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the available evidence suggests that a sizable proportion of undergraduate health sciences students lack good study skills and habits, and that introducing a study skills program could significantly improve students confidence and academic achievement. the undergraduate pharmacy degree programs of the university of zambia comprise a regular five - year bachelor of pharmacy (b. pharm.) degree that primarily admits young high school graduates who have little or no clinical experience, and a parallel program that mostly enrols mature students with a diploma in pharmacy and clinical experience, or similar qualifications. candidates with diploma in pharmacy or first degree qualifications in relevant fields could be admitted to advanced standing in the b. pharm programs. in light of the different student demographics and differences in academic achievement that have been observed between these two programs, this study was designed to analyse the study skills of undergraduate pharmacy students of the school of medicine of the university of zambia, with the goal of identifying areas of deficiency in the domains of time management, note taking, test / examination preparation, motivation, concentration / memory, information processing, reading, and writing skills. the results of this study may help in the design of tailor - made study skills intervention courses to assist individual students. the study population consisted of undergraduate bachelor of pharmacy students in years 3, 4, and 5 of both the regular and parallel programs of the school of medicine, university of zambia, lusaka, zambia. since the parallel program has no year 1 and year 2 students, these levels were omitted from the study. a sample size of 66 was calculated according to yamane formulae based on a total student enrolment of 243 in both programs for the years studied. a demographic questionnaire was used to gather demographic information, while hard copies of the study skills assessment questionnaire (ssaq) developed by houston university counselling services was used to collect data on study skills from the eligible students using a simple random sampling technique. the ssaq instrument contains 64 items grouped into the following eight domains : time management / procrastination ; concentration / memory ; study aids / note taking ; test strategies / test anxiety ; organizing / processing information ; motivation / attitude ; reading / selecting the main idea ; and writing. this instrument uses a four - point likert scale (always, usually, sometimes, and never) to gather respondents views. participants responses were rated as follows : 4, always ; 3, usually ; 2, sometimes ; and 1, never. thus, each domain has a maximum of 32 points and a minimum of 8 points. participants study skills were rated using the following rubric : 1) poor study skills_less than 50% in each domain (24 points) or more than 192 points overall. each consenting student was allowed sufficient time to independently complete the questionnaire and to return it at his / her convenience. the completed and returned questionnaires were scored as described above, and results were analysed using spss version 21 (ibm corp., armonk the mean scores were compared using the student s t - test ; p - values < 0.05 were considered to indicate statistical significance. the reliability of the ssaq among these participants was determined by calculating cronbach s alpha coefficient for internal consistency. ethical approval for this study was obtained from the university of zambia school of medicine research ethics committee (unzasomrec ref # : irb00001131 of ior g0000774). the ssaq results showed that overall, the study skills of the participants were moderate, with a total mean score of 179.7 (range, 138 - 223). forty - five participants (67.2%) had moderate study skills, and 22 participants (32.8%) had good study skills. tables 1 and 2 showed that scores in the subscales varied from moderate to good in the subscales of information processing and test strategies for both regular and parallel participants. although students in the parallel program demonstrated significantly better study skills (mean, 185.414.5) than students in the regular program (mean, 17525.4) (p<0.05), the overall rating for both groups was moderate. statistically significant differences in the ratings within the subscales of time management (p=0.003) and writing skills (p=0.004) were found between the two programs (table 3). the ssaq scores did not significantly vary according to gender, age, year of study, marital status, residence status, or financial support. the results of this study indicated that a majority of the students in both the regular and parallel programs had only moderate study skills, a finding that is consistent with reports from other similar studies in the field of medical and health sciences education. students in the parallel program appear to have had better study skills than regular students. although this discrepancy did not affect the overall rating of the two groups, it may be due to the fact that most of the parallel participants were employed salary earners and more mature, both chronologically and in terms of work experience, than the regular participants. however, the study failed to demonstrate any effect of age or financial support on study skills. therefore, one can only speculate that clinical experience may have contributed to the difference. some studies have found that gender differences affected the academic achievement of both male and female secondary school and undergraduate students, and that such differences may be discipline - specific. this study did not find any differences in the study skills of male and female pharmacy students, nor did it show any significant effects of residence status, marital status, or financial support on study skills. since these findings differed from those of didarloo and khalkhali s study of iranian students of health science programs, it seems likely that other factors were involved in the outcomes of their study. first, only pharmacy students were studied, so the results can only reasonably be applied to similar programs. additionally, the study setting was localised and only a limited number of participants were enrolled. a cross - sectional design implies that internal validity is difficult to assess, and since the answers to the questionnaire were self - reported, it would also be difficult to estimate reporting bias. despite these limitations, the findings of this study significantly contribute to the literature on this topic, and the areas of weakness that were identified may become focal points for designing intervention program to improve study skills in zambia and countries with similar cultures. in conclusion, since time management and writing skills were significantly lower in the regular program pharmacy students than those in the parallel program students, the more intensive training should be done to regular program students to overcome those shortness. | it aimed to compare the study skills of two groups of undergraduate pharmacy students in the school of medicine, university of zambia using the study skills assessment questionnaire (ssaq), with the goal of analysing students study skills and identifying factors that affect study skills. a questionnaire was distributed to 67 participants from both programs using stratified random sampling. completed questionnaires were rated according to participants study skill. the total scores and scores within subscales were analysed and compared quantitatively. questionnaires were distributed to 37 students in the regular program, and to 30 students in the parallel program. the response rate was 100%. students had moderate to good study skills : 22 respondents (32.8%) showed good study skills, while 45 respondents (67.2%) were found to have moderate study skills. students in the parallel program demonstrated significantly better study skills (mean ssaq score, 185.414.5), particularly in time management and writing, than the students in the regular program (mean ssaq score 17525.4 ; p<0.05). no significant differences were found according to age, gender, residential or marital status, or level of study. the students in the parallel program had better time management and writing skills, probably due to their prior work experience. the more intensive training to students in regular program is needed in improving time management and writing skills. |
establish a database of ent, audiology, and speech therapy services in sub - saharan africa.create an awareness in the first world about the status of these services.plan and promote effective, targeted support for these services.gather data to lobby african governments, donor countries, and aid organizations.determine the need for a developing world forum for ent surgery, audiology, and speech therapy. establish a database of ent, audiology, and speech therapy services in sub - saharan africa. plan and promote effective, targeted support for these services. gather data to lobby african governments, donor countries, and aid organizations. determine the need for a developing world forum for ent surgery, audiology, and speech therapy. establish a database of ent, audiology, and speech therapy services in sub - saharan africa.create an awareness in the first world about the status of these services.plan and promote effective, targeted support for these services.gather data to lobby african governments, donor countries, and aid organizations.determine the need for a developing world forum for ent surgery, audiology, and speech therapy. establish a database of ent, audiology, and speech therapy services in sub - saharan africa. plan and promote effective, targeted support for these services. gather data to lobby african governments, donor countries, and aid organizations. determine the need for a developing world forum for ent surgery, audiology, and speech therapy. a questionnaire was distributed by email to an ad hoc group of ent surgeons and audiologists in sub - saharan african countries in which there were known to be ent services. participants were traced through personal contacts and the pan african federation of oto - rhino - laryngological societies (pafos) database, and proved to be challenging or impossible in some countries. questions were asked about the availability of ent, audiology, and speech therapy services and equipment (nil / poor / good / excellent), about training programs for ent surgeons, audiologists, and speech therapists, about the availability of services in rural areas, and about their opinions about how to improve the situation. the number of ent surgeons, audiologists, and speech therapists per country, and compares it to the uk. the uk has the poorest ratio of ent surgeons to population in europe, and is intending to further improve this ratio to 1/75,000 (4). some african countries have no ent, audiology or speech therapy services at all. comparisons of ent surgeons, audiologists, and speech therapists/100 000 people, with the uk. comparisons of ent surgeons, audiologists, and speech therapists/100,000 people, with the uk table 2 presents a summary of ent, audiology, and speech therapy training programs. many countries have no training programs, especially for audiology and speech therapy. if one applies the current ratios of ent surgeons, audiologists, and speech therapists in the uk to the total population (521.7 m) of the countries surveyed, then the shortage of personnel in these 18 countries is as follows : ent : 5,251496 = 4,755 ; audiology : 21,381525 = 20,856 ; speech therapy : 85,5251,182 = 84,343. in all the countries surveyed, the overwhelming majority of people depend on the state health services. table 3 6 present summaries of the levels of clinical services in the state sector in the countries surveyed. countries with access to ent services outside major cities table 4 highlights the poor access to simple, routine audiology, hearing aids and the most basic hearing restoration surgery such as ventilation tubes and tympanoplasties. countries with access to hearing - related services table 5 demonstrates that there is very poor access even for relatively low - cost oncologic surgery, and that laryngectomees are not offered the opportunity of post - laryngectomy voice restoration. countries with head and neck oncologic surgery table 6 presents the poor access that patients have to modern sinus and rhinologic surgery, and the reliance that is still placed on open as opposed to endoscopic sinus surgery. in some countries, countries with sinus and rhinologic surgery table 7 highlights the limitations to delivering a modern ent service due to a lack of equipment. in engaging for health (2006), the world health organization released a plan for global health for the next decade, and proposed the following major policy thrusts (5):investing in health to reduce poverty.building individual and global health security.promoting universal coverage, gender equality, and health - related human rights.tracking the determinants of health.strengthening health systems and equitable access.harnessing knowledge, science, and technology.strengthening governance, leadership, and accountability.there has been an unprecedented global response in the past 10 years, with large increases in funding being directed at global health improvement. recognition of the dire circumstances in africa has placed the subcontinent at the forefront of such efforts through initiatives around aids / hiv, malaria, and tuberculosis. these efforts are however uncoordinated, and the disconnection between vast donor funding and lack of development of health systems is lamentable (6), for in most cases, the financial aid targets particular diseases, such as hiv / aids and malaria ; interventions, such as arv programs or bed nets ; or a list of challenges which are unconnected to strengthening of health systems and indigenous institutions which have the potential for making a sustainable impact on health. a further reality is that the design of several of these initiatives is dominated by the wealthy world, with limited input by those most affected, leading to programs which may not serve short- or, indeed, long - term health development needs. efforts to address specific health challenges lie in the realm of innovative, targeted interventions, based on careful analysis of the problem, and designed by those who have practical experience of, and insight into, appropriate and effective solutions. investing in health to reduce poverty. building individual and global health security. promoting universal coverage, gender equality, and health - related human rights. tracking the determinants of health. strengthening health systems and equitable access. harnessing knowledge, science, and technology. strengthening governance, leadership, and accountability. it is common knowledge that all the countries surveyed in this study have an enormous ent - related bod. this audit presents a disturbing picture of under - resourced, understaffed, and outdated ent, audiology, and speech therapy services in africa. patients still die from simple ent infections and curable cancers, and do not have access to the most basic hearing tests or hearing rehabilitation. the audit also highlights the lack of training facilities, critical to improving the shortage of qualified staff. while we acknowledge the great importance of non - specialist ent, audiology and speech services provided by nurses and other medical personnel, it is only through the leadership, training, research, and specialized clinical services provided by a critical mass of resident, well - trained specialists in these fields, that these services will become self - sustaining and countries may one day become independent of foreign medical assistance. what is clearly needed is a comprehensive, multipronged, multinational program to improve the quality of, and access to, ent, audiology, and speech therapy services in africa. such a program has to be carefully planned to take into consideration political, infrastructural, and staffing realities, and will have to be planned jointly with local medical practitioners, academics, academic institutions, governments, and international donor and health organizations in order to be effective, cost - effective, and sustainable. vorobiof and abratt reported that 21 african countries do not have radiation therapy facilities (7). hence, reliance in these countries has to be placed on a purely surgical approach to cancers of the head and neck. our study also highlights the lack of modern equipment and reliance on outdated surgical techniques. for these reasons, ent surgeons in poorer countries are compelled to practice lower technology, lower cost type surgery compared to their counterparts in the developed world. this chiasm between high technology, high - cost medical practice in the first world versus lower technology, lower cost medical practice in the developing world, is rapidly increasing, and will continue to accelerate. it is unlikely that the poor who live in the developing world will ever have ready access to new, high technology, high - cost medical advances. with changing medical practice in the first world due to advances such as imaging, chemotherapy, irradiation, neuronavigation, robotics, minimally invasive surgery, interventional angiography, genetics, and pharmaceuticals etc, younger generations of first world medical practitioners have become unfamiliar with medical and surgical care appropriate to the developing world, even though it might have been the standard of care in the first world as recently as 10 years ago. diseases such as leprosy, tuberculosis, hiv, malaria, rheumatic fever, complicated mastoiditis and sinusitis, etc. are essentially diseases of the developing world and are rarely encountered in the first world. consequently, medical practitioners in developing world countries can no longer turn to centers of excellence in the first world for guidance about sound, lower technology, lower cost healthcare. it is therefore crucial that we acknowledge the existence of two tracks of medical practice i.e. a higher technology, higher cost first world medical practice versus a lower technology, lower cost developing world medical practice. developing world medicine and surgery should be recognized and fostered as very important fields of clinical practice, teaching, and research that cater for > 50% of the world 's people. centers of excellence in the developing world need to be fostered and need to take the lead in terms of teaching, training, and research in developing world medical practice. a developing world forum for ent surgery, audiology, and speech therapy, directed and driven by africa and the developing world, but supported by the first world, should be established, and should develop a comprehensive intervention to turn around the severe shortage of clinical services and expertise in africa and the developing world. a key element would be to establish and/or strengthen centers of clinical, teaching, and research excellence in the developing world. teaching institutions in the first, but in particular in the developing world should provide specialist training and clinical fellowships to under - resourced countries, with an emphasis on staff training at teaching hospitals. but the answers to the challenge of africa 's health and that of global health inequity also lie with global health policies and practices, including developing new norms and standards for health care which serve the interests of the global community, and are based on the current realities of global health, and which strive to advance efforts to promote health equity. | backgroundburden of disease (bod) is greatest in resource - starved regions such as africa. even though hearing disability ranks third on the list of non - fatal disabling conditions in low- and middle - income countries, ear, nose, and throat (ent) disorders are not specifically coded for within the framework governing global bod estimates, and in discussions about health challenges, non - communicable diseases receive scant attention. implementing cost - effective interventions to address conditions largely neglected by global estimates of bod such as hearing loss are important contributors to health and economic development.objectivesestablish a database of ent, audiology, and speech therapy services in sub - saharan africa ; create awareness about the status of these services ; propose effective intervention ; gather data to lobby african governments, donor countries, and aid organizations ; determine need for developing world forum for ent, audiology, and speech therapy services.designsurvey of ent, audiology, and speech therapy services and training in 18 sub - saharan africa countries.resultsthere is an alarming paucity of services and training opportunities, and there is a large gap between higher technology, expensive health care in high - income countries and lower technology, low - cost practice in low - income countries.conclusionslower technology and lower cost developing world medical practice should be recognized and fostered as a field of medical practice, teaching, and research. developing world centers of excellence must be fostered to take a lead in teaching, training, and research. a developing world forum for ent surgery, audiology, and speech therapy, directed and driven by africa and the developing world, supported by the first world, should be established, to develop a comprehensive intervention to turn around the severe shortage of services and expertise in the developing world. global health policies and practices should include new norms and standards which serve the interests of the global community, and are based on current realities of global health. |
the online version of this article (doi:10.1007/s12576 - 014 - 0350 - 7) contains supplementary material, which is available to authorized users. after muscle injury or change of use, repair starts with an initial acute inflammatory response (typically from 0 to 48 h) followed by a repair and regeneration phase, typically lasting from 48 h to 10 days. ccaat / enhancer - binding protein - beta (c / ebp-, encoded for by cebpb mrna) is a pleiotropic transcription factor that regulates several immune cell functions. c / ebp- has been shown to be central to post - injury muscle repair since cebpb expression is markedly increased during macrophage differentiation towards an anti - inflammatory (m2) repair phenotype that governs the integration of newly formed myoblasts into regenerating muscle tissue. in a murine model of repair after a muscle injury, deletion of camp response element - binding protein (creb) binding sites in the c / ebp- promoter (thereby reducing cebpb expression) in macrophages prevented the transition to an m2 regrowth and repair phenotype and inhibited muscle regeneration, resulting in fibrosis and muscle fibre loss, compromising muscle function. satellite progenitor cells proliferate as myoblasts and fuse into myotubes at the site of damage, where they differentiate into fully regenerated muscle fibres. this sequence is mediated by a cascade of myogenic regulatory factors, which are subject to signalling from resident or infiltrating macrophages, and systemic factors originating in t lymphocytes. the proliferative phase of myoblasts is promoted by pro - inflammatory (m1) macrophages. we have previously demonstrated that the expression of the cebpb gene in blood was the most strongly transcript associated with grip strength in 698 mostly older people in the inchianti study in a transcriptome - wide analysis of human circulating blood leukocytes. a potential mechanism explaining the strength - blood cebpb association may be the role of c / ebp- in muscle regeneration following lifelong muscle stress and injury. contraction - induced damage to muscle is common in everyday life, especially during lengthening contractions, and decreased repair after such damage has been linked to muscle strength, particularly in old age. in the current study, we aimed to establish whether exercise - induced muscle damage (eimd) in humans results in increases in cebpb expression in blood, consistent with cebpb s role in post - injury muscle repair, which typically shows sharply increasing numbers of m2 macrophages at the injury site by 48 h after injury. we also tested whether blood cebpb and related expression and plasma cytokines exhibit changes consistent with the muscle regeneration program in human volunteers following eimd. here we present evidence that cebpb expression is altered with eimd, and cebpb expression is associated with changes in markers characteristic of inflammatory polarisation. sixteen healthy and physically active males [age, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\bar{x}$$\end{document}x (sd) = 43.1 (18.5) years ] undertook three sets of eccentric loading, 80 % 1rm [\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\bar{x}$$\end{document}x (sd) = 25.4 (5.1) kg, each set to task failure, separated by 2 min recovery ] of the elbow flexors. the exercise protocol we used is consistent with inflammation in the biceps brachii and brachialis muscles ; maximal voluntary contraction (mvc) was assessed at 60 of flexion to allow potentially a greater contribution of damaged brachialis muscle than at 90. mvc was assessed (peak torque, 5 3 s isometric contractions/3 s rest) prior to exercise and during recovery on an isokinetic dynamometer (biodex system 3, biodex medical systems, ny, usa). this project was approved by the ethics committee of the department of sport and health sciences, university of exeter. six millilitres of venous blood was collected into a lithium heparin vacutainer (becton - dickinson, usa) and 2.5 ml into a paxgene blood tube (preanalytix gmbh, hombrechtikon, switzerland) prior to and 1, 2, 4 and 7 days post exercise. these time points were chosen to coincide with the m1/m2 phenotype muscle regeneration model described by tidball and villalta. the lithium heparin tube was centrifuged for 10 min at 4,000 rpm ; the supernatant was harvested and analysed immediately for creatine kinase (ck) activity. the paxgene tubes were inverted ten times, incubated at room temperature for ~3 h and stored without separation at 80 c. creatine kinase activity was measured at 37 c using the colorimetric technique described by szasz. absorbance was recorded at 340 nm on a jenway 6300 spectrophotometer (bibby scientific limited, staffordshire, uk) and enzymatic activity quantified using a linear regression equation derived from a 2-point calibration of known standards. th1 and th2 cytokines were quantified in plasma by electrochemiluminescence multiplex assay (th1/th2 10-plex, meso scale discovery, rockville, md, usa). the panel of cytokines measured ifn-, il-1, il-10, il-12 p70, il-13, il-2, il-4, il-5, il-8 and tnf-. for rna extraction the paxgene blood rna kit (qiagen ltd. mrna was reverse transcripted to complimentary dna using superscript vilo (applied biosystems, usa) and amplified. cdna was loaded onto custom taqman low density array (tlda) cards on the abi prism 7900ht platform (life technologies, carlsbad, ca, usa). genes included in the study were quantified relative to an endogenous control gene (ppia). normalisation to ppia precludes the need to correct for leucocytosis and has been well validated for its use as an appropriate housekeeping gene. the quantitative value obtained from taqman real - time rt - pcr is a threshold cycle (ct). the fold differences between time conditions were calculated from the relativised ct values (ct gene x - ct housekeeping gene, ppia) according to the comparative ct method. genes quantified included seven associated with the classical inflammatory t1 phenotype and 7 (including cebpb) with an were compared by paired t - tests for differences at a priori time points relevant to expected outcomes, in particular the rise from baseline to the mean of the subsequent 2 days, during which time m2 polarisation is expected to be induced. relationships between mrna expression and circulating cytokine concentrations were explored by pearson s correlation coefficients. the relationships between blood transcript expression changes and markers of damage severity (loss of function and ck activity) were assessed using robust regression analysis. statistical significance was accepted at p 10,000 peak plasma creatine kinase activity (c, p = 0.006). d shows the participants ' change in cebpb expression (day 2 minus baseline) and peak ck levels time - course and relationship with cebpb expression. isometric strength (nm torque, sem) decreases from baseline (a, p 10,000 peak plasma creatine kinase activity (c, p = 0.006). d shows the participants ' change in cebpb expression (day 2 minus baseline) and peak ck levels overall cebpb gene expression change after exercise (day 2, baseline) did not reach significance (p > 0.05). however in the subset whose peak ck activity exceeded 10,000 u / l, cebpb expression did increase from baseline to 2 days post exercise (t(1,8) = 3.72, p = 0.006, fig. the same change in cebpb expression was observed in a subset of ten subjects whose 24 h post exercise mvc decreased > 17 % (t(1,9) = 2.57, p = 0.03). the repeated measures of cebpb expression over the 7 days did not significantly vary over time (anova p > 0.05) ; only the day 2 change from baseline (in the high ck group) was significant (anova p = 0.014). supplementary table 4 contains the cebpb expression data at each time point and peak ck measure. there were no overall significant changes post exercise in the selected plasma cytokine concentration (table s2) or other transcripts (table s3), and none of these were significant in the high ck subgroup. supporting the concept that cebpb changes are related to t lymphocyte or macrophage differentiation, cebpb expression correlated with il-1 (r = 0.701, p = 4.14 10, fig. 2a), arg1 (r = 0.457, p = 0.004, fig. 2b) and stat1 (r = 0.402, p = 0.008) expression. we also observed positive correlations between cebpb and plasma inf- (r = 0.369, p = 0.013, fig. 2c) and il4 concentration change (r = 0.29, p = 0.031, fig. 2c). = [day 1 + day 2]/2 baseline. after negative changes in cebpb expression had been excluded from the analysis, all the above associations remained significant except for interleukin 4.fig. following exercise - induced muscle damage, cebpb blood transcript expression change shows positive correlations with il1 (a, p = 4.14 10) and arg1 (b, p = 0.004) blood transcript expression change as well as change in circulating inf- (c, p = 0.013) and il-4 concentration in plasma (d, p = 0.031) (t = [day 1 + day 2]/2 baseline, n = 16) associations of cebpb expression to macrophage polarisation - associated markers. following exercise - induced muscle damage, cebpb blood transcript expression change shows positive correlations with il1 (a, p = 4.14 10) and arg1 (b, p = 0.004) blood transcript expression change as well as change in circulating inf- (c, p = 0.013) and il-4 concentration in plasma (d, p = 0.031) (t = [day 1 + day 2]/2 baseline, n = 16) in our previous ageing study, we showed that cebpb expression in circulating leukocytes correlated with muscle strength in older people. we hypothesised that this relationship may reflect the reported critical role cebpb plays in leukocyte mediation of muscle regeneration following injury. we anticipated that a single bout of exercise - induced muscle damage would initiate a leukocyte - governed muscle regeneration response in which cebpb expression would provide the essential switch between m1 and m2 inflammatory phenotypes. thus, in a microcosm, we intended to create a model to assess the relevance of chronic circulating cebpb expression that in future research could be applied to the study of age - related functional impairment. here we present evidence that muscle - damaging exercise resulted in elevated blood cebpb expression in volunteers exhibiting symptoms of more severe damage (elevated ck activity and decreased mvc). although the degree of muscle damage was necessarily very small compared to the significant injuries explored in laboratory models, we found gene expression with similar direction and timing to those seen in models of the th1/th2 transition. we also found suggestive evidence that cebpb expression changes post exercise correlated with changes in th1- and th2-linked cytokine and transcript levels as hypothesised. these findings imply that in the exercise - induced muscle damage model, systemic inflammatory status may play a remote role in the governance of muscle remodelling, although more work is needed on this to provide definitive results. since we measured gene transcripts expressed in blood leukocytes, rather than in leukocytes resident in damaged muscle, we were challenged to justify our reasoning behind linking cebpb expression to muscle remodelling. much of the research into immunomodulatory effects on muscle regeneration has focused on the actions of resident macrophages. the differentiation of macrophages appears to occur in situ in damaged muscle rather than prior to extravasation, and whether activated macrophages re - enter the circulation is unclear. yet there may be a role of systemic t lymphocytes by either influencing myogenesis directly or remote macrophage phenotype induction. indeed, cebpb is involved in the differentiation of th0th1 and th2 cd4+cells, and the molecular cascade that determines the inflammatory phenotype shares similarities with that of the macrophage. the relationships between cebpb and th1/th2 markers may support this. circulating il4 may originate from regenerating muscle, known to stimulate cebpb expression involved in m2 macrophage polarity. cebpb expression is also influenced by il1b, suggesting activation from pro - inflammatory leukocytes. we also report a relationship between cebpb and arg1 expression change ; m2 macrophages produce arginase 1, which competes with inos for l - arginine, thereby decreasing the m1 control of myogenic events. in il4-stimulated macrophages, these relationships provide some support that circulating cebpb expression is related to th1/th2 inflammatory polarity. a role for peripheral factors in age - related muscle regeneration is supported by a murine study that infused serum from young mice into old, boosting the regeneration capacity. chronic low - grade systemic inflammation is well known to be related to decreases in muscle mass and functional capacity in longitudinal studies of aging [1921 ]. evidence of an anti - inflammatory antagonistic effect is sparse, although il-10 is emerging as a key mediator of macrophage inflammatory status, associated with muscle regeneration. as a transcription factor, cebpb responds to environmental cues and elevates the production of il-10, thus acting as an upstream mediator of the anti - inflammatory cascade, potentially ameliorating the negative consequences of inflammation. induction of pro - inflammatory mediators of myoblast proliferation is essential for optimal muscle regeneration ; thus cebpb may possibly be a target for pharmaceutical therapy to facilitate endogenous anti - inflammatory responses. this study has several limitations, perhaps most importantly the relatively small sample size of 16, limiting statistical power. this small sample size likely explains why we did not detect an association between baseline muscle strength and baseline cebpb expression, as previously reported in our analysis in 698 subjects. the observed changes in mvc and ck suggest that structural damage and inflammation occurred in all volunteers, although evidence of inflammatory polarity in circulation was only observed in the most severely damaged individuals. the varying severity of damage and resulting disassociation from a universal inflammatory time course weakened potential main effects. statistical power may be improved in future with the recruitment of a larger sample size or by employing laboratory techniques to improve the resolution of data. creatine kinase is a widely used but imperfect measure of muscle injury, and follow - up studies could be strengthened by direct measures of injury, macrophage infiltration and repair. changes in cebpb expression from baseline to day 2 were variable between study subjects : in the group (n = 9) with ck changes above 10,000 u / l one individual showed essentially no change in cebpb expression from baseline. decreases in expression of cebpb did occur in those with lower peak ck values and are likely to reflect the low baseline expression of this gene, against which small fluctuations in rt - pcr results appear to cause larger relative changes (a low signal - to - noise ratio). we did not account for changes in leukocyte counts and subsets, but instead observed th1/th2 activity via circulatory factors and normalised rather than quantified gene expression. measures of the abundance of specific cell subtypes (especially monocytes and macrophages) and gene expression within these cells could have provided more direct evidence linking cebpb expression and macrophage activation. future work might explore cebpb and related expression after both a first and repeat bouts of exercise, as the latter should attenuate damage. we used t - tests in this analysis focussed, for example, on the change in cebpb expression from baseline to day 2 rather than modelling the whole sequence of expression changes across all measures using a repeated measures anova approach. this is because we expected a pattern of early increase in cebpb expression by day 2 with a subsequent decline thereafter, given the dynamics of immune changes after muscle injury as summarised by tidball and villalta and others. anova tests of baseline and day 2 cebpb expression gave similar results to t - tests, but repeat measures anova for all cebpb measures was not significant. in conclusion, cebpb expression in blood was previously reported to be associated with muscle strength in humans and a suggested mechanism for this is that cebpb expression in macrophages is necessary for repair after muscle injury. we therefore sought to test whether cebpb expression in blood leukocytes is responsive to muscle injury inducing exercise. we have shown that in volunteers who likely sustained more severe muscle injury (i.e. those with ck levels > 10,000 u / l) the expected increase in cebpb expression in blood was present 48 h after exercise. further study is needed to confirm the cellular origin of the cebpb and related expression changes and to confirm the mechanistic relationship with downstream muscular regeneration events. | in mouse models, ccaat enhancer - binding protein beta (cebpb) is necessary for m2 macrophage - mediated regeneration after muscle injury. in humans, cebpb expression in blood was strongly associated with muscle strength. in this study we aimed to test whether cebpb expression in blood in people is increased 2 days after exercise designed to induce muscle damage and subsequent repair. sixteen healthy male volunteers undertook elbow flexor exercises designed to induce acute muscle micro - damage. peripheral blood samples were collected at baseline and days 1, 2, 4 and 7 following exercise. expression of cebpb and related genes were analysed by qrt - pcr. extent of muscle damage was determined by decline in maximal voluntary isometric torque and by plasma creatine kinase activity. nine subjects had peak (day 4) creatine kinase activity exceeding 10,000 u / l. in this subgroup, cebpb expression was elevated from baseline to 2 days post exercise (paired - samples t(1,8) = 3.72, p = 0.006). related expression and selected cytokine changes after exercise did not reach significance. muscle - damaging exercise in humans can be followed by induction of cebpb transcript expression in peripheral blood. associations between cebpb expression in blood and muscle strength may be consistent with the cebpb - dependent muscle repair process.electronic supplementary materialthe online version of this article (doi:10.1007/s12576 - 014 - 0350 - 7) contains supplementary material, which is available to authorized users. |
biomarkers are molecules that represent a pathological signature that is easily identified and quantified for a particular disease or disorder. in recent years biomarkers have proven essential in identifying individuals at risk for disease, tracking disease progression, and selection of therapeutic intervention. despite their clinical importance and extensive genetic studies, there are currently no definitive biomarkers for pd available for use in research or diagnostics. it is estimated that by the time a patient with pd sees a physician for the hallmark motor symptoms of rigidity, resting tremor, bradykinesia, and postural instability, up to 70% of the patient s dopaminergic neurons have already been lost. one of the major aims of biomarker research in pd is to provide both medical professionals and researchers with tools that enable accurate diagnosis before extensive neuronal death has occurred. in addition to monitoring at - risk individuals for early diagnosis, biomarkers may expedite drug discovery and development. current clinical trials for pd use relatively subjective measures such as motor activity, behavior, and mood to assess treatment efficacy. this makes such trials extremely expensive and labor - intensive, especially considering that more than 90% of drugs entering clinical trials for brain diseases do not yield marketable compounds. adding biomarkers to clinical trials may reduce this drug failure rate. in clinical trials for cancers, biomarker - based studies have had considerable success, to the point that there are now biomarker - driven basket trials underway, which enroll patients based on genetic biomarkers independent of tumor histology [5, 6 ]. the use of biomarkers rather histopathological diagnosis in these basket trials has generated enormous interest, because they implement a hypothesis - driven strategy for incorporating precision medicine into clinical trials. in pd, accurate and measurable biomarkers have the potential to add great value to clinical trials by identifying patient subgroups that are more likely to be treatment - responsive and by providing quantifiable measures of disease outcome and treatment response. recent investigations suggest that epigenetic marks may be a new source of biomarkers for pd (see fig. 1). epigenetics refers to heritable and acquired alterations in gene activity and expression, without changes in dna sequence. the epigenome is partially dynamic, such that stable epigenetic changes can occur in fully differentiated, post - mitotic cells in response to environmental signals. epigenetic marks, such as biochemical modifications of dna and of histone proteins, regulate gene expression by controlling dna accessibility and signaling to the transcriptional machinery [8, 9 ]. to date, dna modifications, which includes dna methylation and other covalent cytosine modifications, have been the most commonly investigated epigenetic marks for biomarker discovery studies. historically, dna modifications were thought to occur exclusively within cpg (cytosine - phosphate - guanine) sites, but it is now established that dna modification occurs at non - cpg sites as well, particularly in brain neurons [1012 ]. other epigenetic marks, such as post - translational modification of histones and noncoding rnas, frequently act in concert with dna modifications, affecting chromatin accessibility and structure as well as the recruitment of various transcription factors and protein complexes [1315 ]. by modifying genomic activities, epigenetic mechanisms impact downstream protein amounts, cellular function, and phenotypic outcome. the epigenome also exhibits considerable heterogeneity between tissues, tissue subregions, and cell types within an organism, which enables the divergent biological roles of tissues and cell types. in the brain, epigenetic mechanisms are central to neurodevelopment, synaptic transmission, and plasticity [1618 ]. epigenetic abnormalities have been implicated in the mechanism of numerous brain illnesses, including pd. pd has numerous complex, non - mendelian features that are consistent with the dynamic nature of the epigenome. such features includes the prevalence of sporadic, non- familial pd cases (90%) ; the low concordance rates (only 11%) between monozygotic twins for sporadic pd ; the 1.5 - 2 times higher risk of pd in males relative to females ; diurnal fluctuations in symptom severity (sundowning syndrome) ; and the various agricultural and industrial chemicals associated with increased pd risk [2325 ]. the epigenome is well known to vary between monozygotic twins, respond to sex hormones, exhibit circadian fluctuations and affect neuronal functions following exposure to pd - associated environmental toxins [2931 ]. moreover, epigenetic mechanisms may contribute to the greatest risk factor for pd : aging. epigenetic variation accumulates in aging cells, even between genetically - identical individuals, a phenomenon often referred to as including the brain, there are rapid changes in dna modifications in the early life period, but these changes gradually slow over the life span [12, 3234 ]. epigenetic aging affects genomic locations differentially : promoter - associated cpg islands tend to increase in dna methylation with age, while areas with high dna methylation (i.e. repetitive elements in intergenic regions) tend to lose methylation with age [33, 3537 ] epigenomic alterations during aging are mediated not only by external / environmental factors but by genetic factors as well. there are numerous sites in the genome that exhibit allele - specific epigenetic differences, which have been shown to be haplotype - dependent, highly tissue - specific, and prevalent in the brain [3840 ]. different dna haplotypes can demonstrate markedly different epigenetic changes with age, which affects phenotypic outcome. thus, the epigenome can serve as a convergence point for genetic and environmental risk factors of disease, making it an attractive means of detecting heritable and nonheritable disease risk, disease progression, and treatment efficacy in biomarker applications. recent studies have begun to search for epigenetic - based biomarkers using candidate gene and genome - wide approaches. dna modifications, particularly dna methylation, are being used as successful biomarkers for several types of cancer [42, 43 ], and they show the most promise for epigenetic biomarker development in neurodegenerative disease. effective pd biomarkers based on dna methylation status will greatly depend on the concordance of this epigenetic mark between brain and more easily accessible tissues, such as blood. this is challenging, because epigenetic patterns are significantly different between tissue types (such as brain and blood). indeed, data from the nih epigenomics roadmap and encode projects demonstrate tissue and cell - type specific dna methylation patterns at key genes implicated in pd [13, 45 ]. epigenetic divergence between blood and brain can be further amplified by aging, environmental, and stochastic factors. moreover, capacity to detect reliable epigenetic patterns within blood (and other tissues) can be skewed by variability in cellular composition. this is an important consideration for developing epigenetic biomarkers to examine whole blood, because the sensitivity and reliability of the test needs to exceed circadian fluctuations and inter - individual differences in blood leukocyte populations. thus, the reliability and extent to which peripheral tissues are able to mirror the non - dividing cells of the brain represents a major hurdle for epigenetic biomarker strategies. nonetheless, several studies suggest that dna methylation profiles at certain genes in blood can distinguish control subjects from pd cases, opening a new source for biomarker discovery in pd. at present, the most studied epigenetic - based biomarker for pd is dna methylation in the -synuclein gene. -synuclein is a presynaptic neuronal protein that has been linked to familial and sporadic cases of pd. -synuclein is the principal component of lewy bodies and lewy neurites, the hallmark protein inclusions of pd. increases in -synuclein levels lead to its abnormal aggregation and to neuronal degeneration in vivo [48, 49 ] elevated -synuclein mrna has been observed in individual laser - captured dopaminergic neurons in the substantia nigra of sporadic pd cases. dna methylation in the promoter element of the -synuclein gene (a cpg island located in intron 1) was reported to be essential for the regulation of - synuclein transcription. moreover, dna methylation at the -synuclein intron 1 promoter was reduced in postmortem brain tissue (particularly the substantia nigra) of patients with sporadic pd [51, 52 ]. the change in dna methylation at -synuclein intron 1 in the substantia nigra appears to be specific to pd, as it was not observed in cohorts including individuals with lewy body dementia [52, 53 ]. loss of dna methylation at the -synuclein promoter could explain the increase of -synuclein mrna in sporadic pd, which in turn leads to lewy body formation and neurotoxicity. however, this conclusion merits caution, as the tissues analysed in these epigenetic studies have the potentially confounding factors of neuronal loss, medication differences, and limited sample size. encouragingly, recent studies of blood have supported the reduction in dna methylation at the -synuclein gene in pd cases when compared with healthy controls. a relatively large study of the peripheral blood of 490 sporadic pd patients and 485 healthy controls observed significant hypomethylation at the -synuclein intron 1 promoter in the pd cases. the -synuclein dna methylation patterns in blood mirrored those of the brain of pd patients, suggesting that -synuclein dna methylation signatures in blood may be a good proxy for the brain changes at this loci in pd. however, there has been some conflicting reports on the detection of dna methylation differences at the -synuclein intron 1 promoter in pd [58, 59 ]. though certain studies in the pd substantia nigra reported large dna methylation differences (> 30%) at select cpgs [51, 52 ], studies in blood have detected much smaller dna methylation differences between pd cases and controls (5%) (52,53, 54, 55). low sample numbers (n50) may account for why some studies did not identify dna methylation differences at -synuclein in pd blood [58, 59 ]. discrepancies in the selection of cpg sites examined at the -synuclein promoter and differences in pd clinical subtypes have likely also contributed to the conflicting findings [55, 58, 59 ]. overall, modest dna methylation differences in peripheral tissues and genomic target selection represents significant challenges for epigenetic biomarkers. the robustness and suitability of -synuclein - based epigenetic biomarkers may be improved by further work examining patient and clinical variables, including pd subtypes, symptoms, genetic risk carriers, and age groups. age - dependent changes in dna methylation have also been observed at the - synuclein intron 1 promoter [54, 60 ]. in sporadic pd patients, age of onset was positively correlated with dna methylation at-synuclein intron 1, where the greater the methylation levels, the later the occurrence of motor symptoms. age - dependent differences in dna methylation at -synuclein intron 1 in blood are, however, relatively low (90%) of identified risk snps are located in intergenic regions, and as a result their roles in gene function and disease pathogenesis are not evident. however, several of the top gwas risk snps for pd demonstrated allelic differences in nearby dna methylation levels. furthermore, a gwas risk snp in intron 1 of the -synuclein gene (rs3756063) was found to modify dna methylation at the overlapping - synuclein promoter in the blood and brain of pd patients [54, 56 ]. epigenetic analysis can therefore provide insight into the functional effects of snps, which aids in prioritizing candidate sites for biomarker development. disease - associated snps are, for the most part, in linkage disequilibrium with many other snps. this means that snps identified in gwas are often not the true disease risk factor, but rather one of the co - inherited snps is likely responsible for disease risk. as a result epigenetic profiling of regions surrounding co - inherited snps could be a useful approach to revealing true risk variants. this was shown in an elegant recent study by soldner., who identified a snp allele that can up - regulate the -synuclein gene by 1020%. this snp was identified by intersecting pd- associated snps in the -synuclein locus (463 snps) with publicly available epigenetic data generated by the nih roadmap. this effort revealed two snps in -synuclein intron 4 that overlapped histone marks characteristic of enhancers. enhancers are distal regulatory elements that increase the expression of genes, and enhancer activity is determined by epigenetic status. the authors then tested the effects of the snp alleles using the crispr - cas9 gene editing technique in human pluripotent stem cell - derived neurons. the g - allele of snp rs356168 was found to significantly increase -synuclein mrna expression and was associated with greater pd disease risk. this is one example of a dna variant that modifies disease risk via epigenetically - controlled regulatory elements. however, there are certainly many more, because studies of the adult brain have found that pd - associated snps are enriched in distal enhancers. analysis of active enhancers overlapping pd risk loci have also implicated tissues outside of the central nervous system. such research indicates that epigenetic analysis at pd - associated snps can generate molecular biomarkers for detection of disease vulnerability. in addition, combined epigenetic genetic analysis can pave the way for the development of genetically - modified cell lines useful for drug candiadate screening and therapeutic discovery. although mitochondrial dna (mtdna) represents only 1% of the total cellular dna, mitochondrial gene products are essential for normal cell function. thus, it stands to reason that mtdna would be studied for epigenetic changes, yet only recently have research efforts moved beyond nuclear dna (ndna) and into mtdna. while methylation of ndna is well established, methylation of mtdna has been a controversial matter. arguments against mtdna methylation have included the idea that methylases could not access the mitochondria in eukaryotes, and that mtdna is devoid of histones as it is arranged in nucleoid clusters that adhere to the mitochondrial membrane. recently, however, both methylated mtdna and the dna methyltransferase dnmt1 have been found within the mitochondria [79, 80 ]. in the central nervous system of humans, changes in mtdna methylation have been associated with environmental toxins, oxidative stress, drug treatment, disease, and aging. methylation levels of mtdna were analyzed in the brain of 4- and 24-month old mice, and it was found that hydroxymethylation, but not methylation levels in mtdna decreased with age in the frontal cortex. recently, mitochondrial methylation and hydroxymethylation were examined in the d- loop region (which regulates mitochondrial transcription and replication), and the nadh dehydrogenase 6 (mt - nd6) gene in the substantia nigra of pd cases and healthy controls. strikingly, the d - loop region of mtdna in the pd brain showed a loss of methylation in nearly all cpg and non - cpg sites relative to control samples. methylation levels in the mt - nd6 gene and hydroxymethylation in the d - loop were unchanged in pd cases relative to controls. this supports the hypothesis that reduced methylation levels in the d - loop was not the result of diminished neuronal content in the pd substantia nigra, but rather that there was epigenetic misregulation of a site important to mitochrondrial function in pd. if further research shows concordance between blood and brain, the methylation status of the d - loop of mtdna could become a diagnostic biomarker for pd. in order to develop disease - modifying and neuroprotective therapies, biomarkers that can discern the prodromal phase of pd will be necessary. prodromal pd refers to the stage before the disease has fully manifested, when very early signs and symptoms of the disease are present, but diagnosis using the current motor symptom criteria is not yet possible. prodromal pd involves a range of non - motor symptoms such as sleep dysfunction, severe constipation, late - onset hyposmia, and episodes of major depression that often pre - date the motor symptoms by years. identifying biomarkers for the prodromal period is complicated because the period in which these early symptoms dominate is poorly defined, with estimates ranging from 520 years. furthermore, there is extensive inter - individual variability for many of the non- motor symptoms. at present, there is no accepted combination of symptoms to assist clinicians in definitively diagnosing prodromal pd. one method of predicting whether individuals presenting non - motor symptoms may develop the full illness is by examining their epigenetic clock. it has been repeatedly shown that dna methylation status at specific cpg sites in the genome reliably changes with age such that it can be used to accurately predict chronological age [36, 88, 89 ]. in a study by hovarth, dna methylation datasets for 8,000 samples from various tissues were used to construct and evaluate a predictor of dna methylation age. astonishingly, this age prediction method shows chronological accuracy across both sexes and in most cell and tissue types, including blood, breast, kidney, liver, and brain [89, 90 ]. it detected accelerated epigenetic aging due to progeria, obesity, down syndrome, and hiv infection, and it predicted all - cause mortality even after adjusting for various risk factors. in the blood of pd patients, the epigenetic clock tool found accelerated epigenetic aging. in addition, genes linked to accelerated epigenetic aging in the brain had significant overlap with those implicated in pd and other neurodegenerative diseases. analysis of epigenetic aging in pd also identified striking differences in blood cell type composition between pd cases and controls. specifically, blood from pd patients contained more granulocytes and fewer t - helper and b cells than did control samples. accelerated epigenetic aging in combination with altered immune cell counts may precede the onset of motor and cognitive symptoms in pd, which could be a useful biomarker for predicting progression to pd in prodromal individuals. emerging evidence suggests that disruption of circadian rhythms may not only be a consequence of neuronal loss in pd, but may itself contribute to the neurodegenerative process. circadian rhythms are physiological and behavioral cycles generated by an endogenous biological clock in the suprachiasmatic nucleus. circadian rhythms regulate many physiological and behavioral functions, such as 24-h rhythmicity in rest - activity behavior, body temperature, hormone levels, and homeostasis. numerous studies have reported that the symptoms and behaviors associated with pd, such as disruption in motor activity, sleep dysfunction, and responsiveness to dopaminergic treatments, show diurnal fluctuation. these observations indicate there are circadian influences on the expression of pd s clinical features. several clock genes have been identified including period (per1, per2, and per3), cryptochrome (cry1 and cry2), clock, bmal1, and npas2. histone modifications and dna modifications regulate many of these clock genes and can exhibit circadian fluctuations [28, 98100 ]. in pd, a circadian regulator, the npas2 gene promoter, was shown to have a 13% decrease in dna methylation relative to controls. clock genes are known to greatly interact through complex feedback loops to generate and sustain circadian rhythms. hence, aberrant dna methylation of key clock genes in the pd brain may potentiate widespread circadian deregulation and neuronal dysfunction. while the epigenome has promise for both prognostic and diagnostic biomarkers for pd, it is not without its limitations. the ability to detect these biomarkers using noninvasive means will be crucial, and it is known that epigenetic marks, such as dna methylation, vary widely across tissues. another critical challenge is that the size of the epigenetic differences observed in patients will have to substantially exceed the variation within populations and cell composition of the assay tissue. detection of the epigenetic signal will also have to reliably surpass the technical noise of the assay. although there is now a wide range of tools to measure epigenetic marks, sensitivity and specificity come at a price. many of the current platforms require specialized, expensive equipment that would make the use of these tests cost prohibitive. furthermore, determining which specific genomic locations are most appropriate for epigenetic biomarker development is challenging. detection of histone marks, though not as streamlined and practical for clinical biomarker purposes, could be used to predict which genomic sites have biomarker potential. since there are many types of histone modifications, researchers could use this diversity of histone marks to determine which sites in the genome are most homologous between tissues, such as blood and brain. sites demonstrating consistently similar histone modification profiles between brain and peripheral tissues are likely more reliable for epigenetic (and genetic) biomarker applications. as such, analysis of histone modification patterns can refine the discovery and development of dna modification biomarkers for pd. despite its current limitations, epigenetics represents an auspicious target for pd biomarkers. both stool- and blood - based epigenetic tests are already commercially available for early - stage colorectal cancer, and there are many more epigenetic based biomarkers in clinical studies. since dna methylation patterns at specific genomic sites in the blood of pd patients can mirror those of brain, there is promise for these types of tests for pd. not only could epigenetic marks serve to predict and diagnose patients, but epigenetic information could also help determine which patient subgroups would benefit most from a treatment. for example, in patients newly diagnosed with glioblastoma, mgmt promoter methylation is predictive of a favorable response to temozolomide chemotherapy. epigenetic biomarkers therefore can greatly expand the potential for personalized therapeutics. integrating epigenetic information with existing pd diagnostic tools for example, neuroimaging techniques such as datscan, which is used to detect the density of dopaminergic transporters in the brain, helps clinicians distinguish pd from atypical parkinsonian disorders. epigenetic - based biomarkers could rapidly discern individuals at greater risk, which would prompt clinical monitoring and neuroimaging earlier ; enhancing detection of prodromal pd cases. in addition, the combination of datscan and epigenetic biomarkers could also predict which patients will be most responsive to the main drug for pd, levodopa, given that dopaminergic treatments affect dna methylation at the -synuclein gene. epigenetic biomarkers may also predict therapeutic utility of the newer treatments targeting -synuclein which are currently in clinical trials. finally, epigenetic biomarkers could be used in combination with genetic screens to identify individuals at risk for familial and sporadic forms of pd. recent studies suggest that phenotypic effects of sequence variants can be influenced by accompanying epigenetic signatures, via allele - specific methylation. studies demonstrating the abundance of allele - specific methylation in the brain [39, 103 ] and its presence at pd risk genes may lead to the development of novel combinatorial genetic - epigenetic biomarkers for pd. though still at a very early stage, epigenetic research in pd may unify the aging, environmental and genetic risk factors, and thus change the strategies used in pd diagnosis and treatment. | parkinson s disease (pd) is a prevalent neurodegenerative illness that is often diagnosed after significant pathology and neuronal cell loss has occurred. biomarkers of pd are greatly needed for early diagnosis, as well as for the prediction of disease progression and treatment outcome. in this regard, the epigenome, which is partially dynamic, holds considerable promise for the development of molecular biomarkers for pd. epigenetic marks are modified by both dna sequence and environmental factors associated with pd, and such marks could serve as a unifying predictor of at - risk individuals. epigenetic abnormalities have been detected in pd and other age - dependent neurodegenerative diseases, some of which were reported to occur early on and were reversible by pd medications. emerging reports indicate that certain epigenetic differences observed in the pd brain are detectable in more easily accessible tissues. in this review, we examine epigenetic - based strategies for the development of pd biomarkers. despite the complexities and challenges faced, the epigenome offers a new source of biomarkers with potential etiological relevance to pd, and may expand opportunities for personalized therapies. |
pancreatic cancer is a common gastrointestinal cancer, and the incidence and mortality has been increasing in recent years. in the usa, it has been the fourth leading cause of death. in 2009, approximately 42 000 new patients were diagnosed, and 35 000 americans died of pancreatic cancer.. early diagnosis is difficult because of the lack of clinical symptoms in the early stages, and the 5-year survival rate ranges from 4% to 6% or less. although the incidence and mortality of pancreatic cancer are remarkably high, few treatment options are effective. the primary option is to reduce pancreatic cancer risk by taking preventive measures. in humans, free radicals, which result from polyunsaturated fatty acids reacting with oxygen in the lipid membranes, might be essential for the occurrence of tumors. vitamin e is an effective antioxidant that prevents the occurrence of some tumors by protecting cells and dna from free radical damage. whether vitamin e, as an antioxidant, could reduce the incidence of pancreatic cancer has been under consideration. in 2000, heisler. reported the results from an in vitro study showing that vitamin e could inhibit pancreatic cancer cell line growth. another animal study showed that vitamin e increased the activity of superoxide dismutase (sod) and decreased the level of thiobarbituric- acid - reactive substances (tbars), which might be a mechanism of decreasing liver metastasis in pancreatic cancer. many epidemiological studies have assessed the relationship between vitamin e and the risk of pancreatic cancer. in the 1990s, 1 of 2 case - control studies showed that vitamin e intake was linked to a decreased risk of pancreatic cancer ; however, the other study showed a null association. subsequent epidemiological studies, including case - control and cohort studies, have suggested an inconsistent association between vitamin e intake and the risk of pancreatic cancer [1017 ]. the sample size of each original study was smaller and original study was conducted in a single population, which might be the primary reason for the unsatisfactory results. we conducted a meta - analysis of the relevant studies by combining the results from the published observational studies to assess the relationship between vitamin e intake and the risk of pancreatic cancer. additionally, we examined the influences of various study characteristics on the overall risk estimate. we intended to provide the best available evidence as to whether vitamin e intake has a preventive effect on pancreatic cancer. the web of science, pubmed, and embase databases were used to identify the observational studies that reported the association between vitamin e intake and the risk of pancreatic cancer through december 31, 2014 with the following terms : vitamin e intake, dietary vitamin e and vitamin e in combination with pancreatic cancer and pancreatic carcinoma. the reference lists of the identified studies were searched for potential studies. if necessary, we contacted the authors of the original studies for the required data. the studies that met the following criteria could be included : 1. the study had been published as a full text in the english language ; 3. the study reported the association between the vitamin e intake and the risk of pancreatic cancer ; 4. the study reported the rr and the corresponding 95% ci for the highest vs. the lowest level of vitamin e intake ; 5. if there were duplicate publications on the same study population, we included the most recent one. an article was excluded based on to the following criteria : if it were a review, case report, or animal experiment ; if it were not published as a full text ; or if it reported an exposure factor or endpoint that was not relevant to our study. the following information was collected from the included studies : the last name of the first author, publication year, population, number of cases and controls or total sample size, rr and the corresponding 95% ci from the most fully adjusted model for the highest vs. the lowest vitamin e intake, and the factors of adjustment for potential confounders. all the procedures were conducted by 2 independent authors, and any disagreements were resolved by discussion. the key components of designs (e.g., selection of study populations, ascertainment of exposure and outcome, duration of follow - up) were used to estimate the quality of primary studies rather than reporting the aggregate scores. the pooled relative risk (rr) and the corresponding 95% ci were used to assess the association between vitamin e intake and the risk of pancreatic cancer. a fixed - effects model when low evidence of homogeneity was found or random - effects model when strong evidence of homogeneity was found was used to calculate the combined rr. subgroup analysis was performed to identify the source of heterogeneity, if possible, and the effect of the potential factors on the overall risk estimate. in addition, we conducted a sensitivity analysis to investigate the influence of a single study on the overall risk estimate by omitting 1 study at a time. begg s and egger s tests were used to detect the evidence of a publication bias. in our study, if the p - value were less than 0.05, it was considered statistically significant. the web of science, pubmed, and embase databases were used to identify the observational studies that reported the association between vitamin e intake and the risk of pancreatic cancer through december 31, 2014 with the following terms : vitamin e intake, dietary vitamin e and vitamin e in combination with pancreatic cancer and pancreatic carcinoma. the reference lists of the identified studies were searched for potential studies. if necessary, we contacted the authors of the original studies for the required data. the studies that met the following criteria could be included : 1. the study had been published as a full text in the english language ; 3. the study reported the association between the vitamin e intake and the risk of pancreatic cancer ; 4. the study reported the rr and the corresponding 95% ci for the highest vs. the lowest level of vitamin e intake ; 5. if there were duplicate publications on the same study population, we included the most recent one. an article was excluded based on to the following criteria : if it were a review, case report, or animal experiment ; if it were not published as a full text ; or if it reported an exposure factor or endpoint that was not relevant to our study. the following information was collected from the included studies : the last name of the first author, publication year, population, number of cases and controls or total sample size, rr and the corresponding 95% ci from the most fully adjusted model for the highest vs. the lowest vitamin e intake, and the factors of adjustment for potential confounders. all the procedures were conducted by 2 independent authors, and any disagreements were resolved by discussion. the key components of designs (e.g., selection of study populations, ascertainment of exposure and outcome, duration of follow - up) were used to estimate the quality of primary studies rather than reporting the aggregate scores. the pooled relative risk (rr) and the corresponding 95% ci were used to assess the association between vitamin e intake and the risk of pancreatic cancer. a fixed - effects model when low evidence of homogeneity was found or random - effects model when strong evidence of homogeneity was found was used to calculate the combined rr. subgroup analysis was performed to identify the source of heterogeneity, if possible, and the effect of the potential factors on the overall risk estimate. in addition, we conducted a sensitivity analysis to investigate the influence of a single study on the overall risk estimate by omitting 1 study at a time. begg s and egger s tests were used to detect the evidence of a publication bias. in our study, if the p - value were less than 0.05, it was considered statistically significant. according to the inclusion criteria, 10 observational studies involving 2976 patients and 254 393 participants or controls were included [817 ]. the studies were published from 1991 to 2014. in the 10 studies, 6 were case - control studies [810,12,13,17 ], and 4 were cohort studies [11,1416 ]. four studies were conducted in usa populations, 2 in asian populations (japanese and chinese), and 4 in european populations [11,1315 ] (finnish, italian, dutch, and british). all the studies were adjusted for a wide range of potential confounders, such as age, sex, smoking, and energy intake. in total, 11 data items from the 10 included studies were used to assess the association between vitamin e intake and the risk of pancreatic cancer. of these, 4 results showed that vitamin e intake was linked to a reduced risk of pancreatic cancer. overall, our study found that there was a statistically significant inverse relationship between vitamin e intake and the risk of pancreatic cancer ; the summary rr and corresponding 95% ci of pancreatic cancer for the highest vs. the lowest level of vitamin e intake was 0.81 (0.73, 0.89). there was minimal statistically significant evidence of heterogeneity across these studies (i=19.8%, p=0.255). figures 3 and 4 show the results of the subgroup analyses for the influence of the study design and population on the overall risk estimate, respectively. the results showed that there was an inverse association between the vitamin e intake and pancreatic cancer risk both in the case - control and cohort studies, in which the pooled rr (95% ci) were 0.65 (0.54, 0.77) and 0.88 (0.79, 0.99), respectively. we found no evidence of heterogeneity among the case - control and cohort studies (i=0.0%, pcase - control=0.760 and pcohort=0.868). the inverse association between vitamin e intake and pancreatic cancer was found among people in the usa, asians, and europeans, for which the combined rrs (95% cis) were 0.67 (0.55, 0.82), 0.72 (0.52, 0.99), and 0.87 (0.77, 0.98), respectively. there was minimal evidence across the 3-subgroup analyses (iusa=0.0%, pusa=0.413, iasian=0.0%, pasian=0.454, and ieuropean=0.0%, peuropean=0.410). by removing 1 study at a time, we conducted a sensitivity analysis to assess the influence of each included study on the pooled rr. the combined rrs were similar to each another, and none significantly modified the pooled rr. after removing the study with the highest weight, the pooled rr was not significantly modified. although the funnel plot shows all the included studies symmetrically distributed in the triangle area, begg s and egger s regression tests showed that there was a low probability of publication bias in our study (p=0.049). according to the inclusion criteria, 10 observational studies involving 2976 patients and 254 393 participants or controls were included [817 ]. the studies were published from 1991 to 2014. in the 10 studies, 6 were case - control studies [810,12,13,17 ], and 4 were cohort studies [11,1416 ]. four studies were conducted in usa populations, 2 in asian populations (japanese and chinese), and 4 in european populations [11,1315 ] (finnish, italian, dutch, and british). all the studies were adjusted for a wide range of potential confounders, such as age, sex, smoking, and energy intake. in total, 11 data items from the 10 included studies were used to assess the association between vitamin e intake and the risk of pancreatic cancer. of these, 4 results showed that vitamin e intake was linked to a reduced risk of pancreatic cancer. overall, our study found that there was a statistically significant inverse relationship between vitamin e intake and the risk of pancreatic cancer ; the summary rr and corresponding 95% ci of pancreatic cancer for the highest vs. the lowest level of vitamin e intake was 0.81 (0.73, 0.89). there was minimal statistically significant evidence of heterogeneity across these studies (i=19.8%, p=0.255). figures 3 and 4 show the results of the subgroup analyses for the influence of the study design and population on the overall risk estimate, respectively. the results showed that there was an inverse association between the vitamin e intake and pancreatic cancer risk both in the case - control and cohort studies, in which the pooled rr (95% ci) were 0.65 (0.54, 0.77) and 0.88 (0.79, 0.99), respectively. we found no evidence of heterogeneity among the case - control and cohort studies (i=0.0%, pcase - control=0.760 and pcohort=0.868). the inverse association between vitamin e intake and pancreatic cancer was found among people in the usa, asians, and europeans, for which the combined rrs (95% cis) were 0.67 (0.55, 0.82), 0.72 (0.52, 0.99), and 0.87 (0.77, 0.98), respectively. there was minimal evidence across the 3-subgroup analyses (iusa=0.0%, pusa=0.413, iasian=0.0%, pasian=0.454, and ieuropean=0.0%, peuropean=0.410). by removing 1 study at a time, we conducted a sensitivity analysis to assess the influence of each included study on the pooled rr. the combined rrs were similar to each another, and none significantly modified the pooled rr. after removing the study with the highest weight, the pooled rr was not significantly modified. although the funnel plot shows all the included studies symmetrically distributed in the triangle area, begg s and egger s regression tests showed that there was a low probability of publication bias in our study (p=0.049). our meta - analysis suggested a significant inverse relationship between vitamin e intake and the risk of pancreatic cancer a high level of vitamin e was significantly linked to a decreased risk of pancreatic cancer. although the included studies were conducted in different populations and varied in study design, little evidence of heterogeneity was found across the studies. all the studies were published in english and had a high quality of assessment and the results from each original study were adjusted for a wide range of potential confounders, eliminating the most obvious confounding factors. in the subgroup analyses, the summary risk estimate of 6 case - control studies and 4 cohort studies showed an inverse association between vitamin e intake and a risk of pancreatic cancer. it is well known that a higher level of statistical evidence is detected in cohort studies than in case - control studies, thus, our results were credible, but more cohort studies are needed. additionally, our subgroup analyses showed that this inverse association was not modified by different populations, including asians, europeans, and people in the usa. the subgroup analyses added additional evidence for the association between vitamin e intake and risk of pancreatic cancer. although there is evidence of an association of vitamin e intake with a decreased risk of many cancers [1820 ], the mechanisms are not well understood. the primary mechanism of vitamin e might be as an antioxidant, preventing dna damage by scavenging lipid peroxyl radicals and terminating the lipid peroxidation chain reaction while increasing the activity of superoxide dismutase (sod) and decreasing the level of thiobarbituric - acid - reactive substances (tbars). other potential mechanisms are based on the possibility that vitamin e might decease the activity of the protein kinase c (pkc) pathway, enhance the immune response of the body, suppress cancer cell growth by down - regulation of the phosphoinositide 3-kinase pathway, and increase the expression of anti - oncogene p27. early - stage diagnosis is difficult, and the mortality associated with pancreatic cancer is remarkably high ; there are few effective treatment options to overcome this fatal cancer. our study, which included 10 observational studies with 2976 patients and 254 393 participants or controls, significantly improved the statistical power and found a more reliable association between vitamin e intake and a pancreatic cancer risk. we provided evidence that vitamin e intake is related to a decreased risk of pancreatic cancer, and that vitamin e intake might effectively prevent pancreatic cancer. first, 10 studies with relatively larger sample sizes were included in our studies ; however, many of the studies were case - control studies, and recall bias and selection bias were inevitable. although 4 cohort studies were included, the following factors limited the statistical evidence : a smaller number of sample cases and a lack of groups of patients of different ages. second, most of the included studies did not respectively report the risk among males and/or females, and hormonal factors might lead to the male - to - female differential in the incidence of pancreatic cancer. in general, a high level of estrogen exposure via estrogen - only therapy could significantly reduce the risk of pancreatic cancer. third, vitamin e is a mixture of 8 structurally related and naturally occurring components, including tocopherols (,,,) and tocotrienols (,,,). however, only 1 component is naturally contained in food, and determining the diverse biological activities between the dietary intake and supplementation is challenging. in the included studies, 1 study reported the rrs associated with the intake of various vitamin e isoforms and the risk of pancreatic cancer. most studies did not report the source of vitamin e. fourth, the pooled risk estimate was predominantly based on usa and european populations ; 2 asian populations and no other populations were included in the studies, and additional studies that include other populations are needed. this study showed a significant inverse relationship between vitamin e intake and the risk of pancreatic cancer. | backgroundsome epidemiological studies have suggested that vitamin e intake reduces the risk of pancreatic cancer ; however, this conclusion has not been supported by all the published studies. we conducted a meta - analysis to assess the relationship between vitamin e intake and the risk of pancreatic cancer by combining the results from published articles.material/methodswe searched the published studies that reported the relationship between vitamin e intake and pancreatic cancer risk using the pubmed, web of science, and embase databases through december 31st, 2014. based on a fixed - effects or random - effects model, the rr and 95% ci were used to assess the combined risk.resultsin total, 10 observational studies (6 case - control studies and 4 cohort studies) were included. the overall rr (95% ci) of pancreatic cancer for the highest vs. the lowest level of vitamin e intake was 0.81 (0.73, 0.89). we found little evidence of heterogeneity (i2=19.8%, p=0.255). in the subgroup analyses, we found an inverse association between vitamin e intake and pancreatic cancer risk both in the case - control and cohort studies. additionally, this inverse association was not modified by different populations.conclusionsin our meta - analysis, there was an inverse association between vitamin e intake and the risk of pancreatic cancer. a high level of vitamin e might be a protective factor for populations at risk for pancreatic cancer. |
acquired pure red cell aplasia (prca) is a rare, generally chronic condition of profound anemia characterized by a severe reduction in the number of reticulocytes in the peripheral blood and the virtual absence of erythroid precursors in the bone marrow. these include : drugs - phenytoin, trimethoprim, zidovudine, chlorpropamide, recombinant erythropoietin, and mycophenolate mofetilinfections - parvovirus b19, hiv, and viral hepatitisautoimmune disorders - systemic lupus erythematosus, rheumatoid arthritis, hemolytic anemia, and allogeneic stem cell transplantlymphoid malignancies - chronic lymphocytic leukemia, large granular leukemia, non - hodgkin lymphoma, hodgkin lymphoma, and myelomamyeloid malignancies - chronic myeloid leukemia, myelofibrosis, and prodrome of myelodysplastic syndromeother causes - thymoma, pregnancy, idiopathic, etc. drugs - phenytoin, trimethoprim, zidovudine, chlorpropamide, recombinant erythropoietin, and mycophenolate mofetil infections - parvovirus b19, hiv, and viral hepatitis autoimmune disorders - systemic lupus erythematosus, rheumatoid arthritis, hemolytic anemia, and allogeneic stem cell transplant lymphoid malignancies - chronic lymphocytic leukemia, large granular leukemia, non - hodgkin lymphoma, hodgkin lymphoma, and myeloma myeloid malignancies - chronic myeloid leukemia, myelofibrosis, and prodrome of myelodysplastic syndrome other causes - thymoma, pregnancy, idiopathic, etc. a number of cases of coexisting myasthenia gravis (mg) and prca have been described in the literature ; all were thymoma associated except for one with thymic hyperplasia. as the onset of anemia in prca is insidious, patients may have little signs and symptoms until the anemia becomes severe. extreme pallor or decreased exercise tolerance may be the first sign of this disorder in a previously healthy individual. bone marrow examination shows normal overall cellularity, with complete or virtually complete absence of red cell precursors. there is a well - documented association of prca with thymoma, with an incidence of about 5%. even if a thymoma is present, surgical resection only occasionally results in improvement or cure of the prca, and additional treatment (e.g., glucocorticoids, cyclosporine, and cyclophosphamide) is needed in most cases. even though it is not very common to have acquired prca, documenting its etiology needs extensive and systemic workup, which involves multispecialty approach. we are reporting a similar case who was treated outside our hospital for many years with blood products and hematinic and later got documented thymoma as an etiological cause. a 60-year - old gentleman, who was normotensive, nondiabetic, and euthyroid, presented to our outpatient clinic with features of symptomatic anemia in the form of malaise and poor exercise tolerance. there was no history of apparent blood loss, bleeding diathesis, or poor intake. there was also no history of muscle weakness, diplopia, speech fatigue, or any surgery. he gave a history of repeated blood transfusions, ten such events he could remember and was on iron supplements since 6 years. on clinical examination, he had severe pallor and mild splenomegaly, rest of examination was unremarkable. on evaluation, he had hemoglobin of 2 g / dl with normal red cell indices and raised serum iron indices [table 1 ]. his liver function test, kidney function test, lactate dehydrogenase, electrolytes, calcium, and phosphorous were normal. bone marrow aspiration was done, which revealed hypercellular marrow with absent erythroid precursors, abundant iron stores with normal other cell lines [figure 1 ]. laboratory parameters of patient bone marrow revealing absence of erythroid precursors hepatitis b, c, and a was negative as was epstein connective tissue disease markers such as antinuclear antibody, dsdna, and rheumatoid factor levels were normal. contrast - enhanced computed tomography (ct) scan was done which showed large anterior mediastinal mass with close approximation to great vessels [figure 2 ]. then, ct - guided biopsy of mass was carried out, which was consistent with who class b1 thymoma [figure 3 ]. further immunohistochemistry was carried out, which revealed ck 5/6 positive in plump oval cells, terminal deoxynucleotidyl transferase, and cd3 positive in lymphocytes and ki67 of 50% [figure 4 ]. (b) contrast histopathological examination (hpe) of anterior mediastinal mass revealing small oval to spindle cells admixed with lymphocytes consistent of who type b1 thymoma immunohistochemistry revealing thymoma positive for ck5, cd3, and ki67 is 40% as for as management was concerned, prca is treated with steroids or immunosuppressants, but some patients go into remission on surgery only. patient was not a surgical candidate as he would have persisted with gross residual disease, so we put this patient on multiagent chemotherapy with cisplatin, adriamycin, and cyclophosphamide. plan is to give four cycles of chemotherapy followed by surgery with or without adjuvant radiotherapy. prca, a disorder first characterized in 1922, is a syndrome characterized by severe normochromic, normocytic anemia associated with reticulocytopenia and absence of erythroblasts from an otherwise normal bone marrow. the acquired form of prca presents either as an acute self - limited disease, predominantly seen in children or as a chronic illness that is more frequently seen in adults. as was seen in our patient, he also presented with chronic anemia, had got multiple blood transfusions till we received him. depending on the cause, the course can be acute and self - limiting or chronic with rare spontaneous remissions. in approximately 60% of patients with prca, their serum and its igg fraction inhibit the growth of patient and normal erythroid progenitors in vitro. the target antigen is usually not known ; in a few cases, however, the igg fraction contains an inhibitor of erythropoietin. in other cases of autoimmune prca, suppression of erythropoiesis seems to be mediated by t - lymphocytes. in our patient the net result of the immune attack in prca, which is characterized by marked reduction or absence of all recognizable red cell precursors in the bone marrow and absence of reticulocytes in the peripheral blood. in our patient, net hemoglobin was 2.0 g / dl with normal red cell indices and almost absent reticulocytes in peripheral blood and absent erythroblasts in bone marrow. mg appears in about 20%40% of patients with thymoma, and prca develops in about 2%5% of those patients. on the other hand, thymoma is detected in 10%17% of patients with mg and in 5%13% of patients with prca. however, the simultaneous occurrence of mg, prca, and thymoma is extremely rare. in our patient, chest x - ray had mild mediastinal widening, but ct showed large mediastinal mass with close relation with large arteriovenous system. as per the who histological classification, thymoma is divided into six types as type a, ab, b1, b2, b3, and c. our patient had b1 histology pattern, consisting of lymphocytic rich predominantly cortical organoid cells. it has been seen that type a is associated with prca and type b with mg, but in our patient, this correlation was not seen. patient was inoperable at beginning, and likely fitted in clinical stage ii to iii and complete surgical excision was not possible. hence, our patient was put on chemotherapy at outset and plan is thymectomy after few cycles of chemotherapy. it has been seen that prca improves in 30% of patients after complete thymectomy, and complete surgical excision with adjacent structures is possible in 88% of patients. there are several options for inducing remission of prca after surgery, but many patients with acquired prca require immunosuppressive therapy to maintain remissions. corticosteroids, cyclosporin a, and cyclophosphamide are almost equally effective for inducing remissions of prca, but the most important difference between these agents is the feasibility of long - term maintenance. considering the recurrent nature of acquired prca, it has been suggested cyclosporin a as first - line therapy for these patients at a dose of 2.53 mg / kg twice daily to achieve trough cyclosporin a (csa) levels of 150250 ng / ml for a maximum of 34 months. this trough csa level has been empirically determined according to a consensus from a multicenter randomized study in japan for aplastic anemia. prognosis of thymoma is related to stage of presentation, presence or absence of complete resection, and histological subtype. subtype a and ab behaves as benign tumor, subtype b (b1 and b2) behaves as low - grade tumor, and subtype b3 and c behaves as thymic carcinoma. | pure red cell aplasia (prca) is a known entity in clinical medicine. patients are often transfusion dependent for their whole life. ascertaining its etiology is always a herculean task. we received a similar transfusion - dependent patient, who on evaluation was found to have thymoma as an etiological factor. thymoma presenting as prca is seen in 2%5% patients and evaluating prca for thymoma is seen in 5%13% patient. as per the who histopathological classification, thymoma has six types and type a is associated with prca and type b is associated with myasthenia gravis. this correlation was not seen in our patient, who had type b thymoma. surgical resection of thymus improves 30% of prca and rest needs immunosuppression. our patient was not the surgical candidate, and hence he was put on chemotherapy. |
magnetic resonance myelography (mrm) has been developed to evaluate the spinal subarachnoid space non - invasively. in addition, advantages of mrm as compared to conventional or computed tomography (ct) myelography are that it does not involve the use of intrathecal contrast or radiation.[14 ] on the heavily t2-weighted sequence used in this technique, cerebro - spinal fluid (csf) in the subarachnoid space has hyperintense signal while normal or abnormal soft tissue structures are seen as filling defects or extrinsic compressions. images obtained with single - slice thick - slab techniques require very short time as compared to multi - slice techniques. in addition, single - slice technique of mrm provides excellent suppression of background signals (from fat or paravertebral veins) and has significantly reduced csf flow artifacts.[357 ] mrm has been previously used to assess spinal stenosis and nerve root compression with some benefits.[810 ] the aim of this study is to evaluate the advantages of routine use of 2-d single thick - slice mrm in providing additional information in spinal and extra - spinal regions. the study included all patients who were referred for mr imaging irrespective of age, sex or clinical presentation. the study group included 143 cases that had mild to severe degenerative disease, 13 cases of congenital variants or abnormalities, 17 cases of tuberculous spondylitis, 12 cases of primary or secondary spinal tumors and 35 cases of spinal trauma. the mr imaging examinations were performed by using an 18 channel, 1.5-t avanto system (siemens, erlangen, germany). single thick - slice mrm projection images were obtained in mid - sagittal and coronal planes in addition to the routine mr sequences. t2 half - fourier acquisition single - shot turbo spin - echo (haste) sequence was used with extremely long echo time (te) of 1200 ms and repetition time (tr) of 8000 ms. other sequence parameters were echo train length = 369, slice thickness = 50 mm, field of view = 280 - 400 mm, flip angle = 150, base resolution = 512, phase resolution = 72, signal to noise ratio (snr) = 1 and nex = 2. image matrix was 369 512 with voxel size of 0.8 0.5 50 mm. mrm images for a single region required an imaging time of 34 sec while whole spine mrm required an additional 34 sec. the mrm views of entire spine could be processed in single image using total imaging matrix (tim) technology by stepwise moving the patient table without repositioning the patient or changing the coil. one radiologist evaluated the mrm images while the second radiologist with sub - specialty training in neuroradiology evaluated the routine mr sequences. we tried to grade the additional merits of mrm in spinal and extra - spinal regions by using a 3 point grading scale which was modified from the one used by oconnell. the study included all patients who were referred for mr imaging irrespective of age, sex or clinical presentation. the study group included 143 cases that had mild to severe degenerative disease, 13 cases of congenital variants or abnormalities, 17 cases of tuberculous spondylitis, 12 cases of primary or secondary spinal tumors and 35 cases of spinal trauma. the mr imaging examinations were performed by using an 18 channel, 1.5-t avanto system (siemens, erlangen, germany). single thick - slice mrm projection images were obtained in mid - sagittal and coronal planes in addition to the routine mr sequences. t2 half - fourier acquisition single - shot turbo spin - echo (haste) sequence was used with extremely long echo time (te) of 1200 ms and repetition time (tr) of 8000 ms. other sequence parameters were echo train length = 369, slice thickness = 50 mm, field of view = 280 - 400 mm, flip angle = 150, base resolution = 512, phase resolution = 72, signal to noise ratio (snr) = 1 and nex = 2. image matrix was 369 512 with voxel size of 0.8 0.5 50 mm. mrm images for a single region required an imaging time of 34 sec while whole spine mrm required an additional 34 sec. the mrm views of entire spine could be processed in single image using total imaging matrix (tim) technology by stepwise moving the patient table without repositioning the patient or changing the coil. one radiologist evaluated the mrm images while the second radiologist with sub - specialty training in neuroradiology evaluated the routine mr sequences. we tried to grade the additional merits of mrm in spinal and extra - spinal regions by using a 3 point grading scale which was modified from the one used by oconnell. the utility of mrm was categorized as grade 3 in 10.9% cases (24/220) and grade 2 in 21.8% (48/220) cases [table 2 ]. thus, the overall additional merit of mrm in spine was seen in 32.7% (72/220) of cases. in 67.3% cases (148/220), the mrm did not give any contribution to the final diagnosis (grade 1). mrm has been compared to conventional and ct myelography previously. pictorial quality and resolution of conventional and ct myelography in evaluating thecal sac and nerve roots is better than that of mrm. we accept these facts but conventional and ct myelography are invasive procedures requiring contrast injection into the thecal sac with potential complications.[14 ] multi - slice as well as single - slice techniques have been used for obtaining mrm. multi - slice techniques using fast spin - echo (fse) sequences require long acquisition times and post processing of the data. single - slice mrm has been attempted with various sequences including rapid acquisition with relaxation enhancement (rare), single - shot turbo spin - echo (with long effective te), and t2 half - fourier acquisition single - shot turbo spin - echo (haste). single - slice mrm is faster, does not require any post processing, provides excellent suppression of background signals and has markedly reduced csf flow artefacts. however, limited data is available about the additional benefits of single - slice mrm as a routine sequence in spinal imaging.[810 ] we selected haste for acquiring single thick - slice mrm images in two planes. oconnell., also acquired mrm images using this sequence with following parameters ; slice thickness : 20 mm, echo train length : 256, fov : 25 cm and acquisition time of 3 min 20 sec. we have modified the sequence parameters and used thicker slab (50 mm), and increased the echo train length (369) and field of view (28 - 40 cm). by using this protocol, we were able to obtain good quality images with negligible csf pulsation artefacts and excellent suppression of background signal in a very short time of 34 seconds for single region and 68 seconds for whole spine. the larger fov and thicker slab significantly reduced the image acquisition time and it also became easier for us to screen for abnormal fluid signal in extra spinal regions, nearby organs and joints. mrm has still not been uniformly recommended for routine use in spine because of long acquisition times and presumably limited information although previous studies have evaluated its role in diagnosing various spinal diseases. the potential use of mrm in evaluating fluid signal in extra - spinal regions has not been studied previously. however, by employing this single - slice thick - slab technique of mrm in two planes, we could add diagnostic information in a substantial number of cases without any significant addition of imaging time or cost [figures 19 ]. in our study, conjoined nerve roots and traumatic nerve root avulsions could be diagnosed only on mrm [figure 2, 3b and 5 ]. mrm also demonstrated diagnostic or at least an initial interpretative value in intrathecal vascular congestion, post operative scars, arachnoid adhesions and sequestrated discs [figure 7, 9 ]. the heavily t2-weighted sequence and larger fov employed in our study resulted in prompt detection of additional foci of incidental or abnormal fluid signal in spinal or extra - spinal locations. perineural cysts, synovial neoarthrosis and facetic effusions could be detected on mrm even by the relatively inexperienced observer [figure 1 ]. it was also possible to reveal diaphragmatic hernia, ascites, regional joint pathologies and lymphadenopathy in patients who were just referred for spinal imaging. routine spinal imaging [figure 4 ] sequences were limited in providing information about these extra - spinal findings in the first look due to the limited fov and anterior saturation bands which resulted in a signal drop anterior to the spine. ((a) sagittal image shows synovial neoarthrosis (black arrow), (b) coronal image depicts facetic effusion (broad black arrow), parafacetal cysts(thin black arrow). (a - c) magnetic resonance myelograms in congenital variants/ abnormalities. (a) coronal image demonstrates conjoined nerve roots (leftsided black arrow), tarlov 's cyst (right - sided black arrow), (b) sagittal image shows syrinx in diastematomyelia (black arrow), and (c) sagittal image shows incidental occult sacral meningocele (black arrow). (a - c) (a) sagittal image shows prevertebral and posterior epidural collection in tuberculous spondylitis, (b) coronal image reveals pseudomeningocele secondary to nerve root avulsion, and (c) coronal image demonstrates a well defined intradural filling defect due to meningioma. (a - c) coronal magnetic resonance myelograms depicting various additional extra - spinal findings(black arrows), (a) diaphragmatic hernia and left glenohumeral joint effusion, (b) residual apical scar in chronic pulmonary tuberculosis, and (c) small left - sided iliac lymph nodes. a) coronal magnetic resonance myelogram clearly shows nerve root avulsion (black arrow) compared to b) routine coronal t2 sequence scan. a) coronal and b) sagittal single thick - slice magnetic resonance myelograms show simultaneous first look detection of significant lumbar canal stenosis, spinal arterio - venous malformation (a) and synovial neoarthrosis (b). a) coronal magnetic resonance myelogram reveals intrathecal vascular congestion proximal to the level of spinal canal block (a - white arrow) while on b) coronal t2 weighted image it is difficult to differentiate vessels from nerve roots. spinal intradural metastatic deposits. a) coronal magnetic resonance myelographyshows thickening and nodularity of cauda equina nerve roots (white arrow) which on b) coronal t2 sequence is not discernable. post - contrast fat - suppressed t1 sequence c) coronal and d) sagittal views show nodular enhancement along cauda equina nerve roots on reevaluation (white arrows). a) coronal magnetic resonance myelogram depicts focal asymmetrical irregularity of margin of thecal sac on right side with root sleeve blunting and subtle thickening of cauda equina nerve roots cranially (black arrows). routine t2 images b) sagittal, c) coronal, d) axial views the findings are difficult to delineate. on post - contrast fatsuppressed t1 sequence e) coronal, f) axial no abnormal enhancement is seen. in the single thick - slice technique, images are available in only two planes. at times, this leads to incomplete evaluation of spinal canal stenosis and difficulty in localization of the source of fluid signal. a repetition of the sequence is also required if the plane is not chosen correctly. thicker slab selection can possibly lead to cross talk of the data but it was not experienced in our study. in the single thick - slice technique, images are available in only two planes. at times, this leads to incomplete evaluation of spinal canal stenosis and difficulty in localization of the source of fluid signal. a repetition of the sequence is also required if the plane is not chosen correctly. thicker slab selection can possibly lead to cross talk of the data but it was not experienced in our study. two - dimensional single thick - slice mrm sequence could have additional merits in complimenting routine mr sequences in spinal imaging. we suggest that mrm using this technique be included in the routine spinal mri protocol. it could aid in detection of abnormal fluid signals in the spinal and extra spinal regions and provide additional information beyond the referred region of interest without significantly prolonging overall imaging time. | objective : to validate the additional merits of two - dimensional (2d) single thick - slice magnetic resonance myelography (mrm) in spinal imaging.materials and methods:2d single thick - slice mrm was performed using t2 half - fourier acquisition single - shot turbo spin - echo (haste) sequence in addition to routine magnetic resonance (mr) sequences for spine in 220 patients. the images were evaluated for additional diagnostic information in spinal and extra - spinal regions. a three - point grading system was adopted depending upon the utility of mrm in contributing to the detection of spinal or extra - spinal findings. grade 1 represented no contribution of mrm while grade 3 would indicate that it was essential to detection of findings.results:utility of mrm in spine was categorized as grade 3 in 10.9% cases (24/220), grade 2 in 21.8% (48/220) cases and grade 1 in 67.3% cases (148/220). thus, the overall additional merit of mrm in spine was seen in 32.7% (72/220) of cases. besides in 14.1% cases (31/220) extra - spinal pathologies were identified.conclusion:2d single thick - slice mrm could have additional merits in spinal imaging when used as an adjunct to routine mr sequences. |
enzymes of the eukaryotic protein kinase superfamily phosphorylate serine, threonine or tyrosine residues in proteins. protein kinases and their substrates form complex networks that regulate essentially every eukaryotic cellular process (1). defects in phosphorylation networks result in numerous disease states, making protein kinases important pharmacological targets. to ensure signalling fidelity, protein kinases act on discrete sets of substrates. two major factors are responsible for substrate recognition (2) : substrate recruitment, encompassing any process that promotes kinase - substrate encounters ; and peptide specificity, the preference for particular residues surrounding the phosphorylation site. we have previously developed a method, named predikin, to predict the peptide specificity of protein serine predikin identifies key conserved substrate - determining residues (sdrs) in the protein kinase substrate - binding pocket. the region of the substrate contacted by these residues corresponds to the heptapeptide sequence comprised of positions 3 to + 3 relative to the phosphorylated residue, so the physicochemical properties of sdrs can be used to predict which heptapeptides are the best substrates for a particular protein kinase. we have recently completely revised and expanded the predikin codebase and provided access to predikin via a new webserver. the predikin webserver is built from three components : (i) predikin.pm, a perl module that provides data and methods for the analysis of protein kinase and substrate sequences ; (ii) predikindb, a database of protein kinases and their substrates and (iii) the website user interface. in this article, we provide a brief description of the methods, capabilities and usage of the predikin webserver. a perl module, predikin.pm, provides data and methods for the analysis of both protein kinase and substrate sequences. the protein kinase is analysed using the following methods : (i) assignment of protein kinase type (serine threonine, cmgc or tyrosine kinase) using a regular expression match based on prosite patterns (4) ; (ii) classification by kinase sequence database (ksd) family (5) ; (iii) classification by panther database family (6) and (iv) identification of the key sdrs. the module makes extensive use of the bioperl library (7), hmm libraries and the hmmer package (8). sdrs in the query kinase are located using an alignment of the kinase sequence with a hmm profile model of the kinase catalytic domain (s_tkc, accession sm00220) from the smart database (9). ksd family is assigned using the hmmer tool hmmpfam to compare the kinase sequence with a set of hmms built from ksd family alignments. panther family is assigned using hmm families and the pantherscore program, both obtained from the panther database website. having characterized the catalytic domain of the query kinase, predikin moves to the next step in the procedure : calculation of kinase - specific weight matrices for substrate prediction. the unique feature of predikin compared with existing methods is that it permits substrate prediction based solely on kinase sequence, as opposed to providing predictions only for a kinase family. predikindb contains three linked tables that describe protein kinases, their substrates and phosphorylation sites. the data in predikindb are derived from the uniprot database using custom parsers written in perl. predikindb is updated automatically at regular intervals using a pipeline of scripts that download uniprot files, parse and generate the database tables. the key feature of predikindb is that where possible, phosphorylation sites are linked with the sequence of the kinase acting at the site. this is achieved by parsing the uniprot mod_res line for a kinase name (e.g. by pka) and comparing it with a list of gene names for kinases from the same organism as the substrate sequence. predikindb currently contains 2335 serine, threonine and tyrosine residues that are annotated as phosphoresidues (with uniprot evidence level potential) in 1116 proteins and that are linked to a specific protein kinase sequence. 11 999 sites are also annotated as experimental by the phospho.elm database (10), of which 690 are linked to a kinase. linking phosphorylation sites with kinase sequences allows the retrieval of phosphorylation sites from the database, where (i) the kinase is known ; (ii) the phosphorylation site is annotated with high confidence and (iii) the kinase has similarity in the catalytic domain (as measured by sdrs, ksd or panther family) to those of the query kinase. users can specify a minimum confidence value for the phosphorylation sites used in scoring matrices (phospho.elm experimental ; uniprot experimental, by similarity, probable or potential) and can also specify that only non - redundant sites be retrieved (homology reduction). the sites are then aligned and used to construct position weight matrices (figure 1) by comparing the frequency of an amino acid at each position in the alignment with the frequency in all substrate sequences for the type (serine threonine, cmgc or tyrosine kinase) of the query kinase. the matrices can then be used to score potential phosphorylation sites in putative substrates of the kinase. figure 1.frequency (upper) and weight (lower) matrices generated by predikin to score potential substrates of protein kinase cla4p from saccharomyces cerevisiae, using the method of classification by ksd family. frequency (upper) and weight (lower) matrices generated by predikin to score potential substrates of protein kinase cla4p from saccharomyces cerevisiae, using the method of classification by ksd family. the predikin.pm perl module provides methods to score potential phosphorylation sites using the weight matrices generated for the query kinase sequence. the user uploads putative substrates in fasta format, from which all peptides with the sequence xxx[st]xxx (serine threonine and cmgc kinases) or xxxyxxx (tyrosine kinases) are extracted. these sites can then be scored using one of the sdr, ksd or panther matrices for the query kinase. a cutoff score below which in addition, the disembl (11) and tmhmm (12) packages can be employed as filters to discriminate against putative phosphorylation sites on the basis of low intrinsic disorder and location within a transmembrane helix, respectively. analysis of experimentally validated phosphorylation sites in predikindb shows that over 90% are found in a disordered region as predicted by at least one of disembl 's ; three algorithms and < 0.1% are located in a tmhmm - predicted helix. the output from predikin is a table (figure 2) containing identifiers for the kinase, catalytic domain and substrate, the location of the potential phosphorylated residue, the heptapeptide xxx[sty]xxx and a relative score between 0 and 100 indicating the likelihood of phosphorylation by the kinase. we have performed an evaluation of predikin scores using kinase - substrate pairs from predikindb to determine how well predikin discriminates known phosphorylation sites of a kinase from unknown sites. briefly, sites linked to a kinase were retrieved from predikindb and randomly divided into test (10%) and training (90%) sets. all xxx[sty]xxx sites in each test set substrate were scored by generating a scoring matrix for the corresponding kinase, omitting those sites in the training set linked to the same kinase. known / unknown sites were labelled 1/0, respectively and redundant sites (same peptide, same kinase and so same score) were discarded. the procedure was repeated 100 times to obtain 100 samples of scores and labels for each scoring method / kinase type combination. predikin sdr scores for the protein kinase cla4p from saccharomyces cerevisiae are shown for potential phosphorylation sites in cla4p and yeast protein yol113w. a sample prediction generated by the predikin webserver. predikin sdr scores for the protein kinase cla4p from saccharomyces cerevisiae are shown for potential phosphorylation sites in cla4p and yeast protein yol113w. area under receiver operating curve (aroc) values, obtained by plotting true positive (annotated sites) versus false positive (unannotated sites) rates as the score threshold is successively lowered (13) ranged from 0.71 0.98 sd (tyrosine kinases, ksd scores) to 0.93 0.02 sd (cmgc kinases, sdr scores), depending on the predikin scoring method used and the kinase type. detailed comparison with other methods (gps, kinasephos, netphosk, ppsp and scansite) is beyond the scope of this article ; a preliminary aroc analysis indicates that predikin performs as well or better than other phosphorylation site predictors. however, we emphasize that such comparisons are of limited value, particularly as the other methods can only assign a kinase family to a query substrate, whereas predikin predicts substrates based on solely on query kinase sequence. the predikin webserver user interface is built using the open - source joomla content management system (cms ; http://www.joomla.org). the joomla cms provides a convenient modular approach to website design, making it easy to add features such as user management, custom forms, documentation and discussion forums. joomla is written in php and so the pecl php embedded perl extension (http://pecl.php.net/perl) is employed to allow communication between the webserver and the predikin perl module. the webserver is primarily designed for users interested in a small set of kinases and potential substrates, identified in an experimental screen. however, users can acquire a large set of substrate predictions for a kinase quite rapidly, since all predictions for a session are stored in a temporary database table and can be exported as comma - separated text for easy import to other applications and further analysis. users with more complex requirements (such as genome - scale prediction of substrates for kinases) may wish to use the standalone predikin perl module and are encouraged to contact us for more information. a perl module, predikin.pm, provides data and methods for the analysis of both protein kinase and substrate sequences. the protein kinase is analysed using the following methods : (i) assignment of protein kinase type (serine threonine, cmgc or tyrosine kinase) using a regular expression match based on prosite patterns (4) ; (ii) classification by kinase sequence database (ksd) family (5) ; (iii) classification by panther database family (6) and (iv) identification of the key sdrs. the module makes extensive use of the bioperl library (7), hmm libraries and the hmmer package (8). sdrs in the query kinase are located using an alignment of the kinase sequence with a hmm profile model of the kinase catalytic domain (s_tkc, accession sm00220) from the smart database (9). ksd family is assigned using the hmmer tool hmmpfam to compare the kinase sequence with a set of hmms built from ksd family alignments. panther family is assigned using hmm families and the pantherscore program, both obtained from the panther database website. having characterized the catalytic domain of the query kinase, predikin moves to the next step in the procedure : calculation of kinase - specific weight matrices for substrate prediction. the unique feature of predikin compared with existing methods is that it permits substrate prediction based solely on kinase sequence, as opposed to providing predictions only for a kinase family. predikindb contains three linked tables that describe protein kinases, their substrates and phosphorylation sites. the data in predikindb are derived from the uniprot database using custom parsers written in perl. predikindb is updated automatically at regular intervals using a pipeline of scripts that download uniprot files, parse and generate the database tables. the key feature of predikindb is that where possible, phosphorylation sites are linked with the sequence of the kinase acting at the site. this is achieved by parsing the uniprot mod_res line for a kinase name (e.g. by pka) and comparing it with a list of gene names for kinases from the same organism as the substrate sequence. predikindb currently contains 2335 serine, threonine and tyrosine residues that are annotated as phosphoresidues (with uniprot evidence level potential) in 1116 proteins and that are linked to a specific protein kinase sequence. 11 999 sites are also annotated as experimental by the phospho.elm database (10), of which 690 are linked to a kinase. linking phosphorylation sites with kinase sequences allows the retrieval of phosphorylation sites from the database, where (i) the kinase is known ; (ii) the phosphorylation site is annotated with high confidence and (iii) the kinase has similarity in the catalytic domain (as measured by sdrs, ksd or panther family) to those of the query kinase. users can specify a minimum confidence value for the phosphorylation sites used in scoring matrices (phospho.elm experimental ; uniprot experimental, by similarity, probable or potential) and can also specify that only non - redundant sites be retrieved (homology reduction). the sites are then aligned and used to construct position weight matrices (figure 1) by comparing the frequency of an amino acid at each position in the alignment with the frequency in all substrate sequences for the type (serine threonine, cmgc or tyrosine kinase) of the query kinase. the matrices can then be used to score potential phosphorylation sites in putative substrates of the kinase. figure 1.frequency (upper) and weight (lower) matrices generated by predikin to score potential substrates of protein kinase cla4p from saccharomyces cerevisiae, using the method of classification by ksd family. frequency (upper) and weight (lower) matrices generated by predikin to score potential substrates of protein kinase cla4p from saccharomyces cerevisiae, using the method of classification by ksd family. the predikin.pm perl module provides methods to score potential phosphorylation sites using the weight matrices generated for the query kinase sequence. the user uploads putative substrates in fasta format, from which all peptides with the sequence xxx[st]xxx (serine threonine and cmgc kinases) or xxxyxxx (tyrosine kinases) are extracted. these sites can then be scored using one of the sdr, ksd or panther matrices for the query kinase. a cutoff score below which in addition, the disembl (11) and tmhmm (12) packages can be employed as filters to discriminate against putative phosphorylation sites on the basis of low intrinsic disorder and location within a transmembrane helix, respectively. analysis of experimentally validated phosphorylation sites in predikindb shows that over 90% are found in a disordered region as predicted by at least one of disembl 's ; three algorithms and < 0.1% are located in a tmhmm - predicted helix. the output from predikin is a table (figure 2) containing identifiers for the kinase, catalytic domain and substrate, the location of the potential phosphorylated residue, the heptapeptide xxx[sty]xxx and a relative score between 0 and 100 indicating the likelihood of phosphorylation by the kinase. we have performed an evaluation of predikin scores using kinase - substrate pairs from predikindb to determine how well predikin discriminates known phosphorylation sites of a kinase from unknown sites. briefly, sites linked to a kinase were retrieved from predikindb and randomly divided into test (10%) and training (90%) sets. all xxx[sty]xxx sites in each test set substrate were scored by generating a scoring matrix for the corresponding kinase, omitting those sites in the training set linked to the same kinase. known / unknown sites were labelled 1/0, respectively and redundant sites (same peptide, same kinase and so same score) were discarded. the procedure was repeated 100 times to obtain 100 samples of scores and labels for each scoring method / kinase type combination. predikin sdr scores for the protein kinase cla4p from saccharomyces cerevisiae are shown for potential phosphorylation sites in cla4p and yeast protein yol113w. a sample prediction generated by the predikin webserver. predikin sdr scores for the protein kinase cla4p from saccharomyces cerevisiae are shown for potential phosphorylation sites in cla4p and yeast protein yol113w. area under receiver operating curve (aroc) values, obtained by plotting true positive (annotated sites) versus false positive (unannotated sites) rates as the score threshold is successively lowered (13) ranged from 0.71 0.98 sd (tyrosine kinases, ksd scores) to 0.93 0.02 sd (cmgc kinases, sdr scores), depending on the predikin scoring method used and the kinase type. detailed comparison with other methods (gps, kinasephos, netphosk, ppsp and scansite) is beyond the scope of this article ; a preliminary aroc analysis indicates that predikin performs as well or better than other phosphorylation site predictors. however, we emphasize that such comparisons are of limited value, particularly as the other methods can only assign a kinase family to a query substrate, whereas predikin predicts substrates based on solely on query kinase sequence. the predikin webserver user interface is built using the open - source joomla content management system (cms ; http://www.joomla.org). the joomla cms provides a convenient modular approach to website design, making it easy to add features such as user management, custom forms, documentation and discussion forums. joomla is written in php and so the pecl php embedded perl extension (http://pecl.php.net/perl) is employed to allow communication between the webserver and the predikin perl module. the webserver is primarily designed for users interested in a small set of kinases and potential substrates, identified in an experimental screen. however, users can acquire a large set of substrate predictions for a kinase quite rapidly, since all predictions for a session are stored in a temporary database table and can be exported as comma - separated text for easy import to other applications and further analysis. users with more complex requirements (such as genome - scale prediction of substrates for kinases) may wish to use the standalone predikin perl module and are encouraged to contact us for more information. the predikin webserver provides user - friendly access to the improved and enhanced predikin prediction system. a number of existing tools such as scansite (14), kinasephos (15), netphosk (16), networkin (17), gps / gps2 (18) and ppsp (19) are available to predict protein kinase substrates. the fundamental difference between these tools and predikin is that analysis using the other tools begins with a substrate sequence, which has to be assigned to a limited number of pre - assigned kinase families. predikin, on the other hand, uses the kinase sequence to build scoring matrices based on key residues in the kinase catalytic domain that are known from structural analysis to interact with the substrate phosphorylation site. it can therefore make substrate predictions for any protein kinase based on sequence alone, provided that phosphorylation sites of similar kinases are present in the predikindb database. new features and enhancements provided by the revised predikin code include (i) more reliable determination of sdrs through the use of profile hmm alignments ; (ii) filters to prescreen potential phosphorylation sites based on accessibility and disorder ; (iii) three methods to generate kinase - specific scoring matrices based on sdrs, ksd or panther family and (iv) use of the predikindb database, which is updated continually with new annotated phosphorylation sites and links sites with specific kinase sequences rather than kinase families, so forming the basis for substrate prediction using kinase features. predikin provides a range of applications, such as predicting candidate substrates for a protein kinase, candidate protein kinases for a substrate and the assignment of protein kinases to their substrates in large datasets. | the predikin webserver allows users to predict substrates of protein kinases. the predikin system is built from three components : a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences ; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. the predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate - determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. the web interface provides user - friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. the server is available at http://predikin.biosci.uq.edu.au. |
ret (rearranged during transfection) encodes a membrane receptor tyrosine kinase (rtk) composed of four extracellular cadherin - like motifs and a cysteine - rich region, a transmembrane portion, and an intracellular domain with tyrosine kinase activity. the ret signaling pathways are outlined in (figure 1). ret signals through a ligand / coreceptor / ret multiprotein complex instead of the usual receptor / ligand binding. to date, several ligands of the glial - derived neurotrophic factor (gdnf) family, which include gdnf, artemin, neurturin, and persephin and a family of gpi - linked ret coreceptors (gfr1 - 4), have been identified. the formation of ligand / coreceptor and ret complexes results in ret dimerization and triggers autophosphorylation at intracellular tyrosine residues. phosphorylated tyrosine 687 (y687), serine 696 (s696), y752, y791, y806, y809, y826, y864, y900, y905, y928, y952, y981, y1015, y1029, y1062, y1090, and y1096 constitute docking sites for numerous intracellular adaptor proteins such as rac1-guanine exchange factor (gef), growth factor receptor - bound (grb) docking proteins grb7/10, chicken rous sarcoma virus oncogene (c - src), focal adhesion kinase (fak), phospholipase c- (plc-) and src homologue collagen (shc), insulin receptor substrate 1/2 (irs1/2), fibroblast growth factor substrate 2 (frs2), or downstream of kinase 1/4/5 (dok1/4/5) (reviewed by de groot.). phosphorylation of intracellular target proteins activates several downstream pathways which include mitogen - activated protein kinase cascade : rat sarcoma oncogene / rapidly accelerated fibrosarcoma / extracellular regulated kinase 1/2 (ras / raf / erk1/2), the phosphatidylinositol 3-kinase / protein kinase b pathway (pi3k / akt) [7, 8 ], the c - jun n - terminal kinase pathway (jnk), p38, enigma extracellular regulated kinase 5 (erk5), the camp - responsive element - binding protein, and the signal transducer and activator of transcription 3 (stat3) (for a review see arighi. and de groot.. more recently, gujral. have shown that ret mediates direct tyrosine phosphorylation of beta - catenin, which associated with an induction of the wnt pathway, that accounts for a part of ret tumorigenic ability in vivo. many of the above - mentioned intracellular signalling pathways are otherwise known to be general signal transducing pathways targeted not only by ret, but by other rtks as well. yet, ret is the main rtk targeted for genetic lesions in thyroid cancer. the transforming ability of activated ret, which was actually on the basis of its isolation as an oncogene, could be attributable to the diversity of its signalling which covers several hallmarks of cancer. increased growth signals and proliferation result from the activation of the ras / raf / erk1/2 cascade and phosphorylation of stat3 [14, 15 ]. cell migration is dependent on ret - mediated activation of rac1 and jnk [3, 16 ], and fak is also reported to play a role in cell migration and to be required for invasion and metastatic behaviour [5, 17 ]. inflammation (regarded as the 7th hallmark of cancer) has also been shown to operate as a major component downstream of oncogenic ret mutations. in freshly isolated human thyrocytes, the activation of ret generates a transcriptional program that is similar to that which occurs during inflammation inducing the expression of various inflammatory factors [1921 ]. furthermore, key protein components of the ret - activated inflammatory program were found in tumor specimens taken by biopsy, and larger amounts of these inflammatory molecules were found in the primary tumors of patients with lymph - node metastasis than in primary tumors in the absence of lymph - node metastasis (reviewed in). these and other results ([23, 24 ] ;) connect the activation of ret to inflammation. overall, as stated before, varied signalling properties, covering multiple hallmarks of cancer, might afford explanation for the ability of ret to transform certain cell types. nonetheless, the most solid grounds for the significance of ret as a cancer gene come from the fact that, when inherited, an ret germline point mutation alone primes a specific spectrum of tissues to develop endocrine tumors [26, 27 ]. carriers of ret germline mutations develop hereditary medullary thyroid carcinoma (hmtc) as the first and most common clinical presentation. along with hmtc, patients present with pheochromocytoma (tumor of the adrenal medulla) and parathyroid adenomas. this syndromic condition is referred to as multiple endocrine neoplasia type 2 (men2). penetrance for hmtc is near complete, which highlights the critical role of ret activation in the development of mtc and can be further taken to pinpoint ret as a relevant therapeutic target for mtc. in hmtc, ret mutations occur in a specific spectrum of codons and result in gain of function, increased kinase activity, and receptor activation. mutational hotspots are located at the cysteine - rich region of the extracellular domain and in the intracellular tyrosine kinase domain. the clustering of mutations in hotspots might be explained by the fact that proto - oncogene activation requires changes at residues that specifically interact in specific ways with receptor function, and thus mutations can not occur in a widespread manner. a comprehensive description of all known germline ret variations can be found at the men2 ret database (http://www.arup.utah.edu/database/men2/men2_welcome). the most common ret germline mutations are missense substitutions of extracellular cysteine residues, occurring at cysteine codon 634 in 80% of cases. cysteine codons 609, 611, 618, 620, and 630 are less frequently affected. other noncysteine extracellular domain mutations, located at exons 5 and 8, have been detected. tyrosine kinase domain mutations affect a more varied spectrum of amino acids, and most frequently recurring mutations replace met918, val804, leu790, tyr791, and ala883. less frequently, residues 768, 876, 891, 886, and 912 are affected. rare mutations found in isolated families have been reported, comprising homozygous mutations, duplications, and double mutations. besides the point mutations found in mtc, an alternative somatic genetic event that causes ret activation is found in the papillary type of thyroid carcinoma (ptc). this involves chromosomal translocations between ret and a number of other loci, referred in general as ret / ptc rearrangements, which interestingly occur as alternative events to the v600e somatic braf mutation. in men2 there are consistent genotype / phenotype correlations that underlie aspects such as clinical manifestation, ret activation mechanisms, and disease penetrance, allowing for a mutation - specific classification of men2. in clinical terms, three disease phenotypes can be recognized : men2a, men2b, and a familial form of medullary thyroid carcinoma (fmtc). men2a was found to be associated with substitutions at one of six specific cysteine residues in exons 10 (609, 611, 618, 620) and 11 (630 and 634). men2a cysteine mutations give rise to mtc at young age (onset at 5 to 25 years), along with variable expression of pheochromocytoma (50%) and hyperparathyroidism (1530%). men2b, on the other hand, is mainly caused by a specific missense mutation located at the ret tyrosine kinase domain (met918thr), which accounts for 95% of cases. a second tyrosine kinase domain substitution (ala883phe) has been detected in a small proportion of men2b patients. additionally, double mutations affecting codons 804 and 805 and 804 and 806 were described in individual men2b cases [33, 37 ]. men2b kinase domain mutations give rise to a more complex clinical phenotype characterized by an early onset (sometimes < 1 year old) and very aggressive form of mtc, concomitant with pheochromocytoma in 50% of cases and accompanied by other nonneoplastic features, such as mucosal neuromas of the tongue, lips, and eyelids, ganglioneuromatosis of the gastrointestinal tract, thickening of corneal nerves, and marfanoid habitus. in fmtc the only disease manifestation is mtc, which usually occurs in adult age, with no additional endocrinopathies. ret mutations with low clinical expression, involving codons 321, 533, 768, 790, 791, 804, and 891, may be found in these families. occasionally, patients with these mutations may also develop the men2a phenotype, showing that fmtc and men2a represent a continuum of clinical expression in a common genetically related disorder [3942 ]. age - dependent penetrance for mtc in men2 is also codon specific, and classification of the risk of developing mtc can be done based on the genotype (reviewed by raue and frank - raue in). this is of clinical relevance because the ideal timing of prophylactic thyroidectomy should take into consideration the balance between the adverse effects of thyroidectomy at early ages and the individual risk of developing mtc. comprehensive guidelines have been issued by the american thyroid association concerning this aspect. in general, ret mutations with a very high risk of producing mtc (risk level d), comprising all the men2b mutations, require surgery before 1 year of age. ret mutations at codon cys634 constitute risk level c and are managed by thyroidectomy before 5 years old. level b mutations encompass the changes in the remaining extracellular cysteine codons 609, 611, 618, 620, and 630. in these cases, surgery is advised before 5 years old ; however it can be postponed until calcitonin level rise. risk level a accounts for the fmtc mutations, for which surgery before 5 years old is not required and can be delayed until calcitonin levels rise. aside from germline mutations, a somewhat similar spectrum of somatic mutations is observed in about 50 to 60% of the cases with sporadic mtc. a catalogue of somatic mutations can be found at the cosmic database (http://www.sanger.ac.uk/genetics/cgp/cosmic/). the most frequent somatic lesion is the prototypic men2b met918thr mutation at exon 16, which comprises up to 60% of the mutation positive cases. moreover, patients in which tumors harbor men2b mutations have a higher prevalence and number of lymph node metastases, present more often with multifocal tumors and with persistent disease at advanced stage, indicating that among the sporadic mtcs, cases with somatic men2b mutations are associated with the worst prognosis [45, 46 ]. interestingly, cases with ret mutations at the cysteine cluster have the most indolent course, and those with no ret mutations have an intermediate risk. the functional basis for the differences in clinical expression of distinct ret genotypes might be explained by the recognition of mutation - specific mechanisms of activating the ret proto - oncogene. mutations in the extracellular cysteine - rich region result in the replacement of a cysteine residue by another amino acid, subsequently leading to loss of an intramolecular disulfide bond. as a consequence, one cysteine residue becomes available for the formation of an intermolecular disulfide bond, which results in covalently bound receptors that are constitutively active because of ligand - independent receptor dimerization. in contrast, the intracellular men2b - specific mutations and other tyrosine kinase domain mutations affect receptor activation in a totally different way. by altering the conformation of the catalytic core of the tyrosine kinase domain they increase catalytic activity and alter the spectrum of intracellular substrates, resulting in remarkable changes of the signalling properties of the receptor. these observations highlight that distinct clinical presentations can arise from differences in the ret activation mechanism and the corresponding output in terms of oncogenic signalling. however, not much is known about the specific differences in signalling properties of the different ret mutants. studies have shown that wild - type and mutated ret display differences in the autophosphorylation levels of docking sites, which are likely to lead to differential activation of downstream cascades. support for this paradigm comes from evidence that there are marked differences between men2a and men2b mutations in terms of their capacity for downstream pi3k / akt activation. this pathway seems to be more active in men2b than in men2a, and this difference might be attributed to an enhanced autophosphorylation of y1062 caused by the men2b mutation. however, the fmtc mutants, tyr791phe and ser891ala, seem to do so through a different route and need the involvement of src and jaks in order to constitutively activate stat3. thus, on the basis of the above - mentioned evidence that distinct signalling properties are displayed by ret mutants, it is conceivable that different sensitivity to the action of tyrosine kinase inhibitors can occur due to the potentially different conformations of the receptor in each of the ret mutants. the small molecule tyrosine kinase inhibitors (tkis) mechanism of action is based on the principle that sterically blocking the atp - binding pocket results in impaired phosphorylation activity, inhibits signal transduction, and prevents activation of intracellular signalling pathways relevant to tumor growth and angiogenesis. the finding of various compounds (table 1) capable of inhibiting oncogenic ret (mutated or rearranged), such as pp1 and pp2, zd6474 (vandetanib), rpi-1, cep-701, cep-751, imatinib, sunitinib (su5416, su11248), gefitinib, sorafenib (bay 43 - 9006), motesanib (amg706), axitinib (ag013736) and xl 184, has brought further clinical relevance to the classification of the pharmacological sensitivity of ret mutants, as metastatic mtc is the most common cause of death in patients with men2. in addition, these compounds could find application in radioactive iodine - refractory ptc with ret / ptc rearrangements. the pyrazolopyrimidines pp1 and pp2 and the 4-anilinoquinazoline vandetanib inhibit ret - rearrangement - derived oncoproteins with a half maximal inhibitor concentration (ic50) below 100 nm. these molecules were shown to inhibit ret enzymatic activity and phosphorylation of downstream targets, such as erk1/2. vandetanib has also been found to inhibit ret signalling in two human ptc cell lines and to reduce tumorigenicity of ret / ptc - transformed fibroblasts injected into nude mice. vandetanib blocks in vivo phosphorylation and signalling mediated by ret / ptc3 oncoprotein and of an epidermal growth factor- (egf-) activated egf - receptor / ret chimeric receptor. finally, it blocks anchorage - independent growth of ret / ptc3-transformed nih3t3 fibroblasts and the formation of tumors after injection of nih - ret / ptc3 cells into nude mice. nonetheless, preclinical studies have shown that sorafenib can inhibit the kinase activity and signalling of wild - type and oncogenic ret. sorafenib inhibited oncogenic ret kinase activity at an ic50 of 50 nm or less in nih3t3 cells. it arrested the growth of nih3t3 and rat1 fibroblasts transformed by oncogenic ret and of thyroid carcinoma cells that harbour rearranged. finally, ptc cells carrying the ret / ptc1 rearrangement were found to be more sensitive to sorafenib than ptc cells carrying a braf mutation. there is an ongoing phase ii clinical trial using sorafenib in patients with advanced thyroid cancer. rpi-1 is a 2-indolinone derivative initially shown to inhibit ret / ptc1 activity in an immunokinase assay with an ic50 of 2742 m. it selectively inhibited the anchorage - independent growth of nih3t3-transformed cells expressing the ret / ptc1 gene, and the transformed phenotype of nih3t3ptc1 cells was reverted to a normal fibroblast - like morphology. in these cells, the constitutive tyrosine phosphorylation of ret / ptc1, of the transducing adaptor protein shc, and of a series of co - immunoprecipitated peptides was substantially reduced. activation of jnk2 and akt was abolished, thus supporting the drug inhibitory efficacy on downstream pathways. in addition, cell growth inhibition was associated with a reduction in telomerase activity by nearly 85%. sunitinib was initially described as a tki targeting vegf and pdgfr receptors and also found to inhibit c - kit. it is now approved for the treatment of gist and renal cell carcinoma. in vitro kinase assays showed that sunitinib inhibited the phosphorylation by ret / ptc3 of a synthetic tyrosine kinase substrate peptide in a dose - dependent manner. ret / ptc - mediated y705 phosphorylation of stat3 was inhibited by addition of sunitinib, and the inhibitory effects of sunitinib on tyrosine phosphorylation and transcriptional activation of stat3 very closely correlated with decreased autophosphorylation of ret / ptc. sunitinib caused a complete morphological reversion of transformed nih - ret / ptc3 cells and inhibited the growth of tpc-1 cells that have an endogenous ret / ptc1. treatment of two patients with progressive metastatic thyroid carcinoma (1 ptc and 1 ftc) demonstrated sustained clinical responses to sunitinib over a period of four years. gefitinib was initially approved for nonsmall cell lung cancer since it targets oncogenic egfr. in vitro data conditional activation of ret / ptc oncoproteins in thyroid pccl3 cells markedly induced expression and phosphorylation of egfr, which was mediated in part through mitogen - activated protein (map) kinase signalling. ligand - induced activation of egfr resulted in phosphorylation of a kinase - dead ret, and this effect was entirely blocked by egfr kinase inhibitor. gefitinib also inhibited cell growth induced by various constitutively active mutants of ret in thyroid cancer cells as well as in nih3t3 cells. these pieces of evidence have provided a biological basis for clinical evaluation of gefitinib in thyroid cancer. the results obtained in a phase ii trial showed no objective responses among the 25 thyroid cancer patients treated with gefitinib. effective inhibition of ret phosphorylation in a dose - dependent manner is achieved at concentrations < 100 nm. these compounds also block the growth of mtc cells in culture. these drugs also potentiate the effects of irinotecan treatment in tt cell culture and xenografts and result in durable complete remission in 100% of the mice. cep-751 inhibited the induction of the dna repair program (marked by phospho - h2ax) as well as the checkpoint pathway (marked by the activated chk1). since preclinical models have demonstrated that both cep-751 and cep-2563 have antitumor activity in a variety of tumors, phase i trials were undertaken. several other tki molecules are being evaluated with regard to their efficacy in metastatic mtc treatment with limited published data. axitinib (ag-013736) was assessed in a phase ii study with 60 mtc patients. eighteen cases (30%) presented partial responses, and 23 (38%) had stable disease. motesanib (amg706) was evaluated in differentiated thyroid cancer and in a phase i study in 91 patients with either hereditary (16 cases) or sporadic mtc (75 cases), 2% of the patients showed partial response, and 81% had stable disease. it is a tki that targets vegfr2, ret, and also met and whose efficacy has been demonstrated for several solid tumors, especially thyroid cancer []. in patients with hereditary and sporadic mtc very interesting response rates were obtained with 9/17 patients (53%) showing partial remission. based on these findings, a phase iii registration trial of xl184 as a potential treatment for medullary thyroid cancer (mtc) has been initiated. although a number of patients with refractory mtc have been undergoing treatment with several tkis in the last few years, it is not yet clear whether clinical response to these drugs is actually influenced by the ret genotype of the tumor cells. at this point, indeed, some compounds used against ret seem to confirm the paradigm that certain mutations can render ret resistant to inhibition. this was first illustrated by pp1, pp2, and zd6474 (vandetanib) which, despite being efficient in inhibiting phosphorylation of most of the men2-associated ret mutants (at codons 768, 790, 883, 918, and 634), were incapable of inhibiting men2-associated swap of valine 804 for bulky hydrophobic leucine or methionine within the ret kinase domain. thus valine 804 emerged as a structural determinant amino acid mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines [78, 79 ]. this was also found to be the case for the v804m / e805k tandem lesion, detected in non - met918/ala883 men2b, which was shown to also confer resistance to pp1, suggesting a mode of action different from the classical men2b mutations. however, inhibition of ret phosphorylation and signaling by mutation of the val804 gatekeeper residue was not impaired in cells subjected to sorafenib treatment, indicating that this drug could be a potential therapeutic tool for ret val804 positive thyroid tumors. the fact that using another compound can overcome a mutation - specific primary resistance renders further support to the idea that sensitivity of ret mutants will, in the end, result from mutation - dependent structural determinants of the ret atp - binding site. however, to support the paradigm of an ret pharmacogenetics, much more needs to be evaluated before we can confirm that this concept is useful for the clinical practice. to start, it would be imperative that the mutation status of the tumors from patients included in clinical trials is ascertained and correlated with clinical response. until now, none of the clinical studies have published the mutation status of the patients. on the other hand, we must not forget that despite the in vitro data has proven highly informative for genotype / phenotype correlations, it can not be taken directly to indicate differences in terms of clinical response. in addition, many of these small molecule inhibitors act upon several target rtks, rendering it difficult to ascertain which of the effects over different rtks actually accounts for the observed clinical response. we should also be aware that some of the effects of these compounds may go beyond interference with the atp - binding pocket and may affect ret expression. for instance, sorafenib suppresses ret tyrosine kinase activity by direct enzymatic inhibition and also by promoting ret lysosomal degradation independent of proteasomal targeting. at this point, given that a number of molecules are starting to become available, it would be worth to compare these drugs against each other in their efficacy to inhibit the activity of the most frequent ret genotypes. this may come as a means to define and stratify drugs for use as first - line and second - line treatments on the basis of the ret genotype. as we highlighted before, specific ret mutations may lead stronger induction of specific intracellular signalling targets, many of which have their own dedicated inhibitors under development. in this respect, the information about the specificities in oncogenic signalling of different genotypes might be valuable to design combinatorial therapies employing mutation - specific combinations of inhibitors for treatment. at present, the clinical use of tyrosine kinase inhibitors in patients with thyroid cancer still does not rely in the genetic background of each tumor [58, 61, 81 ]. nonetheless, results from clinical trials suggest that these compounds have a more cytostatic than cytolytic effect, and thus are just adding another step of selective pressure to the progressing tumor (which buys time), but eventually secondary resistance can develop. in models such as abl / cml (imatinib), egfr / lung cancer (gefitinib), or kit / gist (imatinib), prolonged therapy with tkis leads to the acquisition of resistance mutations in the receptors targeted by these drugs, rendering them insensitive to therapy. although no secondary ret mutations have been described thus far, the experience with patients undergoing clinical trials taught that some patients suddenly fail to respond while on treatment. this implies that, in order to translate the use of these inhibitors into increased long - term survival, we may need to perform molecular followup of the progressing lesions, in order to predict resistance and eventually change from one inhibitor to another. finally, to reduce the biology of mtc to ret activation and signaling boosting is almost certainly a simplistic view. likely these tumours also carry mutations in other genes, and possibly one should also know these to think about combinatory therapies. indeed, data is accumulating regarding alternative pathways that contribute to mtc development from precursor c - cell hyperplasia. this is the case of the wnt pathway activation by ret - mediated tyrosine phosphorylation of -catenin and the synergistic effects of p18 and p27, two members of the rb pathway [82, 83 ]. this may provide additional targets for combination of ret inhibitors with other compounds targeting these pathways. also relevant to this the challenge for the years to come is to use the pools of knowledge generated in ret signaling pathways and mtc progression steps to rationalize combinatory therapies, targeting different molecules and different signaling pathways that are relevant in mtc. | the significance of ret in thyroid cancer comes from solid evidence that, when inherited, an ret activating mutation primes c - cells to transform into medullary carcinomas. moreover, environmental exposure to radiation also induces rearranged transforming ret isoforms that are found in papillary thyroid cancer. the ret gene codes for a tyrosine kinase receptor that targets a diverse set of intracellular signaling pathways. the nature of ret point mutations predicts differences in the mechanisms by which the receptor becomes activated and correlates with different forms of clinical presentation, age of onset, and biological aggressiveness. a number of ret - targeting tyrosine kinase inhibitors (tkis) are currently undergoing clinical trials to evaluate their effectiveness in the treatment of thyroid cancer, and it is conceivable that the ret genotype may also influence response to these compounds. the question that now emerges is whether, in the future, the rational for treatment of refractory thyroid cancer will be based on the management of an abnormal ret signal. in this paper we address the ret - targeting tkis and review studies about the signaling properties of distinct ret mutants as a means to predict response and design combinatorial therapies for the soon to be available tkis. |
bile duct cancer has been associated with a poor prognosis ; node metastasis is a particularly important poor prognostic factor [16 ]. despite advances in the diagnosis and treatment of cholangiocarcinoma, such as endoscopic biliary drainage, surgical procedures, and chemotherapy, long - term outcomes several reports of recurrence in long - term survivors have also been published [79 ]. recurrence usually occurs at the margin of the bile duct, in the peritoneum, or in the liver [1, 2 ]. this report describes a patient with hilar cholangiocarcinoma who underwent resection of 11th rib metastasis 10 years after curative resection of the primary tumor. it mainly affected the right hepatic duct, but also extended to the left hepatic duct and proximal common bile duct. percutaneous transhepatic cholangio drainage tubes were inserted into the anterior brunch and posterior brunch approached from the 7th intercostal space and into the posterior brunch approached from the 9th intercostal space. according to the sixth edition of the uicc tnm classification, the tumor was classified as pt3 pn1 pm0 pstage iii. in addition, according to the fifth edition of the general rules for biliary tract cancer by the japanese society of biliary surgery, the tumor was described as bcrism, nodular - infiltrating type, 2.2 cm, moderate- to well - differentiated adenocarcinoma (tub2 > tub1), pat, sci, infb, ly1, v0, pn0, hinf1, ginf0, panc0, du0, hm1, dm1, em1, pv0, a0, n2 (12c 1/1, 12p 1/8), pt4 pn2 m0 fstage ivb, final curability b. four months after the initial operation, the patient underwent eight courses of hepatic arterial injection of fluorouracil (5fu) (1,500 mg) every week as adjuvant chemotherapy. thereafter, three courses of systemic adjuvant chemotherapy with methotrexate (50 mg), cisplatin (20 mg), and 5fu (750 mg) on days 1, 8, and 15 were administered every 6 months, along with radiation therapy to the hilar region at a dose of 56 gy. after these adjuvant therapies, the patient continued to be followed up with serum carcinoembryonic antigen (cea) and carbohydrate antigen 19 - 9 (ca 19 - 9) testing every 2 months and computed tomography (ct) every 6 months for 10 years ; she showed no signs or symptoms of recurrence over the next 10 years. in september 2008, a sudden increase of the serum ca19 - 9 level to 68 u / ml (normal range 5 years) survivors after curative resection for intrahepatic bile duct cancer have been published [79 ]. in most of these cases, the recurrence occurred at the surgical margins of the bile ducts or in the peritoneum(table 1). these reports were subject to intrahepatic bile duct cancer, but no report has published in hilar or extra bile duct cancer. solitary recurrences outside the peritoneal cavity are not common, and bone metastasis is rare. in terms of other gastroenterological cancers, long - term survival rates have been reported in only a few studies [1618 ]. for gastric cancer, moon. reported that a 10-year long - term recurrence after surgery occurred in 2.0 % of patients. in terms of the recurrence pattern, in contrast, distant metastasis was the main relapse pattern during 510 years post - gastrectomy and after 10 years post - gastrectomy. until now, there have been no reports of bone metastasis in long - term survivors by pubmed searches using cholangiocarcinoma, bone metastasis, and long - term survivor as searchable terms in any entire period.table 1the recurrence of bile duct cancer was detected in long - term survivors > 5 years after the curative resectionno. authorage (years)/genderlength (years)primary lesionpathologyadjuvant chemotherapylocation of recurrence1machimotom74/f12hilartub1oral uft for 5 yearsabdominal wall2sasaki45/m9mbdtub2nocholedochojejunostomy region3tanaka53/m10lbdpapnocholedochojejunostomy region4our case60/f10hilartub2hai uft 4 weeks for 1 year11th ribmtx + cddp + 5fu 6 months for 1 year 6 months with radiation for 56 gy mbd middle bile duct, lbd lower bile duct, uft uracil tegafur, hai hepatic arterial injection, mtx methotrexate, cddp cisplatin, 5fu fluorouracil the recurrence of bile duct cancer was detected in long - term survivors > 5 years after the curative resection mbd middle bile duct, lbd lower bile duct, uft uracil tegafur, hai hepatic arterial injection, mtx methotrexate, cddp cisplatin, 5fu fluorouracil recurrences many years after the treatment may be related to long - lasting tumor dormancy being turned on, especially in distant organs [1921 ]. no change ; the tumor does not disappear, but remains at the same size for a long period of time. in many types of cancer, improvements in outcome have been reported when the tumor is in an immutable state. takahashi. reported that tumor dormancy is induced by chemotherapy, and patients can survive for long periods without signs of disease recurrence, even if the tumor remains. in our case, it might be estimated that adjuvant chemotherapy induced tumor dormancy ; therefore, our patient was alive without recurrence for a long period of time. surgical stress might also be associated with tumor metastasis. in this case, although solitary rib metastasis was apparent, some other dormant cancer cells might exist systemically and be activated by the surgical stress. kato. reported efficacy of downsizing chemotherapy in unresectable locally advanced cholangiocarcinoma. these findings seemed to have a choice of resection if the tumor was controlled by chemotherapy several times. our case was not strongly suspicious of metastasis, but chemotherapy may have been a reasonable strategy if cancer had been confirmed in biopsy. steffen. reported that in melanoma, the tumor immune system dynamic is critically important in determining tumor regrowth after resection. adjuvant immunotherapy using polysaccharide - k (krestin) reportedly had a survival benefit in gastric and colorectal cancer, and the host immune status was considered to be one of the prognostic factors. more recently, in ovarian cancer, neurobehavioral stress and stress - associated hormones such as norepinephrine, epinephrine, and cortisol have been shown to be associated with increased tumor growth and metastasis. tumors may rapidly grow from various causes, such as changes in immune function and physical condition. in this case, however, there was no deterioration of immune function, weight loss, or deterioration of nutritional status in the 10 years that the patient was observed. such cases keenly highlight the difficulty of achieving cures in patients with bile duct cancer. although several possibilities have been considered, factors predictive of long - term recurrence of bile duct cancer remain unclear. in cholangiocarcinoma, ca19 - 9 the diagnostic potential for primary lesions is almost equivalent between pet and ct, whereas pet is superior in the diagnosis of new metastatic lesions. anderson. reported that 30 % of patients evaluated for suspected cholangiocarcinoma had their therapy plans altered because of detection of unsuspected metastases on fdg - pet. the present patient was followed up for > 10 years after curative resection of the primary tumor because she had positive nodal metastasis. close follow - up was performed for > 5 years after the initial operation with serum ca19 - 9 testing every 2 months and ct every 6 months. in this case, the metastatic lesion was suspected based on the elevation of ca19 - 9. thus, solitary rib metastasis, which could not be found by ct, was detected by pet. long - term survival patients with cholangiocarcinoma may require continual long - term surveillance of tumor markers such as cea or ca19 - 9, and active implementation of pet might be beneficial when these tumor markers are elevated. in conclusion, in patients with bile duct cancer, it must be emphasized that long - term surveillance is required even in patients without recurrence for > 5 years after curative resection of the primary tumor, even if there are no early signs of recurrence. | abstract a 61-year - old female underwent right hemihepatectomy and caudate lobectomy for hilar cholangiocarcinoma in 1999. ten years later, increasing serum carbohydrate 19 - 9 was detected by routine follow - up. subsequent positron emission tomography revealed an asymptomatic lesion in the right 11th rib. as the mass steadily grew in size, the lesion was resected en bloc with the affected rib and muscle. the histopathological findings closely resembled those of the primary cholangiocarcinoma. thus, the tumor was diagnosed as a metastatic recurrence 10 years after resection of the primary tumor. there have been a few reports of cholangiocarcinoma recurrence in long - term survivors at the surgical margins, peritoneum, or transhepatic drainage route. however, there are no reports of solitary extra - abdominal recurrence. this case highlights the need for careful follow - up of patients with cholangiocarcinoma and nodal metastasis, even in the absence of recurrence for > 5 years after curative resection. |
silica is a toxicant that can stimulate cells to produce various cellular products such as free radicals, cytokines, and growth factors. silica and its induced substances may induce apoptosis to regulate the evolution of silica - induced inflammation and fibrosis. to examine this hypothesis, groups of wistar male rats were intratracheally instilled with different doses of min - u - sil 5 silica (silica, berkeley springs, wv). ten days after the instillation, we obtained cells by bronchoalveolar lavage and placed them on slides by cytospin preparation. the slides were stained with diff - quik (lab aids, sydney, nsw, australia) and examined under oil immersion. a substantial number of cells with apoptotic features were identified in all silica - instilled rats and the apoptosis was confirmed by agarose gel electrophoresis. the number of apoptotic cells was clearly related to silica dosage. engulfment of apoptotic cells by macrophages was also noted. neutrophil influx in silica - instilled rats could be saturated with the increase of silica dosage and the number of macrophages in different dose groups changed in parallel with the proportion of apoptotic cells. fifty - six days after instillation, morphologically apoptotic cells could be identified in granulomatous cells of lung tissue from silica - instilled rats. we conclude that intratracheal instillation of silica could induce apoptosis in both alveolar and granulomatous cells, and the apoptotic change and subsequent engulfment by macrophages might play a role in the evolution of silica - induced effects.imagesfigure 1.figure 3.figure 4.figure 5.figure 7.figure 8. |
