerance testing failed to suppress gh . petrosal sinus sampling showed an approximately six- to sevenfold central - to - peripheral gh gradient ( peripheral = 3.27 ; left = 17.80 ; right = 20.20 ) , consistent with central gh production . coronal ( left ) and sagittal ( right ) magnetic resonance imaging head scans . enhanced fat - suppressed spin echo t1 images through the midsella demonstrate a stable mass in continuity with the infundibulum , extending below the sellar floor and into the widened intersphenoid septum . a midline sphenoid sinus septa was resected , and the anterior wall and floor of the sella turcica were removed . the dura overlying the pituitary gland extended into an expansion of the sphenoid sinus ; this dura was resected , revealing underlying soft tumor tissue that was removed using ring curettage and suctioning . histology confirmed a typical pituitary adenoma , with cells that were positive for both human gh and prolactin by immunohistochemistry ( fig . twenty - four hours after surgery , gh level was normal at 1.65 ng / ml ( 0.053.00 ng / ml ) , and other pituitary hormones remained within normal limits . follow - up igf-1 level 2 months after surgery was 67 ng / ml . formalin - fixed , paraffin - embedded tissue histology demonstrates pituitary adenoma ( a : 1,000 magnification ; hematoxylin eosin stain ) and gh secretion ( b : 1,000 magnification ; polyclonal rabbit antihuman gh antibody ) . acromegaly stems from gh - secreting pituitary tumors in approximately 99% of all cases , and most tumors are visible on mri of the sella.10 only six prior instances of acromegaly occurring with both radiographic ess and an ectopic sphenoid sinus pituitary adenoma have been documented.14 15 16 17 18 19 while our patient 's pituitary location within the walls of the bony intersphenoid septum was suggested by ct and mri studies , it appears to be a unique anatomical variation not previously described . it was recognized as pituitary tissue only retrospectively , on collaborative interdepartmental review of intraoperative findings and imaging , highlighting the difficulty of intrabony adenoma identification . since pituitary remnants laying outside the hypophyseal portal system may be less susceptible to normal hypothalamic regulation,20 it is possible that other cases of intrabony adenomas within the intersphenoid septum have gone unrecognized in patients with pituitary hormone dysregulation and ess / unidentified tumor . ectopic pituitary tissue is most often situated in the sphenoid sinus or nasopharynx14 and is felt to result from faulty embryological migration and invagination course of rathke 's pouch , or mechanical adenoma protrusion through sellar floor defects ( especially in conjunction with higher fluid - filled es / intracranial pressure).19 21 absent , eroded , and intact sellar floors were all described in six previously documented cases of acromegaly with combined ess and sphenoid sinus ectopic adenoma14 ; our patient demonstrated an absent sellar floor . his enlarged sella and absent sellar floor might support a mechanical event perhaps an originally larger macroadenoma which then hemorrhaged , necrosed , and involuted . intracranial hypertension might then have led to an expanded sella along with caudal descent / flattening of the pituitary and extension into the intersphenoid septum , where the remaining tumor ultimately settled . secondary ess ( and often , associated acromegaly remission ) has been a documented sequela to pituitary apoplexy22 23 24 25 26 27 ; however , increased intracranial pressure or aggressive tumor growth would theoretically displace the pituitary lateral to the intersphenoid septum along the path of least resistance , rather than induce a split furthermore , our patient 's earliest baseline mri suggested primary ess with no evidence of hemorrhagic or apoplectic evolution over several years of follow - up imaging , and while his igf-1 levels decreased 5 years into his course , repeat oral glucose tolerance testing confirmed persistent biochemical acromegaly . therefore , we postulate that our patient 's clinical course and split bony septum ( with smooth walls surrounding the ectopic pituitary ) best support an anomalous embryological migration etiology , with baseline sellar floor incompetence also enabling his ultimate pituitary ectopy . treatment of patients with acromegaly and nonvisualized and/or extrasellar sellar tumors poses a challenge to definitive acromegaly management , which generally demands source localization followed by surgical tumor resection . to our knowledge , octreotide scintigraphy has been utilized in only one prior case of ectopic pituitary acromegaly , and that scan successfully identified the sphenoid sinus adenoma.14 however , our patient 's scan was unremarkable , failing to reveal his intersphenoid septum tissue as an acromegaly - causative tumor . his ectopic pituitary was demonstrated on serial cts / mris , but was confirmed only retrospectively as pituitary tissue , due to lack of previously reported bony - encased intrasphenoid septal tumors and unfamiliarity with this diagnosis . therefore , in his case , repeated testing and imaging were necessary to rule out a nonpituitary etiology and to better predict a central pathology amenable to neurosurgical resection . in this patient , a low ghrh ruled out an ectopic ghrh - secreting neuroendocrine tumor ; and repeat.1 11 in - labeled octreotide scintigraphy , pan - ct , and petrosal sinus sampling ruled out a noncentral location . although our patient 's tumor location within a bony septum is unique , its location in the sphenoid sinus was consistent with other extrasellar reports12 of the 14 previously reported cases of ectopic adenoma acromegaly involved tumors within the sphenoid sinus.14 our article demonstrates that in cases of central acromegaly , where imaging shows ess and fails to identify an adenoma in paranasal sinuses or other peripheral locations , it is prudent to review mri scans for a possible intra - intersphenoid septal tumor .

the isolation and study of reactive intermediates provides a wealth of insights into the mechanism and stereochemical course of synthetically useful reactions . of course , many intermediates are not amenable to isolation because of their instability , reactivity or other complicating factors . in these cases , the synthesis of more stable model compounds can often provide useful information about critical structure and reactivity attributes . the isolation of intermediates from catalyzed reactions presents the additional challenges of low concentration and short half - life of relevant species that contain the catalytically active moiety complexed with one or more substrates . here again , the synthesis and study of putative intermediates in stoichiometric reactions representing elementary steps in the catalytic cycle can afford valuable insights provided that proper care is taken to establish the species is on the reaction pathway and is kinetically competent . extensive studies of the kind described above for reactive intermediates in metal - catalyzed cross - coupling reactions have played a significant role in formulating the current understanding of the mechanistic detail of those processes . for two of the elementary steps in the now well - accepted catalytic cycle , oxidative addition and reductive elimination , wide - ranging investigations involving kinetic , spectroscopic , crystallographic , and computational analyses have revealed a clear , consistent picture of the structure of the intermediates and the structural attributes of substrate and reagents that influence the rate of these elementary steps . the third elementary step in the cross - coupling catalytic cycle , namely transmetalation , involves the organometallic donor , and thus , this step both unifies and differentiates the manifold variants of this process . with the exception of the stille reaction , fewer decisive mechanistic studies are extant for this critical step in the catalytic cycle , most likely for several reasons : ( 1 ) its location in the middle of the catalytic cycle makes it difficult to study in isolation , ( 2 ) the nature of the pre - transmetalation intermediates is not well established , and/or ( 3 ) the pre - transmetalation intermediates are highly reactive . as part of our ongoing preparative and mechanistic studies on the palladium - catalyzed cross - coupling of alkenylsilanolate salts ( ii ) , we obtained compelling kinetic evidence for the intermediacy of a discrete pre - transmetalation intermediate iii , a palladium complex containing the silanolate , the electrophile ( from i ) , and ligands ( scheme 1 ) . moreover , our kinetic analysis concluded that the transmetalation step from this neutral ( 8-si-4 ) intermediate was extremely rapid and did not require additional activation via a hypercoordinate siliconate species such as iv . thus , the reigning dogma that mandated a hypercoordinate silicon species ( 10-si-5 ) for transmetalation was challenged . this critical conclusion , based solely on kinetic analysis , demanded additional concrete validation , ideally through isolation of the putative pre - transmetalation intermediate , arylpalladium alkenylsilanolate species iii , followed by a full structural and kinetic characterization . in addition , a thorough understanding of the controlling elements that influence the rate of transmetalation was desired . it is feasible that the nature of the transferable group plays a significant role in determining whether anionic activation is needed . another reasonable hypothesis is that ligands contribute significantly to the course of transmetalation . the challenge to answer these questions was that iii undergoes spontaneous transmetalation at room temperature , thus precluding straightforward isolation and characterization . however , if species such as iii could be isolated and studied , they would provide an unprecedented opportunity to directly interrogate the critical transmetalation step in the catalytic cycle . a program of research was thus formulated to synthesize , isolate , and characterize ( both structurally and kinetically ) arylpalladium(ii ) alkenylsilanolate complexes , stabilized by phosphine ligands of disparate denticity . using these complexes , a detailed comparison of the effects of both phosphine ligands and silanolate structure on the transmetalation pathway more importantly , the kinetic competence of these complexes could be established under various conditions and the comparison of these rates to the preparatively significant catalytic processes studied previously could be made . a complete understanding of the catalytic cycle can guide improvements in substrate selection , reaction design and other crucial parameters for these synthetically useful transformations . any investigation of the mechanistic features of the catalytic cycle in metal - catalyzed cross - coupling reactions must take careful account of the role of the ligands involved . throughout the evolution of metal - catalyzed cross - coupling reactions , ligands have had an enormous impact on many aspects of this process , such as the ability to facilitate the various steps in the catalytic cycle , prolong