a recent systematic analysis showed that in 2011 , 314 ( 296 - 331 ) million children younger than 5 years were mildly , moderately or severely stunted and 258 ( 240 - 274 ) million were mildly , moderately or severely underweight in the developing countries . in iran a study among 752 high school girls in sistan and baluchestan showed prevalence of 16.2% , 8.6% and 1.5% , for underweight , overweight and obesity , respectively . the prevalence of malnutrition among elementary school aged children in tehran varied from 6% to 16% . anthropometric study of elementary school students in shiraz revealed that 16% of them suffer from malnutrition and low body weight . snack should have 300 - 400 kcal energy and could provide 5 - 10 g of protein / day . nowadays , school nutrition programs are running as the national programs , world - wide . national school lunch program in the united states there are also some reports regarding school feeding programs in developing countries . in vietnam , school base program showed an improvement in nutrient intakes . in iran a national free food program ( nffp ) is implemented in elementary schools of deprived areas to cover all poor students . however , this program is not conducted in slums and poor areas of the big cities so many malnourished children with low socio - economic situation are not covered by nffp . although the rate of poverty in areas known as deprived is higher than other areas , many students in deprived areas are not actually poor and can afford food . hence , nutritional value of the nffp is lower than the scientific recommended snacks for this age group . furthermore , lack of variety of food packages has decreased the tendency of children toward nffp . on the other hand , the most important one is ministry of education ( moe ) of iran , which is responsible for selecting and providing the packages for targeted schools . the ministry of health ( moh ) is supervising the health situation of students and their health needs . welfare organizations , along with charities , have the indirect effect on nutritional status of students by financial support of their family . provincial governors have also the role of coordinating and supervising all activities of these organizations . parent - teacher association is a community - based institution that participates in school 's policy such as nffp . in addition to these organizations , nutritional literacy of students , their parents and teachers , is a very important issue , which could affect nutritional status of school age children . therefore , the present study was conducted with the aim of improving the nffp , so that by its resources all poor children will be covered even in big cities . moreover , all food packages were replaced by nutritious and diverse packages that were accessible for non - poor children . according to the aim of this study and multiple factors that could affect the problem , public health advocacy has been chosen as the best strategy to deal with this issue . therefore , the present study determines the effects of nutrition intervention in an advocacy process model on the prevalence of underweight in school aged children in the poor area of shiraz , iran . this interventional study has been carried out between 2009 and 2010 in shiraz , iran . this survey was approved by the research committee of shiraz university of medical sciences . in coordination with education organization of fars province two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . in those schools all students ( 2897 , 7 - 13 years old ) were screened based on their body mass index ( bmi ) by nutritionists . according to convenience method all students divided to two groups based on their economic situation ; family revenue and head of household 's job and nutrition situation ; the first group were poor and malnourished students and the other group were well nourished or well - off students . for this report , the children 's height and weight were entered into center for disease control and prevention ( cdc ) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards . the significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . for implementing the interventions , the advocacy process model weight was to the nearest 0.1 kg on a balance scale ( model # seca scale ) . standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer . advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders . the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts . the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) . accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children . after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . for educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support . healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron . low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions . the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean . each student that was malnourished and poor has been taken into account for free food and nutritious snacks . demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire . this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province . statistical analyses were performed using the statistical package for the social sciences ( spss ) software , version 17.0 ( spss inc . , the results are expressed as mean sd and proportions as appropriated . in order to determine the effective variables on the malnutrition status paired t test was used to compare the end values with baseline ones in each group . in this project , the who z - score cut - offs used were as follow : using bmi - for - age z - scores ; overweight : > + 1 sd , i.e. , z - score > 1 ( equivalent to bmi 25 kg / m ) , obesity : > + 2 sd ( equivalent to bmi 30 kg / m ) , thinness : < 2 sd and severe thinness : < 3 sd . this interventional study has been carried out between 2009 and 2010 in shiraz , iran . this survey was approved by the research committee of shiraz university of medical sciences . in coordination with education organization of fars province two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . in those schools all students ( 2897 , 7 - 13 years old ) were screened based on their body mass index ( bmi ) by nutritionists . according to convenience method all students divided to two groups based on their economic situation ; family revenue and head of household 's job and nutrition situation ; the first group were poor and malnourished students and the other group were well nourished or well - off students . for this report , the children 's height and weight were entered into center for disease control and prevention ( cdc ) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards . the significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . for implementing the interventions , weight was to the nearest 0.1 kg on a balance scale ( model # seca scale ) . standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer . advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders . the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts . the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . duration of intervention was 6 months . education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) . accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . obviously , student 's families had remarkable effect on children 's food habit . it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children . after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support . healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron . low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions . the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean . each student that was malnourished and poor has been taken into account for free food and nutritious snacks . demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire . this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province . advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders . the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts . the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) . accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . obviously , student 's families had remarkable effect on children 's food habit . it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children . after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support . healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron . low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions . the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean . each student that was malnourished and poor has been taken into account for free food and nutritious snacks . demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire . this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province . statistical analyses were performed using the statistical package for the social sciences ( spss ) software , version 17.0 ( spss inc . , chicago , il , usa ) . the results are expressed as mean sd and proportions as appropriated . in order to determine the effective variables on the malnutrition status paired t test was used to compare the end values with baseline ones in each group . two - sided p < 0.05 was considered to be statistically significant . in this project , the who z - score cut - offs used were as follow : using bmi - for - age z - scores ; overweight : > + 1 sd , i.e. , z - score > 1 ( equivalent to bmi 25 kg / m ) , obesity : > + 2 sd ( equivalent to bmi 30 kg / m ) , thinness : < 2 sd and severe thinness : < 3 sd . study population contains 2897 children ; 70.8% were primary school students and 29.2% were secondary school students . 2336 ( 80.5% ) out of total students were well - off and 561 children ( 19.5% ) were indigent . 19.5% of subjects were in case group ( n = 561 ) and 80.5% were in the control group ( n = 2336 ) . the mean of age in welfare group was 10.0 2.3 and 10.5 2.5 in non - welfare group . demographic characteristics of school aged children in shiraz , iran table 2 shows the frequency of subjects in different categories of bmi for age in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model in shiraz , iran . the frequency of subjects with bmi lower than < 2 sd decreased significantly after intervention among non - welfare girls ( p < 0.01 ) . however , there were no significant decreases in the frequency of subjects with bmi lower than < 2 sd boys . when we assess the effect of intervention in total population without separating by sex groups , we found no significant change in this population [ table 3 ] . bmi for age for iranian students aged 7 - 14 years based on gender according to who growth standards 2007 bmi for age for iranian students aged 7 - 14 years according to who growth standards 2007 in non - welfare and welfare groups of total population table 4 has shown the prevalence of normal bmi , mild , moderate and severe malnutrition in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model . according to this table there were no significant differences in the prevalence of mild , moderate and severe malnutrition among girls and boys . table 4 also shows the mean of all anthropometric indices changed significantly after intervention both among girls and boys . the pre- and post - test education assessment in both groups showed that the student 's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 ( p < 0.0001 ) . bmi , height and weight in non - welfare and welfare groups of school aged children separately in males and females before and after a nutrition intervention based on advocacy process model in shiraz , iran according to study 's finding the odds ratio ( or ) of sever thinness and thinness in non - welfare compared with welfare is 3.5 ( or = 3.5 , confidence interval [ ci ] = 2.5 - 3.9 , p < 0.001 ) . furthermore , the finding showed or of overweight and obesity in welfare compared to non - welfare is 19.3 ( or = 19.3 , ci = 2.5 - 3.9 , p = 0.04 ) . the result of this community intervention study revealed that nutrition intervention based on advocacy program had been successful to reduce the prevalence of underweight among poor girls . this study shows determinant factor of nutritional status of school age children was their socio - economic level . according to our knowledge , this is the first study , which determines the effect of a community intervention based on advocacy process on the malnutrition indices in a big city ( shiraz ) in iran . the other program in iran ( nffp ) is specified to deprived area and is not conducted in big cities . allocating millions of dollars to nffp by government , selecting the malnourished students through an active screening system at primary and middle schools , paying attention of policy makers to student 's nutrition have provided the opportunity to combat the problem . however , negligence of under - poverty line , providing poor snacks in terms of nutritional value and lack of variety are the main defects of this program . advocacy by definition is a blending of science , ethics and politics for comprehensive approaching health issues . by using advocacy program in california among the high school students for improving their nutrition and physical activity angeles unified school district participants emphasized on nutrition classes for families as well as students in addition to other interventions . in the present study another study revealed that evaluability assessment gave stakeholders the opportunity to reflect on the project and its implementation issues . it seems that in iran , free food program among the students not only is needed in deprived areas , but also it should be performed in big cities such as shiraz . at baseline , no significant difference was founded among wealthy students between the pre- and post - nutritional status intervention . in contrast , the numbers of students who have malnutrition decreased from 44% to 39.4% , which was identified as a significant among impecunious girls students . there was also a significant increase in the proportion of children with bmi that was normal for age ( 2 to + 1 sd ) most of the published community interventions showed better results among females compared with males . this difference in the impact of nutritional interventions between male and female might be related to the different age of puberty in the female population compared to the male population . in the age range of the present study female although , there is no nffp in big cities of iran , there are some programs for improving the nutritional status such as providing free milk in schools . a recent publication has shown that school feeding programs focus on milk supplementation had beneficial effects on the physical function and school performances specifically among girls in iran . the results of the mentioned study showed an improvement in the weight of children , psychological test 's scores and the grade - point average following this school feeding program . the intervention in the present study had focused on the snack intake in the school time . there are some reports regarding the nutrition transition in iran , which shows the importance of nutrition intervention to provide more healthy eating dietary habits among welfare groups of adolescents . hence , nutrition intervention especially in the form of nutrition education is needed in big cities and among welfare children and adolescents . although a study among iranian adolescents showed that dietary behavior of adolescents does not accord to their knowledge , which emphasize on the necessity of community intervention programs . a recent study regarding the major dietary pattern among iranian children showed the presence of four major dietary patterns , in which fast food pattern and sweet pattern as two major dietary patterns can be mentioned among iranian children . in advocacy program audience 's analysis accordingly , one of the prominent strategies in this study was working with media and was meeting with parent - teacher association that both of them were secondary target audiences . we also took into account policy makers in different levels , from national to local as primary audiences . advocacy team had several meetings with management and planning organization at national level and education organization of the fars province as well as principal of the targeted schools . providing nutritious snacks need contribution of private sector such as food industries or factories , but their benefits should be warranted . another choice was community involvement ; which can be achieved by female health volunteers who are working with the health system . advocacy team by using the support of charities and female health volunteers could establish a local factory that produced student 's snacks based on the new definition . however , there are some challenges on the way of expanding this program . mass production of the proposed snacks according to different desires and cultures and getting involvement of food industries with respect to marketing issues is one of those challenges . moreover , providing a supportive environment in order to change the food habits of the students and their parents among the wide range of the population require a sustainable and continuous inter - sector collaboration . although in a limited number of schools , in our study , interventions and advocacy program was successful , expanding this model to another areas around the country depends on convincing the policy makers at national level . in this regard , advocacy team should prepare evidenced based profile and transitional planning to convince the policy makers for improving the rule and regulation of nffp . the same as this study in other studies have also emphasized that there must be efforts to strengthen the capacity within the schools to deal with the nutritional problems either overweight , obesity or malnutrition by using of educational and nutritional intervention . assessing the dietary adherence is very important in nutrition intervention among population . as this population was children and adolescents we had a limitation in the blood sample collection to assess the subject 's dietary adherence . furthermore , this intervention was only focused on the intake of snack in school time and we did not have comprehensive information on the dietary intake of children and adolescents after school all over the day . the investigators propose further investigation in different areas of the country based on socio - cultural differences in order to make necessary modification and adapt this model to other areas . regarding the nutritional needs of the school age children , provision of a good platform for implementing and expanding this efficient model to the whole country based upon the socio - economic situation of each region is advisable to the moh and the moe . community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children .

background : the present study was carried out to assess the effects of community nutrition intervention based on advocacy approach on malnutrition status among school - aged children in shiraz , iran.materials and methods : this case - control nutritional intervention has been done between 2008 and 2009 on 2897 primary and secondary school boys and girls ( 7 - 13 years old ) based on advocacy approach in shiraz , iran . the project provided nutritious snacks in public schools over a 2-year period along with advocacy oriented actions in order to implement and promote nutritional intervention . for evaluation of effectiveness of the intervention growth monitoring indices of pre- and post - intervention were statistically compared.results:the frequency of subjects with body mass index lower than 5% decreased significantly after intervention among girls ( p = 0.02 ) . however , there were no significant changes among boys or total population . the mean of all anthropometric indices changed significantly after intervention both among girls and boys as well as in total population . the pre- and post - test education assessment in both groups showed that the student 's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 ( p < 0.0001).conclusion : this study demonstrates the potential success and scalability of school feeding programs in iran . community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children .

INTRODUCTION MATERIALS AND METHODS Participants Instruments Procedure First step Second step Third step Forth step Interventions Fifth step (assessment) Data analysis RESULTS DISCUSSION CONCLUSION

it occurs in more than 50% of patients and may reach 90% in certain types of cancers , especially in patients undergoing chemotherapy and/or radiation therapy.1 anemia is defined as an inadequate circulating level of hemoglobin ( hb ) ( hb < 12 g / dl ) and may arise as a result of the underlying disease , bleeding , poor nutrition , chemotherapy , or radiation therapy . preliminary studies suggest that survival and loco - regional control after radiation therapy , especially in head and neck cancers , may be compromised by anemia.24 anemia often worsens symptoms such as fatigue , weakness , and dyspnea , and thus may have a negative effect on quality of life ( qol ) and performance status in patients with cancer . thus , to improve physical functioning , qol , and prognosis in patients with cancer , it would be reasonable to take a proactive approach in identifying populations who need treatment for cancer - associated anemia ( caa ) and provide timely management . blood transfusion is an effective way to replace depleted hb within a short period , but the effect is , unfortunately , temporary and can cause serious adverse risks and increased mortality . in randomized clinical trials in patients with caa , erythropoiesis - stimulating agents ( esas ) produced significant increases in hb level , decreased transfusion requirements , and improved qol.57 however , 30%50% of patients do not respond to such agents . in addition , the use of esas often causes concern about severe adverse reactions.6,8 in several studies , esas were found to shorten overall survival time , or time to tumor progression in patients whose hb level reached more than 12 g / dl . these studies included patients with different primary cancers , such as breast , lung , head and neck , cervix , and lymphomas.911 the lack of response to erythropoietin stimulation in patients with cancer is partly attributed to the functional iron deficiency state , in which the high rate of erythropoiesis exceeds the delivery of usable iron , despite adequate iron stores.12 absolute iron deficiency , in contrast , occurs when iron delivery is impaired because iron stores are depleted ( serum ferritin , < 100 ng / ml ; transferring saturation , < 20%).13 hepcidin , a peptide hormone produced by the liver , is up - regulated in chronic inflammatory states including cancer . hepcidin inhibits iron transport across cell membranes , thus decreasing the accessibility of stored iron and gastrointestinal absorption of dietary iron , leading to an increased frequency of iron - restricted erythropoiesis.1416 many randomized trials examined the role of intravenous ( iv ) iron in addition to esas in the treatment of anemia in patients with cancer . many of these studies showed improvement in esa response , time to maximal response , reduction in esa dose , and improvement in qol parameters ( when measured ) in favor of the combination over esas alone . the observed benefit was independent of baseline iron parameters.1721 one study found a 36% reduction in the number of patients transfused.21 this pilot study assessed the efficacy and feasibility of iv iron monotherapy in patients with cancer who have anemia and who are undergoing treatment with chemotherapy and/or radiation therapy without the use of esas . patients received the study treatment for 12 weeks followed by a 4-week follow - up period . eligible patients were at least 18 years old , about to start a cycle of chemotherapy and/or radiation therapy within 1 week of inclusion , and had a nonmyeloid malignancy , hb levels of 11.0 g / dl or less , a life expectancy of more than 24 weeks , and an eastern cooperative oncology group performance status of 02 . patients were also required to have a serum ferritin level of 100 ng / ml or higher or transferrin saturation ( tsat ) levels of 15% or higher and to have received no esas or iv iron therapy within 30 days and no oral iron therapy ( 27 mg / day or more ) within 7 days before enrollment . patients were excluded for leukoerythroblastic features on blood film , hemolysis , gastrointestinal bleeding , folate or vitamin b12 deficiency , elevated serum ferritin ( 900 ng / ml ) or transferrin saturation ( tsat ) ( 35% ) levels , pregnancy or lactation , liver dysfunction ( grade 2 or higher based on national cancer institute common toxicity criteria ) , renal dysfunction ( serum creatinine levels 2.0 mg / dl ) , active infection requiring systemic antibiotics , personal or family history of hemochromatosis , comorbidities precluding study participation , hypersensitivity to iv iron , red blood cell transfusion within the last 2 weeks , or any investigational agent within 30 days before enrollment . patients were not allowed to take any vitamin , mineral , or herbal supplements containing 27 mg or more of iron per day or 100 mg vitamin c per day during the study or follow - up period . blood transfusions were permitted at the primary physician s discretion if hb levels decreased to 8 g / dl or less , and such patients were considered treatment failures . written informed consent was provided by all patients before study participation , and the protocol and supporting documents were approved by the institutional review board of king hussein cancer center . the study was conducted in accordance with the declaration of helsinki and good clinical practice as contained in the us code of federal regulations that governs the protection of human subjects and the obligations of clinical investigators . patients received 200 mg ferric hydroxide sucrose diluted in 100 ml normal saline and infused over the course of 1 hour weekly for a total of 12 weeks . the first dose was given during the first clinic visit ( 4 days from the initiation of chemotherapy or radiation therapy ) . tsat was monitored , as protocol mandated withholding iron therapy when tsat levels were higher than 50% . at the first clinic visit ( week 1 ; baseline ) , a blood sample was obtained for laboratory assessments before the study treatment was started . patients attended weekly clinic visits for treatment and assessment ; and returned for follow - up visits at week 14 which included a complete physical examination . complete blood count and tsat were done every 3 weeks , and again 2 weeks after last treatment ( week 14 ) . complete laboratory assessment ( hb , serum ferritin , reticulocyte count , transferrin , tsat , serum iron , total iron binding capacity , red cell indices , white blood cell count with differential , platelet count , and serum chemistries ) were done at week 1 and at week 14 ( end of study ) . adverse events were assessed at each clinic visit until study completion or withdrawal , and during the 30 days after the last study treatment . hb test results were presented as mean , median , and range through all 12 weeks . comparison between means of hb level were made between the baseline hb and hb levels in the following weeks , using t - test . a significance criterion of p < 0.05 was used in the analysis . all analyses were performed using sas version 9.1 ( sas institute inc , cary , nc , usa ) . patients received the study treatment for 12 weeks followed by a 4-week follow - up period . eligible patients were at least 18 years old , about to start a cycle of chemotherapy and/or radiation therapy within 1 week of inclusion , and had a nonmyeloid malignancy , hb levels of 11.0 g / dl or less , a life expectancy of more than 24 weeks , and an eastern cooperative oncology group performance status of 02 . patients were also required to have a serum ferritin level of 100 ng / ml or higher or transferrin saturation ( tsat ) levels of 15% or higher and to have received no esas or iv iron therapy within 30 days and no oral iron therapy ( 27 mg / day or more ) within 7 days before enrollment . patients were excluded for leukoerythroblastic features on blood film , hemolysis , gastrointestinal bleeding , folate or vitamin b12 deficiency , elevated serum ferritin ( 900 ng / ml ) or transferrin saturation ( tsat ) ( 35% ) levels , pregnancy or lactation , liver dysfunction ( grade 2 or higher based on national cancer institute common toxicity criteria ) , renal dysfunction ( serum creatinine levels 2.0 mg / dl ) , active infection requiring systemic antibiotics , personal or family history of hemochromatosis , comorbidities precluding study participation , hypersensitivity to iv iron , red blood cell transfusion within the last 2 weeks , or any investigational agent within 30 days before enrollment . patients were not allowed to take any vitamin , mineral , or herbal supplements containing 27 mg or more of iron per day or 100 mg vitamin c per day during the study or follow - up period . blood transfusions were permitted at the primary physician s discretion if hb levels decreased to 8 g / dl or less , and such patients were considered treatment failures . written informed consent was provided by all patients before study participation , and the protocol and supporting documents were approved by the institutional review board of king hussein cancer center . the study was conducted in accordance with the declaration of helsinki and good clinical practice as contained in the us code of federal regulations that governs the protection of human subjects and the obligations of clinical investigators . patients received 200 mg ferric hydroxide sucrose diluted in 100 ml normal saline and infused over the course of 1 hour weekly for a total of 12 weeks . the first dose was given during the first clinic visit ( 4 days from the initiation of chemotherapy or radiation therapy ) . tsat was monitored , as protocol mandated withholding iron therapy when tsat levels were higher than 50% . at the first clinic visit ( week 1 ; baseline ) , a blood sample was obtained for laboratory assessments before the study treatment was started . patients attended weekly clinic visits for treatment and assessment ; and returned for follow - up visits at week 14 which included a complete physical examination . complete blood count and tsat were done every 3 weeks , and again 2 weeks after last treatment ( week 14 ) . complete laboratory assessment ( hb , serum ferritin , reticulocyte count , transferrin , tsat , serum iron , total iron binding capacity , red cell indices , white blood cell count with differential , platelet count , and serum chemistries ) were done at week 1 and at week 14 ( end of study ) . adverse events were assessed at each clinic visit until study completion or withdrawal , and during the 30 days after the last study treatment . hb test results were presented as mean , median , and range through all 12 weeks . comparison between means of hb level were made between the baseline hb and hb levels in the following weeks , using t - test . a significance criterion of p < 0.05 was used in the analysis . all analyses were performed using sas version 9.1 ( sas institute inc , cary , nc , usa ) . twenty - five patients ( 17 women and 8 men ) were eligible , consented , and included in the study ; their mean age ( standard deviation , [ sd ] ) was 56 years ( 13.0 years ) . chemotherapy varied according to the primary cancer and included anthracycline , platinum , taxanes , cyclophosphamide , high - dose ifosfamide , vincristine , vinblastine , bleomycin , and others . many of the included patients had their chemotherapy treatment as second- or third - line therapy . patients characteristics , including age , primary tumor , and active anticancer treatment are summarized in table 1 . one patient died during the study from his tumor ( after week 2 ) , and five patients withdrew from the study because of inconvenience ( three after week 3 , and two after week 4 ) . nineteen ( 76.0% ) patients completed a minimum of three treatments , 15 ( 60.0% ) completed nine treatments , and 14 ( 56.0% ) completed all twelve planned weekly treatments . as seen in table 2 , the mean hb level of the 25 patients at baseline was 9.6 g / dl ( median , 9.9 g / dl ; range , 6.9 g / dl10.9 g / dl ) . for the 15 patients who completed at least nine treatments , the mean change in their hb level was 1.7 g / dl ( median , 1.1 g / dl ; range , 1.9 g / dl to 3.2 g / dl ) . for the 14 patients who completed the whole treatment period ( 12 weeks ) , the mean hb level change was 2.1 g / dl ( median , 1.3 g / dl ; range , 0.2 g / dl to 4.6 g / dl ; p = 0.0007 ) . eight ( 42.1% ) of the 19 patients who completed at least three iron infusions had a more than 1 g / dl increase in their hb level . hemoglobin level changes for the 14 patients who completed twelve iron infusions are shown in figure 1 . no iv iron - related adverse events were reported among patients during the study or the follow - up period . tsat was monitored during the study period , and no patients had tsat levels increase to more than 50% . the highest ferritin level among patients who completed at least nine iv iron treatments was 1,170 ng / ml ; the mean level at the end of study period for the whole group was 379 ng / ml . five ( 20.0% ) patients received blood transfusions and were considered treatment failures ( three after week 3 , transfused at hb levels of 6.9 g / dl , 7.8 g / dl , and 5.4 g / dl ; one after week 4 , transfused at an hb level of 8.2 g / dl ; and one after week 9 , transfused at an hb level of 7.2 g / dl ) . low hb levels are associated with diminished qol and possibly decreased overall survival.2 successful treatment of anemia has undeniable benefits for patients , often yielding dramatic symptomatic improvement . although the role of esas is well - established in treating caa , big concerns were recently raised about the negative effect of esas on survival in some patients with cancer.911 concerns about the risk for thromboembolism in patients with cancer with higher hb levels who are receiving esa were also addressed in many trials.22,23 in addition , the possible immunosuppressive effects of blood product transfusions that may have relevance to neoplasia progression were addressed before.24 , 25 in our pilot study , we tested the feasibility of using iron supplementation alone to treat anemia in patients with cancer who are undergoing chemotherapy without the use of esas or blood transfusion , which could be a valid alternative , especially for patients with curable cancers . oral iron is easier to administer and relatively inexpensive , but low patient adherence , poor enteral absorption , and poor tolerance because of a wide range of troublesome gastrointestinal adverse effects limit its overall effectiveness.26 anemia of chronic disease may occur in patients with cancer and is associated with an increase in hepcidin levels , which decreases oral iron absorption and bone marrow iron use , negating any possible effect of regular doses of oral iron.15 iv iron therapy significantly improves response to epoetin alfa when compared with oral iron or no iron in anemic patients with cancer who are receiving chemotherapy.1721 oral iron supplements with esas showed no significant benefit over esas alone in treating caa.21 sodium ferric gluconate and iron sucrose appear to have more favorable safety profiles over iron dextran . a large prospective safety comparison trial failed to show serious anaphylactoid reactions,27 which is confirmed in our study , in which no patients developed reactions and no patients withdrew from the study because of adverse effects . given that the mean hb increase using esas with iv iron in one large controlled trial was 2.4 g / dl,21 the results obtained in our study are clinically significant . these findings should be further confirmed and better assessed in larger studies , in which questions such as the optimal timing of iv iron therapy with respect to chemotherapy and the optimal total dose of iv iron should be determined . the use of iv iron monotherapy was recently reviewed by a group in germany that studied the use of ferric carboxymaltose to replace esa and blood transfusions as a treatment for caa . iron - deficient patients treated with ferric carboxymaltose alone ( n = 233 ) had a median of 1.4 g / dl increase in hemoglobin levels compared with those receiving additional treatment with esas ( n = 46 ; median , 1.6 g / dl ) . our study , however , is peculiar in using iron therapy in a non - iron - deficiency state.28 iron overload after iv iron therapy , with potential concerns about the risk of developing secondary cancers and infection , might be raised . the highest serum ferritin level in the present study in patients who completed at least 9 weeks of iv iron therapy was 1,170 ng / ml . most of the literature addressing cancer and infections in iron - overloaded patients comes from patients with hemochromatosis or patients who are undergoing hemodialysis . published reviews report an increase in hepatocellular carcinoma only in patients with hemochromatosis after they develop cirrhosis.29 similarly data supporting the association between iv iron therapy and higher infection rate are weak and not well - supported.30 in fact , anemia itself is a risk factor for infections in patients receiving hemodialysis.31 a multivariate analysis of associations between iron and mortality in more than 58,000 patients receiving hemodialysis reported no increased death rate from serum ferritin levels as high as 1,200 ng / ml.30 the increasing cost of therapy in patients with cancer is of grave concern , which could be an additional benefit of iv iron over the use of esas in such patients . to further address many of the questions raised , our team is planning a bigger trial for iv iron in patients with cancer who have anemia to confirm the results discussed in this pilot trial . in addition , we will be looking into predictors of response to iv iron , such as serum hepcidin level . iv iron therapy alone is safe and may be effective in improving hb levels in patients with cancer who are undergoing active anticancer therapy . further randomized trials are needed to address many of the questions raised in our pilot study .

backgroundanemia in patients with cancer who are undergoing active therapy is commonly encountered and may worsen quality of life in these patients . the effect of blood transfusion is often temporary and may be associated with serious adverse events . erythropoiesis - stimulating agents are not effective in 30%50% of patients and may have a negative effect on overall survival.aimsto assess the efficacy and feasibility of intravenous iron therapy in patients with cancer who have non - iron - deficiency anemia and who are undergoing treatment with chemotherapy without the use of erythropoiesis - stimulating agents.methodsadult patients with solid cancers and non - iron - deficiency anemia were included . ferric sucrose at a dose of 200 mg was given in short intravenous infusions weekly for a total of 12 weeks . hemoglobin level was measured at baseline , every 3 weeks , and 2 weeks after the last iron infusion ( week 14 ) . adverse events related to intravenous iron were prospectively reported.resultsof 25 patients included , 19 ( 76.0% ) completed at least three iron infusions and 14 ( 56.0% ) finished the planned 12 weeks of therapy . the mean hemoglobin level of the 25 patients at baseline was 9.6 g / dl ( median , 9.9 g / dl ; range , 6.9 g / dl 10.9 g / dl ) . the mean change in hemoglobin level for the 15 patients who completed at least 9 treatments was 1.7 g / dl ( median , 1.1 g / dl ; range , 1.9 g / dl to 3.2 g / dl ) ; it reached 2.1 g / dl ( median , 1.3 g / dl ; range , 0.2 g / dl to 4.6 g / dl ; p = 0.0007 ) for the 14 patients who completed all 12 weekly treatments . five ( 20.0% ) patients were transfused and considered as treatment failures . no treatment - related adverse events were reported.conclusionintravenous iron treatment alone is safe and may reduce blood transfusion requirements and improve hemoglobin level in patients with cancer who are undergoing anticancer therapy . further randomized studies are needed to confirm these findings .

Introduction Patients and methods Study design Treatment Assessment Statistical analysis Results Discussion Conclusion

tardive dystonia ( td ) , a rarer side effect after longer exposure to antipsychotics , is characterized by local or general , sustained , involuntary contraction of a muscle or muscle group , with twisting movements , generally slow , which may affect the limbs , trunk , neck , or face . td has been shown to develop in about 3% of patients who have had long - term exposure to antipsychotics . . the low risk of td for atypical antipsychotics is thought to result from their weak affinity for dopamine receptors . compared with typical , atypical antipsychotic agents have a greater affinity for serotonin 5-ht2a than dopamine d2 receptors , with a low propensity to induce td . among this olanzapine is thought to have preferential action at mesolimbic over nigrostriatal dopaminergic pathways and is , therefore , associated with a very low incidence of extrapyramidal symptom ( eps ) . furthermore , a retrospective analysis of controlled multicentric trials suggested that olanzapine also improves preexisting symptoms of tardive movements . we report a case of 20-year - old male , belonging to lower socioeconomic class , educated up to 2 standard , presented with severe unilateral dystonic left sided neck movements [ figure 1 ] . careful history exploration revealed he was taking risperidone 2 mg irregularly for 2 months and then olanzapine 5 mg for another 4 months . picture of neck dystonia of patient at 19 years , the patient presented with occasional anger outbursts , getting provoked on small matters and beating family members , running away from home , screaming episodes occasionally , fearfulness , sleep disturbance for 2 days ; which was precipitated after fever . according to the mother , one friend might have threatened / made fun of him actually and after that patient stopped going out of house , and displayed above mentioned symptoms . this was interpreted as psychosis with persecutory ideas , and he was treated with risperidone 2 mg / day for 2 months and then with olanzapine 5 mg / day for 4 months . in last two follow - ups patient did not present himself , and mother reported unusual neck movements , which were taken as a part of his overall psychopathology and not taken seriously , slight intermittent neck movements reported were missed as part of adolescent behavior problems mimicking some hero in movies . as neck dystonia increased , the patient had a severe disability as patient had to keep his hands behind his head for the support . the movement would decrease when the patient was lying down and was absent during sleep . he even stopped taking food due to severe neck movements making chewing and swallowing difficult . his birth and early developmental milestones were normal . during 210 years of age patient was inattentive and mildly hyperactive . other siblings were educated with master 's degree , and patient was also sent to school , but due to inattention and restlessness , he did not pass 2 standard after three attempts . he left the schooling . with average executive functioning and life skills , he worked as an unskilled laborer in the neighborhood shops as a helping hand . , he was found to be getting over familiar , cheerful , moody , and short tempered . sometimes , the patient had inappropriate social judgment ; for which his friends made fun of him , and teased him . on mental status assessment , routine investigations , thyroid function tests , electroencephalogram , fundus examination , cervical x - ray , magnetic resonance imaging brain were normal . after consulting neurophysician , wilson 's disease and other secondary causes of dystonia were ruled out . the patient was treated with clonazepam 1 mg total dissolved solid ( tds ) , tetrabenazine 25 mg tds , trihexiphenidyl 2 mg bipolar disorder ( bd ) . after 2 months , there was some improvement of around 30% . baclofen 10 mg was added ; increased up to 20 mg , trihexyphenidyl reduced to 2 mg . with little improvement after 4 months of treatment for dystonia , levodopa + carbidopa ( 100 + 25 ) was added by neurophysician and increased up to tablet tds and baclofen omitted . after 12 months of treatment , patient has improved around 90% with tetrabenazine 75 mg , levodopa + carbidopa ( 100 + 25 ) - tablet bd , and clonazepam 1 mg bd . earlier case reports reported td developing with high - dose atypical antipsychotics such as olanzapine 20 mg or aripiprazole 15 mg with longer duration of exposure of around 1215 months in established psychiatric illness like schizophrenia or any other psychotic illness . eps in general and tardive dyskinesia , in particular , have been extensively studied in schizophrenia . even though a number of studies suggest that bipolar patients experience higher rates of eps ( parkinsonism , dystonia , akathisia ) and td compared to patients with a diagnosis of schizophrenia , research within the bd population has been limited . the risk is found to be 3 to 5 times higher in elderly patients compared to young patients . in addition to age , the risk is directly proportional to : female gender , daily and total dose of the antipsychotic drug , presence of mood disorder , the use of anticholinergics with neuroleptics , previous physical therapies ( electroconvulsive therapy ) , the presence of other physical illness such as diabetes or an organic disorder , younger age of exposure , and the presence of extrapyramidal symptoms early in treatment . this patient 's severe dystonic neck movements developed within short period of 6 months of exposure to atypical antipsychotics risperidone 2 mg and then olanzapine 5 mg only , which can cause minimal extrapyramidal side effects . in this case , risk factors for developing serious disabling td were neuroleptic exposure , borderline intellectual functioning , externalizing behavior , probable misdiagnosis , and overlooking early indicators of side effects . this case highlights dangers of casually prescribing low dose second generation antipsychotics in patient with hyperthymic temperament and borderline intellectual functioning with vague short lasting presenting complaints ; probably misdiagnosed as psychosis ; leading to such severe adverse effects because patients with organic brain damage are more prone to develop adverse effects like td . thus , judicious use of antipsychotics , with detailed and frequent assessments is important , and emergent stereotyped behavior or unexplained movements must be examined carefully and taken seriously . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

tardive dystonia ( td ) is a serious side effect of antipsychotic medications , more with typical antipsychotics , that is potentially irreversible in affected patients . studies show that newer atypical antipsychotics have a lower risk of td . as a result , many clinicians may have developed a false sense of security when prescribing these medications . we report a case of 20-year - old male with hyperthymic temperament and borderline intellectual functioning , who developed severe td after low dose short duration exposure to atypical antipsychotic risperidone and then olanzapine . the goal of this paper is to alert the reader to be judicious and cautious before using casual low dose second generation antipsychotics in patient with no core psychotic features , hyperthymic temperament , or borderline intellectual functioning suggestive of organic brain damage , who are more prone to develop adverse effects such as td and monitor the onset of td in patients taking atypical antipsychotics .

INTRODUCTION CASE REPORT DISCUSSION Declaration of patient consent Financial support and sponsorship Conflicts of interest

lepidoptera include agricultural pests that , through feeding and other activities , negatively affect stored grains , food and fiber crops [ 2 , 3 ] . since a single lepidoptera adult can produce hundreds of eggs , and their primary food source is typically plant material , they can cause significant damage to agricultural crops . although biological agents can help manage these insect pests , insecticides currently are essential for large - scale effective and economical pest control . these insecticides can also affect non - target organisms , including pollinators , and their application not only disrupts natural ecosystems but also reduces yields of crops that rely on pollination [ 5 , 6 ] . the non - target effect of some pesticides is in part due to their effects on insect immunity , which is necessary for insect survival in natural environments . for example , currently used pesticides have been shown to affect cellular [ 710 ] and humoral [ 11 , 12 ] immune responses and interfere with grooming behavior [ 13 , 14 ] . these effects on immunity are likely non - specific and negatively impact the health of both the target pest and beneficial arthropods . therefore , there is a need for novel target - specific approaches to control insect pests without affecting beneficial arthropods . although immune pathways can be generally and non - specifically inhibited by pesticides , they also are a likely source of candidate molecules that could be inhibited for target - specific insect control since multiple classes of insect immunity genes , including signaling pathways , can be under strong selection for diversification . fundamental mechanisms of innate immunity comprising cellular and humoral pathways are conserved throughout the animal kingdom and are controlled by signaling pathways activated by various stimuli [ 17 , 18 ] , including pathogen recognition by immune surveillance systems . despite this overall conservation , aspects of immune systems are subject to strong selection to evolve in response to varying pathogen exposure and to pathogen evolution of virulence determinants that modulate immunity [ 15 , 1921 ] . such co - evolutionary dynamics can promote diversification of conserved elements of immunity as well as the recruitment of novel effectors . as such , the investigation of insect immune pathways and mechanisms of pathogen modulation can yield insights into components that may be susceptible to inhibition . for example , the insect pathogen xenorhabdus nematophila suppresses cellular and humoral immunity in the lepidopteran moths manduca sexta and spodoptera exigua [ 23 , 24 ] but not in the dipteran fly drosophila melanogaster , suggesting the stage of immunity suppressed by x. nematophila may be absent from d. melanogaster , but present in lepidoptera . since dipteran flies serve as pollinators [ 26 , 27 ] , decomposers , food sources for other animals , and pest control agents , capitalizing on the possible differences between dipteran and lepidopteran immune signaling cascades will help in the identification of targets for pest - specific inhibition . with this knowledge in hand , pest management can be achieved by developing small molecule inhibitors of these targets that will suppress pest insect immunity and lead to increased susceptibility to environmental pathogens . indeed , many insecticides may contribute to insect ( target and non - target ) death by modulating aspects of immunity . the feasibility of targeted pest control via insect immune inhibition has been established for termites ; a small molecule inhibitor of an immune surveillance protein led to faster termite death upon exposure to various pathogens . much of our current knowledge of insect immune signaling pathways and receptor and effector function is based on the premiere model organism d. melanogaster , for which there are extensive genetic tools and several fully sequenced genomes . well - established lepidopteran insect models such as the silkworm bombyx mori and the tobacco hornworm m. sexta also have been widely used to study insect immunity . these organisms have been particularly useful for investigating hemolymph proteins and hemocyte function because of their relatively large larval size and hemolymph volume . many insects in the order lepidoptera are easy to rear in laboratory conditions , and new tools such as rna interference have been implemented successfully to study genetics of their immune systems [ 30 , 31 ] . also , their immune signaling pathways are gradually being revealed by genomic and transcriptomic data [ 3238 ] . based on these model insect systems a fairly detailed picture of immunity , from pathogen detection to effector function , is emerging , though many gaps remain , particularly with regard to components that are unique to different insect orders . here we review aspects of insect immunity with an emphasis on the similarities and distinctions between d. melanogaster and representative lepidoptera . in insects , the cellular immune response includes phagocytosis , nodulation and encapsulation [ 3942 ] and the humoral response involves the expression of antimicrobial peptides ( amps ) [ 43 , 44 ] as well as the pro - phenol oxidase ( propo ) proteolytic cascade that results in formation of melanized nodules and toxic reactive compounds [ 45 , 46 ] . amps are small cationic peptides that insert into and disrupt microbial membranes , thereby killing and clearing pathogens . they are synthesized by hemocytes and to a greater extent in fat body from which they are released into the insect hemolymph rapidly after microbial infection [ 43 , 47 ] . amps are also expressed in extra - embryonic tissues of eggs , which may help protect the developing embryo from infection . amps are a conserved component of immunity in plants and animals and while they have diverse structures most can be assigned to larger families such as cecropins , attacins , defensins and diptericins . their diversity and immune effector function as well as their variant representation among insects ( table 1 ) have made them a central focus in the study of invertebrate pathology [ 30 , 52 ] . in d. melanogaster amp synthesis each of these pathways is activated by detection of microbial components via different pattern recognition receptors ( prrs ) that trigger , through complex regulatory cascades , nuclear factor kappa b ( nf-b ) dependent transcription of the genes encoding amps . after amps are translated in the cytoplasm they are released into the hemolymph where their high concentrations and broad activity are thought to enhance clearance of invading microorganisms from the insect . bioinformatic and experimental data support the existence of the amp - inducing toll and imd pathways in lepidopterans , though not all components have been identified in model organisms such as m. sexta [ 32 , 35 ] . the conserved presence of amps in immunity coupled with the possibility that certain elements of their induction pathways may vary among insects enhances the probability that microbially - induced amp expression could be inhibited in a pest - specific manner . as such , for the remainder of this review we focus on pathways leading to amp gene expression . in d. melanogaster , transcription of amp - encoding genes is activated by the nf-b family transcription factors in response to infection [ 6569 ] with distinct nf-b family transcription factors responding to the toll and immune deficiency ( imd ) signal transduction pathways . in response to toll pathway activation , the nf-b inhibitor cactus is phosphorylated and degraded allowing its targets , the nf-b factors dif and dorsal , to be translocated to the nucleus . imd pathway activity culminates in the nf-b factor relish being activated by a stimulus - induced proteolytic cleavage . in the case of dif and dorsal , gram - positive bacterial and fungal infections primarily serve as the stimuli that induce degradation of cactus through the toll signaling pathway . in general , gram - negative bacterial infections of d. melanogaster stimulate the proteolytic cleavage of relish through the imd pathway . once in the nucleus , these transcription factors drive the transcription of immune effectors , including amp genes whose promoters contain nf-b binding sites [ 70 , 72 ] . overall , nf-b proteins and their dna - binding specificities are conserved among organisms , including those lepidoptera studied to date [ 73 , 74 ] . however , the nfb - binding regions for inhibitor of b ( ib ) proteins ( e.g. , cactus ) are not conserved , suggesting diversification and co - adaptation between ib and nfb pairs . also , recent work indicates that nf-b nuclear co - regulators may contribute to species - specific regulation of amp gene expression . therefore , modulation of inhibitors and nuclear - co - regulators of nf-b - dependent transcription may be one avenue by which target - specific immune suppression could be achieved . in d. melanogaster , nf-b - dependent amp induction through the toll and imd pathways is activated by detection of microbial components via different pattern recognition receptors ( prrs ) . prrs are soluble or membrane - bound proteins that bind to specific microbe associated molecular patterns ( mamps ) such as lipopolysaccharide ( lps ) , lipoteichoic acid ( lta ) , peptidoglycan ( pgn ) or -1,3-glucan that are released from or are found on the cell surfaces of bacteria or fungi . upon interaction with mamps , prrs can directly agglutinate pathogens or trigger proteolytic signaling cascades and cytokine release , which in turn lead to the activation of downstream cellular and humoral pathways , including pro - po activation and amp gene expression [ 16 , 65 , 76 ] . pgn recognition proteins ( pgrps ) and -1,3-glucanase - related proteins ( grps ) were discovered in the lepidopteran silkworm ( b. mori ) by assaying for plasma components that activate the propo cascade . pgrps were subsequently shown to be conserved across mammals and insects , and in d. melanogaster their role in the induction of amp gene expression through toll and imd pathways in response to pgn has been well documented [ 7983 ] . similarly , grps have been shown to induce amp expression through toll pathway in response to fungal infections [ 79 , 84 ] . in contrast , there is a dearth of literature linking specific pgrps or grps to amp induction in lepidoptera . such a link is possible , since pgn and -1,3-glucan can activate amp gene expression in m. sexta and b. mori [ 8590 ] and multiple infection - induced pgrp- and grp - encoding genes have been identified in lepidoptera [ 32 , 38 , 54 , 55 , 9194 ] . however , there are numerous hints that lepidoptera and diptera may have evolved divergent mechanisms of linking pathogen detection to conserved toll and imd signal transduction cascades . first , a genome comparison between b. mori and d. melanogaster failed to identify 1:1 pgrp orthologs . similarly , b. mori gram - negative binding protein ( gnbp ) and m. sexta microbe binding protein ( mbp ) , members of the -1,3-glucanase - related protein superfamily [ 76 , 95 ] , appear to be distantly related to d. melanogaster gnbps , suggesting divergence of this group of proteins . m. sexta mbp expression is strongly up - regulated in fat body after immune challenge and shows specific binding to lta , lps , dap - pgn . also , in contrast to the situation in d. melanogaster , highly purified lps and lta are inducers of amp gene expression in lepidoptera , though not as potently as crude lps ( with contaminating pgn ) or purified pgn [ 85 , 90 , 96 , 97 ] . this raises the possibility that different mamps or combinations of mamps are most efficacious in eliciting amp gene expression in lepidoptera relative to diptera . also , since purified lps can elicit amp expression in lepidoptera but not d. melanogaster , lepidoptera have either a distinct repertoire of prrs responsible for lps - dependent triggering of imd or toll pathways , or an as - yet undiscovered pathway that links lps to amp induction . testing these ideas awaits the identification of the suite of prrs and signal transduction pathways responsible for transducing lps , lta , pgn , or combinatorial microbial signals to amp gene expression . one class of lepidopteran prr that may mediate infection - dependent induction of amps is the c - type lectins ( ctls ) , ca - dependent , secreted proteins that have carbohydrate - binding capabilities . similar to some ctls of d. melanogaster , several ctls of m. sexta and b. mori [ 54 , 99 ] are reported to mediate induction of cellular responses and the propo cascade . although the nomenclature quickly becomes confusing , ctls include lipopolysaccharide - binding protein ( lbp ) . m. sexta iml-1 binds to gram - positive and gram - negative bacteria as well as yeast , iml-2 shows specific binding to lps , iml-3 and iml-4 show specific binding to lps and lta , and iml-3 can also bind laminarin , a -1,3-glucan [ 103 , 104 ] . diversity in ctl carbohydrate - binding specificities may result in lineage - specific pathogen recognition - signal transduction connections . of particular relevance to the theme of this review are prrs present in lepidoptera but not diptera ( table 1 ) . in general , both orders of insects encode grps and pgrps . however , specific representatives of each class are restricted to lepidoptera ( table 1 ) . for example , the lepidopteran grp-2 , which binds fungal cell wall -1,3 glucans and lta , is absent from diptera . such derived grp and pgrps may contribute to lepidopteran - specific transduction of signals to downstream pathways . other lepidoptera - specific prrs are hemolin and hemolymph proteinase-14 precursor ( prohp14 ) ( table 1 ) . like iml c - type lectins , hemolin is an lps- and lta - binding prr with roles in mediating cellular responses and as an opsonin to enhance phagocytosis . hp14 has been shown to detect and bind a broad range of mamps , and may coordinate with grp1 or grp2 to activate propo [ 60 , 106 ] . the potential role of the prrs discussed above in mediating the expression of amp genes remains to be determined , and further study of the lepidoptera - specific immune surveillance proteins and divergent activities of conserved prrs likely will yield novel avenues for pest - control . d. melanogaster has both mamp - dependent and mamp - independent routes to activate the toll pathway . in mamp - dependent toll induction , bacterial lys - pgn ( typical of gram - positive bacteria ) is detected by pgrp - sa or pgrp - sd ( in the presence of gnbp-1 ) , while yeast or fungal -1,3-glucan is detected by gnbp-3 [ 85 , 108 , 109 ] ( figure 1 ) . mamp - independent stimuli are virulence determinants , such as proteases and chitinases , secreted by microbes and dubbed mamps and mamp - independent stimuli each trigger a distinct proteolytic cascade that both culminate in cleavage of the cytokine sptzle by the serine protease sptzle processing enzyme ( spe ) . interaction of active sptzle c - terminal domain ( c-106 ) with the surface - localized toll receptor triggers an intracellular signal transduction terminating in induced expression of amps and cellular responses . some of the basic components of the toll pathway are present in lepidoptera ( figure 2 ) . m. sexta hemocytes express an infection - induced toll - like receptor and the genome of b. mori encodes 14 genes predicted to encode toll - like receptors as well as homologs of each of the intracellular components of toll - dependent signaling [ 85 , 114 , 115 ] . both m. sexta and b. mori encode homologs of the d. melanogaster toll - activating cytokine sptzle ( figure 2 ) . also , for both m. sexta and b. mori there is experimental evidence linking the toll pathway with amp induction [ 116118 ] . in m. sexta , toll pathway results in expression of several antimicrobial peptides , including attacin-1 , cecropin-6 , moricin and lysozyme . in addition , the transcript level of hemolin , a pattern recognition protein exclusive to lepidopterans ( table 1 ) , is induced by injection of activated sptzle - c108 into larvae . despite the conservation of certain aspects of the toll pathway , the extracellular cascades that lead to sptzle activation may have diverged between d. melanogaster and the two lepidoptera ( figure 1 ) . for example , in contrast to what is known in d. melanogaster , the m. sexta toll pathway is activated by gram - negative - associated mamps [ 115 , 118 ] . also , the genome of b. mori lacks 1:1 orthologs of grass , spirit and persephone , the d. melanogaster serine proteases responsible for mamp / prr - dependent and mamp - independent cleavage of spe ( figure 1 ) . progress has been made in identifying a m. sexta proteolytic cascade that results in processing pro - sptzle into its active c - terminal domain ( c-108 ) . the direct cleavage is mediated by hemolymph proteinase ( hp ) 8 [ 11 , 13 ] , a homolog of d. melanogaster spe , in turn , hp8 is processed into its active form by hp6 . hp6 is most similar to d. melanogaster persephone protease , which activates spe in response to mamp - independent stimuli [ 110 , 113 ] . this hemolymph proteinase is activated in response to gram - positive or gram - negative bacteria and in response to -1,3-glucan . however , the prrs and proteolytic cascade that transduce mamp signals to amp induction are unknown ( figure 1 ) . the findings reviewed above demonstrate that while the overall architecture of the toll pathway is conserved among insects , the specific identities of proteolytic cascade members are distinct and many gaps remain in our understanding of toll activation in lepidoptera . filling these gaps should reveal potential lineage - specific molecules that can serve as targets to hinder the activation of the toll pathway in agricultural pests . in d. melanogaster the imd pathway also contributes to amp gene induction and is triggered by direct interaction of dap - pgn , a mamp typical of gram - negative bacteria , with the transmembrane receptor pgrp - lc [ 82 , 83 , 125 , 126 ] . for example , pgrp - le can act as an intracellular receptor for monomeric pgn and its truncated form can enhance pgrp - lc - mediated recognition . dap - pgn / pgrp - lc interaction activates intracellular imd , which then recruits fas - associated death domain ( fadd ) and death - related ced-3/nedd2-like protein ( dredd ) to form a complex [ 129 , 130 ] . current evidence supports the idea that dredd , a caspase - like molecule , cleaves the nf-b transcription factor relish . imd also appears to activate a phosphorelay : the transforming growth factor- ( tgf)-activated kinase 1 ( tak1 ) phosphorylates the ikb kinase ( ikk ) relish cleavage into its activated amino - terminal transcriptional regulator domain allows its translocation into the nucleus , where it activates amp gene expression . the translocation of relish into the nucleus is regulated by two recently discovered components of this pathway : inhibitor of apoptosis 2 ( iap2 ) and transforming growth factor - activated kinase 1 ( tak1)-binding protein 2 ( tab2 ) [ 132 , 133 ] . both , iap2 and tab2 act upstream of relish and downstream of imd , while iap2 functions downstream of tak1 [ 132 , 134 ] . of particular importance to amp gene expression is iap2 , the knockdown of which hampers the sustained expression of amp genes . while iap2 and tab2 are necessary for imd signal transduction , the gene product of pirk , a recently characterized gene , interacts directly with imd and pgrp - lc . pirk overexpression analyses revealed that it acts as a negative regulator by reducing the expression of the amp genes attacin b , cecropin b , and diptericin b , which are all under the control of the imd pathway . most of the information available about the imd pathway in lepidoptera comes from bioinformatics ; orthologs of all intracellular components of the imd pathway have been found in b. mori [ 54 , 137 ] and m. sexta . however , few experiments have been done to characterize the molecular mechanisms leading to activation of the imd pathway in these insects . in m. sexta several genes of the imd pathway , including those encoding imd , fadd , tak1 , dredd and relish are up regulated in fat body of immune challenged 5th instar larvae and in the midgut of b. mori during the wandering stage . genes encoding lysozyme , moricin and defensin amps also were up regulated in the midgut of b. mori in the wandering stage , consistent with the possibility that amp induction is imd - mediated . finally , in the lepidopteran beet armyworm spodoptera exigua , rnai - mediated knockdown of relish expression resulted in loss of cecropin induction upon fungal infection , further strengthening the idea that the imd pathway may contribute to amp gene expression in lepidoptera , though perhaps it is triggered by distinct signals . further study is needed to elucidate imd - mediated amp induction in lepidoptera and to reveal any differences there are in this pathway between diptera and lepidoptera . in d. melanogaster , transcription of amp - encoding genes is activated by the nf-b family transcription factors in response to infection [ 6569 ] with distinct nf-b family transcription factors responding to the toll and immune deficiency ( imd ) signal transduction pathways . in response to toll pathway activation , the nf-b inhibitor cactus is phosphorylated and degraded allowing its targets , the nf-b factors dif and dorsal , to be translocated to the nucleus . imd pathway activity culminates in the nf-b factor relish being activated by a stimulus - induced proteolytic cleavage . in the case of dif and dorsal , gram - positive bacterial and fungal infections primarily serve as the stimuli that induce degradation of cactus through the toll signaling pathway . in general , gram - negative bacterial infections of d. melanogaster stimulate the proteolytic cleavage of relish through the imd pathway . once in the nucleus , these transcription factors drive the transcription of immune effectors , including amp genes whose promoters contain nf-b binding sites [ 70 , 72 ] . overall , nf-b proteins and their dna - binding specificities are conserved among organisms , including those lepidoptera studied to date [ 73 , 74 ] . however , the nfb - binding regions for inhibitor of b ( ib ) proteins ( e.g. , cactus ) are not conserved , suggesting diversification and co - adaptation between ib and nfb pairs . also , recent work indicates that nf-b nuclear co - regulators may contribute to species - specific regulation of amp gene expression . therefore , modulation of inhibitors and nuclear - co - regulators of nf-b - dependent transcription may be one avenue by which target - specific immune suppression could be achieved . in d. melanogaster , nf-b - dependent amp induction through the toll and imd pathways is activated by detection of microbial components via different pattern recognition receptors ( prrs ) . prrs are soluble or membrane - bound proteins that bind to specific microbe associated molecular patterns ( mamps ) such as lipopolysaccharide ( lps ) , lipoteichoic acid ( lta ) , peptidoglycan ( pgn ) or -1,3-glucan that are released from or are found on the cell surfaces of bacteria or fungi . upon interaction with mamps , prrs can directly agglutinate pathogens or trigger proteolytic signaling cascades and cytokine release , which in turn lead to the activation of downstream cellular and humoral pathways , including pro - po activation and amp gene expression [ 16 , 65 , 76 ] . pgn recognition proteins ( pgrps ) and -1,3-glucanase - related proteins ( grps ) were discovered in the lepidopteran silkworm ( b. mori ) by assaying for plasma components that activate the propo cascade . pgrps were subsequently shown to be conserved across mammals and insects , and in d. melanogaster their role in the induction of amp gene expression through toll and imd pathways in response to pgn has been well documented [ 7983 ] . similarly , grps have been shown to induce amp expression through toll pathway in response to fungal infections [ 79 , 84 ] . in contrast , there is a dearth of literature linking specific pgrps or grps to amp induction in lepidoptera . such a link is possible , since pgn and -1,3-glucan can activate amp gene expression in m. sexta and b. mori [ 8590 ] and multiple infection - induced pgrp- and grp - encoding genes have been identified in lepidoptera [ 32 , 38 , 54 , 55 , 9194 ] . however , there are numerous hints that lepidoptera and diptera may have evolved divergent mechanisms of linking pathogen detection to conserved toll and imd signal transduction cascades . first , a genome comparison between b. mori and d. melanogaster failed to identify 1:1 pgrp orthologs . similarly , b. mori gram - negative binding protein ( gnbp ) and m. sexta microbe binding protein ( mbp ) , members of the -1,3-glucanase - related protein superfamily [ 76 , 95 ] , appear to be distantly related to d. melanogaster gnbps , suggesting divergence of this group of proteins . m. sexta mbp expression is strongly up - regulated in fat body after immune challenge and shows specific binding to lta , lps , dap - pgn . also , in contrast to the situation in d. melanogaster , highly purified lps and lta are inducers of amp gene expression in lepidoptera , though not as potently as crude lps ( with contaminating pgn ) or purified pgn [ 85 , 90 , 96 , 97 ] . this raises the possibility that different mamps or combinations of mamps are most efficacious in eliciting amp gene expression in lepidoptera relative to diptera . also , since purified lps can elicit amp expression in lepidoptera but not d. melanogaster , lepidoptera have either a distinct repertoire of prrs responsible for lps - dependent triggering of imd or toll pathways , or an as - yet undiscovered pathway that links lps to amp induction . testing these ideas awaits the identification of the suite of prrs and signal transduction pathways responsible for transducing lps , lta , pgn , or combinatorial microbial signals to amp gene expression . one class of lepidopteran prr that may mediate infection - dependent induction of amps is the c - type lectins ( ctls ) , ca - dependent , secreted proteins that have carbohydrate - binding capabilities . similar to some ctls of d. melanogaster , several ctls of m. sexta and b. mori [ 54 , 99 ] are reported to mediate induction of cellular responses and the propo cascade . although the nomenclature quickly becomes confusing , ctls include lipopolysaccharide - binding protein ( lbp ) . m. sexta iml-1 binds to gram - positive and gram - negative bacteria as well as yeast , iml-2 shows specific binding to lps , iml-3 and iml-4 show specific binding to lps and lta , and iml-3 can also bind laminarin , a -1,3-glucan [ 103 , 104 ] . diversity in ctl carbohydrate - binding specificities may result in lineage - specific pathogen recognition - signal transduction connections . of particular relevance to the theme of this review are prrs present in lepidoptera but not diptera ( table 1 ) . in general , both orders of insects encode grps and pgrps . however , specific representatives of each class are restricted to lepidoptera ( table 1 ) . for example , the lepidopteran grp-2 , which binds fungal cell wall -1,3 glucans and lta , is absent from diptera . such derived grp and pgrps may contribute to lepidopteran - specific transduction of signals to downstream pathways . other lepidoptera - specific prrs are hemolin and hemolymph proteinase-14 precursor ( prohp14 ) ( table 1 ) . like iml c - type lectins , hemolin is an lps- and lta - binding prr with roles in mediating cellular responses and as an opsonin to enhance phagocytosis . hp14 has been shown to detect and bind a broad range of mamps , and may coordinate with grp1 or grp2 to activate propo [ 60 , 106 ] . the potential role of the prrs discussed above in mediating the expression of amp genes remains to be determined , and further study of the lepidoptera - specific immune surveillance proteins and divergent activities of conserved prrs likely will yield novel avenues for pest - control . d. melanogaster has both mamp - dependent and mamp - independent routes to activate the toll pathway . in mamp - dependent toll induction , bacterial lys - pgn ( typical of gram - positive bacteria ) is detected by pgrp - sa or pgrp - sd ( in the presence of gnbp-1 ) , while yeast or fungal -1,3-glucan is detected by gnbp-3 [ 85 , 108 , 109 ] ( figure 1 ) . mamp - independent stimuli are virulence determinants , such as proteases and chitinases , secreted by microbes and dubbed mamps and mamp - independent stimuli each trigger a distinct proteolytic cascade that both culminate in cleavage of the cytokine sptzle by the serine protease sptzle processing enzyme ( spe ) . interaction of active sptzle c - terminal domain ( c-106 ) with the surface - localized toll receptor triggers an intracellular signal transduction terminating in induced expression of amps and cellular responses . some of the basic components of the toll pathway are present in lepidoptera ( figure 2 ) . m. sexta hemocytes express an infection - induced toll - like receptor and the genome of b. mori encodes 14 genes predicted to encode toll - like receptors as well as homologs of each of the intracellular components of toll - dependent signaling [ 85 , 114 , 115 ] . both m. sexta and b. mori encode homologs of the d. melanogaster toll - activating cytokine sptzle ( figure 2 ) . also , for both m. sexta and b. mori there is experimental evidence linking the toll pathway with amp induction [ 116118 ] . in m. sexta , toll pathway results in expression of several antimicrobial peptides , including attacin-1 , cecropin-6 , moricin and lysozyme . in addition , the transcript level of hemolin , a pattern recognition protein exclusive to lepidopterans ( table 1 ) , is induced by injection of activated sptzle - c108 into larvae . despite the conservation of certain aspects of the toll pathway , the extracellular cascades that lead to sptzle activation may have diverged between d. melanogaster and the two lepidoptera ( figure 1 ) . for example , in contrast to what is known in d. melanogaster , the m. sexta toll pathway is activated by gram - negative - associated mamps [ 115 , 118 ] . also , the genome of b. mori lacks 1:1 orthologs of grass , spirit and persephone , the d. melanogaster serine proteases responsible for mamp / prr - dependent and mamp - independent cleavage of spe ( figure 1 ) . progress has been made in identifying a m. sexta proteolytic cascade that results in processing pro - sptzle into its active c - terminal domain ( c-108 ) . the direct cleavage is mediated by hemolymph proteinase ( hp ) 8 [ 11 , 13 ] , a homolog of d. melanogaster spe , in turn , hp8 is processed into its active form by hp6 . hp6 is most similar to d. melanogaster persephone protease , which activates spe in response to mamp - independent stimuli [ 110 , 113 ] . this hemolymph proteinase is activated in response to gram - positive or gram - negative bacteria and in response to -1,3-glucan . however , the prrs and proteolytic cascade that transduce mamp signals to amp induction are unknown ( figure 1 ) . the findings reviewed above demonstrate that while the overall architecture of the toll pathway is conserved among insects , the specific identities of proteolytic cascade members are distinct and many gaps remain in our understanding of toll activation in lepidoptera . filling these gaps should reveal potential lineage - specific molecules that can serve as targets to hinder the activation of the toll pathway in agricultural pests . in d. melanogaster the imd pathway also contributes to amp gene induction and is triggered by direct interaction of dap - pgn , a mamp typical of gram - negative bacteria , with the transmembrane receptor pgrp - lc [ 82 , 83 , 125 , 126 ] . for example , pgrp - le can act as an intracellular receptor for monomeric pgn and its truncated form can enhance pgrp - lc - mediated recognition . dap - pgn / pgrp - lc interaction activates intracellular imd , which then recruits fas - associated death domain ( fadd ) and death - related ced-3/nedd2-like protein ( dredd ) to form a complex [ 129 , 130 ] . current evidence supports the idea that dredd , a caspase - like molecule , cleaves the nf-b transcription factor relish . imd also appears to activate a phosphorelay : the transforming growth factor- ( tgf)-activated kinase 1 ( tak1 ) phosphorylates the ikb kinase ( ikk ) relish cleavage into its activated amino - terminal transcriptional regulator domain allows its translocation into the nucleus , where it activates amp gene expression . the translocation of relish into the nucleus is regulated by two recently discovered components of this pathway : inhibitor of apoptosis 2 ( iap2 ) and transforming growth factor - activated kinase 1 ( tak1)-binding protein 2 ( tab2 ) [ 132 , 133 ] . both , iap2 and tab2 act upstream of relish and downstream of imd , while iap2 functions downstream of tak1 [ 132 , 134 ] . of particular importance to amp gene expression is iap2 , the knockdown of which hampers the sustained expression of amp genes . while iap2 and tab2 are necessary for imd signal transduction , the gene product of pirk , a recently characterized gene , interacts directly with imd and pgrp - lc . pirk overexpression analyses revealed that it acts as a negative regulator by reducing the expression of the amp genes attacin b , cecropin b , and diptericin b , which are all under the control of the imd pathway . most of the information available about the imd pathway in lepidoptera comes from bioinformatics ; orthologs of all intracellular components of the imd pathway have been found in b. mori [ 54 , 137 ] and m. sexta . however , few experiments have been done to characterize the molecular mechanisms leading to activation of the imd pathway in these insects . in m. sexta several genes of the imd pathway , including those encoding imd , fadd , tak1 , dredd and relish are up regulated in fat body of immune challenged 5th instar larvae and in the midgut of b. mori during the wandering stage . genes encoding lysozyme , moricin and defensin amps also were up regulated in the midgut of b. mori in the wandering stage , consistent with the possibility that amp induction is imd - mediated . finally , in the lepidopteran beet armyworm spodoptera exigua , rnai - mediated knockdown of relish expression resulted in loss of cecropin induction upon fungal infection , further strengthening the idea that the imd pathway may contribute to amp gene expression in lepidoptera , though perhaps it is triggered by distinct signals . further study is needed to elucidate imd - mediated amp induction in lepidoptera and to reveal any differences there are in this pathway between diptera and lepidoptera . insecticides are necessary to guarantee effective insect pest management in agricultural settings . however , the cost and off - target effects of these insecticides directly and indirectly increase economic burden ; the latter by affecting beneficial arthropods such as pollinators . the study of insect immunity can provide tools for the development of target - specific cost - effective approaches to control agricultural pests . directed suppression of pest immune defenses is predicted to render them susceptible to environmental and applied biocontrol pathogens , as recently demonstrated in termites by bulmer and colleagues . the studies summarized above suggest that many aspects of insect immunity , including recognition factors and serine proteases , have diverged between d. melanogaster and lepidoptera . continued comparative immunity of a broad array of species from diptera , lepidoptera , and other insect orders will reveal possible candidate immunity factors for target - specific approaches that will enable the effective control of insect pests . however , before such approaches can be realized , the details of lepidopteran immune signaling pathways must be elucidated . the relatively large sizes of last instar larvae of many lepidopteran species will facilitate biochemical approaches to such studies , while the establishment of immune - inducible lepidopteran cell lines such as the uga - cie1 cell line can enable the characterization of molecular mechanisms leading to imd pathway activation and its contribution to amp gene expression . finally , ongoing investigations into the immune - modulatory mechanisms of entomopathogens will help identify key steps in immunity that are susceptible to manipulation , contributing to the development of natural , cost - effective , non - toxic alternatives to chemical insecticides currently used for pest management .

many lepidopteran insects are agricultural pests that affect stored grains , food and fiber crops . these insects have negative ecological and economic impacts since they lower crop yield , and pesticides are expensive and can have off - target effects on beneficial arthropods . a better understanding of lepidopteran immunity will aid in identifying new targets for the development of specific insect pest management compounds . a fundamental aspect of immunity , and therefore a logical target for control , is the induction of antimicrobial peptide ( amp ) expression . these peptides insert into and disrupt microbial membranes , thereby promoting pathogen clearance and insect survival . pathways leading to amp expression have been extensively studied in the dipteran drosophila melanogaster . however , diptera are an important group of pollinators and pest management strategies that target their immune systems is not recommended . recent advances have facilitated investigation of lepidopteran immunity , revealing both conserved and derived characteristics . although the general pathways leading to amp expression are conserved , specific components of these pathways , such as recognition proteins have diverged . in this review we highlight how such comparative immunology could aid in developing pest management strategies that are specific to agricultural insect pests .

1. Introduction 2. Insect Immunity 3. Signaling Pathways Involved in Antimicrobial Peptide Gene Expression 3.1. Induction of AMP Genes by the NF-kB Family of Transcription Factors 3.2. Recognition and Proteolytic Cascades 3.3. Toll Pathway 3.4. IMD Pathway 4. Conclusions

syncope is caused by transient diffuse cerebral hypoperfusion and is characterized by transient loss of consciousness with a rapid onset followed by spontaneous and complete recovery . clinical features of syncope may include myoclonic jerks which are often multifocal and asynchronous , convulsions , and urinary incontinence , making it difficult to differentiate from epileptic seizure by clinical features alone . significant fluctuations in cerebral perfusion pressure are prevented by autoregulation of cerebral circulation , but there may be conditions where such mechanism may not compensate adequately . cough syncope , a rare form of syncope , may be a result of transient failure of the cerebral autoregulatory mechanism to cope with sudden decrease in cerebral blood flow . we present an unusual case of recurrent cough syncope , which was initially diagnosed and treated as seizures , in the context of a left - sided glomus jugulare tumor , a benign paraganglioma . a 43-year - old right - handed woman with history of glomus jugulare tumor in the left jugular fossa with intracranial extension into the posterior cranial fossa was transferred from another hospital for recurrent seizure - like spells . she had a 90% surgical resection of the tumor done in 2011 followed by radiation therapy in september 2012 . her episodes occurred multiple times a day ( 7 per day on average ) during wakeful state . they were triggered by coughing ( usually a bout of cough ) and were characterized by staring and unresponsiveness as well as stiffening of the body with mild shaking of both upper extremities . she was diagnosed with epileptic seizures but continued to have episodes during treatment with the antiepileptic drugs ( aeds ) phenytoin , levetiracetam , and lamotrigine . escalation of aed therapy made her increasingly drowsy , and she was on all three aforementioned aeds at the time of presentation . her physical examination was remarkable for excessive drowsiness , mild dysarthria , right sixth cranial nerve palsy , mild hypertonia with hyperreflexia in the lower extremities ( left more than right ) , and bilateral ( left more than right ) ankle clonus . she had a lumbar puncture done at the outside hospital , and the opening cerebrospinal fluid ( csf ) pressure was reported to be 25 cm . blood work was also unremarkable except for mild anemia ( hemoglobin : 9.4 g / dl ) , mild hyponatremia ( 132 meq / l ) , and mild hypokalemia ( 3.1 meq / l ) . antiepileptic drug levels were within therapeutic range ( free phenytoin : 1.3 g / ml , levetiracetam : 5.9 g / ml , and lamotrigine : 2.3 g / ml ) . all started with a bout of cough when the patient was lying in bed ( in supine or in lateral position ) which was followed by brief ( less than a minute ) distal upper extremity tremor and subtle proximal upper extremity myoclonic jerks and prolonged unresponsiveness for up to 10 min . all of these episodes were associated with hypotension ( 7278/3147 mm of hg as revealed by continuous arterial pressure monitoring ) and bradycardia ( 5459 bpm ) . the eeg during the spells was characterized by generalized synchronous and asynchronous high amplitude 1- to 2-hz delta activity which progressed to generalized attenuation and then transitioned to generalized delta activity again with recovery ( fig . 1 ) . a head ct showed recurrence of the glomus jugulare tumor and communicating hydrocephalus . an external ventricular drain ( evd ) after placement of the evd , her drowsiness gradually started to improve , and episodes decreased in frequency to one per day . 3 showed an enhancing t2 hyperintense left skull base mass in the region of the left jugular foramen with extension into the posterior cranial fossa and below the base of the skull . brain imaging showed evidence of hydrocephalus that had increased compared with her previous brain imaging done 2 months back . her mental status continued to improve , and she had only one mild episode triggered by cough during the next two days before her discharge . repeat surgical resection of the tumor was recommended by the otolaryngology team , which the patient declined . based on the clinical features and eeg findings , the episodes observed in our patient are most consistent with cough syncope . the mechanism underlying cough syncope is not definitively established , but it is postulated that coughing increases intrathoracic and intraabdominal pressures leading to a transient increase in icp . increased icp , in turn , causes a decrease in cerebral perfusion pressure which , if it drops below a critical level , may result in global cerebral hypoperfusion leading to syncope . transient cerebral circulatory arrest has been demonstrated by transcranial doppler measurements during cough syncope . our patient also had a drop in blood pressure and heart rate but probably not sufficient to cause syncope by itself . cough syncope has been associated with posterior fossa mass lesions or tonsillar herniation and with hydrocephalus . it may be speculated that bouts of cough caused transient herniation of cerebellar tonsils obstructing csf flow that further contributed to the increase in icp during coughing . decrease in frequency of events following placement of evd to relieve icp lends support to this notion . paragangliomas are rare tumors of extraadrenal chromaffin cell origin that most commonly occur in the head and neck region . catecholamine - hypersecreting paraganglionomas are uncommon in the head and neck region , and most patients ( 95% ) with hypersecreting paraganglionomas have hypertension . hypotension accompanying syncope observed in our patient was not orthostasis - related ( the patient was always supine during spells ) and was most likely related to cough . identified a subset of patients with cough syncope who lacked a blood pressure overshoot ( expected response ) after the relief of straining during valsalva maneuver . the authors postulate that cough syncope in these patients might be the result of delayed recovery from hypotension that follows a paroxysm of cough , and this was likely contributing to global cerebral hypoperfusion in our patient . this case highlights the fact that cough syncope , a rare form of syncope , may be associated with intracranial mass lesions that indirectly exaggerate the increase in icp in response to cough . glomus caroticum tumor presenting as recurrent unexplained syncope and posterior fossa meningioma presenting as recurrent cough syncope have been described . recurrent cough syncope should trigger search for factors , including brain tumors , with the potential to cause transient elevation in icp . this case also illustrates an important role for ceeg monitoring with video in distinguishing syncope from seizures in cough syncope cases .

we present an unusual case of recurrent cough syncope in a 43-year - old woman , which was initially thought to be seizures . syncopal episodes were triggered by paroxysms of cough and were characterized by unresponsiveness and myoclonic jerks in her extremities . she had a left - sided glomus jugulare tumor that extended into the posterior cranial fossa with evidence of worsening communicating hydrocephalus on brain imaging . we postulate that bouts of cough produced increased intracranial pressure both by raising intrathoracic and intraabdominal pressures as well as by transient obstruction to cerebrospinal fluid flow secondary to intermittent tonsillar herniation during cough . this resulted in diffuse decrease in cerebral blood flow causing syncope . the patient 's syncopal episodes decreased in frequency once an external ventricular drain was placed followed by a ventriculoperitoneal shunt . search for factors that can increase intracranial pressure seems warranted in patients with recurrent cough syncope .

Introduction Case report Discussion Conflict of interest

world - wide , infertility affects 1015% of couples who are trying to conceive , and about 15% of these cases are caused by male factors , which affect 1 out of 20 men in the general population . most cases of male infertility are idiopathic , apart from several etiologies , such as obstruction of deferent duct , varicocele , sexual dysfunction , and cryptorchidism . although assisted reproductive technology ( art ) has helped many sterile couples to conceive , non - obstructive azoospermia ( noa ) , which accounts for a considerable proportion of male infertility , has a dramatically lower rate of sperm retrieval and clinical pregnancy . the etiological mechanism of noa is unknown , but factors such as oxidative stress were considered to have effects on spermatogenesis , and some antioxidants have been effective in protecting spermatogenesis . therefore , it is helpful to explore the underlying pathogenesis of noa in these patients . micrornas are a class of small rnas that do not code amino acid sequences , but they play fundamental roles in regulating gene expression after transcription . lian et al . found 154 down - regulated mirnas and 19 up - regulated mirnas in testes of noa patients compared to fertile males , by using microarray technologies . furthermore , some of the mirnas have been shown to affect the proliferation , apoptosis , and dna damage in germ cells [ 911 ] . mir-210 is one of the 19 up - regulated mirnas in testes of noa patients , located within the genomic loci of transcript ak123483 . it can be induced by hypoxia , and plays an essential role in cell adaptation to hypoxia . mir-210 also affects regulation of diverse physiological processes , such as angiogenesis , cell survival , proliferation , cell cycle arrest , protein modification , and dna damage repair . although mir-210 has been shown to be involved in regulation of physiological processes in various diseases and to be an up - regulated mirna in testes of noa patients , it remains unknown how mir-210 affects spermatogenesis . hence , the aim of this study was to investigate the underlying mechanisms by which mir-210 is involved in the pathogenesis of spermatogenesis . we enrolled 25 patients ( aged 1841 years ) with azoospermia ( proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university ) . pathological examinations were performed on each testicular specimen . combined with clinical features , 4 patients were diagnosed as having sertoli - cell - only syndrome ( scos ) , 7 patients were diagnosed as having maturation arrest ( ma ) , 8 patients were diagnosed as having hypospermatogenesis , and the other 6 patients were diagnosed as having obstructive azoospermia ( oa ) . all patients provided informed consent before their participation in this study . our local medical ethics committee approved this study before it began . to examine the location of insulin - like growth factor ii ( igf2 ) in human testicular tissues , we performed immunohistochemistry staining to detect the igf2 expression . tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax . after the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al . , sections were incubated with igf2 antibody ( abcam ) overnight at 4c and biotinylated secondary antibody ( abcam ) for 2 h at room temperature . to detect expression of mir-210 , rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . . briefly , rna extraction was performed following a standard trizol protocol , real - time pcr was carried out with the abi step one system ( applied biosystems ) , and the sybr premix ex taq ii kit ( takara bio , inc . ) was used . primers for q - rt pcr were as follows : forward primer : 5-caataactgtgcgtgtgacagc-3 reverse primer : 5-tatggttttgacgactgtgtgat-3 forward primer : 5-cagcacatatactaaaattggaacg-3 reverse primer : 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells . anti - igf2 ( abcam ) was used for western blot analysis , and we used -actin as a loading control to detect expression of igf2 . we supplemented the medium with 10% fetal bovine serum ( life technology inc . ) , 1% antibiotics ( 100 units / ml penicillin , and 100 ug / ml streptomycin , life technology inc . ) . cells were incubated at 37c in a humidified incubator with 5% co2 . to transfect oligonucleotides and plasmids into nt-2 cells , lipofectamine rnaimax ( invitrogen ) and fugene hd ( roche ) all processes were performed in accordance with the protocols supplied by manufacturers . in this study , all the experiments were performed independently at least 3 times . we enrolled 25 patients ( aged 1841 years ) with azoospermia ( proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university ) . pathological examinations were performed on each testicular specimen . combined with clinical features , 4 patients were diagnosed as having sertoli - cell - only syndrome ( scos ) , 7 patients were diagnosed as having maturation arrest ( ma ) , 8 patients were diagnosed as having hypospermatogenesis , and the other 6 patients were diagnosed as having obstructive azoospermia ( oa ) . all patients provided informed consent before their participation in this study . to examine the location of insulin - like growth factor ii ( igf2 ) in human testicular tissues , we performed immunohistochemistry staining to detect the igf2 expression . tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax . after the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al . , sections were incubated with igf2 antibody ( abcam ) overnight at 4c and biotinylated secondary antibody ( abcam ) for 2 h at room temperature . rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . . briefly , rna extraction was performed following a standard trizol protocol , real - time pcr was carried out with the abi step one system ( applied biosystems ) , and the sybr premix ex taq ii kit ( takara bio , inc . ) was used . primers for q - rt pcr were as follows : forward primer : 5-caataactgtgcgtgtgacagc-3 reverse primer : 5-tatggttttgacgactgtgtgat-3 forward primer : 5-cagcacatatactaaaattggaacg-3 reverse primer : 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells . anti - igf2 ( abcam ) was used for western blot analysis , and we used -actin as a loading control to detect expression of igf2 . we supplemented the medium with 10% fetal bovine serum ( life technology inc . ) , 1% antibiotics ( 100 units / ml penicillin , and 100 ug / ml streptomycin , life technology inc . ) . cells were incubated at 37c in a humidified incubator with 5% co2 . to transfect oligonucleotides and plasmids into nt-2 cells , lipofectamine rnaimax ( invitrogen ) and fugene hd ( roche ) the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2 , which is only produced from the paternal allele . the maternal allele is transcriptionally silent . to clarify the location of igf2 in human testicular tissues , we found igf2 located in spermatocytes in the testes of patients with oa ( figure 1 ) . because igf2 is located in spermatocytes of the testis , we detected the expression of igf2 in cases with ma , hypospermatogenesis , and oa , but not in the scos patients . we found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients , which was considered as the control group with normal spermatogenesis , although without a significant difference ( figures 2 , 3 ) , possibly because there were fewer samples and longer preservation times of some samples . quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma , hypospermatogenesis , and oa . we found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients ( figure 4 ) . however , due to errors in the rna extraction in the preliminary experiment , 3 testis samples ( 1 each ) from ma , hypospermatogenesis , and oa patients were damaged and were not tested . in the targetscan database , because the 3utr of the igf2-mrna has a putative mir-210-binding site , igf2 was predicted to be a potential target of mir-210 . to identify whether the igf2 gene was targeted by mir-210 directly , renilla luciferase reporters , which include the wild - type full - length 3utr forms of mir-210 seeding sites , figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells , and inhibiting mir-210 expression increased activity of the reporter renilla luciferase . expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells , and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 ( figures 6 ) . the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2 , which is only produced from the paternal allele . the maternal allele is transcriptionally silent . to clarify the location of igf2 in human testicular tissues , we found igf2 located in spermatocytes in the testes of patients with oa ( figure 1 ) . because igf2 is located in spermatocytes of the testis , we detected the expression of igf2 in cases with ma , hypospermatogenesis , and oa , but not in the scos patients . we found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients , which was considered as the control group with normal spermatogenesis , although without a significant difference ( figures 2 , 3 ) , possibly because there were fewer samples and longer preservation times of some samples . quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma , hypospermatogenesis , and oa . we found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients ( figure 4 ) . however , due to errors in the rna extraction in the preliminary experiment , 3 testis samples ( 1 each ) from ma , hypospermatogenesis , and oa patients were damaged and were not tested . in the targetscan database , because the 3utr of the igf2-mrna has a putative mir-210-binding site , igf2 was predicted to be a potential target of mir-210 . to identify whether the igf2 gene was targeted by mir-210 directly , renilla luciferase reporters , which include the wild - type full - length 3utr forms of mir-210 seeding sites , were used . figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells , and inhibiting mir-210 expression increased activity of the reporter renilla luciferase . expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells , and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 ( figures 6 ) . during recent decades several studies have focused on the effects of mirnas on spermatogenesis in male infertility [ 911,17 ] . however , it was not understood how mir-210 , which is one of the up - regulated mirnas in testes of patients with noa , was involved in spermatogenesis in male infertility . the transformation of diploid spermatogonia into mature haploid cells in spermatogenesis is a complex biological process in the testes of males . the insulin / igf system takes part in the processes of cell proliferation , cell growth , differentiation , and survival , which affects nearly every organ in the body . also , insulin / igf plays an important role in the proper function of the testis in males . igf2 binds to igf1r and insr - a with a high affinity and binds to insr - a / igf1r , insr - b / igf1r , but with lower affinity . found that in inactivated insr and igf1r , there was a 79% reduction in daily sperm production in adult mouse testes by a conditional ko approach . taken together , the aforementioned data suggest that igf2 might be involved in the process of spermatogenesis . to examine the specific mechanism by which mir-210 is associated with the process of spermatogenesis , quantitative real - time pcr was performed to detect mir-210 expression . we found that mir-210 was significantly up - regulated in the testes of subjects with ma and hypospermatogenesis patients compared to oa . these results agree with findings of lian et al . using microarray technologies performed in noa and normal controls . several studies have suggested that this mirna could be mediated by hypoxia and participate in various types of regulation of angiogenesis , cell survival , proliferation , cell cycle arrest , and protein modification [ 1214 ] . furthermore , some researchers even found that mir-210 might be considered as one of the indicated markers in some diseases , such as clear cell renal cell carcinoma and acute myeloid leukemia . in the present study we found that igf2 was targeted by mir-210 directly in the in vitro experiment in nt2 cells , and mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility . firstly , as some errors occurred in the rna extraction in the preliminary experiment , mir-210 of 3 testes samples were damaged and not detected in the subsequent quantitative real - time pcr experiment , which might have affected our results . secondly , we did not investigate the functions of mir-210 and igf2 in vitro or in vivo , and we plan to do this in future research . we demonstrated that mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility . future mechanistic studies on the role of mir-210/igf2 in the process of spermatogenesis in male infertility will provide new insights into the diagnosis and management of male infertility .

backgroundmicrornas ( mirnas ) play pivotal roles in spermatogenesis . microrna-210 ( mir-210 ) expression was up - regulated in the testes of sterile men with non - obstructive azoospermia ( noa ) . however , the underlying mechanisms of mir-210 involved in the spermatogenesis in patients with noa are unknown.material/methodsexpression of mir-210 and insulin - like growth factor ii ( igf2 ) in the testes of noa cases ( only including maturation arrest and hypospermatogenesis ) were detected in this study . we carried out in vitro experiments to determine if igf2 was directly targeted by mir-210 in nt2 cells.resultscompared with obstructive azoospermia ( oa ) as normal control , our results suggest that mir-210 was significantly up - regulated in testis of patients with noa ( p<0.05 ) , and igf2 was down - regulated , but without a significant difference . the results also indicated that igf2 was directly targeted by mir-210 in nt2 cells.conclusionsthe results showed that mir-210 was involved in spermatogenesis by targeting igf2 in male infertility .