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the estimated prevalence has been reported from 16.8% to 58%.15 frequent drooling may cause skin maceration and infection, body fluid loss, and recurrent pneumonia.6,7 at school and at home, children with salivary secretions may cause damage to books, teaching materials and furniture, and it even interferes with social relationships.6,8,9 van der burg,10 reported that children with cp that drool are often avoided by other children, and familiar and unfamiliar adults (including their parents). hockstein reported that drooling in children with cp could interfere with their education and increase their dependent level of care. such studies suggest that drooling might be associated with a reduced quality of life among children with cp. previous studies have shown that there are many factors that interfere with the health - related quality of life (hrqol) in children with cp including : motor, cognitive, language, and social impairment.1114 although the prevalence of drooling is high among children with cp, there are few articles regarding its relationship to the hrqol in these children. most prior studies8,10,15 have used modified questionnaires or qualitative methods to evaluate the relationship between drooling and hrqol. in this study, standardized measurement of the hrqol in children with cp, with and without drooling, was investigated. in addition, the relationship between drooling and hrqol was evaluated, as well as the factors that predict the variability of hrqol in these children. children with cp that attended one medical center hospital and two early intervention institutions at daytime for an early intervention and habilitation program in taiwan were enrolled. children with cp, aged 2 to 6 years, without drooling (with a drooling ranking score of 2 according to the drooling rating scale developed by thomas - stonell and greenberg16), were the control group. children with cp, aged 2 to 6 years, with drooling (a drooling ranking score > 2), were the study group. the exclusion criteria were : (1) children with cp combined with other problems such as congenital malformation or metabolic disorder, (2) children taking anticholinergic drugs over the past 2 months, and (3) children with an acute infection or other systemic disease. children with cp were enrolled consecutively from february 2011 to july 2011. the study was conducted according to the criteria of the declaration of helsinki and the review board of the university hospital approved this study. forty - seven children were included in the study : 14 did not drool (mean age : 43.2 13.8 months, diplegia 17%, quadriplegia 12.8%) and 33 did drool (mean age : 48.9 14.4 months, diplegia 10.6%, quadriplegia 59.6%). in the group without drooling, there were eight boys and six girls, and in the group with drooling, there were 22 boys and 11 girls (table 1). previous studies4,17 have reported that drooling in infancy usually resolves by 18 months of age. the children that drool beyond 4 years of age are considered abnormal and require further treatment. therefore, the participants were divided into groups ; those less than 4 years old (> 2 years and 2), were the study group. the exclusion criteria were : (1) children with cp combined with other problems such as congenital malformation or metabolic disorder, (2) children taking anticholinergic drugs over the past 2 months, and (3) children with an acute infection or other systemic disease. children with cp were enrolled consecutively from february 2011 to july 2011. the study was conducted according to the criteria of the declaration of helsinki and the review board of the university hospital approved this study. forty - seven children were included in the study : 14 did not drool (mean age : 43.2 13.8 months, diplegia 17%, quadriplegia 12.8%) and 33 did drool (mean age : 48.9 14.4 months, diplegia 10.6%, quadriplegia 59.6%). in the group without drooling, there were eight boys and six girls, and in the group with drooling, there were 22 boys and 11 girls (table 1). previous studies4,17 have reported that drooling in infancy usually resolves by 18 months of age. the children that drool beyond 4 years of age are considered abnormal and require further treatment. therefore, the participants were divided into groups ; those less than 4 years old (> 2 years and 0.05). there were no significant differences in the hrqol (physical health summary score and psychosocial health summary score) by sex (18.96 17.43 vs 24.49 22.16, t = 0.95, p > 0.05 and 49.20 16.68 vs 43.51 18.31, t = 1.09, p > 0.05, respectively ; table 2) and by age group (25.57 19.66 vs 16.90 28.25, t = 1.57, p > 0.05 and 52.13 16.43 vs 42.75 17.19, t = 1.91, p > 0.05, respectively). the physical health summary scores of the quadriplegic group were significantly lower than those of the diplegic group (34.19 18.69 vs 15.90 17.11, t = 3.20, p 0.05). for the children that drooled, both the physical health summary scores and the psychosocial health summary scores were significantly lower than the scores for the children that did not drool (31.97 22.22 vs 16.29 15.97, t = 2.73, p 0.1). the drooling ranking score and gross motor development predicted 56.6% of the variation of the physical health summary score (r = 0.566 ; p 0.1). the language development level predicted 25.6% of the variation of the psychosocial health summary score (r = 0.256 ; p < 0.01). the frequency of drooling in the quadriplegic children was greater than in the diplegic children. this result is compatible with the findings of hegde and pani3 that showed that patients with quadriplegia had the most severe drooling, followed by children with diplegia and the least affected children had athetoid cp. but other reports showed opposite results and they found the drooling is more prevalent and intense in children with dyskinetic cp than in children with spastic cp.5,20 the main etiologies of drooling include : impaired postural control as well as oral motor and swallowing abnormalities.3,2022 the quadriplegic children with cp are more likely to develop drooling due to their more extensive brain dysfunction and poor oral motor and sensory function compared to the diplegic children with cp. in this paper, there was no dyskinetic - type cp enrolled for study so we can not compare the drooling problem with other cp subtypes. the results of this study showed that the language and cognitive development of the children that drooled was lower than in the children that did not drool ; however, the gross motor development showed no significant difference between these two groups (table 1). these findings are compatible with the report of senner that showed that children who drooled had more severe dysarthria and impaired nonverbal intelligence, but their gross motor status was not more impaired ; although impaired motor control was considered one of the contributing factors of drooling.21,22 children at different developmental stages may have different findings and explanations for their disease.24,25 younger children were less likely to be perceived as performing less well on the hrqol questions than older children.26 it is possible that finding no significant hrqol differences between age groups, in this study, was due to the younger ages of our study groups. previous studies12,14 also revealed that the hrqol (both physical and psychosocial) of quadriplegic children was lower than that of diplegic children. in this study, the physical health summary scores of quadriplegic children were lower than diplegic children ; however, the psychosocial health summary score was not. the possible explanations for this result include : (1) most of the subjects attended early intervention institutions or a hospital program, where they possibly had a more supported and structured environment and received less negative feedback from social interaction;10,14 (2) most of the subjects were young and cognitively impaired ; the impairment was known since birth and they received support from their caregivers to accomplish daily activities, and therefore had fewer experiences of negative psychosocial well - being;11,27 (3) the number of subjects was too small to show a statistically significant difference in psychosocial health scores. the results of this study showed that the physical health summary scores and psychosocial health summary scores were significantly lower in the children with cp that drooled than in the children with cp that did not drool. these results were compatible with previous studies showing that drooling may lead to health - related problems such as skin maceration, recurrent pneumonia, and malnutrition.7,28 although our result showed a significant level of correlation between drooling and psychosocial hrqol, but the correlation coefficient showed only a lower level of correlation (r = 0.381 ; p < 0.01). some studies have reported that drooling was associated with impaired social relationships of these children with adults and their peers ; however, few showed negative emotional reactions due to drooling.8,10,15 van der burg reported that children with cp who drooled showed few negative emotional reactions because they attended special education schools and the drooling problem seems to be acceptable and ignored in these places. although the language and cognitive developmental status was positively correlated with the physical health summary score, these two variables were excluded from the stepwise regression model because of the statistical result of p 0.1. thus, the most important variables left in the stepwise regression model were gross motor development and rank of drooling, and they predicted 56.6% of the variation of the physical health summary score. in predicting the variation associated with the psychosocial health summary score, the level of drooling, gross motor development and cognitive development were excluded from the stepwise regression model (p 0.1) ; the remaining language development predicted 25.6% of the variation of the psychosocial health summary score. dickinson showed that gross motor development correlated most with the physical wellness of children with cp ; intellectual disability and language impairment significantly interfered with psychosocial wellness. however, drooling was not investigated as a problem that interfered with the quality of life in this prior study. in our study, only the ranking of drooling / gross motor development and language development were considered as important factors associated with the physical health summary score and psychosocial health summary score. further investigation of these possible variables (eg, family or institutional factors, gross motor function classification system of children with cp) that might be correlated with the hrqol of children with cp requires further research. although cognitive development was excluded in the stepwise regression model, its importance can not be overlooked due to nearly the same correlation coefficient associated with the psychosocial health summary score as with the language development score. however, the excluded variables did not add much to predicting the variability of the psychosocial health summary score, when it was included. because the subjects in this study were too young or too cognitively impaired, a parent / proxy report was used to assess the drooling and hrqol of the enrolled children. a bias might have been introduced by parents that were bothered by their children s drooling and inclined to rate their children s hrqol lower. as in the study by davis,29 there was discordance between parent / proxy and child self - report because they responded to the hrqol questionnaire items differently. but in this study, the definitions of drooling severity and frequency were clearly defined for parents and primary caregivers. in addition, the standard hrqol questionnaires were used for the hrqol evaluation, and were not likely significantly affected by bias. although a self - reported hrqol is standard for the perceived hrqol, the parents are valuable proxies to assess their children s hrqol if the children are too young or too cognitively impaired to complete a self - reported hrqol.10,30 therefore, the findings of this study based on parent / proxy reports showed that children with cp that drooled had a lower hrqol are important. this was a cross - sectional study and focused only on 2- to 6-year - old children with diplegic and quadriplegic cp from a university hospital and early intervention institutions. because our subjects in this paper were young and recruited from localized areas in taiwan, and contained only two cp subtypes, the results can not be generalized to all ages and all groups of children with cp. this was a cross - sectional study and focused only on 2- to 6-year - old children with diplegic and quadriplegic cp from a university hospital and early intervention institutions. because our subjects in this paper were young and recruited from localized areas in taiwan, and contained only two cp subtypes, the results can not be generalized to all ages and all groups of children with cp. in conclusion, the standard assessment inventory was used to evaluate the correlation of drooling on the hrqol of children with cp. the more severe the drooling was (without considering the type of cp), the lower the physical and psychosocial health quality of life was in the children with cp. the gross motor development level and ranking of drooling predicted the physical health score better, and the language development level predicted the psychosocial health score better. with regard to providing early intervention programs for children with cp, their developmental status should be assessed as well as their drooling problem, which has a negative correlation on their hrqol. the focus of this study was on young children with cp ; further evaluation is needed to determine similar correlations among older children with cp. | objectiveto investigate the association between drooling in children with cerebral palsy (cp) and their health - related quality of life (hrqol), as well as the possible variables that predict their hrqol.methoda cross - sectional design was used for this study. children with cp, without other identified disease, aged 2 to 6 years, who drool (n = 33) or did not drool (n = 14), were included. the dependent variables were the physical health summary scores and the psychosocial health summary scores of the pediatric quality of life inventory version 4.0. the t test, pearson product moment correlation, mann whitney u test and stepwise regression analysis were used for statistical analysis.resultsthe physical health and psychosocial health summary scores of the children that drooled (16.29 15.97 and 42.92 17.57, respectively) were lower than for the children that did not drool (31.97 22.22 and 57.09 12.21, respectively ; p < 0.01). the drooling ranking score was negatively correlated with the physical health summary score (r = 0.355 ; p < 0.05) and the psychosocial health summary score (r = 0.381 ; p < 0.01). the stepwise regression showed that gross motor development and the drooling ranking score predicted 56.6% of the variability of the physical health summary score (r2 = 0.566 ; p < 0.01). the language development score predicted 25.6% of the variability of the psychosocial health summary score (r2 = 0.256 ; p < 0.01).conclusiondrooling was associated with a lower hrqol. prediction of the physical health summary score was more closely associated with gross motor development and the drooling ranking scores. prediction of the psychosocial health summary score was more closely associated with the language development of children with cp aged 2 to 6 years. |
this study was conducted under the ethical guidelines for medical and health research involving human subjects by the health, labor and welfare ministry of japan (http://www.mhlw.go.jp/stf/seisakunitsuite/bunya/hokabunya/kenkyujigyou/i-kenkyu/). according to number 6 (omission of procedures concerning informed consent, etc.), chapter 5, part 12 in this guideline, informed consent was not obtained from the subjects. cbs and the corresponding histological specimens were cut to 4 m thickness and were prepared on silanized glass slides. ihc staining and the dish assay were then performed for both cb and histological specimen sections. the staining and assay were performed with a ventana benchmark ultra (roche diagnostics, basel, switzerland). the 2013 asco / cap criteria for her2 testing in breast cancer1 was used to categorize the results. two cases were not used because of low numbers of cells on the cb slides. the her2 dish assay was performed where there was discrepancy between the ihc results of her2 obtained from cbs and from the corresponding histological specimens. a single specimen was collected from each tumor using a 21gauge needle attached to a 20 ml syringe mounted on an aspiration gun. the cells were fixed in 10% buffered formalin for 1628 hour, processed for cb preparation by the sodium alginate method, and embedded in paraffin. the cb preparation was performed as follows : samplecontaining tubes were centrifuged at 3000 rpm for 5 min, formalin was removed, 1% sodium alginate was added, tubes were centrifuged at 3000 rpm for 5 min, and 1 m calcium chloride was added. the gel pellet formed by this process was used as the histological specimen. the following tumors were included : 49 invasive ductal carcinomas of no special type, two invasive lobular carcinomas, two noninvasive ductal carcinomas, and one mucinous carcinoma. ihc staining was performed on both the cb and histological sections using the ventana iview dab detection kit. the protocol involving heat antigen retrieval was used as recommended by the manufacturer for paraffinembedded sections. for the primary antibody, the antiher2/neu (4b5) rabbit monoclonal primary antibody of ventana iview pathway (roche diagnostics) was used. staining results were scored as 0, 1 +, 2 +, or 3 + according to the following criteria : strong circumferential membranous staining in > 10% of tumor cells was considered as 3 + ; moderate circumferential staining in > 10% of tumor cells or strong circumferential membranous staining in 10% of tumor cells was considered as 2 + ; weak and incomplete membranous staining in > 10% of tumor cells was considered as 1 + ; and the absence of staining or weak and incomplete membranous staining in 10% of tumor cells was considered as 0. the her2 expression was considered as negative if scored as 0 or 1 +, intermediate if scored as 2 +, and positive if scored as 3 +. the inform her2/neu dual ish dna probe cocktail assay was performed on both the cb and tissue sections. the dish assay was performed according to the manufacturer 's recommended protocol for surgical specimens. the standard protocol was initially performed for both types of sections ; however, the protease reaction time was extended if signals were weak. the her2/neu (black) to chromosome enumeration probe 17 (cep17) (red) ratio was manually counted using a light microscope in each specimen by one investigator to avoid subjective bias, and the result was confirmed by a second investigator. the criteria consist of a combination of the her2/cep17 ratio and the average number of her2 signals per cell. the her2 gene amplification was scored as amplified if the case had a her2/cep17 signal count ratio of 2.0 or if the her2/cep17 signal count ratio was 10% of tumor cells was considered as 3 + ; moderate circumferential staining in > 10% of tumor cells or strong circumferential membranous staining in 10% of tumor cells was considered as 2 + ; weak and incomplete membranous staining in > 10% of tumor cells was considered as 1 + ; and the absence of staining or weak and incomplete membranous staining in 10% of tumor cells was considered as 0. the her2 expression was considered as negative if scored as 0 or 1 +, intermediate if scored as 2 +, and positive if scored as 3 +. the inform her2/neu dual ish dna probe cocktail assay was performed on both the cb and tissue sections. the dish assay was performed according to the manufacturer 's recommended protocol for surgical specimens. the standard protocol was initially performed for both types of sections ; however, the protease reaction time was extended if signals were weak. the her2/neu (black) to chromosome enumeration probe 17 (cep17) (red) ratio was manually counted using a light microscope in each specimen by one investigator to avoid subjective bias, and the result was confirmed by a second investigator. the criteria consist of a combination of the her2/cep17 ratio and the average number of her2 signals per cell. the her2 gene amplification was scored as amplified if the case had a her2/cep17 signal count ratio of 2.0 or if the her2/cep17 signal count ratio was < 2.0 but the average number of her2 signals per cell was 6.0. a score of equivocal was given if the case had a her2/cep17 signal count ratio of < 2.0 and the average number of her2 signals per cell was 4.0 and < 6.0. a score of not amplified was given if the case had a her2/cep17 signal count ratio of < 2.0 and the average number of her2 signals was < 4.0. the fleiss cohen 's weighted kappa coefficient was used to assess the correlation between the results from cbs and those from the tissue specimens. the correlation was scored as good if the kappavalue exceeded 0.6 and excellent if it exceeded 0.8. of the 52 cases, 40 cases showed agreement between cbs and corresponding histological sections (concordance rate, 77% ; weighted kappa, 0.818) (table 1). of these, 10 cases were her2 intermediate in cbs but negative in histological sections, and two cases were her2 negative in cbs but intermediate in histological sections. no discrepancy between the two types of specimen was observed in cases where her2 expression was positive. comparison of her2 expression in cbs and histological sections her2, human epidermal growth factor receptor 2. an example of a her2 negative (1 +) case showing consistent results between the cb (a) and histological specimen (b). [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. ] an example of a her2 positive (3 +) case showing consistent results between the cb (a) and histological specimen (b). [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. ] the her2 dish results of the 10 cases where intermediate her2 expression was observed in the cb sections but was negative in histological sections are shown in table 2. eight cases were not amplified by her2 dish in both histological specimens and cbs (fig. the her2/cep17 signal count ratio of this case was 1.3 in the histological specimen and 2.0 in the cb. the weakness of cep17 signals in the cb of this case leads to underestimation of the true cep17 signal count. the last of the 10 discrepant cases was observed as equivocal in the histological specimen but as amplified in the cb. the her2/cep17 signal count ratio of this case was 1.5 in the histological specimen and 2.2 in the cb. an example of a discrepant case of her2 expression, showing her2 intermediate (2 +) in the cb (a) and her2 negative (1 +) in the histological specimen (b). the results by dish assay in both cb (c) and histology are negative (d). [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. ] her2 dish results for cases of intermediate her2 expression on cbs but negative her2 on histological section her2, human epidermal growth factor receptor 2 ; dish, dual in situ hybridization. of the two cases showing her2 negative in cbs but her2 intermediate in histological specimens, one case was not amplified by her2 dish in both histological specimen and cb, and one case was amplified on the histological section but not amplified on the cb section. the her2/cep17 signal count ratio of this latter case was 2.0 in the histological specimen and 1.4 in the cb. of the 52 cases, 40 cases showed agreement between cbs and corresponding histological sections (concordance rate, 77% ; weighted kappa, 0.818) (table 1). of these, 10 cases were her2 intermediate in cbs but negative in histological sections, and two cases were her2 negative in cbs but intermediate in histological sections. no discrepancy between the two types of specimen was observed in cases where her2 expression was positive. comparison of her2 expression in cbs and histological sections her2, human epidermal growth factor receptor 2. an example of a her2 negative (1 +) case showing consistent results between the cb (a) and histological specimen (b). [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. ] an example of a her2 positive (3 +) case showing consistent results between the cb (a) and histological specimen (b). [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. ] the her2 dish results of the 10 cases where intermediate her2 expression was observed in the cb sections but was negative in histological sections are shown in table 2. eight cases were not amplified by her2 dish in both histological specimens and cbs (fig. the her2/cep17 signal count ratio of this case was 1.3 in the histological specimen and 2.0 in the cb. the weakness of cep17 signals in the cb of this case leads to underestimation of the true cep17 signal count. the last of the 10 discrepant cases was observed as equivocal in the histological specimen but as amplified in the cb. the her2/cep17 signal count ratio of this case was 1.5 in the histological specimen and 2.2 in the cb. an example of a discrepant case of her2 expression, showing her2 intermediate (2 +) in the cb (a) and her2 negative (1 +) in the histological specimen (b). the results by dish assay in both cb (c) and histology are negative (d). [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. ] her2 dish results for cases of intermediate her2 expression on cbs but negative her2 on histological section her2, human epidermal growth factor receptor 2 ; dish, dual in situ hybridization. of the two cases showing her2 negative in cbs but her2 intermediate in histological specimens, one case was not amplified by her2 dish in both histological specimen and cb, and one case was amplified on the histological section but not amplified on the cb section. the her2/cep17 signal count ratio of this latter case was 2.0 in the histological specimen and 1.4 in the cb. evaluation of the her2 receptor status at metastatic sites is important for selecting the correct chemotherapy for treatment of recurrent disease.1 cytology can be applied to several types of metastatic lesion from which biopsies may be difficult to obtain, a particular example being from body cavity fluids. the use of cytology can therefore be a rapid, inexpensive, and less traumatic alternative to biopsy in these situations. several studies have reported that hormone receptor status, monitored in various types of cytological specimens, correlates well with the corresponding histological specimen.17, 18, 19 however, there are issues that remain to be resolved regarding her2 testing for cytological specimens before this method can be adopted in routine clinical practice. although the fish assay of cytological specimens demonstrates strong and consistent correlation with the her2 status of original tissue samples,4, 5, 6, 7 this method has some disadvantages for clinical use. in the fish assay, therefore, the fish assay is not suitable for cytological specimens taken from body cavity fluids or aspirates because of the presence of nonneoplastic inflammatory cells. in addition, fluorescence fades quickly, thus fish is unable to provide a durable record. the dish assay could be a reliable and practical method to assess the her2 status of breast cancer cytological specimens. several studies have utilized the her2 dish assay on liquidbased cytology specimens, using the thinprep technique8, 20, 21 and cbs.9, 10, 22 we therefore used the dish assay to assess the reliability of discrepant cases regarding ihc results between cbs and corresponding histological specimens. false positive results of ihc staining for her2 on alcoholfixed cytological specimens are a major problem in smears8 and cbs.14, 15 in a preliminary study at our institution, we encountered difficulties in scoring the her2 status by ihc methods using liquidbased cytological specimens stored in thinprep preservcyt solution (hologic) and found a low concordance rate with the corresponding histological specimens (data not shown). according to the reasoning mentioned above, formalinfixed cbs are recommended for her2 ihc staining.2, 3 however, discordant results are reported between formalinfixed cbs and tissue specimens. one reason is the errors of interpretation on cb analysis, potentially resulting from abundant cytoplasmic as well as background staining.12 also, it appears that her2 expression can be overestimated in small biopsy samples.2, 13 although our study showed excellent agreement between cbs and corresponding histological specimens (weighted kappa, 0.818), there were 12 discrepant cases. the ihc results of cbs in these 12 discrepant cases were her2 intermediate (2 +) or her2 negative (0 or 1 +). the her2 dish assay was used to confirm the results in the cases where her2 expression was intermediate on cbs but negative on histological sections. eight of 10 cases were not amplified by her2 dish in both histological specimens and cbs. one case that was unamplified on the histological section but was amplified on the cb section should be categorized as an unamplified case because the discrepancy was caused by the weakness of the cep17 signals on the cb section. the cause of discrepancy of the other one case was thought to be caused by the distribution of amplified cells. therefore, these cases with intermediate her2 expression by ihc staining on cbs but negative expression on histological specimens should be defined as her2 negative cases, except in one case observed as equivocal in the histological specimen but as amplified in the cb by dish assay. there were two cases showing her2 negative on cb but her2 intermediate on the histological section. one of these cases should be categorized as a her2 negative case because it was not amplified by her2 dish in both the histological specimen and the cb section. the cause of discrepancy of the other case was that the background signals seen on the histological section led to miscounting of the real her2 signal. therefore, these cases showing her2 negative in cbs and her2 intermediate in histological specimens should be categorized as her2 negative cases. two cases were not used because of low numbers of cells on the cb slides. the reason of eliminating these cases is to use good quality samples for the data evaluation. therefore, 100 cells on a slide are enough amount of cells to be evaluable. in summary, ihc staining of her2 from breast cancer biopsies can be performed in cbs in the same way as that in histological specimens, although the number of equivocal cases in cbs may be more than that in histological specimens. | backgroundhuman epidermal growth factor receptor 2 (her2) testing of samples from recurrent or metastatic breast cancer is recommended by the 2013 update of the american society of clinical oncology / college of american pathologists guidelines. although cytological analysis can be applied to several types of metastatic lesions, the practical method for her2 testing of cytological specimens is yet to be resolved. we conducted immunohistochemical (ihc) staining for her2 in breast cancer cell blocks (cbs) and compared the results with those from the corresponding histological specimens. in cases of discrepancy between the two types of specimen, the brightfield her2 dual in situ hybridization (dish) assay was performed.methodscbs were prepared from 54 surgically excised breast cancers. the cells were fixed in 10% buffered formalin and embedded in paraffin. a ventana benchmark ultra (roche diagnostics) with antiher2/neu (4b5) rabbit monoclonal primary antibody and inform her2/neu dual ish dna probe cocktail was used for the assays.resultssuccessful results were obtained in 52 of 54 cbs. forty cases showed agreement between cbs and the histological specimens. no discrepancy was observed between the two types of specimens in cases where her2 expression was positive. ihc results of cb in 12 discrepant cases were her2 intermediate or negative. the dish results of 11 of these cases were negative.conclusionihc staining of her2 for breast cancer cbs can be used in the same way as that used for histological specimens, although the number of equivocal cases in cbs is greater than that in histological specimens. diagn. cytopathol. 2016;44:274279. 2016 the authors diagnostic cytopathology published by wiley periodicals, inc. |
the transamidase complex catalyzes the glycosylphosphatidylinositol (gpi) lipid anchor attachment to substrate proteins of eukaryotes in the lumen of the endoplasmic reticulum (er). it remains one of the poorly understood macromolecular machines, both with regard to the molecular function of its many subunits, as well as their 3d structure, despite more than 25 years of research in the vertebrate, yeast, and trypanosomal model systems. first, a c - terminal propeptide is cleaved from the substrate protein. then in the next step, a peptide bond is formed between the newly established c - terminal residue (called -site) of the substrate protein and a phosphoethanolamine group of the gpi lipid anchor. the c - terminal, 4-partite sequence pattern for gpi lipid anchoring in substrate proteins is well established and can recognize substrate proteins with high sensitivity and low false - positive prediction rate. the gpi lipid anchor pathway has a role in multiple human pathologies including cancer. in human, the known subunits of the gpi lipid anchor transamidase complex are pig - k (gpi8p in yeast), pig - s (gpi17p), pig - t (gpi16p), gpaa1 (gaa1), and the subunit pig - u (cdc91/gab1) was found most recently. subunits pig - k and pig - t were discovered to form a covalent complex via a disulphide bridge. pig - k is a c13-clade cysteine protease with a predicted 3d structure similar to that of gingipain r and caspases. it is known to cleave the c - terminal propeptide from the substrate protein even in the absence of a gpi lipid anchor. the 3d structure of pig - t is predicted to be a c - terminal -propeller complemented with an n - terminal -helical hook that embraces the protease pig - k. it is thought that pig - t shields the active site of pig - k from attacking unrelated proteins. so far, the molecular functions and structures of the remaining 3 subunits remain in the dark. here, we report sequence - analytic evidence that the lumenal domain of gaa1/gpaa1 has a 3d structure similar to that of an m28-type aminopeptidase. we suggest that gaa1/gpaa1 is the prime and only candidate for the missing enzyme that catalyzes the formation of the peptide bond between the -site and a phosphoethanolamine group of the gpi lipid anchor. the sequence architecture of gaa1/gpaa1 provides for an n - terminal transmembrane (tm) region followed by a segment of ~300 residues located in the er lumen and further 6 tm helices. if the lumenal gaa1/gpaa1 segments from a wide variety of taxa are queried with hhpred against the hmm database derived from sequences with known structures (pdb_6feb14), a sub - stretch of ~290 residues generates significant, full - length hits into numerous m28-type peptidase sequences with structures such as 4fuu_a, 3gux_a, 4f9u_a, 3tc8_a, and 1tkj_a (see table 1). with the identical queries, the structure prediction tool phyre2 delivers hits to the same set of proteins with confidence scores in the range 97100%. remarkably, this is despite the fact that the sequence identities of the alignments generated by phyre2 are very low (917% except for the one match between 1tkj_a and the fly gpaa1 np572273, where it is 21% ; to note, sequence identity loses its predictive role for tertiary structure similarity at the threshold ~30%). whereas psi - blast did collect only gaa1/gpaa1 sequences from various eukaryote organisms in 2003, a decade later with much larger sequence databases, it runs into microbial sequences with similarity to m28-type peptidases beginning with round 2 (e.g., the first hit cbh37168 from an unidentified archebacterial species with significance 1e-05). the table presents hits found with hhpred when using the lumenal domain segments of the gaa1/gpaa1 protein sequences of various taxa. ce, ceanorhabditis elegans ; dm, drosophila melanogaster ; hs, homo sapiens ; mm, mus musculus ; pf, plasmodium falciparum ; sc, saccharomyces cerevisiae. the following 5 columns, separately for each structure, present the pdb structure code, the sequence length in the first row, and the e - value of the hit and the aligned segment in each following row. thus, there is no doubt that the lumenal domain of gaa1/gpaa1 has a structure very similar to that of m28 peptidases. as shown in the alignment in figure 1, this is an /-hydrolase fold with a central -sheet consisting of 8 strands surrounded by 7 -helices and a gaa1/gpaa1-specific elaboration closer to the c terminus (n - terminal to -helix 6) with a possible additional -helix. it was serendipitously found with small angle x - ray scattering (saxs) that the gaa1 sub - segment 70247 from baker s yeast forms a compact structure (~two - thirds of the whole domain, corresponding to the stretch with secondary structural elements from 2 up to 5 in fig. 1), and its secondary structural content measured with circular dichroism (28% -helix and 27%-sheet) is quite well approximated by this prediction (25% -helix and 20% -sheet as lower estimates in fig. 1 ; i.e., about equal amounts of both types of secondary structure and each type covering close to a quarter of the total sequence). figure 1. a representative set of lumenal domain segments of gaa1/gpaa1 protein sequences is shown together with sequences from the metalloprotease m28 family of proteins (with protein structure data bank [pdb ] and chain identifiers 4fuu_a, 3gux_a, 4f9u_a, 3tc8_a and 1tkj_a). five sites (numbered 1, 2, 3, 4, and 5 ; for better visibility, surrounded by brackets) indicate conserved polar residue positions that are known to play in role for metal ion binding for various members of this family. the zn2 site conserved among the gaa1/gpaa1 sequences is formed by residues at site positions 2, 3, and 5 (all marked by a star [] on top of the alignment column). we also show the experimentally determined secondary structure of 4f9u_a (-helices as red and -strands as green bars) and the predicted secondary structure for the human gpaa1 (o43292) derived with hhpred. the multiple alignments were created with input from hhpred and muscle, and manually adjusted subsequently. the 2-letter prefix in front of the accession numbers and the sequence ranges denotes the species. bt, bacteroides thetaiotaomicron ; bv, bacteroides vulgatus atcc 8482 ; ce, ceanorhabditis elegans ; dm, drosophila melanogaster ; hs, homo sapiens ; mm, mus musculus ; pd, parabacteroides distasonis ; pf, plasmodium falciparum ; sc, saccharomyces cerevisiae ; sg, streptomyces griseus. m28 family peptidases are metalloenzymes that usually carry 2, sometimes (e.g., in the case of the glutaminyl cyclase) 1 metal ion (most often zinc) in a tetrahedral coordination. three of the 4 ligands required are provided by amino acid residues from the protein structure. these metal ions have catalytic function (see the peptidase database merops). in the case of the 2-ion streptomyces griseus aminopeptidase 1tkj_a, these are residues his85, asp97, and asp 160 for zn1 (sites 1, 2 and 4 in fig. 1) and asp97 (shared among the 2 zincs), glu132, and his247 for zn2 (sites 2, 3, and 5 in fig. 1). although all 5 sequence positions are conserved in the mammalian cyclase sequences, only the second metal ion site is occupied. in gaa1/gpaa1, 4 of the 5 sequence positions (i.e., sites 2, 3, 4, and 5) in the respective 4 loops (c - terminal of 3 and 3-3, 4-4, c - terminal of 5, 8-7 ; see fig. 1) can be aligned with residues suitable for zinc binding, such as asp, glu, his, tyr. given the local sequence conservation pattern, we suggest asp153, asp188, glu226, and tyr328 in human gpaa1 (o43292) as the equivalents of asp97, glu132, asp160, and his 247 from s. griseus. to note, the residues are determined to the accuracy of the loop between secondary structural elements. sometimes there are several candidate residues within the respective loops, where, generally, alignments are quite uncertain ; thus, our assignment might not be the final word in every case. to conclude, we think that gaa1s / gpaa1s carry one metal binding site corresponding to zn2, similar to the glutaminyl cyclase case (sites 2, 3, and 5 in fig. 1). the m28 family of metalloproteases is unusual in that it contains mostly aminopeptidases (cleaving n - terminal amino acids) but also carboxypeptidases (cleaving c - terminal residues ; see the peptidase database merops). it includes also enzymes catalyzing some more exotic chemistry, such as the cyclization of n - terminal glutamine (with the formation of pyroglutamine for the loss of n - terminal basicity). neither of these catalytic options appears of direct relevance in the transamidase context. in particular, the protease function needed for cleaving the c - terminal propeptide from the substrate protein has already been solidly associated with pig - k / gpi8p. inspection of the chemistry linking the substrate protein s -site with the gpi lipid anchor shows that the naturally used adaptor moiety, a phosphoethanolamine, pre - attached to the anchor actually forms a peptide bond with the c - terminal amino acid (fig. 2). since catalyzers facilitate reactions in both directions, with the net result depending on the circumstances, we conclude that the lumenal domain of gaa1/gpaa1 is the enzyme still missing that catalyzes the formation of the peptide bond between the -site and the respective phosphoethanolamine moiety. the peptide bond linking the -site of the substrate protein with the phosphoethanolamine of the gpi lipid anchor. the typical chemical structure of the gpi lipid anchor and its linkage via the -site to the substrate protein for the transamidase reaction are schematically illustrated (drawn with the software suite chembiodraw / perkin elmers). only residues ala, asn, asp, cys, gly, and ser are possible in this position. the peptide bond between the phosphoethanolamine unit and the -site residue (in blue) is marked with an arrow. it is thought that this bond is established with catalytic support from the lumenal domain of gaa1/gpaa1. to emphasize, gaa1/gpaa1 is the most plausible candidate for this function among the remaining 3 transamidase units (including pig - s and pig - u), as previous indirect hints from literature and sequence studies indicate. most importantly, pig - k (gpi8p) and gpaa1 (gaa1) were the first transamidase subunits discovered. the respective mutations led to the accumulation of completely synthesized, free gpi lipid anchors. with hindsight, these 2 transamidase subunits are the enzymes, and they would provide the easiest measurable (all - or - none) effect in a mutation screen. the sub - complex of gpi8p (pig - k), gp16p (pig - t), and gaa1 (gpaa1), the catalytic core of the transamidase, is most tolerant to purification conditions. as a side note it can not be excluded that gaa1/gpaa1 might have some exopeptidase activity when isolated, and this activity could be responsible for the observed instability. the addition of phosphoethanolamine to the tetrasaccharide during synthesis of the gpi lipid anchor was experimentally proven to be absolutely instrumental before attachment of the anchor to the substrate protein can occur. to note, nature uses phosphoethanolamine as adaptor in this case to make the non - peptide gpi lipid anchor appear as the n terminus of a peptide or amino acid, so that a peptidase module can be evolutionarily repurposed for catalyzing the lipid anchor attachment. in this context, it is intriguing that sortase a (srta), a completely different, c60 family (trans-)peptidase from gram - positive bacteria can be used for chemoenzymatic coupling of peptides and proteins to gpi lipid anchors in an artificial system. in the 2003 review, it was hypothesized that gaa1/gpaa1 binds the free gpi lipid anchor for consumption by the transamidase complex. the concept of simple / complex tm regions can be used to distinguish between mere hydrophobic anchors in the membrane (simple tms) in contrast to complex tms that fulfil also other structural and/or functional roles. with the exception of the plasmodium falciparum case, all other gaa1/gpaa1 sequences studied (human, fly, worm, yeast, arabidopsis thaliana, leishmania, trypanosoma) have a least 6 complex tms (as reported by the tmsoc server). it was experimentally shown that the gaa1/gpaa1 tm regions (especially the c - terminal one with a conserved proline) are important for binding the gpi lipid anchor in a functionally productive manner to the transamidase complex. with regard to the other 2, more loosely bound, transamidase components, pig - u would have too small a lumenal domain for exhibiting protease activity, and pig - s appears to carry too few tms (just 2) to hold the gpi lipid anchor moiety. notably, the recently published genome of the fungus glarea lozoyensis atcc 20868 includes the gene coding for the protein epe25974, annotated just as zn - dependent exopeptidase, obviously, by an automated annotation pipeline. apparently, the density of sequences has become large enough toward late 2012 that automated annotation pipelines have recognized the aminopeptidase - like lumenal domain, though the more obvious function as gaa1 became obscured in the process. to summarize, the transamidase subunit gaa1/gpaa1 is the long sought for enzyme that catalyzes the attachment of the gpi lipid anchor to the carbonyl intermediate of the substrate protein at the -site. its lumenal domain is a metallo peptide synthetase with an / hydrolase fold and a central 8-strand -sheet and a single metal (most likely zinc) ion coordinated by 3 conserved polar residues. phosphoethanolamine is used as an adaptor to make the non - peptide gpi lipid anchor look chemically like the n terminus of a peptide. functional characterization of non - understood genome regions, especially of protein - coding genes, is certainly the most pressing task in life sciences today. this discovery of gaa1/gpaa1 s molecular function will help to understand the biochemical mechanisms of gpi lipid anchoring and help to interfere into the process pharmacologically, for example in battling parasites. | the transamidase subunit gaa1/gpaa1 is predicted to be the enzyme that catalyzes the attachment of the glycosylphosphatidyl (gpi) lipid anchor to the carbonyl intermediate of the substrate protein at the -site. its ~300-amino acid residue lumenal domain is a m28 family metallo - peptide - synthetase with an / hydrolase fold, including a central 8-strand -sheet and a single metal (most likely zinc) ion coordinated by 3 conserved polar residues. phosphoethanolamine is used as an adaptor to make the non - peptide gpi lipid anchor look chemically similar to the n terminus of a peptide. |
stage iiia endometrial cancer represents a wide range of tumor involvement, including the uterine serosa, adnexa, parametria, and peritoneal space, based on the staging system of the international federation of gynecology and obstetrics (figo) in 1988 [1 - 3 ]. in addition, the heterogeneity of histology, such as papillary serous or clear cell carcinoma, makes it difficult to compare outcomes between studies. this variability has led to a wide range of 5-year overall survival (os) rates for stage iiia endometrial cancer, varying from 39% to 76% [3 - 6 ]. the optimal adjuvant management for patients with advanced endometrial cancer has yet to be defined, particularly for iiia patients as incidence is low, representing only 2.6% of patients with endometrial cancer. radiotherapy (rt) has traditionally been employed in advanced endometrial carcinoma to improve locoregional control. several studies have evaluated the role of combined modality adjuvant therapy compared with a single modality in advanced endometrial cancer and have suggested that combined chemoradiotherapy (ctrt) can provide additional benefits compared with computed tomography (ct) or rt alone [10 - 13 ]. in contrast, a randomized study failed to show an improvement in disease - free survival (dfs) and os in patients treated with combined ctrt compared with adjuvant rt alone for high - risk endometrial cancer. thus, the effects of different adjuvant therapies for stage iiia endometrial cancer remain controversial. in addition, these previous studies involved a small portion of iiia populations, patient s stage was determined based on the 1988 figo stage, and both endometriod and non - endometrioid adenocarcinoma were included. the current study included a group of patients with stage iiia endometrioid adenocarcinoma with favorable histology only, using the revised 2009 figo stage to overcome the aforementioned variabilities. the revised figo iiia, which was published in 2009, excluded positive peritoneal cytology alone and denoted parametrial involvement separately as stage iiib. several studies have attempted to analyze risk factors for poor outcomes ; however, few studies have evaluated the role of particular adjuvant therapies according to risk group for revised 2009 figo stage iiia diseases. here, we evaluated survival outcomes according to risk factors and sought to determine the benefit of combined modality treatment after surgery in the high - risk subset of stage iiia patients. all patients with stage iiia endometrial cancer treated with surgery, followed by adjuvant rt or combined ctrt, in korea between january 1990 and december 2011 were evaluated. nineteen patients with non - endometrioid type adenocarcinoma, such as papillary serous, clear cell, adenosquamous, or carcinosarcoma were excluded. in addition, five patients with only positive peritoneal cytology, and those who underwent adjuvant ct alone (n=3) or inadequate rt (n=2) were also excluded. the remaining 93 stage iiia patients who underwent surgery followed by adjuvant rt alone or combined ctrt were analyzed retrospectively. after approval by the korean radiation oncology group (krog 13 - 17), the medical and rt records of the patients were reviewed retrospectively. all patients underwent hysterectomy and bilateral salpingo - oophorectomy with pelvic and/or aortic lymphadenectomy, followed by postoperative rt or combined ctrt. pelvic lymph node dissection was performed in 77 patients (82.8%) and paraaortic lymph node (paln) dissection or sampling was performed in 31 patients (33.3%). all patients underwent external pelvic rt, delivered to the tumor bed and regional lymphatics with 10-mv photons using the four - field box technique. the total dose to the pelvis ranged from 45 gy to 54 gy in 1.8 gy daily fractions, 5 days per week. twenty - one patients (21.6%) underwent additional vaginal brachytherapy using fletcher - suit after - loading applicators. two to six fractions of 3 - 5 gy were delivered to the vaginal surface or 5 mm from the vaginal surface. two patients (2.2%) were treated with an extended - field rt encompassing a volume of paln, usually located at the t12-l1 interface. another two patients received total abdominal rt of 30 gy in 1.5 gy fractions followed by a boost to the pelvis with the doses of 19.8 gy or 24 gy in 1.8 gy fractions. thirty patients were treated with concurrent ctrt (83.3%), four patients with ct followed by rt (11.1%), and two patients with rt followed by ct (5.6%). patients receiving adjuvant ct were treated with cisplatin - based ct (n=19, 52.8%), carboplatin plus paclitaxel (n=8, 22.2%), paclitaxel alone (n=6, 16.7%), or other drugs (n=3, 8.3%). adjuvant ct was delivered with a median of six cycles (range, 1 to 9 cycles), although two patients received only one cycle, and another two patients only received two cycles. five patients did not complete the full course of ct because of g3 hematologic toxicity (n=2), patient refusal (n=1), or no available data on toxicity (n=2). in adjuvant rt alone, the average treatment period was 42.9 days (range, 36 to 60 days) and 46.9 days (range, 35 to 117 days) for the ctrt group. no significant difference in the rt period was observed between the two treatment groups (p=0.057). locoregional recurrence was defined as a recurrence in the pelvis, vagina, or paraaortic lymphatic region. failure was defined as biopsy - proven recurrence or progression of disease on serial imaging studies. time to recurrence and death was calculated from the date of surgery until failure or death from any cause. survival curves were calculated using the kaplan - meier method, and comparison of the curves was performed using a log - rank test. the chi - square test, fisher exact test, and independent samples t test were used for comparison of characteristics between the two groups. 21 (ibm co., armonk, ny), and p - values less than 0.05 were considered statistically significant. all patients underwent hysterectomy and bilateral salpingo - oophorectomy with pelvic and/or aortic lymphadenectomy, followed by postoperative rt or combined ctrt. pelvic lymph node dissection was performed in 77 patients (82.8%) and paraaortic lymph node (paln) dissection or sampling was performed in 31 patients (33.3%). all patients underwent external pelvic rt, delivered to the tumor bed and regional lymphatics with 10-mv photons using the four - field box technique. the total dose to the pelvis ranged from 45 gy to 54 gy in 1.8 gy daily fractions, 5 days per week. twenty - one patients (21.6%) underwent additional vaginal brachytherapy using fletcher - suit after - loading applicators. two to six fractions of 3 - 5 gy were delivered to the vaginal surface or 5 mm from the vaginal surface. two patients (2.2%) were treated with an extended - field rt encompassing a volume of paln, usually located at the t12-l1 interface. another two patients received total abdominal rt of 30 gy in 1.5 gy fractions followed by a boost to the pelvis with the doses of 19.8 gy or 24 gy in 1.8 gy fractions. thirty patients were treated with concurrent ctrt (83.3%), four patients with ct followed by rt (11.1%), and two patients with rt followed by ct (5.6%). patients receiving adjuvant ct were treated with cisplatin - based ct (n=19, 52.8%), carboplatin plus paclitaxel (n=8, 22.2%), paclitaxel alone (n=6, 16.7%), or other drugs (n=3, 8.3%). adjuvant ct was delivered with a median of six cycles (range, 1 to 9 cycles), although two patients received only one cycle, and another two patients only received two cycles. five patients did not complete the full course of ct because of g3 hematologic toxicity (n=2), patient refusal (n=1), or no available data on toxicity (n=2). in adjuvant rt alone, the average treatment period was 42.9 days (range, 36 to 60 days) and 46.9 days (range, 35 to 117 days) for the ctrt group. no significant difference in the rt period was observed between the two treatment groups (p=0.057). locoregional recurrence was defined as a recurrence in the pelvis, vagina, or paraaortic lymphatic region. failure was defined as biopsy - proven recurrence or progression of disease on serial imaging studies. time to recurrence and death was calculated from the date of surgery until failure or death from any cause. survival curves were calculated using the kaplan - meier method, and comparison of the curves was performed using a log - rank test. the chi - square test, fisher exact test, and independent samples t test were used for comparison of characteristics between the two groups. 21 (ibm co., armonk, ny), and p - values less than 0.05 were considered statistically significant. the tumor and treatment characteristics of all 93 patients, 57 (61.3%) who received postoperative rt alone and 36 (38.7%) who received ctrt, are shown in table 1. the median age of patients in the ctrt group was older than that in the rt alone group. a larger portion of patients in the ctrt group showed involvement of serosa (27.8% vs. 19.3%, p=0.361) and lymphovascular space (30.6% vs. 15.8%, p=0.214). a higher proportion of patients in the ctrt group underwent paraaortic lymphadenectomy compared to the rt alone group (52.8% vs. 21.1%, p=0.002). the extent of irradiation field of adjuvant rt and vaginal brachytherapy did not differ significantly in the two groups. the median follow - up period was 62 months (range, 3 to 188 months). eight patients (14.0%) in the rt alone group and 11 patients (30.6%) in the ctrt group relapsed during the follow - up period (p=0.054) (table 2). the majority of recurrences were distant metastases including abdominal recurrence, 75% (6/8) in the rt alone group and 81.8% (9/11) in the ctrt group. in the rt alone group, one patient developed combined local and distant metastases with pelvic lymph node and liver metastasis. the 5-year os and dfs were 93.7% and 79.0%, respectively. univariate analyses showed statistically significant difference in dfs with regard to age (p < 0.001), tumor grade (p=0.029), and lymphovascular involvement (p=0.041) (table 3). no significant difference in os (91.9% vs. 96.3%, p=0.262) or dfs (82.4% vs. 74.1%, p=0.130) was observed between the rt alone group and the combined ctrt group. five - year dfs was lower in patients with serosal involvement than in those without, but there was no statistical significance (81.5% vs. 67.2%, p=0.180). of the 93 patients, 57 (61.3%) had solitary adnexal or serosal involvement. patients with serosal involvement had a worse 7-year dfs (66.7%) compared to those with solitary adnexal involvement (90.5%), but this difference did not reach statistical significance (p=0.629). the above mentioned variables (age 60 years, tumor grade 2/3, and lymphovascular space involvement) for prognosis in dfs were defined as risk factors. of 77 patients with available data for risk factors, 25 patients (32.5%) had no risk factors, 30 (38.9%) had one, 16 (20.8%) had two, and six (7.8%) had three. a significant correlation the 5-year dfs was 91.7% in patients with no risk factors, 84.8% in patients with one, 55.0% in patients with two, and 33.3% in patients with three (p=0.001). the 5-year os according to the number of risk factors was 95.0% in patients with no risk factors, 92.0% in patients with one, 93.8% in patients with two, and 80.0% in patients with three (p=0.542). the patients were subgrouped according to low - risk (patients with no or one risk factor) and high - risk (patients with two or three risk factors) groups. the proportion of high - risk patients in the combined ctrt group was more than twice that of the rt alone group (41.9% vs. 19.6%, p=0.033). thirty - seven patients (80.4%) in rt alone were classified as low risk and nine (19.6%) as high risk. in ctrt, 18 patients (58.1%) were classified as low risk, while 13 patients (41.9%) as high risk. a significant reduction in 5-year dfs was observed in the high - risk group (49.0% vs. 88.0%, p < 0.001) compared with the low - risk group (fig. five - year os was not significantly lower in the high - risk group (90.7%) versus the low - risk group (93.5%) (p=0.618). for rt alone, 5-year dfs and os were lower in the high - risk group than in the low - risk group (66.7% vs. 84.6%, p=0.133 and 77.8% vs. 93.5%, p=0.149, respectively), but the differences did not reach statistical significance (fig. the low - risk group showed an improved 5-year dfs (94.4% vs. 38.5%, p=0.001) compared with the high - risk group, but there was no significant difference in os (93.3% vs. 100%, p=0.465). to examine the role of adjuvant multi - modality therapy (84.6% vs. 94.4%, p=0.852) was observed between the rt alone group and the combined ctrt group in the low - risk group. in the high - risk group, combined ctrt showed a trend for improved 5-year os (100% vs. 77.8%, p=0.086) compared with rt alone, but the difference did not reach statistical significance. combined ctrt did not affect 5-year dfs (38.5% vs. 66.7%, p=0.274) compared with rt alone for the high - risk group. in multivariate analysis including age, grade, lympohovascular involvement, number of risk factors, and type of adjuvant modality, more than one risk factor was the only predictor of worse dfs (hazard ratio [hr ], 5.45 ; 95% confidence interval [ci ], 2.12 to 13.98 ; p < 0.001). in multivariate analysis for os, only old age was predictive of os (hr, 5.8 ; 95% ci, 1.15 to 29.61 ; p=0.034). in multivariate analysis, combined ctrt did not show a statistical difference compared to rt alone in dfs or os (table 4). twelve patients (33.3%) in the ctrt group experienced grade 3 - 4 acute toxicity. grade 3 - 4 acute hematologic toxicities were observed in 11 patients in the ctrt group and a grade 3 acute intestinal obstruction was found in one patient. in the rt alone group, one patient (1.8%) treated with extended - field rt had grade 4 acute hematologic toxicity. no patients in the ctrt group had grade 3 - 4 late toxicity and one patient in the rt alone group treated with whole abdominal irradiation had a grade 3 late leg edema. the tumor and treatment characteristics of all 93 patients, 57 (61.3%) who received postoperative rt alone and 36 (38.7%) who received ctrt, are shown in table 1. the median age of patients in the ctrt group was older than that in the rt alone group. a larger portion of patients in the ctrt group showed involvement of serosa (27.8% vs. 19.3%, p=0.361) and lymphovascular space (30.6% vs. 15.8%, p=0.214). a higher proportion of patients in the ctrt group underwent paraaortic lymphadenectomy compared to the rt alone group (52.8% vs. 21.1%, p=0.002). the extent of irradiation field of adjuvant rt and vaginal brachytherapy did not differ significantly in the two groups. the median follow - up period was 62 months (range, 3 to 188 months). eight patients (14.0%) in the rt alone group and 11 patients (30.6%) in the ctrt group relapsed during the follow - up period (p=0.054) (table 2). the majority of recurrences were distant metastases including abdominal recurrence, 75% (6/8) in the rt alone group and 81.8% (9/11) in the ctrt group. in the rt alone group, one patient developed combined local and distant metastases with pelvic lymph node and liver metastasis. univariate analyses showed statistically significant difference in dfs with regard to age (p < 0.001), tumor grade (p=0.029), and lymphovascular involvement (p=0.041) (table 3). no significant difference in os (91.9% vs. 96.3%, p=0.262) or dfs (82.4% vs. 74.1%, p=0.130) was observed between the rt alone group and the combined ctrt group. five - year dfs was lower in patients with serosal involvement than in those without, but there was no statistical significance (81.5% vs. 67.2%, p=0.180). of the 93 patients, 57 (61.3%) had solitary adnexal or serosal involvement. patients with serosal involvement had a worse 7-year dfs (66.7%) compared to those with solitary adnexal involvement (90.5%), but this difference did not reach statistical significance (p=0.629). the above mentioned variables (age 60 years, tumor grade 2/3, and lymphovascular space involvement) for prognosis in dfs were defined as risk factors. of 77 patients with available data for risk factors, 25 patients (32.5%) had no risk factors, 30 (38.9%) had one, 16 (20.8%) had two, and six (7.8%) had three. the 5-year dfs was 91.7% in patients with no risk factors, 84.8% in patients with one, 55.0% in patients with two, and 33.3% in patients with three (p=0.001). the 5-year os according to the number of risk factors was 95.0% in patients with no risk factors, 92.0% in patients with one, 93.8% in patients with two, and 80.0% in patients with three (p=0.542). the patients were subgrouped according to low - risk (patients with no or one risk factor) and high - risk (patients with two or three risk factors) groups. the proportion of high - risk patients in the combined ctrt group was more than twice that of the rt alone group (41.9% vs. 19.6%, p=0.033). thirty - seven patients (80.4%) in rt alone were classified as low risk and nine (19.6%) as high risk. in ctrt, 18 patients (58.1%) were classified as low risk, while 13 patients (41.9%) as high risk. a significant reduction in 5-year dfs was observed in the high - risk group (49.0% vs. 88.0%, p < 0.001) compared with the low - risk group (fig. five - year os was not significantly lower in the high - risk group (90.7%) versus the low - risk group (93.5%) (p=0.618). for rt alone, 5-year dfs and os were lower in the high - risk group than in the low - risk group (66.7% vs. 84.6%, p=0.133 and 77.8% vs. 93.5%, p=0.149, respectively), but the differences did not reach statistical significance (fig. the low - risk group showed an improved 5-year dfs (94.4% vs. 38.5%, p=0.001) compared with the high - risk group, but there was no significant difference in os (93.3% vs. 100%, p=0.465). to examine the role of adjuvant multi - modality therapy, the risk groups were analyzed according to treatment modalities. no significant difference in 5-year os (93.5% vs. 93.3%, p=0.785) or dfs (84.6% vs. 94.4%, p=0.852) was observed between the rt alone group and the combined ctrt group in the low - risk group. in the high - risk group, combined ctrt showed a trend for improved 5-year os (100% vs. 77.8%, p=0.086) compared with rt alone, but the difference did not reach statistical significance. combined ctrt did not affect 5-year dfs (38.5% vs. 66.7%, p=0.274) compared with rt alone for the high - risk group. in multivariate analysis including age, grade, lympohovascular involvement, number of risk factors, and type of adjuvant modality, more than one risk factor was the only predictor of worse dfs (hazard ratio [hr ], 5.45 ; 95% confidence interval [ci ], 2.12 to 13.98 ; p < 0.001). in multivariate analysis for os, only old age was predictive of os (hr, 5.8 ; 95% ci, 1.15 to 29.61 ; p=0.034). in multivariate analysis, combined ctrt did not show a statistical difference compared to rt alone in dfs or os (table 4). twelve patients (33.3%) in the ctrt group experienced grade 3 - 4 acute toxicity. grade 3 - 4 acute hematologic toxicities were observed in 11 patients in the ctrt group and a grade 3 acute intestinal obstruction was found in one patient. in the rt alone group, one patient (1.8%) treated with extended - field rt had grade 4 acute hematologic toxicity. no patients in the ctrt group had grade 3 - 4 late toxicity and one patient in the rt alone group treated with whole abdominal irradiation had a grade 3 late leg edema. the current study showed the impact of a number of risk factors for stage iiia endometrioid adenocarcinoma. patients with more than one risk factor (high - risk group) had a worse prognosis. indeed, more than one risk factor was the only strong negative prognostic factor in multivariate analysis. patients in the high - risk group were 4 to 5 times more likely to develop a distant metastasis compared to those in the low - risk group (40.9% vs. 9.1%, p < 0.001), therefore, the addition of more intense systemic therapy to those in the high - risk group may improve the outcome. the current study showed that combined ctrt showed a trend with an improved 5-year os (100% vs. 77.8%, p=0.086) compared with rt alone, although ctrt did not affect 5-year dfs compared with rt alone for the high - risk group. several studies have suggested that combined ctrt can provide additional benefits compared with ct or rt alone [10 - 13 ]. the retrospective study in stage iii and iv endometrial cancer by alvarez secord. reported that adjuvant ct alone was associated with poor 3-year os (33%) and progression - free survival (19%) compared to either rt alone (70% and 59%) or combined therapy (79% and 62%). in a multicenter retrospective study conducted with 78 patients with stage iii endometrial cancer treated with adjuvant ct and/or rt, the 3-year relapse - free survival rates were 86.5% for the combined ctrt group, 65.8% for the ct alone, and 44.1% for rt alone, suggesting that combined ctrt modality may induce an advantage in relapse - free survival compared to rt or ct alone. the first trial was run by the italian oncology group with 153 patients (iib - iiic, 65% stage iii), and the second by the nordic society of gynaecological oncology / european organisation for the research and treatment of cancer, which included patients with mainly high - risk early stage disease (only 1.6% stage iii). in the italian trial, however, in joint pooled analysis in these two randomized trials, sequential addition of ct to rt improved at least progression - free survival (78% vs. 69%, p=0.009), and trended a benefit of overall survival (82% vs. 75%, p=0.07) compared with rt alone. an important question is whether or not it is reasonable to recommend adjuvant rt alone without chemotherapy in low - risk patients. prior study has suggested that a subgroup of women with stage iiia who have endometrioid tumor, no lymphovascular space involvement, and positive peritoneal cytologic finding only show an excellent prognosis.. found that among 24 patients with peritoneal cytology only, non - serous histology, and grade 1 - 2 disease, no recurrence was found in 12 patients receiving adjuvant treatment, while one patient showed recurrence without adjuvant treatment. our study included patients according to the 2009 figo staging system and excluded those with peritoneal cytology only. for 25 patients with no risk factors, 17 patients (68%) were treated with adjuvant rt alone and eight patients (32%) with adjuvant combined ctrt, resulting in excellent 5-year dfs (91.7%) and os (95.0%), respectively. no difference in dfs and os thus, a subset of stage iiia patients with no risk factors might be treated with adjuvant rt alone. several studies have reported that patients with extrauterine spread limited to the adnexa showed favorable 5-year dfs rates, ranging from 71% to 89.6%. a retrospective study of 15 patients with solitary serosal invasion had a poor 5-year dfs (41.5%) due to the high incidence of distance metastases. a multicenter retrospective comparative analysis in netherlands published the outcomes for 67 patients with stage iiia endometrial carcinoma, evaluating differences in outcome between serosa and adnexal involvement. those with involvement of the serosa alone had a worse 7-year distant metastasis - free survival (58.7%) compared to adnexal involvement alone (72.7%), but this difference was not significant (p=0.399). authors concluded that either adnexal or serosal involvement showed a comparable disease - specific survival and only presence of lymphovascular invasion was significant in multivariate analysis (hr, 3.6 ; p=0.038). the current study was composed of a relatively large homogeneous subgroup of surgically staged iiia patients with only adnexal or serosal involvement. of the 93 patients in our study, 57 (61.3%) had solitary adnexal or serosal involvement. consistent with previous studies, patients with serosal involvement had a worse 7-year dfs (66.7%) compared to those with solitary adnexal involvement (90.5%) (p=0.629). in this multicenter analysis, we failed to demonstrate additional benefit of combined adjuvant ctrt compared with adjuvant rt alone for stage iiia endometrial cancer. while these findings might suggest a comparable survival outcome between combined ctrt and rt alone groups, our study is limited by the difference of high - risk features between the groups. although there was no statistically significant difference in median age, grade, and lymphovascular involvement between the rt alone group and the combined ctrt group, the ctrt groups did show a trend with an older age, higher grade, and more involvement of lymphovascular space than the rt alone group. in addition, a subset of patients with more than one risk factor, the high - risk group, in the combined ctrt group was more than twice that of the rt alone group (41.9% vs. 19.6%, p=0.033). these factors might lead to comparable survival outcomes between the rt alone group and the combined ctrt group. another limitation of this study is the retrospective design with heterogeneity of the ct agent, sequence of combined ctrt and surgical technique with paraaortic lymphadectomy. this variability leads to difficulty in determining the additional benefit of ctrt in stage iiia endometrioid adenocarcinoma. for gynecologic oncology group 258 (gog258), the current phase iii randomized trial for adjuvant modality, the ctrt followed by carboplatin+paclitaxel is being compared to combination of carboplatin and paclitaxel in stage iii and iva patients. multi - center randomized trials are required to further evaluate the role of adjuvant combined modality, according to risk groups, particularly in stage iiia endometrial adenocarcinoma which is an uncommon entity. in conclusion, the current study showed a favorable outcome in a subset of low - risk patients compared to the high - risk group in stage iiia endometrioid adenocarcinoma treated with adjuvant rt alone or adjuvant combined ctrt. we found that patients classified as high risk (2 risk factors) showed a significant reduction in 5-year dfs compared with the low - risk group (49.0% vs. 88.0%, p < 0.001) with an increase in the development of distant metastasis (40.9% vs. 9.1%, p < 0.001). we identified a subset of stage iiia patients without risk factors who might be reasonable candidates for adjuvant rt alone. | purposewe evaluated the role of adjuvant therapy in stage iiia endometrioid adenocarcinoma patients who underwent surgery followed by radiotherapy (rt) alone or chemoradiotherapy (ctrt) according to risk group.materials and methodsa multicenter retrospective study was conducted including patients with surgical stage iiia endometrial cancertreated by radical surgery and adjuvant rt or ctrt. disease - free survival (dfs) and overall survival (os) were analyzed.resultsninety-three patients with stage iiia disease were identified. nineteen patients (20.4%) experienced recurrence, mostly distant metastasis (17.2%). combined ctrt did not affect dfs (74.1% vs. 82.4%, p=0.130) or os (96.3% vs. 91.9%, p=0.262) in stage iiia disease compared with rt alone. patients with age 60 years, grade g2/3, and lymphovascular space involvement had a significantly worse dfs and those variables were defined as risk factors. the high - risk group showed a significant reduction in 5-year dfs (2 risk factors) (49.0% vs. 88.0%, p < 0.001) compared with the low - risk group (< 2). multivariate analysis confirmed that more than one risk factor was the only predictor of worse dfs (hazard ratio, 5.45 ; 95% confidence interval, 2.12 to 13.98 ; p < 0.001). of patients with no risk factors, a subset treated with rt alone showed an excellent 5-year dfs and os (93.8% and 100%, respectively).conclusionwe identified a low - risk subset of stage iiia endometrioid adenocarcinoma patients who might be reasonable candidates for adjuvant rt alone. further randomized studies are needed to determine which subset might benefit from combined ctrt. |
hydatid disease (hd), which is caused by echinococcus granulosus, is a common parasitic infestation especially in endemic regions such as the middle east, mediterranean, and south american countries. even though it mainly involves the liver, it has been reported in nearly all parts of the body. the incidence of the splenic cysts in hd varies from one series to another, ranges 0.54% of all cases of echinococcosis. however, one study showed that hd is common in jordan, but isolated spleen disease is described only in 2.5% of cases. symptoms of splenic hydatidosis is usually mild and is mainly caused due to pressure of adjacent organs such as colon / diaphragm or the presence of complications. cysts may become infected or may rupture into the peritoneal cavity, especially subcapsularly located cysts like our case. the hydatid fluid is antigenic and highly toxic and can cause a potentially fatal anaphylactic reaction in humans. herein, we describe an acute happening of anaphylactic shock due to a spontaneous rupture of the primary isolated hydatid cyst in a 57-year - old woman. a 57-year - old woman was admitted to the emergency department for diffuse maculopapular rashes on the whole body and anaphylactic shock. she had been complaining of sudden- onset of abdominal pain, dyspnea, pruritus, and urticaria. on physical examination, it revealed tenderness, guarding, and rigidity all over the abdomen. the patient recovered after emergency medications and then abdominal computed tomography (ct) revealed a splenic cystic lesion measuring about 9 6 cm in diameters and containing floating membranes. the anterior wall of the cyst showed discontinuity at a point [figure 1 ]. then, the patient underwent a laparoscopic partial cystectomy and omentoplasty, since the spread of the disease into the peritoneal cavity of lesions was strong contraindications for a radical surgical approach. at the time of laparotomy the postoperative course was uneventful, and the patient was discharged 10 days after the surgery. we applied albendazole, 10 mg / kg / day for 6 months, and the patient was followed up with ct that displayed no recurrence of the disease during a follow - up of 6 months [figure 3 ]. computed tomography showed floating membranes in the splenic cystic lesion (long arrow), rupture point (short arrow), and perisplenic fluid (open arrow) intraoperative photograph shows a ruptured outer membrane of an echinococcal splenic cyst follow - up computed tomography image shows no residue or recurrence in spleen with omentopexy (star) hydatid disease is a parasitic infestation caused by the larva of echinococcus, more frequently encountered in endemic areas such as mediterranean countries, south america, africa, and middle east. even though various theories have been published about the pathophysiology of splenic hydatidosis, the most suitable study was published by bourgeon. the development of echinococcal cysts in the spleen is uncommon because hexacanth embryos are usually trapped in the liver (first lemman 's filter) or lung (second lemman 's filter), but will be trapped in the splenic capillaries once in the systemic circulation. splenic echinococcosis may also occur by retrograde reach from the liver to the spleen via the portal and splenic veins in portal hypertension. the spleen may also be affected due to rupture of a hepatic echinococcal cyst into the peritoneal cavity. patients remains asymptomatic for 520 years before the diagnosis, and approximately 30% of splenic cysts are asymptomatic. in these asymptomatic patients, when the cyst reaches a huge size, it presents with a painful mass in the left upper quadrant. if the cyst is infected, or abscess formation has been occurred, left upper quadrant pain, fever, and leukocytosis develop. rupture into the abdominal cavity is a rare but serious complication because the hydatid fluid is antigenic and highly toxic. anaphylactic reaction to the hydatid cyst usually occurs after microscopic or macroscopic rupture of the cyst and leakage of contents into the peritoneum or blood circulation. the response can be vary from a mild hypersensitivity reaction to a fatal anaphylactic shock. rupture of a splenic hydatid cyst occurs commonly secondary to trauma, but it may also rarely occur spontaneously such as our patient. the diagnosis is mainly historical and geographic background of the patient, physical examination, serology, and visualization of peripheral calcification or daughter cysts within the large cystic lesion or coexistent cystic lesions with radiologic examination in the liver or other organs. ultrasound and ct imaging have been used successfully in the detection of a ruptured hydatid cyst. at present, ultrasonography is the most valuable and most widely available imaging modality for the diagnosis, classification, and follow - up of splenic hydatid cysts. ct is more sensitive than plain films or sonograms in depicting subtle cyst wall calcification. ct is an important imaging modality to have the actual number and location of the cysts not only in the spleen, but also in the abdomen. the presence of a cyst in the spleen of a patient who developed anaphylactic reaction with detection of membrane - like structures inside the cyst and discontinuity of the wall are considered diagnostic for ruptured hydatid cysts such as our patient. surgical excision is still the only curative treatment, but good results have been reported with albendazole administration. medical treatment is indicated in cysts inaccessible for surgical removal or as a complementary therapy to prevent recurrence. the surgical procedure consists of removing the cyst and possible daughter cysts spilled into the abdominal cavity in combination with peritoneal washing. total splenectomy is advocated by the majority of surgeons, especially for patients with large cysts located centrally or near the hilus, since it gives rise to minimal risk of recurrence. laparoscopic or laparoscopically assisted splenectomy has also been successfully used for echinococcal disease of the spleen. atmatzidis. showed that the recurrence rate after total splenectomy was not significantly different compared to spleen - preserving surgery, and the complication rate and hospital stay were also comparable between these two groups. in summary, the hydatid cyst is still a serious health problem in endemic areas as well as in our country. although the liver and lung are the most frequently involved organs, primary splenic hydatidosis is quite rare. hydatid cyst must be considered in the differential diagnosis of patients presenting with anaphylactic shock in areas where the disease is endemic and even in nonendemic. | echinococcosis is a helminthic zoonosis mainly caused by echinococcus granulosus and commonly encountered in endemic areas. the liver and lung are the most frequently involved organs. a primary isolated hydatid cyst of spleen is an extremely rare disease even in endemic areas. anaphylactic reaction is a known complication of cystic hydatid disease, a parasitic infestation caused by the larval / cyst stage of e. granulosus that usually occurs after trauma or during interventions. to the best of our knowledge, anaphylaxis with spontaneous rupture of primary isolated splenic hydatidoses had not been reported previously. the main purpose of this report is to highlight life - threatening complications such as anaphylactic shock that should be considered due to primary isolated splenic cyst hydatid rupture in especially endemic regions. |
regional odontodysplasia (ro) is a rare developmental dental anomaly that involves ectoderm and mesoderm derived tissues. the prevalence of this condition is still not clear since the studies reported till date have mainly been based on case reports. although several factors such as local trauma, infection, ischemia, neural damage, and somatic mutations of neural crest cell migration have been advocated, the underlying pathophysiology of the condition remains unclear. the maxillary teeth are more commonly affected, and only four cases of mandibular involvement have been reported so far. the teeth with ro often display a brownish or yellowish discoloration and most frequent clinical symptoms accompanied by this anomaly are failure of eruption and gingival enlargement. radiologically, the affected teeth illustrate hypoplastic crowns and lack of contrast between enamel and dentin is usually apparent. enamel and the dentin are very thin, displaying a ghost - like appearance. the other pathognomic radiological characteristics are enlarged pulp chambers, short roots, and open apices. a 33-month - old boy reported to the department of pediatric and preventive dentistry of our college with the complaint of delayed eruption of teeth in the lower jaw. there was no history of abnormal tooth anomalies on both maternal and paternal part of the family. extra - oral examination revealed normal symmetrical face and normal skin, hair, and nails. the intra - oral examination revealed an adequate maxillary arch with fully erupted primary teeth. all the maxillary teeth appeared normal except for some carious involvements in molars [figure 1 ]. although all the primary teeth were visible in the mandibular arch, they were not fully erupted and seemed to be embedded in abnormal, slightly hyperplastic alveolar mucosa. furthermore, these teeth revealed abnormal crown morphology with yellowish discoloration and hypoplastic enamel [figure 2 ]. normal maxillary arch with a full complement of teeth hypoplastic crowns and gingival hyperplasia in entire mandibular arch the orthopantomogram (opg) [figure 3 ] which was taken after detailed clinical examination, showed a full complement of maxillary deciduous teeth and follicles of permanent teeth except for tooth germs of premolars, which was normal for his age. however, in the mandibular arch, all the primary teeth showed a striking ghost - like appearance [figure 3 ]. the demarcation line between enamel and dentin in these teeth was not clear, and enamel was hypoplastic. none of the permanent tooth germs were seen except for the follicles of first molars. the laboratory investigations showed that the serum calcium, phosphorus, sodium, and potassium levels were in normal range. since the child was only 33 months old, and none of the teeth showed any abnormal mobility, extraction was not carried out. however, gingival biopsy examination revealed the odontogenic tissue in the epithelium and intramesenchymal calcifications. on the basis of clinical and radiographic findings, a diagnosis of ro was proposed. maintenance of proper oral hygiene and regular follow - up examinations for monitoring the developing dentition was advised. considering the age of the child, conservative treatment was preferred over extractions because it preserves oral functionality and esthetics until the end of growth period after which a more definite treatment could be planned. regional odontodysplasia is a rare tooth anomaly that affects maxillary teeth more often with very less tendency to cross the midline. however, cases have been reported with bilateral or multiquadrant involvement. in the present case, clinical and radiographic findings were consistent with that of ro, but this case was notable due to involvement of the entire mandibular arch. till date, only four cases of mandibular involvement have been reported. the cause of the present case also remains unknown because the patient 's past medical history and family history was noncontributory, and no congenital or acquired diseases were reported. regional odontodysplasia has shown to affect both primary and permanent dentition. in the present case, delayed development of permanent tooth germs in the mandibular arch was also evident which was in agreement with some cases reported previously. typical clinical features in this case included gingival hyperplasia and teeth embedded in the gingival overgrowth ; both of these features were consistent with the majority of cases reported previously. a differential diagnosis of ro includes hereditary conditions, such as dentin dysplasia, dentinogenesis imperfecta, and amelogenesis imperfecta as these conditions may present similar features of enamel hypoplasia, abnormal pulp morphology, and calcification. hereditary developmental anomalies of enamel and dentin, however, usually affect the entire dentition rather than segments. dentinogenesis imperfecta type iii bears close resemblance to ro but can be excluded if opalescent dentine, bell - shaped crowns, and family history are absent. factors such as the patient 's age, extent of the lesion, and eruption of the teeth, medical history, and esthetics need to be carefully considered. for the present case, in addition, since the opg showed absence of developing permanent tooth germs, it was thought appropriate to preserve the primary teeth. follow - up examinations were planned to observe the development of mandibular permanent tooth germs. the case presents unique clinical and radiographic features of ro involving the entire mandibular arch in a child aged 33 months. a dental professional must be prepared to manage cases of dental anomalies, providing an opportunity for early diagnosis, adequate monitoring and treatment plans that minimize the after - effects on the patient 's development. in cases of ro, treatment depends on the age of the patient, extent of the involvement of the teeth, and the individual functional and esthetic needs. | regional odontodysplasia (ro) is a rare developmental anomaly involving both mesodermal and ectodermal components in primary or permanent dentition. it affects the maxilla and the mandible or both ; however, maxilla is more commonly involved. this article reports the case of 33-month - old boy who came with the chief complaint of delayed eruption of mandibular teeth. findings of clinical and radiographic examination were consistent with those of ro. maxillary dentition was unaffected. clinical and radiographic features and treatment options are discussed. |
genomic instability, a key hallmark of cancer, is driven in part by oncogene - induced dna replication stress (drs). specifically, in cancer cells, activated oncogenes induce dissociation of the replication machinery from the dna fork (fork collapse), formation of dna double - strand breaks (dsbs), and genomic instability (gorgoulis., 2005, bartkova., 2005,, 2008, negrini., 2010, arlt., 2012, hills and diffley, 2014, macheret and halazonetis, 2015). prolonged exposure to chemical agents that interfere with dna replication can also lead to fork collapse (branzei and foiani, 2010, petermann. following fork collapse, dna replication can be completed by repair of the collapsed forks, by incoming replication forks, or by dormant origin firing (branzei and foiani, 2010, blow., 2011, yeeles., 2013, mayle., 2015). we previously described break - induced replication (bir) as a repair pathway for collapsed dna replication forks in cancer cells (costantino., 2014) and, more recently, the scope of bir in human cells was expanded to include dna replication repair in prophase and alternative lengthening of telomeres (minocherhomji., 2015, bir has been studied extensively in budding yeast, as a homologous recombination (hr)-based repair pathway for one - ended dna dsbs (llorente., 2008, notably, the d loop moves together with the replication fork, and dna replication is conservative (donnianni and symington, 2013, saini. these unique properties distinguish bir - initiated forks from origin - initiated forks and suggest the involvement of different proteins at these two types of forks. indeed, pol32, a nonessential subunit of budding yeast dna polymerase delta, is required for bir, but not for origin - initiated replication (lydeard., 2007). mammalian pold3, the ortholog of budding yeast pol32, is also required for bir, as is pold4, another subunit of mammalian dna polymerase delta that has no apparent ortholog in budding yeast (costantino., 2014, in addition to pol32, bir in yeast requires rad52 (llorente., 2008, 2008). however, the role of rad52 in yeast is not specific to bir ; dna dsb repair by gene conversion (a.k.a. synthesis - dependent strand annealing) and single - strand annealing also require rad52, and yeast mutants lacking rad52 are very sensitive to dna damaging agents (symington, 2002, sugawara., 2003). rad52 is conserved at the amino acid level from yeast to human, but its function is apparently only partially conserved. thus, human rad52 retains the strand - annealing activity (kagawa., 2002, singleton., 2002), 2015). accordingly, whereas homozygous deletion of the brca2 gene in mice leads to embryonic lethality (sharan., 1997), rad52-knockout mice have a normal lifespan and no major phenotype, raising the question of what the physiological function of mammalian rad52 is (rijkers., 1998, yamaguchi - iwai., 1998). we previously performed an sirna screen to identify dna repair genes that are important for cell cycle progression when cyclin e is overexpressed (costantino., 2014). here we performed a more focused screen centering on genes that function in hr, and we identified rad52. further characterization revealed that rad52 has a role in bir, making it a potential target for the development of cancer - specific therapies. in an effort to identify genes that function in bir in human cells, we performed an sirna screen targeting about 70 genes that had previously been linked to dna dsb repair and hr (table s1). the requirement of these genes in bir was examined using u2os cells that overexpress cyclin e in an inducible manner (bartkova., 2005). in these well - characterized cells, cyclin e overexpression leads to drs, and the damaged replication forks are repaired to a significant degree by bir. thus, when bir is inhibited for example, by depleting pold3progression through the cell cycle is delayed (costantino., 2014). to enable monitoring of cell cycle progression, the cells were pulsed consecutively with two thymidine analogs (edu and brdu ; 1 hr pulse each with the two pulses separated by 6 hr) and then examined by flow cytometry (figure 1a and figure s1a). cells that remained in g1 during the 8 hr period would stain negatively for both edu and brdu, whereas cells that transitioned from g1 into s phase would stain negatively for edu and positively for brdu. as observed before (costantino., 2014), the fraction of cells that remained in g1 over the 8 hr period decreased when cyclin e was overexpressed (figure 1a, control sirna ; ne, normal cyclin e expression ; oe, cyclin e overexpression). most of the sirnas tested did not affect the fraction of ne or oe cells that remained in g1 over the 8 hr period (figure 1a and table s1). this included two sirnas that depleted the helicase pif1, even though in budding yeast pif1 is required for bir (wilson., 2013). a small number of sirnas preferentially enhanced the fraction of cells that remained in g1 when cyclin e was overexpressed. these were the sirnas targeting slx4, mus81, smarcal1, tipin, timeless, pold4, and rad52 (figures 1a and 1b). slx4, an adaptor protein that binds mus81 ; mus81, a nuclease ; and smarcal1, a helicase, remodel damaged replication forks (btous., 2012, pepe and west, 2014, sarbajna., 2014) ; tipin and timeless are part of the replication fork protection complex (chou and elledge, 2006, errico and costanzo, 2012), while pold4 functions in bir (costantino., 2014). we decided to pursue the last hit, rad52, which is dispensable for normal development in mice, but whose homolog in budding yeast is important for all forms of hr, including bir. using crispr / cas9 (jinek., 2012), we generated rad52-knockout clones in the context of the u2os cells, in which cyclin e could be inducibly overexpressed. clone 2 g contained two mutant alleles, whereas in clones 3c and 4a only one mutant allele was identified (figure s1b). clone 3c retained a wild - type (wt) allele, whereas in clones 2 g and 4a no wt alleles could be identified. consistent with the sequencing data, rad52 protein was undetectable in clones 2 g and 4a and barely detectable in clone 3c (figure 1c). importantly, all clones retained the capacity to regulate cyclin e levels in a tetracycline (tet)-dependent manner (figure 1c), and all of them displayed decreased progression from g1 into s phase when cyclin e was overexpressed (figures 1d and s1c s1f). we next examined the intracellular localization of rad52 in cells that were either overexpressing cyclin e or exposed to chemical agents, such as hydroxyurea (hu) and camptothecin (cpt), that induce drs. in about 20% of cells overexpressing cyclin e for 4 days, endogenous rad52 localized to drs foci, as marked by staining for rpa and atrip (figures 2a, 2b, and s2a). recruitment of rad52 to drs foci was also observed in about 50% of the cells exposed to hu for 24 hr but was mostly absent in cells exposed to hu for 2 hr (figures 2c, 2d, and s2b). these kinetics parallel the known effects of hu on dna replication forks ; short treatment of cells with hu leads to a reduction in the ribonucleotide pools and fork stalling, whereas forks collapse after prolonged exposure to hu (petermann., exposure of cells to cpt, which induces covalent bonding of topoisomerase i to dna and, subsequently, fork collapse (pommier, 2006), also led to recruitment of rad52 to drs foci ; this was evident within 2 hr of exposure in some cells but was much more evident at 24 hr (figures 2c and s2c). interestingly, in cells overexpressing cyclin e or exposed to either hu or cpt, rad51 foci were less prevalent than rad52 foci (figures 2a and 2c) ; however, the functional significance, if any, of this apparent difference remains to be investigated. exposure of cells to hu for 24 hr was also associated with posttranslational modifications of the chromatin - bound fraction of rad52, as detected by immunoblotting, whereas a high dose (9 gy) of ionizing radiation did not elicit similar modifications (figure 2e). a subset of the rad52 posttranslational modifications induced in response to hu were atr dependent, since they were suppressed when the cells were treated with an atr inhibitor (figure 2f). furthermore, the modifications were sensitive to treatment of the chromatin extracts with lambda phosphatase (figure 2f). taken together if rad52 is important for bir, then its depletion should compromise the repair of collapsed forks and lead to a stronger dna damage response. to explore this possibility, we depleted rad52 by sirna and monitored h2ax phosphorylation both 2 and 24 hr after adding hu to the cells. at the 24 hr time point, the majority of the replication forks were collapsed, whereas at early time points, a large fraction of the forks are stalled and can resume replication upon hu withdrawal (figure s3a). the effects of pold3, mus81, and rad51 depletion on h2ax phosphorylation were also examined (figure s3b). two hours after exposure of the cells to hu, h2ax levels increased modestly and equally in the control, rad52-, pold3-, and mus81-depleted cells (figures 3a and 3b). after 24 hr exposure to hu, h2ax phosphorylation levels increased further, but the increase was much stronger in the cells depleted for rad52 or pold3 (figures 3a and 3b). codepleting rad52 and pold3 had the same effect as depleting only rad52 (figure 3c). these results are consistent with rad52 and pold3 functioning epistatically in repair of collapsed, but not stalled, replication forks. interestingly, depletion of rad51 suppressed h2ax phosphorylation when compared to control sirna - treated cells at both the 2 and 24 hr time points, an observation that merits further study (figures 3a and 3b). to examine whether rad52 facilitates restart of replication after fork collapse, we performed dna fiber analysis of cells exposed to hu for 6 or 24 hr. however, we were concerned that new origin firing near a collapsed fork might be misinterpreted as fork restart if replication from the new origin proceeded all the way to the collapsed fork. we therefore performed the assay in the presence of a cdc7 inhibitor that does not inhibit transcription (menichincheri., 2010, montagnoli., 2010b) after demonstrating that this inhibitor effectively suppressed new origin firing in cells released from a 24 hr hu replication block (figures s3a and s3c). to monitor fork restart, the cells were incubated with cldu for 1 hr, then incubated with hu for 6 or 24 hr in the presence of the cdc7 inhibitor ; finally, after release from the hu block, the cells were incubated with idu for 1 hr again in the presence of the cdc7 inhibitor to prevent new origin firing. the 1 hr idu incubation period was found to be sufficient to observe restart of replication. in the parental u2os cells, replication restart was observed after exposure to hu for both 6 and 24 hr, although this was the case to a greater extent for the shorter incubation period (figure 3d). in the rad52-knockout clone, replication restart was significantly reduced, especially when the cells were exposed to hu for 24 hr (figures 3d and s3d). since the cdc7 inhibitor prevented new origin firing to further monitor the function of rad52 in bir, we employed a green fluorescent protein (gfp)-based reporter assay in which bir - mediated repair of dna dsbs induced by the endonuclease i - scei leads to gfp fluorescence (costantino., 2014). since the initial description of this assay, we have generated a new, stably transfected cell clone expressing the gfp reporter plasmid that provides a better signal - to - noise ratio than the original clone. depletion of rad52 by sirna in the new clone led to a significant suppression of gfp fluorescence, consistent with rad52 functioning in bir (figure 3e). depletion of pold3 and pold4 also suppressed bir in this system, although not as efficiently as rad52. interestingly, codepletion of rad52 and pold3 or rad52 and pold4 suppressed bir, as efficiently as depletion of rad52 alone, further suggesting that pold3, pold4 and rad52 function epistatically (figure 3e). since bir repairs collapsed forks in cells with oncogene - induced drs, its deletion should curtail cancer development and/or progression. first, we examined tumor formation in apc mice rendered heterozygous for apc by constitutively expressing the cre recombinase under the control of a cmv promoter and enhancer. in these mice, the entire coding sequence of the mutant apc allele is deleted by the cre recombinase (cheung., 2010). in regard to rad52, the mice either had two wt rad52 alleles (rad52) or were homozygous for deletion of the rad52 gene (rad52). at 8 months of age, the mice, while still having no overt signs of disease, were sacrificed, and the presence of tumors in the entire small intestine was scored by histology. deletion of rad52 did not affect the number of observed tumors (a total of 46 versus 50 tumors in six rad52 and six rad52 mice, respectively), but resulted in smaller tumor sizes (figure 4a). deletion of rad52 also resulted in an increase in the fraction of tumor cells scoring positive for phosphorylated h2ax, consistent with an inability to repair collapsed replication forks ; however, this difference was only evident in the early stages of tumor development, when tumors were less than 1.5 mm in diameter (figures 4b and s4). the effect of rad52 on cancer progression was further examined in apc heterozygous mice. in a wt rad52 background, the lifespan of these mice is significantly shorter than that of mice bearing the apc allele described above (moser., indeed, lifespan of the apc heterozygous mice was significantly extended from a mean of 127 days for the rad52 mice to 178 days for the rad52 mice (figure 4c). the function of rad52 in dna repair is well established in yeast, but less well defined in higher eukaryotes and mammals (symington, 2002). in fact, mice with homozygous deletion of the rad52 gene are viable and have no obvious phenotype (rijkers., 1998). this could indicate that rad52 serves a backup function that is relevant only when the primary dna dsb repair pathways are inactivated or overwhelmed by excessive dna damage. supporting this model, depletion of rad52 is synthetic lethal with brca2 deficiency in human cell lines (feng., 2011, lok and powell, 2012). also, cells rely on rad52 for repair when extensive dna damage overwhelms the repair capacities of brca1 and 53bp1 (ochs., 2016). however, the findings reported here suggest that rad52 has more than a backup role in dna repair. specifically, we propose that rad52 has a key role in bir repair of collapsed dna replication forks (figure 4d). endogenous rad52 localized to sites of drs in cells with collapsed dna replication forks, depletion of rad52 led to increased levels of dna damage in cells exposed to hu for 24 hr, dna replication restart from collapsed forks was dependent on rad52, and rad52 depletion suppressed repair of dna dsbs by bir in a gfp - based reporter assay. together with the known biochemical function of mammalian rad52 in strand annealing (kagawa. 2002), these results argue that strand invasion by rad52 leads to dna structures that are conducive to initiation of dna replication after fork collapse. in yeast, rad52 is important for rad51-dependent and rad51-independent forms of hr. whereas repair of dna dsbs by gene conversion in yeast is rad51 dependent, bir can be rad51 independent (ira and haber, 2002, payen., 2008). in this case, the strand annealing activity of rad52 is important, and indeed, it is easy to envision the presence of single - stranded template dna to which the invading strand can anneal at collapsed dna replication forks. by analogy, mammalian rad52, with or without rad51, may be involved in the strand - invasion step of bir, as is the case for its ortholog in yeast (llorente., 2008, a role for rad52 in bir can explain why its depletion in cells with drs leads to increased dna damage (wray., 2016 ; figures 3a and 3b), why the rad52 gene is amplified in human cancers, and why its inactivation curtails cancer development (treuner., 2004, cramer - morales., 2013, lieberman., 2016 ; figure 4). bir also mediates dna repair synthesis in mitosis (minocherhomji., 2015), and it is noteworthy that rad52 is essential also in this context (bhowmick., 2016). for the sirna screen, u2os cells engineered to overexpress cyclin e in an inducible (tet - off system) manner (u2os - cyce cells) were plated in the presence or absence of tet, and the next day they were transfected with sirna. then, 3 days later, the cells were pulsed for 1 hr with edu ; 6 hr after that, they were pulsed for 1 hr with brdu and then processed for flow cytometry as described (costantino., 2014). to generate rad52-knockout u2os - cyce cells, two crispr / cas9 constructs targeting exons 3 and 9 of the human rad52 gene, respectively, were used. after transfection, single clones were expanded and characterized by dna sequencing of the targeted alleles and by immunoblotting for rad52 protein. either u2os - cyce cells grown in the presence or absence of tet for 4 days or u2os parental cells treated with 2 mm hu or 2 m cpt were processed for immunofluorescence, as described (costantino., 2014). to monitor h2ax levels by flow cytometry, u2os cells transfected with the indicated sirnas were treated with 2 mm hu for 0, 2, or 24 hr ; fixed in 70% ice - cold ethanol ; and stained using the flowcellect histone h2ax phosphorylation assay kit (millipore). u2os cells were pulse labeled with cldu for 1 hr, then treated with 2 mm hu and cdc7 inhibitor for 6 or 24 hr, and finally pulse labeled with idu for 1 hr in the presence of the cdc7 inhibitor. dna fibers were spread on aps - coated coverslips and visualized using primary antibodies recognizing cldu or idu. see the supplemental information for a full list of antibodies as well as a full list and detailed description of the methods used in this study. proposed experiments, discussed the results, and contributed to the writing of the manuscript. | summaryhuman cancers are characterized by the presence of oncogene - induced dna replication stress (drs), making them dependent on repair pathways such as break - induced replication (bir) for damaged dna replication forks. to better understand bir, we performed a targeted sirna screen for genes whose depletion inhibited g1 to s phase progression when oncogenic cyclin e was overexpressed. rad52, a gene dispensable for normal development in mice, was among the top hits. in cells in which fork collapse was induced by oncogenes or chemicals, the rad52 protein localized to drs foci. depletion of rad52 by sirna or knockout of the gene by crispr / cas9 compromised restart of collapsed forks and led to dna damage in cells experiencing drs. furthermore, in cancer - prone, heterozygous apc mutant mice, homozygous deletion of the rad52 gene suppressed tumor growth and prolonged lifespan. we therefore propose that mammalian rad52 facilitates repair of collapsed dna replication forks in cancer cells. |
boronic acids and their derivatives are versatile reagents in modern organic synthesis, and the hydroboration reaction is a well - established method for generating these building blocks through the addition of b first described by hurd in 1948 and later developed in detail by brown, this reaction has inspired many catalyzed variants. recently, several compelling examples of related b x bond addition reactivity have been reported for x = c, si, sn, s, b, cl, br, and i (figure 1a). many of these transformations proceed through the oxidative addition of a catalytic transition metal such ni(0), pd(0), or pt(0) into the b x bond. (a) previous work developing addition of b x bonds across alkynes. o bonds across alkynes. despite this progress, the corresponding activation of b o bonds and addition to c c multiple bonds alkoxyboration has remained elusive for 65 years. this striking dearth of b o bond activation reactivity may be due to the extremely high strength of the b o bond, 136 kcal / mol, compared to less than 105 kcal / mol for all others entries in the series. this high stability may render the b o bond unreactive toward oxidative addition, thus preventing the successful application of ni, pd, or pt catalysis in an alkoxyboration reaction. organoboron reagents are the building blocks of choice for medicinal chemistry and drug discovery. given that ethers are found in many diverse classes of natural products and in nearly 25% of the top - grossing pharmaceuticals in the united states for 2012, the development of such a transformation allows for the preparation of oxygen - containing building blocks useful in drug discovery and materials science. herein we report the realization of an alkoxyboration reaction of alkynes (figure 1b), through which new o- heterocyclic organoboronate coupling partners are available for downstream functionalization. the high functional group tolerance of this reaction enables downstream divergent synthesis of functionalized benzofurans the ability to access multiple benzofurans from one bench stable precursor. in contrast, current methods for synthesizing benzofurans often rely on harsh conditions that limit compatibility with functional groups desirable for divergent synthesis. we envisioned that the desired alkoxyboration reactivity could be promoted through an activation pathway employing a bifunctional lewis acidic / lewis basic catalyst, which could simultaneously activate both the alkyne and the b o bond partners. we anticipated that this unique strategy could allow for the anti addition of b o bonds across alkynes by circumventing the previous problematic strategy of oxidative cleavage of the b o bond. our optimized one - pot procedure begins with 2-alkynyl phenols (1), which are converted into the requisite boric ester intermediate 2 using the readily available reagent b - chlorocatecholborane (figure 2). treatment of this intermediate with the commercially available lewis acidic gold(i) precatalyst ipraucl and natfa affords alkoxyboration product 3 in good to excellent conversion as determined by the eretic method. interestingly, our examination of alternative -lewis acidic transition metal catalysts revealed no other active catalysts aside from au(i). for synthetic ease, the catechol boronic ester alkoxyboration product 3 was converted into either the organotrifluoroborate or n - methyliminodiacetic acid (mida) boronate derivative, 4, both of which are air stable indefinitely. values in parentheses represent h nmr yields of the corresponding catechol boronic ester 3 versus an external mesitylene standard using the eretic method. organotrifluoroborate 4a is readily isolated in high yield using a chromatography - free purification, making this derivatization method particularly amenable to applying the alkoxyboration reaction on preparative scale. the corresponding mida derivative (4b) provides an option for purification by silica gel chromatography, but this comes at the cost of slightly diminished yield. single - crystal x - ray diffraction analysis of 4b allowed for the unambiguous identification of the alkoxyboration product (figure 3). x - ray structure of 4b with the thermal ellipsoids set at the 50% probability level (b, yellow ; c, gray ; h, white ; o, red ; n, blue). the alkoxyboration reaction is tolerant of a variety of functional groups suitable for downstream reactivity. aryl bromide 4c, silyl - protected alcohol 4d, terminal alkyne 4f, amide 4 g, esters 4h and 4i, and the functionally dense iodonitrile 4j are compatible with the reaction conditions. many of these alkoxyboration reactions proceed smoothly at 50 c, although the reactions generating 4d, 4 g, 4h, and 4j required heating to 90 c in order to affect full conversion. we attribute the relatively slow formation of 4d to the high steric encumbrance from the silyl ether at the 2-position of the benzofuran. the cyclization of substrates containing lewis basic nitrogen atoms (forming 4 g, 4h, and 4j) was likely retarded by reversible n b coordination that was observed by b nuclear magnetic resonance (nmr) spectroscopy. for all substrates, the mass balance was largely attributable to protonolysis of the product c b bond. notably, many of these products contain functional groups incompatible with commonly employed methods of benzofuran synthesis, including via other borylation techniques (figure 4). in one frequently used borylation technique, an aryl lithium intermediate is trapped by a boron electrophile (method 1) ; thus, electrophiles such as carbonyl or nitrile groups and enolizable protons are not generally tolerated due to the highly nucleophilic and basic nature of the requisite organolithium intermediate. the miyaura borylation is a more mild alternative that is compatible with electrophilic functional groups (method 2), but aryl halides are borylated through this pd(0)-catalyzed reaction and are therefore not spectator functional groups under these conditions. finally, the ir - catalyzed c h activation / borylation reaction is an effective means of accessing aryl boronic acid derivatives (method 3), but this reaction is regioselective for either 2- or 7-borylation ; 3-borylated benzofurans such as those available through the alkoxyboration reaction can not be synthesized regioselectively through c h activation / borylation. benzofuran boronic acid derivatives inaccessible using conventional borylation methods but newly accessible using the alkoxyboration reaction. we set out to demonstrate the utility in divergent synthesis of the alkoxyboration products enabled through this synthesis in subsequent divergent functionalization steps (scheme 1). rh - catalyzed conjugate addition of 4i into methyl vinyl ketone using the method developed by batey provides -benzofuranyl ketone 6 in moderate yield. subjection of the same benzofuran trifluoroborate to suzuki - miyaura coupling conditions described by molander and biolatto afforded afford 3-arylated benzofuran 8 with concomitant methanolysis of the ethyl ester. finally, addition of 4i to an iminium ion was used to prepare aminated benzofuran 10. thus, a single bench - stable alkoxyboration product can be functionalized in a variety of ways, which is important in diversity - oriented syntheses to develop compound catalogs for drug discovery. ester - containing phenol 1i was successfully converted to more than 1 g of organotrifluoroborate 4i on a 5 mmol scale with 2.5% gold catalyst (eq 1). full conversion of starting material was affected even with this lower au catalyst loading. this convenient scalability demonstrates that quantities of o - heterocyclic boronic acid derivatives sufficient for multistep synthesis may be prepared using the alkoxyboration method.1 having demonstrated the utility of this transformation in generating members of the benzofuran class of o - heteroaryl boronic acid derivatives, we explored its application to the synthesis of a nonaromatic oxygen - containing heterocycle (eq 2). simple and commercially available homopropargyl alcohol 11 was subjected to standard alkoxyboration reaction conditions to prepare dihydrofuran product 12. a large number of unidentifiable trace coproducts were detected in this reaction, possibly consistent with intermolecular reactivity. this substrate demonstrates the potential for generality in the alkoxyboration reaction : the reaction features low labor setup cost by employing simple, commercially available starting materials to generate highly value - added o - heterocyclic organoboronate compounds in one synthetic step, and the cyclization proceeds without requiring the gain of product aromaticity or the need for a fused ring system that enforces a conformational bias toward cyclization.2 a number of lewis - acidic metal catalysts have been developed for the addition of oxygen - electrophile bonds across alkynes, albeit not with boron and thus not generating these building blocks for downstream functionaliztion. we propose the catalytic cycle shown in scheme 2 featuring bifunctional lewis acidic / lewis basic substrate activation. the bifunctional catalyst iprautfa can be generated in situ from ipraucl and natfa. reaction of the lewis basic trifluoroacetate moiety with electrophilic boric ester 2a gives nucleophilic borate 14. the resulting lewis acidic au(i) cation may then bind to the alkyne (15), increasing its electrophilicity. au complex by the phenol b o bond would provide two neutral intermediates : boron electrophile 16 and organogold nucleophile 17, which could recombine to regenerate 13 with concomitant formation of the observed alkoxyboration product 3a. thus, the iprau moiety of the catalyst activates the alkyne for nucleophilic attack, and the tfa counterion allows for reversible tuning from a boron electrophile to a nucleophilic borate adduct. this reaction manifold is fundamentally unique from the metal - catalyzed addition of b c, b s bonds, which often proceed through oxidative addition of a low - valent metal catalyst into the b x bond. we believe that the new activation strategy employed in the alkoxyboration reaction could be extended to other types of b notably, this approach also suggests a new generalizable mechanism for au catalyst turnover by trapping with electrophilic boron to generate other previously inaccessible organoboron building blocks. this fundamentally new activation is showcased in a mild, scalable technique for the preparation of o - heterocyclic boronic acid derivatives and downstream - functionalized benzofurans. the reaction provides a simple new bond disconnection for constructing these motifs with different regioselectivity and broader functional group compatibility than existing methods. this compatibility yields highly functionalized bench - stable building blocks for divergent synthesis that are not directly accessible using alternative methods. the carbophilic lewis acid activation mechanism for b x addition reactions and the ability to synthesize previously inaccessible organoboron building blocks via this new strategy for turning over gold and other carbophilic metal catalysts. sodium trifluoroacetate was dried at 130 c at 10 mtorr for 18 h before use. toluene and dichloromethane were purified by passage through an alumina column under argon pressure on a push - still solvent system. anhydrous dimethylsulfoxide was obtained by stirring over activity i alumina 18 h under n2 atmosphere, decanting the liquid, and distilling the liquid at 10 torr over cah2. toluene - d8 was dried over cah2, degassed using three freeze pump thaw cycles, and vacuum transferred prior to use. all manipulations were conducted in a glovebox under nitrogen atmosphere or using standard schlenk techniques unless otherwise specified. plates were visualized under uv irradiation (254 nm) and/or using a basic aqueous solution of potassium permanganate. flash chromatography was conducted using a teledyne isco combiflash rf 200 automated flash chromatography system, and teledyne isco redisep 3570 m silica gel. all proton and carbon nuclear magnetic resonance (h and c nmr) spectra were recorded on a bruker drx-400 spectrometer, bruker drx-500 spectrometer outfitted with a cryoprobe, or a bruker avance-600 spectrometer. all boron nuclear magnetic resonance (b nmr) all fluorine nuclear magnetic resonance (f nmr) spectra were recorded on a bruker drx-400. all chemical shifts () are reported in parts per million (ppm) downfield of tetramethylsilane, and referenced to the residual protiated solvent peak (= 7.26 ppm for cdcl3, = 2.50 ppm for dmso - d6, or = 1.94 ppm for cd3cn in h nmr spectroscopy experiments ; = 77.16 ppm for cdcl3, = 39.52 ppm for dmso - d6, or = 1.34 ppm for cd3cn in c nmr spectroscopy experiments). b and f nmr spectroscopy experiments are referenced to the absolute frequency of 0 ppm in the h dimension according to the xi scale. low- and high - resolution mass spectrometry data were obtained at the university of california, irvine. all alkoxyboration reactions were conducted in a n2-filled glovebox due to the high moisture sensitivity of the boric ester intermediate 2. the reaction progress was monitored by removing a small aliquot of the reaction mixture from the glovebox and diluting it in 1:1 etoac : water. this results in rapid hydrolysis of boric ester intermediate 2 back to the phenol starting material 1. thus, co - spotting the reaction mixture versus phenol 1 provides a convenient method for determining whether or not intermediate 2 has been fully consumed. the addition of pph3 to quench the au catalyst between the alkoxyboration step and the formation of the organotrifluoroborate or mida boronate was essential. a solution of phenol 1a (97.0 mg, 0.500 mmol, 1.00 equiv) in 1.0 ml of toluene was added to a flame - dried 10-ml schlenk tube. to this stirring solution was added dropwise at 25 c a suspension of nah (69 wt % purity, 17.4 mg, 0.500 mmol, 1.00 equiv) in 0.5 ml of toluene over 2 min. a suspension of natfa (20 mg, 0.15 mmol, 30 mol %) in 0.5 ml of toluene was added next, and the resulting suspension was stirred for 15 min to affect full deprotonation. to the resulting stirring sodium phenoxide suspension was added at 25 c a solution of b - chlorocatecholborane (77.0 mg, 0.500 mmol, 1.00 equiv) in toluene (1.0 ml), using additional toluene as a rinse to ensure full transfer (2 0.5 ml portions). the resulting suspension was stirred vigorously for 30 min to allow for full conversion to boric ester intermediate 2a. next, a suspension of ipraucl (16 mg, 0.025 mmol, 5.0 mol %) in toluene (0.5 ml) was added to the reaction mixture at 25 c, using additional toluene as a rinse to aid in full transfer (1 0.5 ml portion). the reaction mixture was then sealed with a ground glass stopper and a ptfe sealing ring and placed in a preheated 50 c copper shot heating bath. after 24 h, analysis by tlc (20% etoac / hexanes) indicated full consumption of boric ester intermediate 2a. the reaction mixture was cooled to 25 c, and a solution of pph3 (13 mg, 0.050 mmol, 10 mol %) in toluene (0.5 ml) was added. the resulting suspension was stirred for 22 h at 25 c in order to quench iprautfa before proceeding. the quenched reaction mixture was removed from the glovebox and filtered through a fiberglass filter to remove the suspended solids. the filter was then rinsed with toluene (3 3 ml), and the combined filtrates were concentrated in vacuo to a pale yellow powder, which was suspended in acetone (4.5 ml) and added to a stirring solution of khf2 (140 mg, 1.8 mmol, 3.5 equiv) in water (1.5 ml). the resulting mixture was stirred at 25 c for 30 min, and then concentrated in vacuo to remove the solvents. to this residue was added 2 ml of et2o and the solution was subsequently concentrated at ca. the resulting pale yellow solid residue was washed with et2o (4 2 ml) and extracted with acetone (4 2 ml). the combined acetone extracts were concentrated in vacuo, and the resulting residue was subjected to an additional washing / extraction cycle to yield 4a as a white powder (113 mg, 75% yield) after removing volatiles at 25 c and ca. 10 mtorr for 18 h. h nmr (dmso - d6, 600 mhz) : 8.10 (d, j = 7.4 hz, 2h), 7.88 (d, j = 7.7 hz, 1h), 7.42 (d, j = 8.0 hz, 1h), 7.38 (t, j = 7.6 hz, 2h), 7.26 (t, j = 7.3 hz, 1h), 7.13 (td, j = 6.5 hz, 1.2 hz, 1h), 7.08 (td, j = 7.4, 0.9 hz, 1h). c nmr (dmso - d6, 125 mhz) : 154.7 (q, jc f = 4.6 hz), 153.8, 135.4, 133.3, 127.9, 126.8, 126.7 (q, jc f = 2.3 hz), 124.1 (q, jc [note : as with many organotrifluoroborates, the ipso carbon was not detected, presumably due to broadening through coupling to the 11b nucleus. b nmr (dmso - d6, 193 mhz) : 3.2 (br s). f nmr (dmso - d6, 376 mhz) : 131.9 (br s). hrms (esi-) : calculated for c14h9bf3o ([m k ]), 261.0701 ; found, 261.0706. a solution of phenol 1a (97.0 mg, 0.500 mmol, 1.00 equiv) in 1.0 ml of toluene was added to a flame - dried 10-ml schlenk tube. to this stirring solution was added dropwise at 25 c a suspension of nah (92 wt % purity, 13.0 mg, 0.500 mmol, 1.00 equiv) in 0.5 ml of toluene over 2 min. a suspension of natfa (20 mg, 0.15 mmol, 30 mol %) in 0.5 ml of toluene was added next, and the resulting suspension was stirred for 15 min to affect full deprotonation. to the resulting stirring sodium phenoxide suspension was added at 25 c a solution of b - chlorocatecholborane (77.0 mg, 0.500 mmol, 1.00 equiv) in toluene (1.0 ml), using additional toluene as a rinse to ensure full transfer (2 0.5 ml portions). the resulting suspension was stirred vigorously for 30 min to allow for full conversion to boric ester intermediate 2a. next, a suspension of ipraucl (16 mg, 0.025 mmol, 5.0 mol %) in toluene (0.5 ml) was added to the reaction mixture at 25 c, using additional toluene as a rinse to aid in full transfer (1 0.5 ml portion). the reaction mixture was then sealed with a ground glass stopper and a ptfe sealing ring and placed in a preheated 50 c copper shot heating bath. after 24 h, analysis by tlc (20% etoac / hexanes) indicated full consumption of boric ester intermediate 2a. the reaction mixture was cooled to 25 c, and a solution of pph3 (13 mg, 0.050 mmol, 10 mol %) in toluene (0.5 ml) was added. the resulting suspension was stirred for 16 h at 25 c in order to quench iprautfa before proceeding. anhydrous dmso (2.0 ml) and h2mida (81 mg, 0.55 mmol, 1.1 equiv) were added to the quenched alkoxyboration reaction mixture, and the resulting suspension was stirred at 90 c for 2 h. the reaction mixture was then cooled to 25 c and removed from the glovebox. the resulting semisolid residue was adsorbed onto celite from a mecn suspension and purified by silica gel chromatography using an elution gradient from 100% et2o to 100% mecn. 10 mtorr for 18 h afforded mida boronate 4b as a white powder (101 mg, 58% yield). crystals suitable for x - ray diffraction analysis were prepared by slow diffusion of et2o into a saturated solution of 4b in et2o / acetone at 25 c over 3 days. h nmr (cd3cn, 600 mhz) : 7.72 (dd, j = 7.8 hz, 0.8 hz, 1h), 7.677.65 (m, 2h), 7.55 (d, j = 9.7 hz, 1h), 7.477.44 (m, 3h), 7.357.31 (m, 1h), 7.297.26 (m, 1h), 3.97 (d, j = 17.1 hz, 2h), 3.65 (d, j = 17.1 hz, 2h), 2.56 (s, 3h). c nmr (cd3cn, 125 mhz) : 169.0, 156.0, 133.6, 133.0, 130.6, 130.2, 129.3, 125.2, 123.9, 123.5, 111.8, 63.0, 48.2. [note : no signals were observed for the quaternary c b ipso carbon or the quaternary carbon at the benzofuran 2 position. ] b nmr (cd3cn, 193 mhz) : 11.3 (br s). hrms (esi+) : calculated for c17h19bbrno5 ([m + na ]), 372.1023 ; found, 372.1016. | for nearly 70 years, the addition of boron x bonds to carbon carbon multiple bonds has been employed in the preparation of organoboron reagents. however, the significantly higher strength of boron oxygen bonds has thus far precluded their activation for addition, preventing a direct route to access a potentially valuable class of oxygen - containing organoboron reagents for divergent synthesis. we herein report the realization of an alkoxyboration reaction, the addition of boron oxygen bonds to alkynes. functionalized o - heterocyclic boronic acid derivatives are produced using this transformation, which is mild and exhibits broad functional group compatibility. our results demonstrate activation of this boron o bond using a gold catalysis strategy that is fundamentally different from that used previously for other boron addition reactions. |
with the rising proportion of older adults and increases in life expectancy, there has been increased interest in maintaining and promoting cognitive health in later life. although declines in some domains of cognition are part of the natural course of aging [2, 3 ], sufficient evidence from prospective and observational studies indicates that the trajectories and outcomes of cognitive decline may be mitigated by participating in cognitively stimulating activities [4, 5 ]. recent reviews of cognitive interventions suggest some potential benefits that may improve functioning in healthy older adults or slow decline in individuals with mild cognitive impairment (mci) and those already affected with dementia [69 ]. results from a meta - analysis of randomized controlled trials in healthy aging revealed a strong positive effect on cognition at immediate, medium-, and long - term followup after cognitive training. compelling results from large longitudinal studies have also shown that engagement in everyday cognitive activities predicts preserved cognition [11, 12 ] and decreases in incident alzheimer 's disease. technologies, currently valued at $ 300 million, is projected to swell exponentially within this decade. a facet of this research that is relatively understudied involves examining the degree to which discrete types of everyday cognitive activity relate to change in specific cognitive domains over time. most of the aforementioned trials incorporated training in multiple cognitive abilities and accordingly found support for cognitive training in general, but some reviews report less promising results for domain - specific training. memory is frequently the targeted cognitive domain in many interventions, with training involving efforts to improve recall of newly learned information, including skills training, imagery, and mnemonic strategy use ; however, meta - analyses reveal minimal efficacy for memory - focused techniques. investigations of the effectiveness of training in other cognitive abilities such as executive functions and working memory suggest that these have farther - reaching effects on cognitive function, but results are regarded as preliminary. formal interventions employing cognitive skills training are rarely conducted outside of clinical trials, leaving observational studies as a valuable resource for evaluating potential benefits of everyday cognitive activities. observational studies typically include self - report inventories of activities commonly regarded as cognitively stimulating, such as solving puzzles, listening to the radio, or reading books. while such studies are not experimental by design, they contribute a significant addition to the literature by assessing changes in accessible, everyday cognitive activities as these relate to change in cognitive abilities in a naturalistic setting. despite the promising body of work that has accumulated in recent years, definitive conclusions regarding the benefits of cognitive activity are precluded by several methodological concerns [11, 18 ]. limitations include the inadequacy of activity assessments, psychometric variability of cognitive outcome measures, conceptual differences in the expected relationships between activities and specific cognitive domains, and insufficient assessment of moderating variables such as level of education and sex. while a study in which all these limitations are fully addressed has yet to be conducted, existing data from several longitudinal studies can be leveraged to disentangle some of these effects. in this paper, we intend to demonstrate that a coordinated analysis of four large longitudinal studies of aging can elucidate the benefits of changes in cognitive activity over time to performance trajectories in specific cognitive domains. thus, the purpose of this study was to examine the effects of self - reported everyday cognitive activities and changes in these activities on changes in four domains of cognition (reasoning, fluency, memory, and semantic knowledge) in longitudinal models that incorporate data from the origins of variance in the oldest - old : octogenarian twins study (octo - twin), the long beach longitudinal study (lbls), the seattle longitudinal study (sls), and the victoria longitudinal study (vls). this investigation was part of a larger coordinated effort to examine the effects of lifestyle activities on cognitive function across multiple large - scale longitudinal studies of aging that formed the basis of a meeting of the advanced psychometrics methods in cognitive aging workshop. the aim of this workshop was to use a common analytic protocol across studies from the integrative analysis of longitudinal studies on aging (ialsa) network. these studies were specifically selected based on their collection of cognitive, physical, and social activity data along with a range of cognitive functioning measures over multiple occasions. while the cognitive activity and cognitive function variables are not always identical, the subsets of variables in each study were chosen based on the rationale that they tapped similar domains at the construct level ; specifically, we chose measures thought to tap fluid reasoning (gf, i.e., verbal reasoning, block design, and verbal fluency measures), short - term memory (gsm, i.e., immediate recall of a verbally presented story or word list), and crystallized knowledge (gc, i.e., measures of vocabulary and acquired knowledge). in some cases the measures are the same, but more often they differ, precluding statistical combination of indicators and outcomes between studies. however, we argue that concurrently analyzing the data with the same method provides opportunities for both strict and conceptual replication within the same study thus, our goal was to build and implement a common model to each dataset to enable comparisons across all outcomes for the four longitudinal studies. the two primary hypotheses we tested were whether (1) cognitive activity at baseline would predict the trajectory of cognitive function over time and (2) change in cognitive activity would predict change in cognitive function over time. we report a series of mixed effects models that included baseline and change in cognitive activity predicting cognitive function over up to 21 years of time in four large - scale longitudinal studies of older adults. three of the four studies included in this analysis (lbls, sls, and vls) specifically aimed to study healthy aging and only recruited community - dwelling older adults who were presumed to be cognitively normal at baseline. the fourth study, octo - twin, also included a largely cognitively normal sample of older adults, but those who did have dementia diagnoses at baseline (n = 98) were excluded from the present analysis. in order to model roughly equivalent cognitive outcomes across the four study samples in this coordinated effort, analyses included selected measures of reasoning, fluency, episodic memory, and semantic knowledge from the larger battery of tests included within each longitudinal study sample. in the octo - twin study, there was no fluency measure available, and we thus present only three of the four cognitive outcomes. these cognitive tasks were selected to represent a range of cognitive abilities from basic to more complex functions. study sample characteristics and demographic, cognitive function, and cognitive activity measures are described below (tables 14). the octo - twin study is based on the oldest cohort of the swedish twin registry and includes 702 participants aged 80 years and older at the time of the first examination. all individuals with a dementia diagnosis at baseline the longitudinal design for survivors included a maximum of five measurement times at two - year intervals beginning in 19911993. the average rate of attrition from one test interval to the next was 20% (10% per year), primarily due to death. table 1 provides a summary of participant characteristics for the octo - twin participants included in this study. reasoning was assessed using block design, in which participants are presented with red and white blocks and instructed to assemble the blocks to reproduce a design portrayed on a card within a predetermined time limit. as previously mentioned, the octo - twin study did not have a measure of fluency so this cognitive domain was not analyzed and compared with fluency results from the three other longitudinal studies. memory was assessed using the prose recall test in which participants were asked for immediate free recall of a brief (100 word) story that had a humorous point. responses were coded for the amount of information recalled in a manner similar to the scoring of story units in the wechsler memory scale logical memory test. semantic knowledge was assessed using the swedish version of the wais information task, which requires participants to provide answers to questions assessing acquired knowledge of facts. the cognitive activity measure was based on self - report of engagement in six cognitively stimulating activities including playing games (e.g., chess and bridge), completing crossword puzzles, reading literature, writing, conducting genealogical research, or any otherdocumentation, studies, or other mentally demanding activity (e.g., handicraft), each rated dichotomously as no (0) or yes (1). participants were also asked if they train their memory or keep their mind active rated as no (0), yes, to a certain degree (1), or a composite score for cognitive activity was created by summing responses across items (range = 08). change in cognitive activity was computed by subtracting the cognitive activity score at baseline from all subsequent activity scores. the lbls was started in 1978 in long beach, california, with participants recruited from the family health plan health maintenance organization (hmo) who were primarily from long beach and orange counties. the ethnic composition of the older group (98% caucasian) was similar to the 65 + population for the area based on the 1970 census. panel 2, initiated in 1992, included 633 contacted from the same hmo (64 were excluded due to frank dementia, serious sensory, or neurological problems). in order to include the same measures as those in the seattle longitudinal study, lbls panel 1 (n = 106) and panel 2 (n = 631) data from 1994 to 2003 were used in the current analysis. during this period, data were collected at 3-year intervals. only visits occurring at age of 55 or older were included in this study (baseline n = 561). the table displays the number of participants at each test occasion that completed the four cognitive measures and the retention rates for those measures from one testing to the next (top line). collapsed across measures and testing, the average retention rate from one testing to the next was 55.8% or 17% per year. age and education increased from the first to the fourth test occasions, suggesting that the sample became more selective over time. similar patterns of selection were observed for the cognitive measures of reasoning, memory, fluency, and semantic knowledge. reasoning was indexed as a composite score of the schaie - thurstone adult mental abilities test (stamat) letter and word series tests. in letter series, participants viewed a series of letters (e.g., a b c c b a d e f f) and were asked to discover the rule that governs the series by identifying the letter from an array of four possible responses that should come next in the series. participants were to complete as many of the 30 items as possible within six minutes. word series was a parallel test to letter series but the letters were replaced with months (e.g., january) and days of the week (e.g., monday). fluency was measured using word fluency, in which participants were instructed to write down as many words as possible in five minutes that begin with a specified letter s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words (e.g., if the letter was w and you already said want, you should not also say wants, wanting, or wanted). memory was measured using immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes. participants were given a word and asked to circle a synonym of that word from four possible alternatives. the cognitive activity measure was derived from a modified version of the life complexity scale (lcs), originally developed for the seattle longitudinal study. the modified scale consisted of six items from the lcs : educational activities, leisure reading, playing musical instruments, writing letters, playing games, and cultural activities participants were asked to record the number of hours per week on average they spent doing each activity. due to extreme variability in reported hours observed within and between items, responses were dichotomized for the present analysis with those who reported no time spent on a given activity coded as 0 and those who reported one or more hours of activity coded as 1. items were summed to create a composite measure of cognitive activity (range = 06). change in cognitive activity was computed by subtracting the cognitive activity score at baseline from all subsequent activity scores. the sls was initiated in 1956 in seattle, washington, and includes eight samples recruited from a local hmo at seven - year intervals and followed longitudinally every seven years (total n across all study samples = 4,854). the current analysis includes data from participants in the study from 1984 to 2005 (total n across 19842005 study samples = 2,040) and includes longitudinal data for up to four testing occasions. this subset of the larger study was selected due to changes in measures used over the course of the entire study in order to have equivalent measures of cognition and activity at each time point and with the lbls. only visits occurring at age 55 or older were included in our analyses, yielding a total of 1,649 participants at baseline. baseline was defined as each participant 's first study visit, and time was measured in all analyses as years in study (coded as 0, 7, 14, and 21). see table 3 for sls participant characteristics over the four waves of data analyzed here. reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test (stamat), in which participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format by identifying the rule that governed a series. the test consisted of 30 items, and total score was based on number of correct responses completed in 6 minutes. fluency was assessed with the word fluency test from the primary mental abilities test, in which participants were asked to write down words beginning with the letter s following a rule set (do not use proper nouns and do not use different conjugations of the same word). memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items. semantic knowledge was assessed with the educational testing service (ets) test of advanced vocabulary, in which participants were asked to identify synonyms for printed words from 5 choices. total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes. the cognitive activity measure was derived by summing dichotomized test responses to five cognitive activity items (reading, educational activities, music, writing, and cultural activities) from a modified version of the life complexity scale. cognitive activity change was computed by subtracting baseline activity from each follow - up activity measure. the vls was begun in the 1986 in victoria, british columbia, and consists of three cohorts started in 1986, 1992, and 2001, respectively, followed longitudinally at 3-year intervals. longitudinal data used in this study were from samples 1 (baseline n = 484) and 2 (baseline n = 530). for this investigation, data from seven waves of sample 1 and five waves of sample 2 were included in analyses. approximately 25% of the sample was lost to follow up at each wave or 8% per year. reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule. fluency was measured by performance on a similarities task. in this timed task, participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within 6 minutes. memory was indexed using a 30-item noun list learning task comprised of five semantic categories. participants studied the word list for 2 minutes followed by a 5-minute free recall task. the cognitive activity measure included a subset of items from the vls activity lifestyle questionnaire. the 27 items comprising the novel information processing scale were selected due to the cognitively stimulating nature of the activities. for each item, participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 (i.e., never, less than once a year, about once a year, 2 or 3 times a year, about once a month, 2 or 3 times a month, about once a week, 2 or 3 times a week, and daily). individual item distributions were reviewed, and 11 of the 27 original items with little to no variability were eliminated. the remaining items were a priori hypothesized to fall into three general types of activities : those involving what we termed communication, computations, or confirmatory factor analysis using mplus version 6.0 was conducted to test a three - factor model including six items indexing communication (enrolling in college courses, giving a talk, attending lectures, studying a second language, writing, and writing letters specifically), five items indexing computation (balancing a check book, performing mathematical calculations, working on taxes, engaging in business activity, and using a calculator), and five items indexing conundrums (engaging in crosswords, chess / checkers, knowledge games, word games / scrabble, and jigsaw puzzles). fit criteria were the comparative fit index (cfi) and the root mean squared error of approximation (rmsea), where criteria for excellent fit include cfi > 0.95 and rmsea < 0.05. allowing for within - factor residual correlations, the model demonstrated acceptable fit (cfi = 0.95, rmsea = 0.04). the factor scores generated by this analysis were then used as the primary predictor variables in three separate mixed effects models. the current analysis was conducted as part of a larger effort to examine the effects of lifestyle activities on cognitive function using the same analytic approach across studies from the integrative analysis of longitudinal studies on aging (ialsa) network, and models were selected in part to maintain consistency across lifestyle activities. across all four studies, we examined common demographic covariates including age (in years), years of formal education, and sex (coded as 0 = male, 1 = female). age and education were mean centered to their respective study 's baseline mean value. in order to maximize use of all available data, we defined baseline as the first study visit for each participant with available cognitive activity data. we analyzed the data with mixed effects modeling using stata software, version 12 (statacorp, 2011) and restricted maximum likelihood (reml) estimation, random slopes and intercepts, and an unstructured covariance matrix. in the octo - twin study, participants were nested within their twin pair. in the vls, we controlled for enrolment cohort. model assumptions were verified by examining residuals computed using predicted values that included the random effects. we defined the criterion for significance as p < 0.05. while we recognize that this criterion may be viewed as liberal, given the large number of comparisons across all statistical models in our analysis, we assert that this approach is warranted in this study as we are representing results from four independent longitudinal studies following similar statistical procedures for each. thus, the emphasis in this paper is replication of the pattern of results across the studies. in this way, the strictness of the evaluation of the effects comes from noting whether a particular effect is replicated across the different samples. alpha rate is that which occurs within each study, not across all of them together. an initial 19-term model included the following terms : (1) baseline age, (2) sex, (3) education, (4) baseline activity, (5) baseline activity age, (6) baseline activity sex, (7) baseline activity education, (8) individually defined time since baseline, (9) time baseline age, (10) time sex, (11) time education, (12) time baseline activity, (13) time baseline activity baseline age, (14) time baseline activity sex, (15) time baseline activity education, (16) change in activity from baseline (activity change), (17) activity change baseline age, (18) activity change sex, and (19) activity change education. this full model was evaluated in each study data set independently, and terms that were not significant in any of the four studies were dropped in order to present a parsimonious set of results that retained the fullest set of parameters found in any study. this process eliminated 7 of the 19 terms, including all 3-way interactions, and four of the 2-way interactions, including the interactions between activity change and age, sex, or education, as well as the interaction between baseline activity level and sex. as a proper meta - analytic summary would require identical measures across a larger number of studies, we rely on straightforward comparison of the conclusions derived from each study. there was a significant relationship between self - reported cognitive activity at baseline and baseline performance on tests of cognitive abilities across all measures and studies but the lbls, which did not find this relationship in the reasoning and memory models. overall, these findings suggest that participants who were more cognitively active at baseline tended to have better cognitive performance. one of the studies (vls) included three distinct measures of cognitive activity those involving communication (e.g., writing), computations (e.g., managing finances), and conundrums (e.g., completing crossword puzzles)enabling us to determine if specific cognitive activities were differentially related to the cognitive outcomes. while all three types of cognitive activities showed significant cross - sectional relationships with cognitive outcomes (all p < 0.001), the strongest relationships with cognitive function were found for conundrums, followed by computation and communication. older age was associated with lower baseline performance across all studies on measures of reasoning, fluency, and memory. in contrast, the relationship between age and baseline performance on semantic knowledge measures was inconsistent, with lbls and octo - twin results suggesting lower performance in older age, sls showing no age differences, and vls suggesting that older age was associated with better performance. baseline associations between sex and cognitive performance showed a consistent relationship across studies for all memory outcomes, with women consistently performing higher than similar aged men. sls and lbls women additionally performed higher than men on reasoning and fluency measures. across other cognitive outcomes, baseline associations between performance and sex were less consistent, with vls women performing better on fluency in the computations model and octo - twin women performing lower than men on semantic knowledge. higher education was consistently associated with higher baseline cognitive performance across all studies and cognitive outcomes. two baseline covariate interaction terms were retained in the final model, and both showed inconsistent relationships across studies and outcome measures : the age by baseline cognitive activity interaction term was significant in the vls memory models and the conundrums / semantic knowledge model. there was a similarly significant interaction between baseline age and activity level in the lbls reasoning model (p < 0.05). the education by baseline cognitive activity interaction term in the vls communication models for reasoning, fluency, and semantic knowledge was significant, suggesting that those with lower education had a higher association between baseline activity and cognitive test performance. the vls computation and octo - twin models for semantic knowledge also showed this relationship. across all studies and cognitive outcomes, there was, with one exception (vls computation with reasoning), no evidence for baseline level of cognitive activity predicting change in cognitive outcomes over time. there was, however, a consistent positive relationship between change in cognitive activity from baseline and within person variability in cognitive outcomes across nearly all cognitive outcomes in all four studies. specifically, after accounting for the expected linear within person trajectories, variation in cognitive activity was significantly related to variation in performance on all measures in all studies except reasoning and fluency in lbls and reasoning and memory, in the case of conundrums only, in vls. within - person declines were seen over time across all studies and all cognitive outcomes except lbls fluency. older participants declined faster compared to younger participants on all vls, sls, and lbls cognitive outcome measures except lbls memory. evidence for differential decline in older participants was not seen in octo - twin, which has a much narrower age range. women declined less than men on fluency measures in the sls and vls computations models and on semantic knowledge measures in the sls and octo - twin study. level of education was not a significant predictor of rate of cognitive decline in all but one study (lbls) and one outcome measure (reasoning, coefficient = 0.06, p < 0.01). our results provide compelling evidence across four longitudinal studies that changes in everyday cognitive activity level tracks with variation in multiple aspects of cognitive function. in three of the four studies (octo - twin, sls, and vls), participants reported engaging in fewer cognitive activities over time. in the fourth study (lbls), participants endorsed a slight increase in average number of cognitive activities over time, which was likely due to differential retention of higher functioning individuals. while the overall trend was for participants to report slightly less cognitive activity at each follow - up visit in all but the lbls sample, there was actually considerable variability in activity change scores, with some participants in each study reporting increased cognitive activity at follow - up visits relative to their baseline levels. these results suggest that there is an increased risk of cognitive decline for individuals whose engagement in cognitive activities decreases over time relative to their baseline levels, and, conversely, the results suggest that increases in cognitive activity from baseline are associated with better than expected cognitive performance. cognitive activity change appeared to most consistently track with variation in semantic knowledge, as the activity change term was significant in all six models. strong evidence of activity change tracking with fluctuations in memory and fluency was also indicated, as five of six models had significant activity change terms in the memory models and in four of the five fluency models. activity change was significantly related to variation in reasoning in four of the six models, making the models with reasoning outcomes the least consistent relative to models with the other cognitive outcomes. that two of the four inconsistent findings occurred in lbls, which had the most similarity with sls, in terms of both measures and sampling, suggests that some other factor, such as attrition, may be responsible for these differences. it is interesting to note, however, that the standard deviation of reported activity level did not differ from that of sls. the lack of association with reasoning and memory for one of the three vls activity variables (conundrums) could be due to chance, although this finding may also suggest that changes in level of engagement on tasks involving problem solving are less related to changes in reasoning and memory function than they are to changes in fluency and semantic knowledge. across studies, with the exception of vls computation with reasoning, there were no significant relationships between baseline cognitive activity and change in cognition over time, suggesting that level of cognitive activity at an earlier point in time is not related to subsequent cognitive decline. thus, these results do not demonstrate that level of engagement in cognitively stimulating activities earlier in older adulthood can somehow increase one 's cognitive reserve or ability to maintain cognitive function in spite of age - related brain changes. nonetheless, our results do have important clinical implications in that they suggest that individuals who exhibit changes from a previous level of cognitive activity can be expected to have associated fluctuations in cognitive performance, or vice versa. in terms of cross - sectional relationships, all studies provide evidence for activity / cognition relationships, and the vls results allow us to conclude that level of engagement in cognitive activities involving what we termed conundrums (e.g., playing chess, completing crossword puzzles) are most strongly and consistently related to concurrent function across cognitive domains, but evidence for relationships between engagement in activities involving computations (e.g., balancing a check book) and communication (e.g., writing letters) was also demonstrated. thus, while the data do not provide particularly compelling evidence that engagement in one type of cognitively stimulating activity is preferable, activities involving novel information processing appear to be most related to concurrent cognitive function, a finding that is consistent with the extant literature. the lack of evidence for cognitive activity level at baseline predicting cognitive decline over time in some respects may be interpreted as discouraging, as it implies that older adults who more frequently engage in cognitive activities may not be influencing the trajectory of their cognitive function in the coming years. however, across all studies, change in level of cognitive activity from baseline generally followed a normal distribution, with considerable portions of each sample reporting an increase in level of cognitive activity from baseline levels. the positive association between cognitive activity change and the cognitive outcomes across studies thus suggests that individuals who increase their cognitive activities may be effectively reducing age - related cognitive decline. our results demonstrate that older age is associated with faster decline, which supports the overall validity of our approach and suggests that we are detecting relevant change. the finding that education was not predictive of rate of cognitive decline with one exception (the lbls reasoning model) suggests that education is not protective or predictive of a faster decline in normal aging. these multistudy results build upon findings from a recent paper using data from one of the studies (vls) included in the current paper, in which the authors conclude that the relationship between education and cognitive performance is merely a cross - sectional relationship between level of education and cognitive function, and that longitudinal models that covary for baseline cognitive function are in effect creating a statistical artifact that is seen as an effect of education on rate of decline. however, it is important to note that all studies included in the current analysis were designed to characterize normal cognitive aging, and results are not directly comparable to studies examining the effect of education level or cognitive reserve on the incidence and rate of decline in alzheimer 's disease. the current study has many strengths, including the large sample sizes and multinational representation in our study samples, which improves the generalizability of the findings. in addition, the inclusion of four separate studies with unique sample characteristics, methodologies for recruitment, different methods for measuring cognitive activity and cognitive function, and differing frequency and length of followup, all serve to minimize the likelihood that these findings are spurious. when results across such a coordinated analysis are inconsistent, any one of these differences between studies could be responsible for discrepancies and reflect a limitation of the design. for example, the inconsistencies in the relationships between baseline covariates and their interactions (e.g., sex and age with baseline activity level) highlight a weakness of our study design. inconsistencies could also be attributable to the heterogeneity in the activity measures used across the four longitudinal studies, as the scales included different items with different response ratings, yielding restricted ranges of responses on some measures. it is also possible that the inconsistencies are due to differences in the cognitive outcomes used in the different studies, or any number of other differences in the methodologies across studies. however, it is important to note that when the model results demonstrate consistent patterns across studies despite variations in methodology, the heterogeneity of measures and sampling methods becomes a major strength of the multi - study approach, as there is improvement in the reliability of conclusions that can be drawn from the results, relative to the typical single - study design. perhaps the most obvious limitation inherent in the observational design of all studies included in this investigation is that conclusions implying causality can not be inferred from these results. specifically, while an increase in cognitive activity from baseline was associated with better than expected cognitive performance, and, conversely, activity decrease was associated with worse than expected performance, it is not possible to conclude that change in activity level was the cause for change in rate of cognitive decline. an alternative explanation is that decreases in level of cognitive activity from baseline levels observed in this study result from deteriorating cognitive functions rather than cause it. put simply, this study design does not answer whether completing crossword puzzles reduces one 's risk of cognitive decline or if cognitive decline reduces the likelihood that one will complete crossword puzzles. in addition, this study does not address the protective effects of cognitive activity for incident dementia or alzheimer 's disease. while there is a large body of the literature examining the beneficial effects of cognitive activity in reducing dementia risk (e.g.,), the studies included in the current investigation were based on normal cognitive aging, and individuals with dementia diagnoses were excluded from the present analysis. what these results impart, however, is that regardless of the causal mechanisms underlying these changes, the associations between cognitive activity and cognitive outcomes in this study are in directions that are intuitively and scientifically consistent with prior literature. this fact, coupled with the large - scale naturalistic, observational design of this study, lends credence to the burgeoning literature that directly examines the causal effect of cognitive activity on cognitive outcomes. extension of this work in populations at great risk for dementia, or with individuals already diagnosed with neurodegenerative diseases, remains a worthwhile goal. | engagement in cognitively stimulating activities has been considered to maintain or strengthen cognitive skills, thereby minimizing age - related cognitive decline. while the idea that there may be a modifiable behavior that could lower risk for cognitive decline is appealing and potentially empowering for older adults, research findings have not consistently supported the beneficial effects of engaging in cognitively stimulating tasks. using observational studies of naturalistic cognitive activities, we report a series of mixed effects models that include baseline and change in cognitive activity predicting cognitive outcomes over up to 21 years in four longitudinal studies of aging. consistent evidence was found for cross - sectional relationships between level of cognitive activity and cognitive test performance. baseline activity at an earlier age did not, however, predict rate of decline later in life, thus not supporting the concept that engaging in cognitive activity at an earlier point in time increases one 's ability to mitigate future age - related cognitive decline. in contrast, change in activity was associated with relative change in cognitive performance. results therefore suggest that change in cognitive activity from one 's previous level has at least a transitory association with cognitive performance measured at the same point in time. |
israel s center for disease control seasonal influenza surveillance system operated throughout our 5-year study. the system is based primarily on 1) anonymous patient visits for influenza - like illnesses (ili) to maccabi community clinics, israel s second largest health maintenance organization, insuring 1 of every 4 israelis ; and 2) nasopharyngeal swabs from sample ili patients at designated sentinel clinics countrywide. ili was defined as fever (> 37.8c) with > 1 of the following : cough, coryza, sore throat, or myalgia. swab samples were tested for influenza viruses at the health ministry s central virology laboratory (1) by using multiplex real - time reverse transcription pcr (rt - pcr) (taqman chemistry quantitative rt - pcr) (2). ili rates constituted 3 escalating waves of infection, all at times atypical for seasonal influenza (figure 1). israel s schools close july / august, but children stay together in summer frameworks during july. wave 2 peaked mid - september (week 38), 2 weeks into the school year, declining when schools closed for holidays until the end of week 41. during week 42, the third, largest wave began, peaking mid - november (week 46). rates of weekly visits to community health clinics for influenza - like illness, by age group, june 2009april 2010, compared with the 20052009 average (maccabi health services), with school holidays indicated, israel. the cumulative incidence (cases/10,000 population) of ili in children 018 years of age during the pandemic (week 25, 2009 to week 7, 2010) was 369.3 (95% confidence interval [ci ] 365.7373.1), far higher than average rates documented in earlier influenza seasons (143.4, 95% ci 140.7146.2). incidence was 295.8 (95% ci 285.7306.1) for children 2 years of age had a significantly higher rate of underlying illness compared with children 12 months of age. parents were advised not to attend the clinic for mild disease, although anxiety may have increased visits. there may have been differences between diagnoses of ili among different maccabi physicians. the 2 hospitals studied, which represented 10% of hospitalized children, were selected not as nationally representative but because of the feasibility of viral diagnosis since 2005. influenza detection during the pandemic in patients hospitalized at hadassah was based on pcr ; immunofluorescent antibody assay was used for previous seasons. awareness that pandemic influenza may have unique clinical characteristics, risk factors, and increased incidence, mainly among children 518 years of age, is advocated. because school opening in late summer 2009 triggered the wave of pandemic (h1n1) 2009 influenza (15), closing or delaying opening schools until vaccine is available should be considered among mitigation strategies in future influenza pandemics, especially for more virulent viruses. | during the pandemic (h1n1) 2009 outbreak in israel, incidence rates among children were 2 higher than that of the previous 4 influenza seasons ; hospitalization rates were 5 higher. children hospitalized for pandemic (h1n1) 2009 were older and had more underlying chronic diseases than those hospitalized for seasonal influenza. |