catalyst lifetime , influence site selectivity , or induce enantioselectivity . however , the means by which each ligand accomplishes these feats is associated with the molecular detail of the mechanistic pathways . as many preparative optimization studies have clearly demonstrated , not all ligands provide an effective catalytic process . ligands can impact multiple steps of a catalytic cycle , and thus the need to isolate the influence on each elementary step becomes paramount . whereas data on the influence of ligands and mechanistic studies on oxidative addition and reductive elimination abound , much less is known about the effects on the transmetalation step . thus , the following section will focus on the current state of understanding of this critical elementary step in various cross - coupling processes . as depicted in figure 1 , the currently accepted mechanism of palladium - catalyzed cross - coupling reaction begins with oxidative addition of the carbon following oxidative addition , the organometallic nucleophile approaches the palladium center to which the alkenyl or aryl group is transferred , creating a carbon palladium bond . this process occurs through either coordination to the palladium center or direct attack of a nucleophile on the metal center . carbon bond is formed through reductive elimination from the palladium center with simultaneous regeneration of the pd(0 ) catalyst . the rate - determining step in many of these cross - coupling reactions is believed to be transmetalation , but other factors may influence which step becomes rate determining . the catalytic cycle involves a pd(0)/pd(ii ) cycle ; however , many of the precatalysts used in these cross - coupling are pd(ii ) precursors , which must be reduced to enter the catalytic cycle . previous investigations in these laboratories have identified the detailed mechanism by which alkenylsilanols undergo fluoride - promoted cross - coupling reactions . a bimolecular transmetalation of a fluoride - activated disiloxane 1 with an arylpalladium(ii ) halide has been identified by both kinetic and spectroscopic methods ( scheme 2 ) . the transmetalation step has been identified as the turnover - limiting step , given the second - order dependence on silanol , the first - order dependence on arylpalladium halide , and the inability to saturate the silane cross - coupling partner as the activated disiloxane . early hypotheses on the mechanism of the cross - coupling of alkenylsilanols under activation by brnsted bases proposed that a deprotonated silanolate plays a role similar to that of fluoride by activating silicon toward transmetalation ( iv , scheme 1 ) . however , detailed kinetic analysis revealed the following characteristics : ( 1 ) zeroth - order dependence on silanolate at high concentrations , which provides support for the existence of an intermediate containing a silicon oxygen palladium bond ; ( 2 ) the participation of an activated complex , iv ( scheme 1 ) , is excluded as a result of the first - order dependence on silanolate when [ silanolate]/[pd ] < 1 ; and ( 3 ) rate - determining formation of iii at [ silanolate]/[pd ] < 20 , and a rate - determining intramolecular transmetalation from iii at [ silanolate]/[pd ] > 20 . the preparations of complexes that model the intramolecular transmetalation of a number of organonucleophiles have been reported . echavarren and co - workers described the isolation of a pre - transmetalation intermediate employing both an alkylstannane and an alkylsilane moiety ( scheme 3 ) . treatment of iodophenoxystannane 2 with [ ( dppf)pd(dba ) ] complex results in oxidative addition of the aryl iodide to generate 3 . addition of ag2co3 promotes the intramolecular transmetalation step , and the resulting complex 4 can be isolated . similarly , treatment of iodophenoxysilane 5 with [ ( ph3as)2pd(dba ) ] affords the trans - ligated complex 6 . this complex undergoes transmetalation in the presence of either tbaf or ag2co3 to afford the palladacycle 7 . the isolation of another stable stannylpalladium complex has been achieved employing a pincer - type complex and 2-(tributylstannyl)furan . these systems clearly demonstrate the isolation of competent transmetalation intermediates , but they are not specific to a catalytic process . platinum(ii ) and palladium(ii ) silanolates have been prepared and isolated and are potentially relevant to the cross - coupling of organosilanolates . of these complexes , a majority are derived from triphenylsilanol . the first of these complexes was prepared in 1994 by fukuoka and co - workers by the equimolar reaction of cod - pt(et)br ( cod = 1,5-cyclooctadiene ) and sodium triphenylsilanolate in thf at low temperature . the complexes were isolated from the reaction mixtures and purified by recrystallization , and their structures were confirmed by x - ray analysis . the isolation of organoplatinum(ii ) silanolates from aryl(dimethyl)silanols provides a model for the cross - coupling of aryl(dimethyl)silanols . the reaction of ( 4-trifluoromethylphenyl)dimethylsilanol with phenylplatinum(ii ) iodide in the presence of 1.0 equiv of ag2o provides the corresponding phenylplatinum(ii ) silanolate complex . this species is thermally stable and does not undergo transmetalation to the diorganoplatinum(ii ) complex upon warming to 60 c . this is in contrast to the reaction of a cationic platinum(ii ) complex with an arylsilanol , wherein only the post - transmetalation diorganoplatinum(ii ) complex could be isolated . these experiments highlight the effect of ligands and cationic platinum sources on the transmetalation of aryl(dimethyl)silanols . the above cases represent the isolation of organoplatinum silanolates that are not relevant to the preparative cross - coupling reactions of interest . on the other hand , the first complex was isolated in 1999 by fukuoka and co - workers from the reaction of ( cod)pd(ch3)br and sodium triphenylsilanolate . this complex was isolated and characterized but not investigated for its potential in cross - coupling . the ability to independently prepare competent intermediates along the catalytic cycle for the cross - coupling reaction of alkenylsilanolates creates a unique opportunity to systematically study the features of the transmetalation step in detail . instructive experiments designed to probe the molecular detail of this critical step can be devised , such as ( 1 ) verification of the mechanistic significance of the putative si o pd intermediate identified in previous kinetic studies , ( 2 ) independent isolation and characterization of arylpalladium alkenylsilanolate complexes , ( 3 ) determination of the kinetic competence of these isolable pre - transmetalation intermediates , and ( 4 ) comparison of the kinetic behavior of these complexes with the catalytic reactions . we report herein the successful achievement of these goals through the preparation of a number of arylpalladium(ii ) alkenylsilanolate complexes stabilized by phosphine ligands . these species have been thoroughly studied to elucidate the roles that ligands play in the key transmetalation step . furthermore , these isolable intermediates and their kinetic behavior have further refined the controlling factors in the thermal and anionically activated pathways that have been proposed for the cross - coupling of this class of nucleophiles . the accompanying article details similar , albeit much more extensive studies on the cross - coupling of arylpalladium arylsilanolate complexes . in view of the extensive kinetic analysis already in hand on the cross - coupling of alkenylsilanols , the isolation of a putative transmetalation intermediate involving alkenylsilanolates was investigated first . as was alluded to above , the rapid rate of reaction for these coupling partners presented a serious challenge to the isolation of a pre - transmetalation intermediate . the reaction conditions for the catalytic process employed no ligands ( only iodides had been coupled at that time ) , so the use of strongly coordinating phosphine ligands was investigated to slow the key transmetalation step and allow the intermediate to be intercepted . thus , the independent synthesis of an alkenylsilanolate complex was initially pursued through the introduction of 1,3-(diphenylphosphino)propane ( dppp ) and triphenylphosphine as stabilizing ligands . combination of ( tmeda)(2-tolyl)palladium(ii ) iodide complex 9(19 ) with styrylsilanol 8 in the presence of silver oxide afforded the ( tmeda)(2-tolyl)palladium(ii ) silanolate 10 in quantitative yield ( scheme 4 ) . treatment of 10 with dppp afforded ( dppp)(2-tolyl)palladium(ii ) silanolate 11p in 72% yield , whereas treatment with 2.0 equiv of triphenylphosphine afforded the trans-(ph3p)2(2-tolyl)palladium(ii ) silanolate 11 t in 42% yield . h nmr spectroscopic analysis of complex 11p showed the loss of the tmeda ligand and the characteristic pattern for the styrylsilanol moiety . moreover , the p nmr spectrum of 11p displayed two non - equivalent phosphorus nuclei ( = + 20.1 and 11.7 ppm , doublets , j = 49 hz ) , whereas the p nmr spectrum of 11 t showed a single phosphorus resonance ( 28.0 ppm ) . the structures of complexes 11p and 11 t were confirmed by single - crystal x - ray analysis , which unambiguously established the presence of a si o pd linkage ( figure 2 ) . the pd(1)o(1 ) bond lengths for 11p ( 2.050 ) and 11 t ( 2.053 ) are slightly longer than those for other palladium(ii ) silanolates . the si(1)o(1 ) bond length is typical for organosilanolates , which suggests that the lengthened pd(1)o(1 ) bond may be due to a ligand effect at the palladium center . the trans influence of the 2-tolyl group in 11p is significant , as the p(2)pd(1 ) bond ( 2.348 ) is much longer than the p(1)pd(1 ) bond ( 2.218 ) trans to the silanolate oxygen in 11 t . however , the major difference in these structures lies in the orientation of the styryl group . the two phosphine atoms are 177 disposed , which causes some steric crowding about the palladium center . as shown in figure 2 , the dppp - derived styryl complex places the olefin of the styrene above the square plane of the complex , whereas the steric environment created by the six phenyl rings of 11 t prevents the alkene from approaching the palladium center . the ligand directs the styryl substituent to point away from the metal coordination sphere in the solid state . the ligand effects seen in the solid state may also manifest themselves in changes in the chemical reactivity of these intermediates in solution and play a role in the transmetalation step . structural representations of dppp ( 11p ) and ph3p ( 11 t ) ligated 2-tolylpalladium(ii ) ( e)-styrylsilanolate complexes ( hydrogens omitted for clarity ) . the following studies were directed toward understanding how both the transferable group on silicon and the respective ligands affect the transmetalation step . moreover , these investigations can help establish which mode of transmetalation ( 8-si-4 versus 10-si-5 ) occurs under various reaction conditions . with these overarching goals in mind , each of the complexes prepared in the foregoing section was evaluated for their independent reactivity as well as in quantitative comparisons