Background Material and Methods Human testicular samples Immunohistochemistry Staining RNA extraction and real-time PCR Western blotting Cell lines/cell culture and transfection Statistical analysis Results IGF2 located in spermatocytes in the testis of patients with OA Down-regulation of IGF2 in testes of MA and hypospermatogenesis patients Up-regulation of miR-210 in testis of MA and hypospermatogenesis patients IGF2 was targeted directly by miR-210 Discussion Conclusions

midwife - led primary delivery care for low - risk pregnant women during labor has been reported to have various advantages , such as increased odds of high maternal satisfaction and a decrease of unnecessary medical interventions [ 18 ] . although the maternity care system for low - risk pregnant women peculiar to one country can not easily be compared with those in other countries , consumer demands for the humanization of obstetric care have arisen in various countries [ 18 ] . to date , we have found no evidence that midwife - led primary obstetric care is unsafe for low - risk pregnant women in comparison with obstetric care with the favorable cooperation of obstetricians and midwives in japan [ 912 ] . in addition , about 85% of low - risk pregnant women request that they give birth while receiving midwife - led primary delivery care . therefore , safe midwife - led delivery care with the backup of obstetricians may also be required for low - risk pregnant women in japan . if complications occur or threaten to occur during the primary midwife - led delivery care , the midwives have to refer the woman to obstetricians at the same or a neighboring hospital or private obstetric clinic as soon as possible . this is because , in deliveries managed by independent midwives in japan , many intervention measures , such as oxytocin infusion , epidural anesthesia , episiotomy , suture , and instrumental delivery , are not available based on japanese legal restrictions . in our institute , one of the main tokyo city perinatal centers , there are 3 japanese systems of midwife - led delivery care , as follows : ( 1 ) those intending to give birth at home managed by midwives who do not belong to our hospital , ( 2 ) those planning to give birth on futons ( i.e. , japanese - style bedding ) in japanese tatami mat delivery rooms in our hospital managed by the same midwives who do not belong to our hospital , and ( 3 ) those planning to give birth in japanese tatami mat delivery rooms managed by midwives who belong to our hospital . the objective of this study was to describe trends in transfers and perinatal outcomes among labors using these 3 japanese systems of midwife - led primary delivery care . the protocol for this analysis was approved by the ethics committee of the japanese red cross katsushika maternity hospital . in addition , informed consent for analysis from a retrospective database was obtained from each subject during their hospital visit . in our hospital , pregnant women who are initially considered low - risk at 3436 weeks of gestation can choose freely between the 3 systems of midwife - led care and obstetric shared care . in the midwife - led care units , midwives can practice autonomously and are fully accountable for their own practice , unsupervised by obstetricians . factors used to exclude women from the low - risk group comprise the following [ 912 ] : ( 1 ) medical history : pregnancy - induced hypertension , chronic hypertension , diabetes mellitus , renal disease , idiopathic thrombocytopenia , and other systemic illnesses ; ( 2 ) gynecological history : history of infertility therapies of in vitro fertilization , congenital uterine anomalies , uterine myomatosis , and adnexal anomaly ; ( 3 ) obstetric history : narrowing of the pelvic outlet , cephalopelvic disproportion , previous cesarean section , previous anal sphincter injury , previous postpartum hemorrhage 1,000 ml with blood transfusion , previous manual removal of placenta , previous gestational diabetes , and history of severe preeclampsia ; ( 4 ) complications during the present pregnancy : multiple pregnancy , nonvertex presentation , obesity ( maternal body mass index before pregnancy 25 and/or during the third trimester 28 ) , anemia ( hemoglobin < 9.0 g / dl ) , epilepsy with treatment , polyhydramnios , oligohydramnios , low - set placenta , placenta previa , fetal growth restriction , heavy for date fetus , gestational diabetes , and pregnancy - induced hypertension ; when risk factors are present , those women are managed by obstetricians and midwives ; ( 5 ) complications during labor : intrauterine infection , thick meconium staining , prolongation of labor such as active - phase dilation < 1 cm / hour and duration of second stage of labor 2 hours , prolonged rupture of membranes ( 24 hours ) , uterine inertia , arrest of labor , and fetal heart rate abnormality such as a nonreassuring fetal status . when these factors are present , the women are transferred to be managed mainly by obstetricians ( obstetric shared care ) in a standard western - style delivery room or surgery room in our hospital . a retrospective study was performed to examine trends in transfers and perinatal outcomes among labors that started using the 3 systems of midwife - led primary delivery care . in this study , student 's t - test was used for continuous variables and the test for categorical variables . odds ratios ( ors ) and 95% confidence intervals ( cis ) were also calculated . differences with p between 2009 and 2012 , a total of 678 low - risk women were placed in the 3 forms of midwife - led primary delivery care at the onset of labor pains and/or rupture of membranes at 3741 weeks of gestation . of these , 123 ( 18% ) intended to give birth at home , 88 ( 13% ) planned to give birth in the japanese tatami mat rooms in our hospital managed by midwives who do not belong to our hospital , and 467 ( 59% ) planned to give birth managed by the midwives belonging to our hospital . table 1 shows the clinical descriptions of the 678 pregnant women initially considered as low - risk for receiving our midwife - led primary delivery care systems . there were no significant differences in the maternal age or parity among the 3 groups . table 2 shows the rate of transfers in the 3 groups of the midwife - led primary delivery care systems . the total rate of transfers in the system run by the midwives belonging to our hospital ( 56% ) was higher than in the other 2 systems run by the independent midwives ( 31% in planned home birth : or 1.87 , 95% ci 1.23.0 , p < 0.01 ; 38% in planned hospital birth : or 2.51 , 95% ci 1.73.8 , p < 0.01 ) . in addition , the timing of transfers in the system run by the midwives belonging to our hospital ( before the second stage of labor : 52% ) was earlier than those in the other 2 systems ( 21% in the planned home birth : or 4.12 , 95% ci 2.66.6 , p < 0.01 ; 20% in planned hospital birth : or 4.29 , 95% ci 2.57.4 , p < 0.01 ) . however , if classified into nulliparous and parous women , there were no significant differences in the rate of transfers among the 3 groups , as shown in table 1 . in addition , among the 3 groups there were no significant differences in the rate of the main 2 indications for transfer : fetal heart rate abnormality and failure to progress . the main indications for transfer after delivery were maternal postpartum hemorrhage and neonatal respiratory distress associated with asphyxia . table 3 shows the obstetric and neonatal outcomes in the pregnant women initially considered as low - risk for receiving our midwife - led primary delivery care systems . there were no significant differences in these outcomes among the 3 groups our obstetric care system involves the division of women in labor into low- and high - risk groups [ 912 ] . the women who are initially considered low - risk can choose freely between midwife - led care and obstetric shared care . if complications occur or risk factors arise during labor in the primary midwife - led care , they are transferred to obstetric shared care . this may be the first report concerning the differences in the timing of transfers from midwife - led care to obstetric shared care among the 3 systems of midwife - led primary delivery care in japan . in this study , there was no evidence that the primary midwife - led care is unsafe for low - risk pregnant women in any of these 3 midwife - led delivery care systems . however , there were no significant differences in the timing of referrals from midwife - led care to obstetric shared care between the system led by midwives who belong to our hospital ( hospital midwifery system ) and the systems led by the midwives who do not belong to our hospital . in the hospital midwifery system , the timing of transfers seemed to be the earliest due to the ease of transfer within the same hospital and administrator setting . on the other hand , the rate of transfers after delivery with the other 2 systems was higher than that in the hospital midwifery care . during the period , the main indications for transfers were maternal postpartum hemorrhage and/or neonatal respiratory distress associated with asphyxia . fortunately , the difference was not associated with adverse obstetric or neonatal outcomes ; however , unfortunately , they led to early mother - to - child separation , especially in cases of planned home birth because healthy puerperal women or newborns can not be transferred from home to hospital according to japanese law . although home birth might be very comfortable , those involved must be prepared for mother - to - child separation in cases of referrals after delivery . the major limitations of this study were the small sample size and lack of long - term follow - up of mothers and children to consider the potential of the findings based on our own context . there were no cases of fetal / neonatal death under the midwife - led delivery care . the most evaluated outcome under midwife - led delivery was the satisfaction of pregnant women with the development of mother - child relationships after delivery . in addition , there might be some bias related to the backgrounds in the selection of the systems because this was not a randomized trial study . therefore , a further large prospective study with long - term follow - up may be needed . there were no significant differences in perinatal outcomes among the 3 systems ; however , there were some differences in the status of the transfers to the obstetric shared care .

objective . the objective of this study was to describe the recent clinical characteristics of labor using 3 systems of japanese midwife - led primary delivery care , as follows : ( 1 ) those intending to give birth at home managed by midwives who do not belong to our hospital , ( 2 ) those planning to give birth in our hospital managed by the same midwives , and ( 3 ) those planning to give birth managed by midwives who belong to our hospital . methods . a retrospective cohort study was performed . results . there were no significant differences in the obstetric or neonatal outcomes among the 3 groups . the rate of transfers during labor with the system involving midwives belonging to our hospital was higher than those with the other 2 systems . in addition , the timing of transfers in the system with the midwives belonging to our hospital was earlier than with the other 2 systems . among the 3 groups , there were no significant differences in the rate of the main 2 indications for transfers : fetal heart rate abnormality and failure to progress . conclusion . there were no significant differences in perinatal outcomes among the 3 systems ; however , there were some differences in the status of transfers to obstetric shared care .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

diabetes decreases the overall life expectancy and cause a heavy burden on public health ( 1 ) . moreover , the asia - pacific region is considered to be on the verge of an emerging diabetes epidemic ( 2 ) . the development of type 2 diabetes is affected by genetic and environmental determinants ( 3 ) . recently , one study investigated whether common variants of functional and positional candidate genes , including adrb3 , pparg , enpp1 and capn10 , were determinants of type 2 diabetes ( 4 ) . enpp1 , also called k121q , has a glutamine substitution for lysine at codon 121 ( 5 ) . type 2 diabetes is characterized by insulin resistance ( 6 ) , and enpp1 plays an important role in insulin resistance ( 7 , 8) . enpp1 interacts with -subunit of the insulin receptor to interrupt signaling ( 9 ) . in previous studies , the k121q polymorphism of the human pc-1 gene was strongly associated with insulin resistance ( 1 , 3 , 7 , 10 - 12 ) . however , there was no association between insulin resistance and the k121q variant ( 13 , 14 ) . in addition , there were discrepancies for the impact of enpp1 polymorphism on obesity between ethnic groups ( 5 , 10 , 13 , 15 - 19 ) . obesity increases the concentration of insulin in plasma and is the major contributor of insulin resistance ( 20 ) . obesity appears to be an effect modifier of type 2 diabetes in d1057 carriers ( 21 ) . the association of obesity with the genetic variant of the insulin receptor substrate was identified in other studies ( 21 , 22 ) . in the chinese han population , the pc-1 q121 allele was associated with insulin resistance . in women , carriers of the q allele had an increased risk for obesity development ( 3 ) . in caucasians and african - americans , 121q carriers had an association with increased body mass index ( bmi ) ( 23 ) , and the three - allele risk type haploid qdeltg with the q allele increased the risk for obesity ( 24 ) however , in the danish population , there were no differences in the distribution of frequencies of dominant types ( kk wild type and kq / qq variant type ) and alleles ( 19 ) . the complexity of type 2 diabetes is related to factors such as genetic heterogeneity , interactions between genes , and the modulating role played by the environment ( 4 ) . in spite of these limitations , studies of type 2 diabetes and genetic factors of obesity can predict the risks for development of both type 2 diabetes and obesity in order to assist primary prevention , and korea is an appropriate country for such studies because of the homogeneity of racial composition and lifestyle ( 25 ) . therefore , the aim of this study was to analyze the presence of the enpp1 polymorphism , not studied yet in korean population , to identify the association between genotypes and allele with type 2 diabetes and obesity . this company is an electric power company located at kori , yonggwang , ulchin , wolsung , and seoul in korea . there were 195 male workers ( age 48.26.7 yr , bmi 24.672.64 kg / m ) who were diagnosed as diabetics during medical examinations conducted from march 2004 to october . the 1,750 male workers ( age 45.27.7 yr , bmi 24.772.64 kg / m ) in the control group were selected randomly . subjects were included if they met one of the criteria below and their onset age was older than 20 yr old to exclude type 1 diabetes : 1 ) blood - sugar level before a meal exceeded 126 mg / dl twice or more ; 2 ) blood - sugar level before a meal exceeded 126 mg / dl once or more and blood - sugar level two hours after a meal exceeded 200 mg / dl ; 3 ) those who reported that he had a history of diabetes in the questionnaire and being taken oral hypoglycemic agents . the workers included in the obesity group were those whose bmi was 25 kg / m or more . after collecting a blood sample from vein in fasting ( 8 hr ) status , we measured the blood - sugar level , insulin , and lipid profile . we also measured height and weight to calculate bmi . if fasting glucose levels were greater than 126 mg / dl , we checked the 2 hr post - prandial plasma glucose level at each site within 1 month . height and weight were measured by autoanalyzer ( health guard , fanics , seoul , korea ) . the fasting blood level was analyzed by glucose - oxidase assay using an autochemistry analyzer . to determine the lipid profile , total cholesterol was analyzed by enzyme assay using cholesterol oxidase ( cod ) , high density lipoprotein ( hdl ) cholesterol by glycerol phosphate oxidase assay , low density lipoprotein ( ldl ) cholesterol by direct surfactant assay . we carried out this study under the approval by the ethnics committee of the asan medical center , and obtained written consent from all subjects , providing subjects with sufficient explanation to obtain informed consent . we extracted genomic dna from buffy coats using the generall blood sv kit ( general biosystem , seoul , korea ) and following the instructions suggested by the manufacturer . the method of genotyping used to identify the k121q polymorphism in enpp1 exon 4 was polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) , using dna treated with a restriction enzyme after pcr , based on the paper reported by abate et al . we carried out student 's t - test to analyze the effects of genotypes on biochemical parameters using genotypes as factors . the hardy - weinberg equilibrium was computed based on the goodness - of - fit test . we also investigated the differences in frequencies of genotypes between type 2 diabetic and normal groups , and between obesity and normal groups by fisher 's exact test . the spss 12.0 ( for window ) statistical software package was used for statistical analysis . this company is an electric power company located at kori , yonggwang , ulchin , wolsung , and seoul in korea . there were 195 male workers ( age 48.26.7 yr , bmi 24.672.64 kg / m ) who were diagnosed as diabetics during medical examinations conducted from march 2004 to october . the 1,750 male workers ( age 45.27.7 yr , bmi 24.772.64 kg / m ) in the control group were selected randomly . subjects were included if they met one of the criteria below and their onset age was older than 20 yr old to exclude type 1 diabetes : 1 ) blood - sugar level before a meal exceeded 126 mg / dl twice or more ; 2 ) blood - sugar level before a meal exceeded 126 mg / dl once or more and blood - sugar level two hours after a meal exceeded 200 mg / dl ; 3 ) those who reported that he had a history of diabetes in the questionnaire and being taken oral hypoglycemic agents . the workers included in the obesity group were those whose bmi was 25 kg / m or more . after collecting a blood sample from vein in fasting ( 8 hr ) status , we measured the blood - sugar level , insulin , and lipid profile . we also measured height and weight to calculate bmi . if fasting glucose levels were greater than 126 mg / dl , we checked the 2 hr post - prandial plasma glucose level at each site within 1 month . height and weight were measured by autoanalyzer ( health guard , fanics , seoul , korea ) . the fasting blood level was analyzed by glucose - oxidase assay using an autochemistry analyzer . to determine the lipid profile , total cholesterol was analyzed by enzyme assay using cholesterol oxidase ( cod ) , high density lipoprotein ( hdl ) cholesterol by glycerol phosphate oxidase assay , low density lipoprotein ( ldl ) cholesterol by direct surfactant assay . we carried out this study under the approval by the ethnics committee of the asan medical center , and obtained written consent from all subjects , providing subjects with sufficient explanation to obtain informed consent . we extracted genomic dna from buffy coats using the generall blood sv kit ( general biosystem , seoul , korea ) and following the instructions suggested by the manufacturer . the method of genotyping used to identify the k121q polymorphism in enpp1 exon 4 was polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) , using dna treated with a restriction enzyme after pcr , based on the paper reported by abate et al . we carried out student 's t - test to analyze the effects of genotypes on biochemical parameters using genotypes as factors . the hardy - weinberg equilibrium was computed based on the goodness - of - fit test . we also investigated the differences in frequencies of genotypes between type 2 diabetic and normal groups , and between obesity and normal groups by fisher 's exact test . the spss 12.0 ( for window ) statistical software package was used for statistical analysis . the frequencies of the kk type , kq type , and qq type in enpp1 k121q were 82.1% , 17% and 0.9% , respectively . the homozygous type of q carrier ( qq type ) was added to the heterozygous type ( kq type ) because the frequency of qq type was very low ( 0.9% ) . we investigated the differences in age , blood pressure , bmi , and results of clinical examinations according to genotypes of enpp1 k121q between the type 2 diabetic and non - diabetic groups ( table 1 ) . when type 2 diabetics and non - diabetics were pooled ( n=1,945 ) , there were no significant differences in bmi , systolic pressure , diastolic pressure , glucose value in fasting status , total cholesterol , ldl cholesterol , hdl cholesterol , triglyceride , c - reactive protein , and homa - ir between the kk type and kq+qq type . in addition , there were no significant differences in the same characteristics above between type 2 diabetic group and non - diabetic groups ( table1 ) , and between the obese group and non - obese groups ( table2 ) . the frequencies of genotypes were in accordance with the hardy - weinberg equilibrium ( p=0.85 ) . the odds ratio of having a kq+qq genotype was 0.85 for diabetics versus non - diabetics . the odds ratio of having a q allele was 0.91 for diabetics versus non - diabetics . however , there was no significant difference in the genotypic and allelic distribution between type 2 diabetics and non - diabetics ( table3 ) . the odds of a kq+qq genotype were 0.93 for obese versus non - obese subjects . furthermore , the odds of having a q allele were 0.96 for obese versus non - obese subjects . however , there was no significant difference in the genotypic and allelic distribution between obese and non - obese ( table4 ) . the frequency of the kk type in enpp1 k121q genotypes was 82.1% , that of kq+qq type was 17.9% and that of q allele was 9.4% . there was no statistically significant difference in the distribution among these genotypes and alleles ( p=0.81 , p=0.89 , respectively ) , although the 121q carrier and q allele in obese and/or diabetics seemed to differ slightly from those in the non - obese non - diabetics ( reference group ) . in determining the prevalence of the q allele carriers ( kq and qq subjects ) and q allele , there were no significant differences in the genotypic and allelic distribution with the respect to any phenotypes ( data not shown ) . after adjusting for the effects of obesity , the probability of type 2 diabetes in kq+qq type was 0.858 ( data not shown ) and that in q allele was 1.095 , showing no significant difference ( table 5 ) . moreover , after adjusting for the effects of type 2 diabetes , the probability of developing obesity in the kq+qq type was 0.936 ( data not shown ) and that in the q allele was 1.038 , showing no significant difference ( table 6 ) . the results of studies of the association between enpp1 k121q variants and both type 2 diabetes and obesity in several races are disparate . we carried out this study to investigate the association of k121q variants with type 2 diabetes and obesity in korean male workers . insulin resistance is a major component of the pathogenesis of type 2 diabetes ( 27 ) , and insulin receptor kinase activity is impaired in muscle and other insulin - sensitive tissue of many type 2 diabetic patients ( 28 ) , and then a potential inhibitor of the insulin receptor tyrosine kinase is identified as the plasma - cell membrane differentiation antigen-1 ( pc-1 ) ( 29 ) . therefore , it is significant to analyze enpp1 ( pc-1 ) polymorphism . in the previous studies of the association between type 2 diabetes and polymorphism in enpp1 , the k121q missense mutation increased the odds ratio ( or ) for type 2 diabetes in dominican ( 10 ) , south asian , caucasian ( 16 ) , finnish ( 18 ) , and french populations ( 24 ) . moreover , according to a meta - analysis of the association between enpp1 k121q variant and type 2 diabetes , the odds ratios were 1.30 ( 95% confidence interval [ ci ] , 1.13 - 1.50 ) ( 16 ) and 1.17 ( 95% ci , 1.10 - 1.25 ) ( 19 ) , showing significant association . in this study , 121q was not associated with type 2 diabetes , showing consistent results with those in the japanese population ( 5 ) , danish caucasians ( 13 ) , oji - cree population ( 17 ) , finnish population ( 18 ) , and danish white subjects ( 19 ) . most type 2 diabetes in koreans is characterized by non - obesity , thus the enpp1 k121q mutant relevant to insulin resistance possibly could be a candidate gene that is not appropriate to explain susceptibility to type 2 diabetes . this can be a possible explanation for the lack of association between enpp1 121q carrier and type 2 diabetes in this study . obesity is a main risk factor for the development of type 2 diabetes ( 20 ) and there is linear association between obesity and type 2 diabetes ( 3 ) . in previous studies of the association between obesity and polymorphism in enpp1 , 121q carriers and/or q allele were associated with obesity in the chinese han population ( bmi of obesity group 27 kg / m ) ( 3 ) , caucasians ( bmi of obesity group > 90th percentile ) , african - american adults ( bmi of obesity group > 80th percentile ) ( 23 ) , french population ( bmi of obesity group 95th percentile ) ( 24 ) , and dominican population ( bmi of obesity group 30 kg / m ) ( 10 ) . however , in this study ( bmi of obesity group 25 kg / m ) , there was no difference in distribution between obesity and 121q carriers and presence of the q allele . this result was consistent with those from a study of 7,333 danes ( 19 ) and a spanish population ( 14 ) in which the bmi of the obesity group was 25 kg / m or higher . on the other hand , in matsuoka 's study , the percentage of subjects whose bmi was 30 kg / m or higher was too low ( 2.5% ) to investigate the effect of k121q genotype on obesity . the results of the present study showed that the frequencies of the q allele was 8.7% in the type 2 diabetic group and 9.2% in the obesity group , which was lower than those in finnish and swedish populations ( 12.9 - 15.1% ) ( 11 ) , danish caucasians ( 14 - 16% ) ( 13 ) , south asians in chennai ( 14% ) , caucasians in dallas ( 16% ) , south asians in dallas ( 19% ) ( 16 ) , the dominican population ( 54.2% ) ( 10 ) and , black children ( 77% ) ( 30 ) . the frequencies of the q allele investigated in this study might have a smaller statistical power to explain any association with either type 2 diabetes or obesity with 121q carriers ( kq+qq ) and/or the q allele . in conclusion , the present study suggests that the enpp1 k121q polymorphism was not associated with type 2 diabetes and obesity . the results of negative associations in this study might be attributable to the low prevalence of obesity , relatively younger age , and low frequencies of the 121q carriers . large and prospective studies are needed to confirm this preliminary observation in the korean population .

type 2 diabetes is characterized by insulin resistance , and enpp1 plays an important role in insulin resistance . we investigated the association of the enpp1 k121q polymorphism with both diabetes and obesity ( body mass index [ bmi ] ) in korean male workers . the study design was case - control . subjects were 1,945 male workers ( type 2 diabetes , 195 ; non - diabetes , 1,750 ) of nuclear power plants who received examinations from march to october in 2004 . we collected venous blood samples under fasting ( 8 hr ) conditions , calculated bmi by height and weight , and assessed relevant biochemical factors . the results of this study demonstrated that the enpp1 121q genotype ( kq+qq types ) was not associated with type 2 diabetes ( odds ratios [ or ] , 0.854 ; 95% confidence interval [ ci ] , 0.571 - 1.278 ) or obesity ( or , 0.933 ; 95% ci , 0.731 - 1.190 ) . in addition , the frequency of the q allele was not related to type 2 diabetes ( or , 0.911 ; 95% ci , 0.630 - 1.319 ) or obesity ( or , 0.962 ; 95% ci , 0.767 - 1.205 ) . we concluded that the enpp1 121q allele is not a critical determinant for either diabetes or obesity in korean males . the discordance between the results of this study and those derived from studies of dominican , south asian , caucasian , finnish , and french populations might be due to differences in genetic backgrounds between these populations .

INTRODUCTION MATERIALS AND METHODS Subjects DNA extraction and PCR Statistical analysis RESULTS DISCUSSION

lipid apheresis provides a safe and effective means of treating patients with severe hyperlipidemia . it functions by first separating plasma from blood cells with a cell separator and then using either the adsorption of apolipoprotein b by affinity columns containing anti - apolipoprotein b antibodies or dextran sulphate , or their precipitation at low ph by heparin . lipid apheresis allows patients to attain lower levels of low - density lipoprotein ( ldl ) , which are usually not attainable with traditional drug therapy alone , while leaving high - density lipoprotein ( hdl ) levels generally unaffected . when used in conjunction with statins and other lipid - lowering drugs , lipid apheresis may also induce the regression of coronary atherosclerotic plaque in familial hypercholesterolemia ( fh ) patients . fh is a group of autosomal dominant genetic defects resulting in elevated serum ( ldl ) cholesterol levels . in the heterozygous state , fh is a relatively common but serious genetic disorder , with an incidence of about 1 in 500 persons in the general population . fh has been associated with an increased risk for atherosclerosis , premature coronary heart disease , and heart failure [ 3 , 4 ] . fh is caused by a mutation affecting apolipoprotein b , proprotein convertase subtilisin kexin type 9 ( pcsk9 ; an enzyme involved in ldl receptor degradation ) , or , most commonly , the ldl receptor gene , resulting in defective ldl receptors and/or a diminished number of ldl receptors [ 7 , 8 ] . these mutations cause ldl to be catabolized at a slower rate and thus accumulate in the circulation . currently , fh is treated using a variety of cholesterol - lowering drugs , most notably statins or hmg - coa reductase inhibitors . for many patients , however , statins are not a viable treatment option , because of either intolerance or ineffectiveness . lipid apheresis is an alternative form of treatment for these fh patients as well as those who have persistently elevated ldl levels despite treatment . because apheresis is performed at only a few highly specialized centers in relatively low volume , there is very little literature discussing the effectiveness of lipid apheresis on the reduction of lipid profiles and the prevention of future cardiac events . this study , therefore , reports the experience in a single metropolitan center of treating patients with hyperlipidemia with lipid apheresis . retrospective chart reviews were performed and questionnaire surveys were given to active lipid apheresis patients at the minneapolis heart institute ( mhi ) at abbott northwestern hospital ( anw ) , minneapolis , minnesota . mhi and anw are divisions of allina health , a large healthcare provider in minnesota and western wisconsin . patients were identified through an electronic health record ( ehr ) screen of ambulatory patients representing all patients seen at all allina health metro area and regional locations between 2009 and 2012 ( epic systems , verona wi ) . of these patients , criteria to qualify for apheresis were based on the united states food and drug administration ( fda ) approval recommendations . currently , the fda supports ldl apheresis for patients who , after six months , do not have an adequate response to diet therapy and maximum drug therapy , due to either ineffectiveness or intolerance , and meet the following criteria : functional homozygotes with an ldl cholesterol > 500 mg / dl without cad , functional heterozygotes with ldl cholesterol > 300 mg / dl without cad , functional heterozygotes with ldl cholesterol > 200 mg / dl with documented coronary heart disease . functional homozygotes with an ldl cholesterol > 500 mg / dl without cad , functional heterozygotes with ldl cholesterol > 300 mg / dl without cad , functional heterozygotes with ldl cholesterol > 200 mg / dl with documented coronary heart disease . the date of birth , gender , date of apheresis initiation , lipid disorder diagnosis , apheresis frequency , and family history of cardiac events were recorded . patients were noted as having fh if the active problem list contained a diagnosis of fh . to determine which patients had fh , we used the national lipid association ( nla ) criteria for an 80% probable fh diagnosis , using the highest ldl recorded in the patient chart as follows : age < 20 and ldl > 190 mg / dl , age 2029 and ldl > 220 mg / dl , and age 30 and ldl > 250 mg / dl . potential homozygous fh ( hofh ) patients were defined as having an untreated ldl > 500 mg / dl or a treated ( on statin ) ldl over > 300 mg / dl , in addition to clinical evidence of xanthomas before age of 10 years or having two parents with heart disease or high lipids . identifiable secondary causes for marked hyperlipidemia were excluded from the analysis by examining the ehr chart of each potential homozygote . current cholesterol lowering medications were also recorded , focusing on the use of statins , colesevelam ( welchol ) , ezetimibe ( zetia ) , niacin , and aspirin . a significant cardiovascular event was defined as a myocardial infarction ( mi ) , a percutaneous transluminal coronary angioplasty ( ptca ) or stenting procedure , or a coronary artery bypass graft ( cabg ) using ehr documented icd-9 criteria . cardiac events were separated by their occurrence before or after the patient began apheresis , and the total number of events was recorded for each group . multiple cardiac events occurring at the same hospitalization , such as mi followed by ptca , were counted as a single event for cardiac event rate calculation . pre- and postapheresis cardiac event rates were calculated by adding the total number of cardiac events and dividing by the total person years during each time period . the preapheresis time period describes the time from the first documented ehr visit to the date of apheresis initiation . the postapheresis time period describes the time from the date of apheresis initiation to the study date . unverifiable events noted in the ehr but occurring prior to the first documented ehr visit were noted but excluded from the cardiac event rate calculation . mean acute ldl reductions were calculated by averaging all recorded ldl values prior to and immediately after the treatment sessions . mean acute total cholesterol , hdl cholesterol , and triglyceride reductions were calculated by using lipid profile from the most recent treatment session . ldl apheresis was performed at abbott northwestern hospital using the kaneka liposorber la-15 system ( kaneka medical products ) . the system consists of the kaneka ma-03 machine , the integrated sulflux kp-05 plasma separator , which consists of porous hollow fibers , to separate the plasma from the whole blood , and two disposable liposorber la-15 adsorption columns to adsorb apolipoprotein b - containing lipoproteins from patient plasma . patients confirmed information in their ehr such as risk factors , answered questions relating to their awareness of fh and if their family had been previously tested for it , provided their level of satisfaction with their apheresis program , and indicated their interest in learning more about alternative treatments . the data from the questionnaires was cross - referenced with the data from the patient charts to ensure accuracy . descriptive statistics are displayed as means and sds for continuous variables ; number and percentage with characteristic are given for categorical variables . categorical variables were analyzed using pearson 's chi - square or fisher 's exact tests . continuous variables were analyzed using student 's t - test . a value of p < 0.05 was considered significant , and p values are two - sided where possible . all statistical calculations and plots were done with stata 11.2 ( college station , tx ) . institutional review board approval was obtained for data collection , follow - up , and data analysis . of these , 8 ( 72.7% ) were male , 10 ( 90.9% ) were caucasian , 1 ( 9.1% ) was african american , 10 ( 90.9% ) carried the diagnosis of fh , with 2 ( 18.2% ) patients identified as probable homozygotes , and 1 ( 9.1% ) was diagnosed as having familial combined hyperlipidemia . the average age of patients was 65.6 9.3 years , and patients had been on apheresis for an average of 6.2 7.0 years . four ( 36.4% ) patients were currently on statins while the other 7 ( 63.6% ) had a history of statin intolerance . five of 11 ( 45.5% ) patients were on a nonstatin cholesterol lowering medications , including 1 ( 9.1% ) on colesevelam ( welchol ) , 3 ( 27.3% ) on ezetimibe ( zetia ) , and 1 ( 9.1% ) on niacin . maximum ldl levels ranged from 211 to 448 mg / dl with a mean ( sd ) value of 298 80.7 mg / dl in the study group . since our ehr was implemented in 2005 , it is possible we may be underestimating the highest lifetime ldl for each patient . of the 11 participants , 9 completed the questionnaire in its entirety ; 1 patient provided answers to all questions but did not disclose risk factors and 1 patient did not complete the questionnaire . all of the patients indicated that they were aware that they likely had fh and 7 patients indicated that their immediate family had been tested for fh . the patients self - reported a total of 44 cardiac events before apheresis and 8 cardiac events after apheresis . of the 10 patients that completed the questionnaire , 4 patients were currently on statins while the other 6 were statin intolerant . eight patients ( 72.7% ) had a cardiac event documented by ehr , with 43 cardiac events occurring overall ( table 4 ) . self - reported events which were unable to be verified via the ehr were excluded from the cardiac event rate analysis . thirty - four cardiac events were documented before apheresis in 8 patients compared with 9 events in 5 patients after apheresis . after excluding cardiac events that were unverifiable , 14 cardiac events were documented in the preapheresis time period and 7 were documented in the postapheresis time period . the cardiac event rates were calculated to be 0.23 ( 0.13 , 0.39 ) events per person year in the preapheresis group and 0.10 ( 0.041 , 0.21 ) events per person year in the postapheresis group ( p = 0.064 ) . patients were observed for an average of 7.6 5.9 years before apheresis and 6.2 4.7 years after apheresis , with 60.6 total patient years before apheresis and 67.8 patient years after apheresis . this study was conducted to gain more information on lipid apheresis and evaluate the effectiveness in lowering lipid values . in addition , through chart review and patient survey , we attempted to gain a greater understanding of this patient population in terms of traditional risk factors , family awareness and screening , statin and other cholesterol medication uses , desire for additional treatment options , and ultimately cardiac events . our study shows that apheresis markedly lowers total cholesterol , ldl cholesterol , triglycerides and , to a much lesser degree , hdl cholesterol . there was a small , but statistically significant , reduction in hdl values after apheresis . many of these patients were statin intolerant and some had been using nonstatin cholesterol medications . importantly , 10/11 ( 90.9% ) participants indicated a desire to learn more about other potential treatment options , indicating that this population may indeed experience fatigue of this procedure . although taken from a small study population , our data suggests a reduction in cardiac event rate after apheresis . while not statistically significant , our data shows a strong trend towards event rate reduction . this statistical insignificance is likely explained by the study 's small sample size . with a larger population it is also important to note that the risk for cardiac events increases with age . ldl apheresis has been shown to improve endothelium dependent vasodilation [ 11 , 12 ] , microvascular flow , and myocardial perfusion . some studies [ 2 , 15 , 16 ] have also shown a significant reduction in angiographic cad , but others have not . these studies have been small , primarily nonrandomized trials . the ldl - apheresis atherosclerosis regression study ( laars ) looked at the change in plaque characteristics of patients undergoing apheresis compared to drug therapy over a period of two years . in that period , 7 out of 21 patients on apheresis had a cardiac event compared with 3 out of 21 on medication only . while this study found that apheresis arrested the progression of atherosclerosis the fh regression study found that ldl apheresis combined with simvastatin was more effective than colestipol plus simvastatin in reducing ldl cholesterol and lipoprotein ( a ) but was less effective at influencing coronary atherosclerosis . another study found that , out of 18 patients , 3 had myocardial infarctions , 1 underwent a cabg , and 12 needed coronary angioplasties within two years of beginning a combination therapy of apheresis , statins , and probucol . before beginning the combination therapy , 11 had experienced a mi , 5 had undergone a cabg , and 13 had undergone an angioplasty . the heparin - induced extracorporeal ldl precipitation ( help ) study found that help is suitable for reducing ldl concentrations and may work to reduce the burden of atherosclerosis , as there were no myocardial infarctions and a low coronary intervention rate in patients who began apheresis . due to the expensive nature of apheresis , a randomized , controlled clinical trial is needed to truly gauge the effectiveness of apheresis in reducing the occurrence of cardiac events . if apheresis is not deemed effective or is minimally effective , as some of these studies suggest , other types of treatment , such as lomitapide , mipomersen , or pcsk9 inhibitors , should be pursued . while satisfaction was generally high in our survey , patients specifically cited that this satisfaction was based on the results of apheresis and not on the process itself . many patients complained about the invasive nature of apheresis , citing bruises from the procedure and the inconvenience of reporting for treatment every two weeks . additionally , almost all patients were interested in learning more about alternative treatments , suggesting that they would prefer an alternative treatment which could match the results provided by apheresis . this study had several limitations . since lipid apheresis is an advanced treatment for an uncommon genetic disease , the limited number of patients available to participate in the study led to a small sample size . the event rate reduction was not statistically significant but showed a strong trend toward cardiac event rate reduction before and after apheresis . by defining the observational period initial time point as the first documented ehr visit , we excluded 20 events before apheresis and 2 events after apheresis from the cardiac event rate calculation . the lipid - lowering effects of apheresis are best expressed as reductions in interval means . although lipid apheresis was performed every two weeks , ldl values were not measured ever two weeks due to clinical practices . this inconsistency in measurement intervals prevents the use of more advanced measures to accurately track the effect apheresis has on ldl measurements . finally , this study focused on active apheresis patients and therefore did not include patients who had stopped apheresis or were deceased . lipid apheresis can reliably reduce ldl , non - hdl cholesterol , triglyceride , and total cholesterol levels in fh patients . our data suggest that lipid apheresis shows a strong , but not statistically significant , trend towards the reduction of cardiac events . apheresis is a viable treatment for fh patients , especially those that are statin - intolerant , due to its lipid lowering nature and its apparent reduction of cardiac events . however , there is a need for alternative treatments which are less invasive and provide easier patient access .

lipid apheresis is used to treat patients with severe hyperlipidemia by reducing low - density lipoprotein cholesterol ( ldl - c ) . this study examines the effect of apheresis on the lipid panel and cardiac event rates before and after apheresis . an electronic health record screen of ambulatory patients identified 11 active patients undergoing lipid apheresis with 10/11 carrying a diagnosis of fh . baseline demographics , pre- and postapheresis lipid levels , highest recorded ldl - c , cardiac events , current medications , and first apheresis treatment were recorded . patients completed a questionnaire and self - reported risk factors and interest in alternative treatment . there were significant reductions in mean total cholesterol ( 58.4% ) , ldl - c ( 71.9% ) , triglycerides ( 51% ) , high - density lipoprotein ( hdl ) cholesterol ( 9.3% ) , and non - hdl ( 68.2% ) values . thirty - four cardiac events were documented in 8 patients before apheresis , compared with 9 events in 5 patients after apheresis . our survey showed a high prevalence of statin intolerance ( 64% ) , with the majority ( 90% ) of participants indicating an interest in alternative treatment options . our results have shown that lipid apheresis primary effect is a marked reduction in ldl - c cholesterol levels and may reduce the recurrence of cardiac events . apheresis should be compared to the newer alternative treatment modalities in a randomized fashion due to patient interest in alternative options .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

agenesis of the inferior vena cava ( ivc ) as a cause of recurrent deep vein thrombosis ( dvt ) is uncommon . a 33-year - old male with no family history of thrombophilia , who had experienced multiple recurrent episodes of dvt over a 15-year period of unknown cause , was admitted into our hospital because of cellulitis in the right leg . congenital absence of the ivc could be a rare risk factor for idiopathic dvt , especially in young individuals . venous thromboembolism ( vte ) , which includes deep vein thrombosis ( dvt ) and pulmonary embolism , has an incidence of 1 to 3 per 1000 individuals per year in western populations.1 congenital anomalies of the inferior vena cava ( ivc ) are uncommon , and have been associated with the development of venous thrombosis of the lower limbs.2 congenital anomalies of the ivc has been reported as a risk factor for dvt , especially in individuals < 30 years old , and a concomitant thrombophilic disorder has been found in such individuals.3 we report a case of recurrent dvt in a 33-year - old man with agenesis of the ivc . the patient had experienced recurring episodes of idiopathic dvt in the right leg for 15 years . a 33-year - old man was admitted to the internal medicine department , holy family hospital , nazareth , israel , because of cellulitis in the right leg . one week prior to his admission , he complained about pain and increased local heat in the left ankle and thumb of the right leg . the patient had no history of previous trauma , surgery , insect bites , dysuria , or joint symptoms , and no family history of thrombophilia . he reported that he had ( a ) rheumatic fever without any complications when he was 19 years old , which was treated with penicillin , ( b ) been hospitalized when he was 23 years old because of infected skin ulcers on the right calf , for which he was treated by parenteral antibiotics , and ( c ) recurrent episodes of idiopathic dvt for the last 15 years . he also reported that he had not been treated with warfarin , but he had been on prophylactic enoxaparin therapy for dvt some years ago which has since been stopped and that he had been recently treated with allopurinol and colchicine for a presumed diagnosis of gout . he had been investigated several times for a primary hypercoagulability state , and the results were negative . on examination , the most outstanding clinical findings were swelling of ankles , mild edema , redness , and increased temperature of the right ankle and calf with trophic skin changes ( skin discoloration with ulcers ) , and superficial varicose veins in the lower abdomen ( figure 1 ) . the clinical laboratory findings ( erythrocyte sedimentation rate , leukocyte and platelet counts , and plasma hemoglobulin , plasma protein c , plasma protein s , fibrinogen , and antithrombin iii levels ) , the results of the kidney and liver function tests , and resistance to activated protein c were all normal . polymorphisms of the genes that encode for methylenetetrahydrofolate reductase were not detected , and the factor v leiden and prothrombin mutations g20210a were absent . the results of the clinical immunological studies for complement c3 and c4 and rheumatoid factor were negative , and no circulating titers for antinuclear antibody , antineutrophil cytoplasmic antibody , and cardiolipin antibody were found . cultures from the infected skin ulcers of the right leg were positive for methicillin - resistant staphylococcus aureus ( mrsa ) . ultrasound imaging of the leg veins showed a previous dvt in the right common femoral vein , and dilated superficial inguinal veins . computer tomography with contrast of the abdomen showed agenesis of the infrarenal segment of the ivc ( figure 2 ) with dilated azygos and hemiazygos veins ( figure 3 ) . there were also varicose veins in the abdominal wall and right groin , which were associated with dilated superficial and collateral veins ( figure 4 ) . transthoracic echocardiography of the patient s heart revealed mild atrial enlargement and good systolic function of the left ventricle , and no pathological valvular flows . the patient was diagnosed as having agenesis of the infrarenal segment of the ivc and dvt of the right leg without concomitant risk factors for vte . since we attributed the agenesis of the ivc to be the underlying cause of the recurrent episodes of the dvts , the patient was started on anticoagulant therapy ( subcutaneous enoxaparin 160 mg / day ) for dvt , antibiotic therapy ( intravenous vancomycin 1.5 g / day for mrsa skin infection ) , and referred to a vascular surgeon specialist but the patient refused . at follow - up in the internal medicine clinic , the most outstanding clinical findings were swelling of left ankle , redness , with trophic skin changes , and a mild improvement of the skin ulcers . despite several phone calls for follow - up the normal ivc is composed of 4 segments : hepatic , suprarenal , renal , and infrarenal . since many transformations can occur during the formation of the ivc such anomalies occur in 0.3% of otherwise healthy individuals , and in 0.6% to 2% of patients with other cardiovascular anomalies.4 ruggeri et al reported 10 years ago 4 cases of congenital absence of the ivc in 75 young patients with idiopathic dvt over a 5-year period , and estimated that 5% of young patients with dvt had an anomaly of the ivc.5 venous thrombosis is caused by the presence of isolated or combined risk factors . almost 150 years ago , the nineteenth century pathologist rudolf virchow described 3 critically important causes of venous thrombosis : venous damage , coagulation defect(s ) , and venous stasis.6 individuals with a congenital anomaly of the ivc are typically asymptomatic , and the anomaly is usually detected incidentally during radiological or abdominal procedures . congenital absence of the ivc is infrequently associated with thromboembolic events.5 patients who suffer from congenital anomalies of the ivc usually develop a compensatory circulation through the azygos veins or collateral abdominal veins in order to keep the venous return near normal levels.7 most reported cases of congenital anomalies of ivc cases have been linked to thrombophilia disorders.3,5,7 however , the true prevalence of thrombophilia in congenital anomalies of the ivc is unknown because the screening for thrombophilia in patients with an ivc anomaly was usually incomplete.3 anticoagulants , but not thrombolytic therapy , are usually prescribed for venous thrombosis , but the duration of the anticoagulant therapy is not well established . hence , anticoagulant therapy for an indefinite duration will probably be prescribed , unless vascular reconstructive surgery is done on the anomalous ivc . such surgery has been rarely reported , and its long - term outcome is undetermined.8 congenital anomalies of the ivc may cause recurrent dvt , especially in young individuals .

background : agenesis of the inferior vena cava ( ivc ) as a cause of recurrent deep vein thrombosis ( dvt ) is uncommon.case:a 33-year - old male with no family history of thrombophilia , who had experienced multiple recurrent episodes of dvt over a 15-year period of unknown cause , was admitted into our hospital because of cellulitis in the right leg . computer tomography with contrast of the abdomen showed an absence of ivc.conclusion:congenital absence of the ivc could be a rare risk factor for idiopathic dvt , especially in young individuals .

Background: Case: Conclusion: Background Case Conclusion

an exponential rise in alzheimer 's disease ( ad ) prevalence rates is predicted to parallel the aging of baby boomers creating a potentially unsustainable economic burden to the healthcare system . delaying the onset or progression of ad , even modestly , by earlier pharmacological intervention could substantially reduce the economic and psychosocial impact of the illness [ 1 , 2 ] . unfortunately , many ad patients remain undiagnosed or go undetected until the later stages of disease . insights into the underlying pathological mechanisms involving beta - amyloid plaque deposition within the brain have led to the development of a host of antiamyloid agents that are in various stages of clinical investigation . there is now a scientific consensus that the pathological events in ad initiate decades before clinical symptoms become apparent , and if disease modification is realized in the coming decades , the need for improved methods of early detection prior to the overt clinical signs will be accentuated . traditionally , neuropsychological measures , particularly those that tap cognitive abilities subsumed by the hippocampal formation such as episodic memory , have shown usefulness in identifying cognitively normal elders who subsequently develop ad [ 4 , 5 ] . decrements in semantic memory and concept formation have been shown to occur nearly a decade before the development of ad . performance on visual - spatial and verbal memory measures in midlife have also been shown to predict later memory loss . however , individuals with very high premorbid intellectual abilities experiencing incipient cognitive decline may go undetected , and false positives are possible in individuals with a low level of intellectual abilities . also appropriate interpretation of extensive neuropsychological testing requires a high degree of expertise and training , which limits its use in routine clinical settings . the advancement of molecular imaging tracers that bind to amyloid , such as pittsburgh compound b ( pib ) or longer - lived probes ( e.g. , fddnp ) , offers a non - invasive in vivo method to detect and quantify brain amyloid deposition [ 8 , 9 ] . however , this approach for presymptomatic detection is economically impractical for routine use given the current costs and restrictions on medically necessary use . similarly , biomarkers including a142 and phosphorylated tau ( also implicated in ad pathology ) in cerebral spinal fluid ( csf ) can predict subsequent cognitive decline [ 10 , 11 ] , but lumbar puncture carries risks and is inconvenient for wide - scale use in cognitively impaired elderly subjects . blood - based biomarkers have more practical applicability for routine use and are likely to be more cost effective than both csf and imaging procedures . consequently , measurement of a140 and a142 in blood is increasingly being explored and shows potential in identifying individuals at the preclinical stage of ad [ 1214 ] . it has been reported that csf a levels are subject to high diurnal fluctuations with extremely high variability reported over 12 hours . over days and weeks , furthermore , serum contains more a than plasma , possibly due to the release of bound a during the clotting process . hence , serum a appears suitable for use in predicting mci / ad and optimal sensitivity , and specificity is probably achievable if combined with current diagnostic procedures , such as brief neuropsychological testing . in this study , we examined the usefulness of brief neuropsychological tests in combination with blood a140 and a142 as a predictive test for detecting mci / ad in at - risk older adults at a pre - symptomatic stage . such an approach will be more practical for clinical use and be germane in designing large - scale prevention trials . participants included a subset of subjects enrolled in the alzheimer 's disease anti - inflammatory prevention trial ( adapt ) . adapt was a randomized , placebo - controlled , multicenter primary prevention trial sponsored by the national institute on aging . subjects were randomly assigned to one of three groups : celecoxib ( 200 mg b.i.d . ) , naproxen sodium ( 220 mg b.i.d . ) , or placebo . full details of data collection , measurements , and study procedures are available at http://www.jhucct.com/adapt/manall43.pdf and described elsewhere . the inclusion criteria for adapt subjects were age of 70 or older at enrollment , a self - reported family history of ad - like dementia , and normal cognitive performance on a brief battery of neuropsychological tests . recruitment for adapt began in 2002 , and the study was completed in 2007 . in 2005 , the roskamp institute initiated a proteomic ancillary study ( f. crawford , pi ) involving blood draw from these subjects . the inclusion criteria for this ancillary study stipulated that each subject was an active adapt participant and had met all the adapt inclusion and exclusion criteria . a separate consent was also obtained from each subject who participated in the ancillary study . two hundred and fifteen subjects from the roskamp adapt cohort enrolled in the proteomic ancillary study . at the time of blood draw , subjects maintained cognitively normal status as determined by their performance on an annual cognitive assessment battery . blood was collected during the semi - annual followup visits , and the cognitive assessments were performed at the baseline visit and at the annual visits . the time from baseline cognitive testing to the diagnosis of mci / ad was 4.06 years ( 1.3 sd ) . timeframe from baseline cognitive testing to blood draw was 2.25 years ( 0.71 sd ) and from blood draw to diagnosis was 1.79 years ( 1.2 sd ) . the cognitive measures completed at baseline and annual followup included the modified mini - mental state examination ( 3ms ) ; the hopkins verbal learning test - revised ( hvlt - r ) ; digit span ( forward and backward ) from the wechsler adult intelligence scale - revised ( wais - r ) ; a generative verbal fluency test ( supermarket items ) ; the narratives from the rivermead behavioral memory test ( rbmt ) ; the brief visuospatial memory test - revised ( bvmt - r ) . the mini - mental state examination ( mmse ) was extracted from 3ms . alternate forms were utilized annually for the hvlt - r , rbmt , and bvmt - r on each subsequent annual visit . subjects also completed the 30-item geriatric depression scale and a self - rating scale of memory functions . collateral respondents completed the dementia severity rating scale ( dsrs ) . due to significant intercorrelations between these tests , analyses described below are limited to those baseline cognitive tests that were sensitive to early changes ( i.e. , verbal learning and memory ) associated with mci / ad or tests that were similar to those previously shown to be associated with a levels . normative data from the cache county study was used to develop the standardized cut - off scores utilized in adapt . individuals who scored below the cut scores on annual cognitive assessments underwent further dementia workup including physical and neurological examinations , laboratory studies ( i.e. , cbc , chemistry count , sedimentation rate , vitamin b12 and folic acid levels , thyroid test , and syphilis serological test ) , and neuroimaging ( i.e. , mri or ct ) , as applicable . a more comprehensive neuropsychological assessment was also administered by a neuropsychologist as part of the dementia work - up . this battery of tests consisted of the expanded consortium to establish a registry for alzheimer 's disease ( cerad ) battery ; logical memory i and ii of the wechsler memory scale - revised ; benton visual retention test ( benton ) ; a generative fluency test ( animals ) ; control oral word association test ( cowat ; cfl ) ; the trail making test ; symbol digit modalities test ( smdt ) ; shipley vocabulary . following completion of all components of the dementia work - up , a consensus team determined cognitive status using published diagnostic criteria . the diagnosis of ad was made using nincds - adrda and amnestic mild cognitive impairment ( mci ) using petersen criteria . all mci patients were considered to be amnestic mci , as they only had memory impairment , but maintained normal activities of daily living and overall had a well - preserved cognition in other cognitive domains . ample evidence indicates that amnestic mci patients may be in a transitional stage between normal aging and ad with 85% of these subjects converting to ad over a 7-year period . additional evidence comes from an imaging study which demonstrated that the pattern of brain atrophy in amnestic mci patients is typical of that observed in ad patients . it is then reasonable to combine these diagnoses in a single category , thus allowing a large enough numbers to supply statistical power . of the 215 subjects who gave blood for the ancillary study , two developed non - ad dementia , and of the remaining subject pool of 208 used in these analyses , 28 subjects met criteria for either ad ( n = 10 ) or mci ( n = 18 ) in the two years following blood draw . the serum a content was determined , as per manufacturer 's instructions , using the elisa kits for human a140 and a142 and the inter - assay cv , and the intraassay cv was reported to be 10% ( invitrogen , calif ) . dna was extracted from whole blood for apoe genotyping using pure gene kits ( gentra systems , calif ) , and apoe genotyping was performed using previously established methods , as described elsewhere . apoe genotypes were unavailable for 4 individuals , but these were included in the analyses . the data set was range checked , and prior to analyses , the dependent and independent variables were examined for missing data , outliers , and violations of the normalcy assumption . differences among groups on demographic variables , neuropsychological variables , and serum a140 levels were examined using either the student 's t - test or analyses , depending on the type of variable measurement . time - updated cox regression modeling was used to test whether neuropsychological test scores , a , or a combination of both can predict conversion to mci / ad in individuals who were cognitively normal at baseline . potential confounding variables shown to impact risk for cognitive decline included age , education , gender , apoe status , serum creatinine , triglycerides , presence of apoe 4 allele , and history of vascular disease as determined by treatment with statins or antihypertensive medication which were entered as covariates . the latter variables , coded dichotomously , have been previously shown to impact a levels . because previous analyses revealed a nonsignificant increase of ad risk with naproxen in this cohort , we also controlled for this effect . logistic regression modeling was employed to construct receiver operator curves ( roc ) to examine the predictive performance of neuropsychological measures from the baseline visit and serum a levels in diagnoses of mci / ad . roc curve comparisons were based on area under the curve ( auc ) , se , and the associated 95% confidence interval ( ci ) . we subsequently calculated sensitivity of the various models using the predicted probability of each subject by logistic regression modeling with specificity of at least eighty percent . post hoc power calculations using the g - power software for multivariate regression analyses utilized here suggest a power of nearly 100% at the alpha value 0.05 for the current sample size , total number of predictors , and the observed effect size . the mean age and education of the sample was 76.7 ( sd = 3.9 ) and 14.6 ( sd = 2.8 ) years , respectively . the majority of the sample was caucasian ( 98.1% ) , and 51.9% were male . despite the cohort 's self - report of enriched family history , less than one - third of the total sample ( 31.7% ) carried at least one apoe 4 allele , a frequency similar to the general population . comparisons on variables between subjects who remained cognitively normal and those who declined over the short follow - up period are reported in table 1 . although all subjects at enrollment performed within the normal limits based on the established cut - off scores , those that ultimately declined had generally poorer scores on the 3ms , mmse , and all memory measures . the two groups were also significantly different on serum a142 levels and a142/a140 ratios prior to diagnoses of mci / ad . only 23% of the cognitively normal individuals had serum a142 in the lowest quartile compared to the nearly 50% of the diagnostic group ( 44% of mci subjects and 50% of ad subjects ) . time - dependent cox regression analyses were performed to examine the relationship between these cognitive tests and a on the prediction of subsequent conversion to mci / ad . all neuropsychological analyses were adjusted for age , gender , and education , but no adjustment for the study medications was required as these were baseline scores . cox regression analyses show that the model using neuropsychological tests predicted mci / ad ( 2 log - likelihood = 206.51 , = 52.11 , df = 8 , p < .001 ) . significant individual neuropsychological measures were 3ms ( = 0.25 0.06 , wald = 17.78 , p < .001 ) ; generative verbal fluency ( = 0.12 0.04 , wald = 8.09 , p < .004 ) ; hvlt - r scores ( = 0.24 0.11 , wald = 4.58 p < .032 ) . cox regression analysis showed that a142 measured in the lowest two quartiles compared to the highest quartile was a significant individual predictor of conversion to mci / ad in this model ( 2 log - likelihood = 197.47 , = 38.41 , df = 15 , p < .001 ) . the regression analysis utilizing the a142/a140 ratio found similarly significant results ( 2 log - likelihood = 204.69 , = 36.10 , df = 14 , p < .001 ) with the lowest ratios being most predictive of subsequent conversion to mci / ad . the final full model , adjusting for confound and the study medications , included hvlt - r , fluency , 3ms , a142 levels , and a142 quartiles ( 2 log - likelihood = 166.25 , = 74.55 , df = 18 , p < .001 ) with fluency , 3ms , and a142 in the lowest two quartiles as significant individual predictors of mci / ad in the model . similar results were observed when a140 levels and a142 quartiles were substituted in this model with a142/a140 ratios ( 2 log - likelihood = 168.49 , = 72.90 , df = 17 , p < .001 ) . baseline values for the 3ms , hvlt - r , and generative verbal fluency scores were subtracted from those obtained at the 12-month repeat testing to determine if changes in these measures differ by a142 and a142/a140 ratios . in unadjusted analyses , among subjects who converted to mci / ad , the greatest decline for hvlt - r was observed among individuals with the lowest quartile of a142 ( 1.17 , 2.33 sd ) and a142/a140 ratios ( 0.75 , 2.63 sd ) where individuals in the highest quartile of a142 ( 1.33 , 1.86 sd ) and a142/a140 ratios improved by nearly one point ( 0.6 1.82 sd ) . however , these differences were not statistically significant ( p > .05 ) . for the 3ms scores , among subjects who converted to mci / ad , those with a142 in the lowest quartile declined ( 1.83 1.28 sd ) as compared to the highest quartile ( 4.83 1.35 sd ) , and this difference was statistically significant ( f = 3.42 , p = .033 ) . for mci / ad subjects with the lowest quartile of the a142/a140 ratios , the 3ms values remained ultimately unchanged ( 0.16 1.20 sd ) , while the scores improved among those with the highest quartile of the a142/a140 ratios ( 4.33 1.20 sd ) , and these differences were also statistically significant ( f = 3.10 , p = .046 ) . for generative verbal fluency test , a decline was noted in both the lowest quartile ( 4.17 1.40 sd ) and the highest quartile ( 1.17 2.13 sd ) of a142 , and these differences were marginally significant ( f = 2.63 , p = .073 ) . for a142/a140 ratios , a similar pattern was observed , but this difference was not statistically significant . among individuals who remained cognitively normal , while a similar pattern was observed , those with lowest quartile of a142 and a142/a140 ratios had a larger decline than those with the highest quartile for each hvlt - r ( 0.28 0.27 sd versus . 0.14 0.33 sd , respectively . ) and 3ms ( 1.02 0.51 sd versus 0.39 0.44 sd ) . however , due to the small magnitude of the change in these scores , these differences were not statistically significant . no such change was observed for the generative verbal fluency test ( data not shown ) . examination of sensitivity and specificity using roc analysis revealed the auc for neuropsychological testing with age , education , and gender as covariates was 0.83 ( 95% ci [ 0.750.91 ] , p < .001 ) . for a142 ( adjusted for presence of apoe 4 allele , vascular risk factors , and associated medications ) , the auc was 0.79 ( 95% ci [ 0.700.88 ] , p < .001 ) . when neuropsychological testing ( 3ms , hvlt - r , and generative verbal fluency ) and a142 were combined , the auc was increased to 0.91 ( 95% ci [ 0.860.95 ] , p < .001 ) . for the adjusted ( as above ) a142/a140 ratios alone , .001 ) , and when combined with the neuropsychological measures , auc was 0.91 ( 95%ci [ 0.870.96 ] , p < .001 ) . optimal sensitivities with specificity of at least 80% predicted probabilities are shown in table 2 . the highest sensitivity and specificity was achieved using a combination of cognitive scores and a142/a140 ratio , but this finding was driven by a142 . the pathogenesis of ad is initiated before the clinical symptoms of cognitive impairment and functional decline become apparent in its victims . a simple and pragmatic method for identifying older adults at an increased risk for mci / ad who may benefit from targeted prevention is therefore of importance in reducing the burden of ad . the combination of brief neuropsychological tests along with blood - based biomarkers of ad represents a reasonable approach with a potential for wide - scale use . our findings here provide support for this notion and demonstrate that early prediction of risk for developing mci / ad may be feasible via a combination of brief neuropsychological tests and biomarkers in an at - risk cohort . in this subcohort from adapt , measures of global cognitive function ( 3ms ) , episodic memory ( hvlt - r trial 4 ) , language fluency , and serum a142/a140 ratio achieved an excellent accuracy of 91% . furthermore , sensitivity with specificity of at least 80% for the combined measures was superior to neuropsychological measures or to serum a levels alone . we have recently shown that a levels alone can predict mci / ad , but a levels are influenced by vascular disease and associated medications and require adjustment to observe the full impact of a in predictive modeling . we have also shown that in subjects diagnosed with ad , there is an association between measures of language tests of fluency and object naming and a140 and that memory performance is associated with serum a142 . an association between serum a140 and cognitive measures of memory and language has also been reported in cognitively normal older adults . high baseline a142 and a140 with stable a142 over time is shown to be associated with diminishing cognition . more recently , yaffe and colleagues demonstrated that low a142/a140 ratios predict cognitive decline over 9 years . in our study , we demonstrate that low a142 and a142/a140 ratios are associated with cognitive decline even within one year . this is extremely valuable from the clinical perspective , as the ability to identify at - risk individuals within a year prior to the onset can significantly improve the quality of care and the recruitment strategy for prevention trials by redirecting those individuals who may not benefit from preventive therapies towards more suitable clinical intervention . this is demonstrated by recent adapt findings , which suggest that individuals with low baseline cognitive scores converted soon after the trial initiated and that neither naproxen nor celecoxib intervention was beneficial to these individuals . collectively , these findings suggest that combining cognitive tests with blood a may be useful for predicting future mci / ad , which to date has not been explored , particularly as either a or the cognitive tests alone may not have the desired sensitivity or specificity for prediction of future mci / ad . this current work presented here provides evidence that the combination of brief neuropsychological tests and blood a has potential utility in predicting mci / ad at least 2 to 4 years prior to the clinical classification of mci or diagnosis of ad . in addition , our findings also demonstrate the importance of accounting for factors such as apoe , vascular risk factors , and medications when using a in predicting mci / ad . although at present no studies have reported sensitivity and specificity of csf a142 in predicting mci / ad conversion from normal cognition , a large multicenter study has shown that csf a142 predicts transition from mci to ad , while tau alone achieved a high sensitivity ( 83% ) with acceptable specificity ( 72% ) . it is interesting to note that our findings using blood and cognitive tests , a far less invasive method , resulted in higher sensitivities and specificities for predicting cognitive decline in at - risk cognitively normal older adults . despite the limitation that blood sampling was not conducted at the same time point as the cognitive testing , our data provide strong support for further evaluation of this approach , particularly as we have not seen significant fluctuations in a levels over a one - year period ( pers . our study provides support that blood - based a levels may have diagnostic utility when combined with neuropsychological measures . this proposed method warrants further investigation to determine its practical applicability in specialized clinic setting by allied health personal and in routine primary care clinics .

we examined the usefulness of brief neuropsychological tests and serum a as a predictive test for detecting mci / ad in older adults . serum a levels were measured from 208 subjects who were cognitively normal at enrollment and blood draw . twenty - eight of the subjects subsequently developed mci ( n = 18 ) or ad ( n = 10 ) over the follow - up period . baseline measures of global cognition , memory , language fluency , and serum a142 and the ratio of serum a142/a140 were significant predictors for future mci / ad using cox regression with demographic variables , apoe 4 , vascular risk factors , and specific medication as covariates . an optimal sensitivity of 85.2% and specificity of 86.5% for predicting mci / ad was achieved using roc analyses . brief neuropsychological tests and measurements of a142 obtained via blood warrants further study as a practical and cost effective method for wide - scale screening for identifying older adults who may be at - risk for pathological cognitive decline .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

we compared serum polychlorinated dibenzo - p - dioxins ( pcdds ) and polychlorinated dibenzofurans ( pcdfs ) among residents of two homes to levels among age- and sex - matched comparison subjects . the residents of the two homes consumed contaminated eggs and beef from animals raised at the homes . the animals had greater soil contact than those raised with conventional commercial husbandry practices . the comparison subjects were from a similar rural area , but did not consume home - produced beef and eggs . serum levels of 2,3,7 , 8-substituted tetra- , penta- , and hexacdds and penta- , hexa- , and heptacdfs were increased between 2- and 6-fold in residents from one home ; contaminated eggs and beef were consumed by residents for 2 - 15 years . elevations were less for those in the other index home , where only home - produced eggs were consumed for 2 years ; a 3-fold elevation of 1,2,3,7,8,9-hexacdd as compared to controls was most apparent . very strong bivariate correlations among all of the 2,3,7 , 8 penta- and hexacdds / cdfs were observed . the elevations observed verify that pcdd / pcdf - contaminated food contributed to the body burden of these compounds . the blood levels among the highest exposed participants are generally higher than those observed in other studies of u.s . contaminated - fish consumers and higher than average adipose tissue levels observed in u.s . urban populations . there are sufficient animal toxicologic and human epidemiologic data to recommend that exposures be reduced . in the study area , pentachlorophenol and pentachlorophenol incineration sources have been identified , and the animal contamination and blood elevations probably reflect these sources . soil reference values and site - specific risk assessments should include estimates of exposures to contamination in home - produced animal products . such estimates can be verified with limited pcdd / pcdf sampling of animals and humans.imagesfigure 1figure 2

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the wnt/-catenin pathway has been implicated in the development and progression of melanoma and a wide range of cancer types , including colorectal cancer , breast cancer , esophageal carcinoma , and liver cancer [ 13 ] . under normal conditions , increases in expression and binding of certain wnt ligands to frizzled receptor or mutations in specific components of the -catenin degradation assembly deactivate this regulatory mechanism . nuclear -catenin stimulates transcription of a large number of tcf/-catenin responsive genes that include cyclin d1 , c - myc [ 5 , 6 ] , and the melanocyte - specific gene , microphthalmia - associated transcription factor mitf - m . thus , accumulation of nuclear -catenin as observed in several cancer types is considered a marker of canonical wnt/-catenin pathway deregulation and unfavorable prognosis [ 3 , 8 ] . previous studies have reported an association between nuclear -catenin accumulation and melanoma progression and suggested nuclear -catenin to be a marker of poor prognosis [ 1 , 7 ] . however , recent studies have shown that contrary to breast and colon cancer , metastatic progression of melanoma is associated with decreases in nuclear and cytoplasmic -catenin expression [ 9 , 10 ] . moreover , clinical , genetic , and histological studies suggest that nuclear and cytoplasmic -catenin may be used as biomarkers of good prognosis in melanoma [ 1114 ] . recently , hhr6 , a human homologue of the yeast rad6 gene and a principal component of the postreplication dna repair pathway , has been identified as an important regulator of canonical wnt/-catenin signaling [ 15 , 16 ] . hhr6 , referred hereafter as rad6 , stabilizes -catenin by polyubiquitin modifications that render -catenin resistant to 26s proteasomal degradation . furthermore , rad6 is a transcriptional target of -catenin , thus revealing a positive feedback loop between -catenin - mediated activation of rad6 gene expression and rad6-induced -catenin stabilization . rad6 expression is low in normal breast tissues ; however , increases in rad6 protein expression are detected in hyperplastic , ductal carcinoma in situ ( dcis ) and invasive breast carcinomas . we have previously demonstrated a role for rad6 in breast cancer progression through its regulatory effect on the canonical wnt/-catenin pathway . since the decrease / loss of nuclear -catenin [ 9 , 10 ] , rather than increases as in breast cancer , is linked to melanoma progression , it is not known whether rad6 and -catenin work in concert to promote melanoma pathogenesis . furthermore , rad6 expression in the skin has not been investigated , and there are no data on the role of rad6 in the pathogenesis of benign ( nevi ) and malignant ( melanoma ) melanocytic lesions . it is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because rad6 and -catenin have been identified as therapeutic targets [ 19 , 20 ] . in this study , we examined rad6 and -catenin expressions in serial sections of nevi , primary , and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression . our data suggest that membranous relocation of -catenin and upregulation of rad6 are independent markers of melanoma development and progression . we also offer a hypothesis that explains the role of membranous -catenin relocation and decreasing cytoplasmic -catenin in melanoma development , a phenomenon that has been linked to unfavorable prognosis [ 9 , 21 , 22 ] . cases were retrieved from the files of the pinkus dermatopathology laboratory ( pdl ) , a private dermatopathology laboratory located in monroe , mi . preserved paraffin - embedded tissue specimens collected for each case were assigned an accession code that excluded patient identifier information . nevus and primary melanoma cases were selected for study using random numbers generated by a uniform random number generator ( stata / mp 13.1 ) . the study groups consisted of 30 cases of melanocytic nevi , 29 cases of primary cutaneous melanoma , and 29 cases of metastatic cutaneous melanoma . the study includes all metastatic cutaneous melanoma samples that were archived between 2010 and 2012 . the number of cases for each nevus and primary melanoma subtype was determined to reflect the lesion 's relative representation in cases obtained at the pdl during the above period . atypical nevi were diagnosed using criteria originally proposed by clark and lesion architecture as reviewed by roth et al . . primary antibodies used in the study are as follows : ( i ) anti--catenin ( is702 ) was purchased from dako ( glostrup , denmark ) and used in an undiluted form ; and ( ii ) anti - rad6 ( ab31917 ) was purchased from abcam ( cambridge , ma ) and used at a 1 : 500 dilution . in humans , the yeast homologous rad6 gene is duplicated and the proteins encoded by the two genes hhr6a ( or rad6a ) and hhr6b ( rad6b ) from chromosomes xq24-q25 and 5q23-q31 , respectively , share 95% identical amino acid residues . neither ab31917 , our own rad6 antibody , nor any other commercially available anti - rad6 antibody is currently able to distinguish between rad6a and rad6b proteins . therefore , rather than referring as rad6a or rad6b , we refer to the protein detected by the antibody as rad6 . briefly , five - micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol . for antigen retrieval , sections were microwaved in citrate buffer ph 6.0 ( biogenex , san ramon , ca , usa ) for 12 min at 95c and cooled for 30 min prior to immunostaining . sections were incubated with 3% hydrogen peroxide for 15 min , followed by incubation with primary antibody for 60 min . an automated immunostainer ( i6000 ; biogenex ) was utilized for subsequent incubation steps : sections were incubated in multilink biotinylated anti - igg for 20 min , horseradish peroxidase conjugated secondary antibody for 20 min , followed by development with 3-amino-9-ethyl - carbazole for 10 min ( biogenex ) . all incubation steps were performed at room temperature , and sections were washed with tris - buffered saline between incubations . lung and colon cancer tissues were included as positive controls for immunostaining with anti--catenin antibody , and breast cancer tissues were included as positive controls for staining with anti - rad6 antibody . stained sections were independently enumerated by two coauthors ( d. r. mehregan and m. campbell ) , who were blinded to patient medical records for each case . blinded enumeration was performed under light microscopy at 400x magnification , and an ocular grid consisting of a simple square lattice of 100 test points was utilized to count the number of positively and negatively stained melanomas or nevus cells for each section . per section , the total number of positively and negatively stained cells was counted for each of three sequential horizontal fields . the mean value of the three fields was used to estimate the relative density of cells in a specimen . to increase assessment accuracy , all positively and negatively stained melanomas or nevus cells in a visual field when independent readings for positively stained cells differed by 20% for a given section , both evaluators reviewed the section together to establish a consensus reading . a specimen was considered negative if less than 4% of the cells were immunostained for rad6 or -catenin . a tumor was considered to be stained with high intensity if > 50% of the cells in a specimen expressed rad6 or -catenin , similar to the criteria used by mineta et al . . kruskal - wallis tests were used to compare groups on the basis of continuous variables such as age and percent positive cells . chi - square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and -catenin localization . spearman 's rank correlation was used to assess the pairwise association between age and percent of rad6 positive and -catenin positive cells . multinomial logistic regression was used to assess the simultaneous association of rad6 and age with diagnostic group . adjustments were made for multiple comparisons using wilcoxon rank sum tests with bonferroni correction for pairwise comparisons . our analysis included 30 individuals diagnosed with nevi , 29 with primary melanoma , and 29 with metastatic melanoma ( table 1 ) . these groups differed marginally with respect to gender ( p = 0.08 ) and significantly in age ( p = 0.0001 ) . significant age differences were observed between individuals with nevi and those with either primary melanomas ( p = 0.02 ) or metastatic melanomas ( p = 0.0001 ) . in contrast , no age difference is observed between individuals with primary and metastatic melanomas ( p = 0.27 ) . significant differences in age were also observed between groups defined by -catenin localization ( p = 0.007 ) . individuals with -catenin localized in the cytoplasm were significantly younger than individuals with -catenin localization at the cell membrane ( p = 0.02 ) and marginally younger than individuals with -catenin localization at both the cytoplasm and the cell membrane ( p = 0.05 ) . when age was categorized as < 50 , 5060 , and > 60 years , there were statistically significant differences in rad6 expression between the groups ( p = 0.0008 ) , although there is substantial variability ( table 2 ) . median rad6 is greater in the group of people older than 60 years compared to the 5060 years old group ( p = 0.04 ) and < 50 years old group ( p = 0.001 ) . a 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases , and these cases were evaluated together to establish a consensus reading . melanoma development and progression were not associated with significant changes in percentage of specimens expressing -catenin . -catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas . also , the percentages of nevi ( 93% ) , primary melanoma ( 97% ) , and metastatic melanoma ( 93% ) that expressed -catenin in more than 50% of the cells did not differ significantly ( figures 1 , 2 , and 3 ) . however , significant differences were observed between the percentages of nevi ( 59% ) , primary melanoma ( 90% ) , and metastatic melanoma ( 56% ) that expressed -catenin in more than 90% of the cells ( p = 0.02 ; figure 2 ) . these differences were greatly impacted by the percentage of primary melanomas ( 48% ) that expressed -catenin in 100% of the cells , which was approximately twofold higher than the percentages of nevi ( 21% ) or metastatic melanoma ( 26% ) ( data not shown ) . none of the nevi or melanomas expressed -catenin in the nucleus ( figure 1 ) . the percentage of tumors that expressed membranous -catenin increased dramatically from nevi ( 10% ) to primary and metastatic melanomas ( 83% and 93% , resp . ; concurrently with this increase , the percentage of tumors that expressed cytoplasmic -catenin decreased from nevi ( 90% ) to primary and metastatic melanomas ( 45% and 38% , resp . ; in contrast , no significant differences were observed between the percentages of primary and metastatic melanomas that expressed -catenin at either the membrane ( 83% and 93% , resp . ; p = 0.289 ) or the cytoplasm ( 45% and 38% , resp . ; p = 0.633 ; figure 4 ) . while all four nevi types ( junctional , intradermal , compound , and atypical ) expressed -catenin in the cytoplasm , only junctional and atypical nevi expressed -catenin at the plasma membrane . as opposed to nevi types , the three primary melanoma types examined ( superficial spreading , nodular , and lentigo maligna ) did not differ in -catenin localization as -catenin was localized at the plasma membrane , in the cytoplasm , or in both ( figure 4 ) . conversely , all primary melanomas ( 100% ) and the majority of metastatic melanomas ( 96% ) exhibited greater than 50% rad6 expression . the increase in tumor populations expressing rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant ( p = 0.0001 ; figures 1 , 2 , and 3 ) . melanoma progression from primary to metastatic disease was not associated with changes in the ( i ) percentage of melanomas expressing rad6 ( 100% of primary and metastatic melanomas ) , or ( ii ) percentage of melanomas expressing rad6 in more than 50% of the tumor cells ( 100% and 96% of primary and metastatic melanomas , resp . ) . the increase in proportion of tumor populations , expressing rad6 in more than 50% of the cells in primary melanoma ( 67% ) versus metastatic melanoma ( 79% ) was not significant ( p = 0.37 ; figure 2 ) . this study was not designed to test whether the distribution of the tumor cells positive for rad6 is the same between the subtypes of nevi . however , the percentages of benign tumors that lacked rad6 were similar between atypical nevi ( 62% ) and the group of other three nevi types ( 59% ) . interestingly , only one of the 30 nevi ( atypical nevus , 3% ) expressed rad6 in > 80% of the cells , and none of the primary and metastatic melanomas expressed rad6 in < 40% of the cells ( figure 2 ) . these results prompted us to examine whether rad6 expression can serve as a marker for histological diagnosis of melanoma . using a multiple logistic regression model , we found that the strength of rad6 expression is a strong predictor of melanoma ( p < 0.001 ) even when age group ( p = 0.65 ) and gender ( p = 0.24 ) are included in the model . the model predicts that every 1% increase in rad6 expression results in a 9% increase in the probability that a lesion is melanoma . if we assume that a predicted probability of > 0.5 indicates melanoma , the model with only rad6 has sensitivity of 93% and specificity of 80% . these results are very encouraging ; however , they need to be validated in a larger study . the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin in more than 50% of the cells , whereas only 27% of the same population of nevi expressed rad6 ( figure 2 ) . -catenin and rad6 expressions in nevi were not significantly correlated ( r = 0.06 ; p = 0.77 ) . there is a 2.7-fold difference in the percentage of primary melanomas ( 100% ) expressing rad6 compared to nevi ( 37% ) and virtually no difference in -catenin expression between primary and metastatic melanomas ( 100% ) . accordingly , rad6 and -catenin expressions in primary melanoma were not correlated ( r < 0.001 , p > 0.99 ) . a significant correlation between rad6 and -catenin positive cells however , this association diminished ( r = 0.40 , p = 0.05 ) following the exclusion of two observations which are disproportionally influential ( one with < 50% positive rad6 and one with < 50% positive -catenin ) . this is the first study to characterize rad6 expression in cutaneous benign and malignant melanocytic tumors . in this study , we examined the association between rad6 and -catenin expressions in benign and malignant melanocytic tumors to determine whether rad6 works in concert with -catenin to influence melanoma development and progression . rad6 and -catenin positively regulate each other in breast cancer [ 15 , 18 ] . however , while -catenin has been implicated in the pathogenesis of melanoma and other cancer types , data about the role of rad6 in cancer pathogenesis are mostly limited to breast cancer . therefore , we hypothesized that comparison of rad6 and -catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [ 15 , 28 ] . accumulation of nuclear and cytoplasmic -catenin has been implicated in driving the development and progression of several cancer types ( e.g. , colon and ovarian cancers ) [ 2931 ] . however , our results show that the expression levels of -catenin do not contribute to melanoma initiation and progression since no difference in -catenin levels was found between nevi , primary melanoma , and metastatic melanoma ( 93%97% of all samples expressed -catenin in > 50% of the tumor cells ) . the high expression levels of -catenin are in line with the crucial role of -catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells . also , our findings are in agreement with previous reports of positive -catenin staining in nevi ( 100% ) and primary melanoma ( 95% , 94% ) but are higher than reported in metastatic melanoma ( 75% , 68% ) [ 9 , 21 ] . the variation in expression of -catenin levels in metastatic melanomas between the studies while we studied only melanoma metastases to the skin , other studies either obtained 58% of their specimens from lymph nodes , tonsil , and liver or did not identify the anatomical site of their metastases [ 9 , 21 ] . furthermore , different anatomical sites may regulate dissimilar antigen expressions in metastases that originate from the same primary tumor in the same patient [ 33 , 34 ] . previous studies have shown higher percentages of nuclear -catenin in nevi than in melanoma ( 84% versus 33% , and 44% versus 15% ) [ 9 , 22 ] . those observations provided the basis for the currently held concept that loss of nuclear and cytoplasmic -catenin suggest poor prognosis and decreased overall survival of melanoma patients [ 12 , 22 ] . in light of these data , the absence of nuclear -catenin in all the nevi and melanomas analyzed in our study was surprising . usage of different anti--catenin antibodies may explain in part the discrepancy in nuclear -catenin expression observed between the studies . however , our results are consistent with the lack of nuclear -catenin reported in four studies which comprised 57 nevi , more than 55 primary melanomas , and 20 metastatic melanomas [ 21 , 3537 ] . moreover , nuclear -catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas . in another study of 70 primary melanomas , nuclear -catenin was reported in only 6.4% of the melanomas . finally , in a study of 230 primary and metastatic melanomas , nuclear -catenin was reported in only 13 cases ( 5.6% ) and therefore those cases were excluded from analysis . taken together , the absence or negligible amount of nuclear -catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for -catenin in nevi and melanoma . this notion is supported by a role for cytoplasmic -catenin to execute functions that do not require nuclear translocation ( e.g. , activation of map kinase p38 and nf - kb ) [ 37 , 40 ] . a major finding of this study is the association between melanoma development and intracellular redistribution of -catenin . the percentage of cases that expressed -catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas , respectively . concurrently , the percentage of cases that expressed cytoplasmic -catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas , respectively ( figure 4 ) . we hypothesize that the relocation of -catenin from the cytoplasm to the cell membrane may serve as a deactivating mechanism of canonical wnt/-catenin signaling and that the resulting reduction in cytoplasmic -catenin level may contribute to the malignant transformation of melanocytic nevi . the proposed hypothesis is supported by the following observations : ( i ) as in our study , bachmann et al . also reported an association between nevus to melanoma development and relocation of -catenin to the cell membrane . nevertheless , the authors of that study did not offer an explanation for their observation ; ( ii ) our analysis of the data of kagashita et al . showed -catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in -catenin distribution corresponded with the malignant transition of nevi ; ( iii ) wnt4 signal has been identified as a mechanism that can drive -catenin relocation from cytoplasm to cell membrane ; and ( iv ) -catenin relocation from cytoplasm to cell membrane has been reported to block -catenin signaling in a human embryonic kidney ( hek293 ) cell line . of note , this hypothesis can explain how despite the abundant -catenin expression in melanoma [ 1 , 7 ] , cytoplasmic -catenin is selectively decreased , a phenomenon that has been associated with unfavorable melanoma prognosis [ 9 , 21 , 22 ] . our current efforts are directed towards determining if the increases in membranous -catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated -catenin at the membranous site . rad6 has been implicated in early breast cancer development since an increase in rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue . in contrast , our findings do not support rad6 to play a similar role in nevus formation as in benign breast neoplasia , since 63% of the nevi were negative for rad6 . rad6 has also been implicated in breast cancer progression because rad6 levels increase with progression from ductal carcinoma in situ to invasive primary carcinoma and metastatic cancer [ 15 , 28 ] . in accordance with the upregulation of rad6 in early stages of breast cancer development as compared to benign hyperplasia [ 15 , 17 ] , we observed a striking increase in rad6 expression in primary melanoma when compared to nevi . while all primary melanomas displayed strong rad6 staining ( > 50% of the tumor cells ) , rad6 was negative in 63% of the nevi . these findings suggest that rad6 may play a role in malignant transformation of nevi as in breast cancer . progression of melanoma from primary to metastatic disease was not significantly associated with further changes in the percentage of tumors expressing rad6 or rad expression intensity as > 50% of tumor cells stained positively in 100% and 96% of primary melanomas and metastatic melanomas , respectively . these findings suggest that rad6 may play a sustained role in melanoma metastasis as it does in melanoma development . in benign and malignant breast tumors , rad6 stabilizes -catenin , and , in turn , -catenin positively upregulates rad6 transcription [ 1517 ] . however , this direct positive correlation between -catenin and rad6 expression does not appear to be conserved in melanoma as the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin compared to only 27% of nevi that expressed rad6 in more than 50% of their cells ( figure 2 ) . these observations suggest that the high -catenin expression in nevi is likely driven by regulators other than rad6 . at first glance , it would appear that -catenin and rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas . also , these findings correspond to the 80% correlation between rad6 and -catenin expressions in primary breast cancer . however , it is unlikely that the high rad6 expression in primary melanoma is driven by the concurrent high -catenin expression , because rad6 expression is low in nevi despite the presence of high cytoplasmic -catenin expression that is comparable to primary melanoma . this notion is confirmed by lack of statistical correlation between rad6 and -catenin expressions in primary melanoma . -catenin is not the only activator of rad6 ; for instance , rad6 is activated by nerve growth factor in nervous tissue . therefore , it is conceivable that , in primary melanoma , rad6 expression is regulated by yet unidentified activators . we also demonstrated that further progression of melanoma from primary to metastatic disease is not associated with a correlation between the -catenin and rad6 expressions . taken together , our study does not support a direct positive interaction between -catenin and rad6 in either benign or malignant melanocytic tumors . we characterized for the first time rad6 expression in cutaneous benign and malignant melanocytic tumors . we are showing a striking upregulation of rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas . these findings strongly suggest a role for rad6 in the development of primary melanoma and metastatic disease . we show that in contrast to rad6 , -catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors . taken together , in contrast to the rad6 -catenin positive relationship in breast cancer [ 1517 ] , our study does not support a similar positive interaction between -catenin and rad6 in benign or malignant melanocytic tumors . finally , our findings suggest a role for the cytoplasmic to membrane translocation of -catenin in the development of primary melanoma . future studies will determine whether newly generated -catenin at the membranous site coincide with -catenin translocation from the cytoplasm .

we have previously demonstrated that rad6 and -catenin enhance each other 's expression through a positive feedback loop to promote breast cancer development / progression . while -catenin has been implicated in melanoma pathogenesis , rad6 function has not been investigated . here , we examined the relationship between rad6 and -catenin in melanoma development and progression . eighty - eight cutaneous tumors , 30 nevi , 29 primary melanoma , and 29 metastatic melanomas , were immunostained with anti--catenin and anti - rad6 antibodies . strong expression of rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas . -catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas , and unlike rad6 , in 93% of nevi . none of the tumors expressed nuclear -catenin . -catenin was exclusively localized on the cell membrane of 55% of primary , 62% of metastatic melanomas , and only 10% of nevi . cytoplasmic -catenin was detected in 90% of nevi , 17% of primary , and 8% of metastatic melanoma , whereas 28% of primary and 30% of metastatic melanomas exhibited -catenin at both locations . these data suggest that melanoma development and progression are associated with rad6 upregulation and membranous redistribution of -catenin and that -catenin and rad6 play independent roles in melanoma development .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