with the enactment of section 2991 of the social security amendments of 1972 (public law 92 - 603), full medicare coverage was extended to persons with end stage renal disease (esrd), effective july 1, 1973. to be eligible for medicare esrd benefits, the patient must first be currently or fully insured, be eligible for social security benefits, or be the spouse or a dependent child of such a person. additionally, a physician must certify that the individual requires chronic dialysis or a kidney transplant to maintain life. the medicare program pays a prospectively determined amount for kidney transplants and for certain drug treatments. for example, immunosuppressive drugs prescribed for transplant recipients are covered by medicare for 1 year following discharge from the transplant hospitalization. the drug, epoietin, used to combat anemia in dialysis patients, was added to medicare coverage effective june 1, 1989. integral to the effective management of the esrd program is the operation of a comprehensive data base covering medical and demographic information for the medicare esrd population. this data base, along with other esrd program - related data, is a part of a larger computer system the esrd program management and medical information system. this system served as the principal source for the data used in this article. in this article, trends for the medicare esrd population are examined by comparing : esrd enrollment with the total medicare enrollment (i.e., the count of people enrolled for coverage at a certain point in time). rates of growth for enrollment in esrd by age groups and for the incidence (new cases) of esrd by age groups. (these comparisons are made to provide evidence of the aging of the esrd population.) the comparison of esrd to the total medicare program in terms of numbers of beneficiaries enrolled and expenditures is shown in table 1. this table shows that the esrd population is growing at a much faster rate than the medicare population in general. esrd enrollment increased from 76,412 in 1982 to 124,197 in 1987, an average annual increase of 10.2 percent. for the same period, total medicare enrollment increased from 29,494,219 to 32,411,204, an average annual increase of only 1.9 percent. from 1985 through 1987, however, the annual growth rates for the esrd beneficiaries consistently decreased from 13.1 percent in 1983 to 8.7 percent in 1987. (a summary of these rates of increase, by year, is shown in table 4.) in figure 1, the annual rates of growth of the total medicare population and the esrd population for the years 1983 through 1987 reflect the decreasing differential in annual rate of growth. even though the annual rate of growth for esrd enrollment decreased during this period, esrd beneficiaries continued to increase as a percent of the total medicare population. in table 1, it can be seen that, in 1982, esrd represented 0.26 percent of the overall medicare population. by 1987 expenditures for the esrd population are disproportionately high when compared with the relationship between the total number of esrd beneficiaries and the total number of medicare beneficiaries (table 1). in 1984, $ 2,332.6 million, or 4 percent, of the $ 58,354.2 million total expenditures for medicare were used for esrd beneficiaries. in this same year, the 95,526 esrd beneficiaries represented only 0.31 percent of the total of 30,455,971 medicare beneficiaries. this proportion peaked in 1986 when the esrd expenditures of $ 3,047.6 million represented 4.44 percent of the $ 68,582.5 million total medicare expenditures. however, the 114,306 esrd enrollees were only 0.36 percent of the total of 31,749,708 medicare beneficiaries. by 1987, the esrd expenditures of $ 3,315.4 were 4.39 percent of the $ 75,442.1 million total medicare expenditures, while 124,197 esrd enrollees represented only 0.38 percent of the total of 32,411,204 medicare beneficiaries. the average annual rate of increase for expenditures for the esrd population, from 1983 to 1987, was better than 3 percentage points higher than that for medicare in general (12.4 percent and 8.9 percent, respectively). the enrollment data discussed in this section reflect counts of all beneficiaries who were enrolled for coverage under the esrd program on december 31 of each year. table 2 contains data on esrd beneficiaries by whether they were on dialysis treatment or whether they had a functioning transplanted kidney and by age. in 1982, there were 76,412 esrd beneficiaries enrolled in the medicare program. by 1987, this number had increased to 124,197, a 63-percent increase. however, the growth rate for enrollment, which was 13.1 percent between 1982 and 1983, had decreased to 8.7 percent between 1986 and 1987 (table 4). from 1982 to 1983, the total number of esrd dialysis patients increased from 65,597 to 73,438, an annual rate of increase of 11.9 percent. however, for those 65 years of age or over, the rate of increase was 1.9 times larger at 22.3 percent (18,155 in 1982 to 22,201 in 1983). from 1986 (90,841 enrollees) to 1987 (97,111 enrollees), the annual rate of increase for all dialysis patients had declined to 6.9 percent. for those 65 years of age or over (31,558 in 1986 and 34,911 in 1987), the rate also declined, but at 10.6 percent, it was still 1.5 times larger than the overall rate. the average annual rate of increase from 1982 to 1987 was 8.2 percent for all dialysis patients, but it was 14.0 percent for those 65 years of age or over (table 4). in figure 2, the rates of increase in enrollment for all dialysis patients are compared to the rates of increase in enrollment for those 65 years of age during the period from 1982 - 87. the average annual rate of increase for this same period, 1982 to 1987, for functioning kidney transplants was 20.2 percent. the rate for transplants for those 65 years of age or over was more than double that rate at 45.0 percent (table 4). the number of enrollees in table 2 shows that, in terms of the total dialysis beneficiaries, the group 65 years of age or over increased from 18,155 in 1982 (27.7 percent of the 65,597 total esrd dialysis population) to 34,911 in 1987 (35.9 percent of the 97,111 total). the number of beneficiaries with functioning kidney transplants who were 65 years of age or over increased from 114 (1.1 percent of the 10,815 total esrd transplant population) in 1982, to 731 (2.7 percent of the 27,086 total) in 1987. the total counts of esrd incidence (newly enrolled medicare esrd beneficiaries) by age are shown in table 3. the trend in medicare esrd incidence supports that which was found in esrd enrollment ; that is, the esrd population is aging. in 1982, 21,876 persons were added to the medicare enrollment files as esrd beneficiaries. by 1987, the annual number of new esrd beneficiaries had increased to 33,749, representing an average annual rate of increase over the 6-year period of 9.1 percent. however, there were 6,424 beneficiaries 65 years of age or over added in 1982 and 12,844 added in 1987, which represented an average annual rate of increase of 14.9 percent. (a summary of the rates of increase in incidence of esrd, along with those of enrollment, are included in table 4, for comparison.) from 1986 to 1987, the number of newly enrolled esrd beneficiaries increased by only 7.7 percent, from 31,328 in 1986 to 33,749 in 1987. this annual rate of increase indicates a slowing of overall growth in new cases of esrd, when compared with the average annual rate of increase of 9.1 percent from 1982 to 1987. the annual rate of growth for new cases of esrd among older persons has also slowed, but for the last 3 years shown, it has consistently exceeded the average annual rate of growth of esrd overall. from 1984 to 1985, the annual rate of growth in incidence for all ages was 10.7 percent (26,453 to 29,290, respectively) ; for those 65 years of age or over, it was 15.4 percent (9,100 to 10,498, respectively). from 1985 to 1986, the annual rate of growth in incidence was 7.0 percent (29,290 to 31,328, respectively) ; for those 65 years of age or over it was 11.0 percent (10,498 to 11,648, respectively). from 1986 to 1987, the annual rate of growth in incidence was 7.7 percent 31,328 to 33,749, respectively) ; for those 65 years of age or over, it was 10.3 percent (11,648 to 12,844, respectively). a final indicator of the aging of the esrd population is also found in incidence rates. in 1982, there were 6,424 esrd beneficiaries in the group 65 years of age or over, which represented 29.4 percent of the total incidence of 21,876 for that year. by 1987, the 12,844 new esrd cases in the 65 years of age or over group represented 38.1 percent of the total incidence. it has been shown that the esrd population is growing at a much faster rate than the general medicare population. also, it has been shown that the esrd beneficiaries consistently use a disproportionately higher share of medicare funds compared with their representation in the total medicare population. the esrd statistics also reflect a clear trend in the aging of the esrd population. the reasons for why this may be happening were not examined, but it has been clearly shown that the group 65 years of age or over continues to represent a larger portion of esrd enrollment and of esrd incidence, over time. | a synopsis is given between the relationship of the number of end stage renal disease (esrd) patients to the total medicare population and their associated expenditures. the aging trend within the esrd population is examined in terms of enrollment statistics and incidence (new cases) counts. also, longitudinal trends in expenditures, program enrollment, and incidence of esrd are included. findings indicate that the esrd population is growing at a faster rate than medicare in general. further, within esrd, the beneficiary population is aging. |
the online version of this article (doi:10.1007/s00125 - 010 - 1741 - 9) contains a list of members of adag study group, which is available to authorised users. current understanding of normoglycaemia is largely based on studies of populations without diabetes, often with a small number of glucose measurements per individual. this results in limited insight into patterns of real - life glycaemia as experienced by healthy individuals. as new options in diabetes treatment increasingly focus on specific glucose profiles such as postprandial glycaemia, it is important to have a clearer understanding of what constitutes a normal glucose profile. knowledge of how much time normoglycaemic individuals spend at different levels of glycaemia under real - life conditions is needed to serve as a benchmark for the more detailed study of impaired glycaemic states and the ability of novel treatments to normalise glucose profiles. we therefore studied the glucose profiles of non - diabetic individuals who participated in the a1c - derived average glucose (adag) study. this observational study included continuous interstitial glucose monitoring (cgm) under real - life conditions. our aim was to analyse to what extent individuals without diabetes exceed ogtt thresholds for impaired glucose tolerance (igt) and diabetes. study participants the adag study was conducted at ten centres in the usa, europe and africa from 2006 to 2008. the population for the present analysis consisted of the 80 non - diabetic control participants who completed the intensive glucose monitoring period of 12 weeks. the non - diabetic participants were selected on the basis of having no history of diabetes, a plasma glucose level 5.4 mmol / l (97 mg / dl) after an overnight fast and hba1c 6.1 (110)100 (80)395273688>7.0 (126)99 (79)10945200>7.8 (140)93 (74)311181>11.1 (200)9 (7)8362>16.7 (300)1 (1)32pg level specified in column 1for participants reaching specific pg leveliqr, interquartile range ; pg, plasma glucosefig. 1proportion of individuals spending time with plasma glucose concentrations above selected glucose thresholds (per 24 h monitoring period) of a 7.8 mmol / l, b 11.1 mmol / l and c 16.7 mmol / l time (per 24 h cgm period) spent above selected plasma glucose concentrations pg level specified in column 1 for participants reaching specific pg level iqr, interquartile range ; pg, plasma glucose proportion of individuals spending time with plasma glucose concentrations above selected glucose thresholds (per 24 h monitoring period) of a 7.8 mmol / l, b 11.1 mmol / l and c 16.7 mmol / l we found that nearly all individuals without diabetes exceeded the igt threshold of 7.8 mmol / l (140 mg / dl) at some point during the day and spent a median of 26 min (range 0 min to 6 h 52 min) per day above this level. we also found that one in ten individuals reached diabetic levels (11.1 mmol / l, 200 mg / dl). these findings suggest that glucose levels in persons without diabetes frequently reach igt range concentrations and that a considerable proportion reach diabetic levels. a study in 32 individuals with confirmed normal glucose tolerance found that seven participants (22%) reached glucose concentrations above 11.1 mmol / l (200 mg / dl) during an average of 28 days of cgm and that participants spent on average 42 min / day at glucose concentrations above 7.8 mmol / l (140 mg / dl). in a smaller study, 15 hospital staff without known diabetes and monitored by cgm for 24 h were found to spend an average of 72 min / day with glucose levels higher than 7.0 mmol / l (126 mg / dl). during a standardised ogtt mmol / l in individuals with normal glucose tolerance in the time preceding the 2 h value. however, since the 75 g ogtt is an extreme glucose load compared with an average mixed meal with regard to glucose concentration and the simple carbohydrate content (fast uptake), our results, which were based on real - life monitoring over a prolonged period, add an important dimension. the limitations of the current study include the absence of ogtts to rule out diabetes with certainty or to classify participants as having igt. our fasting plasma glucose exclusion criterion has been shown to be highly specific for ruling out diabetes, but does not rule out the possibility that some of the participants could have had igt. in addition, our hba1c exclusion criterion of 6.5% has recently been proposed as the new diagnostic level for diabetes. at 5.2% (sd 0.3) factors that cause glucose fluctuations, such as food intake, exercise, stress, or beta cell function and insulin sensitivity, were not examined in this study under free - living conditions. however, the present observations are relevant in public health terms, and to clinicians dealing with individuals with a wide, often unknown array of dietary and physical activity patterns. our results confirm that considerable variability of glucose levels exists even among individuals classified as not having diabetes. these findings may lead to speculation on the adequacy of current diagnostic practice and whether the use of multiple glucose measurements at different time points of the day or assessment of hba1c would identify hyperglycaemic individuals more accurately. however, in the absence of data assessing the impact of these glucose profiles on clinical endpoints, the current study can not establish whether periods of transient hyperglycaemia are part of normal variability or whether such periods affect the risk of progression to diabetes and development of diabetic complications. when glucose levels are measured under real - life conditions in non - diabetic individuals, as defined by a very low level of fpg and hba1c levels below 6.5%, these individuals are seen to spend a considerable amount of time with glucose levels classified as dysglycaemic or even diabetic. since chronic glucose exposure is considered to be one of the main mediators of long - term outcomes, including microvascular and cardiovascular disease, our findings demonstrate that exposure to moderately elevated glucose levels remains under - appreciated when individuals are classified on the basis of isolated glucose measurements. if no adverse outcomes attend these periods of hyperglycaemia, our findings suggest that it is common for persons who are regarded as | aims / hypothesisreal - life glycaemic profiles of healthy individuals are poorly studied. our aim was to analyse to what extent individuals without diabetes exceed ogtt thresholds for impaired glucose tolerance (igt) and diabetes.methodsin the a1c - derived average glucose (adag) study, 80 participants without diabetes completed an intensive glucose monitoring period of 12 weeks. from these data, we calculated the average 24 h glucose exposure as time spent above different plasma glucose thresholds. we also derived indices of postprandial glucose levels, glucose variability and hba1c.resultswe found that 93% of participants reached glucose concentrations above the igt threshold of 7.8 mmol / l and spent a median of 26 min / day above this level during continuous glucose monitoring. eight individuals (10%) spent more than 2 h in the igt range. they had higher hba1c, fasting plasma glucose (fpg), age and bmi than those who did not. seven participants (9%) reached glucose concentrations above 11.1 mmol / l during monitoring.conclusions/interpretationeven though the non - diabetic individuals monitored in the adag study were selected on the basis of a very low level of baseline fpg, 10% of these spent a considerable amount of time at glucose levels considered to be prediabetic or indicating igt. this highlights the fact that exposure to moderately elevated glucose levels remains under - appreciated when individuals are classified on the basis of isolated glucose measurements.electronic supplementary materialthe online version of this article (doi:10.1007/s00125 - 010 - 1741 - 9) contains a list of members of adag study group, which is available to authorised users. |
nowadays, with technological advances in medicine, the use of invasive procedures and injections in patients has expanded. as a result, health care staffs are exposed to a high risk of blood - borne pathogens (1). needle stick injury (nsi) is one of the most dangerous occupational situations (2). among 20 types of blood - borne pathogens, which are transferred through nsi to health care workers, viral infections such as hepatitis b, c and human immunodeficiency virus (hiv) studies have shown that being exposed to infectious diseases and nsi can cause stress in nurses (6). this particular type of stress has a negative impact on the individuals and influences their families and colleagues (7). stress represents a major occupational hazard in modern age and has been incriminated for productivity reduction, absence from job, displacement of personnel, work conflicts and higher health care costs for employees (8). in a study on the psychological consequences of nsi, greene and griffiths found that the severity of the illness caused by needle stick is as significant as other psychological traumas. moreover, the depression resulting from this situation is similar to other psychological traumas, and the duration of the condition is associated with the duration of obtaining the result of the second test. this has a far - reaching impact on family relationships, sexual health and presence at the workplace (9). between 1992 and 2003, leigh. stated that injury stress led to the absence of 903 nurses, physicians and workers. moreover, 7% of the injuries resulted in loss of more than 31 working days (10). performed a study, which measured the incidence of post - traumatic stress disorder (ptsd) after injury, on doctors. results showed that 12% were suffering from ptsd reactions, therefore suggesting an emphasis on addressing psychological reactions to nsi (11). despite numerous studies performed on the prevalence and incidence of injuries from sharp objects in iran, no research with specific tools therefore, the aim of this study was to assess the degree of stress in nurses, who have been exposed to sharp objects. the study subjects were nursing staff of several cities that were selected by cluster sampling in three stages. at first, four provinces were randomly chosen from different geographic areas of iran. in the second stage, 11 hospitals of these cities were selected by the same form of simple randomization and finally, the nurses were enrolled through census sampling from every shift. hospitals were located in tehran (four hospitals), isfahan (three hospitals), kerman (two hospitals) and hamedan (two hospitals. data collection was a two - part questionnaire including demographic characteristics and stress of exposure to sharps injury. the questionnaire section related to the stress of exposure to sharp injury contained 20 closed questions and the likert response was classified to five scoring levels : very low (score of 1), low (score of 2), average (score of 3), high (score of 4), and very high (score of 5). the minimum total score that could be obtained was 20 and the maximum score was 100. the stress questionnaire had four dimensions, including safety policy (five questions), occupational safety (five questions), contact nursing (four questions) and mental - environmental conditions (six questions). the cronbach s alpha coefficient of internal consistency of this instrument was 0.92 and interclass correlation coefficient was 0.94 (12, 13). the questionnaire assessed stress levels caused by exposure to sharp objects in different situations or circumstances, as well as the amount of stress for nursing staff. in this study, based on the formula n = and p = 0.3, d = 0.04, the sample size was calculated as 502, and considering the 10% loss, 550 questionnaires were distributed and 527 questionnaires were returned, thus a total of 527 fully completed questionnaires were included in the study. the research objectives and the study methods were approved by the ethical committee of baqiyatallah university of medical sciences, teheran, iran, and the study subjects gave their written consent. the analyses were performed using spss 16.0 (released 2007 ; spss for windows, spss inc., basic descriptive statistics for quantitative variables was presented using mean (sd) and n (%) for qualitative variables. adjusted linear regression model with 95% confidence interval (ci) was used to predict the degree of stress in nurses, who were exposed to sharp objects. after review of the collected 527 questionnaires, the mean age of the study subjects was reported at 35.7 (sd = 8.7) years, and of these, 41.2% were male and 58.8% were female. the average work experience was 12 (sd = 8.3) years, with a minimum of one year and maximum of 40 years. of the subjects, 83.1% had bachelor s degree and 58.3% reported a history of injury with a sharp object. stress from risk of injury by exposure to sharp objects in nurses was reported to be 58.37%. the average stress score in nursing staff was 58.37 (sd = 15.08, 95% ci : 57.08 to 59.66). table 1 confirms that changes in independent variables were significantly able to predict variance in stress scores. the adjusted linear regression model explained 36% of the overall variance in stress score (r2 = 0.60), which was found to significantly predict outcome, f (4, 521) = 8.26, p < 0.001. according to table 1, the adjusted linear regression model showed risk predictors for stress score in nurses, who were exposed to sharp objects according to ward satisfaction, having master of science, age, and number of contacts. the stress of nurses was measured based on different domains of the questionnaire after standardization (figure 1). this study, using a proprietary questionnaire, was conducted to investigate the stress of dealing with sharp objects by nursing staff. regarding the occupational safety domain, one study showed a significant relationship between carelessness of nurses and injuries from sharp objects, especially in the setting of insufficient number of nurses (1.92 times), weariness and excitement (2.16 times), lack in resources support (1.88 times) and inexperience of nurses (1.74 times). considering the actual conditions of a shortage of nurses, there is an increase of the degree and amount of damage secondary to injury (14). the study of torshizi and ahmadi measured the domain of patient care stress in nurses by using two items, exposure to body fluids and risk of infectious diseases transmission, which were insufficient for the evaluation of stress produced by sharp objects (15). the severity and extent of the stress of a contact - care study conducted on two nurses that were injured during care of an hiv patient, in addition to emotional problems during a 22-month follow up, led to depression, anxiety, insomnia and nightmares in the participants, even after receiving negative laboratory results about any possible infection (16). each of the studies focused on only one of the items of the area. in the study of oconnor, it was demonstrated that injury by sharp objects caused leave or absence and prolonged work interruption due to anxiety or stress disorder, with a rate of one out of 20 individuals, resulting in a reduction of the quality of work (17). the study of seng. showed that only 146 cases out of 242 injured health workers reported their injuries to the authorities (4). results of other studies showed that improvement in performance attitudes and increasing awareness and education are essential to control and reduce damage (18). the cause of damage is related to the workplace, including improper conditions for disposal of the needles and sharp instruments, overcrowding, noise, heat, chaos, lack or inadequate protective equipment gloves, goggles and gowns, which account for 27.2% and patient - related factors, such as sudden movements, improper use of equipment designed for patient and disease - related damage by sharp objects, which are responsible for 7.6% of the total causes (19). in the area of safety policy, serinken. showed that personnel - related factors, including failure to use protective equipment, carelessness, lethargy, and lack of proper training, caused 64.9% of incidents (19). it is noteworthy to mention that only 36% of nurses, who were injured with needle stick within the past year, reported this to their supervisor or hospital emergency ward or infection control committee (20)., showed a high level of awareness and knowledge of the universal precautions, however, there s still a gap between science and practice, which leads to the extended rate of damage (21). however, other results showed that health workers required more training (22). in another study, 38.3% of the subjects had a history of injury from needles and sharp objects within the last six months (23). we calculated the incidence of injuries with sharp objects at 58.3%, which is different to the study of sharifian. because of the three - month period of investigation in their study, compared to our study, which was performed during a longer time period (24) however, the results of a study in germany showed a reduced incidence of nsi in nurses inclined to use a safety box. the use of safety equipment reduced the rate of such injuries from 69% to 52% (25). in our study, despite the fact that more than 75% of the participants worked more than the required time, there was no significant difference in the amount of stress. however, a study in baltimore confirmed that working for 13 hours or more during a day marked a significant contribution to the incidence of nsi (26). the results of this study, showed that stress levels in nurses are highly increased by working with sharp objects, an issue, which requires special attention from authorities to implement educational programs and raise the awareness and ability of the staff to prevent injury and to improve the skills of self - control in stressful situations. mastery over the mind, the environment and the health service is the best course, in addition, to ensure risk - free environments, providing facilities to reduce injuries and making managers understand their duties to follow up and provide medical service and prophylaxis in the event of injury, are necessary. | backgroundinjuries caused by sharp objects, which involve biological hazards are considered as one of the most important factors that lead to stress among the nursing staff. contact with sharp objects is a major concern among healthcare workers, especially nurses.objectivesthis study was done to determine the amount of stress caused by exposure to sharp medical instruments among nurses.materials and methodsthis was a cross - sectional research on 527 nurses, working at different medical centers across iran, with a cluster - sampling method. the relevant data was collected with a valid and reliable questionnaire. the cronbach s alpha coefficient of internal consistency of this instrument was 0.92 and interclass correlation coefficient was 0.94resultsthe results showed that ward satisfaction, having master of science, age, and number of contacts were significantly able to predict variance in stress scores. the adjusted line regression model explained 36% of the overall variance in stress score (r2 = 0.60)conclusionsthe results of this study showed that exposure to sharp objects may cause high stress in the nursing staff. considering higher levels of stress in the area of contact care, the provisions on how to deal with patients and safe care can help reduce stress. |
ultracold polar molecules are attracting considerable attention, and their investigation will extend our knowledge on the physics of ultracold quantum gases into new regimes. a very interesting class of molecules in this regard is represented by mixed alkali alkaline earth (ak ake) molecules with both electric and magnetic dipole moments in their 1/2 electronic ground state. such molecules are proposed for engineering the lattice spin models of condensed matter physics, a state of matter with topological order, that can be used for the realization of a new class of quantum computation. further applications for ultracold ak ake molecules are precision metrology with the aim to test fundamental physics constants or quantum state selective ultracold chemistry. the most promising starting point for the production of ultracold ak ake molecules are quantum degenerate mixtures of ultracold alkali and alkaline - earth atoms. though mixtures of homo- and heteronuclear alkali molecules have been studied extensively in the past (see ref (1) and references therein), investigations of ak ake like mixtures have only been reported for liyb and rbyb. recent progress in ultracold atomic physics (e.g. the bose einstein condensation of alkaline earth metal atoms ca and sr) suggests that the formation of ultracold ground state ak ake molecules is within reach, and quantum degenerate mixtures of rb and sr atoms have been successfully realized very recently. the standard methods to overcome the gap between the interatomic pair distance in an ultracold atomic gas and the final binding length of the diatomic molecule are to convert ultracold atoms to molecules by either photoassociation or magneto - association followed by coherent population transfer to produce ground state molecules. this approach has been successfully used for the preparation of ground state cs2, krb, and sr2 molecules by stimulated raman adiabatic passage (stirap). an important requirement for the formation of ultracold molecules is the knowledge of their electronic structure, which is crucial for the search for optimal pathways to couple two colliding atoms with their molecular ground state. hence the theoretical as well as the experimental exploration of excited states can greatly simplify the navigation through complex molecular potentials and can be used for the prediction of transition frequencies and transition probabilities. the preparation of ak ake molecules in molecular beams or heat - pipe ovens is complicated, and despite the rising interest in these molecules, only a few spectroscopic data are available. here we report on a new method for the production of ak ake molecules. our approach uses helium nanodroplets for the isolation of cold (0.37 k) ak ake molecules in their vibronic ground state in a sequential pickup scheme. in the past 20 years, superfluid helium nanodroplets have been established as a matrix for the preparation of tailored molecules. the method of helium nanodroplet isolation spectroscopy has been used previously for the investigation of a class of molecules very similar to ak ake molecules, heteronuclear alkali dimers (and also trimers) on the surface of helium nanodroplets. the interaction between the molecule and helium droplet, which manifests itself typically in an asymmetric broadening of vibronic transitions, is relatively small and allows the extraction of molecular parameters of free molecules. furthermore, the structure of the broadened vibronic transition in the recorded spectrum gives insights into the interaction between molecule and helium droplet. we demonstrate our approach on the example of lica, a molecule where experimental data as well as calculations are available, which allows a detailed comparison with our results. besides liba, lica is the only system where high resolution spectroscopic data have been obtained. in addition to the experiment, we present quantum chemical ab initio calculations of the lica molecule. the majority of available calculations is focused on the ground state because of the outstanding properties and the importance for possible applications of ultracold ground state ak ake molecules. gopakumar. have recently presented calculations of the lowest two excited states. the most extensive calculations of lica potential energy curves, including higher states, have been presented by allouche. and russon., which can be compared to our results. the advantage of our approach compared to previous experiments is that the combination of all ak and ake molecules is possible on the droplet with the same experimental arrangement and requires no additional experimental effort. one of the greatest problems in experiments with heat - pipe oven sources is the large singlet dimer background of the alkali partner of the ak ake molecule. this disadvantage is overcome by helium nanodroplet isolation spectroscopy, which provides a powerful method for the investigation of vibronic transitions of mixed ak ake molecules. this manuscript is organized as follows : upon a brief description of the experimental setup and methods we discuss the experimental results. we present the recorded spectra according to their energetic ordering, starting with the 4 x transition. for this transition, dispersed emission spectra allow us to draw conclusions on the electronic ground state of lica. subsequently the 31/2,3/2 x transition is discussed where isotope shifts and the spin orbit splitting could be studied. the experimental setup is described in detail in refs (31) and (46), and a closer description of the resonance enhanced multiphoton ionization time - of - flight detection can be found in ref (47). to give a short overview : the helium droplets are formed by a supersonic jet expansion ; i.e., precooled helium gas (t0 = 15 k) is expanded through a nozzle (d0 = 5 m) under high pressure (p0 = 60 bar) into the vacuum. the produced droplet sizes obey a log - normal distribution, and the given source conditions lead to a droplet size distribution maximum of n60,15 = 6000, which corresponds to a radius of r60,15 = 40 (assuming spherical droplets). the helium droplet beam is then guided into the pickup chamber where it successively passes through two pickup cells holding small amounts of the corresponding doping elements li and ca. changing the cell temperature affects the probability of each droplet to pick up one or more atoms. the optimum temperatures for the formation of lica are tli = 350 c and tca = 370 c. the excitation spectra were recorded using resonance enhanced multiphoton ionization time - of - flight (rempi - tof) spectroscopy. in this method the lica on the he droplet is excited by a tunable pulsed dye laser (lambda physik fl 3002) and ionized by a fraction of the pump laser power (radiant dyes rd - exc 200 xecl laser, 26 ns pulse duration, 100 hz) for spectra below 19000 cm or by a second photon of the dye laser for spectra above 19000 cm. the ion yield is recorded by a time - of - flight mass spectrometer (jordan d-850 aref) with angular reflectron. in addition, the lowest recorded lica transition (4 x) was investigated by laser induced fluorescence (lif) spectroscopy. fluorescent light either was monitored with a peltier cooled photomultiplier tube (hamamatsu r94301) or, for dispersed fluorescence emission spectroscopy, was sent through a modified mcpherson eu-700 grating monochromator with an attached ccd camera (lot - andor idus du401abr - dd). the excitation spectrum of lica on hen, shown in figure 1, was recorded with rempi - tof spectroscopy in the range 1520025500 cm. the two lowest recorded band systems (the 4 x and 31/2,3/2 x transitions) consist of a progression of vibrational bands with a characteristic asymmetric (lambda - shaped) peak form. for transitions into higher states the vibrational spacing could not be resolved and the bands appear as broadly extended, structureless features in the spectrum. as the density of states increases, the vibrational bands start to overlap and the complexity of the spectra increases. for three transitions, experimental data from molecular beam spectroscopy experiments are available and can be compared to our results. this allows us to draw conclusions on the interaction between the lica molecule and the droplet and the perturbation of molecular states by the droplet. in the following we discuss the recorded transitions in detail, followed by a presentation of calculated potential energy curves and transition dipole moments for lica as well as a comparison of calculations and experiments which allows the assignment of the higher excited states. excitation spectrum of lica on hen as recorded by rempi - tof spectroscopy from 15200 to 25500 cm. four band systems have been assigned to the 4 x, 31/2,3/2 x, 5 x, and 4 x transitions of lica on hen and the peak at 24500 cm is assigned to the two overlapping 6 x and 5 x transitions. the r2pi excitation spectrum of the lica 4 x transition is shown in figure 1 in the range 1520016300 cm. this excited lica molecular state correlates to the li 2s, s + ca 4s3d, d atomic asymptote. the vibrational levels could be resolved and can be followed from = 04. the absence of hot bands reveals that in the presence of the low - temperature hen environment the lica molecules are cooled efficiently to the lowest vibrational level = 0. hence, upon doping with li and subsequently with ca, the molecule is formed in the vibronic ground state x(=0) and the gained bond formation energy is released into the droplet. the efficient formation of lica demonstrates that the two surface bound species, li and ca, find each other on the droplet surface and a large fraction does not desorb despite the released binding energy of 2605.3(100) cm. the excess energy is carried away by evaporated he atoms, causing a droplet shrinking of about 520 he atoms, if 5 cm binding energy of a he atom to the droplet is assumed. the lica molecule is more strongly bound than the alkali triplet molecules, but weaker than, for example, the sodium singlet dimers (5942.6880(49) cm), which have been investigated on the helium droplet surface extensively. the observed peak structure is characteristic for surface located molecules with strongly coupled vibrational motion to the surface of the helium droplet. because of the similarities between li and ca helium interaction energies and the similarities between the spectra of surface bound alkali triplet molecules and the lica spectra we expect the lica molecule to reside on the surface of the helium droplet. to increase the signal - to - noise ratio and to avoid saturation effects, we investigated the 4 x transition additionally with lif spectroscopy. saturation effects can occur due to the relatively high pulse energy of the pulsed dye lasers, which is needed for a reasonable r2pi signal of the molecule on the helium droplet. the lif excitation spectrum is shown in figure 2 and was recorded with a continuous wave (cw) ring dye laser operated with dcm (500 mw). with lif spectroscopy the lif spectrum can be compared with the spectrum in figure 1 in ref (37). the intensities of the = 0 and = 1 peaks in figure 2 match the relative intensities in ref (37), which demonstrates that the franck condon factors (fcf) are not influenced by the interaction with the droplet for this transition. the vibrational levels obtained for the 4 state can be compared to spectroscopic data of free lica molecules. band origins for the free molecule are shown as vertical blue lines in figure 2. it can be seen that the onset of the rising edge of the lambda - shaped peaks coincides with the free molecule value. the broad phonon - wing is caused by the interaction of the excited molecule with the helium droplet. the peaks have a fwhm of 3040 cm and no zero - phonon line could be observed. the peak structure is very similar to the shape of alkali triplet transitions with resolved vibrational levels. also the bandwidths compare to alkali triplet transitions (e.g., for the na2 1g 1u+ 30 cm was reported). for the case of alkali triplet molecules it was concluded that a phonon - wing without zero - phonon lines suggests a strong coupling of the vibrational motion of the molecule to the surface of the helium droplet. hence we conclude that the lica molecule is also strongly coupled to the helium droplet. the data have been offset corrected, and the red line has been smoothed. the blue lines denote the free molecule transitions as found in ref (37). the peaks in the lif spectrum were fitted with an asymmetric 2-function:1 the maximum of the second derivative of the fit function corresponds to the onset of the rising edge of the peak and hence to the origin of the vibrational band. as has been shown in ref (35) for lithium triplet molecules on helium droplets, this procedure can be used to determine molecular parameters of free molecules and the values for the origins of the vibrational bands agree within a few cm. in table 1 we compare the experimentally determined vibrational band origins from refs (37) and (39) with our fit results. we include the values obtained from the r2pi spectra for the 30 and the 40 band origins because they agree well with the literature values, indicating that they are not influenced by saturation effects. as shown in table 1, the results from helium droplet isolation spectroscopy agree very well with the molecular beam and heat - pipe oven experiments, demonstrating the suitability of our method for the determination of molecular parameters and for testing calculated potential energy curves of free ak ake molecules. the molecular parameters te, e, and xee have been calculated from a least - squares fit to the standard expression given in eq 2.2 one standard deviation uncertainties are given in parentheses. figure 3 shows the 4 x emission of lica molecules upon excitation of the 4 = 1, 2, and 3 vibrational levels at 15585, 15857, and 16130 cm, respectively. the cw laser was tuned to the phonon - wing maximum of each transition on the helium droplet. four peaks corresponding to the molecular 01, 00, and 11 transitions and the li 2p 2s transition can be seen in the spectra. the observed fluorescence light in the spectrum originates only from free molecules that leave the droplet upon laser excitation. fluorescence light could only be detected from the lowest two vibrational levels, indicating a droplet mediated cooling of the lica molecules in the excited states prior to the emission. similar results have been reported for the emission spectra after the excitation of alkali dimers. the observation of the li 2p 2s emission indicates that a considerable fraction of the molecules fragments into ca and excited li atoms. note that excited ca atoms could not be detected in this experiment because, in this energy range, only the lowest metastable triplet p states can be populated upon fragmentation. their long lifetime (e.g., 4.2 s for the ca p1 state, the upper state of the strongest intercombination line) forbids a detection in our experiment. a comparison of the three recorded spectra shows that the two signals recorded upon excitation of the = 1 and = 2 levels do not differ strongly, except for an increased li atomic emission at the former. as can be seen from the spectra, the majority of molecules from which fluorescence light could be detected, are in the vibrational ground state = 0. we conclude that the cold helium environment induces a relaxation of the molecules before they leave the droplet. the striking difference at the excitation of = 3 is the absence of transitions originating from = 0 (i.e., the 00 and 01 line) accompanied with an increased li atomic emission. this suggests that in this case for the two competing underlying processes, relaxation and desorption, the latter is faster and prevails. the observation of an increased li atom signal from the d lines is unexpected because predissociation of the 4 state upon interaction with the crossing 1 state was reported to occur above = 9. we think that this observation is related to the interaction between molecule and helium droplet, where due to the presence of the droplet it could also be possible that the 1 state affects the dynamics of the excited molecule. our recorded emission spectra demonstrate that the helium droplet isolation technique can be used for the preparation and investigation of free molecules that have desorbed from the droplets upon excitation. most importantly, the recorded emission spectra give insight into the vibrational levels of the electronic ground state. we think that in further experiments these free molecules can be investigated upon formation on the droplet with additional lasers, allowing spectroscopy of cold tailored molecules without restricted resolution due to the interaction with the droplet. spectra of the 4 x emission of lica molecules formed on helium nanodroplets. emission was collected upon excitation of = 1, 2, and 3 in the 4 state, which is shown as black, blue, and red lines, respectively. four peaks corresponding to the molecular 01, 00, and 11 transitions and the li 2p 2s transition can be seen in the spectra. figure 4 shows a detailed view of the 31/2,3/2 x transition of lica on hen. the 3 excited molecular state adiabatically correlates to the same separated atom limit as the 4 state discussed above (li 2s, s + ca 4s3d, d). the data have been fitted with eq 1, and the band origins of the vibrational bands correspond to the free molecule transitions and have been obtained by calculating the maximum of the second derivative of the fit. the fwhm of the peaks is in the range of 80 cm. in table 2 we compare our results with those of a molecular beam experiment, which are, to our knowledge, the only existing experimental data for this transition. the vibrational spacing as obtained by the fits is within several cm of the literature values. please note that the values for the vibronic transitions are compared to the 31/2 molecular data, which follows from the fit. the molecular constants have been obtained by a least - squares fit to the standard expression, eq 2. in this case the parameter xee was set to zero, because an inclusion of this parameter resulted in large uncertainties of the molecular parameters. the free molecule values for the parameters te and e were calculated from the values given in table 2 in ref (37). as can be seen from table 2, the determined parameters lie well within the one standard deviation interval and differ only a few cm from the free molecule values. the relative intensities of the peaks do not allow us to draw conclusions about the franck condon factors in this case, because the signal has not been normalized with the relative laser pulse energy over the wavelength range. close - up of the rempi - tof signal of the 31/2,3/2 x transition. plot a shows a comparison of the atomic li and ca ion signals to the molecular lica ion signal. plot b shows the signal for the different isotopologues of lica, lica and lica, where the latter signal has been scaled by a factor of 15 because of the low abundance of the li isotope. in plot c the effect of spin orbit splitting can be seen in the form of a slight kink in the rising edge of each peak, which is situated between the corresponding spin orbit split components of the free molecule transitions, indicated by vertical blue lines. calculated from values given in table 2 in ref (37). to highlight the effects that can be seen in the recorded signal, figure 4 has been divided into three sections. plot a shows a comparison of the atomic li and ca ion signal to the lica molecular ion signal. atomic calcium follows the trend of lica, whereas the atomic li signal shows only a very weak structure. the 3 potential energy curve is crossed by the 1 curve, which gives rise to a predissociation of the lica molecule, as has been suggested in refs (3739). the 1 curve is purely repulsive above the lowest vibrational level of the 3 state (case c according to mulliken s classification of predissociation cases), leading to ground state li atoms and ca atoms excited into their 4s4p, p state in the separated atom limit. as suggested in ref (we attribute the observation of the large ca ion signal that follows the lica ion signal to predissociated molecules. however, helium droplets are known to induce relaxation processes, which have been observed in the form of intersystem crossings in alkali triplet dimers and quartet trimers or spin relaxation in atoms doped to helium droplets. the lica molecules will interact with the helium droplets during the predissociation which can lead to atomic fragments in states different from the dissociation products associated with the 1 molecular state. hence predissociation of the lica molecule on a helium droplet can give rise to both excited ca and li fragments. a contribution of ground state li atoms to the li signal is unlikely, because we do not observe a background signal caused by the free atom beam, which is always present in our detection chamber due to our pickup design. in light of this discussion we think that the following effects are responsible for the signals in figure 4a : the majority of molecules is ionized in a two - photon ionization process and form a stable molecular lica ion. a fraction of molecules predissociates, resulting in a ca ion signal and also a weak li ion signal caused by the interaction with the droplet. in both cases, for the excited li and ca, two photons are necessary for the ionization. a competing process is the fragmentation of the molecule upon absorption of a third photon, which will also contribute to both the li and the ca ion signal. at the 3 x lica transition we were able to separate the signals of lica and lica and thus obtain a spectrum for both isotopologues, which is shown in figure 4b. despite the very weak lica signal, due to the low abundance of li (7.4%), the isotope shift can be clearly seen and the trend of an increasing shift for higher excited vibrational levels is obvious. the isotope shift is well comparable to the values given in ref (37), as shown in table 2. also the molecular parameters te and e for both isotopologues are in good accordance with the literature values. as has been shown in ref (orbit (so) constant and hund s case (a) is appropriate for the description of the coupling of spin and orbital angular momentum with the molecular axis. the 3 state splits into two spin orbit sub - bands. due to the broadening of the lines by the interaction with the he droplet, they are hard to resolve. despite this, in our data a small effect of the spin orbit splitting (so constant a0 = 13.3 cm, for = 0) can be seen in the form of a slight kink in the rising edge of each vibrational band in the lica spectrum. this effect is highlighted in the figure 4c, where the kink can be seen for each vibrational level = 03 between the band origins of the two spin orbit split components of the free molecule, indicated by the vertical blue lines. it is remarkable that the value of the spin orbit constant seems to be conserved despite the presence of the droplet. assuming the molecule lies flat on the surface of the droplet, the symmetry of the system will be reduced and an effect on the so constant would be expected. however, the coupling of spin and orbital angular momentum to the intermolecular axis seems to be much stronger than the influence of the droplet which would make the effect very small. if on the other hand the molecular axis of the lica molecule was aligned perpendicular to the droplet surface, the symmetry around the internuclear axis would also be conserved. this would be another explanation for the observed so splitting. in this section the single structures that can be seen in figure 1 in the energy region above 21250 cm are discussed. the band at 22000 cm has been assigned to the 4 x transition, where the upper level adiabatically correlates to li 2s, s + ca 4s3d, d in the separated atom limit. the transition extends from 22150 to 23100 cm and shows a steep rising edge and a high signal on the low energy side and a broad shoulder to the high energy side. the 4 state correlates to a ca singlet state, in contrast to the 4 and 3 states described above, which both correlate to the same ca d state. calculations have shown that the cahe potential for the ca 4s3d, d state has a pronounced minimum for its molecular substate, whereas all molecular substates corresponding to ca 4s3d, d are repulsive or very weakly bound. the lack of vibrational resolution in the 4 x excitation spectra indicates a stronger interaction of the excited molecule with the droplet surface. the steep rising edge of the peak indicates that the laser excitation starts at the lowest vibrational transition, which is confirmed by our calculations. the onset of the rising edge is shifted to the red by 100 cm as compared to the values found in ref (37). our calculations show that the 4 x transition has a very high transition dipole moment with a maximum of the franck condon envelope at the 00 transition, which could cause an easy saturation of this transition. although the shoulder on the high energy side does broaden with increasing laser energy, the steep rising edge does not show a significant energy dependence. an analysis of the atomic signals again shows that ca follows the lica signal ; however, at this level of excitation there is a large number of crossings and avoided crossings of molecular potentials that, together with the influence of the hen environment, increase the possibility of the lica molecule being predissociated. the rempi - tof signal shows a weak cahe ion signal that follows the lica ion signal in the region of the 4 x transition (not shown). we explain this in accordance with the detection of fragments in the 31/2,3/2 x transition : he droplets could act as an intermediate and lead from an excited licahen system via a licahe hen transition state to the formation of cahe exciplexes. the observation that a cahe ion signal is detected at the 4 x transition is in agreement with theoretical results in ref (66) wherein a relatively strong binding of the ca(d)he potential was reported. two more lica transitions have been found, one weak transition between 21250 and 21500 cm and a strong transition between 24000 and 25250 cm. the assignment of these states to molecular transitions is based on our calculations and will be treated in detail below. in addition to the experimental investigation we further examine the excited states of lica by means of molecular - orbital - based quantum chemistry using the molpro software package. the potential energy curves (pec) for the lowest 28 states were obtained from multireference configuration interaction (mrci) calculations based on complete active space self - consistent field (casscf) wave functions. orbitals and occupation schemes are referring to the program - specific internal ordering of the irreducible representation (a1/b1/b2/a2). the 10 innermost electrons of the ca atom were replaced by an effective core potential (ecp) of the stuttgart group (ecp10mdf). further improvement, especially of the energy spacing, was obtained by applying a core polarization potential (cpp) with a static dipole polarizability of 3.522 au and a cutoff radius of 1. because the matching ecp - basis set did not provide enough basis functions for an accurate description of the excited states in the experimentally relevant energy range, we tested several larger all - electron basis sets for compatibility with the ecp. in a series of benchmark calculations on the atomic excitations of ca we identified the cc - pv5z basis set as the most suitable compromise between accuracy and computational effort. the rather unconventional procedure of combining an all - electron basis set with an ecp was justified by the improved reproducibility of atomic excitations deviating from experimental values by less than 3.5%. basis functions with angular momentum larger than g had to be removed due to program - internal limitations. this procedure led to a basis set consisting of (15s9p6d4f3g)/(26s18p8d3f2 g) elementary functions and [7s6p6d4f3 g ] /[8s7p8d3f2 g ] contracted functions for ca / li. the active space consisted of 19 active orbitals (9/4/4/2) filled with three electrons. of the 28 calculated states, (8/5/5/2) states were calculated in the doublet multiplicity and (3/2/2/1) states in the quartet multiplicity. in table 3 our calculations tend to slightly overestimate the potential depth, which is a known consequence of the application of effective core potentials. good agreement is found for our results and the theoretical work of allouche. and recent experiments of stein. particularly large deviations between different theoretical approaches occur for the x ground state. single reference approaches tend to predict shorter bond lengths and reduced binding energies. recently, a potential depth of 2260 cm at a distance of 3.395 was determined at the coupled cluster level of theory. obtained a potential depth of 2607 cm at a distance of 3.364, which is in very good agreement with recent experimental investigations. a comparison to our result for the ground state demonstrates the overestimation of the potential depth. benchmark calculations using an all electron basis set at the casscf / mrci level with douglas kroll correction of the eighth order yielded a potential depth of 2664 cm at a distance of 3.38. a calculation using the ecp gave a potential depth of 3057 cm at a distance of 3.36. inclusion of the cpp finally leads to the improved value listed in table 3. figure 5 contains the pecs of the doublet manifold. for large internuclear distances the 2 and 3 states approach nearly the same value, although the atomic excitations show a difference of about 300 cm. one asymptotic value lies within 0.6% of the experimentally determined value for the li 1s2p, p excitation and remains independent of minor changes to the basis set and changes to the ecp and cpp. the two states are very close and exhibit an avoided crossing for large internuclear separations. the 1 and 2 states show the same behavior for long - range and give the wrong order (figure 5). the calculated asymptotes show a slight deviation from experimental atomic excitation energies (taken from the nist database). all remaining excited states in figure 5 converge to atomic excitations of the ca atom. the states 5 and 6 show an avoided crossing at 4.5. because the energies obtained for large internuclear separation differ from the atomic states, our result for the position (internuclear distance) and energy of the level crossing are not very accurate. the same is valid for the avoided crossing of the states 4 and 5 at 5.5. only after adding an appropriate cpp, the 5 state shows the strong bonding displayed in figure 5, without the cpp it appears weakly bound, similar to the 2 state. the transition dipole moments (tdms) for the doublet states are presented in figure 6. vertical excitation at the equilibrium distance of the ground state into the 4 state has the largest transition dipole moment in the given range. this transition gives rise to a strong signal in the experimental measurements. among the states the 4 state has the largest tdm for a vertical excitation, which agrees well with the experiment. transitions into the 2 and 3 states have also strong tdms, but they are outside the range of the experimental investigation. the tdm for the 1 state is very small, the 2 state lies outside the range of the experimental investigation. transitions into the 5, 6, 3, and 5 states have non - negligible tdms and are experimentally observed. dipole transitions from the ground state into to states are forbidden by selection rules and have negligible tdms. transitions from the x state into quartet states are forbidden and the tdm is zero. core polarization effects were neglected, but because there is a good agreement with previous results, the error is expected to be small. for the ground state the permanent dipole moment increases continuously with decreasing internuclear separation until it reaches a value of 0.61 au (1.55 d) at 2.7, and then the value drops. at the equilibrium distance the permanent dipole moment has a value of 0.50 au, only slightly deviating from the value determined by kotochigova. betafit 2.1 the pecs of experimentally observed states (3, 4, 4, 5, 5, 6) were fitted with an analytical function using 1114 parameters. these potential functions were used in the program level 8.0 to determine the vibronic levels and the franck condon factors. the latter mostly showed strong transitions for the = 0 states with franck condon factors between 0.95 and 0.7. with increasing vibrational quantum number the franck 6 states show a maximum of the franck condon factor of about 0.1 at higher vibrational quantum numbers. the calculated pecs and tdms for the 4 x transition confirm our interpretation of the experimental data. we find good agreement between calculated and experimentally determined band origins with deviations below 20 cm. the trend of decreasing transition probability with increasing vibrational quantum number is in accordance with the experimental findings. the theoretically predicted transition energy for the 31/2,3/2 x transition is too large : the calculated potential energy curve (figure 5) lies above the atomic value in the separated atom limit. despite this inaccuracy in the absolute position, the vibrational spacing and the isotope shifts are well reproduced by the calculations. figure 10 shows the experimental and the calculated excitation spectra in the range between 21100 and 25500 cm. the theoretical spectrum was obtained by multiplying the fcf with the tdm of the respective state at 3.4. the observed bands have been assigned to the 5, 4, 6, and 5 states, respectively. although calculations predict an extremely small tdm for the 5 x transition, a weak signal originating from this transition is experimentally observed. the theoretically predicted values are at slightly higher energies than the experimental values. the next structure in the spectra this state was also investigated experimentally by russon., but due to the interaction with the droplet, the vibrational states could not be resolved in the experiment. for the 4 state the interaction between the helium and the diatomic molecule is relatively strong, which is indicated by the deep potential between a ca atom in this excited state and a he atom, as can be seen in the diatomic potential curves in ref (66). the deviation between the theoretical and experimental line positions can be explained by taking a look at figure 5. the two highest states show an avoided crossing, which means that the 4 state is associated with the p ca atomic limit. the potential at large internuclear separation is about 300 cm below the atomic value, which corresponds to the difference between the theoretical and experimental values for the 4 x transition. on the basis of our calculations, we can assign the structure between 24000 and 25000 cm to the 6 and 5 states. condon factors are in excellent agreement with the experimentally observed structure and the deviation of the position is less than 100 cm. this good correspondence is probably related to the good reproduction of less than 100 cm of the atomic d (ca) state at large internuclear separations. because of an avoided crossing the functions that describe the 5 state at large internuclear separations are the same that describe the 4 state at small internuclear distances. the calculated asymptotic value (p ca state) obtained for the 5 state deviates from the exact value. because of the avoided crossing, we expect that the 4 potential energy curve shows a similar deviation at small internuclear separations. the main features of this transition can be explained by the 5 state, which has a strong transition dipole moment. the vibrational states of this transition are not resolved because of the narrow vibrational spacing of e = 75 cm. the rising edge deviates from the prediction, which can be explained by the additional contribution of the 6 state to the signal. for this structure a significant li ion - signal this can be explained by the interaction with even higher states. for the limit of separated atoms the states li 3s, s + ca 4s, s and li 2p, p + ca 4s4p, p would follow, both including an excited li atom. the pecs of states converging to excited li approach the 6 and 5 states and for the 6 state an avoided crossing is indicated by the potential form and a discontinuity in the transition dipole moment (figures 5 and 6). the red curve shows the smoothed data, the colored vertical lines represent the calculated franck the fcfs have been scaled to fit to the signal ; the scaling factors are given in the legend. the transitions into the 5, 5, and 6 states have not been observed before and are assigned with the help of our calculations. the short black vertical lines above the 4 x transition refer to values from ref (37). the red vertical line near 22000 cm represents our calculated 00 band position. in addition to the experimental investigation we further examine the excited states of lica by means of molecular - orbital - based quantum chemistry using the molpro software package. the potential energy curves (pec) for the lowest 28 states were obtained from multireference configuration interaction (mrci) calculations based on complete active space self - consistent field (casscf) wave functions. orbitals and occupation schemes are referring to the program - specific internal ordering of the irreducible representation (a1/b1/b2/a2). the 10 innermost electrons of the ca atom were replaced by an effective core potential (ecp) of the stuttgart group (ecp10mdf). further improvement, especially of the energy spacing, was obtained by applying a core polarization potential (cpp) with a static dipole polarizability of 3.522 au and a cutoff radius of 1. because the matching ecp - basis set did not provide enough basis functions for an accurate description of the excited states in the experimentally relevant energy range, we tested several larger all - electron basis sets for compatibility with the ecp. in a series of benchmark calculations on the atomic excitations of ca we identified the cc - pv5z basis set as the most suitable compromise between accuracy and computational effort. the rather unconventional procedure of combining an all - electron basis set with an ecp was justified by the improved reproducibility of atomic excitations deviating from experimental values by less than 3.5%. basis functions with angular momentum larger than g had to be removed due to program - internal limitations. this procedure led to a basis set consisting of (15s9p6d4f3g)/(26s18p8d3f2 g) elementary functions and [7s6p6d4f3 g ] /[8s7p8d3f2 g ] contracted functions for ca / li. the active space consisted of 19 active orbitals (9/4/4/2) filled with three electrons. of the 28 calculated states, (8/5/5/2) states were calculated in the doublet multiplicity and (3/2/2/1) states in the quartet multiplicity. we have presented a comprehensive experimental and theoretical study of the lica molecule. we show that these molecules can be formed very efficiently on helium nanodroplets by using a sequential pickup scheme. our results represent the first experimental observation of mixed alkali alkaline earth molecules on helium nanodroplets. a comparison of our experimental results for the x, 4, 3, and 4 states with those of previous molecular beam and recent heat - pipe oven experiments reveal that the determined molecular parameters of lica on hen lie within a few cm of the gas phase values. this demonstrates the capability of helium droplet isolation spectroscopy for the characterization of alkali alkaline earth molecules. the interaction between droplet and molecule manifests itself in the appearance of phonon wings in the spectra. they are caused by the coupling of the vibrational motion of the lica molecule to excitation modes of the helium droplet and extend from the vibronic band origin toward higher energies. for the 4 and 3 states the vibrational spacing in combination with narrow phonon wings allows the separation of vibrational states. the narrow, lambda - shaped peak form, which is typical for surface bound molecules, indicates a surface location of lica. ab initio quantum chemical calculations of potential energy curves, transition dipole moments, franck condon factors, and permanent dipole moments support our spectroscopic study of the lica molecule. the 19 lowest lying potential energy curves were determined by using a multireference configuration interaction calculation. on the basis of our calculations, we were able to identify the previously unobserved transitions into the 5, 5, and 6 states. our results for the lower excited states and the ground state of lica agree well with previous calculations and extend the previous works on lica to higher excited states. despite the perturbation of the molecule by the droplet, the resolution of the experimental spectra obtained for lica is sufficient to test calculated potential energy curves. lica has been taken as an alkali alkaline earth prototype molecule because of the available experimental and theoretical reference data. the experimental results serve as a proof of principle and demonstrate that formation of alkali alkaline earth molecules on helium nanodroplets is possible. our results indicate that the preparation of various tailor - made alkali alkaline earth molecules on helium droplet will be possible, opening a new route for the characterization of these molecules. this could be an important contribution for the preparation of ultracold molecules from ultracold atoms, a process which relies on the knowledge of accurate potential energy curves. both rb and sr atoms are surface bound species and have been well characterized on helium droplets. on the basis of our results for lica, we conclude that the formation of rbsr molecules for the determination of molecular parameter is feasible. beyond the scope of this article, our results suggest that the molecules which desorb upon excitation can be further investigated with additional lasers, which would overcome resolution constraints and lead to ro - vibrationally resolved spectra. | we report on the formation of mixed alkali alkaline earth molecules (lica) on helium nanodroplets and present a comprehensive experimental and theoretical study of the ground and excited states of lica. resonance enhanced multiphoton ionization time - of - flight (rempi - tof) spectroscopy and laser induced fluorescence (lif) spectroscopy were used for the experimental investigation of lica from 15000 to 25500 cm1. the 42+ and 32 states show a vibrational structure accompanied by distinct phonon wings, which allows us to determine molecular parameters as well as to study the interaction of the molecule with the helium droplet. higher excited states (42, 52+, 52, and 62+) are not vibrationally resolved and vibronic transitions start to overlap. the experimental spectrum is well reproduced by high - level ab initio calculations. by using a multireference configuration interaction (mrci) approach, we calculated the 19 lowest lying potential energy curves (pecs) of the lica molecule. on the basis of these calculations, we could identify previously unobserved transitions. our results demonstrate that the helium droplet isolation approach is a powerful method for the characterization of tailor - made alkali alkaline earth molecules. in this way, important contributions can be made to the search for optimal pathways toward the creation of ultracold alkali alkaline earth ground state molecules from the corresponding atomic species. furthermore, a test for pecs calculated by ab initio methods is provided. |
the purposes of this study were to determine the normal retinal microvasculature measurements in human infants who are born at term and to determine whether birth weight influences measurements of retinal microvasculature. retinal arteriole and venule measurements were obtained in a cohort of 24 infants who were born at term. digital images of both the retinas were obtained using a digital retinal camera after pupillary dilation. in all, 24 newborn infants born at term (12 females and 12 males) were analyzed in this study. the measured retinal arteriole diameters were from 66.8 to 147.8 m (mean, 94.219.6 m), and the venule diameters were from 102.0 to 167.8 m (mean, 135.219.1 m). seven babies in the sample had low birth weight (lbw), while 17 babies were born with normal weight. babies with lower birth weights had larger arteriole (113.117.9 m vs 86.414.4 m ; p=0.0009) and venule diameters (151.714.9 m vs 128.416.9 m ; p=0.0040). retinal venules and arterioles in lbw babies are larger compared with those of normal - birth - weight babies. we postulate that the difference observed in our study was due to in utero pathophysiological changes that occurred in the cerebral circulation of growth - restricted fetuses. in utero insults that result in low - birth - weight (lbw) infants (birth weight < 2500 g) are now well recognized as risk factors contributing to the development of vascular - related diseases in adulthood. lbw infants are a heterogeneous group of infants, comprising infants who are premature (< 37 completed weeks of gestation), growth - restricted (weight below the 10th percentile for their gestational age) or a combination of both. the exact mechanism of this phenomenon has yet to be fully understood, but there is increasing evidence to suggest that microcirculatory pathology forms the mechanistic link between fetal insult and the adult manifestation of illness. the retina provides an opportunity for in vivo investigation of human microcirculation, and changes in the retinal vessels have been identified in some individuals who had lbw as infants and later developed hypertension, ischemic heart disease, stroke and renal disease. the ability of retinal - imaging technology to assess and measure the retinal microvasculature makes this a very valuable assessment tool. studies involving young children, adolescents and adults who were born small have shown abnormalities in the retinal vasculature. although the retinal microvascular of premature infants is routinely assessed to detect and treat retinopathy of prematurity, there are no published studies regarding the use of retinal - imaging technology to assess the retinal microvasculature of at - term, growth - restricted infants. the purpose of the present study was to determine normal measurements of the retinal microvasculature in human infants who are born at term. this study was performed in the department of neonatology, the townsville hospital, douglas, qld, australia. the department of neonatology is a tertiary perinatal center catering to more than 10 000 births each year. the study commenced in august 2010, and the data presented in this study are based on patients recruited until may 2011. written parental consent was obtained, and babies with syndromes, prematurity and chromosomal abnormalities were excluded. babies with birth weights of 2500 g were classified as lbw babies, and babies weighing from 2501 to 4500 g were classified as appropriate for gestational age babies. only babies who were born at term (37 weeks of gestation completed) were included in this study. after pupillary dilation, digital images of both the retinas were obtained using a digital retinal camera (retcam, massie laboratories, dublin, ca, usa). measurements of the diameters of retinal vessels were then obtained using a predetermined protocol that first involved the identification of retinal vessels located from 0.5 to 1 disc diameter from the margin of the optic disc (figure 1). measurements of vessel diameter were then obtained using semi - automated software (vesselmap, imedos gmbh, jena, germany). the caliber of directly viewed vessels was determined by the size of the red - cell column, because the vessel walls and peripheral plasma layer are nearly transparent. measurements of vessels from each eye were obtained, and the largest venule and arteriole for each patient was determined. an intra - class correlation coefficient was used to determine the reliability of this technique ; this correlation coefficient was 0.90 (95% confidence interval of 0.75 to 0.96). a previously published study in infants has shown that the blood flow in the central retinal arteries is similar in both the eyes. in adult eyes, correction can be applied to compensate for inaccuracies in the measurements of retinal structure that occur because of refractive error ; this correction requires parameters such as axial length and keratometry (curvature of the anterior surface of the cornea) to be known. in infants, these calculations are more challenging because obtaining these measurements is difficult and the eye is continuing to grow. 11.0 (stata, college station, tx, usa). using student 's t - test, p values a total of 247 babies were admitted to the department during the study period. of these, 99 were suitable for recruitment, and their parents were approached for participation. written consent was obtained for 24. all 24 newborn infants born at term (12 females and 12 males) were analyzed in this study. birth weights ranged from 1845 to 4310 g (mean, 3029649 g) with gestational ages of 37 to 41.6 weeks (mean, 38.71.4 weeks). retinal arteriole diameters were from 66.8 to 147.8 m (mean, 94.219.6 m), and venule diameters were from 102.0 to 167.8 m (mean, 135.219.1 m). the infants were divided into two cohorts based on birth weight (lbw and appropriate for gestational age). babies with lbw had larger arteriole (113.117.9 m vs 86.414.4 m ; p=0.0009) and venule diameters (151.714.9 m vs 128.416.9 m ; p=0.0040). pearson 's coefficient of correlation between retinal arteriole and venule diameter was 0.7522 (95% confidence interval 0.50 to 0.89 ; p<0.0001). to date, studies of retinal vasculature in infants have mainly focused on premature infants and retinopathy of prematurity. to our knowledge, this is the first study to investigate measurements of retinal microvasculature using digital retinal imaging in infants born at term. these measurements could be used as a baseline for future studies that investigate the effects of birth weight on retinal microvasculature. previous studies have shown a strong relationship between lbw and retinal vasculature size in older children, adolescents and adults. however, no published studies utilized baseline measurements of infant retinal vasculature for comparison. for the first time, we were able to measure retinal arteriole and venule sizes in lbw infants during infancy. by contrast, previously published studies of young children have shown that children who were born as lbw infants had narrower retinal arteriolar calibers. narrowing of these vessels has been linked to the development of cardiovascular diseases in adults. only infants born at term were reviewed in this study ; thus, the cause of lbw in this cohort was intrauterine growth restriction. the retinal images in this study were all taken during the first week of life, so we propose that the differences observed in our study were due to pathophysiological changes that occurred in utero. there are many causes of intrauterine growth restriction, but the most common is uteroplacental insufficiency, which results in fetal hypoxia. fetal cerebrovascular responses to hypoxia are fundamentally different from those observed in the cerebral circulation of adults. the vasculature of the immature brain is highly plastic and can respond to hypoxia with robust increases in capillary density. endothelial vasodilator capacity is typically depressed in fetal cerebral arteries, and the endothelium contributes relatively little to hypoxic vasodilatation in the fetus. by contrast, the endothelial contribution to hypoxic vasodilatation increases throughout early postnatal life, becoming quite prominent in the cerebral arteries of adults. the smaller and more peripheral cerebral arteries relax quickly and completely in response to hypoxia, whereas the larger and more proximal arteries, including the common carotid, maintain muscle tone much better and have a more important role in the gradual adjustments of cerebrovascular tissue to resist hypoxia. animal studies have provided insight into some aspects of the basic pathophysiology of intrauterine growth restriction, and studies using technologies such as doppler ultrasound to investigate maternal and fetal vessels have added further information. doppler ultrasound allows for the assessment of the vascular effects of placental dysfunction on the placental and fetal vasculature. in response to hypoxia, the fetus uses a compensatory mechanism to redistribute cardiac output and blood supply to the brain to maintain constant blood delivery to this organ (the head - sparing effect). the result is a decrease in cerebral blood - flow resistance and vasodilatation of the arteries. this effect, which can be measured using doppler ultrasound, shows a decrease in resistance and an increase in blood flow in the middle cerebral artery. studies of growth - restricted fetuses have confirmed dilatation of the middle cerebral artery and the resulting increase in blood flow to the brain compared with fetuses with normal growth. figure 4 shows the close relationship between the retinal artery and the middle cerebral artery. the endothelia of the vessels in the brain and retina are lined with continuous endothelial cells, connected by tight junctions that help to maintain the blood doppler flowmetry data from newborn babies have shown that an increase in blood flow in the middle cerebral and ophthalmic artery is closely followed by an increase in blood flow in the central retinal artery. blood flow in the middle cerebral artery and cerebral blood flow are spatially and temporally coupled to fetal brain function and metabolism, and we postulate that, in a growth - restricted fetus, the same neurovascular coupling extends to the retinal artery. dilation of the middle cerebral artery possibly results in dilatation of retinal vessels in growth - restricted, lbw infants. it was also difficult to account for any refractive error that could have contributed to the results. we plan to follow this cohort over time to identify the changes in retinal vasculature as these infants grow. | objective : the purposes of this study were to determine the normal retinal microvasculature measurements in human infants who are born at term and to determine whether birth weight influences measurements of retinal microvasculature.study design : retinal arteriole and venule measurements were obtained in a cohort of 24 infants who were born at term. digital images of both the retinas were obtained using a digital retinal camera after pupillary dilation.result:in all, 24 newborn infants born at term (12 females and 12 males) were analyzed in this study. the measured retinal arteriole diameters were from 66.8 to 147.8 m (mean, 94.219.6 m), and the venule diameters were from 102.0 to 167.8 m (mean, 135.219.1 m). seven babies in the sample had low birth weight (lbw), while 17 babies were born with normal weight. babies with lower birth weights had larger arteriole (113.117.9 m vs 86.414.4 m ; p=0.0009) and venule diameters (151.714.9 m vs 128.416.9 m ; p=0.0040).conclusion : retinal venules and arterioles in lbw babies are larger compared with those of normal - birth - weight babies. we postulate that the difference observed in our study was due to in utero pathophysiological changes that occurred in the cerebral circulation of growth - restricted fetuses. |
the possibility of controlling the motion of a robotic arm by mere thought, as suggested by popular media since the advent of brain - machine interfaces (bmis), has captured the imagination of fiction writers and science journalists. but is mind control a reasonable or even a desirable practical goal for the future of neuroprosthetics ? if the ultimate clinical objective is to endow amputees and paralyzed people with the ability to act naturally through the interaction of their brain with an artificial limb, then the fact is that, as we carry out the simplest actions, such as operating the handle of a door, we do not occupy our minds with what we are doing. we do not think about opening up the grasp, closing it on the handle, twisting the wrist and so on. this is because motor acts are stored in the brain in hierarchically organized goal - directed actions. the addressing of a given action representation is the only thing the brain must do in order to cause the cascade of events leading to execution. in other words, our nervous systems do all that is needed without loading our thought processes, apart from the explicit activation of a very general action procedure. it is only in the early stages of learning that one must be aware of the details of one 's detailed movements. the goal of this review is to provide a perspective that emerged from work by our group and others on how bmis, based on the bidirectional flow of information between a neural population and a controlled device, may lead to the creation of automatic behavior. these interactions are also a fundamental tool for investigating how information is processed by the brain. in the early 90s, sharp, abbott and marder, introduced a new method to bridge the gap between experimental and computational analysis of neural behavior (sharp. they established a direct dialogue between a computer simulation and a group of neurons in a dish. the technique is called dynamic clamp and is based on an exquisitely simple idea : to simulate on a computer the input / output properties of a membrane conductance by obtaining the input membrane potential from an actual neuron and injecting the output a current into another neuron. to derive the current from the potential, one must integrate a system of ordinary differential equations ; a task that can be done in real - time if the size of the system is within the available computational power. the difference between this and a more standard computer simulation is that the variables in question are exchanged between simulation and real neurons. the dynamic clamp establishes a symbiosis between the artificial computation and the biological element, or, to quote sharp and colleagues (sharp., 1993) : the dynamic clamp behaves as if the channels described by the programmed equations were located at the tip of the microelectrode. the concepts that led to the dynamic clamp can be extended from the cellular to the system 's level of analysis. a number of recent studies provided a similar closed - loop feedback to neural systems involved in motor task learning. in this focused review, we discuss how the physical connection between biological neural systems and artificial computational processes established by bmis may lead to new paths for understanding neural information processing and be harnessed to benefit people suffering from paralysis. we begin by describing a simple neuro - robotic system, in which a small mobile robot provides an artificial body to a brain preparation maintained in a ringer 's solution. we discuss how the analysis of the coupled behavior may provide insight on the connectivity of the neural system that transforms input stimuli into output control signals. then, we review more recent work aimed at characterizing the dynamical behavior of a neural system engaged in a two - way interaction with an external device. this knowledge is likely to be critical, also for pursuing the goal of programming the operation of bmis by gaining control on the plastic properties of neurons. we conclude with a new perspective on tuning the maps implemented by bidirectional interfaces so as to approximate the desired behavior of a control system expressed as a force field. almost three decades ago, valentino braitenberg wrote a small manifesto in semi - fictional form (braitenberg, 1984). he considered a family of hypothetical vehicles, endowed with various sensors and motor - driven wheels, in the form of mobile robots. the book narrates in entertaining but also thoughtful terms, how the electrical connections between sensors and wheels determine a repertoire of different responses to the stimuli in the environment. it presents two distinct viewpoints : one is the viewpoint of an electrical engineer who puts together the wiring scheme starting from a desired behavior of the vehicle ; the other is the analytical viewpoint of a scientist who observes the behavior and attempts to find out how it derives from some possible neural wiring. the insight that we obtained from braitenberg 's vehicles is that neural structures and properties can be established by artificially constraining the relation between neural system and behavior. this guided our group to develop an experimental approach, in which the behavior of a simple artificial device is generated by an isolated neural preparation (reger. the brains of sea lamprey larvae were extracted and placed in a recording chamber where they were maintained at constant physiologically relevant temperature in a ringer 's solution. we placed two stimulation microelectrodes, one on the right and one on the left side of the midline, among the axons of the rhombencephalic vestibular pathways. we also placed two recording glass - electrodes, one on each side of the brainstem 's midline, among visually identified reticulospinal neurons of the reticular formation, which represent the final command neurons to activate and maintain locomotion in vertebrates (grillner., 2008). a simple interface decoder converted the spiking activities detected by the recording electrodes into driving signals for the corresponding wheels of a small robot (a khepera, by k - team). a set of optical sensors on the robot measured the light coming from the right and left side, implementing two very rudimentary electronic eyes. the light intensities were then mapped by the interface encoder into the frequencies of two impulse generators connected to the two stimulating electrodes. this was effectively the first implementation of a bidirectional interface, which closed the loop from recorded neural activities to electrical stimulation via a robotic device. it was quite impressive to see the small robot responding to a shining light by movements that were most often directed toward it. this response is called positive phototaxis and reflects the predominance of excitatory pathways crossing the brainstem 's midline (figure 2). this was indeed one of the first models discussed in braitenberg 's book : if the right sensor is connected to the left wheel and vice - versa, then a light shining on one side will cause the wheel on the opposite side to spin faster. as a result, the vehicle will tend to orient itself toward the light and to proceed in the forward direction. however, positive phototaxis was not the only observed behavior of the neuro - robotic system exposed to a light source. negative phototaxis a tendency to move away from the light source- was observed as well (karniel., 2005) and the general scheme includes a brain model, a communication interface characterized by one coding and one decoding block, and a robotic body. implementation of the first bmi realized at northwestern university : a hybrid neuro - robotic system connecting a lamprey 's brainstem to a small mobile robot. signals from the optical sensors of the robot (bottom) are encoded by the communication interface into electrical stimuli, whose frequency depends linearly upon the light intensity. stimuli are delivered by tungsten microelectrodes to the right and left vestibular pathways (top. the whole brain is immersed in artificial cerebro - spinal fluid within a recording chamber. glass microelectrodes record extracellular responses to the stimuli from the posterior rhombencephalic reticular nuclei (prrn). recorded signals from right and left prrns are decoded by the interface, which generates the commands to the robot 's wheels. these commands are set to be proportional to the estimated average firing rate on the corresponding side of the lamprey 's brainstem. the neural system between stimulation and recording electrodes determines the motions in response to each light source (modified from mussa - ivaldi and miller, 2003). the five light bulbs placed on the circular boundary of the workspace were turned on in sequence. movements toward the lateral lights were curved, with an initial part in the forward direction, followed by a turn toward the light. the four panels on the right show the simulation results obtained after fitting the neural responses of the neural preparation with polynomial surfaces of various degrees, from linear to 4th degree. the data for the fit were generated in a separate session, in which stimulation patterns with different frequencies were applied to the two electrodes placed among vestibular axons. the responses were collected from the two recording electrodes in the posterior rhombencephalic reticular nuclei (prrn) on the right and left side of the midline. thus, the data were a collection of points { xr, i, xl, i, yr, i, yl, i}i=1n (x : stimulus, y : response) from the right and left side. these were used to derive, by least squares, the parameters w in eq. 3. as the robot was exposed to a single source of light, it moved along rather complex and curvilinear pathways. it was immediately evident that the neural circuitry responsible for the observed movements had properties that go beyond the structure of a simple linear feedforward network. a notable feature of this neuro - robotic interaction is that it allowed us to make a direct comparison between behaviors generated by the neural preparation and behaviors generated by a computational model. this was possible (a) because the robotic system was a simple artificial body whose dynamics were simpler and much better known than those of any biological body, and (b) because the interactions between the robot and the neural preparation were confined to a set of well defined signals. the dynamics of the robot were captured by two first - order ordinary differential equations that yield the translational and rotational velocities as functions of the orientation and of the spinning rates of the two wheels. the sensor response to a source of light depended upon the orientation of the robot with respect to the source and was inversely proportional to the square of the distance between the source and the sensor. these are simple relations that allowed us to predict (a) the motion of the robot, given the recorded output activity, and (b) the neural stimulus as a function of the motion of the robot. the remaining, very important, part is the neural tissue between stimulation and recording electrodes. a simple model of the transformation performed by the tissue is a static algebraic non - linear mapping, i.e. where x = (xl, xr) is a vector of input stimuli on the left and right electrodes, y = (yl, yr)is a vector of recorded responses from the two sides and w is a matrix of weights, parametrizing the outputs as functions of the inputs. in a simpler form, this can be a static linear mapping, as in more complex, yet particular, non - linear relations can also be considered. for example polynomials of higher degree, as by analyzing the responses of the neural preparation to stimuli of different frequencies applied to both stimulation electrodes, it was possible to estimate the w parameters in polynomial models (karniel., 2005). then, the models were used to predict the motor behavior of the robot in the presence of a fixed light stimulus. figure 2 shows a comparison between actual trajectories, and trajectories simulated using models from linear to 4th degree. as the polynomial degree increases from linear to cubic, there is a visible increase of the model 's ability to reproduce the data. this kind of failure was due to over - fitting the measured data ; however, more importantly as described below, the performance of these kinds of models is limited since the actual neural system is dynamic rather than static. (2005) modified the linear model (2) by adding a simple first - order dynamic component. the dynamic component was expressed as a linear dependence of the neural output at an instant of time upon the neural output at a previous instant they found that, with this correction the performance of the model was much better than higher order polynomial models, despite a reduced number in free approximation parameters. the interaction between a neural system and an external device provides a framework for further investigating the dynamical properties of a neural system (kositsky., 2003, 2009). the interaction between device and neural tissue is entirely self - contained. to simplify our discussion, we assume that the nervous system and the artificial device are governed by some deterministic dynamics. of course, while the dynamics of the external device are generally well known, the neural dynamics are unknown and are the object of study it can be a computer simulation, for example, of a spring - mass system. the use of simulated devices is particularly useful for investigating specific properties of the neural system. moving along the diagram of figure 3 in a clockwise direction, the device sends an output vector variable to the input interface, which encodes this variable into a stimulus pattern, e.g., a frequency of a pulse train. the neural preparation receives the stimulus and responds to it with a pattern of activities. these are recorded either extracellularly or intracellularly with one or more electrodes, depending on the experimental setup. here, again, we need to make the critical assumption that the recorded activities depend in a deterministic way upon the stimulus. of course, such assumption is likely to be violated in reality and in various ways. in fact, an important but difficult task facing the experimenter is to ensure that the preparation is isolated as much as possible from external influences, which tend to create time - dependent fluctuations in the observed neural activity. and, of course, such fluctuations need to be analyzed as a form of experimental noise. finally, the loop is closed by an output interface, which converts the recorded activity into an input vector to the external device. computational maps associated with an ideal closed - loop interaction between a device and a neural preparation. the external device and the neural tissue dynamics are described by a state equation, yielding the next state as a function of the current state and the input. the output of the device is mapped into a stimulation pattern by the input interface. the recorded neural activities are mapped into a control signal for the external device. by combining these dynamical equations, one obtains an autonomous system [qt+1 = m(qt) ] whose behavior is entirely determined by its initial conditions (from kositsky., 2009). a fundamental parameter of any dynamical system is the minimum number of independent state variables that are needed to predict the response to an external input. more concisely, this is the dimension of the state space, also known as dynamical dimension. a point mass in free space has dimension 6, as its state is determined by 3 position and 3 velocity coordinates. however, the closed - loop system described in figure 3 can be used to estimate it by exploiting the simple fact that the dimension of the neural (s) and artificial (x) component combine by addition to yield the dimension of the closed - loop hybrid system (q) the unknown dimension of the neural system dim(s) is derived by subtracting the known dimension of the external device from the measured dimension of the combined system. the combined system is autonomous by construction, as it does not receive any external input and we make the assumption that its parameters are time - independent (at least within sufficiently long time intervals.) a well known theorem (arnold, 1973) establishes that, under broad conditions of smoothness, the solutions of an ordinary differential equation are unique. this implies that the state - space trajectories of an autonomous system, corresponding to different initial conditions, do not overlap. this fact is exploited by a technique (kaplan and glass, 1992 ; kaplan, 1994) which seeks to find the dimension of a dynamical system by embedding observed trajectories into candidate state - spaces of increasing dimension, until all intersections are removed (figure 4, bottom left panel). (2009) were able to estimate the dynamical dimension of several preparations from the lamprey 's brainstem. importantly, as t is shown in figure 4, the estimated dimension of the neural tissue remained unchanged as the dimension of the simulated external system varied from two to four. the lamprey 's brainstem with a stimulation electrode in the vestibulo - reticular pathway and a recording electrode in the prrn. one of the devices was governed by a 2 order differential equation, the other by a 4th order equation. the trajectories obtained by setting a variety of initial conditions are plotted in different colors, over a time span of 20 s. bottom left : kaplan 's analysis (kaplan 1994). it considers pairs of points (vi, vj), and their successors along corresponding trajectories (vi + 1, vj + 1). if the trajectories do not intersect, small always bear small. surrogate state vectors are constructed from a one - dimensional output signal by combining values of the signal at different delays. so, an n - dimensional state vector s(t) is obtained from the output signal y(t) as s(t)=[y(t)y(t)... y(t(n1))]t. the method consists in building surrogate state vectors of increasing dimension until one can assess that the trajectories do not have self - intersections, using the kaplan 's method. the dynamical dimension of the combined system is assessed as the dimension that makes vanish when approaches zero. the plots on the left are with the 2d external system, those on the right are with the 4d system. this is consistently reflected by the difference in the corresponding estimated dimensions (from karniel., 2005 ; kositsky., bidirectional bmis may lead to a new level of understanding of neural plasticity and its role in shaping new behaviors. while different forms of neural plasticity, such as long term potentiation (ltp) (bliss and lomo, 1973) and long - term depression (ito, 1989), are currently seen as important components of the neurobiological basis for learning and memory, the connection between changes in neural excitability, observable at the cellular level, and purposeful modifications of behavior remains largely unexplored. this is because the macroscopic scale of behavior is often orders of magnitude larger than the cellular scale. bidirectional bmis open new pathways of investigation because they connect observable behaviors with the activities that are recorded from a population of neurons and convey feedback from behavior directly to another population in the proximity of the stimulation electrodes. this provides a new tool for manipulating the mechanisms of hebbian plasticity (hebb, 1949 ; abbott and nelson, 2000) by controlling the relation between presynaptic signals associated with the stimulation and the postsynaptic activities that generate the behavior. (2005) tested the possibility of inducing plastic changes in the lamprey 's -vestibulo - reticular pathways by performing an this is another peculiar opportunity offered by such hybrid systems : they allow us to produce reversible changes in the communications between external device and neural preparation. then, to assess the occurrence of a plastic change in the neural preparation, one can observe the difference between the behavior that takes place after the lesion is reversed and the behavior before the lesion was applied. the investigators performed this experiment by temporarily blinding the left electronic eye of the mobile robot. for this, it was sufficient to reduce the gain of the left optical sensor by a factor of 0.1. then, they exposed the system to random light stimulation for about 20 min. at the end of this period, they restored the initial optical gain and tested the system on a set of standard light sources. exposure to the unilateral reduction of the optical gain was sufficient to induce a sustained tendency of the robot to veer toward the right after the balance between the sensors was reestablished. this effect could be explained in two ways : either by a reduction of the spinning rate in the right wheel or by an acceleration of the left wheel (or both). the comparison of this behavioral observation with the prediction of a simple computational model driven by the recorded stimulus / activity patterns revealed that the main change was likely caused by a reduction of the recurrent dynamical gain which relates the activity of the right population of reticular neurons to their own state of firing (the term vrr in eq. 4). this indicates a general reduction of excitability in the output population contralateral to the lesion and can be attributed to the fact that this population received a reduced input from the lesioned site for an extended period of time. is it possible to modify the connectivity in a biological neural network to achieve a desired behavior ? the theory of artificial neural networks (bishop, 1996) has grown and advanced precisely on this premise. but can we exploit the actual mechanisms of neural plasticity to create a desired behavior of the external device ? this question has not yet been answered ; however, there are signs of progress. different groups around the world (demarse., 2001 ; martinoia., 2004 ; 2009) are working on systems conceptually similar to that described in figure 3, but using a different biological model. the neural preparation in these studies is a culture of dissociated neurons from rat cortices grown onto micro - electrode arrays (meas). each electrode of the mea is able to both record and stimulate the extracellular activity of the cultured network. the external device is a simulated or a real vehicle that navigates over an arena. even with different methods and approaches, these groups succeeded to program the unstructured neuron culture in order to make the vehicle able to solve specific behavioral tasks, such as obstacle avoidance. in one example, the network was programmed by the delivery of tetanic stimulation (chiappalone., 2008) to punish the wrong behavior of the robot in case of a collision with an obstacle. after repeated stimulation, an improvement in the robot 's performances (i.e., a lower number of collisions) was observed (novellino., 2007). while this is still a very preliminary result, it demonstrates that at least in principle it may be possible to reach the goal of programming the behavior of a bidirectional bmi by inducing controlled changes in neural excitability. a critical milestone, still unreached, is the controlled induction of plastic changes in both directions (potentiation and depression) with brief exposure to targeted conditioning signals. most work on bmis, so far, has developed decoding paradigms to translate the neural activities captured by surface electrodes or by meas into commands for an external device. this requires the users to keep a constant focus of attention on the execution of detailed motor commands. in these setups, feedback is limited to vision, which involves long delays and requires gaze to be constantly on the moving device. furthermore, non - kinematic information, such as the weight, rigidity and temperature of a manipulated object, are not directly sensed. these limits have propelled investigations toward the development of goal - decoding interfaces (musallam., 2004) and of bidirectional bmi 's (mussa - ivaldi and miller, 2003 ; fagg., 2007). bmi 's are devices that can not only decode neural activity but also encode external information in the form of brain stimuli. if successful, bmi prosthetic control systems are to be developed which will surely require the use of a trainable bidirectional interface. a bidirectional interface can, in principle, be programmed to implement a pattern of neural stimuli and responses capable of approximating a desired behavior of the controlled system (chao., 2008). mathematically, this process corresponds to translating the behavior of the neural system into a control policy that maps the current observed state of the controlled system into a corresponding action. this concept is closely related to earlier evidence that spinal interneurons organize muscles into synergy groups whose mechanical outputs are force fields acting upon the limbs (bizzi., 1991 ; giszter. these studies demonstrated a simple mechanism of vector summation capable of generating a rep ertoire of control policies out of a small set of non - linear force fields (mussa - ivaldi., 1994 ; mussa - ivaldi and bizzi, 2000). as a future direction, we propose to program bidirectional bmis for generating control policies in the form of force fields acting on the controlled external devices. we call dynamic shaping the interface algorithm that implements this neuro -- mechanical translation (figure 5). dynamic shaping has two -components : (1) a decoder that maps the recorded output activity into a force vector, and (2) an encoder that maps the state of the device into a pattern of stimuli. if the dimension of the vector field is smaller than the number of recorded units, the transformation from recorded activities to force vector involves a dimensionality reduction (e.g., by principal component analysis.) in a dynamically shaped interface, the external neural input sets an initial condition and the dynamic field in the absence of other influences determines the ensuing trajectory. this approach would free the user from the need to guide the connected device instant by instant. at the same time, however, the user would be able to perform a continuous control, thus guiding the device through arbitrary paths. so far, we have implemented and tested dynamic shaping with a simulated neural system, a simple feedforward neural network model of the biological component (figure 5). a simple artificial feedforward neural network is connected to a pattern of stimuli, delivered to 6 binary input units (c ; white : on, black off) and generates responses over a set of 16 output neurons. in this model, there are no hidden units between input and output units but there is a set of four hidden noise units that generate random inputs to the output units. input and output units are connected by a synaptic matrix the output units respond to the weighted sum of their input via a sigmoid transfer function. a set of 21 calibration stimuli (c) are presented in sequence to the input units and 21 responses (a) are recorded from the output units. the calibration stimuli and responses are used to approximate a desired force field (e), acting on a mass - damping system. to perform the calibration, the two top principal components of the 21 neural responses are mapped over the ranges of the force vector components in the desired field. this mapping is performed by the linear decoder. then, for each force vector, the corresponding point of application is determined and is associated to the stimulus pattern that generated the force vector (d). a simple form of the stimulus encoder maps the current location of the mass - damping system into the nearest point determined in the calibration. the resulting field generated by the encoder / decoder system (f) is a fragmented approximation of the desired field. (g) : simulated trajectories of the spring - damper system under the field generated by the interface. as dynamic clamps provide us with the means for isolating particular elements of cellular physiology, such as individual channels, the bidirectional interactions between brain and machines either physical or simulated provide the nervous system with artificial bodies that are endowed with well known properties and that communicate through well defined channels. this marriage of the nervous system with artificial devices offers an unparalleled opportunity to acquire knowledge about neural computation and plasticity while opening a path for restoring functions lost to accident or disease. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | brain - machine interfaces (bmis) are mostly investigated as a means to provide paralyzed people with new communication channels with the external world. however, the communication between brain and artificial devices also offers a unique opportunity to study the dynamical properties of neural systems. this review focuses on bidirectional interfaces, which operate in two ways by translating neural signals into input commands for the device and the output of the device into neural stimuli. we discuss how bidirectional bmis help investigating neural information processing and how neural dynamics may participate in the control of external devices. in this respect, a bidirectional bmi can be regarded as a fancy combination of neural recording and stimulation apparatus, connected via an artificial body. the artificial body can be designed in virtually infinite ways in order to observe different aspects of neural dynamics and to approximate desired control policies. |
gardner and richard in their landmark article described a syndrome consisting of hereditary intestinal polyposis, with osteomas and multiple cutaneous and subcutaneous lesions. after that other dental findings, skin and soft - tissue tumors have been added in the description of this syndrome. the documented frequency of gardner 's syndrome is in between 1 in 1400 and 12000 live births. however, approximately 20% of cases represent spontaneous mutations, with no family history reported. mutation in the adenomatous polyposis coli gene which is present on chromosome 5 leads to this condition. we report a case of gardner 's syndrome in an indian patient which is representing the full continuum of dental, colonic, and extracolonic manifestations. a 52-year - old male patient reported to the outpatient department with a chief complaint of swelling on the right side of the palate for the past 5 years. the patient had undergone surgical treatment for a bony swelling at lower border of the mandible about 30 years back. after that, he was apparently normal until 5 years back when he noticed a small swelling on the palate which was very slowly increasing in size. the past medical history revealed chronic bowel upset in the form of abdominal cramps and diarrhea. family history revealed that both sons of the patient had bony swellings of the jaw and both of them were reluctant to get their screening done for gardner 's syndrome. on examination, on palpation, all the swellings were bony hard in consistency, nontender and were fixed to the underlying bone. (a) extraoral photograph shows fullness of nasolabial folds on both sides with small bony swellings seen on the right temple region. (b) bony swellings seen on the right and left sides of the palate. on the right side, buccal vestibule is obliterated in 14 and 15 region and palatal swelling is extending from the mesial aspect of 16 to the distal aspect of tooth 17 anteroposteriorly. on the left side, the palatal swelling is extending from mesial aspect of tooth 25 to the mesial aspect of 28 anteroposteriorly. dense radio - opacities are seen on the right and left maxilla in premolar - molar region. surgical defect seen on the left lower border of mandible and a well - defined round radio - opacity is seen on the left sigmoid notch intraoral examination revealed swellings on the right and left sides of the palate. on the right side, buccal vestibule was obliterated in 14 and 15 region and palatal swelling was extended from the mesial aspect of 16 to the distal aspect of tooth 17 anteroposteriorly. on the left side, the palatal swelling extended from mesial aspect of tooth 25 to the mesial aspect of 28 anteroposteriorly [figure 1b ]. a diffuse swelling was seen on mandible on the left side, causing buccal vestibular obliteration in 34 and 35 region. there were clinically missing teeth 11, 15, 23, 34, 35, and 45. to see the exact extent of the lesion, a panoramic radiograph was obtained which revealed impacted teeth w.r.t. surgical defect was noted on the left lower border of the mandible and a well - defined round radio - opacity was seen on the left sigmoid notch [figure 1c ]. computed tomography (ct) scan showed multiple dense bony islands within the facial and skull bones. three - dimensional ct images showed multiple small nodular swellings on the frontal, parietal, occipital, temporal, and sphenoid bones. diffuse bony swellings of the maxilla and mandible were appreciated [figure 2d and e ]. (a) axial section shows near total obliteration of the right and partial obliteration of left maxillary sinus. diffuse radiopaque areas are seen in the maxilla and zygomatic bones. (c) sagittal sections of computed tomography scan show dense radio - opacities in the frontal and occipital bones. a well - defined dense round bony exostosis is seen attached to the left condyle. (d and e) three - dimensional reconstruction on computed tomography scan showing multiple osteomas of frontal and parietal bones along with diffuse bony swelling of the maxilla and mandible. a well - demarcated osteoma of left sigmoid notch and a surgical defect on the left lower border of mandible is appreciated although the patient had no family history of major illnesses or disorders, based on our observations of multiple osteomas and the dental findings, he was advised to be evaluated by a gastrointestinal specialist for intestinal polyps to rule out gardner 's syndrome. within a week after our recommendations, barium enema was done [figure 3 ] which showed multiple colonic polyps and suspicious short segment narrowing in the sigmoid colon with irregular margins, which needed additional evaluation with colonoscopy. colonoscopy was done and showed multiple sessile polyps all over the large intestine from rectum to ileocecal junction. to rule out malignancy, biopsy was taken from the polyp which revealed normal colonic mucosa with moderately dense chronic inflammatory infiltrates in the lamina propria. based on this clinical, radiographic, and histopathological evidence, a diagnosis of gardner 's syndrome was given. barium enema shows enhancement of the entire large intestine with narrowing in the sigmoid colon with irregular margins intestinal polyposis is the most serious characteristic feature of this syndrome which may undergo malignant transformation. the polyps are multiple in numbers and scattered in distribution, occur particularly in the distal colon, but may involve any location in the git. passage of blood or mucosa, diarrhea, and cramp - like abdominal pain are the common presenting symptoms. one of the essential components of gardner 's syndrome is osteomas which vary from slight thickening to large masses and may affect all parts of the skeleton. commonly affected sites include angle of the mandible, skull, and the paranasal sinuses. clinical and radiographic evidence of colonic polyposis or gardner 's syndrome may be preceded by osteomas ; therefore, they serve as an indicator for the disease. in the present case, commonly seen abnormalities are congenitally missing teeth, impacted teeth, hypercementosis, and supernumerary teeth. other defects such as fused roots of the first and second molars, long and tapered roots of posterior teeth, dentigerous cyst, and multiple caries are also seen. they may occur on the face, extremities, and scalp and commonly seen around puberty. adrenal adenoma, papillary carcinoma, hepatocellular carcinoma, adenocarcinoma, and osteosarcoma have also been documented. incidence of multiple congenital hypertrophy of retinal pigment epithelium lesions has been associated with the presence and development of polyposis in gardner 's syndrome. surgical excision can opt for osteomas which are interfering with normal function or causing severe deformation. dental managements may include extraction of the impacted teeth and enucleation or marsupialization of the jaw cysts. the patient may be referred to an ophthalmologist for evaluation of retinal anomalies. for esthetic concern, removable partial denture was fabricated for all the missing teeth and the patient is under regular follow - up for colonoscopy. this case represents a classical picture of gardner 's syndrome which is usually overlooked by many medical and dental professionals. this can be very unfortunate as intestinal polyps in this condition have 100% chance of malignant transformation. timely detection of dental, colonic, and extracolonic manifestations may help in halting this dreaded disease from further progression. after the diagnosis of gardner 's syndrome, the patients must be aggressively followed - up since there is a constant threat to their lives at any age. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | gardner 's syndrome is an autosomal dominant disease characterized by the presence of colonic polyposis, osteomas, and a multitude of soft - tissue tumors. dental anomalies are present in estimated 30% of all affected individuals of gardner 's syndrome, so dental professionals play an important role in determining the early signs of the syndrome. the intestinal polyps have a 100% risk of undergoing malignant transformation if not treated thus, early diagnosis and regular surveillance are important. in this report, we describe classical presentation of gardner 's syndrome in a patient who presented with bilateral swellings on palate along with multiple impacted teeth. |
severe alpha 1-antitrypsin (aat) deficiency (pizz) is an autosomal, codominant, hereditary disorder characterized by low aat levels in the serum and lungs. the main function of aat is to inhibit neutrophil elastase and other serine proteases, including proteinase-3 and plasmin activator.1 this genetic defect results in the polymerization of the aat molecule in the hepatocytes, leading to an accumulation of the z - protein within the hepatocytes and a decreased release into the circulation. aat deficiency contributes to protein degradation and increased inflammation because of proinflammatory effects of polymerized aat and the loss of anti - inflammatory and antiproteolytic functions.2 in sweden, the prevalence of aat deficiency is 1/1,600, a figure firmly established by a nationwide screening program of all 200,000 newborns over the period 19721974, when 127 homozygotes were identified.3 major causes of disability and death associated with severe aat deficiency are early - onset panacinar emphysema and liver disease, which typically presents as cholestasis in infancy, and cirrhosis and primary liver carcinoma (plc) in adulthood.1 association with systemic vasculitis (antineutrophil cytoplasmic antibody positive), necrotizing panniculitis, and a variety of different inflammatory and neoplastic diseases have also been reported.1,4,5 previously published mortality studies have shown that severe aat deficiency leads to reduced life expectancy and that respiratory failure and liver disease are the most common causes of death.68 very little is known about mortality due to other diseases, such as cardiovascular disease (cvd), inflammatory diseases, and cancer, except for plc that is a well - known risk in pizz individuals.9 increased risk of lung cancer has also been described in carriers of the aat - deficient z and s alleles.10 the swedish national register of individuals with severe aat deficiency, pizz, has comprehensive physiological data and documents comorbidities by regular follow - ups every 2 years.11 since 1991, all adult individuals with established severe aat deficiency (pizz) in sweden have been invited to participate in the register. after inclusion, the patients are prospectively followed up every 2 years by their attending physician, and the results are reported to the register. the aim of this study was to analyze all - cause mortality and disease - specific mortality in the pizz individuals included in the swedish national aat deficiency register, with particular reference to ischemic heart disease (ihd), pulmonary embolism, lung cancer, and other diseases. we analyzed data from the swedish national aat deficiency register, which has been detailed elsewhere.11 the study population included all pizz individuals included in the swedish national aat deficiency register since 1991. the inclusion criteria in the register are the pizz phenotype diagnosed by isoelectric focusing and age 18 years or older. the register is approved by the ethical review board, lund university, sweden, and by the swedish data inspection board. data on respiratory symptoms, diagnoses, spirometry, and smoking habits were reported by the attending physician to the swedish national aat deficiency register via a questionnaire.11 lung function tests were performed at the local hospitals. the results of the lung function tests are expressed as the percentage of predicted values according to european reference tables.12 the results of the first spirometry at inclusion were analyzed. vital status and causes of death were obtained from the national register of causes of death up to april 2014. the underlying cause of death was coded according to the ninth (before 1996) and the tenth revisions of the who international classification of diseases (icd).13,14 icd codes were grouped (icd-9, icd-10) as respiratory diseases (460519, 786 ; j00j99, r04r06), cvd (390459, 785 ; i00i99, r00, r01), ihd (410414 ; i20i25), heart failure (425, 428 ; i42, i50, i51.7), stroke (430438 ; i60i69), aortic aneurysm (441 ; i71), venous thromboembolism (415, 451 ; i26, i80), digestive organ diseases (520579, 787 ; k00k93), pneumonia (481486 ; j13j18), lung cancer (162 ; c34), colon cancer (153 ; baseline data were tabulated using frequencies and percentages for categorical variables, mean with standard deviation (sd), and median with range or interquartile range (iqr) for continuous variables with normal and skewed distribution, respectively. comparisons of continuous variables with normal distribution were analyzed using analysis of variance (anova). the period of follow - up was from the date of inclusion in the register to the date of death or up to april 1, 2014. excess mortality compared to the swedish general population was calculated as standardized mortality ratios (smrs), with respect to age and calendar year. the smr is defined as the ratio of the observed deaths in the study population to the expected number of deaths. the expected number of deaths was calculated using the age-, sex-, and calendar year - specific mortality rates of the swedish general population, obtained from the swedish national board of health and welfare. smrs were calculated for overall and cause - specific mortality for all patients, and separately for males / females and smokers / never - smokers. smrs were expressed with 95% confidence intervals (cis) calculated from the poisson distribution. statistical analyses were performed with the statistical package for the social sciences (spss), version 22.0 (imb corporation, armonk, ny, usa). we analyzed data from the swedish national aat deficiency register, which has been detailed elsewhere.11 the study population included all pizz individuals included in the swedish national aat deficiency register since 1991. the inclusion criteria in the register are the pizz phenotype diagnosed by isoelectric focusing and age 18 years or older. the register is approved by the ethical review board, lund university, sweden, and by the swedish data inspection board. data on respiratory symptoms, diagnoses, spirometry, and smoking habits were reported by the attending physician to the swedish national aat deficiency register via a questionnaire.11 lung function tests were performed at the local hospitals. the results of the lung function tests are expressed as the percentage of predicted values according to european reference tables.12 the results of the first spirometry at inclusion were analyzed. vital status and causes of death were obtained from the national register of causes of death up to april 2014. the underlying cause of death was coded according to the ninth (before 1996) and the tenth revisions of the who international classification of diseases (icd).13,14 icd codes were grouped (icd-9, icd-10) as respiratory diseases (460519, 786 ; j00j99, r04r06), cvd (390459, 785 ; i00i99, r00, r01), ihd (410414 ; i20i25), heart failure (425, 428 ; i42, i50, i51.7), stroke (430438 ; i60i69), aortic aneurysm (441 ; i71), venous thromboembolism (415, 451 ; i26, i80), digestive organ diseases (520579, 787 ; k00k93), pneumonia (481486 ; j13j18), lung cancer (162 ; c34), colon cancer (153 ; c18), and other cancers (140239 ; c00d48). baseline data were tabulated using frequencies and percentages for categorical variables, mean with standard deviation (sd), and median with range or interquartile range (iqr) for continuous variables with normal and skewed distribution, respectively. comparisons of continuous variables with normal distribution were analyzed using analysis of variance (anova). the period of follow - up was from the date of inclusion in the register to the date of death or up to april 1, 2014. excess mortality compared to the swedish general population was calculated as standardized mortality ratios (smrs), with respect to age and calendar year. the smr is defined as the ratio of the observed deaths in the study population to the expected number of deaths. the expected number of deaths was calculated using the age-, sex-, and calendar year - specific mortality rates of the swedish general population, obtained from the swedish national board of health and welfare. smrs were calculated for overall and cause - specific mortality for all patients, and separately for males / females and smokers / never - smokers. smrs were expressed with 95% confidence intervals (cis) calculated from the poisson distribution. statistical analyses were performed with the statistical package for the social sciences (spss), version 22.0 (imb corporation, armonk, ny, usa). a total of 1,561 pizz individuals (49% males) were included in the study. forty - six percent of the study population was never - smokers and 57% were identified for reasons other than respiratory symptoms. females had smoked significantly less than males, with a mean (range) of 13 (0.177) and 16 (0.2116) pack - years, respectively (p=0.003). during follow - up, lung function tests were available for 1,535 individuals (98%) at inclusion, of whom 792 (52%) fulfilled the criteria for copd with a forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) ratio < 0.70. the median follow - up period was 12 years (iqr = 13) with a total of 18,881 person - years at risk of death. the smr for pizz individuals was higher than that for the matched swedish population (3.6, 95% ci 3.33.9). the main causes of death were copd and its complications such as respiratory failure and infections (n=281, 54%), liver diseases (n=74, 14%), cvd (n=76, 15%), and cancer (n=87, 17%). pizz patients had significantly increased mortality due to respiratory and hepatic diseases, pulmonary embolism, and colon diverticulitis compared with the general swedish population, as shown in table 2. in contrast, they had a reduced risk of mortality due to ihd, and no increased mortality risk due to cvd as a whole. however, mortality due to heart failure was significantly increased. of the 18 patients who died of heart failure, eleven had copd, one had pulmonary fibrosis, and one had marfan s syndrome. complicated colon diverticulitis with peritonitis was the cause of death of five patients ; none of these patients had known inflammatory bowel disease. the mean age at death was lower in males (67 years, sd 11 years) compared with females (69 years, sd 12 years). smr for males was 3.6 (95% ci 3.24.0) and 3.5 (95% ci 3.14.0) for females. twenty males died of plc compared with an expected 0.2 deaths, giving an smr of 100 (95% ci 61154), and seven females compared with an expected 0.09 deaths, giving an smr of 77.8 (95% ci 31.3160.2). there was no difference in mortality due to respiratory diseases, liver failure, and cvd between males and females. during the follow - up period, the mean age at death was lower in ever - smokers compared with never - smokers (65 years, sd 11 and 74 years, sd 11, respectively). the smr for ever - smokers was higher than for never - smokers (4.6 [95% ci 4.15.1 ] and 2.5 [95% ci 2.12.9 ], respectively). mortality due to respiratory diseases was also higher in ever - smokers compared with never - smokers (table 3). no difference was found in mortality due to ihd, pulmonary embolism, and lung cancer between ever - smokers and never - smokers. mortality due to plc tended to be higher in the never - smokers than in the ever - smokers. mortality due to respiratory diseases was five times higher in individuals with copd (fev1/fvc ratio < 0.70) at inclusion compared with those with normal lung function (table 4). an excess mortality due to heart failure was found in aat - deficient individuals irrespective of lung function. individuals who were identified by screening (n=367) had significantly higher overall mortality compared with the general swedish population, smr 2.2 (95% ci 1.63.0). they had significant excess mortality from the following diseases : respiratory disease, smr 27.1 (95% ci 16.342.3) ; plc, smr 42 (95% ci 5.1151.0) ; liver cirrhosis, smr 14.5 (95% ci 1.852.3) ; and heart failure, smr 14.5 (95% ci 4.733.8). mortality due to cvd, all cancer, and lung cancer was similar to that in general population, smr 1.3 (95% ci 0.632.3), smr 0.9 (95% ci 0.31.8), and smr 0.7 (95% ci 0.023.7), respectively. the median follow - up period was 12 years (iqr = 13) with a total of 18,881 person - years at risk of death. the smr for pizz individuals was higher than that for the matched swedish population (3.6, 95% ci 3.33.9). the main causes of death were copd and its complications such as respiratory failure and infections (n=281, 54%), liver diseases (n=74, 14%), cvd (n=76, 15%), and cancer (n=87, 17%). pizz patients had significantly increased mortality due to respiratory and hepatic diseases, pulmonary embolism, and colon diverticulitis compared with the general swedish population, as shown in table 2. in contrast, they had a reduced risk of mortality due to ihd, and no increased mortality risk due to cvd as a whole. however, mortality due to heart failure was significantly increased. of the 18 patients who died of heart failure, eleven had copd, one had pulmonary fibrosis, and one had marfan s syndrome. complicated colon diverticulitis with peritonitis was the cause of death of five patients ; none of these patients had known inflammatory bowel disease. the mean age at death was lower in males (67 years, sd 11 years) compared with females (69 years, sd 12 years). smr for males was 3.6 (95% ci 3.24.0) and 3.5 (95% ci 3.14.0) for females. an excess mortality for plc was observed in both males and females. twenty males died of plc compared with an expected 0.2 deaths, giving an smr of 100 (95% ci 61154), and seven females compared with an expected 0.09 deaths, giving an smr of 77.8 (95% ci 31.3160.2). there was no difference in mortality due to respiratory diseases, liver failure, and cvd between males and females. during the follow - up period, 350 ever - smokers and 174 never - smokers died. the mean age at death was lower in ever - smokers compared with never - smokers (65 years, sd 11 and 74 years, sd 11, respectively). the smr for ever - smokers was higher than for never - smokers (4.6 [95% ci 4.15.1 ] and 2.5 [95% ci 2.12.9 ], respectively). mortality due to respiratory diseases was also higher in ever - smokers compared with never - smokers (table 3). no difference was found in mortality due to ihd, pulmonary embolism, and lung cancer between ever - smokers and never - smokers. mortality due to plc tended to be higher in the never - smokers than in the ever - smokers. mortality due to respiratory diseases was five times higher in individuals with copd (fev1/fvc ratio < 0.70) at inclusion compared with those with normal lung function (table 4). an excess mortality due to heart failure was found in aat - deficient individuals irrespective of lung function. individuals who were identified by screening (n=367) had significantly higher overall mortality compared with the general swedish population, smr 2.2 (95% ci 1.63.0). they had significant excess mortality from the following diseases : respiratory disease, smr 27.1 (95% ci 16.342.3) ; plc, smr 42 (95% ci 5.1151.0) ; liver cirrhosis, smr 14.5 (95% ci 1.852.3) ; and heart failure, smr 14.5 (95% ci 4.733.8). mortality due to cvd, all cancer, and lung cancer was similar to that in general population, smr 1.3 (95% ci 0.632.3), smr 0.9 (95% ci 0.31.8), and smr 0.7 (95% ci 0.023.7), respectively. this study demonstrates that individuals with severe aat deficiency have a reduced mortality due to ihd compared with the swedish general population matched for age, sex, and calendar year. this finding can not be explained by differences in smoking habits, as a similar reduction was seen in both smokers and nonsmokers. we also found that the mortality rate due to cvd as a whole was similar to that in the general population. to our knowledge, this is the first study that analyzes cause - specific mortality in individuals with severe aat deficiency. previous mortality studies have compared mortality either between never- and ever - smoking pizz individuals or between pizz individuals and the general population.68 these studies have demonstrated an increased mortality risk in pizz individuals compared with the general population, and a significantly reduced survival in pizz smokers compared with never - smokers. emphysema and liver cirrhosis have been the predominant causes of death in both ever- and never - smokers. one possible mechanism for the reduced mortality from ihd is that pizz individuals may have lower blood pressure than healthy controls, as reported by dahl.15 hypertension being a major risk factor for both cvd and ihd, a lower level of blood pressure in aat - deficient individuals than in the general population could reduce the risk of cvd and ihd. dichtl found that aat deficiency leads to fewer cleaved fragments of aat in atherosclerotic plaques and thereby reduces atherosclerotic inflammation and risk of ihd. an interesting hypothesis is that a low serum concentration of aat or the z mutation of the aat molecule could provide biochemical or genetic protection against ihd. however, duckers reported increased aortic stiffness in a small group of pizz individuals with copd in comparison with age- and sex - matched controls with normal lung function, indicating increased cardiovascular risk. no patients with usual copd were included in the study, and it therefore remains unclear whether the increased aortic stiffness was related to aat deficiency or copd per se. in our study, we analyzed cause - specific mortality, and therefore it is difficult to compare our results with other studies that analyze risk of disease or clinical signs of diseases. interestingly, the risk of death from pulmonary embolism was markedly increased seven times higher than in the general population. this is a new finding and has previously not been reported in mortality studies.7,8 in our previously published study, six (2%) of 302 deaths were caused by pulmonary embolism.6 a few case - report studies have suggested that aat deficiency may be associated with an increased risk of venous thromboembolism.1820 there are two possible mechanisms for increased thrombophilia in aat deficiency. an unopposed proteolytic activity of plasminogen activator could cause the activation of the coagulation cascade. additionally, unopposed proteinase-3 activity may damage and/or activate endothelial cells, resulting in a prothrombotic state.20 increased mortality from heart failure is easier to explain, because copd and respiratory failure are common causes of right heart failure. because previously published studies have shown that patients with copd have increased mortality due to cvd,21 we also analyzed smr stratified by lung function. we still did not find any increased mortality due to cvd in the individuals with reduced lung function (fev1/fvc ratio < 0.70) compared with those with normal lung function. simultaneously, the decreased mortality due to ihd and the increased mortality due to heart failure and pulmonary embolism remained significant in both the individuals with copd and those with normal lung function. we found that mortality due to respiratory diseases was 14 times higher in aat - deficient individuals with normal lung function at baseline than in the general population. furthermore, aat - deficient individuals with copd at inclusion had a fivefold higher risk of mortality due to respiratory diseases compared with those with normal lung function. increased mortality due to cancer, overall, was driven by increased mortality due to plc, while mortality due to lung, breast, or colon cancer was not increased. previously published studies have indicated that aat deficiency carriers have increased risk of lung and bladder cancer, and malignant lymphoma. yang have reported, in a case - control study, an increased risk of lung cancer in carriers of aat - deficient z and s alleles compared with noncarriers. they concluded that the risk was caused by an imbalance between aat and neutrophil elastase. it is possible that the burden of smoking is lower among the aat - deficient patients than among the patients with usual copd. because no smoking data are available for the general population, this hypothesis can not be tested in this study. stoller have previously reported that diverticulitis was the underlying cause of death in 3% of the 120 decedents with severe aat deficiency in the national heart, lung and blood institute register. in our study, five of the 524 decedents (1%) died of diverticulitis, which was significantly higher than the expected 0.24 deaths in the swedish general population. one of the most important strengths of our study is that all data are collected from a well - established national register of pizz individuals with a correct diagnosis verified by isoelectric focusing. in sweden the majority of individuals in the register had been identified in investigations for reasons other than respiratory symptoms, and 24% were identified by family or population screening. due to these facts, a large number of the study population (48%) had normal lung function at inclusion. another important strength in our study is that it is based on the swedish system of personal identification number, which implies that the vital status was available for all subjects. in the national mortality statistics, the causes of death are recorded for all deceased individuals in sweden. using a relative mortality model and the national mortality statistics made it possible to describe the excess cause - specific mortality among the pizz individuals compared with the matched general population with complete follow - up. the cause - specific estimates could be affected by changes over time in diagnostics as well as changes in coding and classification of the causes of death by physicians and in the swedish causes of death register. it is unlikely that the change from icd-9 to icd-10 in 1997 has affected the reliability of the results, according to analysis performed by the swedish national board of health and welfare.22 the possible bias is unlikely to substantially affect the internal or external validity of the present study. furthermore, we can not completely exclude that observed differences (or lack thereof) between the analyzed patient categories (by sex, smoking, and lung function) may be caused by differences in person - year distribution with respect to age and/or calendar year. third limitation is that no individual data on smoking habits in the swedish general population are available. one of the most important strengths of our study is that all data are collected from a well - established national register of pizz individuals with a correct diagnosis verified by isoelectric focusing. in sweden the majority of individuals in the register had been identified in investigations for reasons other than respiratory symptoms, and 24% were identified by family or population screening. due to these facts, a large number of the study population (48%) had normal lung function at inclusion. another important strength in our study is that it is based on the swedish system of personal identification number, which implies that the vital status was available for all subjects. in the national mortality statistics, the causes of death are recorded for all deceased individuals in sweden. using a relative mortality model and the national mortality statistics made it possible to describe the excess cause - specific mortality among the pizz individuals compared with the matched general population with complete follow - up. the cause - specific estimates could be affected by changes over time in diagnostics as well as changes in coding and classification of the causes of death by physicians and in the swedish causes of death register. it is unlikely that the change from icd-9 to icd-10 in 1997 has affected the reliability of the results, according to analysis performed by the swedish national board of health and welfare.22 the possible bias is unlikely to substantially affect the internal or external validity of the present study. furthermore, we can not completely exclude that observed differences (or lack thereof) between the analyzed patient categories (by sex, smoking, and lung function) may be caused by differences in person - year distribution with respect to age and/or calendar year. third limitation is that no individual data on smoking habits in the swedish general population are available. further studies of cvd and cancer in pizz individuals compared with controls with known smoking habits are needed. | backgroundsevere alpha 1-antitrypsin deficiency (pizz) predisposes to morbidity and mortality due to early - onset emphysema and liver disease. the risk of death from other causes, including cardiovascular disease and cancer, has not been well investigated. we aimed to analyze cause - specific mortality in pizz individuals compared with the general swedish population.methodsdata on 1,561 pizz individuals from the swedish national aat deficiency register, prospectively followed from 1991 to 2014, were analyzed. causes of death according to the swedish national causes of death register for the study group were compared with those for the general swedish population matched for age, sex, and calendar year, with the excess mortality expressed as standardized mortality ratios (smrs) with 95% confidence intervals (cis).resultsthere were 524 deaths during the follow - up period. pizz individuals had excess all - cause mortality compared with the swedish general population (smr 3.6, 95% ci 3.33.9). smr for ischemic heart disease (ihd) was 0.5 (95% ci 0.30.8) and was similar for never and ever - smokers, and in males and females. smr for lung cancer was 0.9 (95% ci 0.41.7). pizz individuals had increased mortality compared with the general population for the following diseases : respiratory disease, smr 48.4 (95% ci 43.054.5) ; primary liver carcinoma, smr 90.0 (95% ci 59.3130.9) ; complicated colon diverticulitis, smr 20.8 (95% ci 6.748.6) ; and pulmonary embolism, smr 6.9 (95% ci 3.312.7).conclusionpizz individuals had a reduced mortality risk of ihd. mortality due to respiratory, hepatic disease, diverticulitis, and pulmonary embolism was markedly increased compared with the age- and sex - matched swedish population. |
a 9-year - old female patient reported complaining of pain in the upper front tooth since 3 days. there was a history of trauma to the same tooth due to fall about 4 days back. on clinical examination, periapical radiograph showed incomplete root formation with wide open apices for the same tooth [figure 1 ]. the working length was established within one mm of the radiographic apex by using size 30 hedstrom file. next, pulp extirpation and complete debridement of the canal was done using h file number 40 followed by copious irrigation with normal saline. after drying of the canal using paper points, calcium hydroxide powder was mixed with normal saline and this mixture was placed into the canal and pushed to the short of apex using plugger., a periapical radiograph was taken, which showed complete formation of the root apex in maxillary right central incisor, without any signs and symptoms and periapical radiolucency. clinically, apical barrier formation was confirmed by using a size 30 gutta - percha (gp) point to check for the presence of a resistant stop and absence of hemorrhage, exudates or sensitivity [figure 3 ]. in the next visit, complete obturation was carried out with gp using lateral condensation technique [figure 4 ] followed by composite restoration. case 1 : periapical radiograph showing wide open apex in relation to 21 (arrow) case 1 : periapical radiograph showing placement of caoh dressing case 1 : periapical radiograph taken after 3 months shows confirmation of apical barrier with gutta - percha point case 1 : radiograph showing complete obturation of 21 an 11-year - old male patient reported with a chief complaint of discolored right maxillary central incisor with a history of trauma 1 year back. the periapical radiograph revealed a large blunderbuss canal of the same tooth [figure 5 ]. on clinical examination, pus was extruded from the root canal immediately after the access preparation ; irrigation was done with saline. calcium hydroxide was placed in the root canal and patient recalled after 5 days. at subsequent appointment, the canal was dried with paper points and mta placed with pluggers until thickness of 6 mm [figure 6 ]. a wet cotton pellet was placed in the canal and access cavity was sealed with temporary cement. in next appointment, case 2 : periapical radiograph showing wide open apex in relation to 11 (arrow) case 2 : radiograph showing placement of mineral trioxide aggregate case 2 : radiograph showing complete obturation of 11 a 9-year - old female patient reported complaining of pain in the upper front tooth since 3 days. there was a history of trauma to the same tooth due to fall about 4 days back. on clinical examination, periapical radiograph showed incomplete root formation with wide open apices for the same tooth [figure 1 ]. the working length was established within one mm of the radiographic apex by using size 30 hedstrom file. next, pulp extirpation and complete debridement of the canal was done using h file number 40 followed by copious irrigation with normal saline. after drying of the canal using paper points, calcium hydroxide powder was mixed with normal saline and this mixture was placed into the canal and pushed to the short of apex using plugger., a periapical radiograph was taken, which showed complete formation of the root apex in maxillary right central incisor, without any signs and symptoms and periapical radiolucency. clinically, apical barrier formation was confirmed by using a size 30 gutta - percha (gp) point to check for the presence of a resistant stop and absence of hemorrhage, exudates or sensitivity [figure 3 ]. in the next visit, complete obturation was carried out with gp using lateral condensation technique [figure 4 ] followed by composite restoration. case 1 : periapical radiograph showing wide open apex in relation to 21 (arrow) case 1 : periapical radiograph showing placement of caoh dressing case 1 : periapical radiograph taken after 3 months shows confirmation of apical barrier with gutta - percha point case 1 : radiograph showing complete obturation of 21 an 11-year - old male patient reported with a chief complaint of discolored right maxillary central incisor with a history of trauma 1 year back. the periapical radiograph revealed a large blunderbuss canal of the same tooth [figure 5 ]. on clinical examination, pus was extruded from the root canal immediately after the access preparation ; irrigation was done with saline. calcium hydroxide was placed in the root canal and patient recalled after 5 days. at subsequent appointment, the canal was dried with paper points and mta placed with pluggers until thickness of 6 mm [figure 6 ]. a wet cotton pellet was placed in the canal and access cavity was sealed with temporary cement. in next appointment, case 2 : periapical radiograph showing wide open apex in relation to 11 (arrow) case 2 : radiograph showing placement of mineral trioxide aggregate case 2 : radiograph showing complete obturation of 11 the goal of apexification is to obtain an apical barrier to prevent the passage of toxins and bacteria into periapical tissues from root canal. in the literature, many materials have been used for apexification, such as calcium hydroxide in combination with sterile water, saline, local anesthetic, cmcp, zinc oxide paste with cresol and iodoform, polyantibiotic paste and tricalcium phosphate. calcium hydroxide is one of the most important medicaments used in treatments of pulp conditions and apical periodontitis. the use of caoh in apical barrier formation has shown promising results. because of its enhanced success rate, easy availability for the clinician and affordability for patients some of the postulated mechanisms of caoh are as follows : presence of high calcium concentration increases the activity of calcium dependent pyrophosphatasedirect effect on the apical and periapical soft - tissuehigh ph, which may activate alkaline phosphatase activityantibacterial activity. presence of high calcium concentration increases the activity of calcium dependent pyrophosphatase direct effect on the apical and periapical soft - tissue high ph, which may activate alkaline phosphatase activity antibacterial activity. sheehy and roberts reported that the use of calcium hydroxide for apical barrier formation was successful in 74 - 100% of cases and the average time for apical barrier formation was ranging from 5 months to 20 months. in the present case, appetite like interfacial deposits form during the maturation of mta result in filling the gap induced during material shrinkage phase and improves the frictional resistance of mta to root canal walls. mta has superior biocompatibility and it is less cytotoxic due to its alkaline ph and presence of calcium and phosphate ions in its formulation results in capacity to attract blastic cells and promote favorable environment for cementum deposition. a total of 5 mm barrier is significantly stronger and shows less leakage than 2 mm barrier. in the present case, mta based on the existing literature and our present cases, both mta and calcium hydroxide can be used efficiently for apexification procedure. considering the time duration for the apex closure mta has superior properties when compared with calcium hydroxide. | the completion of root development and closure of the apex occurs up to 3 years after the eruption of the tooth. the treatment of pulpal injury during this period provides a significant challenge for the clinician. the importance of careful case assessment and accurate pulpal diagnosis in the treatment of immature teeth with pulpal injury can not be overemphasized. the treatment of choice for necrotic teeth is apexification, which is induction of apical closure to produce more favorable conditions for conventional root canal filling. the most commonly advocated medicament is calcium hydroxide, although recently considerable interest has been expressed in the use of mineral trioxide aggregate (mta). we report a case series wherein calcium hydroxide and mta were used successfully for one step apexification in teeth with open apex. |
a literature search using relevant terms such as zn and animal feed, bacteria, (antibiotic resistance or antimicrobial resistance), requirement, bioavailability using the advanced search builder provided by pubmed (www.ncbi.nlm.nih.gov/pubmed) or web of science was performed. a similar search using the same terms, but the reference lists in the selected citation were scrutinized to identify additional articles or reports, overlooked by the searches. the titles of all hits were scanned, and for those that were of potential relevance, the abstracts were also scanned. of these, for those of potential relevance, the full text was obtained and assessed whether it was of relevance to this paper. original and review articles, and textbook content were included as references in this paper. a list of the articles on zn / cu driven co - selection of antibiotic resistance, which fulfilled the inclusion criteria with summary of the findings and main conclusion, is presented in table 1. mrsa : methicillin resistant staphylococcus aureus ; mssa : methicillin sensitive staphylococcus aureus, arg : antimicrobial resistance gene the microbiota associated with animals represents a complex assemblage of microorganisms covering all three domains of life (bacteria, achaea and eukarya) (5). all body sites are colonized, with the lower gastrointestinal tract being the most densely populated. in the number of cells, the microbiota generally outnumbers the host by a factor of 10, while with respect to the number of genes the microbiota contains 100 times more genes than the host. thus, the gut microbiota can be considered an organ in itself (6). the function of the microbiota is to protect the host from pathogen invasion, to train the immune system, to extract energy from low accessible nutrients, and in addition to produce essential vitamins and metabolites needed by the host (7). generally, lower gut microbes are strictly anaerobic due to the anaerobic conditions in that area. the anaerobic conditions are mainly created by microbial respiration (8). the porcine gut microbiota shows a dominance of the phyla firmicutes and bacteroidetes, which is similar to that of humans (9, 10), while the chicken microbiota is lower in bacteroidetes, and higher in lactobacillus and proteobacteria (11). it has been proposed that diet is the main driver for the composition and functioning (nutrient breakdown and metabolite production) of the gut microbiota since microorganisms in the gut can utilize nutrient compounds that the host can not break down (12). mainly due to the complexity, we have very limited knowledge of what shapes the host associated microbiota composition (10). the main unresolved questions are if the host - associated microbiota is shaped by bacterial bacterial competition, or if the host can shape the composition (13). in microbial ecosystems there is a delicate balance between trace metals such as zn and cu as limiting factors, and the toxic effect of these (14). zn and cu are common co - factors in enzymes, while the effects of elevated exposure are more diverse, ranging from the replacement of other trace elements, binding to enzymes, and oxidation. it has been hypothesized that the antimicrobial effect of zn and cu leads to growth promotion in a similar manner as for the effect of antibiotic - based growth promoters (15). for cu, although it has been reported that there is a shift in the microbiota associated with exposure (16), it could be that its main pathogen related effect is through increased host tolerance for lipopolysaccharides (17). a range of pathogens can exploit host responses through inflammation induction (18, 19). for instance, upon inflammation the host will produce chelating agents such as calprotectin that will limit the availability of the trace elements and thereby bacterial growth (20). for zn, however, recent evidence suggests that pathogens can have a competitive advantage over the commensal microbiota under zn limiting conditions, thereby being promoted under an inflamed state (14, 21). since diarrhea in itself can lead to zn depletion and this could also promote the pathogen survival. the potential mechanisms of growth promotional effects of zn / cu are attributed to their antimicrobial activities, similar to that of antibiotics, in that gut microbiota are altered to reduce fermentation loss of nutrients and to suppress gut pathogens (22). the data from (22) illustrated reduced sizes of major groups of bacteria among the porcine gastrointestinal commensals, namely, the lactobacilli and streptococci by elevated doses of dietary zno and cuso4. the reduced level of these commensals in the proximal part of the gastrointestinal tract may benefit the host animal by allocating more feed components for its growth performance. furthermore, feeding the animals high dietary zno doses resulted in an altered pattern of organic acid accumulation, with lower levels of lactate and succinate in the stomach and small intestine and an accumulation of these compounds in the cecum and colon. how this influences the physiology of the animals needs further elucidation in detail, since lactic acid produced in the stomach is normally considered a part of the natural defense mechanism of the host, whereas lactate accumulation in the large intestine has mainly been observed in connection with various disorders. cuso4 reduced the number of coliforms in the large intestine, which may be a part of other mechanisms, such as the suppression of specific pathogens and induced resistance of the animal to pathogen adhesion and invasion as well as pathogen - produced toxins (23). trace elements like zn and cu may be toxic to bacteria and this microbial toxicity may be due to their chemical affinity to the thiol groups of macro - biomolecules but also depends on the solubility of the metal compounds under physiological conditions. to avoid cellular toxicity to elevated trace element exposure, bacteria have evolved mechanisms of metal tolerance. both the mechanisms of toxicity and tolerance to trace elements in bacteria are discussed extensively in the review article of seiler and berendock (24). the authors concluded that in addition to antibiotic agents, heavy metals used in animal farming and aquaculture might promote the spread of antibiotic resistance via co - selection. it has been proved that antimicrobial agents other than antibiotics have the ability to promote a co - selection process, indirectly selecting for antibiotic resistance (25). the trace elements like zn and cu seems to have potential to act as a selective pressure that forces the proliferation and evolution of zn / cu and antibiotic resistance not only at the farm level, but also in the environment. the total amounts and concentrations used of zn and cu in feed may differ among countries, due to f. ex. restrictions imposed by national legislation. as a consequence different selective pressure might be exerted within different countries. data regarding development of resistance against zn and cu in bacteria of human origin is deficient. the average exposure of zn and cu in humans, from food, is probably usually far lower than the exposure in animals fed diets supplemented with these trace elements. in bacterial isolates found in animals (table 1), elevated minimum inhibitory concentration (mic) values to zn and cu were detected in several opportunistic bacterial species compared to background isolates. the data presented in these studies indicate that such elevation in mic - values may be due to elevated exposure of these trace elements in animal feed. mic is defined as the lowest concentration of a given agent that inhibits growth of a microorganism under standard laboratory conditions. testing for susceptibility against zn / cu in various bacterial species was performed using either a micro - dilution technique or an agar - dilution technique and under different methodological conditions (studies listed in table 1). there is currently no standardized and approved method to determine the mic values for zn / cu. among the examined bacterial species, the development of resistance to cu in enterococci is associated with the presence of a cu resistance gene (tcrb), which is often located on a plasmid (3, 2628). in enterococci, the cu resistance gene tcrb was shown to be associated with resistance to the macrolide antibiotic erythromycin (ermb). a conjugation study demonstrated co - transfer of tcrb and ermb genes between e. faecium and e. faecalis (3). transferable cu resistance tcrb has been reported in these enterococci isolated from piglets, calves, poultry, as well as humans in denmark (29). several studies performed in denmark show a link between resistance to cu and resistance to macrolides and also to glycopeptides (vancomycin) in enterococcal isolates of pig origin (30, 31). the authors concluded that there is a frequent occurrence of cu resistance gene in these isolates, where cu sulphate is being used in large amounts as feed additive. the glycopeptide antibiotic avoparcin has been used as a growth promoter in animal production by adding to feed, in many european countries, including norway, in the past (from mid-1970s). however, it has been prohibited since the 1990s because of the development of vancomycin resistance in bacteria, in particular in enterococci. the discontinued use of avoparcin in animal feed has resulted in a reduction in the number of vancomycin - resistant organisms isolated from animals (32, 33). because avoparcin and vancomycin are similar in structure, bacteria resistant to avoparcin are resistant to vancomycin as well. vancomycin - resistant enterococci (vre) spread rapidly and have become a major problem in many countries. the possibility of transfer of vancomycin resistance genes from enterococci to other gram - positive bacteria, like staphylococci, raises significant concerns about the emergence of vancomycin - resistant s. aureus (34). nowadays, vancomycin constitutes one of the last resort antibiotics for treatment of mrsa infection in humans. several studies in table 1 have demonstrated an association between resistance to zn and resistance to methicillin in staphylococci (4, 35, 38). (38) found that mrsa strains from pigs from european countries, canada, and china had a high prevalence of zn resistance (mainly associated with czrc gene), whereas the corresponding mssa were susceptible. similar association between resistance to zn and resistance to methicillin was also observed in samples from veal farms from the netherlands. methicillin is not the drug of choice for treatment of infection in veterinary medicine, neither in norway nor in any countries within eu. there is a lack of knowledge regarding the source of methicillin zn - resistant staphylococci in animals. it is not clear whether the methicillin - resistant staphylococci in animals are of human origin and have been resistant to zn after exposure to feed or the zn - resistant staphylococci have been resistant to methicillin, due to exposure to antibiotic(s). a recent publication from germany (37) showed a higher diversity of e. coli clones in piglets fed with diets supplemented with zn compared to the background control group. the proportion of multiresistant e. coli was significantly increased in the zn group compared to the control group. the authors suggested two possible mechanisms for their results : 1) co - selection via zn resistance as some of the isolates were both zn and antimicrobial resistant ; 2) enhanced plasmid uptake under the influence of zn, as the authors detected several resistance plasmids in isolates of the zn feeding group. there is a lack of data which can demonstrate whether cu / zn - resistant bacteria may acquire antibiotic resistance genes / be antibiotic resistant or antibiotic - resistant bacteria are more capable to be cu / zn - resistant than antibiotic susceptible bacteria. the resistance genes to these trace elements are identified in some bacterial species from animals. resistance to zn / cu and its link to antibiotics resistance in bacterial species originated from animal resistance genes to zn / cu are often located on plasmids, which may be transferable to other bacteria, intra- and inter - species. although overuse of antibiotics in agriculture and medicine is partially responsible for the increased level of antibiotic resistance in bacteria, exposure to trace metals may also contribute to antibiotic resistance, even in the absence of antibiotics themselves. a resistance link between zn and methicillin resistance to cu is often linked to resistance to macrolides (e.g. erythromycin) or glycopeptides (e.g. vancomycin) in enterococci. zn supplementation to animal feed may increase the proportion of multiresistant e. coli in gut microbiota. the transmission of zn / cu - resistant bacteria with resistance to antimicrobial agents to human microbiota can not be discounted. our knowledge regarding mechanisms of induction of zn / cu resistance in bacteria is limited and knowledge on dose - response relations is lacking. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. | farmed animals such as pig and poultry receive additional zn and cu in their diets due to supplementing elements in compound feed as well as medical remedies. enteral bacteria in farmed animals are shown to develop resistance to trace elements such as zn and cu. resistance to zn is often linked with resistance to methicillin in staphylococci, and zn supplementation to animal feed may increase the proportion of multiresistant e. coli in the gut. resistance to cu in bacteria, in particular enterococci, is often associated with resistance to antimicrobial drugs like macrolides and glycopeptides (e.g. vancomycin). such resistant bacteria may be transferred from the food - producing animals to humans (farmers, veterinarians, and consumers). data on dose - response relation for zn / cu exposure and resistance are lacking ; however, it seems more likely that a resistance - driven effect occurs at high trace element exposure than at more basal exposure levels. there is also lack of data which could demonstrate whether zn / cu - resistant bacteria may acquire antibiotic resistance genes / become antibiotics resistant, or if antibiotics - resistant bacteria are more capable to become zn / cu resistant than antibiotics - susceptible bacteria. further research is needed to elucidate the link between zn / cu and antibiotic resistance in bacteria. |
