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as reported previously (drfer et al . 2009), the study had been approved by the imdec international medical & dental ethics commission freiburg (approval number 01 17 04 03). subjects were recruited from the general population between november 10th and 14th 2003, to which the research was advertized by flyers at points highly frequented by the public around the campus. subjects who gave their written informed consent to participate were required to satisfy the study inclusion and exclusion criteria. to qualify for inclusion, the subjects were required to be 1870 years of age, in good general health, with a minimum of 18 scorable teeth (excluding third molars, teeth with orthodontic appliances, bridges, crowns or implants) and with two or more teeth showing recession on the facial surface of at least 2 mm. other main exclusion criteria were as follows: any condition that might preclude normal oral hygiene procedures or study participation; medical condition requiring prophylactic antibiotic coverage prior to dental treatment; therapy with any drugs for at least three consecutive days within the previous 28 days that might affect study outcome; neglected dental health; major hard or soft tissue lesions or trauma at the baseline visit; known allergy to the test products; or participation in any other oral hygiene clinical study within the previous 30 days. if applicants fulfilled all inclusion criteria and had no exclusion criteria, they were consecutively included in the study and randomized to either group. this was a singlecentre, randomized, examinerblind, parallelgroup study of gingival recession that compared the effects on oral tissues of power and manual brushing. subjects brushed twice daily and were assessed after 6 0 months (drfer et al . 2009), and after 12 0 and 18 1 and 35 2 months. an oscillating rotating and pulsating power brush (d17u, oralb professionalcare, procter & gamble, cincinnati, oh, usa), and an american dental association reference flat trim manual brush were used. a standard sodium fluoride dentifrice (blendamed ; however, they had to be informed about the aim of the study and the hypothesized interaction between toothbrushing and recession formation . at baseline, subjects were stratified based on initial preexisting gingival recession, gender and smoking status, and were randomized by the principal investigator to either the power brush group or the manual brush group . also, at this first visit, all subjects were instructed to brush their teeth for 2 min . twice daily throughout the study period using the assigned toothbrush and supplied a standard dentifrice . participants using the manual brush were told to continue brushing as they normally do . in the power group, participants were referred to the written instructions from the toothbrush manufacturer . subjects were instructed to return to the study centre every 3 months and clinical assessments were carried out at baseline, 6 0, 12 0, 18 , 1 and 35 2 months . at baseline, the medical history and details of concomitant medication and oral hard and soft tissue were assessed and any adverse events reported . plaque, gingival health, and periodontal measurements were also made at scheduled clinical assessments . every 3 months, standard fluoride toothpaste ( blendamed regular, procter & gamble, germany) was dispensed along with a new manual brush or power brushhead, and questions about participation were answered. additional supplies of standard toothpaste were provided throughout the study at the study centre whenever requested. clinical measurements for all subjects at all time points were made by the same clinical examiner (dw), who was blinded with respect to the assigned treatment, familiar with the clinical measurements used in the study, and had been calibrated (drfer et al . 2009). study measurements were made in the following order: oral safety assessments of soft and hard tissue, gingival index, plaque index, and periodontal measurements. periodontal measurements were carried out on each scorable tooth at six sites: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual and distolingual. the oral cavity structures examined included the lips, tongue, gingivae, sublingual area, inner surfaces of the cheeks, mucobuccal folds, hard and soft palate, pharyngeal area, and cervical areas of all the teeth. any abnormal findings including observed or voluntarily reported adverse events were recorded. plaque and gingival health were also assessed, using the turesky modification of the quigley and hein plaque index (turesky et al . 2007) and the le and silness gingival index (le & silness 1963) respectively. in order to assess clinical recession, at each site periodontal pocket depths and clinical attachment levels were measured. a periodontal probe marked at each mm (pcpunc15, hufriedy, chicago il, usa) was used to measure probing pocket depths (ppd) and clinical attachment level (cal). after gently positioning the probe, measurements were averaged upward where the gingival margin or the cementoenamel junction was between mm markings. the gingival recession was calculated as difference between cal and ppd. if the cementoenamel junction was covered by a crown or cervical restoration the measurement was taken from the most apical margin of the restoration. the primary outcome variable was the mean recession change at preexisting recession sites at baseline. as secondary endpoints, recession changes were analysed at both the tooth and site level. cal and ppd were reported also as mean standard deviation as well as plaque and gingival index. no power calculation was made prior to the start of this study as no preliminary data were available, but the intention was for 50 subjects per group to have completed 6 months of the study (drfer et al . clinical measurements were made at baseline, 6 0, 12 0, 18 1, and 35 2 months for periodontal pocket depth and clinical attachment levels, and for the plaque and gingival indices for all sites . interim analyses were performed at 6, 12, and 18 months for quality assessment purposes . missing values at all time points were imputed by the baseline data of the respective participant . the statistical unit for the primary outcome was the participating subject ( firstlevel analysis). changes in recession between visits for each group were analysed using anova with post hoc bonferroni corrections for multiple testing. group differences in recession at 12, 18, and 35 months were tested for statistical differences by ttest. a twostep logistic regression analyses was performed to indicate the influence of relevant factors on the in our data set. on the first level, relevant factors from the literature such as age, gender, and smoking status as well as the brush used were entered into the model. on the secondlevel, the local factors type of tooth, mandibular or maxillary, preexisting recession at baseline, local plaque, and local gingivitis at the final visit were added. risks due to the tooth type were reported relative to the second molar, due to the jaw relative to the mandibular and due to the brush used relative to the manual toothbrush respectively. the dependent variable' recession' was dichotomized into either having not changed or increased compared to being improved. statistical testing was twosided and a significance level of = 0.05 was used. as reported previously (drfer et al . 2009), the study had been approved by the imdec international medical & dental ethics commission freiburg (approval number 01 17 04 03). subjects were recruited from the general population between november 10th and 14th 2003, to which the research was advertized by flyers at points highly frequented by the public around the campus. subjects who gave their written informed consent to participate were required to satisfy the study inclusion and exclusion criteria. to qualify for inclusion, the subjects were required to be 1870 years of age, in good general health, with a minimum of 18 scorable teeth (excluding third molars, teeth with orthodontic appliances, bridges, crowns or implants) and with two or more teeth showing recession on the facial surface of at least 2 mm. other main exclusion criteria were as follows: any condition that might preclude normal oral hygiene procedures or study participation; medical condition requiring prophylactic antibiotic coverage prior to dental treatment; therapy with any drugs for at least three consecutive days within the previous 28 days that might affect study outcome; neglected dental health; major hard or soft tissue lesions or trauma at the baseline visit; known allergy to the test products; or participation in any other oral hygiene clinical study within the previous 30 days. if applicants fulfilled all inclusion criteria and had no exclusion criteria, they were consecutively included in the study and randomized to either group. this was a singlecentre, randomized, examinerblind, parallelgroup study of gingival recession that compared the effects on oral tissues of power and manual brushing. subjects brushed twice daily and were assessed after 6 0 months (drfer et al . 2009), and after 12 0 and 18 1 and 35 2 months. an oscillating rotating and pulsating power brush (d17u, oralb professionalcare, procter & gamble, cincinnati, oh, usa), and an american dental association reference flat trim manual brush were used. a standard sodium fluoride dentifrice (blendamed ; however, they had to be informed about the aim of the study and the hypothesized interaction between toothbrushing and recession formation . at baseline, subjects were stratified based on initial preexisting gingival recession, gender and smoking status, and were randomized by the principal investigator to either the power brush group or the manual brush group . also, at this first visit, all subjects were instructed to brush their teeth for 2 min . twice daily throughout the study period using the assigned toothbrush and supplied a standard dentifrice . participants using the manual brush were told to continue brushing as they normally do . in the power group, participants were referred to the written instructions from the toothbrush manufacturer . subjects were instructed to return to the study centre every 3 months and clinical assessments were carried out at baseline, 6 0, 12 0, 18 , 1 and 35 2 months . at baseline, the medical history and details of concomitant medication and oral hard and soft tissue were assessed and any adverse events reported . plaque, gingival health, and periodontal measurements were also made at scheduled clinical assessments . every 3 months, standard fluoride toothpaste ( blendamed regular, procter & gamble, germany) was dispensed along with a new manual brush or power brushhead, and questions about participation were answered. additional supplies of standard toothpaste were provided throughout the study at the study centre whenever requested. clinical measurements for all subjects at all time points were made by the same clinical examiner (dw), who was blinded with respect to the assigned treatment, familiar with the clinical measurements used in the study, and had been calibrated (drfer et al . study measurements were made in the following order : oral safety assessments of soft and hard tissue, gingival index, plaque index, and periodontal measurements . periodontal measurements were carried out on each scorable tooth at six sites : mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual and distolingual . the oral cavity structures examined included the lips, tongue, gingivae, sublingual area, inner surfaces of the cheeks, mucobuccal folds, hard and soft palate, pharyngeal area, and cervical areas of all the teeth . plaque and gingival health were also assessed, using the turesky modification of the quigley and hein plaque index ( turesky et al . 2007) and the le and silness gingival index (le & silness 1963) respectively. in order to assess clinical recession, at each site periodontal pocket depths and clinical attachment levels were measured. a periodontal probe marked at each mm (pcpunc15, hufriedy, chicago il, usa) was used to measure probing pocket depths (ppd) and clinical attachment level (cal). after gently positioning the probe, measurements were averaged upward where the gingival margin or the cementoenamel junction was between mm markings. if the cementoenamel junction was covered by a crown or cervical restoration the measurement was taken from the most apical margin of the restoration. the primary outcome variable was the mean recession change at preexisting recession sites at baseline. cal and ppd were reported also as mean standard deviation as well as plaque and gingival index. no power calculation was made prior to the start of this study as no preliminary data were available, but the intention was for 50 subjects per group to have completed 6 months of the study (drfer et al . clinical measurements were made at baseline, 6 0, 12 0, 18 1, and 35 2 months for periodontal pocket depth and clinical attachment levels, and for the plaque and gingival indices for all sites . interim analyses were performed at 6, 12, and 18 months for quality assessment purposes . final analyses were performed based on the principle of intention to treat . missing values at all time points were imputed by the baseline data of the respective participant . the statistical unit for the primary outcome was the participating subject ( firstlevel analysis). changes in recession between visits for each group were analysed using anova with post hoc bonferroni corrections for multiple testing. group differences in recession at 12, 18, and 35 months were tested for statistical differences by ttest. a twostep logistic regression analyses was performed to indicate the influence of relevant factors on the in our data set. on the first level , relevant factors from the literature such as age, gender, and smoking status as well as the brush used were entered into the model. on the secondlevel, the local factors type of tooth, mandibular or maxillary, preexisting recession at baseline, local plaque, and local gingivitis at the final visit were added. risks due to the tooth type were reported relative to the second molar, due to the jaw relative to the mandibular and due to the brush used relative to the manual toothbrush respectively. the dependent variable' recession' was dichotomized into either having not changed or increased compared to being improved. statistical testing was twosided and a significance level of = 0.05 was used. out of a total of 156 individuals that were screened, 109 subjects fulfilled the inclusion criteria and were enrolled at baseline. a number of subjects withdrew during the course of the study for a variety of reasons, which included pregnancy, moving to another city, and no further interest in participating in the study. in total, 75 subjects (36 female, 39 male) completed the study; 38 in the manual group and 37 in the power group. table 1 shows the mean ages of both groups at baseline and the numbers of subjects assessed at each time point. age of subjects at baseline and numbers of subjects assessed at each study time point sd, standard deviation. varied between 33 and 37 months for individual subjects depending on visit schedule. the mean values at baseline and at 12, 18, and 35 months for periodontal pocket depth, clinical attachment level, plaque scores and gingivitis scores are summarized for all sites in table 2. assessments at all time points for six sites per tooth: ppd (mm), clinical attachment levels (cal, mm), plaque (tqhi), and gingivitis scores (gi) sd, standard deviation; ppd, probing pocket depth; cal, clinical attachment level; tqhi, turesky modification of the quigley heinindex; gi, le and silness gingival index. varied between 33 and 37 months for individual subjects depending on visit schedule. gingival recession measurements at preexisting recession sites for both groups are given in table 3. for both groups, analysis of the differences in values between all time points showed a significant reduction in recession for both brushes. no statistically significant differences between groups were seen for any of the comparisons of changes in recession between study time points. gingival recession for sites with initial recession (mm): mean values at all study time points and changes in recession during study sd, standard deviation; n.s., not significant. group difference non significant for all comparisons (ttest ; p > 0.05). varied between 33 and 37 months for individual subjects depending on visit schedule. the of the logistic regression analysis showed on the tooth level statistically significant increases in risk for canines and first premolars (odds ratios ( or) 1.46 (95%ci 1.101.69), and 1.25 (1.061.47) respectively ) and decreased risks at maxillary teeth compared to mandibular teeth (or 0.66 ( 0.480.92). on the tooth level, also the use of the power toothbrush reduced the risk of progression in preexisting recessions statistically significant (or 0.81 ( 0.690.95), p = 0.011 ) compared to the use of manual toothbrushes. multiple logistic regression analysis of increasing recession over 35 months in a multivariate model on the site level included variables: gender, age, smoking, recess base (recession at baseline), ging 35 (gingivitis score at 35 months), plaq 35 (plaque score at 35 months), manual tb / power tb (manual or power brush with manual as reference), manidbular / maxillary (mandibular or maxillary with mandibular as reference), tooth types from central incisor to second molar (with second molar as reference). examination of the oral cavity at each assessment visit revealed no adverse effects on hard or soft tissues in either group. this controlled, parallelgroup study compared the effect on preexisting gingival recession of brushing with an oscillating rotating power toothbrush (oralb professionalcare) or a manual brush over a period of approximately 3 years and showed no significant group differences in preexisting gingival recession. an unexpected finding from this study was a significant reduction in recession over time that occurred in both groups and led to significantly improved recession values over the complete observation time. the absence of a difference between groups in gingival recession corroborates the findings of the 6month report (drfer et al . although, after 12 months a tendency towards a slight relapse was observed, but the recessions after nearly 3 years were still statistically improved compared to the baseline findings . the stability of the periodontal conditions during the complete course of the study can be seen by the overall probing depths, attachment levels and plaque and gingivitis scores . this is, therefore, the first controlled clinical longterm study showing existing localized gingival recessions may improve when patients ' attention is directed to the potential relationship between toothbrushing and gingival recession . although this is the first study to examine the course of preexisting gingival recessions under different conditions of brushing over such a long time in a randomizedcontrolled trial, the findings are consistent with previous studies on surrogate parameters, none of which recorded greater gingival abrasion or gingival trauma with powered than with manual brushes ( niemi et al . showed for sonic toothbrushes no difference in recession development compared to manual toothbrushes over a period of 12 months. the from the present clinical study showing no group difference can be, therefore, considered a robust finding for reasons stated previously (drfer et al . 2009). mainly, the selection of subjects with preexisting recession, and hence the likelihood of further recession, would have improved measurement sensitivity and enhanced the opportunity for revealing any group differences. 1993 ), which reported recession as a secondary outcome, appear to show that tooth brushing itself using either manual or power brushes does not cause gingival recession. the of those studies together with the improvements found in this study at 6 months (drfer et al . 2009), and shown here to be maintained after 3 years of brushing, for subjects having preexisting recession, clearly contradict the of studies that show tooth brushing per se may play a causal role in the development of gingival recession (serino et al . those studies that have related tooth brushing to gingival recession have, however, been observational and generally not designed to report a causal relationship ; instead they have derived their from correlational findings ( rajapakse et al . for example, in a study with a population that had a high standard of oral hygiene, the subjects were examined at baseline and reexamined after 5 and 12 years ( serino et al . 1994) and although recession was observed to increase with age, the amount of tooth brushing over that study period may not have been the cause of increased recession. instead, an unsatisfactory brushing habit, such as a horizontal scrub technique (tezel et al . 2001), or an ageing process could have been responsible. in support of this suggestion it is worth noting the of an observational study of 100 dental students, who also maintained a high level of oral hygiene (carlos et al . , the authors concluded that the gingival recessions could have been attributed to the wrong brushing technique . other factors identified were too much strength exerted while brushing, over brushing, and the use of hard toothbrush bristles . in a study that considered a history of hard toothbrush use and gingival recession, subjects with a history of hard toothbrush use showed more pronounced gingival recession and had more surfaces with recession associated with increased brushing frequency, than subjects without a history of hard brush use ( khocht et al . 1993). however, in a study again with dental students over 5 years of dental education (daprile et al . 2007) it was shown that recession increased although the use of hard brushes was significantly reduced and an almost perfect brushing technique was achieved over 5 years of observation. the authors conclude that other than the suspected factors such as wrong brushing techniques or too hard brushes may explain their observation. they speculate that pressure, time, and amount of toothpaste may be responsible for progressive recession despite the elimination of the above mentioned factors. it is possible, therefore, that aggressive brushing, rather than the amount of brushing per se, could have caused the recession seen in observational studies. there are clear limitations when attempting to establish causality based on correlational evidence from observational studies alone. in contrast, wellcontrolled prospective experimental studies are able to provide convincing evidence of causation. this was the intention of the present controlled study that had a number of design features to allow findings to be interpreted with greater confidence, while also accounting for apparent discrepancies between the drawn from observational and experimental studies. in this study, subjects were selected in terms of predefined inclusion and exclusion criteria, they were randomly assigned to groups and assessments were made by a trained, calibrated examiner, who was blinded to treatment. the reliability of this methodology was emphasized in the earlier report on the 6month data (drfer et al . scores for probing pocket depth, clinical attachment level, and plaque and gingivitis all reveal the periodontal health of participants, and any changes in periodontal condition over the course of the study can be expected to be reflected in these scores . low plaque and gingivitis scores were seen at baseline and this study did not seek, or expect, evidence for significant changes over time or for group differences . evidence that certain power toothbrushes are superior compared to manual brushes with respect to gingivitis reduction in longterm studies is already available ( yaacob et al . 2014, van der weijden & slot 2015). preexisting gingival recession, however, was of primary interest in this study, and its reversal seen both at 6 months (drfer et al . 2009) and in this report requires an explanation, possibly in terms of some behavioural change on the part of the participating subjects. it is wellknown that the general public does not brush for the recommended 2 min. twice a day (macgregor & rugggunn 1979) and brushing technique is commonly less than ideal (saxer & yankell 1997). in any group of subjects, therefore, there is likely to be plenty of opportunity for improvement in oral hygiene. if the subjects had adopted better brushing behaviour in this study, then this could account for reduced gingival recession. as was proposed to account for the reduction in recession seen in the 6month study, this behavioural change could have arisen simply because these subjects knew they were participating in an investigation. this is an example of the hawthorne effect , familiar to behavioural scientists, according to which, in a wide variety of experiments, the behaviour of human subjects is modified purely as a of knowing that they are experimental subjects (adair 1984). its relevance here is supported by a study showing that by deliberately inducing the hawthorne effect it was possible to improve oral hygiene in a group of noncompliant adolescent orthodontic patients with poor oral hygiene; improvements were seen at both 3month and 6month observation periods (feil et al . , it should be noted that although brushing instructions had not been given at any visit during the study, the regular and repeated visits in circumstances likely to command their attention alone could be expected to change their brushing behaviour towards a gentler attitude . this seemed to work better than a systematic brushing exercise ( slot et al . the hypothesis that changes in brushing behaviour may lead to a decrease in recessions has been at least anecdotally reported ( everett 1968). although it is unlikely due to the amount of effect, the stability over time and the validity of the measurements, the effect seen after 6 months may be influenced in parts by regression to the mean(egelberg 1989). in summary, power toothbrushes with an oscillating rotating action have been shown to be more effective than manual brushes in plaque removal and control of gingivitis (heanue et al . although the total amount of tooth brushing over time has been thought to play a causal role in the development of gingival recession ( hirschfeld 1931), in the present 3year study no group differences emerged and neither the use of a power brush nor a manual brush was accompanied by increased gingival recession. on the contrary it seemed likely that the procedures followed in this study ed in improved tooth brushing behaviour and that this, in turn, reduced gingival recession. it has been suggested that with an improved technique, along with some slight reduction in gingival inflammation, there can be an element of creeping buccal attachment more usually seen after mucogingival surgery ( rajapakse et al. this offers a possible explanation for the reversal of gingival recession found in this study. the findings of this study of the effects of toothbrushing on preexisting gingival recession are clear and compelling: over a period of approximately 3 years of regular twicedaily brushing, there was a significant and sustained reduction in gingival recession in both the group using a power toothbrush and the group using a manual brush.over the same period, there was no difference in the amount of gingival recession between the two groups.longterm reductions in gingival recession may have been caused by sustained improvements in brushing technique due to the manual tooth brushing carried out daily, appears to have no adverse effects on gingival recession; it may even serve to improve the condition. over a period of approximately 3 years of regular twicedaily brushing , there was a significant and sustained reduction in gingival recession in both the group using a power toothbrush and the group using a manual brush. over the same period, there was no difference in the amount of gingival recession between the two groups. longterm reductions in gingival recession may have been caused by sustained improvements in brushing technique due to the power or manual tooth brushing carried out daily, appears to have no adverse effects on gingival recession; it may even serve to improve the condition.	abstractaimto compare longterm effects of brushing with an oscillating rotating power toothbrush or an ada reference manual toothbrush on preexisting gingival recession.materials and methodsin this controlled, prospective, singleblind, parallelgroup study, healthy subjects with preexisting recession were randomized and brushed with a power toothbrush (n = 55) or an ada reference manual toothbrush (n = 54) for a 3year study period. subjects were required to brush their teeth twice daily for 2 min. using a standard fluoride toothpaste. during the study, subjects were assessed for clinical attachment loss and probing pocket depths to the nearest mm at six sites per tooth by the same calibrated examiner. gingival recession was calculated at preexisting sites as the difference between clinical attachment loss and probing pocket depths. hard and soft oral tissues were examined to assess safety.after 35 2 months, mean gingival recession did not differ significantly between groups, but was significantly reduced from baseline (p < 0.001), from 2.35 0.35 mm to 1.90 0.58 mm in the power and from 2.26 0.31 mm to 1.81 0.66 mm in the manual group.gingival recession in subjects with preexisting recession was significantly reduced after 3 years of brushing with either a power or manual toothbrush.
health needs assessment is a process of determining the health and health care needs of any given population or sub - group in an area. it is a complex task requiring epidemiological expertise, the ability to work across organizational boundaries as well as an understanding of, and an ability to engage with all appropriate population groups. health needs assessment process needs to take account of the diversity within these populations. health improvement programs provide opportunities to engage in such assessments in the national and regional population. research has shown that preventive programs can improve community health when properly implemented.1 there are examples of successful prevention programs in local communities. however, many still have significant challenges, which demonstrate a gap between science and practice. though in the united states, there are such common strategies (training programs), to address this issue, outcomes are still unsatisfactory. building the capacity of the community to implement high quality prevention can help communities achieve positive health outcomes, thereby narrowing the gap between theory and practice. while there is ample research on the efficacy of evidence - based programs, there is little on how to improve community capacity to improve the quality of prevention. what is being proposed is a new model of research: one based on community science that improves the latest theoretical understanding of community capacity and evaluates technologies designed to enhance it. in most developing countries, the evolution of health services has been dominated by western models of health care2. these have rarely taken into account how local people explain illness, seek advice, or use traditional healing methods. the emphasis has been on hospitals and curative care rather than on trying to address local health needs equitably and effectively. since the alma ata declaration on primary health care, more attention has been given to increasing coverage of basic services and preventing common diseases. health care needs are changing and new challenges arising from chronic diseases and hiv infection must be faced. better coverage of preventive and essential healthcare services has led to greater emphasis on improving the quality of health care to ensure the efficient and judicious use of scarce resources. for example, infant mortality has fallen dramatically in the past two decades through such interventions as oral rehydration for diarrhea and immunization programs. with fewer children dying, there has been greater emphasis on the need to tackle the causes of infant and child morbidity. the size of families can be reduced with the improvement of the availability of family planning. if health services are to respond to the changing health needs of their local populations, planners and managers would need useful and timely information about the health status of these populations. some of this information can come from routine data sources or may be collected from large, one - off population studies. a confusing number of terms describe similar methods: rapid evaluation methods, rapid appraisal methods, rapid community surveys, rapid rural appraisal, relaxed rural appraisal, participatory rural appraisal3. the development of rapid appraisal methods in the 1980s came in recognition of the time consuming and rigid nature of traditional epidemiological and questionnaire surveys. experience with these appraisal methods showed that when they were perfectly executed, they provided valuable, reliable, and timely information on health status, knowledge, attitudes, and behaviors. more recently, emphasis has been placed on encouraging people to participate in their own appraisal (for example, participatory rural appraisal)4. the hardest part of any needs assessment is translating the into policies and practices to elicit and (or) initiate beneficial change. the involvement of health care workers in techniques such as rapid or rural appraisal will encourage changes at an individual level. local workshops can provide opportunities to review the lessons learnt with other health care workers. if this change is going to be sustainable and adaptable, then the appraisal should be a continuous process with ongoing feedback. implementation of strategic changes can be facilitated if the policy - makers themselves are active in the process. active collaboration between communities and researchers is critical in developing appropriate public health research strategies that address community concerns.5 partners for healthy communities conducted interviews with community members from the ethnically diverse neighborhoods of central and southeast seattle. the suggest that effective community - researcher collaborations require a paradigm shift from traditional practices to an approach that involves: acknowledging community contributions, recruiting and training minority people to participate in research teams, improving communication, sharing power, and valuing respect and diversity. academic institutions have always found it a challenge to persuade community members to participate in academic research projects6. starting an open dialogue is usually the critical first step. to begin this dialogue with community members in dayton, ohio, in 1999, staff from wright state university decided to organize a community forum, the history of health in dayton. the forum was intended as the first project of a new research organization, alliance for research in community health (arch), established with federal funding from the health resources and services administration in 1998. arch was created as a bridge between the department of family medicine of wright state university school of medicine and the center for healthy communities, a health advocacy and service organization committed to health profession education. arch's mission is to improve the health of citizens of dayton through research involving community participation. through arch, community members help researchers define priorities, resolve ethical issues, refine procedures, and interpret . guidelines for participatory research, proposed by the national primary care research group in 1998 adopted by the alliance, emphasize the importance of open dialogue among researchers, subjects, academics, and community members. the initial response to the forum was enthusiastic, with a majority of community residents expressing interest in attending future presentations. barker described the different ways in which academics and community groups may work together, including academic / practice/ community partnerships. several principles of practice for engaging in these research partnerships are presented followed by a description of how these principles have been put into operation in a family violence prevention program7. the principles presented are: identification of the best processes / models to be used based on the nature of the issue and the intended outcome; acknowledgement of most of the differences between community input and active community involvement; development of relationships based on mutual trust and respect; acknowledgment and honoring of the different agendas of partners; consideration of multi - disciplinary approaches; use of evaluation strategies that are consistent with the overall approach taken in the academic / practice/ community partnership; and awareness of partnership maturation and associated transition periods. the limitations of these principles and their application in various settings are discussed. while many members of the public are deeply interested in and supportive of the three traditional missions of academic medicine -- education, research, and clinical care, they also want to know what academic health centers (ahcs) are doing to improve the overall health of their communities8. much is already being done toward this goal, but improving communities health in a measurable way requires a far broader agenda. ahcs must bring together the approaches of medicine and public health, and need to form partnerships with many other players. the author reviewed illustrative and emerging national, state, and local efforts, both public and private; in both medicine and public health, in partnerships with individuals and institutions in the larger community. he also highlights the physician's role in assisting stakeholders efforts to deal with health threats from the environment, and offers advice on how such efforts should proceed. he closes by emphasizing the importance of community - based research in learning about the health status, problems, and resources of particular communities; and presents a set of principles for such community - based research. lillie - blanton and hoffman examined strategies and methodological issues for researchers to consider when conducting community - based research within a racial / ethnic minority group.9 members of minority communities have considerable skepticism about the health care system and researchers who work under its auspices. to facilitate quality research, suggested strategies for accomplishing this, such as searching for information on the social and political forces shaping the community and developing the community's capacity to undertake research of this type, are described. methodological issues include the importance of community input in defining the minority population group and its leadership, the benefits and limitations of conducting comparative analysis, and the need for measurement tools and techniques that are culturally and socially appropriate. minority and non - minority researchers must make a concerted effort to understand and have respect for a community whose culture, values, and beliefs may differ. a recent study reported that nearly a quarter of the children in riyadh contracted diarrhea during the two weeks preceding the data collection, giving about six episodes of diarrhea per child per year10. diarrhea was more common in children over 6 months of age, in children who had no vaccination or follow - up cards, and in those who were being cared for by friends and neighbors as their mothers were working outside home. the mothers of the affected children were young, married before 25 years of age, with 26 years of normal schooling. during diarrheal episodes, about 25% of mothers stopped or reduced breast - feeding, 11.3% reduced the volume of fluids given to their children, and 22.7% of the children were fed less solid / semi - solid foods. mothers used oral rehydration salt in more than 40% of diarrheal episodes and unprescribed antibiotics were used in 17% of cases. the mothers who were not taking appropriate action included young mothers with low level of education and those working outside the home. in response to the need for comprehensive, cost - effective and cost - benefit services, there have been major changes in the health care system of saudi arabia since the early 1980s. currently, there are nearly 1800 governmental phc centers distributed evenly throughout the country, 1707 of which belong to the ministry of health (moh)11. the remainder are run by various health care providers, including universities, the military, the national guard and the security forces. more than 180 secondary and tertiary care hospitals serve as referral units and each group of phc centers is attached to a hospital. a number of themes emerged as important to the impact of health needs assessments on policy and planning12. these included careful design, strict methodology, decisive leadership, good communication, involvement and the ownership of the work from relevant stakeholders, support from senior decision - makers, appreciation of political dynamics, and engagement with local priorities, availability of resources and, finally, an element of chance. these themes can be categorized broadly into contextual factors, and quality or robustness of the work. although this study has demonstrated that there are conditions under which needs assessment are more likely to be effective in terms of its influence on policy and planning, it is clear that it is not central in the decision - making process of the health service, for it remains vulnerable to a range of factors over which those responsible for implementation of the decisions have little or no control. it has been reported that quality of life was unrelated to satisfaction of services, but was strongly associated with unmet needs of mental and physical health, and of rehabilitation.13 the quality of life decreased as needs increased. primary health care can meet the needs of the population in an equitable way only if these needs are known. the who regional office for europe convened a working group to examine the consequences of the assessment of the health needs of the population at the district level for primary care.14 the group discussed a useful model for community - oriented primary care (copc), which involved the delivery of programs tailored to community needs. the group considered needs assessment as the basis for allocating resources, prioritizing the needs of community health programs, planning and evaluating these programs; in the third area, they stressed the need to develop further the model for a community - oriented planning and evaluation cycle (copec). the impact of community needs assessments was used in south australia where the data was collected from regional health planning officers.15 the needs assessments were found to vary from the regional to the locally driven. approaches ensured local involvement, but the process was slower and more arduous for the planner. the use of community health needs assessment was useful, but for greater impact these should not be broad, but focused on feasible changes that the health services could support. other priority - setting techniques, such as marginal analysis, should be used to determine where it could also be used to determine where maximum health gains are possible. an area of controversy of need assessment is that which asks the question whose need? several researchers stress the importance of collecting both quantitative and qualitative data from a variety of sources to ensure that community needs are examined from a variety of perspectives1617. other studies have described four types of need that should be considered in needs assessment. these include comparative, normative, expressed and felt.1819 indicators of normative needs can be seen as the vision of health or benchmarks or targets that are described by experts, task forces, commissions, etc. indicators of comparative needs include information about the determinants of health for the population as they compare with benchmarks of other populations and other areas of health. expressed needs are described as information about demands or as wants to put into action related to health gathered from key informants, survivors, advocacy groups, government directives, etc. felt needs are wants or attitudes related to personnel or the community's visions of health, e.g., we want to feel safe walking alone at night the proposed framework is based on indicators of needs for each of these dimensions. doctors, sociologists, philosophers, and economists all have different views on what needs are.2022 in recognition of the scarcity of resources available to meet these needs, health needs are often differentiated as needs, demands and supply. if health needs are to be identified then an effective intervention should be available to meet these needs and improve health. there will be no benefit from an intervention that is not effective or if there are no resources. demand is what patients ask for; it is the need that most doctors encounter. general practitioners play a key role as gatekeepers in controlling this demand, and waiting lists become a surrogate marker with an influence on this demand. demand for a service from patients can depend on characteristic of the patient or on the media interest in that service. this depends on health interests of the professionals, the priorities of politicians, and the amount of money available. a lead article which introduced the community survey concept, detailed reasons for a community survey, and outlined mythological framework for completing such a study.23 the article defined a community survey as a survey of population researched by health services provider within a defined geographic area such as hospital service area. the most critical step in conducting a community survey is for the manager to specify what types of information are needed and how that will be used. to assess health needs of a rural community , it is also important to include a cross - section of health care providers in the information identification process to avoid focusing only on services offered by major health care providers (i.e. the local hospital). recently, a study reported the awareness of general practitioners (gp) and their experiences of needs assessment.24 most gps were unfamiliar with the concept of needs assessment, and there was no evidence that needs assessment had influenced commissioning decisions. the motivation and attitude of the majority of the gps is a barrier to needs assessment in primary care. gps require more resources and training if they are to bear this responsibility. over the past 20 years , governments throughout western europe and north america have encouraged patients to contribute to the planning and development of health care services.25 in england and wales, the involvement of patients is central to current efforts to improve the quality of health care. underlying these changes, is the belief that involving patients leads to more accessible and acceptable services and the improvement of health and quality of life of patients. rapid appraisal can be used to involve the public in the identification of local health needs and supplement more informal methods of assessing needs.26 rapid appraisal is best used in homogenous communities. the process of rapid appraisal can give structured orientation to new workers in the community. rapid appraisal can be adapted to introduce medical students to the concept of community diagnosis as a natural adjunct to individual clinical diagnosis. palmer identified the need for reproductive health care in a community affected by conflict in southern sudan.27 the study comprised interviews with key informants, in - depth interviews, and group discussions. reproductive health in general, and sexually transmitted diseases in particular were important issues for these communities. perceptions of reproductive issues varied between service providers and community leaders. to improve the health of any population or subgroup of that population these are the health authorities, the local authorities, local business, the voluntary sector, the pharmaceutical industry, and organized groups of the society.28 improving health is far more complex and long - term than the provision of health care, as some of the root causes of ill health (poverty, housing, lifestyle, employment and crime) are beyond the control of health services. occupying an area of about 16000 km and with a population of about one million (1421 census), the jazan region is in the south - western region of saudi arabia. it has three geographically distinct zones: the mountain zone which is 2000 - 2500 m above sea level with > 300 mm of rain / year, the hill zone, 400 - 600 m above sea level with < 300 mm of rain / year, and the coastal zone that is < 400 m above sea level with very little rain. the region is intersected by perennial streams. as a of its special climate and topography, poor sanitation, inadequate water supply and a middle to low socio - economic status of some communities in the mountainous areas, water borne and water associated diseases such as malaria, schistosomiasis, leishmaniasis, hepatitis, typhoid etc. health statistics from the area indicate high rates of morbidity, mortality & low health care coverage compared to other regions of saudi arabia. the close association and proximity of man and animal has ed in endemicity of such zoonotic diseases as brucellosis. the 1999 epidemic of the rift valley fever occurred at the border areas as a of the movements of the border population and the introduction of animals from neighboring countries. the recent ministry of health annual report29 has shown that the prevalence of infectious diseases in jazan region as follows: tb 147/2322 (6.3%), bilharzias 18.35%, malaria 67.48 %, hepatitis a 100\2250 (4.4%), and measles 26/617 (4.2%). there are 135 primary health care centers (phcc), and 13 hospitals, only two of which are specialized. academic public health plays an important role in teaching and research, as well as supporting service departments of public health at national and local levels.30 the academic - service is a continuum and is essential in providing high quality public health delivery for the nation. concerns with health conditions of the population have become a part of a wider concern with the direction of development of human resources. all social and economic development is underpinned by the development of the country's human resources to their full potential. thus, the training of saudi personnel in various health fields is important for the success of the kingdom's plans for health development. the jazan college of medicine was therefore, established in 2001. the expected mission of jazan medical college is to raise the standard of health in this area by training health personnel who would be involved in community programs to combat health problems, and work with other health agents in the region in preventive and curative programs. the jazan medical college conducted a comprehensive health survey of jazan31 which involved three techniques: key informant interviews, focus group interviews and a household survey. the key findings were as follows: the most important perceived health problems in jazan were the shortage of health care providers, and an increased prevalence of communicable diseases and poor environmental health, high level of awareness of communicable diseases with weakness in prevention, most frequently reported chronic conditions: hypertension, bronchial asthma, diabetes mellitus and joint diseases, diarrhea in around 15.6 percent of children, and malaria treatment of 35.9% at the health facility, an estimated 33% of the rural and 37.7% of the urban are current cigarette smokers, and the 61.7% current khat users in rural compared to 45.7% in the urban areas. the data and information generated from this study will be utilized as a baseline and reference information for policy formulation, subsequent planning and cost effective intervention programs, and will be important in the development of the curriculum of jazan medical college. it will also help decision makers in the jazan region in the planning of any future health program. though a combination of quantitative and qualitative approaches is recommended in health service research, it has advantages and disadvantages. the incorporation of the view point of the general population through development in the health service has the potential of improving the relevance and impact of research and the quality of subsequent services provided.3233 a very recent study documented important lessons learned from the use of community key informants in needs assessment surveys to identify health problems and needs. the study suggested the means to control and prevent major health problems in the region of jazan. the study also emphasized the role of the jazan medical college in providing skilled manpower and expertise to improve the health care service delivery in the region of jazan.34 the historical development of health services has been dominated by western models of health care and health beliefs.35 these have rarely taken into account how local people explain illness or seek advice. however, the assessment of health needs is not simply a process of listening to patients or reliance on personal experience and anecdotes. it is a systematic method of identifying unmet need and making changes to fill this need. in public health, it is the need of the population, which perhaps could be that of a district or perhaps a section of the population such as women of childbearing age, which have to be assessed. in : the academic institutions, ministry of health, and other health care institutions should cooperate in looking for innovative approaches to increase awareness of the broader social and public health issues, and increase funding from both regional and national sources to support community based studies.	this paper takes a public health approach to briefly examine: (i) the concept of community health care need assessment; (ii) the roles of academic institutions in health needs assessment; (iii) jazan study to address the health care needs in jazan region, saudi arabia. the methods included an analysis of the literature, distillation of experience from the recently jazan health need assessment survey, and who reports. the most important perceived health problems in jazan region are shortage of health care providers, increased prevalence of communicable diseases and poor environmental health. the academic institutions, ministry of health and other health care institutions need to work together to look for innovative approaches, especially to increase the awareness of the society on public health issues, and give more support to increase national and regional funding for community based studies.the findings of the assessment of the health needs of jazan presented in this review could be utilized as a baseline and reference information for policy formulation, subsequent planning and cost effective intervention programs. it could also be utilized for the curriculum development or review for a community oriented medical schools.
it has been reported that many short children suffer from problems at school and with peer relationships. these difficulties with peer relationships, as well as low self - esteem seem to influence the psychosocial status of some individuals with short stature. additionally, short stature children are often found to exhibit behavioral disturbances including immaturity, inhibition and anxiety and are victims of bullying by peers. often attributed to overprotection by families and aversive social experience related to the child s short stature, these difficulties can lead to impaired emotional and social development, poor - self perceptions of well - being, and a reduced quality of life (qol). in contrast to the well - established physiological benefits of growth hormone treatment (ght), the potential psychological effects of the treatment are less clear. recently, there is increased research focused on the effect of ght on the qol of patients. however, a few studies have shown multiple impairments in qol in short versus normal height children . we undertook a multicenter study to evaluate the psychosocial status of short - stature children and their parents with or without ght. short stature children with and without ght enrolled in the child health and development network were compared. one hundred and thirteen children diagnosed as having growth hormone deficiency were treated with the recommended dose of growth hormone, and 67 children diagnosed with idiopathic short stature were observed without ght. self - administered questionnaires were collected from children with short stature (age range : 3 to 18 yr) and their families who regularly visit the outpatient clinics participating in the child health and development network. numbers of collected answers are shown in table 1table 1 number of cases, while the age distribution of children is shown in fig. black bars indicate patients with ght, and gray bars indicate those without ght.. the mean sd of the height sd scores (htsds) were 1.99 0.98 sd and 2.29 0.74 sd for patients with and without ght, and the difference was not significant (p=0.057). there are peaks in the number of children aged 3, 5, 6 and older than 10 yr. children younger than 6 yr old who could not answer the questionnaire by themselves were excluded. black bars indicate patients with ght, and gray bars indicate those without ght. statistical analysis was performed by fisher s exact test and pearson product - moment correlation coefficient. statistical analysis was performed using graphpad prism (graphpad software, la jolla, ca). medical ethics review committees had not yet been established at out hospital when this study was designed, but informed consent was obtained from all participants. the distributions of the expected adult height of both children and parents with or without ght are shown in figs. black bars indicate patients with ght, and gray bars show those without ght. 3 distribution of parents expected adult height according to ght status of their children. black bars indicate patients with ght, and gray bars show those without ght. , respectively. the half of expected adult height of boys who received ght was around 170 cm, but it varied widely among boys without ght. the median expected adult height of girls who received ght was 160 cm, but it ranged between 150 cm and 160 cm among girls without ght (fig . the expected adult height of boys as estimated by their parents was greater than that the estimation of the boys themselves, regardless of ght . black bars indicate patients with ght, and gray bars show those without ght . black bars indicate patients with ght, and gray bars show those without ght . there was no correlation between the target height and the expected adult heights predicted by the father, mother or by the boys themselves . only the expected adult heights as estimated by the girls with or without ght correlated with the target height ( r=0.003 and 0.093, respectively). as summarized in table 2table 2 responses to questionnaires by children, most parents reported that the current therapy is effective (98% with ght vs. 83% without ght). however, parents of children receiving ght were more satisfied with the therapy than parents of children without ght (79% vs. 50%, respectively). additionally, parents of children not receiving ght were more anxious about therapy than parents of children with ght (58% vs. 31%, respectively). these questions revealed statistically significant differences between parents of children with and without ght (p=0.0005). most parents worried about their children s height (92% with ght vs. 97% without ght). according to parents of children with and without ght, respectively, their children were having no difficulty in their daily lives (89% vs. 95%), were positive (56% vs. 65%), had been bullied in their classroom (25% vs. 25%), and were worried about their own height (61% vs. 63%). children who received ght and those who did not reported a similar likelihood of feeling inferior about their shortness (79% vs. 83%), being unsatisfied with their height (84% vs. 90%), expecting ght to make them taller (81% vs. 75%), having no difficulty in their daily lived (90% vs. 93%), being positive (81% vs. 75%), having been bullied in their classroom (26% vs. 29%), and worrying about their height (56% vs. 55%), respectively. however, children receiving ght showed a greater expectation to be taller versus those without ght (97% vs. 83%, respectively). also, there was a tendency for patients of a younger age and shorter stature to be more likely to be bullied (fig . 4 numbers of children being bullied by peers according to their ght status, age range and height . the numbers are divided by the patient s age ( younger than 9 yr old, age between 10 to 12 yr old and older than 13 yr old). the upper panel shows patients with ght, and the lower panel shows those without ght. black bars indicate numbers of patients that have been bullied and gray bars show the numbers of those who have not been bullied. ). numbers of children being bullied by peers according to their ght status, age range and height. the numbers are divided by the patient s age (younger than 9 yr old, age between 10 to 12 yr old and older than 13 yr old). the upper panel shows patients with ght, and the lower panel shows those without ght. black bars indicate numbers of patients that have been bullied and gray bars show the numbers of those who have not been bullied. lastly, responses to each question by parents of children receiving ght were divided according to their children s height (table 3table 3 responses to questionnaires by children). in this analysis, anxiety regarding treatment and expectations for ght effectiveness were greater among parents of children with an htsds that was still below 2 sd despite ght. the need for medical consultation was also significantly higher in children shorter than 2 sd the subjects in this study are patients who visited the hospital complaining about short stature and did not include individuals who did not visit a hospital despite their shortness. the age distribution of patients at first visit peaks at 3 and 6 yr of age. these are the ages at which children receive public health checks, which are effective for detecting disorders of growth. children younger than 6 yr rarely feel psychosocial stress because of their short stature. however, parents of these young children, especially mothers, feel anxiety regarding their child s stature. thus, in most cases, these children are brought to a clinic not because they want to be taller, but because of parental concern. this may also be true for older children who answered the questionnaires, since parents anxiety for their children s height is much higher than the children s anxiety for their own height. short boys were more than twice as likely to be bullied in comparison with boys of normal height. social rejection, name calling, and bullying by peers may have deleterious effects on social and emotional maturity and may lead to poor adaptation in adult life. in this study , however, most short children appeared to have no difficulty in their daily lives, and their parents recognized this positive attitude. however, approximately 25 to 30% of short children reported experiencing bullying in the classroom. compared qol between short and normal height children and reported that 20% of both groups reported bullying. indeed, in the current study, the percentage of short children being bullied was not significantly greater than that of normal children. although short children have no difficulty in their daily lives, they are not satisfied with their height, feel inferior, and worry about their stature. it has previously been reported that ght ameliorated problems in behavior and improved the psychological problems caused by short stature. in this study , we could not evaluate the effect of ght because we did not collected questionnaires before ght. but this study addressed the psychological problems of short children according to ght status. although parents of both children receiving and not receiving ght have some anxiety about their children s height, the anxiety regarding on - going therapy is greater among parents with children not on ght. it is understandable that growth improvement in short children who are on ght decreases the anxiety of their families and increases the satisfaction and the expectation for on - going therapy. short children receiving ght are more likely to expect to become taller than children not receiving ght. the medians of the expected adult heights for the short boys and girls on ght were 170 cm and 160 cm, respectively. however, the mean achieved adult heights of gh - treated boys and girls were reported to be 160.3 cm and 147.8 cm, respectively. in reality, it is difficult for most patients with ght to reach their expected adult height. therefore, it is important for doctors to inform patients and parents before starting treatment that ght can not guarantee the attainment of their expected adult height. an unrealistically high expectation might in patients disappointment at the end of ght. short children on ght and their parents have high expectations of the treatment. however, parents of children who are still below 2 sd despite active ght worry about the efficacy of ght. , medical counseling is needed to encourage such patients and families to continue ght. additionally, it might be necessary for doctors to increase the gh dosage to ensure the growth promoting effect of ght. in , although short children have no difficulty in their daily lives despite their short stature, they are not necessarily satisfied with their height. short children on ght and their parents are anxious regarding their height and their expectations of the treatment effectiveness. before starting ght , patients should be properly informed by their physicians regarding realistic expectations of the treatment effect. finally, medical consultation is recommended for the patients who remain below 2 sd despite being on ght.	to evaluate the psychosocial status of short children with and without growth hormone therapy (ght) and that of their parents, self - administered questionnaires were collected from patients and parents who regularly visit the outpatient clinics participating in the child health and development network. completed questionnaires were received for one hundred and thirteen patients with ght and 67 patients without ght. according to the parents, both children with ght and without ght have no difficulty in their daily lives (89% vs. 95%) and are positive (56% vs. 65%), respectively. ninety - eight percent of parents of children with ght and 83% of parents of children without ght had expected the current treatment strategy to be effective. parents of children with ght are more satisfied with the current therapy than those without ght (79% vs. 50%), and feel less anxiety about the on - going therapy than (31% vs. 58%, respectively). children treated with or without equally reported having no difficulty in their daily lives (90% vs. 93%), and being positive in their lives (81% vs. 75%, respectively) despite their short stature. although less than one third of the patients have been bullied in their classroom (26% with ght vs. 29% without ght), younger and shorter children tend to be bullied more often. short children undergoing ght and their parents have anxiety regarding their height and expectations of the effect of ght. it is important for doctors to inform their patients regarding realistic height expectations before starting ght. additionally, medical consultation is recommended for patients who remain below 2 sd in height despite ght.
as characterized by hanchanale and coworkers, the perception of botulinum toxin a has been transformed from poison to a healing agent. information concerning botulinum neurotoxin serotypes, molecular structures, substrate specificities, mechanisms of zinc - dependent peptide hydrolysis, ion channel formation, and other detailed topics has been extensively reviewed. of the seven immunologically distinct serotypes (a f) from clostridium botulinum and several other species, type a is the best characterized and is among the most potent of all toxins. the neurotoxin is initially expressed as a single polypeptide of nearly 1300 amino acid residues (mw ~150 kda). crude toxin extracts (mw ranges from ~300 to 900 kda) contain several nontoxic ancillary proteins that form a complex with the neurotoxin. when ingested, these additional proteins are thought to protect the neurotoxin against austere environments such as those found in the certain regions of the gastrointestinal tract. the neurotoxin is posttranslationally modified to form two chains that are covalently bridged with a disulfide bond. the light (l) chain (mw ~50 kda) has zinc - dependent proteolytic activity, while the heavy (h) chain contains the translocation and binding domains. subsequent to binding to specific receptors (sv2) at peripheral cholinergic nerve terminals, the receptor - toxin complex is internalized into a membrane - bound compartment that undergoes a drop in ph. conformational changes occur that allow the insertion of the h - chain into this compartment's membrane. as a , the disulfide bond that links the l and h chains is reduced, an ion channel is formed, and the presumed proteolytic active moiety, the l - chain, is translocated into the neuroplasm. when the type a toxin substrate, snap-25, is selectively cleaved, synaptic vesicle - mediated neurotransmission is blocked that could eventually lead to fatal paralysis. since the 1980s, the therapeutic potential of this toxin has been exploited. extraocular muscles have been injected with the partially purified neurotoxin as an adjunct or alternative to surgical correction in treating strabismus. the chemodenervation effects of this most poisonous of poisons have been used to relax hyperkinetic striated muscle groups to diminish the effects of dystonia and related diseases. food and drug administration (fda) for the following indications: strabismus, blepharospasm, cervical dystonia, upper limb spasticity, maxillary hyperhidrosis, chronic migraine, and urinary incontinence. these indications along with the temporary enhancement in the appearance (cosmesis) with botox cosmetic of moderate to severe wrinkles in adults introduces the theme of muscle immobilization in terms of a desired therapeutic outcome. advantage has also been taken of this toxin's chemoimmobilization property to improve the healing of wounds. in contrast to the relatively vast amounts of information regarding this toxin's structure and mechanism of action, the newer, off - label uses for botulinum toxin have been less extensively reviewed. to gain further insight regarding the scope of these efforts, we have gathered and examined biomedical research articles by conducting systematic searches of the relevant literature in pubmed, in a manner similar to that of steele and madoff. we have examined studies that range from descriptive observations to randomized controlled clinical trials to obtain more information about the components and processes involved in wound healing and the related time courses of action of botulinum toxin a. the processes observed clinically on the wound healing effects of the type a toxin are at an early stage of our understanding. this proposal is substantiated by evidence - based reviews that critically evaluate this toxin's effects with different indications. we previously noted that only a few clinical studies have focused on kinetic analyses. constructing even partial models for the clinically observed effects by this toxin remains a challenge. to advance our understanding, we have selected some of those clinical studies that have examined the timing of this toxin's effects. botxminer, the botulinum reference tool of clostridial neurotoxin citations in entrez - pubmed / medline, was initially used to search in article titles, abstracts, and mesh headings for the words wound and heal or healing. a more extensive list of 29 wound - related keywords was then generated: anal, angiogenesis, collagen, cytokine, fibroblast, fibroblastic, fibrosis, fissure, flap, glycosaminoglycan, heal, healing, hemorrhoid, hemorrhoidectomy, hypertrophic, incision, inflammation, inflammatory, keloid, lesion, repair, scar, scarring, sphincterotomy, surgical, tendon, tensile, ulcer, and wound. this controlled vocabulary was used in the batch mode to search botxminer for additional related citations. another set of filter terms were used to find time - course - related information about this toxin. this set included 26 terms: clearance, day, decay, decline, delay, diffusion, duration, follow up, frequency, hour, hr, interval, kinetic, latency, minute, month, onset, period, persistence, recurrence, repeat, resistance, sec, time, week, and year. within the two lists in the batch matrix search, (a1, a2, , am) and (b1, b2, , bn), the terms undergo the or operation which can be represented in a general form of (a1 or a2 or, etc .). in addition, the lists are combined with the and operation which in a batch query that can be represented by (a1a2am)(b1b2bn). summaries from the two sets of terms were returned by botxminer in the form of tables, histograms, and lists. additional keywords and phrases within the more than 70 downloaded text files were automatically searched with file search assistant (v. 3.1, 2009, aks - labs, raleigh, nc, usa). data from the clinical literature that were analyzed were fitted to an exponential function y = y0+(a)exp(kdecayt), where y is the cumulative number of patients who are free of symptoms at time = t, y0 is the cumulative number of patients who are symptom - free at t = , a is a preexponential constant, and kdecay is the rate constant for the decay of this response. chicago, ill, usa ) was used to conduct a least - squared fit for the values of y0, a, and kdecay. this equation is commonly used for simulating the decay rate of a reactant from a single model compartment. the fda has approved generic, nonproprietary names for commercial formulations of botulinum toxin. for botulinum toxin type a, botox (allergan, calif, usa) is onabotulinumtoxina, dysport (ipsen biopharm limited co., uk) is abobotulinumtoxina, and xeomin (merz pharma gmbh & co kgaa . , germany) is incobotulinumtoxina. for botulinum toxin type b, myobloc / neurobloc (solstice neuroscience, inc . , pa, usa ; eisai ltd ., uk) is now rimabotulinum toxin b. the changes in nomenclature emphasize the different potencies and the noninterchangeable unit dosages of these distinct brand name products. as reviewed by alberto, these distinctions in names emphasize the differences in manufacturing and formulation techniques that may contribute to differences in the pharmacokinetics, efficacy, safety, and antigenicity among these products. in the present paper, partially purified toxin with nontoxic or accessory proteins is referred to as botulinum toxin type a to distinguish it from botulinum neurotoxin a (bont / a), the pure holotoxin. the lists of selective keywords are not intended to be exhaustive but serve as a starting point for the present work. additional terms, such as proctology and coloproctology, can be used in more comprehensive studies. verification confirms that, for example, the equations being coded are producing the correct calculations. software can be validated when it can model the that best fit existing data, and is subsequently reinforced by further data obtained experimentally. for kinetically related clinical problems, the underlying processes that need to be included are still uncertain. furthermore, mathematical models are not designed to replace validation by basic research experiments or clinical observations. rather, models are meant to enhance validation procedures by providing stimuli for new ideas, hypotheses, and perspectives on the problems being examined. a preliminary search of botxminer, using the query wound and (heal or healing) for years 19802010, returned over 150 citations about half of which were true positives due to the large number of references related to tetanus (false positives). from the list of true positive citations, a variety of indications were found, in which botulinum toxin a has been used for wound healing and related conditions. these examples included experimental cutaneous scars in animal models and in clinical studies: chronic anal fissures , cleft lip surgical repair, traumatic head lacerations or elective excisions of forehead masses, focal fold granuloma, hypertrophic scarring, pressure ulcers , raynaud's phenomenon (vasospastic ischemia of the digits, digital ischemia, including chronic ulcers), and self - mutilation injuries in lesch - nyhan syndrome. another healing application of botulinum toxin a, referred to as protective ptosis, is used against persistent corneal ulcers, burns, and other ophthalmic - related problems. conducting a matrix batch search in which tetanus was filtered out, using 26 time - related terms along with the 29 wound healing - related terms, yielded 671 unique citations. from this filtered output, we concentrated on references dealing with wound conditions and indications in which botulinum has been used for therapeutic purposes in which some mechanistic, dosage, and/or kinetic information was also available (methods, figure 1). the normal wound - healing process has been described as being comprised of four overlapping phases: haemostasis, inflammation, tissue proliferation, and remodeling. if any of these processes are disrupted, healing is impeded leading to a chronic wound state. the interference by botulinum toxin in reducing muscle movement has helped to define healing phases further. for example, a vicious cycle involving inflammation, pain, and muscle spasm was first noted to be the underlying cause for the development of chronic anal fissures. subsequently, low blood flow and ischemia were added to this cycle. additional components of the healing process have been identified for other conditions as presented in this section. scar formation is a hallmark of wound healing, and it usually causes significant physical, psychological, and cosmetic problems. hypertrophic scarring is a common, refractory dermal disease that is manifested by the abnormal appearance of wound healing which can be the of different types of injuries. in the study by xiao et al. , 19 patients were treated once a month over 3 months with 1.835 u botulinum toxin a (hengli / cbtx - a, lanzou biochemical co., china). scores were assigned before treatment for the associated erythema, pliability (lesion softening), and the severity of itching. after a 6-month followup of the 19 patients participating in that study, 15 gave an overall assessment of their lesion improvement as good, and seven others rated their improvement as excellent. some critical comments provided by the authors included the lack of control subjects, the study was not double blinded, and a small patient population size. finally, there was only a relatively short follow - up time of 6 months so that no determination of the total time course of toxin action could be established. a number of quantitative parameters may be accessed to evaluate the effectiveness of wound healing by botulinum toxin a. increased metabolic activity and inflammation during the healing process induce muscle contractions around the edges of the skin wound. the major role of botulinum toxin a in this healing process is to prevent the repeated, small contractions that produce microtraumas near the hypertrophic scar and thereby decrease the tensile force (muscle activity) during scar formation. traditional surgical techniques that align incisions along langer's lines do not prevent repeated contractions. the development of fibrosis also involves the deposition of extracellular collagen and glycosaminoglycans that can cause the scar to hypertrophy, invert, and become hyperpigmented ing in poor color matching of this tissue with the neighboring skin. other parameters for wound healing include size of wound, amount of and infiltration of inflammatory cells, blood vessel proliferation, and wound thickness. additional cellular and molecular mechanisms of healing by the formation of scar tissue (traumatic cicatrisation) are beginning to be elucidated. transforming growth factor 1 (tgf- 1) is a fibrotic cytokine that stimulates cellular growth, differentiation, and adherence and leads to the collagen deposition. this cytokine initiates these processes by extracellularly binding to a coupled pair of serine - threonine kinases. on binding, one receptor recruits and phosphorylates the other this signaling pathway eventually stimulates transcription of the collagen gene and the formation of hypertrophic scars. because human fibroblasts derived from hypertrophic scars overexpress and secrete tgf-1, another wound - healing effect of botulinum toxin a has been speculated to be inhibiting the secretion of tgf-1. similarly, suramin, an antifibrotic polysulfonated naphthylurea compound, has been reported to promote wound healing by antagonizing tfg-1 in muscle - derived fibroblasts. the circulatory system is also affected by the apparent ability of botulinum toxin to enhance wound healing. using a rat model for wounds, yoo's group observed that pretreatment with botulinum toxin a increased dorsal skin flap survival and concluded that this process was caused by increased perfusion. because this toxin inhibits secretion of norepinephrine from sympathetic vasodilator and vasoconstrictor neurons , the effect of botulinum toxin a may involve increased perfusion by decreasing sympathetic vasoconstriction in the skin flaps, thus promoting skin flap survival. the protective effects of botulinum toxin - induced ptosis have been used for the conditions of recalcitrant corneal ulcers and other surface disorders as an alternative to the surgical practice of partially sewing the eyelids together (tarsorrhaphy). this secondary healing effect is produced when botulinum toxin a is injected into eye muscles, typically the levator palpebrae superioris (lps) muscles. this therapeutic application has been the subject of studies that have also generated kinetic data. from an open - label, multicentered study with 16 ophthalmic patients who received 5 u botox in the lps muscle, the time to suitable ptosis was 4.0 0.5 days (mean se, range : 28 days), and the duration of this ptosis was 46.0 12 days (1206 days). a similar number of patients who received a single, lower dose (2.5 u) had a comparable mean time to ptosis (3.6 days) and a shorter, mean duration (16 days). although statistical analyses with more patients are required to make more definitive , these trends are not unexpected but are, nevertheless, remarkable because a twofold change of a low dose apparently ed in appreciable differences in duration. chronic anal fissure (caf) is a painful condition caused by spasms of the internal sphincter smooth muscles. the traditional surgical approach has been sphincterotomy that can in the adverse effect of incontinence. traditional nonsurgical approaches that have been used include sitz bath, topical anaesthesia, nitroglycerin, isosorbide dinitrate, nifedipine, diltiazem, l - arginine gel, hyperbaric oxygen, and botulinum toxin a. the first use of botulinum toxin as a medical alternative for caf was conducted by jost and schimrigk. for this indication, we chose what has been described as one of the longest follow - up studies using botulinum toxin (type a) in the treatment of caf. we analyzed data for the time course of recurrence of caf symptoms in patients in a 42-month follow - up study. as illustrated in figure 3, at the beginning of the followup (at t = 0), all 53 patients were symptom - free for 6 months after one to two injections of 1020 u botox. most patients (31 of 53) did not undergo recurrence indicating that they remained healed during this follow - up period. from the nonlinear fit of these data to, the fitted values sems are y0 = 29.2 1.2, a = 24.4 1.3. the kdecay (decay rate constant) is 7.08 10 1.07 10 month, 1.64 10min (see below), or 0.85 years (see table 1). the value of 1/kdecay or is 14.1 months, and the corresponding value of t1/2 (= ln2) is 9.8 months or 294 days. the long - term success rate was only 31/53 (58%) or a 42% rate of recurrence. from arroyo's group, recurrence rate of 12% occurred initially, and a rate of 53% at the 3-year follow - up point. bilateral fissurectomies of the internal anal sphincter combined with toxin injections have been reported to have a high success rate although the follow - up time in that study was limited to 1 year. also, it was not determined whether surgery itself produced a similar rate of healing. an interpretation of the single exponential decay curve during the 42-month followup (figure 3) is that it reflects a zero - order elimination or inactivation step of the persistent, intraneuronally located toxin. this step is described by the rate constant of decay, kdecay, in figure 4 and ke in a previous publication. the table summarizes the processes displayed in figure 4 and highlights the recurrence of symptoms that may be used to gauge the slow elimination or inactivation of the type a botulinum toxin. notably, this rate may be comparable to the slow rates of healing of some chronic wounds. alternatively, the long - lived toxic effect may be related to the rate of restoration of intact snap-25 intracellular levels or a combination of a slow degradation and a persistent inhibitory action of the snap-25 bont / a cleavage product. the value of t1/2 of 294 days is comparable to the recurrence times of 444 132 days (mean s.d . ; range 270718 days) for achalasia patients after receiving a single injection of 80 u botox. on the other hand, our calculated decay rate constant of 1.64 10min is about 1000 times slower than the estimated rate constant for decay of efficacy (1.1 10min) in a single dystonic patient. this difference may be due to several factors, among which are the different patient populations, the muscles being injected, the conditions being treated, and the assessment methods. while the above studies show encouraging trends in support of using botulinum toxin a in wound - healing paradigms, additional studies are necessary. overall, more prospective clinical studies of these treatments with botulinum toxin a are needed. evidence from blinded, randomized, placebo - controlled, multicentered studies will help determine if these toxin treatments have significant benefit and if the minimal adverse reactions can be sustained. future trials should also use larger populations of more homogeneous (standardized) patients and control subjects, plan to examine long - term outcomes, and conduct cost - benefit analyses. although randomized controlled trials are considered the gold standard of clinical research, assessing them using criteria for standardizing phase iii trials remains a substantial challenge. moreover, there is also a need for additional controlled studies to clearly establish an advantage of botulinum products over other methods. an outcome of one of these analyses was the low probability that type a botulinum toxin or calcium channel blockers were found to be more effective in treating caf than nitroglycerin ointment in 182 patients.'s analysis which determined that for 279 caf patients the traditional surgical procedure of lateral internal sphincterotomy (lis) was more effective than botox in healing chronic anal fissure. while lis produced a higher rate of minor anal incontinence, botulinum toxin was associated with a higher rate of recurrent disease. for those patients who had a high risk of incontinence, local injection of the toxin computational modeling and simulation studies at different levels of granularity (i.e., detail) should also be beneficial. starting with existing minimal kinetic models , dose - dependent kinetic models could be developed to predict the time course (onset, duration, and recurrence of symptoms) and the extent of botulinum toxin a's effectiveness. kinetic models could help to identify what research gaps exist and which ones can be experimentally or clinically resolved. one gap that could be experimentally verified is to determine if the intracellular diffusion of botulinum toxin a is influenced by other coinjected materials, for example, epinephrine and local anesthetics (lidocaine, xylocaine), compounds that have been considered in controlling local diffusion and predicting the extent of this toxin's paralytic effect. as more realistic physiological - pharmacological models are developed, more free parameters and more sources of error, assumptions, and caveats will need to be evaluated. potentially confounding factors associated with wounds include cell viability, alkali conditions, the generation of reactive oxygen species, and inflammation that is related to low blood flow and ischemia. changes in the ion flow through transmembrane channels and in metabolism could also have roles in wound healing. another confounding factor is the well - known, persistent muscle weakness (up to 5 years) that from protracted patient immobilization due to critical illness polyneuropathy or myopathy. this persisting weakness and residual neurologic deficits are likely due to denervation, combined with catabolic muscle wasting and potential myopathic changes. it remains for future studies to differentiate these physiological deficits from botulinum toxin - induced effects in either a botulism patient or with therapeutic treatments with this toxin. the tissues involved in the healing process in cytoskeletal architectures and in membrane structures associated with various organs also need to be considered. neurophysiological abnormalities, such as transient denervation - induced muscle fibrillations and presynaptic alterations producing muscle fasciculations, may also need to be considered. other complicating factors in the healing process could also involve the biomechanical dynamic properties of soft tissue (e.g., stiffness) in response to stress and strain, and tissue anisotropy (directionality of nonhomogeneous material). this succinct review examines the soft tissue wound - healing properties of botulinum toxin. when viewed from the perspective of treating neurologic and other disorders , it is noteworthy that the efficacy of this toxin is predicted to be transient as the toxin's effect wanes, while for wound healing, a permanent resolution is expected. further, with respect to wound healing, the concept is described that for some lesions, botulinum toxin interferes with the vicious cycle of muscle spasm, pain, inflammation, decreased blood flow, and ischemia. a reaction pathway scheme is outlined to illustrate botulinum toxin's involvement in stopping the vicious cycle. a minimal kinetic scheme for healing chronic wounds is also presented that includes different macroscopic states of soft tissue conditions (normal, initial wound, and chronic wound), and quantitative estimates for the relevant rate constants are provided. a definitive validation of the , that is, the minimal kinetic model for predicting the beneficial effects of type a toxin, awaits additional clinical data. perhaps the most useful outcome is that our kinetic model is capable of identifying a measurable gap (decay rate of toxin 's effect) in our attempt to comprehend how complex, interacting biological systems respond to environmental stressors.	a relatively new approach in the treatment of specific wounds in animal models and in patients with type a botulinum toxin is the focus of this paper. the indications or conditions include traumatic wounds (experimental and clinical), surgical (incision) wounds, and wounds such as fissures and ulcers that are signs / symptoms of disease or other processes. an objective was to conduct systematic literature searches and take note of the reactions involved in the healing process and identify corresponding pharmacokinetic data. from several case reports, we developed a qualitative model of how botulinum toxin disrupts the vicious cycle of muscle spasm, pain, inflammation, decreased blood flow, and ischemia. we transformed this model into a minimal kinetic scheme for healing chronic wounds. the model helped us to estimate the rate of decline of this toxin's therapeutic effect by calculating the rate of recurrence of clinical symptoms after a wound - healing treatment with this neurotoxin.
esophageal carcinoma is a malignant tumor originating from the epithelium of the esophagus, and it has the characteristics of strong invasiveness and high mortality. around 300 000 people die of esophageal cancer every year around the world, but its incidence and mortality varies greatly in different countries. china has one of the highest incidences of esophageal cancer, at around 150 000 deaths per year. distant metastasis and tumor recurrence are the main causes of death in patients with escc. circulating tumor cells (ctcs) are tumor cells that leave the primary tumor site and enter the bloodstream, where they can spread to other organs. ctcs are very important in the diagnosis, treatment, and prognosis of malignant tumors. abnormal proliferation of tumor cells in decreased adhesion between cells, and between cells and the surrounding matrix. this loss of adhesion allows tumor cells to escape from the primary tumor into the circulatory system and become ctcs with the capacity for invasion and metastasis. compared with lung cancer, gastric cancer, breast cancer, and colorectal cancer , there has been less research into ctcs from esophageal cancer. the ctcs were acquired by a negative enrichment method that used magnetic activated cell sorting (macs). then, the proportion of patients with escc who were positive for ctcs was analyzed, after which the relationship between ctcs and clinical pathological features and the prognosis of patients with escc was evaluated. all patients were treated at the first hospital of lanzhou university for escc and those with complete clinical data were enrolled. all procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. all 140 patients underwent surgical resection, and 16 of them were underwent esophageal cancer palliative resection. among the 140 patients, 124 with a total of 140 patients were included in the present study and they were diagnosed with primary escc based on pathological findings. the patients were 117 men and 23 women, ranging in age from 36 to 78 years (mean sd : 62.88.5 years). the tumor - node - metastasis (tnm) stage of the escc patients was defined according to the 7 edition of the tnm classification of the international union against cancer. in addition, 25 healthy volunteers without cancer were randomly selected as a control group. the clinical and pathological features of the 140 patients with escc are summarized in table 1. blood (5 ml) was collected into citrate anticoagulant blood collection tubes from the patients with escc after fasting. four ml of whole blood was mixed with a buffer to lyse the red cells. then, the labelled magnetic particles (lyle bio pharmaceutical technology co., ltd ., jiangsu, china) was added and centrifuged for 20 min. a mixed solution of 3 ml of metal salts and phosphate - buffered saline (pbs) was added, and it was centrifuged for 5 min at room temperature (300 r / min). the upper 2 layers of solution was collected and placed in a 15-ml centrifugal tube, then we added concentrated (10 times to 14 ml) pbs and bovine serum albumin (bsa) buffer and centrifuged it for 5 min at a speed of 950 r / min at room temperature. the supernatant was removed to make a volume of 300 l after being mixed well. the specimen was transferred to a new centrifuge tube and put on a magnetic frame (promega corporation, madison, wi) for 23 min. the eluted liquid was removed into 1.5-ml centrifuge tubes separately, placed into the top of a 15-ml centrifuge tube, and centrifuged for 5 min at a speed of 2000 r / min for 3 min. the supernatant was removed to make a volume of 100 l, then we added fixing liquid to it and allowed it to dry naturally at room temperature for 24 h. the dried specimen was fixed, aged, dehydrated, covered with a coverslip, and sealed without bubbles. the specimens were placed in an automatic hybridizer for 1.5 h. the specimen then had the coverslips removed and the samples were washed with 0.2% bsa solution. after removal of the bsa solution from the specimen, the rabbit antihuman ck19 polyclonal antibody and mouse antihuman cd45 monoclonal antibody (lyle biological medicine technology co., ltd ., jiangsu, china) was added to the specimen. after incubating it for 1 h in the dark at room temperature, we added 10 l of 4, 6-diamidino-2-phenylindole (dapi ; sigma - aldrich, st . louis, mo) counterstain into the area of the specimen, applied a coverslip, and read the specimen under a fluorescence microscope. the complete morphology and nucleus of the cells could be observed under the light microscope. the interpretation of the of immunofluorescence staining was ck19(+), cd45, and dapi(+). cells with a signal > 2 and without blood - borne leukocyte surface antigen (red circle or a red plate) were counted as positive ctcs. cells positive for the presence of blood - borne cell surface antigen (red circle or sheet) or the signal 2 were counted as negative ctcs. the relationships between ctcs and clinical pathological characteristics of patients with escc were analyzed with the chi - square test. the primary outcomes were disease - free survival (dfs) and overall survival (os). spss v. 17.0 statistical software (spss, chicago, all patients were treated at the first hospital of lanzhou university for escc and those with complete clinical data were enrolled . all procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards . all 140 patients underwent surgical resection, and 16 of them were underwent esophageal cancer palliative resection . among the 140 patients, 124 with a total of 140 patients were included in the present study and they were diagnosed with primary escc based on pathological findings . the patients were 117 men and 23 women, ranging in age from 36 to 78 years ( mean sd : 62.88.5 years). the tumor - node - metastasis (tnm) stage of the escc patients was defined according to the 7 edition of the tnm classification of the international union against cancer. in addition, 25 healthy volunteers without cancer were randomly selected as a control group. the clinical and pathological features of the 140 patients with escc are summarized in table 1. blood (5 ml) was collected into citrate anticoagulant blood collection tubes from the patients with escc after fasting. four ml of whole blood was mixed with a buffer to lyse the red cells. then, the labelled magnetic particles (lyle bio pharmaceutical technology co., ltd ., jiangsu, china) was added and centrifuged for 20 min. a mixed solution of 3 ml of metal salts and phosphate - buffered saline (pbs) was added, and it was centrifuged for 5 min at room temperature (300 r / min). the upper 2 layers of solution was collected and placed in a 15-ml centrifugal tube, then we added concentrated (10 times to 14 ml) pbs and bovine serum albumin (bsa) buffer and centrifuged it for 5 min at a speed of 950 r / min at room temperature. the supernatant was removed to make a volume of 300 l after being mixed well. the specimen was transferred to a new centrifuge tube and put on a magnetic frame (promega corporation, madison, wi) for 23 min. the eluted liquid was removed into 1.5-ml centrifuge tubes separately, placed into the top of a 15-ml centrifuge tube, and centrifuged for 5 min at a speed of 2000 r / min for 3 min. the supernatant was removed to make a volume of 100 l, then we added fixing liquid to it and allowed it to dry naturally at room temperature for 24 h. the dried specimen was fixed, aged, dehydrated, covered with a coverslip, and sealed without bubbles. the specimens were placed in an automatic hybridizer for 1.5 h. the specimen then had the coverslips removed and the samples were washed with 0.2% bsa solution. after removal of the bsa solution from the specimen, the rabbit antihuman ck19 polyclonal antibody and mouse antihuman cd45 monoclonal antibody (lyle biological medicine technology co., ltd ., jiangsu, china) was added to the specimen. after incubating it for 1 h in the dark at room temperature, we added 10 l of 4, 6-diamidino-2-phenylindole (dapi ; sigma - aldrich, st . louis, mo) counterstain into the area of the specimen, applied a coverslip, and read the specimen under a fluorescence microscope. the complete morphology and nucleus of the cells could be observed under the light microscope. the interpretation of the of immunofluorescence staining was ck19(+), cd45, and dapi(+). cells with a signal > 2 and without blood - borne leukocyte surface antigen (red circle or a red plate) were counted as positive ctcs. cells positive for the presence of blood - borne cell surface antigen (red circle or sheet) or the signal 2 were counted as negative ctcs. the relationships between ctcs and clinical pathological characteristics of patients with escc were analyzed with the chi - square test. the primary outcomes were disease - free survival (dfs) and overall survival (os). spss v. 17.0 statistical software (spss, chicago, il) was used for statistical analysis. no positive of ctcs was identified in any blood specimens of the 25 healthy volunteers. positive of ctcs were identified in 62 of 140 patients with escc (44.3%). of the 140 patients with escc, 62 were positive for ctcs according to the current criteria. in this study, ctcs positivity was found in 11.5% of patients with stage i, 21.0% with stage ii, and 48.4% with stage iii iv. the positive rate of ctcs was significantly related with stage status of escc patients (p=0.013), meaning that stage was significantly more advanced among ctcs - negative patients compared with ctcs - positive patients. however, there was no relationship between ctc status and age, sex, smoking history tumor, tumor location, differentiation of tumor, lymphatic invasion, or lymph venous invasion (p>0.05). according to the detection , 140 cases escc patients were divided into 2 groups by ctcs status (positive and negative for ctcs). kaplan - meier analysis showed that ctcs - positive patients had significantly shorter survival time (dfs and os) than ctcs - negative patients (figure 2). os was significantly correlated with differentiation, pt, pn, stage, lymphatic invasion, and ctcs status, and was significantly longer in ctcs - negative patients than in ctcs - positive patients (p=0.013). multivariate analysis demonstrated that stage and ctcs status were significant prognostic factors for os and dfs of patients with escc (table 4). no positive of ctcs was identified in any blood specimens of the 25 healthy volunteers. positive of ctcs were identified in 62 of 140 patients with escc (44.3%). table 2 summarizes the clinicopathological features of the patients with escc. of the 140 patients with escc, ctcs positivity was found in 11.5% of patients with stage i, 21.0% with stage ii, and 48.4% with stage iii iv. the positive rate of ctcs was significantly related with stage status of escc patients (p=0.013), meaning that stage was significantly more advanced among ctcs - negative patients compared with ctcs - positive patients. however, there was no relationship between ctc status and age, sex, smoking history tumor, tumor location, differentiation of tumor, lymphatic invasion, or lymph venous invasion (p>0.05). according to the detection , 140 cases escc patients were divided into 2 groups by ctcs status (positive and negative for ctcs). kaplan - meier analysis showed that ctcs - positive patients had significantly shorter survival time (dfs and os) than ctcs - negative patients (figure 2). os was significantly correlated with differentiation, pt, pn, stage, lymphatic invasion, and ctcs status, and was significantly longer in ctcs - negative patients than in ctcs - positive patients (p=0.013). multivariate analysis demonstrated that stage and ctcs status were significant prognostic factors for os and dfs of patients with escc (table 4). ctcs are tumor cells that enter the peripheral blood circulation from the primary tumor or metastasis. ctcs are also known as rare blood cells, tumor micro - metastasis, latent tumor cells, or circulating epithelial cells. ctcs exist in many forms in the peripheral circulation, either independently or in the form of cell clumps. some ctcs may also be combined with platelets, which form a shell on the ctcs surface. under normal circumstances, however, ctcs combined with platelets forming a shell can escape from the immune surveillance of natural killer cells and become closely associated with endothelial cells, which leads to the occurrence of tumor cell metastasis. the detection of ctcs is mainly divided into 2 steps: enrichment and identification of ctcs. enrichment involves sorting out tumor cells using some physical characteristics such as the size and density of tumor cells or by the specific binding of antigens and antibodies. identification involves counting the number of cells and analyzing the characteristics of the tumor cells using the specific markers in the nucleus or on the surface of tumor cells. in this study , we used an immunomagnetic separation method to enrich for tumor cells, and used ihc to identify the ctcs. ck19 is a sensitive marker of epithelial cells and epithelial tumor cells, so ck19 was used as a marker in the identification of ctcs. in the enrichment process, a small number of white blood cells remained and cd45 may be expressed on the surface of normal white blood cells. most theories of tumor metastasis suggest that tumor cells fall off and migrate into the peripheral circulation during the primary stage of tumor metastasis. for some patients from whom tumor tissue can not be easily obtained, the detection of ctcs in the peripheral circulation can provide a new way to determine their prognosis and may provide new methods for their treatment. therefore, it is of clinical significance to detect ctcs in the peripheral circulation of patients with malignant tumors. the relationship between the prognosis of patients with breast cancer and ctcs has been demonstrated in many previous studies. compared with histological examination of tumor tissue, ctcs are easy to obtain, can be treated repeatedly, and are relatively noninvasive. this may become a new method for performance of tumor biopsies in real - time. in this study, the positive rate of ctcs was related with stage status of escc patients. this means that ctcs - positive escc patients are at a more advanced stage of disease compared with ctcs - negative escc patients. however, there was no relationship between ctcs status and age, sex, smoking, tumor history, tumor location, differentiation of tumor, lymphatic invasion, or lymph venous invasion. recently, good progress has been made in research on ctcs, particularly for patients with carcinoma. explored ctcs levels in pancreatic cancer by using a newly - developed platform - integrated subtraction enrichment and immunostaining - fluorescence in situ hybridization (se - ifish). showed that ctcs could be detected in pancreatic cancer patients in various stages, whether localized, locally advanced, or metastatic. ctcs count could serve as a prognostic marker for metastatic malignant tumors, but the latest studies show that ctcs has positive significance in early recurrence of malignant tumors. some researchers have examined the ctcs levels in patients with colorectal cancer by using a sensitive device. eventually, they found that ctcs may be a simple, independent prognostic marker for non - metastatic colorectal cancer patients who are at high risk of early recurrence. in the present study, we observed that ctcs positivity was associated with advanced tumor stage in escc patients. these suggest that ctcs could be used to predict early recurrence of escc patients who received surgical treatment. the mechanism of formation of ctcs in patients with malignant tumors may be that the tumor cells located in their original position transform from epithelial to mesenchyme cells. they can cross the surrounding matrix, invade blood vessels, and become tumor cells in the peripheral circulation. ctcs can be combined with platelets, which form a layer or shell using tissue factors of the cell surface. this prevents the immune killer cells from recognizing the surface antigens of tumor cells, so that ctcs can escape immune surveillance. some proteins are expressed abnormally, such as integrin proteins, and they are also involved in leading tumor cells into the circulatory system and forming ctcs. for the above reasons, ctcs- positive escc patients are more likely to have regional or distant metastases in the early stages of the disease. multivariate analysis revealed that ctcs positivity is an independent prognostic biomarker that indicates a worse prognosis for patients with escc. we predict that ctcs may be useful in early diagnosis of escc, timely monitoring of the recurrence and metastasis of cancers, accurate assessment of patient prognosis, and guidance of the clinical treatment.	circulating tumor cells (ctcs) are tumor cells that leave the primary tumor site and enter the bloodstream, where they can spread to other organs; they are very important in the diagnosis, treatment, and prognosis of malignant tumors. however, few studies have investigated ctcs in esophageal squamous cell carcinoma (escc). the aim of this study was to investigate the ctcs in blood of escc patients and its potential relevance to clinicopathological features and prognosis.material/methodsctcs were acquired by a negative enrichment method that used magnetic activated cell sorting (macstm). fluorescent immunohistochemistry (ihc) was used to identify the ctcs. then, the positive ctc patients with escc were analyzed, after which the relationship between ctcs and clinicopathologic features was evaluated.in the present study, 62 out of 140 (44.3%) patients with escc were positive for ctcs. the positive rate of ctcs was significantly related with stage of escc patients (p=0.013). however, there was no relationship between ctc status and age, sex, smoking tumor history, tumor location, differentiation of tumor, lymphatic invasion, or lymph venous invasion (p>0.05). kaplan - meier analysis showed that patients positive for ctcs had significantly shorter survival time than patients negative for ctcs. multivariate analysis demonstrated that stage and ctc status were significant prognostic factors for patients with escc.ctcs positivity is an independent prognostic biomarker that indicates a worse prognosis for patients with escc.
diabetes mellitus (dm) and chronic kidney disease (ckd) have become two of the fastest growing pathologies worldwide , while diabetic kidney disease (dkd) is still the leading cause of ckd and end - stage renal disease. population ageing and increase in prevalence of many interrelated comorbidities suggest that these numbers will worsen in the near future. despite emerging strategies and constant investigation, no current single treatment has been able to reverse or at least stop dkd progression. at best, some of the measures can partially slow the speed at which renal function is lost. although albuminuria probably remains as the most influencing prognostic factor, up to one - fourth of normoalbuminuric diabetic patients will eventually develop ckd. this has raised questions about the suitability of albuminuria as a surrogate marker in clinical trials, and renal function decline still remains as the most important target of nephroprotection. on the other hand, a growing body of evidence is uncovering various mechanisms of renal injury in the context of dm, leading to the appearance of potential novel drugs. in this review , we summarize the available evidence regarding classical treatments for diabetic nephropathy, as well as novel agents, paths, and targets under basic and clinical investigation. dkd occurs in approximately 20% of diabetic patients, and it can appear despite a good glycemic control. nevertheless, many important studies have demonstrated that a tighter glycemic control can delay the onset of dkd and slow its progression, beyond its well - known cardioprotective effect. this effect has been proved valid in both type 1 and type 2 dkd and in the short and long terms. however, the risk of severe hypoglycemic adverse events prompted a change in international guidelines, which currently recommend individualization in treatment intensity according to patients' characteristics.. some of them, such as incretin degradation inhibitors or glucagon - like peptide analogues, may have specific nephroprotective effects independent of their glycemic impact, but these require confirmation. given the pathogenetic importance of intraglomerular hypertension in the initiation of dkd, earlier guidelines recommended a stricter blood pressure control in diabetic patients. the latest 2012 kdigo guidelines maintain a tighter blood pressure recommendation for proteinuric patients, regardless of etiology. however, more recent data from several studies in the field of hypertension have evidenced the risks of hypotensive episodes and their vascular consequences. hence, similarly to the evolution of recommendations in glycemic control, a more individual approach to blood pressure targets is advised. overweight and obesity are frequent comorbidities to diabetes and play an important role in the pathogenesis of ckd. this may be due both to a further increase in hyperfiltration and to specific hormonal dysregulations related to adipokines. a decrease in serum creatinine has also been demonstrated in very hypocaloric diets, but this effect could be secondary to muscular mass loss. there is also growing evidence about the beneficial effects of bariatric surgery in morbid obese patients over diabetes, renal function, and albuminuria , but no trial has been yet specifically designed to analyze this effect on dkd. dietary advice in dkd patients is a complex issue: it compels carbohydrate consumption regulation, but the frequent concurrence of comorbidities also requires a low - salt diet for hypertension, fat - free for dyslipidemia, and hypocaloric intake for obesity. there is evidence of the benefits of moderate protein restriction up to 0.8 g / kg / day , and this indication is included in international guidelines at least for patients with reduced glomerular filtration rates (gfr). cigarette smoking has been linked to the appearance and progression of dkd, probably due to oxidative stress stimulation, and the cessation of this habit has also been associated with slower progression of the nephropathy. if not for this reason, strong smoking cessation support should be offered to all diabetic and/or ckd patients as a means to reduce their increased vascular risk. one of the most important risk factors for kidney disease progression in diabetic patients is the onset and persistence of proteinuria. the use of angiotensin converting enzyme inhibitors (acei) or angiotensin ii receptor blockers (arb) to reduce proteinuria this benefit is valid for both type 1 and type 2 diabetic patients, even with low - grade proteinuria and normal gfr. many clinical trials have been performed in this respect. a different approach that has attracted much attention has aimed to demonstrate the usefulness of combining two or even three of these drugs. the efficacy for lowering proteinuria with the combination of renin - angiotensin - aldosterone system blockers is at least the same as using one of them at maximum dosage. however, published studies have not succeeded in demonstrating these positive outcomes with the same adverse effects (renal function decline or hyperkalemia in the combination arms of several trials forced to stop some of them). while using arb or acei to lower proteinuria in dkd and in proteinuric ckd is considered mandatory (evidence grade 1a), the lack of positive studies has encouraged a change in current recommendations against the use of dual blockade. in spite of this fact , dual blockade is spreading more than ever, as shown in a recent retrospective study that included a great number of diabetic patients. in a more detailed analysis of these trials (table 1), acei and arb combined treatment efficiently decreases proteinuria, and adverse events are usually limited to hyperkalemia and renal impairment. for these reasons, we recommend that, in adequately selected cases with very high urinary protein excretion, dual blockade can probably be tried as long as a close monitoring can be ensured. the road study showed that uptitration to the highest tolerated dose can be an interesting strategy to avoid adverse effects while achieving the maximum reduction in proteinuria. in this sense, the use of a combination with equipotent doses of acei and arb is not supported, due to a lack of benefits in terms of proteinuria as shown in the pronedi trial. the controversy about an early treatment of nonproteinuric diabetic patients still remains. in the roadmap trial, proteinuria had a delayed onset in those patients treated with olmesartan, although at the expense of higher rates of cardiovascular events. this benefit in primary prevention of dkd had been previously demonstrated with trandolapril in the benedict trial. a review and meta - analysis of the cochrane database concluded that acei could reduce the risk for new onset of albuminuria, but this effect can not be proved with the use of arb. another option that has been proposed is the use of a selective inhibitor of human renin in combination with an acei, an arb, or an aldosterone blocker. aliskiren is a direct renin inhibitor that has been tested as an antiproteinuric agent in dkd. the avoid trial generated important evidence about the efficacy of this drug with a nonsignificant rise of adverse effects (the aliskiren group developed more hyperkalemia, but the difference did not achieve statistical significance). however, the security profile of this treatment was questioned after the premature stop of the altitude trial due to a higher rate of adverse effects in an intermediate analysis. for this reason, the use of aliskiren in combination with acei / arb is not supported for lowering proteinuria in kidney disease. the benefits of the addition of an aldosterone blocker to the standard therapy in dkd have been noted in some clinical trials. beyond the mere addition of spironolactone or eplerenone, these drugs have demonstrated slight renoprotective superiority in small studies compared to acei or arb therapies. including 136 patients that were using dual blockade with acei and arb demonstrated that the substitution of the first one by spironolactone provides additional benefits in terms of proteinuria reduction with the same profile of adverse effects after 18 months of follow - up. however, a reduction in gfr was noted in the spironolactone group, independent of blood pressure control. in ckd, this drop in renal function has been reported by other authors, but it appears reversible after the first treatment weeks. although this beneficial effect is not well understood, it is hypothesized that these drugs avoid the aldosterone escape that happens in up to 40% of patients treated with an acei (table 1). in an interesting study conducted by sato et al. after 24 weeks, proteinuria was significantly reduced, showing no adverse effects. regarding the use of eplerenone, epstein et al. demonstrated in a randomized double - blind study that eplerenone decreased albuminuria in diabetic patients at 4-, 8-, and 12-week follow - up. no differences in adverse effects were seen. in spite of these good , and given that these trials were only performed in early ckd stages, we must still be cautious until larger studies with long - term follow - up are published. potential adverse events must still be closely monitored, especially hypotension, hyperkalemia, and renal failure. blocking renin - angiotensin system is not always enough to avoid proteinuria, so other approaches have been proposed. the formerly unexplored fields of inflammation and oxidative stress now become more important as targets for new treatments. unfortunately, most of the studies performed yield incomplete or that have not been confirmed in other studies. bardoxolone methyl is an antioxidant agent that activates keap1-nfr2 pathway and regulates inflammation in the kidney. however, the inhibition of this pathway focusing on slowing ckd progression in diabetic animals has produced controversial . nrf2-deficient mice do not develop hyperfiltration in response to hyperglycaemia but experience a faster decline in renal function. in addition, some studies report different degrees of proteinuria in diabetic nrf2 knock - out mice. in humans, the beam trial included 227 ckd patients (gfr between 20 and 45 ml / min/1.73 m) that were randomized to placebo or various doses of bardoxolone for 52 weeks. gfr increased significantly in all the bardoxolone arms, with a peak at 12 weeks that then remained stable. one of the most important of the study regarding renal function was that albumin - to - creatinine ratio (acr) was raised inversely to gfr in the treatment group. besides, adverse effects were more frequent in the bardoxolone groups, especially muscle spasms (that reached 61% in the 75 mg group). the beacon trial was designed to confirm the findings of the beam trial, but it was stopped prematurely due to unacceptable high rates of cardiovascular events in the bardoxolone methyl arm. in this study, 2185 type 2 diabetic ckd patients were randomized to receive placebo or 20 mg / day of bardoxolone methyl. a composite cardiovascular endpoint (nonfatal myocardial infarction, stroke, heart failure, or cardiovascular death) was achieved after a median exposure time to the drug of 7 months, so the trial was terminated due to safety concerns. recently, a derivative of bardoxolone methyl, an nrf2 agonist called dh404, has shown beneficial effects in mice via decreasing inflammation and oxidative stress, but only at low doses. this study reopens the interests on the nrf2 pathway in renoprotection in dkd. vitamin d is a well - known modulator of many different processes, and its deficiency can drive abnormalities in immune system, inflammation, or even cardiovascular events. in addition, lower 25-oh - vitamin d levels have been independently linked to dkd progression in a subanalysis of the pronedi study. the pleiotropic effects of vitamin d receptor activation have aroused a growing interest in some drugs, such as paricalcitol. the presence of vitamin d receptors in podocytes has promoted several clinical studies, with the hypothesis of an effect of podocyte modulation on proteinuria. designed a small trial of 113 diabetic patients randomized to placebo or paricalcitol, demonstrating proteinuria reduction with paricalcitol qualitatively assessed by dipstick. this effect was later confirmed in the vital study, published by de zeeuw et al.. in this study, 281 patients were randomized to receive placebo or 1 or 2 g / day of paricalcitol for 24 weeks. only 40% of the patients were receiving maximum doses of arb or acei, and the median of urinary acr was 612.3 mg / g. proteinuria was not reduced in patients with paricalcitol at any dose when compared to placebo, but albuminuria was significantly reduced in patients with higher sodium intakes. it should be noted that only 58% of the patients assigned to 2 g / day of paricalcitol received the full dose during the whole study, due to adverse effects. a recent paper published by eren et al. demonstrates that the combination of paricalcitol with other drugs such as aliskiren can reduce dkd progression in rats beyond the simple reduction of proteinuria, when the renin - angiotensin system is adequately blocked. in this study, a recent systematic review that included clinical trials about the effect of active vitamin d (both paricalcitol and calcitriol) on the control of proteinuria in ckd concludes that these drugs provide a significant reduction in proteinuria in addition to rennin - angiotensin system blockade. however, except the vital trial, the rest of the included studies were small in sample size, and the underlying conditions differed between them (like the etiology of the proteinuric state). both insulin resistance and diabetes this fact has generated a growing interest in anti - inflammatory therapies to slow diabetes and dkd progression. pentoxifylline is a methylxanthine derivative and a nonspecific phosphodiesterase inhibitor of tumor necrosis factor (tnf-) that has demonstrated an antiproteinuric effect in dkd. however, the heterogeneity and short follow - up of published studies have turned pentoxifylline away from the usual therapeutic arsenal against diabetes. a well - designed long - term trial by navarro - gonzlez et al. has been recently published. one hundred and sixty - nine diabetic patients with 3 - 4 stage ckd were randomized to placebo or pentoxifylline 600 mg daily one month, followed by 600 mg twice daily for 23 more months. all of them were receiving renin - angiotensin system blockers and the median of urinary albumin excretion was 1.1 grams per day. the study concludes that pentoxifylline slows renal disease progression (gfr slope) after the first year of treatment and maintained a statistically significant difference with placebo after 24 months. our group had previously published a small trial including 91 ckd patients, showing that pentoxifylline stabilizes renal function at 12 months, while patients in the placebo arm experienced a decline in renal function (estimated by mdrd). in the predian trial, urinary albumin excretion was reduced (mean of reduction difference of 20.6% between groups) in the pentoxifylline group at 6, 12, 18, and 24 months. surrogate markers of inflammation also decreased at the end of the study in those patients receiving pentoxifylline. these therefore place pentoxifylline as one of the first - line drugs to be used in addition to renin - angiotensin system to avoid or at least decrease residual proteinuria in diabetic kidney disease. some studies have tried to show beneficial effects of other drugs such as statins, aspirin, or rapamycin. these anecdotal should be cautiously managed, until studies designed with hard endpoints reveal further evidence. an increasing knowledge of pathogenic mechanisms in dkd beyond proteinuria has enhanced studies of new molecules that could interfere in ckd progression (table 2). endothelins are small vasoactive peptides that influence hypertension and ckd through various mechanisms, including endothelial dysfunction, vasoconstriction, cell damage, and albuminuria. their action is mediated through two families of receptors: endothelin-1 receptor (eta) has been implied in the deleterious effects of endothelins, while endothelin - b receptors (etb) act in the proximal tubule enhancing sodium excretion. all endothelin inhibitors have demonstrated positive effects on the kidney, by reducing proteinuria and renal function loss. however, the effect of inhibiting etb in inappropriate sodium retention, with more episodes of peripheral edema, congestive heart failure, and cardiovascular events. unfortunately, this mishap happens with all known endothelin inhibitors, since they all have an effect on both eta and etb. although first described with the earlier bosentan, molecules with higher selectivity on eta over etb like sitaxsentan and avosentan also showed these adverse events, which led to early termination of several trials. currently, the sonar study is evaluating the effect of atrasentan on renal endpoints in type-2 diabetic patients, but it excludes patients with a history of peripheral edema or heart insufficiency and those with higher levels of type - b natriuretic peptide, so a limitation in its future indications is expected. several vitamin analogs and other molecules that inhibit redox reactions (such as taurine, luteolin, d - saccharic 1,4-lactone, silybin, or hemin) have proved to diminish kidney damage in animal models by normalizing superoxide dismutase, inducing hemoxygenase, or inhibiting nadph oxidase. n - acetylcysteine, which has been tested in many clinical trials for the prevention of contrast - induced nephropathy with controversial , has not yet been proved effective in dkd, although studies are too small to be conclusive. allopurinol has already shown efficacy in preventing vascular events and slowing kidney function loss in several clinical trials , some of which included diabetic patients. the ongoing clinical trials pearl and feather are currently investigating the specific usefulness of allopurinol, and its novel analogue febuxostat, in type 2 dkd. there are many attempts to interfere with the inflammatory pathways of dkd, aiming to interrupt the fibrotic pathogenesis. some of these attempts are addressed at the earlier phases of the process, by inhibiting several transcription factors. protein - kinase activity is directly related to fibrosis, and several molecules have been studied to inhibit them. ruboxistaurin, a protein - kinase c inhibitor, showed promising initial in the fields of retinopathy and peripheral neuropathy. data on dkd are very scarce with either negative effects or a discreet benefit on protein excretion and gfr loss in the long term. however, these have not been confirmed in larger populations or in patients with a decreased gfr. other protein - kinase inhibitors are under current evaluation after associating renal benefits in animal models: tyrosine - kinase inhibitors imatinib, nilotinib, genistein, and pp2 , rho - kinase inhibitors fasudil and y27632 , p38-mapk inhibitor fr167653 , phosphoinositide 3-kinase (pi3k) inhibitors wortmannin, ic87114, and as101 , or activin - like kinase 3 agonists (alk-3) thr-123. the janus kinase - signal transducer and activator of transcription (jak - stat) system has also been related to kidney damage. baricitinib is a jak inhibitor currently under evaluation or rheumatoid arthritis that is also being studied for dkd. the most studied pathway in this area is vitamin d receptor activation, but other ways are under evaluation. on the one hand, dopamine has been involved in blood flow regulation and hyperfiltration in earlier diabetic kidney disease. experimental antagonism of d3 receptor with d3-ra showed beneficial effects on albuminuria and glomerulosclerosis, but in humans are not yet available. on the other hand , serotonin has also been studied, and 5-hydroxytryptamine receptor antagonist sarpogrelate, which is more known for its antiplatelet action, has also demonstrated renal anti - inflammatory and antiproteinuric effects and is undergoing a phase iv randomized control trial. melanocortin receptor activation has been evaluated in several nondiabetic proteinuric glomerulopathies, and treatment with subcutaneous acth has also shown efficacy in reducing proteinuria in dkd. finally, reinforcement of endogenous mechanisms that are inherently protective against hyperglycemia - derived kidney damage has also been tried. for example, exogenous administration of the adipocytokine apelin or of activated protein c has renoprotective effects in dkd animals models. the exogenous activation of cannabinoid receptors has shown similar . in fact, tnf- inhibition with infliximab or etanercept has been shown to decrease albuminuria and slow ckd progression in animal models, but further investigation in humans is required. transforming growth factor beta (tgf-) blockade has been achieved through pirfenidone, currently approved for lung and liver fibrosis. tranilast, currently approved for allergic states and keloids, showed a reduction in albuminuria in a small pilot study with diabetic patients , but never underwent a larger clinical trial. after promising experimental data, there are several ongoing studies to evaluate the efficacy of specific anti - tgf- monoclonal antibodies, such as fresolimumab, in various proteinuric nephropathies. other tgf- blockers have been tested in animal models but have not yet arrived to human subjects. connective tissue growth factor (ctgf) has also been implied in the process of renal fibrosis in dkd. fg3019 is an anti - ctfg monoclonal antibody that showed albuminuria reduction in dkd but has not been further investigated for this indication. several antagonists of the receptors ccr2 and ccr2/5 of the mcp-1 pathway (such as ccx 140-b, tlk-19705, rs102895, pf-04634817, or bms-813160) have shown positive experimental and some of them are being evaluated in clinical trials. another important family of proteins is that of matrix metalloproteinases (mmps), mainly involved in extracellular matrix regulation. molecules with capacity to inhibit mmps, such as the antibiotic agents doxycycline and minocycline or the newer xl081 and xl874, were expected to have renoprotective effect, but when tested in humans, the impact was limited in magnitude and duration. finally, one of the newest therapeutic approaches is based on developing molecules that target microrna (mirna) pathways. these small noncoding rna fragments are involved in gene expression regulation, and many of them have been identified with both protective and pathogenic roles. growing knowledge in their functions triggers interest in developing new drugs to either silence pathogenic mirnas (via anti - mirna oligonucleotides or similar agents) or to enhance renoprotective mirnas (with mimics, vectors, or exosomes). to date , only one oligonucleotide has been tested in diabetic mice to prove its renoprotective efficacy. hyperglycemia - derived advanced glycation end - products (age) are known to have a pathogenic effect through the activation of their receptor (rage), causing protein dysfunction and altered collagen turnover activating metalloproteinases. several molecules that inhibit age formation, such as pimagedine or pyridoxamine, showed beneficial effects on animal models but negative or unacceptable adverse events in human trials. several oral adsorbents for uremic toxins have been tested, based on the hypothesis that reducing intestinal absorption of some of these toxins would diminish systemic inflammation and immune system activation. the most studied compound has been ast-120, also called kremezin, a spherical carbon preparation. initial studies cast hopeful in early ckd , but randomized clinical trials in moderate - to - severe ckd showed no effect. a recent meta - analysis that included both kremezin and other adsorbents from asian origin like ai xi te and niaoduqing granted a possible benefit in slowing the speed of renal loss, but without clear evidence. finally, other approaches are still in earlier stages of investigation. these include infusion of endovenous mesenchymal precursor cells or modulation of immune response through regulatory t cells or autophagy. many other attempts have revealed unsuccessful, despite arriving to phase ii or iii, clinical trials. this has been the case of palosuran, a urotensin - ii receptor antagonist, or sulodexide, a glycosaminoglycan with anti - inflammatory properties in animal models.	diabetic kidney disease is the leading cause of end - stage renal disease. albuminuria is recognized as the most important prognostic factor for chronic kidney disease progression. for this reason, blockade of renin - angiotensin system remains the main recommended strategy, with either angiotensin converting enzyme inhibitors or angiotensin ii receptor blockers. however, other antiproteinuric treatments have begun to be studied, such as direct renin inhibitors or aldosterone blockers. beyond antiproteinuric treatments, other drugs such as pentoxifylline or bardoxolone have yielded conflicting . finally, alternative pathogenic pathways are being explored, and emerging therapies including antifibrotic agents, endothelin receptor antagonists, or transcription factors show promising . the aim of this review is to explain the advances in newer agents to treat diabetic kidney disease, along with the of the renin - angiotensin system blockade.
a 15-year - old female patient has been referred to the dental hospital in madurai, tamil nadu, india, with a chief complaint of missing maxillary central incisor. past dental history revealed that the patient had earlier visited a dental clinic where she had been diagnosed with a congenitally missing central incisor and was advised to wear a removable partial denture. the patient was wearing the removable partial denture for the past 3 years. for a more definitive treatment, clinical examination revealed orthognathic facial profile and presence of good facial balance in all proportions. an intraoral examination revealed the presence of all permanent teeth except for the right upper central incisor. panoramic (orthopantomogram or opg), periapical radiographs were taken to establish a good idea about the position and morphology of unerupted right permanent central incisor in maxilla. tooth was bulging in the labial sulcus at the mucogingival junction. its position was very high up in the alveolar bone with a thick layer of soft tissue covering the crown in a vertical direction. the largest width of the crown of erupted permanent left central incisor was 8 mm. it was decided to do surgical exposure of impacted tooth and then bond a bracket on the labial surface of the tooth and bring down to its normal position. begg brackets were bonded on permanent maxillary left central incisor, lateral incisor, and left canine and right lateral incisor and right canine. after the crown of the impacted incisor was surgically exposed , a begg bracket was bonded to the exposed incisor and 0.010 a. j. wilcock wire of supreme grade was used to align the right central incisor. the patient was pleased with the esthetic . at 6-month follow - up, the left maxillary incisor remained vital and responded normally to percussion and mobility and sensitivity testing with good width of attached gingiva. an anomaly in the eruption of anterior teeth can affect facial esthetics and may cause psychological problems. if the impacted tooth is extracted, loss of alveolar bone is anticipated. following the healing period , the alveolar ridge becomes thinner and deficient, with these disadvantages in mind, facilitating eruption of the natural tooth and maintaining natural appearance become the goals of orthodontic treatment. as a , several reports have indicated an impacted tooth can be brought into proper alignment in the dental arch. the following factors are used to determine whether successful alignment of an impacted tooth can take place: the position and direction of the impacted tooth, the degree of root completion, the degree of dilacerations, and the presence of space for the impacted tooth. orthodontic and surgical intervention should not be delayed to avoid unnecessary difficulties in aligning the tooth in the arch. two of the most commonly used surgical exposure techniques for labial impacted teeth are: exposure of the entire labial aspect to the anatomic crown with total excision of all keratinized tissue (the window approach) and a technique which exposes only 45 mm of the most superficial portion of the labial aspect of the cusp tip while maintaining 23 mm of keratinized tissues. in this case, the available space for tooth alignment was sufficient and tooth was brought into right anatomical position in the dental arch. approach causes statistically significant loss of attachment, recession and gingival inflammation occur on maxillary canines after surgical exposure. therefore, a part of keratinized gingiva must be preserved or an apical flap should be used. it is important for a tooth to erupt through attached gingival, and not through alveolar mucosa. if the impacted tooth is diagnosed with its root completely formed or if present in an unfavorable position, combination of surgical and orthodontic treatment has to be carried out. the treatment of an unerupted tooth will depend on its state, position, and presence of enough space in the dental arch to accommodate. if eruption is delayed, the permanent tooth should be exposed because it is important to allow the tooth to erupt into correct position as soon as possible. impaction of maxillary permanent incisors is not a frequent case in dental practice, but its treatment is challenging because of the importance of these teeth in facial esthetics.	impaction of maxillary permanent central incisor is not a frequently reported case in dental practice, but its treatment is challenging because of its importance to facial esthetics. early detection of such teeth is most important if complications are to be avoided. we report a case of a 14-year - old female with an impacted central incisor tooth in the maxillary anterior region. the impacted supernumerary tooth which was preventing the eruption of permanent incisor was surgically removed. combined approach with surgical exposure and the application of an orthodontic force brought the impacted left maxillary central incisor down to its proper position in the dental arch.
laminated wood products are becoming popular in various applications such as construction, furniture units, and indoor decorations due to utilization of raw materials with alternative approach. compared with sawn lumber, laminated lumber can be used for creating wood products that are free from defects and are much larger in size than pieces of wood which were sawn. there are also advantageous in that they are dimensionally more stable, have a variable cross - section, and are more attractive than wood products manufactured from solid wood. in recent years, development in lamination technology has played an important role in the expansion of the use of laminated lumber. it has been extensively utilized in the manufacture of window profiles in many european countries because of its efficiency and positive influence on the environment. characteristics of adhesive, properties of raw materials such as grain orientation and density, and their manufacturing methods for lamination processes play an important role in determining its final quality. therefore, it is important to select the right type of adhesive and to control the overall process to obtain laminated products with acceptable strength properties. the quality of window profiles manufactured from laminated material is comparable to those made from solid wood. however, extra attention should be paid to laminating variables such as difference in moisture content between layers, which should not exceed 2% to comply with standards set by german window technical institute (pren 386, 1991). however, water - resistant resin should be used where laminated wood is exposed to high relative humidity. a number of studies was carried out to evaluate lamination techniques and their application as function of species, adhesive type, and layer thickness. laufenberg determined the influence of outdoor conditions on the performance of veneer and solid - wood laminated materials using phenol resorcinol adhesive. schimid proposed that layer thickness should be less than 15 mm to achieve ideal strength properties for the laminated window profiles. lejeune and leclercq utilized various tropical wood species such as merbau and meranti to manufacture laminated window profiles. turkulin (1992 and 1993) reported strength properties of laminated window profiles made from softwood species using polyvinyl acetate (pvac). many studies have been performed in order to improve the wood properties, and these studies have been commonly named wood modification methods. however, heat treatment is one of the processes used to modify the properties of wood. the heat treatments are usually performed to improve swelling - shrinkage properties of woods and therefore to increase its dimensional stability and biological resistance against fungus. industrial - scale heat treatment process for woods was developed at the technical research center of finland in the early 1990s. the total production capacity of the heat - treated wood in 2002 was approximately estimated to be 265.000 m. recent efforts with regards to the thermal treatment of wood have led to the development of several treatment processes, and materials produced through thermal treatments have been introduced to the european market. some of the products developed using thermal treatment include thermowood (stellac) in finland, torrefaction (perdure) in france, and plato - wood in the netherlands. the extent of the improvement in wood properties after the heat treatment depends on many factors such as thermal modification approaches, wood species, treatment time, and temperature. heat treatment of woods over 150 c alters the physical and chemical properties significantly. it was speculated that the higher the treatment temperature, the better the wood's biological durability. therefore, the use of heat - treated wood in load - bearing constructions is restricted. although heat treatments can be reduced, effects on certain mechanical properties of woods, the dimensional stability, and the biological durability of woods can be increased. however, heat - treated wood is an ecofriendly alternative to impregnated wood materials and can be used for aesthetic places such as garden, kitchen, and sauna furniture, cladding on wooden buildings, bathroom cabinets, floor material, musical instruments, ceilings, inner and outer bricks, doors and window joinery, and a variety of other outdoor and indoor wood applications. currently, there is no information about the effects of heat treatment on the strength properties of laminated window profiles manufactured from kosipo wood. therefore, the objective of this study is to evaluate the effects of heat treatment on some mechanical properties of laminated window profiles manufactured from kosipo (entandrophragma candollei harms .) using differenet types of adhesives. improving the characteristics of laminated window profiles produced from kosipo through heat treatment may provide an initial data to the manufacturers and offer interesting opportunities in future. kosipo wood, a tropical species and usually used as a substitute to mahogany in furniture and sawmilling industry, was utilized for producing laminated window products. thirty samples with 76 mm 86 mm 1000 mm for each species were manufactured in l shape as illustrated in figure 1. two types of commercial adhesives, namely, macroplast ur 7221, which is polyurethane - based adhesive, and water - resistant pvac - type dorus md 072 were used for the experiments. istanbul - turkey based on germanwindows technical institute standards using a cold press at a pressure of 600 kpa for 20 min for pvac and ur 7221 adhesives, respectively. specimens were divided into six treatment groups and for each group, a total of 20 test and 20 control samples were used. the samples were subjected to heat treatment at 150 and 180 c (1c) for 2, 6, and 10 h in a small heating unit under atmospheric pressure. the mc of the samples was 0 percent after heat treatment. after heat treatment, samples were conditioned to 12% moisture contents (mc) in a conditioning room at 20 c (2c) and with 65% relative humidity (rh) as specified in standard ts 642 and iso 554. bending strength (bb), modulus of elasticity (moe), and compression strength (db) tests were carried out on an universal testing system equipped with a load cell of 10 tonnes capacity. tests of the compression strength parallel to grain (ts 2595, 1976), bending strength (ts 2474, 1976), and modulus of elasticity in bending (ts 2478, 1976) were carried out and the values were noted as treated and untreated (control) samples. dimensions of the test samples are shown in figure 2. when the experiments were performed, the mcs (the mc deviation from 12 percent) of the specimens were measured according to standard ts 2471, and the strength values were corrected (transformed to 12% moisture content) using the following strength conversion equation: where 12 is the strength at 12% mc (n / mm2), m is the strength at mc that deviated from 12 percent (n / mm2), is the constant value that shows the relationship between strength and mc (= 0.06, 0.04, and 0.02 for db, bb, and moe, respectively), and m2 is the mc during the experiment (%). the experimental were statistically analyzed using analysis of variance (anova), and significant differences between the control and the treated samples were determined using duncan's multiple range test (astm d 3110, 1998). table 1 shows the changes in the compression strength parallel to grain, bending strength, and modulus of elasticity in bending at varying treatment temperatures and durations. kosipo samples manufactured using ur 7221 adhesive had higher strength than those manufactured using pvac. it appears that adhesive type significantly influenced moe values of the samples in both directions. the lower moe value of 1008.99 n / mm was determined for kosipo profiles laminated in perpendicular direction to pvac adhesive. however, average moe value of kosipo samples laminated with ur 7221 was found to be 11.27% greater than that of laminated samples using pvac (1118 n / mm vs. 1026.95 n / mm). this level of treatments was also give the lowest compression strength value of 39.33n / mm and 43.20 n / mm for ur 7221 and pvac, respectively. these indicate 27.70% less strength for ur 7221 and 20.58% for pvac - treated samples. similarly, the lowest bending - strength values were also obtained for the samples treated at 180 c for 10 h. the loss in bending strength for the heat - treated samples was found to be 31.84%, 46.36%, 11.91%, and 29.49% for profile laminated in parallel direction to ur7221 adhesive, profile laminated in perpendicular direction to ur7221, profile laminated in parallel direction to pvac, and profile laminated in perpendicular direction to pvac, respectively. the lowest modulus of elasticity in bending values was also obtained under similar treatment conditions. the decrease in modulus of elasticity in bending was found to be 14.45% for profile laminated in parallel direction to ur7221 adhesive, 22.80% for profile laminated in perpendicular direction to ur7221, 28.54 for profile laminated in parallel direction to pvac, and 24.10% for profile laminated in perpendicular direction to pvac, respectively. it was also realized that lamination adversely influenced the overall strength properties of kosipo specimens compared with those of solid wood. in general, the of this study on the effect of heat treatment on kosipo are compatible with the findings in the literature on the effect of heat treatment on different tree species. reported a study of compression parallel to grain in eucalyptus saligna wood samples heated at 100155 c for 10160 h and they found that the compression strength values generally deteriorated with increase in temperature or exposure time. tangential cross - section configuration using different layer thickness had the highest bending strength with an average of 99.22 n / mm. found that the modulus of elasticity of maritime pine (pinus pinaster ait .) samples laminated with pvac and ur 7221 adhesives were 84.1, 78.8, 85.6, and 79.1 n / mm for directions perpendicular and parallel to lamination directions, respectively. the determined for kosipo was generally consistent the reports found in the literature for various woods. it was found that the compression strengths of laminated samples of maritime pine were 8.3% greater than those of solid wood. however, laminated samples of kosipo had 10.4% lower compression strength properties compared with those of solidwood. this is probably related to the chemical composition of wood samples and their behavior under heat treatment. yildiz conducted a similar study with spruce (picea orientalis l.) and beech (fagus orientalis l.) woods at 200 c for 6-h conditions and reported a 36% decrease in compression strength. hence, loss in compression strength (10.4%) for laminated kosipo can be acceptable. whereas a slight increase in compression strength was observed at 130 c for 6-h conditions. a number of studies were conducted for evaluating the effects of heat treatments on strength properties of woods. consistently, bending strength and modulus of elasticity were reported to decrease up to 3050% under drastic heat - treatment conditions. a number of studies have been conducted on the reasonably explanation for heat treatment of woods. however, most studies have shown that the strength properties of woods are reduced upon heating. consistently, the decrease of the strength properties of heat treated woods because of changes in the basic structure of wood constituents. the decreases in the strength properties depended on the treatment conditions and on the chemical constituents of woods, which is attributed to the depolymerization reactions of wood polymers during the thermal degradation. particularly, lignin and hemicelluloses, which are less resistant to heat than cellulose, are the primary factors for loss in strength in high - temperature treatments. however, it is probable that heat can be plasticizer to cell wall and effects increasing kinetic energy of molecules, hence, strength of laminated wood samples can be decreased. on the basis of the initial , it appears that the production of laminated window profiles from kosipo would be considered as an alternative method to use raw material more efficiently without having any adverse effect on their properties. the findings of the experiments are comparable to those studies previously carried out in such area. both types of adhesives ed in significant differences in their strength characteristics at 95% confidence level. using laminated profiles in window manufacturing would also in sustainable management of forest resources. in further studies, profiles manufactured using various press parameters such as different pressure levels and press time could be desirable to obtain better information about the properties of the profiles.	the mechanical properties of laminated window profiles manufactured using two types of adhesives were determined. the objective of this study is to evaluate the effects of heat treatment on some mechanical properties of laminated window profiles that manufactured from kosipo (entandrophragma candollei harms .) using differenet type adhesives. commercially produced polyurethane based macroplast ur 7221 and polyvinyl acetate (pvac) adhesive were used for experiments. the overall test were found to be comparable to those obtained in the previous studies. both types of adhesives ed in significant differences in their strength characteristics at 95% confidence level. adhesive ur 7221 improved the overall properties of the samples in contrast to pvac.
a 70-year - old woman was admitted to hospital because of bilateral pleural effusion, and referred to our neurology department due to a 2-year history of gait disturbance that occasionally ed in falling down, before which she had been well. six months after the first episode of falling down, her family noticed that she had a masked face, monotonous speech, and bradykinesia in both arms and legs. during the subsequent year she fell down more frequently, and she could not walk without assistance. she had been taking vitamin d and a calcium preparation to treat a 10-year history of idiopathic hypoparathyroidism and recurrent compression fracture of the thoracic and lumbar vertebral bodies. , she was oriented to place and people, but had difficulty copying, calculating, and maintaining attention. a neuropsychological investigation showed a severe impairment in memory function, reduced psychomotor speed, and visuospatial dysfunction (table 1). she had a decreased blink rate, a paucity of movement of the face, and mild dysarthria. horizontal voluntary saccades were slightly limited and hypometric, and vertical upward and downward saccades were extremely limited and slow. she had a full range of eye movement in the doll's head maneuver, demonstrating that her gaze palsy was caused by a supranuclear problem. all four limbs exhibited bradykinesia and rigidity. sitting up in a bed ed in spontaneous retropulsion. she stood on a narrow base, and her stride was reduced with decreased arm swing bilaterally. the deep tendon reflexes were normoactive in all four limbs. however, there was no dystonia, pyramidal signs, or autonomic impairment. laboratory studies at admission showed the following values: 6.6 mg / dl serum total calcium (normal : 8.6 to 10.0 mg / dl), 4.99 mg / dl phosphate (normal : 2.7 to 4.5 mg / dl), 52 u / l alkaline phosphatase (normal : 25 to 100 u / l), and 1.44 pg / ml intact - parathyroid hormone (normal : 13.0 to 54.0 pg / ml). brain computed tomography (ct) demonstrated extensive, bilateral calcification of the basal ganglia, centrum semiovale, and bilateral dentate nuclei of the cerebellum (fig . there were mixed high - and low - intensity signals compatible with the distribution of calcification on ct . 1-b). single - photon - emission computed tomography using tc - hexamethylpropylene amineoxime showed no abnormal perfusion defects in the brain. levodopa was started at 100 mg twice daily, and the dosage was slowly increased to 1000 mg per day. however, this did not produce any clinical improvement. we experienced a case of striopallidodentate calcification associated with idiopathic hypoparathyroidism presenting with prominent oculomotor disturbances. the prominent oculomotor disturbances, in combination with these extrapyramidal features and dementia, suggested a clinical diagnosis of psp.13 the origin of the parkinsonian symptoms and oculomotor disturbances of this patient is difficult to establish. one possibility is that the basal ganglia calcification observed in brain ct was an incidental finding, because asymptomatic basal ganglia calcifications may be seen at autopsy, especially in the elderly.14 however, the probability of such a coincidence is extremely low given the reported prevalence.15,16 in addition, it has been suggested that a metabolic disturbance such as low calcium or a high phosphorous plasma level may cause dopaminergic dysfunction in the substantia nigra pars compacta.17 f - deoxyglucose and positron - emission tomography have shown that glucose metabolism in the basal ganglia and the frontal brain is massively reduced in fahr's disease.9,18 this abnormality possibly from a disruption of frontostriatal circuits, presumably at the level of the basal ganglia.9 the precise mechanism of oculomotor disturbance in our patient was unknown. in view of the co - occurrence of oculomotor abnormalities in other progressive basal ganglia disorders such as parkinson's disease, psp, and huntington's disease, the significant impairment of eye movement in our patient may have been attributable to dysfunction of the basal ganglia in the generation of voluntary saccades.19 in , we have presented a rare case with idiopathic hypoparathyroidism complicated by oculomotor disturbances and parkinsonism with a psp - like phenotype.	we present a 77-year - old woman with levodopa - nonresponsive parkinsonism, dementia, and supranuclear gaze palsy on vertical and horizontal gaze. laboratory findings were consistent with idiopathic hypoparathyroidism, and brain computed tomography showed extensive bilateral calcifications of the basal ganglia, centrum semiovale, dentate nuclei, and cerebellar white matter. these illustrate that striopallidodentate calcification due to hypoparathyroidism may present with symptoms mimicking progressive supranuclear palsy.
dysfunction or weakness of the gluteus medius is related to diverse musculoskeletal diseases, including lumbar pain1, 2. the height of the hip joint is different in a person with lumbar pain, and radiating pain around the hip joint is due to weakness in the gluteus medius3. in a study of the ability to perform wall squat, pelvic drop, and wall press exercises to strengthen the gluteus medius, the muscle activity was highest during the wall press2. samantha et al.4 conducted a lunge, single leg squat, and step up and over study and observed that gluteus medius activity was highest during the single leg squat. lee et al.5 placed a vertical load on the lower extremities during the swing phase of gait and observed changes in the activity of the gluteus medius during the stance phase. they noted that the muscle activity increased when a vertical load weighing 0.5 kg was placed on the lower extremities during the swing phase and that the muscle activity decreased when a 1 kg load was used in place of the 0.5 kg load. in another study, strengthening of the gluteus medius muscle improved the movement of the lower extremities, preventing injury, and possibly decreased pain6. the present study examined the activity and the gait characteristics of the gluteus medius and the trunk stability muscles during the stance phase of gait on level ground when a vertical load corresponding to 0%, 1%, or 2% of body weight was placed on the lower extremities during the swing phase of gait. the exercise methods used in the study can easily be performed in everyday life to selectively strengthen the gluteus medius. the subjects were 40 young male university students aged between 21 and 30 years. a vertical load corresponding to 0%, 1%, or 2% of body weight, which was measured with an electronic scale, was placed bilaterally 3 cm above the upper part of the lateral malleous. the order of the load was decided randomly, and the load was affixed to each subject prior to the measurement. five steps were taken, and after discarding the data of the first and last cycles, the average of the remaining three cycles were calculated. sufficient exercise for natural gait a wireless surface emg telemyo 2400 t (noraxon co., usa) was used to obtain the emg values. the electrodes were symmetrically attached to the gluteus medius, erector spinae, external oblique, and internal oblique muscles7. the activities of the bilateral gluteus medius, erector spinae, external oblique, and internal oblique were compared among and within the different breathing patterns. one - way analysis of variance was used to examine the muscle activity of the lower extremities during the stance phase. as a post hoc test there were significant differences in the activities of the left gluteus medius, bilateral external oblique, and right internal oblique during gait with vertical loads of 0%, 1%, and 2% of body weight (table 1table 1 . comparison of muscle activations of various vertical loads ( unit : % rvc)0%1%2%gmleft483.74 48.70706.97 67.75597.88 48.70right340.58 41.21329.40 36.58365.91 43.51esleft345.39 33.88357.18 37.63361.48 37.56right344.04 27.35358.18 31.27373.27 33.17eoleft211.53 13.14221.89 15.67265.65 19.40right216.79 14.21222.87 18.36276.51 21.94ioleft192.74 16.20181.95 9.46213.81 16.91right176.76 13.82190.38 10.17221.34 14.29p<0.05, mean se, gm: gluteus medius, es: erector spinae, eo: external oblique, io: internal oblique, vl: vertical load. significant difference between 0% and 1% (p<0.05),significant difference between 1% and 2% (p<0.05), significant difference between 0% and 2% (p<0.05). ). * p<0.05, mean se, gm: gluteus medius, es: erector spinae, eo: external oblique, io: internal oblique, vl: vertical load. significant difference between 0% and 1% (p<0.05),significant difference between 1% and 2% (p<0.05), significant difference between 0% and 2% (p<0.05). lee et al.5 investigated changes in the activity of the gluteus medius muscle during the stance phase by placing a vertical load on the lower extremities during the swing phase. they noted that when a vertical load of 0.5 kg was placed on the lower extremities during the swing phase, the muscle activity increased compared to gait with no load, and that muscle activity with a 1 kg load decreased compared to that during gait with a 0.5 kg load. this study also compared the activity of the gluteus medius muscle during the stance phase when a 1 kg load corresponding to 1% body weight was placed on the lower extremities during the swing phase. we found that the gluteus medius muscle was significantly higher during 1% vertical load gait compared to 0% vertical load gait. the muscle activity during gait with the 2% vertical load was higher than during gait with the 0% vertical load, but lower than during the gait with the 1% vertical load. however, there was no significant difference in the gluteus medius activity between the 1% vertical load and the 2% vertical load. this suggests that the activity of the gluteus medius muscle does not increase in proportion to the increase in the vertical load. in contrast, the activities of the internal and external oblique abdominal muscles significantly increased during gait with the 2% vertical load compared to gait with the 0% and 1% vertical loads. the external oblique abdominal muscle plays a role in the bending of the trunk, when it acts bilaterally8. in the present study, the action of the external oblique abdominal muscle was significantly different between gait with the 0% and 2% vertical loads. during gait with the 0% and 1% vertical load , the activity of the gluteus medius muscle may have been more significant than that of the external oblique abdominal muscle, enabling gait with a vertical load on the lower extremities. however, under the 2% vertical load, providing a vertical load that was possibly greater than the muscle strength needed to move the gluteus medius muscle, may have forced the alignment of the trunk to the right in order to maintain gait, making the bilateral external oblique abdominal muscle act as trunk flexors, moving the trunk anteriorly. when the external oblique abdominal muscle and the internal oblique abdominal muscle contract as individual muscles, the external oblique abdominal muscle acts as a rotator muscle on the opposite side of the trunk. simultaneously, the internal oblique abdominal muscle acts in the same direction as the trunk. however, when the two muscles act together, the distance between one shoulder and the iliac crest on the opposite side decreases8. in the present study, the right internal oblique abdominal muscle showed significant activity differences between gait with the 0% and 2% loads. when the left lower extremity was in the stance phase and the right extremity was in the maximal stance phase, the right internal oblique abdominal muscle and the right external oblique abdominal muscle acted together. thus, the gluteus medius muscle regulated the pelvis of subjects at loads corresponding to 1% of their weight. however, when the load on the lower extremity during the swing phase was increased to 2% of the subject s weight, in addition to the gluteus medius muscle regulating the pelvis, the external oblique abdominal muscle and the internal oblique abdominal muscle acted together to stabilize the trunk and to raise the lower extremities. the gluteus medius muscle, which contracts simultaneously with the external oblique abdominal muscle and the internal oblique abdominal muscle, is a postural muscle that is necessary for stability when raising the heels during gait; it also regulates the pelvis in relation to the movement of the lower limbs9. the activation of the gluteus medius was accompanied by changes in the activity of the internal oblique abdominal muscle and the external oblique abdominal muscle to ensure the stability of the trunk under the different conditions of gait at the various vertical loads. gait was only possible with the activity of the gluteus medius muscle and the trunk muscles resisting the different vertical loads rather than activating other muscles of the lower extremities in terms of energy efficiency.	the present study examined the activity and the gait characteristics of the gluteus medius and the trunk stability muscles during the stance phase of gait on level ground when a vertical load corresponding to 0%, 1%, or 2% of body weight was placed on the lower extremities during the swing phase of the gait. the subjects were 40 young males aged between 21 and 30 years. the vertical load, corresponding to 0%, 1%, 2% of weight, which was measured with an electronic scale, was placed bilaterally 3 cm above from the upper part of the lateral malleous. electrodes were symmetrically attached to the gluteus medius, erector spinae, external oblique, and internal oblique muscles. there were significant differences in the activities of the left gluteus medius, bilateral external oblique, and right internal oblique muscles among the vertical loads of 0%, 1%, and 2% during gait. increases in vertical load were accompanied by changes in the activities of the internal and external oblique abdominal muscles to ensure the stability of the trunk under the different loads. gait was only possible with the activity of the gluteus medius muscle and the trunk muscles resisting the different vertical loads rather than activating other muscles of the lower extremities in terms of energy efficiency.
varicose veins occur commonly in the general population but despite much research the etiology of venous disease is still poorly understood. obesity, age, parity, standing for long times, and family history are risk factors. incidence of varicose veins in adult population has been shown to vary among populations (between 10% and 60%) and to increase by age in various studies. the main factors in the etiology of varicose vein are venous dilation and valvular insufficiency that are started by unknown factors. a number of epidemiological studies have shown that, in addition to environmental factors, genetic mechanisms may play a role in determining susceptibility to vascular disease. in particular, abnormalities in the enzymes that control homocysteine metabolism have been shown to cause atherosclerotic vascular disease. since genetic mechanisms may play a role in determining susceptibility to vascular disease, we studied several mutations in patients with varicose veins to look for any association between varicose veins and homocysteine level, protein c, protein s, fv, fviii, d - dimer, vitamin b12 level, folic acid level, mthfr, fvr2, b fibrinogen, fv leiden, and prothrombin mutations besides other possible factors. the present study is planned to determine various risk factors and to analyze the methylenetetrahydrofolate reductase polymorphisms (mthfr-677 and mthfr-1298) and fv leiden mutations in patients with primary varicose veins. the patients considered for the study were those attending ankara education and research hospital for the management of venous disease. the following data were collected: age, height, weight, smoker or nonsmoker, personal and family medical history with a special focus on varicose veins and birth number > 1 (women only), standing for a long time before, and additional diseases. informed written consent this study protocol was approved by the ethics committee of ankara education and research hospital. the diagnosis of primary varicose vein was performed by the combination of clinical and duplex scanning examination. the exclusion criteria for the study were deep vein thrombosis, postthrombophlebitic syndrome, and recent infection. there were 45 patients with superficial venous reflux, 2 patients with superficial and perforator venous reflux, and 2 patients with superficial venous reflux and healed venous ulcer. the effects of the grade and type of the reflux on the were not studied in the paper. we could not evaluate the location of valvular reflux at the various levels, sfj, upper thigh, midthigh, lower thigh, or below the knee, because of outpatient density in the radiology department. since there were not enough patients in the study group, the differences in the gene polymorphism or the procoagulant parameters between c2 and c3 patients versus the control group were not analysed. the control subjects did not have any disease established by careful history, examination, and routine laboratory test including the levels of homocysteine, ferritin, vitamin b12, hemoglobin, sedimentation rate, mean corpuscular volume, low density lipoprotein, and rheumatoid factor. the patients in the observation group and the control group were similar regarding gender, body mass index, smoking habits, medical history, family medical history, and age. we asked patients one by one about how long they stand up all through the day and recorded the duration of time in hours for each patient separately. in the physical examination , subjects stood on a raised platform with their feet in three standard positions during inspection of the legs: facing towards the examiner with heels together and forefeet spread wide apart, facing away from the examiner in a similar position, and facing away from the examiner with feet parallel. they were asked to remain in a standing position for a minimum of two minutes before examination of their veins, to allow the blood to pool in the legs. real - time images of the common femoral, deep femoral, femoral, and popliteal veins were obtained before and after compression. all patients were evaluated with color duplex scanning in a warm, comfortable examination room by a radiology physician. a 512 mhz linear transducer was used to measure the diameters of the great saphenous vein (gsv) and the small saphenous vein (ssv) and to rule out acute or chronic deep venous thrombosis (dvt) in supine position. using color flow imaging in the longitudinal view, the valvular function of the gsv was evaluated at the sfj, upper thigh, midthigh, lower thigh, and below the knee. the valvular function of the ssv was evaluated at the level of the popliteal fossa. the reflux was quantified based on valve closure time, with the doppler spectral tracings obtained in a longitudinal plane. reflux was defined as being present if the valve closure time was greater than 0.5 seconds. examination for reflux was made with the patients standing, with upper body elevation of more than 45, or in reverse trendelenburg position. blood samples were collected into citrate tubes (for protein c, protein s, fibrinogen, and homocysteine) and centrifuged at 4000 rpm (revolutions per minute) for 4 min to obtain plasma and serum fractions. quantitative determination of functional protein c depended on the prolongation of activated partial thromboplastin time. protein s was determined in accordance with the inhibition of activated factor v. the normal ranges of protein c and s antigens in plasma were 70130% and 65140%, respectively. the assay kit is designed to quantitate the true functional fxiii and fxiiia activity in plasma by measuring transglutaminase activity. in this assay, the thrombin - activated fxiiia from the patient plasma binds to a substrate coated plate. next, a horse radish peroxidase- (hrp-) conjugated fxiii is cross - linked to the captured fxiiia. the cross - linked hrp is then detected with a hrp chromogenic substrate at 450 nm. the data was analyzed to evaluate the correlation between fxiii activity and other dic markers which included platelet count, fibrinogen activities, prothrombin time (pt), activated partial thromboplastin time (aptt), and fibrin degradation product (d - dimer). pt, aptt, fibrinogen, and d - dimer were generated with an automated clotting analyzer (sta - r). for the genetic testing, a sample of 10 ml 0.05 m edta - anticoagulated blood was taken. dna was extracted according to a standard salting - out procedure. for dna analysis, commercially available kits were used and the procedure was performed in accordance with the manufacturer's instructions. mutation status (normal, heterozygous, or homozygous) the analysis of factor v leiden and prothrombin g20210a mutations was done. for determination of the mutation status of the mthfr c677 t and a1298c mutations, we used pcr amplification and restriction fragment length polymorphism analysis that was performed according to kim et al. , frosst et al. , frosst et al., and van der put et al. since the control group has less than 30 patients, all parameters in the study were assumed nonparametric and the statistical analyses were chosen for nonparametric tests. nominal parameters were described using frequency analysis, whereas scale parameters were described using mean and standard deviations. comparison between groups was tested by mann - whitney u at 95% confidence interval (ci ; = 0,05 level). gender distribution, age, bmi, smoking, diabetes mellitus, rheumatoid factor positivity, hypertension, family history, long standing duration, and birth of more than one child for patient and control groups are gathered as baseline characteristics of the respondents. the ages of the forty - nine subjects with varicose veins included in the study group ranged between 15 and 85 years (mean 44.04 15,05 years) and the ages of the sixteen subjects in the control group ranged between 22 and 68 years (mean 40,94 13,60 years). the women / men ratio was 30/19 in the study group and 15/11 in the control group. diabetes mellitus, hypertension, family history of varicosis, and birth of more than one child of the study group were higher than of the control group. differences between the two groups were found statistically insignificant (p > 0,05) except for standing for long durations (p < 0,001) and rheumatoid factor positivity that were found to be statistically significant (p < 0,05). it is possible that increasing severity of c class might in greater differences in hematological or biochemical parameters. since the classification of the patient group in the study was most notably between class 2 (c2) and class 3 (c3), the differences were not noted in this study with the increasing c class. according to table 2, ceap class of the study group was changing between class 2 (c2) and class 5 (c5). comparison of hematological and biochemical parameters in the study and the control groups was given in table 3. no statistically significant difference was observed between both groups with respect to haematological and biochemical parameters, including whole blood count, erythrocyte sedimentation rate, prothrombin time, partial thromboplastin time, c - reactive protein level, glucose level, cholesterol profile, electrolyte levels, liver and kidney function tests, homocysteine, vitamin b12, folic acid, factor v, factor viii, and protein c and protein s levels. table 4 shows the comparison of point mutations in the study group and the control group. when we look at the comparison of point mutations between the study group and the control group, heterozygote mutations were more dominant in the study group, but the difference was not statistically significant. homozygote mutations for all parameters were seen to be rare and scattered in both study and control groups. we could say that heterozygote mutations were dominant in the study group compared to the control group, but the difference was not statistically significant. incidence of varicose veins in adult population has been shown to vary among populations (between 10% and 60%) and to increase by age in various studies. the main factors in the etiology of varicose vein are venous dilation and valvular insufficiency that are started by unknown factors. on the basis of experimental and clinical evidence collected for the past decade, it is possible to suggest that the cause of dilatation of the varicose vein is in the vascular wall. valvular theory, suggesting valvular incompetence, has been criticized in a number of biochemical and morphological studies. despite the evidence of a primary defect in the vein wall javien's study showed that varicose veins were more common in women, but female sex was not found to be a strong risk factor. among the risk factors most closely associated with chronic venous insufficiency (cvi) were age, family history of varicose veins, and constipation. this is in keeping with findings from recent epidemiologic studies. obesity and lack of physical activity were strongly associated with cvi in women, more so than in men. a modest association was found with female sex, previous injury in legs (dvt), and remaining in the standing position for a long time, although these parameters are usually among those mostly agreed on as being risk factors. a number of epidemiological studies have shown that, in addition to environmental factors, genetic mechanisms may play a role in determining susceptibility to vascular disease. in particular, abnormalities in the enzymes that control homocysteine metabolism have been shown to cause vascular disease. methylenetetrahydrofolate reductase (mthfr) plays an important role in the folate cycle and contributes to the metabolism of the aminoacid homocysteine. it catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, thus generating the active form of folate required for remethylation of homocysteine to methionine. if there is a genetic mutation in the mthfr gene, homocysteine levels may not be regulated properly. genetic mutations in mthfr are the most commonly known inherited risk factor for elevated homocysteine levels. the most common mthfr mutation is called the mthfr c677 t mutation, or the thermolabile mthfr mutation. even when 2 mthfr mutations are present (e.g., 2 c677 t mutations, or one c677 t mutation and one a1298c mutation), not all people will develop high homocysteine levels. although these mutations do impair the regulation of homocysteine, adequate folate levels essentially cancel out this defect. the mthfr polymorphism, which is associated with a predisposition for elevated plasma concentrations of homocysteine, has been reported to represent a genetic risk factor for occlusive vascular diseases, carotid atherosclerosis, silent brain infarction, and small artery occlusion with ischemic stroke, although these associations remain controversial. in our study we studied whether these mutations are associated with another type of vascular disease in the form of varicose veins because the observation of vascular complications in patients with homocystinuria led to the hypothesis that mild to moderate elevation of plasma homocysteine may be related to changes in the vascular wall. whether mild hyperhomocystinuria is causally related to the development of varicose veins was not known. since genetic mechanisms may play a role in determining susceptibility to vascular disease, we studied several mutations in patients with varicose vein to look for any association between varicose veins and homocysteine level, protein c, protein s, fv, fviii, d - dimer, vitamin b12 level, folic acid level, mthfr, fvr2, b fibrinojen, and fv leiden and prothrombin mutations. in our study, we did not observe high levels of homocysteine in our varicose disease patients compared to control group. the of our study showed that there was no statistically significant difference between patients and control groups based on their baseline characteristics except for standing durations of the patients and rheumatoid factor positivity. age, bmi, gender distribution, ht, family history, and birth of more than one child were not different between groups. although some researches reported that family history of the patients has an effect on varicose veins, we found it noneffective in the study. in addition, it was seen that heterozygote mutation was dominant in both male and female patients in the patient group, but the difference was not significant. a study done on a population of 1684 individuals around turkey demonstrated that the frequency of the c677 t in turkey was 42.9%; of c677c, 47.4%; and of t677 t, 9.6%. the frequency in turkey of a1298c was 43.7%; of a1298a, 46.3%; and of c1298c, 10.0%. the allelic frequencies of the t allele of mthfr 677 and the c allele of mthfr 1298 were 33.34 and 33.16%, respectively. the frequency of c677t / a1298c compound heterozygosity is highest in turkey (21.6%), as compared to canada (15%), the united states (17%), and the netherlands (20%). genetic abnormalities specific to factor v, prothrombin, and homocysteine metabolism were shown to increase the risk for myocardial infarction and ischemic stroke, particularly among younger patients and women in a study. unlike our sverdlova am demonstrated an association between the mthfr genotype and the risk of developing varicose veins in the lower limbs. they found a significantly higher prevalence of subjects with at least one c677 t mthfr allele among those with varicose veins than among a control group (or = 1.74 ; p < 0,005). studies including higher numbers of cases and controls could show more significant relations between them. since the number of cases in the patient and control group is small, we did not take the effect of the type (superficial and deep reflux) and the degree of reflux into consideration in the study. economical factors and the type of rheumatic diseases were not evaluated in the study also. we only studied rheumatic factor positivity which was found to be statistically significant compared to the control group. while there was a trend towards an increase in certain biochemical parameters in our study, ultimately there was no statistically significant difference in hematological or biochemical markers between the subject group and the control group. in this small study, there appear to be no significant genetic differences related to folate metabolism or procoagulant predisposition or medical risk factors including bmi value, smoking, diabetes mellitus, hypertension, family history of varicosis, and birth of more than one child. a history of increased standing and rheumatoid factor positivity were found to be associated with varicose veins. further studies, perhaps with a larger cohort, could show significance of parameters that were found to be insignificant in our study. further studies should also be conducted to find any association between rheumatic diseases and varicose vein development.	the objective of the study is to evaluate a range of potential risk factors in the etiology of varicose veins with superficial venous reflux. forty - nine patients attending a cardiovascular surgery clinic for the management of varicose disease between 2009 and 2010 were enrolled for the study. the age range of the patient group was 44,04 15,05 years and female / male (f / m) ratio was 30/19. twenty - six normal, healthy volunteers with the age of 40,94 13,60 years and with the female / male ratio of 15/11 acted as control subjects. we investigated several parameters including body mass index, age, birth number > 1, standing for a long time (standing for 8 or more hours without taking a break), systemic diseases, family history, venous doppler fndings, the levels of homocysteine, ferritin, vitamin b12, and hemoglobin, sedimentation rate, mean corpuscular volume, low density lipoprotein, and rheumatoid factor of the patient group and the control group. we also determined the contribution of the methylene tetrahydrofolate reductase 677 c > t and 1298 a > c gene polymorphisms and fv leiden in both groups. in this small study , there appears to be no association between varicose veins and body mass index, smoking, type 2 dm, hypertension, family history, and birth number. a history of increased standing duration period (> 8 hours) and rheumatoid factor positivity have association with varicose veins with superficial venous reflux.
over the past 20 years, advances in radiographic imaging and computer technology have allowed for application of image - guided surgery in orbital reconstruction. conversely, volumetric analyses of anatomical structures have been utilized for the design of standardized anatomic implants for orbital reconstruction and custom patient - specific implants for complex orbital and midfacial defects. these advances have allowed for improved efficiency, accuracy, and safety in the surgical management of orbital pathology. the purpose of this review is to discuss the applications of computer - guidance and advanced computed tomography (ct)-imaging in the management of various conditions affecting the bony orbit. four illustrative case examples are presented: orbital decompression for thyroid - eye disease, midface reconstruction following a zygomaticomaxillary complex (zmc) injury, reconstruction of a posttraumatic two - wall orbital defect, and reconstruction of a large orbital floor defect with a custom alloplastic implant. thyroid eye disease (ted) is an autoimmune inflammatory disorder of the orbit that in proptosis, diplopia, eyelid retraction, exposure keratopathy and in severe cases, optic nerve compression.1 the pathogenesis of ted has not been entirely elucidated however it is most likely due to the activation of thyroid stimulating receptors by circulating thyrotropin receptor antibodies. activation of these receptors in the orbit in deposition of hyaluronan and ing extraocular muscle enlargement as well as hypertrophy of orbital adipose tissue.123 as the orbital contents enlarge, the patient is at risk for sight - threatening compressive optic neuropathy. various treatment methodologies including steroids, orbital radiation, immunomodulators, and selenium have been used. however, refractory or sight - threatening cases of ted usually require surgical decompression.3 orbital decompression has been a mainstay of ted treatment since the 1950s. the variability in outcomes particularly in terms of proptosis and ocular alignment has ed in the development of numerous surgical techniques for decompression with no clear consensus on which is most effective.4 the recent development of computer - assisted surgical planning and execution has the potential to in more efficacious, consistent and predictable decompressions. multiplanar ct scans with three - dimensional (3d) reconstruction allow the surgeon to carefully evaluate the individual anatomy and identify specific bone segments for resection. intraoperatively, the ct images and the patient's anatomy are virtually overlapped allowing for stereotactic navigation throughout the case.5 this allows the surgeon to check his / her distance from important anatomical landmarks and to measure the extent of bony resection in order to carefully match preoperative planning.6 postoperative imaging and assessment may be used to objectively quantify the magnitude of decompression and correlate this with clinical outcomes. the objective data provided by computer - assisted orbital decompression has the potential to in safer, more efficacious and consistent decompressions. in the clinical example shown , real - time image guidance was utilized to allow for extensive, asymmetric decompression of the bilateral orbits (left : lateral orbital wall, right : medial and lateral walls). preoperative (top panels) and postoperative (bottom panels) frontal and submentovertex views of a male patient with thyroid orbitopathy who underwent computer - assisted orbital decompression preoperative (left panels) and postoperative (right panels) demonstrating the three - dimensional computer planning for orbital decompression and superimposition of the expected over the postoperative images (right middle and right bottom panels) injuries to the bony orbit are common among patients sustaining craniomaxillofacial trauma.789101112 the external orbital framework is disrupted in several different types of facial fractures (e.g. zmc, frontal bone, le fort iii). the integrity of the internal orbit can likewise be disrupted, either in isolation or as a component of complex midfacial or upper facial injury. the orbital skeleton provides support for the globe and ocular adnexa and houses neurovascular structures critical to normal visual sensory function. while disruption of the orbital framework can compromise these structures, such injuries are, fortunately, rare. conversely, surgical management of orbital skeletal injuries is fraught with potential peril, as dissection, bony mobilization, or placement of intra - orbital hardware may damage the globe or critical neurovascular structures. in this regard, advances in computer imaging have enhanced the surgeon's ability to safely dissect the internal orbit, have allowed for the design and manufacture of standard implants for orbital reconstruction, planning for correction of secondary deformities, quantitative assessment of fracture reduction and volume restoration, and have improved the ability to visualize, in real - time, the orbital anatomy during dissection.13141516171819202122232425262728293031 zmc fractures sometimes termed zygoma fractures or orbitozygomatic fractures, are among the most common type of facial fracture.78910 high - or low - energy mechanisms can disrupt the articulations of the zygomatic bone with the frontal bone (frontozygomatic suture), temporal bone (zygomatic arch), sphenoid bone (sphenozygomatic suture), and maxilla (zygomaticomaxillary buttress). the 3d relationship between these articulations was not initially understood in the management of these injuries, which were commonly referred to as tripod fractures. advances in ct have improved the understanding of these injuries and the complex 3d anatomy that needs to be restored for successful management.78910111213 restoration of the sphenozygomatic articulation in the lateral orbit remains the most reliable predictor of a successful reduction but is difficult to assess without surgical access to the internal orbit.712272829 furthermore, management of the orbital floor remains controversial among patients with zmc fractures.2829 the use of real - time imaging (mini c - arm, ct) has allowed for assessment of anatomic alignment and orbital floor integrity following reduction, improving operating times and decreasing patient morbidity from unnecessary orbital exploration.13202123 in the case presented, an orbitozygomatic fracture was repaired utilizing virtual planning to establish the appropriate position of the right zygoma. to accomplish this, the left zygoma complex was digitally rendered from a 3d ct reconstruction and subsequently mirrored and placed onto the right midface. the reconstituted virtual position of the zygoma served as a template and was then used to guide intra - operative positioning of the displaced fracture. virtual planning also allowed for visualization of the sizable orbital floor defect, which required orbital exploration and reconstruction using an anatomical plate. three - dimensional and axial computed tomography, as used for operative planning for a displaced orbitozygomatic fracture. in the preoperative images (top panels), the displaced zygoma (top left) was repositioned by mirroring the left zygoma and virtually positioning the mirrored bone (top middle and top right panels). the postoperative images (bottom panels) demonstrate anatomic alignment of the zygomatic articulations and reconstruction of the orbital floor injuries to the orbital floor are commonly encountered among surgeons affiliated with trauma centers that treat facial injuries. orbital floor injuries may occur in isolation or in conjunction with higher level le fort injuries, zmc fractures, naso - orbital - ethmoid fractures, panfacial trauma, and often occur with fractures of the medial orbital wall. the primary goal for orbital reconstruction is to restore the premorbid orbital volume. in this regard, image - guidance technology has been useful for the design of anatomic orbital implants, particularly for two - walled defects involving the floor and medial wall.14151617181924 custom implants can also be utilized for reconstruction of irregular defects or when there is a significant bone loss. real - time intra - operative image guidance is a useful to aid in dissection of the orbital floor and medial wall, allowing the surgeon to assess accurately position relative to the superior orbital fissure, optic canal, and the anterior and posterior ethmoidal foramina, thereby decreasing the risk of damage to critical arterial, venous, and nervous structures in these areas. in the first clinical example, virtual planning was used to assess the size and position of the orbital implant required for reconstruction of a posttraumatic defect involving the right medial orbital wall and floor. the unaffected orbit was used for comparison to allow for adequate reconstitution of orbital volume. the preoperative enophthalmos was predictably corrected; the postoperative image fusion demonstrates appropriate positioning of the implant along the orbital floor and medial wall, with a stable landing on the posterior ledge. in the second clinical example, a patient with a large, posttraumatic orbital floor defect with significant volume change and marked enophthalmos underwent presurgical planning and design of a custom titanium / polyethylene implant for orbital reconstruction. the postoperative demonstrates marked improvement in globe position, with excellent positioning of the implant along the fracture margins. preoperative (top panels) frontal and submentovertex views of a male patient with an orbital floor and medial wall fracture who underwent computer - assisted planning for orbital reconstruction. in the postoperative (bottom panels) photos, the enophthalmos has been corrected and the globe position is symmetric preoperative (left panels) and postoperative (right panels) demonstrating the three - dimensional computer planning for orbital reconstruction with an anatomic implant. the implant is rendered within the software and virtually placed within the defect. this allows the surgeon to position the implant and compute the orbital volume relative to the unaffected side. intra - operatively, placement of the implant in the appropriate position can be ensured with the use of real - time navigation preoperative (top panels) frontal and submentovertex views of a female patient with a large right orbital floor fracture with significant enophthalmos. computer - assisted planning was utilized to design a custom implant for reconstitution of orbital volume for enophthalmos correction. postoperatively (bottom panels), the patient has has no orbital dystopia or enophthalmos preoperative (left panels) and postoperative (right panels) images, demonstrating the three - dimensional computer planning and subsequent reconstruction of a large orbital floor defect with an anatomic titanium - alloplastic hybrid implant. the preoperative images are utilized to compute the volume of the defect relative to the unaffected side. the custom implant is then designed to restore the orbital volume and correct the globe position advances in computer technology and imaging have improved the accessibility and efficacy of orbital reconstruction for defects involving the bony orbit. due to the complex shape of the internal orbit, proximity of critical soft tissue structures, and small margins of error, the orbital skeleton is an ideal anatomic region for virtual planning and real - time intra - operative navigation. utilization of this technology has the potential to improve the surgical treatment of common problems, make challenging clinical cases more accessible and predictable, and has enormous utility as an adjunct for trainee instruction.	the advent of computer - assisted technology has revolutionized planning for complex craniofacial operations, including orbital reconstruction. orbital reconstruction is ideally suited for virtual planning, as it allows the surgeon to assess the bony anatomy and critical neurovascular structures within the orbit, and plan osteotomies, fracture reductions, and orbital implant placement with efficiency and predictability. in this article, we review the use of virtual surgical planning for orbital decompression, posttraumatic midface reconstruction, reconstruction of a two - wall orbital defect, and reconstruction of a large orbital floor defect with a custom implant. the surgeon managing orbital pathology and posttraumatic orbital deformities can benefit immensely from utilizing virtual planning for various types of orbital pathology.
photothermal ablation is an exciting noninvasive cancer paradigm,15 which is just beginning to enter the clinical arena for human cancer therapy.6 numerous nanomaterials including nanoshells,1,2 polyhydroxy fullerenes,5 gold speckled silica nanoparticles (nps),7 gold nps,8 gold nanorods,3 nanotubes,9,10 nanocages,11 and quantum dots12 have been investigated as anticancer photothermal agents. numerous studies have demonstrated the efficacy of these nanomaterials for tumor ablation in vitro.2,7,8,13 several studies have focused on the effect of photothermal therapy using these nanomaterials using in vivo models.1,10,13 these in vivo studies have demonstrated that a single photothermal treatment in partial but incomplete tumor destruction based on long term follow - up of tumor growth.10,13 approaches and strategies to improve the efficacy of the photothermal antitumor strategy are needed. understanding the intratumoral fate of nanomaterials after photothermal therapy is essential for furthering the clinical application of nanoparticle - mediated photothermal therapy. to investigate this issue in vivo requires the development and application of nanomaterials that are not only robust photothermal reagents, but also stable bioimaging contrast agents. magnetic resonance imaging,1 optical coherence tomography,13 and photoacoustic imaging14 have been used to guide photothermal treatments in vivo. near - infrared (nir) fluorescence imaging has not been utilized commonly to track photothermal nanomaterials in vivo due to difficulties in combining plasmonic materials with a fluorescent component into single, biocompatible nanoconstructs.15 in this report, novel multidye theranostic nps (mdt - nps) were employed to address the fate of nanomaterials following photothermal ablation. these recently developed nps are biocompatible, have strong and stable nir fluorescence, and robust nir photothermal properties.16 the unique multi - functionality and stability of these novel nanomaterials favor their application to answer these questions regarding the fate of nanomaterials of photothermal therapies. in this report , the ability of mdt - nps to mediate enhanced photothermal tumor destruction when utilized for multiple fractionated ablations is demonstrated. the findings suggest fractionated photothermal therapy as a new paradigm for noninvasive image - guided cancer therapy. nir fluorescent (nirf) mesoporous silica nps16 were synthesized by incorporating a modified, silane - conjugated heptamethine cyanine dye (ir780 ; sigma - aldrich corporation, st louis, mo) during the synthesis of mesoporous silica nps, as described elsewhere.16 briefly, 0.5 g of cetyltrimethylammonium bromide (c16tab ; sigma - aldrich) and 1.75 ml of 2 m sodium hydroxide (fisher scientific co, pittsburgh, pa) were added to 240 ml of nanopure water (fisher scientific) and heated to 80c. once thermal equilibrium was reached, 2.5 ml of tetraethyl orthosilicate (fisher scientific) was added dropwise to this solution. after 23 minutes, the transparent solution turned milky due to the nucleation of silica nps. at this time, 800 l of silane conjugated dye was added. the particle suspension was allowed to stir for 3 hours at constant temperature of 80c. for the removal of surfactant , particles were dispersed in 3 wt% methanolic solution of calcium chloride (fisher scientific) and the suspension was refluxed for 4 hours at 60c. for 1 g particles, 100 ml of calcium chloride - methanol solution was used. these particles were centrifuged (7000 g, 20 min ; beckman model j2 - 21, ape - bridgepath scientific, frederick, md) and resuspended in the calcium chloride - methanol solution with the aid of sonication (crest tru - sweep model 575d ; crest ultrasonics, trenton, nj). the refluxing/ centrifugation / resuspension steps were repeated three times in order to completely remove the surfactant from the pores. finally, these particles were suspended in nanopure water by centrifugation and resuspension using sonication. to confirm the complete removal of surfactant, the nps were incubated with cells and a lactate dehydrogenase assay (roche diagnostics, indianapolis, in) was performed; the assay demonstrated no deleterious effects on cells. for the synthesis of mdt - nps, nirf mesoporous particles were dispersed in chloroform (fisher scientific) to encapsulate the photothermal dye inside the pores. in a typical synthesis, nirf mesoporous silica nps (10 mg) were dispersed in 8 ml chloroform. then, 2 mg of silicon 2,3-naphthalocyanine dihydroxide (sigma - aldrich) in chloroform (concentration 1 mg / ml) was added to the nanoparticle dispersion. the dispersion was stirred overnight to allow for the maximum loading of the dyes into the np pores. measuring the absorbance of the supernatant allowed an estimate of the amount of dye incorporated inside the pores of the nps (11.1%). nanoparticles were characterized for absorbance, emission, and heating using techniques detailed elsewhere.16 the 4t1 murine mammary tumor cell line (tufts university, medford, ma) was orthotopically implanted (1 10 cells) into female 6-week - old balb / c mice (charles river laboratories, wilmington, ma). on postimplantation day eight, 27 mice were equally and randomly distributed to nine groups (three mice per group): mdt - np / no ablation, mdt - np / one ablation, mdt - np / two ablations, mdt - np/ three ablations, mdt - np / four ablations, control / one ablation, control / two ablations, control / three ablations, control / four ablations. only one ablation was performed per day per mouse. at the start of the experiment , mice were initially imaged via fluorescence (710 nm excitation/820 nm emission) with the ivis spectrum (caliper life sciences, hopkinton, ma). for in vivo imaging experiments, 820 nm wavelength was chosen as it provides the greatest signal to noise ratio. this was followed by administration of mdt - nps via intratumoral injection (15 mg / ml, 20 l) or 0.9% sterile saline solution (20 l ; fisher scientific) for control mice. mice then underwent photothermal ablation (continuous wave laser, 785 nm, 625 mw / cm, 5 minute duration, 1.5 cm source - tumor distance). fluorescence imaging was also obtained after each photothermal ablation. for imaging and ablation experiments, mice were anesthetized using 2%3% isoflurane (baxter healthcare corporation, deerfield, il). on day nine, whole animal digital photographs (d90 ; nikon, melville, ny) were obtained before mice were euthanized. severity of treatment effect was based on gross tumor inspection (experiment day nine) and scored in a blinded fashion using a scale of zero (no treatment) to four (maximal treatment effect). tumors were preserved in 10% neutral buffered formalin (fisher scientific) for histological analysis. histological tumor analysis was performed in a blinded fashion by a board certified pathologist (jak). region of interest quantification of fluorescent signal was performed at all imaging points with living image 3.2 or 4.0 software (caliper life sciences). all experiments were performed under protocols approved by the university of florida institutional animal care and use committee. mdt - nps were synthesized using a two - step approach.16 first, nirf mesoporous silica nps were synthesized by incorporating a modified, silane - conjugated heptamethine cyanine dye (ir780) during the surfactant - templated synthesis of mesoporous silica nps. second, for the synthesis of mdt - nps, these nirf mesoporous nps were dispersed in chloroform to encapsulate the nir photothermal silicon 2,3-naphthalocyanine dihydroxide dye inside the pores of the silica matrix. the final nanoconstructs were washed and dispersed in water for the subsequent sequential photothermal ablation experiments and visualization. the photophysical properties (absorbance and fluorescence) of nirf nps are presented in figure 1a. the broad excitation spectra and large stoke s shift of the particles enabled use of a broad range of excitation wavelengths and decreased the extent of self - quenching, respectively. having a porous interior structure, these nps were circa 100 nm, as measured by transmission electron microscope (jeol 2010f, tokyo, japan) (figure 1d). upon loading of a silicon 2,3-naphthalocyanine dihydroxide dye into the pores of nirf nps, their absorption cross - section in the nir region increased (figure 1b); the broad extinction spectra of these mdt - nps displayed their ability to absorb light over the entire nir region. the ability of mdt - nps to absorb nir light and generate heat was tested by measuring the temperature increase of an aqueous dispersion of mdt - nps upon illumination with a low power laser source (785 nm, 625 mw/ cm). the temperature of a solution of mdt - nps (1 mg / ml) increased by approximately 15c after 5 minutes of continuous irradiation. the repeated heating over a period of 3 days ed in only partial loss (~4% decrease in temperature on day three relative to day one) of their heat generating capacity (figure 1c). next, a murine orthotopic model of breast cancer was used to study the fate of mdt - nps following photothermal ablation in vivo. after intratumoral injection of mdt - nps, mammary tumors in mice were exposed to nir laser light once daily for 1, 2, 3, or 4 days. after each nir irradiation event, the fluorescence signal was plotted by normalizing each signal to the intensity of the original injected mdt - nps (day one before ablation, figure 2). following a single injection of mdt - nps, all experimental groups (both with and without laser irradiation) showed a consistent intratumoral fluorescence pattern for 9 days. importantly, mdt - nps could easily been seen in the intratumoral position for the entire 9-day observation period in all groups. figure 3 is a fluorescence imaging sequence over a time course of 9 days of a representative mouse from the mdt - np / four ablations group. after 9 days, mdt - nps retained high fluorescence imaging capacity and remained within the tumor. in order to determine if mdt - nps were accumulating in reticuloendothelial organs (liver and spleen) following photothermal ablation therapy, whole animal organ in situ fluorescence imaging was performed following primary tumor removal on day nine. no mdt - nps were detected beyond the intratumoral injection site in these mice (figure 3). the persistence of mdt - nps within the tumor for 9 days following administration suggests the possibility for fractionated image - guided photothermal therapy after a single administration of mdt - nps to a tumor. increasing the number of ablation fractions corresponded to a significant (p = 0.026) increase in treatment effect as assessed by gross tumor inspection on experiment day nine (figures 4 and 5). control groups demonstrated no effect from irradiation, and tumors in the mdt - np treated / no ablation group were unaltered by the presence of mdt - nps (figure 5). histologically, a congruent pattern was observed: no evidence of thermal injury or other tissue damage was noted in control mice (figure 6a), whereas mice treated with mdt - nps and exposed to four fractionated photothermal ablations demonstrated significant thermal injury with coagulative tumor necrosis, hemorrhage, and edema (figure 6b). for fractionated photothermal therapy to be possible, photothermal nps must maintain their intratumoral location following photothermal activation. to the authors knowledge, the intratumoral fate of photothermal nps following one or more photothermal treatments has not been previously well characterized. mdt - nps are a novel tool which has allowed the intratumoral fate of photothermal nps following photoablation to be studied. multimodal mdt - nps can easily be both tracked using fluorescence imaging and repeatedly excited for photoablation with a nir laser. in the reported experiments, mdt - nps were clearly able the presence of mdt - nps within the tumor following photothermal treatments (as demonstrated by nir imaging) and their ability to be repeatedly heated in vitro (figure 1c) suggests that they may be used for additional photothermal treatments (fractionated photothermal therapy). the ability to image mdt - nps within the tumor over time it was observed that mdt - nps remain in the intratumoral position for days following photothermal treatment in the 4t1 murine mammary carcinoma model. it is possible that the kinetics of nanomaterial extravasation are different for other tumor types with different lymphatic and vascular patterns. intratumoral persistence and function of mdt - nps following photoablation may allow the use of lower laser intensities to treat tumors compared to single ablation strategies. it is noteworthy that in the present study, very low laser intensity (625 mw / cm) was required to achieve fractionated photothermal ablation compared to previous reports of photothermal ablation (4 w / cm).1,10 the minimal number of ablations needed to achieve complete tumor destruction is not investigated in the present study and very likely depends on many factors, such as tumor type, tumor size, tumor location, nanoparticle construct (eg, nir - absorbing component used for ablation), intratumoral distribution of nps, and concentration of mdt - nps within tumors.17 such investigations would represent the next step in advancing the concept of fractionated tumor photothermal ablation toward clinical application. foremost, if complete tumor destruction is not obtained on the initial photothermal treatment, then subsequent successive treatments can be performed using the fractionated approach. absolute dosing of nanomaterials for photothermal therapy may be minimized with the fractionated treatment paradigm. mdt - nps are unique nanomaterials that maintain their fluorescent and photothermal properties after photoactivation. in the 4t1 murine breast cancer model, it has been demonstrated mdt - nps can therefore be utilized as mediators of fractionated photothermal therapy. fractionated treatments can be guided by the ability to image mdt - nps within the tumor using fluorescence imaging techniques. the concept of image - guided, fractionated photothermal ablation is one which has the potential to expand the efficacy of photothermal therapy for clinical application while minimizing the morbidity of cancer care. this new paradigm should allow photothermal treatments to be delivered and modulated depending on tumor response to achieve the desired clinical effect.	purposephotothermal therapy is an emerging cancer treatment paradigm which involves highly localized heating and killing of tumor cells, due to the presence of nanomaterials that can strongly absorb near - infrared (nir) light. in addition to having deep penetration depths in tissue, nir light is innocuous to normal cells. little is known currently about the fate of nanomaterials post photothermal ablation and the implications thereof. the purpose of this investigation was to define the intratumoral fate of nanoparticles (nps) after photothermal therapy in vivo and characterize the use of novel multidye theranostic nps (mdt - nps) for fractionated photothermal antitumor therapy.methodsthe photothermal and fluorescent properties of mdt - nps were first characterized. to investigate the fate of nanomaterials following photothermal ablation in vivo, novel mdt - nps and a murine mammary tumor model were used. intratumoral injection of mdt - nps and real - time fluorescence imaging before and after fractionated photothermal therapy was performed to study the intratumoral fate of mdt - nps. gross tumor and histological changes were made comparing mdt - np treated and control tumor - bearing mice.the dual dye - loaded mesoporous nps (ie, mdt - nps ; circa 100 nm) retained both their nir absorbing and nir fluorescent capabilities after photoactivation. in vivo mdt - nps remained localized in the intratumoral position after photothermal ablation. with fractionated photothermal therapy , there was significant treatment effect observed macroscopically (p = 0.026) in experimental tumor - bearing mice compared to control treated tumor - bearing mice.fractionated photothermal therapy for cancer represents a new therapeutic paradigm enabled by the application of novel functional nanomaterials. mdt - nps may advance clinical treatment of cancer by enabling fractionated real - time image guided photothermal therapy.