to functioning catalytic systems . the rate equation for the catalytic cross - coupling of styryl(dimethyl)silanolate under ligandless conditions was determined to establish if it was operating in the same kinetic regime as the previously studied alkenyl(dimethyl)silanolate , which employed dibenzylideneacetone ( dba ) as the ligand . previous studies had established that the use of allylpalladium chloride dimer ( apc ) affords ligandless palladium(0 ) because the reduction is effected by silanolate displacement of the allyl ligand to produce an allyl silyl ether and potassium chloride . moreover , it was discovered that phosphine oxides serve very effectively as weakly coordinating , stabilizing ligands for the palladium nanoparticles formed and increase the turnover number by preventing precipitation of palladium black . the partial order in each component in the reaction of potassium ( e)-2-phenylethenyldimethylsilanolate ( k8 ) with 2-iodotoluene ( 12 ) catalyzed by apc in the presence of a phosphine oxide additive was determined individually at 40 c in benzotrifluoride ( table 1 ) . equimolar amount of bis - oxide was used to ensure consistent palladium concentrations at lower temperatures . the rate of the cross - coupling reaction is clearly independent of the concentration of k8 and establishes zeroth - order behavior for this component ( table 1 , entries 13 ) . a slope of 0.019 was obtained for the order in 2-iodotoluene at 40 , 80 , and 160 mm , corresponding to no change in reaction rate and , again , established zeroth - order behavior for this component ( entries 2 , 4 , and 5 ) . next , the dependence of the rate constant on the loading of the palladium catalyst was determined by comparison of the initial rates of product formation at 80 mm in 2-iodotoluene ( entries 2 , 68 ) . a slope of 1.105 , obtained from a log(rate ) vs log(conc ) plot of the data , is consistent with a first - order dependence of the observed rate constant on the concentration of palladium . to ensure that the phosphine oxide additive does not impact the reaction rate , the cross - coupling was repeated with increased dppp(o)2 loadings ( entries 1 and 9 ) . no change in the reaction rate was observed , ruling out any kinetically significant role of the phosphine oxide . these data establish the overall rate equation for the reaction of k8 with 18 catalyzed by [ allylpdcl]2 , where k = 8.83 10 s , as1 this rate equation matches that of the alkenylsilanolates reported previously , in which turnover - limiting , direct transmetalation from an arylpalladium(ii ) intermediate ( iii to v ) takes place without anionic activation via iv ( scheme 1 ) . however , this rate equation is also consistent with an activated mechanism where iii is completely saturated as iv . indeed , turnover - limiting , activated transmetalation from k8 will also result in a zeroth - order dependence on k8 . the kinetic analysis of the catalytic reaction with k8 alone does not unambiguously distinguish between the two possible mechanisms . support for the assertion that k8 was reacting via a direct , thermal transmetalation of an 8-si-4 species was obtained from the kinetic analysis of the reaction of the cesium salt ( table 2 ) . reaction of cs8 with 2-iodotoluene ( 12 ) ( with apc and dppp(o)2 in benzotrifluoride at 40 c ) changed the rate equation such that a fractional order ( 0.55 ) in cs8 was observed ( eq 2 ) . apparently , the more nucleophilic cs8 is not operating in the same kinetic regime as k8 and may open an activated pathway for transmetalation . the partial order indicates that two separate mechanistic pathways are operative , involving equilibration of the intermediates xii and xiii ( figure 3 ) . moreover , as the loading of cs8 increased , the rate leveled to a zeroth - order behavior for this component ( table 2 , entries 6 and 7 ) . this is consistent with a regime of complete saturation of xii as xiii , such that only an activated transmetalation pathway is operative.2 average of triplicate runs . summary of kinetic regimes for the transmetalation in the presence of cs8 . when the preformed dppp complex 11p was stirred in either thf or benzotrifluoride at room temperature , no product formation was observed , even after 24 h. remarkably , no product formation was observed until a temperature of > 100 c was reached . in refluxing benzotrifluoride ( bp 102 c ) , the expected stilbene 13 was produced in 82% yield ( gc ) ( scheme 5 ) . given the low temperature for the catalytic reaction , the elevated temperature needed for the isolated complex to undergo transmetalation must be associated with the requirement for dissociation of the strongly binding phosphine ligand . to support this hypothesis , it was necessary to effect the chemical decomplexation of the ligand from 11p and measure the rate of cross - coupling . liebeskind , farina , and co - workers have demonstrated the ability of copper(i ) thiophene carboxylate ( cutc ) to sequester phosphines in palladium - catalyzed cross - coupling reactions . the addition of cutc to an alkenylsilanolate complex should result in the decomplexation of dppp , thus creating a quantity of the 14-electron species from which transmetalation may proceed . indeed , when complex 11p was treated with 10 mol % of cutc in benzotrifluoride at room temperature , 13 was generated in quantitative yield with an initial rate of 2.27 10 mm / s . when the corresponding trans - ph3p complex 11 t was employed under identical conditions , the initial rate of product formation was slightly faster ( 2.75 10 mm / s).these results clearly demonstrate the ability for alkenylsilanolates to undergo transmetalation without the need for external activation at room temperature . to make a direct comparison to the kinetic rate constant for the catalytic reaction , the cu - assisted thermal transmetalation was conducted at 40 c , which resulted in a faster rate of transmetalation ( 11p , 2.71 10 mm / s ; 11 t , 1.20 10 mm / s ) . however , the rates for these processes at 40 c are slightly lower than those for the corresponding catalytic reaction ( 3.25 10 mm / s ) . we assume that the stoichiometric reaction of 11p is 1.25 mm in active pd catalyst ( because it is 1.25 mm in cutc ) , which gives the 2.71 10 mm / s rate . simply dividing the initial rate by [ pd ] gives the principal rate constant for this process , 2.17 10 s. accordingly , the kinetic rate constant for this process at 40 c is lower than that for the corresponding catalytic reaction ( k = 9.04 10 s ) , thus precluding an unambiguous conclusion about the intermediacy of iii . this disparity could arise from either incomplete or slow decomplexation of the ligand from the isolated complexes . the unknown rate and extent of ligand dissociation at lower temperatures prevents the direct rate comparison of catalytic alkenylsilanolate cross - coupling reactions with their putative pre - transmetalation intermediates . the rate equation found from kinetic experiments involving k8 and 12 employing apc and dppp(o)2 k8 at 40 c in trifluorotoluene ( eq 1 ) was identical to that for the cross - coupling of 1-heptenyl(dimethyl)silanolate , where no rate effect was observed with increasing the concentration of silanolate . on the other hand , the use of cs8 ( with 12 ) led to a fractional dependence ( 0.55 ) on the concentration of the silanolate . clearly these two salts are operating ( for the most part ) in different kinetic regimes . the various steps of the catalytic cycle predict different concentration dependences on each component . the experimentally established rate law can eliminate a number of possibilities for the turnover - limiting steps ( figure 4 and table 3 ) . starting from the palladium(ii ) precatalyst [ allylpdcl]2 , reduction to the active pd(0 ) catalyst involves the participation of the silanolate . oxidative addition to pd(0 ) occurs with the aryl halide , producing a palladium(ii ) halide intermediate ( first order in , step a ) . displacement of the palladium iodide proceeds by a nucleophilic attack by an equivalent of m8 and predicts a first - order dependence on m8 ( step b ) . next , if the transmetalation is proceeding via a direct , unactivated process , then a zeroth - order behavior for m8 should arise ( step c ) . alternatively , either a zeroth- or first - order behavior could result from an activation - limiting transmetalation step ( step e ) . finally , if reductive elimination is turnover limiting ( step f ) , then zeroth order in both components , m8 and 12 , will be observed . thus , zeroth - order dependence on m8 and 12 is consistent with step c , e , or f ; however , step f has already been eliminated . the most reasonable explanation is that the styrylsilanolate cross - coupling is proceeding via a rate - limiting transmetalation involving either neutral 8-si-4 or 10-si-5 intermediates , depending upon the nucleophilicity of the silanolate . previous studies on the alkenylsilanolate cross - coupling using pd(dba)2 demonstrated zeroth - order dependence for [ silanolate ] once saturation of the intermediate xiii is achieved . this intermediate undergoes transmetalation at 40 c without activation in the presence of the potassium salt k8 . however , the increased nucleophilicity of the cesium silanolate cs8 opened the possibility for an activated transmetalation pathway via xiii not seen previously . , the rate of cross - coupling represents the intrinsic rate of intramolecular transmetalation via xiii . the fractional order in cs8 most likely represents simultaneous operation of both pathways ( figure 3 ) . the rate of the catalytic reaction employing phosphine oxides was four times faster than the rate obtained by generation of a subligated palladium complex using cutc . the lower temperatures employed for the cutc - assisted transmetalation may decrease the rate of deligation ; therefore , the nominal concentration of subligated palladium is smaller than expected . nevertheless , the ability for the cross - coupling of alkenylsilanolates to proceed via an unactivated , direct transmetalation has been confirmed through both kinetic studies and the isolation of ligand - stabilized complexes . the mechanistic formulation for the cross - coupling of alkenylsilanolates with aryl halides is now substantially understood . the isolation and characterization of two arylpalladium alkenylsilanolate complexes allowed for the unambiguous demonstration of both neutral ( 8-si-4 ) and anionic ( 10-si-5 ) mechanistic pathways for transmetalation from silicon to palladium . in general , potassium salts of alkenylsilanolates react via neutral ( 8-si-4 ) intermediates , whereas the enhanced nucleophilicity of the cesium alkenylsilanolates allows for the reaction to access the 10-si-5 intermediate and proceed via the anionically activated pathway . these conclusions mandate a revision of the paradigm that organosilicon compounds must be anionically activated to engage in transmetalation processes ( hiyama hatanaka paradigm ) . through the agency of intramolecularity , direct transmetalation of silicon to palladium the mechanistic details of the related cross - coupling of arylsilanolates with aryl halides are addressed in detail in the accompanying paper .