study population and sample preparation : one hundred and 24 human subjects were included in the dog - contact risk factor group. in detail, the dog - contact risk factor group was composed of 41 dog owners and 43 veterinarians who regularly contact dogs. in this group, 12 veterinarians who were themselves dog owners. each of the dog owners including veterinarians was matched with their own 39 dogs by labeling for the statistical analyses. forty human subjects with no history of pet ownership were included for the negative dog - contact factor (supplemental fig. 1). the feces and saliva samples were taken by using sterilized cotton swabs and subsequently submerged in 500 l of autoclaved phosphate buffered saline. dna was extracted from 20 to 30 l of each sample by using dneasy tissue kit (qiagen, santa clarita, ca, u.s.a.). the dna samples were eluted in 200 l volume and stored in a 20c freezer until pcr was conducted. genus - specific pcr : each dna sample was amplified on helicobacter 16s rrna gene using c70 and b37 outer primers30, and subsequently, the pcr products were conducted nested pcr using c97 and c98 inner primer pair (supplemental table 1). the pcr mixture, total volume of 20 l contained a final primer concentration of 0.5 m, 1 l dna samples (0.3 l for nested pcr) and 18 l diethyl pyrocarbonate treated water, was added to maxime pcr premix kit (intron biotechnology inc., the pcr samples were heated at 95c for 5 min followed by 30 cycles of denaturation at 94c for 45 sec, annealing at 53c for 45 sec and extension at 72c for 3 min and finally extended at 72c for 15 min using outer primers by using programmed temperature control system (pc808, astec, fukuoka, japan). for nested pcr, the pcr products were heated at 94c for 2.5 min followed by 35 cycles of denaturation at 94c, annealing at 50.5c, extension at 72c for 1 min each and a final extension at 72c for 15 min. the pcr products were electrophoresed by 1.5% agarose gels containing ethidium bromide in 0.5x tbe buffer and visualized on ultraviolet light illuminator. species- specific pcr : pcr amplifications of h. felis and h. bizzozeronii were performed using primer (supplemental table 1) which amplify the urease b gene of them. the total volume of pcr mixture 20 l including a final each primer concentration of 0.5 m, 1 l dna samples (0.3 l for nested pcr) and 18 l diethyl pyrocarbonate treated water was added to maxime pcr premix kit (intron biotechnology inc.). dna extracts from pure cultures of h. pylori(hpktcc h. pylori strain 114), h. felis(atcc 49179) and h. bizzozeronii(atcc 70030) served as positive controls. for pcr amplification of h. pylori, the samples were heated at 95c for 5 min and followed by 35 cycles at 94c for 45 sec, at 59c for 45 sec and at 72c for 45 sec and a final extension at 72c for 10 min using outer primes. second round of pcr was performed at 95c for 5 min, 30 cycles followed at 94c for 45 sec, at 54c for 45 sec and at 72c for 30 sec and final extension at 72c for 10 min. for h. felis - specific pcr, samples were heated at 94c for 2.5 min once, followed by 40 cycles of denaturation at 94c, annealing at 45c, extension at 72c for 1 min each with a final extension at 72c for 15 min using outer primes. second round of pcr was performed at 94c for 2.5 min, 30 cycles followed at 94c for 45 sec, at 50c for 45 sec and at 72c for 45 sec and final extension at 72c for 15 min. the h. bizzozeronii - specific pcr was carried out following conditions, heated at 94c for 2.5 min once and 33 cycles of at 94c for 1 min, at 57c for 1 min and at 72c for 1 min. second round of pcr was performed at 94c for 2.5 min, 30 cycles followed at 94c for 45 sec, at 55.5c for 45 sec and at 72c for 45 sec and final extension at 72c for 15 min. the pcr products were electrophoresed on ethidium - bromide stained 1.5% w / v agarose gels in 0.5x tbe buffer and visualized on ultraviolet light illuminator. nucleotide sequence analysis : in order to confirm the identity of h. pylori, h. felis and h. bizzozeronii specific pcr assay products with their target genes, after the pcr products of the specific size were extracted by commercial gel extraction kit (megaquick - spin, intron, seoul, korea), direct sequencing of the pcr products with specific primer was conducted by abi prism 3730 xl dna analyzer (pe applied biosystems, foster city, ca, u.s.a.). the result of sequencing was compared to those present in databases using blast software. statistical analysis : pearson s chi - square test and fisher s exact test were used to find the dependence between 2 categories. to check for the independence of 2 categories, including positive / negative pcr detection, the results in human subjects / animal subjects were tested by chi - square analysis using contingency tables. after counting the positive results for helicobacter spp., h. pylori, h. felis and h. bizzozeronii as the variables, the possibility of risk of transmission between animal and human subjects was assessed using pearson s chi - square test with yates continuity correction or 2-tailed fisher s exact test for count data to rule out a variety of distribution assumptions. for this reason, pearson s chi - square test is vague when cell counts are less than 5. from the contingency tables, odd ratios for each helicobacter spp statistical analyses were conducted using the conventional statistical software r (version 2.15.2), and the significance was set at p<0.05. risk factor analyses by genus / species - specific pcr : genus / species - specific single pcr was performed using inner primer for 16s rrna gene in helicobacter spp. h. pylori, h. felis and h. bizzozeronii were detected by nested pcr assay (supplemental fig. 2). direct sequencing was performed on 2 of each helicobacter species - specific pcr product in both human and animal, and these pcr products were randomly selected. when the result of sequencing was compared to those present in databases using blast software, above 99% was represented homology to their specific helicobacter species. for the risk factor analyses, 124 eligible human subjects and 39 dogs participated in this study. among the human subjects, 41 dog owners and 43 veterinarians including 12 veterinarians who were themselves dog owners infection diagnosed by genus - specific pcr showed a statistically significant result (p<0.01, table 1table 1.transmitted infection possibility of helicobacter spp., helicobacter pylori, helicobacter felis and helicobacter bizzozeronii in dog - contact risk factorhelicobacter spp. helicobacter pylori helicobacter felis helicobacter bizzozeronii species subtotaltotalnegativepositivenegativepositivenegativepositivenegativepositivedog - contactnegative1921346391241640160positive186631536618513384336a) pearson s test with yates continuity correction : =7.59, p < 0.01, fisher s exact test for count data : odds ratio=3.28, p < 0.01. b) pearson s test with yates continuity correction : =23.23, p = not significant, fisher s exact test for count data : odds ratio=9.50, p = not significant. c) pearson s test with yates continuity correction : =6.09, p < 0.05, fisher s exact test for count data : odds ratio=10.50, p < 0.01. d) pearson s test with yates continuity correction : =0, p = not significant, fisher s exact test for count data : odds ratio=0.97, p = not significant.), but in the relativity analyses between groups of humans with frequent contact with dogs and h. pylori, h. felis and h. bizzozeronii infections diagnosed using species - specific pcr, only h. felis showed a statistically significant result (table 1). although h. pylori infection showed a statistically significant relativity, no statistically significant association was found between the veterinarian subjects and helicobacter. h. felis and h. bizzozeronii infections (table 2table 2.transmitted infection possibility of helicobacter spp., helicobacter pylori, helicobacter felis and helicobacter bizzozeronii in veterinarian risk factorhelicobacter spp.helicobacter pylorihelicobacter felishelicobacter bizzozeronii d)species subtotaltotalnegativepositivenegativepositivenegativepositivenegativepositiveveterinariannegative103120213110281341164positive8351132358232043172a) pearson s test with yates continuity correction : =0.14, p = not significant, fisher s exact test for count data : odds ratio=1.40, p = not significant.b) pearson s test with yates continuity correction : =3.59, p<0.05, fisher s exact test for count data : odds ratio=2.73, p<0.05. c) pearson s test with yates continuity correction : =0.14, p = not significant, fisher s exact test for count data : odds ratio=0.71, p = not significant. d) pearson s test with yates continuity correction : =1.35, p = not significant, fisher s exact test for count data : odds ratio=1.85, p = not significant.). on performing risk factor analyses of hhlo-2 infection by transmission, using matching species, between hhlo-2-positive dog owners and hhlo-2-positive dogs, h. felis infection showed an extremely significant relativity (p<0.0001, table 3table 3.risk factor analyses of helicobacter felis and helicobacter bizzozeronii by matching species between dog - owner and doghelicobacter felis a)dogshelicobacter bizzozeroniidogsnegativepositivenegativepositivedog - ownersnegative230dog - ownersnegative181positive79positive713a) pearson s test with yates continuity correction : =13.79, p<0.0001, fisher s exact test for count data : odds ratio = inf. infinite value describes the faulty cell data by excel program (0/0). b) pearson s test with yates continuity correction : =12.62, p<0.001, fisher s exact test for count data : odds ratio=30.04, p < 0.001.), and h. bizzozeronii may also be a possible significant risk factor (p<0.01, table 3). a) pearson s test with yates continuity correction : =7.59, p < 0.01, fisher s exact test for count data : odds ratio=3.28, p < 0.01. b) pearson s test with yates continuity correction : =23.23, p = not significant, fisher s exact test for count data : odds ratio=9.50, p = not significant. c) pearson s test with yates continuity correction : =6.09, p < 0.05, fisher s exact test for count data : odds ratio=10.50, p < 0.01. d) pearson s test with yates continuity correction : =0, p = not significant, fisher s exact test for count data : odds ratio=0.97, p = not significant. a) pearson s test with yates continuity correction : =0.14, p = not significant, fisher s exact test for count data : odds ratio=1.40, p = not significant. b) pearson s test with yates continuity correction : =3.59, p<0.05, fisher s exact test for count data : odds ratio=2.73, p<0.05. c) pearson s test with yates continuity correction : =0.14, p = not significant, fisher s exact test for count data : odds ratio=0.71, p = not significant. d) pearson s test with yates continuity correction : =1.35, p = not significant, fisher s exact test for count data : odds ratio=1.85, p = not significant. a) pearson s test with yates continuity correction : =13.79, p<0.0001, fisher s exact test for count data : odds ratio = inf. infinite value describes the faulty cell data by excel program (0/0). b) pearson s test with yates continuity correction : =12.62, p<0.001, fisher s exact test for count data : odds ratio=30.04, p < 0.001. the present study investigated the possible role of frequent contact with dogs or dog ownership in the transmission of helicobacter spp. infection in a representative population sample comprising of dogs, dog owners, non - dog owners and veterinarians in korea. we specifically tested the hypothesis that hhlo-2 may be a very important agent causing cross infection, because it has attracted attention as a zoonotic agent in recent studies [11, 17 ]. dog owners and veterinarians were included as the subjects, because of their frequent contact with dogs. overall, we found strong evidence for an increased risk of hhlo-2 infections, such as h. felis and h. bizzozeronii infections, associated with the presence of dogs in the household (dog owners group), and co - infection rates were relatively high in dog - contact group (42.85%) than no dog - contact group (7.50%). in addition, h. felis and h. bizzozeronii co - infection rate was most frequent in both human and animal subject (42.85% in human subject and 55.12% in animal subject). in the previous study about h. pylori, co - infection rate of h. pylori l - form and vegetable - form in human subject results indicated that in the group that was in frequent contact with dogs inside or outside the house (group in frequent contact with dogs), genus helicobacter spp. (p<0.01) and h. felis(p<0.05) infection could be considered as a zoonotic infection. however, frequent contact with dogs did not have relativity as a risk factor in h. pylori and h. bizzozeronii infections, and this result supported the result of the previous studies. interestingly, the veterinary group in korea showed a high prevalence of h. pylori infection with statistical significance as shown in table 2. acquisition of hhlo-2, including h. felis or h. bizzozeronii, may occur through animal contact, contamination of the household environment and also provably through direct or indirect contact inside the house. among these possible transmission routes, possibility of transmission via animal contact through the oral - oral or the fecal - oral route may be highest in dog owners as compared with non - dog owners. a statistically strong positive relation between h. felis infection and contact with dogs was identified in this study. therefore, the potential for zoonosis might be considered in h. felis infection, both from dogs to humans and from humans to dogs, indicating that dogs might be a source for this infection ; and this result is in contrast with the result of previous studies that have considered a low possibility of zoonosis in h. pylori infection in germany. further research should integrate the approaches to the environmental or cultural factors, including diet and other life - style variables with clear roles of individual factors. several reports described the isolation of h. pylori from cats, and a possible non - primate reservoir for h. pylori was also identified [13, 17, 18 ]. h. pylori has been discovered in the stomach of dogs, and h. pylori was found in 3 dogs with no clinical signs according to our pcr results (data not shown). this result may suggest that dogs could be the natural host for h. pylori, or it may be transmitted from other sources especially humans. furthermore, on comparing the type of identified hhlo-2 in owners and their dogs, the dog owners group showed the presence of organisms identical to those in their dogs. it might be speculated that h. felis and h. bizzozeronii could be transmitted both from dogs to humans and from humans to dogs in a strong statistically significant manner. although the other sources of infection, such as contaminated water or food, were considered, the possibility of these sources of infection is comparatively low in h. pylori infection. these data support that dog ownership was linked to a high socioeconomic status in the risk of transmitted hhlo-2 infection. treatment of helicobacter infection consists of combination therapy with antimicrobial and anti - secretory drugs. it is already well - known that helicobacter infection in dogs and cats can be eradicated with treatment, and it is also known which drugs are the best for use in the veterinary medical field. a strong incidence of hhlo-2 infection has been reported in dog owners, suggesting that transmission from animals to humans or from humans to animals is strongly possible. in this report, we have statistically significant supporting results in dog owners and veterinarians of hhlo-2 infection, which should be included as the subjects, because of their frequent contact with dogs. overall, we found strong evidence for an increased risk of hhlo-2 infections, such as h. felis and h. bizzozeronii infections from this research, although infection - positive subjects showed no clinical signs (subclinical infection) mostly. only few human subjects reported extremely sporadic vomit symptoms in the pre - investigation for sampling procedures. in conclusion, we might suggest that hhlo-2 (helicobacter heilmannii - like organisms type 2) infection might be zoonotic, because continuous contact with dogs was proved to be correlated with human h. felis and h. bizzozeronii infections in this study. however, this report never intended to criticize pet ownership, but to carry out more intensive prevention, treatment and socio - epidemologic research of helicobacter felis and h. bizzozeronii infections, which should be considered in both the medical and veterinary fields. | abstracthelicobacter spp. may have multiple routes of transmission. it is unclear, however, whether the agent is zoonotic and therefore transmitted from an animal reservoir, including dogs. the aim of this population - based study was to assess the relationship between pet ownership or frequent exposure to dogs and helicobacter spp. infection, especially focusing on hhlo-2 (helicobacter heilmannii - like organisms type 2) in saliva and feces samples in korea, using non - invasive genus / species - specific pcr. one hundred twenty - four eligible human subjects and 39 dogs participated in this study. relativity of contact with dogs and helicobacter spp. infection diagnosed by genus - specific pcr showed a statistically significant result (p<0.01), but in the relativity analyses between contact with dogs and h. pylori, h. felis and h. bizzozeronii infections diagnosed using species - specific pcr, only helicobacter felis showed a statistically significant result. although h. pylori infection showed a statistically significant relativity, no statistically significant association was found between veterinarian subjects and helicobacter. spp., h. felis and h. bizzozeronii infections. on performing risk factor analyses of hhlo-2 infection by transmission, using matching species, between hhlo-2-positive dog owners and hhlo-2-positive dogs, helicobacter felis infection showed an extremely significant relativity (p<0.0001), and helicobacter bizzozeronii may also be a possible significant risk factor (p<0.01). these results suggest that hhlo-2 infection might be a zoonotic infection, because continuous contact with dogs was proved to be correlated with human h. felis and h. bizzozeronii infections in this study. |
the search for an ideal restorative material is a challenge for which dentistry has yet to find a solution. thus, for each individual clinical situation, dentists must consider certain properties in order to identify the most suitable material. such properties include biocompatibility, adhesion to the dental structure, absence of marginal leakage, wear and pressure resistance, fluoride release, setting time, facilities related to its manipulation and cost. glass ionomer cements (gic) are widely indicated in modern dentistry, especially in atraumatic restorative treatment (art) and pediatric dentistry. the first commercially produced ionomer had poor esthetic properties, reduced working time and very slow final setting. other gic were also introduced to the market, such as silver - modified gic (ketac silver / espe), resin - modified gic (vitremer / 3 m) and, more recently, conventional gic with high powder / liquid proportion (chemflex / dentsply, fuji ixgp / gc corporation, ketac molar / espe, vidrion rcaps / ss white). the latter gic have improved mechanical properties in comparison with their predecessors21 and were especially developed for atraumatic restorative treatment (art). art is a dental care proposal that attempts to control the development of the carious lesion, helping the organism to react against the attack of cariogenic bacteria. it basically consists of partial removal of the carious lesions and subsequent filling of cavities with an adhesive restorative material in association with preventive and educational measures6,7,12. conventional restorative gic are indicated in art for their adhesion capacity and fluoride release properties as well as the chemical setting mechanism, which foregoes technological sophistication, as this treatment was initially proposed for places without the proper infrastructure. however, such materials are as yet too costly for widespread use in developing countries such as brazil, especially those with high powder / liquid proportion18. this leads some professionals to look for alternative materials, such as vidrion r (ss white) and even the temporary restorative material irm (dentsply), when performing art among underprivileged populations, such as those in public institutions. the aim of the present study was to compare the performance of conventional brazilian glass ionomer cements, which are considered much more financially accessible and available in the market, to the performance of imported cements suitable for art with regard to microleakage in class ii restorations of primary molars in an in vitro environment. the experimental protocol was approved by the institutional review board of the federal university of minas gerais (report n. 214/01). with the informed consent of children and their families, 40 clinically sound primary second molars were used in the research. two standard class ii cavity preparations were performed on each tooth just above the cementoenamel junction. the cavities were prepared with a 1.8-mm round - tipped cylindrical diamond bur (microdont 4230) at high - speed under air and water cooling. cavity finishing was performed with the same bur at low - speed as well as with manual instruments. the cavity was cylindrical with approximately 2 mm in diameter and gingival wall situated 1 mm above the cementoenamel junction. restorative procedures were performed by a single operator who did not exceed 3 hours of daily work at 23c + / - 2c. the teeth were randomly assigned to two groups of 20 teeth each, according to the gic mixing method used : hand - mixed or capsulated gic. mesial cavities were restored with brazilian gic and distal cavities were restored with imported gic, according to pin,.18 (1998), culminating in 4 groups with 20 restorations each : group 1 - vidrion dentin conditioner was applied to the cavity with a disposable brush for 30 seconds and rinsed 3 times with a moist cotton pellet. the gic vidrion r was measured, mixed by hand and inserted according to the manufacturer 's instructions with the aid of a blunt hollemback instrument (duflex). group 2 - application of fuji ix liquid to the cavity surface with a disposable brush for 10 seconds, after which the previously described rinsing and drying methods were used. the gic fuji ix were measured, mixed by hand and inserted according to the manufacturer 's instructions with the aid of a blunt hollemback instrument (duflex). group 3 - the conditioning procedures and the stainless steel band adaptation were performed in exactly the same manner as in group 1. the vidrion rcaps capsule was activated and mixed in agreement with the manufacturer 's instructions. the capsule was then opened ; the gic was removed and inserted into the cavity with the aid of a centrix syringe. group 4 - gc cavity conditioner was applied to the cavity with a disposable brush for 10 seconds, after which the previously described rinsing and drying methods were used. the fuji ixgpfast capsule was activated and mixed in agreement with the manufacturer 's instructions. the insertion of gic into the cavity was performed directly through the capsule adapted to a metallic applicator (gc capsule applier). the capsulated gic were mixed using a high - frequency mechanical mixer (capmix / 3 m espe). as soon as the cement began to lose its shiny appearance, pressure was applied for 30 seconds with an amalgam packer (duflex) lubricated with petroleum jelly (vaseline). fifteen minutes after the restorative procedure was completed, the band was cut and the restoration was finished using a surgical blade adapted to a scalpel. a layer of varnish was applied (vidrion v over mesial restorations and fuji varnish over distal restorations). specimens were stored in distilled water at 37c for 24 hours and then polished using paper discs (sof - lex/ 3 m espe). a new layer of varnish was then applied. the materials used to perform the restorations are displayed in table 1, along with the respective manufacturers, batches, features and mixing methods. for the microleakage test, the pulp chambers were filled with ultra fast - setting epoxy resin (araldite) to prevent infiltration of the dye solution. specimens were then entirely covered with two layers of nail varnish, except for the cervical margin of the class ii filling and 1 millimeter beyond it. next, teeth were immersed in 0.5% methylene blue water solution (ph 7.2, at 37c) for 4 hours, rinsed in tap water, dried for 24 hours and embedded in transparent polyester resin (crystal). the specimens were half - sectioned on a precision cutting machine (isomet 1000) with a diamond disk (buehler series 15 hc diamond, n. 11.4215) cooled with distilled water. the cut was meant to coincide with the center of the restorations. nevertheless, anatomical features inherent to deciduous teeth made it impossible to use a single cut in all cases and at times an additional sequential cut was needed. all sections of each restoration obtained after cutting were assessed by three independent calibrated examiners on a stereomicroscope at 30x magnification to verify dye penetration. the following criteria based on the work of yap,.30 (2000) were used to score the extent of leakage at the cervical margin : 0 = no dye penetration ; 1 = dye penetration up to half the extension of the cervical wall ; 2 = dye penetration beyond half up to the whole extension of the cervical wall ; 3 = dye penetration reaching the axial wall. each examiner performed all possible readings for each of the test samples (1 test sample = 1, 2, 3 or 4 readings, depending on the sections made available by the cuts). median values were used between examiners for each section, as the kappa test revealed fair agreement among them (1x2 = 0.61 ; 1x3 = 0.71 ; 2x3 = 0.77). the kruskal - wallis non - parametric test was applied to compare the median scores of cervical leakage obtained by the three examiners for each of the dental sections (1, 2, 3, or 4 section obtained by the cuttings) and revealed no differences with regard to the percentage of maximum scores. the wilcoxon test was used for dependent samples in the comparison of the median scores of microleakage obtained for each material. the material with the best performance was vidrion rcaps (62.5% of the samples with light or absence of leakage). the material with the worst performance was fuji ix (94.1% of the samples restored with this material presented severe microleakage). when the manually mixed and the mechanically mixed correspondents of each gic were assessed as a group, it was observed (table 3) that a greater percentage of maximum scores for cervical microleakage was found when using the imported gic (80.3%, against 55.4% when using the brazilian gic). for the wilcoxon test, an equal number of samples were needed so that n was 21 in all groups. the percentages for the equal samples are not presented due to the similarity obtained between them for the total sample. table 4 displays the wilcoxon test results for comparison of microleakage scores for the different gic. a highly significant difference was observed (p=0.001) in cervical microleakage scores between brazilian and imported materials, with the former ones presenting smaller values. when only fuji ixgpfast capsule and vidrion rcaps were compared, the brazilian material also presented significantly lower (p=0.003) cervical microleakage scores than the values presented by the imported material. however, the difference between these scores could not be considered statistically significant (p=0.102) (table 4). assuming the fact that the main intrinsic features of an effective restorative material related to a good marginal sealing are its adhesion capacity to the tooth structure and its dimensional stability, and that conventional gic present chemical adhesion to the tooth2,13, low linear thermal expansion coefficient - similar to the dental structure4,13- and minimal setting shrinkage2,24, one might expected them to avoid microleakage and all its consequences. in the present work, however, very high cervical microleakage indices were found for all conventional gic assessed. from the total sample, 68% presented a maximum score, 22% presented some microleakage and only 10% of samples were leakage - free. the vast majority of papers in related literature also reveal high cervical leakage indices related to conventional gic in both permanent1,24,30 and deciduous8,28 teeth. a few authors, however, have reported considerable marginal sealing with conventional gic in permanent4 and deciduous27 teeth. the lack of detailed methodological information makes it hard to conclusively assess the reasons why these authors obtained such good results. alperstein,.1 (1983) and yap,.30 (2000) have demonstrated considerable microleakage in gic restorations even when samples were not submitted to thermocycling, which is known to increase microleakage values. this fact raises the question of what factors other than adhesive capacity and dimensional stability account for adequate initial marginal sealing. as in vitro assays presented dye penetration in gic restorations, but the in vivo assays did not detect bacteria under those same restorations, heys and fitzgerald10 (1991) concluded that either the gap between the dentin and the gic was too small for the bacteria and their byproducts but big enough to allow dye penetration, or the bacteriostatic / bactericide properties of the material were sufficient in preventing the penetration of viable bacteria. arcoria,.2 (1990) have reported that the methylene blue dye molecule is much smaller than 0.5 mm, which would be the space needed to permit the passage of bacteria and their products through the dentin / restoration interface. therefore, dye penetration is not an absolute indicator of what could take place in a clinical context and thereby overestimates results on many occasions. although gic does not avoid microleakage in the in vitro tooth / restoration interface, the material may present a good performance in clinical situations due to its fluoride - releasing capacity that can postpone or prevent the development of secondary carious lesions, which represent the real clinical threat of microleakage18,28. furthermore, gic is potentially able to remineralize residual carious dentin to a varying extent in clinical situations such as art in primary molars25. its preventive role makes gic widely indicated in pediatric dentistry when the quality of the restoration is often damaged by unsuitable children 's behavior. the fact that primary teeth have a limited life span also reduces demands on wear resistance, which remains a setback for gic5. it is well known that a few clinical events may enhance it (such as successive dimensional changes of the material caused by sudden temperature changes, mechanical occlusal stress and hygroscopic alterations), whereas other events may decrease it (such as the maturation of the restorative material or prolonged exposure to saliva, causing obliteration of the space between the tooth and the restorative material through the deposition of mineral salts) on a long - term basis9. a number of strategies can be used to simulate these microleakage events in vitro and, when under a very strict control, may actually mimic what occurs in the mouth. nevertheless, it was decided not to adopt any of these simulating strategies in the present study. instead, an attempt was made to control all variables to the utmost for only the appreciation of the initial leakage of different appraised gic. in future studies, we intend to include other factors that are known to be equally relevant. as there are reports in the literature that the mixing technique may significantly alter the final properties of the gic3,11,14,16, we chose to work with brazilian and imported gic used in art, which presented a version in separate bottles and an encapsulated correspondent : vidrion r / vidrion rcaps (commercialized by the brazilian company ss white) and fuji ix / fuji ixgpfast capsule (commercialized by the multinational company gc corporation). brazilian gic presented a more favorable behavior regarding cervical microleakage than the imported gic, although this difference was only statistically significant (p=0.000) when the materials were mechanically mixed. raggio,.20 (2002) also found smaller microleakage values in class v restorations performed with vidrion r in relation to fuji ix in the cervical wall on deciduous teeth, although this difference was also not statistically significant and the authors only evaluated manually mixed gic. the difference between the material setting times may be related to the differences found in microleakage. a very short solidification time may not allow an appropriate flow of gic to the cavity bottom and may therefore disturb the marginal sealing of the restoration. in the present study, gic with a shorter setting time presented higher values of microleakage than gic with a slower setting time (setting time according to the manufacturer 's information : fuji ix = 2 min and 20 s ; fuji ixgpfast capsule = 2 min ; vidrion r = 5 min and 30 s ; vidrion rcaps = 3 min and 30 s to 4 min and 30 s). thus, the reduced setting times of the imported materials, especially the encapsulated form, may have disturbed the appropriate gic / dental surface adaptation in the cervical area. on the other hand, a slow rate of the gic setting reaction is one of the problems associated to clinical use11. thus, a reduction in the setting time of conventional gic is of clinical interest, as the solidification reaction consists of a complex acid / base reaction that is quite sensitive in its initial stage, so that the dehydration or the contamination of the material by humidity during this period can harm the final properties of the gic11,13. this is especially true for art when it is not possible to use a rubber dam or even saliva suction. furthermore, a slow setting time may result in lower overall productivity of clinicians, which is undesirable considering the great demand for dental treatment in countries such as brazil. once the form of insertion of two gic in the cavities was standardized in the most similar way possible for each mixing method, its influence on differences in microleakage was disregarded. peutzfeldt and asmussen17 (1990) and tanumiharja,.26 (2000) demonstrated that variations in the concentrations and in the time of use of polyacrylic acid do not significantly interfere with the resistance adhesive of conventional gic to the dentin, and should also not interfere with microleakage. thus, we disregarded the influence of the different conditioning agents used in the present study with respect to microleakage [liquid of the material itself (40% polyacrylic acid) for fuji ix ; gc cavity conditioner (20 - 25% polyacrylic acid) for fuji ixgpfast capsule ; vidrion dentin conditioner (11.5% polyacrylic acid) for vidrion r and vidrion rcaps ] as well as the different conditioning times employed in accordance with the respective manufacturer 's instructions. considering mechanically mixed gic, a possible explanation for the difference in microleakage may be related to differences between the capsules of the two materials with regard to shape, weight, size and location of the sealing wax that separates the liquid from the powder. these factors may influence the effective contact between powder and liquid, as well as the extension of the mixture and, consequently, the powder / liquid proportion in the final material11,14. according to rupp,.21 (1996), achievement of an appropriate manipulation of encapsulated gic the mixer speed varies due to a number of factors, including the capsule weight. however, more studies are necessary to permit conclusions related to the influence of capsule variations on gic properties, including microleakage. in the present study, mesial cavities were restored with brazilian gic and distal cavities were restored with imported gic, according to pin,.18 (1998). we do not believe that this option had any effect on the results since no differences regarding the direction of enamel prisms at cervical margins, thickness of enamel, aprismatic layer or dentinal tubular pattern have been observed in the related literature between mesial and distal surfaces of second primary molars22. well - adapted restorations with adequate marginal sealing are of extreme importance for the success of atraumatic restorative treatment, as isolating the external media is necessary in order to create the proper conditions for the organism to fight the remaining bacterial infection in the dentinal tissue at the bottom of the cavity, and also to remineralize the local dental structure26. an interesting result originated from the comparison between gic : a better performance was expected from the imported materials to which improved properties are attributed than the other conventional restorative gic due to the high powder / liquid proportion23,29. this seems very appropriate, since they are much more financially accessible products than fuji ix or fuji ixgpfast capsule, allowing their wide use in public health both for art applications and conventional restorative treatment. however, it is important to keep in mind that the present study only assessed microleakage. other characteristics that were not evaluated here, such as bond strength, fluoride release, solubility, compressive strength, surface hardness, tensile strength, wear resistance, porosity, fracture resistance, setting time and practicability are of extreme importance to the clinical success of glass ionomer cements. based on such factors, there may be benefits in using one of the imported materials. according to raggio,.20 (2002), although the brazilian gic are cheaper, they could present some disadvantages, such as a long setting time, accentuated wear and greater solubility over time, so that the final treatment cost may increase if there is need for further intervention. despite the many limitations presented by the in vitro studies failing to precisely reproduce clinical conditions, they are still considered very useful for indicating directions in the properties of new materials18. however, attention should be drawn to the fact that laboratory studies with small sample sizes in carefully controlled and standardized environments, such as the conditions reproduced in this paper, should not be extrapolated to more complex clinical situations. this makes it of paramount importance to pursue further clinical studies in order to compare in vitro test results with the clinical performance of materials. none of the conventional restoring gic for atraumatic restorative treatment assessed in the present work was able to avoid the cervical microleakage in class ii restorations of primary molars in an in vitro environment, and all presented high indices of dye penetration in the tooth / restoration interface. brazilian gic (vidrion r / vidrion rcaps) presented a better performance regarding cervical microleakage in comparison to imported gic (fuji ix / fuji ixgpfast capsule). considering the microleakage alone, it appears viable to use gic vidrion r / vidrion rcaps as an alternative material in atraumatic restorative treatment. | with the aim of assessing the performance of brazilian and imported glass ionomer cements (gic) with regard to microleakage, 40 primary molars received two standard class ii cavity preparations with margins in enamel. twenty cavities were filled with brazilian materials (vidrion r and vidrion rcaps) and the other 20 cavities were filled with imported materials (fuji ix and fuji ixgpfast capsule). all fillings were performed by a single operator according to the manufacturer 's instructions. teeth were immersed in 0.5% methylene blue and half - sectioned. three independent calibrated examiners assessed microleakage using scores (0 - 3). data were submitted to the kruskal - wallis statistical test and wilcoxon analysis. high microleakage indexes were verified for all ionomer cements : 59.5% of the samples restored with vidrion r or vidrion rcaps and 83.4% of the samples restored with fuji ix or fuji ixgpfast capsule obtained the maximum score (3). the brazilian ionomer cements presented less microleakage than imported cements, although this difference was only significant (p=0.003) among the encapsulated materials. |
this manifestation, widely known as post - operative cognitive dysfunction (pocd), is characterized by disordered thinking and impaired consciousness with later onset and fluctuating course [24 ]. pocd, as mentioned above, is common in elderly patients, and probably has a pathogenesis similar to that of ad and may even evolve into ad. unfortunately, it has been demonstrated that 41.4% of aged patients have pocd at hospital discharge. leptin is synthesized and secreted by adipocytes, and has been recognized as having an important role in coordinating the peripheral and central signals, ultimately regulating food intake and body weight [811 ]. although the biological effects of leptin are thought to regulate eating behavior and energy expenditure, a prospective clinical study with 785 participants showed that higher circulating levels of leptin contribute to reduce ad incidence. a preclinical study has shown that leptin can reduce pathology and improve memory in a transgenic mouse model of ad. collectively, these findings indicate that leptin has unique therapeutic effects on cognitive dysfunction, which is the primary pathological feature of ad. in addition to pocd, it is also characterized by cognitive dysfunction and shares similar pathogenesis with ad. we hypothesized that leptin has prophylactic and therapeutic effects on pocd, and that the leptin signaling pathway may be involved in the pathogenesis of pocd. a previous study by doherty. indicated that leptin prevents hippocampal synaptic disruption and neuronal cell death induced by amyloid- (a). a study by marwarha. has shown that leptin treatment reversed the 27-hydroxycholesterol - induced increase in a and tau phosphorylation (p - tau). ad, a progressive neurodegenerative disease, is characterized by the accumulation of a peptide - containing neuritic plaques and neurofibrillary tangles composed of p - tau. in this regard, pocd is also characterized by abnormal deposition of a and p - tau. these findings strongly support the hypothesis that leptin may have beneficial effects for the treatment of pocd by down - regulation of a and dephosphorylation of p - tau. amp - activated protein kinase (ampk), a ser / thr kinase, has a critical role in the maintenance of energy metabolism at cellular and body levels. furthermore, leptin is capable of decreasing the levels of tau phosphorylation by activation of ampk in rat cortical neurons. it is widely known that pocd and ad are both aging - related diseases, and slowing the aging process may have therapeutic effects. ampk is a major regulator, which can activate the autophagic pathway, while activation of ampk inhibits mtor, an inducer of autophagy. our recent study proposed a hypothesis that inhibiting mtor activates the autophagic pathway, thereby leading to therapeutic effects for pocd. leptin probably has prophylactic and therapeutic effects in pocd, and the leptin signaling pathway may be involved in the pathogenesis of pocd. further investigations are needed to determine whether leptin has unique effects in the treatment of pocd, and to make certain whether leptin signaling pathway is involved in the pathogenesis of pocd. | leptin plays a critical role in neuronal development and also promotes structural and functional activities in the central nervous system. recent studies have demonstrated that leptin could produce therapeutic effects for cognitive impairments of patients with alzheimer s disease (ad). post - operative cognitive dysfunction (pocd), defined as a significant dysfunction in cognitive performance for several weeks after surgery, probably has a pathogenesis similar to that of ad. specifically, they are both characterized by cognitive impairment. in this regard, we hypothesized that leptin probably has a therapeutic benefit of alleviating symptoms of patients with pocd, and the leptin signaling pathway may be involved in the pathogenesis of pocd. |
in the mid-1970s, acute myocardial infarction (ami) was identified as being the result of a ruptured atherosclerotic plaque, causing thrombosis and occlusion of the coronary artery. randomized trials have indicated that primary percutaneous coronary intervention (ppci) during the early hours of ami offers certain advantages over tt [25 ]. the major limitation of primary angioplasty as firstline therapy on a community basis is restricted availability of 24-hour cardiac catheterization laboratories staffed with skilled cardiologists. most hospitals do not have facilities for 24 hour coronary angioplasty, and the large majority of patients with ami are admitting to a non - pci capable hospital. tt is, however, the standard of care for patients with ami, because of its widespread availability and its efficacy for reduction of mortality [69 ]. the ischemia grade system (gi) is an electrocardiographic classification of stemi based on st segment and qrs complex changes on baseline ecg. grade1 : tall sharp t waves without st segment elevation ; gi2 : st segment elevation in > 2 contiguous leads without terminal qrs deformation ; and gi3 : st segment elevation with terminal qrs deformation in > 2 contiguous leads. previous studies have shown that patients with gi3 on the presenting ecg have a worse prognosis [1114 ], larger infarct size [11,1518 ], less benefit from tt and less hibernation in the infarcted myocardium than patients with gi2. thus, fibrin - specific regimens should be used in patients with gi3 if there is no possibility for ppci. however, there is no study investigating relation between gi and the effect of fibrin - specific regimens. our study is unique in the literature that compares fibrin and non - fibrin - specific drugs according to gi. the purpose of the study was to evaluate whether the success of reperfusion can be predicted by gi and to consider the effects of tt according to types of tt, gi, and infarct localization. in the current study, we enrolled 229 consecutive patients admitted with the diagnosis of ami and who received tt. patients admitted within 12 hours after the onset of symptoms and chest pain lasting at least 20 minutes with st - segment elevation of at least 0.2 mv in 2 or more adjacent leads on admission ecg were included. the exclusion criteria were isolated posterior ami, left bundle branch block, killip class 2 and 3 at admission, presence of any contraindication for tt, and prior ami. all patients received either t - pa as fibrin - specific or skz as non - fibrin - specific regimen for treatment of stemi. t - pa was administered with a loading dose of 15 mg, then a maintenance dose of 50 mg (0.75 mg / kg not exceeding 50 mg) over 30 minutes and 35 mg (0.50 mg / kg not exceeding 35 mg) over the next 60 minutes. patients were dived into gi2 or gi3 groups according to the grade of ischemia, and as anterior or non - anterior according to the infarct location on baseline ecg and their combinations as gi2 anterior, gi2 non - anterior, gi3 anterior, and gi3 non - anterior. rates of successful reperfusion in the groups and their relations with gi and infarct localization were evaluated. if there was 50% or more st segment resolution at maximally elevated st segment, it was accepted as successful reperfusion. if there was less than 50% st segment resolution, it was accepted as failed reperfusion and the patient was taken into the cardiac catheterization laboratory for rescue percutaneous coronary intervention. the baseline ecgs were performed before tt were analyzed and classified as gi2 or gi3. in ecgs with gi3, qrs configuration was deformed either lack of an s wave in > 2 leads that have a terminal s wave (usually v1 to v3), or j point amplitude > 50% of the r wave amplitude in 2 of all other leads. baseline characteristics of the patients with gi2 and gi3 were compared by the chi - square test for categorical variables. variables included in the regression analysis were : gi, localization, age, kind of tt, preconditioning, and time of onset of symptoms. the baseline ecgs were performed before tt were analyzed and classified as gi2 or gi3. in ecgs with gi3, qrs configuration was deformed either lack of an s wave in > 2 leads that have a terminal s wave (usually v1 to v3), or j point amplitude > 50% of the r wave amplitude in 2 of all other leads. baseline characteristics of the patients with gi2 and gi3 were compared by the chi - square test for categorical variables. variables included in the regression analysis were : gi, localization, age, kind of tt, preconditioning, and time of onset of symptoms. nearly 89.1% of 229 patients with stemi were admitted within the first 4 hours of symptom onset, and 9.2% of patients were admitted at between 4 to 6 hours of symptom onset. the remaining 1.7% of patients were admitted at more than 6 hours of symptom onset. there was no relationship between the gi and gender, hypertension, hyperlipidemia, diabetes, family history, or previous coronary artery disease. rate of successful reperfusion with tt was found to be significantly higher in patients with gi2 than in patients with gi3 (82.4% vs. 64.4%, respectively, p=0.002) (table 2). therefore, gi3 was observed as having negative predictive value for reperfusion (p=0.042) (table 3). according to the infarct localization, higher rates of successful reperfusion with non - anterior than anterior localization were observed (anterior 67.9% & non - anterior 79.8%, p=0.043). patients with gi3 who received the t - pa regimen significantly more than gi2 patients (p=0.022). however, there was no difference between t - pa and skz in successful reperfusion (t - pa 75.2% and skz 73.5%, p=0.77). t - pa achieved success in 80.8% of gi2 patients (p=0.2). while 56.2% of gi3 patients had successful reperfusion by skz, 66.6% of gi3 patients had successful reperfusion by t - pa (p=0.5). there was no significant difference between skz and t - pa in terms of infarct localization according to the success of reperfusion (57.6% by skz and 71.2% by t - pa in anterior [p=0.19 ] and 82.5% by skz & 76.6% by t - pa in non - anterior [p=0.41 ]). the rates of successful reperfusion were significantly higher for patients treated with t - pa than patients treated with skz in the gi3 anterior group (t - pa 63.6% and skz 30%, p=0.061) (table 3). it was observed that the ejection fraction of patients with gi3 was significantly lower than patients with gi2 (gi2 437% and gi3 416.2%, p=0.039). the relationship between successful reperfusion and gender, hypertension, hyperlipidemia, smoking, diabetes, family history, and previous coronary artery disease was not statistically significant. independent predictors of reperfusion by multivariate analysis were gi (p=0.006), infarct localization (p=0.042), preconditioning (p=0.058), and symptom onset (p=0.06) (table 4). it is now widely accepted that for patients with stemi, ppci is the preferred reperfusion strategy if it can be delivered in a timely fashion. although, thrombolytic regimens may be perceived as an old - fashioned treatment of ami, they are still used widely. in fact, most stemi patients present to hospitals without ppci capability and require transfer to pci - capable hospitals. however, after adding pharmaco - invasive therapy for ami to current guidelines, tt has begun to be discussed again. non - fibrin - specific regimens are cheaper than fibrin - specific regimens and their efficacy has been established. thus, non - fibrin - specific regimens like skz are used more than other regimens. however, non - fibrin - specific regimens might be inadequate for some patients, especially those in high - risk groups. the purpose of our study was to identify patients at high risk for failed reperfusion while non - fibrin - specific regimens were administered for treatment of ami. in our study, there was no significant difference in baseline characteristics, except for smoking, between gi3 and gi2. the incidence of hypertension, dyslipidemia, diabetes mellitus, positive family history, previous coronary artery disease, and prior angina did not differ between the groups. in contrast to previous studies [1113,24 ], more patients with gi3 were current smokers. the sub - study from the gusto1 trial indicated that ami develops in smokers at earlier periods of coronary disease without any significant coronary lesion and thus the impact of tt is better in smokers than non - smokers. the current study shows that patients with gi3 and anterior mi localization had less benefit from tt. when we evaluated the rate of successful reperfusion, no significant difference was observed between t - pa and skz in gi3 patients. although t - pa reperfused infarct - related arteries faster than non - fibrin - specific regimens, surprisingly, it was not more successful than skz in our study except in anterior gi3 patients. also, danami2 and gusto 2b sub - studies demonstrated that there was no difference in mortality between fibrin - specific regimens and primary pci in gi3 patients. birnbaum. assessed final infarct size (using predischarge selvester score) by 3 electrocardiographic variables in 267 patients with first anterior wall ami undergoing tt or not. they found that the presence of distortion of the terminal portion of qrs (gi3) on admission ecg were associated with final infarct size. moreover, although tt reduced infarct size (by selvester score) in gi2 anterior patients, tt did not reduce infarct size in gi3 anterior patients. in the retrospective analysis of the gusto2b angioplasty sub - study, it was found that gi3 on admission was associated with higher in - hospital mortality and reinfarction and a trend towards a higher mortality rate within 30 days. the mortality among the gi3 patients was comparable between those treated with ppci and tt. similarly, there was no difference in mortality between ppci and tt among the gi2 patients. the danami2 sub - group study showed that the gi3 on admission ecg in patients with ami is an independent predictor of mortality, regardless of kind of reperfusion treatment. in addition, patients with gi3 achieved the most benefit from ppci if they were treated within 3 hours of symptoms onset. in our study, we found that gi3 and anterior localization are the strongest predictors of failed thrombolysis. therefore, our study supported findings of previous studies showing that gi3 is associated with a worse prognosis, larger infarct size, and less benefit from tt [1118 ]. so, ppci should be the first - choice therapy for patients with gi3 and anterior localization. however, if there is no possibility of ppci or transfer to a pci - capable hospital, fibrin - specific thrombolytics would be preferred. the patients with both gi3 and anterior localization have the lowest chance for reperfusion with thrombolytic regimens ; 55.8% of these patients were successfully reperfused with tt. moreover, non - fibrin - specific regimens are the worst choice for treatment of gi3 anterior patients. while skz achieved successful reperfusion in only 30% of these patients, t - pa achieved successful reperfusion in 63.6%. treatment of ami should be individualized and prompt identification of high - risk criteria at admission has crucial importance. in the current study, we found that gi2 is an independent predictor for successful reperfusion in stemi treated with tt. patients with anterior and gi3 on presenting ecg are accepted as having high risk and fibrin - specific thrombolytics should be preferred for treatment. | backgroundthis study was aimed to determine whether the grade of ischemia can predict the success of reperfusion in patients treated with thrombolytic therapy (tt) for st elevation myocardial infarction (stemi).material / methodswe enrolled 229 consecutive patients with diagnosis of stemi and receiving tt. patients were divided into 2 groups grade 2 ischemia (gi2) and grade 3 ischemia (gi3) according to initial electrocardiogram (ecg). as tt, fibrin - specific (tissue plasminogen activator (t - pa)) or non - fibrin - specific (streptokinase (skz)) regimens were used. successful reperfusion was defined as > 50% resolution of the maximal st segment on 90-min ecg. we tried to evaluate whether the grade of ischemia could predict the success of reperfusion and if there were any differences in terms of successful reperfusion between different thrombolytic regimens.resultsthe successful reperfusion rate was significantly higher in gi2 than gi3 (82.4% vs. 64.4% respectively, p=0.002). the success rate was lowest at anterior gi3 (55.8%). although there was no significant difference between thrombolytic regimens in all groups (p=0.77), t - pa was superior to skz in anterior gi3 (63,6% vs. 30%, p=0.061). in addition, in multivariate analysis, gi and infarct localization were found as independent predictors for successful reperfusion with tt (p=0.006 and p=0.042, respectively).conclusionsin the current study, we found that gi2 is an independent predictor for successful reperfusion in stemi treated with tt. fibrin specific regime should be preferred in anterior gi3. |
endodontic irrigants should have, amongst other properties, a broad antimicrobial spectrum of activity against anaerobic and facultative micro - organisms growing in biofilms and a relative absence of toxicity against periapical tissues and oral mucosa. sodium hypochlorite (naocl) is recommended as the main root canal irrigant because of its broad antimicrobial activity, the capacity to prevent formation of and dissolve the smear layer, in association with chelating agents, and its ability to dissolve tissue remnants. however, naocl has been shown to have a cytotoxic effect on vital tissue and can therefore elicit inflammatory reactions if it reaches the periapex. furthermore, naocl has been shown to cause a change in the force required to fracture dentin, and a reduction of the elastic modulus and flexural strength of dentin. furthermore naocl corrodes protaper niti rotary (dentsply / maillefer, baillagues, switzerland) and carbon steel instruments, cause early fractures of protaper (dentsply / maillefer) instruments and, when heated, reduce resistance to cyclic fatigue of nickel - titanium files. sterilox 's aquatine alpha electrolyte (optident dental, ilkley, west yorkshire, uk) is a super - oxidized water that consists of a mixture of oxidizing substances including hypochlorous acid (hocl) at a concentration of 144 mg / l, with a ph of 5.0 - 6.5 and a redox potential of > 950 mv20. the manufacturer suggests that the production of hocl in the sterilox dental system (optident dental) does not produce free radical cl and that the available free chlorine in the solution is 200 ppm, that is larger than the concentration reported in literature. super - oxidized water has been suggested as an alternative to naocl, as it provides efficient cleaning of root canal walls, and has been recommended for the disinfection of endoscopes, dental unit water lines and dental impression materials. the aim of this study was to compare the antimicrobial action against enterococcus faecalis of naocl, optident sterilox electrolyte solution (optident dental) and sterilox 's aquatine alpha electrolyte when used as irrigating solutions in an e. faecalis infected bovine root canal model. bovine incisors were used throughout this study. the study exerted no influence on the animal 's fate at any stage as they were previously slaughtered in an italian slaughterhouse for commercial purposes. the apical 5 mm and the crown of each incisor were dissected and the remaining root was cut into 1 cm slices with a diamond disc (abrasive technology inc, westerville, oh, usa). subsequently the canal lumen was widened to a minimal diameter of 1.4 mm using the parapost xp endodontic post system drills (coltene / whaledent, konstanz, germany). finally the smear layer was removed via copious irrigation in an edta solution (smear clear, sybronendo, scafati, italy) (4 min) and naocl (teepol bleach, teepol, orpington, uk) (4 min) in an ultrasonic bath. fifteen roots were placed individually in 10 ml of brain heart infusion (bhi) broth (oxoid, basingstoke, uk) and autoclaved. these were left to cool to room temperature and then incubated overnight at 37c to verify the sterility of the samples. the bhi broths containing the roots were inoculated with 100 l of an overnight culture of e. faecalis jh2 - 2 and incubated for 10 days at 37c to allow for bacterial growth, infiltration of the dentin tubules and e. faecalis jh2 - 2 biofilm formation. the roots were divided into 3 groups, according to the irrigant used : group 1 was irrigated with the optident sterilox electrolyte solution (this is essentially saline and was used as our negative control), group 2 was irrigated with 4% naocl (teepol bleach) and group 3 was irrigated with freshly prepared sterilox 's aquatine alpha electrolyte solution. after sealing the apical portion with autoclaved physiowax (ra lamb ltd, eastbourne, uk), 5 cc of the selected irrigant was dispensed using a 27 gauge monoject syringe (kendall, tyco, mansfield, ma, usa) in an up - and - down motion, and left in situ for 3 min. following the removal of the apical seal to allow for the irrigation solution to drain, the coronal 5 mm portion of the specimen was sampled by grinding dentin and canal contents using parapost xp endodontic post system drill (coltene / whaledent) with a diameter of 1.5 mm. debris collected in the flutes of each drill was placed in a 1.5 ml microcentrifuge tube containing 1 ml bhi broth. after vortexing for 10 sec, a serial dilution of the debris containing bhi broth was made and 100 l of neat, 10, 10 and 10 dilutions were plated in duplicate onto fresh bhi agar plates and incubated overnight at 37c. to confirm the morphology and gram group of the bacterial cells, if the carry - over of naocl could prevent the growth of cells in the broths an additional experiment was carried out on sterile bovine teeth. these had been treated the same as the teeth used above except that they had not been inoculated with e. faecalis. after irrigation with naocl, the debris from the drill flutes were put into 900 l of bhi and 100 l of stationary phase e. faecalis culture was added. in addition, one group of teeth was irrigated with sterile water to provide a negative control. colony - forming units (cfu) with too many to count (tmtc) (defined here as > 800 cfu per agar plate) were attributed the highest rank in a non - parametric approach with anova on ranks with a duncan post - hoc. the kruskal - wallis test was used to compare the three groups involved followed by mann - whitney 's test as a post - hoc procedure adjusted with bonferroni correction for multiple comparisons. data were analyzed using spss software v. 15.0 for windows (spss inc., the overnight incubation of the sectioned bovine root canals resulted in no growth in any of the samples. this indicates that all the root sections were sterile at the start of the experiment. gram staining of a number of the resulting colonies showed the presence of gram - positive cocci, consistent with the e. faecalis inoculum. results of the dilution series are presented in table 1, while statistical analysis of the raw data for neat and 10 dilution is presented in figure 1. each group contained 5 roots and serial dilutions and plating were carried out in duplicate. whilst naocl (group 2) was the only irrigant to eradicate the e. faecalis a significant difference was seen between the optident sterilox electrolyte solution (group 1) and the sterilox 's aquatine alpha electrolyte irrigation (group 3) tmtc = too many to count (> 800 cfu per plate). mean number of colony - forming units recovered from debris after dentine grinding and root canal irrigation with naocl (a) - sterilox 's aquatine alpha electrolyte (b) - or optident sterilox electrolyte solution (c) - 1a ; neat broth (no dilution). the vertical dashed line shows significant difference between the groups on either side (p<0.05). naocl was the only irrigant to eliminate all bacteria and was significantly better at killing e. faecalis than both the optident sterilox electrolyte solution (saline) and sterilox 's aquatine alpha electrolyte. additionally sterilox 's aquatine alpha electrolyte solution was superior to optident sterilox electrolyte solution. the experiment to determine the effect of carry - over of naocl resulted in similar colony counts for the samples from teeth irrigated with naocl and water (results not shown) indicating that carry over of naocl had no noticeable effect in our experiment. this study evaluated the antimicrobial action of sterilox 's aquatine alpha electrolyte, a commercially available super - oxidized water in the united kingdom, in bovine root canals. e. faecalis was selected as the test species, because it is commonly detected in asymptomatic, persistent root canal infections. the bovine root model was chosen as it is clinically relevant, although the large root canal preparation size allows for more favorable dynamics of irrigation for the solution tested than is likely to occur in vivo. additionally the number of bacteria present is likely to be artificially high compared to the in vivo situation. despite these limitations, the ex vivo model has been successfully used previously to test the ability of e. faecalis to survive diverse root canal irrigations. our study suggested that the protocol followed was either able to prevent carry - over of the antimicrobial effect of naocl onto the bhi plates, possibly due to drainage of the solutions after irrigation or any carry over had no effect on the viability of the organisms, possibly due to the immediate dilution of the samples in the bhi broth. furthermore, based on pilot studies, it was decided to collect samples at a single depth as no difference was found between different depths of sampling when naocl was used as irrigant ; this is consistent with the results form other investigators on a similar bovine tooth model. sterilox 's aquatine alpha electrolyte is obtained by passing a sodium chloride solution (optident sterilox electrolyte solution) over coated titanium electrodes at 9 amps in a specifically made device (optident sterilox dental generator ; optident dental). optident sterilox electrolyte solution (non - activated) was used as the negative control as we did not expect any antimicrobial action from this irrigant. naocl was tested, because it is largely recommended as the main root canal irrigant. endodontic literature suggests that infection of the root canal at the time of obturation has a negative influence on the prognosis of endodontic treatment ; naocl was the only irrigant tested which was consistently associated with negative cultures in our study. however, sterilox 's aquatine alpha electrolyte might be able to reduce the bacterial load to levels that could influence treatment outcome. these results are not consistent with those of a previous study where different irrigants, including naocl, where tested against e. faecalis in a bovine tooth model, in fact krause,. (2007) suggest that 5.25% naocl was not able to render the dentinal shavings obtained sterile, it was however, significantly more effective than the other solutions tested. the major difference between the models is the difference in volumes of irrigation used ; 60 l twice against 5 cc in our study, therefore suggesting a role for the amount of irrigant used on the ability to eliminate root canal infection in the bovine root model ; in the same way a previous investigation indicates that the volume of irrigation has a significant influence in removing a bio - molecular film from root canal walls. the disinfecting actions of super - oxidized water are heavily reduced in the presence of organic contamination. the model used in our study allows for a greater bacterial growth than one might expect in an in vivo situation. it further excludes the mechanical aspect of root canal preparation, so that the bacterial biomass present in the root canal is likely to be greater than in normal clinical conditions. consequently, its elimination will depend exclusively on the flushing and chemical effects of the irrigation solution tested. the importance of instrumentation in obtaining a significant reduction in bacterial content has been shown. therefore we hypothesize that, in the presence of a reduced bacterial load, as a result of a chemo - mechanical preparation super - oxidized water irrigation might have the ability to eradicate a more clinically relevant root canal infection. the result of a previous study showed that super - oxidized water had no ability to prevent the growth of e. faecalis using paper disks as the delivery method on petri dishes, a protocol more favorable to the irrigant when compared to the bovine root model due to long time of contact with the micro - organisms, absence of interaction with dentin and cells in a metabolically active phase, therefore more susceptible to antimicrobials. nonetheless, a different irrigation source was tested (dermacyn, oculus innovative sciences, petaluma, ca, usa). different super - oxidized waters are produced by a similar electrolysis process but, due to a difference in the active concentration and the ph of the final solution, the product can have a different anti - microbial activity. one of the suggested advantages of super - oxidized water, when compared to naocl, is its level of toxicity. it is worth noting, that the mechanism of action of super - oxidized water involves oxidative damage which might cause ageing and irreversible dysfunctions that eventually produce cellular death. a ph - neutral super - oxidized solution (microcyn ; dermacyn, oculus innovative sciences, petaluma, ca, usa) has been tested. it was found to be significantly less cytotoxic than antiseptic hydrogen peroxide concentrations (used as a positive control for oxidative damage) because it does not induce genotoxicity or accelerated ageing in vitro. however, microcyn has a different ph than sterilox 's aquatine alpha electrolyte and this needs to be taken into account when comparing the two irrigants. under the conditions of this study sterilox 's aquatine alpha electrolyte appeared to have significantly more antimicrobial action when used as an irrigant in the root canal system compared to the non - activated optident sterilox electrolyte solution, but naocl was the only irrigant able to eliminate all bacteria in our experiments. sterilox 's aquatine alpha electrolyte caused a bacterial load decrease although being less effective than naocl. | ideally root canal irrigants should have, amongst other properties, antimicrobial action associated with a lack of toxicity against periapical tissues. sodium hypochlorite (naocl) is a widely used root canal irrigant, however it has been shown to have a cytotoxic effect on vital tissue and therefore it is prudent to investigate alternative irrigants. sterilox 's aquatine alpha electrolyte belongs to the group of the super - oxidized waters ; it consists of a mixture of oxidizing substances, and has been suggested to be used as root canal irrigant. super - oxidized waters have been shown to provide efficient cleaning of root canal walls, and have been proposed to be used for the disinfection of medical equipment.objectiveto compare the antimicrobial action against enterococcus faecalis of naocl, optident sterilox electrolyte solution and sterilox 's aquatine alpha electrolyte when used as irrigating solutions in a bovine root canal model.methodologyroot sections were prepared and inoculated with e. faecalis jh2 - 2. after 10 days of incubation the root canals were irrigated using one of three solutions (naocl, optident sterilox electrolyte solution and sterilox 's aquatine alpha electrolyte) and subsequently sampled by grinding dentin using drills. the debris was placed in bhi broth and dilutions were plated onto fresh agar plates to quantify growth.resultssodium hypochlorite was the only irrigant to eliminate all bacteria. when the dilutions were made, although naocl was still statistically superior, sterilox 's aquatine alpha electrolyte solution was superior to optident sterilox electrolyte solution.conclusionunder the conditions of this study sterilox 's aquatine alpha electrolyte appeared to have significantly more antimicrobial action compared to the optident sterilox electrolyte solution alone, however naocl was the only solution able to consistently eradicate e. faecalis in the model. |
the chemical and physical properties of serpentine and amphibole asbestos are considered in the context of their interaction with tissue of the tracheobronchial tree and lungs. in vitro studies in cultures of several types are evaluated and work with the erythrocyte hemolysis system is reviewed. although fibers of the two major mineral types differ substantially, it is likely they are modified by secretions and membranes of cells after inhalation to the respiratory tract. investigations using virgin asbestos might not provide an accurate picture of events in vitro.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6. |
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pediatric cerebral aneurysms are rare. according to a recent review by rao., these aneurysms constitute 2.9% of all cerebral aneurysms. incidence of these aneurysms in pediatric patients is more in the posterior circulation than in the anterior, when compared to the aneurysms in adults. lasjunias., in a report on 20 pediatric aneurysm cases, suggested that these aneurysms must be the expression of various vessel wall dysfunctions, producing transient or permanent failure to repair a partial insult. we present two case reports of non - giant aneurysms of the anterior circulation in the pediatric group, treated successfully by endovascular technique at our institute. an 8-year - old female child presented with a complaint of persistent headache for 7 days (grade i sub - arachnoid hemorrhage (sah) (hunt and hess scale)). on clinical examination computerized tomography (ct) scan revealed fischer grade-3 sah in the prepontine cistern and right sylvian fissure. pre - procedure routine detailed work was done along with special investigations including antineutrophilic antibody (ana), antineutrophilic cytoplasmic antibody (anca), and antiphospholipid antibody (apla). detailed screening and imaging were done to rule out any associated conditions, such as autosomal dominant polycystic kidney disease (adpkd), fibro muscular dysplasia, co - arctation of aorta, ehlers syndrome, and marfans syndrome. digital subtraction angiography (dsa) showed the presence of a1saccular aneurysm on the right side [figure 1 ab ]. endovascular treatment was planned after analyzing the morphology of the aneurysm and the flow hemodynamics of the aneurysm. high - risk informed consent was obtained from the parents after detailed explanation of the risks versus benefits of the treatment procedure. case 1 : (a) computerized tomography (ct) scan shows subarachnoid hemorrhage (sah) (arrow) in the prepontine and sylvian fissures and (b) pre - procedure digital subtraction angiogram reveals saccular a1 aneurysm (arrow) on the right side under general anesthesia, right 5f common femoral was accessed. standard heparinization (100 units / kg as bolus at the beginning of the procedure followed by 20 units / kg every hour) was given to maintain the activated clotting time of 250 s. guiding catheter (5f envoy, cordis) with nimodipine infusion was placed in the internal carotid artery (ica). microcatheter (echelon-10, ev3) over microwire (expedion 14, ev3) was navigated into the sac of the aneurysm [figure 1 c, d, and e ]. post - procedure angiogram showed near total exclusion of the aneurysm from the circulation and excellent filling of all branches of the right ica, anterior cerebral artery, and middle cerebral artery. postprocedure, the patient was extubated in the cathlab and kept under observation for 3 days in the neurointensive care unit. there were no intra-, peri-, or post - procedural complications and the child was discharged on day 7 post operation. control angiogram done after 2 years showed stable occlusion of the aneurysm with coil mass in situ. (c) microcatheter is shown positioned in the sac of the aneurysm (arrow) and (d) the post - procedure angiogram shows exclusion of the aneurysm (arrow) from the circulation with (e) the coil mass in the aneurysm (arrow) a 11-year - old male child presented with 2 episodes of generalized seizures and severe headache for 2 days. on clinical examination, there was no focal neurological deficit (grade i sah - hunt and hess scale). ct scan [figures 2a ] showed hemorrhage in the left frontal lobe with intraventricular extension (fischer grade iv). dsa was done subsequently which revealed a bilobed ica bifurcation aneurysm on the left side [figure 2b ]. endovascular treatment (balloon - assisted coiling) was planned after discussion with the team of neurosurgeon and neurointensivist. high - risk informed consent was obtained after detailed explanation of the risks versus benefits to the parents. case 2 : (a) computerized tomography (ct) scan shows bleeding in the left frontal region with intraventricular extension. (b) pre - procedural angiogram reveals internal carotid artery (ica) bifurcation aneurysm on the left side. under general anesthesia, standard heparinization (100 units / kg as bolus at the beginning of the procedure followed by 20 units / kg every hour) was given to maintain the activated clotting time of 250 s. guiding catheter (6f envoy, cordis) with nimodipine infusion was placed in the left ica. hyper glide occlusion balloon (3 mm 10 mm, ev3) was placed across the neck of the aneurysm. microcatheter (echelon-10, ev3) over microwire (neuroscout-10, codman) was navigated into the sac of the aneurysm. subsequently, we started packing the aneurysm with the platinum coils using balloon remodeling technique. after placement of the second coil and balloon deflation, a small loop of a previously detached coil was seen projecting into the parent vessel [figure 2 c f ] on the check angiogram. (c) magnified image shows the coil loop in the internal carotid artery (ica) (arrow) (d) enterprise stent(white arrows) deployed from m1 to supraclinoid ica with secured coil mass (black arrow) in the aneurysm sac. (e) postprocedure angiogram shows complete exclusion of aneurysm (arrow) from circulation. (f) follow - up angiogram reveals stable occlusion of the aneurysm in order to secure the aneurysm and prevent untoward effects of the prolapsed coil loop into the parent vessel, we deployed an 4 mm 22 mm enterprise self - expanding stent (codman, johnson and johnson) across the neck of the aneurysm from the right supraclinoid ica to m1. the patient was loaded with antiplatelet drugs before stent deployement through the nasogastric tube (clopidogrel, 225 mg and aspirin, 150 mg) and was also started on infusion of tirofiban which was continued for 4 hours (till the effect of oral antiplatelet agents took over). there were no peri- or post - procedural complications and the patient was discharged on day 7 after operation on double antiplatelet regimen (aspirin,50 mg and clopidogrel,75 mg). clinical follow - up of the child after 1 month showed normal results. on the 6-month follow - up, neurological examination was normal and the patient was continued on only aspirin, 50 mg. follow - up angiogram done after 1 year showed stable occlusion of the aneurysm and normal patency of the stented artery. an 8-year - old female child presented with a complaint of persistent headache for 7 days (grade i sub - arachnoid hemorrhage (sah) (hunt and hess scale)). on clinical examination computerized tomography (ct) scan revealed fischer grade-3 sah in the prepontine cistern and right sylvian fissure. pre - procedure routine detailed work was done along with special investigations including antineutrophilic antibody (ana), antineutrophilic cytoplasmic antibody (anca), and antiphospholipid antibody (apla). detailed screening and imaging were done to rule out any associated conditions, such as autosomal dominant polycystic kidney disease (adpkd), fibro muscular dysplasia, co - arctation of aorta, ehlers syndrome, and marfans syndrome. digital subtraction angiography (dsa) showed the presence of a1saccular aneurysm on the right side [figure 1 ab ]. endovascular treatment was planned after analyzing the morphology of the aneurysm and the flow hemodynamics of the aneurysm. high - risk informed consent was obtained from the parents after detailed explanation of the risks versus benefits of the treatment procedure. case 1 : (a) computerized tomography (ct) scan shows subarachnoid hemorrhage (sah) (arrow) in the prepontine and sylvian fissures and (b) pre - procedure digital subtraction angiogram reveals saccular a1 aneurysm (arrow) on the right side under general anesthesia, right 5f common femoral was accessed. standard heparinization (100 units / kg as bolus at the beginning of the procedure followed by 20 units / kg every hour) was given to maintain the activated clotting time of 250 s. guiding catheter (5f envoy, cordis) with nimodipine infusion was placed in the internal carotid artery (ica). microcatheter (echelon-10, ev3) over microwire (expedion 14, ev3) was navigated into the sac of the aneurysm [figure 1 c, d, and e ]. post - procedure angiogram showed near total exclusion of the aneurysm from the circulation and excellent filling of all branches of the right ica, anterior cerebral artery, and middle cerebral artery. postprocedure, the patient was extubated in the cathlab and kept under observation for 3 days in the neurointensive care unit. there were no intra-, peri-, or post - procedural complications and the child was discharged on day 7 post operation. control angiogram done after 2 years showed stable occlusion of the aneurysm with coil mass in situ. (c) microcatheter is shown positioned in the sac of the aneurysm (arrow) and (d) the post - procedure angiogram shows exclusion of the aneurysm (arrow) from the circulation with (e) the coil mass in the aneurysm (arrow) a 11-year - old male child presented with 2 episodes of generalized seizures and severe headache for 2 days. on clinical examination, there was no focal neurological deficit (grade i sah - hunt and hess scale). ct scan [figures 2a ] showed hemorrhage in the left frontal lobe with intraventricular extension (fischer grade iv). dsa was done subsequently which revealed a bilobed ica bifurcation aneurysm on the left side [figure 2b ]. endovascular treatment (balloon - assisted coiling) was planned after discussion with the team of neurosurgeon and neurointensivist. high - risk informed consent was obtained after detailed explanation of the risks versus benefits to the parents. case 2 : (a) computerized tomography (ct) scan shows bleeding in the left frontal region with intraventricular extension. (b) pre - procedural angiogram reveals internal carotid artery (ica) bifurcation aneurysm on the left side. under general anesthesia, standard heparinization (100 units / kg as bolus at the beginning of the procedure followed by 20 units / kg every hour) was given to maintain the activated clotting time of 250 s. guiding catheter (6f envoy, cordis) with nimodipine infusion was placed in the left ica. hyper glide occlusion balloon (3 mm 10 mm, ev3) was placed across the neck of the aneurysm. microcatheter (echelon-10, ev3) over microwire (neuroscout-10, codman) was navigated into the sac of the aneurysm. subsequently, we started packing the aneurysm with the platinum coils using balloon remodeling technique. after placement of the second coil and balloon deflation, a small loop of a previously detached coil was seen projecting into the parent vessel [figure 2 c f ] on the check angiogram. (c) magnified image shows the coil loop in the internal carotid artery (ica) (arrow) (d) enterprise stent(white arrows) deployed from m1 to supraclinoid ica with secured coil mass (black arrow) in the aneurysm sac. (e) postprocedure angiogram shows complete exclusion of aneurysm (arrow) from circulation. (f) follow - up angiogram reveals stable occlusion of the aneurysm in order to secure the aneurysm and prevent untoward effects of the prolapsed coil loop into the parent vessel, we deployed an 4 mm 22 mm enterprise self - expanding stent (codman, johnson and johnson) across the neck of the aneurysm from the right supraclinoid ica to m1. the patient was loaded with antiplatelet drugs before stent deployement through the nasogastric tube (clopidogrel, 225 mg and aspirin, 150 mg) and was also started on infusion of tirofiban which was continued for 4 hours (till the effect of oral antiplatelet agents took over). there were no peri- or post - procedural complications and the patient was discharged on day 7 after operation on double antiplatelet regimen (aspirin,50 mg and clopidogrel,75 mg). clinical follow - up of the child after 1 month showed normal results. on the 6-month follow - up, neurological examination was normal and the patient was continued on only aspirin, 50 mg. follow - up angiogram done after 1 year showed stable occlusion of the aneurysm and normal patency of the stented artery. pediatric aneurysms constitute 2.9% of all cerebral aneurysms with common location being the posterior circulation and with higher incidence of large and giant complex aneurysm. the ica bifurcation is the commonest location in the anterior circulation. during the past decade, there has been dramatic improvement in endovascular techniques that are increasingly being used in treatment of pediatric age group patients. large and giant aneurysms are more common in the pediatric age group. both our cases were saccular aneurysms in the anterior circulation with grade i sah on presentation. xianli., in their series, reported the use of coils in all saccular aneurysms. in case 1, coiling of the sacculara1 aneurysm was done with dense packing and exclusion of the aneurysm from the circulation. liang., in their series of 24 pediatric patients, concluded that this age group differed in many ways from adults. both microsurgical approaches and endovascular treatment were effective in these cases. in two patients with fusiform giant aneurysms of their series, they used balloon - mounted stents to treat the aneurysm and one of the patients with basilar trunk aneurysm succumbed to bleeding on the 23 day post operation. in one patient in the series, a 10-year - old female child with basilar apexsaccular aneurysm, stent (neuroform, boston scientific) assisted coiling was done with successful outcome (glasgow outcome scale = 5). in case 2, we used a self - expanding stent as a bail out measure (enterprise, johnson and johnson) to secure the neck of the aneurysm and to achieve dense packing of the aneurysm with coils, we did not encounter any untoward effects. however, a close long - term follow - up has been planned for this patient to look for any long - term adverse effects of the stent, if any. pediatric cerebral aneurysms are rare with varying clinical presentation and natural history compared to those encountered in adults. endovascular method is an elegant and minimally invasive alternative treatment of choice in managing these aneurysms and has promising results. however, a larger case series is required to better evaluate the safety and efficiency of the treatment method. | aneurysms in the pediatric age group are rare and have preponderance for the posterior circulation. these aneurysms are more commonly large, giant, and complex. we present two case reports of saccular aneurysms in pediatric patients who were treated successfully by endovascular technique. |
long chain polyunsaturated fatty acids (lcpufa) including docosahexaenoic acid (dha) and arachidonic acid (ara), are essential for normal growth, vision, neurodevelopment and overall health. in utero lcpufa accretion occurs primarily during the last trimester of pregnancy, when maternal levels are high and growth and brain development are rapid. premature infants born before this process is complete are relatively deficient in dha, the most variable of these essential lcpufas.(1) additionally, dha status in very low birth weight infants (vlbws) remains low due to inadequate fat stores, ineffective conversion from precursor fatty acids and a limited nutritional supply.(2) evidence demonstrates that lcpufa supplementation improves neurodevelopmental and visual outcomes in this high risk population.(315) new evidence is emerging to suggest that the benefits of dha supplementation extend beyond the brain. in vitro, animal model, and a few human studies demonstrate a role for improved lcpufa provision in prevention of diseases specific to premature infants, including bronchopulmonary dysplasia (bpd)(11, 1622), necrotizing enterocolitis (nec)(2327), and retinopathy of prematurity (rop).(2834) the purpose of this review article is to encourage further discussion about the recommended provision of dha specifically for premature infants and the need for further study of specific dose, timing, safety and benefits in this high risk population. essential lcpufas are important components of the phospholipid bi - layer of cell membranes, contributing to structural integrity and function throughout the body. in vitro and animal studies, they have highly specialized functional roles making them important for normal signal transduction, neurotransmission and neurogenesis. in tissues throughout the body, they are released from membranes by phospholipases for conversion to important hormones, eicosanoids, lipoxins and resolvins that mediate inflammation, immune function, platelet aggregation and lipid homeostasis. they also serve as local signaling molecules and transcription regulators of genes involved in inflammation, development and metabolism. their ubiquitous arrangement and multifaceted functionality make lcpufas extremely important for normal growth, development, and overall health. humans can synthesize saturated and monounsaturated fas but lack the enzymes required to synthesize omega-3 and omega-6 lcpufas de novo. dha and ara (22- and 20-carbon lcpufas, respectively) may be obtained directly through the diet oily fish for dha, meat and eggs for ara - or from their 18-carbon precursor fas, -linolenic acid (ala) and linoleic acid (la).(figure 1.) the most common essential fa found in the westernized diet is la, an omega-6 fa abundant in vegetable oils, nuts and seeds. ara is found throughout the body in phospholipid membranes and upon activation serves as a precursor to prostaglandins, thromboxane and leukotrienes. the nutritionally less abundant omega-3 fa precursor is ala, found in flaxseed, canola, walnuts and soy. ala can be converted to eicosapentaenoic acid (epa) and dha, but only in small amounts. these omega-3 lcpufas are rapidly and preferentially incorporated into cell membranes where they serve important functional and structural roles in the brain and retina and have anti - inflammatory and metabolic signaling functions in other tissues. an appropriate balance of these pathways is necessary for normal immune function and clotting, however, in excess leads to inflammation. the omega-6 and omega-3 fa families are not interchangeable, making intake from both groups essential. additionally, the conversion to ara and dha from their respective precursors is through the same rate limiting and inefficient desaturase enzyme in the liver. due to the pervasive lack of omega-3 in the typical western diet, there is an increasing dietary imbalance of omega-6 to omega-3 lcpufa which can induce a pro - inflammatory state, attributing to multiple disease states. in a dha deficit, the specialized phospholipid membranes in the retina and brain can become replaced with substitute fas altering function which may affect memory, attention and visual processing. unfortunately, a typical westernized diet contains an abundance of omega-6 with very little omega-3 fas, driving this imbalance bias. because dha can not be synthesized de novo, the developing fetus is dependent on a maternal source. most dha accumulation occurs during the third trimester of pregnancy when growth and brain development are rapid.(35) hormonal changes during pregnancy induce a hyperlipidemic state, increasing the availability of all circulating lipids ; estrogen further increases conversion of precursor ala to dha, sustaining preferential uptake. passive transport is directly dependent on maternal blood levels, while active transport occurs through fa transport proteins which are up - regulated during pregnancy to preferentially transport lcpufas to the fetal blood stream. indeed, preterm infants have lower dha levels than their term peers.(1) furthermore, in very preterm infants (0.32%) may be necessary to correct the relative deficiency and to optimize the benefits for vlbw infants. a relatively new strategy for increasing dha provision to premature infants is through its addition to commercialized human milk fortifiers used to increase calories, protein and mineral content of human milk and meet the needs of vlbw infants. until 34 to 35 weeks gestation, when babies develop a coordinated suck and swallow, they are typically given enteral feedings through a feeding tube. until that time, mothers pump and freeze their milk which is then thawed and fortified with human milk fortifier for feedings. with the growing focus on dha, some fortifiers have now added dha in an attempt to meet the needs of vlbw infants. this unique dosing method provides a higher daily dha dose, but still requires establishment of full enteral feedings and is not uniform due to widely variable dha levels in mother s milk that may be further altered by freezing and storing. to date, no dha supplementation study has attempted to normalize the dha status of preterm infants throughout the critical first weeks of postnatal development (i.e., achieve levels found in term babies). the route and dose provided through either infant formula or breast milk with fortifier relies on the variable ability of the infant s gastrointestinal system to handle full enteral feedings. many vlbws are not fed completely by enteral route for several weeks or longer, and routinely available intravenous lipids do not contain preformed omega-3 fas. due to these factors, the average accumulation of dha during the first month of life in a very preterm infant is roughly 50% of the expected in utero accretion.(2) new parenteral products are being developed to provide improved lcpufa balance by iv route until full enteral provision can be accomplished an alternative approach could be the direct enteral provision of dha, independent of diet. this potentially cost - effective method would allow early intervention even before the infant reaches full feedings or fortification. daily enteral dosing can be easily adjusted, is independent of the need for invasive intravenous access and may be continued beyond parenteral nutrition needs. although there may be benefits to either parenteral or enteral supplementation, careful evaluation of potential adverse consequences or unintended alterations to the balance of the omega-6 : omega-3 ratio will be required. vlbw infants rapidly become and remain dha deficient for an extended period of time due to ineffective conversion from precursor fatty acids, lower fat stores, and a limited nutritional provision of dha after birth. optimizing lcpufa provision postnatally may not only improve vision and neurodevelopment in vlbw infants, but may also reduce the morbidity and mortality from bpd, nec, and rop. | long chain polyunsaturated fatty acids (lcpufa) including docosahexaenoic acid (dha), are essential for normal vision and neurodevelopment. dha accretion in utero occurs primarily in the last trimester of pregnancy to support rapid growth and brain development. premature infants, born before this process is complete, are relatively deficient in this essential fatty acid. very low birth weight (vlbw) infants remain deficient for a long period of time due to ineffective conversion from precursor fatty acids, lower fat stores, and a limited nutritional provision of dha after birth. in addition to long- term visual and neurodevelopmental risks, vlbw infants have significant morbidity and mortality from diseases specific to premature birth, including bronchopulmonary dysplasia (bpd), necrotizing enterocolitis (nec), and retinopathy of prematurity (rop). there is increasing evidence that dha has protective benefits against these disease states. the aim of this article is to identify the unique needs of premature infants, review the current recommendations for lcpufa provision in infants, and discuss the caveats and innovative new ways to overcome the dha deficiency through postnatal supplementation, with the long term goal of improving morbidity and mortality in this at risk population. |
the laser radiation produces many different effects depending on light - beam parameters as well as the tissues that are subjected to irradiation. despite documented therapeutic effect of low - level laser therapy (lllt), there is still much controversy and scientific debate over its effectiveness [13 ]. the variety of procedures and the inconclusive results of the conducted studies mobilized to search for the parameters of laser radiation, which both in vitro and in vivo will result in acceleration of cell proliferation and the expected therapeutic efficacy. laser radiation may affect individual cells only via the structures that absorb the radiation. visible light photoreception (635 nm) occurs at the level of mitochondria [2, 4 ]. consequently, electron transport, adenosine triphosphate nitric oxide release, blood flow, reactive oxygen species increase, and diverse signaling pathways are activated. reports about this subject seem to confirm that the photoinducing electron transfer reactions may initiate the synthesis and conformational changes of proteins and may have an influence on dna and rna synthesis increase. laser therapy has a universal application in the wound healing, especially nonhealing decubitals and burned wounds, and also in the treatment of ulcer - dependent microcirculation disorders such as diabetic foot. despite the proven therapeutic effect, there are still controversies about the mechanisms and nature of the interaction of laser radiation on living tissue. the literature analysis about this problem (this topic) demonstrates that the conducted studies were focused on keratinocytes and fibroblasts involved in tissue healing and only in a little extent on endothelial cells., it plays a fundamental role in many pathological processes including hypertension, diabetes, or inflammatory process. it should be underlined that growth factors produced by endothelial cells (ecs) take a main part in tissue healing and angiogenesis process. wound healing is a multistep process and consists of these stages : primary hemostasis with platelets and secondary, leading to the production of fibrin, the contribution of inflammatory cells that migrate to the wound and cause inflammation, differentiation, proliferation, and migration of mesenchymal cells to the wound, angiogenesis, and reepitelialization. vascular endothelial growth factor (vegf) is one of the key regulators of angiogenesis. the family of this growth factor includes vegf - a, vegf - b, vegf - c, vegf - d, vegf - e, and pigf (eng. vegf - a occurs in several isoforms counting 121, 165, 189, and 206 amino acids. vegf induces its biological effects by binding with high - affinity receptors belonging to the family of tyrosine kinase receptors. two known receptors for vegf identified as vegfr-1 (also known as flt1) and vegfr-2 (also known as kdr) are located in the endothelial cells of blood vessels. vegfr-1 is responsible for the formation of capillary structures, while vegfr-2 is expressed on endothelial cells involved in angiogenesis and circulating endothelial progenitor cells derived from the bone marrow. the presence of soluble forms of receptors svegfr-1 and svegfr-2 with high affinity for vegf was noted. both of the receptors, especially svegfr-1, are able to inhibit vegf - induced mitogenesis and may be a physiological negative regulator of vegf. they reduce the availability of vegf and prevent overgrowth of endothelial cells which is also used in a cancer therapy. vegfr-2initiates endothelial cell proliferation, which shows limited potential growth in the absence of stimulation by this factor. vegfr-2 stimulation on mesodermal stem cells results in the transformation of these cells into endothelial cells. vegf transmits a signal through the vegfr-2 receptor, whose expression is increased on endothelial cells involved in angiogenesis and circulating endothelial progenitor cells derived from the bone marrow. inhibiting vegf or vegfr-2 leads to apoptosis of endothelial cells and decreasing diameter, density, and vascular permeability [12, 13 ]. it is believed that vegf plays a key role in postnatal, physiological (e.g., wound healing), and pathological (e.g., cancer, rheumatoid arthritis, proliferative retinopathy) angiogenesis. to date, only few studies are concerned with the influence of lllt on endothelial cell proliferation and vegf - a secretion [1517 ]. thus, the aim of this study was to assess the impact of low - level laser radiation (lllt) at a wavelength of 635 nm and 1,875 mw / cm power on vascular endothelial cells, secretion of vegf - a, and presence of svegfr-1 and svegfr-2 receptors. the aim of this study was to assess the impact of lllt at a wavelength of 635 nm and power density of 1,875 mw / cm on vascular endothelial cells proliferation in vitro and secretion of vegf - a and its receptors concentration. endothelial cells (human umbilical vein endothelial cell (huvec) line) were derived from human umbilical veins by the enzyme method using collagenase according to the method described by jaffe.. cells were cultured in m199 media supplemented with 20 % fetal bovine serum (fbs), 100 u / ml penicillin (gibco) and growth factors : 50 g / ml endothelial cell growth supplement (ecgs, biomedical technologies inc. the cells of each treatment group and the control group were cultured in the same culture medium, which was changed before each irradiation. the cells were incubated at 37 c in a humidified atmosphere with 5 % co2. after two to four passage and seeding of the cells in 6-well culture plates, cells come from three independent isolations. a semiconductor - based (gaalas) laser (roithner lasertechnik gmbh, austria) was used to generate visible laser beam with the wavelength of 635 nm. in this paper, the authors have used the optoelectronic set for controlled, reproducible exposure of electromagnetic radiation of biological structures in the spectral band of tissue transmission window 6001,000 nm. at the cell - layer level, the power density measured using a laser power meter (gentec, model solo2 r2, canada) was 1,875 mw / cm for 635 nm. the distance between the laser source and the surface of application was 10 cm, application was carried through an optical fiber ; the irradiated area was 80 cm. the experiment was conducted in four groups : i the control group (no radiation) ; ii the energy dose, 2 j / cm ; iii 4 j / cm ; and iv 8 j / cm. the cells were cultured for 6 days, with two radiations on the day nos. 2 and 4 with 1-day break. conditioned medium from each well of culture plates was centrifuged for 10 min at 2,000g and frozen at 86 c. after thawing concentration of vegf - a, svegfr-1 and svegfr-2 in the supernatant were measured by elisa test (r&d company) according to the manufacturer s instructions. the remaining cells on the bottom of each well were harvested using trypsin and counted by buerker hemocytometry. this method uses trypan blue dye according to the method described by basso.. the results of the concentration of the parameters in the supernatant of each well culture plates were analyzed per number of cells in the each well. statistical analysis was performed using statistical 9.1 for windows (stat soft inc.). the one - way anova with post hoc test was used for the assessment differences between the control group and other research groups. the approval of the bioethics commission of the ncu collegium medicum in bydgoszcz was obtained, no kb/135/2009, date 25.03.2009. figure 1 presents the effect of laser radiation with the wavelength of 635 nm (1,875 mw / cm) on number of endothelial cells depending on rising energy dose of radiation. energy doses of 2, 4, and 8 j / cm significantly increased number of cells (p = 0.0041, significant differences between groups : i vs. ii, iii, and iv). the highest value was observed at an energy dose of 2 and 4 j / cm and was about 23 % higher than in the control group. the concentrations of vegf - a and its soluble receptors svegfr-1 and svegfr-2 are presented in figs. 2, 3, and 4. the use of laser radiation of 635 nm (1,875 mw / cm) was associated with a statistically significant lower concentration of svegfr-1. the results of analysis of variance were as follows : vegf - a p = 0.0808, svegfr-1 p = 0.0197 (significant differences between groups i vs. ii, iii, and iv), and svegfr-2 p = 0.2340. 1number of huvecs, depending on lllt dose with the wavelength of 635 nm. asterisk above bars indicate significant differences vs. control group (p < 0.01)fig. 2concentration of vegf - a in the supernatant, depending on lllt dose with the wavelength of 635 nm (p = 0.08). 3concentration of svegfr-1 in the supernatant, depending on lllt dose with the wavelength of 635 nm. asterisk above bars indicate significant differences vs. control group (p < 0.01)fig. 4concentration of svegfr-2 in the supernatant, depending on lllt dose with the wavelength of 635 nm (p = 0.23). values are expressed as the mean sem number of huvecs, depending on lllt dose with the wavelength of 635 nm. asterisk above bars indicate significant differences vs. control group (p < 0.01) concentration of vegf - a in the supernatant, depending on lllt dose with the wavelength of 635 nm (p = 0.08). values are expressed as the mean sem concentration of svegfr-1 in the supernatant, depending on lllt dose with the wavelength of 635 nm. asterisk above bars indicate significant differences vs. control group (p < 0.01) concentration of svegfr-2 in the supernatant, depending on lllt dose with the wavelength of 635 nm (p = 0.23). vegf is the major regulator of angiogenesis ; thus in many studies, the attempt to explain the way of vegf action through vegfr-1 and vegfr-2 was undertaken. the soluble forms of receptors for vegf svegfr-1 and svegfr-2 are physiological inhibitors of ec proliferation. these receptors by binding with vegf inhibit their action to stimulate cell proliferation and angiogenesis [11, 12 ]. in literature, there are a few references about influence of lllt on vegf secretion and angiogenesis [21, 22 ]. the authors, examining the processes of tissue healing with the use of lllt, usually report the stimulation of fibroblast activity [23, 24 ]. silva and colleagues studied the expression of vegf mrna after irradiation lllt wounds at rats. using laser radiation with a wavelength at 780 nm, 70 mw power, and dose of 35 j / cm on the day of surgery and after 2 days (wavelength 660 nm, power 40 mw, dose of 5 j / cm) showed its effect on the increased vegf expression during the process of tissue healing. araujo and colleagues conducted a detailed study of wound healing in mice after irradiation with he - ne laser (632.8 nm) using the exposure parameters : 1 j / cm and exposure time 3 min. autoradiographic and immunohistochemical analysis performed after 15 days of irradiation demonstrated reduction of inflammation, acceleration of reepitelialization, fibroblasts activation, and increased amounts of collagen fibers in the area of exposure. the results were compared with wounds that were not treated with lllt. in our study, varied effects of laser radiation on cells and secretion of angiogenic factors may be due to the different levels of photoreceptive interaction. the use of laser radiation in the visible light conditions resulted in the reduction of vegf - a, svegfr-1, and svegfr-2 in the supernatant (figs. 2, 3, and 4) with simultaneous activation of endothelial cell proliferation (fig. 1). it is known that vegf action is determined by vegf binding with its membrane receptors. that causes receptors expenditure during the stimulation of endothelial cell division. in this way, the reduced amount of vegf - a in the supernatant under the influence of laser radiation may be explained. the lowest vegf - a concentration at doses 2 and 4 j / cm (= 635 nm) at simultaneously the highest level of cell proliferation suggests that in these conditions, the most molecules of vegf - a are connected with receptors that significantly affected the level of ec proliferation. lower concentrations of svegfr-1 and svegfr-2 in the supernatant as compared to the control group could contribute to the increase of cells proliferation by the influence of lllt. fewer vegf - a molecules are blocked by soluble forms of receptors, which are regulators of vegf [10, 11 ] (fig. this mechanism is used in cancer therapy, where the binding of vegf and inhibiting vegf native receptors suppress the new blood vessels creation, which reduces growth of the solid tumors and metastasis [11, 24 ]. in vitro studies conducted by schindl. confirmed the increase in huvec proliferation as a result of laser radiation at the wavelength range 670 nm and doses of 2, 4, 8 j / cm.fig. 5the hypothesis of the action of vegf - a and soluble receptors svegfr-1 and svegfr-2 in the supernatant. a the connection of vegf - a with vegfr-2 receptor located on the cell membrane contributes to endothelial cell proliferation. b competitive vegf - a binding with soluble forms of receptors svegfr-1 and svegfr-2 present in the supernatant leads to reducing endothelial proliferation the hypothesis of the action of vegf - a and soluble receptors svegfr-1 and svegfr-2 in the supernatant. a the connection of vegf - a with vegfr-2 receptor located on the cell membrane contributes to endothelial cell proliferation. b competitive vegf - a binding with soluble forms of receptors svegfr-1 and svegfr-2 present in the supernatant leads to reducing endothelial proliferation this research should be continued, so that the influence of low - level laser radiation on endothelial cell proliferation and secretion of angiogenic factors is fully explained. the possibility of angiogenesis modulation by application laser radiation may contribute to the improvement of its use in the therapy of diseases whose base is the process of blood vessels formation (such as wound healing). low - level laser therapy at 635 nm (1,875 mw / cm) significantly increases the number of huvecs.low-level laser therapy at 635 nm (1,875 mw / cm) significantly decreases the concentration of soluble vegfr- 1. low - level laser therapy at 635 nm (1,875 mw / cm) significantly increases the number of huvecs. low - level laser therapy at 635 nm (1,875 mw / cm) significantly decreases the concentration of soluble vegfr- 1. | growth factors as vascular endothelial growth factor (vegf), produced by the endothelial cells, take an essential part in pathological and physiological angiogenesis. the possibility of angiogenesis modulation by application of laser radiation may contribute to the improvement of its use in this process. thus, the aim of the study was to investigate the influence of low - level laser therapy (lllt) on the proliferation of endothelial cells, secretion of vegf - a and presence of soluble vegf receptors (svegfr-1 and svegfr-2) in the medium after in vitro culture. isolated human umbilical vein endothelial cells (huvecs) were irradiated using a diode laser at a wavelength of 635 nm and power density of 1,875 mw / cm2. depending on radiation energy density, the experiment was conducted in four groups : i 0 j / cm2 (control group), ii 2 j / cm2, iii 4 j / cm2, and iv 8 j / cm2. the use of laser radiation wavelength of 635 nm, was associated with a statistically significant increase in proliferation of endothelial cells (p = 0.0041). moreover, at 635-nm wavelength, all doses of radiation significantly reduced the concentration of svegfr-1 (p = 0.0197). |
the extraction of impacted third molars, which may be prophylactic or therapeutic, is one of the most frequently performed operative procedures in oral and maxillofacial surgery.12 surgical extraction of impacted third molars inevitably results in trauma to soft and hard tissues ; consequently, significant pain, swelling, and trismus may be experienced by patients who undergo such extractions.34 frequently, they experience significant postoperative distress and a decline in quality of life (qol).567 the undesirable consequences of impacted mandibular third molar surgery on the patients ' postoperative qol have been reported to show a 3-fold increase in patients who experience pain, swelling, or trismus alone or in combinations, compared to those who had none of these symptoms.7 various techniques intended to mitigate the detrimental effects of third molar surgery have been proposed, including the use of mouth washes, drains, specific suture techniques, steroids, ice packs, laser, analgesics, and drains, among others.8 the administration of corticosteroids such as dexamethasone and prednisolone considerably reduces the manifestations of inflammation such as swelling, redness, warmth, and tenderness that are frequently observed at the operative site.7 steroids may be administered along with nonsteroidal anti - inflammatory drugs and this combination has been found to have a distinct effect in reducing the severity of postoperative pain, swelling, and trismus.1491011 prednisolone, which is a synthetic analog of cortisol, has a half - life of 2.13.5 h. it is about 4 times as potent as hydrocortisone, has duration of action of 1836 h and quite importantly, a low mineralocorticoid activity.1213 prednisolone has a long record of efficacy and safety.1214 the various routes of administration have been described, which include oral, intramuscular, intravenous, and submucosal routes.1315 majid and mahmood,16 in a randomized controlled clinical trial where dexamethasone was administered intramuscularly and submucosally to two groups of subjects, reported comparable results between both groups with evidence of reduction in the postoperative measurements of pain, trismus, and edema. subjects in the control group were found to have experienced a greater severity of postoperative sequelae in comparison to subjects in the test groups.16 oral administration of prednisolone may have associated gastrointestinal side effects. in addition, systemic availability in oral administration may be unpredictable, and compliance may be a problem in extended usage.4131718 intramuscular administration of prednisolone on the other hand may predispose to the higher frequency of systemic adverse effects.1920 submucosal administration on the other hand is quicker to take effect than when given orally and there is no possibility of associated gastrointestinal disturbances.921 furthermore, submucosal administration exhibits less systemic effects and has effects confined largely to the operative site since the drug is concentrated at the operative site.1621 the objective of this study was to compare the effect of submucosally injected methylprednisolone (adjacent to the surgical site) with that administered orally, on the postoperative sequelae of surgically extracted impacted mandibular third molars in the immediate postoperative period. this was a prospective, randomized controlled clinical study to evaluate the effect of submucosal and oral administration of prednisolone on postoperative sequelae after third molar surgery. ethical approval was obtained from the health research and ethics committee of the lagos university teaching hospital. all potential subjects with known contraindications to the use of steroids such as hypertension, gastrointestinal tract ulcer, diabetes, glaucoma, active bacterial / fungal / viral infections, history of thromboembolic / cardiovascular events, glaucoma, psychosis, were excluded from the study. in addition, pregnant or lactating females and patients determined to be on anti - inflammatory drugs were also excluded from the study. subjects were allocated into three groups (groups a, b, and c) using a computer - generated table of random numbers. subjects in group a received 40 mg prednisolone per oral, which was administered 30 min preoperatively ; group b consisted of subjects who had submucosal administration of 40 mg prednisolone, which was administered preoperatively 5 min after local anesthesia had been achieved [figure 1 ]. subjects in group c did not receive any prednisolone, and they were the control. all subjects received amoxicillin 500 mg orally 8 hourly for 5 days and metronidazole 200 mg perorally 8 hourly for 5 days after surgery ; and ibuprofen 200 mg perorally immediately after the surgery and then 8 hourly for 3 days, regardless of their group. submucosal prednisolone administration all subjects were informed about the procedures and objectives of this study, and a written consent was also obtained from each participant. preoperative data were obtained from the subjects : demographics (age, sex), indications for extraction, location of the third molar (left or right), type of impaction, and the degree of impaction. preoperative and postoperative assessment for facial width / swelling, trismus, and pain was done for all subjects using the same methods and by the same operator. all the impacted mandibular third molars were classified according to winter 's classification and pell and gregory classifications using a standard periapical radiograph. preoperative measurements formed the baseline values for pain, mouth opening, and facial width. the preoperative pain was assessed using the linear 100 mm visual analog scale (vas). the subjects were asked to mark on the line with a pen ; the point they felt was most representative of their pain perception, with the worst imaginable pain and no pain represented by either extreme ends of the scale. subsequently, the vas score for each subject was decided by measuring in millimeters from the left extreme of the line to the point marked by the subject. facial width was determined preoperatively using the tape measuring method described by gabka and matsumura.22 three measurements were made as follows : the tip of the tragus to the soft tissue pogonion ipsilaterally (line a), tip of the tragus to the ipsilateral oral commissure (line b), and lateral canthus of the eye to the angle of the mandible ipsilaterally (line c) [figure 2 ]. markings for facial width measurement preoperative mouth opening measurement was done with the aid of the mono - block basic vernier caliper as the maximum inter - incisal distance [figure 3 ]. where the incisors were absent, the occlusal part of the edentulous ridges using the labial frenum as a guide for centrality was used. this measurement was taken with the subject seated upright and the frankfurt plane parallel to the floor. these measurements were done thrice, and the average was recorded in millimeters. inter - incisal mouth opening measurement with vernier calipers all operations were carried out under local anesthesia with 2% lignocaine hydrochloride with 1:80,000 adrenaline. the inferior alveolar nerve and lingual nerve anesthesia were achieved using the conventional technique, whereas the standard buccal nerve block technique was used to achieve long buccal nerve anesthesia. the buccal guttering technique was used to expose and undermine the tooth under copious irrigation with normal saline. where necessary, sectioning of the tooth was done, and delivery was done with coupland elevator. following tooth delivery, copious irrigation of the surgical site with sterile water postoperatively, all subjects were assessed for pain, facial swelling, and maximal inter - incisal distance using an identical method to that used preoperatively. for pain measurement, each subject was given a postoperative pain assessment form / diary (vas) to be filled each day for 7 consecutive days. the measurements were done and recorded on the postoperative days (pods) 1, 3, and 7. postoperative mouth - opening ability was obtained using maximum inter - incisal distance as described for the preoperative measurement using vernier calipers. the measurements were done and recorded on the pods 1, 3, and 7. data were analyzed using the statistical package for social sciences (spss) for windows (version 16.0, spss inc., the student 's t - test was used in the analysis of measures of pain, inter - incisal mouth opening, and facial swelling. the comparison of scores among the three groups was done using the analysis of variance. all operations were carried out under local anesthesia with 2% lignocaine hydrochloride with 1:80,000 adrenaline. the inferior alveolar nerve and lingual nerve anesthesia were achieved using the conventional technique, whereas the standard buccal nerve block technique was used to achieve long buccal nerve anesthesia. the buccal guttering technique was used to expose and undermine the tooth under copious irrigation with normal saline. where necessary, sectioning of the tooth was done, and delivery was done with coupland elevator. following tooth delivery, copious irrigation of the surgical site with sterile water was done. postoperatively, all subjects were assessed for pain, facial swelling, and maximal inter - incisal distance using an identical method to that used preoperatively. for pain measurement, each subject was given a postoperative pain assessment form / diary (vas) to be filled each day for 7 consecutive days. the measurements were done and recorded on the postoperative days (pods) 1, 3, and 7. postoperative mouth - opening ability was obtained using maximum inter - incisal distance as described for the preoperative measurement using vernier calipers. the measurements were done and recorded on the pods 1, 3, and 7. data were analyzed using the statistical package for social sciences (spss) for windows (version 16.0, spss inc., the student 's t - test was used in the analysis of measures of pain, inter - incisal mouth opening, and facial swelling. the comparison of scores among the three groups was done using the analysis of variance. one hundred and ninety - eight subjects who satisfied the inclusion criteria and consented to participate were recruited for the study. however, 186 subjects out of the 198 participated in all stages of this study and were then included in the final analysis. statistically, 69 were males, and 117 were females, giving a male to female ratio of 1:1.7. overall, the mean age (standard deviation [sd ]) and sex distribution for all the subjects were 28.1 (7.4) years (range, 1851 years) [table 1 ]. the mean age (sd) of subjects was 28.5 (7.9) years, 27 (6.6) years, and 28.8 (7.5) years for groups a, b, and c, respectively (p > 0.05). age and sex distribution of subjects the most frequent type of impaction seen, using winter 's classification, was mesioangular which constituted 40.9% of cases, followed by distoangular (25.8%), vertical (16.1%), recurrent pericoronitis (52.7%) was the most frequent reason for surgical extraction, followed by caries and its sequelae (26.3%), periodontal disease (11.3%), and orthodontics (5.4%). there was no statistically significant difference between groups a, b, and c regarding mean preoperative inter - incisal distance, pain, and facial width (> 0.05) [table 2 ]. preoperative mouth opening, facial width measurement, and pain the highest mean pain score was recorded on pod1 and it gradually decreased over the 7 days postoperative period in all three groups [table 3 ]. the severity was observed to be lower among subjects in groups a and b when compared with those in group c throughout the immediate postoperative period [table 3 ]. both prednisolone groups showed a statistically significant lower pain magnitude in comparison with the control group at all intervals (p < 0.05) [table 3 ]. there was no statistically significant difference in pain perception between subjects in groups a and b at all the postoperative evaluation points [table 4 ]. pain perception by subjects in all groups comparison of pain perception between groups a and b there was a decrease in the mean postoperative inter - incisal mouth opening distance for all groups in the immediate postoperative period in comparison with the preoperative measurements [table 5 ]. furthermore, there was a significant reduction in the mean inter - incisal distance on pod1, pod3, and pod7 in group c when compared with groups a and b (p < 0.05). subjects in group c also exhibited higher mean postoperative swelling on pod1, pod3, and pod7 when compared with group a and b (p < 0.05) [table 5 ]. a comparison of mean inter - incisal distance between groups a and b revealed a statistically significant difference on pod1 and pod3 [table 6 ]. comparison of mean mouth - opening ability among the three groups comparison of mouth - opening ability between groups a and b there was an increase in the mean postoperative facial swelling for all groups on pod1, pod3, and pod7 [table 7 ] in comparison with the preoperative facial width measurement. in all groups, the swelling was most severe on pod1 followed by the pod3, to approximately reach the preoperative measures by the 7 day. subjects in group c exhibited the highest mean postoperative swelling as measured on pod1, pod3, and pod7 [table 7 ]. there was a statistically significant difference in the mean postoperative facial swelling between prednisolone groups (groups a and b) and control (group c) at pod1 and pod3 [table 7 ]. however, a comparison of the postoperative facial swelling between subjects in prednisolone groups (groups a and b) showed no statistically significant difference (pod1, p = 0.8 ; pod3, p = 0.9 ; pod7, p = 0.6). there was an increase in the mean postoperative facial swelling for all groups on pod1, pod3, and pod7 [table 7 ] in comparison with the preoperative facial width measurement. in all groups, the swelling was most severe on pod1 followed by the pod3, to approximately reach the preoperative measures by the 7 day. subjects in group c exhibited the highest mean postoperative swelling as measured on pod1, pod3, and pod7 [table 7 ]. there was a statistically significant difference in the mean postoperative facial swelling between prednisolone groups (groups a and b) and control (group c) at pod1 and pod3 [table 7 ]. however, a comparison of the postoperative facial swelling between subjects in prednisolone groups (groups a and b) showed no statistically significant difference (pod1, p = 0.8 ; pod3, p = 0.9 ; pod7, p = 0.6). the mean age of subjects observed in this study is similar to the reports by majid and mahmood as well as bamgbose., who reported mean ages of 26.9 years and 27.9 years, respectively.116 most of the subjects were in their third or fourth decades of life ; this is in agreement with reports in the literature.2324 some authors have hypothesized that this observation may be due to the fact that most mandibular third molars erupt between ages 17 and 25 years.2526 a slightly higher female prevalence was observed in this study. obiechina.12327 this is in contrast to the report by bui., who reported a male to female ratio of 1.3:1.28 quek. reported no sex prevalence in a study of a singaporean chinese population while a study of a chinese population by mcgrath. reported a clear female preponderance with a male to female ratio of 1:2.9.629 the most common type of impaction in this study was mesioangular impaction this is in agreement with studies by akinbami and ofomala, and adeyemo.3031 however, bui. reported the most common winter 's classifications as vertical and mesioangular 63.9% and 25.7%, respectively.28 ladeinde. reported distoangular impaction (46%) as the most common.32 there is no consensus on the reason for higher prevalence of mesioangular impaction in the literature ; however, some authors have postulated that the primordial tooth germ of mandibular third molar develop high up in the mandibular ramus with its occlusal surface slanting mesially or sometimes horizontally.3334 in the present study, recurrent pericoronitis was the most common indication for the removal of impacted mandibular third molar. ladeinde.31323536 unrestorable caries and its sequelae on impacted mandibular third molar and the adjacent tooth was the second most common indication for extraction (26.3%). this is in consonance with the studies by laureano filho. and adeyemo.3135 periodontal disease accounted for 11.3% of indications for surgical extractions in this study. this is similar to the report by adeyemo., who reported periodontal disease as an indication in 9.2% of cases.36 obiechina. reported pericoronitis and periodontal disease as accounting for 42.92% of cases seen in their study.27 orthodontic reasons for surgical extraction accounted for 5.4% extractions done while other reasons for extraction such as facial neuralgia and prosthodontic reasons accounted for the remaining 4.3%. surgical extractions of third molars for prophylactic reasons are not common in our environment. in a study by adeyemo., it accounted for 0.6% of cases.37 this is in contrast with european studies where it is reported that between 18% and 50.7% undergo surgical extraction of third molars for prophylactic reasons.38 postoperatively, an interwoven cascade of functional and structural changes, which are often expressed as pain, swelling, and trismus, occur.35 these expected sequelae have been reported to be detrimental to the patients ' qol in the immediate postoperative period.6 pain is often experienced by patients after surgical extraction of impacted mandibular third molars. it characteristically increases in intensity in the early postoperative period until it peaks within the first 2448 h postextraction.39 in addition, snyder. reported that patients who experience pain sufficient to prompt taking pain medications experienced a comparative significant interference with recovery for lifestyle and oral function after third molar surgery.40 in this study, the vas was used to evaluate pain perception by the subjects. it has been used in the evaluation of postoperative pain after mandibular third molar surgery by several.2441 the highest pain intensity in this study was recorded on pod1, but it gradually decreased in value in all groups during the immediate postoperative period. higher mean pain scores were observed in the control group throughout the immediate postoperative period than in the oral and submucosal prednisolone groups. these differences in pain scores among the groups were statistically significant all through the immediate postoperative period. the mean pain scores observed in the immediate postoperative period were slightly higher in group a than group b except on pod2. this is in agreement with the findings of tiwana., who found lower pain values in the corticosteroid group in the postoperative period compared with the control.42 in a systematic review by markiewicz., the authors observed that subjects in the corticosteroid group reported less vas points for pain than the control group, 13 days after surgery.43 this decrease in pain may be credited to corticosteroid effects which may have decreased patients ' inflammatory response and reaction to pain by repressing tissue bradykinin and -endorphin intensity.113942 in the present study, there was a significant reduction in postoperative swelling in prednisolone groups (groups a and b) in comparison with the control group on pod1 and pod3. the prednisolone groups also had lower values for facial swelling measurement on pod7 ; however, the difference in values obtained was not statistically significant when compared with the control group. a comparison of the difference in facial swelling measurement values on pod1, pod3, and pod7 for the groups a and b was not statistically significant. this is in agreement with the study by majid, who evaluated the effect of submucosal dexamethasone in subjects undergoing surgical extraction of the lower third molar.41 shah. and hassan also reported similar findings.2144 milles and desjardins performed a crossover study on 11 patients using 16 mg methylprednisolone orally, the evening before surgery. they reported that the low dose of methylprednisolone reduced swelling by 34% at 48 h postoperatively.45 warraich., using a single dose (4 mg) of submucosal dexamethasone preoperatively, reported significantly less swelling in the subjects receiving corticosteroids than the subjects in the control by the 2 postoperative day.7 similar observations were made by grossi. and majid.341 in addition, grossi. also observed that there was no statistically significant difference in postoperative edema measurements between the corticosteroid and control groups.3 furthermore, neupert. reported no statistically significant reduction in postoperative swelling on the 2 and 7 postoperative days between the corticosteroids and control groups.46 mouth - opening ability was evaluated by measuring the maximum inter - incisal distance between the corresponding central incisors with the vernier caliper ; this method has been used in numerous researches.13543 the mean postoperative inter - incisal distance decreased in comparison with the mean preoperative inter - incisal distance in the immediate postoperative period. the severity of the trismus seen is a direct effect of the degree of inflammatory response.947 subjects in group a and b had a less severe limitation in mouth opening in the immediate postoperative period compared to those in group c. this is likely due to the anti - inflammatory effects of prednisolone administered. subjects in group b had a slightly higher mean inter - incisal distance compared with those in group a. this finding is in consonance with reports by tiigimae - saar. and kang., who administered 30 mg and 20 mg of prednisolone, respectively in subjects undergoing third molar extraction.2048 this is possibly because of the local administration of prednisolone in group b subjects which is close to and concentrated at the operative site. in addition, it bypasses the gastrointestinal tract and the liver, thereby increasing the bioavailable fraction of the drug. however, grossi. in their study noted no statistically significant difference between the study and control groups.3 this may have been because of the relatively small sample size of 61 subjects which they randomly divided into three groups. this study has shown that the administration of prednisolone has a significant impact in reducing postoperative pain, edema, and trismus following third molar surgery. in addition, it offers a safe, simple, cost - effective, and painless method, which produces a high concentration of prednisolone at the operative site, thereby lessening the systemic effects. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | background : the aim of the study was to evaluate the effect of preoperatively administered submucosal and oral prednisolone on postoperative pain, facial swelling, and trismus following third molar surgery.patients and methods : this was a randomized controlled trial in which subjects were randomly distributed into three groups. group a consisted of subjects who received 40 mg oral prednisolone ; group b consisted of subjects who received 40 mg submucosal injection of prednisolone while group c consisted of subjects who did not receive prednisolone. each group had 62 subjects. measurements for facial width / facial swelling, pain, and mouth opening were recorded preoperatively and postoperatively. the postoperative evaluation points were postoperative days 1, 3, and 7. these measurements were compared with the preoperative values both within and among the groups.results:most of the subjects were in their third decade of life. a considerable increase in the mean postoperative values for pain, facial width and trismus was observed. notably, subjects who did not receive prednisolone showed comparatively higher values for the measured parameters throughout the postoperative evaluation period. subjects who received submucosal injection of prednisolone showed overall lower values compared to those who received oral prednisolone.conclusion:the results of this study indicate that the administration of prednisolone has a significantly beneficial effect in ameliorating the postoperative sequelae of the third molar surgery. in addition, the effect of submucosally injected prednisolone is comparable to the orally administered prednisolone ; indeed it shows superiority to the latter in a number of dimensions. submucosal injection of prednisolone offers a simple, effective, easy, safe, and minimally invasive option to existing therapeutic methods of reducing these postoperative sequelae. |
penaeid prawns are important subjects of mariculture and coastal fisheries activities throughout the tropics and subtropics [13 ]. traditionally, prawn farms located near the coast used directly coastal seawater to rear the prawns without additional processes. shrimp culture is therefore potentially affected by anthropogenic activities including metal contamination. specifically in coastal mexican waters, a great number of basic metal industries represent the mainly heavy metal (cadmium, lead, nickel, chromium, etc.) source [49 ]. penaeid prawns have revealed great capacity to accumulate and take up metals from solution, either essential or not essential elements and yet survive in these polluted environments [1014 ]. thus, it is of great concern to investigate detoxificatory processes involved in prawns exposed to metals. it is well known that the induction of metallothionein which is a cystein - rich protein and has detoxifying properties, occurs in aquatic invertebrates after in situ or laboratory metal exposure (for review see amiard. and, physiological markers reflecting the energetics of an organism may contribute to the understanding of the mode of the toxicant [1618 ]. the metabolic cost hypothesis suggests that toxic stress induces metabolic changes which may lead to a depletion of its energy reserves resulting in adverse effects on growth and reproduction. comprehension of the mechanisms related to the sublethal effects caused by toxic metals upon shrimp metabolism would help to develop sensitive and diagnostic tools (biomarkers) with a predictable capacity in assessing the toxic effects, thus contributing to better coastal seawater management and sustainable aquaculture. this study is part of a wider investigation of the regulation and accumulation of the trace metals nickel and lead in a penaeid prawn penaeus vannamei ; metals previously detected in coastal mexican waters [48 ]. nickel has occasionally been interpreted as an essential metal [21, 22 ], probably as a consequence of its ability to be substituted for other divalent metal ions, particularly zinc, or as a part of an enzymatic structure [23, 24 ]. this paper specifically investigates both metallothionein - like proteins and energy reserve levels in the hepatopancreas and abdominal muscle of penaeus vannamei, after sublethal exposure of the prawns to dissolved nickel and lead. juveniles of penaeus vannamei (between 13.0 cm total length ; mean dry weight 11.0 8.0 mg ;) were obtained from cultures at aquapacific s.a de c.v., prawns were maintained in artificial sea water (instant ocean (io), aquarium systems) at 25 salinity (ph 7.8), 12 : 12 light / dark periods and 25c as previously described [13, 14 ]. animals were fed every two days with commercial flakes (tetramin) for 15 minutes maximum, before water changes. the use of artificial seawater in all experiments provided physicochemical stability and replicability for trace metal uptake tests. the dissolved metal concentrations used in laboratory exposure of prawns are environmentally realistic for contaminated mexican coasts [27, 28 ]. the experimental design involved nine separate experimental groups containing ten prawns each : controls (not metal exposed ; three groups), prawns exposed to 55.5 g l nickel (two experimental groups), 191 g l lead (two experimental groups), and same concentration of nickel / lead mixture (two experimental groups) in io at 25 salinity and 25c. the hepatopancreas and abdominal muscle were removed from one control group at the beginning of the experiment, as described by nunez - nogueira and rainbow. prawns from the remaining two control and metal - treated groups were dissected after 10 days of exposure. samples destined for biochemical analysis were treated as described by nunez - nogueira.. briefly, samples were transferred into plastic vials to be immediately frozen in liquid nitrogen for a few minutes. after freezing, then, samples were homogenized in a buffer solution (20 mm tris, 10 mm -mercaptoethanol, 150 mm nacl solution adjusted to ph 8.6). the soluble and insoluble fractions were separated by centrifugation at 25,074 g for 55 minutes at 4c (biofuge 28 rs, heraeus sepatech). the cytosolic heat - stable compounds including metallothionein were isolated by centrifugation of the soluble fraction (12000 g for 10 minutes at 4c) after heat - treatment (75c for 15 minutes). in the heat - denaturated cytosol, the amount of mt was determined by differential pulse polarography (dpp) according to thompson and cosson and olafson and olsson. a mde 150 stand polarographic (radiometer copenhagen) tracelab 50, controlled by the computer software tracemaster 5 through a polarographic analyser pol 150 was used. the method of standard addition was used for calibration with rabbit liver mt (sigma chemical co., st louis, mo, lot. polarographic determination in heat - denaturated cytosol is an analytical procedure based on several characteristics of mts, but it does not allow, with certainty, the assertion that the target molecule is a true mt unless purification and sequencing are carried out. strictly, therefore, what has been measured is the concentration of proteins with metallothionein properties, that is, metallothionein - like proteins (mtlp). all glass labware was soaked in 10% hcl, rinsed three times with deionized water and dried in a desiccator protected from atmospheric dust. the insoluble and soluble fractions were heated (75c, 12 h) with suprapure hno3 acid (carlo erba). after digestion, metal levels in these acid solutions were determined after dilution with deionized water by flame aas using the zeeman effect (shimatzu 8600-aa spectrophotometer). standard addition analyses were performed in an isomedium and concentrations of each element were 25 mg metal ml for aas. the analytical methods were validated by external intercalibrations as previously described nunez - nogueira. and use of reference material tort-2 (lobster hepatopancreas) with more than 90% recovery. total metal concentrations were recalculated from summation of quantities of trace elements in soluble and insoluble fractions determined previously, by combine means procedure. the results were expressed in gg dry weight of the organ for ni and ngg in case of pb, respectively. hepatopancreas and abdominal muscle samples were homogenized with a porcelain mortar and pestle at 2c. the powder obtained was homogenized in a motorized grinder with 1.5 ml citrate buffer (ph 5.0) for glycogen and lipid analyses. total lipids were determined by the sulfophosphovanillin reaction, according to frings., while glycogen concentration were determined in two aliquots of the homogenate, using enzymatic digestion by amyloglucosidase, according to carr and neff. olive oil and glycogen from oysters (sigma type iii) were used as standards for each method, respectively. students t - test (p <.05) were performed for protein, lipids, glycogen, and metal concentration comparisons. tests were developed for small samples according to williams, and carried out in statistica 5.1 for windows (statsoft inc.). the mtlp concentrations in hepatopancreas and abdominal muscle of juveniles of penaeus vannamei are illustrated in table 1. mtlp concentrations were higher in hepatopancreas than in abdominal muscle in all experimental organisms (control, ni exposed, pb exposed, ni+pb exposed). in hepatopancreas, the difference in mtlps concentrations between the control and ni - treated shrimps were not significant, while a significant increase in mtlp concentrations was observed in pb - treated and metal mixture - treated organisms versus controls. the mtlp concentration ranged from 6.83 to 31.04 mg g in controls, 9.46 to 62.04 in ni - treated, 31.79 to 121.85 in pb - treated, and 36.11 to 94.83 mg g in metal mixture - treated, respectively. in abdominal muscle, a significant increase in mtlp concentrations was observed in ni - treated and pb - treated prawns in comparison with controls whereas no differences in mtlp concentrations were detected in metal mixture treated organisms versus controls (apparently due to the presence of two muscle samples (out of ten) with higher mtlp concentrations within the pb - treated prawns, suggesting noninfluence of ni and/or pb in mtlp induction in muscle, even when an apparent difference was detected in one metal treated groups). the mtlp concentration in muscle tissue ranged from 0.30 to 0.83 in controls, 0.30 to 0.99 in ni - treated, 0.54 to 1.54 in pb - treated, and 0.51 to 0.60 mg g in metal mixture - treated, respectively. ni and pb concentrations (total, soluble, insoluble) in the hepatopancreas and abdominal muscle of penaeus vannamei are indicated in tables 2 and 3, respectively. ni concentrations were significantly higher (p <.05) in the hepatopancreas versus the abdominal muscle of prawns, pb was found in major quantities in abdominal muscle. the measured total ni concentration in hepatopancreas ranged from 3.67 to 21.37 g g (controls), 1.98 to 11.86 (ni - treated), 1.11 to 22.84 (pb - treated), and 3.86 to 56.23 g g (mixture - treated), respectively. due to a great individual variability, no significant differences were observed among groups, including ni - treated. in abdominal muscle, ni concentration ranged from 0.02 to 0.03 g g in each group and not differences against controls were observed (table 2). the distribution of ni among the soluble and insoluble fractions in the hepatopancreas and the abdominal muscle of prawns are shown in figures 1(a) and 1(b), respectively. in hepatopancreas, this metal was nearly equally distributed between soluble and insoluble fractions (figure 1(a)) whereas in the abdominal muscle (figure 1(b)), the soluble fraction was more important than the insoluble fraction. lead was detected only in the insoluble fraction of both tissues (table 3). the mean pb concentrations in all groups were below 0.17 mg kg in abdominal muscle and 0.003 mg kg in hepatopancreas. a slight increase (not significant) was observed in the hepatopancreas in prawns exposed to this metal, either alone or in a mixture. the concentration of lipids and glycogen, determined in hepatopancreas and abdominal muscle are shown in figures 2 and 3, respectively. lipid concentration in abdominal muscle ranged from 1.08 to 16.45 mg g dw (controls) and from 0.86 to 18.30 mg g (metal - treated). in hepatopancreas, lipid concentration ranged from 1.85 to 48.95 mg g dw (controls) and from 6.53 to 88.66 mg g (metal - treated). no significant differences were observed between controls and treated prawns in the case of lipids. the range of glycogen was from 0.51 to 23.60 mg g (controls) and 1.60 to 15.05 mg g (metal - treated) in abdominal muscle, while it was from 4.72 to 8.89 mg g (controls) and 1.07 to 11.33 mg g (metal - treated) in hepatopancreas, respectively. glycogen concentration in abdominal muscle from pb - treated prawns was the only one significantly (p <.05) different from controls. in this case, different studies have shown the presence of heavy metals, including ni and pb in mexican coastal waters [46 ]. for example, coastal zones in tabasco and veracruz states (gulf of mexico) have shown lead concentration in water between 65 to 210 g l, while others like cr, hg, and cd between 0.5 and 15 g l. these concentrations can be above the national recommended level (local legislation), as have been observed in the mexican pacific coast. the pacific white prawn penaeus vannamei spends part of its life cycle in coastal or estuarine areas, making it suitable for metal exposure. this species showed capacity to induce metallothioneins by metal exposure [36, 37 ] and accumulate metals [12, 25 ]. total ni concentrations found here were in good agreement with previous studies that show ni regulation in decapod crustaceans [38, 39 ], including p. vannamei. nickel was found almost equally distributed between the soluble and insoluble fraction (table 2). these concentrations are within values previously described in penaeid prawns (including p. vannamei) [40, 41 ] for these tissues. the reason of this lack of increase compared to controls must be related to the fact that both metals are regulated or partially regulated (in case of lead as a nonessential metal) by this prawn. previous studies have shown that ni can be regulated by decapods crustacean [39, 42 ] providing a capacity to maintain a minimal trace level concentration in their bodies. in case of pb, vogt, and quinitio suggested that this non - essential metal is eliminated by forming insoluble - lead rich deposits that are excreted after lysosome autolysis, process that can include mt content during protein turnover (see below). due to that metal exposures do not exceed the lc-50 for 96 hours in both cases for the experimental conditions here tested, and are far below these values (60.54 mg l for pb, resp.), suggests that such a trace level of exposures is not enough to reach the threshold level of regulation in case of penaeus vannamei without significant accumulation. according to the results obtained here, mtlp appear to be induced by pb in hepatopancreas under the experimental conditions tested (table 1). prawns exposed only to ni in solution did not show a significant increase in tissue concentration, while prawns treated with pb either alone or in a mixture with ni, almost double the amount of mtlps observed in controls and ni - treated groups (table 1). most of the lead was detected in the insoluble fraction of the hepatopancreatic samples (table 1), suggesting a nonsoluble lead - rich deposit. some authors have suggested that mt 's induction is an intermediate step before insoluble deposits are formed [45, 46 ]. lead - insoluble deposits or granules have been observed in terrestrial and aquatic invertebrates [4751 ]. rainbow highlighted that isopods accumulated metals in detoxified granules (non - soluble) with rapid elimination of lead, compared to other metals (e.g., cadmium). complementary to vogt and quinitio, our results suggest that lead is mainly stored and detoxified within granules, instead to mtlp 's, perhaps at the antennary gland as in penaeus monodon, and excreted as an apocrine secretion in the urine [49, 50 ], not only at high - lead level of exposure, as was proved in p. monodon. in this way, lead appears to be treated as some essential metals, in respect of quelating cytosolic molecules. this difference in detoxification between essential and non - essential metals was already observed in p. vannamei and penaeus indicus, where mtlps appear to be the first detoxification strategy involved in non - essential metals exposure, while essential metals are regulated in cytoplasm by another method (e.g., insoluble deposits). it is also considered that mtlps increase slightly with time and doses as a result of a constant turned over. in the abdominal muscle, slight increase in mtlp concentration was observed in ni- and pb - treated, but not in a mixture (table 1). no significant relationship (not shown) between soluble ni and mtlp was observed. comparing the total metal concentration between controls and treated groups in muscle, no significant increase was detected, suggesting that these increases in mtlp could be related to other factors, like normal homeostasis of other essential metals (e.g., zn or cu) and inflammation process caused during experiment procedure, rather than metal exposure [15, 54, 55 ]. this idea is also supported by the lack of mtlp induction in muscle in metal mixture - treated group (table 1). it has been proved, that longer period of exposure and higher metal concentrations induced mtlps in crustaceans and aquatic or terrestrial invertebrates [36, 5658 ]. our results obtained here suggest that it is necessary to carry out bioassays with different metal concentrations near (below) the lc50, to identify the threshold level for mtlp induction for these metals in this species. a recent work in our lab, revealed that using the following metal concentrations : 15.6, 31.3, 62.5, 125 and 250 mg l for pb, for 96 hours of exposure, are higher than the environmentally realistic metal concentrations tested (55.5 g l for ni and 191.2 g l for pb, resp.), and far below the lethal doses obtained, in good agreement with our results, where the trace level of exposure only inducing minimal or no detectable changes in the metal body concentrations. it is widely recognized that both ni and pb share chemical properties and features with class b and borderline metals, like copper, zinc, arsenic, cadmium, and mercury [59, 60 ]. within these metals, cadmium, copper, mercury, zinc, and arsenic have been identified as metallothionein inductors [56, 57, 6164 ]. due to these the first involves zinc / copper substitution by these metals in the active sites of the cystein - rich proteins (metal - clusters) or secondly by direct generation of metal element responses (mers) inside the cells. in the first case, nickel or lead compete against zinc for the active clusters sites of the mt or generate an exchange ni / pb versus zn, inducing and increase in the zinc intracellular concentration and its bioavailability, promoting an homeostatic response by increasing the amount of mts. this phenomenon has been observed in other invertebrates [56, 63 ] and vertebrates [65, 66 ], and metal substitution in metallothioneins has been registered in different cases [56, 58, 64 ], for example, cadmium in crustaceans. in the case of mers production, the mtlp induction might be related to free - radicals generation and/or oxidative damage that activate a metal transcription factor capable to interact with the promotor region of the mt gene by a positive feedback. these results are in good agreement with previous nickel and lead reports as mt inductors, supporting that this inductive capacity is present not only in in vitro tests [63, 66 ]. one interesting observation relay on the relationship observed between the amount of mtlps concentration in hepatopancreas of controls and the whole body weight, which almost reaches 3% (table 3). metallothioneins are considered as homeostatic proteins, widely present in aquatic and terrestrial invertebrates either from contaminated and noncontaminated sites [56, 61, 6769 ]. an explanation could be related to a reduction in the tissue weight, due to lower quality food used during captivity, or stressful conditions during acclimation, producing a minor difference between tissue mass and mtlp content. weight is a factor proved that affect the mt concentration in soft tissues of aquatic invertebrates [67, 68 ], however, this appears to be scarcely possible due to the fact that mean weight of the hepatopancreas was between 8 and 9 mg (dry weight) in all experimental groups, including controls, representing nearly 6% of the whole body weight, in good agreement to the proportion established in healthy decapods. the hepatopancreas of p. vannamei showed traces of pb and relatively amounts of ni in controls (0.002 and 6.41 g g, resp.), which might be triggering the defensive and regulatory mechanisms of mts, explaining why the mtlp concentration in this group almost reaches 3% of the tissue weight, highlighting that previous metal exposures increase the original amount of mtlps. higher concentrations of energy reserves, mainly lipids, and mtlps in the hepatopancreas are in good agreement with its function of main organ for metabolic reserves and metal - binding proteins. energy reserves are of importance considering (among others) its use in detoxification processes [7274 ]. in this respect, the concentration of lipids (figure 2) and glycogen (figure 3) did not change in the hepatopancreas. in abdominal muscle, a slight increase in the lipid and glycogen fraction occurred, suggesting a possible effect of pb on these reserves, but no correlation was observed with metal concentration in the tissue. toxic pb exposures have been considered a cause for a decrease in metabolic rates in postlarvaes of p. indicus (according to satyavathi and chinni and yallapragada), causing perhaps the higher glycogen concentration observed in the abdominal muscle, as a result of lower degradation rates by glycogen phosphorylase and an increase in the carbohydrate synthesis by the glycogen synthase. however, the low pb concentration detected in the muscle, suggested that other stressful factors could be involved, and not necessarily related to the metal presence in the tissue. previous studies in prawns and other crustaceans, have shown that under some particular stressful conditions (e.g., salinity and metal conditions), the energy budget can be modified in opposite direction. in some cases, energy production can be altered by metals like mercury and zinc by increasing the lactate dehydrogenase activity, enzyme involved in energy production, as was observed in the crab carcinus maenas. the fact that no significant changes were observed in the hepatopancreas, which showed to be the main target organ for pb, suggests that the concentration of metal in solution tested in this study is not sufficient to compromise the amount of energy available in these tissues, and it is enough to cover the detoxification process trigger under the stress simulated in the hepatopancreas, or involves alternative mechanisms to mitigate the adverse effect. it is possible that higher metal concentrations, near lethal concentrations and longer exposure periods, could induce significant changes in the energy reserve, as has been observed in p. indicus exposed to pb, but further studies are required in p. vannamei. this study shows the first results of mtlp quantification and energy reserves in the estuarine prawn p. vannamei exposed in the laboratory to ni and pb. it is assumed that the mtlp induction in the hepatopancreas is caused by metal stress (presence of pb and ni) and did not influence the energy metabolism. this protein induction appears to be related either to the detoxification process of this non - essential metal (pb) and by essential metal regulation (ni). in case of ni, the mtlp concentration could be considered as the basal level for cellular homeostasis of the essential metal. the experimental conditions tested do not modify the energy budget present in both tissues at the sublethal exposure tested. mtlp concentrations could not be considered as a biomarker, at least in the hepatopancreas of p. vannamei, for the realistic metal concentration tested here, in environmental risk studies. further studies involving different metal concentrations (just below the lethal level) and exposure times will help to define the threshold level for mtlps and energy reserves changes in this species. | this study analyzed the changes in metallothionein - like proteins (mtlps) and energy reserves (ers) in hepatopancreas and abdominal muscle of the white prawn penaeus vannamei. realistic metal concentration exposure for 10 days to ni and pb in solution revealed that juvenile prawns partially induce mtlp in hepatopancreas after pb exposure. ni was distributed equally between soluble and insoluble fractions, while pb was present only in the insoluble fraction, suggesting different detoxification strategy. no changes in lipids and glycogen concentration were detected under these experimental conditions in both tissues analyzed. mtlp could not be considered as a suitable indicator for lead exposure in hepatopancreas. |