astrocytes are the most abundant cell type in the central nervous system (cns), providing crucial support for the development and maintenance of cns function. nevertheless, astrocytes are also involved in the injury process and in a variety of neurological disorders, in which they become activated or dysfunctional (sloan and barres, 2014 ; ben haim et al ., 2015 ; khakh and sofroniew, 2015). given this dichotomy, it is important not only to define their phenotypic and functional properties, but also to identify the molecular mechanisms and develop protocols for the preparation of supportive astrocyte subtypes, particularly if these cells are to be used as beneficial transplants in the cns. these supportive astrocytes have numerous beneficial effects in the aftermath of cns injury promoting host axonal regeneration, limiting the inflammatory response, and reducing the inhibitory microenvironment of the injury site to create a permissive environment for axon growth and synaptic connectivity (hill et al ., 2004 ; bonner et al ., 2011 ; jin et al importantly, restoration of functional connectivity after spinal cord injury ( sci) has focused on promoting host axon regeneration and using neuronal relays with graft - derived neurons (bonner and steward, 2015), strategies that are facilitated by the use of supportive astrocytes (figure 1). the objective of this paper is to summarize recent findings pertaining to the properties of glial restricted precursors (grp) and grp - derived astrocytes and discuss how these findings can be translated into a therapeutic strategy that promotes connectivity and recovery following sci. glial restricted precursor (grp) transplants provide a supportive environment following spinal cord injury (sci) and can be used in combinatorial strategies to restore connectivity. the lesion border contains reactive astrocytes and a scar of chondroitin sulfated proteoglycans, while the lesion core is composed of activated microglia and infiltrating macrophages. damaged host axons retract from the site of injury and the physical, chemical, and inflammatory inhibitors of axon growth. (b) transplantation of grp creates a permissive environment that provides for: 1 ) reduction of glial scar formation, 2 ) reduced activation of microglia / macrophages, and 3 ) support for axon growth and regeneration into lesion. (c) transplantation of grp together with nrp allows for the formation of synaptic connections between regenerating sensory host axons and graft - derived neurons. in this case , grp transplants also support: 4 ) survival and differentiation of nrp into neurons and 5 ) synaptic connectivity for the formation of a neuronal relay. during cns development, astrocytes originate from a variety of progenitor cells including grp, which give rise to both astrocytes and oligodendrocytes (rao and mayer - proschel, 1997). grp, as well as neuronal restricted precursors (nrp), can be isolated from the rat spinal cord during embryonic development at day (e) 13.514 or derived in culture from multipotent neuroepithelial stem cells (cai et al ., 2002 when grp are grafted into normal adult spinal cord they show robust survival, proliferation, and migration as well as differentiation into both astrocytes and oligodendrocytes, confirming their progenitor properties ( han et al ., 2004 ; lepore and fischer, 2005). our previous studies have focused on the ability of grp and grp - derived astrocytes to support regeneration and connectivity following sci (figure 1). we and others found that grp and grp - derived astrocytes have remarkable supportive properties that include: 1 ) reduction of glial scar formation as indicated by preventing the upregulation of host glial fibrillary acid protein (gfap) and chondroitin sulfated proteoglycan (cspg) expression, 2 ) supporting axon growth and regeneration, 3 ) promoting the survival and neuronal differentiation of nrp, and 4 ) supporting structural and functional synaptic connectivity and the formation of a neuronal relay (lepore and fischer, 2005 ; bonner et al ., 2011 ; jin et al ., 2011 ; haas and fischer, 2014). in summary, transplantation of grp generates supportive astrocytes, which can be used as a therapeutic platform in strategies designed for sci repair in animal models and eventually translate into human clinical trials. grp are characterized by a refractive, short stellate morphology, the expression of the surface marker a2b5, and the neural precursor marker nestin (rao and mayer - proschel, 1997). traditionally, astrocytes have been defined by their multi - process stellate morphology and expression of gfap, which is commonly used as a marker of mature astrocytes, although other markers such as s100, aldh1l1, and glast have also been used. however, there is growing evidence for a heterogeneous population of astrocytes capable of performing a diverse array of temporally, regionally, and contextually specific functions (zhang and barres, 2010 ; khakh and sofroniew, 2015). our previous studies demonstrated that grp cultured in serum - free, defined media supplemented with basic fibroblastic growth factor (bfgf) maintained their phenotypically undifferentiated state (haas et al ., 2012). furthermore, under such conditions, these cells were capable of extensive proliferation, allowing for the expansion and cryopreservation of large, near - homogenous cell stocks. we found that in vitro differentiation of grp into astrocytes with bmp4 or cntf induced distinct morphological and phenotypic changes, with bmp4 generating a more mature phenotype and cntf an intermediate state that retained progenitor markers. the use of grp for preparation of astrocytes provides considerable advantages over isolation and culturing primary astrocytes from the cns. these advantages are underscored by comparable protocols developed for human grp and the availability of embryonic stem (es) and induced pluripotent stem (ips) cells for preparation of neural progenitors (haas and fischer, 2013 ; roybon et al . , 2013 ; palm et al ., 2015). in vitro co - culture assays are useful tools to evaluate the interactions between cell populations and elucidate the mechanisms of these interactions. such interactions can be studied by a direct co - culture model (analyzing cell - cell interactions) or an indirect model with multi - compartment slides or the use of conditioned medium (analyzing secreted factors). studies using co - cultures of astrocytes and dorsal root ganglion (drg) neurons have demonstrated the permissive properties of astrocytes with respect to their ability to support axon growth (smith et al ., 1990). importantly, in these studies, only immature astrocytes, freshly isolated from early post - natal cerebral cortices, but not mature astrocytes, expanded in culture in the presence of fetal bovine serum or those isolated from the mature cortex, were able to support axon growth (smith et al ., 1990). similar to the effects of immature astrocytes on axon growth, grp and astrocytes pre - differentiated with either bmp4 or cntf also supported axon growth of embryonic and adult rat drg neurons (haas et al ., in addition, conditioned media harvested from high - density grp cultures facilitated the growth of drg axons on an inhibitory cspg - enriched substrate and allowed them to cross onto a cspg - enriched border ( ketschek et al ., 2012). these data indicate that grp and immature astrocytes, isolated from fetal tissue or derived from grp, secrete trophic factors that support and promote axon growth even in the presence of an inhibitory environment, underscoring their potential use in sci. to test the data obtained from in vitro experiments in animal models , we examined the properties of grp using a c4 dorsal column hemisection of sci and found that ascending sensory axons, labeled with cholera toxin b, regenerated into the injury / transplant site as shown in figure 1 (hill et al . importantly, both grp and grp - derived astrocytes prepared with bmp4 or cntf showed comparable effects, creating a permissive environment for host axon growth into but not out of the injury site . the finding that grp transplants were capable of inducing only a modest regenerative response, which did not extent beyond the injury site, may be related to the limited regenerative response of axons in the cns and the permissive nature of the grp for axon growth in absence of directional guidance molecules outside the transplant . it is important to note that the regenerative capacity of the motor system, particularly that of the corticospinal tract ( cst), is poor even in the presence of grp or astrocytes (hill et al ., 2004, and unpublished data). in this setting, grp were only able to prevent the significant cst retraction that occurs in the aftermath of sci. taken together, grp and grp - derived astrocytes create an improved supportive environment for axon regeneration in both in vitro and in vivo systems. the clinical use of cellular products for transplantation requires large - scale preparation, cryopreservation, and a strict process of quality assurance to ensure the standardized properties of cellular therapeutics with minimal lot - to - lot variability. to guarantee a supply of supportive astrocytes it will be necessary to expand grp cultures while ensuring that the therapeutic properties of these cells have not changed. there have been several reports examining the phenotypic and functional properties of neural cells cultured for extensive periods of time with respect to their supportive properties. for example, multipotent neural stem cells isolated from embryonic human spinal cord and grown as neurospheres can be expanded for up to 25 passages (over 350 days) in vitro and maintain their multipotent capacity in vivo when grafted into an sci model (akesson et al ., 2007). similarly, oligodendrocyte precursor cells (opc) can be maintained as oligospheres for several months and retain their ability to differentiate into functional oligodendrocytes capable of myelinating axons of primary cortical neurons in a co - culture assay (zhu et al ., 2014). in contrast, olfactory ensheathing cells (oec), specialized glial cells with schwann cell and astrocyte - like properties, can lose their ability to remyelinate the injured cord after 6 weeks in culture (radtke et al ., 2010). these examples emphasize the importance of testing and identifying the appropriate temporal parameters and culture conditions for expansion of cells to be used in transplantation studies. in a recent study , we examined the long - term properties of grp following expansion of adherent cultures in serum - free, defined media supplemented with the bfgf mitogen (hayakawa et al . , 2015). specifically, we compared the morphological, phenotypic, and functional properties of early (1020 days in culture) and late (120140 days in culture) passage grp. our demonstrated that late passage grp were maintained in an undifferentiated state, and more importantly, showed comparable axon growth - promoting effects on adult drg axon growth in both direct co - culture and indirect conditioned medium experiments (figure 2a) (hayakawa et al ., 2015). taken together, we established an in vitro culture protocol that enables grp to maintain their progenitor characteristics and supportive properties relative to axon growth even after long - term culture. it is now important to confirm that late passage grp maintain their permissiveness in vivo and support axon growth when transplanted into animal models of sci. differences in the phenotypic and the functional properties of grp using in vitro co - culture experiments with dorsal root ganglion (drg) neurons. both early and late passage grp were characterized by high expression of a2b5 and low expression of glial fibrillary acid protein (gfap) typical of an immature state. early and late passage grp promoted comparable axon growth relative to control cultures consisting of drg neurons alone. (b) grp treated with inflammatory factors and their ability to support axon growth. grp were exposed to various concentrations of lipopolysaccharide (lps)/interferon gamma (ifn) for 24 hours and tested in co - culture experiments. grp treated with low concentrations of lps / ifn retained their axon growth supportive properties. in contrast, grp treated with high concentrations of lps / ifn showed a transient gfap expression that was followed by a loss of a2b5 expression, and displayed an attenuation of their axon growth supporting properties. traumatic injury to the spinal cord initiates an inflammatory cascade mediated by a multitude of signaling molecules in a stage - specific manner (alexander and popovich, 2009 ; bowes and yip, 2014). astrocytes respond to injury by undergoing a spectrum of morphological, biochemical, and functional alterations in a process known as reactive astrocytosis (fitch and silver, 2008 ; sofroniew, 2009). during this process, astrocytes become hypertrophic, upregulate gfap expression, and form a glial scar. the consequences of reactive astrocytosis depend on the location, severity, and temporal relationship to the injury, ranging from reversible alterations with preservation of cellular domains and tissue structure to, in severe cases, irreversible changes ing in scar formation with permanent rearrangement of tissue structure. accordingly, there is growing evidence for the presence of different types of reactive astrocytes with diverse morphological, molecular, and functional properties (bowes and yip, 2014 ; khakh and sofroniew, 2015). given the dramatic effects of inflammatory factors on the properties of mature astrocytes, we examined how the inflammatory environment may directly affect the morphological, phenotypic, and functional properties of grp using the drg co - culture system (figure 2). we used a combination of lipopolysaccharide (lps) and interferon gamma (ifn), maximizing the pro - inflammatory effects by signaling through two divergent pathways (schroder et al ., 2006). we found that grp exposed to either low or high concentrations of lps / ifn for 24 hours ed in transient gfap expression, which returned to normal levels by 72 hours after treatment cessation (hayakawa et al ., 2015). although expression of gfap was transient and normalized following withdrawal of the inflammatory factors, the expression of a2b5, a marker of grp, was significantly reduced in grp treated with high concentrations of lps / ifn. these suggest that the exposure of grp to a severe pro - inflammatory microenvironment induces progressive changes in the phenotypic characteristics of these cells in a dose dependent manner. importantly, these phenotypic alterations were mirrored by the reduced ability of grp to support axon growth after exposure to high concentrations of lps / ifn as evaluated by direct co - culture assays (figure 2b). these observations are consistent with reports demonstrating alterations in transcriptional profiles of astrocytes following lps exposure, consistent with reactive astrogliosis (zamanian et al ., 2012). they also highlight the importance of the microenvironment on the properties of transplanted cells and suggest that modulation of the injury environment may be important in creating more favorable conditions for cell transplantation. as we relate these to our previous studies, which demonstrated that grp create a permissive environment when grafted in an acute sci model (bonner et al ., 2011 2012), we need to consider: 1 ) the dynamic nature of the injury environment characterized by a complex cascade of pro - inflammatory and anti - inflammatory signaling molecules, 2 ) the diversity of cells present and/or recruited in the injury site (e.g., resident microglia and recruited macrophages) and their plasticity in response to inflammation that provides both beneficial and detrimental effects, and 3 ) the complex bi - directional interactions between grafted cells and the injured environment. while this complex environment is difficult to recreate in culture conditions, in vitro experiments serve as a model to define not only the direct interactions between cells, but also with signaling molecules, contributing to an understanding of the mechanism of the inflammatory process. the complexity of sci is underscored by changes that occur in distinct spatial and temporal patterns, variations in injury models and experimental design, and the need for combination therapy targeting multiple aspects of the injury. it is therefore important to continue research using not only in vitro systems to identify potential therapeutic candidates and evaluate their mechanism of action, but also in vivo models of sci to validate promising strategies with respect to functional recovery. our work with grp has emphasized the value of grp as a therapeutic platform in the repair of sci. the in vitro co - culture studies identified the axon growth - supporting properties of grp, the means to expand grp cultures to prepare sufficient stocks for transplantation experiments, and the direct impact of pro - inflammatory factors on the phenotypic and functional properties of these cells. subsequently, grp have been evaluated as a therapeutic strategy in animal models of sci, which confirmed the therapeutic potential of these cells and their derived astrocytes with respect to modulation of the injured environment, host axon growth, and synaptic connectivity. it is likely that grp transplants will be used as a therapeutic platform in combination with additional strategies to reconnect the injured nervous system, such as: 1 ) nrp, for relay formation, 2 ) trophic molecules, to enhance grp migration or guide axon regeneration, 3 ) neuronal modification, to enhance the intrinsic regenerative capacity of injured neurons, 4 ) anti - inflammatory agents, to limit the inflammatory microenvironment, 5 ) anti - scar agents, to reduce glial scarring in order to achieve more efficient cell survival and axonal regeneration, and 6 ) biomaterials, to create a niche capable of providing a more permissive environment for transplantation. finally, it is also important to note that the lessons and experience gained from sci studies could also be applied to the treatment of various other neurological diseases, such as amyotrophic lateral sclerosis (als), where transplantation of grp and immature astrocytes could be used to provide neuroprotection and slow progression of the neurodegenerative process (lepore et al ., 2008).	in the aftermath of spinal cord injury, glial restricted precursors (grps) and immature astrocytes offer the potential to modulate the inflammatory environment of the injured spinal cord and promote host axon regeneration. nevertheless clinical application of cellular therapy for the repair of spinal cord injury requires strict quality - assured protocols for large - scale production and preservation that necessitates long - term in vitro expansion. importantly, such processes have the potential to alter the phenotypic and functional properties and thus therapeutic potential of these cells. furthermore, clinical use of cellular therapies may be limited by the inflammatory microenvironment of the injured spinal cord, altering the phenotypic and functional properties of grafted cells. this report simulates the process of large - scale grp production and demonstrates the permissive properties of grp following long - term in vitro culture. furthermore, we defined the phenotypic and functional properties of grp in the presence of inflammatory factors, and call attention to the importance of the microenvironment of grafted cells, underscoring the importance of modulating the environment of the injured spinal cord.
the heat exchange between the human body and the thermal environment can be described in the form of the energy balance equation, an application of the first theorem of thermodynamics applied to the body's heat sources (metabolism and environmental) and the various avenues of heat loss to environment (see method box 1). among the advanced heat budget models, fanger's pmv - equation can be considered as appropriate if gagge et al.'s improvement in the description of latent heat fluxes by the introduction of pmv * is applied. together with a radiation model this approach combines the operational thermal assessment procedure klima michel-model (kmm) of the german national weather service deutscher wetterdienst (dwd) with the output parameter (descriptive term) perceived temperature, pt. pt follows the pet approach, standard effective temperature (set), or outdoor standard effective temperature (out_set) in order to achieve a descriptive term in c. pt is defined as the air temperature of a standard environment (wind calm, air temperature = mean radiant temperature, relative humidity ( rh) = 50%, metabolic rate 2.3 met = 135 wm, which means walking at 4 kmh ) that would produce the same thermal stress as the actual environment (table 1). additionally, optimal behavioural adaptation by clothing (along the scale from the thermal resistance of summer clothing ( 0.5 clo) to winter clothing (1.75 clo) (1 clo = 0.155 kmw) is considered based on iso 9920. the pt scale (table 1) refers to the ashrae - scale (iso 7730) extended to a nine - step scale. metabolic rate (activity) storage (change in heat content of the body) turbulent flux of sensible heat turbulent flux of latent heat (diffusion of water vapour) turbulent flux of latent heat (sweat evaporation) respiratory heat flux (sensible and latent) mean radiant temperature wind velocity relative to the body water vapour pressure the meteorological input variables include air temperature t a, water vapour pressure e, wind velocity v and mean radiant temperature t mrt including short and long - wave radiation fluxes, in addition to metabolic rate and clothing insulation. in eq. the appropriate meteorological variables are attached to the relevant energy fluxes in w / m. the physiological (internal) variables, such as the temperature of the core or the skin, the sweat rate, and the skin wetness, which all interact with the environmental heat conditions, are not mentioned here. an overview on the basics in thermo - physiology and heat exchange modelling with respect to adaptation is given e.g. in. perceived temperature, predicted mean vote (according to ), and corresponding thermal stress (initial conditions without acclimatisation ; negative values are heat load) the pt procedure incorporates the acclimatisation approach health related assessment of the thermal environment (herate). herate is a conceptual model of short - term acclimatisation based on findings in adaptation studies. the procedure modifies the absolute pt thresholds of table 1 by superimposing the (relative) historic experience of the population in terms of pt of the previous weeks. 2 shows how the actual thresholds for the different thermal stress categories change from the initial values of table 1. this procedure has the advantage that the index can be used without modification in different climate regions and during different times of the year without the need to artificially define seasons and calibrate it to a particular locale. example of daily values of perceived temperature at 12 utc (black line) for the weather station lisbon (portugal) from march to september 2003. compared to table 1 the coloured lines indicate the variable thresholds for the different thermal stress categories specifying acclimatisation. for global bioclimate maps however, the necessary meteorological input data for the global scale can be taken either from global gcms or from reanalyses of numerical weather prediction (nwp) models (e.g. european centre for medium - range weather forecasts ( ecmwf), reading / uk, or national centers for environmental prediction (ncep), camp springs / usa ). in this investigation we use two runs of the global gcm echam4 at the relatively high resolution t106, about 100 km grid - point distance in middle latitudes (see method box 2). due to restricted computer power, only two time slices with a length of 10 years each are available in the high - resolution version of the gcm. in the first experiment (control run) the concentration of greenhouse gases is set constant at the observed level 19711980 (recent climate conditions). in the second run the concentration of greenhouse gases is taken from the ipcc - scenario is92a business - as - usual for the period 20412050. this scenario is between the two middle range scenarios a1b and a2 used in the report of the intergovernmental panel on climate change (ipcc). using the model output data of echam4/t106, the values of pt were calculated for the different time periods for each land - grid point and four universal times (utc) at 00.00, 06.00, 12.00 and 18.00. in order to obtain comparable pt values, the predominant effect of the daily thermal variation was considered by recalculating corrected pt values for 12 mean local time (mlt) for all grid points by a nonlinear interpolation procedure centred around the four neighbouring pt values at the given utc times. for the given six - hour intervals the maximum mean error of this procedure is in the order of 1 k (= 1c), which was tested with observational data of a couple of european weather stations with a time resolution of one hour. the global gcm echam4 applies the relatively high resolution t106, about 100 km grid - point distance in middle latitudes. in both experiments (time slices), the surface of the earth is represented by 320160 grid points which means a distance of about 1.1 in zonal and meridional direction. the gcm - data are available at fixed times 00.00, 06.00, 12.00 and 18.00 universal time (utc). the meteorological variables required for the assessment are air temperature t a, dew point t d (to derive water vapour pressure) and wind velocity v, which are directly used to calculate the sensible and latent heat flux. in addition to these, for the parameterisation of the long - wave and short - wave radiant fluxes, cloudiness information such as total cloud cover n and cloud cover in the different tropospheric levels (low, middle and high) are used to calculate mean radiant temperature tmrt. the maps of pt values are related to people staying outdoors at noon (12 mlt). because ptmax usually occurs one or two hours later , the pt value at 12 mlt can be considered as a mean value over a certain time period starting some time before 12 mlt until some time after the same pt value as at 12 mlt occurs once more in the afternoon. from these 12 mlt pt - time - series at every land - grid point the mean annual frequency (number of days p.a .) of comfort conditions (no thermal stress) or different intensities of thermal stress, respectively, has been derived for the two echam4 time slices 19711980 and 20412050, taking acclimatisation into account according to herate. the actual pt value for a certain thermal stress level at a given point and a given date always depends on the previous thermal conditions. the difference between the time periods and can be interpreted as the change in the bioclimate. the heat exchange between the human body and the thermal environment can be described in the form of the energy balance equation, an application of the first theorem of thermodynamics applied to the body's heat sources (metabolism and environmental) and the various avenues of heat loss to environment (see method box 1). among the advanced heat budget models, fanger's pmv - equation can be considered as appropriate if gagge et al.'s improvement in the description of latent heat fluxes by the introduction of pmv * is applied. together with a radiation model this approach combines the operational thermal assessment procedure klima michel-model (kmm) of the german national weather service deutscher wetterdienst (dwd) with the output parameter (descriptive term) perceived temperature, pt. pt follows the pet approach, standard effective temperature (set), or outdoor standard effective temperature (out_set) in order to achieve a descriptive term in c. pt is defined as the air temperature of a standard environment (wind calm, air temperature = mean radiant temperature, relative humidity ( rh) = 50%, metabolic rate 2.3 met = 135 wm, which means walking at 4 kmh ) that would produce the same thermal stress as the actual environment (table 1). additionally, optimal behavioural adaptation by clothing (along the scale from the thermal resistance of summer clothing ( 0.5 clo) to winter clothing (1.75 clo) (1 clo = 0.155 kmw) is considered based on iso 9920. the pt scale (table 1) refers to the ashrae - scale (iso 7730) extended to a nine - step scale. metabolic rate (activity) storage (change in heat content of the body) turbulent flux of sensible heat turbulent flux of latent heat (diffusion of water vapour) turbulent flux of latent heat (sweat evaporation) respiratory heat flux (sensible and latent) mean radiant temperature wind velocity relative to the body water vapour pressure the meteorological input variables include air temperature t a, water vapour pressure e, wind velocity v and mean radiant temperature t mrt including short and long - wave radiation fluxes, in addition to metabolic rate and clothing insulation. in eq. the appropriate meteorological variables are attached to the relevant energy fluxes in w / m. the physiological (internal) variables, such as the temperature of the core or the skin, the sweat rate, and the skin wetness, which all interact with the environmental heat conditions, are not mentioned here. an overview on the basics in thermo - physiology and heat exchange modelling with respect to adaptation is given e.g. in. perceived temperature, predicted mean vote (according to ), and corresponding thermal stress (initial conditions without acclimatisation ; negative values are heat load) the pt procedure incorporates the acclimatisation approach health related assessment of the thermal environment (herate). herate is a conceptual model of short - term acclimatisation based on findings in adaptation studies. the procedure modifies the absolute pt thresholds of table 1 by superimposing the (relative) historic experience of the population in terms of pt of the previous weeks. 2 shows how the actual thresholds for the different thermal stress categories change from the initial values of table 1. this procedure has the advantage that the index can be used without modification in different climate regions and during different times of the year without the need to artificially define seasons and calibrate it to a particular locale. example of daily values of perceived temperature at 12 utc (black line) for the weather station lisbon (portugal) from march to september 2003. compared to table 1 the coloured lines indicate the variable thresholds for the different thermal stress categories specifying acclimatisation. however, the necessary meteorological input data for the global scale can be taken either from global gcms or from reanalyses of numerical weather prediction (nwp) models (e.g. european centre for medium - range weather forecasts ( ecmwf), reading / uk, or national centers for environmental prediction (ncep), camp springs / usa ). in this investigation we use two runs of the global gcm echam4 at the relatively high resolution t106, about 100 km grid - point distance in middle latitudes (see method box 2). due to restricted computer power, only two time slices with a length of 10 years each are available in the high - resolution version of the gcm. in the first experiment (control run) the concentration of greenhouse gases is set constant at the observed level 19711980 (recent climate conditions). in the second run the concentration of greenhouse gases is taken from the ipcc - scenario is92a business - as - usual for the period 20412050. this scenario is between the two middle range scenarios a1b and a2 used in the report of the intergovernmental panel on climate change (ipcc). using the model output data of echam4/t106, the values of pt were calculated for the different time periods for each land - grid point and four universal times (utc) at 00.00, 06.00, 12.00 and 18.00. in order to obtain comparable pt values, the predominant effect of the daily thermal variation was considered by recalculating corrected pt values for 12 mean local time (mlt) for all grid points by a nonlinear interpolation procedure centred around the four neighbouring pt values at the given utc times. for the given six - hour intervals the maximum mean error of this procedure is in the order of 1 k (= 1c), which was tested with observational data of a couple of european weather stations with a time resolution of one hour. the global gcm echam4 applies the relatively high resolution t106, about 100 km grid - point distance in middle latitudes. in both experiments (time slices), the surface of the earth is represented by 320160 grid points which means a distance of about 1.1 in zonal and meridional direction. the gcm - data are available at fixed times 00.00, 06.00, 12.00 and 18.00 universal time (utc). the meteorological variables required for the assessment are air temperature t a, dew point t d (to derive water vapour pressure) and wind velocity v, which are directly used to calculate the sensible and latent heat flux. in addition to these, for the parameterisation of the long - wave and short - wave radiant fluxes, cloudiness information such as total cloud cover n and cloud cover in the different tropospheric levels (low, middle and high) are used to calculate mean radiant temperature tmrt. the maps of pt values are related to people staying outdoors at noon (12 mlt). because ptmax usually occurs one or two hours later , the pt value at 12 mlt can be considered as a mean value over a certain time period starting some time before 12 mlt until some time after the same pt value as at 12 mlt occurs once more in the afternoon. from these 12 mlt pt - time - series at every land - grid point the mean annual frequency (number of days p.a .) of comfort conditions (no thermal stress) or different intensities of thermal stress, respectively, has been derived for the two echam4 time slices 19711980 and 20412050, taking acclimatisation into account according to herate. the actual pt value for a certain thermal stress level at a given point and a given date always depends on the previous thermal conditions. the difference between the time periods and can be interpreted as the change in the bioclimate. the map of the frequency distribution of thermal comfort (i.e. no thermal stress) 19711980 shows that the mid - latitude regions are most comfortable, in particular in the maritime affected areas (fig . this is true for western europe, new zealand, the southern parts of australia, chile and argentina as well as a narrow coast strip in the west of north - america where 300 days with thermal comfort at 12 mlt can be taken as typical . similar conditions can be found in higher elevations of the andes and in the area of the foothills of the himalayas . with increasing continental influence in mid - latitudes or increasing subtropical influence, the number of days with comfort conditions decreases considerably . in the lowlands of the humid tropics thermal comfort this is also true for the ice shield regions of antarctica and greenland due to the limit to maximum clothing insulation of 1.75 clo which is used here . mean annual frequency of comfortable conditions ( no thermal stress) at 12 mlt (= noon) taking acclimatisation into account based on echam4/t106-data 19711980. the general relationship of heat load conditions to latitude (solar climate) is evident (fig . heat load is the predominant thermal state in the tropics throughout the year . with increasing latitude the heat load probability declines, but even in moderate climates a considerable number of days with heat load ( a d) mean annual frequency of exceeding the threshold for (a) heat load, (b) moderate heat load, (c) strong heat load and (d) extreme heat load at 12 mlt taking acclimatisation into account, based on echam4/t106-data 19711980. from a health point of view, higher heat load intensities become more relevant. 4b ) is of course smaller and now almost covers those regions of the world known for tropical or subtropical climate. in the equator region it is interesting, for example, that saudi arabia shows the most days with extreme heat load (> 170 days) while the threshold for at least strong heat load is more frequently exceeded in the amazon basin and in indonesia (> 300 days). the zonal variation in the thermal conditions is also found in the distribution of cold stress (in spite of winter clothing with 1.75 clo! ; fig . while almost every day cold stress occurs in antarctica, followed by less than one - third of the time span of a year over the southern tip of south - america ( tierra del fuego), the northern hemisphere shows a more differentiated pattern due to superimposed topography and position relative to the sea. the frequency of cold stress in the tropical and subtropical regions is zero or close to that. when the thresholds for cold stress are tightened in the given definitions (fig . extreme cold stress can only be found some distance away from the coast over the ice shields of antarctica almost every day ( fig . 5d). in central greenland, the most extreme region in the northern hemisphere, with respect to health consequences it is interesting to note that the regions showing at least moderate cold stress (fig . ( a d) mean annual frequency of exceeding the threshold for (a) cold stress, (b) moderate cold stress, (c) strong cold stress and (d) extreme cold stress at 12 mlt taking acclimatisation into account, based on echam4/t106-data 19711980. the spatial distribution of the thermal conditions across europe looks very similar to maps based on data from 918 european weather stations 1971/2000. the echam4/t106 simulation of the climate in the period 20412050 (future) uses the ipcc is92a business - as - usual scenario. difference of the mean annual frequency of comfortable conditions (no thermal stress) at 12 mlt between future and recent climate taking acclimatisation into account, based on echam4/t106-data (20412050 and 19711980). (a d) difference of the mean annual frequency of exceeding the threshold for (a) heat load, (b) moderate heat load, (c) strong heat load and (d) extreme heat load at 12 mlt taking acclimatisation into account, based on echam4/t106-data (20412050 and 19711980). (a d) difference of the mean annual frequency of exceeding the threshold for (a) cold stress, (b) moderate cold stress, (c) strong cold stress and (d) extreme cold stress at 12 mlt taking acclimatisation into account, based on echam4/t106-data (20412050 and 19711980). both the humid tropics and antarctica are characterised by already lacking comfort conditions (permanent heat load or cold stress conditions, respectively) (fig . 6), and these will remain. the most pronounced decrease in comfort will happen in the subtropics up to the middle latitudes (moderate climate). an increase in comfortable conditions is mainly limited to cold areas with low population density, maybe with the exception of southern scandinavia, parts of russia, china, the northwest of the usa, and british columbia in canada. the distribution of the changes of the annual frequencies of heat load (fig . 7a 7b) as these are already exceeded. there is a considerable increase in heat load in the subtropics and higher latitudes. while the affected areas are becoming smaller with tightening thresholds although the characteristics are different, almost all densely populated areas are affected by the increase of adverse thermal conditions due to climate change. as is nowadays the case , no cold stress will occur in the tropics and subtropics also in a future climate (fig . d). the most densely populated areas will never be affected by extreme or even strong cold stress. in moderate climates a considerable reduction in the frequency of cold stress the at least moderate cold stress situation in the area of the great lake district in north - america, scandinavia and russia will be improved (fig . 8b) while a significant decrease in stronger cold stress will occur in practically non - populated regions (fig . 8c and d). the map of the frequency distribution of thermal comfort (i.e. no thermal stress) 19711980 shows that the mid - latitude regions are most comfortable, in particular in the maritime affected areas (fig . this is true for western europe, new zealand, the southern parts of australia, chile and argentina as well as a narrow coast strip in the west of north - america where 300 days with thermal comfort at 12 mlt can be taken as typical . similar conditions can be found in higher elevations of the andes and in the area of the foothills of the himalayas . with increasing continental influence in mid - latitudes or increasing subtropical influence, the number of days with comfort conditions decreases considerably . in the lowlands of the humid tropics thermal comfort this is also true for the ice shield regions of antarctica and greenland due to the limit to maximum clothing insulation of 1.75 clo which is used here . mean annual frequency of comfortable conditions ( no thermal stress) at 12 mlt (= noon) taking acclimatisation into account based on echam4/t106-data 19711980. the general relationship of heat load conditions to latitude (solar climate) is evident (fig . heat load is the predominant thermal state in the tropics throughout the year . with increasing latitude the heat load probability declines, but even in moderate climates a considerable number of days with heat load ( a d) mean annual frequency of exceeding the threshold for (a) heat load, (b) moderate heat load, (c) strong heat load and (d) extreme heat load at 12 mlt taking acclimatisation into account, based on echam4/t106-data 19711980. from a health point of view, higher heat load intensities become more relevant. 4b ) is of course smaller and now almost covers those regions of the world known for tropical or subtropical climate. in the equator region it is interesting, for example, that saudi arabia shows the most days with extreme heat load (> 170 days) while the threshold for at least strong heat load is more frequently exceeded in the amazon basin and in indonesia (> 300 days). the zonal variation in the thermal conditions is also found in the distribution of cold stress (in spite of winter clothing with 1.75 clo! ; fig . while almost every day cold stress occurs in antarctica, followed by less than one - third of the time span of a year over the southern tip of south - america ( tierra del fuego), the northern hemisphere shows a more differentiated pattern due to superimposed topography and position relative to the sea. the frequency of cold stress in the tropical and subtropical regions is zero or close to that. when the thresholds for cold stress are tightened in the given definitions (fig . extreme cold stress can only be found some distance away from the coast over the ice shields of antarctica almost every day ( fig . 5d). in central greenland, the most extreme region in the northern hemisphere, with respect to health consequences it is interesting to note that the regions showing at least moderate cold stress (fig . ( a d) mean annual frequency of exceeding the threshold for (a) cold stress, (b) moderate cold stress, (c) strong cold stress and (d) extreme cold stress at 12 mlt taking acclimatisation into account, based on echam4/t106-data 19711980. the spatial distribution of the thermal conditions across europe looks very similar to maps based on data from 918 european weather stations 1971/2000. the echam4/t106 simulation of the climate in the period 20412050 (future) uses the ipcc is92a business - as - usual scenario. figs. difference of the mean annual frequency of comfortable conditions (no thermal stress) at 12 mlt between future and recent climate taking acclimatisation into account, based on echam4/t106-data (20412050 and 19711980). (a d) difference of the mean annual frequency of exceeding the threshold for (a) heat load, (b) moderate heat load, (c) strong heat load and (d) extreme heat load at 12 mlt taking acclimatisation into account, based on echam4/t106-data (20412050 and 19711980). (a d) difference of the mean annual frequency of exceeding the threshold for (a) cold stress, (b) moderate cold stress, (c) strong cold stress and (d) extreme cold stress at 12 mlt taking acclimatisation into account, based on echam4/t106-data (20412050 and 19711980). both the humid tropics and antarctica are characterised by already lacking comfort conditions (permanent heat load or cold stress conditions, respectively) (fig . 6), and these will remain. the most pronounced decrease in comfort will happen in the subtropics up to the middle latitudes (moderate climate). an increase in comfortable conditions is mainly limited to cold areas with low population density, maybe with the exception of southern scandinavia, parts of russia, china, the northwest of the usa, and british columbia in canada. the distribution of the changes of the annual frequencies of heat load (fig . 7a there is a considerable increase in heat load in the subtropics and higher latitudes . while the affected areas are becoming smaller with tightening thresholds although the characteristics are different, almost all densely populated areas are affected by the increase of adverse thermal conditions due to climate change . as is nowadays the case , no cold stress will occur in the tropics and subtropics also in a future climate ( fig . d). the most densely populated areas will never be affected by extreme or even strong cold stress. in moderate climates a considerable reduction in the frequency of cold stress the at least moderate cold stress situation in the area of the great lake district in north - america, scandinavia and russia will be improved (fig . 8b) while a significant decrease in stronger cold stress will occur in practically non - populated regions (fig . 8c and d). climate modelling provides global - wide time series of grid data for every day at several (here four) hourly time points, and here we used echam4 time - slice modelling assuming the business - as - usual green house gas scenario. in order to estimate impacts of climate or climate change on human health and well - being, a specific physiologically relevant assessment of the climate data this assessment must be based on heat exchange theory taking all mechanism of heat exchange into account, which in heat budget models such as the pt procedure. such models have the ability to correctly assess the climatological variables: air temperature, mean radiant temperature, wind velocity and water vapour pressure. it is likely that our assessment procedure can be applied across the world because thermo - physiology functions are fundamentally the same in all populations. at the population level (neglecting age, gender and inter - individual differences) the susceptibility to thermal stress is mainly controlled by physiological and behavioural adaptation. the psychological aspects of how people assess and prefer climate sensation (see e.g. 42, 43) are not subject of this study. thermal comfort in our study stands for no stress which means no physiological strain. societies have always adapted their cultures to meet the climate requirements. clothing (see e.g. adaptive model by 45, 46), building design, food and drinking habits, working hours (siesta), avoiding outdoors activities during the hottest time of the day, lowering metabolic rate, etc. if acclimatisation were perfect no adverse health effects of climate would be expected, but numerous publications report health impacts of climate indicating that societies are not able to adapt completely. acclimatisation (= adaptation to climate) is here considered firstly by assuming reasonable behaviour of the target group general population with respect to clothing. the range of clo - values between 0.5 and 1.75 refers to moderate climate experience. although there is some evidence that in cold winter climates the general population is not used to wearing more protective clothing thus limiting obviously their time for staying outdoors, this is certainly not true for people (not considered here) with particular activities such as soldiers, rangers, hunters, skiers who are able to avoid any cold stress by appropriate special clothing. typical inuit clothing (4.0 clo) would actually produce approximate comfort under the predominant conditions in extreme cold winter climates. shorts instead of jeans as often used by people in the tropics reduce the clo - value to 0.4 which would slightly reduce heat load. secondly, the acclimatisation approach herate adjusts the initial thermal thresholds automatically to the prevailing local climate. in the warm humid tropics and in the hot subtropics this often in an increase of the thresholds for the different heat load intensities of 68c. however, possible long - term (over years and decades) acclimatisation has not been considered due to the lack of a quantitative approach. additionally the assessment procedure focuses on the population level which means people walking outdoors with a given metabolic rate of 2.3 met (135 wm). reducing the met activity could be assumed under heat load conditions as an adaptation measure of the population if no specific work intensities are required. on the other hand there are many outdoor working conditions requiring much higher metabolic rates. in tropical and sub - tropical climates the obstruction of heat release from the body can very quickly produce extreme heat load conditions in a working individual which significantly differ from the assessments in our analysis for an assumed general public. with the help of pt and herate, the current and predicted thermal environment is described in health - related frequencies of exceeding locally adapted thresholds based on daily values at a fixed time (noon = 12 mlt) which is representative for a few hours around noon. this makes a big difference to the usual consideration of climate and climate change based on monthly or annual mean values of air temperature. in terms of both comfort and the absence of intense thermal stress, privileged while comfortable or only slight heat load conditions only rarely occur in the humid tropics. the distribution of heat load follows the solar climate to a great extent, while it is modified by maritime or continental influences and altitude. in the tropics and subtropics a huge number of human beings are affected by heat stress, particularly in asia and africa. in tropical and sub - tropical areas, where extreme heat load is already common extreme with significant impacts on health and well - being of populations living in these areas. of course these societies apply some behavioural adaptation measures but the dramatically increased and from a greenhouse gas point of view contra productive use of air - conditioning systems in these countries by those who have the financial capabilities indicates that they are not satisfied with the existing climate. the problem increases because in rapidly growing cities traditional buildings which relied on local experience in climate - related building design, is no longer realised. in addition, the urban heat island effect in large cities is already greater than the predicted climate change until the end of this century, and the public health risks are significant. when considering the fact that (a) almost the entire population of the world lives in areas indicated by yellow or red colours in the presentation of the changes (see fig . 7c) and (b) already exceeding the threshold for moderate heat load would increase mortality (at least based on studies in moderate climates), the public health issue of this aspect of climate change becomes evident. this would also be true if, due to an underestimation of the effect of acclimatisation in the applied procedure, the thresholds for the different heat load intensities were actually somewhat higher. the predominant part of the world's population will belong to the climate losers, being faced with more frequent and more intense adverse thermal conditions, while few countries or areas will belong to the climate winners, a view on the problem that first was introduced by auliciems in 1994. the described assessment procedure can also be applied to regional climate models (see e.g. 48) are nested into global models, thus providing bioclimatological conditions with higher resolution (down - scaling). further improvements of these assessments can be expected by applying the almost finalised universal thermal climate index (utci ; ( cost 730) ), which is based on human response - related thermo - physiological modelling with the help of a multi - segmental, multi - layered representation of the human body, to the new generation of climate simulation models including down - scaling. the application of the thermo - physiological assessment procedure pt to climate data broadens the usual consideration of climate maps presenting only single climate data such as air temperature. the approach in a spatial assessment of thermal stress categories which relate climate to the health and well - being of humans. this provides specific problem - oriented information to the climate and climate change, and human health relationships in general and for the estimation of the vulnerability of different populations. optimal conditions in terms of no or at most slight thermal stress differ significantly between the individual continents. privileged while particularly africa and australia suffer from heat load with highest frequency at the extremes. asia and both the americas lie approximately in between. in the predicted future climate, based on the echam4 simulations, the most pronounced changes will occur at the extreme heat load category which will then become the most frequent condition for america as it is already now for africa and australia. favourable conditions will decrease basically all over the world. a better understanding of acclimatisation as the adaptation to climate and climate change in different time scales remains an important research issue. the authors have not received any funding or benefits from industry to conduct this study.	the close relationship between human health, performance, well - being and the thermal environment is obvious. nevertheless, most studies of climate and climate change impacts show amazing shortcomings in the assessment of the environment. populations living in different climates have different susceptibilities, due to socio - economic reasons, and different customary behavioural adaptations. the global distribution of risks of hazardous thermal exposure has not been analysed before.objectiveto produce maps of the baseline and future bioclimate that allows a direct comparison of the differences in the vulnerability of populations to thermal stress across the world.designthe required climatological data fields are obtained from climate simulations with the global general circulation model echam4 in t106-resolution. for the thermo - physiologically relevant assessment of these climate data a complete heat budget model of the human being, the perceived temperature procedure has been applied which already comprises adaptation by clothing to a certain degree. short - term physiological acclimatisation is considered via health related assessment of the thermal environment.the global maps 19711980 (control run, assumed as baseline climate) show a pattern of thermal stress intensities as frequencies of heat. the heat load for people living in warm humid climates is the highest. climate change will lead to clear differences in health - related thermal stress between baseline climate and the future bioclimate 20412050 based on the business - as - usual greenhouse gas scenario is92a. the majority of the world's population will be faced with more frequent and more intense heat strain in spite of an assumed level of acclimatisation. further adaptation measures are crucial in order to reduce the vulnerability of the populations.this bioclimatology analysis provides a tool for various questions in climate and climate change impact research. considerations of regional or local scale require climate simulations with higher resolution. as adaptation is the key term in understanding the role of climate / climate change for human health, performance and well - being, further research in this field is crucial.
the palmaris longus muscle is one of the flexor muscles of the forearm that originates from the medial epicondyle of the humerus, terminates on the flexor retinaculum and contributes to the palmar fascia. although in the upper limbs its function is considered insignificant, in the event of tendon grafting, it is considerably important. in the anatomical and surgical texts, occurrence of absence of the said muscle is reported to be about 15%. but this prevalence level may not be an indicator for all the world populations. in most races, variation of the occurrence of absence of the palmaris longus muscle has been reported. reported the prevalence of absence of the palmaris longus muscle (30.7%) in the south of iran. in another study, the prevalence of palmaris longus muscle agenesis in tehran (the capital of iran) was reported as 22.8% in medical students, and in an additional study this prevalence was reported as 21.0% in patients referred to one health center. however, no report has been presented in relation to the occurrence of absence of palmaris longus muscle in north of iran. therefore, considering the importance of providing anatomic data that could serve to enhance clinical applications, the aim of this study is to determine the prevalence of agenesis of the palmaris longus muscle and its association with gender in the guilan population in the north of iran. this study was performed on 1,124 upper limbs in 562 consecutive outpatients of the orthopedic ward of poursina hospital in rasht. the patients, who had a record of surgery, abnormality and inborn deformation of the upper limbs, and also children under the age of 10, were excluded. for examining the palmaris longus muscle tendon, standard clinical methods were used. after explaining the study aim to the patients, they were asked to lay their thumb against their little finger and at the same time flex their wrist (schaeffer s test). in this state, the palmaris longus muscle tendon in the front of the wrist will be raised under the skin (figure 1a). if in this test the tendon is not observable or touchable, other tests such as pushpakumar s test (figure 1b), thomson s test (figure 1c) and mishra s test ii (figure 1d) were used for confirmation of the muscle absence. with these methods, existence or absence of the palmaris longus muscle tendon in the two upper limbs were registered. in this study, occurrence of absence of the palmaris longus muscle tendon in men and women, and symmetry of the right and left side were considered. the categorical variables were compared using the chi - squared test. a p value of < 0.05 was considered statistically significant. informed consent was obtained from each patient included in the study, and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki. the age of the patients ranged from 15 to 70 year old; the average age being 35 years. the overall prevalence of palmaris longus agenesis, according to gender and limb , is summarized in table 1. (%). out of a total of 562 patients examined, at least one palmaris longus tendon was present in 540 patients (96.1%), and both the tendons were present in 488 (86.8%). out of those with unilateral agenesis, 29 (5.2%) had left - sided agenesis and 23 (4.1%) had right - sided agenesis. hence, agenesis was seen more commonly on the left side than on the right, and the difference was statistically significant (p < 0.001). on the whole, female subjects had a prevalence of agenesis of the palmaris longus tendon (unilateral and bilateral combined) of 36 out of 281 (12.8%), while in male subjects this prevalence was 38 out of 281 (13.5%). although the proportions of patients with unilateral and bilateral agenesis were comparable (22 patients or 3.9% had bilateral agenesis and 52 patients or 9.3% had unilateral agenesis, p=0.759), the pattern of agenesis of the palmaris longus was different in the two genders. in male subjects, 26 (68.4%) were unilateral, compared to 12 (31.6%) who were bilateral (p = 0.076). female subjects had unilateral absence of the palmaris longus more commonly; 26 (72.2%) were unilateral, compared to 10 (27.8%) who were bilateral (p = 0.040). this implies that there is a 31.6% probability of a male subject with one - sided agenesis to have agenesis on the opposite side as well. in contrast, if the palmaris longus tendon in a female subject is absent on one side, there is a 27.8% chance that it will also be absent on the other side. the palmaris longus is considered one of the superficial flexor muscles of the forearm and one of the most variable muscles of the body. most researchers consider this muscle equivalent to the plantaris muscle in the lower limbs. the palmaris longus muscle is well developed in the animal species, which tolerates more weight on the upper limbs than other species. but in the human species, in which the role of the upper limbs in toleration of body weight has been decreased, the palmaris longus muscle is less developed and uncompleted. despite the fact that this muscle exists in humans, its role in hand function is decreased, and in most cases of limb surgery it is used as a tendon graft. various studies have shown that absence of the palmaris longus muscle will create no disorder in hand function; therefore, in necessary cases, the tendon of this muscle is used in tendon grafting without fear that patients will have decreased ability to clench their fist or perform other hand functions. the ability to estimate palmaris longus muscle absence in different population groups is an added value to most surgeons around the globe, wherever they are increasingly dealing with patients of different ethnic s. compared with the other muscles, because of its unique specifications including length and raised nature, the palmaris longus muscle tendon is well observable and available and has been studied by researchers more frequently than others. absence of the palmaris longus muscle has been the subject of studies performed by many researchers in living and deceased people, and the occurrence percentage was variable in different populations and races. in the current research, absence of the palmaris longus muscle, both unilaterally and bilaterally, in the guilan population in the north of iran has been studied. in addition, the prevalence of absence of the muscle was compared between the two genders. in this study, the overall prevalence of absence of the palmaris longus muscle was 13.2%, which was lower than three studies performed by karimi et al. (13.2% vs. 30.7%, p < 0.001), ashouri et al. (13.2% vs. 22.8%, p < 0.001) and kamrani et al. (13.2% vs. 21%, p = 0.002) in iran. compared to other studies, the prevalence of palmaris longus absence in serbia (22.4% and 37.5% in two studies ), america (25.0%, ), turkey (26.6%, ), india (17.2% to 28.0% in three studies in indian population ) and bahrain (36.4%, ) was higher than our . other studies showed a lower value such as 1.5% in zimbabwe, 4.1% in korea, 5.5% in japanese subjects, 6% in chinese subjects, 7.1% in native american subjects, 11.3% in malaysia and 11.5% in a south african population. therefore, it can be said that the study of one population can not be considered for another population, and absence of the palmaris longus muscle is dependent on race. most researchers reported occurrence of palmaris longus absence in women more than in men and in the left side more than in the right side. but our findings showed absence of the palmaris longus muscle in men was not significantly higher than in women (51.4% vs. 48.6%, p > 0.05), and in the left side was not - significantly higher than in the right side (5.2% vs. 4.1%, p > 0.05). also, there was not a significant difference between the two genders in most previous studies, which is concordant with our findings (table 1). one limitation of our study was that the presence of a palmaris longus muscle (left, right and bilateral existence) was determined by clinical exam, which can be examiner dependent, rather than cadaveric dissection. to mitigate this issue	: the palmaris longus is a degenerating weak flexor muscle in the anterior of the forearm. many techniques for clinically determining the presence of the palmaris longus have been described. ethnic variations in the prevalence of the absence of the palmaris longus are well known.objectives:this study considered the prevalence of absence of the palmaris longus muscle tendon in the north of iran.patients and methods: the presence of the palmaris longus was clinically determined in 562 men and women from the guilan population, using the standard technique (schaeffer s test). in subjects with an absent palmaris longus , three other tests (thompson, pushpakumar and mishra tests) were performed to confirm the absence.:the overall prevalence of right, left, bilateral and total absence of the palmaris longus were 4.1%, 5.2%, 3.9% and 13.2%, respectively. there was no significant difference in its absence with regard to the body side or gender (p > 0.05).: this study demonstrated that the presence of the palmaris longus muscle tendon in the guilan population was considerably higher than the absence of the palmaris longus tendon. the overall prevalence of right, left, bilateral and total absence of the palmaris longus was not significantly different between men and women. the prevalence of the left - absent palmaris longus was more common in the present study.
candida albicans is a major commensal fungus residing in the mucosal surfaces of our bodies. it is mainly found in the gastrointestinal tract, skin and genital tract. when the mucosal barrier is disturbed, c. albicans penetrates into the epithelium and causes systemic infection. conditions associated with immunocomprimise, particularly neutropenia, are a major risk factor for invasive candidiasis. this deep - seated infection occurs, as hyphae spread to internal organs, among which the kidney is the major target. the mortality rate of patients with systemic candidiasis exceeds 30~40% even when potent antifungal agents are administered. like many other pathogens, the host defense against invasive c. albicans infections is a function of immune detection and elimination of the pathogen. this defense strategy is referred to as resistance. however, mortality of the host following c. albicans infection may largely from immunopathology, in which the immune response against c. albicans inflicts tissue damage. mice are known to tolerate high fungal burdens, even though uncontrolled fungal proliferation is lethal. thus, it is important to understand defense mechanisms that reduce host susceptibility to tissue damage caused by fungal infection or related immunopathologies, but that do not affect the fungal burden. , we discuss the role of il-33 in host resistance and tolerance to systemic c. albicans infections. il-33 is a member of the il-1 family of cytokines that delivers its signaling through st2 receptors. endothelial cells and epithelial cells rapidly produce il-33 in response to a variety of intrinsic and environmental insults. however, there are many sources of il-33, and the kinetics of il-33 production vary across types of cells and stimuli. interestingly, the il-33 receptor, st2, is expressed on virtually all types of immune cells and numerous types of nonhematopoietic cells, suggesting that it exhibits a functional diversity. il-33 function is context - dependent and can have opposing effects (e.g., inflammatory or anti - inflammatory) on an immune response. for example, il-33 initiates type 2 immune responses through secretion of il-4, il-5 and/or il-13 by th2 cells and type 2 innate lymphoid cells (ilc2s). il-33 is also associated with autoimmune and inflammatory diseases mediated by th1 and th17 cells. in addition, anticancer and antiviral effects of il-33 require cd8 t cells, nk cells and ilc2s. recent studies have identified anti - inflammation functions of il-33: it plays a critical role in regulatory t cell expansion in acute colitis, asthma, and obesity. one effector molecule produced by regulatory t cells is amphiregulin, which plays a role in tissue repair without influencing antiviral immune responses. thus, the seemingly anti - inflammatory effect of il-33 is in fact a of enhanced tolerance mechanisms. il-33 also may exert its anti - inflammatory effects by suppressing th1 or th17-mediated immune responses. this outcome can be particularly important in inflammatory or infectious diseases in which th1 or th17 cells mediate immunopathology. recent studies have begun to reveal the molecular mechanisms behind these observations. specifically, ifn and il-27 have been shown to counteract the activity of ilc2s mediated by il-33. the participation of il-33 in host resistance to c. albicans infection was initially demonstrated in a fungal peritonitis model. il-33 priming before infection with c. albicans ed in effective fungal clearance and subsequently decreased mortality caused by fungal peritonitis. this priming by il-33 acts on multiple steps of an anti - candida neutrophil response. first, il-33 priming enhances recruitment of neutrophils to the site of infection through two mechanisms: 1 ) it increases production of neutrophil - chemotactic cxcr2 ligands (cxcl1 and cxcl2) by peritoneal macrophages; and 2 ) it suppresses the internalization of surface cxcr2 in neutrophils. il-33 does so by inhibiting tlr2-induced grk2 expression that is required for cxcr2 internalization. the increased phagocytosis of neutrophils occurs because of specific upregulation of the complement receptor cr3 through the synergistic activation of the tlr2 and dectin-1 signaling pathways by il-33. increase in production of reactive oxygen species (ros) is correlated with yeast killing of il-33-primed neutrophils. it is not known why il-33 specifically promotes clearance of opsonized c. albicans, but this occurred under physiological conditions (our unpublished data). mononuclear phagocytes, including resident macrophages and inflammatory monocytes, also play a critical role in candida clearance. administration of il-33 before and during c. albicans infection markedly induces m2 macrophage polarization. il-33-induced m2 polarization is mediated by cd4 t cell - derived il-13. surprisingly, il-33 stimulates bone marrow - derived m2 macrophages to efficiently phagocytize and kill c. albicans. the mechanism of action is not known, but one possibility is that il-33 increases killing by directly upregulating dectin-1 and mannose receptor or by indirectly inducting il-13 production. massive emergency hematopoiesis occurs after infections to satisfy the need for myeloid cells in the periphery. under these conditions, in addition, hematopoietic stem and progenitor cells (hspcs) emigrate from the bone marrow to the periphery and active extramedullary hematopoeisis occurs. injection of il-33 induces massive expansion of myeloid cells, particularly eosinophis, in the periphery. recently, we provided an explanation for how il-33 mediates this phenomenon. vascular endothelial cells produce high levels of ccl7 in response to il-33, and ccl7, in turn, induces emigration of hspcs out of the bone marrow with the help of ccr2. on the other hand, il-33 directs the differentiation of hspcs toward myeolopoiesis in both the bone marrow and periphery by an unknown mechanism. systemic c. albicans infections induce tubular epithelial cells of the kidney to produce il-33 (our unpublished data) and st2 is highly expressed on hspcs. thus, it is tempting to propose that il-33 may be involved in hspc differentiation during c. albicans infection. this hypothesis is substantiated by the observation that c. albicans stimulates differentiation of hspcs toward monocytes. altogether, these strongly suggest that il-33 may control emergency granulopoiesis to supply sufficient numbers of granulocytes to the site of infection, thus, indirectly increasing host resistance to c. albicans infections. although administration of il-33 increases host survival by promoting fungal clearance, the anti - inflammatory activity of il-33 can also contribute to host survival during systemic c. albicans infections. there were significantly lower levels of proinflammatory mediators (il-6, tnf-, il-1, cxcl1, and cxcl2), relatively little neutrophil infiltrate, and m2 macrophage polarization in the kidneys of il-33-primed mice throughout the course of infection. as mentioned previously, il-13 mediates il-33-induced m2 polarization. interestingly, m2 polarization is required not only to keep fungal burden in check in the kidneys but also to maintain persistent immunosuppression associated with reduced candida - induced immunopathology that leads to renal damage. because hyperimmune responses are associated with more fatalities than high fungal burden in systemic c. albicans infections consistent with these , il-33 mice are more susceptible to systemic candida infection compared to wild - type mice, showing impaired fungal clearance and severe renal immunopathology (our unpublished data). severe renal inflammation is associated with a dysregulated differentiation of tnf-- and inos - producing dendritic cells (tip - dcs) in the kidney of candida - infected il-33 mice. in contrast, these mice have a lower number of il-12- and il-23-producing mature dendritic cells (dcs) in their infected kidneys. as reducing tip - dcs decreases renal immunopathology without influencing fungal proliferation in il-33 mice, il-33 is a unique example of a factor that helps to reduce host vulnerability to damage by promoting pathogen clearance and reducing immunopathology (our unpublished data). one important outstanding issue to explore is whether il-33 is involved in types of tolerance other than immunopathology - related tolerance. il-33 for example, il-33 can have direct protective effects on parenchymal cells of organs during a stress response, or il-33 may exert tolerance through activation of cells that have a protective role against tissue injury. il-33 administration expands ilc2s and regulatory t cells that can produce amphiregulin, a molecule critical for tissue repair and host tolerance. two other types of cells that can be targeted by il-33 for tissue protection are m2 macrophages and th17 cells. however, dysregulation of il-33 can in chronic inflammation or fibrosis. whether the tolerance mechanisms mentioned above operate in host defenses against systemic candida infection needs to be tested. we propose that il-33-induced tolerance is associated with an increased capacity for tissue repair and decreased immunopathology. il-33 production reaches a peak in the kidney at day 3 of systemic c. albicans infection, when maximum inflammation and active fungal proliferation are occurring. at this time , il-33 seems to prevent overactivation of an inflammatory response on one hand and to promote fungal clearance on the other hand (fig . 1). the former process may mainly from blocking inflammatory dcs, while the latter may occur at multiple stages of innate and adaptive immune responses to c. albicans infection. il-33 also promotes il-23 production by dcs which in activation of the nk cell - gm - csf - neutrophil axis. finally, dc - derived il-12 and il-23 may be critical in il-33-mediated fungal clearance through the induction of th1 and th17 differentiation, respectively. these beneficial activities of il-33 have therapeutic implications for patients with invasive candidiasis. the ability of il-33 to promote th1 and th17 responses against c. albicans also indicates that il-33 has merits as an anti - candida vaccine adjuvant.	il-33 is a multifunctional cytokine that is released in response to a variety of intrinsic and extrinsic stimuli. the role of il-33 in candida albicans infections is just beginning to be revealed. this cytokine has beneficial effects on host defense against systemic c. albicans infections, and it promotes resistance mechanisms by which the immune system eliminates the invading fungal pathogens; and it also elevates host tolerance by reducing the inflammatory response and thereby, potentially, tissue damage. thus, il-33 is classified as a cytokine that has evolved functionally to protect the host from damage by pathogens and immunopathology.
among the many different symptoms encountered in every day practice, cough is the most common symptom in children visiting the pediatrician. chronic nonspecific cough is defined as a nonproductive cough in the absence of identifiable respiratory disease or known cause persisting for more than 3 to 8 weeks. routine diagnostic tests, such as total serum ige determination, allergen - specific serum ige determination, and skin prick test, can help significantly in diagnosing allergic asthma and distinguishing it from other respiratory diseases as well as from chronic nonspecific cough syndrome. although sensitization is a major risk factor for asthma development, the role of sensitization remains poorly understood and thus is continuously debated in scientific literature. the importance of total serum ige levels in diagnosis of asthma has been verified throughout a number of studies. it is also reported that asthma below certain levels of total ige does not exist. in addition, increased levels of total serum ige at birth and in early life have been associated with an increased risk for the development of persistent asthma later in life. while total serum ige determination is one of the key methods to reveal atopy, the determination of allergen - specific ige (sige) to environmental allergens has diagnostic and therapeutic importance; reveals the patient s sensitization to a certain allergen and enables monitoring of the success of specific immunotherapy (sit). among specific allergens, high sensitization rate have been reported for the house dust mites der p (dermatophagoides pteronyssinus) and der f (dermatophagoides farinae). early sensitization and levels of sige to perennial respiratory allergens have likewise been associated with increased risk of childhood asthma. similarly reported that mite sensitization early in infancy (measured by sige levels) accounts for an up to 19.7-fold increased risk for allergic asthma. also found house dust mite sensitization to be the most important risk for allergic asthma. have shown that asthmatic children with higher asthma severity have a higher serum concentration of both total ige and specific ige to der p. the atopy can also be defined by positive to skin prick test (spt) to a standard panel of allergens. investigating the association of skin test reactivity, total serum ige levels, and peripheral blood eosinophilia with asthma, khadadah et al. proved spt has the best positive predictive value and best efficiency in diagnosing respiratory atopic diseases. comparing spt to total serum ige levels, fajraoui et al. found that the 2 tests agree in 80% of cases. because chronic cough is one of the most common presentations of allergic asthma in children and being sensitized does not automatically include diagnosis of asthma, differentiation between allergic asthma and chronic (nonspecific) cough syndrome remains a relevant clinical problem. even though allergic diseases and diagnostic methods have been investigated during recent years, conclusive guidelines on how to distinguish allergic asthma from chronic (nonspecific) cough in the population of children have not been available. the aim of this study was to determine the diagnostic value of sensitization profile (including serum total, sige determination, and spt) in children with persistent respiratory symptoms to differentiate between children with allergic asthma and children with chronic (nonspecific) cough. this study is an analysis of data collected from patients at children s hospital srebrnjak, department of allergology and pulmonology, zagreb, croatia, during a 6-month period. a total of 131 children, aged 115 years, were included in the study. all of the patients included in the study experienced respiratory symptoms and were sent to our department for further diagnosis. participants underwent the standard allergological examination, including spt to the standard set of inhalatory allergens common for the region, lung function tests, and in vitro diagnostic tests. participants were tested for total ige and 3 allergen - specific ige antibodies against the most prevalent aeroallergens in children in continental region in croatia: hose dust mites (dermatophagoides pteronyssinus), common ragweed (ambrosia artemisifoliae), and timothy grass (phleum pratense) pollen. study participants were divided into 2 groups: children with clearly diagnosed allergic asthma, ie, having at least 3 episodes of wheezing and/or a positive bronchodilatation test (nih gina 2009); n=71, age 215 years, =8 years, 49 (69.01%) males. children with chronic cough, ie, having less than 3 episodes of wheezing, with persistent cough lasting for more than 6 weeks; n=60, age 114 years, =7 years, 40 (66.67%) males. total serum ige concentration was determined by fluoroimmunochemical method (abbot, usa) using an automatic analyzer imx (abbot, usa). for data comparison, 95% central range was used and serum ige concentration was compared to in - house reference values. der p (dermatophagoides pteronyssinus), phl p (timothy grass pollen), and amb a (short ragweed) were determined by immunocap (pharmacia, uppsala, sweden). analysis was performed using a unicap 100 analyzer (pharmacia, uppsala, sweden). the spt was performed with standardized allergens produced by allergopharma, comprising the standard set of inhalatory allergens common for croatia. total and specific ige concentration showed asymmetric distribution and were presented by range and median, and the statistical significance of the difference between the 2 groups was tested using the wilcoxon test. the diagnostic efficiency of total ige and sige determination sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) were estimated for the groups of patients with allergic asthma and chronic cough. receiver operating characteristic (roc) curve analysis was performed using statistica for windows, version 6.0 (statsoft, inc ., this study is an analysis of data collected from patients at children s hospital srebrnjak, department of allergology and pulmonology, zagreb, croatia, during a 6-month period . a total of 131 children, aged 115 years, were included in the study . all of the patients included in the study experienced respiratory symptoms and were sent to our department for further diagnosis . participants underwent the standard allergological examination, including spt to the standard set of inhalatory allergens common for the region, lung function tests, and in vitro diagnostic tests . participants were tested for total ige and 3 allergen - specific ige antibodies against the most prevalent aeroallergens in children in continental region in croatia : hose dust mites ( dermatophagoides pteronyssinus), common ragweed (ambrosia artemisifoliae), and timothy grass (phleum pratense) pollen. study participants were divided into 2 groups: children with clearly diagnosed allergic asthma, ie, having at least 3 episodes of wheezing and/or a positive bronchodilatation test (nih gina 2009); n=71, age 215 years, =8 years, 49 (69.01%) males. children with chronic cough, ie, having less than 3 episodes of wheezing, with persistent cough lasting for more than 6 weeks; n=60, age 114 years, =7 years, 40 (66.67%) males. total serum ige concentration was determined by fluoroimmunochemical method (abbot, usa) using an automatic analyzer imx (abbot, usa). for data comparison, 95% central range was used and serum ige concentration was compared to in - house reference values. der p (dermatophagoides pteronyssinus), phl p (timothy grass pollen), and amb a (short ragweed) were determined by immunocap (pharmacia, uppsala, sweden). analysis was performed using a unicap 100 analyzer (pharmacia, uppsala, sweden). the spt was performed with standardized allergens produced by allergopharma, comprising the standard set of inhalatory allergens common for croatia. total and specific ige concentration showed asymmetric distribution and were presented by range and median, and the statistical significance of the difference between the 2 groups was tested using the wilcoxon test. the diagnostic efficiency of total ige and sige determination sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) were estimated for the groups of patients with allergic asthma and chronic cough. receiver operating characteristic (roc) curve analysis was performed using statistica for windows, version 6.0 (statsoft, inc . median total ige in the group of children with allergic asthma was significantly higher than in the group of children with chronic cough ( 495.0 kiu / l in the allergic asthma group ; 59.0 kiu / l in the chronic cough group, p<0.05 ; table 1) the percentage of patients with elevated concentration of total and specific ige was higher in children with allergic asthma than in children with chronic cough (p<0.0001 ; table 2). comparing the number of patients with elevated total and sige level between the 2 groups , we found an elevated total ige level in 97.2% of children with allergic asthma, whereas elevated ige level was found in 31.7% children with chronic cough. level of sige was elevated in 100% of children with allergic asthma and in 21.7% children with nonspecific cough (=116.258 ; df=2 ; p=0.0000, * * =129.741 ; df=2 ; p=0.0000) comparing levels of sige in sensitized children, we discovered that children sensitized to der p had a higher concentration of sige than children sensitized to phl p and amb a (p<0.05) (table 3). we established that at a cut - off value of 116.6 kiu / l of total ige had 96.8% sensitivity and 77.8% specificity in differentiating allergic asthma and chronic cough. when comparing sige to der p in the group of children with allergic asthma and those in group with chronic cough, we found 89% sensitivity and 97% specificity (table 4). the best diagnostic value of cut - off values for sige was found for der p between children with allergic asthma and children with chronic cough (table 4). the sensitization profile can be defined by total and specific ige determination in serum and skin prick test to common inhalatory allergens. data on exact diagnostic efficiency (a combined measure of sensitivity and specificity) of serum total and sige determination and clinical relevance of ige level determination in differentiating children with asthma and non - specific respiratory symptoms differ from study to study. this study showed that total ige, and particularly sige, clearly distinguishes children with allergic asthma from children with nonspecific respiratory symptoms. this confirms the reports of other groups, who found that ige testing improved the predictive accuracy and patient differentiation. the differences in total ige concentration between the 2 groups (children diagnosed with asthma and children with chronic cough) were confirmed by the values of ige concentration (p<0.05) and roc curve analysis. cut - off values of total ige concentration (116.6 kiu / l asthma vs. chronic cough) were determined with excellent diagnostic efficiency. regarding literature variations of referent values for total ige concentration, the variation is due to different populations included in the studies and the applications of different immunochemical methods for ige determination. another important diagnostic procedure in estimating sensitization of children is the measure of ige to specific allergens in blood. of our study show that children sensitized to the perennial allergen der p showed a higher concentration of sige than in children sensitized to amb a and phl p, which are present only during the blooming season. it is believed that because house dust mites are the most widespread perennial inhalatory allergens, a greater exposure to der p in comparison with other allergens in higher sige concentration. variations between normal and elevated ige in the same individual may occur, depending on the age, the exposure to the specific allergen, and disease development. diagnostic efficiency of sige to pollen allergens could therefore be influenced by the period during which blood was sampled (during or before vs. after the blooming season). it was also confirmed that exposure and sensitization to certain allergens strongly depend on the region and climate. sensitization to aeroallergens in correlation to asthma development is currently a world - wide scientific focus. sensitization is most commonly defined by a positive skin prick test , along with sige in serum. being sensitized does not imply the diagnosis of allergic asthma or any other atopic disease. in 2001, crimi et al. showed that in a population of children proven to be sensitized, a third of them were without any allergic symptoms. it is also important to emphasize the correlation between sensitization, especially to the most common aeroallergens, and asthma in children. in 2010 craig used spt to define sensitization and concluded that about 95% of patients with mild asthma were sensitized, as well as the 90% of those with severe asthma. one of the most frequent sensitizations in children with asthma is to the house dust mite. as in croatia, in many other countries around the world this sensitization is the most common among asthmatic children. in florida, 89.6% of asthmatic children had positive sige to house dust mites, along with 81.9% of chinese asthmatic children and children in zimbabwe. many studies have observed that deteriorating asthma can be related to increased exposure to allergens, particularly allergens from house dust mites, cockroaches, cats, rodents, mold, or pollen. isaac, the international study of childhood asthma and allergies, showed that remission of asthma is 10% yearly and was inversely correlated to sensitization. carroll et al. confirmed that increasing atopic sensitization (estimated by spt and total ige level) is associated with increased disease severity in children with asthma. furthermore, elevated level total serum ige has also been linked to risk of hospital admission. in contrast, jedrychowski et al. related reversibility of asthma with lung function growth there are a number of factors that can influence sensitization, such as location of residence, sex, and ethnicity. with lower prevalence of sensitization, female sex and residence in a rural environment a great number of studies have investigated risk factors for allergen sensitization and asthma development. sporic et al. described a significant relationship between early life exposure to dust mite allergen and asthma at the age of 11 years. studies that are still ongoing, such as the prevention and incidence of asthma and mite allergy study (piama), the manchester asthma and allergy study (maas), and the childhood asthma prevention study, are expected to reveal new information about asthma prevention through prevention of sensitization to specific aeroallergens. in , it is important to emphasize that for a confirmed diagnosis of allergic asthma, determination of serum ige should always be supplemented with a thorough allergy investigation, including a detailed medical history (especially concerning allergen exposure), the presence of other possible immediate hypersensitivity diseases, skin tests, challenges, examinations for eosinophilia in blood and mucous membrane secretions, and in some cases with excluding the ger as a possible risk for asthma development. the of our study show that determination of total ige and sige is a good method for differentiating asthmatic children from those with nonspecific chronic cough. sige determination in children with clinical asthma symptoms had a better diagnostic value than total ige determination. the best diagnostic value of cut - off values for sige was shown for der p. in our study, spt was shown to have lower efficacy (78.82% sensitivity, 91.3% specificity) in distinguishing asthmatic children from children with non - specific chronic cough in comparison with levels of sige. although the differentiation between children with non - specific respiratory problems and children with asthma can be difficult, sensitization profile (total and specific ige levels and spt) facilitates achieving diagnosis. after analyzing 131 patient aged 115 years , we conclude that total ige, and particularly sige, clearly distinguish children with allergic asthma from children with nonspecific chronic cough. the final diagnosis should, however, always be confirmed by a thorough allergy investigation. considering the of our study, as well as the of other studies of sensitization profile and its correlation to asthma, we conclude that standardized procedures such as spt, total ige level, and sige should be performed in patients presenting with chronic cough, while it significantly correlates with asthma and can lead the physician to the final diagnosis of asthma vs. nonspecific cough.	in the subgroup of children with chronic cough, distinguishing children with allergic asthma from those with non - specific respiratory symptoms is difficult. we have focused on determination of diagnostic efficiency of serum total ige, sige, and skin prick test in differentiation of asthmatic children from children with nonspecific respiratory symptoms.material/methodsa total of 131 children with median age of 7.5 years were enrolled in study and divided into 2 groups; children with allergic asthma (n=71) and children with chronic cough (n=60). participants underwent the standard allergological examination, including skin prick test and measurement of total ige, and following 3 allergen - specific ige antibodies against aeroallergens: dermatophagoides pteronyssinus, ambrosia artemisiifolia, and phleum pratense.the percentage of patients with elevated level of total and sige was higher in children with allergic asthma than in children with chronic cough syndrome (p=0.0001). in children with asthma, sige had a better diagnostic value than total ige. the best diagnostic efficiency of cut - off values for sige was shown for der p sige. skin prick test to all allergens had 78.82% sensitivity and 91.3% specificity in differentiating the 2 tested groups. the highest sensitivity and specificity in skin prick test was proved for dermatophagoides pteronyssinus.the sensitization profile consisting of total ige, sige levels, and spt clearly distinguishes children with allergic asthma from children with chronic nonspecific cough, but still with overlap. therefore, diagnosis should always be confirmed by a thorough allergy investigation.
the plan for management of obstructive uropathy is based on precise delineation of both renoureteral units along with accurate estimation of the renal function. usual approach is to combine diagnostic tools which delineates the anatomy like ivu and radioisotope renal scan for assessment of the split renal function which is unfortunately time consuming, expensive and not freely available. nuclear renal scan is currently the gold standard imaging study to determine differential renal function. there are different methods to evaluate the renal function like 24 hours creatinine clearance and ct - estimated gfr. so, the residual parenchymal volume can very readily and accurately measured by 64 slice helical ct scan along with other anatomical informations. we propose helical computerized tomography as a single modality for both the anatomical and functional evaluation of kidney with impaired function. in the present study renal parenchymal volume is measured and percent total renal volume is used as a surrogate marker for differential renal function. the objective of this clinical study is to correlate between differential renal function estimation using ct based renal parenchymal volume measurement with differential renal function estimation using tc - dtpa renal scan. between february 2011 and august 2011, 21 patients with unilateral obstructive uropathy were enrolled in this prospective comparative study. renal units with unilateral obstruction was diagnosed by history, clinical examination, routine laboratory investigations including s. creatinine, plain x - ray of the abdomen (kub)/ultrasonography kub region, ivu and tc - dtpa renal scan. the cases with s.creatinine > 2 mg / dl and acute obstruction are excluded from the study. furthermore, they were subjected to 64 slice helical ct scan (ge medical system, light speed vct ct99). the volume estimation software comes preinstalled with the ge 64 slice helical ct scanner (ge medical system, light speed vct ct99) workstation (adw 4.3). the three dimensional tools that was available in this software package have add object function that was used to build the three - dimensional virtual representation of the kidneys depending on the arterial phase images. first the area of interest of renal parenchyma was delineated manually then by selecting a piece of enhancing renal parenchyma from the area of interest allows the software to identify the remaining surrounding renal parenchyma that have similar voxel values which is then added into the volume voxel to create the three dimensional renal volume. the final 3d model of each kidney was compared with the cross sectional images to ensure that only the renal parenchyma was included and sometimes it was necessary to use the cutting tool to free the margins of kidney from adjacent structures. renal parenchymal volume estimation was done by one radiologist in all the cases who was blinded regarding the nuclear scan report. the right and left percent renal volume was calculated by dividing right and left volumes respectively by the combined right and left renal volume. this percent renal volume was compared with percent renal function determined by tc - dtpa renal scan. correlations between percent renal volume and percent renal function is evaluated using pearson coefficient in all cases. in our study male: female ratio was 1:1.1 with and the age ranged from 20 to 55 years. we evaluated a total 21 (13 left and 8 right) obstructed renal units and 21 nonobstructed renal units i.e 42 renal units. of 21 obstructed renal units 2 had relief of obstruction before dtpa and ct scan. the etiology of chronic obstruction was pelvi - ureteric junction obstruction in four cases, ureteric calculus in nine cases, renal obstructing calculi in seven cases and lower ureteric stricture in one case. the range of s. creatinine was 0.5 - 1.2 mg / dl (0.78). the range of gfr estimated by tc - dtpa in obstructed renal units was between 0 and 27 mg / dl (7.35) and in unobstructed renal units ranged between 23.3 and 61.7 mg / dl (46.43). the parenchymal volume as estimated by 64 slice helical ct scan in obstructed renal units are in the range of 0 - 147.5 cc (31.45) and in unobstructed renal units was 94.2 - 267cc (142.41). the percent renal fuction as estimated by tc - dtpa in obstructed renal units ranges from 0%-39.33% (13.03) and in nonobstructed units ranges from 60.67 to 100% (86.89). the percent renal volume estimated by 64 slice ct scan in obstructed unit ranges from 0 to 35.58% (16.01) and in nonobstructed units ranges from 64.44 to 100% (83.94). we found a strong correlation between percent renal function estimated by tc - dtpa renal and percent renal volume estimated by 64 slice ct scan in obstructed renal units (r = 0.828, p < 0.001, 95% ci : 0.618 - 0.928). positive correlation between % renal volume and % renal function in obstructed renal units raw data for scatter graph (figure 2) we found a strong correlation between percent renal function estimated by tc - dtpa renal and percent renal volume estimated by 64 slice ct scan in nonobstructed renal units (r = 0.827, p < 0.001, 95% ci : 0.615 - 0.927). strong correlation is found between percent renal function estimated by tc - dtpa renal and percent renal volume estimated by 64 slice ct scan on combining both obstructed and non obstructed renal units (r = 0.985, p < 0.001, 95% ci : 0.974 - 0.992). positive correlation between % renal volume and % renal function in nonobstructed renal units data for scatter graph (figure 3) positive correlation between % renal volume and % renal function in both obstructed and nonobstructed renal units obstructive uropathy is a common entity that urologists encounters in daily practice and accurate assessment of differential renal function is vital in the management of these chronically obstructed renal units. twenty - four hours creatinine clearance is one of the methods to evaluate the total renal function but we need to have differential renal function for management of these obstructed units. at present nuclear renal scan is considered as the imaging modality of choice for differential renal function. but this also have some drawbacks like it is expensive and not freely available particularly in a country like india, also it does not give any information regarding the anatomy of the renal units which is of utmost importance for the management of these obstructed renal units. found a strong linear relationship between differential renal function by dynamic ct using modified patlak graphic analysis, nuclear renal scan and 24-hour creatinine clearance. but it has certain disadvantages like it needs assumption of functional renal homogeneity as it involves only a particular section of kidney, complex calculations and nonavailability of the software everywhere. ng et al. in his study compared differential renal parenchymal volume with 24-hr creatinine clearance by percutaneous nephrostomy in obstructed units and found correlation between differential renal function with differential creatinine clearance. herts et al. in their study proposed that estimated gfr by ct - based parenchymal volume can replace gfr measurement by i - iothalamate clearance imaging after studying 244 renal donors. observed contrast enhanced spiral ct is more sensitive than ivp for identifying the cause of chronic obstructive uropathy and it is as accurate as radioisotope renal scan for calculating the total and separate kidney function. so they recommend spiral ct with contrast medium as a single radiological diagnostic modality for the assessment of patients with chronic renal obstruction and normal serum creatinine. feder mt in their study predicting differential renal function using computerized tomography measurements of renal parenchymal area found differential renal parenchymal area measured by ct strongly correlates with differential function on renal scintigraphy and it may obviate the need for nuclear renal scan in some circumstances. morrisroe s n et al. found strong correlations between percent renal function and percent renal volume in all cases (r = 0.90, p < 0.001). they concluded that differential renal volume measured from computerized tomography strongly correlates with differential renal function on nuclear renal scan for normal and chronically obstructed kidneys and ct may serve as a single radiological diagnostic study for anatomical and functional assessment in patients in whom a poorly functioning renal unit is suspected. tc - dtpa renal scan and ct angiography are the investigations that are mandatory to evaluate the donor for the assessment of split renal function and vascular anatomy of the donor kidney. in his study found split renal function based on 3d ct models can provide a one - stop evaluation of both the anatomical and the functional characteristics of the kidneys of living potential kidney donors. differential renal function estimation by ct - based parenchymal volume can not replace nuclear scan in cases of acute cortical dysfunction and for documentation of improved renal function after surgery. the disadvantages of ct estimated differential functions are its inability to be performed in renal insufficiency and false in acute obstruction. according to the health physics society fact sheet 2010: the effective dose of ivu is 3 msv, the effective dose of ct abdomen / pelvis is 10 msvand the effective dose in dtpa scan is 1.8 msv. if we combine ivu and dtpa the radiation dosage is half that of ct scan which is a disadvantage but other factors which are advantages in case of ct have to be considered as well. measurement of differential renal function by ct - based parenchymal volume correlates strongly with differential renal function estimated by tc - dtpa renal scan in both obstructed and nonobstructed renal units. it has the added advantage of precise delineation of the anatomical details of the renoureteral unit. so, helical ct scan can be used as a single modality to asses the functional and anatomical status of the obstructed kidneys. it will help urologists in better management of obstructed renal units and lowering the expenses incurred by the patients. although our study is small it throws light into the efficacy of helical ct in assessment of split renal function. larger studies are needed to ascertain the usefulness of cect as a functional study in patients with severely impaired renal function.	introduction and objective: nuclear renal scan is currently the gold standard imaging study to determine differential renal function. we propose helical ct as single modality for both the anatomical and functional evaluation of kidney with impaired function. in the present study renal parenchymal volume is measured and percent total renal volume is used as a surrogate marker for differential renal function. the objective of this study is to correlate between differential renal function estimation using ct - based renal parenchymal volume measurement with differential renal function estimation using 99mtc - dtpa renal scan.materials and methods: twenty - one patients with unilateral obstructive uropathy were enrolled in this prospective comparative study. they were subjected to 99mtc - dtpa renal scan and 64 slice helical ct scan which estimates the renal volume depending on the reconstruction of arterial phase images followed by volume rendering and percent renal volume was calculated. percent renal volume was correlated with percent renal function, as determined by nuclear renal scan using pearson coefficient. and observation: a strong correlation is observed between percent renal volume and percent renal function in obstructed units (r = 0.828, p < 0.001) as well as in nonobstructed units (r = 0.827, p < 0.001).: there is a strong correlation between percent renal volume determined by ct scan and percent renal function determined by 99mtc - dtpa renal scan both in obstructed and in normal units. ct - based percent renal volume can be used as a single radiological tests for both functional and anatomical assessment of impaired renal units.
a previously healthy 20-year - old man presented to our community emergency department with nausea, vomiting, and generalized weakness for 1 month. he had undergone an extensive outpatient evaluation with normal imaging and laboratory studies and an esophagogastroduodenoscopy revealing only reflux gastritis. vital signs included a blood pressure of 100/73 mmhg, heart rate 105 bpm, and 95% oxygen saturation on room air. poc echocardiography was performed by the medical resident, revealing a severely reduced left ventricular ejection fraction with multiple echogenic structures suspicious for thrombi (fig . several hours later, standard echocardiogram confirmed an ejection fraction of 1015% with three apical thrombi . the patient was then transferred to a specialized cardiomyopathy service where inotropic and diuretic therapy led to resolution of his gastrointestinal symptoms which were presumed to be from intestinal venous congestion . we recently reported another case demonstrating the impact of poc ultrasonography that of an 84-year - old man with a history of esophageal cancer ( in remission) and deep venous thrombosis on warfarin who presented with shortness of breath. echocardiogram on the day of admission revealed a low normal ejection fraction with signs of elevated right - sided pressures and no pericardial effusion. the clinical diagnosis was congestive heart failure and his symptoms improved after several days of diuretics. on the fifth day of hospitalization, laboratory studies revealed a creatinine steadily rising to 3.5 mg / dl, an inr of 3.5, and fractional excretion of sodium of 0.3%. the assessment was that of over - diuresis with prerenal acute kidney injury, but he did not respond to small boluses of intravenous fluids. due to progressive decompensation overnight, poc ultrasonography was performed by the internal medicine resident. unexpectedly, this revealed a moderate pericardial effusion with right atrial and right ventricular collapse consistent with tamponade physiology (fig . the patient was urgently transferred to a tertiary care center where pericardiocentesis yielded 750 cc of frank blood . positron emission tomography / computed tomography ( pet / ct) was negative for any sign of recurrence of his esophageal cancer. warfarin was discontinued and a repeat echocardiogram, 4 weeks later, showed no pericardial effusion. a pericardial effusion is noted with associated right atrial and right ventricular inversion consistent with tamponade physiology. in the last decade, there has been a steadily growing body of research on the utility of ultrasonography for a myriad of conditions commonly seen in internal medicine. in fact, when compared to standard imaging, ultrasound has often yielded similar if not better . this is particularly well demonstrated in the realm of pulmonary disease. a recent meta - analysis including over 1,000 patients demonstrated a high sensitivity (94.1%) and specificity (92.4%) for ultrasonography in the diagnosis of pulmonary edema. ultrasound has also proven to be better than chest x - ray (cxr) and comparable to ct for pneumonia and lung consolidation. two recent meta - analyses, both including over 1,000 patients, confirmed these findings with a sensitivity of 9497% and specificity of 9496% for the diagnosis of pneumonia. the area under the receiver operating characteristic (roc) curve was consistently high (0.980.99) with useful positive and negative likelihood ratios. lung ultrasound can also be used to follow patients for resolution of their pneumonia similar to cxr and ct. with regard to pneumothorax, ultrasound can outperform radiography by several measures and is quite comparable to ct while being much faster. 22 ) have published a well - known algorithm that uses a variety of sonographic lung findings to help narrow the differential diagnosis in patients presenting with respiratory insufficiency. similarly, a recent, large multicenter study demonstrated that ultrasound can distinguish cardiogenic from non - cardiogenic dyspnea better than clinical assessment, cxr, or brain natriuretic peptide levels. this can be particularly difficult to assess outside the intensive care unit (icu) where pulmonary artery catheters and stroke volume monitors are absent. however, multiple studies have demonstrated the usefulness of sonographic inferior vena cava (ivc) and cardiac evaluation in non - icu patients. notably, guiotto et al. showed that ultrasound assessment of the ivc can guide volume removal by ultrafiltration. this can be particularly valuable with hemodialysis patients on the general medical floor in whom the determination of how much volume to remove is not usually done via very objective or precise means. with regard to the abdomen, sonography has exhibited excellent sensitivity and specificity in diagnosis of small bowel obstruction. in addition, sonographic techniques have long been described for diverticulitis and appendicitis and have very good specificity when compared to ct. interestingly, inflammatory bowel disease can be distinguished from non - inflammatory disease by ultrasound as well. in a study by novak et al. , sonography demonstrated high specificity and negative predictive value in comparison with gold standard endoscopy in patients presenting with abdominal pain and diarrhea. likewise, with regard to patients presenting with possible nephrolithiasis, poc ultrasound was similar in accuracy compared to both conventional radiology department ultrasound and ct scan without differences in outcomes. clearly, many of the conditions that are encountered in the internal medicine setting can be evaluated quite well with ultrasonography. the question that naturally follows is whether providers on the general medical floor particularly internal medicine house staff are able to apply this technology at the poc. there is indeed growing literature demonstrating that poc ultrasound performed by medical residents can change the course of patient care. in nearly 200 patients undergoing cardiac and abdominal sonography, andersen et al. showed that approximately one - third had an additional diagnosis uncovered or the primary diagnosis changed completely. in the pulmonary realm, filopei et al. demonstrated how ultrasound increased residents diagnostic accuracy compared to clinical assessment alone particularly for chronic obstructive pulmonary disease (copd), pneumonia, pleural effusion, and pulmonary edema. residents are also able to perform and interpret abdominal sonography of the kidneys comparable to radiologists; they can evaluate the abdominal aorta comparable to vascular surgeons and sonographers as well. similar findings have been shown with estimating volume status and diagnosing various cardiac pathologies compared to history and physical examination. interestingly, in one study, the use of portable cardiac ultrasound by residents changed management in 40% of cases and solidified prior management decisions in 76%. more specifically, residents are able to assess left ventricular function, pleural and pericardial effusion, and even valvular disease accurately compared to cardiologists. what is particularly remarkable is that extensive training is not needed for residents to acquire and interpret images effectively. after training on 20 patients, house staff were able to accurately identify left ventricular dysfunction with sensitivity, specificity, and positive and negative predictive values close to or well over 90%. after a 5-h module, medicine residents could diagnose certain kidney conditions with high sensitivity and specificity compared to attending radiologists. similarly, with a half - day workshop, 84% of residents in one study were able to independently evaluate the abdominal aorta compared to only 16% beforehand. in fact, many studies demonstrating accurate resident - performed ultrasound show that only a cumulative total training time of several hours to at most a few days is needed. understandably, these skills need to be reinforced with regular practice lest they wane over time. given the above, it is not surprising that poc ultrasonography can impact quality measures and costs. when lung ultrasound was implemented, peris et al. demonstrated a clear reduction in the number of cxrs and ct scans performed without any decrement in outcomes in one population. when used to guide thoracentesis, ultrasonography decreases complication rates, costs of hospitalization, and lengths of stay. furthermore, in an age where hospital readmission rates are closely monitored, ivc ultrasound by residents can be predictive and preventative, reducing rates from 30 to 4% in one recent study. demonstrated how residents are able to perform poc echocardiography not only accurately but also nearly a full 24 h before formal echocardiogram are available to confirm their findings. delays in imaging are known to cause increased hospital lengths of stay. synthesizing the aforementioned data , there is likely no more appropriate environment for poc ultrasound training of internal medicine house staff than the community hospital. in these settings, community hospitals are not usually designed to provide highly specialized services such as at a tertiary care center, but it is crucial, nonetheless, to identify patients in need of such therapies. our cases ed in the appropriate and necessary transfer of the patient to a university center. given such experiences at our institution, we are currently working to formalize poc ultrasonography in our house staff curriculum and highly encourage other community hospital programs to do so as well. in the last decade, there has been a steadily growing body of research on the utility of ultrasonography for a myriad of conditions commonly seen in internal medicine. in fact, when compared to standard imaging, ultrasound has often yielded similar if not better . this is particularly well demonstrated in the realm of pulmonary disease. a recent meta - analysis including over 1,000 patients demonstrated a high sensitivity (94.1%) and specificity (92.4%) for ultrasonography in the diagnosis of pulmonary edema. ultrasound has also proven to be better than chest x - ray (cxr) and comparable to ct for pneumonia and lung consolidation. two recent meta - analyses, both including over 1,000 patients, confirmed these findings with a sensitivity of 9497% and specificity of 9496% for the diagnosis of pneumonia. the area under the receiver operating characteristic (roc) curve was consistently high (0.980.99) with useful positive and negative likelihood ratios. lung ultrasound can also be used to follow patients for resolution of their pneumonia similar to cxr and ct. with regard to pneumothorax, ultrasound can outperform radiography by several measures and is quite comparable to ct while being much faster. 22 ) have published a well - known algorithm that uses a variety of sonographic lung findings to help narrow the differential diagnosis in patients presenting with respiratory insufficiency. similarly, a recent, large multicenter study demonstrated that ultrasound can distinguish cardiogenic from non - cardiogenic dyspnea better than clinical assessment, cxr, or brain natriuretic peptide levels. this can be particularly difficult to assess outside the intensive care unit (icu) where pulmonary artery catheters and stroke volume monitors are absent. however, multiple studies have demonstrated the usefulness of sonographic inferior vena cava (ivc) and cardiac evaluation in non - icu patients. notably, guiotto et al. showed that ultrasound assessment of the ivc can guide volume removal by ultrafiltration. this can be particularly valuable with hemodialysis patients on the general medical floor in whom the determination of how much volume to remove is not usually done via very objective or precise means. with regard to the abdomen, sonography has exhibited excellent sensitivity and specificity in diagnosis of small bowel obstruction. in addition, sonographic techniques have long been described for diverticulitis and appendicitis and have very good specificity when compared to ct. interestingly, inflammatory bowel disease can be distinguished from non - inflammatory disease by ultrasound as well. in a study by novak et al. , sonography demonstrated high specificity and negative predictive value in comparison with gold standard endoscopy in patients presenting with abdominal pain and diarrhea. likewise, with regard to patients presenting with possible nephrolithiasis, poc ultrasound was similar in accuracy compared to both conventional radiology department ultrasound and ct scan without differences in outcomes. clearly, many of the conditions that are encountered in the internal medicine setting can be evaluated quite well with ultrasonography. the question that naturally follows is whether providers on the general medical floor particularly internal medicine house staff are able to apply this technology at the poc. there is indeed growing literature demonstrating that poc ultrasound performed by medical residents can change the course of patient care. in nearly 200 patients undergoing cardiac and abdominal sonography, andersen et al. showed that approximately one - third had an additional diagnosis uncovered or the primary diagnosis changed completely. in the pulmonary realm, filopei et al. demonstrated how ultrasound increased residents diagnostic accuracy compared to clinical assessment alone particularly for chronic obstructive pulmonary disease (copd), pneumonia, pleural effusion, and pulmonary edema. residents are also able to perform and interpret abdominal sonography of the kidneys comparable to radiologists; they can evaluate the abdominal aorta comparable to vascular surgeons and sonographers as well. similar findings have been shown with estimating volume status and diagnosing various cardiac pathologies compared to history and physical examination. interestingly, in one study, the use of portable cardiac ultrasound by residents changed management in 40% of cases and solidified prior management decisions in 76%. more specifically, residents are able to assess left ventricular function, pleural and pericardial effusion, and even valvular disease accurately compared to cardiologists. what is particularly remarkable is that extensive training is not needed for residents to acquire and interpret images effectively. after training on 20 patients, house staff were able to accurately identify left ventricular dysfunction with sensitivity, specificity, and positive and negative predictive values close to or well over 90%. after a 5-h module, medicine residents could diagnose certain kidney conditions with high sensitivity and specificity compared to attending radiologists. similarly, with a half - day workshop, 84% of residents in one study were able to independently evaluate the abdominal aorta compared to only 16% beforehand. in fact, many studies demonstrating accurate resident - performed ultrasound show that only a cumulative total training time of several hours to at most a few days is needed. understandably, these skills need to be reinforced with regular practice lest they wane over time. given the above, it is not surprising that poc ultrasonography can impact quality measures and costs. when lung ultrasound was implemented, peris et al. demonstrated a clear reduction in the number of cxrs and ct scans performed without any decrement in outcomes in one population. when used to guide thoracentesis, ultrasonography decreases complication rates, costs of hospitalization, and lengths of stay. furthermore, in an age where hospital readmission rates are closely monitored, ivc ultrasound by residents can be predictive and preventative, reducing rates from 30 to 4% in one recent study. demonstrated how residents are able to perform poc echocardiography not only accurately but also nearly a full 24 h before formal echocardiogram are available to confirm their findings. delays in imaging are known to cause increased hospital lengths of stay. synthesizing the aforementioned data, there is likely no more appropriate environment for poc ultrasound training of internal medicine house staff than the community hospital. in these settings, community hospitals are not usually designed to provide highly specialized services such as at a tertiary care center, but it is crucial, nonetheless, to identify patients in need of such therapies. our cases ed in the appropriate and necessary transfer of the patient to a university center. given such experiences at our institution, we are currently working to formalize poc ultrasonography in our house staff curriculum and highly encourage other community hospital programs to do so as well. our cases demonstrate the diagnostic impact of poc ultrasonography in the hands of trained medical residents especially in the community hospital setting. providers on the general medical floor particularly medical residents are able to use this highly adaptive technology quite effectively, including in settings where urgent formal studies may not be easily accessible. a thorough medical history and detailed physical examination will remain cornerstones of diagnosis but, nevertheless, technology that can improve accuracy should be embraced. rene laennec met some degree of resistance when he introduced the first stethoscope in 1,821 but soon enough this new technology became an icon of the medical profession. similarly, in the training of its newest physicians, the internal medicine specialty needs to accept its newest the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	point - of - care (poc) ultrasonography is considered fundamental in emergency medicine training and recently has become a milestone in critical care fellowship programs as well. currently, there is no such standard requirement for internal medicine residency programs in the united states. we present a new case and briefly review another case at our institution a community hospital in which internal medicine house staff trained in ultrasonography were able to uncover unexpected and critical diagnoses that significantly changed patient care and outcomes. we also review the growing evidence of the application of ultrasound in the diagnosis of a myriad of conditions encountered in general internal medicine as well as the mounting data on the ability of internal medicine residents to apply this technology accurately at the bedside. we advocate that the literature has sufficiently established the role of poc ultrasonography in general internal medicine that there should no longer be any delay in giving this an official place in the development of internal medicine trainees. this may be particularly useful in the community hospital setting where 24-h echocardiography or other sonography may not be readily available.
superoxide radical (o2) is known to be harmful to cellular components and to function as a precursor of other reactive oxygen species (ros), such as singlet oxygen (o2) and hydroxyl radical (oh). a dismutation reaction can in the formation of hydrogen peroxides (h2o2) and o2 from the reaction of o2 with water. a reactive non radical although h2o2 itself is not very reactive, it can convert into more reactive species such as oh, the most reactive and harmful ros, by ultraviolet irradiation, fenton like reactions and the metal ion catalyzed haber - weiss reaction. electron spin resonance (esr) using the spin trapping agent 5,5-dimethyl-1-pyrroline - n - oxide (dmpo) is a technique for the direct detection of unpaired electrons such as o2 and oh, both of which are identified by hyperfine coupling constants assigned to the spin adducts such as dmpo - ooh (an adduct from dmpo and o2) and dmpo - oh (an adduct from dmpo and oh). that is, the primary function is nutritional feature (life support), and the secondary function is gustational feature (taste, flavor, and texture). recently, antioxidant potency has been received much attention as one of the tertiary function of foodstuffs. for instance, human studies with de - alcoholized red but white wine showed short - term cardiovascular benefits, and the specific components of the de - alcoholized wine that are active on cardiovascular endpoints are the polyphenols found in red wine, especially resveratrol. in addition to its potent antioxidant properties, it has recently been reported that resveratrol mimics the anti - ageing effects of calorie restriction in lower organisms, and ameliorates insulin resistance, increases mitochondrial content, and prolongs survival in mice fed a high - fat diet. based on these s , we conducted a large scale screening to search for edible herbal extracts with potent antioxidant activity. in this review, we summarize our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on o2 scavenging activity followed by characterization of antioxidant properties. more than a thousand of herbal ethanol extracts were prepared as shown in table 1. the assay used in this study was essentially identical to that described in our previous papers. in brief, 50 l of 2 mm hypoxanthine (hpx), 30 l of dimethyl sulfoxide (dmso), 50 l of sample dissolved in dmso, 20 l of 4.5 m dmpo, and 50 l of 0.4 u / ml xanthine oxidase (xod) were placed in a test tube and mixed. in the primary screening, all of the herbal extracts were served at a fixed concentration of 25 g / ml in the reaction mixture. the mixture was transferred to an esr spectrometry cell, and the dmpo - ooh spin adduct was quantified 100 s after the addition of xod. when a spin trapping agent, dmpo, was added to a solution of the hpx - xod reaction system, an esr signal with the hyperfine coupling constants of an = 1.37 mt, ah = 1.10 mt, ah = 0.12 this signal was assigned to the spin adduct, dmpo - ooh, by the hyperfine coupling constants. the reduction of the signal intensity of dmpo - ooh is likely reflected by the ability to scavenge o2. polyphenol contents of some herbal extracts were determined by folin - denis method, and were expressed as garlic acid equivalency. table 1 shows a list of all herbs tested and herbs that showed 80% or more reduction of signal intensity of dmpo - ooh measured by esr - spin trapping method at a fixed concentration of 25 g / ml. 1 shows the representative esr spectra of solvent control and ethanol extract with either poor or potent o2 scavenging activity. based on the data, we picked up four edible herbal extracts for further analyses. they are no.a-058 (peel of punica granatum), no.c-037 (bud of syzygium aromaticum), no.i-077 (kernel of mangifera indica), and no.i-079 (fruit of phyllanthus emblica). as shown in fig. 2, these four extracts contained abundantly polyphenols, whilst herbs with poor o2 scavenging activity (5% or less reduction of signal intensity of dmpo - ooh at a fixed concentration of 25 g / ml) contained a little amount of polyphenols. the indicate that the o2 scavenging activity was apparently reflected by the polyphenols content. esr analyses of oh from fenton reaction and from photolysis of h2o2 by uv - irradiation were conducted. the assays used in this study were essentially identical to those described in a previous paper. in brief for the former assay, 50 l of 2 mm h2o2 dissolved in 0.1 m phosphate buffer (ph 7.4), 50 l of 8.9 mm dmpo dissolved in pure water, 50 l of sample dissolved in pure water and 50 l of 0.2 mm feso4 dissolved in pure water were placed in a test tube and mixed. each mixture was transferred to an esr spectrometry cell and the dmpo - oh spin adduct was quantified 113 s after the addition of feso4. for the latter assay, a reaction mixture consisting of 440 l of 100 mm h2o2 prepared in 25 mm phosphate buffer (ph 7.4), 10 l of 111.25 mm dmpo dissolved in pure water and 50 l of sample dissolved in pure water was exposed to 254 nm uv irradiation at 4 w for 1 min at a distance of 12 cm. then the esr spectrum of dmpo - oh was measured. when dmpo was added to a solution of the fenton reaction system, the spin adduct dmpo - oh was formed. a reduction in the signal intensity of dmpo - oh by the addition of selected four herbal extracts likely reflects an ability to scavenge oh. this was also confirmed by the assay of photolysis of h2o2 by uv - irradiation. in the assay, the esr signal of dmpo - oh was reduced by adding any of the four herbal extracts, depending on the concentration. this indicates that any of the extracts has the ability to directly scavenge oh. as for the heat resistance of the antioxidant potency of the four herbal extracts, the effect of heat (100c) exposure on the o2- and oh - scavenging activity of the extracts was examined. while the o2-scavenging activity of 6.25 g / ml l - ascorbic acid as a reference agent, at which concentration l - ascorbic acid exerted scavenging activity comparable to 25 g / ml herbal extract, was completely inactivated after heat exposure for 30 min, the activities of 25 g / ml extracts from p. granatum (peel), m. indica (kernel) and p. emblica (fruit) were reduced by only about 20% even after heat exposure for 120 min. the extract of s. aromaticum (bud) was relatively heat - labile compared with the other three herbal extracts, as its activity was reduced by almost 40% after heat exposure for 60120 min. similar tendency was also observed in the oh - scavenging activity of the four herbal extracts exposed to heat (100c). these indicate that the four herbal extracts chosen from extensive screening possess desirable antioxidant properties. in particular, the extracts of p. granatum (peel), m. indica (kernel) and p. emblica (fruit) are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance.	this paper summarizes our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on superoxide radical (o2) scavenging activity followed by characterization of antioxidant properties. firstly, scavenging activity against o2 was extensively screened from ethanol extracts of approximately 1000 kinds of herbs by applying an electron spin resonance (esr)-spin trapping method, and we chose four edible herbal extracts with prominently potent ability to scavenge o2. they are the extracts from punica granatum (peel), syzygium aromaticum (bud), mangifera indica (kernel), and phyllanthus emblica (fruit). these extracts were further examined to determine if they also scavenge hydroxyl radical (oh), by applying the esr spin - trapping method, and if they have heat resistance as a desirable characteristic feature. experiments with the fenton reaction and photolysis of h2o2 induced by uv irradiation demonstrated that all four extracts have potent ability to directly scavenge oh. furthermore, the scavenging activities against o2 and oh of the extracts of p. granatum (peel), m. indica (kernel) and p. emblica (fruit) proved to be heat-resistant.the of the review might give useful information when choosing a potent antioxidant as a foodstuff. for instance, the four herbal extracts chosen from extensive screening possess desirable antioxidant properties. in particular, the extracts of the aforementioned three herbs are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance.
isolation and cultivation of adipose derived mesenchymal stem cells: the canine ascs used in this study were obtained in the same manner described in previous paper, and these were previously characterized. briefly, adipose tissues were aseptically harvested from gluteal region of 2-year - old beagle dogs, which were sacrificed for other objectives. the procedure was conducted with the approval of the institutional animal care and use committee of seoul national university (snu-120306 - 5). adipose tissues were washed extensively with phosphate - buffered saline (pbs), minced with cut and digested with collagenase type i (1 mg / ml ; sigma - aldrich, st . the tissue samples were washed with pbs solution and centrifuged at 300 g for 10 min . the pellets were resuspended, filtered with a 100 m nylon mesh and incubated overnight in low - glucose dulbecco s modified eagle medium ( low glucose - dmem, gibco, life technologies, grand island, ny, u.s.a .) supplemented with inactivated 10% fetal bovine serum (fbs, gibco, life technologies) and 100 units / ml penicillin-100 mg / ml streptomycin at 37.0c, in a 5% co2 incubator. after 24 hr, unattached cells and residual non - adherent red blood cells were removed by washing with pbs solution. the medium was changed every 2 days until the cells reached 8090% confluence. at passage 3, the cells were used for the following experiments. reconstruction of dermal papilla - like tissues: after canine ascs were cultured until 80% confluence, the culture medium was changed from low glucose - dmem to dermal papilla forming media (dpfm) that contained 10 ng / ml hydrocortisone (hydrocortisone, sigma - aldrich), 5 ml insulin - transferrin - selenium liquid media (its, gibco, life technologies), 100 units / ml penicillin, 100 mg / ml streptomycin and 20 ng / ml recombinant human hepatocyte growth factor (hgf, gibco, life technologies). the last changed medium contained green fluorescent nanoparticles (cellstalker ii - csf, biterials, seoul, korea) at concentration of 0.2 mg / ml to label the cells. after that, the cultured cells were treated with the cell dissociation reagent (accutase, gibco, life technologies) to detach the cells from the culture dish (detach - attach step). cells were re - applied at concentrations of 8 10 cells / cm. cells were undergone redistribution, and aggregation appeared 24 hr after the detach - attach step. cell aggregates became suspended 2448 hr and then isolated from the culture medium by centrifugation at 500 rpm for 3 min. all procedures used for reconstruction of dplts were based on and modified from previous reports. immunohistochemistry of dplts: to observe the structure and protein expression state of dplts, they were harvested and embedded in collagen gel. according to the manufacturer s instructions, seven parts of 0.5% collagen solution (matrixen - psc, bioland ltd ., gyeonggi, korea), two parts of 5x dmem (gibco, life technologies) and one part of 0.05 n naoh solution containing 2.2% sodium bicarbonate and 200 mm hepes were mixed and blended with reconstructed dplts on ice. this mixture was then placed in a humidified incubator (95% air/5% co2 at 37c) for 1 hr until gelation. after the gel containing dplts was fixed with 4% paraformaldehyde solution overnight, that was embedded in paraffin. sections were cut in 4 m and stained with hematoxylin and eosin (h&e). immunohistochemistry for laminin (rabbit polyclonal antibody, ab11575, 1:100, abcam, cambridge, ma, u.s.a .), vimentin (mouse monoclonal antibody, ab8069, 1:100, abcam), versican (rabbit polyclonal antibody, ab19345, 1:50, abcam) and vascular endothelial growth factor (rabbit polyclonal antibody, ab46154, 1:100, abcam) were performed. the sections were exposed to horseradish peroxidase (hrp)-conjugated goat anti - rabbit igg (g21234, 1:500, molecular probes, eugene, or, u.s.a .) or goat anti - mouse igg (g21040, 1:500, molecular probes) and were visualized by a reaction with 3,3-diaminobenzidine (dab) tetrahydrochloride (immpact dab peroxidase substrate, sk-4105, vector laboratories, burlingame, ca, u.s.a .). canine skin tissue was used as the positive control, and 5% normal goat serum was applied instead of primary antibodies as the negative control. alkaline phosphatase activity: alkaline phosphatase (alp) activity of asc and dplts was measured with a colorimetric method using a tracp & alp assay kit (# mk301, takara bio, tokyo, japan). undifferentiated ascs were washed with 0.9% sodium chloride, and floating dplts were collected with culture medium and washed. equal parts of the cell lysate and substrate solution containing p - nitro - phenyl phosphate (pnpp) were mixed and reacted in a 96-well plate for 30 min. absorbance at 415 nm of each well was measured with a microplate reader (model 680, bio - rad laboratories, burlingame, ca, u.s.a .). protein concentration in the cell lysate was measured at the same time with a bradford assay for normalizing the alp activity. preparation of the experimental animal model: the hair inducing activity of dplts and their fate were evaluated in vivo. thirty athymic nude mice (balb / cslc - nu / nu, six weeks old, female, 1720 g, japan slc inc ., shizuoka, japan) were used in this study, which were divided equally in each group (control or dplts group ; 10, 20 and 30 days). mice were anesthetized by intraperitoneal injection of a tiletamine / zolazepam combination (30 mg / kg, zoletil50, virbac korea, seoul, korea) and xylazine (10 mg / kg, rompun, bayer korea, seoul, korea). the scalp of mice was prepared aseptically, and full thickness skin wounds were created with a 6 mm biopsy punch. in the dplts group, 400 dplts mixed with 50 l of pbs were injected into the inner - dermis at the border between the wound and the normal skin. on the other hand, only 50 l of pbs were injected in the same manner to mice in the control group. paul, mn, u.s.a. ) was used for wound protection until wound healing was completed. the animals were housed in a specific pathogen - free (spf) room at seoul national university, and all of the procedures were approved by the institutional animal care and use committee of seoul national university (snu-140917 - 2 - 3). evaluation of hair growth in gross and histological examinations: the wounds were photographed using a digital camera at 0, 10, 20 and 30 days after transplantation of dplts. the mice were euthanized by cervical dislocation at 10, 20 and 30 days from the time of dplts injection. the skin around the wounds was harvested and divided into two parts with wound as a center: one part for histologic evaluation and the other part for western blotting. the sampled skin was fixed in 10% neutral formalin solution and embedded in paraffin. the number of hair follicles and sebaceous glands in the regenerated wounds, and vascularization were evaluated. immunohistochemistry for endothelial cells with anti - cd31 antibody (rabbit polyclonal antibody, ab28364, 1:50, abcam) was used to visualize the vessels. the positive control (murine skin tissues) and negative control were also applied in the same manner. vascularization was evaluated by the number of blood vessels in the high - power field using five randomly selected fields from each skin section. for immunofluorescence , the sections were incubated with rabbit anti - vascular endothelial growth factor (vegf) antibody (ab46154, 1:200, abcam). then, cytm3-conjugated affinipure donkey anti - rabbit igg (h+l) (711 - 165 - 152, 1:500, jackson immunoresearch, west grove, pa, u.s.a .) was applied at room temperature for 1 hr. western blotting: the tissue extracts were prepared from the skin with the pro - prep protein extraction solution (intron biotechnology, gyonggi - do, korea). the protein concentrations were determined with the bradford assay (bio - rad laboratories). the extracted proteins (20 g) were mixed with laemmli s sodium dodecyl sulfate (sds)-sample buffer (gendepot, baker, tx, u.s.a .) and boiled for 5 min before loading. samples were run on a 12% sds - polyacrylamide gel and transferred onto a nitrocellulose membrane. the membranes were blocked with 5% skim milk for 1 hr at room temperature and incubated overnight at 4c with rabbit polyclonal anti - vegf antibody (ab46154, 1:1,000, abcam). anti - rabbit igg - hrp was used as a secondary antibody for 1 hr at room temperature. anti--actin antibody (sc-47778, 1:1,000, santa cruz biotechnology, santa gruz, ca, u.s.a .) was used for a loading control. blots were visualized with an enhanced chemiluminescence kit (ecl kit, invitrogen, life technologies) and visualized under chemidoc. the optical densities were quantified using image analyzer software (image j, u.a . statistical analysis : statistical analyses were performed using spss, version 22.0 ( spss inc ., tokyo, japan). a mann - whitney u test was used to assess the changes or differences between the control group and the dplts group. data are reported as the mean values, with the variability expressed as standard deviation. reconstruction of dplts: ascs underwent morphological changes by dpfm from a spindle shape to a polygonal shape, which started approximately 24 hr after changing media. after the detach - attachment step, cell distribution was changed, and groups of cells began to pull apart and form aggregates; cell aggregates started to float after 2448 hr. the morphology and the size of dplts were consistent with the previous study. structure and protein expression of dplts: the compact structure of the dplts, which had closely connected cells, was observed by h&e staining (fig . 1.analysis of the structure and the protein expression of collagen embedded dplts by histological ( a), immunohistochemical (ihc) staining (b d) and alkaline phosphatase activity (e). dplts were immunostained by versican (b), laminin (c) and vimentin (d). (e) intracellular alp concentration was significantly (* * p<0.01) higher in dplts compared to ascs. reconstructed self - aggregated dplts had significantly higher levels of alp compared to the ascs (fig . analysis of the structure and the protein expression of collagen embedded dplts by histological ( a), immunohistochemical (ihc) staining (b d) and alkaline phosphatase activity (e). dplts were immunostained by versican (b), laminin (c) and vimentin (d). (e) intracellular alp concentration was significantly (* * p<0.01) higher in dplts compared to ascs. hair inductivity: approximately 10 days after wound induction, wound healing was completed in all groups. new hair fibers on the regenerated skin were observed at 1520 days after injection, especially in the dplts group (fig . photographs of the wounds at days 10, 20 and 30 from the control and the dplts groups . hair growth around and at the wound bed was remarkable in the dplts group compared to the control group at day 20 .). regenerated hairs at the wound bed in the control group were short and thin as well as mainly observed around the periphery of the wound, whereas the central wound region had regenerated hairs in the dplts group. intriguingly, the number of hairs at the wound bed slightly decreased at day 30. photographs of the wounds at days 10, 20 and 30 from the control and the dplts groups. hair growth around and at the wound bed was remarkable in the dplts group compared to the control group at day 20. the difference between the groups was more apparent in the histological evaluation. at day 10 and day 20, the skin tissues from the dplts group (4.6 2.19 and 10.7 2.89/wound bed) contained significantly more regenerated hair follicles in the wound bed than those from the control group (0.5 1.00 and 4.0 1.00/wound bed) (fig . microphotographs of wounds and outside the wound bed at days 10, 20 and 30 ( a). trichogenicity was assessed by the number of regenerated hair follicles (b) and regenerated sebaceous glands (c) per wound bed at a section. the number of regenerated hair follicle and sebaceous gland was significantly (* p<0.05) higher in the dplts group versus the control group at days 10 and 20. at days 30, the number of regenerated hair follicles was slightly decreased. and, between the groups, the differences in the number of hair follicles and sebaceous glands were unremarkable. ). newly formed hair follicles increased until day 20, and then, the trends changed by 30 days. the number of regenerated hair follicles was slightly decreased in both groups, and the difference between two groups was not significant at day 30. microphotographs of wounds and outside the wound bed at days 10, 20 and 30 (a). trichogenicity was assessed by the number of regenerated hair follicles (b) and regenerated sebaceous glands (c) per wound bed at a section. the number of regenerated hair follicle and sebaceous gland was significantly (* p<0.05) higher in the dplts group versus the control group at days 10 and 20. at days 30 between the groups, the differences in the number of hair follicles and sebaceous glands were unremarkable. the number of sebaceous glands in the wound bed observed at days 10 and 20 was significantly higher in the dplts group (2.2 0.84 and 2.3 0.50/wound bed) compared to the control group (0.0 0.00 and 0.3 0.50/wound bed) (fig . however, similar to the hair follicles, there was no significant difference between two groups in the number of sebaceous glands at day 30 . in addition, the majority of the pre - existing hair follicles outside the wound bed were in the anagen phase at day 20, which consisted of multilayered cells and located deeply in the subcutaneous region . while, most pre - existing hair follicles at day 30 were in the telogen phase ( fig . angiogenesis was evaluated by counting the number of vessels in the slides, which were immunostained against cd31 at day 10 ( fig . microphotographs of the wounds at 10 day in the control group ( a) and the dplts group (b). the number of vessels at the wound per five fields at days 10, 20 and 30 (c). significantly (* * p<0.01) more vessels were observed in wound bed from the dplts group than from the control group. ). the number of cd31-positive vessels at the wound per five fields was significantly higher in the dplts group (48.0 15.05/hpf) relative to the control group (16.2 5.54/hpf) at day 10. microphotographs of the wounds at 10 day in the control group (a) and the dplts group (b). the number of vessels at the wound per five fields at days 10, 20 and 30 (c). significantly (* * p<0.01) more vessels were observed in wound bed from the dplts group than from the control group. the role of dplts in hair inductivity: under fluorescence microscopy, it is confirmed that injected fluorescence - labeled dplts did not constitute regenerated dps. instead of structural function, we performed immunohistochemistry on collagen - embedded dplts and mice skin tissues at day 10 in order to identify the paracrine effect of dplts on hair regeneration. several vegf - positive dplts were detected in both samples, which indicates that dplts could secrete vegf in vitro and in vivo (fig . 5a and 5bfig . 5.in vitro ( a) and in vivo (b) vegf secretion of dplts and quantitative evaluation of vegf from the wound at day 10 (c). (a) notice strong positive cytoplasmic immunostaining for vegf of collagen embedded dplts. (b) ihc of injected dplts (green - labeled) for vegf (red) at 10 day. (c) quantitative evaluation of vegf protein from the wound at day 10 by western blotting. western blotting was performed to quantify the expression level of vegf at day 10. the larger amount of vegf was detected in the dplts group compared with the control group, though the difference in the thickness of the bands was not significant (fig . ( a) and in vivo (b) vegf secretion of dplts and quantitative evaluation of vegf from the wound at day 10 (c). (a) notice strong positive cytoplasmic immunostaining for vegf of collagen embedded dplts. (b) ihc of injected dplts (green - labeled) for vegf (red) at 10 day. (c) quantitative evaluation of vegf protein from the wound at day 10 by western blotting. our previous study demonstrated that dplts could be successfully reconstructed from canine ascs. in succession, reconstructed dplts were evaluated as an alternative to actual dps by rna and protein expression through the previous and the present studies. our immunohistochemistry data revealed versican in the dplts, and alp activity is significantly elevated in dplts than in ascs. moreover, the compact spheroid - forming structure of the cells was observed in h&e staining, and the extracellular matrix formed during aggregation of dplts was immunostained by laminin and vimentin. versican and alp are key characteristic molecular markers of dps, which are specifically expressed in dps during the anagen phase and absent in the telogen phase. consistent with our findings, the levels of versican and alp are closely associated with in vivo hair inducing activity of the dpcs , and a distinct tendency to form cell aggregates of dermal papilla cells indirectly indicates the in vivo trichogenic property of cells. in vivo trichogenesis was evaluated by injecting dplts into a full - thickness skin wound on the head of nude mice. athymic nude mice, which were used in this study, are characterized by disturbed development of hair follicles and dysgenesis of the thymus , which is a suitable animal model to evaluate hair regeneration with a xenograft of canine cells. yoo et al. transplanted dplts to inner - dermis in nude mice with outer root sheath cells (orscs) as a source of epithelial cells. by contrast , we injected dplts as a mesenchymal component at the full - thickness skin wound, anticipating the wound epithelium would interact with dermal components as epithelial components as previously described. gross and microscopic findings at days 10 and 20 indicate that dplts play a crucial role in contributing to hair regeneration. regeneration of sebaceous gland was evaluated with that of hair follicle in this study. the of the present study indicated that not only hair follicles, but also the complete pilosebaceous units were regenerated by dplts. the number of hair follicles was slightly decreased at day 30 compared to that of at day 20, and the difference between the groups also decreased. there are two possible explanations for the decrease in regenerated hair follicles at day 30. first, hair inducing role of dplts could be influenced by the hair cycle of mice. nude mice are not actually nude; they also have hair follicles and a hair cycle, which is synchronized and repeated with a 3-week interval. this is consistent with our observation, and anagen phase proceeded during days 1020, whereas at days 30, most hair follicles outside the wound bed were in the telogen phase, which was characterized by shrunk hair bulb. in addition, our findings demonstrated small amounts of injected dplts could survive for 30 days, and the effect of dplts was minimal at day 30. moreover, in the histological examination, the number of vessels in the wound bed was higher in the dplts group. especially, statistically significant difference between groups was observed at day 10, which suggests that implanting dplts on the injection site may enhance the neovascularization. the decrease in the number of vessels in the dplts group could be explained by the decrease of granulation tissue as time passed. in addition, as the survival rate of injected dplts declined over time, the amount of vegf secreted from dplts also diminished. the transition of the hair cycle (physiological hair loss) and alopecia (pathological hair loss) were not only controlled but also characterized by dps and peri - papillary vascularization. in addition, the close connection between hair regeneration and neovascularization around hair follicles has been identified under various treatments for alopecia including when medical treatments and laser therapy were applied. while mesenchymal stem cells have been observed to have a stimulatory effect on hair regeneration due to the paracrine secretion of growth factors in previous studies , we expected dplts to be the direct structural components of the hair follicle as a substitute for dps under control of emi. therefore, emi with reconstructed canine dplts is not fully evaluated in the present study, and a further study is needed. instead of a direct structural role, growth factor secretion of dplts although polyclonal antibody used in this study detected both murine and canine vegf in an in vivo study, merged signals of dplts and vegf could prove the presence of growth factors secreted from dplts. these are consistent with the fact that the mesenchymal component, including dpcs, strongly expresses vegf compared to other components of the hair follicle. in other words, the vegf autocrine effect of dps is also identified on our reconstructed dplts. vegf stimulates the proliferation of dpcs in vitro as well as stimulates hair regrowth and increases the size of the hair follicles and hair shafts in vivo. as a of western blotting, the amount of vegf tended to be higher in the dplts group than in the control group, although no significant difference was observed between groups. these could be explained by the survival rate of dplts on wound bed. a further study and application of scaffold or biomaterials with dplts which act as a delivery vehicles and physical support, first of all, structural role of dplts was not proved in the study, where activated epithelial cells through the wound healing had been expected to interact with dplts by emi. a further study, where epithelial component should be injected with dplts (dermal component) without wound, was needed to evaluate emi. second, residual dplts and the amount of vegf secreted by dplts were not constant by time according to our observation. the survival rate of cells on the injected site is the major concerns in regenerative medicine. biomaterials which act as a scaffold for extending the survival time and maintaining 3d structure of dplts should be evaluated. although only paracrine effect of dplts was proved in the present study, dplts also have the several positive effect on hair regeneration similar to dps. the dps have a stem cell - like role to differentiate into multi - lineage in itself, as well as the dps contact the bulge and activate the bulge stem cells at the early anagen to form the the new hair follicles after remaining intact during the catagen and the telogen phases. furthermore, dps help neighboring epidermal cells to differentiate into cells of hair follicle lineages. other paracrine and signaling pathways, such as wnt/-catenin / bmp, fgf and igf, in dplts also should be evaluated in a further study. in , this study demonstrates the possibility of dplts made from canine ascs as a substitute for dps in alopecic animals. in addition to the previous in vitro study, the in vivo indicate that this engineered construct could enhance the hair follicle regeneration in an athymic nude mouse model by increasing neovascularization on the wound bed. to our knowledge, the development of reconstructed dps from mesenchymal stem cells from animals has not been previously reported. despite its imperfections, engineered canine dplts have the feasibility to be a useful treatment option for alopecic animals as an easily obtained source of dpcs as well as a study model to further characterize dpcs.	the purpose of this study was to investigate the protein expression pattern and the in vivo trichogenicity of dermal papilla - like tissues (dplts) made from canine adipose - derived mesenchymal stem cells (ascs) in athymic nude mice. canine ascs were isolated and cultured from adipose tissue, and differentiation was induced by culturing ascs in dermal papilla forming media. dplts were embedded in collagen gel, and their structural characteristics and protein expression were evaluated by hematoxylin and eosin stain and immunohistochemistry. athymic nude mice were divided into two groups (control and dplts groups), and dplts were injected in skin wounds of mice in the dplts group. the trichogenicity of dplts was assessed by gross and histological evaluations for 30 days. the fate and the growth factor - secretion effect of dplts were evaluated by immunohistochemistry and western blotting. dplts have a compact aggregated structure, form extracellular matrix and highly express the protein specific for dermal papillae, including alp and versican. new hair follicle formation was remarkable in nude mice of the dplts group in gross findings and h&e stain. vascularization was increased in the dplts group, which was the effect of vascular endothelial growth factor secreted by dplts in vitro and in vivo. these data suggest that engineered canine dplts have characteristics of dermal papillae and have a positive effect on hair regeneration by secreting growth factors.
the accurate treatment of the adsorption of molecules on surfaces is a major challenge of materials modeling, with important applications in nanotechnologies and science: heterogeneous catalysis, sensors, corrosion, lubrication, friction, and coatings, to name but a few. clay minerals are natural aluminosilicates that find use in a wide variety of fields such as medicine, adhesives, paints, and oil drilling. they also act as catalysts to ice nucleation in the atmosphere and help cleanse soils and groundwater through adsorption of pollutants. a clear understanding of how molecules interact with the surfaces of clays is of the utmost importance to understand, improve, and control such processes. reliable reference data from theory and simulation are of intrinsic value and often important as a complement to experiments. computer simulations of water surface interactions, at the molecular level, are often based on force fields (ff) and density functional theory (dft) approaches. although these techniques are incredibly powerful and useful, there are cases where their accuracy is not satisfactory. ff potentials have parameters that have to be fit in order to reproduce experimental or higher lever theoretical benchmarks, and this is not always straightforward. unfortunately, dft are highly sensitive to the choice of the exchange - correlation (xc) functional used, and nowadays there are countless xc functionals to choose from. in the field of materials science, the description of weak bonding interactions, and in particular london dispersion forces, is one of the most important challenges. immense progress has been made in this area recently; however, there is no rigorous way to systematically improve xc functionals, and as a validation with alternative methods is needed. of the various high level reference methods available, quantum monte carlo (qmc) is a powerful approach for obtaining benchmark values for solids, surfaces, and large molecular systems. qmc, within the fixed node diffusion monte carlo (dmc) approach, has already been used to tackle interesting materials science problems that have been beyond the reach of dft (see, e.g., refs ). this has provided reference data which have exposed shortcomings in existing ff models and dft xc functionals, which in turn aids the development of such approaches. in this paper we will use two qmc approaches to investigate molecular adsorption on a clay surface, namely, dmc and lattice regularized diffusion monte carlo (lrdmc). the particular clay we will examine is kaolinite (al4si4o10(oh)8 ), as shown in figure 1. since the first outline of the kaolinite crystal structure by pauling in 1930, numerous structural studies using x - ray and neutron powder diffraction, x - ray single crystal, and electron diffraction methods, as well as theoretical studies have been carried out on kaolinite. consequently it is one of the most suitable aluminosilicate clays to assess the performance of various theoretical methods. in addition, when looking at adsorption processes on kaolinite, cleavage along the basal plane leads to exposure of either aluminate or silicate faces (figure 1). the aluminate (aloh) face is terminated in hydroxyl groups and as a is regarded as hydrophilic, whereas the silicate (sio2) face which exposes saturated si the distinct chemical nature of these two surfaces means that adsorbates will interact differently with them, making kaolinite an interesting case study for understanding the role of vdw forces on the adsorption at clay mineral surfaces. the hydrogens are sketched in white, the oxygens in red, the silicons by yellow tetrahedra, and the aluminums by pink octahedra. the figures on the right are the hydroxyl - terminated face (top) and the silicate - terminated face (bottom). various adsorption sites on the hydroxyl- and silicate - terminated face are labeled. in what follows , we will provide benchmark values for the adsorption of water and methanol molecules at the pristine hydroxyl- and silicate - terminated faces of kaolinite, by using dmc and lrdmc. then, we probe dft xc functionals by considering a range of generalized gradient approximation (gga) functionals, hybrid functionals, and dispersion - corrected density functionals which account for vdw forces. in the case of molecules adsorbed on kaolinite we find that the bare ggas and hybrids are quite unreliable: as expected adsorption energies are underestimated, but more importantly, the relative adsorption energies of water versus methanol do not even agree with qmc. moreover, on the silicate - terminated face the molecules hardly bind at all and move quite far from the surface during geometry optimizations. accounting for vdw forces improves adsorption energies significantly and stabilizes the structures. however, most of the vdw - corrected and vdw - inclusive functionals predict adsorption energies which are slightly too large compared to qmc. this indicated that there remains room for improvement in terms of how vdw forces are handled in dft. since a range of techniques has been used, for brevity the more detailed descriptions of the computational setups are provided in the supporting information. qmc and dft evaluations of the adsorption of water and methanol at both faces of kaolinite are reported and discussed in section 3. adsorption was examined on a single layer of kaolinite, a system with 2d periodicity along the a and b axes as indicated in figure 1. the simulated supercell was 1 2 the conventional unit cell of bulk kaolinite (ca . 10.38 9.01) and is comprised of 8 aluminums, 8 silicons, 36 oxygens, and 16 hydrogens for the kaolinite slab plus the atoms of the adsorbed molecule. note that with ca. 300 electrons the simulations are large for qmc calculations. the adsorption energy, eadsm@x, of the molecule m at the face x of the kaolinite layer, where m can either be the water (h2o) or the methanol (meoh) and x can be the hydroxyl - terminated (aloh) face or the silicate - terminated (sio2) face, can be evaluated in two ways: the first method, hereafter called complex - minus - fragments, is computed as1where eslab+m@x is the total energy for the system with m at the x - face of the kaolinite slab, and eslab and em are the total energies of the isolated slab and the isolated molecule m, respectively. the second method, hereafter called complex - minus - far, is computed as2where eslab m is the total energy of a system where the kaolinite slab and the molecule m are far enough apart that their interaction is negligible. the two methods are equivalent if and only if: (i) the size effects due to the periodicity of the system are negligible and (ii) the electronic structure calculations are performed with methods that are exactly size consistent. if these conditions are not satisfied in general we have that eslab m em + eslab, meaning that eqs 1 and 2 provide different evaluations of the adsorption energies. in particular, whenever size effects are detected, the complex - minus - far method usually benefits from a larger error cancellation. on the other hand, in cases where size effects are negligible and electronic structure methods are size consistent, there are no residual interactions between the molecule and the periodic partners, and then the complex - minus - fragments method is usually to be preferred. the reason for that is the computational cost: for a system with n electrons the computation is proportional to n, with > 1 (e.g., in dft is typically between 2 and 3 and in qmc between 3 and 4), so the cost for calculations of eslab and em is cheaper than eslab moreover, when several adsorption energies need to be evaluated, eslab is calculated only once, whereas a different calculation of eslab m has to be performed for each molecule m. the two qmc approaches used are dmc and lrdmc. they are both projection monte carlo methods: they can access the electronic ground state energy of the system by iteratively projecting an initial trial wave function t into the ground state, with the constraint that the projected wave function has the same nodal surface of an appropriately chosen guiding function g (fixed node approximation). both the trial and the guiding wave functions are parametrized functions, and they have to be the best approximation of the ground state that we can provide (given the constraint of their ansatz) thus, usually they are taken such that t = g = vmc, where vmc is the best function obtained within a variational monte carlo approach, with the variational parameters optimized in order to minimize either the variational energy or the variance. whenever g has the exact nodal surface, the approach is exact; otherwise, it gives the best approximation of the ground state given the fixed node constraint. in projection monte carlo approaches there is a second approximation in how the projection is performed, and it is different in dmc and lrdmc. the projection in dmc comes from the imaginary time schrdinger equation; it is implemented as an imaginary time evolution, where a time step has to be chosen. the chosen is a trade - off between accuracy and computational cost: exact are obtained for 0, but the computational cost is 1/. the finite time - step error can be controlled by performing several calculations with different values of and finally extrapolating to the continuum limit 0. however, in big systems like those considered here, the extrapolation is impractical and sometimes unfeasible or unreliable, but it is sufficient to consider the for a small enough that the expected finite - time error is smaller than the required accuracy. here , we have chosen in order to have an expected time - step error smaller than the stochastic error of the evaluations (see section i in the supporting information). on the other hand, lrdmc is based on the spatial discretization of the molecular hamiltonian on a lattice of mesh size a, and it resorts to the projection scheme used also in the green function monte carlo algorithm. the error induced by the finite mesh size a is analogous to the time - step error appearing in standard dmc calculations. lrdmc preserves the variational principle even when used in combination with nonlocal pseudopotentials (pps), and it is size consistent for any value of the mesh a, maintaining its efficiency even for systems with a large number of electrons. both dmc and lrdmc provide excellent benchmark values for weakly interacting systems, as established in a number of studies. we used here a standard setup, described in detail in the supporting information. the stochastic error associated with the qmc evaluations of the adsorption energy is ca. the systems under consideration are too large for a qmc - based structural optimization, even at the variational level, so the reference structures were those obtained from the pbe - d3 functional, as described in section 3.1. as we will see in section 3, pbe - d3 configurations are in good agreement with those obtained by all the other vdw - inclusive functionals, thus the bias given by the use of pbe - d3 configurations is expected to be small compared to the stochastic error of the dmc evaluation. there is one aspect of qmc simulations that deserves special care in this specific system, namely, the finite - size errors (fses). qmc is a many - body method, and in contrast to (effective) one - particle methods such as dft, qmc can not simply exploit bloch s theorem in calculations for extended periodic systems. fses can be taken into account by performing simulations in larger periodic supercells, through the twist - average method, corrections to the ewald energy, or the kwee, zhang, krakauer (kzk) method. in this work we have used the kzk method (see section 3 in the supporting information). there is by now an almost limitless variety of dft xc functionals that we could examine. here we restrict ourselves to the lda functional; two gga functionals, pbe and rpbe; two hybrid functionals, pbe0 and b3lyp; three vdw - corrected pbe functionals pbe - d2, pbe - d3, (both from grimme, d3 correction with zero - dampling), and pbe+vdw(ts) from tkatchenko and scheffler; two vdw - corrected hybrid functionals, pbe0-d3 and b3lyp - d3 (both from grimme); and four self - consistent nonlocal functionals (often called vdw - inclusive functionals), the original vdw - df from dion (also named revpbe - vdw), the second generation vdw - df2, as well as optpbe - vdw and optb86b - vdw from klime et al. we stress that the latter four vdw - inclusive functionals are actually based on ggas and basically differ from the vdw - corrected gga functionals (e.g., pbe - d2, pbe - d3, and pbe+vdw(ts) ) only in the way the dispersion energy is approximated. other functionals and vdw corrections have been tested, and obtained using a comprehensive set of approaches is reported in table s1 of the supporting information. adsorption energies were evaluated using the complex - minus - fragments method (see eq 1), but the are the same as those obtained with the complex - minus - far method, as expected. further details about the setup of the dft calculations are reported in section 4 of the supporting information. we also performed a series of molecular dynamics simulations using classical force fields for aqueous water the kaolinite slab was modeled as a single sheet of kaolinite (approximately 31 36) using the clayff force field, and the oh bond lengths were constrained using the p - lincs algorithm. above this slab 538 tip4p/2005 water and 230 opls / ua methanol molecules were randomly placed in order to create a liquid film on the kaolinite surface. the standard lorentz berthelot mixing rules were used to compute cross - interactions, except to adjust the adsorption energies as detailed below. using the gromacs 4.5 simulation package, constant volume and temperature dynamics were propagated using a leapfrog integrator and a nos - hoover chain thermostat, along with replica - exchange among eight replicas with temperatures ranging from 275 to 310 k in 5 k intervals. real - space interactions were truncated at 9 with corrections to the energy applied, and particle - mesh ewald was used to account for long - range electrostatics with the corrections for the slab geometry of the system. a time step of 2 fs was used and molecular dynamics simulations performed for at least 11 ns, with the first nanosecond being disregarded as equilibration. adsorption energies were computed after geometry optimization using the complex - minus - fragments method. this was done in three ways: first, the adsorption energy was computed by applying the standard mixing rules. this yielded adsorption energies of 642 and 640 mev for water and methanol, respectively, and eads = 2 mev. second, the strength of the lennard - jones interaction between the ch3 group of methanol and the oxygen atoms of the kaolinite oh groups was adjusted such that eads matched that of pbe. finally, the same was done to match eads obtained by dmc. water adsorption on the hydroxyl - terminated face of kaolinite has been studied experimentally and theoretically, whereas adsorption on the silicate - terminated face is less well studied. very little is known about methanol adsorption on either face. in the following, the most stable adsorption structures identified for water and methanol on the two kaolinite surfaces are presented. on each surface a range of adsorption sites were considered, as indicated in figure 1. according to the number of h - bonds formed between the adsorbate and the surface, the adsorption sites can be classified into three categories: 3-fold, 2-fold, and 1-fold sites. the most stable configurations obtained, using dft with the pbe - d3 functional, are shown in figures 2a2h. these structures have been taken as the reference for dmc, lrdmc, and the other dft calculations. in addition, starting from the reference pbe - d3 structures, we have relaxed the geometries for each of the different functionals considered, as shown in figures 3a3d. figure 3e compares the distance of the molecules from the slab as obtained with different functionals. adsorption of water and methanol on the hydroxyl - terminated and the silicate - terminated faces of kaolinite (side view in first row, top view in second row). geometries are relaxed using the pbe - d3 functional and have been taken as reference for the other calculations. the adsorbed molecule on kaolinite is depicted in cyan and gray, and the h - bonds are represented by the blue dashed lines. panels a, b, c, and d show the most stable dft structures for the adsorption of water and methanol on the hydroxyl- and silicate - terminated faces of kaolinite, provided by the different xc functionals considered. the color scheme for the various functionals is blue for pbe, cyan for rpbe, white for pbe - d2, black for pbe - d3 (that is also the reference for qmc calculations), pink for pbe+vdw(ts), violet for vdw - df, green for vdw - df2, gray for optpbe - vdw, and yellow for optb86b - vdw. panel e shows the height of the center - of - mass of the adsorbed molecules from the average surface plane defined by the surface oxygens for the different xc functionals. the four dashed horizontal lines correspond to the values for the reference pbe - d3 structures. concerning water at the hydroxyl - terminated face (h2o on aloh), all structures initially put in 2-fold and 1-fold sites (a5a8) moved to the 3-fold site a1. this preference for the a1 site agrees with previous dft studies with local and semilocal xc functionals. in the most stable configuration, shown in figures 2a and 2e , the c2 axis of the water molecule lies almost parallel to the plane of the surface. the water molecule donates one h - bond (oh distance of 1.69) to and accepts two h - bonds (2.01 and 2.04) from the surface (pbe - d3 values). let us now consider the adsorption of methanol at the hydroxyl - terminated face (meoh on aloh). one way of viewing methanol is as a water molecule with one of its hydrogen atoms replaced by a methyl group. this leads to two possible types of interaction with the surface: (i) hydrogen bond formation with the hydroxyl functional group and (ii) dispersion interactions arising from the ch3 group. all calculations in which the methanol began parallel to the surface ended with the methanol perpendicular to the surface, maximizing the distance between the ch3 group and the kaolinite. the ch3 group can therefore be considered a spectator group that does not participate directly in the adsorption on the surface. the adsorption of methanol is therefore very similar to that of water, and indeed, we find that a1 is the most favorable site, with the methanol donating one h - bond to and accepting two from the surface. as was the case for the water structure, the h - bond donated by the methanol is much stronger than the two h - bonds it accepts: 1.68 vs 1.97 and 2.03 , respectively, with the pbe - d3 functional. the most stable configuration is shown in figures 2b and 2f. as noted above, adsorption of water at the silicate - terminated face (h2o on sio2) of the six adsorption sites (s1s6) considered here, the 1-fold s5 site turned out to be the most stable at the gga level, and the 2-fold s1 generally is the most stable for the vdw - corrected and vdw - inclusive functionals. the most stable structure found for methanol at the silicate - terminated face (meoh on sio2) using the pbe - d3 functional is shown in figures 2d and 2h. the leading interaction here is dispersion; there is no h - bond - like interaction because the oh group of the methanol is parallel to the surface of the slab. the dmc and lrdmc for water and methanol adsorption on the two faces of kaolinite are reported in table 1. as mentioned in the previous section, dmc and lrdmc evaluations have to be corrected for finite - size effects, and in our lrdmc simulations there is also an unphysical dipole interaction between slabs due to the 3d periodicity employed. the dmc calculations have been performed with 2d periodicity and so do not suffer from the latter problem. the bare and corrected it can be seen that our best estimates of the adsorption energy of water on the hydroxyl - terminated face are 648 18 mev with dmc and 675 14 mev with lrdmc. for methanol our best estimates of the adsorption energy are 694 18 mev with dmc and 701 13 mev with lrdmc. we notice that we are in the chemisorption regime both for water and for methanol, although the adsorption energy of methanol is slightly larger. note that for both molecules the dmc and lrdmc evaluations are in good agreement, with the differences falling within the stochastic error of the evaluations. this shows that fixed - node projection qmc schemes are robust approaches: they are only slightly affected by the actual computational setup and implementation. nonetheless, two slightly different adsorption energies for each case are obtained, and we should choose only one of them to use as our benchmark. we feel that in the specific case considered here the dmc values are likely to be more reliable since they have been obtained in 2d, as opposed to the lrdmc which have been corrected for the dipole in the 3d cell. moreover, the reported lrdmc evaluations use eq 1, which has larger fse than the reported dmc evaluations, which use eq 2. as discussed in the text, bare dmc and lrdmc are affected by finite - size errors (see section 3 and table s2 in the supporting information) that we have estimated and corrected the adsorption energies for accordingly. dipole interaction between the periodic slabs (because in this case 2d periodicity was not available, and we had to use 3d periodicity), thus we have included a dipole interaction correction. having compared the of the two qmc approaches on the hydroxyl - terminated face, we have only performed a dmc evaluation on the silicate - terminated face. the dmc adsorption energy at the silicate - terminated face is 184 23 mev for water and 250 18 mev for methanol. the methanol adsorbs more strongly than water, as for the hydroxyl - terminated face; however, in this case the adsorption is weaker, and we are in the physisorption regime. we now examine how the various dft xc functionals considered in this study perform for water and methanol adsorption on the two faces of kaolinite. in table 2 and figure 4 we summarize the adsorption energies obtained with the different density functionals. at the gga level pbe and rpbe give significantly different adsorption energies, with rpbe providing a value that is roughly 50% that of pbe. in line with the smaller adsorption energy we also see that the bonds the molecule makes with the surface with the rpbe functional are considerably longer than what is obtained with pbe. specifically, with rpbe the two h bonds accepted from the surface are 2.30 and 2.36 , and the one donated is 1.81 , versus 2.03, 2.06, and 1.70 with pbe. including dispersion interactions does not drastically change the geometry of the adsorbed water monomer at the hydroxyl - terminated face: the bond lengths at the pbe - d2, pbe - d3, pbe+vdw(ts), and opt - b86b - vdw level are slightly shortened, but they remain within 0.05 of the pbe structure. pbe - d2 predicts the shortest distance from the surface and the shortest h - bonds. the other functionals give h - bond lengths between the values provided by pbe and rpbe. from the shortest to the longest interaction distance, the functionals are ranked in the following order: pbe - d2 < pbe - d3 optb86b - vdw pbe+vdw(ts) < pbe < optpbe - vdw < vdw - df2 < vdw - df < rpbe. this trend roughly follows the sequence of adsorption energy predicted by the functionals and is also consistent with previous studies of dft xc functionals for hydrogen - bonded systems. relaxation from the pbe - d3 geometry (performed for all the gga and gga+vdw functionals) in rather small increases in adsorption energies. the maximum difference is observed for vdw - df, with an increase of 36 mev upon relaxation. energies have been obtained on pbe - d3 optimized structures, but in parentheses we also report the adsorption energies when geometries are fully relaxed consistently for each gga and gga+vdw functional. adsorption energies on kaolinite obtained by various xc functionals and dmc, for water on the hydroxyl - face (h2o on aloh, blue upper triangles), methanol on the hydroxyl - face (meoh on aloh, red squares), water on the silicate face (h2o on sio2, cyan lower triangles), and methanol on the silicate face (meoh on sio2, orange diamonds). filled points represent the values with the reference structures (obtained using pbe - d3), and empty points (reported only for gga and gga+vdw functionals) correspond to relaxed structures for the specific functional. the solid lines are the reference dmc adsorption energies, and the shaded areas show the stochastic error. a comparison with the qmc adsorption energies shows that vdw - df2 and optpbe - vdw yield the best agreement, with the former providing a slightly underestimated adsorption energy (by 32 18 mev) and the latter a slightly overestimated one (by 41 18 mev). it also appears that the two gga functionals (pbe and rpbe), the two hybrid functionals (pbe0 and b3lyp), and vdw - df underestimate the interaction energy, whereas all the other functionals (pbe - d2, pbe - d3, pbe+vdw(ts), optb86b - vdw, pbe0-d3, and b3lyp - d3 ) overestimate the interaction. in particular , evaluations of the adsorption using vdw - corrected hybrid functionals do not seem to improve significantly compared to the gga+vdw approaches. a careful investigation shows that the ordering of the functionals according to the h - bond lengths and to the adsorption energy is almost the same as that observed for water. the only exception is vdw - df, which for methanol gives a larger adsorption energy than that obtained with pbe. the comparison with dmc confirms, as for water, that the best performing functionals are vdw - df2 and optpbe - vdw. again the gga functionals and vdw - df underestimate the adsorption energy, while all the other functionals (pbe - d2, pbe - d3, pbe+vdw(ts), optb86b - vdw, pbe0-d3, and b3lyp - d3 ) overestimate the interaction. as for the previous system, vdw - corrected hybrid functionals do not seem to improve significantly with respect to the gga+vdw approaches. before discussing adsorption on the silicate face of kaolinite an important finding from the presented in table 2 is that with the gga functionals the adsorption energies of water and methanol are similar (e.g., eads, pbeh2o = 608 mev and eads, pbemeoh = 616 mev), but upon inclusion of dispersion interactions methanol is stabilized to a greater extent (e.g., eads, optpbe vdwh2o = 699 mev and eads, optpbe this is apparent in figure 5, where the difference in adsorption between water and methanol is plotted . therefore, even though the methyl group is considered a spectator, its vdw interaction with the surface is non - negligible, and it is clearly desirable to properly account for dispersion interactions in these systems . dmc confirms the reliability of the vdw - inclusive functionals on this issue as dmc also finds that methanol binds more strongly than water . difference in adsorption energy, between water and methanol on the hydroxyl - terminated ( left panel) and silicate - terminated (right panel) faces of kaolinite, as obtained from various xc functionals and dmc. filled points represent the values for the configurations optimized using pbe - d3, and empty points (reported only for gga and gga+vdw functionals) correspond to relaxed structures for the specific functional. the solid lines are the reference dmc adsorption energies, and the shaded areas show the stochastic error. in table 3 we present the from all the functionals for adsorption on the silicate - terminated face. irrespective of which functional is used the adsorption energies obtained are in the physisorption regime. consequently, the inclusion of vdw forces is expected to have a more obvious impact than on the hydroxyl - terminated face. energies have been obtained on pbe - d3 optimized structures, but in parentheses we also report the adsorption energies when geometries are fully relaxed consistently for each gga and gga+vdw functional. as on the hydroxyl - terminated face, rpbe gives an adsorption energy that is noticeably less exothermic than pbe. in the case of the vdw - dfs we see an across - the - board stabilization relative to the gga functionals. like at the hydroxyl - terminated face, vdw - df and vdw - df2 the pbe - d2 and pbe+vdw(ts) functionals give the strongest overall adsorption energy, with 276 and 273 mev, respectively, followed by optpbe - vdw, pbe - d3, and optb86b - vdw with values close to 250 mev. overall, the spread of the vdw - based evaluations is much smaller than for the hydroxyl - terminated face. whereas at the hydroxyl - terminated face the adsorption structure was altered only moderately upon inclusion of vdw, at the silicate - terminated face more significant changes are observed. specifically, the gga functionals predict the molecule to be much further away from the surface than the vdw inclusive functionals do. this difference is also reflected in figure 4, if we consider the difference between the adsorption energies of the gga functionals at the pbe - d3 geometry and when the structures are relaxed. on the other hand, the geometries provided by the vdw - inclusive approaches are in very good agreement with pbe - d3, so eads evaluated on either the pbe - d3 geometry or on the relaxed structures are similar. comparison with dmc supports the general reliability of the vdw - corrected and vdw - inclusive approaches over the bare gga and hybrid functionals. however, it also shows that almost all the gga+vdw and the two hybrid+vdw functionals overestimate the adsorption energy. similar overestimates have been seen recently for physisorbed water on hexagonal boron - nitride. on this surface the best performance is seen for the vdw - df and the vdw - df2 functionals, both of them being in agreement with dmc, given the dmc stochastic error. it is also interesting to note that even though water still binds preferentially to the hydroxyl - terminated face the relative adsorption strengths are significantly altered: the ratio eadsh2o@aloh / eadsh2o@sio2 is 6.6 for pbe, 9.3 for rpbe, ca. 3 for the vdw - inclusive functionals, and 3.5 0.5 at the dmc level. similar to water, we again expect dispersion interactions to play more of a role at the silicate - terminated than at the hydroxyl - terminated face. indeed, we again observe big differences between functionals including or not the vdw interaction. in the most stable structure found using the pbe - d3 functional there is no hydrogen - bond - like interaction, and methanol is parallel to the surface of the slab (see figure 2d). the geometry at the gga level has the methanol molecule found at a much larger distance from the surface, as depicted in figure 3d. as on the hydroxyl - terminated face, the degree of stabilization due to dispersion interactions is greater for methanol than it is for water. at the gga level, water and methanol bind with similar interaction strengths (methanol binds more strongly by 27 mev at the pbe level), but when vdw is accounted for in the functional we observe that methanol binds more strongly by 64 mev (for pbe - d2) to 121 mev (for optpbe - vdw), as shown in figure 5. the comparison with dmc shows that in this case the gga functionals underestimate the adsorption energy and that all the gga+vdw and hybrid+vdw functionals overestimate eads. the best agreement is again obtained for the vdw - df and vdw - df2 functionals, the former overestimating the interaction by 47 18 mev and the latter by 42 18 mev. the ratio eadsmeoh@aloh / eadsmeoh@sio2 is 5.2 for pbe, 2.1 for rpbe, between 2.1 and 2.6 for the vdw - corrected and vdw - inclusive functionals, and 2.8 0.2 at the dmc level. in this paper we have used qmc to examine the adsorption of water and methanol on the hydroxyl- and silicate - terminated faces of kaolinite. the qmc on the hydroxyl - terminated face have been obtained independently with two different fixed - node projection qmc methods: dmc and lrdmc. the two methods differ in terms of algorithms (dmc is based on a time - discretization approximation, lrdmc on a space - discretization approximation), implementation (dmc calculations have been performed using the casino code ; lrdmc using the turborvb code), and setup (for instance, different pps, basis sets, and jastrow terms). nonetheless, both approaches produce in good agreement with the small differences between the approaches coming within the stochastic error of the evaluations. qmc indicate that both water and methanol adsorb on the hydroxyl - terminated face, forming three h - bonds, with an interaction energy larger than 0.6 ev. the adsorption on the silicate - terminated face is much weaker, smaller than 0.3 ev. in both cases as discussed, the qmc provide a benchmark that can help in further understanding of how other computationally cheaper methods perform for adsorption. specifically, we have compared them with the provided by a selection of commonly used xc functionals in dft (covering gga, hybrid, vdw - corrected gga, vdw - corrected hybrid, and vdw - inclusive functionals). this shows that the vdw - corrected and vdw - inclusive functionals predict adsorption energies that are considerably larger than those calculated using the bare gga or hybrid functionals, but the degree of stabilization is system dependent. as discussed, in the systems under consideration in this work the qmc references indicate that bare gga and hybrid - based predictions are often underestimated, whereas approaches that account for the vdw interaction yield in qualitative agreement with qmc, although the absolute value of the adsorption energy can be overestimated, particularly on the silicate - terminated face. overall, the best are provided by vdw - df2, and among the vdw - corrected approaches we notice good performance from pbe - d3. inclusion of exact exchange does not appear to lead to any improvement for the systems considered here; for instance, from pbe - d3 and pbe0-d3 are almost identical. the gga+vdw functionals, although based on gga, perform better than the bare ggas also in terms of geometries. indeed, on the silicate - terminated face (where the interaction is weaker) structure relaxation performed with the vdw - corrected and vdw - inclusive functionals leads to very similar configurations, whereas with the ggas the adsorbates sometimes strayed away from the surface. looking forward there certainly still seems to be scope for further improvements in the treatment of these systems with dft. of the functional considered vdw - df2 offers the best performance, but it does not convincingly deliver chemical accuracy for all four adsorption scenarios considered. approaches such as hamada s revised vdw - df2 functional or tkatchenko s many - body dispersion would be interesting to explore. the comparison between the adsorption of water and methanol is also interesting. at the gga level there is very little difference in adsorption energies, whereas methanol becomes more strongly bound when vdw interactions are accounted for. as clay minerals can cleanse groundwater through the uptake of pollutants, the relative adsorption energies with respect to water is a highly important quantity. even for methanol, which is one of the simplest organic molecules able to form a hydrogen bond , we see that including vdw interactions can significantly alter the adsorption energy relative to water; on the hydroxyl - terminated face, water and methanol bind with similar energies, but inclusion of dispersion forces tips the balance in favor of methanol. before closing we note that we have examined the consequences of altering the relative interaction strength of water and methanol with kaolinite through a series of classical molecular dynamics simulations of liquid water specifically in figure 6b we show obtained with water and methanol interaction parameters that use standard lorentz berthelot mixing rules, values matching pbe, or values matching dmc. as can be seen figure 6b, with the dmc value of eads the adsorption of methanol yields a density profile with a much more pronounced first peak compared to the eads, corresponding to pbe or standard lorentz thus, we see that the standard approach for exploring aqueous solutions at a clay surface with force fields leads to a rather poor description of the interface. this effect is likely to become even more significant as the size of the organic tail of the adsorbate increases and demonstrates the importance of an accurate modeling of dispersion interactions when exploring wet interfaces of environmental relevance. the ability to accurately incorporate nonlocal dispersion interactions is therefore extremely important if one aims to model environmentally relevant adsorption processes on kaolinite and other clays. molecular dynamics simulations of a water methanol mixture on the hydroxyl - terminated face of kaolinite. panel a: snapshot of the simulation system, where the methanol molecules are shown in red and the water molecules as glassy blue circles, and the kaolinite slab is the same as in figure 1. panel b: density profiles of methanol above the kaolinite surface, for the eight replicas with temperatures ranging from 275 to 310 k, obtained for values of eads (namely, the difference between adsorption energy of water and methanol ( see text) ) corresponding to lorentz z is the distance from the average position of the top layer of oxygen atoms in the kaolinite surface. it is clear that an appropriate choice of eads can significantly affect the density of the water	clay minerals are ubiquitous in nature, and the manner in which they interact with their surroundings has important industrial and environmental implications. consequently, a molecular - level understanding of the adsorption of molecules on clay surfaces is crucial. in this regard computer simulations play an important role, yet the accuracy of widely used empirical force fields (ff) and density functional theory (dft) exchange - correlation functionals is often unclear in adsorption systems dominated by weak interactions. herein we present from quantum monte carlo (qmc) for water and methanol adsorption on the prototypical clay kaolinite. to the best of our knowledge, this is the first time qmc has been used to investigate adsorption at a complex, natural surface such as a clay. as well as being valuable in their own right, the qmc benchmarks obtained provide reference data against which the performance of cheaper dft methods can be tested. indeed using various dft exchange - correlation functionals yields a very broad range of adsorption energies, and it is unclear a priori which evaluation is better. qmc reveals that in the systems considered here it is essential to account for van der waals (vdw) dispersion forces since this alters both the absolute and relative adsorption energies of water and methanol. we show, via ff simulations, that incorrect relative energies can lead to significant changes in the interfacial densities of water and methanol solutions at the kaolinite interface. despite the clear improvements offered by the vdw - corrected and the vdw - inclusive functionals, absolute adsorption energies are often overestimated, suggesting that the treatment of vdw forces in dft is not yet a solved problem.