end - stage renal disease ( esrd ) is associated with higher incidence of pulmonary arterial hypertension ( pah ) , leading to increased chances of mortality , morbidity and early rejection in patients slated for renal transplantation . the prevalence of pah among the patients receiving long - term haemodialysis ( hd ) ranges from 16% to 58% . early renal transplantation is required in these patients as the pro - inflammatory proteins and oxidative stress markers are not adequately excreted through hd , which is the cause for cardiovascular compromise . in this article , we report management of a patient with pah for renal transplantation and stress on the need for early renal transplantation and tailored anaesthetic management . a 51-year - old nigerian male with esrd and associated systemic hypertension belonging to new york heart association functional classification iii was scheduled for live - related renal transplantation . he was on regular hd thrice weekly through left radio - cephalic arteriovenous ( av ) fistula . on routine pre - transplant evaluation , his resting blood pressure was 220/110 mmhg and he was receiving multiple oral antihypertensives including amlodipine 10 mg twice daily , clonidine 0.2 mg four times daily , telmisartan 40 mg once daily , prazosin 5 mg twice daily and metoprolol 50 mg twice daily . he had features suggestive of right heart failure such as raised jugular venous pressure , hepatosplenomegaly , moderate ascites and basal crepitations . echocardiography showed severe concentric left ventricular hypertrophy , grade i left ventricular diastolic dysfunction with pulmonary arterial systolic pressure ( pasp ) 70 mmhg and left ventricular ejection fraction ( lvef ) 55% . his contrast tomogram pulmonary angiogram showed mildly dilated central pulmonary arteries with no evidence of pulmonary thrombo - embolism . in addition , our patient was retroviral positive on anti - retroviral therapy ( art ) . all haematological and biochemical parameters were within normal range . following a detailed evaluation by a multidisciplinary team which included the nephrologist , transplant surgeons , anaesthesiologist , cardiologist and pulmonologist , renal transplantation was planned under epidural anaesthesia . after connecting standard monitors , intravenous ( iv ) with patient in sitting position , under local anaesthesia , an 18-gauge epidural catheter was placed in t9t10 interspace . a total of 10 ml was given over 15 min , after which epidural infusion of 0.25% bupivacaine and 2 g / ml fentanyl was started at 35 ml / h . right radial artery and right internal jugular vein were cannulated with 20-gauge arterial catheter and triple lumen 7.5 fr catheter , respectively under local anaesthesia with ultrasound guidance . a loading dose of dexmedetomidine 1 g / kg was infused over 20 min and then titrated at 0.20.5 g / kg / h to reach a ramsay sedation score 4 . systemic and pulmonary hypertensions were managed with nitroglycerin ( 510 g / min ) and milrinone ( 0.375 g / kg / min ) infusion titrated to keep target mean arterial pressure around 90 mmhg . intraoperative blood loss was about 300 ml and 2 l of balanced salt solution was transfused to maintain central venous pressure around 810 mmhg . dexmedetomidine and nitroglycerin infusions were stopped at the end of surgery , and he was shifted to the intensive care unit . epidural infusion of 0.125% with 2 g / ml of fentanyl was continued into the post - operative period for pain management . on the 1 post - operative day ( pod1 ) , echocardiography showed pasp 45 mmhg . saturation on room air was 98% and echocardiography showed pasp 25 mmhg and lvef 50% . pah is defined as persistent elevation of the mean pulmonary arterial pressure ( mpap ) > 25 mmhg at rest or > 35 mmhg with exercise . mpap of 2540 mmhg is classified as mild pah , 4155 mmhg is moderate pah and > 55 mmhg is severe pah . pulmonary hypertension is seen in patients with esrd and the causes are said to be prolonged duration of renal replacement therapy , extra - osseous pulmonary calcification , high cardiac output due to av fistula , anaemia , fluid overload , hormonal and metabolic imbalances associated with uraemia and impaired endothelial function . human immunodeficiency virus - related pah is a rare complication of hiv infection and the incidence is estimated to be 0.5% , which is approximately 2500 times greater than that of primary pulmonary hypertension ( pph ) in the general population . highly active art down - regulates viral replication leading to a decrease in abnormal t - cell activation . art by decreasing chronic stimulation of the immune system might also decrease the ( still unknown ) driving force behind the development of pph . in esrd , there is significant elevation of the pro - inflammatory proteins interleukin-6 , tumour necrosis factor- and c - reactive protein . in addition , oxidative stress markers such as plasma protein carbonyls and f2-isoprostanesare are also elevated . hd is ineffective in controlling the levels of these factors thus leading to significant rise in pulmonary pressure . after renal transplantation , due to the use of immunosuppressant and restoration of normal kidney function , these levels decline over a period of 2 months . our patient underwent dialysis through av fistula and had high cardiac output in a non - compliant heart , hypertrophied left ventricle and enlarged left atrium with pulmonary congestion , which has led to inevitable severe pulmonary hypertension . medical line of management for pulmonary hypertension in esrd has minimal role , and thus , it was decided to treat the cause surgically . recommended intraoperative treatment for pulmonary hypertension are inhaled nitric oxide at 1040 ppm , milrinone ( phosphodiesterase 3 inhibitor ) used as infusion of 0.250.75 g / kg / min ( initial 50 g / kg bolus ) , inhaled epoprostenol ( continuous ) 1050 ng / kg / min and iv prostacyclin 410 ng / kg / min . our anaesthetic goals in this patient were to maintain systemic vascular resistance and optimal preload to the heart , to maintain sinus rhythm and to avoid rise in mpap due to hypoxaemia , hypercarbia and pain . in severe pulmonary hypertension , direct laryngoscopy during general anaesthesia causes increased sympathetic outflow leading to increased pulmonary vascular resistance and thereby precipitating acute right heart failure . over use of narcotics , inhalational agent or muscle relaxants to control sympathetic response we could successfully manage this patient with severe pulmonary hypertension using epidural anaesthesia and dexmedetomidine infusion to sedate and allay anxiety . to control his pulmonary vascular resistance , adequate ventilation and oxygenation were ensured , and milrinone infusion in titrated doses was instituted till pod 2 . following renal transplantation , our patient showed significant decrease in mpap from 70 mmhg to 25 mmhg over a period of 6 days . regional anaesthesia either alone or in combination with plays an important role in the anaesthetic management of complex patients with pulmonary hypertension presenting for renal transplantation .

a significant number of patients with diabetes remain poorly controlled , mainly as a result of poor diet compliance . the important choices that affect blood glucose control in people with diabetes are made by themselves , and not by their physicians or other medical professionals . people with diabetes are advised to adopt an appropriate diet including dietary habits and meal patterns on a lifelong basis . traditionally , recognition of the relationship between dietary constituents and glucose tolerance has contributed to the development of nutritional prescriptions . such strategies aim to restrict energy intake and provide macronutrient balance . however , some diabetic patients may have difficulty understanding diets based on a restrict energy intake , as well as changing their daily food habits . hyperglycemia is associated with increased risk for atherosclerosis by suppressing endothelium - dependent vasodilation , activating thrombosis , and increasing oxidative stress in people with type 2 diabetes mellitus ( t2d ) , with impaired glucose tolerance ( igt ) , and even in subjects with normal glucose tolerance ( ngt ) . a reduction in postprandial hyperglycemia by an -glucosidase inhibitor delayed progression of carotid intima - media thickening in subjects with t2d . vegetables , including seaweed and mushrooms , before carbohydrates , including potatoes , pumpkin , and corn , etc . on the reduction in postprandial glucose levels . we reviewed the effect of eating vegetables before carbohydrates on postprandial plasma glucose and insulin in patients with t2d . we conducted a randomized crossover study in 15 outpatients with t2d controlled by diet ( male / female ; 7/8 , age ; 61.7 11.6 y , duration of diabetes ; 5.3 8.8 y , body mass index ( bmi ) ; 24.7 4.3 kg / m , hba1c ; 6.4 0.6% , fasting plasma glucose ( fpg ) ; 6.28 1.12 mm ) . patients ate test meals consisting of 150 g white rice and vegetable salad ( sliced tomato and cabbage with olive oil dressing ) , eating either vegetables before carbohydrates or vice versa . plasma glucose and serum insulin levels were evaluated at 0 , 30 , 60 and 120 min after each test meal . plasma glucose and serum insulin levels between eating vegetables before carbohydrates and the reverse regimen were examined using the student s paired t test with spss 15.0 for windows ( spss inc , chicago , il ) . postprandial plasma glucose levels in those following the vegetables before carbohydrates regimen were reduced at 30 and 60 min compared to the reverse regimen ( fig . postprandial serum insulin decreased significantly at 30 and 60 min in the vegetables before carbohydrates regimen ( fig . a continuous glucose monitoring system ( cgms ) is capable of detecting hypoglycemia and hyperglycemia that may be undetectable by self monitoring blood glucose and glycated hemoglobin . particularly , the mean amplitude of glycemic excursions ( mage ) is a significant determinant of overall metabolic control , as well as increased risk of diabetes complications . we examined whether eating vegetables before carbohydrates could reduce the daily postprandial glucose excursions assessed by cgms in japanese patients with t2d and subjects with ngt . all participants were assigned to cgms ( medtronic minimed gold , northridge , ca ) for 72-h by eating test meals of vegetables before carbohydrates and carbohydrates before vegetables on the 2nd and the 3rd day in a randomized crossover design . all study protocols were approved by the ethics committee of the school of comprehensive rehabilitation at osaka prefecture university , and each subject provided written informed consent before enrollment in the studies . the test meals consisted of rice / bread , meat / fish , 500 g of vegetables ( tomato , spinach , broccoli , and radish , etc . ) , and contained 21 g of dietary fiber and 125.6 kj kg per day . energy intake was adjusted by the amount of rice and bread for each participant and the rest of the meals were identical . the energy ratio of protein , fat , and carbohydrates was 17% , 25% , and 58% , respectively . the subjects ate the first dishes of vegetables for 5 min , then the main dish for 5 min , and consumed rice or bread for 5 min successively within a 15 to 20 min total eating time for each meal , and vice versa . nineteen outpatients with t2d ( male / female ; 6/13 , age ; 65.5 9.4 y , duration of diabetes ; 16.4 10.2 y , bmi ; 22.5 3.1 kg / m , hba1c ; 7.2 1.0% , fpg ; 8.06 2.67 mm , diet / oral hypoglycemic agents / insulin treatment ; 3/3/13 : mean sd or n ) and 21 subjects with ngt ( male / female ; 2/19 , age ; 29.8 11.3 y , bmi ; 20.8 3.0 kg / m , hba1c ; 5.4 0.6% , fpg ; 4.89 0.50 mm ) were enrolled in the study . glycemic parameters between the day of eating vegetables before carbohydrates and the day of eating carbohydrates before vegetables were examined using the student s paired t test with spss 15.0 for windows . the mean of the daily glucose values was plotted to show the reduction in glucose excursions of eating vegetables before carbohydrates compared to the reverse regimen in subjects with t2d and ngt ( fig . 2 ) . the levels of sd , mage , large amplitude of glycemic excursions ( lage ) , mean 1-h postprandial glucose ( ppg ) , mean incremental area under the glucose curve0 - 3h ( iauc0 - 3h ) , and mean incremental glucose peak ( igp ) were all significantly reduced when the participants ate vegetables before carbohydrates compared to the reverse regimen in both subjects with t2d and ngt ( table 1 ) . the mean blood glucose levels did not show any difference between the two treatments because the hyperglycemia and hypoglycemia were suppressed when subjects ate vegetables before carbohydrates . in addition to lowering acute postprandial glucose levels , we studied whether educating diabetic patients to eat vegetables before carbohydrates was effective on long - term glycemic control . to test this hypothesis , we carried out a retrospective study in patients with t2d that compared changes in hba1c as the primary outcome and changes in weight , serum lipids , and blood pressure as the secondary outcomes . a total of 333 outpatients were divided into two groups to receive instructions about eating vegetables before carbohydrates ( educational group , n = 196 ) or a control group ( n = 137 ) who underwent a medical examination by a doctor . all patients were scheduled for return visits every 4 weeks with a physical examination and patients in the educational group were routinely scheduled to see dietitians at every visit . depending on the patient s current dietary intake , intervention aimed to encourage increased consumption of vegetables , including seaweed and mushrooms , and eating them first for 5 min , then the main dishes ( meat , fish , soybeans , etc . ) for 5 min , and rice or bread , including potatoes , pumpkin , corn , etc . , successively within a 15 to 20 min eating time using an original educational brochure in the educational group ( fig . 3 ) . the patients in the control group were given general information about lifestyle and diabetes risk by either the doctor or nurses at every visit . student s t tests were used to assess the difference between the clinical parameters in the two study groups and paired t tests were performed to analyze them within groups over time . improvements in glycemic control were observed in patients in the educational group after the intervention and the levels of blood pressure , total cholesterol , and ldl cholesterol decreased significantly in both groups after 1 and 2.5 y ( table 2 ) . after the intervention , dietary energy intake , protein , fat , and carbohydrates decreased in the educational group . these reductions in dietary nutrients were due to a decrease in the consumption of grain ( rice ) , fruits , oil , sweets , and beverages . on the other hand , consumption of vegetables increased after intervention in the educational groups . this evidence supports the effectiveness of eating vegetables before carbohydrates on glucose excursions in the short - term and glycemic control in the long - term in patients with t2d . dietary carbohydrates consumed after vegetables were digested slowly and required less insulin for subsequent metabolic disposal by the dietary fiber in the vegetables . other factors may influence the glycemic response and digestion of carbohydrates in the small intestine , including the rate of digestion , cooking method , transit time , and rate of intestinal absorption . similar to our study , horowitz et al . reported that whey protein and olive oil consumed before meals resulted in a substantial reduction in postprandial glycemia in patients with diabetes . protein and oil given before carbohydrates stimulate glucagon - like peptide 1 ( glp-1 ) secretion , and delay gastric emptying which leads to a reduction in glycemic excursions . however , in contrast to the reduced insulin secretion observed after eating vegetables , whey protein augmented insulin secretion , possibly by a combination of the incretin effect and the direct stimulation of -cells by absorbed amino acids . effects of eating vegetables before carbohydrates are probably similar to the gut peptide - based therapies for diabetes which may act predominantly by slowing gastric emptying , particularly when eating vegetables and protein before the carbohydrates . dietary intervention should aim not only to suppress hyperglycemia and hypoglycemia , but also glucose excursions in order to reduce vascular damage . the approach of eating vegetables before carbohydrates supports the concept of emphasizing food choices , what to eat first , how to eat and not just to concentrate on energy intake . the most important point was that the method made it easy to make the appropriate behavioral changes , to increase the consumption of vegetables and to reduce the consumption of rice and sweets for patients . as one aspect of nutritional education , eating vegetables before carbohydrates should be advised to patients with t2d and this advice could even be applicable to healthy people in order to prevent future cardiovascular events .