the multiple myeloma is a hematological malignancy characterized by the development of a clone of plasma tumor invading hematopoietic marrow. in its usual form, it involves bone marrow plasma cell infiltration, a monoclonal immunoglobulin in blood and/or urine, and bone involvement. meningeal localization of myeloma is a rare presentation and poor prognosis 14. a high myeloma burden, stage iii disease, high labeling index, circulating plasma cells in the peripheral blood, and iga or igd m protein were the most frequent clinical features of these patients. we report a case of a patient who developed meningeal involvement 3 months after achieving complete remission after autologous stem cell through multiple myeloma revealed by a nasopharyngeal mass and discuss the presumed mechanism of meningeal involvement. we also speculate that meningeal involvement may occur also in cases with a low myeloma mass and with a disease apparently well controlled by high - dose therapy 4,5, but who had initially an infiltration from contiguous structures when lytic lesions erode skull and dura mater 6. we report a case of a 41-year - old man who was admitted in our institution for multiple igg kappa myeloma iss3 score, in a context of general alteration with 9 kg weight loss, dysphagia to solids, a left laryngeal immobility. the physical examination found a large mass in the nasopharyngeal. brain magnetic resonance imaging (mri) rating extensive homogeneous cell mass developed at the expense of nasopharyngeal extending into the oropharynx, invading the soft parts of the cranio - spinal shower hinge, part of the skull base, the predominant left temporal fossae, large sphenoid wings being crossed by the tumor process (fig.1). the microscopic examination of the tumor 's biopsy showed a fragment consisting of nasopharyngeal cohesive sheets of small cells overwritten. immunohistochemistry noted of cd145 + cells, cd20, cd3, cd138 +, monotypic lambda with an estimated of 90% proliferation index. the pet scanner showed a large mass developed at the expense of nasopharyngeal extending the skull base and oropharynx. sagittal view of a 3d - t1-weighted mri with gadolinium shows extensive homogeneous cell mass invading the soft parts of the cranio - spinal shower hinge, part of the skull base, the predominant left temporal fossae, large sphenoid wings being crossed by the tumor process. the complete blood count on presentation demonstrated a white blood cell count of 8.1 g / l (segmented neutrophils 61% ; band neutrophils 0% ; lymphocytes 28% ; monocytes 9% ; eosinophils 1% ; basophils 1%) with hemoglobin level of 9.8 g / dl, and platelets levels of 283 bone marrow aspiration indicated marrow involvement due to the presence of 40% of plasma cells. lumbar puncture including electrophoresis of proteins of the cerebrospinal fluid (csf) was normal. the patient was treated with vcd regimen, melphalan autograft, radiotherapy of nasopharyngeal and spine. autograft was complicated by (1) pulmonary infection, (2) a significant mucositis grade 4 with major malnutrition, and (3) weight loss of 10 kg. the patient was readmitted to the emergency department with intensive headache, dysarthria, unsteady gait, and limb weakness, without nuchal rigidity or fever. a brain mri demonstrated a contrast - enhanced dural multinodular thickening next to the right parietal lobe, with focal extensions to the subarachnoid space and vasogenic edema of the underlying brain parenchyma (fig.2). the adjacent inner table and diploe of the skull were not involved. a bilateral parenchymal enhancement of the superior cerebellar peduncle was also observed. axial view of a 3d t1-weighted mri with gadolinium (a) and flair (b) shows an enhanced dural thickening (arrowheads) with a parenchymal edema of the underlying cortex and subcortical white matter (double arrow). coronal view (c) also reveals an involvement of the subarachnoid space (arrowhead). an axial view of the brain stem shows bilateral parenchymal enhancement of the cerebellar peduncles (arrows). a lumbar puncture was performed and showed a low level of glucose (0.4 mmol / l), a high protein level (2.1 g / l), and a high white cell count composed almost exclusively by plasmas cells (fig.3), all with neoplastic phenotype cd138 positive. plasma cells in the cerebrospinal fluid may grunwald giemsa (mgg) 100. he received intrathecal administration of 12 mg of methotrexate, 40 mg of cytarabine, and 40 mg of prednisolone, three times per week, and pad regimen (bortezomib adriamycine about 65 cases were reported in the literature 13 and reviewed by peterson. the largest series including 14 patients was reported by chamberlain. 1. a high myeloma burden, stage iii disease, high labeling index, circulating plasma cells in the peripheral blood, and iga or igd m protein were the most frequent clinical features of these patients. a hematogenous spread can be hypothesized in plasma cell leukemia, and an infiltration from contiguous structures is possible when lytic lesions erode skull and dura mater 6. otherwise, a few patients with a cns relapse had circulating plasma cells (pc) at the first recognition of multiple myeloma (mm) : in these patients, pc might have infiltrated the meninges at diagnosis and might have grown during the course of the disease since most of the drugs used in mm treatment and even high - dose melphalan can not overcome the blood brain barrier. this complication appeared in the majority of the patients as a terminal event 7, but it was also reported as a presenting feature of mm 5. moreover, a few cases of meningeal involvement were reported in patients who had been successfully treated with high - dose therapy and stem cell rescue and had no overt medullary plasmacytosis 4,5. four patients were described : all presented meningeal involvement a few months (2 or 3) after an autologous stem cell transplant (asct) with the attainment of a complete remission and no evidence of residual disease in the bone marrow. the conditioning regimen was high - dose melphalan in three cases and total body irradiation (tbi) plus melphalan in the fourth. these data confirm that meningeal involvement may occur also in cases with a low myeloma mass and with a disease apparently well controlled by high - dose therapy. our report describes a patient with meningeal involvement who had not detectable disease after vcd regimen, melphalan autograft, radiotherapy of nasopharyngeal and spine but who had initially a nasopharyngeal mass invading the base of the skull. although pc can be seen in the csf in other conditions, both infectious and noninfectious 8, the presence of pc in the csf of a patient with mm supports the diagnosis of myelomatous meningitis. the definitive proof is the definition of monoclonality of the pc obtained by immunophenotyping or the immunoelectrophoresis of csf showing an m component 9. the prognosis of meningeal and cerebral involvement of mm is very poor. on the basis of the 65 cases of meningeal myelomatosis reported mainly as case reports in the literature, with available information about survival, the median overall survival (kaplan meyer) from the time of the diagnosis of the meningitis was 6 weeks 4. the it given included methotrexate, cytarabine, thiotepa, and hydrocortisone and in some cases it was associated with systemic chemotherapy. in conclusion, the occurrence of neurological symptoms in a patient with myeloma requires an accurate evaluation with mr and lumbar puncture to detect a possible meningeal or cerebral involvement, when metabolic factors (hypercalcemia, uremia), hyperviscosity, or medullary compression can be excluded. herein, we confirmed that this hypothesis has to be considered also in patients with a disease apparently responsive to standard or high- dose therapy. there is no consensus toward the modalities of treatment ; in our experience, it chemotherapy and systemic chemotherapy did not have the expected result. | key clinical messagea patient with multiple myeloma with a mass in the nasopharyngeal was diagnosed. he received melphalan autograft and radiotherapy, and obtained complete remission. he relapsed 3 months later, with meningeal involvement and without systemic relapse. he received intrathecal and systemic chemotherapy, without neurological improvement and died 4 weeks after relapse. |
the flash electroretinogram (erg) provides an excellent noninvasive means to assess retinal function. the largest feature of this response is the b - wave, which follows the initial negative a - wave. the b - wave corresponds to the granit 's pii component and is believed to be generated by the activity of on - bipolar cells. however, a number of studies have suggested that the pii component may arise from several sources, thus complicating the interpretation of b - wave loss in studies of retinal disease. potential contributors to the pii include rod and cone bipolar cell responses, with the later showing faster rise and decay kinetics. on- and off - responses can also be visualized in erg responses from primates, rabbits, cats, and guinea pigs [47 ]. the responses of on- and off - bipolar cells may have distinct temporal signatures in the b - wave as on - bipolar cells have been shown to be more sustained compared with their off - counterparts in turtle retina. more recent studies suggest that bipolar cells appear to segregate into those producing transient and sustained responses [9, 10 ]. have attributed sustained and transient off - bipolar cell responses to differences in ionotropic glutamate receptors subtypes : kainate and ampa receptors on sustained and transient off - bipolar cells, respectively. the temporal characteristics of bipolar cell responses are also thought to be modified by inhibitory processes [1113 ]. a number of studies have shown that inhibition of glycine and gaba receptors can modify bipolar cell output [14, 15 ]. for example, inhibition of gabac receptors produces more sustained on bipolar cell responses [1618 ] as well as smaller and slower erg b - wave [17, 19, 20 ]. more recently, herrmann and coworkers showed that rod bipolar cell and the dark - adapted b - wave dynamic range in mice are extended by gabac - mediated sustained chloride current. whether a similar effect is seen in the rat erg has yet to be defined. first, the putative pii was isolated by removing photoreceptoral and associated glial and epithelial responses. second, response amplitudes are considered as a function of stimulus luminance systematically across a range of fixed times. finally, by applying this analytical approach to data collected following pharmacological manipulation, it is possible to show that the rat erg b - wave contains distinct fast and slow components. all animal experimental procedures were conducted with approval from our institutional animal experimentation ethics committee (a04001) and in accordance with the australian code of practice for the care and use of animals for scientific purposes. adult long evans rats between 10 and 12 weeks of age (180270 g, monash animal services, clayton, vic, aust) were maintained at 22c in a 3070 lux environment with a 12-hour light / dark cycle (on at 8 am). animals were dark adapted overnight (> 12 hours) and prepared under dim red light (max ~ 650 nm). anesthesia was induced with intramuscular injections of 60 mg / kg ketamine (ketamil 100 mg / ml, troy laboratories, smithfield, australia) and 5 mg / kg xylazine (xylazil 100 mg / ml, troy laboratories). corneal anesthesia was achieved by instillation of one drop of proxymetacaine (opthetic 0.5%, allergan, frenchs forest, nsw, australia). mydriasis was induced with one drop of 0.5% tropicamide (mydriacyl, alcon laboratories, frenchs forest, nsw, australia), giving complete pupil dilation (4 mm) within 15 minutes that was maintained for the duration of experiments. body temperature was maintained (37 0.5c) by placing the rat over a circulating - water heat pad (mgw lauda, lauda - knigshoffen, germany) and covering the animal with an insulating blanket. the animal and heat pad were mounted on a custom - built platform with the rat 's neck and abdomen lightly secured to the stage with velcro to minimize movement during recordings. the active electrode was placed at the center of the cornea, and the reference was a circular scleral electrode placed around the equator. a stainless steel needle (f - e2 - 30, grass - telefactor, west warwick, ri, usa) inserted subcutaneously into the tail served as the ground. following electrode placement 1.0% carboxymethylcellulose sodium (celluvisc, allergan, irvine, ca, usa) was applied to aid electrical conductivity. the signals were band - pass filtered (0.3 hz1 khz) over a 2560 (640 ms) epoch with a 4 khz sampling rate. a 50 hz notch filter was applied post hoc to eliminate line noise. stimulus duration of 256 ms was used to better visualize potential slow and fast components. white flashes (5 watt leds, luxeon, philips lumileds lighting co., ca, usa) were delivered via a ganzfeld integrating sphere (total diameter of 36 cm and 13 cm diameter aperture ; photometric solutions international, huntingdale, vic, australia). stimulus energy was calibrated using an il-1700 research radiometer (international light, newbury port, ma, usa) to return values in scotopic cdm. erg responses were obtained for flash luminances from 2.58 to 2.82 log cdm (2.58, 2.26, 1.94, 1.54, 0.95, 0.83, 0.62, 0.28, 0.04, 0.36, 0.76, 0.97, 1.27, 1.68, 2.18, 2.53, and 2.82 log cdm). the interstimulus interval was increased progressively from 45 to 240 s, to allow for complete recovery of b - wave amplitudes as established in pilot trials. as previously described [23, 24 ], pharmacological agents were injected into the vitreal chamber via a 30-gauge needle attached with polyethylene tubing (inner diameter = 0.38 mm) to a hamilton syringe (sge syringes, ringwood, australia). the needle was introduced through the pars plana, approximately 1 mm posterior to the superior limbus and at an angle of 45 degrees to avoid the lens. contact with the lens resulted in opacification, and data from such animals were excluded (2 log cdm). cnqx application resulted in a second phase of amplitude growth that was approximately 1 log unit more sensitive compared with the control data. figure 3(a) shows representative pii waveforms following inhibition of gabac receptors (black line) compared with controls (thin grey line). the offset relaxation was less apparent following tpmpa treatment, consistent with smaller onset amplitudes. analysis of the intensity - response characteristics at a50 (during light step, figure 3(b)) and a400 (after light step, figure 3(c)) shows that tpmpa resulted in changes that are qualitatively similar to cnqx treatment (see figure 2). specifically, at a400, tpmpa resulted in reduced amplitudes as well as a more sensitive second phase of amplitude increase. figures 4(a) and 4(b) show average amplitudes at selected fixed times optimized with a hyperbolic function (representative fixed times of a90 and a220). at these fixed times, a simple hyperbolic function (thick line) fails to fully describe the data, suggesting the presence of more than one component. the same data are better modeled by the summation (thin solid line) of two hyperbolic functions (dashed and grey lines), both having their exponents constrained to one. an f - test showed that a significantly better fit to the data could be achieved with the two hyperbolic functions (p 170 ms), another feature appears to grow in amplitude to contribute ~20% to the pii. this secondary amplitude growth, designated as m(3), may reflect the presence of an additional slower component or the relaxation of m(1) to a baseline during a light step. the k50 for m(1) (figure 4(d)) shows a transition at 170 ms, to become less sensitive (arrows) consistent with the possibility of intrusion from another component. its intensity - response function reveals a low sensitivity, suggesting the possibility that it might arise from the cone pathway. the waveform designated m(2) shows an increase in sensitivity to plateau at later times, consistent with a single component. figure 5(a) applies the same modeling approach to amplitudes at various fixed times following stimulus offset. the example shown here is for the fixed time of a400, which shows an initial growth in amplitude, followed by a reduction in magnitude between 0.62 and 1.27 log cdm and finally a second phase of amplitude growth. this intensity - response profile can not be modelled using one or even two hyperbolic functions. indeed, it can only be adequately modeled with three functions (figure 5(a)), the two negative components designated m(4) and m(5), with the addition of a positive component designated m(6). the merit function (log sum of square (ss) error) for the two- and three - component models was compared using an f - ratio. figure 5(b) shows that beyond a fixed time of 340 ms, the addition of a third positive hyperbolic function provides a statistically smaller ss compared with a two - function model. the vmax parameters for the three components increase with longer fixed times as shown in figure 5(c). figure 5(d) shows that the k50 parameters for the three components are distinct from each other and remain relatively constant across the range of fixed times after a light step. figure 6 shows the effect that cnqx and tpmpa have on the parameters of the putative m(1) and m(2) components. for vmax, m(1) was reduced by cnqx treatment (~59%) at early fixed times (180 ms). figure 6(d) shows that cnqx and tpmpa had little effect on the k50 of m(2). as was the case for the control data, those recorded following cnqx and tpmpa application were evaluated to consider if three components are evident following a light step. in both cnqx- and tpmpa - treated data, the log merit function shows that there was not a significant reduction in error to warrant the use of the more complex three hyperbolic function models ; thus, only two are needed to explain the trends in the drug - treated eyes. figures 7(a) and 7(b) show vmax after a light step derived from control and drug - treated data for m(4) and m(5). eyes treated with cnqx or tpma returned smaller amplitudes compared with controls. the reductions in amplitudes after a light step may simply reflect drug - induced reductions in amplitudes during the light step. the k50 is shown for each function in figure 7(d). a t - test shows for m(4) that the cnqx k50 is larger than that of controls (control : 2.55 0.04, cnqx : 2.12 0.05 ; p < 0.001), whereas tpmpa was similar. for m(5), both cnqx and tpmpa treatments produced k50 's that were smaller than controls (control : 0.78 0.02, cnqx : 0.39 0.04, tpmpa : 0.47 0.05 ; p < 0.001). we find that the isolated pii (control waveform apb / cnqx sensitive waveforms) in the pigmented rat electroretinogram has complex luminance response characteristics during and after a 256 ms light step. our approach of modeling the intensity - response function across a range of fixed times revealed that during a light step, the rat pii contains transient and sustained responses, which relax to baseline after the light step. in addition, there was a slow corneal positive response that follows stimulus offset. consistent with previous studies of rodent b - waves [28, 30 ], we also find that the isolated pii amplitude (in our case for a fixed time of 50 ms, a50) intensity - response function returned a slope of 0.99 (0.89, 1.09) (2.5%, 97.5%). thus, a single hyperbolic function can describe the earliest portions of the rat pii leading edge. however, a single hyperbolic function can not fully describe the intensity - response data for later fixed times after stimulus onset. such complex behavior has previously been attributed to interactions between negative piii and positive pii components [3133 ]. however, even after subtracting corneal negative waveforms isolated using apb / cnqx, the pii intensity - response function retains its complex profile, suggesting that there are multiple components underlying the response. to model the amplitude, intensity - response function extracted at fixed times after stimulus onset required two hyperbolic functions (figure 4), which segregate into processes that have high and low sensitivities to light (figure 4(d)) and are sustained (m(2)) and transient (m(1)) (figure 4(c)), respectively. this is consistent with previous studies suggesting that the b - wave contains sustained and transient responses (also termed slow and fast pii) [1, 9, 34, 35 ]. here, we show that these sustained and transient components do not arise from interactions with corneal negative responses. application of cnqx to block ionotropic glutamate receptors does not completely remove either m(1) or m(2), indicating that the transient and sustained responses do not arise simply from on- and off - bipolar. in addition to these sustained and transient components, there appears to be a third longer latency (140190 ms) component (designated as m(3)). this component has low amplitude (~ 20%, figure 4(c)) and is less sensitive than m(1) for times longer than ~140 ms (figure 4(d)). treatment with either cnqx or tpmpa removes the transition of k50 from a more sensitive m(1) to a less sensitive m(3), producing a k50 profile that resembles a single mechanism (figure 6(c)). this outcome suggests that m(3) is likely to be a continuation of m(1) during a light step. lateral elements in the ipl, in particular amacrine cells, can modulate the on - bipolar cell response and the erg b - wave [11, 17, 19, 36, 37 ]. inhibitory input has been shown to confer transient and sustained characteristics to bipolar cell neurotransmitter release [11, 38 ]. recordings from single cells suggest that such inhibition occurs via gabaa and gabac receptors onto rod and cone on - bipolar cells in rats. show that higher proportions of gabac to gabaa receptors - mediated inputs produce more prolonged inhibition, whereas a relative higher proportion of gabaa to gabac results in more transient inhibition. this is consistent with the finding that inhibition of gabac receptors using tpmpa makes bipolar cell responses in mouse, rat, and amphibian retina more sustained [16, 39 ] with a smaller response range. a less hyperpolarized resting membrane potential arising from loss of a tonic gaba - mediated current [21, 41 ] would account for smaller and more sustained b - wave. provide compelling evidence that dopamine - mediated gaba release possibly from horizontal cells, acting on both gabaa and gabac receptors, modulates the dynamic range of rod bipolar cells via a sustained cl current. activation of gabac receptors increases presynaptic cl influx, hyperpolarizing bipolar cell and thereby inhibiting ca entry via l - type ca - channels [11, 21, 38 ]. studies have shown that l - type ca channels may be differentially modified as a function of time following light onset [38, 42 ], implying that the on - bipolar cell responses may be both enhanced and inhibited at different times. in rat retina, a17 amacrine cells are known to make reciprocal synapses onto rod bipolar cells [11, 43 ]. it has been suggested, based on data from tiger salamander retina, that a glycinergic amacrine cell initially inhibits the a17 amacrine, thereby delaying its action at gabac receptors on on - bipolar cell axon terminals. in this way, amacrine cell inhibition of bipolar cells will occur only after the glycinergic amacrine cell inhibition onto the a17 amacrine cell is removed (estimated to be ~150 ms,). this mechanism provides an explanation for our finding that gabac - mediated inhibition produces a more transient pii component, by differentially modifying the pii at early and later times. specifically, our tpmpa data shows that there is amplitude enhancement and increased light sensitivity from 70 to 160 ms after onset of the light step, as well as a decrease in light sensitivity for times later than 180 ms (figure 6(c)). moreover, that cnqx results in the same qualitative outcome is consistent with amacrine cell involvement, as ampa / ka inhibition will affect both glycinergic and gabaergic amacrine cells.. suggest that dopamine released from amacrine cells might also modulate the tonic gaba currents at the level of rod bipolar cell dendrites. thus, modification of bipolar cell responses might occur at both outer and inner plexiform layers. it is possible that transient and sustained components described here can reflect both cone and rod bipolar cell responses. the difference in response range of m(2) and m(1) might suggest rod and cone on - bipolar cell contributions to these components, respectively. previous work by nixon and colleagues has shown that the rod b - wave is ~1.5 log units more sensitive than the cone b - wave for short duration flashes. this difference is comparable to that found at early fixed times for m(1) and m(2) seen here (< 120 ms, ~1.5 log units). however, interpreting m(1) and m(2) to be cone and rod mediated needs to guarded as responses from single rat rod bipolar cells to a step of light have a faster onset than the m(2) components isolated in this study. the response following the offset of a light step could not be modeled with two components and required a third hyperbolic function (figure 7). at very dim intensities however, with brighter intensities, two additional components become apparent, which are opposite in polarity (m(5) negative and m(6) positive). these offset components indeed, the k50 values for m(4) and m(5) correlate well with those for m(3) and m(2) (compare figure 7(d)to 6(c) and figure 7(d)to 6(d), respectively, consistent with this proposal. this possibility is also supported by the findings that cnqx (reduction at a5050.7 1.5% and a400 56.1 4.1%, p = 0.19) and tpmpa (reduction at a50 41.9 2.5% and a400 41.1 5.0%, p = 0.40) induce comparable reductions in amplitude for both onset (a50) and negative offset components (a400). a corneal positive component (figure 7(c)) apparent following stimulus offset (here designated m(6)) was only present in control eyes. naarendorp and williams have shown that positive off - component arising after a light step in rat was abolished after inhibition of ionotropic glutamate receptors, indicating an origin from hyperpolarizing bipolar cells. our finding that the m(6) was removed by cnqx, which inhibits ionotropic receptors, correlates well with this interpretation. hence, this component may also be modulated by disruption to inner retinal feedback as it is known that cnqx also modifies amacrine cell function. putative components m(1) to m(6) are plotted in figure 8 to yield the composite pii waveform in response to a 256 ms light step. based on our analysis, we believe that the isolated scotopic pii in rats contains at least three separate components. during a light step, there is an initial transient - on response, which is made more transient by gabac - mediated inhibitory processes. the majority of the pii for times greater than ~100 ms reflects a sustained - on component, likely arising from rod bipolar cells. after a light step, transient - on and sustained - on components relax to baseline. in addition, there is a positive sustained - off component that is likely to arise from the off - pathway, but it may also be modulated by gabac receptors. thus, assessing the intensity - response functions at early and later times during a light step provides a means by which transient and sustained contributions to the rat pii erg can be identified. | the most dominant feature of the electroretinogram, the b - wave, is thought to reflect on - bipolar cell responses. however, a number of studies suggest that the b - wave is made up of several components. we consider the composition of the rat b - wave by subtracting corneal negative components obtained using intravitreal application of pharmacological agents to remove postreceptoral responses. by analyzing the intensity - response characteristic of the pii across a range of fixed times during and after a light step, we find that the rat isolated pii has 2 components. the first has fast rise and decay characteristics with a low sensitivity to light. gabac - mediated inhibitory pathways enhance this transient - on component to manifest increased and deceased sensitivity to light at shorter (< 160 ms) and longer times, respectively. the second component has slower temporal characteristics but is more sensitive to light. gabac - mediated inhibition enhances this sustained - on component but has little effect on its sensitivity to light. after stimulus offset, both transient and sustained components return to baseline, and a long latency sustained positive component becomes apparent. the light sensitivities of transient - on and sustained - off components are consistent with activity arising from cone on- and off - bipolar cells, whereas the sustained - on component is likely to arise from rod bipolar cells. |
cat scratch disease (csd) is a self - limiting disease in immunocompetent patients. other bartonella spp. known to cause systemic and ocular manifestation are bartonella quintana, bartonella grahamii, and bartonella elizabethae.1,2 csd is transmitted to humans through a cat scratch, cat bite, cat saliva, or cat flea bite. csd may present in a wide spectrum of systemic manifestations, including endocarditis, hepatitis, encephalopathy, meningitis, transverse myelitis, hemolytic anemia, glomerulonephritis, and osteomyelitis. in immunocompromised patients, bartonella infection can cause a vasoproliferative response as seen in bacillary angiomatosis which resembles kaposi s sarcoma histologically.3,4 rarely, csd with ocular bartonellosis manifests as parinaud s oculoglandular conjunctivitis and intraocular inflammation (uveitis) in the form of neuroretinitis or retinochoroiditis. neuroretinitis was previously described as leber s idiopathic stellate neuroretinitis in 1916.5 it is characterized by optic disc swelling with partial or complete macular star,5 and occurs in 1%2% of patients with csd.6 neuroretinitis in association with serologic evidence of bartonella infection was documented in 1994.79 serologic tests for bartonella became readily available in recent years and are a means of establishing csd diagnosis. diagnosis of csd is mainly by clinical presentation and is confirmed with positive serologic analysis for bartonella. an increase in immunoglobulin (ig) m titer with a significant increase in igg antibodies titer can be considered an acute bartonella infection.10 the aim of this retrospective interventional case series is to describe features of neuroretinitis and serologic verification of b. henselae for ocular bartonellosis. this is a retrospective interventional case series of four patients who presented in the ophthalmology clinic at the hospital universiti sains malaysia, kubang kerian, malaysia from june 2012 to march 2013. all four patients had a history of contact with cats ; had fever prior to ocular symptoms ; and presented with optic disc swelling and macular edema, and had positive serology for b. henselae. table 1 shows the summary of the four patients concerning age and sex, systemic and ocular findings, and serologic analysis for b. henselae, comparing visual outcome and treatment given to the patients. a 15-year - old boy presented with gradual - onset, painless reduced vision of his left eye of 7 days duration. he had high - grade fever for 1 week prior to eye symptoms, which had resolved spontaneously. ocular examination revealed positive relative afferent pupillary defect (rapd) in his left eye. best corrected visual acuity (bcva) of the right eye was 6/9, and was 6/90 in the left eye. fundus examination showed features of bilateral neuroretinitis characterized by optic disc swelling with partial macular star (figure 1a and b). optical coherence tomography (oct) showed presence of bilateral subretinal fluid with loss of foveal depression (figure 1c and d). blood analysis of full blood count, renal profile, and liver function test were within normal limits, and erythrocyte sedimentation rate (esr) was 48 mm per hour. biochemistry, antinuclear antibodies (ana), complement studies, and rheumatoid factor studies were normal. serologic studies of cytomegalovirus (cmv), human immunodeficiency virus (hiv), toxoplasma, leptospira, and venereal disease research laboratory (vdrl) were also negative. serology analysis showed strongly positive igm (titers 1:384) and igg (titers 1:512) for b. henselae. the boy was treated empirically with oral azithromycin 250 mg daily for 6 weeks. the patient was also treated with oral prednisolone 60 mg daily for 1 week ; the oral prednisolone treatment was tapered down slowly within the next 1 month. after 6 weeks of systemic azithromycin therapy and oral prednisolone, there was improvement of visual acuity in the left eye (right eye : 6/9, left eye : 6/9) and bilateral resolving of optic disc swelling and macular star (figure 2a and b). there was resolution of panuveitis and subretinal fluid in both eyes (figure 2c and d). a 42-year - old malay lady was referred to our clinic from a district hospital for sudden onset painless blurring of vision in the left eye of 5 days duration, which was associated with floaters and a central scotoma. examination of the left eye showed optic disc swelling inferiorly and it was associated with multiple flame - shaped retinal hemorrhages. blood examination of full blood count, liver function test, renal profile, and esr were within normal limits. a serologic test detected toxoplasma igg antibody, but its avidity was 63.5, which indicates past infection. b. henselae ig titers were positive for igm (titers 1:20) and igg (titers 1:256). the patient was treated with oral hypoglycemic agent for her newly diagnosed diabetes mellitus. for the ocular bartonellosis, she was treated with azithromycin 250 mg daily for 4 weeks. follow - up at 1 month showed regression of optic disc swelling and resolving of macular edema with formation of partial macular star (figure 3c). two months after the initial visit, the visual acuity of the left eye had improved to 6/6 with complete resolution of optic disc swelling and macular edema (figure 3d). repeat oct of the left eye showed resolution of the subretinal fluid (figure 3e). a 23-year - old malay man presented with sudden - onset blurring of vision in the right eye of 2 weeks duration, which was associated with a central scotoma and retrobulbar pain fundus examination of the right eye showed optic disc swelling associated with flame - shaped retinal hemorrhages. there was presence of focal retinitis lesion at the temporal and superior margins of the optic disc (figure 4a). blood examination of full blood count, liver function test, renal profile, and esr were normal. serology analysis for b. henselae was positive (igm titers 1:96, igg titers 1:512). the patient was also treated with oral prednisolone 60 mg daily for 1 week ; the oral prednisolone treatment was tapered down slowly within the next 1 month. there was formation of macular star of the right eye at 1-week follow - up (figure 4c). at follow - up at 6 weeks, there was a slight improvement in visual acuity of the right eye, from counting fingers to 6/90. repeat oct of the right eye showed resolution of the subretinal fluid (figure 4e). a 27-year - old malay woman complained of painless, sudden blurred vision in the right eye of 1 day s duration, which was associated with a superotemporal scotoma. however, there were no floaters or flashes of light and nor was there any eye redness or eye discharge. she reported having been scratched by her cat at home 1 week prior to visual loss. she had a history of fever 3 days prior to the eye symptoms. physical examination revealed a body temperature of 37.0c with presence of right submandibular lymphadenopathy. however, there was no skin lesion. fundus examination of the right eye showed optic disc swelling at the inferior disc margin associated with macular star (figure 5a). blood examination of full blood count, liver function test, and renal profile were normal. serologic studies of cmv, hiv, toxoplasmosis, leptospira, and vdrl were also negative. serology analysis for b. henselae was positive for igm and igg (1:96 and 1:512, respectively). the patient was treated with oral doxycycline 100 mg 12-hourly for 4 weeks. at follow - up at 1 month, there was improvement of visual acuity of the right eye, from 6/30 to 6/7.5, with regression of optic disc swelling and prominent macular star (figure 5c). repeat oct of the right eye showed resolution of the subretinal fluid (figure 5d). a 15-year - old boy presented with gradual - onset, painless reduced vision of his left eye of 7 days duration. he had high - grade fever for 1 week prior to eye symptoms, which had resolved spontaneously. ocular examination revealed positive relative afferent pupillary defect (rapd) in his left eye. best corrected visual acuity (bcva) of the right eye was 6/9, and was 6/90 in the left eye. fundus examination showed features of bilateral neuroretinitis characterized by optic disc swelling with partial macular star (figure 1a and b). optical coherence tomography (oct) showed presence of bilateral subretinal fluid with loss of foveal depression (figure 1c and d). blood analysis of full blood count, renal profile, and liver function test were within normal limits, and erythrocyte sedimentation rate (esr) was 48 mm per hour. biochemistry, antinuclear antibodies (ana), complement studies, and rheumatoid factor studies were normal. serologic studies of cytomegalovirus (cmv), human immunodeficiency virus (hiv), toxoplasma, leptospira, and venereal disease research laboratory (vdrl) were also negative. serology analysis showed strongly positive igm (titers 1:384) and igg (titers 1:512) for b. henselae. the patient was also treated with oral prednisolone 60 mg daily for 1 week ; the oral prednisolone treatment was tapered down slowly within the next 1 month. after 6 weeks of systemic azithromycin therapy and oral prednisolone, there was improvement of visual acuity in the left eye (right eye : 6/9, left eye : 6/9) and bilateral resolving of optic disc swelling and macular star (figure 2a and b). there was resolution of panuveitis and subretinal fluid in both eyes (figure 2c and d). a 42-year - old malay lady was referred to our clinic from a district hospital for sudden onset painless blurring of vision in the left eye of 5 days duration, which was associated with floaters and a central scotoma. she had a cat at home but there was no history of cat scratch. physical examination revealed a body temperature of 37.0c with no lymphadenopathy. examination of the left eye showed optic disc swelling inferiorly and it was associated with multiple flame - shaped retinal hemorrhages. blood examination of full blood count, liver function test, renal profile, and esr were within normal limits. a serologic test detected toxoplasma igg antibody, but its avidity was 63.5, which indicates past infection. b. henselae ig titers were positive for igm (titers 1:20) and igg (titers 1:256). the patient was treated with oral hypoglycemic agent for her newly diagnosed diabetes mellitus. for the ocular bartonellosis, follow - up at 1 month showed regression of optic disc swelling and resolving of macular edema with formation of partial macular star (figure 3c). two months after the initial visit, the visual acuity of the left eye had improved to 6/6 with complete resolution of optic disc swelling and macular edema (figure 3d). repeat oct of the left eye showed resolution of the subretinal fluid (figure 3e). a 23-year - old malay man presented with sudden - onset blurring of vision in the right eye of 2 weeks duration, which was associated with a central scotoma and retrobulbar pain. fundus examination of the right eye showed optic disc swelling associated with flame - shaped retinal hemorrhages. there was presence of focal retinitis lesion at the temporal and superior margins of the optic disc (figure 4a). blood examination of full blood count, liver function test, renal profile, and esr were normal. serology analysis for b. henselae was positive (igm titers 1:96, igg titers 1:512). the patient was also treated with oral prednisolone 60 mg daily for 1 week ; the oral prednisolone treatment was tapered down slowly within the next 1 month. there was formation of macular star of the right eye at 1-week follow - up (figure 4c). at follow - up at 6 weeks, there was a slight improvement in visual acuity of the right eye, from counting fingers to 6/90. repeat oct of the right eye showed resolution of the subretinal fluid (figure 4e). a 27-year - old malay woman complained of painless, sudden blurred vision in the right eye of 1 day s duration, which was associated with a superotemporal scotoma. however, there were no floaters or flashes of light and nor was there any eye redness or eye discharge. she reported having been scratched by her cat at home 1 week prior to visual loss. she had a history of fever 3 days prior to the eye symptoms. physical examination revealed a body temperature of 37.0c with presence of right submandibular lymphadenopathy. however, there was no skin lesion. fundus examination of the right eye showed optic disc swelling at the inferior disc margin associated with macular star (figure 5a). blood examination of full blood count, liver function test, and renal profile were normal. serologic studies of cmv, hiv, toxoplasmosis, leptospira, and vdrl were also negative. serology analysis for b. henselae was positive for igm and igg (1:96 and 1:512, respectively). the patient was treated with oral doxycycline 100 mg 12-hourly for 4 weeks. at follow - up at 1 month, there was improvement of visual acuity of the right eye, from 6/30 to 6/7.5, with regression of optic disc swelling and prominent macular star (figure 5c). repeat oct of the right eye showed resolution of the subretinal fluid (figure 5d). neuroretinitis affects 1%2% of patients with b. henselae,6 and is characterized by optic disc swelling with partial or complete macular star.5 typically, patients present with unilateral neuroretinitis,11 and bilateral neuroretinitis is a rare presentation. in our study, one patient (case 1) had bilateral csd neuroretinitis and the remaining three patients had unilateral csd neuroretinitis. however, ocular bartonellosis with neuroretinitis may be complicated by retinal vascular occlusion causing permanent visual loss.12 other posterior segment presentations of csd include central retinal artery and vein occlusion, neovascular glaucoma, macular hole, choroiditis, retinal microgranuloma, serous macular detachment, and papillary vasoproliferative changes.1315 diagnosis of csd is based on epidemiology of exposure to cats, fundus finding, and positive serology or culture of b. henselae. approximately two - thirds of patients have positive serologic testing.16 serologic testing for bartonella by indirect immunofluorescence to detect serum anti - b. henselae antibodies has high sensitivities and specificities in immunocompetent patients.16 isolation of bartonella from blood or biopsy specimens is possible but difficult to yield, and growth of bacteria may require up to 4 weeks. cross - reactions are well known between bartonella spp. and, in the last 20 years, new zoonotic species were identified.17 epidemiological evidence18 and experimental studies19 have demonstrated the important role of fleas in the transmission of b. henselae to cats. high clinical suspicion of csd as a cause of vision - threatening intraocular inflammation is necessary to initiate prompt antibiotic treatment. early antibiotic treatment seems to improve visual outcome and hasten visual recovery.8,20 recommended antibiotics for ocular bartonellosis include gentamicin, doxycycline, azithromycin, trimethoprim / sulfamethoxazole, ciprofloxacin, and rifampicin, which have been reported to have good efficacy.20 patients treated with corticosteroids for ocular bartonellosis had a good response in several studies.2123 in our case series, two patients (case 1 and case 3) had poor vision at initial presentation that warranted the use of steroids. moreover, a study done involving csd - associated neuroretinitis in japan reported a good outcome with antibiotics and steroids, and no relapse occurred in their patient.24 ocular bartonellosis is a rare manifestation of csd, and neuroretinitis is a typical presentation.. vision - threatening ocular manifestation of csd can be improved with systemic antibiotics and steroids. | we report a case series of neuroretinitis in ocular bartonellosis and describe the serologic verification for bartonella henselae. this is a retrospective interventional case series of four patients who presented in the ophthalmology clinic of hospital universiti sains malaysia from june 2012 to march 2013. all four patients had a history of contact with cats and had fever prior to ocular symptoms. each patient presented with neuroretinitis characterized by optic disc swelling with macular star. serology analysis showed strongly positive for b. henselae in all of the patients. all patients were treated with oral azithromycin (except case 4, who was treated with oral doxycycline), and two patients (case 1 and case 3) had poor vision at initial presentation that warranted the use of oral prednisolone. all patients showed a good visual outcome except case 3. vision - threatening ocular manifestation of cat scratch disease can be improved with systemic antibiotics and steroids. |
disposing the 3d structure of small drug - like molecules can be critical for several computational approaches such as in silico screening (1,2), either ligand - based (35) or receptor structure - based (69) employed prior to or to complement experimental screening for hit identification, lead optimization or chemical biology purposes. in addition, for some of the methods, like rigid ligand docking or 3d ligand - based screening, a multiple conformer ensemble is required. chemical compounds are often distributed by chemical vendors in 1d smiles (simplified molecular input line entry system), 1d cansmiles (canonical smiles) (10) or in 2d sdf (11) (structure data file) formats. generating an accurate 3d structure for a small chemical compound is a complex task (12). different techniques using rule - based or data - based methods, building linker regions on pre - generated fragments or stochastic procedures up to quantum mechanical methods (12,13) have been developed. numerous studies have been carried out to compare the existing approaches and to analyze the small molecule conformations experimentally observed (14,15). they revealed that for a satisfactory sampling of the conformational space, the most important parameters to be optimized are the energy window with respect to the global minimum and the root mean square deviation (rsmd) value. several well established commercial packages such as corina (corina molecular networks, gmbh computerchemie langemarckplatz 1, erlangen, germany, 2000), omega, catalyst (14) or med-3dmc (16) generate multiple ensemble conformations of small molecules. in addition, several utilities like zinc (17), faf - drugs (18) or pubchem (18) take advantage of commercial software to propose pregenerated collections of compounds in 3d. yet, very few free tools are available for a single or multiple conformation generation. for instance, balloon (13) using a multi - objective genetic algorithm approach and multiconf - dock http://dock.compbio.ucsf.edu/contributed_code/index.htm (20) using a systematic search are freely available. the open source program dg - ammos (21) based on a distance geometry approach and molecular mechanics optimization has been recently reported. a practical alternative of the standalone packages, in particular for non - advanced users, are the web services which can provide direct 1d/2d to 3d facilities, such as openeye 's omega, molsoft, corina and from some academic sites such as at cbs. three years ago we developed and reported the web - service frog (22) (http://bioserv.rpbs.univ-paris-diderot.fr/frog.html) providing an on - line generation of a single or ensembles of 3d conformations for drug - like compounds. frog is a mixed rule - based data - based approach based on frowns (a chemoinformatics toolkit available at http://frowns.sourceforge.net/) to which several functionalities have been added to allow the generation of 3d structures starting from smiles or sdf data input. here, we describe a new version of frog, frog2, which is able to (i) generate single or ensembles of low to medium energy 3d conformations starting from 1d/2d or 3d input structure, to (ii) fully or partially disambiguate compound stereochemistry including chiral sites with a user - defined maximum number of generated conformers, and to (iii) minimize the energy of the generated conformers using ammos (18) if the user requires. the following important improvements are achieved in frog2 compared to frog1 : (i) generation of compound rings not available in the fragment database of frog1 using dg - ammos (17) ; (ii) significantly improved diversity of the generated conformer ensembles, and (iii) considerable increase of the computation speed. in addition, several ancillary tools are provided, such as the format interconversion using openbabel (23) or energy minimization of 3d conformations via ammos (24). frog2 still does not allow ring flexibility during the multiple confirmation generation which is under development. in this study, in addition to describing the web - service frog2, we validate frog2 on compounds from the astex dataset (25). the comparison of frog2 with frog1 and the commercial package omega (http://www.eyesopen.com) shows persuasive performance of frog2. the new and the optimized modules of frog2 are shown in the gray white boxes. overall, the frog internal 3d generation is based on a graph decomposition of the compound (22) coupled with an identification of the stereo centers for which the chirality is unspecified. once these sites have been identified, the combinatorial of the unambiguous isomers for which the chirality of all the identified stereo centers is completely specified is generated, but randomly truncated to a maximum of eight chiral centers for 3d generation. for each of these, a starting 3d conformation is generated. during this generation, frog2 takes advantage of dg - ammos to generate on the fly missing rings and adds them to the ring library, thus escaping a major limitation of frog1. instead, it relies on openbabel (23) to generate hydrogen coordinates for a standard protonation state. another frog2 major improvement comes from the optimization of the conformation ensemble generation engine. as in frog1 the first stage explores the conformational flexibility based on a limited number of representative dihedral angular values depending on atom types. this monte - carlo step includes a procedure to avoid conformation redundancy : new conformations that correspond to previously visited combinations of dihedral values are forbidden. also, the possibility of biasing the exploration of dihedral values depending on their position relative to the center of the compound has been introduced. for this, the probability that a step affects a particular dihedral is not uniform, but depends on the number of flanking atoms at each side, using the following weight :, where (resp.) right) of the bond undergoing the rotation, is the total number of atoms, so that dihedrals located at terminal positions have smaller weights compared to dihedrals having a balanced number of atoms each side. the second - stage monte carlo uniformly considers small rotations within the stage one conformations, so as to refine them. in order to prune the combinatorial exploration, this described conformation ensemble generation engine is supplemented by a standard divisive hierarchical clustering using an approach similar in essence to that used for fingerprints (26). this approach guarantees that all conformers in a class are below a fixed rmsd threshold and that the rmsd between selected conformers are always above that threshold. finally, in order to speed the computations, the rank of the conformers is based on an internal score of frog2 taking into account only van der waals interactions, which might be insufficient to prevent some geometry distortions during the assembly process. to overcome this limitation, it is possible to minimize the conformers using the ammp force field as implemented in ammos (24). frog2 accepts as input three formats widely used by the community : smiles, sdf and mol2 formats. since the sdf and mol2 formats can correspond to both 2d or 3d descriptions of a compound, the user must be precise among the two possible types of input : 1d/2d or 3d. specifying the 1d/2d type, all 3d information of the input will be discarded and the 3d generation will be performed from scratch. specifying the 3d type, frog2 will simply call the conformation ensemble generation engine, not considering stereoisomery and taking the input coordinates as a starting point for the conformation ensemble generation. parameters related to the generation of ensembles correspond to a maximal number of conformations, and energy thresholds to define the allowed energy window referred to the lowest energy conformation generated. it is also possible to specify a minimal rmsd value for geometric clustering of the conformers, and to invoke the minimization of the compounds. the energy minimization takes into account the valence, angle and van der waals components, not considering electrostatics terms. it is, however, possible to choose between mol2, sdf or pdb formats, thanks to openbabel. the new and the optimized modules of frog2 are shown in the gray white boxes. overall, the frog internal 3d generation is based on a graph decomposition of the compound (22) coupled with an identification of the stereo centers for which the chirality is unspecified. once these sites have been identified, the combinatorial of the unambiguous isomers for which the chirality of all the identified stereo centers is completely specified is generated, but randomly truncated to a maximum of eight chiral centers for 3d generation. for each of these, a starting 3d conformation is generated. during this generation, frog2 takes advantage of dg - ammos to generate on the fly missing rings and adds them to the ring library, thus escaping a major limitation of frog1. instead, it relies on openbabel (23) to generate hydrogen coordinates for a standard protonation state. another frog2 major improvement comes from the optimization of the conformation ensemble generation engine. as in frog1 the first stage explores the conformational flexibility based on a limited number of representative dihedral angular values depending on atom types. this monte - carlo step includes a procedure to avoid conformation redundancy : new conformations that correspond to previously visited combinations of dihedral values are forbidden. also, the possibility of biasing the exploration of dihedral values depending on their position relative to the center of the compound has been introduced. for this, the probability that a step affects a particular dihedral is not uniform, but depends on the number of flanking atoms at each side, using the following weight :, where (resp.) right) of the bond undergoing the rotation, is the total number of atoms, so that dihedrals located at terminal positions have smaller weights compared to dihedrals having a balanced number of atoms each side. the second - stage monte carlo uniformly considers small rotations within the stage one conformations, so as to refine them. in order to prune the combinatorial exploration, this described conformation ensemble generation engine is supplemented by a standard divisive hierarchical clustering using an approach similar in essence to that used for fingerprints (26). this approach guarantees that all conformers in a class are below a fixed rmsd threshold and that the rmsd between selected conformers are always above that threshold. finally, in order to speed the computations, the rank of the conformers is based on an internal score of frog2 taking into account only van der waals interactions, which might be insufficient to prevent some geometry distortions during the assembly process. to overcome this limitation, it is possible to minimize the conformers using the ammp force field as implemented in ammos (24). frog2 accepts as input three formats widely used by the community : smiles, sdf and mol2 formats. since the sdf and mol2 formats can correspond to both 2d or 3d descriptions of a compound, the user must be precise among the two possible types of input : 1d/2d or 3d. specifying the 1d/2d type, all 3d information of the input will be discarded and the 3d generation will be performed from scratch. specifying the 3d type, frog2 will simply call the conformation ensemble generation engine, not considering stereoisomery and taking the input coordinates as a starting point for the conformation ensemble generation. parameters related to the generation of ensembles correspond to a maximal number of conformations, and energy thresholds to define the allowed energy window referred to the lowest energy conformation generated. it is also possible to specify a minimal rmsd value for geometric clustering of the conformers, and to invoke the minimization of the compounds. the energy minimization takes into account the valence, angle and van der waals components, not considering electrostatics terms. it is, however, possible to choose between mol2, sdf or pdb formats, thanks to openbabel. we first assessed the frog2 performance for finding conformation similar to bioactive ones among the generated multiconformation ensemble. figure 2 shows the results for generating multiconformation ensembles of the astex dataset (25) containing 85 diverse drug - like molecules using frog2, frog1 and omega 2 [openeye scientific software (http://www.eyesopen.com) ]. the same input parameters were applied to run frog2, frog1 and omega, namely : up to 50 conformers, rmsd threshold of 0.8.. in order to make possible the comparison with frog1, using these input parameters, applying unambiguation for the stereoisomery and allowing the stage two monte - carlo, frog2 finds conformations closer to the bioactive ones than frog1 for most of the astex molecules. on average, the frog2 bioactive ' closest conformation is at rmsd values whereas frog1 shows an average rmsd of. interestingly, frog2 performs better than frog1 even without employing the stage two monte carlo with an average rmsd of, respectively. similar results in terms of rmsd are obtained using frog2 and omega when a maximum of 50 conformers were generated on the astex dataset with a slight, outperformance of omega with average values being of (wilcoxon signed rank test shows no significant difference). for a maximum of 50 conformers per molecule, omega and frog2 found conformations within an rmsd with the bioactive one of 1.5 for 82 and 79 compounds out of 85, respectively. that can be considered as an acceptable accuracy keeping in mind that the required rmsd for clustering the similar conformations was set to 0.8. finally, figure 2c illustrates the impact of increasing the maximal number of conformers up to 100. examples demonstrating the conformational diversity achieved by frog2 and omega when 50 conformers generated for two molecules of the astex dataset can be seen in figure 3. according to the computed rmsd values and visual analysis, one can conclude that both frog2 and omega explore quite well the conformational space and are able to generate conformations that are close to the bioactive one. in addition, the user has the possibility to energy - minimize the generated conformers via frog2 or to minimize a own compound library in 3d. our assessment of the impact of the minimization on the frog2-generated astex conformers does not show a significant improvement by means of finding the bioactive conformations. on average, the rmsd from the bioactive conformations was of, i.e. similar results to the ones obtained by frog2 without a final minimization stage. in addition, we validated the frog2 performance on a larger collection of compounds taken from the pdbbind database (27). the compounds contatining up to 15 rotatable bonds and not including large bridged rings (more than 30 atoms, see the discussion about frog2 limitations above) were retained. the distribution of the resulted test - set compounds depending on the number of rotatable bonds is given in the supplementary data. the results for rmsd between the best generated and experimental structures are also given in the supplementary data. the conformer ensembles were generated for a maximum of 50 conformers, enabling stage two monte carlo, and without applying minimization. as can be expected, the performance (in terms of rmsd) decreases with increasing molecular flexibility. the median rmsd to the experimental conformation is below 1 up to 7 rotatable bonds, and frog2 results remain however acceptable up to 15 rotatable bonds. figure 2.rmsd between the best - fitted conformers and the x - ray structures for conformers generated by : (a) frog1 versus frog2 for series of up to 50 conformers ; (b) frog2 versus omega for series of up to 50 conformers. pdb codes denote (i) the compounds with poorest relative performance, i.e. deviating the most from the diagonal (1y6b, 1xoz) and (ii) best performing for both frog2 and omega (1gpk, 1u4d, 1sqn) ; and (c) frog2 for series of up to 50 conformers versus frog2 for series of up to 100 conformers. figure 3.conformational ensembles for two small molecules of the astex dataset generated by frog2 (all atom colors, carbons in yellow) and omega (all atom colors, carbons in light pink : (a) pdb code 1jla ; and (b) pdb code 1meh. the experimental structure of the co - crystallized ligands are also shown (all atom colors, carbons in magenta). rmsd between the best - fitted conformers and the x - ray structures for conformers generated by : (a) frog1 versus frog2 for series of up to 50 conformers ; pdb codes denote (i) the compounds with poorest relative performance, i.e. deviating the most from the diagonal (1y6b, 1xoz) and (ii) best performing for both frog2 and omega (1gpk, 1u4d, 1sqn) ; and (c) frog2 for series of up to 50 conformers versus frog2 for series of up to 100 conformers. conformational ensembles for two small molecules of the astex dataset generated by frog2 (all atom colors, carbons in yellow) and omega (all atom colors, carbons in light pink : (a) pdb code 1jla ; and (b) pdb code 1meh. the experimental structure of the co - crystallized ligands are also shown (all atom colors, carbons in magenta). finally, we assessed the gain in speed of frog2 compared to frog1. on a computer with intel xeon processors at 2 ghz, omega - generated conformers for all the astex set in 6 min. in addition, the computational time can be reduced to 5 min if frog2 is run without the two monte carlo stage. in summary, frog2 generates better conformational quality and diversity than frog1 and is nine times faster than frog1 using conditions strictly comparable. frog2 still outperforms frog1 when employing the faster approach without two - stage monte carlo in terms of conformational diversity and is 20 times faster. this dramatic decrease in execution time without affecting the quality of the generated conformations demonstrates the impact of the optimizations implemented into frog2. we should note that activating the minimization option leads to significant increase of the computational time. over the astex set, using the disambiguation option and stage two monte carlo, the calculation times increased from 11 min up to 207 min, i.e. close to 20 times slower. however, the minimization may be required to energy - minimize some generated structures with detected structural inaccuracies, or for a small number of compounds. the main goal of the frog2 development was to overcome several limitations of frog1 and to increase the overall quality and speed performance. over these, rings were a central concern. indeed, frog2 dependency on a ring library is much less critical since frog2 takes the advantage of dg - ammos to generate on the fly missing rings. furthermore, frog2 addresses a frog1 issue problem related to rings ' protonation now generated standardly via openbabel. as a result, used over 10 000 compounds randomly selected out of 40 0000 cmps from the chembrdge diverset (http://www.chembridge.com/), frog2 only failed to generate conformers for < 2% of the compounds. as illustrated from the tests on the astex set, frog2 reaches a good structure quality. the frog2 development permitted to better control the diversity in the conformation ensemble generation and frog2 reaches extensive conformational diversity. further improvements are expected. in particular, better consideration of symmetry is under investigation. indeed, in a context of virtual screening experiments, where millions of compounds can be considered, the time to generate libraries can become a concern. one can suppose that once generated in 3d, a chemical compound collection can be in silico screened for different projects, however, more and more generation of focused libraries, either target - based or ligand - based, is required to increase the efficiency of discovery programs. in this respect, frog2 brings significant improvement since it is able to generate satisfactory quality conformations 20 times faster than frog1. we should note that a balance between the speed and conformational diversity can be achieved depending on the project purposes and the number of compounds to treat. inactivating the stage two monte carlo, operational on the server, will result in a faster generation, but a reduced conformational diversity will be reached. for applications requiring finally, frog2 takes advantage of ammos to offer the possibility to minimize the conformations that can be very helpful in some particular cases, even its use results in dramatically larger computational cost. several limitations are still present in frog2. the first one is related to the ring rigidity that can affect the quality or the diversity of the conformations generated in some cases. particularly, compounds including large bridged rings for which flexibility impacts the conformational search are presently out of frog scope. also, a side effect of the ring library strategy is that frog2 is presently not able to treat properly some cases of stereoisomery involving stereo centers in rings. finally, some errors in the generation of some particular groups can be presently observed since frog2 builds from scratch the linkers, at a single atom level, i.e. not combining pregenerated fragments. in addition, the implementation of some ring flexibility is under development. despite of the minor current limitations, the use of frog2 could help for various in silico studies, from ligand - based or structure - based virtual screening, to lead compounds optimization etc. thus, the frog2 server offers a very valuable tool which provides efficient features to the community for an extremely competitive computational time. | frog is a web tool dedicated to small compound 3d generation. here we present the new version, frog2, which allows the generation of conformation ensembles of small molecules starting from either 1d, 2d or 3d description of the compounds. from a compound description in one of the smiles, sdf or mol2 formats, the server will return an ensemble of diverse conformers generated using a two stage monte carlo approach in the dihedral space. when starting from 1d or 2d description of compounds, frog2 is capable to detect the sites of ambiguous stereoisomery, and thus to sample different stereoisomers. frog2 also embeds new energy minimization and ring generation facilities that solve the problem of some missing cycle structures in the frog1 ring library. finally, the optimized generator of conformation ensembles in frog2 results in a gain of computational time permitting frog2 to be up to 20 times faster that frog1, while producing satisfactory conformations in terms of structural quality and conformational diversity. the high speed and the good quality of generated conformational ensembles makes it possible the treatment of larger compound collections using frog2. the server and documentation are freely available at http://bioserv.rpbs.univ-paris-diderot.fr/frog2. |
highly chemoselective direct reduction of primary, secondary, and tertiary amides to alcohols using smi2/amine / h2o is reported. the reaction proceeds with c n bond cleavage in the carbinolamine intermediate, shows excellent functional group tolerance, and delivers the alcohol products in very high yields. the expected c o cleavage products are not formed under the reaction conditions. the observed reactivity is opposite to the electrophilicity of polar carbonyl groups resulting from the nx c = o (x = o, n) conjugation. mechanistic studies suggest that coordination of sm to the carbonyl and then to lewis basic nitrogen in the tetrahedral intermediate facilitate electron transfer and control the selectivity of the c n / c o cleavage. notably, the method provides direct access to acyl - type radicals from unactivated amides under mild electron transfer conditions. |
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screening with mammography has been shown to reduce mortality from breast cancer [1, 2 ]. however, the sensitivity to non - palpable cancer of screening mammography in radiographically dense - breasted women is as low as 3048%. extremely dense - breasted women have an 18-fold increase in interval cancer found between annual mammograms, compared with fatty - breasted women. magnetic resonance imaging (mri) has been demonstrated to be, and recommended as, an efficacious adjunct to mammography for very high - risk, dense - breasted women [5, 6 ]. three limitations to mri screening for breast cancer are cost, intravenous injection of gadolinium - containing contrast medium, and lower specificity of mri compared with mammography with increased false positive callbacks and biopsies. for radiographically dense - breasted women, whole - breast ultrasound as an adjunct to screening mammography has shown promise. berg. increased cancer discovery 42% by adding handheld whole - breast ultrasound performed by radiologists. kelly. used an automated whole - breast ultrasound (awbu) device capturing a cin loop of 2d breast images. this blinded study of mostly dense - breasted women showed a 100% increase in cancer detection, and a 200% increase in discovery of invasive cancers 1 cm or less, compared with mammograms alone. these cin loops were recorded and are available for reader trials similar to those performed for comparison of screening mammography with and without computer - aided detection (cad). for awbu to be a useful adjunct to screening mammography for dense - breasted women, interpretation of examinations must be shown as beneficial, when performed by community radiologists. this paper evaluates the performance of such radiologists in detection before and after awbu is added to a test set of screening mammograms of radiographically dense - breasted women. mammograms standard cranio - caudal (cc) and medio - lateral oblique (mlo) views of each breast were available for all cases. if implants were present, displacement views were included. original analog films (66 cases) or prints of digital films (36 cases) all cases used in the study provided informed consent, and the protocol was approved by the institutional review boards at each hospital, or the western institutional review board. awbus automated whole - breast ultrasound (awbu) is a computer - based system for performing, recording, and reading whole - breast ultrasound examinations similar in appearance to 2d freehand imaging (sonocine, reno, nv). the transducer is attached to a computer - guided mechanical arm that acquires images in cc rows overlapping 7 to 10 mm insuring complete coverage of both breasts. the awbu software creates a cin loop for review of approximately 3,000 images, simulating real - time imaging. the windows-based reading station uses a high - definition 1,600 1,200 monitor and special software to increase cancers conspicuity. twelve board - certified breast radiologists who use breast ultrasound in their practices were recruited as readers for the trial. one had reviewed limited awbus 8 years earlier during the developmental phase of the technology. each reader had a 4-h tutorial with one author (kk) explaining the awbu reading station operation. the readers reviewed and discussed approximately 12 awbus with known cancers, not part of the test set. they were not in the test set because either palpable findings were present or there were no concurrent mammograms. nothing concerning the study was discussed, other than the use of the data form (appendix a) and the number of cases to be reviewed. a set of 51 malignant cases (3 cases with bilateral cancers), including invasive and in situ cancer were collected for the trial (table 1). screening mammography and awbu the mammograms were heterogeneously dense or extremely dense breast tissue (birads 3 or 4) on the original reports. twelve cancers were included that were not prospectively reported on either imaging technique, but are visible in retrospect. four of these became palpable within 1 year, three in more than 1 year ; five were discovered in a subsequent screening round, three by awbu only, and two by both awbu and mammography. table 1pathological diagnosis of 51 positive cases (54 cancers)1 cm>1 to 2 cm>2 cmtotaldcis2046idc1719541ilc3216mixed idc and ilc0101total22221054dcis ductal carcinoma in situ, idc invasive ductal carcinoma, ilc invasive lobular carcinoma pathological diagnosis of 51 positive cases (54 cancers) dcis ductal carcinoma in situ, idc invasive ductal carcinoma, ilc invasive lobular carcinoma fifty - one normal cases performed from 2003 to 2008 were matched with each of the positive cases for the following factors : facilitydigital or analog mammogramultrasound machine modelamerican breast cup size (a dd)acr birads breast densityimplant (saline or silicone) and location (pre- or retropectoral)breast cancer historyagethe normal case matching factors 1 to 7 closest to the age of the positive case was matched as the normal partner case. the mean difference in age between the positive case and its matched normal was 31 days. digital or analog mammogram ultrasound machine model american breast cup size (a dd) acr birads breast density implant (saline or silicone) and location (pre- or retropectoral) breast cancer history testing occurred on a subsequent date at each reader s own site with only the reader and a research assistant (monitor) present. she had no knowledge of the test set makeup, had no mammography or ultrasound training, reviewed the test data forms in real - time for completeness, and transferred the data to the study database. at each test site 102 mammograms were placed on a film alternator in random order, generated once, and used for all readers. excluding breaks, the test subject s time for review was recorded. the upper half of a data form (appendix a) was completed for each case, checked by the monitor, and entered into the database. four questions were asked : would you request further evaluation based on this mammogram, or recommend routine screening?where is / are the most suspicious (up to 3) lesions, identifying their location by breast and clock face position?what would be your prediction of the final acr birads after any needed diagnostic workup was completed?what is the reader s confidence level that the woman has or does not have cancer (dmist likelihood scale) ? where is / are the most suspicious (up to 3) lesions, identifying their location by breast and clock face position ? what would be your prediction of the final acr birads after any needed diagnostic workup was completed ? what is the reader s confidence level that the woman has or does not have cancer (dmist likelihood scale) ? the american college of radiology breast imaging reporting and data system (birads) is a seven - point scale (0 = incomplete, needs additional assessment ; 1 = normal ; 2 = benign ; 3 = probably benign ; 4a = possible malignancy ; 4b = probable malignancy, or 5 = highly suggestive of malignancy) designed to categorize the results of mammography and other imaging studies [3, 11 ]. similar to the dmist, readers were asked to predict a birads score before any diagnostic workup. the dmist likelihood rating is a seven - point scale to express the confidence of the diagnosis, and ranges from definitely not cancer to definitely cancer [3, 11, 12 ]. a correct location response was recorded for an hour position marked within the half of the breast centered at the middle of the cancer. a true positive (tp) was recorded for mammography for any malignant case if callback was marked for mammography and any correct tumor location was identified. a tp was recorded for mammography plus awbu if callback was marked on either or both halves of the form in the malignant cases, with at least one correct location identified. thus, a correctly identified tp found with mammography would remain tp even were it not identified again on awbu. awbu findings could change the outcome to tp if a cancer was correctly identified with awbu, but missed with mammography. we evaluated readings on a per - case (i.e., per - patient) basis rather than a per - score basis because screening serves as a go no - go gatekeeper for subsequent workup. a true negative (tn) was recorded for mammography for any normal case if callback was not marked for mammography. a tn was recorded for mammography plus awbu for any normal case if callback was not marked on the second half of the form. this allowed the reader to reverse a callback for an asymmetric density seen mammographically but cleared by the awbu as no suspicion. to validate tn cases, all cases were followed for at least 1 year or more. a false positive (fp) was recorded for mammography in two situations : callback was marked for mammography in a normal case.callback was marked for mammography in a cancer case, but none of the marked locations corresponded to the cancer. callback was marked for mammography in a cancer case, but none of the marked locations corresponded to the cancer. an fp was recorded for mammography plus awbu in the same two situations as above when callback was marked for awbu. a false negative (fn) was recorded for mammography when callback was not marked in a cancer case in the mammography portion of the form. similarly, an fn was recorded for mammography plus awbu when callback was not marked in a cancer case in either portion of the form. the 102 abwus were reviewed by readers on a review station brought by the research assistant acting as a monitor. they worked approximately 8 h daily for 3 days, with breaks at the readers choosing. the readers were given the corresponding mammograms with each awbu and completed the second half of the data sheet with the knowledge from the mammogram - only evaluation available. the same questions were answered for awbu and the reading time of each awbu recorded. analyses were conducted in a multi - reader multi - case (mrmc) framework where each reader screened all cases and each case contained both screening techniques. the mrmc design efficiently reduces the number of readers and cases needed to detect improvements across techniques. analyses appropriate for an mrmc design were chosen both to correctly model correlations between readings on the same case across readers and to estimate correctly standard errors. unless specified otherwise, analyses were conducted in sas software version 9.2 (sas institute inc. we present f statistics, shown as f(numerator degrees of freedom, denominator degrees of freedom), and p values for comparisons between mammography plus awbu and mammography alone. cases identified for further imaging were assessed by four binary measures : sensitivity = number of tp / number of cancer cases ; specificity = number of tn / number of non - cancer cases ; positive predictive value (ppv) = number of cancer cases/(number of tp + fp cases) ; and negative predictive value (npv) = number of non - cancer cases/(number of fn + tn). random - effect logistic regression models were used to test whether each binary measure differed significantly between mammography plus awbu versus mammography alone. to account for the mrmc framework accuracy was assessed through birads ratings and dmist likelihood scores, comparing two commonly used indicators of accuracy between mammography plus awbu versus mammography alone : areas under the curve (auc) and figures of merit (fom). the fom incorporates information from each reader on the region of suspected malignancy, as well as their confidence level in the finding, incorporated in an auc. because it includes both confidence level and location accuracy, the fom is more powerful than auc in detecting differences between techniques. we include both analyses, as described below : areas under the curve (auc) were estimated in dbm mrmc 2.1 (available from http://perception.radiology.uiowa.edu) using the trapezoidal / wilcoxon method. we also present reader - averaged receiver operating characteristic (roc) curves ; average values were calculated from separate roc analyses conducted on each reader in the proc logistic procedure. figures of merit (fom) were estimated by using jackknife alternative free - response receiver operating characteristic methodology as implemented in jafroc version 1.0 (available from http://www.devchakraborty.com). the fom is defined as the probability that a cancer on an abnormal image is scored higher than a falsely marked location on a normal image and is analogous to the roc curve ; a higher fom indicates improvement in reader performance. confidence in identification of cases for further imaging we used linear regression, comparing birads ratings and dmist likelihood scores across the two screening techniques among tp cases ; mean ratings and scores are estimated by the regression for each screening technique. to account for the mrmc framework, we included random effects similar to the dbm model ; the model included a fixed effect for technique, classified as mammography plus awbu or mammography alone, and random effects for readers and cases. mammograms standard cranio - caudal (cc) and medio - lateral oblique (mlo) views of each breast were available for all cases. if implants were present, displacement views were included. original analog films (66 cases) or prints of digital films (36 cases) all cases used in the study provided informed consent, and the protocol was approved by the institutional review boards at each hospital, or the western institutional review board. awbus automated whole - breast ultrasound (awbu) is a computer - based system for performing, recording, and reading whole - breast ultrasound examinations similar in appearance to 2d freehand imaging (sonocine, reno, nv). the transducer is attached to a computer - guided mechanical arm that acquires images in cc rows overlapping 7 to 10 mm insuring complete coverage of both breasts. the awbu software creates a cin loop for review of approximately 3,000 images, simulating real - time imaging. the windows-based reading station uses a high - definition 1,600 1,200 monitor and special software to increase cancers conspicuity. twelve board - certified breast radiologists who use breast ultrasound in their practices were recruited as readers for the trial. one had reviewed limited awbus 8 years earlier during the developmental phase of the technology. each reader had a 4-h tutorial with one author (kk) explaining the awbu reading station operation. the readers reviewed and discussed approximately 12 awbus with known cancers, not part of the test set. they were not in the test set because either palpable findings were present or there were no concurrent mammograms. nothing concerning the study was discussed, other than the use of the data form (appendix a) and the number of cases to be reviewed. a set of 51 malignant cases (3 cases with bilateral cancers), including invasive and in situ cancer were collected for the trial (table 1). screening mammography and awbu the mammograms were heterogeneously dense or extremely dense breast tissue (birads 3 or 4) on the original reports. twelve cancers were included that were not prospectively reported on either imaging technique, but are visible in retrospect. four of these became palpable within 1 year, three in more than 1 year ; five were discovered in a subsequent screening round, three by awbu only, and two by both awbu and mammography. table 1pathological diagnosis of 51 positive cases (54 cancers)1 cm>1 to 2 cm>2 cmtotaldcis2046idc1719541ilc3216mixed idc and ilc0101total22221054dcis ductal carcinoma in situ, idc invasive ductal carcinoma, ilc invasive lobular carcinoma pathological diagnosis of 51 positive cases (54 cancers) dcis ductal carcinoma in situ, idc invasive ductal carcinoma, ilc invasive lobular carcinoma fifty - one normal cases performed from 2003 to 2008 were matched with each of the positive cases for the following factors : facilitydigital or analog mammogramultrasound machine modelamerican breast cup size (a dd)acr birads breast densityimplant (saline or silicone) and location (pre- or retropectoral)breast cancer historyagethe normal case matching factors 1 to 7 closest to the age of the positive case was matched as the normal partner case. the mean difference in age between the positive case and its matched normal was 31 days. digital or analog mammogram ultrasound machine model american breast cup size (a dd) acr birads breast density implant (saline or silicone) and location (pre- or retropectoral) breast cancer history testing occurred on a subsequent date at each reader s own site with only the reader and a research assistant (monitor) present. she had no knowledge of the test set makeup, had no mammography or ultrasound training, reviewed the test data forms in real - time for completeness, and transferred the data to the study database. at each test site 102 mammograms were placed on a film alternator in random order, generated once, and used for all readers. excluding breaks, the test subject s time for review was recorded. the upper half of a data form (appendix a) was completed for each case, checked by the monitor, and entered into the database. four questions were asked : would you request further evaluation based on this mammogram, or recommend routine screening?where is / are the most suspicious (up to 3) lesions, identifying their location by breast and clock face position?what would be your prediction of the final acr birads after any needed diagnostic workup was completed?what is the reader s confidence level that the woman has or does not have cancer (dmist likelihood scale) ? where is / are the most suspicious (up to 3) lesions, identifying their location by breast and clock face position ? what would be your prediction of the final acr birads after any needed diagnostic workup was completed ? what is the reader s confidence level that the woman has or does not have cancer (dmist likelihood scale) ? the american college of radiology breast imaging reporting and data system (birads) is a seven - point scale (0 = incomplete, needs additional assessment ; 1 = normal ; 2 = benign ; 3 = probably benign ; 4a = possible malignancy ; 4b = probable malignancy, or 5 = highly suggestive of malignancy) designed to categorize the results of mammography and other imaging studies [3, 11 ]. similar to the dmist, readers were asked to predict a birads score before any diagnostic workup. the dmist likelihood rating is a seven - point scale to express the confidence of the diagnosis, and ranges from definitely not cancer to definitely cancer [3, 11, 12 ]. a correct location response was recorded for an hour position marked within the half of the breast centered at the middle of the cancer. a true positive (tp) was recorded for mammography for any malignant case if callback was marked for mammography and any correct tumor location was identified. a tp was recorded for mammography plus awbu if callback was marked on either or both halves of the form in the malignant cases, with at least one correct location identified. thus, a correctly identified tp found with mammography would remain tp even were it not identified again on awbu. awbu findings could change the outcome to tp if a cancer was correctly identified with awbu, but missed with mammography. we evaluated readings on a per - case (i.e., per - patient) basis rather than a per - score basis because screening serves as a go no - go gatekeeper for subsequent workup. a true negative (tn) was recorded for mammography for any normal case if callback was not marked for mammography. a tn was recorded for mammography plus awbu for any normal case if callback was not marked on the second half of the form. this allowed the reader to reverse a callback for an asymmetric density seen mammographically but cleared by the awbu as no suspicion. to validate tn cases, all cases were followed for at least 1 year or more. a false positive (fp) was recorded for mammography in two situations : callback was marked for mammography in a normal case.callback was marked for mammography in a cancer case, but none of the marked locations corresponded to the cancer. callback was marked for mammography in a cancer case, but none of the marked locations corresponded to the cancer. an fp was recorded for mammography plus awbu in the same two situations as above when callback was marked for awbu. a false negative (fn) was recorded for mammography when callback was not marked in a cancer case in the mammography portion of the form. similarly, an fn was recorded for mammography plus awbu when callback was not marked in a cancer case in either portion of the form. the 102 abwus were reviewed by readers on a review station brought by the research assistant acting as a monitor. they worked approximately 8 h daily for 3 days, with breaks at the readers choosing. the readers were given the corresponding mammograms with each awbu and completed the second half of the data sheet with the knowledge from the mammogram - only evaluation available. the same questions were answered for awbu and the reading time of each awbu recorded. analyses were conducted in a multi - reader multi - case (mrmc) framework where each reader screened all cases and each case contained both screening techniques. the mrmc design efficiently reduces the number of readers and cases needed to detect improvements across techniques. analyses appropriate for an mrmc design were chosen both to correctly model correlations between readings on the same case across readers and to estimate correctly standard errors. unless specified otherwise, analyses were conducted in sas software version 9.2 (sas institute inc., we present f statistics, shown as f(numerator degrees of freedom, denominator degrees of freedom), and p values for comparisons between mammography plus awbu and mammography alone. cases identified for further imaging were assessed by four binary measures : sensitivity = number of tp / number of cancer cases ; specificity = number of tn / number of non - cancer cases ; positive predictive value (ppv) = number of cancer cases/(number of tp + fp cases) ; and negative predictive value (npv) = number of non - cancer cases/(number of fn + tn). random - effect logistic regression models were used to test whether each binary measure differed significantly between mammography plus awbu versus mammography alone. to account for the mrmc framework accuracy was assessed through birads ratings and dmist likelihood scores, comparing two commonly used indicators of accuracy between mammography plus awbu versus mammography alone : areas under the curve (auc) and figures of merit (fom). the fom incorporates information from each reader on the region of suspected malignancy, as well as their confidence level in the finding, incorporated in an auc. because it includes both confidence level and location accuracy, the fom is more powerful than auc in detecting differences between techniques. we include both analyses, as described below : areas under the curve (auc) were estimated in dbm mrmc 2.1 (available from http://perception.radiology.uiowa.edu) using the trapezoidal / wilcoxon method. we also present reader - averaged receiver operating characteristic (roc) curves ; average values were calculated from separate roc analyses conducted on each reader in the proc logistic procedure. figures of merit (fom) were estimated by using jackknife alternative free - response receiver operating characteristic methodology as implemented in jafroc version 1.0 (available from http://www.devchakraborty.com). the fom is defined as the probability that a cancer on an abnormal image is scored higher than a falsely marked location on a normal image and is analogous to the roc curve ; a higher fom indicates improvement in reader performance. confidence in identification of cases for further imaging we used linear regression, comparing birads ratings and dmist likelihood scores across the two screening techniques among tp cases ; mean ratings and scores are estimated by the regression for each screening technique. to account for the mrmc framework, we included random effects similar to the dbm model ; the model included a fixed effect for technique, classified as mammography plus awbu or mammography alone, and random effects for readers and cases. sample subjects averaged 59.4 years of age (sd = 10.2 ; range = 4183). the 51 cancer patients and 51 normal subjects were well - matched with an insignificant mean difference of 31.0 days in age between abnormal and normal cases (t test = 1.47, df = 50, p = 0.15). identification of cases for further imaging table 2 details individual performance in the identification of cancer cases for further imaging. mean sensitivity increased from 50% to 81%, an improvement of 63% in the number of cancer cases identified (25.4 vs. 41.4, f(1, 1,161) = 165.95, p 1 to 2 cm>2 cmtotal#%#%#%#% # of cancers1710022100610045100mean cancers by mammography4.42613.5613.05020.946mean additional cancers by awbu6.7396.6302.03315.334mean total cases detected11.16520.1915.08336.280% improvement compared to mammography alone151%49%67%73%for cases with more than one invasive tumor, the larger of the two was used. for interval cancers after imaging, size is the greatest diameter of the tumor seen retrospectively on the awbu or mammogram, otherwise the diameter is that reported by pathological diagnosis reader performance categorized by imaging technique (n = 102, 51 positive cases) m mammography, m+a mammography plus automated whole - breast ultrasound (awbu) reader # presented by best to worst performance based on sensitivity on m+a reader performance with 45 invasive cases for cases with more than one invasive tumor, the larger of the two was used. for interval cancers after imaging, size is the greatest diameter of the tumor seen retrospectively on the awbu or mammogram, otherwise the diameter is that reported by pathological diagnosis accuracy the roc area was greater for mammography plus awbu for both birads (0.808 versus 0.701 ; f(1, 123) = 14.79, p < 0.001) and likelihood scores (0.810 versus 0.703 ; f(1, 85) = 17.88, p < 0.001) as estimated by multi - reader multi - case analyses. this is highlighted in fig. 1 by roc curves that are generated by averaging the results of separate roc analyses for each reader. the birads and likelihood auc curves for mammography and mammography plus awbu in both cases almost superimpose when confidence in malignancy by mammography is high, but when confidence in malignancy by mammography is low, as in the lower portions of the graphs, the curves in both cases diverge significantly. in both cases the mammography plus awbu approaches the y - axis indicating better cancer recognition. 1receiver operating characteristic curves averaged across 12 readers for mammography alone (circles and dashed line) and mammography plus awbu (triangles and solid line)figure 2 shows the areas under the roc curves for each reader and for the average of all readers as estimated by multi - reader multi - case analyses. these individual line graphs mirror the improvement in reader performance shown in table 2. 2changes in areas under the receiver operating characteristic curve(s) for each reader (hollow circles) and averaged across 12 readers (solid circles)similar to roc areas, the figures of merit (fom) were higher for mammography plus awbu across all readers, compared with mammography alone using both the birads scores (0.786 versus 0.613 ; f(1, 270) = 34.1, p < 0.001) and dmist likelihood scores (0.791 versus 0.614 ; f(1, 238) = 37.9, p receiver operating characteristic curves averaged across 12 readers for mammography alone (circles and dashed line) and mammography plus awbu (triangles and solid line) changes in areas under the receiver operating characteristic curve(s) for each reader (hollow circles) and averaged across 12 readers (solid circles) confidence in identification of cases for further imaging readers reviewing cancer cases were more confident in correctly identifying cases for further imaging, i.e., tp reading, using mammography plus awbu compared with mammography alone. on average, both birads scores (mean = 4.8 versus 4.2, f(1, 740) = 81.91, p < 0.001) and dmist likelihood scores (mean = 4.8 versus 4.1, f(1, 740) = 82.21, p < interpretation times average reading time per study for the 102 awbus was 7 min 58 s (7:58) varying from 5:54 to 12:51. the difference in review time was unrelated to the number of cancers identified by each reader (correlation = 0.02, p = 0.96). this is shown by a 63% increase in callbacks of cancer cases with only a 4% decrease in correct identification of the true negative cases. the confidence of the diagnoses of the 102 cases with predictive birads and dmist likelihood scales was confirmed by using auc and fom methodology. with a short training period experienced radiologists using 2d awbu significantly improve their ability to diagnose cancer in dense - breasted women. the slower transducer speed enforced by the awbu decreases inter - image distance, allowing the reader more time to identify small masses. at a review speed of 10 images per second the observer has 0.5 s to identify a 5-mm mass. a high - resolution computer screen, along with a post - processing technique to expand the grayscale at the black end of the spectrum, results in visually sharper margins and more contrast of masses against the background tissue. this automated process for breast ultrasound eliminates operator variability, provides greater consistency, and ensures reproducibility of quality images. study radiologists increased discovery of t1a and t1b invasive cancers 150% over mammography alone (table 3). the average review time per awbu study was about 11 min shorter than the 19 min for radiologists in the acrin 6666 trial of handheld screening ultrasound. as half of our test set subjects had cancers, it would be expected that the average review time we observed for awbu would be significantly longer than in a typical screening population with mostly normal studies. although the test set was confidential, the readers probably quickly realized that it was enriched. a false increase in tps would occur if all the correctly identified cancers were not subsequently confirmed with biopsy. also, analysis was performed on a case basis in the three patients in whom cancers were present in both breasts ; it was assumed if one of the cancers was identified, the cancer in the other breast would be found by the subsequent workup. any of the following factors could have decreased the readers accuracy with awbu (decreased true positives and negatives, and increased false positives and negatives) compared with a normal screening situation. some readers do not perform screening ultrasound.limited experience with awbu this was the first exposure to awbu for 11 of the readers.unfamiliarity with some ultrasound formats images from many different manufacturers were used. limited experience with awbu this was the first exposure to awbu for 11 of the readers. unfamiliarity with some ultrasound formats images from many different manufacturers were used. in spite of these hindrances our observations clearly show that radiologists improve detection of cancers, especially small invasive ones, by adding awbu to mammography findings. radiologists will significantly improve their cancer detection rates in dense - breasted women by adding awbu to mammography. this procedure has the potential for both standardizing the performance of whole - breast ultrasound and shortening the time required for radiologists. | objectiveradiologist reader performance for breast cancer detection using mammography plus automated whole - breast ultrasound (awbu) was compared with mammography alone.methodsscreenings for non - palpable breast malignancies in women with radiographically dense breasts with contemporaneous mammograms and awbu were reviewed by 12 radiologists blinded to the diagnoses ; half the studies were abnormal. readers first reviewed the 102 mammograms. the american college of radiology (acr) breast imaging reporting and data system (birads) and digital mammographic imaging screening trial (dmist) likelihood ratings were recorded with location information for identified abnormalities. readers then reviewed the mammograms and awbu with knowledge of previous mammogram - only evaluation. we compared reader performance across screening techniques using absolute callback, areas under the curve (auc), and figure of merit (fom).resultstrue positivity of cancer detection increased 63%, with only a 4% decrease in true negativity. reader - averaged auc was higher for mammography plus awbu compared with mammography alone by birads (0.808 versus 0.701) and likelihood scores (0.810 versus 0.703). similarly, fom was higher for mammography plus awbu compared with mammography alone by birads (0.786 versus 0.613) and likelihood scores (0.791 versus 0.614).conclusionadding awbu to mammography improved callback rates, accuracy of breast cancer detection, and confidence in callbacks for dense - breasted women. |
uveitis, especially noninfectious uveitis, is more common in women than in men, in most large surveys, presumably because of the greater frequency of autoimmune diseases in women. sex - determined biological variations in type or intensity of response to infections may exist, just as they appear to exist in noninfectious and autoimmune disorders. if anything, the sex differences in infectious uveitis are likely to be greater than in autoimmune disorders because exposure to infections involves behavioral and cultural issues not encountered with the other uveitides. differences in sexual behaviors between men and women would predict that uveitis associated with sexually transmissible diseases such as hiv and syphilis would be even more likely to show gender disparities than infections transmitted through environmental and occupational exposures. monogamy and strict adherence to ideals of chastity and faithfulness in some cultures are important social factors that would likely reduce the prevalence of sexually transmitted infectious diseases in women. examples that would increase risk of infection among women are sex work and heterosexual transmission from bisexual or promiscuous male partners. greater exposure increases the risk of infectious uveitis, even though uveitis typically arises in only a small percentage of infected individuals. it is unknown whether men or women would have greater susceptibility on a biological basis to uveitis associated with infectious diseases. in general, men seem to be more susceptible to infections in multiple species. the development of uveitis may depend on other factors that would skew prevalence toward one sex or the other. for nonsexually transmitted types of infectious uveitis, the difference between male and female prevalence seems to be small indicating that large hormonal influences are unlikely. for sexually transmitted diseases, behavioral factors are likely to overshadow any biological effects related to sex - specific gene expression. calculation of odds ratios based on proportions of women with sexually transmitted infectious diseases only versus those with both systemic and ocular manifestations would require more detailed data than is currently available. in addition to unequal transmission of infections predisposing to infectious uveitis, women have additional concerns related to vertical transmission of infections during pregnancy, most commonly those infections included in the torch spectrum (toxoplasmosis, cytomegalovirus, and herpes simplex) which can have devastating ocular consequences. it is the purpose of this review to examine infectious uveitis from the standpoint of its relationship to occupational transmission, sexual transmission, and vertical transmission of pathogens. a medline search was conducted for peer - reviewed articles in english published from 1990 to 2014 that concerned infectious uveitis qualified by search terms such as prevalence, female, congenital, transmission, and hiv. occupational exposure to pathogens that have a high penetrance of ocular involvement may display unequal sex ratios as dramatic as those related to sexually transmitted disease. leptospira uveitis in india is often associated with farming or other exposures to animals in rural areas, occupations more likely undertaken by men. other epidemiological patterns include exposure to ground water in tropical climates and rodents in urban areas, which are more likely to affect the general population including women and children. it is estimated that up to 10% of patients with the systemic disease will have ocular manifestations. the male to female ratio of leptospiral uveitis was 3 : 1 in one study. sex differences in brucellar uveitis are another example of differing occupational exposures to animal vectors that result in infectious uveitis. men aged 2045 years, engaged in butchering or rendering animal carcasses, seem to be at special risk because of exposure to b. abortus or b. suis (http://www.who.int/csr/resources/publications/brucellosis.pdf, accessed 12 april, 2014). in contrast, women in peru are twice as likely as men to have brucellar uveitis. this is because the manufacture, distribution, or consumption of sheep and goat milk products places more women and children at risk of exposure to a more virulent species, b. melitensis, (http://www.who.int/csr/resources/publications/brucellosis.pdf, accessed 12 april, 2014). in western iran, brucellosis is more common in housewives than in farmers ; however, in both western and central iran, the male : female ratio was 2.1. ophthalmic manifestations, especially in chronic brucellosis, include posterior uveitis in about 40% of patients and anterior, intermediate, or panuveitis in another 15% each. hunters who field dress animals may acquire toxoplasmosis and, unlike the other zoonoses or lyme disease from ticks, then transmit the disease through household exposure to the meat. viable t. gondii was isolated from 17%29% of white - tailed deer hunted in the united states. women who assume traditional roles of food preparation can be exposed when handling the meat ; a social history should include the possible exposure to infected wild meat. in endemic areas of toxoplasmosis in brazil, the production and ingestion of contaminated sausage may be a factor in the very high prevalence of toxoplasma chorioretinitis in that population. clustering of toxoplasma seropositivity among all ages and sexes sharing the same household suggests that foodborne transmission is important in endemic areas of brazil. differences in prevalence of nonsexually transmitted infectious uveitis between the sexes would depend on the amount, type, and infectivity of the activities to which each sex was typically exposed in their culture. when all persons are exposed mainly through food, an equal sex ratio would be expected. the most striking differences between prevalence in men and women would be expected in diseases that are sexually transmitted because exposure involves a large behavioral component. in 2011 in the usa, there were 8.3 per 100,000 primary and secondary syphilis infections in men versus 1.0 per 100,000 in women. a large imbalance in syphilitic uveitis would also be expected. (http://www.cdc.gov/std/stats11/tables.htm, accessed 01 sep, 2013, 2:00 pm.) the virulence and persistence of syphilis to the point of causing central nervous system or ocular manifestations is influenced by concomitant hiv infection. among hiv infected patients, syphilitic uveitis is almost exclusively seen in men ; in one meta - analysis, 97 of 101 patients were male. this balance may change as the proportion of hiv - infected women grows relative to men. among non - hiv infected patients the imbalance in syphilitic uveitis is less striking. a small chinese case series of 14 non - hiv infected patients with syphilitic uveitis showed only a slight male predominance. syphilis may be more evenly distributed between men and women in china because of fewer men having sex with men or other behavioral factors. access to care for early treatment or frequency of screening may differ between men and women. penetrance of syphilis infection in the chinese population may also be less overall than in the west, resulting in a skewed sample from a very small number of patients ; epidemiological information about syphilis in china could not be obtained from online resources. in the united states, the cdc does not separately tally syphilitic uveitis, but only tallies total cases of syphilis in men and women. conversely, cases of syphilitic uveitis are usually reported without background information regarding the number of cases without ocular disease from the same population and without concurrent controls that have syphilis but not uveitis and might also show gender imbalances from which susceptibility to symptomatic ocular disease could be ascertained. presumably intraocular involvement is considerably rarer than the systemic infection that causes it in any cultural setting, although the uveitis or optic neuropathy can be most symptomatic manifestation of untreated latent disease. an international series of syphilitic uveitis of the posterior placoid variant recorded 9 of 60 (15%) of the newly reported and previously published patients to be females. hiv infection was confirmed in 9 of the current and 14 of the historical patients (38%). among hiv negative patients there was a much higher number of women : 8 of 37 hiv negative patients were women (24.3%) versus 1 of the remaining 23 patients (4.3%). the relatively low prevalence of hiv infection in this series of syphilitic uveitis raises the issue of whether the specific ocular manifestations of syphilitic uveitis, such as the posterior placoid variant, could be influenced by transmission to either a healthy or immunocompromised host, with healthier individuals perhaps more likely to have a limited posterior infection without panuveitis, figure 1. understanding the relative influences of immune status, particularly hiv infection, sex, and sexual behavior on syphilitic uveitis would depend on publication of more cases from populations with known seroprevalence of prior syphilitic infection and disease frequencies of symptomatic late manifestations. prospective studies in sexually transmitted disease clinics are not feasible due to the early treatment of most patients and reduction in their risk of later manifestations of infection, such as uveitis. cytomegalovirus retinitis (cmvr) is of particular interest because the virus is sexually transmitted as well as transmitted by body fluids and was the predominant cause of blindness and visual disability among hiv - infected patients prior to the initiation of highly active antiretroviral treatment (haart) in 1996. prior to the introduction of haart, the incidence of aids indicator infections was dependent on the degree of immunodeficiency rather than sex. positive women in the early years of the aids epidemic, only those who had nonsexually transmitted hiv such as injection drug - use had an increased risk (odds ratio 1.43) for cytomegalovirus disease. after the introduction of haart, data from a large multicenter study of the ocular complications of aids documented that the percentage of incident cases of cmvr in women more than doubled after the introduction of haart (35.4% versus 15.3%), a statistically significant change. reanalysis of data in 2012 from the same cohort no longer found a difference in the incidence of cmvr between men and women. globally, testing and treatment for hiv infection is now more readily available to women, which may help reduce risk of opportunistic infections such as cmvr. cmv can also be transmitted through household contacts, placing women at special risk if they care for infected young children. in general, unlike syphilis, and independent of hiv status, healthy women are more likely to be infected with cmv than men (or 1.17 [1.141.21 ]). infectious uveitis caused by nonsexually transmitted pathogens would be predicted to be associated with fewer sex differences. nonetheless, infectious uveitis of any type is a concern in hiv patients, the sex ratio of which varies according to geographic region therefore variably exposing women. in a large cohort study of hiv - infected individuals, herpes class viruses other than cmv (simplex, zoster), biologic differences related to sex were therefore not apparent in these variably immunocompromised individuals, although case numbers were low. a large series of 111 non - hiv infected turkish patients, with herpetic iridocyclitis, showed a slight female predominance of 1.2 : 1.0. in the united states, women are more commonly affected than men with herpes simplex 2 (http://www.cdc.gov/std/herpes/stdfact-herpes.htm, accessed 01 sept, 2013 2:00 pm). a similar situation may have influenced the sex disparity in the turkish series ; patients were not typed as having hsv 1 or 2. chronic anterior uveitis, associated with rubella, herpes, and cytomegalovirus, was slightly more common in men than women in a cross - sectional study of 166 saudi patients ; population seroprevalence of the candidate viruses was not reported. it is unclear whether small differences of this type are due to the prevalence of the primary infection or somehow related to a sex - based susceptibility to the eye disease. for tuberculosis, there is male predominance although it is among the top three causes of death for women world - wide. some of this imbalance may be due to the 13% of tb cases that are in hiv - positive individuals ; however, most of these are in the african region where the sex balance in hiv infection is more equal than in the european or american regions. in saudi arabia, a large survey of uveitis etiologies revealed presumed tuberculous uveitis to be the most common type of uveitis. male and female prevalence was essentially equal, whereas some immunological causes of uveitis were statistically more common (vogt - koyanagi - harada and multiple - sclerosis related) or less common (behet) in women than in men. an excellent review of prior publications in tb uveitis from the same group summarizes clinical manifestations, most commonly posterior, panuveitis, or occlusive retinal periphlebitis. women also vertically transmit infections during pregnancy that may cause peri- or postnatal infectious uveitis in their children. a recent series of herpes simplex 2 associated acute retinal necrosis in children identified maternal factors such as birth history with the possibility of direct infection through the birth canal or maternal antibodies in the majority of cases. congenital syphilis remains relatively common in the united states if considered in the light of the good surveillance and treatment of syphilis during pregnancy. the number of ocular infections among the 350 annual cases (about 1 in 10,000 live births) is unknown. cataract, chorioretinal scarring, and optic neuropathy seem to be rare and do not appear in uveitis surveys. women with positive treponemal tests but negative nontreponemal tests seem to be at a low risk of transmission of congenital syphilis to their children. rubella infection currently occurs in less than 1 case per 10,000,000 population in the united states. in oman where rubella is incident in 0.6 of 1000 live births, bilateral chorioretinitis was the most common manifestation. a historical series from the united kingdom in 1993 also found bilateral retinopathy to be the most common manifestation of congenital rubella syndrome, although it was not related to vision loss. interestingly, new diagnoses of fuchs uveitis syndrome, virologically related to rubella, declined in us - born patients after institution of the vaccination program in 1959 whereas the percentage of new fuchs patients that were foreign born increased. congenital cmv infection is not specifically related to maternal hiv infection and is more common than congenital hiv. the incidence rate of cmvr in hiv infected children was low in the pre - haart era (0.5 per 100 person - years) and has fallen further in the post - haart era. congenital cmvr is therefore not a specifically hiv - related problem. among non - hiv infected persons, nonwhite women and those in lower socio - economic groups have higher frequencies of cmv seropositivity and therefore are at greater risk of transmitting cmv prenatally if the primary infection occurs during pregnancy. unlike syphilis, chorioretinitis or other manifestations affecting the visual pathways are present in almost all of the symptomatic congenital cmv infants, who are 5 to 15% of the total born with serological evidence of congenital disease. preconception immunity does not protect against transmission to the fetus : about half of children with congenital cmv infection are born to preimmune mothers. the ability to infect a fetus even if the primary maternal infection does not occur in pregnancy may relate to periodic reactivations of lifelong cmv infection, similar to other herpes class viruses such as simplex and zoster. the greatest amount of information about vertically transmitted infectious uveitis relates to congenital toxoplasma chorioretinitis. screening of pregnant women is sometimes undertaken proactively in countries with high frequencies of congenital toxoplasmosis, such as france. this research enabled the establishment of antibiotic regimens for primary prevention of toxoplasma infection in the fetus, if seroconversion occurs during pregnancy. diagnosis by ocular screening of mothers is not efficient because only 3.8% to 13.7% of mothers of children with congenital toxoplasmosis have chorioretinal lesions consistent with healed toxoplasmosis. as for other infections termination of pregnancy is not usually recommended for all women who become infected with toxoplasmosis during the first trimester as only a low percentage of the fetuses will have symptomatic disease. igg avidity testing can be used to exclude infections that occurred more than 4 months previously despite the persistence of igm production. intrauterine sampling can be used to determine if the fetus is infected and ultrasound can detect malformations [42, 43 ]. if seroconversion is detected in the first trimester, treatment of the mother with antibiotics during pregnancy and the child during infancy can result in good visual outcomes. postnatal treatment did not prevent the development of new fundus lesions in 34 of 108 (31%) [2341, 95% c.i. ] of affected children but did reduce the incidence of new lesions compared to 18 of 25 (72%) congenitally infected but untreated children. in both groups, about half of the new lesions appeared at age 10 or older, figure 2. although in general preimmune women are felt to be incapable of transmitting toxoplasmosis to a fetus, a case has been reported of transmission from a mother who had reactivation of chorioretinitis during pregnancy. as for cmv lymphocytic choriomeningitis (lcm) virus is a less known congenital infection that produces chorioretinal scarring and vision loss ; in one study in chicago, antibodies against lcm were encountered more frequently in severely retarded children with chorioretinal scars than toxoplasmosis, rubella, cmv, or herpes simplex. in some children chorioretinitis was present but serology did not identify candidate infections indicating the likelihood that other infections can produce fetal ocular infections. syphilitic uveitis is also strongly associated with hiv infection but can also occur in immunocompetent women. women are also at risk of transmitting infections such as herpes simplex, cytomegalovirus, toxoplasmosis, and lymphocytic choriomeningitis virus, and herpes simplex that can cause chorioretinitis in neonates. robust screening and treatment programs for vertical transmission of toxoplasmosis have reduced the impact of toxoplasma chorioretinitis on children. | current data permit only speculations regarding sex differences in the prevalence of infectious uveitis between women and men because uveitis case surveys do not uniformly report gender data. differences in prevalence that are reported in the literature could relate to simple differences in the number of women and men at risk for infection or to biological differences between men and women. compared to other types of uveitis, infectious uveitis may be directly related to occupational exposures or sexual behaviors, which differ between women and men, and may mask actual biological differences in susceptibility to ocular manifestations of the infection and its prognosis. in infectious uveitis for which there is no element of sexual transmission and data is available, prevalence of ocular disease is roughly equal between women and men. women also have a unique relationship with infectious uveitis in their role as mothers. vertical transmission of infections such as herpes simplex, toxoplasmosis, and cytomegalovirus can produce severe chorioretinitis in neonates. |
the incidence of malignant melanoma in fair - skinned patients has increased dramatically in most parts of the world over the past few decades. because the prognosis of melanoma depends almost entirely on tumor thickness, early detection of thin melanoma is important for the survival of patients [1, 2 ]. the diagnostic accuracy of the clinical examination of pigmented skin lesions, however, is still rather poor. literature results arise the evidence that the ability of general practitioners to early diagnose cmm with the naked eye is very low ; the ability of dermatologists to early diagnose cmm with the naked eye ranges from 50% to 75% ; there is a high rate of false positive (causing unneeded surgical excision). in the last decade dermoscopy is a noninvasive technique that enables the clinician to perform direct microscopic examination of diagnostic features, not seen by the naked eye, in pigmented skin lesions. this technique is more accurate than naked eye examination for the diagnosis of cutaneous melanoma, in suspicious skin lesions when performed in the clinical setting. several rather successful computer programs have been implemented to the aim of an automatic analysis of melanocytic lesions and their discrimination between naevi and melanomas (see, e.g., [48 ] ; see also [9, 10 ] for a comparison between automatic and human performance). most of them keep into account the traditional abcde parameters used by dermatologists : asymmetry (of boundary, texture, and color), boundary (irregularity and dishomogeneity), color (presence of several colors), dimension, and evolution. in particular, asymmetry is generally based on quantitative comparison of the two parts into which a lesion image is split by its principal axes. here we have developed a new method for comparing in a qualitative, yet precise way the two parts of a lesion at the sides of a splitting line. the mathematical tool for comparison is the theory of size functions, applied to three features : boundary shape, mass, and color distribution. for each splitting line of a pencil we get an asymmetry measure, so forming a map (two for each of the three features). some characteristic numbers of the six maps are finally fed to a support vector machine. a classification experiment has been led on data set of 977 lesions with very good results. the whole research is a follow - up of the adam project of the european union. we are well aware that qualitative measure reads like an oxymoron ; of course, we mean that we compute a precise, objective, repeatable measure of the difference between the two half images ; yet, this difference is of a qualitative kind, in that it is not bound to geometric deformations, superimpositions or the like. size functions (sfs) are modular invariants of whatever signal the user is interested in ; in the present case, the concerned features are boundary shape, mass, and color distribution. they depend on two inputs : an object (e.g., a lesion boundary) and a real map, called measuring function defined on it (e.g., distance from the center of mass). essentially, the sf registers the behavior of the measuring function by using morse theory (see). sfs are qualitative not only in that they are topological in nature, but also in that a similarity based on them depends on the user 's choice of a measuring function and of a distance between sfs adapted to the context. let us recall the definition of an sf, adapted from the more general setting of, where measuring functions are allowed a multidimensional range. consider a continuous real - valued function : m r, defined on a subset m of a euclidean space. the size function of the pair (m,) is a function (m,) : r n { }. for each pair (x, y) r, consider the set mx = { p m : (p) x}. the value (m,)(x, y) is defined to be the number of the connected components of my which contain at least one point in mx. the discrete version of the theory substitutes the subsets of the plane with a graph g = (v, e), the function : m r with a function : v r and the concept of topological connectedness with the usual connectedness notion for graphs. figure 1 shows the size function obtained from a curve with the ordinate as measuring function. sfs have a standard structure, the one of superimposed triangles already apparent in figure 1. this has an important outcome, in that the relevant information can be condensed in the vertices of those triangles. comparison of two images (as far as the criterion intrinsic to the measuring function is concerned) can then be carried out by comparing the sets of these points. several distances can be defined on the set of sfs ; one which is very successful is the matching distance (see figure 2). unfortunately, there do not exist archetypal naevi or melanomas, so the task is harder than for classical classification problems. statistical learning comes into play ; vectors of characteristic numbers are the input of a support vector machine. the first processing step is segmentation, that is, the isolation of the skin lesion from its background. this is carried out with well - tested methods depending on several parameters, most of which have been fixed by experiment. this is notoriously a serious problem in the processing of dermatological images, and has been solved by the operations of erosion and dilation coming from mathematical morphology. the experience of dermatologists suggests that a major criterion for suspecting malignancy is the asymmetry of various aspects of the lesion. we have followed this suggestion by splitting each lesion in two halves by a straight line passing through the center of mass. comparison of the two halves is then performed by computing the distance between their size functions. this represents a definite progress with respect to classical methods for detecting asymmetry ; these detected only geometrical asymmetry, while distances of size functions determine also qualitative asymmetry. we repeat the splitting for 45 equally spaced radial lines, so getting distance as a function of angle (see figure 4). from this curve the software extracts a set of characteristic numbers : min, max, average, min plus the value at 90 from min, integral, first moment, variation, min derivative, max derivative, integral of absolute value of derivative, and variation of absolute value of derivative. a support vector machine with a third - order kernel is fed with these numbers, computed for each measuring function. actually, the vectors also contain three more parameters : area, perimeter, and a bumpiness measure coming from the sf of the whole lesion, with distance from center of mass as the measuring function. an initial set of experiments had been carried out with 90 lines instead of 45, but the hit ratio was just slightly higher, while almost doubling computing time. we have used six measuring functions to distil the structure of boundary, mass distribution, and color distribution, respectively. the third sums distances of colors (in rgb space) of consecutive pixels along segments orthogonal to the splitting line. our initial experiments used just these three measuring functions. adding their three opposite functions improved the hit ratios of 2 to 5 percentage points. the acquisition setup consists of an leica 650 m stereomicroscope and a sony 3ccd-930 color video camera. the illumination of the stereomicroscope consists of a 12 v/50 w halogen lamp that creates a bundle of light perpendicular to the area of interest. the digital images have been archived by means of the dbdermo mips software package (dell'eva - burroni, siena). over half of the data set used in the present research, had already been the subject of a formal study of clinical diagnostic validation using also the local population - based cancer registry (i.e., registro tumori romagna) to cross - check for possible false negatives, published on. the data set comes from the daily practice of one of us (stanganelli) ; of course, only interesting naevi had been acquired. all melanomas and several naevi have been subjected to histological test ; all remaining naevi have been subjected to follow - up. we have selected 977 images of melanocytic lesions (melanomas and naevi) acquired in epiluminescence microscopy with a fixed 16-fold magnification. the data set contains 50 melanomas (28 of them with thickness less than 0.75 mm) and 927 naevi. every second image was assigned to the training set (melanomas were listed consecutively). in tests r1 and r2, a training set of 25 melanomas and 500 naevi was randomized from the data set. the test set was formed by the complement (the remaining 25 melanomas and 427 naevi). a fourth test (s) was performed without cross - validation, with the whole data set both as training and test set ; we interpret the not much higher scores of test s as a proof of stability. in table 1 we report, for each of tests h, r1, r2, and s, the specificity and sensitivity of what we judge to be the best performances. as a further information, in test s a 100% specificity was attained only at cost of 4% sensitivity, but the decrease of specificity to 93.64% yielt a jump to 70% sensitivity. our system is not intended to be provided to the public as a yes / no diagnostic tool ; it yields a risk index in the following way. two classifiers, one tuned at high sensitivity, the other at fairly good specificity, give their response ; if they agree to classify the lesion as a naevus (resp., a melanoma) then a low (resp., high) risk is stated ; if they disagree, the output is of middle risk. a comparison has been done between the output of this compound classifier and the judgement of an expert dermatologist, who had classified the lesions as sure melanomas, sure naevi and uncertain. the percentages reported in table 2 refer to the fractions of the three classes (as classified by the human expert) labeled by the machine with the three risk levels. as stressed in, there are quite different selection criteria, melanomas / naevi ratios, data set sizes, analysis methods. instead of reporting selected results of competitors, we refer to table 1 of that thorough paper. we just would like to comment on very high sensitivity scores (over 95%). with the noticeable exception of seidenari., such scores seem to have been attained either with very small data sets, or with high melanoma percentages, so in situations which appear to be rather far from real - world ones. even counting them, the result of our cross - validated test r1 is placed in the top third of the reported scores. of course, the single - set test s places us at an even higher rank. it would be interesting to compare as suggested by a referee the asymmetry assessment given by our method with the one given by an expert dermatologist. this is unfortunately not possible, since our evaluation does not consist of a single measure, but of 66 (see section 5), what compelled us to use support vector machines for classification. in a comparison of the performance of our system and of human operators (three dermatologists and three general practictioners) the true novelty of the presented method consists in the use of a qualitative but objective mathematical tool, the size functions, to evaluate asymmetry (of boundary, color, and mass distribution). three experiments with 977 lesions, carried out under cross - validation, show very good performances. but its good hit ratio, together with the complete independence from the competitors ' tools, make our method a tempting candidate for integration. in this line of thought, | size functions and support vector machines are used to implement a new automatic classifier of melanocytic lesions. this is mainly based on a qualitative assessment of asymmetry, performed by halving images by several lines through the center of mass, and comparing the two halves in terms of color, mass distribution, and boundary. the program is used, at clinical level, with two thresholds, so that comparison of the two outputs produces a report of low - middle - high risk. experimental results on 977 images, with cross - validation, are reported. |
in recent years, nursing staff shortages have become a major challenge for health care systems around the world, including iran. in 2008, iranian hospitals needed approximately 220,000 nurses ; nonetheless, there were just about 90,000 nurses. this shortage is related to decreased satisfaction among nurses and posed a potential threat to the quality of patient care. in the study which was conducted in 12 countries in europe and the usa, the range of nurses burnout was from 10% in the netherlands to 78% in greece, job dissatisfaction was from 11% in the netherlands to 56% in greece, and intention to leave was from 14% in the usa to 49% in finland and greece. nursing shortages are a symptom of inadequate policies on recruitment and retention of nurses in the world. also, in a nationwide study, iranian nurses reported an average level of job satisfaction. another important challenge of iranian health care system is poor quality of patient care, which is influenced strongly by nurses motivation. nurses are the main group of the human resources in the health care and the quality of health care is highly dependent on an adequate recruit and supply of qualified and motivated nursing personnel. motivation is an important factor that influences nurses job satisfaction and their intention to work. therefore, understanding nurses motivators can enhance job satisfaction, quality of care, and productivity. identifying nurses motivators can help nurse mangers to find a way to motivate nursing staffs. but the focus of previous studies has been mainly on the association of job motivation with different variables such as evaluation outcomes, psychiatric nurses anger, job satisfaction, innovation, and individual and organizational factors. in general, therefore, psychologists have extensively studied about motivation and have provided theories about the reasons for motivation. motivation is defined as a process responsible for making the efforts strong, directed, and continuous to achieve the goals. in general, motivation originates from the needs that must be met. russell and swansburg believed that motivation is a concept used to describe an external condition stimulating a special behavior and internal responses revealing that behavior. many theories were applied to explain motivation at work, such as the needs theory (maslow 1954 ; herzberg. 1959), expectancy theory (vroom 1964), equity theory (adams 1965), and goal setting theory (campbell and pritchard 1976) ; however, there is no single theory describing motivation comprehensively. it can be one of the possible reasons for the lack of consensus in an instrument to measure motivation. this study was conducted to identify the motivating factors among the hospital nurses of tehran university of medical sciences. this is a descriptive study in which the motivation of 310 nurses working at 14 hospitals of tehran university of medical sciences was evaluated in 2010. nurses who had full - time work experience of at least 1 year in clinical practice in nursing and also had associate degree or higher educational qualification in nursing were included in the study. according to our given population size (n = 2427), however, to ensure obtaining an adequate sample size, with likelihood of 10% incomplete questionnaires, 330 questionnaires were delivered to nurses. the strata are formed based on members shared attributes or characteristics. in this study, each hospital was considered as a stratum and the samples were selected based on the proportion of the number of nurses in each hospital from the sampling frame. the researcher explained the purpose of the study and freedom for joining in the study to the potential participants. so, if the selected nurses did not wish to participate in the study or complete the delivered questionnaire, researchers selected the next nurse from the list. the analysis was done for the data of 310 nurses in the sample because 20 questionnaires were incompletely filled. demographic information questionnaire was used to measure the variables of age, gender, marital status, educational level, income status, work position, having overtime, and working in other hospitals. in spite of the availability of instruments to measure motivation, we developed a new questionnaire because of iranian hospitals context and socio - cultural differences. the instrument was developed by an extensive review of the literature including previous studies conducted in iran. after the initial draft of the instrument was developed, its content validity was evaluated by the nursing scholars who have experiences regarding the topic. in this stage, two items were omitted and a number of them were modified. also, the content validity index (cvi) was estimated and all items were approved based on their cvi > 0.85. then, for face validity, the instrument was distributed to 10 nurses and their views on its simplicity and comprehensibility were taken and the required changes were made. after exploratory factor analysis, 30 items were reduced to 25 and classified into four factors : career development (7 items), job characteristics (9 items), job authority (5 items), and recognition (4 items). the factors were named after the item with the highest standardized factor loading within the domain. factor loadings and cronbach alpha coefficient for nursing motivation questionnaire for reliability, 30 nurses completed the instrument and the cronbach alpha coefficient was calculated as 0.81. the items were scored on likert scale, with scores ranging from very low (1) to very high (5). distribution of scores from the instrument and its factors was categorized based on the range of possible scores as follows : low (033%), medium (3366%), and high (66100%). also, to compare different factors, the mean score of each factor was obtained by dividing the sum of scores of each factor by the number of its items. statistical package for social sciences (spss) version 16.0 for windows (spss inc. descriptive statistics and independent t - test, analysis of variance, tukey post - hoc test, chi - square and fisher 's exact test were used. a significance level of p < 0.05 was considered. the mean age of the participants was 35.07 years [standard deviation (sd) = 7.46 ; range : 2253 ]. the length of the participants work experience ranged from 1 to 30 years, with a mean of 11.48 years (sd = 7.25). characteristics of the participants (n=310) the motivation of most of the subjects in career development (52.6%) and job authority (64.5%) was at a medium level, while the motivation in job characteristics (61.3%) was at a high level. half of the subjects had a high motivation in the factor of recognition. the findings of this study showed that the least mean of the factor score, considering the number of items, was 3.23 for career development, while the highest mean was 3.81 for job characteristics. scores of motivating factors and total motivation independent t - test showed that there was no statistically significant difference in the total motivation score between males and females (p = 0.381) and in terms of being employed in other hospital or none (p = 0.599). also, independent t - test showed that total motivation of nurses who had overtime was higher than that of others (p = 0.031). also, the results of one - way analysis of variance (anova) showed that there was no difference in total motivation in terms of marital status and educational level. there was a statistically significant difference in the total motivation of nurses in terms of their positions (p = 0.007). tukey post - hoc test showed that the motivation of head nurses was significantly higher than that of staff nurses (p = 0.005). the mean score of total motivation among nurses who had inadequate income was significantly less than those who had adequate income (p < 0.001). the chi - square test showed no statistically significant relationship of career development with gender, working overtime, and working in other hospital. this factor had a statistically significant relationship to income (p = 0.001), age (p = 0.035), and work position (p = 0.04). fisher 's exact test showed that the factor job characteristics was significantly related to the work position (p = 0.021), job authority was only related to overtime (p = 0.035), and also recognition was significantly related to overtime (p = 0.043). the present study was conducted to identify the motivating factors among iranian nurses. in this study, the factors of career development, job characteristics, job authority, and recognition were considered as the aspects of motivation in nurses. shattuck., in a systematic review, identified seven major motivational themes including financial rewards, career development, continuing education, hospital infrastructure, resource availability, hospital management, and recognition / appreciation. except two factors, career development and recognition / appreciation, which are completely similar with the themes identified in the mentioned review, the findings showed that the least mean score of motivation, considering the number of items, was related to career development, while the highest one was related to job characteristics. these results are comparable with the results of a study conducted by mahmoudi., who reported the nature of work among the internal factors and supervision among the external factors as the most important factors of job motivation in iranian critical care nurses. in the mentioned study, the ability to serve the community and the value of nursing among people as work nature factors have had the highest effect on job motivation. several previous studies have identified accomplishing jobs, working independently, and attaining interpersonal relationships as the most important factors that motivate nurses. the findings showed that the motivation score of 55.5% of nurses was at a medium level. similarly, jodat. reported that nurses overall score for job motivation was moderate. also, this finding could be compared with the findings of the study conducted by taghavi larijani., in which the motivation of 65% of iranian nurses working in medical - surgical wards was reported as low. this controversy can be explained by the difficulties confronted by nurses in the iranian health care system. iranian hospitals deal with challenges including inadequate staffing, limited equipments available to nursing care, lack of authority to change practice, and organizational cultures rewarding routine and task - based practice. low levels of motivation among nurses are important in view of the fact that the largest subgroup of human resources in the health care system consists of nurses. it becomes even more important considering that staff 's motivation is a predictor of their productivity, and the studies conducted on productivity of iranian nurses show that only 7.5% of iranian nurses have shown the desired productivity. one of the main challenges in providing good - quality nursing care in iran and around the world is shortage of nurses. studies show that this shortage has a domino effect in nurses loss of knowledge and motivation, exhaustion, burnout, and severe stress. furthermore, job stress, lack of autonomy, and nurse physician collaboration are the most important factors that contributed to nurses job dissatisfaction and retention in the nursing literature. reported that job motivation of nurses is an important factor in the intent of nurses to leave nursing. the findings of the present study showed a significant relation of age, work position, and the income status with the first factor (career development), work position with the second factor (job characteristics), and finally, working overtime with the third and fourth factors (job authority and recognition, respectively). several researchers have shown a positive significant association between the age of nurses and their job motivation. although there was no significant association between gender and total motivation score in this study, franco. reported that female health workers in jordan had lower ratings to motivational determinants, and there were almost no differences related to gender or age in georgia. found that nurses younger than 30 years were more motivated than older nurses, and higher university degrees were related to higher motivation in nurses. de cooman. stated that male and female nurses are motivated for similar characteristics of nursing. in a study by the mixed method in which the motivation of non - medical staff of tanzania was examined, the findings showed that though non - financial factors are of great importance for motivation, financial satisfaction is a prerequisite of any intervention for increasing the motivation by non - financial motivators. also, the study of lambrou. showed the managerial position was associated with job characteristics among greek nurses. the importance of the supervision factor becomes less while the importance of the factor of appreciation and recognition is increased. this means that the sample covered nurses who work in public university hospitals in tehran, the capital and largest city of iran ; therefore, future study should be conducted to have a clear understanding about the motivating factors among nurses in public / private sectors of urban / remote areas. in addition, the questionnaire of this study was developed by the authors and used for the first time ; hence, its criterion validity including concurrent and predictive validity should be evaluated in future studies. our findings showed that the factor of career development had the maximum potential ability to inspire nurses in their profession. so, it is necessary that nursing managers pay more attention to use the element of mentioned factors such as effective supervision, encouragement, and job enrichment. however, other motivating factors including job characteristics, job authority, and recognition should not be neglected to motivate nurses. specifically, the findings of this study imply that the importance of the factor of job characteristics, such as the use of various and high - level nursing skills, feedback, and critical thinking, as the first ranked motivating factor in the motivation of nurses needs to be emphasized. moreover, the medium level of motivation in participants indicates the necessity of paying more attention to the motivating factors to improve motivation in nurses. the results of the current study can be used in programs designed to improve nurses work motivation. | background : one of the most important challenges of iranian health care system is quality of care, and it is assumed that motivated nurses are more ready to provide better care. there are limited studies investigating iranian nurses motivations ; however, factors which motivate them have not been studied yet. identifying the motivating factors enables nurse managers to inspire nurses for continuous quality improvement. the aim of this study was to identify motivating factors for iranian hospital nurses.materials and methods : this is a cross - sectional descriptive study in which 310 nurses working at 14 hospitals of tehran university of medical sciences were selected by proportionate stratified random sampling. data were collected in 2010 by a researcher - developed questionnaire. descriptive statistics and independent t - test, analysis of variance, tukey post - hoc test, chi - square and fisher 's exact test were used for statistical analysis by statistical package for social sciences (spss) version 16.results:the mean score of motivation was 90.53 10.76 (range : 59121). four motivating factors including career development (22.63 5.66), job characteristics (34.29 4), job authority (18.48 2.79), and recognition (15.12 2.5) were recognized. the least mean of the motivation score, considering the number of items, was 3.23 for career development, while the highest mean was 3.81 for job characteristics.conclusions:the findings showed that motivation of nurses was at a medium level, which calls for improvement. the factors that have the greatest potential to motivate nurses were identified in this study and they can help managers to achieve the goal of continuous quality improvement. |
urodynamic testing has greatly improved our diagnostic capabilities for patients with complex neurogenic bladder dysfunction. however, despite years of refinements in technical standards and recommendations, urodynamics require experience - based individual interpretation and lack established sensitivity and specificity data. detrusor external sphincter dyssynergia (desd) is defined as : a detrusor contraction concurrent with an involuntary contraction of the urethral and/or periurethral striated muscle in a patient with a known neurologic condition. diagnosis is made by a combination of a clinical history of a known or potential upper tract lesion, increase in visual or audible electromyography (wire or patch), fluoroscopic visualization of a dilated bladder neck and proximal urethra to the external urethral sphincter (eus), or a urethral pressure profile with interruption of flow (when present) with increased detrusor pressure during interruption. most urodynamic centers in the united states employ patch electrodes rather than needles for electromyography. the international continence society (ics) has published statements in order to standardize reporting of many components of urodynamic results and technique [2, 3 ]. certain parameters for diagnosis of desd have been noted, such as an electromyography (emg) recording minimum of 20 khz, and the suggestion that quantitative measurement may be supplemented by imaging (videourodynamics). however, standard methodology and recommendations such as the type of needle and needle versus surface patch electrodes for the recording of external sphincter activity has not been published. the importance of each diagnostic test or parameter is left to the individual physician and published studies vary widely in emg technique and reporting. some of the discordance between variables is intuitive ; for example, a closed bladder neck on voiding cystourethrography (vcug) prohibits visualization of the external urethral sphincter. in the case of emg, electrode placement is operator dependent and the patch introduces additional factors due to interference from the perianal musculature. to evaluate the concordance of the two modalities most commonly used in clinical practice, we prospectively examined the question of diagnostic discordance employing patch emg compared to vcug for the diagnosis of desd. patients were prospectively evaluated by a single urodynamicist (ed) and entered into an institutional database over a 24-month period. this database represented all patients offered urodynamics in an academic referral - based continence center. the urodynamic readings were then retrospectively re - evaluated by an independent urodynamicist (ck) for agreement with the original diagnosis of desd. the presence of desd was determined by increased patch emg activity and/or (figure 1), a dilated bladder neck and proximal urethra on multichannel videourodynamics during detrusor contraction (figure 2). in the absence of valsalva, pelvic floor dystonia (dysfunctional voiding), or attempt to inhibit voiding, for example guarding during uninhibited contraction (uic). isolated mild emg elevation in the absence of flow interruption, elevated detrusor pressure, or neurological disease was interpreted as dysfunctional voiding. patients with history of sphincterotomy or urethral stent or whose urodynamics lacked the relevant data points (e.g., omission of fluoroscopy in severe contrast allergy) were excluded from analysis. the urethral pressure profile was examined for increase in detrusor pressure and drop in flow when present. additional data points analyzed include age, sex, supine position, permissive voiding or uic, postvoid residual, other source of obstruction, and neurologic diagnosis. urodynamics were meticulously performed according to the international continence society guidelines and recorded using multichannel technique on the laborie triton pro 835 urodynamics system, lab850 urodynamics software (laborie medical technologies inc., briefly, two patch emg electrodes (cleartrode, conmed corp., utica, ny, usa) were placed at the 2 and 10 o'clock position around the anus and a third ground patch electrode was placed over the adductor tendon on the medial aspect of the patient 's left knee. the electrodes were then covered with tape to prevent them from becoming wet or dislodged. covington, ga, usa) was primed with contrast and, using aseptic technique, was introduced per urethra. was then placed per rectum with 2.5 cc in the 10 cc balloon and taped in position (cat510 silicone rectal catheters, laborie medical technologies inc., after confirming brisk and yoked response of the catheters to cough, the system was zeroed to atmospheric pressure. the volume in the rectal balloon was adjusted to achieve a detrusor pressure (pdet) of between 0 and 4 cmh20. filling cystometry was initiated using contrast at a rate of 30 cubic centimeters per minute. minimal acceptable criterion for agreement between the emg and vcug was set at 70% concordance. binomial test was employed to establish the presence or absence of agreement. for continuous variables the comparisons between groups was calculated by the nonparametric kruskal - wallis test for data not normally distributed. of the 376 patients prospectively entered into the database 52 were given a diagnosis of desd. after retrospective review of readings, 6 were eliminated for having only one modality available (e.g., noncontrast exam) or other source of obstruction (e.g., urethral stricture), leaving 46 patients with desd and no other source of obstruction. of these 46 patients, 25 were diagnosed by both tests, 11 by vcug alone and 10 by patch emg alone (table 1). the agreement between the two diagnostic modalities was compared, assuming that the patients identified in the database adequately represent patients diagnosed with desd by patch emg and vcug in a referral clinical practice. setting a minimal acceptable criterion of 70% agreement for two diagnostic tests, we found that patch emg and vcug do not agree for a positive diagnosis of desd. the proportion of agreement was 54% (95% ci 39% to 68%), which is significantly less than our criteria for agreement of 70% (table 2). patients with desd had a statistically higher pvr (214 33) compared to patients without desd (56 22, p = 0.001) and higher mean detrusor pressure (47.3 6.3) versus (33.4 3.5, p = 0.045) during contraction. of these 46 patients with desd, of the two tests, male patients were more likely to be diagnosed by emg alone, while female patients were more likely to be diagnosed by vcug alone. (table 1) the average age of patients with desd was (49 16), not statistically different from the general database (56 15). in the recordings, a pressure flow variation was clearly seen in 14 tracings, unobtainable (due to supine position or lack of flow) in 13, obscured in 6 and not present in 4 patients assigned a diagnosis of desd. the neurologic diagnoses are presented in table 3 with some patients carrying more than 1 diagnosis. the most common diagnoses were multiple sclerosis (13) and spinal cord injury or disease (11 total, 7 with paraplegia and 4 with quadriplegia). our study was designed to provide information about the diagnostic congruence of the two most commonly used tests performed in the united states for diagnosis of desd during urodynamic testing. we are not promoting surface electrodes as a recommended modality for diagnosis but intended it would be most useful to investigate what our patients are seeing in practice. our findings demonstrate a 54% agreement and 46% disagreement between patch emg and vcug for diagnosing desd. the visualization of the external sphincter by vcug is precluded by bladder neck dyssynergy or outlet obstruction from the prostate, which would explain the higher incidence in male patients. likewise artifact from the urinary stream tracking down the perineum can preclude a clear emg reading in female patients. the discordance found in this study between patch emg and vcug in a voiding dysfunction practice is similar to our previous results with wire needle emg in a rehabilitation hospital practice, which demonstrated a 60% agreement and 40% disagreement for desd by vcug. while previous studies have shown improved emg measurements using needles, patch emg is more widely used due to patient tolerance, ease of electrode placement and greater freedom of movement. our previous paper discusses the wide variation in type of emg used throughout the urodynamic literature, including the limitations of in use of the external anal sphincter as a proxy [4, 610 ]. it is not the aim of the current study to compare or assess the validity of two approaches but rather to point out that there is significant discordance between vcug and the most commonly used emg technique for diagnosis of desd. technical factors with eus emg placement are of course suggested in the cases lacking and emg diagnosis. it is well known that patch emg suffers from many limitations even when identified and addressed. most urodynamicists pay attention to multiple additional factors during interpretation including suspicion for the diagnosis (presence of an upper motor neuron lesion interrupting the spinobulbar pathways), obstruction of flow (if the patient leaks or voids), elevated detrusor pressure (diagnosis of desd implies high voiding or storage pressures and potential for damage to the upper urinary tracts) and increased auditory feedback for those who perform oscilloscopy. the technologies for diagnosis are only tools to inform the observer. we argue that interpretation is aided by the presence of the urodynamicist during the procedure. for example, if there is an uninhibited contraction and emg / eus tone increases, the increase could be due to voluntary guarding. increased eus activity during detrusor contraction in a neurologically intact woman who is nervous about the test is interpreted differently than increased activity in a patient with quadriplegia and no bladder sensation in this example. the examiner can instruct the sensate patient to relax the pelvic floor and allow a void during the next urge on repeat cystometrogram. the permissive void will allow for better assessment of eus coordination. in this and the 2005 article regarding wire needle emg, the significant amount of disagreement between modalities suggests that the routine combination of emg and vcug will identify more cases of desd than either modality alone. we include our data on voiding pressures and flow interruption to support the diagnosis of desd. these types of observations also inform the clinical impression. given the nature of urodynamic testing, it is unlikely a gold standard could be agreed upon for the diagnosis of desd. better understanding of the interplay and relative importance of the techniques should be pursued as guidelines to the benefits and limitations of each modality contributing to the diagnosis could improve objective assessment of desd. in this prospective study employing consistent technique and using patch emg electrodes, we found only 54% (95% ci 39% to 68%) agreement between patch emg and vcug in the diagnosis of desd. this reinforces our prior similar findings using needle emg and expands the applicability of our data to the majority of clinicians who use patch emg electrodes. this further supports the idea that the combined use of emg and vcug or other modalities for diagnosis can identify more cases of desd than either modality alone and opens the stage for guidelines regarding the diagnosis of desd. | introduction. the diagnosis of detrusor - external sphincter dyssynergia (desd) is a clinically relevant finding during urodynamic testing. however, there is no consensus regarding diagnostic specifics of electromyography (emg) or voiding cystourethrography (vcug). we evaluated the concordance of the two modalities most commonly used in clinical practice for the diagnosis of desd. methods. patients were prospectively evaluated by a single urodynamicist at an academic center and retrospectively re - evaluated by an independent urodynamicist for agreement. desd was determined by increased patch emg activity or a dilated bladder neck / proximal urethra on vcug during detrusor contraction. minimal acceptable criterion for agreement was set at 70%. results. forty - six patients were diagnosed with desd with both modalities available. of these 46 patients, 25 were diagnosed by both tests, 11 by vcug alone and 10 by patch emg alone. binomial testing demonstrated the proportion of agreement was 54% (95% ci 39% to 68%). conclusion. we found significant disagreement between the two modalities, similar to previously reported findings using needle emg, and we expand the applicability of our data to the majority of clinicians who use patch emg electrodes. this further supports the idea that the combined use of emg and vcug for diagnosis can identify more cases of desd than either modality alone. |
with the widespread use of laparoscopy in the 1990 's, many patients with heartburn and sequelae from gastroesophageal reflux disease (gerd) are undergoing repair of their hiatal hernias. although there is a greater than 85% success rate, complications and failures occur following surgery (table 1). one of the major concerns during the operative procedure is safe placement of a dilator or bougie. many times the passage of the bougie is not performed by the operating surgeon but by a surrogate, like the anesthesiologist. it is ironic that most surgeons are not concerned by the anesthesiologist placing an orogastric tube for intraoperative gastric decompression and rarely is this 18f tube a cause of a complication, like perforation. with this in mind, a dual tube system was developed to decrease the incidence of esophageal perforation and allow safe dilator placement. the first portion of the system is a standard 18f single lumen levine - type tube. the length is marked to assist placing the tip of the tube through the mouth into the stomach. it is extended with a screw cap to double the length and allow easy threading of the bougie over the levine tube. the second portion of the system is a clear dilator measuring 48f or 58f where the tip is bored out to allow passage of an 18f tube. it has marks indicating distance from the tip to guide the operator and surgeon while advancing the bougie down the esophagus and into the stomach. the surgeon using the laparoscope should observe the dilator pass into the stomach as the operator indicates the length of bougie passed. the surgeon may need to grasp the stomach as the bougie reaches the gastroesophageal junction. the bougie can be pulled back or readvanced, as needed, during the hiatal hernia repair as sutures are placed or as the wrap is pulled behind the esophagus. the 18f orogastric tube has a dual purpose to suction out the stomach and to act as a guide for the dilator to be passed directly into the stomach (figure 1). at the completion of the surgery, the 18f orogastric tube may be replaced through the nose for postoperative gastric decompression. laparoscopic repair of hiatal hernias was introduced at the baptist : hospital laparoscopic surgery center in 1994. since that time, 145 patients have had the procedure performed. during the first 3 years of performing these procedures (102 patients), there were 5 perforations of the esophagus and 3 wraps that were redone for being too tight. there was difficulty with this system in getting the stiff tapered tip past the oropharynx. this system was abandoned for a softer, blunt - tipped system much like a there was one stomach perforation from an orogastric tube, but it was not related to the dilator system. this perforation was recognized and repaired at the time of the hiatal hernia repair and caused no postoperative sequelae. the average age of all patients was 51 years, which did not change over the 4-year period. operative time has declined to 132 minutes, after initial times began at 210 minutes. length of hospital stay was 2.3 days for the first 3 years and, during 1997, had decreased to 1.66 days. since the introduction of the new dilator system, there have been 5 postoperative complications of which 3 were atelectasis, the one recognized gastric perforation noted above, and one patient returned to the or on the same admission to have a heller myotomy following a toupet fundoplication. intraoperative esophagogastroscopy was performed by one of the operating surgeons or the gastroenterologist who had referred the patient for surgery. it confirmed proper wrap size and placement, as well as testing the security of the sutures holding the wrap in place. the stomach was distended with air and submerged in saline during the gastroscopy to look for air bubbling indicating a perforation. reports in the literature have quoted excellent control of reflux symptoms after laparoscopic nissen fundoplication from 85 to 91%. however, with any surgical operation there are well recognized complications. there are numerous reports in the literature discussing the benefits justifying laparoscopic fundoplication for the treatment of gerd. a few of the reports have been examined and the perforation rates and dysphagia rates identified (table 2). weerts and dallemagne reported 1 perforation, 2 lacerations of the gastroesophageal junction and 1 wrap disruption in 132 patients.. reported on 17 perforations in 364 patients of which 5 were due to the passage of the bougie. one of the first reports in the literature in 1994 was by hinder who noted 3 perforations in the first 198 patients. swanstrom and pennings discussed safe dissection and had 2 complications from the bougie in 152 procedures. reporting on 300 patients over a 4 year period, hunter. the rate of esophageal perforation is probably higher in those institutions that are not performing this surgery at the same number of cases, much like our data. it is imperative to take every precaution to perform safe laparoscopic dissection of the gastroesophageal junction. the ge junction in these patients is at high risk of perforation because of the chronic irritation from reflux juices and the angle of the junction below the crura. there are very few reported or noted perforations from standard orogastric or nasogastric decompression tubes. the seldinger technique for placement of catheters is well known to surgeons. with this in mind, the cook esophageal dilator set has been shown in this limited study to be safe and effective for placing a bougie during laparoscopic repair of hiatal hernias. | the increased use of laparoscopy for treatment of reflux esophagitis has been associated with a 1 - 8% complication rate. perforation of the esophagus from bougie placement, wrap breakdown or too tight a wrap are some of the complications seen from this surgery. an esophageal dilator system was developed to overcome these problems. thirty patients had an esophageal dilator system used whereby a 48f or 58f dilator was placed over a 18f orogastric tube. intraoperative gastroscopy documented a properly created wrap. there were no esophageal perforations or morbidity associated with the dilator. |