it is caused by viruses of the genus flavivirus, family flaviviridae, and group iv ssrna. dengue is transmitted to humans by the mosquito aedes aegypti and is one of the most rapidly spreading viral infections. there has been a 30-fold increase in its incidence in the last 50 years coupled to increasing migration from rural to urban areas. annually dengue remains a major health concern for southeast asian countries with cyclic epidemics. in india multiple viral serotypes are circulating and some regions have case fatality rates of 35% in general population, which is much higher than other southeast asian regions (1%). literature suggests an increased risk of maternal hemorrhage, preterm labour, and oligohydramnios. clinical presentation of dengue may be confused with hellp syndrome, both conditions having elevated liver enzymes, haemolysis, and low platelet counts; however serology helps in distinguishing the two conditions. as reported in previous studies, dengue is associated with preterm births, low birth weight, fetal deaths, and vertical transmission leading to neonatal thrombocytopenia requiring platelet transfusions. in a quest to answer vital issues we critically analysed dengue antigen positive cases in our institute and studied maternal and fetal outcomes. this study was conducted in the department of obstetrics and gynaecology of a tertiary level government hospital, delhi, india. during the months of september and october 2015 , 60 pregnant women were seen in the emergency room with complaint of fever and were admitted for evaluation. as per protocol, dengue pcr (ns1ag) clinical grading was done according to who classification and case definitions. an acute febrile illness with two or more clinical manifestations like headache, retroorbital pain, myalgia, arthralgia, rash, hemorrhagic manifestation, or leukopenia and positive serology or occurrence at a time of dengue outbreak was taken as the definition of dengue fever (df). dengue hemorrhagic fever (dhf) was classified as fever, hemorrhagic tendencies, thrombocytopenia, evidence of plasma leakage, association of hepatomegaly, and circulatory disturbances. increase in serial hematocrit was taken as evidence of plasma leakage and ultrasound showing pleural effusion, ascites, and gall bladder edema were taken as supportive evidence. dengue shock syndrome (dss) was classified when dhf symptoms included rapid and weak pulse, narrow pulse pressure of less than 20 mmhg, and hypotension. patients were managed with antipyretics, adequate hydration, and blood product transfusion as necessary. obstetric data, clinical, laboratory parameters, and maternal and fetal outcomes of women are depicted in table 1. a majority of women were unbooked and presented with classical symptoms of high grade fever, myalgia, headache, and rashes to the emergency room. all women were young, age ranging from 22 to 32 years; mean age was 25 years. out of 16 cases 13 presented in third trimester and two in second trimester and one was referred after lscs was done in another hospital. she presented with large rectus sheath hematoma following caesarean section needing surgical exploration and drainage. none of the women had any underlying diseases or comorbidity except one (# case 5) who had moderate anemia on admission. in the current study, eight women (50%) had dengue haemorrhagic fever (dhf). ten women required transfusion to maintain platelet count in desired range; on average each woman required six platelet transfusions. most women had low platelet counts at the time of admission (mean 81,000/mm); 62% had platelets less than 50,000/mm. platelet count less than 30,000/mm was seen in dengue shock syndrome with increased risk of bleeding manifestations. a sri lankan case series reported 10 women with dhf but only eight required platelet transfusion. three women succumbed to dengue shock syndrome in current study with a mortality rate of 18% compared to 35% case fatality rate in rest of the india in nonpregnant population. in another study done in india by agrawal et al. symptomatic dengue infection may increase the risk of preterm labour and, hence, low birth weight (lbw) as suggested by previous studies. in the study under discussion most women presented near term (n = 10) and of the four who presented in early gestation two had preterm labour. preterm births reported were as low as 11% in the french guiana study versus 41% in the study by basurko et al. one woman had spontaneous abortion (6%) and seven (43%) pregnancies were complicated with oligohydramnios in present study compared to 52% rate of oligohydramnios from a study in india by agrawal et al.. bleeding manifestations were seen in seven women and three (19%) had postpartum hemorrhage (pph) (# 2, 11, and 13). one of them was diagnosed as dss and the other two were diagnosed as df. both the patients did not have any other bleeding manifestations and signs of plasma leakage hence were classified as only df. in retrospective study by basurko et al. on 53 pregnant women, pph was reported in 10% of the cases compared to 19% in our study. there were six neonatal intensive care unit (nicu) admissions (37%), three intrauterine deaths (iud) (18%), and one neonatal death (6%). none of the neonates in our study showed symptoms of dengue such as thrombocytopenia, anemia, fever, or bleeding manifestations. there are a number of studies on poor fetal outcome following dengue infection in parturient females. they also reported iud rate of 3.8% and neonatal death rate of 1.9% while carles et al. pregnant women with dengue fever should be considered for admission early because of its unpredictable course. oligohydramnios is an ominous sign when seen concomitantly with dengue infection because the high fetal mortality was associated with it. the exact cause of oligohydramnios is not known but dehydration associated with dengue fever may be contributory. hydration should be maintained by encouraging oral intake of oral rehydration solution (ors), fruit juice, and other fluids containing electrolytes and glucose for replacing losses from fever and associated vomiting. another dilemma faced was in differentiating dengue infection from hellp syndrome as both have somewhat similar laboratory parameters; however serology will help clinch the diagnosis. careful examination and high index of suspicion by the treating obstetrician are required to diagnose and manage such cases especially during epidemics. our knowledge on effect of dengue on pregnancy is somewhat limited. in this study dengue awareness programs and medical education programs on the management of the dengue in pregnancy should be initiated especially during outbreaks to provide quality care.	dengue is a vector transmitted viral infection; tropical and subtropical countries see outbreaks of dengue each year. there is a paucity of literature on effects of dengue infection on pregnancy outcome and this prompted us to undertake a study for better understanding of pregnancy implications with dengue infection. pregnant women admitted during the seasonal outbreak of dengue between september 2015 and october 2015 were studied and maternal and fetal outcomes in sixteen ns1ag positive women were analysed. out of sixteen women diagnosed with dengue fever, three had dengue shock syndrome (dss) and eight had dengue haemorrhagic fever (dhf). the most common obstetric complication seen in 43% of the cases was oligohydramnios. bleeding manifestations occurred in seven women and there were three maternal deaths. perinatal complications included three intrauterine deaths, six nursery admissions, and one neonatal death. thus dengue infection was associated with high maternal and perinatal mortality. in view of poor obstetric outcomes, this viral infection warrants early admission and prompt management.
approximately 70% of patients with multiple myeloma have urinary m protein spike and about 2025% have kidney disease at the time of diagnosis. up to half of myeloma patients demonstrate renal involvement during the course of their disease. myeloma kidney is implicated in about 41% of cases and is distinctive for multiple myeloma. monoclonal immunoglobulin deposition disease, primary amyloidosis, proximal and distal tubulopathy, renal vein thrombosis, type 1 cryoglobulinemia, proliferative glomerulonephritis, interstitial nephritis, plasma cell infiltration, urate deposition disease, pyelonephritis and nephrocalcinosis represent less common forms of renal involvement. rapidly progressive glomerulonephritis in multiple myeloma is rare; cases reported thus far typically demonstrate proliferative glomerulonephritis and/or glomerular deposits on renal biopsy. we here report a case of antineutrophil cytoplasmic antibodies (anca)-negative, pauci - immune, chronic sclerosing, crescentic glomerulonephritis without deposits on electron microscopy, which was discovered at the time the patient presented with multiple myeloma and which ed in end - stage kidney disease. a 57-year - old hispanic woman was admitted with nausea, vomiting and weakness of 1 week duration. she described herself to be in good health but had not seen a doctor for 15 years. her history was only significant for pregnancies and visits to the emergency room for epistaxis 3 years prior to admission and after a fall 6 years prior to admission. her father died from malignancy, type unknown. physical examination on admission was normal except for pallor. the patient was started on hemodialysis on admission and was transfused with 2 units of packed red blood cells for severe anemia. urinalysis was positive for microscopic hematuria, and the patient had positive urine m spike with faint light chain on serum immunofixation (table 1). renal biopsy on day 4 of admission was consistent with chronic sclerosing pauci - immune glomerulonephritis (fig . initial bone marrow biopsy showed monoclonal plasmacytosis ( 12%) consistent with plasma cell myeloma. there was no evidence of increased blasts, monoclonal b - cell or aberrant t - cell process. the patient was eventually discharged home with hematology / oncology follow - up and remains on outpatient hemodialysis. she was started on high - dose dexamethasone a few days after discharge and bortezomib 1.3 mg / m twice weekly after hemodialysis approximately 1 month after discharge. she was readmitted 3 months later with fever and symptoms of upper respiratory tract infection and was treated with antibiotics. the immunological investigations were repeated because of the rarity of multiple myeloma presenting with pauci - immune crescentic glomerulonephritis. however, repeat serum protein electrophoresis showed no m spike, and serum immunofixation showed a polyclonal pattern. spot urine protein was 834 mg / dl, the urine creatinine concentration was 24 mg / dl, the urine albumin concentration was 35 mg / dl and the urine protein / creatinine ratio was 35. a repeat bone survey showed mild generalized osteopenia, but it revealed no focal lesions. a follow - up bone marrow biopsy after 4 months of bortezomib and she continues to require hemodialysis despite reduction of the free light chain burden and disappearance of monoclonal plasma cells in the bone marrow. crescentic glomerulopathy is a rare complication of multiple myeloma first described by kaplan and kaplan in 1970 in a 49-year - old patient with renal failure due to igg myeloma. three cases of extracapillary proliferative glomerulonephritis were reported by meyrier et al., of which two were due to plasma cell dyscrasias and one was due to waldenstrom's macroglobulinemia. described a patient with diabetes mellitus who developed rapid progressive renal failure over the course of 5 weeks that led to chronic hemodialysis caused by light chain myeloma. reported a 60-year - old male patient who had a relapse of rapidly progressive glomerulonephritis 10 months after an initial episode that responded to cyclophosphamide and steroid therapy. this time, he had an extensive investigation that revealed light chain monoclonal gammopathy treated with 2 injections of rituximab followed by 2 cycles of bortezomib therapy ing in clinical remission. perhaps, if she had presented early, she may have recovered her renal function with bortezomib and dexamethasone treatment. the exact mechanism of renal injury in this case remains unclear and needs further investigation since there were no demonstrable immune complexes, monoclonal deposition disease or cast nephropathy. one possible theory could be a form of direct toxic injury to the glomerular basement membrane by the free light chains with subsequent disruption of the basement membrane and formation of fibrocellular crescents. whether this is preceded by extracapillary and/or endocapillary there could also be concurrent direct toxic injury to tubular cells by light chains and interstitial nephritis. this case highlights an atypical renal pathologic manifestation of multiple myeloma and underscores that multiple myeloma should be considered in the differential diagnosis of rapidly progressive glomerulonephritis, especially if there is heavy proteinuria. in such patients, early screening for serum free light chains followed by renal biopsy and bone marrow biopsy can establish the diagnosis and guide therapy the authors declare that no financial or other conflict of interest exists in relation to the content of the article.	pauci - immune crescentic glomerulonephritis (picgn) is most commonly associated with antineutrophil cytoplasmic antibodies (anca). we report a case of chronic, sclerosing anca - negative picgn discovered when a patient presented with multiple myeloma. a 57-year - old woman presented with complaints of nausea, emesis and weakness. she was found to be in renal failure with a serum creatinine of 9.4 mg / dl, mild hyperkalemia and acidosis. she was noted to have normochromic, normocytic anemia with normal platelet and white cell counts, normal plasma proteins and serum protein electrophoresis. further studies revealed increased concentrations of and light chains in a ratio of 34.89; a bone marrow biopsy found 12% plasma cells. serum protein electrophoresis revealed no spike. anca, anti - glomerular basement membrane, antineutrophil antibody, hepatitis panel and serum complements were normal. a kidney biopsy showed chronic sclerosing picgn plus tubular necrosis, severe tubular atrophy, interstitial fibrosis and severe arteriosclerosis. congo red stains were negative and electron microscopy showed no intraglomerular deposits. the patient was subsequently treated for myeloma with bortezomib and dexamethasone with good hematologic response but never recovered renal function. she remains on outpatient hemodialysis. renal manifestations of myeloma often involve glomerular deposition disease, tubulointerstitial disease, with characteristic proteinaceous casts, or both. in contrast, our patient demonstrated neither of these findings but had chronic sclerosing picgn. crescentic glomerulonephritis occurring in patients with plasma cell dyscrasias has been previously reported, but the association remains extremely rare.
methods of topical fluoride application include professional topical application and home use of fluoride dentifrice and fluoride mouth rinse. professional topical application is a means of preventing dental caries in both deciduous and permanent teeth and is performed by dental specialists (dentists and dental hygienists) in dental clinics, health care facilities, and other appropriate settings specializing in dental care. advantages of professional topical fluoride application include the ability to treat patients starting from a very young age, immediately after the deciduous anterior teeth erupted, in addition to the fact that application is necessary only two to four times a year. in addition, the ingestion of fluoride from the professional topical fluoride application is not considered to be a risk factor in dental fluorosis. the fluoride agents in professional topical application are available in liquid, gel, and foam types. because the gel and foam are generally applied using a tray, these have an advantage in that the fluoride can be applied to all of the teeth simultaneously. in the united states and a number of other countries, the tray method using acidulated phosphate fluoride (apf) foam is widely used, and the foam application procedure, including the amount of foam used, is standardized. in japan, the ministry of health and welfare has issued guidelines governing professional topical application of fluoride, which describes a method for applying the neutral and acidic naf liquids that were initially introduced in japan in the 1960s. a method for applying apf gel, which was subsequently introduced in the 1980s, has also been established based on fundamental research on factors, such as the amount of gel to be used and the residual amount in the oral cavity. strongly acidic apf has an advantage in that a large amount of uptake into the tooth substance can be expected, but there are also reports expressing concerns about damage occurring to several types of restorative material. these reports underscore the necessity for topical application of neutral naf, and as a , neutral naf foam is now available on the market in japan. however, no information is currently available on aspects such as methods for applying this neutral naf foam or the amount of foam that remains in the oral cavity after application. first, various amounts of foam (butler fluodent foam n, sunstar inc ., osaka, japan) in the deciduous and permanent dentition study model trays (butler tray, sunstar inc ., osaka, japan) were prepared to apply to the deciduous and permanent dentition study models (study model pe - ana004 and study model pe - ana002, respectively, nissin dental products inc ., initially, the weight of tray without the foam was measured by an electronic balance ( libror aeg-45sm, shimazu co., kyoto, japan), then the foam was placed, and the total weight was measured. the amount of fluoride foam was calculated by subtraction of the weight of tray from the total weight. to sufficiently cover all the dried teeth surfaces of the study models and to minimize leakage outside of the dentition, the appropriate amount of foam (aaf) was filled to be adjusted to a level approximately 2 mm below the tray. using the deciduous and permanent dentition study models, after the foam application, the tray was removed from the study model immediately, and the net weight of foam remained in the tray (trf : tray - remained foam) and the net weight foam adhering to the study model were measured, respectively. then the foam adhering the area of mucosa of study model was wiped with a wiping paper (kimwipe s-200, nippon paper crecia co., ltd, tokyo, japan), and the net weight of foam adhering to the surface of study model (trsf : tooth - surface retained foam) was measured. the weight difference between above study models was defined as overflowed foam (of). based on the procedure of experiment 1, an application experiment was carried out in human subjects. eight healthy participants (three males and five females), ranging in age from 21.3 to 24.8 years, were recruited from the students at kanagawa dental college. each subject signed the informed consent form, which had been approved by the ethics committee of kanagawa dental college (no . 44). the inclusion criteria in this study were: with at least 28 natural teeth; unstimulated saliva flow rate > 0.3 ml / min; no orthodontic appliance in their oral cavity. the exclusion criteria were: clinically detectable caries; periodontitis; history of allergies and metabolic diseases such as diabetes; other medical condition that could interfere with the study. subjects were instructed to brush their teeth using a nonfluoride toothpaste starting 3 days before the day of the experiment until the end of the experiment, and all other fluoride applications were suspended. the experiment began at 2:00 p.m. before the naf foam was applied to subjects, their teeth were dried with the compressed air. the trays were filled with naf foam to a level approximately 2 mm below the edge of the trays and were placed over the teeth, the subject was instructed to close the jaws with the trays in contact for 4 minutes. the subject was instructed not to swallow but to allow the saliva to dribble into a 500-ml plastic beaker held directly under the mouth. at the end of the topical application, the trays were removed from the mouth and placed in the same beaker, and the subject expectorated the mixture of saliva and foam into the same beaker immediately after removal of the tray and expectorated once again 30 seconds later. whole saliva was collected into a separate 50-ml plastic vessel for 5 minutes a total of eight times (immediately prior to application, 5 minutes afterwards, 15 minutes afterwards, 30 minutes afterwards, 60 minutes afterwards, 120 minutes afterwards, before the subjects went to bed, and immediately after they awoke the following day). the saliva samples obtained up to 30 minutes afterwards were diluted by double - deionized water at the ratio of 1: 10, and others were not diluted. total ionic strength adjustment buffer (tisab) (tisab ii, orion research inc ., beverly, mass, usa) were added to all saliva samples as the ratio of 1: 1, and the fluoride concentration of saliva was analyzed as ppm using the ion - specific electrode (orion 9609bnwp combination electrode, thermo electron corp . the subject was instructed not to swallow but to allow the saliva to dribble into a 500-ml plastic beaker held directly under the mouth . at the end of the topical application , the trays were removed from the mouth and placed in the plastic beaker, and the subject expectorated the mixture of saliva and foam into the same beaker and expectorated once again 30 seconds later . total amount of 500 ml double - deionized water was added to the beaker . the solution was stirred by the magnetic stirrer until no traces of the foam were visible . then the solution was analyzed for fluoride concentration using the ion - specific electrode ( orion 9609bnwp combination electrode, thermo electron corp . the total amount of fluoride recovered to the oral cavity was calculated as the product of the fluoride concentration and volume . the weight of residual fluoride was calculated by subtracting the weight recovered from the weight applied . in addition, the ratio of residual fluoride in the oral cavity was determined from the percentage of fluoride remaining in the oral cavity in relation to the amount of fluoride used . the test of population mean for the fluoride concentrations in saliva following the foam application and the test of coefficient of correlations between with aaf, trf, trsf, and of in the study models were analyzed ( jmp, ver . using deciduous and permanent dentition study models, we found that in order to sufficiently cover the dentition when applying the naf foam and to minimize leakage outside of the dentition, the tray needed to be filled to a level approximately 2 mm below the tray edge . using this method the showed significant variation in mean appropriate amount of foam ( aaf); that of the deciduous dentition study model was 0.57 0.17 (0.360.86) g (table 1), while that of the permanent dentition study model was 0.96 0.24 (0.681.39) g (table 2). the mean of the amount of foam retained on the tooth surfaces (tsrf : tooth - surface - retained foam) of the deciduous and permanent dentition study models were 0.03 0.01 (0.010.05) g and 0.12 0.06 (0.030.20) g, respectively, (tables 1 and 2). analysis of the coefficient of correlations between the appropriate amount of foam (aaf) and the amount of foam adhering to and recovered from the tray (trf : tray - retained foam), aaf and the amount of foam adhering to the mucosa other than the tooth surface on the models (of : overflowed foam), and aaf and the tooth surface retained foam (tsrf) showed a strong positive correlation between aaf and trf and between aaf and of in both the deciduous and permanent dentition study models (table 3). the mean amount of naf foam applied in each adult subjects was 0.81 0.20 (0.541.12) g. the mean fluoride concentration in saliva following application was 35.95 28.84 (7.4885.80) ppm, which was the maximum value 5 minutes after application, and by 30 minutes after application had dropped sharply. it continued to drop gradually thereafter, but the concentration of fluoride in saliva measured at the time subjects awoke the following morning was relatively high, at 0.12 ppm (figure 1). there were significantly differences in salivary fluoride concentrations up to 120 min following the application compared with 0.05 ppm as criterion value (test of population mean, p < .05). the mean amount of naf foam used was 0.80 0.22 (0.561.16) g. the mean amount of fluoride used was 7.18 1.94 (5.0010.47) mg, and the mean amount of fluoride used per kilogram body weight was 0.14 0.04 (0.090.19) mg f / kg. the amount of fluoride recovered was 5.44 1.75 (3.658.70) mg, and the amount and ratio of residual fluoride in the oral cavity after foam application were 1.74 0.47 (1.322.44) mg and 24.9 5.80 (16.934.40) %, respectively. the amount of residual fluoride in the oral cavity per kilogram body weight was 0.033 0.012 (0.0170.053) mg f / kg (table 4). a strong correlation was observed between the amount of fluoride used per kilogram body weight and the amount of residual fluoride in the oral cavity per kilogram body weight (r = 0.71, p < .05). the fluoride agents in professional topical application are available in liquid, gel, and foam types. currently, the fluoride agents in professional topical application are available in liquid or gel types in japan. accompanying documentation specifies that the amount of professional topical application to be used is 2 ml or less using the paint - on technique. this applies when a liquid or a gel is used but does not apply to foams. in an experiment conducted by whitford et al. in 46 children between the ages of 8 and 12 years , the appropriate amount of apf foam or apf gel used to cover the dentition without leaking into the oral cavity was one - third the depth of the tray, which was 0.89 0.02 g of foam and 3.86 0.06 g of gel. with these , the amount of foam needed to sufficiently cover the dental surface was 23.1% that of gel. using apf gel, the amount of gel used with a deciduous dentition study model was 2.13 0.77 g and that for a permanent dentition study model was 3.94 1.38 g, while the amounts of residual gel on the dental surface after wiping away gel that had leaked from the dentition study models were 0.06 0.03 g and 0.18 0.08 g, respectively. in our experiment, we initially prepared enough foam to reach the tray edge, meaning an amount of foam equivalent to the capacity of the tray. when the prepared foam was applied to the study model, a large amount of foam overflowed from the tray and the model. as a , it was determined that the appropriate amount of foam should be filled to be a level approximately 2 mm below the edge of the tray. the mean amount of foam used with the deciduous dentition study model was 0.57 (0.360.86) g (table 1) and that with the permanent dentition study model was 0.96 (0.681.40) g (table 2). these are 26.7% and 24.9%, respectively, of the amounts of gel reported by arakawa et al. and are largely consistent with the reported by whitford et al.. for the amounts of foam remaining on tooth surfaces, the were 0.03 (0.010.05) g for the deciduous dentition study model and 0.12 (0.030.20) g for the permanent dentition study model (table 4), which was approximately half the amount of gel reported by arakawa et al. in the present experiments, the variations in the appropriate amount of foam were comparatively wide, even though the tray was filled with foam to a level approximately 2 mm below the tray edge, using the same study model and using foam from the same container. the reason of this phenomenon is not clear. in experiments 2 and 3, the tray was filled with foam to a level approximately 2 mm below the tray edge. the mean amount of foam used for the eight adult subjects was approximately the same, 0.80 g. this was largely consistent with the amount of foam used by whitford et al.. this suggests that with the neutral naf foam used in the present experiments, the amount used can be set at a lower amount than that for gel. in research conducted by sudo et al., in which apf gel was applied using the toothbrush method in subjects 1.5 years of age, the mean amount of gel applied was 0.66 g, and the mean amount of residual fluoride in the oral cavity per kilogram body weight was 0.19 mg / kg. the mean residual ratio was 25.8%. in our experiments, the mean amount of residual fluoride in the oral cavity following application of naf foam was 1.74 mg, and the amount per kilogram body weight was 0.033 mg / kg, with a mean residual ratio of 24.9% (table 4). comparing the of this study, the residual ratios in our experiments were largely the same, but because our experiments targeted adult subjects, the residual amount of fluoride per kilogram body weight was extremely small. the amount of fluoride that causes acute toxicity is 1.35 to 1.8 mg / kg or higher. the of our experiment indicate that the maximum value would be 0.16 mg / kg, even in a child weighing 15 kg; therefore, this amount assures adequate safety. where the application of highly acidic dental coatings such as apf is cited as possibly causing corrosion of dental restorative materials such as glass - containing resin composites, glass - ionomer cement, and porcelain, 0.9% neutral naf foam is reported to have little effect on surface hardness of the materials. moreover, in patients who have restorative materials, use of a neutral naf coating is recommended in order to avoid corrosion, discoloration, and other problems with the restorative materials. earlier studies have reported that when fluoride products such as dentifrice, rinse, and gels are used, a high fluoride concentration in the saliva was initially obtained, subsequently it dramatically reduced with time. the of this research showed that the concentration of fluoride in saliva immediately following foam application was extremely high and still be elevated at 1.2 ppm f in the next morning (figure 1). the retention of fluoride in saliva may be very important in the prevention or reversal of caries. / l ) in saliva are sufficient to effectively inhibit demineralisation and/or enhance remineralisation of enamel. it seems that this 2% neutral naf foam is effective in prevention of caries immediately after the application because the salivary fluoride concentration up to 120 min is higher than 0.05 ppm. based on the of the research described here, using a 2% neutral naf foam enables application using the tray method with smaller amounts of fluoride. foam types of fluoride agents are readily dispersed within the oral cavity and are easier to apply than other forms, even when patients have fixed orthodontic appliances present. furthermore, in vitro research has shown no significant differences between apf gel and foam in the amount of uptake into enamel, and while foam could provide an effect similar to that of gel in terms of preventing dental caries , there has not yet been any clinical research showing that the professional topical application of fluoride using 2% neutral naf foam prevents dental caries. in this research, the number of subjects might not be enough large, it caused the large values of standard deviation in the . further research which included not only adults but also children as subjects is necessary to investigate the appropriate frequency of use and the effects in preventing dental caries. the present found the following: when carrying out professional topical fluoride application using 2% neutral naf foam with a dedicated tray, filling the tray with foam to a level approximately 2 mm below the tray edge was appropriate, and this amount made it possible to adequately cover all the dentition. the appropriate amount of 2% neutral naf foam used for permanent dentition was approximately 0.8 g on average, which was approximately one - fifth the amount of gel ordinarily used with the tray method. the residual ratio of fluoride in the oral cavity following a 4-minute application of 2% neutral naf foam was approximately 25% of the amount of foam used, and a strong correlation was observed between the amount of fluoride used per kilogram body weight and the amount of residual fluoride in the oral cavity per kilogram body weight.	the objective of the present research was to determine the appropriate amount of fluoride to use professional topical application and the residual amounts of fluoride in the oral cavity using a 2% neutral sodium fluoride (naf) foam with a dedicated tray. using dentition study models, a method for determining the appropriate amount of naf foam was investigated. in eight adult subjects, the appropriate amount of naf foam, the concentration of fluoride in the saliva following professional topical application, and the amount of residual fluoride in the oral cavity following professional topical application was examined. the indicated that the appropriate amount of naf foam was approximately 0.8 g, the amount of residual fluoride in the oral cavity was approximately 25% of the amount of foam used.
iran by a population of 78 million, 30 provinces, 400 districts and more than 65,000 villages comprises of a 65% of the population that are living in urban areas. according to the 4 5-year national development plan the family physician reform should be extended to the whole country. therefore, family physician program was launched for the first time in 2005 in rural areas and cities with a population below 20,000. after that and toward extension of this program in urban areas, two provinces of fars, in the south of iran with a population around 4.4 million, and mazandaran, in the north of iran with an approximate population of 3 million, were selected for pilot this program from 8 of july 2012. similar to any other project, family physician program has its advantage and disadvantages which have to be evaluated in order to get an optimum . this study as the first population - based study in this issue, aimed to measure the knowledge and practice of people lives in shiraz toward family physician program to present an evidenced - based feedback to national and regional policy makers to improve planning and management of this program. this cross - sectional, questionnaire - based study was conducted from october to december 2014 in shiraz, south of the iran. the sample size was calculated as 1382, supposed level of knowledge of people toward family physician program as 50%, dropout rate of 20%, design effect of 3, 5% precision level and a confidence level of 95%. multistage randomized and proportional to size sampling was used. in each address, one person who was at least 18 years and was a resident at shiraz for at least 2 years was asked to fill the questionnaire. the coded anonymous questionnaire comprised of a brief introductory paragraph about title, aims identification and phone call number of the executor of this study, followed by consent form that emphasized on voluntary participation and keeping confidentiality. they asked about their demographic and socioeconomic information including age, gender, level of education, marital status, job status, position in the family (as breadwinner of family or other family member), number of family members and monthly income. being under the coverage of main and supplementary insurance systems and also family physician program was queried. the questionnaire contained questions about knowledge and items about practice of people regarding urban family physician program. in the knowledge section, participants were asked about choosing and changing family physician, family physician addresses, tasks of family physician, work time of family physician in holidays and nonholidays, address of reference and proper action in cases of having complaints or need to more information, electronic record form, referral form and visit - fee. in the practice section, reference to family physician and nonfamily physician, waiting time in the family physician's waiting room, phone counseling with family physician, average of payment upon each referral to family physician, having problem in obtaining prescribed drugs by family physician, having problem in accessing the specialist family physician and being interviewed and examined completely by family physician in each visit were queried. the questionnaire was validated by two experts in family physician program and its reliability according to cronbach's alpha was calculated as 0.64 through pilot testing of the questionnaire. all data were entered into spss version 20 software (spss, chicago, il, usa). the accuracy of data entry was ensured by randomly selecting and checking completed questionnaires against their corresponding data in the spss software. chi - squared, t - tests, pearson correlation and stepwise linear regression model were used. voluntary participation in this study, designing of an anonymous questionnaire, possibility of access to executives of this study via two exclusive phone lines and keeping confidentiality in all aspects of research were some ethical aspects that were applied. furthermore, the research protocol as described here was approved by the ethics committee of the health policy research center affiliated with shiraz university of medical sciences. voluntary participation in this study, designing of an anonymous questionnaire, possibility of access to executives of this study via two exclusive phone lines and keeping confidentiality in all aspects of research were some ethical aspects that were applied. furthermore, the research protocol as described here was approved by the ethics committee of the health policy research center affiliated with shiraz university of medical sciences. participation rate of participants was 1257 of 1382 (90.9%) and 997 (79.3%) filled the questionnaire at home addresses. mean age of participants was 38.1 13.2 years and of total 1257, 634 (50.4%) were men, 882 (70.2%) were married and 474 (37.7%) had associate or bachelor degree of education. social and demographic characteristics of participants in the population - based study aimed to determine correlates of knowledge and practice toward urban family physician program in shiraz, southern iran (n=1257) six hundred seventeen (49.1%) had job with mean income 1000$/month. the mean family member was 3.9 1.5 and 539 (42.9%) were breadwinners of their families. one thousand hundred nineteen (89%) and 479 (38.1%) were under the coverage of one of the main and supplementary insurance systems respectively. one thousand fifty - eight (84.1%) of respondents and 1012 (80.5%) of their families members were under the coverage of urban family physician program. peoples total knowledge toward urban family physician program was 5 2.7 of 19, showed that 1121 (89.2%) had a low level of knowledge. southern iran (n=1257) of total, 879 (69.9%) of people knew about family physician choosing rules but 880 (69.9%) did not know that, it is possible to change their family physician and 59 (4.6%) stated that family physicians should present both preventive and medical services to their clients. four hundred seventy - fours (37.7%) knew about their family physicians substitutes and 58 (4.6%) were informed about where they should refer in the absence of their family physician. three hundred and fifty - three (28%) and 2 (0.1%) knew correctly about how much they should pay to general and specialist family physician in each visit, respectively. of 1257, 22 (1.8%) knew that where they should refer if need any information or have any complaint about family physician program while 1173 (93.2%) did not know or could not correctly tell the 4 digits phone number of unit responding to complaints about family physician program. a few of people were informed about referral form (233 ; 18.5%), about what they should do with filled referral forms (154 ; 12.3%) and about electronic health record (11 ; 0.8%). family physician office distance to home was less that 1 km in 538 (42.8%) of the responders. univariate analysis showed that knowledge toward family physician program was lower in younger than 30 and older than 60 years people, in males, in singles, in whom with < 8 years of education, in whom that were not under coverage of main or supplementary insurance systems and in whom were not under the coverage of family physician program. single variable analysis of correlates of knowledge and practice of people toward urban family physician program in shiraz south of iran stepwise linear regression model showed that peoples total knowledge toward their rights in urban family physician program by adjusted r 0.18 and constant = 8.5 (95% confidence interval = 7.69.4, p < 0.001) was in order correlated to being under coverage of urban family physician program (= 2, 95% ci = 1.42.5, p < 0.001), being other family member (s) under the coverage of urban family physician program (= 1.1,95% ci = 0.71.6, p < 0.001), being under the coverage of one of the main insurance systems (= 0.5, 95% ci = 0.011, p = 0.04) and being married (= 0.4, 95% ci = 0.10.8, p = 0.002). peoples practice toward urban family physician program has gained mean 2.3 0.9 of total score 7 in this study, showed that 942 (74%) had poor performance while 86 (6.8%) had moderate and 3 (0.2%) had expected level of practice. of total 1257, 882 (70.2%) stated that they became sick during previous year of this study and 700 (55.6%) referred to their family physician, showing 700 of 882 (79.3%) referral rate to family physicians. eighty (6.4%) had phone counseling with their family physician in the similar period. mean number of references to family physicians in whom that were under the coverage of family physician program was 2.4 3.6 in the previous year of this study while mean number of references to nonfamily physicians in whom that were not under the coverage of family physician program was 2.4 4.3 in the same year (p = 0.3). one hundred seventy - eight (14.2%) had changed their family physicians during last year of this study and 152 (20.6%) paid higher than legally approved visit - fee to their family physician. two hundred twelve (16.9%) had problems in providing drugs that were prescribed by their family physicians and 342 (27.2%) had problems in access to specialist family physician. practice and problems of people toward urban family physician program in shiraz, southern iran (n=1257) univariate analysis showed that practice toward family physician program was lower in men, in whom which were not under the coverage of main insurance systems and in whom that were not covered by family physician program. stepwise linear regression model showed that total practice of people toward their rights in urban family physician program by adjusted r square 0.54 and constant (= 4.6, 95% ci = 4.34.8, p < 0.001) was in order correlated to being under coverage of urban family physician program (= 1.2, 95% ci = 0.91.4, p < 0.001), being other family members under coverage of urban family physician program (= 0.9, 95% ci = 0.71.1, p < 0.001) and having higher than 1000 $ income monthly (= 0.2, 95% ci = 0.050.3, p = 0.008). after 9 years of establishment and modest achievements in rural family physician program in iran, thought and policy of extension of this system to urban settings has been dominating in recent years as evidenced in health sector reform of this country. therefore, as the pilot, this national project was launched in 2012 in two provinces of iran, including 4.5 million populated fars provinces in the south of the iran. in shiraz, the capital city of fars province, with a population 1.5 million, 650 general family physicians and 300 specialist family physician were included in the family physician program. considering that urban family physician program is a complex and multi - disciplinary structure, it is necessary to monitoring its performance periodically from different aspects. one of the important aspects of the monitoring could be regarded as the assessment of the trend of knowledge and practice of the people toward this program and before and after implemented interventions. therefore, after 2.5 years of starting this program and as the first official report, this study was conducted to evaluate the knowledge and practice of the people toward family physician program. present study demonstrated that the knowledge of the people about their rights in this program is generally low. the also showed that only few people knew about what to do when they had any question or any complain about the program. this should be considered as an important obstacle toward improvement of the program since the policy makers may hardly get access to the voice of the people. furthermore, most of the people did not know what to do when their family physicians are absent and how to find an alternative one. this leads to ignore or delay to visit by the family physician and gradually in mistrust to and outgoing from the program. the of the present study revealed that the lack of knowledge was more common among those who were not under coverage of any health insurance system and also among single people. furthermore, remarked that the practice of the people toward urban family physician program was so weak. a significant portion of the people had problems with providing drugs that were prescribed by their family physicians and also to access specialist family physicians. furthermore, about one fifth of the people complained that they had paid higher that legally approved visiting fees. this matter could be solved if people get more informed about their rights meanwhile teach them how send their feed backs and complains. however, establishment of an effective and continuous supervision system may also come to help in this regard. another achievement of this survey was that, poor practice is common among those with lower outcome. we found that low knowledge toward this program was not related to level of income, therefore above pointed that low economics may suffer from weaker infrastructures of family physician system in their areas although other studies are needed to prove such claim. another finding was that practice of people toward urban family physician system had a poor correlation with their knowledge as it was endorsed in previous studies with emphasis on that educating alone may not lead into a better good level of practice. hence, strengthening the software and hardware resources are mandatory for the sake of good performance of this system. present study marked that the reference rate of the patients to family physicians is high in the current system. however, this was not so different from those who were not under coverage of family physician and were referred to out of the family physician system doctors. there are few studies that demonstrate that the number of unnecessary patients referrals to pharmacies, laboratories, and radiology centers has been increased as a of running family physician program, it is needed to perform other studies about the cost effectiveness of the urban family physician program in our setting, as well as the efficiency of monitoring - evaluation system. saying about limitations in this study, it should be clear that despite our effort to design the questionnaire simple and user friendly and also providing prepaid envelope for resending the filled questionnaires, approximately 10% of the people did not answer the questionnaire. it was half of dropout rate of 20% that we assumed for estimation of sample size and we also noticed that the nonrespondents did not show statistically difference among different postareas, therefore it is unlikely that it could have influence on the and their representativeness. another point was that this study was conducted in shiraz, the most populated city of the fars province that does not have exactly the same situation as the small cities of this province. however, by choosing participants randomly and from different socioeconomic classes, the possible discrepancy may be faded. this study showed that the knowledge and practice of the people toward family physician program are weak. therefore, continuous education and effective training of people about their rights in this program could lead to a better performance of them and come into play for future outcome of this program. last but not least, lessons from this project could help policymakers at national level before any decision to extension this program to whole the country or even may be, followed by neighboring and regional countries that look to iran as a hub for regional health sector reforms.	: urban family physician program has been launched as a pilot in fars and mazandaran provinces of iran since 2012. attitudes of policy makers and people toward urban family physician program have become challenging. this study shows what people know and practice toward this program.methods:this cross - sectional population - based study was conducted by a multistage randomized sampling in shiraz, southern iran. knowledge and practice of adults toward urban family physician program were queried through filing the questionnaires. single and multiple variable analyzes of data were performed.:participation rate was 1257 of 1382 (90.9%), and the mean age of the respondents was 38.1 13.2 years. of 1257, 634 (50.4%) were men and 882 (70.2%) were married. peoples total knowledge toward urban family physician program was 5 2.7 of 19, showed that 1121 (89.2%) had a low level of knowledge. this was correlated positively and in order to being under coverage of this program (p < 0.001), being under coverage of one of the main insurance systems (p = 0.04) and being married (p = 0.002). the mean score of people's practice toward the program was 2.3 0.9 of total score 7, showed that 942 (74%) had poor performance, and it was correlated positively and in order to being under coverage of this program (p < 0.001) and having higher than 1000 $ monthly income (p = 0.004). correlation of people's knowledge and practice toward the program was 24%.: current evidences show a low level of knowledge, poor practice and weak correlation of knowledge - practice of people toward urban family physician program.
diphyllobothriasis is caused by the adult tapeworm (diphyllobothrium spp .), and is contracted by consuming raw or undercooked fish in northwest europe and east asia. many patients with diphyllobothriasis are asymptomatic, while other patients develop gastro - intestinal discomfort, diarrhea, or abdominal pain. of the 18 diphyllobothrium spp. known to cause human diphyllobothriasis, diphyllobothrium latum and diphyllobothrium nihonkaiense are usually reported in republic of korea. these 2 diphyllobothrium species have similar morphologic features, and thus, clinicians are not able to differentiate the 2 parasites by naked eyes. as an alternative, genetic studies can be used to identify the species. recently, several nucleotide sequencing analysis studies revealed that previous identifications of d. latum based on morphologic characteristics were incorrect and that the parasite was in fact d. nihonkaiense. although the chemotherapy required for both species is identical, the correct identification of diphyllobothrium species is required from the perspectives of epidemiology and public health promotion, particularly by providing accurate information on intermediate hosts. in this regard , we used nucleotide sequencing analysis of the mitochondrial cytochrome c oxidase subunit i (cox1) gene to identify diphyllobothrium species in 2 korean male patients. on april 3rd and 12th of 2013, 2 patients visited kyungpook national university hospital with a whitish yellow tapeworm segment. the patient that visited on april 3 was a healthy 50-year - old man (patient a), who visited the emergency room after finding a part of a tapeworm while defecating. he had not experienced any abdominal discomfort or pain, and had not visited any foreign country recently, and reported infrequent consumption of raw salmon and trout in side dishes. he also felt a foreign body while defecating, and visited the same emergency room. no abnormal clinical signs, such as, abdominal discomfort or pain, were observed, and he has taken a vermicide during september 2012. the specimens obtained from the 2 patients were forwarded to our laboratory with a request to identify the parasite species concerned. the parasite samples from the 2 patients consisted of a number of proglottids; gravid proglottids showed a rosette - shaped uterus as shown in fig. 1a and e. testes were follicular and ovaries were kidney - shaped in the posterior region. in longitudinal sections of gravid proglottids, uterine and genital pores were separated at midline, and the uterus was filled with eggs (b, c, f, and g in fig . 1 ; h&e stain), which were oval and enclosed by a shell (d and h in fig . total genomic dna was extracted from each specimen, and oligonucleotide primers were designed to amplify the 1,480-bp product of the mitochondrial cytochrome c oxidase subunit 1 ( cox1) gene (forward primer, 5'-atgactaatcttaaagtttt-3 ' ; reverse primer, 5'-taaagccaacatactataatc-3 '). pcr was performed as follows; initial denaturation at 96 for 5 min, followed by 30 amplification cycles (96 for 1 min, 58 for 1 min, and 72 for 1 min), and a final extension step at 72 for 10 min. the purified pcr - amplified fragments obtained were cloned into pgem-t easy vector (promega, madison, wisconsin, usa), and the inserted plasmids (cox1 gene - pgem-t easy vector) were sequenced using a big - dye terminator sequencing kit (applied biosystems, foster city, california, usa) on an automated dna sequencer (applied biosystems). (clc bio, aarhus, denmark), and similarities in genbank were examined using blastn. the cox1 sequences (1,480 bp) of the 2 parasites showed similarities of 99.9% (1,478/1,480 bp), 95.1% (1,407/1,480 bp), 93.1% (1,378/1,480 bp), and 92.6% (1,371/1,480 bp) with d. nihonkaiense (genbank no . the patients were treated with a single oral dose of praziquantel ( 15 mg / kg) and no evidence of recurrence has been noticed. d. nihonkaiense was first identified by yamane et al. in 1986 by comparing its morphologic characteristics, such as egg size, the pit shape of eggshells, and the angle between the long axis of the cirrus sac and seminal vesicle, with those of other diphyllobothrium species. d. nihonkaiense and d. latum had distinct morphologic characteristics. specifically, the average size of d. nihonkaiense eggs was 55.5 (1.0) 40.5 (1.5) m, which is smaller than the average size of d. latum eggs (greater than 60.0 45.0 m). the eggshells of d. nihonkaiense had shallower pits distributed on the smooth surface than the eggshells of d. latum did. the cirrus sac is situated obliquely in d. nihonkaiense, and the angle between the long axis of the cirrus sac and seminal vesicle was sharper in d. nihonkaiense than in d. latum. in addition to these distinct morphologies, d. nihonkaiense and d. latum have different intermediate host specificities. the intermediate hosts of d. nihonkaiense are salmon, such as cherry, pink, chum, and sockeye salmon (onchorhychus masou, o. gorbuscha, o. keta, and o. nerka, respectively), which are found in the northern pacific, whereas the intermediate hosts of d. latum are perch, pike, burbot, and walleye. based on his history, patient a likely contracted parasites from these fish. on the other hand, patient b had no history of raw salmon consumption, and similarly, several other cases of d. nihonkaiense infection not associated with salmon consumption have been reported. accordingly, accumulating evidence indicates d. nihonkaiense has another intermediate host and that further research on the intermediate hosts of d. nihonkaiense in korea is needed. previously, most cases of diphyllobothriasis in korea were thought to be caused by d. latum. reported occurrence of d. nihonkaiense in korea (35th annual meeting of the korean society for parasitology, kwangju, rok). owing to the publication of the complete mitochondrial genome of d. nihonkaiense, d. nihonkaiense can now be accurately identified by dna sequencing of the mitochondrial cox1 gene. in 2009, jeon et al. reported that 62 cases diagnosed as d. latum infection were actually caused by d. nihonkaiense, which were identified by genetic analysis. moreover, another case of diphyllobothriasis was recently confirmed as d. nihonkaiense infection by cox1 gene analysis. these findings suggest that d. nihonkaiense has been misdiagnosed as d. latum and that d. latum might not exist in korea. the present study, in which 2 korean male patients were diagnosed as d. nihonkaiense by cox1 gene sequencing, supports this suggestion. from the clinical point of view, this specific diagnosis might seem academic because diphyllobothriasis can be easily cured with praziquantel. nevertheless, specific diagnosis at the species level is important in the contexts of epidemiology and public health, and would provide further insight into distribution of different taxa and improve our understanding of imported cases and outbreaks of diphyllobothriasis.	diphyllobothrium latum and diphyllobothrium nihonkaiense are the 2 reported main causes of human diphyllobothriasis in the republic of korea. however, the differentiation of these 2 species based on morphologic features alone is difficult. the authors used nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene to diagnose diphyllobothrium spp. two patients visited the emergency room at kyungpook national university hospital on 3 april and 12 april 2013, respectively, with fragments of parasites found while defecating. the parasites were identified as diphyllobothrium spp. based on morphologic characteristics, and subsequent cox1 gene sequencing showed 99.9% similarity (1,478/1,480 bp) with d. nihonkaiense. our findings support the hypothesis that d. nihonkaiense is a dominant species in korea.
the fetus enjoys special privileges that minimize the risk of being rejected by the maternal immune system during pregnancy. the maternal immune system is alerted and responds actively to the fetal invasion, but the type of inflammation generated is not a milieu in which rejecting t cell responses are favored. at the fetal maternal interface , the decidua serves as an immunologically privileged tissue playing essential functions in pregnancy maintenance. during the first trimester of pregnancy, the majority of leucocyte populations in the human decidua is composed of 70% natural killer (nk) cells, and 1020% antigen presenting cells (apcs), whereas t cells are sparse and b cells are virtually absent. dendritic cells (cd11cdcs) are the key professional apcs representing 510% of all hematopoietic uterine cells. dcs are not only essential for the induction of primary immune responses but also important for the establishment of immunological tolerance. the local microenvironment influences the functions and differentiation of dcs with tolerogenic activities that play a prominent role in dictating the quantity and quality of immune responses. two different myeloid dc subsets, bdca-1 and bdca-3, were detected in normal human first trimester decidua. bdca-1 decidual cells express hla - dr, cd80 and cd86 at low levels, consistent with the immature characteristics of myeloid dcs. in addition, kammerer et al. have shown that early human pregnancy decidua harbors c - type lectin - expressing cells (dc - sign) that show functional features of immature dcs. during human pregnancy, non - classical hla class i hla - g proteins, specifically expressed in the trophoblasts, contribute to the establishment of immune tolerance. seven different isoforms of hla - g exist, four of which are membrane - bound (hla - g1 to -g4) and three are soluble forms (hla - g5 to -g7). hla - g locus is low polymorphic in the coding region, but polymorphisms that can regulate its expression are present at both 5 up - stream regulatory region (urr) and 3 un - translated region (utr) non - coding regions. the immune - regulatory properties of hla - g from interactions with diverse inhibitory receptors: directly via ig - like transcript (ilt)2 expressed on myeloid and lymphoid cells, ilt4 specifically expressed on apcs, including dcs, and kir2dl4 on nk cells and cytotoxic t lymphocytes (ctl); indirectly via cd94/nkg2a on nk cells. myeloid apcs may express hla - g and its expression is greatly enhanced by interferon-, il-10 and maturation stimuli. the expression of membrane - bound hla - g and the secretion of soluble hla - g by myeloid apcs contribute to the generation of a tolerogenic microenvironment that may alter the functions of hla - g - expressing myeloid apcs (hla - g apcs) themselves, in a feedback loop. thus, myeloid hla - g apcs may be viewed as suppressor cells capable of inhibiting other effector cells and of generating regulatory cells, such as tolerogenic dcs and regulatory t cells (tregs). recently, a subset of il-10-producing human dc (dc-10) has been characterized in the peripheral blood. these cells secrete high levels of il-10, express membrane - bound hla - g, ilt2, ilt3, ilt4, and are potent inducers of adaptive il-10-producing type 1 tregs (tr1) in vitro through the il-10-dependent ilt4/hla - g pathway. cd4 t cells constitutively expressing hla - g have been shown to accumulate at sites of inflammation. it has been demonstrated that cd4hla - g cells suppress t cell proliferation via a reversible non - contact il-10- and soluble hla - g5-dependent process that leads to regulation of tissue inflammation at the target organ. in the present study we identify for the first time the presence of dc-10 and cd4hla - g t cells at the fetal maternal interface where they may contribute to the tolerance establishment and maintenance in the first trimester decidua. first trimester decidua (n = 10) at 612 weeks of gestational age were obtained from caucasian women with clinically normal pregnancies which were scheduled for elective abortion due to social or psychological reasons. peripheral blood was collected soon before the aspiration procedure. decidual tissues were taken through the cervix during dilatation and aspiration according to formal clinical procedures. informed consent was obtained from all subjects, and the investigation was approved by the ethical committee of fondazione irccs ca granda, milan, italy. human peripheral blood was obtained from healthy donors upon informed consent in accordance with local ethical committee approval (tiget periblood) and with the helsinki declaration. peripheral blood mononuclear cells (pbmc) were isolated by centrifugation over lymphoprep ficoll gradients (fresenius kabi norge as, halden, norway). this study was approved and monitored by the internal ethical committee of the san raffaele scientific institute and the ethical committee of fondazione irccs ca granda, milan, italy. decidual tissues were dissected into small pieces and enzymatically dissociated in rpmi 1640 medium containing collagenase type iv (1 mg / ml ; sigma aldrich, carlsbad, ca), and dnase i (final 0.01%, invitrogen) for 90 min at 37 c with gentle shaking. the suspension was filtered through a 40-m nylon mesh (cell strainer, bd biosciences, san jose, ca). decidual mononuclear cells (dmnc) were separated by density gradient centrifugation over lymphoprep ficoll gradients (fresenius kabi norge as, halden, norway). dmnc and pbmc were initially incubated for 15 min at room temperature with fcr blocking reagent (miltenyi biotech, germany) and stained for additional 30 min at room temperature with the following human monoclonal antibodies (mabs): cd1c (bdca-1) and cd83 (miltenyi biotech, germany), cd14, cd16, dc - sign, cd11c, cd4, cd8 (bd bioscience, ca), cd45 (biolegend, usa), ilt4 and cd56 (beckman coulter, france) and hla - g (mem - g9, exbio, praha, czech republic). samples were acquired using a facs canto ii flow cytometer (becton dickinson, mountain view, ca), and data were analyzed with fcs express (de novo software). significance was defined as p 0.05, p 0.005, p 0.0005, and p < 0.0001. first trimester decidua (n = 10) at 612 weeks of gestational age were obtained from caucasian women with clinically normal pregnancies which were scheduled for elective abortion due to social or psychological reasons. peripheral blood was collected soon before the aspiration procedure. decidual tissues were taken through the cervix during dilatation and aspiration according to formal clinical procedures. informed consent was obtained from all subjects, and the investigation was approved by the ethical committee of fondazione irccs ca granda, milan, italy. human peripheral blood was obtained from healthy donors upon informed consent in accordance with local ethical committee approval (tiget periblood) and with the helsinki declaration. peripheral blood mononuclear cells (pbmc) were isolated by centrifugation over lymphoprep ficoll gradients (fresenius kabi norge as, halden, norway). this study was approved and monitored by the internal ethical committee of the san raffaele scientific institute and the ethical committee of fondazione irccs ca granda, milan, italy. decidual tissues were dissected into small pieces and enzymatically dissociated in rpmi 1640 medium containing collagenase type iv (1 mg / ml ; sigma aldrich, carlsbad, ca), and dnase i (final 0.01%, invitrogen) for 90 min at 37 c with gentle shaking. the suspension was filtered through a 40-m nylon mesh (cell strainer, bd biosciences, san jose, ca). decidual mononuclear cells (dmnc) were separated by density gradient centrifugation over lymphoprep ficoll gradients (fresenius kabi norge as, halden, norway). dmnc and pbmc were initially incubated for 15 min at room temperature with fcr blocking reagent (miltenyi biotech, germany) and stained for additional 30 min at room temperature with the following human monoclonal antibodies (mabs): cd1c (bdca-1) and cd83 (miltenyi biotech, germany), cd14, cd16, dc - sign, cd11c, cd4, cd8 (bd bioscience, ca), cd45 (biolegend, usa), ilt4 and cd56 (beckman coulter, france) and hla - g (mem - g9, exbio, praha, czech republic). samples were acquired using a facs canto ii flow cytometer (becton dickinson, mountain view, ca), and data were analyzed with fcs express (de novo software). significance was defined as p 0.05, p 0.005, p 0.0005, and p < 0.0001. during pregnancy, subpopulations of leukocytes present at the fetal - maternal interface change participating in a coordinated manner to orchestrate sequential, reproductive events that in the maintenance of pregnancy. during the first trimester, nk cells represent the most abundant immune cells in the decidua. these cells are cd56cd16, are distinct from peripheral blood nk subsets, and are critical for the maintenance of pregnancy since their cytotoxicity is inhibited by the expression of hla - e and hla - g on trophoblasts. as expected, 58.3 4.6%, (mean sem, n = 10) of the human decidual mononuclear cells were cd56 nk cells, of which > 90% were cd16 (fig . the remainder decidual leukocyte population included t and b lymphocytes, cd14 monocytes, cd11b and cd11c myeloid cells . in comparison with peripheral blood lymphocytes, the proportion of cd4 t cells infiltrating human decidua was significantly lower ( 16.01 1.64%, mean sem, n = 10, vs. 39.12 3.83%, mean sem, n = 9, p = 0.0009) (fig . no differences in the percentages of cd8 t cells, cd19 b cells, and cd14 monocytes were observed between the human decidua and the peripheral blood . human decidual leukocytes contained also 6.4 1.8% ( mean sem, n = 8) of cd11b cells, 49.95 6.07% (mean sem, n = 9) of cd11c cells, and 3.54 1.2%, (mean sem, n = 9) of bdca-1 (cd11ccd1c) cells (fig . the frequencies of decidual myeloid cd11c and bdca-1 cells were significantly higher than their counterpart in the peripheral blood of pregnant women ( 13.12 1.74%, mean sem n = 9, p = 0.0027, and 0.69 0.27, mean sem, n = 9, p = 0.0047, respectively) (fig . our data are in line with a previous report demonstrating the presence of several subsets of dcs, including bdca-1 ( 0.2%), in the human first trimester decidua; however, we found a higher percentage of decidual bdca-1 cells (3.5%). this may be due to the method used for isolating decidua - infiltrating cells: ban et al. used a non - enzymatic method, while we isolated immune cells from decidual tissues by collagenase - mediated digestion. in addition, in the previous study the percentage of bdca-1 cells was calculated based on total decidual cells, whereas we evaluated the frequency of bdca-1 cells by gating on cd45 cells. in our setting, the percentage of cd45 cells ranged from 10% to 70% of the total decidual cells. human decidua harbored a significant population of dc - sign cells that represents 610% of decidual cells. decidual dc - sign cells have been shown to be immature myeloid cells and have been proposed to play a role in normal and pathological pregnancy outcome. in our setting, the percentage of decidual dc - sign cells was 2.9 0.6% (mean sem, n = 8), and their frequency was significantly higher than that observed in the peripheral blood of pregnant women (1.12 0.23%, mean sem n = 9, p = 0.01) (fig . dc-10 are tolerogenic dcs characterized by their ability to secrete il-10 and by the high expression of membrane - bound hla - g . since dc-10 are highly potent in inducing tr1 cells, we hypothesized that these cells might represent one of the major subsets of apcs present in the decidua involved in promoting and maintaining tolerance during pregnancy . dc-10 were identified by the concomitant expression of cd14, cd16 and cd83 on human decidual leucocytes ( cd45). interestingly, a significantly higher percentage of dc-10 was present in the human decidua than in the peripheral blood of pregnant women (2.29 0.75%, mean sem, n = 10 vs. 0.25 0.07%, mean sem, n = 8, p = 0.0014) (fig . human decidual dc-10 expressed similar levels of hla - g, but significantly lower levels of ilt4 ( 80.38 2.45%, mean sem, n = 10 vs. 96.38 1.01%, mean sem, n = 8 ; p = 0.0003) as compared to circulating dc-10 (fig . similarly, inflammatory monocytes ( cd14cd16) present in the human decidua expressed similar levels of hla - g but lower amounts of ilt4 as compared to their circulating counterpart (56.86 4.89%, mean sem, n = 7 vs. 94.57 1.23%, mean sem, n = 9 ; p = 0.001, fig . the expression of hla - g and ilt4 on decidual bdca-1 cells was comparable to that observed in their peripheral blood counterpart ( fig . 2c). the increased frequency of dc-10 observed in the decidua may be dependent on several mechanisms: dc-10 can be recruited from the peripheral blood, resident decidual dcs can be converted into dc-10, or the decidual microenvironment can promote the de novo induction of dc-10. the decidual microenvironment is enriched of several chemokines, including ccl2 and cx3cl1 that have a role in tissue remodeling and in the recruitment of immune cells. peripheral blood dc-10 express ccr2 and cx3cr1, thus it can be hypothesized that they are attracted and accumulated in the decidua. several cytokines including il-4, il-10, and gm - csf as well as growth factors and hormones with anti - inflammatory properties are present at the decidual level (reviewed in). this pro - tolerogenic microenvironment is known to promote alternatively activated macrophages and tolerogenic dcs. in particular, the high levels of il-10 may promote the up - regulation of hla - g, ilt2 and ilt4 on resident decidual immature dcs converting them into dc-10. alternatively, the presence of gm - csf, il-4 and il-10 can allow, through the il-10 dependent ilt4/hla - g pathway, the de novo induction of tolerogenic dc-10. further functional studies are required to better define the origin and the role of dc-10 in the decidua. in addition to dcs, cd4cd25foxp3 tregs are enriched at the fetal - maternal interface and are expanded accordingly to gestational stages. we confirmed that cd4cd25foxp3 tregs cells are present in the human decidua, and no differences in the percentages of these cells were observed between human decidua and peripheral blood in our cohort of pregnant women (data not shown). in addition to cd4cd25foxp3 tregs, other tregs either naturally occurring or induced have been described. among them , we focused our attention on a new type of naturally occurring cd4 and cd8 tregs that constitutively express hla - g. cd4hla - g and cd8hla - g tregs are present in the peripheral blood. moreover, cd4hla - g tregs have been shown to accumulate at sites of inflammation, and are potent suppressors of t cell proliferation in vitro. to date, there is no evidence of the presence of hla - g - expressing cd4 or cd8 t cells in the peripheral blood and the decidua during pregnancy. we first investigated the frequency of circulating cd4hla - g t cells in pregnant women as compared to healthy donors. showed a significantly higher frequency of hla - g expressing cd4 t cells in the peripheral blood of pregnant women (pbmc pw) compared to that of healthy donors (pbmc hd) (5.29 1.3%, mean sem, n = 8 vs. 2.13 0.28%, mean sem, n = 53, respectively, p = 0.0053) (fig . interestingly, a distinct population of cd4 t cells expressing hla - g was detected in the human decidua ( 18.97 2.98% mean sem, n = 9) (fig . 3a and b). the frequency of cd8hla - g t cells in the human decidua was comparable to that observed in the peripheral blood of pregnant women (data not shown). similarly to what has been described in the central nervous system, cd4hla - g t cells can be recruited to the human decidua in the first phases of embryo implantation to control immune responses against the semi - allogeneic fetal antigens. it still remains to be defined whether decidual cd4hla - g t cells express ccr5 or other chemokine receptors, which may allow their recruitment into the decidua, in analogy to what has been described for the same cells during the active inflammatory phase of multiple sclerosis. we can not exclude that cd4hla - g t cells could be directly induced in the decidua by local il-10 as it is known that il-10 promotes hla - g expression in cd4 t cells. moreover, we previously demonstrated that during tr1 cell induction via dc-10, il-10-derived from dc-10 induces hla - g expression on cd4 t cells that become anergic t cells, and subsequently tr1 cells. in this scenario, it can be speculated that naive cd4 t cells, once recruited in the decidua, can be activated via dc-10, up - regulate hla - g expression and then, in the continuous presence of il-10 and chronic allo - specific stimulation, they become allo - specific tr1 cells. it still remains to define whether cd4hla - g t cells are precursors of tr1 cells or represent a distinct subset of tregs, which co - operate with other tregs, including cd4cd25foxp3 tregs, in promoting and maintaining fetal - maternal tolerance. in addition to an overall improvement of the knowledge on the biological mechanisms underlying tolerance associated with hla - g expression, our findings may have different potential clinical implications. the ultimate goal of this study is the development of a risk assessment of women with a history of recurrent implantation failure who undergo in vitro fertilization (ivf). from a diagnostic point of view , the tolerance status of women with recurrent embryo implantation failure after ivf can be evaluated. the analysis of dc-10 and hla - g tregs and their potential association with hla - g polymorphisms will allow the identification of specific hla - g genotypes that could be included among the pre - ivf screening tests. from a therapeutic point of view , the study will open new perspectives for the modulation of immune responses in the early phases of pregnancy with the aim of minimizing the negative effects on embryo implantation associated with a reduced maternal tolerance status.	multiple mechanisms underlie the surprising willingness of mothers to tolerate the semi - allogeneic fetal tissues during pregnancy. chief among these is the expression of the hla - g molecules that has been largely demonstrated to be responsible for reprogramming the local maternal immune response towards tolerance. we recently identified a subset of tolerogenic dendritic cells, dc-10 that secrete high amounts of il-10 and express high levels of hla - g and its ligand ilt4. dc-10 are present in the peripheral blood and are essential in inducing adaptive regulatory t cells. we investigated the presence of dc-10 and hla - g - expressing cd4 + t cells in human decidua in the first trimester of pregnancy. showed that these cells are highly represented in human decidua as compared to the peripheral blood. this is the first report describing decidual dc-10 and cd4+hla - g+ t cells, strongly suggesting that they may accumulate or be induced at the fetal maternal interface to promote tolerance.
in esrd patients on renal replacement therapy, k homeostasis is mainly maintained on adequate dialytic k removal and adapted extrarenal k regulation1 ). however, due to the different dialysis modes between hd and capd, i.e. the episodic (3 - 4 times / wk) in hd versus the daily continuous dialysis in capd, hd patients do not achieve a steady blood leading to usually highest before dialysis and lowest immediately after dialysis while capd patients approach a steady state for it's level, so that wide variations in blood are not present. excessive dietary k gain before dialysis in hd can temporarily contribute to the development of hyperkalemia but poor dietary k intake in capd may lead to hypokalemia. therefore, hyperkalemia would be more frequent in hemodialysis than in capd, especially predialysis stage, and hypokalemia more frequent in capd than in hd. the k imbalance in dialysis patients seems to be primarily related to poor dietary compliance. in fact, too much k intake with poor compliance to diet in interdialytic interval in hd despite adequacy of hd contributes to the high frequency of hyperkalemia (10 - 24%) responsible for the most common cause of death (3 - 5%) among electrolyte imbalances, whereas malnutrition with poor k intake in capd is a well known factor for the high prevalence of hypokalemia (10 - 36%) leading to the higher cardiovascular mortality than normokalemic capd patients in a recent study2 ). however, besides dietary factors, there are other important factors to be considered among the causes of k imbalance in dialysis patients regardless of dialysis modes, since they are mostly remediable with identifying and then avoiding or removing them. those are clinical conditions causing the derangement of the external k regulation including abnormal colonic k secretion, i.e. constipation or diarrhea, as well as administration of drugs affecting internal k balance in k homeostatic mechanism related to neurohormones such as insulin, catecholamines, and renin - angiostensin - aldosterone (ras), such as non - selective bet - ablockers, ace - inhibitors and/or arbs, and aldosterone receptor blocker (spironolactone, eplerenone). as an another factor for k homeostasis in the patients on capd , daily continuous infusion of standard glucose containing dialysates might be involved in intracellular k shift related to insulin hormone and showed high intracellular k contents in muscles as shown in a few studies. recently, icodextrin with glucose - polymer solution leading to less glucose load and less insulin secretion has been replaced for the patients on capd with ultrafiltration failure during long - overnight dwell, which might affect k distribution in them3 ). in this paper, the etiologies responsible for hyperkalemia in hd and for hypokalemia in capd with evaluation of their prevalence and incidence as well as their treatments based on the contributing factors for the k imbalances are reviewed with collected data from the literature and our data. hd patients with esrd are continuously exposed to the risk of hyperkalemia due to episodic removal of body k through dialysis for 3 to 4 hrs with 3 or 4 times per week in frequency rather than continuous k excretion by the intact kidneys. however, the degree of hyperkalemia in hd during the interdialytic perod depends on several factors: 1 ) a dietary k intake, 2 ) the amount of dialysate k removal by hd related to the gradient and to the k passive refilling fro icf to ecf, 3 ) increased intestinal excretion of k as an adaptive extrarenal k homeostasis4 ), and 4 ) the transcellular k shifts as in healthy subjects. hyperkalemia was pointed out as a " potential silent killer " and suggested even one of the leading causes of sudden death in hd patients5 ). in our survey of hd patients during past 10 yrs, 108 out of 548 patients (20%) expired, and its most common causes are cardiovascular disorder (44%) and infection (17%) same as reported in the world literature, but other notable causes included unknown etiologies (22%) and hyperkalemia (6%)6 ). in the admitted patients with severe hyperkalemia, the most common causes of hyperkalemia were related to inadequate dialysis with noncompliance to hd frequency per week (55%) and excessive k intake with noncompliance to dietary regimen (35%) followed by others including medications7 ). nonselective beta - blockers have been shown to increase serum in patients with esrd due to preventing intracellular k shift by the inhibition of beta-2 adrenergic receptors.8 - 9 however, nonselective beta - adrenergic blocker with a selective alpha-1 blocking activity, carvediolol, did not enhance the hyperkalemic effect of moderate physical exercise on hd, but in our observation predialysis - serum with carvediolol was significantly higher than that without it (5.131.0 meq / l vs 5.71.0 meq / l)10 ). though renin - angiotensin - aldosterone system (raas) blockers such as acei, arb and spironolactone or eplerenone currently are recommended in hd patients for the cardio - protective effects, it has been reported to be independently associated with an increased risk of developing hyperkalemia11 ). recently, low - dose spironolactone, 25 mg / d12 ), and even the combined therapy of ras blockers with ace plus arb failed to show any significant increase in frequency of hyperkalemia and in the higher mean serum k level when compared to basal values without their exposure in our study (no - exposure, 5.540.67 meq / l ; acei alone, 5.540.75 meq / l ; arb alone, 5.500.66 meq / l ; acei plus arb, 5.420.66 meq / l, p = ns)13 ). these contradictory data suggest that the cautious trial of ras blockers, either alone or combined, would be justified with the overweighing benefits of the cardioprotective effects for the patients on hd rather than their hyperkalemic risks. before the initiation of hd as the most efficient treatment of hyperkalemia is available, other conservative managements of the emergency treatment of hyperkalemia consists of four basic modalities as monotherapy including calcium gluconate, insulin with glucose, nebulized beta-2 agonists, intravenous nahco3 and oral or rectal cation - resins. however, the effect of nahco3 for the treatment of hyperkalemia is uncertain and its infusion in varying amount failed to lower serum in esrd patients14 ), but when combined with insulin with glucose or beta-2 agonists (nahco3 alone, -0.130.06, p = ns ; salbutamol alone, -0.570.03 meq / l, p<0.02 ; nahco3 plus salbutamol, -0.960.08 meq / l, p=0.000), their hypokalemic effect were enhanced probably due to the activation of na - k pump with acute correction of underlying metabolic acidosis in the combined regimen with bicarbonate as dual therapy15, 16 ). also, other study introduced the efficacious and safe modalities for the acute treatment of hyperkalemia in esrd patients with the combined regimens as dual therapy, insulin with glucose plus beta 2-agonists17 ). therefore, once pseudohyperkalemia also present in hd patients as in other general patients is excluded18 ), the temporizing measures of transcellular k shifting regimens can be initiated alone or combined therapy to buy time until hemodialysis can be initiated with the assessment of ekg at the outset as shown in the flow chart (fig . 1)19 ). as a long - term treatment plan of hyperkalemia in hd, the correction of the underlying pathophysiology responsible for hyperkalemia should be started with removal or cautious trials of medications inducing hyperkalemia with balancing the ratio of their benefits and risks in addition to improving the compliance to dialysis and diet. among other therapeutic regimen for hyperkalemia on the long - term basis, the administration of laxative such as bisacodyl increasing colonic k seretion20 ), and exogenous mineralocorticoid or mineralocorticoid - like substance may provide simple means of reducing interdialytic hyperkalemia in hd21, 22 ). in our analysis of serum profile in stable capd patients (n=253)23 ), normokalemia (83%) and hypokalemia (16%) were much more frequent than hyperkalemia (1%), being similar to the recent data of chinese capd patients, i.e. normokalemia (80%), hypokalemia (20%), and none in hyperkalemia2 ). the chinese data revealed that serum is correlated with serum albumin level (r=0.17 ; p=0.005) suggesting close relation between hypokalemia and malnutrition. also, on analysis of serum profile in admitted capd patients for 3 yrs, we observed the significant differences between groups of hypokalemia (11/48, 23%) and without hypokalemia (37/48, 77%) in serum albumin, bun, and creatinine levels. furthermore, the history of poor dietary intake was more prominent in hypokalemic patients (9/11 vs 6/37, p<0.01), but daily k loss via dialysates was similar in two groups. these data support strongly that high prevalence of hypokalemia in capd as well as the close correlation of hypokalemia with malnutrition and poor dietary k intake24 ). however, one study investigated for the pathogenetic mechanism of hypokalemia in capd with measuring the amount of k intake minus that of urinary and peritoneal k excretion and considering stool k losses of around 6 - 50 meq / day. external k imbalance (i.e. k losses) alone was not enough to justify the hypokalemia. therefore, the deranged internal k balance, i.e. intracellular k shift by mostly insulin hormone stimulated by the continuous glucose peritoneal infusion in capd, was proposed as a contributory factor for the development of hypokalemia even in the context of a low k excretion25 ). in our recent study, the mean serum was higher in the group of glucose - free dialysates (icodextrin) than in the group of standard glucose dialysates for long overnight - dwell (4.60.1 meq / l vs 4.40.1 meq / l, p<0.05).13 for k supplementation in the management of hypokalemia furthermore, k mixed with dialysate solution would be administered thru intraperitoneal route upto 40 meq in 2 l dialysate safely. in hypokalemic patient (< 3.5 meq / l), the degree of k absorption via peritoneum and that of intracellular shift of absorbed k were 766% and 7419%, respectively, and the increment of serum was 0.80.2 meq / l.26 a recent study of comparison of intraperitoneal k administration (40 meq mixed in 2 l dialysate) after one exchange between standard 2.5% glucose and glucocose - free (icodextrin) dialysates revealed the less increase in serum (0.180.1 meq / l vs 0.560.1 meq / l, p<0.05), despite the less increment of insulin level from basal value (-199% vs 3712%) in the latter and similar blood glucose concentrations in two different dialysates13 ). undoubtedly, acute administration of k in hypokalemic patients in capd regardless its routes would be necessary for its correction to avoid neuromuscular weakness and serious cardic arrhythmia. nevertheless, in the chronic long - term basis, further investigation may be required to investigate the benefits of k supplementation alone, because hypokalemia may simply be surrogate marker of malnutrition and/or other serious comorbid illness. in dialysis patients, i.e. hd or capd, k imbalance should be always considered as a common condition related to either a potentially lethal condition itself or the important contributory factor for their morbidity and mortality. the pathophysiologic factors for hyperkalemia in hd are well understood, and accordingly its managements are somewhat straightforward, while the pathogenetic mechanisms of hypokalemia in capd need further clarification for the role of intracellular k redistribution. for the management of k imbalance in dialysis, the foremost step is to improve compliance to dietary k intake and dialysis regimen besides the reversal or removal of pathophysiologic factors and other potential sources including drugs affecting k imbalance should be always considered. however, cautious administration of certain beneficial drugs such as raas blockers could be justified in esrd on dialysis with tremendous burden with cardiovascular morbidity and mortality. as temporizing acute k - lowering regimens in severe lifethreatening hyperkalemia, dual - therapeutic regimens, i.e. nahco3 plus insulin with glucose or insulin with glucose plus beta-2 agonist, could be recommended as an efficacious modality. in capd patients with hypokalemia, for the long - term management, the correction of malnutrition would be required as one of the essential managements and the selection of glucose - free dialysates such as icodextrin could be considered rather than the conventional glucose dialysates. also, as short - term management, k supplements could be administered safely and efficiently via intraperitoneal route, if required.	in end - stage renal disease (esrd) patients regardless of dialysis modes, i.e. maintenance hemodialysis (hd) and continuous ambulatory peritoneal dialysis (capd), potassium (k) homeostasis is regulated primarily via dialysis and extrarenal k regulation in the diverse daily k intake. however, k metabolism has been known to differ greatly between the two main methods of dialysis. hyperkalemia is a common complication (10 - 24%) and the most common cause of the death (3 - 5%) among electrolyte disorders in patients on maintenance hd. on the contrary, hypokalemia (10 - 36%) is responsible for a rather common complication and independent prognostic factor on capd. although excessive k intake or inadequate dialysis on maintenance hd and poor nutritional k intake on capd are accused without doubts upto 50% of esrd patients as a primary cause of the k - imbalance, i.e. hyperkalemia on hd and hypokalemia on capd, other contributory factors including certain medications and unknown causes remain still to be resolved. accordingly, the effects of medications as another source of k - imbalance on hd with ras blockades and beta blockers as well as those of conventional and glucose - free dialysates (icodextrin) for internal k - redistribution on capd were evaluated with reviewing the literatures and our data. furthermore, new developments in the clinical managements of hyperkalemia on hd following the exclusion of pseudohyperkalemia before the initiation of dialysis were suggested, especially, by the comparison of the effects between mono- and dual - therapy with medications for transcellular k shifting in the emergent situation. also, the intraperitoneal k administration via conventional glucose - containing (2.5%) and glucose - free dialysates (icodextrin) as a specific route of k - supplementation for hypokalemia on capd was examined for its efficiency and the degree of intracellular k shift between these two different types of dialysates.
pure red cell aplasia (prca) is a rare adverse reaction occurring in patients with chronic kidney disease (ckd) who are treated with erythropoiesis - stimulating agents (esa). it is generated by epoetin - induced antibodies (ab) that neutralize all the exogenous erythropoietin (epo) and cross - react with endogenous epo. in the early 2000s a peak increase in the incidence rate of prca was observed after a change in the formulation of epoetin alpha produced outside the usa. following reinforcement of the cold storage chain, a shift towards the intravenous administration route and the elimination of uncoated rubber stoppers, the incidence of prca has markedly decreased. biosimilars of epoetin alpha have been approved in the european union since 2007, and following the expiration of the us patent, they will enter the us market soon. we report on a patient who developed prca following the subcutaneous administration of epoetin zeta, which is one of the two biosimilars of epoetin alpha licensed in europe. to our knowledge a 72-year - old caucasian female was regularly followed up in the nephrology outpatient clinic of versilia hospital because of a slowly progressive ckd. in 1990 the patient underwent left nephrectomy for severe nephrolithiasis, complicated by acute pyelonephritis and sepsis. in 1991, she developed new stones in the right kidney and received extracorporeal shock wave lithotripsy. in 1995 since then, she has been under regular cardiologic follow - up. from 2007 until 2014 kidney function slowly deteriorated, reaching stage iv v ckd. in june 2014 , she attended the day hospital of the nephrology unit for severe hip arthrosis. her (hb) was 9.7 g / dl with adequate iron stores; she was prescribed epoetin zeta 4000 iu twice a week subcutaneously (figure 1) and antalgic therapy for hip pain. rbc, red blood cells. in december 2014, she was hospitalized for 4 days in the nephrology department due to the persistence of severe hip pain. the patient's serum creatinine was 3.7 mg / dl and hb was 10.2 g / dl. given that occult blood was detected in three stool samples, colonoscopy was suggested. in february 2015, the patient arrived at the emergency unit because of severe asthenia and mild shortness of breath. she received blood transfusion (six units of packed red cells) and was admitted again to the nephrology department. colonoscopy showed diverticulosis, but was not diagnostic for incomplete cleaning. at 2 weeks after admission, at discharge her hb was 9.3 g / dl; epoetin zeta dose was increased to 4000 iu three times per week. in april 2015, she was hospitalized again for 8 days because of severe anaemia (hb 6.2 g / dl, reticulocytes 0.1%, 2500/l). the patient underwent bone marrow biopsy showing severe hypoplasia of the erythroid line (cd34 blasts < 5%) with hyperplasia of the megakaryocytic and granulocytic lines. table 1.main laboratory parameters at onset of prca25/02/201509/04/2015haemoglobin (g / dl)3.99.36.27.3reticulocytes (/l)na2500leucocytes (n / mmc)68508680platelet count136 000137 000serum iron (g / dl)227218transferrin saturation (%) 80.582.2serum ferritin (ng / ml)268512serum creatinine (mg / dl)3.53.7c - reactive protein (mg / l)na0.3parathyroid hormone (pg / ml)na155na, not available.post-transfusion. main laboratory parameters at onset of prca in may 2015, at the time when bone marrow biopsy became available, the patient had become transfusion dependent and epoetin zeta administrations were immediately interrupted. assays for antinuclear ab, anti - dna ab and hepatitis b ab were normal; parvovirus b19 dna and hepatitis b antigen were not detected. anti - epo ab were tested by ipm biotech gmbh, hamburg, germany and found positive in three different samples by a screening assay, followed by specificity confirmation testing. the patient was prescribed oral prednisone (0.5 mg / kg / day) for 3 months and progressive decrease in the need for blood transfusions was observed. in september 2015, she received her last blood transfusion and steroid therapy was interrupted. since then, her hb values have stabilized (last available hb value of 11.5 g / dl in february 2016). no worsening of renal function had occurred over the period (in february 2016, serum creatinine was 3.9 mg / dl). retesting of anti - epo ab titre has already been planned, in the case the patient needs re - challenging with esa in the future. in the late 1980s, epoetin alfa was developed thanks to the recombinant dna technique in chinese hamster ovary cells. this is a delicate manufacturing process, which invariably leads to heterogeneity of the final product. given that the drug is poorly water - soluble, excipients are required to enhance dispersion or inhibit precipitation when mixed with water. as a , all esa molecules are delicate and fragile, require cold storage and are sensitive to changes in the stabilizer and/or manufacturing processes. of note, the interruption of the cold chain may be less problematic with long - acting esa, as they have a longer stability at room temperature compared with short - acting esa. the upsurge of prca cases with eprex was preceded by the substitution of human serum albumin with polysorbate 80. the hypothesis that this stabilizer may elicit the formation of immunogenic epoetin - containing micelles, possibly increasing immunogenicity, has been questioned. the uncoated rubber stoppers in the pre - filled syringes could have possibly increased immunogenicity as well. this may be due to the shift from the subcutaneous to the intravenous route of administration, the reinforcement of the product cold chain or the elimination of uncoated rubber syringe stoppers. in 2008 regulatory authorities readmitted the subcutaneous use of eprex in the absence of a vascular access for intravenous administration. more recently, an unexpected increase in local cases of prca associated with subcutaneous administration of eprex was described in singapore. the introduction of biosimilars in the eu market has brought savings of around 1530%, together with a price reduction of the originator. considering that the manufacturing process of epoetin alfa is owned by the producer of the reference product and can not be precisely duplicated, companies developing biosimilars have to implement a new manufacturing process. this is true also in highly regulated countries such as those of the eu, or the usa, where high - quality processes are used. some years ago, one randomized clinical trial using hx575 was halted for safety reasons following one prca case and one anti - epo ab positivity. the potential cause of immunogenicity was identified in soluble tungsten, most likely derived from the pins used to manufacture the syringes, causing unfolding and aggregation of hx575 in pre - filled syringes. considering that only 160 patients were treated with hx575 in the trial over a follow - up of 52 weeks, the incidence of anti - epo neutralizing ab in this study is particularly high. epoetin theta is an analogue of epoetin beta, which was approved as an originator by european medicine agency in 2009. data from one clinical trial administering epoetin zeta subcutaneously to 230 ckd patients did not report cases of anti - epoetin ab or prca. similarly, no cases had occurred during a large post - authorization observational study involving more than 1600 ckd patients receiving epoetin zeta intravenously. our patient had been enrolled in a second, ongoing, post - authorization observational study to estimate the incidence of prca and/or neutralizing ab during treatment with epoetin zeta administered subcutaneously in more than 6000 ckd patients. to our knowledge, this is the first prca case that has been described for epoetin zeta. our patient suddenly developed severe hypo - proliferative anaemia after 7 months from the start of epoetin zeta administered subcutaneously. this is in line with data in the literature describing a median of 7 months (range 1 month to 5 years) from the beginning of therapy until the diagnosis of prca. the severity of anaemia, which had become unresponsive to esa therapy, is testified by the very low reticulocyte count and by the dependence on blood transfusions. prca diagnosis was supported by the findings of the bone marrow biopsy, showing severe hypoplasia of the erythroid line, and by the presence of high - titre anti - epo neutralizing ab. no other causes of prca, such as parvovirus or hepatitis b infection, thymoma, lymphoproliferative disorders, drugs or autoimmune disease were identified. however, the timing from drug exposure and the fact that the patient received uniquely epoetin zeta makes the relationship between the drug and the occurrence of prca extremely likely. considering its rarity, it is difficult to distinguish whether this is a sporadic case or the expression of increased immunogenicity of epoetin zeta. the calculation of prca incidence based on length of exposure to epoetin zeta would be of interest, but is beyond the scope of this case report. however, in the absence of randomized clinical trial, there are insufficient data to provide guidance on the preferred immunosuppressive agents or treatment regimen. this is in line with other reports in the literature. interestingly, after therapy her hb stabilized without the need to challenge her with new esa therapy. it is possible that the iron load she received with blood transfusion had contributed to hb stability after the recovery of prca. one possible limitation of this case report is that reticulocytes were not tested at onset and that prca diagnosis was made with a certain delay. while this does not reduce the importance of the relationship between epoetin zeta use and the occurrence of prca, we acknowledge that an earlier testing of reticulocyte values would have prevented needless workup of anaemia with endoscopies and ct scans, and directed us towards bone marrow biopsy sooner. a more timely diagnosis would also have prevented the increase in epoetin zeta dose, which have possibly enhanced anti - epo ab production. to conclude, we report for the first time a patient who developed ab - mediated prca after receiving epoetin zeta subcutaneously. the patient described in this paper was enrolled in the post - marketing observational study epoe-09 - 11 pasco ii, which is sponsored by hospira. hospira has been informed about this case and the intention to publish it. v.p. f.l. was a member of an advisory board and/or speaker at meetings supported by akebia, amgen, astellas, janssen, gsk, hospira, pharmacosmos, roche and sandoz.	pure red cell aplasia (prca) may develop in patients with chronic kidney disease receiving erythropoiesis - stimulating agents (esa). we report on a 72-year - old patient who developed hypo - proliferative anaemia unresponsive to esa following the administration of epoetin zeta subcutaneously for 7 months. on the basis of severe isolated hypoplasia of the erythroid line in the bone marrow and high - titre neutralizing anti - erythropoietin antibodies (ab) , a diagnosis of ab - mediated prca was made. epoetin zeta was discontinued and the patient was given steroids. this was associated with anaemia recovery. to our knowledge this is the first prca case related to epoetin zeta.
to define pd predisposition genes and loci, omim (http://www.ncbi.nlm.nih.gov/omim) was searched with the words parkinson disease and genes and locus / loci. the somatic cm mutation data used in this article merged melanoma exome / genome sequencing from different sources as described elsewhere. all mutational data from 6 different whole - exome / genome sources were collated from 4 published studies and unpublished data. the data were formatted so that all positional data were mapped to the same genome build. in this case, any data that were on hg18 were lifted over to hg19 using the lift genome annotations tool available from ucsc (http://genome.ucsc.edu/cgi-bin/hgliftover, uc santa cruz software, the regents of the university of california, santa cruz, ca). in some cases, as data were merged, it became necessary to eliminate redundant information. for instance, with some samples, both a tumor and a cell line derived from it were sequenced and the overwhelming majority of mutations were shared. this is also true in the case of samples that were sequenced in more than one study and for multiple metastases extracted from the same patient in another study. when removing these duplicates and redundancies, all mutations were retained at a count of one and the sample name was merged into a single entry. this step was taken to ensure that the number of recurrent positions was not inflated in later analysis. once the list of mutations was established, the positional data and changes were formatted to an oncotator input format and annotated using the web - based version of oncotator (http://www.broadinstitute.org/oncotator, cambridge, ma). the next step taken was to remove any samples that were listed as acral, mucosal, or uveal melanoma subtypes, to ensure focusing on cm. in the final step, any samples in the initial publication that did not include a matched normal genotype were also removed. the data were arranged in a table, where the rows represent genes and the columns are cm tissue samples. the entries are the number of somatic mutations per each combination of gene and tissue sample. the somatic cm mutation platform was cross - referenced with the list of the defined park genes and loci (park1 to park20). to assess cm - related specificity of the findings, identical analyses were performed for adenocarcinoma of lung (adenoca - lung) and squamous cell carcinoma of lung (squamca - lung), based on data derived from the cosmic database (studies cosu417 and cosu418). the data format of the cm mutation data set was compatible with the cosmic data sets. splice variants are annotated in cosmic and are used if a mutation arises in a region that maps to the coding domain of a gene and the predicted protein is different. , we analyzed each gene individually, and in the second step, we analyzed the park genes combined as a group. the question of interest was whether single park genes harbor a relatively high number of somatic mutations, when compared with other genes in cm tissue. to answer this question, 2 measures were defined: the sum of mutations in each gene (smut) and the number of tissue samples in which a given gene was mutated at least once (ssampl). (note that the difference between smut and ssampl is that in ssampl, each sample is counted at most once .) analysis was performed by the following steps: computation of the empirical distributions for smut and ssampl for all analyzable genes, determination of the percentiles (75th, 90th, and 95th) of the empirical distribution, and determination of the location of the known park genes in the above empirical distributions. for comparison, and to ensure cm specificity of the findings, identical analyses were applied for adenoca - lung and squamca - lung in cosmic data sets of studies cosu417 and cosu418. lung carcinomas were chosen as control tissues because the number of somatic mutations in lung cancer is roughly similar to that of cm (1213/megabase in lung vs 1018/megabase in cm). because some samples harbored mutations in more than 1 park gene, we analyzed the distribution of the number of mutated park genes in cm using 2 methods: we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test.here, the question of interest was how often the 15 park genes are mutated as a group, relative to a randomly chosen group of 15 genes. we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test. here, the question of interest was how often the 15 park genes are mutated as a group, relative to a randomly chosen group of 15 genes. using a bootstrap - like procedure, we generated the empirical distributions of the variables of interest (smut, ssampl), aggregated over the 15 park genes. the aggregated variables are denoted by smutgroup, which is the total number of mutations of the 15 park genes as a group in all samples, and ssamplgroup, which is the number of samples in which at least one gene from the group was mutated. the procedure was performed as follows: randomly select a group of 15 genes.compute smutgroup and ssamplgroup for the selected group.repeat steps 1 and 2, ten thousand times.obtain the empirical distributions of smutgroup and ssamplgroup. we note that the 15 park genes were excluded from the pool of genes (18,758 15 = 18,743 genes). the same analysis was applied to each of the 3 cancer types. once the empirical distributions of these variables were generated for random gene groups, we verified the locations of the group of 15 park genes in these distributions, for smutgroup and ssamplgroup and for each cancer type. to define pd predisposition genes and loci, omim (http://www.ncbi.nlm.nih.gov/omim) was searched with the words parkinson disease and genes and locus / loci. the somatic cm mutation data used in this article merged melanoma exome / genome sequencing from different sources as described elsewhere. all mutational data from 6 different whole - exome / genome sources were collated from 4 published studies and unpublished data. the data were formatted so that all positional data were mapped to the same genome build. in this case, any data that were on hg18 were lifted over to hg19 using the lift genome annotations tool available from ucsc (http://genome.ucsc.edu/cgi-bin/hgliftover, uc santa cruz software, the regents of the university of california, santa cruz, ca). in some cases, as data were merged, it became necessary to eliminate redundant information. for instance, with some samples, both a tumor and a cell line derived from it were sequenced and the overwhelming majority of mutations were shared. this is also true in the case of samples that were sequenced in more than one study and for multiple metastases extracted from the same patient in another study. when removing these duplicates and redundancies, all mutations were retained at a count of one and the sample name was merged into a single entry. this step was taken to ensure that the number of recurrent positions was not inflated in later analysis. once the list of mutations was established, the positional data and changes were formatted to an oncotator input format and annotated using the web - based version of oncotator (http://www.broadinstitute.org/oncotator, cambridge, ma). the next step taken was to remove any samples that were listed as acral, mucosal, or uveal melanoma subtypes, to ensure focusing on cm. in the final step, any samples in the initial publication that did not include a matched normal genotype were also removed. the data were arranged in a table, where the rows represent genes and the columns are cm tissue samples. the entries are the number of somatic mutations per each combination of gene and tissue sample. the somatic cm mutation platform was cross - referenced with the list of the defined park genes and loci (park1 to park20). to assess cm - related specificity of the findings, identical analyses were performed for adenocarcinoma of lung (adenoca - lung) and squamous cell carcinoma of lung (squamca - lung), based on data derived from the cosmic database (studies cosu417 and cosu418). the data format of the cm mutation data set was compatible with the cosmic data sets. splice variants are annotated in cosmic and are used if a mutation arises in a region that maps to the coding domain of a gene and the predicted protein is different. , we analyzed each gene individually, and in the second step, we analyzed the park genes combined as a group. the question of interest was whether single park genes harbor a relatively high number of somatic mutations, when compared with other genes in cm tissue. to answer this question, 2 measures were defined: the sum of mutations in each gene (smut) and the number of tissue samples in which a given gene was mutated at least once (ssampl). (note that the difference between smut and ssampl is that in ssampl, each sample is counted at most once .) analysis was performed by the following steps: computation of the empirical distributions for smut and ssampl for all analyzable genes, determination of the percentiles (75th, 90th, and 95th) of the empirical distribution, and determination of the location of the known park genes in the above empirical distributions. for comparison, and to ensure cm specificity of the findings, identical analyses were applied for adenoca - lung and squamca - lung in cosmic data sets of studies cosu417 and cosu418. lung carcinomas were chosen as control tissues because the number of somatic mutations in lung cancer is roughly similar to that of cm (1213/megabase in lung vs 1018/megabase in cm). because some samples harbored mutations in more than 1 park gene, we analyzed the distribution of the number of mutated park genes in cm using 2 methods: we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test.here, the question of interest was how often the 15 park genes are mutated as a group, relative to a randomly chosen group of 15 genes. we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test. here, the question of interest was how often the 15 park genes are mutated as a group, relative to a randomly chosen group of 15 genes. using a bootstrap - like procedure , we generated the empirical distributions of the variables of interest (smut, ssampl), aggregated over the 15 park genes. the aggregated variables are denoted by smutgroup, which is the total number of mutations of the 15 park genes as a group in all samples, and ssamplgroup, which is the number of samples in which at least one gene from the group was mutated. the procedure was performed as follows: randomly select a group of 15 genes.compute smutgroup and ssamplgroup for the selected group.repeat steps 1 and 2, ten thousand times.obtain the empirical distributions of smutgroup and ssamplgroup. we note that the 15 park genes were excluded from the pool of genes (18,758 15 = 18,743 genes). the same analysis was applied to each of the 3 cancer types. once the empirical distributions of these variables were generated for random gene groups, we verified the locations of the group of 15 park genes in these distributions, for smutgroup and ssamplgroup and for each cancer type. the question of interest was whether single park genes harbor a relatively high number of somatic mutations, when compared with other genes in cm tissue. to answer this question, 2 measures were defined: the sum of mutations in each gene (smut) and the number of tissue samples in which a given gene was mutated at least once (ssampl). (note that the difference between smut and ssampl is that in ssampl, each sample is counted at most once .) analysis was performed by the following steps: computation of the empirical distributions for smut and ssampl for all analyzable genes, determination of the percentiles (75th, 90th, and 95th) of the empirical distribution, and determination of the location of the known park genes in the above empirical distributions. for comparison, and to ensure cm specificity of the findings, identical analyses were applied for adenoca - lung and squamca - lung in cosmic data sets of studies cosu417 and cosu418. lung carcinomas were chosen as control tissues because the number of somatic mutations in lung cancer is roughly similar to that of cm (1213/megabase in lung vs 1018/megabase in cm). because some samples harbored mutations in more than 1 park gene, we analyzed the distribution of the number of mutated park genes in cm using 2 methods: we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test.here, the question of interest was how often the 15 park genes are mutated as a group, relative to a randomly chosen group of 15 genes. we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test. here, the question of interest was how often the 15 park genes are mutated as a group, relative to a randomly chosen group of 15 genes. using a bootstrap - like procedure , we generated the empirical distributions of the variables of interest (smut, ssampl), aggregated over the 15 park genes. the aggregated variables are denoted by smutgroup, which is the total number of mutations of the 15 park genes as a group in all samples, and ssamplgroup, which is the number of samples in which at least one gene from the group was mutated. the procedure was performed as follows: randomly select a group of 15 genes.compute smutgroup and ssamplgroup for the selected group.repeat steps 1 and 2, ten thousand times.obtain the empirical distributions of smutgroup and ssamplgroup. randomly select a group of 15 genes. compute smutgroup and ssamplgroup for the selected group. we note that the 15 park genes were excluded from the pool of genes (18,758 15 = 18,743 genes). the same analysis was applied to each of the 3 cancer types. once the empirical distributions of these variables were generated for random gene groups, we verified the locations of the group of 15 park genes in these distributions, for smutgroup and ssamplgroup and for each cancer type. a total of 15 park genes and 20 park - associated loci were identified (table 1, first column). somatic cm analysis ed in 315,914 mutations in 18,758 genes by whole - exome sequencing in 246 metastatic cm tissue samples. table 1 summarizes the observed number of mutations and the number of samples harboring park gene mutations and the percentiles of the empirical distribution of mutations. as shown in the table, park8 (shown as straight bold numbers) was mutated in cm, as counted by smut, 55 times, thus being above the 95th percentile of the empirical distribution (which is 49 times) and in ssampl = 40 cm samples, again above 36, the 95th percentile of number of samples with mutations. park mutations in cutaneous melanoma, adenocarcinoma, and squamous cell carcinoma of lung three additional park genes (park2, park18, and park20, shown in table 1 as italic bold numbers) were mutated above the 90th percentile of the empirical distributions of smut and ssampl. figure 1 summarizes park genes found above the 90th percentile of the empirical distribution of tissue samples (ssampl) in cm, adenoca - lung, and squamca - lung, and table 1 depicts the distributions of park gene mutations for the smut and ssampl variables in the 3 cancer types. venn diagram summarizing mutated park genes observed above the 90th percentile of the empirical distribution in melanoma, adenocarcinoma, and squamous cell carcinoma of lung. overall, 48% of cm samples had a mutation in at least 1 park gene and 25% had mutations in multiple park genes (28 mutated genes) (table 2). samples with mutations in multiple park genes (25 mutated genes) were found in only 6% of adenoca - lung and in 7% of squamca - lung samples. comparison of the distribution of the number of mutated park genes in the 3 cancer types is shown in table 2. significantly more cm samples harbored multiple park gene mutations compared with squamca - lung (p = 0.0026) and with adenoca - lung (p < 0.0001) (kolmogorov - smirnov test). this finding was significant after bonferroni correction for multiple comparisons (required p value < 0.025 = 0.05/2). number of samples with mutations in i mutated park genes, i = 1, ,8, by cancer type the number of samples carrying diverse combinations of multiple park gene mutations in cm samples is depicted in table 3. the higher frequencies of some of the combinations in cm samples (in particular, those involving park2, park8, park18, and park20, table 3) vs the paucity of such samples in adenoca - lung (table e-1 at neurology.org/ng) and squamca - lung (table e-2) are clearly notable. frequencies of combinations of park gene mutations in cutaneous melanoma (246 samples) to further study the park genes as a group, we used the bootstrap - like permutation method (see statistical analysis). smutgroup and ssamplgroup of park genes shown as the vertical lines appearing to be located in the upper end of the distribution of genes in cm (figure 2a) and squamca - lung (figure 2c) and not for adenoca - lung (figure 2b). for cm (figure 2a), the location of smutgroup for the 15 park genes as a group was at the 83.5th percentile and that of ssamplgroup was at the 90.8th percentile. for squamca - lung (figure 2c), the location of smutgroup was at the 92.8th percentile and that of ssamplgroup was at the 94.1th percentile. for adenoca - lung (figure 2b), the respective sums were located at the 76.3rd percentile for smutgroup and at the 60.8th percentile for ssamplgroup. histograms of the empirical distributions of the sums of mutations in groups of 15 genes, generated by randomly selecting 10,000 groups of 15 genes in (a) cutaneous melanoma, (b) adenocarcinoma of lung, and (c) squamous cell carcinoma of lung (smutgroup upper panel, ssamplgroup lower panel). the vertical lines depict the locations of the sums of mutations (smutgroup) in the park genes. a total of 15 park genes and 20 park - associated loci were identified (table 1, first column). somatic cm analysis ed in 315,914 mutations in 18,758 genes by whole - exome sequencing in 246 metastatic cm tissue samples. table 1 summarizes the observed number of mutations and the number of samples harboring park gene mutations and the percentiles of the empirical distribution of mutations. as shown in the table, park8 (shown as straight bold numbers) was mutated in cm, as counted by smut, 55 times, thus being above the 95th percentile of the empirical distribution (which is 49 times) and in ssampl = 40 cm samples, again above 36, the 95th percentile of number of samples with mutations. park mutations in cutaneous melanoma, adenocarcinoma, and squamous cell carcinoma of lung three additional park genes (park2, park18, and park20, shown in table 1 as italic bold numbers) were mutated above the 90th percentile of the empirical distributions of smut and ssampl. figure 1 summarizes park genes found above the 90th percentile of the empirical distribution of tissue samples (ssampl) in cm, adenoca - lung, and squamca - lung, and table 1 depicts the distributions of park gene mutations for the smut and ssampl variables in the 3 cancer types. venn diagram summarizing mutated park genes observed above the 90th percentile of the empirical distribution in melanoma, adenocarcinoma, and squamous cell carcinoma of lung. overall, 48% of cm samples had a mutation in at least 1 park gene and 25% had mutations in multiple park genes (28 mutated genes) (table 2). samples with mutations in multiple park genes (25 mutated genes) were found in only 6% of adenoca - lung and in 7% of squamca - lung samples. comparison of the distribution of the number of mutated park genes in the 3 cancer types is shown in table 2. significantly more cm samples harbored multiple park gene mutations compared with squamca - lung (p = 0.0026) and with adenoca - lung (p < 0.0001) (kolmogorov - smirnov test). this finding was significant after bonferroni correction for multiple comparisons (required p value < 0.025 = 0.05/2). number of samples with mutations in i mutated park genes, i = 1, ,8, by cancer type the number of samples carrying diverse combinations of multiple park gene mutations in cm samples is depicted in table 3. the higher frequencies of some of the combinations in cm samples (in particular, those involving park2, park8, park18, and park20, table 3) vs the paucity of such samples in adenoca - lung (table e-1 at neurology.org/ng) and squamca - lung (table e-2) are clearly notable. frequencies of combinations of park gene mutations in cutaneous melanoma (246 samples) to further study the park genes as a group, we used the bootstrap - like permutation method (see statistical analysis). smutgroup and ssamplgroup of park genes shown as the vertical lines appearing to be located in the upper end of the distribution of genes in cm (figure 2a) and squamca - lung (figure 2c) and not for adenoca - lung (figure 2b). for cm (figure 2a), the location of smutgroup for the 15 park genes as a group was at the 83.5th percentile and that of ssamplgroup was at the 90.8th percentile. for squamca - lung (figure 2c), the location of smutgroup was at the 92.8th percentile and that of ssamplgroup was at the 94.1th percentile. for adenoca - lung (figure 2b), the respective sums were located at the 76.3rd percentile for smutgroup and at the 60.8th percentile for ssamplgroup. histograms of the empirical distributions of the sums of mutations in groups of 15 genes, generated by randomly selecting 10,000 groups of 15 genes in (a) cutaneous melanoma, (b) adenocarcinoma of lung, and (c) squamous cell carcinoma of lung (smutgroup upper panel, ssamplgroup lower panel). the vertical lines depict the locations of the sums of mutations (smutgroup) in the park genes. in the present study, we observed that genes associated with pd predisposition (park genes) are somatically mutated above random occurrence in cm. it is possible that the co - occurrence of mutations in multiple park genes has a cumulative contribution beyond the influence of a single gene dysfunction. the most commonly mutated park gene observed herein in cm (15% of tumors) was park8 or lrrk2, a gene that accounts for the most common autosomal dominant form in pd. lrrk2, leucine - rich repeat kinase 2, is a large protein displaying dual enzymatic functions, a protein kinase effector domain, and a ras - oncogene like gtpase domain, in addition to multiple protein interaction domains. the g2019s missense mutation, the most common lrrk2 germline mutation in pd, in gain of function. downregulation of lrrk2 in cultured renal and thyroid cancer cells compromised met activation and reduced downstream signaling, suggesting that lrrk2 and met cooperate in controlling tumor growth. patients with ashkenazi - jewish pd, who carry the germline g2019s*lrrk2 mutation, were reportedly at higher risk for developing mainly hormone - related cancers, breast and prostate, but not for melanoma or any skin cancer compared with pd patients who do not carry this mutation. it is of interest to note that a new bioinformatics tool used for detecting somatic activating mutations identified the lrrk2 locus as a possible oncogene, albeit analyzed only in nonskin cancers, and not cm. the second most commonly mutated gene in our study was park2 encoding for parkin, an ubiquitin ligase 3. park2-inactivating germline mutations underlie the most common form of autosomal recessive early - onset pd. several studies have shown that park2 is a potential tumor suppressor, inactivated in many cancers including glioblastoma, renal cell carcinoma, colon, lung, and pancreatic cancers. germline park2-inactivating mutations were recently reported to be more frequently encountered in a cm cohort than in controls (odds ratio = 4.93). furthermore, parkin was underexpressed in melanocytes whereas reexpression of parkin in melanoma cell lines ed in reduction of cell proliferation rates. in that study, 43% of primary and 66% of metastatic cm cell lines harbored an inactivating park2 mutation. the third most frequently mutated gene in cm in the present study was park18 or eif4g1. eif4g1 is a member of the translation initiation complex eif4f and plays a central role in eif4f complex formation. the effect of eif4g1 mutations on cap complex assembly and initiation of protein synthesis remains currently unknown. notably, lrrk2 is involved in protein translation and cross - talks with elongations factors, one of which is encoded by eif4g1. the possible association of this gene with carcinogenesis is indirectly inferred from abnormal expression patterns in cancers such as squamca - lung, inflammatory breast cancer, and nasopharyngeal cancer. the fourth most commonly mutated gene somatically in cm is park20 or synj1, a gene whose biallelic mutations are associated with an uncommon form of autosomal recessive pd. recently, functional protein - protein interactions of eif4g1 with synj1 were reported. synj1 has not been shown to be involved in cancer pathogenesis, but its homolog, synj2, was reportedly overexpressed in breast cancer. one caveat to these and the putative specificity of park genes targeted somatically in cm is the fact that cm displays much higher somatic mutation rates than other cancer types: somatic mutation frequency per megabase is 2 in ovarian cancer, 34 in breast cancer, vs 1018 in cm. to account for these different mutation rates, we chose to compute the relative location of park genes in cm, in comparison with the empirical distributions of the plethora of mutations in cm itself and were able to dissect park genes mutated above these distributions. in addition, we chose lung cancer, which has the closest mutation load (1213/megabase) to cm as control tumor tissue and showed that cm has significantly more mutated park genes compared with adenoca - lung and squamca - lung. the strength of our study is its approach focusing on all park genes and in the use of big data. our study relies exclusively on bioinformatics findings, and there is no assignment of the somatic sequence variants as pathogenic or nonpathogenic and there are no experimental data to prove (or disprove) the possible mechanisms of association of genetic variants with phenotypes. additional concerns involve the use of stringent statistical nonparametric analysis that may only be able to detect high odds and omit moderate contributions of single genes or subsets and thus may leave true associations undetected. the present study focused only on park genes that are somatically mutated in cm. yet, the possible functional and genetic intersections between cm and pd are, in all likelihood, more complex. notably, the reported list of somatic cm mutations encompasses several genes that are functionally associated with pd. for example, the alpha - synuclein gene (snca) is associated with 2 autosomal dominant inherited forms of pd (park1 and park4). snca accumulates in neurons and forms the main component of the lewy bodies, the pathologic hallmark of familial and sporadic pd. snca is highly expressed in both primary and metastatic melanoma tissues, but not in nonmelanocytic cells. another example is pla2g6 (park14), a gene related to nevus count, whose germline mutations cause familial pd and whose variability has been associated with increased risk for developing melanoma. a common denominator between the cells affected in cm and pd is their shared neural crest origin. the initiation of cm malignant transformation process occurs presumably early in life and is associated in a number of cases with sunburn episodes during early childhood. pd genotype may be more prone to uv - induced mutations, increasing the likelihood of acquiring driver this may be suggestive of some genomic instability that ultimately predisposes to pd and cm. the present study highlights an association between somatic pd - related gene mutations and cm. although the functional consequences of the mutations observed in shared genes in both diseases remain to be elucidated, our observation suggests shared pathways and offers one plausible explanation for the observed cm - pd relationship. rivka inzelberg: drafting / revising the manuscript, study concept or design, analysis or interpretation of data. yardena samuels: drafting / revising the manuscript, study concept or design, analysis or interpretation of data, acquisition of data. edna schechtman: drafting / revising the manuscript, analysis or interpretation of data, statistical analysis. eitan friedman: drafting / revising the manuscript, study concept or design, analysis or interpretation of data. y. samuels is supported by the israel science foundation grants 1604/13 and 877/13, the erc (stg-335377), the henry chanoch krenter institute for biomedical imaging and genomics, the estate of alice schwarz - gardos, the estate of john hunter, the knell family, the peter and patricia gruber award, and the hamburger family. dr. eytan domany has served on the editorial board of bmc bioinformatics journal of statistical mechanics: theory and experiment, holds a patent for serologic diagnosis of pemphigus vulgaris, has received research support from the leir charitable foundation, has received royalty payments for technology invention (immunarray co.), and holds stocks / stock options in immunarray.	objective: to assess whether parkinson disease (pd) genes are somatically mutated in cutaneous melanoma (cm) tissue, because cm occurs in patients with pd at higher rates than in the general population and pd is more common than expected in cm cohorts.methods:we cross - referenced somatic mutations in metastatic cm detected by whole - exome sequencing with the 15 known pd (park) genes. we computed the empirical distribution of the sum of mutations in each gene (smut) and of the number of tissue samples in which a given gene was mutated at least once (ssampl) for each of the analyzable genes, determined the 90th and 95th percentiles of the empirical distributions of these sums, and verified the location of park genes in these distributions. identical analyses were applied to adenocarcinoma of lung (adenoca - lung) and squamous cell carcinoma of lung (squamca - lung). we also analyzed the distribution of the number of mutated park genes in cm samples vs the 2 lung cancers.:somatic cm mutation analysis (n = 246) detected 315,914 mutations in 18,758 genes. somatic cm mutations were found in 14 of 15 park genes. forty - eight percent of cm samples carried 1 park mutation and 25% carried multiple park mutations. park8 mutations occurred above the 95th percentile of the empirical distribution for smut and ssampl. significantly more cm samples harbored multiple park gene mutations compared with squamca - lung (p = 0.0026) and with adenoca - lung (p < 0.0001).: the overrepresentation of somatic park mutations in cm suggests shared dysregulated pathways for cm and pd.
what was different in pci between the two merged analysis described above?14,31 we may argue that patients treated with dess had more three - vessel disease (87.3% vs. 36.1% with bms) and perhaps a higher syntax score than those treated with bms (in fact, we will never know which was the syntax score for them). however, on the other hand, bms pooled data also included diabetic population (18.1% vs. 0% with dess) and more frequently compromise of proximal lad stenosis (90% vs. 59% with dess).14,31 the fact that first - generation dess were used in syntax trial could be one of the most attractive explanations; in fact, syntax patients with definitive set (6.8%) had 35.4% occurrences of cardiac death.24 however, that was not the case with ees used in the best trial, which significantly improved safety compared to the first designs. therefore, perhaps, stent design itself is not solely the reason for the poorer long - term outcome data of these two rcts. if we analyze the trial methods,21,30 in spite of different des designs, both studies share similar pci strategies, meaning that the goal was to achieve complete anatomic revascularization defined by authors as not any residual stenosis 50% in any major coronary artery or their large branches30; consequently, we can assume that many intermediate lesions were stented and that concurs with the similarity of stent length in both studies. when we analyze completeness of revascularization with pci,1 we should take into account several different situations such as the amount of myocardium at risk or stenosis severity of the lesions not included in the revascularization strategy. prognosis in patients with incomplete revascularization should be different if the not - attempted vessels had a complete chronic closure with collateral circulation (fig . 1a), high - degree steno - sis in a large vessel (fig . differences in the amount of completeness of revascularization with both, pci and cabg, was present in all rcts since the first study was performed ; however, in the past, such differences were not associated with poorer outcome4,14 ( 53.2% with pci vs. 82.7% with cabg, p = 0.0003, see table 1). furthermore, the fact that only 56.7% and 50.9% of syntax and best patients, respectively, in the pci arm achieved the goal of complete revascularization demonstrated how difficult it is for pci to achieve such aim.31 therefore, improved des design is only one face of the problem, while changing pci strategies using a more conservative policy during des implantation would be the other. the eraci iv study,32,33 with a patient population of multiple vessel disease and left main stenosis, used a second - generation des and a conservative pci strategy, defined as stenting only severe lesions (visually 70%) in large vessels. intermediate (> 50 to < 70) lesions in small or large vessels or severe lesions in small vessels (< 2.0 mm) were not included. this pci strategy of not scoring lesions not included in the revascularization strategy allowed to build a new scoring system where low syntax score was found in 54% of patients and only 17.2% of patients persisted with high syntax score.34 investigators of eraci iv, at more than two years of follow - up, reported remarkable low rates of adverse events including death / mi / stroke of 3.9%, unplanned new revascularization of only 4%, and death / mi / stroke / target vessel revascularization (tvr) of 6.7%. it is important to note that there was no major penalty for this conservative policy as it was reflected by the low tvr rate in the not - stented intermediate lesions (1.3%). we do not know whether these will remain at a five - year follow - up, although at the present time, the low rate of events in the not - stented lesions supports the pci strategy of this study. in agreement , fame investigators largely demonstrated that nonischemic lesions had better outcome when they were not treated with dess.35,36 we have to take into account that, even in the era of safer dess, neoatherosclerosis as a consequence of des implantation has not disappeared.28 to recap, interventional cardiologists have been doing a lot of work during this 25-year journey trying to close the safety / efficacy gap between pci and cabg in patients with multiple vessel disease and, looking at long - term from rcts with the old and new dess, we may conclude that improved stent design alone is not enough to narrow the gap between pci and cabg.37 new revascularization strategies during pci and the search for a more functional revascularization avoiding unnecessary des implantation should be the new goal for future randomized comparisons between pci and cabg.	randomized clinical trials (rcts) with first- and second - generation drug - eluting stents (dess) confirmed the superiority of coronary artery bypass surgery (cabg) in patients with multiple vessel disease. in spite of different des designs, investigators in these trials used similar percutaneous coronary intervention (pci) strategies hoping to achieve complete revascularization, meaning that all intermediate lesions would be stented. one of these studies also included small vessels in the revascularization policy. on this revision, authors searched for a potential explanation of these intriguing findings and also for solutions to this problem, not seen years ago when other rcts compared cabg with pci in the previous des era. after they revised old and new scientific data, they concluded that improved des design is not itself enough to narrow the gap between pci and cabg and that in the future rcts we should institute more conservative strategies avoiding unnecessary multiple des implantation.
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it only restricts reproduction of the content for commercial gain. this restriction is an essential component of the business models of the vast majority of publishers, which remain subscription based. cc: creative commons a nonprofit organization that provides standardized copyright licenses, which define the terms of reuse of creative work. by: attribution must be given to the original work. nc: noncommercial published work can be reused without permission, as long as it is for noncommercial purposes. sa: share alike any subsequent distribution must follow the rules set out in the original license. this provides easy access to a machine - readable form of the content for text and data miners. we encourage all publishers to participate in this service. if all participants would agree that text and data mining constitute fair use, the content could be mined by any third party regardless of the copyright / licensing terms attached to it. the name open access subset is actually a misnomer, as the content does not have to be available on an immediate basis (or under a cc - by license) to be included. efforts to open up citation data for mining are commendable if one is trying to break the stranglehold on citation data by large commercial entities, but citations are only a small portion of the textual data in an article. there are no licensing or technical barriers to making all of the text and data published by subscription - based publishers available for mining.	the existing public access policy for our three journals the journal of cell biology, the journal of experimental medicine, and the journal of general physiology is fully compliant with new policies from the research councils uk (rcuk) and the wellcome trust. in addition to mandating public access, the new policies specify licensing terms for reuse of content by third parties, in particular for text and data mining. we question the need for these specific terms, and we have added a statement to our licensing policy stipulating that anyone, including commercial entities, is permitted to mine our published text and data.
full - thickness cartilage defects remain one of the most common, yet difficult challenges faced by orthopedic surgeons today. curl et al. reported in over 31,000 knee arthroscopies that 63% of patients had a grade iii or iv articular cartilage lesion. these lesions demonstrate limited healing potential, with full - thickness cartilage lesions healing with both types i and ii cartilage, and lower than normal concentrations of proteoglycans. the formation of structurally inferior fibrocartilage leads to premature irregularities of the joint surface over time, meniscal tears, and, eventually, degenerative arthritis. microfracture was developed by steadman et al. as a novel technique to treat full - thickness cartilage lesions. the technique of microfracture uses an awl to penetrate the subchondral plate, which leads to migration of mesenchymal stem cells into the cartilage defect and reconstitution of the injured area with a fibrocartilage repair with varying amounts of types i, ii, and iii collagen content. microfracture reportedly in 70% to 90% of patients having short - term improvement in function. however, a small but substantial percentage has only moderate to poor fill and worse subjective and objective clinical outcomes. despite the clinical success of microfracture in healing small (< 2 cm) cartilage defects in the knee , penetration of the subchondral bone allows for mesenchymal stem cells to enter into a localized defect and produce fibrocartilage. current surgical techniques typically allow for complete fill in 54%, partial fill in 29%, and poor fill in 17%. not surprisingly, there is a correlation between amount of fill and clinical outcome. as the of microfracture have been variable, there have been many attempts to increase the likelihood of complete lesion fill with hyaline cartilage. previous animal studies have utilized bone morphogenetic proteins (bmps), transforming growth factor-, and insulin - like growth factor-1 to stimulate cartilage healing with promising . in a recent study, morgan et al. found bmp-7, which induces cartilage formation, worked synergistically with microfracture to heal full - thickness cartilage defects in a rabbit model. despite the early success of these studies in animal models, the role of exogenous parathyroid hormone (pth) in fracture healing continues to be elucidated. alkhiary et al. originally reported that intermittent administration of pth enhanced fracture healing in a rat fracture model. the authors found that treatment with 80 g / kg pth increased callus area and strength, with an increase in the amount of new bone formed with histologic analysis. reported increased expression of sox9 and procollagen -1 and increased chondrocyte proliferation in animals receiving intermittent pth. sox9 is a high - mobility group domain transcription factor expressed in chondrocytes during cartilage formation and is essential for cartilage formation in sox9 transgenic mice. expression of sox9 is decreased in cartilage defects that failed to heal, highlighting the importance of this transcription factor in healing of cartilage lesions. increased expression of wnt 4, 5a, 5b, and 10b in animals treated with intermittent pth to stimulate fracture healing. taken together, these studies suggest that exogenous pth stimulates fracture healing by increasing the activity of genes such as sox9 and the wnt family of proteins to increase chondrogenesis and promote cartilage formation. the purpose of this study was to evaluate the effects of exogenous intermittent pth administration on the gross and the microscopic cartilage healing in a rabbit microfracture model. based on the improved chondrogenesis seen with the use of pth in a fracture healing model, we hypothesized that the same mechanisms would stimulate improved cartilage formation in a microfracture setting we specifically aimed to measure the effect that exogenous, intermittent pth administration has following 1- and 4-week treatment periods on the gross and histologic appearance of cartilage defects treated by microfracture compared to animals treated with microfracture alone. female adolescent new zealand white rabbits (n = 12), weighing from 3.1 to 3.5 kg, were utilized for this study. the animals were divided into three groups: microfracture alone, microfracture + 10 g / kg human recombinant pth for 7 days, and microfracture + 10 g / kg recombinant pth for 28 days (fig . 1). the pth was administered subcutaneously on a daily basis commencing on the day of surgery and continued until the specified day. standard anteroposterior and lateral radiographs of the affected knee were obtained at the time of sacrifice to identify any osseous changes. the research protocol was approved by the animal ethics committee of the hospital for special surgery and animal care and experimental procedures were approved by the animal institutional review board. 1a c diagrams illustrate the experimental design for the pth treatment of 6-mm - diameter full - thickness defects of articular cartilage. a saline was administered as a control, b 10 g / kg pth was administered daily for 1 week, or c 10 g / kg pth was administered daily for 4 weeks. materials and methods section a c diagrams illustrate the experimental design for the pth treatment of 6-mm - diameter full - thickness defects of articular cartilage. a saline was administered as a control, b 10 g / kg pth was administered daily for 1 week, or c 10 g / kg pth was administered daily for 4 weeks. a midline incision was made over the knee and a medial parapatellar arthrotomy was performed to expose the knee. a 6-mm full - thickness defect was made on the weight - bearing surface of the trochlea using a curette. the microfracture holes punctured the subchondral plate and blood was seen to exit each of the holes. at the sacrifice, the soft tissue was removed from the distal femur, and the femur was sectioned approximately 1 cm above the joint. we scored the gross appearance of the lesion based on the fill of the lesion (1 = no fill ; 2 = minimal fill ; 3 = near - complete fill ; 4 = complete fill), quality of the tissue within the lesion (1 = no tissue ; 2 = soft tissue within the lesion ; 3 = normal appearing tissue within the lesion), and surrounding changes (1 = substantial degenerative cartilage changes surrounding the lesion with loss of cartilage down to bone ; 2 = slight changes in the surrounding cartilage without loss of cartilage to bone ; 3 = no changes). this allowed for a calculation of a gross score with a maximum of 10 points. following gross examination, the distal femurs were fixed in 4% paraformaldehyde at room temperature and then transferred to 10% ethylenediaminetetraacetic acid for 3 weeks for decalcification. five - micrometer transverse sections were taken through the center of the defect and stained with hematoxylin and eosin or alcian blue. for semiquantitative analysis of cartilage restoration, we (bf, zd) examined the sections in a blinded manner and scored according to the grading scale of pineda et al. with modifications as described by mizuta et al. the maximum score with this scale is 14, indicating complete fill with reconstitution of the osteochondral junction, normal staining of the matrix, and normal cell morphology. table 1scoring system for the histological appearance of full - thickness cartilage defectscharacteristicscoredefect fill125%3100%475%350%225%10%0reconstitution of osteochondral junctionyes3almost2not close1matrix stainingnormal4reduced3faint2minimal1none0cell morphologynormal4mostly hyaline and fibrocartilage3mostly fibrocartilage2some fibrocartilage1nonchondrocytic cells only0 scoring system for the histological appearance of full - thickness cartilage defects data are presented as the mean standard deviation. differences between groups in both the gross and histologic score were evaluated with a one - way analysis of variance. at the time of sacrifice, there were no complications due to pth administration, and all animals appeared to tolerate the treatment well. one animal developed ossification around the knee and a flexion contracture during the last week of the study. this animal was treated with a microfracture alone, and therefore, this was not a complication of pth administration. administration of pth inhibited or delayed the regeneration of cartilage in the microfracture sites compared to the untreated animals. grossly, all animals in the microfracture alone group appeared to have completely healed their defects with normal - appearing cartilage in three of four animals. the overall gross score was 9.1 0.8. in the 1-week pth group, there was minimal or no new cartilage present and the defects remained present in all the animals. the overall gross score was 2.3 1.4 (p < 0.001 versus control). in the 4-week pth group, there was minimal cartilage formed in two of the four animals although the cartilage felt soft. the overall gross score was 3.6 2.5 (p < 0.001 versus control ; p = ns versus 1-week pth ; fig . 2). 2a c photographs show the gross appearance of representative specimens at the time of sacrifice. a in the microfracture alone specimen, there is reconstitution of the lesion with near - complete fill and normal - appearing cartilage. b in the microfracture + 1-week pth specimen, the lesion is still clearly evident with minimal fill. c in the microfracture + 4-week pth specimen, similar to the 1-week treatment group, the lesion remains evident with minimal fill within the lesion. a in the microfracture alone specimen, there is reconstitution of the lesion with near - complete fill and normal - appearing cartilage. b in the microfracture + 1-week pth specimen, the lesion is still clearly evident with minimal fill. c in the microfracture + 4-week pth specimen, similar to the 1-week treatment group, the lesion remains evident with minimal fill within the lesion. there appears to be some mild erosive as well histologic analysis confirmed the findings of the gross analysis further suggesting that pth administration inhibited cartilage regeneration in the microfracture sites. there was near - complete fill of the defect in the microfracture alone group, although there were fewer cells and reduced matrix staining within the microfracture lesion compared to control contralateral femurs (fig . , there was no fill of the lesion and there appeared to be thinning of the surrounding cartilage in all the samples ( fig . similar were found in the 4-week pth group, with minimal fill of the defect and thinning of the surrounding cartilage and decreased matrix staining ( fig . 3c). the average histologic score in the microfracture alone group was 10.3 3.4, compared to 2.7 2.5 in the 1-week pth group (p < 0.001 versus microfracture alone) and 3.4 2.5 in the 4-week pth group (p < 0.001 versus microfracture alone ; fig . 4). 3a c photomicrographs show the histologic appearance of representative specimens at the time of sacrifice (stain, hematoxylin and eosin, alcian blue ; original magnification, 10). a in the microfracture alone specimen, there is reconstitution of the lesion with near - complete fill, although there are fewer than normal cells and the matrix staining is less than normal. b in the microfracture + 1-week pth specimen, the lesion is still clearly evident with minimal fill. c in the microfracture + 4-weeks pth specimen, no cartilage formation is evident within the lesion, and the surrounding cartilage appears to be thinned as wellfig. p < 0.001 versus control animals. mfx microfracture a c photomicrographs show the histologic appearance of representative specimens at the time of sacrifice (stain, hematoxylin and eosin, alcian blue ; original magnification, 10). a in the microfracture alone specimen, there is reconstitution of the lesion with near - complete fill, although there are fewer than normal cells and the matrix staining is less than normal. b in the microfracture + 1-week pth specimen, the lesion is still clearly evident with minimal fill. c in the microfracture + 4-weeks pth specimen, no cartilage formation is evident within the lesion, and the surrounding cartilage appears to be thinned as well a graph shows the histologic score as described by pineda et al. intermittent pth administration can enhance fracture healing in an animal model. despite the success of exogenous pth on fracture healing and spine fusion, we investigated the role of intermittent administration of exogenous recombinant pth on healing of a microfracture model. based on the improved chondrogenesis seen with the use of pth in a fracture healing model, we hypothesized that the same mechanisms would stimulate improved cartilage healing in a microfracture setting. contrary to our hypothesis, we found that intermittent pth given for 1 or 4 weeks was effective at inhibiting cartilage production in this model. although disappointing from a cartilage repair standpoint, our are interesting as it increases our understanding of the role of pth in bone and cartilage formation. first, since we designed a preliminary study, we used no alternate doses or administration time courses. a dose of 10 g / kg was chosen based on its success in previous studies that it found successfully improved bone formation via chondrogenesis. likewise, a 1- and 4-week time course of pth administration was chosen based on its success in previous studies. it is possible that a shorter period of time (i.e., 25 days) would be sufficient to stimulate cartilage formation without leading to inhibition of osteochondral healing. second, the underlying mechanism of inhibition of cartilage formation with intermittent pth is not clearly understood. with continuous pth administration, there is downregulation of the pth / pth - related protein (pthrp) receptors, but it is unknown if intermittent pth functions by the same mechanism to inhibit cartilage repair. clearly, these factors should be clarified in subsequent studies. based on the promising with other growth factors and the enhanced chondrogenesis seen using recombinant intermittent pth in a fracture healing model, we presumed that administration of 10 g / kg intermittent pth would enhance cartilage formation in a microfracture model. however, we found that intermittent pth actually nearly completely inhibited the formation of new cartilage within the defect in our model. with 1 week of pth , there was no cartilage formed within the defect, a finding that was clearly evident on both gross and histologic examination. the findings were similar in animals treated for 4 weeks of intermittent pth. on gross examination, there was minimal or no fill in almost all the animals treated with intermittent pth, suggesting an inhibition of chondrocyte proliferation. a smaller proportion of the animals also demonstrated adjacent cartilage breakdown after treatment with pth. interestingly, there appeared to be decreased staining of the surrounding cartilage matrix in those animals treated with pth, suggesting an alteration of collagen or proteoglycan concentration in these animals. these clearly demonstrate that intermittent pth administration is not an effective means to improve the ability of microfracture to heal chondral defects. two previous studies utilizing continuous pth administration in a chondral defect model have demonstrated similar to ours. reported that pth, when given continuously via an osmotic pump into the knee, completely inhibited chondrogenesis. the authors found that, although proliferation of the chondral precursors was normal, pth administration effectively inhibited chondrogenic differentiation. in a subsequent study, the authors found that continuous pth administration for 4 weeks was able to irreversibly block expression of pth / pthrp receptor, suggesting cells that migrate into the chondral defect maintain their chondrogenic potential for no more than 2 weeks. our data suggest that even intermittent pth administration in a similar outcome in this model. this is surprising as it is traditionally thought that continuous and intermittent pth function in separate ways in a fracture healing model. these demonstrate that it is vital to more completely understand the mechanisms that govern the outcome of pth administration in osteochondral injury models. although it may be alluring to use pth to improve fracture healing in these difficult to treat fracture models, it is possible that the pth would have a deleterious effects on the cartilage injury and in a much poorer overall outcome. we found that intermittent pth did not improve healing of full - thickness chondral defects in a rabbit microfracture model and in fact inhibited cartilage formation within the defect. although these did not support our hypothesis, they do highlight the importance of understanding the molecular mechanisms that govern the relationship between pth and chondrogenesis. future studies focusing on the molecular mechanisms of pth - mediated chondrogenesis and the time course of gene expression may help elucidate why pth is effective in stimulating cartilage formation in a fracture healing model yet is unable to stimulate cartilage in a full - thickness chondral injury model.	intermittent parathyroid hormone administration can enhance fracture healing in an animal model. despite the success of exogenous parathyroid hormone on fracture healing and spine fusion, few studies have examined the role of parathyroid hormone on cartilage formation. we determined the effects of intermittent parathyroid hormone on cartilage formation in a rabbit microfracture model of cartilage regeneration. twelve rabbits were divided into three equal groups: microfracture alone, microfracture + parathyroid hormone daily for 7 days, and microfracture + parathyroid hormone for 28 days. nonoperated contralateral knees were used as controls. the animals were sacrificed at 3 months and gross and histologic analysis was performed. the microfracture alone group demonstrated the most healing on gross and histologic analysis. treatment with either 1 or 4 weeks of parathyroid hormone inhibited cartilage formation. although discouraging from a cartilage repair point of view, this study suggests that the role parathyroid hormone administration has in clinical fracture healing must be examined carefully. although parathyroid hormone is beneficial to promote healing in spine fusion and midshaft fractures, its deleterious effects on cartilage formation suggests that it may have adverse effects on the outcomes of periarticular fractures such as tibial plateau injuries that require cartilage healing for a successful clinical outcome.
the main endothelium derived factor is nitric oxide (no) which is not only a potent vasodilator but also inhibits platelet aggregation, smooth muscle cell migration and proliferation, monocyte adhesion and adhesion molecule expression, thus protecting the vessel wall against the development of atherosclerosis and thrombosis. endothelium - dependent vasodilation in response to substances, such as acetylcholine, bradykinin and substance p and reactive hyperemia, was reduced in brachial, coronary, renal arteries and femoral areteries in patients with essential hypertension. impairment of endothelial function has been shown to play an important role in the development and maintenance of hypertension. therefore, an important aim of antihypertensive therapy would be not only to normalize blood pressure values but also to reverse endothelial dysfunction by restoring no availability. several studies have demonstrated the restoration of endothelial function in essential hypertensive patients through the administration of antihypertensive agents, while others have shown that effective antihypertensive therapy did not restore impaired endothelium - dependent vasodilation in the forearm circulation of hypertensive patients. the effects of antihypertensive agents on endothelial function have not been fully evaluated in human hypertension and data on the forearm circulation of humans are controversial. it may be clinically important to select an appropriate antihypertensive agent that is effective in improving endothelial dysfunction in patients with established essential hypertension. recently, the role of superoxide anion and its interaction with nitric oxide has been investigated. under physiological conditions, these oxygen - free radicals are potent chemical inactivators of no and the balance between no and superoxide is more important than the absolute levels of either alone. vitamin c is an important antioxidant in human plasma, capable of scavenging oxygen - free radicals and sparing other endogenous antioxidants from consumption. therefore, the aim of this study was to evaluate the endothelial function in hypertensive patients to investigate whether vitamin c administration has any benefit on the endothelial function and to determine whether treatment with calcium antagonist improves endothelial dysfunction in hypertensive patients. eight hypertensive patients (age range 35 to 73 years) were recruited. they had a clinical blood pressure reading (the average of 3 different sphygmomanometric measurements, each performed on 3 separate days) of > 140/90 mmhg. the possibility of secondary causes of hypertension was excluded by standard clinical and laboratory tests. exclusion criteria were 1 ) evidence of overt atherosclerotic disease, i.e., coronary artery disease, peripheral vascular disease, stroke, etc. 2 ) having other risk factors of atherosclerosis, i.e., current smoking and smoking within 1year, severe hypercholesterolemia (> 240 mg / dl) and diabetes mellitus 3 ) advanced organ failure 4 ) malignancy. in all patients, subjects were required to refrain from drinking alcohol or caffeine - containing beverage for 12 hours before the study. the protocol of the study was approved by the ethics committee of our institution and informed consent was obtained from each participant. the procedures followed were in accordance with institutional guidelines. the examination was done at supine position. a mercury - filled silastic strain gauge was placed around the thickest part of the forearm. the size of the strain gauge was selected to be about 2 cm less than the maximal forearm circumference. the strain gauge was connected to plethysmograph (hokanson ec - r5, issaquah, washington) to record the forearm volume change. a rapid cuff inflator (hokanson e-10) was used to inflate the arm cuff to 40 mmhg instantaneously, thus occluding venous return from the forearm. a wrist cuff was inflated to 20 mmhg above the systolic pressure to cut off the arterial flow to hand. intra - arterial pressure was measured continuously (transpac ; abbot laboratories) throughout the study. the measurement of forearm volume change was repeated 7 times for each stage. between infusions, the cuffs were deflated, allowing at least 15 min for forearm blood flow to recover from the preceding infusion and before further baseline measures were recorded. all solutions were prepared aseptically from sterile stock solutions or ampoules immediately before infusion into brachial artery. acetylcholine and l - nmma had been diluted in distilled water and filtered through 0.22 m filter. an intra - arterial infusion of 5% dextrose solution was begun at 1 ml / min and continued throughout drug infusion. basal measurement was obtained during the infusion of dextrose solution. acetylcholine (sigma chemical) acetylcholine was infused for 5 minutes for each dose level and forearm blood flow was measured during the last 2 minutes of each dose. after 15 minutes of wash - out period, intra - arterial infusion of vitamin c at 24 mg / min was done for 10 minutes. with continued infusion of vitamin c at the same rate, acetylcholine was infused at the same incremental doses as the previous stage and measurement of forearm blood flow was made during the last 2 minutes of each dose. after another 15 minutes of rest period, intra - arterial infusion of vitamin c at the same rate, plus infusion of l - nmma (ng - monomethyl - l - arginine, sigma chemical), an inhibitor of nitric oxide synthesis, was begun at 100 g / min and continued for 5 minutes. then infusion of acetylcholine at incremental dose and measurement of forearm blood flow were done as in previous stages. forearm blood flow was measured and forearm blood flow changes during acetylcholine infusion were expressed as percentage changes from the baseline immediately preceding each drug administration. forearm blood flow changes were compared between normal and hypertensive groups with and without vitamin c infusion. comparison was also made between before and after antihypertensive treatment in hypertensive group and forearm blood flow was compared in hypertensive group with and without vitamin c infusion. 6.12 all values were expressed in mean standard error of mean and p<0.05 was considered significant. eight hypertensive patients (age range 35 to 73 years) were recruited. they had a clinical blood pressure reading (the average of 3 different sphygmomanometric measurements, each performed on 3 separate days) of > 140/90 mmhg. the possibility of secondary causes of hypertension was excluded by standard clinical and laboratory tests. exclusion criteria were 1 ) evidence of overt atherosclerotic disease, i.e., coronary artery disease, peripheral vascular disease, stroke, etc. 2 ) having other risk factors of atherosclerosis, i.e., current smoking and smoking within 1year, severe hypercholesterolemia (> 240 mg / dl) and diabetes mellitus 3 ) advanced organ failure 4 ) malignancy. in all patients, subjects were required to refrain from drinking alcohol or caffeine - containing beverage for 12 hours before the study. the protocol of the study was approved by the ethics committee of our institution and informed consent was obtained from each participant. a mercury - filled silastic strain gauge was placed around the thickest part of the forearm. the size of the strain gauge was selected to be about 2 cm less than the maximal forearm circumference. the strain gauge was connected to plethysmograph (hokanson ec - r5, issaquah, washington) to record the forearm volume change. a rapid cuff inflator (hokanson e-10) was used to inflate the arm cuff to 40 mmhg instantaneously, thus occluding venous return from the forearm. a wrist cuff was inflated to 20 mmhg above the systolic pressure to cut off the arterial flow to hand. intra - arterial pressure was measured continuously (transpac ; abbot laboratories) throughout the study. the measurement of forearm volume change was repeated 7 times for each stage. between infusions, the cuffs were deflated, allowing at least 15 min for forearm blood flow to recover from the preceding infusion and before further baseline measures were recorded. all solutions were prepared aseptically from sterile stock solutions or ampoules immediately before infusion into brachial artery. acetylcholine and l - nmma had been diluted in distilled water and filtered through 0.22 m filter. an intra - arterial infusion of 5% dextrose solution was begun at 1 ml / min and continued throughout drug infusion. acetylcholine (sigma chemical) was infused intra - arterially at 7.5, 15 and 30 g / min. acetylcholine was infused for 5 minutes for each dose level and forearm blood flow was measured during the last 2 minutes of each dose. after 15 minutes of wash - out period, intra - arterial infusion of vitamin c at 24 mg / min was done for 10 minutes. with continued infusion of vitamin c at the same rate , acetylcholine was infused at the same incremental doses as the previous stage and measurement of forearm blood flow was made during the last 2 minutes of each dose. after another 15 minutes of rest period, intra - arterial infusion of vitamin c at the same rate, plus infusion of l - nmma (ng - monomethyl - l - arginine, sigma chemical), an inhibitor of nitric oxide synthesis, was begun at 100 g / min and continued for 5 minutes. then infusion of acetylcholine at incremental dose and measurement of forearm blood flow were done as in previous stages. forearm blood flow was measured and forearm blood flow changes during acetylcholine infusion were expressed as percentage changes from the baseline immediately preceding each drug administration. forearm blood flow changes were compared between normal and hypertensive groups with and without vitamin c infusion. comparison was also made between before and after antihypertensive treatment in hypertensive group and forearm blood flow was compared in hypertensive group with and without vitamin c infusion. 6.12 all values were expressed in mean standard error of mean and p<0.05 was considered significant. eight hypertensive patients and eight normotensive controls (age range 35 to 73 years) were recruited. there were not any significant differences in cholesterol levels between normotensive controls and hypertensive patients. only systolic and diastolic blood pressure differed in the two groups (table 1). after 2-month antihypertensive treatment, there were significant decreases in systolic and diastolic blood pressure (mean blood pressure ; 169/98 mmhg 131/74 mmhg, p<0.05). absolute fbf data recorded in the infused limbs at baseline and during the infusion of acetylcholine at three dose levels before and after antihypertensive treatment are presented in table 2. endothelium - dependent vasorelaxation (vasodilatory response to acetylcholine) was significantly greater in normal control group compared to hypertensive group before antihypertensive treatment (maximum forearm blood flow in nc : 44863%, ht : 30258%, p<0.05, figure 2). forearm blood flow response to acetylcholine was significantly enhanced with intra - arterial infusion of vitamin c in hypertensive group before antihypertensive treatment (maximum forearm blood flow in vit c : 30258%, vit c (+): 44643%, p<0.05, figure 3a ). such an enhanced response was not observed in normal control group (maximum forearm blood flow in vit c : 44863%, vit c (+): 38351%, p=0.11, figure 3b ). co - infusion of l - nmma, an inhibitor of nitric oxide synthase, blunted forearm blood flow response to acetylcholine (maximum forearm blood flow in vit c ( +): 44643%, vit c + l - nmma (+): 22923%, p<0.05, figure 3c ). after antihypertensive treatment with amlodipine for 2 months in hypertensive group, endothelium - dependent vasorelaxation (vasodilatory response to acetylcholine) was significantly improved in amlodipine treated group compared to before treatment (maximum forearm blood flow in before treatment : 30258%, after treatment : 53981%, p<0.05, figure 4a). intra - arterial infusion of vitamin c in amlodipine treated hypertensive group did not change the forearm blood flow response to acetylcholine (maximum forearm blood flow in vit c : 53981%, vit c (+): 44878%, p=0.21, figure 4b ). co - infusion of l - nmma, an inhibitor of nitric oxide synthase, blunted forearm blood flow response to acetylcholine in amlodipine treated hypertensive group (maximum forearm blood flow in l - nmma : 53981%, l - nmma (+): 24022%, p<0.05, figure 4c ). endothelium - dependent vasorelaxation (vasodilatory response to acetylcholine) was significantly greater in normal control group compared to hypertensive group before antihypertensive treatment (maximum forearm blood flow in nc : 44863%, ht : 30258%, p<0.05, figure 2). forearm blood flow response to acetylcholine was significantly enhanced with intra - arterial infusion of vitamin c in hypertensive group before antihypertensive treatment (maximum forearm blood flow in vit c : 30258%, vit c (+): 44643%, p<0.05, figure 3a ). such an enhanced response was not observed in normal control group (maximum forearm blood flow in vit c : 44863%, vit c (+): 38351%, p=0.11, figure 3b ). co - infusion of l - nmma, an inhibitor of nitric oxide synthase, blunted forearm blood flow response to acetylcholine (maximum forearm blood flow in vit c ( +): 44643%, vit c + l - nmma (+): 22923%, p<0.05, figure 3c ). after antihypertensive treatment with amlodipine for 2 months in hypertensive group, endothelium - dependent vasorelaxation (vasodilatory response to acetylcholine) was significantly improved in amlodipine treated group compared to before treatment (maximum forearm blood flow in before treatment : 30258%, after treatment : 53981%, p<0.05, figure 4a). intra - arterial infusion of vitamin c in amlodipine treated hypertensive group did not change the forearm blood flow response to acetylcholine (maximum forearm blood flow in vit c : 53981%, vit c (+): 44878%, p=0.21, figure 4b ). co - infusion of l - nmma, an inhibitor of nitric oxide synthase, blunted forearm blood flow response to acetylcholine in amlodipine treated hypertensive group (maximum forearm blood flow in l - nmma : 53981%, l - nmma (+): 24022%, p<0.05, figure 4c ). the present study demonstrates that the endothelium - dependent vasodilation is impaired in essential hypertensive patients as compared with normotensive control subjects. in addition, short - term, intra - arterial administration of the antioxidant vitamin c restores endothelium - dependent vasodilation in patients with essential hypertension. a nitric oxide (no) synthase inhibitor, l - nmma, blunted the improvement of endothelium - dependent vasodilation. these findings suggest that oxygen - derived free radicals may decrease the bioavailability of endothelium - derived nitric oxide and impair endothelium - dependent vasodilation in patients with essential hypertension. these were consistent with previous observations that, in essential hypertensive patients, impaired endothelium - dependent vasodilation of forearm circulation could be improved by the antioxidant vitamin c. solzbach et al demonstrated that impaired endothelium - dependent vasodilatory function of human coronary arteries in hypertensive patients could be improved by the administration of the antioxidant vitamin c. assessment of the clinical relevance of the present should take into account the fact that superoxide anion production has been found to cause endothelial dysfunction in the presence of several cardiovascular risk factors. in patients with coronary artery disease, both single dose (2 g po) and long - term (500 mg / d) administration of vitamin c reverse endothelial vasomotor dysfunction in the brachial circulation. with regard to mechanism , it has been proposed that ascorbic acid improves no action by scavenging superoxide anion and preventing inactivation of no. this explanation is attractive because atherosclerosis and hypercholesterolemia are linked to excess generation of superoxide, and superoxide reacts with no and eliminates its biological activity. in addition to scavenging superoxide anion or inhibiting ldl oxidation, vitamin c could alernatively improve no action by sparing intracellular glutathione which, together with vitamin c, is the primary regulator of intracellular redox state. in essential hypertension, impaired endothelium - dependent vasodilation seems to be a primary phenomenon and the endothelial vasomotor dysfunction is not normalized by the mere reduction of blood pressure. several investigators have addressed the possibility that antihypertensive treatment could restore or at least improve endothelial function. calcium antagonists have been shown to be effective in reversing endothelial dysfunction of angiographically normal and stenotic epicardial coronary vessels in essential hypertension. in agreement with these , but several other studies have demonstrated that the treatment with long - term calcium antagonist did not improve forearm vasodilator response to reactive hyperemia. hirooka et al also reported that single dose administration of nifedipine did not alter forearm vasodilation with acetylcholine in hypertensive patients. in our findings, prolonged (8 weeks of oral treatment) amlodipine administration could improve endothelium -dependent vasodilation in essential hypertensive patients. intra - arterial infusion of vitamin c to the treated patients did not increase forearm vasodilation in response to acetylcholine. these data indicate that amlodipine is effective in improving endothelial dysfunction in essential hypertension and amlodipine appears to act specifically on the no pathway by a mechanism that is probably related to antioxidant activity. experimental data indicate that calcium antagonists exert an antioxidant effect and therefore, could protect endothelial cells against free radical injury and diminish oxidative breakdown of no. an insufficient blood pressure reduction with an antihypertensive drug with antioxidant activity would not improve the endothelial function. so, a sufficient blood pressure reduction with an antihypertensive drug with antioxidant activity would be required. all the hypertensive patients in this study have normalized their systolic and diastolic blood pressure. even though the relative importance of the various possible mechanisms leading to depressed endothelial function in essential hypertension remains to be elucidated, our study shows that vitamin c or amlodipine in demonstrable improvement by a mechanism that is probably related to antioxidant activity.	the effects of antihypertensive agents on endothelial function have not been fully evaluated in human hypertension and data on the forearm circulation of humans are controversial. the aim of this study was to evaluate the endothelial function in hypertensive patients to investigate whether vitamin c administration has any benefit on the endothelial function and to determine whether treatment with calcium antagonist improves endothelial dysfunction in hypertensive patients.methodsthe endothelial function was estimated using venous occlusion plethysmography (vop) in 8 hypertensive patients and 8 healthy volunteers. the patients in the hypertension group were treated with amlodipine, then examined again. the change of forearm blood flow (fbf) was measured with acetylcholine infusion through brachial artery and also with intra - arterial vitamin c.forearm blood flow response to acetylcholine was significantly enhanced with intra - arterial infusion of vitamin c in hypertensive group before antihypertensive treatment. co - infusion of l - nmma, an inhibitor of nitric oxide synthase, blunted forearm blood flow response to acetylcholine. after treatment with amlodipine for 2 months in hypertensive group, endothelium - dependent vasorelaxation to acetylcholine was significantly improved compared to pretreatment, and vitamin c did not affect the improved endothelial function by amlodipine treatment.vitamin c (acutely) and amlodipine (chronically) improved endothelial function in hypertensive patients. these suggest that increased oxidative stress, at least in part, may be involved in the decreased endothelial function in hypertension.
an estimated 1,000 cases of spinal cord injury (sci) related to athletics are treated yearly in the united states, representing 8.9% of total sci cases,1 2 and although much has been written about both surgical and nonsurgical treatment methods, the literature guiding return to function is scant. transient neurapraxia and cervical cord neurapraxia (ccn) have been written about extensively, but still only very low - level medical evidence exists regarding management.3 4 5 6 the risk of recurrence after return to sports has been debated in the literature, and there is no definitive universally accepted guideline.1 7 various criteria based on numerous retrospective studies for return to play (rtp) have been suggested by torg,8 watkins,9 cantu et al,10 bailes et al,11 and torg and ramsey - emrhein6; however, the decision regarding rtp is individualized to the specific athlete, the level of function, and the expectations of the athlete.7 12 13 14 15 moreover, these return criteria are subjective, difficult to apply clinically, and poorly validated on prospective studies. completion of a randomized trial regarding rtp may not ever become possible, and instead information on medical decision - making must be found in other sources. a recent meta - analysis of literature evaluating evidence for rtp with various injury patterns concluded that only grade c or d practice recommendations supported by level iii evidence at best were available for rtp after sci.16 that is, evidence supporting interventions is compiled from limited cohort studies and clinical trials, ing in clinicians using significant judgment of the current evidence to make patient treatment decisions. in patients with sci, the data are clear that improved function is dependent upon early initiation of rehabilitation17; however, in patients with minimal or no deficits, the expectations are greater than in patients with a severe or complete sci. once the patient has fully recovered from the injury, decisions must be made to release the patient to a specific level of activity, with return to sports being one of the more demanding levels of function. consensus opinion regarding rtp may provide better evidence than individual case reports or small case series regarding opinions about return to sporting activity. consensus regarding expert opinion can be especially useful in guiding surgical decision - making where other types of evidence are absent or interpretation of available evidence is subjective.18 19 this approach has been widely used in the spine literature for interpreting guidelines and assessing interobserver reliability in clinical decision - making.20 21 22 23 24 this study was designed to identify the maximum level of sporting activity that a group of experienced spine trauma surgeons would allow patients to participate in after recovery as a guide to making general recommendations, which from a medical perspective is essentially a subjective determination of the likelihood that an individual who returns to sports will suffer an additional significant injury directly related to the original trauma. whether a patient can function at the preinjury performance level would depend on many factors and can not be addressed in this article. a treating physician is frequently faced with the decision to release a patient to a given level of contact and currently must make a decision based on his or her own anecdotal experience with little supportive literature. the aim of this study is to offer the treating physician a consensus analysis of expert opinion regarding rtp that can be incorporated with the unique factors of the case for the final individual decision. twenty - five spine surgeons in the spine trauma study group (stsg) who consider spine trauma to be a significant component of their practice, all from separate level 1 trauma centers, were surveyed.20 21 22 23 24 the survey was administered at a national meeting discussing guidelines for cervical neurapraxia. the stsg was founded in 2004 and consists of 50 surgeons from 12 countries specifically focused on the study of traumatic spine injuries. each surgeon was presented 10 common case scenarios involving cervical injuries and asked to identify the level of sports participation they would allow following recovery (tables 1 and 2). the levels of sports were defined as high - contact, intermediate - contact, noncontact, or no sports, representing the frequency and severity of expected stress on the spine. cases were categorized into those involving cervical neurapraxia and stenosis, atlantoaxial cervical injury, subaxial cervical injury, and general cervical injury. participants were also asked how soon after recovery they would allow a return to activity and what would be the minimum imaging studies necessary to make the decision. the surveys were completed simultaneously after an explanation of each scenario, the definition of level of play, the use of time to return as that of return to maximum participation, and the use of imaging tools. abbreviations: acdf, anterior cervical diskectomy and fusion; ncaa, national collegiate athletic association; mri, magnetic resonance imaging. each brief case scenario (table 3) represented increased levels of injury and assumed that the patient has fully recovered subjectively and objectively. full clinical recovery was defined as minimal to no neck pain, return of full or near - full range of motion, and return of normal motor and sensory function. after the recommendations for full recovery were collected, the surgeons were presented with the same scenarios but instructed that the patient had persistent symptoms, such as moderate neck pain, upper extremity paresthesias, or single - level radicular weakness (table 4). because this study focused on cervical injury, the levels of contact were stratified by the frequency and severity of head / neck impact (table 1). the surveyed surgeons were given seven specific time frames within which they would allow the patient to return to the maximum level of play: same game, 1 to 2 days, 1 week, 2 to 4 weeks, 2 to 3 months, 3 to 6 months, or greater than 6 months (table 4). finally, the surgeons were asked what imaging studies were the minimum necessary to make the final rtp decision after a history and physical exam plain radiographs, computed tomography (ct) scanning, and/or magnetic resonance imaging (mri) scanning. no difference between orthopedic surgeons and neurosurgeons recommendations seen, mann - whitney u test. no difference between orthopedic surgeons and neurosurgeons recommendations seen, mann - whitney u test. after the surveys were completed, the data was tabulated for analysis of each case scenario. the responses from the orthopedic surgeons were compared with those from the neurosurgeons to determine whether any significant differences in recommendations existed using the kruskal - wallis and mann - whitney u test. a p value < 0.05 was considered significant. fourteen orthopedic surgeons and 11 neurosurgeons who were members of the former stsg completed the survey. a summary of the level of play recommendations for each scenario with full recovery is shown in table 3. there were no differences between orthopedic and neurosurgeons ( not shown), so the data was analyzed as one group, which is consistent with a previous finding from this group of fellowship - trained spine surgeons regarding evaluation of cervical dislocation injuries.20 the scenarios in which the surgeons most consistently allowed for return to high - contact sports were the episodes of ccn with early resolution of symptoms and no stenosis on mri (case 1, 88%, p < 0.0001 and case 3, 64%, p = 0.03). in the setting of diffuse stenosis and early resolution of ccn, the percentage of surgeons allowing return to high - contact sports dropped to 35% (case 2, p = 0.6) and 27% (case 4, p = 0.6), with greater heterogeneity of opinions. cases in which some surgeons favored allowing return to high - contact sports included c1 ring or c2 hangman's fractures that healed nonoperatively (67%, case 5, p = 0.005), c5c6 unilateral facet dislocation presenting neurologically intact with subsequent posterior fusion (56%, case 8, p = 0.02), and herniated disks repaired operatively (71%, case 9, p = 0.003). the surgeons generally favored a return to a less vigorous level of activity such as intermediate or noncontact sports when the case involved ccn with os odontoideum and c1c2 fusion (82%, case 6, p = 0.04), c5 flexion compression of american spinal injury association grade d with c4c6 anterior cervical corpectomy and fusion (80%, case 10, p = 0.06), and c5 burst fracture treated nonoperatively (52%, case 7, p = 0.03). good consensus was seen for these cases in favoring return to a degree of activity less than high contact. changes in the clinical scenario to indicate a lack of patient symptom resolution ed in greater variation and less consensus of recommendations and a lower number of physicians recommending return to high - contact sports ( not shown). with a lack of resolution of symptoms, only case 2 (p = 0.03) and case 4 (p = 0.02) showed significant consensus in recommending noncontact or no sports activity. table 4 outlines the time frame within which surgeons would allow rtp at the level they recommended. a relative consensus in recommending return after 2 to 4 weeks was seen in cases 1 to 4 involving cervical neurapraxia and stenosis. all surgeons recommended waiting 2 to > 6 months for cases 5 to 10, involving atlantoaxial, subaxial, or general cervical spine injury patterns. there was no difference in the recommendations of orthopedic surgeons and neurosurgeons for level of activity or time to rtp ( not shown). recommendations for x - rays (64 to 92%), cts (20 to 84%), and mris (50 to 100%) varied among different cases, indicating a high consensus for obtaining imaging in making recommendations ( not shown). there were also no differences in recommendations between orthopedic surgeons and neurosurgeons for imaging prior to making rtp decisions. most surgeons favored extensive imaging with radiographs, ct, and mri before allowing rtp. decisions about safe rtp after cervical injury can be difficult because there are opposing forces to be considered, such as the patient's desires and the medicolegal implications. other than for ccn, there is almost no literature to determine an appropriate strategy for return to sports after cervical injury. torg et al reported that cervical stenosis was predictive of another episode of ccn (53%) but not predictive of catastrophic injury25; however, repeated episodes of neurapraxia and cord contusions were thought to be a relative contraindication for rtp. more recently, brigham and capo followed four professional athletes who had cord contusions from presumed hypermobility at c3c4 or a disk herniation elsewhere in the spine.7 despite the positive findings on radiographic images, the players were completely asymptomatic after stabilization and subsequently returned to professional sports. the authors emphasize close observation, careful assessment, and thorough counseling of the patients. the of studies of return to professional sports after treatment of cervical injury by hsu and colleagues supported rtp after treatment of cervical disk herniations, with a greater rate of return for operatively managed cases12 26; however, the authors discussed the unclear consensus in management of patients with herniated disks and concomitant cervical stenosis. despite evidence to suggest that athletes with spinal stenosis may return to high - contact sports if they are asymptomatic,3 6 the current study reveals the prevailing caution regarding rtp when an athlete has lost the space available for the spinal cord. morganti et al completed a similar rtp survey based on case scenarios of cervical trauma administered to members of the cervical spine research society, the herodicus sports medicine society, and members of the authors' own department.27 an evaluation of 113 responses (32.7% response rate) demonstrated that the consensus on rtp was poor and that most of the differences were based on type of subspecialty interest (i.e., spine or sports) and seniority. although 49% of respondents reported using guidelines at the time for decision - making, only 1 of 10 survey cases was evaluated as appropriate in this manner. the of the study presented a question of how recommendations for rtp could be made in the face of limited data. in contrast, our study was directed specifically at spine surgeons at level 1 trauma centers who include trauma as a significant component of their practice. the surgeons surveyed for this study had a thorough understanding of the available literature on transient neurapraxia, including a recent issue of spine that was focused on spine trauma and included an evidence - based review of rtp after transient neurapraxia.28 the of that review were based on a consensus of members of the stsg, as well as available literature.5 because there is so little literature available on the other scenarios, most of those recommendations were based on expert opinion from clinical experience. some important differences can be observed in the rtp recommendations after transient neurapraxia. if a patient had congenital stenosis after an episode of neurapraxia, then experienced trauma spine surgeons were distinctly less willing to allow that patient to return to a high - contact sport. if the neurapraxia was brief, 88% would allow return to high - contact sports if the athlete did not have congenital stenosis whereas only 35% would allow the return if congenital stenosis existed. if the deficit lasted for a few hours rather than resolving quickly, only 64% would allow return to high - contact sports even if the patient did not have congenital stenosis and just 27% would allow return to high - contact sports if congenital stenosis existed on mri. thus, it appears that duration of neurapraxia, perhaps indicating a more significant injury, was important in the rtp decision, as was the presence of congenital stenosis. our study did not address the issue of repetitive neurapraxia, which could also influence decision - making just as repeated cerebral concussions do.29 the case scenarios set forth do not cover the entire gamut of potential cervical injuries but they were thought to include a broad representation of the common injury patterns so that some extrapolation to other injury patterns may be made. for example, a single - level posterior fusion for facet fracture without dislocation could be assessed in a similar fashion to a single - level posterior fusion for a unilateral facet dislocation in scenario 6. the only other injury patterns in which a majority of those surveyed would allow return to high - contact sports were in the scenarios that ed in a stable single - level subaxial fusion, either anterior or posterior, and nondisplaced, healed upper cervical fractures. in the scenarios of a healed posterior c1c2 fusion (even with normal neurology), those with two - level fusions, and those in which patients had an incomplete sci (excluding transient neurapraxia), only a small minority of less than 20% would consider return to high - contact sports. absolute contraindications to high - contact sports have generally included patients with odontoid abnormalities, atlanto - occipital fusion, klippel - feil fusions above c3, and acute fracture with instability.16 limitations of this study include the reliance on expert opinion for surgical recommendations because of the difficulty of studying these injury patterns in the active athlete. the findings are highly dependent on individual interpretation of data and personal experience; however, in the face of convincing data, consensus opinion can be a source of guidance on patient management as well as starting point for further exploration. other limitations of the study include an inability to totally account for all mechanisms and types of cervical sport injury in making practice recommendations. clinicians applying this survey may not find every scenario fits their specific patient, although the principles evaluated in each case may be widely applied. although this study will not completely answer the questions about rtp, it establishes a reasonable consensus of expert surgeon opinion with substantial experience to guide the treating physician involved in similar case scenarios. it can also serve as a basis upon which future prospective, multicenter studies can be designed to confirm or disprove current dogma.	study design survey. objective sports - related spinal cord injury (sci) represents a growing proportion of total scis but lacks evidence or guidelines to guide clinical decision - making on return to play (rtp). our objective is to offer the treating physician a consensus analysis of expert opinion regarding rtp that can be incorporated with the unique factors of a case for clinical decision - making. methods ten common clinical scenarios involving neurapraxia and stenosis, atlantoaxial injury, subaxial injury, and general cervical spine injury were presented to 25 spine surgeons from level 1 trauma centers for whom spine trauma is a significant component of their practice. we evaluated responses to questions about patient rtp, level of contact, imaging required for a clinical decision, and time to return for each scenario. the chi - square test was used for statistical analysis, with p < 0.05 considered significant. evaluation of the surgeons' responses to these cases showed significant consensus regarding return to high - contact sports in cases of cervical cord neurapraxia without symptoms or stenosis, surgically repaired herniated disks, and nonoperatively healed c1 ring or c2 hangman's fractures. greater variability was found in recommendations for patients showing persistent clinical symptomatology. this survey suggests a consensus among surgeons for allowing patients with relatively normal imaging and resolution of symptoms to return to high - contact activities; however, patients with cervical stenosis or clinical symptoms continue to be a challenge for management. this survey may serve as a basis for future clinical trials and consensus guidelines.
virus isolation was performed in the bsl-4 laboratory at the centers for disease control and prevention in atlanta. vero e6 cells were inoculated and observed for characteristic cytopathic effect, syncytium formation. nv was isolated from 2 oropharyngeal swabs (spb200401066, spb200406506), 1 cerebrospinal fluid (spb200401617), and 1 urine specimen (spb200405758) from human patients, and isolation was confirmed by reverse transcription polymerase chain reaction (rt - pcr). two isolates were from rajbari, and 1 was from faridpur; the fourth isolate, from the rajshahi district (100 km from rajbari), was not linked to the other 2 outbreaks. the complete genomic sequence of the first viral isolate (spb200401066) from rajbari was derived and submitted to genbank (accession no . the sequences of the open reading frame ( orf) coding for nucleoprotein (n) were obtained for the other 3 isolates. the methods used for rt - pcr, sequencing, cdna cloning, rapid amplification of cdna ends (race), and sequence analysis were previously described. the genome of nv - b is 18,252 nt in length, 6 nt longer than nv - malaysia (nv - m), the prototype strain of nv (spb199901924). the additional 6 nt map to the 5 nontranslated region of the fusion protein (f) gene. the length of the nv - b genome is evenly divisible by 6, suggesting that nv - b follows the " rule of six ". the gene order and sizes of all the orfs except v are conserved between nv - b and nv - m (table, figure, a). the overall nucleotide homology between the genomes of nv - b and nv - m is 91.8%, but the changes are not uniformly distributed throughout the genome. nucleotide homologies are higher in the protein coding regions than in the noncoding regions, although the sizes of the nontranslated regions remain highly conserved (table). the predicted amino acid homologies between the proteins expressed by nv - m and nv - b are all > 92% (table). * nv, nipah virus.. percentage amino acid identity or nucleotide homology after sequences were aligned by using gap from gcg. nv - malaysia gene lengths and 5 and 3 nontranslated sequences and gene lengths were obtained from genbank accession no. open reading frames (orfs) are indicated by shaded boxes: n, nucleoprotein; p, phosphoprotein; m, matrix protein; f, fusion protein; g, attachment protein; l, polymerase protein. a phenogram of the n orfs of members of this subfamily was created by using maximum parsimony analysis with paup 4.02 (sinauer associates, sunderland, ma, usa). abbreviations and accession numbers: hpiv-1, human parainfluenza virus, d01070; sendai, x00087; hpiv-3, d10025; cdv, canine distemper virus, af014953; pdv, phocine distemper virus, x75717; rpv, rinderpest virus, x68311; mv, k01711; dmv, dolphin morbillivirus, x75961; ndv, newcastle disease virus, af064091; gp, goose paramyxovirus, af473851; hpiv-4b, m32983; hpiv-4a, m32982; tioman, af298895; menangle, af326114; hpiv-2, m55320; simian virus 5 (sv5), m81442; mumps, d86172; nv - umcc1, ay029767; nv - malaysia, af212302; nv - p. hypomelanus, * af376747; nv - bangladesh; hendra virus, af017149; tupaia paramyxovirus, af079780; mossman virus, ay286409; and salem virus, af237881. c ) the phylogenetic relationship between the n gene sequences of the 4 human nv isolates from the bangladesh outbreak in 2004 and the n gene sequences from pig and human nv isolates from malaysia. accession numbers for the pig isolates of nv are aj627196, aj564622, and aj564621. hypomelanus is sequence from a virus isolated from pteropus hypomelanus, the island flying fox. overall, the predicted amino acid homologies of the surface glycoproteins, f and g, of nv - b and nv - m are high (table). in the f protein, the predicted cleavage site, f1 amino - terminal domain, transmembrane domain, and predicted n - glycosylation sites are identical in nv - b and nv - m. four of the 9 predicted amino acid changes occur in the first 11 amino acids (aa) of the precursor of the f protein, f0, which fall within the predicted signal peptide and would be cleaved from the mature protein. within the g proteins, the predicted transmembrane domains, and the positions of all 17 cysteine residues are conserved between nv - b and nv - m. of 8 predicted n - linked glycosylation sites in the g protein of nv - m, 6 are conserved in nv - b and in hv. the coding strategy of the p gene is identical in nv - b and nv - m. in these viruses, the p gene contains the c, v, and w orfs in addition to the p orf. like most other paramyxoviruses, the henipaviruses have a conserved ag - rich region that acts as an editing site to facilitate the addition of nontemplated g residues into the transcripts of the p gene. the edited transcripts encode 2 proteins, v and w, which are co - amino - terminal with p but have unique carboxy termini. the addition of 1 g residue generates the mrna for the v protein, and the addition of 2 g residues produces the mrna for the w protein. sequence analysis of multiple cdna clones containing the editing site of nv - b identified edited transcripts that encoded both the v and w proteins (data not shown). the conserved 20-nt region encompassing the editing site is identical in nv - m, hv, and measles virus (mv); however, the editing of site of nv - m (uggguaauuuuucccguguc) differs from nv - b (gggauaauuuuucccguguc) at 2 nt positions (underlined) all of the cysteine residues are conserved in the v proteins of nv - b and nv - m; however, the unique portion of the v protein of nv - b is predicted to be 55 aa, 3 aa longer than the v protein of nv - m. the predicted w protein of nv - b is identical in size and sequence to the w protein of nv - m. recently, aa 100160 and 230237 of the v protein of nv - m have been identified as necessary for inhibition of interferon signaling. these regions are highly conserved in the v protein of nv - b, which has 4 predicted amino acids substitutions (1 conservative) between positions 100160 and no predicted substitutions between positions 230237. in addition, the v protein of nv - b has 3 predicted amino acid substitutions in the crm1-dependent nuclear export signal that was identified between aa 174193 in the v protein of nv - m. the l proteins of nv - b and nv - m had a high level of predicted amino acid conservation (table). the 6 highly conserved domains of viral polymerases, originally described by poch et al. , remain largely unchanged between nv - b and nv - m. domains 1, 2, and 5 have 1 conservative amino acid change each and domain 3, which is considered the most conserved domain within the l proteins of paramyxoviruses, has 2 aa changes. the 4 motifs identified in domain 3, including the qgdne motif, which is assumed to be the active site of the polymerase, are identical between the nv - b and nv - m, as is the predicted nucleotide - binding motif in domain 6. the cis - acting control sequences are highly conserved in the genomes of nv - b and nv - m. as in nv - m, the intergenic sequences in nv - b are gaa, with the exception of the sequence between the g and l genes, uaa, which is unique among the henipaviruses. however, the intergenic sequence between the g and l genes is gaa in the second isolate from bangladesh. the transcriptional start and stop signals of each gene of nv - b are highly conserved in relation to the other henipaviruses. the 3 leader sequence of nv - b is identical in length to those of all other paramyxoviruses and has nucleotide changes at positions 14 and 47 compared to nv - m. the 5 trailer of nv - b is identical in length and sequence to nv - m. phylogenetic analysis was used to compare the sequence of the n orf of nv - b to the sequences of the n orfs from other members of the subfamily paramyxovirinae. the confirmed the of the sequence comparisons, which show that nv - b is most closely related to the henipavirus nv - m, and support the that nv - b should be regarded as new strain of nv (figure, b). phylogenetic analyses conducted with the sequences of the other genes produced similar (data not shown). the sequences of the n orfs of 4 nv isolates from bangladesh share 99.1% nt homology (figure, c) but exhibited more interstrain nucleotide heterogeneity than the sequences of the human isolates in malaysia, which were nearly identical. these varying amounts of genetic variability may reflect differences in the mode of transmission of nv in the 2 countries. in malaysia , molecular evidence suggests that at least 2 introductions of nv into pigs occurred (figure, c). however, the nearly identical sequences of human and pig isolates from the later phase of the outbreak suggest that only 1 of the variants spread rapidly in pigs and was associated with most human cases. in contrast, the sequence heterogeneity observed in bangladesh may be the of multiple introductions of nv into humans from different colonies of fruit bats. this first look at strain variation in nv indicates that viruses circulating in different areas have unique genetic signatures and suggests that these strains may have coevolved within the local natural reservoirs. until 2004, the isolation and genetic characterization of nv - b confirm that nv was the etiologic agent responsible for these outbreaks. note: after this article was accepted for publication, nipah virus was isolated from pteropus lylei in cambodia. phylogenetic analysis of the n gene sequences demonstrated that this virus is more closely related to nipah - malaysia than to nipah - bangladesh and represented another lineage of nipah virus.	until 2004, identification of nipah virus (nv)-like outbreaks in bangladesh was based on serology. we describe the genetic characterization of a new strain of nv isolated during outbreaks in bangladesh (nv - b) in 2004, which confirms that nv was the etiologic agent responsible for these outbreaks.