by the age of 80 years , humans generally lose 3040% of their skeletal muscle fibers , particularly type ii fibers . the decline in muscle mass and strength with age is well documented.2 , 3 in general , the muscle mass of 6070-year - olds decreases to 7080% of that of younger people ( < 60 years old).4 , 5 the main features associated with aging skeletal muscles are muscle weakness , decreased flexibility , vulnerability to certain types of injury , and impaired functional restoration , which result in the deterioration of physical performance and function . lower extremity strength decreases linearly with age in both men and women , as shown by numerous cohort studies.7 , 8 , 9 physical performance , with respect to balance and gait speed , for example , is closely linked with muscle mass and strength . therefore , age - related changes in muscle mass and strength should be carefully monitored for signs of decline . although functional decline with increased age is typical , the mechanisms underlying this decline , and the features that characterize it , are complex and variable . therefore , it is important to develop specific interventions that will prevent or delay functional decline based on understanding age - related changes in the muscular systems of older adults . concerning exercise interventions that have therapeutic potential specifically for age - related muscle atrophy , recent studies have focused on evidence - based interventions aimed at preventing mobility decline or enhancing physical performance in older adults.11 , 12 , 13 several different treatment modalities , other than traditional strengthening programs , have been designed to manage age - related functional decline more effectively.12 , 14 in this study , we reviewed the literature to delineate the role of physical function in disability and falls in older adults , and to assess the therapeutic potential of eccentric exercises for age - related muscle atrophy . sarcopenia is defined as the loss of skeletal muscle mass and strength with increased age.15 , 16 , 17 this results in weakness , limited mobility , and increased susceptibility to injury . several operational definitions have been used to diagnose sarcopenia , of which the most prevalent is an appendicular skeletal muscle mass ( asm ) value 2 standard deviations below the asm of young adults , divided by height squared ( kg / m ) or weight . as a rule , the relationship between mass and strength is linear , as is that between muscle strength and performance . however , there is no linear correlation between muscle mass and function . we found that muscle mass was not associated with physical performance in weak older adults , suggesting that measures of muscle strength may be of greater clinical importance in this population than muscle mass per se . the correlation between muscle mass and functional performance was significant in the higher strength group , but not in the weaker group . this is because fatty or connective tissue infiltration into muscle tissues are included in muscle mass in those patients such that we can not distinguish abnormal intramuscular changes by evaluating muscle mass alone , particularly in patients with mobility impairments . therefore , the functional status of intramuscular tissues should be included in any assessment of sarcopenia.8 , 18 mobility decline among the aging population is closely linked with changes in the muscle force velocity relationship . such changes have functional implications , e.g. , slower walking speeds . in recent studies , gait speed , or walking velocity , was highlighted as a major indicator of mobility decline or sarcopenia in geriatric populations.20 , 21 , 22 predicted years of remaining life , for both sexes , increases commensurate with gait speed regardless of age , according to a pooled analysis of nine cohort studies on walking speed and survival . gait speeds of 1.0 m / s or higher were consistently associated with longer survival than would be expected based on age and sex alone . according to the consensus in european group for determining sarcopenia , gait speed should be measured initially , and mass profiles should be evaluated when gait speed is abnormal . the mechanical properties of muscles can be assessed according to their active , contractile characteristics , and passive tension or stiffness . intrinsic contractile characteristics related to the cross - bridge mechanics of single fibers change with aging . some studies have reported that both a decrease in contractile materials and a reduced force - generating capacity per cross - bridge affect the contractile properties of aging muscles . a selective loss of type ii ( fast twitch ) muscle fibers is associated with the age - related decline in strength . cross - sectional dissections of whole thigh muscles of older cadavers have also shown an 18% decline in total muscle area and a 25% decrease in the total number of muscle fibers , with a particular decrease in the number of type ii muscle fibers . these results are also supported by other cross - sectional studies that have shown that the area of type i mean fibers is preserved with aging , whereas type ii fibers show atrophy . in addition to changes in muscle fiber size and number with advancing age , specific changes in the intrinsic ability of aged muscles to generate force have also been observed . in humans , decreased specific force ( i.e. , force normalized by cross - sectional area [ csa ] ) and unloaded shortening velocity in the type i and iia fibers of older sedentary males have been reported relative to young sedentary controls.28 , 29 these changes are also consistent with those observed in rat skeletal muscle throughout life . thompson et al reported decreases in the specific force and unloaded shortening velocity of the soleus muscles of aging rats . they further noted a dissociation between loss of muscle csa and a decline in maximal force . these human and rat data provide evidence that intrinsic changes in muscle quality may also be important for understanding age - related changes in the function of skeletal muscle . future studies should evaluate the relationship between the intrinsic force and shortening velocity of aging skeletal muscle , and the relationship between these factors and age - related declines in whole muscle strength and peak power . changes in both the fiber elasticity and contractility of type i and type iia fibers have been assessed in terms of instantaneous stiffness , that is , according to the ratio between force changes and corresponding length changes . as an active component of elasticity , instantaneous stiffness reflects the elastic characteristics of muscle fibers but has some limitations with respect to indexing passive components . passive elastic properties are instead assessed in terms of passive stiffness , or tension , which is thought to be related to sarcolemma , connective tissues , and titin filaments . passive stiffness is measured by the tensile force associated with displacement during stretching of the skeletal muscles . increased stiffness , or decreased extensibility , of the muscle or muscle - tendon unit has been reported in aging skeletal muscles . increased stiffness , along with decreased size and contractility , stiff collagen tissue , fatty changes , and changes in muscle cells are major determinants of passive stiffness in aging muscles . at a cellular level , sarcomere proteins such as titin , nebulin , and intermediate filaments control elastic muscle functions . there has been some controversy regarding the increase in muscle stiffness due to aging : in a previous study , muscle stiffness remained unchanged with older age ; however , it accounted for a greater proportion of total tension because of significant declines in muscle mass and contractile tension with age . due to physiological and structural changes , the force velocity relationship of human muscles changes with age , and muscular strength and power are reduced at all contraction speeds.35 , 36 , 37 , 38 however , older adults show a relatively preserved capacity for eccentric strength . compared with concentric force , eccentric force shows relatively less decline with age ( 248% ) . thus , preservation of eccentric strength in elders underpins the therapeutic potential of eccentric exercises in this population.37 , 39 , 40 eccentric muscle contraction involves a lengthening contraction that has greater force than shortening contraction with less energy expended per unit of muscle force ( fig . eccentric muscle contraction is very useful when a high degree of muscle strength is required . many functional activities require eccentric contraction , although delayed - onset muscle soreness can be problematic . although the mechanisms underlying eccentric contraction remain unclear , they appear to originate in the muscle itself , where both passive and active elements may regulate muscle stiffness . increased passive stiffness due to age - related accumulation of noncontractile material confers a mechanical advantage during eccentric contraction.33 , 39 ochala et al reported greater instantaneous stiffness per force unit , indicating greater active stiffness in the cross - bridge , in the fibers of older versus younger men ; this may represent a compensatory mechanism against less efficient muscle contraction with aging . in older muscle , a relative increase in active stiffness in the cross - bridge may be a mechanism underlying reduced muscle contraction efficiency . in a study on skeletal muscle fiber , we measured residual force enhancement ( rfe ) after lengthening contraction in aged rats . rfe is a well - known phenomenon that the isometric steady - state force at the stretched length after lengthening contraction is greater than the steady - state isometric force at that same length for a purely isometric contraction . we found that rfe was greater in older versus younger muscle fibers ( unpublished data ) . we concluded that preservation of eccentric strength likely contributed to the observed increase in rfe in aged skeletal muscle fibers . however , further studies are required to elucidate the mechanism underlying the preservation of eccentric strength . although the mechanisms remain unclear , an understanding of why older adults show this relative maintenance of eccentric strength could be relevant for practical applications , such as training and rehabilitation of elderly people many trials have aimed to understand how to prevent or delay functional decline , as well as the development of disability , in older adults . it is clear that exercise can play a key role in this respect , but issues remain regarding the specific interventions that are most effective and practically useful for functionally impaired individuals . many types of exercise can be used to enhance the physical function of older adults . the exercise recommendations of the american geriatric society have been widely accepted as being applicable to older adults . however , although the guidelines may be effective and safe for the healthy aging population , they may be too general for the mobility - impaired aging population . aging is associated with a decrease in the proportion of type ii muscle fibers , a decrease in fiber csa , and a change in contractile properties such as p0 , p0/csa , and v0.29 , 42 type ii fibers seem to be more affected by aging than other fibers , because they show greater decreases in csa , which in turn leads to increases in the relative muscle area occupied by type i fibers . several recent studies have suggested that , when the training stimulus is of an appropriate intensity ( i.e. , 7090% of the one - repetition maximum [ 1rm ] ) , gains in muscle strength and size in older healthy and frail individuals are comparable to the gains observed in young individuals . the difference in strength gains ( 28227% ) reported by various studies may be related to several factors , such as the participants initial strength , age , comorbidities , and differences in the muscles trained . effects of resistance training on the single muscle fibers of older individuals were detected in both type i and iia fibers in males , but not in females.43 , 44 moreover , the contractility of single - fibers might be sensitive to changes in physical activity levels , rather than aging per se . in several studies , progressive resistance training was associated with minimal improvements in peak power ; therefore , training interventions need to be designed to maximize peak power in older individuals.11 , 14 , 44 regarding the muscle force velocity relationship , power output is derived from the force velocity curve using hill 's equation ( fig . , there was a greater decrease in power output than in force in the former group . power output , which is the maximum capacity to perform muscular work per unit time , is a critical variable in muscle impairment , functional limitation , and subsequent disability.44 , 45 , 46 bean et al47 , 48 reported