an epileptic seizure is defined as a transient symptom of abnormal excessive or synchronous firing of some or many of the brain s cells, or neurons. the outward effect can be as dramatic as convulsions with wild thrashing movements (tonic - clonic seizure) or as mild as a brief loss of awareness (absence seizure). sometimes seizures consist of repeated full body slumps, with the person simply losing body control and crashing to the ground. it has been found that initial, thoughtfully chosen medication can make almost 50 percent of patients seizure - free for extended periods of time. if an initial drug fails, another well - chosen drug may make an additional 14 percent of patients seizure - free. if that drug fails, too, then the likelihood of rendering someone with epilepsy seizure - free is poor. more than 30 percent of patients with epilepsy will not have seizure control even with the best available medications. despite the introduction of many new anticonvulsant medications, these figures have remained consistent over time. working with families whose children had epilepsy, we realized that there were no books written for parents to help them cope. to fill the void, my colleague, eileen vining, m.d., my coordinator / counselor, diana pillas, and i decided to team up to write one. the result was seizures and epilepsy in childhood : a guide for parents,3 published by the johns hopkins press in 1993. it contained three pages on the ketogenic diet. sensing a need for a separate book that focused solely on the diet, the dietitian millicent kelly and i collaborated with my daughter, jennifer freeman, a freelance writer, to write the epilepsy diet treatment : an introduction to the ketogenic diet, a shorter book specifically about the ketogenic diet. it was 1993, and charlie abrahams, the two - year - old son of jim abrahams, the hollywood producer of airplane and naked gun, continued to suffer many uncontrollable drop seizures each day, despite extensive medical intervention. as abrahams has stated, after thousands of epileptic seizures, an incredible number of drugs, dozens of blood draws, eight hospitalizations, a mountain of eegs, mris, cat scans, and pet scans, one fruitless brain surgery, five pediatric neurologists in three cities, two homoeopathists, one faith healer, and countless prayers, charlie s seizures continued unchecked, his development delayed, and he had a prognosis of continued seizures and progressive retardation. researching epilepsy treatments himself, abrahams found our book, seizures and epilepsy : a guide for parents,3 with its three pages on the ketogenic diet. after a phone call, he brought charlie to johns hopkins, where the toddler first fasted, according to our protocol. within several days gradually taken off his medications, charlie has remained seizure - free, on no medications, for the past 19 years. jim was outraged that in all his conversations with medical experts and other parents, he had never been told of the diet. determined to make information about the ketogenic diet available to parents and physicians, he was instrumental in bringing the 1994 dateline nbc news magazine program, an introduction to the ketogenic diet, featuring his friend, the actress meryl streep, to the public. the foundation funded a seven - year study and published 2,500 copies of our shorter book, which sold quickly and attracted a more established publisher, demoshealth. the foundation also funded the production of several dvds explaining the diet to parents, dietitians, and physicians. despite offering them free to physicians at national and regional epilepsy meetings, there were few takers a sign that much work still needed to be done. a big breakthrough in promoting the diet finally came in 1997, when an abrahams - directed, made - for - tv movie, first do no harm, also with streep, was followed by a flood of thousands of phone inquiries and about 150 patients seeking help with the diet from johns hopkins. the new patients allowed us to gather important data about the diet s effectiveness and its side effects. the reports from johns hopkins were the first of an avalanche of abstracts and articles on the clinical outcomes of children who were treated, including outcomes of the diet s various aspects and modifications. the huge increase in the number of clinical abstracts was presented annually at american epilepsy society meetings. the foundation has since been the moving force behind increasing physicians knowledge about the diet and in training parents and dietitians in its use. at the third international conference on dietary therapies for epilepsy and other neurological disorders, organized by the foundation in 2012, close to 500 physicians, dietitians, and parents from 30 countries around the world honored streep for her role in reintroducing the diet. the conference promoted in the foundation s newsletter, ketonews included cooking demonstrations and exhibits, testimonials from parents, and a professional symposium. at first, most epileptologists did not believe that a diet could be as effective as drugs, although multiple large studies documented the diet s effectiveness. but these studies were uncontrolled, and since many were based on the large johns hopkins patient population, there was a tendency for physicians to discount the results as biased by enthusiasts. finally, two blinded crossover studies one from the cross group in england4 in 2008, the other funded by the national institutes of health at johns hopkins in 2009documented the diet s effectiveness in children in a controlled fashion. although there are currently no large studies in adults, anecdotal reports indicate the diet s effectiveness, although it appears adults have more difficulty adhering to its rigidity.5 the outcome of children with uncontrolled seizures who are placed on the diet is shown in the figure (see below), which summarizes data from johns hopkins studies6 and is similar to many reports from other centers. it is notable that 33 percent of the children with intractable seizures were seizure - free, or had only rare seizures, after being on the diet for one year, and 27 percent of the children whose seizures had previously been uncontrollable by medications had no seizures or only rare seizures three to six years after initiating the diet, although by that time most of the children were off the diet and all medications. some with continued seizures remain on the diet because it has decreased the number of medications the children need to take as well as the consequent side effects. no anticonvulsant drugs have been studied for that duration or have shown that rate of beneficial effects. the diet is rarely used as the initial treatment for epilepsy, but should be strongly considered when two anticonvulsants, properly used, have failed. however, the diet may be the initial treatment of choice in infantile spasms and other despite considerable recent research, how the diet exerts its beneficial effects remains unknown.6 it is not solely the ketosis, the accompanying acidosis, the lipidosis, or any of the other chemical changes that have been investigated that are responsible. a recent study9 suggests that episodic fasting, in addition to the diet, is even more effective than the in controlling seizures. learning the mechanisms by which the diet controls epilepsy may give us a better understanding of epilepsy itself. modifications of the ketogenic diet, such as the modified atkins diet, the medium - chain triglyceride (mct) diet, and the low - glycemic diet (lgd) have been developed as alternatives to the rigidity of the classic version. these diets, all of which have been found to have degrees of effectiveness, may be more acceptable to adolescents and adults.5 although large studies of these diets have yet to be performed, uncontrolled studies suggest that they may be effective. if tried, and the individual s seizure control is less than satisfactory, the more rigid, classic ketogenic diet is recommended.1 while the classic ketogenic diet may cause complications1 such as kidney stones,9 lipidemia, and gastrointestinal symptoms, problems are rarely serious and easily managed. vomiting is common in the early stages of fasting and may be relieved with small doses of glucose. constipation is common and may be relieved with small amounts of medium - chain triglyceride oil (mct) or a readily available laxative such as miralax. carnitine is rarely needed, but sugar - free multivitamins and minerals such as calcium are recommended. calcium oxalate and uric acid kidney stones occur in 15 to 20 percent of patients and can be treated or prevented by the administration of potassium citrate. plasma lipids are also known to rise slightly, but they return to normal levels after six months. the rare patient with familial dyslipidemias has been seen, and therefore lipid levels should be checked occasionally.1 generally, children whose seizures are controlled by the diet are tapered off it after two years. but if seizures continue, or recur, the child should remain on the diet longer. some patients have remained on the diet for more than 25 years without adverse effects. the resurgence of interest in the ketogenic diet has led to some very preliminary studies of its use in conditions other than epilepsy. neurodegenerative disorders provide a unique opportunity to study cellular protection through diet.6.9,11 animal models and anecdotal human reports suggest that glucose restriction and the ketogenic diet may have beneficial effects on brain tumors.7,8 tumors rapidly metabolize glucose but are unable to utilize ketones as an energy source. brain tissue, on the other hand, is able to use both. glioblastoma implanted in rodents rapidly regresses on a glucose - restricted ketogenic - like diet. anecdotal reports and preliminary studies in humans have found tumor regression on ketogenic diets. the dramatic findings in glioblastoma may also be true of other brain tumors and perhaps other systemic tumors. there are also anecdotal reports of the diet s benefits6,9,11 in modifying alzheimer s disease, parkinsonism, amyotrophic lateral sclerosis, and possibly posttraumatic brain injury, stroke, and severe hyperactivity. however, such studies are needed to prove or disprove the diet s usefulness. the diet, or a modified form of it, may also be useful in the management of diabetes. preliminary reports of its use in inflammatory disease and the management of pain are also of interest and deserve further study.6 although it has taken 20 years to reintroduce the once - abandoned ketogenic diet, it has become an important new therapy especially for difficult - to - control seizures in children. as more dietitians are trained, and more physicians become aware of the diet, it is used increasingly throughout the world.1 guides to its use are now available in many languages and in many countries, thanks to the charlie foundation and its british cousin, matthew s friends. the idea of food as medicine has been a controversial topic in this country for many years. but the statistics do nt lie, nor do the hundreds of young people who will tell you how their lives were changed because of it. | editor s note : epilepsy and seizures affect nearly 3 million americans of all ages. the incidence is greater in african - americans and in socially disadvantaged populations, and about 200,000 new cases of epilepsy are diagnosed each year. despite these alarming figures, no magic pill exists to eliminate convulsions. while drugs work for some, others find them ineffective. what seems to work just as well, if not better, especially in children, is a relatively unknown, high - fat diet. the author, john m. freeman, m.d., one of the nation s leading advocates for its use, writes about the evolution of the diet and its struggle for acceptance. |
during a first step (november 2009february 2010), seroprevalence rates for a(h1n1)pdm09 virus were assessed in 120 breeding pigs (> 4 years old) from 57 farms. blood was obtained from randomly selected pigs at the only slaughterhouse on the island, where pigs are held for 20) ; the range of positive titers was 40640, and 54.2% of the samples expressed high hi titers (160640). of the 98 serum samples, 5 reacted at low titer and with only 1 european a (h1n1) swine virus (titer 4 dilutions lower than for a(h1n1)pdm09 virus, indicating cross reactivity (6). thus, pigs from 47 (82.4%) of 57 tested farms had been infected by a(h1n1)pdm09 virus ; the seroprevalence rate was 81%100% for pigs on 79.0% of the farms. farms with affected pigs were located throughout the island (figure). hi, hemagglutination inhibition. hi tests were performed according to standard procedure (5).titers are expressed as the reciprocal of the highest dilution of serum that inhibits 4 hemagglutination units of virus. a(h1n1)pdm09 lineage. location of farms tested for antibodies against influenza a(h1n1)pdm09 virus in serologic surveys, runion island, 20092011. blue dots, seronegative farms ; red dots, seropositive farms. in a second step (june 2010, when a(h1n1)pdm09 infection was no longer detected among humans), we tested whether the virus was still circulating among pigs born that year. to obtain nasal swab and blood samples for testing, we randomly selected 390 fattening pigs (2527 weeks old) at the slaughterhouse ; the pigs originated from 45 farms. at the time of sampling, the veterinary surveillance system did not report any clinical signs suggesting virus circulation among herds. however, 3.5% of the serum samples (9% of tested farms) contained antibodies to a(h1n1)pdm09 virus (hi titers 20160). nasal swab specimens from 6.7% (26/390) of pigs were positive for a(h1n1)pdm09 virus as determined by using a specific real - time reverse transcription pcr (rrt - pcr) ; the pigs originated from 13 (28.8%) farms (7). two strains, a / sw / la reunion/0164/10 and a / sw / lareunion/110348/10, were isolated onto mdck cell cultures (5). during july december 2010, 11 farms reported influenza - like clinical signs in pigs, and proof of a(h1n1)pdm09 virus infection was established on 3 farms (farms a fattening pigs on farm a were seronegative for a(h1n1)pdm09 virus. in july, when acute respiratory disease was reported among pigs, 12 of 39 fattening pigs (1821 weeks old) sampled on farm a were still seronegative for a(h1n1)pdm09 virus ; however, rrt - pcr results were positive for a(h1n1)pdm09 virus. four weeks later, when pigs had recovered from influenza, only 7.7% (3/39) of sampled pigs on farm a had rrt - pcr results positive for a(h1n1)pdm09 virus, and all 39 were seropositive for the virus. high rates of rrt - pcr positivity were also noted for pigs on farms b (17/30 pigs) and c (6/15 pigs). two a(h1n1)pdm09 strains (a / sw / lareunion/0167/10 and a / sw / la reunion/110194/10) were isolated from pigs on farms a and b, respectively. four influenza virus strains were isolated from pigs, and all induced a cytopathic effect and displayed hemagglutinating activity on chicken erythrocytes ; all 4 were confirmed as a(h1n1)pdm09 virus by specific rrt - pcrs. in addition cross - hi assays (5) revealed that these strains exhibit antigenic relationships with swine influenza a(h1n1) viruses from classical and avian - like lineages, although they reacted most strongly with a(h1n1)pdm09 virus (table 1). genome sequencing of these strains showed high (> 98%) nucleotide sequence homology to the corresponding genes of a / california/04/09 and 2009 human strains from runion island, suggesting human - to - swine transmission (h. pascalis, unpub. data). in a third step (march, july august, and october 2011), 3 other sampling campaigns were conducted at the slaughterhouse, including 831 fattening pigs from 104 farms. nasal swab samples for 7 (8.4%) pigs from 3 (2.9%) farms still had rrt - pcr positive results. however, serologic analyses revealed that pigs on 40% of the farms (distributed throughout the island) were seropositive for a(h1n1)pdm09 virus, indicating continuing circulation of the virus in swine herds (table 2). seroprevalence rates are % farms positive at the herd level among all farms (n) and % pigs positive at the animal level among all animals (n). consistent with findings elsewhere (8), our results show that a(h1n1)pdm09 virus has substantially affected swine herds in runion island. results of our long - term (2 years) investigation show that a(h1n1)pdm09 virus has circulated in pigs beyond the 5-week epidemic among humans during the austral winter 2009 (3) and has become a novel enzootic pathogen in runion island. several facts may account for the heavy human - to - swine transmission of a(h1n1)pdm09 virus. first, the reassortant pandemic virus contains genomic segments originating from swine influenza viruses established in pigs (1). second, pigs are highly susceptible to experimental inoculations with a(h1n1)pdm09 virus and support high intraspecies transmissibility (9). third, the pressure of infection caused by a(h1n1)pdm09 virus among humans in runion island was high but most infections were mild or asymptomatic (3) ; therefore, people pursued their professional activities, acting as silent spreaders of the virus. last, pigs on runion island had no history of previous passages of swine influenza viruses ; thus, the lack of specific immunity to influenza a viruses would have contributed to the high sensitivity of the pigs to infection, as described (10,11). despite serologic proof of large numbers of infected pigs during late 2009early 2010 in runion island, influenza - like signs were not exhibited and reported until july 2010 ; this finding was similar to that in new caledonia (8). in july 2010, several herds showed symptomatic changes in infection that could indicate either a change in virulence of the circulating strain or the intervention of co - infecting pathogens or some other environmental factor(s). mycoplasma hyopneumoniae and pasteurella multocida were co - detected on farms with pigs with signs of infection (data not shown). co - infection with swine influenza virus and these bacteria is known to contribute to severe respiratory disorders among pigs ; thus, these bacteria may have enhanced pathogenicity of a(h1n1)pdm09 virus on affected farms (12). because specific immunity to a(h1n1)pdm09 virus will decline over time when the virus is no longer circulating among humans, persistence of the virus in an animal reservoir raises concerns about the risk for genetic evolution of the virus and retransmission back to humans of variants with potentially increased virulence. as an example, during the 2011 austral winter, only influenza a(h3n2) and b viruses were recorded (13). novel reassortant viruses containing genomic segments from a(h1n1)pdm09 and enzootic swine influenza viruses have been isolated in pigs (14). such a reassortant was responsible for several cases of influenza among humans in 2011 (15) ; these cases were mild, but other, more virulent pathogenic viruses could emerge. hence continuous surveillance of a(h1n1)pdm09 infection in pigs | during 2009, pandemic influenza a(h1n1)pdm09 virus affected humans on runion island. since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. continuous surveillance of a(h1n1)pdm09 infection in pigs is recommended. |
semiconductor quantum dots are excellent fluorescent nanoprobes for biological and biomedical applications because of their unique size - dependent optical and electronic properties (penn 2003 ; alivisatos these nanoparticles (nps) possess unique optical properties in comparison with traditional organic dyes including size- and composition - tunable emission, narrow emission spectra, and excellent photostability (hotz 2005). recent improvements in surface chemistry have augmented the ability to make functionalized nps to create bioconjugated nps that allow for specific targeting or signal enhancement (farokhzad 2006 ; fujiwara 2006 ; goodman 2006 ; ipe 2006 ; suk 2006). bioconjugated nps, coupled with spectroscopy or other spectral imaging tools, eg, flow cytometry and histological techniques, provides a novel and powerful platform for mapping the molecular and disease profiles associated with infection by different pathogens (zhao 2004 ; driskell 2005 ; yao 2006 ; mcveigh 2006). another advantage of bioconjugated nps over conventional pathogen detection methods is that the nps may be conjugated with many biomolecules thereby enhancing the signal and the binding avidity by mitigating a multivalency effect. this is particularly important for targeting nps and for increasing the delivery or efficacy of bioconjugated molecules. however, only limited reports are available which address the utility of nps for detection or diagnosis virus infection (agrawal 2005, 2006 ; bentzen 2005 ; driskell 2005 ; li, cu, 2005 ; li, liu, 2005 ; wabuyele and vo - dinh 2005 ; wan 2005 ; fuentes 2006 ; liu, cao, 2006 ; souza 2006). respiratory syncytial virus (rsv) is a single - stranded negative sense rna virus in the paramyxoviridae family that is the primary cause of morbidity and life - threatening lower respiratory tract disease in infants and young children worldwide, as well as an important pathogen of the elderly and immune compromised (bader and mckinsey 2005 ; ebbert and limper 2005 ; mejias 2005) the disease burden associated with rsv infection is considerable as rsv is a leading cause of hospitalization for infants and young children worldwide having infection rates approaching 70%80% in the first year of life with many patients requiring hospitalization (mccarthy and hall 2003 ; leung 2005). unfortunately, despite five decades of research, no safe and effective rsv vaccine is available and few effective prophylactic or therapeutic treatments are available. thus, there is a critical need for the development of novel tools and platforms to augment virus diagnosis and foundational studies required for development of disease intervention strategies. the surface topography of viruses lends to detection by antibodies, a feature that has been successfully used for bio - conjuagted np detection of rsv (agrawal 2005, 2006 ; bentzen 2005). rsv has two major surface proteins, ie, g and f proteins, that are primarily responsible for virus attachment and fusion, respectively (tripp 2005). both g and f proteins are recognized by the immune system as neutralizing antigens ; thus a majority of antibodies are directed toward these proteins (tripp 2005). the rsv f protein is more conserved than the g protein among rsv strains (tripp 2005), thus antibodies reactive to the f protein are ideal target developing bioconjugated nanoparticles. taking advantage of the composition - tunable emission of nps and their multivalent specificity when conjugated to monoclonal antibodies, we tested the ability of these bioconjugated nps to detect rsv infection in vitro and in vivo using in a single - step format. we show that bioconjugated nps can be used to rapidly and sensitively detect rsv infection in both cell lines and mice, and that the detection can be done in a single step format. this single - step format dramatically improves virus detection time, potentially saves costs, and reduces background staining that is currently associated with conventional virus detection methods. to develop bioconjugated nanoparticles to detect rsv infection, semiconductor cadmium telluride (cdte) quantum dots (qds) were synthesized as previously described (wang 2002 ; liu, chen, 2006). qds have been reported to be an excellent quantum particle for use in biological systems based on very high photoluminescence quantum efficiencies, and the ability to cover the whole visible spectral range depending on particle size (liu, chen, 2006). thus, two different sized cdte qds with emission peaks at 585 nm and 540 nm were prepared (figure 1) and used in the preparation of bioconjugated nanoparticles (nps) for detection of rsv infection. these particles were prepared for bioconjugation as previously described (wang 2002), and conjugated to monoclonal antibodies reactive to rsv f protein, clone 131 - 2a. briefly, thioglycolate (tga)-coated cdte particles were mixed 1:1 with rsv anti - f protein monoclonal antibody (4 mg / ml) and the solution was stabilized with 10x phosphate - buffered saline (pbs), ph 7.2 to a final 1 x pbs concentration. edc / nhs (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hcl / n - hydroxysulfosuccinimide) was added to bring to qd solution to a final concentration of 50 mm/5 mm, respectively. the reaction was incubated for 2 h at room temperature, then overnight at 4 c. precipitated bioconjugated np aggregates were separated by centrifugation to remove unwanted aggregates from single, bioconjugated nanoparticles. the final concentration of the purified np - antibody conjugate stock solution was 0.625 mm. vero cells (african green monkey kidney cells) were grown in dulbecco s modified eagle s media (dmem) containing 10% fetal bovine sera (dmem-10%). for some experiments involving nps, vero cells were propagated on lab - tek chamber slides to 80%90% confluency using dmem-10%. respiratory syncytial virus strain a2 (rsv) rsv was diluted in dmem and the cells infected at a multiplicity of infection (moi) of 1. the virus was allowed to adsorb for 2 h at 37 c after which dmem-10% was added and the cells incubated at 37 c for 4 days. at day 4 post - infection (pi), the virus was recovered by removing the cell culture supernatant, freeze - thawing the infected cells, and centrifuging the cell lysate to remove debris and recover the virus from the cell lysate supernatant. the bioconjugated nanoparticles conjugated to anti - rsv f protein monoclonal antibody (rsv - nps), ie, both 585 nm and 540 nm qds, were tested for their ability to detect rsv infection of vero cells. rsv - infected vero cells were propagated on lab - tek chamber slides to 80%90% confluency and subsequently infected with rsv at a moi = 1 as previously described (tripp 1999). at day 4 pi, the media from the cells was removed, the cells were washed gently with pbs, fixed with acetone : methanol (60:40), and air dried. rsv - nps (diluted 1:10,000) were added to the cells and incubated for 1 hour at 37 c, followed by three washes with pbs/0.05% tween 20. cells were visualized by fluorescent microscopy (40x) on an olympus ckx41 inverted microscope (olympus, center valley, pa), and photographed with an olympus q - color3 digital camera. the most widely used assay to quantitate virus titers in both the laboratory and clinical setting is the virus plaque assay. virus plaques on the cell lawn are enumerated by immunostaining to specifically detect virus plaques due to confounding background staining that is often associated with the counterstain procedure. rsv - nps were evaluated for detection of rsv infection using a modification of an immunostaining plaque assay as previously described (tripp 1999). briefly, the modification involved substituting rsv - nps in place of the primary (anti - rsv monoclonal antibody : clone 131 - 2a), secondary (anti - mouse whole molecule immunoglobulin g (igg)-alkaline phosphatase conjugated), and detection substrate, eg, vector black alkaline phosphatase, to generate a single - step nanoparticle - based assay. in this modified assay, vero cells were plated onto 24-well flat - bottom plates 24-h prior to rsv infection. subsequently, the cell supernatants were removed and rsv diluted in dmem was added to the cells at a moi = 1. the virus was allowed to adsorb for 1 h at 37 c after which the cells were overlayed with 2% methylcellulose in dmem-10% as previously described (tripp 1999), and cells incubated for 6 days at 37 c, 5% co2. at day 6 pi, the overlays were removed, and cells were fixed with acetone : methanol (60:40), and allowed to air dry. cells were blocked for 30 minutes with powerblock (10% casein in pbs) at room temperature. rsv - nps, either 540 nm or 585 nm bioconjugated qds, were diluted 1:1,000 from 0.625 mm stock solution in pbs. for positive control wells, anti - rsv monoclonal antibody (clone 131 - 2a) was diluted 1:500 in pbs containing powerblock, added to the appropriate wells, and incubated 1 hour for rsv - nps, or 2 hours for the primary monoclonal antibody at 37 c. cells were washed 3 times with pbs/0.05% tween. for standard immunostaining plaque assay, secondary antibody (anti - mouse igg - alkaline phosphatase conjugated) was diluted 1:500 in powerblock, added to cells, incubated for 1 hour at 37 c, washed twice with pbs, and developed using a vector black alkaline phosphatase substrate kit according to the manufacturer s protocol. for rsv - nps, the plates were directly scanned for virus plaques using an amersham biosciences typhoon 9210 scanner with a fluorescence excitation (532 nm) and emission (526 sp) filter. the virus plaques were enumerated and compared to plaque numbers obtained by standard counting using an olympus sz dissecting scope. to determine the ability of rsv - nps to detect rsv infection in lung tissue, the natural site of rsv infection and replication (tripp 2005), 68 week old female balb / c mice were intranasally infected with 10 plaque - forming units (pfu) of rsv as previously described (tripp 1999). at day 4 pi, rsv - infected or nave balb / c mice were intravenously administered by tail vein 0.05 ml of rsv - nps, either 540 nm or 585 nm bioconjugated qds, diluted 1:1000 in pbs from a 0.625 mm stock solution. at day 5 pi, the lungs from rsv - np - treated or pbs carrier - treated mice were removed and prepared for thin sectioning and immunohistochemistry (ihc) analysis as previously described (li, sarmento, 2005). briefly, lung tissue was frozen as tissue blocks and sectioned (20 m) on a cryostat. the frozen sections were placed on slides which were immersed in acetone at 4 c for 15 min, air dried for 30s, and then stored in pbs until immunostaining was performed. briefly, slides were stained with anti - rsv monoclonal antibody (clone 131 - 2a) diluted 1:500 in pbs and incubated for 2 h at 37 c. the slides were washed 2 times with pbs, and a secondary antibody (fitc conjugated anti - mouse igg) diluted 1:500 in pbs was added to slides and incubated for 1 h at 37 c, and washed twice with pbs. the lung sections from rsv - np - treated mice and untreated mice that were immunostained as noted above were directly visualized using a fluorescent microscope. to develop bioconjugated nanoparticles to detect rsv infection, semiconductor cadmium telluride (cdte) quantum dots (qds) were synthesized as previously described (wang 2002 ; liu, chen, 2006). qds have been reported to be an excellent quantum particle for use in biological systems based on very high photoluminescence quantum efficiencies, and the ability to cover the whole visible spectral range depending on particle size (liu, chen, 2006). thus, two different sized cdte qds with emission peaks at 585 nm and 540 nm were prepared (figure 1) and used in the preparation of bioconjugated nanoparticles (nps) for detection of rsv infection. these particles were prepared for bioconjugation as previously described (wang 2002), and conjugated to monoclonal antibodies reactive to rsv f protein, clone 131 - 2a. briefly, thioglycolate (tga)-coated cdte particles were mixed 1:1 with rsv anti - f protein monoclonal antibody (4 mg / ml) and the solution was stabilized with 10x phosphate - buffered saline (pbs), ph 7.2 to a final 1 x pbs concentration. edc / nhs (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hcl / n - hydroxysulfosuccinimide) was added to bring to qd solution to a final concentration of 50 mm/5 mm, respectively. the reaction was incubated for 2 h at room temperature, then overnight at 4 c. precipitated bioconjugated np aggregates were separated by centrifugation to remove unwanted aggregates from single, bioconjugated nanoparticles. the final concentration of the purified np - antibody conjugate stock solution was 0.625 mm. vero cells (african green monkey kidney cells) were grown in dulbecco s modified eagle s media (dmem) containing 10% fetal bovine sera (dmem-10%). for some experiments involving nps, vero cells were propagated on lab - tek chamber slides to 80%90% confluency using dmem-10%. respiratory syncytial virus strain a2 (rsv) was propagated in vero cells as previously described (tripp 1999). rsv was diluted in dmem and the cells infected at a multiplicity of infection (moi) of 1. the virus was allowed to adsorb for 2 h at 37 c after which dmem-10% was added and the cells incubated at 37 c for 4 days. at day 4 post - infection (pi), the virus was recovered by removing the cell culture supernatant, freeze - thawing the infected cells, and centrifuging the cell lysate to remove debris and recover the virus from the cell lysate supernatant. the bioconjugated nanoparticles conjugated to anti - rsv f protein monoclonal antibody (rsv - nps), ie, both 585 nm and 540 nm qds, were tested for their ability to detect rsv infection of vero cells. rsv - infected vero cells were propagated on lab - tek chamber slides to 80%90% confluency and subsequently infected with rsv at a moi = 1 as previously described (tripp 1999). at day 4 pi, the media from the cells was removed, the cells were washed gently with pbs, fixed with acetone : methanol (60:40), and air dried. rsv - nps (diluted 1:10,000) were added to the cells and incubated for 1 hour at 37 c, followed by three washes with pbs/0.05% tween 20. cells were visualized by fluorescent microscopy (40x) on an olympus ckx41 inverted microscope (olympus, center valley, pa), and photographed with an olympus q - color3 digital camera. the most widely used assay to quantitate virus titers in both the laboratory and clinical setting is the virus plaque assay. virus plaques on the cell lawn are enumerated by immunostaining to specifically detect virus plaques due to confounding background staining that is often associated with the counterstain procedure. rsv - nps were evaluated for detection of rsv infection using a modification of an immunostaining plaque assay as previously described (tripp 1999). briefly, the modification involved substituting rsv - nps in place of the primary (anti - rsv monoclonal antibody : clone 131 - 2a), secondary (anti - mouse whole molecule immunoglobulin g (igg)-alkaline phosphatase conjugated), and detection substrate, eg, vector black alkaline phosphatase, to generate a single - step nanoparticle - based assay. in this modified assay, vero cells were plated onto 24-well flat - bottom plates 24-h prior to rsv infection. subsequently, the cell supernatants were removed and rsv diluted in dmem was added to the cells at a moi = 1. the virus was allowed to adsorb for 1 h at 37 c after which the cells were overlayed with 2% methylcellulose in dmem-10% as previously described (tripp 1999), and cells incubated for 6 days at 37 c, 5% co2. at day 6 pi, the overlays were removed, and cells were fixed with acetone : methanol (60:40), and allowed to air dry. cells were blocked for 30 minutes with powerblock (10% casein in pbs) at room temperature. rsv - nps, either 540 nm or 585 nm bioconjugated qds, were diluted 1:1,000 from 0.625 mm stock solution in pbs. for positive control wells, anti - rsv monoclonal antibody (clone 131 - 2a) was diluted 1:500 in pbs containing powerblock, added to the appropriate wells, and incubated 1 hour for rsv - nps, or 2 hours for the primary monoclonal antibody at 37 c. secondary antibody (anti - mouse igg - alkaline phosphatase conjugated) was diluted 1:500 in powerblock, added to cells, incubated for 1 hour at 37 c, washed twice with pbs, and developed using a vector black alkaline phosphatase substrate kit according to the manufacturer s protocol. for rsv - nps, the plates were directly scanned for virus plaques using an amersham biosciences typhoon 9210 scanner with a fluorescence excitation (532 nm) and emission (526 sp) filter. the virus plaques were enumerated and compared to plaque numbers obtained by standard counting using an olympus sz dissecting scope. to determine the ability of rsv - nps to detect rsv infection in lung tissue, the natural site of rsv infection and replication (tripp 2005), 68 week old female balb / c mice were intranasally infected with 10 plaque - forming units (pfu) of rsv as previously described (tripp 1999). at day 4 pi, rsv - infected or nave balb / c mice were intravenously administered by tail vein 0.05 ml of rsv - nps, either 540 nm or 585 nm bioconjugated qds, diluted 1:1000 in pbs from a 0.625 mm stock solution. at day 5 pi, the lungs from rsv - np - treated or pbs carrier - treated mice were removed and prepared for thin sectioning and immunohistochemistry (ihc) analysis as previously described (li, sarmento, 2005). briefly, lung tissue was frozen as tissue blocks and sectioned (20 m) on a cryostat. the frozen sections were placed on slides which were immersed in acetone at 4 c for 15 min, air dried for 30s, and then stored in pbs until immunostaining was performed. briefly, slides were stained with anti - rsv monoclonal antibody (clone 131 - 2a) diluted 1:500 in pbs and incubated for 2 h at 37 c. the slides were washed 2 times with pbs, and a secondary antibody (fitc conjugated anti - mouse igg) diluted 1:500 in pbs was added to slides and incubated for 1 h at 37 c, and washed twice with pbs. the lung sections from rsv - np - treated mice and untreated mice that were immunostained as noted above were directly visualized using a fluorescent microscope. current antibody - based methods used for virus detection are cumbersome, have limited sensitivity due to substantial background staining, and are costly as they often require multiple antibodies and enzyme substrate for detection. in an effort to ameliorate these issues, and reduce the time for virus detection, we synthesized semiconductor cdte qds with emission at either 540 nm or 585 nm to be conjugated to monoclonal antibodies to detect rsv - infected cells. these bioconjugated nps (rsv - nps) that are specific to rsv f protein were tested for their ability to detect rsv infection of cells both in vitro and in vivo. as expected, no differences in virus detection were observed using either 540 or 585 nm nps. as shown in the high resolution transmission electron microscopy (hrtem) images in figure 1, the green emission cdte nps had an average size of approximately 3 nm (a) and the orange cdte nps of 5 nm (b). given the reported difficulties conjugating proteins to cdte nps (tan 2004 ; gao 2005 ; mcveigh 2006), the cdte nps were produced with a thioglycolate coating to allow direct anti - rsv f protein monoclonal antibody conjugation via an edc / sulfo - nhs conjugation reaction. these bioconju - gated nps (rsv - nps) were stable for > 1 year at 4 c, and did not precipitate in the pbs diluent at room temperature or at 4 c. to determine the ability of the rsv - nps to detect rsv infection, in vitro studies were performed using rsv - infected vero cells. in these studies, we compared rsv detection using conventional antibody staining methods, ie, primary and secondary antibody staining plus enzyme substrate, to that of a single - step detection method using rsv - nps. conventional antibody staining methods produced substantial background staining in mock - infected vero cells (figure 2a), however did detect rsv plaque formation in the vero cell lawn (figure 2b). in contrast, rsv - nps used in a single - step method produced little background (figure 2c), and readily detected rsv plaques (figure 2d). these results show that rsv - nps can be used to detect rsv in a single - step fashion, and that this method can be used to greatly reduce the amount of time and reagent needed for rsv detection compared to conventional immunostaining procedures (tripp 1999). extending the in vitro detection findings (figure 2), we determined if rsv - nps could be used to quantitate rsv titers in a single step fashion, thereby shortening the detection time needed to quantitate virus titers by plaque assay (figure 3). in these studies, we compared a conventional immunostaining plaque assay which requires 5 or 6 days of in vitro culture post - rsv infection (tripp 1999) to rapid single - step detection by rsv - nps. for comparison of the different plaque assay methods, virus titers were assessed at days 2, 3, 5, or 6 pi. the results showed that at days 5 or 6 pi, conventional immunostaining (figure 3a) and the single - step rsv - np detection method (figure 3b) were similarly sensitive for detecting rsv plaques in the vero cell lawn. however, rsv - nps could detect rsv plaques earlier (days 2 or 3 pi) compared with conventional immunostaining which was comparatively ineffective (figure 4), indicating that the single - step rsv - nps procedure was more sensitive at detecting limiting levels of rsv. this result is not unexpected given the multivalent properties of rsv - nps. to evaluate the efficacy of rsv - nps to detect rsv infection in vivo, balb / c were either intranasally infected with 10 pfu rsv or mock - treated, and at day 4 pi, rsv - infected or mock - treated mice were intravenously administered 0.05 ml of rsv - nps from the stock solution (0.625 mm) by tail vein. at day 5 pi, the lungs from rsv - np - treated or mock - treated mice were removed, frozen, and prepared for thin sectioning and ihc analysis using conventional methods, i.e. primary and secondary antibody staining, or directly visualized by fluorescence microscopy in the case of rsv - np treatment. treatment with rsv - nps provided clear and rapid detection of rsv - infected lung tissue (figure 5a) with very limited background staining as assessed in lung tissue from mock - treated mice similarly administered rsv - nps (figure 5b). importantly, the magnitude and location of rsv - nps staining along the epithelial cells of the alveoli in the lungs was similar to that of conventional ihc staining (figure 5c), and the background staining for rsv - nps was lower in nave mice compared to conventional ihc staining (figure 5d). the selective staining by rsv - nps of only rsv - infected lung epithelial cells suggests antibody - mediated specific targeting to sites of infection. these results suggest that rsv - nps may be useful to determine the progression of rsv infection in the lungs, and as a result, aid our understanding of sites of infection, and help to define disease intervention strategies that employ bioconjugated np targeting strategies. the results from this study show that rsv - nps can be used to detect rsv infection both in vitro and in vivo and suggest that beyond diagnostics, bioconjugated nps may be useful for multiplexed detection of viral and/or host cell antigens, as well as for targeting sites of virus infection with antiviral agents that may be linked to the nps. indeed, bioconjugated nps have been used to identify the presence and progression of rsv infection in a human epithelial cell line, ie, hep-2 cells (bentzen 2005), as well as used in a variety of intracellular tracking and related studies (alivisatos 2005 ; bruchez 2005 ; gao 2005 ; hotz 2005 ; shenoy 2006 ; mcveigh 2006). importantly, we show here that rsv - nps can be applied as tools to enhance virus detection by increasing sensitivity of virus detection while decreasing the detection time. application of rsv - nps for virus detection in animal models can provide a path to assist our ability to dissect important parameters of rsv disease pathogenesis such as determining sites of virus infection and virus load in the lungs of infected animals. | the integration of nanotechnology with biology has produced major advances in molecular diagnostics, therapeutics, and bioengineering. recent advances have led to the development of functionalized nanoparticles (nps) that are covalently linked to biological molecules such as antibodies, peptides, proteins, and nucleic acids. these functionalized nps allow for development of novel diagnostic tools and methods, particularly for pathogens, as rapid and sensitive diagnostics are essential for defining the emergence of infection, determining the period that preventive measures should be applied, for evaluating drug and vaccine efficacy, and for controlling epidemics. in this study, we show that functionalized nps conjugated to monoclonal antibodies can be used to rapidly and specifically detect respiratory syncytial virus in vitro and in vivo. these results suggest that functionalized nps can provide direct, rapid, and sensitive detection of viruses and thereby bridge the gap between current cumbersome virus detection assays and the burgeoning need for more rapid and sensitive detection of viral agents. |
se presentan dos casos de hepatitis viral en los cuales la terapia convencional no fue efectiva. ambos fueron tratados posteriormente segn protocolos de uso de medicamentos homeopticos, como se describe a continuacin. ambos pacientes registraron una remisin sostenida durante 2 aos, luego de consumir medicamentos naturales ultradiluidos y de que se descontinuaran sus correspondientes tratamientos convencionales. el protocolo de tratamiento incluy como medicamentos principales chelidonium majus (celidonia mayor) 6x y thuja (tuya) 30c. se confirmaron casos de desarrollo de anti - cuerpos estndares contra la hepatitis y mediciones de carga viral. se realiz un seguimiento de los pacientes durante ms de 2 aos con mediciones de carga viral, enzimas hepticas y otros marcadores biolgicos relevantes de enfermedades hepticas. ambos pacientes viven y se desenvuelven normalmente en sus hogares, luego de transcurridos ms de 2 aos desde el inicio del tratamiento. discusin : analizamos la informacin relacionada con los medicamentos principales empleados en estos casos y descubrimos que tienen efectos teraputicos conocidos y demostrados que sugeran mecanismos de accin convincentes aplicables a estos casos. los ensayos clnicos de este protocolo de tratamiento homeoptico se deben llevar a cabo para analizar los efectos teraputicos potenciales de este tipo de medicamentos para el tratamiento de la hepatitis viral. on a routine health maintenance visit in 1994, a 37-year - old woman was found to have elevated liver enzymes. her first liver biopsy in january 1998 showed grade 1 (of 4) inflammation and stage 1 to 2 (of 4) delicate bridging fibrosis. subsequent hepatitis c antibody testing revealed chronic hepatitis c. genotyping of the virus in 1998 revealed type 1b with a viral count of 33 000 000 iu / ml. she enrolled in a clinical trial of pegylated inter - feron (peg - inf) subcutaneously 1.5 [.proportional]g / kg once a week for 4 weeks followed by peg - inf 0.5 [.proportional]g / kg once a week for 44 weeks along with ribavirin 1000 mg orally daily, which was reduced to 600 mg daily due to anemia at treatment week 30. her virologic response using polymerase chain reaction (pcr)based assay for hepatitis c virus rna showed a temporary response. posttreatment liver biopsy performed 6 months after completing treatment (in july 2000) was scored as grade 3 of 4 inflammation and stage 1 of 4 fibrosis with piecemeal necrosis consistent with relapse. a biopsy conducted in december 2003 showed inflammation grade 3 and fibrosis stage 3 of 4. in april 2004, she started a second course of peg - inf with ribavirin after undergoing whole - body hyperthermia. after 6 months, she was found to have no response to the interferon, and the drugs were discontinued. a biopsy in november 2005 showed stage 3 of 4 fibrosis and moderate (3 of 4) portal inflammation. viral count in july 2006 was 14 250 000 iu / ml, and the patient was found to have persistent stage 3 of 4 fibrosis and grade 3 of 4 inflammation with bridging necrosis. in august 2006, the following protocol was used : chelidonium 6x twice a day, thuja 30c twice a day, and kalium muriaticum 3x and ferrum phosphoricum 3x twice a day. the 6x potency is the 6th decimal potency that is achieved by serial dilution and agitation of the mother tincture, or alcoholic extract, of the root of the plant chelidonium majus. thuja 30c is likewise the 30th centesimal serial dilution and agitated product ; here, the alcoholic extract is from the fresh leaves and small twigs of the young thuja occidentalis plant. the kali muriaticum 3x and ferrum phosphoricum 3x are triturations of the substances to the 3rd decimal potency. the medicine was procured from reputable homeopathic drug manufacturers and manufactured as per the homeopathic pharmacopoeia of india. chelidonium 6x and thuja 30c are our standard protocol for cases of chronic viral hepatitis. chelidonium has a strong body of research supporting its use for liver disease, and thuja is effective in treating a wide variety of viral infections (see discussion section). the combination of kali muriaticum and ferrum phosphoricum is our standard protocol for treatment of anemia, which this patient experienced as a side effect of interferon / ribavirin therapy. the patient adhered to this protocol for 2 years and was rebiopsied in the united states in december 2008. her inflammation was reduced to stage 1 of 4, and her fibrosis had regressed to stage 01a of 4. as of june 2011, she remained in remission and continued treatment with chelidonium 6x twice a day. case 1 : chronic active hepatitis c homeopathic treatment initiated august 2006 ; as of june 2011, patient remained in remission. abbreviations : hcv, hepatitis c virus ; rna, ribonucleic acid ; pcr, polymerase chain reaction. in late november 2007, a 28-year - old male was admitted to the premier indian medical institution, the all india institute of medical science (aiims) in delhi, for a case of hepatitis b virus (hbv)related chronic liver disease decompensated by acute hepatitis e virus (hev) infection. his clinical history included a rapidly progressing jaundice followed by pedal edema, ascites, fever, and abdominal tenderness. viral antibody testing revealed a positive australia antigen (hepatitis b surface antigen), negative immunoglobulin m for hepatitis b core antigen, hbv dna 1300 copies / ml, and positive immunoglobulin m antibody for hev. at aiims, he was treated with intravenous glycyrrhizin (0.2%) 60 ml daily for 6 weeks, and then the dose was reduced to 3 times a week. additionally, he received daily diuretic treatment with spironolactone / furosemide (lasilactone, sanofi - aventis) 50 to 75 mg per day, 20% albumin 100 ml intravenously daily for the first 2 months of hospitalization, cefuroxime axetil (ceftum, glaxosmithkline) 500 mg twice a day for 4 weeks, and lamivudine - hbv 100 mg daily. after 6 weeks of hospitalization and treatment at aiims, the patient 's serum bilirubin continued to be markedly elevated and alanine transaminase was continuously 75 times normal, indicating failure of conservative treatment. they refused to have him placed on the transplant list, and he was discharged in january 2008 and returned to kolkata. after repeated episodes of spontaneous bacterial peritonitis requiring multiple hospitalizations in kolkata, he developed right hepatic hydrothorax. at this point, the patient sought treatment at pbhrf. on first presenting at pbhrf on august 22, 2008, he had severe ascites, dyspnea without exertion, abdominal pain, and 4 + pitting edema in the lower extremities. treatment was initiated with the following protocol : chelidonium 6x 3 drops alternating 3 times a day with carduus marianus (milk thistle) mother tincture 10 drops, thuja 30c 2 pills once every evening, lycopodium clavatum 30c 3 drops 3 times a day, and belladonna 3c alternating with carduus marianus mother tincture every 10 minutes as needed for pain. in addition to our first - line agent, chelidonium, we added carduus marianus, as it has a long history of use in traditional herbal medicine for support of liver problems. lycopodium is our first - line agent for treatment of edema or fluid retention of any kind. belladonna is one of our first - line agents for pain, particularly pain of visceral origin. when the patient 's condition did not improve, on september 15, 2008, myrica (bayberry) mother tincture was added, alternating every 3 hours with chelidonium 6x, and carduus was discontinued. on september 27, acetic acid 30c, another of our prime medicines for water retention and effusions, 3 drops 3 times a day replaced the lycopodium for management of the ascites. by december 13, 2008, the patient 's pleural effusion was clearing, ascites had decreased substantially, and urine output improved significantly. clinic notes from january 7, 2009, reported worsening of the pleural effusion and ascites, and treatment exclusively by pbhrf continued with the expectation that improvement was likely to recur. by march 2009, the patient was reporting a sustained improvement in symptoms, and the pleural effusion and ascites had almost completely subsided. on june 3, 2009, the hbv dna count was 5.82 copies, and the patient was feeling well. blood work done on may 9, 2009, revealed liver function tests generally near the normal range, as indicated in table 2. table 2 also shows the continued improvement in liver function tests when retested in december 2009. as of june 2011, the patient continued to feel well, having been in remission for 2 years. case 2 : hepatitis b virus and hepatitis e virus homeopathic treatment initiated august 2008 ; as of june 2011, the patient remained in remission. abbreviations : alk phos, alkaline phosphatase ; alt, alanine transaminase ; ast, aspartate transaminase ; ggt, gamma - glutamyl transferase ; inr, international normalized ratio ; tbili, total bilirubin. the protocols used in these cases were developed based on the extensive experience of the physicians at pbhrf, which spans several generations, as well as the known actions of its specific component medicines. chelidonium majus (greater celandine) is an herb with documented hepatotoxic properties in its undiluted tincture or herbal form, but it has also been shown to have hepatoprotective, antitumor, and immunostimulatory actions. thuja and its related species also have been reported to have antiviral and antimetastatic properties. myrica, or bayberry, is a common herb that is high in tannins ; there is virtually no research documenting its effectiveness in treatment of liver disease, but standard homeopathic references all list jaundice as one of its principle indications. the conclusion of this rigorous review was that milk thistle could potentially affect alcoholic and/or hepatitis b or c virus liver diseases. therefore, large - scale randomized clinical trials on milk thistle for alcoholic and/or hepatitis b or c liver diseases versus placebo are needed. the larger issue is how ultradilute, serially agitated preparations of these biologically active substances are able to exert therapeutic effects even when the dilutions exceed avogadro 's number, which is the case for the dilutions of 30c used in the banerji protocol for hepatitis. the preparations of chelidonium are diluted to a factor of 1/1 000 000 ; this explains the lack of toxicity observed in the normally hepatotoxic chelidonium when in its crude form but does not explain its effectiveness as a hepatoprotectant. the emerging disciplines of complexity, nanoscience, and materials science offer some hypotheses on how these ultradilute medicines may still maintain biological activity. one research team advocated the hypothesis based on available scientific evidence and logic that one major pathway of ultra - dilute homeopathic drugs could possibly be through regulation of expression of relevant genes. a recent study by frenkel provided solid support for this hypothesis. the medicines used by pbhrf for treatment of breast cancer were tested in vitro at the university of texas md anderson cancer center, houston. the remedies exerted preferential cytotoxic effects against 2 breast cancer cell lines, causing cell cycle delay / arrest and apoptosis. the researchers demonstrated a clear biological activity of the tested natural products (phytolacca, carcinosin, conium, and thuja) when present at ultradiluted doses. despite the lack of a proven explanation for how these ultradilute medicines exert their effects, there is significant laboratory evidence that highly dilute toxins can paradoxically protect the very tissues they harm in macrodoses. there are several reports of liver damage reversal in mice with ultradilutions of arsenic trioxide after exposure to toxic doses of the same substance. one randomized double - blind placebo - controlled human study documented favorable improvements in multiple markers of arsenic toxicity after 2 months of treatment with a serially agitated dilution (1:100 dilution 30 times) of arsenic, or the 30th centesimal potency. a recent review of the in vitro research on serially diluted and agitated solutions concluded that even the studies with high methodological standards demonstrated an effect of these solutions. a number of articles have been published in medical journals denouncing categorically the use of homeopathic medicine, claiming that there is no evidence to support any further research into their therapeutic effects. clearly, even in this very brief research review and in these case reports, there is enough to suggest that this is an area that should be further explored. the era of nanomedicine is upon us and requires a fresh look at medicines that are ultradiluted. a major advantage of treating disease with ultradilute solutions is that adverse effects are virtually eliminated. the case reports in this article will, hopefully, inspire a fresh interest and further research in this fascinating and controversial area of therapeutics. | abstractintroduction : two cases of viral hepatitis that had failed conventional therapy are presented. both were subsequently treated with protocols using homeopathic medicines as detailed below. both patients sustained remissions for 2 years after taking ultradilute natural medicines after their conventional treatment had been discontinued.methods:the treatment protocol included chelidonium majus 6x and thuja 30c as the main medicines. other homeopathic medicines were used as detailed below. cases were confirmed with standard hepatitis antibody and viral measurements. patients were followed for more than 2 years with measurements of viral counts, liver enzymes, and other relevant biomarkers of liver disease.results:both patients are alive and functioning normally in their home environments more than 2 years after treatment initiation.discussion:we review the literature related to the chief medicines used in these cases and find that they have known and demonstrated therapeutic effects suggesting plausible mechanisms of action in these cases.conclusions:clinical trials of this homeopathic treatment protocol should be conducted to explore the therapeutic potential of these medicines for treatment of viral hepatitis. |
odontoma that was previously considered as tumor of odontogenic origin is now considered as hamartomatous dental malformation formed by the overgrowth or transitory of complete dental tissue. odontomas are usually asymptomatic and generally consist of unerupted or impacted teeth, retained deciduous teeth, swelling and evidence of infection. the incidence of compound odontoma ranges between 9% to 37% and that of complex odontoma between 5% to 30%, respectively. odontomas are usually discovered during second and third decades of life with some cases in first decade of life too. the compound odontoma is more common than complex odontoma, which in turn is more common than ameloblasticodontoma. majority of odontomas in anterior segment of jaws are compound odontoma (61%) ; whereas in posterior segment, complex odontomas are common (34%). interestingly, it has been observed that both type of odontomas occurred more frequently on right side of jaw than left, (compound 62% and complex 68%). the compound odontoma frequently occurs in incisor - cuspid region of maxillary arch in contrast to complex odontoma that is usually found in premolar - molar region of mandible. in this paper, a rare case of massive compound odontoma with 36 denticles in 9 years boy is discussed that was diagnosed accidentally and treated for the presentation of condition with relevant literature review. a 9-year boy reported to the department of pediatric and preventive dentistry, with a chief complaint of pain and swelling in mandibular left posterior region since 1 year. the swelling was small initially that increased gradually over a period of time to present size with pain aggravating on mastication. intra - oral examination of affected side revealed presence of a grossly carious, retained 74. an oval shaped swelling was noticed over buccal gingiva extending from mesial margin of 33 to distal margin of 35 anteroposteriorly measuring approximately 3 cm and superoinferiorly 1.5 cm in dimension. adjacent 33 was partly erupted and revealed slight mesial displacement as compared to contralateral 43. patient was advised an intra - oral periapical radiograph (iopar) and orthopantomograph (opg) that revealed presence of multiple dense radio - opaque structures contained in a radiolucent cavity surrounded by a corticated border in relation to apices of carious 74 [figure 1a and b ]. based on clinical signs and radiographic findings, the condition was provisionally diagnosed as compound odontoma. differential diagnosis of this condition included odontoma, ameloblastic fibro - odontoma, ameloblastic fibroma, dentinoma, third stage cementoblastoma and odonto - ameloblastoma. (a) intra - oral periapical radiograph showing multiple radio - opaque tooth - like structures below 74, (b) orthopantomograph showing multiple denticles surrounded by a narrow radiolucent zone treatment of choice for management of odontoma comprise extraction of retained primary tooth, followed by complete surgical enucleation of denticles and associated soft tissues. after all investigations, profuse local anesthesia of associated area was achieved by mandibular nerve block and local infiltration technique. the retained 74 was extracted and full thickness trapezoidal mucoperiosteal flap from mesial margin of 33 to distal margin of 35 was reflected to visualize the area. a bony window was prepared through occlusal cortical bone and about 36 denticles were removed carefully along with the capsule [figure 2 ]. the unerupted 34 was exposed to facilitate its eruption and sharp bony margins were rounded off [figure 2 ]. the patient was prescribed suitable antibiotics and analgesics for 5 days and recalled after 1 week for suture removal. gross specimen of surgically removed odontomas showing denticles and surgically exposed 34 after removal of all denticles the excised tissue was sent to department of oral pathology and microbiology for histopathological evaluation. macroscopically extracted denticles were independent as well as fused with morphological resemblance to normal tooth. dimensions of these denticles were variable ; from 2 mm 3 mm to 8 mm 12 mm and shapes from regular droplet to bizarre peg like [figure 2 ]. decalcified hematoxylin and eosin stained section of the specimen showed central zone of pulp spaces and areas of active mineralization [figure 3a ]. areas of primary dentin were observed juxtaposed between outer empty spaces of partially demineralized enamel / primary cementum and pulp [figure 3b ]. ground section of denticles showed areas of enamel, dentin and pulp in coronal region as well as cementum, dentin and pulp spaces in transmitted light [figure 3c ]. (a and b) decalcified h and e stained odontoma showing enamel (e), dentin (d) and pulp space (p) with connective tissue stroma and cementum (c) (10 view). (c) ground section of odontoma showing tooth - like arrangement of enamel (e), dentin (d) and pulp space (p) (10 view) healing of surgical wound was uneventful and by means of secondary intension. the patient is on periodic observation for 6, 12, and 24 months interval. radiographic observations after 6 months revealed erupting 34 with space loss due to drifting of adjacent teeth [figure 4 ]. follow - up radiographic evaluation (opg and iopar, respectively) of erupting 34 shows eruptive tooth movement [figures 5 and 6 ]. orthopantomograph showing 6 months follow - up and erupting 34 follow - up orthopantomograph radiograph of patient after 9 months showing eruptive tooth movement follow - up intra - oral periapical radiograph of 34 after 9 months showing eruptive tooth movement treatment of choice for management of odontoma comprise extraction of retained primary tooth, followed by complete surgical enucleation of denticles and associated soft tissues. after all investigations, profuse local anesthesia of associated area was achieved by mandibular nerve block and local infiltration technique. the retained 74 was extracted and full thickness trapezoidal mucoperiosteal flap from mesial margin of 33 to distal margin of 35 was reflected to visualize the area. a bony window was prepared through occlusal cortical bone and about 36 denticles were removed carefully along with the capsule [figure 2 ]. the unerupted 34 was exposed to facilitate its eruption and sharp bony margins were rounded off [figure 2 ]. the patient was prescribed suitable antibiotics and analgesics for 5 days and recalled after 1 week for suture removal. gross specimen of surgically removed odontomas showing denticles and surgically exposed 34 after removal of all denticles the excised tissue was sent to department of oral pathology and microbiology for histopathological evaluation. macroscopically extracted denticles were independent as well as fused with morphological resemblance to normal tooth. dimensions of these denticles were variable ; from 2 mm 3 mm to 8 mm 12 mm and shapes from regular droplet to bizarre peg like [figure 2 ]. decalcified hematoxylin and eosin stained section of the specimen showed central zone of pulp spaces and areas of active mineralization [figure 3a ]. areas of primary dentin were observed juxtaposed between outer empty spaces of partially demineralized enamel / primary cementum and pulp [figure 3b ]. ground section of denticles showed areas of enamel, dentin and pulp in coronal region as well as cementum, dentin and pulp spaces in transmitted light [figure 3c ]. (a and b) decalcified h and e stained odontoma showing enamel (e), dentin (d) and pulp space (p) with connective tissue stroma and cementum (c) (10 view). (c) ground section of odontoma showing tooth - like arrangement of enamel (e), dentin (d) and pulp space (p) (10 view) healing of surgical wound was uneventful and by means of secondary intension. the patient is on periodic observation for 6, 12, and 24 months interval. radiographic observations after 6 months revealed erupting 34 with space loss due to drifting of adjacent teeth [figure 4 ]. follow - up radiographic evaluation (opg and iopar, respectively) of erupting 34 shows eruptive tooth movement [figures 5 and 6 ]. orthopantomograph showing 6 months follow - up and erupting 34 follow - up orthopantomograph radiograph of patient after 9 months showing eruptive tooth movement follow - up intra - oral periapical radiograph of 34 after 9 months showing eruptive tooth movement odontoma is a type of dental malformation resulting from growth of both epithelial and mesenchymal components of dental lamina remnants. these components demonstrate complete differentiation, resulting in functional ameloblasts and odontoblasts forming enamel and dentin. odontomas are inherited through postnatal mutant gene interference that control tooth development. in humans, mutation of these persistent dental lamina cells or tooth germ epithelial cells may trigger their inherent capacity to transform into cap and bell stages necessary for tooth formation. moreover, retained ability of these tissues may stimulate mesenchymal cell differentiation, obligatory for hard tissue formation. comparison of normal tooth forming and odontogenic tumor cells revealed presence of common molecules, expressed in both types of cells. a very likely explanation for this is recapitulation and over expression of tumor specific genetic programs transcribed at an undetectable level during normal odontogenesis and detectable level by odontogenic tumor epithelial cells in an abnormal manner. odontoma is usually asymptomatic ; however, occurrence of odontoma is associated with multiple factors such as trauma and infection at the affected site offering ideal conditions for its initiation. pressures due to growth, trauma, infection, presence of mature ameloblasts, cell rests of serres (dental lamina remnants) and extraneous odontogenic epithelial cells may be regarded as sources of disturbances in the mechanism of development. a developing odontoma can be detected by routine radiography and sometimes may cause difficulty in identification due to inadequate calcification. however, a mature compound odontoma appears as collection of tooth - like structures surrounded by a narrow radiolucent zone. the compound odontoma appears as a collection of numerous radio - opaque, miniature tooth - like structures known as denticles. occasionally, they may become large and produce expansion of bone with consequent facial asymmetry. in most of the cases, pathologic alterations observed in neighboring teeth include devitalization, malformation, aplasia, malposition or impaction. increase in the size of odontoma leads to sequestration or resorption of overlying bone, thus causing its occlusal movement. degree of morpho - differentiation and histodifferentiation of dental hard tissue is essential for making differential diagnosis of odontoma. although ameloblastic odontoma, ameloblastic fibro - odontoma bear greater radiographic resemblance to odontoma, histopathological examination for definitive diagnosis of the condition is always recommended. some investigators propose that ameloblastic fibroma and ameloblastic fibro - odontoma both developmentally and histomorphologically represent early stages of odontoma formation. in earlier days ; odontoma as tumor of odontogenic origin, was treated with radical resection of the affected area. however, changed concept of odontomas as hamartomatous malformations has modified treatment plan to conservative modality including selective removal of denticles with more emphasis on enucleation of connective tissue capsules, as its remnants left behind can predispose to cystic change, interfere with eruption of permanent teeth and cause considerable destruction of bone due to recurrence. a thorough visual, manual as well as radiographic examination should be performed for all the patients before and after surgical enucleation of odontomas. if associated permanent tooth is impacted due to inadequate space and possibility of its eruption is limited, then orthodontic treatment for eruption guidance is advised in patients those presenting with clinical evidence of delayed eruption, missing tooth or temporary tooth displacement, with or without history of trauma. in the present case, the occurrence of compound odontoma was detected in the first decade of life that is uncommon finding. unlike the documentation in literature, compound odontoma in this case was detected in mandibular premolar region on left side. thus, the odontoma excised from its site can be classified as intraosseous, geminated, denticulo - particulate, variably shaped, mature compound odontoma approximately 36 in number. for every pediatric patient those presenting clinical evidence of delayed tooth eruption, transient tooth displacement or retained deciduous teeth with or without a history of previous dental trauma careful radiographic examination and early diagnosis should be performed. correct diagnosis of the condition facilitates the clinician to adopt a simpler and less complex approach of treatment and ensures better prognosis of the disease. in spite of low frequency, care should be taken for complete surgical enucleation of odontomas to avoid relapse as well as displacement or devitalization of adjacent teeth. | the purpose of this paper is to describe the case of surgical management of massive compound odontoma with 36 denticles in a 9-year - boy who presented with a complaint of pain and swelling in mandibular left posterior region and retained 74. the denticles were removed after the removal of retained 74 completely and wound healing was observed. odontomas are considered as hamartomatous dental malformation rather than true neoplasm of odontogenic origin. they are the most commonly occurring abnormally formed dental tissues that interfere with eruption of associated teeth. the eruption disturbances seen due to odontomas are delayed eruption or deflection of associated teeth. these malformations are usually asymptomatic and discovered during routine radiographic investigations. correct diagnosis followed by proper treatment plan results in a favorable prognosis. |
the global hcc bridge (bridge to better outcomes in hcc) study was the first multiregional, large - scale, longitudinal cohort study to document the hcc patient experience from diagnosis to death.the objective was to provide an improved understanding of global patterns of hcc therapy and associated outcomes across real - world clinical practice.the study showed the pattern of initial and second recorded treatments in real practice.these results confirm previously reported regional trends in patient demographic characteristics and hcc risk factors, document treatment heterogeneity across regions / countries, and underscore the need for earlier hcc diagnosis worldwide. the global hcc bridge (bridge to better outcomes in hcc) study was the first multiregional, large - scale, longitudinal cohort study to document the hcc patient experience from diagnosis to death. the objective was to provide an improved understanding of global patterns of hcc therapy and associated outcomes across real - world clinical practice. these results confirm previously reported regional trends in patient demographic characteristics and hcc risk factors, document treatment heterogeneity across regions / countries, and underscore the need for earlier hcc diagnosis worldwide. the bridge study was a real - world, observational, longitudinal cohort study, with data collected from 1 january 2005 to 30 september 2012. the primary objective was to assess current treatment approaches and associated clinical outcomes in hcc. secondary objectives were to assess and compare the characteristics of patients with hcc treated with sorafenib or with other therapies in the same time period, and to evaluate the treatment pattern and resource use. the study was done in accordance with ethical principles based on those in the current declaration of helsinki, and was consistent with international conference on harmonization good clinical practice guidelines, good epidemiology practices and applicable regulatory requirements. eligible patients were male or female ; aged 18 years or older ; newly diagnosed with hcc between 1 january 2005 and 30 june 2011 in accordance with aasld, easl or comparable local guidelines (2,9,21,22) ; and who received or were receiving hcc treatment through a selected study site. patients whose primary treatment was via participation in a randomized clinical trial were excluded ; similarly, patients who, at a later point in time consented to take part in a clinical trial, were withdrawn, with the exception of patients entering single - arm trials or adjuvant treatment trials. other exclusion criteria were unknown date of hcc diagnosis or unknown date of first visit for hcc at a given study site. sites were instructed to enrol all eligible patients on a sequential basis, with data to be extracted on a rolling basis from patient charts by personnel at the study site. a selection scheme was employed to cap enrolment at specific sites when the number of eligible patients exceeded the number allowed by power calculation. seasonality was avoided by distributing the number of patients entered in a given cohort year based on month of diagnosis (e.g. if 120 patients were entered as part of a cohort, the first 10 eligible patients diagnosed each month would be entered). study data were entered into a web - based, electronic data - capture system developed by outcome sciences, inc. (cambridge, ma, usa), and subject to rigorous monthly monitoring and cleaning. key data collected included patient demographics ; hcc risk factors ; selected laboratory values required to stage patients ; tumour characteristics ; hcc - directed therapy ; and outcomes. data on resource use in addition to treatment (e.g. physician visits, type and date of assessments) were also collected. criteria for site selection included tertiary referral centre providing surgical and routine follow - up care of hcc ; oncology centres treating patients with hcc ; patient population with hcc aetiologies consistent with the national average (by type and proportion) ; and centres utilizing hcc screening practices in accordance with national standards. centres with a patient population previously used to represent the national population for other research purposes (i.e. development of staging systems, or determination of national incidence or prevalence rates) were also considered. the results reported here were based on the final data set, including all available data as of september 30, 2012. all eligible patients enrolled in the study were included in the analysis population, with information from patients who did not complete follow - up included in the analyses, unless the patient requested otherwise. the primary measure of treatment outcome was os, as measured from date of first hcc treatment to death (to be consistent with clinical trial data). secondary measures of treatment outcome included evidence of disease progression (yes / no), systemic treatment - limiting adverse event and systemic treatment failure, as well as the time to each of these events (also measured from date of starting treatment). patient follow - up was defined as date of hcc diagnosis or first date on record at the site where the patient was seen for hcc, whichever was earlier, until death or end of study, whichever came first. an ongoing effort to limit the amount of missing data was made by alerting sites to missing data identified during monthly monitoring and cleaning. results are presented as descriptive statistics, based on patients for whom data were available ; results for which data are missing for > 30% of patients are noted. os was estimated using kaplan - meier methods, with analyses by bclc stage and by region reported here. data are available to perform analyses on os by treatment type ; however, initial results suggested the need for further study, which was considered to be beyond the scope of this initial report. cox proportional hazards models were used to test for significance and all reported p - values are two - sided. the bridge study was a real - world, observational, longitudinal cohort study, with data collected from 1 january 2005 to 30 september 2012. the primary objective was to assess current treatment approaches and associated clinical outcomes in hcc. secondary objectives were to assess and compare the characteristics of patients with hcc treated with sorafenib or with other therapies in the same time period, and to evaluate the treatment pattern and resource use. the study was done in accordance with ethical principles based on those in the current declaration of helsinki, and was consistent with international conference on harmonization good clinical practice guidelines, good epidemiology practices and applicable regulatory requirements. eligible patients were male or female ; aged 18 years or older ; newly diagnosed with hcc between 1 january 2005 and 30 june 2011 in accordance with aasld, easl or comparable local guidelines (2,9,21,22) ; and who received or were receiving hcc treatment through a selected study site. patients whose primary treatment was via participation in a randomized clinical trial were excluded ; similarly, patients who, at a later point in time consented to take part in a clinical trial, were withdrawn, with the exception of patients entering single - arm trials or adjuvant treatment trials. other exclusion criteria were unknown date of hcc diagnosis or unknown date of first visit for hcc at a given study site. sites were instructed to enrol all eligible patients on a sequential basis, with data to be extracted on a rolling basis from patient charts by personnel at the study site. a selection scheme was employed to cap enrolment at specific sites when the number of eligible patients exceeded the number allowed by power calculation. seasonality was avoided by distributing the number of patients entered in a given cohort year based on month of diagnosis (e.g. if 120 patients were entered as part of a cohort, the first 10 eligible patients diagnosed each month would be entered). study data were entered into a web - based, electronic data - capture system developed by outcome sciences, inc. (cambridge, ma, usa), and subject to rigorous monthly monitoring and cleaning. key data collected included patient demographics ; hcc risk factors ; selected laboratory values required to stage patients ; tumour characteristics ; hcc - directed therapy ; and outcomes. data on resource use in addition to treatment (e.g. physician visits, type and date of assessments) were also collected. criteria for site selection included tertiary referral centre providing surgical and routine follow - up care of hcc ; oncology centres treating patients with hcc ; patient population with hcc aetiologies consistent with the national average (by type and proportion) ; and centres utilizing hcc screening practices in accordance with national standards. centres with a patient population previously used to represent the national population for other research purposes (i.e. development of staging systems, or determination of national incidence or prevalence rates) were also considered. the results reported here were based on the final data set, including all available data as of september 30, 2012. all eligible patients enrolled in the study were included in the analysis population, with information from patients who did not complete follow - up included in the analyses, unless the patient requested otherwise. the primary measure of treatment outcome was os, as measured from date of first hcc treatment to death (to be consistent with clinical trial data). secondary measures of treatment outcome included evidence of disease progression (yes / no), systemic treatment - limiting adverse event and systemic treatment failure, as well as the time to each of these events (also measured from date of starting treatment). patient follow - up was defined as date of hcc diagnosis or first date on record at the site where the patient was seen for hcc, whichever was earlier, until death or end of study, whichever came first. an ongoing effort to limit the amount of missing data was made by alerting sites to missing data identified during monthly monitoring and cleaning. results are presented as descriptive statistics, based on patients for whom data were available ; results for which data are missing for > 30% of patients are noted. os was estimated using kaplan - meier methods, with analyses by bclc stage and by region reported here. data are available to perform analyses on os by treatment type ; however, initial results suggested the need for further study, which was considered to be beyond the scope of this initial report. cox proportional hazards models were used to test for significance and all reported p - values are two - sided. as of september 30, 2012, a total of 42 sites in 14 countries had participated in the study (fig.1). data were available for a total of 18 031 patients treated for hcc [asia : 15 sites, n = 12 031 (67% of patients) ; europe : 23 sites, n = 3673 (20%) and north america : four sites, n = 2326 (13%) ]. because of the large percentage of patients from china, and also because of substantial differences in risk factors and treatment patterns between all four asian countries included, results for the asian countries are presented separately (either by all four countries separately or by china, separately from grouped taiwan, south korea and japan). the study included a total of 8683 patients from china (72% of asian patients and 48% of all patients) ; 1587 patients from taiwan (13% of asian patients) ; 1227 patients from south korea (10% of asian patients) ; and 534 patients from japan (4% of asian patients). the most common risk factor for hcc was hcv in north america, europe and japan, and hbv in china, south korea and taiwan (table1). alcoholic liver disease was a substantially higher risk factor in north america and europe than in any asian country. in north america, europe and south korea, at least 40% of patients reported past or current alcohol abuse, and more than 50% of patients reported past or current tobacco use ; these rates were lower in china, taiwan and japan. patients from north america, europe, taiwan and japan had median afp in the range of 1725 ng / ml, while median afp was 101 ng / ml for south korean patients and 219 ng / ml for chinese patients. patient demographics and clinical characteristics at diagnosis (n = 18 031) afp, alpha - fetoprotein ; ald, alcoholic liver disease ; bclc, barcelona clinic liver cancer ; ecog / who, eastern cooperative oncology group / world health organization ; hbv, hepatitis b virus ; hcc, hepatocellular carcinoma ; hcv, hepatitis c virus ; nash, non - alcoholic steatohepatitis ; sd, standard deviation. percentages were calculated among patients evaluated for hcc risk factors ; patients who were not evaluated had missing data and were not included in the calculations. data missing in > 30% of patients. includes patients with missing number of measurable lesions who had values for largest diameter in liver. a greater ecog / who performance status grade indicates worse health status (5 = death ; 0 = asymptomatic). a greater karnofsky score indicates better health status (100 = normal, no complaints, and no evidence of disease ; 0 = death). among patients with known staging information, the most common bclc stage at diagnosis was stage c in north america, europe, china and south korea, and stage a in taiwan and japan (see table1). in taiwan and japan, approximately 70% of patients were diagnosed with hcc at bclc stage 0 or a, and less than 20% were diagnosed at bclc stage c or d. in all other regions or countries (north america, europe, china and south korea), more than 50% of hcc cases were stage c or d at diagnosis. using the child - pugh scoring system, the most common status at diagnosis was a across all regions and countries, although the proportion of a was much higher in china, taiwan and japan (90%) compared with north america and europe (70%). median tumour diameter at diagnosis ranged from 2.56.7 cm, with the largest median tumour diameter observed in chinese patients. the highest incidences of portal vein invasion or thrombosis and extrahepatic spread occurred in south korea (29 and 10%, respectively), followed by china and north america. across regions / countries, most patients had eastern cooperative oncology group / world health organization (ecog / who) performance status grade (23) of 0 or 1 (87% per region) and karnofsky scores (24) of 80100 (79% per region). first recorded hcc treatment varied substantially between regions (fig.2a). across all disease stages, tace was most frequently used first in north america, europe, china and south korea, while pei or rfa were most frequently used first in japan ; in taiwan, resection was the most common first treatment (see fig.2a). for patients with bclc stage 0c at diagnosis, resection, tace and pei or rfa were the most frequently used first treatments, while palliative care was most frequently used in patients with stage d disease (fig.2b). first recorded hcc treatment by country / region (a) and bclc stage (b). any systemic therapy other than sorafenib, e.g., doxorubicin, gemcitabine, cisplatin, or other cytotoxic or biological agent. any locoregional therapy not clearly pei / rfa or tace, e.g., transarterial radioembolization (tare) or cryoablation. percentages are based on number of patients with data available ; total may add up to > 100% if more than one treatment was started concurrently. pei, percutaneous ethanol injection ; rfa, radiofrequency ablation ; tace, transarterial chemoembolization. the most common second treatment following first treatment with resection, tace or pei / rfa varied by region, but was most often another non - systemic therapy (fig.3). after resection, tace was the most frequently recorded second treatment for hcc in all analysis groups apart from europe, where pei / rfa were used more frequently. tace was also the most common second treatment for hcc after pei / rfa in all regions apart from north america, where liver transplant was more common. second treatments showed greatest variation by region after first - line tace ; transplant was most frequently used in north america, pei / rfa in grouped taiwan, south korea and japan, sorafenib in europe, and palliative care in china. second recorded hcc treatment after first recorded resection, tace, or pei / rfa. combination therapy was not defined in the bridge data ; however, patients treated with either pei or rfa were pooled together. includes grouped patients from taiwan (n = 1587 ; 47%), south korea (n = 1227 ; 37%), and japan (n = 534 ; 16%). pei, percutaneous ethanol injection ; rfa, radiofrequency ablation ; tace, transarterial chemoembolization. median os was not reached for bclc stage 0, and was 80, 27, 15 and 4 months for bclc stages a, b, c and d respectively (p 30% of patients. includes patients with missing number of measurable lesions who had values for largest diameter in liver. a greater ecog / who performance status grade indicates worse health status (5 = death ; 0 = asymptomatic). a greater karnofsky score indicates better health status (100 = normal, no complaints, and no evidence of disease ; 0 = death). among patients with known staging information, the most common bclc stage at diagnosis was stage c in north america, europe, china and south korea, and stage a in taiwan and japan (see table1). in taiwan and japan, approximately 70% of patients were diagnosed with hcc at bclc stage 0 or a, and less than 20% were diagnosed at bclc stage c or d. in all other regions or countries (north america, europe, china and south korea), more than 50% of hcc cases were stage c or d at diagnosis. using the child - pugh scoring system, the most common status at diagnosis was a across all regions and countries, although the proportion of a was much higher in china, taiwan and japan (90%) compared with north america and europe (70%). median tumour diameter at diagnosis ranged from 2.56.7 cm, with the largest median tumour diameter observed in chinese patients. the highest incidences of portal vein invasion or thrombosis and extrahepatic spread occurred in south korea (29 and 10%, respectively), followed by china and north america. across regions / countries, most patients had eastern cooperative oncology group / world health organization (ecog / who) performance status grade (23) of 0 or 1 (87% per region) and karnofsky scores (24) of 80100 (79% per region). first recorded hcc treatment varied substantially between regions (fig.2a). across all disease stages, tace was most frequently used first in north america, europe, china and south korea, while pei or rfa were most frequently used first in japan ; in taiwan, resection was the most common first treatment (see fig.2a). for patients with bclc stage 0c at diagnosis, resection, tace and pei or rfa were the most frequently used first treatments, while palliative care was most frequently used in patients with stage d disease (fig.2b). first recorded hcc treatment by country / region (a) and bclc stage (b). any systemic therapy other than sorafenib, e.g., doxorubicin, gemcitabine, cisplatin, or other cytotoxic or biological agent. any locoregional therapy not clearly pei / rfa or tace, e.g., transarterial radioembolization (tare) or cryoablation. percentages are based on number of patients with data available ; total may add up to > 100% if more than one treatment was started concurrently. pei, percutaneous ethanol injection ; rfa, radiofrequency ablation ; tace, transarterial chemoembolization. the most common second treatment following first treatment with resection, tace or pei / rfa varied by region, but was most often another non - systemic therapy (fig.3). after resection, tace was the most frequently recorded second treatment for hcc in all analysis groups apart from europe, where pei / rfa were used more frequently. tace was also the most common second treatment for hcc after pei / rfa in all regions apart from north america, where liver transplant was more common. second treatments showed greatest variation by region after first - line tace ; transplant was most frequently used in north america, pei / rfa in grouped taiwan, south korea and japan, sorafenib in europe, and palliative care in china. second recorded hcc treatment after first recorded resection, tace, or pei / rfa. combination therapy was not defined in the bridge data ; however, patients treated with either pei or rfa were pooled together. includes grouped patients from taiwan (n = 1587 ; 47%), south korea (n = 1227 ; 37%), and japan (n = 534 ; 16%). pei, percutaneous ethanol injection ; rfa, radiofrequency ablation ; tace, transarterial chemoembolization. median os was not reached for bclc stage 0, and was 80, 27, 15 and 4 months for bclc stages a, b, c and d respectively (p 30%. in addition, it is possible that the results are not generalizable because the study was performed at tertiary referral centres, which might be expected to provide the best care available in each country. in an attempt to maximize generalization of results across a particular country, study sites were selected where patient populations had hcc etiologist consistent with previously reported national patterns. in the case of single sites within one country (taiwan, south korea and japan), sites were additionally chosen to be representative of the practice of other centres in the country. however, it is possible that more treatable patients with better liver function and good performance status may have been enrolled, thereby introducing a selection bias. in particular, the lack of enrolled bclc stage d patients in china (2%), as well as the low proportion with child - pugh stage b and c (12 and 1%, respectively), suggest that such patients were not seen at the participating sites and may therefore not have been included in the study. similarly, the relatively low rates of patients reporting alcohol abuse in china, taiwan and japan suggest that such patients also may not have been treated at the participating sites for some reason (for example, such patients might be expected to be less likely to seek and receive care at a tertiary centre, and less likely to be supported in doing so). these low rates could also, however, reflect under - reporting because of the possible stigma associated with admitting such abuse in some countries. who estimates of per capita alcohol consumption (litres of pure alcohol) for 2011 were 9.4 for the united states, 12.2 for europe, 5.9 for china, 14.8 for south korea and 8.0 for japan (40). given these figures and the known strong interaction between alcohol abuse and other risk factors for hcc, the reported rates of alcohol abuse for china (24%), taiwan (18%) and especially japan (2%) seem unexpectedly low. missing advanced - stage patients may help to explain the possibly higher - than - expected median os reported here for china. they could also contribute to the superior os seen here for taiwan and japan, but this more likely can be attributed to the aforementioned lead - time bias, as well as, particularly in the case of taiwan, artificial inflation because of the effects of censoring, which makes the os reported here increasingly less reliable with time. finally, interactions between variables in such a large sample size as the population in this study are hard to control, and our results must accordingly be interpreted with caution. in conclusion, these real - world findings from the bridge study provide a broad overview of the current state of hcc treatment and document the heterogeneity of treatment approaches across regions and in different countries. results from the study confirm previously reported regional trends in patient demographic characteristics and hcc risk factors, underscore the need for earlier diagnosis of hcc worldwide, and also suggest that treatment guidelines may benefit from re - evaluation. the data from taiwan and japan, in particular, suggest it may be possible to improve outcomes by focusing on identifying high - risk individuals and then following them with surveillance to achieve early detection. it is hoped that information obtained from the bridge study will help identify unmet clinical needs and contribute to the development of new treatment paradigms that ultimately improve outcomes in patients with hcc. the study has generated a very large dataset which could potentially be used to address unanswered research hypotheses and is available for further analysis by interested investigators. additional analyses of potential value could include the aforementioned survival by treatment type, including systemic vs. non - systemic therapy, assessment of regional practice vs. regional guidelines, as well as exploratory identification of possible predictors of survival, such as changes in tumour size or afp levels over time. professional medical writing and editorial assistance was provided by stemscientific, funded by bristol - myers squibb. m. colombo : grant and research support merck, roche, bristol - myers squibb, gilead sciences ; science advisory committees merck, roche, novartis, bayer, bristol - myers squibb, gilead sciences, tibotec, vertex, janssen cilag, achillion, lundbeck, abbott, boehringer ingelheim, glaxosmithkline, genspera, abbvie ; speaking and teaching tibotec, roche, novartis, bayer, bristol - myers squibb, gilead sciences, and vertex. l. r. roberts : grant and research support bristol - myers squibb, gilead sciences, inova diagnostics, and wako diagnostics. m. schwartz : has been a consultant / advisor for bayer and onyx and has received research funding from bristol - myers squibb. j. chen : steering committee meeting glaxosmithkline ; advisory board bayer, bristol - myers squibb, glaxosmithkline, and roche ; research grants to his institution bristol - myers squibb ; speakers bureau bristol - myers squibb ; travel / accommodations / meeting expenses to international symposiums bayer, bristol - myers squibb, and gilead sciences. m. sherman : has been a consultant / advisor and given expert testimony for bristol - myers squibb. additional supporting information may be found in the online version of this article : table s1. | background & aimshepatocellular carcinoma (hcc) is the second most common cause of cancer deaths worldwide. the global hcc bridge study was a multiregional, large - scale, longitudinal cohort study undertaken to improve understanding of real - life management of patients with hcc, from diagnosis to death.methodsdata were collected retrospectively from january 2005 to september 2012 by chart reviews of eligible patients newly diagnosed with hcc at participating institutions.resultsforty-two sites in 14 countries contributed final data for 18 031 patients. asia accounted for 67% of patients, europe for 20% and north america for 13%. as expected, the most common risk factor was hepatitis c virus in north america, europe and japan, and hepatitis b virus in china, south korea and taiwan. the most common barcelona clinic liver cancer stage at diagnosis was c in north america, europe, china and south korea, and a in taiwan and japan. across all stages, first hcc treatment was most frequently transarterial chemoembolization in north america, europe, china and south korea, percutaneous ethanol injection or radiofrequency ablation in japan and resection in taiwan. survival from first hcc treatment varied significantly by region, with median overall survival not reached for taiwan and 60, 33, 31, 24 and 23 months for japan, north america, south korea, europe and china respectively (p < 0.0001).conclusionsinitial results from the bridge study confirm previously reported regional trends in patient demographic characteristics and hcc risk factors, document the heterogeneity of treatment approaches across regions / countries and underscore the need for earlier hcc diagnosis worldwide. |
several multifunctional proteins are involved in both transcriptional and translational control (wilkinson and shyu, 2001). here we focus on a family of such multifunctional proteins, the y - box protein family, in terms of its significance in cell proliferation and cancer. as different aspects of the y - box proteins have already been reviewed (matsumoto and wolffe, 1998 ; swamynathan, 1998 ; evdokimova and ovchinnikov, 1999 ; kohno, 2003), we briefly appraise the structure and functions of the y - box proteins with the emphasis on recent findings. we then summarize the role of a y - box protein yb-1 in cancer and its use in the clinical setting. y - box proteins (or y - box binding proteins) are so named because they were originally identified as dna binding proteins that are capable of associating with the y - box (inverted ccaat - box) sequence of the major histocompatibility complex class ii gene. y - box proteins have thus far been known to regulate positively or negatively a number of genes, such as multidrug resistance 1, cyclin a, cyclin b1, matrix metalloproteinase 2 and collagen alpha2(i) (higashi, 2003a ; jurchott, 2003 ; kohno, 2003). however, members of y - box protein family are also found in the cytoplasm and associated with mrnas as major components of messenger ribonucleoprotein particles. y - box proteins regulate translation in a dose - dependent manner ; low concentrations of y - box proteins activate translation and high concentrations repress it (evdokimova and ovchinnikov, 1999). collectively, y - box proteins have been implicated in the regulation of mrna metabolism in multiple steps in both the nucleus and the cytoplasm, including transcription, splicing, mrna stability and translation. y - box proteins consist of three domains : the n - terminal domain, the cold shock domain (csd) and the c - terminal tail domain (figure 1). the csd is a highly conserved nucleic acid binding domain that confers rna- and single - stranded and double - stranded dna binding activities to the y - box proteins. both the short n - terminal and the c - terminal tail domains are less conserved among the y - box proteins. the charged c - terminal tail domain of vertebrate y - box proteins, consisting of alternating clusters of acidic / aromatic and basic amino acids, is likely to account for its rna - binding activity and ability for associating with various proteins. y - box proteins have been shown to interact with a number of cellular and viral proteins that are involved in various cellular processes (figure 2). + + and indicate clusters of basic and acidic / aromatic amino acids. numbers in parentheses indicate the references as follows : (1) shnyreva, 2000, (2) kohno, 2003 ; swamynathan, 1998 and references therein, (3) higashi, 2003b, (4) safak, 1999, (5) zou, 1997, (6) kojic, 2004, (7) chansky, 2001, (8) moraes, 2003, (9) funke, 1996, (10) raffetseder, 2003, (11) wilhelm, 2000, (12) matsumoto, 2005, (13) balda, 2003, (14) moorthamer, 1999, (15) matsumoto and wolffe, 1998 ; evdokimova and ovchinnikov, 1999 and references therein, (16) balda and matter, 2000, (17) frankel, 2005. in a variety of cell types, y - box proteins are predominantly found in the cytoplasm. however, given that y - box proteins regulate transcription, they are expected to localize to the nucleus. y - box proteins are translocated into the nucleus by a number of conditions and mechanisms, including uv irradiation, hyperthermia, interferon - gamma treatment, adenovirus infection, interaction with p53 and a splicing factor srp30c and high levels of ectopic yb-1expression (higashi, 2003a ; kohno 2003 ; raffetseder, 2003 and references therein ; zhang, 2003). both the csd and the tail domains are implicated in nuclear localization of yb-1 ; the tail domain seems to contain a non - canonical nuclear localization signal and the isolated csd also contributes to nuclear retention (bader and vogt, 2005). y - box proteins are capable of nucleocytoplasmic shuttling, which allows them to contribute to the coupling control of transcription and translation. y - box proteins become associated with nascent transcripts cotranscriptionally and are presumed to accompany mrna into the cytoplasm (soop, 2003). interestingly, a mouse y - box protein msy2 preferentially associates with mrnas that are transcribed from genes containing y - box sequences in their promoter regions and stores those mrnas in male germ cells (yang, 2005a). experiments with overexpression or down - regulation of the y - box proteins in cultured cells or animals have shown that the amount of y - box proteins must be precisely controlled (see below). therefore, it is important to understand how the synthesis of y - box proteins is regulated. recent data have shown that the synthesis of a y - box protein, yb-1 (y - box binding protein-1), is regulated both at transcriptional and post - transcriptional levels. yb-1 mrna accumulates when cells are treated with cisplatin or uv irradiation (ohga, 1996). transcription of the yb-1 gene is stimulated by p73 through an enhanced recruitment of the c - myc - max complexes to e - box sequences in the yb-1 promoter (uramoto, 2002). yb-1 protein represses translation of yb-1 mrna by binding to specific elements in the 5- and 3-untranslated regions (fukuda, 2004 ; skabkina, 2005). this self - regulation may contribute towards maintaining the concentration of yb-1 protein optimal in a cell. in human and mouse, there are three y - box proteins, two of which are expressed in both somatic cells and germ cells (table 1). the most extensively studied y - box protein, yb-1 human contrin and mouse msy2 are germ cell - specific members of the y - box protein family. analyses of the effects of targeting y - box genes in chicken cells and mice have been widely carried out in the last three years. chicken b - cell lymphoma dt40 cells are widely used to study functional consequences of disrupting specific genes because of the high frequency of homologous recombination. the yb-1 (or yb-1b) gene in dt40 cells has been disrupted by two independent groups of investigators ; one group reported that yb-1 cells show slow - growth phenotype and increased dna content (swamynathan, 2002). the other group of researchers found that heterozygous disruption resulted in no growth defects but homozygous gene disruptants exhibited a slow and cold - sensitive growth phenotype (matsumoto, 2005). one research group tried to disrupt yb-1 gene in mice but encountered difficulties in disrupting both alleles of the yb-1 gene (shibahara, 2004). however, as was the case in chicken cells, another group recently reported homozygous yb-1gene disruption, showing the importance of yb-1 in late stages of embryonic development (lu, 2005). they observed developmental defects of yb-1embryos after embryonic day 13.5 including craniofacial lesions, hemorrhage and respiratory failure, with yb-1 mef cells showing premature senescence and hypersensitivity to different cellular stresses. the reason for the presence or absence of the haplo - insufficient phenotypes is currently unknown. in mice lacking msy2, both male and female homozygotes are sterile, a consequence of disturbed spermatogenesis due to reduction of postmeiotic germ - cell mrnas in male and oocyte loss in female (yang, 2005b). overall, studies designed to reduce or deplete a y - box protein in cells or whole organisms underscore the significance of y - box proteins in appropriate cell growth, stress responses and development. y - box proteins in human and mouse the role of yb-1 in cancer progression has attracted attention in recent years.yb-1 has been found to be upregulated during prostate cancer tumor progression (gimenez - bonafe, 2004). increased yb-1 expression has been correlated with dna topoisomerase ii and proliferating cell nuclear antigen expression in human lung cancer (gu, 2001) and colorectal cancer (shibao, 1999) and linked to markers of cellular proliferation in osteosarcoma (oda, 1998). yb-1 has been identified as a cell cycle stage - specific transcription factor (jurchott, 2003). nuclear accumulation of yb-1 in hela cells was demonstrated to transcriptionally activate cyclin a and b1 genes, which are crucial for cell cycle progression. increase in cyclin a has been reported to be associated with poor clinical outcome in breast cancer (michalides, 2002). in addition, yb-1 is believed to promote metastasis by enhancing the transcription of gelatinase a, a matrix metalloproteinase that facilitates cell migration (cheng, 2002). recently, berquin. (2005) has also shown that yb-1 may induce epidermal growth factor (egf) independence in mammary epithelial cells via activation of the egf receptor pathway, thereby contributing to breast tumor aggressiveness. in yet another recent paper, bergmann and colleagues (2005), using a transgenic mouse model, showed that overexpression of yb-1 may cause breast cancer through the induction of genetic instability. on the other hand, yb-1 may have anti - oncogenic activity as it is reported to be capable of blocking oncogenic cell transformation (bader and vogt, 2005). the phosphoinositide 3-kinase (pi 3-kinase) pathway is known to show gain of function in human cancers (bader and vogt 2004). the catalytic subunits of pi 3-kinase, p110 (of which p3k is a homolog) and akt are oncoproteins and yb-1 is specifically known to inhibit p3k and akt - induced transformation involving protein synthesis (bader., 2003). yb-1 may interefere with the synthesis of growth - related proteins including growth factors, receptors, kinases, transcriptional regulators and cell cycle proteins associated with p3k and akt pathways (zimmer, 2000 ; bader and vogt 2004). a seminal paper describing yb-1 expression in cancer tissues was first reported by royer 's group in breast cancer (bargou, 1997). the pathological significance of yb-1 in a variety of cancers is shown in table 2. overexpression of yb-1 and pathological significance in human cancers substantial yb-1 expression was demonstrated in multidrug - resistant breast, gastric and pancreatic cell lines (holm, 2004). altered drug sensitivity to cisplatin, a very potent and widely used anti - cancer agent and mitomycin c has been observed following treatment of cells with antisense yb-1 (ohga, 1996 ; torigoe, 2005). expression of yb- 1 protein has been reported to reflect the chemosensitivity of ovarian serous adenocarcinoma (kamura, 1999) and breast cancer (janz, 2002 ; huang, 2005). increased nuclear localization of yb-1 has been observed in acquired cisplatin - resistant ovarian cancer (yahata, 2002). yb-1 expression has also been shown to be associated with p - glycoprotein (pgp) expression in breast cancer cells resulting in drug resistance (bargou, 1997 ; saji, 2003 ; huang, 2005). pgp, encoded by the mdr1 gene, is a member of the atp - binding cassette transporter superfamily of proteins involved in the protection of cells from xenobiotics and drugs (kuwano, 2003). pgp has become an important molecular target for limiting chemoresistance as it plays a major role in the development of multidrug - resistant tumor type and is known to mediate resistance to a wide range of anticancer agents (kuwano, 1999). bay and co - workers have recently demonstrated a direct interaction between yb-1 and pgp using the computer - based resonance recognition model (huang, 2005). the same investigators observed the occurrence of raised recurrence rates in breast tumor patients with high yb-1 expression who underwent a chemotherapy regime which contained anthracycline (a pgp substrate). besides breast cancer, yb-1 has been correlated with pgp in ovarian cancer (huang, 2004), prostate cancer (gimenez - bonafe, 2004) and osteosarcoma (oda, 1998). yb-1 has been shown to bind p53 (okamoto, 2000) and interaction with p53 could be necessary for the self - defense of cells exposed to dna - damaging agents (kuwano, 2003). as mentioned earlier, p73, a close relative of the p53 family, has also been observed to stimulate transcription of the yb-1 promoter by enhancing the recruitment of the cmyc - max complex to its target gene (uramoto, 2002). c - myc, an oncogene with a dual function in cell proliferation and apoptosis can confer resistance to cisplatin. p73 is known to induce apoptosis (irwin, 2000). and p73 overexpressing clones have been observed to be cisplatin resistant (gong, 1999). hence, c - myc and p73 may form a complex necessary in yb-1 mediated drug resistance (uramoto, 2002). y - box proteins consist of three domains : the n - terminal domain, the cold shock domain (csd) and the c - terminal tail domain (figure 1). the csd is a highly conserved nucleic acid binding domain that confers rna- and single - stranded and double - stranded dna binding activities to the y - box proteins. both the short n - terminal and the c - terminal tail domains are less conserved among the y - box proteins. the charged c - terminal tail domain of vertebrate y - box proteins, consisting of alternating clusters of acidic / aromatic and basic amino acids, is likely to account for its rna - binding activity and ability for associating with various proteins. y - box proteins have been shown to interact with a number of cellular and viral proteins that are involved in various cellular processes (figure 2). + + and indicate clusters of basic and acidic / aromatic amino acids. numbers in parentheses indicate the references as follows : (1) shnyreva, 2000, (2) kohno, 2003 ; swamynathan, 1998 and references therein, (3) higashi, 2003b, (4) safak, 1999, (5) zou, 1997, (6) kojic, 2004, (7) chansky, 2001, (8) moraes, 2003, (9) funke, 1996, (10) raffetseder, 2003, (11) wilhelm, 2000, (12) matsumoto, 2005, (13) balda, 2003, (14) moorthamer, 1999, (15) matsumoto and wolffe, 1998 ; evdokimova and ovchinnikov, 1999 and references therein, (16) balda and matter, 2000, (17) frankel, 2005. in a variety of cell types, y - box proteins are predominantly found in the cytoplasm. however, given that y - box proteins regulate transcription, they are expected to localize to the nucleus. y - box proteins are translocated into the nucleus by a number of conditions and mechanisms, including uv irradiation, hyperthermia, interferon - gamma treatment, adenovirus infection, interaction with p53 and a splicing factor srp30c and high levels of ectopic yb-1expression (higashi, 2003a ; kohno 2003 ; raffetseder, 2003 and references therein ; zhang, 2003). both the csd and the tail domains are implicated in nuclear localization of yb-1 ; the tail domain seems to contain a non - canonical nuclear localization signal and the isolated csd also contributes to nuclear retention (bader and vogt, 2005). y - box proteins are capable of nucleocytoplasmic shuttling, which allows them to contribute to the coupling control of transcription and translation. y - box proteins become associated with nascent transcripts cotranscriptionally and are presumed to accompany mrna into the cytoplasm (soop, 2003). interestingly, a mouse y - box protein msy2 preferentially associates with mrnas that are transcribed from genes containing y - box sequences in their promoter regions and stores those mrnas in male germ cells (yang, 2005a). experiments with overexpression or down - regulation of the y - box proteins in cultured cells or animals have shown that the amount of y - box proteins must be precisely controlled (see below). therefore, it is important to understand how the synthesis of y - box proteins is regulated. recent data have shown that the synthesis of a y - box protein, yb-1 (y - box binding protein-1), is regulated both at transcriptional and post - transcriptional levels. yb-1 mrna accumulates when cells are treated with cisplatin or uv irradiation (ohga, 1996). transcription of the yb-1 gene is stimulated by p73 through an enhanced recruitment of the c - myc - max complexes to e - box sequences in the yb-1 promoter (uramoto, 2002). yb-1 protein represses translation of yb-1 mrna by binding to specific elements in the 5- and 3-untranslated regions (fukuda, 2004 ; skabkina, 2005). this self - regulation may contribute towards maintaining the concentration of yb-1 protein optimal in a cell. in human and mouse, there are three y - box proteins, two of which are expressed in both somatic cells and germ cells (table 1). the most extensively studied y - box protein, yb-1, is ubiquitously expressed in various tissues. human contrin and mouse msy2 are germ cell - specific members of the y - box protein family. analyses of the effects of targeting y - box genes in chicken cells and mice have been widely carried out in the last three years. chicken b - cell lymphoma dt40 cells are widely used to study functional consequences of disrupting specific genes because of the high frequency of homologous recombination. the yb-1 (or yb-1b) gene in dt40 cells has been disrupted by two independent groups of investigators ; one group reported that yb-1 cells show slow - growth phenotype and increased dna content (swamynathan, 2002). the other group of researchers found that heterozygous disruption resulted in no growth defects but homozygous gene disruptants exhibited a slow and cold - sensitive growth phenotype (matsumoto, 2005). one research group tried to disrupt yb-1 gene in mice but encountered difficulties in disrupting both alleles of the yb-1 gene (shibahara, 2004). however, as was the case in chicken cells, another group recently reported homozygous yb-1gene disruption, showing the importance of yb-1 in late stages of embryonic development (lu, 2005). they observed developmental defects of yb-1embryos after embryonic day 13.5 including craniofacial lesions, hemorrhage and respiratory failure, with yb-1 mef cells showing premature senescence and hypersensitivity to different cellular stresses. the reason for the presence or absence of the haplo - insufficient phenotypes is currently unknown. in mice lacking msy2, both male and female homozygotes are sterile, a consequence of disturbed spermatogenesis due to reduction of postmeiotic germ - cell mrnas in male and oocyte loss in female (yang, 2005b). overall, studies designed to reduce or deplete a y - box protein in cells or whole organisms underscore the significance of y - box proteins in appropriate cell growth, stress responses and development. the role of yb-1 in cancer progression has attracted attention in recent years.yb-1 has been found to be upregulated during prostate cancer tumor progression (gimenez - bonafe, 2004). increased yb-1 expression has been correlated with dna topoisomerase ii and proliferating cell nuclear antigen expression in human lung cancer (gu, 2001) and colorectal cancer (shibao, 1999) and linked to markers of cellular proliferation in osteosarcoma (oda, 1998). yb-1 has been identified as a cell cycle stage - specific transcription factor (jurchott, 2003). nuclear accumulation of yb-1 in hela cells was demonstrated to transcriptionally activate cyclin a and b1 genes, which are crucial for cell cycle progression. increase in cyclin a has been reported to be associated with poor clinical outcome in breast cancer (michalides, 2002). in addition, yb-1 is believed to promote metastasis by enhancing the transcription of gelatinase a, a matrix metalloproteinase that facilitates cell migration (cheng, 2002). (2005) has also shown that yb-1 may induce epidermal growth factor (egf) independence in mammary epithelial cells via activation of the egf receptor pathway, thereby contributing to breast tumor aggressiveness. in yet another recent paper, bergmann and colleagues (2005), using a transgenic mouse model, showed that overexpression of yb-1 may cause breast cancer through the induction of genetic instability. on the other hand, yb-1 may have anti - oncogenic activity as it is reported to be capable of blocking oncogenic cell transformation (bader and vogt, 2005). the phosphoinositide 3-kinase (pi 3-kinase) pathway is known to show gain of function in human cancers (bader and vogt 2004). the catalytic subunits of pi 3-kinase, p110 (of which p3k is a homolog) and akt are oncoproteins and yb-1 is specifically known to inhibit p3k and akt - induced transformation involving protein synthesis (bader. yb-1 may interefere with the synthesis of growth - related proteins including growth factors, receptors, kinases, transcriptional regulators and cell cycle proteins associated with p3k and akt pathways (zimmer, 2000 ; bader and vogt 2004). a seminal paper describing yb-1 expression in cancer tissues was first reported by royer 's group in breast cancer (bargou, 1997). the pathological significance of yb-1 in a variety of cancers is shown in table 2. substantial yb-1 expression was demonstrated in multidrug - resistant breast, gastric and pancreatic cell lines (holm, 2004). altered drug sensitivity to cisplatin, a very potent and widely used anti - cancer agent and mitomycin c has been observed following treatment of cells with antisense yb-1 (ohga, 1996 ; torigoe, 2005). expression of yb- 1 protein has been reported to reflect the chemosensitivity of ovarian serous adenocarcinoma (kamura, 1999) and breast cancer (janz, 2002 ; huang, 2005). increased nuclear localization of yb-1 has been observed in acquired cisplatin - resistant ovarian cancer (yahata, 2002). yb-1 expression has also been shown to be associated with p - glycoprotein (pgp) expression in breast cancer cells resulting in drug resistance (bargou, 1997 ; saji, 2003 ; huang, 2005). pgp, encoded by the mdr1 gene, is a member of the atp - binding cassette transporter superfamily of proteins involved in the protection of cells from xenobiotics and drugs (kuwano, 2003). pgp has become an important molecular target for limiting chemoresistance as it plays a major role in the development of multidrug - resistant tumor type and is known to mediate resistance to a wide range of anticancer agents (kuwano, 1999). bay and co - workers have recently demonstrated a direct interaction between yb-1 and pgp using the computer - based resonance recognition model (huang, 2005). the same investigators observed the occurrence of raised recurrence rates in breast tumor patients with high yb-1 expression who underwent a chemotherapy regime which contained anthracycline (a pgp substrate). besides breast cancer, yb-1 has been correlated with pgp in ovarian cancer (huang, 2004), prostate cancer (gimenez - bonafe, 2004) and osteosarcoma (oda, 1998). yb-1 has been shown to bind p53 (okamoto, 2000) and interaction with p53 could be necessary for the self - defense of cells exposed to dna - damaging agents (kuwano, 2003). as mentioned earlier, p73, a close relative of the p53 family, has also been observed to stimulate transcription of the yb-1 promoter by enhancing the recruitment of the cmyc - max complex to its target gene (uramoto, 2002). c - myc, an oncogene with a dual function in cell proliferation and apoptosis can confer resistance to cisplatin. p73 is known to induce apoptosis (irwin, 2000). and p73 overexpressing clones have been observed to be cisplatin resistant (gong, 1999). hence, c - myc and p73 may form a complex necessary in yb-1 mediated drug resistance (uramoto, 2002). expression of the yb-1 protein has a prognostic significance in determining disease progression in human cancers. perhaps more importantly, yb-1 has the potential to be a biological marker which predicts chemotherapy resistance and aid in the selection of appropriate adjuvant chemotherapy. there has been cumulative evidence in the literature to suggest that yb-1 is involved in pleiotropic resistance to different classes of dna - targeting drugs (levenson, 2000). as clinical drug resistance hampers effective chemotherapy, a recent focus in cancer therapeutic strategy is to develop molecular cancer therapeutics (kuwano, 2003 ; holm, 2004). in this regard, yb-1 holds promise as target molecule for the development of novel approaches in overcoming multidrug resistance in cancer chemotherapy (janz, 2002). | y - box proteins belong to the cold shock domain family of proteins that are known to be involved in both transcriptional and translational control. here, we give a brief overview of the structure, regulation and physiological functions of the y - box proteins. this is followed by examining the role of y - box protein 1 (yb-1), the most extensively studied of the y - box protein in tumorigenesis, and its clinicopathological significance. yb-1 has the potential to be a prognostic marker and predictor of chemoresistance in human cancers. |
it is not a specific disease entity, but a gingival response associated with a variety of conditions. these desquamative lesions represent oral manifestations of various dermatoses which include lichen planus, pemphigus vulgaris, erythema multiform, lupus erythematosus, dermatitis herpetiformis, and chronic ulcerative stomatitis. hence, it is of paramount importance to ascertain the identity of the disease responsible of desquamative gingivitis to establish the appropriate therapeutic approach and management. this paper reports a case of rare desquamative lesion involving the gingiva in a 66-year - old female patient. a 66-year - old female patient reported to department of periodontics, m. s. ramaiah dental college and hospital, bangalore, with the complaint of burning sensation in the gums for the past one month which had started spontaneously, not associated with pain and aggravated on taking spicy foods and relieved gradually. she also had bleeding gums for the past one month, while brushing and eating. her personal history reveals that she had no habits and she used medium brush with non - medicated paste. on extra oral examination, she had some obvious skin lesions on the scalp which appeared as depigmented scarring alopecia of the scalp [figure 1 ]. depigmentation with scarring alopecia of the scalp hyperkeratotic plaque present on the posterior auricle of the left ear intra oral examination of gingiva revealed reddish marginal, attached gingiva and interdental papilla in both maxillary and mandibular anterior region [figure 3 ]. desquamation of epithelium was present in the attached gingiva of maxillary right first molar region, mandibular right canine, between maxillary right premolars on the palatal aspect. desquamation of the gingiva -maxillary and mandibular anterior region desquamation present palatal desquamation present lingual nikolsky 's sign - positive the h and e section showed parakeratinized stratified squamous epithelium overlying the connective tissue. the connective tissue showed dense inflammatory infiltrate beneath the basement membrane chiefly composed of lymphocytes [figure 7 ]. hematological examination included total white blood cell (wbc) count, differential white blood count, erythrocyte sedimentation rate, hemocrit, bleeding time, and clotting time were within normal limits. based on the clinical and histopathologic features, lesion was suggestive of discoid lupus erythematosus. hand scaling was performed as gently as possible in order to minimize tissue disruption. hand scaling was preferred, soft tooth brush and mouth rise (chlorhexidine 0.12%) was recommended. triamcinolone acteonide oral paste was advised to apply two to three times per day and was recalled for follow - up and maintenance. lupus erythematosus is an auto immune connective tissue disease which consists of discoid lupus erythematosus (dle) and systemic lupus erythematosus (sle). one to five percent of patients with discoid lupus may develop systemic lupus erythematosus and 25% of patients with sle may develop typical chronic discoid lesions at some time during their illness. early classic dle lesions typically evolve into sharply demarcated, coin shaped (i.e., discoid) erythematous plaques covered by a prominent, adherent scale that extends into the orifices of dilated hair follicles. the lesions typically expand with erythema and hyperpigmentation at the periphery leaving hallmark atrophic central scarring, telangiectasia, and hypopigmentation. when the adherent scale is lifted from more advanced lesions, keratotic spikes similar in appearance to carpet tacks can be seen to project from the under surface of the scale (i.e., the carpet tack sign). affected sites include face, scalp, ears, v area of the neck, and extensor aspects of the arms. a symmetric, hyperkeratotic, butterfly - shaped plaque is occasionally found over the malar areas of the face and bridge of the nose. the margins of the lesions are not sharply demarcated, but frequently show the formation of narrow zone of keratinization, hyperemia with edema, superficial, painful ulceration may occur with crusting, but no actual scale formation as is seen on the skin. it is important to remember that patients with lupus erythematosus may be taking, systemic steroids or / and other anti - inflammatory medications such as aspirin or non steroidal anti inflammatory drugs (nsaid 's) consequently these patients may be at risk of developing adrenal insufficiency and bleeding disorders. in these situations, oral lesions may be the first clinical manifestation of dle, so proper diagnosis is required for early detection and to establish better treatment planning, this promotes resolution of established lesions and prevents scarring of skin lesions and also, alleviates discomfort of the patients. | desquamative gingival lesions are non - plaque induced inflammatory gingival lesions. it is a clinical description and not a diagnosis. these desquamative lesions represent oral manifestations of various dermatoses. discoid lupus erythematosus is one of the rare dermatoses which show desquamative lesions as oral manifestations. this article presents a rare case report of discoid lupus erythematosus with oral lesions involving gingiva of a 66-year - old female patient. |
rehabilitation for the cerebral palsied children should be given thought a team work including parents as a model refers to patient centered treatment regimens1,2,3. this idea encourages considering parent s opinions about physiotherapy and rehabilitation for their children with cp. for this reason, health providers, especially physiotherapists (pts), should talk with the parents of a disabled child before planning a specific treatment or intervention in order to establish the most suitable program for the child4,5,6,7. the parents of disabled children seek to learn all details, both positive and negative aspects, about a physiotherapy and rehabilitation program that will be applied to their children. this is necessary for the parents to understand the mission and goal of the program. moreover, the pts and mother (m) or father of a disabled child should evaluate the child together so that they can define the needs of the child. for this reason, the parents of children with cp should also be included in making decisions in the rehabilitation process4, 5, 8, 9 the purposes of this research were (1) to understand perceptions of ms and pts regarding the rehabilitation programs their children receive and (2) to report the current knowledge of ms and pts, highlighting consensus and disagreement. one hundred and thirty children (75 boys, 55 girls) who were being treated in special education centers in different parts of turkey and their ms and 130 pts who were treating them were included in the study. informed consent was obtained from ms and pts, written approval was obtained for the study from the managers of the schools, and the study was completed in accordance with the principles of the helsinki declaration. the inclusion criteria were as follows : all participants agreed to participate, the children had been diagnosed with cp by a pediatric neurologist, and the caregivers of the children had to be ms. demographic data of children, ms, and psts were recorded. the gross motor function classification system (gmfcs) was used to determine the functional level appropriate for the age of the child and score it between 1 and 5. while a score of 1 indicates that the child may easily achieve indoor ambulation without the need for adjunctive mobilization devices, a score of 5 indicates that the child is totally dependent for mobilization. the reliability and validity of the classification system have been determined for children aged between 2 months and 12 years, and studies have also been done for adults with cp10, 11. the questionnaire form was composed of 6 open - ended questions asking about the expectations and opinions of the ms and pts with regard to the physiotherapy and rehabilitation programs being used. the questionnaire form was created by experienced pts who were working in the pediatric rehabilitation units of university hospitals and special education rehabilitation centers. in the present study, power analysis revealed that 90% power would be obtained with a reliability of 95% if 130 people were included in the study. characteristics of the children with cp, ms, and pts are presented as mean standard deviation, numbers, and percentages. the kappa coefficient (k) was used to analyze agreement with regard to the views of the pts and ms. a kappa coefficient for two values of between 0.0 and 0.20 was considered to indicate statistically insignificant concordance, and one between 0.21 and 0.40 was considered to indicate statistically moderate concordance. the mean age of the children who participated in the study (75 boys, 55 girls) was 89.8052.05 months, and the mean duration of treatment was 73.6242.11 months. the mean age of the pts was 28.077.28 years, and their mean number of working years was 6.847.51. of the ms, 83.1% had 12 years or less of education, 14.6% had 12 or more years of education and 2.3% were illiterate (table 1table 1. characteristics of the children with cp, ms, and ptschildren with cpxsdmin maxage (months)89.8052.0518300treatment period (months)73.6242.1118240gendern%boys7557.70girls5542.30motherxsdmin - maxage (years)35.475.792349physiotherapistxsdmin - maxage (years)28.077.282251length of service (years)6.847.51130education level of mothern%12 years or less10883.112 years or more1914.6illiterate32.3clinical types of the children with cpn%spastic11185.4dyskinetic75.4ataxic64.6hypotonic43.1mixed type21.5gmfcslevel 1107.7level 22116.2level 33829.2level 43627.7level 52519.2). the distribution of clinical types of cp cases and levels according to the gmfcs are shown in table 1. according to the results of the questionnaire that asked about the opinions of the ms and pts, while 33.1% (n=43) of the pts and 32.3% (n=42) of the ms defined the health status of the children as good, the concordance was found to be 13.1%. in addition, while 38.5% (n=50) of the pts and 39.2% (n=51) of the ms defined the health status of the children as moderate, the concordance was found as 18.5%. statistically insignificant correlation was found (k=0.129 and p=0.015) (table 2table 2. description of the health of the children with cp, awareness about the received treatments, and views about the appropriateness of the treatmentsphysiotherapistmother physiotherapist- mother agreement description of the health of the children with cpn%n%n%perfect43.132.310.8very good129.21310.010.8good4333.14232.31713.1moderate5038.55139.22418.5bad2116.22116.275.4awareness about the received therapiesbobath11386.95240.04736.2vojta21.543.110.8special education53.85038.543.1reflexology53.843.121.5botox32.321.5 - -i have no idea21.51813.8 - -views about the appropriateness of the treatmentyes12394.610883.110782.3no32.396.932.3i have no idea43.11310.021.5statistically significant (p < 0.05), kappa coefficient ; statistically significant (p < 0.01), kappa coefficient). statistically significant (p < 0.05), kappa coefficient ; statistically significant (p < 0.01), kappa coefficient when the awarenesses of the ms and pts about the therapies the children received were analyzed, 86.9% (n= 113) of the pts and 40% (n=52) of the ms stated that the children were receiving bobath therapy, and the concordance was found to be 36.2%. statistically insignificant concordance was found between the ms and pts (k=0.077 and p=0.016) (table 2). while 94.6% (n=123) of the pts and 83.1% (n=108) of the ms stated that they found the therapy appropriate, the concordance was found to be 82.3%. moderate concordance was found between the pts and ms when all answers about the appropriateness of therapy were evaluated (k=0.338 and p=0.0001) (table 2). when asked about the ability to walk with / without help, 31.5% (n= 41) of the pts and 38.5% (n=50) of the ms stated that they wanted the children with cp to walk with / without help, and the concordance was found to be 17.7%. statistically insignificant concordance was found between the pts and ms when all expectations were evaluated (k=0.187 and p=0.0001) (table 3table 3. the views of the physiotherapists and the mothers about the expectations from the treatment the children with cp receive, additional therapies and effectiveness of the physiotherapy and rehabilitation programphysiotherapistmotherphysiotherapist - mother agreementexpectations from the treatmentn%n%n%walking with / without help4131.55038.52317.7standing with / without help1511.586.243.1sitting with / without help3123.81310.086.2independency in daily life activities2015.44635.486.2increased balance and postural control2317.71310.053.8additional therapiesnot necessary2418.54232.386.2speech therapy3426.23728.51410.8water exercise2317.72418.596.9special education and psychosocial support2015.41410.864.6reflexology129.253.821.5vojta32.332.3 - -surgical53.832.3 - -sensory perception motor education96.921.5 - -effectiveness of the physiotherapy and rehabilitation programperfect1310.0129.232.3very good3325.44030.81310.0good6953.15945.43526.9moderate107.7118.521.5bad53.886.232.3statistically significant (p < 0.01), kappa coefficient). statistically significant (p < 0.01), kappa coefficient when asked about additional therapy, 18.5% (n=24) of the pts and 32.3% (n=42) of the ms stated that it was not necessary. statistically insignificant concordance was found between the pts and ms with regard to additional therapies (k=0.136 and p=0.001) (table 3). finally, when questioned about the efficacy of the physiotherapy and rehabilitation programs, 25.4% (n=33) of the pts and 30.8% (n=40) of the ms defined the applied therapy as very good, and the concordance was found to be 10.0%. statistically insignificant concordance was found between the pts and ms in terms of opinions about the efficacy of the programs (k=0.141 and p=0.009) (table 3). the results of our study showed that while statistically insignificant concordance (k=0.00.20) was found between the pts and ms regarding the definition of the health statuses of the children, treatment methods applied to the children, required additional therapies, and appropriateness of the rehabilitation programs, statistically moderate concordance was found regarding the appropriateness of the therapies (k=0.210.40). for families with a disabled child, it is quite difficult to accept the disability and rearrange lifestyles to adapt to the child s condition12,13,14. ms who are the primary caregivers of disabled children and interact more often with them represent a risk group for mental health due to anxiety and worries about the problems of their disabled child15, 16. the significance of participation of the family in treatment and education of a disabled child is emphasized in the literature17,18,19. it has been emphasized that early application of physiotherapy to a child with cp are important for motor development of the child and that the mother perceives the condition of the child20. considering that all rehabilitation processes should be realized in the natural environment of the child, involving the family in this process is inevitable. informing the family about the care and rehabilitation of the child and providing help are as effective as directly educating the child21. according to the results of our study, we consider that it is necessary to increase the contribution of mothers to treatment, to educate them in order to apply the therapies at home and to better understand their children, and to increase the cooperation with pts. in a study of karaduman families graded the treatment methods applied to their children as special education, physiotherapy, speech therapy, drug therapy, and surgical therapies, respectively22. in our study, the mothers stated that bobath therapy and special education treatment were the most appropriate and most beneficial treatment types for their children. low concordance was found between pts and ms in terms of the therapies applied to the children and degree of benefit from treatment. in the study of karaduman., the outcomes expected by families from treatment were attainment of the most efficient level of the disability or total elimination of the disability22. in our study, 38.5% of ms wanted their children to walk with / without help, and 35.4% wanted their children to perform daily activities independently. high concordance was found between pts and ms in terms of expectations from therapies. however, 67.72% of the ms and 81.5% of the pts considered alternative treatment methods to be necessary (table 3). the importance of a high education level of families for adequate care and treatment of a disabled child is known. in studies investigating the relations between families and health teams in early and later periods, ms stated that they did not understand the explanations their child s disease23, 24. in our study, 83.1% of ms had 12 years of education or less (table 1). we consider that this resulted from the fact that families are not sufficiently informed about their child s health and treatments or that they can not understand enough of the information they are given. emphasized that having enough knowledge about the condition of the child is important for adaptation to the child and care, education, and rehabilitation of the child25. one of the most important factors that positively affects the ability to cope with difficulties is education26. in the study of singer. conducted with families of disabled children, families with a higher education level reportedly cared for their children better ; in other words, awareness about the disease was higher27. in our study, the concordance between ms and pts regarding the appropriateness of therapy was 82.3%, and it was found to be 36.2% for treatment awareness. these concordance rates show the importance of informing and educating the mothers of children with cp. in conclusion, participation of the ms in the treatment program and awareness about the treatment are important. we consider that health staff involved in the care of disabled children should consider the opinions of the ms about the therapy when determining the most appropriate and most beneficial treatment. we consider that ms should cooperate with pts in the course of rehabilitation and receive education about the condition of their children in order to increase their awareness of their child s disease, find the most appropriate treatment option, practice the therapy at home in the most effective and most appropriate way, and obtain better results from rehabilitation programs. | [purpose ] the aim of this study was to examine the agreement between physiotherapists (pts) and mothers (ms) about the treatment of children with cerebral palsy (cp) who received treatment in special education and rehabilitation centers. [subjects ] ms of 130 children with cp (75 boys, 55 girls) and 130 pts who applied rehabilitation programs were interviewed. [methods ] clinical types and gross motor function levels of the children were recorded. a questionnaire consisting of 6 open - ended questions was used to describe the expectations and views of the pts and ms about the physiotherapy and rehabilitation programs for the children. [results ] the mean age of the children was 89.8052.05 months. the mean treatment period for the children was 73.6242.11 months. the mean age of the mothers was 35.475.79 years, and the mean age of the pts was 28.077.28 years. we found a statistically moderate level of agreement between the pts and ms regarding the appropriateness of the treatment provided to the children. there was statistically insignificant agreement regarding the applied treatment methods and the appropriateness of the applied rehabilitation programs. [conclusion ] we believe that the views and expectations of the ms should be taken into account by the pts when preparing a treatment program for children with cp. |
intoxication due to carbon monoxide (co) is one of the most common types of poisoning, and one of the most important toxicological global causes of morbidity and mortality (1). a weak association between carboxyhemoglobin (cohb) level and patients clinical picture is documented (2). co binds rapidly to hemoglobin with greater affinity than oxygen (o2) and forms cohb, which leads to a decrease in the o2 carrying capacity of the blood resulting in tissue hypoxia. therefore, organs with high oxygen demand, such as heart, brain and lungs are most sensitive to hypoxia (3 - 6). cardiac effects of cohb range from simple arrhythmias to myocardial infarction (7 - 9). cardiac troponins (ctn) the release of ctn into the circulation occurs as a consequence of cardiomyocyte injury (10). highly sensitive - ctn (hs - ctnt) assays are developed recently, enabling measurements of concentrations that are 100-fold lower than those of the ones previously measurable (11). the current study aimed to assess whether or not myocardial damage occurs in patients with co poisoning. the study investigated the relationship between blood carboxyhemoglobin and hs - ctnt level with a highly sensitive assay in patients with acute co poisoning. the current retrospective study was conducted at the sevket yilmaz training and research hospital that is a state tertiary hospital with 1050 beds, located in the eastern bursa, turkey. the study used the data available in the hospital clinical data warehouse, a centralized data repository integrating information in several databases including the order entry database and the laboratory results database of the hospital. patients diagnosed with co poisoning were included in the study and their corresponding electronic charts were reviewed for data collection. prescription data were linked to detailed clinical information including patient demographics, diagnosis, clinical characteristics (including past medical history, smoking status), co source (charcoal or fire), vital signs at presentation, physical examination characteristics, cohb levels, treatment therapy, complications in erectile dysfunction (ed), and laboratory data ; the latter included specimen collection date, time, and location (for example : intensive care unit). two - hundred - seventeen cases admitted to the emergency medicine unit of the hospital in 2012, (january 2012 - january 2013) with the diagnosis of acute co intoxication seventy - six patients whose additional diagnoses indicated chronic ischaemic heart disease, heart failure, myocarditis, muscular dystrophy, polymyositis, implanted cardiac resynchronization device, chronic inflammatory disease, and chronic renal failure were excluded. the overall study population included 141 subjects (87 females (62%) and 54 males (38%) ; 70% of the poisonings occurred in the winter, 24% in spring and 6% in autumn. the study was approved by the sevket yilmaz research and education hospital ethics committee (no. 2013/7 - 4) and was in compliance with the helsinki declaration ; informed consent was not assumed necessary because of the retrospective observational nature of the study and all steps were taken to ensure the anonymity of the data. blood samples were first collected (on admission) in the emergency department and four hours later in the intensive care unit (icu). routine laboratory data (cohb, creatine kinase - myocardial band (ck - mb) and high - sensitivity cardiac troponin t (hs - tnt) were recorded. cohb levels were measured by a blood gas analyzer (omni s, roche diagnostics penzberg, germany) supported by a co - oximetry panel. hs - ctnt and ck - mb levels were determined by an elecsys 2010 autoanalyzer (roche diagnostics, penzberg, germany) using commercial assays. according to the manufacturer of the hs - ctnt stat assay, ng / l, and the 99th percentile in healthy volunteers was 14 ng / l. for quality assurance purposes, the laboratory participates in an external quality assessment scheme run by labquality, helsinki, finland. at the time of the present study, the qc program reported average values as part of the treatment, all the patients inhaled high flow normobaric oxygen and were monitored and followed up in icu. treatment continued until clinical findings stabilized and serum cohb levels decreased to target levels of 5%. the patients intoxicated with co were divided into three groups depending on cohb levels : group i, mild cohb level 15% ; group iii, severe acute co intoxication cohb levels > 25%. samples with hs - ctnt below the limit of blank (i e, 3 ng / l) were assigned a value of 1 ng / l. the comparisons between the medians of three groups were performed by the kruskal - wallis test and the post - hoc dunnett tests were used to examine the significance levels between groups. spearman rank analysis was used to assess associations between cohb, ck - mb and hs - tnt levels (spss, chicago, il) and p values of 15% ; group iii, severe acute co intoxication cohb levels > 25%. samples with hs - ctnt below the limit of blank (i e, 3 ng / l) were assigned a value of 1 ng / l. the comparisons between the medians of three groups were performed by the kruskal - wallis test and the post - hoc dunnett tests were used to examine the significance levels between groups. spearman rank analysis was used to assess associations between cohb, ck - mb and hs - tnt levels. (spss, chicago, il) and p values of 25% (n = 45) (table 1) (12). abbreviations : ck - mb, creatine kinase - myocardial band ; cohb, carboxyhemoglobin ; hs - tnt, high - sensitivity cardiac troponin t. values are expressed as median (interquartile range) or mean sd. median hs - ctnt increased with increasing cohb level (kruskal - wallis p = 0.05 ; table 1). when the post - hoc dunnett test was performed, hs - ctnt levels were not statistically different between the groups. ck - mb levels did not differ between the three groups (kruskal - wallis ; p = 0.48). cohb levels with hs - tnt values were weakly correlated (r = 0.173, p = 0.041) ; on the other hand, ck - mb levels were not correlated with those of the cohb (r = 0.013, p = 0.883) (table 2). abbreviations : ck - mb, creatine kinase - myocardial band ; hs - tnt, high - sensitivity cardiac troponin t. on admission, 5 of the 141 patients had elevated serum ck - mb levels and 20 had elevated serum hstnt levels (> 14 ng / l), only three of the patients with cardiac markers were elevated on the follow - up period. the findings of the cohort current study of patients with cohb intoxication hs - ctnt levels were slightly higher in severe toxicated patients than in mild toxicated patients without significant correlation between cohb and hs - ctnt levels. cardiac troponins are components of the contractile apparatus of cardiomyocytes and are released during myocardial necrosis. serum troponin elevation is a specific and well established myocardial necrosis biomarker, and can detect extremely small amounts of myocardial necrosis (< 1.0 g) (12, 13). very low, but detectable amounts of hs - ctnt levels may reflect a normal biological process of myocyte turnover and it may also be associated with an increased cell turnover (14, 15). the proposed mechanisms of cardiac troponin release include apoptosis, cellular release of proteolytic degradation products, increased cell wall permeability, and formation and release of membranous blebs (11). many acute diseases are associated with elevated ctn in the absence of acute ischemic heart disease that can occur for many reasons (16). direct toxic effects of circulating cytokines and chemotherapies can cause severe myocardial toxicity as severe sepsis and septic shock (12, 16). recent data showed that one can detect the effects of some toxic chemotherapy by monitoring ctn (16). low levels of co activate soluble guanylate cyclase which in turn exerts beneficial effects such as vasodilatation and inhibition of platelet aggregation (7). in the current study, hs - ctnt levels were higher in patients with severe co toxicity compared to the ones with mild co intoxication. although ultramicroscopic changes are reported in cases of co toxicity, its relative effects need to be documented (17). a direct toxic effect of cohb is discussed as a consequence of experimental studies on cytochrome oxidase (17). co binds with cardiac myoglobin causes a rapid decrease in myocardial oxygen reserves (18). when the energy source is blocked, then the function of the myoglobin is diminished (19, 20). myocardial fiber necrosis and other changes observed with electron microscopy are associated with impaired energy metabolism (22, 23). however, cardiac troponins are released without electron microscopic changes (24, 25). in this study ck - mb levels did not differ between the three groups and did not correlate with cohb. patients with detectable troponin, but no ck - mb, in the blood may exhibit microscopic zones of myocardial necrosis (microinfarction) (13). (26) showed that in co poisoning, patients without known underlying significant coronary artery disease with cohb levels of up to 60% do not develop myocardial damage but they used only cardiac biomarkers ck - mb and ctnt. the current study did not find any significant elevation in ck - mb levels, but in contrast a significant increase in hs - ctnt levels was found, which might be because of the microscopic myocardial necrosis (26). myocardial injury, documented with elevations in cardiac biomarkers, could be present in about one - third of the patients with serious co poisoning and it was associated with mortality (7). the severity of myocardial injury depended on the duration and amount of co exposure (27). moreover, the level of co in the tissues may have an equal or greater impact on the clinical status of the patient than does the blood level of co (28). the primary limitation of the current small study group was the method of data collection. there was no information on the length of co exposure and the timing of the blood cohb levels in relation to the co exposures. further studies are necessary in this regard. another limitation was the retrospective nature of the study. the data were reviewed by one researcher who avoided the selection bias ; however, misclassification, may still exist, which can not be verified or validated. these results apply only to the elecsys hs - ctnt and ck - mb (roche diagnostics) assays, and may not be generalized to other high sensitivity assays by other manufacturers. finally, the study design was based on the data available in one hospital, and the obtained results may not necessarily be generalized. in conclusion, in patients without clear signs of myocardial infarction, even mild co poisoning is associated with quantifiable circulating levels of hs - ctnt when tnt is measured using a highly sensitive assay and cardiac complications should be considered in such patients. plasma levels of the hs - tnt and ck - mb assays were not correlated with the cohb levels. in conclusion, in patients without clear signs of myocardial infarction, even mild co poisoning is associated with quantifiable circulating levels of hs - ctnt when tnt is measured using a highly sensitive assay and cardiac complications should be considered in such patients. plasma levels of the hs - tnt and ck - mb assays were not correlated with the cohb levels. | backgroundintoxication due to carbon monoxide (co) is one of the most common types of poisoning. cardiac effects of carboxyhemoglobin (cohb) range from simple arrhythmias to myocardial infarction.objectivesthe current study aimed to investigate the relationship between blood carboxyhemoglobin and high - sensitivity cardiac troponin t (hs - ctnt) level with a highly sensitive assay in patients with acute carbon monoxide poisoning.patients and methodsthis retrospective study was conducted on 141 (54 males and 87 females) patients, with acute co intoxication, admitted to the sevket yilmaz research and education hospital emergency unit during a one - year period (january 2012 - january 2013). the patients were divided into three groups based on cohb levels : group i, mild cohb level 25%. cohb, hs - ctnt (stat), creatine kinase (ck) and creatine kinase - myocardial band (ck - mb) levels were measured on admission.resultsthe mean age of the patients was 38 16 years. cohb levels ranged from 8 to 35. hs - ctnt levels on inclusion in this study were slightly different between the groups (p = 0.05). cohb levels with hs - ctnt values were weakly correlated (r = 0.173, p = 0.041) ; on the other hand, ck - mb levels were not correlated with cohb (r = 0.013, p = 0.883).conclusionsin patients without clear signs of myocardial infarction, even mild co poisoning was associated with quantifiable circulating levels of hs - ctnt when tnt was measured using a highly sensitive assay in the current study patients. plasma levels of the hs - tnt and ck - mb assays were not correlated with the cohb levels in the current study patients. |
fifteen tri - colored guinea pigs were obtained from the animal experiments laboratory of north sichuan medical college and were reared in large cages. one eye of the guinea pigs was randomly selected and treated with 7.00d lenses. the method of cycloplegia was induced with three drops of tropicamide, and the refraction status was measured by means of streak retinoscopy in hand - held, awake animals. the axial length of the eyes was measured by an a - scan ultrasound while the animals were anesthetized with ketamine (80 mg / kg) by intramuscular injection. ocular refraction and axial length were performed before the experiment and 1, 2, and 3 weeks after minus lens intervention. the measurement was repeated at least three times for each eye, and the refraction and axial length of each guinea pig at every measurement were averaged. anova, with repeated measures design, was used to investigate the influence of the intervention on the axial length and refraction. a one - way anova, followed by student 's unpaired t - test, with bonferroni correction for multiple testing, was used to analyze ocular parameters between groups. this study was complied with the tenets of the association for research in vision and ophthalmology statement for the use of animal in ophthalmic and vision research. after the refraction and axial length measurements at 1, 2, and 3 weeks of minus lens intervention, five guinea pigs were randomly selected and were deep anesthetized with 0.03 ml / kg chloral hydrate by intraperitoneal injection. with the animals under deep anesthesia, both eyes were enucleated at a similar time point (between 4:00 and 6:00 pm) to minimize the effect of diurnal variation on gene expression. a circumferential incision was made along the limbus, followed by removal of the cornea, crystalline lens, and vitreous body. the entire retina was separated from the choroid while the sample was soaked in iced neutral buffer formaldehyde solution. twenty - four hours later, the retina was embedded in paraffin to detect expression of mfn1 with immunohistochemistry (streptavidin - perosidase (sp) three steps). before the intervention, there were no statistically significant differences between the two groups in terms of the refraction (p = 0.860) and axial length data (p = 0.115). however, repeated measures anova [tables 1 and 2 ] revealed a statistically significant effect of minus lens intervention and a significant minus lens intervention by time interaction for the axial length and refraction from baseline (p = 0.000). the lens - induced myopia (lim) eyes became more myopic by 4.70d and had an increase of axial length by 0.46 mm after lens induction for 3 weeks [table 3 ]. the average increase of axial length was 0.46 mm in lens - induced eyes and 0.18 mm in the normal control eyes [table 4 ]. the results of axial length analysis by anova (mm) the results of refraction analysis by anova the refraction data measured in the two eyes the results of axial length in both eyes by t - test the results of protein expression [figs. mfn1-positive cells could be observed in the retina of both eyes, mainly in the cytoplasm and cell membrane of the ganglion cells ; mfn1-positive cells appeared brownish or yellow. in the lim eyes, the immunopositive cells were staining deep, and more positive cells could be observed ; however, mfn1-positive cells scattered expressed in the ganglion cell layer in the control eyes. furthermore, mfn1-positive expression could be seen mainly in ganglion cells at the 1 week of treatment ; with the extension of lens induction time, many mfn1-positive cells also appeared in the bipolar cell layer and the rod - cone cell layer, and this phenomenon could not be found in normal control eyes. (a and b) one week after treatment mitochondrial fusion protein 1-positive cells were observed in the retina of both eyes, mainly in the cytoplasm and cell membrane of the ganglion cells, and the positive cells seem staining deep in the lens - induced eyes (a and b) two weeks after treatment the positive cells in the lens - induced eyes were staining deep, abundant - positive cells could be observed in the ganglion cell layer, and many mitochondrial fusion protein 1-positive cells could also be seen in the bipolar cell layer and rod - cone cell layer ; however, positive cells only scattered in the ganglion cell layer in the control eyes (a and b) three weeks after treatment mitochondrial fusion protein 1-positive cells scattered expressed in the ganglion cell layer and the bipolar cell layer of the control eyes, while many positive cells appeared in the rod - cone cell, the bipolar cell and ganglion cell layer in the lens - induced eyes to the best of our knowledge, this is the first study that describes the mfn1 gene expression and myopia. in a previous british study, five single nucleotide polymorphism (snp) loci around the mfn1 gene were found strongly associated with myopia (including high, medium, and low myopia). especially, the snp locus of rs6794192 and rs7618348 showed lower p values. in our recent study, we genotyped rs3976523 which was linked with rs6794192 (r = 0.942) in the myopia population, and no correlation was found between rs3976523 and myopia. for rs7618348, we found that the p values in the allele difference between controls and myopia patients were more than 0.05. however, we also genotyped another two other loci in the mfn1 gene (rs6762399 and rs13098637) in our previous study. interestingly, we found that rs13098637 locus located in an intron at the center of the mfn1 gene was significantly correlated to myopia. although human studies have revealed mfn1 as a candidate gene of myopia, the relationship between the mfn1 expression and myopia has not been investigated. in this study, we further investigated the expression of mfn1 in the retina, so as to get a better understanding about its possible involvement in the occurrence or development in the mammalian myopia. guinea pig is a kind of mammals, the eyeball structure is similar to the human 's, and myopia animal model of guinea pig has successfully been established. all guinea pigs were raised with a monocular 7.00d lens, and the fellow eye was untreated and served as the self - control group. during the 1 week, both axial length (0.04 mm in the control eyes and 0.11 mm in the experimental eyes) and refraction data changed in both groups (0.25d in the control eyes and 0.43d in the experimental eyes), but the refraction data did not show any statistically significant difference (p = 0.388) whereas the axial length showed a statistically significant difference (p = 0.008). the axial length in the lens - induced eyes increased rapidly when compared to control group, owing to the minus lens intervention. the refraction data are the combined effect of axial length and the refractive components of the eye, but we suspect that the refractive components did not change to compensate for the axial length change during the 1 week in this research. this could be the reason for the refraction data did not show a significant difference between the two groups during the 1 week while the axial length showed a significant difference. over the time of intervention, about 2 weeks later, the refraction data and axial length were both significantly different between the two groups. three weeks after the intervention, the lens - induced eyes became more myopic, and the mean refraction was 1.78d in lim eyes whereas it was + 1.05d in control eyes, which indicated that myopia animal model had been successfully established by a 7.00d minus lens. in the current study, mfn1-positive cells could be observed in the retina of both eyes. in the lim eyes, the immunopositive cells were staining deeper and more positive cells could be observed while mfn1-positive cells only scattered expressed in the ganglion cell layer of the control eyes. in addition, in the lim eyes, 1 week after minus lens intervention, we could find that mfn1 expression positive cells mainly located in the ganglion cell layer, with the intervention time extension, more and more positive cells occurred in the bipolar cell layer and the cone - rod cell layer, especially in the cone - rod cell layer, abundant - positive cells could be seen and the dyeing color was very deep. these results indicated that mfn1 overexpression in the retina of the experimental eyes might be associated with the development of lim. from both the mfn1 expression results and refraction data, we also speculated that minus lens intervention may interrupt the emmetropization in experimental eyes of animals and also the physiological control of gene expression., the investigation of mfn1 expression in the retina had not been extensively studied ; in addition, the sample size in this experiment was a little small, and so this investigation could only be looked as a preliminary exploratory research. unfortunately, in the present study, only according to the images, we only could qualitatively describe this interesting phenomenon on mfn1 expression. hence, mfn1 expression in the retina should be quantitatively studied and analyzed in future research. we found some rough alterations of mfn1 expression in the lim eyes of the guinea pigs. by combining our previous study which found that rs13098637 locus within the mfn1 gene was related to myopia, we speculated that mfn1 genetic variants might be likely to influence the development of myopia and that the relationship between myopia and mfn1 should be worthy of further investigation. this study was supported by the national nature science foundation of china (81341105) and by the research funds from the health department of sichuan province (120442). this study was supported by the national nature science foundation of china (81341105) and by the research funds from the health department of sichuan province (120442). | purpose : the aim of this study is to preliminarily investigate the expression of mitochondrial fusion protein 1 (mfn1) in a lens - induced animal myopia (lim) model and to explore the relationship between mfn1 and the visual development.materials and methods : mfn1 gene expression in guinea pigs was examined during the development of minus lim, 15 tri - colored guinea pigs were obtained, and one eye of each pig was randomly selected and treated with 7.00d lenses. ocular refraction and axial length were collected before intervention and 1, 2, and 3 weeks after intervention. after the refraction and axial length measurements at 1, 2, and 3 weeks of lens intervention, five guinea pigs were randomly selected. mfn1 expression in the retina of both eyes was tested by immunohistochemistry technique.results:mfn1-positive cells could be observed in the retina of both eyes. the positive cells in the lim eyes were staining deeper, and much more positive cells could be observed. furthermore, mfn1-positive expression could be seen mainly in ganglion cells after 1 week of minus lens intervention, and with time extension, more and more positive cells appeared in the rod - cone cell and bipolar cell layer, and this phenomenon could not be found in the normal control eyes.conclusion:this study suggested that mfn1 might be correlated to the development of myopia. |