a 72 year - old, 150 cm, 57 kg, female patient presented with a neck mass that developed a few years earlier. she had no specific medical records except for taking medication for hypertension for 2 years. a physical examination revealed a palpable, broad, and firm tumor around both thyroid glands, but she did not complain of any symptoms. neck ct detected a thyroid neoplasm on both sides (about 6 3 cm on the right thyroid gland ; about 7 7 cm on the left thyroid), which revealed the presence of tracheal stenosis. the region of tracheal constriction narrowed from 3.6 mm below the glottis to the sternal notch, in which the narrowest lumen of the trachea was approximately 3.9 21 mm in diameter and appeared to be compressed by the tumors of both sides (fig . a thyroid papillary carcinoma was diagnosed by an aspiration biopsy, and on thoracic ct, a concern of metastasis into the lung urged immediate treatment . arterial blood gas analysis ( abga) showed a ph of 7.424, paco2 of 43.1 mmhg, pao2 of 74.8 mmhg, and base excess of 2.9. the distance between the glottis and constriction site was so short that an endotracheal tube could not to be placed above the constriction region. however, there were no episodes of respiratory disturbance despite the severe tracheal obstruction, nor was there any increase in the paco2. the partial pressure of oxygen (pao2) also was in the normal range for her age. upon auscultation, normal breathing sounds were heard, which made us suspect that the tracheal obstruction was mobile. after explaining the potential difficulty of endotracheal intubation due to the tracheal stenosis one day before surgery, the patient provided informed consent for the procedure. a flexible fiberoptic bronchoscopy and laryngeal mask airway (lma) were prepared, and uncuffed endotracheal tubes, 4.0, 4.5, 5.0 in size, and cuffed endotracheal tubes, 4.0 to 7.0 in size, were ready for use. for pre - anesthesia medication, 0.5 mg of atropine was administrated 30 minutes before the onset of anesthesia. an electrocardiogram (ecg), non - invasive blood pressure (nibp) monitor, and pulse oximetry were placed after arriving in the operating room. her vital signs indicated a bp, heart rate and oxygen saturation of 145/85 mmhg, 92/min and 99%, respectively, and she did not complain of any discomfort in the supine position. before the onset of anesthesia, denitrogenation was induced with 100% oxygenation at 6.0 l / min for 5 minutes. because she had no dyspnea during sleep midazolam 3 mg was administered and the patient fell asleep. however, when a bronchoscope intubation was attempted, her spontaneous respiration stopped and support ventilation was provided. when assisted ventilation with 100% oxygen was confirmed to function well, thiopental 100 mg was administered and the ventilation function was reconfirmed. succinylcholine 50 mg was then administrated and endotracheal intubation was attempted. after inserting an uncuffed endotracheal tube of 4.0 without resistance, the tube was withdrawn while keeping the bronchoscope in place. when a cuffed endotracheal tube (size 5.0) was inserted without difficulty, rocuronium 30 mg was then injected into the vein and anesthesia was maintained with both oxygen and nitrous oxide of 1.5 l / min, and sevoflurane 1.0 - 2.5 vol%, while remifentanil was administrated continuously as a supplement. the tidal volume (tv), respiratory rate (rr) and peak inspiratory pressure (pip) was 500 ml, 10/min, and 18 cmh2o, respectively. the operation was started after placing a catheter into the left dorsalis pedis artery for continuously monitoring of the arterial blood pressure and abga. extubation was performed after confirming that the patient's spontaneous breathing was restored and she had reached consciousness. she was transferred to the ward after sufficient monitoring in the post - anesthesia recovery unit. a 66 year - old, 175 cm, 73 kg, male patient was referred to our institution from a clinic due to an episode of dyspnea over the last 3 - 4 months. he was diagnosed with bronchial asthma and received medical treatment, but there were no favorable changes and he was admitted to our hospital. he had no other abnormalities in his medical records other than taking medication due to a 20-year history of hypertension. he often experienced respiratory disturbances in the left lateral decubitus or supine position, whereas there were no complaints of dyspnea in the right lateral decubitus or semisupine position. a physical examination revealed a firm tumor, approximately 3 cm in size, below the right thyroid, but a simple chest x - ray did not show any abnormalities. the abga were ph 7.413, paco2 44.0 mmhg, pao2 106.4 mmhg, and base excess 2.4. the pulmonary function test showed mild lung impairment with a mild obstructive pattern (fvc 3.8 l / min , fev1 2.53 l / min). cervical magnetic resonance imaging (mri) detected an ill - defined tumor in the lower part of the right thyroid and showed that the tumor had invaded the trachea and formed a polypoid tumor in the postro - internal direction. the tumor was distributed from approximately 2 - 3 cm below the glottis to the sternal notch with the narrowest region being 2.79 mm in diameter at approximately 4 cm below the glottis (fig . a partial resection of the thyroid and trachea was determined after diagnosing thyroid papillary carcinoma by an aspiration biopsy . the distance from the glottis was so short that a tracheal tube was not placed above the region of the tracheal obstruction . extracorporeal circulation was planned under the suspicion that a metastasis of the tumor into the trachea would cause a high airway pressure, and extracorporeal circulation would be safer than laryngeal mask airway due to the symptoms of dyspnea . after consultation regarding our plan, his attending surgeon strongly recommended endotracheal intubation on the grounds that the base of the polypoid legion was so broad that the tissue would be far from being damaged or cut off and that there would be no bleeding or necrosis . therefore, it was decided to attempt endotracheal intubation through a bronchoscope under the sleep state . in case of failure of endotracheal intubation, a lma was provided and endotracheal tubes ( size 4.0 and 4.5 without a cuff and sizes 4.0 to 7.5 with a cuff) were set. one day before the operation, the patient was given an explanation of the potential difficulty in endotracheal intubation due to the presence of the tumor inside the trachea, the process of intubation under conscious sedation or in the awakened state, and the possibility of extracorporeal circulation. there was no pre - operative medication and the upper body was elevated to approximately 30 after arriving in the operating room. the bp measured immediately after entering the or was 180/112 mmhg, which necessitated the administration of hydralazine 10 mg. a catheter was placed into the right dorsalis pedis artery to monitor the arterial bp and perform abga. propofol and remifentanil were administered continuously for sedation to maintain normal breathing, and oxygenation was provided by a mask. when respiration became shallow, assisted ventilation was performed and the ventilation rate was increased until the patient's respiration had returned to normal, and reached a proper sleep state and did not respond to hearing stimulus. a size 5.0 cuffed endotracheal tube was threaded over a 4.0 mm flexible bronchoscope and the bronchoscope was inserted carefully into the region of the tumor. although there was a cough attack when the bronchoscope was passed over the glottis, the bronchoscope was advanced uneventfully up to the tumor location in a single attempt. after confirming the tumor, the bronchoscope was advanced further to confirm the carina. to prevent damage to the tumor, the tracheal tube threaded over the bronchoscope was advanced with a 180 turn in order for its bevel to be directed toward the right. the length of the 5.0 tracheal tube was 22 cm, and it was pushed up as much as possible to allow the adapter part of the tube to be attached to the patient's incisor. the tip of the tube was confirmed to be placed above the carina. before removing the bronchoscope, we checked for any damaged tissue, and after removing it, suction was performed twice to remove the unfound tissue debris. mannual ventilation was provided with 100% oxygen at 4 l / min and sevoflurane, using the anesthesia machine. a cuffed 6.0 endotracheal tube was inserted through a tube exchanger because the pip increased sharply to 35 cmh2o and the length of the tube was too short to be advanced further on resection of a part of the trachea. a bronchoscope was inserted to again confirm the removal of fragments due to tumor damage, followed by further suction. no fragments of damaged tissue were found, and the administration of protofol was ceased. anesthesia was maintained with both 500% oxygen and nitrous oxide 1.5 l / min, respectively and sevoflurane supplemented with the continuous administration of remifentanil. a 72 year - old, 150 cm, 57 kg, female patient presented with a neck mass that developed a few years earlier. she had no specific medical records except for taking medication for hypertension for 2 years. a physical examination revealed a palpable, broad, and firm tumor around both thyroid glands, but she did not complain of any symptoms. neck ct detected a thyroid neoplasm on both sides (about 6 3 cm on the right thyroid gland ; about 7 7 cm on the left thyroid), which revealed the presence of tracheal stenosis. the region of tracheal constriction narrowed from 3.6 mm below the glottis to the sternal notch, in which the narrowest lumen of the trachea was approximately 3.9 21 mm in diameter and appeared to be compressed by the tumors of both sides (fig . a thyroid papillary carcinoma was diagnosed by an aspiration biopsy, and on thoracic ct, a concern of metastasis into the lung urged immediate treatment . arterial blood gas analysis ( abga) showed a ph of 7.424, paco2 of 43.1 mmhg, pao2 of 74.8 mmhg, and base excess of 2.9. the distance between the glottis and constriction site was so short that an endotracheal tube could not to be placed above the constriction region. however, there were no episodes of respiratory disturbance despite the severe tracheal obstruction, nor was there any increase in the paco2. the partial pressure of oxygen (pao2) also was in the normal range for her age. upon auscultation, normal breathing sounds were heard, which made us suspect that the tracheal obstruction was mobile. after explaining the potential difficulty of endotracheal intubation due to the tracheal stenosis one day before surgery, the patient provided informed consent for the procedure. a flexible fiberoptic bronchoscopy and laryngeal mask airway (lma) were prepared, and uncuffed endotracheal tubes, 4.0, 4.5, 5.0 in size, and cuffed endotracheal tubes, 4.0 to 7.0 in size, were ready for use. for pre - anesthesia medication, 0.5 mg of atropine was administrated 30 minutes before the onset of anesthesia. an electrocardiogram (ecg), non - invasive blood pressure (nibp) monitor, and pulse oximetry were placed after arriving in the operating room. her vital signs indicated a bp, heart rate and oxygen saturation of 145/85 mmhg, 92/min and 99%, respectively, and she did not complain of any discomfort in the supine position. before the onset of anesthesia, denitrogenation was induced with 100% oxygenation at 6.0 l / min for 5 minutes. because she had no dyspnea during sleep midazolam 3 mg was administered and the patient fell asleep. however, when a bronchoscope intubation was attempted, her spontaneous respiration stopped and support ventilation was provided. when assisted ventilation with 100% oxygen was confirmed to function well, thiopental 100 mg was administered and the ventilation function was reconfirmed. succinylcholine 50 mg was then administrated and endotracheal intubation was attempted. after inserting an uncuffed endotracheal tube of 4.0 without resistance, the tube was withdrawn while keeping the bronchoscope in place. when a cuffed endotracheal tube (size 5.0) was inserted without difficulty, rocuronium 30 mg was then injected into the vein and anesthesia was maintained with both oxygen and nitrous oxide of 1.5 l / min, and sevoflurane 1.0 - 2.5 vol%, while remifentanil was administrated continuously as a supplement. the tidal volume (tv), respiratory rate (rr) and peak inspiratory pressure (pip) was 500 ml, 10/min, and 18 cmh2o, respectively. the operation was started after placing a catheter into the left dorsalis pedis artery for continuously monitoring of the arterial blood pressure and abga. extubation was performed after confirming that the patient's spontaneous breathing was restored and she had reached consciousness. she was transferred to the ward after sufficient monitoring in the post - anesthesia recovery unit. a 66 year - old, 175 cm, 73 kg, male patient was referred to our institution from a clinic due to an episode of dyspnea over the last 3 - 4 months. he was diagnosed with bronchial asthma and received medical treatment, but there were no favorable changes and he was admitted to our hospital. he had no other abnormalities in his medical records other than taking medication due to a 20-year history of hypertension. he often experienced respiratory disturbances in the left lateral decubitus or supine position, whereas there were no complaints of dyspnea in the right lateral decubitus or semisupine position. a physical examination revealed a firm tumor, approximately 3 cm in size, below the right thyroid, but a simple chest x - ray did not show any abnormalities. the abga were ph 7.413, paco2 44.0 mmhg, pao2 106.4 mmhg, and base excess 2.4. the pulmonary function test showed mild lung impairment with a mild obstructive pattern (fvc 3.8 l / min , fev1 2.53 l / min). cervical magnetic resonance imaging (mri) detected an ill - defined tumor in the lower part of the right thyroid and showed that the tumor had invaded the trachea and formed a polypoid tumor in the postro - internal direction. the tumor was distributed from approximately 2 - 3 cm below the glottis to the sternal notch with the narrowest region being 2.79 mm in diameter at approximately 4 cm below the glottis (fig . a partial resection of the thyroid and trachea was determined after diagnosing thyroid papillary carcinoma by an aspiration biopsy . the distance from the glottis was so short that a tracheal tube was not placed above the region of the tracheal obstruction . extracorporeal circulation was planned under the suspicion that a metastasis of the tumor into the trachea would cause a high airway pressure, and extracorporeal circulation would be safer than laryngeal mask airway due to the symptoms of dyspnea . after consultation regarding our plan, his attending surgeon strongly recommended endotracheal intubation on the grounds that the base of the polypoid legion was so broad that the tissue would be far from being damaged or cut off and that there would be no bleeding or necrosis . therefore, it was decided to attempt endotracheal intubation through a bronchoscope under the sleep state . in case of failure of endotracheal intubation, a lma was provided and endotracheal tubes ( size 4.0 and 4.5 without a cuff and sizes 4.0 to 7.5 with a cuff) were set. one day before the operation, the patient was given an explanation of the potential difficulty in endotracheal intubation due to the presence of the tumor inside the trachea, the process of intubation under conscious sedation or in the awakened state, and the possibility of extracorporeal circulation. there was no pre - operative medication and the upper body was elevated to approximately 30 after arriving in the operating room. the bp measured immediately after entering the or was 180/112 mmhg, which necessitated the administration of hydralazine 10 mg. a catheter was placed into the right dorsalis pedis artery to monitor the arterial bp and perform abga. propofol and remifentanil were administered continuously for sedation to maintain normal breathing, and oxygenation was provided by a mask. when respiration became shallow, assisted ventilation was performed and the ventilation rate was increased until the patient's respiration had returned to normal, and reached a proper sleep state and did not respond to hearing stimulus. a size 5.0 cuffed endotracheal tube was threaded over a 4.0 mm flexible bronchoscope and the bronchoscope was inserted carefully into the region of the tumor. although there was a cough attack when the bronchoscope was passed over the glottis, the bronchoscope was advanced uneventfully up to the tumor location in a single attempt. after confirming the tumor, the bronchoscope was advanced further to confirm the carina. to prevent damage to the tumor, the tracheal tube threaded over the bronchoscope was advanced with a 180 turn in order for its bevel to be directed toward the right. the length of the 5.0 tracheal tube was 22 cm, and it was pushed up as much as possible to allow the adapter part of the tube to be attached to the patient's incisor. the tip of the tube was confirmed to be placed above the carina. before removing the bronchoscope, we checked for any damaged tissue, and after removing it, suction was performed twice to remove the unfound tissue debris. mannual ventilation was provided with 100% oxygen at 4 l / min and sevoflurane, using the anesthesia machine. a cuffed 6.0 endotracheal tube was inserted through a tube exchanger because the pip increased sharply to 35 cmh2o and the length of the tube was too short to be advanced further on resection of a part of the trachea. a bronchoscope was inserted to again confirm the removal of fragments due to tumor damage, followed by further suction. no fragments of damaged tissue were found, and the administration of protofol was ceased. anesthesia was maintained with both 500% oxygen and nitrous oxide 1.5 l / min, respectively and sevoflurane supplemented with the continuous administration of remifentanil. a tracheal obstruction has many symptoms according to the location, degree and cause, and is generally treated according to the symptoms. the airway obstruction is caused by primary tumors in the trachea, or secondarily by compression on the trachea or metastasis into the trachea by a neighboring tumor or by granuloma growth from a previous history of tracheal intubation, injury, or inflammation. from the location, tracheal obstructions are divided into two groups: obstructions inside the thorax and obstructions outside the thorax. in the case of obstructions inside the thorax , it is essential to determine if the obstruction occurs in the trachea or in the bronchus. regardless of the cause, a bronchial obstruction is less life - threatening than a tracheal obstruction. a tracheal obstruction in the posterior mediastinum caused by the compression of a tumor inside the thorax does not carry a large risk. on the other hand, an obstruction in the anterior mediastinum may impede the maintenance of proper ventilation by increasing the compression on the trachea by the tumor when the patient's spontaneous respiration disappears or muscle relaxation is achieved. tumors in the anterior mediastinum require a prompt warning of severe post - general anesthesia complications if measurements of the peak expiratory flow rate and tracheal area are < 50% of the expected one. in the case of a tracheal obstruction outside the thorax , it is essential to confirm whether it is caused by neighboring tumors, intratracheal tumors, or scars ing from trauma or inflammation. a tracheal obstruction caused by trauma or inflammation, regardless of its position, allows only endotracheal tubes with a smaller size than the measured diameter. therefore, it is essential to make an accurate estimate of the extent of tracheal obstruction. in cases of difficult endotracheal intubation due to a severe obstruction, the anesthetic method should be decided according to whether the obstruction site is above or below a possible site of the tracheostomy. a tracheal obstruction can also be caused by tissue fragments from a primary intratracheal tumor during or after tracheal intubation. although such obstructions are rare, it is important to understand the features and characteristics of tumors even in smaller ones. reported that a huge thyroid tumor did not raise the issue of difficult endotracheal intubation (dei), and some cases with dei did not show any difficulty when the endotracheal tube was passed over the compressed region. by contrast, the degree of thyroid tumor progression is a more dangerous factor for dei because fibroblastic proliferation caused by the progression of thyroid cancer decreases the mobility of the larynx. regarding the anesthetic methods for patients with tracheal obstruction, there are several options worth considering. these include placement of a tracheal tube above the tracheal obstruction site, placement of a tube with a smaller diameter being passed over the obstruction site, tracheostomy, use of a lma, high frequency jet ventilation, high frequency positive pressure ventilation, extracorporeal circulation, etc. all these methods can secure the airway, but each method is vulnerable to certain complications. in any patient in whom a tracheal obstruction is suspected, anesthesia should be induced with the preparation of all necessary tools and equipment ready in case of emergency. in the present cases, thyroid cancer obstructed the trachea by compression or invasion. in case 1, the tumor grew in both thyroid glands and compressed the trachea on both sides; the narrowest diameter of the trachea was approximately 3.9 21.2 mm. abga revealed normal findings, whereas the trachea with an anterioposterior diameter of approximately 21.2 mm was compressed by the tumor on both sides but the thyroid tumor was believed not to be fixed to the surrounding tissues and was mobile. her surgeon stated that a tracheotomy under local anesthesia would be difficult because the tumor was large enough to cover the tracheotomy site. taking the use of lma or extracorporeal circulation into account, the relevant clinical reports were reviewed, which showed that endotracheal tubes with diameter a larger than the tracheal diameter could inserted in cases of a tracheal obstruction caused by the compression of thyroid cancer. therefore, a flexible fiberoptic bronchoscope, lma, and a variety of endotracheal tubes of different sizes were made ready in case of emergency, and the induction of general anesthesia was scheduled after making preparations for an otolaryngologist to perform an emergency tracheostomy. although tracheal intubation under consciousness is safer, the patient did not have any symptoms of dyspnea during sleep, so a sleep state was induced with midazolam, and we tried to attempt tracheal intubation during sleep under a bronchoscope. manual ventilation was needed due to the occurrence of apnea during the induction of sleep, and tracheal intubation was performed under the direct laryngoscope. the tracheal obstruction was so severe that the endotracheal tubes were changed gradually to a larger size from 4.0, 5.0, to 6.5, while checking the airway resistance to reduce the potential damage caused by intubation. in retrospect, it may have been worth attempting to insert a 6.5 size tube next to a 4.0 size one to save time and trouble. as expected, an endotracheal tube with a diameter larger than the measured inside diameter (i d) was easily inserted. in this case, a partial tracheostomy was not scheduled because there were no signs of endotracheal metastasis, so lma was not employed because it was believed that it would not be able to maintain anesthesia for more than 6 hours until the thyroid tumor had been removed completely. however, a plan for lma and tracheostomy was made as a contingency plan for difficult endotracheal intubation (dei). extracorporeal circulation also was not attempted due to the risk of blood loss because this method was rather invasive and required heparin despite its advantage of enabling anesthesia without touching the trachea when it is difficult to secure the airway. in case 2, to avoid damage to the tumor, a 4.0 mm flexible fiberoptic bronchoscope was inserted through a cuffed 5.0 size endotracheal tube to reduce the space between the bronchoscope and tube while a 6.5 size endotracheal tube was inserted for replacement with its bevel being directed toward the tumor lesion. 3 ), but the anesthetic method in case 2 did not appear to be the proper choice in retrospect. this is because naked eye observation does not ensure that there is no tumor tissue debris remaining and the presence of such fragments may be another issue of bronchial obstruction. the reason why the method in this case did not present any problems is that the endotracheal tube with a diameter larger than the measured i d could be inserted because the trachea compressed by the thyroid tumor was not constricted by foreign surrounding tissue. in , endotracheal tubes with a diameter larger than the measured i d were used despite the severe tracheal obstruction due to the absence of dyspnea or very mild symptoms in patients showing normal abga and pulmonary function test . in addition, the normal of these two tests demonstrated that the trachea compressed by the foreign tumor outside the thorax was mobile. as suggested in our cases, anesthesia should be initiated only after making all preparations for securing the trachea, even when easy tracheal intubation is anticipated. as in case 2,	to achieve safe airway management, it is essential first to predict whether there will be difficulties in intubating or ventilating the patient's airway. an enlarged thyroid mass can produce a tracheal obstruction by compression or intraluminal invasion or both. we report two patients with thyroid cancer that obstructed the trachea by compression or invasion. there was no difficulty in endotracheal intubation of the patients with marked thyroid enlargement or in securing passage of the endotracheal tube through the compressed or narrowed portion of the trachea.
adhesion involves interactions at the interface between materials and depends of several factors as cleanliness, composition, and roughness of adherent surface. mechanical bonding is the most effective means of creating strong joints. in this type of adhesion , the material penetrates into the adherent, becoming mechanically interlocked at some level. to attain effective adhesive bonding irrigating solutions used in endodontics clean the dentin surface, and may interfere with the chemical structure of dentin, changing the calcium / phosphorus (ca / p) ratio of the surface. these alterations can increase the surface roughness, which may affect the sealing ability and adhesion of dental materials - such as resin - based cements and root canal sealers - to dentin and alter the nature of adhesion and the adhesion strength of various bacteria. sodium hypochlorite solutions (naocl) are the most common irrigating agents used in biomechanical preparation based on their excellent microbicidal activity and tissue - dissolving capabilities. however, despite these properties, this solution only removes the organic structure of the smear layer produced during mechanical instrumentation, and combining it with chelating agents is necessary to remove the inorganic phase of this layer. various chelating agents can be used for this purpose, such as ethylenediaminetetraacetic acid (edta), citric acid (ca), mtad (mixture of doxycycline, citric acid and tween 80), and chitosan. recently, etidronate (hebp), a substance that prevents bone resorption has been used in medicine for patients suffering from osteoporosis or pagets disease, and was suggested as substitute for traditional chelators due to fewer effects observed on dentin structure. it is considered the unique chelator that can be mixed with naocl without interfering with its antimicrobial property. however , chelating solutions also remove calcium ions from the dentin surface, exposing the collagen matrix, which may contribute to bacterial adherence in recontaminations, as with enterococcus faecalis. the use of naocl solutions has been suggested as a strategy to remove this exposed collagen matrix in a process called deproteination, which restores to the surface characteristics of untreated dentin. the importance of roughness studies is supported due to the strong relationship between surface topography and its influence on dentin wettability, a property that directly influences the bonding of the dental materials and microorganism adhesion. the increase in roughness could be potentiated by the combination of the irrigation solutions and, to date, there have been no studies evaluating the effect of hebp and different irrigation regimens on the roughness of root canal dentin. therefore, the purpose of this in vitro study was to evaluate the effects of naocl, edta, hebp and ca associated with different irrigation regimens on root dentin roughness. solutions of 2.5% (wt / vol) and 5% naocl, 10% ca (sigma - aldrich, st louis, missouri, usa), 9 and 18% hebp (zschimmer & schwarz mohsdorf gmbh & co kg, burgstdt, sn, germany) were prepared using pure chemicals dissolved in deionized water. the 17% edta solution (sigma aldrich) was prepared as previously described in other study. all solutions were stored at 5c in airtight dark containers between experiments; prior to being used, the solutions were removed from the refrigerator and stored for 60 min at room temperature. a fresh 1:1 mixture of 5% naocl and 18% hebp was prepared immediately before the experiments, producing a solution that contained 2.5% naocl and 9% hebp. remnants of debris and soft tissue on the tooth surfaces were removed, and all teeth stored in 0.1% thymol at 9c until use. tooth crowns were removed at the cement - enamel junction using a low - speed diamond disk (kg sorensen ind . the canals were explored with a size 15 k - type file ( dentsply maillefer, ballaigues, switzerland) until the tip of the instrument was adjusted to the apical foramen. at this point, another sectioning was performed to standardize the root apical limit and ensure that only the canal dentin was analyzed. subsequently, each root was sectioned longitudinally in the buccolingual direction to expose the entire canal extension, and the pulp tissue was removed. the tooth halves thirds were transversely marked with the aid of a digital caliper pd-150 (vonder, curitiba, pr, brazil), and then the halves were horizontally sectioned into apical, middle and cervical thirds. each segment was identified and horizontally mounted in autopolymerizing acrylic resin (dentbras ind . the specimens were wet polished on a circular grinding machine with a series of ascending grades ( 400, 600, 1200, and 2000) of silicon carbide abrasive papers (3 m do brasil ltda ., sumar, sp, brazil) under water coolant to achieve a standard surface roughness. then, the samples were thoroughly washed and sonicated in distilled water to remove residual particles. the surface roughness of each sample was determined in ra (arithmetic average roughness - m) with a portable digital roughness tester sj 301 (mitutoyo, tokyo, japan) within a pre - established cut - off (distance traveled in each reading) of 0.8 mm. specimens with similar surface roughness were included in the study; the other samples were again subjected to polishing until they reached the roughness standard mean (0.10 - 0.14 ra) determined by the pilot test. the 45 specimens of each third were randomly divided into groups as shown in figure 1. the specimens were immersed in irrigating solutions for 30 minutes to simulate the biomechanical preparation. in stage 2, the inorganic phase of the smear layer was removed through the application of chelating agents, and in step 3, deproteination was performed with the use of 2.5% naocl solution for 3 min. the irrigation solutions were renewed every 5 min to ensure their chemical effectiveness. after each step, the specimens were washed for 1 min with distilled water in an ultrasonic tub to avoid residual effects from the solutions. the measurements were performed after each stage in the same manner as the initial measurements, and the roughness values were recorded. twenty - seven specimens of each root third were subjected to naocl and then distributed according to the irrigation regimens used. to avoid false - positive inflation, the roughness values of only 9 specimens the nonparametric wilcoxon test (<0.05) was used to compare the dentin surface roughnesses before and after treatments, and the friedman test (<0.05) was used to detect differences among the root thirds. solutions of 2.5% (wt / vol) and 5% naocl, 10% ca (sigma - aldrich, st louis, missouri, usa), 9 and 18% hebp (zschimmer & schwarz mohsdorf gmbh & co kg, burgstdt, sn, germany) were prepared using pure chemicals dissolved in deionized water. the 17% edta solution (sigma aldrich) was prepared as previously described in other study. all solutions were stored at 5c in airtight dark containers between experiments; prior to being used, the solutions were removed from the refrigerator and stored for 60 min at room temperature. a fresh 1:1 mixture of 5% naocl and 18% hebp was prepared immediately before the experiments, producing a solution that contained 2.5% naocl and 9% hebp. remnants of debris and soft tissue on the tooth surfaces were removed, and all teeth stored in 0.1% thymol at 9c until use. tooth crowns were removed at the cement - enamel junction using a low - speed diamond disk (kg sorensen ind . the canals were explored with a size 15 k - type file ( dentsply maillefer, ballaigues, switzerland) until the tip of the instrument was adjusted to the apical foramen. at this point, another sectioning was performed to standardize the root apical limit and ensure that only the canal dentin was analyzed. subsequently, each root was sectioned longitudinally in the buccolingual direction to expose the entire canal extension, and the pulp tissue was removed. the tooth halves thirds were transversely marked with the aid of a digital caliper pd-150 (vonder, curitiba, pr, brazil), and then the halves were horizontally sectioned into apical, middle and cervical thirds. each segment was identified and horizontally mounted in autopolymerizing acrylic resin (dentbras ind . the specimens were wet polished on a circular grinding machine with a series of ascending grades ( 400, 600, 1200, and 2000) of silicon carbide abrasive papers (3 m do brasil ltda . , sumar, sp, brazil) under water coolant to achieve a standard surface roughness. then, the samples were thoroughly washed and sonicated in distilled water to remove residual particles. the surface roughness of each sample was determined in ra (arithmetic average roughness - m) with a portable digital roughness tester sj 301 (mitutoyo, tokyo, japan) within a pre - established cut - off (distance traveled in each reading) of 0.8 mm. specimens with similar surface roughness were included in the study; the other samples were again subjected to polishing until they reached the roughness standard mean (0.10 - 0.14 ra) determined by the pilot test. the 45 specimens of each third were randomly divided into groups as shown in figure 1. the specimens were immersed in irrigating solutions for 30 minutes to simulate the biomechanical preparation. in stage 2, the inorganic phase of the smear layer was removed through the application of chelating agents, and in step 3, deproteination was performed with the use of 2.5% naocl solution for 3 min. the irrigation solutions were renewed every 5 min to ensure their chemical effectiveness. after each step , the specimens were washed for 1 min with distilled water in an ultrasonic tub to avoid residual effects from the solutions. the measurements were performed after each stage in the same manner as the initial measurements, and the roughness values were recorded. twenty - seven specimens of each root third were subjected to naocl and then distributed according to the irrigation regimens used. to avoid false - positive inflation, the roughness values of only 9 specimens the nonparametric wilcoxon test (<0.05) was used to compare the dentin surface roughnesses before and after treatments, and the friedman test (<0.05) was used to detect differences among the root thirds. the median and interquartile range of roughness values of root canal dentin before and after treatment with irrigation regimens in cervical, middle, and apical thirds are summarized in tables 1, 2 and 3, respectively. a significant increase in dentin roughness was observed following the treatment with the different irrigation regimens, except when saline (g1) or naocl (g3) were used alone. median (med) and interquartile range (iqr) and p values (wilcoxon test) for roughness analysis in the cervical third before (t0) and after (t1) the application of the irrigation regimens median (med) and interquartile range (iqr) and p values (wilcoxon test) for roughness analysis in the middle third before (t0) and after (t1) the application of the irrigation regimens median (med) and interquartile range (iqr) and p values (wilcoxon test) for roughness analysis in the apical third before (t0) and after (t1) the application of the irrigation regimens the data for initial roughness revealed no statistically significant difference among the different thirds (table 4). after treatments, the different thirds behaved the same way, except in g2 in which the apical third had a lower increase in surface roughness than did the other thirds (p=0.0043). the p values (friedman test) of the roughnesses before (t0) and after (t1) the same treatment on different thirds ct = cervical third; mt = middle third; at = apical third; p=0.0043: ct x mt p = not significant; ct x at p<0.05; and mt x at p<0.05 the irrigation solutions might influence the physicochemical properties of human root canal dentin, including microhardness, permeability, solubility, wettability and roughness. in endodontics, an increase in surface roughness could be clinically beneficial because it may enhance the micromechanical bonding of root canal sealers, which requires irregularities on the surface of the adherent for penetration. however, too much roughness can facilitate bacterial adhesion, which might lead to plaque formation. in the present study , naocl treatment did not modify the roughness of the dentin surface when used before or after the chelating agents, consistent with previous findings that naocl does not cause decalcification or changes in dentin wettability, a factor that has been correlated with dentin roughness. however, these are in contrast to those of other studies, most likely due to the standardization of the initial polishing used here; this procedure was not reported in the other studies. initial roughness standardization is recommended in studies of chemical conditioning because it offers a controlled reference point for the correct and unambiguous assessment of the morphological effects induced by subsequent chemical treatments. the regimens that employed ca (g5 and g8) and hebp mixed with naocl (g2) demonstrated greater increases in roughness values than did other groups. the strong activity of ca is most likely due to its greater capacity for demineralization that, in addition to removing the smear layer, can also cause extensive demineralization in dentinal tubules and peritubular dentin. when mixed, the hebp causes some reduction in the activity of naocl after 1 h, but the two substances remain active in fresh mixture. the observed in g2 are most likely due to the ability of naocl to create deproteination canals in the dentin, which may increase the area of action available for hebp. associated with this factor, the longer use of this weak chelating agent for 30 min also may have potentiated its effect. the smallest changes in surface roughness after the use of a chelating agent were observed in regimens that employed 9% hebp for 5 min after naocl treatment (g6 and g9). these findings confirm that hebp is a weak chelating agent that attacks less dentin surface than other commonly used chelators, such as edta, but the hebp solutions need 300 seconds to completely remove the smear layer. in terms of the behavior of different root thirds in each of the irrigation regimens, it was observed that, despite the heterogeneous structure of these regions, they exhibit similar behaviors in response to direct contact between the irrigation agent and the root surface (table 4). one exception was the apical third in g2, possibly because the greater percentage of sclerotic dentin in this area reduced the number of deproteination canals created by naocl, decreasing the area of action available for hebp. the of this study showed the effects of direct contact from different irrigation regimens using different auxiliary chemical substances on dentin surface roughness. however, these can not be extrapolated to clinical practice, because it is difficult to introduce irrigation solution to the apical region, which, associated to the use of endodontic instruments, may change these values. however, the analysis of the behavior of different root thirds into direct contact with the irrigating solutions is relevant to assess if, besides the difficulty of the irrigators to reach the apex, the dentin composition also influences the increase in roughness. it was possible to observe that only the irrigation regimens that employed chelating solutions increased the surface roughness and the use of naocl before the chelating agents or as a final flush did not modify the dentin roughness. further studies should be performed to evaluate not only the effect of different irrigation regimes on the dentin structure but also the effect of these protocols on the adhesion of root canal sealers and bacteria in recontaminations. the findings of this study indicated that only the irrigation regimens that used chelating agents were capable of increasing the roughness of root canal dentin. this study was supported in part by capes - coordination of higher education and graduate training - and by cnpq - national council for scientific and technological development.	an increase in dentin roughness, associated with surface composition, contributes to bacterial adherence in recontaminations. surface roughness is also important for micromechanical interlocking of dental materials to dentin, and understanding the characteristics of the surface is essential to obtain the adhesion of root canal sealers that have different physico - chemical characteristics.objectivesto evaluate the effects of sodium hypochlorite (naocl), ethylenediaminetetraacetic (edta), etidronic (hebp), and citric acid (ca) associated with different irrigation regimens on root dentin roughness.material and methodsforty - five root halves of anterior teeth were used. the root parts were sectioned in thirds, embedded in acrylic resin and polished to a standard surface roughness. initially, the samples of each third were randomly assigned into 3 groups and treated as follows: g1 - saline solution (control); g2 - 5% naocl+18% hebp mixed in equal parts; and g3 - 2.5% naocl. after initial measuments, the g3 samples were distributed into subgroups g4, g5 and g6, which were subjected to 17% edta, 10% ca and 9% hebp, respectively. following the new measuments, these groups received a final flush with 2.5% naocl, producing g7, g8 and g9. the dentin surface roughness (ra) was determined before and after treatments using a profilometer. the wilcoxon test (<0.05) was used to compare the values before and after treatments, and the friedman test (<0.05) to detect any differences among root thirds. (i) naocl did not affect the surface roughness; (ii) there was a significant increase in roughness after the use of chelating agents (p<0.01); and (iii) only the g3 group showed a difference in surface roughness between apical third and other thirds of the teeth (p<0.0043).only the irrigation regimens that used chelating agents altered the roughness of root dentin.
hemolytic uremic syndrome (hus) is a hematological disease characterized mainly by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. it is commonly caused by infections of shiga - like toxin producing bacteria, such as escherichia coli strain o157:h7, o111:h8, o103:h2, o123, and o26.1 diarrhea is often present in such cases which can also be referred to as typical hus. on the other hand, atypical hus (ahus) is characterized by the absence of diarrheal illness and can be acquired, genetic, or idiopathic. ahus represents approximately 10% of all hus cases.2 currently, plasma exchange and/or plasma infusions is the recommended first - line management for ahus.3 this paper presents the technical challenges faced while treating a patient who had ahus with therapeutic plasma exchange (tpe). a 21-year - old female with a known history of ahus presented to our emergency department in september 2012. she had three relapses before when she was aged 8, 10, and 20. she presented with 3 days of fever and sore throat followed by dark urine. on examination , she was found to be afebrile, alert, oriented, and she was talking in full sentences. her sitting blood pressure was 140/90 mmhg and her heart rate was 70 beats per minute with an oxygen saturation of 99% on room air. hematological investigations revealed a hemoglobin (hb) count of 96 g / l, platelet count 16 10/l, red cell count 3.01 10/l, and a hematocrit of 0.27 there was also marked thrombocytopenia and microangiopathic anemia on the film, features suggestive of relapse of known hus. biochemistry showed a lactate dehydrogenase (ldh) level of 1,201 u / l, haptoglobin < 0.06 g / l, potassium 3.7 mmol / l, urea 20.7 mmol / l, creatinine 202 umol / l, estimated glomerular filtration rate 27 ml / minute, and c - reactive protein (crp) of 27 mg / l. high levels of ldh and low haptoglobin levels pointed to severe hemolysis while the renal markers, such as creatinine, urea, and estimated glomerular filtration rate showed a deteriorating renal function. earlier attempts to perform the biochemical tests had been futile due to severe hemolysis. after a multidisciplinary team collaboration that involved the intensive care unit, hematology, nephrology, and the emergency department, a left femoral vascath was inserted with the view of commencing tpe using 3 l of fresh frozen plasma (ffp). the procedure was scheduled to be done in the emergency department using a membrane based tpe machine (mtpe). plasma flux psu 2s plasma exchange filters manufactured by fresenius se & co (bad homberg, germany) were used. these filters have a surface area of 0.6 m, blood priming volume of 70 ml, and a plasma sulphone membrane. filtration is primarily based on pressure gradients allowing filtration of molecules of up to 1,000 kda including immunoglobulins, complement factors, and albumin. soon after commencing tpe , the machine showed a blood leak alarm and it was evident that the membrane had ruptured gauging by the color of the effluent (figure 1). the filter was replaced with another one which also did not last long before the machine showed a blood leak alarm. an attempt was made to resume treatment with a centrifuge based tpe machine (ctpe). the centrifugal device we used was a spectra optia apheresis system, a product of terumo bct (lakewood, co, usa). this machine operates by separating blood products according to their specific gravity using centrifugal force. the spill over alarm persisted on ctpe and red blood cell detected was shown on the machine. at this point, it was agreed to stop tpe due to the nature of the technical problems which were attributed to severe hemolysis. on day 2, it was agreed that the patient could be treated with eculizumab (complement c5 blocker), but the drug was not available for compassionate access. disable the red blood cell detector and ctpe was initiated successfully using a very low inlet flow rate ranging from 2070 ml / minute with an anticoagulant infusion rate of 1.0 ml / minute. the patient continued to receive daily ctpe until day 16 when a decision was made that she had reached clinical remission, and she was discharged home. her hematological (table 1) and biochemical (figure 3) continued to improve. a follow up of this patient after 6 weeks revealed that she was clinically well and had resumed her normal daily routines. however, plans had been made for her to have a permanent vascular access in the form of an arterio - venous fistula (avf) in the setting of these recurrent ahus episodes which seem to resolve after aggressive tpe treatment. both machines (mtpe and ctpe) were fitted with safety mechanisms to prevent loss of red blood cells during treatment and if there was a blood leak, an alarm was triggered and the machine would stop automatically. interestingly, in our case, we knew that the effluent was very rich in hemolysis products and the machine that won the day was one that we could manipulate easily to tailor our treatment plan; this was the ctpe. furthermore , another dilemma with using the mtpe is that plasma has different optical properties from ultrafiltrate, which can set off the blood leak alarm and stop the pump even if there is no actual blood leak. in this respect, nursing staff were not comfortable overriding the mtpe blood leak alarm since it is challenging to determine whether there is an actual blood leak or not. the ctpe also gave us an opportunity to assess the color changes of the patient s effluent and this boosted our confidence as we visually noticed some positive of the treatment. we believe that our patient received the best tpe treatment we could offer since some clinical cross over trials have reinforced the notion that ctpe allows a higher plasma exchange rate compared to mtpe without adverse effects on treatment quality and tolerability.4 this sentiment has been shared by another researcher who has found that plasma removal is more efficient with ctpe compared to mtpe systems, which have a lower plasma extraction ratio and therefore require longer procedure times.5 the ctpe treatment also ensured that our patient received the 3 l of ffp that was ordered and that an equal volume of patient plasma was removed. from our experience with dialysis, in patients whose post dialysis weight does not correlate with the reported ultrafiltration, the preciseness of the built - in ultrafiltration controller in the mtpe machine is questionable. ffp was used as a substitution solution during tpe despite the high rate of side effects, such as anaphylactic reactions6,7 that may actually require cessation of plasmatherapy.8 we used ffp because it provides normal amounts of complement factors (cfh, cfi, cfb, and c3) and functional proteins9 which are vital for treating ahus. it was unfortunate that our patient could not access eculizumab, which is an anti - complement factor 5 (c5) monoclonal antibody that binds to c5 thereby preventing activation of the terminal complement cascade. recent impressive improvement in the management of ahus has been reported with the use of eculizumab which is becoming the new breakthrough treatment option for patients with primary ahus, providing improved control of the disease over plasma exchange, with a good safety profile.10 however, it is still not quite clear whether life - long or recurrence - specific treatment is necessary and how genetics may or may not impact care of persons on eculizumab.11 what is known, however, is that defects in more than one complement regulator in ahus cases may pose significant therapeutic challenges.12 recent reports indicate that mutations in dgke (which encodes diacylglycerol kinase) were found in several ahus patients and this may have an impact on the successful treatment of individuals with eculizumab.13 nevertheless, we are delighted that we successfully delivered first line treatment8 for ahus through ctpe. traditionally, ctpe is the method preferred by hematology or blood bank based physicians for plasma exchange while, on the other hand, nephrology based physicians prefer mtpe. from our experience, it may be better to individualize these treatment options since patient needs vary. for a patient with ahus, treating with centrifuge devices such as the spectra optia apheresis system (terumo bct) may in better outcomes. clinicians also seem to be more comfortable managing the technical challenges, such as the continuous activation of blood leak alarms caused by severe hemolysis for patients on ctpe compared to mtpe.	atypical hemolytic uremic syndrome (ahus) is a very rare, life - threatening, progressive disease that frequently has a genetic component and in most cases is triggered by an uncontrolled activation of the complement system. successful treatment of ahus with plasma infusions and therapeutic plasma exchange (tpe) is well reported. tpe has been the treatment of choice in most adult patients with ahus. however, due to severe hemolysis, which is common among ahus patients, there are some technical challenges that can affect tpe treatment such as the continuous activation of the blood leak alarm due to hemolysis. our experience shows that such patients can be managed better on a centrifuge based tpe machine compared to a membrane based tpe machine.
polyamidoamine (pamam) dendrimers were purchased from sigma aldrich (poole, dorset, uk) and used without further purification. the pamam dendrimers with amino and amidoethanol surface groups were diluted to a concentration of 10 pmol/l in solutions of methanol (fisherscientific, loughborough, uk) to water (j. t. baker, middlesex, uk) to acetic acid (fisherscientific) (49:49:2, vol / vol). the 0.5-generation pamam dendrimer with sodium carboxylate surface groups was prepared (10 pmol/l) in methanol: acetic acid (98:2) solution. mass spectrometry analysis was performed on a thermo finnigan ltq ft mass spectrometer (thermo fisher scientific, bremen, germany). samples were injected by use of an advion biosciences triversa nanomate electrospray source (advion biosciences, ithaca, ny, usa). data acquisition and analysis were conducted using the xcalibur 2.0 (thermo fisher scientific) software. precursor ions were selected and isolated for ecd in the linear ion trap before transfer to the icr cell. electrons were generated on the surface of an indirectly heated barium tungsten cylindrical dispenser cathode (5.1 mm diameter ; heat wave labs, inc ., watsonville, ca, usa), situated 154 mm from the cell, 1 mm off - axis. the current across the electrode was about 1.1 a. ions were irradiated with electrons for 70 ms. cid experiments were performed in the front - end linear ion trap and the fragments transferred to the icr cell for detection. cid experiments were performed with helium gas at a normalized collision energy of 35%. each cid scan consisted of five coadded microscans. all tandem mass spectrometry (ms / ms) spectra were averaged over 30 scans and analyzed manually. ecd fragments were assigned based on the nomenclature devised by oh and colleagues (see scheme 2). assignments are given in the form gn(m), where subscript n refers to the generation in which fragmentation takes place and m describes the type of fragmentation; a / x, b / y, c / z. for example, fragment g1(y) was derived from cleavage of the amide bond in generation 1. gn(in) and gn(out) notations concern fragmentation that takes place core - side of the tertiary amines. the second - generation pamam dendrimer with amidoethanol surface groups and ethylenediamine core is a symmetrical molecule that contains three tertiary amine branches (generations 0, 1, and 2) and 16 neutral alcohol surface groups. electrospray ionization of this pamam dendrimer leads to the formation of multiply protonated molecular ions through. the most abundant multiply protonated molecular ions of the pamam dendrimer, , and ions were isolated and subjected to ecd. the ecd ms / ms spectrum of precursor ions is shown in figure 1 (top). all fragments detected are described in supplementary table s-1, which can be found in the electronic version of this article. the mass spectrum shows peaks corresponding to triply charged fragments g1(in), g0(in) singly charged gcore(in), and quadruply charged g1(in). in addition, g(out) fragments, which also from cleavage at tertiary amines but with the charge retained toward the surface of the dendrimer, were observed. these fragments also undergo secondary fragmentations, ing in singly charged fragment ions at m / z 160.1205, at m / z 389.2624, and at m / z 459.2915. the c / z cleavages that are the most abundant in the ecd of peptides / proteins were found here only as minor channels: g1(z) at m / z 274.1757 and g0(z) at m / z 732.4592. as mentioned earlier, similar fragmentation patterns were seen for each charge state of pamamg2oh. in each case, fragmentation within these cations occurred in the innermost generations, i.e., the core, g0 or g1. electrospray ionization of the generation 1 pamam dendrimer, containing ethylenediamine core, two tertiary amine branches (generations 0 and 1), and eight primary amino surface groups, in the formation of multiply protonated molecular ions through. mass spectra shown here were obtained from the most abundant charge state, i.e., ions. the ecd mass spectrum of the ions of pamamg1nh2 is shown in figure 2 (top) and the fragments observed are detailed in supplementary table s-2. the peaks at m / z 592.9225 and 289.2342 can be assigned to g0(in) and g0(y) fragments, respectively. other abundant fragment ions at m / z 388.3021 and 159.1366 are attributed to secondary fragmentation and can be assigned to and , respectively. these fragments are presumably the of cleavage of the amide bond followed by loss of co, together with cleavage at the tertiary amine. comparison of the ecd mass spectra obtained from pamamg1nh2 and pamamg2oh dendrimers leads to the that, despite the different chemical properties, the nature of the surface groups does not affect the ecd fragmentation behavior of these polymers. in both cases the mechanism of ecd is bound up with protonation of the tertiary amines and the presence of amide functionalities in the polymer backbone and can be explained on the basis of a charge - solvation model (proton sharing). in both cases the ecd mass spectra are dominated by fragments that come from the inner generation(s). the most abundant fragments are of the same type for each dendrimer; gn(y), gn(in) and gn(out), where n stands for the inner generation(s) (n = 0, 1, in the case of pamamg2oh ; n = 0 in the case of pamamg1nh2). assignments are given in form agn, bgn, and cgn, where subscript gn refers to the generation in which cleavage takes place. note that bgn is equivalent to ecd assignment gn(kout), but the multiplicity of the cid fragmentation (see following text) demands simpler nomenclature. the collision - induced dissociation of ions of pamamg2oh (figure 1, bottom), is dominated by the loss of two neutral fragments, ag2 (m = 103.0633) and bg2 (m = 115.0633) and combined losses of the two in various stoichiometries. the precise connectivity of this fragment is unknown: the proton localized on the tertiary amine can be bound to the oxygen atom from carbonyl group. as a of proton transfer and rearrangement, the fragment ch2=c(oh)n(h)ch2ch2oh may be formed. neutral fragment bg2 is the of cleavage at the tertiary amine in the second generation and has the formula ch2=ch c(o)n(h)ch2ch2oh. fragment ions are observed following cid that from loss of the same neutral, but differ by charge. for example, the most abundant peak (m / z 511.6547) can be assigned to the loss of two ag2 neutrals; i.e., and the peak at m / z 613.7838 can be assigned to. in general, we observe series of peaks that can be assigned to combined losses of various stoichiometries of ag2 and bg2 for charge states + 6 through + 3. for example, for the + 5 charge state, the following ions were detected: , , , , , , , , , , , and. in all dissociation reactions, hydrogen migration followed by cleavage at tertiary amine (fragments bg2, bg1, bg0) or between two carbon atoms (fragments ag2 comparison of our for ions with those obtained for both singly and doubly charged precursors by brodbelt et al . suggests that cid of pamam dendrimers does not depend on the number of mobile protons present . the collision - induced dissociation of ions of pamamg1nh2 ( figure 2, bottom), is dominated by loss of water; loss of two neutral fragments ag1 and bg1; and combined losses of water and ag1 and bg1 in various stoichiometries (see supplementary table s-4). such fragmentation behavior is quite surprising, particularly with regard to the cid of pamamg2oh, in which no loss of water was observed. we speculate that the unusual cid behavior of pamamg1nh2 dendrimer proceeds as shown in scheme 4. the loss of water is pronounced probably because p is particularly stable as a of charge delocalization between the two nitrogen atoms. this idea is supported by a recent study of deamination and dehydration processes of n - terminal glutamine in cid of protonated peptides. when mobile protons are present the predominant neutral loss process from n - terminal glutamine is elimination of water because of formation of a protonated five - member aminopyrroline ring. when no mobile protons are present, deamination is observed as a of formation of a neutral pyrrolidinone ring. both reactions depend on the charge state and stability. in the present case of quapruply protonated pamamg1nh2 ions, four protons are present and a maximum of four molecules of water are eliminated during cid. in contrast, cid of both singly and doubly charged pamamg1nh2 ions ed in loss of ammonia but not loss of water. neutral losses observed in the cid of pamamg1nh2 depend on the charge state; for higher charge states, the dominant process is loss of water and, for lower charge states, elimination of nh3 is observed. unlike ecd, the of the cid experiments performed for pamamg2oh and pamamg1nh2 suggest a strong dependence of the dissociation processes on the nature of the surface groups. for pamamg1nh2, the pamam dendrimer, generation 0.5, with eight sodium carboxylate surface groups and ethylenediamine core (pamamg0.5coona), forms multiply charged anions via negative electrospray. the dendrimer ions have mixtures of sodium and proton counterions because protons present in the meoh / nh4oh solution compete with sodium ions associated with the surface groups. within each charge state, mixtures of counterions were found according to the formula , where n is charge state (n = 24), and m is the number of protons replacing sodium in the surface groups. figure 3 shows the edd and cid mass spectra obtained from ions. the edd mass spectrum (figure 3, top) is dominated by peaks corresponding to the loss of one, two, and three t fragments (m = 71.01333), which from cleavage at an outer tertiary amine. loss of t fragments is also observed following cid (figure 3, bottom). the only peak present in the edd mass spectrum, but not present in the cid mass spectrum, occurs at m / z 497.7054 and can be assigned to the fragment (m / zcalc 497.7080). this fragment from cleavage at the tertiary amine, which was also a major fragmentation channel in the ecd of pamam cations. no fragments corresponding to cleavage of the amide bond or c / z - type cleavages were observed following edd. we have investigated the electron - mediated dissociation (ecd and edd) of three pamam dendrimers with the aim of determining the effect of the macromolecular properties (number of generations) and the nature of the surface group. in all cases, fragmentation was dominated by cleavage at the tertiary amines with some amide bond cleavages (ecd). the c / z - type dissociation, prevalent in ecd of peptides and proteins, is observed only as a minor channel. the suggest that ecd (and edd) are independent of the nature of the surface group, but tend to occur within the innermost generations. in contrast, cid of the pamam dendrimers tends to occur in the outermost generation and is strongly dependent on the nature of the surface group. in comparison with cid the demonstrate the potential utility of ecd as a tool for the structural analysis of pamam dendrimers.	here, we investigate the effect of the structure (generation) and nature of the surface groups of different polyamidoamine (pamam) dendrimers on electron - mediated dissociation, either electron capture dissociation (ecd) or electron detachment dissociation (edd), and compare the fragmentation with that observed in collision - induced dissociation (cid). ecd and edd of the pamam dendrimers ed in simple mass spectra, which are straightforward to interpret, whereas cid produced complex mass spectra. the show that electron - mediated dissociation (ecd and edd) of pamam dendrimers does not depend on the nature of the surface group but tends to occur within the innermost generations. cid of the pamam dendrimers showed a strong dependence on the nature of the surface group and occurred mostly in the outer generation. the demonstrate the potential utility of ecd and edd as a tool for the structural analysis of pamam dendrimers.
sex steroid hormone receptors are located in various human ocular tissues, such as cornea, iris, ciliary body, lens, conjunctiva, retina, lacrimal and meibomian gland, in both males and females.1,2 epidemiological studies show that sex hormones act differently according to sex and increase the incidence of age - related cataract, glaucoma, dry eye, neovascular age - related macular degeneration, central serous chorioretinopathy, etc.35 there are variations in the levels of the sex steroid hormones during the menstrual cycle. thus, some researchers have shown that these hormone fluctuations show correlation with the ocular tissue variables.6,7 on the other hand, some researchers advocate that these fluctuations do not affect ocular variability significantly.8 the ocular biometric parameters may change during the different phases of the menstrual cycle. they found that ophthalmic artery perfusion is increased after administration of intranasal 17-beta - estradiol in postmenopausal women. the lenstar ls 900 optical biometer is a device that is based on optical low- coherence reflectometry (olcr) technology. the lenstar biometer measures central corneal thickness (cct), anterior chamber depth (acd), lens thickness (lt), axial length (al), keratometry, pupil diameter, and white - to - white distance.10 the aim of our study was to evaluate the ocular biometric characteristics during the menstrual cycle using the lenstar ls 900 biometer. twenty - two healthy women between the ages of 19 and 36 years with regular menstrual cycles were enrolled in this prospective study. the study protocol was approved by adnan menderes university s ethics committee and complied with the tenets of the declaration of helsinki. subjects with irregular menstrual cycles, those taking contraceptive pills, those with a history of ocular surgery or trauma, and those unable to cooperate with the biometry device were excluded from this study. subjects were asked to define the phases by counting forward the days from the start of menses. also, biometric measurements (cct, al, acd, lt, and keratometric values) were made at the same time using the olcr device. both eyes were measured, but only the right eye was taken to the study. two - way analysis of variance for intergroup comparisons was used for normally distributed variables and descriptive statistics are shown as mean standard deviation. for variables which were not in accordance with normal distribution , the friedman test was used for intergroup comparisons, and descriptive statistics are shown in median (25th to 75th percentile) format. two - way analysis of variance for intergroup comparisons was used for normally distributed variables and descriptive statistics are shown as mean standard deviation. for variables which were not in accordance with normal distribution, the friedman test was used for intergroup comparisons, and descriptive statistics are shown in median (25th to 75th percentile) format. the mean cct, al, acd, lt, and kerato - metric measurements (steep and flat keratometry readings) of the right eye are summarized in table 1 (two - way analysis of variance). measurements were made at the beginning of the cycle (13 days), at ovulation (1216 days), and at the end of the cycle (2632 days), on different menstrual cycle days, for 1 month (table 2) (two - way analysis of variance). the cct and al were thinnest at the beginning of the cycle and increased steadily until the end of the cycle, but these differences were not statistically significant (p=0.498 and p=0.421, two - way analysis of variance). conversely, the lt was the thickest, and steep and flat keratometry readings were the highest, at the beginning of the cycle and decreased regularly at the end of the cycle (p=0.179, p=0.892, p=0.434, two - way analysis of variance). the acd was reached the thickness value at the middle of the cycle (table 2) (friedman test). changes in the anterior segment parameters during the menstrual cycle are referred to in the literature. some researchers claim that these variations are significant, while others advocate that these differences are inconsiderable. they reported the cornea was thickest at the end of the cycle and thinnest at the beginning.11 on the other hand, hashemi et al investigated corneal thickness, corneal curvature, and acd during the menstrual cycle using the scheimp - flug imaging technique.12 they found no significant difference in measurements during the menstrual cycle period. corneal biomechanical parameters like corneal resistance factor and corneal hysteresis are determined by the ocular response analyzer. goldich et al reported that corneal resistance factor and corneal hysteresis decreased at the ovulation phase of the menstrual cycle.13 the cct was thickest at the end of the cycle and thinnest at the beginning.13 seymenolu et al determined corneal biometric properties during the menstrual cycle by using the ocular response analyzer.14 in contrast to goldich et al s study, they could not find differences in corneal biomechanical properties and intraocular pressure during the menstrual cycle. the lenstar is an optical biometer that provides cct, acd, lt, al, and keratometric measurements. the lenstar uses the olcr measurement principle and allows fast, comfortable, noncontact, less user - dependent, highly reliable, and reproducible measurements.15 recently, uakhan et al compared the corneal curvature and acd measurements using the lenstar ls 900, pentacam (oculus, wetzlar, germany), and a manual keratometer in healthy eyes.16 the authors reported good correlation between the lenstar and pentacam for measuring the acd and corneal curvature. another study, by cruysberg et al evaluated the reproducibility of the lenstar ls 900.17 the cct and acd measurements were compared between the lenstar ls 900 and visante as - oct. acd and al measurements, keratometric readings, and chamber depth measurements were compared between the lenstar ls 900 and iol master. they found differences between the lenstar ls 900, iol master, and visante as - oct, and they did not recommend the use of these three optical devices interchangeably. on the other hand, they showed that the lenstar ls 900 is an excellent reproducible optical biometric device for all reported measurements. in our study, cct, al, acd, lt, and keratometric values were found to change during the menstrual cycle. some measurements were highest at the beginning of the cycle (lt and keratometric values), some measurements were highest at the middle of the cycle (acd), and other measurements were highest at the end of the cycle (cct and al). secondly, different phases of the menstrual cycle should be taken into consideration to avoid inconsistent of changes in ocular variables. lastly, we could not perform a multivariate regression model in order to evaluate the role of confounding factors (cardiovascular risk factors, the pharmacological history, etc) on . biometric measurement is important in refractive surgery calculation, in intraocular lens selection, prior to cataract surgery, for eyeglass or contact lens prescription, etc. the lenstar ls 900 biometric measurement device is not affected by the variations of hormonal changes. therefore, measurement with the lenstar ls 900 can be used reliably in healthy women without reckoning with variations of hormonal changes during the menstrual cycle.	purposeto determine the ocular biometric characteristics during the menstrual cycle using the optical low - coherence reflectometry (olcr) biometry.methodstwenty-two healthy women between the ages of 19 and 36 years with regular menstrual cycles were enrolled in this prospective study. subjects with irregular menstrual cycles, those taking contraceptive pills, those with a history of ocular surgery or trauma, and women unable to cooperate with the ocular biometry device were excluded from this study. a complete ophthalmic examination was performed between 8.30 and 10.30 am for all participants. also, central corneal thickness, axial length, anterior chamber depth, lens thickness, and keratometric measurements were made at the same time using the olcr device. measurements were taken at the beginning of the cycle (13 days), at ovulation (1216 days), and at the end of the cycle (2632 days).the mean age of the participants was 22.864.22 (range : 1836) years. the difference in central corneal thickness, axial length, anterior chamber depth, lens thickness, and keratometry values were not statistically significant during the menstrual cycle.the ocular biometric parameters did not significantly vary during the menstrual cycle according to the olcr biometry.
among the different entities causing rhinoliquorrhea in closest proximity to the sphenoid bone, trigeminal meningoceles (tms), lateral sphenoidal meningoceles (lsms), and the persistence of the lateral craniopharyngeal canal (sternberg cruveilhier canal, sc) have been described. tms have only been reported several times in the literature, and nomenclature is still heterogeneous. in general, reports cover various mass lesions in the meckel cave, ranging from lipoma and meningioma to schwannoma. only a few deal with meningoceles in the pterygopalatine region, such as in patients with typical stigmata of neurofibromatosis type 1 (nf 1).1 2 tms are localized in the lateral sphenoid wing lateral to v2 and the foramen rotundum underneath the semilunar ganglion, reaching to the superior orbital fissure, pterygopalatine fossa, and medial orbital space, mostly emerging from an enlarged meckel cave and giving rise to osseous erosion of the sphenoid wing. as a different and more frequent reason for rhinoliquorrhea, lsms have been described. they lie medially to v2 near the base of the sphenoidal bone3 4 5 6 7 8 and can be distinguished from tms by the absence of orbital or pterygopalatine fossa involvement. the third dura leak associated pathology of this region is the developmental anomaly of a persistent sc embryonal canal (lateral craniopharyngeal canal). still controversially discussed in literature, it has been found in some cases as an underlying condition for lsms.9 10 11 the canal lies medially to the foramen rotundum and extends from the maxillary nerve root (v2) to a recess of the lateral sphenoid wall, opening into the sphenoid sinus. from an anatomical point of view, there are several weak spots in the embryonic development of the sphenoidal bone, which can explain the occurrence of meningoceles or spontaneous rhinoliquorrhea, especially in the area of the trigeminal ganglion and the carotid artery. the cartilage precursor of the skull base has to form around the cranial nerves, arteries, and veins while respecting their lumen. this can lead to thinned cranial base structures that might be eroded during a lifetime by inflammatory processes or increased intracranial pressure.12 the bone formation itself is inhomogeneous. the collision zone between endochondral ossification (lesser wing) and intramembranous ossification (greater wing) lies laterally to the foramen rotundum, extending to the region of the foramen ovale (fig . study, 25 patients with lateral sphenoidal cerebrospinal fluid ( csf) leaks were all shown to have the bony defects in this area, lateral to the foramen rotundum.8 cranial base in a 6-month - old embryo shown from above (modified from fenart and landouzy) depicting the four main parts forming the sphenoid bone. cranial nerves: ii, v1, v2, v3, vii, ix, x, xi, xii. alisph, alisphenoid; boc, basioccipitale; bsph, basisphenoid; fm, foramen magnum; fz, fusion zone between (lateral) and endochondral (medial) ossification; orbsph, orbitosphenoid; petr, petrous bone; prsph, presphenoid; scc, site of the sternberg cruveilhier canal. the sutures between the ossification centers can leave small basal clefts, such as the lateral craniopharyngeal canal (sc). the canal represents a remnant of the fusion zone between the alisphenoid (greater wing) and the basisphenoid (fig . 1).13 development of the sphenoid bone is crucial for the whole cranial base.14 15 16 distorted midline structure development can in encephaloceles and palate cleft malformations,17 18 19 and midline basal encephaloceles are known as very rare malformations.11 20 lateral sphenoid malformations give rise to lateral sphenoid meningoceles, as well as to meningoceles of the trigeminal nerve. the meningoceles seem to be associated with sphenoid bone malformations, increased intracranial csf pressure, and accompanying erosive processes. as shown in the following case description, tms can in rhinoliquorrhea and csf leakage to the pterygopalatine fossa and periorbital fatty tissue, followed by exophthalmos and conjunctivitis together with conjunctival edema. in literature, many different terms have been employed to describe the pathology of a tm. arachnoid cyst of meckel's cavity appears to be imprecise, because lsms also arise from it. the terms transalar sphenoid meningocele and transsphenoidal and transethmoidal meningoceles have been used synonymously in literature.19 20 we suggest the term lateral sphenoidal meningocele to describe pathologies medial to v2 and the term trigeminal meningoceles as anatomically more precise for lesions lateral to the foramen rotundum and v2. she also reported a left - sided orbital swelling, especially when bending forward or in prone position. her first meningitis had been at the age of 17 years, a second occurred a few years later, and both had been treated successfully with antibiotics. a former traumatic head injury was denied and basal cranial fractures had been ruled out with multiple imaging techniques. extended neuroradiological imaging included cisternographic magnetic resonance (mr) scan after intrathecal gadolinium - application in prone position. it revealed a left - sided csf leak along a csf - containing enlargement of the temporal fossa that extended into the pterygopalatine fossa. origin of the fistula was suspected in the temporopolar parasellar region in close proximity to the cavernous sinus and to the meckel cavity. the clivus, sellar area, and ethmoidal cells appeared to be anatomically altered in the ct - scan (fig . ( a) preoperative magnetic resonance (mr) t1-weighted axial images enhanced by cisternography. (b) oblique reconstruction parallel to the optic canal, (c) axial and (d) coronal reconstruction. the surgical strategy was discussed in the multidisciplinary skull - base board and under suspicion of a temporomedial meningocele of the maxillary nerve, a pterional craniotomy with transsylvian approach and closure of the parasellar entry point of the meningocele was advised. in a first operation, the suspected entry point of the fistula the patient then decided to be treated in another neurosurgical department, where a second pterional operation was performed without relieving the symptoms. about 2 years after first surgery, the patient decided to restart treatment in our institution. intrathecal contrast - enhanced ct revealed the refilled fistula and an enlarged, csf - containing space in the paraclival region, close to the maxillar nerve and the meckel cave. a third pterional exploration was proposed, but the patient opted for conservative therapy. only after increasing orbital swelling and reappearance of rhinoliquorrhea fusion of the ct dataset with neuronavigation pictures allowed identification of the entrance of the meckel cavity. the wall of the cavity showed a cisternlike arachnoid covering in which the nerve fibers crossing the cavity were partly adhering to the basal arachnoid layer of the cyst and were spread apart. two walls of the cavity were found, corresponding to arachnoid cystic structures and dural tissue, thus displaying typical criteria for meningoceles. to close the fistula, abdominal fat and muscle tissue were harvested and the periarachnoidal space of the meckel cave was filled in proximal and distal direction. hereafter, fat tissue was positioned in the remaining arachnoid space rostrally. between fat and muscle tissue, liquid dura glue (duraseal xact, covidien, mansfield, massachusetts, usa) the opening of the cavity was sealed with tachosil (takeda pharmaceuticals ; zurich, switzerland) and surgicel (ethicon, somerville, new jersey, usa). after this intervention, rhinoliquorrhea and orbital swelling disappeared and the third nerve palsy nearly completely recovered within 3 months. however, about 4 months postoperatively the patient experienced an intermittent csf leak. shortly thereafter, orbital swelling and conjunctivitis reoccurred. t2- and ciss 3d t2-scans revealed a partly occluded, yet csf - containing, meningocele. the patient refused any further interventions, especially implantation of a ventriculoperitoneal (vp) shunt to reduce the intracranial csf pressure, and she was lost to follow - up. review of the literature was performed in the medline database using the search terms trigeminal, encephalocele, meckel's cave, sphenoid, pterygopalatine, csf fistula, and sternberg in all combinations of two keywords. search ed in 38 relevant cases from 23 reports, ranging from 1888 to 2011 (table 1). within these 38 reports, patients presented with rhinoliquorrhea in 92.1% (35 patients), headaches in 34.2% (13 patients), and meningitis in 15.7% (6 patients). abbreviations: csf, cerebrospinal fluid; lsm, lateral sphenoid meningocele; sc, sternberg cruveilhier canal; sof, superior orbital fissure; tm, trigeminal meningocele. out of 38 patients, 6 had a tm, whereas a persistent sc or lsm was found in 32 patients. a total of 29 patients underwent endoscopic surgery for lsms, whereas in 3 cases the transcranial approach was preferred. in two cases4 transcranial reoperation was performed after endoscopic surgery due to recurrence of csf leaks.4 recurrence rate was 13.7% (4 of 29 cases). from the six tms, three were treated by an endoscopic transsphenoidal approach, with recurrence of csf leaks in two of them (66%). one of three patients undergoing craniotomy and open repair experienced recurrent therapy - resistant csf leakage (present case, 33%). apart from persistent rhinoliquorrhea, the most common complications in the endoscopic group were meningitis (6.3%) and maxillary nerve irritation (3.1%). in the craniotomy group, the case presented here illustrates a rare tm extending from an enlarged meckel cave into the medial cranial and pterygopalatine fossa, causing therapy - resistant csf leakage and orbital affection with an unusual collection of csf in the temporal muscle (fig . although the predominant symptoms of csf leaks such as rhinoliquorrhea, headache, and meningitis do not help in localizing the dural defect, clinical appearance may vary with the localization of the specific arachnoidal cyst, meningocele, or encephalocele . for example, arachnoid cysts of the region of the meckel cavity often become symptomatic with facial numbness or pain due to their relation to the trigeminal nerve.21 the therapeutic difficulties of that unusual entity have not been solved . in contrast to the far lateral tms, lsms and persistent sc can be treated successfully with a transsphenoidal approach . this may be caused by their more medial localization, medially to the foramen rotundum and v2 at the sphenoid base . patients with tms are likely to have a greater benefit from a transcranial approach, which provides better access to the lateral aspects of the meckel cave and the semilunar ganglion . this is underlined by the higher recurrence rate when using the transsphenoidal approach ( table 1). alternatively, transmaxillar transpterygoid approaches may be employed for tms with a limited pterygoid fossa leakage.1 csf leaks associated with tms can be assumed to be of idiopathic origin with an elevated intracranial pressure (icp) as an additional factor. thus, icp recording and icp normalization should be taken into account, and routine use of lumbar drainage as a diagnostic and therapeutic measure has been proposed in lateral sphenoid csf leaks.22 in light of the reviewed literature, together with the presented therapy - resistant case, alternative strategies such as implantation of a vp shunt or temporary lumbar drainage to reduce csf pressure have to be discussed. the occurrence of the spontaneous csf leak as part of an idiopathic hydrocephalus syndrome should be considered, with acetazolamide or furosemide being a treatment option.22 however, the one case treated conservatively experienced persistence of symptoms, similar to our patient, who experienced recurrence after deciding for nonsurgical follow - up (table 1). midfacial degloving has been suggested as approach to the pterygopalatine fossa meningoceles and remains an option for recurrence.21 a staged treatment algorithm using lumbar csf drainage, icp recording, and invasive location of the skull base defect with intrathecal administration of contrast medium seems to be most promising. depending on the location and anatomical shape of the tm, open craniotomy or endoscopic transpterygoid approach can be selected.6 7 8 22 whereas for medial pathologies (sc, lsm) endoscopic transsphenoidal approaches appear to be advantageous, tms should be primarily considered for an open craniotomy. alternatively to transcranial approaches, transfacial or transmaxillary / transpterygoid approaches have to be considered as treatment for recurrent, extended tms involving the pterygopalatine fossa, in which transsphenoidal endoscopic techniques have limited success rates. in case of therapy - refractory csf leaks and marked elevation of icp, vp shunt placement and/or medical treatment should be considered before reintervention.22 compared with the spontaneous rhinoliquorrhea caused by a persistent sc or lsms, the point of leakage in tms lies laterally to v2 and can not always clearly be visualized, even by sophisticated neuroradiological techniques. this can in considerable technical difficulties and therefore therapeutic decisions should follow the advice of a multidisciplinary skull base team after taking into account the individual anatomic situation of a patient, preceding operations, and specific risk factors for elevated icp.	trigeminal meningoceles, lateral to the maxillary nerve (v2), have seldom been reported as underlying pathology for spontaneous rhinoliquorrhea. in contrast to sphenoid meningoceles arising from a persistent lateral craniopharyngeal canal (sternberg cruveilhier, medial to v2), their occurrence seems to be generated by addition of erosive processes to the constitutively thin bony shell underneath the semilunar ganglion, lateral to the round foramen (and v2).the developmental and anatomical relationships of trigeminal meningoceles to the sphenoid bone are depicted, and in a review of the literature we present the different surgical approaches employed for sealing the dura leak. in view of these techniques we discuss an unusual case of therapy - resistant rhinoliquorrhea with left - sided trigeminal meningocele involving the meckel cave at the lateral sphenoid and reaching the superior orbital fissure and the medial orbital space.in contrast to patients who have lateral sphenoidal meningoceles with a persistent lateral craniopharyngeal canal (sternberg cruveilhier), who can be treated successfully using an endoscopic transsphenoidal approach (recurrence rate 13.7%), the recurrence rate of cerebrospinal fluid (csf) efflux for trigeminal meningoceles lies much higher (endoscopically 66%, open craniotomy 33%). the surgical strategy thus has to be chosen individually, taking into account specific anatomical situations and eventually preceding operations.
the palate is a complex area of the mouth, with a variety of native tissue types that give rise to a plethora of pathological conditions, both benign and malignant. they are generally classified as either hodgkin lymphoma or non - hodgkin lymphoma (nhl) and may be of either b - lymphocyte or t - lymphocyte origin. lymphoma is the second most common neoplasm of the head and neck region after squamous cell carcinoma. nearly 24%48% of nhl can arise in extranodal locations, and 3%5% are primarily located in the oral cavity. differential diagnosis of palatal swellings oral lymphomas are relatively rare and are often difficult to diagnose as they may mimic other pathologies such as periodontal diseases, osteomyelitis, or some other malignancy. lymphoid lesions of the palate can be divided into three categories, the management and prognosis of each category being different: primary lymphoma of the palate, with no other lymphomatous lesion detected elsewhere in the bodylymphoma of the palate occurring as one of the lesions in a case of disseminated lymphomabenign lymphoid hyperplasia (blh) of the palate primary lymphoma of the palate, with no other lymphomatous lesion detected elsewhere in the body lymphoma of the palate occurring as one of the lesions in a case of disseminated lymphoma benign lymphoid hyperplasia (blh) of the palate in addition, histologically, lymphoid lesions of the palate may be misinterpreted as inflammatory in nature. the purpose of our report is to present a case of b - cell lymphoma on the palate and distinguish it from benign lymphoid hyperplasia (blh). a 40-year - old man with a swelling in the right palatal region was referred to the department of oral pathology for evaluation and diagnosis. intraoral examination exhibited a firm, exophytic, oval mass with an intact overlying mucosa in the region of the right hard palate measuring 3 4.5 cm in size. there were no signs of ulceration, bleeding, discharge, or numbness in the area. the patient did not have the habit of chewing tobacco or betel nut. on general examination , he was found to be afebrile, with no palpable lymph nodes in the head and neck region. he did not mention any sudden weight loss in the recent past and the medical history was noncontributory. clinical photograph showing swelling on the palate ct scan revealed a mass on the right side of the hard palate, with no involvement of the maxillary sinus. computed tomography scan of patient an excisional biopsy was performed under local anesthesia. and a bony crater - like defect was seen on the palatal bone after soft tissue removal. histopathological examination of sections of the resected specimen revealed an intact stratified squamous epithelium with underlying vaguely follicular and diffuse proliferation of lymphoid cells. the follicle - like structures were composed of central large cells (giving a washed - out appearance) surrounded by a thin rim of small, round lymphocytes. the central large cells had abundant pinkish cytoplasm and convoluted nuclei with inconspicuous nucleoli. epimyoepithelial islands were also observed. on microscopic examination of hematoxylin and eosin (h and e)stained sections , there was a conflict of opinion over the distinction between benign (reactive) lymphoid hyperplasia (pseudolymphoma) and non - hodgkin lymphoma. the large lymphoid cells showed immunoreactivity for cd20 and the rim of small lymphocytes were positive for cd5. the large lymphoid cells were negative for cd5, bcl-2, and cd10. photograph of gross excisional tissue (a) the overlying epithelium is seen separated from follicles. the histological picture in this view gives the impression of reactive follicular hyperplasia of the lymphoid tissue (h and e, stain ; original magnification, 2.5). (b) high - power view of the follicular pattern gives the impression of a reactive lesion (h and e, stain ; original magnification, 10) magnified view showing central larger cells with mitotic figures, convoluted nuclei, and inconspicuous nucleoli. peripherally, small round lymphocytes are seen (h and e, stain ; original magnification, 20) the large lymphoid cells show positive immunoreactivity for cd20, whereas the peripheral small lymphocytes are negative. inset shows control stain (original magnification, 10) the peripheral small lymphocytes show positive immunoreactivity for cd5, whereas the central large cells are negative. inset shows control stain (original magnification, 10) the peripheral small lymphocytes show positive immunoreactivity for bcl2, whereas the central large cells are negative. inset shows control stain (original magnification, 10) negative immunoreactivity for cd10 (original magnification, 10) immunohistochemistry profile showing very low ki67 proliferative index (original magnification, 20) the patient was referred to a general physician for systemic evaluation and was found to be free of other systemic manifestations. a final diagnosis of low - grade b - cell lymphoma of mucosa - associated lymphoid tissue (malt) was accorded based on the clinical, radiographic, histopathologic, and immunohistochemical investigations. lymphoma constitutes a diverse and complex group of malignancies of lymphoid histogenesis. according to the revised european american classification of lymphoid neoplasms (real), classification the category of marginal zone b - cell lymphoma includes lymphomas that were originally designated as monocytoid b - cell lymphoma and low - grade b - cell lymphoma of malt. both these neoplasms were thought to represent different clinical presentations of a b - cell lymphoma believed to arise from normal marginal zone b - cells in the lymph node or their extranodal counterparts, respectively. the most frequent location of extranodal lymphoma in the head and neck is the palate. many salivary gland lymphocytic infiltrates of the palate are usually non - hodgkin b - cell lymphomas of malt. extranodal marginal b - cell lymphomas of malt (b - malt) are known to show histologic features similar to benign lymphoid hyperplasias (blh). in the past, many low - grade lymphomas were probably misdiagnosed and inappropriately categorized as blh. extranodal infiltration composed of small round or slightly irregular lymphoid cells often admixed with plasma cells, histiocytes and lymphoid follicle were classified as pseudolymphomas since clinical studies showed that in patients with these lesions, the disease pursued an indolent clinical course. the term pseudolymphoma / benign lymphoid hyperplasia should be restricted to tumefactive lesions with prominent lymphoid infiltrate that by routine morphology appears to be reactive and that on ancillary immunophenotypic and molecular genetic analysis lacks evidence of clonality. the subtle distinctions in interpretation of the histological characteristics between benign and malignant lymphoid proliferations makes the diagnosis of these lesions difficult. several papers have dealt with the histologic features of blh and nodular lymphoma. fortunately, with the advent of advanced immunophenotyping and molecular genetic techniques, it is now possible to differentiate the two lesions. table 2 lists out the essential features that help us distinguish blh from b - cell malt lymphomas. lymphoid hyperplasia vs b.cell malt lymphoma blh is characterized by numerous well - demarcated germinal centers of varying size and shape often rimmed by a mantle of small lymphocytes. while many b - cell lymphomas have a nodular growth pattern, these nodules are neoplastic and should not be considered germinal centers. in addition, in lymphomas, these neoplastic nodules tend to be ovoid to round and show little variation in size. in blh , the cells demonstrate all stages of follicular center cell transformation, including plasma cells. mitosis may be abundant, although never atypical and never seen outside the germinal centers. the cells comprising malignant lymphomas, on the other hand, are neoplastic and do not show the wide range of cell types seen in reactive lesions. nuclear atypia as well as mitosis (occasionally atypical) may be seen, but the mitotic rate rarely approaches that seen in benign lesions. in our case, the follicle - like structures showed variation in size and were composed of central large cells surrounded by a thin rim of small, round lymphocytes. mitotic activity was found to be quite low. hence, it was quite difficult to give a confirmative diagnosis solely on the basis of routine histopathologic examination. therefore we performed immunohistochemical analysis on the lesional tissue for the following markers: cd20, cd5, cd10, bcl2, and ki-67. the was positive for cd20 and negative for cd5, cd10, and bcl2, while ki-67 showed positivity of 1%. immunophenotypic studies have shown that low - grade b - cell lymphomas of malt typically do not express the b cell - associated antigens cd10, cd21, or cd23, or the t - cell antigens (including cd5). these findings were consistent with the immunohistochemistry profile of our case and thus confirmed our diagnosis of low - grade b - cell lymphoma of malt. patients with localized low - grade b - cell lymphomas follow a very indolent clinical course. therapeutic strategies for this kind of disease are not standardized yet due to the small number of malt lymphomas described in the head and neck region. remedial measures include combined radiochemotherapy for advanced cases and either radiation therapy or surgery for small lesions. our patient underwent surgical excision of the lesion and continues to be well after 6 months of follow - up. a number of factors can make the diagnosis of oral lymphoma difficult, despite the fact that the histological features are the same as at other sites. many lymphomas are extranodal and there is almost always a prominent superimposed, nonspecific, inflammatory response. clinically obvious lymphomas present primarily to the medical rather than the dental practitioner or oral surgeons. hence to see a spectrum of lymphomas in oral pathology practice is unusual. early recognition and biopsy are extremely important because the lymphoma may be confined entirely to the palate in the early stages and such localized palatal lymphomas respond well to irradiation, whereas disseminated disease necessitates chemotherapy. malignant b - cell lymphoma mimics blh, both clinically and histologically. the pathologist must be familiar with the features that distinguish between these two diseases.	diagnosis of palatal swellings is a challenge. benign and malignant tumors may be misinterpreted as lesions of inflammatory origin. we present a case of b - cell non - hodgkin lymphoma on the palate of a 40-year - old male. a number of factors can make the diagnosis of oral lymphoma difficult. many lymphomas are extranodal, there is usually a prominent superimposed nonspecific inflammatory response and thus they mimic benign lymphoid hyperplasia. it is important for the pathologist to be familiar with features that distinguish benign from malignant lymphoid proliferations.
clear cell odontogenic carcinoma (ccoc) is a rare jaw tumor that was first described by hansen et al.1 in 1985, in which patients presented with local bony invasions without metastasis. at first, ccoc was called a clear cell odontogenic tumor or clear cell ameloblastoma, and was considered more aggressive than ameloblastoma. the potential for ccoc to metastasize was initially unclear; however, ccoc was defined as a benign tumor in the 1992 world health organization (who) classification2. the who reclassified ccoc as a malignant tumor of odontogenic origin in 20053 because of its aggressive and destructive growth capacity and metastasis to the lungs and lymph nodes4,5,6,7,8. about onethird of ccoc cases were initially misdiagnosed, and the primary diagnosis was ameloblastoma which lead to inadequate treatment for some patients. in this case report, we describe a case of a ccoc, which presented in a similar manner to a cystic lesion; the patient was misdiagnosed and received insufficient treatment. we introduce this case to discuss differential patient presentations to reduce the rate of malignancy misdiagnosis. a 66-year - old female visited the department of oral and maxillofacial surgery at yonsei dental hospital due to swelling and pain on the right premolar maxillary area. clinical examination showed fluctuating swelling on the right maxillary vestibule with tenderness to palpation, severe tooth mobility and loss of vitality on the first and second premolars and no appreciable cervical lymphadenopathy. a panoramic radiograph revealed an approximately 2729 mm well - defined radiolucency in the apex of the maxillary right premolars with root resorption.(fig . 1) a computed tomography (ct) scan showed a low attenuated cystic lesion with a volume of 7,419 mm (simplant software ; materialise, leuven, belgium).(fig . 2) at first, the lesion was diagnosed as infected odontogenic cyst (radicular cyst), the patient received decompression in order to separate the cyst from maxillary sinus wall. two months after decompression, a panoramic radiograph showed that the lesion size was reduced to approximately 2526 mm.(fig . 3) the lesion was reduced to a volume of 4,797 mm (simplant software) on the 3-month follow - up conebeam ct.(fig . 4) the measurement and comparison may not be precise because of the differences in ct imaging; however, there was definite shrinkage of the lesion.(fig . 5) because there was not a change in the lesion size, a cyst enucleation was performed and specimens were sent to oral pathology. b ) magnetic resonance imaging of the neck and positron emission tomography ct, were taken to examine the lymph nodes and distant metastasis.(fig . an obturator was placed at the time of surgery and a 2.42.0 cmsized ccoc with a positive basal resection margin, massive bone marrow infiltration and lymphovascular permeation was reported.(fig . nearly 100 cases of ccoc have been reported in the englishlanguage literature to date, and these tumors have a higher reported incidence in females with an male / female ratio of 1 : 1.8 . most cases of ccoc have been diagnosed in patients over the age of 40, with an average age of 54 years . the mandible is more frequently involved than the maxilla, and the posterior of the jaw is a more frequent site than the anterior site9,10,11 . radiographic findings show mainly radiolucent lesions with or without regular margins, although some cases exhibited a mixed radiolucent - radiopaque lesion . the likelihood of the recurrence depended on the method of initial therapy and the extent of the tumor invasion . three - quarters of the patients who were treated with conservative care ( curettage or enucleation) had recurrence and onethird of the patients who were treated with resection experienced a recurrence. conservative care (curettage or enucleation) and presence of soft tissue involvement were associated with a higher recurrence rate10,12. the ideal treatment approach for ccoc a wide resection with at least 1 cm of a tumor - free margin is recommended and when there is evidence broad soft tissue invasion, palpable neck lymph node, perineural invasion or tumor removal without free margins, adjuvant neck dissection and/or radiotherapy should be considered10,12,13. however, except for squamous cell carcinoma and high - grade central mucoepidermoid carcinoma, neck dissection is rarely required for maxillary carcinomas without nodal metastases12,13,14. in our case, the patient had swelling, incision and drainage history and a well - defined unicystic radiolucent lesion, that was comparable with a cystic lesion. at first we misdiagnosed the lesion as an infected cyst, performed decompression and were unable to send specimen for pathologic examination. our treatment was limited because we did not make a correct diagnosis when the patient first presented at our clinic. when decompression was performed, the clinical findings were similar to a cystic lesion and during follow the up period we found that lesion size decreased. currently, mass properties have not been described in previous reports, and our case study presented with both radiographic and clinical similarities to cystic lesion symptoms; for these reasons our diagnosis was delayed. additional reports have indicated that many cases of ccoc are misdiagnosed because of its rarity and similarity to cystic lesions11,13. in this report , we discuss a patient that presented with a painful cystic lesion, jaw enlargement jaw and loosening teeth. similar cases should be considered for the possibility of malignant ccoc to identify and treat patients with ccoc.	clear cell odontogenic carcinoma (ccoc) is a rare jaw tumor that was classified as a malignant tumor of odontogenic origin in 2005 by the world health organization because of its aggressive and destructive growth capacity and metastasis to the lungs and lymph nodes. we report a case of a 66-year - old female who had swelling, incision and drainage history and a well - defined unicystic radiolucent lesion that was comparable to a cystic lesion. at first , the patient received decompression, and the lesion size decreased. three months after decompression, cyst enucleation was performed. the pathologic indicated that the lesion was ccoc. in this report we emphasize that patients with painful cystic lesions in addition to jaw enlargement and loosening teeth should be considered for the possibility of malignancy.
according to the international agency for research on cancer, there were an estimated 482,000 incident cases of esophageal cancer (ec) with high mortality (84%) around the world in 2008 1. ec often infiltrates neighboring organs and easily metastasizes to lymph nodes, and as a , the prognosis of locally advanced patients is extremely poor, with a 5-year survival of 15 - 34% 2. more recently, preoperative chemoradiotherapy (crt) has gained popularity with physicians, as its tolerance is better than postoperative crt, permits downstaging and higher respectability in the subsequent surgery, and may eradicate occult distant disease. several randomized clinical trials have testified a significant survival benefit for neoadjuvant crt in patients with squamous - cell carcinoma or adenocarcinoma of the esophagus 2 - 4. 5 recently conducted a meta - analysis, which included 24 clinical trials and 4188 patients with resectable esophageal carcinoma. the showed that neoadjuvant crt provided an 8.7% absolute survival benefit at 2 years after surgery alone and 5.1% survival benefit after neoadjuvant chemotherapy. a proportion of patients show minor or no response to crt and are merely exposed to its toxicity. furthermore, neoadjuvant treatment produces a pathological complete response, and outcomes are better 6. one retrospective study from stahl et al. 7 showed that the overall survival was significantly hampered in patients with residual tumor in their resected specimen compared with patients who showed a pathological complete tumor remission (overall survival rate at 3 years 25.2% versus 65.6% ; hazard ratio = 3.50 ; 95% confidence interval 1.91 - 6.44 ; p < 0.0001) it is vital to select patients who will experience survival benefit from receiving neoadjuvant crt before treatment. furthermore, several studies have reported that surgery can be omitted in patients that achieved pathological complete response (pcr) after neoadjuvant crt 8, 9. in this article , we summarize the recent literature with respect to the predictive factors of neoadjuvant crt response and provide a review the progress of the field and future challenges to be expected. predictive biomarkers and functional imaging have become a hot technique for the individualized treatment of cancer. however, traditional clinical factors, such as tumor stage, patient age, and performance status, are still used to select the best therapy for a particular patient. 10 initially found that tumor invasion depth was the only clinical factor significantly correlated with response to preoperative chemotherapy for thoracic esophageal squamous cell carcinoma (escc). 12 identified that age, baseline hemoglobin level, smoking habit, and tumor length were important pcr predictors in escc. in patients with esophageal adenocarcinoma, patel et al. 13 found that signet ring cell histology on pretreatment biopsy predicts a decreased likelihood of pcr and survival. 14 created a regression classification based on two parameters (histomorphologic tumor regression and postoperative pathological node stage) to predict the complete resections following neoadjuvant crt for ec patients. the following parameters were incorporated into this model: post - chemoradiation positron emission tomography (pet) standardized unit value (suv), post - chemoradiation biopsy, sex, histologic tumor grade, and baseline endoscopic ultrasonography tumor stage. the area under the receiver - operating characteristic curve was 0.72 (95% ci : 0.662 - 0.787). this model needs to be prospectively validated before it can be used in clinical practice. serum c - reactive protein (crp) as an inflammatory factor has also been evaluated in the treatment response prediction of ec patients. 16 was the first to demonstrate that serum crp levels during crt were closely associated with the pathological response, particularly in patients with elevated crp prior to crt, a decrease in crp within normal ranges 2 - 3 weeks following crt initiation predicted a favorable pathological response with the highest accuracy. a large spectrum of biomarkers at the level of alterations of genomic dna, gene expression of messenger rna (mrna), micro - rna (mirna), and protein expression have been identified and analyzed to predict the response of neoadjuvant therapy. cisplatin (cddp) and 5-fluorourcil (5-fu) based neoadjuvant chemotherapy have been widely used in clinical practice. the excision repair cross - complementing 1 (ercc1) gene codes for a nucleotide excision repair protein involved in the repair of radiation- and chemotherapy - induced dna damage. 18 testified that ercc1 mrna expression and ercc1 (rs11615) gene polymorphisms correlated with treatment response to cddp - based chemotherapy. furthermore, rna expression levels of 5-fu metabolism - associated genes, thymidylate synthase (ts), dihydropyrimidine dehydrogenase (dpd), thymidine phosphorylase (tp), methylenetetrahydrofolate reductase (mthfr), as well as of cddp and taxane - related genes gluthatione s - transferase (gstp-1), caldesmon, and multi - drug resistance gene (mrp1) have been testified to be predictors of response to neoadjuvant therapy 19 - 22. mirnas are small noncoding rnas, which are involved in the regulation of gene expression by inhibiting messenger rna translation 23. 24 conducted a comprehensive mirna profiling in 16 specimens with pre - neoadjuvant and post - neoadjuvant therapy, and the selected mirnas were verified in 80 ec patients. the showed that mir-192 and mir-194 in pre - therapeutic biopsies are considered indicators of major histopathologic regression. moreover, in vitro assays showed that mir-296 and mir-200c expression correlated with chemotherapy resistance 25, 26. furthermore, mir-148a has been reported to improve response to chemotherapy in sensitive and resistant esophageal carcinoma cells 27. long non - coding rnas (lncrnas) are a new class of non - protein - coding rnas, which are longer than 200 bases 28. 29 initially explored the relationship between lncrna loc285194 and the response to neoadjuvant crt in escc and showed that the decreased expression of loc285194 indicated crt resistance and poor prognosis. other biomarkers, such as epidermal growth factor receptor (egfr), vascular endothelial growth factor (vegf), proliferating cell nuclear antigen (pcna), p53 status, p21 status, bcl-2, ki-67, transcription factor nuclear factor kb (nf - kb), and rad51 have been shown to correlate with response to neoadjuvant therapy in ec 30 - 39 (table 1). 30 adopted the proximity ligation assay (pla) followed by enzyme - linked immunosorbent assay (elisa) to identify serum biomarkers that predict treatment response of neoadjuvant crt in 79 escc patients. both methods testified that low pretreatment serum vascular endothelial growth factor - a (vegf - a) significantly correlated with pcr. however, the predictive value of transforming growth factor (tgf)-1 was not validated by elisa. 22 also revealed that ts and dpd rna expression in the peripheral blood of ec patients could be highly specific predictors to identify a subset of patients who do not benefit from neoadjuvant chemoradiotherapy. it has been reported that chemoradiotherapy induces cancer cell death through tumor antigen - specific t cell response 40. 41 conducted serum profiling of 84 cytokines in escc patients who received neoadjuvant crt plus surgery and revealed that increased serum soluble interleukin-6 receptor was correlated with a poor response to preoperative therapy. it is recognized that multiple gene alterations are involved in the development and progression of ec 42. markers originating from different molecular levels, such as gene expression, mrna expression, protein expression, epigenetic modification, and mutation, have always been validated independently in separate studies. given that tumor cells interact at different levels in the organism, which interferes with angiogenesis, dna repair and apoptosis, cell cycle control pathways, or cell - to - cell communication pathways, analysis of one pathway alone can not cope with the complexity of the interacting tumor cells. whole genome microarray technology allows for high - throughput identification of gene expression profiles in cancers 43. this approach had already been used to identify genes that could serve as biomarkers of neoadjuvant crt response prediction. table 2 summarizes the whole genome profile - related studies regarding the neoadjuvant crt response prediction. 44 initially identified a combination of three differentially expressed genes (perp, s100a2, and sprr3) that allowed for the discrimination between pcr and < pcr with sensitivity and specificity of 85% after profiling pretreatment cancer biopsies from 19 ec patients that received neoadjuvant crt. 45 performed cdna microarrays of 46 pretreatment endoscopic biopsy samples and identified a 32-gene classifier that can be used to predict the response to crt in escc. 46 performed gene expression profiling on pretreatment samples of escc patients who received chemotherapy, and constructed a diagnostic system with 199 most informative genes that showed 82% accuracy. 47 proved that the ephrin b3 receptor, a differentially expressed gene via microarray, is related to the neoadjuvant chemotherapy response. 48 identified two novel markers, cul2 and stk11 using human genome microarrays, for response prediction in ec. 49 established a five gene based model that predicted the response to neoadjuvant crt with 95% accuracy in 74% of ec patients. 50 also performed gene expression profiling on pretreatment biopsies from 28 esccs who received neoadjuvant crt in a phase iii clinical trial and developed a prediction model based on three genes (mmp, limch1, clorf226) with 81% accuracy in the validation cohort (table 2). first, most markers presented within this review were mainly generated by focusing on relatively small cohorts within retrospective analyses. the are mostly preliminary and require further validation. for popularization and application of these biomarkers, large prospective trials are warranted. finally, the crt or chemotherapy response must be evaluated pathologically. conventional imaging modalities (endoscopy, endoscopic ultrasonography, computed tomography, and magnetic resonance imaging) can not reliably differentiate between viable tumor and inflammatory reactions, edema, and scar tissue 51, 52. positron - emission - tomography with the glucose analog fluorodeoxyglucose (fdg - pet) is a functional imaging modality that can detect changes in tissue metabolism. current evidence has shown that tumor metabolic activity has been proven to correlate with histopathologic response in ec 53. however, pet - based parameters to stratify prognosis in the literature has varied from different studies, including pre - radiation standard unit value (suv), post - radiation suv, a percentage decrease of suv, pet - tumor length, and pet - tumor volume based parameters. the pretreatment pet evaluation is quite important in developing a strategy to identify the value of therapy in its early stage. 54 showed that pretreatment suv is a reliable predictor of response to definitive crt in escc. 55 also demonstrated that an initial suv higher than the median (10.1) was associated with a better pathologic response. more attention has been paid to the dynamic changes of suv and the timing of post - radiation fdg - pet imaging. several studies testified that the decrease of suv post - neoadjuvant therapy or preoperatively can be useful for predicting pathologic response 58 - 61. however, swisher et al. 56 found that post - crt suv was predictive of pathologic response with a relatively low specificity. this high false positive rate may be due to the inflammatory changes following radiotherapy, which leads to falsely elevated suv values because of the presence of metabolically active leukocytes and macrophages. therefore, the timing of rechecking fdg - pet in the course of therapy may be critical because the false positive rate appears to decrease with time 57. to address this question, wieder et al. 58 studied the time course of changes in tumor fdg - uptake in patients with escc patients treated with preoperative therapy. the showed that metabolic changes within the first 2 weeks of therapy are slightly better predictors compared with later changes. this observation is most likely related to the complex proinflammatory and anti - inflammatory effects of radiation, which are strongly dependent on time and dose. the authors conceived that the cytotoxic effects of radiotherapy on radiosensitive cells, such as lymphocytes, limit the intensity of inflammatory reactions in the tumor tissue during and early after completion of therapy. additionally, in consideration of the application of suv in clinical practice, relative changes are better predictors of crt response than absolute suvs, as absolute suvs are much more sensitive to differences in data acquisition, image reconstruction, and data analysis than relative changes 59. except for the suv value, other pet image - derived parameters, such as tumor longitudinal length (tl) and volume (tv) and total lesion glycolysis (tlg = tv suv mean), 62 investigated the predictive value of baseline fdg - pet image - derived parameters regarding therapy response in ec patients. for study purposes, the tumor was automatically delineated on the baseline pet image using an adaptive threshold and the automatic fuzzy locally adaptive bayesian (flab) methodologies to attract the tl, tv, suv, and the derived tlg values. the showed that commonly used parameters, such as suvs, were not significant predictive factors of the response; parameters related to tumor functional spatial extent (tl, tv, tlg) could significantly differentiate histological response with sensitivity above 75% and specificity above 85%, regardless of the functional volume delineation strategy. 63, who revealed that baseline mtv and tlg were not found to be predictors of response to neoadjuvant therapy in ec patients, although a trend towards a correlation between response to crt and smaller mtv was observed. this discrepancy may be related to the limited accuracy and reproducibility of the available tumor delineation methods, small sample size, and different response evaluation criteria in both studies. more recently, the spatial - temporal fdg - pet has gained popularity with physicians, which offers more information, including intensity features, texture features (spatial patterns), geometry features, and geometry - intensity features (total glycolytic volume), compared to the conventional pet measures with suv 64. 65 initially built a predictive model using multiple, comprehensive tumor response measures, including conventional fdg - pet measures, clinical parameters, and demographics, and spatial - temporal fdg - pet features. this model achieved very high accuracy (100% sensitivity and 100% specificity) for prediction of pathologic tumor response to crt in 20 patients with ec. this model needs to be validated with a large and prospective patient cohort. in , conventional fdg - pet image - derived parameters, especially the relative changes of suv values, have been proven a significant predictor of treatment response. furthermore, spatial - temporal fdg - pet offers more information about the intensity, texture, geometry, and geometry - intensity features compared with the conventional pet measures with suvs and will be useful for differentiating the responders to neoadjuvant crt. nevertheless, there are significant issues to be resolved with regard to the standardization of pet imaging protocols, image - processing methods and the time point for repeat imaging. diffusion - weighted magnetic resonance imaging (dwmri) is also a functional imaging that is based on the microscopic random translational motion of water molecules in biological tissues. the magnitude of this motion is characterized by its apparent diffusion coefficient (adc) values. recently, a few studies have been published to evaluate the efficacy of adc values in predicting neoadjuvant crt response. 66 analyzed the pretreatment adc values of 80 patients with escc and found that an adc value of 1.10 10 mm /s can differentiate crt responders from non - responders (a high adc group responded better to crt than did a low adc group) with a sensitivity, specificity, and accuracy of 73.8%, 86.8% and 80.0%, respectively. similarly, imanishi et al. 67 reported that the adc at the time of 20 gy and the increased rate of the adc at the time of 20 gy were significant predictors of treatment response in locally advanced escc. computed tomography perfusion (ctp) images can quantify tumor vascularity by measuring the temporal changes in tissue attenuation following intravenous contrast administration, which has been reported to associate with tumor characterization, survival, and therapy response in ec 68 - 70. the tumor blood flow was closely related to tissue oxygen status and tumor microcirculation, which was demonstrated to be an important factor for determining chemoradio - sensivity. thus, ctp parameters will provide an important insight into the individualized treatment of ec. published data demonstrate that clinic - histopathological factors, molecular biomarkers, and functional imaging are predictive of neoadjuvant therapy. these clinical factors and biomarkers need to be further validated and novel biomarkers warrant additional exploration. fdg - pet image - derived parameters, especially the relative changes of suv values, have been proven a significant predictor of treatment response. nevertheless, there are significant issues to be resolved with regard to the standardization of pet imaging protocols, image - processing methods and the time point for repeat imaging. in addition, it is noteworthy that there is currently no comprehensive clinical study that incorporates clinical factors, biomarkers, and functional imaging to identify patients that may benefit from receiving neoadjuvant therapy. therefore, a predictive model based on these factors needs to be established with a large, prospective, and homogeneous patient cohort in the near future. standardization of staging, biomarker detection method, and image acquisition protocol will be critical for the generalization of this model. aside from these outstanding discoveries, prospective, multi - center controlled trials, which stratify patients according to these predictive factors, will help guide individualized treatment strategies for patients with ec.	currently, the most promising strategy to improve the prognosis of advanced esophageal cancer is neoadjuvant chemoradiation (crt) followed by surgery. however, patients who achieved pathological complete response can experience more survival benefit. therefore, it is critical to identify the responders early in the course of treatment. published data demonstrate that clinic - histopathological factors, molecular biomarkers, and functional imaging are predictive of neoadjuvant therapy. the existing biomarkers, including epidermal growth factor receptors, angiogenetic factors, transcription factors, tumor suppressor genes, cell cycle regulators, nucleotide excision repair pathway, cytokines, and chemotherapy associated genes, need to be validated and novel biomarkers warrant further exploration. positron emission tomography (pet) is useful for differentiating the responders of neoadjuvant crt. the most valuable parameters and the time point of performing pet in the course of treatment remains to be elucidated. furthermore, predictive models incorporating the multiple categories of factors need to be established with a large, prospective, and homogeneous patient cohort in the future. standardization of staging, biomarker detection method, and image acquisition protocol will be critical for the generalization of this model. prospective, multi - center controlled trials, which stratified patients according to these predictive factors, will help guide individualized treatment strategies for patients with esophageal cancer.
type 2 diabetes mellitus is a polygenic disorder that is caused by a metabolic and/or hormonal imbalance between insulin secretion from cells and insulin sensitivity in peripheral tissues, both of which might be modified by genetic and environmental factors. the decreased sensitivity to insulin leads to an increased requirement for insulin and is often associated with obesity in which metabolic disturbances are marked in insulin - target organs, such as the liver, muscle, and adipose tissues. obesity plays key roles in the pathophysiology of several metabolic diseases and is a risk factor for diabetes mellitus and for dyslipidemia. diabetic animal models play critical roles in the elucidation of the mechanisms of diabetes mellitus and the complications and in the development of novel drugs as treatments. based on the previously mentioned concept, a novel model of obesity - related diabetes they established a congenic line of the spontaneously diabetic torii (sdt) rat by introducing the fa allele of the zucker fatty rat into the sdt rat genome via the speed congenic method using a pcr technique with dna markers. they have normal body weights, blood glucose levels, insulin levels, and lipid levels until about 16 weeks of age and, thereafter, develop hyperglycemia associated with hypoinsulinemia, which from the degeneration of pancreatic beta cells. as a of chronic hyperglycemia, the sdt rats develop profound complications in eyes, peripheral nerves, kidneys, and bones. the fa / fa (sdt fatty) rats of both sexes became overtly obese and showed a significant hyperphagia. also, the bmi (body mass index) was greater in sdt fatty rats (mean value, 0.91 and 0.87 g / cm in males and females, resp .) than that in lean rats (0.75 and 0.58 g / cm in males and females, resp .) at 14 weeks of age. both visceral and subcutaneous fat weights were significantly higher in sdt fatty rats, but, especially, the visceral fat weight was markedly elevated with aging (table 1 and figure 1). serum glucose levels in sdt fatty rats of both sexes were elevated from 6 weeks, and lipid parameters such as serum triglyceride and total cholesterol levels in the rats were elevated from 4 weeks of age. the hyperglycemia and hyperlipidemia were sustained for a long time afterwards. the male sdt fatty rats showed hyperinsulinemia from 4 to 8 weeks of age, but after 16 weeks their insulin levels decreased to levels similar to those in sdt rats. in the female rats, hyperinsulinemia was shown from 4 to 12 weeks of age, and the insulin levels decreased gradually. also, a remarkable rise in renal parameters such as urine volume and urine protein was shown in sdt fatty rats of both sexes. effect of food restriction in sdt fatty rats was investigated. body weights of the pair - fed rats were similar with those of sdt rats. improvement of hyperglycemia or hypertriglyceridemia was observed, but hypercholesterolemia was not entirely improved (figure 2). the visceral / subcutaneous (v / s) fat ratio decreased in the pair - fed rats (mean standard deviation : control rats, 2.04 0.66 ; pair - fed rats, 1.28 0.19), although the total fat (visceral fat and subcutaneous fat) weight did not change (mean standard deviation : control rats, 137.5 52.5 g ; pair - fed rats, 135.2 10.4 g). cell size of the epididymal fat in the pair - fed rats tended to decrease, and glucose oxidation level in epididymal fat in the pair - fed rats was recovered to a similar level with that in sdt rats. in the glucose tolerance test conducted at 9 weeks of age, sdt fatty rats showed higher serum glucose levels after glucose loading without any response of plasma insulin. those impaired glucose tolerance and insulin secretion were deteriorated with aging. in pancreatic islets of female sdt fatty rats, pathological findings such as vacuolation, hypertrophy, and hemorrhage were observed from 8 weeks of age, and findings such as atrophy and fibrosis in the islets were observed from 24 weeks of age (figure 3). in sdt rats, glucose intolerance was observed in prediabetic stage. the histological features in the pancreas of sdt rats were as follows: in 1020 weeks of age, slight changes such as hemorrhage, hemosiderin deposition, inflammatory cell infiltration, and fibrosis in and around the islets; in 25 weeks of age, hemosiderin deposition, cellular infiltration with lymphocytes and macrophages, and fibrous tissue proliferation in and around the islets. nonfasted serum parameters, such as leukocyte count (wbc), erythrocyte count (rbc), hemoglobin (hb), and hematocrit (ht) level, were examined at 6 and 12 weeks of age. also, differential counts of leukocytes, such as neutrophils, eosinophils, basophils, monocytes, and lymphocytes, were determined, respectively. the levels were measured using an automatic analyzer (advia 120 hematology system ( siemens ag), erlangen, germany ). wbc and ht levels in sdt fatty rats were significantly higher as compared with those in sd rats at both 6 and 12 weeks of age. in differential counts of leukocytes, the monocyte count was significantly higher at 12 weeks of age in sdt fatty rats. it is reported that type 2 diabetes mellitus or obesity is a chronic inflammatory state aggravated by factors that promote inflammation at the level of vasculature and adipose tissue. since the monocyte count in wbc is elevated, in the future, it is necessary to investigate the inflammatory state in sdt fatty rats. rbc and hb levels in sdt fatty rats were comparable to those in sd rats at 6 and 12 weeks of age. we examined blood pressure, known to be a risk factor for metabolic syndrome, in sdt fatty rats. in male sdt fatty rats, blood pressure was significantly higher from 8 to 24 weeks of age, as compared with age - matched sd rats. furthermore, we confirmed a new insight regarding blood pressure in sdt fatty rats: a high sensitivity to sodium. a 1% nacl solution was given to male sdt fatty rats for 8 weeks, from 4 to 12 weeks of age. the systolic blood pressure in sdt fatty rats was significantly higher as compared with that in control - sdt fatty rats (mean standard deviation : nacl - sdt fatty rats, 200.2 22.0 mmhg ; control - sdt fatty rats, 137.2 13.0 mmhg). in other words, an elevation of blood pressure in the rats was more prominent after sodium loading. with early incidence of diabetes mellitus, diabetes - associated complications in sdt fatty rats were seen at younger ages compared to those in the sdt rats. in male sdt fatty rats, histopathological examination of the kidneys revealed changes in the glomeruli from 16 weeks and in the renal tubules from 8 weeks of age. in the glomeruli, glomerulosclerosis was observed from 16 weeks of age, and the sclerosis progressed with aging. nodular lesions were observed at 40 weeks of age. in the renal tubules, glycogen deposition in the tubular epithelium (armanni - ebstein lesions) and tubular dilation were noted from 8 weeks of age, and the change progressed from 8 to 16 weeks of age. in female sdt fatty rats, the female rats revealed changes in the glomeruli from 32 weeks of age, and in the renal tubules from 16 weeks, and the changes progressed with aging. furthermore, we investigated histopathological characteristics of the kidneys in male and female sdt fatty rats at 60 weeks of age. diffuse glomerulosclerosis, including increased mesangial matrix and glomerular hypertrophy, was severely progressed in the sdt fatty rats. moreover, tubular and interstitial lesions, including fibrosis and inflammatory cell filtration, were progressed in the sdt fatty rats (figure 4). histopathological findings in lens, including hyperplasia of epithelium, vacuolation of fiber, and occurrence of morgagnian globules, were observed from 8 weeks of age in male sdt fatty rats, and these changes progressed with aging. also, retinal lesions, such as folding and thickening, were observed with aging in male and female sdt fatty rats. diabetic peripheral neuropathy was evaluated at 8, 24, and 40 weeks of age in male sdt fatty rats. tail motor nerve conduction velocity (mncv) in the sdt fatty rat was delayed at 24 weeks of age and was further decreased at 40 weeks of age (figure 5). histopathologically, at 40 weeks of age, the fiber number was significantly decreased, and sdt fatty rats revealed significant atrophy in myelinated nerve. six - week treatment of pioglitazone, a peroxisome proliferator - activated receptor (ppar)- agonist, lowered blood glucose level and prevented delay of sciatic mncv in sdt fatty rats. we investigated the effects of obese type 2 diabetes on bone turnover, bone mass, and bone strength in sdt fatty rats. both serum osteocalcin, a bone formation marker, and urine deoxypyridinoline, a bone resorption marker, levels were lower in male sdt fatty rats compared to age - matched sd rats from 8 to 40 weeks of age (figures 6(a) and 6(b) ). the early onset of diabetes and obesity in male sdt fatty rats induced decreases in both serum osteocalcin and urine deoxypyridinoline and ed in low bone turnover at a young age, 8 weeks of age. male sdt fatty rats showed lower bone mineral density (bmd) and bone mineral content (bmc) of the whole tibia (figures 7(a) and 7(b) ) and shortening of the tibia and femur compared to age - matched sd rats. deterioration in bone geometrical properties of the femur midshaft, such as cortical thickness and minimum moment of inertia, was observed in male sdt fatty rats. furthermore, trabecular bone volume of the distal femur was lower in the sdt fatty rats. these negative effects on bone in the sdt fatty rats caused severe decreases in maximum load, stiffness, and energy absorption of the femur. in addition, serum levels of homocysteine, a candidate bone fragility marker, were elevated in male sdt fatty rats compared to age - matched sd rats (mean standard deviation : sdt fatty rats, 14.4 3.4 nmol / ml ; sd rats, 6.6 1.7 nmol / ml). we also investigated changes in bone metabolism and bone quantity at 10, 15, 25, and 40 weeks of age in female sdt fatty rats. serum osteocalcin and urine deoxypyridinoline levels were lower at 10 weeks of age in the sdt fatty rats compared to those in sd rats, but only the urine deoxypyridinoline levels were elevated in the sdt fatty rats from 25 weeks of age (figures 6(c) and 6(d) ). female sdt fatty rats showed lower bmc and bmd of the whole tibia from 8 to 25 weeks of age (figures 7(c) and 7(d) ). sdt fatty rat is a useful model to investigate bone abnormalities in obese type 2 diabetes. since sdt fatty rats (fa - homozygous) are infertile in both males and females, we used the sdt - fa/+ rats (fa - heterozygous) for reproduction. the reason the rats show infertility is unknown, but some functional disorders related to reproduction are observed. we monitored the estrus cycle by vaginal smear cytology between 12 and 17 weeks of age and compared them with normal sd rats used as control. sd rats showed a regular 4-day estrus cycle, whereas sdt fatty rats showed an irregular 4- to 5-day estrus cycle with persistent estrus stage and metestrus extension. weight of reproductive organs (ovary, uterus, and vagina) in each estrus cycle stage was measured at 12 weeks of age, and the organs were examined by histopathological analysis. relative weights of ovary, uterus, and vagina of sdt fatty rats were significantly lower than those of sd rats. in sdt fatty rats, histopathological changes, such as atrophy in uterus and inflammation in vagina, were observed (figures 8 and 9). irregular estrus cycle, increased leukocytes in vagina, or dysgenesis of reproductive organs might cause the infertility in female sdt fatty rats. moreover, testosterone levels in male sdt fatty rats tended to be lower as compared with those in sd rats at 8 and 24 weeks of age (mean standard deviation : sdt fatty rats, 212.2 84.4 pg / ml ; sd rats, 960.4 802.7 pg / ml ; at 8 weeks of age, resp .). since the testosterone levels in male sdt fatty rats are low, the reproductive function is considered to be decreased. in histological analysis of testis in the sdt fatty rats in further study, it is necessary to elucidate the mechanism of hypogonadism in sdt fatty rats. in 2004, dr. masuyama and dr. shinohara (research laboratories of torii pharmaceutical co., ltd ., japan) established the congenic type 2 diabetes model spontaneously diabetic torii fatty (sdt fatty) rat by introducing the fa allele of the zucker fatty rat into the genome of the original sdt rat. clea japan has received right of production and sales from japan tobacco inc. and has distributed the animals as sdt fatty rats since 2012. diabetes mellitus and diabetic complications in sdt fatty rats were found at a younger age than those in sdt rats. the early onset of diabetes or diabetic complication has advantages for the use of sdt fatty rats in diabetes research. furthermore, not only the male rats but also the female rats developed diabetes mellitus at a young age. female sdt fatty rat has the potential to become an important animal model of type 2 diabetes mellitus with obesity, especially in women, where few models currently exist. since sdt fatty rats showed a hypertension with obesity, hyperglycemia, and hyperlipidemia, the rat might have potency to be established as a metabolic syndrome model. also, it is interesting that sdt fatty rats showed various diabetic complications, such as osteoporosis and hypogonadism, with microangiopathy. use of sdt fatty rats will assist in the further elucidation of the pathogenesis of human diseases related to metabolic disorder and in discovery of new drugs.	spontaneously diabetic torii leprfa (sdt fatty) rat, established by introducing the fa allele of the zucker fatty rat into sdt rat genome, is a new model of obese type 2 diabetes. both male and female sdt fatty rats show overt obesity, and hyperglycemia and hyperlipidemia are observed at a young age as compared with sdt rats. with early incidence of diabetes mellitus, diabetic complications, such as nephropathy, retinopathy, and neuropathy, in sdt fatty rats were seen at younger ages compared to those in the sdt rats. in this paper, we overview pathophysiological features in sdt fatty rats and also describe new insights regarding the hematology, blood pressure, renal complications, and sexual dysfunction. the sdt fatty rats showed an increase of leukocytes, especially the monocyte count, prominent hypertension associated with salt drinking, end - stage renal disease with aging, and hypogonadism. unlike other diabetic models, the characteristic of sdt fatty rat is to present an incidence of diabetes in females, hypertension, and retinopathy. sdt fatty rat is a useful model for analysis of various metabolic disorders and the evaluation of drugs related to metabolic disease.
this short invited article reviews diffusion techniques used to infer sizes of structures in samples that lead to restricted diffusion. then it explains how the time dependence is used to infer sizes of structures, such as axon diameters, in the sample. it then discusses the limitations of current methods inferring small sizes and briefly explains one possible alternative to overcome these limitations. their mean square displacement depends on the diffusion time, , as described by einstein s relation < r > = 2d (in one dimension) where d is the diffusion coefficient.1 molecules diffusing in a uniform medium with no barriers experience unrestricted diffusion. in non - uniform media (eg, porous samples and cellular tissues) barriers restrict molecular displacements so that the diffusion depends on the time scale of the study and the permeability of the barriers. assuming einstein s relation, < r > during the time can be used to find an apparent diffusion coefficient (adc) that is less than d because of restrictions or hindrances to motion. understanding the influence of the restrictions or hindrances on the adc gives information about the geometry of the boundaries of the surrounding medium which, for mri, could be tissues, rocks, concrete, cement, polymers, gases, etc.2 as an example, the adc in a simple system consisting of molecules with diffusion coefficient d that are entirely restricted to a single pore of diameter a will depend on. for a/(2d), all molecules, regardless of their starting position, will be found anywhere in the pore.3 measurements as a function of thus provide information about the structure in which the molecules are diffusing. specifically, there will be a change in the adc when measuring through the time = a/(2d) which can be used to determine the pore size. traditional mr measurements of the adc in different samples use the pulsed gradient spin echo (pgse) sequence,4 see figure 1. after excitation, a magnetic field gradient pulse is applied to the sample for a short time. this causes the spins to obtain a phase based on their position at the time of the pulse. a rf pulse is then applied to the system which reverses the phase of the spins. another identical gradient pulse is applied to the sample which changes the phase of the spins based on their position at the time of the second pulse . if no diffusion has occurred, the phase acquired from the second pulse will be equal and opposite to the phase of the spin just before the pulse, ing in a net phase of zero. if diffusion occurs, the mean squared phase of all spins will be nonzero and cause a loss in mr signal which can be used to calculate the adc.4 alternatively, callaghan proposed q - space imaging using pgse with short gradient pulses to measure the conditional probability, p(rr,), that a spin initially at r has migrated to r over the time. by measuring the signal as a function of gradient strength, one can calculate the probability distribution of displacements of spins.5 using this probability distribution, one can infer from mr images tissue geometric information, such as axon diameter distributions and axonal and cellular volume fractions or densities.59 it is difficult, however, to find p in white matter because of the variability of axon diameter.2 in vivo measurements using echo planar imaging (epi) sequences are used to acquire images more rapidly.2 this allows full diffusion tensor imaging (dti) in vivo although the resolution is often not as good as standard imaging sequences.10 perfusion can also be measured using mri and the effects of perfusion on diffusion measurements must be taken into account.1114 many changing cellular structures, due to disease or injury, have already been shown to affect adc measurements. specifically for white matter imaging, some have measured changes in dti metrics with changing myelin content or myelin damage.2,1522 there are several methods for estimating axon diameter distributions and densities using mri and most use pgse with adc or q - space. one method, axcaliber, uses a framework that combines composite hindered and restricted models of water diffusion (charmed) and pgse measurements.23 this method uses one fixed gradient direction and multiple diffusion times and gradient strengths to make the charmed model more sensitive to axon diameter.8 it fits the mr signal to an equation which has components due to restricted diffusion, hindered diffusion, and later free diffusion.24 water in each axon size will experience restricted diffusion at different. for example, water in an axon with a small diameter will experience restriction for much smaller than water in a larger axon.2 thus by shortening , smaller and smaller axons move from the restricted component to the hindered component allowing the inference of axon diameter of smaller and smaller axons. the framework used in axcaliber for dividing the models into different types of diffusion was based on another model, which describes bovine optic nerve tissue as a three - compartment system, (axons, glial cells, and extracellular space). each compartment has its own diffusion coefficient, size, volume fraction, membrane permeability, and nmr relaxation times. it uses multiple s and gradient strengths in a pulse sequence similar to pgse (stimulated echo) which allows for longer diffusion times and measures the mean axon diameter, but not the distribution.25 another method, activeax, extended and optimized these methods to determine the accuracy and precision with which this important new biomarker, axon diameter, can be estimated in live human subjects.9 the work from this group combined a simplified version of charmed with high - angular - resolution diffusion imaging (hardi) and a model with a single axon diameter.9 it has been modified to be robust in the presence of orientation dispersion.26 the same group has also combined two - shell hardi with a three - compartment tissue model to create neurite orientation dispersion and density imaging (noddi).27 table 1 summarizes the literature for some of the axon diameter measurements made with mri. diffusion times used ranged from 7 to 305 ms measuring diameters from 0.414 m. measuring similar restriction sizes in porous objects at room temperature would likely need longer imaging times because of the slower diffusion at the lower room temperature compared to body temperature. measuring axon diameter distributions with mri, even with intact ex vivo brains, has advantages over traditional ex vivo histological techniques. ex vivo histological techniques are cumbersome, require tissue sectioning and are subject to inaccuracies such as cell shrinkage.9 with mri, the brain remains intact and images can provide measurements over large regions of the brain. because of the difficulties with ex vivo histological measurements of axon diameter distributions, the variation of fiber composition over the population and during development is largely unstudied.9 although fixation can affect samples, studies have been done to compare adc and other dti metrics between in vivo and fixed ex vivo samples.22,2830 while absolute values of adc change, partially due to the temperature of the sample, fractional anisotropy is similar.31 thus ex vivo measurements of axon diameters on intact fixed brains can still offer important information. the pgse sequence requires, however, that be large with respect to the restriction sizes in biological tissues, thus limiting the information that could be obtained from the measurements as will now be explained. the signal from a pgse sequence is given by ln (s / s0) = g (/3) d = bd, where is the gyromagnetic ratio of the hydrogen nucleus, is the duration of the gradient pulse, and g is the amplitude of the gradient pulse. in order to measure the diffusion coefficient, the difference in signal with, s, and without, s0, given that small s are desired, and < for pgse, the only remaining factor that can be increased is the gradient strength. with q - space imaging, the smallest displacements which can be probed depend on the largest q which can be used.8 again, to make a large q, large gradient strengths need to be used. moreover, for small duration, large amplitude gradient pulses, there will be a large change in magnetic field experienced by the subject in a short amount of time. this induces an electric field in the subject, which could cause twitching of the skin, pain, or interference with the function of the heart or brain.32 thus, in practice, it is difficult to make measurements at short diffusion times using the pgse sequence. pgse signals from water molecules in small axons do not change with diffusion time in the typical range used. thus methods to determine axon diameter from these pgse measurements are insensitive to these small axons. shortening would allow the inference of restriction sizes, for example rodent sized axon diameters, and surface - to - volume ratios in samples. in 1969, an oscillating gradient spin echo (ogse) sequence was proposed to make measurements at short diffusion times,34 see figure 2. in this sequence, the trapezoidal gradient pulses of the pgse sequence are replaced with sinusoidally varying gradient pulses. each period of the sine wave acts as a diffusion weighting so that the magnetic moments are dephased by the first lobe of the sine wave and rephased by the second lobe. these sine waves are repeated multiple times so that sufficient diffusion weighting can be obtained. the first measurements were made of surface - to - volume ratios from packed beads in water33 and in vegetables.36 however, only in the case of unrestricted brownian motion can these multiple diffusion - weighting periods be considered independent.37,38 for this reason, the signal attenuation should be described in terms of a frequency spectrum rather than simply a scalar value . more complicated gradient modulations that selectively sample a narrow frequency domain of the diffusion spectrum have been proposed.39 these provide a straightforward means of characterizing the diffusion spectrum, but are difficult to implement accurately.35 thus double - sine- and apodized cosine - modulated gradient waveforms, which are modified forms of ogse, have been used and are still termed ogse sequences.35 in this so - called temporal diffusion spectroscopy, the effective diffusion times are changed by varying diffusion gradient frequencies, and thus a spectrum of diffusion rates, which describe the biological tissue microenvironment, can be measured. these measurements are different from q - space imaging where the propagator is usually defined at a given long diffusion time and has spatial and directional dependences over a large range of q - space.5,40 the contrast between tissues might be greater at a discrete, moderately high frequency than at low frequencies, as suggested by theoretical consideration of simple geometries and preliminary studies in tumors41 and rat brain.42 using ogse sequences might improve axon measurements. as alexander notes: more significant improvements may come from replacing the standard pgse sequence with other diffusion - sensitive sequences such as oscillating gradient combination of the experiment design with these other pulse sequences should allow the a priori range of axon diameters to extend to include smaller diameters. this should provide protocols with sensitivity to wider ranges and provide more discriminative axon diameter indices.9 this same group performed experiments with optimized gradient waveforms (gen) and were able to make axons with smaller radii more distinguishable with gen than with pgse.43,44 they still suggest that oscillating waveforms would provide valuable information for in vivo studies.43 developing dti techniques to visualize better smaller fibers is considered an active area of research45 and creating new techniques using short diffusion times will cause water in the larger fibers to become less restricted allowing for easier visualization of the restricted diffusion of water in the smaller fibers. extending the measurements made with ogse from studying the variation of adc with diffusion time or frequency to create a method which uses this variation to infer restriction sizes using the shortest possible diffusion times could allow for the probing of smaller axon diameters. in theory , this method overcomes problems with high gradient strengths necessary in current mr diffusion methods (pgse and q - space) to allow smaller restriction sizes to be distinguished.35 the ogse and pgse methods could be used together to distinguish a large range of restriction sizes from very small (ogse) to fairly large (pgse). combining the two methods might allow for a more complete understanding of the geometry of the sample.	this article reviews a new concept in magnetic resonance as applied to cellular and biological systems. diffusion weighted magnetic resonance imaging can be used to infer information about restriction sizes of samples being measured. the measurements rely on the apparent diffusion coefficient changing with diffusion times as measurements move from restricted to free diffusion regimes. pulsed gradient spin echo (pgse) measurements are limited in the ability to shorten diffusion times and thus are limited in restriction sizes which can be probed. oscillating gradient spin echo (ogse) measurements could provide shorter diffusion times so smaller restriction sizes could be probed.
schizophrenia and bipolar disorder are severe and chronic psychiatric disorders, both considered as major psychosis that are thought to share several pathogenetic factors involving a dysfunctional gene x environment interaction. epigenetic events in telencephalic gabaergic and glutamatergic neurons contribute to the pathogenesis of psychotic symptoms in patients affected by schizophrenia. atypical antipsychotics and valproic acid, drugs commonly used to treat psychosis, have been demonstrated to exert a potential effect on epigenetic mechanisms and to modulate chromatin structure remodeling by inducing dna demethylation and histone modifications. refers to stable alterations in chromatin structure and genes expression that occur without modifying the underlying dna sequence. these alterations strongly depend on gene - environment interactions, can be transmitted through generations of cell divisions and may induce phenotypic modifications in the organisms. epigenetic modifications of the genome may occur via different pathways: (i) cytosine dna methylation at specific clusters of dinucleotyde sequence (cpg) by a family of enzymes called dna methyltransferases (dnmts); (ii) covalent modifications of histones at their amino (n)-terminal tails, the most important of which being acetylation and methylation; (iii) rna interference (fig . , an increasing interest arose among the scientific community on this topic : epigenetic mechanisms have been identified in the pathogenesis of many diseases, including psychiatric diseases , and the development of drugs with specific ability to modulate the epigenetic machinery is at the moment a very intriguing and promising therapeutic strategy . evidence from neuroimaging, neuropathology, and epidemiological studies has led to the that schizophrenia ( sz) and bipolar (bp) disorders are likely to be neurodevelopmental disorders that originate before birth. moreover, an association between prenatal stress and risk for developing sz and associated psychiatric disorders including anxiety and affective disorders has been established. adult offspring of mice exposed to repeated episodes of restraint stress during pregnancy, here defined as prenatally restraint stressed mice or prs mice, exhibit a sz - like behavioral phenotype characterized by hyperactivity, stereotyped and compulsive behaviors, deficits in social interaction, pre - pulse inhibition (ppi), fear conditioning, and object recognition, and hypersensitivity to n - methyl d - aspartate (nmda) receptor blockers. this behavioral phenotype recapitulates positive and negative symptoms, as well as cognitive dysfunction displayed by patients affected by sz or bp disorder. prs mice also show a deficit in cortical gabaergic innervation , which is expected to cause abnormal synchronization of the firing rate of pyramidal neurons, a putative electrophysiological substrate of cognitive dysfunction in psychotic patients and neurodevelopmental animal models of sz. from a molecular standpoint, prs mice show a disrupted signature of chromatin remodeling at genes typically expressed in gabaergic and glutamatergic neurons, i.e., genes encoding for glutamate decarboxylase-67 (gad1), reelin (reln) and brain derived neurotrophic factor (bdnf) respectively. these molecular changes in prs mice are similar to those observed in the brain of sz and bp patients, strongly suggesting that aberrant epigenetic gabaergic / glutamatergic mechanisms may underlie psychotic symptoms. the epigenetic endophenotypes common to the frontal cortex (fc) and hippocampus of prs mice and the brain of patients affected by sz and bp disorder are the following: (i) increased mrna and protein levels of the dna methylating enzymes, dna methyltransferase 1 (dnmt1) and ten - eleven metylcytosine dioxygenase -1 (tet1); (ii) enhanced dnmt1 binding to gad1, reln, and bdnf - ix promoters; (iii) increased levels of 5-methylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc) at gad1, reln, and bdnf promoters; (iv) increased binding of methyl cpg binding protein (mecp2) to gad1 and reln promoters; and (v) a reduced expression of gad1, reln and several bdnf - splice variants (fig . different studies have shown that the sz - like behavioral alterations present in neurodevelopmental rodent models of sz are corrected by systemic administration of valproate and clozapine . in the present review, we address the question of whether prs mice may provide a behavioural and molecular epigenetic animal model of sz valuable for the study of the antipsychotic - like activity of types-2/3 metabotropic glutamate ( mglu2/3) receptor agonists and their interaction with typical or atypical antipsychotic drugs. metabotropic glutamate receptors are gtp - binding - protein (g - protein) coupled receptors that modulate both excitatory and inhibitory synaptic transmission in the cns, and are considered as candidate drug targets for neurological and psychiatric disorders. in the last decade, a role for mglu2/3 receptors in schizophrenia has been suggested by preclinical and clinical studies. pharmacological activation of mglu2/3 receptors decreases glutamate release in the cns, thereby reducing synaptic firing. this effect is shared with atypical antipsychotic drugs such as clozapine, which suppress serotonin - induced post - synaptic excitation in different brain regions (fig . 3). both orthosteric mglu2/3 receptor agonists and selective mglu2 receptor enhancers reverse the effect of nmda receptor antagonists on working memory, sensorimotor gating, locomotor activity, and cortical glutamate efflux in rodents. in addition, drugs that activate mglu2/3 receptors restrain the electrophysiological and behavioral effects of agents acting on 5-ht2a serotonin receptors, i.e. hallucinogens. in patients affected by schizophrenia, systemic treatment with pomeglumetad methionyl, an oral prodrug of the mglu2/3 receptor agonist, ly404039, displayed significant antipsychotic activity in a phase-2 clinical trial, but not in subsequent trials. perhaps a reason for these contrasting findings was the recruitment of patients that had been treated with atypical antipsychotic drugs, which are known to epigenetically down - regulate mglu2 receptors in mice. thus, mglu2/3 agonists might still be promising for the treatment of targeted subpopulations of patients affected by sz. as outlined above, prs mice exhibit a sz - like phenotype characterized by alterations in in social interaction, prepulse inhibition, and locomotor activity, and, by an epigenetic down - regulation of reelin, bdnf, and gad67 proteins in the frontal cortex and hippocampus. interestingly, prs mice also showed significant reductions in mglu2 and mglu3 receptor mrna and protein levels in the frontal cortex, which was manifest at birth and, at least for mglu2 receptors, persisted in adult life. this reduction was associated with an increased binding of dnmt1 to cpg - rich regions of the grm2 and grm3 gene promoters and an increased binding of mecp2 to the grm2 receptor promoter. more recently, we have found a significant increase of tet1 (the enzyme that catalyzes the transformation of 5-methylcytosine ( 5-mc) into 5-hydroxymethylcytosine (5-hmc) in the frontal cortex of prs mice. all epigenetic and behavioral changes of prs mice were reversed by treatment with the mglu2/3 receptor agonist, ly379268. a reduced expression of dnmt1 and dnmt3a was also observed in response to ly354740 (another potent mglu2/3 receptor agonist) (authors unpublished observation). in addition ly379268 increased both mrna and protein levels of growth arrest and dna damage 45- (gadd45-), a protein involved in the regulation of dna demethylation, in mice frontal cortex and hippocampus. the binding of gadd45- to specific promoter regions of reln, bdnf, and gad1 genes was also increased after treatment with ly379268. the effects of mglu2/3 receptor agonists in prs mice were similar to those produced by the atypical antipsychotic clozapine and by valproate, a drug that has multiple mechanisms of action including the inhibition of hdacs. in contrast, the classical antipsychotic, haloperidol, had no effect on prs mice. all together, these findings suggest that treatment with mglu2/3 receptor agonists or mglu2 receptor pams might correct epigenetic alterations typical of patients affected by sz. a large body of evidence suggests that 5-ht2a and mglu2 receptors tightly interact in regulating the response of cortical pyramidal neurons to thalamic inputs in the prefrontal cortex, and that this interaction is relevant to the pathophysiology of schizophrenia. activation of 5-ht2a receptors enhances glutamate release in the apical dendritic region of cortical layer v pyramidal cells, and this effect is blocked by pharmacological activation of presynaptic mglu2/3 receptors. gonzales - maeso and his associates have shown that mglu2 physically associates with 5-ht2a receptors via specific amino acid residues located at the c - terminal end of the 4 transmembrane (tm) domain, and that activation of mglu2 receptors restrains specific signaling pathways activated by hallucinogens acting at 5-ht2a receptors. the finding that expression of mglu2 receptors is reduced, and expression of 5-ht2a receptors is increased, in post - mortem cortical tissue of patients affected by sz suggests that an imbalance between mglu2 and 5-ht2a receptors contributes to the pathophysiology of sz and can be targeted by therapeutic intervention. this imbalance was also shown in prenatally stressed mice, and in the adult progeny of dams exposed to viral infection during pregnancy. in both models, early life stress caused a down - regulation of mglu2 receptors and an up - regulation of 5-ht2a receptors in the cerebral cortex in the adult life. these changes in receptor expression might not be interrelated because it is the blockade or genetic deletion of 5-ht2a receptors that epigenetically down - regulate mglu2 receptors. 5-ht2a receptor knockout mice showed a reduced expression of mglu2 receptors in the frontal cortex, which was associated with a reduced h3 and h4 histone acetylation and an increased h3-k27 tri - methylation at the grm2 gene promoter, two epigenetic changes that cause gene repression. similar changes were found in normal mice chronically treated with atypical antipsychotic drugs inhibiting 5-ht2a receptors. all these findings suggest that mglu2 and 5-ht2a receptors are specifically targeted by a pathological epigenetic programming that is triggered by adverse events occurring early in life and that ultimately in a long - lasting dysfunction of thalamic - cortical transmission, which is a key event in the pathophysiology of sz. pharmacological activation of mglu2 receptors seems to have a favorable impact on epigenetic changes associated with sz, but the effect of mglu2/3 receptor agonists or mglu2 receptor pams might be limited by the down - regulation of mglu2 receptors, a process associated with the disorders and even amplified by the use of atypical antipsychotics. thus, mglu2 receptors should be targeted in drug - nave patients, preferentially at the first episode of psychosis, or at least in the early phase of the disease. one can easily predict that epigenetic drugs acting at the grm2 gene promoter to enhance the expression of mglu2 receptors may have a permissive effect with mglu2 receptor agonists / pams in the treatment of sz. accordingly , pharmacological inhibition of hdacs has been shown to reverse the epigenetic down - regulation of mglu2 receptors caused by atypical antipsychotics. l - acetylcarnitine enhances mglu2 receptor expression via a mechanism mediated by acetylation of histones or transcription factors. l - acetylcarnitine is currently marketed for the treatment of neuropathic pain and shows an excellent profile of safety and tolerability. it will be interesting to examine whether l - acetylcarnitine is able to reverse the epigenetic programming triggered by early life stress by enhancing the expression of mglu2 receptors. if so, a combination of mglu2/3 receptor agonists (or mglu2 receptor pams) and l - acetylcarnitine might be proposed as a valuable strategy in the treatment of sz.	schizophrenia and bipolar disorder are chronic psychiatric disorders, both considered as major psychosis; they are thought to share some pathogenetic factors involving a dysfunctional gene x environment interaction. alterations in the glutamatergic transmission have been suggested to be involved in the pathogenesis of psychosis. our group developed an epigenetic model of schizophrenia originated by prenatal restraint stress (prs) paradigm in mice. prs mice developed some behavioral alterations observed in schizophrenic patients and classic animal models of schizophrenia, i.e. deficits in social interaction, locomotor activity and prepulse inhibition. they also showed specific changes in promoter dna methylation activity of genes related to schizophrenia such as reelin, bdnf and gad67, and altered expression and function of mglu2/3 receptors in the frontal cortex. interestingly, behavioral and molecular alterations were reversed by treatment with mglu2/3 agonists. based on these findings, we speculate that pharmacological modulation of these receptors could have a great impact on early phase treatment of psychosis together with the possibility to modulate specific epigenetic key protein involved in the development of psychosis.in this review, we will discuss in more details the specific features of the prs mice as a suitable epigenetic model for major psychosis. we will then focus on key proteins of chromatin remodeling machinery as potential target for new pharmacological treatment through the activation of metabotropic glutamate receptors.
there are various pathophysiologic reasons for coronary heart disease (chd) including atheroma, also known as atherosclerosis, which is critical for chd occurrence. of these, oxidative stress is one of the most significant aspects in the pathogenesis of chd, which is thought to play an important role in the progression of atherosclerosis. several studies suggest that gamma - glutamyl transferase (ggt) is at the pathophysiological in the precipitation and progression of atherosclerosis. the ggt is regarded as a biomarker of hepatobiliary disease and alcohol consumption or abuse. however, it has been recently demonstrated that the activity of ggt in serum is a sensitive marker of oxidative stress associated with concomitant risk factors, such as obesity, fatty liver, hypertension, dyslipidemia, diabetes, and metabolic syndrome. serum ggt levels are also related positively to novel cardiovascular risk factors like c - reactive protein (crp), and fibrinogen. many studies were executed in order to prove that the higher level of serum ggt within normal range is related to higher incidence of hypertension, diabetes mellitus, fatty liver, and metabolic syndrome, especially in men. however, limited data exist on the significance of serum ggt level within the reference range with the increased risk of chd prediction in men. this study aimed at determining whether serum ggt within its reference range in korean men is associated with the risk of chd prediction. the framingham risk score (frs) modified by the national cholesterol education program (ncep) adult treatment panel iii (atp iii) guidelines from the korea national health and nutrition examination surveys (knhanes) from 2010 to 2011 was employed for computation. the knhanes has been conducted periodically by the korea centers for disease control and prevention since 1998 and provides comprehensive information on health status, health behavior, nutritional status, and sociodemographics in 600 national districts. the cross - sectional analysis used samples containing serum ggt taken from knhanes data (knhanes v-1, 2010 and v-2, 2011). about 5932 out of 8983 people were excluded due to missing data on frs, smoking, or alcohol history. moreover, approximately 1750 subjects with diabetes mellitus, chd, hepatobiliary disease, positive tests for antibody to hepatitis b surface antigen, antibody to hepatitis b virus core antigen or anti - hepatitis c virus, and patients taking drugs influencing liver function and lipid - lowering drugs were excluded. subjects that had been smoking cigarettes regularly 1-year before the time of survey were considered as current smokers. the weekly alcohol intake was calculated and converted based on grams of ethanol consumed. a reference sample constituted by healthy subjects was obtained according to the following inclusion criteria: no smoking, no heavy alcohol intake (< 70 g of ethanol per week), and absence of cardiovascular disease, hypertension, diabetes, obesity, renal diseases, metabolic syndrome, and dyslipidemia. the final reference serum ggt was drawn from the population, and reference range between 6 and 65 iu / l, which was a statistical interval representing 95% or 2 standard deviations. before collection of blood samples, each subject fasted for more than 10 h. after overnight fasting, a venous blood sample was obtained between 08:00 and 10:00 a.m. to measure ggt and fasting blood glucose (fbg), liver enzymes, total cholesterol, triglycerides, high - density lipoprotein (hdl) cholesterol, and low - density lipoprotein (ldl) cholesterol. serum ggt was assayed by the standard method recommended by the international federation for clinical chemistry using l - f - glutamyl-3-carboxy-4-nitroanilide as substrate with a toshiba 200fr autoanalyzer. in addition, the fbg, liver enzymes, and lipid levels were assayed using a toshiba-200fr automatic analyzer (toshiba medical systems, tokyo, japan). blood pressure (bp) was measured using a standard mercury manometer with the participant in a sitting position for 5 min prior to measurement, where the average measurement was recorded. hypertension was defined as a systolic bp (sbp) 140 mmhg or a diastolic bp 90 mmhg or by the use of antihypertensive medication. body mass index (bmi) was calculated as weight (kg) divided by height squared (m). the distribution of ggt values and weekly alcohol consumption were right - skewed; therefore, a natural log - transformation was applied. after assessment, the general characteristics were presented, and the study subjects were grouped into quartiles according to levels of serum ggt. analysis of variance (anova) trend analysis using polynomial contrasts was adapted to perform tests for trends. in order to evaluate the relationship between serum ggt activity within reference range and the individual components of the frs, spearman's correlation analysis was employed. the frs was calculated from the ncep atp iii algorithm based on six coronary risk factors: age gender, total cholesterol, hdl - cholesterol, sbp, and smoking habit. among these factors, framingham risk equations were used to predict the risk of developing coronary disease events (myocardial infarction or chd death) over the next 10-year for adults aged 20 and older without heart disease or diabetes. participants were divided into three groups: low risk (< 10% risk of developing a chd event over the next 10-year), intermediate risk (1020% risk), and high risk (> 20% risk). however, the number of high - risk group was so small that it included the high - risk group into intermediate - risk group and beyond. smoking status was classified as either current smoker or nonsmoker. for analysis relating to serum ggt within reference interval to the intermediate - risk group and beyond, we constructed adjusted logistic regression analyses that considered bmi, weekly alcohol intake, and ldl cholesterol. the of group data were expressed as mean standard error (se). data were analyzed using pasw spss version 18.0 (spss inc ., chicago, il, usa). the knhanes has been conducted periodically by the korea centers for disease control and prevention since 1998 and provides comprehensive information on health status, health behavior, nutritional status, and sociodemographics in 600 national districts. the cross - sectional analysis used samples containing serum ggt taken from knhanes data (knhanes v-1, 2010 and v-2, 2011). about 5932 out of 8983 people were excluded due to missing data on frs, smoking, or alcohol history. moreover, approximately 1750 subjects with diabetes mellitus, chd, hepatobiliary disease, positive tests for antibody to hepatitis b surface antigen, antibody to hepatitis b virus core antigen or anti - hepatitis c virus, and patients taking drugs influencing liver function and lipid - lowering drugs were excluded. subjects that had been smoking cigarettes regularly 1-year before the time of survey were considered as current smokers. the weekly alcohol intake was calculated and converted based on grams of ethanol consumed. a reference sample constituted by healthy subjects was obtained according to the following inclusion criteria: no smoking, no heavy alcohol intake (< 70 g of ethanol per week), and absence of cardiovascular disease, hypertension, diabetes, obesity, renal diseases, metabolic syndrome, and dyslipidemia. the final reference serum ggt was drawn from the population, and reference range between 6 and 65 iu / l, which was a statistical interval representing 95% or 2 standard deviations. before collection of blood samples, each subject fasted for more than 10 h. after overnight fasting, a venous blood sample was obtained between 08:00 and 10:00 a.m. to measure ggt and fasting blood glucose (fbg), liver enzymes, total cholesterol, triglycerides, high - density lipoprotein (hdl) cholesterol, and low - density lipoprotein (ldl) cholesterol. serum ggt was assayed by the standard method recommended by the international federation for clinical chemistry using l - f - glutamyl-3-carboxy-4-nitroanilide as substrate with a toshiba 200fr autoanalyzer. in addition, the fbg, liver enzymes, and lipid levels were assayed using a toshiba-200fr automatic analyzer (toshiba medical systems, tokyo, japan). blood pressure (bp) was measured using a standard mercury manometer with the participant in a sitting position for 5 min prior to measurement, where the average measurement was recorded. hypertension was defined as a systolic bp (sbp) 140 mmhg or a diastolic bp 90 mmhg or by the use of antihypertensive medication. body mass index (bmi) was calculated as weight (kg) divided by height squared (m). the distribution of ggt values and weekly alcohol consumption were right - skewed; therefore, a natural log - transformation was applied. after assessment, the general characteristics were presented, and the study subjects were grouped into quartiles according to levels of serum ggt. analysis of variance (anova) trend analysis using polynomial contrasts was adapted to perform tests for trends. in order to evaluate the relationship between serum ggt activity within reference range and the individual components of the frs, spearman's correlation analysis was employed. the frs was calculated from the ncep atp iii algorithm based on six coronary risk factors: age gender, total cholesterol, hdl - cholesterol, sbp, and smoking habit. among these factors, framingham risk equations were used to predict the risk of developing coronary disease events (myocardial infarction or chd death) over the next 10-year for adults aged 20 and older without heart disease or diabetes. participants were divided into three groups: low risk (< 10% risk of developing a chd event over the next 10-year), intermediate risk (1020% risk), and high risk (> 20% risk). however, the number of high - risk group was so small that it included the high - risk group into intermediate - risk group and beyond. smoking status was classified as either current smoker or nonsmoker. for analysis relating to serum ggt within reference interval to the intermediate - risk group and beyond , we constructed adjusted logistic regression analyses that considered bmi, weekly alcohol intake, and ldl cholesterol. the of group data were expressed as mean standard error (se). data were analyzed using pasw spss version 18.0 (spss inc ., chicago, il, usa). the range of the first - to - fourth quartiles of serum ggt values was 613, 1418, 1928, and 2965 participants in higher ggt quartiles were older and had more general chd risk factors such as hypertension, dyslipidemia, more alcohol consumption, and current smoking history. in addition, higher quartiles of serum ggt concentration were significantly associated with increasing trends in framingham point scores and the risk of 10-year chd prediction (p for trend < 0.05). the general characteristics of the study subjects according to serum ggt grading data are expressed as mean se after data weighting in complex sample analysis. ggt: gamma - glutamyl transferase; bw: body weight; bmi: body mass index; wc: waist circumference; sbp: systolic blood pressure; dbp: diastolic blood pressure; hdl - c: high - density lipoprotein cholesterol; ldl - c: low - density lipoprotein cholesterol; tg: total glycerides; ast: aspartate aminotransferase; alt: alanine aminotransferase; chd: coronary heart disease; se: standard error; q1: 1 quartile; q2: 2 quartile; q3: 3 quartile; q4: 4 quartile. the amount alcohol consumption calculation formula: 10 g shots frequency / week. the correlation between log - transformed ggt and 10-year chd risk was r = 0.237 (p < 0.001). log - transformed ggt was also well - correlated with individual risk factor scores including age, smoking, total cholesterol, hdl - cholesterol, and sbp (p < 0.05 for all). the spearman's rank correlation coefficients relating individual components and total framingham risk score to log - transformed ggt ggt: gamma - glutamyl transferase; hdl - c: high - density lipoprotein cholesterol; sbp: systolic blood pressure; chd: coronary heart disease. table 3 shows the odds ratio (or) for intermediate - risk and beyond for chd in relation to quartiles of serum ggt. the 10-year chd risk was significantly associated with increasing quartiles of serum ggt (p for trend < 0.05). compared with individuals with lowest quartile of serum ggt, the nonadjusted or (95% confidence interval) was 1.18 (0.781.78) for the second quartile, 1.42 (0.94 2.16) for the third quartile, and 2.77 (1.74 4.40) for the highest quartile. after adjustment of bmi, the amount of alcohol intake and ldl cholesterol, the or (95% ci) for intermediate risk and beyond of 10-year chd prediction with lowest quartile of participants was 1.21 (0.78 1.87) for second quartiles, 1.39 (0.88 2.21) for third quartiles, and 2.03 (1.23 3.34) for highest quartiles. in addition, the or for intermediate risk and beyond of 10-year chd prediction showed an increased tendency as the serum ggt quartile gets greater (p for trend < 0.05). the or of intermediate - risk and beyond for chd (10-year risk 10%) by ggt multivariate logistic regression model was used after data weighting in complex sample analysis. model 1: unadjusted; model 2: after adjustment for bmi, the amount of alcohol intake and ldl - c. ggt: gamma - glutamyl transferase; chd: coronary heart disease; or: odds ratio; ci: confidence interval; bmi: body mass index; ldl - c: low - density lipoprotein cholesterol; q1: 1 quartile; q2: 2 quartile; q3: 3 quartile; q4: 4 quartile. in the use of serum ggt as a test variable to predict the presence of intermediate risk beyond a 10-year chd prediction, a receiver - operating characteristic (roc) curve was prepared. based on the roc curve, the best serum ggt cut - off obtained was 28.5 iu / l that predicted the intermediate risk, beyond a 10-year chd prediction with sensitivity of 58%, specificity of 52%, positive predictive value of 54.7%, and negative predictive value of 55.3%. we investigated the association between the ggt level in normal range and the possible prevalence of chd in 10-year, using frs calculated from the ncep atp iii algorithm in korean men. frs is one of the numbers of scoring systems used to determine an individual's chances of developing chd. cardiovascular risk scoring systems presents an estimate of the probability that a person will develop chd within a given time - frame, usually 1030 years. in this study, a higher quartile of serum ggt level, even within the reference range, was found to be significantly related to the elevated risk of chd which ed from the calculation of frs in men. moreover, this association persisted even after adjusting established cardiovascular risk factors such as ldl - cholesterol and bmi, which were not used in the necp atp iii and confounding factors like the amount of alcohol intake. the or of a 10-year chd risk prediction increased in dose - dependent manner with increasing quartiles of serum ggt activity. the best serum ggt cut - off was 28.5 kim et al. previously demonstrated that multivariable - adjusted ors for frs > 20% were significantly increased from the lowest to highest ggt quartiles, compared to the lowest baseline ggt category. however, it was a single center - based study and did not target reference value of serum ggt. in other words, the present study derives its significance in the aspect that the association between serum ggt activity within reference value and a 10-year chd prediction risk was evaluated. the present study also concluded that individuals with higher quartiles of serum ggt were well correlated with individual factors such as higher bmi, waist circumference, sbp, lipid levels, and fbg, which include frs components. , the prevalence rate of metabolic syndrome increased significantly in relation to serum ggt level within normal range in korean men. demonstrated that serum ggt level even within the reference interval was correlated with nonalcoholic fatty liver disease (nafld) and the or of ggt activity for nafld was elevated according to elevation of the ggt grading. liu et al. also mentioned that ggt was strongly consistent with cardiovascular and metabolic variables in the cross - sectional study among 616 young healthy participants. this tendency has been proven not only in asian studies but also in previous studies targeting caucasians, africans, and other countries. they indicated that individuals with metabolic syndrome, impaired fasting glucose, and nafld had considerably elevated levels of serum ggt regardless of any ethnicity. increased serum ggt level has been traditionally understood as a marker of alcohol abuse and/or liver damage however, a large number of studies suggested that serum ggt is not only a marker for oxidative stress but also a relative factor of cardiovascular disease and metabolic syndrome. despite the fact that serum ggt activity which reflects the risk of chd is not completely understood, there are several possible mechanisms that support the hypothesis. the first probable mechanism is that ggt is regarded as a biomarker for oxidative stress. in other words, the oxidative stress could be a crucial factor in the pathophysiology of cardiovascular disease and ggt has an important role in maintaining intracellular glutathione transport into most types of cells. elevated ggt could be the expression of subclinical inflammation because serum ggt is highly associated with white blood cell count and some features of low - grade inflammation. furthermore, excess reactive oxygen species and superoxide which is generated by oxidative stress and low - grade inflammation recapitulate not only endothelial dysfunction but also cardiovascular dysfunction. the third acceptable mechanism could be a strong correlation of ggt with various atherosclerotic risk factors. previous studies reported that serum ggt concentrations were related with hypertension, metabolic syndrome, and diabetes. serum ggt level was also found to increase insulin resistance and be positively correlated with risk factors of atherosclerotic chd and inflammation such as male gender, crp, total cholesterol and uric acid level. reversely, as also shown in this study, serum ggt was negatively correlated to hdl - cholesterol level which is a well - known negative risk factor of chd. the origins of ggt in plaques could be through the influx of lipoproteins which carry it into lesions. one of the products of glutathione hydrolysis which were produced by ggt is cysteinyl - glycine. it is also observed in the studies which examined the enzymatic activity of ggt in the coronary and carotid atheroma. furthermore, the data may guarantee that it represent the whole population because it was derived from a random selection. therefore, it is reasonable for the to be generalized into the korean population. in addition, a 10-year chd risk prediction was computed using the ncep atp iii. however, even though this study showed a positive relationship between serum ggt level within the reference range concentration and a 10-year chd risk prediction, there are several limitations. this study was cross - sectional, and we did not measure oxidative stress directly. additionally, the end point of this study is a mathematical substitute for the presence of chd, even though frs is a generalized tool. furthermore, the statistical analysis to serum ggt concentration in women was not applied because the number of subjects with intermediate - risk and beyond for chd (frs 10%) in this gender group was too small. therefore, further longitudinal cohort studies are needed to evaluate the predictive value of biomarkers for the increased risk of chd in the korean population. in , this study suggests that serum ggt activity, within its reference range, is a useful predictor of a 10-year chd risk using frs calculated by ncep atp iii. therefore, serum ggt within the reference range could be a helpful tool and an additional marker in the prediction of chd risk in korean men.	: limited data exist on the association of serum gamma - glutamyl transferase (ggt) level within the reference range with the increased risk of coronary heart disease (chd) prediction in men. the study examined the association between serum ggt concentration within the reference range and the chd risk prediction in korean men.methods:the study employed data from korean national health and nutrition examination survey (v-1, 2010 and v-2, 2011) where a total of 1301 individuals were analyzed. a 10-year chd risk prediction was computed using the framingham risk score (frs) modified by the national cholesterol education program (ncep) adult treatment panel iii (atp iii).: positive correlations were established between log - transformed ggt concentration and frs (r = 0.237, p < 0.001). after adjustment of body mass index, the amount of alcohol intake and low - density lipoprotein - cholesterol, the odds ratio (95% confidence interval) for intermediate risk and beyond of 10-year chd prediction (10-year risk 10%) with lowest quartile of participants was 1.21 (0.781.87) for second quartiles, 1.39 (0.882.21) for third quartiles and 2.03 (1.233.34) for highest quartiles.:higher serum ggt within its reference range was significantly correlated with a 10-year chd risk prediction estimation using ncep atp iii in korean men.
lichen planus is an inflammatory mucocutaneous disease; it is a very common disease in adults but uncommon in children. an autoimmune basis has been proposed; however, there is evidence suggesting that lp is not a true autoimmune disease but rather a chronic, cell - mediated immune disorder, involving activated lymphocytes and up - regulating cytokine production. several retrospective reviews have estimated that only 1% 16% of lp patients are younger than 15 years. factors responsible for the rarity of juvenile olp include a low incidence of autoimmune diseases, systemic diseases, precipitating factors such as stress and lp related infections in children. the purpose of this paper is to report two cases of childhood olp and review the literature in this regard. an 8 year - old male patient was referred to the department of pedodontics, pms college of dental science and research with the complaint of pain and white discoloration under the tongue. the patient had difficulty tolerating spicy food. the medical history revealed that the boy had been vaccinated against japanese encephalitis one year earlier as part of school immunization program. almost 10 months after this, fluid - filled blisters appeared on both feet and ruptured after 23 days. these were associated with severe itching for which he had taken ayurvedic medicine. on examination, 1 ), on the ventral tongue mucosa and also on the labial mucosa (fig . white interlacing striae on the left buccal mucosa striae on the upper labial mucosa ( case 1) on palpation, these lesions were non - tender and non - scrapable and the buccal mucosae appeared rough and corrugated. differential diagnoses included olp, lichenoid reaction and leukoplakia and a provisional diagnosis of lp was made by correlating the clinical appearance with the age of the patient. the provisional diagnosis was confirmed by histopathological examination following incisional biopsy from the left buccal mucosa. the biopsy specimen showed typical features of lp including the saw - toothed rete pegs, hyperkeratosis and lymphocytic infiltrate (fig . 3). pictomicrograph of case 1 showing saw - toothed rete pegs and lymphocytic infiltration as the lesions were symptomatic, topical anti - fungal treatment (clotrimazole mouth paint twice daily) was prescribed for the first two weeks. on the recall session, the burning sensation had reduced considerably and the clinical appearance of the lesions had improved with regard to severity. an 8 year - old male patient was referred from a local clinic with burning sensation while eating spicy food. they gave a history of burning sensation since three months earlier after which a local dentist replaced an amalgam restoration with glass ionomer in the left posterior mandible. due to no relief, the patient was referred to a hospital. on examination, the patient had a glass ionomer restoration on tooth # 75, and all other teeth were caries - free. white interlacing striae were noted on the left buccal mucosa (unilaterally) (fig . 5) revealed saw - toothed epithelial rete pegs, basal cell degeneration with subepithelial lymphocytic infiltrates and melanin incontinence, which confirmed the diagnosis of lp. after the biopsy, the lesion and the symptoms subsided; thus, no treatment was warranted. striae on the left buccal mucosa (case 2) pictomicrograph of case 2 showing saw - toothed rete pegs and lymphocytic infiltration an 8 year - old male patient was referred to the department of pedodontics, pms college of dental science and research with the complaint of pain and white discoloration under the tongue. the patient had difficulty tolerating spicy food. the medical history revealed that the boy had been vaccinated against japanese encephalitis one year earlier as part of school immunization program. almost 10 months after this, fluid - filled blisters appeared on both feet and ruptured after 23 days. these were associated with severe itching for which he had taken ayurvedic medicine. on examination, 1 ), on the ventral tongue mucosa and also on the labial mucosa (fig . white interlacing striae on the left buccal mucosa striae on the upper labial mucosa ( case 1) on palpation, these lesions were non - tender and non - scrapable and the buccal mucosae appeared rough and corrugated. differential diagnoses included olp, lichenoid reaction and leukoplakia and a provisional diagnosis of lp was made by correlating the clinical appearance with the age of the patient. the provisional diagnosis was confirmed by histopathological examination following incisional biopsy from the left buccal mucosa. the biopsy specimen showed typical features of lp including the saw - toothed rete pegs, hyperkeratosis and lymphocytic infiltrate (fig . 3). pictomicrograph of case 1 showing saw - toothed rete pegs and lymphocytic infiltration as the lesions were symptomatic, topical anti - fungal treatment (clotrimazole mouth paint twice daily) was prescribed for the first two weeks. on the recall session, the burning sensation had reduced considerably and the clinical appearance of the lesions had improved with regard to severity. an 8 year - old male patient was referred from a local clinic with burning sensation while eating spicy food. they gave a history of burning sensation since three months earlier after which a local dentist replaced an amalgam restoration with glass ionomer in the left posterior mandible. due to no relief, the patient was referred to a hospital. on examination, the patient had a glass ionomer restoration on tooth # 75, and all other teeth were caries - free. white interlacing striae were noted on the left buccal mucosa (unilaterally) (fig . 5) revealed saw - toothed epithelial rete pegs, basal cell degeneration with subepithelial lymphocytic infiltrates and melanin incontinence, which confirmed the diagnosis of lp. after the biopsy, the lesion and the symptoms subsided; thus, no treatment was warranted. striae on the left buccal mucosa (case 2) pictomicrograph of case 2 showing saw - toothed rete pegs and lymphocytic infiltration lichen planus is a chronic inflammatory mucocutaneous disease most frequently seen in the middle aged and the elderly populations with a female to male ratio of approximately 2:1. childhood lp is common in the tropics, and children of asian origin may be more prone to this condition. this possibly indicates that a specific genetic predisposition (hla - dependent) in the asian race, in spite of a negative family history, may be involved in the pathogenesis of olp. the etiology of lp remains obscure, but recently pathogenic mechanisms with an immune - mediated basis have been proposed. causes such as allergy to dental restorative materials, local trauma (koebner phenomenon) and several infections (plaque causing microorganisms) have also been reported. also, lp has been reported as a complication of hepatitis b vaccination in both children and adults. reactions to measles - mumps - rubella (mmr) and diphtheria - tetanus pertussis - polio (dtap - ipv) vaccinations can also be related to this disease. moreover, our first case gave a positive medical history of vaccination against japanese encephalitis within the previous year. but this association can not be confirmed conclusively because there is no available literature regarding development of lp following this particular vaccination. this agrees with nnoruka, who reported a relationship between this phenomenon and the occurrence of lp. in our second case , the etiology could be the amalgam restoration but persistence of symptoms even after replacement of amalgam with glass ionomer cement led to the diagnosis of idiopathic lp. some patients report roughness of the lining of the mouth, sensitivity of the oral mucosa to hot or spicy foods, presence of red or white patches or oral ulcerations. but in our second case, unilateral presence of the lesion led us to a differential diagnosis of lichenoid reaction. the only difference is in the site of involvement like the palate, which is atypical of lp. in most cases, the cause for lichenoid reactions can not be identified; hence the diagnosis by exclusion is idiopathic lp. although the differential diagnoses were considered, histopathological features confirmed the diagnosis of lp in both cases. use of topical corticosteroids is the most widely accepted treatment for olp because it reduces patients pain and inflammation. several therapies, including intra - lesional injection, retinoids, dapsone, tacrolimus, and ultra - violet light have been tested with varying . clinical improvement with relief of symptoms has been reported following the use of amphotericin - b, nystatin and azole antifungals. miconazole gel is useful as an adjunct to topical steroids. in our first case, since the patient complained of severe burning sensation, immediate treatment with topical antifungals was done and there was marked reduction in the symptoms. while in our second case, since the lesion subsided spontaneously after the incisional biopsy, no treatment was warranted. there are a number of studies on olp with regard to its malignant transformation in the past few decades although olp is considered rare in children, presence of a non - scrapable white lesion should alert the clinicians to consider lp in the list of differential diagnoses. timely diagnosis with proper treatment oriented towards the etiological factor	lichen planus (lp) is a chronic mucocutaneous disease widely recognized in adults, but uncommon in children. the purpose of this paper is to report two cases of lp in children. the diagnosis was made based on clinical and histopathological findings. the treatment consisted of antifungal and multivitamin therapy. regression of lesions was observed. the patients are still under follow - up. although lp is uncommon in children, it is necessary to have adequate knowledge about this condition for proper diagnosis and treatment.