the guanine heterocyclic ring is rich in chemical reactivity toward oxidants and adduct - forming species . in dna , 2-deoxyguanosine ( dg , mass = m ) is the chief site for base oxidation due to its low redox potential leading to many products that are characterized by their mass signatures . the principal products characterized with one - electron oxidants in aqueous solutions can be grouped based on the site of reaction on the radical intermediate resulting from one - electron oxidation of dg . the 2-deoxyribonucleoside products arising from initial reactivity at c5 of dg include a four - electron oxidation product , an imidazolone ( diz , m-39 ) that hydrolyzes to an oxazolone ( dz , m-21 ) , and the two - electron oxidation product 5-carboxamido-5-formamido-2-iminohydantoin ( d2ih , m+34 ; scheme 1 ) . when the initial reaction occurs at c8 of dg , either 2,6-diamino-4-hydroxy-5-formamidopyrimidine ( fapy - dg , m + 18 ) is observed under reducing conditions or 8-oxo-7,8-dihydroguanine ( dog , m+16 ) is observed under oxidizing conditions , in which the latter compound is a key marker for monitoring oxidative stress in cells ( scheme 1 ) . the two - electron oxidation product dog is stable but is highly susceptible to further oxidation leading to two hydantoin compounds . the yield for the thermodynamically preferred product spiroiminodihydantoin ( dsp , m + 32 ) is greatest in nucleoside reactions at ph > 6 or in unencumbered reaction contexts ( i.e. , single - stranded dna or g - quadruplexes ) , while the yield of 5-guanidinohydantoin ( dgh , m + 6 ) is greatest in nucleoside reactions at ph < 6 or in sterically demanding duplex contexts ( scheme 1 ) . the hydantoins are also readily formed from direct four - electron oxidation with o2 . this list of products represents those that are consistently observed from many oxidant systems conducted by several laboratories ; however , other compounds have been reported in lower yield . in the cellular context , oxidation reactions with dg can have other nucleophilic participants such as amines or phenols . in most cases , when amines or phenols participate in the reaction , products are observed with similar backbone structures as characterized with water serving as the nucleophile . for example , when dg is oxidized in the presence of lysine , products retaining the dg heterocycle and those with a core similar to dgh or dsp have been observed . the adducts characterized by our laboratory and others show lysine competing with water for sites of covalent bond formation , where a single lysine is observed at c8 analogous to dog , at either c5 or c8 of a spirocyclic core similar to dsp , and last a bis - adduct at c5 and c8 with a spirocyclic ring structure has been identified ( figure 1 ) . the lysine adducts are all stable allowing their quantification and characterization . as a last example , the polyamine spermine has been adducted to dg and dog under oxidative conditions . in the dg oxidation studies , a spermine adduct at c8 is observed retaining the dg heterocyclic core , while dog oxidations yield an adduct at c5 that does not undergo acyl migration to create a spirocycle , but instead the polyamine can generate an unstable hemiaminal intermediate that decomposes leaving a ribosylurea lesion at the adduct formation site . moreover , the facile formation of spermine adducts to dog oxidation intermediates has been harnessed for quantification of dog from dna samples . in contrast to amines , phenols ( i.e. , tyrosine ) are more redox active than amines and better able to compete with dg for oxidant during reactions . the products observed when phenols participate with dg during oxidation have more variability in their structures compared to amine adducts . these model studies aid in understanding the chemical nature of dna protein cross - links that are detrimental to cellular processes . in the current work , oxidations that allowed dg to react with nh3 were conducted while the full product and diastereomer distributions were monitored . after careful characterization of the nucleoside reactions , similar oxidations were conducted in the single - stranded and duplex oligodeoxynucleotide ( odn ) contexts . the purified ammonia adducts were also studied with respect to their decomposition pathways leading to end products that have not been previously determined . lastly , polymerase insertion studies were conducted to test the hypothesis that amine adducts might have altered base - pairing preferences ; every substitution of an oxo group on a base for an amino group converts a hydrogen bond acceptor into a potential hydrogen bond donor . these results provide a fundamental understanding of the stability and base - pairing properties of amine adducts to dg that result during oxidation , which may occur in vivo due to the high concentration of nucleophilic amines in the vicinity of the genome . adducts observed when dg and lysine are allowed to react in the presence of oxidant . the unencumbered nucleoside dg ( 1 mm ) was chosen for the initial oxidation reactions where nh4cl ( 20 mm ) provided the source of nucleophilic nh3 . all reactions were conducted with 75 mm napi buffer at ph 7.4 at 22 c . the oxidants chosen include the photooxidants riboflavin and rose bengal and the one - electron oxidant na2ircl6 . reactions were conducted in triplicate to achieve 70% conversion to products in each reaction , and the products ( scheme 2 ) were analyzed by a dual hplc method . the first round of hplc analysis utilized a reversed - phase column to identify dog ( m+16 ) and 8-amino - dg ( m+15 ) , while all other products eluted in the void volume . the void volume from the previous run was collected and analyzed on a hypercarb hplc column that allowed analysis of the hydantoins , ammonia adducts , and dz . however , diz hydrolyzes to dz that was detected on the hypercarb column ; thus , formation of diz is inferred from quantification of dz . moreover , the hypercarb hplc column also allowed separation and quantification of all spirocyclic diastereomeric pairs of products . the dgh diastereomers are interconvertible , and thus , their diastereomer ratios are not reported ( see the supporting information for complete experimental details ) . lastly , a test reaction with na2ircl6 was conducted in which half was directly analyzed by the hypercarb hplc column and the other half was analyzed by the dual hplc method outlined above . the product distributions observed from these comparative studies were within 3% of one another . the photooxidant riboflavin led to the largest number of different products and was used to obtain suitable amounts of material for characterization . esi - ms ( figure s1 , supporting information ) , and the adducts observed that did not involve participation of nh3 during product formation included dz ( m - 21 ) , dog ( m + 16 ) , dgh ( m + 6 ) , and dsp ( m + 32 , scheme 2 ) . confirmation of their structures was achieved by esi - ms / ms fragmentation of the free bases while monitoring the daughter fragments . the esi - ms / ms experiments were conducted on hplc purified nucleosides in which the n - glycosyl bond was cleaved in the ionization source to liberate free bases that were further fragmented in the cid chamber to generate the daughter fragments used in identification of the structures . the ms / ms fragmentation spectra were compared to literature values for the two dsp diastereomers ( figure 2 ) and dz to further confirm their structural assignments ( figures s2 and s3 , supporting information ) . the structure for dsp has been further established by x - ray crystallography and nmr . ammonia adducts observed included two pairs of chromatographic peaks for the diastereomers of spirodi(iminohydantoin)-2-deoxyribonucleosides ( dsi ) , whose names are based on the site of nh3 attachment to guanine , thus 5-dsi ( m + 31 ) and 8-dsi ( m + 31 ) ( scheme 2 , see reference cited for the correct dsi iupac nomenclature ) . to confirm the identities of the 5- and 8-dsi constitutional isomers and their respective diastereomers , esi - ms / ms of the free bases were conducted ( figure 2 ) . comparison of the esi - ms / ms spectrum for 5-dsi with dsp gave a pair of peaks for 5-dsi that established c5 as the site of nh3 attachment ( m / z [ m + h ] = 140 and 96 , figure 2 ) . as for 8-dsi , comparison of its esi - ms / ms spectrum with those obtained from dsp and 5-dsi identified new masses that are best explained by nh3 attachment at c8 ( m / z [ m + h ] = 141 and 97 , figure 2 ) . on the basis of the proposed mechanism ( scheme 2 ) , a product that includes bis - addition of nh3 at c5 and c8 of an oxidized guanine to yield a spirocycle ( 5,8-dsi , m+30 ) is possible ; however , this adduct was not observed for reasons that will be elaborated on below . additionally , nh3 adducts with a dgh core were not observed most likely due to the fact that the reactions were performed at ph 7.4 where yields of dgh are minimal . lastly , the yields of dog ( m + 16 ) and 8-amino - dg ( m + 15 ) were very low ( < 1% ) as determined by lc - esi - ms ( figure s1 , supporting information ) ; hence , their quantities are not reported . the nucleosides dog and c8 amine - adducted dg have redox potentials that are 600 mv below the parent nucleoside dg causing them to be much more susceptible to further oxidation . further , one - electron oxidized dog was observed to have an even lower redox potential than the parent compound dog , which means that once oxidation of dog , and likely 8-amino - dg , occurs product formation is inevitable due to the 70% conversion to product that yielded the spirocyclic compounds dsp , 5-dsi , or 8-dsi . esi - ms / ms spectra for dsp ( top ) , 5-dsi ( middle ) , and 8-dsi ( bottom ) . the data provided was collected on the first eluting diastereomer of each spirocycle from a hypercarb hplc column . data for the second eluting diastereomer for dsp , 5-dsi , and 8-dsi can be found in the supporting information ( figures s2 , s4 , and s5 ) . the heavy lines represent the fragment observed and the thin lines represent the portion of the molecule lost upon fragmentation . relative product distributions observed with each oxidant system were determined by integration of the hplc peak areas measured at 240 nm followed by normalization via each molecule s unique extinction coefficient ( 240 nm ) . extinction coefficients for 5-dsi and 8-dsi are not known but were determined from experiments that are reported below . in the riboflavin - mediated oxidations , this result was anticipated because riboflavin is a type i photooxidant and effects oxidation by electron transfer from dg yielding o2 under aerobic reaction conditions . oxidation of dg by one - electron and proton transfer yields an intermediate radical ( dg ) that only couples with o2 to initially yield diz that is prone to hydration leading to dz ( scheme 2 ) , the species quantified . the adducts derived from the nucleophile trapping of electrophilic dg oxidation intermediates show nh3 participation through the products 5-dsi ( 29% ) that was the highest yielding nh3 adduct followed by nearly 3-fold less 8-dsi ( 11% ) . the participation of h2o as the trapping nucleophile was identified by the lower yields of dsp ( 11% ) and dgh ( 4% ) . the nucleophilicity of nh3 is much greater than h2o ; therefore , as expected , adducts resulting from nh3 participation dominated over those derived from h2o . relative product distributions observed when dg reacted with nucleophilic h2o or nh3 in oxidation reactions . the oxidants include the photooxidants riboflavin and rose bengal , as well as na2ircl6 . reactions were conducted with 1 mm dg , 20 mm nh4cl , in 75 mm napi buffer ( ph 7.4 ) at 22 c . ( 1 ) photoactivation of riboflavin ( 200 m ) was achieved with 350 nm light for 3 h , ( 2 ) photoactivation of rose bengal ( 100 m ) was achieved with 350 nm light for 3 h , and ( 3 ) oxidation with na2ircl6 ( 10 mm ) was achieved by bolus addition of the salt . results represent the average of triplicate trials and the error is 10% of each reported value . the second oxidant studied was rose bengal , a type ii photooxidant ( o2 ) that furnished 5-dsi as the major product ( 62% ) followed by a 3-fold lower amount of dsp ( 31% ) , and low yields of dz ( 5% ) and dgh ( 2% ) were observed to complete the mass balance ( figure 3 ) . as expected , this oxidant did not yield 8-dsi based on its mechanism of oxidation . oxidation of dg by o2 proceeds by 4 + 2 cycloaddition to the imidazole ring , followed by ring opening to yield 8-hoo - g that eliminates water giving the proposed electrophile dog . next , dog is trapped by nucleophiles at c5 leading to dsp with h2o or 5-dsi with nh3 ( scheme 3 ) . support for exclusive nucleophilic attack at c5 under o2 oxidations was derived from h2o studies followed by mapping the labeled site by esi - ms / ms ; the current observation that the only nh3 adducts were the 5-dsi diastereomers further support this previously proposed mechanism , and further confirms our structural assignments for these peaks . comparisons of the product distributions from the photooxidants riboflavin and rose bengal show a dramatic difference with respect to the major product observed ( figure 3 ) . in the riboflavin oxidation , dz was the major product while in the rose bengal oxidation , 5-dsi was the major product , and these observations can be ascribed to each oxidant s unique mechanism of oxidation ( schemes 2 and 3 ) . product distributions from this oxidant included nearly equivalent distributions of dsp ( 50% ) and 5-dsi ( 42% ) with the mass balance completed by dgh ( 8% , figure 3 ) . in this oxidation reaction , the distribution did not yield nh3 adducts as the major products , which was unexpected on the basis of nh3 being the better nucleophile compared to h2o . comparison of the product distributions observed with the one - electron oxidants riboflavin and na2ircl6 identified both nh3- and h2o - adducted compounds . the current results differ from those previously reported by our laboratory when lysine was adducted to dg in analogous oxidation reactions . previously , in riboflavin oxidations spirodihydantoins with lysine at c8 were 2-fold greater than those with lysine at c5 ( figure 1 ) , and in na2ircl6 oxidations a c8 lysine adduct was observed . in contrast to these results , riboflavin oxidations in the presence of nh4cl gave more c5 adducts ( 29% ) than c8 adducts ( 11% ) , and na2ircl6 oxidations did not yield detectable amounts of c8 adducts . we propose the difference in the current ammonia results compared to the lysine data can be attributed to a difference in the mechanism of product formation for the c8 adduct in the nucleoside context . the former work from our laboratory proposed that c8 amine adducts result from oxidation of the amine to the aminyl radical that adds at the c8 carbon of dg followed by oxidation leading to product formation ( scheme 4 ) . in nucleoside dg , this proposed mechanism best supports the observation of ammonia adducts at c8 , because upon one - electron oxidation of dg the initial radical cation ( dg ) formed is very acidic ( pka 3.9 ) and rapidly deprotonates to the neutral radical ( dg ) that is not susceptible to nucleophilic attack . thus , dg reacts with o2 yielding diz / dz and not with amines or h2o . formation of amine adducts at c8 must result from a difference in the amine reactivity . the key difference between lysine and ammonia resides in their standard reduction potentials . in general , primary amines ( 1.0 v vs nhe , ph 10 ) have a lower redox potential than ammonia ( > 1.3 v vs nhe , ph 9 ) ; this trend should scale down to ph 7 , in which the oxidations were conducted . thus , oxidation of lysine to an aminyl radical that adds to c8 of dg is possible with na2ircl6 ( 0.9 v vs nhe , ph 7 ) and riboflavin ( 1.7 v vs nhe , ph 7 ) ; in contrast , the analogous reaction does not readily occur for nh3 because at ph 7 dg is the dominant site of oxidation due to its lower redox potential leading to products other than 8-dsi . in summary , one - electron oxidant driven oxidations of dg in the presence of nh3 lead to spirocyclic adducts at c5 and c8 that are in competition with h2o adducts of the same core structure . as the next step , we set out to determine the decomposition products and pathways through which the ammonia adducts proceed . hplc was used to provide diastereomerically pure 5-dsi and 8-dsi samples that were subjected to conditions of ph 3 ( 0.1% formic acid ) or ph 10 ( 20 mm napi ) at 22 c for 30 min or 10 h. not only could the decomposition products be determined , but having diastereomerically pure starting material also allowed us to probe the mechanism of decomposition , specifically for 5-dsi . first , both 5- and 8-dsi were stable at ph 10 during this time frame , while 5-dsi readily hydrolyzed to dsp at ph 3 in 30 min and 8-dsi hydrolyzed to dsp after 10 h. for 5-dsi , deamination of the amine group can occur by two possible mechanisms : ( 1 ) a retro - acyl migration can occur followed by loss of nh3 to yield an electrophilic intermediate ( dog ) susceptible to h2o attack followed by a second acyl migration back to dsp or ( 2 ) acid - catalyzed deamination can occur directly to furnish dsp ( scheme 5 ) . if the retro - acyl migration proceeds on the diastereomerically pure sample , it is expected that a mixture of dsp diastereomers would be observed , and if direct deamination occurs , then only one dsp diastereomer would be predicted . upon incubation of one diastereomer of 5-dsi at ph 3 , only one diastereomer of dsp was observed , supporting the direct deamination mechanism . moreover , the early eluting 5- and 8-dsi nh3 adducts decomposed to the early eluting dsp isomer and the late eluting adducts deaminated to the later eluting dsp isomer ( figure s6 , supporting information ) . these results aid in establishing the absolute configuration of the 5-dsi and 8-dsi diastereomers that will be discussed below . moreover , these results also allude to an explanation as to why the bis - ammonium adduct was not observed in any of the reactions . based on these data , the bis - ammonium adduct , if formed , could rapidly deaminate to yield either 5-dsi or 8-dsi , although this hypothesis could not be further validated . utilization of the hypercarb hplc column for analyzing the product distributions of these spirocycles allowed determination of the diastereomer ratios . it has previously been determined that ( r)-dsp elutes first from this column and ( s)-dsp elutes second . the absolute stereochemistry for the diastereomers of the ammonia adducts had not yet been determined . the results of these studies identified ( r)-dsp and ( s)-dsp yields to be nearly equal . also , for the 5-dsi and 8-dsi ammonia adducts nearly equal yields for the diastereomers were observed . on the basis of these results , the defining point of the reaction to determine product stereochemistry must not be very sterically impeded in order to lead to such a small diastereomer preference . as stated above , the diastereomerically pure ammonia adduct samples were determined to decompose to give a single diastereomer of dsp ; therefore , on the basis of hplc elution order , the absolute configuration for the diastereomers of the two dsi constitutional isomers can be determined . the first - eluting 5-dsi and 8-dsi diastereomers decomposed to the first dsp diastereomer ( r ) , and the late - eluting diastereomer decomposed to the late - eluting dsp diastereomer ( s ) ; therefore , for 5-dsi the r diastereomer elutes first and the s diastereomer elutes second , because the r and s assignments are the same between dsp and 5-dsi . in contrast , the r and s assignments for the 8-dsi diastereomers are opposite those of dsp ; thus , ( s)-8-dsi elutes first and ( r)-8-dsi elutes second and from the hypercarb hplc column ( figure 4 ) . these examples provide a fascinating case outlining how the movement of ring substituents affects the r and s stereochemical assignments . assignment of absolute configurations for the diastereomers of dsp , 5-dsi , and 8-dsi based on their elution profile from a hypercarb hplc column and their ecd spectra . the r and s assignments for the diastereomers of 8-dsi are the opposite of those for the dsp and 5-dsi isomers with the same geometric configuration of the spirocyclic ring , due to a change in the cahn ingold the diastereotopic ammonia adducts were then probed by electronic circular dichroism spectroscopy ( ecd ) . previously , ecd was used in tandem with vibrational circular dichrosim spectroscopy , nmr , and x - ray crystallography to establish the absolute configuration for dsp . the ecd spectra for ( r)- and ( s)-dsp isomers gave three lobes that were mirror images of one another , as expected ( figure 4 ) . critical for assigning the absolute configuration for dsp was the low energy lobe , in which the s isomer gave a positive rotation at 258 nm , and the r isomer gave a negative rotation at 259 nm . in comparison to these results , the 5-dsi diastereomers gave ecd spectra that were different than dsp but showed a similarity in the low energy lobe ( figure 4 ) . for ( r)-5-dsp , the lobe at 258 nm gave a negative rotation and ( s)-5-dsi gave a positive rotation at 258 nm . the higher energy lobes observed in the ecd for the 5-dsi diastereomers were different than those measured for dsp ( figure 4 ) . as a last comparison , the 8-dsi diastereomer ecd spectra were compared to those recorded for dsp ( note : 8-dsi and dsp isomers with the same geometric configuration of the spirocyclic ring give opposite r and s assignments due to a difference in the cahn the low energy lobe gave a negative rotation at 262 nm similar to ( r)-dsp , and ( r)-8-dsi gave a positive rotation similar to ( s)-dsp . furthermore , the 8-dsi diastereomers also gave similar rotations and energies as their analogous dsp diastereomers at the 238 nm lobe , and 8-dsi and dsp gave similar rotations at the highest energy lobe , but the absolute energy was different ( i.e. , 202 nm for 8-dsi and 211 nm for dsp ; figure 4 ) . the similarity in the critical low energy lobe further supports the absolute configuration assignments that were made from the deamination studies monitored by hplc . future computational studies to model these ecd spectra will help solidify these conclusions , and may address some of the challenges that occurred during modeling of the ecd spectra for the dsp diastereomers . the ability to deaminate the dsi adducts to dsp allowed the determination of the extinction coefficients for 5- and 8-dsi relative to dsp , in which these were the values used to determine the relative yields . peak areas for identically pure 5-dsi samples were measured before and after incubation in formic acid and the change in peak area measured was used to determine the relative 240 nm compared to the value established for dsp . a similar experiment was conducted for the 8-dsi diastereomers . from these experiments , the 240 nm for the 5-dsi and 8-dsi diastereomers were determined to be 3800 and 3500 lmolcm , respectively , which are both slightly greater than that determined for the dsp diastereomers ( 3300 lmolcm ) . the next step from the nucleoside studies was to explore the context dependence of nh3 adduct formation in single- and double - stranded odns ( ssodn and dsodn ) . the photooxidant riboflavin was chosen for the studies because it gave the most diverse distribution of products in the nucleoside reactions and might provide the most insight into context effects . for these studies , the ssodns selected for analysis were the 18-mers odn-1 and odn-2 , and the dsodn context was studied via the duplex formed from these two single strands ( odn-12 ) . after oxidation of these odn systems , they were digested with a suite of nucleases and phosphatase to the nucleosides followed by analysis using the previously described hplc methods . previously , our laboratory demonstrated that the digestion method used provides complete degradation of dsp - containing odns to nucleosides ; it is assumed that the dsi adducts are equally digested to completion . the nuclease digestion conditions were modified to include ammonium salts for buffer to prevent loss of 5-dsi via deamination to dsp ( scheme 5 ) , controls conducted with sodium salts for buffers did not detect 5-dsi due to deamination to dsp during the nuclease digestion ( 18 h at ph 5.4 , scheme 5 ) . a comparison between the contexts studied when dg was allowed to react with nh3 in the presence of the photooxidant riboflavin is provided in figure 5 . the first observation from these data was with respect to dz distributions that dramatically decreased when proceeding from the nucleoside to odn contexts ; specifically , the dz distribution in the dsodn context ( 2% ) was 10-fold less than the ssodn context ( 20% ) and 20-fold less than that observed in the nucleoside context ( 40% ) . this observation has already been reported in the literature and is proposed to result from the odn context increasing the lifetime of the dg that reacts with nucleophilic h2o to give the c8 product dog while decreasing the lifetime of dg that reacts with o2 to yield dz ( schemes 2 and 6 ) . with respect to the yields of the hydantoins , more dsp relative to dgh was observed in the nucleoside context ( 11% vs 4% , respectively ) versus the dsodn context ( 9% vs 21% , respectively ) . again , this trend follows those in the literature in which the duplex context favors the less sterically demanding product dgh . the nh3 adducts 5-dsi and 8-dsi provided some interesting context - dependent product distributions . the yield of 5-dsi was greatest for the nucleoside ( 29% ) and decreased by half in all odn contexts studied ( 14% ) . in contrast , the yield of 8-dsi was smallest for the nucleoside ( 11% ) and increased by more than 3-fold in the ssodn context ( 39% ) , and over half the products observed in the dsodn context were 8-dsi ( 54% ) . as previously stated , the lifetime of the electrophilic dg is longer in the duplex context and the superior nucleophilicity of nh3 compared to h2o greatly increased the yield of 8-dsi in dsodn oxidations ( scheme 6 ) . furthermore , the increase in 8-dsi yield from the dsodn oxidations further supports this product resulting from nucleophilic addition of nh3 at c8 leading to 8-dsi , and not the nh3 aminyl radical adding at c8 to yield the same product . these results clearly demonstrate a dependence on the reaction context in formation of dg oxidation products and nh3 adducts . a look at the distribution of diastereomers resulting from oxidation of the odn contexts gave nearly equal amounts of both r and s diastereomers . this observation parallels our previous work looking at dsp diastereomer formation in single - stranded and duplex contexts . coupled together , these observations support a state of disorder in the duplex at the time of nucleophilic attack at c5 of oxidized dog or 8-amino - dg ( likely intermediates leading to the spirocycles ) that leads to nearly equal covalent bond formation from both the re and si faces of these electrophiles . relative product distributions measured when dg was allowed to react with nh3 in the presence of photoexcited riboflavin in various contexts . reactions were conducted in 75 mm napi buffer ( ph 7.4 ) at 22 c , and 20 mm nh4cl . in the nucleoside studies , 1 mm dg was studied , and in the odn contexts 20 m ssodn or 10 m dsodn was studied . ( 1 ) photoactivation of riboflavin ( 200 m ) was achieved with 350 nm light for 3 h in the nucleoside studies and 30 min in the odn studies . these conditions achieved 70% conversion to product in the nucleoside reactions and 50% in the odn studies . it must first be noted that the bis - ammonium adduct could not be quantified due to its instability , but if formed , the adduct is most likely deaminated to either 5-dsi or 8-dsi . keeping this limitation in mind , the oxidation of dg by riboflavin initially yields dg that rapidly deprotonates to dg in the nucleoside contexts ( pka 3.9 ) , dominating the product - forming step of the reaction . in contrast , dg in the dsodn context retains more cationic character because the acidic proton is h - bonded with 2-deoxycytosine in a base pair ; therefore , the product - forming steps of the reaction are determined by the radical cation intermediate . as shown in scheme 2 , dg could couple with radicals ( e.g. , o2 ) to ultimately yield dz , while dg reacts with nucleophiles ( e.g. , h2o and nh3 ) at c8 , ultimately yielding a spirocyclic product after completion of the four - electron oxidation . thus , the yield of 8-dsi was anticipated to be greatest in the duplex context , as indeed it is . furthermore , these results support the original hypothesis by steenken that the duplex context would favor the radical cation nature of one - electron oxidized dg , which is still the focus of many current research efforts . because of the limitation imposed by the inability to detect the bis - ammonium adduct , or to understand its decomposition pathway , any comparison between the ammonia adduct distributions would be based on poorly defined assumptions ; consequently , further discussion of these results is not provided . for the last set of studies , we were inspired by the observation that the a ring of dsp mimics the h - bonding pattern of thymidine , while the a ring of 5-dsi mimics the h - bonding pattern of 2-deoxycytidine ( figure 6 ) . on the basis of this observation , polymerase insertion assays opposite dsp vs 5-dsi were conducted to determine if these h - bonding schemes applied during the selection of a nucleotide to insert opposite these spirocycles . comparison of h - bonding patterns between dsp vs t and 5-dsi vs dc . site - specific synthesis of dsp or 5-dsi was achieved by synthesizing dog into an odn by solid - phase synthesis and then oxidizing this strand with or without nh4cl present with the one - electron oxidant na2ircl6 to furnish the desired products . upon ion - exchange hplc purification , the absolute stereochemical assignments for the dsp isomers have been established for the odns , and those for 5-dsi are assigned in this work to be analogous based on the mechanism of deamination discussed above ( see figure s7 , supporting information , for more details ) . standing start polymerase studies were conducted while providing the enzyme only one type of nucleotide per reaction . on the basis of previous studies , insertion of dttp opposite dsp was not observed ; therefore , to ensure that only one nucleotide was inserted opposite the lesion , and no extension past the lesions occurs , the sequence had an a placed 5 to the spirocycle ( figure 7 ) . klenow fragment exo- was selected as the polymerase , and after the extension reactions , polyacrylamide gel electrophoresis ( page ) was conducted to determine the amount of datp , dttp , dgtp , or dctp incorporated opposite the dsp or 5-dsi diastereomers . the polymerase did not select either pyrimidine for insertion opposite the two lesions ( figure s7 , supporting information ) ; thus , only data for purine insertion opposite the lesions is provided in figure 7 . with respect to insertion opposite the dsp diastereomers , ( r)-dsp was observed to give a slight preference for insertion of datp opposite , and ( s)-dsp gave nearly equal insertion of datp and dgtp . in comparison to the dsp results , the 5-dsi isomers showed that ( s)-5-dsi gives similar amounts of datp and dgtp insertion , while ( r)-5-dsi yields a slight preference for datp insertion . unfortunately , these results do not support the hypothesis that h - bonding in the a ring of the spirocycle is the key parameter for nucleotide selection , but do show that stereochemistry may be important for selecting a base pairing partner by this polymerase . sequence studied for polymerase dntp insertion studies and the percent dntp insertions opposite the diastereomers of dsp and 5-dsi . ( a ) sequence for the dsodn construct used in the polymerase insertion assays and ( b ) comparison of the percent purine dntp insertion opposite the lesions dsp and 5-dsi by the dna polymerase klenow fragment exo- . the present studies mapped the pathways and quantified the products observed when dg was allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal , as well as the one - electron oxidant na2ircl6 . on the basis of the analysis of the products in the nucleoside context , the major products were oxidant dependent . the major product for riboflavin was dz , rose bengal yielded the 5-dsi diastereomers , and na2ircl6 gave the dsp diastereomers as the major products ( figure 3 ) . further analyses determined that 5-dsi and 8-dsi decompose via an acid - catalyzed deamination pathway leading to dsp as an end product ( scheme 5 ) . oxidations of dg in the nucleoside , ss- and dsodn contexts with photochemically activated riboflavin demonstrated three major context effects : ( 1 ) the yield of dz was highest in the nucleoside studies and decreased dramatically in dsodn contexts ; ( 2 ) when nh3 participated as the nucleophile , 5-dsi was obtained in the highest yield in nucleoside studies , while the yield of 8-dsi was highest in dsodn contexts ; and ( 3 ) when h2o was the nucleophile in nucleoside reactions , dsp presented in the highest yield , while in dsodn contexts dgh was obtained in the highest yield ( figure 4 ) . furthermore , a combination of mapping the decomposition pathways of the dsi compounds to dsp , in which the absolute configuration has been assigned , allowed the determination of the absolute configurations for the diastereomers of 5- and 8-dsi . these studies provide fundamental chemical insight into formation of amine adducts to dg and their stability . further , these studies insert a cautionary note to researchers who purify their odns using ammonium salts and then conduct oxidation reactions after purification . ammonia is a better nucleophile and out - competes water for the electrophilic intermediates derived from dg or dog oxidation , resulting in a new mass of dg + 31 or dog + 15 , respectively . a similar observation has been highlighted for oxidations occurring in tris buffer that generate tris adducts to dg . oxidations were conducted with 2-deoxyguanosine ( dg ) at 1 mm concentration in 75 mm napi buffer ( ph 7.4 ) and 22 c . reactions with 20 mm nh4cl and without this salt allowed product profile comparisons . the oxidants and specific reaction conditions were achieved as follows : ( 1 ) riboflavin oxidations were initiated by adding 200 m riboflavin while exposing the samples to 350 nm light for 3 h. ( 2 ) rose bengal oxidations were achieved by adding 100 m rose bengal and exposing these samples to 350 nm for 3 h. the light source for the riboflavin and rose bengal reactions came from a sun lamp that was placed 7 cm above the reaction eppendorf tubes . the tube lids were left open to allow all wavelengths of light to pass into the reaction samples . ( 3 ) the na2ircl6 oxidations were initiated by a bolus addition of the oxidizing salt with a final concentration of 10 mm , after a 30 min reaction the samples were quenched with 50 mm edta ( ph 8) . the reaction products and their distributions were determined by a dual hplc method following a previously reported set of protocols ( specific details can be found in the supporting information file ) . the first rp - hplc run allowed analysis of dog and 8-amino - dg ( both observed in < 1% yield ) , while all other products eluted in the void volume of this run . the void volume was collected , dried , and reinjected on a hypercarb hplc column to analyze the diastereomers of dgh , dsp , 5-dsi , and 8-dsi , as well as the product dz , while monitoring their absorbance at 240 nm . to determine product distributions the peak areas were integrated and normalized by each compound s 240 nm ( values provided below ) . esi - ms , and then each compound was hplc purified for further structural analysis . the dgh diastereomers were previously characterized by nmr , dz was also previously characterized by nmr , and the dsp diastereomers have been characterized by x - ray crystallography . structural characterization of 5-dsi and 8-dsi by nmr was not conducted because of the instability of these compounds toward deaminating to dsp , and the lack of nonexchangeable protons on the ring of either ammonia adducts makes them very challenging for structural analysis . furthermore , if deamination of either 5-dsi or 8-dsi to dsp occurs in the nmr tube during analysis , the peaks observed would be similar and challenging to interpret ; therefore , the best method for obtaining structural data on the diastereomers of 5-dsi and 8-dsi is via the esi - ms / ms that was conducted ( figure 2 ) , and provided satisfactory results to determine their structures . characterization for each molecule is as follows , the mixture of the dgh diastereomers ( not resolvable ) gave tr = 6 min ; lc esi - ms m / z [ m + h ] calcd 274.3 , found 274.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c9h15n5o5na 296.0971 , found 296.0979 ; uv vis 240 = 2400 l molcm.(r)-dsp : tr = 11 min ; lc esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit . 184 , 156 , 141 , 114 , 113 , 99 , 86 , found 184 , 156 , 141 , 114 , 113 , 99 , and 86 ; uv vis 240 = 3,300 lmolcm ; cd ( c 1.24 10 m in ddh2o ) nm 259 ( 8.7 ) , 236 ( + 35.1 ) , and 211 ( 37.4 ) . ( s)-dsp : tr = 18 min ; lc esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit . 184 , 156 , 141 , 114 , 113 , 99 , 86 , found 184 , 156 , 141 , 114 , 113 , 99 , and 86 ; uv vis 240 = 3300 lmol cm ; cd ( c 1.30 10 m in ddh2o ) nm 258 ( + 8.7 ) , 234 ( 32.9 ) , and 212 ( + 40.5 ) . ( r)-5dsi : tr = 9 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , found 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0920 ; esi - ms / ms m / z [ m + h ] found values 183 , 166 , 140 , 123 , 113 , 96 , and 86 ; uv vis 240 = 3800 lmolcm ; cd ( c 1.50 10 m in ddh2o ) nm 258 ( 8.2 ) , 242 ( + 20.8 ) , and 216 ( + 61.8 ) . ( s)-5dsi : tr = 12 min ; lc - esi - ms m / z [ m + h ] calcd 299.3 , found 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0916 ; esi - ms / ms m / z [ m + h ] found 183 , 166 , 140 , 123 , 113 , 96 , and 86 ; uv vis 240 = 3800 lmolcm ; cd ( c 1.45 10 m in ddh2o ) nm 258 ( + 8.2 ) , 242 ( 20.1 ) , and 216 ( 62.2 ) . ( r)-8dsi : tr = 7 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0924 ; esi - ms / ms m / z [ m + h ] found 183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv vis 240 = 3500 lmolcm ; cd ( c 1.30 10 m in ddh2o ) nm 261 ( + 20.0 ) , 237 ( 43.2 ) , and 203 ( + 18.6 ) . ( s)-8dsi : tr = 14 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0926 ; esi - ms / ms m / z [ m + h ] found 183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv vis 240 = 3500 lmolcm ; cd ( c 1.24 10 m in ddh2o ) nm 262 ( 13.7 ) , 239 ( + 46.0 ) , and 201 ( 33.1 ) . dz : tr = 27 min ; lc esi - ms m / z [ m + h ] calcd 247.3 , found 247.3 ; hrms ( esi - tof ) m / z [ m + na ] for c8h14n4o5na calcd 269.0862 , found 269.0870 ; esi - ms / ms m / z [ m + h ] lit . 131 and 117 , found 131 and 117 ; uv vis 240 = 1800 lmolcm . the odns were hplc purified using an ion - exchange hplc column , and the purification salts ( naoac ) were removed by dialysis prior to their oxidation following previously reported methods . the riboflavin oxidations were conducted similarly to those reported for the nucleoside studies , with the following exceptions : the ssodn oxidations were conducted on 20 m samples while the dsodn oxidations were conducted on 10 m samples . in addition , the reaction times were decreased for the odn reactions to 30 min . after the oxidations , the odns were digested with a suite of nucleases and phosphatases to nucleoside samples following a previously established protocol , with the exception that all buffers during the digestion process were comprised of ammonium salts . next , the digested mixture was analyzed by the hplc method used in the nucleoside studies . the polymerase insertion assays were conducted on duplex odn samples that had site specific incorporation of dsp or 5-dsi in the template strand . the site - specific synthesis was commenced on odns that had a dog phosphoramidite synthesized at the desired site of modification within the sequence 5-cgt tax ggc gca act gga aa-3 where x = dog . the modifications were synthesized by taking 1 nmol of the dog - containing odn and placing it in 100 l of reaction buffer ( 75 mm napi , ph 7.4 ) with or without 2 mm nh4cl . the reaction without nh4cl gave the dsp diastereomers and the reaction with nh4cl gave the 5-dsi diastereomers . the individual diastereomers were purified using an ion - exchange hplc column running naoac as the resolving salt and characterized via digestion of the odn to nucleosides followed by hplc analysis ( purification details can be found in the supporting information ) . the primer template duplex for the insertion studies was made by annealing 125 nm primer ( 5-tt tcc agt tgc gcc-3 ) with 156 nm lesion - containing template ( 5-cgt tax ggc gca act gga aa-3 where x = ( r)-dsp , ( s)-dsp , ( r)-5-dsi , or ( s)-5-dsi to obtain 100 nm duplex in klenow fragment exo- buffer ( 50 mm tris , 50 mm nacl , 5 mm mgcl2 , 1 mm dtt at ph 8) . to a 25 l reaction 20 l of the annealed duplex was added , 1 l of klenow fragment exo- ( 0.2 units/l ) , 0.5 l of dntp ( 500 m stock solution ) , and 8.5 l of klenow buffer to obtain a 100 nm duplex solution with 10 m dntp and 0.2 u of polymerase . the reaction was incubated at 37 c for 30 min , after which loading dye ( 95% dmf plus 0.025% bromophenol blue , and 0.025% xylene cylanol ) was added to the samples and they were heated at 95 c for 20 min to quench the reaction and denature the dna the denatured samples were loaded on a 20% page gel and electrophoresed for 2 h at 45 w. upon completion of the electrophoresis , the gel was placed in a phosphor screen overnight and imaged by storage phosphor autoradiography .

upon oxidation of the heterocyclic ring in 2-deoxyguanosine ( dg ) , the initial electrophilic intermediate displays a wide range of reactivities with nucleophiles leading to many downstream products . in the present study , the product profiles were mapped when aqueous solutions of dg were allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal as well as the diffusible one - electron oxidant na2ircl6 . product characterization identified the 2-deoxyribonucleosides of spiroiminodihydantoin , 5-guanidinohydantoin , and oxazolone resulting from h2o as the nucleophile . when nh3 was the nucleophile , a set of constitutional isomers that are diastereotopic were also observed , giving characteristic masses of dg + 31 . esi+-ms / ms of these nh3 adducts identified them to be spirocycles with substitution of either the c5 or c8 carbonyl with an amine . the nh3 adducts exhibit acid - catalyzed hydrolysis to spiroiminodihydantoin . quantification of the nh3 and h2o adducts resulting from oxidation of dg in the nucleoside , single - stranded , and duplex oligodeoxynucleotide contexts were monitored allowing mechanisms for product formation to be proposed . these data also provide a cautionary note to those who purify their oligonucleotide samples with ammonium salts before oxidation because this will lead to unwanted side reactions in which ammonia participates in product formation .

Introduction Results and Discussion Conclusion Experimental Section

a review of the literature and an extensive medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility . he reported an inability to conceive with his wife after 18 months of unprotected intercourse . a semen analysis was performed that included spermatozoa count , liquefaction , morphology , motility , viscosity and volume . two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility . evidence for the effectiveness of guaifenesin is almost entirely anecdotal . given the mechanism of action of guaifenesin , it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . additional studies of the effects of guaifenesin on male fertility could yield information of the medication s effect on men with normal or decreased total sperm counts . there are currently anecdotal reports and popular news media stories on the use of guaifenesin , particularly the brand name product robitussin ( pfizer , inc . , new york , ny ) , for use in treating both male and female infertility.14 guaifenesin is an expectorant medication sold over the counter and usually taken by mouth to assist expectoration of phlegm from the airways in acute respiratory tract infections . its mode of action in treating infertility is not well understood , but it appears to decrease mucus viscosity . a 32-year - old male patient presented to his primary care provider for an infertility evaluation . the patient is a nonsmoker , who consumes little or no alcohol with no known allergies . a recent screening exam for pulmonary tuberculosis was negative and the patient had recently undergone a required military service physical exam . he reported an inability to conceive with his wife after 18 months of unprotected , regular intercourse . as part of a routine infertility evaluation a semen analysis was performed that included spermatozoa count , liquefaction , morphology , motility , viscosity and volume ( cpt code 89320 ) . initial results of the semen analysis demonstrated low sperm count and motility ( table 1 ) . this sample , as well as the follow - up sample , were obtained through masturbation and provided to the lab within 30 minutes of collection . the patient s primary care provider offered treatment with guaifenesin 600 mg extended release tablets twice daily . the repeat semen analysis demonstrated marked improvement in both total sperm count and motility ( table 1 ) . the patient made no other significant lifestyle changes during the treatment course with guaifenesin . at the time of writing this case report describes the semen analysis laboratory results in a male patient who was given guaifenesin . guaifenesin is a mucolytic agent usually taken orally to assist the expectoration of phlegm from the airways in acute respiratory tract infections . scientific evidence for the effectiveness of guaifenesin is almost entirely anecdotal ; a review of medical literature revealed very limited data on use of guaifenesin for infertility.5,6 there appeared to be some improvement in a small study without controls of female infertility related to hostile cervical mucus.5 check regards guaifenesin as the simplest but least effective method of improving cervical mucus.7 given the proposed mechanism of action of guaifenesin , it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . additional study of the effects of guaifenesin on male fertility suggests the need to conduct a more rigorous placebo - controlled clinical trial that could yield information of the medication s effects on men with normal or decreased total sperm counts .

backgrounda review of the literature and an extensive medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility.casea 32-year - old male presented for an infertility evaluation . he reported an inability to conceive with his wife after 18 months of unprotected intercourse . a semen analysis was performed that included spermatozoa count , liquefaction , morphology , motility , viscosity and volume . initial results of the semen analysis demonstrated low sperm count and motility . the provider offered treatment with guaifenesin 600 mg extended release tablets twice daily . two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility.conclusionevidence for the effectiveness of guaifenesin is almost entirely anecdotal . given the mechanism of action of guaifenesin , it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . additional studies of the effects of guaifenesin on male fertility could yield information of the medication s effect on men with normal or decreased total sperm counts .

Background Case Conclusion Background Case Conclusion

agriculture has been one of the primary economic avenues in the philippines contributing to about 20% to the gross domestic product ( gdp ) . crops comprise about 47.56% of the total agricultural sector and have contributed to about 510 billion pesos ( p510b ) to the country 's national income . benguet is a province in the northern portion of the philippines belonging to the cordillera administrative region . there are about 27.5 thousand farms covering 30 thousand hectares of agricultural land in benguet . it is also the largest producer of vegetables and fruits , supplying the capital cities in the philippines . the province is known as the salad bowl of the philippines as its major crops are tubers , roots , bulbs , leafy vegetables , stems , and flowers . in 2005 , benguet was the top producer of brocolli and carrots producing about 1.2 thousand and 13.7 metric tons contributing to 87.4% and 81.4% , respectively , to the national output . however , growing vegetables is considered a risk occupation in some areas in developing countries . soogarun et al . in 2003 found significantly low / abnormal mean blood cholinesterase levels among vegetable growers in thailand . health impacts of pesticide misuse on the other hand greatly affect the farming communities in the philippines questioning the economic advantages of its use . many researchers have correlated the extent of direct and indirect pesticide exposure and health hazards such as increased mortality , dermal contamination , depression in cholinesterase level , fetal abnormalities , and spontaneous abortion among pregnant women [ 36 ] . it is a discouraging fact though , that with knowledge of health risks , many filipino families still perceive that crop yield outweighs the health risks associated with pesticide use . pesticide poisoning is one of the most prevalent health problems in the philippines . in a study by the department of health ( doh ) from 19911995 , organophosphates accounted for the highest number of poisoning cases while organochlorines caused the most number of deaths . cheng in 1994 studied 2000 benguet vegetable farmers and found that the most common complaints were allergic reactions both in the skin and the eyes , abdominal pain , dizziness , chest pain , headache , and nose bleed . meanwhile , a study on pesticide poisoning in selected hospitals in four philippines regions in 2001 found that cases of acute poisoning were more prevalent than chronic cases . this study aimed to identify the pesticide exposure and risk factors among vegetable farmers . the data can be used as baseline data on the vegetable industry in the philippines . this was a cross - sectional study to investigate the prevalence of pesticide exposure and its risk factors . target population consisted of vegetable farmers in the largest vegetable producing community in the philippines . the inclusion criteria were farmers living in the community for at least one year from the time of interview , and practicing farmers who own or work a farm in the community . those who were involved in organic farming and the migrant farmers who have been in the area for less than one year were excluded . there were 211 respondents from the identified municipalities selected as the study population using cluster sampling . the sampling size calculated with p = .05 was 211 vegetable farmers and 37 farms . data gathering was done using the following : ( 1 ) questionnaire structured personal interview with farm workers / farmers was done by research assistants who were trained prior to the data collection . details included personal information , health history , pesticide usage , work practices , work conditions , risk factors associated with pesticide exposure , and health data ; ( 2 ) exposure assessment monitoring on work conditions , work practices , and pesticide concentration ; ( 3 ) work analysis in each farm was also done to validate work practices related to pesticide preparation and application . recall bias was dealt with by confining the health data questionnaire to the last one year from the time of interview . the health data were also collected by medical doctors who simultaneously conducted physical assessment of the farmers . this project study was collaborated with the local agencies coordinating with farmers in the vegetable industry in benguet . ethical clearance was given by the research , information and dissemination office of the proponent 's institute . the study included 127 males ( 60.2% ) and 84 females ( 39.8% ) with ages ranging from 16 to 72 ( mean = 45 12 ) showing a relatively adult population . seventy one percent ( 71% ) were married and majority were working as agricultural workers ( 82% ) , and the remaining were pesticide applicators , mixers , and loaders ( 18% ) . the respondents were living in their present address for an average of 34.76 years ( sd = 16.72 ) with a mean distance of 3163 meters ( sd = 36539.13 ) from the vegetable plantation or farm . few farmers reported history of smoking ( 16.2% ) , and 7% claimed they smoked and 2% had a history of chewing tobacco . the average number of cigarettes and tobacco consumed in a week were 12 sticks and < 1 tobacco , respectively . the farmers used pesticides in their farms in an average of 1.9 days per week . the mean total application time was 3.47 hours ( mean = 3.47 2.09 ) . the mean amount of pesticide used in an application was 21.35 l per application ( mean 21.35 48.17 ) . the farmers also reported that in an average year , there were 2.3 ( mean = 2.3 0.53 ) cropping seasons with a mean of 3.84 ( mean = 2.3 0.53 ) months per cropping season ( table 1 ) . 44.5% reported that they wiped their sweat with a contaminated piece of fabric , 41.7% re - entered recently sprayed area , 37.4% had exposure because of damaged backpack sprayer , and 31.8% were exposed when they sprayed against the wind ( table 2 ) . however , further analysis shows that they did not frequently use such equipment nor had adequate gear to fully protect themselves . the same pattern was seen among all kinds of personal protective equipment ( ppe ) with the exception of boots which was frequently used by 77.5% of farmers . 94% said that they have worked with or used pesticides in their lifetime , and 16.4% from this population used pesticides in their own households . the vegetables commonly grown in the area were potatoes ( 67.4% ) , cabbage ( 63.7% ) , and carrots ( 36.8% ) . majority ( 87% ) the most common route of pesticide entry in the study was respiratory ( 68.9% ) followed by dermal and ocular entry ( 60.5% and 38% , resp . ) . sumicidine was the most commonly used pyrethroid which contains fenvalerate as its active ingredient . table 3 shows that 37% and almost 14.7% of the study population used pesticides with active ingredients of fenvalerate and cypermethrin , respectively . although pyrethroid was the most frequently used pesticide , it is organophosphate that consisted the largest amount of exposure among farmers at 210.02 liters , followed by pyrethorid at 151.4 liters , and carbamates at 32.16 liters for the entire one year . the pesticide exposure of the farmers measured in table 4 as dependent variable was related to the amount of pesticide used in liters , frequency of use and duration of use . all the independent variables , except amount and years of pesticide use were categorical variables . those who used more pesticides over a longer period of time had higher total pesticide exposure . furthermore , a farmer who was a pesticide applicator , mixer , loader and who wiped sweat with contaminated piece of fabric , and who had not been given instructions through training association was at risk of having higher pesticide exposure . seventy four percent ( 74% ) of the respondents became ill because of work for the last 12 months preceding the study . the most common symptoms were headache ( 64.1% ) , muscle pain ( 61.1% ) , cough ( 45.5% ) , weakness ( 42.4% ) , eye pain ( 39.9% ) , chest pain ( 37.4% ) , and eye redness ( 33.8% ) . a subsequent study is recommended to focus on adverse health effects of these farmers and association with certain risk factors . the results of this study identified pesticide exposure and farming practices of farmers in the largest vegetable producing area in the philippines . this has also been documented in other countries . in the study of coble et al . in 2005 , and thompson et al . in 2003 , unsafe practices like re - entry of recently spayed area , use of damaged backpack sprayer and wiping sweat with a contaminated piece of fabric were identified in this study . re - entering a recently sprayed area , as mentioned in the study of tielemans et al . in 1999 is an important determinant of dermal exposure to specific chemicals such as captan and tolylfluanid . a pesticide 's formulation is a significant factor for human exposure , with greater risks present among aqueous and emulsifiable concentrates because it impairs the protective function of chemically protective gloves . according to wolfe in 1993 , pesticides may react through chemical and biotic processes . however , pesticides may undergo activation processes unexpectedly and may be broken down to equally or more potent and mobile toxic compounds posing a greater threat to nontarget organisms . it is then advised to limit or decrease the frequency or duration of staying in the contaminated crops right after pesticide application . organophosphates , carbamates and pyrethroid pesticides were the most commonly used type of pesticides among the farmers in this study . the same was seen in the study of clarke et al . in 1997 in ghanna where organophosphates consisted of the most commonly used pesticides followed by carbamates and organochlorines . the same trend was seen among farmers in sri lanka and in brazil [ 15 , 16 ] . the farmers in this study used pesticides in their farms with a mean application time of 3.47 hours ( mean = 3.47 2.09 ) . the mean amount of pesticide used in an application was 21.35 liters per application ( mean 21.35 48.17 ) . the number of spray operations per week has been proven to have significant association with the likelihood of experiencing neurobehavioral , respiratory , or intestinal symptoms in a study among indonesian farmers . in a study among north carolina growers and agents , it was found that the study population perceived that once the pesticide is diluted and reentry intervals are observed , the risk it poses becomes diminished . sumicidine , which was the most commonly used pesticides among the benguet farmers contains fenvalerate . fenvalerate induces numbness , itching , tingling , and burning sensations in exposed workers that developed after a latent period of approximately 30 minutes , peaked by 8 hours , and disappeared within 24 hours . additional data among chinese workers demonstrated that fenvalerate decreased the semen quality of occupational workers . on the other hand , other active ingredients like cypermethrin , mancozeb , and skin sensations were reported to occur among field workers and usually lasted only for a few hours and did not persist for more than one day after exposure to cypermethrin . for mancozeb , prolonged low - level exposure to mancozeb affected several aspects of immune functioning and moderate association existed between mancozeb and neural tube defects . most often , gloves were the most commonly used personal protective equipment because the hands were the most exposed areas [ 23 , 24 ] . many circumstances contributed to nonadherence to proper use of ppe like extreme heat during pesticide application , uncomfortable to use , few resources to afford new ppe , peer - related factors , and increasing age [ 18 , 2527 ] . the study also showed certain risk factors associated with pesticide exposure such as re - entering recently sprayed area , spraying against the wind , use of damaged backpack sprayer , spills on the back , spills while mixing pesticides , among others . aside from direct pesticide use , the different agricultural tasks mentioned above may also contribute as risk factors to pesticide exposure . re - entering a recently sprayed area , as mentioned in the study of tielemans et al . in 1999 is an important determinant of dermal exposure to specific chemicals such as captan and tolylfluanid . there is evidence that weight loss could be a possible health effect of chronic pesticide poisoning . decreased body mean mass accompanied by reduced cholinesterase activities among seven farm workers was documented . also , kackar et al . in 1999 found that when rats were administered orally with mancozeb ( ethylenebisdithiocarbamate ) , dose - dependent signs of poisoning , weight loss , and mortality developed . senthilselvan et al . found a significant association between carbamate exposure and prevalence of asthma among those non - asthmatic farmers and lower mean lung function variables among those with asthma . this study has shown the pesticide exposure of farmers in the largest vegetable producing area in the philippines . it is vital that a sequential exposure assessment be done in order to come up with a correlation study between pesticide exposure and health problems . the study showed that pesticide use is prevalent among farmers in benguet which is the largest vegetable producer in the philippines . it is also suggested that chronic effects of pesticide cited in certain studies [ 31 , 32 ] such as carcinogenic effects , poor reproductive outcomes , neurologic and respiratory disorders , impairments of the immune system and birth defects should also be investigated in future studies . this manuscript adds to existing literature on pesticide exposure in the philippines which are so far mainly descriptive in nature . this paper also identifies risk factors such as work practices and designs of containers / sprayers that may increase pesticide exposure among farmers . this also calls for a local level policy research for program intervention among vegetable farmers using pesticides .

this was a cross - sectional study that investigated pesticide exposure and its risk factors targeting vegetable farmers selected through cluster sampling . the sampling size calculated with p = .05 was 211 vegetable farmers and 37 farms . the mean usage of pesticide was 21.35 liters . risk factors included damaged backpack sprayer ( 34.7% ) , spills on hands ( 31.8% ) , and spraying against the wind ( 58% ) . the top 3 pesticides used were pyrethroid ( 46.4% ) , organophosphates ( 24.2% ) , and carbamates ( 21.3% ) . those who were exposed to fungicides and insecticides also had higher total pesticide exposure . furthermore , a farmer who was a pesticide applicator , mixer , loader , and who had not been given instructions through training was at risk of having higher pesticide exposure . the most prevalent symptoms were headache ( 64.1% ) , muscle pain ( 61.1% ) , cough ( 45.5% ) , weakness ( 42.4% ) , eye pain ( 39.9% ) , chest pain ( 37.4% ) , and eye redness ( 33.8% ) . the data can be used for the formulation of an integrated program on safety and health in the vegetable industry .

1. Introduction 2. Methodology 3. Results and Discussion 4. Discussion 5. Conclusion

evans , using a mouse mutant for the lim homeodomain transcription factor ( islet1 , isl1 ) , demonstrated that an isl1 positive ( isl1 + ) population is localised in the secondary heart field ( shf ) , contributing to the outflow tract , right ventricle , atria and ( to a small extent ) the left ventricle . isl1 is considered a marker of shf cells that contribute to the venous and arterial poles of the cardiac tube , while the other cells from the shf contribute only to the arterial pole . the shf or anterior heat field ( ahf ) was identified as a splanchnic mesoderm underlying the caudal pharynx , which provides myocardium to the outflow tract during tube looping . in 2005 , the same research group also identified isl1+/c - kit negative ( c - kit-)/stem cell antigen-1 negative ( sca-1- ) progenitors in the postnatal heart , demonstrating that these cells persist after birth and that these organ - specific progenitor cells may be isolated , purified , expanded and differentiated into mature cardiac myocytes for future cardiac applications . our research group identified isl1 + cells during gestation until the postnatal age , and demonstrated the contemporary expression of isl1 and c - kit in the interstitial cells in the ventricular myocardium in human foetal and postnatal hearts . this paper demonstrated , by confocal analysis , that all isl1 + cells were also c - kit+ , while c - kit+ cells were not necessarily isl1 + , as if isl1 + cells were a subpopulation of c - kit+ cells . it has been supposed that there may be a primordial cardiovascular precursor in the shf that expresses the three markers isl1 , nkx2.5 and fetal liver kinase 1 ( flk1 ) and can generate three lineages : cardiac muscle , smooth muscle and endothelium . the shf cooperates with the first heart field ( fhf ) and the cardiac neural crest cells during the development of the vertebrate heart . the fhf lineage forms the early cardiac tube and generates the left ventricle , while the shf contributes additional cells to the maturing heart and , along with the cardiac neural crest cells , generates the outflow tract vessels and valves . the presence of cardiovascular progenitor cells positive for isl1 and/or nkx2.5 in the shf has been well confirmed by a recent work published in science . the authors suggested that there are two distinct populations , one for the shf and the other for the fhf ; the first population is clearly positive for isl1 and expresses mirna 199a / b , while the second has no distinct markers and expresses mirna 200a / b . progenitor cells of the shf depend on the cardiac specific transcription factors isl1 and nkx2.5 , which play a key role at an early stage of cardiac progenitor formation . their expression is regulated by wnt / beta - catenin signalling , which in turn also regulates bmp4 signalling . this last signalling pathway activates gata4 and srf . despite the key role of isl1 , this transcription factor is not considered an indubitable marker of this subpopulation of shf progenitor cells . it has been demonstrated very recently that isl1 labels both shf precursors and some cardiac neural crest cells and that mef2c is a more efficient marker of the shf progenitor cells in labelling experiments . this paper indirectly suggests both a review of past literature in view of these new data and using a different marker in studies of the shf progenitor cells . moreover , considering heart development in different species , such as chicken , mouse and xenopus , it appears that isl1 is expressed in a common cardiac cell population that splits into fhf and shf lineages during heart development and that this common progenitor also contributes cells to the cardiovascular system . anversa s research group described rat cardiac stem cells for the first time , many research groups have claimed to have discovered new and important cardiac progenitor / stem cells in the adult heart .. since 2005 , the scientific community talked about adult c - kit+ and/or sca-1 + and/or mdr-1 + cells , and embryonic isl1 + cells . these two populations have always been considered different entities and are described separately in many research papers and reviews , although expression of the isl1 transcription factor by cardiac precursor cells has also been reported in adult hearts . di nardo , described for the first time the concomitant expression of the three markers , i.e. , c - kit , sca-1 and isl1 , in the same cardiac precursor cell . prof . di nardo s research group described the concomitant expression of the isl1 , c - kit and sca-1 markers in adult mouse cardiac progenitor cells in 2008 , while in 2009 our research group confirmed the expression of the three markers in adult rat cpcs . in a systematic work published in 2011 about the identification of isl1 + cells in the mouse heart from postnatal day 1 to young adulthood in different strains , the authors found clusters of positive cells in the cardiac ganglia of the studied strains , while found very a few clusters of isl1 + cardiac precursors only in 129svj or balb / c strains and in animals not older than 4 months . recently genead et al . demonstrated the contemporary expression of c - kit and isl1 markers in rat adult hearts in normal , pregnant and infarcted individuals . they reported the expression of both markers in the entire heart and in the right ventricle , the left ventricle , the outflow tract and the peri - infarct and peri - ischemia regions . unfortunately , the study was based on real - time pcr analysis , and whenever immunocytochemistry was shown , no double staining for both markers had been performed . other evidence supports the hypothesis that isl1 + cells are not a different population from sca-1 + cpcs . the contemporary expression of sca-1 and isl1 has been described both in a subpopulation of sca-1+/c - kit- cells identified and isolated from adult mouse hearts and in sca-1 + cardiosphere - derived cells obtained from cardiac explants from normal , sham - operated or post - myocardial infarct hearts . in this study , only sca-1+/cd45- cells were also positive for isl1 and increased in number only after an acute myocardial infarct . considering that other authors identified isl1 + cells in adult murine and rat hearts , weinberger et al . used heterozygous isl1-lacz mice , a more sensitive genetic approach , to investigate the presence and localisation of these precursor cells in 30 animals at different time points after birth ( 10 weeks to 18 months ) . they found four different populations of isl1 + cells : i ) clusters of isl1 + neurons were found in the cardiac parasympathetic ganglia of the posterior side of the heart and in the nervous plexus surrounding the pulmonary veins ; ii ) clusters of isl1 + smooth muscle cells were found in the muscular layer above the aortic and pulmonary valve in the proximal part of the aorta and the trunk of the pulmonary artery ( only a few positive cells were present in the aortic valve leaflets ) ; iii ) clusters of isl1 + cardiomyocytes were found in the left ventricular outflow tract region ; iv ) clusters of isl1 + sinoatrial node ( san ) cells were found in the muscular wall between right atrium and vena cava superior . these results support the hypothesis that in the adult heart many cell populations may derive from isl1 + embryonic precursors , as smooth muscle cells , parasympathetic neurons , san cells , but also that , even if reduced in number , isl1 + cardiomyogenic progenitors , may be present in the adult myocardium . many papers have been published on cpcs , and many research groups claimed to have discovered different populations of progenitor cells . it is strange to think that the cardiac tissue possesses a larger number of stem / progenitor cells compared to the other tissues found in our body , and it is hopeful that cpcs may vary in number with external stimuli or with age as it happens for other adult stem cells . the hypothesis that the myocardium is a non - regenerative tissue is not valid anymore , and much evidence supports the new idea that there is at least one cardiac progenitor cell . isl1 + and c - kit+/sca-1 + cells were considered to be two different populations until 2012 , when prof . sussmann , citing our recent paper published in the european journal of histochemistry in an editorial in circulation research , suggested considering the possibility of studying a population of cells positive for both isl1 and c - kit instead of choosing to study only one side of the coin . in the review and in the research paper published in 2009 , we reported the concomitant expression of isl1 , c - kit and sca-1 in a population of cpcs isolated without any sorting or selection and suggested for the first time that there could be a unique population of cells with several subpopulations and that the several markers identified could be only the effect of different cultivation conditions in the different laboratories . other research groups later described the co - expression of isl1 , sca-1 and/or c - kit and we further demonstrated that , in samples from foetal and postnatal human hearts , there was also an isl1 + subpopulation of c - kit+ cells . anversa among the authors , on a study of the presence of c - kit+ cardiac stem cells in embryonic , foetal and neonatal mouse hearts . they demonstrated that these cells can form cardiomyocytes and coronary vessels and , as we have demonstrated in our paper , they are more abundant than isl1 + cells . moreover , they stated that isl1 + cells are present only in the outflow tract , atria and part of the right ventricle and that they are a population of committed cells derived from an immature primitive cell . we can say that there is strong evidence suggesting that c - kit , sca-1 and isl1 may be markers of subpopulations of cpcs that are present in the embryonic , foetal and neonatal heart and persist from after birth until adulthood . the identification of the real immature cardiac precursor expressing the four cell markers ( c - kit , sca-1 , mdr-1 and isl1 ) and above all the identification of a unique isolation protocol is very important in the clinical practice . this immature common cardiac precursor may represent the perfect candidate for cardiac tissue engineering and , hopefully , may be driven pharmacologically to one phenotype or the other . to identify a standardised protocol is extremely important to go further in the studies on cardiac tissue regeneration , and common guidelines are needed in cardiac stem cell therapy . the isolation of stem cells by cell sorting or immunobead selection using surface markers such as c - kit , sca-1 or mdr-1 is important to identify a standardised protocol of isolation , however isl1 may be useful in basic research to follow the fate of cpcs in heart development and myocardium differentiation . moreover , it is extremely important to understand which is the predominant precursor population in the different stages of life and which population should be used in a paediatric patient instead of an adult one . if fate of immature cardiac precursors may be determined pharmacologically , the possibility to regenerate san cells is very important in curing arrhythmia s both in patients with ischemia and malformations . the identification of the different subpopulations in the developing heart may be a fundamental step in identifying the optimal cell line for cell - based therapies .

cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells , endothelial cells and cardiomyocytes . the embryonic origin of these cells has not yet been demonstrated , but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest . in the last decade , two different populations of cardiac progenitor or stem cells have been identified and isolated , i.e. , the islet1 positive ( isl1 + ) and c - kit positive ( c - kit+)/stem cell antigen-1 positive ( sca-1 + ) cells . until 2012 , these two populations have been considered two separate entities with different roles and a different origin , but new evidence now suggests a connection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells , derived from a common immature primitive cell . to find a common consensus on this concept is very important in furthering the application of stem cells to cardiac tissue engineering .

Islet1 positive cells during heart development Isl1 positive cells in the adult heart Concluding remarks

primary diffuse leptomeningeal gliomatosis ( pdlg ) is a rare neoplasm with a short survival time of a few months . we report on a 53-year - old male patient who presented with epileptic seizures , gait disturbance , paraparesis and sensory deficits in the dermatomes t8 - 10 . magnetic resonance imaging ( mri ) revealing numerous spinal and cranial gadolinium - enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with who grade iii astrocytic cells . consecutively , the patient underwent craniospinal radiotherapy ( 30 gy ) and 11 sequential cycles of temozolomide . thirteen months later , spinal mri revealed tumor progression . second - line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration . the patient died twenty - two months after diagnosis , being the longest survival time described thus far with respect to pdlg consisting of astrocytic tumor cells . radiochemotherapy including temozolomide , as established standard therapy for brain malignant astrocytomas , might be valid as a basic therapeutic strategy for this pdlg subtype . primary diffuse leptomeningeal gliomatosis ( pdlg ) is diagnosed when a glioma is located in the subarachnoid space , while intraparenchymal tumor lesions are absent . pdlg must be distinguished from secondary meningeal gliomatosis resulting from a primary gliomatous cns tumor . pdlg , especially when caused by malignant astrocytic cells , is associated with very poor survival . we describe a patient with pdlg consisting of malignant astrocytic cells who underwent combined radio- and chemotherapy leading to the longest survival time described in the literature thus far . a 53-year - old man was referred to the dept . of neurology due to a generalized epileptic seizure and five months later he presented with bilateral sensory deficits at dermatome levels t8 - 10 , paraparesis and gait disturbance . cerebrospinal fluid ( csf ) analysis revealed a high cell count ( 300/3 cells/l ; reference range : 5/3 cells/l ) with atypical cells that could not be further characterized . t1-weighted images ( t1-wi ) revealed spinal and cranial gd - enhancing nodules in the leptomeninges ( figures 1a - e ) and on t2-weighted images ( t2-wi ) an intramedullary edema ( figure 1c ) . note gd - enhanced supra- and in - fratentorial meningeal thickenings of the brain in sagittal t1-w image ( a ) . prior to ra - diochemotherapy , t1-wi reveals lep - tomeningeal gd - enhancement ( b ) and t2-wi shows edema of the myelon on levels c7 , t1 - 2 and t5 - 6 due to minor tumor extensions in the adjacent spinal cord ( c ) . following radiochemotherapy , t1-wi displays reduced leptomeningeal gd - enhancement ( d ) while t2-wi shows less myelon spinal edema on those levels ( e ) . three weeks later , intradural biopsy from a gd - enhancing nodule at l2 - 3 level revealed fibrously - thickened meninges infiltrated by malignant astrocytic tumor cells ( figures 2a - c ) . the tumor was categorized as primary diffuse leptomeningeal gliomatosis ( pdlg ) confirmed by the reference center for brain tumors in dsseldorf , germany . intradural lumbar biopsy displays fibrously - thickened lep - tomeninges infiltrated by pleomorphic neoplastic astrocytic cells ( a ) . the astrocytic tumor cells reveal intensive intracytoplasmic staining with anti - glial fibrillary astrocytic protein . ki67 ( mib-1 ) staining reveals a high proliferation index of up to 20% ( c ) . prior to radiotherapy of the craniospinal axis ( 4 2.5 gy / week , total dose : 30 gy ) , the patient underwent 3 cycles of temozolomide ( tmz ) ( cycle 1 : 150 - 200 mg / mtzm , d1 - 5 , q28d ) . on re - staging , spinal t1-weighted images showed tumor - lesion regression on l5 and spinal t2-wi images demonstrated spinal - edema regression ( compare figures 1c - d with figures 1a - b ) . three weeks after the completion of radiotherapy the patient received eight additional cycles of tmz ( 200 mg / mtzm , d1 - 5 , q28d ) , during which meningeal thickening of the brain regressed . however , spinal mri thirteen months after starting tmz therapy revealed meningeal tumor progression at the lesions on spinal levels c1 - 2 , c7-t2 and t5 - 8 . five cycles of second - line chemotherapy with irinotecan and bevacizumab failed to halt further clinical deterioration . the patient died twenty - two months after the diagnosis of pdlg and exhibited until dead an encouraging karnovsky performance status of 60% . the diagnosis of pdlg is usually established by autopsy but rarely diagnosed prior to death . pdlg of the oligodendroglial and that of the well - differentiated astrocytic tumor type are associated with a considerably longer median survival time than that of the malignant astrocytic tumor type . pdlg was diagnosed in the aforementioned case , as we observed on histopathology leptomeningeally - encapsulated malignant astrocytic cells without primary attachment to the spinal cord or brain parenchyma and gd - enhancing leptomeningeal thickening at the base of the brain and spinal level in mri . various treatment modalities were used for 14 patients suffering from pdlg with malignant astrocytic cells reported in the literature so far ( cf . table 1 ) , demonstrating the lack of a standardized treatment regimen of pdlg with malignant astrocytic cells . although the number of cases is small , the data on their survival times ( cf . table 2 ) might suggest that radiotherapy and temozolomide ( tmz ) as established treatment for newly diagnosed recurrent anaplastic astrocytomas also seem to be valid for pdlg with malignant astrocytic cells . this concept is supported by the observation that radiotherapy alone can prolong the median survival time of five months for patients without any specific therapy to a median survival of 12 months and that integration of tmz in the chemotherapy alone may lead to a prolonged median survival of 15 months . the importance of tmz in the chemotherapy treatment of pdlg is supported by the observation that the median survival time fell to 3 months when the integration of tmz was omitted . clinico - pathological characteristics and treatment of adult patients suffering from pdlg with malignant astrocytic cells including our case 5-fu = 5-fluouracil , adr = adriamycin , auc 5 = carboplatine , bcnu = carmustine , brs = brainstem , bvz = bevacizumab , c = cycle , ca = cytarabine , ccnu = lomustine , cr = cranial , cyc = cyclophosphamide , ddp = cisplatin , eto = etoposide , inn = topotecan , int = irinotecan , irp = raised intracranial pressure , it = intrathecally , mcnu = ramustine , mtx = methotrexate , sc = spinal cord , tmz = temozolomide , top = thiotepa , vincristine , prednison summary of the literature : pdlg with malignant astrocytic cells the prolonged survival of our patient may be due to the addition of tmz to radiation therapy as it resulted in an extent of the median survival time for high grade glioma patients . in addition , the hypofractionated radiotherapy regimen used is supposed to be more effective than conventional fractionated irradiation . additionally , the patient 's good karnofsky performance status may have contributed to our patient 's prolonged survival . in conclusion , we report on partial regression and long survival in a patient with pdlg of the malignant astrocytic type following hypofractionated radiotherapy and tmz . an immediate radiochemotherapy seems crucial for a prolonged survival of pdlg patients in good general condition . the authors disclose no or potential conflict of interest including any financial , personal or other relationships with other people or organizations within the three years of beginning the submitted work that could inappropriately influence or be perceived to influence their work .

objectiveprimary diffuse leptomeningeal gliomatosis ( pdlg ) is a rare neoplasm with a short survival time of a few months . there is currently no standardized therapeutic approach for pdlg.materials and methodswe report on a 53-year - old male patient who presented with epileptic seizures , gait disturbance , paraparesis and sensory deficits in the dermatomes t8-10.resultsmagnetic resonance imaging ( mri ) revealing numerous spinal and cranial gadolinium - enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with who grade iii astrocytic cells . consecutively , the patient underwent craniospinal radiotherapy ( 30 gy ) and 11 sequential cycles of temozolomide . this regimen led to partial tumor regression . thirteen months later , spinal mri revealed tumor progression . second - line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration . the patient died twenty - two months after diagnosis , being the longest survival time described thus far with respect to pdlg consisting of astrocytic tumor cells.conclusionsradiochemotherapy including temozolomide , as established standard therapy for brain malignant astrocytomas , might be valid as a basic therapeutic strategy for this pdlg subtype .

Objective Materials and methods Results Conclusions Introduction Case Report Discussion Conflict of interests

in past years , numerous studies have described the role of microalbuminuria ( mau ) as a predictor of cardiovascular disease ( cvd ) and death among subjects with type 2 diabetes ( t2d).13 mau is one of the earliest clinical signs for diabetic nephropathy and a significant risk factor for progression to proteinuria.1 additionally , hypertensive t2d individuals with mau have an increased risk of developing end - stage renal disease ( esrd).4 risk factors known to be associated with cvd and diabetic nephropathy are high blood pressure ( bp ) and elevated glycosylated hemoglobin ( a1c).5,6 achieving adequate bp and glycemic control ( gc ) plays an essential role in preventing renal and cvd events in individuals with t2d . a current secondary analysis from the hispanic health and nutritional examination survey ( hhanes)7 indicated that cuban americans have higher serum cholesterol and systolic bp than puerto ricans and mexican americans . furthermore , compared with other hispanic subgroups , cuban americans have the highest proportion of hypertension ( htn ) and mean serum creatinine levels.8 smith and barnett9 examined the national center for health statistics ( nchs ) from 1996 to 1997 and concluded that cuban americans 35 years of age and older have the highest percentage of diabetes - related deaths compared with other hispanics . although previous studies have shown significant differences and diversity within hispanics , further studies conducted in the cuban american population are scarce . over the past decade , the prevalence of t2d has increased , especially among cuban americans who have a higher incidence of diabetes ( 8.2% ) compared with 6.6% of non - hispanic whites.10 the high incidence of t2d combined with an increased risk for developing diabetes complications warrants further examination of the cuban american population . screening for mau can detect individuals at risk for renal dysfunction and cvd events and possibly reduce the burden associated with diabetes complications . therefore , the purpose of this study was to investigate to what degree the coexistence of htn and poor gc influences the likelihood of having mau among cuban americans with t2d . it was hypothesized that individuals with t2d , htn , and poor gc will have an increased likelihood to test positive for mau . it was further hypothesized that this association will be stronger after controlling for confounding variables . this was a cross - sectional study conducted in cuban americans with and without t2d . data from a complete sample set of cuban americans with and without t2d were used in the present study . recruitment of participants was conducted in alternate phases of potential subjects with and without t2d , age matching subjects by age groups . during a 1-year period , approximately 10,000 letters outlining the study were mailed to subjects aged 30 years or older with and without diabetes . letters were sent in english and spanish and included an invitation flyer to which interested participants could respond . the participants were initially recruited by random selection ( every tenth address ) from a randomly generated mailing list . this company provided a mailing list of cuban americans identified as with and without t2d from miami - dade and broward counties , florida . three percent ( n = 300 ) of the letters were returned due to unknown addresses . from interested participants were initially interviewed on the phone , at which time the study purpose was explained and the age and gender of the responders were determined . to ascertain t2d status , only 18 subjects did not qualify for the study for not being cuban americans ( n = 2 ) , age younger than 30 years ( n = 9 ) , and having other chronic illnesses ( n = 7 ) . if a subject was determined to be eligible , then their participation was requested at the human nutrition laboratory at florida international university ( fiu ) . participants were instructed to refrain from smoking , consuming any food and beverages except water , and doing any unusual exercise for at least 8 hours prior to their blood collection . the purpose and protocol of the study were explained to the subjects , and their written consent , either in spanish or english , was obtained prior to the commencement of the study . seven participants reported not having diabetes but were reclassified because their lab results classified them as having t2d according to american diabetes association ( ada ) standards . for the data analysis , subjects with caloric intakes > 5000 kcals ( n = 2 ) and missing a1c levels ( n = 2 ) were excluded . for two participants , we were unable to perform a1c analysis . in total , we included only the data from subjects with t2d ( n = 179 ) who were aged 30 years and older . a sociodemographic questionnaire was given to each participant to complete , which included questions related to age , gender , smoking status , medications for diabetes , htn , and cholesterol . height and weight were measured using a seca balance scale ( seca corp , columbia , md , usa ) . body mass index ( bmi ) was calculated as weight in kg / height in m. bp was measured twice then averaged in participants in a sitting position after a 15-minute rest using a random zero sphygmomanometer ( tycos 5090 - 02 welch allyn pocket aneroid sphygmomanometer , arden , nc , usa ) and a stethoscope ( littmann cardiology , 3 m , st paul , mn , usa ) . htn was defined as follows : systolic bp 140 mm hg systolic or diastolic bp 90 mm hg or using antihypertensive treatment.11 dietary intake was measured using a validated semiquantitative food frequency questionnaire ( ffq ) developed by willett et al.12 this ffq has also been validated by nath and huffman13 exclusively for the cuban american population . participants self - reported average consumption of specified amounts of various foods over the past year and chose from frequency responses ranging from never to six or more servings per day . in addition to food items , the ffq included questions about type and duration of vitamin / mineral supplement use , alcohol consumption , and specific details about fat , salt , and sugar used in cooking and as condiments . macro- and micronutrient intake was calculated by multiplying frequency of consumption by the nutrient value of the food item obtained from the harvard university food composition database ( boston , ma , usa ) . venous blood ( 20 ml ) was collected from each subject after an overnight fast ( at least 8 hours ) by a certified phlebotomist using standard laboratory techniques . blood samples were collected into a vacutainer serum separator tube ( sst ) ( becton , dickinson and company , franklin lakes , nj ) for analysis of lipids and another tube containing ethylenediamine tetraacetic acid to analyze a1c . after coagulation was completed ( 3045 minutes ) , the sst was centrifuged at 2500 rpm for 30 minutes . lipid panel was assayed by enzymatic methods , and a1c percentages were measured from whole blood samples with the roche tina - quant method by laboratory corporation of america , miami , fl , usa ( labcorp ) . poor gc was defined according to the ada standards ( a1c > 7%).14 fresh , single - voided , first morning urine samples were collected from each participant to determine mau by a semi - quantitative assay ( immunodip , diagnostic chemicals limited , oxford , ct , usa ) . immunodip urinary albumin test uses a monoclonal antibody against human serum albumin to detect mau . the study conducted by davidson et al15 was designed to evaluate the clinical performance of the immunodip dipstick compared with a reference measure recommended by the ada for detecting mau ( albumin : creatinine ratio < 30 ug / mg [ negative ] ; > 30 ug / mg [ positive ] ) determined by laboratory techniques ( quest hitachi 717 autoanalyzer ) . additionally , results from the immunodip were compared with quantitatively measured albumin concentrations as a secondary outcome . urinary albumin concentrations were considered < 18 mg / l ( negative ) and > 18 mg / l ( positive ) . screening for mau with immunodip exhibited a sensitivity of 96% and specificity of 80% when compared with albumin : creatinine ratio > 30 ug / mg . when immunodip was examined against quantitatively measured albumin concentrations , the dipstick yielded a sensitivity of 95% and specificity of 94% . recommendations from the national academy of clinical biochemistry ( nacb ) for the diagnosis and management of diabetes indicated that a useful semiquantitative screening test for mau should have a sensitivity > 95%.16 the immunodip dipstick fulfilled the requirements from the nacb as a screening tool to detect mau . in our study , this cut - off value was established by the manufacturer ( immunodip , diagnostic chemicals ltd ) and corresponded to albumin : creatinine ratio > 30 ug / mg values for mau detection.15 all analyses were performed using spss version 17 ( spss inc . , chicago , il , usa ) . t - tests and chi - square tests were performed to compare means and proportion differences between subjects with and without mau . unadjusted odds ratios and logistic regression analysis were conducted to investigate the extent to which htn and gc are associated with an increased likelihood of having mau . controlled variables included in the logistic regression analysis were age , gender , bmi , known duration of diabetes , total cholesterol levels , diabetes and cholesterol medications , smoking , total kcal and protein intake , and intake of potassium , phosphorous , and sodium . this was a cross - sectional study conducted in cuban americans with and without t2d . data from a complete sample set of cuban americans with and without t2d were used in the present study . recruitment of participants was conducted in alternate phases of potential subjects with and without t2d , age matching subjects by age groups . during a 1-year period , approximately 10,000 letters outlining the study were mailed to subjects aged 30 years or older with and without diabetes . letters were sent in english and spanish and included an invitation flyer to which interested participants could respond . the participants were initially recruited by random selection ( every tenth address ) from a randomly generated mailing list . this company provided a mailing list of cuban americans identified as with and without t2d from miami - dade and broward counties , florida . three percent ( n = 300 ) of the letters were returned due to unknown addresses . from the remaining delivered mail , 4% ( n = 388 ) responded . interested participants were initially interviewed on the phone , at which time the study purpose was explained and the age and gender of the responders were determined . to ascertain t2d status , only 18 subjects did not qualify for the study for not being cuban americans ( n = 2 ) , age younger than 30 years ( n = 9 ) , and having other chronic illnesses ( n = 7 ) . if a subject was determined to be eligible , then their participation was requested at the human nutrition laboratory at florida international university ( fiu ) . participants were instructed to refrain from smoking , consuming any food and beverages except water , and doing any unusual exercise for at least 8 hours prior to their blood collection . the purpose and protocol of the study were explained to the subjects , and their written consent , either in spanish or english , was obtained prior to the commencement of the study . seven participants reported not having diabetes but were reclassified because their lab results classified them as having t2d according to american diabetes association ( ada ) standards . for the data analysis , subjects with caloric intakes > 5000 kcals ( n = 2 ) and missing a1c levels ( n = 2 ) were excluded . for two participants , in total , we included only the data from subjects with t2d ( n = 179 ) who were aged 30 years and older . a sociodemographic questionnaire was given to each participant to complete , which included questions related to age , gender , smoking status , medications for diabetes , htn , and cholesterol . height and weight were measured using a seca balance scale ( seca corp , columbia , md , usa ) . body mass index ( bmi ) was calculated as weight in kg / height in m. bp was measured twice then averaged in participants in a sitting position after a 15-minute rest using a random zero sphygmomanometer ( tycos 5090 - 02 welch allyn pocket aneroid sphygmomanometer , arden , nc , usa ) and a stethoscope ( littmann cardiology , 3 m , st paul , mn , usa ) . htn was defined as follows : systolic bp 140 mm hg systolic or diastolic bp 90 mm hg or using antihypertensive treatment.11 dietary intake was measured using a validated semiquantitative food frequency questionnaire ( ffq ) developed by willett et al.12 this ffq has also been validated by nath and huffman13 exclusively for the cuban american population . participants self - reported average consumption of specified amounts of various foods over the past year and chose from frequency responses ranging from never to six or more servings per day . in addition to food items , the ffq included questions about type and duration of vitamin / mineral supplement use , alcohol consumption , and specific details about fat , salt , and sugar used in cooking and as condiments . macro- and micronutrient intake was calculated by multiplying frequency of consumption by the nutrient value of the food item obtained from the harvard university food composition database ( boston , ma , usa ) . venous blood ( 20 ml ) was collected from each subject after an overnight fast ( at least 8 hours ) by a certified phlebotomist using standard laboratory techniques . blood samples were collected into a vacutainer serum separator tube ( sst ) ( becton , dickinson and company , franklin lakes , nj ) for analysis of lipids and another tube containing ethylenediamine tetraacetic acid to analyze a1c . after coagulation was completed ( 3045 minutes ) , the sst was centrifuged at 2500 rpm for 30 minutes . lipid panel was assayed by enzymatic methods , and a1c percentages were measured from whole blood samples with the roche tina - quant method by laboratory corporation of america , miami , fl , usa ( labcorp ) . fresh , single - voided , first morning urine samples were collected from each participant to determine mau by a semi - quantitative assay ( immunodip , diagnostic chemicals limited , oxford , ct , usa ) . immunodip urinary albumin test uses a monoclonal antibody against human serum albumin to detect mau . the study conducted by davidson et al15 was designed to evaluate the clinical performance of the immunodip dipstick compared with a reference measure recommended by the ada for detecting mau ( albumin : creatinine ratio < 30 ug / mg [ negative ] ; > 30 ug / mg [ positive ] ) determined by laboratory techniques ( quest hitachi 717 autoanalyzer ) . additionally , results from the immunodip were compared with quantitatively measured albumin concentrations as a secondary outcome . urinary albumin concentrations were considered < 18 mg / l ( negative ) and > 18 mg / l ( positive ) . screening for mau with immunodip exhibited a sensitivity of 96% and specificity of 80% when compared with albumin : creatinine ratio > 30 ug / mg . when immunodip was examined against quantitatively measured albumin concentrations , the dipstick yielded a sensitivity of 95% and specificity of 94% . recommendations from the national academy of clinical biochemistry ( nacb ) for the diagnosis and management of diabetes indicated that a useful semiquantitative screening test for mau should have a sensitivity > 95%.16 the immunodip dipstick fulfilled the requirements from the nacb as a screening tool to detect mau . in our study , this cut - off value was established by the manufacturer ( immunodip , diagnostic chemicals ltd ) and corresponded to albumin : creatinine ratio > 30 ug / mg values for mau detection.15 t - tests and chi - square tests were performed to compare means and proportion differences between subjects with and without mau . unadjusted odds ratios and logistic regression analysis were conducted to investigate the extent to which htn and gc are associated with an increased likelihood of having mau . controlled variables included in the logistic regression analysis were age , gender , bmi , known duration of diabetes , total cholesterol levels , diabetes and cholesterol medications , smoking , total kcal and protein intake , and intake of potassium , phosphorous , and sodium . mau was present in 26% ( n = 47 ) of cuban americans with t2d . there was a significantly higher percentage of subjects with mau classified as hypertensive ( p = 0.038 ) and taking diabetes medication ( p = 0.039 ) compared with those without mau . additionally , subjects who tested positive for mau had significantly higher a1c levels ( p = 0.002 ) than those with normoalbuminuria ( table 1 ) . unadjusted odds ratios indicated that subjects with poor gc were 3.96 times more likely to have positive mau if they had htn compared with those without htn ( p = 0.014 ; 95% confidence interval [ ci ] 1.25 , 12.5 ) ( figure 1 ) . logistic regression analysis showed that after controlling for covariates , subjects with poor gc were 6.76 times more likely to have mau if they had htn compared with those without htn ( p = 0.004 ; 95% ci 1.83 , 23.05 ) ( table 2 ) . the results of this study showed that htn and poor gc are major contributors to increasing the likelihood of having mau among cuban americans with t2d . the combination and/or interaction of these factors over time might increase the risk for progression to proteinuria / esrd and cvd in this population . our findings are supported by the multicenter study conducted in europe that found that the likelihood of having mau increases in patients when htn and poor gc are present along with other coexisting risk factors for cvd.17 ravid et al5 reported from their longitudinal study that a combination of risk factors , including abnormal bp , plasma cholesterol , a1c levels , high bmi , and male gender , identifies a group of individuals for poor renal and cardiovascular outcomes . maintaining adequate the uk prospective diabetes study ( ukpds),18 a longitudinal study with a 10-year median follow - up , showed that exposure over time to hyperglycemia was associated with diabetes complications in subjects with t2d . this study also indicated that for every 1% reduction in a1c level , the risk for microvascular complications decreased by 37% and diabetes - related death by 21% . a study by thomaseth et al19 showed that in hypertensive t2d subjects with incipient diabetic nephropathy , both tight bp control and optimal gc delay the progression of glomerular filtration rate deterioration . additionally , hypertensive individuals with t2d under tight bp control experience a reduction in risk for microvascular and macrovascular complications.20 the mechanism and pathways involving mau with diabetic nephropathy and cvd are not fully understood . they may be interrelated with endothelial dysfunction and inflammation.21 stehouwer et al22 examined the relationship between endothelial dysfunction and inflammation with mau and risk of death in a prospective study among subjects with t2d . the results of that study showed that those participants with mau , endothelial dysfunction , and inflammation had an increased risk in mortality ; however , the associations of these variables with risk of mortality were independent from each other . hyperglycemia and obesity were associated with an increase in markers of endothelial dysfunction and inflammation activity,22 possibly indicating that the combinations of these factors may be interrelated in increasing the risk of death . the usual course of mau is progressive ; however , not all t2d individuals with mau will develop macroalbuminuria . several authors have documented remission and/or regression of mau in subjects with t2d.2327 antihypertensive therapy has been shown to reduce or slow the progression of diabetic nephropathy . in a 6-year prospective study , remission and regression to normoalbuminuria were observed in about 50% of t2d individuals.23 other factors independently linked with remission and/or regression of mau were proper bp and gc and short duration of mau . the study by chan et al24 with a 5-year mean follow - up of t2d patients with mau treated with angiotensin - converting enzyme ( ace ) inhibitor medications showed a 13% reduction in urinary albumin excretion ( uae ) . in addition , the authors pointed out the main role that bp and gc play in renal function . t2d individuals treated with angiotensin receptor blockers ( arbs ) ( eg , irbesartan and valsatran ) experienced a 38% and 44% reduction in uae over 2-year and 6-month follow - ups , respectively.25,26 evidence from another prospective study with a 7.8-year follow - up indicated that 30% of the participants on antihypertensive therapy achieved remission to normoalbuminuria.27 additionally , the odds for remission to normoalbuminuria increased in these participants with every 1% decrease in a1c level . furthermore , in a study conducted by mogensen et al,28 urinary albumin : creatinine ratio was decreased by 50% after a 3-month combination treatment with arb and ace inhibitors in hypertensive t2d individuals with mau . remission and/or regression of mau may not only conserve renal function but also reduce the risk of cvd . testing for mau has been recommended by the ada in individuals with t2d to be performed at diagnosis of diabetes and every year afterwards.29 the immunodip dipstick is a rapid and easy screening test to perform , does not involve equipment and/or skilled personnel , exhibits a high sensitivity , requires only a random urine sample ( first morning urine is recommended ) , and has a relatively low cost . however , this test does not quantify urinary albumin values and requires confirmation with a secondary analysis . this method can be used in a physician s office and/or research setting as a first screening tool to detect the presence of mau . the clinical significance of early screening and monitoring for mau is possibly to improve individuals prognosis for microvascular and macrovascular complications , especially among t2d individuals with other concomitant conditions . first , due to the cross - sectional design of the study , single - voided urine was collected to measure mau , and we were not able to determine the cause second , due to the relatively small sample size in this study , our sample of cuban americans with t2d is not representative of the entire cuban american population living in the usa . nevertheless , to our knowledge , this is the first and only study that has examined the relationship between mau , htn , and gc in this hispanic subgroup , which has an increased risk for t2d and cvd . first , due to the cross - sectional design of the study , single - voided urine was collected to measure mau , and we were not able to determine the cause second , due to the relatively small sample size in this study , our sample of cuban americans with t2d is not representative of the entire cuban american population living in the usa . nevertheless , to our knowledge , this is the first and only study that has examined the relationship between mau , htn , and gc in this hispanic subgroup , which has an increased risk for t2d and cvd . early detection of mau in this population may provide more valuable treatment and improve individuals renal and cvd outcomes . the therapeutic goals and strategies for mau should focus on preventing long - term complications associated with t2d , such as kidney and heart diseases . further investigations need to be carried out to fully understand the mechanism and absolute cvd risk that an individual with t2d has when mau coexists with other comorbidities .

purpose : to investigate to what degree the presence of hypertension ( htn ) and poor glycemic control ( gc ) influences the likelihood of having microalbuminuria ( mau ) among cuban americans with type 2 diabetes ( t2d).methods : a cross - sectional study conducted in cuban americans ( n = 179 ) with t2d . participants were recruited from a randomly generated mailing list purchased from knowledge - base marketing , inc . blood pressure ( bp ) was measured twice and averaged using an adult size cuff . glycosylated hemoglobin ( a1c ) levels were measured from whole blood samples with the roche tina - quant method . first morning urine samples were collected from each participant to determine mau by a semiquantitative assay ( immunodip).results : mau was present in 26% of cuban americans with t2d . a significantly higher percentage of subjects with ma had htn ( p = 0.038 ) and elevated a1c ( p = 0.002 ) than those with normoalbuminuria . logistic regression analysis showed that after controlling for covariates , subjects with poor gc were 6.76 times more likely to have mau if they had hypertension compared with those without hypertension ( p = 0.004 ; 95% confidence interval [ ci ] : 1.83 , 23.05).conclusion : the clinical significance of these findings emphasizes the early detection of mau in this hispanic subgroup combined with bp and good gc , which are fundamentals in preventing and treating diabetes complications and improving individuals renal and cardiovascular outcomes .

Introduction Methods Design Study population Study variables Dietary assessment Blood collection Urinary albumin Data analysis Results Discussion Limitations of the study Conclusion

cancer is the second leading cause of mortality and morbidity in both developed and developing countries . in india , cancer prevalence is estimated around 2.5 million , with over 0.8 million new cases and 0.5 million deaths occurring each year . there is an increase in the incidence of breast cancer and found to be gradually overtaking cancer of the cervix . breast self - examination ( bse ) is an important screening measure for detecting breast cancer . there is evidence that women who correctly practice bse monthly are more likely to detect a lump in the early stage of its development , and early diagnosis has been reported to influence early treatment , to yield a better survival rate . thus the present study aimed at identifying the level of knowledge and practice of bse among degree female students who are the citizen of the future and they can teach their family members , neighbors , friends and the community which helps the people to detect breast cancer in early stage . thus the morbidity or mortality can be reduced . in the current study only one participant was practicing bse occasionally so incorporating the bse concept in the degree education curriculum is very useful and helpful . the present study aimed at assessing the level of knowledge and the effectiveness of planned teaching program among degree female students on bse . it is a patient - centred , inexpensive and noninvasive method of screening for breast cancer . based on increased incidence of breast cancer and unawareness of bse among young women , researcher felt a need to provide awareness of bse among young women and can be reduced the incidence and prevalence of breast cancer in future . the objectives of the study were to : to assess the level of knowledge of degree college female students on bse.to determine the effectiveness of planned teaching program among degree college female students on bse.to find the association between pretest knowledge and selected demographical variables . to assess the level of knowledge of degree college female students on bse . to determine the effectiveness of planned teaching program among degree college female students on bse . the study attempted to test following hypotheses all the hypotheses were tested at 0.05 level of significance . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students.there will be a significant association between pretest knowledge score and selected demographical variables . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students . the study assumed that : the degree college female students will have some knowledge on bse.feel free to express their attitude toward bse.bse helps in early detection of breast cancer . the degree college female students will have some knowledge on bse . feel free to express their attitude toward bse . independent variables : teaching program on bse . selected variables : age , education , parent 's education and exposure to mass media . the present study aimed at assessing the level of knowledge and the effectiveness of planned teaching program among degree female students on bse . it is a patient - centred , inexpensive and noninvasive method of screening for breast cancer . based on increased incidence of breast cancer and unawareness of bse among young women , researcher felt a need to provide awareness of bse among young women and can be reduced the incidence and prevalence of breast cancer in future . the objectives of the study were to : to assess the level of knowledge of degree college female students on bse.to determine the effectiveness of planned teaching program among degree college female students on bse.to find the association between pretest knowledge and selected demographical variables . to assess the level of knowledge of degree college female students on bse . to determine the effectiveness of planned teaching program among degree college female students on bse . the study attempted to test following hypotheses all the hypotheses were tested at 0.05 level of significance . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students.there will be a significant association between pretest knowledge score and selected demographical variables . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students . the study assumed that : the degree college female students will have some knowledge on bse.feel free to express their attitude toward bse.bse helps in early detection of breast cancer . the degree college female students will have some knowledge on bse . feel free to express their attitude toward bse . independent variables : teaching program on bse . selected variables : age , education , parent 's education and exposure to mass media . a pre - experimental one group pretestpost - test study was conducted among degree female students from selected colleges of udupi district . a cluster sampling technique was used to select the college and 40 students selected by convenient method from all the streams of study . demographic proforma consisted of age , stream of study , education and occupation of parents , income of parents and sources of information on bse . a structured questionnaire consisted of 25 multiple choice questions ( mcq ) was developed to assess the knowledge on bse . knowledge scores were categorized into poor ( 0 - 8 ) , average ( 9 - 16 ) and good ( 17 - 25 ) . planned teaching program . lesson plan with power point presentation given for validation contained basic anatomy and physiology , risk factors for breast cancer and steps of bse . validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . data was collected by administering knowledge questionnaire on bse and planned teaching program was introduced for them . on 8 day post - test was done by administering the same tool . the data was analyzed using descriptive ( frequency and percentage ) and inferential statistics based on the objectives and hypotheses . demographic proforma consisted of age , stream of study , education and occupation of parents , income of parents and sources of information on bse . a structured questionnaire consisted of 25 multiple choice questions ( mcq ) was developed to assess the knowledge on bse . knowledge scores were categorized into poor ( 0 - 8 ) , average ( 9 - 16 ) and good ( 17 - 25 ) . planned teaching program . lesson plan with power point presentation given for validation contained basic anatomy and physiology , risk factors for breast cancer and steps of bse . validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . demographic proforma consisted of age , stream of study , education and occupation of parents , income of parents and sources of information on bse . a structured questionnaire consisted of 25 multiple choice questions ( mcq ) was developed to assess the knowledge on bse . knowledge scores were categorized into poor ( 0 - 8 ) , average ( 9 - 16 ) and good ( 17 - 25 ) . planned teaching program . lesson plan with power point presentation given for validation contained basic anatomy and physiology , risk factors for breast cancer and steps of bse . validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . administrative permission was obtained from the principal of selected colleges . written consent was obtained from the study participants . data was collected by administering knowledge questionnaire on bse and planned teaching program was introduced for them . on 8 day post - test was done by administering the same tool . the data was analyzed using descriptive ( frequency and percentage ) and inferential statistics based on the objectives and hypotheses . the data presented in table 1 shows that among 40 samples , the majority ( 52.5% ) of the samples were in the age group of 18 - 19 years and majority ( 90% ) of them were studying in basic science group . sample characteristics only 35% of them were heard about bse through mass media and 8.5% of participant ( only one participant ) were practiced bse only one time at the time of study . the description of knowledge scores shows that 72.5% of students had average knowledge on bse in pre test and 85% of students had good knowledge score in post - test [ figure 1 , table 2 ] . t test computed to test the effectiveness of planned teaching program on bse ( t=12.46 ) shown in table 3 . percentage distribution of sample based on knowledge score frequency and percentage of knowledge score computation of effectiveness of planned teaching program the computed to find the association between knowledge and selected variables shows no significant association between knowledge and selected variables . hence the null hypotheses was accepted and research hypotheses was rejected shown in table 4 . a survey conducted among nurses and midwives in turkey found that among 80 samples only 52% of samples performed bse , no significant relation was found between sociodemographic factor and bse . a study conducted in chennai by s. aruna supported this study as no significant association found between demographic variables and level of knowledge of breast cancer and bse among working women . study conducted among resettlement colony women , shows that they have poor knowledge on breast cancer and risk factors , warning signs and early detection procedure . the world health organization stresses on promoting awareness in the community and encouraging early diagnosis of breast cancer , especially for women aged 40 - 69 years who are attending primary health care centres of hospitals for other reason , by offering clinical breast examination . there are other many methods to detect the breast cancer but those are expensive for the community . breast - related matters are sensitive issues for few females ; unless the breast lesions starts bothering them they may not seek medical attention . bse is the cheapest and convenient method to detect the breast cancer in the early stage . in this study majority of the samples acquired good knowledge on bse . bse plays a major role in early detection and prevention or prompt treatment of breast cancer . by giving teaching to the young girls , they can teach their mother and siblings so that the incidence of the breast cancer may be reduced . it is essential task of each and every woman to do bse and protect herself and her family . the nurse has to play an important role in health promotion and it is only possible when she teaches or educates her client . however , the teaching of bse can help women to be alert to any abnormal changes in their breasts and seek medical advice immediately .

introduction : breast cancer accounts for 19 - 34% of all cancer cases among women in india . there is high mortality due to late stage diagnosis as patients usually present at an advanced stage because of lack of awareness and nonexistent breast cancer screening programs . early detection and prompt treatment offer the greatest chance of long - term survival and breast self - examination ( bse ) seems to be a important viable optional substitute for early detection of cancer.objectives:1 ) to assess the level of knowledge of degree college female students on bse . 2 ) to determine the effectiveness of planned teaching program among degree college female students on bse . 3 ) to find the association between pretest knowledge and selected demographic variables.materials and methods : pre - experimental one group pretestpost - test design was carried out among 40 degree female students by using cluster sampling method from selected colleges of udupi district.results:the data analyzed showed that majority ( 52% ) of them was in the age group of 18 - 19 years and 72% of them were had average knowledge on bse in the pretest score . out of 40 participants only one student was performing bse occasionally.conclusions:awareness regarding breast self examination among young generations is useful and it is the most important viable tool for early detection .

INTRODUCTION Purpose Objectives Hypotheses Assumption Variables MATERIALS AND METHODS Data collection instruments Part 1 Part 2 Part 3 Data collection procedure RESULT DISCUSSION CONCLUSIONS

der p 1 was isolated from house dust mite fecal pellets ( allergon ) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti der p 1 mab ( clone 4c1 ; indoor biotechnologies ) , removal of contaminating serine proteases on immobilized soybean trypsin inhibitor ( sigma chemical co. ) , and finally fast protein liquid chromatography ( fplc ) to remove low - molecular - mass contaminants . the purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer ( applied biosystems , inc . ) , sds - page analysis ( 15% gel ) , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 ( l - trans - epoxysuccinyl - leucylamido [ 4-guanidino]butane ) . protein concentration was determined using a bicinchoninic acid ( bca ) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e ( 280 nm ) = 16.4 . before use , der p 1 was preactivated with 5 mm cysteine ( sigma chemical co. ) to regenerate its thiol group , which becomes oxidized during purification . the catalytic activity of der p 1 was ascertained in a continuous rate ( kinetic ) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl ( boc)-gln - ala - arg7-amino-4-methyl - coumarin ( amc ; reference 6 ) . to block the proteolytic activity of cysteine - activated der p 1 , 1,000-fold molar excess of e-64 ( sigma chemical co. ) was used ; a similar molar ratio of the cysteine protease inhibitor iodoacetamide ( sigma chemical co. ) was used as another sulfhydryl reactive agent . the cells ( 2 10 ) were suspended in rpmi ( gibco life technologies ) and stimulated for 3 d at 37c with con a ( 5 g / ml final concentration ) in the presence of il-2 ( 100 u / ml ) in a humidified atmosphere of 5% co2 . cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 ( preactivated with 5 mm cysteine ) for 1 h at 37c in a total volume of 200 l serum - free aim v medium ( gibco life technologies ) . the cells were then resuspended in rpmi containing 2% fcs , stained for 45 min at room temperature in the dark with fitc - labeled anti mouse cd25 mab ( clone amt-13 ; sigma chemical co. ) , and fixed with 5% formaldehyde . the expression of other t cell surface markers , namely cd3 , cd4 , and cd8 , was monitored in the same way using appropriate pe- or fitc - labeled antibodies ( clone kt3 , beckman coulter ; and clones yts191.1 and kt15 [ serotec ltd . five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1 , 10 g of e-64blocked der p 1 , 10 g of iodoacetamide - blocked der p 1 , 10 g of ova ( as proteolytically inactive antigen ; sigma chemical co. ) , or 10 g of ova with e-64 , respectively . a tail bleed was obtained 1 wk before the start of immunization ( prebleed ) , and a total bleed was obtained by cardiac puncture 1 wk after the last injection ( final bleed ) . the proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above . serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass . the optimal dilutions used here were 1/10 for detecting total ige , der p 1specific ige , and ova - specific ige and 1/20,000 , 1/40,000 , and 1/250 for detecting der p 1specific igg , igg1 , and igg2b , respectively . mouse ige ( clone r35 - 72 ; pharmingen ) as capture antibody and a second biotinylated monoclonal anti der p 1specific ige , ova - specific ige ( measured using samples that have been depleted of igg on a protein g column [ pharmacia ] ) , and der p 1specific igg , igg1 , and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated ( for ige clone r35 - 118 and igg1 clone a85 - 1 [ pharmingen ] and for igg2b clone ab275 [ the binding site ] ) or alkaline phosphatase conjugated isotype - specific antibodies ( for igg ; sigma chemical co. ) . alkaline phosphatase conjugated extravidine ( sigma chemical co. ) was used in conjunction with biotinylated antibodies . unpaired student 's t test was used to compare levels of antibody responses between the different immunization groups ; p < 0.05 was considered significant . der p 1 was isolated from house dust mite fecal pellets ( allergon ) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti der p 1 mab ( clone 4c1 ; indoor biotechnologies ) , removal of contaminating serine proteases on immobilized soybean trypsin inhibitor ( sigma chemical co. ) , and finally fast protein liquid chromatography ( fplc ) to remove low - molecular - mass contaminants . the purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer ( applied biosystems , inc . ) , sds - page analysis ( 15% gel ) , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 ( l - trans - epoxysuccinyl - leucylamido [ 4-guanidino]butane ) . protein concentration was determined using a bicinchoninic acid ( bca ) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e ( 280 nm ) = 16.4 . before use , der p 1 was preactivated with 5 mm cysteine ( sigma chemical co. ) to regenerate its thiol group , which becomes oxidized during purification . the catalytic activity of der p 1 was ascertained in a continuous rate ( kinetic ) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl ( boc)-gln - ala - arg7-amino-4-methyl - coumarin ( amc ; reference 6 ) . to block the proteolytic activity of cysteine - activated der p 1 , 1,000-fold molar excess of e-64 ( sigma chemical co. ) was used ; a similar molar ratio of the cysteine protease inhibitor iodoacetamide ( sigma chemical co. ) was used as another sulfhydryl reactive agent . spleen t cells were obtained from c57bl/6j mice using standard procedures . the cells ( 2 10 ) were suspended in rpmi ( gibco life technologies ) and stimulated for 3 d at 37c with con a ( 5 g / ml final concentration ) in the presence of il-2 ( 100 u / ml ) in a humidified atmosphere of 5% co2 . cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 ( preactivated with 5 mm cysteine ) for 1 h at 37c in a total volume of 200 l serum - free aim v medium ( gibco life technologies ) . the cells were then resuspended in rpmi containing 2% fcs , stained for 45 min at room temperature in the dark with fitc - labeled anti mouse cd25 mab ( clone amt-13 ; sigma chemical co. ) , and fixed with 5% formaldehyde . the expression of other t cell surface markers , namely cd3 , cd4 , and cd8 , was monitored in the same way using appropriate pe- or fitc - labeled antibodies ( clone kt3 , beckman coulter ; and clones yts191.1 and kt15 [ serotec ltd . five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1 , 10 g of e-64blocked der p 1 , 10 g of iodoacetamide - blocked der p 1 , 10 g of ova ( as proteolytically inactive antigen ; sigma chemical co. ) , or 10 g of ova with e-64 , respectively . a tail bleed was obtained 1 wk before the start of immunization ( prebleed ) , and a total bleed was obtained by cardiac puncture 1 wk after the last injection ( final bleed ) . the proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above . serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass . the optimal dilutions used here were 1/10 for detecting total ige , der p 1specific ige , and ova - specific ige and 1/20,000 , 1/40,000 , and 1/250 for detecting der p 1specific igg , igg1 , and igg2b , respectively . mouse ige ( clone r35 - 72 ; pharmingen ) as capture antibody and a second biotinylated monoclonal anti der p 1specific ige , ova - specific ige ( measured using samples that have been depleted of igg on a protein g column [ pharmacia ] ) , and der p 1specific igg , igg1 , and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated ( for ige clone r35 - 118 and igg1 clone a85 - 1 [ pharmingen ] and for igg2b clone ab275 [ the binding site ] ) or alkaline phosphatase conjugated isotype - specific antibodies ( for igg ; sigma chemical co. ) . alkaline phosphatase conjugated extravidine ( sigma chemical co. ) was used in conjunction with biotinylated antibodies . unpaired student 's t test was used to compare levels of antibody responses between the different immunization groups ; p < 0.05 was considered significant . der p 1 is a 25-kd cysteine protease whose structure has been modeled 7 on the crystal structure of papain , with which it shows considerable sequence similarities , most notably for residues involved in the enzyme active site 8 . the proteolytic activity of der p 1 can be inhibited by e-64 , the class - specific inhibitor of microbial origin 9 . this inhibition is brought about when cysteine within the der p 1 active site forms a thioether covalent bond with the epoxy group of e-64 . this is an irreversible process that does not lead to significant structural changes , as evidenced by crystallographic studies of a papain e-64 complex 10 . we have purified der p 1 from fecal pellets using a multistep procedure and confirmed its purity by nh2-terminal sequencing , sds - page analysis , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and inhibited by e-64 and iodoacetamide ( fig . 1 ) . we have recently shown that der p 1 selectively cleaves human cd25 from the surfaces of peripheral blood t cells 4 . here we demonstrate that der p 1 also selectively cleaves cd25 from cultured mouse spleen t cells ( fig . 2 ) , which is not surprising given the high degree of sequence homology that exists between human 11 and mouse 12 cd25 . this observation has therefore provided the justification for using this animal species for testing our hypothesis , namely that the proteolytic activity of der p 1 is a major contributor to its allergenicity . intraperitoneal immunization of groups of 10 cba / j mice with either proteolytically active or inactive ( e-64blocked ) der p 1 over a 6-wk period showed a statistically significant enhancement in total ige ( p < 0.01 ) and der p 1specific ige ( p < 0.02 ) responses in animals immunized with proteolytically active der p 1 . this effect was ige specific , as der p 1specific igg , igg1 , and igg2b responses increased to the same extent with proteolytically active or inactive der p 1 ( fig . we are not sure why the igg1 response did not follow that of ige , as these two isotypes are considered to be coregulated in the mouse . however , the ige - restricted enhancement seen in response to immunization with proteolytically active der p 1 does suggest a mechanism that is unique to ige isotype switching / synthesis . furthermore , our control experiments clearly show that the ige - specific effect observed here is not due to e-64 exerting a suppressive influence on ige production by a mechanism that is independent of its binding to the der p 1 enzyme active site ( fig . , suppression of total ige ( p < 0.04 ) and der p 1specific ige ( p < 0.05 ) productions was also obtained when the proteolytic activity of der p 1 was blocked with iodoacetamide , another sulfhydryl reactive agent . second , the ige antibody response to ova , a proteolytically inactive antigen , was not suppressed when the animals were immunized with ova plus e-64 . our results are direct evidence that the cysteine protease activity of der p 1 induces a significant increase in ige responses . such an effect is clearly consistent with the ability of der p 1 to proteolytically cleave mouse cd25 and induce a th2 response by modulating the balance between il-4 and ifn- 13 . the recent demonstration in mice that leishmania mexicana cysteine proteinase deficient mutants potentiate a th1 response , compared with the th2 response normally seen in response to infection with wild - type parasite 14 , is also of great relevance here . allergic individuals could potentially be achieved by administering der p 1 in a catalytically inactive ( mutant ) form . on the other hand , exploring the potential th2 adjuvant property of the proteolytic activity of der p 1 would have important implications in defining principles for modulation of th1-mediated pathological conditions . our demonstration that the cysteine protease activity of der p 1 enhances total ige production , apart from increasing der p 1-specific ige , suggests that this allergen may play a central role in destabilizing the microenvironment within target tissues to one that is proallergic and thus aids in the initiation and propagation of the allergic cascade . in other words , the proteolytic activity of der p 1 may exert an ige - specific adjuvant effect . the in vivo relevance of the proteolytic activity of der p 1 is further highlighted by reports demonstrating that it increases the permeability of the human respiratory epithelium to macromolecules 1516 . such observations , together with our current findings showing a direct effect on the immune system , indicate that the proteolytic activity of der p 1 is a major contributor to its allergenicity .

the house dust mite dermatophagoides pteronyssinus allergen der p 1 is the most immunodominant allergen involved in the expression of dust mite specific immunoglobulin ( ig)e mediated hypersensitivity . the reason for this potent ige - eliciting property of der p 1 remains unknown , but there is mounting in vitro evidence linking the allergenicity of der p 1 to its cysteine protease activity . here we demonstrate for the first time that immunization of mice with proteolytically active der p 1 results in a significant enhancement in total ige and der p 1specific ige synthesis compared with animals immunized with der p 1 that was irreversibly blocked with the cysteine protease inhibitor e-64 . we conclude that the proteolytic activity of der p 1 is a major contributor to its allergenicity .