we initiated our study by investigating the reactivity of substrates derived from geraniol under our previously reported hydroamination conditions (table 1). a variety of leaving groups, including alkoxy, silyloxy, carbonate, phosphate, and carboxylates, provided the desired product with high levels of enantioselectivity (entries 16). the stable and readily - prepared allylic benzoate was found to provide the desired product in high yield. incorporation of an electron - withdrawing group on the benzoate to increase the nucleofugality of the leaving group was found instead to significantly reduce the yield (entry 7). in contrast, the use of electron - donating substituents on the benzoate, such as a 4-dimethylamino group (entry 8), produced near quantitative yield of the -chiral amine (s)-3a. an evaluation of ligands revealed dtbm - segphos to give superior results compared to all others tested (entry 8 vs. entries 912). under the optimized conditions, the scope of allylic benzoates that could be transformed was investigated (table 2). a variety of substrates were converted into the corresponding chiral amines with high enantioselectivity and in moderate to excellent yields. a variety of 3,3-dialkyl substituted allylic 4-(dimethylamino)benzoates were first investigated (table 2a). when the isomeric nerol - derived 4-(dimethylamino)benzoate containing a (z)-configured allylic double bond was exposed to the optimized conditions, the opposite enantiomer (r)-3a was obtained with slightly lower yield and enantioselectivity (3a vs. 3a). bulky groups at the 3-position of the allylic system were tolerated and provided chiral amine products in moderate to good yields and exceptionally high enantioselectivity (3b d). a variety of functional groups were readily accommodated, including a ketal (3d), an aryl group (3e), a sulfonamide (3f), an alkyl chloride (3 g), ethers (3h, 3j), an ester (3k), a free alcohol (3i) and an unprotected secondary amine (3l). when an aldehyde - containing substrate was employed, not surprisingly, reduction to the alcohol was observed to produce the amino alcohol product (3 m). protected amino acid - containing substrate 1n could be transformed to -chiral amine (3n, 3n) without carbonyl reduction or epimerization, reflecting the mildness of the reaction conditions. additionally, 3-aryl - substituted allylic benzoates could also undergo reductive relay hydroamination (table 2b). substrates bearing electron - rich (3p, 3u) and electron - poor (3qs) aryl substituents were tolerated. a 3-thienyl substituted substrate (3 t), as well as a chromane - derived bicyclic substrate (3u), were also compatible with these conditions. likewise, a silyl - substituted allylic benzoate (table 3a, 1v) was converted into the -chiral silylamine (3v). in addition to 3,3-disubstituted allylic benzoates, this protocol was also applicable to racemic 1-aryl-3-alkyl - substituted allylic benzoate (table 3a, 1w), which reacted in an enantioconvergent manner to provide the -branched chiral amine product (3w). we reasoned that substrates containing an appropriate duo of vicinal allylic and homoallylic substituents would undergo sequential insertion / elimination sequences before undergoing hydroamination. such a cascade process would lead to the formation of -chiral amines. to test this idea, allylic epoxide (table 3a, 1x) and allylic acetonide (table 3a, 1y) substrates as predicted, racemic epoxide 1x reacted in an enantioconvergent manner to provide the -chiral amine 3x with high enantioselectivity. likewise, enantioenriched acetonide 1y also underwent the cascade hydroamination process to afford either enantiomer (3y, 3y) of the -chiral amine product under catalyst control. the successful extension of the reductive relay hydroamination protocol to this complex cascade serves to illustrate the flexibility and generality of this strategy. in some cases, the free allylic alcohols could be used directly in the current catalytic system (table 3b). by adding an extra equivalent of silane the use of the free alcohol did not affect the enantioselectivity of the process, although the yields obtained in this extended cascade were somewhat diminished relative to the use of the corresponding (4-dimethylamino)benzoate esters (table 2a, 3a, 3e, 3 g). to demonstrate the scalability of this process under slightly modified reaction conditions, a catalyst loading of 0.5 mol% proved sufficient for a reaction on this scale (table 3c). as first observed by lipshutz, the inclusion of triphenylphosphine as an additive led to improved stability of the active catalyst, allowing higher catalyst turnover numbers to be attained without any significant effect on the enantioselectivity. the utility of this catalytic system was further demonstrated through the use of a number of hydroxylamine esters as aminating reagents (table 4). despite the presence of a stereocenters adjacent to the nitrogen atom of the electrophilic aminating reagent, the hydroamination reaction proceeded in a completely catalyst - controlled manner (4a, 4a) to form a minimally differentiated stereocenter (methyl vs ethyl) with excellent selectivity. in addition, acyclic (4b), cyclic (4d), and sterically hindered (4c) hydroxylamine esters could be utilized, as could one containing a carbamate protecting group (4d). furthermore, substrates containing heterocycles, such as pyridine (4e), pyrimidine (4f), benzothiadiazole (4 g), and piperazine (4f, 4 g) were handled without event. finally, duloxetine, a drug used to treat depression and generalized anxiety disorder, could be functionalized with complete control of diastereoselectivity (4h, 4h), demonstrating the potential utility of this protocol in the late - stage functionalization of complex molecules. we investigated the reactivity of substrates containing multiple c c double bonds and found that the hydroamination protocols described here and previously exhibited excellent chemoselectivity when different types of double bonds were present. in general, styrenyl and terminal double bonds both react in preference to trisubstituted allylic esters, and this difference in reactivity was exploited for sequential chemoselective hydroamination. for example, sequential hydroamination of diolefin 5 containing a trisubstituted allylic ester and terminal olefin with two different aminating reagents resulted in the formation of diamine 7 with excellent chemo- and enantioselectivity (fig. furthermore, by simultaneously taking advantage of the difference in reactivity between mono- and dialkyl aminating reagents, as well as between styrenyl double bonds and trisubstituted allylic esters, a three component coupling of two aminating reagents with diolefin 8 could be achieved with good chemo-, diastereo-, and enantioselectivity (fig. importantly, this strategy can also be employed to form all four stereoisomers of a diamine product in uniformly high stereoselectivity depending on the enantiomer of chiral ligand used in each step (17:1 to 28:1 dr, > 99% ee) (fig. 2c, 11a - d). in summary, we have developed a cuh - catalysed reductive relay process to access - and -chiral amines. this method allows for the installation of a stereocenter and a distal amino group in a single operation under mild conditions. excellent enantio-, regio-, and chemoselectivity were observed for a broad range of substrates with high functional group tolerance. furthermore, this system was found to be applicable to the late - stage modification of a pharmaceutical agent and was suitable for large - scale synthesis. lastly, we also demonstrated that the cuh - catalysed protocol could be applied to the chemoselective sequential amination of substrates containing more than one olefin. the expansion of this relay strategy to other areas including drug and natural product synthesis is currently underway and will be reported in due course. we initiated our study by investigating the reactivity of substrates derived from geraniol under our previously reported hydroamination conditions (table 1). a variety of leaving groups, including alkoxy, silyloxy, carbonate, phosphate, and carboxylates, provided the desired product with high levels of enantioselectivity (entries 16). the stable and readily - prepared allylic benzoate was found to provide the desired product in high yield. incorporation of an electron - withdrawing group on the benzoate to increase the nucleofugality of the leaving group was found instead to significantly reduce the yield (entry 7). in contrast, the use of electron - donating substituents on the benzoate, such as a 4-dimethylamino group (entry 8), produced near quantitative yield of the -chiral amine (s)-3a. an evaluation of ligands revealed dtbm - segphos to give superior results compared to all others tested (entry 8 vs. entries 912). under the optimized conditions, the scope of allylic benzoates that could be transformed was investigated (table 2). a variety of substrates were converted into the corresponding chiral amines with high enantioselectivity and in moderate to excellent yields. a variety of 3,3-dialkyl substituted allylic 4-(dimethylamino)benzoates were first investigated (table 2a). when the isomeric nerol - derived 4-(dimethylamino)benzoate containing a (z)-configured allylic double bond was exposed to the optimized conditions, the opposite enantiomer (r)-3a was obtained with slightly lower yield and enantioselectivity (3a vs. 3a). bulky groups at the 3-position of the allylic system were tolerated and provided chiral amine products in moderate to good yields and exceptionally high enantioselectivity (3b d). a variety of functional groups were readily accommodated, including a ketal (3d), an aryl group (3e), a sulfonamide (3f), an alkyl chloride (3 g), ethers (3h, 3j), an ester (3k), a free alcohol (3i) and an unprotected secondary amine (3l). when an aldehyde - containing substrate was employed, not surprisingly, reduction to the alcohol was observed to produce the amino alcohol product (3 m). protected amino acid - containing substrate 1n could be transformed to -chiral amine (3n, 3n) without carbonyl reduction or epimerization, reflecting the mildness of the reaction conditions. additionally, 3-aryl - substituted allylic benzoates could also undergo reductive relay hydroamination (table 2b). substrates bearing electron - rich (3p, 3u) and electron - poor (3qs) aryl substituents were tolerated. a 3-thienyl substituted substrate (3 t), as well as a chromane - derived bicyclic substrate (3u), were also compatible with these conditions. likewise, a silyl - substituted allylic benzoate (table 3a, 1v) was converted into the -chiral silylamine (3v). in addition to 3,3-disubstituted allylic benzoates, this protocol was also applicable to racemic 1-aryl-3-alkyl - substituted allylic benzoate (table 3a, 1w), which reacted in an enantioconvergent manner to provide the -branched chiral amine product (3w). we reasoned that substrates containing an appropriate duo of vicinal allylic and homoallylic substituents would undergo sequential insertion / elimination sequences before undergoing hydroamination. such a cascade process would lead to the formation of -chiral amines. to test this idea, allylic epoxide (table 3a, 1x) and allylic acetonide (table 3a, 1y) substrates as predicted, racemic epoxide 1x reacted in an enantioconvergent manner to provide the -chiral amine 3x with high enantioselectivity. likewise, enantioenriched acetonide 1y also underwent the cascade hydroamination process to afford either enantiomer (3y, 3y) of the -chiral amine product under catalyst control. the successful extension of the reductive relay hydroamination protocol to this complex cascade serves to illustrate the flexibility and generality of this strategy. in some cases, the free allylic alcohols could be used directly in the current catalytic system (table 3b). by adding an extra equivalent of silane the use of the free alcohol did not affect the enantioselectivity of the process, although the yields obtained in this extended cascade were somewhat diminished relative to the use of the corresponding (4-dimethylamino)benzoate esters (table 2a, 3a, 3e, 3 g). to demonstrate the scalability of this process under slightly modified reaction conditions, a catalyst loading of 0.5 mol% proved sufficient for a reaction on this scale (table 3c). as first observed by lipshutz, the inclusion of triphenylphosphine as an additive led to improved stability of the active catalyst, allowing higher catalyst turnover numbers to be attained without any significant effect on the enantioselectivity. the utility of this catalytic system was further demonstrated through the use of a number of hydroxylamine esters as aminating reagents (table 4). despite the presence of a stereocenters adjacent to the nitrogen atom of the electrophilic aminating reagent, the hydroamination reaction proceeded in a completely catalyst - controlled manner (4a, 4a) to form a minimally differentiated stereocenter (methyl vs ethyl) with excellent selectivity. in addition, acyclic (4b), cyclic (4d), and sterically hindered (4c) hydroxylamine esters could be utilized, as could one containing a carbamate protecting group (4d). furthermore, substrates containing heterocycles, such as pyridine (4e), pyrimidine (4f), benzothiadiazole (4 g), and piperazine (4f, 4 g) were handled without event. finally, duloxetine, a drug used to treat depression and generalized anxiety disorder, could be functionalized with complete control of diastereoselectivity (4h, 4h), demonstrating the potential utility of this protocol in the late - stage functionalization of complex molecules. we investigated the reactivity of substrates containing multiple c c double bonds and found that the hydroamination protocols described here and previously exhibited excellent chemoselectivity when different types of double bonds were present. in general, styrenyl and terminal double bonds both react in preference to trisubstituted allylic esters, and this difference in reactivity was exploited for sequential chemoselective hydroamination. for example, sequential hydroamination of diolefin 5 containing a trisubstituted allylic ester and terminal olefin with two different aminating reagents resulted in the formation of diamine 7 with excellent chemo- and enantioselectivity (fig. furthermore, by simultaneously taking advantage of the difference in reactivity between mono- and dialkyl aminating reagents, as well as between styrenyl double bonds and trisubstituted allylic esters, a three component coupling of two aminating reagents with diolefin 8 could be achieved with good chemo-, diastereo-, and enantioselectivity (fig. importantly, this strategy can also be employed to form all four stereoisomers of a diamine product in uniformly high stereoselectivity depending on the enantiomer of chiral ligand used in each step (17:1 to 28:1 dr, > 99% ee) (fig. 2c, 11a - d). in summary, we have developed a cuh - catalysed reductive relay process to access - and -chiral amines. this method allows for the installation of a stereocenter and a distal amino group in a single operation under mild conditions. excellent enantio-, regio-, and chemoselectivity were observed for a broad range of substrates with high functional group tolerance. furthermore, this system was found to be applicable to the late - stage modification of a pharmaceutical agent and was suitable for large - scale synthesis. lastly, we also demonstrated that the cuh - catalysed protocol could be applied to the chemoselective sequential amination of substrates containing more than one olefin. the expansion of this relay strategy to other areas including drug and natural product synthesis is currently underway and will be reported in due course. to an oven - dried 4 ml screw - cap vial equipped with a magnetic stir bar was added cu(oac)2 (2.05.0 mol%) and (r)-dtbm - segphos (2.25.5 mol%). the tube was sealed with a teflon - lined screw cap, evacuated, and backfilled with argon (this process was repeated a total of three times) by piercing with a needle attached to a schlenk line. anhydrous thf (1.0 ml) was added by syringe, and the mixture was stirred for 10 min at room temperature. at this time diethoxymethylsilane (560720 l, 3.54.5 mmol, 3.54.5 equiv) was added by syringe and stirring was continued for another 5 min. into a separate oven - dried medium - sized screw - cap test tube was added -disubstituted allylic benzoate (1.0 mmol, 1.0 equiv) and o - benzoyl - n, n - dibenzylhydroxylamine (381 mg, 1.2 mmol, 1.2 equiv). the tube was sealed with a teflon - lined screw cap, evacuated, and backfilled with argon (this process was repeated a total of three times). the catalyst solution was then transferred via syringe to the reaction tube containing the substrates, and the reaction mixture was stirred at 4050 c for up to 36 h. after the reaction was complete, the reaction mixture was allowed to cool to room temperature and was directly filtered through a short pad of silica gel (using ethyl acetate in hexanes) to give the crude product. 1,1,2,2-tetrachloroethane (84 mg, 0.50 mmol) was added as internal standard for h nmr analysis of the crude material. the product was purified by chromatography on silica gel or by acid - base extraction as indicated for each substrate. the enantiomeric excesses (% ee) were determined by hplc analysis using chiral stationary phases as described in the supplementary information. the x - ray crystallographic coordinate for 3 g is deposited at the cambridge crystallographic data centre (ccdc) under deposition number ccdc 1413400. to an oven - dried 4 ml screw - cap vial equipped with a magnetic stir bar was added cu(oac)2 (2.05.0 mol%) and (r)-dtbm - segphos (2.25.5 mol%). the tube was sealed with a teflon - lined screw cap, evacuated, and backfilled with argon (this process was repeated a total of three times) by piercing with a needle attached to a schlenk line. anhydrous thf (1.0 ml) was added by syringe, and the mixture was stirred for 10 min at room temperature. at this time diethoxymethylsilane (560720 l, 3.54.5 mmol, 3.54.5 equiv) was added by syringe and stirring was continued for another 5 min. into a separate oven - dried medium - sized screw - cap test tube was added -disubstituted allylic benzoate (1.0 mmol, 1.0 equiv) and o - benzoyl - n, n - dibenzylhydroxylamine (381 mg, 1.2 mmol, 1.2 equiv). the tube was sealed with a teflon - lined screw cap, evacuated, and backfilled with argon (this process was repeated a total of three times). the catalyst solution was then transferred via syringe to the reaction tube containing the substrates, and the reaction mixture was stirred at 4050 c for up to 36 h. after the reaction was complete, the reaction mixture was allowed to cool to room temperature and was directly filtered through a short pad of silica gel (using ethyl acetate in hexanes) to give the crude product. 1,1,2,2-tetrachloroethane (84 mg, 0.50 mmol) was added as internal standard for h nmr analysis of the crude material. the product was purified by chromatography on silica gel or by acid - base extraction as indicated for each substrate. the enantiomeric excesses (% ee) were determined by hplc analysis using chiral stationary phases as described in the supplementary information. the x - ray crystallographic coordinate for 3 g is deposited at the cambridge crystallographic data centre (ccdc) under deposition number ccdc 1413400. | amines with remote stereocenters (stereocenters that are three or more bonds away from the c n bond) are important structural elements in many pharmaceutical agents and natural products. however, previously reported methods to prepare these compounds in an enantioselective manner are indirect and require multistep synthesis. here we report a copper hydride - catalysed, enantioselective synthesis of - or -chiral amines from readily available allylic alcohols, esters, and ethers using a reductive relay hydroamination strategy (a net reductive process in which an amino group is installed at a site remote from the original c c double bond). the protocol was suitable for substrates containing a wide range of functional groups and provided remote chiral amine products with high levels of regio- and enantioselectivity. sequential amination of substrates containing several carbon - carbon double bonds could be achieved, demonstrating the high chemoselectivity of this process. |
the plan of treatment of deformities of the jaws includes pre - surgical orthodontic treatment designed to eliminate dental compensations present (i.e., the anomalies of the teeth that have taken place) and the preparation of the dental arches in relation to the scheduled surgery. in the field of orthodontics and surgery, studies have been carried out to improve some characteristics like the modulus of elasticity and frictional modulus of orthodontic appliances. although the le fort i osteotomy is a safe surgical technique, many complications have been reported with a low incidence of occurrence. these include swelling, haemorrhage, infections, nerve injuries, bone necrosis, tmj problems, periodontal disease, ophthalmic and middle ear disorders, dysphagia and psychological problems. in particular, vascular complications can arise from direct trauma to the vessels due to the blind nature of the osteotomy in the maxillary posterior area. unwanted fracture lines may also extend to the pterygopalatine fossa, skull base and to the orbit. as a rule, the osteotome position during pterygomaxillary separation is contiguous to the internal maxillary artery (i m a), therefore, complications are rare. the entire course of internal maxillary artery (i m a) from its retro - mandibular origin to its termination within the pterygopalatine fossa is vulnerable to iatrogenic injury. therefore, to avoid possible delayed complications and facilitate their management, continuous postoperative monitoring is essential. the descending palatine artery is the vessel most often involved in traumatic injuries during orthognatic surgery of the maxilla. less often, the maxillary artery and its terminal branches, the pterygoid venous plexus, the internal carotid artery, and internal jugular vein may be damaged. we present a case of an extended cervico - facial haematoma due to delayed bleeding from the terminal branches of the maxillary artery after orthognatic surgery. a 23-year - old man was referred to the department of maxillofacial surgery, university of verona, italy for the surgical correction of a class iii asymmetric malocclusion [figure 1a ]. the patient underwent a le fort i osteotomy, with impaction of the maxilla associated with an epker mandibular bilateral sagittal split ramus osteotomy, with maxillary advancement and rigid internal fixation of the mandible with four miniplates (osteomed), and four miniplates for the upper maxilla as well. class iii asymmetric malocclusion the patient had a medical history of one wisdom tooth extraction, performed under local anaesthesia. preoperative vital parameters were : temperature, 36.4c ; heart rate, 62 bpm ; blood pressure, 120/65 mmhg ; respiration rate, 17 breaths per minute and 100% oxygen saturation. preoperative laboratory tests showed rbc, 4.86 e + 12/l ; hb, 14.1 g / dl ; ht, 43.1% ; coagulation tests were normal (prothrombin time 1.05 inr, activated partial thromboplastin time 1.18 and fibrinogen 241 mg / dl, clauss method 7.11 the patient received a single - shot antimicrobial prophylaxis using 1 gm of amoxicillin, in addition to a single dose of corticosteroids (1 gm of methylprednisolone) one hour before the operation. the surgical procedure was performed in general hypotensive anaesthesia, with the following parameters : blood pressure, 100/70 mmhg ; heart rate, 80 beats per minute and 100% oxygen saturation. sevoran, fentanile, remifentanyle, 1500 ml of crystalloids and 500 ml of colloids were infused. the first post - surgery day, the patient developed a gradual dispnea together with neck swelling. a cat scan with contrast agent was performed, showing a parapharyngeal thickening, with dislocation and compression of upper airways ; laboratory values were ht, 34.9 % ; hb, 12.3 g / dl ; rbc count, 3.81 e + 12/l. after 5 hours, a progressive worsening of the general condition led to immediate intubation. laboratory results in the emergency room were ht, 28.2% ; hb, 9.9 g / dl ; rbc count, 3.06 e + 12/l ; prothrombin time, 1.20 inr, activated partial thromboplastin time, 1.18 and fibrinogen, 452 mg / dl. the patient was sedated with midazolam and diprivan and was given amoxicillin vial gm, 1 fl every 8 hour a day and methylprednisolone vial 125 : 1 vial 3/die. the same night, a cat scan showed a wider extension of the haematoma on the upper and lower airways, together with a decrease in the diameter of the parapharyngeal space [figure 1a and b ]. the patient underwent a bilateral carotid arteriography [figure 2 ], and a slight bleeding from the palatine branches of the left ascending pharyngeal artery which was probably closed by the arterial spasm induced by the catheter was revealed. other cat slices did n't show a significant difference in comparison with the first cat. haematoma on the upper and lower airways the patient underwent a bilateral carotid arteriography on the second postoperative day, the patient 's general condition improved with a progressive normalization of laboratory tests values. on the fourth postoperative day, total recovery from haematoma was achieved within two months and the final clinical check showed a healthy appearance with good occlusion. le fort i osteotomy is a standard orthognatic surgery procedure used for the correction of dentofacial deformities. it was first described by vom lagenbeck in 1859 and it was used for the first time in 1927 by wassmund. axhasen in 1934 and schuchardt in 1942 described the use of an osteotome to separate the pterygomaxillary suture. bell studied the biological basis for maxillary osteotomy, bone healing and revascularization after corticotomy. it is of great importance to understand the mechanism of the complications in order to minimize the potential risks. vessel injury may occur when the maxillary tuberosity is separated from the pterygoid plates with an osteotome, or during the downfracture procedure. precious reported that pterygomaxillary separation can be achieved safely and easily by leverage alone without the use of a pterygoid chisel. he described the use of tessier 's spreaders, together with digital manipulation to achieve pterygomaxillary separation followed by maxillary mobilization. smith 's 3-prong spreaders, turvey 's maxillary expanders, and modified mobilisation forceps are also used to aid leverage. in le fort i osteotomy, most often, a haemorrhage arises from the branches of i m a.. collaterals of the i m a include the posterior, superior, alveolar, infraorbital, greater palatine, lesser palatine, ascending pharyngeal, vidian, and sphenopalatine. the descending palatine artery may be a common source of bleeding during and after le fort i osteotomy because of its anatomic location in the posteromedial wall of the maxillary sinus. arterial haemorrhage tends to be more persistent and can be recurrent, which makes it more difficult to manage. bleeding can occur intraoperatively during the osteotomy, but also within the first 2 weeks following surgery and generally presenting as epistaxis. in some cases, even months after the procedure, there can be a delayed presentation of pseudoaneurysm. in order to investigate the major cause of bleeding after a le fort osteotomy a cat scan is useful to define the newly established bone anatomy, the location of the pathology, and the vessel relationships to the fracture line. a cat scan with contrast agents provides faster, easily detectable and interesting clues for the type of pathology of the affected side. treatment modalities used to arrest postoperative haemorrhage include nasal packing, packing of the maxillary antrum, reoperating with clipping or electrocoagulation of the bleeding vessels, the use of topical haemostatic agents in the pterygomaxillary region and selective embolization of the maxillary artery and its terminal branches. according to literature, different embolization materials have been used with various degrees of success for pseudoaneurysm treatment : gelfoam, gianturco coils, stainless steel coils with gelatin or dacron fibres, guide wires, detachable balloons, n - butyl cyanoacrylate, autologous clot, polivinil alcohol, complex platinum coils, guglielmi detachable coils. concerns about the risk of haemorrhage as a result of pterygomaxillary separation led to the development of an osteotomy technique anterior to the pterygomaxillary suture. rohner described an endoscopically assisted le fort i osteotomy to ensure preservation of the descending palatine artery. ueki. recently reported the use of an ultrasonic bone curette to mobilize the pterygoid process, thus avoiding the damage to the descending palatine vessels. for the authors, the ultrasonic bone curette offers a safe procedure for performing pterygoid process fractures, without damaging the surrounding tissues. this technique allows surgeons to perform procedures in the pterygomaxillary area to safely mobilize the pterygoid process. during the intraoperative phase a straight follow - up of the patients is important in cases who may show vascular hypersensivity, able to cause a vessel spasm release the day after the operation. new trends for ambulatory surgery reduce costs for patients but can increase medical responsibility. for legal reasons, even if it is necessary to mention typical complications during preoperative counselling, it is ideal to follow up on patients with standard checkups. not only should the patient be informed about the frequency of complications, but he should also be told about its implications in later life. once a frequent complication, haemorrhage has become rare when the surgical technique is handled by an experienced orthognatic surgeon. sometimes, despite preoperative planning and a careful surgical technique it is important to check immediate or delayed postoperative bleeding also, using a protocol that can take care of day surgery patients. an increased knowledge of the basic biology of the le fort i osteotomy, the development of instruments specially designed for this le fort i procedure and the use of hypotensive anaesthesia have dramatically reduced the morbidity and duration of this procedure. | although the le fort i osteotomy is a safe surgical technique, many complications have been reported. we present a case of an extended cervico - facial haematoma due to delayed bleeding from the terminal branches of the maxillary artery after orthognatic surgery. a 23-year - old man was referred to our observation for the surgical correction of a class iii asymmetric malocclusion. the patient underwent a le fort i osteotomy, with impaction of the maxilla, associated with an epker mandibular bilateral sagittal split osteotomy, with maxillary advancement and rigid internal fixation of the mandible with four miniplates and another four for the upper maxilla as well. the first post - surgery day, the patient developed a gradual dispnea together with neck swelling. by second postoperative day, the patient 's general condition improved with a progressive normalization of laboratory tests values. the computerised axial tomography (cat) scan confirmed a decrease in the parapharyngeal thickening. total recovery was achieved within two months, the final clinical check showed a healthy appearance with good occlusion. an increased knowledge of the basic biology of the le fort i osteotomy, the development of instruments specially designed for the le fort i procedure and the use of hypotensive anaesthesia could reduce the morbidity and duration of this procedure. |
this article is a summary of our experience with the treatment of a patient with an extraordinarily large deep burn (99.5% tbsa and 23% fourth degree burn) by using the microskin autografting and alloskin repeated grafting method to close the deep burn wound because of scarcity of skin sources of the patient. the patient has been observed for 2 years, and is able to face the reality of life peacefully with the support of his family. the salvage rate of large burns has improved steadily in the past 10 years in china. however, in patients with a scarcity of skin sources, treatment of large fourth degree burns remains a great clinical challenge. for extensive severe burn wounds repair, there are 3 major solutions : meek skin grafting, chinese - originated micro - skin autografting, and transplantation of cultured epidermal cells. meek skin grafting can expand the donor skin by 39 times, and micro - skin autografting can do this 1015 times larger. therefore, micro - skin autografting is more suitable for application to severely burned patients who have a scarcity of skin sources. autologous transplantation of cultured keratinocytes can provide adequate skin sources for wound closure, but the long cultivation time, high cost and rigorous technology requirements limit its application in developing countries. even though the patient is saved, the patient s quality of life remains an increasing concern, both on the part of clinicians and society. this article is a summary of our experience with the treatment of a patient with an extraordinarily large deep burn (99.5% tbsa and 23% fourth degree burn) by using the microskin autografting and alloskin repeated grafting method to close the wound because of scarcity of skin sources. the patient was observed for 2 years, and the quality of life is evaluated. the patient is a 29-year - old male who was seriously burned by molten steel (about 1,500c) from the top of the head throughout the body, in an accidental furnace explosion. thirty hours after fluid resuscitation in a local hospital, he was referred to our hospital, when the patient was conscious and the vital signs were relatively stable. except for a small piece of unburned skin on the posterior head measuring about 0.5% tbsa, the rest of the scalp and the face suffered deep second degree burns, and the trunk and the 4 extremities were full of black eschar. when the 4 extremities were incised to reduce tension, muscular eversion and necrosis were seen in part of the median sides of the upper limbs and thighs, and lateral sides of the calves, the left arm being the most serious. the fingers of both hands and toes of both feet were carbonized, presenting as branch - like dry necrosis (figure 1). a diagnosis was made of 99.5% tbsa burn wound, 5.5% deep second degree, 71% third degree and 23% fourth degree, and left eye burn with corneal perforation. fluid resuscitation, anti - infection therapy, mechanical ventilation, and maintenance of internal environmental stability were continued on admission to protect organ functions. the patient overcame the shock phase in stable condition. on the 3 day after the injury, escharectomy of the 4 extremities and trunk was performed, involving 65% tbsa, and the wound was covered with nitrogen - preserved alloskin. as there was only 0.5% tbsa normal scalp available, we used the microskin autografting and alloskin repeated grafting method to repair the wound as soon as possible for the sake of preventing infection, which included using alloskin to cover and protect the wound temporarily, and repeated microskin grafting to sequentially close the wound. from the 5 day after the injury, the well preserved scalp skin was dissected 5 times for microskin grafting of the right thigh, right upper arm, left thigh, anterior trunk and posterior trunk, closing about 10% tbsa wound. the scalp skin was taken 6 times for stamp - like autoskin grafting to close the residual wound surfaces. the entire course of treatment lasted 120 days, during which 14 operations were done. most of the wound was healed and repaired (table 1). as the 4 extremities of the patient sustained large areas of fourth degree burns with muscular necrosis and liquification, infection was likely to occur. in addition, myoglobin produced by decomposition of the necrosed muscle was liable to impair renal function and seriously threaten the patient s life. on the basis of careful intra - operative observation, those definitely necrosed tissues were resected without delay by incising the deep fascia and sarcolemma, and the exposed deep tissues were covered with alloskin. as the muscle of the left upper arm was seriously necrosed, amputation was performed on the 10 day after the injury. as large amounts of muscle of both calves were necrosed, the tibia was exposed after clearing the necrosed tissue. as we were not able to cover it with flaps, holes were drilled on the exposed tibia to penetrate the cortex and reach the medulla for the sake of promoting granulation formation in the spaces created by the holes. when the formed granulation tissue covered the exposed tibia in about 4 months, stamp - like autoskin grafting was performed to close the wound of the right calf (figure 3). although the left tibia was covered by granulation tissue, bone marrow infection occurred in the long course of dressing changes. in addition, the left ankle completely lost its function, and the left foot was deformed due to scar contraction. the lower leg and 1/3 of the upper leg had to be amputated. the fingers and toes of the patient were also affected by deep burns, for which exposure therapy was used to keep the eschar dry. in about 3 months the eschar was lysed and fell off. the scars were red and hard, and softened gradually over the course of 2 years. scars in the perineum and right armpit were adhered due to contraction, for which scar dissolution was performed, and acellular dermal matrix and auto scalp skin were grafted. the condition was ameliorated to some extent, but the patient was still handicapped by severe dysfunction, presenting as right finger defection and palm contraction, so that the patient lost the grasping, holding and lifting functions. in addition, further plastic surgery was also difficult for him because there was no supply of quality skin sources from his body. in the course of treatment, though perceiving the magnitude of his disease, the patient held fast to hope of survival and showed gratitude to the medical staff. currently, there is no obvious functional damage to the patient s heart, lung, liver, kidney and other organs. particularly, our patient showed no sign altered thinking, language skills and audition between pre - injury and now. unfortunately, due to amputation and scar contraction, our patient lost his ability to walk and to hold objects, among other survival skills. in addition, his left eye suffered severe vision impairment, and post - traumatic stress syndrome sometimes troubled him, such as depression and nightmares. however, with the support of his family, he preserved the will to live and face reality. he actively listened to the daily news, watched entertainment tv programs, and showed enthusiasm for life. this was a rare case of burn injury involving 99.5% of tbsa, including 23% tbas fourth degree burn. the key to successful treatment of such patients depends on correct management and timely closure of the wound. for repair of large burns, most developed western countries use the meek technique of skin expansion, or culture and transplantation of human auto epidermal cells. although the meek technique has unique advantages of mild scar formation and good functional recovery, expansion of an area of skin is limited, usually to 69 times greater, which may be insufficient for patients with a scarcity of skin sources. epidermal cell culture is able to produce large areas of cell membranes in vitro in 34 weeks, but survival of the transplanted cell membranes is unstable. in addition, serious scar formation and high cost are problems that limit its routine use in developing countries. knowing that our patient was seriously short of endogenous skin sources, and that the above method was inapplicable to the patient, we made full use of the only remaining 0.5% tbsa normal scalp by means of the microskin autografting and alloskin repeated grafting method to repair the wound. the scalp served as a constant skin source at a mean interval of 10 days. skin cutisection should not be too deep, otherwise healing of the donor area may be affected. microskin grafting is a skin transplantation technique of chinese origin, and contributes much to maintaining china s first - class success rate in curing patients with large burns. in the present case, we used the 0.5%tbsa scalp to expand the skin by 1020 times and close the 510% tbsa wound. most of the wounds were closed by 5 episodes of microskin grafting, and the rest were treated by autoskin stamp grafts. wounds that were not covered with autoskin were covered with alloskin to reduce exudation and evaporation, and to prevent infection. this not only provided favorable conditions for autoskin grafting, but also benefited improvement of the general condition and maintenance of organ functions of the patient. rejection reaction developed 2030 days after alloskin coverage, for which the alloskin was replaced in time to prevent the wound from exposing. although the course of treatment of the patient was 4 months, no significant wound infection, disturbance of the internal environment and organ dysfunction occurred. this should be primarily attributed to timely and effective closure of the wound by use of autoskin and alloskin techniques. management of fourth degree burns is a huge clinical challenge. for small fourth degree burn areas, flap repair is a routine solution, but in this patient, the fourth degree burn area was large and the other areas were also deeply burned, so flap repair was inapplicable. after repeated debridement, we used stamp - like autoskin grafts to close the wound after granulation tissue formation. the disadvantage was that it needed multiple operations performed over a long period of time. the quality of life of burn patients has long been a great concern both on the part of clinicians and society. a survey of delegates at the 39 annual meeting of the british burns association showed that there are no conclusive criteria for abandonment of treatment of severe burn patients. quality of life of burn patients is related not only to the severity of burn, but is also significantly related to educational backgrounds and family and social support. in china, treatment of severe burn patients for example, we have successfully saved many patients with greater than 80% tbsa burns, and most of them can take care of themselves and live a high - quality life. on admission of this rare case of severe burn, the doctors were fully aware of problems that the case might entail even though it was healed, such as deformity due to scar contraction, amputation, and loss of daily living abilities. although the patient experienced severe disability and dysfunction after healing, he is able to keep calm and face reality peacefully with the support, companionship and attention of his family. | summarybackgroundtreatment of extraordinarily large deep burns remains a huge clinical challenge.case reportthis article is a summary of our experience with the treatment of a patient with an extraordinarily large deep burn (99.5% tbsa and 23% fourth degree burn) by using the microskin autografting and alloskin repeated grafting method to close the deep burn wound because of scarcity of skin sources of the patient.conclusionsthe patient has been observed for 2 years, and is able to face the reality of life peacefully with the support of his family. |
tanzania has experienced tremendous social changes and this has affected the health status of vulnerable populations over the last few years (mujinja and kida 2014). rural women especially have been placed in a worse health - care position compared with urban women (tanzania gender networking program and macro 2007). since the 1990s, despite the fact that free medical services are provided to mothers and children, indicators such as nutritional status of mothers, anc attendance and facility - based delivery have not correspondingly improved (national bureau of statistics tanzania and icf macro 2000 ; maletnlema 2002 ; abubakar. the situation is worse in rural tanzania because of poor sociocultural, physical and financial accessibility (kruk. tanzania has a network of health facilities at national, regional, district, divisional, ward and village level enabling > 90% of population to be within 10 km of a health facility (mella 2003). overall 60% of health services are provided by public sector and the government exempts the poor and vulnerable groups from user - fees in essential maternal and child health services (ministry of health 2003). in urban areas, health - care services is more easily accessible than in rural areas due to available and reliable transportation and higher coverage of health facilities (pfeiffer and mwaipopo 2013). maternal mortality is one of the key indicators of women s health status. despite the global efforts to improve maternal health, the mortality rate is still unacceptably high in tanzania. according to a world health organization (2012) report, the maternal mortality ratio in 2010 was 460 per 100 000 live births and annually, around 8500 women die from pregnancy - related causes. because most of the maternal deaths occur during and immediately after childbirth (abouzahr 1998), delivery in a health facility has been suggested as the key component for prevention of pregnancy - related death. there are four groups of determinants of delivery service use : (1) sociocultural, (2) perceived need, (3) economic accessibility and (4) physical accessibility (gabrysch and campbell 2009). these factors are found to influence use of delivery care at community and individual level (gage and guirlne calixte 2006 ; asp. 2014). among individual factors, attending anc visits at least two or three times is known to be a strong predictor of facility delivery along with the mother s age, parity, her education and that of her partner and frequent media exposure (amooti - kaguna and nuwaha 2000 ; simkhada. 2008 ; gabrysch and campbell 2009 ; anyait. 2012 ; pervin. according to previous reports, more than three anc visits is related to an higher probability of subsequent facility delivery compared with only two anc visits and this relationship is affected by various factors across the different geographical regions (pervin. is explained by factors at the area level (gage 2007), this persistently steady change in the facility - based delivery could be explained better with regional or systemic factors such as a regional - anc rate. generally, at least four anc visits are recommended for safe motherhood (world health organization, department of making pregnancy safer 2006). most of the policies to promote the mother s health have focused on improving economic and physical accessibility to increase the number of anc visits at least four times (olsen. 2005 ; finlayson and downe 2013). in 2002, tanzania s ministry of health and social welfare implemented focused antenatal care (fanc) model from world health organization. the principle of this model was to integrate anc through health promotion, disease prevention, detection and treatment of diseases and birth preparedness (von both. the fanc had been expected to serve as a mechanism for increasing facility - based deliveries (kearns. 2014). during the past two decades, however, the percentage of women who had four or more anc visits has declined, regardless of whether they are from urban or rural areas despite all the efforts made to encourage them (national bureau of statistics tanzania and icf macro 2011). nevertheless, the prevalence of facility delivery has remained around 4050% with little change (tanzania gender networking program and macro 2007). these findings suggest four times of anc visits would not always associate with higher probability of facility - based delivery. considering the variations in reasons women give for delivering at home depending on the different settings (kitui. 2013), the relationship between the number of anc visits and facility - based delivery needs to be explored (exavery. rural differentials have been concealed by aggregate figures. according to the tanzania demographic and health survey (tdhs) 200405, 2 or 4 and those delivered in health facility, nationwide (tdhs 19912010). proportion of women with anc visits > 2 or 4 and those delivered in health facility, nationwide (tdhs 19912010). table 1 summarizes key indicators according to different periods of the survey in urban and rural areas. the mean age, number of live births were higher in rural than urban area in all timeframes. on the other hand, percentage of media exposure, secondary education and facility delivery of women who gave birth within 5 years of the survey, as well as the proportion of nulliparous women, were consistently higher in urban than in rural areas throughout the study period. when the percentages of specific numbers of anc visits were compared, the proportion of women who had two, three or more anc visits has been higher in rural than in urban areas since 1996. for the percentage of anc 4, there were no significant differences between urban and rural areas for time points 14. since 2004, some rural areas showed a higher proportion of women who had at least four anc visits than in urban districts ; this was the case in dar es salaam (63.8 in rural vs 55.5% in urban), dodoma (56.3 in rural vs 47.4% in urban) and town west (55.7 in rural vs 64.1% in urban ; data of each district is not shown). table 1.comparison of maternal age, number of children, percentage of anc, media exposure, education, nulliparity and facility delivery, urban and rural areaperiod regional indicatorstime 1 (199192)ptime 2 (1996)ptime 3 (1999)ptime 4 (200405)ptime 5 (200910)purbanruralurbanruralurbanruralurbanruralurbanruralmaternal age (years)26.5 0.327.8 0.1 2 or 4 and those delivered in health facility, urban and rural area (tdhs 19912010). table 3.adjusted risk ratio of facility - based delivery according to percentage of women who had > 14 anc visits in three models, urban and rural area (tdhs, 19912010)percentage of women in the areaurbanruralmodel 1model 2model 3model 1model 2model 3anc 11.121.101.120.930.930.93anc 21.141.131.140.920.920.92anc 31.221.111.110.940.920.92anc 41.051.05 1.05 0.930.940.94 p 14 anc by 10%. all risk ratios were adjusted for covariates (time points, mean maternal age, mean number of total live birth, secondary education of women and partner, proportion of media exposure and nulliparous women). proportion of women with anc visits > 2 or 4 and those delivered in health facility, urban and rural area (tdhs 19912010). adjusted risk ratio of facility - based delivery according to percentage of women who had > 14 anc visits in three models, urban and rural area (tdhs, 19912010) p 14 anc by 10%. all risk ratios were adjusted for covariates (time points, mean maternal age, mean number of total live birth, secondary education of women and partner, proportion of media exposure and nulliparous women). for the exploration of the reasons for this gap between anc and facility delivery, the responses to the question why did nt you deliver in a health facility? in tdhs 200910 were analysed (table 4). among the 5244 women who had anc visits at least once during their latest pregnancy in 200910, 2388 (45.5 %) reported that they did not deliver the baby in a health facility. in both urban and rural areas, too far / no transportation was the most frequent reason. and not necessary, not customary and cost were the other major inhibitory factors. when the proportions were compared, rural women reported more partner and familial factors than urban women (p = 0.04). table 4.reasons of home delivery in women with anc visit more than once (n = 2388), urban and rural area (tdhs, 200910)q : why did nt you deliver in a health facility ? (multiple responses)% urban women (weighted n = 189)% rural women (weighted n = 2356)p valuehusband / family did not allow0.452.690.04facility not open2.751.890.54cost too much7.907.840.98too far / no transportation33.1742.930.09no female provider at the facility0.001.410.28not necessary15.8919.360.36dont trust facility / poor quality service1.831.570.83not customary5.969.470.26pearson s chi - squared test with yates continuity correction. reasons of home delivery in women with anc visit more than once (n = 2388), urban and rural area (tdhs, 200910) pearson s chi - squared test with yates continuity correction. this study used the dhs data for tanzania from the united states agency for international development from five time points. tdhs is a nationally representative survey of over 10 000 households selected from sample points. the sampling design and survey method of this national representative survey are described elsewhere (canavan. individual women s self - reported data were also included to calculate the regional data of each time dimension. to obtain the most recent information, only the women who delivered their babies within 5 years of the survey were included for the analysis. of those who had more than one delivery in the last 5 years therefore, the values of several indicators in this study could be different from those of official dhs reports, which include all reported pregnancy cases. to explore the association between regional factors and longitudinal changes in facility delivery rate, the regional mean level of indicators was used as panel data. because the set of observations is not from identical individuals across all waves, this kind of data aggregation over a time period has been referred to as repeated cross - sectional (rcs) or pseudo - panel data (lebo and weber 2014). this rcs design was also used in a recent study on the relationship between sugar and occurrence of population - level diabetes (basu. there are five time points according to the year of survey : time point 1 covers dhs data from 1991 to 1992 ; 1996 is time point 2 ; 1999 is time point 3 ; time point 4 is 200405 and time point 5 is 200910 according to the survey year. the survey is conducted every 5 years taking 35 months at each time period (national bureau of statistics tanzania and icf macro 2000, 2005, 2011). key variables were included in reference to the results of previous studies and availability of the data. the mean age of the women, mean number of total live births, percentage of women and partners who had completed secondary education, proportion of number of anc visits made, proportion of women who were exposed to mass media and the percentage of nulliparous women were calculated by region. those women who read newspapers or listened to radio or watched television at least once a week were assumed to be exposed to mass media. because the variables for level of household wealth and type (public / private and health centre / dispensary) of anc providers were only available in time points 4 and 5, these factors could not be considered in the analyses. finally, responses to the question why did nt you deliver in a health facility?, which was only included in tdhs 200910, were analysed to explore the reasons why some women who had visited for anc at least once gave birth to their babies at home. because the study utilizes only publicly available, anonymous data, the institutional review was not required in our institution. the calculation and comparison of each variable between urban and rural areas was done using weighted values. because the regional rate of facility delivery is not normally distributed and the mean value is relatively large, the log - transformed value was used in the analysis. regional factors related with facility - based delivery rate were analysed in urban and rural areas separately. linear mixed model was used in exploring the association between proportion of anc visits (14 times) and regional facility - based delivery rates to consider the correlation structure within and between the repeated regional data of facility delivery in different time points. model 1 indicates the random coefficient model without exploring the error ; model 2 explores the correlation structures within the regional facility delivery rate and model 3 contains the covariance between and within the regional facility delivery rate. regional - level covariates were applied in the multivariable models to identify the adjusted effect of proportions of anc 2. the akaike information criteria (aic) of each linear mixed model were calculated by the method recommended by diggle and wolfinger using the restricted maximum likelihood for variance covariance structure selection (wolfinger 1996). the analyses were done using sas software, version 9.3 (sas institute inc., cary, nc). a total of 30 830 women who delivered their babies within 5 years at the time of survey in up to 52 districts were included for the analysis. between 1991 and 2010, the national proportion of women who had more than four anc visits during their last pregnancy had decreased from 56.1% to 33.3% as shown in figure 1. in the same period, there was a decreasing trend in facility delivery followed by a relatively modest increase in the latter timeframe (50.3% in 199192, 38.4% in 1999 and 45.1% in 200910). figure 1.proportion of women with anc visits > 2 or 4 and those delivered in health facility, nationwide (tdhs 19912010). proportion of women with anc visits > 2 or 4 and those delivered in health facility, nationwide (tdhs 19912010). table 1 summarizes key indicators according to different periods of the survey in urban and rural areas. the mean age, number of live births were higher in rural than urban area in all timeframes. on the other hand, percentage of media exposure, secondary education and facility delivery of women who gave birth within 5 years of the survey, as well as the proportion of nulliparous women, were consistently higher in urban than in rural areas throughout the study period. when the percentages of specific numbers of anc visits were compared, the proportion of women who had two, three or more anc visits has been higher in rural than in urban areas since 1996. for the percentage of anc 4, there were no significant differences between urban and rural areas for time points 14. since 2004, some rural areas showed a higher proportion of women who had at least four anc visits than in urban districts ; this was the case in dar es salaam (63.8 in rural vs 55.5% in urban), dodoma (56.3 in rural vs 47.4% in urban) and town west (55.7 in rural vs 64.1% in urban ; data of each district is not shown). table 1.comparison of maternal age, number of children, percentage of anc, media exposure, education, nulliparity and facility delivery, urban and rural areaperiod regional indicatorstime 1 (199192)ptime 2 (1996)ptime 3 (1999)ptime 4 (200405)ptime 5 (200910)purbanruralurbanruralurbanruralurbanruralurbanruralmaternal age (years)26.5 0.327.8 0.1 2 or 4 and those delivered in health facility, urban and rural area (tdhs 19912010). table 3.adjusted risk ratio of facility - based delivery according to percentage of women who had > 14 anc visits in three models, urban and rural area (tdhs, 19912010)percentage of women in the areaurbanruralmodel 1model 2model 3model 1model 2model 3anc 11.121.101.120.930.930.93anc 21.141.131.140.920.920.92anc 31.221.111.110.940.920.92anc 41.051.05 1.05 0.930.940.94 p 14 anc by 10%. all risk ratios were adjusted for covariates (time points, mean maternal age, mean number of total live birth, secondary education of women and partner, proportion of media exposure and nulliparous women). proportion of women with anc visits > 2 or 4 and those delivered in health facility, urban and rural area (tdhs 19912010). adjusted risk ratio of facility - based delivery according to percentage of women who had > 14 anc visits in three models, urban and rural area (tdhs, 19912010) p 14 anc by 10%. all risk ratios were adjusted for covariates (time points, mean maternal age, mean number of total live birth, secondary education of women and partner, proportion of media exposure and nulliparous women). for the exploration of the reasons for this gap between anc and facility delivery, the responses to the question why did nt you deliver in a health facility? in tdhs 200910 were analysed (table 4). among the 5244 women who had anc visits at least once during their latest pregnancy in 200910, 2388 (45.5 %) reported that they did not deliver the baby in a health facility. in both urban and rural areas, too far / no transportation was the most frequent reason. and not necessary, not customary and cost were the other major inhibitory factors. when the proportions were compared, rural women reported more partner and familial factors than urban women (p = 0.04). table 4.reasons of home delivery in women with anc visit more than once (n = 2388), urban and rural area (tdhs, 200910)q : why did nt you deliver in a health facility ? (multiple responses)% urban women (weighted n = 189)% rural women (weighted n = 2356)p valuehusband / family did not allow0.452.690.04facility not open2.751.890.54cost too much7.907.840.98too far / no transportation33.1742.930.09no female provider at the facility0.001.410.28not necessary15.8919.360.36dont trust facility / poor quality service1.831.570.83not customary5.969.470.26pearson s chi - squared test with yates continuity correction. all the calculations were done with weighted value. reasons of home delivery in women with anc visit more than once (n = 2388), urban and rural area (tdhs, 200910) pearson s chi - squared test with yates continuity correction. the findings in this study support the hypothesis that four or more anc visits are not always associated with higher probability of facility - based delivery. increase of anc visits > 24 times was associated with higher facility delivery rate only in urban area. the proportion of at least four anc visits was not related to facility delivery rates at the district level in rural tanzania. this difference in the association between anc visits and facility - based delivery rate could be one of the systemic factors causing persistent urban rural disparity in facility - based delivery rate. contrary to the studies that have reported the positive relationship between anc visits and facility delivery, rockers. (2009) showed that a considerable proportion of women who had anc did not go to a health facility for delivery in rural tanzania. in addition, the declining trend in the rate of adequate anc visits was not accompanied by a corresponding change in facility delivery incidence. especially in rural areas, the percentages of women who had 14 anc visits in the region were found to have no significant contribution to facility - based delivery rates. the gap between those who had anc visits at least once and those who underwent facility - based delivery has been unexpectedly large nationally. more than 90% of women reported that they had visited a health facility (private or public) for anc at least once, irrespective of urban or rural residence (national bureau of statistics tanzania and icf macro 2011). the reason why some women who received anc from a skilled provider, but did not deliver their babies in a health facility, is not yet clear. limited evidence from tdhs 200910 shows several factors such as distance, cost and sociofamilial cultures as the leading cause of this gap. the findings of this study suggest that pregnant women in rural areas, who are found to have no abnormal findings during anc visits, might not be motivated enough to visit a health facility given the expense and trouble. according to a recent quantitative study conducted in rural northeastern tanzania, between 19 and 28% of women who had delivered in health facilities experienced disrespectful and/or abusive treatment from health providers during childbirth (kruk. generally positive attitude about anc in this country despite its poor quality might have affected on the high prevalence of anc visits < 4 times (mwifadhi. 2009 ; nyamtema. this quality of care issue could have been one of the major inhibitory factors to facility - based delivery in rural settings. because more than one - third (35.0%) of responses reported other reasons for home delivery than these, further study using qualitative methods would explain the differences between urban and rural areas. rural differentials in facility - based delivery could be beyond the problem of accessibility. in rahman s (2008) research, the partner s secondary education was a significant determinant in urban areas, while women s secondary education and whether they read newspapers were found to be significant factors in rural bangladesh. in a survey of rural uganda, mass media exposure has no significant association with birth preparedness and showed a similar result as our study (asp. (2013) reported that nulliparous women are more likely to deliver their babies in health facilities than multiparous women in rural tanzania. the proportion of nulliparous women was also found to be related with variation in regional - facility delivery rates over time in our rural data. this study suggests policies focusing on improving accessibility to anc to increase the number of anc visits could be insufficient to promote facility delivery in rural setting. in urban area, expanding the coverage of anc visits 2 times could be effective in increasing facility delivery rate. the barriers in the way from anc to facility delivery and different determinants between urban and rural area should be investigated and considered when the policies are decided and implemented. as a longitudinal study of data for the past two decades, this study has several limitations. the effects of education and media exposure could be associated with the level of household wealth which could not be included in the analyses. recent study in the three districts (urban and rural) of tanzania indicated women in wealthiest quintile were three times more likely to have had delivered at health facilities (exavery. on the other hand, survey research in rural area revealed that parity determines the odds of institutionalized delivery more than household poverty or education (ndao - brumblay. the association between socioeconomic position and facility childbirth was not significant in a rural district of kenya (nganjo phiri. household wealth data of further time points would enable us to measure the effect of longitudinal change of regional level of wealth. a study using pseudo - panel data such as this one has its own limitations of unmeasured commonalities of units within the time points (lebo and weber 2014). this study used data collapsed into regional mean values at five time points to avoid debatable individual - level analyses. in addition, a relatively small number of cases in the first wave could be another limitation. despite these weaknesses, as the first longitudinal study on the relationships between the number of anc visits and facility delivery using rcs data for two decades, the findings of this study may contribute to future investigation on the longstanding gap between anc visits and facility delivery. undertaking more than one, two, three or even four anc visits was not significantly associated with facility - based delivery in rural tanzania. different regional factors mediate the longitudinal changes in facility delivery rates between urban and rural areas. for more effective strategies to improve access to facility - based delivery and subsequent reduction of maternal mortality only publicly available, anonymous data was used, the institutional review was not required. | there is a known high disparity in access to perinatal care services between urban and rural areas in tanzania. this study analysed repeated cross - sectional (rcs) data from tanzania to explore the relationship between antenatal care (anc) visits, facility - based delivery and the reasons for home births in women who had made anc visits. we used data from rcs demographic and health surveys spanning 20 years and a cluster sample of 30 830 women from 52 districts of tanzania. the relationship between the number of anc visits (up to four) and facility delivery in the latest pregnancy was explored. regional changes in facility delivery and related variables over time in urban and rural areas were analysed using linear mixed models. to explore the disconnect between anc visits and facility deliveries, reasons for home delivery were analysed. in the analytic model with other regional - level covariates, a higher proportion of anc (> 24 visits) and exposure to media related to an increased facility delivery rate in urban areas. for rural women, there was no significant relationship between the number of visits and facility delivery rate. according to the fifth wave result (200910), the most frequent reason for home delivery was physical distance to facility, and a significantly higher proportion of rural women reported that they were not allowed to deliver in facility. the disconnect between anc visits and facility delivery in rural areas may be attributable to physical, cultural or familial barriers, and quality of care in health facilities. this suggests that improving access to anc may not be enough to motivate facility - based delivery, especially in rural areas. |
optical coherence tomography (oct) allows automated quantification of both peripapillary retinal nerve fiber layer (rnfl) and ganglion cell - inner plexiform layer (gcipl) macular thicknesses, providing information on the probability of an abnormality being present after comparison with an internal normative database. several known factors can affect these measurements such as age, ethnic background, refractive errors, optic disc area, and foveal - disc angle.16 recently, longer axial length has been significantly associated with an increased incidence of false - positives (fps) on ganglion cell analysis (gca) and rnfl maps after uni- and multivariate analyses.37 caution is recommended in moderate myopic individuals because a high percentage can be misclassified as abnormal by rnfl and gca cirrus oct maps.4 a study has reported that spectralis oct (heidelberg engineering, dossenheim, germany) might be more specific than cirrus (39% vs 18%) when evaluating rnfl thickness for caucasians and moderate myopic population.5 axial length, mean spherical equivalent, presence of peripapillary atrophy, and tilted disc were significantly related to the rnfl fp occurrence displayed by both the devices ; therefore, the degree of myopic optic disc tilt should be considered when interpreting the rnfl thickness measured by oct.5,710 clinical features of tilted disc overlap with other conditions, such as myopic disc and glaucoma, but the role of conventional oct imaging technologies for the optic nerve head (onh) is limited.4,5 currently, rim width measurement method uses the bruch s membrane opening (bmo) as the anatomical border of the rim, referenced to a bmo horizontal reference plane, termed as bmo - horizontal rim width (bmo - hrw).11 in contrast, the spectralis oct glaucoma module premium (gmp) edition provides a new, objective method of onh analysis using bmo, but the neuroretinal rim assessment is performed from the bmo to the nearest point on the internal limiting membrane (ilm), and this shortest distance measurement is referred to as bmo minimum rim width (bmo - mrw). this parameter considers the orientation of the rim tissue relative to the point of measurement, and the highly variable anatomy of the onh both within and between individuals, and quantifies the rim width perpendicular to the trajectory of axons. moreover, the new software provides an anatomic positioning system (aps) where acquisition of data is based on fovea - to - bmo - center axis, reducing the interindividual variation.1113 recently, a higher sensitivity of bmo - mrw compared with bmo - hrw methods has been reported.11 moreover, the structure function relationship was enhanced due to its geometrically accurate properties, indicating mrw as a new structural marker for the detection and risk profiling of glaucoma.11,14,15 the aims of the current study were to evaluate and to compare the rates of fp results in eyes with tilted optic disc regarding the color code classification of rnfl and bmo - mrw using the new gmp edition (spectralis heidelberg engineering, dossenheim, germany), as well as the macular gca by cirrus oct (carl zeiss meditec, dublin, ca, usa). this study is based on the bmo - mrw imaging study, an ongoing prospective study of patients with glaucoma and other optic neuropathies and healthy volunteers at the glaucoma and neurophthalmic department of ramon y cajal university hospital. the study protocol was approved by the ethics committee of hospital universitario ramn y cajal, and the study adhered to the tenets of the declaration of helsinki. after a discussion of the nature and purpose of the study, thirty healthy individuals with tilted disc aged 18 years without other ocular pathology were invited to participate and were recruited between july 2014 and february 2015. eligible participants underwent a thorough ophthalmic examination (gr) to confirm the lack of ocular pathology other than tilted optic disc. this examination included measurement of snellen visual acuity, noncycloplegic refraction using an autorefractor, intraocular pressure by applanation tonometry, axial length using partial laser interferometry (iol master ; carl zeiss meditec), slit - lamp biomicroscopy with a plus 90d lens, and automated visual field (vf) testing (humphrey field analyzer with swedish interactive thresholding algorithm standard 24 - 2 test program). vf was classified as normal when there was a mean deviation or a pattern standard deviation within the 95th percentile ; a normal glaucoma hemifield test and absence of a cluster of three or more non - edge points on the pattern deviation plot with a probability of occurring in 6.0 diopters (d) of spherical equivalent or 3.0 d of astigmatism, any history of ocular surgery, ocular disease, best corrected visual acuity as poor as 20/40, intraocular pressure (iop) 18 mmhg, past history of raised iop, evidence of increased or asymmetric cupping (interocular asymmetry 0.2), neuroretinal rim notching, or optic disc hemorrhages. digital fundus images were used to assess the disc characteristics, and all the optic discs were evaluated by a single experienced examiner (ac). tilted disc was defined as an index of tilt 25. to determine the frequency of fp results, the sector maps of spectralis for each participant s eye were examined. a yellow or red color - coded average thickness and a sector map with 1 yellow- or red - colored sectors were considered as abnormal in both rnfl and bmo - mrw classifications. the bmo area and this protocol performs 512 horizontal a - scans and 128 vertical b - scan lines within a 66 mm cube of acquired signal data centered on the fovea. the gcipl software algorithm automatically identifies the outer boundary of the rnfl and the outer boundary of the inner plexiform layer and measures macular gcipl thickness within an annulus with inner vertical and horizontal diameters of 1 and 1.2 mm, respectively, and outer vertical and horizontal diameters of 4 and 4.8 mm, respectively. the gca provides quantitative assessment of the overall average, minimum thickness (the lowest gcipl thickness over a single meridian crossing the annulus), and 6 sector areas (superotemporal, superior, superonasal, inferonasal, inferior, and inferotemporal). those that are abnormally decreased at the 5% and at the 1% level are represented by yellow and red backgrounds, respectively. to determine the fp rate and average and minimum thickness, the sector and deviation maps were all considered. two independent observers (gr and ac) identified the fp color codes on gca maps. an abnormal result on the cirrus gcl deviation map was defined and categorized according to kim s criteria.6 eyes with abnormal gca deviation maps were categorized into the following 3 groups based on the shape and location of abnormal gcipl color - coded area : group a (donut - shaped round color pattern around the inner annulus) ; group b (island - like isolated color pattern) ; and group c (diffuse and circular color pattern with an irregular inner margin in either or both hemifields).6 the frequency of fp results was calculated by the number of eyes with abnormal mrw, rnfl, or gca maps divided by the total number of eyes. for the overall fp rate for rnfl and mrw classifications, the number of eyes with 1 average and sector map with abnormal color codes was determined. the overall fp rate for gca maps, the number of eyes with 1 gca (average, minimum, sector, and deviation) maps with abnormal color codes was assessed. comparative analysis between the fp rates of each oct protocol was performed using mcnemar test. a generalized estimating equations (gee) model was used to compare demographic and clinical factors between the eyes with normal findings and eyes with abnormal results. variables assessed in this study included age, gender, eye side, spherical equivalent, axial length, presence of peripapillary atrophy, tilted index, bmo area, and fovea - to - bmo - center axis.1719 student s t - test for independent samples was used to compare age and ovality index between non / low myopia and moderate myopia. data were analyzed using statistical software spss version 20.0 (ibm corporation, armonk, ny, usa) and stata software version 12.0 (statacorp., college station, tx, usa). this study is based on the bmo - mrw imaging study, an ongoing prospective study of patients with glaucoma and other optic neuropathies and healthy volunteers at the glaucoma and neurophthalmic department of ramon y cajal university hospital. the study protocol was approved by the ethics committee of hospital universitario ramn y cajal, and the study adhered to the tenets of the declaration of helsinki. after a discussion of the nature and purpose of the study, thirty healthy individuals with tilted disc aged 18 years without other ocular pathology were invited to participate and were recruited between july 2014 and february 2015. eligible participants underwent a thorough ophthalmic examination (gr) to confirm the lack of ocular pathology other than tilted optic disc. this examination included measurement of snellen visual acuity, noncycloplegic refraction using an autorefractor, intraocular pressure by applanation tonometry, axial length using partial laser interferometry (iol master ; carl zeiss meditec), slit - lamp biomicroscopy with a plus 90d lens, and automated visual field (vf) testing (humphrey field analyzer with swedish interactive thresholding algorithm standard 24 - 2 test program). vf was classified as normal when there was a mean deviation or a pattern standard deviation within the 95th percentile ; a normal glaucoma hemifield test and absence of a cluster of three or more non - edge points on the pattern deviation plot with a probability of occurring in 6.0 diopters (d) of spherical equivalent or 3.0 d of astigmatism, any history of ocular surgery, ocular disease, best corrected visual acuity as poor as 20/40, intraocular pressure (iop) 18 mmhg, past history of raised iop, evidence of increased or asymmetric cupping (interocular asymmetry 0.2), neuroretinal rim notching, or optic disc hemorrhages. digital fundus images were used to assess the disc characteristics, and all the optic discs were evaluated by a single experienced examiner (ac). tilted disc was defined as an index of tilt 25. to determine the frequency of fp results, the sector maps of spectralis for each participant s eye were examined. a yellow or red color - coded average thickness and a sector map with 1 yellow- or red - colored sectors were considered as abnormal in both rnfl and bmo - mrw classifications. the bmo area and this protocol performs 512 horizontal a - scans and 128 vertical b - scan lines within a 66 mm cube of acquired signal data centered on the fovea. the gcipl software algorithm automatically identifies the outer boundary of the rnfl and the outer boundary of the inner plexiform layer and measures macular gcipl thickness within an annulus with inner vertical and horizontal diameters of 1 and 1.2 mm, respectively, and outer vertical and horizontal diameters of 4 and 4.8 mm, respectively. the gca provides quantitative assessment of the overall average, minimum thickness (the lowest gcipl thickness over a single meridian crossing the annulus), and 6 sector areas (superotemporal, superior, superonasal, inferonasal, inferior, and inferotemporal). those that are abnormally decreased at the 5% and at the 1% level are represented by yellow and red backgrounds, respectively. to determine the fp rate and average and minimum thickness, the sector and deviation maps were all considered. two independent observers (gr and ac) identified the fp color codes on gca maps. an abnormal result on the cirrus gcl deviation map was defined and categorized according to kim s criteria.6 eyes with abnormal gca deviation maps were categorized into the following 3 groups based on the shape and location of abnormal gcipl color - coded area : group a (donut - shaped round color pattern around the inner annulus) ; group b (island - like isolated color pattern) ; and group c (diffuse and circular color pattern with an irregular inner margin in either or both hemifields).6 the frequency of fp results was calculated by the number of eyes with abnormal mrw, rnfl, or gca maps divided by the total number of eyes. for the overall fp rate for rnfl and mrw classifications, the number of eyes with 1 average and sector map with abnormal color codes was determined. the overall fp rate for gca maps, the number of eyes with 1 gca (average, minimum, sector, and deviation) maps with abnormal color codes was assessed. comparative analysis between the fp rates of each oct protocol was performed using mcnemar test. a generalized estimating equations (gee) model was used to compare demographic and clinical factors between the eyes with normal findings and eyes with abnormal results. variables assessed in this study included age, gender, eye side, spherical equivalent, axial length, presence of peripapillary atrophy, tilted index, bmo area, and fovea - to - bmo - center axis.1719 student s t - test for independent samples was used to compare age and ovality index between non / low myopia and moderate myopia. data were analyzed using statistical software spss version 20.0 (ibm corporation, armonk, ny, usa) and stata software version 12.0 (statacorp., college station, tx, usa). this study included 50 eyes from 30 healthy participants ; 29 eyes had non / low myopia (2 d, range : + 2.13 to 2 d) and 21 had moderate myopia (2 d (0, 1, and 1.5 d) and one eye (25%) had 4.75 d. one case was observed in the superotemporal sector and 3 in the inferonasal sector. no eye was abnormal at the 2 d (0, 1, and 1.5 d) and one eye (25%) had 4.75 d. one case was observed in the superotemporal sector and 3 in the inferonasal sector. no eye was abnormal at the 6.0 d of spherical equivalent were excluded because bmo - mrw normative database just included subjects with refraction between + 6 and 6 d ; therefore, data for high myopia or more severe tilting can not be extrapolated of these findings. another possible source of bias is that the definition of healthy subjects was based on the vf parameters. some eyes with very early optic nerve abnormality might be misclassified, but this is an unavoidable limitation of this type of study, and it can only be answered with a longitudinal follow - up. furthermore, since only specificity was evaluated in the current study, it is not possible to make any meaningful conclusion about the overall diagnostic performance of the new bmo - derived parameters in tilted discs with and without glaucoma. these findings suggest that the new mrw classification might be more specific for caucasians and moderate healthy myopic population with tilted disc than rnfl thickness measurements despite to be calculated according to the fovea - to - bmo - center axis. furthermore, the new mrw map might be more specific than gca analysis provided by cirrus ; however, further studies with larger sample sizes are necessary. the results indicate that bmo - mrw thickness measurements had a higher overall specificity than peripapillary rnfl measurements by gmp - spectralis in eyes with tilted disc with low and moderate myopia, and it performed better than gca analysis by cirrus in eyes with tilted disc and moderate myopia. | background and objectiveto investigate and compare the false - positive (fp) diagnostic classification of the bruch s membrane opening minimum rim width (bmo - mrw) and retinal nerve fiber layer (rnfl) thickness in healthy eyes with tilted optic disc.materials and methodsfifty healthy eyes of 30 participants with tilted optic disc underwent bmo - mrw and rnfl scanning using spectralis and macular cirrus optical coherence tomography (oct) scans.resultsthe overall fp rate was significantly lower using bmo - mrw map compared with both rnfl map by spectralis (8% vs 62%, respectively, p<0.001) and ganglion cell analysis (gca) map by cirrus (8% vs 50%, respectively, p<0.001). specificity was significantly higher using bmo - mrw than rnfl in eyes with low (89.7% vs 41.4%, p<0.001) and moderate myopia (95.2% vs 33.3%, p<0.001).conclusionoct - derived bmo - mrw analysis provides significantly greater specificity than rnfl in tilted disc irrespectively of the refractive error, and it is more specific than gca analysis in tilted disc with moderate myopia. |
caffeine (1,3,7 trimethylxanthine) is one of the most widely used psychoactive drug in the world and consumed in various forms like tea, coffee, and colas. the half life of caffeine is 3 - 7 hours and its significant levels can be detected in the brain after 5 minutes of oral intake, with the peak levels reaching in about 30 minutes. caffeine 's main mechanism of action is by blocking the adenosine receptors and altering the levels of various neurotransmitters like dopamine, adrenaline, serotonin, and acetylcholine. other mechanisms like mobilization of calcium, inhibition of phosphodiesterases, and binding to benzodiazepine receptors have also been postulated.[46 ] studies have been done using questionnaires, electroencephalography, reaction time tests, and evoked potentials to elucidate the effects of caffeine on central nervous system (cns). hollingsworth as early as in 1912, reported that consumption of 65 - 130 mg caffeine increased typing speed, but a dose as high as 390 mg impaired the motor performance. demonstrated a decrease in the reaction time and error rate with increase in amplitude of event related evoked potential p3 following intake of caffeine. similar results have also been reported by dixit in 2006. in another study dixit have shown that caffeine intake changes transmission of impulses in the auditory pathway. besides physiological tests, neuropsychological tests have also been used to understand the influence of caffeine. one such task is the stroop task, first described by john ridley stroop in 1935. the stroop task is believed to be the gold standard of attentional measures aimed at studying the interference of a stimulus of one dimension with recognition of stimulus of another dimension. the conventional color word version task consists of words like blue, red, green and yellow written in another color or are incongruent (e.g., red is written with blue ink) or symbols like xxx in different colors or are neutral. the time taken to perform the task in the two conditions (incongruent and neutral) is recorded and the difference between the two represents interference. similarly the time difference between congruent (red written in red ink) and neutral is an indication of facilitation. foreman in 1989 evaluated the effect of caffeine on numerical version of stroop task by giving subjects no caffeine or 125 mg or 250 mg caffeine in decaffeinated coffee and found slower responses with a dose of 250 mg. however, there was no significant difference in the error rates of the three groups. hasenfratz and battig studied the effect of nicotine and caffeine on numerical stroop task and reported an improvement after ingestion of caffeine. but their subjects were in a state of nicotine deprivation (as they were smokers) and hence their results can not be attributed purely to caffeine. edwards. studied the effect of 125 mg and 250 mg caffeine on both classical and numerical stroop task and did not report any significant change in the performance in the two versions of the task. kenemans. evaluated the effect of a dose of 250 mg caffeine on stroop task. they used two versions of the stroop task : one in which the subjects had to indicate the numerosity of digits and the second was a color - word task. they found a decrease in the error rate in the first task along with a decrease in the reaction time after caffeine consumption. in the color - word task, it is thus clear that there is conflicting evidence with regards to the effect of caffeine on the classical color - word stroop task. this study evaluated the effect of caffeine on classical color word stroop task in order to have further insight into attentional processes. thirty healthy male medical student volunteers in the age group of 18 - 25 years (mean age 20.211.32 years) were recruited after explaining the procedure and taking written consent. the subjects were asked to refrain from caffeine or any other stimulant intake for at least 12 hours prior to the study. they were asked to report to the lab at 9 a.m on the day of testing after having proper night sleep. the exclusion criterion for subjects were : history of medical illness especially neurological diseaseshistory of smoking, alcohol or any other drug consumptionsubjects on any medications during last two weeks. history of medical illness especially neurological diseases history of smoking, alcohol or any other drug consumption subjects on any medications during last two weeks. color- word stroop task : the stroop task was administered using psych / lab for windows. each block had three conditions each of which had 72 trials : neutral condition- xxxx were printed in red, green, blue and yellow colorsincongruent condition- red, green, blue, and yellow were printed in different ink colorcongruent condition- red, green, blue, and yellow were printed in same ink color as that indicated by the word neutral condition- xxxx were printed in red, green, blue and yellow colors incongruent condition- red, green, blue, and yellow were printed in different ink color congruent condition- red, green, blue, and yellow were printed in same ink color as that indicated by the word the subjects in all the three conditions had to respond to the color of the text. the response buttons to be pressed for the color were : z for red, x for green,. for blue and after the first session, the subjects were given caffeine (caffeine pure from loba chemie pvt. ltd.) in a dose of 3mg / kg body weight along with milk powder and sugar in water. the data obtained was analyzed by 2 within factor repeated measures anova using spss 17 followed by tukey 's test. the data obtained was analyzed by 2 within factor repeated measures anova using spss 17 followed by tukey 's test. the analysis of data revealed no significant difference in the reaction time for the three blocks, thereby showing that there was no effect of practice on the stroop task. within a block, there was a significant difference between the three conditions (p<0.001) showing the presence of interference and facilitation effects. also, there was a significant decrease (p<0.001) in the reaction time in all the three conditions after caffeine intake showing that there was modulation of the attentional processes by caffeine. reaction time (in ms) before and after caffeine ingestion correct responses before and after caffeine ingestion the number of correct responses failed to show any significant change from one block to another or one condition to another or before and after caffeine intake. the absolute decrease in interference after caffeine intake was 24.898.71 ms and the increase in facilitation was 8.594.32 ms. the present study evaluated the effect of caffeine, a known cns stimulant on the color - word stroop task. there was a decrease in interference with an increase in facilitation as evident by the decrease in the reaction time after caffeine ingestion. however, there was no effect of practice on the performance of stroop task. our findings of no change in performance due to practice were similar to that of shor. and in contrast to that of edwards., edwards. had given only 10 stimuli, whereas in this study an entire block consisting of 72 trials for each condition was given. hence, it is possible that the subjects had habituated to the stimuli and hence practice had no effect on performance. the fact that there was interference suggests that there was conflict in the attentional resources for processing of the two dimensions, i.e., naming of word and naming of color. when the word and the ink color were same (congruent), there was faster processing of the two dimensions, an indication of facilitation. however, studies done by lyvers. and deslandes. did not find any significant effect of caffeine on stroop task the change in interference in our study was similar to that reported by hasenfratz and batting and kenemans. but the difference between their study and ours was that they had smokers as their subjects who were in a state of nicotine deprivation when the testing was done and also they had used numerical stroop task and not the color word task. kenemans in their study performed two experiments to evaluate the effect of caffeine. in the first experiment they reported no consistent effects of caffeine on reaction time along with a significant decrease in error rate following caffeine administration and suggested that caffeine led to suppression of irrelevant information. in their second experiment, they reported a decrease in reaction time with significant decrease in interference effects following caffeine consumption. however, there was no significant reduction in error rates. based on their experiments, kenemans hogervorst. in their study found that caffeine 's effect was more on complex task reaction time than on baseline test reaction time. it has been suggested that subjects tend to develop a strategy for responding when the stimuli are presented in blocks. kenemans. in their study tried to address this issue by using both blocked and mixed conditions and found no evidence to suggest that the changes in reaction time by caffeine had some contribution from strategy to respond developed by the subjects. we believe that even if the subjects had adopted some strategy for the blocks, the same strategy was being used before and after caffeine intake and thus would have been common to both states. our study found no significant differences in the number of correct responses before and after caffeine consumption. it can be reasonably argued that caffeine was not altering the suppression of irrelevant information, because if that had been the case, the number of correct responses would have changed following caffeine intake. rather, caffeine, promotes faster processing of relevant information along neuronal pathways as evident from the significant reduction in reaction time in the three conditions. studies using evoked potentials have reported that caffeine led to faster information processing and increased arousal levels. caffeine at doses comparable to daily consumption acts via blocking adenosisne receptors which have widespread distribution in the brain.[2527 ] adenosine is formed by action of amp selective 5 nucleotidase and acts as a general cns depressant. leung. demonstrated changes in anterior cingulate, insula, frontal, parietal, and mid temporal regions by incongruent stimuli. thus, it is evident that a number of networks between various areas of brain are functional in the stroop task. we hypothesize that caffeine by altering the levels of neurotransmitters leads to processing of relevant information in the classical color word stoop task. | background : caffeine is a pyschostimulant present in various beverages and known to alter alertness and performance by acting on the central nervous system. its effects on central nervous system have been studied using eeg, evoked potentials, fmri, and neuropsychological tests. the stroop task is a widely used tool in psychophysiology to understand the attention processes and is based on the principle that processing of two different kinds of information (like the word or colour) is parallel and at different speeds with a common response channel.aim:to study the effect of caffeine on classical color word stroop task.materials and methods : this study was conducted on 30 male undergraduate students by performing a test before and 40 minutes after consuming 3 mg / kg caffeine and evaluating the effect of caffeine on stroop interference and facilitation.results:the results revealed that practice has no effect on the performance in a stroop task. however, there was reduction in stroop interference and increase in facilitation after consumption of caffeine as was evident by changes in the reaction times in response to neutral, incongruent, and congruent stimuli.conclusion:we hypothesize that caffeine led to faster processing of relevant information. |
ubiquitination of specific cellular proteins serves as a signal for protein degradation, chromatin remodeling, dna repair, vesicular transport, and changes in protein localization and/or activity depending on the number and structure of the ubiquitin modification. protein ubiquitination is highly regulated and requires a cascade of enzymes that culminates in a substrate and site - specific modification. similarly, deubiquitinating enzymes (dubs) that remove ubiquitin (or ubiquitin - like modifiers like sumo or nedd8) from substrate proteins to allow recycling of ubiquitin and/or modulation of signaling pathways must be tightly controlled. ubiquitination and kinase cascades intersect on multiple levels and together they orchestrate key cellular events including endocytosis, cell cycle progression, and growth factor signaling. kinases activate e3 ubiquitin ligases (e.g., the anaphase promoting complex / cyclosome) which in turn ubiquitinate kinases (e.g., polo) or kinase regulatory subunits (e.g., the cyclin subunit of cyclin - dependent kinases (cdk)). kinases also regulate protein turnover by marking substrates for phosphorylation - dependent ubiquitin - mediated degradation (e.g., by the scf ubiquitin ligase).(14) there are many other examples of cross - regulation of ubiquitin and kinase signaling networks, including phosphorylation of deubiquitinating enzymes (e.g., cyld).(15) here we set the stage for understanding how dubs might be regulated by kinases and phosphatases by cataloging phosphorylation sites of all s. pombe dubs. dubs are a highly conserved family of proteases involved in : (1) processing of ubiquitin precursor proteins, (2) recovery of modified ubiquitin trapped in inactivatable forms, (3) cleavage of ubiquitin from target proteins, and (4) recycling of monoubiquitin from free polyubiquitin chains. the diversity of dub functions is reflected in the number of dubs (95 predicted human dubs), the variety of catalytic domains ubiquitin c - terminal hydrolases (uch), ubiquitin - specific proteases (usp), ovarian tumor proteases (otu), machado - joseph disease proteases (mjd) and jab1/mpn / mov34 metalloenzymes (jamm)(16) and dub domain architecture.(1) s. pombe is an amenable organism in which to conduct a global study of dub function and regulation because of the limited number of dubs containing the required catalytic residues (20), the diversity and conservation of catalytic domains (4 of 5 classes, see table 1), and the genetic tractability of yeast. we recently reported the cellular localization, enzymatic activity profiles and protein interaction networks of the entire s. pombe dub family.(1) a few phosphorylation sites for some s. pombe dubs have been reported in large - scale phosphoproteomics studies, but a detailed analysis of dub phosphorylation is lacking. to begin to understand how phosphorylation impacts dub regulation, we examined the phosphostatus of the entire s. pombe dub family and their binding partners using tandem affinity purification (tap) followed by multidimensional lc - ms / ms (mudpit) from asynchronous and mitotic cell cultures (figure 1). here, we present the global phosphorylation status of the s. pombe dubs and their partners and discuss the implications of these modifications on dub regulation in eukaryotes. experimental scheme for dub purifications, lcms / ms analysis, and phosphopeptide identification and verification. endogenously tagged strains (supplemental table 1, supporting information) were grown in yeast extract (ye) media. for expression of n - terminally tagged proteins, strains were transformed with prep expression vectors, containing a thiamine repressible promoter, using a standard sorbitol transformation procedure.(21) transformed strains were first grown on minimal media containing thiamine to suppress expression and then, to induce expression, cells were grown in minimal media lacking thiamine for 18 h.(22) cell cultures used for tap purifications were grown in 2 l of 4 ye media (c - terminally tap tagged proteins) or in 8 l of minimal media supplemented with the appropriate nutrients (n - terminally tap tagged proteins). all 20 dubs were tagged endogenously at the 3 end with tap or linker - tap as previously described.(23) the linker sequence in the linker - tap cassettes translates to ilgapsgggatagaggaggpagli.(24) n - tap cassettes for ubp1, ubp7, and ubp11 were constructed as previously described.(1) for mitotic purifications of the nuclear dubs (ubp6, ubp8, ubp9, ubp12, ubp14, ubp15, ubp16, uch1, uch2, otu1, and rpn11), log phase cells containing dub tap tags were blocked using a cold sensitive allele of -tubulin (nda3-km11, prometaphase) and/or released for 30 min (anaphase). cells were snap frozen in a dry ice ethanol bath and subjected to tap / lc - ms / ms as described below. cell pellets were frozen in a dry ice / ethanol bath and lysed by bead disruption in np-40 lysis buffer under native (figure 2c) or denaturing conditions (figure 2a / b) as previously described,(25) except with the addition of 0.1 mm diisopropyl fluorophosphate (sigma - aldrich). proteins were immunoprecipitated by igg sepharose beads (ge healthcare) or anti - gfp (roche). for phosphatase collapse, immunoprecipitated proteins were incubated with lambda phosphatase (new england biolabs) in 25 mm hepes - naoh ph 7.4, 150 mm nacl, and 1 mm mncl2 for 30 min at 30 c. immunoblot analysis was performed as previously described(26) except that secondary antibodies were conjugated to alexa fluor 680 (invitrogen) and visualized using an odyssey infrared imaging system (li - cor biosciences). for the block and release experiment, a temperature sensitive strain (cdc2522 ubp9-tap) was grown overnight at 25 c and then shifted to the nonpermissive temperature (36 c) for 3 h to block cells in g2. the cells were then released to the permissive temperature and 20 od pellets were collected every 15 min. lysates and immunoprecipitations were performed as described above except igg coated dynabeads (invitrogen) were used for immunoprecipitation. proteins were purified by tap as described,(27) or using a one step dynabead purification as follows : tosylactivated m-280 dynabeads were coupled to rabbit igg (invitrogen) and used to pull down tap tagged proteins from native lysates (as in tap protocol) and then the proteins were eluted using high ph. the purified proteins were then tca precipitated and digested with trypsin (promega), chymotrypsin (princeton separations), and/or gluc (thermo) and the resulting peptides were subjected to mass spectrometric analysis on a thermo ltq as previously detailed. thermo raw files were converted to mzml files using scansifter (software developed in - house at the vanderbilt university medical center). the s. pombe database (http://www.sanger.ac.uk, october 2009) was searched with the myrimatch algorithm(30) v1.6.33 on a high performance computing cluster (advanced computing center for research & education at vanderbilt university). we added contaminant proteins (e.g., keratin, igg) to the complete s. pombe database and reversed and concatenated all sequences to allow estimation of false discovery rates (10 186 entries). myrimatch parameters were as follows : strict tryptic cleavage ; modification of methionine (oxidation, dynamic modification, + 16 da), s / t / y (phosphorylation, dynamic modification, + 80 da) and cysteine (carboxamidomethylation, static modification, + 57 da) was allowed ; precursor ions were required to be within 0.6 m / z of the peptide monoisotopic mass ; fragment ions were required to fall within 0.5 m / z of the expected monoisotopic mass. ambiguous ids per result 2, min peptide length per result 5, min distinct peptides per protein 3, min additional peptides per protein group 2, minimum number of spectra per protein 3, indistinct modifications m 15.994 da, c 57.05 da and distinct modifications s / t / y 80 da. idpicker results were processed in excel (microsoft) to generate phosphopeptide lists for the dubs and their binding partners. spectra were manually inspected and annotated in seems and a related program called ptmdigger, software developed by in - house (surendra dasari, matthew chambers, and david tabb, vanderbilt university medical center). supplemental figure 1 (supporting information) was generated using software developed in - house (zeqiang ma, surendra dasari, matthew chambers, and david tabb, vanderbilt university medical center). dubs and partners were purified with sequence coverage (%) as follows : otu1 51, otu2 35, ubp1 67, ubp2 95, ubp3 64, ubp4 42, ubp5 72, ubp6 56, ubp7 81, ubp8 62, ubp9 67, ubp11 71, ubp12 67, ubp14 80, ubp15 88, ubp16 44, rpn11 63, sst2 65, uch1 84, uch2 82, ucp6 78, nxt3 71, sfp47 46, ftp105 58, bun62 57 and bun107 64. note that mildly overexpressed n - terminal tap fusions were used for the low abundance dubs ubp1, ubp7 and ubp11. for complete protein identification information for each tap, see a previous publication.(1) using the stringent filter of fdr 20% of the neutral loss peak (3) contained two or more sequential fragments (b and/or y) bracketing the phosphorylation site(s) ; 1242 spectra met these criteria. phosphorylation sites were assigned based on the presence of sequential fragment ions surrounding the modification ; if these ions were missing, the phosphorylation site(s) were assigned to multiple sites ambiguously. deubiquitinating enzymes are present in nearly every cellular compartment(1) (table 1) and participate in essential cellular processes including regulation of endocytosis, protein degradation, transcription, dna repair, and protein localization and/or activity.(18) we and others have shown that dubs are regulated by interaction with protein partners and now we have assessed the phosphostatus of the s. pombe dub family to set the stage for understanding the interplay of phosphorylation and ubiquitination. each of the 20 s. pombe dubs was purified two or more times from asynchronous cultures using an endogenous c - terminal tap tag or an inducible n - terminal tap tag (see experimental methods for details).(1) we also performed purifications of the (partially) nuclear dubs (ubp6, ubp8, ubp9, ubp12, ubp14, ubp15, ubp16, uch1, uch2, rpn11, and otu1) from cells arrested in prometaphase using the tubulin mutation, nda3-km11, and released for 30 min into anaphase to enrich our data set with mitotic phosphorylation events (denoted in tables 2, 3 and 4). each purification was precipitated, digested, and analyzed on a thermo ltq using a mudpit protocol (see experimental methods for details). the resultant mass spectra were processed using software developed at vanderbilt medical center (figure 1) to identify phosphorylation sites. over 1500 mass spectra (fdr > 0.5%) indicative of phosphorylation (+ 80 da) were identified from our bioinformatic analysis (figure 1) and manual validation showed 1242 spectra corresponding to phosphorylation sites (for criteria see experimental methods). the overall spectral quality and peptide sequence coverage is illustrated with two examples of parent and daughter spectra (supplemental figures 2 and 3, supporting information). in total, we identified over 130 phosphorylation sites in over half (12/20) of the s. pombe dubs and dub partners (tables 2, 3, and 4). only ca. 15% of the phosphosites we identified have been previously reported (see supplemental table 2 for details, supporting information). we confirmed biochemically that upb9, bun107, ftp105 and sfp47 are phosphoproteins by lambda phosphatase collapse and western blot (figure 2a). the other dubs exhibited no discernible gel shift after phosphatase treatment (data not shown), but gel conditions were not optimized for each protein. biochemical analysis of dub phosphorylation a) lambda phosphatase collapse for ubp9, bun107, sfp47, and ftp105 b) phosphorylation status of ubp9 in the presence or absence of its wd partners and c) block and release experiment illustrating the cell cycle dependency of ubp9 phosphorylation (see experimenal methods for details). the mitotic purifications revealed upregulated [st]p proline - directed kinase consensus sites, as one might expect for modification by mitotic cdk. phosphorylation sites detected in ubp6, ubp9 and its partner bun107 were much more abundant in the mitotic purifications (denoted in tables 3 and 4), suggesting that these dubs are cell cycle regulated. ubp6 is recruited to the proteasome under conditions of ubiquitin stress(34) which was not the case for our experiments, but it is possible that mitotic phosphorylation plays some role in localization or activity of ubp6. phosphorylation of ubp9 is clearly cell cycle dependent based on block and release experiments (figure 2c) and enrichment of s11 phosphopeptides identified from mitotic cells ; thus, phosphorylation may alter the affinity of ubp9 for its substrates and/or enhance its catalytic activity rather than affect its cellular localization (which is regulated by its wd partners, see discussion below). all components of the ubp9 complex are phosphorylated (this study) and conserved throughout eukaryotes.(35) the larger wd partner, bun107, contains multiple phosphorylation sites consistent with cdk phosphorylation based on amino acid sequence and increased abundance in mitotic purifications (table 4). cross - regulation between ubiquitination and phosphorylation appears to be a common theme for dub complexes. over half of the dubs interact with protein partners near stoichiometric ratios(1) and most of these dubs and their partners are phosphorylated (tables 14 and figure 3), signifying that kinases and phosphosphatases regulate dubs. ubp9, a dub present in the nucleus and at cell tips and septa, is part of a complex containing two wd proteins (bun62 and bun107). both wd partners are required for ubp9s dub activity and regulate its cellular localization.(1) the ubp9 complex shuttles between the nucleus and cytoplasm, but at steady state, accumulates at active sites of endocytosis (cell tips and septa). when bun62 is deleted, ubp9 localizes to cell tips and septa, but not the nucleus, whereas deletion of bun107 causes retention of ubp9 in the nucleus.(1) we have discovered that ubp9 and both of its partners are phosphorylated (tables 3 and 4 and figures 2 and 3). to investigate how phosphorylation might impact ubp9 localization or function, we examined the phosphostatus of ubp9 in strains where each partner had been deleted individually and in combination (figure 2b). when either wd partner is lost, ubp9 is no longer efficiently phosphorylated (figure 2b), suggesting that ubp9 is not competent for phosphorylation unless it is in complex with its partners. both partners of four other dub complexes are phosphorylated, including two cytoplasmic dubs ubp2 and ubp3 and their partners ucp6 and nxt3, respectively, and two endocytic dubs ubp4 and ubp5 and their partners sfp47 and ftp105, respectively (tables 2 and 4, figures 2 and 3). sfp47, an sh3 domain protein, and ftp105, a putative transmembrane protein, recruit their respective dub partners to specific cellular locations (endosomes for ubp4 and the golgi for ubp5).(1) phosphorylation and dephosphorylation cycles may modulate complex formation, cellular localization, dub activity and/or substrate specificity. domain architecture and mapping of detected phosphorylation sites within the s. pombe dubs and their partners. the following domains were found : usp (ubiquitin - specific proteases) jamm (jab1/mpn / mov34 metalloenzymes), dusp (domain in ubiquitin - specific proteases), math (meprin and traf homology), ubl (ubiquitin - like), znf (ubiquitin carboxyl - terminal hydrolase - like zinc finger), uba (ubiquitin - associated). phosphosites are denoted by vertical black lines. surprisingly, most of the dub phosphorylation sites map to the catalytic dub domains (figure 3). in fact, all detected sites for ubp7 are within its extended usp domain, suggesting that its catalytic activity and/or structure could be regulated by phosphorylation. one within their usp domain and one near the n - terminus ; perhaps this arrangement allows tuning of dub cellular localization, substrate binding or catalytic activity by kinases and phosphatases. finally, two endocytic dubs ubp5 and ubp9 have two clusters of sites at their n- and c- termini, respectively, predominately outside the usp domains. as discussed above, the cellular localization of these two dubs is regulated by protein partners(1) and so phosphorylation may add another layer of regulation for substrate binding and/or catalytic activity. the phosphosites detected for the dub partners also cluster within or very near domains (e.g., ubcp6 and ftp105) or in regions predicted to be intrinsically disordered (e.g., sfp47 and bun107) (figure 3). these sites may regulate the availability of specific protein domains for interaction with other partners or the catalytic activity of the holo dub complex. given the diversity of dub cellular localization and function, it is not surprising that the dub phosphosites match consensus sequences for multiple protein kinases. the majority of dub phosphopeptides are products of proline - directed kinases (e.g., map kinases or cdk) and many others match consensus sites for casein - type kinases (cki, ckii, see tables 14 and supplemental table 2, supporting information). phosphosites detected in the exclusively cytoplasmic dubs also include sequences consistent with pikk and gsk3 consensus sites and the cellular localization of these kinases (table 2). while the nuclear dub sites are primarily proline - directed sites, the partially nuclear dubs, ubp9 and ubp12, have phosphopeptides consistent with phosphorylation by pka / pkc (table 3, supplemental table 2, supporting information). there are also many phosphorylation - dependent ww class iv ligand motifs present in both the cytoplasmic and nuclear dubs (tables 2 and 3), suggesting that dub interactions with ww domain - containing proteins could be controlled by phosphorylation. for instance, the hect - type e3 ub - ligases, pub1, pub2 and pub3 and multiple components of the spliceosome contain ww domains and are likely regulated by a combination of kinases and dubs. the cytoplasmic dub ubp2 and endocytic dubs ubp5 and ubp9 are phosphorylated on sites that match the fha domain consensus binding motif that may function in localization and/or substrate recognition. our results show that the majority of dubs and most dub partners are phosphorylated, some in a cell cycle - dependent manner. the phosphosites identified for s. pombe dubs and their partners provide a foundation for understanding the interplay of ubiquitination and phosphorylation in this enzyme class in higher eukaryotes because sites identified in conserved proteins may be conserved or mimicked in higher eukaryotes. future studies aimed at understanding the intersection of ubiquitination and phosphorylation will be useful for understanding dub regulation and, more broadly, the cross - regulation of kinase and ubiquitin signaling networks. | ubiquitination plays a role in virtually every cellular signaling pathway ranging from cell cycle control to dna damage response to endocytosis and gene regulation. the bulk of our knowledge of the ubiquitination system is centered on modification of specific substrate proteins and the enzymatic cascade of ubiquitination. our understanding of the regulation of the reversal of these modifications (deubiquitination) lags significantly behind. we recently reported a multifaceted study of the fission yeast schizosaccharomyces pombe dubs including characterization of their binding partners, in vitro enzymatic activity and subcellular localization.(1) over half of the 20 fission yeast dubs have a stable protein partner and some of those partners regulate the localization and/or activity of their cognate dub. as a next step in understanding how dubs might otherwise be regulated, we investigated the phosphostatus of the entire fission yeast dub family using lcms / ms, and here we discuss the possible implications of phosphoregulation. |
mast cells are integrally involved in cellular based immune responses to pathogens as well as inflammatory reactions prompted by pathogens or toxins [1, 2 ] and have been suggested for several years to play a part in the acute phase of multiple sclerosis (ms) [38 ]. albeit the mechanisms by which mast cells influence ms are yet to be fully understood, trypsin - like proteases released from degranulating mast cells have been shown to trigger demyelination in a mouse model for ms - experimental autoimmune encephalomyelitis (eae). (2000), furthermore, support that mast cells are involved in the pathogenesis of eae. the same group has more recently suggested a significant role for mast cells in activation of inflammasomes localized within meninges. in contrast, other groups have challenged the contribution of mast cells in eae [1113 ]. in particular, feyerabend and colleagues albeit targeting mast cells as a viable approach to alleviate the disease remains debatable, the contribution of mast cell - derived proteases in eae is still largely uninvestigated. one of the many mast cell - expressed proteases that potentially may account for the contribution of mast cells to ms / eae is mouse mast cell protease 4 (mmcp-4), a -chymase predicted to be the murine functional counterpart to the single human chymase (cma1) based on deduced amino acid sequence, tissue localization, and serglycin storage dependence [14, 15 ]. in support for a role of this chymase in mast cell - dependent inflammatory conditions, mmcp-4 plays a protective role in a mouse model of mechanically induced cerebral trauma, yet it is detrimental in lung inflammation and immune complex - induced glomerulonephritis [17, 18 ]. in further support for a role of mmcp-4 in regulating inflammatory mediators, our group has reported that mmcp-4 generates endothelin-1 (et-1) from its precursor big - et-1 [19, 20 ] and that mmcp-4 knockout (ko) mice display a 40% reduction in pulmonary et-1 levels when compared to wild type (wt) congeners. the role of et-1 as a marker in the etiology of ms has only been explored in a limited fashion. (2001) reported a significant increase in et-1 plasma levels in untreated ms patients, an observation confirmed by pache and colleagues (2003). in treated ms patients however, in further support for a role of et-1 in ms, et-1 was shown to be overexpressed in a murine model of eae. based on the above - suggested links between et-1 and ms and between chymase and et-1 generation, respectively, we here asked whether chymase might have a role in eae and whether that enzyme in this experimental setting has a regulatory effect on et-1 production. indeed, the findings presented here suggest that mmcp-4 has a significant detrimental impact on the course of eae and plays role in the generation of et-1 in this mouse model for ms. the impact of mmcp-4 in eae introduces a potential role for mast cell chymase in ms and thereby identifies the inhibitors of this particular enzyme as potential targets for therapy of ms. c57bl/6 mice (i.e., wild type (wt)) were purchased from charles river canada (montral, qc, canada) and housed in our local facility. the mmcp-4 ko mice have been backcrossed for over 10 generations with c57bl/6 congeners and are therefore highly congenial with the later strain. finally, the genotype of mmcp-4 ko mice used in the present study was confirmed by polymerase chain reaction (pcr) (as shown in supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/9797021) via the use of primers described in supplemental table 1. we had previously reported, in mmcp-4 ko mice in vivo as well as in tissues or mastocytes derived from this mouse strain, the complete loss of chymase - dependent hydrolytic activity [19, 20 ]. all animals were kept at constant room temperature (23c) and humidity (78%) under a controlled 1014 h light / dark cycle. animal care and experimentation were approved by ethics committee on animal research of the universit de sherbrooke in accordance with the guidelines of the canadian council on animal care. induction of eae was performed according to the protocol of miller and karpus (2007). in brief, a 1 : 1 emulsion mixture of myelin oligodendrocyte glycoprotein (mog3555) (genemed synthesis inc., san antonio, tx, usa) and complete freund 's adjuvant (cfa) (sigma - aldrich, st. louis, mo, usa) supplemented with 100 g of heat - killed mycobacterium tuberculosis h37ra (difco laboratories, detroit, mi, usa) was prepared. female mice, at 810 weeks old, were subcutaneously injected in two sites (100 l by site) adjacent to the tail with the emulsion. campbell, ca, usa) was administered intraperitoneally on the same day of immunization. mice were scored daily with the following scale to assess clinical scores : 0, no sign of clinical disease ; 0.5, partial tail paralysis (loss of tip tail tonus) ; 1, tail flaccidity or hind limb weakness ; 2, limp tail and weakness in limb ; 3, partial hind limb paralysis ; 4, total hind limb paralysis ; and 5, moribund state or death. the immunized mice were anesthetized by intraperitoneal injection of 2,2,2- tribromoethanol (avertin) (approximately 240 mg / kg) (sigma - aldrich, st. louis, mo, usa), prepared in tert - amyl alcohol and diluted in 0,9% saline solution. the mice were then perfused with ice - cold pbs buffer (wisent, st. bruno, qc, canada), and the spleen, cervical, and thoracic spinal cords and left and right brain hemispheres were collected and stored at 80c immediately, whereas lumbar spinal cords were placed in 10% buffered formalin phosphate (fisher scientific, waltham, ma, usa) before being embedded in paraffin and cut into 5 m sections. slides were deparaffinised in xylene (electron microscopy sciences, hatfield, pa, usa) and hydrated in 100, 95, and 70% ethanol gradient followed by water. slides were incubated 2 - 3 minutes at room temperature in toluidine blue (sigma - aldrich, st. louis, mo, usa) working solution prepared by dilution of stock solution (1% in ethanol 70%) in sodium chloride 1% ph 22.5. the slides were rinsed in water and quickly dehydrated in 95100% ethanol before being rinsed with xylene and coverslipped with permount (fisher scientific, ottawa, on, canada). slides were incubated overnight at 56c in 0.1% luxol fast blue solution (electron microscopy sciences, hatfield, pa, usa) in 95% alcohol w / acetic acid. the slides were then rinsed in distilled water before being differentiated in lithium carbonate 0.05% and in 70% ethanol for approximately 30 seconds each, since the grey matter is clear and white matter is sharply defined. the slides were rinsed in water and dehydrated in 100% ethanol before being cleared in xylene and coverslipped with permount. all histological slides were scanned with a nanozoomer 2.0-rs digital slide scanner (hamamatsu photonics, shizuoka, japan) before being treated with the ndp.view2 viewing software and paint.net software and staining density was quantified by imagej 1,49v (wayne rasband, nih, usa). prior to measuring immunofluorescence, with a sequenze slide rack and coverplate system (ted pell inc, redding, ca, usa), an antigen unmasking was performed by a 10 minutes ' incubation in 10 mm sodium citrate buffer ph 6.0 (sigma - aldrich, st. the slides were then washed in 0.1% triton x-100 in pbs solution and blocked in 5% fetal bovine serum (fbs) supplemented with 0.1% triton x-100 in pbs for one hour before being incubated overnight at 4c with primary antibody (1 : 1000), against the rabbit anti - glial fibrillary acidic protein (gfap) (cedarlane, burlington, on, canada) or against the rabbit anti - ionized calcium binding adaptor molecule 1 (iba1) (wako, osaka, japan). a 2 hours ' incubation at room temperature with the secondary antibody (1 : 2000), alexa fluor 488 affinipure goat anti - rabbit igg (h + l) (jackson immunoresearch laboratories inc. the slides were mounted with dapi fluoromount - g (southernbiotech, birmingham, al, usa) and photomicrograph pictures were taken with retiga srv mono cooled numerical camera attached to zeiss axioskop 2 microscope. the pictures were stitched with adobe photoshop cs3, and stain density was quantified with image - pro plus 5.1 (media cybernetics inc., the left parts of brain from healthy or 1, 2, or 3 weeks post - eae - induced mice were homogenized in a chloroform : methanol (1 : 4) solution and then purified on a dsc-18 solid phase extraction column (supelco, bellefonte, pa, usa) and eluted in acetonitrile : water : trifluoroacetic acid (acn 60% : h2o 40% : tfa 0,1%). the collected eluates were then speed vac - dried overnight before reconstitution in pbs supplemented with 1/32 mouse plasma and endogenous et-1 was measured by quantikine elisa kit from r&d systems (r&d systems, minneapolis, mn, usa) according to the manufacturer 's instructions. briefly, frozen thoracic spinal cords were weighed and homogenized in 0.5 ml of ice - cold lysis buffer (cell signaling technology, beverly, ma, usa) supplemented with protease inhibitors (roche diagnosis, mannheim, germany) by rapid agitation for 2 minutes in the presence of 3 mm stainless beads. the tissue lysate was centrifuged for 20 minutes at 13,000 g at 4c, and the supernatant was transferred to a new tube. the tissue levels of ifn were determined using murine elisa development kits (peprotech, rocky hill, nj, usa), according to the manufacturer 's instructions. the level of ifn was reported as pg / mg of tissue. rna from the right brain hemisphere derived from healthy or 1 or 2 weeks post - eae - induced mice were extracted using ribozol reagent (amresco inc., baker, central valley, pa, usa) was added to each tube per 1 ml of ribozol and incubated at room temperature for 3 minutes followed by centrifugation at 12,000 g for 15 minutes at 4c. nonopaque supernatants were collected and 500 l of isopropanol (fisher scientific, ottawa, on, canada) was added for rna precipitation and incubated 10 minutes at room temperature followed by centrifugation at 12,000 g for 10 minutes at 4c. pellets were washed with addition of 1 ml ethanol 75% followed by 7,500 g centrifugation at 4c for 5 minutes before being redissolved in 50 l of depc water and an incubation at 55c for 10 minutes. cdna was synthesized using oligo(dt)1218 primers (invitrogen, carlsbad, ca, usa), dntps mix 10 m each (thermo scientific, waltham, ma, usa), in superscript iii buffer with dtt, rnaseout, and superscript iii (invitrogen, carlsbad, ca, usa). quantitative pcr was performed for actin and mmcp-4 by monitoring in real time the fluorescence increase of the sybr green in the perfecta sybr green supermix, low rox (quanta biosciences, gaithersburg, md, usa) using the mx3000p multiplex quantitative pcr system (agilent technologies, santa clara, ca, usa). primers (idt, coralville, ia, usa) were used at final concentration of 50 nm per primer and sequences were designed as follows : mmcp-4 f : 5-ctctctccaagctgtgaccgac-3, mmcp-4 r : 5-ctatgagctccaagggtgaca-3, -actin f : 5-gatcaagatcattgctcctcctgagc-3, -actin r : 5-gcagctcagtaacagtccgcctag-3.mmcp-4 ko mice samples were tested as negative controls. as -actin levels were stable between healthy and immunized mice, the latter mrna was used as internal control for normalization and relative expression of chymase mmcp-4 was calculated using the 2 method. mmcp-4 f : 5-ctctctccaagctgtgaccgac-3, mmcp-4 r : 5-ctatgagctccaagggtgaca-3, -actin f : 5-gatcaagatcattgctcctcctgagc-3, -actin r : 5-gcagctcagtaacagtccgcctag-3. all statistical analyses were conducted using graphpad prism 6 software (graphpad software, la jolla, ca, usa). value was below 0.05 and determined using one - way anova kruskal - wallis followed by bonferroni for eae clinical score or based on one - way or two - way anova and multiple student 's t - test for all other analyses. an emulsified mixture of mog3555 in cfa was subcutaneously injected in wt or mmcp-4 ko mice. wt mice developed the disease approximately after 9 days whereas the mmcp-4 ko mice demonstrated a substantial delay in the appearance of clinical symptoms, being detectable starting from ~day 12 after injection (figure 1(a)). moreover, the development of severe disease (disease score 2.5) was markedly delayed in mmcp-4 ko mice up until the third week posttreatment as indicative of tail and back limb weaknesses. however, 4 weeks after eae, wt and mmcp-4 ko mice exhibited similar disease scores with no evidence of total hind limb paralysis. a summation of the total clinical scores, on the other hand, supported a significant role of mmcp-4 as a pathogenic factor in eae (figure 1(b)), even up to the 4th week of observations (wt 41.64 3.02 ; mmcp-4 ko 30.93 2.48, p < 0.05, n = 7 mice). to assess, on the other hand, whether mmcp-4 has an impact on the immune response, spleen weights of wt and mmcp-4 ko mice were monitored up to 4 weeks after immunization however, as seen in figure 2, spleen weights did not differ between the two genotypes except in healthy mice where it was significantly increased in mmcp-4 ko congeners (p < 0.05). in response to spinal cord insults, gfap (an intermediate filament protein expressed by astrocytes and ependymal cells among others) is upregulated in the cns. as another sign of cns damage, the reactive microglial response can be measured by the extent of upregulation of iba1. to evaluate whether mmcp-4 can influence the levels of these markers of cns damage, the spinal cords of 2 weeks after eae immunized mice were extracted and stained for gfap and iba1 (figure 3(a)). mmcp-4 ko mice and wt littermates showed no significant differences in percent level of astrogliosis (gfap staining intensity) and microgliosis (iba1 staining intensity) in the total area of the lumbar spinal cord or in the white matter (figure 3(b)). in contrast, significant reductions in percentages of microgliosis and astrogliosis were found in the grey matter of mmcp-4 ko mice when compared to wt congeners, 2 weeks after eae immunization (in wt and mmcp-4 ko mice, respectively. ; astrogliosis in percentage : 0.87 0.17 and 0.30 0.04, p < 0.05 ; microgliosis in percentage : 1.61 0.18 and 0.55 0.12, p < 0.01) (figure 3(b)). to assess whether the absence of mmcp-4 has an influence on the number of mast cells, lumbar spinal cords from wt and mmcp-4 ko mice were stained with toluidine blue, both at baseline and after induction of eae. as seen in figure 4, the numbers of mast cells were similar in spinal cords from wt and mmcp-4 ko mice at the baseline state (healthy) and 2 - 3 weeks after induction of eae. notably though, there was a significant elevation of the numbers of mast cells 1 week after eae (in wt and mmcp-4 ko mice, respectively ; in percentage, healthy state : 2.30 0.07 and 2.47 0.17, 1 week after eae : 2.93 0.14 and 3.74 0.27, p < 0.05, 2 weeks after eae : 2.73 0.25 and 2.93 0.20, and 3 weeks after eae : 3.20 0.13 and 3.29 0.17). to assess whether chymase has an impact on the levels of intact myelin, lumbar spinal cords from wt and mmcp-4 ko mice were extracted and stained with luxol fast blue, a dye that stains myelin (figure 5). quantification of the luxol fast blue staining intensity revealed a significantly higher myelin content in mmcp-4 ko mice as compared with wt congeners, in both nonimmunized (healthy) mice and immunized mice 1 week after eae induction (figure 6) (in percentage, healthy wt mice : 31.62 0.46 and healthy mmcp-4 ko mice : 39.70 1.03, p < 0.001 ; wt 1 week after eae : 27.37 0.29 and mmcp-4 ko mice 1 week after eae : 33.88 0.50, p < 0.001). a subsequent decline in spinal cord myelin was seen in both wt and mmcp-4 ko mice 2 to 3 weeks after immunization as well as a loss of significant differences in myelin content between the two strains of mice (figure 6). to investigate if eae is associated with an induction of the mmcp-4 gene, right brain homogenates derived from wt mice were analysed for mmcp-4 mrna levels by rt - qpcr. as seen in figure 7(a), a 2.2-fold increase in mmcp-4 mrna expression (as compared with baseline levels in healthy mice) was found in wt mice one week after eae immunization but not at later time points. finally, eae immunization promoted a twofold increase in mature et-1 levels in left brain homogenates of wt but not mmcp-4 ko mice, one but not two weeks after eae when compared to healthy wt mice (figure 7(b)). to investigate the immune response to eae induction, we proceeded to elisa quantification of ifn in thoracic spinal cord in healthy and 1 or 2 weeks after eae. as shown in figure 7(c), the levels of this cytokine are increased after eae compared to healthy basal levels in both strains of mice, but significantly only in wt mice 2 weeks after eae (quantities in pg / mg of tissue, healthy wt mice : 20.77 1.84 ; wt 1 week after eae : 23.30 2.11 ; wt 2 weeks after eae : 32.07 2.11, p < 0.001 and healthy mmcp-4 ko mice : 19.23 1.65 ; mmcp-4 ko mice 1 week after eae : 24.66 4.24 ; mmcp-4 ko mice 2 weeks after eae : 26.47 3.39). the main results of the present study are that mmcp-4 ko mice subjected to eae show a reduced clinical score as well as brain levels of immunoreactive et-1 when compared to wt congeners. in addition, the ko mice show reduction in percentages of microgliosis and astrogliosis as well as grey matter alterations. these results therefore support our hypothesis that this particular chymase isoform plays a significant role in the early development of ms in the mouse eae model. in our hands, the ko of mmcp-4 significantly reduced clinical scores up until 21 days after eae induction. it is noteworthy that mature mast cells can express numerous tryptases (having trypsin - like cleavage specificity) and chymases (having chymotrypsin - like cleave specificities) as well as other types of proteases. out of these, mmcp-4 was shown here to have a major impact on the course of eae development. this suggests that mmcp-4 accounts to a major extent for the demyelination attributed to mast cells in eae. this does not exclude, on the other hand, the involvement of other mast cell - derived mediators in this mouse model of ms. it is also of interest that eae prompted a significant increase in et-1 brain levels one week after eae induction in wt but not in mmcp-4 ko mice. notably, brain levels of this potent vasoactive peptide progressively returned to basal levels on the second and third weeks after eae (results not shown for the 3rd week). these results suggest that et-1 may be either an early marker or, alternatively, an early mediator of the inflammatory reaction occurring in the eae model. (2001) reported that an eta antagonist, bq-123, reduced the duration of paralysis, in eae - induced rats. the same authors also reported high levels of immunoreactive et-1 in spinal cord - localized inflammatory and neuroglial cells. (2014) have recently shown that et-1 is a negative regulator of oligodendrocyte progenitor cell - dependent repair of demyelinated lesions. in support of the latter observation, myelin contents were, in the present study, found to be higher in naive and even one week after eae immunized mmcp-4 ko mice than in their wt congeners. thus, interfering with the chymase - dependent production of et-1 in ms may be a relevant therapeutic strategy in autoimmune diseases such as ms. noteworthy, chymase inhibitors have been shown to possess anti - inflammatory and antiproliferative properties in other settings [3437 ]. albeit still controversial, one should also note the beneficial effects of et-1 antagonists on cerebral blood flow of ms patients. thus, et-1 antagonists may have beneficiary effects not only by repressing cellular events in eae but by improving overall brain blood circulation as well. further studies currently ongoing in our laboratory should shed further light on the contribution of et-1 via one eta and/or etb receptors and their associated mechanisms, in the etiology of eae in the mouse model. albeit we show in the present study that mmcp-4 is detrimental in an experimental model of ms, the same concept can not be extended to all types of experimental injuries of the cns. for example, hendrix and colleagues (2013) reported the protective role of mast cells (in part due to mmcp-4) in a mouse model of mechanically induced brain injury. noteworthy, in the same study the authors reversed the protective role of mmcp-4 with a general chymotrypsin inhibitor, chymostatin, which is nonspecific for chymase - dependent processes. (2004) that, in contrast to spinal cord injury models, eae - induced lesions may facilitate additional levels of axonal reorganization based on the unlesioned fibers of the tract. these observations support the concept that neuroinflammatory and surgical hemisections of the spinal cord, in the mouse model, alter in opposite fashion the reserve capacity of the cns to regenerate. thus, we suggest that immune - related inflammatory reactions and surgical trauma in the cns prompt opposite roles for mmcp-4. interestingly, monitoring of inf in our model suggests an enhanced inflammatory reaction in the spinal cord of wt but not mmcp-4 ko mice. we observed a significantly higher concentration of ifn in the spinal cords of wt compared to mmcp-4 ko mice 2 weeks after immunization. the decreased inflammatory response is correlated with the observation that mmcp-4 ko mice have decreased proliferation of microglia and astrocytes. it can nonetheless be explained by decreased responses of cns inflammatory cells in mmcp-4 ko mice which lead to the decreased concentration of proinflammatory molecules, decreased chemotactic gradient, decreased influx of th1 cells, decreased ifn, and, as a result, decreased demyelination. on the other hand, preserved integrity of bbb may lead to reduced influx of th1 cells from the periphery, a decreased activation of astrocytes and microglia, a decreased overall inflammation in the cns, and a decreased demyelination. based on the above observations, the present study reinforces the hypothesis suggested by scandiuzzi. (2009) [17, 18, 40 ] that mmcp-4 activates cells involved in tissue specific autoimmune reactions. overall we show in the present study that a single mouse chymase isoform, mmcp-4, contributes significantly to the etiology and early symptoms associated with a mouse model of ms, namely, eae. if what is reported here in the mouse model can be extended to the clinical situation, it is suggested that targeting chymase rather than the overall mastocytic activity in that particular autoimmune disease will constitute an added value within the currently available therapeutic arsenal against this neurodegenerative disease. | experimental autoimmune encephalomyelitis (eae) is a mouse model that reproduces cardinal signs of clinical, histopathological, and immunological features found in multiple sclerosis (ms). mast cells are suggested to be involved in the main inflammatory phases occurring during eae development, possibly by secreting several autacoids and proteases. among the latter, the chymase mouse mast cell protease 4 (mmcp-4) can contribute to the inflammatory response by producing endothelin-1 (et-1). the aim of this study was to determine the impact of mmcp-4 on acute inflammatory stages in eae. c57bl/6 wild type (wt) or mmcp-4 knockout (ko) mice were immunized with mog3555 plus complete freund 's adjuvant followed by pertussis toxin. immunized wt mice presented an initial acute phase characterized by progressive increases in clinical score, which were significantly reduced in mmcp-4 ko mice. in addition, higher levels of spinal myelin were found in mmcp-4 ko as compared with wt mice. finally, whereas eae triggered significant increases in brain levels of mmcp-4 mrna and immunoreactive et-1 in wt mice, the latter peptide was reduced to basal levels in mmcp-4 ko congeners. together, the present study supports a role for mmcp-4 in the early inflammatory phases of the disease in a mouse model of ms. |
the association of drinking water hardness and cardiovascular diseases (cvds) has been studied since more than five decades ago. a study in england and wales showed that cardiovascular death - rates had a favorable effect in towns with harder water and had an adverse effect in towns with softer water. another studies showed that high levels of drinking water hardness can be protective against cvd. however, a review of ecological studies showed that the results are inconsistent between studies. a meta - analysis revealed a negative association between concentration of magnesium in water and cvd mortality. moreover, another review showed protective role of magnesium concentration in water against cvd in some case - control studies and one cohort study, but the analytical studies showed little evidence about the association of calcium and magnesium levels in drinking water and cvd risk. water hardness may be also associated with cvd risk factors, for instance positive correlations of water magnesium and calcium with blood pressure is documented. in the netherlands, another study found no overall association between calcium, magnesium or total hardness and ischemic heart disease or stroke mortality. water hardness may be also associated with cvd risk factors, for instance positive correlations of water magnesium and calcium with blood pressure is documented. case - control and cohort studies are more useful than ecological epidemiologicalstudies for investigating cause- and - effect relationships. the guidelines for drinking - water quality of the world health organization in 2011 reported that one of the case - control studies addressed the association between calcium and acute myocardial infarction and three reported the association between calcium and death from cvds. the aim of this study is to investigate the association of calcium and magnesium concentration of drinking water with cvds in urban and rural areas of a city in iran. this case - control study was conducted in 2012 in khansar county in isfahan province, iran. its area is 900 km and it includes 18 towns, 3 villages and one central city. we used the official data of the provincial health center regarding the chemical analysis data of urban and rural areas including the hardness, calcium and magnesium content of drinking water. data of patients hospitalized for cvd in the only specialty hospital of the city was gathered. by using the population of these areas, we calculated the relative frequency of cvd in urban and rural areas. we compared the frequency and relative frequency of cvds in the population studied according to the concentrations of ca and mg hardness in drinking water in 2010 and 2011. we compared the frequency and relative frequency of cvds in the population studied according to the concentrations of ca and mg hardness in drinking water in 2010 and 2011. the findings of the study in 2010 and 2011 are reported in table 1. in 2010, the increase in the calcium hardness above 72 mg / l, the prevalence of cvds in 1000 population decreased [figure 1a ] ; in 2011 this decrease in cvds was observed for calcium hardness of more than 75 mg / l [figure 1b ]. in 2010, the level of mg hardness in water ranged from 23 to 57 mg / l. by increasing mg hardness level above 31 mg / l in 2010 [figure 2a ] and above 26 mg / l in 2011 [figure 2b ], the number of cvd in 1000 people decrease. characteristics of variables studied in 2010 and 2011 cardiovascular patients in 1000 people according to the calcium hardness in water in (a) 2010 and (b) 2011 cardiovascular patients in 1000 people according to the magnesium hardness in water in (a) 2010 and (b) 2011 our study suggests favorable protective effects of water hardness, mainly water magnesium content, on cvds. several epidemiological studies have confirmed a negative association between water hardness and cvd among adults. however, some other studies did not confirm such relationship controversial results exist on the association of water hardness with cvd and their risk factors among the adult population. the health effects of hard water are mainly considered to be because of the effects of its dissolved salts, primarily ca and mg. some studies documented that the softer the water, the higher the cvd death - rates and suggested that water ca may have such protective role. most previous studies showed a significant inverse association between mortality from cvd and water levels of mg, but not ca levels. a review of the current literature reported that 10 of the 19 geographical studies published since 1979, found a significant association, but two geographical studies from sweden, reported no association. moreover, one of the six case - control studies that considered ca, found significant protective effect of high ca levels (> 70 mg / l) in drinking water. mg is a protective element against soft - tissue calcification and has a role in prevention from myocardial infarction. many, but not all, of the geographical correlation, cohort and case - control studies suggested that a high mg water concentration protects against cvd mortality. likewise, a recent review confirmed the protective effects of water hardness, notably its ca level against many chronic diseases including cvds. the main limitation of the current study is its cross - sectional nature, thus the associations documented in this study should be considered with caution. the main limitation of the current study is its cross - sectional nature, thus the associations documented in this study should be considered with caution. water hardness, as well as calcium and magnesium content of drinking water may have a protective role against cvds. further experimental studies are necessary to determine the underlying mechanisms and longitudinal studies are required to study the clinical impacts of the current findings. | background : the aim of this study is to investigate the association of calcium and magnesium concentration of drinking water with cardiovascular disease (cvds) in urban and rural areas of a city in iran.methods:this case - control study was conducted in 2012 in khansar county in isfahan province, iran. we used the official data of the provincial health center regarding the chemical analysis data of urban and rural areas including the hardness, calcium and magnesium content of drinking water. data of patients hospitalized for cvd in the only specialty hospital of the city was gathered for the years of 2010 and 2011.results:in 2010, water calcium content above 72 mg / l was associated with reduced number of cvds in 1000 population ; whereas in 2011 this decrease in cvds was observed for calcium levels of more than 75 mg / l. in 2010, the level of water mg content ranged from 23 to 57 mg / l. by increasing mg hardness level above 31 mg / l in 2010 and above 26 mg / l in 2011 were associated with decreased number of cvds in 1000 people. decrease.conclusions:our study suggests favorable protective effects of water hardness, mainly water magnesium content, on cvds. water hardness, as well as calcium and magnesium content of drinking water may have a protective role against cvds. further experimental studies are necessary to determine the underlying mechanisms and longitudinal studies are required to study the clinical impacts of the current findings. |
malignant tracheoesophageal fistula (tef) is a serious complication of thoracic malignant diseases and is more common in esophageal cancer than in lung cancer. tef sometimes results in aspiration pneumonia, sepsis, and malnutrition associated with trouble in eating. without proper and prompt treatment, life expectancy is estimated at about 6 weeks. stent intubation into the trachea and/or esophagus is a reliable treatment leading to improved quality of life. surgical bypass with gastrostomy is another option, though it might be too invasive in some patients with advanced cancer. chemoradiotherapy (crt) is a controversial option, as it has the potential to induce or worsen tef as a side effect, especially in lung cancer. in esophageal cancer, meanwhile, some reports have described closure of tefs after crt [3, 4 ]. our group experienced a rare case of tef that closed after crt in a patient with poorly differentiated lung carcinoma. crt seems to have good potential as a palliative treatment for tef in lung cancer, as well. a 45-year - old man with complaints of cough, dyspnea, weight loss, and difficulty in swallowing was referred to our hospital. the symptoms had emerged 3 months earlier and he had been completely unable to eat for several days before his admission. on physical examination, a solid mass at the right neck was palpable, the right palpebral fissure was narrowed, and inspiratory stridor was auscultated. an enhanced chest ct scan revealed a small mass in the superior sulcus invading into the mediastinum (fig. 1). the sagittal view revealed a cavity within the mass and compression of the stenotic trachea by the mass, but there was no apparent communication between the esophagus and trachea. laboratory data indicated mild normocytic anemia and elevated c - reactive protein. among tumor markers, an elevation of cytokeratin 19 fragment and neuron - specific enolase (table 1) was noted. the patient was diagnosed with poorly differentiated lung carcinoma by percutaneous needle biopsy (fig. the patient 's respiratory condition was too fragile to permit endoscopic examination at this point. radiation therapy was commenced immediately, before confirmation of the pathological diagnosis, for fear of sudden asphyxiation by the occupying mass. chemotherapy with cisplatin and docetaxel was added, but was discontinued when the patient developed febrile neutropenia requiring granulocyte - stimulating factor and antibiotics for several days. when his respiratory condition improved enough to permit bronchoscopy and upper gastrointestinal endoscopy, these modalities revealed a hole with ulcerative edges in the trachea and upper esophagus (fig. we surmised that palliative treatment would prevent complications of tef, such as aspiration pneumonia. as the fistula was located in the upper esophagus (20 cm below the incisive papilla), we anticipated that intubation of an expandable metallic stent to the esophagus would be deeply uncomfortable and intolerable. with airway stenting, an accidental insertion of the stent into the cavity inside the tumor was a concern. as the initial effect of radiation had improved the compression of the tumor to the trachea, the therapy was continued up to a total dose of 61 gy. about 2 weeks after the radiation therapy was completed, the patient 's severe cough subsided for several days. encouraged by the improvement, he attempted to eat sweets and managed to do so without coughing. a bronchoscopy a few days later revealed only one slit on the trachea, and upper gastrointestinal endoscopy showed a covering of normal epithelial tissue over the fistula (fig. 3). in the ensuing weeks he fully regained the ability to eat orally, traumas and malignancy, conditions that may be associated with side effects of crt, are common causes of acquired tef. as treatment, curative resection followed by repair of the trachea and/or esophagus surgery is usually considered inappropriate in malignant tef, as the patients tend to be in a bad condition and near the end of their lives. palliative therapy should be the primary choice in terms of quality of life, and the therapeutic goals should be to restore the patency of the trachea by reducing the extrinsic compression and to enable these patients to take nutrition and fluid. intubation of a stent to the tracheobronchial tree and/or esophagus is somewhat safer than the other procedures and good for symptomatic relief. some papers report high success rates in double stenting. when it is difficult to place a double stent at the same time, a tracheal stent has priority because an esophageal stent alone may compress the trachea and elicit respiratory distress. we abandoned tracheal stenting in the current case for fear of a sudden impaction of the stent into the cavity. to avoid the risk of tracheal compression by the rapidly growing mediastinal tumor, we decided to administer crt therapy in spite of the danger that the tef would worsen. with recent progress in the treatment of advanced and/or metastatic non - small - cell lung cancer, molecular targeted agents such as gefitinib, erlotinib, and bevacizumab play significant roles in improving patient survival. but the standard therapy for locally advanced tumors not suited for surgery is radiotherapy combined with chemotherapy based on a platinum - containing drug. it remains controversial whether such agents should be added to radiotherapy in locally advanced cases. chemotherapy combined with the angiogenesis inhibitor bevacizumab, a monoclonal antibody to human vascular endothelial growth factor (vegf), improves overall survival in colorectal cancer and non - small - cell lung cancer [8, 9 ]. although bevacizumab is recognized to be relatively safe, it is associated with the risk of hypertension, proteinuria, delayed wound healing, bowel perforation, and so on. a recent report suggests that bevacizumab added to crt is associated with a higher incidence of tef formation in both small - cell and non - small - cell lung carcinomas. most of the published papers on tef usually address the condition in patients with esophageal cancer. reported that crt brought about fistula closure in 17 of 24 (70.8%) patients with esophageal cancer and led to restored oral alimentation in 16 of those 17 (94.1%) patients. esophageal tissues around the fistula appear to epithelialize in response to crt, but the mechanisms behind this effect are unclear. the major pathology of esophagus cancer is squamous cell carcinoma, a tumor with relatively high radiosensitivity., transforming growth factor- (tgf-) is a key cytokine that stimulates the production of surrounding extracellular matrix and heals the injured tissue. given that radiation often induces tgf- signaling, we also speculate that this type of wound healing mechanism might underlie the epithelialization. tef formation in lung carcinoma is rarely reported. to our knowledge, this is the first report of primary lung cancer with tef successfully treated with crt. the therapeutic agents for tef should be selected by carefully considering the patient 's particular condition and the risk of aggravating the tef. though the usefulness and safety must be confirmed through further clinical research, crt may prove to be effective as a palliative treatment for malignant tef in lung cancer. | a 45-year - old man complaining of cough, dyspnea, and difficulty in swallowing was referred to our hospital. chest ct scan showed a mediastinal mass compressing the trachea. he was diagnosed with poorly differentiated lung carcinoma by percutaneous needle biopsy. bronchoscopy and upper gastrointestinal endoscopy revealed a tracheoesophageal fistula (tef). long - lasting febrile neutropenia made it impossible to continue chemotherapy, but a course of radiotherapy (total 61 gy) was completed. the next endoscopy revealed closure of the tef. chemoradiotherapy (crt) has been reported to close tef in esophageal cancer, but the risk of a crt - induced worsening of the fistula has dissuaded physicians from using crt to treat tef in lung cancer patients. crt may serve as a palliative treatment for tef in lung cancer as well as esophageal cancer. |
bardet - biedl syndrome (bbs) is an autosomal recessive disorder characterized by central obesity, mental impairment, rod - cone dystrophy, polydactyly, hypogonadism in males, and renal abnormalities.1,2 the causative genes have been identified as bbs1 - 14 genes that encode proteins possibly linked to cilia function, but more than 20% of patients have no mutations found.3 the diagnosis is made only by the clinical phenotype with the presence of at least three major symptoms, however, it is often difficult partly because of age - dependent development of some symptoms. in the western countries, the prevalence of this disease ranges from 1/13,500 to 1/160,000.3 by contrast, only a few japanese patients have been reported in the english - language literature.46 renal fibrosis is one of the most devastating symptoms, ultimately leading to chronic renal failure requiring hemodialysis.7 the incidence of renal dysfunction or anomalies in previous reports varies considerably ranging from 20% to universal occurrence.2,7 an early detection of such abnormalities may be important for patients and guardians to prepare them. it may also be useful for prompt correct diagnosis of bbs, since the diagnosis of this disease is based on the accumulation of major symptoms as described above. we now report that two japanese patients with bbs had normal bun and creatinine level but elevated levels of cystatin c, a sensitive marker of glomerular filtration rate (gfr). a 20-year - old man (patient 1) had mental retardation (minimental state examination 23 ; normal > 24), rod - cone dystrophy, central obesity (height 158 cm, weight 63 kg, and bmi 25.2) and hypogonadism since the age of 5 years. his blood pressure was 131/85 mmhg, and his heart rate was 61 beats / min.. a 16-year - old boy (patient 2), the younger brother of patient 1, had polydactyly in addition to the symptoms described above (height 165 cm, weight 93 kg, and bmi 34.2). his blood pressure was 128/61 mmhg, and his heart rate was 77 beats / min. the symptoms of patients and probable autosomal recessive inheritance fulfilled the diagnostic criteria for bbs5. after obtaining informed consent, (tartu, estonia). the dna chip (version 5) covered 305 mutations from 14 genes causative for bbs and related diseases (bbs1, bbs2, bbs3, bbs4, bbs5, bbs6, bbs7, bbs8, bbs9, bbs10, bbs12, phf6, alms1, and gnas1), but identified no pathological alterations. nevertheless, because about one fifth of patients with clinically definite bbs have no identifiable mutations as described above and because the chip covered only mutations previously reported to be pathogenic, these results could not rule out the possibility of a diagnosis of bbs in our family. to detect morphological renal abnormalities, the patients underwent abdominal ct scans and abdominal sonography, with no apparent anomalies. blood and urine tests routinely performed in japan failed to identify any obvious abnormalities (table 1, upper rows). other laboratory data of the elder and younger patients included normal blood sugar levels (78 mg / dl and 81 mg / dl, respectively), normal total cholesterol levels (144 mg / dl and 131 mg / dl, normal 120220 mg / dl), unelevated triglyceride levels (28 mg / dl and 72 mg / dl, normal 30150 mg / dl), negative serum crp, and negative urine occult blood or glucose. the results showed elevated cystatin c concentrations in both patients and microalbuminuria in the elder patient (table 1, lower rows). cystatin c levels of the age- and sex - matched controls were also examined, the result of which showed 0.86 mg / l for an elder control and 0.91 mg / l for a younger control. we describe abnormal levels of serum cystatin c in two patients with bbs (table 1). cystatin c is a plasma protein with a molecular weight of 13.4 kda and belongs to the cysteine protease inhibitors.8 it is constantly synthesized in all types of cells, excreted into plasma, and filtered completely by the glomeruli. measurement of cystatin c more sensitively detects mild gfr abnormalities than that of creatinine, a more common but less sensitive marker of gfr,8 probably because the lower molecular weight of creatinine (113 da) facilitates its easier filtration in the glomeruli. in addition to the sensitivity, cystatin c is a more reliable marker than creatinine for detection of chronic renal disease, since creatinine levels are affected by many extra - renal patient - related factors such as muscle mass and consumption of cooked meat that is a source of creatinine.8 our patients had only mild increases in cystatin c. nevertheless, because cystatin c levels age - dependently increase with decreasing gfr, the values of our young patients seem sufficiently high for their ages.8 a urine albumin level increased only in the elder patient. patients with bbs occasionally manifest proteinuria,7 suggesting that patients had not only decreased gfr but also increased protein leakage. urine albumin is used to detect early phases of diabetic or hypertensive nephropathy.9 because neither of our patients showed apparent proteinuria, the elder patient may be in an early phase of protein leakage. in diabetes mellitus, timely treatment with an angiotensin - converting enzyme inhibitor, independently of rise in arterial blood pressure, is considered if improvement of glycaemic control and moderate decrease of dietary protein intake for 612 months have failed to reduce the albumin excretion rate.9 screening programs for microalbuminuria and early intervention can substantially modify the natural history of diabetic renal involvement and disease and possibly reduce the incidence of end - stage renal failure.9 in bbs, although such intervention has not been tested yet, we may consider similar protective methods for renal dysfunction. in conclusion, patients who have bbs with apparently normal kidney functions may have abnormal levels of cystatin c, facilitating an early detection of kidney dysfunctions that might be helpful for prompt correct diagnosis of bbs. however, because our study is based on the results of the small number of patients, conclusion must await further studies. | bardet - biedl syndrome (bbs) is an autosomal recessive disorder characterized by central obesity, mental impairment, rod - cone dystrophy, polydactyly, hypogonadism in males, and renal abnormalities. the causative genes have been identified as bbs1 - 14. in the western countries, the prevalence of this disease ranges from 1/13,500 to 1/160,000, while only a few japanese patients have been reported in the english - language literature. the incidence of renal dysfunction or anomalies in previous reports varies considerably ranging from 20% to universal occurrence. we here report that two japanese patients who had bbs with normal bun and creatinine levels had elevated levels of cystatin c, a sensitive marker of glomerular filtration rate. a urine albumin level increased only in the elder patient. thus, cystatin c may be useful for detecting renal abnormalities in patients with an apparent normal renal function. because this disease is diagnosed by accumulation of symptoms, such a sensitive marker might help early diagnosis of bbs. |