pancreatic development is a complex process. however, pancreatic congenital anomalies are a rare entity. among these anomalies, it can be asymptomatic and diagnosed incidentally during investigations carried out for unrelated causes, or symptoms including abdominal pain, diabetes mellitus, jaundice, and weight loss might present initially. other anomalies might be present as well, in association with dorsal agenesis of the pancreas such as polysplenia, ectopic spleen, mesenteric malrotation, bicornuate uterus, and cardiac anomalies 25. to our knowledge, around 58 cases of dorsal agenesis of the pancreas have been reported from 1913 till 2015, and around nine cases only were associated with pancreatic tumor. we present a case of dorsal agenesis of the pancreas associated with pancreatic tumor, along with ectopic splenia, mesenteric malrotation, and vascular malformations. this is the case of a 29yearold male patient presenting to the hospital with a 2month history of abdominal pain, worsening progressively then localizing in the right upper abdominal quadrant and radiating to the back over the last 2 weeks. patient admits one episode of abdominal pain a year ago, due to which he underwent an endoscopic gastroduodenoscopy that revealed diffuse mild gastritis and no evidence of helicobacter pylori infection. patient was treated with a 3month course of protonpump inhibitors with clinical improvement in his pain. he has no past medical or surgical history and no allergies, does not consume alcohol and is not a smoker. ultrasound of the abdomen was performed showing a right subhepatic complex cystic lesion 12 7 6 cm containing dense debris inside and a thick wall. magnetic resonance cholangiopancreatography (mrcp) was performed showing a loculated mass, 5.6 9.7 cm, located behind the head of the pancreas, displacing it anteriorly and to the right. (mrcp): cuts showing the pancreatic mass (white arrows) as it appeared on mrcp in an axial (a) and coronal fashion (b). abdominopelvic ct scan was performed showing a multiloculated cystic retroperitoneal lesion at the level of the second portion of the duodenum in continuity with the pancreatic head which is displaced anteriorly and to the right (fig . 2) associated with a mild malrotation of the mesentery and proximal small bowels (fig . ( ct scan): (a and b) the multiloculated cystic retroperitoneal lesion (white arrows), at the level of the second portion of the duodenum in continuity with the pancreatic head which is displaced anteriorly and to the right. (a) coronal cut from the abdominopelvic ct scan, showing bowels malrotation and an inferiorly located hepatic flexure (white arrow). (b) 3d reconstruction of the scan performed with iv contrast showing the vascular variation where the sma branches from the celiac trunk (thick white arrow), giving off the gastroduodenal artery (thin white arrow). (ct scan): (a) presence of the ectopic spleen (white arrow); (b) cut showing the absence of pancreatic tissue anterior to the splenic vein, where it is usually located (white arrows). laboratory studies showed the following: hemoglobin 12.8 g / dl, white blood cells 5000/mm, neutrophils 53%, platelets 345,000/mm, pt inr 1.1, sgpt (serum glutamicpyruvic transaminase) 10 /l, sgot (serum glutamicoxaloacetic transaminase) 10 /l, direct bilirubin 0.18 mg / dl, total bilirubin 0.45 mg / dl, alkaline phosphatase 101 /l, amylase 77 /l, lipase 55 /l, cea (carcinoembryonic antigen) 2.09 ng / ml, ca199 19.95 /ml, and ggt (gammaglutamyltransferase) 45 /l. analysis of the aspirate revealed the following: culture: escherichia coli growth, resistant to ampicillin.pathology: necrotic inflammatory smear with no suspicious content, poorly cellular with no ductal cells, no atypical cells.amylase = 982 /l; lipase = 4498 /l.cea = 700 ng / ml; ca199 = 10.8 /ml. culture: escherichia coli growth, resistant to ampicillin. pathology: necrotic inflammatory smear with no suspicious content, poorly cellular with no ductal cells, no atypical cells. cea = 700 ng / ml; ca199 = 10.8 /ml. based on the data collected at that point, the cyst was considered benign and cystojejunostomy was planned. in the operating room, a right subcostal incision was made, and inspection and palpation of the abdominal organs revealed no evidence of metastatic lesions or carcinomatosis. chevron incision was then performed, and the transverse colon was not in its anatomical position, with the hepatic flexure located inferiorly around the right gutter. cyst fluid was suctioned with care taken to avoid any spillage; then, intraoperative wedge biopsy was performed and sent to the pathology laboratory as frozen. came back positive for mucinous cystadenocarcinoma, moderately to poorly differentiated cells, ductal in origin. at this point, dissection was started at the hepatic pedicle, identification of the common hepatic duct, portal vein, and right hepatic artery. the anatomical variation at this level was as follows: superior mesenteric artery branching off the celiac trunk, superior mesenteric vein superficially located with a malrotation of the mesentery, and a rightsided ligament of treitz. common hepatic duct was divided and enterectomy was performed, and retroportal pancreatic dissection with ligation of venous tributaries and total pancreatectomy were performed. lymph node dissection around the hepatic artery and celiac trunk was performed, and hepaticojejunostomy and rouxeny gastrojejunostomy were performed as well. macroscopically, the pancreatic piece was 14 7.5 7.5 cm showing on cut section a partly necrotic partly cystic surface, and microscopically, the cyst located in the lateral peripancreatic region is composed of dense mesenchymal tissues (muscle, fat, cartilage) admixed with neural elements on a of dense lymphoid tissue with lymphoid follicles, all lined by mature squamous epithelium. pancreatic head shows numerous cystic dilated ducts lined by flattened epithelium alternating with small cystic spaces lined by tall columnar epithelium exhibiting dysplasia. common bile duct lining epithelium shows marked atypia and is surrounded by numerous clusters and neoplastic glands invading the underlying pancreas (figs 5, 6, 7, 8). cyst lined by mature squamous epithelium continued by either columnar or at places by cuboidal epithelium. cyst wall composed of mature mesenchymal tissue: cartilage, muscle, and clusters of benign salivarytype glands. major pancreatic duct (mpd) surrounded by small irregular neoplastic glands (adenocarcinoma). diagnosis: pancreatic adenocarcinoma, moderately differentiated mucin secreting arising in common bile duct region, infiltrating pancreatic head with absence of body and tail, measuring 6 4 4 cm with free surgical margins. ct scan (computed tomography scan) was then performed day 7 postoperation showing no intraabdominal collections, drains were removed, patient was started on liquid diet, and no complications were encountered. patient's diet was progressed and he was discharged home on insulin replacement therapy and pancreatic enzyme supplementation. being part of the gastrointestinal organs system, the pancreas develops from the endoderm. between the sixth and eighth week of embryonic life, two evaginations, dorsal and ventral, the ventral evagination arises from a liver diverticulum and will eventually form the posterior part of the pancreatic head and the uncinate process. on the opposite site of the foregut, the dorsal evagination develops and will eventually form the tail and body of the pancreas. later on, ducts begin to develop in a treelike fashion, endocrine cells appear, and the islets of langerhans emerge with time 1, 2. the pancreas is a retroperitoneal organ, extending from the duodenal arc to the spleen. it harbors two ducts: the main duct of wirsung and the duct of santorini 3, 4. with the advancement of imaging techniques, and increasing diagnostic workups made, pancreatic malformations are more described. total pancreatic agenesis has been described in the literature but is an extremely rare entity, usually incompatible with life, and associated mainly with sever intrauterine growth retardation 5, 6. other types of congenital anomalies of the pancreas are more frequently encountered such as pancreas divisum, common bile duct syndrome, ectopic pancreas, accessory pancreatic lobe, annular pancreas, and agenesis of the dorsal pancreas (table 1). pancreatic malformations 7 over the last 100 years, 58 cases were reported with agenesis of the dorsal pancreas, whether partial or complete, with the latter being less frequent, and around nine cases only were associated with pancreatic tumor (table 2). it has been associated with pancreatic bud primary dysgenesis, ischemic insult to the organ during its development, and an autosomal dominant genetic mode of transmission 1, 9, 10. cases of agenesis of the dorsal pancreas with associated pancreatic tumors found in the literature agenesis of the dorsal pancreas is a rare congenital malformation, and its association with tumors makes it a hard condition to deal with. many studies are trying to identify the reason behind this anomaly, with findings suggestive of genetic inheritance in an autosomal dominant mode or xlinked mode 11, 19. other hypotheses state a genomic activation process, notably the sonic and indian hedgehog genes 20, hnf1 gene 18, and others. as demonstrated by the literature review done, the cornerstone for curing tumors in association with dorsal agenesis of the pancreas is surgery. most of the time, the lack of sufficient pancreatic tissue makes it hard to achieve safe margins without undergoing a total pancreatectomy. adenocarcinoma associated or arising from previous teratoma tumors is by itself a rare entity 21, 22, 23, 24. this constellation of presenting tumors with a rare congenital anomaly makes the genetic hypothesis in the frontline of investigations. until today , the proper management of tumors associated with agenesis of the dorsal pancreas consists of surgical intervention for malignant cases and/or symptomatic benign cysts, with or without chemotherapy regimens with respect to the pathology finding. the additional challenge comes afterward involving pancreatic endocrine and exocrine replacement therapies. such cases should be handled in referral center where patient's monitoring can be achieved optimally on the surgical and medical level. furthermore, associated genes should be evaluated in such patients and on a larger scale as well. the approval of the university of balamand faculty of medicine irb was obtained (irb / o/03116). the datasets supporting the of this article are included within the article and its references.	key clinical messagedorsal agenesis of the pancreas is a rare congenital anomaly. fiftyeight cases were reported from 1913 till 2015, nine of which were associated with tumors. we present the 10th case, the first to be associated with pancreatic mucinous adenocarcinoma and cystic teratoma, successfully managed by whipple procedure and total pancreatectomy.
cysticerci, the larval forms of taenia solium, develop in the muscles of the pig, an intermediate host. inadequately heated or raw pork containing adult worms is the main source of human infection. other sources of human infections with c. cellulosae occur by way of 1 ) ingestion of the eggs found on contaminated hands or in food, 2 ) self - contamination by people who have the adult worm in their intestines, and 3 ) internal autoinfection in which the eggs of the adult worm residing in the upper gastrointestinal tract are returned to the stomach by reverse peristalsis. the eggs are hatched in ugi to the embryos which penetrate the intestinal wall and are then carried along blood vessels to all parts of the body, where they soon become cysticerci. cysticerci most frequently invade the intermuscular and subcutaneous tissues; next, the eye and then the brain. they may also invade the heart, liver, abdominal cavity, kidneys, and adrenals, as well as the lungs. it is so rare that, worldwide, only a few cases have ever been reported, with none in korea. among those few cases reported in other areas, this report, then, together with relevant literature, cites another one of the few documented cases of pulmonary cysticercosis and further cites the only case of pulmonary cysticercosis ever documented or reported in korea. a 65-year - old man was admitted to the hospital because of generalized weakness, coughing, and sputum production. two months before his admission, weakness, sweating and anorexia had developed together with an intermittent cough and whitish sputum production. the patient had eaten raw pork several times 20 years ago, but there was no specific family history. vital signs were as follows: temperature 36, pulse 98/min, and respiration 20/min. the blood pressure was 110/75 mmhg. on examination the patient appeared weak and chronically ill. an abdominal examination disclosed mild tenderness to deep palpation in the right upper quadrant, without diminution of bowel sounds; the liver was palpated by four finger breadths. laboratory data on admission were as follows: hemoglobin and hematocrit were 13.9 g / dl and 41.8% on admission; white cell count was 8700 with 4% eosinophils; urine and stool specimens gave normal ; blood urea nitrogen 5 mg / dl; creatinine 1.3 mg / dl; total protein 8.1 g / dl; albumin 4.3 g / dl; and sgot / gpt 42/32 u / dl. the stain for acid fast bacilli and cytologic examination of sputum did not reveal anything microscopically. the pulmonary function test was within normal range; the fev1 was 4.1, the fev1/fvc was 87%. an x - ray film of the chest showed multiple nodular densities throughout both lower lung fields (fig . 1). a computed tomographic scan of the brain and chest revealed multiple nodular and small calcified densities (fig . 3, 4). a biopsy of a subcutaneous nodule was done on the 4th hospital day, and upon microscopic examination, a bladder worm (fig . 6) and parenchymatous portion with spinal canal and separated bladder of the cysticercus (fig . 7)was observed. on the 12 th hospital day, the presence of cysticercus was confirmed by an open lung biopsy (fig . afterward, the patient was treated with praziquantel 50 mg / kg / day for 15 days and discharged on the 18 th hospital day, even though he still reported some weakness, cough, and sputum production . an x - ray film of the chest revealed nearly normal conditions in the lungs(fig . taenia solium has been recognized from the time of hippocrates but was never specifically differentiated from the beef tapeworm, t. saginata, until the time of goseze. leukart first worked out the life cycle and demonstrated that the bladder worm in the tissues of the pig was in the larval stage and infective for man. taenia solium has a world wide distribution but is most commonly found in the soviet union, asia, africa and in central and south america. the eggs of taenia solium and taenia saginata in korea were found in 1.1% of the population, according to a broad parasite study done on 35,018 people in 1981. in 1986, the infection rate for the both parasites decreased to 0.27% (119 of 43,590 people). the eggs of taenia solium are spherical or subspherical in shape, measure 31 to 43 um in diameter and can not be microscopically distinguished from those of t. saginata. cysticerci, the larval forms, are subspherical to ovoid, have milky white bladders with heads invaginated into the bladders, and are approximately 5 mm wide and 8 to 10 mm long. after human ingestion, the eggs develop in the stomach and intestine into embryos, which penetrate the intestinal mucosa, then circulate in the entire body through the venous system of the mesentery and are finally distributed in the muscles and other tissues where development into cysticerci takes place over a period of 60 to 70 days. the fibers of the involved muscles atrophy and their function decreases. in the subcutaneous tissues, however, only a mild tissue reaction occurs, despite the swollen appearance of the protruded skin. the clinical picture of brain cysticercosis varies according to the involved area of the brain cortex and can easily be mistaken for a brain tumor because of those symptoms which involve the central nervous system. if cysticerci develop in the ventricles of the brain, headache, nausea and vomiting occur due to increased intracranial pressure. precysticercus larvae lodged in the brain produce little disturbance during their life spans, but as soon as the larvae begin to lose vitality and parts of them die, dead larvae and their remnants are sensitized as foreign materials and evoke a great variety of brain symptoms, such as allergic reactions. when examples of pulmonary cysticercosis are found, cysticerci may be assumed to have also invaded many other organs like the brain, subcutaneous tissues and muscles of humans or animals as well. the radiological appearance of cysticercosis in the lungs can not be differentiated from other parasitic infections, e.g., ecchinococcosis, pentastomiasis, paragonimiasis, and histoplasmosis, or other conditions such as tuberculosis, alveolar carcinoma and metastases. this is due to the varying reactions of the lung tissues and to the difference in size of the larvae. the reason for the rarity of lung involvement is probably explained by the life cycle of the taenia solium parasite. humans may serve as intermediate hosts for the adult larvae which favor muscle and brain tissue to complete their life cycle. another reason for the rarity of lung involvement may be that pulmonary lesions are overlooked because of the asymptomatic clinical features and because patients usually present with neurological symptoms. although rare, pulmonary cysticercosis does occur; therefore it should not be overlooked in differential diagnosis of multiple lung opacities just because they are relatively frequent in korea. like brain cysticercosis, which is treated with praziquantel (50 mg / kg / day) for 15 days, pulmonary cysticercosis, in this case, was treated completely by the same dose, even though no regimen has yet been established.	cysticercosis, which has a worldwide distribution is found in man, who is usually infected by eating inadequately cooked pork or other contaminated food. cysticercosis develops most commonly in the muscles and brain. pulmonary involvement is very rare and also difficult to recognize because pulmonary lesions caused by the presence of cysticerci are difficult to discern from pulmonary infiltrates, because other parasitic infestations or tuberculosis, as well as metastatic lesions, produce similar chest x - ray findings and similar clinical symptoms.we experienced a case of pulmonary cysticercosis confirmed at gyeongsang national university hospital by means of an open lung biopsy and treated successfully with praziquantel (50 mg / kg per day for 15 days).this case seems to indicate that pulmonary cysticercosis should be considered as a diagnostic possibility in patients with nodular infiltrates in the lungs, especially in endemic areas, until such infiltrates are otherwise explained.
the word pterygium is derived from the greek word pterygion , meaning wing. it refers to an elevated, superficial, external ocular triangular fibro vascular mass that usually forms over the perilimbal conjunctiva and extends onto the corneal surface.1 pterygia are reported to affect males twice as frequently as females and it is uncommon for patients to present with pterygia prior to the age of 20 years.2 countries nearer the equator have higher rates of pterygia, especially in rural areas. a possible reason for this geographic variation is that ultraviolet light may be a risk factor for the development of pterygia. other potential risk factors include ocular dryness, inflammation, occupational exposure to irritants, and ocular dominance where the dominant eye tends to be kept open more in bright sunlight.3 pterygia may be classified clinically as being active or inactive. the progression of active pterygium onto the cornea can lead to both significant corneal distortion and the development of corneal astigmatism.4 patients with pterygia might present with a variety of complaints, ranging from no symptoms to significant redness, swelling, itching, irritation, and blurring of vision associated with elevated lesions of the conjunctiva and contiguous cornea in one or both eyes.5,6 it is more common for the pterygium to present on the nasal side, although it can present temporally as well as in other locations.7 patients with pterygia can be observed without intervention unless the lesions exhibit growth towards the center of the cornea or if the patient exhibits symptoms of significant redness, discomfort, or alterations in visual function.8 medical treatments include artificial tears to lubricate the ocular surface and anti - inflammatory drops to reduce any inflammation. major indications for surgery include involvement of the visual axis, progressive growth that threatens the visual axis, induced irregular astigmatism that reduces vision, and restriction of ocular motility. it is best to excise a pterygium before it reaches a point where it obscures part of the pupil that is why a pterygium extending 3 mm or more from the limbus is a major indication for surgery.9,10 minor indications for surgery include contact lens intolerance, chronic irritation, and disfigurement. while pterygium surgery is often considered a simple office procedure, the decision to operate, especially for minor indications, must weigh the risk of complications or recurrence against the degree to which the symptoms trouble the patient.10 the major endpoints of pterygium removal relate to recurrence, which may be corneal or conjunctival.11 there are other minor less common endpoints such as ocular motility restriction, cosmetic appearance, or tear outflow.1214 simple excision of pterygium (bare sclera technique) is associated with a high rate of recurrence (40%80%) that may be more aggressive than the initial lesion.15,16 other surgical techniques such as conjunctival graft or rotation flap, amniotic membrane graft, thermal cautery, or beta irradiation are associated variably with lower recurrence rates.10,16 the use of medical adjuvant agents capable of stopping pterygium regrowth, such as 5-fluorouracil (5-fu) and mitomycin c (mmc), have had extensive practice worldwide with variable .10 in this study we evaluated the efficacy and safety of the use of antimetabolites as adjunctive medical therapy after simple excision of primary pterygium. the current prospective, randomized clinical trial study was conducted on 50 patients (30 males, 20 females) with bilateral primary pterygium during a 3-year period. the studied patients fulfilled the following inclusion criteria: age > 20 years, living in rural areas or working in outdoor conditions, and pterygium size of 2.5 mm or more measured from the limbus to the cornea. the patients were grouped into two groups: group 1 (25 patients) underwent excision of pterygia with a bare sclera technique for one eye and mmc was applied intraoperatively for the other eye. in group 2 (25 patients), 5-fu was used instead of mmc. after applying an eyelid speculum, the pterygium head was lifted off the corneal surface by blunt dissection from the head in a smooth plane to bare sclera at the limbus, then the pterygium base was excised at its nasal end. approximately 3 mm of bare sclera was left in one eye; in the fellow eye the antimetabolite was applied intraoperatively at the scleral bed after the excision for 3 minutes using a weck - cel sponge (beaver - visitec international, inc, waltham, ma) soaked in 0.5 mg / ml mmc solution or 50 mg / ml 5-fu solution. extreme care was taken not to apply the antimetabolite on the cornea and thorough irrigation with at least 30 ml balanced salt solution was used to make sure that all the antimetabolite was washed out. all patients received ciprofloxacin (antibiotic) and dexamethasone (steroid) eye drops for four weeks postoperatively. chi square test, odds ratio (or), and frequency distribution were used to determine significance levels; p - values < 0.05 were considered statistically significant. after applying an eyelid speculum, the pterygium head was lifted off the corneal surface by blunt dissection from the head in a smooth plane to bare sclera at the limbus, then the pterygium base was excised at its nasal end. approximately 3 mm of bare sclera was left in one eye; in the fellow eye the antimetabolite was applied intraoperatively at the scleral bed after the excision for 3 minutes using a weck - cel sponge (beaver - visitec international, inc, waltham, ma) soaked in 0.5 mg / ml mmc solution or 50 mg / ml 5-fu solution. extreme care was taken not to apply the antimetabolite on the cornea and thorough irrigation with at least 30 ml balanced salt solution was used to make sure that all the antimetabolite was washed out. all patients received ciprofloxacin (antibiotic) and dexamethasone (steroid) eye drops for four weeks postoperatively. chi square test, odds ratio (or), and frequency distribution were used to determine significance levels; p - values < 0.05 were considered statistically significant. age of the patients ranged from 2340 years with a mean age of 36.4 years. this study showed that patients who underwent primary pterygium excision without using antimetabolites as adjunctive agents developed higher recurrence rates than those who received them (mmc in group 1, 5-fu in group 2). or = 2.3 in general. use of mmc was superior to 5-fu in prevention of recurrence (or = 5.4 for mmc and only 1.4 for 5-fu ; table 2). pterygium is a common external ocular problem that is closely related to exposure to uv light, especially in hot, dusty, and dry climates. true estimates of its prevalence in iraq are not well known but having a risky environment makes it a very frequently encountered disease in our daily practice; furthermore, recurrent cases after surgical excision are expected because of the environmental risks. pterygium is a proliferative disease with hyperplastic growth of the corneo - conjunctival fibro vascular tissue onto the cornea.17 surgical therapy can be used to successfully manage pterygia; however, recurrence remains a problem.18 recurrence of the pterygium is usually defined as a corneal recurrence that is evidenced by growth of fibrovascular tissue across the limbus onto the cornea. this usually excludes the persistence of deeper corneal vessels and corneal scaring, which may be left even after adequate pterygium removal. 12 bunching of conjunctiva and formation of parallel loops of vessels, which aim almost like an arrowhead at the limbus, usually denotes a conjunctival recurrence; although, this appearance is sometimes determined by the method of removal. for example, a simple conjunctival closure that may lend itself more to the appearance of a conjunctival recurrence than a broad conjunctival autograft.13,14 cosmetic appearance is another postoperative endpoint; however, as long as there are no structural changes which affect vision or movement and as long as the patient is asymptomatic with respect to irritation then the issue of cosmetic appearance could not realistically be defined in terms of what can be seen at the slit - lamp magnification, but rather what the patient observes by looking in the mirror or other relatives and friends observe by looking at the patient s eye.19 simple excision leaving the sclera bare is the simplest approach for surgical management of pterygium but with the highest recurrence rate.20 the frustration of watching the rapid, inexorable progression of recurrent pterygium after what seemed to be an adequate surgical excision has provided investigators with strong motivation to find a medical adjunctive treatment capable of preventing pterygium regrowth. two such agents, 5-fu and mmc, are being used extensively worldwide with variable success. 5-fu is a pyrimidine analogue that inhibits dna synthesis and is active on the s phase (synthesis phase) of the cell cycle. its effect is most pronounced on rapidly proliferating cells that occur in response to inflammation. it is an alkylating agent rather than an antimetabolite, and selectively inhibits dna replication, mitosis, and protein synthesis. mmc inhibits proliferation of fibroblasts, suppresses vascular ingrowths, and is much more potent than 5-fu.16 fortunately, we did not face any of the expected serious complications secondary to the use of the antimetabolites during the follow - up period. looking at the recurrence rates in the treated groups, simple excision leaving the sclera bare yielded about 33% recurrence rate in all patients. mitomycin was utilized as an ancillary treatment due to its antiproliferative effects; it reduced the recurrence rate successfully to 8% in our series. the antifibroblastic activity of mmc is known to be more than that of 5-fu; accordingly, we observed that the recurrence rate with mmc was much less than that of 5-fu (table 2). variable recurrence rates of pterygia have been reported and surgical are undoubtedly influenced by several factors, including type of pterygium, size, surgical technique, geographical location, and the surgeon s skill. hameed reported a 20% recurrence rate after simple pterygium excision.20 in the usa, vastine reported up to 60% recurrence rate after simple excision leaving the sclera bare, and zloty was faced with only two recurrent cases after performing 400 pterygium exisions with mmc application in an average follow - up of 3 years (cited in gans).10 literature reveals 0%10% recurrence rates after using intraoperative mmc adjunctively with surgical excision.16 although the use of 5-fu as an adjunctive medical therapy to reduce the failure rates of trabeculectomy has been widely explored, to our knowledge no sufficient data are available for comparison with regards to the use of 5-fu as an adjuvant therapy after surgical excision of pterygium. optimum concentration and exposure time for mmc is not well known and vary between 0.10.5 mg / ml and 15 minutes. in general, low or intermediate risk of recurrence indicates the use of a low concentration (0.2 mg / ml), whilst high risk implies the need for a higher concentration (0.40.5 mg / ml). higher concentrations and extended exposure times are associated with an increased risk of complications.15,21 few data are available to compare regimens, and most surgeons increase concentration or duration based on risk factors for recurrence. gans reported a safe dose of 0.3 mg / ml for 5 minutes, and that zloty applied 0.4 mg / ml for 60 seconds only.10 the concentration we used was 0.5 mg / ml (0.05%) for 3 minutes because all of our patients were defined as having high risks of recurrence, all were young patients with relatively large pterygia, and they share the same exposure risks to environmental factors. this concentration yielded only 8% recurrences and there were no serious side effects or signs of toxicity during the whole follow - up period. the mean follow - up period in the current study was 18.8 months; all our recurrent cases were reported within the first 48 months postoperatively. table 3 shows a summary of the of other studies with comparable follow up periods in terms of numbers of eyes with primary pterygium, mmc concentration, intraoperative exposure time, recurrence rates, and mean follow - up periods.22 even when used in correct dosages for brief periods, mitomycin has been associated with prolonged, irreversible stem cell damage with ant chronic keratopathy and toxic keratoconjunctivitis. it is important to note that any use of topical mmc can be toxic and may cause visually significant complications such as aseptic scleral necrosis and infectious sclerokeratitis, secondary glaucoma, uveitis, cataract, corneal edema, corneal perforation, and endophthalmitis.16,23 moreno et al reported corneal melting 2 weeks after the use of mmc in surgical management of pterygium and mentioned that scleral thinning can occur as early as 1 week postoperatively or even years after the use of mmc (cited in menghini et al).21 the critical point regarding these complications is that they may occur many months, or even years, after the use of mmc. the statistically significant reduced recurrence rate that we found and the rarity of postoperative complications during a reasonably long follow up period justify the use of mmc as an adjuvant drug to be applied during surgical excision of pterygium. however, it is not advisable to think about using such medication topically postoperatively because of the possible serious complications that might be encountered during long periods of topical administration or because of drug abuse. intraoperative use should be controlled with regards to the dose and time of application, unlike postoperative topical application which would be dependent on patient administration and overdosing might be encountered, increasing the potential of developing side effects secondary to local tissue damage. upon reviewing the characteristics of recurrent cases in our series, the recurrent pterygia after using mmc were atrophic with scanty vascular components (figure 2a) and they were hardly noticeable by the patients (ie, cosmetically insignificant). on the other hand, the recurrent cases following application of 5-fu shared a highly vascular mass with variable rate of regrowth (figure 2b), which suggests that the role of such an agent in vascular growth suppression is negligible when compared to the antifibroblastic activity. several studies have recently emphasized the importance of vascular endothelial growth factor in the development and recurrence of pterygium.21 therefore, the timely recognition and treatment of an impending recurrence with subconjunctival anti - vegf drugs injection is crucial in stopping recurrences, especially when unusual vascular growth is noticed. the intraoperative application of antimetabolites is of value in reducing the recurrence rate after simple primary pterygium excision. both mmc and 5-fu were safe during the follow up period but a statistically significant high success rate and more cosmetically acceptable appearance after mmc use justifies recommending its use to be superior to 5-fu as a medical adjuvant in the surgical management of primary pterygium.	pterygium is a proliferative disease with hyperplastic growth of corneoconjunctival fibro vascular tissue onto the cornea. surgical therapy can be used to successfully manage pterygia; however, recurrence remains a problem. to reduce recurrence , surgical management may include autoconjunctival grafting, lamellar keratoplasty, amniotic membrane transplantation, and intraoperative antimetabolites application.purposeto assess the safety and the efficacy of intraoperative mitomycin c (mmc) and 5-fluorouracil (5-fu) application in preventing recurrence of pterygium after excision.patients and methodsthe study design is a prospective, randomized clinical trial. a total of 50 patients with bilateral pterygium were recruited for the study. the first group of patients underwent surgical excision of the pterygium with bare sclera in one eye and mmc was applied as adjunctive therapy for the other eye. in the second group 5-fu was used instead of mmc. recurrences and postoperative complications were measured in the two groups. the mean follow up period of the patients was 18.8 months. chi square test, odds ratio, and frequency distribution were used to determine significance levels; p - values < 0.05 were considered statistically significant.in group 1 the recurrence rate was 8% for the mmc treated eyes and 32% for their fellow eyes (p = 0.03). in group 2 the rate was 18% for the 5-fu treated eyes and 34% for their fellow eyes (p = 0.07). no serious complications were recorded in either group.both mmc and 5-fu reduce the recurrence rate of pterygium after simple surgical excision; statistically, the effect of the former was significant, but insignificant for the latter. both antimetabolites were safe during the whole study period, but 5-fu recurrent cases showed cosmetically unacceptable appearances with excessive vascularization. mmc, but not 5-fu, is recommended as an adjunctive therapy to prevent recurrence of pterygium after surgical excision.
symptoms include discomfort or burning sensation, photophobia, blurred or abnormal vision, and eye watering, and the longer - term sequelae can be serious.1 factors contributing to dry eye are numerous, and include aging, autoimmune disorders, infection, abnormalities of the lipid tear layer, menopause, wearing contact lenses, exposure to air conditioning, and use of computers. a modern understanding of dry eye includes etiologic factors such as inflammatory mediators, instability of the tear film, meibomian gland dysfunction, and hyperosmolarity of the tear film.2 the incidence of dry eye has risen considerably in recent years,3,4 partly due to increased computer use in air - conditioned offices. often known as office eye syndrome,5 it from exposure of the precorneal tear film, corneal epithelium, and conjunctiva to artificial heating and dehumidified air provided by air conditioning and is likely amplified by the reduced eye blink rate and increased tear film evaporation consequent to extended viewing of computer screens. the everyday use of computers may be specifically related to the increased reporting of dry eye syndrome in younger populations,6 as may routine contact lens use, which frequently in changes in the corneal epithelium and lacrimal film. a variety of treatments are available for dry eye, including surgery, moisture - retaining spectacles, and anti - inflammatory drugs, but artificial tear preparations are the cornerstone of dry eye management across the severity spectrum and almost 20 of these products are currently available in russia.7 they are widely used for the prolonged treatment of patients with early - stage dry eye syndrome and for improving ocular comfort in patients with transient secondary tear film instability (eg, office workers). some such preparations have been developed based on the naturally occurring polysaccharide, hyaluronic acid. this has excellent water - retaining and lubricant properties, as well as viscoelastic effects that aid vision during blinks but maintain hydration and lubrication between blinks.8,9 studies have shown good efficacy in patients with dry eye syndrome.1014 hyperosmolarity has recently been identified as an important factor in the etiology of dry eye; tear instability and hyperosmolarity are now considered to be interacting and key mechanisms in dry eye and precursors to inflammatory processes and corneal damage.15 indeed, the most recent definition of dry eye by the dry eye workshop makes specific mention of tear hyperosmolarity. clinical studies also suggest that hypotonic eye drops have an advantage over isotonic eye drops in the treatment of dry eye.1619 whilst the efficacy of hyaluronate - containing tear substitutes has been well demonstrated, there remain issues over the use of preservatives in such therapies, particularly when the treatment is likely to be long - term. it is now generally recognized that elimination of preservatives such as benzalkonium is important in the long - term safety and tolerability of ocular preparations,20,21 to the extent that some authorities consider the elimination of preservatives from tear substitutes as one of the most critical advances in the treatment of dry eye.22 the preservative - free hyaluronic acid preparation, hylabak (laboratoires thea, clermont ferrand, france), has recently been developed to address this issue. hylabak comprises hyaluronic acid 0.15% and actinoquinol in a hypo - osmolar, preservative - free abak bottle (laboratoires thea). sterility of the open container is assured by the abak system, that consists of a multidose eye drop dispenser closed by an adapted, small - pore sterilizing filter.23 we present here the findings from four recently published studies with broadly comparable methodologies conducted in russia that have assessed hylabak for the treatment of dry eye.2427 patients enrolled in these studies had a range of symptoms and etiologies ranging from contact lens use and visually intensive occupations to persistent meibomian blepharitis, sjgren s syndrome, and the sequelae of ocular surgery. patient characteristics and a methodologic summary of the four individual studies are shown in table 1. one study,25 however, compared its findings with those from 25 patients treated with tear naturale (0.1% dextran 70, 0.3% hydroxypropyl methylcellulose, preserved with benzylalkonium , alcon laboratories, fort worth, tx, usa) in previous studies.28 a total of 134 children and adults (aged 755 years) were enrolled in the studies. there were various etiologies accounting for the dry eye syndrome, including contact lens use, intensive office work, adenovirus eye infection, postmenopausal status, persistent meibomian blepharitis, sjgren s syndrome, phacoemulsification with intraocular lens implantation, and refractive surgery (table 2). the patients were treated with hylabak for 2 weeks to 2 months, depending on the study, and assessments were performed at various times during treatment. all studies assessed subjective sensations / complaints using a 04 scale (0, absence of symptoms ; 1, slight feeling of discomfort ; 2, evident feeling of discomfort ; 3, the worst feeling of discomfort), tear production (schirmer s test, without anesthesia) and tear stability / break - up time (norn s test);29 other assessments (impression cytology and biomicroscopy, staining, tear osmolarity) performed in the individual studies are shown in table 1. all four studies showed an improvement in dry eye syndrome of varying etiologies with hylabak treatment (table 2). a brief summary of the from each of the individual studies is given below. maychuck and yani24 conducted an open study in 40 patients with dry eye syndrome (adenovirus infection, soft contact lens use, phacoemulsification with intraocular lens implantation, and refractive surgery) who were treated for 28 days. assessments were performed on days 1, 7, 14, 21, and 28, and included subjective complaints, schirmer s test, norn s test, meniscometry, osmometry, and conjunctival xerosis. for patients divided into those with mild or moderate disease showed that subjective complaints were reduced or had disappeared from the first days of treatment irrespective of symptom severity. complaints were typical of dry eye syndrome and included feelings of a foreign body in the eye, eye reddening, eyelid edema, itching, burning sensation, and variation in visual acuity.. tear production (schirmer s test) and precorneal tear break - up time (norn s test) were increased at each assessment in patients with both mild and moderate disease (table 2). similarly, there was an improvement to near normal values in tear osmolarity from 327 0.6 and 336 1.2 mosm / l in the mild and moderate groups, respectively, on day 1 to 286 1.7 and 302 1.5 mosm / l, respectively, on day 28. in both mild and moderate groups combined, the height of the tear meniscus increased markedly from 0.38 0.2 mm on day 1 to 0.58 0.2 mm on day 28, and conjunctival xerosis fell from 4.55 0.5 points on day 1 to 0.4 0.4 points on days 28. the authors concluded that hylabak showed good therapeutic efficacy in the treatment of dry eye syndrome of varying etiologies and severity, and that it was well tolerated during prolonged use. brjesky et al25 conducted an open study in 32 patients with dry eye syndrome (deficiency of tear film production, persistent meibomian blepharitis, sjgren s syndrome) who were treated for 28 days, and the were compared with those from 25 patients treated with tear naturale in previous studies. assessments were performed on days 3, 7, 14, and 28, and included subjective discomfort, schirmer s test, norn s test, tear meniscus index, and basal and total tear production. subjective discomfort, measured on a four - point scale from 0 (no symptoms) to 3 (worst feelings of discomfort), was significantly (p < 0.05 versus baseline) reduced by hylabak from day 7 onwards in postmenopausal women (2.0 0.2 at baseline versus 1.2 0.1 on day 28) and from day 3 onwards in patients with persistent meibomian blepharitis (2.1 0.2 at baseline versus 0.6 0.1 on day 28), or sjgren s syndrome (2.6 0.2 at baseline versus 1.4 0.2 on day 28) (figure 1). this compares with findings using tear naturale in previous studies in which significant differences were reported from day 7 onwards in postmenopausal women (2.0 0.1 at baseline versus 1.4 0.2 on day 28) and those with meibomian blepharitis, and from day 14 onwards (1.9 0.2 at baseline versus 0.8 0.1 on day 28) in those with sjgren s syndrome (2.6 0.2 at baseline versus 1.7 0.1 on day 28). objective signs (four - point scale) were significantly (p < 0.05) reduced by hylabak from day 14 onwards in postmenopausal women (1.4 0.1 at baseline versus 0.3 0.1 on day 28), in patients with sjgren s syndrome (2.1 0.2 at baseline versus 1.3 0.1 on day 28), and from day 7 onwards in those with meibomian blepharitis (1.1 0.1 at baseline versus 0.6 0.1 on day 28, figure 2). in contrast, no significant changes were seen with tear naturale in patients with meibomian blepharitis (1.2 0.1 at baseline versus 0.7 0.2 on day 28) or sjgren s syndrome (2.1 0.3 at baseline versus 1.5 0.1 on day 28), and a significant decrease was seen only on day 28 in post - menopausal women (1.3 0.2 at baseline versus 0.5 0.1 on day 28). tear film stability (norn s test) was significantly increased from day 3 onwards in all groups of patients given hylabak, and in postmenopausal women and patients with sjgren s syndrome given tear naturale; patients with meibomian blepharitis given tear naturale had a significant improvement only from day 7 onwards. tear meniscus index significantly improved from day 3 onwards in all groups of patients, irrespective of treatment. increases from 1.1 0.1, 1.9 0.1, and 1.1 0.1 at baseline to 2.1 0.2, 2.6 0.1, and 2.2 0.1 at day 28 were observed with hylabak in postmenopausal women and those with meibomian blepharitis or sjgren s syndrome, respectively. the corresponding increases in patients given tear naturale were from 1.1 0.1, 1.8 0.2, and 1.1 0.1 at baseline to 1.9 0.1, 2.4 0.1, and 1.9 0.1 on day 28. basal and total tear production was not significantly changed by either treatment in any group of patients. the showed that both hylabak and tear naturale were effective in treating the various etiologies of dry eye syndrome, but that benefits with regard to subjective discomfort, objective signs, and tear film stability were more marked with hylabak (although the differences between the groups did not reach statistical significance). in an open study by petrayevsky et al,26 32 women with dry (office) eye syndrome due to intensive use of personal computers in an air - conditioned office environment were treated for 2 weeks. assessments performed before and after treatment included recording of subjective complaints, schirmer s test, norn s test, and cytologic analysis of the bulbar conjunctiva. subjective signs and symptoms, measured on a four - point scale from 0 (no symptoms) to 3 (severe), were improved after hylabak treatment in patients with mild or moderate symptoms (figure 3). patients with mild symptoms showed nonspecific objective symptoms (eg, local edema of the bulbar conjunctiva involving the free edge of the eyelid and mild hyperemia), whilst those with moderate symptoms showed both specific (eg, reduction of tear meniscus at the lid margin) and nonspecific objective symptoms, all of which were improved or resolved after hylabak treatment. functional tests (schirmer s test and tear break - up time assessed by norn s test) were also significantly (p < 0.01) improved after hylabak (table 2), with the exception of schirmer s test in patients with mild symptoms. cytologic examination of the conjunctiva showed that 50% of patients with mild symptoms had early loss of goblet cells and the remainder had total loss of goblet cells, all without keratinization (tseng stage 1 and 2, respectively). there was a marked improvement after hylabak treatment, with 28% of patients showing normalization and 61% with tseng stage 1 (mild dry eye population). among the patients with moderate symptoms, tseng stage 1 was seen in 36% and tseng stage 2 in 64% at baseline. the authors concluded that hylabak ed in subjective and objective improvement, as well as normalization of functional tests and the cytologic profile of the conjunctiva, in a population suffering from office eye syndrome. thirty children and adolescents were enrolled in this open study by nagorsky et al,27 and were assessed according to whether they wore soft contact lenses during the day or orthokeratologic lenses at night. assessments performed before treatment and after 1 and 2 months included subjective sensations, schirmer s test, norn s test, meniscometry and conjunctival xerosis. amongst these 20 patients, there was a marked improvement in subjective complaints after one month of hylabak treatment, that was increased further after 2 months (figure 4). when asked about the duration of the most comfortable period of wearing their contact lenses this was supported by improvements in objective measures to within the normal range (table 3). there was also a significant (p < 0.05) increase in the height of the tear meniscus, from 0.62 0.20 mm to 0.95 0.20 mm after 2 months, and a reduction in the intensity of corneal xerotic changes. there was also a reduction in corneal xerotic change intensity with hylabak treatment in the 10 patients wearing orthokeratologic lenses, and patients reported that it was faster, easier, and more comfortable to manipulate their lenses. there were no cases of unpleasant sensation, discomfort, or prolonged blurred vision, and most patients noted improved comfort compared with previous eye drops. the authors concluded that hylabak had good therapeutic efficacy and was easy to use, cost - effective, and well tolerated in this group of young patients. maychuck and yani24 conducted an open study in 40 patients with dry eye syndrome (adenovirus infection, soft contact lens use, phacoemulsification with intraocular lens implantation, and refractive surgery) who were treated for 28 days. assessments were performed on days 1, 7, 14, 21, and 28, and included subjective complaints, schirmer s test, norn s test, meniscometry, osmometry, and conjunctival xerosis. for patients divided into those with mild or moderate disease showed that subjective complaints were reduced or had disappeared from the first days of treatment irrespective of symptom severity. complaints were typical of dry eye syndrome and included feelings of a foreign body in the eye, eye reddening, eyelid edema, itching, burning sensation, and variation in visual acuity. this was confirmed by objective measures. tear production (schirmer s test) and precorneal tear break - up time (norn s test) were increased at each assessment in patients with both mild and moderate disease (table 2). similarly, there was an improvement to near normal values in tear osmolarity from 327 0.6 and 336 1.2 mosm / l in the mild and moderate groups, respectively, on day 1 to 286 1.7 and 302 1.5 mosm / l, respectively, on day 28. in both mild and moderate groups combined, the height of the tear meniscus increased markedly from 0.38 0.2 mm on day 1 to 0.58 0.2 mm on day 28, and conjunctival xerosis fell from 4.55 0.5 points on day 1 to 0.4 0.4 points on days 28. the authors concluded that hylabak showed good therapeutic efficacy in the treatment of dry eye syndrome of varying etiologies and severity, and that it was well tolerated during prolonged use. brjesky et al25 conducted an open study in 32 patients with dry eye syndrome (deficiency of tear film production, persistent meibomian blepharitis, sjgren s syndrome) who were treated for 28 days, and the were compared with those from 25 patients treated with tear naturale in previous studies. assessments were performed on days 3, 7, 14, and 28, and included subjective discomfort, schirmer s test, norn s test, tear meniscus index, and basal and total tear production. subjective discomfort, measured on a four - point scale from 0 (no symptoms) to 3 (worst feelings of discomfort), was significantly (p < 0.05 versus baseline) reduced by hylabak from day 7 onwards in postmenopausal women (2.0 0.2 at baseline versus 1.2 0.1 on day 28) and from day 3 onwards in patients with persistent meibomian blepharitis (2.1 0.2 at baseline versus 0.6 0.1 on day 28), or sjgren s syndrome (2.6 0.2 at baseline versus 1.4 0.2 on day 28) (figure 1). this compares with findings using tear naturale in previous studies in which significant differences were reported from day 7 onwards in postmenopausal women (2.0 0.1 at baseline versus 1.4 0.2 on day 28) and those with meibomian blepharitis, and from day 14 onwards (1.9 0.2 at baseline versus 0.8 0.1 on day 28) in those with sjgren s syndrome (2.6 0.2 at baseline versus 1.7 0.1 on day 28). objective signs (four - point scale) were significantly (p < 0.05) reduced by hylabak from day 14 onwards in postmenopausal women (1.4 0.1 at baseline versus 0.3 0.1 on day 28), in patients with sjgren s syndrome (2.1 0.2 at baseline versus 1.3 0.1 on day 28), and from day 7 onwards in those with meibomian blepharitis (1.1 0.1 at baseline versus 0.6 0.1 on day 28, figure 2). in contrast, no significant changes were seen with tear naturale in patients with meibomian blepharitis (1.2 0.1 at baseline versus 0.7 0.2 on day 28) or sjgren s syndrome (2.1 0.3 at baseline versus 1.5 0.1 on day 28), and a significant decrease was seen only on day 28 in post - menopausal women (1.3 0.2 at baseline versus 0.5 0.1 on day 28). tear film stability (norn s test) was significantly increased from day 3 onwards in all groups of patients given hylabak, and in postmenopausal women and patients with sjgren s syndrome given tear naturale; patients with meibomian blepharitis given tear naturale had a significant improvement only from day 7 onwards. tear meniscus index significantly improved from day 3 onwards in all groups of patients, irrespective of treatment. increases from 1.1 0.1, 1.9 0.1, and 1.1 0.1 at baseline to 2.1 0.2, 2.6 0.1, and 2.2 0.1 at day 28 were observed with hylabak in postmenopausal women and those with meibomian blepharitis or sjgren s syndrome, respectively. the corresponding increases in patients given tear naturale were from 1.1 0.1, 1.8 0.2, and 1.1 0.1 at baseline to 1.9 0.1, 2.4 0.1, and 1.9 0.1 on day 28. basal and total tear production was not significantly changed by either treatment in any group of patients. the showed that both hylabak and tear naturale were effective in treating the various etiologies of dry eye syndrome, but that benefits with regard to subjective discomfort, objective signs, and tear film stability were more marked with hylabak (although the differences between the groups did not reach statistical significance). in an open study by petrayevsky et al,26 32 women with dry (office) eye syndrome due to intensive use of personal computers in an air - conditioned office environment were treated for 2 weeks. assessments performed before and after treatment included recording of subjective complaints, schirmer s test, norn s test, and cytologic analysis of the bulbar conjunctiva. subjective signs and symptoms, measured on a four - point scale from 0 (no symptoms) to 3 (severe), were improved after hylabak treatment in patients with mild or moderate symptoms (figure 3). patients with mild symptoms showed nonspecific objective symptoms (eg, local edema of the bulbar conjunctiva involving the free edge of the eyelid and mild hyperemia), whilst those with moderate symptoms showed both specific (eg, reduction of tear meniscus at the lid margin) and nonspecific objective symptoms, all of which were improved or resolved after hylabak treatment. functional tests (schirmer s test and tear break - up time assessed by norn s test) were also significantly (p < 0.01) improved after hylabak (table 2), with the exception of schirmer s test in patients with mild symptoms. cytologic examination of the conjunctiva showed that 50% of patients with mild symptoms had early loss of goblet cells and the remainder had total loss of goblet cells, all without keratinization (tseng stage 1 and 2, respectively). there was a marked improvement after hylabak treatment, with 28% of patients showing normalization and 61% with tseng stage 1 (mild dry eye population). among the patients with moderate symptoms, tseng stage 1 was seen in 36% and tseng stage 2 in 64% at baseline. the authors concluded that hylabak ed in subjective and objective improvement, as well as normalization of functional tests and the cytologic profile of the conjunctiva, in a population suffering from office eye syndrome. thirty children and adolescents were enrolled in this open study by nagorsky et al,27 and were assessed according to whether they wore soft contact lenses during the day or orthokeratologic lenses at night. assessments performed before treatment and after 1 and 2 months included subjective sensations, schirmer s test, norn s test, meniscometry and conjunctival xerosis. amongst these 20 patients, there was a marked improvement in subjective complaints after one month of hylabak treatment, that was increased further after 2 months (figure 4). when asked about the duration of the most comfortable period of wearing their contact lenses, the patients reported a more than three - fold increase with hylabak treatment. this was supported by improvements in objective measures to within the normal range (table 3). there was also a significant (p < 0.05) increase in the height of the tear meniscus, from 0.62 0.20 mm to 0.95 0.20 mm after 2 months, and a reduction in the intensity of corneal xerotic changes. there was also a reduction in corneal xerotic change intensity with hylabak treatment in the 10 patients wearing orthokeratologic lenses, and patients reported that it was faster, easier, and more comfortable to manipulate their lenses. there were no cases of unpleasant sensation, discomfort, or prolonged blurred vision, and most patients noted improved comfort compared with previous eye drops. the authors concluded that hylabak had good therapeutic efficacy and was easy to use, cost - effective, and well tolerated in this group of young patients. all four studies found that hylabak ed in a marked improvement in patients with dry eye syndrome of heterogeneous etiology. subjective sensations and complaints were reduced, and these findings were supported by from a wide range of objective measures, including schirmer s test, norn s test, impression cytology and biomicroscopy, staining, and tear osmolarity. complete resolution of signs and symptoms and normalization of functional tests and cytologic profile was reported in some patients. hylabak ed in rapid relief from symptoms. in one study assessing effects during the first week of treatment, there were statistically significant improvements in subjective discomfort, tear film stability, and tear meniscus index from day 3 onwards, with objective signs improving significantly from day 7 in some patients. in contrast, significant improvements with tear naturale did not occur until later in treatment. in addition, benefits on subjective discomfort, objective signs, and tear film stability were more marked with hylabak than with tear naturale. not all artificial tear preparations are suitable for all types of dry eye syndrome patients. whilst preserved artificial tears may be more convenient for short - term use, for example, a preservative - free formulation would be more appropriate where treatment is likely to continue on a long - term basis. a review of the patients included in the four studies discussed here shows that hylabak was safe and effective in a wide range of dry eye syndrome etiologies including contact lens use, intensive exposure to air - conditioning and computer use, eye infection, postmenopausal status, meibomian blepharitis, sjgren s syndrome, phacoemulsification with intraocular lens implantation, and refractive surgery. there was also a wide age range, with one study enrolling children from 7 years of age. hylabak proved to be well tolerated, effective and easy to use in these young patients, and patients reported that they found contact lens use faster, easier, and more comfortable compared with their previous eye drops. treatment was given daily in all four studies, suggesting that hylabak is suitable for patients with secondary transitional dry eye syndrome, such as office workers who require regularly improved ocular comfort. the findings in this wide - ranging population suggest that hylabak is an ideal choice for first - line treatment of dry eye syndrome, irrespective of the etiology of the disorder. in , hylabak provided rapid and safe relief from the signs and symptoms of dry eye syndrome, as well as improvement in objective measures, in patients with a wide range of dry eye etiologies.	artificial tear preparations are important in the management of dry eye syndrome. we present the findings from four recently published studies conducted in russia assessing hylabak (marketed as hyabak in europe), a preservative - free hyaluronic acid preparation, for the treatment of dry eye syndrome. all studies had an open, noncomparative design, but one compared the findings with those from 25 patients treated with tear naturale in previous studies. a total of 134 children and adults were enrolled, and the etiologies of dry eye syndrome included contact lens use, intensive office work, adenovirus eye infection, postmenopausal status, persistent meibomian blepharitis, sjgren s syndrome, phacoemulsification with intraocular lens implantation, and refractive surgery. the patients were treated with hylabak for 2 weeks to 2 months. all studies showed that hylabak ed in marked improvement as assessed by subjective sensations / complaints, schirmer s test, norn s test, impression cytology and biomicroscopy, staining, and tear osmolarity. greater benefits were also reported compared with tear naturale, including a faster onset of action. hylabak was well tolerated. in , hylabak provided rapid and safe relief from the signs and symptoms of dry eye syndrome, as well as improvement in objective measures, in a wide range of patients.
healthy leaves of capsicum annuum, sasa borealis, potentilla fragarioides and viola mandshurica collected from an arable site and its surrounding field areas in goesan - gun, chungbuk, korea (128 1'e, 36 46'n). only green leaves without signs of insect or microbial injury were used for fungal isolation. collected leaves were gently washed with ddh2o and cut with a sterile scalpel into a small piece (1 1 cm). the pieces were surface - sterilized with 5% sodium hypochlorite (5 min) and 70% ethyl alcohol (1 min). after surface sterilization, four pieces on 0.5% malt extract agar (mea) medium and the medium was incubated in dark at 25 for four weeks. fungal hyphae and structures from slide cultures were observed under a light microscope. the size of condia was measured and the shape was carefully described. for molecular identification, total dna of the fungi was extracted from cultures isolated from each plant using dneasy plant mini kit (qiagen science, usa) and was amplified using pcr. the partial internal transcribed spacer (its) region including 5.8 s ribosomal dna (rdna) was amplified with a universal primer set, its1f and its4, for fungi (gardes and bruns, 1993). the thermal cycler was programmed for 1 cycle of 3 min at 94, and 30 cycles of 1 min at 94 denaturing, 1 min at 55 annealing and 1 min at 72 extension, and finally 1 cycle of 5 min at 72 for hold. nucleotide sequences were determined using abiprism 377 automated sequencer (perkin - elmer, usa). a sequence similarity search of the national center for biotechnology information (ncbi) database was conducted using the basic local alignment search tool (blast) algorithm. two species of pestalotiopsis (p. sp1 and p. sp2) were identified using morphological and molecular characteristics (table 1). spores of four species of amf, acaulospora longula, glomus mosseae and archeospora leptotica were extracted from pure cultures using wet - sieving and sucrose density gradient centrifugation methods (daniels and skipper, 1982). the extracted spores were observed under light microscopes and identified based on their morphological characteristics such as color, shape, surface ornamentation, contents and wall structures of spores (schenck and perez, 1990). am fungal spores were surface - sterilized for inoculation (hildebrandt et al ., 2002). three treatments by types of fungal inoculum were designed in this study; amf only, endophytes only, and combination amf and endophytes. no inoculum on plants was included as control. for root inoculation with amf, plastic pots (50(w) 38(l) 8(h) cm ) were filled with the sterilized vermiculites and sands (1 : 1, v / v). seeds of five plant species (oenothera odorata, plantago asiatica, trifolium repens, isodon japonicas and aster yomena) were obtained from a commercial seed source (seedkorea co., ltd ., a potting medium were mixed with four species of amf spores, and 400 spores of each amf species were contained in a pot . for leaf inoculation, newly emerged leaves of healthy plants were inoculated with mycelium of four species of endophytes by brushing without wounds . each treatment was replicated four times at the same time . after four months of growth in a greenhouse, effects of both symbiotic fungi on species diversity, species composition, and productivity of plant community within the microcosm were examined . dry weights of plants were measured after dehydration in a drying oven at 70 for 48 hours . shannon - wiener index of species diversity ( h ') was calculated for plant community analysis (magurran, 1988). all data was analyzed with one - way analysis of variance (anova) using statistical package spss - win. the mean values were compared by fisher's least significant difference test (lsd, p < 0.05). healthy leaves of capsicum annuum, sasa borealis, potentilla fragarioides and viola mandshurica collected from an arable site and its surrounding field areas in goesan - gun, chungbuk, korea (128 1'e, 36 46'n). only green leaves without signs of insect or microbial injury were used for fungal isolation. collected leaves were gently washed with ddh2o and cut with a sterile scalpel into a small piece (1 1 cm). the pieces were surface - sterilized with 5% sodium hypochlorite (5 min) and 70% ethyl alcohol (1 min). after surface sterilization, four pieces on 0.5% malt extract agar (mea) medium and the medium was incubated in dark at 25 for four weeks. fungal hyphae and structures from slide cultures were observed under a light microscope. the size of condia was measured and the shape was carefully described. for molecular identification, total dna of the fungi was extracted from cultures isolated from each plant using dneasy plant mini kit (qiagen science, usa) and was amplified using pcr. the partial internal transcribed spacer (its) region including 5.8 s ribosomal dna (rdna) was amplified with a universal primer set, its1f and its4, for fungi (gardes and bruns, 1993). the thermal cycler was programmed for 1 cycle of 3 min at 94, and 30 cycles of 1 min at 94 denaturing, 1 min at 55 annealing and 1 min at 72 extension, and finally 1 cycle of 5 min at 72 for hold. nucleotide sequences were determined using abiprism 377 automated sequencer (perkin - elmer, usa). a sequence similarity search of the national center for biotechnology information (ncbi) database was conducted using the basic local alignment search tool (blast) algorithm. two species of pestalotiopsis (p. sp1 and p. sp2) were identified using morphological and molecular characteristics (table 1). spores of four species of amf, acaulospora longula, glomus mosseae and archeospora leptotica were extracted from pure cultures using wet - sieving and sucrose density gradient centrifugation methods (daniels and skipper, 1982). the extracted spores were observed under light microscopes and identified based on their morphological characteristics such as color, shape, surface ornamentation, contents and wall structures of spores (schenck and perez, 1990). am fungal spores were surface - sterilized for inoculation (hildebrandt et al ., 2002). three treatments by types of fungal inoculum were designed in this study; amf only, endophytes only, and combination amf and endophytes. no inoculum on plants was included as control. for root inoculation with amf, plastic pots (50(w) 38(l) 8(h) cm ) were filled with the sterilized vermiculites and sands (1 : 1, v / v). seeds of five plant species (oenothera odorata, plantago asiatica, trifolium repens, isodon japonicas and aster yomena) were obtained from a commercial seed source (seedkorea co., ltd ., a potting medium were mixed with four species of amf spores, and 400 spores of each amf species were contained in a pot . for leaf inoculation, newly emerged leaves of healthy plants were inoculated with mycelium of four species of endophytes by brushing without wounds . each treatment was replicated four times at the same time . after four months of growth in a greenhouse, effects of both symbiotic fungi on species diversity, species composition, and productivity of plant community within the microcosm were examined . dry weights of plants were measured after dehydration in a drying oven at 70 for 48 hours . shannon - wiener index of species diversity ( h ') was calculated for plant community analysis (magurran, 1988). all data was analyzed with one - way analysis of variance (anova) using statistical package spss - win. the mean values were compared by fisher's least significant difference test (lsd, p < 0.05). experimental microcosms contained an assemblage of five species of plants treated with amf and endophytes were set up in a greenhouse. after four months of growth, effects of the fungi on plant productivity were examined. the responses of plant growth to each treatment showed variation among plant species (table 2). growth of o. odorata, p. asiatica, i. japonicus and a. yomena in the community inoculated with amf was significantly increased compared to controls, while no significant growth effect of amf was detected in t. repens. also, microsms inoculated with both symbiotic fungi, the growth of o. odorata, p. asiatica, i. japonicus and a. yomena was significantly increased compared to controls. however, in the communities inoculated with endophytes, a significant increase of dry weights was observed only in p. asiatica and i. japonicas. in plants with amf, mean biomass of all 5 plant species were significantly higher than biomass of control for each plant species, indicating positive mycorrhizal responsive (mr, table 3). mr showed significant variation among plant species; highest in p. asiatica and lowest in a. yomena. the total biomass of plants in a microcosm was significantly higher in communities with amf or with both symbiotic fungi than control (table 2). however, a significant increase of growth was not observed in the microcosms inoculated with endophytes. the species diversity index (h ') was significantly increased in the plant community inoculated with symbiotic fungi (fig . the number of species in the microcosm was not changed during the period of this study, but relative abundances of each species consisting of a community were changed ( table 2). there was a positive correlation between species diversity and total dry weight of plants per microcosm (fig . however, influence of amf and endophytes to host plants is not fully understood yet . in this study, the effects of foliar endophytes and amf on plant community in experimental microcosms containing an assemblage of five species of plants were investigated . the in this study showed that amf and endophytes affect growth of host plants and plant community structure . for example, the nutrition and inter - specific relationships of plants were affected by microbes of root systems ( stanton, 1988) and therefore composition of the plant community was affected by mycorrhizas (allen and allen, 1984 ; bever, 1994). nutrients flowing through the hyphal connection plant to plant affect both growth and competition ability of plants ed in influence of the species composition in plant community. also, studies were reported that host plant species differed in their dependency on amf (hetrick et al . 1998) as in this study (table 3) showing variation among the plant species in mycorrhizal response of each plant species. although amf increased the growth of all plants in the community, they could influence relative abundance of each species in the community due to the interspecific variation of response to the mycorrhizas. as a , in this study, amf would contribute to raise the species evenness of plant community. mycorrhizal symbiosis can strongly influence the patterns and intensity of both intraspecific density effects and interspecific competition of plants (grime et al . mycorrhizas were played a vital role in the maintenance of biological diversity on the plant community . a significant increase was not observed in the productivity of the community with endophytes in this study . however, relative abundance of plant species in the communities inoculated with endophytes was changed and the species evenness of plant community was increased with inoculation of endophytes, indicating endophytes increased species diversity of plant community through increasing species evenness . in this study , endophytes changed the relative abundance by increasing growth of two species, p. asiatica and i. japonicus, in the community and the endophytes infected to these plants could promote resistance of the host plants to various environmental stresses ( west et al ., 1993). it has been observed that plants infected with the endophytic fungi had greater resistance to the drought and high temperatures (marks and clay, 1996). the increased resistance to the environmental stresses with infection of endophytes could raise inter - specific competitive ability of the plants and these factors could influence the composition of plant community (hill et al . the community treated with both symbiotic fungi showed significant differences in growth responses of plant in the community from the community with only amf and endophytes, respectively . the biomass of p. asiatica in the community treated with both symbiotic fungi was lower than when treating only amf, suggesting that the endophytes could negatively influence amf of p. asiatica . if plants were infected with endophytes, the movement of photosynthetic products toward a root could be limited, and it would be possible to affect growth of amf ( clay, 1992). also, toxic metabolites produced by endophytes may inhibit colonization of mycorrhizal fungi. in this study , the plant species diversity and the total dry weight of plants showed a significant positive correlation. these were supported by preceding studies showing that the productivity in the plant community was increased with the increased plant diversity (van der heijden et al ., 1998). of this study indicate that both symbiotic fungi may significantly affect the plant community structure. the interactions between the symbiotic fungi and host plants might increase the species diversity and plant productivity. the of study would increase the scope of understanding with respect to the role of symbiotic fungi on the plant community.	this study was conducted to investigate the effects of foliar endophytic fungi and arbuscular mycorrhizal fungi (amf) on plant community structure in experimental microcosms containing an assemblage of five species of plants (oenothera odorata, plantago asiatica, trifolium repens, isodon japonicas and aster yomena). leaves of sasa borealis, potentilla fragarioides, and viola mandshurica were collected in chungbuk, korea. endophytic fungi were isolated from the surface sterilized leaves and identified to species level using molecular and morphological techniques. four isolates of the endophytic fungi were inoculated to the leaves of host plants in the microcosms. also, three species of amf spores were extracted from pure cultures and the mixture of the three species inoculated to the roots of the plants. after four months of growth in a green house, effects of both symbiotic fungi on plant species diversity, community composition and productivity were examined. the plant species diversity showed significant differences with inoculation of the symbiotic fungi. indicate that amf significantly affect plant productivity and plant community structure.
chronic pulmonary hypertension (ph) is a disease characterized by a sustained pulmonary arterial pressure with increases in pulmonary vascular resistance. the pathogenesis of ph has been ascribed to two mechanisms, the initial event of vasoconstriction followed by remodeling of small- and medium - sized pulmonary arteries which is a hallmark of severe and advanced pulmonary hypertension. the main pathological change related to vascular remodeling is an abnormal pulmonary artery smooth muscle cells (pasmcs) hypertrophy and proliferation ing in obstruction of small pulmonary arteries. recently, more attention has been given to the facts that pulmonary inflammation could contribute to hypoxic vasoconstriction and remodeling. it has been reported that inflammatory cell infiltrates in the areas of plexiform lesions in human severe chronic pulmonary arterial hypertension. circulating inflammatory and/or progenitor cells contribute to hypoxia - induced pulmonary vascular remodeling. in addition, accumulating evidence confirms that chronic hypoxia in the increased expression of lung inflammatory cytokines and chemokines, including interleukin-1 (il-1), interleukin-6 (il-6), tumor - necrosis - factor- (tnf-), and fractalkine, which may potentiate the development of ph. for instance, il-6 promotes the development and progression of pulmonary vascular remodeling and ph through proproliferative antiapoptotic mechanisms. therefore, there is a growing interest in inflammatory mediators in the pulmonary hypertensive process. macrophage migration inhibitory factor (mif) was originally identified as a t - cell - derived cytokine that inhibits the random migration of macrophages. currently, mif is proved to be an important proinflammatory cytokine, secreted by most of the cells including t cells, macrophages / monocytes, endothelial cells, and smooth muscle cells and induce the production of a large number of inflammatory mediators, such as tnf-, il-1, il-6, and il-8. thus, many studies revealed mif to be involved in the pathogenesis of inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, sepsis, asthma, and acute respiratory distress syndrome. besides, mif may act beyond inflammatory cytokine, as suggested by its proliferative effect on vascular smooth muscle cells demonstrated in atherosclerosis. the evidence provides an idea that mif might play a role in the vascular disease. however, there is no report that mif participates in the pulmonary vascular remodeling during exposure to chronic hypoxia. in the present study for that purpose, the expression of mif was examined in the lungs of hypoxic pulmonary hypertensive (hph) rats. then we investigated the effect of mif on hypoxia - induced pasmcs proliferation and the underlying mechanism. all of the experimental procedures were approved by the animal use and care committee for research and education of the fourth military medical university. recombinant mouse mif was purchased from r&d systems (minneapolis, mn, usa). mtt, pd 98059, sb 203580, and sp 600125 were from sigma (st . louis, mo, usa). chuan - min hu, the third military medical university, chong qing, china. antibodies to erk1/2 and phospho - erk1/2, jnk and phospho - jnk were from cell signaling (beverly, ma, usa). the final amount of dmso in the bath solution was less than 0.1%. according to the previous reports, rats were housed intermittently in a chamber containing 10% oxygen, for exposure to continuous hypobaric hypoxia. the intermittent regime consisted of 10 hours in the hypoxic chamber followed by 14 hours in room air (21% oxygen). the normoxic control rats were housed continuously in room air. at the end of hypoxia exposure, measurement of the right ventricle systolic pressure (rvsp) and the ratios of right ventricle/ (rv/) weight were determined. briefly, as soon as median sternotomy was performed, lungs were removed with hearts in fresh pbs. under a dissecting microscope, the 2nd-3rd - division (external diameter < 300 m) pulmonary arteries were isolated carefully. after the adventitial layers together with the surrounding tissue and endothelium were removed, the pulmonary arteries were dissected into small pieces and cultured in dmem supplemented with 100 u / ml penicillin, 0.1 mg / ml streptomycin, 2 mm l - glutamine, and 10% fbs and grown in humidified incubators at 37c in 95% o2 and 5% co2. cells were used for experiments between passages 3 and 6. in the hypoxic groups, pasmcs were transferred into a hypoxic chamber containing 2% o2, 5% co2, and 93% n2 for 24 hours. before exposure to hypoxia or treatment with different agents, lungs were homogenized, and total rna was extracted from the lung tissues by using the rneasy total rna isolation kit (qiagen, valencia, ca). the primers for the rat mif gene were sense, 5-tctccgccaccatgcctatg-3, and antisense, 5-gggtcgctcgtgccactaaa-3, and for the housekeeping gene -actin were sense, 5-atcatgtttgagaccttcaaca-3, and antisense, 5-catctcttgctcgaagtcca-3. pcr reaction was carried out under the following conditions: 30 cycles of denaturation at 94c for 30 s, annealing at 56c for 30 s, and extension at 72c for 30 s. a final extension was performed at 72c for 1 min. lung homogenates were prepared in ripa lysis buffer, containing 50 mm tris (ph 7.4), 150 mm nacl, 1% triton x-100, 1% sodium deoxycholate, 0.1% sds, 2 mm naf, 5 mm edta (ph 8.0), and 1 mm sodium orthovanadate (beyotime inc, jiangsu, china). the protease inhibitor of phenylmethylsulfonyl fluoride (pmsf, 1 mm) was added to the ripa buffer in advance. equivalent amounts of protein (30 g) from each sample were separated on 12% sds - polyacrylamide gels and then transferred onto 0.22 m nitrocellulose filter membranes (millipore, bedford, usa). the membrane was blocked and incubated with primary antibodies for mif, or phosphospecific erk1/2, erk1/2, phospho - jnk, and jnk. the levels of proteins and phosphoproteins were detected with enhanced chemiluminescent substrate (pierce, rockford, il, usa). mif (50, 100, 200 ng / ml) or pd 98059 (20 m), sb 203580 (20 m) and sp 600125 (20 m) and iso-1 (10, 50, 100 m) were added, respectively. after being cultured for 24 hours under normoxic condition or hypoxia exposure, solution mtt was added into each well with a 5 mg / ml concentration. cells were cultured for another 4 hours, and then dimethyl sulfoxide (dmso) was added in. after vibrating for 10 minutes, the optical density values were detected at 490 nm wavelength by using a spectrophotometer (bio - tek power wave xs, usa). cells were stimulated with mif or inhibitors as described above, respectively, and after 24 hours they were harvested by mild trypsinization and counted with a hemocytometer. statistical analysis was processed by using one - way anova, followed by lsd test for post hoc multiple comparisons (spss for windows version 16.0, chicago, usa). rats exposed to hypoxia for 28 days developed pulmonary hypertension (table 1), as demonstrated by an increase in rvsp (48 2.8 mmhg in hypoxic rats versus 23 0.5 mmhg in control rats) (n = 8, p < 0.05) and the ratios of rv/(lv+s) weight (0.41 0.05 in hypoxic rats versus 0.26 0.02 in control rats) (n = 10, p < 0.05). lung homogenates from hypoxic rats showed increases in both mif mrna (figure 1(a) ) and protein (figure 1(b) ) compared with control rats. as the immunohistochemistry show, there was mif staining in bronchial epithelial cells (figure 2(b) ), but no positive immunoreactivity for mif in the pulmonary vasculature in control rats (figures 2(a) and 2(b) ), while intense mif staining of smooth muscle cells of large pulmonary arteries (diameter > 100 m) (figure 2(d) ), endothelial cells of smaller pa (diameter < 100 m), and bronchial epithelial cells (figure 2(e) ), as well as the inflammatory cells around the alveoli (figure 2(f) ), was observed in hypoxic lungs. it is known that hypoxia exposure significantly increases the pasmcs proliferation, but we found that this proliferation was obviously inhibited by three various concentrations of mif antagonist iso-1 (figure 3, p < 0.05). iso-1 had no effect on normoxic control pasmcs proliferation. since we have established that hypoxia enhanced mif expression and mif contributed to the hypoxia - induced proliferation of pasmcs, we further tested the direct effect of mif on pasmcs proliferation. both mtt assay and cell counting showed that higher concentration (100, 200 ng / ml) of mif could directly stimulate pasmcs proliferation (figure 4). as shown in figure 5(a), 100 ng / ml mif - induced pasmcs proliferation was significantly blocked by specific mek inhibitor pd 98059 and jnk inhibitor sp 600125 (p the similar were found as evaluated by cell counting ( figure 5(b) ). 100 ng / ml mif - induced erk1/2 activation was increased in a rapid time - dependent manner with maximal at 30 min and followed by a downregulation of erk1/2 phosphorylation after 60 min (figure 6(a) ). mif could also activate phosphorylation of jnk with peaks at 60 min (figure 6(b) ). treatment with iso-1 (50 m), mif - induced both erk1/2 and jnk activation was attenuated markedly (figure 7). chronic hypoxic exposure induces changes in the structure of pulmonary artery which is associated with increased pulmonary vascular resistance, pulmonary hypertension, and right heart failure. the present study provides evidence that mif is upregulated in the lungs of hph rats and stimulates rats pasmcs proliferation, indicating a possible role for this cytokine in the pathogenesis of ph. investigations revealed that chronic hypoxia induced upregulation of gene expression of a wide spectrum of proinflammatory mediators, including chemokines and their receptors, cytokines, growth and differentiation factors, and adhesion and fibrosis - associated molecules. it is increasingly appreciated that inflammatory mediators could directly contribute to the pulmonary vascular remodeling. they have significant effects on the local vascular wall cells, including increases in proliferation and matrix protein production. it is reported that il-6 may affect pulmonary vascular remodeling via direct stimulation of vascular smooth muscle cell (smc) migration or by indirect effects on vascular smc proliferation. in addition, increased serum level of il-1 was observed in the serum of patients with severe primary pulmonary hypertension. these findings suggest that inflammatory mediators are closely associated with pulmonary hypertensive process. as a critical proinflammatory cytokine , mif is constitutively expressed in a variety of immune and nonimmune cells and also effectively secreted from various phenotype cells into the circulation. upon secretion, mif exhibits broad regulatory properties, including stimulation of the growth of a number of cell lines, except for a key mediator in a number of immune and inflammatory diseases. yang detected that mif was a potent human - endothelial - cell - growth - promoting agent. neutralizing mif bioactivity in atherosclerosis - susceptible mice reduces vascular smc proliferation and neointimal thickening. additionally, fu et al. found that mif mediated the hypoxia response of vascular smc, including cell migration and proliferation. in human, it is reported that patients with ph showed higher mif levels than patients without these manifestations in systemic sclerosis. however, no studies have yet addressed the role of mif in pasmcs proliferation in the pulmonary circulation exposed to hypoxia. therefore, it will be of interest to explore whether mif affects pasmcs proliferation during exposure to chronic hypoxia and contributes to the pulmonary vascular remodeling. in the current study , we found that both mif mrna and protein expression were increased in the lung tissues from hph rats. furthermore, the increased mif mostly located in smooth muscle cells of large pulmonary arteries and endothelial cells of smaller pa, as well as the inflammatory cells and bronchial epithelial cells. these indicated that mif overexpression under hypoxia exposure condition might influence the cells in the vascular walls. due to the key role of pasmcs in hph as the show, the isolated pasmcs proliferated in response to hypoxia, which was consistent with previous reports. iso-1 is a small molecule inhibitor targeting mif which inhibits the catalytic site of mif and leads to a marked reduction in the biological function of mif. next, to observe the direct stimulatory effect of mif on pasmcs growth, recombinant mif was administrated to cells however, there is evidence showing that proliferation of pasmcs was not affected by exogenous mif.. we also added 1% fbs to the culture medium which may contribute to the stimulated effect of mif. thus, our findings add mif to the list of proliferative agents which may contribute to pasmcs proliferation in ph. although mif is a weak stimulator of proliferation, it might work in combination with something else such as other inflammatory mediators, or its receptor expression is altered during hypoxia exposure, which needs to be further studied. mitogen - activated protein kinases (mapks), including extracellular signal - regulated kinase (erk), c - jun nh2-terminal kinase (jnk), and p38 map kinase (p38), play the critical role in cell proliferation, survival, or apoptosis. studies have implied that mif - mediated signaling is associated with a sustained phosphorylation and activation of the p44/p42 erk1/2 subfamily of mapk. on the other hand, mif could mediate phosphorylation of jnk in septic shock and utilizes the jnk pathway in t cells and fibroblasts. also, mif treatment strongly activated erk1/2 and p38 mapk in endometriotic cells. showed that mif mediated adhesion molecule expression via the promotion of p38 mapk activation in human endothelial cells. although mif has been reported to activate mapk signaling in several phenotype cells, there is no available information on the role of mapk pathway in mif - induced pasmcs response. here, we demonstrated that mek and jnk inhibitors block mif - stimulated pasmcs growth, respectively. thus, our findings support the idea that erk1/2 and jnk signaling participate in the mif proliferative pathway. but our findings also show that p38 mapk did not appear to contribute to mif - promoted pasmcs proliferation. the previous findings of amin reported that mif - induced migration of human dermal microvascular endothelial cells was not blocked by inhibitors of src and p38 mapk. it indicated that mif mediated the various effects through different signal transduction pathways in different cell types. in summary, the current study showed that mif expression was increased in the lungs from hph rats. this cytokine may act as a growth factor for pasmcs partially through erk1/2 and jnk pathway without the involvement of p38 mapk. we conclude that mif contributes to the hypoxic pulmonary hypertension, but further studies will be needed to extend these findings, with the aim of identifying new therapeutic targets for treatment of pulmonary hypertension.	pulmonary hypertension (ph) contributes to the mortality of patients with lung and heart diseases. however, the underlying mechanism has not been completely elucidated. accumulating evidence suggests that inflammatory response may be involved in the pathogenesis of ph. macrophage migration inhibitory factor (mif) is a critical upstream inflammatory mediator which promotes a broad range of pathophysiological processes. the aim of the study was to investigate the role of mif in the pulmonary vascular remodeling of hypoxia - induced ph. we found that mif mrna and protein expression was increased in the lung tissues from hypoxic pulmonary hypertensive rats. intensive immunoreactivity for mif was observed in smooth muscle cells of large pulmonary arteries (pas), endothelial cells of small pas, and inflammatory cells of hypoxic lungs. mif participated in the hypoxia - induced pasmcs proliferation, and it could directly stimulate proliferation of these cells. mif - induced enhanced growth of pasmcs was attenuated by mek and jnk inhibitor. besides, mif antagonist iso-1 suppressed the erk1/2 and jnk phosphorylation induced by mif. in , the current finding suggested that mif may act on the proliferation of pasmcs through the activation of the erk1/2 and jnk pathways, which contributes to hypoxic pulmonary hypertension.
the angiomyolipoma of the liver (aml) is a very infrequent benign tumor characterized by three components: adipose, vascular, and muscular. progresses achieved in imaging techniques have substantially increased the number of correct preoperative diagnoses of aml. the publications in the medical literature of some cases that confirm a malign transformation of aml have made the surgical indications for this pathology a controversial issue. laparoscopic approach, when feasible, is a technique of choice that is gaining ground and which is commonly performed with the use of 4 or 5 trocars. we describe here a new case concerning a laparoscopic resection with only three trocars; we have revised the literature and discussed surgical indications. a 65-year - old male patient presented with epigastric pain, with no medical records of interest, and was not affected by tuberous sclerosis. ultrasonography and abdominal ct (figure 1) revealed an adipose - looking lesion of around 4 cm located on the left lateral hepatic sector. the patient was placed in the french position: the surgeon between his legs and the assistant on the left side of the patient. three trocars were placed: a 10 mm trocar in umbilical position for the 30 camera, a 5 mm trocar placed in the right hypochondrium, and another 10 mm trocar (operator 's trocar) in the left hypochondrium (see figure 2). pringle's maneuver was prepared passing a nylon loop, although no vascular control was applied. a partial left sectionectomy was carried out, sealing the parenchyma with ligasure 5 mm (figure 3). the piece was removed in a bag through the umbilical trocar, widening the incision up to 2 cm. the procedure lasted 40 minutes, and the loss of blood amounted to 75 cc. macroscopically, it is a well - delimited 4 cm lesion, although not encapsulated. the histological study revealed a mesenchymal lesion with a muscular, adipose, and vascular component. immunohistochemical study was negative for pankeratin, ae1-ae3, and cd117 but positive for s-100 with reference to the adipose tissue; hmb45 was positive within the cytoplasm of spindle - shaped and epithelioid cells and also for actin and desmin in the vascular component of the thick wall (figure 4), so the final diagnosis confirmed a benign mixed angiomyolipoma. the radiological control carried out 12 months later was normal and did not show any relapse, and the patient is now free of symptoms. regularly, the hepatic angiomyolipoma (aml) is a solitary mesenchymal tumor, not encapsulated and of a variable size (1 to 36 cm) that is composed of three variable tissues: muscular cells, thick - wall vessels, and mature adipose tissue. it occurs more frequently in women, with no age preference, and has a low incidence. it was described for the first time by ishak in 1976, and there are only 300 cases published ever since. commonly, it is a sporadic tumor, but it is associated with tuberous sclerosis in 6% of patients. this predisposition suggests that genes involved in the tuberous sclerosis (tsc1 and tsc2) may be instrumental in aml pathogenesis. aml muscular cells are called pec (perivascular epithelioid cell), while identical cells are seen in other tumors (lung lymphangiomatosis, lung or pancreas clear cells, cardiac rhabdomyoma, etc .), so it has been suggested to include these tumors in a family called pecomas, although this idea is not widely accepted. there are 3 types of aml, histologically speaking, depending on the amount of fat they contain, which are called lipomatous tumors (> 70% of fat), myomatous (< 10%), and angiomatous and mixed variants. the immunohistochemical study of aml is always positive for hmb45 and frequent for s100 and actin. the aml has always been regarded as benign tumor, with a slow growth and with no chances of a malign transformation , but four works published from 2000 to now report the occurrence of 4 malignant aml cases that have or can develop the capacity to relapse and lead to vascular invasion. the combined use of abdominal ultrasonography, ct, and mri has increased preoperative diagnostic certainty of aml, especially in those cases when a fat component and some central and prominent abnormal vessels can clearly be detected; however the correct preoperative diagnosis does not exceed 50%. the ultrasonography reveals a heterogeneous hyperechoic mass that sometimes is difficult to distinguish from a hemangioma. the ct shows an aml with two parts: a peripheral angiomatous component and a lipomatous one with a low attenuation. the mri of the aml reveals an intense signal in t1 and t2, and it seems that a somewhat higher specificity is obtained when compared with other imaging systems. a differential diagnosis is suggested in the event of hepatocarcinoma and other liver tumors that may include a variable fat content (adenoma, lipoma, liposarcoma, sarcoma, gist, metastasis, etc .). the fnap technique may yield a low rate of correct diagnoses, so its utility is reduced. aml is generally asymptomatic, but big - sized tumors may produce compressive symptoms, such as abdominal pain in superior hemiabdomen, plenitude sensation after intake of food, palpable mass, and other symptoms that include weight loss or fever. there seems to be a correlation between a higher size than 5 cm and the occurrence of symptoms. general indications for the resection of benign liver tumor (blt) include diagnostic doubt, occurrence of symptoms, or onset of complications. there is currently a tendency to perform these resections by laparoscopy when it is possible, although this laparoscopic approach should not increase the amount of surgery indications for blt. accepted indications for resection of aml include symptomatic patients, those cases where malignancy can not be excluded, rapid growth tumors, and lesions with an exophytic component, as this last increases the risk of rupture. some authors recommend the resection of all amls larger than 5 cm. the most accepted criteria for a preserving management comprise aml cases that are smaller than 5 cm, asymptomatic, whenever their histology has been tested through fnap, and uninfected by hepatotropic viruses, as it could lead to an erroneous diagnosis of hepatocarcinoma. the above - mentioned existence of malignant cases has reopened the debate on the need to approach all aml cases. the only medical available treatment for aml although no randomized trial has been carried out yet is based on sirolimus, as it seems to reduce the aml size by inhibiting mtorc1. our opinion is that asymptomatic aml, less than 5 cm, in a noncirrhotic liver could be observed but after explaining to the patient the very low (3%) but possible incidence of cancer. the rest of the aml, symptomatic, quick growing, bigger than 5 cm, not of clear diagnosis, should be resected. the first laparoscopic resection of the liver (lrl) was performed in 1992, but it had no exponential growth until the last five years when it coincided with a better technology that did not exist previously. the more frequent lrl (65%) comprises minor hepatectomies (left lateral sectionectomy and atypical resections), and segments ii to vi are regarded as the ideal ones for lrl. a progressive experience on lrl has made possible a higher number of resections on segments that initially were more complex and major hepatectomies. rlr on blt is an excellent indication since it rules out the risk of tumor dissemination, presents the benefits of the laparoscopic surgery for usually young patients, and reduces the mean in - hospital stay and recovery time. the potential disadvantages of lrl include slow progress of the learning curve, bleeding, inaccurate assessment of lesions, which may go unnoticed, and the risk of air embolism. in 2009, an exhaustive worldwide revision was published on 2801 patients who had undergone lrl, out of which 44.7% (n = 1253) of lrl procedures dealt with blt cases. there are, though, few publications devoted exclusively to the implementation of lrl on blt cases; the two most numerous series on this issue totalized 70 patients and only two of them belonged to the aml group. the percentage of complications concerning lrl on blt varies between 10 and 20%, while mortality is 0%, a fact that proves that lrl is feasible and safe the classical lrl technique employs 5 trocars (two of 12 mm, one of 10 mm, and two of 5 mm) and systematic portal clamping. however, the published series reveal that in a percentage of patients, which varies between 12.5% and 46%, the technique was performed without pringle's maneuver. our posture is that in peripheral resections concerning small tumors, or in segmentectomies, it is not so strictly necessary to perform the portal clamping, although it is advisable to be prepared for any contingency. there is only one publication that mentions the use of three trocars in 9 lrl cases. all patients had malign tumors (8 hepatocarcinomas and one hepatic metastasis), superficially located from segments ii to vi and viii and exhibited a mean size of 3 cm. the mean time of procedures was 2 hours, pringle's maneuver was not used, blood loss amounted to 75 ml, and morbidity - mortality was negative. our opinion is that in selected patients those peripheral lesions smaller than 5 cm can be resected with only three trocars instead of the classical 5 ones. the main problems for liver sils surgery are new learning curve, loss of instrumental triangulation, vision problems because camera and instruments are parallel, expensive costs and incisional hernias. after an extensive bibliographical search, only 9 liver sils surgeries have been found, two laparoscopic fenestration of simple cyst and 7 liver resections. lesions were always located in left lateral segment. in every case, the surgeon's opinion is that liver sils surgery is complex, technically demanding, and not always feasible. besides, in some cases additional ports are required. the advantages of 3 trocars technique versus sils are conventional devices, more anatomical vision, nonlearning curve, and costs. so, three - port laparoscopic resection is safe and feasible in some liver lesions. aml is a rare neoplasm of the liver with not well - defined malignant potential so surgical resection in suitable patients is indicated.	angiomyolipoma of the liver (aml) is an infrequent neoplasm composed of three tissues (adipose, muscle and vessels). in spite of advances in radiology, preoperative correct diagnosis is difficult. clasically, a conservative management strategy was adopted in patients with asymptomatic tumors less than 5 cm with undoubtful diagnosis. but after publishing some few cases of malignant angiomyolipoma a more radical has been advocated. laparoscopic resection of liver tumors is becoming a excellent approach for operating on benign liver tumors. usually is performed using five trocars but in some cases a less invasive technique with three trocars could be used. we present a laparoscopic resection of liver angiomyolipoma in a 65 year - old male using only three trocars and also discuss the optimal management of aml and technical tips of three - trocar technique.
the incidence of myopia in the usa and europe is reported to be around 30%, and in asian countries it affects around 60% of the general population. pathologic myopia of more than 6.0 d can be found in 1215% of all myopic patients. low to moderate degrees of myopia can be easily corrected using optical or refractive surgical means. pathologic myopia, while also correctable using these optical approaches, is of major concern because of sight - threatening consequences such as retinal detachment, macular schisis, and macular degeneration. all these complications are associated with progressive axial elongation which can not be treated by refractive means. the pharmacological treatment currently in use is topical atropine, which is accompanied by numerous side effects and offers little benefit for already highly myopic eyes. surgical approaches include scleral reinforcement surgery in which donor sclera or synthetic bands are placed around the back of the globe and sutured to the sclera to provide scleral support or injection of a polymeric composition forming a gel under tenon's capsule and inducing scar tissue to prevent axial elongation. however, the outcomes of all of these surgical therapies are controversial and surgical trauma also should be taken into consideration. there is strong evidence from clinical and experimental studies indicating that the biochemical and biomechanical properties of the sclera play a major role in the progression of myopia. thinning of the sclera and weakened biomechanical properties, particularly at the posterior pole of the eye, have long been known to be an important feature in the development of high myopia in human and mammalian models. thus, the sclera has been considered to be a prime target for therapeutic manipulation regarding myopia progression. as mentioned above surgical treatments based on sclera reinforcement because of the controversial and complications, the various surgical approaches have not been widely applied in clinical practice. animal studies involving tree shrews have shown that impaired collagen cross - linking is an important factor in the weakening process of the myopic sclera. indeed, collagen cross - linking has been successful in treating progressive keratoconus in which the cornea undergoes a thinning process and exhibits weakened biomechanical properties, as in the myopic sclera. several studies have attempted to increase the rigidity of the sclera through collagen cross - linking. collagen cross - linking induced by the photosensitizer riboflavin and ultraviolet a (uva) has been shown to lead to a significant increase in young's modulus for treated porcine, rabbit, and human sclera. however, scleral cross - linking using riboflavin and uva requires an operation entailing surgical exposure of the posterior sclera and has a potential cytotoxic risk for the retina. an alternative method of chemical cross - linking using glyceraldehyde has been shown to significantly increase young modulus in porcine sclera in vivo and in rabbit sclera in vitro. another study involving the rabbit demonstrated that the efficacy of glyceraldehyde in increasing scleral biomechanical strength can extend over a period of 8 months. glyceraldehyde is used for tissue engineering in the pharmaceutical and food industry and is generally considered nontoxic. the safety of glyceraldehyde treatment on eyes has also been studied by wollensak and iomdina. light microscopy examination revealed that there were no abnormalities in the optic nerve and retina, and only some moderate infiltration of neutrophils and bleeding adjacent to the injection site were found in several animals. unlike riboflavin cross - linking and sclera reinforcement surgery, glyceraldehyde can be easily administered by means of sequential parabulbar injections and have large treatment area. consequently, scleral cross - linking with glyceraldehyde may be a promising method for the treatment of myopia. although glyceraldehydes could improve sclera rigidity in normal animal eyes, its effect on progressive myopic eyes has not yet been studied. the aim of the present study was to investigate the effectiveness of collagen cross - linking using glyceraldehyde in increasing scleral rigidity and retarding the axial elongation in form - deprived myopia (fdm) of guinea pig. this study was approved by the animal care and ethics committee at tianjin medical university (tianjin, china). the treatment and care of animals were conducted according to the arvo statement for the use of animals in ophthalmic and vision research. thirty - six pigmented guinea pigs (cavia porcellus ; approximately 3 weeks old) were obtained from the animal breeding unit at tianjin medical college and randomly assigned to the following four groups with nine animals per group: fdm (form - deprived myopia); fdmg (form - deprived myopia treated with glyceraldehyde); fdms (form - deprived myopia treated with 0.9% isotonic sodium chloride); and normal control (free of form deprivation). all animals underwent biometric measurement (refraction and axial length) prior to the experiment. the right eye in the fdmg group was treated using a sub - tenon injection of 0.5 m glyceraldehyde; 0.9% isotonic sodium chloride was administered to the right eye in the fdms group using the same method. biometric measurement was undertaken at four time points (0, 2, and 4 weeks). following the final ocular measurements, the right eyes were enucleated after the animal had received an overdose of anesthesia. the stress - strain of the sclera was measured and histological examination was undertaken with light microscopy. fdm was achieved using a latex facemask (oujie, suzhou, china) covering the right eye. the left eye, nose, and both ears remained exposed, as described by lu et al. the facemasks were examined once daily to ensure that they were in place and fitted well. after topical anesthesia using 0.5% proparacaine hydrochloride (alcon, purrs, belgium), sub - tenon injections of 0.05 ml of 0.5 m glyceraldehydes (sigma - aldrich, steinheim, germany) dissolved in physiologic saline solution were administered as a depot with the injection site 3.0 mm behind the limbus in the superonasal and inferotemporal quadrant, respectively; then the injection site was transferred to the other two quadrants for every other injection; a 1.0 ml tuberculin syringe with a sharp 25-gauge injection needle was used. the first injection was given at day 1 just before achieving fdm and the injections were given twice a week. 0.05 ml of 0.9% isotonic sodium chloride solution (dazhong, tianjin, china) was administered in the same way. ofloxacin eyedrops (santen, osaka, japan) were applied four times a day. retinoscopy for all animals was performed by the same optometrist in a dark room using a streak retinoscope. before examination , 1% cyclopentolate hydrochloride (alcon) was topically administered to the eye every 5 min for four repetitions to achieve a completely dilated pupil. the refraction was recorded as the mean value of the horizontal and vertical meridian. after topical anesthesia with 0.5% proparacaine hydrochloride (alcon), ocular axial length was measured using an a - scan ultrasonography (cinescan a / b, quantel medical, clermont ferrand, france). sound velocities were assumed to be 1557.5 m / s for the anterior segment, 1723.3 m / s for the lens, and 1540 m / s for the vitreous humor. the animals were sacrificed by overdose anesthesia with 160 mg / kg pentobarbital sodium (sigma - aldrich, steinheim, germany). after making a complete circular incision located at a distance 1 mm anterior the limbus, two scleral strips of width 2 mm were dissected sagittally using a double - shade shaver, from the nasal and temporal margin of the optic nerve to the anterior end almost at 1 and 11 o'clock. care was taken to insure that all strips were cut in a similar orientation to minimize any differences ing from possible anisotropy. scleral strips with a width of 2 mm were clamped horizontally with a distance of 5 mm between the jaws of the microcomputer - controlled biomaterial tester (shanghai university ; shanghai ; china). strain was increased linearly with a velocity of 1 mm / min and stress was measured up until tissue rupture (figure 2). the strain - stress curve was recorded and the parameters stress (mpa) and young's elastic modulus (mpa) from 2% to 14% of the strain of the samples were used for analysis. all the eyes of fdms and fdmg group perform histological examination. after the scleral strips used for biomechanical test were removed carefully, the left eye cups were fixed in 10% neutral buffered formalin for at least 24 hours for light microscopy. the specimens were embedded with paraffin. 4 mm thin sections were cut within and adjacent to the treatment area and stained with hematoxylin - eosin. the slides were examined using a light microscope (leica dm4000b ; germany) at 401000 magnification. the refraction and axial length of the right eye were statistically compared to the left eye within the same group prior to the experiment using the paired sample t - test. the biometric and stress - strain for the right eye were compared between the different groups using one - way analysis of variance (anova) with bonferroni correction. prior to the form deprivation (0 time - point ; table 1), there was no significant difference between the right eye and the left eye of the animals regarding refraction and axial length within each individual group (p > 0.05 ; paired sample t - test). the difference in refraction and axial length of the right eyes was not significant among all the four groups (refraction, p = 0.685 ; axial length, p = 0.949 ; one - way anova with bonferroni correction). all of the right eyes of animals in the fdmg, fdm, and fdms groups developed significant myopia in 4 weeks. similar to the fdm group, the biometric parameters of the right eyes of animals in the fdmg and fdms groups kept developing towards myopia during the 4-week observation period. data regarding the development of refraction and axial length in the four groups is detailed in table 1. at all time points the development of refraction and axial length in the deprived eyes of animals in the fdm, fdmg, and fdms groups was similar but was significantly faster than the normal control group; the were presented in tables 1 and 2. when compared with the normal control eyes, statistically significant changes occurred in the biomechanical parameters of the deprived eyes of animals in the fdmg, fdm, and fdms groups (table 3). the stress - strain curves and young's elastic modulus - strain curves for all of the four groups are presented in figures 3 and 4, respectively. for each group the stress - strain curve approximated to a straight line; however, when the thickness of the sclera was considered, there was not a single young's elastic modulus at different strain levels. the elastic modulus increased from 2% strain and reached a value that was almost stable at 6% strain; the difference between the groups also reached almost stable value at 6% strain. the elasticity of the sclera in the eyes of animals in the fdm group increased; the stress in the deprived eyes of animals in the fdm group was significantly lower than normal control eyes, with the exception of the 2% and 4% strain levels. in contrast with the stress , although young's elastic modulus for the deprived eyes of animals in the fdm group was lower than in the normal control eyes, the difference was not significant at all strain levels. saline injection could improve the biomechanical properties of the sclera in the form - deprived eyes. the stress and young's elastic modulus for the right eyes of animals in the fdms group were both significantly higher than in the fdm group, with the exception of 2% and 4% strain (stress : fdms versus fdm, 4% strain, p = 0.052, 6% strain p = 0.01, and 8% strain, p = 0.009 ; young 's elastic modulus : fdms versus fdm, 4% strain, p = 0.072, 6% strain, p = 0.014, and 8% strain, p = 0.042 ; one - way anova with bonferroni correction). however, when compared to normal control eyes, there were no significant differences between the groups at all strain levels (table 3). for the right eyes of animals in the fdmg group, the stress and young's elastic modulus at all strain levels were significantly higher than for the right eyes of animals in all of the other groups (p < 0.01 ; one - way anova with bonferroni correction). the stress in the right eyes in animals in the fdmg group was 1.69 times greater and young's elastic modulus was 1.08 times greater than in the right eyes of animals in the fdm group at 6% strain. the comparison of stress and young's elastic modulus between fdmg and normal control group was showed in table 3. light microscopy examination showed that, in 7 eyes of fdmg group and 6 eyes in fdms group, mild inflammatory infiltration or hemorrhage was seen in the episcleral tissue in the injection area. it adhered to the sclera tightly, but no scar has been observed (figure 5). the optic nerve and retina in all samples were without abnormalities (figure 6). in the present study, it was confirmed that form deprivation can cause significant myopia; in the eyes of guinea pigs with form - deprived myopia the sclera had weakened biomechanical properties. the stress in the eyes of animals in the fdm group was lower than that in the eyes of animals in the normal control group at all strain levels, and the difference was significant at 6% strain. young's elastic modulus for the sclera was lower in the eyes of animals in the fdm group than in the normal control group, but the difference was not significant. these were similar to those reported by phillips and mcbrien. in their study, the sclera extended over a distance that was 25% greater than in the controls at a load corresponding to 20 mmhg intraocular pressure; however, when the thickness of the sclera was taken into consideration, young's elastic modulus was similar between the myopic and normal eyes under physiological pressure. consequently, it was concluded that the alterations in elasticity were mainly the of a thinner sclera in the eyes with myopia. it is believed that scleral thinning in myopia is not the of passive stretch of the sclera but is caused by active tissue remodeling. decrease in the amount of collagen present and fibril diameter are two critical events that lead to scleral thinning and weakening. in an eye with a weakened sclera , physiological intraocular pressures may be sufficient to induce progressive ocular enlargement given sufficient time. consequently, treatment targeting scleral collagen may be effective in retarding the progression of myopia. experiments both in vivo and in vitro have demonstrated that the biomechanical parameters of sclera in normal eyes could be improved by treatment with glyceraldehyde. wollensak and iomdina have demonstrated in vivo in rabbit sclera after glyceraldehyde treatment for 14 days that the ultimate stress increased by 409.7% and young's modulus increased by 1027%; the ultimate strain decreased by 48.2%. in vitro after treatment with glyceraldehyde, the stress of treated porcine and human sclera increased by 487% and 34%, respectively. in our study, it was found that in form - deprived eyes the biomechanical rigidity of the sclera could also be increased by treatment with glyceraldehyde. the stress and elastic modulus in the eyes of animals treated with glyceraldehydes was significant higher than in the eyes of animals in the normal control and fdm groups at all strain levels. this indicates that injected glyceraldehydes could overwhelm the scleral biomechanical changes caused by form deprivation. through the maillard reaction cascade , glyceraldehyde can be added to the ends of protein molecules and can be further transformed to more stable molecules called advanced glycation end products; this in covalent collagen cross - links that can promote increased tissue stiffness and resistance to enzymatic degradation. it had been demonstrated by ultrasound sub - tenon injections can reach posterior sclera immediately and then disperse into surrounding tissue. in this study repeated injections in four quadrants, respectively, facilitated glyceraldehydes reaching the whole posterior sclera which play most important role in the axial elongation. abnormalities of the retina and optic never have not been observed in our study; the same have also been reported by wollensak and iomdina. the tenon capsule of the fdmg eyes became compact and adhered to the sclera tightly that may be caused by injections and glyceraldehydes. the of these changes has not been studied furtherly, but there was no scar observed and the injections have been done well in this study. in the current study, although the injection of glyceraldehydes increased scleral rigidity, the development of myopia was not retarded in experimental eyes relative to the normal control eyes. all of the biometric parameters regarding the eyes of animals in the fdmg group were not significantly different from those in the fdm group at all time points. the of other studies aimed at evaluating the efficiency of sclera reinforcement have been controversial. scleral strengthening by means of polymer injection to slow ocular elongation has been studied in animal models and humans; the were reciprocal. su et al. reported that polymeric hydrogels, either implanted or injected adjacent to the outer scleral surface, could not slow ocular elongation in chicken eyes, although the sclera thickness was significantly increased. the authors considered that a possible reason for this relates to the bilayered structure of the chick sclera; the stiffer cartilage component of the chick sclera may determine the rate of elongation. in mammalian and primate eyes that have monolayered fibrous scleras, the addition of the fibrous capsule is likely to have a greater impact on scleral biomechanical properties and could possibly slow ocular relongation. indeed, it has been reported by avetisov et al. that repeated injections of liquid polymeric composition could promote collagen formation in 146 rabbit eyes and slow myopia progression in 240 human eyes. this finding is at odds with our observations regarding form - deprived myopia in the guinea pig. the axial elongation could not have been retarded by increased scleral stiffness in our study. one possible reason may be that myopia was progressive and more pronounced in the former study. in addition to this, a reduction in collagen fibril diameter has been reported to occur at the posterior pole. in experimental myopia, the collagen fibril diameter has been found to decrease only after a long period of myopia (3 months in the tree shrew). therefore, collagen change may not be the most important factor that contributes to the progression of short - term experimental myopia. the improvement in scleral rigidity based on collagen cross - linking may be more effective in long - term progressive myopia. on the other hand, the of our study may indicate that enhancement of scleral biomechanical properties does not alone guarantee slowed eye growth in the fdm guinea pig eye model, consistent with suggestions by mcbrien and norton. they found that form - deprived myopia could be increased by administration of aminopropionitrile, which could prevent collagen cross - linking; at the same time it was also found that eyes treated with aminopropionitrile without form deprivation were no different from normal untreated eyes. it appears that focused images falling on the retina could control ocular development in a growing eye despite the presence of structural changes involving collagen. several factors are involved in the progression of myopia, a predisposition to myopia or other error signals combined with abnormal collagen structure, and work together to produce myopia. it is reasonable to assume that if the reinforcement of sclera reaches a sufficiently high level it can retard myopia by overwhelming all of the factors leading to its progression. but to make the sclera more rigidity may be combined with more side effects. in summary, the of the present study represent proof that the scleral biomechanical properties of form - deprived eyes can be improved using sub - tenon injection of glyceraldehydes; however, the rate of ocular elongation was not affected. because of differences in high and moderate myopia, a follow - up study involving progressive high and long - term myopia in a mammalian animal models will be necessary to establish their suitability for myopia control with collagen cross - linking.	to investigate the effects of collagen cross - linking using glyceraldehyde on the biomechanical properties of the sclera and the axial elongation of form - deprived myopia in the guinea pig. thirty - six guinea pigs were randomly assigned to four groups: fdm (form - deprived myopia); fdmg (form - deprived myopia treated with glyceraldehyde); fdms (form - deprived myopia treated with 0.9% isotonic sodium chloride); and normal control (free of form - deprivation). fdm was achieved in the right eye using a latex facemask. the right eye in fdmg was treated with a posterior subtenon injection of 0.5 m glyceraldehyde; 0.9% isotonic sodium chloride was administered to the right eye in fdms group using the same method. axial length, refraction, and stress - strain of the sclera were measured at scheduled time points. the treated eyes were also examined histologically by light microscopy. it was found that glyceraldehyde treatment significantly increased the stiffness of the sclera in the fdm eyes and abnormalities have not been observed in the retina and optic nerve of the treated eyes. but the development of myopia was not affected.
glaucoma is an irreversible optic neuropathy characterized by specific optic disc changes and corresponding visual field alterations which are often associated with elevated intraocular pressure (iop). mechanistic theory proposes that increased iop is the most important risk factor in glaucomatous optic neuropathy; however, optic neuropathy can be worsened in patients with well - controlled primary open - angled glaucoma and low iop. vascular dysregulation ing from vascular endotheliopathy is the most important factor in decreased ocular blood flow. in addition to responses to several vasoactive agents and hormones, endothelium itself also releases substances relaxing or constricting vascular smooth muscles such as nitric oxide (no) or endothelin-1 (et-1), respectively. it was found that level of et-1 was increased in the aqueous fluid samples of patients with glaucoma. endothelium - dependent fmd by occlusion at brachial artery is a noninvasive method which is previously used in the measurement of endothelial function in patients with risk factors (dm, hypertension, hypercholesterolemia, homocysteinemia, etc .) for coronary artery disease. the aim of the present study using vascular endothelial function measurement to assess fmd was to evaluate relationship between cases with primary open - angled glaucoma and fmd. the study included 20 patients with poag and 30 age- and sex - matched healthy controls. the diagnostic criteria for paog included iop > 22 mmhg without treatment, open angle at gonioscopy, glaucomatous optic disc changes (cup / disc > 0.7 and thinning or pitting at rim), and characteristic visual field defects corresponding to optic disc. the cases with poor sita reliability indices (false negative errors, false positive errors, and fixation loss exceeding 20%) were excluded. also, patients with previous ocular trauma or surgery, those with history of steroid use, or those having an eye disorder other than glaucoma were excluded. in addition, patients with systemic diseases such as hypertension, congestive cardiac failure, hypercholesterolemia, diabetes mellitus, cerebrovascular event, or autoimmune diseases were excluded. control group included healthy individuals without history of medication who presented for routine ophthalmological visits. the study was approved by the local ethics committee and was conducted in accordance with the declaration of helsinki, good clinical practice guidelines. the participants were informed of the nature of the study, and informed consent was obtained from each patient. in all subjects, blood samples were drawn for measurements of fasting blood glucose, lipid profile, sedimentation rate, and c - reactive protein (crp). fmd measurements in brachial artery were performed by a cardiologist blinded to clinical characteristics of the patients with 2-dimensional high - resolution ultrasound device (7.013.0 mhz, siemens medical sol . ten minutes resting at supine position was provided before measurements and ecg monitoring was applied throughout measurements . radial artery measurement was performed at a point 35 cm superior to antecubital fossa . transition zone was set at a depth between area at 3 cm depth and closer wall . by magnifying image, intermediate zone between tunica media and adventitia on pulse doppler sonography, blood flow velocity and volume were assessed through two - dimensional images generated by signal obtained from center of artery with a sampling angle of 6570 and sampling distance of 1 mm . a 5-minute occlusion was obtained by inflating cuff up to 250 mmhg at the level of brachial artery . radial artery measurements were recorded during r wave on ecg ( end - diastole). response of brachial artery diameter to hyperemia was calculated as percent increase by using the following formula: 100x. statistical analysis was performed by using spss for windows version 17.0 (statistical package for social science, chicago, il, usa). student's t - test was used to assess differences in mean values between groups. table 1 summarizes demographic characteristics and blood measurements of poag and control groups. there were no significant differences between the groups regarding age, sex, and biochemical measurements. percent of fmd increases was 11.9 4.2% and 12.3 4.4% in poag and control groups, respectively (p = 0.86). glaucoma represents a group of eye disorders in which many risk factors play a role in its development and progression. iop is the best known risk factor and reducing iop is the only available method in the treatment of glaucoma. thus, it is needed to investigate ocular dynamic in patients with glaucoma to further improve our understanding of ocular dynamics. previous studies have reported the role of vascular factors in the development and progression of glaucomatous optic neuropathy in paog. these previous works focused on ocular blood blow. in the present study, it was revealed that there is no impairment in systemic vascular endothelial function in patients with poag when compared to controls by using brachial artery fmd measurements. currently, fmd is the gold standard used in the measurement of arterial endothelial function in clinical trials. fmd is high - frequency sonographic visualization of brachial artery that shows endothelial - dependent flow - mediated vasodilation. hyperemia - induced transient ischemia leads to increased stress in brachial artery wall and it causes vasodilation by promoting endothelial no release. however, previous studies showed that inter- and intraobserved differences are minimal without significant effect on . in our study, measurements were performed by an experienced cardiologist. fmd measurement is considered an important marker in establishing prognosis and treatment response in coronary artery disease. , et-1 causes decreased retinal, choroidal, and optic nerve head blood flow and vasoconstriction at posterior ciliary artery. increased et-1 levels can play a role in the astrocyte proliferation seen in glaucomatous optic neuropathy. increasing et-1 levels in the aqueous humor of patients with poag reduces outflow of aqueous fluid by causing contraction in trabecular network. in studies on normotensive glaucoma (ntg), it was found that et-1 levels were increased in both serum and aqueous humor, suggesting both ocular and systemic vascular dysfunction in ntg. in studies on patients with paog , et-1 levels were found to be increased in aqueous humor, while no change was observed in serum et-1 levels. this suggests that the role of et-1 in vascular dysfunction in poag is localized to tissue. reported increased serum et-1 levels in selected cases with disease progression despite well - controlled iop. in the previous 3 studies using fmd, it was reported that fmd was decreased in patients with poag. however, there are inconsistencies among these studies. in the study by su et al., in which patients with ntg and poag were compared, it was reported that mean percent of fmd increase in patients with ntg was lower than that in patients with poag. found no significant difference between patients with paog and ntg; even authors reported lower percent of fmd increase in poag group when compared to ntg group. in the study by cellini et al patient group consisted of those with progressive visual field defect despite well - controlled iop; thus, they emphasized vascular theory. in our study, it was demonstrated that there is no impairment in systemic vascular function of cases with poag. we think that, in contrast to ntg with systemic component, poag is a disease specific to the eye. valuable information could be obtained about systemic vascular function in patients with poag by designing studies with larger sample size which demonstrate indirect vascular endothelial function such as et-1 level and number of endothelial progenitor cells.	objective. we aimed to assess peripheral vascular endothelial function in open - angle glaucoma (poag) by measuring flow - mediated dilatation (fmd). materials and methods. the study included 20 cases with poag (group 1, mean age 58.68 13.3 years) and 30 healthy individuals (group 2, mean age 58.68 13.6 years). in all cases, responses of endothelial function were assessed by a cardiologist through measurement of fmd following brachial artery occlusion. . mean percent of fmd, an indicator of endothelial function, was found to be 11.9 4.2% in group 1 and 12.3 4.4% in group 2 (p = 0.86). . no impairment in systemic vascular function of cases with poag suggests that poag could be a local disorder rather than being a component of systemic disease.
oesophageal atresia (oa) is a congenital condition of oesophageal discontinuity that in proximal oesophageal obstruction and a tracheo - oesophageal fistula (tof) is an abnormal communication between the oesophagus and the trachea. oa and tof can occur alone or in combination. congenital tof without oa (h - type tof) account for < 4% of congenital oesophageal anomalies the case presented here is distinctive because of (a) the rarity of an h - type tof in adults and (b) the utilization of a thoracoscopic approach to repair it. a 23-year - old man was investigated for complaints of persistent night - time regurgitation for 8 years without any dysphagia. an upper gastrointestinal (ugi) endoscopy revealed a fistulous opening in the oesophagus 20 cm from the incisors. a computed tomography (ct) scan with reconstruction confirmed an h - type tof in the mid - oesophagus at t3 level and ruled out any extrinsic mediastinal pathology. a virtual bronchoscopy showed the fistula to be 3 cm above the carina. oesophageal manometry demonstrated a lower oesophageal sphincter pressure to be 4.9 cm of h2 o and ineffective peristalsis in 30% of the wet swallows. the patient was counselled regarding a thoracoscopic repair of the tof in the first instance followed by reassessment of his reflux symptoms after surgery. (b) view of the carina obtained upon passing the endoscope through the fistulous tract. (d) virtual bronchoscopic image demonstrating the tracheal opening (arrow) the surgery was performed under double - lumen endotracheal anaesthesia with the patient placed in the left lateral decubitus position. a 10 mm port was established in the 6 intercostal space in the midclavicular line, and two 5 mm ports were placed on either side in the 5 and 7 intercostal space. the parietal pleura overlying the oesophagus above the level of the azygous vein was incised widely, and a pleural flap was harvested for later use. the trachea was identified, and the fistulous tract extending from the oesophagus was dissected. vascular slings were passed to encircle the broad fistulous tract and the oesophagus distal to it. the 10 mm port was exchanged for a 12 mm endopath xcel port (ethicon endosurgery, mumbai, india). the nasogastric tube was withdrawn, an endoscopic stapler with a blue cartridge (3.5 mm staple depth) was locked on to the fistula, and the sling isolating the fistula was removed. the oesophageal staple line was oversewn with a 3 - 0 polyglycolic acid (vicryl, johnson and johnson, mumbai, india) suture. the pleural flap was interposed between the two staple lines and sutured to the trachea in a similar manner. a 32f intercostal drain was placed through the 12 mm port site, and the incisions were closed. (a) sling around the fistula (f) between the oesophagus (o) and trachea (t). (b) arrow points to the tracheal end of the staple - divided fistula post - operatively the patient was prescribed parenteral analgesics for the first 48 h, and he was kept nil by mouth for 4 days. an esophagogram obtained on the 5 day showed smooth passage of contrast into the stomach without any leakage. it is proposed to assess his reflux symptoms a year after surgery to determine whether he would merit long - term proton - pump inhibitor therapy or anti - reflux surgery. benign, acquired tofs are rare in adults and may as a complication of inflammatory disorders, foreign body ingestion, trauma or prolonged tracheal intubation. congenital h - type tofs presenting in adulthood are even rarer and only around 16 cases have been documented in the surgical literature. the minimal symptoms in many patients may be explained on the basis of the oblique passage (an n rather than h configuration) of the fistulous tract leading to a valve - like action that in the occlusion of its lumen during the passage of the bolus of food or peristaltic wave. presence of a membrane covering the opening or contraction of the of the muscle wall of the fistulous tract are considered to be the other likely explanations for the delay in onset of symptoms until adulthood. a high index of suspicion is needed to diagnose a tof in adults, in whom the respiratory symptoms are far commoner than the gastrointestinal symptoms. the commonest complaint is cough, while eating or drinking (ohno 's sign) followed by recurrent bouts of respiratory tract infection. development of tof in utero disrupts the normal development of the oesophageal myenteric plexuses ing in dysmotility. this may lead to gastroesophageal reflux (as seen in our patient) and esophagitis. the fistulous opening is often apparent during a ugi endoscopy, but may be missed if covered with a membrane or located on the anterior wall of the upper third of the oesophagus. similarly, the lower positioning of the oesophageal opening in relation to the one in the trachea may preclude outlining of the tract on a contrast swallow. unless the fistula is wide (as was the case in our patient) coronal sections of the ct scan may fail to identify it. sagittal views along with a three - dimensional reconstruction are more likely to demonstrate the fistula. bronchoscopy is useful for identifying the fistulas not seen on ugi endoscopy and ruling out intrinsic trachea - bronchial pathology. as in neonates, surgery is the only definitive treatment for tof presenting in adults. traditionally, either a cervical route (for a fistula above the t2 level) or thoracotomy (for fistulas at or below t3 level) is employed for the repair. the repair involves ligation of the fistula or its division, followed by repair of the trachea and oesophagus. a muscle or pleural flap is usually interposed between the divided ends. over the past decade, the feasibility and safety of thoracoscopic repair of the congenital tofs in neonates has been well documented. a recent meta - analysis comparing open and thoracoscopic surgery in the treatment of oa and tof in neonates indicated that the outcomes of the two approaches were comparable. in the only previously reported case of thoracoscopic repair of tof in an adult, staple - divided a mid - thoracic level tof in a 79-year - old female. thoracoscopic approach affords excellent magnification for safe visualization, dissection and division of the tof, even when fistula is a narrow one. this complex surgery is best - undertaken either by a thoracic surgeon well versed with thoracoscopy or (as was the case here) by an experienced minimal access surgeon teamed up with a paediatric surgeon. thoracoscopic repair of a tof may not necessarily shorten the hospital stay, as it is customary to commence oral intake around the 5 day after confirming integrity of the staple / suture line with a contrast study. however, these patients do enjoy the other well - proven benefits of thoracoscopic surgery such as reduced pain, speedy recovery and reduction in wound - related and pulmonary morbidity. thoracoscopic repair appears to be a feasible and safe option for the treatment of tof presenting in adulthood, which is an extremely rare clinical entity.	congenital trcheo - oesophageal fistula (tof) without oesophageal atresia is usually diagnosed and managed in the neonatal period. its presentation in adulthood is a rarity. traditional treatment of a tof in adults involves its repair via a thoracotomy. we report the case of a 23-year - old man diagnosed with an h - type tof during workup undertaken for his symptoms of gastro - oesophageal reflux. this fistula located at the level of third thoracic vertebra was repaired successfully using a thoracoscopic approach.
endometriosis involving the intestines occurs in 5% of premenopausal women. of these, 70% present with large bowel obstruction. we present a case of endometriosis presenting as large bowel obstruction in a woman of childbearing age. she had no previous symptoms to suggest endometriosis and on presentation urgent surgery was required. no cases of endometriosis confined to this sigmoid colon without pelvic involvement were noted in the literature. when women of childbearing age seek medical attention for signs and symptoms of intestinal obstruction, and there is no obvious etiology, intestinal endometriosis should be considered as a differential diagnosis. a 37 year - old housewife and mother of a 10 year - old by caesarian section, presented with a history of generalized, colicky abdominal pain and constipation for 6 days, with bilious vomiting and distention for 2 days. she had normal, regular menses with no history of dyspareunia, dysmenorrhea, abdominal pain, constipation, diarrhea or rectal bleeding. on examination, she was in painful distress, ill looking and mildly dehydrated, with a tachycardia of 108/min. the abdomen was distended and tympanitic with generalized mild tenderness but no peritonism or palpable mass. ct - scan showed grossly distended large intestine from the caecum to the sigmoid colon, with no air in the rectum ( see fig. the complete blood count, liver function test and carcino - embryonic antigen were all within normal limits. at surgery, the large intestine was grossly distended from the caecum to sigmoid colon where there was a palpable solid tumor in the wall of the bowel. the pelvis, ovaries, tubes and uterus were grossly normal and there were no other intra - abdominal abnormalities. with an operative diagnosis of carcinoma, sigmoid colectomy with primary anastomosis the cut specimen revealed an obstructing tumor on the mesenteric border, within the wall of the sigmoid colon without involvement of the mucosa ( see fig. she was subsequently referred to the gynecologist for treatment and remains asymptomatic 15 months later. intestinal involvement by endometriosis occurs in 5% of premenopausal women. of these, 70% present with large bowel obstruction. however, the vast majority of these patients reported are known cases of endometriosis, having complaints of pelvic pain, dyspareunia and/or dysmenorrhea. many of them also have a history of infertility, for which they were subjected to investigation, such as laparoscopy, hence, presenting with a previous diagnosis of endometriosis. our patient had no history to suggest the condition with no visible endometrioma, ovarian or pelvic abnormality indicative of endometriosis. the pathophysiology of endometriosis has been explained by various theories; direct myometrial extension, coelomic metaplasia, lymphatic and hematogenous metastasis, reverse menstruation and implantation during salpingography or due to operative spillage. isolated endometriosis within the muscular layer of the bowel with no involvement of mucosa or any pelvic organ remains unclear. although seat belt trauma had, in one case, been suggested as an etiologic factor, no clear evidence was provided for this. in our patient when the initial presentation of endometriosis is intestinal obstruction with no previous history and no suspicious findings at surgery, the diagnosis is unlikely to be made preoperatively. in most reported cases, the patients had a known history of endometriosis or the surgical findings were very suggestive of it. both these observations were absent in our patient. in the non - obstructed case, colonoscopy, endoscopic we believe that one should always maintain a high level of suspicion of endometriosis, when a woman of childbearing age presents with intestinal obstruction and there is no other obvious cause. if the diagnosis is made preoperatively, surgery may be avoided in the non - obstructed case and only a limited resection done in the event of obstruction. when a woman of childbearing age presents with large bowel obstruction, one should always entertain a possible diagnosis of endometriosis whether or not the patient has other evidence of the disease. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. all the authors of this article contributed to the study design, data collection, data analysis and writing.	introductionisolated endometriosis of the intestine causing large bowel obstruction is rare.presentation of casewe present a case of endometriosis presenting as large bowel obstruction in a woman of childbearing age. she had no previous symptoms to suggest endometriosis and on presentation urgent surgery was required. the diagnosis of endometriosis was made only after pathological evaluation of the specimen.discussionno cases of endometriosis confined to this sigmoid colon without pelvic involvement were noted in the literature.the diagnosis of endometriosis should be entertained when women of childbearing age presents with large bowel obstruction, whether or not the patient has other evidence of the disease.
influenza a virus (iav), a member of the orthomyxoviridae family, is an enveloped, single - stranded rna virus with a genome of 8 negative - sense segments, with most encoding a single gene. these eight segments give rise to 10 or 11 distinct proteins depending on the strain (na, ha, ns1, ns2/nep, m1, m2, np, pa, pb1, pb1-f2, and pb2), and these have been shown to interact extensively with each other and with host cell proteins throughout the virus lifecycle. some interactions elicit alterations in the host proteome, as exemplified by the virus s ability to both induce and evade a host immune response, to influence autophagy and apoptosis, and to increase viral protein synthesis while shutting down host protein synthesis. numerous host proteins may also be important for influenza replication, as recently shown in whole genome sirna screens that identified 100 genes in drosophila, 120 genes in u2os cells, as well as 295 genes and 287 genes in the a549 human carcinoma lung cell line. current anti - influenza therapies consist of only two classes of treatments, and these each target a viral protein: zanamivir and oseltamivir are neuraminidase inhibitors, while amantidine and rimantidine target the m2 protein. recent and currently circulating strains from the 20102011 season have remained susceptible to m2 inhibitors; however, resistance to neuraminidase inhibitors is emerging (www.cdc.gov/flu/weekly/). thus, new targets for which resistance will not quickly be developed are needed, and host cell proteins essential for viral replication represent one option. host interactions is increasingly reliant on quantitative proteomic techniques such as 2d - dige, isotope - encoded affinity tag (icat), and stable isotope labeling of amino acids in cell culture (silac). for example, silac profiling was used to determine differences in expression between hiv- and mock - infected t - cells and the response of several host cell types to hepatitis c infection. quantitative proteomics has also been applied to influenza host interactions, including several studies probing primary macrophages with itraq and 2dige - ms / ms and multiple studies using similar techniques in continuous epithelial cell lines such as ags, mdck, a549, and calu-3 cells. several genomic and proteomic studies have also been carried out in influenza - infected macaques. a novel aspect of the current study is the use of more relevant human primary bronchial epithelial cells, a model that more closely mimics in vivo infection conditions. it has been shown that the response to influenza depends not only on the viral strain but also the host species, cell type and location in the lung, and the state of cell differentiation. while the effect of these different parameters have been characterized in part by measuring secreted cytokines and the kinetics of viral replication, very little is known about differences in host similarly, few studies have compared the effect of iav infection in continuous and primary cell lines of specific cell types. therefore, it is important to extend our previous silac studies using continuous human lung a549 cells, a type ii alveolar epithelial cell, to primary human tracheobronchial airway epithelial (hbae) cells to compare pathways affected by influenza replication, and identify pathophysiologically relevant responses. influenza virus strain a / pr/8/34 (h1n1 ; pr8), an attenuated mouse - adapted strain, was grown in embryonated hens eggs from laboratory stocks, after which chorioallantoic fluid was harvested, aliquoted, and titered in mdck cells by standard procedures. primary normal human broncho - tracheal epithelial cells were obtained from healthy donors by lonza inc. and were certified as mycoplasma-, hiv-, hbv-, and hcv - negative. notably, these cells were capable of > 10 cell doublings, which made the six doublings needed for silac labeling possible. cells were cultured in bronchial epithelial growth media (begm, lonza inc .), which consists of bronchial epithelial basal media (bebm, lonza inc .) supplemented with singlequots (lonza inc .) containing bovine pituitary extract, hydrocortisone, hegf, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, and gentamicin-1000. cells were maintained as monolayers in 10% co2 at 37 c and passaged by trypsinization at 8090% confluence. for silac labeling, cells were grown in silac media that consisted of arginine- and lysine - free begm supplemented with either c12 (light) or c13 (heavy) lysine (29 mg / l) (mass difference of 6.0 da) and c12/n14 (light) or c13/n15 (heavy) arginine (348 mg / l) (mass difference of 10.0 da). light amino acid containing media were prepared from regular stock powder (sigma, st, louis mo), and heavy amino acids were obtained from the silac phosphoprotein identification and quantification kit (invitrogen canada inc . ; burlington, ontario). separate silac labeling and infection experiments were performed four times. for silac experiments, all cells were grown in a pair of t75 flasks in silac media for six cell doublings; after the sixth cell doubling, cells were allowed to reach near confluency. in three replicates c12/n14-light cells were infected with egg - grown pr8 virus that was diluted in gel saline to achieve a multiplicity of infection (moi) of 7 plaque forming units (pfu) per cell. an equivalent number of c13/n15-heavy cells were mock infected as a control using only gel saline. previous studies indicate the small amount of egg protein makes no significant measurable contribution to or . label swapping was performed for the fourth replicate; the c13/n15 heavy cells were infected, and the c12/n14 light cells were mock infected. for all other infections, cells were grown to near confluency and infected at various mois as indicated. in order to synchronize infections, virus- and mock - infected cells were placed at 4 c for the 1 h virus adsorption, after which inoculum was removed and cells were overlaid with appropriate prewarmed light or heavy silac media. infected and mock - infected cell cultures were then kept at 37 c until analysis of viral replication and host protein expression. efficiency of infection was always confirmed by a standard plaque assay as described previously. twenty - four hours postinfection, cells grown in c12/n14-light or c13/n15-heavy begm were collected by brief trypsinization and counted, and then equivalent numbers (roughly 3 10 cells) of each group were mixed together. to verify infection status of each culture , aliquots of all separate cultures were saved for virus titration by plaque assay as mentioned above. mixed cells were washed 3 times in > 50 volumes of ice - cold phosphate - buffered saline (pbs) and lysed for cytosolic proteins by adding np-40 buffer (10 mm trishcl ph 7.4, 3 mm cacl2, 2 mm mgcl2, 0.5% np-40, 1.1 m pepstatin a) to the cells and incubating them for 30 min on ice. nuclei were pelleted at 5000 g for 10 min and the supernatant was saved as cytosol. protein content in the cytosolic fractions were measured using a protein assay kit (biorad) and bovine serum albumin standards. three hundred micrograms of each was reduced, to which 6 the volume of 100 mm ammonium bicarbonate was added. then 100 mm dithiothreitol (dtt) in 100 mm ammonium bicarbonate was added to the peptide mixture, and samples were incubated for 45 min at 60 c. iodoacetic acid (500 mm in 100 mm ammonium bicarbonate) was added to each tube for alkylation, and the tubes were incubated for a further 30 min (room temperature, in the dark). samples were digested overnight at 37 c with 6 g of sequencing grade trypsin (promega, madison, wi) and then stored at 80 c until further processing. peptide fractionation was carried out using a 2d rp (reversed - phase) high ph rp low ph peptide system as described previously. in short, lyophilized tryptic digests were dissolved in 200 l of 20 mm ammonium formate ph 10 (buffer a), injected onto a 1 100 mm xterra (waters, milford, ma) column and fractionated using a 0.67% acetonitrile per minute linear gradient (agilent 1100 series hplc system, agilent technologies, wilmington, de) at a 150 l / min flow rate. sixty 1-min fractions were collected and concatenated using procedures described elsewhere; the last 30 fractions were combined with the first 30 fractions in sequential order (i.e., no . combined fractions were vacuum - dried and redissolved for the second dimension rp separation ( 0.1% formic acid in water). the second dimension was run on a splitless nanoflow tempo lc system (eksigent, dublin, ca) with 20 l sample injection via a 300 m 5 mm pepmap100 precolumn (dionex, sunnyvale, ca) and a 100 m 200 mm analytical column packed with 5 m luna c18 (phenomenex, torrance, ca). both eluents a (water) and b (acetonitrile) contained 0.1% formic acid as an ion - pairing modifier. a 0.33% acetonitrile per minute linear gradient (030% b) was used for peptide elution, providing a total 2-h run time per fraction in the second dimension. a qstar elite mass spectrometer (applied biosystems, foster city, ca) was used in a data - dependent ms / ms acquisition mode. one - second survey ms spectra were collected (m / z 4001500) followed by ms / ms measurements on the 3 most intense parent ions (80 counts / s threshold, + 2 to + 4 charge state, m / z 1001500 mass range for ms / ms), using the manufacturer s smart exit previously targeted parent ions were excluded from repetitive ms / ms acquisition for 60 s (50 mda mass tolerance). files from analyst were submitted simultaneously to protein pilot 3.0 (applied biosystems) for relative quantification and protein identification using the paragon algorithm as the search engine. each ms / ms spectrum was searched against a database of human protein sequences (ncbinr, released march 2008, downloaded from ftp://ftp.ncbi.nih.gov/genomes/h_sapiens/protein/). the search parameters allowed for cysteine modification by iodoacetic acid and biological modifications programmed in the algorithm (i.e., phosphorylations, amidations, semitryptic fragments, etc .). the threshold for detecting proteins (unused protscore ( confidence) ) in the software was set to 2.0 to achieve 99% confidence, and identified proteins were grouped by the progroup algorithm (applied biosystems, foster city, ca) to minimize redundancy. the protein sequence coverage was calculated using peptides identified with > 95% confidence. a decoy database search strategy (ncbinr homo sapiens with all protein sequences reversed) was also used to estimate the false discovery rate (fdr), defined as the percentage of reverse proteins identified against the total protein identification. for our data, the estimated fdr was 0.56%, which is low compared to other studies and indicates a very high reliability of the proteins identified. for relative quantitation, only peptides unique for a given protein were considered, thus excluding those common to other isoforms or proteins of the same family. proteins were identified on the basis of having at least one peptide with an ion score above 99% confidence. among the identified peptides, some of them were excluded from the quantitative analysis for one of the following reasons: (a) the peaks corresponding to the silac labels were not detected. (b) the peptides were identified with low identification confidence (< 1.0%). (c) either the same peptide sequence was claimed by more than one protein or more than one peptide was fragmented at the same time because of shared ms / ms spectra. (d) the sum of the signal - to - noise ratio for all of the peak pairs was 6 for the peptide ratios. in order to compare multiple biological replicates, protein ratios within each replicate were converted to a z - score that allowed protein ratios to be normalized to the mean and standard deviation of its individual experiment. thus, a protein with a z - score > 1.960 indicates that protein s differential expression lies outside the 95% confidence level, 2.576 indicates 99% confidence, and 3.291 indicates 99.9% confidence; z - scores > 1.960 were considered significant. the weighted average z - score was then calculated for each protein found in multiple replicates. gi numbers of all significantly regulated proteins were submitted to and analyzed by the david bioinformatic suite at the niaid, version 6.7, and gene ontologies were examined with the fat and panther databases. the gi numbers were also submitted to, and pathways constructed with, ingenuity pathway analysis software (ipa). mock- and influenza - infected hbae cells were scraped into cold pbs at 24 hpi, pelleted at 5000 g for 5 min, and lysed with lysis buffer (20 mm tris ph 7.5, 100 mm nacl, 0.5% np-40, 0.5 mm edta, 1 antiprotease cocktail ( pierce), 1 phosphatase inhibitor cocktail (pierce) ). forty micrograms of protein was loaded per lane into sds - page gels, separated, and transferred to nitrocellulose membranes. membranes were probed with primary antibodies for viral np (in - house antibody), viral ns-1 (in - house antibody), samd9 (sigma), ifit1 (epitomics), e - cadherin (cell signaling), glg1 (sigma), isg15 (rockland), mxb (santa cruz), rsad2 (abcam), stat1 (cell signaling), -tubulin (cell signaling), ppia (epitomics), oasl (epitomics), -actin (sigma), and gapdh (santa cruz) and rabbit hrp - conjugated secondary antibodies. bands were detected with ecl (amersham) and the alphainnotech fluorchemq multiimage iii instrument, quantitated using alphaease software and virus - to - mock ratios reported without normalization. hbae cells were grown to 80% confluence on 25 mm coverslips and infected or mock - infected at moi = 7. mock and 0, 12, and 24 h infected cells were fixed in 3% paraformaldehyde for 15 min, permeabilized with 0.3% triton - x100 in 3% paraformaldehyde for 15 min, and blocked with 1% bsa in pbs. cells were treated with a primary antibody for np (made in - house) and a cy3-conjugated rabbit secondary antibody (jackson immuno research); all antibodies were diluted in 1% bsa and pbs. coverslips were then mounted onto slides using dapi - prolong gold antifade, dried, and sealed. fluorescent images were obtained using a computer - controlled olympus ix70 microscope (20x objective) equipped with ccd camera and nis - element software. actively proliferating primary epithelial cell cultures are not commonly used for influenza studies; thus, we initially determined optimal conditions for influenza replication in nondifferentiated hbae. a low (0.01) and a high moi were initially chosen to measure the amount of infectious progeny virus released into the supernatant over time. at the low moi, viral replication was most efficient in mdck cells, as peak titers reached almost 10 pfu / ml by 48 hpi, whereas maximum titers of 10 pfu / ml were attained by 48 hpi in a549 cells and no detectable virus was recovered from hbae cells (figure 1a). at moi = 7, mdck and a549 cells reached similar peak titers but at earlier time points, 24 and 31 hpi, respectively; hbae produced detectable virus by 12 hpi and reached a maximum titer of 10 pfu / ml by 24 hpi (figure 1b). influenza replication in hbae at moi = 7 was further confirmed by western blotting for the viral non - structural-1 protein (ns1), which is expressed only during active viral replication. ns1 was clearly expressed in hbae cells by 6 hpi and increased in abundance at both 12 and 24 hpi (figure 1b). additionally, we found that an moi = 7 was sufficient to demonstrate active and productive infection in approximately 50% of cells by 12 hpi, while > 95% of cells demonstrated productive infection by 24 hpi (figure 1c). for silac experiments we therefore chose to study cells 24 h after infection to maximize the amount of time for cellular proteomic changes to occur while retaining active virus replication and minimizing any cytopathic effect (figure 1d). in addition, to verify that hbae cells could be labeled, we analyzed only heavy - labeled cells using lc , we determined that 91% of all peptides contained the c13-label modification (supplementary figure 1). the c13-containing peptides were highly enriched for high h: l ratios; 66% of these peptides had an h: l ratio > 9 (> 90% incorporation), and 92% had an h: l ratio > 2 (> 67% incorporation). in contrast, the peptides that did not contain a c13 label were enriched for peptides with low h: l ratios, e.g., 75% of these peptides had an h: l ratio < 1 (< 50% incorporation), and virtually all unlabeled peptides belonged to keratins. therefore, we concluded that the six hbae cell doublings were sufficient to label the cell proteome. (a) hbae, a549, and mdck cells were infected at moi = 0.01 (left) and 7 (right) pfu / cell to monitor and compare efficiency of infectious progeny virus production. supernatants were collected and titered for progeny virus production by standard plaque assay at indicated time points. (b) hbae cells were infected with influenza a / pr/8/34 at moi = 7 pfu / cell, and protein lysates were assayed for accumulation of viral ns-1 protein at 6, 12, and 24 h post infection by western blotting. (c) hbae cells were infected for 0, 12, and 24 h at moi = 7 pfu / cell. after fixation, the percentage of cells infected by virus was shown using immunocytochemistry for the viral nucleoprotein. (d) phase - contrast images of hbae cells at 24 and 48 h post infection and mock infection. we performed four separate replicate experiments using primary human bronchial epithelial cell cultures from the same donor in which the light - labeled culture was infected in three replicates and the heavy - labeled culture was infected for the fourth replicate. this allowed us to demonstrate that the heavy isotopes had no effect on the virus replication. several proteins were found significantly up - regulated in the light - labeled infections but significantly down - regulated in the heavy - labeled infection. these proteins, which therefore probably represent contaminants, include several keratin species, dermcidin, transferrin, thyroid hormone receptor associate protein 3, and nipsnap2 and were excluded from subsequent analysis. overall, a total of 3740 unique cytoplasmic proteins were detected and quantified; 2282 (61%) of these proteins were detected in 2 or more replicates, 1453 (39%) were found in 3 or more replicates, and 621 (17%) were common to all four replicates (figure 2a). as these proteins were identified using a human database and rely upon the presence of these proteins in both cultures, no viral proteins were identified during mass spectrometry. to confirm that the samples were infected, supernatants were titered for progeny virus using a standard plaque assay on mdck cells (figure 2b). distributions of total, up - regulated, and down - regulated proteins identified. (a) a total of 3740 proteins were identified from four separate biological replicates; 30% of all proteins were found in every run, 53% were found in two or more runs, and 46% were unique to each run. (b) up- and down - regulated proteins were determined statistically using a frequency distribution curve. in order to compare the four replicate runs, infected: mock ratios for each run were normalized by converting them to z - scores, a measure of deviation from the average. therefore, positive values represent proteins that are up - regulated in virus - infected cells, and negative values represent down - regulated proteins. to determine which proteins are significantly altered in abundance we used different confidence intervals as indicated. most proteins were not significantly altered (z - score between 1 and 1); however, a small subset of proteins were strongly up- or down - regulated (z - score > 1.96 and ( c) after silac - labeled hbae cells were infected for 24 hpi, the supernatant was collected and titered by plaque assay to demonstrate that the cell monolayer had been infected and had productively produced progeny virus. (d) the number of up- and down - regulated protein pairs identified in each trial at a confidence level of 95% (z - score > 1.96). specific peptides identified, measured, and used to measure up - regulated and down - regulated proteins are listed in supplementary table 3. comparing the abundance of proteins in infected cultures to uninfected cultures revealed that 97% of all proteins were present in approximately equal amounts (near a 1:1 ratio), thus indicating that iav infection does not alter the expression level of most proteins in host cells. to determine cutoff values for identifying significantly up- and down - regulated proteins, ratios for infected: uninfected in each trial were first converted to z - scores to normalize each run to its own mean and standard deviation, thus allowing us to compare multiple runs. by converting an average z - score back into an average protein ratio , we determined that a z - score of 1.96 (95% confidence interval) corresponded to a 2-fold change. similarly, z - scores of 2.58 (99% confidence interval) corresponded to a protein ratio change of 2.5-fold and z - scores of 3.29 (99.9% confidence interval) corresponded to a 3.0-fold change in protein ratio (figure 2c). further bioinformatic analyses were primarily performed with the 95% confidence data set but were also compared to more restricted data sets based on 99% and 99.9% confidence intervals (not shown). proteins that were identified with only a single peptide were excluded from our data set if they were detected in only a single run; however, any protein identified in multiple trials was included. we also excluded proteins from further analysis if they were not consistently up- or down - regulated between replicate runs. some proteins identified in the current study were unaltered in at least one replicate and up- or down - regulated in other replicate(s). for these scenarios we grouped ratios into three groups: (a) z - scores 0.09 to 0.9 (not altered), (b) 1.0 to 1.96 (moderately altered), or (c) greater than 1.96 (highly altered). we then included only those proteins that had significant values (group c) in at least two runs or one of two runs; additionally, any trial with a nonsignificant value had to have that value still fall within the 80% confidence range (group b) to be considered. outliers could be found at both the peptide and the protein level such that peptides and proteins that were found only in the virus or mock sample were labeled 9999 or 0000 , respectively. we did not remove any outliers at the peptide level but found that they made up a very small percentage of the peptides in our lists of differentially regulated proteins (the details may be found in supplementary table 2) and removing them had minimal impact on the overall protein ratio. for proteins that had a ratio of 9999 and 0 we arbitrarily assigned a z - score of 50 and 50, respectively. these outliers were not included in the statistical analysis but were reincorporated into data sets for further bioinformatic and biological analyses. in summary, we used stringent parameters to generate a data set of proteins of interest based on 99% confidence in protein identification; protein identification based on 2 or more peptide pairs; 95% confidence interval for altered regulation; and the necessity for proteins to show consistent infected: unifected ratios. in this manner we identified 52 up - regulated proteins and 41 down - regulated proteins from pr8-infected primary hbae cells (figure 2c and listed in tables 1 and 2). the identities, associated z - scores and protein percent coverage of the 52 up - regulated and 41 down - regulated proteins are listed in supplementary table 1, and the identities, confidences and other statistical parameters of the specific peptides used for quantitation are listed in supplementary table 2. several of the up - regulated and nonregulated proteins that were identified in the silac analysis were confirmed by western blotting (figure 3), and most western blot confirmed silac - determined regulation status. only one protein, ppia, that was shown to be up - regulated in silac, was not validated using western blotting. this may be caused by inherent differences in sampling (partially degraded proteins would not be measured by western blot but their peptides would be detected by ms) or by inherent differences in the different methods' levels of sensitivity. hbae cells were harvested and lysed with 0.5% np-40 detergent, nuclei were removed, and cytosolic fractions were dissolved in sds electrophoresis sample buffer, resolved in 8%, 10%, or 15% mini bands were visualized and intensities were measured with an alpha innotech fluorchemq multiimage iii instrument. molecular weight standards are indicated at left and ratios of each protein (infected divided by mock - infected) are indicated for each protein at right, along with silac - measured ratios (far right). western blot ratios for host proteins that were detected only in infected cells (undetectable in mock) are designated n.a.. silac ratios are an average of the four replicate runs performed, and western blotting ratios are from a single experiment in a different donor than was used for silac. bands from western blotting were quantitated using alphaease software and are reported without normalization. *: no viral proteins measured by silac because not present in mock - infected samples. gene ontology analyses using go and panther classification terms were determined for proteins that were up - regulated in response to influenza infection and compared at three different confidence intervals: 95% (z - score > 1.96), 99% (z - score > 2.58) and 99.9% (z - score > 3.29) (figure 4a). additionally, canonical pathways and networks that were represented by influenza - altered proteins were constructed with ipa at the 95%, 99%, and 99.9% confidence intervals. approximately half of the proteins up - regulated in response to influenza infection in hbae could be attributed to interferon and other known host cell defense responses. these proteins were identified by several gene ontology categories including defense response, immune response, response to virus and defense response to virus (figure 4a). collectively, these categories included proteins isg15, rsad2, mx1, mx2, ifit1, ifit2, ifit3/4, ifitm1, samd9, samhd1, oasl, oas3, tlr2, stat1, gbp-1, and hla - b / c. pathway analyses also identified immune systems and interferon signaling (figure 5, supplementary figure 2a). proteins belonging to endocytic and intracellular protein trafficking pathways were also indicated as up - regulated (figure 4a) and included -actin, mx1, mx2, and ppia. related canonical pathways that were identified by ipa include caveolar - mediated endocytosis, viral entry via endocytic pathways, and mechanisms of viral exit from host cells (supplementary figure 3b). a third category that was identified among influenza - up - regulated proteins was the presence of gtpase activity, which included proteins mx1, mx2, and gbp1 (figure 4a). lists of (a) up- and (b) down - regulated protein ids were uploaded into david separately and analyzed for enrichment of categories belonging to biological processes, cellular components, and molecular functions. additionally, lists of proteins determined at different confidence intervals (95%, 99%, 99.9%) were compared. interactions between up- and down - regulated proteins. protein ids and ratios from tables 1 and 2 (95% confidence interval) were combined and imported into the ingenuity pathways analysis (ipa) tool from which interacting pathways were constructed. up- and down - regulated proteins are denoted in red and green, respectively; gray proteins indicate that they were detected in our study but not regulated; white proteins interact with many proteins in the network but were not detected in this study. any known direct connections between these proteins are indicated by solid lines; indirect interactions are not shown here. networks are titled (a) infection, gene expression, antimicrobial processe; (b) cancer, dermatological conditions, cellular development; and (c) connective tissues, genetics, cv system development and function. many biological processes were represented among proteins down - regulated at the 95% confidence level. notably, many of these are not cytosolic proteins but were mainly transmembrane (fads2, odz2, fat1, f3, glg1, tnfrsf10a, jag1, fdft1, slc16a2, tnfrsf10b) and extracellular matrix - related proteins (col12a1, lama3, lama5, fn1, adamts1, bigh3) (figure 4b). cellular functions that were attributed to these proteins included cell adhesion (col12a1, fat1, fn1, glg1, lama3, lama5) (figure 4b) as well as signal transduction (odz2, collagen, fat1, fn, f3, bigh3, tnfrsf10a, tnfrsf10b, adamts1) (figure 4a). trail signaling, a process involved in inducing cell death, was also prominently identified (tnfrsf10a, tnfrsf10b) (figures 4b and 5b, supplementary figures 2a and 3d). third, several enzymes involved in lipid metabolic processes were consistently down - regulated including sterol metabolism (fdft1) and fatty acid metabolism (fads2, fads3) (figures 4b and 5b, supplementary figures 2b and 3d). our lab has previously used silac to study a549 alveolar epithelial cell host pathways that are altered in response to infection by influenza virus a / pr/8/34, a highly attenuated mouse - adapted strain of influenza virus. we have now extended this work to include primary human airway epithelial cells, a model that is more closely related to the in vivo situation and that offers opportunity to assess the global relevance of proteomics data obtained using transformed human cell lines. using a human database, we identified 3740 cytosolic hbae protein pairs, of which only a small fraction was altered in response to a / pr/8/34 influenza infection. proteins that were up- and down - regulated were determined statistically using different confidence intervals for which the 95% confidence interval corresponded to a 2-fold or more difference, the 99% limit to at least a 2.5-fold difference, and 99.9% confidence to a 3-fold or greater change. since many previous studies have used either a 2-fold or 1.5-fold change in protein abundance to create data sets of infection - altered proteins, the cutoff values used here are comparable. as viral proteins are not detected by human database searches, we titered the amount of virus secreted by our silac labeled cells at 24 hpi to ensure that viral infection and replication had been successful. it is worth noting that the maximum virus titers produced by hbae vary from donor to donor. for example, the particular cells used in figure 1a produced the highest titers we have observed in hbae, whereas the cells used for the silac experiments (figure 2c) produced considerably lower titers. however, despite these differences, we find robust viral protein expression in these cells from 6 to 72 hpi and can find comparable proteomic expression patterns among hbae from different donors (data not shown). the current study consists of the cytoplasmic cellular fraction, which includes all np40 soluble components such as proteins of the cell membrane, cytoplasm, and cytoplasmic organelles, thereby excluding mainly nuclear proteins. we recognize that the nuclear sample is also an important component of the cell to analyze, particularly because influenza virus replicates within the nucleus, and anticipate analyzing these fractions in future studies. however, the primary purpose of the present study was to compare hbae to a549 , and the later study had only been performed with the np-40-soluble fraction. it is well documented that in many cell types interferon expression is a central cellular response to viral infection. this is thought to be key in developing an antiviral state in both infected and neighboring noninfected cells to limit viral replication. consistent with this paradigm, in our current study up - regulated proteins in hbae were chiefly enriched for host cell defense responses such as interferon (inf) and the jak / stat pathway (figures 4a and 5a, supplementary figures 2a and 3a). of note, influenza a can also antagonize interferon signaling via the viral ns-1 protein through multiple mechanisms including blocking host translation and sequestering viral rna in order to prevent its detection by host pathogen recognition processes. however, the extent of this inhibitory effect is strain - dependent, and since the ns1 protein produced by the a / pr/8/34 influenza strain is generally thought to be a less potent inhibitor of interferon production than other strains ns1 protein, this correlates well with our data. some of these findings were also unique to hbae compared to our previous a549 study. interestingly, neither go nor ipa identified interferon signaling in pr8-infected a549 cells, whereas it was highly significant in hbae cells (p - value = 0.001). in addition, while a few interferon - induced proteins (rsad2, mx1, mx2, and isg15) were highly up - regulated in both cell types, several (stat1, rig - i, samd9, samhd1, ifit1, ifit2, ifit3) were highly up - regulated in hbae but unaltered or undetected in a549. indeed, we were able to confirm with western blotting that cytosolic stat1 abundance was increased in hbae after influenza infection, whereas levels in a549 remained relatively unchanged. samd9, a molecule downstream of stat1, was more highly induced in hbae than a549 after infection, whereas ifit1 was detected only in hbae after infection (figure 6). this suggests that less interferon is produced by a549 than hbae cells and that this difference is dependent either on the cell type (a549 are alveolar epithelial in origin whereas hbae are from airways) or properties of the cell line (i.e., primary versus transformed) rather than the virus strain. notably, a difference in interferon signaling and production could explain the ability of a549, but not hbae, to support replication of the pr8 virus when infected at a low moi (0.01 pfu / cell). these observations provide rationale for future studies assessing the precise role(s) of interferon - associated proteins in preventing viral replication in primary epithelia and mechanisms that suppress this effect in transformed or alveolae - derived epithelial cells. comparison of jak / stat signaling molecule abundance in a549 and hbae cells after influenza infection. cells were harvested and lysed with 0.5% np-40 detergent, nuclei were removed, and cytosolic fractions were dissolved in sds electrophoresis sample buffer, resolved in 10% or 12% mini sds - page, transferred to pvdf, and probed with indicated antibodies. bands were visualized and intensities were measured with an alpha innotech fluorchemq multiimage iii instrument. molecular weight standards are indicated at left, and ratios of each protein (infected divided by mock - infected) are indicated for each protein at right. protein ratios are an average of three experiments each from a549 cells and a single hbae donor and are reported after normalization to gapdh. the interferon - induced proteins commonly up - regulated in both a549 and hbae (rsad2, mx1, mx2, and isg15) were also identified in numerous other influenza studies. for example, myxovirus resistance host proteins mx1 and mx2 are up - regulated in a variety of cell models including mdck and a549 cells, as well as in vivo in studies with macaques. isg15 and rsad2, interferon - induced proteins with anti - influenza activity, were also up - regulated in a549 cells and primary macrophages. these proteins have also been identified in proteomics studies of human host cell responses to infection with other viruses such as rsv, adenovirus, and sindbis virus and hsv. interferon - induced tetratricopeptide repeat proteins (ifit13) have also been found in multiple proteomic studies of influenza involving primary macrophages, the polarized calu-3 airway epithelial cell line and animal models. interestingly, these three ifit proteins have been discovered to form an antiviral complex against 5-triphosphate rna, which is thought to be present during an influenza infection. it is likely that these similar responses to different types of infection reflect the induction of a nonspecific innate immune response against rna viruses by the host cells. we found 19 proteins that were consistently and strongly down - regulated in primary hbae cells in response to influenza infection, and these were mainly linked to cell adhesion, death receptor signaling, and lipid metabolism. cellular adhesion is necessary for the survival of primary cells, and previous studies have found that a reduction in adhesion molecules precedes and may possibly be instrumental in inducing cell death. in epithelial cells adherens junction proteins are essential for cell adhesion and these are largely composed of integrin, cadherin, catenin, and actin complexes. many proteins belonging to these complexes and downstream signaling pathways were detected in our silac data among which integrin 4 was down - regulated 2.4-fold, glg1 2.5-fold, and thbs1 2.6-fold. other adhesion - related proteins such as fibronectin, laminin, tgfbi, and tnc were down - regulated > 2.0-fold. however, cell death processes were not identified by any of our analyses, except for the down - regulation of two trail receptors, tnfsfr10a and tnfsfr10b (figures 4b and 5b). tnfrsf10a, tnfrsf10b and f3 are additionally associated with caspase activity induction, which was similarly indicated as down - regulated (figure 4b). in contrast, tnfsfr10d was found up - regulated 1.4-fold in pr8-infected a549 cells at the same time point. given that trail signaling is important in clearing influenza - infected epithelial cells, it would be interesting to further investigate the mechanism by which trail - receptors are down - regulated in hbae. as cholesterol and lipids are key to maintaining the integrity of the cell membrane and the organization of lipid rafts, it was interesting to find that several proteins related to lipid biosynthesis were down - regulated in response to influenza. for example, a previous study indicated that rsad2 interferes with cholesterol synthesis and subsequent lipid raft integrity by binding and inhibiting fpps, an enzyme in the cholesterol biosynthetic pathway. our silac / hbae data found rsad2 highly up - regulated (> 3-fold) and fpps moderately up - regulated (1.5-fold) (supplementary figure 1c). importantly, fpps is involved not only in the production of cholesterol but also other intermediates, such as isoprenoids fpp and ggpp, which serve as essential activators of gtpase proteins. our silac data identified a number of proteins from this pathway, of which fdft1 was most significantly altered (2.9-fold down - regulated). fatty acid synthesis was also affected through a strong downregulation of fads2 (3.45-fold). a second study has also found that interferon signaling can suppress sterol biosynthesis in macrophages, suggesting that this may be a protective response induced by the host cell rather than by the virus. while none of fpps, fdft1, nor fads2 were altered in a549 cells at 24 hpi, ipa indicated other lipid - related pathways as down - regulated in a549 cells including sphingolipid metabolism and vdr / rxr signaling. collectively, these data suggest that lipid metabolism may be inhibited in epithelial cells during influenza infection. overall this proteomics study provides a broad and unbiased profile of cytoplasmic host cell proteins that are up- and down - regulated in response to an h1n1 mouse - adapted influenza virus in primary human airway epithelial cells. while some of our findings correlate with previous proteomic studies involving influenza or other viruses, we have identified many proteins that have not been previously studied with respect to influenza. thus, our work has expanded the knowledge base for understanding host cell response to influenza infection. of note, we performed our studies using relevant primary human bronchial epithelial cells, whereas many previous studies have been conducted using transformed cell lines such as alveolar a549 cells or nonlung hek293 cells. a comparison of functional categories and pathway alterations between our studies with a549 and hbae identified some common characteristics such as downregulation of cell adhesion and lipid metabolism, as well as upregulation of host defense responses against influenza infection. further validation and investigation into the functions of the candidate proteins we have identified will contribute to understanding novel virus - host interactions and permit integration of this information with previous large - scale analyses of influenza to identify new directions for developing anti - influenza therapies.	influenza a virus exerts a large health burden during both yearly epidemics and global pandemics. however, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. we have previously measured host protein responses in continuously cultured a549 cells infected with mouse - adapted virus strain a / pr/8/34(h1n1 ; pr8). we here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (hbae) cells. a total of 3740 cytosolic hbae proteins were identified by 2d lc ms / ms, of which 52 were up - regulated 2-fold and 41 were down - regulated 2-fold after pr8 infection. up - regulated hbae proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down - regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. comparison to influenza - infected a549 cells indicated some common influenza - induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. however, hbae - specific alterations consisted of interferon and cell death signaling. these data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells.