that peak power in lower extremities was closely associated with functional limitations and self - reported disability in community - dwelling men and women . strength ( i.e. , force ) and muscle power are both closely linked to functional performance ; power output has a strong correlation with performance in a number of functional tasks , such as climbing stairs , rising from a chair , and walking , which shows that power is a better indicator of poor mobility compared with hand grip , knee extensor torque , and muscle mass . therefore , interventions capable of enhancing or maintaining power output in older adults will be more effective . regarding ways to improve the force velocity relationship in old age , raj et al reviewed studies on the effects of different exercises on the force velocity relationship . traditional resistance training ( trt ) , such as the slow lifting and lowering of heavy loads according to the 1rm , had a significant positive effect on strength , but only modest effects on functional measures such as gait speed ; furthermore , changes in the force velocity curve were not significant . in other studies , power training ( pt ) was more effective for improving muscular power and functional capacity in older adults , and was also associated with significant improvements in isometric and concentric strength , as well as power , compared with trt.11 , 46 concerning power - oriented exercises , high velocity , low - resistance exercise can be used for pt . when using an exercise machine , around 40% 1rm represents a suitable external force for an effective increase in exercise velocity . reid et al reported several positive effects of power - specific exercise interventions in community - dwelling older women . however , a study that compared trt with pt found that they were equally effective for improving muscular strength . another study reported changes in peak leg extensor power in response to 12 weeks of resistance training in older women ; increases in strength of 2227% were observed , in addition to a nonsignificant increase in leg extensor power . one potential intervention to enhance force and function in aging muscles is eccentric muscle exercise . eccentric exercise has several important therapeutic advantages for aging populations , including low metabolic cost and minimal cardiorespiratory burden . elastic strain energy is used to resist the external force during eccentric contractions , such that muscle exertion requires minimal energy from adenosine 5-triphosphate ( atp ) breakdown ; although some atp is used during eccentric contractions , more is used during concentric and static contractions . therefore , chronic eccentric exercises can be easily applied to elderly and functionally limited patients , because these exercises do not require high energy consumption . furthermore , such individuals may be so limited in their ability to perform exercises that walking may represent the limit of , or even exceed , their aerobic capacity , thereby precluding exercises performed at intensities sufficient to prevent muscle wasting ( sarcopenia ) . eccentric exercise can cause substantial biologic changes in intramuscular structure and functional capability ( e.g. , increased muscle fascicle length ) . muscle fascicle length is known to decrease following immobilization , as well as with aging , but it can be increased by lengthening contractions.19 , 54 eccentric resistance training alone has been reported to increase vastus lateralis fascicle length in elderly people to a greater extent than trt . carlsson et al reported that myotilin , which plays a key role in the dynamic molecular events that mediate myofibrillar assembly , may also be involved in the development of new sarcomere structures after stretching stimulation . another reason for the usefulness of eccentric exercise is a less delayed onset of muscle soreness in older populations.56 , 57 age - related muscle atrophy tends to occur predominantly in type ii fibers . choi et al found that resistance of the myofilament lattice to mechanical strain was reduced in type iia and type iia / iix , but not in type i , fibers , which indicates that aging muscle may be less vulnerable to eccentric exercise - induced muscle injury . as mentioned above , compared with the decline in concentric force that occurs with age , eccentric force is relatively unimpaired . this preservation of eccentric strength in elders underlies the therapeutic potential of eccentric exercises , which could be used to specifically target this facet of strength . the potential advantages of eccentric resistance training might be particularly important during the initial stages of resistance training interventions for older adults with poor muscle strength . eccentric resistance training is generally conducted using specialized equipment , such as an isokinetic dynamometer . in a recent study , resistance exercises involving negative , eccentrically induced work were applied to older cancer survivors using a specially designed , motor - driven ergometer . at only low levels of exertion , these exercises can produce a relatively high muscle workload via the motor - driven eccentric exercise machine . in addition , reverse cycling of electric - powered pedals can induce biological overload through progressive increases in eccentric power . during this exercise , consistent with the evidence presented above , a number of studies have reported advantages of eccentric versus concentric resistance training . however , other studies have reported little or no additional benefit of eccentric resistance training over conventional training . in addition , the traditional negative standpoint on damage induction by eccentric contraction still remains an academic barrier to its clinical application . accordingly , further research is needed to optimize eccentric resistance training for older adults , particularly by improving the physical function domain . to be more effective , exercise interventions for older adults should focus on enhancing the muscle force velocity relationship . physiological and biomechanical evidence for an effect of lengthening contraction , as well as eccentrically biased training , on skeletal muscle properties and function has accumulated . there is a need to develop exercises that can be performed easily and utilize eccentric strength , which is relatively spared during the aging process , to improve both force and velocity in people with age - related muscle atrophy .

liver cirrhosis ( lc ) is a major , life - threatening health problem worldwide . lc results from liver injuries of numerous different etiologies , causing hepatocyte damage , hepatic inflammation , and fibrogenesis . lc is histologically characterized by increased deposition in and altered composition of the extracellular matrix ( ecm ) and the appearance of regenerative nodules . the destruction of the normal architecture of the liver and the loss of its functional hepatocytes prevent the liver from performing its normal detoxification , synthesis , and metabolic functions , eventually leading to portal hypertension and liver failure . from a clinical standpoint , lc is regarded as an end stage disease that leads to death , unless a liver transplant is performed . however , several problems are associated with liver transplantation , such as a shortage of donors , post - transplant rejection , operative risk , and high costs . recently , it has become increasingly clear that hepatic fibrosis is reversible if its causative agents are successfully targeted ; this has proved to be the most effective treatment for lc thus far . however , the underlying causative agents are treatable only in subsets of patients with liver disease . although there has been considerable research on liver fibrosis , there are no specific treatments for this condition . an ideal anti - fibrosis therapy would be specific for fibrogenic cells in the liver and be effective in attenuating excessive ecm deposition . myofibroblasts are the main effector cells in the fibrotic liver . in both experimental and clinical liver fibrosis cases , the activation of ordinarily quiescent hepatic stellate cells ( hscs ) into myofibroblasts is considered a major pathway of hepatic fibrogenesis associated with liver injury and has thus dominated the focus of studies on liver fibrosis . the activated hscs or their resulting myofibroblasts were the first major cell type in the liver to be identified as prominent in producing ecm in the injured liver . the hepatic resident mesenchymal cells , consisting of the quiescent hscs and the portal fibroblasts , can differentiate into myofibroblasts . then , bone - marrow derived cells , consisting of fibrocytes and mesenchymal stem cells in the peripheral blood , can be recruited to the injured liver to differentiate into myofibroblasts . recent studies have demonstrated that bone - marrow derived cells make only a small contribution to the myofibroblast population in experimental liver fibrosis . instead , fibrocytes may play a crucial role in the initiation of immune response during the earliest phases of tissue injury [ 8 ] . finally , hepatic progenitor cells , hepatocytes , cholangiocytes , and hepatic sinusoidal endothelial cells have been proposed to differentiate into myofibroblasts through epithelial or endothelial mesenchymal transition ( emt ) . although primary hepatocytes can undergo emt in vitro , it is extremely hard to detect hepatic myofibroblasts originating from epithelial or endothelial cells through emt in vivo . thus , the main sources of hepatic myofibroblasts in liver fibrosis are the hepatic resident mesenchymal cells , consisting of the hscs and portal fibroblasts . the most widely used and accessible marker of myofibroblasts are the de novo expression of -smooth muscle actin ( -sma ) . however , no reliable markers have yet been identified for distinguishing hscs from portal fibroblasts after myofibroblastic differentiation . the contribution of portal fibroblasts to hepatic fibrosis is not well understood mainly because of the difficulties in distinguishing and isolating them . development of liver fibrosis and cirrhosis is associated with deposition of ecm , in which collagen type i is the most abundant . transgenic colagen-1(i)-gfp mice have been generated a decade ago . in these mice expression of gfp is driven by collagen-1(i ) promoter , and therefore , expression of gfp is observed in cells that upregulate collagen type i. our current review will summarize the recent results obtained from transgenic reporter mice and novel flow cytometry protocols developed to distinguish hsc- and portal fibroblast - derived myofibroblasts and quantify their relative contributions to hepatic fibrosis . once hepatic epithelial cells ( hepatocytes and/or cholangiocytes ) are damaged by any cause , inflammatory mediators are released to initiate a series of responses to liver injury . inflammatory cells recruited to the site of injury phagocytose necrotic or apoptotic cells and amplify the inflammatory response by releasing pro - inflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-6 ( il-6 ) , and interleukin-1 beta ( il-1 ) , and by recruiting t cells . the hepatic mesenchymal precursor cells of myofibroblasts are activated and differentiated by growth factors and cytokines including transforming growth factor - beta ( tgf- ) , platelet - derived growth factor ( pdgf ) , and interleukin-13 ( il-13 ) . pdgf stimulates hsc proliferation through its positive feedback mechanism involved in the autocrine and paracrine effect . immune cells play a pivotal role in the development of hepatic fibrosis . in experimentally induced fibrosis , for example , c57bl/6 mice ( in which a th1 cell response predominates ) have a lower fibrotic reaction than balb / c mice ( in which a th2 cell response predominates ) . recently , increasing evidence has suggested an emerging novel role of t cell subsets , including th17 , treg , and t cells , in the fibrotic process . however , further studies are needed to fully elucidate their functions . hepatic fibrosis is a dynamic process and can be considered a part of the healing response to liver injury . the ecm is not stable , but is constantly synthesized and degraded by proteolytic enzymes such as the matrix metalloproteinases ( mmp ) or collagenases . the reversibility of mild to moderate hepatic fibrosis is now a reality in patients whose etiology has been successfully treated . in clinical practice , studies of antiviral treatments for hepatitis c have showed that fibrosis is reversible after a sustained virologic response . however , there is no unequivocal evidence for a complete reversal of severe cirrhosis with regenerative nodules and dense fibrotic septa . current clinical studies based on liver biopsies have showed that the matrix enzyme lysyl oxidase like-2 ( loxl2 ) increased in the fibrotic liver and was limited in the healthy liver . the extensiveness of the crosslinks observed in severe