Materials and Methods Der p 1 Preparation. CD25 Cleavage. Immunization Protocol. Antibody Detection. Results and Discussion

given the risk of paralysis associated with cervical transforaminal injection , is it time to reconsider transforaminal injections of the lumbar spine ? arguments for discontinuing lumbar injections have been discussed in the anesthesia literature , raising concern about the risks of epidural steroid injections ( esis ) . in a 47-year - old man , paraplegia of the lower extremities developed , specifically conus medullaris syndrome , after he underwent an esi for recurrent pain . the patient felt his legs going dead ; paraplegia of the lower extremities was noted . an initial magnetic resonance imaging study performed after the patient was transferred to the emergency department was unremarkable . however , a later neurosurgical evaluation showed conus medullaris syndrome , and a second magnetic resonance imaging study showed the conus infarct . we conducted a search of the pubmed database of articles from 2002 to 2011 containing the following keywords : complications , lumbar epidural steroid injection(s ) , cauda equina syndrome , conus medullaris infarction , spinal cord infarction , spinal cord injury , paralysis , paresis , plegia , paresthesia , and anesthesia . summarizing this case and 5 similar cases , we weigh the potential benefits and risks of esi . although one can safely assume that this severe , devastating complication is rare , we speculate that its true incidence remains unknown , possibly because of medicolegal implications . we believe that the rarity of this complication should not preclude the continued use of transforaminal esi ; rather , it should be emphasized for discussion with patients during the consent process . a 47-year - old man who had undergone an l4/l5 laminectomy and discectomy 5 years earlier presented with recurrent pain in the left buttock and posterior leg . after receiving an esi by a local anesthesiology group within the cincinnati , ohio , area , the patient was transferred from the outpatient pain clinic to our institution . information regarding his medical history and prior treatment , as well as details of the procedure , was limited to that provided by the patient himself or the accompanying procedure report . the patient reported that he had previously received esis on the left side of his lower back ; this was the second injection in a series of 3 . correct needle placement in the left l5-s1 neural foramen was verified with epidurography immediately after the injection , the patient felt his legs going dead ; paraplegia of the lower extremities was noted . because of the concern for intrathecal injection with resultant motor blockade , the patient was monitored for 4 hours . when no clinical improvement was observed , he was transferred to a nearby community hospital emergency department for neurologic examination . although the initial magnetic resonance imaging ( mri ) study performed in the emergency department was unremarkable ( fig . however , a second mri study to evaluate the possibility of vascular complications obtained 48 hours after the injection showed a conus infarct ( fig . 2 ) . five hours after esi in a 47-year - old man , magnetic resonance imaging scans performed at the emergency department were unremarkable , showing a normal appearing conus . ( a ) ( b ) t1-weighted sagittal image ( repetition time , 416.7 ; echo time , 15.0 ) . magnetic resonance images ( about 48 hours after the procedure ) showing extensive signal abnormalities within the lower thoracic spinal cord and conus compatible with the clinical diagnosis of conus infarct . ( a ) t1-weighted sagittal image ( repetition time , 675.0 ; echo time , 9.6 ) . ( b ) t2-weighted sagittal image ( repetition time , 3640.0 ; echo time , 102.0 ) . ( c ) short tau inversion recovery ( stir ) sagittal image ( repetition time , 4000.0 ; echo time , 58.0 ) . at 1-month follow - up , the patient could walk without assistance with a slow and calculated gait , had symmetric lower - extremity strength , and had dorsiflexion strength of 4 of 5 bilaterally . his urinary urge sensation had returned , and occasional episodes of fecal incontinence occurred in relation to bladder overdistention . our search of the pubmed database ( 20022011 ) included the keywords complications , lumbar epidural steroid injection(s ) , cauda equina syndrome , conus medullaris infarction , spinal cord infarction , spinal cord injury , paralysis , paresis , plegia , paresthesia , and anesthesia . esi is regarded as an effective and conservative means of treating low - back pain resulting from nerve root inflammation . recent case reports of post - procedural conus medullaris syndrome after spinal vascular compromise raise the question regarding safety . our case represents a sixth patient in whom conus medullaris syndrome developed after sustaining an acute spinal cord infarct during esi . in 5 other case reports ( table 1 ) , this complication affected patients ( aged 4271 years ) who had previous lumbar surgery and then underwent a transforaminal steroid injection . of note , findings of spinal cord injury were not detectable on the initial mri study within the first 24 hours in 1 case . injections are typically evaluated first by aspiration and injection of contrast . a nonvascular penetrating injection of contrast media verifies safe needle placement . however , in a series of 761 lumbosacral transforaminal injections performed , furman et al . reported that the sensitivity of positive flash or actual aspiration was only 44.7% , because only 38 of 85 patients had shown either flash or actual aspiration before injection . specifically , they showed that there was an 11.2% rate of vascular injection in their series . in evaluating correct placement of 316 caudal - approach esis , implicated aspiration as an errorprone method for verifying needle placement , reporting a 9.2% incidence of vascular violation despite negative findings on aspiration . blood is supplied to the caudal - most portion of the spinal cord by the anterior spinal artery , 2 posterior spinal arteries , segmental radiculomedullary arterial branches , and most importantly , the artery of adamkiewicz . the location of the artery of adamkiewicz , which is the primary blood supply to the conus medullaris , is fairly unpredictable . it travels with the nerve through the foramen at or near the level of its origin from the thoracolumbar segmental arteries . . showed that the artery of adamkiewicz originated from the left t9 - 12 posterior intercostal arteries in 75% of cases and from the l1 - 2 lumbar arteries in 10% of cases . in 31 cadavers biglioli et al . located this artery between t12 and l3 in 26 cases ( 83.9% ) . in a 2002 review of more than 4000 spinal angiograms , lo et al . examined the variability of the artery of adamkiewicz , noting that it originated at l2 in 1% of cases and l4 in 0.075% of cases . of the proposed mechanisms by which spinal cord medullary infarction may occur , one explanation may be the combined effect of an undetected direct arterial injection into a low - lying artery of adamkiewicz and the resultant embolic incident from the injectate . houten and errico proposed that the collaterals surrounding the cord at the level of this artery were proximal to the injection site and thus allowed direct passage of the injected material into the conus . however , as discussed by lo et al . considering this observation , we believe that it is unlikely that our patient had this low - lying artery that could have then been injected with an epidural steroid at this precise location . another plausible explanation is that an inadvertent sacral radicular artery injection carried injection material distally to the spinal cord . reported that material injected into the abdominal aorta below the level of the artery of adamkiewicz would appear in the conus through collateralization . the most likely cause is injection of steroid particulate through either the artery of adamkiewicz or collateral radiculomedullary arterial branches . prior lumbar surgery may lead to compromise of normal vascular supply to the spinal cord , thus making it more susceptible to vascular injury . subsequent thrombus formation would result in an embolic infarct in the spinal cord supplied by the affected artery . florey noted that such vasoconstriction was a local effect lasting 5 seconds to 10 minutes at the site of injury only . found that fine needle ( 30-gauge ) arterial puncture in rhesus monkeys induced intense vasospasm , typically lasting anywhere from 4 hours to 4 days . many studies have examined mechanically induced vasospasm of the cerebral vasculature and may be indirectly suggestive that a similar phenomenon can occur in the spinal cord and conus medullaris . of the multiple recommendations made to avoid such devastating complications as paralysis , first and foremost is the strict adherence to the standard and widely accepted techniques of transforaminal esis . these guidelines include the use of multiplanar fluoroscopy or computed tomography guidance together with contrast material to prevent complications . if recovery of neurologic function fails to occur in a patient within a 2- to 3-hour time period after esi , an initial mri study may be obtained to exclude an epidural hematoma . repeat mri after 24 hours should be performed as well . in our patient , as well as 1 other reported case , a delayed effect was observed with mri signal changes occurring after 24 hours . given the risk of paralysis associated with cervical transforaminal injection , is it time to reconsider transforaminal injections of the lumbar spine ? although the risk of permanent neurologic deficit is negligible , arguments for discontinuing lumbar injections have been presented in the anesthesia literature . however , when one considers the potential benefit of this noninvasive treatment modality , abandoning such injections may be premature . however , we speculate that more than 6 cases of conus medullaris syndrome after esi have occurred , and medicolegal considerations may explain why they may go unreported . at the very least , patients should be clearly informed regarding the potential risks of esi , including paralysis . although one can safely assume that this severe , devastating complication is rare , its true incidence remains unknown . in our opinion , the rarity of this complication should , at this time , not preclude the continued use of transforaminal esi for relief of pain in select patients but should be included in the consent process .

backgroundgiven the risk of paralysis associated with cervical transforaminal injection , is it time to reconsider transforaminal injections of the lumbar spine ? arguments for discontinuing lumbar injections have been discussed in the anesthesia literature , raising concern about the risks of epidural steroid injections ( esis).methodsin a 47-year - old man , paraplegia of the lower extremities developed , specifically conus medullaris syndrome , after he underwent an esi for recurrent pain . correct needle placement was verified with epidurography . immediately after the injection , the patient felt his legs going dead ; paraplegia of the lower extremities was noted.resultsan initial magnetic resonance imaging study performed after the patient was transferred to the emergency department was unremarkable . however , a later neurosurgical evaluation showed conus medullaris syndrome , and a second magnetic resonance imaging study showed the conus infarct . we conducted a search of the pubmed database of articles from 2002 to 2011 containing the following keywords : complications , lumbar epidural steroid injection(s ) , cauda equina syndrome , conus medullaris infarction , spinal cord infarction , spinal cord injury , paralysis , paresis , plegia , paresthesia , and anesthesia.conclusionssummarizing this case and 5 similar cases , we weigh the potential benefits and risks of esi . although one can safely assume that this severe , devastating complication is rare , we speculate that its true incidence remains unknown , possibly because of medicolegal implications . we believe that the rarity of this complication should not preclude the continued use of transforaminal esi ; rather , it should be emphasized for discussion with patients during the consent process .

Background Methods Results Conclusions Case report Literature review Discussion Conclusion

because of rampant concern that estrogenic chemicals in the environment may be adversely affecting the health of humans and wildlife , reliable methods for detecting and characterizing estrogenic chemicals are needed . it is important that general agreement be reached on which tests to use and that these tests then be applied to the testing of both man - made and naturally occurring chemicals . as a step toward developing a comprehensive approach to screening chemicals for estrogenic activity , three assays for detecting estrogenicity were conducted on 10 chemicals with known or suspected estrogenic activity . the assays were 1 ) competitive binding with the mouse uterine estrogen receptor , 2 ) transcriptional activation in hela cells transfected with plasmids containing an estrogen receptor and a response element , and 3 ) the uterotropic assay in mice . the chemicals studied were 17 beta - estradiol , diethylstilbestrol , tamoxifen , 4-hydroxytamoxifen , methoxychlor , the methoxychlor metabolite 2,2-bis(p - hydroxyphenyl)-1,1,1-trichloroethane ( hpte ) , endosulfan , nonylphenol , o , p'-ddt , and kepone . these studies were conducted to assess the utility of this three - assay combination in the routine screening of chemicals , or combinations of chemicals , for estrogenic activity . results were consistent among the three assays with respect to what is known about the estrogenic activities of the chemicals tested and their requirements for metabolic activation . by providing information on three levels of hormonal activity ( receptor binding , transcriptional activation , and an in vivo effect in an estrogen - responsive tissue ) , an informative profile of estrogenic activity is obtained with a reasonable investment of resources.imagesp1296-afigure 1.figure 2.figure 3.figure 4.figure 5 .

Images

alzheimer 's disease ( ad ) is a neurodegenerative disorder that is clinically characterized by progressive mental decline and histopathologically defined by highly abundant amyloid deposits and neurofibrillary tangles in the brain parenchyma . the identification of mutations within the amyloid precursor protein ( app ) and presenilin ( ps ) genes that cause autosomal dominantly inherited ad and that result in increased production of amyloid - prone forms of a established beyond doubt that the processing of app and the production of a peptides are intimately involved in the disease process and led to the proposal and the reinforcement of the alzheimer amyloid cascade hypothesis [ 1 , 2 ] . the role of amyloid in neuronal dysfunction has recently been extended by the discovery of small , soluble , oligomers of the a peptide , some forms of which have been termed addls ( a-derived diffusible ligands ) , protofibrils , or a*56 [ 36 ] . these a oligomers are not only potential intermediates in the formation of amyloid filaments , but they also have been shown to be neurotoxic themselves and to inhibit long - term potentiation ( ltp ) , a cellular model of memory , in hippocampal slices [ 4 , 7 , 8 ] . thus , the amyloid cascade hypothesis now includes the essential role of a oligomers in the neurodegeneration process . despite its strength , the amyloid cascade hypothesis is incomplete without including the essential role of amyloid - associated inflammatory proteins . for example , biochemical and histological studies first showed that , in addition to a , amyloid deposits also contained the inflammation / acute phase protein 1-antichymotrypsin ( act ) and , later , apolipoprotein e ( apoe ) [ 10 , 11 ] , which were both hypothesized to serve as catalysts or pathological chaperones of amyloid formation [ 9 , 11 , 12 ] . these and other results also indicated that alzheimer 's disease and its manifestation in middle - aged down syndrome may include an inflammatory process , for both act and apoe are inflammatory and/or acute phase proteins in other contexts , and both are overexpressed in affected regions of the ad brain ( for reviews see [ 1315 ] ) . indeed , alzheimer himself first identified the inflammatory component of alzheimer 's disease when he described reactive astrocytes and microglia in affected brain regions of his first patient . however , until inflammatory proteins such as act , il-1 , hla , and apoe were found to be overexpressed in ad and ds brains , the term inflammation was explicitly excluded from the clinical and pathological description of ad because of the lack of edema and lymphocyte infiltration [ 911 , 17 , 18 ] . the significance of these biochemical results instigated and was reinforced by parallel genetic discoveries implicating a role for inflammation in ad . in particular , inheritance of the apoe 4 allele was found to be the strongest known risk factor for ad besides age , with one copy increasing ad risk 35-fold and two copies over 10-fold [ 1921 ] . because of apoe 's essential genetic , and therefore presumably biochemical , contribution to ad pathology and cognitive decline , it is critical that its role in the ad pathogenic pathway / amyloid cascade be elucidated in order for therapeutics based on apoe to be designed . while recent excellent and encyclopedic literature reviews describe the many potential roles that apoe plays in ad [ 2326 ] , this focused review will concentrate on the interaction between a and apoe and other inflammatory proteins , on the effects of such interactions , and on their implications for designing apoe - based ad therapies . the central question we try to answer is whether increasing or decreasing apoe level and/or function will serve best to reduce ad / ds pathology and cognitive decline . lack of a clear answer may lead to the development of drugs that , rather than serving as an ad therapy , instead potentially exacerbate the disease . to determine whether inflammation contributes to alzheimer 's disease rather than being merely a correlative pathological feature in the ad brain , we and others tested the hypothesis that act and/or apoe serve as amyloid catalysts or pathological chaperones . numerous in vitro and in vivo studies showed that mature amyloid deposition and the associated cognitive decline is strongly stimulated by apoe and act in a dose - dependent and isoform - specific manner , with apoe4 being the strongest promoter of a polymerization and apoe2 being an inhibitor , paralleling the effect of these two isoforms in humans [ 2738 ] . indeed , without one or the other of these amyloid catalysts expressed in the brain , amyloid deposition is profoundly delayed in app transgenic mice and does not become filamentous . such app+/apoe ko animals also exhibit normal cognition despite levels of a expression equal to the apoe - expressing app animals . elegant work by manelli and colleagues also showed that native lipidated apoe4 from transgene replacement astrocytes increases a neurotoxicity compared to apoe3 or e2 , indicating that apoe4 provides a negative gain of function . finally , jones and colleagues recently showed that apoe4 also promotes the conversion and enhanced synaptic localization of a as oligomers , the most neurotoxic form of the alzheimer amyloid peptide [ 40 , 41 ] . these recent studies extended prior work showing that apoe copurifies with a during biochemical isolation of amyloid from human brains , and that apoe preferentially interacts with a peptides in a -sheet structure [ 4245 ] . together these results show that inflammatory proteins , particularly apoe , are integral parts of the amyloid cascade , and that without them the cascade would be arrested at the level of the harmless a monomer , and no ad would ensue . the view of apoe as an integral and pathological part of the amyloid cascade has been shaken by experiments that suggest that apoe , far from being an amyloid catalyst , serves to clear a from the brain . under this view , apoe is protective , with human apoe4 being less protective than apoe3 or e2 ( for the most recent discussion , see and commentary at http://www.alzforum.com/ ) . the first experiments that suggested apoe 's role as a neuroprotector examined the pathology and cognition of app transgenic mice carrying a second transgene expressing one or another human apoe isoform . contrary to expectations , amyloid deposition in these mice was inhibited by the human apoe transgene , as though human apoe was protective . ultimately , the mice did develop amyloid , with the apoe4-expressing strain accumulating earlier and more extensive pathology [ 33 , 34 , 48 , 49 ] . it was proposed that human apoe might serve to inhibit a clearance from the brain compared to mouse apoe , with apoe4 inhibiting clearance the most . other experiments showed that indeed , clearance of a species was inhibited by complexing with apoe , especially apoe4 [ 46 , 50 ] . the possibility that interaction with apoe modulated an a clearance mechanism appeared to be supported by the finding that introduction of anti - a antibodies or other a-binding proteins such as gelsolin , led to a reduced amyloid load in the brain and rapidly improved cognition , with little evidence of a-binding agents invading the brain parenchyma [ 4154 ] . we also introduced apoe itself into the circulation via parabiosis and found that it induced amyloid clearance without entering the brain in ad model mice . thus the peripheral sink hypothesis became a viable alternative or addition to the amyloid cascade hypothesis , with apoe potentially playing an additional role as an a-binding peripheral protein . most recently , an approach to therapy has been investigated in ad mice that is based on activating the liver x receptor ( lxr ) , which also exists on other cells including microglia [ 5557 ] . activation of lxr results in increased expression of many proteins including apoe and its lipidating enzyme , atp - binding cassette transporter a1 ( abca1 ) . the results indicate that activating lxr with the ligand gw3965 or the fda - approved antiskin cancer drug bexarotene reduces soluble and insoluble a and improves cognition in app tg mice , while knocking out the abca1 gene in app mice showed a tendency to reduced amyloid load . because apoe expression and lipidation is stimulated by lxr activation , the results were interpreted as proof that increased apoe levels help microglia clear a and amyloid , as indeed some earlier cell culture experiments had suggested . however , it has also been shown that genetic overexpression of abca1 reduces amyloid deposition in mice where the apoe levels are unchanged . hence , because lxr stimulation influences the levels of many proteins , it is problematic to definitively link its in vivo action to the altered level of one particular protein . furthermore , the increased levels of abca1 induced by bexarotene enhance apoe lipidation , a change that is known to alter apoe / a interactions . hence , it is important to consider the lipidation state of apoe , which affects its function , in addition to the absolute levels of apoe . when trying to distinguish and weigh the value of two hypotheses , it is instructive to consider their testable predictions . if apoe is an amyloid catalyst , then reducing apoe levels or function in the brain should result in reduced amyloid deposition and reduced cognitive decline . if on the other hand , apoe is involved in a clearance with human apoe4 being a greater inhibitor of clearance ( or poorer clearer ) , then reducing apoe levels or apoe binding to a should increase amyloid deposition and cognitive decline . all experiments carried out so far in vitro or in transgenic mice indicate that the ability of a to form neurotoxic filaments or oligomers and cause cognitive decline are increased in the presence of apoe , particularly mouse apoe and human apoe4 , with apoe2 being protective . in contrast , in the complete absence of apoe , the mutant app gene and its product a are harmless , generating neither amyloid deposits , synaptic disfunction , or cognitive decline , with one copy of apoe having an intermediate effect , as discussed above . the in vitro experiments in particular indicate that apoe likely acts catalytically to promote a polymerization , as the molar ratio of a to apoe of about 200/1 was appropriate for the formation of neurotoxic products [ 2730 ] . most recently , earlier work showing that mice expressing only one apoe gene accumulated less amyloid than those with two apoe genes ( 32 ) was repeated in two different laboratories using human apoe knock - in mice , and the same result was found , that is , lower doses of apoe3 or apoe4 led to reduced amyloid deposition [ 59 , 60 ] . the simplest interpretation of the in vitro , cell culture , and transgenic mouse data is that apoe is necessary for a to polymerize into neurotoxic oliogomers / filaments , probably by binding to a and thus altering its structure more toward the -sheet and more easily allowing successive a peptides to add on to the growing chain . the recent finding that apoe promotes a oligomer formation in vivo reinforces this interpretation [ 40 , 41 ] . whether apoe is only needed to initiate the polymerization or also to prepare each peptide for addition to the growing filament is not yet known . even though the key predictions of the polymerization hypothesis , that is apoe serving as an a filament catalyst , have been borne out , the compelling experiments demonstrating that human apoe inhibits filament formation in a mouse background require explanation . furthermore , data from ladu and colleagues and by others have shown that lipidated apoe , presumably the prevalent form in vivo , binds a with an affinity of e2 > e3 finally , the elegant and thorough experiments of castellano and colleagues show very convincingly that expression of a human apoe4 transgene ( in the absence of mouse apoe ) leads to a longer half - life , ( i.e. , slower clearance ) of a in the brain interstitial fluid compared to e2 or e3 . the apoe - a binding studies might be interpreted as support for apoe functioning in a clearance because apoe2 , for example , would bind a tightly and could thereby promote its removal from the interstitial fluid via lrp receptors [ 50 , 6164 ] . however , an important feature of any catalyst is that it must bind its substrate only tightly enough to convert it to the transition state structure and then release it as the reaction is completed [ 65 , 66 ] . if a mutation leads to an overly tight substrate binding , then no further reaction can occur . thus apoe2 could indeed bind a most tightly , and thereby not only prevent apoe4 from binding and promoting a oligo / polymerization , but also prevent the spontaneous polymerization of the peptide . the ability of different apoe isoforms to bind a with different strengths can also explain why human apoe isoforms slow amyloid deposition in the presence of the endogenous mouse apoe , for they may bind a more tightly or differently than mouse apoe and slow the catalytic conversion of a into oligomers / polymers in the mouse background . the data showing that human apoe inhibits a clearance can also be interpreted as reflecting apoe 's role in catalyzing a oligo / polymerization . pathologic macromolecular structures are often resistant to various clearance mechanisms designed for monomeric species , whether by intracellular proteasome degradation or cross - membrane / bbb transfer , thus allowing their accumulation . only when oligo / polymeric structures are anticipated and physiological clearance mechanisms are in place to handle them , as for antibody - antigen complexes , will clearance be facilitated by conversion to larger structures . because apoe clearly has the ability to catalyze the conversion of a into oligomeric and polymeric structures , it is reasonable to assume that those structures will be more difficult to clear , and that such difficulty will be detected as clearance inhibition in the brain , for instance , by apoe4 , in pulse chase type experiments , while the higher apoe levels in blood may aid the clearance of a from the circulations ( figure 1 ) . finally , the ability of gw3965 and bexarotene to reduce soluble and insoluble a in the brain of tg app mice and improve cognition is most easily understood as resulting from a general activation of the phagocytic activity of microglia . previous work showed that activation of microglia by acute intracerebral treatment of app mice with lps or with granulocyte - macrophage stimulating factor can similarly reduce amyloid load and improve cognition [ 6769 ] but that long - term peripheral treatment with lps exacerbated amyloid deposition in an apoe - dependent manner . stimulation of microglial activity via induction of toll - like receptor 9 ( tlr9 ) has also been shown to greatly reduce amyloid load and improve cognition . clearly the interaction of neuroinflammation , microglia , and amyloid load is complex , and the fact that bexarotene cures ad in mice is more likely to be despite , rather than because it stimulates expression of apoe . a good test of any hypothesis about the pathogenesis of a disease is whether it successfully predicts how the pathogenesis can be inhibited or reversed . for example , small fragments of a corresponding to the amino acid sequences to which act ( a1 - 12 ) and apoe ( a12 - 28 ) bind can serve as decoy peptides that prevent the binding of apoe to a and its catalysis of a into neurotoxic species . this early in vitro work has recently been repeated and confirmed in other laboratories [ 72 , 73 ] . the decoy principal was extended in vivo by preparing a version of a12 - 28 that has a better plasma 1/2 life and is nonfibrillogenic / nontoxic . it was shown that this peptide could be peripherally introduced into a transgenic app mouse , where it effectively entered the brain and prevented / reversed oligomer formation , amyloid deposition , and cognitive decline [ 7476 ] . similarly , amyloid plaques in app mice contain mouse act and injecting a1 - 11 into one side of the app mouse brain to block act 's binding site with endogenous a rapidly reduced amyloid load compared to the other , vehicle - injected side of the brain . furthermore the inflammatory cytokine il-1 that is overexpressed in ad brain induces astrocyte expression of act , and blocking il-1 expression in app transgenic mice by ibuprofen treatment , thereby reducing mouse act expression , lowers amyloid formation and restores cognition . evidently , blocking act or apoe expression or function , both in vitro or in vivo , successfully prevents a pathology and neurotoxicity . apolipoprotein j also binds a and can be shown to aid its passage across the blood brain barrier [ 7983 ] . interestingly , knocking out either apoj or apoe reduces amyloid deposition in app transgenic animals , yet knocking out both leads to robust amyloid deposition at an even earlier age than arises in nonmanipulated app animals . this result may reflect the ability of mouse act to promote amyloid formation , but that in the presence of the stronger binding apoe and apoj proteins mouse act is prevented from exhibiting its catalytic activity . the role of apoe and act in the alzheimer pathogenic pathway has potentially general implications . one of the most studied classes of a binding proteins are specific anti - a antibodies , which form the basis of both passive and active immunization therapies for alzheimer 's disease ( for review see ) . the finding that apoe and act can catalyze a oligo / polymerization begs the question of whether a antibodies might also promote or inhibit a polymerization . indeed we found that two a antibodies , 6e10 which is directed to the same the n - terminal sequence bound by act , and 13 m , which binds to the c - terminus , function very differently in the in vitro a polymerization assay . 6e10 inhibits act - catalyzed polymerization of a while 13 m inhibits act catalysis much less and even promotes some polymerization itself . interestingly , the n - terminus of a is also the target of many attempts at ad immunotherapy with the aim of inducing microglial phagocytosis of neurotoxic a species . yet removing the microglial - binding fc portion of 3d6 antibodies to a1 - 5 to generate fab'2 fragments does not reduce the antibody 's ability to remove diffuse amyloid in app mice . evidently , only its a-binding feature is required to allow the antibody to remove amyloid . a possible explanation for this result is that the antibody functions by blocking a interaction with mouse act . the consequent suppression of act - catalyzed oligo / polymerization could thus tilt the dynamic process of plaque development toward depolymerization . these results illustrate the fact that a-binding proteins can have multiple effects on polymerization and that their full range of activities must be considered when using them as potential targets or tools for therapeutic intervention . although a oligomers have been shown to be highly neurotoxic in vitro and in vivo , and their formation is promoted by apoe4 , the mechanism of their toxicity is still being elucidated . the data reviewed above coupled to other recent findings suggest a novel mechanism for a toxicity that encompasses the essential role of apoe . specifically , a oligomers bind to and inhibit certain microtubule motors that are essential for the function and stability of the mitotic spindle similar motors , including kinesin 5 , are also present in mature neurons [ 88 , 89 ] . we have found recently that inhibition of mt motor function by a or by the specific kinesin 5 inhibitor monastrol prevents the efficient transport of receptors such as the ldlr , the nmda neurotransmitter receptor , and the p75 neurotrophin receptor to the cell surface , resulting in reduced function (; in preparation ) . similarly , apoe , particularly apoe4 , has been shown to reduce the cell surface levels and function of nmda , ampa , and apoer2 receptors in neurons . this latter finding can now be understood as potentially reflecting the ability of apoe4 to promote the conversion of endogenous neuronal a into oligomers , which then inhibit mt - based transport of key cellular components such as receptors to their functional location . in sum , it appears that the preponderance of the data can be most consistently interpreted as showing that the brain inflammatory protein apoe plays a catalytic role in the ad / ds amyloid cascade and consequent cognitive decline , with binding and clearance differences between the apoe isoforms reflecting their differing abilities to bind to a and catalyze its conversion into neurotoxic macromolecular species ( figure 2 ) . this conclusion , and the in vivo demonstration that blocking apoe - a interaction prevents ad in a mouse model , suggests that this decoy approach should be translatable into human patients and serve as an effective new approach to ad therapy . other a-binding proteins may be similarly manipulated by a decoy approach to reduce oligomerization and polymerization of a into neurotoxic species . however , the finding that different antibodies to a can both inhibit act - catalyzed a polymerization and promote polymerization of a itself , argues that immunotherapy must be approached with care to avoid the use or induction of antibodies that can catalyze further oligo / polymerization of a , instead of inducing its phagocytosis and removal . furthermore , human and mouse intracerebral environments may differ in important ways with respect to the pattern and activities of a-binding proteins and may also respond differently to intervention or inflammation . such differences may explain why so many treatments that were successful in reducing amyloid - dependent cognitive decline in transgenic mice have failed to translate into human ad patients . finally , the ability of a oligomers to inhibit key microtubule motors and prevent the transport of neurotrophin , neurotransmitter , and other receptors to the cell surface may underlie their neuronal toxicity . it is apparently the apoe- , especially e4-dependent formation of such a oligomers , that constitutes the key catalytic step in the ad pathogenic pathway .

the amyloid cascade hypothesis remains a robust model of ad neurodegeneration . however , amyloid deposits contain proteins besides a , such as apolipoprotein e ( apoe ) . inheritance of the apoe4 allele is the strongest genetic risk factor for late - onset ad . however , there is no consensus on how different apoe isotypes contribute to ad pathogenesis . it has been hypothesized that apoe and apoe4 in particular is an amyloid catalyst or pathological chaperone . alternatively it has been posited that apoe regulates a clearance , with apoe4 been worse at this function compared to apoe3 . these views seem fundamentally opposed . the former would indicate that removing apoe will reduce ad pathology , while the latter suggests increasing brain apoe levels may be beneficial . here we consider the scientific basis of these different models of apoe function and suggest that these seemingly opposing views can be reconciled . the optimal therapeutic target may be to inhibit the interaction of apoe with a rather than altering apoe levels . such an approach will not have detrimental effects on the many beneficial roles apoe plays in neurobiology . furthermore , other a binding proteins , including act and apo j can inhibit or promote a oligomerization / polymerization depending on conditions and might be manipulated to effect ad treatment .

1. Introduction 2. Background: ApoE as Amyloid Catalyst 3. Background: ApoE in A 4. Synthesis 5. A 6. Potential Efficacy and Dangers of A 7. Potential Toxic Mechanism of ApoE-Induced A 8. Conclusion

musculoskeletal tissues show increased bone fragility , loss of cartilage resilience , reduced ligament elasticity , loss of muscular strength , and fat redistribution decreasing the ability of the tissues to carry out their normal functions . the loss of mobility and physical independence resulting from arthropathies and fractures can be particularly devastating in this population , not just physically and psychologically , but also in terms of increased mortality rates . the aim of this article is to present some of the most frequent musculoskeletal disorders of the elderly , including some misleading presentations . we used the following terms and some of its combinations : elderly , aged , epidemiology , fracture , osteoporosis , vertebral , hip , pelvis , arthritis , neoplasm , malignancy , myeloma , paget , gout , infection , microcrystal , and radiology . articles without english abstracts were excluded . besides , certain relevant rheumatological , orthopedic , and radiological books were also used . fractures are frequent in the elderly and result mainly from the effects of falls and osteoporosis . low - impact falls , even from standing height , are the most common cause of injury in geriatric patients . falling is a multi - factorial problem due to both extrinsic ( e.g. , environmental and housing conditions ) and intrinsic risk factors ( e.g. , impaired mobility , loss of muscular strength , poor visual acuity , and medication , for instance corticoids).[46 ] osteoporosis , which is characterized by qualitatively normal , but quantitatively deficient bone , leads to bone fragility and increased risk of fractures . the general prevalence of osteoporosis in women is approximately 50% at the age of 85 years , while in men the prevalence is about 20% at that age . ethnic differences also exist : mineral bone density is higher in black women and lower in asian women , while in white women evidence shows an intermediate value . due to its precision , the most widely used quantitative technique is dual energy x - ray absorptiometry , which makes it possible to diagnose osteoporosis early , predict the risk of fracture , determine therapeutic intervention , and monitor response to treatment . this method grades bone loss according to standard deviations ( sd ) from the mean young adult value . a value less than 2.5 times the sd below the young adult mean , is considered to indicate osteoporosis . the main radiographic features of osteoporosis are increased radiolucency and cortical thinning , mainly in the spine , giving rise to a well - demarcated outline of the vertebral body , which has been described as , picture framing. besides , in the spine , an increased biconcavity of the vertebral end plates , with a protrusion of the intervertebral disk into the vertebral body can be observed . however , early radiographic changes are subtle , as a bone loss of approximately 30% must occur before it can be detected . vertebral compression fractures are the most common osteoporotic fractures , with a greater incidence in women over 60 years of age . they can be observed in up to 25% of women over 70 years of age and 40% of those over 80 years . after one episode , the risk of subsequent vertebral fracture increases at least four - fold , with substantially increased rates during the first year . these fractures are associated with higher mortality and significant morbidity , even though they are afforded little clinical attention . they often appear as multiple fractures with anterior wedging , and may be associated with significant deformity , leading to kyphosis . they are usually well demonstrated on radiographs , but can be better assessed with computed tomography ( ct ) or magnetic resonance ( mr ) imaging , when necessary . the latter imaging modality may be indicated in case of associated neurological symptoms a rare event or when a vertebroplasty is discussed , in order to better localize the recently fractured vertebra . vertebral compression fractures in the elderly may involve several problems : first is differentiating between osteoporotic and malignant vertebral collapse ( vc ) , particularly metastatic vc , as the latter is by far the most common malignant tumor affecting the skeleton in the elderly . in most patients , multiple , relatively symmetrical thoracolumbar vcs , associated with diffuse increased radiolucency are suggestive of osteoporosis [ figure 1a ] . the whole vertebral endplate is impacted , sometimes in association with increased or decreased radiolucency of the subjacent trabecular bone ( band - like distribution of the abnormalities under the fractured vertebral end plate ) . gas can also be seen under the fracture and is nearly pathognomonic of the benign nature of the collapse . there is no cortical osteolysis , although fractures can be identified with retropulsion of a bone fragment . conversely , the following are a cause for concern : a single vc , involvement of the cervical spine , heterogeneous increased or decreased radiolucency that can not be explained by an underlying end plate fracture , a focal collapse ( on the ap or lateral view ) , or a bulging of the vertebral body or cortical osteolysis [ figure 1b ] . if in doubt or for a better assessment of the spinal canal and soft tissue , ct , or better yet , an mri can be performed , in order to differentiate between these two disorders . in benign fractures , an mri may show a band distribution of abnormal signal intensities on t1 and t2 weighted images , spared normal bone marrow signal intensity , retropulsion of a posterior bone fragment , and multiple compression fractures ; on the contrary , signs such as a convex posterior border of the vertebral body , abnormal signal intensity of the posterior elements , an epidural mass or a focal paraspinal mass are suggestive of spinal metastasis.[1921 ] second , vertebral fractures can be missed following moderate trauma , because the fracture may be difficult to identify in an osteoporotic radiolucent bone and / or because the pain is attributed to another painful condition , such as degenerative changes . this is observed in particular in the cervical spine , particularly at c2 , and may lead to secondary neurological complications [ figure 2 ] . ankylosis may be related to ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis , which especially affect males and they have an estimated frequency of 5 to 15% in the elderly.[2225 ] these ankylosed spines become increasingly susceptible to injury , even in the event of low - energy impacts , and are prone to unstable vertebral fractures with an increased frequency of neurological complications , either primary or secondary , after unprotected transfers and manipulation . these lesions may be missed on plain films when non - displaced or mildly displaced , especially in hyperextension , whereas , they are well demonstrated on ct or mr imaging . however , the mri features can sometimes be misleading and mimic infectious spondylitis when signal abnormalities of the disco - vertebral junction are present . caution is required when an elderly patient presents with an ankylosed spine , minor trauma or acute pain , especially if signal abnormalities of the anterior and posterior spine are observed [ figure 3 ] . usual benign vertebral compression ( a ) multiple levels , diffuse radiolucency , and whole vertebral endplate involvement ( arrows ) ; usual malignant vertebral compression characteristics ( b ) single vertebral involvement , focal and bulging of the posterior wall ( arrowheads ) . missed fracture of the odontoid process and vertebral body of c2 ( arrows ) in a patient with a history of fall one month before . transverse fracture of thoracic vertebral body ( arrow ) and posterior arch ( arrowhead ) with posterior epidural hematoma ( curved arrow ) in an ankylosed spine depicted on ( a ) t1- and ( b ) t2-weighted sequences . other important sites of fractures are the hip and the pelvis , which are associated with increased mortality and specific diagnostic problems in the elderly population . they are common and frequently result from lateral falls directly on the greater trochanter , in the case of hip fractures , and forward or backward falls in the case of pelvic fractures . most fractures are easily diagnosed by conventional radiography , particularly fractures of the proximal femur . however , non - displaced fractures may be negative or equivocal , particularly if the bone is osteoporotic . if a clinical suspicion of a fracture persists , ct or better still mr imaging is advocated , as this modality can show the fracture line surrounded by bone marrow edema . they may occur everywhere , but are particularly common in the hip and pelvis , the majority being located in the sacrum and pubic ramus [ figure 4 ] . radiographic findings are often subtle or unremarkable , and in suspected cases mr imaging is indicated due to its superior accuracy as compared to other radiological methods . all the images should be carefully analyzed , as multiple insufficiency fractures are frequent [ figure 5 ] . subchondral fractures are particularly difficult to identify on radiographs , but the thin hypointense line that represents the fracture , surrounded by a variable degree of edema , is well demonstrated on an mri [ figure 6 ] . this kind of fracture can be complicated by a necrosis of the subchondral bone , which is hypointense on t2 weighted images , and is not enhanced following gadolinium administration . insufficiency fractures ( arrows ) located in the sacrum with adjacent edema ( arrowheads ) , better depicted on ( a ) t1- and ( b ) t2- weighted sequences , respectively . multiple fractures ( white arrows ) in ( a ) the proximal femur and ( b ) pubic ramus on coronal t1-weighted images subchondral fracture of the femoral head : ( a ) conventional radiography and ( b ) fat saturated sagittal t2-weighted image ( white arrow ) . osteoarthritis ( oa ) can be defined as a group of distinct , but overlapping diseases , which may have different etiologies , but similar biological , morphological , and clinical outcomes affecting the articular cartilage , subchondral bone , ligaments , joint capsule , synovial membrane , and periarticular muscles . pathological changes can include fibrillation of cartilage , disruption of collagen fibers , changes in proteoglycan staining , chondrocyte proliferation , necrosis , and neovascularization . oa is the most common joint disease in persons 65 years of age and above , with a radiographic prevalence as high as approximately 90% in women and 80% in men.[3335 ] its etiology is not fully understood , although there are several related factors , such as female gender , genetics , metabolism , and excessive mechanical stress . it frequently leads to decreased function and loss of independence . although the joints of the hand are the most commonly affected , they are less likely to be symptomatic than the knee or hip [ figure 7 ] . the diagnosis of oa is primarily based on clinical history and physical examination . plain radiographs can help confirm both the diagnosis and grade the severity of the condition . the cardinal radiographic features of oa are focal / non - uniform narrowing of the joint space in the areas subjected to the most pressure , subchondral cysts , subchondral sclerosis , and osteophytes . however , the severity of the spondylolisthesis of the radiographic changes does not correlate with the clinical symptoms . shoulder oa and rotator cuff disease are also fairly common in the elderly and are associated with significant shoulder pain and disability related to decreased shoulder movement . direct signs of oa and indirect signs of large rotator cuff tears can be seen on plain films , including superior humeral head migration and remodeling of the acromion and the greater tuberosity . ultrasound is another valuable tool for easy assessment of the rotator cuff tendons due to its good sensitivity and specificity [ figure 8 ] . however , when surgical treatment is considered , arthro - ct , or mri may be preferred , in order to improve its planning . the lumbar and cervical spine is also frequently affected , with degenerative changes of the disk spaces , facet joints , and spinous processes , and sometimes with progressive scoliosis . although the intervertebral disks are not synovial joints , the pathological changes are similar to those seen in the articular cartilage . ct and mri are routinely used for the evaluation of narrowing of the central canal , lateral recesses , and neural foramina , when such an assessment is required ( percutaneous or surgical treatment ) . besides , mri can be useful in evaluating marrow changes in the vertebral plate , especially inflammatory changes ( type i modic ) , which present a positive correlation with low back pain . typical osteoarthritis of ( a ) the distal and proximal interphalangeal joints and ( b ) hip joint : joint space narrowing ( arrows ) , subchondral cysts ( arrowheads ) , osteophytes ( curved arrows ) , and subchondral sclerosis ( thick arrow ) . ultrasound images in ( a ) the long and ( b ) short axis of the supraspinatus tendon demonstrating a complete tear ( arrowheads ) . the frequency of microcrystal disorders also increases with age ; this is mainly true of gout and calcium pyrophosphate dihydrate deposition ( cpdd ) arthropathy . gout is the most common inflammatory arthritis in the elderly and is characterized by a disturbance of purine metabolism , with deposits in the joints , cartilage , and kidneys . the first metatarsophalangeal joint is the most common site of involvement in gouty arthritis [ figure 9 ] , but any joint can be involved . the typical radiographic features include eccentric nodular soft tissue masses ( particularly suggestive of tophus when they are dense ) , close erosions ( also suggestive when located at a distance from the joint and when associated with an elevated bony margin ) , preservation of the joint space , exuberant bony proliferation , and lack of periarticular osteoporosis . when the clinical , biological , and radiographic findings are uncertain , an ultrasound may be particularly helpful , as this technique may demonstrate more tophi [ figure 10 ] and erosions than plain films , more synovitis than a clinical examination , suggestive hyperechoic aggregates in the synovium or in the joint fluid , which may be aspirated , and the double contour sign ( an irregular hyperechoic band over the superficial margin of the articular cartilage , related to crystal deposition).[4244 ] calcium pyrophosphate dihydrate deposition ( cpdd ) disease is characterized by articular and periarticular tissue deposits ( hyaline cartilage , fibrocartilage , and other soft - tissue structures ) , even in the spine [ figure 11 ] . unlike gout , in which the increase in serum urates leads to supersaturation and deposits in the joints , in cpdd , the calcium deposits usually appear in the cartilage in the absence of any serum abnormality . even though such deposits may be seen in asymptomatic patients , they may be associated with the development of severe arthropathies or with acute pain related to the migration of the crystals in the synovial fluid , mimicking septic arthritis . gout of the first mtp joint in conventional radiography : excentric dense nodular soft tissue mass ( arrows ) , large erosions ( arrowhead ) , lack of periarticular osteoporosis , and exuberant bony proliferation ( curved arrow ) . gout involvement of the mtp ( a ) shows the tophus ( arrow ) with hyperechogenic microcrystal deposits ( arrowheads ) and a thickened synovium ( curved arrows ) . another patient with tophus deposition in the dorsal midfoot ( b ) demonstrates microcrystal deposits ( arrowhead ) with acoustic shadows ( thick arrows ) . calcium pyrophosphate dihydrate deposition disease demonstrated by deposits of microcrystals in the menisci ( arrow ) , articular cartilage ( arrowhead ) , and periarticular soft tissue ( curved arrow ) . elderly patients are more prone to infection because of an increased incidence of predisposing disorders ( diabetes mellitus , peripheral vascular disease , poor dentition , ) , immunosuppression and surgical procedures in this population ( dental extractions , open heart surgery , prosthetic joint replacement , ) . the infectious agents affecting elderly patients are similar to those found in the younger population ; although in the elderly , evidence shows an increased incidence of infection by nosocomial organisms ( due to institutionalization and hospitalization ) . these infections can have a relatively benign course , as in the case of cellulitis , or serious consequences , as with necrotizing fasciitis . in patients over 80 years of age , the knee , shoulder , and hip are the most frequently affected joints . another peculiarity of the elderly , uncommon in adults , is acute hematogenous osteomyelitis that mainly affects the spine . attention should , moreover , be paid to the diagnosis of tuberculosis , as early manifestations are subtle and advanced disease mimics other infections , granulomatous diseases , and malignancy . tuberculosis may affect the spine ( about 50% of cases ) , with potential neurological consequences , or any joint , causing deformity [ figure 12 ] . it should be noted that elderly men may present with bone lesions , as a manifestation of reactivation of the disease . as there are no pathognomonic imaging signs of musculoskeletal tuberculosis , the most conclusive means of reaching a diagnosis is by biopsy and culture , as chest radiographs and skin tests may not be positive in the elderly . as regards imaging techniques , magnetic resonance imaging is the modality of choice for musculoskeletal infection , as it best delineates the extension of soft tissue infections . computed tomography , ultrasound , radiography , and nuclear medicine studies are considered to be ancillary . in some circumstances ultrasound can be very useful , as it allows the aspiration of fluid collected in the joints or soft tissue , the evaluation of structures adjacent to orthopedic hardware , or the assessment of small peripheral joints . besides , in the case of debilitated elderly patients , a mobile ultrasound device can be easily moved close to their beds . ( a ) on conventional radiography there is mild osteoporosis , extensive soft tissue swelling , and large marginal erosion ( arrowhead ) , ( b ) on doppler ultrasound the synovitis ( arrows ) and bone erosion ( arrowhead ) are well - depicted . paget 's disease is a bone disorder characterized by an increase in osteoclast - mediated bone resorption accompanied by osteoblast - mediated formation of new bone of inferior quality . the frequency of this condition is decreasing , but it still exists in 1 5% of persons over 50 years of age , varying according to geographic areas.[5356 ] the radiographic signs include an advancing wedge of bone resorption , an accentuation and coarsening of the bone trabeculae along lines of stress , cortical thickening , and enlargement of the bone [ figure 13 ] . as a result , secondary osteoarthritis , insufficiency fractures , bowing of the bones , and even spinal cord or nerve root compression can be observed . in a minority of cases , sarcomatous degeneration may develop , mainly in the humerus , pelvis , and proximal femur , with a frequency believed to range from 0.9 to 2% . paget 's disease affecting the iliac bone demonstrated with coarsening of the bone trabeculae and cortical thickening ( arrows ) . tumors : tumors can be revealed by bone pain , pathological fractures , or disability , or be discovered as an incidental finding . primary bone tumors in elderly patients are not common , but , due to the risk of metastases , any newly discovered bone lesion or one developing within a known pre - existing lesion must be assumed to be malignant until proven otherwise . metastases : bone metastases are a common complication of malignant disease , especially of the breast , prostate , and lung . although they are often multiple , a solitary metastasis is still more common than a primary neoplasm . plain films are relatively insensitive for the detection of bone metastases , especially subtle lesions . mr imaging is superior to ct in the detection of malignant marrow infiltration , and it has a better contrast resolution for visualizing soft tissue and spinal cord lesions . even with the advances in mri , especially for bone marrow screening , bone scintigraphy continues to be used as an effective method for initial evaluation of the whole body for bone metastases . sarcomas : in the elderly population , many sarcomas are secondary to a pre - existing disorder of the bone , such as paget 's disease , or lesions treated by radiation . chondrosarcoma must also be kept in mind , especially when isolated osteolytic lesions of the pelvis are seen , as this is the most common primary sarcoma of bone in adults . the cartilaginous nature of the lesion may be suggested by the presence of calcifications or by a lobulated hyperintense lesion in an mri [ figure 14 ] . chondrosarcoma of the pelvis in ( a ) conventional radiography appears as an isolated osteolytic lesion ( arrows ) , ( b ) coronal t2-weighted image demonstrates a lobulated hyperintense lesion ( arrowheads ) . myeloma : myeloma is a characteristic disease in the elderly population , with a peak incidence in the eighth decade that results from an unregulated , progressive proliferation of neoplastic monoclonal plasma cells that accumulate in the bone marrow , leading to anemia and marrow failure . osteolytic lesions may be present on plain films , but differentiating osteoporotic vertebral compression fractures from those associated with myeloma is a common diagnostic dilemma , as bone loss may be the first sign revealing the latter disease . indeed , widespread osteoporosis , due to cytokine - mediated osteoclast activation , is common in patients with myeloma . this possible association must be kept in mind , particularly if other features are present , including unexplained back or bone pain . we used the following terms and some of its combinations : elderly , aged , epidemiology , fracture , osteoporosis , vertebral , hip , pelvis , arthritis , neoplasm , malignancy , myeloma , paget , gout , infection , microcrystal , and radiology . articles without english abstracts were excluded . besides , certain relevant rheumatological , orthopedic , and radiological books were also used . fractures are frequent in the elderly and result mainly from the effects of falls and osteoporosis . low - impact falls , even from standing height , are the most common cause of injury in geriatric patients . falling is a multi - factorial problem due to both extrinsic ( e.g. , environmental and housing conditions ) and intrinsic risk factors ( e.g. , impaired mobility , loss of muscular strength , poor visual acuity , and medication , for instance corticoids).[46 ] osteoporosis , which is characterized by qualitatively normal , but quantitatively deficient bone , leads to bone fragility and increased risk of fractures . the general prevalence of osteoporosis in women is approximately 50% at the age of 85 years , while in men the prevalence is about 20% at that age . ethnic differences also exist : mineral bone density is higher in black women and lower in asian women , while in white women evidence shows an intermediate value . due to its precision , the most widely used quantitative technique is dual energy x - ray absorptiometry , which makes it possible to diagnose osteoporosis early , predict the risk of fracture , determine therapeutic intervention , and monitor response to treatment . this method grades bone loss according to standard deviations ( sd ) from the mean young adult value . a value less than 2.5 times the sd below the young adult mean , is considered to indicate osteoporosis . the main radiographic features of osteoporosis are increased radiolucency and cortical thinning , mainly in the spine , giving rise to a well - demarcated outline of the vertebral body , which has been described as , picture framing. besides , in the spine , an increased biconcavity of the vertebral end plates , with a protrusion of the intervertebral disk into the vertebral body can be observed . however , early radiographic changes are subtle , as a bone loss of approximately 30% must occur before it can be detected . vertebral compression fractures are the most common osteoporotic fractures , with a greater incidence in women over 60 years of age . they can be observed in up to 25% of women over 70 years of age and 40% of those over 80 years . after one episode , the risk of subsequent vertebral fracture increases at least four - fold , with substantially increased rates during the first year . these fractures are associated with higher mortality and significant morbidity , even though they are afforded little clinical attention . they often appear as multiple fractures with anterior wedging , and may be associated with significant deformity , leading to kyphosis . they are usually well demonstrated on radiographs , but can be better assessed with computed tomography ( ct ) or magnetic resonance ( mr ) imaging , when necessary . the latter imaging modality may be indicated in case of associated neurological symptoms a rare event or when a vertebroplasty is discussed , in order to better localize the recently fractured vertebra . vertebral compression fractures in the elderly may involve several problems : first is differentiating between osteoporotic and malignant vertebral collapse ( vc ) , particularly metastatic vc , as the latter is by far the most common malignant tumor affecting the skeleton in the elderly . in most patients , multiple , relatively symmetrical thoracolumbar vcs , associated with diffuse increased radiolucency are suggestive of osteoporosis [ figure 1a ] . the whole vertebral endplate is impacted , sometimes in association with increased or decreased radiolucency of the subjacent trabecular bone ( band - like distribution of the abnormalities under the fractured vertebral end plate ) . gas can also be seen under the fracture and is nearly pathognomonic of the benign nature of the collapse . there is no cortical osteolysis , although fractures can be identified with retropulsion of a bone fragment . conversely , the following are a cause for concern : a single vc , involvement of the cervical spine , heterogeneous increased or decreased radiolucency that can not be explained by an underlying end plate fracture , a focal collapse ( on the ap or lateral view ) , or a bulging of the vertebral body or cortical osteolysis [ figure 1b ] . if in doubt or for a better assessment of the spinal canal and soft tissue , ct , or better yet , an mri can be performed , in order to differentiate between these two disorders . in benign fractures , an mri may show a band distribution of abnormal signal intensities on t1 and t2 weighted images , spared normal bone marrow signal intensity , retropulsion of a posterior bone fragment , and multiple compression fractures ; on the contrary , signs such as a convex posterior border of the vertebral body , abnormal signal intensity of the posterior elements , an epidural mass or a focal paraspinal mass are suggestive of spinal metastasis.[1921 ] second , vertebral fractures can be missed following moderate trauma , because the fracture may be difficult to identify in an osteoporotic radiolucent bone and / or because the pain is attributed to another painful condition , such as degenerative changes . this is observed in particular in the cervical spine , particularly at c2 , and may lead to secondary neurological complications [ figure 2 ] . ankylosis may be related to ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis , which especially affect males and they have an estimated frequency of 5 to 15% in the elderly.[2225 ] these ankylosed spines become increasingly susceptible to injury , even in the event of low - energy impacts , and are prone to unstable vertebral fractures with an increased frequency of neurological complications , either primary or secondary , after unprotected transfers and manipulation . these lesions may be missed on plain films when non - displaced or mildly displaced , especially in hyperextension , whereas , they are well demonstrated on ct or mr imaging . however , the mri features can sometimes be misleading and mimic infectious spondylitis when signal abnormalities of the disco - vertebral junction are present . caution is required when an elderly patient presents with an ankylosed spine , minor trauma or acute pain , especially if signal abnormalities of the anterior and posterior spine are observed [ figure 3 ] . usual benign vertebral compression ( a ) multiple levels , diffuse radiolucency , and whole vertebral endplate involvement ( arrows ) ; usual malignant vertebral compression characteristics ( b ) single vertebral involvement , focal and bulging of the posterior wall ( arrowheads ) . missed fracture of the odontoid process and vertebral body of c2 ( arrows ) in a patient with a history of fall one month before . transverse fracture of thoracic vertebral body ( arrow ) and posterior arch ( arrowhead ) with posterior epidural hematoma ( curved arrow ) in an ankylosed spine depicted on ( a ) t1- and ( b ) t2-weighted sequences . other important sites of fractures are the hip and the pelvis , which are associated with increased mortality and specific diagnostic problems in the elderly population . they are common and frequently result from lateral falls directly on the greater trochanter , in the case of hip fractures , and forward or backward falls in the case of pelvic fractures . most fractures are easily diagnosed by conventional radiography , particularly fractures of the proximal femur . however , non - displaced fractures may be negative or equivocal , particularly if the bone is osteoporotic . if a clinical suspicion of a fracture persists , ct or better still mr imaging is advocated , as this modality can show the fracture line surrounded by bone marrow edema . they may occur everywhere , but are particularly common in the hip and pelvis , the majority being located in the sacrum and pubic ramus [ figure 4 ] . radiographic findings are often subtle or unremarkable , and in suspected cases mr imaging is indicated due to its superior accuracy as compared to other radiological methods . all the images should be carefully analyzed , as multiple insufficiency fractures are frequent [ figure 5 ] . subchondral fractures are particularly difficult to identify on radiographs , but the thin hypointense line that represents the fracture , surrounded by a variable degree of edema , is well demonstrated on an mri [ figure 6 ] . this kind of fracture can be complicated by a necrosis of the subchondral bone , which is hypointense on t2 weighted images , and is not enhanced following gadolinium administration . insufficiency fractures ( arrows ) located in the sacrum with adjacent edema ( arrowheads ) , better depicted on ( a ) t1- and ( b ) t2- weighted sequences , respectively . multiple fractures ( white arrows ) in ( a ) the proximal femur and ( b ) pubic ramus on coronal t1-weighted images subchondral fracture of the femoral head : ( a ) conventional radiography and ( b ) fat saturated sagittal t2-weighted image ( white arrow ) . osteoarthritis ( oa ) can be defined as a group of distinct , but overlapping diseases , which may have different etiologies , but similar biological , morphological , and clinical outcomes affecting the articular cartilage , subchondral bone , ligaments , joint capsule , synovial membrane , and periarticular muscles . pathological changes can include fibrillation of cartilage , disruption of collagen fibers , changes in proteoglycan staining , chondrocyte proliferation , necrosis , and neovascularization . oa is the most common joint disease in persons 65 years of age and above , with a radiographic prevalence as high as approximately 90% in women and 80% in men.[3335 ] its etiology is not fully understood , although there are several related factors , such as female gender , genetics , metabolism , and excessive mechanical stress . although the joints of the hand are the most commonly affected , they are less likely to be symptomatic than the knee or hip [ figure 7 ] . the diagnosis of oa is primarily based on clinical history and physical examination . plain radiographs can help confirm both the diagnosis and grade the severity of the condition . the cardinal radiographic features of oa are focal / non - uniform narrowing of the joint space in the areas subjected to the most pressure , subchondral cysts , subchondral sclerosis , and osteophytes . however , the severity of the spondylolisthesis of the radiographic changes does not correlate with the clinical symptoms . shoulder oa and rotator cuff disease are also fairly common in the elderly and are associated with significant shoulder pain and disability related to decreased shoulder movement . direct signs of oa and indirect signs of large rotator cuff tears can be seen on plain films , including superior humeral head migration and remodeling of the acromion and the greater tuberosity . ultrasound is another valuable tool for easy assessment of the rotator cuff tendons due to its good sensitivity and specificity [ figure 8 ] . however , when surgical treatment is considered , arthro - ct , or mri may be preferred , in order to improve its planning . the lumbar and cervical spine is also frequently affected , with degenerative changes of the disk spaces , facet joints , and spinous processes , and sometimes with progressive scoliosis . although the intervertebral disks are not synovial joints , the pathological changes are similar to those seen in the articular cartilage . ct and mri are routinely used for the evaluation of narrowing of the central canal , lateral recesses , and neural foramina , when such an assessment is required ( percutaneous or surgical treatment ) . besides , mri can be useful in evaluating marrow changes in the vertebral plate , especially inflammatory changes ( type i modic ) , which present a positive correlation with low back pain . typical osteoarthritis of ( a ) the distal and proximal interphalangeal joints and ( b ) hip joint : joint space narrowing ( arrows ) , subchondral cysts ( arrowheads ) , osteophytes ( curved arrows ) , and subchondral sclerosis ( thick arrow ) . ultrasound images in ( a ) the long and ( b ) short axis of the supraspinatus tendon demonstrating a complete tear ( arrowheads ) . the frequency of microcrystal disorders also increases with age ; this is mainly true of gout and calcium pyrophosphate dihydrate deposition ( cpdd ) arthropathy . gout is the most common inflammatory arthritis in the elderly and is characterized by a disturbance of purine metabolism , with deposits in the joints , cartilage , and kidneys . the first metatarsophalangeal joint is the most common site of involvement in gouty arthritis [ figure 9 ] , but any joint can be involved . the typical radiographic features include eccentric nodular soft tissue masses ( particularly suggestive of tophus when they are dense ) , close erosions ( also suggestive when located at a distance from the joint and when associated with an elevated bony margin ) , preservation of the joint space , exuberant bony proliferation , and lack of periarticular osteoporosis . when the clinical , biological , and radiographic findings are uncertain , an ultrasound may be particularly helpful , as this technique may demonstrate more tophi [ figure 10 ] and erosions than plain films , more synovitis than a clinical examination , suggestive hyperechoic aggregates in the synovium or in the joint fluid , which may be aspirated , and the double contour sign ( an irregular hyperechoic band over the superficial margin of the articular cartilage , related to crystal deposition).[4244 ] calcium pyrophosphate dihydrate deposition ( cpdd ) disease is characterized by articular and periarticular tissue deposits ( hyaline cartilage , fibrocartilage , and other soft - tissue structures ) , even in the spine [ figure 11 ] . unlike gout , in which the increase in serum urates leads to supersaturation and deposits in the joints , in cpdd , the calcium deposits usually appear in the cartilage in the absence of any serum abnormality . even though such deposits may be seen in asymptomatic patients , they may be associated with the development of severe arthropathies or with acute pain related to the migration of the crystals in the synovial fluid , mimicking septic arthritis . gout of the first mtp joint in conventional radiography : excentric dense nodular soft tissue mass ( arrows ) , large erosions ( arrowhead ) , lack of periarticular osteoporosis , and exuberant bony proliferation ( curved arrow ) . gout involvement of the mtp ( a ) shows the tophus ( arrow ) with hyperechogenic microcrystal deposits ( arrowheads ) and a thickened synovium ( curved arrows ) . another patient with tophus deposition in the dorsal midfoot ( b ) demonstrates microcrystal deposits ( arrowhead ) with acoustic shadows ( thick arrows ) . calcium pyrophosphate dihydrate deposition disease demonstrated by deposits of microcrystals in the menisci ( arrow ) , articular cartilage ( arrowhead ) , and periarticular soft tissue ( curved arrow ) . elderly patients are more prone to infection because of an increased incidence of predisposing disorders ( diabetes mellitus , peripheral vascular disease , poor dentition , ) , immunosuppression and surgical procedures in this population ( dental extractions , open heart surgery , prosthetic joint replacement , ) . the infectious agents affecting elderly patients are similar to those found in the younger population ; although in the elderly , evidence shows an increased incidence of infection by nosocomial organisms ( due to institutionalization and hospitalization ) . these infections can have a relatively benign course , as in the case of cellulitis , or serious consequences , as with necrotizing fasciitis . in patients over 80 years of age , another peculiarity of the elderly , uncommon in adults , is acute hematogenous osteomyelitis that mainly affects the spine . attention should , moreover , be paid to the diagnosis of tuberculosis , as early manifestations are subtle and advanced disease mimics other infections , granulomatous diseases , and malignancy . tuberculosis may affect the spine ( about 50% of cases ) , with potential neurological consequences , or any joint , causing deformity [ figure 12 ] . it should be noted that elderly men may present with bone lesions , as a manifestation of reactivation of the disease . as there are no pathognomonic imaging signs of musculoskeletal tuberculosis , the most conclusive means of reaching a diagnosis is by biopsy and culture , as chest radiographs and skin tests may not be positive in the elderly . as regards imaging techniques , magnetic resonance imaging is the modality of choice for musculoskeletal infection , as it best delineates the extension of soft tissue infections . computed tomography , ultrasound , radiography , and nuclear medicine studies are considered to be ancillary . in some circumstances ultrasound can be very useful , as it allows the aspiration of fluid collected in the joints or soft tissue , the evaluation of structures adjacent to orthopedic hardware , or the assessment of small peripheral joints . besides , in the case of debilitated elderly patients , a mobile ultrasound device can be easily moved close to their beds . ( a ) on conventional radiography there is mild osteoporosis , extensive soft tissue swelling , and large marginal erosion ( arrowhead ) , ( b ) on doppler ultrasound the synovitis ( arrows ) and bone erosion ( arrowhead ) are well - depicted . paget 's disease is a bone disorder characterized by an increase in osteoclast - mediated bone resorption accompanied by osteoblast - mediated formation of new bone of inferior quality . the frequency of this condition is decreasing , but it still exists in 1 5% of persons over 50 years of age , varying according to geographic areas.[5356 ] the radiographic signs include an advancing wedge of bone resorption , an accentuation and coarsening of the bone trabeculae along lines of stress , cortical thickening , and enlargement of the bone [ figure 13 ] . as a result , secondary osteoarthritis , insufficiency fractures , bowing of the bones , and even spinal cord or nerve root compression can be observed . in a minority of cases , sarcomatous degeneration may develop , mainly in the humerus , pelvis , and proximal femur , with a frequency believed to range from 0.9 to 2% . paget 's disease affecting the iliac bone demonstrated with coarsening of the bone trabeculae and cortical thickening ( arrows ) . tumors : tumors can be revealed by bone pain , pathological fractures , or disability , or be discovered as an incidental finding . primary bone tumors in elderly patients are not common , but , due to the risk of metastases , any newly discovered bone lesion or one developing within a known pre - existing lesion must be assumed to be malignant until proven otherwise . metastases : bone metastases are a common complication of malignant disease , especially of the breast , prostate , and lung . although they are often multiple , a solitary metastasis is still more common than a primary neoplasm . plain films are relatively insensitive for the detection of bone metastases , especially subtle lesions . mr imaging is superior to ct in the detection of malignant marrow infiltration , and it has a better contrast resolution for visualizing soft tissue and spinal cord lesions . even with the advances in mri , especially for bone marrow screening , bone scintigraphy continues to be used as an effective method for initial evaluation of the whole body for bone metastases . sarcomas : in the elderly population , many sarcomas are secondary to a pre - existing disorder of the bone , such as paget 's disease , or lesions treated by radiation . chondrosarcoma must also be kept in mind , especially when isolated osteolytic lesions of the pelvis are seen , as this is the most common primary sarcoma of bone in adults . the cartilaginous nature of the lesion may be suggested by the presence of calcifications or by a lobulated hyperintense lesion in an mri [ figure 14 ] . chondrosarcoma of the pelvis in ( a ) conventional radiography appears as an isolated osteolytic lesion ( arrows ) , ( b ) coronal t2-weighted image demonstrates a lobulated hyperintense lesion ( arrowheads ) . myeloma : myeloma is a characteristic disease in the elderly population , with a peak incidence in the eighth decade that results from an unregulated , progressive proliferation of neoplastic monoclonal plasma cells that accumulate in the bone marrow , leading to anemia and marrow failure . osteolytic lesions may be present on plain films , but differentiating osteoporotic vertebral compression fractures from those associated with myeloma is a common diagnostic dilemma , as bone loss may be the first sign revealing the latter disease . indeed , widespread osteoporosis , due to cytokine - mediated osteoclast activation , is common in patients with myeloma . this possible association must be kept in mind , particularly if other features are present , including unexplained back or bone pain . special attention is required in this population , as an early diagnosis can avoid delay in treatment , which is associated with increased morbidity and mortality . besides , a better understanding of musculoskeletal diseases can lead to the implementation of effective preventive measures , thus reducing public health expenditure , and improving the quality of life in the elderly .

musculoskeletal disorders are among the most common problems affecting the elderly . the resulting loss of mobility and physical independence can be particularly devastating in this population . the aim of this article is to present some of the most frequent musculoskeletal disorders of the elderly , such as fractures , osteoporosis , osteoarthritis , microcrystal disorders , infections , and tumors .

INTRODUCTION Search criteria Fractures Osteoarthritis and associated conditions Microcrystal disorders Infection Paget's disease CONCLUSION

migraine is the leading neurological cause for seeking medical care , and is associated with significant disability in the sufferer . the greatest impact is on migraineurs with headaches on more days than not , a condition defined as chronic migraine ( cm ) . cm is defined as at least 15 days of headache per month in which at least eight of the days fulfill migraine criteria and/or are treated with specific migraine medications , in the absence of a diagnosis of medication overuse headache . patients with cm often had a history of episodic migraine that began in adolescence or early adulthood , reporting a process of transformation marked by headaches that become more frequent over years . among migraineurs , defining risk factors for cm , or for the progression of episodic migraine to cm , identified risk factors include medication overuse , obesity , sleep problems , and psychiatric comorbidity [ 712 ] . studies in both community and tertiary settings have demonstrated an association between migraine and several psychiatric conditions [ 8 , 9 ] . however , the frequency of psychiatric disorders in both setting has not been compared before in a single study . furthermore , differences in methods of studies based in community or tertiary centers prevent appropriate comparison . population studies fail to conduct face - to - face assessments , while clinic - based studies carry the potential for selection bias . studies focusing on best methods to address this gap are of interest , and one strategy is to compare data obtained from the community with those from specialty care , where methods of collection have been virtually identical , and that was the scope of this study . we compared demographic data and psychiatric comorbidity in a sample of individuals with cm from the community with another from a tertiary care clinic . in light of the fact that patients suffering from migraine and comorbid psychiatric disorders are greater health - care service users , we hypothesized that the frequency of psychiatric disorders , notably depression , is higher in patients followed in tertiary care . community data were gathered in capela nova , a city from the state of minas gerais , brazil . according to the 2000 brazilian census , its population was 2,066 inhabitants ( 1,631 over the age of 10 years ) . the present study is part of an observational , cross - sectional and population - based study conducted in two phases [ 14 , 15 ] . initially , community health workers from the family health program directly interviewed all inhabitants aged 10 years or older for headache symptoms in the previous year . the family health program works through family health - care teams which are composed of one physician , one nurse , one auxiliary nurse , and four to six community health workers , and are assigned to specific geographical areas with defined populations of 6001,000 families . activities provided by family health - care teams take place at primary care facilities , in patients homes , and in the community . in the first phase of our study ( screening phase ) , trained community health workers screened for the occurrence of headaches using the following question : have you had any headache episode over the last 12 months ? those who screened positive were asked about headache frequency in the past month , and those reporting 15 days or more of headache were offered in - person assessment by neurologists with expertise in headache medicine . three neurologists independently examined participants , and headaches were diagnosed according to the second edition of the international classification of headache disorders . subsequently , all individuals aged 18 years or older with cm were assessed for psychiatric comorbidities . psychiatric assessment was performed by an experienced psychiatrist using the mini international neuropsychiatric interview ( mini ) . the same investigators involved in the community - based assessment used the very same procedures to diagnose consecutive patients attended at a university - based headache center in the first half of 2006 . this center is located in belo horizonte and is the only headache clinic in the state of minas gerais , brazil . the study followed the guidance of the regulatory norms of the brazilian national health council ( resolution 196/1996 ) which is in accordance with the helsinki declaration . the protocol and all forms were reviewed and approved by the local ethics research committee . data were transferred to epi - info 2000 by a study coordinator and analyzed using spss 12.0 . the relative frequencies of psychiatric comorbidities were stratified by headache type , and confidence intervals were calculated . discrete data were compared between groups using the chi - square test or the fisher test ( when anticipated values were small ) . for continuous non - parametric variables , the mann community data were gathered in capela nova , a city from the state of minas gerais , brazil . according to the 2000 brazilian census , its population was 2,066 inhabitants ( 1,631 over the age of 10 years ) . the present study is part of an observational , cross - sectional and population - based study conducted in two phases [ 14 , 15 ] . initially , community health workers from the family health program directly interviewed all inhabitants aged 10 years or older for headache symptoms in the previous year . the family health program works through family health - care teams which are composed of one physician , one nurse , one auxiliary nurse , and four to six community health workers , and are assigned to specific geographical areas with defined populations of 6001,000 families . activities provided by family health - care teams take place at primary care facilities , in patients homes , and in the community . in the first phase of our study ( screening phase ) , trained community health workers screened for the occurrence of headaches using the following question : have you had any headache episode over the last 12 months ? those who screened positive were asked about headache frequency in the past month , and those reporting 15 days or more of headache were offered in - person assessment by neurologists with expertise in headache medicine . three neurologists independently examined participants , and headaches were diagnosed according to the second edition of the international classification of headache disorders . subsequently , all individuals aged 18 years or older with cm were assessed for psychiatric comorbidities . psychiatric assessment was performed by an experienced psychiatrist using the mini international neuropsychiatric interview ( mini ) . the same investigators involved in the community - based assessment used the very same procedures to diagnose consecutive patients attended at a university - based headache center in the first half of 2006 . this center is located in belo horizonte and is the only headache clinic in the state of minas gerais , brazil . the study followed the guidance of the regulatory norms of the brazilian national health council ( resolution 196/1996 ) which is in accordance with the helsinki declaration . the protocol and all forms were reviewed and approved by the local ethics research committee . data were transferred to epi - info 2000 by a study coordinator and analyzed using spss 12.0 . the relative frequencies of psychiatric comorbidities were stratified by headache type , and confidence intervals were calculated . discrete data were compared between groups using the chi - square test or the fisher test ( when anticipated values were small ) . for continuous non - parametric variables , the mann in the community of 1,605 interviewed inhabitants , 57 ( 3.6 % ) had headaches on at least 15 days for at least three consecutive months . 43 ( 75.4 % ) had cm and 41 consented to being assessed by the psychiatrist ( 95.3 % participation rate ) . in the headache center , 43 patients had cm ( n = 453 , 9.5 % ) and all consented in participating in the psychiatric assessment . sociodemographic profiles were similar between groups with the exception of the mean number of years of formal education , lower in the community relative to the headache center ( table 1).table 1demographic characteristics in individuals with chronic migraine from the community and a clinic - based samplecommunity ( n = 41)headache center ( n = 43)p valuegender men7 ( 17.1 % ) 2 ( 4.7 % ) 0.09 * * women34 ( 82.9 % ) 41 ( 95.3 % ) education ( years of study ) 826 ( 78.8 % ) 5 ( 11.6 % ) < 0.001 * 9115 ( 15.2 % ) 15 ( 34.9 % ) 122 ( 6.1 % ) 23 ( 53.5 % ) age mean ( sd)41.2 ( 17.2)35.7 ( 12.6)0.19 * * * range13731863 * chi - square , * * fisher , * * * mann whitney demographic characteristics in individuals with chronic migraine from the community and a clinic - based sample * chi - square , * * fisher , * * * mann whitney among individuals from the community , 65.9 % of cases were diagnosed with any current psychiatric disorder , relative to 83.7 % in those from the headache center ( p = 0.06 ) . the relative frequencies of some specific diagnoses were remarkably high in both groups , despite not being statistically different . in the headache center , the most prevalent disorders were simple phobia ( 41.9 % ) , generalized anxiety disorder ( 34.9 % ) and major depression ( 32.6 % ) . in the community , the same disorders were also the most common ones : generalized anxiety disorder ( 39.0 % ) , phobias ( 29.3 % ) , and major depression ( 29.3 % ) . bipolar disorder was not seen in the community and was diagnosed in two cases from the headache center . the frequency of antidepressants use was similar in the headache center ( 51.2 % ) and in the community ( 44.4 % ) ( p = 0.51 ) . table 2 summarizes these data.table 2current psychiatric comorbidities in individuals with chronic migraine from the community and a clinic - based sampleheadache center ( n = 43)community ( n = 41)p valueany diagnosis36 ( 83.7 % ) 27 ( 65.9 % ) 0.06*one or two psychiatric diagnoses21 ( 48.8 % ) 16 ( 39.0 % ) 0.36three or more psychiatric diagnoses15 ( 34.9 % ) 11 ( 26.9 % ) 0.42major depression14 ( 32.6 % ) 12 ( 29.3 % ) 0.74*dysthymia9 ( 20.9 % ) 9 ( 22.0 % ) 0.99**bipolar disorder2 ( 4.7 % ) 0 ( 0 % ) 0.23**generalized anxiety disorder15 ( 34.9 % ) 16 ( 39.0 % ) 0.69*specific phobia18 ( 41.9 % ) 12 ( 29.3 % ) 0.23*obsessive compulsive disorder9 ( 20.9 % ) 10 ( 24.4 % ) 0.70*somatization7 ( 16.3 % ) 3 ( 7.3 % ) 0.31**eating disorders2 ( 4.7 % ) 1 ( 2.5 % ) 0.99*alcohol abuse0 ( 0.0 % ) 2 ( 4.9 % ) 0.23 * * * chi - square , * * fisher current psychiatric comorbidities in individuals with chronic migraine from the community and a clinic - based sample * chi - square , * * fisher to the best of our knowledge this is the first study to compare the frequency of psychiatric comorbidity of cm in community and tertiary care clinic samples . the frequency of psychiatric disorders in cm was elevated in both settings , tending to be higher in the tertiary care sample . while psychiatric comorbidity in episodic migraine has been well established in the literature , psychiatric disorders have been less studied in cm . only a few studies have addressed psychiatric comorbidities of cm in population - based samples , finding increased levels of depression and anxiety disorders even in comparison with episodic migraine patients [ 11 , 20 ] . one limitation of these studies was the use of self - report questionnaires rather than clinical interview in ascertaining psychiatric diagnosis . in the present study we found that up to a third of our patients in each setting had depression . a similar rate was described in the american migraine prevalence and prevention ( ampp ) study , a population - based survey based on mailed questionnaires . in that study , depression was assessed by self - report of a physician diagnosis and by the patient health questionnaire ( phq-9)depression module . one interesting result from the ampp study was that cm patients were twice as likely to have depression as assessed by phq-9 in comparison with episodic migraine patients [ 30.2 and 17.2 % respectively ; or ( 95 % ci ) = 2.0 ( 1.67 to 2.40 ) , p < 0.001 ] . cm patients were also approximately twice as likely to report anxiety [ 30.2 vs. 18.8 % respectively ; or ( 95 % ci ) = 1.8 ( 1.51 to 2.15 ) , p < 0.001 ] . the international burden of migraine study ( ibms ) also found higher levels of anxiety and depression in cm when compared with episodic migraine . regarding anxiety syndromes , generalized anxiety disorder and phobias seem to be comorbid with the migraine spectrum [ 2024 ] . interestingly the frequency of obsessive compulsive disorder was significantly high ( between 20 and 25 % ) in cm patients in comparison with its prevalence in the general population ( 2 % ) . only few previous studies have pointed out this association between obsessive compulsive disorder and migraine that may be associated with underlying serotonin system dysfunction . bipolar disorder is also comorbid with migraine ; migraineurs without aura are 2.4 times more likely to have bipolar disorder type 1 , and the ratio increases to 7.3 when the diagnosis is migraine with aura [ 13 , 22 ] . for bipolar disorder type 2 , we failed to detect this association , likely because of small size of the samples . drug abuse has not been traditionally associated with migraine [ 22 , 24 ] , but a recent study reported that illicit drug abuse may be more frequent in migraine patients with depression or post - traumatic stress disorder . demographic profiles were similar in both groups , and the vast majority of individuals with cm were women . it must be highlighted that the assessed community was from a small city , while the patients at the headache center mainly came from a large urban center . nonetheless , studies of migraine that have enrolled subjects with different demographic features also found striking similarities regarding the risk of psychiatric comorbidities , once more pointing to shared biological factors as a plausible mechanism for the comorbidity [ 22 , 24 ] . specific genotypes coding d2 dopaminergic receptors , dysfunction in tyramine conjugation , changes in the metabolism of serotonin and catecholamines and in estrogen levels have been considered to explain the comorbidities [ 2730 ] . it is worth mentioning , however , that we have comprehensively and systematically assessed almost all patients with cm from an entire population of a small city . we did not assess the differential disability associated with headache and psychiatric disorders in the individuals . finally , our findings were not adjusted for other confounding factors , such as parameters of headache severity ( intensity , duration associated symptoms ) , obesity , sleep disorders , use of psychotropic medication and household income . we partly justify these latter limitations by arguing that the demands on patients and resources in conducting these missing assessments could jeopardize the community assessment , since most interviews were conducted in participant households . in conclusion , the present study suggests that psychiatric comorbidity in cm is elevated in the community and clinical settings , tending to be more common in cm patients from a headache center .

although the association between episodic migraine and psychiatric comorbidities is well documented , few studies have focused on the comorbidity with chronic migraine ( cm ) and discrepancies exist between population - based and clinic - based data . the objective of this study is to compare demographic and psychiatric comorbidity correlates between cm samples drawn from the community and tertiary care . all inhabitants from a city borough were interviewed for the presence of headaches occurring 15 or more days per month . cm was diagnosed after subjects had been interviewed and examined by a headache doctor . participants were also assessed with a structured interview by a psychiatrist , who assigned diagnoses based on the dsm - iv . the same investigators assessed all patients consecutively seen in a university - based outpatient headache center over a 4-month period . the samples consist of 41 individuals from the community and 43 from the headache center . sociodemographic profiles were similar between groups with the exception of the mean number of years of formal education . among individuals from the community , psychiatric diagnoses were present in 65.9 % of cases , relative to 83.7 % in those from the headache center ( p = 0.06 ) . phobias ( 41.9 vs. 29.3 % ) and depression ( 32.6 vs. 29.3 % ) were more frequent in patients from the headache center , but this difference did not reach statistical significance . thus the frequency of psychiatric disorders in patients with cm was elevated in both settings , being higher in the specialty care clinic .

Introduction Methods Population study Clinic study Statistical analysis Results Discussion

m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one ) is one of many endogenous exocyclic lesions derived from the reaction of dna with bifunctional electrophiles generated by lipid , protein , or dna peroxidation . being an endogenous constituent of human and rodent genomes , m1dg is mutagenic in bacterial and mammalian cells and leads to mispairing when replicated in vitro by multiple different dna polymerases . we have previously developed a monoclonal antibody against m1dg and incorporated the antibody into analytical schemes using immunoaffinity purification of m1dg followed by mass spectrometric detection . these studies indicated that adult humans excrete m1dg at a rate of 12 fmol / kgd in urine . subsequent investigations into the metabolism and elimination of m1dg revealed that administration of m1dg to rodents at doses from 8 mg / kg to 6 pg / kg leads to its rapid disappearance from plasma due to oxidation and formation of the single , stable metabolite , 6-oxo - m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione ) . in both humans and rats , xanthine oxidase appears to be responsible for this oxidation , while aldehyde oxidase also plays a role in m1dg metabolism in humans . because 6-oxo - m1dg is the sole metabolite of m1dg in rats , it appears to be an ideal surrogate for m1dg as an in vivo biomarker for oxidative stress . all studies of m1dg metabolism in vivo have been conducted with exogenously administered deoxynucleoside . thus , it is not known if 6-oxo - m1dg is actually present in unadulterated intact animals . in order to address this question , we developed a highly specific monoclonal antibody ( mab ) against 6-oxo - m1dg and covalently linked the antibody to sepharose beads . the resultant antibody - sepharose matrix ( the gel ) was used for the immunoaffinity purification of 6-oxo - m1dg from urine and feces followed by lc - ms / ms quantification against the stable isotope - labeled internal standard , [ n5]-6-oxo - m1dg . utilizing this method , we report for the first time that 6-oxo - m1dg is endogenously produced in rodents and is excreted in urine and feces . na2co3 , nahco3 , nacl , water , acetonitrile , mariculture keyhole limpet hemocyanin ( mcklh ) , hcl , acetic acid , and methanol were obtained from thermo fisher scientific ( waltham , ma , usa ) . abts ( diammonium salt ) , thimerosal , kh2po4 , na2hpo4 , kcl , 30% h2o2 , tween 20 , bovine serum albumin ( bsa ) , dimethylformamide ( dmf ) , dimethylsulfoxide ( dmso ) , k2co3 , sodium periodate , sodium methoxide , nabh4 , hypoxanthine , xanthosine , xanthine , ethyl cis-3-iodoacrylate , ethyl cis-3-bromoacrylate aminopterin , ethanolamine , cyanogen bromide - activated sepharose beads , diethylene glycol , 2-deoxyguanosine , sodium hydroxide , formic acid , sodium azide , glycine , and citric acid were obtained from sigma - aldrich ( st . louis , mo , usa ) . horseradish peroxidase - conjugated goat antimouse igg ( h + l ) [ 0.8 mg / ml ] and igg ( fc ) were purchased from jackson immunoresearch ( west grove , pa , usa ) . phosphate buffered saline ( pbs ) was purchased from invitrogen ( carlsbad , ca , usa ) . the synthesis of 6-oxo - m1guo was based on a previously described synthetic scheme with some modifications . guanosine was dissolved in a minimum amount of a dmf / dmso mixture ( 1:1 , v / v ) . k2co3 ( 1.5 equiv ) was added to the solution and the reaction mixture was held at 6065 c for 18 h. ethyl cis-3-iodoacrylate ( 0.25 equiv per h for 5 h ) was added to the reaction mixture . the residue was dissolved into a minimum amount of methanol , and sodium methoxide ( 1.5 equiv ) was added ( dropwise in a 0.5 m solution ) . 6-oxo - m1guo was purified on biotage sp1 flash chromatography system ( biotage , uppsala , sweden ) using the following gradient ; 1% to 5% acetonitrile in 30 column volumes followed by a 5% to 15% gradient in 10 column volumes . separation was achieved on a 12 + m c18 column , and the purified compound showed a single sharp peak by reverse - phase hplc . h nmr ( 600 mhz , dmso - d6 ) : 8.65 ( d , 1h , h8 , j = 7.8 hz ) , 8.29 ( s , 1h , h2 ) , 6.24 ( d , 1h , h7 , j = 8.4 hz ) , 5.82 ( d , 1h , h1 ) , 5.48 ( d , 1h , h2oh ) , 5.20 ( d , 1h , h3oh ) , 5.03 ( d , 1h , h5oh ) , 4.49 ( d , 1h , h2 ) , 4.15 ( d , 1h , h3 ) , 3.91 ( d , 1h , h4 ) , 3.633.55 ( m , 2h , h5 ) . the h nmr may be seen in figure a of the supporting information . 6-oxo - m1dg and its stable isotope - labeled analogue , [ n5]-6-oxo - m1dg , were synthesized as previously described . briefly , anhydrous dg ( 1.6 g , 6 mmol ) and anhydrous k2co3 ( 0.93 g , 6.75 mmol ) were dissolved in anhydrous dmf ( 18 ml ) . one milliliter of a 0.55 m solution of ethyl cis-3-bromoacrylate in anhydrous dmf was added to the reaction every 15 min for 4 h. the reaction was stirred for an additional 2 h then cooled and filtered . the filtrate was evaporated under high vacuum and the residue was dissolved in water ( 4 ml ) and 5% acetic acid ( 4 ml ) . the product was purified by reversed - phase ( c18 ) medium pressure liquid chromatography with a biotage sp1 apparatus ( biotage ) using a prepacked 25 75 mm biotage flash 25 + s cartridge ( kp - c18-hs ) and a flash 25 c18 samplet for sample loading . a pure final product , 6-oxo - m1dg ( 0.92 g , 50.6% ) , was obtained . [ n5]-6-oxo - m1dg was synthesized by substituting [ n5]-dg for dg ( cambridge isotope laboratories , andover , ma , usa ) and employing the scheme described above . the h nmr of 6-oxo - m1dg and its stably labeled analogue matched the spectrum previously reported for this compound . mass spectrometric analysis of 6-oxo - m1dg showed an [ m + h ] peak at m / z 320 , while analysis of [ n5]-6-oxo - m1dg showed an [ m + h ] peak at m / z 325 . the m / z = 325 corresponds to the 6-oxo - m1dg value plus 5 additional mass units from the 5 n atoms incorporated into the [ n5]-dg starting material . a m / z 320 peak was not observed for [ n5]-6-oxo - m1dg , indicating that no unlabeled 6-oxo - m1dg was present in the internal standard . 6-oxo - m1guo ( 12 mg ) was dissolved in 750 l of 100 mm aqueous sodium periodate . protein ( 20 mg of bsa or mcklh ) was reconstituted with 700 l of pbs preadjusted to a ph of 9.5 with 5% k2co3 . the 6-oxo - m1guo solution and the protein after 1 h , 45 l of a 1 m diethylene glycol solution was added to the mixture ( to quench excess oxidizing agent ) followed by 700 l of 0.45 m nabh4 ( aqueous ) . after another 12 h , the ph of the mixture was adjusted to 7.0 with 1.0 m formic acid . the mixture was kept at this ph for 1 h. then , the ph was increased to 8.5 by the careful addition of 1 m aqueous ammonium hydroxide solution . this mixture was dialyzed against pbs buffer twice for 24 h. the sample was lyophilized and stored at 4 c . scheme 1 shows the conjugation reaction of 6-oxo - m1guo with a lysine residue of the carrier protein . figure b of the supporting information depicts the scheme for preparation of the conjugated protein . four balb / cj mice and four a / j mice ( the jackson laboratory , bar harbor , me , usa ) were injected subcutaneously with 50 g of 6-oxo - m1guo - klh and freund s complete adjuvant ( primary boost ) . four wk after the initial immunization , the mice were boosted subcutaneously ( first boost ) with the same dose of conjugate , but with the substitution of incomplete adjuvant ( which was also used in subsequent boosts ) . two wk after the first boost , the mice were tail bled and antibody titers were assessed by direct and competitive elisa as described below . a second boost was administered 4 wk subsequent to the first , and after 2 wk , sera were again extracted and subjected to elisa evaluation . a third boost was administered 25 wk after boost two , and the sera were collected and screened approximately 2 wk later . on the basis of the cumulative elisa data , a single balb / cj mouse ( balb / cj r ) showing the most selective and concentrated anti-6-oxo - m1dg titer was chosen for a fourth and final boost given intraperitoneally and lacking adjuvant to prepare it for splenocyte extraction . four days after the final boost , the mouse was sacrificed by cervical dislocation and the spleen harvested . splenocytes were isolated and subjected to polyethylene glycol - mediated fusion with both sp/20 and ns1 murine myeloma cells ( obtained from the university of virginia and the university of nebraska medical univeristy school of dentistry , respectively ) and allowed to recover for 24 h in liquid culture . the products of the fusion were evenly distributed into 24 96-well plates , with 12 plates corresponding to fusions of sp/20 cells and splenocytes and 12 plates corresponding to fusions of ns1 cells and splenocytes . cells were grown in iscove s modified dulbecco s medium with 20% fetal bovine serum ( invitrogen ) . hybridomas were selected by growing in the presence of aminopterin ( 5 10 m , sigma ) and ht supplement ( 1:100 dilution , invitrogen ) for 14 days with media and aminopterin replenishment every 3 days . supernatant was removed and screened for antigen - specific antibodies by elisa , with 6-oxo - m1dguo - bsa conjugate used as the antigen ( described below ) . positive hybridoma supernatants were rescreened in the presence of xanthine and 6-oxo - m1dg . xanthine was employed as a competitor to identify antibodies not specific to 6-oxo - m1dg . clones exhibiting anti-6-oxo - m1dg activity and specificity in addition to positive growth and productivity were selected and plated on 24-well plates . of these , five clones were chosen based on activity , specificity , growth , and productivity as determined by direct and competitive elisa analysis ( described below ) and then subsequently subjected to two rounds of subcloning . the clones were subsequently cryopreserved . under vanderbilt institutional care and use committee protocol # m-07 - 109 , the vanderbilt antibody and protein resource core is permitted to use freund s complete adjuvant in the manner described above . the following solutions were prepared as follows and their use is described below : ( a ) carbonate - bicarbonate coating buffer ( ph 9.6 ) was prepared from na2co3 ( 1.59 g / l ) , and thimerosal ( 0.10 g / l ) ; ( b ) pbs - tween ( ph 7.4 ) was prepared from nacl ( 8.00 g / l ) , tween 20 ( 1.00 ml / l ) , and thimerosal ( 0.10 g / l ) ; ( c ) bsa ( 5.0 g ) mixed in pbs - tween ( 500 ml ) ; ( d ) 1 nm abts solution in 70 mm citrate - phosphate buffer ( ph 4.2 ) was prepared from citric acid ( 5.64 elisa quality microtiter plates ( thermo fisher scientific ; 96-well immulon 2hb flat bottom microtiter plates or 384-well 4hb flat bottom plates ) were coated with 50 , 75 , or 100 l of a 10 g / ml solution of 6-oxo - m1guo - bsa antigen in carbonate - bicarbonate coating buffer ( a ) and incubated at 4 c overnight . plates were washed three times with 100 l of pbs - tween ( b ) using a bio - tek elx405 automatic microplate washer ( winooski , vt , usa ) , then incubated with 100 l of pbs - tween ( b ) for 30 min at 37 c . aliquots of murine serum dilutions , hybridoma supernatants , or purified antibody ( depending on the stage of the antibody development process ) were then incubated in the coated wells with or without the addition of varying concentrations of 6-oxo - m1dg and structural analogues ( figure 1 ) in a final volume of 100 l pbs for 60 min at 37 c . for wells that included 6-oxo - m1dg or structural analogues , the serum dilution used was 1:5000 in pbs - tween ( b ) , and the compounds depicted in figure 1 were preincubated in the dilute murine serum for 45 min prior to the serum addition step . this allowed the nonspecific antibodies to bind the structural analogues , theoretically leaving behind only 6-oxo - m1dg - specific antibodies . initial analysis revealed that xanthine was recognized nearly as well as 6-oxo - m1dg , which led to the use of this structural analogue in further competition studies . 6-oxo - m1dg , m1dg , and structural analogues that were used as competitive antigens during elisa analysis of murine sera , hybridomal supernatant , and purified antibodies . the plates were then washed three times with pbs - tween ( b ) . aliquots of diluted horseradish peroxidase - conjugated goat antimouse igg fc region - specific or igg ( h + l ) secondary antibody ( diluted 1:5000 in pbs - tween / bsa ) were added , and the plates were incubated at 37 c for 60 min . after 60 min , the plates were washed three times and 100 l of a freshly prepared abts solution ( 1.8 l of h2o2 per 1 ml of abts ) was immediately added to each well . to determine peroxidase activity , the absorbance at 414 nm was measured after 15 and 30 min for each well using a bio - tek powerwave ht 340 plate reader with gen5 software . selected clones were scaled up , inoculated into one - liter bioreactors ( wilson wolf manufacturing , new brighton , mn , usa ) and grown for 34 wk . purification of mab from the bioreactor supernatants was achieved by affinity chromatography on protein - g sepharose ( ge healthcare , piscataway , nj , usa ) followed by a final desalting step into pbs . purified mab were isotyped and subsequently quantified by sds - page electrophoresis followed by infrared coomassie staining . purified anti-6-oxo - m1dg mab was loaded onto pd-10 columns ( ge healthcare ) equilibrated with 25 ml coupling buffer ( 0.1 m nahco3 , 0.5 m nacl , ph 8.0 in water ) . cyanogen bromide - activated sepharose beads ( 3.75 g ) were prepared for conjugation by rapid swelling in 1 mm hcl followed by filtering , rinsing with 400 ml of 1 mm hcl and then 10 ml of coupling buffer . the sepharose beads were transferred to the antibody solution and gently rotated at 4 c for about 20 h. the mixture was then filtered and washed with 500 ml of coupling buffer , and the residue was divided into four parts . each quarter was placed in 14 ml of ethanolamine solution ( 1.0 m , ph 8.0 ) and gently rotated overnight at 4 c . the sepharose beads were then filtered and washed with a low ph acetate buffer ( 0.1 m acetic acid , 0.1 m naoh , and 0.5 m nacl ) followed by a 0.1 m tris hcl buffer ( 0.5 m nacl , ph 8.0 ) . each wash used 75 ml of buffer , and the wash cycle was repeated two more times . the resulting residue was suspended in 20 ml of 0.1 m tris buffer with 0.5 m nacl and 0.2% nan3 ( ph 8.0 ) , and the final product ( the gel ) was stored at 4 c . the gel was used to recover 6-oxo - m1dg and [ n5]- 6-oxo - m1dg from various fluids by the following general procedure . an aliquot of gel was prepared for use by rinsing with the following solutions : 50 ml of 0.1 m glycine ( ph 2.7 ) , 20 ml h20 , 50 ml methanol , and 50 ml pbs . this cleaning procedure was necessary to remove any 6-oxo - m1dg that had bound to the mab during preparation . the ph of the fluid to be analyzed was adjusted to 7.08.0 with acetic acid or ammonium hydroxide . an aliquot of the washed and reconditioned gel was added to the sample , and the suspension was mixed gently for 14 h. the sample / gel suspension was filtered through a precleaned empty polypropylene spe cartridge fitted with a polypropylene frit ( sigma - aldrich ) . the filtered gel was washed with 10 ml of pbs , and the analytes were eluted with 2 ml of methanol . the eluant was dried under n2 gas , capped , and stored at 20 c . the sample was reconstituted in 9:1 water / methanol ( v / v ) immediately prior to lc - ms / ms analysis . urine and feces were collected at 24 h intervals and stored at 20 c following collection . urine samples were analyzed by thawing , adding 200500 fmol of [ n5]-6-oxo - m1dg , and centrifuging the samples at 4000 g for 25 min at 4 c . an aliquot of supernatant was diluted 1015-fold with pbs , and the ph of the final solution was adjusted to 7.5 0.5 with either acetic acid or sodium hydroxide . the gel ( prepared as described above ) was introduced after ph adjustment , and the suspension was rotated gently end - over - end for 14 h. the suspension was filtered and the analyte eluted from the gel as described above . the weighed sample was transferred to a clean vessel with 40 ml of pbs , and 200500 fmol of [ n5]-6-oxo - m1dg were added . dispersion of the sample was accomplished by rotating end - over - end for 23 h followed by sonication for 35 min . the suspension was then filtered through a mesh screen , with the collected solid material rinsed with 10 ml pbs . the supernatant was transferred to a clean vessel , diluted roughly 5 times with pbs , and the antibody gel ( prepared as described above ) was added . the suspension was rotated gently end - over - end for 14 h , filtered , and the analyte eluted from the gel as described above . final lc - ms / ms analysis of gel - purified samples was accomplished on a thermo surveyor autosampler and ms pump in - line with a thermo quantum triple quadrupole mass spectrometer ( thermo fisher scientific ) . the mass spectrometer was equipped with an electrospray source and operated in positive ion mode . 6-oxo - m1dg and its internal standard , [ n5]-6-oxo - m1dg , were detected via selected reaction monitoring ( srm ) with the following transitions , respectively ; m / z 320 204 and 325 209 . in both cases , the transition corresponds to cleavage of the glycosidic bond and loss of the deoxyribose moiety , with the positive charge remaining on the base . the analytes were chromatographed on the following reverse - phase gradient system ; 2% b to 15% b in 5 min , 15% b to 80% b in 1.5 min , followed by a 1 min hold at 80% b. component a was water and component b was methanol / acetonitrile ( 3:1 ) , and each component contained 0.1% acetic acid ( v / v ) . the column used was a phenomenex c18 ( phenomenex , torrence , ca , usa ; 10.0 0.2 cm , 3 m ) held at 3540 c . the column was equilibrated at the initial conditions for 3 min prior to each injection . 6-oxo - m1dg was quantitated by stable isotope dilution against [ n5]-6-oxo - m1dg . lc - ms / ms data was acquired and processed by xcalibur software ( thermo fisher scientific ) . the overall scheme of the antibody generation and isolation process is depicted in figure 2 . eight mice ( four balb / cj and four a / j ) were injected with 6-oxo - m1 g conjugated to klh as described in the materials and methods section . at 6 , 10 , and 35 wk after the initial innoculation , the mice were tail bled , and direct and competitive elisa analyses of the sera were performed ( figure 2 , step 1 ) . all three bleeds from all 8 mice showed robust responses against the 6-oxo - m1g - bsa antigen , indicating that the immune systems of the mice were producing significant amounts of antibodies against the 6-oxo - m1 g portion of the innoculant . competitive elisa screenings were executed to assess antibody specificity , with 6-oxo - m1dg and a series of structural analogues ( figure 1 ) serving as competing antigens . eight mice were inoculated with adduct / protein conjugate , followed by tail bleeding and testing for the presence of 6-oxo - m1dg antibodies using direct elisa analysis ( step 1 ) . splenocytes from a single mouse , balb / cj r , were fused with myeloma cells , plated on twenty - four 96-well plates , and screened using direct and competitive elisa analyses ( step 2 ) . from this screen , five parental cell lines were chosen for subcloning and further screening ( step 4 ) . on the basis of these final screenings , the cell line 6c9ba4c12 was chosen for scale - up and antibody purification ( step 5 ) . figure 3 depicts a competitive elisa analysis of the 35-week serum from mouse balb / cj r. this analysis employed 6-oxo - m1dg as the competing antigen and a robust concentration dependent decrease in optical density is observed , indicating the presence of anti-6-oxo - m1dg antibodies in the sera from this mouse . the results of similar analyses of the sera of all 8 inoculated mice can be seen in supporting information figure c. supporting information figure d displays the results from competitive elisa analyses of the 35-week serum from all 8 inoculated mice where m1dg is employed as the competing antigen . increasing amounts of m1dg do not result in a decrease in optical density , indicating that m1dg does not bind to the anti-6-oxo - m1dg antibodie(s ) present in the sera . 6-oxo - m1dg antibodies are present in the serum of mouse balb / cj r. the serum of mouse balb / cj r was subjected to competitive elisa analysis with 6-oxo - m1dg used as the competitor . the displayed results represent the average of spectroscopic readings at 15 and 30 min postaddition of abts substrate . on the basis of the elisa analysis of murine sera , a single mouse ( balb / cj r ) was chosen for hybridoma production . splenocytes from balb / cj r were fused with both sp/20 and ns1 myeloma cells ( figure 2 , step 2 ) , and 1670 hybridomas were formed from 2304 fusions wells . elisa analysis revealed that of the 1670 hybridomas , 180 showed production of anti-6-oxo - m1dg antibodies . these were grown in 24-well plates ( figure 2 , step 3 ) , and the supernatants were subjected to further elisa screenings . on the basis of the screening data , five hybridomas were selected for subcloning and further screenings ( figure 2 , step 4 ) . figure 4 shows a representative elisa analysis of the supernatants from two subclones ( b and e2 ) of parental hybridoma cell line 6c9 in which xanthine and 6-oxo - m1dg are employed as competing antigens . the presence of unbound 6-oxo - m1dg decreases the optical density of both b and e2 supernatants , indicating the presence of anti-6-oxo - m1dg antibodies . for subclone e2 , xanthine has a similar effect , demonstrating that e2 s mab are not specific to 6-oxo - m1dg . however , xanthine has no effect on the optical density of subclone b supernatant , suggesting that mab from subclone b are specific to 6-oxo - m1dg . on the basis of these data , subclone b was subjected to further subcloning but e2 was not . supporting information figure e illustrates that cell line 6c9ba4 , a subclone of hybridoma 6c9b , retained activity against 6-oxo - m1dg but did not react to xanthine . the specificity of antibodies produced by two hybridoma cell lines ( b and e2 ) subcloned from the parental hybridoma cell line 6c9 were assessed using competitive elisa analysis . the displayed results represent the average of spectroscopic readings of subclones e2 and b at 15 and 30 min postaddition of abts substrate . on the basis of a review of the cumulative elisa data , a final daughter cell line was selected from each parental hybridoma line , and these daughter cell lines were cryopreserved . the elisa data indicated that antibodies from the cell line 6c9ba4c12 displayed the most promising expression and specificity . thus , this cell line was subjected to large - scale expression and antibody purification . figure 5 shows the elisa responses of the mab purified from 6c9ba4c12 in the presence of 6-oxo - m1dg and structural analogues . only 6-oxo - m1dg attenuated the optical density , indicating a specificity of the purified mab for 6-oxo - m1dg over close structural analogues . purified antibodies from the hybridomal subclone ( 6c9ba4c12 ) were screened for specificity for 6-oxo - m1dg in the presence of structural analogues using competitive elisa analyses . the purified anti-6-oxo - m1dg mab was covalently linked to sepharose beads as described above . the purification and analysis protocol outlined in materials and methods was used to quantify aliquots of 10 mm potassium phosphate ( ph 8.0 ) spiked with varying amounts of 6-oxo - m1dg ( 0.25 , 2.5 , and 25 pmol ) and 1.0 pmol of the internal standard , [ n5]-6-oxo - m1dg ( n = 3 for each concentration of 6-oxo - m1dg ) . as shown in table 1 , the experimentally determined amount of 6-oxo - m1dg was within 12% of the known amount at each level . the average percent recovery of 6-oxo - m1dg ranged from 94% to 74% , while the internal standard was recovered at an average rate of 79% . thus , the gel was able to bind 6-oxo - m1dg from an aqueous solution and released the analyte in the presence of methanol . the amount detected , % accuracy , and % recovery values are shown as the mean s.d . the limit of detection ( lod ) of the described lc - ms / ms system was established at approximately 10 fmol 6-oxo - m1dg on - column ( supporting information figure f , top ) by analyzing a series of increasingly dilute 6-oxo - m1dg solutions . given this lod and the percent recovery observed from urine and fecal samples ( approximately 30% ) , the limit of quantification of the assay was estimated to be 50 fmol per sample . additionally , the lc - ms / ms system provided a linear response to 6-oxo - m1dg solutions over a 5000-fold concentration range and a single [ n5]-6-oxo - m1dg concentration ( supporting information f , bottom ) . these results indicate that the gel provided a sufficient level of recovery of the analyte and its internal standard as well as sufficient capacity for the expected levels of 6-oxo - m1dg present in rat urine and feces . they also demonstrate that the lc - ms / ms analysis of the purified sample provided sensitive and accurate quantification . urine and feces collected from male sprague dawley rats over 3 days were analyzed as described in the materials and methods section . 6-oxo - m1dg was detected in urine from only one subject , animal 3 , at a rate of 188 fmol / kgd . however , 6-oxo - m1dg was eliminated in feces in all the animals at a rate of 3501893 fmol / kgd . thus , it appears fecal elimination is the main route by which that 6-oxo - m1dg is excreted from the body . values are given as the mean amount observed in fmol / kgd s.d . ( n = 35 for feces and 2 for urine ) . while the amounts of 6-oxo - m1dg are low , the assay provided a sufficient signal - to - noise ratio to permit accurate quantification . the upper trace ( m / z 320 204 ) shows the analyte , while the bottom trace ( m / z 325 209 ) represents the [ n5]-6-oxo - m1dg internal standard . the inset of figure 6 is the internal standard alone after recovery from pbs . there is no peak in the 6-oxo - m1dg trace of the inset , which is illustrative of the fact that there was no isotopic impurity in the internal standard that could contribute to the 6-oxo - m1dg signal . a representative lc - ms / ms chromatogram of 6-oxo - m1dg and the internal standard [ n5]-6-oxo - m1dg isolated from rat feces is displayed . m1dg is an endogenous dna adduct detectable in the genomic dna of humans and rodents . its presence is associated with peroxidative damage to cellular constituents , particularly polyunsaturated fatty acids , and dna . although many studies have shown that the lipid peroxidation product , malondialdehyde , can react in vitro with deoxyguanosine to form m1dg , recent studies in e. coli , with defined polyunsaturated fatty acid content in membrane phospholipids , indicate that this is not a major source of m1dg . rather , the dna peroxidation products , base propenals , appear to account for nearly all of the m1dg generated when bacteria are exposed to peroxynitrite ( onoo ) . thus , although m1dg appears to be a reliable marker of oxidative damage to cells , it primarily reflects oxidative damage to dna . m1dg is repaired by nucleotide excision repair and is excreted in urine at rather low levels in humans ( 12 3.8 fmol / kgd ) . a major contributor to the low levels of m1dg is its oxidative metabolism to 6-oxo - m1dg . this suggests 6-oxo - m1dg may be superior to m1dg as a noninvasive marker of oxidative damage . in the present manuscript , we describe the generation of a monoclonal antibody that exhibits sensitive and specific binding to 6-oxo - m1dg and its use in a quantitative assay suitable for analysis of urine and feces . in the assay , the sepharose - bound mab is used to isolate 6-oxo - m1dg from urine and feces . the purified sample is subjected to lc - ms / ms analysis where 6-oxo - m1dg is quantified via stable isotope dilution against the synthetic internal standard , [ n5]-6-oxo - m1dg , which is introduced to the sample prior to antibody purification . the antibody gel shows excellent recovery of 6-oxo - m1dg from buffer solutions . the analyte is recovered at 74% or greater from 10 mm potassium phosphate over a 100-fold range of 6-oxo - m1dg , 0.25 pmol to 25.0 pmol . the internal standard was recovered at 79% ( 1.0 pmol ) from these same solutions , and spiked buffer solutions were quantified accurately to 12% . these data demonstrate that 100 l of the antibody gel is able to bind at least 26 pmol of 6-oxo - m1dg from solution , and lc - ms / ms detection of the purified sample is quantitative . the recovery of [ n5]-6-oxo - m1dg from saline solutions was greater than 78% but its recovery from urine and feces was only 29% . this suggests a matrix effect where other nucleosides , deoxynucleosides , and/or endogenous congeners compete with 6-oxo - m1dg for antibody binding sites . although there was minimal cross - reactivity of the antibody with individual members of a panel of exocyclic adducts and purine oxidation products ( figures 1 and 5 ) , it is possible that high levels of low - avidity nucleosides in urine and feces compete with the low levels of endogenous 6-oxo - m1dg for binding sites on the mab . 6-oxo - m1dg was detected in the feces of each of three different sprague dawley rats but was detected in the urine of only one of the rats ( table 2 ) . the fecal levels of 6-oxo - m1dg ( 3501893 fmol / kgday ) were significantly higher than the urinary levels ( 188 fmol / kgday ) . this is consistent with previous findings that demonstrate m1dg is excreted mainly in urine , whereas 6-oxo - m1dg is excreted mainly in feces . because 6-oxo - m1dg is present endogenously in rats , it suggests that its metabolic precursor , m1dg , is also produced endogenously in rats and metabolized to 6-oxo - m1dg . this idea is consistent with the finding that m1dg is present in rodent and human genomic dna and with studies from this laboratory demonstrating that 6-oxo - m1dg is the sole metabolite of m1dg . it may be argued that endogenous 6-oxo - m1dg is generated in the nucleoside pool rather than arising from direct damage to dna . however , this seems unlikely if one considers that base propenals are the principal precursors to m1dg . base propenal formation is triggered by abstraction of the hydrogen from the c-4 position of the deoxyribosyl unit of double - stranded dna . the base propenal produced then reacts with a deoxyguanosine residue in duplex dna to form m1dg . in comparison , the yields of base propenals are much lower when deoxynucleosides are oxidized instead of duplex dna . furthermore , m1dg formation as a reaction of base propenals and free deoxyguanosine is a bimolecular process . if base propenals are formed in duplex dna , they are generated in close proximity to deoxyguanosine residues in the dna duplex , which should maximize the probability of their reaction with a deoxyguanosine residue . one or both of these compounds may have been ingested , then excreted or metabolized . it was not possible to control for this possibility under the reported experimental regime , so future studies with animals fed nucleic acid - free diets will be required to test this hypothesis . the ability to measure excreted 6-oxo - m1dg has the potential for clinical relevance in assessing the levels of oxidative dna damage in humans . since the basal levels of both deoxynucleoside adducts are low , they should provide high sensitivity to increases triggered by oxidative damage . given that the analytical method described herein is applicable to feces and urine , the possibility exists for preclinical or clinical studies conducted in a noninvasive fashion .