plasmodium falciparum has traditionally been the main focus of malaria control programs worldwide, mainly because this parasite is the major cause of severe morbidity and mortality in tropical africa. however, at a time when global eradication is advocated as the ultimate goal of malaria control strategies worldwide, p. vivax needs to be given much more attention from researchers, policy makers, and funding agencies. p. vivax is a major public health challenge in central and south america, the middle east, central, south, and southeast asia, oceania, and east africa, where 2.85 billion people are currently at risk of infection and as many as 250 million infections may be due to this species each year. the emergence of drug - resistant strains and severe (sometimes fatal) disease challenges the traditional view of vivax malaria as a benign infection. although cytoadhesion of p. vivax - infected erythrocytes to endothelial cells has been recently demonstrated and might contribute to the pathogenesis of severe vivax malaria, this phenomenon is likely to be much less common than with p. falciparum infections. compared to p. falciparum, p. vivax has a slightly longer incubation period (12 days to several months) and a similarly phased erythrocytic cycle (4248 hours) that yields fewer merozoites per schizont. however, the distinct ability of p. vivax to stay dormant in host's liver cells and cause relapses weeks or months after the primary infection is the most striking difference between p. vivax and p. falciparum. although the molecular mechanism of relapse remains undisclosed, progression of the parasite through its life cycle is fairly well described. a proportion of sporozoites remain dormant in the liver, as hypnozoites, for prolonged periods of time before developing and causing recurrent infection. the ability to relapse in fact, in areas where both species are present, act (artemisinin - based combination therapy) campaigns have had a greater impact on p. falciparum than on p. vivax prevalence. we demonstrate how this is evident when contemplating elimination scenarios, with p. vivax elimination being extremely difficult to achieve by mass drug administration. epidemiological studies have accumulated evidence that clinical (antidisease) and antiparasite immunity is attained at younger ages for p. vivax, when compared to p. falciparum, under similar infection rates. we put forward an alternative hypothesis arguing that differences between the observed age profiles lie in the characteristic life cycles of both parasites. explicitly, we propose that the ability of p. vivax to relapse can accelerate the piecemeal acquisition of clinical immunity. we developed a model representing the transmission dynamics of p. vivax by adding new elements to the foundation laid by previous work in p. falciparum. we have thus a model structure for p. vivax transmission, represented by figure 1(a), which contains the topology representing p. falciparum as a submodel (figure 1(b) ). the falciparum dynamics are retrieved by equating p1 to 1 in the p. vivax model. natural history of p. vivax infection is generally similar to that of p. falciparum, with a few but crucial idiosyncrasies. susceptible individuals (s) are subject to a certain rate of infection (here represented by the force of infection), which depends on local environmental and socioeconomic factors. in p. vivax, after a mosquito infectious bite, an indeterminate proportion of the inoculated sporozoites remains dormant in the liver, whilst the remaining develops into erythrocyte invading merozoites. the model describing the transmission dynamics of p. vivax must then include a latent class, representing those individuals who, after recovering from infection, keep a remnant of dormant liver forms, called hypnozoites (subject to reactivation at rate) rather than clearing all parasites while acquiring clinical immunity. reactivation is still a rather cryptic process, and most relapses seem to from activation of heterologous hypnozoites, which suggests that genotype - specific immunity somehow modulates the occurrence of relapses, much to the resemblance of how the clinical outcome of a given infection is determined. here, parameter p1 accounts for episodes not followed by a relapse, either because no hypnozoites were formed or because the remaining hypnozoites do not reactivate during their lifespan. for illustration purposes, throughout the paper we keep p1 = 0.25 for p. vivax (and p1 = 1 for p. falciparum). it is generally accepted that the severity of malaria episodes decreases as the host accumulates exposures to the parasite. we implement this aspect of malaria immunity by discretizing the malaria clinical spectrum into two compartments: i1 represents the severe end of the spectrum, and is labeled clinical malaria, " while i2 represents the less severe part and is labeled asymptomatic malaria ". naturally, there is a degree of arbitrariness in this compartmentalization, and the should be interpreted in this context. we consider that immunologically nave individuals will display clinical symptoms when infected (i1). although p. vivax infections are generally not as severe as those caused by p. falciparum, they are far from benign. community studies have revealed that the proportion of p. vivax infections presenting with fever is similar to the one registered for p. falciparum. the probability of clinical outcome upon relapse in individuals that kept hypnozoites (l1) from a previous clinical infection is determined by parameter p2. latent individuals carrying hypnozoites are subject to reinfection at rate , with the ant infection phenotype being determined by parameter p2 as well. individuals who have just recovered from a clinical malaria episode are said to have acquired temporary clinical immunity (r). this means that they do not display clinical symptoms upon reinfection and that, unless they are challenged again within a given time frame, they will lose that clinical protection. in fact these infections are crucial in boosting acquired clinical immunity, and the interplay between the rate of infection in clinically immune individuals and the rate of clinical immunity loss is fulcrum in determining the number of expected clinical malaria cases during one's lifespan. as such, those recovering from an asymptomatic infection are also subject to loss of immunity at rate. these include individuals that either clear all parasite forms and return to r (a proportion p1) or keep a remnant of hypnozoites and go to the l2 class. we consider that, in the latter case, subsequent infections and relapse will give rise to asymptomatic malaria. the described dynamics can be written as the following system of differential equations: st+sa=r((a)+)s,i1t+i1a=(a)s+p2(+(a))l1(1+)i1,rt+ra = p11i1+p12i2(+(a)+)r,i2t+i2a=(a)r+(+(a))l2 + (1p2)(+(a))l2(2+)i2,l1t+l1a=(1p1)1i1+l2(+(a)+)l1,l2t+l2a=(1p1)2i2(+(a)++)l2, with boundary conditions at age a = 0: s(t, 0) = and ii(t, 0) = li(t, 0) = r(t, 0) = 0 for i = 1,2. the p. falciparum transmission dynamics are retrieved by making p1 = 1: st+sa=r((a)+)s,i1t+i1a=(a)s(1+)i1,rt+ra=1i1+2i2((a)++)r,i2t+i2a=(a)r(2+)i2. the force of infection was constructed as an age - dependent parameter (a)=0(1ceka). the function is strictly increasing with age, with a minimum 0(1 r) (at age zero) converging asymptotically to 0 as age increases. parameter k determines how steeply the force of infection increases with age, and r controls the magnitude of that increase. a summary measure of transmission is obtained by integrating the force of infection over age as =(a)p(a)da, where p(a) = e is the total population distributed over age and is the birth and death rate. adopting standard assumptions, is proportional to the frequency of infectious individuals, the proportionality constant being the transmission coefficient, =i1+i2. this standard assumption allows us to analyze how the equilibrium behavior of the system depends on , which is a critical transmission parameter, representing the sylvatic portion of the classical macdonald formulation of the basic reproduction number for vector - borne diseases. the basic reproduction number for the p. vivax model presented here assumes the form r0=(p2n1+1p1n2+2p1n3+(+)n4+1n5)2d1+2d2+1d3+12p1d4+2d5+3d6, where n1=1(1p1)+2,n2=p2,n3=++p2,n4=(+)++2,n5=+(p1+p2)+,d1=1+2p11p2,d2=(p2+p1p1p2)+1(1+p1p2+p1p2),d3=(+2p2+p2p1)+2p1,d4=2p22+p2+p22p1,d5=(1+)(+2)+2(+p1) + 1(2p2p2p1+2),d6=+2++1 + 2. epidemiological changes are often attributed to thresholds in transmission, and these are detected through longitudinal trends and comparative studies across multiple communities. we consider the transmission coefficient, (or the basic reproduction number, r0) as a control parameter and describe the significance of these indices of transmission on selected epidemiological variables. to do so, we calculated the endemic equilibria for systems and, without age dependence. age profiles were obtained by following a cohort under the pressure of an age - dependent force of infection defined by, for 20 years. the common variables between systems and have the same boundary conditions. we used the escalator boxcar train (ebt) technique to simulate the dynamics in our age - structured population. to assert the benefits of specific interventions we first simulate our age - structured model in equilibrium conditions to obtain the age profile of clinical disease prevalence without intervention. we use that age profile as the initial condition for the simulation in which the drug administration trial is in vigor. in such a setup, asymptomatic malaria is treated as effectively as clinical malaria, and thus the recovery rate from an asymptomatic infection, 2, takes the same value as the recovery rate from a clinical infection, 1. the expected prevalence of malaria cases of each plasmodium species is highly dependent on local environmental and socioeconomic factors that can be summarized into some transmission index. in figures 2(a) and 2(c), infectious proportions are plotted in terms of a transmission coefficient, , which encapsulates information on contact rates and infectivity. comparing the model outputs for the p. falciparum (red) and p. vivax (blue) systems, we verify that, if a significant proportion of individuals recovers from infection with dormant forms of the parasite which reactivate later on, it is easier for the parasite to be transmitted in a sustainable manner (figure 2(a) ). latency then generates a mechanism by which the parasite population can be maintained in scenarios where vectors are not very abundant or where human - mosquito contacts are sparse. strikingly, for any value of transmission coefficient that sustains both species, the proportion of individuals with a clinical episode due to p. vivax is lower than that due to p. falciparum (figure 2(a) ), while the overall parasite prevalence (measured as proportion of individuals in the population which carry parasites in the blood stream and are thus potentially infectious) is expected to be higher for p. vivax (figure 2(c) ). figures 2(b) and 2(d) display how equilibrium solutions depend on the basic reproduction number, r0, as a proxy for transmission. figure 3 simulates the introduction of malaria control measures in an area supporting p. vivax transmission versus in an area supporting p. falciparum transmission, with the same parasite prevalence. the intervention consists of applying an mda (mass drug administration) campaign using act aimed at reducing the infectious period of asymptomatic infections, making all infections, regardless of clinical outcome, last the same. figure 3 portrays that by treating asymptomatic infections equally for both species one can reduce falciparum prevalence (red lines) to a much greater extent than vivax prevalence (blue lines) within the same time frame (dot - dashed lines). this intervention alone leads to sustained elimination of p. falciparum if implemented for 274 days (time for the system to enter the basin on attraction of the disease - free equilibrium). in the case of p. vivax , elimination is only possible through a stochastic event, and even then the risk of reemergence will be high as the disease - free equilibrium is unstable for this system. if a drug that can eliminate the dormant forms of the parasite (primaquine) is included in the mda strategy, then the p.. however, once the intervention is halted the risk for reemergence would still be much higher for p. vivax due to instability of the disease - free equilibrium. in figure 4 , we simulate the age profiles for p. vivax and p. falciparum for an equal risk of infection. the predicted age profiles for p. vivax (blue) display a higher value for clinical malaria cases at the peak, when compared with p. falciparum (red), and reveal a decrease in the average age at infection. this means that the risk of a p. vivax episode relative to the risk of having a p. falciparum episode is greater in very young children and lower throughout childhood. this is consistent in the two transmission settings chosen for this illustration as well as for the entire transmission spectrum. the true impact of p. vivax transmission on human populations stands in the shadow of the overwhelming mortality and morbidity burden exerted by p. falciparum worldwide. p. vivax importance has been increasingly recognized over the years, and new estimates of the global malaria burden revealed that there are slightly more people at risk of having a p. vivax infection than a p. falciparum infection. however, probably more interesting is to consider the importance and impact of p. vivax under the scope of its ecological interactions with p. falciparum, especially considering that coinfection with these species might somehow modulate the clinical outcome of infection , and that there might be cross - specific immunity. understanding the transmission dynamics of p. vivax is crucial to understand the current epidemiological scenario and the potential long - term impact of control interventions. we have previously developed a mathematical model to represent the dynamics of p. falciparum transmission in human populations. the model was calibrated on hospitalization data from 8 endemic regions in sub - saharan africa, estimating a fundamental difference between the duration of clinical and asymptomatic infections. this led to the identification of a deterministic elimination threshold for p. falciparum malaria in areas of low to moderate transmission. we adopted the same generic model for p. vivax while adding latency classes to represent those individuals who recover from infection with a remnant of dormant parasites, called hypnozoites. model outputs were generated and compared with and without latent classes, to mimic vivax and falciparum, respectively, while all other features were unchanged. the expected levels of clinical episodes of both p. vivax and p. falciparum for the same levels of the transmission coefficient, , indicate that p. vivax transmission can be sustained for much lower values of this parameter, when compared with p. falciparum (figure 2(a) ). this becomes intuitive in light of p. vivax's ability to relapse, which can transform a single infectious bite into more than one malaria episode leading to higher parasite prevalence in the p. vivax system (figure 2(c) ). these relationships are inverted, however, when parasite prevalence is represented against the basic reproduction number, r0, (figures 2(b) and 2(d) ) attesting the importance of standardizing transmission indices. more importantly the deterministic elimination threshold described for the p. falciparum system is no longer present under the conditions simulated for p. vivax. the parameter regime sustaining the bistability phenomenon that gives rise to the elimination threshold is contracted when latency comes into play. a major advance in the search for effective malaria drug treatment, following the demise of most known drugs in the battle against resistant parasites, came in the form of artemisinin, a very potent and effective drug against chloroquine and sulphadoxine - pyrimethamine - resistant infections, which can clear parasites and resolve fever faster than any other licensed antimalarial. however, artemisinin derivatives have a very short half - life, translating into substantial treatment failures when used as monotherapy, which motivated the combination of artemisinin with longer - lasting partner drugs in the so - called artemisinin combination therapies (acts), assuring that there is substantial antimalarial pressure to deal with the residual parasite biomass that may persist when the artemisinin derivatives have fallen below therapeutic levels. curiously, empirical studies have revealed that the deployment of act as a control strategy affects p. falciparum transmission much more than it does p. vivax transmission. furthermore, unexpected resurgences of p. vivax malaria in areas where elimination attempts were thought to have been successful question to what extent p. vivax control is sustainable. our simulations suggest that, by treating all infections (equally for both species) with an act therapy, one can reduce falciparum prevalence to a much greater extent than vivax prevalence (in the same time scale). this is intrinsically associated with act's inability to kill the dormant forms of p. vivax, which escape drug action, subsist, and can cause a relapse later on, thus sustaining the parasite pool. in such scenario one should invoke the use of drugs targeting hypnozoites (primaquine is the only approved and available drug at the moment) as a means of counteracting the parasite's ability to relapse. if everyone was to receive a dose of primaquine to kill the hypnozoite parasite forms, the dynamics would be similar to that of p. falciparum. a remaining difference, however, is that p. vivax elimination is predicted to be unstable, meaning that any perturbation in the system (introduction of infectious individuals from neighboring populations for instance) would drive it back to the endemic equilibrium. another epidemiological observation that deserves careful consideration is the differential speed at which complete clinical immunity is attained when comparing p. vivax to p. falciparum for scenarios of equal risk of infection. while others have interpreted this phenomenon as an evidence of there being different mechanisms by which immunity is acquired, our suggest that the difference in age profiles of clinical malaria can be accounted for by the intrinsic transmission dynamics inherent to the natural history of infection of each species. in figure 4 , we can clearly see that the model topologies we used to describe the p. vivax and p. falciparum transmission dynamics can account for differences in the age profiles of clinical malaria, specifically for low levels of p1 and p2. we then propose that differences in age profiles of clinical malaria can be explained solely by p. vivax's ability to relapse, which converts a single infectious mosquito bite into one or more malaria infections, thus accelerating the acquisition of clinical immunity. relapse also serves as an immunity boosting mechanism that prevents onsets of malaria episodes in older ages. on a final note, we recall that all parameters other than those governing hypnozoite formation and relapse were unchanged between the two scenarios. p. vivax and p. falciparum transmission dynamics might, however, be modulated by a number of biological characteristics such as gametocyte production and antigenic variation. although our model was not designed to explore these processes, some analogies can be made as detailed in the supplementary material available online at doi:10.1155/2012/921715. first, it should be acknowledged that p. vivax produces gametocytes earlier than p. falciparum during a human infection and these gametocytes are shorter lived in the bloodstream. as a consequence , vivax patients are expected to have transmitted more by the time malaria infection is confirmed and treatment is provided. second, the acquisition of immunity to p. vivax relies on expectations regarding strain specificity of natural immunity and antigenic similarity between primary infections and relapses that deserve further attention.	often regarded as benign, plasmodium vivax infections lay in the shadows of the much more virulent p. falciparum infections. however, about 1.98 billion people are at risk of both parasites worldwide, stressing the need to understand the epidemiology of plasmodium vivax, particularly under the scope of decreasing p. falciparum prevalence and ecological interactions between both species. two epidemiological observations put the dynamics of both species into perspective: act campaigns have had a greater impact on p. falciparum prevalence. complete clinical immunity is attained at younger ages for p. vivax, under similar infection rates. we systematically compared two mathematical models of transmission for both plasmodium species. simulations suggest that an act therapy combined with a hypnozoite killing drug would eliminate both species. however, p. vivax elimination is predicted to be unstable. differences in age profiles of clinical malaria can be explained solely by p. vivax's ability to relapse, which accelerates the acquisition of clinical immunity and serves as an immunity boosting mechanism. p. vivax transmission can subsist in areas of low mosquito abundance and is robust to drug administration initiatives due to relapse, making it an inconvenient and cumbersome, yet less lethal alternative to p. falciparum.
age - related macular degeneration (amd) is one of the most common reasons of visual impairment in the western world. the prevalence of amd is approximately 2% at the age of 40, with an increase to about 17%35% at the age of 80. patients with amd experience irreversible and progressive visual loss, leading to reduced quality of life , reduced health, and increased risk of mortality. in step with visual deterioration amd patients report additional complaints, such as loss of postural stability , fall accidents, and neck / scapular area complaints. in primary health care these symptoms are often downplayed as part of the aging processes and not typically associated with visual impairments. however, research indicates that people suffering from amd may pose a higher risk of developing visual, musculoskeletal, and balance complaints. genetics, lifestyle, body mass index, and smoking have all been found to increase the risk of developing amd. amd is identified by retinal changes beyond what are age - normal. early stages are associated with minor visual disturbance, followed by increased retinal changes during the intermediate stages and the development of pronounced visual deterioration at the late stages. amd follows no particular schedule, but studies show that approximately 20% of cases escalate from intermediate to late stage within 6 years. as vision deteriorates the visual system this may adversely affect visual performance and further distort visual feedback to other systems that are normally supported by visual inputs such as the musculoskeletal and the postural systems. as a consequence there are a number of enhancing devices to help amd patients adjust to their deteriorated vision. although magnifying aids are aimed at facilitating everyday life, they come with some drawbacks such as reduced field of view or the need to use both hands or adopt awkward postures. straining conditions such as orbicularis squinting (to increase effective focus imaging) or unfavorable gaze - angles (to locate the best viewing visual field or facilitate convergence movements) may also occur as the person struggles to acquire an acceptable image, which may in turn impose increased strain in the muscles used for positioning the head. visual deterioration also negatively influences postural control as this is largely based on adequate visual feedback. hence, visual deterioration and increased use of visual aids may increase the risk of musculoskeletal and balance complaints. however, our knowledge of what influence visual loss in amd may pose on musculoskeletal and balance complaints is limited. previous research addressing the impact of visual loss in amd patients on visual, musculoskeletal, and balance complaints is limited or has used cross - sectional design. this research has identified concurrent relationships; however, less is known about the influences of visual decline in amd on visual, musculoskeletal, and balance complaints across time. by using a longitudinal cohort design these issues can be addressed. therefore studies using longitudinal design are warranted in order to investigate to what extent visual deterioration influences visual, musculoskeletal, and balance complaints. the present study aims to identify risk markers for increased visual, musculoskeletal, and balance complaints and perceived general health in patients with amd by using a longitudinal cohort design. based on the previous research described above , we hypothesized that visual, musculoskeletal, and balance complaints shouldincrease more in amd patients than in age - matched individuals with age appropriate vision, increase with increased visual deterioration, negatively affect perceived general health. increase more in amd patients than in age - matched individuals with age appropriate vision, increase with increased visual deterioration, negatively affect perceived general health. this study has a prospective longitudinal case / control design, in which a group of amd patients and an age- and sex - matched reference group with normal vision were assessed twice. at baseline, amd patients were recruited in consecutive order from the queue system of the low vision centre at region rebro county, sweden. inclusion criteria were being diagnosed with late- or intermediate - stage amd according to an ophthalmologic examination at a hospital eye clinic with best corrected visual acuity (bcva) worse than 0.5 logmar and no additional eye disease. the patient should have had the amd diagnosis for at least one year in order to adapt to the visual impairment and become accustomed with use of magnifying visual aids. individuals in the reference group were recruited from relatives and companions of the amd patients visiting the clinic. inclusion criteria included age - normal bcva defined as better than 0.10 logmar with correction if needed and without any known eye disease. actual refraction and bcva were tested in both groups in an eye - examination conducted by an optometrist at the low vision clinic. individuals in either group were excluded at baseline or at follow - up if they were medically diagnosed with musculoskeletal or balance disorders, such as whiplash, arthritis, myalgia, or parkinson's disease. the study included 88 individuals, 64 cases (43 women/21 men, mean age 78.6 years, sd = 5.81, and range 61.885.9 years), and 24 referents (11 women/13 men, mean age 73.9 years, sd = 6.08, and range 64.983.0 years). in 2012, a mean of 3.8 years and sd = 0.46 years later, the former participants were contacted by telephone to schedule the follow - up appointment. at follow - up 55 individuals remained, including 37 cases (28 women/9 men, mean age 81.1, sd = 5.43, and range 67.289.2 years) and 18 referents (11 women/7 men, mean age 77.6, sd = 5.60, and range 69.078.1 years). none of the amd patients were diagnosed with wet neovascular amd at baseline; however, six amd patients have had ranibizumab injections prior to baseline. twenty amd patients (31%) had reached late amd at baseline according to clinical classification of age - related macular degeneration. two participants (one amd patient and one referent) have had monocular cataract surgery with a replaced intraocular lens. among the 27 cases not participating in the follow - up 16 were diseased, 5 were infirm or suffered from dementia and were not able to participate, 2 had moved, and 4 declined without giving any specific reason. among the 6 referents not participating in the follow - up, one was deceased, one had moved, one had acquired amd, and 3 declined without giving any specific reason. consort flow chart is shown in figure 1. informed consent was collected from all participants at baseline and at follow - up. best corrected visual acuity (bcva) using habitual visual aids (ordinary spectacles or contact lenses) was assessed under monocular and binocular viewing conditions using the early treatment diabetic retinopathy study (etdrs) test chart. if va was very low, the bailey - lovie letter - by - letter chart was used to capture va beyond 1.0 logmar. critical print size was assessed by the smallest font size that could be read fluently / best acceptable reading pace, with the use of the participants' normal visual aids. cps was measured in points (p), where 1 p = 1/72 of an inch. a font size of 8 p is commonly used in newspapers and is equivalent to a snellen notation of n8 or 1 m. cps was assessed as participants read the appropriate printed texts, ranging from 4 p to 64 p. reading distance was assessed by measuring the distance in cm between the text and the eyes when the participant was reading the near charts with assistance of their normal visual aids. visual aids contained head - worn visual aids (single vision reading glasses, bifocals, or progressives) used alone or combined with handheld magnifiers or closed circuit television (cctv). the magnification used while reading was estimated by summing up the dioptric power (d) of those visual aids that normally were used simultaneously. the amount of dioptres was then transformed into units of magnification by dividing the sum of d by 4 based on a simplified commonly used nominal magnification transformation formula (m = f/4). this calculation does not give a perfect value of the provided enlargement , as it is not based on the equivalent viewing distance. for example, if reading glasses of + 8 d were used, this refers to a magnification of 8/4 = 2x, but combined with a handheld magnifying aid of + 20 d, this refers to a magnification of 8/4 + 20/4 = 7x. this assumes a reading distance of less than 20 cm, whereof at least half the distance refers to the distance between the specs and the magnifying aid. in clinical practice, most of the elderly have difficulties accomplishing a shorter reading distance than what is provided by reading glasses above + 8.0, which is the reason why the additive handheld magnifying aid is often combined with their best reading glasses to overbridge effects from the continuous visual deterioration and does not shorten the reading distance any further. controls also used near visual aids, in order to compensate for the distance, which was estimated in the same manner. near visual function was assessed on the near activities subscale of the national eye institute - visual function questionnaire 25 (nei - vfq 25). the visual function questionnaire - near activities subscale (vfq - nas) consists of six questions and has shown excellent internal consistency and reliability (cronbach alpha 0.91) as well as convergent validity with bcva and health - related quality of life among patients with amd. the first five alternatives describe the quality of visual function, ranging from 0 to 100 at equal steps (i.e., 0, 25, 50, 75, and 100). the sixth alternative, stopped doing this for other reasons or not interested in doing this, is not related to the quality of visual function and therefore does not contribute to the total vfq - nas score. the sum score from the six questions was divided by the number of contributing questions to form a total near activity visual function score. generally, a total score above 80 indicates minor visual problems and a score of 70 or less is considered clinically significant. visual, musculoskeletal, and balance complaints were measured on the visual, musculoskeletal, and balance complaints questionnaire (vmb). it consists of 15 questions, with five questions each in visual (vmb - v), musculoskeletal (vmb - m), and balance (vmb - b) domains. the questions are rated on visual analogue scales, ranging from 0 (no problem at all) to 10 (problems all the time), with verbal anchors at 3 (occasionally) and 7 (quite often). the sum of the five scores in each domain is then calculated, with scores ranging from 0 to 50. the present study was performed with the use of the original vmb - scale, which was used at baseline and then at follow - up allowing for detection of individual changes using gee. the participant could choose from two alternatives, feeling healthy or not feeling healthy. data analyses were performed using ibm spss statistics version 22 (ibm corp, armonk, ny). most of the data were positively skewed, which does not necessarily indicate a problem with the scales but reflects the underlying nature of the construct in focus. wilcoxon signed rank test was used to compare differences across time and mann whitney u test was used to compare differences between groups. generalized estimation equation (gee) was used to obtain robust parameter estimates and standard errors and to further estimate the correlation of multiple observations for each subject over time. the gee is an extension of generalized linear models, which facilitate regression analyses of dependent variables that are not normally distributed. by using marginal models the analysis gives an average response for observations sharing the same covariates as a function of the covariates; that is, for every one - unit increase in a covariate across the population in focus, gee tells the user how much the average response should change; gee can thus account for correlations in repeated measures, and the interpretation of the estimates is much the same as that in ordinary least squares regression when the dependent variable is normally distributed. in the present study, each predictor variable was regressed separately on each of the vmb to identify significant risk markers for visual, musculoskeletal, and balance complaints. since the vmb variables were positively skewed, the gee regression models were specified with a log link function. the influences of predictor variables on participants' perceived health (dichotomous data) were evaluated using gee logistic models, which were expressed in odds ratios (or) and 95% confidence intervals (95% ci). at baseline there was no significant difference in sex ratios between cases and referents, but there was a trend for a difference at follow - up (= 5.33, p = 0.069). this difference was mainly due to a larger number of dropouts among men with amd (57%) than among women with amd (35%) at follow - up. there was also a difference in age between groups at baseline (u = 445, p = 0.002) but not at follow - up (u = 256, p = 0.10). as shown in table 1, cases had worse bcva at baseline than referents, needed larger font size when reading, used shorter reading distances, and needed greater magnification. fewer cases than referents used progressives, but several more of them used a combination of different near visual aids. cases also reported lowered near visual function, lowered perceived general health, and more visual and balance complaints, but not significantly more musculoskeletal complaints than referents. at follow - up, within group analyses revealed that both groups showed deteriorated bcva and visual function (table 2). cases needed larger font size when reading and more magnification, but referents did not. however, referents needed significantly shorter reading distances at follow - up, which was not found for cases, although cases still needed shorter reading distances than referents (u = 49.5, p < 0.001). at follow - up cases no longer used bifocals or progressives as a basic solution, and none of them reported that they solely relied on these visual aids. instead cases reported significantly more visual, musculoskeletal, and balance complaints at follow - up than at baseline, but no change in the level of complaints was found for the referents (figure 2). complaints increased among cases from 3 times (vmb - m) to 10 times (vmb - v) as much as among referents. general health decreased in cases, where fewer reported good general health at follow - up than at baseline, whereas there was no significant deterioration of perceived general health found in referents. in regard to the first aim, gee analysis of complaints showed significant group influences, indicating that the increase of visual, musculoskeletal, and balance complaints from baseline to follow - up was larger in cases than in referents (b = 15.177, 95% ci : 11.467 ; 18.888, p < 0.001, b = 4.180, 95% ci : 0.018 ; 8.342, p = 0.049, and b = 18.862, 95% ci : 14.373 ; 23.351, p < 0.001, resp .). in support for the second aim, and as shown in table 3, visual and balance complaints increased in step with deteriorating va in cases, along with the need for larger critical print size. the analyses also revealed that all three complaints increased with increased use of magnification and deteriorating visual function. also in referents, visual complaints increased with declining va, ing in more visual and balance complaints as visual function deteriorated (measured by vfq - nas), but there were hardly no changes in magnification and thereby no association between magnification and any of the three vmb complaints. to further evaluate influences from use of visual aids, types of aid were regressed on visual, musculoskeletal, and balance complaints, serving as dependent variables. as shown in table 3 , these analyses showed that in cases reduced use of bifocals and progressives was associated with decreased visual complaints, while increased use of cctv was associated with increased visual, musculoskeletal, and balance complaints. in referents, the use of reading glasses was associated with decreased visual complaints and use of bifocals was associated with increased visual and balance complaints. in support for the third aim, gee analyses showed that cases perceived poorer health with increasing visual and musculoskeletal complaints (table 4). the need for larger font size was also a marker for poorer perceived health in cases. this study followed a group of amd patients and age - matched referents responding to reported change in perceived complaints during visual decline over a period of four years. that is, amd patients were more at risk of increased visual, musculoskeletal, and balance complaint than similarly aged individuals with age - normal vision. additionally, visual deterioration was a risk marker for increased visual and balance complaints both in cases and in referents. in referents this decrease must be considered consistent with the age - normal decline. finally, increased visual, musculoskeletal, and balance complaints constituted risk factors for decreased perceived general health. at baseline amd patients had significantly worse conditions in all assessed areas compared to referents, except for reported musculoskeletal complaints. most of these group differences remained or increased at follow - up, which supports the hypothesis that having amd entails a greater risk of poorer health. it can be hypothesized that, in pace with visual function decline and increased need for support and assistance, low vision patients may successively abandon physical and social activities ing in a less satisfactory quality of life. visual deterioration, such as worsening bcva, need of larger print size, larger magnification when reading, and worsened near visual functioning were risk markers for increased visual and balance complaints among amd patients. interestingly, the referents showed a similar pattern to amd patients, in regard to bcva and near visual functioning. thus, visual deterioration seems to be a risk marker for increasing visual and balance complaints regardless of initial vision status (normal or impaired). these are of course consistent with general deterioration of sensory function with age; however, the increased magnitude of complaints is more profound among amd patients, which may put them at higher risk of developing lower health and lower quality of life than those with same age but with age - normal vision (table 3). reduced near vision function and increased need of magnification were the most prominent risk markers for musculoskeletal complaints among amd patients. this is consistent with previous research showing that during near work tasks an increased use of optical enlargement in visual enhancing devices may adversely in restricted postures that subsequently can lead to increased musculoskeletal complaints as well as balance complaints. magnifying aids facilitate everyday life but may have side effects such as limited and restricted field of view and the need to adopt awkward (nonneutral) head and body postures to see as well as possible. as a consequence, magnifying aids may lead to increased strain on the visual and musculoskeletal systems, subsequently ing in increased complaints. when the portable visual aids are not sufficient any longer, stationary visual aids as cctv may be needed, on the cost of increasing strain on the visual and musculoskeletal systems. in the present study, the increased use of visual enhancing devices such as cctv was related to increased visual and musculoskeletal complaints in amd patients. thus, as vision deteriorates increased visual inputs from use of specialized visual enhancing aids, such as hyperoculars, magnifiers, and cctv, are needed, where amd patients need to adopt certain postures to adjust the eye to the best position for viewing. these adopted postures can be compared in many ways to those found in people using visual display units (vdu) at work, where visual ergonomics are monitored carefully in order to prevent neck / scapular complaints. however, visual ergonomic guidelines might be overlooked when providing cctv, especially as they are situated in old patients' homes (or nursing homes) with limited ability for adequate adjusting. one should, however, bear in mind that cctv is typically prescribed in situations when near vision is severely impaired and when the enlargement gained by ordinary optical enhancing devices is insufficient. cctv is thus often one of a very few remaining solutions when extreme magnification is needed. normally a two times increase in dioptric power represents three line improvements on the near reading test chart but may not be applicable as the distance between the eye and the magnifier may vary. our estimates of magnification at use do not intend to estimate angular enlargement but give a hint of the increasing limits of high powered visual aids. at baseline, fewer cases, proportionately, than referents perceived themselves as healthy. at follow - up, the number of cases perceiving themselves as unhealthy had increased, while there was no significant change among referents. visual function (need for a larger font size), visual complaints, and musculoskeletal complaints constituted risk markers for perceived unhealthiness in amd patients, and visual and balance complaints were significant risk markers among referents. thus, aspects of visual loss affect perceived health as people grow older, irrespective of whether they have visual impairment. however, the magnitude of complaints is larger among amd patients, thus increasing the risk of developing lower health. our are in line with studies showing that visual functional loss is associated with depression and that loss of vision is one of the most - feared disabilities. this is in line with findings in a previous study describing decreased health and increased need of health care in people with neck or back pain. also the association of increased balance complaints and unhealthiness is consistent with previous research. this was somewhat unexpected since both groups showed increased balance complaints and decreased general health over time. the lack of association found between balance complaints and health in amd patients may be statistical in nature and due to ceiling effect. that is, because amd patients already had a greater level of balance problems than referents (by four times) the increase of balance complaints from an already high level may have less effect on perceived health than a similar magnitude of increase from much lower levels, as in the control group. the overall dropout rate was 38%, with a larger dropout rate in the amd group. given that this study was conducted in a group of elderly participants who have an increased risk of illness and mortality, this rate was to be expected. the larger proportion of dropouts due to mortality in amd patients is consistent with research showing that among the elderly late - stage amd is associated with increased risk of all - cause mortality. since the mortality rate is probably larger among those with the most complaints and the poorest health, the effect of this attrition bias would probably decrease any differences between amd patients and referents; thus, differences between the groups may be larger than those reported here and observed differences are probably underrated. it should be noted that six amd patients had ranibizumab injections at the low vision clinic prior to registration and two participants (one amd patient and one referent) had been treated with monocular cataract surgery between registration and follow - up. this may have slowed down their visual deterioration and thus any potential influences on visual, musculoskeletal, or balance complaints in the amd group. therefore, the observed level of complaints may underestimate the level in an amd population not being treated with eye surgery or receiving ranibizumab injections. we hypothesized that magnifying visual aids may give rise to suboptimal ergonomic head postures that affect the muscles in the neck scapular area, ing in subsequent musculoskeletal complaints and increasing the risk of more balance complaints. at the same time , we can not neglect the fact that in late - stage amd visual performance may not be sufficient for adequate visuomotor support. research in this area is limited and future research is warranted. it should be noted that our estimates of magnification do not reflect the exact equivalent viewing power (evp) as we did not collect all required distances for this estimate, that is, the distances between the naked eye / reading glass and the magnifying aid compared to evp. the magnification estimate can therefore be somewhat overestimated. however, the magnification estimate reflects the use of higher dioptric power under conditions of deteriorating visual function. in pace with need of higher amounts of magnification , the reading distance variation gets more limited and the focal depth gets more specific ing in the need for adopting a more constricted and static posture. it was not possible to separate the influences of visual aids from the influences of loss of central vision in the present data because these factors are intertwined. that is, amd patients start increasing the use of visual aids when they start losing their central vision. future research measuring the amount of time in visual aid use might shed light on the magnitude of influence from visual aids and from loss of central vision this study had a longitudinal design where temporal order of causality was met. however, the that visual decline evoked increased visual, musculoskeletal, and balance complaints does not reveal the mechanisms behind these findings or whether there are other variables involved in this process, such as personality, depression, or socioeconomic status. prevalence and intervention studies could shed light on how best to prevent increased complaints in people with amd and on treatments to decrease complaints among those affected. the present study demonstrated that visual, musculoskeletal, and balance complaints increase with visual decline and increase more among people with amd than among people with normal vision. increased visual, musculoskeletal, and balance complaints were also found to negatively affect general health. the study showed that visual decline and increased use of greater magnification in visual aids are important risk factors for increased complaints. the from this study show that amd patients' use of magnifying visual aids has side effects that optometrists and low vision staff must be aware of when prescribing visual aids. as a preventive measure, optometrists may suggest alternative use of both optical and technical visual enhancing aids as well as using devices with text - to - speech function (e.g., electronic readers) when applicable. visual aids are important, especially in elderly people with severe amd, as these aids enable users to continue daily activities and maintain their quality of life. optometrist may also refer patients to a physiotherapist for investigation, treatment, and preventive training. this calls for coordinated actions at an early stage in order to prevent visual - related musculoskeletal and balance complaints in amd patients to minimize the further risk of more serious complaints.	purpose. to investigate whether patients with age - related macular degeneration (amd) run a potentially higher risk of developing visual, musculoskeletal, and balance complaints than age - matched controls with normal vision. methods. visual assessments, self - rated visual function, self - rated visual, musculoskeletal, and balance complaints, and perceived general health were obtained in 37 amd patients and 18 controls, at baseline and after an average of 3.8 years later. . at follow - up both groups reported decreased visual acuity (va) and visual function, but only amd patients reported significantly increased visual, musculoskeletal, and balance complaints. decreased va, need for larger font size when reading, need for larger magnification, and decreased self - rated visual function were identified as risk markers for increased complaints in amd patients. these complaints were also identified as risk markers for decreased health. for controls, decreased va and self - reported visual function were associated with increased visual and balance complaints. . visual deterioration was a risk marker for increased visual, musculoskeletal, balance, and health complaints in amd patients. specifically, magnifying visual aids, such as cctv, were a risk marker for increased complaints in amd patients. this calls for early and coordinated actions to treat and prevent visual, musculoskeletal, balance, and health complaints in amd patients.
the search for novel tools for early diagnosis is one of the major issues in medical research. the discovery of biomarkers in biological fluids and blood is especially challenging due to the tremendous number of biomolecular species, which differ by many orders of magnitude in their relative abundance.1,2 of the several approaches proposed in the last few years, the study of the proteome seems to hold the greatest potential.2,3 proteomics is a quickly developing area of biochemical investigation. the basic aim of proteomic analysis is the identification of specific protein patterns from cells, tissues, and biological fluids related to physiological or pathological condition.2,4 it provides a different view from that of gene expression profiling, which does not evaluate post - transcriptional and post - translational modifications, or protein compartmentalization and half - life changes (eg, ubiquitination and proteosome - driven degradation). all these characteristics make the protein profile much more complex but more informative than gene expression profiling. several approaches can be used to perform proteomic analysis; among these, the most common are methods based on 2d - polyacrylamide gel electrophoresis (2d - page) and mass spectrometry (ms).59 it is now well accepted that the low - molecular - weight (lmw), low - abundance fraction of biological fluids might contain the most informative source of novel biomarkers. the conventional analytical methods mentioned above do not seem to reach a sufficient degree of resolution and sensitivity to reliably detect and identify lmw and low - abundance peptides. therefore, research has recently focused on the development of innovative devices that are able to enrich this fraction of body fluid proteome, making it available for use in common analytical tools. harvesting and enrichment of candidate biomarkers from complex protein mixtures can be pursued in different ways. usually, in the plasma or serum, in which albumin and immunoglobulin alone account for > 90% of total protein content, these species are conventionally removed prior to 2d - page and ms by immunoaffinity depletion columns. however, it must be emphasized that many potentially informative biomarkers, represented by very small peptides noncovalently associated with the carrier protein albumin, are lost following this procedure. many commercial albumin removal kits based on different methods are available, but it has been demonstrated that they can cause loss of several low - abundance proteins, including albuminome.1012 moreover, most of these sieving / filtering systems do not allow sufficient flexibility of the whole process. therefore, nanotechnology methods appear to offer a promising and powerful strategy for overcoming these limitations.1316 discovered over 40 years ago, porous silicon (psi) has attracted increasing attention in many fields of research for its interesting features. in particular, the demonstration of its biodegradability in physiological environments has opened up new perspectives for biomedical application.17 the desired dissolution rate can be obtained through the accurate control of the morphology, pore size, and ph. the typical dissolution rate in alkaline conditions ranges from a few minutes to up to a few days. moreover, the well - known functionalization processes of the porous surface provide further control of bioreactivity and hydrophobicity.1821 various surface derivatization has been reported in the literature and hundreds of different cross - linking agents are now available to selectively bind the target molecules.22 in this paper, we present a direct approach to harvesting the lmw fraction of a complex solution that relies on 3 important properties of silicon nanoporous nanoparticles (npnps): (a) they can act as nanosponges and absorb small molecules depending on nanopore size; (b) they can be separated from solution through efficient centrifugation (the nanoparticle density is higher than that of the solvent); (c) they can be dissolved in water. this last property is relevant because the filtration process can be carried out in physiological solution, without the need for introducing particular solvents, which can contaminate, denaturate, or degrade the potential biomarkers. the whole process is depicted in figure 1 (figures 1a c fabrication process and figures 1c h harvesting process). the starting solution is incubated with npnps which, bcause of size - exclusion, can absorb only the lmw fraction into the nanopore. afterwards, npnps can easily be recovered from solution by means of centrifugation, and resuspended in water, or other solvents in which they can be dissolved. the harvested molecules are then available, in their native state, for further analyses. this easy, cheap, and fast process enables the harvesting of peptides < 13 kda from raw serum. modern biology demands not only fast and easy techniques but also full compatibility with existing protocols. the present approach can be mixed and matched with the majority of current investigation protocols. npnps were fabricated by ultrasonication of a thin film of nanoporous silicon.1324 psi was obtained by anodization of a boron - doped silicon wafer (resistivity 510 cm) of crystal orientation, using an electrolyte binary mixture of hydrofluoric acid (25%), water (25%), and ethanol (50%). the psi film was oxidized in an oven at 200c for 2 hours. in order to obtain npnps, the psi film was sonicated in dimethylformamide for about 60 minutes and then, after washes in ethanol, ultrasonicated (5 w) in water for 10 minutes at a constant temperature of 4c, and finally filtered to eliminate impurities > 500 nm. an ad hoc protein mixture was prepared by mixing 50% (v / v) human serum albumin (mw 66,000 da ; sigma - aldrich, st . louis, missouri, usa); 30% (v / v) bovine plasma gamma globulin (heavy chain mw 45,000 da ; light chain mw 30,000 da ; biorad, berkeley, california, usa); and 20% (v / v) aprotinin (6,500 da ; sigma - aldrich). all proteins were dissolved at a concentration of 1 mg/ ml in 100 mm sodium phosphate buffer and 9% (w / v) sodium chloride ph 7.4 (pbc) to reproduce physiological conditions. human serum was obtained from a healthy anonymous male donor and collected in accordance with human proteome organization (hupo) plasma proteome project guidelines.25 approximately 8 ml of blood were drawn by venipuncture and collected in tubes without additive and allowed to clot at room temperature for 40 minutes. the sample was centrifuged within 2 hours of collection at 1300 g for 10 minutes, aliquoted into silicon tubes, and stored at 80c. the fabricated npnps were deposited onto a glass substrate and characterized using scanning electron microscopy (sem) and fluorescence microscopy. the pores of the particles are too small to be shown by sem (figures 2 a and b), but the typical emission spectrum peak at around 620 nm (figures 2c and d) indicates a pore size of about 23 nm (excitation wavelength 408 nm). the nanoparticle diameter is about 200 nm, and it can be adjusted by changing the power and duration of the sonication process. we studied the interactions of the nanoparticles with complex biological fluids in different environmental conditions. here we report 3 experiments with fluids of increasing complexity in order to show the splitting capability of the npnps: experiment 1. interaction of a complex mixture of proteins with a wide range of mws simulating a biological fluid. npnps were incubated with 2 solutions of fluorescent polymer of different mws (6 kda and 14 kda dextran - fluorescein isothiocyanate 10 mg, npnp 5 mg, water 10 ml, 1 hour). after incubation, the npnps were separated from supernatant (centrifugation), dropped on a slide, dried, and analyzed with fluorescence microscopy. optical and fluorescence images collected on the npnps incubated with polymers of 14 and 6 kda are reported in upper and lower panels, respectively; for higher mw polymer there is no trace of absorption, and only a weak blue fluorescence coming from salt residue is visible. in contrast, green fluorescence emitted by npnps incubated with the lower mw polymer indicates good absorption. after incubation and centrifugation, the recovered npnps can be dissolved in water at a rate depending on the temperature and acidity of the medium. at ph 8 and 90c , the dissolution takes a few minutes, but it can be also carried out at room temperature in a few hours, if nondenaturing conditions are needed. at ph < 5, the npnps do not dissolve, allowing their long - term storage in water at or above room temperature. in experiment 1, the amount of harvested fraction of dextran - fitc (mw 6 kda) can be evaluated by comparing the total fluorescence intensity of 2 solutions (harvested vs supernatant). the 2 solutions show very similar fluorescence intensity values, indicating that, under the experimental conditions described above, 50% of the molecules were harvested, whereas the remaining 50% were left in solution. this shows that the loading capacity is very high, about 1 mg of harvested molecules for each mg of npnps. in the second experiment, the ability of npnps to enrich the lmw fraction of a complex mixture was tested with an ad hoc protein mixture (see materials and methods). for this purpose, the effects of several parameters such as ph, osmolarity, temperature, and incubation time were studied, and conditions were optimized, and, finally, a volume of 200 ml of protein mixture was incubated with 5 mg of npnps (about 10 particles) at room temperature for 1 hour. npnps were subsequently separated from supernatant by centrifugation, and washed once with pbc buffer. the sodium dodecyl sulfate - page (sds - page) in figure 4 (panel a) clearly shows that nanoparticles selectively retain small molecules as aprotinin (mw 6,500 da), whereas proteins with higher mw are completely excluded from the nanopores. we noted that during the incubation a small fraction of the nanoparticles dissolve releasing silicic acid into the solution (about 8% of npnp volume under our conditions).26 this amount can be decreased by lowering the incubation temperature, ph, or incubation time when possible. in the third experiment, the same protocol for experiment 2 was applied to raw human serum to demonstrate that npnps are able to selectively enrich lmw serum proteome (lmwp). after 1 hour of incubation (100 ml of serum sample diluted 1:2 with pbc buffer, 5 mg of npnps), the lmw fraction was recovered by dissolving the nanoparticles in pbc buffer for 12 hours at 37c. we noted that the dissolution process can be hastened by heating the solution and adding a base (to increase ph) or other solvent. an aliquot of serum, before and after incubation, was analyzed by sds - page using a 16.5% ready prepared tris - tricine / peptide gel. the efficiency of npnps to selectively enrich the lmwp is clearly demonstrated in the sds - page analysis shown in figure 4 (panel b): in lines 2 and 3, crude human serum and supernatant are represented. in line 4, the lmw fraction of human serum extracted from npnps is visible: no molecules > 12 kda are still present, except for a very small trace of albumin. from densitometric analysis of lane 4 , it was estimated that > 50% of total pixel volume was from lmw species, whereas in lane 2 (unprocessed serum) the same area represented < 1% of the total pixel volume. the enrichment factor was thus estimated to be > 50. in order to characterize the lmw protein species extracted by incubation with npnps, gel bands from sds - page - separated extracts (figure 4b, lane 4) were cut and processed for tryptic digestion according to the protocol shown in the supplementary information, section 1. based on mw marker information, the 3 processed gel bands corresponded to mw intervals of 1520 kda, 1015 kda and < 10 kda. tryptic peptides were analyzed by nanoscale liquid chromatography interfaced with tandem ms (nanolc - ms / ms, supplementary information, section 2).27,28 in an alternative approach, proteins extracted by incubation with npnps were directly digested in solution by trypsin and analyzed by nanolc - ms / ms. validated protein identifications are reported in 2 tables (supplementary information, section 3). besides abundant lmw serum proteins, such as apolipoprotein a - ii and transthyretin, a database search identified a number of lmw serum proteins that are not considered abundant, such as tetranectin, platelet basic protein, and dermcidin. furthermore, lc - ms / ms analysis identified several high - mw proteins in the in - gel - digested sds - page protein bands. such identifications confirm that the lmw proteome is also populated with fragments of abundant high - mw serum proteins.29 a comparison of the of these experiments with those reported in the literature shows many remarkable characteristics of the npnps: easy and cheap production. easy recovery by centrifugation, long - term storage wet (water solution of ph < 5) or dry. we note that such properties are very attractive also for drug delivery applications, for which the use of nanocarriers is attracting a lot of interest. we report a straightforward tool relying on water - soluble silicon npnps used to harvest the lmw molecules in their native state from a complex fluid. the method is based on the porosity of the nanoparticles, which act as a molecular sieve, and their solubility in a physiological environment. the proposed approach can be mixed and matched with currently available techniques and protocols, and does not require high temperature, denaturing solvents, or other contaminants. a cut - off of about 13 kda was demonstrated for crude human serum. the ability to tune pore size, combined with the availability of hundreds of biomolecule cross - linkers, opens up new perspectives on complex biofluid analysis, discovery of biomarkers, and in situ drug delivery. gel bands below 25 kda were excised and digested by trypsin incubation; selected bands were punched out manually and placed in a silicon eppendorf tube. gel pieces were washed once with 150 ml of deionized water and then destained by 3 washes of 150 ml 35% acetonitrile in 25 mm nh4hco3 buffer. after destaining, trypsin digestion was performed overnight at 37c with modified trypsin (sigma - aldrich, st louis, mo, usa) 0.2 mg / ml. the ing tryptic peptides were acidified, purified by ziptips c18 (millipore, billerica, ma) according to the manufacturer s procedure and eluted with 2 l of a 1:1 mixture of acetonitrile and 0.1% trifluoro acetic acid (v / v). 28 l of loading pump solvent (see below) were added, and 10 l were injected for nanolc - ms / ms analysis. 500 ng of sequencing grade modified trypsin (sigma - aldrich) were added, and digestion was allowed to proceed for 16 h. the ing tryptic peptides were fractionated by off - gel eletrophoresis before nanoscale lc - ms / ms analysis, in order to achieve a 2-dimensional fractionation of the peptide mixture. an agilent 3100 off - gel fractionator (agilent technologies, santa clara, ca) was used. the peptide mixture was diluted with carrier ampholyte mixture (3.6 ml final volume, 10% carrier ampholyte concentration). the sample was then loaded on separate immobiline drystrip, linear ph range 3.010, 18 cm long, purchased from ge healthcare (chalfont st . peptides were focused at a constant temperature of 20c, and at constant current intensity of 50 a . after focusing was complete, fractions were collected . in order to improve peptide recovery, sample wells were washed with 100 l of a water / methanol / formic acid mixture, 49:50:1 ( v / v / v). the wash solution was added to each well and allowed to incubate for 90 minutes before being collected and pooled with the corresponding fraction supernatant. pooled supernatants were reduced to a volume of approximately 10 l in a vacuum centrifuge. a 1:4 mixture of concentrated eluates and loading pump solvent of the nanolc - ms / ms system (see below) was injected for nanolc - ms / ms analysis. considering the injection volume of 10 l, approximately 1/5 of each oge fractions was injected for nanolc - ms / ms. chromatography was performed on an ultimate nanolc system from dionex (sunnyvale, ca, usa), using a valveless setup.1,2 the peptide extracts were redissolved in 30 l of loading pump solvent (see below) and 10 l were loaded onto an in - house packed 100 m i.d. , integra frit (new objective, cambridge, ma) trapping column (packing bed length 1.5 cm) at 10 l / min of loading pump solvent, consisting of h2o / acetonitrile / trifluoroacetic acid (tfa) 97.95:2:0.05 (v / v / v). after 4 minutes of column washing, the trapping column was switched on - line to the analytical column: an in - house packed 50 m i.d. , pico frit column (new objective), filled with the same stationary phase used for the trapping column packing: 3 m c18 silica particles (dr maisch, entringen, germany). mobile phase a was h2o / acetonitrile / formic acid / tfa 97.9:2:0.09:0.01 (v / v / v / v); mobile phase b was h2o / acetonitrile / formic acid / tfa 29.9:70:0.09:0.01 (v / v / v / v). after 10 minutes at 95% b, the column was re - equilibrated at 5% b for 20 minutes before the following injection. ms detection was performed on a qstar xl hybrid lc - ms / ms from applied biosystems (foster city, ca, usa) operating in positive ion mode, with nesi potential at 1300 v, curtain gas at 15 units, cad gas at 3 units. information - dependent acquisition (ida) was performed by selecting the 2 most abundant peaks for ms / ms analysis after a full tof - ms scan from 400 to 1600 m / z lasting 4 seconds. both ms / ms analyses were performed in enhanced mode (3 seconds / scan). ms / ms data were converted to mascot generic format (mgf) by the analyst software 1.1 (applied biosystems). data were searched on the mascot search engine (www.matrixscience.com), version 1.9, against the international protein index database (ipi version 3_38) using the following parameters: ms tolerance 30 ppm; ms/ ms tolerance 0.2 da; variable modifications methionine oxidized; enzyme trypsin; max. missed cleavages 1. ms/ ms identifications were validated by using the transproteomics pipeline.3 peptide identifications with a minimum probability score of 0.7 were retained (4% false discovery rate). proteins identified with a minimum of 2 peptides were retained (protein probability score > 0.9). proteins identified by in - gel digestion and nanolc - ms / ms of sds - page - isolated bands notes: in gray, protein identification obtained with a single hit. those hits were validated by visual inspection. ms / ms data are reported in supplementary information. mascot score () is reported for identifications which have not passed tpp validation, but were above mascot threshold of 29 and for which manual validation was undertaken. additional proteins identified by direct in - solution digestion of silicon nanopraticle extracts notes: in gray, protein identification obtained with a single hit. gel bands below 25 kda were excised and digested by trypsin incubation; selected bands were punched out manually and placed in a silicon eppendorf tube. gel pieces were washed once with 150 ml of deionized water and then destained by 3 washes of 150 ml 35% acetonitrile in 25 mm nh4hco3 buffer. after destaining, trypsin digestion was performed overnight at 37c with modified trypsin (sigma - aldrich, st louis, mo, usa) 0.2 mg / ml. the ing tryptic peptides were acidified, purified by ziptips c18 (millipore, billerica, ma) according to the manufacturer s procedure and eluted with 2 l of a 1:1 mixture of acetonitrile and 0.1% trifluoro acetic acid (v / v). 28 l of loading pump solvent (see below) were added, and 10 l were injected for nanolc - ms / ms analysis. 500 ng of sequencing grade modified trypsin (sigma - aldrich) were added, and digestion was allowed to proceed for 16 h. the ing tryptic peptides were fractionated by off - gel eletrophoresis before nanoscale lc - ms / ms analysis, in order to achieve a 2-dimensional fractionation of the peptide mixture. an agilent 3100 off - gel fractionator (agilent technologies, santa clara, ca) was used. the peptide mixture was diluted with carrier ampholyte mixture (3.6 ml final volume, 10% carrier ampholyte concentration). the sample was then loaded on separate immobiline drystrip, linear ph range 3.010, 18 cm long, purchased from ge healthcare (chalfont st . peptides were focused at a constant temperature of 20c, and at constant current intensity of 50 a . after focusing was complete, fractions were collected . in order to improve peptide recovery, sample wells were washed with 100 l of a water / methanol / formic acid mixture, 49:50:1 ( v / v / v). the wash solution was added to each well and allowed to incubate for 90 minutes before being collected and pooled with the corresponding fraction supernatant. pooled supernatants were reduced to a volume of approximately 10 l in a vacuum centrifuge. a 1:4 mixture of concentrated eluates and loading pump solvent of the nanolc - ms / ms system (see below) was injected for nanolc - ms / ms analysis. considering the injection volume of 10 l, approximately 1/5 of each oge fractions was injected for nanolc - ms / ms. chromatography was performed on an ultimate nanolc system from dionex (sunnyvale, ca, usa), using a valveless setup.1,2 the peptide extracts were redissolved in 30 l of loading pump solvent (see below) and 10 l were loaded onto an in - house packed 100 m i.d., integra frit (new objective, cambridge, ma) trapping column (packing bed length 1.5 cm) at 10 l / min of loading pump solvent, consisting of h2o / acetonitrile / trifluoroacetic acid (tfa) 97.95:2:0.05 (v / v / v). after 4 minutes of column washing, the trapping column was switched on - line to the analytical column: an in - house packed 50 m i.d., pico frit column (new objective), filled with the same stationary phase used for the trapping column packing: 3 m c18 silica particles (dr maisch, entringen, germany). mobile phase a was h2o / acetonitrile / formic acid / tfa 97.9:2:0.09:0.01 (v / v / v / v); mobile phase b was h2o / acetonitrile / formic acid / tfa 29.9:70:0.09:0.01 (v / v / v / v). after 10 minutes at 95% b, the column was re - equilibrated at 5% b for 20 minutes before the following injection. ms detection was performed on a qstar xl hybrid lc - ms / ms from applied biosystems (foster city, ca, usa) operating in positive ion mode, with nesi potential at 1300 v, curtain gas at 15 units, cad gas at 3 units. information - dependent acquisition (ida) was performed by selecting the 2 most abundant peaks for ms / ms analysis after a full tof - ms scan from 400 to 1600 m / z lasting 4 seconds. both ms / ms analyses were performed in enhanced mode (3 seconds / scan). ms / ms data were converted to mascot generic format (mgf) by the analyst software 1.1 (applied biosystems). data were searched on the mascot search engine (www.matrixscience.com), version 1.9, against the international protein index database (ipi version 3_38) using the following parameters: ms tolerance 30 ppm; ms/ ms tolerance 0.2 da; variable modifications methionine oxidized; enzyme trypsin; max. missed cleavages 1. ms/ ms identifications were validated by using the transproteomics pipeline.3 peptide identifications with a minimum probability score of 0.7 were retained (4% false discovery rate). proteins identified with a minimum of 2 peptides were retained (protein probability score > 0.9). proteins identified by in - gel digestion and nanolc - ms / ms of sds - page - isolated bands notes: in gray, protein identification obtained with a single hit. those hits were validated by visual inspection. ms / ms data are reported in supplementary information. mascot score () is reported for identifications which have not passed tpp validation, but were above mascot threshold of 29 and for which manual validation was undertaken. additional proteins identified by direct in - solution digestion of silicon nanopraticle extracts notes: in gray, protein identification obtained with a single hit. ms / ms data were converted to mascot generic format (mgf) by the analyst software 1.1 (applied biosystems). data were searched on the mascot search engine (www.matrixscience.com), version 1.9, against the international protein index database (ipi version 3_38) using the following parameters: ms tolerance 30 ppm; ms/ ms tolerance 0.2 da; variable modifications methionine oxidized; enzyme trypsin; max. missed cleavages 1. ms/ ms identifications were validated by using the transproteomics pipeline.3 peptide identifications with a minimum probability score of 0.7 were retained (4% false discovery rate). proteins identified with a minimum of 2 peptides were retained (protein probability score > 0.9). proteins identified by in - gel digestion and nanolc - ms / ms of sds - page - isolated bands notes: in gray, protein identification obtained with a single hit. those hits were validated by visual inspection. ms / ms data are reported in supplementary information. mascot score (*) is reported for identifications which have not passed tpp validation, but were above mascot threshold of 29 and for which manual validation was undertaken. additional proteins identified by direct in - solution digestion of silicon nanopraticle extracts notes: in gray, protein identification obtained with a single hit.	human serum has the potential to become the most informative source of novel biomarkers, but its study is very difficult due to the incredible complexity of its molecular composition. we describe a novel tool based on biodegradable nanoporous nanoparticles (npnps) that allows the harvesting of low - molecular - weight fractions of crude human serum or other biofluids. npnps with a diameter of 200 nm and pore size of a few nm were obtained by ultrasonication of nanoporous silicon. when incubated with a solution, the npnps harvest only the molecules small enough to be absorbed into the nanopores. then they can be recovered by centrifugation and dissolved in water, making the harvested molecules available for further analyses.fluorescence microscopy, gel electrophoresis, and mass spectrometry were used to show the enrichment of low - molecular - weight fraction of serum under physiological conditions, with a cut - off of 13 kda and an enrichment factor > 50.from these findings, we conclude that ability to tune pore size, combined with the availability of hundreds of biomolecule cross - linkers, opens up new perspectives on complex biofluid analysis, discovery of biomarkers, and in situ drug delivery.
dilated cardiomyopathy (dcm) is a rare disease in children, with an annual incidence of 0.34/100 000 persons. recent studies have suggested that multiple neuroendocrine factors, including the reduction in parasympathetic activity and activation of sympathetic nervous and renin - angiotensin - aldosterone (raa) system, play an important role in the genesis and progression of congestive heart failure (chf). such observations have led to the use of neurohormonal antagonists such as aldosterone antagonists, angiotensin - converting enzyme inhibitors (acei), and -adrenergic blockers for the treatment of chf. reduced measures of heart rate variability (hrv), considered a marker of tonic sympathetic and vagal outflow, several investigators have demonstrated that measurements of hrv can be used to evaluate the effect of acei and -adrenergic receptor antagonists on cardiac autonomic activity. dysregulation of autonomic nervous system function and impaired homogeneity of myocardial repolarization are 2 major mechanisms for the genesis of ventricular arrhythmias. increased qt - interval dispersion (qtd) and decreased hrv have been reported in adult patients with chf, and are considered as potential markers for arrhythmogenicity and for use in predicting mortality. it decreases the chronic adrenergic overstimulation of the myocardium and improves myocardial function, and has been shown to improve survival, decrease morbidity, and improve quality of life in adults with chf. although some studies have suggested that carvedilol therapy decreased qtd and had a beneficial effect on hrv parameters in adult patients, the effects of carvedilol therapy on inhomogeneity of ventricular repolarization and autonomic nervous regulation are not clear. to the best of our knowledge , there have been few published studies about the effect of carvedilol on arrhythmias in dilated cardiomyopathy in pediatric patients, and there are no published data available about the effects of carvedilol therapy on heart rate dynamics and arrhythmia markers in children. therefore, the aim of this study was to examine the effects of carvedilol therapy on autonomic control of the heart and qt - interval dispersion as an arrhythmia marker among children with dcm whose symptoms were not adequately controlled with standard chf therapy. after obtaining approval from the institutional review board of our institution, we retrospectively reviewed the hospital records of the 34 patients who were followed - up in our pediatric cardiology clinic with dcm and treated with carvedilol in addition to standard therapy of digoxin, diuretics, and acei. data collected included: age at diagnosis, therapy prior to carvedilol, time between diagnosis and initiation of carvedilol, age, weight, symptoms at carvedilol initiation and last follow - up, dosage, and adverse effects of the drug. all patients who received carvedilol in addition to standard therapy were followed - up for at least 6 months. the diagnosis of dilated cardiomyopathy was defined as a child having both left ventricular (lv) contractility 2 sd below the normal mean and lv end - diastolic dimension 2 standard deviations above the normal mean, which was not caused by dysrhythmia or any other structural heart disease. we used a modified scoring system of chf signs and symptoms described by ross and reithmann et al. (table 1). each sign or symptom was graded on a scale of 0, 1, or 2 points according to the severity. the sum of points formed the clinical score (range 012 points), with a higher score corresponding to more severe heart failure. the inclusion criteria were: patients with dcm whose symptoms were not adequately controlled with standard chf therapy (clinical score of 5 or more), and whose systemic ventricle is morphologic left ventricle and left ventricular ejection fraction (lvef) 0.40. the exclusion criteria were: congenital heart defect, atrial fibrillation, sustained or symptomatic ventricular dysrhythmias, sinus or av node dysfunction, bradycardia, acute myocarditis, bronchial asthma, obstructive or severe regurgitative valvular disease, significant renal, hepatic, gastrointestinal disease, endocrine disorders, taking any drugs influencing qt dispersion, and use of antiarrhythmic drugs. patient characteristics including age, sex, weight, height and concomitant medications were collected for all patients. clinical, echocardiographic, electrocardiographic parameters, and 24-h holter records of patients were retrospectively evaluated before and after carvedilol treatment. echocardiographic data obtained before and after carvedilol therapy were retrospectively reviewed from patient medical records. for estimates of left ventricular dimension, function and shortening fraction were reported with the standard method of 2-dimensionally directed m - mode measurements. standard 12-lead electrocardiography was obtained simultaneously using a recorder set at 50 mm / s paper speed and calibration of 1milivolt / centimeter, in a comfortable supine position. qt intervals (qtc = qt/rr, maximum and minimum qtc intervals) were also measured. qt dispersions were manually measured in all electrocardiograms (ecg) by the same investigator. all measurements were repeated by a second investigator who was blinded to the demographic information and therapy. qt intervals were measured from the beginning of the qrs complex to the end of the t wave, which was defined as return to baseline in each ecg lead. when u waves were present, the qt interval was measured to the nadir of the curve between the t and u waves. for each lead, 2 or more consecutive cycles were measured and the arithmetic mean of the qt interval for that lead was used in all calculations for qtd. qtd was calculated as the difference between the longest and shortest qt interval measured in each individual ecg lead. premature ventricular contractions (pvcs) were characterized by the following: ectopic, premature, and bizarrely shaped qrs complexes, usually wider than 120 msec; absence of p waves preceding a qrs complex; the t wave is usually large, and its direction is opposite the major deflection of the qrs. couplets were characterized by 2 consecutive premature ventricular contractions. nonsustained ventricular tachycardia (vt) was characterized by 3 or more consecutive beats lasting less than 30 seconds, at a rate > 100/min. all records of the 24-holter monitoring before and after carvedilol therapy were obtained from the computer - based electronic holter archive of our institution. holter studies were considered adequate for interpretation if there was greater than 16 hours of analyzable data for the 24-hour recording. the tapes were manually reviewed by an independent observer who was blinded to the patient s identity and study treatment. total numbers of pvc and number of episodes of vt were calculated. we also performed hrv analysis by using 24-hour holter ecg monitoring (dms 300 holter recorder ; dms inc . abnormal beats and areas of artifact were automatically and manually identified and excluded from the analysis . we analyzed hrv in the time domain by the following 5 standard 24-hour time - domain measures : sdnn ( standard deviation of all normal sinus r - r intervals during 24 hours), sdnni (mean of the standard deviation of all normal sinus r - r intervals for all 5-minute segments), sdann (standard deviation of the average normal sinus r - r intervals for all 5-minute segments), rmssd (root mean square of the successive normal sinus r - r interval difference), and pnn50 (percentage of successive normal sinus r - r intervals longer than 50 ms). changes from pre- to post - carvedilol therapy were assessed using paired t - tests for continuous variables when the differences were approximately normally distributed, and by using wilcoxon signed rank tests for differences with skewed distributions. the relationship among variables was evaluated by pearson s correlation coefficient. a p - value < 0.05 was considered to be significant. analyses were performed with the software package spss 11.0 (spss, inc, chicago, il, usa). after obtaining approval from the institutional review board of our institution, we retrospectively reviewed the hospital records of the 34 patients who were followed - up in our pediatric cardiology clinic with dcm and treated with carvedilol in addition to standard therapy of digoxin, diuretics, and acei. data collected included: age at diagnosis, therapy prior to carvedilol, time between diagnosis and initiation of carvedilol, age, weight, symptoms at carvedilol initiation and last follow - up, dosage, and adverse effects of the drug. all patients who received carvedilol in addition to standard therapy were followed - up for at least 6 months. the diagnosis of dilated cardiomyopathy was defined as a child having both left ventricular (lv) contractility 2 sd below the normal mean and lv end - diastolic dimension 2 standard deviations above the normal mean, which was not caused by dysrhythmia or any other structural heart disease. we used a modified scoring system of chf signs and symptoms described by ross and reithmann et al. was graded on a scale of 0, 1, or 2 points according to the severity. the sum of points formed the clinical score (range 012 points), with a higher score corresponding to more severe heart failure. the inclusion criteria were: patients with dcm whose symptoms were not adequately controlled with standard chf therapy (clinical score of 5 or more), and whose systemic ventricle is morphologic left ventricle and left ventricular ejection fraction (lvef) 0.40. the exclusion criteria were: congenital heart defect, atrial fibrillation, sustained or symptomatic ventricular dysrhythmias, sinus or av node dysfunction, bradycardia, acute myocarditis, bronchial asthma, obstructive or severe regurgitative valvular disease, significant renal, hepatic, gastrointestinal disease, endocrine disorders, taking any drugs influencing qt dispersion, and use of antiarrhythmic drugs. patient characteristics including age, sex, weight, height and concomitant medications were collected for all patients. clinical, echocardiographic, electrocardiographic parameters, and 24-h holter records of patients were retrospectively evaluated before and after carvedilol treatment. echocardiographic data obtained before and after carvedilol therapy were retrospectively reviewed from patient medical records. for estimates of left ventricular dimension, function and shortening fraction were reported with the standard method of 2-dimensionally directed m - mode measurements. standard 12-lead electrocardiography was obtained simultaneously using a recorder set at 50 mm / s paper speed and calibration of 1milivolt / centimeter, in a comfortable supine position. qt intervals (qtc = qt/rr, maximum and minimum qtc intervals) were also measured. qt dispersions were manually measured in all electrocardiograms (ecg) by the same investigator. all measurements were repeated by a second investigator who was blinded to the demographic information and therapy. qt intervals were measured from the beginning of the qrs complex to the end of the t wave, which was defined as return to baseline in each ecg lead. when u waves were present, the qt interval was measured to the nadir of the curve between the t and u waves. for each lead, 2 or more consecutive cycles were measured and the arithmetic mean of the qt interval for that lead was used in all calculations for qtd. qtd was calculated as the difference between the longest and shortest qt interval measured in each individual ecg lead. premature ventricular contractions (pvcs) were characterized by the following: ectopic, premature, and bizarrely shaped qrs complexes, usually wider than 120 msec; absence of p waves preceding a qrs complex; the t wave is usually large, and its direction is opposite the major deflection of the qrs. nonsustained ventricular tachycardia (vt) was characterized by 3 or more consecutive beats lasting less than 30 seconds, at a rate > 100/min. all records of the 24-holter monitoring before and after carvedilol therapy were obtained from the computer - based electronic holter archive of our institution. holter studies were considered adequate for interpretation if there was greater than 16 hours of analyzable data for the 24-hour recording. the tapes were manually reviewed by an independent observer who was blinded to the patient s identity and study treatment. we also performed hrv analysis by using 24-hour holter ecg monitoring (dms 300 holter recorder ; dms inc . abnormal beats and areas of artifact were automatically and manually identified and excluded from the analysis . we analyzed hrv in the time domain by the following 5 standard 24-hour time - domain measures : sdnn ( standard deviation of all normal sinus r - r intervals during 24 hours), sdnni (mean of the standard deviation of all normal sinus r - r intervals for all 5-minute segments), sdann (standard deviation of the average normal sinus r - r intervals for all 5-minute segments), rmssd (root mean square of the successive normal sinus r - r interval difference), and pnn50 (percentage of successive normal sinus r - r intervals longer than 50 ms). changes from pre- to post - carvedilol therapy were assessed using paired t - tests for continuous variables when the differences were approximately normally distributed, and by using wilcoxon signed rank tests for differences with skewed distributions. the relationship among variables was evaluated by pearson s correlation coefficient. a p - value < 0.05 was considered to be significant. analyses were performed with the software package spss 11.0 (spss, inc, chicago, il, usa). a total 34 patients (18 male, 16 female, mean age : 7.44.3 years, range 32 months to 14 years) with dcm were analyzed in the study. all patients had undergone carvedilol therapy in addition to standard therapy for at least 6 months. the median follow - up period was 9.5 months (range 6.413.7 months) after the initiation of carvedilol therapy. the initial mean carvedilol dose was 0.140.07 mg / kg / day, and 0.460.28 mg / kg / day at 6 months. as standard treatment, 34/34 (100%) were on digoxin, 32/34 (94.1%) were on furosemide, and 32/34 (94.1%) were on acei. the average heart rate was significantly reduced after carvedilol treatment (11423 vs. 8921 beat / min, p=0.008). systolic blood pressure tended to decrease after carvedilol therapy, but did not reach statistical significance. after 6 months of carvedilol treatment, the ross clinical score significantly improved from 6 to 3 (p=0.03) (table 3). the most common adverse events were dizziness (22%), vomiting (13%), hypotension (8%), and headache (5%). lvef significantly increased from 34.77.6% (range 2240%) to 45.29.6% (range 2961%) following carvedilol treatment (p=0.002). after carvedilol treatment, the left ventricular fractional shortening (lvfs) significantly increased from 16.49.7% (range 1033%) to 23.97.7% (range 1637%) (p=0.016), left ventricle end - diastolic dimensions (lvedd) significantly decreased from 45.78.1 mm (range 3160 mm) to 41.46.5 mm (range 2658 mm) (p=0.026) and the left ventricle end - systolic dimensions (lveds) significantly decreased from 39.46.9 mm (range 2447 mm) to 34.26.0 mm (range 2043 mm) (p=0.047) (table 3). there were significant increases in mean sdnn, sdann, rmssd, and pnn50 after carvedilol therapy (p=0.002, p=0.001, p=0.008, and p=0.026, respectively). a trend toward an increase in sdnni after carvedilol therapy, sdnn was correlated with the clinical score of chf, heart rate, lvef, lvsf, and total pvcs. in addition, rmssd and pnn50 were correlated with heart rate, lvef, and lvsf after carvedilol therapy. the comparison of clinical, hemodynamic, heart rate variability, and ventricular arrhythmia parameters of patients receiving carvedilol therapy at baseline and after the treatment are shown in table 3, and the correlation between changes in hrv in the time domains and changes in hemodynamic parameters are presented in table 4. intra- and interobserver variability were assessed in 18 randomly chosen patients; all intra- and interobserver variability for ecg parameters ranged from 3.1 to 4.8%. a significant reduction was observed in maximum and minimum qtc interval, qtc, and qtd values after carvedilol treatment. qtd was slightly higher in patients with a lower clinical score than in those with a higher clinical score, but the difference was not statistically significant. qtd was significantly related to total pvcs, but qtd was not related to age, sex, clinical score of chf, heart rate, lvef, lvsf, or lvedd (table 4). although 8 patients (23.5%) had pvcs before treatment, they disappeared in 4 patients and pvc decreased in 2 patients after treatment. sustained ventricular tachycardia was not observed in any patients. before carvedilol therapy, 5 patients (14.7%) had ventricular couplets and 2 patients (5.8%) had nonsustained ventricular tachycardia. a trend toward a decrease in ventricular couplets and nonsustained ventricular tachycardia did not reach statistical significance after 6 months. patients with dcm who had arrhythmic events during follow - up had significantly greater qtd than those without arrhythmic events (59.716.8 vs. 51.415.6, p=0.026). the initial mean carvedilol dose was 0.140.07 mg / kg / day, and 0.460.28 mg / kg / day at 6 months. as standard treatment, 34/34 (100%) were on digoxin, 32/34 (94.1%) were on furosemide, and 32/34 (94.1%) were on acei. the average heart rate was significantly reduced after carvedilol treatment (11423 vs. 8921 beat / min, p=0.008). systolic blood pressure tended to decrease after carvedilol therapy, but did not reach statistical significance. after 6 months of carvedilol treatment, the ross clinical score significantly improved from 6 to 3 (p=0.03) (table 3). the most common adverse events were dizziness (22%), vomiting (13%), hypotension (8%), and headache (5%). lvef significantly increased from 34.77.6% (range 2240%) to 45.29.6% (range 2961%) following carvedilol treatment (p=0.002). after carvedilol treatment, the left ventricular fractional shortening (lvfs) significantly increased from 16.49.7% (range 1033%) to 23.97.7% (range 1637%) (p=0.016), left ventricle end - diastolic dimensions (lvedd) significantly decreased from 45.78.1 mm (range 3160 mm) to 41.46.5 mm (range 2658 mm) (p=0.026) and the left ventricle end - systolic dimensions (lveds) significantly decreased from 39.46.9 mm (range 2447 mm) to 34.26.0 mm (range 2043 mm) (p=0.047) (table 3). there were significant increases in mean sdnn, sdann, rmssd, and pnn50 after carvedilol therapy (p=0.002, p=0.001, p=0.008, and p=0.026, respectively). a trend toward an increase in sdnni did not achieve statistical significance after 6 months. after carvedilol therapy, sdnn was correlated with the clinical score of chf, heart rate, lvef, lvsf, and total pvcs. in addition , rmssd and pnn50 were correlated with heart rate, lvef, and lvsf after carvedilol therapy. the comparison of clinical, hemodynamic, heart rate variability, and ventricular arrhythmia parameters of patients receiving carvedilol therapy at baseline and after the treatment are shown in table 3, and the correlation between changes in hrv in the time domains and changes in hemodynamic parameters are presented in table 4. intra- and interobserver variability were assessed in 18 randomly chosen patients; all intra- and interobserver variability for ecg parameters ranged from 3.1 to 4.8%. a significant reduction was observed in maximum and minimum qtc interval, qtc, and qtd values after carvedilol treatment. qtd was slightly higher in patients with a lower clinical score than in those with a higher clinical score, but the difference was not statistically significant. qtd was significantly related to total pvcs, but qtd was not related to age, sex, clinical score of chf, heart rate, lvef, lvsf, or lvedd (table 4). although 8 patients (23.5%) had pvcs before treatment, they disappeared in 4 patients and pvc decreased in 2 patients after treatment. sustained ventricular tachycardia was not observed in any patients. before carvedilol therapy, 5 patients (14.7%) a trend toward a decrease in ventricular couplets and nonsustained ventricular tachycardia did not reach statistical significance after 6 months. patients with dcm who had arrhythmic events during follow - up had significantly greater qtd than those without arrhythmic events (59.716.8 vs. 51.415.6, p=0.026). the neurohumoral mechanisms of chf involve activation of the sympathetic nervous system and the raa system, leading to intrinsic myocardial dysfunction, apoptosis, and remodeling. stimulation of -receptors increases oxygen consumption of the myocardium by increasing the afterload, which causes peripheral and coronary vasoconstriction that in accumulation of calcium in the myocyte, leading to cell death, and contributes to remodeling of the heart with fibrosis and hypertrophy. carvedilol is a third - generation -blocking agent that at therapeutic target doses blocks all 3 adrenergic receptors that decrease the chronic adrenergic overstimulation of the myocardium and improve myocardial function, and it has been shown to inhibit free radical induced cardiac contractile dysfunction. therefore, it is important to examine the clinical effect of a -adrenergic blocker therapy on dcm (e.g., carvedilol) to verify its efficacy in children. we have shown that oral carvedilol added to standard drug therapy improved ventricular function and clinical symptom scores in children with dcm, and we also found a significant correlation between changes in lvef and time domain parameters of hrv, including sdnn, rmssd, and pnn50. in accordance with the obtained from adults, the improvement of the autonomic function seen after the initiation of carvedilol therapy is likely to play an important role in children with dcm. recently, some authors have observed a statistically significant association between sudden cardiac death and depressed sdnn in patients with dcm. however, bilchick et al. demonstrated that sdnn has a strong and independent association with mortality in patients with moderate - to - severe chf. in this context, the uk - heart prospective study has recently demonstrated that reduced sdnn was the best noninvasive independent predictor of cardiac death in patients with chf. in our study, sdnn and sdann were found to be increased after the addition of carvedilol to standard medical therapy; these may have important clinical implications. the pnn50 and rmssd predominantly reflect parasympathetic activity and are independent of long - term trends. in the present study, improvements in rmssd and pnn50 were noted, and pnn50 and rmssd correlated to improvement in lvef. our findings confirm that carvedilol treatment has a beneficial effect on the mechanisms that sustain the harmful hyperadrenergic state and may improve prognosis in children with dcm. qtd has been found to be a significant, noninvasive prognostic marker of inhomogeneity of myocardial repolarization in several disease settings, and increased qtd may predispose to arrhythmic events. in a retrospective study of adult patients with chf, fu et al. found a larger qtd in patients who died suddenly or had spontaneous ventricular tachycardia than in survivors. however, only limited data is available regarding the effects of carvedilol on qtd in children with dcm. one retrospective study reported significant increases in qtd values in patients with lv systolic dysfunction. our study showed that carvedilol therapy decreased qt dispersion and qtc parameters, and improved ventricular repolarization characteristics in children with dcm after 6 months of follow - up. a randomized trial in adult patients with dcm showed a significant effect of carvedilol in reducing ventricular arrhythmias. our study suggest that the increase in sdnn and reduction in qtd are related with decrease in total pvcs. the increase in sdnn and reduction in qtd under carvedilol treatment may be partly due to an adrenergic blocking effect. moreover, antiapoptotic effects and inhibition of chronic remodeling of the myocardium may indirectly contribute to the observed homogenization of the ventricular repolarization process and prevention of induction of arrhythmia in patients with chf. we observed that ventricular ectopic beats disappeared in 4 patients and decreased in 2 patients with carvedilol treatment. reduced heart rate at rest may lead to better oxygen supply, lower the risk for life - threatening arrhythmia, and slow the myocardial remodeling process; thus, carvedilol may have also been effective in the control of ventricular ectopic beats. our data support the evidence that the increase in heart rate variability reflects improved autonomic regulation of heart rate, and show a significant correlation between heart rate variability changes and hemodynamic improvement with carvedilol therapy in children with dcm. we conclude that the addition of carvedilol to standard medical regimens can improve clinical symptoms and heart rate variability in association with improved left ventricular function, and reduce arrhythmia markers in children with dcm. a randomized, controlled, prospective trial is required to determine the true efficacy of carvedilol on the progression of congestive heart failure and to more clearly define its role in the cardiac autonomic dysfunction and rhythm disorders of children with dcm.	the purpose of this study was to examine the effects of carvedilol therapy on autonomic control of the heart and qt - interval dispersion (qtd) among children with idiopathic dilated cardiomyopathy (dcm) whose symptoms were not adequately controlled with standard congestive heart failure therapy.material/methodspatients with dcm who were treated with carvedilol were enrolled in the study. all patients had undergone carvedilol therapy in addition to standard therapy for at least 6 months. clinical, echocardiographic, and electrocardiographic parameters, and 24-h holter records of patients were retrospectively evaluated before and after carvedilol treatment.a total 34 patients (mean age : 7.44.3 years) with dcm were analyzed in the study. the median follow - up period was 9.5 months. after the 6 months of carvedilol therapy the clinical score significantly improved, left ventricular ejection fraction (lvef) and fractional shortening (lvfs) significantly increased, and left ventricle end - diastolic dimensions and end - systolic dimensions significantly decreased. there were statistically significant increases in mean sdnn, sdann, rmssd, and pnn50 (p=0.002, p=0.001, p=0.008, and p=0.026, respectively). after the carvedilol therapy, sdnn was correlated with the clinical score, heart rate, lvef, lvfs, and total premature ventricular contractions (pvcs). in addition , rmssd and pnn50 were correlated with heart rate, lvef and lvfs. a significant reduction was observed in qtc - minimum, qtc - maximum, and qtd values (434.940.7 vs. 416.136.5, 497.843.6 vs. 456.341.7, 58.617.1 vs. 49.315.6 ; p<0.001, p=0.001, and p=0.008, respectively). qtd was significantly related to pvcs (r=0.62, p=0.02).we conclude that the addition of carvedilol to standard therapy can improve clinical symptoms and heart rate variability, and reduce in arrhythmia markers in children with dcm.
in chapter 5 of integrity in scientific research, the institute of medicine (iom) committee described a theory - driven, evidence - based approach to designing instruction in the responsible conduct of research that would maximize the likelihood that education would influence individuals and institutions rather than merely satisfy an item on a check - off list for that institution. the recommended model for education included these principles: the educational program should be built around the development of abilities that give rise to responsible conduct. these include the ability to: 1 ) identify the ethical dimensions of situations that arise in the research setting and the laws, regulations, and guidelines governing one s field that apply (ethical sensitivity); 2 ) develop defensible rationales for a choice of action (ethical reasoning); 3 ) integrate the values of one s professional discipline with one s own personal values (identity formation) and appropriately prioritize professional values over personal ones (showing moral motivation and commitment); and 4 ) perform with integrity the complex tasks (e.g., communicate ideas and , obtain funding, teach, and supervise) that are essential to one s career (survival skills).the program should be designed in accordance with basic principles of adult learning.the instruction should be provided as much as possible by faculty who are actively engaged in research related to that of the trainees. the educational program should be built around the development of abilities that give rise to responsible conduct. these include the ability to: 1 ) identify the ethical dimensions of situations that arise in the research setting and the laws, regulations, and guidelines governing one s field that apply (ethical sensitivity); 2 ) develop defensible rationales for a choice of action (ethical reasoning); 3 ) integrate the values of one s professional discipline with one s own personal values (identity formation) and appropriately prioritize professional values over personal ones (showing moral motivation and commitment); and 4 ) perform with integrity the complex tasks (e.g., communicate ideas and , obtain funding, teach, and supervise) that are essential to one s career (survival skills). the instruction should be provided as much as possible by faculty who are actively engaged in research related to that of the trainees. in chapter 5, the four abilities drawn from rest s four component model of morality (fcm)are operationally defined. each is seen as a mix of cognitive and affective processes that contribute to the component s primary function. following the definitions, research conducted in professional education settings is summarized, and teaching strategies, assessment methods, and guidelines for designing cases to promote development of the abilities in the research setting are described. since the iom report also addressed the institutional culture that either enables or impedes researchers ability to act at the leading edge of their ethical competence, a separate appendix described outcome measures that could be used or adapted (a) to study organizational culture and (b) to study the ethical competences of individuals. potential outcome measures referenced in chapter 5 are fully described in appendix b. following publication of the iom report, michael zigmond and i prepared a document (appendix 1) to illustrate how the guidelines for designing cases to promote the four abilities could be applied to a set of circumstances that might arise in the research environment. since publication of the iom report, new findings have emerged that have implications for the structuring of a professional ethics curriculum. early studies cited in the iom report had shown that the abilities were independent of one another, as rest had predicted. in other words, competence in one did not predict competence in another, and a shortcoming in a single ability could account for a moral failing. this paper provides summaries of findings for each ability as well as new evidence as to their interconnectedness. following implications for education, the summary and section expands on earlier recommendations for structuring education and assessment to promote the responsible conduct of research. studies using well validated measures of ethical sensitivity illustrate that competence in the ability to interpret the moral dimension of professional problems is distinct from the ability to apply professional norms and values to determine what ought to be done. what is clear from the research is that if one fails to see the moral issue in a professional problem, competence in reasoning and problem solving, even if well - developed, are not brought to bear. further, just as students and professionals show remarkable variability in their level of moral reasoning development (see next section), they also vary greatly in their ability to interpret patient / client characteristics and professional responsibilities embedded in ethical sensitivity tests. finally, ethical sensitivity can be influenced by educational interventions, and, in some settings, researchers have observed small, though statistically significant gender difference favoring women. in a meta - analysis of ethical sensitivity research, researchers identified 37 studies in which 23 measures were described to assess ethical sensitivity in professional settings (e.g., dentistry, medicine, nursing, professional psychology, business, and science). after classifying the measures along several dimensions, including the extent to which the construct was elicited by the stimulus materials, they concluded that only seven of the measures met criteria, and most had not been extensively validated. examples of validated measures that elicit the process include the dental ethical sensitivity test (dest) designed for dentistry and the racial ethical sensitivity test (rest) designed for counseling psychology. what distinguishes measures like the rest and dest is the extent to which the stimulus presents clues to one or more moral problems without ever signaling either the moral issues at stake or the particular professional responsibilities called for. by way of contrast, some test designers seem to conceptualize ethical sensitivity as the ability to simply name the moral issue when a condensed synopsis of a moral problem is presented. examples in appendix 1 show how distinctly different the stimulus for assessing ethical sensitivity is from the stimulus for assessing moral reasoning development. the iom report described the usefulness of the defining issues test (dit) for assessing students capacity for reasoning about moral issues. subsequent reviews update that literature and describe use of the dit to assess (a) the reasoning development across and within professions, and (b) the effectiveness of interventions to promote moral judgment development. as described below, the measure is used in remediation programs to identify shortcomings in ethical decision making of practicing dentistry and medical students. strategies for assessing students and giving individualized feedback on reasoning development are included in chapter appendices. in addition to life - span measures of moral judgment development like the dit, researchers have designed measures of ethical reasoning for specific educational contexts. referred to as intermediate concept measures (icms), because they assess ethical concepts in the intermediate zone between broad moral ideals and profession - specific codes of conduct (e.g., professional autonomy, informed consent), icms present discipline - specific problems and assess agreement between respondents and experts as to the appropriateness or inappropriateness of particular action choices and justifications. it is important to note that test developers engage respondents from the discipline to generate items. an analysis of from multiple icm measures across populations (adolescents, young adults, and professionals) revealed that irrespective of the sample, age group, or icm concept assessed, respondents had more difficulty identifying the bad choices / justifications than the good items. in addition, the obtained mean differences suggested different levels of performance, as there was little to suggest that these findings were due to methodological issues. further, it appeared that identifying inappropriate and appropriate choices or justifications were not poles on a single dimension, as participants seemed not to apply information and experience gained on one set of considerations to the other. perhaps instruction focuses on what one ought to do and what are appropriate choices, and individuals must then infer what is inappropriate and poorly conceived. if so, ethics education may benefit by expanding its focus on inappropriate choices in order to provide more guidance in developing an experiential base to support more optimal moral decisions. when rest proposed his fcm of moral functioning in the early 1980s, moral motivation was featured, though less well articulated than the other three components. rest thought that moral motivation influenced moral action directly and in interaction with the other components of the moral system. in the last decade, considerable attention has been given to the study and measurement of lifespan identity formation particularly in the professions. in an edited volume on moral motivation, researchers summarize early and more recent efforts to operationally define and measure moral motivation. measures include the professional role orientation inventory (proi) described in the iom report, and the theoretical importance of a newer measure (the professional identity essay) used to assess life - span identity formation in dentistry and law and recently adapted for ethics remediation in medical education. note: in addition to providing guidance on the use of the dit, chapter appendices include the pie, adapted for medicine, and criteria for assessing and strategies promoting identity formation. in the same edited volume, researchers summarize evidence from dentistry, medicine, law, and the military that supports constructivists theoretical understanding of a developmental continuum of moral motivation and commitment (rest s component 3). the continuum proceeds from self - interest and concreteness of thought characteristic of entering professionals including entrants into doctoral education programs to more other - oriented and abstract ways of making sense of the self in relation to others. at advanced levels of moral motivation, the exemplary individual s personal and professional moral values are fully integrated, as evidenced by behavior that is consistent across contexts and situations. for example, the exemplary dental professionals studied by rule and bebeau were able to articulate the public duties of their profession, integrate them with personal value frameworks, and regularly and consistently engage in socially responsible actions. the identity of such exemplary professionals was contrasted with the identities of entering students and entering professionals across several professions (reviewed by bebeau and monson) and with professionals who have been disciplined by a licensing board. a consistent shortcoming of all but two of 41 professionals referred for an ethics assessment because they violated laws governing professional practice was in the ability to articulate professional duties and responsibilities. whereas referrals demonstrated a great deal of variability on measures of ethical sensitivity, moral reasoning, and ethical implementation, they consistently demonstrated only vague notions of their responsibilities to patients, their profession, their community, and society. the importance of professional identity formation to professional practice is further supported by a reexamination of data from a study of gender differences on measures of the four ethical abilities (ethical sensitivity, reasoning, role concept, and ethical implementation) for five cohorts who participated in a well - validated four - year dental ethics education program. the researchers (bebeau and thoma, in preparation) noted that those graduates who had developed a mature professional identity by graduation also had developed high levels of competence on measures of the other three components (ethical sensitivity, reasoning, and implementation). taken together, evidence from the three samples (exemplary professionals, disciplined professionals, and recent graduates who completed an ethics curriculum) strongly suggests that development of a moral identity that is consistent with the norms and values of the profession is the driving force that gives rise to the development of other abilities that account for responsible professional conduct. whether ethical sensitivity, reasoning, and competence in implementing ethical solutions develop in tandem with the development of a moral identity is a question for further research. as noted in the iom report, developing self - regulation, self - efficacy and implementation abilities is necessary for effective and responsible professional practice what fisher and zigmond describe as survival skills in the responsible conduct of research. a wide variety of assessment strategies is possible. in the dental ethics curriculum, competence in ethical implementation was based on performance score for eight complex cases presented during the last two years of the dental ethics curriculum. stimulus for assessment was similar to dest cases in that students needed to: 1 ) interpret the facts that must be addressed if the problem is to be resolved efficiently; 2 ) design an action plan; and 3 ) create a dialogue to illustrate effective plan implementation. all responses were assessed by the same rater, and students could challenge the assessment, revise, and resubmit. like other assessments used in the curriculum, an unexpected finding was an observed gender difference in ethical implementation for the 60 men and 60 women randomly selected from five cohorts to explore previously observed gender differences (favoring women) in ethical sensitivity and moral reasoning. no gender difference was observed on ethical sensitivity, and pretest and posttest differences on moral reasoning were trivial. whereas men and women had similar scores on the responsibility dimension of moral motivation (proi scores) at entrance to professional school, at graduation, an effect size of 0.75 for women versus 0.5 for men indicated greater change for women. a statistically significant gender difference, favoring women, was also evident on the measure of moral implementation, though pretest data were not collected to judge the role of general implementation abilities (e.g., interpersonal effectiveness, problem solving, etc .). an effect size of 0.57 suggested that the women s ability to implement more effective action plans was not a trivial difference. recall that a reanalysis of this data set (bebeau and thoma, in preparation) suggests that those students with a highly developed sense of professional identity also scored high on the measure of ethical sensitivity, reasoning, and implementation. given that the observed gender differences were not anticipated, replication with other cohorts and measures is indicated. an ethics education program of moderate duration can facilitate development of the four abilities described by the iom report, provided attention is directed to the educational principles the report outlines. in addition to the effect sizes reported for the undergraduate dental ethics curriculum, enhanced ethical competence was also achieved for professionals referred for assessment / instruction as a condition for license renewal. of the 41 dentists referred over a 20-year period, two were exempt from instruc tion based on pretest performance on the five well - validated measures of the fcm (the dest, dit, derjt, proi, and role concept essays) and 38 completed an individualized course designed to remediate deficiencies in ethical abil ities identified at the pretest. statistically significant pre- to posttest changes (effect sizes ranging from 0.55 to 5.0) were reported for ethical sensitivity (dest scores), moral reasoning (dit scores), and role concept (rce essays and proi scores). analysis of the relationships between ability deficiencies and disciplinary actions supported the explanatory power of rest s fcm. of particular interest was the way the model helped the referrals to deconstruct a summary judgment about their character (as unethical or unprofessional) and to see the self as lacking in particular capacities or abilities that could be further developed. finally, though time - consuming to implement, the individualized remediation programs were highly effective in promoting ethical competencies, in reducing recidivism, and in influencing practitioner perceptions of the programs value. an examination of the extensive practitioner self - assessment data provides guidance for structuring instruction. beginning the instructional process with a discussion of the distinguishing features of a profession and the expectations that follow further, the use of cases to assess and facilitate ethical sensitivity and reasoning was viewed as relevant to professional practice. of particular interest instead of focusing on what is happening? and what ought to be done? as is typical of much ethics instruction, the courses emphasized how to implement an action plan, including what to say and how to say it. with the exception of fisher and zigmond s work on survival skills, this appears to be an often neglected area of ethics instruction. for students and practitioners alike, there is a clear hunger for help with strategies and language to deal with human interaction problems that have clear ethical implications. for further discussion of curriculum and resources to promote ethical implementation, newly designed resources for assessing and promoting identity formation are provided in a chapter, in remediation in medical education. the authors coach educators to use measures of moral reasoning and professional identity formation to provide a diagnostic assessment of a student s strengths and shortcomings in their understanding of the ethical and moral dimensions of professionalism. next, the authors describe a remediation curriculum developed for a group of students who violated professional norms. this program has also been used to address individual transgressions (e.g., cheating, subpar behavior in practice). lastly, the authors highlight strategies they have found effective in therapeutic interactions with individual students who present particular challenges. studies using well validated measures of ethical sensitivity illustrate that competence in the ability to interpret the moral dimension of professional problems is distinct from the ability to apply professional norms and values to determine what ought to be done. what is clear from the research is that if one fails to see the moral issue in a professional problem, competence in reasoning and problem solving, even if well - developed, are not brought to bear. further, just as students and professionals show remarkable variability in their level of moral reasoning development (see next section), they also vary greatly in their ability to interpret patient / client characteristics and professional responsibilities embedded in ethical sensitivity tests. finally, ethical sensitivity can be influenced by educational interventions, and, in some settings, researchers have observed small, though statistically significant gender difference favoring women. in a meta - analysis of ethical sensitivity research, researchers identified 37 studies in which 23 measures were described to assess ethical sensitivity in professional settings (e.g., dentistry, medicine, nursing, professional psychology, business, and science). after classifying the measures along several dimensions, including the extent to which the construct was elicited by the stimulus materials , they concluded that only seven of the measures met criteria, and most had not been extensively validated. examples of validated measures that elicit the process include the dental ethical sensitivity test (dest) designed for dentistry and the racial ethical sensitivity test (rest) designed for counseling psychology. what distinguishes measures like the rest and dest is the extent to which the stimulus presents clues to one or more moral problems without ever signaling either the moral issues at stake or the particular professional responsibilities called for. by way of contrast, some test designers seem to conceptualize ethical sensitivity as the ability to simply name the moral issue when a condensed synopsis of a moral problem is presented. examples in appendix 1 show how distinctly different the stimulus for assessing ethical sensitivity is from the stimulus for assessing moral reasoning development. the iom report described the usefulness of the defining issues test (dit) for assessing students capacity for reasoning about moral issues. subsequent reviews update that literature and describe use of the dit to assess (a) the reasoning development across and within professions, and (b) the effectiveness of interventions to promote moral judgment development. as described below, the measure is used in remediation programs to identify shortcomings in ethical decision making of practicing dentistry and medical students. strategies for assessing students and giving individualized feedback on reasoning development are included in chapter appendices. in addition to life - span measures of moral judgment development like the dit, researchers have designed measures of ethical reasoning for specific educational contexts. referred to as intermediate concept measures (icms), because they assess ethical concepts in the intermediate zone between broad moral ideals and profession - specific codes of conduct (e.g., professional autonomy, informed consent), icms present discipline - specific problems and assess agreement between respondents and experts as to the appropriateness or inappropriateness of particular action choices and justifications. it is important to note that test developers engage respondents from the discipline to generate items. an analysis of from multiple icm measures across populations (adolescents, young adults, and professionals) revealed that irrespective of the sample, age group, or icm concept assessed, respondents had more difficulty identifying the bad choices / justifications than the good items. in addition, the obtained mean differences suggested different levels of performance, as there was little to suggest that these findings were due to methodological issues. further, it appeared that identifying inappropriate and appropriate choices or justifications were not poles on a single dimension, as participants seemed not to apply information and experience gained on one set of considerations to the other. perhaps instruction focuses on what one ought to do and what are appropriate choices, and individuals must then infer what is inappropriate and poorly conceived. if so, ethics education may benefit by expanding its focus on inappropriate choices in order to provide more guidance in developing an experiential base to support more optimal moral decisions. when rest proposed his fcm of moral functioning in the early 1980s, moral motivation was featured, though less well articulated than the other three components. rest thought that moral motivation influenced moral action directly and in interaction with the other components of the moral system. in the last decade, considerable attention has been given to the study and measurement of lifespan identity formation particularly in the professions. in an edited volume on moral motivation, researchers measures include the professional role orientation inventory (proi) described in the iom report, and the theoretical importance of a newer measure (the professional identity essay) used to assess life - span identity formation in dentistry and law and recently adapted for ethics remediation in medical education. note: in addition to providing guidance on the use of the dit, chapter appendices include the pie, adapted for medicine, and criteria for assessing and strategies promoting identity formation. in the same edited volume, researchers summarize evidence from dentistry, medicine, law, and the military that supports constructivists theoretical understanding of a developmental continuum of moral motivation and commitment (rest s component 3). the continuum proceeds from self - interest and concreteness of thought characteristic of entering professionals including entrants into doctoral education programs to more other - oriented and abstract ways of making sense of the self in relation to others. at advanced levels of moral motivation, the exemplary individual s personal and professional moral values are fully integrated, as evidenced by behavior that is consistent across contexts and situations. for example, the exemplary dental professionals studied by rule and bebeau were able to articulate the public duties of their profession, integrate them with personal value frameworks, and regularly and consistently engage in socially responsible actions. the identity of such exemplary professionals was contrasted with the identities of entering students and entering professionals across several professions (reviewed by bebeau and monson) and with professionals who have been disciplined by a licensing board. a consistent shortcoming of all but two of 41 professionals referred for an ethics assessment because they violated laws governing professional practice was in the ability to articulate professional duties and responsibilities. whereas referrals demonstrated a great deal of variability on measures of ethical sensitivity, moral reasoning, and ethical implementation, they consistently demonstrated only vague notions of their responsibilities to patients, their profession, their community, and society. the importance of professional identity formation to professional practice is further supported by a reexamination of data from a study of gender differences on measures of the four ethical abilities (ethical sensitivity, reasoning, role concept, and ethical implementation) for five cohorts who participated in a well - validated four - year dental ethics education program. the researchers (bebeau and thoma, in preparation) noted that those graduates who had developed a mature professional identity by graduation also had developed high levels of competence on measures of the other three components (ethical sensitivity, reasoning, and implementation). taken together, evidence from the three samples (exemplary professionals, disciplined professionals, and recent graduates who completed an ethics curriculum) strongly suggests that development of a moral identity that is consistent with the norms and values of the profession is the driving force that gives rise to the development of other abilities that account for responsible professional conduct. whether ethical sensitivity, reasoning, and competence in implementing ethical solutions develop in tandem with the development of a moral identity is a question for further research. as noted in the iom report, developing self - regulation, self - efficacy and implementation abilities is necessary for effective and responsible professional practice what fisher and zigmond describe as survival skills in the responsible conduct of research. a wide variety of assessment strategies is possible. in the dental ethics curriculum, competence in ethical implementation was based on performance score for eight complex cases presented during the last two years of the dental ethics curriculum. stimulus for assessment was similar to dest cases in that students needed to: 1 ) interpret the facts that must be addressed if the problem is to be resolved efficiently; 2 ) design an action plan; and 3 ) create a dialogue to illustrate effective plan implementation. all responses were assessed by the same rater, and students could challenge the assessment, revise, and resubmit. like other assessments used in the curriculum, an unexpected finding was an observed gender difference in ethical implementation for the 60 men and 60 women randomly selected from five cohorts to explore previously observed gender differences (favoring women) in ethical sensitivity and moral reasoning. no gender difference was observed on ethical sensitivity, and pretest and posttest differences on moral reasoning were trivial. whereas men and women had similar scores on the responsibility dimension of moral motivation (proi scores) at entrance to professional school, at graduation, an effect size of 0.75 for women versus 0.5 for men indicated greater change for women. a statistically significant gender difference, favoring women, was also evident on the measure of moral implementation, though pretest data were not collected to judge the role of general implementation abilities (e.g., interpersonal effectiveness, problem solving, etc .). an effect size of 0.57 suggested that the women s ability to implement more effective action plans was not a trivial difference. recall that a reanalysis of this data set (bebeau and thoma, in preparation) suggests that those students with a highly developed sense of professional identity also scored high on the measure of ethical sensitivity, reasoning, and implementation. given that the observed gender differences were not anticipated, replication with other cohorts and measures is indicated. an ethics education program of moderate duration can facilitate development of the four abilities described by the iom report, provided attention is directed to the educational principles the report outlines. in addition to the effect sizes reported for the undergraduate dental ethics curriculum, enhanced ethical competence was also achieved for professionals referred for assessment / instruction as a condition for license renewal. of the 41 dentists referred over a 20-year period, two were exempt from instruc tion based on pretest performance on the five well - validated measures of the fcm (the dest, dit, derjt, proi, and role concept essays) and 38 completed an individualized course designed to remediate deficiencies in ethical abil ities identified at the pretest. statistically significant pre- to posttest changes (effect sizes ranging from 0.55 to 5.0) were reported for ethical sensitivity (dest scores), moral reasoning (dit scores), and role concept (rce essays and proi scores). analysis of the relationships between ability deficiencies and disciplinary actions supported the explanatory power of rest s fcm. of particular interest was the way the model helped the referrals to deconstruct a summary judgment about their character (as unethical or unprofessional) and to see the self as lacking in particular capacities or abilities that could be further developed. finally, though time - consuming to implement, the individualized remediation programs were highly effective in promoting ethical competencies, in reducing recidivism, and in influencing practitioner perceptions of the programs value. an examination of the extensive practitioner self - assessment data provides guidance for structuring instruction. beginning the instructional process with a discussion of the distinguishing features of a profession and the expectations that follow further, the use of cases to assess and facilitate ethical sensitivity and reasoning was viewed as relevant to professional practice. of particular interest was the value practitioners placed on the curriculum s emphasis on ethical implementation. instead of focusing on what is happening? and what ought to be done? as is typical of much ethics instruction, the courses emphasized how to implement an action plan, including what to say and how to say it. with the exception of fisher and zigmond s work on survival skills, this appears to be an often neglected area of ethics instruction. for students and practitioners alike, there is a clear hunger for help with strategies and language to deal with human interaction problems that have clear ethical implications. for further discussion of curriculum and resources to promote ethical implementation, newly designed resources for assessing and promoting identity formation are provided in a chapter, in remediation in medical education. the authors coach educators to use measures of moral reasoning and professional identity formation to provide a diagnostic assessment of a student s strengths and shortcomings in their understanding of the ethical and moral dimensions of professionalism. next, the authors describe a remediation curriculum developed for a group of students who violated professional norms. this program has also been used to address individual transgressions (e.g., cheating, subpar behavior in practice). lastly, the authors highlight strategies they have found effective in therapeutic interactions with individual students who present particular challenges. evidence from the cited studies adds weight to earlier recommendations for structuring educational programs that use active learning strategies to promote the development of competent, thoughtful, and responsible scientists. it is natural to assume that students who apply for graduate education are mature individuals of good character, and our intention is not to undermine confidence in students personal integrity. yet the evidence shows that entering students, across professions, have rather vague notions of the norms and values of their chosen profession and seem not to pick them up from role models during the course of their education. more importantly, recent studies exploring competence on each of the four ethical abilities defined by rest s four component model of morality illustrate the critical role of professional identity formation. because the formation of a professional identity seems to be the primary driver behind responsible conduct, a first step in designing an educational program in responsible research conduct is to begin by addressing the expectations of a scientist. ask students to respond to open - ended questions in writing which they can reflect upon as they learn about the values and normative practices that guide the research enterprise. by asking students to express the concepts in their own words, and in writing, misperceptions can be identified and addressed before they become an issue. educators may wish to modify the pie applied in medicine and adapt the reflective learning activities for rcr education. as argued in the iom report, to develop thoughtful and responsible scientists who act with integrity and have a broad understanding of their role and a commitment to integrity in science, educators must do more than teach the rules and policies that apply to the conduct of research. once the expectations of the scientist have been clarified, it is important to engage students in active learning (using cases, if possible) to facilitate the abilities that are necessary conditions in addition to a professional identity ethical sensitivity, reasoning, and problem solving for effective moral action. when selecting or designing case materials, the materials must be carefully structured to elicit the process of concern. as argued above, too often cases are written and participants are asked: what should the protagonist do? such a question focuses on problem solving rather than problem identification or moral reasoning. whereas a skilled facilitator may be able to redirect focus to reasoning or problem identification, it is sometimes more difficult. for this reason, we provided an example (appendix 1) to show how to design stimulus materials that focus on the particular skill needed for effective problem identification, reasoning, or implementation. with rather carefully targeted courses of moderate duration, it is possible to show gains in each of the abilities that give rise to responsible conduct. our goal is not to develop the more advanced skills in ethical reasoning that might from courses in moral philosophy. yet evidence shows that problem - based practice (using cases) can be especially effective in helping students recognize and subsequently avoid personal interest arguments while strengthening awareness and adherence to the rules of responsible research conduct.	drawing from multiple sources of evidence, this paper updates previous descriptions (iom, 2002) of measurement strategies and teaching techniques to promote four theoretically derived abilities thought to be necessary conditions for the responsible conduct of research. data from three samples (exemplary professionals, professionals disciplined by a licensing board, and graduates who completed an ethics program designed to promote the four interrelated abilities) suggest that development of a moral identity that is consistent with the norms and values of a profession is the driving force that gives rise to ethical sensitivity, ethical reasoning, and ethical implementation. evidence from the cited studies supports the usefulness of the theoretical model to (a) deconstruct summary judgments about character and see them as abilities that can be assessed and developed; (b) guide the design assessments that are sensitive to the effects of interventions; and (c) augment previous iom recommendations for the development of meaningful learning activities.
the global alarming increase in the prevalence of chronic diseases, as the most leading cause of death, has attracted considerable attention over the past decades. a strikingly high prevalence of chronic diseases and their risk factors has been indicated among iranian adults. findings from numerous studies have confirmed the crucial role of life - style factors, in particular dietary intakes, on the etiology and management of chronic diseases; however, the optimal assessment of dietary intakes as the principal challenge in this regard still remains unresolved. so far, various methods such as dietary recalls, food records, diet histories and food frequency questionnaires (ffqs) have been proposed for dietary intake assessment. recall bias, embarrassment of participants, requiring well - trained interviewers and food coding in the dietary recalls might affect its validity and utilization. despite the accuracy of food records , some weaknesses such as the possibility of change in the usual eating habits and the necessity of substantial training of participants can not be excluded. most importantly, dietary recalls and records do not reflect participants usual intakes. to obtain long - term dietary intakes, the potential bias due to an un - standardized interview approach, time - consuming, lengthy and open - ended food lists in the diet histories, have considerably limited the application of this method. now - a - days, ffqs are the most widely used dietary assessment tool in large - scale epidemiological studies. the acceptability of ffqs is due to several important advantages of this method, such as low cost, ease of administration, computer processing, considering within - person variation and reflecting long - term intakes. some characteristics of ffqs, such as their length and complexity, which is mainly due to asking for detailed information on a long list of food items, portion sizes and food preparation methods could affect the accuracy of collected information. on the other hand, poorly designed ffqs without comprehensive food lists may lead to incorrect information and misclassification, which in turn could in masking diet - disease relations. thus, the development of a comprehensive and appropriate ffq is the cornerstone of nutritional epidemiological studies. so far all previously used ffqs in iranian settings have mainly been consisted of the extensive list of foods and followed a food - based design. however, the limitations and pitfalls in the accuracy of information that is provided using such ffqs have been described before. worldwide, many epidemiological studies, including korean, japanese, brazilian, swiss, bangladeshi and zimbabwean studies have used dish - based ffqs. typical iranian dietary pattern is characterized by various mixed dishes, which contains several ingredients prepared in different ways. with no doubt, it is very difficult to estimate usual intake of single ingredients from various mixed iranian dishes. to the best of our knowledge, no earlier study in iran has used a dish - based ffq or a ffq with multiple - choice frequency response options. furthermore, all previous studies in the country have used the interviewer - administered ffqs. in the framework of the study on the epidemiology of psychological, alimentary health and nutrition (sepahan) project , we designed a dish - based machine - readable semi - quantitative food frequency questionnaire (dfq) with multiple choice frequency response options for the first time in iran. the aim of the present paper is to describe how this dietary assessment tool was developed. to develop the dfq, we used the harvard ffq as a model, which assesses the dietary intake of a person in the preceding 12 months. the following steps were taken to develop the questionnaire: construction of a list of commonly consumed iranian foods, definition of portion sizes, determining the frequency response options for each food item and finally testing the face validity of the questionnaire in a pilot setting to check for the comprehension and feasibility. as the initial step, a comprehensive list of foods was prepared based on the information provided by local experienced nutritionists, considering all commonly consumed iranian foods and mixed dishes. to avoid missing major food items, we listed foods commonly consumed by iranians as meal (breakfast, lunch and dinner) or snack. from the comprehensive list of foods and mixed dishes, we selected those that were nutrient - rich (such as liver), consumed reasonably often (such as different types of bread), or contributed to between - person variations (such as sausage). owing to the high consumption of mixed dishes among iranian population, it is difficult for people to estimate usual intake of ingredients of those dishes. in contrast to previously designed ffqs in the country, we focused on foods and mixed dishes rather than single ingredients. for example, iranians consume potato through several mixed dishes such broth, olviyeh (potato salad), cutlet, different stews, etc. , certainly, it is too hard to estimate total potato intake of a person by asking participants to remember the frequency consumption of potato in the preceding 12 months. participants may get confused while trying to add their usual potato intake that comes from different sources and it is highly possible that some potato - containing dishes be forgotten or ignored. however, it would be much easier for them to make an estimate of the amount and frequency of consuming every single dish. it is generally assumed that questionnaire length has a significant effect on the survey response rate as respondents get tired, bored and/or distracted by external factors. furthermore, a lengthy questionnaire is less likely to be completed and returned. as we attempted to design a self - administered questionnaire, we pilot tested the comprehensive list of food items / dishes among a group of individuals (n = 35) in order to exclude some foods that were consumed rarely or never. finally, these exclusions ed in a list of 106 food items or dishes in the questionnaire. to simplify the completion of the questionnaire, we categorized food items into five major groups: mixed dishes (cooked or canned : 29 items); grains (different types of bread, cakes, biscuits and potato : 10 items); dairy products (dairies, butter and cream : 9 items); fruits and vegetables (22 items); and miscellaneous food items and beverages (including sweets, fast foods, nuts, desserts and beverages : 36 items). although portion sizes in ffqs are generally poorly estimated and do not contribute significantly to the between - person variations in dietary intakes, their quantification in a questionnaire will help better ranking of individuals in terms of their dietary intakes. in the dfq , portion sizes for food items and mixed dishes were defined based on the most commonly consumed portion size for each item among iranian general population. to increase precision and accuracy of estimates , we attempted to provide the portion sizes of foods and mixed dishes as a unit with the same perception for all people. to reach these portion sizes, we used reported portion sizes in dietary recalls and food records in our earlier studies. furthermore, we pilot tested the preliminary ffq by administering it in a group of adults (n = 35) to determine the most appropriate portion size for every single food item. a group of nutrition experts also discussed about the portion sizes to finalize the most suitable choice for each food item. loaf for breads, medium - sized plate for cooked rice, tablespoon for raisins and bag for potato chips. similar to the harvard ffq, we used nine multiple - choice options (for most food items) varying from never or less than once per month to 12 or more times per day to estimate average frequency of different food intakes. the frequency response options for each food item were defined separately in a row against the food list. this is in contrast to the harvard ffq in which the frequency response options are indicated in the top of a column for all food items. for all frequency response options , we also mentioned the portion sizes repeatedly to simplify responding. the number of frequency response options was not constant for all foods. for foods consumed infrequently, we omitted the high - frequency options while for highly consumed foods, the number of multiple - choice options were increased. for instance, the frequency response for tuna consumption included six options, as follows: never or less than once per month, 1 - 3 times per month, 1 time per week, 2 - 4 times per week, 5 - 6 times per week and 1 - 2 times per day. however, for tea which is consumed more frequently, the frequency response included nine options, as follows: never or less than 1 cup per month, 1 - 3 cups per month, 1 - 3 cups per week, 4 - 6 cups per week, 1 cup per day, 2 - 4 cups per day, 5 - 7 cups per day, 8 - 11 cups per day and 12 cups per day ). the pilot study on 35 subjects, which was conducted to test the face validity of the dfq, assisted us to select proper number of response options for each food item in the list. sample of questions and layout of the harvard food frequency questionnaire (a) in comparison with the dfq (b). different number of response options for each food item, including portion sizes that are more familiar to iranians, and including the response options of consumption frequencies for each food item are among the major features that have been considered in designing the dfq using the dfq, a daily value for each item can be calculated based on food composition, specified portion size and the average of reported frequency. for example, if a person reports to consume macaroni 2 - 4 plates per week, we can compute the daily intake of ground beef from this item according to this formula: 0.43 (3 ( average number of plates per week) divided by 7 (number of days per week) ) multiplying 50 (grams of ground beef in each plate of macaroni). moreover, other mixed dishes in this ffq contain ground beef such as barbecue, cutlet, etc. , therefore, total ground beef intake for a participant will be calculated by the sum of his / her daily ground beef intake from different food items in the list. the food composition of mixed dishes was determined based on common recipes consumed in the country. in addition, a group of five nutrition experts collected 15 home or restaurant recipes for each iranian mixed dish that was included the dfq. finally, mean values of different ingredients of a mixed dish was considered as the final food composition details of that dish. given the seasonal variation in the dietary intakes of some fruits and vegetables, in the dfq participants are asked to mention the consumption frequency of such items during the months that those products are widely available in the market. in the calculation of fruit and vegetables, as the initial step, a comprehensive list of foods was prepared based on the information provided by local experienced nutritionists, considering all commonly consumed iranian foods and mixed dishes. to avoid missing major food items, we listed foods commonly consumed by iranians as meal (breakfast, lunch and dinner) or snack. from the comprehensive list of foods and mixed dishes, we selected those that were nutrient - rich (such as liver), consumed reasonably often (such as different types of bread), or contributed to between - person variations (such as sausage). owing to the high consumption of mixed dishes among iranian population, it is difficult for people to estimate usual intake of ingredients of those dishes. in contrast to previously designed ffqs in the country, we focused on foods and mixed dishes rather than single ingredients. for example, iranians consume potato through several mixed dishes such broth, olviyeh (potato salad), cutlet, different stews, etc. , certainly, it is too hard to estimate total potato intake of a person by asking participants to remember the frequency consumption of potato in the preceding 12 months. participants may get confused while trying to add their usual potato intake that comes from different sources and it is highly possible that some potato - containing dishes be forgotten or ignored. however, it would be much easier for them to make an estimate of the amount and frequency of consuming every single dish. it is generally assumed that questionnaire length has a significant effect on the survey response rate as respondents get tired, bored and/or distracted by external factors. furthermore, a lengthy questionnaire is less likely to be completed and returned. as we attempted to design a self - administered questionnaire, we pilot tested the comprehensive list of food items / dishes among a group of individuals (n = 35) in order to exclude some foods that were consumed rarely or never. finally, these exclusions ed in a list of 106 food items or dishes in the questionnaire. to simplify the completion of the questionnaire , we categorized food items into five major groups: mixed dishes (cooked or canned : 29 items); grains (different types of bread, cakes, biscuits and potato : 10 items); dairy products (dairies, butter and cream : 9 items); fruits and vegetables (22 items); and miscellaneous food items and beverages (including sweets, fast foods, nuts, desserts and beverages : 36 items). although portion sizes in ffqs are generally poorly estimated and do not contribute significantly to the between - person variations in dietary intakes, their quantification in a questionnaire will help better ranking of individuals in terms of their dietary intakes. in the dfq, portion sizes for food items and mixed dishes were defined based on the most commonly consumed portion size for each item among iranian general population. to increase precision and accuracy of estimates , we attempted to provide the portion sizes of foods and mixed dishes as a unit with the same perception for all people. to reach these portion sizes, we used reported portion sizes in dietary recalls and food records in our earlier studies. furthermore, we pilot tested the preliminary ffq by administering it in a group of adults (n = 35) to determine the most appropriate portion size for every single food item. a group of nutrition experts also discussed about the portion sizes to finalize the most suitable choice for each food item. loaf for breads, medium - sized plate for cooked rice, tablespoon for raisins and bag for potato chips. similar to the harvard ffq, we used nine multiple - choice options (for most food items) varying from never or less than once per month to 12 or more times per day to estimate average frequency of different food intakes. the frequency response options for each food item were defined separately in a row against the food list. this is in contrast to the harvard ffq in which the frequency response options are indicated in the top of a column for all food items. for all frequency response options , we also mentioned the portion sizes repeatedly to simplify responding. the number of frequency response options was not constant for all foods. for foods consumed infrequently, we omitted the high - frequency options while for highly consumed foods, the number of multiple - choice options were increased. for instance, the frequency response for tuna consumption included six options, as follows: never or less than once per month, 1 - 3 times per month, 1 time per week, 2 - 4 times per week, 5 - 6 times per week and 1 - 2 times per day. however, for tea which is consumed more frequently, the frequency response included nine options, as follows: never or less than 1 cup per month, 1 - 3 cups per month, 1 - 3 cups per week, 4 - 6 cups per week, 1 cup per day, 2 - 4 cups per day, 5 - 7 cups per day, 8 - 11 cups per day and 12 cups per day ). the pilot study on 35 subjects, which was conducted to test the face validity of the dfq, assisted us to select proper number of response options for each food item in the list. sample of questions and layout of the harvard food frequency questionnaire (a) in comparison with the dfq (b). different number of response options for each food item, including portion sizes that are more familiar to iranians, and including the response options of consumption frequencies for each food item are among the major features that have been considered in designing the dfq using the dfq, a daily value for each item can be calculated based on food composition, specified portion size and the average of reported frequency. for example, if a person reports to consume macaroni 2 - 4 plates per week, we can compute the daily intake of ground beef from this item according to this formula: 0.43 (3 ( average number of plates per week) divided by 7 (number of days per week) ) multiplying 50 (grams of ground beef in each plate of macaroni). moreover, other mixed dishes in this ffq contain ground beef such as barbecue, cutlet, etc. , therefore, total ground beef intake for a participant will be calculated by the sum of his / her daily ground beef intake from different food items in the list. the food composition of mixed dishes was determined based on common recipes consumed in the country. in addition, a group of five nutrition experts collected 15 home or restaurant recipes for each iranian mixed dish that was included the dfq. finally, mean values of different ingredients of a mixed dish was considered as the final food composition details of that dish. given the seasonal variation in the dietary intakes of some fruits and vegetables, in the dfq participants are asked to mention the consumption frequency of such items during the months that those products are widely available in the market. in the calculation of fruit and vegetables, this factor has also been taken into account. in the present article, we described the development and design of a dish - based, machine - readable, semi - quantitative ffq (called dfq) for the iranian adult population. to the best of our knowledge, this is the first time that such a dietary assessment tool has been created in the country. in general, ffqs have been proposed as the optimal instruments for dietary intake assessments in large epidemiological studies. data from ffqs can be used to elucidate diet - disease relations through ranking participants based on their usual dietary intakes. as shown by earlier investigations, along with all dietary assessment methods, some potential disadvantages could also be noted about ffqs including recall bias, overestimation of dietary intakes particularly for rarely - consumed and healthy - perceived foods (e.g., fruit and vegetables), bias of current intake, misclassification and bias of pre - established food listing. in the dfq, we focused on compiling a comprehensive list of foods to capture total energy intake. as discussed by well - known researchers in the field of nutritional epidemiology, total energy intake must be controlled for in almost all epidemiological studies. moreover, the main objective of the current study was to develop an easy - to - use ffq for future epidemiological studies in iran. given that longer questionnaires may cause respondent fatigue and poorer quality of gathered information, we focused on nutrient - rich frequently consumed foods and dishes. we also included those foods that could explain the between - person variations in diet. earlier ffqs that have been used in iranian epidemiological studies were developed for the tehran lipid and glucose study (tlgs), the isfahan health heart program (ihhp) and the golestan cohort study. the difference in food items could be explained by the inclusion of food items in earlier questionnaires but dishes in the dfq. compared with previous iranian ffqs, the principal discriminatory feature of dfq was incorporating iranian mixed dishes instead of including their ingredients. since typical iranian dishes usually consist of various ingredients, it is difficult for respondents to estimate their total intake of one ingredient which may be used in several mixed dishes. inclusion of mixed dishes instead of their ingredients in a ffq will not only facilitate participants responding, but also will reduce the length of the questionnaire. using the current approach of including mixed dishes instead of their ingredients could shorten the list of food items in the dfq, shorten the time required to fill the questionnaire, decrease participants boredom and increase accuracy of dietary intake assessment. furthermore, as cooking and other food processing methods affect the nutritional value of foods, including mixed dishes instead of food ingredients can better elucidate the relationship between diet and diseases. some investigators believe that shortening of food list and using a unique recipe for all participants may lead to decreased accuracy and failure to estimate the total energy intake. although open - ended frequency responses would lead to enhanced precision and continuous variables instead of categorical ones, it has been shown that using multiple - choice frequency response options increases clarity and reduces errors. however, it must be kept in mind that frequency options must be designed in a manner that covers all possible choices of consumption frequency responses in participants. the number of options should range between 5 and 10; broadened response options with less than five choices would significantly limit the discrimination capacity, while excessive options may be confusing. between - persons variation for frequently and rarely consumed foods is distinguished at the high and low end of the scale, respectively. the dfq is the first ffq in the country with multiple - choice frequency response options. we believe that the multiple - choice frequency response options will facilitate the completion of the dfq as it can be filled by participants without the help of an interviewer (i.e., self - administered questionnaire). the frequency response choices in the dfq are different from those in the harvard ffq. as consumption frequency of food items may vary in a broad range, we decided to include a unique set of frequency response options for each food item in the dfq. as generally portion sizes are poorly estimated, the inclusion of portion sizes in ffqs is still controversial. it seems that the large percentage of between - persons variation could be explained by consumption frequency, rather than portion sizes. estimating portion size of foods is difficult for most participants. however, it is important to recognize that calculation of absolute nutrient intake is impossible when information on portion sizes is not available. some investigators have suggested to use the commonly consumed portion sizes for calculating nutrient intakes in case of missing portion sizes in a ffq. for example, the correlations of nutrient intake obtained from ffqs with and without portion sizes were over 0.9. presumably, inclusion of pre - specified portion sizes make questionnaire easier to complete by respondents. it has been proposed to include the most frequently consumed or traditional portion sizes in ffqs. although earlier ffqs in iran have included portion sizes, this information was mostly based on serving sizes recommended by the u.s. department of agriculture food guide pyramid, not on the most frequently locally used portion sizes. as previously mentioned, the dfq is a machine - readable questionnaire. for this purpose , we developed an optical mark recognition (omr) system that can distinguish the selected answer for each food item in the scanned pictures of questionnaires. furthermore to convert selected consumption frequency responses option to exact intake of a food item, appropriate software was developed. from the pilot study showed the very high accuracy of the developed computerized systems. for large epidemiological studies, using this system will have a crucial role in reducing the expenses (e.g., staff, time) and errors that are inevitable while extracting data manually. various approaches have been used to assess the performance of ffqs including classic validation study and evaluation of the ability to predict expected diet - diseases correlation. although, a classic validation study has not been conducted for the dfq so far, we have recently indicated several established relationships between dietary factors and diseases using this tool. the dfq could be an appropriate dietary assessment tool for future epidemiological studies in iran. however, before its application in large epidemiological studies, the validity and reliability of this newly developed ffq should be assessed among different iranian populations.	: earlier forms of food frequency questionnaire (ffq) used in iran have extensive lists of foods, traditional categories and food - based design, mostly with the interviewer - administered approach. the aim of the current paper is to describe the development of a dish - based, machine - readable, semi - quantitative food frequency questionnaire (dfq).methods: within the framework of the study on the epidemiology of psychological, alimentary health and nutrition project, we created a novel ffq using harvard ffq as a model.:the following steps were taken to develop the questionnaire: construction of a list of commonly consumed iranian foods, definition of portion sizes, design of response options for consumption frequency of each food item and finally a pilot test of the preliminary dfq. from a comprehensive list of foods and mixed dishes, we included those that were nutrient - rich, consumed reasonably often or contributed to between - person variations. we focused on mixed dishes, rather than their ingredients, along with foods. to shorten the list, the related food items or mixed dishes were categorized together in one food group. these exclusions ed in a list of 106 foods or dishes in the questionnaire. the portion sizes used in the ffq were obtained from our earlier studies that used dietary recalls and food records. the frequency response options for the food list varied from 6 - 9 choices from never or less than once a month to 12 or more times per day.:the dfq could be a reasonable dietary assessment tool for future epidemiological studies in the country. validation studies are required to assess the validity and reliability of this newly developed questionnaire.
improved control of blood pressure in patients with hypertension is a key requirement to reduce cardiovascular and renal morbidity and mortality.1,2 despite the effectiveness of the currently available antihypertensive agents, hypertension remains inadequately controlled, with slightly less than half of patients who receive treatment successfully achieving the goals for systolic and diastolic blood pressure.3,4 the renin angiotensin system (ras) plays a central role in the pathophysiology of hypertension, cardiovascular and renal disease.5,6 it contributes to the increase of blood volume and arterial pressure, to the alterations of endothelial function, vascular reactivity, fibrosis, tissue remodeling, oxidative stress and inflammation which may predispose to the development of cardiovascular disease.57 while there are many drug classes available to reduce blood pressure, pharmacological agents that modulate the ras are more commonly chosen as the first drug or in combination therapy because of their efficacy and the lowest side effect profile among the antihypertensive agents. the development of angiotensin - converting enzyme (ace) inhibitors has represented a cornerstone for the treatment of various pathological cardiovascular conditions, including hypertension. however ace inhibitors only partially inhibit the formation of angiotensin ii (ang ii). from this point of view , angiotensin - receptor blockers (arbs) provide a more rational review tool to inhibit ras activity since they block selectivity the coupling of ang ii to type 1 angiotensin ii receptor (at1r). for this reason they have been developed and increasingly used in the clinical management of hypertension and cardiovascular diseases. among the compounds that antagonize the ras , arbs (which include several molecules such as losartan, candesartan cilexetil, valsartan, irbesartan, telmisartan, eprosartan and olmesartan medoxomil, and the newest azilsartan medoxomil) represent today the best - tolerated class among antihypertensive agents.8 the clinical efficacy of arbs has been established in hypertensive patients, particularly in terms of reduction in cardiovascular morbidity and mortality, prevention and regression of end organ damage and the slow progression of nephropathy. ras blockers provide independent actions on end - organ protection, beyond their blood pressure lowering effect.9 this has been shown with both ace inhibitors and arbs. a large body of evidence indicate that ace inhibitors are effective for the treatment of various pathological conditions including arterial hypertension, diabetes, and cardiovascular or cerebrovascular diseases.1013 however ace inhibitors antagonize the ras only in part. indeed, these drugs partially inhibit plasma ace and ang ii generation, even at high dosages.14 nevertheless, ang ii can be produced by alternative pathways (chymases, caspases, elastases) in the cardiovascular system.15,16 therefore, the selective blockers of the at1r, have been developed and progressively used in the clinical management of hypertension and cardiovascular diseases. moreover, some of the molecules may have ancillary effects such as the increased urinary uric acid excretion (losartan in particular) and may activate peroxisome proliferator - activated receptor (ppar)- which may contribute to a favorable metabolic profile. this latter effect has been described for the arbs telmisartan, irbesartan, candesartan, and losartan.17 randomized clinical trials have proven benefits for the therapy with arbs in primary and secondary prevention in several pathological conditions including hypertension, coronary artery disease, congestive heart failure, and renal disease. arbs have shown cardioprotective effects in patients with hypertension and additional risk factors. in the losartan intervention for endpoint reduction (life)18 the arb losartan was compared to atenolol in over 9000 patients with moderate - to - severe hypertension and left - ventricular hypertrophy. losartan improved the primary composite endpoint, which included cardiovascular mortality, stroke, and myocardial infarction, independently by blood pressure reduction. the losartan treatment was also associated with a 25% lower incidence of new - onset diabetes. this was observed also in the placebo - controlled kyoto heart study19 in which valsartan used as add - on therapy reduced the incidence of new onset of diabetes as well as cardiovascular outcomes in about 3000 hypertensive patients with additional risk factors. a recent meta - analysis has also proven the effectiveness of ras blockers in reducing the occurrence of new - onset diabetes. hence the ace inhibitors or arbs should be preferred in patients with clinical conditions that may increase the risk of developing diabetes.20 both hypertension and diabetes may induce renal damage in patients at risk for cardiovascular diseases. in turn, renal disease may increase the cardiovascular risk even at a preclinical stage (ie, presence of microalbuminuria). furthermore, diabetic nephropathy is responsible for the majority of end - stage renal disease in most countries. in particular, agents that block the ras can delay or prevent the diabetic nephropathy even at the initial stages as well as reduce the degree of albuminuria and the progression to advanced renal disease. in the randomised olmesartan and diabetes microalbuminuria prevention (roadmap) study, the arb olmesartan was associated with a delayed onset of microalbuminuria, although the drug did not reduce the cardiovascular complications associated with the diabetic state and the number of cardiovascular events.21 the arb irbesartan has been shown to delay the progression from microalbuminuria to overt proteinuria and to restore normoalbuminuria in a significant proportion of patients with hypertension and type 2 diabetes.22 moreover, irbesartan protected against advanced nephropathy in hypertensive patients with type 2 diabetes, independently by blood pressure control.23 ventricular dysfunction and heart failure may develop in the progression of the cardiovascular disease in high cardiovascular risk patients, which are generally older than the patients at the early stages of the cardiovascular continuum and have often several comorbidities.24 arbs were equal to ace inhibitors in reducing all - cause mortality in patients with left - ventricular dysfunction or heart failure postmyocardial infarction2426 as well as in chronic heart failure patients.27,28 in patients at higher cardiovascular risk, arbs have been shown to induce similar cardiovascular protective effects. telmisartan was proven to have cardioprotective effects in high cardiovascular risk patients who were intolerant to ace inhibitors.29 in over 25,000 patients with coronary, peripheral or cerebrovascular disease and diabetes with end organ damage the ongoing telmisartan alone and in combination with ramipril global endpoint trial (ontarget)30 has shown that telmisartan had similar effect to ramipril on the primary composite end point (including cardiovascular death, myocardial infarction, stroke or hospitalization for heart failure) and death from any cause. this was the first trial to compare an arb with the ace inhibitor ramipril, which has been proven to improve cardiovascular outcomes in the hope trial.31 in hypertensive patients with a history of a cerebral event within the previous 24 months eprosartan showed to be superior to the calcium - channel blocker nitrendipine for the secondary prevention of morbidity and mortality after stroke.32 however, valsartan was comparable to amlodipine on the primary composite end - point in the valsartan antihypertensive long - term use evaluation (value) trial which included over 15,000 patients with hypertension and additional risk factors (coronary heart disease, diabetes, high cholesterol).33 thus the clinical experience with arbs consistently indicates that this class of drugs represents an effective, safe and well tolerated therapeutic alternative for the prevention and care of cardiovascular disease, even though there is no proven superiority as compared to ace inhibitors except for the well documented better tolerability. caution should be used in considering arbs interchangeably, although a class effect can be advocated for the clinical effectiveness of arbs. therefore, in the clinical practice it is preferable to choose the arb that is proven effective, based on specific evidence derived from clinical studies. while arbs are effective in clinical practice and well tolerated, the extent to which they can reduce blood pressure is eventually considered insufficient and a combination therapy is required in a significant percentage of hypertensive patients.34 moreover most arbs may not completely inhibit the at1r at the approved clinical doses. it is a prodrug that is quickly hydrolyzed to the active moiety azilsartan, a potent and highly selective arb with estimated bioavailability of 60% and elimination half - life of 12 hours.35 findings from pharmacokinetic and dose - ranging studies have assessed that the effective therapeutic antihypertensive dosages of azilsartan medoxomil in humans vary from 40 to 80 mg once daily.35 experimental and clinical studies have shown that this new arb induces a potent and long - lasting antihypertensive effect. in conscious spontaneously hypertensive rats (shrs) and renal hypertensive dogs, azilsartan medoxomil induced more potent and longerlasting antihypertensive effects than olmesartan medoxomil, which is the newest to the market and has been reported to be most effective among arbs in terms of blood pressure reduction. the persistent durability of the antihypertensive effects of azilsartan medoxomil may reduce the variations in blood pressure during the day. this may contribute potential protective effects on cardiovascular consequences.36 clinical trials have compared azilsartan medoxomil with other arbs in the class, by studying patients with primary hypertension in randomized, double - blind, multicenter studies using ambulatory (abpm) and clinic blood pressure measurements (table 1). intraclass differences in blood pressure - lowering effects within the arb class can not be recognized using clinic blood pressure measurements and may be better assessed with abpm.37 moreover, mean 24-hour systolic blood pressure has been shown to correlate with cardiovascular morbidity in patients with hypertension.38 phase iii studies evaluated and compared the efficacy of 24-hour mean systolic blood pressure and the safety of azilsartan (80 mg once daily) with placebo and the maximal, approved doses of olmesartan medoxomil (40 mg once daily) and valsartan (320 mg once daily) in hypertensive patients (stage 12 of hypertension) using ambulatory and automatically measured clinic blood pressure monitoring.39 azilsartan medoxomil at a dose of 80 mg once daily for 6 weeks showed superior efficacy to the top approved doses for hypertension of both valsartan and olmesartan. moreover, also at the dosage of 40 mg azilsartan medoxomil lowered clinic systolic bp to a greater extent than the other arbs, suggesting that this novel agent has a greater potency than these other molecules at the dosages used in the study. moreover, azilsartan was well tolerated and there was no increase in adverse events during this short - term trial. across the effective dose range azilsartan showed superior efficacy compared to the arb valsartan at its maximal recommended dose40 without any significant increase in adverse events. in particular, the antihypertensive effects, safety and tolerability of azilsartan medoxomil were compared to those of valsartan in patients with stage 1 or 2 hypertension in a 24-week randomized, double - blind, parallel - group, multicenter trial.40 on the basis of either abpm or automatically measured clinic blood pressure measurements azilsartan medoxomil at a dose of 40 mg or 80 mg once daily showed greater efficacy (about 10% in absolute rate) than a 320 mg dose of valsartan, the highest approved dose for this drug.40 these findings suggest that azilsartan medoxomil can lower 24-hour blood pressure more effectively than maximally recommended doses of other arbs. this suggests that there may be a measurable hierarchal response in the arb class, as far as the blood pressure levels are considered. azilsartan medoxomil is expected to be able to control the blood pressure for a 24-hour period, which may contribute to the prevention of cardiovascular events. indeed, elevations in blood pressure around midnight and early morning are important predictors of central nervous system and cardiovascular outcomes in hypertensive patients.41,42 azilsartan medoxomil is highly potent in inhibiting the specific binding of 125i - sar1-ile8-ang ii to human at1r, and it is a slowly dissociating ang ii receptor blocker. indeed the inhibitory effect of azilsartan medoxomil persisted after washout of the free compound when compared to other arbs (including olmesartan, telmisartan, valsartan, and irbesartan) which presented attenuated inhibitory effects with washout. in this regard, the inhibitory effects of azilsartan on ang ii - induced contractile response persisted after washout in vascular strips and chinese hamster ovary (cho) cells which overexpress the human at1r.43 thus azilsartan medoxomil may prove to provide a more complete antagonism against endogenous ang ii. this may explain at least in part the greater blood pressure reduction associated with azilsartan. however, these in vitro determinations are yet to be supported in the whole animal or in human studies. hypertension is often associated with insulin resistance which predisposes to the development of metabolic syndrome and/or diabetes. blockade of ras / at1r signaling has been shown to improve the metabolic syndrome in clinical and experimental studies.44 some arbs including losartan, irbesartan, and telmisartan have been shown to improve insulin sensitivity in rodents and humans,45,46 suggesting the possible involvement of the excess of ang ii in the development of insulin resistance. olmesartan medoxomil produced dose - related improvements in the insulin sensitivity of shrs.36 candesartan cilexetil improved the insulin sensitivity of essential hypertensive patients.47 most recently, azilsartan medoxomil has been proven to improve insulin sensitivity in hypertensive rats.36 interestingly, it has been shown that azilsartan medoxomil is more effective than candesartan in reducing plasma concentrations of glucose and fatty acids in normotensive mice. furthermore this novel arb decreases adipose tissue weight and adipocyte size and increases adipose expression of ppar- and its target gene adiponectin, independently of its effects on blood pressure and plasma insulin concentrations.48 it has also been shown that azilsartan medoxomil induces insulinsensitizing effects in obese koletsky rats, independently of decreases in food intake and body weight increase or of the activation of adipose ppar-, the master regulator of adipogenesis. 49 in particular azilsartan treatment decreased the hyperinsulinemia, improved the homeostasis model assessment (homa - ir) index and suppressed the over - increase in plasma glucose and insulin concentrations during oral glucose tolerance tests in obese koletsky rats. in the same rat model, it reduced the basal plasma concentrations of glucose, triglyceride, and nonesterified fatty acids (nefa). it has also been reported that azilsartan medoxomil improved insulin sensitivity in shrs and reduced urinary protein excretion more potently than olmesartan medoxomil.37 taken together, this evidence suggests the possible usefulness of azilsartan in the treatment of insulin resistance / metabolic syndrome, and its potential contribution to reduce the cardiovascular risk linked to glucose and lipid metabolism abnormalities in high risk individuals. indeed, azilsartan medoxomil modulates other metabolic functions which can be involved in the atherosclerotic process. in cultured preadipocytes, azilsartan enhanced adipogenesis and induced the expression of adipokines, including leptin, adipsin, and adiponectin, and enhanced the expression of ppar- and -, at a greater extent than valsartan.50 hypertensive and/or diabetic patients often present microalbuminuria or overt proteinuria which are considered major risk factors for progression to end - stage renal disease and the development of cardiovascular disease.51 reduction and normalization of proteinuria by drug treatment including the arbs is associated with decreased risk for adverse renal outcomes,52 as previously discussed. evidence from experimental studies suggest that similarly to other arbs, azilsartan medoxomil may induce urinary albumin and protein excretion levels. this may possibly occur through the activation of several candidate mechanisms, including normalization of glomerular capillary pressure, inhibition of podocyte injury, inhibition of the proliferation of mesangial cells and inhibition epithelial although the exact mechanisms are unknown.36 azilsartan medoxomil may modulate the cell growth, as it is indicated by the observation that it blocked the ang ii -induced activation of mitogen - activated protein kinases in vascular smooth muscle cells. furthermore azilsartan medoxomil is a potent inhibitor of vascular cell proliferation even at low dosages. this effect is also evident in cells lacking at1r.50 increased expression of plasminogen activator inhibitor type - i (pai-1) in the vessel wall seems to accelerate atherosclerosis and it is involved in increasing the instability of atherosclerotic plaque.53 azilsartan medoxomil reduced the expression of pai-1 in the aortic wall of transgenic mice which overexpressed pai-1 in vsmcs and were prone to atherosclerosis secondary to genetically determined apoe deficiency.54 this was associated with a more stable atherosclerotic plaque. in the same mouse model azilsartan medoxomil reduced the pai-1 levels also in the heart. therefore it may contribute to reduce this profibrotic factor that is associated with the negative left - ventricular remodeling and the development of heart failure after myocardial infarction.55 these evidence suggest that azilsartan could exert pleiotropic cardioprotective effects beyond the expected beneficial effects of the potent and sustained blood pressure - lowering action. azilsartan medoxomil is a new compound proposed for the treatment of stage 12 hypertension, with its potent blood pressure - lowering ability associated with considerably better rates of hypertension control compared with other antihypertensive drugs including arbs at standard doses. in fact even a reduction of systolic blood pressure of 2 mmhg to 3 mmhg or more is associated with greater cardiovascular risk reduction as supported by in epidemiologic reports and interventional trials.56 the pharmacodynamic and pharmacokinetic properties suggest that azilsartan medoxomil should be used as an alternative agent for mild - to - moderate hypertension, particularly when other antihypertensive agents are not well tolerated or as an adjunctive drug in hypertensive patients not controlled with other antihypertensive agents. evidence from experimental studies suggests that azilsartan medoxomil may have cardioprotective properties, through a number of other actions which are independent of effects on blood pressure. at present , there are no data available on the effects of azilsartan on cardiovascular morbidity and mortality as well as on key intermediate endpoints or disease markers.	renin angiotensin system (ras) activation plays a key role in the development of hypertension and cardiovascular disease. drugs that antagonize the ras (angiotensin - converting enzyme inhibitors and angiotensin receptor blockers) have proven clinical efficacy in reducing blood pressure values and cardiovascular morbidity and mortality. ace inhibitors partially inhibit plasma ace, and angiotensin ii generation. thus, arbs, which block selectively type 1 angiotensin ii receptor (at1r), have been developed and used in the clinical management of hypertension and cardiovascular disease. experimental and clinical trials with arbs indicate that this class of drug represents an effective, safe and well tolerated therapeutic option for the prevention and care of hypertension, even though there is no proven superiority as compared to ace inhibitors except for the better tolerability. most arbs may not completely inhibit the at1r at the approved clinical doses. azilsartan medoxomil is a newly approved arb for the management of hypertension. this arb induces a potent and long - lasting antihypertensive effect and may have cardioprotective properties. this article reviews the current evidence on the clinical effectiveness of azilsartan in hypertension.
fetal echocardiography performed at 28 weeks of gestational age in a full - term 2,780-g male neonate showed a double - outlet right ventricle (dorv). the patient was born through a cesarean section with apgar scores of 7 and 9 at 1 and 5 minutes, respectively. the heart rate was 170 beats / min, and the respiratory rate was 54 beats / min. the blood pressure was 71/42 and 43/21 mmhg in the right and the left upper extremities, respectively. the left radial pulse was weak, but the right radial and femoral pulses were normal. two - dimensional echocardiography demonstrated the right aortic arch, anterior malalignment ventricular septal defect, overriding aorta (60%), and pulmonary and infundibular stenosis. patent ductus arteriosus (pda) arose from a tortuous abnormal artery with bidirectional shunting. the left subclavian artery (lsca) was not seen arising from the aorta. however, three - dimensional 64-row multidetector computed tomography (mdct) showed the lsca arising from the left pulmonary artery via ductus arteriosus (fig . associated non - cardiac anomalies were also determined and included inguinal hernia, polydactyly, and syndactyly . the fluorescent in situ hybridization test was positive for the digeorge syndrome ( 22q11 deletion). the patient's age at the time of surgery was 28 days, and his weight was 3.8 kg. the aberrant lsca was divided, and its pulmonary stump was oversewn. after appropriate trimming , the lsca was directly reimplanted into the left common carotid artery (lcca) with 8 - 0 polypropylene sutures (surgipro ; tyco healthcare ussc, norwalk, ct, usa). the left cerebral oxygen saturation was maintained above 80% of the baseline value during the clamping of the lcca. fifteen months later, the patient underwent intraventricular tunnel repair of the dorv at 16 months of age. three - dimensional 64-row mdct at the time revealed the lsca arising from the lcca without stenosis (fig . the patient is now 18 months old and has normal left arm function and growth . the right aortic arch with isolation of the lsca is an uncommon arch anomaly in which the lsca arises exclusively from the pulmonary artery via ductus arteriosus ( da) or ligamentum arteriosum without communication with the aorta. the development of the aortic arch and its branches takes place during the third week of gestation. a common arterial trunk arises from the primitive heart and divides into six paired aortic arches that fuse and form bilateral dorsal aorta which, in turn, fuse caudally into the descending aorta. the persistence or regression of these arches may lead to various arch anomalies. edwards' embryologic model of aortic arch malformation explains this lsca isolation by the interruption of the left aortic arch at two locations: 1 ) between the left carotid and subclavian arteries, and 2 ) between the left ductus arteriosus and the left dorsal aortic root. the isolation of the lsca is commonly associated with congenital heart disease (chd) and 22q11 deletion. luetmer et al. reported associated chd in 23 of the 39 cases (59%) of isolated lsca. further, the tetralogy of fallot was the most common (14 of the 23 cases). double - outlet right ventricle and d - transposition of the great artery have also been reported in a few cases. the clinical presentations of patients with isolated lsca depend on the patency of the da. the isolation of the lsca is usually asymptomatic and is usually discovered during the evaluations of the associated cardiac anomalies or when reduced blood pressure is detected in the left arm. in these patients, if the direction of blood flow is from the left vertebrobasilar artery to the pulmonary artery (pulmonary steal syndrome) or the subclavian artery (subclavian steal syndrome), the patient has vertebrobasilar insufficiency. symptoms may include the disturbance of vision, faintness, syncope, and headache that can be exacerbated by exercising the left upper limb, ing in increased left arm circulation. ischemic symptoms of the left arm, including pain, weakness, coldness, and a reduced limb length, may be present. in a review of the 39 cases described by luetmer and miller , we found that 5 patients had ischemic symptoms of the left arm and 5 had vertebrobasilar insufficiency. they reported that the ages of the symptomatic patients ranged from 22 to 53 years, and the symptom duration before diagnosis ranged from less than 1 year to 11 years. the authors suggested that initially asymptomatic patients may lose the ability to compensate for the steal phenomenon and that eventually symptoms may develop with age. simple ligation of the lsca, surgical reimplantation, device occlusion of the pda, and follow - up have been described as therapeutic options. however, persistent isolated lsca, which was not surgically reimplanted into the aorta, provides an anatomic substrate for a subclavian steal syndrome. although this anomaly may be clinically asymptomatic, we recommend surgical correction before significant vertebrobasilar insufficiency or arm ischemia occurs. in our case, the lsca was directly anastomosed with the lcca to prevent symptomatic subclavian or pulmonary steal. we think that very low operative mortality rates and high success rates justify a surgical approach in patients with isolated lsca. further, surgical reimplantation provides complete abolition of the anatomic substrate for the subclavian steal syndrome.	anomalous aortic origin of the left subclavian artery (lsca) from the left pulmonary artery (lpa) is a rare congenital cardiac malformation. we describe a case of lsca from the lpa via ductus arteriosus in association with a double - outlet right ventricle, which never has been reported previously in korea.
sturge weber syndrome, an encephalo - trigeminal angiomatosis is a rare, congenital neurocutaneous syndrome characterized by unilateral facial cutaneous vascular malformation (port wine stain) associated with ipsilateral leptomeningeal angiomatosis involving parietal or occipital regions. it presents with focal seizures and recurrent episodes of transient hemiparesis either due to acute vascular events or of postictal in origin. we here report a 25-month - old male child born by full - term vaginal delivery presented to us with sudden onset of left - sided hemiparesis lasting 2 hours, not preceded by constitutional symptoms or seizure activity. he had similar episodes of transient hemiparesis on either side in past which relieved spontaneously without any neurological deficit. frequency of seizures increased and child was started on antiepileptic drugs. since age of 11 months, child had recurrent episodes of transient hemiparesis on either side with or without associated seizures, lasting 2 - 3 days and relieved by itself. there was history of developmental delay noticed after the age of 4 months and presently he could only walk with support corresponding to developmental age of 11 months. on examination he was afebrile with normal vitals. there was port - wine stain of 4 1 cm in an atypical site involving midline of forehead extending over nose. his modified gcs was 13/15, had umn type left facial nerve palsy with left - sided hemiparesis. contrast ct scan of brain showed linear hyperdense area in right high parietal lobe with gyriform enhancement along with enlargement of right choroid plexus and left frontal atrophy. clinical photograph of patient showing port - wine stain of 4 1 cm in an atypical site involving midline of forehead extending over face axial contrast ct brain showed linear hyperdense area in right high parietal lobe with gyriform enhancement along with enlargement of right choroid plexus and left frontal atrophy coronal contrast ct brain showed linear hyperdense area in right high parietal lobe with gyriform enhancement along with enlargement of right choroid plexus and left frontal atrophy eeg done urgently revealed no ictal activity, excluding postictal hemiparesis. child was started on aspirin to prevent further recurrences of these thrombotic events. on 3 day of admission , child developed right - sided tonic clonic seizures with facial twitching lasting 15 minutes, and loss of consciousness followed by right - sided hemiparesis. a concurrent eeg revealed epileptic activity with focus on left side, suggesting hemiparesis of postictal origin. child remained seizure - free thereafter until discharge and his residual deficits resolved within 24 hours. on 6 day of admission, child was discharged on anticonvulsants and aspirin prophylaxis. on follow - up up to 1 year features suggestive of sturge weber syndrome in this child were facial port - wine stain, focal seizures, hemiparesis or paroxysmal vascular events and neuroimaging findings. sws is a phakomatosis characterized by port - wine stain over trigeminal nerve area, leptomeningeal angiomatosis and ocular abnormalities caused by abnormal persistence of embryonal vascular system, which is localized around the cephalic portion of neural tube. normally this vascular plexus forms in sixth week and regresses around ninth week of gestation. failure of normal regression in residual vascular tissue, which forms angiomata of face, ipsilateral eye and leptomeninges. neurological dysfunction from secondary effects on surrounding tissue, including hypoxia, ischemia, venous occlusion, thrombosis, infarction and vasomotor phenomenon. type i (classic) is the common form with both facial and leptomeningeal angiomas and glaucoma may be present, type ii with facial angioma and glaucoma but without evidence of intracranial disease and type iii with leptomeningeal angiomas without facial nevus with usually no ocular manifestation. and gomez reported that venous occlusion might actually cause the initial neurological event, either a seizure, transient hemiparesis or both, thereby beginning the process of vascular steal phenomenon developing around the angioma, ing in cortical ischemia. recurrent seizures, status epilepticus, intractable seizures and recurrent vascular events may aggravate this steal further with increase in cortical ischemia, ing in progressive calcification, gliosis and atrophy, which in turn increase the chance of seizures and neurological deterioration. neurological outcome in sws ranges from minimal or no neurological signs to devastating impairment with uncontrolled seizures, hemiparesis, visual field defects and progressive mental retardation. seizure is a common feature, often occurs during first year of life and from cortical irritability caused by angioma through the mechanism of hypoxia, ischemia and gliosis. about 80% of affected persons have focal seizures involving the contralateral side of the port wine stain. developmental delay and mental retardation as postictal event may also present as hemiparesis, it is important to differentiate the two. pet and spect scan can also guide to differentiate both, which can reveal decreased perfusion and metabolism during vascular event. calcification is unusual before 2 years of age, usually involves high parietal or occipital lobes. mri demonstrates thickened cortex, decreased convolutions, abnormal white matter and gadolinium enhancement of leptomeningeal angioma. aspirin prophylaxis reduces these vascular phenomenon, thereby such episodes of transient hemiparesis and overall neurological deterioration. any child with a facial nevus and hemiparesis with or without seizures and/or glaucoma should arouse a suspicion of sws.	sturge weber syndrome (sws) is a rare, sporadically occurring neurocutaneous disorder with a frequency of approximately 1 per 50,000. the hallmark is an intracranial leptomeningeal vascular angioma in association with a port wine nevus, usually involving ophthalmic or maxillary distribution of trigeminal nerve. other clinical findings associated with sws are seizures, glaucoma, hemiparesis and mental retardation. the radiological hallmark is tram - line or gyri - form calcification. 25 to 56% of patients experience recurrent episodes of paroxysmal focal neurological deficits in form of transient hemiparesis, which may be due to vascular ischemia or postictal in origin. eeg helps to differentiate the exact etiology, as it is normal in former. aspirin prophylaxis in those, due to ischemia decreases their recurrences and improves overall neurological prognosis. we report a 25-month - old child of sws with recurrent episodes of transient hemiparesis and atypical midline location of facial vascular nevus.