fibrosis and cirrhosis prevent their degradation by collagenases . hepatic myofibroblasts , characterized by expression of -sma and production of ecm , are mainly found in chronically injured livers , irrespective of the etiology , and are morphologically defined as large and spindle - shaped cells with cytoplasmic stress fibers running parallel to the long axis . myofibroblasts are characterized by several common features based on their ultrastructural analysis , including a prominent rough endoplasmic reticulum , a golgi apparatus producing collagen , peripheral myofilaments , well - developed cell - to - stroma attachment sites ( fibronexus ) , and gap junctions [ 17 , 18 ] . the process of myofibroblast differentiation leads to a highly proliferative , migratory , and contractile phenotype . myofibroblasts can release a number of pro - inflammatory molecules and directly contribute to this continuous inflammation [ 10 , 19 , 20 ] . in both experimental and clinical liver fibrosis , there is a close correlation between the regression of liver fibrosis and the disappearance of myofibroblasts . previous studies have demonstrated that some myofibroblasts undergo cell death by apoptosis , while other myofibroblasts are restored to their quiescent - like state [ 21 , 22 ] . this phenomenon has been identified recently , but has a great potential for anti - fibrotic therapy . however , the mechanism underlying inactivation of hsc / myofibroblasts in response to toxic liver injury remains unknown . future investigations are required to determine why a half of hsc / myofibroblasts apoptose during regression of liver fibrosis , while the other half of myofibroblasts survives and undergoes inactivation . identification of the mechanism of hsc / myofibroblast inactivation , may provide new targets for anti - fibrotic therapy . the two most common methods for modeling experimental liver fibrosis in mice are the administration of carbon tetrachloride ( ccl4 ) and bile duct ligation ( bdl ) . administration of ccl4 leads to centrilobular necrosis , and eventually leads to liver fibrosis and cirrhosis . ccl4 causes damage of hepatocytes , in which highly reactive free radical metabolites are formed by the mixed function oxidase system , including a cyp2e1-mediated reaction . fibrosis first develops in pericentral areas and secondarily between central and portal areas , which is called bridging fibrosis . the hepatic injury induced by bdl in mice is similar to the condition of human secondary biliary cirrhosis ; it is characterized by cholestasis , hepatic inflammation , neutrophil infiltration in the portal tracts , proliferation of cholangiocytes , and portal tract fibrosis . in bdl mice , serum bile acid levels increase by dozens of fold . bile acids are pro - oxidants directly causing tissue damage mediated by reactive oxygen species ( ros ) , or indirectly through activation of kupffer cells to release ros . the overspill of bile acid stimulates the proliferation of cholangiocytes , resulting in a ductular reaction accompanied by portal inflammation and fibrosis [ 25 ] . previous studies have showed the importance of portal fibroblasts as contributors to fibrosis in the bdl model . hscs are intralobular connective tissue cells representing less than ten percent of the total number of liver cells . under physiological conditions , hscs reside in the space of disse and serve as a major storage of vitamin a in the mammalian body . hscs also participate in the homeostasis of the intrahepatic ecm protein turnover by secreting the sufficient amount of ecm molecules required for tissue repair and by releasing mmp and their inhibitors . by virtue of the contractility of their long cytoplasmic processes encircling the sinusoid , the liver is the main storage organ for dietary vitamin a. vitamin a includes numerous retinoid forms such as retinyl esters , retinol , retinal , retinoic acid , and several provitamin a carotenoids . dietary retinoids are absorbed in the small intestine , where they are packaged into chylomicrons for transportation to the lymphatic circulation system . the retinoid - containing chylomicrons are taken up by hepatocytes , wherein retinoids are hydrolyzed to retinol , and bound retinol - binding protein ( rbp ) , to transfer to the hscs for storage . hscs are the central cellular site for retinoid storage in healthy animals , accounting for as much as 5060 % of the total retinoid present in the entire body . retinoids are stored in the form of retinyl esters in the lipid droplets , which are characteristic of hscs . in response to liver injury , quiescent hscs activate and release some of the vitamin a droplets . upon activation , hscs change their morphology , migrate to the site of injury , and upregulate mesenchymal markers such as -sma , collagen 1(i ) , and fibronectin . portal fibroblasts are resident fibroblasts with a spindle shape which are present in very small numbers in the mesenchyme surrounding the bile ducts . under normal conditions , portal fibroblasts almost certainly give rise to myofibroblasts during the development of cholestatic liver injury ( but not toxic liver injury , ) . in response to hepatic injury induced by bdl in mice , portal fibroblasts proliferate and are activate to produce ecm at the periphery of the bile ducts . portal fibroblasts can be distinguished from hscs due to the lack of oil droplets , including vitamin a. in addition , they express elastin and thy-1 ; elastin , fibulin 2 , gremlin 1 , and mesothelin ( a novel marker ) have also been identified as markers of portal fibroblasts [ 32 , 33 ] . thy1 is a t cell marker , which is particularly abundant on the surface of thymocytes and peripheral t cells . therefore , the question is , what are the specific markers for portal fibroblasts , and how portal fibroblasts can be distinguished from other myofibroblasts in fibrotic liver . in chronic cholestatic disorders , the fibrotic tissue is initially located around portal tracts . histological findings from fibrotic livers suggested that portal fibroblasts contribute to the overall fibroblasts observed in cholestatic liver injury . however , their role in liver fibrosis is still unclear because of the lack of markers that can definitively determine the presence of portal fibroblasts from the pool of hepatic myofibroblasts . this problem is further complicated by a recent report by asahina et al . , suggesting that portal fibroblats and hscs may originate from a common progenitor during the embryonic development . in recent years , manipulation of mouse genetics has been remarkably progressed and provided tools that have greatly facilitated the studies designed to dissect many biological processes in mammalian body , including liver fibrosis . thus , development of collagen-1(i)-gfp mice became one of the useful tools to study liver fibrosis [ 9 , 17 , 21 , 35 ] . our group has also utilized the collagen-1(i)-gfp transgenic mouse in which green fluorescent protein ( gfp ) is upregulated in hepatic myofibroblasts in response to fibrogenic liver injury . these mice can undergo chronic liver injury with repeated ccl4 injections or bdl to induce liver fibrosis , after which their collagen - producing cells express gfp , which is easily identified by its gfp fluorescence . the expression of collagen-1(i)-driven gfp in these mice closely correlates with the expression of -sma , a general marker for myofibroblasts . our strategy to detect hepatic myofibroblasts was based on the investigation of gfp - expressing cells in nonparenchymal fractions of ccl4-treated or bdl collagen-1(i)-gfp mice . the study of the cell fate mapping of hscs had demonstrated that although there is a decrease in the amount of vitamin a upon hsc activation , the vitamin a - specific autofluorescence excited with uv can be still detected in all hscs by flow cytometry [ 35 , 37 ] . whereas the gfp is expressed in all myofibroblasts , the presence of droplets containing of vitamin a is solely and exclusively attributed to hsc - derived myofibroblasts [ 35 , 37 , 38 ] . to distinguish hscs from hepatic myofibroblasts of other origins , the flow cytometry has been reported to be a method of choice to distinguish and quantify the contribution of hscs and portal fibroblasts to liver fibrosis induced by either ccl4 treatment or bdl . next , the presence of vitamin a was used to identify myofibroblasts originated from hscs , while all other gfp + vitamin a- myofibroblasts were attributed to myofibroblasts of all other origins . surprisingly , this gfp + vitamin a- fraction was composed mostly by thy1 and te-1 ( elastin ) positive cells , while cd45 + collagen-1(i)-gfp + fibrocytes [ 39 ] constituted only 4 % of total gfp + fraction . taken together , the flow cytometry - based quantification analysis of hepatic myofibroblasts activated in fibrotic liver in response to different types of liver injury ( toxic and cholestatic ) has demonstrated that hscs are the major source of myofibroblasts in ccl4-incuced liver fibrosis . however , portal fibroblasts are the major source of myofibroblasts at the onset of bdl - induced liver injury , within a week of bdl . the relative contribution of portal fibroblasts decreases upon chronic cholestatic injury , as hscs become progressively activated and contribute to the myofibroblast population . remarkably , the phenotype of bdl - activated hscs has more similarities with bdl - activated portal fibroblasts rather than with ccl4-activated hscs , suggesting that portal fibroblasts might affect ( or even regulate ) activation of hscs in bdl - injured liver . both of these cellular populations were isolated from mouse liver by flow cytometry and the gene expression profile was determined for gfp + vitamin a + and gfp + vitamin a- populations from ccl4- and bdl - injured mice . gene expression profiling and complimentary immunohistochemistry revealed that myofibroblasts derived from hscs are positive for desmin , and myofibroblasts derived from portal fibroblasts express thy1 , elastin , and mesothelin . mesothelin is a membrane glycoprotein that is expressed in normal mesothelial cells ; however , its function is not clear . in our study , mesothelin was highly expressed in myofibroblasts derived from portal fibroblasts , such that mesothelin may serve as a novel marker of portal fibroblasts . despite this finding , the function of mesothelin in mice or humans is not yet clear . in addition , recent studies have suggested that liver capsule ( which may also express mesothelial markers ) can contribute to hepatic myofibroblasts in response to fibrogenic liver injury [ 7 , 40 ] . at this time , it remains unclear if the mesothelin+ myofibroblasts represent heterogeneous population of hepatic mesenchymal cells that emerge in the damaged liver in response to chronic injury , or is comprised by the same cell type at different stages of activation . taken together , there might be two major sources of hepatic myofibroblasts in fibrotic liver [ 39 ] . 1 ) , but they exhibit unique properties , and can be distinguished from each other based on their gene expression profile . therefore , we emphasize that the composition of myofibroblasts varies depending on the etiology of the hepatic injury , and the origin of myofibroblasts may determine the personalized anti - fibrotic therapy of patients with liver fibrosis of different etiologies [ 35 , 41].fig . 1strategy to analysis myofibroblasts by flow cytometry : myofibroblasts expressing collagen-1(i)-driven gfp+ are identified in nonparenchymal fraction by argon laser at 488 nm wavelength and further fractionated to vitamin a+ and vitamin a cells by uv laser . hsc - derived myofibroblasts are sort - purified as the gfp+ and vitamin a+ fraction . portal fibroblast - derived myofibroblasts are sort - purified as the gfp+ and vitamin a fraction strategy to analysis myofibroblasts by flow cytometry : myofibroblasts expressing collagen-1(i)-driven gfp+ are identified in nonparenchymal fraction by argon laser at 488 nm wavelength and further fractionated to vitamin a+ and vitamin a cells by uv laser . hsc - derived myofibroblasts are sort - purified as the gfp+ and vitamin a+ fraction . portal fibroblast - derived myofibroblasts are sort - purified as the gfp+ and vitamin a fraction the contribution of portal fibroblasts to liver fibrosis has not been well characterized because of the difficulties in cell sorting - purification and the lack of identifiable and specific markers for portal fibroblasts . our novel flow cytometry method makes it possible to distinguish hsc- and portal fibroblast - derived myofibroblasts from the nonparenchymal cell fraction of the fibrotic liver in mice . it is also able to identify a novel specific marker , mesothelin , which is specific to portal fibroblasts . a detailed investigation of myofibroblasts , particularly using new methods such as ours , will provide insight into the mechanisms underlying liver fibrosis , and may lead to the development of more effective therapy .