oxidative stress triggers dna and lipid peroxidation , leading to the formation of electrophiles that react with dna to form adducts . a product of this pathway , ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one ) , or m1dg , is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway . in vivo , m1dg is oxidized to a primary metabolite , ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione , or 6-oxo - m1dg , which is excreted in urine , bile , and feces . we have developed a specific monoclonal antibody against 6-oxo - m1dg and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo - m1dg from biological matrices . the purified analyte is quantified by lc - ms / ms using a stable isotope - labeled analogue ( [ 15n5]-6-oxo - m1dg ) as an internal standard . healthy male sprague dawley rats excreted 6-oxo - m1dg at a rate of 3501893 fmol / kgd in feces . this is the first report of the presence of the major metabolite of m1dg in rodents without exogenous introduction of m1dg .

Introduction Materials and Methods Results Discussion

a 60-year - old woman ( height , 162 cm ; weight , 61 kg ) visited pain clinic with tactile allodynia and electric shock - like pain in the left dorsal scapular area around the t3 dermatome , which had been diagnosed as phn about 1 month previously and attack of the herpes zoster was 1 year ago . the 100-mm visual analogue scale ( vas ) of allodynia and electric shock - like pain was rated between 70 and 80 mm on a scale from 0 ( no pain ) to 100 ( worst pain imaginable ) . the interlaminar epidural block was performed at the t3 - 4 space by the paramedian approach with 5 ml of 0.2% ropivacaine and 20 mg of triamcinolone acetate . pregabalin and morphine at doses of 150 mg and 10 mg , respectively , twice a day , amitriptyline at a dose of 10 mg before sleep and topical lidocaine patches were prescribed . dosages of all drugs were adjusted depending on the side effects during the follow - up period . epidural blocks were repeated twice with a 1-week interval and the continuous intravenous infusion of ketamine ( 60 mg ) was performed over a period of 1 hour twice a week under careful monitoring . after 1 month elapsed , electric shock - like pain was reduced to a vas score of 30/100 , but allodynia was not diminished ( vas score of 70/100 ) . after 4 months elapsed , we decided to administer magnesium sulfate via the intravenous route . and it was done with continuous intravenous infusion of 1,000 mg of magnesium sulfate in 50 ml of normal saline for 1 hour . before and after infusion , the serum magnesium levels were checked . after magnesium therapy , she felt very good about her pain and the vas of allodynia was reduced to 40 - 50 . at 1-week follow - up , she was very satisfied with the treatment and reported the reduction of allodynia on the dorsal scapular area of up to 50% ( vas 25 - 30/100 ) . however , the serum magnesium level had increased above normal range ( 2.3 meq / l to 2.9 although it was below the serum level reveals of the adverse effect , we decided to stop intravenous infusion of magnesium sulfate . for more accurate and safe delivery of magnesium to the target site , we applied magnesium using the transforaminal epidural injection technique . with the patient 's informed consent , left t3 tfemi identification of the t3 nerve root sheath and epidural space was performed using contrast media ( fig . 1 ) . then , 100 mg of magnesium sulphate and 1 ml of 0.2% ropivacaine ( total volume , 2 ml ) was carefully injected . tfemi was repeated twice with a 1-week interval ( total of three times ) and the degree of pain decreased gradually during the follow - up period . one week after the last procedure , the vas score of allodynia decreased to 15/100 and all medications except pregabalin were discontinued . the vas was 10/100 throughout 1-month follow - up , and pregabalin had also been tapered . to our knowledge , no previous report has described about the magnesium administration by the transforaminal epidural route in patients with neuropathic pain . here , in our report of phn patient , this treatment resulted in effective pain relief . previous studies have demonstrated the anti - allodynic effects of nmda receptor antagonists in neuropathic pain disorders . among the currently available nmda receptor antagonists , ketamine is the most widely used one for the treatment of neuropathic pain . however , ketamine is not always effective and psychomimetic side effects are frequent . magnesium can antagonize nmda receptor channels by blocking calcium influx in a voltage - gated manner . intravenous administration of magnesium is efficacious in the management of various conditions associated with neuropathic pain , including phn . demirkaya and colleagues revealed 1 g i.v . mg sulfate is effective in the treatment of migraine attacks and collins and colleagues reported that 70 mg / kg magnesium sulphate infusions in 4 hours for 5 days reduced pain in patients with complex regional pain syndrome . whether intravenous administration of magnesium can achieve a sufficient concentration in the cerebrospinal fluid to block nmda receptors is unclear and studies have reported on the limited efficacy of magnesium when administered via the intravenous route . furthermore , even if the dose of intravenously administered magnesium is not sufficient to present toxicity , patients are still at risk of magnesium overdose . neuraxial administration of magnesium is an " off - label " use , and the safety of this technique in human subjects is still undetermined . however , animal studies showed that intrathecally administered magnesium was free of neurotoxicity , and recent studies have demonstrated the safety of magnesium administration via the epidural or intrathecal route in humans . in fact , the exact site of action of epidurally administered magnesium ( i.e. , spinal or supraspinal ) remains unclear . however , comparison with previous reports regarding intravenous magnesium administration suggested that the low dose epidural magnesium used in our patient was unlikely to result in systemic effects . in conclusion , tfemi showed a favourable result in the treatment of intractable allodynia associated with phn . this study was performed in only a single case , and further investigations are required to determine the efficacy of tfemi in the management of allodynia in patients with phn .

although postherpetic neuralgia ( phn ) is a common chronic pain syndrome , the pathophysiology of this disorder is not well known and management is often very difficult . n - methyl - d - aspartate ( nmda ) receptor antagonists are known to be effective in phn , and magnesium , a physiological blocker of nmda receptors , is widely used to treat various chronic pain disorders . here , we present a case of the phn refractory to conventional treatment , which was treated successfully with transforaminal epidural injection of magnesium sulphate at the affected dermatome .

CASE REPORT DISCUSSION

central retinal vein occlusion ( crvo ) is a common sight - threatening vascular disease with prevalence rates ranging from 0.1%1 to 0.5%2 in the older adult population . crvo is characterized by occlusion of the central retinal vein and consecutive damming of the venous blood flow . the occlusion may occur at the level of or posterior to the lamina cribrosa.3 despite its prevalence , the pathogenesis of crvo is not completely understood . a combination of vascular , anatomic , and inflammatory factors may contribute to its pathophysiology.4 current treatment options include intravitreal injections of steroids5 or anti - vascular endothelial growth factor ( anti - vegf ) agents.68 fluorescein angiographic ( fa ) assessment is the most important tool for an adequate evaluation of the disease severity and for proper classification between the two types of crvo , ischemic and nonischemic . ischemic crvo is associated with a significant upregulation of vegf , a poor prognosis for visual acuity , and has a higher incidence of secondary neovascular glaucoma ( nvg ) ranging from 20% to 60% compared to nonischemic crvo with no risk of developing nvg.9 given these significant differences with various outcomes and risk profiles , it is crucial to differentiate between ischemic and nonischemic crvo . conventional fa images the retinal circulation within the posterior pole . however , it is not known if conventional fa devices are able to capture areas of interest regarding the intraocular metabolism of vegf , such as the peripheral retina . earlier findings suggested an association between peripheral retinal ischemia and an increased production of vegf.10,11 currently , two ultrawide - field systems are commercially available to perform fa and image the peripheral retina . one method uses a wide - field contact lens system ( ocular staurenghi 230 slo retina lens ; ocular instruments , inc , bellevue , wa , usa ) with the heidelberg retina angiograph ( hra ; heidelberg engineering gmbh , dossenheim , germany).12,13 the second available system uses the optos scanning laser ophthalmoscope ( optos plc , dunfermline , uk ) , which is a special non - contact - lens - based system that provides visualization up to the peripheral retina.1416 it has been shown that ultrawide - field fa ( uwfa ) is useful for the assessment of several retinal pathologies17 including retinal vein occlusions,18 diabetes,19,20 uveitis,21 retinal vasculitis,22 choroidal masses,23,24 retinal detachment,25 and retinopathy of prematurity.26 the aim of this study was to evaluate a possible association between peripheral retinal nonperfusion obtained with uwfa and the number of intravitreal ranibizumab injections needed in patients with crvo . this prospective interventional study was performed between june 1 , 2012 , and february 1 , 2014 . the institutional review board of ludwig - maximilians university in munich approved the study design , and patients care adhered to the tenets of the world medical association declaration of helsinki . all patients gave written informed consent for both participation in the study and for fa . inclusion criteria were diagnosis of crvo ( as revealed by retinal hemorrhages and dilated retinal veins in all four quadrants of the fundus ) with active center - involving macular edema and central subfield thickness > 250 m detected by spectral - domain optical coherence tomography ( sd - oct , heidelberg engineering , heidelberg , germany ) . patients without macular edema , with previous focal or panretinal photocoagulation , and with degenerative disorders of the posterior pole and/or retinal periphery were excluded . additional intravitreal injections were administered in the presence of active center - involving macular edema and central subfield thickness > 250 m , as determined by sd - oct . all patients underwent comprehensive ophthalmologic examination that included best - corrected visual acuity ( bcva ) measurement , slit - lamp biomicroscopy , applanation tonometry , indirect ophthalmoscopy , and sd - oct before injections and at each follow - up visit . uwfa using the optos 200t imaging system was obtained in each case before the first injection . all included patients received three intravitreal injections of 0.50 mg ranibizumab ( lucentis , genentech , inc . , south san francisco , ca , usa and novartis pharma ag , basel , switzerland ) every four weeks and then were examined monthly with follow - up oct scans of the macula . they received additional intravitreal injections if macular edema was detected by sd - oct scans at follow - up visits . sd - oct volume scans ( 2015 with 19 horizontal sections , art 9 , sd - oct , heidelberg engineering , heidelberg , germany ) of the macula were obtained for each study eye to measure the central subfield thickness ( cst ) in m by using heidelberg sd - oct software ; significant macular ischemia was ruled out by uwfa . fa images were acquired approximately one minute ( arteriovenous phase ) and 45 minutes ( late venous phase ) after intravenous injection using the optos 200t scanning laser ophthalmoscope ( optos plc ) after standard intravenous infusion of 5 ml of sodium fluorescein ( 10% ) by one experienced technician for all included cases . fa images were compressed into high - quality jpeg files ( figures 1 and 2 ) and analyzed for retinal nonperfusion by two experienced ophthalmologists ( kaa and fs ) . the far peripheral retina was defined as the area between the ampullae of the vortex veins and the ora serrata . the wide - field color images and wide - field fa images were used to identify the vortex vein ampullae , which were located near the ocular equator.27 based on another published work , we defined retinal nonperfusion as at least five disc areas of hypofluorescence28 ( representing retinal nonperfusion or capillary dropout ) or areas of microvascular pathology ( multiple microaneurysms and significant perivascular leakage ) on uwfa . five disc areas was also the mean size of peripheral retinal nonperfusion of all our patients with crvo . because of the effect of upper and lower eyelid and eyelashes , the far periphery in the horizontal plane alone ( the nasal and temporal retina ) was analyzed . collected parameters included demographic information , previous ocular history , number and dates of intravitreal injections , central subfield thickness , peripheral retinal ischemia , central intraretinal fluid , visual acuity , and intraocular pressure ( throughout the study period ) as well as the occurrence of any complications . data were collected and analyzed using spss software ( version 20.0 , ibm corporation , armonk , ny , usa ) . spearman rho test was used for correlation analysis . a p - value of < 0.05 was considered statistically significant . this prospective interventional study was performed between june 1 , 2012 , and february 1 , 2014 . the institutional review board of ludwig - maximilians university in munich approved the study design , and patients care adhered to the tenets of the world medical association declaration of helsinki . all patients gave written informed consent for both participation in the study and for fa . inclusion criteria were diagnosis of crvo ( as revealed by retinal hemorrhages and dilated retinal veins in all four quadrants of the fundus ) with active center - involving macular edema and central subfield thickness > 250 m detected by spectral - domain optical coherence tomography ( sd - oct , heidelberg engineering , heidelberg , germany ) . patients without macular edema , with previous focal or panretinal photocoagulation , and with degenerative disorders of the posterior pole and/or retinal periphery were excluded . additional intravitreal injections were administered in the presence of active center - involving macular edema and central subfield thickness > 250 m , as determined by sd - oct . all patients underwent comprehensive ophthalmologic examination that included best - corrected visual acuity ( bcva ) measurement , slit - lamp biomicroscopy , applanation tonometry , indirect ophthalmoscopy , and sd - oct before injections and at each follow - up visit . uwfa using the optos 200t imaging system was obtained in each case before the first injection . all included patients received three intravitreal injections of 0.50 mg ranibizumab ( lucentis , genentech , inc . , south san francisco , ca , usa and novartis pharma ag , basel , switzerland ) every four weeks and then were examined monthly with follow - up oct scans of the macula . they received additional intravitreal injections if macular edema was detected by sd - oct scans at follow - up visits . sd - oct volume scans ( 2015 with 19 horizontal sections , art 9 , sd - oct , heidelberg engineering , heidelberg , germany ) of the macula were obtained for each study eye to measure the central subfield thickness ( cst ) in m by using heidelberg sd - oct software ; significant macular ischemia was ruled out by uwfa . fa images were acquired approximately one minute ( arteriovenous phase ) and 45 minutes ( late venous phase ) after intravenous injection using the optos 200t scanning laser ophthalmoscope ( optos plc ) after standard intravenous infusion of 5 ml of sodium fluorescein ( 10% ) by one experienced technician for all included cases . fa images were compressed into high - quality jpeg files ( figures 1 and 2 ) and analyzed for retinal nonperfusion by two experienced ophthalmologists ( kaa and fs ) . the far peripheral retina was defined as the area between the ampullae of the vortex veins and the ora serrata . the wide - field color images and wide - field fa images were used to identify the vortex vein ampullae , which were located near the ocular equator.27 based on another published work , we defined retinal nonperfusion as at least five disc areas of hypofluorescence28 ( representing retinal nonperfusion or capillary dropout ) or areas of microvascular pathology ( multiple microaneurysms and significant perivascular leakage ) on uwfa . five disc areas was also the mean size of peripheral retinal nonperfusion of all our patients with crvo . because of the effect of upper and lower eyelid and eyelashes , the far periphery in the horizontal plane alone ( the nasal and temporal retina ) was analyzed . collected parameters included demographic information , previous ocular history , number and dates of intravitreal injections , central subfield thickness , peripheral retinal ischemia , central intraretinal fluid , visual acuity , and intraocular pressure ( throughout the study period ) as well as the occurrence of any complications . data were collected and analyzed using spss software ( version 20.0 , ibm corporation , armonk , ny , usa ) . each graded variable was tested for normal distribution . spearman rho test was used for correlation analysis . a p - value of < 0.05 was considered statistically significant . fifty - four eyes of fifty - four consecutive patients with treatment - nave crvo were enrolled in this study . twenty - eight patients ( 52% ) were male , and twenty - three eyes ( 42.6% ) were located on the right side . seven patients ( 12.9% ) had known glaucoma , 36 patients ( 66.6% ) had systemic hypertension , and 22 patients ( 40.7% ) were pseudophakic . all patients had center - involving macular edema confirmed by macular leakage seen in fa and retinal thickening in sd - oct images . tables 1 and 2 show the baseline and final bcva and cst . as shown in the tables , there were significant improvements in the visual acuity within and between groups , whereas changes in cst were not significant . twenty - four eyes ( 44% ) showing less than five disc areas of retinal nonperfusion ( group 1 ) that received a mean number of 4.122.73 intravitreal ranibizumab injections . thirty eyes ( 56% ) showed more than five disc areas ( group 2 ) of retinal nonperfusion in uwfa and received a mean number of 9.323.84 intravitreal ranibizumab injections ( p<0.001 , mann whitney u - test ) . there was a significant positive correlation between the size of peripheral retinal nonperfusion measured in terms of disc areas and the number of injections in each group . there was also a significant correlation between the size of retinal nonperfusion and final bcva in each group . there was no significant correlation between the size of retinal nonperfusion and final cst in each group . we did not observe any eye with neovascularization of the disc or neovascularization elsewhere during the observation period ; therefore , panretinal photocoagulation was not performed during this study . furthermore , no cases of injection - related adverse events like retinal detachment or endophthalmitis were encountered . venous occlusive disease of the retina is the second most common retinal vascular disorder after diabetic retinopathy.29 it typically affects patients between 40 and 80 years of age.30 usually , a decrease of visual acuity is the result of macular edema , which can lead to permanent visual loss and even legal blindness in severe cases . applying uwfa , we found a significant correlation between the total number of intravitreal ranibizumab injections and the size of nonperfusion in the peripheral retina : crvo patients with peripheral retinal nonperfusion received more intravitreal injections for treatment of their macular edema than patients without peripheral retinal nonperfusion . we hypothesized that nonperfused peripheral retina could be a source of increased intraocular vegf levels with consecutive macular edema . this observation could explain the requirement for more frequent intravitreal anti - vegf injections of in crvo patients with macular edema and peripheral retinal nonperfusion . this finding is important because an evaluation of the peripheral retina using uwfa may be a prognostic factor and allows the ophthalmologists to estimate the requirement for more or fewer intravitreal injections based on the peripheral retinal perfusion status . looking at the perfusion of the peripheral retina in those patients , a more precise evaluation of the estimated treatment costs within a defined health care system may be feasible , including a better evaluation of injection - related complications such as endophthalmitis or other complications . interestingly , the lack of observed neovascularizations in any of the patients even with extended areas of peripheral retinal nonperfusion might be attributed to good patient compliance , strict follow - up visits every 4 weeks , and immediate treatment in case of detected macular edema . the results of our study differ from those published by spaide31 and singer et al.32 spaide evaluated 22 patients treated with ranibizumab for crvo and found that the area of peripheral nonperfusion was not correlated with the number of injections . the follow - up time of his study was longer than ours , but the sample size was smaller . singer et al32 evaluated 32 patients with retinal vein occlusion and refractory macular edema using sd - oct and uwfa . those patients were retreated with intravitreal injections with anti - vegf and with dexamethasone intravitreal implant . there was no significant difference in the number of anti - vegf ( 1.8 vs 1.6 , p=0.438 ) or dexamethasone implant ( 1.4 vs 1.6 , p=0.364 ) treatments given between the > 10% and 10% nonperfusion groups , nor was there any difference regarding the time to recurrence ( 3.4 vs 4.3 months , p=0.440 ) . in singer et al s series , there were 13 crvo patients with previously failed contiguous therapy with intravitreal ranibizumab and dexamethasone implant . in contrast , we enrolled previously untreated ( treatment - nave ) patients ; furthermore , the number of our cases was higher than in the abovementioned studies . we previously reported that peripheral retinal nonperfusion correlates significantly with intravitreal ranibizumab injections in patients with brvo and macular edema.33 rehak et al34 evaluated 22 crvo patients in a randomized clinical trial and suggested the selective laser photocoagulation of peripheral areas of nonperfusion may further improve the visual outcome and decrease the number of needed ranibizumab reinjections in crvo patients . there are only few published articles regarding the size of peripheral retinal nonperfusion using uwfa . we calculated the mean size of peripheral retinal nonperfusion for all enrolled patients , which was five disc areas , and then the patients were divided into two groups according to that cut - off point . however , the authors totally agree that this approach is not an established step due to lack of evidence . moreover , there are several problems in the interpretation of fluorescein angiograms . the grading of the ultrawide - field angiograms is not uniform because there are eyelash artifacts that influence the entire amount of interpretable fundus , and the graders can also change the clarity of the images that may cause intergrader variability . another drawback of our study design was that we performed uwfa for each patient at only one single time point . thus , we were not able to demonstrate potential dynamic angiographic changes in nonperfusion areas over the follow - up time . in conclusion , uwfa is a precise tool for the detection and quantification of retinal nonperfusion which correlated with the number of needed intravitreal ranibizumab injections in our patients with crvo . retinal nonperfusion could be considered as a prognostic factor for a more precise patient management .

purposeto evaluate the association between the size of peripheral retinal nonperfusion and the number of intravitreal ranibizumab injections in patients with treatment - nave central retinal vein occlusion ( crvo).methodsfifty - four patients with treatment - nave crvo and macular edema were included . each patient underwent a full ophthalmologic examination including optical coherence tomography imaging and ultrawide - field fluorescein angiography . monthly intravitreal ranibizumab injections were applied according to the recommendations of the german ophthalmologic society . two ophthalmologists quantified the areas of peripheral retinal nonperfusion ( group 1= less than five disc areas , group 2= more than five disc areas ) . correlation analyses between the size of nonperfusion with best - corrected visual acuity , central subfield thickness , and the number of intravitreal injections were performed.resultsbest-corrected visual acuity improved significantly after intravitreal injections ( p<0.001 , both groups ) . final central subfield thickness after treatment did not significantly differ between both groups ( p=0.92 , p=0.96 , respectively ) . mean number of injections in group 1 and group 2 was 4.122.73 and 9.323.84 , respectively ( p<0.001 ) . there was a significant positive correlation between areas of nonperfusion and the number of injections in each group . ( r=0.97 , p<0.001 ; r=0.94 , p<0.001 , respectively).conclusionperipheral retinal nonperfusion in patients with crvo correlates significantly with the number of needed intravitreal ranibizumab injections . ultrawide - field fluorescein angiography is a useful tool for detection of peripheral retinal ischemia , which may have direct implications in the diagnosis , follow - up , and treatment of these patients .

Introduction Methods Patient selection Image acquisition Image processing and analysis Data collection Statistical analysis Results Discussion Conclusion

small bowel obstruction ( sbo ) is one of the most feared complications after a gastric bypass . it can be chronic or acute and can range from a nuisance to the patient to a life - threatening emergency sbos related to adhesions occur after laparoscopic roux - en - y gastric bypass ( lgb ) , but ih formation , secondary to the reconstruction of the small intestine , is also of concern to bariatric surgeons . multiple defects in the mesentery of the bowel can occur and may lead to ih and , in an antecolic bypass , a defect is associated with the roux limb passing over the transverse colon . the reported incidence of sbo after lgb varies widely , perhaps because the technique used in the operation also varies widely , not only in placement of the roux limb , but regarding closure or nonclosure of the mesenteric defects . the disparity in reports leads to confusion regarding the true incidence and even the causes of sbo after lgb . in addition , the closure of mesenteric defects is questioned in a substantial body of literature . the objective of this study was to examine the incidence and characteristics of sbo after antecolic antegastric bypass with nonclosure of the mesenteric defect of the jejunojejunal ( jj ) anastomosis . following health insurance portability and accountability act guidelines , the author performed a retrospective chart review of a series of consecutive lgbs performed over a 3-year period and recorded in a prospectively maintained database . the follow up of the patients included office visits at 1 week , 1 month , 3 months , 6 months , and then , yearly . all procedures were performed by the same surgeon using the antecolic antegastric technique . in no case was the mesenteric defect closed . any patients presenting with signs and symptoms of an sbo were emergently taken to the operating room . the policy of our bariatric program has always been to explore aggressively any patient with suspicion of an sbo , whether based on physical examination , radiographic studies , or history . patients with an sbo were usually taken to the operating room from the emergency department . those who underwent revisional bariatric surgery or conversion to open operation during the primary surgery were excluded . revisional procedures were not included because adhesions may have formed from the previous bariatric surgery that could have confounded the results . all patients had given informed consent prior to surgery for their deidentified data to be used for study purposes . the gastric pouch was sized to 20 ml with an orogastric balloon with 4 to 5 staple loads with seamguard ( w. l. gore , flagstaff , arizona ) staple - line reinforcement ( slr ) and the echelon 60-mm stapler ( ethicon endo - surgery , cincinnati , ohio ) . the small bowel was divided 40 cm from the ligament of treitz , and the mesentery had minimal division . the only division of the mesentery was from the same staple load that divided the small bowel . the roux limb was measured to 100 to 120 cm , depending on the body mass index ( bmi ) of the patient . the gastrojejunal anastomosis was hand sewn in 2 layers with absorbable sutures over a 34 french bougie . the jejunojejunostomy was formed with a single firing of the same stapler , and the enteroenterostomy was closed with the stapler . in all cases , an intraoperative endoscopy was used to check the gastrojejunal anastomosis , and drains were used in all male patients because of higher technical difficulty and higher rates of mortality and morbidity ; in patients with a bmi over 50 , for the same reasons ; and in selected cases . there was one conversion to an open operation for an incomplete malrotation ; there was no mortality . the gastric pouch was sized to 20 ml with an orogastric balloon with 4 to 5 staple loads with seamguard ( w. l. gore , flagstaff , arizona ) staple - line reinforcement ( slr ) and the echelon 60-mm stapler ( ethicon endo - surgery , cincinnati , ohio ) . the small bowel was divided 40 cm from the ligament of treitz , and the mesentery had minimal division . the only division of the mesentery was from the same staple load that divided the small bowel . the roux limb was measured to 100 to 120 cm , depending on the body mass index ( bmi ) of the patient . the gastrojejunal anastomosis was hand sewn in 2 layers with absorbable sutures over a 34 french bougie . the jejunojejunostomy was formed with a single firing of the same stapler , and the enteroenterostomy was closed with the stapler . in all cases , an intraoperative endoscopy was used to check the gastrojejunal anastomosis , and drains were used in all male patients because of higher technical difficulty and higher rates of mortality and morbidity ; in patients with a bmi over 50 , for the same reasons ; and in selected cases . there was one conversion to an open operation for an incomplete malrotation ; there was no mortality . there were 249 primary lgbs performed in the study period ( january 1 , 2011 , through december 31 , 2013 ) . sbo in 4 cases was caused by ih ( incidence of 1.6% ) and by adhesions in 11 ( 73% ) ( figure 1 ) . in the cases with ih , the most common locations were from the jj to the roux limb ( n = 5 ) , followed by those forming from the jj to the abdominal wall ( n = 3 ) , and 1 each from the jj to the colon and from the jj to the common channel ( figure 2 ) . in all cases , the adhesions were treated with laparoscopic lysis . the average time to sbo was 8.1 months ( range , 121 ) from the initial surgery . sbo is common after lgb and can be easily managed or can just as easily lead to disaster if not recognized and treated promptly . the surgical literature has many articles that discuss ih formation , and an area of contention has been closure versus nonclosure of the mesenteric defects . most authors seem to agree that if a retrocolic gastric bypass is performed , the mesocolic defect must be closed , which can lead to a 0% rate of ih formation , although that number can climb as high as 15% . elms et al showed that , in almost 2 400 patients who underwent antecolic antegastric bypass , ih formed in only 1.1% , primarily at the mesenteric defect . in those cases the defects had all been closed . cho et al had a 0.2% ih formation rate in 1 400 patients who underwent antecolic antegastric bypasses with no mesenteric closure and no division of the mesentery . rodriguez et al also reported that minimal division of the mesentery could lead to a decrease in ih formation . they showed that , with closure of the defect but with wide opening of the mesentery , there was a 14.4% ih rate ; if the defect was closed and the mesentery was not widely opened , the ih rate dropped to 1.1% . abasbassi et al showed a lower ih formation rate with no division of the mesentery , but also no closure of the mesentery . this cumulative experience could indicate that a way to decrease ih formation is to perform a minimal division of the mesentery . obeid et al used mixed techniques in both retro- and antecolic bypasses , some with closure of defects , some without . in 679 patients with an antecolic roux limb , the majority of all the defects closed . in the retrocolic group , almost 4% of these had closure of the mesenteric defect , 8.4% had nonclosure ; 3.8% were in antecolic and 8.5% in retrocolic bypasses . this wide range of techniques makes an overall analysis of the literature difficult . in 2010 in jsls , hope and colleagues examined the incidence of ih formation after documented closure of mesenteric defects . permanent sutures were used to close these defects , but 15 of 18 patients in the series presented with open defects , causing ih . these outcomes indicate that closure of the defects may not be as permanent as surgeons would like to believe , and indeed this very situation was the catalyst for this paper . in our lgbs , we were closing all defects , but our ih formation rate seemed to be climbing . we noticed most of the mesenteric defects were open at reoperation , and so we switched to nonclosure . as demonstrated here , the published papers on the subject have reported a wide range of ih incidence , from 0% to 15.5% . another complicating factor may be that many of the papers published in this area are from surgical programs training fellows , and the association of postsurgical complications with performance of operations by fellows is not known . in this study , the author specifically examined the incidence and cause of sbo in antecolic antegastric bypasses . in no case was either the mesenteric or antecolic defect closed . the interesting finding was that , of an overall incidence of sbo of 6% , only 1.6% of the total was caused by ih formation , all at the jj mesenteric defect . this outcome compares favorably with the ih formation rate reported by other authors who did or did not close the defects . also , the incidence of sbo caused by adhesions is higher in the current study than in some others . elms et al reported that 47.6% of the sbos in their series were secondary to adhesions , mostly at the jj , compared with our rate of 73% . the opposing end of the adhesion had multiple locations , but the staple line of the jj was always at one end , perhaps because of the intense inflammatory reaction that it generates . a hand - sewn technique could be used to close the common enterotomy and might reduce the incidence of sbo slightly , but the cut end of the biliopancreatic limb still has an exposed staple line . the strength of this study is in the homogeneity of the technique used , as this was a single - surgeon series . however , its strength is also its chief weakness , as the results may not be reproducible by others . the number of sbos is also small enough to raise the possibility of a type ii error . the author lives in a geographically isolated part of the country , with no other surgeons who are willing to care for bariatric patients , and so it was more likely that all the sbos would be treated by the same bariatric surgeon . another limitation of the study is that it was a small series with a short follow - up . although only the years of 2011 through 2013 were examined , and the average time to sbo was 8.1 months , it is possible that the sbo rate will climb in these same patients as time passes , and some of the patients are likely to be lost to follow - up . another confounding factor is that an slr was used early in the series at the jj , but not in later operations . there may be an association of slr with adhesion formation , but the connection is not clear . the most common location of adhesions was at the jj , were slr was used , but about half of the sbos caused solely by adhesions to the jj occurred after the use of slr was abandoned , so there are probably not enough cases to tell if the use of slr increases sbo . the usual weaknesses of a retrospective study apply to this paper as they do to all other studies about ih formation . surgeons should remember that the mainstay of treatment of sbo after lgb is an operation . an isolated roux limb obstruction can be decompressed by vomiting , and the placement of an ngt involves risk of a perforation . the surgeon should move the patient quickly to the operating room once an sbo is diagnosed or even suspected , as bowel ischemia can lead to extensive bowel resection and nutritional debility or even death . these operations can often be performed laparoscopically , but laparotomy is also safe and effective . the incidence of sbo caused by ih with nonclosure of the mesenteric defect is similar to that in other series where the defect was closed . regardless of the cause of the sbo , operation remains the definitive treatment and should not be delayed in the gastric bypass patient .

background and objectives : there is a wide variation of reported incidence of small bowel obstruction ( sbo ) after laparoscopic roux - en - y gastric bypass ( lgb ) . there is also wide variation in technique , not only in placement of the roux limb , but also regarding closure or nonclosure of the mesenteric defects . the objective of this study was to examine the incidence and characteristics of sbo after antecolic antegastric bypass with nonclosure of the mesenteric defect of the jejunojejunal anastomosis.methods:this is a retrospective review of a series of consecutive lgbs over a 3-year period . all procedures were performed by the same surgeon using the same technique . in no case was the mesenteric defect closed . a prospectively maintained database was used for data collection . patients who returned with an sbo were the study group , and those who underwent revisional bariatric surgery or conversion to open operation were excluded.results:there were 249 primary lgbs performed during the study period ; 15 of the operations were followed by sbo , for an incidence of 6.0% . four cases were caused by an internal hernia ( ih ) , for an incidence of 1.6% , and 11 were caused by adhesions , which accounted for 73% of the sbos.conclusions:sbo after lgb is a relatively common complication . the incidence of sbo from ih with nonclosure of the mesenteric defect is similar to that in other series where the defect is closed . regardless of the cause of the sbo , operative treatment of the patient who has a gastric bypass remains the definitive standard and should not be delayed .

INTRODUCTION METHODS Surgical Technique RESULTS DISCUSSION CONCLUSIONS

the following section describes the treatment procedure for patients with deep infection following tka using modified static spacers . the original prosthesis was removed , followed by intensive irrigation and wide debridement of the infected soft tissue . a 36 fr - diameter straight thoracic catheter ( mallinckrodt medical , athlone , ireland ) and a steinmann pin measuring 3.0 mm in diameter and 22 cm in length vancomycin ( 2 g ) was then added to the gentamicin bone cement ( depuy , warsaw , in , usa ) . at the late liquid stage of the cement , the steinmann pin was inserted into the tube , and the prepared cement was delivered through the tube . during this procedure , the tube was removed from the cement rod using a surgical knife . finally , a cement rod measuring 9 mm in diameter and 22 cm in length was formed ( fig . an entry hole was created at the center of the distal femur and proximal tibia for the insertion of the cement rod . the rod was then inserted into the femur and tibia through this hole . during insertion , it was important to place the center of the cement rod on an imaginary joint line . the proximal medullary canal of the tibia was filled with antibiotic - impregnated cement up to the surface of the proximal tibia , while the surgical assistant maintained proper anatomic alignment and joint space . the space between the cement of the distal femur and the proximal tibia was filled with more antibiotic - impregnated cement . finally , the suprapatellar pouch and medial and lateral gutter space were filled with antibiotic - impregnated cement to reduce soft tissue adhesion ( fig . cylinder splint immobilization was required for three days after the operation , after which a fixed - angle knee brace was used and toe - touching ambulation was allowed until the reimplantation surgery . from april to november 2007 , the authors performed the static technique in four patients using the novel antibiotic - impregnated cement rod for the treatment of infected tka ( culture : staphylococcus in three cases and no bacteria in one case ) . follow - up laboratory studies , including erythrocyte sedimentation rate and c - reactive protein , culture study via knee aspiration and a frozen biopsy from the second - stage operation ( < 5 polymorpho - leukocytes / high power field ) were performed to confirm successful eradication of the infection . the second - stage reimplantation was performed when all the criteria for the validation of infection control were met ( fig . the second - stage reimplantation was performed using the rectus snipping approach , and more than 90 of flexion was obtained intra - operatively . in all four patients , no re - infection was evident after two and a half years of follow - up . the range of motion of the knee joints were respectively improved from 50 to 80 , 95 , and 100 , and the knee society scores were 70 , 86 , 65 , and 84 , respectively , in the last follow - up evaluation . fehring et al.8 ) emphasized the importance of resting the joint in septic joint conditions . others also reported that the static spacer technique provides more stability than the mobile spacer technique in patients with severe bone loss.5,8 ) the main advantage of this technique is the maintenance of a normally aligned lower limb during the interval period . this maintains knee stability in combination with early muscle strengthening exercises , including quadriceps setting exercise , and enables the patient to comfortably dress and manage him / herself during the intervening period . the cement rod and static spacer provide a stable gap between the femur and tibia , thereby minimizing soft tissue contracture and shortening of the lower limb . in addition , symmetric maintenance of the soft tissue of both medial and lateral gutters requires no additional soft tissue balancing at the second - stage reimplantation . the additional cost of a metal nail , however , and the difficulty in infection control due to biofilm formation around the metal nail can be troublesome . in addition , a metal nail can not be removed easily at the second stage reimplantation due to its hardness , and soft tissue adhesion of the femoral or tibial medullary canals . on the other hand , an antibiotic - impregnated cement rod enjoys advantages , such as inexpensive antibiotic delivery to the marrow spaces and easy removal by using a hercules cutter . in addition , a static spacer is anchored to the cement rod , which can prevent spacer migration and bone erosion . this feature is believed to generate less cement wear debris than with the conventional static spacer or mobile articulating spacer technique . during the conduct of the antibiotic - impregnated cement rod technique , antibiotic - impregnated cement was applied to the proximal tibia , distal femur , joint gap space , suprapatellar pouch , and both gutters in a stepwise manner . this technique enables easy removal of the cement and reduced soft tissue adhesion at second - stage reimplantation .

the two - stage exchange arthroplasty ( one- or two - stage ) is believed to be the gold standard for the management of infections following total knee arthroplasty . we herein report a novel two - stage exchange arthroplasty technique using an antibiotic - impregnated cement intramedullary nail , which can be easily prepared during surgery using a straight thoracic tube and a steinmann pin , and may provide additional stability to the knee to maintain normal mechanical axis . in addition , there is less pain between the period of prosthesis removal and subsequent reimplantation . less soft tissue contracture , less scar adhesion , easy removal of the cement intramedullary nail , and successful infection control are the advantages of this technique .

SURGICAL TECHNIQUE DISCUSSION

mental fatigue is common and can be a disabling long - time condition following a stroke . it has been estimated that about 3070% of stroke survivors complain of fatigue [ 17 ] . even for those with an almost recovered stroke and without neurological and neuropsychological impairments , the person who suffers from mental fatigue is able to perform mental effort just for short periods , and , notably , it will take longer than normal to regain energy after being exhausted . accompanying symptoms , such as irritability , sensitivity to stress , concentration difficulties , and emotional instability may further impair social interactions [ 811 ] . however , attention has been paid to poststroke fatigue during the last 10 years , and fatigue is now generally held as a separate phenomenon [ 46 , 1214 ] . few investigations have been carried out to evaluate fatigue and cognitive functions following a stroke . leegard reported that fatigue is frequent following a stroke but did not find any related impaired cognitive functions . van zandvoort and coauthors investigated lacunar infarct and reported frequent difficulties relating to fatigue and a decreased cognitive performance under more demanding conditions . with the intention to increase knowledge about mental fatigue and cognitive difficulties related to stroke , we examined well - rehabilitated stroke participants , who had suffered from long - term mental fatigue for at least one year prior to examination . the subjects were examined for subjective self - reporting of mental fatigue , depression , and anxiety symptoms . neuropsychological tests were aimed at evaluating information processing speed , attention , and working memory . twenty - four participants , having recovered from neurological symptoms , but suffering from pathological mental fatigue for at least one year following a stroke and 24 healthy controls were included in the study . the study persons were recruited from an advertisement in a local , daily newspaper or from the neurological clinic at the local university hospital and were later included in intervention studies . the stroke subjects should have been healthy and at work before the stroke meaning that they had no known diseases but hypertonia was present among some participants . the type of stroke was obtained from medical records and self - reports ( see table 1 ) . the control participants were recruited from the local community , with no history of brain injury , stroke , psychiatric or neurological disorder , and no drug abuse , and they were fully able to work . they performed cognitive tests focused on information processing speed , attention , and working memory . the self - assessment for mental fatigue is a multidimensional questionnaire containing 15 questions and is adapted from rdholm et al . . the self - reported questionnaire covers the most common symptoms occurring after brain injury , stroke , or other neurological disorders affecting the brain [ 11 , 17 ] . the self - assessment scale for mental fatigue and related items has been evaluated , and the 14 questions had adequate internal consistency with a cronbach 's alpha of 0.94 . the questions concern fatigue in general , lack of initiative , mental fatigue , mental recovery , concentration difficulties , memory problems , slowness of thinking , sensitivity to stress , increased tendency to become emotional , irritability , sensitivity to light and noise , and decreased or increased duration of sleep as well as 24-hour variations . this cprs scale is used here for self - assessment of depression and anxiety . the neuropsychological tests included digit symbol - coding from the wais - iii , measuring information processing speed , digit span from the wais - iii , measuring attention and working memory ; verbal fluency test , fas ; trail making test ( tmt ) a and b , measuring visual scanning , divided attention and motor speed . in order to evaluate higher demands such as dual tasks , a series of two new trail making tests was constructed with three and four factors , respectively . months were added in part c and both months and days of the week in chronological order in part d. in the latter , the order of letters and digits was switched . a new computer test was constructed in our department including a single and a complex subtest . the single test included speed of mouse click in four squares , located in each corner of a bigger square ( 6 6 cm ) on the computer screen and was performed in a clockwise order . a mouse click outside the square was recorded as a miss , and a new click was necessary to do in order to be able to go on with the test . the complex sub - test also included the same mouse clicking procedure , but at the same time , the subject was asked to count how many instances of a specific digit between zero and nine , randomly chosen , he / she could see . after the 30 seconds , the subject was asked to report how many of the specific digit he / she had seen . it was possible to measure the difference in speed between a single and a complex task , variability over time , and errors made in counting digits . in the complex test , it was necessary to attend to both the square and the digits . a comparison between groups was done by t - test and analysis of covariance ( ancova ) . the mann - whitney u test was used when analyzing separate items included in the self - assessment scales . pearson 's correlation and linear regression were used for analysis of connections between variables . spss 16.0 for windows the control group had significantly more years of education than the stroke group ( t - test , p = 0.001 ) , and age was almost significantly different when comparing the two groups ( p = 0.055 , table 1 ) . accordingly , ancova , controlling for the variance for education and age , was conducted for all the variables analysed . the only significant gender difference found was that the control females were faster in the digit symbol - coding test , and an ancova , also controlling for sex , was conducted for this variable . a significant difference was found between the groups for the total sum of scores for mental fatigue . the mean value for the stroke group was 18.4 with a 95% confidence interval of between 16.420.5 . the mean value for the control group was 4.0 , with a 95% confidence interval of between 2.95.0 ( see figure 1 ) . the total score for the cprs scale , taken from the depression and anxiety subscale , was also rated significantly higher in the stroke group compared to controls ( see table 2 , figure 1 ) . the significant effect for mental fatigue remained after adjustment for depression ( p < 0.001 ) . all the separate items in the self - assessment scale for mental fatigue were rated significantly higher for the stroke group compared to controls . the cprs gave the following findings : without taking into account overlapping items , the items relating to sadness , emotional involvement , pessimistic thoughts , and zest for life rated significantly higher for the stroke group ( adjusted for multiple comparisons , as shown in figure 2 ) . among the stroke subjects , 74% reported a clear 24-hour variation with morning most frequently reported to be the best time of the day and afternoon and evening the worst . the participants from the stroke group were significantly slower on the test measuring information processing speed , primarily digit symbol - coding , and also reading speed and number of mouse clicks in the computer test . the stroke group was also significantly slower and made more errors in tmt d , the most demanding of the tmt tests . they were also significantly slower on tmt b , and produced fewer words on the verbal fluency test . the result for the simple mouse click sub - test was found to be significantly faster for the controls compared with the stroke subjects . the computer test which placed a simultaneous demand on speed , attention , and working memory , resulted in a fairly good speed for the stroke subjects , with no detected difference in speed compared to the controls . on the contrary , the stroke subjects made significantly more errors compared to the controls ( table 2 , figure 3 ) . the cognitive tests with significant results ( p < 0.05 , see table 2 ) were included in a linear regression model , using the enter method . digit symbol - coding ( p = 0.004 ) and the scores for number of errors in the computer test ( p = 0.018 ) were significant predictors for mental fatigue , while the other scores were not significant predictors for mental fatigue . digit symbol - coding and number of errors in the computer test also correlated significantly to the mental fatigue sum of scores ( r = 0.59 and r = 0.46 ) . the total scores for cprs , depression , and anxiety were on a significantly higher level compared to controls . the madrs , which provides a separate scale , has the same format as the cprs self - assessment scale depression , except that the items are graded in a different way . in the cprs scale , depression , the highest level is three for each separate cprs item , while the levels for the alternatives in the madrs scale are double for each separate item , with the highest level being 6 . according to madrs , a score between 12 and 20 is regarded as mild depression and 21 and above indicates a probable true depression . the mean level of eight for the stroke subjects corresponds to 16 on madrs , indicating an overall mild level of depression . there was a highly significant difference between the groups on their self - assessment of mental fatigue , with a mean value of 18 reported for the almost recovered stroke group and a mean value of four for the control group . the self - assessment scale has no cut - off value for fatigue , but our experience shows that a value of 15 and above indicates a clear problem with mental fatigue [ 9 , 23 ] . the clear difference in 24-hour variation also showed the specific exhaustion mental fatigue subjects experience during the active time of the day . it should be noticed that stroke victims were included due to the presence of mental fatigue during one year or more following the stroke . the data we have presented in our study do not include an indication of the frequency of mental fatigue after different types of stroke . however , on an individual basis , according to madrs , eight participants were not depressed , seven had an indicated mild depression , and nine had a probable , true depression . among the controls , two participants scored on a mild depression level , while the remainder scored below this level . the total sum of scores on the depression scales may be deceptive if a person is complaining of concentration difficulties and fatigue , but does not have a depressed mood , nor a lack of interest in and enjoyment of daily activities . in this study , three items were overlapping between mental fatigue and depression , and these items were rated on a higher level than the corresponding specific items on the depression sub - scale ( see figure 2 ) . depression and mental fatigue can occur on their own , but they sometimes occur simultaneously , as shown in this study . accordingly , we suggest mental fatigue and depression to be independent phenomena following a stroke . this also conforms with the findings from other studies [ 46 , 12 , 13 ] . the distressing exhaustion along with the bad memory , concentration difficulties , and not being able to perform simultaneous tasks are the phenomena many subjectively complain of following a stroke . it is important to include cognitive tests in the examinations in order to better understand the difficulties connected to mental fatigue and for the purpose of recommending treatment strategies . in this study , physically well - recovered stroke subjects , who had no medical problems except long - term mental fatigue , also showed decreased information processing speed and made more errors in demanding cognitive tasks compared to the control subjects . processing speed is also fundamental and important when considering cognitive functions of a higher order . few studies have been carried out which cover fatigue and cognitive performance following a stroke . however , studies have shown the connection between fatigue and decreased cognitive performance under more demanding conditions , while no cognitive impairments were detected , despite frequent fatigue . with more demanding and sensitive tests , including processing speed and complex attention , it may be possible to detect cognitive impairments accompanying mental fatigue . this is important , as mental fatigue and cognitive deficits will be an obstacle to almost recovered stroke subjects who are on their way to a return to work and previous activities , as every - day life today is complex , with high demands being placed on simultaneous and rapid decisions . in conclusion , mental fatigue is disabling for many people following an almost recovered stroke , and this is suggested to be related to cognitive impairments , primarily information processing speed , and attention . mental fatigue should also be treated as a separate phenomenon and should be differentiated from , and not confused with , depression . today , no specific guidelines exist for the treatment of mental fatigue , and research is urgently needed for this common yet distressing symptom .

mental fatigue is for many a distressing and long - term problem after stroke . this mental fatigue will make it more difficult for the person to return to work and previous activities . the intention with this study is to investigate mental fatigue in relation to depression and cognitive functions . we examined 24 well - rehabilitated stroke subjects , who suffered from mental fatigue one year or more after a stroke , and 24 healthy controls . subjects were examined using self - assessment scales for mental fatigue , depression and anxiety , and cognitive tests . the results showed a highly increased rating for mental fatigue for the stroke group ( p < 0.001 ) . these participants also had a significantly higher rating on the depression ( p < 0.001 ) and anxiety ( p < 0.001 ) scales . furthermore , they had a slower information processing speed ( p < 0.001 ) and made more errors in a demanding attention and speed test ( p < 0.05 ) . among the cognitive tests , processing speed and errors made in an attention and speed test were significant predictors for mental fatigue . we suggest mental fatigue following a stroke to be related to cognitive impairments , primarily information processing speed . mental fatigue should also be treated as a separate phenomenon and should be differentiated from , and not confused with , depression , even if overlapping symptoms exist .

1. Background 2. Materials and Methods 3. Results 4. Discussion

cigarette smoke ( cs ) is associated with the development of inflammation - related diseases such as chronic obstructive pulmonary disease and vascular diseases , including atherosclerosis and stroke . several studies have revealed that cs is a major contributor to vascular diseases because it accelerates the development of atherosclerotic plaques . the relationship between cs and the increased incidence of atherosclerosis has been reported [ 57 ] , which may be a consequence of direct endothelial damage , increased proliferation of smooth muscle in atherosclerotic lesions , and/or decreased vasodilation . endothelial damage has also been suggested as the initial cause of development of vascular diseases . in a previous study , it was shown that inhibition of oxidative stress exerts protection in human endothelial cells , which could be an effective strategy in the treatment of vascular diseases . a number of studies support that reactive oxygen species ( ros ) causing oxidative stress may play an essential role in mediating endothelial cell death . oxidative stress is a major factor in vascular diseases such as hypertension , stroke , and atherosclerosis . korean red ginseng ( krg ) is a popular traditional herbal medicine that has been widely used to treat several diseases such as cancer and vascular diseases . recent research shows that ginseng may have therapeutic potential in the treatment of alzheimer 's disease , diabetes , cancer , and cardiovascular diseases , through its antioxidant , antithrombotic , antihyperlipidemic , and anticancer effects [ 1215 ] . in endothelial cells , krg simulates no production in vivo and in vitro , suggesting that krg has antihypertensive effects . krg also promotes angiogenesis through the activation of the signaling pathway , indicating that krg can be implicated in potential angiogenic therapies for improving tissue repair , wound healing , and cardiovascular diseases . in addition , our previous study suggested that krg exerts a cytoprotective effect through the induction of heme oxygenase ( ho)-1 expression , suggesting a possible therapeutic mechanism of krg in cardiovascular diseases . it is well known that chronic inflammation contributes to the pathogenesis of many human diseases such as atherosclerosis . accumulating evidence suggests that krg is involved in the regulation of inflammatory responses , suggesting an anti - inflammatory effect of krg . cyclooxygenase ( cox ) catalyzes the conversion of arachidonic acid to prostaglandins that play vital roles in multiple physiological and pathophysiological processes , including inflammation . in particular , cox-2 is normally undetectable in most tissues and is induced in response to numerous stimuli . vascular diseases may , in part , be caused by cox-2 upregulation at sites of inflammation and vascular injury . cox-2 plays an important role in inflammation , therefore , inhibition of cox-2 expression may participate in the treatment of inflammation - related diseases such as vascular diseases . the objective of our study was to investigate the vascular protective effect of krg in acrolein - stimulated human umbilical vein endothelial cells ( huvecs ) . therefore , we examined the involvement of cox-2 expression via p38 mitogen - activated protein kinase ( mapk ) , intracellular ros , and apoptosis in acrolein - stimulated huvecs . krg powder was obtained from the korea ginseng corporation ( daejeon , korea ) . m199 medium and fetal bovine serum krg powder was soaked in water ( 1:25 , w / w ) for 3 h , and boiled for 40 min . following centrifugation at 1,900 g for 60 min , supernatants of ginseng extract were further centrifuged at 10,000 g for 30 min and lyophilized . the general composition of the product offered by the korea ginseng corporation is as follows : moisture 36% , solid volume 64% , ash 2.5% , total fat 0.05% , total crude saponin 70 mg / g , and total ginsenosides 20 mg / g . huvecs were maintained in m199 medium and supplemented with 10% fetal bovine serum , 1% penicillin and streptomycin , 10 ng / ml human fibroblast growth factor , and 18 mu / ml heparin . huvecs were grown to 80% confluence , maintained with fresh medium described above , and subcultured every 2 or 3 d. the cells were used within nine passages during these experiments . we applied 20 or 40 g of the whole cell lysate proteins to each lane and analyzed them with western blotting . western blotting was performed using primary antibodies as follows : anti - cox-2 , p38 mapk , phopho - p38 , cyclic amp - responsive element - binding protein ( creb ) , phospho - creb ( cell signaling , danvers , ma , usa ) and anti - glyceraldehyde 3-phosphate dehydrogenase ( abfrontier , seoul , korea ) . horseradish - peroxidase - conjugated anti - igg antibodies were used as the secondary antibody to detect the above - mentioned protein bands by enhanced chemiluminescence westsave - up ( abfrontier ) . the rna pellets were washed in 70% ethanol , dried completely , and dissolved in diethylpyrocarbonate to inhibit rnase . total rna was quantified using a nd-100 spectrometer ( nanodrop technologies , wilmington , de , usa ) . polymerase chain reaction ( pcr ) was performed using the synthesized cdna as a template and using specific primers for cox-2 or -actin as a loading control . the primer sequence for human cox-2 was 5-gacagtccaccaacttacaat-3 ( forward ) and 5-catctctccatcaattatctgat-3 ( reverse ) . the amplified products were resolved by 1% agarose gel electrophoresis , stained with ethidium bromide , and photographed under ultraviolet light . huvecs were cultured in a glass culture chamber slide ( falcon plastics , london ontario , canada ) and processed for immunofluorescence analysis . the amount of prostaglandin ( pg)e2 in the culture medium was measured using the pge2 eia kit according to the manufacturer 's protocol ( cayman chemical company , ann arbor , mi , usa ) . samples as well as standards were applied to a 96-well plate , precoated with goat anti - mouse igg , and incubated with pge2 acetylcholinesterase tracer and pge2 antiserum . all the wells were emptied , rinsed five times , and incubated with ellman 's reagent for 60 min in the dark with gentle rocking to produce 5-thio-2-nitrobenzoic acid , which has a strong absorbance at 405 nm ; the plate was read at 405 nm in an enzyme - linked immunosorbent assay reader ( el 800 ; bio - tek , winooski , vt , usa ) . we calculated the results using the standard curve , which were expressed as picograms per milliliter . intracellular ros in acrolein - stimulated huvecs is analyzed using a fluorescent dye , 2,7-dichlorofluorescein diacetate ( dcf / da ) . in the presence of oxidants , after 18 h incubation with 25 m acrolein in the presence or absence of krg , cells were stained with 10 m dcf / da , and fluorescence was analyzed by a facs vantage flow cytometer ( becton dickinson , san jose , ca , usa ) and fluorescence microscopy ( eclipse 50i ; nikon , japan ) . to clarify whether krg - mediated inhibition of acrolein - induced cox-2 expression plays a significant role in cytoprotection against oxidative stress , acrolein - stimulated cells were pretreated with krg ( 1 mg / ml ) or untreated , and cell death was measured by in situ terminal transferase dutp nick end labeling ( tunel ) assay . to measure dna fragmentation , the commercially available in situ death detection kit ( roche diagnostics , mannheim , germany ) huvecs were cultured in a glass culture chamber slide and fixed for 30 min in 10% neutral buffered formalin solution at room temperature . a tunel assay system was used , according to the manufacturer 's instructions , for examination under a fluorescence microscope , with excitation at 488 nm and emission at 525 nm . fluorescein isothiocyanate ( fitc ) ( bd pharmingen , san diego , ca , usa ) and propidium iodide ( pi ) staining of necrotic and apoptotic cells . cells were washed in pbs , resuspended in 100 l binding buffer containing 5 l annexin v fitc and 1 g / ml pi , and incubated for 10 min at room temperature in the dark . positioning of quadrants on the annexin v / pi dot plots was performed as previously described . data were expressed as mean standard deviation . statistical analysis was performed using one - way analysis of variance ( graphpad prism version 4 ; graphpad software , san diego , ca , usa ) followed by bonferroni 's multiple comparison test . a previous study found that acrolein in cs induces cox-2 expression in human endothelial cells . krg inhibited acrolein - induced cox-2 protein expression in a concentration - dependent manner ( fig . after pretreatment of acrolein - stimulated cells with krg , the cells were fixed , and cox-2 localization in huvecs was observed by immunofluorescence staining with an anti - cox-2 antibody followed by a fluorescence - tagged secondary antibody . immunofluorescence analysis showed that acrolein - induced cox-2 protein levels were inhibited in huvecs after treatment with krg ( fig . the induction of cox-2 expression is known to be responsible for pge2 release in the culture medium of cells stimulated with acrolein . acrolein increased pge2 secretion , which was dramatically reduced by krg ( fig . 2 ) . this result indicates that krg leads to the reduction of cox-2 protein expression and subsequently pge2 biosynthesis in acrolein - stimulated huvecs . thus , we examined the effect of krg on ros production in acrolein - stimulated cells . the shift to the right of the curve due to increased fluorescence indicates an increase in the intracellular levels of ros . the results indicate that ros generation in cells treated with acrolein increased compared to untreated cells , whereas krg inhibited acrolein - induced ros generation ( fig . these results indicate that krg may play a role in the inhibition of cox-2 expression via reduction of acrolein - generated ros in acrolein - stimulated huvecs . thus , to determine the upstream signaling pathway involved in krg - mediated cox-2 inhibition , we measured the activation of p38 and creb by detecting increased phospho - p38 and phospho - creb levels in acrolein - stimulated cells and found that phosphorylation of p38 and creb was strongly reduced by krg in acrolein - stimulated cells ( fig . 4 ) . these results demonstrate the role of p38 and creb signaling in the inhibition of acrolein - mediated cox-2 induction . fluorescence - activated cell sorting showed that while the number of apoptotic cells increased following treatment with acrolein , pretreatment with krg reduced the number of apoptotic cells ( fig . to confirm this result , we evaluated the presence of dead cells by tunel staining , which is widely used in detecting dna fragmentations in situ . the tunel assay indicates cell death , including apoptosis , by detection of the appearance of intensely stained nuclei , which indicates incorporation of labeled dutp into the 3-end of fragmented dna derived from apoptotic nuclei . as illustrated in fig . 5b , acrolein treatment significantly increased the proportion of tunel - positive cells , which was restored by krg pretreatment . these results revealed that the vascular protective effect of krg is mediated by the inhibition of cox-2 expression in acrolein - stimulated huvecs . in this study , we explored the inhibition of an inflammatory mediator , cox-2 , by krg water extract in huvecs . we found that krg inhibited both mrna and the protein level of cox-2 and its cytoprotective effect in acrolein - stimulated huvecs . there is increasing evidence that ,-unsaturated aldehydes in cs , including acrolein and crotonaldehyde play an important pathophysiological role in vascular diseases such as atherosclerosis and alzheimer 's disease . exposure to ,-unsaturated aldehydes is critical to the inflammatory response via activation of the proinflammatory signaling pathway and redox - sensitive transcription factors . furthermore , ,-unsaturated aldehydes increase oxidative stress , which plays a crucial role in the pathogenesis of vascular diseases via direct injury to the endothelium . cox-2 , a key enzyme for prostaglandin biosynthesis , is an inducible enzyme that is rapidly induced during inflammatory reactions . numerous studies have reported the involvement of cs in vascular diseases through cox-2 and endothelial no synthase activity . chronic inflammation plays an important role in vascular diseases , therefore , cox-2 may participate in the development of inflammation - related diseases , including vascular diseases . ginseng has been used as a general tonic for > 2000 years in east asia , and it has become a famous herbal medicine for treatment of various diseases , including vascular disorders . krg has been reported to have effective pharmacological activities , including antioxidant , anticarcinogenic , and ameliorative effects on blood circulation . recently , the diverse effects of several constituents of krg , including ginsenoside , on endothelial cells have been extensively studied . hien et al demonstrated the anti - inflammatory and antiatherosclerotic activities of ginsenoside rg3 in human endothelial cells , with a decrease of cell adhesion molecules and proinflammatory cytokines . moreover , the cytoprotective effect of ginsenoside rb1 in endothelial cell damage mediated by oxidized low - density lipoprotein has been reported . several constituents of red ginseng have been reported to regulate proliferation and migration and to protect oxidative stress - mediated damage in human endothelial cells . there is evidence demonstrating the presence of major ginsenosides including rb1 and rg1 in krg water extract . thus , these components could also contribute to the diverse retinue of protective actions of krg . a previous study showed that the induction of ho-1 expression may exert protective effects in krg - treated human endothelial cells . however , there have been no reports revealing the mechanism underlying krg - inhibited cox-2 expression in acrolein , ,-unsaturated aldehydes in cs , stimulated huvecs . we have established that the major signaling pathway of cox-2 ( i.e. , p38 mapk creb ) and intracellular ros generation are involved in this inhibition of cox-2 expression in acrolein - stimulated huvecs by krg . as mentioned above therefore , the inhibition of cox-2 expression following krg water extract treatment may be associated with its strong protective effect in acrolein - stimulated huvecs . in conclusion , we propose that the krg water extract may exert a cytoprotective effect through the inhibition of cox-2 induction and that this reduction of cox-2 in acrolein - stimulated huvecs is mediated by the p38 mapk

cigarette smoke is considered a major risk factor for vascular diseases . there are many toxic compounds in cigarette smoke , including acrolein and other ,-unsaturated aldehydes , which are regarded as mediators of inflammation and vascular dysfunction . furthermore , recent studies have revealed that acrolein , an ,-unsaturated aldehyde in cigarette smoke , induces inflammatory mediator expression , which is known to be related to vascular diseases . in this study , we investigated whether korean red ginseng ( krg ) water extract suppressed acrolein - induced cyclooxygenase ( cox)-2 expression in human umbilical vein endothelial cells ( huvecs ) . acrolein - induced cox-2 expression was accompanied by increased levels of phosphorylated p38 in huvecs and krg inhibited cox-2 expression in huvecs . these results suggest that krg suppresses acrolein - induced cox-2 expression via inhibition of the p38 mitogen - activated protein kinase signaling pathway . in addition , krg exhibited an inhibitory effect on acrolein - induced apoptosis , as demonstrated by annexin v propidium iodide staining and terminal deoxynucleotidyl transferase - mediated dutp nick end - labeling assay . consistent with these results , krg may exert a vasculoprotective effect through inhibition of cox-2 expression in acrolein - stimulated human endothelial cells .

Introduction Materials and methods Results Discussion Conflicts of interest

4cl4-coumarate : coa ligasecas9crispr - associated protein 9crisprclustered regularly interspaced short palindromic repeatsctcondensed tanningrnaguide rnammillionpamprotospacer adjacent motifpol iiirna polymerase iiirnairna interferencernaseribonucleasesnpsingle nucleotide polymorphisms / g ligninsyringyl - to - guaiacyl lignin ratiotrnatransfer rna 4-coumarate : coa ligase crispr - associated protein 9 clustered regularly interspaced short palindromic repeats protospacer adjacent motif single nucleotide polymorphism syringyl - to - guaiacyl lignin ratio since the first report of its programmable gene editing potential , the crispr / cas9 technology is revolutionizing all facets of biology from medicine to agriculture ( jinek et al . , 2012 ) . with its efficiency and simplicity , crispr / cas9 has quickly displaced its predecessors ( e.g. , zinc finger and transcription activator - like effector nucleases ) as the method of choice for genome editing ( carroll , 2014 ) . for agricultural applications that have traditionally depended on gene - silencing for rna modification , crispr / cas9 is a game changer . the previous methods , such as antisense or rna interference ( rnai ) , leave much to be desired as the degree , specificity and stability of gene silencing are not always predictable . this necessitates screening and characterization of a large number of transgenic lines for the desired trait . by contrast , crispr / cas9 is guided to disrupt the reading frame thereby protein function of the target gene by dna editing . 2012 ) , and both monoallelic and biallelic mutations are reported ( bortesi and fischer , 2015 ) . with biallelic editing , null mutations are readily obtained in primary transformants , though with varying efficiencies ( brooks et al . , 2014 ; zhang et al . , 2014 ; zhou et al . , 2014 , 2015 ) , interestingly , the most efficient and consistent crispr / cas9 editing was reported in hybrid populus tremula x alba clone 717 - 1b4 ( 717 ) , with biallelic mutations detected in all independent transgenic lines examined ( zhou et al . , 2015 ) . for woody perennials such as forest trees , fruit / nut trees and woody ornamentals with highly heterologous genomes and long generation times , crispr / cas9 affords a facile means to accelerate genetic improvement . the first application of crispr / cas9 genome editing in populus targeted the 4-coumarate : coa ligase ( 4cl ) gene family involved in phenylpropanoid metabolism ( zhou et al . , 2015 ) . one of the genes , 4cl1 ( potri.001g036900 ) , has been extensively characterized for its involvement in lignin biosynthesis . down - regulation of 4cl1 or its orthologs leads to reduced lignin content and altered lignin structure in a number of species ( boerjan et al . , thus , crispr / cas9 editing of 4cl1 served as a proof - of - concept study , allowing an assessment of its efficacy in perturbing lignin biosynthesis relative to previous methods . we generated 36 independent transgenic lines , and amplicon - sequencing of randomly selected events confirmed biallelic mutations in all cases ( zhou et al . , 2015 ) . as frequently reported for crispr / cas9 genome editing , small indels , especially 1-bp modifications , were the predominant patterns and predicted to disrupt the reading frame . consistent with this prediction , lignin content was reduced by 23% with a concomitant decrease of s / g lignin ratio by 30% in all transgenic plants ( zhou et al . , 2015 ) . wood discoloration is a known side effect of lignin modification frequently observed in transgenic plants with suppressed lignin gene expression . however , the coloration patterns reported in those cases lack the uniformity we observed for the crispr / cas9 mutants . for example , antisense downregulation of 4cl1 in populus resulted in patchy wood discoloration in only 5 of 14 transgenic lines ( voelker et al . , wood discoloration patterns of transgenic populus with down - regulated expression of cinnamoyl - coa reductase were highly variable among vegetatively propagated plants ( van acker et al . , 2014 ) , presumably due to the unstable nature of sense- or antisense - mediated post - transcriptional gene silencing . rnai - mediated gene suppression is considered a more effective alternative , with improved trait stability ( li et al . , 2008 ) . for instance , rnai - silencing of 4-coumaroyl - coa 3-hydroxylase in transgenic populus resulted in lignin reductions among 9 independent events that ranged from no change to 1560% ( coleman et al . , similarly , rnai silencing of 4cl in pinus led to wood discoloration in only 2 of 12 viable transgenic lines ( wagner et al . , 2009 ) . by contrast , the homogeneity of wood discoloration both within and across independent crispr / cas9-edited populus mutant lines is consistent with a null 4cl1 , and resembles that of naturally occurring brown midrib mutants of maize and sorghum ( sattler et al . , 2010 ) . the results serve as testament to the superiority of crispr / cas9 over previous gene silencing methods . ( 2015 ) with permission . a second gene , 4cl2 ( potri.019g049500 ) that is phylogenetically distinct from lignin - associated 4cls ( chen et al . , 2014 ) . has long been suggested to participate in flavonoid biosynthesis , based solely on its preferential expression in epidermis and roots ( harding et al . , 2002 ) . crispr / cas9 mutation of 4cl2 was therefore expected to yield functional evidence to substantiate this role . as with the 4cl1 case , biallelic mutations were observed in all independent transgenic lines surveyed by amplicon - sequencing ( zhou et al . , the metabolic consequence was examined in roots where flavonoid - derived condensed tannins ( cts ) are known to accumulate to high levels ( 20% dry weight ) in the experimental poplar genotype ( chen et al . , 2014 ) . crispr / cas9 mutations of 4cl2 resulted in significantly reduced levels of condensed tannins in roots , providing reverse genetics evidence in support of class ii 4cl function in flavonoid biosynthesis . after our initial publication , successful crispr / cas9 editing was reported for populus tomentosa ( fan et al . , 2015 ) . that study targeted a phytoene desaturase ( pds ) gene for mutagenesis by multiplexing 4 grnas , and observed a 50% frequency of albino phenotypes . the lower mutation rates compared to our study may be attributed to gene redundancy and sequence heterozygosity that were not accounted for during grna design ( see discussion below ) . regardless , selected albino transgenic lines were sequence - confirmed to harbor biallelic mutations ( fan et al . , 2015 ) , providing additional support for the superior phenotypic uniformity of crispr / cas9 mutants . a major concern in genome editing is the specificity of the dna modification , as off - target cleavage due to non - specific crispr / cas9 activity may cause unintended mutations . this is especially true for plant genomes that are characterized by recurring episodes of whole - genome , segmental and tandem duplications . we assessed crispr / cas9 specificity in populus by analyzing its on - target versus off - target activities among duplicated genes . the grna for 4cl1 was designed to discriminate against the paralogous 4cl5 ( potri.003g188500 ) , with 3 mismatches in the target sequence and one mismatch in the protospacer adjacent motif ( pam ) ( zhou et al . , 2015 ) . by designing consensus primers flanking the target site , we were able to sequence both 4cl1 and 4cl5 amplicons simultaneously to assess the specificity of crispr / cas9 . a custom program , ageseq ( analysis of genome editing by sequencing ) , was developed to facilitate variant detection ( xue and tsai , 2015 ) . no off - target cleavage in the 4cl5 locus was detected in 4cl1-edited lines , suggesting that crispr / cas9 editing in populus is highly specific ( zhou et al . , 2015 ) . negative experiment , where mismatches between a 4cl5-grna , designed based on the p. trichocarpa reference genome ( phytozome v3 ) , and the corresponding sequence of p. tremula x alba 717 used for transformation abolished cleavage in all transgenic lines examined ( zhou et al . , 2015 ) . in this case , the mismatches were in the form of 2 single nucleotide polymorphisms ( snps ) , one per allele , located near or in the pam . these results , together with the highly heterologous nature of woody perennials , suggest that off - target cleavage of crispr / cas9 is likely to be low in these species . the ability of crispr / cas9 to discriminate single - base differences makes it a powerful tool to investigate functional redundancy of highly homologous genes that are otherwise difficult to discern using gene silencing approaches . for instance , 4cl1 is the most abundantly expressed isoform in populus xylem , constituting > 85% of 4cl transcripts , with 4cl5 being a distant second , based on rna - seq data ( swamy et al . , 2015 ) . however , the crispr / cas9 knockout of 4cl1 reduced lignin content only by 23% ( zhou et al . , 2015 ) . because the 4cl5 locus was unmodified , it is likely involved in the residual lignin biosynthesis of the 4cl1-knockout mutants . in the populus study that targeted pds , 4 grnas were designed based on one gene model ( potri.014g148700 ) without considering the genome duplicate ( potri.002g235200 ) that shares a high degree ( 93% ) of coding sequence identity . while the genome sequence of p. tomentosa is not yet available , analysis of the p. trichocarpa and p. tremula x alba orthologs revealed different degrees of sequence variation between the 2 duplicates in the grna target regions . these differences ( plus unknown allelic heterozygosity ) likely contributed to the variation in the reported editing efficiencies of different grnas ( fan et al . , 2015 ) . examples include designing multigene - targeting grnas based on conserved sequences of homologous genes ( brooks et al . , 2014 ; jacobs et al . , 2015 ) , or multiplexing individual grna cassettes ( brooks et al . , 2014 ; fan et al . , 2015 ; zhang et al . , 2014 a recent study hijacked the trna processing machinery for efficient processing of up to 8 , and potentially more , grnas from one single construct ( xie et al . , the grna is positioned downstream of trna as a polycistronic gene with repeating trna - grna units for multiplexing ( xie et al . , 2015 ) . because mature grnas are released following cleavage of the polycistronic transcript by the trna processing machinery , there is no sequence constraint at the 5 end of grnas ( xie et al . , 2015 ) . this greatly simplifies grna design , as any sequences upstream of pam ( ngg ) can be used ( e.g. , n20-ngg or other truncated lengths ( fu et al . , 2014 ) ) , unlike with the standard design where grna is transcribed directly from the pol iii u6 or u3 promoter using a specific transcription initiation sequence ( e.g. , gn19-ngg or an19-ngg , respectively ) ( wang et al . , 2008 ) . in some cases , grna stacking , either by re - transformation or via controlled crosses of primary transformants may be necessary in order to discern individual gene functions . as discussed above , numerous studies have exploited sequence polymorphisms for developing gene- or allele - specific grnas to achieve precision editing by crispr / cas9 . however , few studies have addressed the added complexity of sequence polymorphisms on crispr / cas9 genome editing , probably because the common plant models , such as arabidopsis , rice , soybean and tomato , are selfing species with homozygous genomes . we argue that sequence polymorphisms are of concern in genome editing of most flowering plants , especially woody perennials that are outcrossing in nature . this is supported by the high frequency of genic snps reported for trees , one per 60 bp in populus tremula ( ingvarsson , 2005 ) or one per 16 - 33 bp in eucalyptus depending on the species ( klheim et al . , 2009 ) . a recent population genomics study of 544 populus trichocarpa individuals identified 17.9 million ( m ) snps by whole - genome resequencing ( evans et al . , 2014 ) . using a similar approach with both resequencing and rna - seq data , we uncovered over 10 m snps from a single individual , p. tremula x alba clone 717 that is commonly used in populus transformation ( xue et al . , 2015 ) . given the high levels of heterozygosity in tree species , the need to consider sequence polymorphisms in genome editing experiments can not be over - emphasized . in fact , most published genome sequences are , by default , consensus sequences of their ( pseudo)haploid genomes . thus , while several web - based grna design programs are available for plants , such as crispr - plant ( xie et al . , 2014 ) . and crispr - p ( lei et al . , 2014 ) , their utility for outcrossing species is limited because the preloaded genome sequences lack biallelic ( or multiploid ) coverage . furthermore , it is often the case as in populus that the species or genotype chosen for whole genome sequencing ( e.g. , , p. trichocarpa nisqually-1 ) is different from that routinely used for genetic transformation ( e.g. , p. tremula x alba 717 ) . to facilitate crispr / cas9 genome editing in 717 , we constructed a custom 717 genome by substituting > 10 m sequence variants into the p. trichocarpa reference genome ( xue et al . , 2015 ) . we then assessed the impact of sequence polymorphisms on grna specificity for 1000 randomly selected genes . grnas were designed based on the haploid genome of ( 1 ) p. trichocarpa or ( 2 ) 717 , and then cross - checked for snps or indels in the 717 target sequences . from the first analysis , we found that more than 57% of the grnas designed based on the p. trichocarpa genome correspond to regions that harbor snps or indels in 717 ( xue et al . the rates were slightly lower but nevertheless high ( 42 - 44% ) in the second analysis when the custom 717 genome was used , owing to allelic variations ( zhou et al . , 2015 ) . together , these data suggest that standard grna designs may suffer from a high probability of failure in crispr / cas9 genome editing , due to the frequent occurrence of snps / indels in outcrossers . the 717 sequences and associated blast and gene model query tools are available via aspendb ( http://aspendb.uga.edu/s717 ) . tool is particularly useful for screening custom grnas or pcr primers against known snps / indels in the p. trichocarpa or the custom 717 genome ( xue et al . , 2015 ) . a set of pre - selected grnas with no known sequence variants for 38,509 populus genes is provided ( zhou et al . , 2015 ) . the modified , variant - sensitive pipeline for grna design , called aspen crispr designer , is also available at aspendb and can be applied to other species . for outcrossing species lacking a genomic variant database , several approaches can be applied to identify snps / indels in the gene(s ) of interest prior to grna selection . rna - seq data from the transformation genotype , when available , is an excellent source for high - confidence snp discovery . provided that the gene of interest is reasonably expressed ( e.g. , , with a high read coverage ) , manual inspection of candidate grnas against rna - seq alignments using programs such as the popular integrative genomics viewer ( http://www.broadinstitute.org/igv ) is usually effective to confirm their specificity . in the absence of deep - sequencing data , pcr primers ( with or without degeneracy ) can be designed to amplify the target gene coding region based on a closely - related reference genome , or on conserved amino acid sequences of orthologs as in conventional rt - pcr cloning ( hu et al . , 1998 ) . as pam ( ngg)the only sequence constraint in grna design occurs frequently , it is not necessary to obtain full - length gene sequences . however , the use of multiple primer pairs is recommended to increase the likelihood of capturing both alleles . direct sequencing of pcr products to identify sequence variants is an established practice in molecular ecology research , and several programs have been developed to decode overlapping sanger chromatograms ( dmitriev and rakitov , 2008 ) . for researchers with convenient access to high - throughput sequencing , sequence reads can then be processed by ageseq to search for potential variants in the target gene sequences ( xue and tsai , 2015 ) . ageseq is available as a standalone program or a galaxy - compatible tool ( https://toolshed.g2.bx.psu.edu ) to support web - based data analysis . it should be noted that pcr amplification of noncoding sequences can be tricky due to the lower degree of sequence conservation ( xue et al . , 2015 ) . thus , for crispr / cas9 applications that target promoter or other noncoding sequences ( e.g. , transcriptional activation or chromatin modification ( sander and joung , 2014 ) ) , resequencing of the transformation clone for unbiased variant calling , as discussed above for populus 717 ( xue et al . , 2015 ) , is recommended . the populus study extended the ever growing list of plant species successfully genome - edited by crispr / cas9 ( bortesi and fischer , 2015 ; kumar and jain , 2015 ) to woody perennials . woody perennials suffer as experimental systems due to the lengthiness of genetic transformation and plant characterization . many forest and horticultural tree species and genotypes are notoriously recalcitrant to tissue culture regeneration with low transformation efficiencies ( litz and padilla , 2012 ; busov et al . , 2005 ) . limitations associated with other established technologies , such as incomplete silencing , position effects or epigenetic modifications of transgenes ( matzke and matzke , 1998 ) , further impede progress in transgenic tree research . even for species with a facile transformation system , such as populus tremula x alba 717 , the common practice of generating multiple independent events for preliminary screening prior to in - depth characterization of selected lines is resource - demanding and time - consuming ( fig . 2 ) . the ability of crispr / cas9 to generate null mutations in primary transformants , and the promise of phenotypic consistency among independent biallelic mutants as demonstrated in the populus study make a compelling argument that it may soon become acceptable to characterize only a few transgenic lines with sequence - verified mutations . high - throughput methods for mapping genome editing patterns ( xue and tsai , 2015 ) and transgene ( e.g. , t - dna harboring cas9 , grna and selectable marker ) insertion sites ( kanizay et al . , 2015 ) are expected to facilitate this effort . as depicted in figure 2 , primary transformants with confirmed editing can be used directly for in - depth characterization when multiple independent events are available as biological replicates ( zhou et al . , 2015 ) . even when only a few mutation events are obtained ( for difficult - to - transform species or from suboptimal transformation trials ) , the ability to bypass rna - level screening and to obtain stable mutations still confers advantages over the gene - silencing approaches . crispr / cas9 thus offers the enticing prospect of streamlined transgenic characterization and expedited large - scale functional characterization . figure 2.schematic comparisons of experimental approaches involving gene silencing vs. crispr / cas9 genome editing . ( a ) for gene silencing by antisense , sense or rnai approaches , knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation . expression knowledge is not necessary for crispr / cas9 editing at the dna level , and vectors with a pol iii promoter ( e.g. , u6 or u3 ) for grna expression and a cas9 under control of a constitutive ( e.g. , 35s ) promoter are widely applicable . ( b ) following regeneration of putative transgenic plants , conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired ( or maximum ) levels of gene suppression . it is common to screen a large number of events ( 10 ) to identify a minimum of 3 for subsequent analysis . this process can be greatly simplified with crispr / cas9 mutants , as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis . ( c ) for species with a robust transformation system , multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . in case of difficult - to - transform species for which few biallelic events may be obtained , micropropagation is necessary to obtain biological replicates before in - depth characterization , as is typically done using gene silencing . ( a ) for gene silencing by antisense , sense or rnai approaches , knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation . expression knowledge is not necessary for crispr / cas9 editing at the dna level , and vectors with a pol iii promoter ( e.g. , u6 or u3 ) for grna expression and a cas9 under control of a constitutive ( e.g. , 35s ) promoter are widely applicable . ( b ) following regeneration of putative transgenic plants , conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired ( or maximum ) levels of gene suppression . it is common to screen a large number of events ( 10 ) to identify a minimum of 3 for subsequent analysis . this process can be greatly simplified with crispr / cas9 mutants , as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis . ( c ) for species with a robust transformation system , multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . in case of difficult - to - transform species for which few biallelic events may be obtained , micropropagation is necessary to obtain biological replicates before in - depth characterization , as is typically done using gene silencing . biallelic mutations of the target gene(s ) in conjunction with the hemizygous nature of transgene ( e.g. , t - dna ) integration in primary transformants ( t0 ) means that dna - edited but transgene - free progenies can be readily obtained from t1 segregants , as demonstrated in annual species ( zhang et al . trait stacking with early flowering induction is thus highly desirable , for example by over - expression of flowering locus t ( ft ) as demonstrated in prunus ( plum ) , populus and eucalyptus ( hoenicka et al . , 2014 ; another concern for outcrossing species is that the progenies will be different from the parents , which necessitates additional screening and selection . we note , however , that introgressing transgenic / mutant events into elite genotypes is an established practice in crop and tree breeding programs , and hence not unique to crispr / cas9-edited mutants . a well - known tree example is the loblolly pine ( pinus taeda ) clone 756 that harbors a naturally occurring null allele of cinnamoyl alcohol dehydrogenase involved in lignin biosynthesis , and that is extensively used as a parent in several breeding programs in the us ( gill et al . , 2003 ) . another example is early - flowering ft - plum that serves as a parent in the fasttrack breeding program to expedite development of improved commercial cultivars ( callahan et al . , various molecular and genomics tools are available to facilitate progeny selection in transgenic breeding programs . recently , virus - based systems have been explored for transient expression of genome - engineering reagents ( ali et al . however , such systems still utilize t - dna to express the repurposed viral genome components , and the resulting plants are not transgene - free without crossing . these methods also depend on agroinfiltration to deliver the vector into plant cells , which unfortunately is not applicable to most woody species with an open minor vein structure in their leaves ( gamalei , 1989 ) . clearly , development of innovative methods will be necessary for facile removal of t - dna from the genome - edited plants without crossing . regardless , null segregants of crop cultivars derived from genome editing are indistinguishable from naturally occurring or chemically - induced mutants , and may therefore be exempt from gmo regulation in countries that adhere to product - based regulations ( voytas and gao , 2014 ) . this is expected to significantly reduce the timeline and financial burden associated with developing new crop varieties , thereby encouraging adoption of crispr / cas9-based genome editing for the forest products , fruit / tree nuts , and woody ornamental industries . the research in the tsai laboratory discussed in this article was supported by the department of energy , office of biological and environmental research ( grant no . de - sc0008470 ) , the department of agriculture , national institute of food and agriculture ( grant no . 2015 - 67013 - 22812 ) , and by the georgia research alliance - hank haynes forest biotechnology endowment .

abstractthe crispr / cas9 technology is a welcome breakthrough for genome editing , owing to its precision , efficiency , versatility and ease of adoption . we recently reported the first application of crispr / cas9 for biallelic mutations in stably transformed populus , extending the species range of this powerful technology to woody perennials . an underappreciated obstacle in genome editing of outcrossing species is the frequent occurrence of sequence polymorphisms that can render crispr / cas9 unproductive . we discuss experimental evidence as well as genome - wide computational analysis to demonstrate the sensitivity of crispr / cas9 to allelic heterozygosity , and highlight tools and strategies that can help deal with such sequence polymorphisms . with its specificity , crispr / cas9 offers a less equivocal means than previous approaches for discerning functional redundancy of paralogous genes that are prevalent in plant genomes . continuing improvements of the crispr / cas9 system for multiplex genome engineering should facilitate these efforts . the paradigm shift brought about by crispr / cas9 promises to accelerate not only basic research but also applied crop improvement progress .