since the discovery of the human immunodeficiency virus-1 (hiv-1) in 1983 and the beginning of the battle against acquired immunodeficiency syndrome, the development of antiretroviral medication has succeeded substantially. the development and use of antiretroviral therapy (arv) started in the mid to end of the 1980s with the first class of substances, the nucleoside reverse transcriptase inhibitors (nrtis).1 in the following years, additional substances and classes have been introduced into clinical practice. until now , 5 classes of arv with more than 20 licensed drugs have been established for therapeutic use. most of the proven substances inhibit the reverse transcriptase (rt) either as nucleoside antagonists or as non - nrtis; others work as protease inhibitors (pis) or integrase inhibitors by modifying the viral enzymatic activity. however, these substances are underlying a risk to select resistant viral mutants due to the variability of the virus under pharmacological pressure. the only antiretroviral class not interfering with the enzymes of the virus is the group of entry inhibitors. different substances, each with a specific mode of action, are represented within this class. the potency of this fusion inhibitor is based on its ability to block the conformational change of gp41 and to avert the approach of virus and cell membrane.2 despite the proven efficacy, enfuvirtide was not established as a routinely used drug because of its application mode as subcutaneous injections twice daily and the ant side effects predominantly present as cutaneous irritations and painful indurations. lacking the disadvantages of enfuvirtide, maraviroc was the first, and is still the only, approved entry inhibitor in oral formulation that showed potency and sustained efficacy to suppress hiv-1 viral load. the mechanism of action of maraviroc is based on the need of hiv-1 to use a main receptor (cd4) and a coreceptor (ccr5 or cxcr4) on the surface of the target cells. this is followed by a conformational change of gp120 and the coreceptor binding, which allows for the next step, the fusion of virus and cell membrane mediated by gp41.3,4 in 1996, huang et al5 demonstrated that a homozygous, 32-base pair mutation in the gene for ccr5 protects against hiv infection by coding for a dysfunctional protein, which is not expressed on the cell surface. the later genetic studies demonstrated relatively high rates of 15%20% of heterozygous 32 mutations in caucasian populations, but rare cases in other populations. in 1996, liu et al6 estimated that approximately 1% of the caucasian population appeared to be homozygous for this 32 mutation. in fact, some of these individuals remained hiv - negative despite repeated exposure to hiv. based on this knowledge, it was found that maraviroc inhibits the attachment of hiv to its target cell via an allosteric modification of the ccr5 on the surface of the cd4 cells. it works as a small molecular ccr5 inhibitor through a binding in a cavity in the transmembrane ccr5 receptor. this fixation of maraviroc changes the geometry of the transmembrane protein, which is originally needed for the binding of gp120 and ccr5.710 although hiv-1 predominantly uses the ccr5 coreceptor for cell infection, it is not the only possibility to operate. some viral strains have the ability to use a second coreceptor called cxcr4 to infect the target cells, and some viruses may use both ccr5 and cxcr4 receptors. epidemiological studies revealed that hiv-1 strains in arv - naive patients predominantly exhibit ccr5 tropism (r5 viral variants).1113 more than 80% of treatment - naive hiv-1-infected individuals carry r5 viruses, whereas approximately 20% show dual - tropic or mixed - tropic viruses (r5/x4 variants). only a minority (< 1%) of viral strains are capable of using cxcr4 coreceptors exclusively (x4 variants). in treatment - experienced populations, r5 variants still account for 48%62% of isolates and r5/x4 variants are found in 34%50% of these patients, whereas viruses that exclusively use the cxcr4 coreceptors for cell entry are seen in only 2%4% of the pretreated population.1416 the clinical relevance of ccr5 receptor variants, especially the homozygous one, is still unclear. most recent studies suggest an association of 32 mutation and elevated mortality in cases of west nile virus infections.17,18 in contrast, earlier studies suggested protection against chronic hepatitis b infection and rheumatoid arthritis or prolonged survival of renal transplant individuals.1921 additionally, an evaluation date back to the end of the last millennium discussed prevention of yersinia infections (bubonic plague).22 in fact, many issues have been discussed but a final of the individual relevance of the ccr5 has not been established. as maraviroc can not prevent cell infection in x4 or r5/x4 variants, it is necessary to detect viral tropism carefully in each individual before using it for arv. as there are different methods to determine the tropism of the virus, it is still unclear which test is the most appropriate for routine clinical use. phenotypic assays are based on the transfection of the virus into the cell culture, to mark the cell with a luciferase reporter gene, and on the determination of lyzed ccr5 or cxcr4 positive cell, in which a single viral cycle has been completed. finally, the luciferase activity is measured by relative light units.23,24 despite being considered to be the gold standard for the measurement of hiv-1 coreceptor usage, the phenotypic method has some disadvantages that should be kept in mind: the method is technically complex, expensive, and laborious. in fact, performance of a phenotypic tropism test takes up to 4 weeks from blood drawing and costs between $ 750 and $ 1000. in contrast, genotypic assays may be performed within some days, and the cost is restricted to the performance of a gene amplification, mostly of the v3 region. subsequently, the amplification products are analyzed in a sequencer, and the generated sequences are correlated with several standard sequences. for interpretation, some of the algorithm systems predicting coreceptor usage are available online. these systems, such as webcat, webpssm, and geno2pheno, are accessible via the internet. established laboratories use these systems to determine the coreceptor usage. a restriction of these systems is the relatively low sensitivity and specificity, but combining 2 or 3 systems enhances the recall ratio.2527 because of the importance of coreceptors for viral entry and the knowledge of research in other entry inhibitors, the pfizer global research and development discovered maraviroc (uk-427,857) as a highly promising substance to block the ccr5 receptor effectively. clinical studies started with dose - finding and safety issues as short - term monotherapy administration in treatment - naive patients and in antiretroviral treatment (art)-experienced patients, who had to be on treatment for at least 8 weeks (a4001007 and a4001015). participation was restricted to individuals with confirmed r5 viral variants, viral load > 5,000 hiv-1 copies / ml, and moderate immunodeficiency with still more than 250 cd4 cells / mm. of these trials demonstrated efficacy of maraviroc in both naive and experienced patients with r5 but not with x4 viruses.28 following trials (motivate-1 and motivate-2) evaluated the efficacy of maraviroc in treatment - experienced patients harboring r5-tropic variants. both trials were double - blind, placebo - controlled, multicenter phase 2b/3 studies, investigating maraviroc plus optimized therapy (obt) vs placebo plus obt in viremic patients (viral load > 5000 copies / ml) carrying ccr5-tropic virus. motivate-1 was conducted in the united states and canada, whereas the identically designed motivate-2 trial enrolled patients in europe, australia, and north america. the primary end point of both studies was viral load change in hiv-1 rna from baseline to week 48. according to the inclusion criteria, all patients had to be triple - class experienced. the showed a significant advantage for patients in the maraviroc groups: viral load declined by 1.66 log10 copies / ml and 1.82 log10 copies / ml for once - daily and twice - daily administration of drug, respectively, whereas in the placebo arm, it declined only by 0.80 log10 copies / ml in motivate-1. nearly the same could be found in motivate-2: 1.72 and 1.87 log10 reduction vs 0.76 log10 reduction, respectively. the study data also showed a superior response of cd4 cells in the treatment arms: the mean increase of cd4 cells in motivate-1 was 113 and 122 cells/l for once - daily and twice - daily administration, respectively, vs 54 cells/l in the placebo arm. in motivate-2, the cd4 cells increased by 122 and 128 cells/l for once - daily and twice - daily administration, respectively, (verum) and 69 cells/l (placebo). the pooled safety analysis of both studies demonstrated no statistical significant differences in treatment - related adverse events, which indicates a good compatibility of maraviroc.2931 the of the motivate studies led to the approval of maraviroc, with the restriction for use in pretreated patients harboring only r5-tropic viral strains. a further study called merit compared maraviroc plus zidovudine / lamivudine with efavirenz plus the same backbone in arv - naive patients. the original study missed its goal to demonstrate noninferiority of maraviroc correlated to efavirenz according to the rate of viral load < 50 copies/l after 48 weeks. however, it demonstrated the very good compatibility of the ccr5-inhibitor: patients following the efavirenz - containing regimen discontinued their therapy thrice, more often than maraviroc recipients did. later analyses verified that the original goal of noninferiority was missed because of the lacking sensitivity of the primarily used test to detect viral tropism. a retrospective analysis, with an exclusion of the additionally found x4-tropic virus in patients, by using an advanced version of the tropism test demonstrated the expected noninferiority of maraviroc. additionally, patients in the maraviroc arm demonstrated a significantly higher cd4 cell increase compared with the efavirenz arm. nonetheless, one of the maraviroc arms was conducted as once - daily administration of study drug. this arm was discontinued prematurely due to a lack of efficacy.32 a compilation of maraviroc - associated data of the motivate and merit trials is shown in table 1. one of the most problematic issues in battling hiv is the development of resistances to antiretroviral drugs. regularly, selection of resistant mutants develops under pharmaceutical pressure, while viral replication occurs due to the irregular intake of medication and/or suboptimal drug levels. in the rt, the protease, and the integrase, occurrence of mutations may be strongly associated with the drug used: eg, lamivudine regularly causes a m184 v mutation in the rt gene locus, which is associated with a resistance to lamivudine but reduces viral fitness significantly.33 on the other hand, some protease - associated mutations, such as l90 m, may be initiated by 1 drug (eg, saquinavir), but may have impact on other drugs of the same class (eg, atazanavir and nelfinavir).34 these mechanisms of direct or cross - resistance are characteristic for drugs interfering with viral enzymes. in ccr5 inhibitors, there are more escape mechanisms for the virus; primarily, the use of the above - mentioned coreceptor may change: a r5-using virus might be successfully defeated but the virus may switch to use cxcr4 as coreceptor. in an in vitro study recently conducted, it has been shown that the presence of maraviroc in a cell culture does not lead to a switch of r5 viral strains to x4 variants. thus, it appears not to be common for a virus to switch from its origin to another tropism, it seems to be rather unusual. therefore, another possibility appears to be more reliable with respect to the development of resistance: a preexisting x4 virus minority may get selected due to the r5 suppression, and as may lead to resistance against ccr5 inhibitors. however, there are also other mechanisms forcing resistance to maraviroc: as maraviroc directly interacts with the human ccr5, the viral correlate, the env gene with its gp120 and gp41, may mutate. studies showed the changes of the v3 loop of gp120 lead to resistance, but mutations have also been reported in other regions of gp120 and gp41 due to the enormous variability of the env gene. additionally, the development of resistance in the presence of ccr5 inhibitors does not select always the same mutations.3539 consequently, the prediction of resistance in ccr5 inhibitors may be more complicated and more difficult to interpret than it is with the rt or pis. a third possibility for the virus to attain infectiveness of its combatant maraviroc or other ccr5 inhibitors is the ability to bind to the coreceptor despite specific drug is bound, whether through a competitive replacement due to higher affinity than the drug or because of the possibility to bind despite a drug - bound receptor.40,41 taking the above - mentioned facts into account, the consequences of therapeutic use still remain elusive. nearly, 80% of all antiretroviral naive patients harbor r5-tropic virus, and therefore, would be eligible for using this drug. however, until 2009, the worldwide approvals for maraviroc indicate its use only for pretreated patients with r5-tropic hiv-1, partly with this requirement alone or partly with the necessity of resistances to other antiretroviral drug. since 2009, maraviroc is licensed in the united states under a statement, for combination antiretroviral treatment of adults infected with only ccr5-tropic hiv-1 (us food and drug administration approval november 20, 2009), which means that it can be used also in naive patients. however, this statement is directly followed by a special note: more subjects treated with selzentry experienced virologic failure and developed lamivudine resistance compared to efavirenz , which puts the indication into perspective. hence, the use of maraviroc in treatment - naive individuals would be according to the applicable label, but may induce a higher risk for the patients to develop virological failure and acquiring resistances. therefore, maraviroc should be the first choice in treatment - naive patients only under special conditions and thorough surveillance. in treatment - experienced subjects, the motivate studies demonstrated a benefit of maraviroc compared with the placebo arms in virological and immunological responses. however, 955 individuals (29.4%) of 3,244 screened patients were directly excluded from the studies due to the occurrence of x4 or dual / mixed tropism. overall, in only 61% of the initially tested patients, an r5 tropism could be clearly demonstrated.29 these were generated with a later revised phenotypic assay (trofile, monogram bioscience, south san fransico, california, usa). unfortunately, the possible subsequently generated data obtained using the enhanced trofile assay, which is more sensitive, are not applicable. nonetheless, the virological activity of maraviroc and the good compatibility could be demonstrated in the studies and in clinical practice. however, before starting arv, it needs to be demonstrated that the patient is harboring r5-tropic virus, exclusively. the mechanism of action of maraviroc is based on the need of hiv-1 to use a main receptor (cd4) and a coreceptor (ccr5 or cxcr4) on the surface of the target cells. this is followed by a conformational change of gp120 and the coreceptor binding, which allows for the next step, the fusion of virus and cell membrane mediated by gp41.3,4 in 1996, huang et al5 demonstrated that a homozygous, 32-base pair mutation in the gene for ccr5 protects against hiv infection by coding for a dysfunctional protein, which is not expressed on the cell surface. the later genetic studies demonstrated relatively high rates of 15%20% of heterozygous 32 mutations in caucasian populations, but rare cases in other populations. in 1996, liu et al6 estimated that approximately 1% of the caucasian population appeared to be homozygous for this 32 mutation. in fact, some of these individuals remained hiv - negative despite repeated exposure to hiv. based on this knowledge, it was found that maraviroc inhibits the attachment of hiv to its target cell via an allosteric modification of the ccr5 on the surface of the cd4 cells. it works as a small molecular ccr5 inhibitor through a binding in a cavity in the transmembrane ccr5 receptor. this fixation of maraviroc changes the geometry of the transmembrane protein, which is originally needed for the binding of gp120 and ccr5.710 although hiv-1 predominantly uses the ccr5 coreceptor for cell infection, it is not the only possibility to operate. some viral strains have the ability to use a second coreceptor called cxcr4 to infect the target cells, and some viruses may use both ccr5 and cxcr4 receptors. epidemiological studies revealed that hiv-1 strains in arv - naive patients predominantly exhibit ccr5 tropism (r5 viral variants).1113 more than 80% of treatment - naive hiv-1-infected individuals carry r5 viruses, whereas approximately 20% show dual - tropic or mixed - tropic viruses (r5/x4 variants). only a minority (< 1%) of viral strains are capable of using cxcr4 coreceptors exclusively (x4 variants). in treatment - experienced populations, r5 variants still account for 48%62% of isolates and r5/x4 variants are found in 34%50% of these patients, whereas viruses that exclusively use the cxcr4 coreceptors for cell entry are seen in only 2%4% of the pretreated population.1416 the clinical relevance of ccr5 receptor variants, especially the homozygous one, is still unclear. most recent studies suggest an association of 32 mutation and elevated mortality in cases of west nile virus infections.17,18 in contrast, earlier studies suggested protection against chronic hepatitis b infection and rheumatoid arthritis or prolonged survival of renal transplant individuals.1921 additionally, an evaluation date back to the end of the last millennium discussed prevention of yersinia infections (bubonic plague).22 in fact, many issues have been discussed but a final of the individual relevance of the ccr5 has not been established. as maraviroc can not prevent cell infection in x4 or r5/x4 variants, it is necessary to detect viral tropism carefully in each individual before using it for arv. as there are different methods to determine the tropism of the virus, it is still unclear which test is the most appropriate for routine clinical use. phenotypic assays are based on the transfection of the virus into the cell culture, to mark the cell with a luciferase reporter gene, and on the determination of lyzed ccr5 or cxcr4 positive cell, in which a single viral cycle has been completed. finally, the luciferase activity is measured by relative light units.23,24 despite being considered to be the gold standard for the measurement of hiv-1 coreceptor usage, the phenotypic method has some disadvantages that should be kept in mind: the method is technically complex, expensive, and laborious. in fact, performance of a phenotypic tropism test takes up to 4 weeks from blood drawing and costs between $ 750 and $ 1000. in contrast, genotypic assays may be performed within some days, and the cost is restricted to the performance of a gene amplification, mostly of the v3 region. subsequently, the amplification products are analyzed in a sequencer, and the generated sequences are correlated with several standard sequences. for interpretation, some of the algorithm systems predicting coreceptor usage are available online. these systems, such as webcat, webpssm, and geno2pheno, are accessible via the internet. established laboratories use these systems to determine the coreceptor usage. a restriction of these systems is the relatively low sensitivity and specificity, but combining 2 or 3 systems enhances the recall ratio.2527 because of the importance of coreceptors for viral entry and the knowledge of research in other entry inhibitors, the pfizer global research and development discovered maraviroc (uk-427,857) as a highly promising substance to block the ccr5 receptor effectively. clinical studies started with dose - finding and safety issues as short - term monotherapy administration in treatment - naive patients and in antiretroviral treatment (art)-experienced patients, who had to be on treatment for at least 8 weeks (a4001007 and a4001015). participation was restricted to individuals with confirmed r5 viral variants, viral load > 5,000 hiv-1 copies / ml, and moderate immunodeficiency with still more than 250 cd4 cells / mm. of these trials demonstrated efficacy of maraviroc in both naive and experienced patients with r5 but not with x4 viruses.28 following trials (motivate-1 and motivate-2) evaluated the efficacy of maraviroc in treatment - experienced patients harboring r5-tropic variants. both trials were double - blind, placebo - controlled, multicenter phase 2b/3 studies, investigating maraviroc plus optimized therapy (obt) vs placebo plus obt in viremic patients (viral load > 5000 copies / ml) carrying ccr5-tropic virus. motivate-1 was conducted in the united states and canada, whereas the identically designed motivate-2 trial enrolled patients in europe, australia, and north america. the primary end point of both studies was viral load change in hiv-1 rna from baseline to week 48. according to the inclusion criteria, all patients had to be triple - class experienced. the showed a significant advantage for patients in the maraviroc groups: viral load declined by 1.66 log10 copies / ml and 1.82 log10 copies / ml for once - daily and twice - daily administration of drug, respectively, whereas in the placebo arm, it declined only by 0.80 log10 copies / ml in motivate-1. nearly the same could be found in motivate-2: 1.72 and 1.87 log10 reduction vs 0.76 log10 reduction, respectively. the study data also showed a superior response of cd4 cells in the treatment arms: the mean increase of cd4 cells in motivate-1 was 113 and 122 cells/l for once - daily and twice - daily administration, respectively, vs 54 cells/l in the placebo arm. in motivate-2, the cd4 cells increased by 122 and 128 cells/l for once - daily and twice - daily administration, respectively, (verum) and 69 cells/l (placebo). the pooled safety analysis of both studies demonstrated no statistical significant differences in treatment - related adverse events, which indicates a good compatibility of maraviroc.2931 the of the motivate studies led to the approval of maraviroc, with the restriction for use in pretreated patients harboring only r5-tropic viral strains. a further study called merit compared maraviroc plus zidovudine / lamivudine with efavirenz plus the same backbone in arv - naive patients. the original study missed its goal to demonstrate noninferiority of maraviroc correlated to efavirenz according to the rate of viral load < 50 copies/l after 48 weeks. however, it demonstrated the very good compatibility of the ccr5-inhibitor: patients following the efavirenz - containing regimen discontinued their therapy thrice, more often than maraviroc recipients did. later analyses verified that the original goal of noninferiority was missed because of the lacking sensitivity of the primarily used test to detect viral tropism. a retrospective analysis, with an exclusion of the additionally found x4-tropic virus in patients, by using an advanced version of the tropism test demonstrated the expected noninferiority of maraviroc. additionally, patients in the maraviroc arm demonstrated a significantly higher cd4 cell increase compared with the efavirenz arm. nonetheless, one of the maraviroc arms was conducted as once - daily administration of study drug. this arm was discontinued prematurely due to a lack of efficacy.32 a compilation of maraviroc - associated data of the motivate and merit trials is shown in table 1. one of the most problematic issues in battling hiv is the development of resistances to antiretroviral drugs. regularly, selection of resistant mutants develops under pharmaceutical pressure, while viral replication occurs due to the irregular intake of medication and/or suboptimal drug levels. in the rt, the protease, and the integrase, occurrence of mutations may be strongly associated with the drug used: eg, lamivudine regularly causes a m184 v mutation in the rt gene locus, which is associated with a resistance to lamivudine but reduces viral fitness significantly.33 on the other hand, some protease - associated mutations, such as l90 m, may be initiated by 1 drug (eg, saquinavir), but may have impact on other drugs of the same class (eg, atazanavir and nelfinavir).34 these mechanisms of direct or cross - resistance are characteristic for drugs interfering with viral enzymes. in ccr5 inhibitors, there are more escape mechanisms for the virus; primarily, the use of the above - mentioned coreceptor may change: a r5-using virus might be successfully defeated but the virus may switch to use cxcr4 as coreceptor. in an in vitro study recently conducted , it has been shown that the presence of maraviroc in a cell culture does not lead to a switch of r5 viral strains to x4 variants. thus, it appears not to be common for a virus to switch from its origin to another tropism, it seems to be rather unusual. therefore, another possibility appears to be more reliable with respect to the development of resistance: a preexisting x4 virus minority may get selected due to the r5 suppression, and as may lead to resistance against ccr5 inhibitors. however, there are also other mechanisms forcing resistance to maraviroc: as maraviroc directly interacts with the human ccr5, the viral correlate, the env gene with its gp120 and gp41, may mutate. studies showed the changes of the v3 loop of gp120 lead to resistance, but mutations have also been reported in other regions of gp120 and gp41 due to the enormous variability of the env gene. additionally, the development of resistance in the presence of ccr5 inhibitors does not select always the same mutations.3539 consequently, the prediction of resistance in ccr5 inhibitors may be more complicated and more difficult to interpret than it is with the rt or pis. a third possibility for the virus to attain infectiveness of its combatant maraviroc or other ccr5 inhibitors is the ability to bind to the coreceptor despite specific drug is bound, whether through a competitive replacement due to higher affinity than the drug or because of the possibility to bind despite a drug - bound receptor.40,41 taking the above - mentioned facts into account, the consequences of therapeutic use still remain elusive. nearly, 80% of all antiretroviral naive patients harbor r5-tropic virus, and therefore, would be eligible for using this drug. however, until 2009, the worldwide approvals for maraviroc indicate its use only for pretreated patients with r5-tropic hiv-1, partly with this requirement alone or partly with the necessity of resistances to other antiretroviral drug. since 2009, maraviroc is licensed in the united states under a statement, for combination antiretroviral treatment of adults infected with only ccr5-tropic hiv-1 (us food and drug administration approval november 20, 2009), which means that it can be used also in naive patients. however, this statement is directly followed by a special note: more subjects treated with selzentry experienced virologic failure and developed lamivudine resistance compared to efavirenz , which puts the indication into perspective. hence, the use of maraviroc in treatment - naive individuals would be according to the applicable label, but may induce a higher risk for the patients to develop virological failure and acquiring resistances. therefore, maraviroc should be the first choice in treatment - naive patients only under special conditions and thorough surveillance. in treatment - experienced subjects, the motivate studies demonstrated a benefit of maraviroc compared with the placebo arms in virological and immunological responses. however, 955 individuals (29.4%) of 3,244 screened patients were directly excluded from the studies due to the occurrence of x4 or dual / mixed tropism. overall, in only 61% of the initially tested patients, an r5 tropism could be clearly demonstrated.29 these were generated with a later revised phenotypic assay (trofile, monogram bioscience, south san fransico, california, usa). unfortunately, the possible subsequently generated data obtained using the enhanced trofile assay, which is more sensitive, are not applicable. nonetheless, the virological activity of maraviroc and the good compatibility could be demonstrated in the studies and in clinical practice. however, before starting arv, it needs to be demonstrated that the patient is harboring r5-tropic virus, exclusively.	maraviroc is the first and, so far, the only licensed representative of the class of chemokine receptor type 5 (ccr5) inhibitors used for the treatment of human immunodeficiency virus (hiv) infection. its safety and efficacy were demonstrated in several clinical trials, and its use was approved in 2007 by the responsible authorities. some specific issues are correlated with maraviroc and its use. it is the only drug in the antiretroviral armamentarium, which does not interact with the viral enzymes but with a human receptor. hence, it is able to be long - term effective only if the infecting virus uses, exclusively, the ccr5 receptor. occurrence and detection of the ccr5 tropism are some of the great challenges of maraviroc use in treatment - experienced patients. although up to 80% of naive patients harbor ccr5-tropic virus, the occurrence of cxcr4 or other tropisms increases with the duration of hiv infection and treatment. nonetheless, maraviroc is a potent medication for eligible patients and helps to improve the outcome of antiretroviral treatment (art) of hiv infection.
rectal cancer is often camouflaged to be part of colorectal cancer with australian statistics approximating 14 225 cases of bowel cancer in 2008 with a higher predominance among men and those above 50 years of age. meanwhile, the american counterpart recorded rectal and colon cancer to be the third leading malignancy plaguing both sexes in 2012 whereby 9% of deaths in each gender group was attributed to the disease. the risk of death before age 75 has declined over the years from a high of 1 in 50 in 1987 to 1 in 91 in 2007 and this is attributed to better early detection of precancerous lesions and management of the disease. treatment of rectal cancer is primarily surgical and is highly dependent on the preoperative staging. early cancers, especially t1 tumors, have been controversially proposed for local excision either via transanal excision or transanal endoscopic microsurgery (tems). while some report good outcomes measurable to radical surgery, others beg to differ prompting the need for salvage therapy. later stages of cancer usually follow the traditional approach of abdominoperineal resection (apr) or anterior resection and its success at reducing local recurrence is improved by total mesorectal excision (tme). however, these procedures are associated with their own set of morbidity and mortality. other therapeutic modalities such as chemotherapy and radiotherapy have been included in conjunction with surgery particularly in advanced cancers. they can be used preoperatively or postoperatively either to increase the chances of a sphincter - preserving procedure to allow better quality of life or with a curative intent. an overall cancer - specific 5-year survival of 77% has been reported. in this paper , we seek to discuss the modern approach to rectal cancer surgery at all disease time points from preinvasive and early rectal cancer, resectable rectal cancer, and locally advanced and metastatic rectal cancer with an emphasis on presenting some of the controversies and the accepted standards of treatment. the advent of endoscopic mucosal resection (emr) for rectal tumours could not have been timelier as the race to provide a treatment that is highly efficacious and of low morbidity continues. emr has been in practice for a few decades now and is commonly used to address gastrointestinal pathologies such as a barrett's oesophagus, and early gastric cancers (egc) with a reported local recurrence rate of 2% in egc. the procedure focuses on removal of diseased mucosal tissue or at most, the superficial submucosa instead of a full - thickness excision. it can be done by means of a strip biopsy, local injection of hypertonic saline - epinephrine in the inject, lift and cut technique, cup and suction otherwise known as the emr - cap technique or emr with band ligation. if necessary, more than one of the aforementioned methods can be applied. emr is an alternative to the locally - approached transanal excision (tae) and transanal endoscopic microsurgery (tems). generally, local excision of rectal cancer is indicated for early disease that encompasses its precursor (dysplasia or adenoma), t1 tumours that display moderate - to - well differentiation and no lymphatic or vascular invasion. at present , it is known that emr can be performed on flat, sessile, or lateral spreading rectal tumours. the success of emr is reflected through its high overall cure rate with some reporting 91% for treatment - nave patients and others at 96%. one of the common consequences of emr is bleeding which occurs from 1%45% of cases. the bleed can be minor and is easily manageable with haemostatic clips or severe requiring blood transfusions or even surgery. 0.7% to 4% of cases are complicated with perforation which can appear later as abdominal distension and pain necessitating further operation. while emr is an excellent therapeutic option for rectal tumours, it is operator dependent. identifying the lesion especially if it is a lateral spreading tumour can be challenging to the untrained eye as it displays subtle changes but a meta - analysis showed that the chances of a complete en bloc resection is directly proportional to experiences if the operator. the concern is that complete resection and a proper histological evaluation is rendered difficult with larger lesions, thus running a risk of local recurrence. in spite of this, some studies showed that the success of the resection is not statistically different between the two techniques. complicating failure of the mucosa to lift during the inject, lift and cut technique may occur secondary to submucosal fibrosis. the success rate of a repeated resection is reduced from 91.0% in a treatment - nave patient to 74.5% in a previously attempted lesion. it is reported that the time for conversion of a precursor lesion to cancer is variable, with dwelling times in the adenoma state ranging from 4 years to as long as 48 years. in fact, flat adenomas need not necessarily be treated as it is almost always noninvasive. this questions the need for emr in such lesions and more so, the implication on the patient who is no doubt heading for months of unwarranted anxiety. nevertheless, emr is a safe and effective treatment modality that is minimally invasive and only requires conscious sedation. it is also efficient with moss et al. reporting a mean procedural duration of 25 minutes while another study accounted a range of 25137 minutes. when possible, patients can be treated as an outpatient with discharges within the same day. a comparison between tae and endoscopic resection showed that the latter was associated with a significantly shorter hospital stay of 2.7 1.1 days with a mean difference of 6.2 days. similarly, tems recorded a range of 044 days of hospitalization while emr had a range of 027 days. emr per se is not as costly as the other procedures available, thus allowing a cheaper and less invasive option for the patient. this translates into reduced expenditure for the spender. on the other hand, tems and tae lesions larger than 8 cm in diameter can be removed with good effect via tems. where emr may fail to completely remove the tumour, both tems and tae first attempt of tems for large rectal adenoma is reported to have a lower early local recurrence rate at 10.2% compared to emr at 31.0% (p < 0.001). meanwhile, low risk - t1 tumour recurrence rate following tems is 010% while santos et al. tae is estimated to have a 15% local recurrence rate of the malignancy at five years. it would seem that the recurrence rate following tae is high compared to emr but contradicting this is a 32-patient study by lee et al. which showed no recurrences of cancer following either endoscopic resection or tae at a median followup of 15 months (range 699). studies evaluating the disease - specific and overall survival rates (tems versus radical surgery and tae versus radical surgery) showed that the values were not statistically significant to one another indicating the efficacy of these procedures. emr appears to be favourable as tems and tae have the added adverse effects including wound breakdown, incontinence, urgency, strictures, neuralgia, and anovaginal fistula. barendse et al. studied both emr and tems and discovered that the former was associated with half the percentage (12%) of postoperative complications compared to the latter (24%). however, given the variable recurrence rates, there is some apprehension towards emr. in view of this instead, it is a judgment call by the surgeon and more importantly the patient himself based on the local expertise and the pros and cons of each option available. as we push the fort of combination treatment, it is likely that in the future, local excisional strategies will become a more commonly adopted strategy in complete or subcomplete responding tumors after chemoradiotherapy. however, the limitation of mesorectal sampling may mean that more intensive followup is required to detect recurrence at the local excisional site and/or mesorectal lymph nodes. its proximity to the anal sphincter also makes this a major consideration into the surgical approach towards resection. in the localized setting, a multimodality approach has in recent years been developed and investigated in trials to improve local recurrence, disease - free and overall survival using a variety of sequences of chemotherapy and radiotherapy. prior to surgery, adequate local staging is paramount in the surgical planning and accurate prediction of the extent of bowel wall involvement may be obtained through endorectal ultrasonography and magnetic resonance imaging may serve the similar purpose but also identify the involvement of lymph node metastases. the aims of rectal cancer surgery are to remove the tumor with an adequate distal margin of a minimum of 2 cm in the case of a low rectal tumor with sphincter preservation or 5 cm in the case of a rectosigmoid / upper rectal tumor with restoration of intestinal continuity through an anastomosis. if a 2 cm distal margin can not be secured, an abdominoperineal excision with enbloc resection of the entire anorectum and an end colostomy is required. surgery should be performed using a no - touch technique with high ligation of the inferior mesenteric artery to achieve adequate lymphatic sampling through harvesting of the sigmoid mesentery and mesorectum. tumors of the middle and lower rectum require a total mesorectal excision (tme). tme reduces the risk of local recurrence and although a prospective randomized trial has not been conducted to verify its efficacy, longitudinal data derived from the netherlands where a tme trial was conducted following rigorous training of colorectal surgeons demonstrated that there was an observed reduction in rate of local recurrence from 16% to 9% with tme surgery being an independent predictor of overall survival. further, data from the mrc cr07 and ncic - ctg co16 randomized trials demonstrated that the plane of surgery achieved in patients undergoing rectal cancer surgery impacted on local recurrences with a 3-year local recurrence rate of 4% for patients whose surgery was completed with achievement of the mesorectal plane, 7% for intramesorectal plane and 13% for muscularis propria plane. pertinent also in low rectal cancers requiring abdominoperineal excision, to avoid a coning effect in the deep pelvis as the tumor is approached from both the abdomen and perineum, extended abdominoperineal excision incorporating resection of the levator muscles to reduce inadvertent bowel perforation and breaching of the circumferential resection margin. today, in the current era of laparoscopic surgery where shorter postoperative hospital stays, reduction in pain scores, shorter time of return of bowel function, lower treatment cost, and improved cosmesis may be achieved, these standardized surgical resections have been demonstrated to be feasible in the laparoscopic approach. initially, the laparoscopic approach was first examined in colon cancer with at least 4 large randomized trials; cost study group trial from the usa, color trial from europe, mrc clasicc trial from the uk, and a trial in rectosigmoid cancers from the prince of wales hospital in hong kong demonstrating equivalent oncologic efficacy with similar overall survival, disease - free survival and local and distant recurrences. these studies although predominantly examined in the setting of colon cancer were quick to translate into standard practice for rectal cancer despite limited large scale prospective randomized trials. however, there remain concerns over the ability to achieve adequate mesorectal excision and clear surgical margins in laparoscopic rectal cancer surgery. in the uk mrc clasicc trial , there was a 34% conversion rate from laparoscopic to open surgery in the rectal cohort, increase performance of tme surgery to ensure adequacy of the distal resection margin in the laparoscopic group because of the inability to palpate the tumor for localization. nonetheless, there was no difference in positive circumferential resection margin, local recurrence, disease - free, and overall survival in both the laparoscopic versus open anterior resection and abdominoperineal resection groups. the prince of wales hospital group from hong kong reported a small prospective randomized trial of laparoscopic assisted versus open abdominoperineal resection for low rectal cancer randomizing 99 patients (51 lap - assisted and 48 open) demonstrating earlier return of bowel function, decrease time to mobilization, lesser analgesia requirement, longer operative time, and higher direct cost in the lap - assisted group without difference in morbidity and mortality. in an update of this trial, after 10 years of followup, the authors reported higher rates of bowel obstruction requiring hospitalization and intervention in the open group but similar oncologic outcomes were shown with 10-year survival of 83.5% and 78% (p = 0.595) and 10-year disease - free survival 82.9% and 80.4% (p = 0.698) in the lap - assisted and open group, respectively. in the corean trial that randomized 170 patients in each arm to laparoscopic and open surgery for mid or low rectal cancer after preoperative chemoradiotherapy, the laparoscopic group had lower amount of blood loss, longer operative time, quicker recovery of bowel function, and a lesser amount of analgesic requirement. surgical quality indicators including the circumferential resection margin, macroscopic quality of the tme specimen, number of harvested lymph nodes, and perioperative morbidity were similar between groups. in a spanish randomized trial of 204 patients of whom 78.6% in the open group and 76.2% in the laparoscopic group underwent sphincter - preserving surgery; blood loss was greater in the open surgery group, operative time was longer in the laparoscopic group, and return to diet and hospital stay was longer in the open surgery group. complication rates were similar between groups but a larger number of lymph nodes were isolated in the laparoscopic group. together, these three small randomized trials suggest that the laparoscopic approach achieves improved short - term outcomes without compromising the surgical quality of rectal cancer operations in skilled hands but a longer operative time is required. longer followup of these trials and of ongoing larger trials will confirm the long - term oncologic outcomes of laparoscopic rectal cancer surgery. thorough preoperative clinical staging is paramount in the sequencing of multimodality therapy for rectal cancer. for smaller tumors t1/t2 , surgery alone with wide surgical resection of low anterior resection or abdominoperineal resection for distal lesions not amendable to low anterior resection may be performed. this allows sampling of mesorectal lymph node for accurate pathological staging. in patients who are medically unfit or who adamantly refuse to undergo standardized resectional surgery, local excision with or without chemoradiotherapy this strategy fails to sample the mesorectal lymph nodes that are essential in disease staging. in a large single institution cohort study comparing their retrospective experience of 350 with stage i rectal cancer of whom 283 patients (80.9%) underwent standardized resection and 67 patients (19.1%) undergoing local excision, 5-year local recurrence was 14.1% in the local excision group compared to 3.3% in the standardized resection group. this significantly higher local recurrence rate may then be salvaged through multimodality approach combining preoperative chemoradiotherapy and surgery. however, at times, these local recurrences may not be resected with standardized resectional surgery and may require radical surgeries such as a pelvic exenteration. for clinically staged t3/t4 rectal tumors without clinically identified nodal disease (stage ii) who undergo tme surgery with either a low anterior resection or abdominoperineal resection with harvesting of at least 12 lymph nodes examined and staged as pn0 chemoradiation is not required. chemoradiation may be considered in the setting of pt3n0 tumors with adverse pathologic features, non - tme surgery or in those with fewer than 12 lymph nodes harvested. for t3/t4 tumors with lymph node metastases (stage iii) identified clinically, treatment involves both chemoradiation with fluorouracil (5-fu) and total mesorectal excision (tme) based surgery. postoperative chemoradiation was shown to achieve superior over postoperative radiation alone with a 34% reduction in recurrence rate with reductions observed for local recurrences and distant metastasis. when postoperative chemotherapy was compared to radiotherapy in the nsabp r-01 randomized trial, postoperative chemotherapy appeared to improve survival and radiotherapy reduced the incidence of locoregional recurrence without survival improvements. given the benefits of chemoradiation in achieving local control as an adjunct to surgery, the german rectal cancer study group then conducted a randomized trial of 421 patients to determine the perisurgical sequencing of chemoradiation for rectal cancer. these investigators compared preoperative to postoperative chemoradiation and demonstrated that the 5-year cumulative incidence of local recurrence was 6% in the preoperative arm compared to 13% in the postoperative arm (p = 0.006) with fewer grad 3/4 acute and long - term toxic effects of chemoradiation observed in the preoperative arm compared to the postoperative arm. in a smaller korean trial, the improved effects of local control was not demonstrated in the preoperative compared to the postoperative chemoradiation arm, however, it was shown that an increased rate of sphincter preservative surgery could be achieved. the exact type of preoperative therapy was also recently debated with a short course radiation (25 gy in 5 fractions) followed by surgery a week after or a long course chemoradiation (50.4 gy in 28 fractions combined with systemic chemotherapy) followed by surgery four to six weeks after. the brief use of radiotherapy in the short course setting has been argued upon its role in providing adequate tumor response to allow sphincter preservative surgery. two randomized trials; polish (n = 312) and australian (n = 326) compared these two regimens and both trials showed a lower rate of early acute toxicity and reduce cost of treatment without any difference in long - term oncologic outcomes. after preoperative chemoradiation, guidelines recommend adjuvant chemotherapy for patients with node positive disease. however, the eortc 22921 trial of 785 clinically staged t3/t4 rectal cancer patients randomized to receive adjuvant fluorouracil based chemotherapy after preoperative (chemo) radiotherapy and surgery showed no survival benefit of chemotherapy on disease - free survival. however, specific subgroup analysis was performed to determine the appropriate role of adjuvant chemotherapy and showed that pathologically staged t0 - 2 (ypt0 - 2) patients appeared to benefit in terms of both disease - free and overall survival from adjuvant chemotherapy compared to ypt3/t4 patients. importantly, adjuvant chemotherapy did not appear to demonstrate any difference in outcomes of patients with ypn0 or ypn+ disease. this demonstrates that further benefits of adjuvant chemotherapy are only observed in responding patients (ypt0 - 2). further trials are required in this area to determine the appropriate role of adjuvant chemotherapy and the selection of high - risk populations who may then benefit from other modern adjuvant agents. tumors extending beyond the rectal wall with invasion into surrounding viscera are considered locally advanced rectal cancer. often in patients who develop local recurrence, recurrent disease often similarly involve adjacent structures where the previously excised rectal tumor was located. although its incidence has decreased following total mesorectal excision and the incorporation of preoperative chemoradiation, when it occurs, it remains a debilitating condition that is difficult to treat. palliative radiotherapy may provide brief symptom relief for an average of 3 months with median survival in these patients being between 12 and 24 months. surgery may provide a long - term palliation to the debilitating symptoms of pelvic recurrences. in the curative setting, delivery of long course preoperative chemoradiotherapy may induce tumor down - staging to facilitate surgical resection. in a randomized trial comparing neoadjuvant radiotherapy to chemoradiotherapy in 207 patients with locally unresectable t4 primary rectal or local recurrent rectal cancer, chemoradiotherapy compared to radiotherapy facilitated higher potential for an r0 resection (84% versus 68% ; p = 0.009), improved local control in patients who underwent a r0 or r1 resection (82% versus 67% at 5 years ; p = 0.03), improved time to treatment failure (63% versus 44% ; p = 0.003), cancer - specific survival (72% versus 55% ; p = 0.02) and overall survival (66% versus 53% ; p = 0.09). the surgery involved necessitated pelvic exenteration where adjacent organs are resected with an aim to achieve clear margins. in patients with primary t4 rectal cancer and recurrent rectal cancer, 28% and 20% of patients in the chemoradiotherapy arm and 27% and 46% in the radiotherapy arm, respectively, required exenteration. intraoperative radiotherapy (external - beam) (iort) is another approach to improve local control. this treatment modality has been investigated in patients with locally advanced unresectable rectal cancer after chemoradiotherapy down - staging and surgery. valentini et al. reported 100 patients with t4m0 tumors undergoing r0 resection after down - staging by chemoradiotherapy and showed that 5-year local control was 90% in patients with r0 surgery and 100% in patients with r0 surgery and iort. further, iort did not appear to compensate for suboptimal surgery with 5-year overall survival of 68% observed in patients with r0 surgery compared to 22% in r1 or r2 surgery. such radical surgery however is not widely performed and only available in specialized institutions. in a pattern of care study of the united states population through data identified from the surveillance, epidemiology and end (seer) registry, only 33% of patients with locally advanced adherent colorectal cancer underwent multivisceral resection for which was shown to be associated with improved overall survival. in patients with synchronous rectal cancer with liver metastases , there remains an enigma over the appropriate sequencing of chemotherapy, radiotherapy, and surgery. in a study from the erasmus university, van der pool et al. reported a consecutive series of 57 patients of whom 29 patients underwent resection of the primary tumor first, 8 patients underwent simultaneous rectal and liver resection and 20 underwent a liver - first approach achieving a median survival of 47 months and 5-year survival of 38%. this was achieved in a multidisciplinary setting where an individualized approach towards treatment was taken. in general, if resection of both primary tumor and liver metastases may be completed in one surgery, this approach may be favored. if the liver metastases are not completely resectable during the rectal surgery or are too advanced for hepatectomy irrespective, neoadjuvant chemotherapy is preferred followed by a liver - first approach followed by restaging and preoperative radiotherapy and rectal surgery. in patients with metachronous liver metastases, of large clinical series have shown that median survival range from 43 months to 64 months with 5-year survival ranging between 37% to 51%. of note, in a large international multi - institutional registry study, rectal primary tumor were associated with extrahepatic recurrences after hepatectomy for liver metastases, hence emphasizing the importance of local control in the pelvis. however, there is no difference in colon or rectal based primary tumor site impacting outcomes after hepatectomy for colorectal liver metastases. epidemiological and observatory data from the followup of patients with curatively treated colorectal cancer have shown that rectal cancer patients have a higher preponderance to developing recurrence in the lungs. in a large population based study of 30 years in burgundy (france), mitry et al. reported that lung metastases often accompanied liver metastases with synchronous lung metastases being more common in left colonic and rectal cancers. surgery for lung metastases is indicated if the lung metastases are the only site of disease and a complete resection may be achieved. where extrapulmonary metastases are present, a highly selective approach should be taken often after adequate tumor response to systemic chemotherapy to select patients whose disease is amendable to resection of both lung and extra - pulmonary metastases. the highest level of evidence for resection of lung metastases comes from a large systematic review of 20 published studies for pulmonary metastasectomy for colorectal lung metastases. report a median 5-year overall survival of 40% in this selected group of patients who underwent surgery. however, given that the liver will often be involved when there is lung metastases, it is important that the selection of patients for pulmonary metastasectomy should include a sufficient disease - free interval from previous liver resection, use of prethoracotomy cea levels and the absence of mediastinal lymph node involvement as a separate selection criteria in this group of patients. presently, a randomized trial (pulmicc) funded by cancer research uk is seeking to investigate if pulmonary metastasectomy contributes to improved survival of patients with colorectal lung metastases by randomizing patients with a history of resected colorectal cancer who are found to have pulmonary metastases to be randomly allocated to active monitoring or active monitoring with pulmonary metastasectomy with overall survival, relapse - free survival, lung function, and patient - reported quality of life as endpoints of this clinical trial. shedding of tumor during the difficult abdominoperineal resection, low anterior resection operation, or invasion of a large t3/t4 tumor in the upper rectum above the peritoneal reflection may in the shedding of free peritoneal tumor cells within the abdominopelvic peritoneal cavity. the growth and implantation of these cells may in the development of peritoneal metastases (carcinomatosis). of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (hipec) have shown that this combined modality technique allows complete excision of peritoneal tumors with locoregional control achieved through the chemoperfusate. in colorectal cancer, a randomized trial comparing cytoreductive surgery and hipec demonstrated a median survival of 22.3 months compared to 12.6 months in patients receive systemic chemotherapy with or without palliative surgery. however, in this trial of 105 patients, only 12 patients had rectal cancer. again, in a large registry study of the french experience of hipec in colorectal cancer, of 523 patients included, only 36 patients (7%) had primary colorectal tumor of rectal origin. in another international registry of 506 patients with colorectal peritoneal metastases undergoing cytoreductive surgery and hipec, there were 40 patients (8%) with the primary tumor of rectal origin and the median survival of these patients was 19.2 months compared to 24 months in patients with tumors of sigmoid origin, 17 months for patients with tumors of the right colon and 20 months for patients with tumors of the left colon. these inform us that peritoneal metastases from rectal cancer is less common but prevent us from drawing any meaningful on whether there may be disparate survival outcomes for colon and rectal cancer patients with peritoneal metastases. based on the currently available evidence, selected rectal cancer patients with limited peritoneal disease burden may be considered for cytoreductive surgery and hipec. rectal cancer surgery has made significant advancement at all - time points of the natural history of this disease. there are now minimally invasive local excision options that are currently being tested for efficacy as we await further clinical trials to verify its efficacy for pre - invasive and early lesions. laparoscopic rectal cancer surgery incorporating total mesorectal excision is now emerging as the standard surgical approach with ongoing clinical trials that will confirm its short and long - term oncologic efficacy. there is now evidence based from clinical trials for both preoperative short and long - course chemoradiation prior to surgery for resectable tumors for which has been shown to improve local control of disease albeit its aim achieving sphincter preservative surgery where possible. ultimately, the goal of this being to achieve adequate sampling of mesorectal lymph nodes to provide adequate information for tumor staging and prediction of local and distant recurrences for which will guide treatment decisions. there is now evidence for resecting metastases from rectal cancer from local recurrences, liver, lung, and peritoneal metastases based on a body of retrospective clinical data with long - term followup.	rectal cancer is a distinct subset of colorectal cancer where specialized disease - specific management of the primary tumor is required. there have been significant developments in rectal cancer surgery at all stages of disease in particular the introduction of local excision strategies for preinvasive and early cancers, standardized total mesorectal excision for resectable cancers incorporating preoperative short- or long - course chemoradiation to the multimodality sequencing of treatment. laparoscopic surgery is also increasingly being adopted as the standard rectal cancer surgery approach following expertise of colorectal surgeons in minimally invasive surgery gained from laparoscopic colon resections. in locally advanced and metastatic disease, combining chemoradiation with radical surgery may achieve total eradication of disease and disease control in the pelvis. evidence for resection of metastases to the liver and lung have been extensively reported in the literature. the role of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal metastases is showing promise in achieving locoregional control of peritoneal dissemination. this paper summarizes the recent developments in approaches to rectal cancer surgery at all these time points of the disease natural history.
atypical hemolytic uremic syndrome (ahus) is a rare, and life threatening, disease. in most cases, the first line of treatment for ahus was plasmapheresis until september 2011, when the food and drug administration approved eculizumab for the treatment of pediatric and adult patients with ahus. eculizumab has been available in the united states since 2007 for the treatment of paroxysmal nocturnal hemoglobinuria. this additional indication makes eculizumab the first drug to be approved for the treatment of ahus. we describe here for the first time in india, use of eculizumab in a 12-year - old boy with ahus, and the challenges faced. a 12-year - old caucasian boy was admitted in september 2014, with complaints of pain abdomen for 4 days, petechial spots over face for 3 days, cola - colored urine and decreased urine output for 1 day. on evaluation, the child had hypertension (blood pressure : 140/90), anemia (hemoglobin = 9 g / dl), severe thrombocytopenia (platelet count 20,000/mm), acute kidney injury (blood urea 183 mg / dl ; serum creatinine 4 mg / dl), lactate dehydrogenase 10,000 iu / ml, and schistocytes on peripheral smear suggestive of hemolysis. malarial antigen, dengue antigen, widal, leptospiral serology, and all cultures were negative. there was no history of diarrhea, dysentery, or fever prior to this illness. a diagnosis of ahus was made and plasma exchange (pe) initiated within 24 h of admission. following three daily pes, his hematuria, platelet count, serum creatinine, and urine output improved. initially, the pe was done daily for 6 days, followed by alternate days for 2 weeks, then thrice a week for 2 weeks, according to the european pediatric study group guidelines for hus. the child was discharged on day 7 of admission, and the rest of the pes were done on an out - patient basis. a complement factor assay was sent to a collaborative laboratory in france which was found to be normal. since it was an unexplained ahus, the need for long - term pes or eculizumab and a complete genetic sequencing workup was discussed with the family. the treatment could only be stopped once the complete genetic work - up was negative. since the child was an american citizen, the american embassy, insurance agency and alexion pharmaceuticals, germany were contacted. the drug is not available in india and a special acquiring permission from the drug controller of india followed by custom duty exemption was taken. the child was already immunized for meningococci according to centers for disease control and prevention schedule. trend of hemolytic parameters with plasma exchanges and eculizumab of the complement exploration performed in the family members subsequently, the child was started on eculizumab (soliris) 1200 mg q 2 weekly, along with penicillin prophylaxis for meningococci. after three doses, the child developed leukopenia (total leukocyte count 3500/mm and absolute neutrophil count 800/mm). the child was put on cotrimoxazole and acyclovir prophylaxis, and the dose interval was increased to q 3 weekly. the child also developed hallucinations after three doses of eculizumab, which could be attributed to intensive care unit psychosis / drug, which improved on adding risperidone and increasing the drug interval. the child was continued on q 3 weekly eculizumab, with normal renal functions, and a close monitoring for ch50 and hemolysis. a trough level of eculizumab at 3-week dose interval was 135.8 mcg / ml (normal trough used in paroxysmal nocturnal hemoglobinuria 100 mcg / ml). the gene sequencing were available in february 2015 and it was normal. it was discussed with the family that the possibility of finding a major genetic defect has been ruled out, though there is still a possibility of relapse. hence, eculizumab was stopped in april 2015, in view of the absence of any symptoms and normal complement factors and gene sequencing. currently the child is 9 months off eculizumab and is doing well, with a normal renal function, no proteinuria, and a normal urine examination with no relapses. despite poor quality evidence, the mainstay of treatment for ahus was plasma therapy till 2009. the outcome for children with ahus is poor, and because of the rarity of these disorders, clinical experience is scanty. the guideline for the investigation and initial therapy in ahus published by the european pediatric study group for hus in 2009, based on anecdotal case reports, retrospective series and expert consensus, advocated early, frequent, and high volume pes. the recently published audit of this guideline indicated considerable morbidity associated with plasma therapy in children. recently, eculizumab has been proposed as the first - line treatment for ahus, to avoid pe and the complications of central venous double lumen catheters. when possible, eculizumab treatment should be initiated within 2448 h of onset or admission. if eculizumab is not (or not immediately) available, pe (or plasma infusions if pe is not possible) should be started as recommended in the 2009 guideline. we switched our patient to maintenance eculizumab after the drug was procured from germany after getting all the necessary legal permissions. current evidence - based management of atypical hemolytic uremic syndrome (adapted from loirat et al .) eculizumab is a monoclonal antibody that binds to the complement protein c5, inhibiting its cleavage to c5a and c5b. this prevents circulation of the pro - inflammatory c5a peptide and generation of the cytotoxic terminal complement complex c5b-9 (referred to as the membrane attack complex). the most common adverse effects reported by the 37 adults and adolescents receiving eculizumab in the two prospective ahus studies were hypertension (in 35% of patients), headache (30%), anemia (24%), leukopenia (16%), diarrhea (32%), vomiting (22%), and nausea (19%). our patient has significant leukopenia requiring increasing the dose interval and adding cotrimoxazole and acyclovir prophylaxis. the reduction in mac formation produced by eculizumab eliminates the body's defense mechanisms against neisseria meningitidis under normal conditions. patients should be immunized with meningococcal vaccine at least 2 weeks prior to administering eculizumab unless the risks of delaying therapy outweigh the risk of a meningococcal infection. the risk of relapse in patients with ahus is influenced by the genetic , and the interval between flares is extremely difficult to predict. the issue of the optimal duration of eculizumab treatment has not yet been properly addressed. the european medicines agency has approved life - long eculizumab treatment for patients with ahus. however, the high cost of the drug and the uncertainties surrounding the natural history of ahus in patients for whom eculizumab prevented the progression to end - stage renal disease, raise the question of whether life - long treatment is warranted for all patients with ahus. given that the natural history of ahus differs depending on the underlying genetic abnormalities, treatments could be tailored on the basis of an individual's complement genetics. life - long treatment may be appropriate in patients with ahus who have mutations associated with poor outcomes (e.g. cfh or c3/cfb gain - of - function mutations). we also carefully planned to stop eculizumab in our patient, since he had no major genetic mutations, with a normal complement panel. there is a need to completely evaluate all patients with ahus for complement abnormalities and antibodies to factor h. these patients should get early pe / eculizumab. hus being a common cause of acute kidney injury in children in india should get timely and appropriate management with lifesaving eculizumab or pes. to improve the availability of the drug in indian market, and subsequently decrease the cost of drug, collaboration among medical experts and health authorities must occur in order to implement a feasible plan of action.	much progress has been made in understanding the pathophysiology and treatment of atypical hemolytic uremic syndrome (ahus). plasma therapy is the mainstay of treatment for ahus. the availability of the first effective anti - complement therapeutic agent, eculizumab, has dramatically changed the outlook of this disease. however, its use in clinical practice raises important questions, such as who should receive the drug, when to start such therapy, and is it safe to stop treatment once the disease is controlled. we describe here for the 1st time in india, use of eculizumab in a 12-year - old boy with ahus. we also describe in this report challenges faced in procuring the drug, and an ideal, evidence - based method of treating ahus in children.
the diaphragm, the primary respiratory muscle, has been reported to act as one of spinal stability muscles and other spinal stability muscles are also activated to increase respiratory capacity1,2,3,4,5,6,7. the muscles used in both spinal stability and respiratory tasks include the diaphragm, transversus abdominis (tra), intercostals muscles, internal oblique muscle (io) and pelvic floor muscles (pfm)1,2,3, which are also known as core muscles. the spinal stability provided by these muscles is derived from their co - contraction which increases intra - abdominal pressure4. these muscles function as respiratory muscles by increasing their activities when respiratory demand increases1,2,3, 5,6,7. we speculated that increasing the strengths of the respiratory muscles would have positive effects on spinal stability. the question was whether forced breathing pattern increased the spinal stability muscle activities enough to strengthen them. bridging exercises was the commonly used methods to increase spinal stability muscle strengths8, 9 and prone position bridging exercise was the most effective among in other positions10. the previous study showed % mvic of these muscles with forced breathing patterns were as high as with bridging exercises. in addition, the synergy ratios of these muscles with forced breathing patterns were similar with those with bridging exercises8, 9, 11,12,13. however, these previous studies were performed in the standing position and it is not clear whether the increased muscle activations were due to increased postural demand or increased respiratory demand or both. in addition, it is necessary to determine whether there are any interaction effects between position and breathing patterns to suggest forced breathing pattern as one way to strengthen spinal stability muscles. thirty three subjects who had no history of low back pain within the last six months, musculoskeletal impairments of the lower limbs, or neurological or respiratory pathology were enrolled in this study. subjects with a cold, or excessive abdominal fat or a current or previous swimming habit were excluded. the data of 33 subjects (16 males, 17 females, 20.33 2.10 years old, height 1.66 0.08 m, weight 59.83 9.60 kg, bmi 21.57 2.31 kg / m) were used for data analysis. prior to their participation , all the participants read and signed an informed consent form, in accordance with the ethical principles of the declaration of helsinki. the protocol for this study was approved by the ethics committee of catholic university of daegu. each participant performed five different breathing patterns in two positions, sitting and standing. the five different breathing patterns were quiet breathing (qb) and forced respiratory maneuvers (frm): combination breathing (cb), diaphragmatic breathing (db), pulsed lip breathing (plb) and respiratory muscle endurance training (rmet). % mvic of internal oblique abdominis / transversus abdominis (io / tra)14, external oblique abdominis (eo)15, rectus abdominis (ra)15, erector spinae (es)16 and multifidus (mf)8 were measured for comparison of muscle activities. measurements and data collection were performed following the procedures described in a previous study13. two - way repeated anova was used to determine the main effects of breathing pattern and position as well as interaction effects between them. two - way repeated anova was performed in order to evaluate the effects of position and breathing pattern on five spinal stability muscle activities. the within - subjects factors were position (two levels : sitting and standing) and breathing patterns (five levels : qb, cb, db, plb and rmet). the main effects of position on % mvic of io / tra, eo, ra, es and mf were significant. in addition, the main effects of breathing patterns on % mvic of io / tra, eo, ra, es and mf were significant. the interaction effects were tested and were significant on only io / tra, eo and mf,. the paired t - tests was performed to examine the significant main effects of position after controlling for the family - wise error rate across these tests using holm s sequential bonferroni approach. differences in mean % mvic of io / tra, eo, ra, es and mf between the two positions were significant: t= 9.61, p<0.001; t= 10.06, p<0.001; t=5.53, p<0.001; t= 8.82, p<0.001; t= 5.27, p<0.001; respectively. of particular interest, mean % mvic of io / tra, eo and mf were higher in standing than in sitting, whereas % mvic of ra and es were lower in standing than sitting. one - way anova was performed to examine the significant main effects of breathing patterns after controlling for the family - wise error rate cross these tests using holm s sequential bonferroni approach (table 1table 1. of the tests for the significant main effect of breathing patterns . unit ( % mvic)io / traeoraesmfqb5.816.12 * 2.811.25 * 2.361.13 * 3.401.70 * 2.891.37cb8.318.404.002.01 2.761.293.841.86 * 3.461.61db9.388.834.292.262.791.243.851.87 * 3.271.56plb6.776.27 * 3.231.52 * 2.511.19 * 3.611.89 * 3.001.31*rmet12.9110.336.123.733.441.565.522.714.011.86p < 0.001 different from rmet, * * p=0.003 different from rmet, * * * p=0.002 different from qb ). for io / tra, ra and mf, rmet induced significantly greater activation of muscles than qb (p < 0.001) and plb (p<0.001, p < 0.001, p=0.002, respectively). eo and es in rmet showed significantly higher % mvic than in cb, db, plb and qb (p<0.001). in addition, p < 0.001 different from rmet, * * p=0.003 different from rmet, * * * p=0.002 different from qb finally, the paired t - test was performed to examine differences among breathing patterns in each position, controlling for the family - wise error rate using holms sequential bonferroni approach. the mean % mvic of io / tra in plb and rmet showed a significantly greater increase in standing than sitting compared to qb (p=0.002, p=0.004, respectively). the mean % mvic of mf in cb showed a significantly greater increase in standing than in sitting compared to db and plb (p=0.003, p=0.002, respectively), however the mean % mvic of mf in qb showed a significantly greater increase in standing than in sitting compared to cb (p<0.001). the purposes of this study were to investigate the influences of position on % mvic of spinal stability muscles and to find the most effective breathing pattern for the activation of spinal stability muscles. the of this study show that the positions and breathing patterns had important influences on spinal stability muscle activities and their influence was different depending on the muscles. the deep muscles and eo showed greater activation in standing than in sitting, while the superficial muscles showed greater activation in sitting than in standing. it is well - known that the deep muscle group provide segmental stability while the superficial muscle group provide torque production and general trunk stability17. to maintain a standing posture, increased segmental stability might be needed rather than increased torque, which is provided by the superficial muscle group. some studies have reported that activation of the deep muscle group is more effective at increasing spinal stability than activation of the superficial muscle group17, 18. in other studies, subjects demonstrated optimal lower back stabilization during exercises with appropriate deep muscle activation19, 20. in addition, sitting posture showed increased trunk sway with decreased compensatory postural control21; therefore, more torque might be needed to minimize trunk sway in sitting, possibly explaining the increase in superficial muscles activities in sitting compared to standing. we suggest that the reason for this is that the lower extremities are unavailable to compensate for respiratory perturbation in sitting. in this study, the spinal stability muscles, except mf, were activated the most by rmet, db, cb, plb and qb in declining order, whereas mf was activated the most by rmet, in the order of cb, db, plb and qb. among the different breathing patterns, rmet induced significantly greater activation of the spinal stability muscles than the other breathing patterns. for io / tra, % mvic in db increased as much as in rmet, and % mvic in db was significantly higher than that in plb or qb. for mf, % mvic in cb was not significantly different from that in rmet, even though % mvic in cb was significantly higher than that in plb or qb. rmet was the breathing pattern that induced the greatest activation of the spinal stability muscles. however, for subjects who have difficulty performing rmet, db or cb could be used as an alternative method for activation and strengthening of the spinal stability muscles. although, position and breathing patterns showed meaningful impacts on spinal stability muscle activation, interaction effects between position and breathing patterns were found for a few muscles in a few breathing patterns. in general, % mvic changes in the spinal stability muscles induced by frm were independent of position with a few exceptions: io / tra in plb and rmet showed increased activation that was greater than the possible position effect compared to the change in qb, while mf in qb was activated more than the possible position effect compared to the change in cb. however, absolute % mvic of both of these muscles with rmet was the highest. in , increased respiratory demands of frm induced greater activation than qb, and rmet was found to be the most effective breathing pattern for increasing the activities of the spinal stability muscles regardless of position. in rmet, the increases in muscle activities induced by increase in respiratory demand was not different between sitting and standing. therefore, it can be expected that rmet in a sitting position activates the spinal stability muscles in a manner comparable to that of standing. subjects with difficulty in maintaining a standing posture could start a spinal stability muscle strengthening program using rmet in a sitting position and progress to rmet in a standing position as spinal stability improves. a previous study showed lumbar stability improved with increased tra contractility after 4 weeks of deep breathing exercises22. however, further research will be required in order to determine whether spinal stability muscles are strengthened by rmet in sitting, and whether a spinal stability strengthening program with rmet can improve spinal stability.	the purposes of this study were to investigate the influences of position on % mvic of spinal stability muscles to establish for the most effective breathing pattern for activation of spinal stability muscles in order to provide an additional treatment method for use in spinal stability exercise programs. thirty - three healthy subjects performed quiet breathing and four different forced respiratory maneuvers (frm); in both standing and sitting positions. % mvic of them (the multifidus ( mf), erector spinae (es), internal oblique / transversus abdominis (io / tra), external oblique (eo), rectus abdominis (ra) measured. io / tra, mf and eo showed greater activation in standing than in sitting, while ra and es showed greater activation in sitting than in standing. rmet induced significantly greater activation of spinal stability muscles then other breathing patterns. % mvic changes of muscle activities induced by frm were independent of position with a few exceptions. the increased respiratory demands of frm induced greater activation of spinal stability muscles than qb. rmet was found to be the most effective breathing pattern for increasing the activation of the spinal stability muscles.
pulmonary complications are frequently encountered after cardiac surgery with cardiopulmonary bypass (cpb), and atelectasis is believed to be a major etiology. atelectasis in a decrease in lung compliance and adversely affects oxygenation. significant lung collapse following cardiac surgery in intrapulmonary shunting and hypoxemia. conventional mechanical ventilation might have additional deleterious influence on these atelectatic lungs, particularly with employment of high tidal volumes and pressures ing in pulmonary hyperinflation. however, if a patient is hypoxemic, he will require positive pressure ventilation to sustain oxygenation. addition of continuous positive airway pressure in such patients, might improve functional residual capacity, and place the patient on a favorable part of the pressure - volume curve. if, however, this fails to improve oxygenation, then it becomes necessary to initiate positive pressure ventilation. it would be ideal, therefore, to ventilate patients at the top of the volume - pressure curve, at high lung volumes, but without accompanying phasic changes. in the absence of facilities for oscillatory ventilation, increasing mean airway pressure without increasing peak pressure can only be achieved by prolonged inspiratory time (ti) in a pressure control mode. prolonged ti improves oxygenation. conversely, when ti exceeds the expiratory time, carbon dioxide removal is adversely affected leading to hypercarbia and respiratory acidosis. we know that generally patients tolerate respiratory acidosis very well, and we could allow this to happen as long as it does not affect the pulmonary vascular resistance significantly, which could be a factor in patients undergoing cardiac surgery. pressure control ventilation (pcv) previously used to be the last resort in ventilating postcardiac surgery patients with very noncompliant lungs and significantly high airway pressures on synchronized intermittent mandatory ventilation (imv). that has changed in most cardiac surgical intensive care units, with pcv now viewed as a good primary ventilation mode in patients with poor lung compliance, especially when combined with longer inspiratory times (inverse ratio pcv, or pcirv). tidal volume of approximately 6 ml / kg ideal body weight is now standard of care in mechanical ventilation of patients with reduced lung compliance. there is increasing recognition of the fact that tidal volumes of 10 ml / kg or more predispose cardiac surgical patients to organ failure and increased ventilation days, even in patients who do not have acute respiratory distress syndrome. especially, susceptibles are women and obese patients who tend to receive greater tidal volumes and have a greater incidence of consequent lung injury. intraoperative ventilation with low tidal volumes has also been suggested to be protective in noncardiac surgery settings. whether these benefits could also be translated to the cardiac surgical patient pcirv should probably be instituted earlier rather than later in postcardiac surgery patients with pulmonary complications, despite a paucity of studies confirming a substantial benefit from this mode. this mode is extremely uncomfortable for patients, who generally need to be heavily sedated, and often paralyzed. intensivists, however, prefer patients to be awake and interacting with the ventilator, leading to the development of newer modes to enable patients to breathe spontaneously even on pcirv. airway pressure release ventilation (aprv) was described in 1987 by stock et al. as a mode for acute lung injury while limiting airway pressures. bilevel ventilation is a mode in which spontaneous ventilation could be achieved in both phases of the high - low positive airway pressure cycle. the goal is to allow unrestricted spontaneous breathing so that need for excessive sedation or in some cases muscle relaxation can be avoided, thus enabling faster separation from mechanical ventilation. a study by rathgeber et al. compared duration of weaning between bilevel positive airway pressure (synonymous with bilevel), volume controlled (vc) imv, and vc continuous mandatory ventilation in patients undergoing cardiac surgery and demonstrated a marginal, yet significant decrease in weaning time. there appear to be no large published studies comparing aprv to conventional modes such as pressure support ventilation or t - piece, or to alternative modalities such as automatic tube compensation, proportional assist ventilation (pav), adaptive support ventilation (asv), or smartcare. however, putensen et al. have documented the benefits accrued from aprv, which include improvements in respiratory system compliance, pao2, cardiac index, and delivery of oxygen, in comparison to patients subjected to conventional mechanical ventilation with muscle paralysis. the increase in cardiac output in patients breathing spontaneously might presumably be due to decreased pleural pressure and elevated abdominal pressure. this in redistribution of splanchnic blood from abdominal viscera to inferior vena cava, ing in enhanced venous return. unlike other modes in which the physician presets a specific tidal volume or pressure, pav lets the patient determine the inspired volume and the flow rate. this mode mandates real - time estimation of resistance and compliance from which it determines the pressure to be generated. however, unlike the more commonly employed asv mode which can be used both in passive as well as actively breathing patient, pav can only be used in active patients. while to the best of our knowledge, there are no published studies exploring the effects of pav in patients undergoing cardiac surgery, in at least one randomized controlled study, asv appreciably reduced the time spent on mechanical ventilation in a population of postcardiac surgery patients and also reduced the incidence of unnecessary alarms and ventilator resetting by clinicians, leading to better utilization of resources. slutsky and ranieri indicate that the ventilator - induced lung injury may be reduced using lung protective mechanical ventilation. as research focuses on newer modes of mechanical ventilation that provide oxygenation and ventilation while reducing collateral pulmonary injury, there have evolved several advanced pressure control modes which seek to provide the benefits of both volume and pressure controlled ventilation. like vc ventilation, delivery of a reasonable tidal volume is guaranteed, and at the same time, like pressure controlled ventilation, this is done with an adjustment of the flow rate to avoid deleterious increases in plateau pressure. surprisingly, however, there are very few clinical trials related to their use in cardiac surgery, despite some evidence that some of them might limit the duration of postoperative mechanical ventilation and its attendant complications. while it is entirely possible to infer that unfamiliarity with many of these modes, as well as lack of substantial differences in tangible clinical end points from the noncardiac surgery settings might explain the reticence of researchers to explore these modes in the postcardiac surgery arena, larger studies are needed to clearly identify strategies that will in improved survival, decreased duration of mechanical ventilation, earlier icu discharge, earlier discharge from hospitals, and economic benefits. clinically, relevant differences in these parameters are likely to be easier to identify in a subset of critically ill patients undergoing cardiac surgery who have a higher risk of requiring prolonged mechanical ventilation including those with extended cpb runs, undergoing complex cardiac repairs, and those with preexisting comorbidities affecting the lungs such as chronic obstructive pulmonary disease. it would be worthwhile pursuing work in these difficult patients in whom these modes might have clinically noteworthy benefits. it would be ideal, therefore, to ventilate patients at the top of the volume - pressure curve, at high lung volumes, but without accompanying phasic changes. in the absence of facilities for oscillatory ventilation, increasing mean airway pressure without increasing peak pressure can only be achieved by prolonged inspiratory time (ti) in a pressure control mode. prolonged ti improves oxygenation. conversely, when ti exceeds the expiratory time, carbon dioxide removal is adversely affected leading to hypercarbia and respiratory acidosis. we know that generally patients tolerate respiratory acidosis very well, and we could allow this to happen as long as it does not affect the pulmonary vascular resistance significantly, which could be a factor in patients undergoing cardiac surgery. pressure control ventilation (pcv) previously used to be the last resort in ventilating postcardiac surgery patients with very noncompliant lungs and significantly high airway pressures on synchronized intermittent mandatory ventilation (imv). that has changed in most cardiac surgical intensive care units, with pcv now viewed as a good primary ventilation mode in patients with poor lung compliance, especially when combined with longer inspiratory times (inverse ratio pcv, or pcirv). tidal volume of approximately 6 ml / kg ideal body weight is now standard of care in mechanical ventilation of patients with reduced lung compliance. there is increasing recognition of the fact that tidal volumes of 10 ml / kg or more predispose cardiac surgical patients to organ failure and increased ventilation days, even in patients who do not have acute respiratory distress syndrome. especially, susceptibles are women and obese patients who tend to receive greater tidal volumes and have a greater incidence of consequent lung injury. intraoperative ventilation with low tidal volumes has also been suggested to be protective in noncardiac surgery settings. whether these benefits could also be translated to the cardiac surgical patient pcirv should probably be instituted earlier rather than later in postcardiac surgery patients with pulmonary complications, despite a paucity of studies confirming a substantial benefit from this mode. this mode is extremely uncomfortable for patients, who generally need to be heavily sedated, and often paralyzed. intensivists, however, prefer patients to be awake and interacting with the ventilator, leading to the development of newer modes to enable patients to breathe spontaneously even on pcirv. airway pressure release ventilation (aprv) was described in 1987 by stock et al. as a mode for acute lung injury while limiting airway pressures. bilevel ventilation is a mode in which spontaneous ventilation could be achieved in both phases of the high - low positive airway pressure cycle. the goal is to allow unrestricted spontaneous breathing so that need for excessive sedation or in some cases muscle relaxation can be avoided, thus enabling faster separation from mechanical ventilation. a study by rathgeber et al. compared duration of weaning between bilevel positive airway pressure (synonymous with bilevel), volume controlled (vc) imv, and vc continuous mandatory ventilation in patients undergoing cardiac surgery and demonstrated a marginal, yet significant decrease in weaning time. there appear to be no large published studies comparing aprv to conventional modes such as pressure support ventilation or t - piece, or to alternative modalities such as automatic tube compensation, proportional assist ventilation (pav), adaptive support ventilation (asv), or smartcare. however, putensen et al. have documented the benefits accrued from aprv, which include improvements in respiratory system compliance, pao2, cardiac index, and delivery of oxygen, in comparison to patients subjected to conventional mechanical ventilation with muscle paralysis. the increase in cardiac output in patients breathing spontaneously might presumably be due to decreased pleural pressure and elevated abdominal pressure. this in redistribution of splanchnic blood from abdominal viscera to inferior vena cava, ing in enhanced venous return. pav allows automated modulation of airway pressure according to force generated by the patient. unlike other modes in which the physician presets a specific tidal volume or pressure this mode mandates real - time estimation of resistance and compliance from which it determines the pressure to be generated. however, unlike the more commonly employed asv mode which can be used both in passive as well as actively breathing patient, pav can only be used in active patients. while to the best of our knowledge, there are no published studies exploring the effects of pav in patients undergoing cardiac surgery, in at least one randomized controlled study, asv appreciably reduced the time spent on mechanical ventilation in a population of postcardiac surgery patients and also reduced the incidence of unnecessary alarms and ventilator resetting by clinicians, leading to better utilization of resources. slutsky and ranieri indicate that the ventilator - induced lung injury may be reduced using lung protective mechanical ventilation. as research focuses on newer modes of mechanical ventilation that provide oxygenation and ventilation while reducing collateral pulmonary injury, there have evolved several advanced pressure control modes which seek to provide the benefits of both volume and pressure controlled ventilation. like vc ventilation, delivery of a reasonable tidal volume is guaranteed, and at the same time, like pressure controlled ventilation, this is done with an adjustment of the flow rate to avoid deleterious increases in plateau pressure. surprisingly, however, there are very few clinical trials related to their use in cardiac surgery, despite some evidence that some of them might limit the duration of postoperative mechanical ventilation and its attendant complications. while it is entirely possible to infer that unfamiliarity with many of these modes, as well as lack of substantial differences in tangible clinical end points from the noncardiac surgery settings might explain the reticence of researchers to explore these modes in the postcardiac surgery arena, larger studies are needed to clearly identify strategies that will in improved survival, decreased duration of mechanical ventilation, earlier icu discharge, earlier discharge from hospitals, and economic benefits. clinically, relevant differences in these parameters are likely to be easier to identify in a subset of critically ill patients undergoing cardiac surgery who have a higher risk of requiring prolonged mechanical ventilation including those with extended cpb runs, undergoing complex cardiac repairs, and those with preexisting comorbidities affecting the lungs such as chronic obstructive pulmonary disease. it would be worthwhile pursuing work in these difficult patients in whom these modes might have clinically noteworthy benefits. it would be ideal, therefore, to ventilate patients at the top of the volume - pressure curve, at high lung volumes, but without accompanying phasic changes. in the absence of facilities for oscillatory ventilation, increasing mean airway pressure without increasing peak pressure can only be achieved by prolonged inspiratory time (ti) in a pressure control mode. prolonged ti improves oxygenation. conversely, when ti exceeds the expiratory time, carbon dioxide removal is adversely affected leading to hypercarbia and respiratory acidosis. we know that generally patients tolerate respiratory acidosis very well, and we could allow this to happen as long as it does not affect the pulmonary vascular resistance significantly, which could be a factor in patients undergoing cardiac surgery. pressure control ventilation (pcv) previously used to be the last resort in ventilating postcardiac surgery patients with very noncompliant lungs and significantly high airway pressures on synchronized intermittent mandatory ventilation (imv). that has changed in most cardiac surgical intensive care units, with pcv now viewed as a good primary ventilation mode in patients with poor lung compliance, especially when combined with longer inspiratory times (inverse ratio pcv, or pcirv). tidal volume of approximately 6 ml / kg ideal body weight is now standard of care in mechanical ventilation of patients with reduced lung compliance. there is increasing recognition of the fact that tidal volumes of 10 ml / kg or more predispose cardiac surgical patients to organ failure and increased ventilation days, even in patients who do not have acute respiratory distress syndrome. especially, susceptibles are women and obese patients who tend to receive greater tidal volumes and have a greater incidence of consequent lung injury. intraoperative ventilation with low tidal volumes has also been suggested to be protective in noncardiac surgery settings. whether these benefits could also be translated to the cardiac surgical patient pcirv should probably be instituted earlier rather than later in postcardiac surgery patients with pulmonary complications, despite a paucity of studies confirming a substantial benefit from this mode. this mode is extremely uncomfortable for patients, who generally need to be heavily sedated, and often paralyzed. intensivists, however, prefer patients to be awake and interacting with the ventilator, leading to the development of newer modes to enable patients to breathe spontaneously even on pcirv. airway pressure release ventilation (aprv) was described in 1987 by stock et al. as a mode for acute lung injury while limiting airway pressures. bilevel ventilation is a mode in which spontaneous ventilation could be achieved in both phases of the high - low positive airway pressure cycle. the goal is to allow unrestricted spontaneous breathing so that need for excessive sedation or in some cases muscle relaxation can be avoided, thus enabling faster separation from mechanical ventilation. a study by rathgeber et al. compared duration of weaning between bilevel positive airway pressure (synonymous with bilevel), volume controlled (vc) imv, and vc continuous mandatory ventilation in patients undergoing cardiac surgery and demonstrated a marginal, yet significant decrease in weaning time. there appear to be no large published studies comparing aprv to conventional modes such as pressure support ventilation or t - piece, or to alternative modalities such as automatic tube compensation, proportional assist ventilation (pav), adaptive support ventilation (asv), or smartcare. however, putensen et al. have documented the benefits accrued from aprv, which include improvements in respiratory system compliance, pao2, cardiac index, and delivery of oxygen, in comparison to patients subjected to conventional mechanical ventilation with muscle paralysis. the increase in cardiac output in patients breathing spontaneously might presumably be due to decreased pleural pressure and elevated abdominal pressure. this in redistribution of splanchnic blood from abdominal viscera to inferior vena cava, ing in enhanced venous return. unlike other modes in which the physician presets a specific tidal volume or pressure, pav lets the patient determine the inspired volume and the flow rate. this mode mandates real - time estimation of resistance and compliance from which it determines the pressure to be generated. however, unlike the more commonly employed asv mode which can be used both in passive as well as actively breathing patient, pav can only be used in active patients. while to the best of our knowledge, there are no published studies exploring the effects of pav in patients undergoing cardiac surgery, in at least one randomized controlled study, asv appreciably reduced the time spent on mechanical ventilation in a population of postcardiac surgery patients and also reduced the incidence of unnecessary alarms and ventilator resetting by clinicians, leading to better utilization of resources. slutsky and ranieri indicate that the ventilator - induced lung injury may be reduced using lung protective mechanical ventilation. as research focuses on newer modes of mechanical ventilation that provide oxygenation and ventilation while reducing collateral pulmonary injury, there have evolved several advanced pressure control modes which seek to provide the benefits of both volume and pressure controlled ventilation. like vc ventilation, delivery of a reasonable tidal volume is guaranteed, and at the same time, like pressure controlled ventilation, this is done with an adjustment of the flow rate to avoid deleterious increases in plateau pressure. surprisingly, however, there are very few clinical trials related to their use in cardiac surgery, despite some evidence that some of them might limit the duration of postoperative mechanical ventilation and its attendant complications. while it is entirely possible to infer that unfamiliarity with many of these modes, as well as lack of substantial differences in tangible clinical end points from the noncardiac surgery settings might explain the reticence of researchers to explore these modes in the postcardiac surgery arena, larger studies are needed to clearly identify strategies that will in improved survival, decreased duration of mechanical ventilation, earlier icu discharge, earlier discharge from hospitals, and economic benefits. clinically, relevant differences in these parameters are likely to be easier to identify in a subset of critically ill patients undergoing cardiac surgery who have a higher risk of requiring prolonged mechanical ventilation including those with extended cpb runs, undergoing complex cardiac repairs, and those with preexisting comorbidities affecting the lungs such as chronic obstructive pulmonary disease. it would be worthwhile pursuing work in these difficult patients in whom these modes might have clinically noteworthy benefits.	lung atelectasis ing after cardiopulmonary bypass (cpb) can in increased intrapulmonary shunting and consequent hypoxemia. advanced pressure control modes of ventilation might have at least a theoretical advantage over conventional modes by assuring a minimum target tidal volume delivery at reasonable pressures, thus having potential advantages while ventilating patients with pulmonary atelectasis postcardiac surgery. however, the utility of these modes in the post - cpb setting have not been widely investigated, and their role in cardiac intensive care, therefore, remains quite limited.
since the 19th century, the natural environment has been considered important for ensuring a greater level of physical and mental health. gatherer past, present day humans have an innate affiliation with nature and living things. consequentially, nature is conducive to involuntary attention and does not require our directed attention, allowing recovery from mental fatigue and facilitating attention restoration. in the past decade, epidemiological studies in the netherlands have identified a positive correlation between improved health outcomes and amount of surrounding green space. subsequently, the diverse health benefits that maybe engendered by nature have become a focal point for research. two recent systematic reviews have concluded that exposure to nature is associated with improved mental well - being in comparison to indoor environments and synthetic or built environments. further support of these has been found in single studies where improvements in self - esteem, positive and negative mood, anxiety levels, and feelings of calmness and comfort have been observed. the studies reviewed by thompson - coon et al. did not report physiological variables, and bowler et al. both systematic reviews conclude that investigation regarding physiological changes during experiencing nature is lacking. however, there are individual studies investigating exposure to nature that identify changes in physiological health markers including decreased heart rate (hr) and decreased systolic (sbp) and diastolic blood pressure (dbp). further, changes in endocrine markers such as reduced adrenaline, noradrenaline, and cortisol, as well as enhanced autonomic control (indirectly measured using heart rate variability, hrv) have also been reported. these findings have also been observed in a controlled and simulated environment indoors where potential confounding factors such as weather, climate, sounds, and smells are eliminated. there is, however, a lack of rigorous research providing empirical evidence of the physiological mechanisms that exist with exposure to nature. thus, the impact of environment on cardiovascular health needs to be explored further using controlled environments and outcome measures which reflect such physiological changes, e.g., autonomic control. hrv provides a measure of autonomic nervous system (ans) functioning and can be used to explore physiological changes associated with nature exposure. the ans is important in controlling many bodily functions, and alterations are prevalent during relaxation and arousal. hrv is an easy to obtain, established noninvasive scientific and clinical measure and has prognostic value regarding cardiovascular health. it requires the measurement of interbeat differences in hr using either electrocardiogram (ecg) or mobile r - r interval monitors. the analysis of hrv reflects ans function by assessing the parasympathetic and sympathetic contributions to sino - atrial node regulation of hr. a higher hrv suggests an increased adaptability of the ans and is associated with better health. previous work that has observed hrv indicates that there is a tendency for higher hrv when nature is viewed in situ or simulated using projected images. the ans plays a central role in governing the response to stress and how the body recovers following a stressor. indeed, lane and thayer utilized functional magnetic resonance imaging (fmri) to examine the hypothesized heart brain connection and found concurrent associations between vagal influenced hrv and changes in blood flow through areas of the brain known to be involved in emotional responses, attention, and working memory. additionally, the prefrontal cortex has been observed to play a role in the top - down regulation of hrv, as demonstrated using direct current stimulation of the dorsolateral area of the prefrontal cortex during viewing of negative images compared to neutral images (images of nature were not used in this study). the relaxation and restorative effect of nature might help combat the rising incidence of psychological stress by providing a potential resilience tool. previous analysis of the restorative effects of nature suggest that participants recover faster from induced stress (in terms of hr) when, during the recovery, they view nature through a window or view projected scenes of natural environments. hr recovery in the latter study was purported to occur due to, in part, an enhanced recovery of parasympathetic activity. it is, however, unknown whether the benefits of nature on cardiovascular reactivity continue to occur after exposure has ceased. it is further unknown whether a longer exposure to nature elicits greater responses during the exposure. during nature exposure, the first 5 min elicits the greatest improvements in mood and self - esteem. physiological alterations in hrv also occur during the first 5 min of viewing slides simulating nature. to date , there is no evidence to suggest that longer exposure times elicit better physiological responses and in particular greater changes in hrv during nature exposure. this time course is important to establish; nature could be potentially used prior to a stressor and thus alter physiological outcomes in recovery from a stressor without the need of exposure to nature during the recovery period; i.e., nature could improve cardiovascular reactivity following a stressor by speeding the recovery process. the primary aim of this study was to investigate the effect that prior viewing of nature scenes had on ans function during recovery from a stressor. the hypothesis was that viewing nature scenes (composed of trees, grass, fields) prior to a stressor will lead to higher hrv in recovery when compared to viewing scenes of built environments (composed of man - made, urban scenes lacking natural characteristics). a secondary aim was to compare ans function during the first and second 5 min of a 10 min exposure to nature images to see if hrv changes were sustained. ethical approval from the university ethics committee was granted, and participants (n = 25, 7 males, 18 females) were recruited from university support staff who were independent from the research group conducting the study. informed consent was obtained from all participants who ranged in age from 19 to 65 years (mean sd, 36.08 10.42), with stature 169.05 7.33 cm (mean sd) and mass 71.66 13.11 kg (mean sd). due to a technical problem, data were not recorded for two participants (1 female, 1 male) during the stressor segment. the participants remaining (n = 23) were aged 36.91 11.09 years (mean sd), with stature 168.35 7.52 cm (mean sd) and mass 70.38 12.42 kg (mean sd). the study was a within - subject randomized crossover design with all participants visiting the laboratory on two occasions one week apart. visits were at the same time of day to eliminate any effect of circadian rhythm on the dependent variables. participants were free from symptoms of disease and were not using medication that would affect the cardiovascular or ans. room temperature was set at 21 c; artificial lighting was used, and blinds were drawn to avoid direct sunlight entering the room. each visit consisted of viewing one of two different groups of slides before being exposed to a mental stressor. participants were randomly assigned an order to either view the built scenes first or the nature scenes first. on arrival, participants completed a battery of psychological questionnaires (described below). following this, participants were told to rest for 15 min in the semisupine position, allowing hr and blood pressure (bp) measures to stabilize. following the period of rest, participants viewed a set of slides (either nature or built environment scenes) for a 10 min period. immediately poststressor, a further 5 min of physiological data were recorded to capture recovery. after recovery, participants completed the same set of psychological questionnaires. at the next visit, a week later, participants repeated this protocol and viewed the other set of slides. the slideshows depicting scenes of nature and built environments involved still photographs representing each of the environments. the photographs were chosen from a central pool of photographs used within the research group. the photographs (figure 1) depicting natural environments included natural elements, e.g., trees, grass, or plant life, and were devoid of any man - made structures, e.g., buildings, vehicles, roads. in contrast, the photographs depicting built environments contained man - made structures, e.g., houses, flats, office buildings, brick walls. a critical element of the scenes of built environments was the lack of natural features. twenty photographs were selected and collated in a microsoft powerpoint slideshow, with each slide being shown for 30 s. each slideshow lasted a total of 10 min, and photographs were shown in the same order for every participant. before viewing the slideshow , participants were given the instructions to try and imagine they were in the environment depicted on the screen. the term condition will be used from this point to describe the viewing of either the nature or built area slide sets. the two slide sets will be referred to individually as the nature condition and built condition. examples of images used in the slideshows to depict scenes of nature environments (a and b) and scenes of built environments (c and d). the mental stressor comprised a forward digit span test with an accompanying socio - evaluative threat and was designed specifically to elicit cardiovascular stress responses. a series of six numbers were displayed on a screen in front of the participant. after each series of six numbers, the participant had 10 s to write the numbers down in the correct order. the test is cognitively demanding and was designed to cause a stress response by adding a socio - evaluative component which has previously been reported to cause a stress response. to ensure a socio - evaluative threat, participants were informed that they would be carefully monitored during the test by the experimenter and a buzzer would sound when an incorrect answer was given. the buzzer was used twice in each session in order to achieve consistency irrespective of whether the participant had provided an incorrect answer. participants were informed at the end of the study that this was the case. they were asked if they were aware that they had in fact got the answer correct. all participants reported that they could not be sure that they had and had assumed they had answered incorrectly. an ecg modified lead ii configuration was used to record interbeat data throughout the protocol. bp oscillations were measured continuously throughout the protocol from a finger bp cuff attached to the middle finger of the nondominant hand (portapres, fms, finapres medical systems bv, netherlands). continuous recording of blood pressure in this manner allows mean blood pressure to be calculated and is advantageous over using momentary measures which are susceptible to white coat syndrome or cuff response. sbp and dbp were calculated using labchart 7 software (adinstruments, uk) to transform the waveform data. sbp is recorded as the highest pressure and dbp as the lowest pressure during a single cardiac cycle. respiratory frequency and depth were measured using a respiratory strap (pneumotrace, adinstruments, uk) fitted around the participant s chest. all data were measured at 1000 hz and collected by a powerlab 8sp (model ml785, adinstruments, uk) using labchart 7 software. data were then analyzed and averaged out into 5 min segments as it is most appropriate for hrv comparison that sections are equal in duration. these segments are categorized into: baseline (base), first 5 min of viewing slides (slides 1), second 5 min of viewing slides (slides 2), mental stressor (stress), and in recovery (recv). in the time - domain standard deviation of r - r intervals (sdrr) was chosen to reflect overall hrv contributed to by both sympathetic and parasympathetic systems. to reflect parasympathetic system activity alone, the root - mean - square of successive differences (rmssd) was used. responses were coded on a 03 scale giving a range for total self - esteem score of 0, representing the lowest level of self - esteem, and 30, the highest level of self - esteem. although designed as a trait measure of self - esteem, rosenberg s scale has previously demonstrated changes as a state measure of self - esteem due to exposure to natural environments. mood - related adjectives (e.g., enthusiastic, inspired, hostile, afraid) were rated on a scale of 1 to 5 for how well each adjective described participants current mood (1 = very slightly or not at all, 5 = extremely). self - esteem and mood were measured at the start and end of the protocol immediately after the recovery period. to test the primary hypothesis, a repeated measures anova was used to identify whether recovery relative to baseline differed between conditions for rmssd, sdrr, hr, sbp and dbp. for the secondary question, changes from baseline values were first calculated to explore if the expected short - term effect during the first 5 min of viewing is repeated in the second 5 min. data were not normally distributed (assessed by kolomogorov - smirnov test for normality); therefore, the friedman s test was performed with posthoc wilcoxon tests to explore significant differences. repeated measures anova was also used to examine whether the stress response (hr and bp only) compared to baseline differed between conditions. to assess if breathing frequency and depth was kept constant throughout the protocol, repeated measures anova was used. to identify whether changes in psychological measures changed from baseline to poststress and whether this differed between viewing condition, paired t tests were used to identify if performance in the mental stress task differed between condition and visit order. significance was set at an alpha of 0.05, where appropriate posthoc paired t tests were used to investigate significant effects between conditions with a bonferroni corrected alpha level. all data are normally distributed except for rmssd (assessed by kolomogorov - smirnov test for normality). planned paired t tests on baseline data (table 1) showed there were no differences between the viewing conditions at baseline for rmssd, sdrr, hr, sbp, dbp, or breathing frequency or depth (p > 0.05). therefore, any subsequent differences are likely attributed to interventions and actions within the protocol. in all analyses, rmssd, root mean square of successive differences; sdrr, standard deviation of r - r intervals; self - esteem quantified using rosenberg s self - esteem, low scores = low self - esteem, range 030; negative and positive mood taken from the positive and negative affect scale (panas), low scores = low negative or positive mood, range 040. for the primary research question, repeated measures anova revealed an interaction effect for rmssd between the nature condition and the built condition over time (f1, 22 = 8.72, p = 0.007, p = 0.28). this demonstrates increased levels above baseline during recovery for the nature condition compared to recovery in the built condition (figure 2) where levels dropped below baseline. repeated measures anova on sdrr determined a main effect of view (f1, 22 = 11, p = 0.003, p = 0.33) and time (f1, 22 = 7.7, p = 0.011, p = 0.26), with levels increasing during recovery irrespective of view and higher sdrr in the nature condition irrespective of time. mean (sd) heart rate and heart rate variability recovery from stress compared to baseline: , main effect for time (p < 0.05); , interaction effect (p < 0.05); , main effect for view (p < 0.05). for the secondary research question, exposure time was split in half to see if ans function during the first 5 min differed to the last 5 min according to condition (figure 3). test for multiple comparisons identified significant differences for sdrr during views ( = 8.06, p = 0.045 ). change in sdrr from baseline was found to be significantly greater in the nature condition (median = 1.20) compared to the built condition (median = 2.44) during the first 5 min (z = 2.56, p = 0.011). heart rate and heart rate variability means sd as change from baseline for the first 5 min and last 5 min of viewing: , significant difference between conditions. repeated measures anova revealed a main effect for time on hr (f1, 22 = 27.3, p = 0.001, p = 0.10) describing an increase from baseline to stress period (67.8 1.8 vs 73.4 1.8 bpm). there was also a main effect of time on sbp (f1,22 = 34.1, p = 0.001, p = 0.61) and dbp (f1,22 = 9.7, p = 0.005, p = 0.31) both revealing an increase from baseline to stress period (117.6 2.6 vs 128.8 3.1 mmhg and 60.1 1.4 vs 63.1 1.5 mmhg, respectively). change in these parameters supports the effectiveness of the mental stressor. repeated measures anova determined that time had an effect on breathing rate (f4, 84 = 6.2, p = 0.001, p = 0.23). pairwise comparisons revealed that only the stress period was different from baseline (18.3 0.7 vs 14.3 0.7, p < 0.0001). repeated measures anovas revealed no main effect for condition on hr (f1, 22 = 1.9, p = 0.187, p = 0.08), sbp (f1, 22 = 1.5, p = 0.231, p = 0.07), dbp (f1, 22 = 0.0003, p = 0.985, p = 0.00001), or breathing rate (f1, 21 = 0.9, p = 0.356, p = 0.04) suggesting that viewing condition did not influence the stress responses. a paired t test showed no difference was observed in the scores for the mental task between the conditions. however, a paired t test revealed that the second visit showed a significant improvement in the population mean score from 91.1 to 97.7, (t22 = 4.12, p = 0.001) out of a possible 102. a repeated measures anova determined an interaction effect on self - esteem (f1, 21 = 5.4, p = 0.03, p = 0.21) describing an enhanced self - esteem from baseline to poststress for the nature condition (table 2). conversely, the built condition showed deterioration in self - esteem from baseline to poststress values. repeated measures anova revealed a main effect of time on negative mood scores (f1, 21 = 7.3, p = 0.013, p = 0.26) describing a decrease in negative mood from baseline to poststress (table 2). self - esteem, quantified using rosenberg s self - esteem, low scores = low self - esteem, range 030; both positive and negative mood quantified using positive and negative affect scale (panas), low scores = low positive or negative mood, range 1050. significant interaction effect (p < 0.05). significant main effect for time, pre to post (p < 0.05). the majority of research exploring the impact of nature on ans mechanisms has included an exercise component which limits the drawn about the contribution of the nature component. as exercise has such positive effects on health parameters, it is often hard to isolate the effects solely from the nature component. therefore, this study looks at the nature contribution to ans function without complicating the effects by combining it with exercise. the main finding of this study was that hrv as a marker of ans function increased during stress recovery, if nature scenes were viewed prior to a stressor, compared to built scenes. this is the first study to suggest that simply viewing scenes of nature prior to a stressor enhances recovery of ans function poststressor. the interaction of nature and recovery from stress has been studied previously. however, previous research has assessed the restorative effects of viewing or interacting with nature during the actual recovery period following a stressor. for example, viewing video footage of nature scenes for 10 min directly after being exposed to a film of stressful images increased heart period (i.e., decreased hr), suggested to be due to enhanced parasympathetic system activity. viewing nature through a window during a 5 min rest period following cognitive tasks was also more effective at reducing hr. both authors postulate that these observations are a consequence of cognitive recovery or attention restoration occurring while nature scenes are viewed. the of the present study suggest greater hrv during viewing nature scenes as contributed to by both sympathetic and parasympathetic systems. it is only during the recovery period that parasympathetic activity alone is greater in the nature views condition. therefore, viewing nature scenes may encourage future healthy stress responses and recovery patterns and could act as a vital tool in preventive health. research shows that a 5 min dose of nature offers the greatest increases in self - esteem and mood. physiologically, 5 min of viewing images of nature is known to increase hrv. however, it is unknown whether an additional 5 min would enhance the initial changes that occur in the first 5 min dose. within a laboratory setting , it appears that the second 5 min of exposure is less effective in inducing hrv changes. the strength of the current study is the use of 5 min segments for hrv analysis which is recommended in short - term analysis, i.e., less than 24 h. furthermore, the division of exposure to nature images into 5 min segments enabled the observation of increased hrv as anticipated in an initial 5 min dose, thus supporting previous work. comparisons in the current study suggest an additional 5 min of exposure to nature does not enhance the greater hrv seen during the initial 5 min dose. in agreement with previous research, this finding suggests enhanced self - esteem associated with viewing nature shows robustness against exposure to a mild stress. in the current study, there was no change in mood associated with condition although this has been shown in previous studies. the changes in mood observed in these studies were measured using the profile of mood states (poms). poms contains 5 subscales associated with negative mood and 1 subscale for positive mood. this restricts poms to predominantly reflect changes of negative mood rather than positive mood. in the current study, negative affect, measured using panas, showed negative mood improved irrespective of condition, therefore not replicating previous observations using poms the use of positive affect scores from panas was to ascertain if changes in positive mood occur that may not be so clearly identified using poms. positive mood did not differ between conditions nor did it change over time. in contrast, a meta - analysis of five studies did observe improvements in positive affect suggesting the manipulation in the current study was not strong enough to elicit positive changes. the use of panas in laboratory research of this nature might not be appropriate. previous literature lacks discussion as to the potential mechanisms behind observed changes in physiological function while viewing nature. one potential mechanism to explain alterations in physiological measures could be attributable to the restorative properties of the nature scenes. the concept of attention restoration occurring after exposure to nature has previously been demonstrated by way of improved performance in attention related cognitive tasks. viewing scenes of nature for 10 min, following a period of mentally fatiguing tasks, improved performance in a backward digit - span memory task. in the current study, there were no such observations of altered cognitive ability, i.e., performance in the mental task, with different viewing conditions. this may be attributable to the lack of a mentally fatiguing task prior to the intervention, but the nature scenes acted as an effective buffer to ans function during recovery. to date, there are only a handful of studies that have measured or inferred changes in the ans associated with nature. we suggest that the different components of the environmental stimulus, e.g., visual, cognitive, emotional, and restorative properties, induce changes in the regulation of different areas throughout the brain thus altering ans function. the findings of this study, alongside previous studies, suggest a top - down mechanism originating in higher centers of the brain. evidence for this was in part obtained from a study which utilized fmri while viewing urban scenes. the urban scenes caused increased activity in the amygdala compared to viewing nature scenes. this action is likely to cause alterations in ans control such as those seen in the current study, through changes in parasympathetic and sympathetic outputs. inhibition of the parasympathetic nervous system arises from the frontal cortex, and the pathways pass through the amygdala and then to the nucleus tractus solitarii and nucleus ambiguus. the prefrontal cortex is prominent in threat - avoidance situations, causing inhibition on hr via the vagus nerve. therefore, during periods of threat, parasympathetic activity is decreased (increasing hr). the of the current study suggest the absence of threat during nature viewing, without decreases in parasympathetic activity, while during built views overall variability decreased in the first 5 min. this interaction may be primarily due to alterations in both the frontal cortex and the amygdala. visual properties of an image may also play a role, as the composition of a picture can alter activity in the visual cortex. images of nature are less aversive and uncomfortable when examining their spectral properties compared to built images. indeed, recent research suggests that the primitive characteristic of color, in particular the greenness , of a nature image is associated with improved mood. through color perception and reduced impact on the visual system, images of nature may evoke lower activity in the amygdala and visual cortex culminating in increased parasympathetic activity as seen in the current study. it is unknown how long the physiological changes that nature evokes are maintained, but it will be vital to explore this further, especially if nature is to be considered as a therapy. in the current study, unlike previous studies, nature exposure was experienced 10 min before the stressor, not during the stressor or immediately following the stressor. the evidence from this study suggests that there does indeed appear to be a buffering effect of nature. a stronger stimulus, i.e., within the environment itself, may prolong the buffering effect and also induce greater changes in cardiovascular measures. this may also be the case when nature is combined with exercise (green exercise). exercising while viewing nature reduces bp in the 5 min following the exercise period in comparison to viewing built images. these effects again may be even greater following real exposure to nature. indeed, recent research suggests that adrenaline, noradrenaline, and bp still remain reduced in the evening following a daytime walk in a forest field. interestingly, forest walking increases natural killer cell activity for a period of 30 days in males and 7 days in females. the impact of individual beliefs on the regulation of emotions when viewing the different environments, and how this effects physiological modulations, is unknown and could pose a mediating factor to the effectiveness of viewing nature on improving stress recovery. in order to quantify individual relationships with nature, the nature relatedness scale could be used to indicate experience, beliefs, and contact with nature. complementary information could be gained by noting home postcode, and thus, the surrounding area could be assessed in terms of land usage to explore the potential impact it may have on participants perception of nature. the present study collected postcode data but does not have a sufficient population size to draw about all potential subsets. the majority of studies to date, including the present study, use extreme examples to depict natural and urban environments in order to examine the influence of nature. investigating a greater variety of environments (including more urban green spaces) would add population level validity to the and account for individual landscape preferences. the restorative properties of nature (images or within the location itself) may evoke different psychological, cognitive, and physiological responses. further studies would benefit from the inclusion of a questionnaire to assess how restorative the scenes or places are perceived to be, as suggested by hartig, mang, and evans, in combination with physiological and psychological responses to these different images. the current study suggests that nature itself may evoke physiological responses, which may be in part driven by psychological reactions and restorative properties of nature. furthermore, the increase in parasympathetic activity in the recovery from a stressor may help to counteract a buildup of psychological stress and thus reduce the impact of stress on physical and mental health. this would likely occur by nature images encouraging a healthier stress recovery pattern. if nature increases autonomic recovery to stress and thus is an effective coping mechanism, this provides an argument for the need for more nearby nature. a green view through a workplace window , small pockets of greenspace in the home and workplace, and accessible local parks could be effective tools in altering ans control of the heart. the buffering effect of nature could have particular relevance for the workplace where it may be beneficial to utilize nature during the lunch break, prior to a stressful afternoon, to help enhance recovery of autonomic function.	a randomized crossover study explored whether viewing different scenes prior to a stressor altered autonomic function during the recovery from the stressor. the two scenes were (a) nature (composed of trees, grass, fields) or (b) built (composed of man - made, urban scenes lacking natural characteristics) environments. autonomic function was assessed using noninvasive techniques of heart rate variability; in particular, time domain analyses evaluated parasympathetic activity, using root - mean - square of successive differences (rmssd). during stress, secondary cardiovascular markers (heart rate, systolic and diastolic blood pressure) showed significant increases from baseline which did not differ between the two viewing conditions. parasympathetic activity, however, was significantly higher in recovery following the stressor in the viewing scenes of nature condition compared to viewing scenes depicting built environments (rmssd ; 50.0 31.3 vs 34.8 14.8 ms). thus, viewing nature scenes prior to a stressor alters autonomic activity in the recovery period. the secondary aim was to examine autonomic function during viewing of the two scenes. standard deviation of r - r intervals (sdrr), as change from baseline, during the first 5 min of viewing nature scenes was greater than during built scenes. overall, this suggests that nature can elicit improvements in the recovery process following a stressor.
trocar - site incisional hernias and their complications are reported in 1% to 6% of patients. such hernias are attributed to the difficulty of applying standard suturing techniques to wound closure. the deschamps needle has a handle and a tip (sharp or blunt), with an opening to pass suture. disposable needles are obviously sharp, but can bend on the needle holder and break in a deep, small incision. the deschamps needle is a rigid, noncutting instrument that can be forced through fascia and peritoneum (around the surgeon 's fingertip) avoiding loss of pneumoperitoneum. we have used the deschamps needle since 1992 in all laparoscopic procedures. we close 10-mm and 5-mm trocar sites and have not observed wound dehiscence or hernias at these sites. disposable, single - use devices vary in price from $ 30 to $ 75 each. the deschamps needle is sold in italy at approximately $ 35 each. considering that it may have been in the trays of most operating rooms for years (as in our case), and the number of procedures performed, we conclude that the real cost of this instrument is almost negligible with the wide diffusion of laparoscopic surgery for many abdominal procedures, trocar site incisional hernias have become more frequent (1% to 6%), along with their related complications (bowel or omentum incarceration and richter 's hernia). most complications occur with 10-mm trocars, but some have occurred at 5-mm trocar sites. the occurrence of these complications has been attributed to the difficulty in applying standard suturing techniques for wound closure. every surgeon who has performed traditional closure of fascia and peritoneum has found that closure of all layers in small, deep wounds is sometimes impossible, frequently unsafe, and never quick and easy. this is especially true in obese patients. immediately after the appearance of these reports of trocar site complications in gynecologic, urologic, and general surgery literature, many authors began to publish papers regarding original techniques and new devices to obviate the problem some have suggested that a foley catheter, spinal needle, hypodermic needle, or urologic instrument could solve the difficulties in closing trocar sites; others developed new devices. the debut of numerous different approaches to closing trocar sites could have been controversial and disorientating to some, but elashry and associates in 1996, published a perspective, randomized trial that compared many of these techniques. their final statement concluded that the preferred method of trocar site closure was one that utilized a new, disposable instrument. several companies have developed safer and smaller trocar tips while others have focused on developing new, disposable (and frequently expensive) wound closure instruments. at the beginning of our laparoscopic experience in 1990 we faced the problem of how to close trocar sites as did many of our colleagues. we struggled, we were frustrated, we doubted, and sometimes we renounced the techniques then available. although we experienced only two uncomplicated umbilical hernias at trocar sites in our initial period (both following wound infections), we were very concerned about this problem. then one day the chief nurse of our operating room came with an old instrument from the forgotten storeroom. this instrument was a deschamps needle (figure 1), which was extensively used in the past for en masse ligature of pedicles. as shown in figure 2, it has a handle and a tip, sharp or blunt, with a hole to pass a suture. it is probably present, and maybe forgotten, in most of the operating rooms in europe. the blunt type is very effective, in our experience, for the closure of trocar wounds. disposable needles although obviously sharp, can bend on the needle holder, and can sometimes break in a deep, small incision. the deschamps needle is a rigid, noncutting instrument that can be forced through the fascia and peritoneum in and out, turning (bending) around the finger tip of the surgeon. loss of pneumoperitoneum is thus avoided (figures 3a, 3b, 3c, 3d) and a full thickness closure of the trocar sites is accomplished. several techniques are available that are suitable for use in 5 mm incisions and in the deep fascial wounds of obese patients. the closure is performed under direct vision through the scope, and the tactile feedback provided by the surgeon's finger allows quick, safe passage of the needle. the last trocar site, after removing the scope, is closed in the same manner, facilitated by maintenance of the pneumoperitoneum. no omentum or bowel is included in the suture, because passage of the instrument is felt by the surgeon's finger. deschamps needle passes through the fascia and peritoneum in and out around the finger tip of the surgeon. since 1992, we have used the deschamps needle in all of our laparoscopic procedures, even when only 5-mm trocars were used. to date, we have had no cases of wound dehiscence or herniation in approximately 1400 procedures. concerning financial matters, disposable, single - use devices have a price that varies from $ 30 to $ 75 per unit, and reusable instruments, frequently sold in a set of various sizes, can cost up to $ 2,500. the deschamps needle is sold in italy at an average cost of $ 35 each. when one considers that the deschamps needle has been on the trays of most operating rooms for many years (as in our case) and when one considers the total number of procedures already performed, the real impact of this instrument on surgical costs is inconsequential. we conclude that the deschamps needle is a safe, cost - effective, readily available device to accurately close fascial defects that arise from the use of trocars and cannulas in laparoendoscopic surgery.	objective: trocar - site incisional hernias and their complications are reported in 1% to 6% of patients. such hernias are attributed to the difficulty of applying standard suturing techniques to wound closure. we report our experience with a simple device, the deschamps ligature needle.methods:the deschamps needle has a handle and a tip (sharp or blunt), with an opening to pass suture. the blunt tip is very effective for closing trocar sites. disposable needles are obviously sharp, but can bend on the needle holder and break in a deep, small incision. the deschamps needle is a rigid, noncutting instrument that can be forced through fascia and peritoneum (around the surgeon 's fingertip) avoiding loss of pneumoperitoneum. a full - thickness closure is accomplished. we perform closure under direct vision through the scope. tactile sense is provided by the surgeon's finger. the last trocar site is closed in the same manner without the scope.:we have used the deschamps needle since 1992 in all laparoscopic procedures. we close 10-mm and 5-mm trocar sites and have not observed wound dehiscence or hernias at these sites.:the deschamps needle is effective in preventing incisional hernias and wound dehiscence. it is cost - effective. disposable, single - use devices vary in price from $ 30 to $ 75 each. the deschamps needle is sold in italy at approximately $ 35 each. considering that it may have been in the trays of most operating rooms for years (as in our case), and the number of procedures performed, we conclude that the real cost of this instrument is almost negligible
chagas disease, also known as american trypanosomiasis, an endemic parasitic disease caused by a flagellate protozoan (trypanosoma cruzi), is highly prevalent throughout latin america. its major clinical manifestation is the chronic chagasic cardiomyopathy (ccc), which affects 1/3 of the chronically infected patients and may present severe symptoms and signs, such as congestive heart failure, thromboembolic phenomena, cardiac arrhythmias, and sudden death. pathological processes in the heart include mononuclear inflammatory infiltration, focal myocarditis, epicarditis, and neuroganglionitis, associated with variable focal fibrosis and a paucity of parasites, which is poorly correlated with myocardial inflammatory infiltration. the pathogenesis of ccc includes the balance between parasite invasiveness and the host immune response, particularly the th1/th2 balance, which affects the resistance / susceptibility to t. cruzi infection. even though an imbalanced, excessive production of th1 proinflammatory cytokines is critical to control of the parasite levels in blood and tissue, this type of response can also be capable of destroying functional cardiomyocytes and intracardiac autonomic neurons this autonomic denervation, involving mainly parasympathetic postganglionic neurons causes a marked cardiac autonomic dysfunction , which may be involved in initiating life - threatening cardiac arrhythmias and sudden death. curiously, this vagal parasympathetic autonomic dysfunction is strongly associated with inflammatory infiltration and immune activation not only in chagas heart disease but also in other types of cardiopathy. although these data support a direct role of the immune system in mediating both cardiac and autonomic nervous disturbances, recent data indicate that changes in the autonomic nervous system also affect the pattern of immune response and inflammation in several cardiac and noncardiac disease states , including chagas disease. both the sympathetic and parasympathetic branches of the autonomic nervous system can exert substantial modulatory effects on the immune response, mainly by inhibiting the th1 response profile. this new evidence also indicates that cardiac sympathetic and particularly parasympathetic autonomic denervation / dysfunction may also contribute to an increased inflammatory response and possibly to enhanced parasite elimination. data from our laboratory collected in the context of acute chagas disease in mice has recently confirmed that cardiac autonomic denervation / dysfunction might contribute to increased inflammation. taking into account the concepts described above, new therapies based on manipulations of vagal neuroimmunomodulation of the heart may be beneficial for treating heart diseases in general and chronic chagasic cardiomyopathy in particular. for example, therapeutic approaches for increasing cardiac vagal function, thereby potentiating or stimulating the vagal anti - inflammatory reflex, might have a positive impact on chronic chagasic cardiomyopathy. in fact, for other cardiopathies, these strategies, in both an experimental and clinical context, including chronic electric vagal stimulation or pharmacological potentiation, have successfully improved cardiac outcomes. pyridostigmine bromide, an anticholinesterasic agent that has been used for many years to treat myasthenia gravis, exhibits protective cardiovascular effects during short - term administration that lead to a reduction of cardiovascular risk markers and an improvement of autonomic dysfunction. by potentiating vagal parasympathetic function, this compound is thought to provoke sinus bradycardia, to reduce av nodal conduction and the refractory period of action potentials and to increase the cardiac excitation threshold, among other direct cardiac effects. it is also believed that pyridostigmine bromide, via potentiation of the cholinergic anti - inflammatory pathway, causes a reduction in myocardial inflammation and fibrosis associated with an improvement in cardiac hypertrophy and remodeling. however, there have been no reports regarding the effects of pyridostigmine bromide on the chronic phase of chagas heart disease. therefore, the main aim of the present study was to evaluate the effects of cholinergic potentiation with the anticholinesterase agent pyridostigmine bromide on electrocardiographical, cardiac autonomic, histological (inflammatory infiltration and fibrosis), immunological, and parasitological parameters in c57bl/6j mice infected with the romildo strain of trypanosoma cruzi during the chronic phase of chagas heart disease. all experiments were performed on wild - type c57bl/6j mice obtained from the animal facility of the department of physiology of triangulo mineiro federal university, uberaba, mg. all animals were males, weighed 2030 g, and were maintained in the animal facility of the department of physiology at the triangulo mineiro federal university on a rodent diet (nuvilab cr1, nuvital nutrientes ltda, curitiba, pr, brazil) and were given water ad libitum until the beginning of the experimental protocols. all the experiments were carried out according to the principles of laboratory animal care formulated by the national society for medical research, england, and the guide for the care and use of laboratory animals published by the us national institutes of health (nih publication number 85 - 23, revised in 1996). all procedures were also submitted to and approved by the commission for ethics in the use of animals in research of the triangulo mineiro federal university. to induce experimental chagas disease, mice were intraperitoneally inoculated with 15,000 trypomastigote forms of the romildo strain of t. cruzi. an additional set of animals matched for gender and weight received intraperitoneal injections of the vehicle, and these were designated as the control noninfected group. after inoculation, all of the animals were observed at least twice daily to monitor their general state and to assess mortality during the acute phase. on the 12th day after inoculation, levels of parasitemia were measured by the microhematocrit method for the peripheral tail blood of all inoculated animals, according to brenner's technique, to confirm infection. all subsequent surgical procedures and experimental protocols were performed at the 5th and 6th months of infection, during the chronic phase of infection, which is characterized by light tissue invasion and low mortality. the experimental groups were divided according to the presence of chagas disease and the administration of pyridostigmine bromide during the month from the 5th to the 6th month of observation (chronic phase), as described below. the following groups were investigated: group i con - nt: wild - type c57bl/6j mice were injected with vehicle as a control (con), not treated (nt) with pyridostigmine bromide and evaluated after six months at the end of the observation period; group ii con - pyrido: wild - type c57bl/6j were injected with vehicle as a control (con) and treated with 30 mg / kg pyridostigmine bromide (pyrido), an anticholinesterase agent, dissolved in tap water, for 30 days from the 5th to the 6th month of observation; group iii chg - nt: wild - type c57bl/6j mice were inoculated with 15,000 trypomastigote forms of the romildo strain of t. cruzi (chg), not treated (nt) with pyridostigmine bromide and evaluated after six months at the end of the observation period; group iv chg - pyrido: wild - type c57bl/6j mice were inoculated with 15,000 trypomastigote forms of the romildo strain of t. cruzi (chg) and treated with 30 mg / kg pyridostigmine bromide (pyrido), an anticholinesterase agent, dissolved in tap water, for 30 days from the 5th to the 6th month of observation. the solution drinking volume was monitored daily, and the pyridostigmine bromide concentration (approximately 0.084 mg / ml) was adjusted daily according to the drinking volume to maintain a daily mean ingested dose of 30 mg / kg. immediately prior to inoculation (ecg1), at the 5th (ecg2, immediately prior to the pyridostigmine bromide treatment) and at the 6th months of observation (ecg3, at the end of the experimental protocol, prior to the surgical procedures), all of the animals were submitted to a conventional ecg study under tribromoethanol (250 mg / kg, i.p .) anesthesia. needle electrodes were placed under the skin to record the conventional bipolar limb leads (i, ii, and iii), the unipolar limb leads (avr, avl, and avf), and the unipolar precordial (chest) leads (va : the needle was placed immediately to the right of the sternum in the 4th intercostal space ; vb : the needle was placed just to the left of the sternum in the 4th intercostal space ; and vc ; the needle was positioned in the 5th intercostal space at the midaxillary line). to avoid errors in the positioning of the leads, the ecg was recorded using an ecg amplifier (model 8811a, hewlett - packard med . ma, usa) coupled to a 12-bit analogue - to - digital interface (di-720-usb, dataq instruments, inc ., akron, oh, usa) at the sampling rate of 3 khz on an ibm personal computer with the analysis performed by windaq - pro+ software (dataq instruments, inc ., the intervals and wavelengths ( ms) were calculated automatically using customized software following wave identification and cursor placement. the ecg tracings were consistently analyzed by the same individual, who was blinded to the study protocol. the following ecg parameters were examined: rr interval (rri), p wave duration (pd), pr interval (pr), qrs duration (qrsd), qt interval (qt), and corrected qt interval (cqt, defined as the qt interval corrected for heart rate using bazett 's equation, where the corrected qtc was equal to qt ( in s)/rr (in s) ). in contrast to humans, the t wave in small rodents is not well characterized and appears as a shoulder of the qrs complex. accordingly, to measure the qt interval, we used the apex of the t wave, which can be determined with high accuracy. the ecg parameters were determined from each lead and were averaged. additionally, the presence of cardiac arrhythmias and atrioventricular or intraventricular blockades, among other alterations, was analyzed by visual inspection of ecg tracings by a blinded ecg specialist. after the third ecg recording, at the 6th month of observation, the animals were reanesthetized with tribromoethanol (250 mg / kg, i.p .), and a pair of stainless steel electrodes were implanted inside the subcutaneous tissue to collect chronic recordings of conventional bipolar limb ecg lead ii. the animals were also cannulated with polyethylene tubing placed in the jugular vein for drug administration. after the surgical procedures, the animals were left to recover in individual cages for at least 4872 h. after 4872 h of surgical recovery and in the absence of anesthesia, the electrodes were connected to an ecg amplifier (model 8811a, hewlett packard, waltham, ma, usa), and the baseline ecg was sampled continuously (3 khz) for a period of 30 minutes with a personal computer (ibm / pc) equipped with a 12-bit analogue - to - digital interface (di-720-usb, dataq instruments, inc ., all animals were always recorded between 8:00 am and 5:00 pm in a quiet condition and in a freely moving state . the time series of rr intervals derived from these chronic ecg recordings were used to study the cardiac autonomic modulation in heart rate variability . from the baseline chronic ecg recordings ( 30 minutes), the rr interval time series were derived automatically by the detection of the r wave peaks using customized linear analysis software, which was kindly provided by dr. the time series of the rr intervals were divided into contiguous segments of 300 beats overlapping by half (welch protocol). after calculating the mean and variance for each segment, a model - based autoregressive spectral analysis was performed, as described elsewhere. briefly, a model of the oscillatory components present in the stationary segments of the beat - to - beat time series of the rr intervals was calculated based on the levinson - durbin recursion, and the order for the model was chosen according to akaike's criterion. this procedure allows for the automatic quantification of the center frequency and power of each relevant oscillatory component in the time series. the oscillatory components were labeled as very low frequency (vlf), low frequency (lf), or high frequency (hf) when the central frequencies were within the bands of 0.010.10 hz, 0.101.00 hz, or 1.005.00 hz, respectively. the power of the lf and hf components of the heart rate variability was also expressed in normalized units, which were obtained by calculating the percentage of the lf and hf variability with respect to the total power after subtracting the power of the vlf component (frequencies < 0.10 hz). the normalization procedure tends to minimize the effect of changes in the total power on the absolute values of the lf and hf variabilities. after 30 minutes of baseline chronic ecg recording, half of the animals were given an intravenous injection of atropine sulfate (1 mg / kg, i.v .) followed by an injection of propranolol (1 mg / kg, i.v .) 15 minutes later, whereas the other half received injections in the reverse sequence (propranolol followed by atropine sulfate). this procedure allowed for the quantification of the cardiac parasympathetic and sympathetic autonomic effects, in the former and latter group, respectively, measured as the differences between heart rate (hr) after atropine and baseline hr (parasympathetic effect) or as the differences between hr after propranolol and baseline hr (sympathetic effect), as well as the measurement of intrinsic pacemaker heart rate (ihr), quantified as the heart rate after the double blockade with atropine sulfate followed by propranolol or propranolol followed by atropine sulfate. at the end of the experimental protocol , all animals were euthanized with an excess dose of sodium thiopental (100 mg / kg, resp ., i.p .), and the chest cavity was then opened to remove the heart for histopathological analysis. to evaluate the extent of inflammatory infiltration, tissue damage, fibrosis, and parasite nests, excised hearts from all animals were cleaned in 0.9% saline solution and fixed in phosphate - buffered 10% formalin solution for 48 h. after embedding the samples in paraffin, five 57 m thick longitudinal (four - chamber) sections of the hearts were stained with hematoxylin - eosin and analyzed using an upright light microscope (axiolab, carl zeiss inc . the inflammatory infiltration and parasite nests were characterized using a semiquantitative approach and the scoring system described by chapadeiro et al . . a global myocardial inflammation score was defined for each animal as the sum of the scores from different regions of the heart . to quantify the fibrotic area in the myocardium, contiguous longitudinal sections of the hearts were stained with picrosirius red, which binds the collagen present in the tissue matrix, and the slides , germany). to enhance the visualization of parasite nests or antigens in cardiac tissue, an immunohistochemistry technique based on the detection of the diaminobenzidine (dab-) the chromogen was generated by a secondary antibody labeled with peroxidase, which binds to a primary antibody against t. cruzi antigens. after antibody labeling, peroxidase reaction, and costaining with hematoxylin, slides were analyzed with an upright light microscope (axiolab, carl zeiss inc . serum samples for each animal were collected by means of cardiac puncture to perform cytokine profiling ( ifn-, tnf-, il-2, il-4, il-5, and il-10) via elisa or the cytometric bead array (cba) technique serum concentrations of ifn- and il-10 were measured by elisa using pairs of monoclonal antibodies in accordance with the manufacturer's specifications (bd pharmingen). briefly, high - affinity 96-well plates (nunc, roskilde, denmark) were sensitized with cytokine - specific monoclonal antibodies followed by blocking with pbs containing 2% bsa (sigma). the sera and recombinant cytokines were then added, and the plates were incubated for 4 h at room temperature. the plates were washed and incubated with 1 g / ml biotinylated anticytokine monoclonal antibody at 37c for 2 h followed by washing and incubation with alkaline phosphatase - conjugated streptavidin at 37c for 2 h. the reaction was developed using disodium - p - nitrophenyl phosphate (sigma) in diethanolamine buffer. absorbance was measured at 405 nm in a microplate reader (bio - rad, 2550 reader eia, ca, usa). the cytokine concentration was calculated using a linear regression analysis of the absorbance values obtained for the recombinant cytokines, and it was expressed as pg / ml. the sensitivity of the tests ranged from 2 to 20 pg / ml. measurement of the cytokines tnf- and il-2 (th1 cytokines) and il-4 and il-5 (th2 cytokines) in serum samples was performed using the cytometric bead array (cba) kit (bd biosciences, usa) according to the manufacturer's instructions. briefly, 50 l of bead populations with discrete fluorescence intensities and coated with cytokine - specific capture antibodies was added to 50 l of mice sera and 50 l of phycoerythrin - conjugated anti - mouse th1/th2 cytokine antibodies. simultaneously, standards for each cytokine (05000 pg / ml) were mixed with cytokine capture beads and the phycoerythrin - conjugated reagent. the vortexed mixtures were incubated for 3 h. beads were washed and analyzed using flow cytometry (facscalibur, bd biosciences, usa). the quantity (pg / ml) of each cytokine was calculated using cellquest software (bd biosciences, usa). the lower limit of detection ranged from 1 to 2.1 pg / ml for different cytokines. for parasite dna isolation from the blood, total blood samples of mice were collected in tubes containing 6 m guanidine hcl with 0.2 m edta (ph 8) (v / v). dna was extracted using the phenol - chloroform - isoamyl alcohol method according to macedo et al.. for parasite dna isolation from the heart, half of the hearts excised at the end of the experimental protocol were homogenized, and dna was extracted by the alkaline lysis method. pcr specific for t. cruzi was performed according to wincker et al. using the following primers to amplify a fragment of 330 bp: 121 (5-aaa taa tgt acg gg(g / t) gag atg cat ga-3 ) and 122 (5-ggt tcg att ggg gtt ggt gta ata ta-3) from a nonvariant region of the kinetoplast dna minicircles of t. cruzi. the cycling conditions were as follows: 95c for 5 minutes followed by 35 cycles at 95c for 1 minute and 65c for 1 minute. the products of the reaction were revealed by electrophoresis in a 6.0% polyacrylamide gel and stained with silver nitrate, which was photographed with a digital camera. all numerical data are expressed as the means (s.e.m .), whereas the semiquantitative data from the histological examinations are expressed as the medians and the 25th and 75th percentiles. according to the normality and variance homogeneity of the distribution, a parametric statistic, such as two - way anova followed by tukey's multiple comparison test, or a nonparametric test, such as the mann - whitney test, parasite detection in peripheral tail blood samples by the microhematocrit technique performed on the 12th day after infection was positive for all t. cruzi - inoculated animals and negative for all control noninfected mice. electrocardiographical parameters measured in the first ecg recording (ecg1) at the beginning of the experimental protocol before t. cruzi inoculation and treatment did not show any significant difference among all experimental groups, as expected. however, after five months of infection and before the pyridostigmine bromide treatment, chagasic animals (from chg - nt and chg - pyrido groups) presented significant elongations of pd, qrsd, qt, and cqt (table 1), indicating a global functional disturbance of heart. interestingly, as shown in table 2, after one month of the pyridostigmine bromide treatment and six months of infection, chg - pyrido mice presented a significant reduction in pd (an atrial parameter) and cqt (a ventricular parameter), suggesting an improvement in the electrical function of the heart. in contrast, in chg - nt mice, most of the ecg parameters were significantly different compared to con - nt mice. it is worth noting that, in the con - pyrido group, a significant increase in pr was observed, as expected, because pyridostigmine bromide might be increasing vagal neural transmission in the atrioventricular node, with a consequent reduction in the conduction velocity of the action potential. these suggest that pyridostigmine bromide treatment can be effective in improving electrical disorders induced by chronic chagasic cardiomyopathy. the of heart rate variability analysis in time- (variance) and frequency - domain (spectral) parameters are shown in table 3. a significant reduction in the absolute values of total variability (variance) and the spectral components vlf, lf, and hf was observed in chg - nt mice compared to con - nt and con - pyrido mice. the lf and hf components expressed in normalized units and the lf / hf ratio were not different from con - nt mice. in contrast, chagasic mice treated with pyridostigmine bromide (the chg - pyrido group) presented values of variance and vlf and hf spectral components that were significantly higher compared to chg - nt mice, suggesting an improvement of some autonomic parameters that changed during chronic chagasic cardiomyopathy (table 3). the autonomic dysfunction observed in the heart rate variability analysis of chg - nt mice and its improvement in chg - pyrido mice was confirmed by pharmacological autonomic blockade with atropine sulfate or propranolol. the use of these autonomic blockers showed that vagal parasympathetic effects significantly decreased without a change in sympathetic effects in chg - nt mice compared with con - nt mice (figure 1). chagasic mice treated with pyridostigmine bromide showed values of vagal parasympathetic effects that were significantly higher compared to chg - nt mice and similar to those found in con - nt mice (figure 1). additionally, noninfected control mice treated with pyridostigmine bromide showed vagal parasympathetic effects that markedly increased without any changes in sympathetic effects compared with con - nt mice (figure 1). no changes in the intrinsic pacemaker heart rate (ihr) were found in any of the experimental groups after the six - month observation period. the relative cardiac weight of chg - nt mice was significantly higher than that observed in the other groups (figure 2), indicating that cardiac hypertrophy was induced by chagas disease in mice and that pyridostigmine bromide treatment was able to reverse or impair the development of cardiac hypertrophy in chagasic mice. a semiquantitative examination of cardiac inflammatory infiltration indicated discrete to mild diffuse myocarditis (average inflammatory score was equal to 0.5, p25% = 0.5, and p75% = 0.5, p < 0.0001 versus con - nt), which was observed in the atria (figures 3(g) and 3(h) ), the ventricles (figures 3(e) and 3(f) ), and the neural ganglia (figure 3(g) ) in the chg - nt mice in comparison to normal con - nt mice (figures 3(a), 3(b), 3(c), and 3(d) ), which presented no inflammatory infiltration (average inflammatory score was equal to 0.0, p25% = 0.0 and p75% = 0.0). in chg - pyrido mice, reduced inflammatory infiltration, which was categorized as very discrete to discrete (average inflammatory score was equal to 0.125, p25% = 0.0, and p75% = 0.25, p < 0.001 versus chg - nt), was observed in the atria, the ventricles (figures 3(i), 3(j), 3(k), and 3(l) ), and the neural ganglia. as expected in this mouse model of chronic chagas disease, no amastigote nests in the myocardium were found in either chg - nt or chg - pyrido mice. in different sections of the heart stained by picrosirius red, the morphometry of fibrosis revealed a marked and diffuse increase in the fibrotic area in both absolute (m) and relative (%) values in the chg - nt group compared to chg - nt mice (figure 4). interestingly, chg - pyrido mice presented significantly reduced fibrotic areas in all cardiac chambers (figure 4) compared to chg - nt mice. despite these reduced values, the fibrotic areas were still larger than those found in con - nt mice (figure 4). the serum levels of the cytokines il-2, il-4, il-5, and tnf-, measured with the cba technique, did not reveal any change in any of the experimental groups. in fact, the serum levels of il-2 and il-4 were undetectable, with values near 0 pg / ml. however, using the elisa technique, serum levels of ifn- were significantly higher in chg - nt mice compared to chg - pyrido mice (p < 0.05). serum levels of il-10, measured with elisa technique, were significantly higher in both chg - nt and chg - pyrido mice compared to con - nt mice (p < 0.05). no differences were found in the serum il-10 levels between chg - nt and chg - pyrido mice (figure 5). the immunohistochemical technique using peroxidase to label t. cruzi parasite nests or antigens revealed a complete absence of parasites in control noninfected mice, whereas t. cruzi labeling was positive for 62.50% of chg - nt and 81.81% of chg - pyrido mice (p = 0.603, not significant using the fisher exact test). the parasite antigens were diffusely distributed throughout the heart tissue (figure 6). using a conventional pcr method to detect parasite dna in blood and heart during the chronic phase of chagas disease in mice, a complete absence of parasite dna in noninfected control mice was observed, whereas t. cruzi dna was found in 16.67% and 42.9% (p = 0.559) in the blood of chg - nt and chg - pyrido mice, respectively, and in 72.73% and 86.67% (p = 0.614) of heart samples from chg - nt and chg - pyrido mice, respectively. to our knowledge, the present study is the first to analyze the effects of the deliberate potentiation of cholinergic signaling using pyridostigmine bromide, an anticholinesterase agent, during the chronic phase of experimental chagas disease in mice. furthermore, this study evaluated the effects of pyridostigmine bromide treatment on electrocardiographic, autonomic, histopathological, immunoinflammatory, and parasitological parameters of chagas disease. the present study is, to our knowledge, the first to demonstrate autonomic dysfunction induced by chronic chagas disease in an experimental mouse model. this dysfunction was characterized by a marked reduction in heart rate variability, with reduced variance (time - domain parameter) and the vlf, lf, and hf components (frequency - domain parameters) of heart rate variability, as well as a concurrent reduction in the cardiac vagal effect, without an apparent change in sympathetic effects, as measured by pharmacological blockade in chronic control chagasic animals. only morphological reports describing the ganglia and nerve lesions in chagasic mice this finding follows our previous report of a similar autonomic dysfunction in mice with acute chagas disease. this autonomic dysfunction was accompanied by electrocardiographic changes associated with mild diffuse inflammatory infiltration of mononuclear cells as well as fibrosis and hypertrophy in the atrial and ventricular myocardium and the elevation of ifn- and il-10 in the blood serum of wild - type and untreated chagasic c57bl/6j mice, which confirms the presence of chronic myocarditis six months following infection with t. cruzi. this increase in serum levels of ifn- and il-10 in chronic chagasic mice matches similar previous reports in human beings. additionally, parasite antigens and dna were detected by immunohistochemical labeling in the heart and by pcr in blood and heart, respectively, reinforcing the role played by parasites in inducing and/or maintaining chronic infection. because our findings are consistent with previous observations in hamsters, rabbits, dogs, and humans during the chronic phase of chagas disease , the mouse model used here appears to represent a suitable tool for future experimental studies in chagas heart disease. during the chronic phase of chagas heart disease, this report revealed a significant increase in cardiac vagal parasympathetic autonomic modulation associated with a significant reduction of inflammatory infiltration in the myocardium of infected mice treated with pyridostigmine bromide during the last month of a six - month observation period. because pyridostigmine bromide is an anticholinesterasic agent, which increases the bioavailability of acetylcholine in the synaptic varicosities, the increase in cardiac vagal parasympathetic autonomic function, demonstrated by the higher heart rate variability and cardiac vagal effect, was expected and was verified after treatment. the anti - inflammatory effect of pyridostigmine bromide was associated with a marked reduction of myocardial fibrosis and hypertrophy. serum levels of ifn- also decreased, whereas tna- trended to decrease, both of which are th1 proinflammatory cytokines, without any change in the augmented serum levels of il-10. the combined analysis of these suggests that pyridostigmine bromide may act on the heart, at least partially increasing the vagal parasympathetic activity, via the accumulation of acetylcholine in the myocardium. this accumulation was confirmed by improvements in heart rate variability and vagal parasympathetic effect, which, acting on immune cells, may exert immunomodulatory effects, thereby shifting the immune response toward the predominance of an anti - inflammatory response. these new findings in the chronic chagas disease context reinforce the idea of an intimate interplay between the immune and autonomic nervous systems and the potential use of parasympathetic stimulation to treat chagasic myocarditis. additionally, the improvement of cardiac inflammation, fibrosis, and hypertrophy in pyridostigmine bromide - treated chronic chagasic animals suggests that the decrease of vagal autonomic function due to ganglionic neuronal lesions and denervation, which occur precociously during the acute phase, may play an important immunomodulatory role in the increased th1 immune response and inflammatory infiltration, as verified during the chronic phase of the disease. even though our support the immunomodulatory role played by the cholinergic vagal parasympathetic nervous system in the heart, we can not rule out a possible role of pyridostigmine bromide in increasing local acetylcholine levels via its release from nonneural local structures, such as endothelial cells, cholinergic lymphocytes, and cardiomyocytes. in fact, these nonneural sources of acetylcholine, potentiated by pyridostigmine bromide treatment, may explain the substantial improvement in ecg abnormalities, inflammation, fibrosis, and hypertrophy observed in both ventricles of the heart, which are poorly innervated by vagal parasympathetic fibers. data from the immunohistochemical labeling of t. cruzi antigens and t. cruzi dna detection by pcr showed that parasites are present in the heart tissue and blood of the chagasic mice six months after infection, confirming previous reports. the number of animals positive for parasite antigens or dna did not differ between pyridostigmine bromide - treated and nontreated chagasic animals. the persistence of the parasite in the chronic phase, even at a very low level, seems to play an important role in the pathogenesis of chronic chagas disease because the low parasite load may continuously stimulate an immune response. although antigens or dna from t. cruzi were detected in the present study, no parasite pseudocysts or amastigote forms were observed in the heart tissue in either treated or nontreated chagasic mice. this lack of entire parasites at the site of cardiac lesions suggests the possibility of a remote niche for the parasites, such as smooth muscle cells, adipocytes, or skeletal muscle cells. in , our support the notion that autonomic dysfunction is a primary cause of the pro-/anti - inflammatory imbalance observed in inflammatory diseases, favoring the shift of immune response toward the predominance of proinflammatory response, thereby contributing to the pathogenesis of these diseases in general and chagas heart disease in particular. additionally, our findings showed the potential beneficial effects of anticholinesterasic agents, particularly pyridostigmine bromide, in increasing cardiac autonomic modulation and reducing inflammatory response to the heart.	the aim of the present study was to assess the effects of an anticholinesterase agent, pyridostigmine bromide (pyrido), on experimental chronic chagas heart disease in mice. to this end, male c57bl/6j mice noninfected (control : con) or chronically infected (5 months) with trypanosoma cruzi (chagasic : chg) were treated or not (nt) with pyrido for one month. at the end of this period, electrocardiogram (ecg); cardiac autonomic function; heart histopathology; serum cytokines; and the presence of blood and tissue parasites by means of immunohistochemistry and pcr were assessed. in nt - chg mice, significant changes in the electrocardiographic, autonomic, and cardiac histopathological profiles were observed confirming a chronic inflammatory response. treatment with pyrido in chagasic mice caused a significant reduction of myocardial inflammatory infiltration, fibrosis, and hypertrophy, which was accompanied by a decrease in serum levels of ifn with no change in il-10 levels, suggesting a shift of immune response toward an anti - inflammatory profile. lower nondifferent numbers of parasite dna copies were observed in both treated and nontreated chagasic mice. in , our findings confirm the marked neuroimmunomodulatory role played by the parasympathetic autonomic nervous system in the evolution of the inflammatory - immune response to t. cruzi during experimental chronic chagas heart disease in mice.
an 85-year - old man presented with an 8-month history of slowly increasing diffuse yellow skin lesions on the torso and upper arms. he had been unaware of the discoloration until his internist noted it and referred him to dermatology. the patient had been followed by a hematologist for paraproteinemia that had not been treated. the examination showed well - demarcated yellow patches and plaques covering large portions of his upper trunk with some islands of sparing (figure 1, 2). the dermoscopic evaluation showed a reticular pattern of yellow amorphous homogeneous structures with overlying branched and linear vessels (figure 3). a skin biopsy showed scattered foamy histiocytes within the reticular dermis, which were consistent with a plane xanthoma (figures 4, 5, 6). a complete blood count, comprehensive metabolic panel, fasting lipid panel were within normal range. diffuse normolipemic plane xanthoma (dnpx) was first described by altman and winkelmann in 1962. dnpx is characterized by xanthelasma palpebrarum; diffuse plane xanthoma of the head, neck, trunk, and extremities; and normal plasma lipid levels. xanthelasma typically appears first, followed by involvement of the lateral parts of the neck and upper trunk. clinically, the dermatosis is characterized by the presence of symmetric yellowish - orange plaques that favor the neck, upper trunk, flexural folds and periorbital region. histologically, foam cells (macrophages that have engulfed lipid droplets) and variable numbers of touton giant cells, lymphocytes, and foamy histiocytes can be seen. while not all cutaneous xanthomas are associated with systemic diseases, dnpx has been associated with systemic diseases, particularly multiple myeloma and monoclonal gammopathy. however, other malignant hematological or lymphoproliferative disorders associated with dnpx include acute monoblastic leukemia, chronic myelomonocytic leukemia, chronic myloid leukemia, chronic lymphatic leukemia, non - hodgkin s lymphoma, adult t - cell lymphoma / leukemia, sezary syndrome, waldenstrom s macroglobulinemia, cryoglobulineimia and castleman s disease. the pathogenesis of dnpx has not been fully elucidated, however, in gammopathy - associated dnpx, monoclonal igg is thought to bind to circulating ldl, rendering the antibody ldl complex more susceptible to phagocytosis by macrophages. dnpx can precede such disorders by several years; therefore close follow - up with periodic laboratory tests for myeloproliferative disorders should be performed. the erbium: yag laser has been used successfully to treat facial xanthomas in one patient. in this patient, currently there are no associated systemic symptoms. , this case shows that dermatological lesions can be the first manifestation of important hematological diseases and so physicians should be familiarized with this rare entity.	diffuse normolipemic plane xanthoma (dnpx) was first described by altman and winkelmann in 1962. it is a rare and non - inherited form of xanthomatosis. clinically, the dermatosis is characterized by the presence of symmetric yellowish - orange plaques that favor the neck, upper trunk, flexural folds and periorbital region. it has been recognized to be associated with hematological diseases, especially with multiple myeloma and monoclonal gammopathy. we present a patient with diffuse plane xanthoma, normal lipid level, and monoclonal gammopathy.
angle region fracture is the most common fracture in developing countries, accounting 30% of all fractures. mandibular angle fracture line involves junction of ramus and body of the mandible to the third molar region and traverses through the inferior alveolar canal to reach the inferior border.. the traditional biomechanical model comprising tension at the superior border and compression at the lower border has been challenged, and it has been found that these tension compression zones reverse as the load position moves posteriorly. fracture involving inferior alveolar canal - associated nerve paresthesia may be transient or permanent type. thurmuller et al. reviewed the literature on nerve injuries in facial trauma and reported the overall incidence of inferior alveolar nerve (ian) injury in mandibular fractures with associated paresthesia to be 5.758.5% after injury without treatment and fractures in the mandibular body and angle region to be 4658.5%. the reported postoperative incidence of ian injury in fractures of the nerve - bearing area of the mandible (angle, body) was 76.191.3%. various factors such as fracture displacement, type of fixation, site, and type of fracture have been reported to influence the incidence of ian injury in mandibular fractures. the actual amount of fracture displacement due to trauma is not accurately estimated, and radiographs only demonstrated the posttrauma tissue recoil bony fragments position. this is not clear why some minimally displaced fractures ed in permanent numbness. in blunt trauma involving the thorax, it is well known that major bony and visceral displacements occur in high - velocity impact, with recoil back to their original position soon after, nevertheless, ing in injury. there is not much published literature supporting that greater fracture displacement can cause more nerve injury or vice versa. in this study, thirty patients of displaced mandibular angle fracture which are traversed by mandibular canal were included in the study. the university ethical board clearance was taken. patients who were reported at the oral and maxillofacial (omf) unit of trauma center and the outpatient department of omf surgery department of king george's medical university were included in the study after their informed consent. in addition to demographic information (i.e. gender age, cause), the following data of all the patients were also collected: location of fracture - left or right, presence of tooth in the fracture line, displacement of the inferior alveolar canal, and any major postsurgical complication; routine antibiotics therapy and use of elastic for slight malocclusion were not considered in complication. clearance to conduct the study was obtained from the university ethical committee. written informed consent to participate in the study was obtained from each patient before enrolling in the study. patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram (opg)displacement of the fractured fragments was measured on opg and was classified as 03 mm, 35 mm, and > 5 mm displacement between the fracture fragments. patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram (opg) displacement of the fractured fragments was measured on opg and was classified as 03 mm, 35 mm, and > 5 mm displacement between the fracture fragments. patients not willing to participate in the studypathological fracture, fractures with major tissue lossprevious surgery (orthognathic surgery, implant surgery, surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery ing numbness)panfacial trauma. patients not willing to participate in the study pathological fracture, fractures with major tissue loss previous surgery (orthognathic surgery, implant surgery, surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery ing numbness) these thirty patients were divided into two groups, 15 patients in each group. in group b, the patients were treated using 2-mm three - dimensional (3d) plate. group a patients were treated with six - hole preangulated plate as was applied along external oblique ridge transbucally. in group b, patients underwent osteosynthesis using 3d titanium strut plate transbucally. postoperatively, pain, swelling, and mobility between fracture fragments, nerve paresthesia, and recovery were assessed between two groups at various periods of follow - up. patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram (opg)displacement of the fractured fragments was measured on opg and was classified as 03 mm, 35 mm, and > 5 mm displacement between the fracture fragments. patients with at least one fracture passing through the inferior alveolar canal on orthopantomogram (opg) displacement of the fractured fragments was measured on opg and was classified as 03 mm, 35 mm, and > 5 mm displacement between the fracture fragments. patients not willing to participate in the studypathological fracture, fractures with major tissue lossprevious surgery (orthognathic surgery, implant surgery, surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery ing numbness)panfacial trauma. patients not willing to participate in the study pathological fracture, fractures with major tissue loss previous surgery (orthognathic surgery, implant surgery, surgery for mandibular pathology involving mandibular canal region and mandibular impacted third molar surgery ing numbness) these thirty patients were divided into two groups, 15 patients in each group. in group b, the patients were treated using 2-mm three - dimensional (3d) plate. group a patients were treated with six - hole preangulated plate as was applied along external oblique ridge transbucally. in group b, postoperatively, pain, swelling, and mobility between fracture fragments, nerve paresthesia, and recovery were assessed between two groups at various periods of follow - up. a total of thirty patients of displaced angle fracture treated with different types of fixation were studied. there was significantly more prevalence of fracture in male patients with a mean age of 25 years. road traffic accident was the major cause (74%) of fracture of the mandible and interpersonal violence and fall from height was less, incidence - wise. the mean duration of time lapse between injury and definitive treatment in both groups was 68 days. the failure to recognize or detect nerve paresthesia initially is due to poor cooperation of patients due to facial edema, lacerations and associated injury, or unconsciousness. nerve sensory function was evaluated by a light touch with cotton wool and two - point discrimination on the skin of chin and lip. postoperatively, there was significant in pain on a visual analog scale between both groups at different intervals. a decreasing trend was found with the time interval in both groups. at 6-week follow - up, the patients in both groups were pain - free. there was a gradual change in molar bite force from 7 day onward at different intervals in both groups; however, in group a, this change was less than group b. preoperatively the mean displacement in group a was 7.86 6.75 mm and in group b was 8.43 6.65 mm preoperatively. the mean displacement in group a was 0.20 0.41 mm and in group b was 0.66 0.61 mm. the mean change was 7.66 4.48 mm and 7.76 6.54 mm, respectively. there was a significant change observed postoperatively in both groups; t and p values were statistically significant, 2.43 and 0.02, respectively. paresthesia was present in 46.6% cases of group a and 40% cases of group b preoperatively. relation of paresthesia with degree of displacement of fractured fragments in both groups at 7 day postoperatively, 33.3% cases in group a and 40% cases in group b had paresthesia. at the end of 4 weeks, 20% cases in group a and 26% cases in group b had paresthesia while at the end of 12 weeks, 20% cases in group a and 26.6% cases in group b had paresthesia. there was no statistical significance (p < 0.05) in anesthesia / paresthesia between both the groups at different time intervals. however, the presence of paresthesia was lower in group a than in group b. one patient in group a and two patients in group b had persistent paresthesia postoperatively at 12 weeks. paresthesia was present 28.5% cases when displacement was 13 mm, 62% cases when displacement was 39 mm, and 71% cases when displacement between fragments was > 9 mm. there was a complete recovery in both groups a and b when displacement was < 5 mm and persistent paresthesia 33% in group a and 66% in group b when displacement was > 9 mm after 12-week follow - up. comparison of the persistence of paresthesia in both groups after fixation, showing greater percentage of recovery in group a the mandibular angle fracture usually ed in pain, swelling, bleeding, and disturbed occlusion due to the displacement of fractured fragments. displaced fractures generally ed in paresthesia, which causes problems such fluid drooling or food escape or accidental lip biting and occasionally shaving and applying makeup even kissing become difficult. angle fracture can be displaced by a number of ways as an open book, vertical overlap, and laterally. this can be explained on the basis that etiology of fracture shifting toward assault or interpersonal violence with a blow to side of the face. there is no accurate method to access the ian injury, but radiographically fractured fragments separation / gap and misalignment of inferior alveolar canal only suggest the ian injury. manipulation of fracture fragments during reduction and stabilization or extraction of the third molar and even screws placement may in inferior nerve injury. persistent mobility at fracture site even in minimally displaced fracture can cause further nerve injury. in this study, group a patients had pretreatment sensory disturbance 46.6% paresthesia preoperatively, and postoperatively, 33% and 20% at 1 week, 4 weeks, and at the end of 12 weeks respectively. while in group b (matrix plate), 40% patients had paresthesia preoperatively, and postoperatively, 26% and 26.6% at 4 weeks and the end of 12 weeks, respectively. postoperative difference in nerve injury recovery may be explained on the basis of fixation, that provides more firm fixation, which helps in early nerve recovery in group a than group b. similar observation was also reported by thurmuller et al. manipulation and reduction of fracture fragments can further in starching or injury to the nerve. nagadia et al. found by computed tomography (ct) imaging that the mean distance from the outer buccal cortex to the inferior alveolar canal in chinese mandible was 6.97 mm (min 4.8 mm); the use of > 6 mm of monocortical screws can cause injury to nerve in this region. levine et al. reported that the distance of buccal cortical margin to the mandibular canal was 4.9 mm using ct imaging. demyelinating nerve lesion generally recovers during the first 4 months of injury; however, after sustained axonal nerve injury, the nerve conduction velocity can also slow down permanently due to the schwann cell regeneration having shorter internodal interval than before injury and it may never reach baseline as happened probably in our few cases. due to wide separation of fractured fragments with persistent mobility at fractured may causes, repeated nerve injuries in the already stretched nerve. there is no direct evidence that suggested the relationship of fracture fragments displacement and time lapse in treatment ed in paresthesia. early reduction and fragments fixation of fracture fragments may offer early nerve chances of nerve recovery margins. al - jandan et al. reported cone - beam ct observation that horizontal distances at the canine, first premolar, and second molar and reported that using 6 mm screw can cause 56% ian injury and 7 mm can cause 78% injury. hence, the routine use of 57 mm screws would have a high risk of injury to the root apex and the inferior alveolar canal if miniplate was placed along champy's line of osteosynthesis. in this study when fractured fragment displacements were > 5 mm, then rate of recovery was poor as 33% in group a and 66% in group b, with persistent paresthesia at the end of 12-week follow - up. this may be explained on the basis that prolonged stretching of fragments may cause more nerve damage. while in < 5 mm displaced group, patients had complete nerve recovery; robinson reported that most of the improvements of nerve injury occur in initial 4 weeks, but long - term 12-month follow - up will definitely be helpful. another study by queral et al. reported that fracture displaced > 5 mm had a 7-fold increased risk of ian after treatment compared with patients with < 5 mm fracture displaced. reported postoperatively 81% had poor sensation to thermal stimuli and 67% to von frey tactile stimulation. ardary and raveh et al. reported that the rate of postoperative sensory deficit has been lower varying from 0.9% to 34%. this variation may be explained on the basis as paresthesia diminished with the time and different follow - up of studies, and possibility studies had included fractures of noninferior canal region (parasymphysis and symphysis). this study suggests that more stable fixation helps in early nerve recovery in displaced angle fracture. permanent ian neurosensory deficits may complain as pain, paresthesia dysesthesia, hypoesthesia, or anesthesia involving the chin, lower lip, and gums. early manual reduction, and temporary stabilization of fracture fragments will definitely prevent mobility of fragments and reduce bleeding, pain, and swelling causing injury to nerve.	objective: the objective of this study was to assess the effectiveness of different types of fixation in the enhancement of posttraumatic inferior alveolar nerve (ian) recovery in displaced mandibular angle fracture and to establish.patients and methods: thirty patients of displaced mandibular angle fracture were treated with preangulated plate and three - dimensional (3d) matrix plate in two groups and were observed during follow - up at 04,06 and 12 weeks along with other parameters.:fifteen patients were treated with preangulated plate and 15 patients with 3d matrix miniplate. there was early nerve recovery in group a than group b, with residual paresthesia 20% in group a and 26.6% in group b at the end of 12-week follow-up.:the displaced mandibular angle fracture with posttraumatic ian paresthesia treated with preangulated plate has shown evidence of early nerve recovery than those fractures were treated with matrix miniplate. the fracture fragments displaced more than 9 mm have shown poor nerve recovery in both groups.
obesity is now a global public health issue and has nearly doubled worldwide since 1980. global obesity estimates from 2008 reported that 35% of adults over the age of 20 were overweight and 11% were considered obese. in addition, obesity rates continue to rise and remain epidemic in the united sates, as well. more than two - thirds of american adults (68.8%) are considered overweight or obese and more than one - third (35.7%) of adults are obese, constituting more than 78 million american men and women. individuals who are considered morbidly, or clinically severely, obese (those with bmi of 35 in addition to documented health problems or those with a bmi over 40) are considered potential candidates for bariatric surgery. individuals elect to undergo bariatric surgery hoping to achieve long - term weight loss and to improve obesity - related comorbid conditions. in 2008, nearly 350,000 bariatric procedures were performed globally, with an estimated 220,000 in the united states alone. however, questions still arise on long - term success and its effect on weight loss maintenance and prevention of the return of lifestyle diseases. previous research indicated that, about a year and a half to two years after bariatric surgery, weight loss from surgery stops and a significant number of individuals begin to regain the weight they had lost. this is when support is needed most; nevertheless, many longitudinal follow - up bariatric surgery studies are short - termed and incomplete. bariatric surgery is a life - changing procedure that requires a lifelong commitment from the individual limited research exists examining personal and psychosocial experiences, including satisfaction with weight loss and challenges patients experience following the first year after bariatric surgery. using a qualitative approach to explore the meaning of the bariatric experience and sharing it with others provides an opportunity for future patients, families of patients, and healthcare providers to gain an understanding of what it is like to undergo a bariatric procedure and to live with the persistent change that accompanies it, after the first two years post - surgery and beyond. this study sought to answer the question: what is the experience of bariatric patients who are at least two years post - surgery? this study used a qualitative research design, guided by a phenomenological approach, to investigate and describe the meaning and essence of experience of patients at least two years after bariatric surgery. phenomenological studies are primarily open - ended, searching for themes of meaning in participants' lives. the researcher seeks to understand the deep meaning of a person's experience and how one describes this experience. exploring the lived experience of patients at least two years after bariatric surgery provided a deeper understanding and awareness of the impact of weight - loss surgery. through the use of interview questions as a guide, the method of in - depth, phenomenological interviews applied to a sample of participants who have all experienced similar social conditions gives power to the stories of relatively few participants, such as life experience. the researchers used purposeful convenience sampling, which enabled relatively easy access to participants. by contacting participants who were acquainted with the researchers and met inclusion criteria for this study, the researcher was able to conduct semistructured interviews with nine bariatric surgery patients at least two years post - surgery. criteria for inclusion was set so that the participants who were at least two years after operation and over the age of 18 were recruited through email / telephone regardless of gender, age, surgery type, ethnicity, or socioeconomic status. the study sample included nine women who ranged from 27 to 57 years of age (m = 42). seven participants had the vertical sleeve gastrectomy (vsg) surgery and 2 had the roux en y gastric bypass (rnyb). self - identified ethnicities of participants were caucasian (n = 5), hispanic (n = 2), and african - american (n = 2). participants ranged from two to seven years after operation (m = 3) (table 2). weight before surgery following the preoperative diet ranged from 199 pounds to 298 pounds (m = 247.4). initial weight loss after surgery ranged from 58 pounds to 116 pounds (m = 88). weight gain after reaching plateau ranged from none to 48 pounds (m = 8.7) (table 3). data analysis included the use of field notes, audiotapes, a reflexive journal, and peer debriefing. each audiotaped interview was transcribed verbatim and field notes were taken during each interview. after transcription, each interview was printed for clarity. the first transcript reading helped develop the coding categories, and then the second reading was conducted to start formal coding in a systematic way using colored post - it notes to group related data. the second transcript aided in identifying new categories of information and the two lists the transcript of the third interview was compared to the previous code list to see if new categories emerged and this process continued comparing each subsequent transcript for coding categories. themes were eventually developed into a written description of the participants' experience with bariatric surgery to answer the research question. the researchers personal experience as bariatric patients included preconceived beliefs that bariatric surgery should be for obese individuals who had failed attempts at losing weight through traditional methods with diet and exercise and who need an additional weight - loss tool to aid in achieving a healthy body weight. being part of the weight - loss surgery community comes with a very pro - surgery mentality that the researchers acknowledged. to focus on the participants' experience after bariatric surgery hays and wood define bracketing as setting aside any assumptions made in everyday life and expressed the need for the researcher to reserve all prejudgments of their experience and rely on intuition and imagination to obtain the picture of the experience. establishing trustworthiness of the findings was utilized by engaging in peer debriefing of instrument protocol and through prolonged discussions of the research project with peers. researcher reflexivity was engaged by keeping a journal and field notes, and simultaneous data collection and analysis, which involved collecting and analyzing data simultaneously. review of the data, including individual interviews and field notes, was conducted to analyze and identify themes of the participants' experience with food after bariatric surgery. two main themes emerged from the data: (a) food after the first year post - op and (b) bariatric surgery is not a magic pill. the overarching theme of viewing weight - loss as work after the first year emerged from the data as participants described the mounting difficulty of adhering to the recommended post - op diet once the first year had passed. in addition, tendencies towards using food as comfort and emotional eating were still a struggle that many participants experienced.i know i have to keep staying on top of it, it's just that i'm sick of protein, i'm sick of water, i'm sick of working out, i'm just sick of it. i just want eat normal sometimes, but i know i just need to suck it up and deal with it. finding that balance after the first year i know i have to keep staying on top of it, it's just that i'm sick of protein, i'm sick of water, i'm sick of working out, i'm just sick of it. i just want eat normal sometimes, but i know i just need to suck it up and deal with it. finding that balance after the first year the recommended diet after bariatric surgery varies among surgeons and across different bariatric surgeries, but there are some commonalities that all participants experienced. generally agreed upon nutritional guidelines are that supplemental minerals and vitamins should be taken daily, small bites of food should be chewed thoroughly before swallowing, liquids should be either ingested well before meals or at least 30 minutes afterwards, and at least 60 grams of proteins should be preferentially eaten before fats and carbohydrates.consuming the right food and the vitamins and doing the right thing with food exist on a continuum it's not a matter of being all right or all wrong or all good or all bad every day you're striving to make the best choices you can in that moment. in the second year, i noticed i had to be very careful about what i ate and i had to really focus on my vitamins every day and make sure to get my protein and water in every day the first year, you did not really have to think much and you'd still lose weight. now i have to focus and pay attention to what i put in my mouth i am more aware of what i'm eating now because my intake is so small, you really have to pay attention to what you're putting in your body. it's quality now, i'm going for the filet minion before the ground hamburger. consuming the right food and the vitamins and doing the right thing with food exist on a continuum it's not a matter of being all right or all wrong or all good or all bad every day you're striving to make the best choices you can in that moment. in the second year, i noticed i had to be very careful about what i ate and i had to really focus on my vitamins every day and make sure to get my protein and water in every day the first year, you did not really have to think much and you'd still lose weight. now i have to focus and pay attention to what i put in my mouth i am more aware of what i'm eating now because my intake is so small, you really have to pay attention to what you're putting in your body. it's quality now, i'm going for the filet minion before the ground hamburger. potential long - term dietary complications include dumping syndrome (e.g., sweating, weakness, hypoglycemia, vomiting, diarrhea, and nausea) related to eating foods high in concentrated sugar or fat. all participants expressed problems with dumping syndrome and/or intolerances to various foods, namely, refined carbohydrates and sugar. most participants talked about food intolerances, especially to sugar.i think the one good thing is that i can not eat a huge amount of sweets without being nauseated i can eat a little that's more cracker - ish or cookies but, like, big bakery pastries or pies, or a big hunk of cake or something, well, that's not going to work very well besides sweets, i have problems with all white carbs like rice, bread, and pasta. i think the one good thing is that i can not eat a huge amount of sweets without being nauseated i can eat a little that's more cracker - ish or cookies but, like, big bakery pastries or pies, or a big hunk of cake or something, well, that's not going to work very well besides sweets, i have problems with all white carbs like rice, bread, and pasta. all participants discussed the amount of food they were able to consume in some way, noting reduced intake even years after surgery compared to presurgery amounts.as far as eating i can eat many more calories at one time than i used to be able to eat when recently post - op. you do have more hunger, though you still can not eat like you used to and i have a bit more of a desire to eat than i did the first year i'm still restricted in how much i can eat and eat about 3 ounces of dense protein and 1 ounce of vegetables and 1 ounce of starch for a meal. as far as eating i can eat many more calories at one time than i used to be able to eat when recently post - op. you do have more hunger, though you still can not eat like you used to and i have a bit more of a desire to eat than i did the first year i'm still restricted in how much i can eat and eat about 3 ounces of dense protein and 1 ounce of vegetables and 1 ounce of starch for a meal. many participants were identified as still grappling with food as a means to cope and it can be a challenge to find new ways to channel emotions without turning to food. participants also identified that it was easier to manage the food cravings after surgery as well as the head hunger which involves not having physical hunger but the desire to eat anyway.i'm realizing now that there is a real emotional and physical effect that different foods have on your body. for example, the effect that sugar might have on your emotions or energy level, the way alcohol affects your body almost immediately post - op i still have those gnawing cravings from time to time and i just find it easier to say you do not need it right now, you can wait. it's not like before where i had to have the food otherwise i was gon na die. if i'm going to have something bad like sugar or something, i make sure i have protein first so i'm full, then it's not such a big deal. i try not to tell myself no on anything because the minute i say no, i'm feeling depressed today and i wish i could just go eat a couple of pieces of cheesecake and that would only make me happy temporarily i'm realizing now that there is a real emotional and physical effect that different foods have on your body. for example, the effect that sugar might have on your emotions or energy level, the way alcohol affects your body almost immediately post - op i still have those gnawing cravings from time to time and i just find it easier to say you do not need it right now, you can wait. it's not like before where i had to have the food otherwise i was gon na die. if i'm going to have something bad like sugar or something, i make sure i have protein first so i'm full, then it's not such a big deal. i try not to tell myself no on anything because the minute i say no, then i start have mental issues with it. i'm feeling depressed today and i wish i could just go eat a couple of pieces of cheesecake and that would only make me happy temporarily it kind of conditioned me to not do that anymore like, hey, this is bad for you this theme was common across all participants and it deserves to be noted that many patients who passed the first two years of surgery realize (e.g., by experiencing weight regain) that bariatric surgery is intended to be used only as a tool to aid in weight loss or to reduce physical hunger. all participants offered words of advice and/or caution to those considering bariatric surgery.i think that the surgery is not a fix all it is a tool to help you achieve weight loss, but it is only a tool. it helps you with the amount you can eat and can definitely help you lose weight but if you still do not pick the right choices to eat and exercise, it's still not going to do you any good in the long term. i mean, everybody has this misconception that you have surgery and then get magically skinny and that's not how it works. you still have to put in the work and your everything into this every day. i think people have it in their heads that you go into surgery and have it done and come out instantly thin. they do not realize that you can not eat and drink at the same time or you can only eat 3 ounces of food at once nobody talks about all that and all the working out and that you've been gutted like a fish, they just think you're instantly skinny within a month i think that the surgery is not a fix all it is a tool to help you achieve weight loss, but it is only a tool. it helps you with the amount you can eat and can definitely help you lose weight but if you still do not pick the right choices to eat and exercise, it's still not going to do you any good in the long term. weight loss is not something that's going to happen on its own. i mean, everybody has this misconception that you have surgery and then get magically skinny and that's not how it works. you still have to put in the work and your everything into this every day. i think people have it in their heads that you go into surgery and have it done and come out instantly thin. they do not realize that you can not eat and drink at the same time or you can only eat 3 ounces of food at once nobody talks about all that and all the working out and that you've been gutted like a fish, they just think you're instantly skinny within a month experience of participants was profiled in an attempt to share their qualities through thick description. the experience of all participants' lives after bariatric surgery provided a glimpse of what role food now plays after the first two years after operation. food was still considered a huge part of life, though limited in quantity, even years after bariatric surgery. participants discussed not consuming their recommended daily intake of protein and not taking their vitamins. low iron, b12, and vitamin d were consequences of poor supplementation. participants compared food cravings to addictions to alcohol or drugs and there was still grief felt in regard to being unable to enjoy the same meals socially and with family as before surgery. participants adjusted to changes in diet in different ways, and some were more successful than others, though many still fight the urges to use food to cope in certain situations. many found eating a simple food such as a cracker (carbs) or any refined sugar may make them become unpleasantly ill. these patients face medical and psychosocial challenges that require continuing care for years after surgery, and the hard work begins post - surgery. it is also pointed out that not much has been written on the topic of follow - up care, but rather the focus has been on who is a good candidate for surgery rather than what patients go through after the procedure. a qualitative study using phenomenological approach was used for this study in order to gain a better understanding of the experience of participants who were at least two years after bariatric surgery. the use of semistructured interviews allowed the participants to express their feelings and experience of life after bariatric surgery which many follow - up surveys lack and may expand the knowledge base from the participants own lived encounters with bariatric surgery. the themes around food generated from the data collected in this study provided a glimpse of life once the magic of the first year has passed and the reality of the surgery had set in. additional emerging themes from this study to be analyzed include the role of social support and perceptions of self and society. while obesity threatens to explode into a reverberating pandemic, there is a need to understand and support those individuals who have made the decision to have bariatric surgery. studies such as this can offer health care professionals needed insight and information to be supportive and provide the care necessary to this vulnerable population. one participant who has struggled more than two years post - surgery with weight regain, and who stands apart from the others in current postoperative bmi (table 3), summed up the lifelong impact of diet for the bariatric patient.it's lifelong and a lifestyle change i mean you can not have your stomach cut out and then think you can eat crap food like you did before. i noticed that it is very easy to eat junk food like chips and soda these foods go down easily also if you drink your calories i just think that if you want long - term success with bariatric surgery then it's a complete lifestyle change and it's not a temporary thing, i know that now. you can not lose all the weight and then think you can go back to unhealthy eating like you did before surgery. i mean you can not have your stomach cut out and then think you can eat crap food like you did before. i noticed that it is very easy to eat junk food like chips and soda these foods go down easily also if you drink your calories i just think that if you want long - term success with bariatric surgery then it's a complete lifestyle change and it's not a temporary thing, i know that now. you can not lose all the weight and then think you can go back to unhealthy eating like you did before surgery.	purpose. obesity has reached epidemic proportions in the u.s. and has nearly doubled worldwide since 1980. bariatric surgery is on the rise, but little focus has been placed on the psychosocial impacts of surgery. the purpose of this study was to explore experiences of patients who have undergone bariatric surgery at least two years before to gain an understanding of the successes and challenges they have faced since surgery. methods. this study used a phenomenological approach, to investigate the meaning and essence of bariatric patients with food after surgery. semi - structured interviews were conducted on a sample of nine participants who had undergone surgery at least two years prior. findings. two main themes regarding food intake emerged from the data: (a) food after the first year post - surgery and (b) bariatric surgery is not a magic pill. upon further analysis, food after the first year post - surgery had four subthemes emerge: diet adherence after the first year post - surgery, food intolerances, amount of food, and tendencies toward coping with food do not magically disappear. . findings revealed that post - operative diet and exercise adherence becomes increasingly difficult as weight loss slows. many participants find that only after the first year after surgery the work really begins.
ultrasound showed a 1.7-cm, well demarcated, heterogeneous, low echoic solid mass in the lower pole of the right thyroid (fig . 1). a computed tomography scan of the neck revealed a 3.3-cm, exophytic, low - attenuated mass in the lower pole of the right thyroid. the fna biopsy of the mass was performed, which was accompanied by the standard papanicolaou stain. the smear was composed of not only cohesive three dimensional clusters and sheets but also singly scattered cells (fig . the tumor cells showed mild anisonucleosis and high nuclear : cytoplasmic ( n / c) ratios. nuclei were round to ovoid with irregular nuclear contours and small, distinct nucleoli (fig . some tumor cells showed dense orangeophilic cytoplasm, which is suggestive of individual cell keratinization ( fig . 2c). some lymphocytes were found on the and within clusters, which was accompanied by the presence of a few stripped nuclei (fig . the aspirate was interpreted as a high - grade malignant thyroid neoplasm without further definitive classification . the mass had a lobular, tan - colored cut surface and a firm consistency ( fig . the tumor was well - circumscribed and it was composed of variably sized and irregularly shaped lobules of cohesive polygonal tumor cells which were separated by bands of dense fibrous stroma ( fig . the tumor cells had high n / c ratios, eosinophilic cytoplasm and ill - defined cell borders . the tumor cells showed positivity for cd5, carcinoembryonic antigen, high molecular weight keratin ( hmwk), cytokeratin 5 (ck5), and p63. one regional lymph node was found and involved by tumor. over a 27-month follow - up period, the cytological diagnoses were mentioned in previous 14 reports consisting of 40 cases. malignant tumor or poorly - differentiated carcinoma were the most frequent cytological diagnosis in cases of castle, except for six cases (table 1).2 - 15 one case was diagnosed as castle for which no cytological findings have been described.11 in previously reported cases of castle, the most common cytological findings include tight clusters and sheets of round tumor cells with high n / c ratios, vesicular nuclei, prominent nucleoli, amphophilic cytoplasm, and lymphocytic , which correspond well with the present case.2 - 4,6 in the present case and those of hirokawa et al.,2 keratinized cells were present. youens et al.6 reported indistinct cell borders, intranuclear grooves and sparse mitotic figures, which were also seen in the present case. youens et al.6 reported not only intranuclear cytoplasmic inclusions and papillary - like structures but also granular chromatin and rosette - like structures. the differential diagnoses of castle include papillary carcinoma, hrthle cell neoplasm, undifferentiated carcinoma, medullary carcinoma and metastatic lymphoepithelioma - like carcinoma. castle may show intranuclear pseudoinclusions or papillary - like structures, but it does not show fine pale chromatin and monolayer sheets of cells with dense cytoplasm. although hrthle cell neoplasm contains cellular aggregates with abundant cytoplasm and prominent nucleoli, lymphocytic of castle is lacking. it is very difficult to distinguish castle from metastatic lymphoepithelioma - like carcinoma, which also shows sheets of poorly differentiated round cells intermixed with small lymphocytes. subtle differences, such as frequent mitotic activity and a polymorphous inflammatory , favor metastatic lymphoepithelioma - like carcinoma.4 castle may resemble undifferentiated thyroid carcinoma with squamous differentiation, but it does not show marked cytologic atypia, frequent mitotic activity and necrotic characteristic of undifferentiated carcinoma. although castle may show rosette - like structures and granular chromatin, along with an abundance of single tumor cells as in medullary carcinoma, it is lacking of typical plasmacytoid cells, pink cytoplasmic granules and amyloid deposition.6 immunocytochemical study for cd5 is helpful to differentiate castle from other malignant thyroid neoplasm. cd5 is a surface glycoprotein expressed on mature t cells and a subset of b cells.16 thymic carcinoma cells are also known to be positive for cd5.17 cd5 is almost always expressed in castle. castle is positive for hmwk, ck5, and p63 because it is also a squamous cell carcinoma. however, thyroid squamous cell carcinoma and poorly - differentiated carcinoma were negative for cd5.17 ito et al.11 reported that the sensitivity and specificity for cd5 immunohistochemistry were 82% and 100%, respectively, in making a diagnosis of castle. in , the presence of poorly - differentiated tumor cells with a focal keratinization and a lymphocytic on the fna biopsy is suggestive of castle.	carcinoma showing thymus - like differentiation (castle) is a rare carcinoma of the thyroid or adjacent soft tissue of the neck with a histologic resemblance to thymic epithelial tumors. although the fine - needle aspiration (fna) plays a central role in the initial evaluation of thyroid nodules, few reports about the cytologic findings of castle have been found according to a review of literatures. we report cytologic findings of a case of castle. a 34-year - old woman presented with a 2-month history of sore throat. the fna showed that the smear was composed of three dimensional clusters and sheets. the tumor cells were round to ovoid with high nuclear: cytoplasmic ratios. the nuclei were vesicular with small nucleoli. there were some tumor cells showing keratinization. some lymphocytes were found on the and within clusters. the presence of poorly - differentiated tumor cells with a focal keratinization and a lymphocytic on the fna is suggestive of castle.