the neural - cadherin ( cdh2 ) gene encodes the n - cadherin protein , a member of the cadherin gene family that mediates cell adhesion , differentiation , embryogenesis , and invasion1 . it has been described in a number of organs1,2 and malignancies derived from these3,4,5 . although the distribution of n - cadherin in the normal testis is known6 , there is no information regarding its presence in testicular germ cell and gonadal stromal tumors . the purpose of this study was to determine the presence and distribution of this protein in testis tumors compared to the distribution in the immature and mature testis . the testis of one 32-week - old fetus and orchiectomy specimens of 105 patients with germ cell tumors as well as 12 gonadal stromal tumors from the testicular tumor registry of the armed forces institute of pathology were used . the h&e stained sections of each patient were reviewed and 1 representative section per case was selected for this study . following deparaffinization , the sections were dehydrated and blocked in 0.6% hydrogen peroxide in methanol for 20 minutes . sections were processed for antigen retrieval in citrate buffer ( ph 6.0 ) for 25 minutes in a microwave followed by 25 minutes of cooling in room temperature . sections were then blocked in 1% horse serum for 40 minutes , followed by incubation with commercial monoclonal mouse anti - human n - cadherin clone 6g11 , isotype igg1 , kappa ( dako cytomation , inc . , carpinteria , ca ) as the primary antibody at a dilution of 1:120 for 60 minutes at room temperature . following primary antibody incubation , sections were incubated with the biotinylated horse anti - mouse antibody at a dilution of 1:200 ( vector burlingame ) for 30 minutes followed by treatment with the abc kit ( vector ) for 30 minutes . the color detection was achieved by treatment with vip ( vector ) for 5 minutes . positive reaction for n - cadherin was scored as membranous , cytoplasmic or cytoplasmic and membranous and recorded . the percentage of cells positive was scored : up to 25% , > 25%-50% , > 50%-75% , and > 75% . the staining intensity was scored as 1 + ( weak ) , 2 + ( moderate ) , or 3 + ( strong ) . the testis of one 32-week - old fetus and orchiectomy specimens of 105 patients with germ cell tumors as well as 12 gonadal stromal tumors from the testicular tumor registry of the armed forces institute of pathology were used . the h&e stained sections of each patient were reviewed and 1 representative section per case was selected for this study . following deparaffinization , the sections were dehydrated and blocked in 0.6% hydrogen peroxide in methanol for 20 minutes . sections were processed for antigen retrieval in citrate buffer ( ph 6.0 ) for 25 minutes in a microwave followed by 25 minutes of cooling in room temperature . sections were then blocked in 1% horse serum for 40 minutes , followed by incubation with commercial monoclonal mouse anti - human n - cadherin clone 6g11 , isotype igg1 , kappa ( dako cytomation , inc . , carpinteria , ca ) as the primary antibody at a dilution of 1:120 for 60 minutes at room temperature . following primary antibody incubation , sections were incubated with the biotinylated horse anti - mouse antibody at a dilution of 1:200 ( vector burlingame ) for 30 minutes followed by treatment with the abc kit ( vector ) for 30 minutes . the color detection was achieved by treatment with vip ( vector ) for 5 minutes . sections were counterstained in hematoxylin for 1 minute , dehydrated , cleared and mounted . positive reaction for n - cadherin the percentage of cells positive was scored : up to 25% , > 25%-50% , > 50%-75% , and > 75% . the staining intensity was scored as 1 + ( weak ) , 2 + ( moderate ) , or 3 + ( strong ) . the percentage of cells positive and intensity of staining for n - cadherin are seen in table 2 for tumors of one histological type and in table 3 for tumors of more than histological type . in the normal testis , n - cadherin was consistently seen in a membranous pattern in sertoli cells of the immature ( fig . 1a , 1b ) and mature testis ( fig . 2a , 2b ) , and served as a built - in positive control . it was also detected in a similar pattern in the rete ( fig . however , n - cadherin expression was limited to the proximal portion of the epididymis ( fig . the distal part of the epididymis and the vas were negative for n - cadherin as were normal stromal components including smooth muscle and endothelial cells . occasionally , leydig cells were diffusely positive . in sections that included the mesothelial surface , 1a , 1b ) and spermatogonia and spermatocytes of the adult testis is difficult to separate from the associated sertoli cells . the unclassified type of intratubular germ cell neoplasia also has membranous positivity with 2 + intensity ( fig . six of the 8 pure seminomas and 11 of 15 seminomatous components in mixed germ cell tumors were positive in a characteristic linear , zipper - like pattern restricted to areas of cell to cell adhesion referred to as a n - cadherin was observed in less than 50% of cells with 2 + intensity ( tables 2 and 3 ) . in the two spermatocytic seminomas , the n - cadherin was identified in a membranous pattern with moderate intensity in 25% to 50% of tumor cells ( fig . 6a , 6b ) and all but one of yolk sac tumor components in 76 mixed germ cell tumors ( fig . the staining was membranous and cytoplasmic and mainly 3 + intensity . in only 8 of 67 mixed tumors , foci of embryonal carcinoma were focally 1 to 2 + positive for n - cadherin ( fig . 2 were positive for n - cadherin , and in 57 mixed tumors with teratoma , 42 were positive for n - cadherin in neuroectodermal elements , glial cells , or glandular epithelium ( fig . the 3 cases with foci of choriocarcinoma were negative as were the syncytiotrophoblastic cells in 20 of the 24 cases . yolk sac tumor was by far the most consistently and strongly immunoreactive germ cell tumor type . four of the six sertoli cell tumors were positive with weak to moderate membranous staining ( fig.11a , 11b ) and one with leydig and sertoli cell differentiation in membranous and cytoplasmic pattern . the distribution of n - cadherin in the immature and mature testis in this study is similar to that reported by others2 , 6 . considering the origin of these tumors from the primordial germ cell8 , 9 , n - cadherin expression in the unclassified type of intratubular germ cell neoplasia and all of the basic germ cell tumor types of the adult is not surprising . the membranous , junctional expression pattern in seminoma reflects the close relationship between seminoma and both the intratubular unclassified type of germ cell neoplasia and the primordial germ cell . the spermatocytic seminoma stains in a pattern similar to the spermatocytes of the adult testis . the preferential staining of glandular and neuroectodermal teratomatous elements is consistent with the n - cadherin expression in tissues of neuroectodermal10 and endodermal origin2 . the embryonal carcinoma has features of an undifferentiated carcinoma and the n - cadherin expression is infrequent , less organized , and very limited . it is possible that the absence of n - cadherin in embryonal carcinoma reflects a lack of differentiation11 . in this study , the n - cadherin expression in 98.7% ( 82 of 83 ) of yolk sac tumor samples was the most consistent . all but one case , which contained a minute focus of yolk sac tumor , stained intensely positive for n - cadherin in the majority , highlighting the different histologic patterns . more than half of adult testicular germ cell tumors consist of more than one cell type . yolk sac tumor is present in approximately 40% of mixed germ cell tumors but may be difficult to recognize because it displays several microscopic patterns . in about 75% to 90% of cases , -fetoprotein can be detected in the serum or by positive staining in the tumor . the focal positivity can make it difficult to identify and differentiate the yolk sac tumor component . as such , additional markers of endodermal differentiation would be beneficial for the diagnosis and classification of these tumors . therefore , n - cadherin could be a useful additional marker for the detection of even minute foci of yolk sac tumor in the setting of a mixed germ cell tumor , which can be easily overlooked in h&e stained sections . compared to n - cadherin , afp is present in 92% of yolk sac tumors , only focally and less extensive . another excellent marker , helpful to identify minute yolk sac tumor elements , is glypican-3 ( gpc3)12 , 13 , 14 , 15 . sal - like protein 4 ( sall4 ) , a stem cell marker , is positive in all germ cell tumor types and is more sensitive in detecting yst than afp or gpc3 , similar to n - cadherin16,17 . unlike sall4 , the embryonal carcinomas were only rarely and focally positive for n - cadherin . the solid yst can resemble a seminoma in the h&e stained sections , but the characteristic linear positivity differs strikingly from the membranous and cytoplasmic pattern in ysts . in metastatic cases , there was one yolk sac tumor and one teratoma . both stained positive for n - cadherin with the yst staining > 75%/3 + and the teratoma staining 50%/2 + . in contrast to the strong membranous reactivity for n - cadherin in immature and mature sertoli cells , the reaction in sertoli cell tumors is generally membranous , but weak and heterogeneous . cdh2 expression in testicular tumors is not restricted to the germ cell tumor variants but is also observed in gonadal stromal tumors albeit more limited and less intense . the strong and consistent positive reaction for n - cadherin in yolk sac tumor was unexpected and potentially helpful . the opinions and assertions contained herein represent the personal views of the authors and are not to be construed as official or as representing the views of the department of the army , the department of defense , or the united states government .