ABBREVIATIONS None HIGH-EFFICIENCY GENOME EDITING OF PRECISION EDITING BY CRISPR/CAS9 HARNESSING SEQUENCE POLYMORPHISMS OF OUTCROSSING SPECIES FOR EFFICIENT CRISPR/CAS9 EDITING CONCLUDING REMARKS AND FUTURE PROSPECTS DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST Funding

like co@fe3o4 , which recently underwent extensive study to elucidate both the oxidative stability of the core and the phase of the shell , fept@fe3o4 core shell nanoparticles can be used as building blocks to form nanocomposites with enhanced magnetic properties with the potential for novel applications . these include magnetic data storage , catalysis , and targeted drug delivery . in terms of nanomagnetics specifically , our hope is that the presence of an inert oxide shell may function to inhibit agglomeration of the cores upon annealing ; this is a necessary step in creating an ordered l10 fept bimetallic structure with a higher magnetic coercivity . the determination of the core composition is important for tailoring their synthesis in order to ultimately achieve the desired 50:50 fept alloy composition . a solution of core shell particles was drop - cast onto a 3 mm holey carbon copper grid . edx data were acquired using an fei osiris tem equipped with a high brightness schottky x - feg gun and a super - x edx system comprising four silicon drift detectors , each approximately 30 mm in area and arranged symmetrically around an optic axis to achieve a collection solid angle of 0.9 sr . edx data were collected in the form of spectrum images , in which a focused electron probe was scanned in a raster across a region of interest in the scanning tem ( stem ) . at each point in the scan , structural information was obtained from the electron scattering incident on a high angle annular dark field ( haadf ) detector , and simultaneously , an edx spectrum was obtained by collecting x - rays emitted from the local volume probed by the electron beam . the resulting edx spectrum image was a three - dimensional data set whose ( x , y ) axes correspond to the position of the probe and whose z axis corresponds to the energy of the detected x - ray . spectrum images were acquired with a probe current of approximately 0.7 na , an acceleration voltage of 200 kv , a spatial sampling of between 0.5 and 1 nm / pixel and 50100 ms / pixel dwell times . x - ray intensities were obtained by fitting a model of the edx spectra to the experimental data using weighted least - squares and atomic fractions were quantified from intensities using the cliff lorimer quantification . the edx model and the quantification were implemented in hyperspy and will be available in future releases of the software . figure 1a displays a haadf stem image obtained during the acquisition of a spectrum image enclosing a cluster of 13 co@fe3o4 nanoparticles . although the particle morphologies are seen to vary slightly from one particle to another , the majority of particles have a round core approximately 20 nm in diameter surrounded by a thin shell approximately 5 nm in thickness . the edx elemental maps for cobalt , iron , and oxygen ( figure 1b d ) , obtained by integration of the element s background - subtracted k - line x - ray peak , show that individual particles are comprised of a cobalt core surrounded by a shell composed of iron and oxygen , as expected . the largest particle in the lower left region of the map appears to have more iron in the core compared to the smaller particles . these conventional edx element maps show the location of the various elements , but the composition of the particle core , for example , can not be determined by elemental mapping due to the presence of the shell above and below the core in projection . elemental maps of ( b ) cobalt , ( c ) iron , and ( d ) oxygen display the location of the various elements with respect to the particle morphology ( scale bar = 50 nm , greyscale = x - ray counts ) . the edx spectrum image data of the co@fe3o4 nanoparticle cluster shown in figure 1a was subsequently processed using bss methods in hyperspy . first , the spectral dimension in the data set was binned by four from 5 ev / channel to 20 ev / channel in order to increase the number of counts per channel . next , we note that the binning step is necessary in order to optimize the accuracy of the variance stabilization channel . the first three principal components , pc#0 , pc#1 , and pc#2 , exhibited significantly greater variance than the remaining components ( figure 2a ) , which suggests that there are only three phases present in the sample . that being the case , those three pca components should be a linear combination of the spectra and distribution maps of those phases , but the mixing matrix is unknown . next , we compute numerically the first derivative of the pca spectral components in order to diminish the correlation caused by the edx background , and we use fastica to estimate the mixing matrix and compute the independent components ( ics ) ic#0 , ic#1 , and ic#2 ( figure 2b ) and their distribution maps ( figure 2c e ) from the pca results . component independence is a much more stringent property than uncorrelatedness imposed by pca . if we disregard the small copper peaks contained in all the independent components , likely originating from the copper support mesh , we see that ic#0 contains cobalt x - ray peaks , ic#1 iron and oxygen peaks , and ic#2 a carbon peak . the three ics appear to belong to the three phases present in the originally scanned area : the core , shell , and supporting film . this hypothesis is explored further in the next section . at this point , however , it is important to note that , unlike in the conventional edx mapping shown in figure 1 , no elements were selected prior to performing ica , and thus , the analysis is free of external bias , except for the choice of the number of components . result of bss by pca and ica of an edx si of a co@fe3o4 core ( b ) corresponding independent component spectra contain the expected x - ray lines for the elements present . independent component maps ( c e ) show that ( c ) ic#0 is concentrated in the nanoparticle cores , ( d ) ic#1 in the shells , and ( e ) ic#2 everywhere on the carbon supporting film ( scale = 50 nm , greyscale = normalized weighting ) . we now move on to the analysis of a second cluster of particles comprised of a bimetallic iron / platinum core surrounded by an iron oxide shell . a crystalline core surrounded by a polycrystalline oxide shell is observed in the representative high - resolution stem haadf image shown in figure 3 . the particle morphologies were found to have a mean core diameter of approximately 3.3 nm and mean shell thickness of approximately 1.7 nm ( figure 4a ) . also visible were pure iron oxide particles ( in the lower right - hand corner of figure 4a ) . ica of a cluster of bimetallic platinum / iron nanoparticle seeds coated by fe3o4 shells . ( b ) scree plot of the first 50 principal components showing the first three components lying above the noise . ( c e ) element maps of ( c ) platinum , ( d ) iron , and ( e ) oxygen . ( f h ) . the ic maps ( f ) ic#0 , ( g ) ic#1 , and ( h ) ic#2 and ( i ) the corresponding ic spectra . from the selected element maps ( figure 4c e ) , it is clear that , with the exception of the two particles in the lower right - hand corner , the particles are comprised of a platinum rich core surrounded by an iron oxide shell . however , it is not clear from the maps alone whether iron is present in the core . by conventional elemental mapping , one can not tell whether the particles contain a pure platinum core surrounded by an iron oxide shell or whether iron is alloyed with platinum to form a bimetallic core . we now address these questions by performing bss on the same edx spectrum image using the same procedure detailed before . once again , by inspecting the scree plot ( figure 4b ) , we find that the sample consists of three phases . the spatial distribution of ic#0 is concentrated in the particle cores , ic#1 in a shell around the cores , and ic#2 is approximately uniformly distributed over the map ( figure 4f h ) . if we again disregard the spurious copper peak , ic#0 contains iron and platinum x - ray peaks , ic#1 iron and oxygen peaks , and ic#2 a single carbon peak ( figure 4i ) . from this analysis it appears that the ica components represent the different phases present in the edx spectrum image ; ic#0 , ic#1 , and ic#2 genuinely represent the bimetallic fept cores , iron oxide shells and the carbon support , respectively . in order to verify the accuracy of the ica results , we evaluated whether ic#0 represented the true composition of the core by analyzing bare fept seed particle clusters obtained from the same chemical synthesis but extracted prior to the shell addition step . being made from the same synthesis , the composition of the bimetallic seed particles are expected to match to the composition of the bimetallic cores in the core shell particles analyzed in figure 4 . a total of 12 edx sis were acquired in order to capture and analyze multiple fept bimetallic seed clusters . the segmentation of one of the edx sis is shown in figure 5a , b . to accurately extract the intensity of the fe k and pt l peaks , a model composed of one gaussian per x - ray line and a background based on kramers and small expressions as developed elsewhere was used , as shown in green in figure 5c . the only free parameters of the model were the area of the gaussian and the height of the background , which was negligible . the mean reduced over the fit of all particles was 1.01 , indicating that the discrepancies between the model and the data are in accordance with the poisson noise variance . the intensities of fe k and pt l peaks in the fitted model were quantified using the cliff lorimer method . the obtained compositions are plotted for each particle in figure 5d along with the fitting error estimated from poisson statistics . the raw data were decomposed using pca on all fept seed data and the first three components were retained for noise reduction . the 103 individual fept bimetallic seeds analyzed were found to have a mean composition of 82.0 at . % pt , which lies well within one standard deviation of the average bimetallic seed composition . the data points in figure 5d are displayed in order of ascending particle size . as such , the calculated compositions on the right - hand side tend to have a smaller error bar on account of the signal noise ratio being higher for larger particles . the compositions of the larger seed particles are also closer to the mean composition and to the composition of ic#0 , confirming the homogeneity of the composition and the validity of the bss analysis . summary of the composition of 103 bare fept bimetallic nanoparticles extracted from a synthesis prior to the shell addition step . ( b ) segmentation of the edx spectrum image prior to quantification of each particle . ( c ) fitting of the edx spectrum from a single seed ( circled in ( b ) ) to a model spectrum to determine the fe k and pt l peak intensities . ( d ) particle seed compositions obtained by quantifying the fitted intensities from 103 different particles ( error bars = 1 standard deviation ) . a comparison of raw edx spectra extracted from fept bimetallic seed particles and from pure fe3o4 particles with ic#0 and ic#1 , respectively , is provided in figure 6 . in both cases , the ics were scaled by a constant to obtain a best fit to the raw spectra . despite the strong overall agreement in each case , the carbon peak difference in both cases is caused by the separation of carbon into a different component ( ic#2 ) . the difference in the shell cu peaks are not due to a compositional difference as the cu signal is spurious in origin . the iron oxide particle spectrum also contains small silicon and sulfur peaks which likely originate from residue on the carbon film . the difference in the pt m core peak may be due to the attenuation of pt m x - rays in the shell of the core shell nanoparticles and in nearby particles along the trajectory to the detector . the strong overall similarity between the raw and ic spectra provide direct evidence showing that the spectral components extracted by ica from the core shell spectrum image data are strongly representative of the buried core , surrounding shell , and carbon support compositions . comparison between raw edx spectra extracted from a fept bimetallic seed particle ( top ) and from an iron oxide particle ( bottom ) with ic#0 and ic#1 , respectively . the fept seed and fe3o4 x when analyzing beam - sensitive materials , the main limitation to the accuracy of the bss analysis method that we propose is the intensity of the edx signal achievable without inducing significant sample damage . in our case , despite the use of a high efficiency edx system , the number of collected x - rays is low . in order to avoid the artifacts that arise when using the variance - stabilizing transformation out of its domain of application , we have binned the data by four in the spectral dimension from 5 ev / channel to 20 ev / channel . given that the resolution of our edx detector is approximately 130 ev at mn k and that there were no overlapping x - ray lines , in our case , the increase in the number of counts per channel ( and hence in the accuracy of the analysis ) comes without significant resolution loss and , therefore , should not have any adverse effect in the analysis . a blind source separation method based on pca and ica has been applied to the analysis of edx spectrum images of core the analysis has accurately determined the number of phases in the analyzed volume ( core , shell , and supporting film ) as well as their spectra and distribution maps . we have confirmed the accuracy of the analysis by comparing the calculated spectra from the platinum iron core and the iron oxide shell to those obtained from these structures in isolation , and the excellent agreement suggests that bss , therefore , can be used to accurately analyze edx data . the use of ica on edx spectrum image data promises to be a powerful technique for extracting buried compositions at the nanoscale in a variety of materials , and further testing on the method s applicability to different systems is now being initiated .

the chemical composition of core shell nanoparticle clusters have been determined through principal component analysis ( pca ) and independent component analysis ( ica ) of an energy - dispersive x - ray ( edx ) spectrum image ( si ) acquired in a scanning transmission electron microscope ( stem ) . the method blindly decomposes the si into three components , which are found to accurately represent the isolated and unmixed x - ray signals originating from the supporting carbon film , the shell , and the bimetallic core . the composition of the latter is verified by and is in excellent agreement with the separate quantification of bare bimetallic seed nanoparticles .

Materials and Methods Results Discussion Conclusions

ionic calcium ( ca ) controls multiple cellular signaling processes in all eukaryotic cells , including proliferation , gene expression , and neurotransmitter release . ca - binding proteins such as calmodulin play a pivotal role in ca signal transmission and amplification . increases in the concentration of intracellular ca activate specific protein targets , among which the ca / calmodulin - dependent protein kinase ii ( camkii ) is a critical signal mediator . in response to an increase in intracellular ca , thr-286 of camkii the coupling of ca - calmodulin to one of the camkii subunits allows for the phosphorylation of an adjacent subunit at thr . calmodulin trapping , confers ca - calmodulin - independent kinase activity to the complex and thus prolongs the ca signal . thus , calmodulin trapping represents a molecular mechanism of memory , which is defined as the capacity to acquire , store ( consolidate ) , and retrieve ( evocate ) information . camkii is a major synaptic protein that is activated during the induction of long - term potentiation ( ltp ) by ca influx through n - methyl - d - aspartate ( nmda ) receptors . calmodulin trapping allows camkii to remain activated long after the initial ca signal has dissipated , suggesting that camkii is a memory molecule crucial for ltp . consistent with this notion , camkii - null mice present with impaired memory formation , and camkii is essential for genesis and maintenance of ltp in postsynaptic neurons . following presynaptic stimulation , camkii is activated in postsynaptic neurons , which creates a physiological imprint of the initial ca signal , and increases translocation of nmda receptors to the plasma membrane . because of its capacity to remain activated long after the initial pulse of ca signaling , camkii perpetuates ca effects and modulates gene expression and the epigenetic profile of postsynaptic neurons . camkii also participates in glucose - stimulated insulin secretion ( gsis ) , as multiple insulin secretagogues increase camkii activity . in the perfused rat pancreas , the dynamics of camkii activation correlate with the amplitude of gsis , and camkii activation is temporally associated with insulin secretion . camkii is essential for appropriate gsis and is involved in several steps of this process , including the synthesis of insulin granules , the modulation of cytoplasmic content of atp , and the activation of synapsin . importantly , camkii also regulates transcription factors central for - cell function such as creb and mafa . here , we investigated whether pancreatic - cells , like neurons , acquire and store the information contained in calcium pulses as a form of metabolic memory . indeed , we find that - cells retain memory of prior activation , and describe the molecular mechanism that contributes to this memory . min6 insulinoma cells or mouse or human pancreatic islets were distributed into three groups : control , pulse , and pulse + kn93 ( 10 m ) . initially , all groups were maintained in the same medium with 5.6 mm glucose for 24 h. after this acclimation period , the control group was exposed to 3-mm glucose for 24 h , while the pulse and pulse + kn93 ( 10 m ) groups were exposed to 30 mm of glucose for four 1-h periods intercalated with 7-hour periods of 3 mm glucose . kn93 ( a camkii inhibitor ) at 10 m was only present during the 30 mm glucose pulses . all groups were then maintained at 3 mm glucose for a 24-h consolidation period . insulin secretion of min6 cells and mouse pancreatic islets was conducted following standard procedures , and insulin was measured by ria . one hundred and fifty islets were handpicked under a light microscope and pre - incubated for 1 h in krbb ( krebs bicarbonate buffer ) containing 0.3 the medium was discarded and the islets incubated for an additional hour in 500-l krbb containing 2.8 or 16.8 mm glucose . subsequently , the supernatant was collected to evaluate insulin secretion by ria . for perifusion assays , 150 treated human islets were placed into a perifusion chamber ( millipore , billerica , ma , usa ) . a computer - controlled fast - performance hplc system allowed programmable rates of flow and concentrations of the appropriate solutions held in a 37 c water bath . islets were perfused for 80 min with krbb in the absence of glucose , followed by krbb with increasing concentrations of glucose ( glucose was ramped from 0 to 30 mm at 0.75 mm / min ) . at the end of each experiment , islets were tested for maximal insulin secretion by adding 30 mm kcl to the perifusate . samples were collected at 2 ml / min and insulin content was determined by using ria . statistical analyses were performed using student 's t - test or a two - way anova with a bonferroni 's posttest , as required . statistical significance was set at p < 0.05 . in order to investigate metabolic memory of insulin - producing - cells , we first had to develop a paradigm in which such memory was evident . as detailed below , we established that exposure of cultured min6 - cells to four 1-h pulses of high glucose , interspersed with 7-h intervals of low glucose and thus mimicking postprandial glucose spikes in vivo , were able to elicit robust metabolic memory . the detailed experimental paradigm is outlined in figure 1a . as shown in figure 1b , min6 - cells exposed to the glucose pulse regimen showed higher insulin secretion both when exposed to low ( 2.8 mm ) or high ( 16.8 mm ) glucose than - cells , which had been cultured continuously at low glucose . this increase in insulin secretion was accompanied by a significant rise in the levels of phosphorylated camkii , consistent with the notion that camkii might be a molecular mediator of this metabolic memory ( figure 1c and d ) . importantly , both the increases in basal and stimulated insulin secretion in the glucose pulse group were abolished by kn93 ( figure 1b ) , a specific camkii inhibitor . next , we evaluated the kinetics of the - cell response to acute glucose exposure , and observed that the pulse group secreted more insulin at basal ( time zero ) and stimulatory concentrations of glucose ( 16.7 mm ) between 5 and 60 min following stimulation ( figure 1e ) . this effect persisted even after glucose concentrations were returned to 2.8 mm . in parallel , the phosphorylation of camkii was higher in the glucose pulse group at both glucose concentrations , and remained elevated even after the glucose concentration was reduced to 2.8 mm ( figure 1f ) . thus , min6 - cells had acquired metabolic memory of prior exposure to glucose pulses , which they retained through a 24-h consolidation period . it was important to determine if metabolic memory is confined to min6 insulinoma cells , or if it is also a property of primary human - cells . therefore , we cultured human islets from multiple non - diabetic deceased organ donors ( see table 1 for donor information ) and exposed them to pulses of 30 mm glucose in the same paradigm as described above . pulse treatment increased insulin secretion at basal ( 2.8 mm ) and stimulatory ( 16.8 mm ) glucose concentrations ( figure 2a ) . as was the case in min6 - cells , the pulse group showed increased camkii phosphorylation at both basal and stimulatory glucose concentrations ( figure 2b ) . next , we determined the human islet response to a glucose challenge in the islet perifusion assay . islets from the glucose pulse group showed both increased first and second phase insulin secretion compared with islets from the control group ( figure 2c and d ) . as before , these effects were abolished by the camkii inhibitor kn93 ( figure 2a d ) . we also tested metabolic memory of - cells using isolated mouse pancreatic islets , with similar outcome ( extended data figure 1 ) . we also found , using live cell calcium imaging of mouse islets , that the increases in cytoplasmic calcium levels following acute glucose stimulation , representing the penultimate step in insulin secretion , are significantly higher in the pulse group compared with controls ( extended data figure 1c ) . to investigate the molecular mechanisms by which the glucose pulse paradigm induces memory in - cells , we evaluated the expression of essential mediators of the insulin secretory response . the glucose pulse paradigm induced expression of glucokinase ( gck ) , the main glucose sensor of - cells ( figure 3a ) , the voltage - gated ca channel ( cav1.2 ) ( figure 3b ) , snap25 , an essential component of the exocytotic machinery ( figure 3c ) , and mafa , a key transcription factor of the insulin gene and the mature - cell phenotype in general ( figure 3d ) . all the effects of the pulsed glucose paradigm on protein levels were reduced or abolished by treatment with kn93 during the high glucose exposure only . finally , we asked if metabolic memory of - cells is dependent on glucose metabolism itself , or if this memory can be produced by membrane depolarization alone . to this end , we treated human islets with pulses of 30 mm kcl , which causes membrane depolarization , opening of voltage - gated ca channels , and insulin secretion without a prior increase in intracellular atp levels . as shown in figure 3e , depolarization by potassium chloride during the acquisition period produced similar , if somewhat lower , increases in pcamkii and psynapsin levels 24 h after the last kcl pulse . thus , increases in intracellular calcium elicited by membrane depolarization are sufficient to activate camkii and produce metabolic memory . total camkii protein levels were not affected by any of the treatments used ( data not shown ) . glucose - stimulated insulin secretion is a complex process that translates glycolytic flux and elevated atp production to increased cytoplasmic ca levels and finally fusion of insulin granules with the plasma membrane . this process is accompanied by increased phosphorylation of camkii ( extended data figure 2 ) , a process now shown to be part of the establishment of metabolic memory in - cells . cerasi and colleagues had previously reported time - dependent potentiation of insulin secretion , in that exposure of rat islets to high glucose ( 27.7 mm ) up to 60 min prior to the test stimulation with glucose increased subsequent insulin secretion . however , these studies focused on analyzing acute effects of high glucose pre - treatment , and did not yet investigate the molecular mechanism underlying the phenomenon . here , we demonstrate that human and mouse pancreatic - cells are able to acquire , consolidate , and retrieve information , induced by high glucose exposure , and determined that this ability is dependent on camkii . in conclusion , we have shown here that , like neurons , human and mouse - cells are able to acquire , store , and retrieve information in a process similar to neuronal ltp . this process , presented schematically in figure 4 , is dependent on the activation of camkii , as is the case for neuronal memory . these findings provide further evidence that the similarity of neurons and - cells on the transcriptome and epigenome level is not accidental , but an important aspect of the biology of these embryologically distinct cell types . metabolic memory of - cells likely represents a useful evolutionary adaptation to variation in food availability . as periods of food abundance in nature can vary dramatically in length , metabolic memory of repeated carbohydrate loads in - cells ensures higher insulin secretion and thus more efficient uptake of excess glucose into skeletal muscle and adipose tissue for long - term storage , contributing to the adaptation to periods of starvation . given the pivotal role of altered insulin secretion in metabolic disorders , future work will need to establish to what extent metabolic memory of pancreatic - cells contributes to the pathophysiology of these diseases .

ca2+/calmodulin - dependent protein kinase ii ( camkii ) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators . therefore , we hypothesized that pancreatic - cells acquire and store the information contained in calcium pulses as a form of metabolic memory , just as neurons store cognitive information . to test this hypothesis , we developed a novel paradigm of pulsed exposure of - cells to intervals of high glucose , followed by a 24-h consolidation period to eliminate any acute metabolic effects . strikingly , - cells exposed to this high - glucose pulse paradigm exhibited significantly stronger insulin secretion . this metabolic memory was entirely dependent on camkii . metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and ca2 + -dependent vesicle secretion , and by elevated levels of the key - cell transcription factor mafa . in summary , like neurons , human and mouse - cells are able to acquire and retrieve information .

Introduction Materials and methods Results and discussion

health is not only related to the absence of the disease , therefore we need to conceptualize and operationalize what health is . increasingly , we have come to understand that information about functional status is needed in order to appreciate the full picture regarding the health of an individual or a population . an individual 's health fundamentally includes their capacity to carry out the full range of actions , activities and tasks required to fully engage in all areas of human life . the health state of a person can be described in terms of capacity to carry out a set of tasks or actions . in addition , the health state also includes changes in body functions and/or structures arising from a health condition . the impact of the health state on a person 's life can be understood by measuring performance of tasks and actions in the person 's real - life or actual environment . the full picture of the health experience can further be appreciated by taking into cognizance the value that people place on levels of functioning in given domains in association with a health condition . plainly , the concept of functional status is integral to health and its achievement . two individuals with identical diagnoses may have utterly different levels of functioning that determine their actual health status . without fsi , our picture of the health of an individual , or a population , is flawed and incomplete . fsi has , of course , long been collected in various ways and used clinically , especially in rehabilitative medicine ; physical , occupational and speech and language therapy ; and in nursing home and home care settings . fsi is essential for needs assessment as well as the development and monitoring of rehabilitative interventions to restore or maintain functions . it is also essential in this area of health care because the aim of therapy is to assist patients in maximizing their capacities to perform activities needed for their lives . although no one doubts that restoring functioning is restoring health ( the ultimate purpose of all forms of health care ) some clinicians , focusing exclusively on acute - care needs , do not see the need to collect or utilize fsi . in most countries with a sophisticated health administrative data collection and utilization infrastructure , a wide variety of information what is often missing is information that would link diagnosis and treatment with health outcomes that are fully meaningful to the patient 's life , namely information about the presence of decrements in capacity to carry out tasks and actions in areas of life as well as how these decrements play out in the person 's actual , real - life environment ( deyo and patrick , 1989 ; lubetkin et al . , 2003 ) . there is growing recognition that there is a gap in health administrative records : the failure to collect or disseminate fsi across all health care settings . unless fsi becomes an essential part of administrative records , the potential value of these data will be lost , not merely to clinicians , but to health administrators concerned about management and quality of care issues , health researchers , and public health agencies . this insight is clearly expressed in a report by the national committee on vital and health statistics ( ncvhs ) ( 2001 ) : without functional status information , the researchers , policymakers , and others who are already using administrative data have at best a rough idea of how people , individually and collectively , are doing and at worst they are making erroneous assumptions and decisions . the report outlines in some detail the benefits of routinely collecting fsi across the entire health care delivery system and throughout all care settings . fsi can serve management needs of all the stakeholders in the health care system clinicians , providers , payers , patients , and government regulatory bodies . this is true especially with respect to evaluating outcomes , comparing treatment modalities , and predicting and managing costs . this links directly to debates of modes of service provision , single or multiple payer , managed care , fee - for - service , or some hybrid mixture . the policy and research applications of fsi are evident for local health management and quality control , and in the broader arena of public health . policy decisions about priorities must be made at the level of individual clinics or hospitals , local or regional health care agencies , or at the level of government planning and budgeting . given the importance of getting the complete picture of health outcomes , fsi is an essential input into evidence - based policy decisionmaking . researchers in all areas of health and social policy , at all levels , need valid and reliable data about functional status in order to make informed decisions . for example , it is a matter of debate whether , as the world 's population lives longer and ages , they will be unhealthy and pose a greater burden on health systems . there is some evidence suggesting that elderly persons today are functioning at higher levels than before . without reliable information on levels of functioning , this debate would be unresolvable because it would not be possible to detect functional status , since the disease morbidity may not have changed very much . compression of morbidity occurs when disability or decrement in functioning is postponed more than longevity is extended , as for example with the effects of exercise or better eating habits . the direct test of compression ( or extension ) of morbidity depends on the effects of reduced health risks on cumulative lifetime disability ( fries , 1980 , 2001 ; vita et al . , , fsi is a crucial element for the description of health states and quantification of overall health status in individuals that can be aggregated to a summary measure of population health . at the who , the use of fsi is in this area , in particular , because this data ( collected by the world health survey now in the field in more than 70 countries ) feeds into ongoing endeavors to determine levels and distributions of health this survey would be inconceivable without information on health outcomes that describe health on multiple dimensions in terms of levels of functioning in a parsimonious set of domains . it is commonly known that the demographic trends toward an older population , at least in developed counties , will create unprecedented burdens on all age - sensitive social policies , such as social security and other pensions , retirement , unemployment , and long - term care . aging , according to a recent organization for economic cooperation and development ( oecd ) ( 2001 ) report , is the principal factor currently driving pension spending costs . since age - sensitive social programming constitutes between 40 and 60 percent of total public spending , the impact of aging is considerable . to comprehend the nature and magnitude of its social impact , those responsible for policies from transportation and housing to employment and taxation , will need reliable data on functional status and how it plays out in the lives of the aging population . for fsi to be available for this wide variety of uses , however , it must be routinely and consistently collected across the entire health care delivery system , preferably in some electronic format . nonetheless , before contemplating the systemwide changes required to collect fsi , a classification that provides a common language and framework to describe the universe of functioning and disability is required . in order to complement the classification scheme , a comprehensive coding system that creates consistent and comparable data across all settings of care and a method of routinely capturing and disseminating these data ( in a mode and manner consistent with social interests in preserving privacy ) linked to measurement tools for clinical and related encounters the foundation of a new structure for collecting fsi is , therefore , a standard classification and coding system that will make it feasible for fsi to be included in administrative data . as the ncvhs report stated : while the international classification of diseases ( icd ) has served us well for more than a century in characterizing diagnoses , it is now time to complement it with a parallel system for characterizing functional status . although the committee argued that more research , analysis , testing , and demonstration projects are required before final recommendations can be made , it concluded that : the concepts and conceptual framework of the icf have promise as a code set for reporting functional status information in administrative records and computerized medical records . in the committee 's view , the icf is the only existing classification system that could be used to code functional status across the age span . in this article , we want to briefly describe the extensive international developmental process that lead to the revision of the original international classification of impairments , disabilities and handicaps ( icidh ) ( world health organization , 1980 ) and produced the icf . we also want to describe the basic principles and structure of the icf , in particular , to show its value in the context of collecting fsi for administrative records . the primary mandate of who is the production and dissemination of reliable and timely information about the health of populations . who 's 1947 constitution requires that : each member shall provide statistical and epidemiological reports in a manner to be determined by the health assembly . countries have long reported causes of death or mortality statistics based on who 's ( 1992 ) international statistical classification of diseases and related health problems ( icd-10 ) . though useful for calculating life expectancy for different countries , however , who recognized that these data did not capture the overall health status of living populations . missing was information about non - fatal health outcomes , i.e. , functioning and disability across all areas of life . to meet this need , who ( 1980 ) issued a tool for the classification of the consequences of disease , namely the icidh . a considerable academic literature built up around clinical and other uses of the icidh , but much of this literature was critical of the underlying model of disability . responding to these critiques and an international call for an updated version , who launched a revision process in 1993 to address what many viewed as an urgent international need for a framework for measuring and reporting the health as functional status at both individual and population levels . over the next 10 years , who 's international collaborating centers and governmental and non - governmental organizations , including groups representing persons with disabilities , engaged in the systematic revision of the icidh . from an exhaustive literature search of existing classifications and assessment tools , the who revision team developed a 3,000-plus item pool of potential classification domain names for areas of human functioning at the body , person , and social levels . all efforts were made to ensure that the icidh-2 , as it was initially named , would be a suitable classification for all domains of functioning associated with both physical and mental health conditions . adopting the strategy of computer software development , alpha and beta drafts were prepared from 1996 forward . the original 1980 icidh had only been approved for field - trial purposes . in light of that , the who team felt for icidh-2 to have the necessary credibility and legitimacy to serve as the international standard language of health and functioning , that the revision process should include several years of field trials and other tests . the first phase of field trials concentrated on the cross - cultural and linguistic applicability of the model and classificatory structure and language of the icidh-2 . the intent of this phase of field trials was to establish the conceptual and functional equivalence of the items contained within the classification . stn et al.(1999a , b ; 2000 ) provide the rationale for the methodologies and presentation and analysis of the 15-country field trials . these results fed into further international collaboration in which the who team relied on a global network of who collaborating centers , non - governmental organizations , disability groups , and individual experts and key informants . the next revision phase began in 1999 when a series of expert drafting teams were assembled in geneva to produce the beta 2 draft . this draft was used for the second round of international field trials , these focusing on questions of reliability , utility , and feasibility of use . once the results of these tests were collected and analyzed , a pre - final draft was produced in early fall 2000 as a result of an intensive editing process grounded in the expert input being received from around the world . the icidh-2 , unlike its predecessor , was from the outset developed in multiple languages , primarily to identify and respond to cross - cultural and linguistic differences that might affect the usefulness of the classification . the collaborating centers and others provided constant input at this stage as the language and classification structures were redrafted and refined in multiple iterations . the draft was put on the internet for comment from a wide range of individuals , including both providers and consumers . after presentation before the executive board in december 2000 , the classification was put on the agenda of the fifty - fourth world health assembly and renamed the icf . the new title reflected the philosophy of moving beyond the consequence of disease approach and highlighted functioning as a component of health . in may 2001 , it was unanimously endorsed , member states were urged to use the icf in their research , surveillance and reporting as appropriate . with its approval , the icf became a member of the who family of international classifications . whereas icd-10 provides the codes for mortality and morbidity , icf provides the codes to describe the complete range of functional states that capture the complete experience of health . the icd-10 and icf are , therefore , complementary and who encourages users to utilize both together , wherever applicable . this will ensure a more meaningful and complete picture of the health of people or populations . soon after its official release , who 's director general , gro harlem bruntland , announced that the icf is who 's framework for measuring health and disability at both the individual and population levels . who has already implemented icf as the basis for its extensive world health survey program , demonstrating its use as a global and universal tool . to improve health , tools are needed to measure health , and in particular to measure the changes in health brought about by interventions . icf is the ruler with which we will take precise measurements of health and disability . ( brundtland , 2002 . ) from the public health perspective , the usefulness of icf goes beyond that of the measuring of population health and the effectiveness of internationally coordinated interventions funded by initiatives , such as the global fund to fight aids , tuberculosis and malaria . in addition , with the icf as their framework , countries will be able to identify social factors such as education , transportation , or housing , both as determinants of health , and social factors influenced by improvements in health . making these links will further support the relationship between health and economic development . in short , we have in the shape of a little red book , an extraordinarily versatile tool a swiss army knife for health ministries , researchers and decision - makers . ( brundtland , 2002 . ) undoubtedly the primary reason that icf can plausibly claim to be a universal tool for classifying states of functioning and disability is that the underlying model of the icf reflects our best understanding of the complex phenomena of functioning and disability in a manner that is , to the greatest extent possible , theory - neutral and therefore compatible with whichever theoretical account of how disability arises , at the individual and population levels , that evidence may confirm . it is the conceptual basis for the definition , measurement , and policy formulations for all aspects of disability . a paradigmatic shift in the thinking with regard to disability that is captured in the icf is the stress placed on health and levels of functioning . heretofore , disability has been construed as an all or none phenomenon : a distinct category to which an individual either belonged or not . the icf , on the other hand , presents disability as a continuum , relevant to the lives of all people to different degrees and at different times in their lives . disability is not something that happens only to a minority of humanity , it is a common ( indeed natural ) feature of the human condition . the icf is for all people , not just people traditionally referred to as disabled and isolated as a separate group . icf thus mainstreams the experience of disability and recognizes it as a universal human experience . by shifting the focus from cause to the full range of lived experiences , it places all health conditions on an equal footing , allowing them to be compared using a common metric the ruler of health and disability . from emphasizing people 's disabilities , and labeling people as disabled , we now focus on the level of health and functional capacity of all people . decrements in functioning may be the result of decrements in intrinsic capacity or problems with body functions or structures ; or they can result from features of the person 's physical , human - built or social environment that lead to problems in performance over and above decrements in capacity . very likely , decrements in functioning are the result of both processes . yet , the extent to which intrinsic decrements in capacity or environmental factors are the cause is not a matter that can be determined a priori . moreover , icf is grounded in the principle of universality , namely that functioning and disability are applicable to all people , irrespective of health condition , and in particular that disability or decrement in functioning at one or more levels is not the mark of a specific minority class of people , but is a feature of the human condition , which is , epidemiologically speaking , over the lifespan , a universal phenomena . in addition , icf is committed to the principle of parity , which states that the functional status is not determined by background etiology , and in particular by whether one has a physical rather than mental health condition . much time , effort , and international collaboration has gone into the development of the icf . it is no longer plausible to insist that the icf is a medical classification of people with disability , that it reduces all issues of functional status to underlying medical conditions , that it ignores the often salient role of the physical and social environment in the creation of restrictions of participation experienced by persons with functional problems . the revision process has produced a classification that has already stood up to rigorous tests of validity , reliability , and cross - cultural applicability . it is , as the ncvhs has concluded , the only existing classification system that could be used to code functional status across the age span . we now turn to the structure of icf as a classification system , in part to show why the committee has correctly assessed the value of the icf as a coding system for functional status , suitable for use in administrative records . the model that informs icf , portrays functioning and decrements in functioning , or disability , as a dynamic interaction between health conditions ( diseases , disorders , and injuries ) and contextual factors . contextual factors include environmental factors , that is , all aspects of the physical , human - built , social , and attitudinal environment that create the lived experience of functioning and disability . although not classified in icf , contextual factors also include personal factors such as sex , age , coping styles , social background , education , and overall behavior patterns that may influence how disability is experienced by the individual . the terms functioning and disability in the icf are the general or umbrella terms for , respectively , the positive and negatives aspects of the interaction between an individual ( with a health condition ) and that individual 's contextual factors ( environmental and personal factors ) . in the icf , health condition is the umbrella term for disease ( acute or chronic ) , disorder , injury or trauma . a health condition may also include other circumstances such as pregnancy , aging , stress , congenital anomaly , or genetic predisposition . the icf interactive model identifies three levels of human functioning : functioning at the level of body or body part , the whole person , and the whole person in their complete environment . these levels in turn define three aspects of functioning : body functions and structures , activities , and participation . disability similarly denotes a decrement in functioning at one or more of these levels that is , an impairment , activity limitation or participation restrictions . table 2 shows the complete list of all of the chapters found in the three classifications included in icf . under each of these chapters are second , third , and in some instances , fourth levels of categories , arranged in a hierarchical , tree - branch - stem - leaf , arrangement . this structure makes it possible for icf to be used as a classification tool for systematically describing situations of human functioning and problems with functioning . this complex information is organized by icf by means of a hierarchical coding system , thereby creating a common international language for functioning and disability . icf organizes information by means of several classifications distributed into two parts : ( 1 ) a component of functioning and disability that includes the component of the body with the body function and body structure classifications , and the component of activities and participation that includes all domains denoting aspects of functioning from an individual and social perspective organized into a single classification , and ( 2 ) a component of contextual factors that has a list of environmental factors organized from the individual 's most immediate to the wider environment . the classifications in the first part identify all of the domains of functioning from basic physiological functions and body structures , to simple and complex actions , tasks , social performances and relationships . the environmental factors list provides a tool for identifying those features of a person 's physical , human - built , social and attitudinal environment that , in interaction with the domains of functioning , constitute the complete lived experience of human functioning and disability . within the contextual factors part , besides the environmental factors , the icf recognizes the existence of personal factors as another component , but provides no classification of these . domains are a practical , meaningful set of related physiological functions , anatomical structures , actions , tasks , or areas of life . domains make up the different chapters and blocks within each component ( world health organization , 2001 ) . in order for these domains to capture descriptive information about functioning and disability in particular cases , they must be used in conjunction with qualifiers that record the presence and severity of a problem or decrement in functioning at the body , person , and social levels . for the classifications of body function and structure , the primary qualifier indicates the presence of an impairment and , on a five - point scale , the degree of the impairment of function or structure ( no impairment , mild , moderate , severe , and complete ) . in the case of the activity and participation list of domains , two essential qualifiers are provided to capture the full range of relevant information about disability . the performance qualifier is used to describe what an individual does in their current or actual environment , including whatever assistive devices or other accommodations the person may use to perform actions or tasks and whatever barriers and hindrances exist in the person 's actual environment . because the current environment always incorporates the overall social context , performance might be understood as involvement in the lived experience of disability . the capacity qualifier describes an individual 's inherent ability to execute a task or an action . operationally , this qualifier identifies the highest probable level of functioning of a person in a given functional domain at a given moment without any specific assistance . for measurement purposes , this level of capacity presumes a standardized assessment environment , namely one that reveals the inherent capacity of a person in a specific functional domain without any particular enhancements . the environmental factors list can be used to describe such a standard assessment environment in order to ensure that results across different studies can be compared by holding this environment constant . intuitively , the performance qualifier captures what people actually do in their lives , whereas the capacity qualifier identifies the person 's inherent capacity without explicit environmental facilitation ( or hindrance ) . who is developing a standard application guide that will operationalize the constructs of capacity and performance with respect to individual items that form the classification . table 3 shows how data can be organized to reflect the role of these two qualifiers used for the domains of the activity and participation classification . as a general matter of describing functioning and disability phenomena fully and accurately , the performance / capacity having access to both performance and capacity data enables icf users to determine the gap between capacity and performance . if capacity is less than performance , then the person 's actual or current environment has enabled him or her to perform better than what data about their capacity would predict : the environment has facilitated performance . on the other hand , if capacity is greater than performance , then some aspect of the environment is acting as a barrier to a level of performance that is feasible in a more suitable environment . icf thus makes it possible to measure the effect of a person 's environment on their decrement in functioning , given their health condition . the environmental factors classification can be used to identify specific features of the person 's actual environment that are barriers or facilitators in general for the person or with specific regard to each item of the person 's body functions , body structures or activities and participation that have been described . it can also be used , as previously stated , to describe specific testing environments where capacity has been measured . for its use as a classification of functional status relevant for health administrative records , icf provides a complete classification of both body and person level domains of functioning . given that it has been designed for a multiplicity of uses and users , there is far more in icf than could ever be plausibly integrated into a viable coding system for health records , although it remains the ultimate lexicon to which any coder , for clinical or research purposes , could turn . clearly , for implementation purposes in this area , a simplified checklist of items is needed . such a checklist was produced and used during the beta 1 and 2 field - testing phase in the revision process ( world health organization , 2001 ) . this checklist , which takes less than 30 minutes to complete , is currently being extensively tested in clinical studies in different disorders in order to study its feasibility , reliability , and concurrent validity with existing assessment instruments as part of a larger project to define core sets of items that may be used in rehabilitation settings for specific conditions and across several disorders ( stucki et al . , 2002 ) . the core sets of items with their corresponding scales could also be then converted into even shorter assessment instruments . the challenge for incorporating the icf into clinical and administrative records beyond a lexicon and framework lies in identifying this parsimonious set of domains or items that captures decrements in functioning across different health conditions and a smaller subset of domains or items that uniquely describe the decrements of functioning that typify a given health condition . in addition , the mapping of instruments ( that measure functioning and disability that are already in use ) onto icf categories will allow a ready crosswalk between measurements already being made at points of encounter to a common framework ( cieza et al . , 2002 ) . the use of the icf in larger population based surveys will also provide data on norms and distributions of health , functioning and disability that will enable the setting of appropriate thresholds for a multitude of purposes . table 4 maps the domains of the icf that have been included in different waves of the world health survey that ought to be included as a minimum or ideal set for information systems . these domains are also included on the icf checklist , which is designed to be a clinical tool . primary data collection strategies with regard to functional status , in a manner that is truly comparable , are in their infancy especially for international use and for use across population groups . further tools need to be developed , and standards and procedures established , so that these data become meaningful and usable . as a final issue , it must be mentioned that the icf has been conceived as a dynamic classification that will not only serve multiple users requiring different levels of detail , but also will continue to evolve with advancements in science . the classification is flexible in its structure such that it can be expanded in the level of detail ( for example , the fourth level ) for specific uses , or new codes added where gaps have been left in the numbering system . a set of operational rules will specify the procedure for this evidence - based expansion , adaptation , or revision of the classification . a common language for describing fsi is the key to ensuring comparability of data from a myriad of sources as well as in providing users with a tool for precise and accurate communication with each other . the recognition that a description of health and health - related outcomes must go beyond a narrow view of health restricted to the absence of disease , as well as that the definition of disability must move beyond the narrow impairment - based view that has been traditionally adopted to define a minority population , will go a long way in bridging the gap between health and disability data . it will also fill the void in existing health outcomes data while measuring the impact of interventions and monitoring them over time . health records must include functioning information in order to ensure a complete description of health states . the icf is the common language and framework that users will employ from now on . in the same way that all languages grow , evolve , and flourish over time and are adapted and modified to express new ideas , the icf will have a multitude of applications where it will be creatively used such that it continues to be a living classification . as with all new languages , it will be important to develop tools to learn this new language . toward this end , who is developing standardized application manuals and web - based learning courses that will use state - of - the art pedagogic methodology to assist end users . its usefulness in describing functional health status information will be one of the measures of its success .

a common framework for describing functional status information ( fsi ) in health records is needed in order to make this information comparable and of value . the world health organization 's ( who 's ) international classification of functioning , disability and health ( icf ) , which has been approved by all its member states , provides this common language and framework . the biopsychosocial model of functioning and disability embodied in the icf goes beyond disease and conceptualizes functioning from the individual 's body , person , and lived experience vantage points , thereby allowing for planning interventions targeted at the individual 's body , the individual as a whole or toward the environment . this framework then permits the evaluation of both the effectiveness and cost effectiveness of these different interventions in devising programs at the personal or societal level .

Introduction New Language of Health and Functioning Classification of Functional Status Conclusion

the centers for disease control and prevention ( cdc ) and united states preventive services task force ( uspstf ) recommend universal hiv screening for all pregnant women entering prenatal care [ 1 , 2 ] . this screening enables hiv - infected women and their infants to benefit from appropriate and timely interventions such as antiretroviral medications . when the recommended antiretroviral and obstetric interventions are used , a woman who knows of her hiv infection early in pregnancy now has a less than 2% chance of delivering an hiv - infected infant . without intervention , this risk is approximately 25% in the united states [ 36 ] . testing for hiv began in 1985 with the introduction of the enzyme immunoassay ( eia ) . in order to account for false positive results using screening tests in a low - prevalence population , confirmatory testing has been implemented using a western blot or immunofluorescence assay . in a low - prevalence population , the false positive rate using the eia is increased compared to a high - prevalence population , and the positive predictive value of any test will always depend on the prevalence of the condition in the population tested . in testing performed by the cdc , the eia positive predictive value ranges from 71 to 83% in populations with hiv prevalence from 0.5 to 1% . some investigators believe that the presence of alloantibodies account for the increased false positive rate associated with pregnancy , transfusions , transplantation , and autoimmune diseases . however , risk factors specific to pregnancy that account for this are poorly understood . conversely , a recent large retrospective study found that the false positive hiv eia rate was lower in pregnant women compared to nonpregnant individuals ( 0.14% versus 0.21% ) . our objective was to determine if any maternal characteristics correlated with false positive hiv eia testing at delivery . this was a review of all women who delivered at parkland memorial hospital in dallas , tex , from october 1 , 2005 through september 30 , 2008 . all women routinely received serum hiv testing at their initial prenatal visit and at time of presentation to labor and delivery for delivery via the opt - out approach with the abbott commander hiv ab hiv-1/hiv-2 ( rdna ) eia ( abbott laboratories , abbott park , ill ) . hiv test results performed at the time of delivery were analyzed in this study . a woman was considered hiv negative if eia testing was negative . positive test results were confirmed with the fluorognost immunofluorescent assay ( ifa ) hiv-1 ( sanochemia corp , stamford , conn , usa ) . women were considered to be falsely positive if eia results were positive and the ifa was negative . women delivered in this time period were identified through the obstetric operations database and linked to the pathology database for hiv , hepatitis b surface antigen ( hbsag ) , and rapid plasma reagin ( rpr ) results . maternal characteristics , including race , parity , age , singleton versus multiple gestation , and a diagnoses of diabetes or hypertension were obtained using the obstetrics operations database . laboratory results drawn 28 days prior to delivery through seven days after delivery were included . the study was approved by the institutional review board at the university of texas southwestern medical center . categorical data were reported as frequencies , and statistical significance was determined using analysis . statistical analyses were performed using sas , version 9.2 ( sas institute , cary , nc ) . a total of 47,794 women were identified who delivered during the study time frame . compared to hiv negative patients , false positive patients were more likely to be nulliparous ( 43% versus 31% , p < 0.001 ) and younger ( mean age 23.9 5.7 versus 26.2 5.9 , p < 0.001 ) . hiv positive patients were significantly older than false positive patients ( 27.4 versus 23.9 , p < 0.001 ) and hiv negative patients ( 27.4 versus 26.2 , p = 0.012 ) . of the 47,794 women , 47,391 ( 99% ) were hiv negative , 145 ( 0.3% ) had a false positive test , 172 ( 0.4% ) were true positives , and 86 ( 0.2% ) tested equivocal or were missing hiv results . the specificity of the hiv eia test was 99.7% with a positive predictive value of 54.3% . the sensitivity of the eia test was presumed to be near 100% , as the false negative rate using the eia has been previously demonstrated to be negligible in studies performed by the manufacturer . table 2 shows the prevalence of diabetes , hypertension , hepatitis b , and results of syphilis testing in the study population . hiv positive women were more likely to test positive for hepatitis b surface antigen ( 1% versus 0% , p = 0.002 ) and rpr ( 2% versus 0% , p = 0.02 ) . there was no significant difference in the prevalence of diabetes or hypertension between the three groups . there was also no significant difference in the rate of hbsag and rpr positivity between the hiv negative and false positive groups . when evaluating the interaction between nulliparity and age , there is a significant correlation between the two variables ; that is , parous patients are likely to be older and , therefore , nulliparity and age are not independent predictors of hiv false positivity . however , when the hiv testing groups ( positive , false positive , negative ) are stratified by parity , the comparison of age across the three groups remains significant only in nulliparous women ( table 3 , p = 0.0003 ) . the interaction between parity and age means that the difference in age between nulliparous and parous women is different depending on the hiv status . for example , in the hiv positive group the nulliparous and parous women are closer in age than in the false positive group . this was the first population - based study to evaluate risk factors for hiv false positivity in pregnant women presenting for delivery at a large urban institution . we found that younger and nulliparous women were more likely to have false positive testing using the hiv eia . the observed positive predictive value ( ppv ) of 54.3% was lower than the previously reported by the cdc ( ppv 7183% ) in a nonpregnant population , suggesting that pregnancy may be associated with a higher rate of false positivity . the low positive predictive value of the hiv eia in our study may have been impacted by the relatively low hiv prevalence ( 0.4% ) in our population . this information could be useful for counseling women who test positive for hiv at delivery and emphasizes the limitations of eia testing if used as a rapid test to determine the need for intrapartum and neonatal antiretroviral prophylaxis . the false positive rates of currently available rapid hiv tests have been reported to be much lower than has been found with eia testing in this study . evaluated the hiv false positive rate in over 900 pregnant women , most of whom were hispanic , using both the eia and a rapid point - of - care ( poct ) test , oraquick . they found that while there were no false positive tests with the oraquick , there were seven false positives using the eia ( ppv 100% versus 35.7% ) . in the mother - infant rapid intervention at delivery ( miriad ) study , jamieson et al . found that the ppv of the oraquick test was 90% while the ppv of the eia was 74% [ 12 , 13 ] . current recommendations by the cdc and the american college of obstetricians and gynecologists include rapid hiv screening for women presenting to labor and delivery with undocumented hiv status and for repeat hiv testing in the third trimester for women at high risk or who live in areas with high hiv prevalence [ 7 , 14 , 15 ] . a woman testing preliminarily positive for hiv in labor should be counseled that she may have hiv infection and that her neonate may be exposed , and immediate antiretroviral prophylaxis should be recommended without waiting for confirmatory test results . the results of our study may aid in counseling women if they test preliminarily positive for hiv using the eia , while awaiting confirmatory testing results . it remains to be determined whether the same risk factors for false positive hiv eia testing apply to the poct tests used in a real life setting . our study found that the positive predictive value of eia testing was only 54.3% and that younger nulliparous women were more likely to test falsely positive . importantly , our data may represent the real world performance of the eia testing in a large obstetric population - based setting . investigators also have noted a significant relationship between influenza vaccination and false positive screening for hiv antibodies [ 1618 ] . a potential reason for this cross reactivity is that there are similarities in homology between the transmembrane domains of the influenza envelope protein hemagglutinin and the hiv-1 envelope proteins . in our population , there were no significant differences in age or parity and influenza vaccination acceptance rates ( unpublished data ) . women in our study were from a single institution , with a predominantly hispanic background , and therefore the results may not be applicable to all populations . reasons for false positive hiv serology may vary depending on the geographical region . while we did find a significant association between young age , nulliparity , and hiv false positive testing , our study does not have the capability to identify a causal relationship or explain why this association may exist . in addition , our study can not address if these same risk factors for false positive testing apply to all other hiv tests . further studies are needed to confirm our findings , elucidate the biological mechanisms for increased hiv eia false positivity in young , nulliparous women , and compare this conventional testing method with contemporary rapid screening assays , including poct .

objective . to examine risk factors for false positive hiv enzyme immunoassay ( eia ) testing at delivery . study design . a review of pregnant women who delivered at parkland hospital between 2005 and 2008 was performed . patients routinely received serum hiv eia testing at delivery , with positive results confirmed through immunofluorescent testing . demographics , hiv , hepatitis b surface antigen ( hbsag ) , and rapid plasma reagin ( rpr ) results were obtained . statistical analyses included pearson 's chi - square and student 's t - test . results . of 47,794 patients , 47,391 ( 99% ) tested negative , 145 ( 0.3% ) falsely positive , 172 ( 0.4% ) positive , and 86 ( 0.2% ) equivocal or missing hiv results . the positive predictive value of eia was 54.3% . patients with false positive results were more likely nulliparous ( 43% versus 31% , p < 0.001 ) and younger ( 23.9 5.7 versus 26.2 5.9 years , p < 0.001 ) . hiv positive patients were older than false positive patients and more likely positive for hbsag and rpr . conclusion . false positive hiv testing at delivery using eia is associated with young maternal age and nulliparity in this population .

1. Introduction 2. Materials and Methods 3. Results 4. Comment

childhood obesity is a serious and growing public health problem that has arisen over the past three decades . the increasing occurrence of disorders such as type 2 diabetes during childhood is believed to be a consequence of this obesity epidemic . in addition to several behavioral and dietary risk factors , genetic predisposition is an important factor in the pathogenesis of childhood obesity . it is estimated that 4070% of adiposity variance can be explained by direct or indirect genetic factors . growth arrest - specific 6 ( gas6 ) , cloned in 1988 and characterized in 1993 , is a secreted vitamin k - dependent protein present in the human circulatory system [ 4 , 5 ] . initially , gas6 was shown to be upregulated in growth - arrested fibroblasts , suggesting that it plays a protective role in certain cellular stresses such as during apoptosis . gas6 expression is widespread in many tissues , including immune cells , endothelial cells , vascular smooth muscle cells , and adipocytes [ 79 ] . the protein is also a ligand for the tam ( tyro-3/axl / mer ) family tyrosine kinase receptor . the gas6/tam system has been implicated in cell survival and proliferation , cell adhesion and migration , hemostasis , and inflammatory cytokine release [ 4 , 10 ] . recently , the gas6/tam pathway was found to be involved in mediating adipocyte survival and proliferation in vitro [ 11 , 12 ] . experiments with mice fed a high - fat diet indicated that overexpression of gas6 might enhance body - fat accumulation , but blocking gas6 signaling using an axl antagonist could reduce body - fat mass and body weight . interestingly , transgenic animals that ectopically express the axl tyrosine kinase receptor also develop progressive obesity with elevated circulating proinflammatory cytokines and severe systemic insulin resistance . this protein - array study also revealed higher levels of axl mrna in subcutaneous adipose tissue of obese humans than their lean control counterparts had . this indicates that the axl receptor may be involved in the development of human obesity . in addition , some studies in transgenic mice indicate that gas6/axl signaling might recruit macrophages and other immune cells into the adipose tissue resulting in the production and secretion of proinflammatory mediators . this suggests that the gas6/axl signaling might play a role in the pathogenesis of obesity - associated systemic inflammation [ 8 , 16 , 17 ] . recent studies have indicated that systemic inflammation , a hallmark of childhood and adult obesity , is a pivotal mechanism linking obesity to insulin resistance and type 2 diabetes [ 1821 ] . although gas6 gene polymorphisms are reported to be associated with stroke , acute coronary syndrome , and type 2 diabetes [ 2224 ] , to our knowledge , both gas6 and axl gene polymorphisms associated with childhood obesity have not yet been identified . in order to address this issue , we conducted a community - based study to determine whether common variations in the gas6 and axl genes correlate with adiposity , systemic inflammation , insulin resistance among adolescents . the taipei children heart study - iii was an epidemiological survey that investigated obesity and cardiovascular disease risk factors among adolescents in taipei city during 2006 . the sampling method and results briefly , the survey included junior high school students in taipei city to collect a representative distribution of demographic , lifestyle , and biochemical characteristics to measure their risk for cardiovascular disease . those with autoimmune diseases , cancers , or active infection and those taking medications known to interfere with insulin or glucose metabolism were excluded . excluding any missing data , 727 adolescents ( 358 boys and 369 girls ) the institutional review board of the tri - service general hospital approved these studies and obtained informed consent from both parents and adolescents . all the participants completed a structured questionnaire detailing their gender , age , puberty development , and lifestyle characteristics ( including cigarette smoking , alcohol consumption , and physical activity ) . based on their responses , the subjects were divided into young adolescents with history of smoking , those without , and those who currently smoke . physical activity was divided into 5 levels based on amount of exercise per week : less than 1 h , 13 h , 35 h , 57 h , and over 7 h. survey questions concerning puberty onset included the development of the penis / testis and pubic hair for boys and development of breasts and pubic hair for girls . body weight was measured of barefoot students wearing light indoor clothing and was rounded to the nearest 0.1 kg . waist circumference ( wc ) was measured at the midway point between the inferior margin of the last rib and the crest of the ilium in a horizontal plane and was recorded to the nearest 0.1 cm . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . to reduce extraneous variation between subjects , we collected blood samples from the students after 12 h fasting and who had consumed their usual diet for the previous 3 days . children who had recently attended a holiday or family celebration were contacted for a blood sample several weeks after the event . plasma glucose concentrations were determined by the glucose oxidase method by using the beckman glucose analyzer ii ( beckman instruments , fullerton , ca ) . the intra- and interassay coefficients of variation ( cvs ) for glucose were 0.6% and 1.5% , respectively . plasma insulin was measured using a commercial immunoradiometric kit ( biosource europe , nivelles , belgium ) . serum levels for high - sensitivity c - reactive protein ( hscrp ) were measured using the tina - quant ( latex ) high - sensitivity assay ( roche , mannheim , germany ) . serum il-6 concentrations were determined using a human high - sensitivity enzyme - linked immunosorbent assay ( elisa ) ( innotest , besancon , france ) . serum tnf- was measured with the biotrak high - sensitivity human elisa kit from amersham biosciences ( buckinghamshire , uk ) . insulin resistance was assessed using the homeostasis model assessment ( homa ) , in which the homa of insulin resistance ( homa - ir ) = fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 . gas6 protein concentration was measured using a sandwich elisa and a polyclonal mouse anti - human gas6 antibody ( r&d systems , lille , france ) as a catcher and a biotinylated goat antiserum as a detector ( r&d systems ) , using previously described methods . the technique has been validated by food and drug administration guidelines in a previous study ( intra- and interassay cvs of 6.5% and 8.5% , resp . ; mean recovery on 10 patients of 97% ; lower limit of quantification 0.26 ng / ml ) . dna was isolated from buffy coats using the qiaamp dna blood kit and following the manufacturer 's instruction ( qiagen , valencia , ca , usa ) . the qualities of isolated genomic dnas were quantified using agarose gel electrophoresis , and the quantities were determined using a spectrophotometer . snps rs8191973 and rs8191974 in gas6 , as well as rs4802113 and rs2304232 in axl , were genotyped using a taqman assay with allele - specific probes on the abi prism 7900ht sequence detection system ( applied biosystems , foster city , ca , usa ) according to the standardized laboratory protocols . descriptive results of continuous variables were expressed as means sd . prior to statistical analysis , the normal distribution and homogeneity of the variables were evaluated using the levene test for quality of variance , and the variables were then given a base logarithmic transformation if necessary . the parameters homa - ir , triglycerides , hscrp , il-6 , and tnf- were analyzed and tested for significance using a log scale . the studied adolescents were categorized into subgroups based on their gas6 rs8191973 genotype ( cc , cg , and gg ) , gas6 rs8191974 genotype ( gg , ga , and aa ) , axl rs4802113 genotype ( cc , ct , and tt ) , and axl rs2304232 ( aa , ag , and gg ) with gender specification . the differences between anthropometric and biochemistry data across genotypes were analyzed using a general linear model after adjusting for age , tanner stages , smoking status , drinking status , and physical activity . chi - square tests were used to determine the genotype distributions for the hardy - weinberg equilibrium and to compare the proportions of abnormal anthropometric and biochemistry variables across genotypes . we tested different genetic inheritance models , and a recessive model was applied in the final analyses for gas6 and axl . to determine whether the gas6 and axl snps are predictors of obesity and obesity - associated complications , logistic regression analysis was used to calculate the odds ratio ( or ) and 95% confidence interval ( ci ) for each genotype and combined genotypes . a two - sided p - value of < 0.05 was considered statistically significant . all statistical analyses were performed using pasw statistics 18.0 software ( spss inc . , chicago , il , usa ) . in total , 727 adolescents ( 358 boys and 369 girls ) were included in this study . the mean age of all adolescents in this study was 13.3 years ( range , 1215 ) and was similar between boys and girls . in general , boys had higher bmi ( 22.3 4.0 versus 21.2 3.3 kg / m ) , wc ( 80.0 10.1 versus 75.1 8.1 cm ) , hscrp ( 0.9 1.3 versus 0.6 0.9 mg / l ) , and glucose levels ( 93.8 6.3 versus 91.5 6.5 mg / dl ) than the girls ( all p < 0.001 ) . however , girls had higher tanner stages ( 3.2 0.5 versus 3.0 0.4 ) than the boys ( p < 0.001 ) . there was no statistically significant difference in the ages , tnf- , il-6 , and insulin levels , and homa - ir between the boys and girls . the allele frequency for the least frequent allele in boys was 12.6 , 22.1 , 41.9 , and 29.7% , and 13.4 , 19.6 , 32.7 , and 33.5% in girls for the gas6 rs8191973 , gas6 rs8191974 , axl rs4802113 , and axl rs2304232 polymorphisms , respectively . there was no significant difference in allele or genotype distribution between boys and girls at the 4 polymorphisms . no statistically significant association between anthropometric characteristics , biochemistry data , and the gas6 rs8191973 genotypes was observed in the boys and girls ( table 1 ) . however , there were significantly different hscrp levels between gg , ga , and aa genotypes of gas6 rs8191974 in boys , regardless of their age , tanner stages , smoking status , drinking status , or physical activity ( table 2 ) . moreover , boys with the gg genotype of gas6 rs8191974 had significantly higher bmi , wc , and hscrp levels than those carrying the a allele . the gas6 rs819174 genotypes were not significantly associated with any anthropometric characteristics and biochemistry in girls . the p - values of all comparisons between anthropometric and biochemistry data across gas6 genotypes were presented in supplemental tables 1 and 2 available online at http://dx.doi.org/10.1155/2014/674069 . in addition , the association between circulating gas6 protein levels and gas6 polymorphisms was investigated . we found that the gas6 rs8191973 or rs8191974 genotypes were not significantly associated with circulating gas6 protein levels in both the sexes . there were significantly different hscrp levels between tt , tc , and cc genotypes of axl rs4802113 in boys , independent of their age , tanner stages , smoking status , drinking status , or physical activity ( table 3 ) . in addition , boys with the gg genotype of axl rs2304232 had significantly higher il-6 and insulin levels and increased homa - ir than those carrying the a allele ( table 4 ) . however , in girls , axl rs4802113 or rs2304232 polymorphisms were not significantly associated with any anthropometric characteristics or biochemistry ( tables 3 and 4 ) . the p - values of all comparisons between anthropometric and biochemistry data across axl genotypes were presented in supplemental tables 3 and 4 . boys with the gg genotype of gas6 rs8191973 were 1.87-fold more likely to have higher hscrp levels than the c allele carriers . even after adjusting for age , tanner stage , smoking status , drinking status , and physical activity , a significant relationship between the gg genotype of gas6 rs8191973 and higher hscrp levels was still observed in boys ( table 5 ) . moreover , boys with the gg genotype of gas6 rs8191974 exhibited a 1.40-fold greater risk for developing high bmi , a 1.58-fold greater risk for developing high wc , and a 2.68-fold greater risk to have higher il-6 levels than the a allele carriers . even after adjusting for all possible confounding factors including age , tanner stage , smoking / drinking status , and physical activity , the relationship between the gg genotype of gas6 rs8191974 , higher bmi / wc , and higher il-6 levels still remained significant in boys . however , the axl rs4802113 or rs2304232 polymorphisms showed no significant association with abnormal adiposity , inflammatory markers , and homa - ir in boys or girls ( see supplemental table 5 ) . logistic regression analyses were applied to evaluate whether the combination of the gas6 rs8191974 and rs8191973 polymorphisms is a stronger risk factor for high adiposity , inflammatory markers levels , and homa - ir than when alone . the combined effects of the 2 gas6 gene polymorphisms in the risk of high bmi , wc , il-6 , and hscrp levels are shown in figure 1 . after adjusting for the relevant confounding factors , we still observed that boys with the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited a 47-fold higher risk of high bmi , wc , il-6 , and hscrp levels than the individuals with both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 did ( or = 4.92 , 95% ci : 1.0823.6 , p = 0.018 ; or = 4.18 , 95% ci : 1.0522.5 , p = 0.016 ; or = 4.08 , 95% ci : 1.0628.56 , p = 0.015 ; or = 7.22 , 95% ci : 1.4635.72 , p = 0.010 , resp . ) . however , for girls , there was no statistically significant association between the combination of the gas6 rs8191974 and rs8191973 polymorphisms and abnormal variables . in addition , we evaluated the combined effect of the gas6 rs8191973 or rs8191974 marker with axl gene polymorphisms and its association with risk of high adiposity , inflammatory marker , and homa - ir . however , combinations of gas6 markers with axl gene polymorphisms were not found to be significantly associated with any abnormal variables in both boys and girls ( data not shown ) . in this study , a strong association between gas6 and axl polymorphisms with body adiposity , systemic inflammation , and insulin resistance was identified among boys . the risk of possessing high adiposity and inflammatory markers levels was higher in boys carrying the gg genotype with gas6 rs8191973 or rs8191974 than the noncarriers . moreover , the combination of both gas6 polymorphisms had an additive effect on the development of obesity and obesity - associated inflammation in boys . these data strongly suggest that gas6 and axl genes play a role in the pathogenesis of childhood obesity and its associated complications . gas6 was originally identified as a gene that is expressed in fibroblasts and increases with serum starvation and contact inhibition ; gas6 is also a potential growth factor for fibroblasts . maquoi and colleagues demonstrated that when fed with a high - fat diet , gas6-deficient mice had significantly less fat than their wild - type counterparts . the authors also reported the expression of gas6 and its 3 receptors ( tyro-3 , axl , mer ) in murine adipose tissues , thus suggesting that gas6 may act in an autocrine and/or paracrine manner to promote murine adipose tissue development . previous experiments in transgenic mice demonstrate that gas6 might also induce obesity - associated inflammation via recruiting immune cells into the adipose tissue to producing and secreting proinflammatory cytokines [ 8 , 16 , 17 ] . our recent clinical study found that circulating gas6 protein levels are associated with adiposity and inflammatory markers in overweight / obese adolescents . in this study , gas6 is further implicated as a candidate susceptibility gene for obesity and systemic inflammation . however , the association between gas6 genotypes and circulating gas6 protein levels was not observed among adolescents . we hypothesize that gas6 polymorphisms could affect the biology of the gas6 protein itself rather than its transcription or process rate , thus influencing adiposity regulation and systemic inflammation . to validate this , recent studies demonstrated that gas6/tam signaling is involved in releasing inflammatory cytokines ( such as il-6 and hscrp ) in diverse human diseases [ 23 , 31 , 32 ] . in addition , the gas6/tam signaling is also known to be involved in several inflammation - related systems , including maturation of immune cells , endothelial activation , and immunoregulation . our present study found that the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 are strongly associated with higher circulating il-6 and hscrp levels in boys . therefore , the gas6 polymorphisms presumably influence gas6/tam signaling and could further activate inflammatory reactions and result in releasing circulating il-6 and hscrp . however , the comprehensive effects of the gas6 polymorphisms in regulation of inflammatory cytokines still remain to be determined by more researches . interestingly , a previous study found that the a allele or the aa genotype of gas6 rs8191974 is associated with decreased risk of stroke . moreover , the a allele and the aa genotype are also thought to be related to a lower risk of developing acute coronary syndrome or type 2 diabetes , suggesting that this genotype exhibits protective activities against developing acute coronary syndrome and type 2 diabetes [ 23 , 24 ] . we also observed similar results in those with the a allele or aa genotype of gas6 rs8191974 . these subjects exhibited a lower risk for developing obesity and systemic inflammation than those with the gg genotypes . together , these findings suggest that the gas6 rs8191974 polymorphisms play an important role in the development of obesity and obesity - associated complications ( e.g. , type 2 diabetes , cerebrovascular , and cardiovascular diseases ) . the protective role of the aa genotype of gas6 rs8191974 against the developing childhood obesity and obesity - associated complications requires further study . the axl protein is a membrane - bound receptor that belongs to the tam family of receptor tyrosine kinases . axl exhibits the highest affinity for gas6 as compared to the other members of the tam family , whereas protein s predominantly binds mer and tyro-3 . gas6/axl signaling has been shown to be involved in the pathogenesis of obesity and systemic inflammation [ 1315 ] . however , our study demonstrates that axl polymorphisms are associated with systemic inflammation rather than childhood obesity . moreover , the combination of gas6 and axl gene polymorphisms is not significantly associated with any variables in adiposity among adolescents . our findings indicated that axl gene polymorphisms might not play a significant role in childhood obesity . recently , scroyen and colleagues have published similar findings indicating that deficiency in a single axl receptor did not significantly affect adipogenesis or adipose tissue development in mice . this is because an axl deficiency can be partially compensated by other tam family members ( tyro-3 and mer ) via gas6 interaction . further studies are needed to investigate the effect of tyro-3 and mer receptors on the development of childhood obesity . in addition , our present study also indicates that gender disparity exists regarding the effects of the gas6 polymorphisms on anthropometric characteristics and inflammatory markers . we found no significant difference in genotype frequencies between boys and girls ; however , the effects of the gas6 polymorphisms , individually or combined , only manifest in boys . the gas6 gene contains an estrogen response element in its promoter and is upregulated by estrogen via an activated estrogen receptor in mammary epithelial cells . therefore , we hypothesized that the gender - specific effect of the gas6 polymorphisms on childhood obesity might be due to a disparity in sex hormone distributions . this has been previously reported to be associated with gas6 expression and body composition [ 40 , 41 ] . despite these results , our study does have certain limitations . first , this was a cross - sectional study , as such we might not be able to assess gas6 polymorphisms on weight dynamics and the development of obesity - associated complications throughout life . second , because of the limitations of our questionnaire , we were not able to comprehensively estimate every adolescent 's daily intake . the impact of dietary energy intake on genetic susceptibility also requires further investigation to better understand any confounding effect . in conclusion , we indicate an association between the gas6 and axl polymorphisms with adiposity , circulating inflammatory markers , and insulin resistance of adolescents , especially in boys . moreover , the gg genotype of gas6 rs8191973 or rs8191974 strongly correlates with susceptibility to develop obesity and systemic inflammation in boys . nonetheless , these results together with those from studies in cellular and animal models encourage the study of the gas6/tam system in childhood obesity and its potential complications and further support the hypothesis that modulation of gas6 activity may indeed provide an important intervention point for future therapies .

the present study was designed to explore the effects of gas6 and axl gene polymorphisms on adiposity , systemic inflammation , and insulin resistance in adolescents . after multistage sampling from the data of the taipei children heart study - iii , we collected 358 boys and 369 girls with an average age of 13.3 years . we genotyped the adolescents ' gas6 rs8191973 , gas6 rs8191974 , axl rs4802113 , and axl rs2304232 polymorphisms . significantly higher body mass index ( bmi ) , waist circumference ( wc ) , and hscrp levels were found in boys with the gg genotype of gas6 rs8191974 than a allele carriers ; higher il-6 and insulin levels and increased homa - ir were found in boys with the gg genotype of axl rs2304232 than the a allele carriers . there was a significant difference in hscrp levels of boys with the tt , tc , and cc genotypes of axl rs4802113 . boys with both the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited higher bmi , wc , il-6 , and hscrp levels than the boys carrying both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 . in conclusion , gas6 and axl polymorphisms are associated with adiposity , systemic inflammation , and insulin resistance in adolescents , especially in boys .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

infertility is one of the crucial and critical events in sex life which engages 1015% of couples all over the world , and as one of the most distressful life experiences , exposes couples to social and psychological problems . couples , who are faced with this critical situation , are prone to depression , anxiety , loss of self - esteem and dissatisfaction with their sex life more than others . however , the intensity of psychological problems resulting from cultural - social circumstances varies in different societies , to the point that the frequency of anxiety in infertile couples has been reported in a wide range from 48% to 96% . although , the advances in assisted reproductive techniques have opened new doors for infertile couples , but studies have shown that these treatments are accompanied with stress , anxiety and depression . but the intensity of these disorders could be defined by the cause of the infertility . evaluating the related factors of depression and anxiety among iranian infertile couples showed that women who were infertile themselves showed higher levels of anxiety and depression than women with male factor infertility . however , in societies that define women as the main source of fertility , starting assisted reproductive treatments with limited successful outcomes could be harmful to these women 's mental health . utilization of assisted reproductive techniques requires processes such as daily injection to stimulate ovulation , vaginal ultrasound and painful processes such as oocyte aspiration which are all too expensive and alongside with the fear of failure could become a harmful condition . although , fertile women , who start assisted reproductive treatments due to their husband 's infertility have natural biological conditions to get pregnant , but they have to tolerate these critical processes due to male factor infertility . also , women are under more cultural and social pressures . these conditions could affect the vulnerability of fertile women during assisted reproductive treatment ; therefore the aim of this study was to evaluate the effect of assisted reproductive treatments on fertile women 's mental health . thus , the prospective study conducted on 70 fertile women who referred to receive assisted reproduction treatments ( in vitro fertilization / intracytoplasmic sperm injection ) due to male infertility . subjects had referred to the fertility and infertility center of esfahan from september 2013 to march 2014 . the number of samples was calculated based on 80% test power and 95% confidence level . inclusion criteria were primary infertility , not having any corporal and mental illnesses , not being a drug addict according to individual 's report , not having a history of mental illnesses , and not experiencing severe distress over 1-month prior to the treatment based on holmes - rahe scale . exclusion criteria included experiencing distressful conditions during the study , discontinuation of the treatment and the cancellation of egg harvesting . the study was approved by the medical ethics committee of the isfahan university of medical sciences . mental health evaluation tool was the valid 28-questioned general health questionnaire that measures mental health on likert scale ( 03 ) in hypochondriasis , anxiety , social impairment , and depression dimensions . in this method gaining the score of 5 in each subscale and 23 in all dimensions together was defined as having a mental disorder . sampling was conducted through simple sampling ; that is , all persons , who referred to fertility and infertility center of esfahan for treatment and to start the process protocol due to male factor infertility were interviewed . then by referring to their medical records and conducting holmes - rahe scale , their eligibility for the study was assessed . after being considered eligible for entering the study , their demographical features including age , educational level of couples , economic condition , and the duration of their infertility were recorded . ovulation was stimulated through the long protocol in all the participants . during the treatment period , ovarian reaction conditions and number of injections employed for ovarian stimulation were followed - up . 3 h after the pickup process , and under appropriate circumstances , the questionnaire for general health was completed again . if the circumstances were inappropriate filling of the questionnaire was delayed for 2 h. research data were analyzed using spss software version 16 [ spss inc . : chicago , il , usa ] , statistical paired t - tests and multi - variable linear regression tests . seven qualified subjects quit the study because of the unwillingness to continue ( three persons ) and ovarian hyper stimulation ( four persons ) . demographic and clinical data results showed no significant difference in the mean score of different dimension of mental health before ovulation induction and after oocyte harvesting [ table 2 ] . also , the rate of psychological disorder in the anxiety dimension showed no significant different before the induction ( 71.4% ) and after the pickup ( 66.7% ) ( p = 0.19 ) . but the level of depression after pickup ( 31.7% ) was significantly lower than before the induction ( 39.7% ) ( p = 0.007 ) . comparison of mental health dimensions b and a results showed no significant difference in the dimension of hypochondriasis before the induction ( 61.9% ) and after the harvesting ( 66.7% ) ( p = 0.07 ) . also , the level of social impairment showed no significant difference before the induction ( 84.1% ) and after the pickup ( 87.3% ) ( p = 0.08 ) . the results of multi - variable linear regression to evaluate the relation between underlying variables and mental health condition before starting the process with the level of depression and anxiety after the harvesting are shown in table 3 . the relation between mental health dimensions and background variables after ovulation stimulation the level of depression after egg harvesting was related to physical condition , depression and social impairment , independent from underlying variables . also , the level of depression after egg harvesting was related to the economic condition , it also showed a significant relation with social impairment and hypochondriasis [ table 3 ] . the present study aimed to assess the level of mental health in fertile women undergoing assisted reproductive treatments as a result of male factor infertility , at the beginning of ovulation stimulation process , and the effect of this process on their mental health . results showed that these women do not start assisted reproduction treatment under appropriate mental health conditions . but the level of depression and anxiety disorders at the beginning of ovulation stimulation was higher in the present research than other reports . this would indicate that the process of treatment in fertile women is also a critical one and might affect their mental health . in a pursuit , to achieve a conceptual model for the effect of ovulation induction on women 's health , its negative effects on women 's functioning and health have been shown . a qualitative study showed that iranian infertile couples show emotional reactions such as fear , concern , anxiety and depression as a result of fear of the failure of treatment during fertility treatment . however , these mental disorders may be harmful even for women with appropriate fertility potentials . also being angry with their husbands due to their infertility could be another explanation for women 's mental reactions . positive correlation between the level of depression and anxiety and other dimensions of experiences during infertility treatment might be accompanied with social impairment , thus affecting the levels of depression and anxiety . efforts to conceal infertility and efforts to get treatment as a result of concerns about being labeled as infertile , are common among iranian couples . this , by itself , could increase the level of anxiety among women when treatment starts . another reason for the high level of depression and anxiety among women at the beginning of the ovulation stimulation might be the negative impact of gonadotropin - releasing hormone ( gnrh ) agonists on the mental health . although , this research did not evaluate mental health before and after the beginning of gnrh agonists , but this might explain women 's psychological disorders before ovulation stimulation regarding high frequency of mental disorders at the time of ovulation stimulation . with regards to the negative impact of anxiety and depression during the treatment on the result of assisted reproduction treatment , it is necessary that this group of women would be examined before the treatment process , since they are exposed to risks of mental health disorders , and be treated in case of high levels of depression and anxiety . following the assessment of the main hypothesis of the research , that is the effect of ovulation stimulation on women 's mental health , study results showed that ovulation stimulation does not affect mental health ; the level of depression and anxiety , and the measure of psychological disorders in these two - dimensions did not show a significant difference before the ovulation and after the harvesting process . on the contrary , the depression level was decreased after the harvesting process . the negative impact of distress of infertility treatment on the level of depression has already been reported . the cessation of treatment distress with the conclusion of ovulation stimulation and harvesting processes might be a reason for the decrease of depression level in subjects under study . furthermore , results showed that fertile women 's level of depression and anxiety does not depend on the reaction of the ovary to the ovulation stimulation , and as these women have no problem in ovarian response to ovulation stimulation , their mental health depends more on other factors . in addition , all subjects , who sustained the research were those who reached the stage of the harvesting process ; the potential for ovum production was proven for these women . this would enhance the self - esteem of subjects and decrease their concern over ovarian nonresponsiveness . but direct correlation between the level of anxiety and depression in the stage after harvesting process with social impairment and hypochondriasis indicates that in this stage also the awareness of social dysfunction , independent from financial status , will have its own effect on mental health . factors other than ovulation stimulation process which might influence the individuals levels of depression and anxiety have not been assessed in the present research . depression and anxiety might result from a variety of factors , which require more extensive research . another result of this research was that financial status , independent from the quantity of follicle obtained from ovulation stimulation , showed a reverse correlation with anxiety level after harvesting process . in ozken 's research , also financial status was a factor determining the anxiety in infertile couples . regarding financial pressures resulting from high costs of assisted reproduction treatments , which lead to the success of only 35% for each cycle , women 's concerns over the success of results in fertilization as an outcome of infertility of the partner detected after harvesting process while insemination in laboratory are not far from expectation . nevertheless , although women are healthy in terms of fertility in couples undergoing male factor infertility treatment , effort to have babies not by normal methods and facing difficult , costly treatment protocols might threaten their evaluation of their feminine role , thus reducing their self - esteem . another finding of this research was the existence of correlation between depression level after treatment process and anxiety level at the first stage , while this correlation was observed between depression level after treatment process and anxiety level at the second stage . this finding indicates that women , who enter the process of treatment with a higher anxiety level are prone to a higher level of depression ; therefore necessary measures should be taken to prevent it . one of this study 's limitations was that mental health assessment was conducted after starting the primary stages of the treatment therefore , the stress of this stage could affect the mental health during the whole process . this study showed that fertile women who undergo assisted reproduction treatments do not start the treatment process in an appropriate mental health condition , and this condition would continue during the whole process until egg harvesting . therefore , it is suggested that before starting the treatment the mental health of these women should be evaluated using screening tools and counseling sessions would be applied for vulnerable women .

introduction : the process of assisted reproductive treatment is a stressful situation in the treatment of infertile couples and it would harm the mental health of women . fertile women who started infertility treatment due to male factor infertility have reported to experience less stress and depression than other women before the assisted reproductive process but considering the cultural and social factors and also the etiology of the assisted reproductive process , it could affect the metal health of these women . therefore , this study was conducted to evaluate the mental health of fertile women who undergo assisted reproductive treatment due to male factor infertility.materials and methods : this study was a prospective study on 70 fertile women who underwent assisted reproductive treatment due to male factor infertility . the exclusion criterion was to stop super ovulation induction . to assess mental health , anxiety and depression dimensions of the general health questionnaire were used . before starting ovulation induction and after oocyte harvesting , the general health questionnaire was filled by women who were under treatment . data were analyzed using multi - variable linear regression , paired t - test , and chi-square.results:the results showed that the mean score of depression and anxiety before ovulation induction and after oocyte harvesting were not significantly different ; but the rate of mental health disorder in the depression dimension was significantly decreased after oocytes harvesting ( 31.7% vs. 39.7% ) . also , there was a significant relation between the level of anxiety and depression before ovulation induction and after oocyte harvesting ( p < 0.05 ) . the anxiety level after oocyte harvesting had a positive and significant correlation with the economic situation ( p < 0.05).conclusion : this study revealed that the process of assisted reproductive treatment does not affect the mental health in fertile women independently , but these women start assisted reproductive process with high levels of depression and anxiety . therefore , prior to the assisted reproductive treatment mental health consultation is needed .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS Financial support and sponsorship Conflicts of interest

balance impairment is an important fall - risk factor , and increases in range of postural sway in the mediolateral direction in older adults are associated with increased fall - risk and rates . postural sway has been shown in older adults to be strongly related to other measures of balance . multivariate analysis reveals serum vitamin d levels as an independent variable associated with postural sway . in individuals with suboptimal levels of vitamin d , epidemiological studies have shown that vitamin d levels show seasonal variation [ 7 , 8 ] . lowest levels of serum vitamin d are recorded towards the end of winter , approximately four weeks after the shortest day of the year . overall , vitamin d supplementation did not reduce rate of falls ( rar 1.00 , 95% ci 0.90 to 1.11 ; seven trials ; 9324 participants ) or risk of falling ( rr 0.96 , 95% ci 0.89 to 1.03 ; 13 trials ; 26 , 747 participants ) but may do so in people with lower vitamin d levels before treatment . older adults are at risk for lower levels of serum vitamin d because of age - related changes in uvb absorption and skin capacity to synthesize vitamin d , reduction in activation in the kidneys , and reduced expression of vitamin d receptors in tissues . there are many factors affecting fall - risk for older individuals , and although these may be different for inside and outside falls , strength and balance remain two important physical fall - risk factors . a recently published overview of the literature supports an assertion that age - related changes in postural reactions may be related to vitamin d status - mediated through either central nervous system integration or antigravity muscles as the effectors in postural responses . despite changes in vitamin d across the seasons , muscle strength in the quadriceps muscles has been shown to remain stable . the relationship of postural sway to any potential increased winter fall rate and decreased levels of vitamin d has not been investigated . the winter season sees an increase in injuries from falls and in the number of accidental deaths from falls . fracture rates from falls in older adults also increase at the end of the winter season , following two to eight weeks after the nadir in serum vitamin d levels . some studies report an increased rate of falls , for both inside and outside falls [ 13 , 14 ] ; however , significant seasonal variation in fall rates was not found in a three - year study while , in a second study , seasonal variation in fall rates was reported in women but not in men . coincident static balance changes with any potential increased fall rates in winter have not been previously reported . the data presented here forms part of a larger study , from which two other papers with the same clinical trial registration have been published [ 12 , 16 ] . the aim of this study was to determine differences in static balance ( postural sway ) , vitamin d , incidence of falls , and type of fall serially at the end of each season over a 12-month period , in older community - living adults . we hypothesised that postural sway , falls , and vitamin d would show a seasonal variation and that there would be an inverse relationship between vitamin d and the other variables . at the end of consecutive seasons , static balance , vitamin d status , and fall rate were measured within a longitudinal study design . no intervention was implemented by study researchers , so that the study could identify natural variations that occur over the seasons . data was collected over a three - week period in each season from end of spring 2009 to the end of spring 2010 , with collection of data timed to coincide with expected peaks and troughs in serum vitamin d levels in australia at latitude 41 degrees south ( tasmania ) . after each assessment , participants were given an appointment for the next collection block in three months ' time . independently living community - dwelling adults aged between sixty and eighty - five years were recruited through local print media and community clubs . daily intake of oral supplementation of vitamin d of greater than 800 international units was also an exclusion criterion . participants were also excluded if they had a history of neurological disease and were withdrawn if they suffered a medical condition while participating in the study that would impact on their ability to perform the physical tests . liver and kidney disease both impact vitamin d metabolism , and any potential participants with either of these conditions were excluded . a priori sample size calculation was based on a previous study reporting mediolateral sway range in a sample of community - dwelling older adults , and this indicated a minimum requirement of 81 completed participants ( minimum effect size 2.5 mm sway ; standard deviation ( sd ) 8 mm ; power 0.8 , alpha 0.05 ) . ninety - eight participants were recruited with the anticipation of a 15% drop - out rate . this repeated measures cohort study was designed to be able to detect differences in postural sway . this project received ethical approval from the human research ethics committee ( tasmania ) network ( h0010561 ) . postural sway range in the medio - lateral sway direction was measured using a force platform ( amti accugait pjb 101 , massachusetts , usa ) for thirty seconds under conditions of eyes open and closed , as well as with the additional challenge of using a 6.5 cm foam cushion ( eyes open and eyes closed ) ( airex elite balance pad ag , switzerland ) . participants were asked to remain stationary with their arms by their sides and look straight ahead while standing on the force platform . foot position ( bare feet ) was standardised with heels 4 cm apart using marked placement for the feet to ensure repeatability between testing occasions . venous blood samples were collected and clotted , then centrifuged ( 1610 relative centrifugal force ) for 15 minutes . serum concentration of 25-hydroxy vitamin d was measured by direct , competitive chemiluminescent immunoassay in a commercial accredited laboratory , using liaison method ( diasorin inc . , participants received an individually coded calendar for 12 months of the study on which to report any falls and associated details by date , including information regarding the location and cause of the fall . information about the type of fall , any injuries that resulted , and if medical attention was sought was recorded . this study utilised the who definition of a fall as an event which results in a person coming to rest inadvertently on the ground or other lower level . annual cyclic trends were investigated by fitting a sine wave formula to data for postural sway and vitamin d , with the amplitude of the seasonal variation ( in percentage change ) and the annual mean values were estimated using repeated measures nonlinear regression , adjusted for age and gender . for secondary analysis the three monthly data were interpolated linearly to estimate intermediate values to correspond with the observed fall incident data . mixed - methods poisson regression was used to determine associations between falls , fall injuries , and postural balance and vitamin d. the association between postural balance and vitamin d and season was estimated using mixed - methods linear regression , adjusted for age , gender , and strength . for comparison , seasonal data for falls was grouped into autumn and winter and compared to spring and summer . physical activity using the champs questionnaire and muscle strength using the physiological profile assessment tools were recorded , and this information has been published elsewhere but forms an integral part of the meta - analysis of this paper . data from eighty - eight participants ( 70% females ) are included in the final analysis . five people did not attend appointments , and five people could not complete testing because of medical events . the participants had a mean ( sd ) age of 69.2 ( 6.5 ) years and body mass index 27.4 ( 3.9 ) kg m. all participants were living in their own homes independently , with only 10% being sole occupants . common chronic controlled health conditions included cardiovascular disease ( 39% ) and arthritis ( 14% ) . twenty - six percent of the participants reported the use of more than 4 medications . all four balance measures had the highest sway scores ( poorest balance ) at the first end of spring measurement . all other seasonal measures were significantly different from this first time point ( all p < 0.05 ) , but no subsequent significant difference was seen after any other seasonal measures , indicating a lack of seasonal variation in this outcome ( p > 0.05 ) . no associations between postural sway and vitamin d were observed ( all p > 0.05 ) . increased postural sway was associated with fall injuries ( irr 1.59 ( ci 1.14 to 2.24 ) ( p = 0.007 ) but not fall rates ( irr 1.36 ( ci 0.95 to 1.97 , p = 0.09 ) . there was 15% variation in this variable over the year , with a peak at the end of summer and the lowest values at the end of winter . seventy - five percent of fall diaries were posted on schedule while the remaining diaries were returned at the subsequent assessment appointment , resulting in a compliance of 100% . thirty - three percent of the cohort ( 29 people ) fell at least once , with 10% of the whole group falling multiple times ( 8 people ) . over the duration of the study , 48 falls were recorded : 14 of these occurred inside the house , and 34 occurred outside . six falls were due to fainting or dizziness and forty due to trip - related events , with one categorised as being pushed over ( by a horse ) and one not able to be categorised . twenty - eight falls resulted in injury , with only four requiring medical treatment ( including one fracture ) . further details on season variation in location and type of fall are provided in table 2 . there were significantly fewer falls during spring than any other season ( p = 0.01 ) , with no other differences between the seasons recorded . when falls data were combined from autumn and winter seasons and compared to the combined spring and summer seasons , there were more falls reported in the combined autumn and winter seasons ( 30 compared to 18 ) . less injuries from falls were recorded in spring than any other season ( p = 0.02 ) , with no other seasonal differences recorded . this is the first cohort study to determine that no seasonal variation of postural sway occurs across 12 months in our population . significant seasonal variation in serum vitamin d levels , with higher serum levels in summer , was recorded . there was a significant relationship between postural sway and the number of injurious falls observed , with lower values for sway range ( i.e. , better stability ) associated with less fall injuries . a significant learning effect was seen with all measures of mediolateral postural sway with time point one having larger ranges than all of the other time points ( all p > 0.05 ) . further to this , there was no seasonal variation in postural sway under any of the four static balance test conditions measured ( eyes open and closed on a firm surface or foam surface ) . postural sway range has been used to identify those people with balance impairment , and it may be useful in describing the fall - risk status of a particular individual . our data indicates that postural sway does not appear to be subject to changes across the year within a participant . it has been suggested that this measure is important in describing sensorimotor deficits or disability rather than functional abilities and hence may not be subject to changes that may occur due to altered patterns of activity or sunlight exposure seen seasonally . the stability of this measure in a cohort across a year provides important information for researchers planning interventions designed to impact postural sway and clinicians who are measuring the effectiveness of their interventions . mediolateral sway range has been shown to be an independent fall - risk factor for indoor falls . in our study , a lower proportion of falls occurred indoors ( 29% ) compared to outdoors ( 71% ) , and although this is similar to other studies where more healthy samples have been reported as having a greater proportion of falls being outdoor falls ( 74% ; ) , it may be a reason why no association between sway range and fall incidence was seen . a trend for this association was evident and a larger sample size may have found a significant relationship between these two variables , as this study was powered to determine mediolateral sway changes not fall rates . an association between increased sway range and rate of injurious falls was recorded in our study , reinforcing the importance of this measure for those most at risk of injury . overall there was no significant relationship found between postural sway and vitamin d. as increased postural sway is linked to low levels of vitamin d , it may be that the levels of vitamin d were sufficient for this parameter even at their lowest levels in participants in this study and did not influence postural sway . if this threshold situation is true , it may be that seasonal variation in postural sway may be present in a population with much lower levels of vitamin d , but this is outside the scope of the current study . annual rates of falling for adults over 65 have been reported up to 40% , and although our cohort includes some adults between the ages of sixty and sixty - five , with a mean age of 69 years and a fall rate of thirty - three percent , our population appears to be representative of older community - dwelling adults in terms of fall rate . fall rates in older - old adults ( over 75 years ) have been shown to vary seasonally , but consistent data for the general population of healthy older community - dwelling adults has not been previously reported . one previous study grouped the peak seasons of winter and autumn together and found differences in fall rates . manipulation of our data in a similar way reveals that there were more falls during the autumn / winter half year compared to spring / summer ( table 2 ) . these seasonal differences may be related to intrinsic factors that may be subject to seasonal variation ( e.g. , vitamin d , physical activity , and muscle strength ) as well as seasonally related environmental factors ( e.g. , weather , temperature ) . further research needs to investigate interventions to address potentially modifiable factors to reduce the increased falls risk in the autumn / winter period . our data indicates a higher rate of falls in summer than has previously been reported , perhaps due to activity characteristics of our cohort . summer and winter falls differ between the genders , with more men falling due to slips in winter and more women falling due to trips in summer . the high proportion of women in our study ( 79% ) may be a factor in the high rate of summer falls observed . another factor to consider is the relationship between fall status and vitamin d. although 60 nmol / l has been determined to be the cutoff for fall - risk function , 16/48 falls ( 33% ) occurred in participants in this study whose vitamin d was above that cutoff level . this may be explained by the higher proportion of summer time falls observed in our study . during summer months , with longer hours of daylight at this latitude , a large proportion of the falls occurred in the outside ( 13 of the 14 falls 93% ) ( table 2 ) . by contrast , in winter out - of - doors fall rate was reduced to 64% , indicating a higher winter time proportion of inside falls . for our generally healthy study population , it is likely that our participants were engaged in outdoor activities with higher associated risk of falls during the warmer weather ; for example , several summer falls occurred while bushwalking . fall injuries , especially fractures , have been found to increase in winter ; this includes both inside ( hip ) and out - of - doors falls ( wrist ) . studies in the area of seasonal variation in fracture rates provide good evidence for increased fracture rates from falls in winter , but these appear in populations with older participants than ours ( mean age over 75 years ) [ 8 , 13 ] . our study recorded few injuries that required medical attention , hence making it difficult to compare serious fall injury data . although this study aimed to recruit independently living community - dwelling older adults , bias in the sample may be present , as volunteers to this type of research project may be more robust than the community members at large . the lack of more frail subgroups , including those with cognitive impairment , depression , or using walking aids , limits the generalisability of this study . this study provides some evidence that measurements of postural sway may be affected by some effect of test - retest learning , and this needs to be considered in future research . this is the first study to investigate the effects of season and postural sway and found that postural sway remained stable over the 12 months . this study provides important evidence for clinicians and researchers that postural sway remains stable over an annual cycle but may be influenced by a learning effect .