insertional mutagenesis after gene transfer in animal models1,2 as well as in x - scid clinical trials35 has led numerous laboratories to focus on the safety of the virus - based vectors used to transfer and express genes of interest. a better understanding of the mechanisms that define the characteristics of the integration of virus - based vectors should allow the development of new vectors with increased biosafety. taking advantage of the subgroup c feline leukemia virus (felv - c) as a platform for efficient transduction of target human cell types, including cd34 + stem and progenitor cells hek293 cells were engineered to stably package momlv vectors with felv 61e gag - pol gene products, in addition to felv - c sarna env, ing in a completely felv - derived packaging system6. in the current study we assessed the integration profile of catpac vector in comparison to the same momlv vector packaged with momlv gag - pol and amphotropic env in order to elucidate the influence of the felv component packaging proteins on viral integration. both primary vector sequences as well as integrase proteins have been implicated in genomic integration site selection for retroviruses, and thus we asked whether this hybrid vector might have a potentially less genotoxic integration pattern than standard momlv, due to the presence of the alternative felv integrase machinery. felv - c and momlv are both members of the mammalian c - type retrovirus family, but there is no prior information on the integration profile for either felv - c or hybrid momlv / felv packaging and vector systems. two rhesus macaques (rq4984 & rq4972) were treated with a combination of g - csf and scf and mobilized circulating hematopoietic progenitor and stem cells were collected by apheresis. pbmncs contained in apheresis product were purified by density gradient centrifugation over lymphocyte separation media (lsm, mp biomedicals) and cd34 + cells were positively immunoselected using the 12.8 igm anti - cd34 biotinylated antibody and macs streptavidin microbeads (miltenyi biotec). the purified cd34 + cells were stimulated for 48 hrs in dmem supplemented with 10% fcs, scf, flt3-l, and tpo and then transduced twice on fibronectin - treated plates (retronectin, takara) with the catpac or control momlv vector supernatants, using our standard in vitro transduction conditions8. the vectors contained the gfp marker gene, allowing assessment of transduction efficiency by flow cytometry. in both animals, the transduction efficiencies of cd34 + cells for catpac and control momlv packaging systems were equivalent: for rq4984 8.1 % and 8.8 % of gfp positive cells for catpac and momlv vectors respectively, and for rq4972 39.7 % and 20.3% respectively. genomic dna of transduced cd34 + cells was isolated using qiagen dneasy blood & tissue kit (# 69506) and lam - pcr was carried out as previously described9 with 100 ng of total genomic dna. the first exponential pcr was carried out with primer lci (5-gacccgggagatctgaat-3) and ltr - r1 primer (5-cagctgttccatctgttc-3), whereas the second exponential pcr was carried out with lciii (5-agtggcacagcagttagg-3) and ltr - r2 primer (5-gctagcttgccaaaccta-3). sequences obtained were aligned by blat or blast to the rhesus macaque genome assembly (mmul 1.0, jan 2006). we obtained 200 and 187 valid integration sites (is) for catpac and momlv vectors respectively (supplemental information). sites mapping to 2 or more genomic positions as well as sites mapping within repetitive genomic sequences were omitted to finally obtain 184 and 175 unique is respectively for catpac and momlv vectors. we also compared both profiles to in silico - randomly generated is sets as previously described9. briefly, 10 000 sets of 184 or 175 random is were designed in silico as follows: an aatt (tasi) site in the genome was selected at random using a random number generator. the in silico is was placed either upstream or downstream (p=0.5) of the aatt site, at a distance matching the size of one of the sequences obtained experimentally. the in silico is was validated only when a blast alignment of the genomic sequence between the aatt and the is returned a unique sequence in the genome. this operation was repeated 184 times and 175 times respectively for catpac and momlv vectors to obtain a single matching random dataset. these control datasets were subjected to the same analyses as the experimental datasets, and the were used to generate empiric p - values. we generated two groups of control genomic coordinates, one with 10 000 sets of 184 coordinates each to mimic the catpac vector is, and one with 10 000 sets of 175 coordinates each to mimic the momlv is. for gene annotation, ensembl release 54 (may 2009) comprising 38 146 predicted gene transcripts was used. the association between the catpac and momlv vectors integration sites was tested using a chi - square test. the obtained for momlv are comparable to previously reported integration patterns for standard momlv derived vectors10,11. we confirmed the preference of momlv for integrating near the tss, a feature also shared by catpac vector. like other retroviral vectors already studied such as momlv10,11, aslv12, siv10,13, and hiv12,14, catpac vector exhibited a profile of integration significantly different from random integrations as can be seen in all the tables and figures presented in this report. as shown in figure 1, out of the 114 catpac vector and 112 momlv is located in a window of 60 kb centered on transcription start sites (tss), 44% (catpac vector) and 35% (momlv) occurred in a 10 kb window centered on tss, implicating a preference for integration near tss. with regard to inter or intragenic insertions, table 1 shows that approximately half of the vector integrations were found inside of a gene (49.5 and 54.3% for catpac vector and momlv vectors respectively). one quarter of all integrations were found within a 30 kb window upstream of a gene (table 1). in these upstream regions as shown in table 2 integration of both vectors in or around (1 kb and 5 kb) cpg islands was significantly different from the random datasets. a quarter of the insertion sites were found 5 kb around cpg islands for catpac (27.7 %) and momlv (25.7 %) vectors. however, this higher percentage probably represents the propensity of catpac and momlv vectors to integrate close to the tss, rather than a preference for cpg islands. looking at integrations with regard to gene density , table 2 shows that less than half of the integrations for both vectors were found in lower gene - density regions (0 to 10 genes / mb) of the genome, and thus 53.8 % and 57.7 % of integration respectively for catpac and momlv vectors in gene dense regions (11 genes / mb and up). although this integration profile is significantly different from in silico - generated integrations, there were no obvious differences in the integration pattern between catpac and momlv vectors with respect to gene density. theoretically, virus - based vectors can lead to tumor formation by insertional mutagenesis either by activating an oncogene or by disrupting a tumor suppressor gene15. most, if not all, cases of malignant disease development following gene therapy for hematological disease have been associated with the activation of a proto - oncogene 25. we therefore looked at integration of catpac and momlv vectors in the proximity of oncogenes (table 3). we assessed both the proximity of proto - oncogenes to is, and conversely the proximity of is to proto - oncogenes. we defined proto - oncogenes based on the most recent version of the sanger oncogene file (dated 2008 - 12 - 16, at http://www.sanger.ac.uk/genetics/cgp/census/). three hundred eighty four human entries were mapped to 368 unique ensembl rhesus macaque gene identifiers. as shown in table 3, with this size dataset, the momlv integrations were not notably different from randoms with regard to proximity to oncogenes. however, the catpac vector integrations were more likely than the random datasets to be in proximity to oncogenes. the 12 catpac vector is in or within a 30 kb window of an oncogene were located: upstream of mllt11, mutyh, cytsb, and lmo2; downstream of arnt; in the intron of lasp1, q4w6x8_macmu, akap9, erg, cbfb, and pcm1; and in the exon of sufu. the 7 momlv is were located: upstream of mllt11 and tal1; downstream of kras and stil; in the intron of cdk6, pms1, and ciita. the only oncogene in proximity to both catpac vector and momlv integrations was mllt11, myeloid / lymphoid or mixed - lineage leukemia translocated to chromosome 11, also termed af1q. expression of this gene has been shown to be linked to poor prognosis in aml (reviewed in16) and has a function in apoptosis and drug resistance. we did not find any integrations in or near the mds1-evi1 gene complex, previously reported to be over - represented in engrafting human, murine, and rhesus macaque hematopoietic stem and progenitor cells transduced with momlv vectors17. interestingly, in the 12 oncogenes targeted by the catpac vector we found an integration 715 nucleotides upstream of lmo2, the gene activated in x - scid trials and causing leukemia through a t cell clonal expansion35. lmo2 was also reported as an mlv insertion site in the ada - scid and cgd gene therapy trials, but so far, no clonal expansion has been observed1820. in order to analyze if there was a significant clustering of vector insertions we used the definition of common integration sites (cis) reported by suzuki and coworkers21. as shown in figure 2, there is a difference between experimental data obtained for catpac vector and random integrations. we found 7 second order cis for catpac vector, a higher value than the frequency of second order cis in the matching random datasets. none of the cis identified were localized within 200 kb of a known proto - oncogene. these investigations contribute to knowledge regarding the determinants of retrovirus integration into the genome. at least two vector - related elements are involved: the integrase / capsid proteins and/or the primary sequence of the vector backbone itself. the relative contributions of each element in determining integration preferences remain under intense investigation, but it appears that the integrase core region is critical. swapping the integrase between even closely related viruses, such as hiv and fiv, in a change in the integration pattern determined primarily by the integrase, along with a contribution from gag - encoded capsid proteins22,23. the path of entry into the cell, determined by the env gene product, appears to have little impact on integration preferences24. in a previous study, pseudotyping of a standard momlv retroviral vector with the felv - c env protein only ed in more efficient transduction of primitive cd34 + cells compared to a standard galv env pseudotype, but no change in integration profile, based on a limited number of integration sites retrieved from myeloid cells following in vivo engraftment25. in the present study, we went on to study the impact of using felv gag and pol to package an momlv backbone and compared it to an momlv backbone packaged with standard momlv components, to determine if the integrase and capsid proteins of felv would change the integration pattern of momlv vector backbones. utilization of felv gag, pol, and env components did not alter the integration profile of momlv vectors, in contrast to the prior comparing the impact of swapping integrase between hiv and fiv viruses. both standard momlv and catpac vectors integrated within genes and in areas of high gene density, near tss. other studies have indicated that vector backbone sequences are also important determinants of integration patterns. the pre - integration complex contains viral integrase and capsid proteins, reverse - transcribed vector dna, and host cell factors, all implicated in both site - selection and the actual integration process2628. there are no large - scale integration profiles published for felv, however cis identification in tumors induced by felv infection indicate activation of an overlapping but not identical set of genes by felv as compared to momlv29. swapping the u3 region of the molv ltr with the same region of felv in a partial shift in cis in tumors30. however, the integration profile of cis in tumors may relate more to the enhancer activity of a specific u3 region in a target cell type as opposed to actual integration characteristics, since analysis of tumor cis relies to a large degree on in vivo selection of clones with activating insertions, from an initial highly polyclonal pool of cells. in , retroviral vectors packaged using the catpac system integrate into the genome in a non - random fashion, with a general profile very similar to vectors packaged using standard momlv gag and pol gene products, unfortunately characterized by a preference for integration within genes and near tss. although the study of a larger number of integration sites might uncover subtle differences between felv and momlv integrase and capsid determinants of integration, it is clear from the data we obtained in this pilot study that their general profiles are similar and characterized by the same risks. therefore , while catpac vector may have advantages in terms of ease of high titer vector production and potential improved efficiency of gene transfer to some target cell populations, the integration profile we have characterized does not suggest any increased safety of these vectors compared to standard retrovirus packaged with momlv components.	adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. the integration profiles of vectors based on mlv, aslv, siv, and hiv have all been shown to be non - random, and novel vectors with a safer integration pattern have been sought. recently we developed a producer cell line called catpac that packages standard momlv vectors with felv gag, pol and env gene products. we now report the integration profile of this vector, asking if the felv integrase and capsid proteins could modify the momlv integration profile, potentially ing in a less genotoxic pattern. we transduced rhesus macaque cd34 + hematopoietic progenitor cells with catpac or standard momlv vectors, and determined their integration profile by lam - pcr. we obtained 184 and 175 unique integration sites (is) respectively for catpac and standard momlv vectors, and these were compared to 10 000 in silico - generated random is. the integration profile for catpac vector was similar to momlv and equally non - random, with a propensity for integration near transcription start sites and in highly dense gene regions. we found an is for catpac vector localized 715 nucleotides upstream of lmo-2, the gene involved in the all developed by x - scid patients treated via gene therapy using momlv vectors. in , we found that replacement of momlv env, gag, and pol gene products with felv did not alter the basic integration profile. thus there appears to be no safety advantage for this packaging system. however, considering the stability and efficacy of catpac vectors, further development is warranted, utilizing potentially safer vector backbones, for instance those with a sin configuration.
there is evidence that the production and consumption of beer began in egypt in the early dynastic period (55003100 bce). high because yeast floats to the top of the tank at a temperature between 15 c and 25 c during production of ale beer with the yeast saccharomyces cerevisiae. this yeast is capable of fermenting at a temperature lower than 10 c and flocculates at the bottom of the tank. the saccharomyces sensu stricto complex includes six parented species: s. cerevisiae, saccharomyces bayanus, saccharomyces cariocanus, saccharomyces kudriavzevii, s. mikatae and s. paradoxus; however, it has been observed that lager - brewing yeast is a hybrid species of two combined genomes of s. eubayanus and s. cerevisiae ,,,,,,,. this provides an important source of chromosomal rearrangements, leading to the gene number and the size of the complete genome,,,. it has been proposed and recently demonstrated that lager yeast is the product of two independent hybridization events that can be divided into two groups: saaz and frohberg, or group i and group ii, respectively,,,,. with the use of next generation sequencing (ngs) technologies, such as the illumina platform, 40,175 prokaryote and eukaryotes genomes have been reported, including 210 different strains of the saccharomyces complex (http://www.ncbi.nlm.nih.gov/genome/browse/ - revised july 22, 2015). to obtain a higher level of understanding of the sequenced organism the brewing yeast saccharomyces sp. strain 790 and a reference sequence of 76 scaffolds from s. eubayanus de c.v. the s. cerevisiae s288c reference genome sequence was retrieved from the yeast genome database (www.yeastgenome.org). the brewing yeast genome was sequenced using the flx 454 titanium (roche) and miseq (illumina) massive sequencing platforms according to the manufacturer's protocols. we obtained 0.8 million reads from flx 454 titanium (454 life sciences, branfort, ct) with an average size of 400 bp; 6 million pair - end reads from illumina (illumina, san diego, ca) with an average size of 150 bp; 5 million mate - pair reads from illumina with an insert size of 350 bp and a size of 101 2 bp; and 11.7 million mate - pair reads from illumina with an insert of 8 kb and a size of 51 2 bp. approximately 454 illumina pair - end reads were assembled with a newbler denovo assembler (roche). this whole genome shotgun project has been deposited at ddbj / ena / genbank under the accession lsmh00000000. the sequencing quality data were analyzed with fastqc 0.10.1 software with a value q30. likewise, alignments were made against reference sequences (s. cerevisiae s288c and s. eubayanus) with the mummer 3.23 software package. the bioinformatics analysis for the annotation was performed with maker v2.31.8 software (university of utah). the brewing yeast saccharomyces sp. strain 790 and a reference sequence of 76 scaffolds from s. eubayanus de c.v. the s. cerevisiae s288c reference genome sequence was retrieved from the yeast genome database (www.yeastgenome.org). the brewing yeast genome was sequenced using the flx 454 titanium (roche) and miseq (illumina) massive sequencing platforms according to the manufacturer's protocols. we obtained 0.8 million reads from flx 454 titanium (454 life sciences, branfort, ct) with an average size of 400 bp; 6 million pair - end reads from illumina (illumina, san diego, ca) with an average size of 150 bp; 5 million mate - pair reads from illumina with an insert size of 350 bp and a size of 101 2 bp; and 11.7 million mate - pair reads from illumina with an insert of 8 kb and a size of 51 2 bp. approximately 454 illumina pair - end reads were assembled with a newbler denovo assembler (roche). this whole genome shotgun project has been deposited at ddbj / ena / genbank under the accession lsmh00000000. the sequencing quality data were analyzed with fastqc 0.10.1 software with a value q30. likewise, alignments were made against reference sequences (s. cerevisiae s288c and s. eubayanus) with the mummer 3.23 software package. the bioinformatics analysis for the annotation was performed with maker v2.31.8 software (university of utah). the read assembly yielded 133 scaffolds with a ~ 70 depth and a n50 of 568,800 bp, suggesting a complete genome size of ~ 22.7 mbp (table 1), similar to previous reports of other lager beer yeasts,,,. approximately 65/133 scaffolds had a size > 10 kbp, which represents 99.667% of the assembled genome (table 2). table 3 shows a comparison of the assembly level of the sequenced genomes of the saccharomyces species (as of august 2015). the alignments against the reference genome, s. cerevisiae s288c, assigned scaffolds to each of its 16 chromosomes, and some scaffolds covered different portions of more than one chromosome; for example, scaffold01 (sf01) aligns with two chromosomes: a small portion with chromosome 1 and with chromosome 12. the estimated size matches the previous and known information; this is due to the presence of 16 chromosomes of the s. cerevisiae sub - genome and 16 of s. eubayanus. likewise, its size is close to the sum of the aforementioned genomes (~ 12 mbp each). , who reported the sequence and assembly of the lager brewing yeast genomes saccharomyces carlsbergensis (78 scaffolds, 29 chromosomes with a 19.5 mbp length), and saccharomyces pastorianus weihenstephan 34/70 (985 scaffolds, ~ 29 chromosomes, and 22.9 mbp). the annotation yielded 9939 cds and a gff file with their locations in the scaffolds of the assembly (fig . the protein and transcript sequences, were subjected to a local alignment with the blast tool against a local database using the sequence of s. cerevisiae s288c as a reference . the transcripts were considered to be genes because previous reports showed that only approximately 5% of the yeast genome contains introns, . the scaffolds were classified using the obtained from blastn according to the mean identity percentage in all of the genes contained in the same scaffold, as follows ( table 4): % i d > 99.0% and e value < 10 = scaffold belongs to s. cerevisiae. % i d < 90.0% and e value < 10 = scaffold does not belong to s. cerevisiae. 99.0% > % i d > 90.0% and e value < 10 = hybrid scaffold. our identity criterion was validated by subjecting the gene sequences from s. cerevisiae s288c to a local alignment against s. eubayanus, and we found that the % i d between these strains was < 90% (supplementary table s1) and the average size of the cds was 1550 bp. approximately 96.8% of the genome was annotated; 53.93% corresponded to s. cerevisiae, 42.86% were non - cerevisiae and 3.20% remained un - annotated fig. its nuclear genome consists of approximately 32 chromosomes, 16 of which correspond to the s. cerevisiae genome and 16 to the s. eubayanus genome, without considering ploidy. nine scaffolds presented continuous translocations (scaffolds 1, 4, 6, 23 for the s. cerevisiae sub - genome and 26, 11, 17, 22 and 32 for the s. eubayanus sub - genome), which indicate homologous recombination events. data on the chromosome number and size, as well as the number of scaffolds obtained, are consistent with previous reports on lager yeast,,. the following are the supplementary data related to this article.supplementary table s1comparative analysis of saccharomyces cerevisiae s288c and saccharomyces eubayanus.supplementary table s1supplementary table s2saccharomyces sp. 790 and saccharomyces cerevisiae comparative .supplementary table s2 comparative analysis of saccharomyces cerevisiae s288c and saccharomyces eubayanus.	the genome of lager brewer's yeast is a hybrid, with saccharomyces eubayanus and saccharomyces cerevisiae as sub - genomes. due to their specific use in the beer industry , relatively little information is available. the genome of brewing yeast was sequenced and annotated in this study. we obtained a genome size of 22.7 mbp that consisted of 133 scaffolds, with 65 scaffolds larger than 10 kbp. with respect to the annotation, 9939 genes were obtained, and when they were submitted to a local alignment, we found that 53.93% of these genes corresponded to s. cerevisiae, while another 42.86% originated from s. eubayanus. our confirm that our strain is a hybrid of at least two different genomes.
	fragments of tracheal epithelium alone or in continuity with connective tissues, can be maintained in culture medium and used for short term or long term studies of toxicity of a variety of chemicals. large numbers of uniform cultures are prepared with the aid of a slicing device or by application of simple method for dissecting sheets of epithelium free from underlying cartilage. the cultures may be placed in an exposure chamber - incubator mounted on a microscope stage and monitored continually for ciliostasis and exfoliation of cells. morphology is further studied by fixation of selected specimens and preparation for light microscopy and electron microscopy. synthetic functions are evaluated by autoradiographic measurement of incorporation of radioactive precursors into macromolecules and other dynamic features are indirectly assessed by histochemical and histoenzymatic methods. short - term studies using these several techniques have shown that ciliostasis does not correlate with cell injury in all instances, and a long - term study has demonstrated dose dependence of a cytotoxic agent when duration of culture viability is measured. the method lends itself to a broad range of investigations in which dose, period of exposure, and role of cofactors must be independently and quantitatively assessed.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12.
pain is one of the most prevalent symptoms in patients with primary bone sarcomas and with the distant metastases of nonbone primary tumors. studies have indicated that bone cancer pain has complex and unique mechanisms that may involve both inflammatory and neuropathic pain. clinically, most patients with bone cancer have already passed the optimal time for radical surgery and multidisciplinary therapies. however, with radiotherapy, majority of these patients experience pain relief, control of tumor growth, and prolonged survival. radiotherapy is not only an effective method in the clinical treatment of bone cancer, but also an important approach for treatment of the severe pain associated with bone cancer. however, mechanisms underlying radiotherapy for bone cancer pain have not been well investigated and remain elusive. cyclic adenosine monophosphate - protein kinase a (camp - pka) signaling pathway plays important roles in a number of cellular processes, including immune function, growth, differentiation, and metabolism, and is essential to the plasticity in neural synapses in cns. activation of camp - pka pathway has been reported to enhance presynaptic neurotransmitter synthesis and vesicular transportation probably through phosphorylation of key transcriptional factors (i.e., camp response element - binding protein) and synaptic vesicle proteins. in addition, recent studies found that camp - pka signaling pathway is involved in both inflammatory pain and neuropathic pain. the peripheral hyperalgesic actions of inflammatory mediators are mediated by the camp - pka signaling pathway. we have recently demonstrated that the camp - pka pathway is crucial for the maintenance of dorsal root ganglia (drg) neuronal hyperexcitability and behaviorally expressed hyperalgesia, in an in vivo neuropathic pain animal model of chronic compression of the drg (ccd model), as well as in an in vitro model of acute drg dissociation. recently, we have further found that activation of the camp - pka signaling pathway plays an important role in both induction and maintenance of bone cancer pain in rats. however, it remains unknown whether and then how camp - pka signaling would contribute toward radiotherapy treatment for bone cancer pain. this study provides evidence supporting an idea that radiotherapy may suppress bone cancer pain through inhibition of abnormal activation of camp - pka signaling pathway in drg and the spinal cord. female adult sprague - dawley rats (160180 g at the start of the experiment) were housed in a controlled lighting environment with free access to food and water. all experiments were approved by the institutional animal care and use committees in oriental hospital and conducted in accordance with the declaration of the national institutes of health guide for the care and use of laboratory animals (publication number 85 - 23, revised 1985). surgery was performed under anesthesia with intraperitoneal injection of sodium pentobarbital (50 mg / kg, i.p .). the protocols of the bone cancer pain model were similar to that described previously. in brief, following induction of general anesthesia with intraperitoneal injection of sodium pentobarbital, rats were placed abdominal side up. after disinfecting with 75% v / v ethanol , a one - centimeter rostrocaudal incision was made in the skin directly above the top half of the tibia. tumor cells (1 10 cells/l, 5 l), extracted from the ascetic fluid of female rats that received walker 256 mammary gland carcinoma cells, were injected (tumor cell implantation, tci) into the intramedullary space of the right tibia to induce bone cancer. rats in the sham group were injected with the same number of boiled tumor cells. rats were immobilized in an acryl jig, and a dose of 6 gy was delivered to the right tibia area using a collimator system with 6 mv x - rays. sham radiotherapy applied in tci rats was using the same protocol, except that they were not given the real x - ray radiation. on the 17th day after tci treatment, radiographic images were taken (exposure setting : 12 ms, 31 kvp) using a philips digital radiographer system (digital diagnost vm ; philips medical systems dmc gmbh, hamburg, germany). bone destruction was evaluated on a scale of 05: 0 = normal bone structure without any sign of deterioration; 1 = small radiolucent lesions in the proximal epiphysis (<3); 2 = increased number of radiolucent lesions (> 3) indicating loss of medullary bone; 3 = loss of medullary bone, plus erosion of the cortical bone; 4 = full - thickness unicortical bone loss; and 5 = full - thickness bicortical bone loss and displaced fracture. thermal hyperalgesia was indicated by a significantly shortened latency of foot withdrawal in response to heat stimulation. to determine thermal hyperalgesia, a radiant heat source was focused and delivered on a portion of the hind paw; the thermal stimuli shut off automatically when hind paw moved (or after 20 s to prevent tissue damage). mechanical allodynia was indicated by a significant decrease in the threshold of paw withdrawal to mechanical indentation of the plantar surface of each hind paw, with a sharp, cylindrical probe. the probe was applied to 6 designated loci distributed over the plantar surface of the foot. the minimal force that induced paw withdrawal was read off the display. the experimenters who performed these behavioral tests were blinded to the treatment condition of the animals. the total rna isolated with trizol reagent (invitrogen, usa) was reverse transcribed using m - mlv reverse transcriptase (takara). primer sets were synthesized by integrated dna technologies (sangon biotech); their sequences are shown in table 1. the amplification conditions were set as follows: 94c for 2 min and 30 cycles of 94c for 30 s and 58c for 40 s. pcr products were analyzed on agarose gel electrophoresis and were verified by dna sequencing. the gels were imaged with tanon 2500 imaging systems and analyzed by analysis software imagej 1.48u. the drg and the spinal cord at segments of l4-l5 ipsilateral to tci were collected on postoperative days 10 and 14 for further neurochemical analysis. commercial enzyme - linked immunosorbent assay kits were used to determine the concentrations of camp, il-1, and tnf- and activity of pka, according to the manufacturers' instructions. elisa kit for camp was purchased from cayman chemical (ann arbor, michigan, usa); elisa kits for il-1, tnf-, and activated pka were purchased from r&d systems (minneapolis, minnesota, usa). all statistical analyses were carried out using statistical product and service solutions, ver. 15.0 (spss inc ., alterations in camp mrna and concentration, pka activity, and the levels of il-1 and tnf- were tested using one - way analysis of variance ( anova) followed by bonferroni post hoc tests. two - way repeated - measures anova (days groups) was used to test the behavioral responses to thermal and mechanical stimuli, followed by bonferroni post hoc tests.. the thresholds of thermal and mechanical withdrawal ipsilateral, but not contralateral, to the tci treatment decreased approximately by 50% during postoperative days 917 compared to sham control. to determine whether radiotherapy treatment affects bone cancer pain, a single dose of x - radiation (6 gy) was applied on the 9th day after operation. such radiotherapy produced long - lasting inhibition of tci - induced thermal hyperalgesia and mechanical allodynia. the inhibition lasted for a week with 1 d delay following the x - radiation application. the peak values of inhibition of the thermal hyperalgesia and mechanical allodynia were approximately 2550%. the thermal and mechanical withdrawal of the hind paw contralateral to tci treatment was not altered following the radiotherapy. no radiographic change (score = 0, no bone destruction) was found in the group of sham without radiotherapy. bone destruction was seen clearly in groups of tci with and without radiotherapy, respectively. score of the tci group with sham therapy was 3.75 0.43 (range : 35). the score in the group of tci with radiotherapy dropped to 2.38 0.52 (range : 13), which was significantly less than the score in tci group with sham therapy. our previous studies have shown that expression of pka - rii and pka - c mrnas was increased after tci treatment in a time - dependent manner. to test whether radiotherapy has an effect on this tci - induced pka mrna expression , we measured the levels of pka - rii and pka - c mrnas on day 1 and day 5 after radiotherapy treatment (postoperative days 10 and 14, resp .) using rt - pcr. we found that radiotherapy treatment greatly inhibited tci - induced increase of expression of pka - rii and pka - c mrna. the expression of pka - rii and pka - c mrna in the tci + radiotherapy group was significantly reduced compared with the tci + sham group (figure 3). we have previously shown that camp concentration and pkg activity in drg and the spinal cord were significantly increased in a time - dependent manner after tci treatment. to test the hypothesis that camp - pka signaling pathway might be altered by radiotherapy, we measured camp level and pka activity in drg and the spinal cord following radiotherapy treatment. the showed that radiotherapy treatment significantly reduced tci - induced increase of camp concentration as well as pka activity in drg and the spinal cord (figure 4). we have recently shown that il-1 and tnf- levels in the spinal cord were significantly increased in a time - dependent manner after tci treatment. to test whether radiotherapy could affect tci - induced increase of il-1 and tnf-, we measured levels of il-1 and tnf- in the spinal cord after radiotherapy on postoperative days 10 and 14 (1 and 5 days after radiotherapy). our showed that radiotherapy treatment significantly reduced tci - induced increase of il-1 and tnf- in the spinal cord (figure 5). this study demonstrates that radiotherapy is an effective treatment approach for treating bone cancer pain. the camp - pka signaling pathway may be a mechanism that underlies the analgesic effect of radiotherapy in bone cancer pain. radiotherapy suppresses tci - induced painful behaviors, thermal hyperalgesia, and mechanical allodynia and alleviates tci - induced massive bone destruction. radiotherapy reduces tci - induced increased expression of pka mrnas in drg as well as the increased level of camp concentration and pka activity in both drg and the spinal cord. in addition, radiotherapy in a significant decrease of il-1 and tnf- activity in the spinal cord. these findings suggest that radiotherapy treatment may suppress tci - induced hyperalgesia and allodynia by inhibiting the camp - pka signaling pathway in drg and the spinal cord. approximately 50% of these patients experience moderate to severe pain. mechanisms of cancer pain are thought to be complex and may involve a combination of inflammation, nerve injury, and other unique factors. considerable evidence of cancer pain treatment shows that radiotherapy effectively relieves pain in up to 95% of patients and can maintain the level of analgesia in more than 70% of the patients for up to three months. previous studies have shown that activation of the camp - pka signaling pathway contributes to noxious stimulus - induced peripheral and central sensitization. we have found that in vivo chronic compression of drg or in vitro acute drg dissociation ed in activation of the camp - pka signaling pathway. continued activation of the camp - pka signaling pathway is required to maintain hyperexcitability of the drg neurons and behaviorally expressed hyperalgesia in these two different injury - related stress conditions. recently, we have confirmed that activation of the camp - pka pathway plays an essential role in the induction and maintenance of bone cancer pain. here, we provide direct evidence that the elevated activity of camp - pka signaling pathway is significantly decreased by radiotherapy in a bone cancer pain model. these support an idea that radiotherapy may suppress bone cancer pain through inhibition of abnormal activation of camp - pka signaling pathway, suggesting a new mechanism for the radiotherapy of bone cancer pain. the proinflammatory cytokines are activators of the camp - pka pathway in primary afferent neurons. during tumor growth and development of bone cancer pain, certain proinflammatory cytokines such as tnf- and il-1 are activated and released from the astrocytes and microglial cells and contribute to bone cancer pain. radiotherapy can reduce these proinflammatory cytokines and inhibit activation of the camp - pka signaling pathway and thus in relief of bone cancer pain. these findings suggest that the cytokines are also targets that may be responsible for radiotherapy - induced analgesia. in addition, our showed that radiotherapy ed in less bone loss in tci rats. this is consistent with the previous finding that radiotherapy with high dose may kill part of the tumor cells or reduce their activation and thus delay the destruction of the bone structure. these findings indicate that radiotherapy may reduce both pain and loss of bone structure following tci treatment. our demonstrate that radiotherapy can effectively suppress bone cancer pain probably through inhibition of activation of camp - pka signaling pathway in the primary sensory neurons and the spinal cord. this study may suggest a new mechanism underlying radiotherapy - induced analgesia of bone cancer pain and support the clinical use of radiotherapy in treatment of certain cancer pain conditions.	radiotherapy is one of the major clinical approaches for treatment of bone cancer pain. activation of camp - pka signaling pathway plays important roles in bone cancer pain. here, we examined the effects of radiotherapy on bone cancer pain and accompanying abnormal activation of camp - pka signaling. female sprague - dawley rats were used and received tumor cell implantation (tci) in rat tibia (tci cancer pain model). some of the rats that previously received tci treatment were treated with x - ray radiation (radiotherapy). thermal hyperalgesia and mechanical allodynia were measured and used for evaluating level of pain caused by tci treatment. pka mrna expression in dorsal root ganglion (drg) was detected by rt - pcr. concentrations of camp, il-1, and tnf- as well as pka activity in drg and the spinal cord were measured by elisa. the showed that radiotherapy significantly suppressed tci - induced thermal hyperalgesia and mechanical allodynia. the level of pka mrna in drg, camp concentration and pka activity in drg and in the spinal cord, and concentrations of il-1 and tnf- in the spinal cord were significantly reduced by radiotherapy. in addition, radiotherapy also reduced tci - induced bone loss. these findings suggest that radiotherapy may suppress bone cancer pain through inhibition of activation of camp - pka signaling pathway in drg and the spinal cord.
creatine kinase (ck) (atp : creatine kinase n - phosphotransferase, ec 2.7.3.2) is thought to be crucial for intracellular transport and the storage of high energy phosphate because it catalyzes the reversible transfer of a phosphoryl group from mgatp to creatine, which leads to the creation of phosphocreatine and mgadp. ck plays an important role in the cellular energy metabolism of vertebrates, and it is widely distributed in tissues that require a lot of energy. several types of ck are expressed in various tissues: the muscle and brain types of ck are the most common, and three different isoenzymes that include ck - mm (the muscle type homodimer), ck - bb (the brain type homodimer), and ck - mb (the muscle plus brain type heterodimer) originate from these two common types. various types of cks (the muscle, brain, and mitochondrial types) are thought to be important not only in the diagnosis of myocardial infarction, cardiac hypertrophy, and muscular dystrophy but also for studies of some other serious diseases, including alzheimer's disease, parkinson's disease, and psoriasis. ck - bb is associated with several pathologies, including neurodegenerative and age - related diseases. recently, chang et al. reported an important role for ck - bb in osteoclast - mediated bone resorption, which was found using a proteomics approach. they found that ck - bb is greatly increased during osteoclastogenesis and suggested that it represents a potential target for antiresorptive drug development. ck - bb interacts with the potassium - chloride cotransporter 3, which is involved in the pathophysiology of hereditary motor and sensory neuropathy with agenesis of the corpus callosum. previous studies have reported that ck - bb is involved in alzheimer's disease (ad) as an oxidatively modified protein. this suggests that oxidatively damaged ck - bb may be associated with aging and age - related neurodegenerative disorders such as ad. ck - mm is a good model to use for studying folding pathways because of several characteristics: (i) it is a dimer that consists of two identical subunits, each with an n - terminal domain with about 100 residues and a c - terminal domain with about 250 residues connected by a long linker; (ii) extensively denatured ck can be renatured spontaneously with restoration of its enzymatic activity in the absence of any external assistance; (iii) its folding pathway is complicated and involves several intermediates; (iv) conformational changes of the secondary and tertiary structures can be easily measured by monitoring activity changes; (v) protein - protein interactions, including molecular chaperones, are observed during refolding. in this study , we obtained computational predictions of the binding proteins by using two types of ck (ck - bb and ck - mm) as hub proteins in bioinformatic algorithms. as a , we obtained 208 protein lists in the interaction networks via application of both muscle and brain types of ck. determination of the binding factors and functions of ck can further promote our understanding of the physiological roles of ck. we present the functionally classified protein - protein interactions on the basis of the cell cycle, cell transport, oxidoreductase, and apoptosis. the protein interaction resources included six databases: dip, bind, intact, mint, hprd, and biogrid. they are protein structural interactome map (psimap), a method that uses the structural domain of the scop (structural classification of proteins) database and protein experimental interactome map (peimap), a common method that uses public resources of experimental protein interaction information such as hprd, bind, dip, mint, intact; and biogrid. the basic procedure of psimap is to infer interactions between proteins by using their homologs. interactions among domains or proteins for known pdb (protein data bank) structures are the basis for the prediction. if an unknown protein has a homolog to a domain, then psimap assumes that the query has the probability to interact with its homolog's partners. we carried out a redundancy check to remove identical protein sequences from the source interaction databases. we identified potential candidates through protein - protein interaction predictions made using various protein interaction resources. by analyzing the hub protein of the networks with metrics such as degree and centrality , we detected 123 potential candidates for ckb interacting (direct or indirect) factors and 85 candidates for ckm. in figure 1, interacting factors such as nfkb1 (np_003989, nuclear factor of kappa light polypeptide gene enhancer in b - cells 1), myoc (np_000252 ; myocilin, trabecular meshwork inducible glucocorticoid response), myom2 (np_003961 ; myomesin ( m - protein) 2, 165 kda ), fhl2 (np_001034581, four - and - a - half lim domains 2), hif1an (np_060372, hypoxia - inducible factor 1, alpha subunit inhibitor), asb9 (np_076992, ankyrin repeat and socs box - containing 9), and ckm (np_001815, creatine kinase, muscle) were elucidated. interestingly, nfkb1 was detected as a hub protein interacting with ck - bb in our . in figure 2, we obtained similar to those from figure 1, where nfkb1, myoc, myom2, fhl2, hif1an, asb9, and ckm were detected as interacting factors that were directly or indirectly associated with ckb. nfkb1, ckm, and asb9 interacted with ckb directly. in the same way, we detected the ckm - associated proteins as shown in figure 3 with 80% sequence identity. as a , we found that ckb, fhl2, myoc, asb9, hif1an, nfkb1, ttn (np_596870, titin), myh9 (np_002464, myosin, heavy chain 9, non - muscle), and itga7 (np_002197, integrin, alpha 7) mainly interacted with ckm at 80% sequence identity. at the level of 100% identity, we found that myom2, ckb, fhl2, and myoc directly interacted with ckm as shown in figure 4. in addition to these factors, complete lists of factors that interacted with ckb and ckm in a direct or indirect manner are shown in tables 1 and 2. after overlapping the from figures 1to 4, we found that nfkb1, fhl2, and myoc were still detected as hub proteins in figure 5. nfkb1 (also known as p50 or nf - kappab) is a well - known transcription regulator that is responsible for the expression and regulation of many genes for immune response, cell adhesion, differentiation, proliferation, angiogenesis, and apoptosis. it translocates into the nucleus and stimulates the expression of many genes involved in various biological functions. nfkb1 is also associated with a number of inflammatory diseases such as lymphoma, alzheimer disease, psoriatic arthritis, breast cancer , and rheumatoid arthritis. activation of nfkb1 requires binding of nf - kappab essential modulator (nemo) to ubiquitinated substrates. with respect to an association with ck , it has been reported that nfkb1 is mostly associated with myocardial ischemia / reperfusion. during reperfusion, the absence of poly(adp - ribose) polymerase-1 (parp-1) leads to a reduction of myocardial apoptosis, which is associated with reduced nfkb1 activation , and proteasome inhibition ablates activation of nfkb1 in myocardial reperfusion and reduces reperfusion injury. myocardial injury was assessed by measuring the serum levels of ck, and ck was reduced in serum along with reduction of nfkb1 activation. fhl2 is a member of the human four - and - a - half - lim - only protein family, which consists of the members fhl1, fhl2, fhl3, fhl4, and act. these proteins function in various cellular processes, including regulation of cell survival, transcription, and signal transduction. fhl2 contains an lim domain, one of the protein - protein interaction motifs, which allows specific proteins to combine with certain partners. the specificity of a protein - protein interaction can be obtained by an interaction code predicted by conserved amino acid sequences. the interaction of fhl2 with transcription factors and other proteins involved in cancer development was examined. since transcription factors control all fundamental developmental and homeostatic processes, transcriptional cofactors such as fhl2 are likely to contribute to human carcinogenesis and are of clinical importance in various forms of cancer, including leukemia. with respect to an association with ck, chung et al. reported that fhl2 (developmentally enhanced phosphotransfer enzyme - anchoring protein) amalgamated the myofibrillar ck metabolic signaling circuit, providing an energetic continuum between mitochondria and the nascent contractile machinery in a murine embryonic stem cell cardiac differentiation model. they reported that ck - m clustered around developing myofibrils, sarcolemma, and the perinuclear compartment, whereas ck - b was tightly associated with myofibrillar alpha - actinin, forming wire - like structures extending from the nuclear compartment to the sarcolemma. fhl2 was also increased in myocardial ischemia - reperfusion injury, where il-6 and il-8 mrna are upregulated in human cardiac myocytes. the ankyrin repeat domains of asb9 can associate with the substrate binding site of ck in a socs box - independent manner. asb9 is a member of the ankyrin repeat and is a suppressor of the cytokine signaling (socs) box protein family. it can interact with the socs box domain of the elongin b - c adapter complex and can further complex with the cullin and ring box proteins to form e3 ubiquitin ligase complexes. these complexes may be involved in specific substrate - recognition for ubiquitination and degradation and mediate the substrate - recognition of the e3 ubiquitin ligases. however, myoc has a cytoskeletal function, and this implies that it may interact with ck somehow. myoc is expressed in many ocular tissues including the trabecular meshwork, which is a specialized eye tissue that is essential in regulating intraocular pressure. myoc mutations have been identified as the cause of hereditary juvenile - onset open - angle glaucoma. researchers could apply computational prediction by ppi mapping to help determine target proteins. since the next step in the functional study of interesting proteins / genes is a time- and cost - consuming process, the number of target proteins is limited; hence, for the right choice, computational prediction on the basis of database information could be critical at this step. functional studies can be further conducted using a mouse model and a large number of clinical samples. final confirmation and ck mechanisms could then be more clearly evaluated for developing drugs to effectively treat ck - related diseases. the functions of most of the candidate proteins predicted in this study have not been well reported in skin diseases or in the pathogenesis of other diseases. we provide new information regarding these candidate proteins' interaction with ck, as well as the involvement of several hub proteins such as nfkb1, fhl2, asb9, and myoc. although we do not suggest a direct role of any candidate protein in skin diseases, we provide candidate proteins to be targeted in further studies of ck - associated diagnostic markers and/or treatment of corresponding skin conditions. furthermore, we also provide some insights into understanding the responses of ck in skin.	creatine kinase (ck ; ec 2.7.3.2) is related to several skin diseases such as psoriasis and dermatomyositis. ck is important in skin energy homeostasis because it catalyzes the reversible transfer of a phosphoryl group from mgatp to creatine. in this study, we predicted ck binding proteins via the use of bioinformatic tools such as protein - protein interaction (ppi) mappings and suggest the putative hub proteins for ck interactions. we obtained 123 proteins for brain type ck and 85 proteins for muscle type ck in the interaction networks. among them, several hub proteins such as nfkb1, fhl2, myoc, and asb9 were predicted. determination of the binding factors of ck can further promote our understanding of the roles of ck in physiological conditions.
we herein describe a series of three cases of acrometastasis to the hand. we have also reviewed the pathogenesis, clinical presentation, and therapeutic management of acrometastasis to the hand. a 60-year - old gentleman presented to our clinic with the chief complaints of a nonhealing ulcer in the right groin for the last 8 years and a growth in the right thumb for the last 6 months. on examination, an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin was noted. there was a presence of two firm, mobile, and tender lymph nodes (4 4 cm and 2 2 cm, respectively) in the left horizontal inguinal chain. besides, a 3 3 cm ulceroproliferative growth involving the distal phalanx of the right thumb with subungual and periungual involvement was seen. an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin a 3 3 cm ulceroproliferative growth involving the distigal phalanx of the right thumb with subungual and periungual involvement fine needle aspiration cytology (fnac) of the left inguinal lymph node was suggestive of metaststic squamous cell carcinoma. a skiagram of the hand revealed a lobulated, homogenous soft tissue mass around the distal phalanx of the right thumb with no definite evidence of bony erosion. a contrast - enhanced ct scan of the chest and whole abdomen was unremarkable excepting the findings of enlarged left inguinal lymphadenopathy. a short course of palliative radiation 30 gy/10 fractions for 2 weeks to the primary lesion and metastatic lymphadenopathy was delivered. this was followed by radiation of 54 gy/27 fractions for 5.5 weeks to the site of acrometastasis. a 55-year - old gentleman was diagnosed with carcinoma supraglottic larynx (stage t4n1m0). he was treated with combined modality therapy: radiation 66 gy/33 fractions for 6.5 weeks with concurrent cisplatin 100 mg / mq 3 week. subsequently, he presented with swelling of the tip of all fingers of the left hand with nail bed involvement , and multiple subcutaneous nodules in the upper and lower limb. fnac of acral lesions was suggestive of metastatic carcinoma. a contrast - enhanced ct scan of the chest, abdomen, and pelvis revealed metastasis in bilateral lungs and liver in view of the poor performance status (ecog 3) and widespread dissemination of disease, he was offered best supportive care. metastatic swelling of all five distal phalanges of the left hand multiple bilateral lung metastasis multiple liver metastasis a 52-year - old gentleman presented to our clinic with the complaints of dysphagia to solids progressing to liquids, headache, and a nodule in the little finger of the left hand for the last 6 months. on endoscopy, a friable tumor involving half of the circumference of the esophageal lumen was seen 25 - 30 cm from the incisors. endoscopic biopsy revealed moderately differentiated squamous cell carcinoma. a contrast - enhanced ct scan of the chest showed circumferential mural thickening of the midesophagus with mediastinal lymphadenopathy. fnac of the nodule involving the distal phalanx of the little finger of the left hand was suggestive of metastatic squamous cell carcinoma. he underwent palliative radiation to esophagus and acrometastasis, 30 gy/10 fractions for 2 week. a 60-year - old gentleman presented to our clinic with the chief complaints of a nonhealing ulcer in the right groin for the last 8 years and a growth in the right thumb for the last 6 months. on examination, an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin was noted. there was a presence of two firm, mobile, and tender lymph nodes (4 4 cm and 2 2 cm, respectively) in the left horizontal inguinal chain. besides, a 3 3 cm ulceroproliferative growth involving the distal phalanx of the right thumb with subungual and periungual involvement was seen. an 8 8 cm ulcer in the right inguinal area adjoining the root of the penis with a necrotic base and rolled up margin a 3 3 cm ulceroproliferative growth involving the distigal phalanx of the right thumb with subungual and periungual involvement fine needle aspiration cytology (fnac) of the left inguinal lymph node was suggestive of metaststic squamous cell carcinoma. a skiagram of the hand revealed a lobulated, homogenous soft tissue mass around the distal phalanx of the right thumb with no definite evidence of bony erosion. a contrast - enhanced ct scan of the chest and whole abdomen was unremarkable excepting the findings of enlarged left inguinal lymphadenopathy. a short course of palliative radiation 30 gy/10 fractions for 2 weeks to the primary lesion and metastatic lymphadenopathy was delivered. this was followed by radiation of 54 gy/27 fractions for 5.5 weeks to the site of acrometastasis. a 55-year - old gentleman was diagnosed with carcinoma supraglottic larynx (stage t4n1m0). he was treated with combined modality therapy: radiation 66 gy/33 fractions for 6.5 weeks with concurrent cisplatin 100 mg / mq 3 week. subsequently, he presented with swelling of the tip of all fingers of the left hand with nail bed involvement , and multiple subcutaneous nodules in the upper and lower limb. fnac of acral lesions was suggestive of metastatic carcinoma. a contrast - enhanced ct scan of the chest, abdomen, and pelvis revealed metastasis in bilateral lungs and liver there was however no evidence of locoregional disease on clinical examination and laryngoscopy. in view of the poor performance status (ecog 3) and widespread dissemination of disease metastatic swelling of all five distal phalanges of the left hand multiple bilateral lung metastasis multiple liver metastasis a 52-year - old gentleman presented to our clinic with the complaints of dysphagia to solids progressing to liquids, headache, and a nodule in the little finger of the left hand for the last 6 months. on endoscopy, a friable tumor involving half of the circumference of the esophageal lumen was seen 25 - 30 cm from the incisors. endoscopic biopsy revealed moderately differentiated squamous cell carcinoma. a contrast - enhanced ct scan of the chest showed circumferential mural thickening of the midesophagus with mediastinal lymphadenopathy. fnac of the nodule involving the distal phalanx of the little finger of the left hand was suggestive of metastatic squamous cell carcinoma. he underwent palliative radiation to esophagus and acrometastasis, 30 gy/10 fractions for 2 week. due to the increasing longevity of patients afflicted with cancer, there is a surge in the metastatic dissemination of disease, often to unusual sites. the underlying mechanism of the deposition of metastatic cells within the hand is unclear, but an increase in vascularity and trauma has been suggested in the past. it is because of the above - mentioned reasons, healey and colleagues have reported an increased incidence of acrometastasis to the dominant hand. in most patients, the tumor initially metastasizes to the bone and subsequently spreads to the adjacent soft tissue, though the reverse may also be occasionally observed. in a review of 257 cases of acrometastasis to the hand by flynn and colleagues, the median age at presentation was noted to be 58 years and men were twice likely to be affected compared to females. the most common primaries were in the lung (44%), kidney (12%), and breast (10%) whereas the remaining cases had primaries in the colon, stomach, liver, prostate, and rectum. the common sites of involvement in the hand were noted to be distal phalanx (74 lesions), metacarpals (56 lesions), proximal phalanx (26 lesions), and middle phalanx (16 lesions). bronchogenic carcinoma usually led to monoostotic lytic lesions, whereas polyostotic sclerotic, lytic, or mixed lesions were preponderant in carcinoma of the breast. acrometastasis to the hand can be asymptomatic or can present with painful swelling and movement restriction. subungual metastasis may present with a painful erythematous enlargement of the distal digit, or a red, violaceous nodule leading to nail dystrophy. the common differentials include acute infection in the form of abscess, felon, or paronychia; underlying osteomyelitis; or metabolic conditions like gout and pseudogout. as acrometastasis generally accompanies widespread disease, the prognosis is poor with an anticipated survival of 6 months. amputation, wide excision, curettage, cementation, radiotherapy, and chemotherapy are the therapeutic options in this rare presentation. solitary acrometastasis to the thumb from cutaneous squamous cell carcinoma of the groin is indeed a rarity. hematogenous dissemination to the skin and subcutaneous tissue of the thumb without underlying osseous involvement is all the more striking. tumor implantation due to contact is not unknown in squamous cell carcinoma and is another possibility. taking into account the advanced age, performance status (ecog 2), and lack of family support, a course of palliative radiation to the primary and acrometastasis was considered in the patient. cutaneous metastasis has been reported to occur in 12 % of patients with squamous cell carcinoma of the head and neck and accounts for less than 10% of all distant metastasis in such cases. a review of the surgical literature revealed only seven previously reported cases of cutaneous metastases from squamous cell carcinoma of the larynx. the common sites of cutaneous metastasis include neck, chest, scalp, face, lips, axilla, areola, back, arms, and digits. it is evident on the literature search that multiple metastases from laryngeal carcinoma involving all five distal phalanges of hand, bilateral lungs, and liver have not been reported till date. considering the poor performance status and widespread metastases in the second case, the best supportive care was offered to the patient. the third patient in the series had an esophageal primary with simultaneous acrometatsasis. following palliative radiation to esophagus and acrometatsasis, a brief course of systemic chemotherapy the patient had progressive disease and the best supportive care was considered in his case. acrometastasis to the hand is an unusual presentation which might mimic an infectious or inflammatory pathology. clinical awareness of unusual sites of metastatic dissemination is a must in the face of increasing cancer survivorship. acrometastasis portends a poor prognosis with limited survival, and optimal integration of the best supportive care is mandatory.	acrometastasis to the hand is an unusual presentation which might mimic an infectious, inflammatory, or a metabolic pathology. we herein describe a case series of three patients of acrometastasis to the hand. we encountered three cases of acrometastasis to the hand attending the departmental clinics from 2007 to 2010. the median age at presentation was noted to be 55 years. all were males. the primaries included squamous cell carcinoma of the skin, larynx, and esophagus. in two patients, acrometastasis was detected at presentation and in one it was detected 2 years postcompletion of radical therapy. two patients were offered palliative radiation to acrometastasis, and best supportive care was given to one. palliation achieved after radiation was noted to be modest to good. the brief report highlights the importance of the clinical awareness of metastatic dissemination to unusual sites in the face of increasing cancer survivorship. acrometastasis portends a poor prognosis with limited survival, and optimal integration of the best supportive care is mandatory. a short course of hypofractionated palliative radiation therapy in modest to good palliation.
hydrogenated nanocrystalline silicon (nc - si : h) has been the subject of intense scientific and technological interest over the past decade, mainly due to its reduced photo - induced degradation, efficient visible photoluminescence, tailored optical band gap, increased conductivity and greater doping efficiency. it has been highlighted that these unique features are a direct cause of the quantum size effects of the silicon nano - crystallites. these improvements make nc - si: h a potential candidate for application in photovoltaic and opto - electronic devices. the hot - wire chemical vapour deposition (hwcvd) technique, based on the catalytic decomposition of the precursor gasses by a heated transition metal filament, has been established as a viable deposition technique for nc - si: h thin films. the structural and opto - electronic properties of the thin films are dependent on the deposition parameters, of which the hydrogen dilution and substrate temperature are the most crucial. it has been established that the etching effect of atomic hydrogen, created by the catalytic decomposition of h2, is responsible for the termination of weak si si bonds from the surface and sub - surface regions and that the nucleation of the nano - crystallites are improved by increasing the hydrogen dilution. it has also been reported that the hydrogen dilution during deposition determines the concentration and the distribution of hydrogen in nc - si: h, which is closely related to the nano - structural features; i.e. crystallite size and crystalline volume fraction. in particular, the quantum size effects of the si nano - crystallites and the hydrogen concentration have a strong correlation with the optical band gap. an investigation into the role of hydrogen in nc - si: h is therefore crucial for the understanding of its relation to the nano - structure and the optical properties. in this contribution, we investigate the effects of the hydrogen concentration and bonding configuration in nc - si: h deposited by hwcvd on the nano - structural features and the optical properties. the nc - si: h thin film was deposited by the hwcvd process simultaneously on single - side polished 100 crystalline silicon and corning 7059 glass substrates, using a mixture of 4 sccm sih4 and 26 sccm h2 decomposed by seven parallel tungsten filaments, 15 cm apart and 36 cm away from the substrates. the filament temperature, substrate temperature and deposition pressure were fixed at 1600 c, 420 c and 60 bar, respectively. the as - deposited nc - si: h thin film was ~1140 nm - thick, as measured using a veeco profilometer. subsequent annealing was performed under high - purity, flowing n2gas in a tube furnace at annealing temperatures (ta) ranging from 200 to 700 c in 100 c increments. the n2flow rate, heating rate and dwell time for all temperatures amounted to 300 sccm, 10 c / min and 30 min, respectively. after each annealing temperature , the thin film was allowed to cool to room temperature in the tube furnace, while maintaining the n2flow rate. thereafter the required analytical techniques were performed. fourier transform infrared (ftir) absorption spectra were collected in transmission geometry from 400 to 4000 cmwith a spectral resolution of 1 cm, using a perkin - elmer spectrum 100 ftir spectrophotometer. the structural properties were investigated using a jobin - yvon hr800 micro - raman spectrometer in backscattering geometry at room temperature. the raman spectra were collected in the region 1001000 cmwith a spectral resolution of 0.4 cm, using an excitation wavelength of 514.5 nm. x - ray diffraction (xrd) spectra were collected in reflection geometry at 2-values ranging from 10 to 90 with a step size of 0.02, using a phillips pw 1830 x - ray powder diffractometer operating at 45 kv and 40 ma. copper k1radiation with a wavelength of 1.5406 was used as the x - ray source. optical transmission spectra were measured from 200 to 900 nm with a spectral resolution of 1 nm, using a perkin - elmer lamda 750s uv / vis spectrophotometer. hydrogen bonding configurations and to calculate the hydrogen concentration in nc - si: h and related material. the ftir absorption spectrum of the sample in the as - deposited state is shown in fig. the strong absorption bands in the region 9201250 cm is associated with the asymmetric si o si stretching vibration, whereas the peak centred around 2250 cm is assigned to the h sio3 vibration. this is indicative of an oxidation effect caused by its porous - like microstructure, which is a typical feature for nc - si: h thin films. the enhanced absorption band centred around 640 cm is attributed to the rocking vibrations of all bonding configurations of si hx. the absorption bands in the region 19002150 cm is a of the convolution of several absorption bands associated with the stretching vibrations of si hx in different configurations. the absorption peaks centred around 1985 cm and 2090 cm are assigned to the stretching vibrations of si h monohydrides in the amorphous network (isolated) and on the surface of the si nano - crystallites (clustered), respectively,. the weak absorption band centred at ~2130 cm is assigned to the existence of (= si = h2)n polyhydride complexes on si nano - crystallite grain boundaries. ftir absorption spectrum of the as - deposited sample and the deconvolution of the stretching vibrations (insert) to quantify the fraction of h bonded on the surface of nano - crystallites in nc - si: h, we define a structure factor, where i denotes that integrated intensity of each decomposed peak. the total bonded hydrogen concentration (ch) was estimated from the integrated absorption of the 640 cm rocking mode using previous reported procedures. in the as - deposited state, ch amounts to ~2 at.%, characteristic for nc - si: h deposited with high hydrogen dilution, where ~66% thereof is bonded on the surface of the nano - crystallites. we propose that this relatively high value for rs is indicative of a high crystalline volume fraction. figure 2shows the plots of the hydrogen concentration and the structure factor as a function of annealing temperature. the hydrogen concentration and structure factor are relatively constant at temperatures below 400 c, demonstrating that the nano - structure is stable in this temperature regime. after annealing at 400 c most of the (= si = h2)npolyhydride bonds on the grain boundaries of the si nano - crystallites have been terminated and consequently in an increase in the structure factor. annealing at higher temperatures induce a significant decrease inch, coupled with an increase inrs. we propose that the instability induced atta400 c is related to the growth of the native nano - crystallites and to the nucleation of nano - crystallites in the amorphous network, thereby ing in an increase in the crystalline volume fraction. it should be noted that no si hxabsorption peaks were identified after annealing at 700 c. ahydrogen concentration andbthe structure factor as a function of annealing temperature raman spectroscopy provides direct nano - structural information quantitatively related to the average nano - crystallite size and the crystalline volume fraction in nc - si: h. figure 3shows the raman spectra of the sample in the as - deposited state and after annealing at specific temperatures. all spectra display the following main features: (i) a sharp peak centred around 515 cm, associated with the transverse optic (to) mode of the nc - si phase; (ii) the broad shoulder centred around 480 cm, due to the to - mode of the amorphous silicon (a - si) phase; and (iii) a smaller shoulder around 505 cm, corresponding to the distribution of crystalline grain boundaries in the sample. raman spectra of the sample in the as - deposited state and after annealing at specific temperatures the crystalline volume fraction, can be estimated from the integrated areas of the afore - mentioned deconvoluted gaussian peaks. the crystallite size is empirically calculated from where is the shift of the 515 cm peak relative to the c - si peak at 520 cm and b = 2.0 cm. the quantitative raman are summarized in table 1. in the as - deposited state, the average crystallite size and the crystalline volume fraction amounts to about 3.9 nm and 53%, respectively, and remain relatively constant after annealing at 300 c. these observations reiterate that the nano - structure of the sample remains stable at temperatures below 400 c. a blue shift of the nc - si to - peak, accompanied with a reduction in the intensity of the a - si to - peak is observed at annealing temperatures 400 c, indicative of an increase in the crystallite size and the crystalline volume fraction, respectively, and supports the claims based on the ftir . crystallite size, crystalline volume fraction and optical properties after specific annealing temperatures xrd was employed as a complimentary method to qualitatively probe the changes in the crystallinity as a function of annealing temperature (see fig . 4). three preferential orientations in the 111, 200 and 311 directions are observed. the crystallite size in the as - deposited state, estimated from the full - width - half - maximum (fwhm) of the-peak, amounts to ~19.5 nm. a narrowing in the fwhm of the-peak, accompanied with an increase in its intensity is observed with an increase in annealing temperature. this confirms the increase of the crystallite size and crystalline volume fraction, as probed by raman spectroscopy. xrd spectra of the sample in the as - deposited state and after annealing at specific temperatures the thermally induced nano - structural changes of the nc - si: h thin film can be interpreted as follows, based on the variation of the si the crystalline volume fraction is relatively large and therefore the majority of h is bonded to the surface of the nano - crystallites. the nano - structural properties are stable at temperatures below 400 c, attributed to its large crystalline volume fraction. an initial increase in the native crystallite size is observed after annealing at 400 c, ing in the removal of hydrogen from the grain boundaries. it is also feasible that smaller crystallites have coalesced into larger crystallites. at higher temperatures , hydrogen is removed preferentially from the amorphous phase, indicative of the nucleation of smaller nano - crystallites of size <3 nm in the amorphous network, undetected by raman spectroscopy and xrd. the optical properties were determined from uv visible transmission measurements performed on the thin film deposited on the corning 7059 glass substrate, using the method proposed by swanepoel. the thickness of the as - deposited sample was calculated to be ~1180 nm, which concurs to that measured by profilometry. the refractive index n of a material is an important optical parameter, since it is directly proportional to density. figure 5 shows the spectral dependence of the calculated refractive index for the sample in the as - deposited state and after annealing at specific temperatures. a slight increase in the refractive index is observed after annealing at 400 c, followed by a decrease at higher temperatures. the initial increase can be ascribed to the increase in the native crystallite size and possibly due to the coalescence of smaller nano - crystallites. furthermore, the presence of (= si = h2)n complexes in the as - deposited state is indicative of a disordered, porous material and the removal thereof after 400 c would therefore in a more compact material. the subsequent decrease of the refractive index at higher temperatures is attributed to a more porous structure, possibly caused by the nucleation of smaller nano - crystallites in the amorphous network. similar trends in the refractive index at zero photon energy (no) are observed (see table 1). refractive index spectra of the sample in the as - deposited state and after annealing at specific temperatures detailed analysis of the refractive index spectra were performed using the model suggested by wemple et al.. at energies below than of the optical band gap, the refractive index is related to the square of the photon energy (h) by: where em and ed is the average gap and dispersion energy, respectively. the plot of 1/n-1 ] versus (h) allows for the determination of emed and no. the extrapolated of no and em, calculated from the linear fit through the data, are listed in table 1. the spectral dependence of the absorption coefficient for the sample in the as - deposited state and after annealing at specific temperatures is depicted in fig. the optical band gap, referred to as e04, is defined as the photon energy where = 10 cm, and the values are reported in table 1. a red shift in e04 is observed for ta 600 c followed by an unexpected blue shift after annealing at 700 c. it is established that the optical band gap of hydrogenated amorphous silicon (a - si : h) deposited by hwcvd and pecvd increases with an increase in the hydrogen concentration. it should be noted that the optical band gap for the sample in the as - deposited state is larger than that of a - si: h with similar ch values. this discrepancy is due to the presence of nano - crystallites in the amorphous network, which lowers the absorption in nc - si: h and shifts the optical band gap towards higher energies. the quantum size effect size also predicts that an increase in crystallite size is associated with a decrease in the optical band gap. the initial decrease in e04 after 400 c is due to the combined effect of the decreased ch and the growth in the crystallite size. after annealing at 600 c the initial hydrogen concentration has decreased by ~90% with about the same incremental increase in the crystallite size as at 400 c, and therefore a more notable decrease in e04 is expected. however, a minute 0.03 ev decrease in e04 is observed and is attributed to the nucleation of smaller nano - crystallites in the amorphous network, which explains the competing increasing effect on e04. after annealing at 700 c, where no hydrogen was detected by ftir spectroscopy, this effect is more pronounced in that an increase in the optical band gap is observed. absorption coefficient spectra of the sample in the as - deposited state and after annealing at specific temperatures the optical band gap and the average gap (em) have similar behaviours with respect to annealing temperature, thereby implying that the growth of the native nano - crystallites and the nucleation of smaller crystallites in the amorphous network have similar effects on the band edges and on the conduction and valence bands. therefore, the average gap can be used to describe the thermal induced changes in the optical properties of nc - si: h. hydrogen bonding configurations and to calculate the hydrogen concentration in nc - si: h and related material. the ftir absorption spectrum of the sample in the as - deposited state is shown in fig. the strong absorption bands in the region 9201250 cm is associated with the asymmetric si o si stretching vibration, whereas the peak centred around 2250 cm is assigned to the h sio3 vibration. this is indicative of an oxidation effect caused by its porous - like microstructure, which is a typical feature for nc - si: h thin films. the enhanced absorption band centred around 640 cm is attributed to the rocking vibrations of all bonding configurations of si hx. the absorption bands in the region 19002150 cm is a of the convolution of several absorption bands associated with the stretching vibrations of si hx in different configurations. the absorption peaks centred around 1985 cm and 2090 cm are assigned to the stretching vibrations of si h monohydrides in the amorphous network (isolated) and on the surface of the si nano - crystallites (clustered), respectively,. the weak absorption band centred at ~2130 cm is assigned to the existence of (= si = h2)n polyhydride complexes on si nano - crystallite grain boundaries. ftir absorption spectrum of the as - deposited sample and the deconvolution of the stretching vibrations (insert) to quantify the fraction of h bonded on the surface of nano - crystallites in nc - si: h, we define a structure factor, where i denotes that integrated intensity of each decomposed peak. the total bonded hydrogen concentration (ch) was estimated from the integrated absorption of the 640 cm rocking mode using previous reported procedures. in the as - deposited state, ch amounts to ~2 at.%, characteristic for nc - si: h deposited with high hydrogen dilution, where ~66% thereof is bonded on the surface of the nano - crystallites. we propose that this relatively high value for rs is indicative of a high crystalline volume fraction. figure 2shows the plots of the hydrogen concentration and the structure factor as a function of annealing temperature. the hydrogen concentration and structure factor are relatively constant at temperatures below 400 c, demonstrating that the nano - structure is stable in this temperature regime. after annealing at 400 c most of the (= si = h2)npolyhydride bonds on the grain boundaries of the si nano - crystallites have been terminated and consequently in an increase in the structure factor. annealing at higher temperatures induce a significant decrease inch, coupled with an increase inrs. we propose that the instability induced atta400 c is related to the growth of the native nano - crystallites and to the nucleation of nano - crystallites in the amorphous network, thereby ing in an increase in the crystalline volume fraction. it should be noted that no si hxabsorption peaks were identified after annealing at 700 c. ahydrogen concentration andbthe structure factor as a function of annealing temperature raman spectroscopy provides direct nano - structural information quantitatively related to the average nano - crystallite size and the crystalline volume fraction in nc - si: h. figure 3shows the raman spectra of the sample in the as - deposited state and after annealing at specific temperatures. all spectra display the following main features: (i) a sharp peak centred around 515 cm, associated with the transverse optic (to) mode of the nc - si phase; (ii) the broad shoulder centred around 480 cm, due to the to - mode of the amorphous silicon (a - si) phase; and (iii) a smaller shoulder around 505 cm, corresponding to the distribution of crystalline grain boundaries in the sample. raman spectra of the sample in the as - deposited state and after annealing at specific temperatures the crystalline volume fraction, can be estimated from the integrated areas of the afore - mentioned deconvoluted gaussian peaks. the crystallite size is empirically calculated from where is the shift of the 515 cm peak relative to the c - si peak at 520 cm and b = 2.0 cm. the quantitative raman are summarized in table 1. in the as - deposited state, the average crystallite size and the crystalline volume fraction amounts to about 3.9 nm and 53%, respectively, and remain relatively constant after annealing at 300 c. these observations reiterate that the nano - structure of the sample remains stable at temperatures below 400 c. a blue shift of the nc - si to - peak, accompanied with a reduction in the intensity of the a - si to - peak is observed at annealing temperatures 400 c, indicative of an increase in the crystallite size and the crystalline volume fraction, respectively, and supports the claims based on the ftir . crystallite size, crystalline volume fraction and optical properties after specific annealing temperatures xrd was employed as a complimentary method to qualitatively probe the changes in the crystallinity as a function of annealing temperature (see fig . 4). three preferential orientations in the 111, 200 and 311 directions are observed. the crystallite size in the as - deposited state, estimated from the full - width - half - maximum (fwhm) of the-peak, amounts to ~19.5 nm. a narrowing in the fwhm of the-peak, accompanied with an increase in its intensity is observed with an increase in annealing temperature. this confirms the increase of the crystallite size and crystalline volume fraction, as probed by raman spectroscopy. xrd spectra of the sample in the as - deposited state and after annealing at specific temperatures the thermally induced nano - structural changes of the nc - si: h thin film can be interpreted as follows, based on the variation of the si the crystalline volume fraction is relatively large and therefore the majority of h is bonded to the surface of the nano - crystallites. the nano - structural properties are stable at temperatures below 400 c, attributed to its large crystalline volume fraction. an initial increase in the native crystallite size is observed after annealing at 400 c, ing in the removal of hydrogen from the grain boundaries. it is also feasible that smaller crystallites have coalesced into larger crystallites. at higher temperatures , hydrogen is removed preferentially from the amorphous phase, indicative of the nucleation of smaller nano - crystallites of size <3 nm in the amorphous network, undetected by raman spectroscopy and xrd. the optical properties were determined from uv visible transmission measurements performed on the thin film deposited on the corning 7059 glass substrate, using the method proposed by swanepoel. the thickness of the as - deposited sample was calculated to be ~1180 nm, which concurs to that measured by profilometry. the refractive index n of a material is an important optical parameter, since it is directly proportional to density. figure 5 shows the spectral dependence of the calculated refractive index for the sample in the as - deposited state and after annealing at specific temperatures. a slight increase in the refractive index is observed after annealing at 400 c, followed by a decrease at higher temperatures. the initial increase can be ascribed to the increase in the native crystallite size and possibly due to the coalescence of smaller nano - crystallites. furthermore, the presence of (= si = h2)n complexes in the as - deposited state is indicative of a disordered, porous material and the removal thereof after 400 c would therefore in a more compact material. the subsequent decrease of the refractive index at higher temperatures is attributed to a more porous structure, possibly caused by the nucleation of smaller nano - crystallites in the amorphous network. similar trends in the refractive index at zero photon energy (no) are observed (see table 1). refractive index spectra of the sample in the as - deposited state and after annealing at specific temperatures detailed analysis of the refractive index spectra were performed using the model suggested by wemple et al.. at energies below than of the optical band gap, the refractive index is related to the square of the photon energy (h) by: where em and ed is the average gap and dispersion energy, respectively. the plot of 1/n-1 ] versus (h) allows for the determination of emed and no. the extrapolated of no and em, calculated from the linear fit through the data, are listed in table 1. the spectral dependence of the absorption coefficient for the sample in the as - deposited state and after annealing at specific temperatures is depicted in fig. the optical band gap, referred to as e04, is defined as the photon energy where = 10 cm, and the values are reported in table 1. a red shift in e04 is observed for ta 600 c followed by an unexpected blue shift after annealing at 700 c. it is established that the optical band gap of hydrogenated amorphous silicon (a - si : h) deposited by hwcvd and pecvd increases with an increase in the hydrogen concentration. it should be noted that the optical band gap for the sample in the as - deposited state is larger than that of a - si: h with similar ch values. this discrepancy is due to the presence of nano - crystallites in the amorphous network, which lowers the absorption in nc - si: h and shifts the optical band gap towards higher energies. the quantum size effect size also predicts that an increase in crystallite size is associated with a decrease in the optical band gap. the initial decrease in e04 after 400 c is due to the combined effect of the decreased ch and the growth in the crystallite size. after annealing at 600 c the initial hydrogen concentration has decreased by ~90% with about the same incremental increase in the crystallite size as at 400 c, and therefore a more notable decrease in e04 is expected. however, a minute 0.03 ev decrease in e04 is observed and is attributed to the nucleation of smaller nano - crystallites in the amorphous network, which explains the competing increasing effect on e04. after annealing at 700 c, where no hydrogen was detected by ftir spectroscopy, this effect is more pronounced in that an increase in the optical band gap is observed. absorption coefficient spectra of the sample in the as - deposited state and after annealing at specific temperatures the optical band gap and the average gap (em) have similar behaviours with respect to annealing temperature, thereby implying that the growth of the native nano - crystallites and the nucleation of smaller crystallites in the amorphous network have similar effects on the band edges and on the conduction and valence bands. therefore, the average gap can be used to describe the thermal induced changes in the optical properties of nc - si: h. the effect of isochronal annealing on the nano - structural and optical properties of nc - si: h, with the emphasis on its relation to the hydrogen distribution and concentration, was investigated. initial changes in the nano - structure are observed after annealing at 400 c, as evident by termination of (= si = h2)npolyhydrides from the grain boundaries caused by the growth of the native nano - crystallites. at higher temperatures, a further increase in the native nano - crystallite size and the crystalline volume fraction is observed, accompanied with the nucleation of smaller nano - crystallites and the subsequent removal of hydrogen from the amorphous network. at temperatures 600 c the nucleation of the smaller nano - crystallites in a porous material with an increased optical band gap and average gap, explained by the quantum size effect. the authors acknowledge the financial assistance of the department of science and technology, the national research foundation and the council for scientific and industrial research (project no : hgera2s) of south africa.	we report on the thermally induced changes of the nano - structural and optical properties of hydrogenated nanocrystalline silicon in the temperature range 200700 c. the as - deposited sample has a high crystalline volume fraction of 53% with an average crystallite size of ~3.9 nm, where 66% of the total hydrogen is bonded as si h monohydrides on the nano - crystallite surface. a growth in the native crystallite size and crystalline volume fraction occurs at annealing temperatures 400 c, where hydrogen is initially removed from the crystallite grain boundaries followed by its removal from the amorphous network. the nucleation of smaller nano - crystallites at higher temperatures accounts for the enhanced porous structure and the increase in the optical band gap and average gap.
candida genus, in particular candida albicans, is the one of the common cause of yeast - associated oral infections. there occurrence has been widely described in the literature. over the last four to five decades the main reason that might attribute for such an increase in its incidence might be the increase in the prevalence of human immunodeficiency virus (hiv) and other immuno - compromising states and diseases the distribution of all the members of this genus is ubiquitous and consists of inhabitation of soil as saprophytes, aquatic environment, and even colonization of various animal reservoirs. at 37c , the growth of most of candida spices is prohibited, and therefore, human colonization is not associated with it under normal conditions. within humans, several species persist as commensal microorganism and can act as potential opportunistic pathogen in compromised body status. with respect to the treatment of these oral fungal infections, the most common line of treatment is the removal of the etiologic factor along with suitable anti - fungal therapy. there is a paucity of data regarding the comparative evaluation of the abovementioned anti - fungal agents in the treatment of oral candidiasis. hence, we planned this study to evaluate the effectiveness of fluconazole and clotrimazole in the treatment of patients suffering from candidiasis. the present study was conducted in the department of oral medicine and radiology of the dental institute and included assessment of 180 participants with chief problem of oral candidiasis from 2010 to 2015. ethical approval was obtained from the institutional ethical committee and written consent was obtained after explaining in detail the entire research protocol. complete physical examination of the patients was done before starting the treatment therapy of the patients. recording of the time duration of the candidiasis was done along with other personal details of the patients. exclusion criteria for the present study included: pregnant patients, patients who underwent any kind of anti - fungal therapy in the past 1 month, patients who were on barbiturates or anticoagulants in the past 1 month, patients with any known drug allergy, patients with alcohol history, patients with history of any psychiatric disorder. patients who underwent any kind of anti - fungal therapy in the past 1 month, patients who were on barbiturates or anticoagulants in the past 1 month, patients with any known drug allergy, patients with alcohol history, patients with history of any psychiatric disorder. group i included patients who were under treatment with fluconazole therapy whereas group ii included patients who were on clotrimazole therapy. based on the clinical history, visual examination of the lesion area and presence of clinical signs and symptoms, diagnosis of the candidiasis both the signs and symptoms of candidiasis were graded by patients into the following categories based on the severity of the lesion and individual perception: mild, moderate, andsevere. symptoms severity was categorized on the basis patient's response to discomfort whereas sign's severity was categorized on the basis of extent of lesion. mild referred to cases which involved localized involvement to one or two oral sites, moderate referred to localized involvement of more than two oral sites, whereas severe cases involved generalized oral candidiasis. swab was taken from the lesion area of the patients and was transferred to the microbiological laboratory in the transport medium for culturing. they were incubated in sabouraud's dextrose agar medium for assessment of culture growth characteristics. i patients, preparation of fluconazole suspension was done and were given to all the patients in the form of prepared moth rinse. patients were instructed to use the suspension mouth rinse three times a day. in group ii patient, suspension of clotrimazole was given in the form of moth paint and patients were instructed to use it thrice daily. recalling of the patients was done after 2 weeks of continuation of the treatment therapy and was examined thoroughly for the presence of clinical signs and symptoms; microbial growth was assessed by culturing swab specimens as done earlier before the starting of the treatment. chi - square test and student's t - test was used for the assessment of level of significance. all samples were examined twice to avoid intraobserver variability and kappa value came out to be 0.8. in both the study groups, prolonged antibiotic therapy was the most common compromised state associated with the presence of fungal infections. mean age of the patients in groups i and ii was 49.5 years and 51.2 years, respectively. in group i, 65% of the patient population was of males whereas remaining 35% were females. among group ii, 62% of patient population was of males while the reaming was of females. among group i patients, before starting the treatment, 81% of the patient population had moderate severity of clinical symptoms, whereas in group ii, 75% of the patient population had moderate severity of symptoms. among group i participants, after commencing the treatment, in 98% of the patients, clinical signs and symptoms were absent whereas in group ii participants, 88% of the patients showed absence of clinician signs. significant were obtained while comparing the signs and symptoms of the patients in the two study groups after the treatment. in group i patients, before and after the treatment, the colony count was 1325.25 and 9.51, respectively, whereas in the group ii patients, the mean colony count was 996.52 and 21.82, respectively. significant were obtained while comparing the mean colony count before and after the treatment in both study groups. distribution of all the medically compromised patients in the study groups comparative assessment of the two study groups in terms of various parameters comparative assessment of the two study groups in terms of various parameters comparison of various clinical signs and symptoms in between the patients of the two study group comparison of various clinical signs and symptoms in between the patients of the two study group comparison of colony counts before and after the treatment therapy over the past few decades, there has been an increase in the incidence of occurrence of fungal infections. furthermore, there has been as simultaneous increase in the incidence and occurrence of the predisposing factors. some of the important predisposing factors included xerostomia, antibiotic therapy for prolonged period of time, local chronic trauma or irritation, endocrinal disorders, medically compromised states, etc. treatment of the fungal infections included removal of the etiologic systemic or local factor along with antifungal therapy. fluconazole and clotrimazole are the two commonly used anti - fungal agents for the treatment of fungal lesions. hence, we aimed to evaluate the effectiveness of fluconazole and clotrimazole in the treatment of patients suffering from candidiasis. in the present study, we observed significant while comparing the mean difference of colony culture growth before and after the treatment in both the study groups. similar were obtained by sholapurkar et al. who observed similar findings in their study. o - prasertsawat et al. comparatively evaluated the effectiveness of fluconazole and clotrimazole in the treatment of vulvovaginal candidiasis. they assessed 103 female patients in a single blinded randomized trial and divided them broadly into two study groups. first group consisted of 53 patients and included participants in whom treatment was done by fluconazole, while the other group consisted of 50 patients and included those in which treatment was done by clotrimazole. they did not observe any significant difference in relation to the clinical characteristic in between the two study groups. they observed approximately 79% and 80% mycological cure rates in the two study groups, respectively. they concluded that for the treatment of cases of vulvovaginal candidiasis, fluconazole can be given as an alternative line of treatment. comparatively evaluated the effectiveness of single dose of fluconazole and intravaginal clotrimazole 200 mg per day for 6 days in the patients undergoing treatment for the acute episode of vulvovaginal candidiasis (vvc). they prospectively analyzed 142 patients with were diagnosed with vvc and divided them randomly into two study groups. first group consisted of 70 patients and included those patients who received intravaginal tablet whereas the other group included 72 patients and consisted of participants who were given single dose oral fluconazole. they observed that, at the time of follow - up, during the second visit of the patients, approximately 85% and 81% of the patients of the group receiving fluconazole were cured clinically and mycologically, respectively. similarly in the other study group, approximately 83% and 70% of the study group patients were cured clinically and mycologically, respectively. from the , they concluded that for the treatment of cases of vvc, oral fluconazole single dose appeared to be a valid mode of treatment. goins et al. compared the effectiveness of nystatin and fluconazole in treating the cases of oral candidiasis. one of the common conditions affecting the young infant group of individuals is oral thrush. one of the frequent problems associated with the administration of nystatin is the frequent cases of recurrence and difficulty encountered in its administration. they randomly analyzed 34 infants and were randomly subjected to either nystatin oral suspension four times a day for 10 days or fluconazole suspension 3 mg / kg in a single daily dose for 7 days. they observed that among the patients treated with nystatin, the clinical curing rate was 32%, while in the other group, a success rate of 100% was observed. significant were obtained while comparing the two anti - fungal agents. from the assessed the efficacy of fluconazole mouthrinse and clotrimazole mouthpaint in the treatment of cases of oral candidiasis. they observed significant difference while comparing the fungal growth on culture media before and after the treatment. authors advocate the use of fluconazole and clotrimazole for treating oral candidiasis patients, thereby helping in improving the quality of life of the patients. sample size was smallunderlying etiologic factors for the occurrence of candidiasis were not explored. sample size was small underlying etiologic factors for the occurrence of candidiasis were not explored. the treatment therapy might differ depending upon the etiologic cause of candidiasis which was not taken as a parameter for the present study. authors advocate the use of fluconazole and clotrimazole for treating oral candidiasis patients, thereby helping in improving the quality of life of the patients. sample size was smallunderlying etiologic factors for the occurrence of candidiasis were not explored. sample size was small underlying etiologic factors for the occurrence of candidiasis were not explored. the treatment therapy might differ depending upon the etiologic cause of candidiasis which was not taken as a parameter for the present study. from the above , the authors obtained significant while using both the fluconazole and clotrimazole in treating patients with oral candidiasis. however, future studies are required in the same field for better exploration of .	aims: one of the most common fungal infections infecting humans is candidiasis. belonging to the group of opportunistic infections, it often affects individuals with various debilitating diseases. fluconazole and clotrimazole are two of the commonly used anti - fungal agents for the treatment of oral candidiasis. hence, we planned this study to evaluate the effectiveness of fluconazole and clotrimazole in the treatment of patients suffering from candidiasis.materials and methods: a total of 180 participants were enrolled in the present study. all the patients of candidiasis were divided broadly into two study groups. group i included patients who were treated with fluconazole mouthrinse whereas group ii included patients who were treated with clotrimazole mouth paint. grading of patient discomfort was done as noted from readings given by the patients. specimen was collection by a swab from the lesional area of the oral cavity from the patients and were incubated in sabouraud's dextrose agar medium and assessed. all the patients were treated with medication as give to their respective groups. patients were recalled as assessed. all the readings were recorded and analyzed.:for group i patients, the fungal eradication was 89.5%, whereas for group ii patients, the fungal eradication was 86.7%. no significant were obtained while comparing the mycological eradiation in patients of the two study groups.:approximately similar effectiveness in terms of treatment was noted with fluconazole and clotrimazole in treating patients with candidiasis.
alterations in left ventricular structure and function have been reported among the cardiac manifestations of systemic lupus erythematosus (sle), especially in those who have renal complications. these alterations include echocardiographic evidence of increases in lv wall thicknesses and mass, a decrease in lv ejection fraction, and impaired diastolic filling. however, it is currently uncertain whether these abnormalities are disease - related effects or a of other predisposing conditions, such as inflammation, hypertension, anemia, and disorder of mineral metabolism. in recent years , there has been a growing interest in the hypothesis that atherosclerosis may be an inflammatory disease. it has been noted that c - reactive protein (crp), a marker of the reactant plasma protein component of the inflammatory response, is a major predictor of cardiovascular disease (cvd) in apparently healthy subjects. previous reports have found the association between crp and left ventricular hypertrophy (lvh) in several pathologic states such as hypertension, insulin resistance, and chronic kidney disease (ckd). in this study , we investigated the potential interrelationships among hs - crps, a more sensitive marker of systemic inflammation and lv mass index (lvmi) in patients with lupus nephritis (ln) by using the clinical cutoff levels of crp. a total of 287 incipient ln patients were consecutively enrolled from january 2005 to december 2008. all participants met the diagnostic criteria of the american college of rheumatology. exclusion criteria included ischemic heart disease, acute coronary syndrome, congestive heart failure (chf) (new york heart association ( nyha) class ii or greater ), old cerebral infarction, history of transient ischemic attack, secondary hypertension, receipt of any immunosuppressant and/or an anti - inflammatory drug (aspirin or nonsteroidal anti - inflammatory drug ( nsaid) ), chronic infection, cancer, and pregnancy. the study protocol was approved by the local ethics committee, and all participants gave their written informed consent to participate in this study. after fasting overnight, bp was measured with an appropriate arm cuff and a mercury column sphygmomanometer on the left arm after a resting period of at least 10 min in the supine position. the following parameters were also determined: serum creatinine, serum lipids including cholesterol, triglyceride, and lipoprotein(a), measurement of serum complement c3 and c4, high - sensitivity c - reactive protein, and antibody testing. high - sensitivity crp (hs - crp) was measured by autoimmune scattering rate nephelometry (bnp nephelometer, dade behring). double - stranded dna (ds - dna) was detected by farr assay (euroimmun ag, germany), and antiphospholipid antibodies (acl) were measured by enzyme - linked immunosorbent assay (elisa) (euroimmun ag, germany). echocardiography was performed by an experienced research technician using standard techniques who was unaware of the clinical characteristics of the patients. studies were performed using phased - array echocardiography with m - mode, 2-dimensional, pulsed, and color - flow doppler capabilities. lv mass (lvm) was calculated using the following formula: lvm = 0.8 (1.04 ( lvst+lvpwt+lvdd)lvdd)+ 0.6, where lvst is lv septal wall thickness, and lvpwt is lv posterior wall thickness, lvdd is lv diastolic diameter. lvmi was indexed for body surface area (bsa), and lvh was defined by an lvmi of over 110 g / m in women and 125 g / m in men. data were described as means sds for those with normal distribution and as medians and interquartile ranges for asymmetrical distribution. comparisons between patients divided by crp cutoff level and with or without lvh were performed by unpaired t - tests in normally distributed data and by nonparametric mann - whitney test in asymmetrically distributed data, or by x test in categorical data. the cut - off level of hs - crp was defined according to the aha / cdc recommendations, in which crp levels 3 mg / l were defined as average- and high - risk groups for cvd. all variables that had significant relations were evaluated for inclusion in a model predicting lv mass using multivariable regression analysis; unstandardized regression coefficients all of the statistics were performed by spss version 13.0, and a 2-tailed p <.05 was considered to indicate statistical significance. the 287 subjects were predominantly female (91.29%), with a mean age of 38.5 13.3 at their entry. totally 223/239 patients (93.30%) showed positive ana, 177/200 patients (88.50%) had positive ds - dna, and 29/153 patients (18.95%) showed acl antibodies. renal biopsy was obtained from 135 (47.04%) patients, which showed minimal mesangial ln (class i) in 4 (3.0%), mesangial proliferative ln (class ii) in 7 (5.2%), focal ln (class iii) in 16 (11.9%), diffuse ln (class iv) in 77 (57.0%), membranous ln (class v) in 28 (20.7%), and advanced sclerotic ln (class vi) in 3 (2.2%) patients according to international society of nephrology / renal pathology society (isn / rps) 2003 classification. we compared the baseline characteristics of patients with and without lvh, as showed in table 1. patients with lvh were much older, had significantly elevated hs - crp level and higher uric acid level, lower hemoglobin level, and egfr. however, bmi, blood pressure, and serum lipids were not significantly different between the two groups. meanwhile, autoantibody parameter positive incidence including ds - dna, ana, and acl did not differ between patients with and without lvh. to further explore the extent to which inflammation augment lvh, the patients were subdivided into low- and average - to - high risk groups according to hs - crp cutoff level. among those who had higher hs - crp levels (3 mg / l), lvmi was significantly increased (132.68 57.84 versus 113.67 29.17, p = .018) (figure 1). in addition, these patients had elder age (39.37 14.09 versus 34.97 11.55, p = .02), lower hemoglobin level (93.04 24.91 versus 106.57 23.65, p < .001), lower cholesterol level (5.60 1.89 versus 6.41 2.76, p = .018), higher esr (56.00 ( 31.5083.00) versus 32.00 (19.7558.50), p <.001 ) and higher serum fibrinogen level (4.17 1.59 versus 3.54 1.12, p = .004) (table 2). in univariate analysis involving the entire sample (table 3), significant correlates of lvmi included age, body mass index, blood pressure, hemoglobin level, hs - crp, uric acid level, and egfr. after introducing all these significant variables into multivariate regression analysis, hs - crp (= 0.228, p = .009), along with uric acid (= 0.382, p < .001), was further confirmed to have positive associations with lvmi. our cross - sectional study revealed a linear relationship between low - grade chronic inflammation estimated by high - sensitivity crp levels and lvmi, independent of several other important covariates, such as adipose tissue distribution bmi, bp levels, serum lipids, renal function, age, and gender. the observation of this independent association between hs - crp level and lvmi is consistent with previous findings , and the present study extended to ln patients. as far as we know, these findings are new and potentially important for refining cvd risk stratification in this population. at the initial stage of the atherosclerotic process , systemic inflammation would appear most importantly associated with subclinical cardiovascular disease development, such as lvh occurrence. a raised baseline crp value has been associated with inflammation, endothelial dysfunction, obesity, the metabolic syndrome , diabetes mellitus, insulin resistance, and severity of hypertension, and thus, various metabolic disorders may occur by increasing crp level and simultaneously promote an increase in lv mass. on the other hand, local crp synthesis and secretion by smooth muscle cells, including those of the human coronary artery, it is possible to speculate that crp may play a direct role in promoting lvh through these mechanisms, including increasing phosphatidylinositol3- kinase activity; upregulating inducible nitric oxide synthase, certain cell signal transduction pathways including the mitogen - activated protein kinase pathway, and nuclear factor -b; upregulating angiotensin ii type 1 receptor in vascular smooth muscle cells, and directly quenching the production of nitric oxide by endothelial cells , ing in increased production of endothelin-1; elevation of von willebrand factor, which is known to be associated with endothelial dysfunction. thus, cardiac hypertrophy may be, at least in part, attributable to an increase in crp itself, via activated transcriptional regulatory mechanisms, proinflammatory and proatherogenic effects, and stimulation of endothelial dysfunction first, it is not possible to conclude from this observational research whether crp stimulates higher lvmi or whether crp is increased before the development of lvh. the cross - sectional design prevents the demonstration of the mechanisms by which lvh is related to inflammation. second, it may be better to introduce sledai score into multivariate regression analysis to further estimate the effect of disease itself on lvh, and inflammation status as well. third, it is very regrettable that some of our patients' autoantibodies data were missing and incomplete. this cohort will be followed and expanded to further observe the prevalence and correlative factors of lvh, especially after intervention therapy. in , in ln subjects initially free of cvd, hs - crp showed a significant association with lvmi, which suggested that assessment of hs - crp level may help to refine cvd risk stratification in this population.	objective. to determine the prevalence of left ventricular hypertrophy (lvh) and its associated risk factors in lupus nephritis (ln) patients. methods. 287 ln patients (age : 38.54 13.31, 262 female) were recruited. echocardiography and serum high - sensitivity c - reactive protein (hs - crp) were measured. their relationship was evaluated by univariate correlation analysis and multivariate regression analysis. . the prevalence of lvh in this cohort was 21.25% (n = 61). serum hs - crp level was significantly elevated in patients with lvh compared to those without (8.03 ( 3.2230.95) versus 3.93 (1.489.48) mg / l, p <.01 ), and correlated with left ventricular mass index (lvmi) (r = 0.314, p = .001). multivariate regression analysis further confirmed that hs - crp was an independent risk factor (= 0.338, p = .002) for lvh in patients with ln. . our findings demonstrated that serum hs - crp level is independently correlated with lvmi and suggested that measurement of hs - crp may provide important clinical information to investigate lvh in ln patients.
in recent years the problem of " cutaneous ulcers " (venous, arterial, diabetic, and pressure sores) has become increasingly important, in particular because of the progressive increase in the elderly population and, therefore, of chronic disorders. chronic cutaneous ulcers of the comorbid elderly patient tend to resist on standard therapy and therefore the management is difficult, time - consuming and expensive. optimally, a short period of advanced therapy to stimulate healing can in the transition of an intractable wound to a treatable one, which can then be treated with less expensive, standard care. platelet - rich plasma (prp) is an autologous preparation of platelets in concentrated plasma and contains various growth factors. due to the presence of high concentrations of these growth factors , prp has been used in a wide variety of surgical procedures and clinical treatments. we herein report a comorbid patient with refractory cutaneous ulcer which did not respond to formal therapeutic methods. the patient was treated with autologous prp and could achieve complete response of the skin lesion within 3 months. on november 25, 2011, a 94 yr - old woman was referred for the management of erythematous to violaceous patches with bullae on dorsum of left foot. she repeatedly hit her left foot in which intravenous line was situated with the opposite side for days. she had a various underlying disease including alzheimer's dementia, angina pectoris, type 2 diabetes, hypertension, chronic kidney disease (stage iv) and was in a bed ridden state. because of the poor oral intake and noncompliance to the levin - tube, the percutaneous endoscopic gastrostomy tube had been inserted on her abdomen and only minimal amount of nutrition was maintained. despite of the daily simple dressing and intermittent debridement about 2 weeks, the status of the wound was deteriorated and advanced to painful ulcerative patches with eschar and granulation tissues (fig . autologous prp were prepared for the alternative management of her worsening wound . 12 ml peripheral autologous blood was obtained and collected into tubes containing acid - citrate - dextrose solution formula ( acd - a) anticoagulant. subsequently, the yellow plasma (containing buffy coat with platelets) was separated from other components. the upper 3 to 4 ml plasma called platelet poor plasma was removed by micropipette. the patient underwent a total of 7 prp treatments over a span of 8 weeks (performed twice a week until 5th treatment and then weekly until 7th treatment). as described above, these treatments involved topically application of activated prp as well as packing of the wound with aseptic film for the sustained effect of growth factors. the film was removed on next day and the top layer was covered with hydrofoam dressing material until subsequent prp treatment. over the course of the prp treatment, the of the changes in wound surface area and depth are shown in fig. the wound was essentially filled in with granulation tissue after seven prp treatments with only subtle epithelialization of the new tissue remaining. there were neither signs of infection and allergic reaction nor discomfort of the patient during the treatment. after 2 months from the last (7th) treatment, the complete epithelialization was seen and no further simple dressing was required for the wound. the complete response of the recalcitrant cutaneous ulcer using prp therapies implies that this stimulation therapy could be an appropriate alternative modality when the degree of healing with conventional treatment appears to be unsatisfactory. the ulcer bed more rapidly demonstrated margin in - growth, granulation tissue development, vascularization and epithelialization. first of all, the growth factors and cytokines released from prp are able to stimulate the proliferation, migration and differentiation of dermal fibroblasts and endothelial cells. prp also increased type i collagen and mmps gene expression, suggesting that prp also have the potential to promote the tissue remodeling of aged skin. the second component is the mesh of fibrin that constitutes the " structure " of the platelet gel: this mesh forms a biological scaffold that helps and guides the migration of the mesenchymal cells, derived from populations of resident stem cells or circulating precursors, from the base and the margins of the wound. the third component is the antibacterial properties of the factors released by the platelets and, probably, by the leucocytes contained in the prp. on the basis of these molecular understandings, prp therapy has been successfully used in the treatment of various dermatological defect including pressure ulcer, diabetic ulcer vascular ulcers, post - traumatic ulcers and lipodermatosclerosis. however, a few studies have reported in the treatment of severe cutaneous ulcers associated with old age, hypomobility and many underlying diseases. although further studies are required for better understandings of the mechanism, efficacy, and safety of autologous prp therapy, it could be considered as an effective alternative option for the recalcitrant cutaneous ulcer with various comorbidities.	the platelet - rich plasma (prp) has been advocated as a way to introduce increased concentrations of growth factors and other bioactive molecules to injured tissues in an attempt to optimize the local healing environment. a 94-yr - old woman with various comorbidities presented with a two - week history of severe cutaneous ulcer on the left dorsum of foot. it was caused by recurrent mechanical trauma and did not respond to several wound debridement and simple dressings. however, after she was completed on seven times of autologous prp treatments, we observed complete healing of the skin lesion within 3 months. herein, we report a case of recalcitrant cutaneous ulcer with various comorbidities and discuss about the promising possibility of autologous prp as an effective alternative therapeutic modality.
recent progress and technical advances in catheter ablation have dramatically improved the success rate and safety of pulmonary vein isolation (pvi) for atrial fibrillation (af). realtime monitoring of tiptotissue contact force (cf) is a useful technique for confirming that the ablation electrode is applying appropriate pressure. the development of cf has allowed control of the quality of lesions during radiofrequency (rf) ablation.1, 2 a low cf during catheter ablation is associated with ineffective lesion formation, whereas excessively high cf may in an increased risk of steam pop, thrombus formation, or cardiac perforation,3 particularly an atrioesophageal fistula in the posterior wall.4 a recent study reported that cf and the force time integral (fti) during rf ablation are predictors of transmural lesion, with the best cutoff fti value of > 392 gramseconds (gs).5 the efficas i study reported that ablation with a minimum fti of < 400 gs showed an increased likelihood of reconnection and that gap occurrence showed a strong trend with lower average cf and average fti6; however, the left atrial (la) wall under the catheter ablation line is not of uniform thickness, and it can be particularly thick in the left lateral ridge (llr). although the llr does not only compose the myocardial fiber,7 it has been reported that llr thickness is associated with the recurrence of af after pvi.8 for these reasons, we speculated that the optimal cf parameters for each lesion may differ according to the anatomical site and atrial wall thickness. the aim of this study was to evaluate the optimal cf or fti for anatomical ipsilateral pvi based on la wall thickness under the catheter ablation line. the study participants were 59 consecutive patients (118 ipsilateral veins) with symptomatic drugrefractory af who were referred to our hospital between september 2014 and august 2015 for rf catheter ablation for their first procedure. we included patients who underwent anatomical ipsilateral pvi and who were assessed for dormant conduction (dc) by an intravenous bolus of adenosine. the study was approved by the ethics committee at the national hospital organization, kanazawa medical center. prior to the procedure, la thrombus was excluded using laboratory data and cardiac computed tomography (ct) angiogram or transesophageal echocardiogram. antiarrhythmic agents were discontinued before the procedure, allowing a washout period of 5 halflives, although atrioventricular blocking agents were permitted in symptomatic patients. we excluded cases that required rf application in carina other than the pulmonary vein (pv) antrum to achieve pv isolation. a multielectrode catheter was transvenously inserted and positioned in the coronary sinus. a 10f intracardiac echocardiography catheter (64element, 5.510.0 mhz, soundstar ; biosense webster) threedimensional ultrasound images of the left atrium and pvs were acquired and processed using the carto 3 system (biosense webster). the reconstructed 3dimensional ct data sets were merged with the 3dimensional ultrasound derived geometries. after a double transseptal puncture was performed under intracardiac ultrasound guidance, heparin was intravenously administered to maintain activated clotting time of > 300 seconds. through transseptal accesses, nonsteerable sheaths jude medical, inc ) were placed into the left atrium. the irrigated cf ablation catheter (navistar thermocool smarttouch ; biosense webster) was advanced into the left atrium through a steerable sheath, and extensive encircling pvi was performed guided by the 3dimensional ultrasound geometries and 3dimensional merged ct images. ablations were performed in a temperaturecontrolled mode with the temperature limited to 43c and the power limited to 25 w in the posterior segments and 30 w in the other segments. luminal esophageal temperature was monitored using a luminal esophageal temperature probe (sensitherm ; st . jude medical), and we considered a luminal esophageal temperature of > 39c as requiring cessation of ablation. the irrigation flow rate was set to 2 ml / min during mapping and 1730 ml / min during ablation. ablations were performed with continuous dragging or point by point except for the regions of the posterior wall where the esophagus makes contact. the operator's decision to move 1 site to the next ablation site was based mainly on electrogram abatement and luminal esophageal temperature increase. the circumferential isolation was first performed anatomically without the circular catheter (lasso) or the 1mm multielectrodemapping catheter (pentaray), using an exclusively anatomical approach. the rf application time at each site varied, and pv antrum isolation was verified (as the absence of any pv potential or la potential in the antral ablation area) using the lasso or pentaray catheter and/or the ablation catheter electrograms. all examinations were performed using a 64detectorrow ct (multidetector ct) scanner (brilliance ct 64 ; philips electronics). for the contrastenhanced scan, 2021 mg of iodine per kg / s of contrast medium the scan was started with a delay of 11 seconds after the detection of contrast in the main pulmonary artery. volume data were reconstructed into axial images with a slice thickness of 0.9 mm and were transferred to a workstation for postprocessing (ziostation2 ; ziosoft, inc). for each pv , we divided the pv antrum into 8 segments under the ablation line, and quantitative measurements for each region were performed, as reported previously.8 at the llr and the carina between the superior and inferior pvs, we used images orthogonal to each region. the transmural myocardial thicknesses of the llr at the superior and inferior pv were each calculated as the mean of 2 measurements taken from 2 directions. the carina when several branches were present on the sameside pv, we adopted the thickest value of the carina. the pv antrum was defined as the region consisting of a 10mm space between the pv ostium and the left atrium. in the segments other than the llr, we measured the wall thickness of the pv antrum estimated to be on the catheter ablation line, using sagittal section. after ipsilateral anatomical pv antrum isolation, all pvs were mapped to detect ablation gaps of pvtoleft atrium conduction. in the absence of gaps, the presence of dcs was assessed in each vein with an intravenous administration of 20 mg adenosine. the positions where rf application ed in a change of sequence or abolition of the pv potential were defined as gap sites. a dc was defined as the reappearance of pv conduction, demonstrated by associated pv spikes of > 1 beat, as recorded by the circular catheter. the positions where rf application ed in a change of sequence or the abolition of dc were defined as dc sites. when 1 gap or dc site included multiple ablation points with the smallest vigitag (biosense webster), we counted all of these points as gaps or dcs. ablation points were assigned to each segment of the pv antrum where they were located. fti, maximum cf, average cf, minimum cf, rf duration, and rf power were obtained for each ablation point. when one ablation site overlapped to the next site, even with the smallest vigitag size, the higher fti site was selected as an ablation point. the fti for each ablation point was divided by the wall thickness of the pv atrium segment where the ablation point was located to calculate the ablation fti required for every 1 mm of wall thickness (fti / wall thickness). the predictive values of different thresholds of cf and fti for gaps and dcs were assessed using sensitivity, specificity, and receiver operating characteristic (roc) curve analysis. all procedures were performed under conscious sedation or general anesthesia. a multielectrode catheter was transvenously inserted and positioned in the coronary sinus. a 10f intracardiac echocardiography catheter (64element, 5.510.0 mhz, soundstar ; biosense webster) threedimensional ultrasound images of the left atrium and pvs were acquired and processed using the carto 3 system (biosense webster). the reconstructed 3dimensional ct data sets were merged with the 3dimensional ultrasound derived geometries. after a double transseptal puncture was performed under intracardiac ultrasound guidance, heparin was intravenously administered to maintain activated clotting time of > 300 seconds. through transseptal accesses, nonsteerable sheaths jude medical, inc ) were placed into the left atrium. the irrigated cf ablation catheter (navistar thermocool smarttouch ; biosense webster) was advanced into the left atrium through a steerable sheath, and extensive encircling pvi was performed guided by the 3dimensional ultrasound geometries and 3dimensional merged ct images. ablations were performed in a temperaturecontrolled mode with the temperature limited to 43c and the power limited to 25 w in the posterior segments and 30 w in the other segments. luminal esophageal temperature was monitored using a luminal esophageal temperature probe (sensitherm ; st . jude medical), and we considered a luminal esophageal temperature of > 39c as requiring cessation of ablation. the irrigation flow rate was set to 2 ml / min during mapping and 1730 ml / min during ablation. ablations were performed with continuous dragging or point by point except for the regions of the posterior wall where the esophagus makes contact. the operator's decision to move 1 site to the next ablation site was based mainly on electrogram abatement and luminal esophageal temperature increase. the circumferential isolation was first performed anatomically without the circular catheter (lasso) or the 1mm multielectrodemapping catheter (pentaray), using an exclusively anatomical approach. the rf application time at each site varied, and pv antrum isolation was verified (as the absence of any pv potential or la potential in the antral ablation area) using the lasso or pentaray catheter and/or the ablation catheter electrograms. all examinations were performed using a 64detectorrow ct (multidetector ct) scanner (brilliance ct 64 ; philips electronics). for the contrastenhanced scan, 2021 mg of iodine per kg / s of contrast medium the scan was started with a delay of 11 seconds after the detection of contrast in the main pulmonary artery. volume data were reconstructed into axial images with a slice thickness of 0.9 mm and were transferred to a workstation for postprocessing (ziostation2 ; ziosoft, inc). for each pv , we divided the pv antrum into 8 segments under the ablation line, and quantitative measurements for each region were performed, as reported previously.8 at the llr and the carina between the superior and inferior pvs, we used images orthogonal to each region. the transmural myocardial thicknesses of the llr at the superior and inferior pv were each calculated as the mean of 2 measurements taken from 2 directions. the carina when several branches were present on the sameside pv, we adopted the thickest value of the carina. the pv antrum was defined as the region consisting of a 10mm space between the pv ostium and the left atrium. in the segments other than the llr , we measured the wall thickness of the pv antrum estimated to be on the catheter ablation line, using sagittal section. after ipsilateral anatomical pv antrum isolation, all pvs were mapped to detect ablation gaps of pvtoleft atrium conduction. in the absence of gaps, the presence of dcs was assessed in each vein with an intravenous administration of 20 mg adenosine. the positions where rf application ed in a change of sequence or abolition of the pv potential were defined as gap sites. a dc was defined as the reappearance of pv conduction, demonstrated by associated pv spikes of > 1 beat, as recorded by the circular catheter. the positions where rf application ed in a change of sequence or the abolition of dc were defined as dc sites. when 1 gap or dc site included multiple ablation points with the smallest vigitag (biosense webster), we counted all of these points as gaps or dcs. ablation points were assigned to each segment of the pv antrum where they were located. fti, maximum cf, average cf, minimum cf, rf duration, and rf power were obtained for each ablation point. when one ablation site overlapped to the next site, even with the smallest vigitag size, the higher fti site was selected as an ablation point. the fti for each ablation point was divided by the wall thickness of the pv atrium segment where the ablation point was located to calculate the ablation fti required for every 1 mm of wall thickness (fti / wall thickness). the predictive values of different thresholds of cf and fti for gaps and dcs were assessed using sensitivity, specificity, and receiver operating characteristic (roc) curve analysis. of the 59 patients, 20 (33.9%) were shown during the ct scan to have af. complete pvi was achieved in 23 patients (39%) after ablation of single continuous circular lesions around ipsilateral pvs. all targeted pvs (118 ipsilateral veins) were successfully isolated by the end of the procedures. characteristics of the study population af indicates atrial fibrillation; bnp, btype natriuretic peptide; egfr, estimated glomerular filtration rate; mdct, multidetector computed tomography; noac, nonvitamin k antagonist oral anticoagulants; rf, radiofrequency. as shown in figure 1, the llr was the thickest part of the ablation line, with mean thicknesses of 3.90.9 mm for the left lateral superior ridge, 3.90.8 mm for the left lateral inferior ridge, and 4.30.9 mm for the left lateral carina ridge. the thinnest atrial wall was the left posterior wall (1.80.3 mm in the left posterior superior and 1.90.3 mm in the left posterior middle and the left posterior inferior). the wall thickness of each pv segment was not significantly affected by the presence of af rhythm during ct scanning (figure s1). myocardial thickness of each segment under the ablation line (lower panels) and distribution of acute gaps and dormant conductions (dcs) after pulmonary vein isolation (upper panels). libt indicates left inferior bottom; lipv, left inferior pulmonary vein; llcr, left lateral carina ridge; llir, left lateral inferior ridge; llsr, left lateral superior ridge; lpi, left posterior inferior; lpm, left posterior middle; lps, left posterior superior; lspv, left superior pulmonary vein; lsrf, left superior roof; rac, right anterior carina; rai, right anterior inferior; ras, right anterior superior; ribt, right inferior bottom; ripv, right inferior pulmonary vein; rpi, right posterior inferior; rpm, right posterior middle; rps, right posterior superior; rspv, right superior pulmonary vein; rsrf, right superior roof. the highest average cf (23.810.2 g) was applied to the right anterior inferior, and the lowest average cf was applied to the left lateral carina ridge (15.86.2 g). rf duration time was the shortest and fti was the lowest of all segments at the left posterior inferior (15.46.4 seconds and 234.7100.0 gs, respectively) as a of avoiding an increase in luminal esophageal temperature. fti / wall thickness was higher in the anterior wall of the right pvs and lower in the llr. the distribution of gaps and dcs was concentrated in the llr and right anterior wall (figure 1). cf parameters for each segment under the ablation line cf indicates contact force; fti, force time integral; gs, gramseconds; max, maximum; min, minimum; pv, pulmonary vein; rf, radiofrequency. all cf parameters including cf, rf duration, fti, and fti / wall thickness were significantly lower in the lesions with gaps or dcs compared with the lesions without them. these correlations were similarly observed within each patient. the delivered rf power was not significantly different between the 2 groups (p=0.08) (table 3). ablation parameters at each point with a gap or dc compared with those without cf indicates contact force; dc, dormant conduction; fti, force time integral; gs, gramseconds; rf, radiofrequency. roc curves were made for fti, average cf, maximum cf, rf duration, and fti / wall thickness to determine the thresholds that best predict gaps or dcs (figure 2). fti / wall thickness showed the best prediction value with an area under the curve of 0.9242 (95% ci 0.90600.9425). the areas under the curve for fti, average cf, maximum cf, and rf duration were 0.8101, 0.7046, 0.6246, and 0.7161, respectively. the best threshold for predicting gaps or dcs, defined by the position on the roc curve at the minimum distance from the left corner of the roc space, was an fti / wall thickness of 76.4 gs / mm (sensitivity 88.0% ; specificity 83.6%). an fti / wall thickness of < 101.1 gs / mm was highly predictive of gaps or dcs (sensitivity 97.0% ; specificity 69.6%). an fti / wall thickness of < 127.3 gs / mm predicted gaps or dcs with 100% sensitivity (specificity 55.01%). conversely, an fti of < 475.5 gs showed high sensitivity but low specificity (sensitivity 97.0% ; specificity 26.6%). subgroup analysis revealed that fti / wall thickness also showed the best prediction value with an area under the curve in the patients whose ct scan was obtained during af rhythm (figure s2). receiver operating characteristic curve analysis for acute gap and dormant conduction (dc) predictability. fti / wall thickness showed the best prediction value with an area under the curve (auc) of 0.9242 (95% ci 0.90600.9425, p<0.001 vs aucs of fti and the other contact force parameters). fti, average cf, maximum cf, and rf duration had aucs of 0.8101, 0.7046, 0.6246, and 0.7161, respectively. the best threshold for fti / wall thickness for predicting acute gaps or dcs was 76.4 gs / mm (sensitivity 88.0% ; specificity 83.6%). an fti / wall thickness of < 101.1 gs / mm was highly predictive of acute gap or dc (sensitivity 97.0% ; specificity 69.6%). avg indicates average; fti, force time integral; gs, gramseconds; max, maximum. as shown in figure 1, the llr was the thickest part of the ablation line, with mean thicknesses of 3.90.9 mm for the left lateral superior ridge, 3.90.8 mm for the left lateral inferior ridge, and 4.30.9 mm for the left lateral carina ridge. the thinnest atrial wall was the left posterior wall (1.80.3 mm in the left posterior superior and 1.90.3 mm in the left posterior middle and the left posterior inferior). the wall thickness of each pv segment was not significantly affected by the presence of af rhythm during ct scanning (figure s1). myocardial thickness of each segment under the ablation line (lower panels) and distribution of acute gaps and dormant conductions (dcs) after pulmonary vein isolation (upper panels). libt indicates left inferior bottom; lipv, left inferior pulmonary vein; llcr, left lateral carina ridge; llir, left lateral inferior ridge; llsr, left lateral superior ridge; lpi, left posterior inferior; lpm, left posterior middle; lps, left posterior superior; lspv, left superior pulmonary vein; lsrf, left superior roof; rac, right anterior carina; rai, right anterior inferior; ras, right anterior superior; ribt, right inferior bottom; ripv, right inferior pulmonary vein; rpi, right posterior inferior; rpm, right posterior middle; rps, right posterior superior; rspv, right superior pulmonary vein; rsrf, right superior roof. the highest average cf (23.810.2 g) was applied to the right anterior inferior, and the lowest average cf was applied to the left lateral carina ridge (15.86.2 g). rf duration time was the shortest and fti was the lowest of all segments at the left posterior inferior (15.46.4 seconds and 234.7100.0 gs, respectively) as a of avoiding an increase in luminal esophageal temperature. fti / wall thickness was higher in the anterior wall of the right pvs and lower in the llr. the distribution of gaps and dcs was concentrated in the llr and right anterior wall (figure 1). cf parameters for each segment under the ablation line cf indicates contact force; fti, force time integral; gs, gramseconds; max, maximum; min, minimum; pv, pulmonary vein; rf, radiofrequency. all cf parameters including cf, rf duration, fti, and fti / wall thickness were significantly lower in the lesions with gaps or dcs compared with the lesions without them. these correlations were similarly observed within each patient. the delivered rf power was not significantly different between the 2 groups (p=0.08) (table 3). ablation parameters at each point with a gap or dc compared with those without cf indicates contact force; dc, dormant conduction; fti, force time integral; gs, gramseconds; rf, radiofrequency. roc curves were made for fti, average cf, maximum cf, rf duration, and fti / wall thickness to determine the thresholds that best predict gaps or dcs (figure 2). fti / wall thickness showed the best prediction value with an area under the curve of 0.9242 (95% ci 0.90600.9425). the areas under the curve for fti, average cf, maximum cf, and rf duration were 0.8101, 0.7046, 0.6246, and 0.7161, respectively. the best threshold for predicting gaps or dcs, defined by the position on the roc curve at the minimum distance from the left corner of the roc space, was an fti / wall thickness of 76.4 gs / mm (sensitivity 88.0% ; specificity 83.6%). an fti / wall thickness of < 101.1 gs / mm was highly predictive of gaps or dcs (sensitivity 97.0% ; specificity 69.6%). an fti / wall thickness of < 127.3 gs / mm predicted gaps or dcs with 100% sensitivity (specificity 55.01%). conversely, an fti of < 475.5 gs showed high sensitivity but low specificity (sensitivity 97.0% ; specificity 26.6%). subgroup analysis revealed that fti / wall thickness also showed the best prediction value with an area under the curve in the patients whose ct scan was obtained during af rhythm (figure s2). receiver operating characteristic curve analysis for acute gap and dormant conduction (dc) predictability. fti / wall thickness showed the best prediction value with an area under the curve (auc) of 0.9242 (95% ci 0.90600.9425, p<0.001 vs aucs of fti and the other contact force parameters). fti, average cf, maximum cf, and rf duration had aucs of 0.8101, 0.7046, 0.6246, and 0.7161, respectively. the best threshold for fti / wall thickness for predicting acute gaps or dcs was 76.4 gs / mm (sensitivity 88.0% ; specificity 83.6%). an fti / wall thickness of < 101.1 gs / mm was highly predictive of acute gap or dc (sensitivity 97.0% ; specificity 69.6%). avg indicates average; fti, force time integral; gs, gramseconds; max, maximum. we evaluated the utility of an optimal value for fti in relation to the wall thickness under the pvi ablation line to predict gaps or dcs. our major findings were as follows: mdct analysis revealed that the wall thickness was different at each part of the ablation line and was thickest at the llr; the gaps or dcs were significantly associated with low cf, rf duration, fti, and fti / wall thickness; and roc curve analysis identified fti / wall thickness as the best predictor for gaps and dcs. in this study, the wall thicknesses of the llr, the right anterior carina, and the posterior walls were 4, 3.4, and < 2 mm, respectively. these are generally consistent with previous reports regarding japanese patients.8 in european studies using heart specimens by dissection and histological sections or by magnetic resonance angiography, the mean width of the llr was greater.7, 9 although the modalities used to measure wall thickness in those studies differed from ours, the different width may be related to race, age, or sex. a lower average cf has been reported as a strong predictor of gap formation.6, 10, 11 it has been reported that cf at the llr tends to be low12 and that the majority of conduction gaps after single continuous circular lesions around ipsilateral pvs were located at the llr and the anterior wall of the right pv.13 in the present study, the cf for the llr was low, and most of the gaps and dcs were located at the llr or the right anterior carina, which was consistent with previous reports.12, 13 we speculate that the lower cf and insufficient fti against the thick atrial walls at the llr and anterior right pv wall led to the formation of gaps and dcs. although rf power is a known predictor of lesion size,3, 14, 15, 16 it had no significant impact on gap or dc formation in this study. this might be derived from the fixed power output setting, which is uniform across the ablation line (25 w in posterior wall segments and 30 w in the other segments). further study is required to test whether the addition of a power output parameter improves prediction performance of fti / wall thickness. it has been reported that a minimum fti of 400 gs for each lesion was necessary to avoid reconnection6, 10 or to create transmural lesions in pvi.5 conversely, the relationship between fti and electrogram attenuation plateaued at 500 gs, and fti and impedance drop also plateaued at 500 gs.17 beyond this plateau point, continuation of ablation is unlikely to produce further gains but may increase the potential risk of complications such as perforation, steam pops, or damage to extracardiac structures.17 nevertheless, these studies did not take wall thickness into account, and it differs at each ablation point. in the present study, most of the gaps or dcs were concentrated at the llr and the right anterior carina regardless of high fti. conversely, few gaps or dcs were observed at the posterior walls, where fti were relatively low. these observations imply that fti sufficient for creating transmural legions is higher for the thicker wall. conversely, excess fti for thin walls may bring a potential risk of damage to extracardiac structures such as the esophagus at the posterior wall of the lpv. in this study, an average rf application number was 90 points per patient. considering that even a few remaining gaps or dcs from these ablation points can lead to af recurrence , more sensitive fti / wall thickness values should be targeted to avoid formation of gaps and dcs. although fti / wall thickness of > 76.4 gs / mm has a best predictive value on the basis of roc analysis, with the best balance between sensitivity and specificity, fti / wall thickness of 101.1 gs / mm with higher sensitivity could be a more suitable target to avoid formation of gaps and dcs. first, this study was a singlecenter retrospective study with a relatively small number of patients. our participants were relatively old japanese patients with small physiques, and it is not known whether the present would be applicable to other races. second, our measurements of wall thickness were made on the basis of the assumed ablation lines on mdct data and might not match the actual ablation line. in addition, the af rhythm observed in 33.9% of the patients during ct scanning may have affected the quality of ct images and the measurements of wall thickness. third, the number of gaps and dcs observed in the present study is greater than the number observed in the previous report13; however, complete pvi was achieved in 39% of the patients after ablation of single continuous circular lesions around ipsilateral pvs, which is consistent with the previous report. we suppose that the larger number of gaps and dcs was related to our definition. in this study, when 1 gap or dc site included multiple ablation points with the smallest vigitag size, we counted all of these points as gaps or dcs. finally, we evaluated the effect of fti only on acute success and the elimination of dcs. although it has been reported that the presence of dcs is associated with a higher risk of recurrence after pvi,18 it is still unclear whether the higher fti / wall thickness actually leads to the better outcome of af ablation. first, this study was a singlecenter retrospective study with a relatively small number of patients. our participants were relatively old japanese patients with small physiques, and it is not known whether the present would be applicable to other races. second, our measurements of wall thickness were made on the basis of the assumed ablation lines on mdct data and might not match the actual ablation line. in addition, the af rhythm observed in 33.9% of the patients during ct scanning may have affected the quality of ct images and the measurements of wall thickness. third, the number of gaps and dcs observed in the present study is greater than the number observed in the previous report13; however, complete pvi was achieved in 39% of the patients after ablation of single continuous circular lesions around ipsilateral pvs, which is consistent with the previous report. we suppose that the larger number of gaps and dcs was related to our definition. in this study, when 1 gap or dc site included multiple ablation points with the smallest vigitag size, we counted all of these points as gaps or dcs. finally, we evaluated the effect of fti only on acute success and the elimination of dcs. although it has been reported that the presence of dcs is associated with a higher risk of recurrence after pvi,18 it is still unclear whether the higher fti / wall thickness actually leads to the better outcome of af ablation. this study demonstrated that the optimal fti for achieving effective ablation for ipsilateral anatomical pvi varies at each point along the ablation line. atrial wall thickness is not uniform under the ablation line, and fti / wall thickness is a strong predictor of gap and dc formation. fti / wall thickness 100 gs / mm could be a suitable target value to achieve effective ablation. figure s1. the effect of atrial fibrillation (af) during computed tomography scanning on the measured myocardial wall thickness of each segment under the ablation line. the wall thicknesses of the pulmonary vein segments were not significantly different between the sinus group and the af group. figure s2. the impact of af during computed tomography scanning on predictability of acute gap and dc. fti / wall thickness showed the best prediction value, with an auc of 0.9325. the best threshold of the fti / wall thickness for predicting acute gaps or dcs was 76.4 gs / mm (sensitivity 93.2% ; specificity 83.0%). the lower panel shows roc curve analysis in af rhythm. in af rhythm, fti / the best threshold of the fti / wall thickness for predicting acute gaps or dcs was 89.7 gs / mm (sensitivity 92.6% ; specificity 77.6%). af indicates atrial fibrillation; auc, area under the curve; dc, dormant conduction; fti, force time integral; gs, gramseconds; roc, receiver operating characteristic.	low contact force and force time integral (fti) during catheter ablation are associated with ineffective lesion formation, whereas excessively high contact force and fti may increase the risk of complications. we sought to evaluate the optimal fti for pulmonary vein (pv) isolation based on atrial wall thickness under the ablation line.methods and contact force parameters and fti during anatomical ipsilateral pv isolation for atrial fibrillation and atrial wall thickness were assessed retrospectively in 59 consecutive patients for their first pv isolation procedure. the pv antrum was divided into 8 segments, and the wall thickness of each segment under the ablation line was determined using multidetector computed tomography. the fti for each ablation point was divided by the wall thickness of the pv antrum segment where each point was located to obtain fti / wall thickness. in total, 5335 radiofrequency applications were delivered, and 85 gaps in pv isolation ablation lines and 15 dormant conductions induced by adenosine were detected. the gaps or dormant conductions were significantly associated with low contact force, radiofrequency duration, fti, and fti / wall thickness. among them, fti / wall thickness had the best prediction value for gaps or dormant conductions by receiver operating characteristic curve analysis. fti / wall thickness of < 76.4 gramseconds per millimeter (gs / mm) predicted gaps or dormant conductions with sensitivity (88.0%) and specificity (83.6%), and fti / wall thickness of < 101.1 gs / mm was highly predictive (sensitivity 97.0% ; specificity 69.6%). fti / wall thickness is a strong predictor of gap and dormant conduction formation in pv isolation. an fti / wall thickness 100 gs / mm could be a suitable target for effective ablation.
obesity and metabolic - related disorders are considered major health issues worldwide. over the last decade, the incidence of obesity and overweight has almost doubled in developed countries and the trend is mirrored in developing nations that are transitioning to first - world economies. obesity from an interaction of many factors including genetic, physiologic, behavioural, and environmental influences. however, the rapid increases in the rates of obesity suggest that environmental and behavioural influences, rather than genetic causes, are fuelling the present epidemic. increasing evidence from both clinical and animal studies has highlighted the link between altered maternal nutrition and the risk of offspring developing obesity and the metabolic syndrome. initial epidemiological studies suggested that fetal growth restriction is correlated with later disease, implying that fetal nutritional deprivation may be a strong stimulus for developmental programming. however, although maternal nutrient deprivation has been well characterized in this context, in many societies, maternal and postnatal nutrition can be excessive. as a , excessive weight gain and/or obesity are common nutritional problems complicating pregnancy in developed countries. as such, there is now accumulating evidence from human and animal studies suggesting that excess maternal caloric intake has adverse effects on the health and well - being of offspring, independent of postnatal diet and exerted transgenerational effects. maternal obesity has many adverse outcomes, including labour and delivery complications, fetal and neonatal death, maternal hypertension, and preeclampsia and gestational diabetes. in addition to acute risks to the obese mother, negative outcomes extend to offspring, including obesity and cardiovascular disease in adulthood. on one hand, while a shared postnatal environment and genetic susceptibility are likely contributors, maternal bmi is reported in some cohorts as having greater influence on offspring than paternal bmi, highlighting the independent influence of the intrauterine environment on offspring adiposity. in a rodent model , we recently reported that offspring born to high - fat - fed mothers showed an obese phenotype in adulthood characterised by hyperinsulinemia and hyperleptinemia, independent of postnatal diet. although the mechanistic drivers underlying this obesogenic phenotype are still unclear, experimental data in rodents suggest that leptin plays a critical role in underpinning early life influences on postnatal phenotypic development. early studies demonstrated that leptin, an adipokine produced primarily by adipocytes, plays a key role in regulation of energy homeostasis and food intake via its action on specific hypothalamic nuclei. leptin has been since demonstrated to exert other important functions, including its regulation of bone growth, skeletal metabolism, and linear growth via direct effects on osteoblast and osteocalcin release and growth hormone secretion, respectively. in rodents, a characteristic of the neonatal period is a leptin surge, which normally peaks in the second week of neonatal life. it has been shown that early life nutritional insults affect this surge ing in altered hypothalamic development. maternal undernutrition in rats has been shown to in a blunted and altered timing of the leptin surge in neonatal pups, and leptin administration during the neonatal period in ob / ob mice normalised hypothalamic development and partially normalised orexigenic behaviour. importantly, postweaning leptin administration had no effect, emphasising that, in the rat, the critical stage of hypothalamic leptin regulation is during the first 2 weeks of neonatal life. our group has previously shown that neonatal leptin treatment can reverse the deleterious effects of maternal undernutrition on postnatal outcomes in male and female offspring. in contrast, kirk et al. reported that maternal high - fat diet led to an amplified leptin surge in neonatal pups during the first 2 weeks of life, ing in altered hypothalamic regulation of food intake. there is growing evidence that leptin plays a significant role in the development of an obesogenic phenotype after early - life exposure to an imprudent diet. despite this , no studies have investigated whether maternal high - fat - diet - induced changes in neonatal leptin action regulate this association. we, therefore, hypothesized that leptin blockade, using a specific leptin antagonist, during the critical neonatal period of leptin sensitivity would ameliorate maternal high - fat - induced obesogenic effects on offspring. we investigated the effect of leptin antagonist administration during the time of the critical neonatal leptin surge on weight gain, food intake, and body composition in male offspring born to mothers fed either a control or a high - fat diet. the animal model of maternal high - fat nutrition has been described in detail previously. briefly, female wistar rats were time - mated using a rat estrus cycle monitor (ec40, fine science tools, foster city, ca, usa) to assess the stage of estrus of the animal before introducing the male. upon confirmation of mating, rats were randomly assigned to one of two maternal diets: the control chow diet throughout pregnancy and lactation (con, n = 11 litters, diet 2018, harlan - teklad, oxon, uk) or a high - fat diet (mhf, n = 13 litters, 45% kcals from fat, d12451, research diets, new brunswick, nj, usa) to be fed ad libitum throughout pregnancy and lactation. females were housed individually, with free access to water, and bodyweight and food intakes were measured every two days until the end of lactation. at birth, pups were weighed, and on postnatal day 2, litter size was adjusted to 8 pups per litter to ensure adequate and standardized nutrition until weaning. at postnatal day 3 , mhf and con litters were randomly assigned to receive either saline (s) or pegylated rat leptin antagonist (la, mutant l39a / d40a / f41a, protein laboratories rehovot, israel). the la or saline was administrated by subcutaneous injection at postnatal days 3, 5, and 7 at a dose of 12.5 g / g. dosage and timing of la administration was derived from calculated half - life (approximately 20 hours) and prior cited publications. male pups were weighed daily during the treatment period and then every 2 days thereafter until weaning (p22). at weaning, saline- and la - treated con and mhf male offspring were housed two per cage, and randomly placed on either the control rat chow (c) or high - fat (hf) diet until the completion of the trial (day 110). body weights and caloric intakes were recorded in offspring every 3 days until the end of the study. body composition (fat mass, bone mineral content ( bmc), and bone mineral density (bmd) ) was measured by dual - energy x - ray absorptiometry (dexa) at p100 under light isoflurane (2%) anaesthesia and using a dedicated small animal software package (lunar hologic, waltham, ma). rats were culled at p110 by decapitation following anaesthesia with sodium pentobarbitone (60 mg / kg). a tail blood sample was taken for fasting glucose and -hydroxybutyrate (bhb) measurements (roche accucheck) and a rectal temperature measurement recorded. trunk blood was collected into heparinised vacutainers, centrifuged and plasma stored at 20c for later analysis. all animal work was approved by the animal ethics committee of the university of auckland. plasma leptin and insulin concentrations were analysed using commercially available rat - specific elisas (no . cary, nc, usa) and r software (v.2.9.0, r foundation for statistical computing, vienna, austria) for windows. all models were statistically validated for assumptions of normality of residuals and absence of heteroscedasticity. nonnormal data were log transformed to normalize where necessary. maternal pregnancy data and neonatal data at birth were analysed using one - way anova. maternal caloric intakes; weight and body composition during lactation was analysed by two - way anova with maternal diet and treatment group as factors and litter as covariate. although the growth analysis was performed on absolute body weight data, growth figures are shown as relative changes for sake of clarity given the number of experimental groups involved. preweaning data for pups was analysed by two - way factorial anova with maternal diet and la administration as factors, and their interactions. data from adult offspring were analyzed by three - way factorial anova with maternal diet, postweaning diet, and la treatment as factors, and the interaction between these factors (litter included as a covariate). consistent with our previous observations, maternal hf diet during pregnancy and lactation ed in a transient increase in caloric intake from day 2 (p < 0.001) to day 15 of gestation when intakes returned to levels similar to those observed in con dams (data not shown). increased caloric intake in mhf dams was reflected in an increased maternal weight gain by gestational day 7 (p = 0.04, figure 2), which persisted until birth. mhf dams remained heavier than controls from the early neonatal period until mid - lactation when body weights returned to match those of controls. there was no overall significant effect of neonatal la administration on weight or caloric intake of dams during pregnancy and lactation. at weaning, despite a similar maternal body weight, total fat mass (%) and fat: lean ratios (f / l) were significantly increased in mhf dams compared to con (% fat : con 14.7 1.7% versus mhf 22.9 2.3, p = 0.01 ; f / l : con 0.18 0.08 versus mhf 0.31 0.16, p = 0.01). there was no significant effect of neonatal la treatment or an interaction between treatment and maternal diet, on body composition of dams. birthweights were slightly but significantly reduced in male offspring of mhf dams compared to con (con 6.2 0.1 g ; mhf 5.9 0.1 g, p < 0.001). at p3 and prior to start of la administration, pups born to mhf mothers remained lighter than con neonates (con 7.1 0.1 g ; mhf 6.8 0.1 g, p < 0.05). la administration leads to an increased neonatal weight gain in con and mhf offspring compared to their saline treated counterparts (p < 0.005, figure 3). the increased weight gain in la - treated neonates was more pronounced in offspring of mhf dams reflected in a maternal diet la treatment interaction (p < 0.005). by weaning (p22), mhf offspring were slightly but significantly heavier than con offspring and la treatment further increased weaning weights in cont and mhf offspring (p22 : con - s 59.9 0.9 g, con - la 62.0 1.1 g, mhf - s 61.8 1.7 g, mhf - la 66.3 1.1 g, p < 0.05 for effect of maternal diet and la treatment, no interactions). an mhf diet had no significant effect on adult body weight at postnatal day 110 but ed in significantly increased total percent body fat and decreased lean body weight percentage compared to con animals as quantified by dexa scanning (table 1). a postweaning hf diet increased body weight and total body fat mass in all hf - fed groups. neonatal la treatment had a significant overall effect on reducing total percent body fat mass, increasing lean mass and a decreased fat: lean ratio (table 1). a significant maternal diet la treatment postnatal diet interaction (p < 0.001) revealed that body weights were significantly reduced in offspring of con dams that were treated as neonates with la and fed a postweaning hf diet as compared to saline treated con offspring fed the hf diet (figure 4(c) ). la treatment in con neonates reduced hf diet - induced obesity by approximately 10% and equated to an absolute bodyweight difference of 72.6 g (figure 4(a) ). dexa analysis of body fat content showed that this reduction in body fat was paralleled by a reduction in fat mass in these animals compared to saline treated (figure 4(d) ). this effect was not observed in mhf offspring where neonatal la failed to significantly impact on postweaning hf - induced changes in final bodyweight or fat mass (figures 4(b) and 4(d) and table 1 ). this may reflect a more marked increase in relative lean mass in la - treated con - hf offspring as compared to la treated mhf - hf offspring compared to relative saline - treated groups (table 1). there were no significant effects of mhf diet, neonatal la treatment, or postweaning hf diet on total caloric intake (expressed as kcals consumed per gram body weight) across any of the treatment groups (figures 5(a) and 5(b) ). there were significant overall effects of maternal diet and la treatment on nose - anus (na) length (table 1). post hoc analysis revealed la treatment increased na length in mhf offspring but not con offspring as reflected in a maternal diet la treatment interaction (p < 0.05). a postweaning hf diet increased na length only in mhf offspring (table 1). maternal diet had no overall effect on nose - tail (nt) length (table 1). there were overall significant effects of neonatal la treatment and postweaning hf diet on increasing nt length. a significant maternal diet la treatment interaction revealed that increases in nt length as a of neonatal la treatment were greater in mhf offspring compared to con offspring for both chow and postweaning hf diets (table 1). there was no effect of mhf diet on bmd (table 1). neonatal la treatment reduced bmd in all treatment groups and a postweaning hf diet increased bmd in all offspring. an mhf diet had the overall effect of increasing bmc in all offspring (table 1). neonatal la treatment reduced bmc in all treated groups and a postweaning hf diet increased bmc in all offspring. a postweaning hf diet had no significant effect on rt (p = 0.092). an mhf diet significantly increased plasma leptin levels in all mhf offspring (table 2). there was a strong trend toward increased fasting plasma insulin levels in mhf offspring, but this difference did not reach statistical significance (p = 0.068). a postweaning hf diet increased plasma insulin levels in all hf - fed groups (table 2). fasting blood bhb levels were not altered by mhf diet or neonatal la treatment but were significantly increased in all hf - fed offspring compared to chow - fed offspring (table 2). these have demonstrated for the first time that early - life manipulation of the leptin axis via neonatal leptin antagonism can exert marked effects on growth and body composition, which are dependent upon prior maternal nutrition status and postweaning diet. investigators using neonatal leptin treatment given to offspring of normally fed dams have shown increased adiposity and leptin and insulin resistance in offspring in later life. control offspring, given a leptin antagonist prior to being fed an obesogenic hf diet postweaning, show an amelioration of a diet - induced fat accumulation and reduced linear body growth. the marked contrast in adult phenotype in offspring of normally nourished mothers, based on exposure to either leptin or leptin antagonism during early - life development, further serves to highlight how critical the maintenance of leptin threshold levels is during this period of developmental plasticity. as we have shown previously, maternal high - fat nutrition ed in increased adiposity, leptin, and insulin concentrations in offspring compared to offspring of control mothers, independent of postweaning diet. there is a well - characterized leptin surge in the first two weeks of life in the rodent although the source of the leptin is yet to be defined with the surge occurring independently of changes in neonatal body weight trajectory and milk leptin intake. this concurs with the increased sensitivity to body weight gain in mhf neonates treated with the la as compared to con offspring. in other studies, hypoleptinemic offspring of mothers undernourished during pregnancy have either a delayed or premature leptin surge. however, there is little known about the leptin surge in models of maternal obesity. recently reported that rat offspring of mothers fed an obesogenic diet had normal serum leptin levels at birth but displayed an amplified and prolonged neonatal leptin surge, which was accompanied by an elevation in leptin mrna expression in abdominal white adipose tissue. however, it is unknown whether the leptin surge in the mhf offspring of the present study is altered. although inborn leptin deficiency causes weight gain, it is unclear whether induced leptin deficiency in adult wild - type animals would be orexigenic. leptin antagonists have only recently become commercially available and provide an invaluable tool for investigating central and peripheral leptin deficiency and exploring the involvement of leptin in metabolic processes. previous reports using a nonpegylated leptin antagonist have been problematic. the extremely short half - life of the antagonist necessitated administration of supraphysiological doses to induce a clinical response and was not sufficient to induce a true metabolic state of leptin deficiency. hormones with molecular masses similar to that of leptin are cleared primarily via the kidney with a half life of only 830 minutes. the effect of early postnatal leptin blockade in normal rat neonates has previously been reported in the study by attig et al.. in this work, the authors studied the long - term effect of neonatal therapy with a non - pegylated leptin antagonist (day 2 to day 13) in female wistar rats. in contrast to the present study, they showed that leptin antagonism induced a decrease in neonatal weight gain, which has previously been commonly associated with neonatal leptin treatment. later in life, the leptin disruption led to a higher sensitivity to diet - induced obesity, as shown by a higher body weight gain when challenged with a high - energy diet, associated with increased adiposity and leptinemia. these animals also displayed a phenotype of leptin resistance at 4 months, characterized by the inability of treated animals to respond to leptin by failing to reduce food intake and showing reduced birth weight. overall, the long - term effect in the attig study was paradoxically similar to that reported for rats treated with leptin during neonatal life. importantly, the molecule used as the leptin antagonist was different from the one used in our study. indeed in this work, the authors used the leptin mutein, a molecule acting as an antagonist, with in vivo effects previously validated only using intracerebroventricular, but not subcutaneous, administration. furthermore, this antagonist, obtained by alanine mutagenesis of amino acids 39 to 41 - 42, has an extremely short half - life and high doses are required to produce a clinical response that is similar to a true metabolic state of leptin resistance. the present study utilised a recently developed rat - specific pegylated la whereby the attachment of polyethylene glycol increased the overall molecule size to 70 kda. pegylation of the la in an approximate 30-fold increase in in vivo half - life, thus true states of induced leptin deficiency are possible at physiologic doses. to date, only one prior study has examined the effects of the pegylated la moiety, albeit in normal postweaning animals where it was shown that treatment with the pegylated la to postweaning mice in a rapid and dramatic increase in food intake and weight gain. the blood brain barrier (bbb) in the neonatal rat is relatively immature; pegylated leptin antagonist has been shown to block circulating leptin from crossing the bbb, an action that would attenuate the anorexigenic effect of leptin. the windows of treatment are different, bbb permeability is at different developmental stages (neonatal versus postweaning), and offspring responsiveness to leptin intervention is known to elicit sexually dimorphic responses. in addition, the work in the mouse examined the immediate phenotypic response to la treatment, whereas the present study examines a postnatal phenotype derived from an early - life neonatal intervention. however, consistent with the reports from mice, the present demonstrated that la treatment induced a significant increase in body weight over the neonatal treatment period. in the present study, neonatal leptin antagonism, despite having significant effects on pre - weaning weights in offspring of mhf mothers, had no effect on postnatal weight gain in con or mhf offspring fed the standard chow diet. there was, however, a marked effect of neonatal la treatment in reducing body weight gain in con offspring fed the hf diet after weaning. conversely, la treatment to mhf offspring subsequently fed the hf diet had no significant effect on body weight; independent of changes in body weight and circulating plasma leptin concentrations. interestingly, neonatal la treatment did not alter postweaning caloric intake, thus the observed changes in body weight gain are independent of food intake and suggest a lack of effect of la administration on the arcuate nucleus and related feeding circuitry, as has been reported with neonatal leptin treatment in the ob / ob mouse. nose - anus lengths were increased in mhf offspring but not con offspring, which may suggest that altered effects on the growth - hormone- (gh-) insulin - like growth factor (igf) axis are mediated by neonatal la exposure. the observed change in tail length in con and mhf la - treated offspring was unexpected but may have ed from altered thermoregulatory set - point processes as reflected in the significant differences in basal body temperature. in the rat , a significant portion of total body heat loss occurs through sympathetically mediated changes in tail blood flow. however, since rectal temperature was decreased in la - treated con and mhf offspring, it is difficult to explain the disparate changes in tail length to thermoregulatory processes and, as with nose - anus length, may reflect la - induced alterations in the gh - igf axis in mhf offspring as compared to controls or development of a thrifty metabolic phenotype as regards thermogenesis and energy expenditure. future independent studies looking at brown fat thermogenesis and uncoupling proteins may further explain this observation. bone morphology was significantly altered in adult offspring following neonatal la treatment with overall significant reductions in bmc and bmd. it is well established that leptin treatment can in enhanced bone formation and promotion of pro - osteogenic factors in bone marrow , and the current data suggests that the reverse holds true for leptin antagonism and further work investigating specific bone markers is now warranted. this is the first study designed to examine the efficacy of neonatal leptin antagonism following altered maternal nutrition and its interaction with differing levels of postweaning nutrition, on offspring phenotype development. responsiveness to neonatal leptin antagonism is dependent upon both maternal and postweaning nutrition, with minimal efficacy in chow - fed offspring of either con or mhf mothers. more studies are now required to further understand the mechanistic underpinnings of the present observations, including characterization of the effects of leptin antagonism on the timing and magnitude of the leptin surge in offspring of mothers with different dietary s. however, it is important to recognise that leptin - mediated development of feeding circuits occurs postnatally in the rodent and occurs in utero in primates, including humans, and thus timing of intervention strategies may be different. nonetheless, taken together, the data on both neonatal leptin treatment and leptin antagonism in the setting of both normal and nutritionally challenged pregnancies serves to highlight the important role of leptin regulation during critical early - life windows of development on lasting growth and metabolic function in offspring.	epidemiological and experimental studies report associations between overweight mothers and increased obesity risk in offspring. it is unclear whether neonatal leptin regulation mediates this association between overweight mothers and offspring obesity. we investigated the effect of neonatal treatment with a leptin antagonist (la) on growth and metabolism in offspring of mothers fed either a control or a high fat diet. wistar rats were fed either a control (con) or a high fat diet (mhf) during pregnancy and lactation. male con and mhf neonates received either saline (s) or a rat - specific pegylated la on days 3, 5, and 7. offspring were weaned onto either a control or a high fat (hf) diet. at day 100, body composition, blood glucose, -hydroxybutyrate and plasma leptin and insulin were determined. in con and mhf offspring, la increased neonatal bodyweights compared to saline - treated offspring and was more pronounced in mhf offspring. in the post - weaning period, neonatal la treatment decreased hf diet - induced weight gain but only in con offspring. la treatment induced changes in body length, fat mass, body temperature, and bone composition. neonatal la treatment can therefore exert effects on growth and metabolism in adulthood but is dependent upon interactions between maternal and post - weaning nutrition.
morton s neuroma is a common paroxysmal neuralgia affecting the forefoot, typically in the third interdigital space. the clinical syndrome of morton s neuroma was described over a century ago, but its etiopathology remains poorly understood. recent studies suggest that morton s neuroma is a mechanically induced degenerative neuropathy which has a strong predilection for the third common digital nerve. clinical examination is the most sensitive and specific, and is superior to imaging modalities like ultrasound or magnetic resonance imaging scan. various treatment options have been described in the literature for the treatment of morton s neuroma, including infiltration of a local anaesthetic and steroid combination into the interdigital space, sonography - guided alcohol injection, and surgery. the of local steroid injections and ultrasound - guided alcohol injections are comparable to those of surgery, but limited literature is available on long - term . some authors still recommend therapeutic injections prior to surgery. however, surgical resection has shown good long - term , with improvement in 80 % of cases. rheumatoid synovitis and nodules producing symptoms mimicking morton s neuroma have been reported in the literature , but are still rare. most of the cases reported in the literature were found in rheumatoid arthritis patients. we report three patients (five feet) presenting with symptoms and signs of morton s neuroma due to underlying rheumatoid nodules. histology of the excised tissue showed the presence of a rheumatoid nodule and morton s neuroma in four feet and a rheumatoid nodule and unremarkable nerve bundles in one. our patients were rendered asymptomatic with surgical treatment and went on to have appropriate management of rheumatoid arthritis. informed consent was obtained from the patients prior to being included in the case series. case 1. a 38-year - old lady presented to the foot and ankle clinic with pain and a burning sensation over the third intermetatarsal space of the right foot and the second intermetatarsal space of the left foot. clinical examination revealed fullness in the interdigital space with splaying of the corresponding toes (fig . 1) and tenderness over the plantar aspect of the corresponding intermetatarsal space with a positive mulder s click. the patient underwent surgery through a single dorsal linear incision in the corresponding intermetarsal space. intraoperatively, a large soft tissue mass 2 cm in size involving the digital nerve and capsule of the second metatarsophalangeal (mtp) joint was noted on the left side, along with necrosis of the underlying fat pad. a soft tissue swelling originating from the dorsum of the capsule of the third mtp joint, filling the intermetatarsal space, was noted on the right side.fig. 1fullness in the second intermetatarsal space, with splaying of the surrounding toes fullness in the second intermetatarsal space, with splaying of the surrounding toes histology of the operative specimen revealed fibrinoid necrosis rimmed by palisaded histiocytes and fibroblasts, confirming the diagnosis of rheumatoid nodule (fig . 2). it also showed thickened nerve bundles and blood vessels which were surrounded by fibrous tissue consistent with morton s neuroma (fig . 3) in the left foot, and a rheumatoid nodule and unremarkable digital nerve bundles in the right foot.fig. 2hematoxylin and eosin 100. 3hematoxylin and eosin 25. thickened blood vessels and nerve bundles that are rimmed by fibrous tissue consistent with morton s neuroma hematoxylin and eosin 100. central fibrinoid necrosis rimmed by palisaded fibroblasts and histiocytes indicating a rheumatoid nodule hematoxylin and eosin 25. thickened blood vessels and nerve bundles that are rimmed by fibrous tissue consistent with morton s neuroma she was subsequently tested for rheumatoid arthritis, and gave an equivocal on ra assay and a strong positive for anti - mutated citrullinated vimentin antibodies (anti - mcv) suggestive of seropositive rheumatoid arthritis. during follow - up, she developed symptoms of rheumatoid arthritis seven months after her first attendance at the foot and ankle clinic. case 2. a 37-year - old lady with a past medical history of rheumatoid arthritis presented to the foot and ankle clinic with pain and swelling in the second intermetatarsal space bilaterally. clinical examination revealed splaying of the 2/3 toes bilaterally, with a positive mulder s click. a clinical diagnosis of morton s neuroma was made and the feet were operated on, one side at a time. intraoperatively, large fluid - filled bursae were noted in the intermetatarsal spaces bilaterally, with nerves lying underneath and tethered to bursae. case 3. a 38-year - old lady presented to the foot and ankle outpatient clinic with an 18-month history of pain over the plantar aspect of the third and fourth metatarsal heads. a clinical diagnosis of synovitis of the third and fourth metatarsophalangeal joints was made, and an ultrasound scan was organized. her blood work was normal, with an erythrocyte sedimentation rate (esr) of 2, c - reactive protein (crp) of 3, and a normal white cell count. the ultrasound scan revealed a hypoechoic mass in the third intermetatarsal space suggestive of a morton s neuroma. histology revealed the presence of morton s neuroma and a rheumatoid nodule in the specimen. she was tested positive on ra assay and was referred to rheumatologists for further management. between january 2004 and october 2008, 101 cases of morton s neuroma were operated on by two orthopedic surgeons with a special interest in foot and ankle surgery. among these, histology revealed rheumatoid nodules in five cases. morton s neuroma can be a local manifestation of a generalized disease such as rheumatoid arthritis. the incidence of interdigital neuroma in rheumatoid arthritis patients was reported to be 1 in 520, with a female preponderance. rheumatoid synovitis and nodules producing symptoms mimicking morton s neuroma have been reported in the literature, but most of the cases were found in previously diagnosed rheumatoid arthritis patients. awerbuch et al. presented a series of 50 patients with morton s neuroma, and found that 24 % had rheumatoid arthritis at the time of diagnosis, 16 % developed rheumatoid arthritis over a follow - up period ranging from two months to 15 years, and an inflamed bursa in the intermetatarsal space was the first sign of rheumatoid arthritis in 8 %. in this series, out of 20 patients, a histological diagnosis of rheumatoid arthritis without any evidence of morton s neuroma was made for ten patients, of which seven were asymptomatic at presentation but three developed symptoms with four years of the initial presentation. suggested seeking histological evidence of rheumatoid disease in all tissues excised in the surgical treatment of morton s neuroma, as they believed that rheumatoid disease is the basic etiology in a significant number of patients. in the senior author s view, this is a rather interesting and unusual finding, and may be due to selection bias. in a series by vainio, performing prophylactic metatarsophalangeal synovectomy in rheumatoid arthritis patients ed in a decrease in morton s neuroma operations. the distribution of the location of the neuroma in rheumatoid arthritis patients in this series was almost the same (in interspaces ii iii and iii iv), in contrast to nonrheumatoid patients, in whom the iii iv interspace is more common. vainio also suggested that metatarsalgia associated with rheumatoid arthritis should preferably be called morton s metatarsalgia, as the nerve changes in this group of patients are not typical of neuroma. in our case series, out of three patients, one had a history of rheumatoid arthritis, and two patients were later diagnosed with seropositive rheumatoid arthritis after rheumatoid nodules were identified on a histological examination of the excised tissue. our case series demonstrates that a rheumatoid nodule presents with symptoms similar to morton s neuroma. however, the number of cases in our series is too small to suggest that patients with ii iii intermetatarsal neuralgia may be rheumatoid in origin, although the literature seems to suggest that view. we therefore suggest that a nodule of rheumatoid origin should be considered in the differential diagnosis of morton s neuroma, not only in patients with a known history of rheumatoid arthritis but also in patients who present with symptoms of morton s neuroma and have atypical intraoperative findings. the presence of a rheumatoid nodule in the foot can be the first manifestation of rheumatoid arthritis, and requires prompt referral to the rheumatology team.	among 101 feet that presented with symptoms and signs similar to morton s neuroma, intermetatarsal rheumatoid nodules were found in five feet (three patients). two patients had bilateral involvement. histology of the excised tissue showed the presence of a rheumatoid nodule and morton s neuroma in four feet and a rheumatoid nodule with unremarkable nerve bundles in one. a rheumatoid nodule can coexist with morton s neuroma, as seen in our patients, and the presentation is often similar to that of a morton s neuroma. our patients were rendered asymptomatic with surgical treatment and went on to have appropriate management of rheumatoid arthritis. rheumatoid nodule should be considered in the differential diagnosis of morton s neuroma in not only rheumatoid arthritis patients but also asymptomatic patients who have never been tested for rheumatoid antibodies.
pfeiffer syndrome is a rare autosomal dominant disorder, characterized by premature fusion of cranial sutures that prevents the skull from growing normally and affects the shape of the head and face, ing in brachycephaly, hypoplastic maxilla, shallow orbits, proptosis, and exophthalmos, and accompanied with broad and deviated thumbs and big toes1 ). mutations in the fibroblast growth factor receptor (fgfr1) gene2 ) or fgfr2 gene3 ) cause pfeiffer syndrome. here we describe a case of pfeiffer syndrome with a novel c833_834gc > tg mutation (encoding cys278leu) in the fgfr2 gene, with an everted coccyx. his mother's obstetric history was gravida 1, para 0, and was unremarkable. his parents were both korean, phenotypically normal, and did not have any history of consanguinity. he weighed 3,360 g (75 - 90 percentile), was 50 cm long (75 percentile), and had a head circumference of 34 cm (75 - 90 percentile). he had brachycephaly, maxillary hypoplasia, exophthalmos, proptosis, low - set ears, preauricular skin tags, atresia of the external auditory canal, high arched palate, radially deviated broad thumbs and medially deviated big toes, ankylosed elbows, and a mass on the coccygeal area (fig . three dimensional skull ct revealed closure of the bilateral coronal sutures and left lambdoidal suture, midface hypoplasia, and shallow orbits ( fig . a lateral spine view noted the disappearance of normal curvature of spine ; spinal magnetic resonance imaging showed an outwardly everted coccyx ( fig . 2c), and plain radiographs of his hands and feet showed widening of both 1st phalangeal bones, metacarpal bones, and metatarsal bones. with informed consent, pcr was performed for exon 7 and exon 8 to analyze major mutation of fgfr2 gene, and revealed a novel amino acid substitution of leucine coded by tks, for cysteine coded by tgc, at codon 833_834 (cys278leu) of the fgfr2 gene (fig . pfeiffer syndrome was first described by pfeiffer in 1964, and 60 cases have been reported . it affects about 1 in 100,000 individuals1), but is more rare in the asian population, with only a few cases reported in korea5 - 8 ). pfeiffer syndrome involves the cranial bones and thumbs and great toes, which are broad and bend away from the other digits, occasionally accompanied by an ankylosed elbow or radiohumeral synostosis9 ). type 1 consists of brachycephaly, a hypoplastic mid - face, and finger and toe abnormalities with normal to near - normal intelligence (classic type). type 2 is characterized by a cloverleaf - shaped head, severe proptosis, and central nervous system involvement caused by more extensive fusion of the skull bones, with potential elbow ankylosis or synostosis. type 3 is similar to type 2 but without a cloverleaf - shaped head. both types 2 and 3 are more severe and have poor neurodevelopmental outcomes. the patient in this case report was pfeiffer syndrome type 1, the classic type. cranial sutures normally close in a synchronized manner after birth, allowing the skull to achieve normal size and shape. when this normal development is disrupted, premature cranial fusion or delayed cranial closure occurs. mutations in fgfr1, 2, or 3 can affect craniofacial and skeletal development10 ). more than 60 mutations in fgfr, a majority of which occur in fgfr2, are associated with craniosynostosis syndrome such as antley - bixler syndrome, apert syndrome, bearse - stevenson syndrome, crouzon syndrome, muenke syndrome, and pfeiffer syndrom11 ). mutations in the fgfr1 at chromosome 8p11.2-p12 were only detected in pfeiffer syndrome type 22, 12 ). mutations in the fgfr2 at chromosome 10q25-q26 were reported in all three subtypes13 ), including 1036t->c3 ) and 1037g->a which were also detected in crouzon syndrome14 ). we report a novel c833_834gc > tg mutation (encoding cys278leu) in pfeiffer syndrome. spinal anomalies such as the cervical spine fusion are rare in pfeiffer syndrome15 ), but coccygeal anomaly occurred in one other case in korea7 ). 90% of alp in young infants is produced from bone, and reflects osteoblastic activity, bone turnover, and rapid bone growth, and positively correlates with bone mineral accretion. alp is normal at birth and rises gradually in the first 2 - 3 weeks of life16 ). osteoblast progenitors in bone marrow were significantly increased in fgfr2cc342y/+ heterozygote mice, which had a phenotypically shortened face, protruding eyes, and premature fusion of cranial sutures17 ). patients with apert syndrome with the s252w mutation in the fgfr2 gene enhanced osteoblastic marker gene expression and alp activity18 ). increased alp from birth might reflect prenatally increased osteoblastic marker gene expression ing from mutations in fgfr2 gene, but further study is needed. the prognosis of pfeiffer syndrome depends on accompanying anomalies, and multiple surgeries are needed to release the prematurely closed sutures19, 20 ). in , this report describes a case of pfeiffer syndrome with a novel c833_834gc > tg mutation (encoding cys278leu) in the fgfr2 gene, with an everted coccyx resembling a tail and increased alp activity.	pfeiffer syndrome is a rare autosomal dominant disorder characterized by coronal craniosynostosis, brachycephaly, mid - facial hypoplasia, and broad and deviated thumbs and great toes. pfeiffer syndrome occurs in approximately 1:100,000 live births. clinical manifestations and molecular genetic testing are important to confirm the diagnosis. mutations of the fibroblast growth factor receptor 1 (fgfr1) gene or fgfr2 gene can cause pfeiffer syndrome. here, we describe a case of pfeiffer syndrome with a novel c833_834gc > tg mutation (encoding cys278leu) in the fgfr2 gene associated with a coccygeal anomaly, which is rare in pfeiffer syndrome.
in vitro maturation (ivm) is a technique described in the 90s primarily as alternative technique to conventional stimulation for patients with polycystic ovaries syndrome (pcos) because the decreased risk of ovary hyperstimulation. however, ivm has additional benefits such as a reduced cost compared to regular in vitro fertilization (ivf) procedures and represents a friendly approach for female patients. recent reviews of the literature confirmed that ivm is inferior to conventional stimulation in terms of pregnancy and take - home baby rates. this is why ivm is considered as an experimental procedure according to the american society for reproductive medicine and its application is limited in the united states. despite ivm is routinely applied in some european countries and it is a flagship for the mild approaches movement. one potential application is the use of ivm as a less expensive alternative to stimulated ivf in case of severe male infertility. the experience of using ivm with non - ejaculated sperm is limited, case reports of pregnancies are published from sperm obtained by testicular biopsy, testicular fine - needle aspiration, percutaneous epididymal sperm extraction, and microtesticular sperm extraction. nevertheless, some of the reports used a low stimulation including low doses of follicle - stimulating hormone (fsh), menotropins, or clomiphene citrate that constitute mild stimulation cycles and not ivm. in our private center in leon mexico, we started to offer ivm routinely in 2007. in this report , we describe a case where human chorionic gonadotropin (hcg)-primed ivm was performed for a male infertility indication using a testicular sperm sample from a patient with nonobstructive azoospermia. a 28-year - old male with nonobstructive azoospermia with normal karyotype, no y chromosome microdeletions, or common mutations in cftr gene and fsh 12 mui / ml, luteinizing hormone (lh) 9.0 mui / ml, and testosterone 300 ng / ml. female partner 34 years with primary infertility, normal menstrual cycles, basal fsh 7.4 mui / ml, lh 2.5 mui / l, e2: 22.5/l, testosterone 0.2 ng / ml, 14 follicles at antral follicle count, and endometrial thickness of 9 mm. taking into account that no female factors were detected, and the prognosis was good, we decided offer ivm as treatment option to the couple. the couple had chosen ivm for their desire of a lower cost in ivf procedures. a transvaginal ultrasound scan on day 3 revealed ten antral follicles. on day 7 of the cycle, a follow - up of follicle growth when one follicle reached 1214 mm and endometrial thickness was 7 mm, the patient received a single intramuscular injection of 250 g of recombinant hcg (ovidrel, merck serono, switzerland). thirty - six hour after hcg injection, oocyte retrieval was performed under patient intravenous (iv) sedation using a 17-gauge 20 single lumen needle (kitazato oocyte pickup needle). fourteen cumulus - oocytes complexes were retrieved, cumulus cells were removed using hyaluronidase, no mature (metaphase ii) oocytes were found. immature oocytes (germinal vesicle or metaphase i oocytes) were cultured in ivm media (sage) supplemented with 0.75 iu / ml of recombinant fsh (rfsh) and 0.75 iu / ml of recombinant lh. testicular biopsy was performed under iv sedation 1 day after oocyte capture, fresh spermatozoa obtained by testicular biopsy. the testicular tissue was minced into small pieces using two scalpels in human tubal fluid (htf)-hepes, the supernatant was centrifuged at resuspended in fresh htf until intracytoplasmic sperm injection (icsi). injected oocytes were transferred to global medium (lifeglobal, usa) incubated at 37c in 6% co2. embryos were culture in 20 l droplets of global medium at 37c in 6% co2, three cleavage embryos in day 2 were transferred. a singleton live birth at 37 weeks was obtained with a weight of 2700 g and length of 50 cm, and apgar score was 9/9/9. a 28-year - old male with nonobstructive azoospermia with normal karyotype, no y chromosome microdeletions, or common mutations in cftr gene and fsh 12 mui / ml, luteinizing hormone (lh) 9.0 mui / ml, and testosterone 300 ng / ml. female partner 34 years with primary infertility, normal menstrual cycles, basal fsh 7.4 mui / ml, lh 2.5 mui / l, e2: 22.5/l, testosterone 0.2 ng / ml, 14 follicles at antral follicle count, and endometrial thickness of 9 mm. taking into account that no female factors were detected, and the prognosis was good, we decided offer ivm as treatment option to the couple. the couple had chosen ivm for their desire of a lower cost in ivf procedures. a transvaginal ultrasound scan on day 3 revealed ten antral follicles. on day 7 of the cycle, a follow - up of follicle growth when one follicle reached 1214 mm and endometrial thickness was 7 mm, the patient received a single intramuscular injection of 250 g of recombinant hcg (ovidrel, merck serono, switzerland). thirty - six hour after hcg injection, oocyte retrieval was performed under patient intravenous (iv) sedation using a 17-gauge 20 single lumen needle (kitazato oocyte pickup needle). fourteen cumulus - oocytes complexes were retrieved, cumulus cells were removed using hyaluronidase, no mature (metaphase ii) oocytes were found. immature oocytes (germinal vesicle or metaphase i oocytes) were cultured in ivm media (sage) supplemented with 0.75 iu / ml of recombinant fsh (rfsh) and 0.75 iu / ml of recombinant lh. testicular biopsy was performed under iv sedation 1 day after oocyte capture, fresh spermatozoa obtained by testicular biopsy. the testicular tissue was minced into small pieces using two scalpels in human tubal fluid (htf)-hepes, the supernatant was centrifuged at resuspended in fresh htf until intracytoplasmic sperm injection (icsi). injected oocytes were transferred to global medium (lifeglobal, usa) incubated at 37c in 6% co2. embryos were culture in 20 l droplets of global medium at 37c in 6% co2, three cleavage embryos in day 2 were transferred. a singleton live birth at 37 weeks was obtained with a weight of 2700 g and length of 50 cm, and apgar score was 9/9/9. in this report, a live birth from an hcg - primed ivm using testicular sperm is reported, this as part of routine offer of ivm to selected patients in our clinic in mexico. because pregnancy rates are lower that for conventional ivf, it is difficult to introduce this procedure in private practice where patients cover completely with the cost of assisted reproduction procedures. however, ivm offers several advantages for the case of male infertility such as a friendly procedure for a female partner, the absence of risk related with ovary stimulation in otherwise healthy women, and the reduction in costs of treatment. the published experience of ivm using testicular sperm is quite heterogeneous, and in some reports, the stimulation is more intense than simple hcg priming, contradicting the main benefit of ivm. on the other hand, apparently ivm in normo - ovulatory women and pregnancy rates the biggest experience of icsi with testis biopsies from non - obstructive azoospermia in normo - ovulatory women using rfsh + hcg indicated that pregnancy rates are similar. however, in pcos patients using only hcg lower fertilization and pregnancy rates are reported. in the present case, pregnancy was obtained at the first ivm attempt; but further research is required to understand factors impacting the of ivm, such as hormonal priming, ovary function, and the etiology of azoospermia when ivm oocytes are fertilized using testicular spermatozoa. summarizing ivm may be an option for normo - ovulatory women in cases of male infertility that requires icsi with sperm obtained from testicular biopsies. repa is supported by conacyt and elphinstone scholarships and a fellowship from ibsa foundation. repa is supported by conacyt and elphinstone scholarships and a fellowship from ibsa foundation.	in this report, we present a case of in vitro maturation (ivm) with surgical retrieved testicular sperm in a normo - ovulatory female. human chorionic gonadotropin - primed ivm, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. fourteen cumulus - oocyte complexes were obtained in germinal vesicle or metaphase i stage, eight oocytes reached metaphase ii, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. a single healthy newborn was obtained. our suggest that ivm may be an alternative for in vitro fertilization in normo - ovulatory women even if surgical retrieval of sperm is needed. further research is required to depict contributing factors to the success of ivm in indications different from polycystic ovaries syndrome and the role of male gamete.