introduction . low serum vitamin d levels are associated with increased postural sway . vitamin d varies seasonally . this study investigates whether postural sway varies seasonally and is associated with serum vitamin d and falls . methods . in a longitudinal observational study , eighty - eight independently mobile community - dwelling older adults ( 69.7 7.6 years ) were evaluated on five occasions over one year , measuring postural sway ( force platform ) , vitamin d levels , fall incidence , and causes and adverse outcomes . mixed - methods poisson regression was used to determine associations between measures . results . postural sway did not vary over the year . vitamin d levels varied seasonally ( p < 0.001 ) , peaking in summer . incidence of falls ( p = 0.01 ) and injurious falls ( p = 0.02 ) were lower in spring , with the highest fall rate at the end of autumn . postural sway was not related to vitamin d ( p = 0.87 ) or fall rates , but it was associated with fall injuries ( irr 1.59 ( ci 1.14 to 2.24 , p = 0.007 ) . conclusions . postural sway remained stable across the year while vitamin d varied seasonally . participants with high values for postural sway demonstrated higher rates of injurious falls . this study provides important evidence for clinicians and researchers providing interventions measuring balance outcomes across seasons .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Limitations 6. Conclusion

since the dawn of history , nature and natural sources , such as plants , animals , microbes , and minerals have remained a veritable source of bioactive compounds with medicinal values . among these sources , plants have been the most explored and exploited for their bioactive medicinal components . lead compounds or templates for the rational development of drugs are of more specific efficacies and fewer side effects ( 1 ) . one of the botanicals of interest in which the abortifacient claims in the folk medicine have been substantiated by scientific evidence ( 2 ) but with no documentation in the open scientific literature on its bioactive abortifacient agent(s ) is senna alata . senna alata ( linn . ) roxb ( leguminosae ) , also known as craw - craw plant or ringworm plant ( english ) , asunwon oyinbo ( yoruba - western nigeria ) , nelkhi ( igbo - eastern nigeria ) , filisko or hantsi ( hausa - northern nigeria ) is an erect tropical , annual herb which grows up to 0.15 m high . the fruit is a pod , while the seeds are small and square in shape . the plant has been claimed to be used in the management of several diseased conditions such as hepatitis , dermatitis , jaundice , gastroenteritis , eczema , constipation and diarrhoea ( 3 , 4 ) . previous studies have shown that s. alata have antifungal , antibacterial and antioxidant activities ( 3 , 58 ) . furthermore , yakubu et al . ( 2 ) reported that the aqueous leaf extract of the plant contained saponins ( 1.22% ) , flavonoids ( 1.06% ) , cardiac glycosides ( 0.20% ) , phenolics ( 0.44% ) , alkaloids ( 0.52% ) , cardenolides and dienolides ( 0.18% ) . aqueous leaf extract of s. alata has also been scientifically validated for its acclaimed use as an abortifacient ( 2 ) . however , there is no study in the open scientific literature that has reported on the exact bioactive abortifacient agent(s ) in s. alata leaves . therefore , the present study aimed to validate the speculation that the alkaloids in aqueous extract of s. alata leaves are responsible for its abortifacient activity . the focus on alkaloid was a follow - up from our previous study that speculated on alkaloids to be responsible for the abortifacient activity of the crude extract of s. alata leaves and from several studies that implicated the phytochemical property of colchicine , quinazoline alkaloids , e.g. vasicine and vasicinone in several botanicals such as xylopia aethiopica , peganum harmala epigeal parts , areca catchu nuts and gloriosa superba roots as abortifacient bioactive agents and/or their role in the contraction and relaxation of uterine muscles ( 912 ) . the plant leaves , obtained from herb sellers at oja tuntun , ( new market ) in ilorin , nigeria , was authenticated at the herbarium unit of the forestry research institute of nigeria ( frin ) in ibadan , nigeria . a voucher specimen ( fhi 10845 ) was deposited at the herbarium of the institute . assay kits for both glucose and cholesterol were products of randox laboratories , ltd , united kingdom , while those of progesterone , follicle stimulating and luteinizing hormones were products of inteco uk ltd , united kingdom . thin layer chromatographic ( t lc ) plates and silica gel were products of merck ( darmstadt , germany ) . para - nitrophenyl phosphate and other reagents were products of sigma- aldrich inc . , st . male and female wistar rats ( rattus norvegicus ) weighing 178.913.07 and 143.99 1.21 g , respectively , were obtained from the animal holding unit of the department of biochemistry , university of ilorin , ilorin , nigeria . the animals which were housed individually in plastic cages and placed in a well - ventilated room ( temperature : 28 - 31c ; photoperiod : 12 hr natural light and 12 hr darkness ; humidity : 50 - 55% ) were provided with unrestricted access to rat pellets ( bendel feeds and flour mills , ewu , nigeria ) and water . the animals were also handled according to the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ets 123 ( 13 ) . the leaves of senna alata which were oven - dried at 40c for 48 hr were pulverised using a mikachi blender ( mk-1830 , china ) . alkaloids were extracted from the powder according to the procedure described by manske ( 14 ) . a known amount ( 500 g ) of the powder was extracted in 1.2 l of hexane for 72 hr and filtered with whatman no the hexane extract ( containing fats , oils , terpenes , waxes ) were discarded and the resulting residue extracted again in 1.2 l of methanol for a week and subsequently filtered . the filtrate was evaporated using a rotavapor ( r110 , gallenkamp , england , uk ) and the process was repeated two more times . the three filtrates were combined , concentrated to give a methanolic , green slime ( 90 g ) which was treated with 1 m hcl and then basified by adding 5 m naoh with continuous stirring until a cloudy precipitate appeared . a known volume ( 500 ml ) of chloroform and 200 ml of 1 m nacl were added and the process was repeated three more times . equal volumes ( 150 ml ) of 1 m nacl and 5 m naoh were added to the organic layer in a separating funnel after which the mixture was evaporated to yield brownish - black slurry ( 18 g ) of alkaloids that corresponded to a yield of 3.60% that was used for subsequent experiments . the procedure described by singh and sahu ( 15 ) was adopted for the preparation of thin layer chromatography ( tlc ) plates . furthermore , about 10 l of the test solution ( extract ) was spotted onto the thin layer plate using a micropipette after which the plates were developed in chloroform : methanol ( 10:2 ) in which 0.01 g / ml of butylated hydroxyl toluene and butylated hydroxyl anisole were added to prevent oxidation ( which may lead to increase in the number of bands with time ) ( 16 ) . the relative values related to the solvent front ( rf ) of the spots were also computed . twenty - four pregnant rats were allocated in a complete randomize design to four groups ( a , b , c and d ) consisting of six animals each . animals in group a ( controls ) , orally received 0.5 ml of distilled water with the aid of an oropharyngeal cannula while those in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration was done once daily from day 10 until day 18 of pregnancy ( period of organogenesis in wistar rats ) ( 18 ) . by the 19th day ( 24 hr after their last dose ) the following parameters were recorded / computed : number of live fetuses ; number of dead fetuses ; average weight of live foetuses ; survival ratio ( % ) = ( number of live fetuses/ number of live + dead fetuses ) 100 ; number of rats that aborted ; percentage of rats that aborted= ( number of rats that aborted / number of rats assessed ) 100 ; number of rats with vaginal bleeding ; number of implantation sites ; number of corpora lutea ; implantation index=(total number of implantation sites / number of corpora lutea ) 100 ; pre - implantation loss=(number of corpora lutea number of implantation sites / number of corpora lutea ) 100 ; post - implantation loss= ( number of implantation sites number of live fetuses / number of implantation sites ) 100 ; number of resorption sites = number of implantation sites in the control animals number of implantations in the test animals ; resorption index=(total number of resorption sites / total number of implantation sites ) 100 . the weights of the animals both before pairing and prior to sacrifice , as well as feed and water intake were also recorded . the in vivo estrogenic / anti - estrogenic response of the rats to the alkaloids was evaluated by adopting the procedure described by kanno et al . twenty - four ovariectomized , female rats ( 159.677.12 g ) were allocated to four groups ( a , b , c and d ) of six animals per group . group a ( controls ) , orally received 0.5 ml of distilled water while animals in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration which commenced from the eighth day of ovariectomy , lasted for another seven days . on day 16 , the weights of the animals were determined prior to sacrifice and uterine to body weight ratio was computed . the uterine protein , glucose , cholesterol , and alkaline phosphatase activity were determined using standard procedures ( 2023 ) . the serum and uterine homogenates were prepared according to the procedures described by yakubu and bukoye ( 24 ) . the procedures outlined in the manufacturer 's protocol were adopted for the quantitative determination of progesterone , follicle stimulating and luteinizing hormones in the serum of the animals . data which were expressed as the meansd of six independent replicates were statistically analyzed using one - way analysis of variance and duncan multiple range test . the plant leaves , obtained from herb sellers at oja tuntun , ( new market ) in ilorin , nigeria , was authenticated at the herbarium unit of the forestry research institute of nigeria ( frin ) in ibadan , nigeria . a voucher specimen ( fhi 10845 ) was deposited at the herbarium of the institute . assay kits for both glucose and cholesterol were products of randox laboratories , ltd , united kingdom , while those of progesterone , follicle stimulating and luteinizing hormones were products of inteco uk ltd , united kingdom . thin layer chromatographic ( t lc ) plates and silica gel were products of merck ( darmstadt , germany ) . para - nitrophenyl phosphate and other reagents were products of sigma- aldrich inc . , st . male and female wistar rats ( rattus norvegicus ) weighing 178.913.07 and 143.99 1.21 g , respectively , were obtained from the animal holding unit of the department of biochemistry , university of ilorin , ilorin , nigeria . the animals which were housed individually in plastic cages and placed in a well - ventilated room ( temperature : 28 - 31c ; photoperiod : 12 hr natural light and 12 hr darkness ; humidity : 50 - 55% ) were provided with unrestricted access to rat pellets ( bendel feeds and flour mills , ewu , nigeria ) and water . the animals were also handled according to the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ets 123 ( 13 ) . the leaves of senna alata which were oven - dried at 40c for 48 hr were pulverised using a mikachi blender ( mk-1830 , china ) . alkaloids were extracted from the powder according to the procedure described by manske ( 14 ) . a known amount ( 500 g ) of the powder was extracted in 1.2 l of hexane for 72 hr and filtered with whatman no . 1 filter paper . the hexane extract ( containing fats , oils , terpenes , waxes ) were discarded and the resulting residue extracted again in 1.2 l of methanol for a week and subsequently filtered . the filtrate was evaporated using a rotavapor ( r110 , gallenkamp , england , uk ) and the process was repeated two more times . the three filtrates were combined , concentrated to give a methanolic , green slime ( 90 g ) which was treated with 1 m hcl and then basified by adding 5 m naoh with continuous stirring until a cloudy precipitate appeared . a known volume ( 500 ml ) of chloroform and 200 ml of 1 m nacl were added and the process was repeated three more times . equal volumes ( 150 ml ) of 1 m nacl and 5 m naoh were added to the organic layer in a separating funnel after which the mixture was evaporated to yield brownish - black slurry ( 18 g ) of alkaloids that corresponded to a yield of 3.60% that was used for subsequent experiments . the procedure described by singh and sahu ( 15 ) was adopted for the preparation of thin layer chromatography ( tlc ) plates . furthermore , about 10 l of the test solution ( extract ) was spotted onto the thin layer plate using a micropipette after which the plates were developed in chloroform : methanol ( 10:2 ) in which 0.01 g / ml of butylated hydroxyl toluene and butylated hydroxyl anisole were added to prevent oxidation ( which may lead to increase in the number of bands with time ) ( 16 ) . the relative values related to the solvent front ( rf ) of the spots were also computed . twenty - four pregnant rats were allocated in a complete randomize design to four groups ( a , b , c and d ) consisting of six animals each . animals in group a ( controls ) , orally received 0.5 ml of distilled water with the aid of an oropharyngeal cannula while those in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration was done once daily from day 10 until day 18 of pregnancy ( period of organogenesis in wistar rats ) ( 18 ) . by the 19th day ( 24 hr after their last dose ) the following parameters were recorded / computed : number of live fetuses ; number of dead fetuses ; average weight of live foetuses ; survival ratio ( % ) = ( number of live fetuses/ number of live + dead fetuses ) 100 ; number of rats that aborted ; percentage of rats that aborted= ( number of rats that aborted / number of rats assessed ) 100 ; number of rats with vaginal bleeding ; number of implantation sites ; number of corpora lutea ; implantation index=(total number of implantation sites / number of corpora lutea ) 100 ; pre - implantation loss=(number of corpora lutea number of implantation sites / number of corpora lutea ) 100 ; post - implantation loss= ( number of implantation sites number of live fetuses / number of implantation sites ) 100 ; number of resorption sites = number of implantation sites in the control animals number of implantations in the test animals ; resorption index=(total number of resorption sites / total number of implantation sites ) 100 . the weights of the animals both before pairing and prior to sacrifice , as well as feed and water intake were also recorded . the in vivo estrogenic / anti - estrogenic response of the rats to the alkaloids was evaluated by adopting the procedure described by kanno et al . twenty - four ovariectomized , female rats ( 159.677.12 g ) were allocated to four groups ( a , b , c and d ) of six animals per group . group a ( controls ) , orally received 0.5 ml of distilled water while animals in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration which commenced from the eighth day of ovariectomy , lasted for another seven days . on day 16 , the weights of the animals were determined prior to sacrifice and uterine to body weight ratio was computed . the uterine protein , glucose , cholesterol , and alkaline phosphatase activity were determined using standard procedures ( 2023 ) . the serum and uterine homogenates were prepared according to the procedures described by yakubu and bukoye ( 24 ) . the procedures outlined in the manufacturer 's protocol were adopted for the quantitative determination of progesterone , follicle stimulating and luteinizing hormones in the serum of the animals . data which were expressed as the meansd of six independent replicates were statistically analyzed using one - way analysis of variance and duncan multiple range test . the alkaloids yielded 0.30 g which corresponds to 1.50% of the starting material of 500 g. the five different spots gave rf values of 0.28 , 0.33 , 0.39 , 0.47 and 0.55 . the spots gave positive reaction with both meyer 's and wagner 's reagents producing a creamy precipitate and reddish - brown spots on a grey background ( tlc ) . the alkaloid truncated the development of the fetuses as none of them survived in the experiment groups as against the average number of live fetuses of 10.26 in the distilled water - treated control animals ( table 1 ) . the average weight of live fetuses in the controls was 4.92 g against none in all the extract - treated animals . the percentage of fetal death in the 250 , 500 and 1000 mg / kg body weight of the alkaloid - treated animals was 6.03 , 6.00 and 6.50 , respectively . there was neither episode of abortion nor vaginal bleeding in all the alkaloid - treated animals . both the number of implantation sites and corpora lutea decreased significantly ( p < 0.05 ) . while the implantation index was similarly high in all the alkaloid - treated animals , the resorption index , as well as pre- and post- implantation losses were many fold higher than the controls ( table 1 ) . although , all the pregnant rats gained weight at the end of the experimental period ( final maternal weight ) compared with their weight prior to pairing with the males ( initial maternal weight ) ( table 1 ) , the weight gained by the alkaloid - treated animals was about 50% less than the control animals . furthermore , the feed and water intake by the animals treated by different doses of the alkaloid decreased significantly ( p < 0.05 ) . effect of the alkaloids from senna alata leaves on some abortifacient parameters of pregnant rats values are expressed as meansd of six independent determinations ; test values carrying superscripts different from the control for each parameter in the same row are significantly different ( p < 0.05 ) . maternal weights of the animals before pregnancy were compared with their corresponding weights after pregnancy for each treatment group ( p < 0.05 ) all different doses of the alkaloids significantly ( p < 0.05 ) decreased the serum concentrations of the follicle stimulating hormone , luteinizing hormone and progesterone in the pregnant animals ( table 2 ) . effect of alkaloids from senna alata leaves on some female reproductive hormones of pregnant wistar rats values are expressed as meansd of six independent determinations ; test values carrying superscripts different from the control for each parameter in the same row are significantly different ( p < 0.05 ) the alkaloids also decreased ( p < 0.05 ) the absolute weight of the uterus , computed uterine to body weight ratio , and concentrations of uterine glucose and cholesterol ( table 3 ) . in contrast , the concentration of uterine protein and the activity of alkaline phosphatase increased significantly ( p < 0.05 ) . furthermore , the alkaloids did not provoke vaginal opening or cornification in any of the animals ( table 3 ) . effect of alkaloids from senna alata leaves on some indices of oestrogenicity in pregnant rats values are expressed as meansd of six independent determinations ; test values carrying superscripts different from the control for each parameter in the same row are significantly different ( p < 0.05 ) analysis of the tl chromatogram indicated that the mixture consisted of five alkaloids as evidenced by the creamy precipitates and reddish- brown spots produced with meyer 's and wagner 's reagents , respectively . in the present study , alkaloids from s. alata leaves significantly affected the fetal and maternal parameters of the animals . for instance , the death of the fetuses in the alkaloid - treated pregnant rats may not only suggest inhibition of mitotic division of the fetuses ( 25 ) , since the animals were exposed during the period of organogenesis , but may also show its relevance to the reduction in the concentrations of gonadotropins and progesterone in the present study . ( 2 ) on the aqueous leaf extract of the plant where only the 500 and 1000 mg / kg body weight of the extract produced fetal death . furthermore , the absence of abortion and vaginal bleeding in the alkaloid - treated animals which were hitherto observed with the crude extract in our previous study suggest zero abortifacient activity for the alkaloid . therefore , the abortifacient activity of the aqueous extract of the plant leaf reported earlier ( 2 ) is not due to the alkaloidal content alone , but to some other phytochemicals such as saponins and flavonoids which may act synergistically or additively to produce the desired result . it is interesting to note that both the number of implantation sites and corpora lutea decreased in the alkaloid - treated animals when the process of implantation ought to have been completed ( implantation takes place normally within 5 - 6 days post - coitus in rats ) before the exposure of the animals to the alkaloid mixture ( treatment commenced from day 10 of pregnancy ) . the reason for this decrease is not immediately known but may not be unconnected with the consequence of some general hormonal effect ( reduced progesterone ) and/or absence of conceptuses growth ( 26 ) . it is also possible that blastocytes were not activated and well - positioned before implantation , probably due to impaired muscular activity of the uterus ( 27 ) . the implantation index and pre - implantation loss evaluates the number of blastocysts implanted in the uterus while the resorption index and post - implantation loss relate to the number of implanted blastocysts and those that have not developed ( 18 , 28 ) . therefore , the high implantation index , and pre- and post- implantation losses suggest that pregnancy was interrupted by the alkaloid , probably creating an environment that was not conducive for the fertilized eggs . normally , an abortion in most cases is accompanied by vaginal bleeding and when this is absent , the resorption will increase . therefore , the increase in the resorption index in the alkaloid- treated animals confers antifertility effects ( such as anti - implantation , anti - blastocystic and antizygotic ) of the alkaloid . the alkaloid did not exhibit complete abortifacient effect since the pregnant animals exposed to the alkaloid presented with closed vagina just like the controls . the findings in the present study is similar to the report by elbetieha et al . ( 29 ) where the administration of 200 , 400 and 800 mg / kg body weight of ethanolic extract of salvia fruticosa did not cause pregnancy failure but increased the number of resorption in the pregnant rats . the alkaloids reduced the sense of taste and appetite of the animals as evidenced by the decrease in feed and water intake . such reduction may account for the decrease in the computed percentage gain of maternal weight and may also be a consequence of impaired growth and development of the uterine contents ( 30 ) . all these findings except those of feed and water intake , as well as maternal weight gain , contrast the previous report by yakubu et al . it is well - known that for the implantation of the fetus and sustenance of pregnancy , an exact equilibrium must exist between the secretion of estrogen and progesterone ; this regulation is controlled by luteinizing and follicle stimulating hormones ( 31 ) . thus , the reduction in the gonadotropins may equally be responsible for the reduced concentration of progesterone in the present study and this may not only account for the death of the fetuses , but also for the increase in the resorption sites . the reduction in progesterone may suggest impaired endometrium function , which will adversely affect the normal secretion of special proteins required to nourish an implanted fertilized egg , and consequently , pregnancy failure . thus , it is possible that the alkaloids posses anti - gonadotropic and anti - progestogenic activities which are inimical to the continued development of the fetuses . many plant extracts with anti - fertility properties are known to exhibit estrogenic activity by increasing protein synthesis , uterine weight , water uptake and retention of fluid leading to ballooning of the uterus , uterine content of glucose , cholesterol , glycogen and alkaline phosphatase activity , thereby changing the uterine milieu and creating non - receptive conditions in the uterus ( 32 ) . therefore , the contrasting effects the alkaloids on some of these parameters in the present study suggest that estrogenic activity was not total but selective . the alkaloids exhibited more anti - oestrogenic activity ( 71.43% ) than oestrogenic ( 28.57% ) . overall , the alkaloid from s. alata leaves at the oral doses of 250 , 500 and 1000 mg / kg body weight on daily basis from days 10 until day 18 post - coitum exhibited several potential effects on the maternal and fetal outcomes of pregnant rats ( anti - implantation , anti - gonadotropic , anti - progesteronic , selective estrogenic , embryonic resorption and fetotoxic activities ) , but it could not induce abortion in the animals . therefore , the alkaloids may not alone be responsible for the abortifacient effects of the crude extract of s. alata which had been reported earlier . finally , work is in progress on isolating the other phytochemicals ( saponins and flavonoids ) and evaluating their synergistic effects in pregnant animals .

backgroundthe abortifacient claim of senna alata ( s. alata ) was scientifically validated recently with alkaloids speculated to be the bioactive agent . this speculation is yet to be substantiated or refuted by scientific evidence . the present study was aimed to investigate the pregnancy terminating effects of the alkaloids from s. alata leaves.methodstwenty four pregnant rats ( 143.991.21 g ) allocated randomly to four groups : a , b , c and d respectively received , 0.5 ml of distilled water , 250 , 500 and 1000 mg / kg body weight of the s. alata extracted alkaloids orally , once daily from day 10 until day 18 post - coitum . the indices of abortifacient were evaluated at the end of the exposure period . the results were analyzed by both the analysis of variance and duncan 's multiple range test and p < 0.05 was considered as statistically significant.resultsthin-layer chromatographic separation produced five spots with rf values of 0.28 , 0.33 , 0.39 , 0.47 and 0.55 which gave positive reaction with meyer 's and wagner 's reagents , respectively . the number of implantation sites and corpora lutea , as well as the concentrations of fsh , lh , progesterone , weight of uterus , uterine/ body weight ratio , glucose and cholesterol decreased significantly ( p < 0.05 ) whereas the resorption index , pre- and post - implantation losses , uterine protein content and alkaline phosphatase activity increased significantly . none of the alkaloid treated animals presented with provoked vaginal opening or bleeding except fetal deaths . the alkaloid decreased the maternal weight gain , as well as feed and water intake.conclusionoverall , the alkaloids from s. alata leaves exhibited anti - implantation , anti - gonadotropic , anti - progesteronic , embryonic resorptive , feto - maternal toxic activities but not complete abortifacient . the alkaloids alone may not be the sole abortifacient bioactive agent in the leaf extract .

Introduction Methods Plant material and authentication Chemicals and reagents Animals Extraction of alkaloids Analysis of the alkaloids Analysis of abortifacient activity Determination of estrogenic/anti-estrogenic activities Preparation of serum and uterine homogenates Determination of some reproductive hormones Statistical analysis Results Discussion Conclusion Conflict of Interest

erg protein expression has been recently suggested to be reflective of erg gene rearrangements in prostate cancer ( pca ) documenting remarkable concordance between the two [ 16 ] . the rearrangements between the androgen receptor - regulated gene tmprss2 ( 21q22.3 ) and members of the ets family member of transcription factor gene , most commonly erg ( 21q22.2 ) , are among the most common genetic alterations detected in prostate cancer [ 711 ] . erg gene rearrangements have been detected in roughly half ( 4060% ) of pca of surgical cohorts compared to a rate of 12%15% in incidental or watchful waiting cohorts [ 7 , 1218 ] . previous studies investigating the prognostic significance of erg gene rearrangements have revealed mixed results [ 1922 ] . however , it is becoming more evident that erg gene rearrangements signify a molecular subtype of pca . some studies investigating the significance of erg protein expression in localized pca failed to show association with adverse clinical outcome [ 23 , 24 ] . however , a recent report by our group demonstrated an association of erg expression with lethal disease in patients with unsuspected and advanced / castrate resistant disease who were treated by transurethral resection of prostate . moreover , we documented significant association between erg expression and both gleason score and tumor volume . studies from our group and others have also linked erg status to responsiveness to hormonal therapy , and longer progression time to castration resistant disease , compared to men with no erg expression [ 24 , 25 ] . in the current study , we investigated the association of erg protein expression to clinical - pathological parameters in a cohort of men with localized prostate cancer . the study cohort consisted of 198 patients who were treated by retropubic radical prostatectomy for localized prostate cancer with a mean followup of 4.8 years ( range 015.8 ) . clinical progression was defined as a postoperative serum psa elevation of > 0.2 ng / ml . prostate samples were embedded onto three tissue microarray ( tma ) blocks using a manual tissue arrayer ( beecher instruments , silver spring , md , usa ) . one to nine cores ( average 3.3 ) , 0.6 mm in diameter , were sampled including benign , high grade intraepithelial neoplasia ( hgpin ) , and prostate cancer ( pca ) . after construction , 4 m sections were cut and stained with haematoxylin and eosin on the initial slides to verify the histological diagnosis . briefly , 4 m thick sections from formalin - fixed paraffin - embedded tissue blocks were stained with ventana autostainer . prior to the staining , heat - induced antigen retrieval was carried out by vegetable steamer in sodium citrate antigen retrieval buffer ( 10 mm ph 6.0 ) for 40 minutes and then cooled down to room temperature for about 20 minutes . the slides were incubated for 60 minutes at 37c with erg rabbit monoclonal antibody ( epitomics , clone epr 3864 ) at 1 : 50 dilution . a ventana iview dab detection kit ( ventana tucson , az , usa ) the diagnoses of all tma cores were confirmed by the three study pathologists ( lht , cw , and tab ) . for each patient , the two predominant patterns were sampled and included on the tmas for analysis . erg protein expression was assessed semiquantitatively using 3-tiered system ( 0 , negative ; 1 , low ; 2 , high ) . cases with either 1 or 2 intensity were considered positive based on previous correlation with erg gene rearrangement as detected by fluorescent in situ hybridization ( data not shown ) . the erg antibody was consistently strongly expressed in endothelial cells , which acted as internal control for expression and intensity level . patient characteristics were presented as frequencies and percentages for categorical variables and as means and ranges for continuous variables . chi - square tests were used to test for associations between erg protein expression and gleason score , surgical margin , and pathological stage . the kaplan - meier approach along with the log - rank test was used for the survival analyses to test the association between erg expression and serum psa relapse . in all statistical tests , mean patients ' age of this cohort was 64 years ( range 42.780.5 years ) with average follow - up time of 4.8 years ( range 0.015.8 months ) . table 1 demonstrates patients ' demographics of the study cohort with respect to erg expression . overall , there were no significant differences between the two subgroups ( erg pos & erg neg ) of patients except for pathological stage , with 37% of erg positive tumors detected in pt3 versus 24% in pt2 . to investigate erg expression in different diagnostic categories , we characterized erg expression based on individual cores sampled . when we accounted for foamy type pca morphology , the rate of erg expression was 15/84 ( 17.9% ) , compared to 302/704 ( 42.9% ) in acinar pca ( cases with no foamy type morphology ) ( p < 0.001 ) . there was no difference in high erg intensity between foamy type and acinar pca ( data not shown ) . however , mean intensity level of erg in acinar pca was significantly higher than foamy type pca , 1.01 1.27 versus 0.37 0.83 ( p < 0.001 ) . erg intensity levels in hgpin were comparable to those in foamy type pca but slightly lower ( 0.13 0.56 ) but significantly lower than those in acinar pca ( p < 0.001 ) ( figure 1 ) . erg expression was noted in 106/280 ( 37.8% ) , 175/463 ( 37.8% ) , and 37/108 ( 34.2% ) of gleason scores 6 , 7 , and 810 , respectively . figure 2 demonstrates examples of erg expression in tissue samples and the distribution of erg in relation to gleason score . when investigating relations between erg expression and pathological parameters , there was significant association between erg expression and higher disease stage . in this cohort , erg expression was present in 50/131 ( 38.1% ) patients with pt2 versus 30/55 ( 54.5% ) patients with pt3 ( p = 0.04 ) . a similar association was also noted between erg expression and extra - capsular extension . in this cohort , 52/134 ( 38.8% ) erg positive patients demonstrated organ confined disease versus 29/53 ( 54.7% ) erg positive patients showing extra - capsular extension ( p = 0.04 ) . similar trends were noted between erg expression and seminal vesicle invasion , but this was not statistically significant ( p = 0.10 ) ( data not shown ) . no significant association was noted between overall erg positivity and positive surgical margins ( table 1 ) . similarly , no association was observed with postsurgical psa levels when assessed by univariate or multivariate analysis ( figure 3 ) . although not informative due to limited patients ' numbers , the association between erg expression and higher stage disease was more pronounced in patients with higher gs . in this cohort , none of the patients with gs > 7 and erg positive ( 0/7 ) were of stage pt2 compared to 47% ( 7/15 ) of erg negative patients who were of pt2 stage ( p = 0.02 ) ( table 2 ) . this study reports on the potential significance of erg protein expression in localized prostate cancer . erg gene rearrangements and erg expression have been documented in roughly 50% of localized prostate and locally advanced castrate resistant prostate cancer compared to 12%15% in watchful waiting or incidental cohorts [ 16 , 17 , 22 , 25 , 29 ] . published reports on the significance of erg expression to patients ' outcome are conflicting with some showing association with adverse outcome , while others document no association . some suggest that it indicates a better prognosis [ 16 , 22 , 25 , 2933 ] . however , it is proposed that erg signifies a molecular class of prostate cancer and may play a role in disease progression within those tumors . this pathway is closely linked to increased rate of pten genomic deletions as well as increased erg expression or erg gene rearrangements [ 34 , 35 ] . erg gene rearrangements and erg expression are associated with adverse outcome and lethal disease in watchful waiting or expectant cohorts [ 16 , 25 ] . moreover , patients with erg overexpression demonstrated shorter progression times to castrate resistance , needing surgical intervention ( channel turp ) . in localized prostate cancer , the majority of the reported data suggest no prognostic implication for erg rearrangements or erg overexpression in relation to psa relapse or the recurrence of local disease [ 22 , 36 , 37 ] . however , two earlier reports suggested adverse association of erg rearrangements with psa relapse after radical prostatectomy [ 20 , 31 ] . in nam 's study , yashimoto 's group noted that the adverse prognostic association was linked to pten genomic deletions . of note , a study by reid et al . documented that erg gene rearrangements in addition to pten genomic deletions had actually favorable outcomes compared to pten deletion alone . these observations suggest that the method utilized in determining erg status , the inclusion or exclusion of other genomic aberration , and the cohort chosen may be the reason for the different results obtained in various studies investigating the role of erg in prostate cancer . our results support previous observations showing no prognostic relationship between erg overexpression and clinical outcome in localized prostate cancer . however , in contrast to earlier studies , we document significant association between erg expression and some pathological parameters . in this cohort , patients whose tumors were erg positive were at higher risk of exhibiting extra - prostatic extension and increased disease stage compared to patients whose tumors did not express erg . specifically , all patients with gs > 7 and erg expression showed pt3 stage disease compared to none of pt2 . however , this data is limited by the number of patients within each gs subgroup and need further confirmation . more importantly , in addition to not documenting any clinical prognostic significance for erg expression , we did not observe any association between erg expression and other pathological parameters such as gleason score or surgical margins , which further diminishes any potential prognostic significance for erg expression , at least in localized pca . another issue worth mentioning regarding erg is that , erg gene rearrangements have been previously associated with specific histopathological features and detected more frequently in some morphologic variants of prostate cancer than others . in this study , we confirm lower rates of erg expression in tumors with foamy / xanthomatous morphology which demonstrated lower mean expression intensities compared to acinar pca . the significance of this is not yet known , but suggests that those two types of tumors may be different at the molecular level . in conclusion , this study demonstrates significant association between erg expression and extra - prostatic extension and higher pathological stage in localized prostate cancer . moreover , the lack of association with any other pathological parameters also significantly diminishes any potential clinical application of erg expression , at least in men with localized prostate cancer .

background . the prognostic significance of erg expression in prostate cancer ( pca ) has generated mixed results . we sought to investigate the prognostic significance of erg expression in a localized cohort of men with pca . material and methods . we investigated erg protein expression in a cohort of 198 men with localized pca . erg expression was correlated with patients ' clinical outcome and several pathological parameters , including gleason score ( gs ) , pathological stage , surgical margin , and extra - capsular extension . results . erg expression was detected in 86/198 ( 43.4% ) patients exclusively in neoplastic epithelium . overall , erg mean expression intensity was 1.01 1.27 versus 0.37 0.83 in acinar pca compared to foamy type pca ( p < 0.001 ) . in hgpin , erg intensity levels were comparable to those in foamy type pca ( 0.13 0.56 ) but significantly lower than those in acinar pca ( p < 0.001 ) . erg expression was significantly associated with extra - prostatic extension and higher pathological stage and showed a trend toward seminal vesicle invasion . herein , erg expression was documented in 50/131 ( 38.1% ) patients with pt2 versus 30/55 ( 54.5% ) patients with pt3 ( p = 0.04 ) . erg association with higher pathological stage was more pronounced in patients with gs > 7 . grouping patients into those with gs 7 versus > 7 , there was no significant association between erg expression and gs . similarly , no association was present in relation to either surgical margins or postsurgical serum psa levels . conclusion . we report significant association between erg protein levels and extra - prostatic extension and higher pathological stage . erg expression is not associated with adverse clinical outcome and is of limited prognostic value in localized pca .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

the three main arteries of the leg , the anterior tibial artery ( ata ) , posterior tibial artery and peroneal artery , form a dense vascular network around the distal leg , ankle , and foot , which ramifies perforators as the basis of all kinds of pedicle flaps such as lateral or medial malleolar perforator flaps . there is plenty of literature describing the anatomy and clinical application of perforators of the peroneal artery and posterior tibial artery around the distal lower leg . however , literature regarding perforator flaps of ata or anterior supramalleolar artery ( asma ) , one of the major branches of distal ata , are scant . these papers either mention the existence of the perforators from ata or asma , or describe their communication with the medial and lateral supramalleolar arteries . none of them have a detailed anatomical study on asma . the objective of this study was to identify the anatomic parameters of the asma and its perforators that would enable microsurgeons to harvest potentially multi - paddle flaps or composite flaps from the foot and ankle region . we try to preserve the ata intact after we harvest the composite flaps around the ankle using asma . we also want to use the ata as the only one pedicle and harvest multi - paddle flaps , decreasing the microsurgical risk greatly and minimizing the sacrifice of the blood supply to the ankle and foot . after the study was approved by the university of louisville irb ; 24 lower extremities of fresh human cadavers were studied . the ata was cannulated and injected with 20 ml of red latex ( microfilm , flow tech , inc . , ) at the mid - point of lower leg where ata can be identified between the extensor digitorum longus and tibialis anterior muscles . then , careful dissection along the distal 1/3 of the lower leg , the ankle , and the proximal foot ( 8 - 10 cm above the lateral malleolus and 1 - 2 cm anterior to the edge of the fibula ) was performed to observe the origin and the course of the asma . the distance between the origin of asma and the extensor retinaculum ( o - r ) was recorded . the diameters of the asma proper as well as its branches ( a set of fixed - width hooks was used for measurement ) , and the length of asma were measured . the collaterals between asma and the medial and lateral supramalleolar arteries and the number of perforators were observed . chi - square test was used for this study to analyse the variation of the o - r . fasciocutaneous flaps with multiple skin paddles were elevated based on the perforators to demonstrate its potential application . we classified the asma into four types according to the pattern of origin from the ata [ figures 1 - 4 ] . type a ( n = 10 ) asma originates from the ata with a 1 - 1.5 cm long main trunk and then gives off its lateral and medial branches [ figure 1a and b ] ; each consists of 2 - 3 perforators . in type b ( n = 7 ) , the asma does not have a main trunk , and its lateral and medial branches stem directly from ata with the sites of origin at the same level [ figure 2a and b ] . type c ( n = 6 ) is similar to type b , with the lateral and medial branches arising from the ata but at different origins [ figure 3a and b ] . type d ( n = 1 ) has 2 medial , and 1 lateral branch arising from the ata [ figure 4a and b ] . ( a ) type a the anterior supramalleolar artery originates as an artery stem . ( 1 ) short artery stem ; ( 2 ) lateral branches ; ( 3 ) medial branches ; ( 4 ) descendent perforators ; ( 5 ) ascendant perforators . ( 6 ) proximal end of anterior tibial artery ( ata ) ; ( 7 ) distal end of ata ; ( 8) blood supply to tibia ; ( 9 ) extensor retinaculum ( b ) the anterior supramalleolar artery originates as a 1 - 1.5 cm short artery stem and then ramifies lateral and medial branches . the medial branch gives off ascendant perforators and descendent perforators ( a ) type b the anterior supramalleolar artery originates as lateral and medial artery branches at the same point . ( 1 ) medial branches ; ( 2 ) ascendant perforators . ( 3 ) descendent perforators ; ( 4 ) lateral branches ; ( 5 ) proximal end of anterior tibial artery ( ata ) ; ( 6 ) distal end of ata ; ( 7 ) extensor retinaculum ; ( 8) blood supply to tibia ( b ) the anterior supramalleolar artery orients as lateral and medial artery branches at the same point . the medial branch gives off ascendant perforators and descendent perforators ( a ) type c the anterior supramalleolar artery originates as lateral and medial artery branches at the different point . ( 1 ) medial branches ; ( 2 ) ascendant perforators . ( 3 ) descendent perforators ; ( 4 ) lateral branches ; ( 5 ) proximal end of anterior tibial artery ( ata ) ; ( 6 ) distal end of ata ; ( 7 ) extensor retinaculum ( b ) the anterior supramalleolar artery orients as lateral and medial artery branches at different point . the medial branch gives off ascendant perforators and descendent perforators ( a ) type d - the anterior supramalleolar artery originates as lateral and two medial artery branches at the different point . ( 1 ) medial branches ; ( 2 ) lateral branches ; ( 3 ) proximal end of anterior tibial artery ( ata ) ; ( 4 ) distal end of ata ; ( 5 ) extensor retinaculum ( b ) the anterior supramalleolar artery orients as one lateral and two medial arteries branches at different point the mean distance between the origin of the proximal branch of asma and the superior margin of the extensor retinaculum ( o - r ) was 2.0 0.8 cm . the mean diameters of the medial branch ( d1 ) , lateral branch ( d2 ) and the main trunk of the asma ( d3 ) ( only in type a ) were 1.0 0.2 mm , 0.8 0.3 mm and 1.1 0.2 mm , respectively . the mean feasible length of the pedicles of the lateral flap ( l1 ) and medial flap ( l2 ) were 5.1 1.0 cm and 3.7 0.6 cm , respectively . bi - foliate fasciocutaneous flaps can be harvested based on the medial and lateral branches of the type a asma with preservation of the ata [ figure 5 ] . data of 24 specimens of the anterior supramalleolar artery the biofoliate flap using type a anterior supramalleolar artery huber dissected 200 feet and was the first to describe the asma as a vessel arising about 5 cm above the ankle joint . wee described that this septocutaneous branch of ata runs through the anterior crural septum between the tendons of tibialis anterior and extensor hallucis longus and gives out 3 perforators . further named the reverse - flow flap based on the perforators of this artery as anterior tibial type iii . later research showed that asma is always found in the distal 1/3 of the lower leg and appears 8 - 10 cm above the lateral malleolus , 1 - 2 cm anterior to the edge of the fibula . in 2003 , koshima dissected 4 ankles and described that the asma has 1 - 2 perforators just above the extensor retinaculum and gives off the anterolateral and anteromedial branches . however , we still lack detailed anatomical knowledge of its origin , calibre and so on . the diameters of the lateral and medial branches of asma are 0.8 0.3 mm and 1.0 0.2 mm , respectively . the feasible pedicle lengths of these two branches are 5.1 1.0 cm and 3.7 0.6 cm , respectively . thus , an island flap can be harvested based on one or both of the branches for defect coverage around the ankle . in addition , due to its collateral connection with the posterior tibial artery and peroneal artery , a reverse - flow anterolateral or anteromedial flap can also be designed as described in literature . the flaps with asma are very useful especially for patients who suffer from refractory malleolar ulcers due to paralysis , diabetes mellitus , venous stasis , or vasculitis . for patients who have cosmetic consideration , perforator - based adipofascial flap is also a good option with the use of asma . the perforators of the asma are suitable for a bi - foliate design in situations such as simultaneous defects of the volar and dorsal aspects of the hand . the flap based on asma is pliable and thin , which makes it an excellent option for hand and wrist coverage . if there is a simultaneous metacarpal deficiency at the same time , which can present especially in high - energy injuries , vascularised bone graft can also be harvested easily supplied by 1 - 2 perforators according to research by gilbert et al . an additional advantage of this perforator flap is that the ata can be preserved in type a asma , and morbidity of the donor site can be minimised . based on different types of asma , its lumen size can vary from 0.8 0.3 to 1.1 0.2 mm , which is still suitable for microvascular anastomosis . a difficult problem that a hand surgeon encounters conventional methods include inserting the hand into an abdominal pocket for several weeks followed by skin graft , using a large flap to cover all the defects together , or covering the wounds with several free flaps at the same time . often , these patients will still exhibit stiff digits and a bulky hand . and combined multiple single flaps would increase the surgical risk exponentially . in this instance , we propose a novel idea of using a multiple - foliate flap based on different perforators of the lateral and medial branches of the asma to cover multiple digits at the same time . these branches all derive from the same trunk - the ata and its terminal branch . this design provides the capability of harvesting up to 2 - 3 skin paddles based on one sizable pedicle , the ata . the terminal branches of the peroneal artery to ankle and foot , as well as the branches of posterior tibial artery to ankle and foot are also preserved . in this way , the vascular network of the ankle and foot is still preserved , and donor site morbidity is significantly decreased . there are several factors which should be kept in mind during the clinical application of asma based flaps . when the skin is opened , the lateral and medial branches of the asma are generally visible . if we want only the stem artery and their branches , we can just follow the course of these branches and easily find the origin of these branches . . however , in any situation it would be more effective to repair the extensor retinaculum carefully and immobilize the foot for 3 - 4 weeks . further study and long - term follow - up are needed to understand the blood supply of the ankle and foot after harvesting the asma - based flap . its sizable diameter and lengthy pedicle make it suitable as bi - foliate fasciocutaneous flaps or composite flaps .

purpose : a further understanding of the anterior supramalleolar artery ( asma ) and its potential applications in reconstructive surgery.materials and methods : a total of 24 fresh lower limbs from fresh cadavers were injected with red latex for dissection . the type of origin , course , diameter of the pedicle , and the distance between the origin of the asma from the anterior tibial artery to the extensor retinaculum ( o - r ) were recorded . bi - foliate fasciocutaneous flaps were harvested using the branches of the asma.results:we found four types of origin of the asma , and we have accordingly classified them into four types . 10 of them were type a , 7 were type b , 6 were type c and 1 was type d. the mean o - r ( origin of asma to retinaculum ) distance was 2.0 0.8 cm . the diameter of the medial branch ( d1 ) , the diameter of the lateral branch ( d2 ) , and the diameter of artery stem ( d3 ) ( only in type a ) were 1.0 0.2 mm , 0.8 0.3 mm , 1.1 0.2 mm , respectively . the mean pedicle length of the lateral flap ( l1 ) and medial flap ( l2 ) were 5.1 1.0 cm and 3.7 0.6 cm , respectively.conclusions:the asma exists constantly with four different types of origin . its sizable diameter and lengthy pedicle make it suitable for bi - foliate fasciocutaneous flap transfer .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

ankle dorsiflexion passive range of motion ( df prom ) measurements are performed in the field of physical therapy to estimate ankle motion during functional activities1 and to prevent lower extremity injuries2 . although in the clinical setting , ankle df prom is frequently measured under non - weight - bearing ( non - wb ) conditions1 , 3 , 4 , many researchers have stated that the wb position is more appropriate for estimating the amount of ankle df motion during functional activities5 , 6 . therefore , wb ankle df prom should be measured during interventions focused on increasing ankle df prom . limited ankle df prom with knee extended may result from gastrocnemius tightness and insufficient posterior talar glide7 . thus , gastrocnemius stretching and talocrural joint mobilization have been performed as intervention strategies to increase ankle df prom3 , 8 , 9 . previous studies have reported a significant increase in ankle df prom after these interventions3 , 8 , 9 ; however , to our knowledge , no study has demonstrated the combined effect of both interventions on wb ankle df prom . therefore , the aim of the present study was to examine the influence of gastrocnemius stretching combined with joint mobilization on wb ankle df prom . in total , 11 male subjects with bilateral limited non - wb ankle df prom with knee extended ( mean age , 22.82 3.09 years ; mean height , 175.91 3.39 cm ; mean weight , 69.55 3.78 kg ; mean non - wb ankle df prom , 4.17 2.48 ) participated in this study . inclusion criteria were 1 ) ankle df prom with knee extension < 10 ; 2 ) ankle df prom with knee flexion > 10 ; and 3 ) > 5 difference in ankle df prom between knee extension and knee flexion conditions on bilateral sides in non - wb positions3 . subjects with a history of surgery on the lower extremity , fracture , or neurological diseases were excluded from this study . all participants signed an informed consent form approved by the institutional research review committee of inje university prior to participation in this study . the study protocol of this study complies with the ethical standard of the declaration of helsinki . wb ankle df prom with knee extended was measured following the procedures suggested by munteanu et al10 . subjects stood in front of a wall and placed the leg being tested behind the contralateral leg in a lunge posture . subjects were asked to place both hands on the wall and then lean forward without heel - off and knee flexion until maximum stretch was felt in the gastrocnemius on the tested leg . the force applied to the tested leg was maintained at 60 5% of the subject s weight using scales11 . an examiner determined the maximum tibial inclination using an inclinometer to measure the wb ankle df prom with knee extended . measurements of wb ankle df prom were repeated 3 times for each ankle under pre- and post - intervention conditions . the mean value of 3 trials was used for data analysis . for gastrocnemius stretching combined with joint mobilization , subjects leaned forward against the wall in the same lunge posture as that during measurement of wb ankle df prom with knee extended until the maximum gastrocnemius stretch was felt . subjects held the end - range posture while an examiner provided the talus of the tested leg with sustained anterior - to - posterior gliding force . an intervention trial was performed for 30 s , and 10 trials were repeated with 30-s rest periods for each ankle . the difference in wb ankle df prom with knee extended between pre- and post - intervention conditions was analyzed using a paired t - test . wb ankle df prom with knee extended was significantly increased in post - intervention compared with pre - intervention conditions ( 42.60 5.49 versus 38.24 4.69 , p < 0.001 ) . our findings demonstrate that gastrocnemius stretching combined with joint mobilization significantly increases wb ankle df prom with knee extended . stretching exercises increase tolerance , resulting in increased rom12 . additionally , increased displacement of the myotendinous junction ( mtj ) after gastrocnemius stretching for 5 min was found in a previous study13 . therefore , the change in tolerance and/or increase in mtj displacement might have influenced our findings . the addition of talocrural joint mobilization to gastrocnemius stretching is another possible explanation for our findings . previous research by dinh et al.3 showed a 4.25 increase in wb ankle df prom with knee extended after gastrocnemius stretching alone for 3 weeks . although gastrocnemius stretching combined with joint mobilization was applied for 5 min in the present study , the amount of increase in wb ankle df prom after intervention ( i.e. , 4.36 ) was similar to that found previously . considering this outcome , despite the relatively short period of intervention in the present study , it may be inferred that the addition of talocrural joint mobilization might maximize the effects of general gastrocnemius stretching . thus , we conclude that gastrocnemius stretching combined with joint mobilization might decrease gastrocnemius tightness and increase posterior talar gliding movement , which effectively increases wb ankle df prom with the knee extended . first , although non - wb ankle df prom was used as an inclusion criterion , changes in non - wb ankle df prom after intervention were not measured . however , we believe that wb ankle df prom is clinically more important because most functional activities are performed under the wb condition . second , our study included only male subjects , and the results can not be generalized to women .

[ purpose ] the purpose of this study was to investigate the effect of gastrocnemius stretching combined with talocrural joint mobilization on weight - bearing ankle dorsiflexion passive range of motion . [ subjects ] eleven male subjects with bilateral limited ankle dorsiflexion passive range of motion with knee extended participated in this study . [ methods ] all subjects received talocrural joint mobilization while performing gastrocnemius stretching . ankle dorsiflexion passive range of motion was measured using an inclinometer under weight - bearing conditions before and immediately after intervention . a paired t - test was used to analyze the difference between weight - bearing ankle dorsiflexion passive range of motion pre- and post - intervention . [ results ] a significant increase in weight - bearing ankle dorsiflexion passive range of motion was found post - intervention compared with pre - intervention . [ conclusion ] these findings demonstrate that gastrocnemius stretching combined with joint mobilization is effective for increasing weight - bearing ankle dorsiflexion passive range of motion .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

dox is one of the key chemotherapeutic drugs for cancer treatment , but its use is limited by chronic and acute toxic side effects . dox is an antibiotic anthracycline that was isolated from a pigment of streptomyces peucetius in the early 1960s and it had been employed for more than 30 years in the battle against cancer , but it is now chemically synthesized . renal dox - induced toxicity may be part of a multiorgan damage mediated mainly through free radical formation eventually leading to membrane lipid peroxidation . induction of apoptosis and modulation of nox are mechanisms that are involved in toxic adverse effects associated with dox therapy . in addition , dox has a direct renal damaging effect as it accumulates preferentially in the kidney . dox has toxic effects on other organs such as heart and liver which may lead to modulation of blood supply to the kidney and alter xenobiotic detoxification processes , respectively , thus indirectly contributing to dox - induced nephropathy . dia is a new anti - inflammatory , analgesic , and antipyretic drug that was developed specially for the treatment of osteoarthritis . it is highly effective in relieving the symptoms of osteoarthritis and may be able to modify the course of the disease . interleukin 1 is a proinflammatory and proapoptotic agent that induces cytokine production by activating nfb and mitogen activated protein kinase signaling . a major cause of dox - induced nephrotoxicity is the production of reactive oxygen species which induce cytokines , including interleukin 1 [ 6 , 9 , 10 ] . the aim of the present study was to study the effect of the interleukin 1 receptor antagonist diacerein ( dia ) on dox - induced nephropathy . dia powder was from eva pharma company and it was dissolved in 1% carboxymethylcellulose . dox hydrochloride 10 mg vial ( pharmacia italia , spa , italy ) , polyclonal rabbit / antirat caspase-3 , tnf , and nfb antibody ( lab vision , usa ) , biotinylated goat anti - rabbit secondary antibody ( transduction laboratories , usa ) , urea , gsh , sod , and catalase kits ( biodiagnostic , egypt ) , and creatinine ( humen , germany ) were purchased . adult male wistar rats weighing about 250350 g were obtained from the animal research centre , giza , egypt . animals were kept in standard housing conditions in cages and were left to acclimatize for one week . this work was conducted in the pharmacology department , faculty of medicine , el - minia university , egypt , and the animal experimental protocol was approved by the faculty board . rats were randomly assigned into 6 groups ( n = 6 each ) as follows . group i received vehicle ( 1% carboxymethylcellulose ) for 15 days and ip saline at day 11 . group ii was treated with dld ( 25 mg / kg / d orally ) for 15 days and ip saline at day 11 . group iii was treated with dhd ( 50 mg / kg / d orally ) and ip saline at day 11 . group iv was treated with vehicle for 15 days and dox ( 15 mg / kg ) at day 11 . group v was treated with dld ( 25 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . group vi was treated with dhd ( 50 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . the doses of dox and dia were based on the previous studies [ 4 , 11 ] . group i received vehicle ( 1% carboxymethylcellulose ) for 15 days and ip saline at day 11 . group ii was treated with dld ( 25 mg / kg / d orally ) for 15 days and ip saline at day 11 . group iii was treated with dhd ( 50 mg / kg / d orally ) and ip saline at day 11 . group iv was treated with vehicle for 15 days and dox ( 15 mg / kg ) at day 11 . group v was treated with dld ( 25 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . group vi was treated with dhd ( 50 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . the doses of dox and dia were based on the previous studies [ 4 , 11 ] . after 4 days of dox injection , each rat was weighed then anesthetized with ip injection of urethane ( 25% in a dose of 1.6 gm / kg ) and then sacrificed . a longitudinal section of the left kidney and one half was fixed in 10% formalin then embedded in paraffin for histopathological and immunohistochemical examinations . the rest of the kidneys were snap frozen in liquid nitrogen and kept at 80c . briefly , the method is based on the fact that sulfhydryl group of gsh reacts with 5 , 5-dithiobis ( 2-nitrobenzoic acid ) ( ellman 's reagent ) and produces a yellow colored 5-thio-2-nitrobenzoic acid which was measured colorimetrically at 405 nm using beckman du-64 uv / vis spectrophotometer , usa . assessment of renal catalase antioxidant enzyme activity was determined from the rate of decomposition of h2o2 at 510 nm after the addition of tissue homogenate as described by colorimetric kit . the assessments of sod levels were based on the ability of the enzyme to inhibit the phenazine methosulfate - mediated reduction of nitroblue tetrazolium dye and the results were expressed as unit / g tissue . the renal contents of lipid peroxides were assayed by a spectrophotometric method based on the reaction between mda and thiobarbituric acid . the absorbance values of the samples and the blank were determined at 535 nm using a ( beckman du-64 spectrophotometer , usa ) and then blank absorbance value was subtracted from the sample absorbance value . from a standard curve , mda concentration in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol / gm tissue by multiplying in the tissue dilution factor . nitric oxide ( no ) in the form of nitrite was determined with spectrophotometric method using griess reagent systems . the stable oxidation end products of no , nitrite ( no2 ) , and nitrate ( no3 ) were used as indicators of no production . nox was measured after the reduction of nitrate to nitrite by copperized cadmium granules in glycine buffer at ph 9.7 . quantification of no2 was based on the griess reaction , in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of n - naphthylene diamine and sulfanilamide . the absorbance of the sample and the blank were measured at 545 nm using ( beckman du-64 spectrophotometer , usa ) . the blank absorbance is then subtracted from the sample absorbance . from a standard curve , nox content in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol / g tissue . renal tissue was fixed in 10% formalin , embedded in paraffin , sectioned by a microtome at 5 m thickness and stained with hematoxylin and eosin for routine histopathological assessment . the renal tissues were examined in random microscopic areas semiquantitatively under 40 high power fields and the number of changes was assessed by the counting of 3 nonoverlapped fields for the same slide of each animal . the frequency and the severity of lesions in the kidneys were assessed semiquantitatively as follows : score : assigned normal , score + : in between normal and mild , score + + ( mild level ) : less than 25% of the examined fields revealed histological alterations , score + + + ( moderate level ) : less than 50% of the examined fields revealed histological alterations , and score + + + + ( severe level ) : less than 75% of the total fields examined revealed histological alterations . the caspase-3 , tnf , and nfb immunolabeled cells were counted . in each animal , 3 sections were examined and the cells were counted in 3 adjacent nonoverlapping fields levels . immunohistochemical staining was performed for caspase-3 , tnf , and nfb using polyclonal rabbit / antirat antibody according to previously published protocol [ 20 , 21 ] , respectively . table 1 shows the results of the effect of dia on serum creatinine and urea . rats receiving a single dose of dox ( 15 mg / kg , ip ) showed a significant increase in serum creatinine and urea levels compared to control group . both doses of dia resulted in significant decrease in serum creatinine compared to dox treated rats . dia 50 mg / kg / day but not 25 mg / kg / day resulted in significant decrease in serum urea compared to dox treated rats . renal mda was evaluated as an indicator of kidney lipid peroxidation and nitrites and nitrates as an indicator of renal nox levels ( table 1 ) . dox ( 15 mg / kg ) significantly increased renal mda and nox levels compared to control group . administrating both doses of dia to dox treated rats significantly decreased mda and nox compared to dox treated group . treatment with dox ( 15 mg / kg ) caused significant decrease in renal gsh , sod , and catalase levels compared with untreated control group ( table 2 ) . concomitant treatment of dox with dia significantly increased the levels of renal gsh , sod , and catalase compared to dox treated group . the histological study of the rat renal cortical tissue of control group ( figure 1(a ) ) , dld ( 25 mg / kg / day ) group ( figure 1(b ) ) , and dhd ( 50 mg / kg / day ) group ( figure 1(c ) ) showed normal architecture of renal glomeruli and tubules . dox treated group ( figure 1(d ) ) showed marked enlargement of some vascular glomeruli which tightly fill the renal corpuscles . dox + dld group ( figure 1(e ) ) showed amelioration of the damaging effects of dox . there were less tubular distortion , narrow bowman 's spaces , and fewer cytoplasmic vacuolations of renal corpuscle and tubular cells were also observed . dox + dhd group ( figure 1(f ) ) had more obvious decrease in the morphological changes caused by dox exposure . the severity of the morphological changes was assessed semiquantitatively ; dox exposed group showed increase in the glomerular and tubular morphological changes at the light microscopic levels when compared with control group . these changes were suppressed by the administration of both doses of dia , but the high dose showed marked improvement than the low dose ( table 3 ) . administration of dox caused significant increase in the immunoreactivity of caspase-3 , nfb , and tnf ( figures 2 , 3 , and 4 and table 4 ) respectively , which were highly expressed in both renal glomeruli and tubules cytoplasmically and in some nuclei . administration of both doses of dia concomitantly with dox decreased the expression of them , compared to dox group . administration of both doses of dia in vehicle treated rats alone and control groups showed no expression . effective anticancer therapy with anthracyclines as dox is limited because of its toxicity to various organs including kidneys . nephrotoxic action of dox is also considered to be via drug - induced free radical generation . the formation of free radicals induces the production of proinflammatory cytokines as interleukin 1 initiating the biological effects associated with inflammation . this directed our attention to investigate the role of dia which is interleukin 1 receptor antagonist as a possible nephroprotective agent against dox - induced renal damage . induction of dox nephrotoxicity was detected in our study by significant elevation of serum urea and creatinine levels which were confirmed by toxic histopathological changes compared to control group . urea and serum creatinine are the most sensitive markers of nephrotoxicity implicated in the diagnosis of renal injury [ 24 , 25 ] . the nephrotoxic effect of dox is characterized by decreasing glomerular filtration rate leading to a rise in serum urea and creatinine . our results are in good agreement with the previous studies [ 22 , 26 ] . improvement of dox - induced nephrotoxicity was previously tried by compounds that partially succeeded in preserving normal renal function and structure probably through their antioxidant and anti - inflammatory effects as caffeic acid phenethyl ester , zingiber officinale roscoe , and solanum torvum so that we investigated the role of another antioxidant and anti - inflammatory drug as dia on dox - induced nephrotoxicity . dia could significantly decrease serum urea and creatinine compared to dox treated group . that is due to the anti - inflammatory and antioxidant effects of dia which suppress dox mediated oxidative stress , inflammation , and tissue damage . our histopathological changes showed that dox treated group presented with marked damage of renal tubules . these results are in agreement with zhao et al . who detected the protective effect of rhein ( the active metabolite of dia ) on acetaminophen induced hepatotoxicity and nephrotoxicity in rats . they found that serum urea and creatinine significantly decreased in rhein and acetaminophen coadministration compared to acetaminophen group and normalization of toxic histopathological changes . the elevated levels of gsh could effectively provide thiol group for the possible gsh mediated detoxification reactions of gpx ( glutathione peroxidase ) and gst ( glutathione - s - transferase ) which is involved in the scavenging of o2 generated from the dox . our findings are consistent with the previous reports that showed that gsh concentration is significantly decreased upon dox treatment compared to control group [ 4 , 22 ] . sod extensively distributes in all cells and has a significant shielding role against oxidative injury induced by reactive oxygen species . in our study , the activities of sod and catalase significantly decreased in dox treated rats in kidney as compared to control rats . the accumulation of these highly reactive free radicals leads to the reduction of the activity of sod and catalase which in turn results in damaging effects in the form of loss of cell membrane integrity and function . the decrease in the sod and catalase activities related to the increase in the intracellular levels of h2o2 . catalase has been reported to be responsible for the detoxification of h2o2 , which is an effective inhibitor of sod . coadministration of dia significantly improved sod , gsh , and catalase levels compared to dox treated group . these results may be due to antioxidant effect of dia which was approved previously by tamura et al . who indicated the inhibitory effect of dia on indomethacin - induced gastric ulceration which could be mediated by the suppression of reactive oxygen species production based on its inhibition of neutrophil activation and antioxidant activity . in addition , hu et al . investigated the protective effects of rhein lysinate ( rhl ) , against kidney impairment in senescence - prone inbred strain 10 ( samp10 ) mice . treatment of samp10 mice with rhl significantly increased the sod and gpx levels in the kidneys . o2 reacts with lipid to form lipid peroxides followed by -oxidation to form mda . that was detected in our study which showed significant increase of mda level in dox treated group compared to control group . these results are in agreement with el - sheikh et al . and yagmurca et al . the high production of nox results in peroxynitrite formation which is a potent and aggressive cellular oxidant and is involved in dox toxicity . the current findings showed that dox administration significantly increased renal level of nox compared to control group and that is in agreement with other studies [ 26 , 37 ] . these results are in agreement with zhao et al . who detected the protective effect of rhein on acetaminophen induced nephrotoxicity in rats which was approved by significant decrease of mda and nox on coadministration of rhein plus acetaminophen group compared to acetaminophen group . our results are in agreement with martel - pelletier and pelletier who reported that no is produced through the activity of inducible nitric oxide synthase and it is a major catabolic factor involved in the pathophysiology of oa . our results are consistent with hu et al . who investigated the protective effects of rhein lysinate ( rhl ) , against kidney impairment in senescence - prone inbred strain 10 ( samp10 ) mice . induction of p53 mediates cell apoptosis through activation of caspase-3 family of proteases and apoptotic cell death . our study is showing significant increase in caspase-3 expression in dox treated group in comparison with control group . our study is in consistence with torina et al . who showed that treatment with dia once a day for 4 weeks after myocardial infarction improved ventricular remodeling by partial blockage of the proinflammatory cytokines which led to lower caspase-3 activity and nfb p65 transcription b pathway . dox - induced superoxide anion production which was reported to be responsible for tnf-induced nuclear factor ( nf ) activation that increases nf and tnf over expression . our study showed significant increase in tnf and nfb expressions in dox group compared to control group and the same results were found with al - saedi et al . . coadministration of dia significantly decreased tnf and nfb expression compared to dox treated group that is in agreement with gadotti et al . who showed that dia inhibits neuropathic pain by decreasing proinflammatory cytokines as tnf and nf. also , hu et al . the active metabolite of dia is rhein which possesses anti - inflammatory activity and may be effective in suppressing the inflammatory cytokines contributing to the pathogenesis of diabetic nephropathy . . demonstrated that rhein had protective effect in different models of nephropathy as iga induced nephropathy , obstructive nephropathy , chronic allograft nephropathy , and high glucose and angiotensin ii induced nephropathy . oral administration of rhein ( 150 mg / kg / d ) ameliorated renal lesions . rhein was capable of protecting against renal injury by decreasing the activities of nfb and caspase-3 in the early phase of glomerulosclerosis . our results are consistent with meng et al . who reported that rhein possesses various pharmacological activities , including anti - inflammatory , antioxidant , and antitumor . in their study , a model of hyperuricemia and nephropathy induced by adenine and ethambutol in mice was established . the results demonstrated that rhein significantly improved the symptoms of nephropathy through decreasing the production of proinflammatory cytokines , including interleukin 1 , prostaglandin e2 , and tnf. yu et al . aimed to explore the effect of rhein on sepsis - induced acute kidney injury by injecting lipopolysaccharide ( lps ) and cecal ligation and puncture ( clp ) in vivo and on lps - induced hk-2 cells in vitro . rhein could significantly decrease concentration of serum urea and creatinine and level of tnf , nfb , and il-1 in two different mouse models of experimental sepsis . in conclusion , dia protected against dox - induced nephrotoxicity in rats most probably due to its antioxidant and anti - inflammatory activities . however , dhd ( 50 mg / kg / day ) showed more protective effect than dld ( 25 mg / kg / day ) .

nephrotoxicity is one of the limiting factors for using doxorubicin ( dox ) . interleukin 1 has major role in dox - induced nephrotoxicity , so we investigated the effect of interleukin 1 receptor antagonist diacerein ( dia ) on dox - induced nephrotoxicity . dia ( 25 and 50 mg / kg / day ) was administered orally to rats for 15 days , in the presence or absence of nephrotoxicity induced by a single intraperitoneal injection of dox ( 15 mg / kg ) at the 11th day . we measured levels of serum urea , creatinine , renal reduced glutathione ( gsh ) , malondialdehyde ( mda ) , total nitrites ( nox ) , catalase , and superoxide dismutase ( sod ) . in addition , caspase-3 , tumor necrosis factor alpha ( tnf ) , nuclear factor kappa b ( nfb ) expressions , and renal histopathology were assessed . our results showed that dox - induced nephrotoxicity was ameliorated or reduced by both doses of dia , but diacerein high dose ( dhd ) showed more improvement than diacerein low dose ( dld ) . this protective effect was manifested by significant improvement in all measured parameters compared to dox treated group by using dhd . dld showed significant improvement of creatinine , mda , nox , gsh , histopathology , and immunohistochemical parameters compared to dox treated group .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

suicide is a leading cause of death among adolescents and has become a significant public health issue globally ( 1 ) . statistics korea report 2014 showed that suicide is the cause of 28.4% of adolescent deaths in 2013 , and it was the number one cause of death among adolescents ( 2 ) . furthermore , suicide is the second to third cause of mortality among adolescents in the western world and is an important concern in developing countries ( 3 ) . numerous causes of suicidal behavior during adolescence have been reported , including mental disorders , peer affiliations , and level of achievement in school ( 4 ) . additionally , weight - related concerns are a common cause of suicidal behaviors ( 56 ) . this transition often leads to confusion about personal identity , especially in the context of enduring developments in identity and cognition as well as physical growth ( 7 ) . adolescents typically enter a period of a high level of self - consciousness about their body shape and size ( 8) . they have a greater tendency to consider how they look to others at this life stage than during any other period of life ( 9 ) . these weight - related issues are influenced by many factors including friends , peers , social norms , and media ( 10 ) . the media is a particularly strong influence because it has become a general source of information and adolescents are easily exposed to it ( 11 ) . the media encourages as " ideal " slim and thin body shapes for girls and muscular bodies for boys ( 1213 ) , and provides abundant information on the newest diet methods and quick fixes for weight control ( 14 ) . this may induce adolescents , who have a particularly keen interest in their figure , to distort their body image and to engage in unneeded or inappropriate weight control behaviors ( wcb ) ( 15 ) . many adolescents engage in inappropriate wcb such as vomiting , taking nonprescription diet pills , and taking laxatives or diuretics . among korean adolescents engaging in wcb , 13.4% of boys and 18.8% of girls took inappropriate methods to control their weight ( 16 ) . according to a study using data from national surveys , similar rates were also observed in the united states , with 10% of boys and 21.3% of girls conducting at least one inappropriate wcb ( 17 ) . inappropriate wcb are a harmful influence on adolescents ' physical and psychological development ( 12 ) . for instance , a 5-year longitudinal study reported that engagement in inappropriate wcb can be a predictor of obesity and eating disorders ( 18 ) . moreover , adverse outcomes of inappropriate wcb also include the onset of depression ( 19 ) and suicide ideation ( 20 ) . due to the high prevalence of and the possible negative side effects associated with inappropriate wcb , it is a growing serious public health concern . therefore , the prevention of inappropriate wcb among adolescents is imperative to protect them from negative physical and psychosocial consequences . to date , studies about inappropriate wcb merely suggest the association between inappropriate wcb and negative psychosocial consequences ( 212223 ) . in the present study , we investigate not only the association between inappropriate wcb and suicide ideation and attempt , but also the difference in this association with body mass index , body weight perception , and body shape misperception . moreover , we investigate the top five wcb combinations used by korean adolescents and examine the association between these five wcb combinations and suicide ideation and attempt . we used a cross - sectional design ; thus , the results should be interpreted with caution due to possibility of a bi - directional effect . we used data from the 2014 korea youth risk behavior web - based survey ( kyrbs ) . the kyrbs has been conducted annually since 2005 by the korean centers for disease control and prevention ( kcdc ) , the korea ministry of education , science and technology , and the korea health and human services . the data were collected via an ongoing , anonymous web - based survey in a self - reporting format that was conducted on a nationally representative sample of middle- and high - school students . it aimed to plan and assess the korean adolescent health promotion policies by investigating health - related behaviors and status . in the 2014 survey , 799 middle and high schools were selected , including 72,060 students in grades 7 to 12 ( stratified ) on a national scale . however , we excluded 2,334 individuals due to missing data for variables used in this study ; therefore , our cohort ultimately comprised 69,726 adolescents ( 35,224 boys and 34,361 girls ) . suicide ideation and attempt were measured via responses to the following questions : " have you seriously considered suicide during the past 12 months ? " and " have you tried suicide during the past 12 months ? " the possible responses to both questions were " yes " or " no . " we characterized wcb via responses to the following multi - part question : " have you experienced the following weight control methods during the past 30 days , with the following activities listed : 1 ) did regular exercise , 2 ) fasted at least 24 hours , 3 ) ate less , 4 ) took prescription diet pills , 5 ) took nonprescription diet pills , 6 ) took laxatives or diuretics , 7 ) vomited , 8) ate only one food , 9 ) took oriental medicine , and 10 ) ate diet food . " the possible responses to all questions were " yes " or " no . " if participants responded ' yes ' to at least one of 2 ) , 5 ) , 6 ) , 7 ) , and 8) , we classified them into the " inappropriate wcb " group ( 16 ) . if participants responded ' no ' at all of 2 ) , 5)-8 ) , we classified them into the " appropriate wcb " group . if participants responded ' no ' to all activity choices , we classified them into the " nothing " group . body mass index ( bmi ) was calculated by self - reported height and weight . bmi percentiles for age and gender were calculated according to the 2007 standard growth charts for korean children and adolescents by the korean pediatric society ( 24 ) , and four categories were created : underweight ( 15th percentile ) , normal weight ( 16th to 84th percentile ) , overweight ( 85th to 95th percentile ) , and obese ( 95th percentile or bmi 25 ) . we further grouped overweight and obese groups into a single overweight group , thus using three bmi categories body weight perception ( bwp ) was split into five categories : very underweight , underweight , normal weight , overweight , and very overweight . we further grouped these categories into three groups : thin , normal , and obese . body shape misperception ( bsm ) was determined by an agreement between bmi and bwp categories . participants perceiving their weight at least one category below their actual bmi categories were designated part of the " underestimate " group , while those perceiving their weight at least one category above their actual bmi were designated part of the " overestimate " group ( i.e. , if participants whose bmi was a normal weight perceived their body weight as obese , they were placed in the " overestimate " group ) ( 25 ) . control variables included age , household economic status , parents ' presence , residential area , school level , academic achievement in school , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress level , and depression . we performed statistical analyses of the survey data using sas version 9.4 ( sas inc . , the relevant primary sampling units , sample weights , and stratification were considered in the analysis given that the kyrbs was designed as a complex sample . a pearson 's test was used to determine significant differences in the distribution of each variable . next , a logistic regression analysis was conducted to determine the association between wcb and suicide ideation and attempt . additionally , we performed a set of subgroup analyses using bwp , bmi , and bsm to determine whether body related variables led to differences in the association between wcb and suicide ideation and attempt . results were presented as adjusted odds ratios ( ors ) with 95% confidence intervals ( 95% cis ) . the ethics approval for the publicly open kyrbs data was waived by the institutional review board ( irb ) . after the purpose of the survey was fully explained to the students , written informed consent was provided . students were assured that they could refuse or withdraw from the research at any stage ( 16 ) . we used data from the 2014 korea youth risk behavior web - based survey ( kyrbs ) . the kyrbs has been conducted annually since 2005 by the korean centers for disease control and prevention ( kcdc ) , the korea ministry of education , science and technology , and the korea health and human services . the data were collected via an ongoing , anonymous web - based survey in a self - reporting format that was conducted on a nationally representative sample of middle- and high - school students . it aimed to plan and assess the korean adolescent health promotion policies by investigating health - related behaviors and status . in the 2014 survey , 799 middle and high schools were selected , including 72,060 students in grades 7 to 12 ( stratified ) on a national scale . however , we excluded 2,334 individuals due to missing data for variables used in this study ; therefore , our cohort ultimately comprised 69,726 adolescents ( 35,224 boys and 34,361 girls ) . suicide ideation and attempt were measured via responses to the following questions : " have you seriously considered suicide during the past 12 months ? " and " have you tried suicide during the past 12 months ? " the possible responses to both questions were " yes " or " no . " we characterized wcb via responses to the following multi - part question : " have you experienced the following weight control methods during the past 30 days , with the following activities listed : 1 ) did regular exercise , 2 ) fasted at least 24 hours , 3 ) ate less , 4 ) took prescription diet pills , 5 ) took nonprescription diet pills , 6 ) took laxatives or diuretics , 7 ) vomited , 8) ate only one food , 9 ) took oriental medicine , and 10 ) ate diet food . " if participants responded ' yes ' to at least one of 2 ) , 5 ) , 6 ) , 7 ) , and 8) , we classified them into the " inappropriate wcb " group ( 16 ) . if participants responded ' no ' at all of 2 ) , 5)-8 ) , we classified them into the " appropriate wcb " group . if participants responded ' no ' to all activity choices , we classified them into the " nothing " group . body mass index ( bmi ) was calculated by self - reported height and weight . bmi percentiles for age and gender were calculated according to the 2007 standard growth charts for korean children and adolescents by the korean pediatric society ( 24 ) , and four categories were created : underweight ( 15th percentile ) , normal weight ( 16th to 84th percentile ) , overweight ( 85th to 95th percentile ) , and obese ( 95th percentile or bmi 25 ) . we further grouped overweight and obese groups into a single overweight group , thus using three bmi categories body weight perception ( bwp ) was split into five categories : very underweight , underweight , normal weight , overweight , and very overweight . we further grouped these categories into three groups : thin , normal , and obese . body shape misperception ( bsm ) was determined by an agreement between bmi and bwp categories . participants perceiving their weight at least one category below their actual bmi categories were designated part of the " underestimate " group , while those perceiving their weight at least one category above their actual bmi were designated part of the " overestimate " group ( i.e. , if participants whose bmi was a normal weight perceived their body weight as obese , they were placed in the " overestimate " group ) ( 25 ) . control variables included age , household economic status , parents ' presence , residential area , school level , academic achievement in school , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress level , and depression . we performed statistical analyses of the survey data using sas version 9.4 ( sas inc . , the relevant primary sampling units , sample weights , and stratification were considered in the analysis given that the kyrbs was designed as a complex sample . a pearson 's test was used to determine significant differences in the distribution of each variable . next , a logistic regression analysis was conducted to determine the association between wcb and suicide ideation and attempt . additionally , we performed a set of subgroup analyses using bwp , bmi , and bsm to determine whether body related variables led to differences in the association between wcb and suicide ideation and attempt . results were presented as adjusted odds ratios ( ors ) with 95% confidence intervals ( 95% cis ) . the ethics approval for the publicly open kyrbs data was waived by the institutional review board ( irb ) . after the purpose of the survey students were assured that they could refuse or withdraw from the research at any stage ( 16 ) . the demographics of the 35,224 boys and 34,361 girls in this study are listed in table 1 . of the participants , 10.6% ( n = 3,709 ) of boys and 15.0% ( n = 5,172 ) of girls reported that they experienced suicide ideation , while 2.1% ( n = 722 ) of boys and 3.4% ( n = 1,151 ) of girls reported attempting suicide . 33.5% ( n = 11,827 ) of boys and 45.2% ( n = 15,489 ) of girls reported that they engaged in appropriate wcb , and 4.2% of boys ( n = 1,466 ) and 9.6% of girls ( n = 3,324 ) reported that they engaged in inappropriate wcb . boys who had engaged in inappropriate wcb were more likely to report suicide ideation than those not engaging in wcb group ( or , 1.57 , p < 0.001 ) . girls engaging in inappropriate wcb also had a high suicide ideation rate ( or , 1.32 , p < 0.001 ) . boys engaging in inappropriate wcb were also more likely to attempt suicide than those not engaging in wcb ( or , 2.49 , p < 0.001 ) . girls engaging in appropriate wcb ( or , 1.21 , p = 0.02 ) or inappropriate wcb ( or , 1.92 , p < 0.001 ) were more likely to attempt suicide than girls not engaging in wcb at all , and the association was stronger among the latter comparison group . the association between suicidal behavior and wcb stratified by bmi , bwp , and bsm is shown in table 3 . boys with an underweight bmi and that engaged in inappropriate wcb experienced the highest rate of suicide ideation ( or , 2.22 , p < 0.05 ) compared to other bmi groups . girls with an underweight bmi and that engaged in inappropriate wcb were 4.53 times more likely to attempt suicide than girls who did not engage in wcb . girls who perceived their weight as normal and engaged in inappropriate wcb were more likely to experience suicide ideation ( or , 1.50 , p < 0.001 ) and attempt ( or , 2.31 , p < 0.001 ) than girls who did not engage in wcb . considering bsm , boys who distorted their body shape and who engaged in inappropriate wcb were more likely to experience suicide ideation ( underestimate : or , 1.44 , p < 0.01 , overestimate : or , 1.91 , p < 0.01 ) and attempt ( underestimate : or , 1.96 , p < 0.01 , overestimate : or , 2.94 , p < 0.01 ) than boys who do not engaged in wcb . girls who underestimated or overestimated their body shape and engaged in inappropriate wcb showed significant association with suicide attempt ( underestimate : or , 1.89 , p < 0.001 , overestimate : or , 1.73 , p < 0.001 ) compared to girls who do not engaged in wcb . adjusted for age , sex , household economic status , parents presence , residential area , school level , academic achievement , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress status , and depression . can not be calculated due to the lack of number . table 4 lists the odds ratios of the top five wcb combinations engaged in by korean adolescents and their association with suicide behaviors . conducting regular exercise and eating less together with fasting was significantly associated with suicidal ideation ( boys : or , 1.70 , p < 0.01 , girls : or , 1.33 , p < 0.05 ) and attempt ( boys : or , 1.84 , p < 0.05 , girls : or , 2.24 , p < 0.001 ) among both boys and girls , and girls had higher rates of attempted suicide . adjusted for age , sex , household economic status , parents presence , residential area , school level , academic achievement , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress status , and depression . in the present study , we investigated association between inappropriate wcb and suicide ideation and attempt by korean adolescent by using nationally represented data . inappropriate wcb was significantly associated with suicidal behaviors in both boys and girls , and with a higher rate in boys . our study findings of a higher likelihood of suicidal behaviors among adolescents engaging in wcb ( especially inappropriate wcb ) , confirm results described in previous studies . adolescents using inappropriate methods to control weight are highly likely to experience psychological symptoms including anxiety , fatigue , and impaired concentration ( 26 ) . additionally , there is a prospective association between wcb and depression ( 27 ) . depression in adolescence is a serious public health concern due to the increased risk of suicide among depressed youth ( 28 ) . moreover , wcb can cause eating disorders , with fasting an especially high risk factor for development of eating disorders ( 29 ) . another study among adolescents and with a 5 year follow up reported that unhealthy wcb were the most powerful predictors of eating disorders ( 18 ) . eating disorders have adverse consequences on adolescents ' physical and mental health ( 30 ) , and can cause suicide ideation and attempt ( 31 ) . therefore , one possible interpretation of this finding is that wcb represent a risk factor for suicide ideation and attempt by causing depression or eating disorders , both of which have serious consequences on adolescents ' mental health . furthermore , our study demonstrated stronger associations among boys than girls , which is corroborated by other studies ( 623 ) . girls are more sensitive to their weight and easily engage in inappropriate wcb ( 32 ) , whereas boys are not as sensitive about their weight and have a lower likelihood of engaging in inappropriate wcb ( 7 ) . therefore , it is possible that boys who consider controlling their weight in inappropriate ways may already be suffering from severe psychological distress due to experiencing immense trauma from teasing or other adverse events . we investigated differences in association between suicide ideation and attempt and wcb by conducting subgroup analyses using bmi , bwp , and bsm . in the context of bmi , underweight boys engaged in inappropriate wcb experienced the highest rate of suicide ideation and attempt than other bmi groups , and underweight girls were considerably more likely to attempt suicide . additionally , suicidal behavior was also associated with boys and girls of a normal weight engaged in inappropriate wcb . this suggests that adolescents who do not need to control their weight but engage in unnecessary wcb nonetheless are at higher risk for experienced adverse consequences ( 12 ) . with regards to bsm , the results of this study showed that misperception of body shape is also associated with suicide ideation and attempt . the media continues to portray slender bodies for girls and muscular bodies for boys as ideal body shapes , and this could have an effect on body shape misperception among adolescents ( 33 ) . this mismatch contributes to adolescent depression , which in turn increases the risk of suicide among youth ( 34 ) . moreover , this mismatch could be related to adolescents ' unnecessary weight control behaviors to obtain the ideal body shape . eventually , the effect of inappropriate wcb aggravates adverse effects from a distorted body image , and it seems that this psychological burden causes adolescents to think about or attempt suicide . we also extracted the five most common combinations of weight control methods used by korean adolescents and investigated the association between these wcb and suicidal behaviors . only conducting regular exercise was the most frequent wcb among boys and the third most common among girls . however , this combination was not significantly associated with suicidal behaviors in boys or girls . the most common wcb engaged in by girls was ' regular exercise and eating less ' , and it also did not show any significant association with suicidal behaviors . interestingly , both boys and girls engaging in ' regular exercise , fasting , and eating less ' were more likely to experience suicidal behaviors compared to those who did not conduct any wcb . although regular exercise and eating less are both appropriate wcbs , when fasting , which is classified as an inappropriate wcb , is added , this combination could contribute to adolescents ' suicidal behaviors . according to a previous study , fasting for weight control showed an association with depression when compared with other wcbs ( 35 ) . in practice , fasting causes the blood glucose concentration to fall below normal , and this status releases the hormone cortisol , which is associated with anxiety and negative feelings ( 36 ) . therefore , it seems that this combination influences suicidal behaviors due to the strong influence of fasting on depressive symptoms . this study was designed as cross - sectional study ; therefore , we could not exclude the potential bi - directional effect . in addition , the time frames of wcb ( within the past 30 days ) and suicide ideation and attempt ( within the past 12 months ) differed . therefore , a reverse - time order could not be clearly excluded ( 6 ) . moreover , responses from the kyrbs represent self - reported data , and therefore may be inaccurate . additionally , we could not obtain detailed descriptions of each wcb including frequency or amount . however , despite the limitations , this study can be generalized by using nationally representative data . additionally , compared to previous studies that only reported associations between wcb and suicidal behavior , we further investigated differences in this association in the context of bmi , bwp , and bsm . moreover , this is the first study to investigate common wcb combinations and to determine the association between the top five such combinations and suicide ideation and attempt . considering that the high incidence of suicide and the severe negative impact of adolescents ' inappropriate wcb on their health , our findings represent an important motivation for health policy makers to identify solutions for controlling adolescents ' suicide problem . inappropriate wcb among adolescents is a serious public health concern considering its widespread prevalence and harmful influence on growth , physical health , and psychosocial growth . additionally , as shown here , wcb can be associated with health compromising behaviors including suicide ideation and attempt . therefore , to protect adolescents ' healthy psychological status in the context of weight concerns , policy makers and health professionals must endeavor to correct the distorted body perception experienced by adolescents . additionally , they should help adolescents develop skills for avoiding inappropriate wcb , and encourage appropriate methods of weight control such as physical activity , fruit and vegetable consumption , and the avoidance of fatty and sweet foods . future studies should be designed regardless of gender or weight status because boys and normal or underweight adolescents are currently low priority even though they are also at risk for adverse effects due to wcb according to the present study . therefore , broader viewpoints and approaches are needed when considering adolescents ' weight - related behaviors .

suicide is a leading cause of death among adolescents globally , and body weight is also a recognized reason for adolescent suicide . therefore , we investigated the association between weight control behaviors ( wcb ) and suicide ideation and attempt , focusing on inappropriate weight control measures . we used data from the 2014 korea youth risk behavior web - based survey , representing a total of 35,224 boys and 34,361 girls aged 12 to 18 years . adolescents were classified into groups based on wcb : appropriate wcb , inappropriate wcb , and no wcb . we performed logistic regression models to examine associations between wcb and suicide ideation and attempt , controlling for covariates . both boys and girls with inappropriate wcb were more likely to report suicide ideation and attempt . underweight and normal weight boys with inappropriate wcb were more likely to think or attempt suicide , and underweight girls with inappropriate wcb were also more likely to attempt suicide . among five common wcb combinations , the combination of " regular exercise , fasting , eating less " was highly associated with suicide ideation and attempt . we confirmed that inappropriate wcb is associated with suicide ideation and attempt among korean adolescents . given the high incidence rate of suicide among adolescents and the adverse effect of inappropriate wcb , encouraging adolescents to control their weight in healthy ways is imperative .

INTRODUCTION MATERIALS AND METHODS Data and participants Variables Statistical analyses Ethics statement RESULTS DISCUSSION

in the past 15 years , green fluorescent protein ( gfp ) has changed from a nearly unknown protein to a commonly used molecular imaging tool in biology , chemistry , genetics , and medicine . in 2006 , more than 10 000 papers gfps and gfp - like proteins ( i.e. , chromoproteins and fluorescent proteins ) are particularly useful due to their stability and the fact that the chromophore ( see figure 1 ) is formed in an autocatalytic cyclization of the 65syg67 sequence that does not require a cofactor . this means that unlike most other bioluminescent reporters , gfp fluoresces in the absence of any other proteins , substrates , or cofactors . furthermore , it appears that fusion of gfp to a protein does not alter the function or location of the protein . by changing residue 66 and/or the amino acid residues around the chromophore (n1c1c2c3 ) and (c1c2c3c4 ) dihedral angles of the gfp chromophore . in the protein , r1 is gly67 and r2 is ser65 , and in hbdi , an often - used model compound , r1 = r2 = ch3 . in one - bond flips ( -obf ) the dihedral rotation occurs around the torsional angle , in a -obf it is around the dihedral angle , and in a positively correlated hula - twist ( + ht ) the and dihedral angles concertedly rotate in the same direction ( as shown above ) , while in a negatively correlated hula twist ( ht ) they concertedly rotate in opposite directions . a plot of the and dihedrals for a perfectly correlated negative ht will have a slope of 1 . if the chromophore cavity is complementary with a planar chromophore , then the and best fit line will pass through the origin and all the and angles will be centered around the origin . a nonzero intercept along the or axis ( see , for example , figure 4 ) or and dihedrals centered in quadrant ii ( > 0 ; < 0 ) or quadrant iv ( < 0 ; > 0 ; see , for example , figure 8) are indications of a cavity that is not complementary with a planar chromophore . the fluorescent emission of the chromophore within gfp occurs with high efficiency ( quantum yield fl = 0.8 ) and a respectable fluorescence lifetime ( 3 ns).(19 ) when the protein is denatured the fluorescence yield decreases by at least 3 orders of magnitude.(20 ) model compounds of the chromophore do not fluoresce in solution ( quantum yield fl < 10 ) , unless the rotation of the arylalkene bond is restrained.(21 ) fluorescence can , however , be obtained by lowering the temperature to 77 k ; this freezes the solution . it has been suggested that twisting between the phenolate and imadazolidinone groups of the chromophore is the mechanism for the ultrafast fluorescence quenching internal conversion process . neutral form ( a ) of the chromophore , with the phenolic oxygen protonated , can convert to the anionic species ( b ) by going through an intermediate state ( i ) . the change from forms a to i is solely a protonation change , while the change from i to b is a conformational change with most changes occurring at thr203 . upon excitation of the a state , an excited - state proton transfer ( espt ) occurs in which the proton is transferred from the chromophore to glu222 in a time scale of the order of picoseconds . following radiative relaxation from the excited - state intermediate ( i * ) , the systems returns to the ground state a through the ground - state intermediates i1 and i2.(29 ) excitation of the anionic b state results in direct emission from the excited state ( b * ) at 482 nm . recently , a nonfluorescent dark state , state c , has been observed that is distinct from states a and b and absorbs at higher energies.(30 ) the c state , perhaps the neutral trans form of the chromophore , may be populated by nonradiative decay from a * and it may be depopulated by excitation to the excited c * state with transcis isomerization to repopulate state a. fluorescent blinking has been ascribed to nonadiabatic crossing and conversions between the neutral and anionic states,(31 ) or a possible dark ( z ) zwitterionic state . while the ground - state minimum of the gfp chromophore is close to planar , this is not necessarily so for the excited state ; in fact , in some cases the excited state has an energy minimum with a twisted chromophore in which both rings are 90 to each other.(32 ) according to quantum mechanical calculations , the ground and excited states for the one - bond flip ( obf ) and hula twist ( ht ) in the neutral form ( a ) and the obf in the zwitterionic form come very close to each other . it has been proposed that in the absence of the protein matrix , which surrounds the chromophore and prevents twisting , this process can lead to fluorescence quenching internal crossing;(32 ) see figure 3 . recent calculations on the gfp chromophore model compound hbdi suggest that the anionic form of hbdi may also undergo a -obf that leads to a favored radiationless decay channel , which is particularly efficient in solvent.(34 ) ( a ) model compounds of the gfp chromophore in the ground state ( s0 ) can be excited to the first singlet state ( s1 ) in which a ht or obf can freely occur . upon reaching the perpendicularly twisted conformation , fluorescence quenching nac ( nonadiabatic crossing ) occurs . ( b ) in the ground state ( s0 ) , the residues surrounding the gfp chromophore exert a twisting force on the chromophore ( ) . upon excitation , the conjugation across the ethylenic bridge of the chromophore is reduced and it will twist ; however , the protein matrix prevents the chromophore from reaching the perpendicularly twisted conformation ( ) and fluorescence quenching internal crossing is prevented . recently , the results from a number of interesting experiments that provide evidence for chromophore twisting and/or fluorescence quenching internal crossing ( ic ) have been published . for example , a molecular dynamics simulation of the chromophore of cyan fluorescent protein found that the average dihedral angle was about 0 ; however , when the surrounding protein was considered in the simulation the average shifted by approximately 5 , showing that the protein matrix of cfp twists the chromophore . on the basis of their calculations , the authors concluded that the driving force for this twist comes from the strong short - range repulsion by four residues ( ile167 , val150 , phe165 , and thr203 ) surrounding the average position of the chromophore.(46 ) photoswitching fluorescent proteins can be switched back and forth between the naturally occurring green state and a dark state by 405 nm irradiation ( e.g. , dronpa , mtfp0.7 , kfp1 ) . a cistrans isomerization of the chromophore has been proposed as the structural basis for the photoswitching observed in dronpa.(47 ) this is supported by the fact that mutating either val157 or met159 with smaller residues accelerates photoswitching , presumably by decreasing steric hindrance to cistrans isomerization.(48 ) the m159 t and v157 g mutations also decrease the quantum yield of dronpa from 0.85 to quantum yields of 0.23 and 0.77 , respectively.(48 ) recently , it has been suggested that adoption of a trans configuration can not solely be responsible for the nonfluorescent form.(49 ) on the basis of nmr analyses , miyawaki et al . propose that the fluorescence of the protein is regulated by the degree of flexibility of the chromophore but is not necessarily accompanied by cistrans isomerization.(49 ) interestingly , gfp is not unique in photoactive yellow protein ( pyp ) the protein matrix also prevents the chromophore from adopting a completely planar structure . in pyp the asymmetric proteinchromophore interaction probably serves as the initial accelerant for the light induced photocycle,(50 ) which ultimately leads to a cistrans isomerization.(51 ) the chromophore in wild - type gfp is planar due its extended system ( see figure 1 ) ; however , the energy barrier to deformation is low and the protein matrix in wild - type gfp exerts some strain away from planarity . when the chromophore is computationally permitted to freely rotate , it will adopt a conformation that complements the protein matrix . recently , we have used computational methods to show that wild - type gfp is not an anomaly and that all gfp and gfp - like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore.(54 ) in most cases the freely rotating chromophore undergoes rotations of at least 20. in some cases these rotations are accompanied by an equal but opposite rotation of the dihedral angle ( a negatively correlated ht ) . none of the proteins examined have a cavity that causes a rotation solely around the dihedral angle.(54 ) these calculations were done by minimizing , with freely rotating and dihedral angles , the crystal structure of 38 gfp analogues and mutants found in the pdb . they found the energy minimum conformation of a freely rotating chromophore in the protein matrix of the gfp mutant or gfp - like protein examined ; however , they did not provide any information about the range of low - energy conformations available to a freely rotating chromophore . to get this information , we ran molecular dynamics simulations of some of the interesting gfp mutants in the protein databank.(56 ) by running molecular dynamics simulations , with freely rotating and dihedrals , we have been able to determine the range of conformations available to chromophores with complete rotational freedom . the coordinates of three crystal structures ( 1gfl,(57 ) 1myw,(58 ) 2emd(59 ) ) were obtained from the protein data bank ( pdb),(56 ) and hydrogen atoms were added to protein and solvent atoms as required . the opls_2005 force field of macromodel v9.0016(60 ) was used . starting structures for mutants for which no crystal structure has been determined were calculated by graphically mutating a known structure and undertaking a conformational search . the flexible dihedral angles of all the side chains of residues that are within 8.00 of the chromophore were randomly rotated by between 0 and 180 , and all solvent molecules in that sphere were randomly rotated and translated by between 0 and 1.00 in each monte carlo ( mc ) step.(63 ) 15 000 mc steps were taken in each search . structures within 50 kj / mol of the lowest energy minimum were kept and a usage - directed method(62 ) was used to select structures for subsequent mc steps . structures found in the conformational search were considered unique if the least - squared superimposition of equivalent non - hydrogen atoms found one or more pairs separated by 0.25 or more . the lowest energy structure obtained in the monte carlo torsional and molecular position variation search the final structures obtained from the mc search ( monte carlo torsional and molecular position variation and large - scale low mode ) or fully minimized pdb structures were used to initiate md simulations with freely rotating and dihedral angles ( v1 = v2 = v3 = 0.000 ) . the predynamics simulation was set for 100 ps and the full md simulation for 5000 ps . the cambridge structure database ( csd ) v5.29 was released in january 2008 ; it comprises 436 384 small molecule crystal structures . the area of the convex hull of the and graphs was calculated as was the smallest convex set containing the region . molecular mechanics and dynamics calculations have been used to examine the steric environment of the chromophore in gfp in its ground state . they are techniques that are based on classical physics and were designed to model structural and not electronic properties ; therefore , molecular mechanics and dynamics simulations of gfp can not examine the excited state of the chromophore . quantum calculations are an excellent technique to determine the energy profiles of the ground and excited states of the chromophore ; however , they are cpu - intensive and it would be cost prohibitive to do conformational searches on the excited state of a series of gfp and gfp - like proteins . therefore , in an attempt to supplement the quantum calculations , we have examined the conformational space available to the chromophore within gfp using a freely rotating chromophore that is an approximation ( based on qm calculations ) of the conformational space available to the chromophore in its excited state . as shown in figure 2 , gfp adopts two states , the neutral state ( a ) of the chromophore with the phenolic oxygen protonated , and the anionic species ( b ) the change from forms a to i is solely a protonation change , while the change from i to b is a conformational change with most changes occurring at thr203.(67 ) in order to see whether the gfp chromophore has different dihedral freedom in forms a , b , and i , we conducted molecular dynamical simulations with a freely rotating chromophore , as described in the . not surprisingly , table 1 and figure 4 show that forms a , b , and i of gfp have similar dihedral freedom and that all undergo similar negatively correlated ( same slopes of best fit lines in figure 4 ) hula twists . plot of the vs dihedral angles ( see figure 1 for nomenclature ) for the 2000 gfp - a ( pink ) , gfp - b ( green ) , and gfp - i ( light - blue ) structures obtained in the freely rotating molecular dynamics simulation . however , there are subtle differences ; forms a and i are closer to the planar ground - state conformation , while the b form adopts conformations further from planarity when given rotational freedom around the and dihedrals ( intercept further from = 0 ) ; and the anionic b form has the most freedom ( largest area , and and range ) , while the neutral a form has the least freedom . the a form has robust hydrogen bonds between both arg96 and gln94 and the imidazolidinone carbonyl group that remain intact throughout the simulations . in the intermediate ( i ) form these hydrogen bonds are supplemented by an additional hydrogen bond between the phenolic oxygen of the chromophore and his148 ( see table 2 ) . the main difference between the anionic b form and the intermediate form is that thr203 has to rotate in order to hydrogen bond to the phenolic oxygen in form i , otherwise they have the same hydrogen - bonding interactions throughout the simulation . the y66h mutant of gfp exhibits blue fluorescence and has therefore been termed blue fluorescent protein . the crystal structure of bfp ( not to be confused with blue fluorescent protein from aequorin bfp - aq(68 ) ) has been solved at ph 4.5(27 ) and ph 8.5;(59 ) the overall fold of the protein is identical to wild - type gfp and consists of a chromophore surrounded by an 11-stranded -barrel . while gfp has absorption maxima at 395 and 475 nm and emits at 508 nm , bfp absorbs at 382 nm and emits at 448 nm.(9 ) unfolding of bfp results in an absorption red shift of 15 nm,(27 ) and quantum mechanical calculations suggest that the 15 nm shift might be due to the protein - induced nonplanarity of the chromophore.(69 ) blue fluorescent protein ( bfp ) has a much lower fluorescence quantum yield than gfp ( fl = 0.20 vs 0.80 ) . it has been suggested that this is due to the fact that his66 ( bfp - chromophore ) forms fewer hydrogen bonds with the surrounding protein than tyr66 ( gfp - chromophore ) does , and that the smaller imadazole ring ( his66 ) in bfp may have more conformational freedom than the larger phenol ( tyr66 ) , which leads to more intersystem crossing.(27 ) in a very elegant series of experiments , boxer et al . have shown that the fluorescence quantum yield of blue fluorescent protein increases from fl = 0.20 to 0.35 when the pressure is increased from atmospheric pressure to 570 mpa.(70 ) analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes . temperature - dependent fluorescence measurements reveal two barriers ( 19 and 3 kj / mol , respectively ) for the transition into nonfluorescing states . these steps are probably linked with dissociation of the hydrogen bond between the chromophore and his148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state , respectively.(70 ) in order to establish the consequences of the y66h mutation on the flexibility of the chromophore , molecular dynamics simulations of the ph 8.5 bfp crystal structure ( pdb code 2emd with neutral imidazole rings for his66 and his148 ) with a freely rotating chromophore were run . [ we thank one of the referees for suggesting the ph 8.5 ( 2emd ) over the ph 4.5 ( 1bfp ) structure . ] figure 5 and table 1 show that the chromophore in bfp has significantly more rotational freedom than that available to the chromophore in gfp , and that the bfp cavity is less complementary to a planar chromophore than the gfp cavity is ; that is , the bfp sampled structures are further from a planar chromophore ( = = 0 ) and the intercept for the best fit line through the bfp structures is further from the origin than that of gfp . plot of the vs dihedral angles for the 2000 bfp ( blue ) and gfp - a ( green ) structures obtained in the freely rotating molecular dynamics simulation . analysis of the 2000 bfp structures revealed a hydrogen bond between the nh of the his66 imidazole ring and glu222 , which is retained through - out the entire simulation ( see figure 7 ) . the smaller imadazole ring of the bfp chromophore results in more dihedral freedom for the chromophore in bfp than phenol in gfp ; however , there seems to be no large difference in the number and stability of hydrogen bonds formed by the chromophore in the two fps . recently , two new blue fluorescent proteins ( azurite and a5 ) with enhanced brightness and photostability were created . the methodology applied to find the brighter bfp mutants was based on the concept that replacing the residues surrounding the chromophore with bulkier amino acids would constrain the chromophore s motion and thereby increase the proteins brightness . the crystal structure of a5 has yet to be solved . in order to find a starting conformation for the md simulation , the 2emd crystal structure was graphically mutated to a5 and a thorough conformational search was undertaken ( see ) . given the high structural similarity between all the solid state structures of gfp mutants in the protein databank , a thorough conformational search should find the lowest energy conformations of a5 by making the relevant mutations to the 2emd structure . figure 6 and table 1 show that the conformational space available to the chromophore in bfp is larger than that in a5 and that the bfp conformations tend to be further from planarity ; i.e. , for a5 median = 2.92 , median = 2.22 and intercept of best - fit line is 4.62 vs for bfp median = 27.59 , median = 10.18 and intercept of best - fit line 14.27 . similar results were found for md simulations carried out from all of the five lowest energy conformational families sampled in the conformational search of a5 and in a md simulation initiated from an a5 structure obtained by taking the 2emd structure , graphically mutating it to a5 , and thoroughly minimizing the structure without undertaking a conformational search . plot of the vs dihedral angles for the 2000 bfp ( dark blue ) and a5 ( pink ) structures obtained in the freely rotating molecular dynamics simulation . the n of the his66 imidazole ring hydrogen bonds with water305 ( 2emd numbering ) in all the sampled structures . this water connects his66 to the surface of the protein via a robust hydrogen - bonding network to his148 and is also hydrogen bonded to glu222 . in half the structures sampled , the n of the his66 imidazole ring hydrogen bonds to water302 which is in an extensive hydrogen - bonding network with val68 , arg224 , and gln69 . in contrast to 2emd there are no hydrogen - bonding interactions with the n hydrogen ; see figure 7 . hydrogen - bonding interactions ( --- ) for 2emd ( top ) and a5 ( bottom ) chromophore . these results seem to indicate that the enhanced brightness of a5 is at least in part due to the fact that its chromophore movement is restricted relative to bfp . this restriction can be due to both steric factors and the increased hydrogen - bonding networks in a5 . in the first rationally designed mutant based on the crystal structure of gfp - s65 t , it was decided to mutate t203 into a tyrosine so that it could stack with the phenolic group in the chromophore . ( 73 ) the resultant mutant , yfp , is red - shifted by 16 nm relative to gfp - s65 t and does indeed have a stacking interaction between the chromophore and tyr203.(74 ) on the basis of the crystal structure of yfp , remington et al . proposed that the red shift was due to the additional polarizability of the -stacked tyr203 , the hydrogen - bond pattern around the chromophore , or an out - of - plane distortion of the chromophore ( in analogy with out - of - plane distortions in porphyrin systems).(74 ) comparison with the t203v mutant revealed that the t203y substitution leads to a significant i - form population and only about 10 nm of the shift can be ascribed to the interaction.(75 ) a new varient of yfp , venus , with improved brightness and maturation properties as well as a reduced environmental sensitivity was developed and crystallized.(58 ) since some researchers have argued that the presence of tyr203 leads to a decrease in chromophore flexibility ( van der waals volume for tyr = 141 vs 93 for thr ) , and others argue that this is not necessarily so,(32 ) we have examined the conformational space available to a freely rotating chromophore in yfp ( pdb code 1myw ) with the phenol of tyr66 in both the protonated and unprotonated forms . both table 1 and figure 8 show that the chromophore ( in the phenolic form ) in gfp and yfp has differing rotational freedom . in yfp the chromophore has approximately 1.2 times more dihedral freedom than in gfp and although the dihedrals undergo negatively correlated hula - twisting in both proteins , in yfp the average conformation sampled is further from planarity than that found in gfp ; i.e. , the majority of the sampled conformations are located in quadrant iv . plot of the vs dihedral angles ( see figure 1 for nomenclature ) for the 2000 structures obtained in the freely rotating molecular dynamics simulation of gfp ( dark blue ) and yfp ( pink ) in their phenolic forms . the majority of the yfp conformations sampled are nonplanar ( i.e. , in quadrant iv < 0 and > 0 ) . during the entire simulation the hydrogen bonds between tyr66 and ser205 , arg96 and the imidazolone carbonyl , as well as between tyr203 and water354 remain stable , while those between tyr203 and gln69 were less stable . we also examined the distance between the centroid of tyr66 ( the choromophore ) and tyr203 . the distance is fairly short and does not change much during the simulation ( minimum = 3.43 , maximum = 4.18 , average = 3.75 , sd = 0.103 ) while the angle between the planes of the phenols varies between 0.0 and 22.5 ( average = 6.9 , sd = 4.11 ) . the short distance between the phenol rings is indicative of stacking , which may be responsible for the red shift observed in yfp ; however , the variability in the angle between the planes of the phenols shows that tyr203 does not significantly restrict the rotational freedom of the excited chromophore . the cambridge structural database ( csd)(77 ) v5.29 comprises 436 384 small molecules crystal structures . it has 5551 structures with two phenol rings in separate molecules that have centroids within 5.00 of each other . the closest ones are 3.16 from each other , and only 832 structures are within 3.75 ; i.e. , the average distance between the phenol centroids in the freely rotating yfp . a plot of the distance vs the angle between the phenol planes in our freely rotating simulation and the csd structures shows that the protein matrix of yfp restricts the distance between tyr66 and tyr203 from being larger than 4.18 , but that the angles between the two phenols have as much freedom in yfp as in the small molecule structures with intermolecular distances of less than 4.18 ( see figure 9 ) . it does not seem as if the increased fluorescence lifetime of yfp is due to a decrease in the dihedral freedom of the chromophore . it is more likely due to electronic effects such as those proposed by jung et al.(78 ) they suggested that the anionic form of the chromophore can be described by two mesomeric forms , the benzoidal and the quinoidal structure . the benzoidal form with the majority of its negative charge on the phenol is stabilized by hydrogen bonding to thr203 , which is not possible in yfp and therefore the t203y mutation favors the quinoidal form , which may have a longer fluorescence lifetime.(78 ) distance vs angle between the phenol planes in yfp ( ) and all structures in the csd that have two unconnected phenols separated by less than 5.00 ( + ) . some have suggested that the crystal structures of strongly fluorescent gfp and gfp - like proteins have their chromophores in a cis configuration , while those of the nonfluorescent proteins are in a trans configuration . however , the trans - planar form of eqfp611 is an important exception : it is fluorescent.(82 ) remington et al . have suggested that coplanarity might be more important than isomer configuration in determining the efficiency of fluorescence . most current theories ( see ) suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield . our calculations show that this is the case for the systems examined . for bfp , a5 , yfp , and gfp , there is an inverse correlation between the dihedral freedom of the chromophore and the quantum yield ; see table 1 . in all simulations , the protein matrix is not complementary with a planar chromophore , and in all cases the freely rotating chromophore undergoes a negatively correlated hula twist ( also known as a bottom hula twist mechanism ) .

green fluorescent protein ( gfp ) and gfp - like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore . according to quantum mechanical calculations , the excited state of chromophore model systems has significant dihedral freedom , which may lead to fluorescence quenching intersystem crossing . molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green , yellow , and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state . most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield . according to our calculations , the dihedral freedom of the systems studied ( bfp > a5 > yfp > gfp ) increases in the inverse order to the quantum yield . in all simulations , the chromophore undergoes a negatively correlated hula twist ( also known as a bottom hula twist mechanism ) .

Introduction Experimental Section Results and Discussion Conclusion

fnas represent an advantageous , effective means to obtain diagnostic cellular material as they can often sample a wide area of the target lesion , acquire tumor cells with lower contamination by background stromal connective tissue elements , and allow for immediate assessment during rapid on - site evaluation ( rose ) . during the on - site assessment , a member of the cytopathology team is present and prepares direct smears , using the contents expelled from the needle , at the location of the procedure . this is advantageous for three reasons : each needle pass can be examined to determine tumor cell adequacy ; there is an opportunity to engage the clinical care provider in a conversation regarding the preliminary diagnosis and relevant molecular diagnostic tests ; and the cytopathology team member can help ensure that the specimen is processed in a manner that optimizes judicious triage for ancillary tests , including molecular studies . fnas can be performed without rose and in this setting , the contents of the fna needles are typically expelled and rinsed in a cell preservative solution for use in liquid - based cytology ( lbc ) preparations . examples of lbc preparations include thinprep ( hologic , bedford , ma , usa ) , surepath ( becton dickinson , franklin lakes , nj , usa ) , thin - layer advanced cytology assay system ( tacas , mbl , tokyo , japan ) , and liqui - prep ( lgm international inc . , nonetheless , rose is an effective means to maximize the chances of success in acquiring adequate tumor cells for diagnosis and anticipated molecular studies . the ultimate goal is to prevent unnecessary repeat procedures to obtain additional tissue just for molecular studies which can lead to delays in treatment . for patients afflicted with lung cancer at our institution , we perform rose for cytopathologist - performed fnas and for ebus - guided fnas , performed by our clinical colleagues . the contents of each needle pass are expelled onto a slide which is utilized to prepare direct smears . the needle is then rinsed into buffered media ; we utilize rpmi media for this purpose . commonly , a pair of direct smears is prepared per needle pass ; one is air - dried and the other is immediately alcohol - fixed . the air - dried smear is stained on - site with the diff - quik ( romanowsky ) stain and the stained slide can be examined under the microscope immediately thereafter . the alcohol - fixed smear is stained later in the cytopathology laboratory with the papanicolaou stain . alternatively , the needle contents can be distributed over multiple smears allowing for flexibility in the utilization of direct smears for cytomorphologic evaluation and ancillary studies ( fig . based on the findings in the diff - quik stained smears , the determination can be made to perform additional needle passes to obtain more tumor cells for diagnosis and/or anticipated ancillary studies while the patient is still accessible . for instance , additional direct smears can be prepared and/or dedicated needle passes for the rinse solution ( i.e. , the needle contents are rinsed in the rpmi solution without the preparation of smears for those needle passes ) obtained for the cell block preparation . this overall approach is flexible , forgiving , and engages the cytopathology team in maximizing the chance of success in obtaining adequate cellular material for diagnosis and ancillary studies . this approach can help minimize the chances of encountering the scenario in which diagnostic cellular material is present on only one smear ; microdissection of the cells for molecular studies can result in sacrificing this only diagnostic slide in this context , which can have medico - legal consequences . if this scenario is encountered , however , this risk can be mitigated by digital archiving prior to microdissection either via digital slide scanning and/or obtaining photomicrographs . after immediate assessment of the cytomorphologic findings on the diff - quik stained smears , a preliminary diagnosis can be rendered by the cytopathologist and communicated to the clinical care providers . after the conclusion of the procedure , the needle rinse solution , containing a suspension of aspirated cells , is centrifuged in the cytopathology laboratory to pellet the cells . once the supernatant is removed , the cell pellet is congealed in a matrix ; an agar - like substance such as histogel ( thermo scientific , waltham , ma , usa ) can be used or the cells can be mixed with plasma and thrombin to create a clot . at our institution , we employ histogel for this purpose . commonly , this cassette is fixed in formalin and processed to ultimately create a formalin - fixed , paraffin - embedded ( ffpe ) cell block . this is analogous to a ffpe block of small biopsy tissue , which can be sectioned for evaluation via the hematoxylin and eosin stain and ancillary tests such as immunocytochemistry or molecular diagnostic assays . it should be noted that heavy metal fixatives such as zenker s fixative and acidzinc - formalin , acidic fixatives such as bouin s solution , or decalcification solutions should generally be avoided as these render specimens unusable for molecular testing . the main advantage of using cell blocks is that the majority of ancillary tests are validated for ffpe sections ; ffpe cell blocks are treated similarly to traditional surgical pathology ffpe blocks . furthermore , multiple serial sections from cell blocks can be utilized to perform a battery of ancillary studies . first , adequate cellularity in cell blocks is not guaranteed at the time of the procedure . performing dedicated needle passes , for the needle rinse solution during the fna procedure , may improve the chances of obtaining a sufficiently cellular cell block but still does not guarantee success . in addition , for hypocellular cell blocks , there is a risk of depleting the cells of interest upon deeper sectioning for molecular tests . second , the cell block is derived from a needle rinse solution which is a pooled specimen that contains contents of all the needle passes . thus , if one or more needle passes contain tumor cells but other needle passes contain abundant benign background cellular elements , the tumor cells will be diluted by these benign cells in the cell block . in addition to the above disadvantages , dna extracted from ffpe cell blocks may produce sequencing artifacts as formalin fixation leads to the crosslinking of nucleic acids and proteins and the possibility of sequence alterations . recently , given the shortcomings of cell blocks , there has been an increasing appreciation of alternative cytopreparatory platforms for molecular testing such as direct smears and lbc samples . in the subsequent sections below , the utilization of these cytopreparatory platforms will be discussed in the context of the clinically relevant mutations and translocations analyzed during molecular diagnostic testing of nsclcs . the epidermal growth factor receptor ( egfr ) gene encodes a transmembrane growth factor receptor that exhibits tyrosine kinase activity . upon activation , intracellular signaling is mediated by cytoplasmic effectors in the ras - raf - mek - erk , pi3k - akt , and stat pathways . egfr mutations are predominantly observed in lung adenocarcinomas ; the l858r substitution and small in - frame deletions in exon 19 are the most commonly observed mutations and account for up to 90% of all egfr mutations in this setting . these mutations are more commonly associated with east asian ethnicity , female gender , and non - smoking history . however , these are not absolute rules and clinical characteristics should not be used to exclude lung cancer patients from mutation testing . lung adenocarcinomas harboring these sensitizing mutations in egfr have been shown to respond to egfr tyrosine kinase inhibitors ( tkis ) . , tki therapy has been demonstrated to result in improved progression - free survival , compared to standard chemotherapy , for patients with lung adenocarcinoma harboring egfr mutations [ 11 - 16 ] . egfr mutation analysis is commonly performed via polymerase chain reaction ( pcr ) and sequencing - based approaches ; advances in the development of testing modalities have afforded a multitude of methodologies . sanger sequencing is considered the gold standard as this involves direct dna sequence acquisition and can provide information regarding the presence of all potential mutations including common , known mutations and novel mutations . nonetheless , this test requires a relatively higher enrichment of tumor cell dna content in the sample . the typical analytic sensitivity for sanger sequencing is 15%20% mutant allele , which equates to 30%40% tumor cells assuming that the genetic mutation is a heterozygous event without amplification . this can be problematic in both small biopsy and cytology specimens , especially cell blocks , in which the tumor cell population can be diluted by background benign cellular elements such as inflammatory cells , bronchial epithelial cells , and/or stromal mesenchymal cells . especially in this setting , a negative mutation result can be either due to the true absence of the mutation in the tumor cells or insufficient percent tumor cellularity that falls below the analytic sensitivity threshold thereby resulting in the failure to detect the mutation even despite the presence of the mutation . therefore , often times , there is more reliance on tumor cell enrichment by either macrodissection or microdissection to obtain a reliable result . sanger sequencing is also relatively more labor intensive and time consuming than targeted methods and can lead to longer turnaround times . in contrast to the general sanger sequencing approach , targeted mutation detection methods such as pcr - restriction fragment length polymorphism , real - time pcr , pyrosequencing , high resolution melting analysis ( hrma ) , and pcr fragment analysis can be utilized . the advantages of these approaches include their improved analytic sensitivity and less time - consuming nature leading to reduced turnaround times . at our institution , we utilize a multiplex pcr fragment analysis assay for egfr mutation testing ; this allows for the simultaneous assessment of the two most commonly observed egfr mutations ( fig . 2 ) . the analytic sensitivity of this method is better than that of sanger sequencing ; a minimum of only 10% tumor cells is required . in the past decade , myriad studies have been reported demonstrating that a variety of cytologic samples and cytopreparatory platforms can be effectively utilized for egfr mutational analysis . these have been reviewed elsewhere but salient examples will be discussed below . as mentioned previously , ffpe cell blocks represent the traditional cytopreparatory platform on which ancillary molecular diagnostic tests are performed . much of the reasoning behind this lies in that ffpe cell blocks best approximate ffpe blocks containing tissue specimens and the majority of the molecular assays are validated using ffpe material . nonetheless , in light of the inherent disadvantages of cell blocks mentioned previously , investigation into other cytopreparatory platforms for molecular diagnostic assays have been performed by our group and many others . cytopathology specimen preparation is diverse and versatile ; the consequence of this is that each type of cytologic analyte platform needs to be carefully validated for any given molecular test . cytologic direct smears may ultimately prove to be the cytologic platform best suited for pcr - based analysis due to the high quality of nucleic acids and immediate nature of specimen assessment for tumor cell adequacy . cells on direct smears are not exposed to formalin but rather undergo an alcohol - based fixation process prior to staining ; thus , the negative effects of formalin fixation on nucleic acid quality is non - contributory for direct smears . furthermore , the tumor cells of interest can be directly visualized on the direct smear and isolated for nucleic acid extraction and molecular analysis ; in essence , what you see is what you get . we have previously demonstrated that uncoverslipped and previously coverslipped / decoverslipped diff - quik stained smears are robust sources of cellular material for pcr - based mutational analysis such as egfr mutation testing of lung adenocarcinoma and braf mutation testing of metastatic melanoma fna specimens . we and others have observed that destaining the smears is not necessary for successful dna extraction and pcr . our preference in utilizing diff - quik stained smears over papanicolaou stained smears is rooted in the following observations : ( 1 ) cellular material on a diff - quik stained smear can be easily visualized without a coverslip and immediately triaged for molecular diagnostic testing ; and ( 2 ) the report by killian et al . that nucleic acids extracted from diff - quik stained smears show better preservation and integrity than dna extracted from papanicolaou stained smears . with respect to the latter point , others have pointed out that papanicolaou stained smears are just as feasible for pcr - based molecular tests . multiple groups have successfully demonstrated the utilization of cytologic direct smears for egfr mutation testing of lung adenocarcinomas [ 2,9,25 - 36 ] with low failure rates and high degree of concordance with egfr molecular testing results from corresponding histologic specimens , when available . in our experience , 100 to 200 tumor cells are sufficient for successful dna isolation and egfr mutation analysis and this is congruent with the observations from others . lbc samples have also been investigated for egfr mutation testing and have been shown to be an effective analyte for this purpose . this is valuable when a member of the cytopathology team is not available for rose . several lbc technologies have been developed including thinprep , surepath , tacas , and liqui - prep . of these , the emerging literature , to date , on the utilization of lbc samples for molecular testing of nsclc have predominantly focused on cytolyt ( cytyc corp . , marlborough , ma , usa ) cell suspensions and thinprep slides . collecting fna samples in the methanol - based cytolyt solution results in reduction of background blood as the cytolyt solution exhibits hemolytic properties . the cell suspension can be divided and an aliquot is used to prepare a thinprep slide that is stained via the papanicolaou method , similar to alcohol - fixed papanicolaou stained direct smears . tumor cells can be isolated from the thinprep slide and used for dna isolation and egfr mutation analysis . furthermore , the cell suspension aliquot that is not used to prepare a thinprep slide can be centrifuged and the cell pellet itself can be used as an analyte for mutation testing [ 38 - 40 ] . similar to the needle rinse cell suspension utilized to prepare cell blocks , the cytolyt cell suspension is a pooled specimen if multiple needle passes are expelled and rinsed into cytolyt . thus , if one or more needle passes are high in tumor cell content whereas other needle passes contain a high proportion of benign cellular elements , the tumor cells in the final pooled cell suspension will be diluted . first , targeted methods for detecting egfr mutations with improved analytic sensitivity , relative to sanger sequencing , can be utilized . second , tumor cell enrichment from the thinprep slide can be accomplished by laser capture microdissection ( lcm ) . to illustrate these points , malapelle et al . directly compared the performance of egfr mutation analysis by sanger sequencing using paired samples for each case analyzed : ( 1 ) pelleted cells from the cytolyt suspension and ( 2 ) tumor cells obtained from the thinprep slide via lcm . they observed that egfr mutations were more reliably identified in the latter rather than the former . this difference was minimized when utilizing more sensitive egfr mutation analytic approaches which included hrma and pcr fragment analysis assays . based on these studies , the authors speculated that coupling the use of highly sensitive egfr mutation detection assays and cytolyt derived cell pellets may be sufficient obviating the need for microscopy . nonetheless , it must be emphasized that any negative molecular testing result should carefully be reconciled with the analyte input utilized for the mutation assay . this is essential to determine whether the negative result represents a true negative or potentially a false - negative , necessitating possible retesting . in this regard , the utilization of microscopy still remains an essential pre - analytic quality assurance activity to best ensure that the input analyte is of sufficient tumor cellularity to maximize confidence in the results of the mutation assay . of note , a follow - up study by bellevicine et al . they observed that the direct smears exhibited significantly higher cellularity than thinprep slides , on average . accordingly , the average yield of dna extracted from direct smears was significantly higher than that from thinprep slides . as the l858r substitution and deletions in exon 19 represent approximately 90% of all egfr mutations in lung adenocarcinoma , one antibody is specific for the exon 21 l858r mutation and the other specific for the 15-base pair/5-amino acid deletion ( e746_a750del ) in exon 19 ( clone 6b6 ) . the immunohistochemical approach to detecting mutant egfr proteins using these antibodies has been examined in several studies on lung cancer tissues as well as cytologic and small biopsy samples [ 42 - 48 ] . the sensitivity of these assays range from 47%92% but their high positive predictive value and specificity supports the feasibility of utilizing this approach as a first - line screening approach . first , immunohistochemistry should be performed after careful validation and formulation of immunostain scoring criteria . the significance of how best to interpret equivocal staining results should be clarified as overinterpreting weak immunoreactivity as a positive mutation result can lead to increased false positives and decreased specificity . second , the clone 6b6 antibody best detects the 15-base pair ( e746_a750del ) egfr deletion mutant protein but demonstrates variable immunoreactivity for the egfr mutant proteins resulting from non-15-base pair deletions in exon 19 . the anaplastic lymphoma kinase ( alk ) protein is a receptor tyrosine kinase and rearrangements involving the alk gene locus is observed in approximately 5% of lung adenocarcinomas . these mutations are more commonly observed in younger age patients who are never - smokers . however , there can be exceptions to this and clinical characteristics should not be used to exclude lung cancer patients for alk rearrangement testing . most alk rearrangements in lung adenocarcinoma result from interstitial deletions and small inversions within the short arm of chromosome 2 . this results in the fusion of portions of the echinoderm microtubule - associated protein - like 4 ( eml4 ) and alk genes . other less common alk rearrangements involve fusions between alk and other genes , such as kif5b and tfg . alk rearranged lung adenocarcinoma has been recognized as a legitimate target for small molecular inhibitor therapy ; crizotinib was shown to be efficacious in treating patients with these cancers . in a phase 1 study evaluating 143 patients , an overall response rate of 61% and estimated overall survival rates of 74.8% at 12 months therefore , the evaluation of lung adenocarcinoma fna samples for alk rearrangements , in addition to egfr mutations , has become increasingly incorporated into patient management algorithms . fish is currently the preferred approach to assaying lung adenocarcinomas for alk rearrangements according to expert recommendations based on review of the literature . in the united states of america , there is only one test approved by the food and drug administration ( fda ) for this purpose . this assay utilizes a dual alk breakapart probe strategy in which orange and green labeled probes hybridize to the highly conserved translocation breakpoint region in the alk gene . alk gene loci that have not undergone rearrangement typically display fused orange and green signals ( yellow ) or juxtaposed touching orange and green signals . when an alk rearrangement occurs , the orange and green signals become separated . however , as the majority of the alk rearrangements involve a small inversion within chromosome 2p , rather than a rearrangement involving another chromosome , the extent to which the two signals are split is finite . in order to score a nucleus as positive for the alk rearrangement , the orange and green signals must be separated by a distance of > 2 signal diameters ( fig . 3 ) ; a nucleus can also be scored positive if a single orange signal without a corresponding green signal is observed . typically , up to 100 tumor cell nuclei are scored in this assay and a lung adenocarcinoma is considered positive for the alk rearrangement if at least 15% of the nuclei are scored as positive for the rearrangement . the fda has approved this dual breakapart probe set for use on paraffin embedded tissue sections . as mentioned previously , paraffin embedded cell blocks are processed similarly to paraffin embedded tissue blocks ; thus , cell blocks are traditionally used for the performance of alk rearrangement fish assays . nonetheless , there is an observed noticeable failure rate when using and relying on cell blocks for these assays due to insufficient tumor cell material in a significant proportion of cases . thus , we and others have investigated alternative cytopreparatory platforms for alk fish . in our study , we demonstrated the effective use of diff - quik stained direct smears for this purpose and observed that the performance of this assay using direct smears was better than the performance using cell blocks . . also examined the use of papanicolaou stained direct smears and observed that the failure rate due to insufficient cellularity was significantly higher for cell blocks than for direct smears . therefore , the utilization of both diff - quik and papanicolaou stained smears are feasible analytes for alk fish . recently , thinprep slides have also been shown to be a feasible platform for alk fish . the advantage of utilizing cytologic preparation platforms such as direct smears and thinprep slides for this purpose is that entire tumor cell nuclei are being analyzed . fish evaluation on paraffin sections derived cell blocks and other ffpe blocks are prone to signal loss in some of the tumor cells due to section truncation artifacts . in addition , alternative methods to fish have been the subject of recent investigation . immunohistochemistry utilizing antibodies directed against alk is an attractive alternative as it is simpler , quicker , and less expensive . the challenge associated with this approach is that the alk protein is expressed at much lower levels in alk rearranged lung tumors than in anaplastic large cell lymphoma , the prototypical alk rearranged tumor . fortunately , monoclonal anti - alk antibodies ( clones d5f3 , d9e4 , and 5a4 ) have been shown to exhibit high sensitivity and specificity . immunohistochemistry using the d5f3 and 5a4 monoclonal antibodies on cytologic specimens have been recently described and shown to exhibit a high degree of concordance with alk fish testing . this supports the feasibility of utilizing an immunohistochemical approach as a first - line screening methodology to select specimens for alk fish testing . of note , the mouse monoclonal anti - alk1 antibody ( cd246 ) typically used for the diagnosis of anaplastic large cell lymphoma is less reliable for identifying alk rearrangements in lung adenocarcinoma ; this is most likely attributable to the limited sensitivity of this particular antibody in detecting the lower expression levels of the alk fusion proteins in lung adenocarcinoma relative to anaplastic large cell lymphoma . finally , the application of reverse transcriptase pcr ( rt - pcr ) to cytologic direct smears for alk rearrangement analysis has been recently reported . when evaluating a cohort of paired cytologic - histologic specimens by rt - pcr , mitiushkina and nonetheless , as a cautionary note , there is a high degree of variability in eml4-alk fusion events with at least 13 variants of eml4-alk being reported . furthermore , other fusion partners to alk such as tfg and kif5b can be observed . therefore , an rt - pcr approach may not capture all clinically relevant alk rearrangements . while egfr mutations and alk rearrangements represent the two best characterized , clinically actionable molecular alterations in nsclc , other molecular markers are becoming increasingly appreciated and investigated . ros1 encodes a receptor tyrosine kinase and is rearranged in approximately 2% of lung adenocarcinomas . met encodes another receptor tyrosine kinase , hepatocyte growth factor receptor , and is amplified in a subset of nsclc ; a significant proportion of these cases are seen in context of acquired resistance to egfr tkis . as crizotinib also targets this receptor , studies are under way to determine whether this agent will be effective in met amplified lung cancers . therapeutic agents targeting braf , such as dabrafenib , are currently being investigated in clinical trials . in addition , gene rearrangements involving the ret tyrosine kinase gene ( e.g. , kif5b - ret ) have been observed in approximately 2% of lung adenocarcinomas ; ret specific tkis such as sunitinib , sorafenib , and vandetanib may be useful in treating patients with these lung cancers . finally , amplification of fgfr1 and mutations in pik3ca have been observed in some lung squamous cell carcinomas and agents targeting these gene products are also under investigation . the above mentioned molecular genetic markers highlight the distinct molecular profiles that are becoming increasingly appreciated for different subtypes of nsclc , especially adenocarcinomas and squamous cell carcinomas . therefore , efforts to correctly subtype cases of nsclc are important , including challenging cases of poorly - differentiated nsclc . at our institution , if the subtyping of nsclc is deemed to be challenging based on cytomorphologic evaluation alone , immunocytochemistry is utilized ; we have demonstrated that this can be accomplished using smears as well as cell blocks . for example , napsin - a and thyroid transcription factor-1 are often positive in lung adenocarcinomas and can be utilized for confirming this subtype of nsclc . in contrast , p63 and p40 are useful markers for confirming a diagnosis of squamous cell carcinoma . ultimately , utilizing fna samples of nsclc for immunocytochemistry must be judiciously leveraged to ensure that sufficient material still exists for molecular ancillary testing . given the continuously evolving landscape in our understanding of the genetic events responsible for lung cancer pathogenesis , the integration between anatomic pathology , particularly cytopathology , and molecular diagnostics will become even more essential . emerging molecular technological advances , such as next generation sequencing ( ngs ) , that allow for high - throughput , high - sensitivity molecular analyses will likely play an important role in the management of patients with nsclc . cytologic specimens , based on recent reports , represent a robust source of cellular material for ngs [ 62 - 65 ] .

in this era of precision medicine , our understanding and knowledge of the molecular landscape associated with lung cancer pathogenesis continues to evolve . this information is being increasingly exploited to treat advanced stage lung cancer patients with tailored , targeted therapy . during the management of these patients , minimally invasive procedures to obtain samples for tissue diagnoses are desirable . cytologic fine - needle aspirates are often utilized for this purpose and are important not only for rendering diagnoses to subtype patients lung cancers , but also for ascertaining molecular diagnostic information for treatment purposes . thus , cytologic fine - needle aspirates must be utilized and triaged judiciously to achieve both objectives . in this review , strategies in utilizing fine - needle aspirates will be discussed in the context of our current understanding of the clinically actionable molecular aberrations underlying non - small cell lung cancer and the molecular assays applied to these samples in order to obtain treatment - relevant molecular diagnostic information .

FINE-NEEDLE ASPIRATION SPECIMEN PROCESSING EPIDERMAL GROWTH FACTOR RECEPTOR ANAPLASTIC LYMPHOMA KINASE EMERGING MOLECULAR GENETIC BIOMARKERS CONCLUDING THOUGHTS

these tumors are classified as typical net ( tnet ) , goblet cell carcinoid ( gcc ) , and atypical gcc histologies . ex - goblet or composite - goblet are further classified into signet ring cell carcinoid ( srcc ) and poorly differentiated adenocarcinoid of the appendix . these tumors have a distinctive morphology showing tight clusters of cells with compact nuclei and abundant intracytoplasmic mucin resembling goblet or signet ring cells often with admixed enterochromaffin cells . pathologic features of gcc include presence of large mucin filled cells with crescent nuclei arranged in small clumps or rosettes mixed with cells of typical carcinoid appearance that stain positive for chromogranin a . patients with tnet have a 5-year survival ranging from 60% to 84% , with the most common site of metastasis being the liver . on the other hand , atypical gcc have a more aggressive clinical course with an increased incidence of lymph node and distant metastases , along with a lower 5-year survival ranging from 36% to 56% [ 5 - 7 ] . current management of gcc and atypical gcc is based on very limited data from small single institutional experiences . the rarity of appendiceal net , gcc , and srcc limits the ability to conduct appropriate randomized clinical trials to explore the optimal management . to assess the role of clinicopathologic features in survival of net , gcc , and srcc patients , the outcome of cases reported to the national cancer institute s surveillance epidemiology and end results ( seer ) program was evaluated . furthermore , to characterize the management of net , gcc , and srcc , a treatment strategy based on results of current analysis , published literature and institutional experience is suggested . seer data collected between 1973 and 2011 was used to identify cases with appendiceal tnet , gcc , and srcc . the seer registry data collection began in the early 1970s and gradually expanded from the original nine to the current 18 registries that now account for over a quarter of the united states population . eligibility criteria included international classification of diseases for oncology third edition ( icd - o-3 ) codes for primary site of appendix and histologic types of tnet ( 8240 , 8241 , 8242 , 8246 , and 8270 ) , gcc ( 8243 , 8244 , 8245 , and 8249 ) , and srcc ( 8490 ) . information regarding age at diagnosis , sex , race , year of diagnosis , grade , histology , tumor location , type of surgery , vital status and duration of follow - up , was extracted from the seer database . the patients characteristics were compared among three histologic types : tnet , gcc , and srcc by chi - square test for race , sex , and stage , and kruskal - wallis test for age . survival functions were estimated by the kaplan - meier method and log - rank test was used to assess the difference in overall survival ( os ) between three histologic types : tnet , gcc , and srcc . univariate survival analysis for each covariate was carried out using the cox proportional hazards model . the multivariate survival analysis of histology was conducted after adjusting for age , race , and sex using a backward variable selection method with an alpha level of removal of 0.1 . the model was stratified by stage since there was an interaction effect between histology and stage on os . 9.3 ( sas institute inc . , cary , nc ) was used for data analyses . null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 . seer data collected between 1973 and 2011 was used to identify cases with appendiceal tnet , gcc , and srcc . the seer registry data collection began in the early 1970s and gradually expanded from the original nine to the current 18 registries that now account for over a quarter of the united states population . eligibility criteria included international classification of diseases for oncology third edition ( icd - o-3 ) codes for primary site of appendix and histologic types of tnet ( 8240 , 8241 , 8242 , 8246 , and 8270 ) , gcc ( 8243 , 8244 , 8245 , and 8249 ) , and srcc ( 8490 ) . information regarding age at diagnosis , sex , race , year of diagnosis , grade , histology , tumor location , type of surgery , vital status and duration of follow - up , was extracted from the seer database . the patients characteristics were compared among three histologic types : tnet , gcc , and srcc by chi - square test for race , sex , and stage , and kruskal - wallis test for age . survival functions were estimated by the kaplan - meier method and log - rank test was used to assess the difference in overall survival ( os ) between three histologic types : tnet , gcc , and srcc . univariate survival analysis for each covariate was carried out using the cox proportional hazards model . the multivariate survival analysis of histology was conducted after adjusting for age , race , and sex using a backward variable selection method with an alpha level of removal of 0.1 . the model was stratified by stage since there was an interaction effect between histology and stage on os . 9.3 ( sas institute inc . , cary , nc ) was used for data analyses . null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 . the seer database yielded 1,021 tnet patients , 1,582 with gcc , and 534 srcc patients . between 1973 and 2011 , the incidence of tnet , gcc , and srcc increased . in 2011 , the incidence of tnet reached 6.7 , gcc was 0.3 , and srcc was two patients per 100,000 persons . baseline characteristics are compared between appendiceal tnet , gcc , and srcc in table 1 . significant differences in age at presentation ( p < 0.001 ) , sex distribution ( p < 0.001 ) , surgery ( p < 0.001 ) , type of surgery ( appendectomy , right hemicolectomy , and surgery not otherwise specified ) ( p < 0.001 ) , and stage ( p < tnet patients presented younger ( median age of 41 vs. 54.5 in gcc and 57 years in srcc ) . female patients constituted a higher proportion among tnet cases ( 66.2% vs. 61.4% of srcc and 48.5% of gcc ) . advanced stage disease was more common in srcc patients ( 60.9% vs. 14.1% in tnet and 10.4% in gcc ) . a higher proportion of white patients was observed in all three histologies , but the difference was not statistically significant ( p=0.11 ) . surgery was performed in 839 ( 82.6% ) of the tnet patients , 1,365 ( 86.4% ) of the gcc , and 419 ( 78.6% ) of the srcc patients , and these differences were statistically significant ( p < 0.001 ) . appendectomy was performed in 52 of patients with tnet ( 5.9% ) as compared to 54 of the gcc ( 3.9% ) , and 9 of the srcc ( 2.1% ) . right hemicolectomy was performed in 484 of the tnet ( 54.9% ) , 1,208 of the gcc ( 86.4% ) , and 301 of the srcc patients ( 70.7% ) . the differences in type of surgery were significantly different among the three histologies ( p < 0.001 ) . the mos for gcc and tnet were not reached at the time of this analysis . comparing survival among the different histologies , tnet had a survival advantage over gcc ( hazard ratio [ hr ] , 0.56 ; 95% ci , 0.45 to 0.69 ; p=0.005 ) , which had a better survival over srcc histology ( hr , 0.22 ; 95% ci , 0.19 to 0.26 ; p < 0.001 ) ( table 3 ) . in each stage , tnet and gcc had significant survival advantages over srcc ( figs . 1 - 3 ) . in the localized stage , mos was not reached for any of the three histologies . comparing srcc , the hr for survival was 0.26 ( 0.15 - 0.46 ; p < 0.001 ) for tnet and 0.42 ( 0.26 - 0.69 ; p < 0.001 ) for gcc . for regional stage disease , the mos for srcc was 35 months ( 95% ci , 30 to 45 ) , while mos for gcc and tnet were not reached . the tnet regional disease stage patients had survival advantage over gcc ( hr , 0.37 ; 95% ci , 0.24 to 0.59 ; p < 0.001 ) . gcc had better survival compared to srcc patients with regional disease stage ( hr , 0.29 ; 95% ci , 0.21 to 0.40 ; p < 0.001 ) . for distant disease stage , mos was 32 ( 95% ci , 13 to not reached ) for tnet , 23 ( 95% ci , 18 to 28 ) for gcc , and 15 months ( 95% ci , 13 to 18 ) for srcc . in this group , tnet patients had survival advantage over gcc ( hr , 0.61 ; 95% ci , 0.44 to 0.83 ; p=0.002 ) . gcc patients survived better compared to srcc patients with distant disease ( hr , 0.73 ; 95% ci , 0.60 to 0.90 ; p=0.003 ) . after adjusting for age , stage , and histology , there was a statistically significant difference in survival for srcc patients treated with hemicolectomy compared to appendectomy ( p=0.01 ) . there was no significant difference in survival for tnet and gcc patients based the type of surgery ( p=0.21 and p=0.94 , respectively ) . in the multivariate analysis stratified by stage and age , there was a statistically significant difference in survival favoring tnet ( hr , 0.41 ; 95% ci , 0.31 to 0.55 ) and gcc ( hr , 0.59 ; 95% ci , 0.48 to 0.72 ) over srcc . the seer database yielded 1,021 tnet patients , 1,582 with gcc , and 534 srcc patients . between 1973 and 2011 , the incidence of tnet , gcc , and srcc increased . in 2011 , the incidence of tnet reached 6.7 , gcc was 0.3 , and srcc was two patients per 100,000 persons . baseline characteristics are compared between appendiceal tnet , gcc , and srcc in table 1 . significant differences in age at presentation ( p < 0.001 ) , sex distribution ( p < 0.001 ) , surgery ( p < 0.001 ) , type of surgery ( appendectomy , right hemicolectomy , and surgery not otherwise specified ) ( p < 0.001 ) , and stage ( p < tnet patients presented younger ( median age of 41 vs. 54.5 in gcc and 57 years in srcc ) . female patients constituted a higher proportion among tnet cases ( 66.2% vs. 61.4% of srcc and 48.5% of gcc ) . advanced stage disease was more common in srcc patients ( 60.9% vs. 14.1% in tnet and 10.4% in gcc ) . a higher proportion of white patients was observed in all three histologies , but the difference was not statistically significant ( p=0.11 ) . surgery was performed in 839 ( 82.6% ) of the tnet patients , 1,365 ( 86.4% ) of the gcc , and 419 ( 78.6% ) of the srcc patients , and these differences were statistically significant ( p < 0.001 ) . appendectomy was performed in 52 of patients with tnet ( 5.9% ) as compared to 54 of the gcc ( 3.9% ) , and 9 of the srcc ( 2.1% ) . right hemicolectomy was performed in 484 of the tnet ( 54.9% ) , 1,208 of the gcc ( 86.4% ) , and 301 of the srcc patients ( 70.7% ) . the differences in type of surgery were significantly different among the three histologies ( p < 0.001 ) . the mos for gcc and tnet were not reached at the time of this analysis . comparing survival among the different histologies , tnet had a survival advantage over gcc ( hazard ratio [ hr ] , 0.56 ; 95% ci , 0.45 to 0.69 ; p=0.005 ) , which had a better survival over srcc histology ( hr , 0.22 ; 95% ci , 0.19 to 0.26 ; p < 0.001 ) ( table 3 ) . in each stage , tnet and gcc had significant survival advantages over srcc ( figs . 1 - 3 ) . in the localized stage , mos was not reached for any of the three histologies . comparing srcc , the hr for survival was 0.26 ( 0.15 - 0.46 ; p < 0.001 ) for tnet and 0.42 ( 0.26 - 0.69 ; p < 0.001 ) for gcc . for regional stage disease , the mos for srcc was 35 months ( 95% ci , 30 to 45 ) , while mos for gcc and tnet were not reached . the tnet regional disease stage patients had survival advantage over gcc ( hr , 0.37 ; 95% ci , 0.24 to 0.59 ; p < 0.001 ) . gcc had better survival compared to srcc patients with regional disease stage ( hr , 0.29 ; 95% ci , 0.21 to 0.40 ; p < 0.001 ) . for distant disease stage , mos was 32 ( 95% ci , 13 to not reached ) for tnet , 23 ( 95% ci , 18 to 28 ) for gcc , and 15 months ( 95% ci , 13 to 18 ) for srcc . in this group , tnet patients had survival advantage over gcc ( hr , 0.61 ; 95% ci , 0.44 to 0.83 ; p=0.002 ) . gcc patients survived better compared to srcc patients with distant disease ( hr , 0.73 ; 95% ci , 0.60 to 0.90 ; p=0.003 ) . after adjusting for age , stage , and histology , there was a statistically significant difference in survival for srcc patients treated with hemicolectomy compared to appendectomy ( p=0.01 ) . there was no significant difference in survival for tnet and gcc patients based the type of surgery ( p=0.21 and p=0.94 , respectively ) . in the multivariate analysis stratified by stage and age , there was a statistically significant difference in survival favoring tnet ( hr , 0.41 ; 95% ci , 0.31 to 0.55 ) and gcc ( hr , 0.59 ; 95% ci , 0.48 to 0.72 ) over srcc . the analysis of the seer registry sample confirms the differences in presentation and outcome of these diseases . tnet tend to occur at a younger age and has a less aggressive clinical course , early stage at presentation and significantly improved os . at the other end of the disease spectrum , the aggressive nature of srcc is reflected by the high risk of distant metastasis at diagnosis , and worse survival outcomes even when controlled for stage . the reported sex distribution in tnet ranges from a female preponderance ( 63% to 73% ) to evenly distributed . similarly for gcc , literature reports range from female gender predominance , to equal distribution . most of these series are based on small numbers of patients from single institution studies . the current report confirms , using a large national database , that there are gender differences in distribution of tnet , gcc , and srcc ( p < 0.001 ) . for srcc histology , we confirmed a higher prevalence amongst women in this seer analysis as compared to gcc histology , consistent with a previous report . additionally , appendiceal srcc appear to be clinically similar to right - sided , microsatellite unstable , signet ring cell colon adenocarcinoma with respect to gender distribution , age of diagnosis and outcome . this raises the question whether appendiceal srcc and right colon adenocarcinomas behave more like a single disease entity distinct from other colorectal cancer ( crc ) adenocarcinoma and appendiceal gcc . this apparent difference in clinical behavior will require further characterization through genomic analysis of crc srcc , appendiceal srcc and typical gcc . hence , a molecularly based classification may facilitate prognostication and perhaps target identification for future clinical trials . the analysis of the seer data confirms that histology and stage at presentation are the major determinants of outcome . the high risk of metastatic disease in the srcc patients confirms the aggressive clinical behavior of this tumor . the rate of metastasis at diagnosis in srcc histology ranges from 14% to 63% . the survival advantage for tnet and gcc over srcc persisted after controlling for the difference in stage distribution . similar observations were reported with 5-year survival of patients with advanced stage gcc , srcc and poorly differentiated adenocarcinoid tumor being 100% , 38% , and 0% , respectively . given their rarity , there is no consensus on the management of appendiceal tnet , gcc , or srcc . the treatments employed included surgical resection and cytoreduction of primary tumor and metastatic sites , intraperitoneal chemotherapy and systemic fluorouracil - based chemotherapy . debulking surgery and hyperthermic intraperitoneal chemotherapy treatment is not well - established in appendiceal net , gcc , and srcc tumors , although it improves appendiceal mucinous adecarcinoma . clinically , the management decisions are usually based on histologic subtype and stage , as well as the patient s performance status . early stage tnet , gcc , or srcc should be managed surgically , although the extent of surgery is an open question . historically , the accepted surgical dogma was that simple appendectomy is sufficient for resection of tnet less than 2 cm . recent reports demonstrated that appendiceal tnet measuring less than 2 cm may have regional nodal involvement , raising the question whether a more extensive surgery than simple appendectomy is required irrespective of size . these observations are further supported by a seer analysis where 32.8% of patients with net less than 2 cm in size ( 20/61 ) had lymph node metastasis . the north american neuroendocrine tumor society ( nanets ) and european neuroendocrine tumor society ( enets ) guidelines suggest right hemicolectomy for all tumors > 2 cm or presence of deep mesoappendiceal invasion , positive or unclear margins , higher proliferative rate ( grade 2 ) , angi olymphatic invasion , and mixed histology , irrespective of tumor size . while simple appendectomy may be adequate for early stage gcc , cecal involvement or high mitotic count index should be an indication for right hemicolectomy . surgery for srcc should always involve a right hemicolectomy due to the high likelihood of lymph node metastasis , irrespective of the size of the primary appendiceal mass . this is supported by our analysis of the seer data that indicates superior survival for patients with srcc who undergo right hemicolectomy . the role of surgical resection of the ovaries in female patients with localized srcc or gcc remains controversial . a summary of the nanets and enets guidelines is provided in table 4 . unlike crc adenocarcinoma , the role of adjuvant therapy is not established for tnet , gcc , or srcc . based on our analysis , patients with tnet have an excellent os and therefore adjuvant therapy is not indicated . on the other hand , given the high risk of systemic metastasis in srcc , we recommend offering adjuvant therapy for all patients undergoing resection . in our analysis of the seer sample , the outcome of patients with early stage gcc appears favorable , suggesting that patients with gcc should be considered for adjuvant therapy only if they have high risk features such as cecal invasion , perforation , or lymph node involvement . for gcc patients with advanced stage disease , options of treatments include peritoneal debulking with intraperitoneal chemotherapy for patient with limited peritoneal disease or systemic fluorouracilbased chemotherapy . for srcc patients , our recommendations include treating with systemic fluorouracil - based chemotherapy initially and only consider peritoneal resection for patients with good response to initial therapy . a summary of the proposed treatment algorithm is provided in fig . predictive and prognostic biomarkers are not well defined for these diseases . in an attempt to identify gene variations between appendiceal mucinous adenocarcinoma and gcc , we profiled nine gcc samples using second - generation gene sequencing . only one patient with gcc ( 11% ) had kras mutation , and two had tp53 mutation ; myc , smad4 , and apc mutations were absent in gcc . proved the absence of kras mutations in 16 and 14 gcc samples . in the light of its rarity , molecular differentiation markers for appendiceal tnet , gcc , and further confirmation with larger studies is needed to evaluate pathologic genomic sequencing for the three histologies in order to detect differences in survival and possible future biomarkers that predict response to specific target treatments . the seer registry findings confirm the distinct clinical entities of tnet , gcc , and srcc with respect to presentation and outcome .

purposeappendiceal tumors are a heterogeneous group of diseases that include typical neuroendocrine tumors ( tnet ) , goblet cell carcinoids ( gcc ) , and atypical gcc . atypical gcc are classified into signet - ring cell cancers ( srcc ) and poorly differentiated appendiceal adenocarcinoids . the prognosis and management of these diseases is unclear because there are no prospective studies . the aim of this study is to assess the characteristics and outcome of appendiceal tnet , gcc , and srcc patients.materials and methodsappendiceal tnet , gcc , and srcc patients diagnosed between 1973 and 2011 were identified in the surveillance epidemiology and end results ( seer ) database . demographics , type of surgery , and clinicopathologic characteristics were collected . survival functions were estimated by the kaplan - meier method , and log - rank test was used to assess the difference in overall survival ( os ) among the three histologies.resultsthe seer database yielded 1,021 tnet patients , 1,582 with gcc , and 534 srcc patients . tnet presented at a younger age ( p < 0.001 ) . patients with srcc presented with advanced stage disease ( p < 0.001 ) . the median os ( mos ) for gcc and tnet patients was not reached ; mos for srcc was 24 months . multivariate analysis stratified for stage revealed significantly longer survival for tnet and gcc than srcc ( p < 0.001).conclusionthis is the largest report to date for appendiceal neuroendocrine tumor patients , suggesting a spectrum of diseases with different characteristics and outcomes . in this report , we present a treatment approach for this complex spectrum of disease , based on the experience of ohio state and emory universities investigators .

Introduction Materials and Methods 1. Patients 2. Statistical analysis Results 1. Characteristics of patients with TNET, GCC, and SRCC 2. Survival outcome of patients with TNET, GCC, and SRCC Discussion Conclusion

we have prepared two new diastereoisomeric 2-aza-5-phosphabicyclo[2.2.1]heptanes from naturally occurring trans-4-hydroxy - l - proline in six chemical operations . these syntheses are concise and highly efficient , with straightforward purification . when we used these chiral phosphines as catalysts for reactions of -substituted allenoates with imines , we obtained enantiomerically enriched pyrrolines in good yields with excellent enantioselectivities . these two diastereoisomeric phosphines functioned as pseudoenantiomers , providing their chiral pyrrolines with opposite absolute configurations .

Supplementary Material

igg4-rd is a newly recognized fibroinflammatory condition of unknown etiology characterized by tumefactive lesions , dense lymphoplasmacytic infiltration rich in igg4-positive plasma cells , storiform fibrosis and an elevated serum igg4 level [ 13 ] . the kidney is one of the organs commonly affected by igg4-rd , and tubulointerstitial nephritis ( tin ) with infiltration of numerous igg4-positive plasma cells is the predominant type of kidney lesion [ 47 ] . high levels of igg and igg4 are striking and characteristic features of renal involvement such as multiple lesions with low attenuation demonstrated by contrast - enhanced computed tomography are often evident [ 47 ] . although tin is the main feature of the kidney lesion in igg4-rd , some glomerular lesions have also been described , and membranous nephropathy ( mn ) has been recognized as the most common form [ 48 ] . in previously reported cases of mn associated with igg4-rd , mn was diagnosed together with , or after the onset of igg4-rd [ 912 ] . here , we report a case of idiopathic mn as a preceding disease before the onset of igg4-rd . he had previously been well and had no significant medical history . his serum total protein level was 46 g / l ( 4.6 g / dl ) , serum albumin 17 g / l ( 1.7 g / dl ) , urinary protein excretion 4.3 g / day without occult blood and the 24-h creatinine clearance was normal at 1.51 ml / s ( 90.5 ml / min ) ( table 1 ) . the anti - nuclear antibody was negative , and serum immunoglobulins g , a and m were all within normal limits at 11.98 g / l ( 1198 mg / dl ) , 11.37 complement levels were also within normal limits : c3 1.12 g / l ( 112 mg / dl ) , c4 0.314 g / l ( 31.4 mg / dl ) and ch50 30 u / ml . contrast - enhanced computed tomography ( ct ) demonstrated small , non - specific nodules in the right lung , and a hemangioma of the liver . table 1.clinical course and laboratory findings in the present casemay 2008january 2009july 2009july 2010december 2010march 2011august 2011eventdiagnosed as mnworsening of nsdiscontinuation of psladmission to our hospitaltreatmentdiureticspsl , furosemidefurosemide , olmesartanfurosemide , olmesartanfurosemide , olmesartanfurosemide , olmesartanfurosemide , olmesartantp ( g / l)46566071778592alb ( g / l)17152931303124igg ( g / l)12nanananana39urinalysis ( protein)3 + 3 + 3 + 3 + 3 + 3 + 3+up ( g / day)4.33.03.1nanana2.124 h ccr ( ml / s)1.511.121.37nanana1mn , membranous nephropathy ; ns , nephritic syndrome ; psl , prednisolone ; tp , total protein ; alb , albumin ; up , urinary protein excretion ; ccr , creatinine clearance ; na , data not available . clinical course and laboratory findings in the present case mn , membranous nephropathy ; ns , nephritic syndrome ; psl , prednisolone ; tp , total protein ; alb , albumin ; up , urinary protein excretion ; ccr , creatinine clearance ; na , data not available . percutaneous kidney biopsy was performed , and the histology revealed diffuse membranous changes on the glomerular basement membrane without tubulointerstitial lesions ( figure 1a and b ) . an immunofluorescence study showed diffuse , fine - granular staining of igg , c3 , fibrinogen , kappa- and lambda - chain in the glomerular capillary loops . there were no deposits in other areas including mesangeal matrix , subendothelial aspects and tubular basement membrane . an additional immunofluorescence study of igg subclasses showed predominant positive staining for igg4 and igg1 and weak positive staining for igg2 and igg3 in the glomeruli ( figure 1c ) . an electron microscopy examination revealed diffuse , homogenous , small electron - dense deposits in the subepithelial aspects of the glomerular basement membrane , which corresponded to ehrenreich and churg stage 2 . subendothelial deposits were not present , and endothelial cells did not contain any tubuloreticular structure . the mesangial matrix and tubular basement membrane did not contain electron - dense deposits . 1.histopathologic findings of kidney biopsy at the previous hospital at the time of onset of idiopathic mn . ( a ) light microscopy shows no significant mesangial cell or matrix proliferation in glomeruli , and no interstitial lesions . ( b ) diffuse , small deposits are evident in the subepithelial aspects of the glomerular basement membrane . ( c ) an immunofluorescence study of igg subclasses shows predominant positive staining for igg1 and igg4 and weak positive staining for igg2 and igg3 in glomeruli . histopathologic findings of kidney biopsy at the previous hospital at the time of onset of idiopathic mn . ( a ) light microscopy shows no significant mesangial cell or matrix proliferation in glomeruli , and no interstitial lesions . periodic acid - schiff stain , 200 . ( b ) diffuse , small deposits are evident in the subepithelial aspects of the glomerular basement membrane . ( c ) an immunofluorescence study of igg subclasses shows predominant positive staining for igg1 and igg4 and weak positive staining for igg2 and igg3 in glomeruli . a systemic work - up revealed no other disease or malignancy , and the patient prednisolone was administered at 40 mg daily , but the patient suffered repeated cerebral infarction attacks after starting the therapy . prednisolone was tapered and discontinued 6 months after the start of the therapy , without any significant effect . the patient 's proteinuria persisted at around 3 g / day and he was followed up while receiving diuretics and the angiotensin receptor blocker , olmesartan . two years after discontinuation of glucocorticoid therapy , the patient developed epigastric discomfort and was hospitalized again . laboratory examinations revealed elevated serum levels of ast , alt , alp , total bilirubin and -gtp [ 64 iu / l , 106 iu / l , 1948 iu / l , 111 mol / l ( 6.5 mg / dl ) and 1544 iu / l , respectively ] accompanied by a slight increase in serum creatinine at 100 mol / l ( 1.13 mg / dl ) and a normal serum amylase level of 111 iu / l . abdominal ct revealed swelling of the pancreatic head , dilatation of the main pancreatic duct and stenosis of the common bile duct . tumor markers including cea and ca19 - 9 were all within the normal ranges , while hypergammaglobulinemia was observed and the serum igg level was elevated at 39.21 g / dl ( 3921 mg / dl ) with marked elevation of igg4 at 19.2 hypergammaglobulinemia persisted , and serum iga , igm and complement studies showed that all were within normal limits . serum anti - nuclear antibody was negative , whereas the rheumatoid factor was positive at 120.6 iu / ml . urinary protein excretion was 2.1 g / day without occult blood and 24-h creatinine clearance was mildly deteriorated at 1 ml / s ( 60.0 ml / min ) ( table 1 ) . excretion of urinary 2-microglobulin ( 2-mg ) and n - acetyl--d - glucosaminidase was markedly elevated at 11 636 g / day and 25.0 iu / day , respectively . contrast - enhanced ct showed bronchial thickening in the bilateral lungs , pancreatic swelling with stenosis of the main pancreatic duct and bile duct , bilateral swollen kidneys with multiple lesions exhibiting low attenuation , periaortic and periarterial mass lesions , diffuse swelling of the prostate gland and systemic lymphadenopathy , compatible with the radiologic abnormalities characteristic of igg4-rd ( figure 2 ) . 2.contrast-enhanced ct at our hospital at the time of onset of igg4-rd . marked swelling of pancreatic head and bilateral swollen kidneys with multiple lesions exhibiting low attenuation were observed . contrast - enhanced ct at our hospital at the time of onset of igg4-rd . marked swelling of pancreatic head and bilateral swollen kidneys with multiple lesions exhibiting low attenuation were observed . percutaneous kidney biopsy was not performed since we could not obtain the patient 's agreement . instead , percutaneous needle biopsies from the pancreas and prostate gland specimens obtained by biopsy from the prostate gland and pancreas revealed dense lymphoplasmacytic infiltration with storiform fibrosis and infiltration of numerous igg4-positive plasma cells ( igg4/ igg - positive plasma cell ratio > 50% ) without malignancy ( figure 3 ) . 3.histopathologic features of a specimen obtained from the prostate gland by percutaneous needle biopsy at our hospital at the time of onset of igg4-rd . there is no evidence of cancer cells and there is a typical swirling pattern of fibrosclerosing inflammation with infiltration of numerous lymphocytes , the so - called storiform fibrosis. ( b ) immunohistochemical staining for igg in a needle biopsy specimen from the prostate gland , 150 . immunohistochemical staining for igg4 in a needle biopsy specimen from the prostate gland , 150 . the igg4-positive plasma cell / igg - positive plasma cell ratio was over 50% in tissue from the prostate gland . histopathologic features of a specimen obtained from the prostate gland by percutaneous needle biopsy at our hospital at the time of onset of igg4-rd . there is no evidence of cancer cells and there is a typical swirling pattern of fibrosclerosing inflammation with infiltration of numerous lymphocytes , the so - called storiform fibrosis. ( b ) immunohistochemical staining for igg in a needle biopsy specimen from the prostate gland , 150 . immunohistochemical staining for igg4 in a needle biopsy specimen from the prostate gland , 150 . the igg4-positive plasma cell / igg - positive plasma cell ratio was over 50% in tissue from the prostate gland . western blot analysis of the patient 's serum for the detection of serum autoantibodies against the m - type phospholipase a2 receptor ( anti - pla2r ) was performed , as described previously , and was negative . oral administration of prednisolone was initiated at 40 mg daily , and the patient 's clinical symptoms including jaundice and anorexia improved rapidly . the laboratory data and multiple organ lesions also improved , while proteinuria persisted at around 3 g / day . it has been reported that mn is present in 7% of kidney biopsy specimens in igg4-rd . most cases of mn associated with igg4-rd are accompanied by igg4-related tin [ 46 , 9 , 10 ] , although recent reports have also documented mn without tin but with other features of igg4-rd ( 8 , 11 , 12 ) . in mn associated with igg4-rd , positive deposition of igg4 in the glomerular basement membrane has been observed , either predominantly or together with other igg subclasses [ 812 ] . originally , the immunofluorescent examination of igg subclasses was known as a very useful tool to distinguish the primary mn from the secondary mn . predominant deposition of igg4 , often accompanied by other subclasses such as igg1 , was one of the well - known features of idiopathic mn . moreover , idiopathic mn and mn associated with igg4-rd share an essential immunologic feature known as th2 cytokine - mediated immune reaction [ 14 , 15 ] , suggesting that these two disorders share common pathophysiologic processes for increased production of th2 cytokines and subsequent increase of igg4 . on the other hand , these two disorders differ in many other features , including clinical symptoms , laboratory parameters and radiographic findings . an anti - pla2r antibody , which has been described as a major target antigen involved in idiopathic mn and seen in 7080% of patients with idiopathic mn [ 13 , 1618 ] , has been reported to be negative in all cases of igg4-rd and also cases of mn associated with igg4-rd [ 8 , 10 , 12 ] , suggesting no involvement of this antibody in the development of igg4-rd . in the present case , there was no evidence of any association of igg4-rd based on the clinical symptoms , serological data and imaging findings when the patient developed nephrotic syndrome . therefore , it was reasonable to have diagnosed the present case as idiopathic mn at the time of onset of nephrotic syndrome . the anti - pla2r antibody is described as negative in all cases evaluated in mn associated with igg4-rkd and was also negative in this patient , although the result was obtained from the serum 3 years after the diagnosis of idiopathic mn and after corticosteroid therapy . however , 2025% of patients with idiopathic mn are reported to be negative for the antibody [ 1618 ] . the absence of the anti - pla2r antibody is thus only partly helpful to distinguish from idiopathic mn . interestingly , the patient 's total serum protein level increased gradually without any significant improvement of proteinuria or elevation of the serum albumin level ( table 1 ) , and eventually the clinical symptoms and laboratory abnormalities of igg4-rd became evident 3 years after the onset of idiopathic mn . accordingly , it is possible that some conditions underlying the persistent predominance of th2-type immune responses during the 3 years of follow - up might have led to the development of igg4-rd . currently , idiopathic mn is defined as mn without any other disorder that could cause secondary mn , with predominant deposition of igg4 in the glomerular basement membrane . however , mn associated with igg4-rd has also been revealed to be igg4-predominant and , interestingly , the igg subclass of the anti - pla2r antibody seen in idiopathic mn is predominantly igg4 . although the pathogenesis of igg4-rd is poorly understood , it may share some common mechanisms with idiopathic mn , and thus a comparative study of the two diseases might be helpful for clarifying their pathogenesis . consideration for igg4-rd in mn , including systemic evaluation or measurement of serum igg4 level , would be necessary in addition to the evaluation of the anti - pla2r antibody and igg subclasses in the immune complex deposited on the glomerular basement membrane .

we report a case of igg4-related disease ( igg4-rd ) diagnosed after 3 years of follow - up for idiopathic membranous nephropathy ( mn ) . mn has been considered as glomerular lesion of igg4-related kidney diseases in recent years and was diagnosed simultaneously with or after a diagnosis of igg4-rd in previously reported cases . in the present case , igg4-rd developed 3 years after the diagnosis of idiopathic mn , indicating a possible relationship between idiopathic mn and igg4-rd through common underlying mechanisms of development .

Background Case report Discussion

treatment duration lasts for months , and is often fraught with potentially dangerous side effects . some of these side effects , such as influenza - like illness , myalgias , depression , and cytopenias can be intolerable resulting in premature treatment cessation , but have no lasting effects . ocular complications are among the complications of interferon therapy and include retinal hemorrhages , cotton wool spots , loss of color vision , cataracts , glaucoma , and occasionally retinal artery or vein obstruction . although the incidence of ophthalmological disorders while on interferon therapy is low , this can result in loss of vision.411 in this prospective case series study , one hundred patients with chronic hepatitis - c under interferon ( inf ) therapy were consecutively recruited from patients attending the outpatient ophthalmic clinic at the university hospital , faculty of medicine , al - azhar university , assuit . the study protocol was approved by the ethics committee at al - azhar university hospital . written informed consent was obtained from all patients included in the study , and then in addition to the medical and laboratory examination , all patients were subjected to full ophthalmological assessment to evaluate the retinopathy associated interferon therapy . inclusion criteria were : ( 1 ) seropositivity for anti - hcv antibodies determined using elisa ; ( 2 ) first use of interferon therapy . exclusion criteria were : ( 1 ) previous interferon therapy ; ( 2 ) patients with hepatitis b ; ( 3 ) patients with decompensated liver ; ( 4 ) patients with renal failure ; ( 5 ) patients with cardiovascular disorders such as coronary insufficiency , congestive heart failure , and ischemic stroke ; ( 6 ) patients with a dense cataract , visual field abnormality , glaucoma , or any other ocular abnormalities . evaluation before treatment : history of visual loss , hazy vision , and ocular disease or ocular surgeries ophthalmological examination : ( 1 ) best corrected visual acuity ( bcva ) measured using landolt s broken rings ; ( 2 ) pupillary reaction ( direct and consensual ) and if there is afferent pupillary defect ; ( 3 ) full slit - lamp examination ; ( 4 ) intraocular pressure ( iop ) measured by goldmann s applanation tonometer ; ( 5 ) fundus biomicroscopy ( carried out after maximum pupillary dilation by a combination of tropicamide 1% eye drops and phenylephrine 2.5% eye drops using both slit - lamp biomicroscopy with noncontact + 90 d volk lens and/or goldmann contact lens ) ; ( 6 ) examination of the retinal periphery using an indirect ophthalmoscope . medical examinations and abdominal ultrasonography investigations : ( 1 ) ocular , both colored fundus photography and fundus fluorescein angiography ( ffa ) ; ( 2 ) laboratory ( including enzyme - linked immunosorbent assay ( ellsa ) test for hepatitis surface ag and ab ; polymerase chain reaction ( pcr ) for hepatitis c virus rna ; full blood count ; prothrombin time ; direct and indirect bilirubin ; fasting blood sugar ; serum creatinine ; liver enzymes ( serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase ) ; alanine aminotransferase . patients were treated with pegylated interferon -2a at a dose of 180 mcg injected subcutaneously once weekly for 48 weeks . all patients underwent clinical and laboratory assessments , ophthalmic examination , at 15 days , 1 month , 2 months , 3 months , 6 months , 8 months , and 9 months after the start of ifn treatment . patients were treated with pegylated interferon -2a at a dose of 180 mcg injected subcutaneously once weekly for 48 weeks . all patients underwent clinical and laboratory assessments , ophthalmic examination , at 15 days , 1 month , 2 months , 3 months , 6 months , 8 months , and 9 months after the start of ifn treatment . initially , 120 patients fulfilled the criteria for ifn therapy , but 20 of them were excluded due to non - compliance with the follow - up visits and due to the presence of diabetic or hypertensive retinopathy . also , two patients died during the study time , due to a disease not related to hepatitis , so these patients are not included . therefore , a total of 100 patients ( 68 male and 32 female ) , with chronic hcv were selected . after the start of interferon therapy , 16 out of 100 patients ( 16% ) developed retinopathy which was bilateral in 12 ( 75% ) patients and unilateral in 4 ( 25% ) patients . retinopathy was initially diagnosed by the appearance of a cotton wool spot in 14 patients . in four of the 14 patients , retinal hemorrhage two of the 16 patients who developed retinopathy were diagnosed by retinal hemorrhage without cotton wool spots . one patient ( not diabetic or hypertensive ) had cystoid macular edema , this was the only patient who complained of the visual disturbance due to interferon . all patients had bcva of ( 6/6 ) during ifn treatment except for three patients : the one who had cystoid macular edema ( bcva was 6/12 and returned to 6/6 after cessation of treatment ) and two patients with ametropic amblyopia . in addition , we confirmed that all patients with hypertension had no retinopathy using the standard classification . to exclude the possibility of the development of diabetic or hypertension retinopathy after ifn treatment , we deleted the data obtained from patients who had diabetes or hypertension and any retinopathy 2 months after the cessation of ifn treatment . retinopathy was first diagnosed 214 weeks after the start of treatment and disappeared in 11 of the 16 patients despite the continuation of therapy . a few cotton wool spots , retinal hemorrhages , or both were observed in the remaining five patients at the end of treatment but resolved within 1 month after treatment was stopped . no other ocular complications ( ie , cataracts , glaucoma , retinal artery , or vein obstruction ) were observed . the mean hemoglobin values , white blood cell , red blood cell , and platelet counts decreased significantly ( p < 0.0001 ) during ifn treatment . the clinical profiles and laboratory data of the patients are shown in tables 1 and 2 , respectively . interferon - associated retinopathy was first recognized in 1990 when ikebe and associates reported a 39-year - old patient who developed retinal hemorrhages and cotton wool spots following intravenous administration of interferon.12 the underlying mechanisms of interferon - induced retinopathy are not completely understood . guyer et al13 had proposed an immune complex deposition causing occlusion of retinal capillaries leading to cotton wool spot formation . on the other hand , an ischemic insult , similar to that seen in hypertensive and diabetic patients could be another potential mechanism by which these changes develop.14 some studies15,16 have shown a relatively higher incidence of retinopathy ( symptomatic or asymptomatic ) ranging from 19% to 64% during treatment with interferon . panetta and gilani17 and cuthbertson et al14 show relatively lower incidences of retinopathy ( 3.8% and 6.25% , respectively ) . similar to other studies,4,5,9 our study also shows that retinal changes usually develop within the first 3 months of interferon treatment , a fact that may favor an immune etiology rather than a cumulative effect of interferon leading to the development of retinopathy . retinopathy developed by week 14 in most of the patients ( 13/16 , 81.25% ) after the start of therapy and disappeared in the majority of the patients ( 11/16 , 68.75% ) during the 48 week period in which the patients were receiving the treatment . this suggests that treatment can be continued despite the development of retinopathy in many patients . however , two patients who developed cotton wool spots early in the therapy ( 2 weeks ) thereafter suffered from retinal hemorrhage in a prolonged manner . therefore , patients who develop cotton wool spots early on during therapy should be carefully monitored . however , as reported in previous studies,4,13,14 most of the patients with retinopathy in this study were asymptomatic . this study confirms previous reports that retinopathy is a temporary and asymptomatic complication of interferon therapy . therefore there is no rule for suspending the use of interferon in patients who develop retinopathy or any other ocular side effects . it s commonly agreed among authors that careful monitoring should be performed in the presence of any ocular sign , even without symptoms.18

purposeto evaluate retinopathy associated with interferon therapy in patients with chronic hepatitis c.methodsone hundred patients with chronic hepatitis c undergoing interferon therapy were examined for the presence of cotton wool spots , retinal hemorrhages , cystoid macular edema , capillary non - perfusion , and arteriolar occlusion . complete ophthalmological examination including indirect ophthalmoscopic fundus examination was carried out for all patients and colored fundus photography and fluorescein angiography were carried out for the patients with positive fundus findings . the follow - up period was 9 months.resultssixteen percent of patients developed retinopathy in the form of cotton wool spots , retinal hemorrhages , cystoid macular edema , and capillary non-perfusion.conclusioninterferon therapy can lead to retinopathy which is mostly reversible and dose related . periodic fundoscopic examinations help in early detection and prevent progression to permanent visual loss .

Introduction Patients and methods Treatment protocol Follow-up Results Discussion Conclusion

nathan , as an oncology fellow , knew well that white blood cells fought infections . this was an experiment repeated in front of us all the time , he says : chemotherapy lowered his patients ' white blood cells and increased their risk of infections . mackaness had shown that macrophage activation did not depend on direct contact with t cells ( 1 ) , suggesting the possibility of a secreted factor . when nathan tested the supernatant from activated t cells , he saw that it did indeed induce macrophage activation ( 2 ) . nathan got a rough idea of the molecular weight ( 3 ) , but that was the best anyone could do , he says . protein separation methods were primitive , and cloned proteins and monoclonal antibodies would only become available a decade later . henry murray , one of nathan 's collaborators , sums up the feeling of frustration : we were all nibbling at the edges of the same problem . nathan therefore changed tack to take a closer look at the activated macrophages . short - lived neutrophils were known to produce hydrogen peroxide , and nathan found the same was true of longer - lived activated macrophages ( 4 ) . unlike previous signs of macrophage activation increased spreading , phagocytosis , and glucose metabolism this so - called respiratory burst ifn had been on the cover of time magazine , and recombinant murine ifn was found to induce macrophages to kill tumor cells ( 5 ) . nathan , now a faculty member in zanvil cohn 's macrophage factory at rockefeller university ( new york , ny ) , thought ifn might also activate macrophages to kill intracellular parasites . consistent with this idea , ifn was made by antigen - stimulated t cells and was associated with defense from infection . now the respiratory burst gave nathan an assay , berish rubin ( down the street at the new york blood center ) supplied an ifn monoclonal antibody , and a phone call to genentech yielded recombinant ifn. in a seminal paper published in the journal of experimental medicine in 1983 , nathan was thus able to show that depleting ifn from unpurified t cell supernatants decreased the respiratory burst activity and the killing of intracellular protozoa in human macrophages . adding back recombinant ifn into this mix restored macrophage activation ( 6 ) . i had an assay , a hunch , a history of purifying proteins that did this , and the serendipity of meeting with people nearby who had the antibody . nathan next showed that ifn worked in people . injecting recombinant ifn directly into cutaneous lesions of lepromatous leprosy patients induced macrophage infiltration , hydrogen peroxide production , and killing of the causative pathogen , mycobacterium leprae ( 7 ) . in the 1990s , the macrophages of children with ifn receptor deficiencies were shown to be defective in killing mycobacteria ( 8) . tracing the pathway from t cells to macrophages to bacteria started , for nathan , in 1967 , and he says we still haven't finished making the molecular links .

t cells tell macrophages when to start making the toxic soup of lysosomal enzymes , reactive oxygen species , and nitric oxide that destroys intracellular pathogens . in 1983 , carl nathan proved that this start signal comes in the form of the secreted cytokine ifn.

The elusive eluate Back on track

was initiated by our team about a decade ago , and this has been continued at the medical research center , kasturba health society , mumbai . the comparative effects of three plant products on perimenopausal symptoms , quality of life ( qol ) and clinical biochemistry have been reported by our group earlier . our observation of frequent mood changes and sleep disturbances in women in the premenopausal group prompted us to undertake a study of cyclic symptoms in maitreyi hcp . in this communication , we describe the preliminary findings from the analysis of premenstrual symptoms in 200 consecutive women who underwent a comprehensive check - up in the maitreyi hcp . diagnosis of premenstrual tension syndrome ( pmts ) is based on the following criteria as per the american association of obstetricians and gynecologists : a 30% increase in the intensity of symptoms of premenstrual syndrome ( measured using a standardized instrument ) from cycle days 510 as compared with the 6-day interval before the onset of menses and documentation of these changes for at least two consecutive cycles . at least one of the following affective and somatic symptoms during the 5 days before menses in previous cycles . affective symptoms : depression , angry outbursts , irritability , anxiety , confusion , social withdrawal . somatic symptoms : breast tenderness , abdominal bloating , headache , swelling of extremities ; symptoms relieved from days 4 through 13 of the menstrual cycle . to determine the prevalence of premenstrual cyclic symptoms in women who attended the maitreyi hcp . to determine the prevalence of premenstrual cyclic symptoms in women who attended the maitreyi hcp . maitreyi 's hcp for women above 35 includes comprehensive health questionnaire , gynecological and physician 's check - up , biochemical investigations of complete blood count , glucose tolerance test , lipid profile , liver functions , serum creatinine and serum thyroid stimulating hormone . pap smear , pelvic sonography and urine and stool examination for occult blood are also included . a multidisciplinary trans - system team offers this community - oriented programme to women with voluntary services by the consultants . bone mineral density and mammography this analysis is a preliminary report restricted to the premenstrual cyclic symptomatolgy in 200 consecutive women who attended this program over 2 years . a special case record form has been used and a detailed medical and surgical history is included in the same . twelve women who continued to menstruate beyond 50 , up to 55 years , have been included in the analysis . only those who had symptoms at least during two consecutive cycles or more were included in the analysis . the american society of obstetricians and gynecologists and the american psychiatric society have now brought out the consensus criteria for inclusion of the condition in pmts or premenstrual dysphoric disorder ( pmdd ) , the latter usually requiring treatment for neuropsychiatric symptoms . accordingly , pmts is said to be present when the woman reports three or more of the following symptoms in two cycles or more[46 ] : there are 36 symptoms as listed below in the sf-36 mental component . in the present analysis , the symptoms were classified as per the medical outcomes study short form-36 for assessment of premenstrual symptoms , and the presence of three of these occurring over two or more cycles was labelled as pmts : anxiety , irritability , depression , tension , mood swings , loss of self - control , difficulty in concentration , insomnia , confusion , headache , crying attacks , fatigue , aches , breast tenderness , cramps , bloatedness , food craving , visual disturbances , tiredness , fatigue , puffiness of the face , weight gain , altered libido , aggression , nausea , vomiting , acne , skin rashes , constipation , diarrhea , joint stiffness , backache , abdominal cramps / pain , anger attacks , aggravation of epilepsy , aggravation of asthma . of 107 women , 26 ( 24.3% ) did not report any premenstrual symptoms at all and 81 ( 75.7% ) reported at least one symptom [ table 1 ] . number of symptoms experienced by women in different age groups in two or more cycles maximum frequency was seen in women from 35 to 50 years of age . forty women ( 37.4% ) who reported premenstrual symptoms had three or more cyclic symptoms for more than two cycles and could be classified as those with pmts . fifteen ( 14% ) had five or more symptoms cyclically in at least two cycles . almost 10% reported five or more symptoms inclusive of anger attacks and could be labelled as pmdd . mastalgia was the most common symptom ( 50.5% ) , followed by mood changes ( 46.7% ) , depression ( 7.5% ) and anger attacks ( 6.5% ) . pmdd was present in 10% of the cases who were referred to a clinical psychologist or a psychiatrist . many women have premenstrual cyclic symptoms of psychological or physical nature and , sometimes , these limit their functional capacity . the symptoms due to neurohormonal imbalance or ageing may start as early as 35 years of age . pmts is an entity that causes considerable morbidity , and in about 3% of the women , it may disrupt the woman 's life when severe symptoms recur . when there are severe symptoms like anger attacks , depression and suicidal thoughts , special attention including psychiatric consultation may be required , i.e. the pmdd . the prevalence of pmts in general is about 210% for disabling symptoms , while minor symptoms may be present in up to 85% of the women.[468 ] in the present series , 75.7% reported at least one symptom cyclically . in the present series halbreich has reported that in about 20% of the women , the symptoms are severe enough to warrant treatment . dean et al . have reported a prevalence of 1930% when all women from 18 to 45 years of age were screened . in a recent survey of 3,913 women of 15 - 54 years of age , tschudin et al . observed that about 10% reported pmts and 3.1% reported pmdd . when the indian scenario is considered , chaturvedi et al . in the 1990s have reported a prevalence of 20% in a general population and severe symptoms in 8% . the same authors have , in a later study , reported suicidal ideas and/or death wish during the premenstrual period in 10% of the subjects , more so among college students and industrial working women as compared with housewives . banerjee et al . , in a group of 62 women , reported pmdd in 6.4% . there is a close association between mood disorders and the sleep rhythm and disturbances in sleep pattern in premenopausal age.[31314 ] many women and their physicians regard pmts as a physiological phenomenon and do not think treatment is necessary . however , it does affect qol in some and it is therefore essential to identify which women will benefit from lifestyle management and which will require pharmacological intervention . those who want to reach their full functional potential and who find that the symptoms are hampering their progress or interfering with normal daily routine should be certainly offered safe pharmacological interventions provided that they come for routine follow - ups . because there are many etiological factors that have been described as probably causative , and multiple causes may be present in an individual woman , there is a wide variety of treatment options and combinations depending on specific symptoms and their severity . the postulated causes range from hormonal imbalances like progesterone deficiency , prolactin excess , thyroid hypofunction , fluctuation in circulating level of estrogen ; electrolyte disturbances like rennin - angiotensin alterations , antidiuretic hormone excess , decreased colloidosmotic pressure ; neurotransmitter disturbances with serotonin or gamma - amino - butyric - acid , or -endorphin activity alternations , serotonin metabolism alternations or prostaglandin imbalance ; cytokine imbalances like excess of interleukin ( il)-6 , il-1 and tumor necrosis factor ( tnf)- ; nutritional deficiencies like vitamin b or vitamin d deficiency ; lack of exercise or psychosocial disturbances.[8101315 ] the treatment therefore will depend on the particular symptom complex in the individual woman . in concordance with other authors , lifestyle measures like regular exercise , balanced nutritious diet and regular hours of sleep will benefit all , and may be adequate for some.[1921 ] for others , pharmacological interventions may be helpful if required . the therapy is multipronged and has to be individualized . while simple measures like the institution of physical exercises , relaxation techniques like yoga and vitamin and mineral supplements are adequate for most women,[1924 ] some require hormonal therapy with combined oral contraceptives or regulation of fluid and electrolytes . as a matter of fact , combined oral contraceptives are used frequently in the more severe cases and , drosperidone , containing newer combinations , is relatively free from side - effects . selective estrogen modulators , including nonsteroidal agents like centchroman , also could be useful , particularly in women with contraindications to hormones . few women need psychiatric consultation and will benefit from antidepressants , particularly sertraline or fluoxetine , while some respond to prolactin inhibitors like bromocriptine or cabergoline . complementary and alternative therapies like soy , hypericum perforatum and gingko biloba have a definite role to play.[242931 ] evening primrose oil does not have significant efficacy . we have observed relief of mild to moderate premenstrual symptoms with soy , gycyrrhiza glabra and withania somnifera . we have also observed complete relief of pmts in two women treated with an extract rich in saraca asoka , prescribed for menorrhagia , which was also controlled . there is need for more research in this area because commonly used drugs may have side - effects that may prevent regular long - term use [ table 2 ] . common side - effects of existing pharmacologic therapies for pmts recently , two well - designed studies have shown that pmts leads to loss of work hours and economic losses , and does not allow women to reach their full potential.[3436 ] effective and safe therapy is indicated . it is also important to bear in mind that other chronic diseases like migraine or depression or irritable bowel syndrome could be exaggerated during the premenstrual phase , and a multidisciplinary team should evaluate and manage such cases . many women and their physicians regard pmts as a physiological phenomenon and do not think treatment is necessary . however , it does affect qol in some and it is therefore essential to identify which women will benefit from lifestyle management and which will require pharmacological intervention . those who want to reach their full functional potential and who find that the symptoms are hampering their progress or interfering with normal daily routine should be certainly offered safe pharmacological interventions provided that they come for routine follow - ups . because there are many etiological factors that have been described as probably causative , and multiple causes may be present in an individual woman , there is a wide variety of treatment options and combinations depending on specific symptoms and their severity . the postulated causes range from hormonal imbalances like progesterone deficiency , prolactin excess , thyroid hypofunction , fluctuation in circulating level of estrogen ; electrolyte disturbances like rennin - angiotensin alterations , antidiuretic hormone excess , decreased colloidosmotic pressure ; neurotransmitter disturbances with serotonin or gamma - amino - butyric - acid , or -endorphin activity alternations , serotonin metabolism alternations or prostaglandin imbalance ; cytokine imbalances like excess of interleukin ( il)-6 , il-1 and tumor necrosis factor ( tnf)- ; nutritional deficiencies like vitamin b or vitamin d deficiency ; lack of exercise or psychosocial disturbances.[8101315 ] the treatment therefore will depend on the particular symptom complex in the individual woman . in concordance with other authors , lifestyle measures like regular exercise , balanced nutritious diet and regular hours of sleep will benefit all , and may be adequate for some.[1921 ] for others , pharmacological interventions may be helpful if required . the therapy is multipronged and has to be individualized . while simple measures like the institution of physical exercises , relaxation techniques like yoga and vitamin and mineral supplements are adequate for most women,[1924 ] some require hormonal therapy with combined oral contraceptives or regulation of fluid and electrolytes . as a matter of fact , combined oral contraceptives are used frequently in the more severe cases and , drosperidone , containing newer combinations , is relatively free from side - effects . selective estrogen modulators , including nonsteroidal agents like centchroman , also could be useful , particularly in women with contraindications to hormones . few women need psychiatric consultation and will benefit from antidepressants , particularly sertraline or fluoxetine , while some respond to prolactin inhibitors like bromocriptine or cabergoline . complementary and alternative therapies like soy , hypericum perforatum and gingko biloba have a definite role to play.[242931 ] evening primrose oil does not have significant efficacy . we have observed relief of mild to moderate premenstrual symptoms with soy , gycyrrhiza glabra and withania somnifera . we have also observed complete relief of pmts in two women treated with an extract rich in saraca asoka , prescribed for menorrhagia , which was also controlled . there is need for more research in this area because commonly used drugs may have side - effects that may prevent regular long - term use [ table 2 ] . recently , two well - designed studies have shown that pmts leads to loss of work hours and economic losses , and does not allow women to reach their full potential.[3436 ] effective and safe therapy is indicated . it is also important to bear in mind that other chronic diseases like migraine or depression or irritable bowel syndrome could be exaggerated during the premenstrual phase , and a multidisciplinary team should evaluate and manage such cases . it is very important to ensure that the first option is nonpharmacological as many women may respond to it . pharmacotherapy can be added in recalcitrant cases and must be individualized as per the woman . great care should be taken to avoid side - effects and drug interactions because multiple therapies may be used . medicinal plants may be a safe alternative in some women . because pmts could be a predictor for menopausal syndrome , it will be useful to warn women with pmts and their families of a flare up or the need for long - term follow - up . have reported pmts to be predictive of menopausal syndrome in a long - term follow - up study and have shown the association of hormonal imbalance with depressive symptoms in women .

objective : to determine the prevalence of premenstrual cyclic symptoms in perimenopausal age.subjects and methods : women attending bhavan 's sparc maitreyi 's health care programme ( hcp ) for women around 40 years of age were included in the study . last 200 women who attended from april 2002 to october 2004 are included for analysis . out of these 107 qualified for final analysis as others were post hysterectomy or post menopausal . thirty five symptoms listed under premenstrual tension syndrome were analysed.results:forty one women ( 38.3% ) had 3 or more symptoms whilst 15 ( 14.0% ) had 5 or more cyclic symptoms . five women ( 4.7% ) reported that the symptoms were severe . eleven women had seeked treatment for premenstrual tension syndrome ( pmts ) . the commonest symptom was mastalgia or heaviness of breasts . next was whilst also was reported by several women . women reported anger attacks and reported depression.conclusion:pmts was common between 36 and 55 years . about half of them have experienced 3 more symptoms and 1 in 20 may require treatment .

INTRODUCTION Objective METHODS RESULTS DISCUSSION Management Strategies Effect on Work and QOL CONCLUSION