decades of targeted studies and recent progress in phosphoproteomics has resulted in a large body of protein phosphorylation data (2). determining how these phosphorylation sites change through time, for example during the cell cycle or following exposure to extracellular stimuli is now possible with techniques such as quantitative mass spectrometry (3). however, it remains difficult to determine which of the 518 human kinases is responsible for the phosphorylation of an observed site ; a glance at the phospho.elm database reveals that only about a quarter of known in vivo phosphorylation sites have been assigned as substrates of a specific kinase, and this fraction is constantly decreasing (2). this has motivated the development of numerous computational methods for predicting kinase substrate relations, for example, scansite (4), netphosk (5,6), predikin (7), predphospho (8) gps (9), ppsp (10) and kinasephos (11). these methods all rely on consensus sequence motifs recognized by the active site of the enzymes, represented by either position - specific scoring matrices (pssms), neural networks, support vector machines or other machine - learning representations. however, kinase specificity is known to also depend on other factors, such as auxiliary protein interactions, scaffolds, coexpression and colocalization (collectively referred to as we recently introduced a computational framework, networkin, which uses a probabilistic protein association network [string (12) ] to model the context of kinases and substrates ; combined with consensus sequence motifs, this gave a 2.5-fold leap in prediction accuracy over previous methods (13). here, we present a database of predicted kinase substrate relations based on the latest human phosphoproteome and protein association network from the phospho.elm (2), phosphosite (14) and string (12) databases. this database is available via a web interface at http://networkin.info, which enables the user to query the database for any kinases or substrates of interest, to submit new substrates and to explore the evidence underlying a prediction. the foundation of the networkin algorithm is the fact that signalling proteins are modular in nature, that is they consist of discrete functional modules, such as protein kinase domains and the linear peptide motifs they recognize and phosphorylate. this makes it possible to model the behaviour of such proteins by coupling the prediction of linear motifs to that of identifying the corresponding binding module in a network context. due to the improved capability of mass spectrometry to identify phosphorylation sites and other post - translational modifications, the scope of modelling these events has changed from predicting what could get phosphorylated to predicting what kinase phosphorylates which sites. the networkin algorithm is designed to work from a set of experimentally identified in vivo phosphorylation sites (although the algorithm can also be used ab initio). the precomputed results in the database are based on the latest human phosphoproteome from the phospho.elm and phosphosite databases (2,14) (figure 1). the release cycle of the database is approximately every 3 months due to the high throughput of mass - spectrometry - driven proteomics, and we intend to keep networkin up - to - date with future releases of phospho.elm and phosphosite. networkin starts from a set of known in vivo phosphorylation sites, which are obtained and kept synchronized with the phospho.elm and phosphosite databases to facilitate reciprocal database cross - links. we first compare these phosphorylation sites to consensus sequence motifs from the scansite and netphosk resources in order to predict possible kinase families responsible for the phosphorylation. second, we capture the kinase and substrate context using a probabilistic functional association network from the string database (12). substrate interaction network is stored in a sql database, which is accessible via a web interface. networkin starts from a set of known in vivo phosphorylation sites, which are obtained and kept synchronized with the phospho.elm and phosphosite databases to facilitate reciprocal database cross - links. we first compare these phosphorylation sites to consensus sequence motifs from the scansite and netphosk resources in order to predict possible kinase families responsible for the phosphorylation. second, we capture the kinase and substrate context using a probabilistic functional association network from the string database (12). substrate interaction network is stored in a sql database, which is accessible via a web interface. these data are processed through the networkin algorithm, which is implemented in python and c. the sites are classified by matching them to a motif collection (figure 1) based on the position - specific scoring matrices from scansite (4) (http://scansite.mit.edu) and the artificial neural networks from netphosk (5) (http://www.cbs.dtu.dk/services/netphosk). each consensus sequence motif is considered to be a representative for a family of closely related kinases ; for example, the netphosk cdk5 predictor is used for predicting possible phosphorylation sites for all cyclin - dependent kinases. within a proteome, kinases are identified and assigned to these families based on their best hit in a blastp (15) sequence similarity search against a set of 82 representative kinase domain sequences, which have been manually assigned to families. only hits with an e - value better than 10 and with at least 50% sequence identity are considered. to capture the biological context of a substrate, we use a probabilistic network of functional associations extracted from the string database (12) (http://string.embl.de, figure 1). this network is based on four fundamentally different types of evidence : genomic context (gene fusion, gene neighbourhood and phylogentic profiles), primary experimental evidence (physical protein interactions and gene co - expression), manually curated pathway databases, and automatic literature mining. we showed that the three latter evidence types are of comparable importance, whereas genomic context methods contribute very little towards the predictions made by networkin (13). as the curated pathway databases generally contain few errors, a confidence score of 0.9 is assigned to this type of evidence. the best candidate kinases within the appropriate kinase families are identified from a protein network of functional associations [generated using the string database (12) ] by calculating the proximity to the substrate for all kinases, defined as the probability of the most probable path connecting them (floyd warshall algorithm). the context is thus used as a filter that eliminates many of the false - positive predictions obtained from the sequence motifs and hence improves the prediction accuracy. however, the current algorithm is unable to recover sites that are missed by the sequence motifs (i.e. false - negative predictions). the database also contains cross - references to the phospho.elm (2), phosphosite (14) and string (12) databases. this database can be accessed via a web interface, which consists of a collection of cgi scripts, that query the database backend and format the results as xhtml for display in a web browser., we will explain the various features of the web interface, using the tumour suppressor 53bp1 as an example. for large - scale analysis or visualization, most users will probably prefer to download the complete set of predictions for human phosphoproteins, which is available in tab - separated and cytoscape format. for all other users, the primary entry point to networkin is its search interface shown in figure 2a. the user can select a specific substrate and/or kinase to view the corresponding subset of predictions ; in our example, we query for 53bp1 as the substrate and use the wildcard to obtain predictions for all kinases. the web interface also offers an advanced search form, which enables the user to pose much more refined queries. in either case, the search results will be presented as a table in which each row shows a predicted relation between a kinase and a specific phosphorylation site in a substrate. in case of 53bp1, we get a list of 78 predictions for 39 sites and 12 kinases ; the first 10 of these predictions are shown in figure 2b. for each prediction we list two scores, namely the context score and the motif score, both of which should preferably be high. it should be noted that the motif scores for different kinase families are not comparable ; in particular, motif scores from netphosk should not be compared with motif scores from scansite. for this reason, as the results of a single query may be extensive, the results can also be downloaded in the formats mentioned previously., this protein is chosen as the substrate protein to query the database for predictions relating any kinase to specific phosphorylation sites within 53bp1 (a). the system returns a total of 78 relations involving 12 different kinases that are predicted to phosphorylated 39 different sites, which are presented in a tabular view (b), (only the first 10 predictions are shown). these predictions can now be investigated in further detail by following either the links to the phospho.elm and phosphosite databases for curated knowledge related to the sites (c), or by following the links to the string network viewer to visualize the most probable path in the protein assocation network, which connects the kinase and the substrate (d). a researcher is interested in the 53bp1 tumour surpressor. from the homepage of networkin, this protein is chosen as the substrate protein to query the database for predictions relating any kinase to specific phosphorylation sites within 53bp1 (a). the system returns a total of 78 relations involving 12 different kinases that are predicted to phosphorylated 39 different sites, which are presented in a tabular view (b), (only the first 10 predictions are shown). these predictions can now be investigated in further detail by following either the links to the phospho.elm and phosphosite databases for curated knowledge related to the sites (c), or by following the links to the string network viewer to visualize the most probable path in the protein assocation network, which connects the kinase and the substrate (d). furthermore, the user can investigate the predictions in greater detail via the web interface. for each substrate, we link to phospho.elm or phosphosite where the user can find manually curated information on in vivo phosphorylation sites including, when known, the kinase(s) involved (figure 2c). to allow the user to investigate how a specific prediction was made by networkin, we provide a link to the string network viewer, in which the most probable path connecting the kinase and the substrate will be highlighted (figure 2d). alternatively, the user can select multiple predictions and display the network context for all the proteins involved. from the network viewer, this ability to thoroughly investigate individual predictions is particularly useful for interpreting non - obvious cases, which are often based on indirect links between the kinase and the substrate. although phospho.elm, phosphosite and hence networkin are kept up - to - date with new published phosphorylation sites, many researchers will be interested in predictions for their own, unpublished sites. we thus allow users to submit protein sequences and a corresponding set of phosphorylation sites for analysis ; although possible, we discourage submitting sequences without prior knowledge on phosphorylation. after uploading the data, the user will be presented with a confirmation page where potential data entry errors can be detected and fixed. the final predictions will be presented in a tabular format similar to the one used when querying the precomputed results in the database. many users are interested in specific kinases or substrates ; however, others may want to get an overview of the complete phosphorylation network. to facilitate this, all kinases and substrates are shown using a colour scale to signify their connectivity, namely the number of substrates for a given kinase or the number of kinases for a given substrate. by selecting one or more kinases, deselecting one kinase will deselect only the substrates specific for that kinase, keeping the other ones. we find this approach to be an intuitive way to gain insight into pleiotropic properties of kinases. similar to the search interface, map selections can be visualized in their network context. in the future, we intend to keep networkin up - to - date with the latest data on phosphorylation sites from phospho.elm and phosphosite, functional associations from string and consensus sequence motifs from scansite and other sources. furthermore, the algorithm will be extended to take into account docking motifs (e.g. for map kinases) and phosphorylation - dependent binding modules (e.g. sh2, ptb and brct domains), which is expected to both improve the prediction accuracy and facilitate more comprehensive modelling of signalling networks. we also intend to extend the method to include phosphatases as soon as data for this is available to us. although, networkin is so far specifically aimed at protein phosphorylation, many other post - translational modifications are mediated by enzymes that recognize short linear motifs. for example, the modifications of histone tails through acetylation, methylation and phosphorylation has been shown to be context dependent (16), and acetylated or methylated sites in turn bind interaction domains, such as bromo- or chromodomains. extending the resource to cover also other post - translational modifications is thus a long - term goal. | protein kinases control cellular responses by phosphorylating specific substrates. recent proteome - wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. systematically assigning all 518 human kinases to all these sites is a challenging problem. the networkin database (http://networkin.info) integrates consensus substrate motifs with context modelling for improved prediction of cellular kinase substrate relations. based on the latest human phosphoproteome from the phospho.elm and phosphosite databases, the resource offers insight into phosphorylation - modulated interaction networks. here, we describe how networkin can be used for both global and targeted molecular studies. via the web interface users can query the database of precomputed kinase substrate relations or obtain predictions on novel phosphoproteins. the database currently contains a predicted phosphorylation network with 20 224 site - specific interactions involving 3978 phosphoproteins and 73 human kinases from 20 families. |
high - density lipoprotein (hdl), known as antiatherosclerotic lipoprotein, is also involved in innate immunity. for instance, hdl binds to lipopolysaccharides (lps) and lipoteichoic acid (lta) derived from microorganisms and neutralizes the physiological activity of these molecules [2, 3 ]. additionally, hdl is known to have a bactericidal activity toward yersinia enterocolitica serotype o:3 ; this activity is mediated by the complement system. mycobacterium avium (m. avium) is a species of nontuberculous mycobacteria and causes opportunistic infections in immunocompromised hosts. although the incidence of m. avium infection is increasing worldwide, its pathogenicity remains poorly understood unlike that of m. tuberculosis., cholesterol performs an important function in the invasion and survival of mycobacteria inside macrophages [711 ]. cholesterol accumulation in the host cell membrane is observed at the entry point of m. tuberculosis and of other mycobacteria, and cholesterol depletion inhibits the invasion of cells by mycobacteria. after phagocytosis of mycobacteria, they are enveloped by the cell membrane containing cholesterol - rich domains ; tryptophan aspartate - containing coat protein (taco) is recruited to the phagosomes and prevents the fusion of these organelles with lysosomes. consequently, the engulfed mycobacteria evade degradation by lysosomes and survive inside the host cell. the invading mycobacteria modulate lipid metabolism in the host cell and promote formation of lipid droplets (lds), which are mainly composed of neutral lipids such as triacylglycerol and cholesteryl ester. lds physiologically contribute to lipid storage and lipid metabolism and are available to mycobacteria as a carbon source [10, 14 ]. scavenger receptor type b1 (sr - b1), a receptor of apolipoprotein a - i (apoa - i) present in hdl as a main component, is generally known to perform the function of transferring esterified cholesterol from matured hdl into the cytosol in accordance with the cholesterol gradient. sr - b1 was also identified as a nonopsonic phagocytic receptor for mycobacteria because suppression of sr - b1 expression attenuates phagocytosis of mycobacteria [16, 17 ]. we screened various bacteria for binding with hdl and found that m. avium exhibits a stronger interaction with hdl than do other gram - negative bacilli. the fact that hdl binds to m. avium has never been reported and the effect for innate immunity is unclear. the aim of the present study was to elucidate the molecular mechanism of hdl 's binding to m. avium and the physiological meaning of this interaction. m. avium (atcc 700737) was cultured for 3 weeks in middlebrook 7h9 broth (difco) supplemented with the oleic acid - albumin - dextrose complex (oadc ; becton dickinson). gram - negative bacteria (isolated at the clinical laboratory, medical hospital of tokyo medical and dental university) were cultured on trypticase - soy - agar with 5% sheep blood (becton dickinson) before use. the thp-1 cell line was obtained from atcc (manassas, va) and maintained at 210 10 cells / ml in the rpmi 1640 medium (sigma - aldrich) supplemented with 10% heat - inactivated fetal bovine serum (invitrogen), with a penicillin - streptomycin - l - glutamine solution (wako), and 1x nonessential amino acids (gibco). the thp-1 cells were induced to differentiate into thp-1 macrophages by phorbol myristate acetate (pma, sigma - aldrich) for 72 h. after washing with phosphate - buffered saline (pbs), the thp-1 macrophages were incubated in the serum - free rpmi 1640 supplemented with nutridoma - sp (roche) and then infected with m. avium (multiplicity of infection [moi ] 20 : 1) with or without hdl (50 g protein / ml) for 24 h at 35c in a humidified atmosphere containing 5% co2. hdl (1.0631.210 g / ml) hdl and apoa - i were dialyzed against pbs and stored at 4c and 20c, respectively, until use. bacteria (3 10) were washed with pbs and then incubated with 40 l of normal human serum (nhs), heat - inactivated serum (his), hdl, apoa - i, or pbs for 10 min at 37c. after washing with pbs, the bacteria were mixed with lysis buffer (50 mm tris - hcl ph 8.0, 150 mm nacl, 0.1% sds, and 0.5% sodium deoxycholate). the lysates were then analyzed by electrophoresis on 12.5% sds - polyacrylamide gels, and the separated proteins were transferred onto polyvinylidene fluoride (pvdf) membranes (millipore). apoa - i was detected with a goat anti - apoa - i polyclonal antibody (academy bio - medical company) followed by a peroxidase- (pod-) conjugated rabbit anti - goat igg antibody (medical & biological laboratories). finally, apoa - i was visualized using hydroperoxide and 3,3-diaminobenzidine as the substrate. this analysis was carried out using a previously described method with a slight modification. lipids were extracted from the outer cell wall of m. avium by means of folch 's extraction procedure and were then spotted onto a nitrocellulose membrane. the membrane was sequentially incubated with 5% (w / v) skim milk and 50 g / ml hdl in 10 mm tris - hcl (ph 8.0) containing 140 mm nacl and 0.1% tween 20 (tbs - t) for 1 h and 5 h, respectively, at room temperature. the membrane was washed with tbs - t and then incubated with a pod - conjugated anti - apoa - i polyclonal antibody (cosmo bio). the lipids obtained from m. avium were spotted onto three silica gel 60 plates (merck millipore). the tlc plates were simultaneously developed with chcl3/ch3oh (95 : 5, v / v). after the run was completed, one of the plates was treated with 20% sulfuric acid solution to visualize the separated lipids, and the other 2 were subjected to tlc blot analysis according to a previously described method. one membrane was incubated with 1% bovine serum albumin (bsa) in tbs - t and subsequently incubated with 83 g / ml of apoa - i in tbs - t at 4c overnight. the membrane was washed with tbs - t and incubated with the pod - conjugated anti - apoa - i polyclonal antibody (binding site). apoa - i was visualized as described above. to analyze by matrix - assisted laser desorption ionization time - of - flight mass spectrometry (maldi - tof ms), the lipid bound to apoa - i was extracted from the other membrane by means of chcl3/ch3oh (2 : 1, v / v) according to comparison with the position of the apoa - i spot on the membrane visualized by the tlc blotting described above. live m. avium cells (3 10/ml) were briefly sonicated to disperse clumps and mixed with the middlebrook 7h9 broth supplemented with oadc with or without hdl (50 g protein / ml). absorbance of the medium at 530 nm was monitored for 144 h. this assay was carried out according to the previously described method [22, 23 ]. briefly, m. avium was autoclaved and stained with 0.5 mg / ml fluorescein isothiocyanate (fitc, dojin laboratories) in pbs for 30 min. after exhaustive washing, we resuspended the stained m. avium at 3 10/ml in pbs and then stored the suspension at 80c until use. thp-1 macrophages (10/well) were cultured with the fitc - conjugated m. avium in the presence of hdl (50 g protein / ml), apoa - i (50 g / ml), or bsa (50 g / ml). after quenching extracellular fluorescence using trypan blue (1.2 mg / dl), we fixed the cells with cellfix (becton dickinson) and subjected them to flow cytometric analysis on a navios flow cytometer (beckman coulter). the fitc - conjugated e. coli, which was prepared by the similar method described above, was also analyzed by the phagocytosis assay as the reference. thp-1 macrophages (10/well) were infected with live m. avium (moi 20 : 1) with or without hdl (50 g protein / ml) for 24 h. the cells engulfing m. avium were washed three times with pbs and further cultured in the absence of m. avium and hdl for 0, 24, and 48 h. then, thp-1 macrophages were lysed with pbs containing 0.1% sds to recover the engulfed m. avium. the lysates diluted 100-fold with pbs were cultured on middlebrook 7h11 agar plates (difco) supplemented with oadc. thp-1 macrophages (5 10/well) were infected with live m. avium (moi 20 : 1) with or without hdl (50 g protein / ml) for 24 h. after being fixed with a 10% formaldehyde solution for 15 min, they are stained with oil red o (oro) for 5 min. the cells were counterstained with mayer 's hematoxylin for 2 min in samples to be examined under a light microscope. for quantification of lds amount, infected thp-1 macrophages (5 10/well) were stained with only oro as described above. oro was then extracted in 500 l of 100% isopropyl alcohol, and absorbance was measured at 540 nm as described previously. all of the values were shown by the mean standard error of the mean (sem). cfus, the number of lds, and the others were statistically analyzed by paired student 's t - test, kruskal - wallis test, and multifactorial analysis of variance (anova) followed by tukey post hoc tests, respectively. the protocol of this study was approved by the research ethics committee of tokyo medical and dental university (decision number 1491). to screen the bacteria for interaction with hdl, some species of bacteria that were incubated with normal human serum (nhs) were analyzed by western blotting with an anti - apoa - i antibody (figure 1(a)). the apoa - i signal was well visible in the lysate of m. avium incubated with nhs but only barely visible in samples from other gram - negative bacteria. to confirm the direct interaction between apoa - i and m. avium, we carried out western blotting of the lysate of m. avium incubated with heat - inactivated serum (his), nhs, isolated hdl, or purified apoa - i (figure 1(b)). the apoa - i signal was well visible after incubation not only with nhs but also with the his, hdl, and apoa - i, indicating that hdl bound directly to m. avium via apoa - i. we hypothesized that apoa - i interacts with the abundant lipids on the m. avium cell wall because of amphipathic -helices present in the apoa - i molecule. to test this hypothesis, we performed a western blot assay of the lipids extracted from the outer cell wall of m. avium (figure 2(a)). apoa - i was detected in the spots of the extracted lipids incubated with hdl and the intensity of signals increased with the amount of spotted lipids (at a constant hdl concentration). the tlc blot assay was carried out to determine whether apoa - i binds to a specific lipid molecule of m. avium (figure 2(b)). several lipid spots on the tlc plate were nonspecifically visualized by a charring reagent (figure 2(b), left). one of those spots visibly interacted with apoa - i by tlc blot analysis (figure 2(b), right), indicating that apoa - i formed a complex with this specific lipid of m. avium. the lipids extracted from the apoa - i - positive spot of the tlc blot were analyzed by maldi - tof ms (supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4353620). two prominent peaks at m / z 1243.8 and m / z 1271.8 were observed along with the several peaks with an interval of 28 da, suggesting that the lipid that bound to apoa - i included a variety of fatty acid moieties. the growth curve analysis of m. avium in the presence or absence of hdl was performed to determine whether hdl directly kills m. avium (figure 3). no significant difference was observed in the absorbance of the medium with or without hdl for 6 days. this finding indicated that hdl itself had no bactericidal activity toward m. avium. to find out whether hdl participates in the nonopsonic phagocytosis of m. avium by thp-1 macrophages, we used flow cytometry to analyze the effect of hdl on the number of thp-1 macrophages engulfing fitc - conjugated m. avium (figure 4(a)). the percentage of fitc - positive cells decreased with the addition of hdl and apoa - i but did not change significantly after addition of bsa. the percentage of fitc - positive cells which is obtained using fitc - conjugated e. coli indicated a similar result to that of fitc - conjugated m. avium ; however the addition of hdl did not decrease the percentage of fitc - positive cells. when the number of fitc - positive cells of control samples was set to 100%, hdl and apoa - i decreased the number of fitc - positive cells to 68.7% 1.5% and 83.3% 0.4%, respectively (figure 4(b)). bsa did not affect the engulfment of m. avium by thp-1 macrophages (101.8% 2.0%). in contrast, fitc - positive cells did not decrease, if anything increased, in the case of fitc - conjugated e. coli. assay of cfus was performed to assess the number of live m. avium inside thp-1 macrophages by means of m. avium recovered from the lysed thp-1 macrophages (figure 5(a)). the results that were expressed in cfu / well were (8.5 1.2) 10 and (11.7 0.6) 10 (mean se) in the presence and the absence of hdl, respectively. this observation is consistent with the result obtained by the flow cytometric analysis (figure 4), namely, that hdl attenuated the engulfment of m. avium by thp-1 macrophages. to assess the survival rate of m. avium inside thp-1 macrophages, the infected thp-1 macrophages were further cultured in the absence of m. avium and hdl for 24 and 48 h. the number of cfus per well indicated a tendency to decrease in a time - dependent manner (figure 5(b)). however, no significant difference was observed in the decreasing profiles between the presence and the absence of hdl, suggesting that the coexistence of hdl could not affect bacterial killing after ingestion by thp-1 macrophage. after the infection of thp-1 macrophages with live m. avium, lds inside the cells were stained with oil red o (oro) (figure 6(a)). the numbers of lds per cell were significantly increased by the coexistence of live m. avium and hdl (figure 6(b)). lds amounts were also estimated by the absorbance at 540 nm of the extracted oro (figure 6(c)). the existence of hdl or m. avium alone indicated no significant effect in the quantity of oro compared with the control. however, the coexistence of hdl and m. avium caused an increase in the lds amount. in this study, we found that hdl binds to m. avium via the interaction between apoa - i (the main structural component of hdl) and a specific lipid molecule of m. avium. although a structure of the specific lipid molecule was analyzed by maldi - tof - ms / ms (data not shown), we failed to identify it among the known lipid molecules of m. avium. it should be noted that m. avium causes inflammation and increases permeability of blood vessels, increasing the chance of interaction with leaked hdl. if this specific lipid of m. avium plays a role in host pathogenesis, hdl may neutralize the activity of this lipid, just as in the case of lps and lta. it could be important to identify the lipid molecule in order to elucidate a participation of hdl in m. avium infection. we explored the influence of hdl 's binding to m. avium on an innate immunity in experiments with thp-1 macrophages. hdl does not directly kill m. avium, but the presence of hdl significantly decreases the number of thp-1 macrophages engulfing m. avium, and hdl indicated no effect on bacterial killing after ingestion. sr - b1, known as an apoa - i receptor, also plays a role in nonopsonic phagocytosis of mycobacteria [16, 17 ]. according to these observations, we can hypothesize that hdl competitively inhibits the binding of m. avium to sr - b1. in this study, we did not analyze sr - b1, and further research is needed to assess the interaction between m. avium and hdl, including the participation of sr - b1. when mycobacteria are recognized by toll - like receptors 2 or 6 (tlr2/6), lds formation is enhanced by tlr signaling [25, 26 ]. moreover, some researchers proposed a mechanism behind the enhancement of lds formation during mycobacterial infection : upregulation of a series of scavenger receptors including sr - b1. in our study, m. avium infection did not apparently upregulate lds formation under serum - free condition ; however, the coexistence of hdl increased the number and the amount of lds in thp-1 macrophages, suggesting that hdl - cholesterol is utilized as a raw material for lds formation. two mechanisms may explain this observation. according to one mechanism, hdl - cholesterol is internalized together with m. avium via phagocytosis. according to the other, although approximately 30% of the engulfment of m. avium by thp-1 macrophages was attenuated by the coexistence of hdl (figure 4), the hdl - cholesterol influx is enhanced by upregulation of sr - b1 in thp-1 macrophages engulfing m. avium. consequently, tlr signaling and hdl - cholesterol influx via sr - b1 may promote lds formation in the presence of both m. avium and hdl. on the basis of the present study, we believe that hdl plays a crucial role in the infection of thp-1 macrophages by m. avium. identification of the lipid molecule of m. avium that binds to hdl via apoa - i and the analysis of the expression of the scavenger receptor, sr - b1, in an upcoming project, should shed some light on the pathogenesis of m. avium infections and on the relevant immune response. hdl affects m. avium infection through the binding between apoa - i, the main component of hdl, and a specific lipid of m. avium. | high - density lipoprotein (hdl) is involved in innate immunity toward various infectious diseases. concerning bacteria, hdl is known to bind to lipopolysaccharide (lps) and to neutralize its physiological activity. on the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. however, the pathogenicity of mycobacterium avium (m. avium) infection, which tends to increase worldwide, remains poorly studied. here we report that hdl indicated a stronger interaction with m. avium than that with other gram - negative bacteria containing abundant lps. a binding of apolipoprotein (apo) a - i, the main protein component of hdl, with a specific lipid of m. avium might participate in this interaction. hdl did not have a direct bactericidal activity toward m. avium but attenuated the engulfment of m. avium by thp-1 macrophages. hdl also did not affect bacterial killing after ingestion of live m. avium by thp-1 macrophage. furthermore, hdl strongly promoted the formation of lipid droplets in m. avium - infected thp-1 macrophages. these observations provide new insights into the relationship between m. avium infection and host lipoproteins, especially hdl. thus, hdl may help m. avium to escape from host innate immunity. |
the promotion as an associate professor (ap) represents the final academic step followed only by a full professorship at german universities. the academic degree of an associate professor is invested by faculties to persons, which have received a doctorates degree, have qualified as a professor by writing a habilitation thesis and obtained postdoctoral lecture qualification. the academic degree as an ap can not be distinguished by outsiders to a full university professor as the special prefix however, at most of the german medical faculties the nomination for an associate professor does not only involve the completion of a certain time frame, but is also connected to a continuous and outstanding research performance as well as academic teaching. as pabst and strate have already noticed in 2000 and as an actual wikipedia description of german associate professors (auerplanmiger professor) further delineates, that the title of an associate professor is invested above average especially in medicine, next to medical doctorates (dr.med.) and assistant professors (privatdozent) (http://de.wikipedia.org/wiki/professur),. the requirements for assistant professors to become an associate professor, however, differ substantially between the 35 german medical faculties. in the year 2000 several points for a standardization and simplification of the preconditions to become an associate professor have already been discussed. the basis for these postulations has been a first appraisal of different parameters of the regulations for associate professors at german medical faculties at that time. to establish a solid comparability, we performed a systematic qualitative as well as quantitative scoring of different parameters of the requirements to become an associate professor at german medical faculties for the first time. inquiries of the regulations to become an associate professor (ap) the ap - regulations have been downloaded of the respective internet sites of the different german medical faculties or have been ordered and sent via e - mail. in the case that special information could not be gained from the regulations, medical faculties have again been contacted via e - mail or telephone for respective details. using this technique 35 out of 36 (97%) ap - regulations could be included into this evaluation. in one case, the authors could not obtain the regulation neither electronically nor via telephone as the respective document must be used as an internal university document for assistant professors only. the following seven parameters are preconditions for the appointment of an ap at german medical faculties, which have been analyzed, evaluated and described. the regulations have been evaluated with special interest to the quality of the performance of the candidates. the respective criteria have been summarized in groups of similar requirements and have then been assessed with a simple scoring system with increasing precision of the information or requirement description as similarly published recently,. herein, the ap - regulation of the medical faculties could reach scores between 0 and 20 points. scientific publication 0 points : no detailed information available1 point : no information of necessary numbers 2 points : information on quality published in appropriate scientific journals or listed in current contents 3 points : information on authorship and quality preferably as single author or manuscripts with creativity that attest scientific competence 4 points : information on authorship and quality sufficient and/or adequate quantity of publications in renowned international scientific journals 6 - 12 original articles in renowned international scientific journals 5 points : detailed information on quantity, quality and authorships 10 publications, thereof 6 as first author in peer reviewed journals 8 - 10 high ranked original articles predominantly as first author 10 publications as first author, thereof 5 in renowned scientific peer reviewed journals 12 original articles, 8 first authorships, impact factor 10 in renowned scientific peer reviewed journals 0 points : no detailed information available 1 point : no information of necessary numbers 2 points : information on quality published in appropriate scientific journals or listed in current contents 3 points : information on authorship and quality preferably as single author or manuscripts with creativity that attest scientific competence 4 points : information on authorship and quality sufficient and/or adequate quantity of publications in renowned international scientific journals 6 - 12 original articles in renowned international scientific journals 5 points : detailed information on quantity, quality and authorships 10 publications, thereof 6 as first author in peer reviewed journals 8 - 10 high ranked original articles predominantly as first author 10 publications as first author, thereof 5 in renowned scientific peer reviewed journals 12 original articles, 8 first authorships, impact factor 10 in renowned scientific peer reviewed journals 2. research performance 0 points : no detailed information available1 point : unclear description of achieved research work2 points : qualified research work or successful research for several years 3 points : qualified research work with raised third - party funds4 points : qualified research work with raised third - party funds and presentation of future research projects and possible perspectives at the respective institution in research and teaching 0 points : no detailed information available 1 point : unclear description of achieved research work 2 points : qualified research work or successful research for several years 3 points : qualified research work with raised third - party funds 4 points : qualified research work with raised third - party funds and presentation of future research projects and possible perspectives at the respective institution in research and teaching 0 points : no detailed information available or optional documentation1 point : documentation without specifications 2 points : with quality requirements (successful in teaching)3 points : with quantitative requirements such as one semester with documentation of at least 15h lecture activity 4 points : with quantitative requirements such as several semesters with at least 4 semesters required courses or 4 years of active practical activity in teaching 5 points : with quantitative requirements such as several semesters and additional evaluation of the didactic ability by students or faculty members 0 points : no detailed information available or optional documentation 1 point : documentation without specifications 2 points : with quality requirements (successful in teaching) 3 points : with quantitative requirements such as one semester with documentation of at least 15h lecture activity 4 points : with quantitative requirements such as several semesters with at least 4 semesters required courses or 4 years of active practical activity in teaching 5 points : with quantitative requirements such as several semesters and additional evaluation of the didactic ability by students or faculty members 4. scientific conference presentations /posters 0 points : no detailed information on scientific presentations or posters1 point : list on scientific presentations or posters2 points : detailed list on scientific presentations or posters with information on quantity, quality and authorship or specification on the five most important presentations 0 points : no detailed information on scientific presentations or posters 1 point : list on scientific presentations or posters 2 points : detailed list on scientific presentations or posters with information on quantity, quality and authorship or specification on the five most important presentations 5. reduction of the minimum time as an assistant professor 0 points : no reduction possible or no description1 point : the reduction of the minimum time as an assistant professor is possible by being listed on the nomination list to a full university professorship or other excellent or extraordinary achievements 0 points : no reduction possible or no description 1 point : the reduction of the minimum time as an assistant professor is possible by being listed on the nomination list to a full university professorship or other excellent or extraordinary achievements 6. recommendation of candidates by faculty members 0 points : recommendation is not necessary ; application by candidate itself is possible1 point : the recommendation of respective candidates by one or more members of the faculty is expected 0 points : recommendation is not necessary ; application by candidate itself is possible 1 point : the recommendation of respective candidates by one or more members of the faculty is expected 7. evaluation of the capabilities being an associate professor 0 points : no evaluation necessary or no detailed information available 1 point : evaluation by to internal reviewers of the faculty 2 points : evaluation by several reviewers, thereof one reviewer has to be from another university or medical school (external review) 0 points : no evaluation necessary or no detailed information available 1 point : evaluation by to internal reviewers of the faculty 2 points : evaluation by several reviewers, thereof one reviewer has to be from another university or medical school (external review) the ap - regulations of all 35 german medical faculties in 2010 have been 3.70.6 years old ; with the oldest one being 15 years old and four faculties presented actual regulations from the year 2010. from the 35 included ap - regulations 7 have been valid for the entire university (20%), while the majority (n=28) has been especially designed for medical faculties (80%). the total score for the requirements to become an associate professor at german medical faculties is 13.50.6 (95% confidence interval 12.2 14.7), while three faculties could reach 19 of 20 possible scoring points. the 35 included ap - regulations of german medical faculties have been analyzed and scored for seven main items as summarized in table 1 (tab. 1). in agreement with all analyzed actual regulations, only the criterion of a respective teaching activity has been designated by all medical faculties (100%). in 94% candidates mandatorily need adequate research achievements. furthermore, 29 out of 35 medical faculties claim an evaluation of the candidates and at 24 locations (69%) the candidate may only apply for a nomination with a recommendation of a faculty member. reduction of the minimum time as an assistant professor is possible at 16 medical faculties after second to third place listing on the nomination list in an appointment process for a full university professorship. scientific publication. in only two ap - regulations most of the medical faculties (51%) could reach the highest score value (5 points) in this category, with detailed information on quantity, quality and first or last authorships (see table 2 (tab. are requested of the assistant professors for a successful application in the respective minimum time. three medical faculties (8.6%) do not give any information on the required research work (0 points) and in seven ap - regulations (20%) only imprecise information is given (1 point ; 20%). after reaching successful postdoctoral lecture qualification or nomination for an assistant professorship, eight medical faculties (22.9%) claim ongoing scientific work for several years. detailed information on the respective research performance with successful raise of third - party funds is given in 31.4%. four medical faculties additionally demand a perspective concept on future research and teaching performance of the candidates at the respective institutions (5 points ; see table 2 (tab. 2)).teaching activity. the actual evaluation of the teaching activity in the included ap - regulations feature detailed information on form, extent and content of the required teaching modalities for becoming an associate professor. while ten years ago only ten medical faculties (28%) have given some information on the minimum teaching qualifications, the actual analysis of the ap - regulations reveals, that specific information is given in more than 80% (n=28) of the regulations, reaching score values of 3 (see table 2 (tab. the candidates are allowed to perform every kind of teaching (lectures, seminars, practical courses). at six medical faculties, the ap - regulations claim an additional evaluation of the lectures by students or other faculty members (see table 2 (tab. the minimum time requirements, however, differ substantially and are markedly increased at some universities since the year 2000, ranging from 1.5 to 4 semester periods per week (spw). at some medical faculties, candidates, which act predominantly in patient care, are allowed to reduce the minimum spw by 50%. a further criterion for a successful teaching performance is the mentoring of doctorate or diploma theses. at 21 medical faculties this is a favored or demanded issue, correlating with an increase of 87.5% in comparison to the year 2000 data,.scientific conference presentation / posters. an additional criterion for the successful nomination of an associate professor are scientific presentations or posters. 54.3% of medical faculties do not attach importance on the active participation on scientific congresses or meetings (see figure 1 (fig. a (detailed) listing of all oral or poster presentations is required for the ap - application. the nomination of an associate professor can be invested from universities to persons, who have shown outstanding performance and qualifications. the nomination requires a successful and independent performance in research and teaching over several years. after the nomination for an assistant professor or reaching postdoctoral lecture qualification, german medical faculties expect different minimum times before these candidates may apply for the associate professor nomination, averaging 4.50.3 years (95% confidence interval 3.8 5.8). this period of time might be reduced for candidates, which have shown extraordinary scientific performance at 54.3% of german medical faculties. this is especially the case, if the candidates have been listed on the nomination list in an appointment process for a full university professorship (secundo et tertio loco). furthermore, it can also be reduced, when an outstanding scientific price could be won in the area of expertise or in medicine in general. at one medical school, the ap - regulations point out that the rejection of a first place listing (primo loco) in an appointment process might directly lead to the nomination as an associate professor.recommendation of candidates by faculty members. at 25 medical faculties (28.6%) assistant professors, which have fulfilled the respective requirements, are not allowed to apply for the ap - nomination. the application process may only start by a recommendation of members or representatives of the faculty or full university professors. these persons are requested to write a recommendation letter or favor the candidates with corresponding documents for the ap - nomination. in all other faculties the candidates are allowed to hand in all documents which are required for the nomination process on their own (see figure 1 (fig. the abilities and performance for assistant professors, which apply for the nomination to become an associate professor, have to be evaluated by reviewers at most medical faculties. an evaluation is not necessary or is not described in the ap - regulations (0 points) at five medical faculties. the evaluation of the candidates by internal reviewers only, is necessary at three faculties (1 point). the majority (n=27), however, requires an evaluation process, which is supported by an external reviewer from a different university. on average 21 reviewers have to evaluate the candidates, focusing not only on the required issues, but also on the general ability for a nomination as an associate professor. scientific publication. in only two ap - regulations most of the medical faculties (51%) could reach the highest score value (5 points) in this category, with detailed information on quantity, quality and first or last authorships (see table 2 (tab. are requested of the assistant professors for a successful application in the respective minimum time. three medical faculties (8.6%) do not give any information on the required research work (0 points) and in seven ap - regulations (20%) only imprecise information is given (1 point ; 20%). after reaching successful postdoctoral lecture qualification or nomination for an assistant professorship, eight medical faculties (22.9%) claim ongoing scientific work for several years. detailed information on the respective research performance with successful raise of third - party funds is given in 31.4%. four medical faculties additionally demand a perspective concept on future research and teaching performance of the candidates at the respective institutions (5 points ; see table 2 (tab. the actual evaluation of the teaching activity in the included ap - regulations feature detailed information on form, extent and content of the required teaching modalities for becoming an associate professor. while ten years ago only ten medical faculties (28%) have given some information on the minimum teaching qualifications, the actual analysis of the ap - regulations reveals, that specific information is given in more than 80% (n=28) of the regulations, reaching score values of 3 (see table 2 (tab. the candidates are allowed to perform every kind of teaching (lectures, seminars, practical courses). at six medical faculties, the ap - regulations claim an additional evaluation of the lectures by students or other faculty members (see table 2 (tab. 2)). the minimum time requirements, however, differ substantially and are markedly increased at some universities since the year 2000, ranging from 1.5 to 4 semester periods per week (spw). at some medical faculties, candidates, which act predominantly in patient care, a further criterion for a successful teaching performance is the mentoring of doctorate or diploma theses. at 21 medical faculties this is a favored or demanded issue, correlating with an increase of 87.5% in comparison to the year 2000 data, an additional criterion for the successful nomination of an associate professor are scientific presentations or posters. 54.3% of medical faculties do not attach importance on the active participation on scientific congresses or meetings (see figure 1 (fig. 1)). in other respects, at 16 medical faculties a (detailed) listing of all oral or poster presentations reduction of the minimum time for the nomination as an associate professor. the nomination of an associate professor can be invested from universities to persons, who have shown outstanding performance and qualifications. the nomination requires a successful and independent performance in research and teaching over several years. after the nomination for an assistant professor or reaching postdoctoral lecture qualification, german medical faculties expect different minimum times before these candidates may apply for the associate professor nomination, averaging 4.50.3 years (95% confidence interval 3.8 5.8). this period of time might be reduced for candidates, which have shown extraordinary scientific performance at 54.3% of german medical faculties. this is especially the case, if the candidates have been listed on the nomination list in an appointment process for a full university professorship (secundo et tertio loco). furthermore, it can also be reduced, when an outstanding scientific price could be won in the area of expertise or in medicine in general. at one medical school, the ap - regulations point out that the rejection of a first place listing (primo loco) in an appointment process might directly lead to the nomination as an associate professor. assistant professors, which have fulfilled the respective requirements, are not allowed to apply for the ap - nomination. the application process may only start by a recommendation of members or representatives of the faculty or full university professors. these persons are requested to write a recommendation letter or favor the candidates with corresponding documents for the ap - nomination. in all other faculties the candidates are allowed to hand in all documents which are required for the nomination process on their own (see figure 1 (fig. evaluation of the capabilities being an associate professor. the abilities and performance for assistant professors, which apply for the nomination to become an associate professor, an evaluation is not necessary or is not described in the ap - regulations (0 points) at five medical faculties. the evaluation of the candidates by internal reviewers only, is necessary at three faculties (1 point). the majority (n=27), however, requires an evaluation process, which is supported by an external reviewer from a different university. on average 21 reviewers have to evaluate the candidates, focusing not only on the required issues, but also on the general ability for a nomination as an associate professor. the 35 included ap - regulations of german medical faculties have been analyzed and scored for seven main items as summarized in table 1 (tab. 1). in agreement with all analyzed actual regulations, only the criterion of a respective teaching activity has been designated by all medical faculties (100%). in 94% candidates mandatorily need adequate research achievements. furthermore, 29 out of 35 medical faculties claim an evaluation of the candidates and at 24 locations (69%) the candidate may only apply for a nomination with a recommendation of a faculty member. reduction of the minimum time as an assistant professor is possible at 16 medical faculties after second to third place listing on the nomination list in an appointment process for a full university professorship. scientific publication. in only two ap - regulations most of the medical faculties (51%) could reach the highest score value (5 points) in this category, with detailed information on quantity, quality and first or last authorships (see table 2 (tab. are requested of the assistant professors for a successful application in the respective minimum time. three medical faculties (8.6%) do not give any information on the required research work (0 points) and in seven ap - regulations (20%) only imprecise information is given (1 point ; 20%). after reaching successful postdoctoral lecture qualification or nomination for an assistant professorship, eight medical faculties (22.9%) claim ongoing scientific work for several years. detailed information on the respective research performance with successful raise of third - party funds is given in 31.4%. four medical faculties additionally demand a perspective concept on future research and teaching performance of the candidates at the respective institutions (5 points ; see table 2 (tab. 2)).teaching activity. the actual evaluation of the teaching activity in the included ap - regulations feature detailed information on form, extent and content of the required teaching modalities for becoming an associate professor. while ten years ago only ten medical faculties (28%) have given some information on the minimum teaching qualifications, the actual analysis of the ap - regulations reveals, that specific information is given in more than 80% (n=28) of the regulations, reaching score values of 3 (see table 2 (tab. the candidates are allowed to perform every kind of teaching (lectures, seminars, practical courses). at six medical faculties, the ap - regulations claim an additional evaluation of the lectures by students or other faculty members (see table 2 (tab. the minimum time requirements, however, differ substantially and are markedly increased at some universities since the year 2000, ranging from 1.5 to 4 semester periods per week (spw). at some medical faculties, candidates, which act predominantly in patient care, in addition, one medical school demands the further education in didactics. a further criterion for a successful teaching performance is the mentoring of doctorate or diploma theses. at 21 medical faculties this is a favored or demanded issue, correlating with an increase of 87.5% in comparison to the year 2000 data,.scientific conference presentation / posters. an additional criterion for the successful nomination of an associate professor 54.3% of medical faculties do not attach importance on the active participation on scientific congresses or meetings (see figure 1 (fig. a (detailed) listing of all oral or poster presentations is required for the ap - application. the nomination of an associate professor can be invested from universities to persons, who have shown outstanding performance and qualifications. the nomination requires a successful and independent performance in research and teaching over several years. after the nomination for an assistant professor or reaching postdoctoral lecture qualification, german medical faculties expect different minimum times before these candidates may apply for the associate professor nomination, averaging 4.50.3 years (95% confidence interval 3.8 5.8). this period of time might be reduced for candidates, which have shown extraordinary scientific performance at 54.3% of german medical faculties. this is especially the case, if the candidates have been listed on the nomination list in an appointment process for a full university professorship (secundo et tertio loco). furthermore, it can also be reduced, when an outstanding scientific price could be won in the area of expertise or in medicine in general. this may reduce the time by four years. at one medical school, the ap - regulations point out that the rejection of a first place listing (primo loco) in an appointment process might directly lead to the nomination as an associate professor.recommendation of candidates by faculty members. at 25 medical faculties (28.6%) assistant professors, which have fulfilled the respective requirements, are not allowed to apply for the ap - nomination. the application process may only start by a recommendation of members or representatives of the faculty or full university professors. these persons are requested to write a recommendation letter or favor the candidates with corresponding documents for the ap - nomination. in all other faculties the candidates are allowed to hand in all documents which are required for the nomination process on their own (see figure 1 (fig. the abilities and performance for assistant professors, which apply for the nomination to become an associate professor, have to be evaluated by reviewers at most medical faculties. an evaluation is not necessary or is not described in the ap - regulations (0 points) at five medical faculties. the evaluation of the candidates by internal reviewers only, is necessary at three faculties (1 point). the majority (n=27), however, requires an evaluation process, which is supported by an external reviewer from a different university. on average 21 reviewers have to evaluate the candidates, focusing not only on the required issues, but also on the general ability for a nomination as an associate professor. scientific publication. in only two ap - regulations most of the medical faculties (51%) could reach the highest score value (5 points) in this category, with detailed information on quantity, quality and first or last authorships (see table 2 (tab. are requested of the assistant professors for a successful application in the respective minimum time. three medical faculties (8.6%) do not give any information on the required research work (0 points) and in seven ap - regulations (20%) only imprecise information is given (1 point ; 20%). after reaching successful postdoctoral lecture qualification or nomination for an assistant professorship, eight medical faculties (22.9%) claim ongoing scientific work for several years. detailed information on the respective research performance with successful raise of third - party funds is given in 31.4%. four medical faculties additionally demand a perspective concept on future research and teaching performance of the candidates at the respective institutions (5 points ; see table 2 (tab. the actual evaluation of the teaching activity in the included ap - regulations feature detailed information on form, extent and content of the required teaching modalities for becoming an associate professor. while ten years ago only ten medical faculties (28%) have given some information on the minimum teaching qualifications, the actual analysis of the ap - regulations reveals, that specific information is given in more than 80% (n=28) of the regulations, reaching score values of 3 (see table 2 (tab. the candidates are allowed to perform every kind of teaching (lectures, seminars, practical courses). at six medical faculties, the ap - regulations claim an additional evaluation of the lectures by students or other faculty members (see table 2 (tab. however, differ substantially and are markedly increased at some universities since the year 2000, ranging from 1.5 to 4 semester periods per week (spw). at some medical faculties, candidates, which act predominantly in patient care, a further criterion for a successful teaching performance is the mentoring of doctorate or diploma theses. at 21 medical faculties this is a favored or demanded issue, correlating with an increase of 87.5% in comparison to the year 2000 data, an additional criterion for the successful nomination of an associate professor are scientific presentations or posters. 54.3% of medical faculties do not attach importance on the active participation on scientific congresses or meetings (see figure 1 (fig. 1)). in other respects, at 16 medical faculties a (detailed) listing of all oral or poster presentations is required for the ap - application. the nomination of an associate professor can be invested from universities to persons, who have shown outstanding performance and qualifications. the nomination requires a successful and independent performance in research and teaching over several years. after the nomination for an assistant professor or reaching postdoctoral lecture qualification, german medical faculties expect different minimum times before these candidates may apply for the associate professor nomination, averaging 4.50.3 years (95% confidence interval 3.8 5.8). this period of time might be reduced for candidates, which have shown extraordinary scientific performance at 54.3% of german medical faculties. this is especially the case, if the candidates have been listed on the nomination list in an appointment process for a full university professorship (secundo et tertio loco). furthermore, it can also be reduced, when an outstanding scientific price could be won in the area of expertise or in medicine in general. at one medical school, the ap - regulations point out that the rejection of a first place listing (primo loco) in an appointment process might directly lead to the nomination as an associate professor. at 25 medical faculties (28.6%) assistant professors, which have fulfilled the respective requirements, are not allowed to apply for the ap - nomination. the application process may only start by a recommendation of members or representatives of the faculty or full university professors. these persons are requested to write a recommendation letter or favor the candidates with corresponding documents for the ap - nomination. in all other faculties the candidates are allowed to hand in all documents which are required for the nomination process on their own (see figure 1 (fig. evaluation of the capabilities being an associate professor. the abilities and performance for assistant professors, which apply for the nomination to become an associate professor, an evaluation is not necessary or is not described in the ap - regulations (0 points) at five medical faculties. the evaluation of the candidates by internal reviewers only, is necessary at three faculties (1 point). the majority (n=27), however, requires an evaluation process, which is supported by an external reviewer from a different university. on average 21 reviewers have to evaluate the candidates, focusing not only on the required issues, but also on the general ability for a nomination as an associate professor. in 2000, a qualitative evaluation of the ap - regulations at german medical faculties revealed many differences and a great variety of requirements. one of the major findings in the actual evaluation is that the regulations among the medical faculties became more homogenous (small 95% confidence interval) and but also increased markedly in a 10 year interval (mean score 13.5 points). as nagelschmidt and colleagues already discussed in their analysis of habilitation theses, the same holds true for the ap - regulations, that the available requirements does not display the reality at the respective locations, but are rather a guideline. high scoring points might display apparent high requirements for the ap - nomination as well as clearly and definitely formulated ap - regulation at the respective faculty. low scoring points display unclear and imprecise regulations, however, giving the nomination committee the much wider leeway in decision - making but does also hinder the standardization process for ap - regulations in germany. the evaluation by pabst and strate discussed three main points of critique of the ap - regulations from the year 2000, which will be highlighted with the results of the actual analysis : time as an assistant professor : in the last 10 years we could not find any standardization in that issue. the minimum times for assistant professors still vary from two to six years until an ap - application may be possible. however, the possibility of a time reduction by a nomination listing seems to be established at more then 50% of german medical faculties.scientific publication : the research performance might be best documented by the respective numbers of publications of the ap - candidates. this is mentioned by 88% of the ap - regulations in the actual evaluation and is therefore the third most important precondition for an ap - nomination. while in 2000 only 17 medical faculties gave detailed information on this issue, only two of 35 ap - regulations do not describe this in the here presented analysis. furthermore, the fixed orientation on impact factors as predominant criterion for the evaluation of the publication performance in 2000 plays an only subordinated role today. the actual evaluation of publications complies rather to peer reviewed articles as well as the numbers of first or last authorships. due the achievement - oriented granting of funds at german medical faculties and german universities, which is geared to quantifiable parameters, it seems to be only of less interest for authors to publish in journals without an impact factor. evaluation of the candidates and their performance : as a last point of criticism the external evaluation of the candidates abilities and performance has been mentioned in 2000 involved with the fact to reduce the time liability and to perform the evaluation only by the internal ap - board of the respective medical school. in a 10-years comparison this issue seems not to be provided at all, although only three medical faculties did not change their ap - regulations in that time. in the course of an objective nomination process, the external review of the candidates still seems to be of great importance. time as an assistant professor : in the last 10 years we could not find any standardization in that issue. the minimum times for assistant professors still vary from two to six years until an ap - application may be possible. however, the possibility of a time reduction by a nomination listing seems to be established at more then 50% of german medical faculties. scientific publication : the research performance might be best documented by the respective numbers of publications of the ap - candidates. this is mentioned by 88% of the ap - regulations in the actual evaluation and is therefore the third most important precondition for an ap - nomination. while in 2000 only 17 medical faculties gave detailed information on this issue, only two of 35 ap - regulations do not describe this in the here presented analysis. furthermore, the fixed orientation on impact factors as predominant criterion for the evaluation of the publication performance in 2000 plays an only subordinated role today. the actual evaluation of publications complies rather to peer reviewed articles as well as the numbers of first or last authorships. due the achievement - oriented granting of funds at german medical faculties and german universities, which is geared to quantifiable parameters, it seems to be only of less interest for authors to publish in journals without an impact factor. evaluation of the candidates and their performance : as a last point of criticism the external evaluation of the candidates abilities and performance has been mentioned in 2000 involved with the fact to reduce the time liability and to perform the evaluation only by the internal ap - board of the respective medical school. in a 10-years comparison this issue seems not to be provided at all, although only three medical faculties did not change their ap - regulations in that time. in the course of an objective nomination process, the external review of the candidates still seems to be of great importance. in the classic thought of an academic career at german universities, the nomination as an associate professor displays the next - to - last step. to become an associate professor does not only stand for a certain quality characteristic to the community, but is also of great importance for the career chances of the respective persons. the frequency with which german medical faculties perform nomination processes for a doctorates, assistant as well as associate professors degree is suspiciously surveyed by other faculties and sometimes deprecatingly commented,,. in this context it was also argued of an inflation and loss of value of the invested academic degrees in medicine. furthermore, the academic proof of the qualification for assistant professors or postdoctoral lecturers has been critically challenged and their abrogation has been discussed. this problem and discussion, however, led to the point that academic medicine itself addressed this problem and performed corresponding studies,,,,,. independent studies could show that the achievement of academic degrees significantly supports the personal career. their abrogation, however, was not favored by a majority, despite considerable quantitative and qualitative preconditions and a strong integration into direct patient care,. on the contrary, an analysis of 616 medical assistant professors from 1998 resulted in the wish of an urgent reform of the current system of ap - requirements by 80% of the interviewees,. the first steps in standardization of the requirements, especially for assistant professors, are currently launched and despite significantly increasing requirements, the number of successful nomination processes for assistant professors is still constantly high (http://www.landkarte-hochschulmedizin.de/home.aspx). in addition, 48% (n=2998) of all assistant and associate professors in 2009 served in the section of medicine and health care sciences. the frequency of successful academic nomination processes might also be based on the above - average scientific activity of medical faculties, underscoring the favorable granting of german medical faculties in the initiative of excellence of the german federal government in 2006/2007, in which the german university medicine participated disproportionately high. as long as the requirements for specific academic degrees in medicine itself are not regulated uniformly, a fair and constructive comparability and criticism to other areas of expertise can not be established. taken together, the requirements for the nomination of associate professors have been markedly increased and got much more detailed in a ten years analysis, although there is no scoring data available from the year 2000. here, the total score might imply an only small range of preconditions for becoming an associate professor, however, a deeper look inside the currently valid ap - regulations of german medical faculties shows an exceeding heterogeneity. in this context it should be clarified whether the high location bound requirements and the general heterogeneity of the ap - regulations are admissible and justified in the focus of the career chances and equality of opportunities of the candidates. furthermore, the equalization of the preconditions for assistant professors is on a good way. based on a standardized quantitative and qualitative scoring system, this work might be the basis for the ongoing discussion and consensus declarations of german medical faculties on this topic. | background : first quantitative evaluation of the requirements for the promotion as associate professor (ap) at german medical faculties material and methods : analysis of the ap - regulations of german medical faculties according to a validated scoring system, which has been adapted to this study.results : the overall scoring for the ap - requirements at 35 german medical faculties was 13.50.6 of 20 possible scoring points (95% confidence interval 12.2 - 14.7). more than 88% of the ap - regulations demand sufficient performance in teaching and research with adequate scientific publication. furthermore, 83% of the faculties expect an expert review of the candidates performance. conference presentations required as an assistant professor as well as the reduction of the minimum time as an assistant professor do only play minor roles. conclusion : the requirements for assistant professors to get nominated as an associate professor at german medical faculties are high with an only small range. in detail, however, it can be seen that there still exists large heterogeneity, which hinders equal opportunities and career possibilities. these data might be used for the ongoing objective discussion. |
in the present work, the possibility of increasing the lifespan of mammalians and humans by transplantation of the genetically identical (or similar) stem cells with the lower number of the genomic errors to the old recipients is investigated. the idea of this method follows directly from informational theory of aging in which the degradation (error accumulation) of the genetic information in cells is considered a basic reason of aging (see more detailed information in discussion). informational theory of aging links the accumulation of genomic errors in cells with a decrease of their functionality, which in turn leads to decreasing functionality of whole organism (vitality) and finally to increasing probability of its death. it is well known that the stem cells are a source for many different types of cells. as a result of the transplantation of the stem cells of young donors with the low number of genomic errors the increase of the number of various cells with the reduced number of genomic errors and improving of the overall functionality of the organism (vitality) can be expected. however, where can one get the genetically identical stem cells with the lower number of the genomic errors ? the cryopreservation of the animal 's (human 's) own stem cells that have been isolated at a young age is one of the possible techniques. indeed, it seems obvious that in the young cells stored in cryogenic conditions the number of genomic errors will be lower than in the cells of the old organism. as the source of the various types of stem cells (mesenchymal and hematopoietic) the bone marrow (bm) can be used. it is necessary to emphasize that all the basic operations of this variant of our method (figure 1):isolation (collection) of bm, cryopreservation of bm, bm autotransplantation to recipient in old age, are well established procedures, with many years of application experience and are approved for medical use in most of countries [27 ]. isolation (collection) of bm, cryopreservation of bm, bm autotransplantation to recipient in old age, unfortunately, a very long time would be required for the direct verification of this method for large animals and humans because of their large initial life expectancy. use of laboratory animals with the small lifespan (mice and rats) is faced with a problem of another kind. because of the small size of these animals it is difficult to isolate (take) their bm without significant damage to their health. this reason has forced us to modify the scheme of our experiment. instead of application of the autologous transplantation (figure 1(a)) we used the bm transplantation from genetically similar animals of inbred groups (figure 1(b)) with the various levels of genetic similarity (syngeneity). such scheme of experiments gives the following additional possibilities : first, we can study the increase of lifespan as the function from the level of genetic similarity (syngeneity) of donors and recipients and second the engraftment of the transplanted cells can be observed if the transgenic animals are used (gfp mice). the mouse strain c57bl/6-tg(actbegfp)1osb / j carries a transgene of an enhanced green fluorescent protein in chromosome 15 (briefly egfp c57bl/6 strain) and was received with the assistance of a. m. malashenko (scientific center of biomedical technologies, russian academy of medical sciences) from jackson laboratory, bar harbor, united states, and thanks to the kind permission of a. v. chervonskii it was used in the experiment. in the egfp c57bl/6 mouse strain, some mice carry a green fluorescent protein gene (egfp c57bl/6 mice), which is a vital label allowing unambiguous identification of donor cells following their transplantation into the recipient organism that does not carry this gene [8, 9 ]. mice carrying the green fluorescent protein gene were selected as donors for performing both syngeneic and allogeneic transplantation. when syngeneic transplantation was performed, the donor - recipient pair was taken from the same strain, with the recipients being mice not carrying the egfp transgene (egfp c57bl/6 mice) ; moreover, the donor and recipient were of the same sex. when allogeneic transplantation was performed, female mice of the balb strain were used as recipients. mice were kept in the vivarium of the institute of cell biophysics, russian academy of sciences, in standard conditions. the animals were additionally fed with grain mix consisting of wheat, barley, red millet, sunflower seeds, corn, and grass granules. bm was isolated from two femur bones via grounding in a porcelain mortar with a phosphate saline buffer (600 l). the obtained suspension was filtered through a nylon filter with a pore size of 70 m. transplantation was performed by administering of 100 l (2 10 cells) of the whole fraction of bm immediately after its isolation into a side tail vein with an insulin syringe. the tail of the animal was preheated at 4045c with warm water. to study the problem, how the level of genetic similarity (syngeneity) of donors and recipients influences the lifespan extension, three groups of mice within inbred strain egfp c57bl/6 the fourth group of mice with minimal level of genetic similarity was formed for allotransplantation (balb mice were recipients and egfp c57bl/6 mice were donors). for syngeneic transplantation, we formed three experimental groups of egfp c57bl/6 mice. females were as the recipients and donors in two groups and in the third group males were as recipients and males and females were as donors. in three control groups of egfp c57bl/6 mice the number of animals was 11, 10, and 21 mice in the first, second, and third experimental groups and 12, 13, and 12 mice in three control groups, respectively. before transplantation works, we obtained progeny of 3 - 4 subsequent generations from the same female and her young male offspring or descendant of next 2 - 3 generations to reach high level of syngeneity. in the second and third experimental and control groups additional inbreeding for the first and second experimental groups, donors were selected within offspring of the recipient mouse. the females of the experimental group were mated with male mouse (brother or son) to obtain offspring with a high degree of inbreeding, starting at 58 months of age with intervals of 3 months. from the resulting litter, for old females who were unable to produce offspring, the young egfp donors, the descendants of these females of the second and subsequent generations, were selected. in the control groups of females, for the third experimental group consisting of males, we have chosen as donors the males and females of 1.53 months carrying the gene of green fluorescent protein regardless of the relationship degree. thus, the first experimental group had the highest level of syngeneity between the donor of bm and the recipient (because of additional inbreeding procedure and choosing the donors among progenies of the females). the second group had the middle level of syngeneity (there was no additional inbreeding but the donors were progenies of the females). the third group consisting of males had the minimal level of syngeneity (the donors were not progenies of the males). in all three experiments, bm transplantation (2 10 cells) was performed with the intervals of 3 months to the death of the animal. experiments were started at age of 7 - 8 months for the recipients of the first and the second experimental groups and at age of 610 months for those of the third group. experiment was carried out using 1.53-month - old egfp c57bl/6 mice as donors and 13 - 14-month - old balb mice (14 females) as recipients. transplantation of bm in the amount of 2 10 cells was performed periodically, starting from 13 - 14 months of age, and repeated every 3 months until the death of the animal. the balb / c mice (14 females) were used in the control group, for which transplantation was not performed. to assess the engraftment of egfp bm cells, the dynamics of egfp cell numbers in recipient bm, spleen, thymus, and blood by fluorescence, the 1.52-month - old egfp mice were used as donors for egfp young group (3 - 4-month - old recipients) and 1.53-month - old egfp donor mice were used for egfp old group (1014-month - old recipients). in experiments on allogeneic transplantation, 1.52-month - old egfp c57bl/6 mice were used as donors for 3 - 4-month - old balb mice. the investigation was carried out for 1417 days following bm transplantation in the syngeneic transplantation experiments and for 7 days in the experiments on the allogeneic transplantation until egfp cells were detectable in the investigated organs. the recipient mice were euthanized using the cervical dislocation technique after certain times following the bm administration. during autopsy, next, organs were pulped with a scalpel through a 70 m pore size nylon filter into a 1 or 2 ml volume of the phosphate saline buffer for the thymus and spleen, respectively. a 60 l aliquot of blood was collected from thoracic cavity into a tube containing 15 l of 0.5 m edta. 15 l aliquot of blood, suspension of bm, thymus, and spleen were placed onto microscope slides coated with polylysine (thermo scientific, germany) and covered with a 20 20 mm cover slips. the presence of egfp cells in them was revealed with an axiocam z1 fluorescence microscope equipped with an axiocam mrc5 color digital camera (carl zeiss, germany). the egfp protein has a fluorescence maximum at 508 nm in the green region of the spectrum when illuminated with light in the wavelength region of 395475 nm. for estimation of the egfp cells quantity in the sample, they were counted on eight horizontal lines from the left to the right edge of the cover slip uniformly distributed within the sample. the number of the cells was estimated using arbitrary units from 1 to 5 (1 : from 5 to 40 cells ; 2 : from 41 to 160 cells ; 3 : from 161 to 400 cells ; 4 : from 401 to 600 cells ; and 5 : more than 600 cells in the sample). for staining of cell surface proteins, bm or spleen cell suspensions isolated using the technique described above were incubated with phycoerythrin- (pe-) coupled antibodies against mouse cd117 (biolegend, san diego, ca, usa) and cd45r / b220 (bd biosciences, san jose, ca, usa) for 1 h at 4c in the dark. confidence interval for the difference in the mean lifespan of mice between the control and test groups was determined using a standard student 's t - test. for approximation of experimental data, polynomial functions of the first in the present study three groups of experimental animals for the syngeneic transplantation (two consisted of females and one consisted of males) were formed. experiments were designed to reduce the immune response to a donor 's cell as result of some variation of alleles of non - major histocompatibility complex genes. for this purpose, bm cells were isolated from the young offspring of the first and subsequent generations of the recipients. transplantation has been repeated every 3 months until the death of recipients. on figures 2(a) and 2(b) the dependence of survival on age of the first and the second experimental and control groups of mice is presented. for the first experimental group of mice, the mean lifespan is 34% longer (20.6 2.2 versus 15.4 2.6 months ; p = 0.05) and for the second experimental group the mean lifespan is 19% longer than for the control ones (22.6 1.6 versus 18.9 1.4 months ; p = 0.20). it should be noted that such difference in the increment of mean lifespan between the first and the second experimental groups is a natural result and can be explained by previously carried out additional inbreeding within the first group of mice. on figure 2(c) the survival curves for the first and second experimental groups together and total control groups are shown. the mean lifespan in the experiment was 21.5 1.6 months, that is, 25% more than in the control group (17.2 1.8 months ; p = 0.05). the combined data allow us to improve the statistical reliability of the result, obtained by increasing the number of animals in the groups, and to draw a conclusion about the possibility of a significant extension of the lifespan of animals with syngeneic bm transplantation. for the third experimental group, increase of the lifespan value reached only 10% and was 18.6 0.6 months compared with 16.8 0.8 months in the control group (p = 0.4) (figure 2(d)). the dependence of the balb mice survival on age after allogeneic bm transplantation (egfp c57bl/6 mice are as donors) is presented in figure 2(e). it is evident that the dynamics of survival of the experimental and control groups differ slightly. they reach 18.3 2.2 months for the experimental group and 18.4 2.4 months for the control group (p = 0.05). to determine the engraftment degree of donor cells in the host organism, quantity of egfp cells was estimated in bm, spleen, thymus, and blood of 3 - 4-month - old recipient mice on different days after transplantation of bm suspension (2 10 cells). the dependence of change of the egfp cells amount in mice organs on time after syngeneic and allogeneic transplantation is presented in figure 3. the maximum time of egfp cells engraftment was established after syngeneic transplantation and on the 12th day the donor cells are still registered in bm, spleen, and thymus (figure 3(a)). after allogeneic transplantation, the maximum stay of egfp cells was 5 days for the balb strain (figure 3(b)). after the syngeneic and allogeneic transplantation, the greatest number of donor cells was recorded in bm and spleen. in thymus and blood, after allogeneic transplantation, for the first 2 - 3 days only individual cells were recorded. in older recipients (1014-month - old) after the syngeneic transplantation, the engraftment dynamic of the blood system was similar to that for young recipients, and the maximum time of registration of donor cells (17 days) exceeded that of the young group by 40% (not shown in the figure). in figure 4, the cells of bm (a, b) and spleen (c, d) in the recipient at the time of maximum engraftment are presented. the differential interference contrast (dic) microscopy (a, c) shows all the cells in the suspension. only host hematopoietic progenitor cells (cd117 expression, orange fluorescence) are visible in bm (figure 4(b)). in the spleen both donor and host cells highly express cd45, which are revealed with bright orange fluorescence (figure 4(d)). the main experimental result of this work is finding the relation between the lifespan increase and genetic similarity (syngeneity) of donors and recipients within the framework of the proposed method. the value of lifespan increase in our experiments varies from 34% for high - level syngeneic transplantation to 0% for allogeneic transplantation. one can note that our results are in good agreement with the results of earlier works [1118 ] (table 1), where the transplantation with the low levels of syngeneity is used. it seems important to explain these experimental results within the framework of our informational theory of aging in more detail. there are many experimental works in which the fact of accumulation of lesions in the genetic material of the cells of various organisms in the course of aging is directly established [1922 ]. however, the absence of a clear answer to the question why the genomic errors do not accumulate in germ line cells from generation to generation [2328 ] induces search for other basic mechanisms of aging, such as epigenetic, that are not related with the genome damage by random factors [2935 ]. in 2009 we proposed a solution for the problem of escaping germ line from aging within the framework of a wide range of hypotheses based on the aging notion as a process of accumulation of genomic errors [2427, 3638 ]. it has been found that the mechanism providing correction of genomic errors in germ line cells involves two elements that are well known and common practically for all eukaryotic organisms. it is well known that as a result of gene recombination (crossing over) in cells of germ line the haploid daughter cells - precursors are replicated. these haploid daughter cells (gametes) contain 1/2 of the genetic information of next generation organism. we need to note here that the density of gamete 's genomic errors differs from the density of genomic errors in germ line cells of maternal organism. indeed, the stochastic nature of the location of genomic errors and the stochastic nature of gene recombination (crossover) leads to the random distribution of errors between gamete 's genomes as shown in figure 5. in addition, it is important that the density of the genomic errors in some of them be lower than in the cells of germ line of maternal organism (e.g., gamete 2 in figure 5). the second element of the given mechanism of correction of the genomic errors is the process of selection of precursor cells (gametes) with minimal error density. moreover, the haploidy of such cells raises the efficiency selection inasmuch as it elevates the specific contribution of every error into the overall reduction of gamete functionality. although each element of this mechanism (gene recombination + gamete selection) was in itself well known and studied, the conclusion that the reduction of the density of genomic errors comes out from their joint action was a priority of our previous works. this solution of the paradox of the not aging germ line rehabilitates a wide range of aging hypotheses united by the idea of degradation of the genetic information by the aging [2427, 3638 ]. the conception of aging as the process of accumulation genomic errors (degradation of the genetic information) in the most general case, supplemented with the above described mechanism of correction genetic lesions in germ line cells, has the name of an informational theory of aging. to test the workability of the informational theory of aging, we have built the simple simulation model. in spite of simplicity of this model those phenomena are reproduced in the model : the mortality curve for model organisms (figure 6(a));gompertz curve (figure 6(b));accumulation of the genomic errors in old age (figure 6(c)). the mortality curve for model organisms (figure 6(a)) ; gompertz curve (figure 6(b)) ; accumulation of the genomic errors in old age (figure 6(c)). it should be noted here that the gompertz curve describes aging dynamics of wide class of eukaryotic organisms from the colonies of yeast up to the higher mammals including humans. it is interesting that the model reproduces not only the linear part of the gompertz curve but also the phenomenon of neonatal mortality and the phenomenon of relative stabilization of mortality in elder age groups. the basis of the model is a population genome matrix g~(t)=g(i, p, m, s, t) ; the element is taking the value of 1 if the gene is intact or 0 if it contains an error ; the indices numerate the organism in the population i = (1,, ni), the cell in the organism p = (1,, np), the gene in the genome m = (1,, nm), and the genome copy in the diploid cell s = (1, 2) ; t is the discrete time parameter. this matrix evolves in time in accordance with three main algorithms : aging (accumulation of errors in genomes);death of old age (upon exceeding a certain threshold of genomic errors);birth of a new organism (rejuvenation of genome). aging (accumulation of errors in genomes) ; death of old age (upon exceeding a certain threshold of genomic errors) ; birth of a new organism (rejuvenation of genome). (1) aging (defect accumulation) is realized as follows : m quartets { i, p, m, s } are randomly chosen at every modeling step t, and the corresponding genes are considered to be defective : (1)gi,p,m,s,t+t=0. an important feature of the model (it explains the use of the term informational theory of aging) is the absence of mechanisms able to correct the informational errors in the genome. it is different, in principle, from the more general class of genome damage for which the molecular mechanism of correction (repair) may exist. (2) death of model organism happens when the number of its functional cells falls below a critical level n < nmin. in the model, loss of cell function occurs when even one gene has a defect in both copies of genome. that is, cell p0 of organism i0 is considered nonfunctional if, for at least one gene m, (2)gi0,p0,m,1,t = gi0,p0,m,2,t=0. upon death of i0, the corresponding block g~(t) (3) birth of a new organism involves several steps as follows:(3.1)a pair of parental cells (i1, p1), (i2, p2) is randomly chosen among viable cells of different organisms (i1, i2) above a certain age tmin.(3.2)each of parental cells produces an even number 2nj of gametes with a single set of genes (formulae for both cells are identical):(3)g~gi1,p1=ggi1,p1m, j = gi1,p1,m, sj, m, tggi1,p1m, j+nj = gi1,p1,m,3sj, m, t, where j = (1,, nj). the random matrix s(j, m), taking values (1, 2), determines crossover procedure in the model. figure 5 shows the 14-gene diploid genome of parent with four errors (two in each set). gene recombination (crossover) (s(1, m) = { 1,1, 1,1, 2,2, 2,2, 2,2, 1,1, 1,1 }, nj = 1) yields two gametes : one with all four errors (j = 1) and the other without any (j = 2).(3.3)upon forming two sets of gametes with genomic matrices g~gi1,p1 and g~gi2,p2, one gamete from each set is chosen with a probability proportional to the viability of this gamete 's v(j) and v(j), which itself depends on the number of genomic errors as (identically for v(j))(4)vi1,p1j = expm=1nmggi1,p1m, j1, where is a parameter specifying the extent to which genomic damage affects gamete viability, j = (1,, 2nj). in essence, this defines a process of selection of gametes for the minimal amount of errors : g~gi1,p1j1 ; g~gi2,p2j2, where j1, j2 denote the gametes obtained from parents i1, i2.(3.4)finally, the matrices of the two selected mating gametes (j1, j2) are combined in a new block of the population matrix to represent the organism that replaces the dead i0:(5)gi0,p, m, s, t+t = ggis, psm, js. a pair of parental cells (i1, p1), (i2, p2) is randomly chosen among viable cells of different organisms (i1, i2) above a certain age tmin. each of parental cells produces an even number 2nj of gametes with a single set of genes (formulae for both cells are identical):(3)g~gi1,p1=ggi1,p1m, j = gi1,p1,m, sj, m, tggi1,p1m, j+nj = gi1,p1,m,3sj, m, t, where j = (1,, nj). the random matrix s(j, m), taking values (1, 2), determines crossover procedure in the model. figure 5 shows the 14-gene diploid genome of parent with four errors (two in each set). gene recombination (crossover) (s(1, m) = { 1,1, 1,1, 2,2, 2,2, 2,2, 1,1, 1,1 }, nj = 1) yields two gametes : one with all four errors (j = 1) and the other without any (j = 2). upon forming two sets of gametes with genomic matrices g~gi1,p1 and g~gi2,p2, one gamete from each set is chosen with a probability proportional to the viability of this gamete 's v(j) and v(j), which itself depends on the number of genomic errors as (identically for v(j))(4)vi1,p1j = expm=1nmggi1,p1m, j1, where is a parameter specifying the extent to which genomic damage affects gamete viability, j = (1,, 2nj). in essence, this defines a process of selection of gametes for the minimal amount of errors : g~gi1,p1j1 ; g~gi2,p2j2, where j1, j2 denote the gametes obtained from parents i1, i2. gametes (j1, j2) are combined in a new block of the population matrix to represent the organism that replaces the dead i0:(5)gi0,p, m, s, t+t = ggis, psm, js. when all organisms that died within the given time step (t t + t) have been replaced with newborn ones (which technically means the formation of a new population genome matrix and zeroing of the corresponding age counters), the model goes to the next time step (t + t t + 2t). once the model is formulated, the process of accumulation and correcting of genomic errors can be viewed in more detail. figure 7 displays the steady - state distribution of the number of genomic errors (nonfunctional genes in percentage) for cells of different age groups and type. as noted above, this simple model correctly reproduces the survival curve (gompertz curve) for the eukaryotic organisms (figure 6). it should be noted that within the framework of the model that has been formulated we could not only reproduce known results but also perform such experiments in the model populations that are difficult (impossible) to carry out in real system. for example, we can turn off the process of gene recombination (crossing over). the key role of gene recombination during gametogenesis for stabilizing of the model 's population is demonstrated in figure 8. the steady - state with a 20% level of the genomic errors is reached in 1015 of time steps in the model with the active algorithm of the gene recombination (crossover) and can be maintained indefinitely long (curve 1). in model in which the algorithm of the gene recombination is blocked, the model 's population will end quite soon (110 of time steps) because of accumulation of genomic damage (curve 2). unfortunately, this model is too simple to explain the results of our experiments, in which the rise on 3 - 4 months of life expectancy of laboratory animals has been observed (table 1), whereas bone marrow cells were detected in the recipient only about 0.5 months (figure 3). on the other hand, the total residence time of donor cells, considering the number of bone marrow injection, was about 2.5 months (5 injections 0. 5 months), which is much closer to the experimental values of lifespan increase on 3 - 4 months. to understand this fact, we need to develop the more advanced model within the framework of the informational theory of aging. the more general description will include the following principals (not the full list):(1)in somatic cells of eukaryotic organism, each of the two copies of genetic information g^1pt, g^2pt throughout the cell lifespan accumulates defects (errors):(6)rg^1pt+trg^1pt;rg^2pt+trg^2pt, where r(g^sp(t)) is the number of defects in the genomic copy s = (1, 2) of somatic cell p at a moment t.(2)after a mitotic division p p, q the number of defects in daughter cells p, q will be not smaller than in mother 's p:(7)rg^spt+trg^spt;rg^sqt+trg^spt.(3)accumulation of the genomic errors rg^sp(t) decreases the viability of individual cells vp(g^sp(t)) and viability of the organism(8)vt = vg^spt.(4)with viability of the organism becoming less able to withstand any adverse influence and the probability of its death p(v(t)) rising,(9)ddtvt0,vpvt0ddtpvt0. in somatic cells of eukaryotic organism, each of the two copies of genetic information g^1pt, g^2pt throughout the cell lifespan accumulates defects (errors):(6)rg^1pt+trg^1pt;rg^2pt+trg^2pt, where r(g^sp(t)) is the number of defects in the genomic copy s = (1, 2) of somatic cell p at a moment t. after a mitotic division p p, q the number of defects in daughter cells p, q will be not smaller than in mother 's p:(7)rg^spt+trg^spt;rg^sqt+trg^spt. accumulation of the genomic errors rg^sp(t) decreases the viability of individual cells vp(g^sp(t)) and viability of the organism(8)vt = vg^spt. with viability of the organism becoming less able to withstand any adverse influence and the probability of its death p(v(t)) rising,(9)ddtvt0,vpvt0ddtpvt0. the principles that were introduced above will be the basic for wide class of models in the information theory of aging. however, the exact determination of the specific values entered in the various models may be different. the model in (brief description and results are given in section 4.2) solves the problem of demonstrating the correctness of information theory of aging in general and is greatly simplified for this purpose. it is necessary to note two important simplifications : the model in assumes that all cells of the organism are equivalent;the functional of vitality that was inversely proportional to the amount of genomic errors is also an oversimplification. the model in assumes that all cells of the organism are equivalent ; the functional of vitality that was inversely proportional to the amount of genomic errors is also an oversimplification. in a more general class of models within the framework of the informational theory of aging a multicellular organism should be considered as a set of cell populations, each of which is characterized by the average number of errors r(t) as well as the number of cells in the population n(t), where c indicate cell population (cluster). in addition, it is obvious that the vitality of the organism will depend not only on the average number of errors and on the number of cells in the corresponding cell populations but also on environmental conditions:(11)vt = vn^t, r^t, e^t, where e^t is matrix of environmental parameters. changing the number of cells in a given population, in turn, will depend on same parameters:(12)dn^tdt = d^n^t, r^t, e^t, where d^n^t, r^t, e^t is matrix of dynamics of cells populations (types of cells). under this approach, it becomes possible to explain the results of our experiments. indeed, donor 's cell, taking some of the functions of recipient 's ones, apparently allows increasing the corresponding population of recipient 's cells. in this case, the increasing vitality of the organism will have prolonged nature, remaining elevated even after all the donor cells will be removed from the recipient 's organism. however, in order to examine in more detail this question, it is necessary to perform additional experiments including quantitative measurements of parameters of cell populations (of hematopoietic and mesenchymal stem cells, for example) in the recipient that would define the parameters of advanced models (articles on identification of systems) [3942 ]. the authors are ready to cooperate and can provide a useful guide to set up a problem and to analyze the data obtained. biological aspects. an approach presented here for explanation of aging phenomenon gives the opportunity of a fresh look at a number of well - known facts in biology and clarifies their biological sense.so, for example, the well - known mechanism of gene recombination (crossing over) within the framework of our approach is not only a mechanism of optimizing the evolutionary process of eukaryotic organisms but also an important mechanism of supporting stability of their genome and preventing population degeneracy.moreover, the advantage of eukaryotic organization of the highly developed living forms becomes more clear. the ordinary evolutionary mechanism of individual organism elimination with genetic errors acts only when the mathematical expectation of the appearance of a genetic error during the whole life cycle is less than one.such mechanism is realized in bacteria with small genome and short life cycle. the long - lived prokaryotic organism with the complex (large) genome would unavoidably accumulate the genetic errors from the foregoing generation to the following one which would lead at last to collapse of all population.multiple repetitions of genes in the genomes of eukaryotes enhance resistance to genetic errors. we believe that the lifespan of the eukaryotic organism is longer when a degree of such repetitions is higher. the flowering plants with a potentially very long lifespan (and a large genome) are characteristic example. we consider here not only champions like sequoia or baobab but also ability of many short - lived species to reproduce themselves by vegetative way (without crossing over) during many years.the role of stem cells for highly organized organisms becomes more clear.it is more preferable for such organisms to have the special cells (long - term stem cells) with a decreased level of metabolism which leads to reducing the number of appearing free radicals and thus leads to decreasing rate of accumulation of genetic errors. the rate of accumulation of genetic errors in the stem cells together with the degree of repetition of genetic information determines species lifespan of different highly organized organisms (excluding a number of cases when a death of an organism has a programmed nature as the death of the salmon). so, for example, the well - known mechanism of gene recombination (crossing over) within the framework of our approach is not only a mechanism of optimizing the evolutionary process of eukaryotic organisms but also an important mechanism of supporting stability of their genome and preventing population degeneracy. moreover, the advantage of eukaryotic organization of the highly developed living forms becomes more clear. the ordinary evolutionary mechanism of individual organism elimination with genetic errors acts only when the mathematical expectation of the appearance of a genetic error during the whole life cycle is less than one. the long - lived prokaryotic organism with the complex (large) genome would unavoidably accumulate the genetic errors from the foregoing generation to the following one which would lead at last to collapse of all population. we believe that the lifespan of the eukaryotic organism is longer when a degree of such repetitions is higher. the flowering plants with a potentially very long lifespan (and a large genome) are characteristic example. we consider here not only champions like sequoia or baobab but also ability of many short - lived species to reproduce themselves by vegetative way (without crossing over) during many years. it is more preferable for such organisms to have the special cells (long - term stem cells) with a decreased level of metabolism which leads to reducing the number of appearing free radicals and thus leads to decreasing rate of accumulation of genetic errors. the rate of accumulation of genetic errors in the stem cells together with the degree of repetition of genetic information determines species lifespan of different highly organized organisms (excluding a number of cases when a death of an organism has a programmed nature as the death of the salmon) conclusion about impossibility of cardinal (significant) prolongation of human life by the pharmacological ways and by methods of epigenetic rejuvenation of the own stem cells of a patient is one of the important conclusions based on the theory presented here. indeed, if we are to accept that genetic errors accumulation in the organism cells is the crucial factor of aging, it will be obvious that there are no biochemical procedures or chemicals which may restore the lost genetic information. in some sense our approach is one of the theoretical substantiations of a skeptical viewpoint on feasibility of rejuvenation human organism therapy. at the same time, this skepticism should not be mechanically expanded on our proposed method, since it considerably differs from the earlier proposed methods and is aimed at compensation of age - specific changes associated with decreasing cell functionality, which is a common phenomenon for the completely different organisms. it should be noted that the experiments carried out by us are preliminary ones from the medical viewpoint due to experimental subject choice (mouse) and due to the obtained meaning of reliability (p = 0.05) which is quite sufficient for the biological researches but which is insufficient for the medical applications. in addition, the scheme of our present experiment (figure 1(b)) differs from the method of rejuvenation proposed for the medical application (figure 1(a)). the next concrete experiments may be proposed and may be interesting for achieving our purposes : carrying out works that will be analogous to these described in this paper, but in which cryopreserved bone marrow will be used;using relatively big mammals (rabbit, dog) to realize fully autologous transplantation (figure 1(a));carrying out autologous transplantation using cloned animals (mouse);transplantation of the different types of the stem cells;induction of immune tolerance for using the donor stem cells at allogeneic transplantation. carrying out works that will be analogous to these described in this paper, but in which cryopreserved bone marrow will be used ; using relatively big mammals (rabbit, dog) to realize fully autologous transplantation (figure 1(a)) ; carrying out autologous transplantation using cloned animals (mouse) ; transplantation of the different types of the stem cells ; induction of immune tolerance for using the donor stem cells at allogeneic transplantation. the significant increase of lifespan for inbred laboratory animals demonstrates the efficiency of the proposed method for lifespan extension of multicellular organisms (human) in general and confirms the basis of the informational theory of aging. it allows developing the various methods of cellular therapy without risk of fatal consequences of immunological incompatibility and the methods of lifespan extension by using cryopreservation of stem cells taken in young age for autotransplantation in old age. in addition, in particular we want to note two experimental facts obtained in our work : the genetically not - identical bone marrow transplantation provides the significant increase of laboratory animal lifespan (up to 34%);the strong dependence of the life expectancy increase from the level of genetic similarity (syngeneity) of donors and recipients exists.from these facts the possibility of much greater increase of lifespan (than 34%) follows. it would be provided using the autotransplantation or the transplantation of the genetically identical stem cells (bone marrow) with the low level of genomic errors. the genetically not - identical bone marrow transplantation provides the significant increase of laboratory animal lifespan (up to 34%) ; the strong dependence of the life expectancy increase from the level of genetic similarity (syngeneity) of donors and recipients exists. | the method of lifespan extension that is a practical application of the informational theory of aging is proposed. in this theory, the degradation (error accumulation) of the genetic information in cells is considered a main cause of aging. according to it, our method is based on the transplantation of genetically identical (or similar) stem cells with the lower number of genomic errors to the old recipients. for humans and large mammals, this method can be realized by cryopreservation of their own stem cells, taken in a young age, for the later autologous transplantation in old age. to test this method experimentally, we chose laboratory animals of relatively short lifespan (mouse). because it is difficult to isolate the required amount of the stem cells (e.g., bone marrow) without significant damage for animals, we used the bone marrow transplantation from sacrificed inbred young donors. it is shown that the lifespan extension of recipients depends on level of their genetic similarity (syngeneity) with donors. we have achieved the lifespan increase of the experimental mice by 34% when the transplantation of the bone marrow with high level of genetic similarity was used. |
oligochaete worms, as the second largest group of the annelida, occur in a wide variety of freshwater environments. within this diverse group, limnodrilus hoffmeisteri claparde, 1862 (oligochaeta : tubificinae), is one of the most common and abundant aquatic oligochaetes. like all other oligochaetes, the species is a hermaphrodite, with a complex reproductive system. typically, each body segment possesses 4 bundles of chaetae (chitinous bristles projecting from the body). the chaetae, vary considerably in size and shape between families, and consequently are used extensively in identification. the life cycle of this species includes a stage that involves shedding of eggs into aquatic or moist environments with subsequent development into earthworms. these worms are capable of producing high population densities in paddy fields, eutrophic lakes, and rivers. thus, bathing, picnicking, or field working without protection measures may bring the potential risk of ingress. along with helminths and parasitic nematodes, occasional cases of non - parasitic worm infections (caused by enchytraeus sp. however, aquatic oligochaete human infections have not been reported in china up till now. in this study, we are reporting a case of oligochaete nasal infection in a 25-year - old chinese man. after detailed microscopic examinations and molecular identification, the infected specimen was identified as l. hoffmeisteri. the infectious agent was eliminated and was no longer detectable in the patient s nasal cavities after treatment with zentel (albendazole). these data will assist clinicians to be aware of this diagnosis and properly guide preventable measures to those who are in risk (such as aquaculture farmers, fishery workers, and ornamental fish hobbyists). the patient was a 25-year - old male residing in zhangjiakou city, hebei province, china employed by a coalmine machinery factory. he initially presented with severe nasal itching, rhinorrhea, and continuous sneezing followed by a nose bleed 2 days prior to consultation. mucosal damage to kiesselbach s area (on the antero - inferior part of the nasal septum), was diagnosed by nasal endoscopy. the worms extracted from the nasal discharge of the patient were identified by using morphological and molecular methods. the observed worm length was 1545 mm, and worm width was 0.60.8 mm (fig. the prostomium of the observed worms was cone - shaped, and contained about 110 proglottids (fig. long and thin bifid chaetae with a curved distal end were observed on the dorsal pre- and post - clitellar regions (fig. the ratio of the length of the dorsal bifid chaetae to the ventral chaetae was approximately 1.15. the penile sheath was long and tubular, and the length to width ratio ranged from 10.3 to 11.3 (fig. the morphological characteristics of the species were consistent with a previously published description of l. hoffmeisteri. furthermore, genomic dna was extracted from individual worms using a genomic dna purification kit (tiangen, china) for amplification of the small subunit ribosomal rna (rrna) region (fig. 2). the sequences obtained showed 99% identity with l. hoffmeisteri ncbi genbank entries (nos. a neighbor - joining (nj) tree constructed from ssu sequences grouped the worm isolated from the patient into the same cluster as l. hoffmeisteri (fig., the morphological characteristics and molecular data confirmed that the species isolated from the patient was l. hoffmeisteri. fortunately, the oligochaete worms were no longer detectable in the patient s nasal cavities following treatment with 400 mg zentel (albendazole) and yunnan baiyao (also called yunnan paiyao, a hemostatic powdered medicine) once daily for 6 days. l. hoffmeisteri is the best known and is considered to be the dominant species of the genus. although uncommon, the infection can occur in people after exposure to contaminated water. in the present study, for the first time we have reported the case of l. hoffmeisteri infection in the nasal cavity of a chinese man. as a result of this study, total 444 live red worms were isolated and examined from the nasal discharge of the patient by the first affiliated hospital of hebei north university. measurements of l. hoffmeisteri, including the body and penile sheath size, prostomium shape, and the ratio of the length of the dorsal bifid chaetae to the ventral chaetae, were performed. the specimens were identified as microdrile tubificid l. hoffmeisteri claparde, 1862 (tubificidae) and megadrile dichogastrid dichogaster bolaui michaelsen, 1891 (octochaetidae), respectively. in our study, we demonstrated a case of human nasal infection with l. hoffmeisteri in china. the likely source of infection of the chinese patient was fish bait used by the patient to feed domestically maintained tropical fish. it is likely that finger - to - nose contact with the fish bait gave rise to the associated infection. a second potential infection source may be a local reservoir used by the patient for recreational purposes 6 months prior to the infection. such opportunistic infection by l. hoffmeisteri occurs more frequently in individuals who are exposed to aquatic environments through labor occupation or leisure activities, usually with skin or mucosal lesions, paresthesia or itching at the ingress site, as well as associated systemic manifestations. accurate identification of pathogenic agents of these opportunistic infections is critical for the proper selection of clinical complementary tests and detailed anamnesis. to summarize, this is the first report of nasal infection with l. hoffmeisteri in a human in china. this l. hoffmeisteri nasal infection can serve as a reminder that oligochaete worms can result in human infections. this also demonstrates that appropriate hygiene precautions are important during field working and leisure activities. | the infection by limnodrilus hoffmeisteri claparde, 1862 (oligochaeta : tubificinae) in humans is relatively uncommon. the present report is to describe an incidental human infection with oligochaetes in the nasal cavity of a chinese man, a 25-year - old man residing in zhangjiakou city, hebei province, china presenting with nose bleed, severe itching, continuous sneezing, and rhinorrhea. a lot of oligochaete worms were found in the nasal discharge of the patient. the detected worms were identified as limnodrilus hoffmeisteri (annelida : oligochaeta) based on morphological and molecular characteristics. this incidental l. hoffmeisteri nasal infection is the first case in china and indicates that oligochaete worms can be encountered in humans. |
plantarflexor muscles play a critical role in ankle stabilization, and their combined eccentric contraction decreases the rate of tibial advancement during midstance and causes the foot and tibia to roll off over the forefoot rocker during terminal stance1. limited ankle dorsiflexion range of motion (rom) is associated with various cumulative injuries and also causes compensatory changes during walking2. for normal walking, proper ankle dorsiflexion rom is necessary to absorb the body weight and contributes to the forward body movement during the stance phase of the gait cycle1. ambulation on a sloped surface requires a shift towards increased ankle dorsiflexion and more muscle activation of the knee and ankle joint for stability3. deficits in ankle dorsiflexion rom can come from restraints in contractile or noncontractile tissue, and abnormal tissue flexibility requires greater force, which leads to energy dissipation1, 4. mobilization with movement (mwm) is widely used to improve the accessory motion of joints ; however, it has a limitation in that it can only be applied by a therapist in a physical therapy room7. recently, mwm has been modified by using tape to provide a posterior inferior talar glide, which can cause passive movement by substituting for a therapist 's force8. a lot of physical therapists have often employed mwm using tape to increase ankle dorsiflexion, but how incline walking with modified mwm using tape influences the foot and ankle biomechanics is still unclear, and no information has been gathered about the muscle activities. an inclined surface is often used as a therapeutic intervention because it requires additional muscle action and increases the concentric activity of the plantarflexor muscles9. biomechanical alterations resulting from clinicians ' utilization of modified mwm using tape have the potential to change the plantarflexor and dorsiflexor muscle activities. therefore, the purpose of this study was to investigate the effect of modified mwm using tape on the talus on the gcm and ta muscle activities during incline treadmill walking in women with limited ankle dorsiflexion. fifteen women (a total of 22 feet with limited ankle dorsiflexion) were recruited for this study. the mean age, height, and weight of the subjects were 28.13.0 years (meansd), 161.96.9 cm, and 54.55.0 kg, respectively. our eligibility criteria were as follows : 1) passive ankle dorsiflexion rom of less than 8 degrees when measured in a non - weight - bearing condition with the knee extended and ankle dorsiflexion rom with the knee flexed to at least 5 degrees greater than with the knee extended in the subtalar neutral position ; 2) leg length discrepancy of less than 2 cm ; 3) no lower extremity injury within 6 months prior to participation in the study ; 4) no history of neurological dysfunction ; and 5) ability to ambulate without pain. we chose experimental feet with a passive ankle dorsiflexion rom of 8 degrees or less with the knee extended6. a vicon mx - t20 motion analysis system (vicon motion system, ltd., oxford, uk) (sampling rate 100 hz) with eight cameras was used to record the location of reflective skin markers over the lateral calcaneus ; this was used to determine the time of heel strike and heel off. the activities of the medial gcm and ta muscles of the lower extremity with limited ankle dorsiflexion were measured by a wireless electromyography (emg) system (delsys, inc., boston, ma, usa) using surface electrodes with a fixed interelectrode distance of 10 mm. the emg signals were rectified and low - pass filtered using a zero - lag butterworth filter with a cutoff frequency of 10 hz. the data processing was controlled by the nexus software (ver. gait cycle events were used to determine heel strike, heel off, and toe off events for each gait trial. prior to incline walking, electrodes were attached to the medial gcm and ta muscles. a sampling rate of 1,000 hz was used, and the root mean square (rms) values of the raw data were calculated. for normalization, maximal emg signals were acquired during a maximal voluntary isometric contraction (mvic) maneuver, which was performed for 5 seconds in the manual muscle - testing position described by kendall. each subject was instructed to walk with and without modified mwm using tape on an inclined treadmill (model 1100, hebb industries, inc., tyler, texas, usa) set to a grade of 6 degrees at a speed of 1.25 m / s for 5 minutes. the modified mwm using taping was applied by one examiner 's maximal force using a piece of tape. each participant placed their foot on a box and positioned closed - packed dorsiflexion as tibia forward from the talus anterior progressing inferiorly and posteriorly. one examiner attached the tape (a mueller kinesiological tape with a width of 37 mm) to the plantar surface of the calcaneus from the lateral to medial side. the two tasks (incline walking with and without modified mwm using tape) were administered randomly, and each subject rested for 30 minutes between tasks in order to minimize any learning effect. statistical analysis was performed using a statistical package (pasw statistics, 18.0, ibm corporation, ny). the paired t - test was used to examine differences in emg activities of the gcm and ta muscles with and without modified mwm using tape, and significance was defined as p<0.05. there were significant differences with and without the modified mwm using tape in terms of the normalized mean emg activity of the gcm (p=0.001) and ta (p=0.049) between heel strike and heel off. the normalized mean emg activities of the gcm during incline walking with and without the modified mwm using tape were 67.6321.48%mvic and 54.5617.68%mvic, respectively. the normalized mean emg activities of the ta during incline walking with and without the modified mwm using tape were 24.8515.97%mvic and 27.5215.14%mvic, respectively. there were no significant differences among tasks in terms of the emg activities of the gcm and ta between heel off and toe off. the normalized mean emg activities of the gcm during incline walking with and without the modified mwm using tape were 56.2429.79%mvic and 52.5619.05%mvic, respectively. the normalized mean emg activities of the ta during incline walking with and without the modified mwm using tape were 26.8616.48%mvic and 24.9712.54%mvic, respectively. this study investigated the effect of modified mwm using tape on muscle activities during inclined walking in women with limited ankle dorsiflexion. we found that modified mwm using tape during incline walking increased the activity of the medial gcm muscle and decreased the activity of the ta muscle between heel strike and heel off, while there was no difference between heel off and toe off in the two muscles. we consider that applying tape to the talus during incline walking may make the talus move in the posterior and inferior directions. it is also possible that the force of the tape toward the posterior and inferior directions on the talus and a repetitive activity involving 5 minutes of incline walking performed additively could induce the talus to roll and glide posteriorly, which may affect ankle arthrokinematics, resulting in a gain in the normal dorsiflexion rom. active physiological movements of the ankle joint under weight - bearing activity could increase ankle dorsiflexion rom, while changed ankle mechanics could alter muscle activities11. therefore, regaining of ankle dorsiflexion rom by modified mwm using tape could change the flexibility of the gcm muscle ; enhanced muscle flexibility, in turn, could allow more efficient movements, which might increase the emg activity of the gcm. incline walking with the modified mwm using tape involves active physiological motion, and repetitive motion for 5 minutes could change gcm muscle activity. we think that incline walking with the modified mwm using tape could be applied for effects similar to those of dynamic stretching, which consists of performing movements12. we observed that there was decreased emg activity of the ta muscle between heel strike and heel off during inclined walking with modified mwm using tape compared with that without using tape. at least 10 degrees of ankle dorsiflexion is needed during the stance phase of the gait cycle ; this contributes to forward body movement for normal walking. the ta muscle is active at heel strike and continues until the loading response ; initially, eccentric activity and concentric activity in the second half of the loading response move the tibia forward1. to compensate for the limited ankle dorsiflexion rom, the activity of the ta muscle may increase during the early stance phase13. incline walking with the modified mwm using tape could be helpful to in increasing ankle dorsiflexion rom, which may contribute to controlling the compensatory strategy and preventing pronation, thereby resulting in decreased activity of the ta muscle. the emg activities in both the gcm and ta muscles did not change between heel off and toe off in this study. we focused on the mean emg activity of the gcm prior to the pre - swing phase because plantarflexion primarily arises from the recoil of the tendon, and actually, the gcm seldom moves between heel off and toe off1. our data were obtained from a small sample of only young women, so it is difficult to standardize the change in the activities of the lower extremity muscles. further study with a larger number of subjects should be performed to analyze the kinetics and kinematics during incline walking in a synchronous fashion. in addition, we need to investigate the long - term effect of modified mwm using tape through various exercise frequencies. we concluded that incline walking with modified mwm using tape increased the emg activity of the medial gcm muscle and decreased the activity of the ta muscle between heel strike and heel off. therefore, our results suggest that modified mwm using tape during incline walking could be an effective intervention for altering the emg activities of medial gcm and ta muscle in people with limited ankle dorsiflexion. | [purpose ] this study compared the emg activities of the plantarflexor and dorsiflexor muscles during inclined walking with and without modified mobilization with movement (modified mwm) using tape in women with limited ankle dorsiflexion. [subjects ] fifteen women with limited dorsiflexion in their feet (22 feet in total) were recruited for this study. [methods ] the subjects walked with and without modified mwm using tape on a treadmill at 6 degrees with a speed of 1.25 m / s for 5 minutes. the emg activities of the medial gastrocnemius (gcm) and tibialis anterior (ta) muscles were measured using a surface emg system. [results ] during incline walking with modified mwm using tape, the mean emg activity of the gcm significantly increased, and that of the ta decreased between heel strike and heel off. there was no difference between heel off and toe off in the two muscles. [conclusions ] modified mwm using tape on the talus during incline walking could alter the muscle activities of the gcm and ta between heel strike and heel off in women with limited ankle dorsiflexion. |
since bacteria gain access to the periodontal pocket, they can invade the epithelium, connective tissue and radicular hard tissue, such as cementum and dentin, of periodontally diseased teeth1516,18. these sites can serve as bacterial reservoirs from which periodontal bacteria recolonize root surfaces after periodontal therapy16. depth of bacteria penetration into the dentinal tubules is variable and frequently can not be reached by mechanical periodontal therapy. bacterial invasion of dentinal tubules commonly occurs when dentin is exposed following a breach in cementum integrity during periodontal disease12,7,11. bacterial products can diffuse through the dentinal tubules towards the periodontal pocket and produce inflammatory changes that will influence periodontal repair and may be responsible for persistence of the periodontal infection. this study was undertaken in an effort to discuss the importance of dentinal tubule invasion by bacteria originating from periodontal pockets in periodontally diseased human teeth. the experimental group consisted of 10 periodontally diseased human teeth with extensive loss of periodontal supporting tissue, which were extracted from 10 fifty - year - old male patients, randomly selected from a private dental office. all participants were fully informed on research purposes and methodology and signed an informed consent form for participation in the study. care was taken to avoid mechanical damage to root surface. immediately after extraction, blood and saliva the specimens were fixed in a 10% neutral buffered formalin solution and decalcified in formic sodium citrate acid. root segments were prepared for histological analysis in the usual manner, i.e., paraffin embedding, serial sectioning to an average thickness of 6 m and staining by the brown and brenn technique4. the histological examination of periodontally diseased human teeth demonstrated that loss of cementum due to periodontal disease exposed the dentinal tubules, allowing penetration of bacteria. bacterial invasion in dentin occurred to such a depth that would be hard to reach by periodontal therapy (figure 1). no contamination was observed in dentinal tubules when cementum was present over the dentin layer (figure 2). an essential change in the bacterial flora of the gingival margin during plaque development is necessary to produce clinical gingivitis. nevertheless, gingival alterations are dispersed throughout the dentition, and the time required to develop clinical gingivitis varies considerably, despite the fact that bacterial colonization of gingival margin is almost the same in any individual17. although there is a general agreement that bacteria colonizing and growing at the gingival margin in the gingival sulcus cause inflammation, the mechanism that conducts gingivitis to periodontitis is not completely understood17,27. it seems that gingivitis is almost always dependent on endogenous bacterial plaque accumulation and probably aggressive anaerobic bacteria are not predominant in the initial phase of periodontitis. on the other hand, in the periodontal pocket, exogenous anaerobic bacterial plaque is always present and predominant19. as plaque age increases, nevertheless, other etiologic factors have necessarily to act together with bacteria in order to induce periodontitis only in a determined tooth or tooth region14,17. this means that if periodontal pocket were promoted only by bacteria, periodontal disease would probably be active in all teeth at the same time, producing the same pocket depth with the same bacterial plaque, or only in areas with poor plaque control. the multifactorial etiology of periodontal disease, however, clearly indicates that bacteria alone are not sufficient to produce periodontal pocket. it seems that the initial phase of destructive periodontitis is dependent on the association between endogenous bacteria and other etiologic factors to produce the ideal environment as periodontal pocket to be infected by exogenous anaerobic bacteria. in fact, periodontal disease produces destruction and deep pockets only in some regions (i.e., around a single tooth, in an interproximal space or at one side of a single tooth), which are under influence of other predisposing etiological factors5,22. it is thus difficult to find generalized destruction caused by periodontal disease in all teeth of the same patient12,21. when severe periodontitis - induced loss of supporting bone is localized in one tooth, the bacterial species involved in this deep pocket are very different from those of the gingival sulcus of the adjacent teeth10,17,20,23,24,26. this phenomenon may be attributed to the fact that contaminant bacteria require an anaerobic favorable environment, such as periodontal pocket, to survive. when an established gingivitis becomes a destructive periodontitis, pocket formation is the most important clinical and pathological alteration associated with inflammatory periodontal disease27. in this stage, etiologic factors of periodontal disease could change mainly the contaminant bacteria that colonize periodontal pockets, as a result of repeated infection by the various species or combinations of species. considerable variation has been reported in the profile of bacterial species present in subgingival plaque samples from different individuals and from sites of the same individuals. discreet complexes of bacterial species have been described in association with periodontal disease status and progression26. a shift towards increasing numbers of gram - negative species occurs in samples from subjects with plaque - induced gingivitis28. colonization with a specific periodontal pathogen seems necessary but not sufficient for periodontal disease progression, since most colonized sites remain quiescent for long periods9,25. however, some of these gram - negative anaerobic or facultative anaerobic bacterial species themselves may destruct host defensive cells3. periodontal tissues are destructed during the short inflammatory acute phase of periodontitis. however, the most prevalent inflammatory phase is chronic and reparative25, which always allows repair of the attached connective tissue zone that separates the underlying destructed alveolar bone from the apical end of the pocket epithelium, and also permits repair of the cortical bone inside the bone defects created by periodontal disease12. because of this reparative process, osseous graft surgical procedures require mechanical debridement with curettes and fine rotary instruments in order to promote an intra - marrow penetration into periodontal bone defects, allowing the vascularization ingress and access to osteogenic bone cells in the marrow bone and periodontal ligament before grafting29. although several bacteria that act as primary etiologic factors are present in a stable periodontal pocket, it is likely that they can only restart periodontal destruction if the bacteria - induced inflammatory acute phase occurs at exactly the same time as the inflammatory acute phase determined by other predisposing etiological active factors. all these inflammatory acute processes must act simultaneously to alter the zone of gingival attached connective tissue (biological width), which is the main barrier against destructive phase of periodontal disease. to be coincidental, these acute phases could be aided by other debilitating conditions, such as alterations in host defense mechanisms or even systemic, emotional and local factors. bacteria may be inaccessible to mechanical periodontal therapy6 in concavities, lacunae and especially in dentinal tubules. dentinal tubules ' microflora associated with a periodontal pocket could act as a reservoir for pocket recolonization12,11. most species retrieved from radicular dentin are gram - positive bacteria, such as p. micros, s. intermedius, a. naeslundii, with smaller numbers of gram - negative organisms, such as p. gingivalis, p. intermedia, bacteroides forsythus, f. nucleatum and v. parvulla 11. bacterial invasion of dentinal tubules commonly occurs when dentin is exposed following physicochemical and structural alterations of the cementum, such as localized resorptive lacunae or demineralization promoted by bacterial enzymes and acidic metabolites8. the depth of bacterial invasion in dentin, which depends in part on the dentinal tubule diameter, may be of such an extent that can not be reached by conventional mechanical and chemical periodontal therapy. bacteria that are able to invade radicular dentin tubules from the periodontal pocket may release bacterial products that diffuse through the dentinal tubules towards the pocket and evoke an inflammatory response, contributing to infection persistence13. the conditions under which established but stable periodontitis develops into destructive periodontitis are among the most interesting research issues of periodontology because, in some cases, periodontal lesions remain quiescent for a long time, while in others they cause aggressive breakdown16,25. this study discussed about bacteria, which are primary etiological factors of periodontal disease and will always be present in the mouth, having the potential to invade dentinal tubules during periodontal disease and being hard to eliminate in some cases. however, in spite of the persistence of bacteria in the periodontal tissues, periodontal disease may either be stable and quiescent or cause destruction, depending on whether or not other predisposing etiologic factors act together with the pathogens to develop a coincidental inflammatory destructive acute phase. preventing and treating periodontitis are thus of key importance to understand, diagnose, control and/or eliminate bacteria and other predisposing etiologic factors implicate in this disease. bacteria may invade dentinal tubules exposed to periodontal pocket and are very hard to be eliminated by conventional periodontal therapy. contaminated dentinal tubules of periodontally diseased teeth can thus act as active bacterial reservoirs to promote recolonization of mechanically treated root surfaces, which could interfere with the periodontal healing and progression of periodontal disease. | this study demonstrated that a significant number of bacteria is present in the radicular dentinal tubules of periodontally diseased human teeth. ten periodontally diseased teeth were prepared and stained by brown and brenn technique for histological examination. bacteria were detected in all teeth. it is suggested that bacteria may invade dentinal tubules exposed to periodontal pocket and are very hard to be eliminated by conventional mechanical and chemical periodontal therapy. contaminated dentinal tubules of periodontally diseased teeth can thus act as active bacterial reservoirs to promote recolonization of mechanically treated root surfaces, which could interfere with the periodontal healing and progression of the disease. |
about 30 % of people with congenital hearing loss are syndromic and the remaining 70 % are non - syndromic. in addition, most elderly people develop age - related (late - onset) hearing loss [13 ]. in general, these hearing losses have been classified as different diseases due to distinct pathogeneses [1, 2 ]. sensorineural hearing losses are caused by impairments of inner ears and are difficult to cure due to the location and complex morphology of inner ears [1, 2 ]. sensorineural hearing loss is a clinically heterogeneous disease leading to negative impacts on quality of life (qol) in all generations. inner ears have been analyzed in order to clarify the pathogeneses of sensorineural hearing losses. the organ of corti contains two kinds of sensory cells [inner hair cells (ihcs) and outer hair cells (ohcs) ] and plays an important role in mechanotransduction, by which sound stimuli are converted into electric stimuli. auditory information from the sensory cells is transferred to spiral ganglion neurons (sgns) as the primary carriers and is eventually transferred to the auditory cortex in the cerebrum [1, 2 ]. the sv consists of marginal cells, melanocytes (also known as intermediate cells) and basal cells, and has been shown to maintain high levels of potassium ion for endocochlear potential (ep) [4, 5 ]. melanocytes in the inner ear are located specifically in the sv, and defects in melanocytes lead to impaired ep levels resulting in hearing loss. thus, disturbance of these constituent cells in inner ears has been shown to cause hearing losses. vestibular hair cells covered with otoconia play an important role in mechanotransduction, by which gravity impulses are converted into neural impulses. thus, the vestibule containing hair cells and an otolith is one of the organs responsible for balance. impairments of hearing and balance both major problems in the field of occupational and environmental health are caused by the intricate interplay of genetic, aging and environmental factors [13 ]. this review focuses on hearing impairments caused by neurodegeneration of sgns due to impairments of hearing - related genes (c - ret and ednrb) and by environmental stresses [low frequency noise (lfn) and heavy metals ]. glial cell line - derived neurotrophic factor (gdnf)one of the ligands for c - ret exerts its effect on target cells by binding to a glycosyl phosphatidylinositol (gpi)-anchored cell surface protein (gfr1). this binding facilitates the formation of a complex with the receptor tyrosine kinase c - ret. formation of this complex activates c - ret autophosphorylation as a trigger for c - ret - mediated signaling pathways to give positive signals for cell survival [912 ]. previous studies have also indicated that gdnf stimulates a ret - independent signaling pathway [10, 13, 14 ]. tyrosine 1062 (y1062) in c - ret plays an important role in kinase activation as one of the autophosphorylation sites, and is also a multi - docking site for several signaling molecules including shc, a transmitter for c - ret - mediated signaling pathways [13, 15, 16 ]. in both mice and humans, c - ret has been shown to be essential for the development and maintenance of the enteric nervous system (ens) [13, 15 ] and to be the most frequent causal gene of hirschsprung disease (hscr ; megacolon disease) (in 2025 % of cases) in humans [17, 18 ]. in fact, severe hscr (e.g., total intestinal agangliosis and impaired development of the kidney) has been shown to develop in homozygous knock - in mice in which y1062 in c - ret was replaced with phenylalanine (c - ret - ki - mice), while heterozygous c - ret y1062f knock - in mice (c - ret - ki - mice) are reported to have no hscr - linked phenotypes. thus, the results of previous studies indicate that hscr in mice develops recessively, while hscr in humans has been shown to develop dominantly due to ret mutations. as described above, c - ret and c - ret are crucial genes for hscr ; however, there had been no direct evidence to link c - ret and c - ret to hearing impairments in mice or humans. our recent studies have shown that complete unphosphorylated y1062 in c - ret, with no change in expression level, caused congenital hearing loss in c - ret - ki - mice, while partially unphosphorylated c - ret led to normal hearing development until 1 month of age but then accelerated age - related hearing loss in c - ret - ki - mice. thus, impairments of c - ret phosphorylation monogenetically result in early - onset syndromic hearing loss as well as late - onset non - syndromic hearing loss. our results correspond in part to the results of previous studies demonstrating that c - ret, gfr1 and gdnf are expressed in auditory neurons [22, 23 ] and that gdnf has a protective effect on antibiotic - mediated ototoxicities [2427 ]. waardenburg - shah syndrome (ws type iv, ws - iv), which is caused by mutations in the transcription factor sox10, cytokine endothelin (et)-3 and its receptor endothelin receptor b (ednrb), is characterized by hypopigmentation, megacolon disease and hearing loss. the incidence of ws is 1 per 10,000 to 20,000 people. endothelin receptor b (ednrb / ednrb) belongs to the g - protein - coupled receptor family that mediates the multifaceted actions of endothelins [32, 33 ]. mutations of ednrb / ednrb have been shown to cause embryonic defects in melanocytes and enteric ganglion neurons derived from the neural crest, resulting in hypopigmentation, megacolon disease and congenital hearing loss. in previous studies with animal models, both piebald - lethal rats in which ednrb is spontaneously mutated and ednrb homozygous knock - out [ednrb(/) ] mice have been shown to have typical ws - iv phenotypes. thus, previous studies indicate that ednrb is a key regulatory molecule for embryonic development of melanocytes and peripheral neurons, including neurons in the ens. previous studies also demonstrated that impairments of ednrb / ednrb cause syndromic hearing loss due to congenital defects of melanocytes in the stria vascularis of the inner ear [30, 3235 ]. in our previous study, ednrb protein was expressed in sgns from wild - type (wt)-mice on postnatal day 19 (p19), while it was undetectable in sgns from wt - mice on p3. correspondingly, ednrb homozygously deleted mice [ednrb(/)-mice ] developed congenital hearing loss (fig. 1). thus, expression of ednrb expressed in sgns in the inner ears is required for postnatal development of hearing in mice. a therapeutic strategy for congenital hearing loss in ws - iv patients has not been established. ednrb expressed in sgns could be a novel potential therapeutic strategy for congenital hearing loss in ws - iv patients.fig. 1schematic summary of congenital deafness caused by neurodegeneration of spiral ganglion neurons (sgns) in c - ret - knock - in - mice and ednrb - knock - out - mice. triangles rosenthal s canals in wild - type (wt) (light gray background), or homozygous c - ret - knock - in (ret - ki) and homozygous ednrb - knock - out - mice (ednrb - ko) (white background) ; gray circles / no outline immature sgns ; gray circles / thin outline sgns ; gray circles / bold outline sgns with dark gray circles / dotted outline sgns with decreased phosphorylation of y1062 in c - ret or decreased expression of ednrb. a c - ret - ki- and ednrb - ko - mice suffer from congenital deafness with neurodegeneration of sgns. bc - ret - ki - mice showed no y1062-phosphorylated sgns even on p8, although y1062-phosphorylated sgns began to appear in wt mice from p8. ednrb - ko - mice also showed undetectably low expression level of ednrb in sgns on p8, although ednrb - positive sgns began to appear in wt mice from p8 schematic summary of congenital deafness caused by neurodegeneration of spiral ganglion neurons (sgns) in c - ret - knock - in - mice and ednrb - knock - out - mice. triangles rosenthal s canals in wild - type (wt) (light gray background), or homozygous c - ret - knock - in (ret - ki) and homozygous ednrb - knock - out - mice (ednrb - ko) (white background) ; gray circles / no outline immature sgns ; gray circles / thin outline sgns ; gray circles / bold outline sgns with dark gray circles / dotted outline sgns with decreased phosphorylation of y1062 in c - ret or decreased expression of ednrb. a c - ret - ki- and ednrb - ko - mice suffer from congenital deafness with neurodegeneration of sgns. bc - ret - ki - mice showed no y1062-phosphorylated sgns even on p8, although y1062-phosphorylated sgns began to appear in wt mice from p8. ednrb - ko - mice also showed undetectably low expression level of ednrb in sgns on p8, although ednrb - positive sgns began to appear in wt mice from p8 phosphorylation of y1062 in c - ret has been shown to mediate several biological responses, including development and survival of neuronal cells [13, 37 ]. in our recent studies, c - ret - ki - mice developed severe congenital deafness with neurodegeneration of sgns on postnatal day (p) 8 - 18, while c - ret - ki - mice showed morphology of sgns comparable to that in wt mice on p2 - 3. phoshorylation of y1062 in c - ret of sgns from wt mice on p2 - 3 was below the limit of detection, while that on p8 - 18 was clearly detectable. thus, it is thought that sgns from c - ret - ki - mice developed normally at least until p3 after birth, when y1062 in c - ret of sgns from wt mice is unphospholylated. however, in c - ret - ki - mice, phosphorylation of y1062 is no longer maintained by p8p18, when y1062 in c - ret of sgns from wt mice exhibits significant phosphorylation. furthermore, partially unphosphorylated y1062 in c - ret of sgns accelerated age - related hearing loss with accelerated reduction of sgns from 4 months of age, while normal hearing and normal density of sgns were observed at least until 1 month of age, when hearing has matured. on the other hand, ednrb protein was expressed in sgns from wt - mice on postnatal day 19 (p19), while it was undetectable in sgns from wt - mice on p3. correspondingly, ednrb(/)-mice with congenital hearing loss showed a decreased number of sgns (fig. 1) and thus, our results show that ednrb expression in sgns in inner ears is required for postnatal survival of sgns in mice. the neurodegeneration of sgns from c - ret - ki - mice and ednrb(/)-mice did not show typical apoptotic signals and did not involve disturbance of hair bundles of ihcs and ohcs [20, 36 ]. the congenital hearing loss involving neurodegeneration of sgns as well as megacolon disease in ednrb(/)-mice were improved markedly by introducing an ednrb transgene under the control of the dopamine beta - hydroxylase promoter (ednrb(/) ; dbh - ednrb - mice). neurodegeneration of sgns was restored by introducing constitutively activated in the case of c - ret - mediated hearing loss. thus, our results indicate that c - ret and ednrb expressed in sgns could be molecular targets in the prevention of hearing impairments. exposure to noise is recognized as one of the major environmental factors causing hearing loss. noise consists of sound with broad frequencies, but there is limited information about the frequency - dependent influence of noise on health. low frequency noise (lfn) is constantly generated from natural and artificial sources. the frequency range of lfn is usually defined as being below 100 hz, while that of infrasound is usually below 20 hz. in our recent study, we found that chronic exposure to lfn at moderate levels of 70 db sound pressure level (spl) causes impaired balance involving morphological abnormalities of the vestibule with increased levels of oxidative stress (fig. 2). previous studies have shown that behavioral impairments induced by antibiotics involved degeneration of vestibular cells and oxidative stress [40, 41 ]. in addition, a previous study has shown that antioxidant compounds prevent noise - induced hearing loss. ototoxicity caused by oxidative stress in inner ears has been shown to accompany impairment of antioxidant enzymes. thus, existing studies indicate the necessity for further investigation of a causal molecule related to oxidative stress in vestibular hair cells affected by lfn, and of the preventive effect of antioxidants on impaired balance caused by lfn exposure. on the other hand, exposure to heavy metals including mercury, cadmium and arsenic has been suggested to cause impairments in balance and hearing [4446 ] in humans and experimental animals. childhood exposure to heavy metals has been shown to sensitively affect hearing development in humans [4850 ]. aging has also been shown to affect sensitivities to ototoxic factors in mice. therefore, further studies are needed to determine the age - specific susceptibilities to environmental stresses, including heavy metals, in terms of ototoxicity in mice and humans.fig. 2schematic summary of impaired balance in mice caused by exposure to low frequency noise (lfn). chronic exposure to low frequency noise (lfn, 0.1 khz) at moderate levels of 70 db sound pressure level (spl) causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress schematic summary of impaired balance in mice caused by exposure to low frequency noise (lfn). chronic exposure to low frequency noise (lfn, 0.1 khz) at moderate levels of 70 db sound pressure level (spl) causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress our studies provide direct evidence that c - ret and ednrb expressed in sgns are novel targets for hearing loss. these studies underline the importance of considering the activity as well as the expression of the target molecule in order to elucidate the etiologies of hereditary deafness. in addition, environmental stresses, including exposure to noise and heavy metals, can cause impairments of hearing and balance that are affected intricately by aging and genetic factors. information obtained in previous studies prompts further investigation of the influence of environmental stresses on the impairment of hearing and balance with consideration of aging and genetic factors to develop new diagnostic, preventive and therapeutic strategies against impairment of hearing and balance. | impairments of hearing and balance are major problems in the field of occupational and environmental health. such impairments have previously been reported to be caused by genetic and environmental factors. however, their mechanisms have not been fully clarified. on the other hand, the inner ear contains spiral ganglion neurons (sgns) in the organ of corti, which serve as the primary carriers of auditory information from sensory cells to the auditory cortex in the cerebrum. inner ears also contain a vestibule in the vicinity of the organ of corti one of the organs responsible for balance. thus, inner ears could be a good target to clarify the pathogeneses of sensorineural hearing losses and impaired balance. in our previous studies with c - ret knock - in mice and endothelin receptor b (ednrb) knock - out mice, it was found that syndromic hearing losses involved postnatal neurodegeneration of sgns caused by impairments of c - ret and ednrb, which play important roles in neuronal development and maintenance of the enteric nervous system. the organ of corti and the vestibule in inner ears also suffer from degeneration caused by environmental stresses including noise and heavy metals, resulting in impairments of hearing and balance. in this review, we introduce impairments of hearing and balance caused by genetic and environmental factors and focus on impairments of sgns and the vestibule in inner ears as the pathogeneses caused by these factors. |
multiple myeloma is a malignant b - cell disorder characterized by proliferation of atypical plasma cells in bone marrow with or without the presence of monoclonal immunoglobulin protein in serum and/or urine. multiple myeloma has correlation with plasmacytoma, which is a mass of plasma cells found outside of bone marrow that needs medical intervention with radiotherapy or chemotherapy. while multiple myeloma frequently accompanies plasmacytoma at the time of diagnosis, plasmacytoma precedes multiple myeloma in some cases. the disease entity called solitary plasmacytoma exists in 4% of plasma cell tumors [5, 6 ], and approximately 4050% of patients with solitary plasmacytoma will develop multiple myeloma. hence, plasmacytoma is an early form or an accompanying disease of myeloma, and the data regarding the clinical behavior of plasmacytoma are quite accumulated. however, not much is known about the cellular biology of plasmacytoma per se. fortunately, we previously succeeded in establishing two cell lines with different tropism from a single patient. briefly, 14 weeks after injecting mononuclear cells obtained from multiple myeloma patient 's bone marrow in a nrg / scid mouse via tail vein, tumor developed at subcutis of the mouse. the engraftment of myeloma cells into mouse bone marrow (bm) was also observed. after separation and culturing cells from subcutis and bm, we succeeded in the establishing of two cell lines (snu_mm1393_sc and snu_mm1393_bm) from a single patient with different tropism. snu_mm1393_bm and snu_mm1393_sc showed a high degree of resistance against bortezomib compared to u266 cell line. snu_mm1393_bm had the greater lethality compared to snu_mm1393_sc. from genetic perspective, we believe that unique somatic mutations may allow tumor cells to adapt and survive in tumor microenvironment. in other words, there may be specific genetic changes that contribute to tumor tropism. hence, in this study, we tried to characterize genomic profile specific for snu_mm1393_bm and snu_mm1393_sc to understand genetic background of difference in these cell lines. we were particularly interested in genetic changes specific for snu_mm1393_sc, because we thought these snu_mm1393_sc specific genetic changes would reveal genetic background for plasmacytoma which exhibit tropism for extramedullary space. to find these genetic changes, we performed whole exome sequencing (wes) using dna of both cell lines. as it is well known, wes allows comprehensive characterization of genomic changes in individual tumors. japan) was used to extract dna according to the manufacturer 's recommendations. for wes, we sequenced exome using the solexa sequencing technology platform (hiseq2000, illumina, san diego, ca, usa) following the manufacturer 's instructions. we randomly sheared 3 ug of genomic dna using covaris system to generate about 150 bp inserts. the fragmented dna was end - repaired using t4 dna polymerase and klenow polymerase, and illumina paired - end adaptor - oligonucleotides were ligated to the sticky ends. we analyzed the ligation mixture by electrophoresis on an agarose gel, sliced and purified fragments with 200250 bp sizes. purified dna library was hybridized with sureselect human all exon v4 probes set (agilent, santa clara, ca, usa) to capture 50 mb targeted exons following manufacturer 's instruction. we prepared the hiseq2000 paired - end flow cell to the manufacturer 's protocol using captured exome library. clusters of pcr colonies were then sequenced on the hiseq2000 platform using recommended protocols from the manufacturer. fastq files were aligned to human reference (human_g1k_v 37.fasta) by using the burrows - wheeler aligner (bwa-0.7.5) to make sam file. sortsam in picard - tools-1.68 was used to convert to bam file and sort with chromosome and went through a pcr duplicate marking process, which enables the genome analysis toolkit (gatk-1.6.5) to ignore duplicates in subsequent processing. we performed a local realignment prior to recalibration, which gives the most accurate quality scores for each sample. for single nucleotide variant (snv) and small indel calling, we used varscan2 (http://genome.wustl.edu/). because germline control was absent, we used pilup2snp command for single sample calling using human_g1k_v 37 as reference genome. we set the following options : (1) minimum coverage above value of 20, (2) minimum variant allele frequency above value of 0.1, and (3) default p value below 0.05 other option value set as default values. to select unique mutation, we performed comparison between two calling results. for functional annotation and prediction of variant effect, we used annovar with polyphen database version 2.2.2. for comparing public data with results in this study, we used datasets from tcga (https://tcga-data.nci.nih.gov/tcga/tcgahome2.jsp), cbioportal for cancer genomics (http://www.cbioportal.org/public-portal/), and kegg database for pathway analysis (http://david.abcc.ncifcrf.gov/). the raw data size of snu_1393mm_bm and snu_1393mm_sc was 9,090 mb and 8,979 mb, respectively. approximately 99.00% of the targeted reads (165483843 reads) were covered sufficiently to pass our thresholds for calling variants (mapq > 20 by ngs qc toolkitv2.3). mapq distribution following that above 30 was 98.2% (164088367), above 20 was 0.8% (1395476), and below 20 of mapq was under 10%. for snu_1393mm_sc, mapq distribution following that above 30 was 98.1% (159871347), above 20 was 0.8% (154084), and below 20 of mapq was around 10%. when snv calling was performed using varscan, a total of 18573 snvs were found in snu_mm1393_sc. their distribution according to the functional consequences was as follows : 8595 (46.2%) nonsynonymous, 9575 (51.5%) synonymous, 68 (0.003%) stop - gain, and 6 (0.0003%) stop - loss. in snu_mm1393_bm, a total of 18781 snvs were found and their distribution was as follows : 8694 (46.2%) nonsynonymous, 9667 (51.5%) synonymous, 75 (0.004%) stop - gain, and 5 (0.0003%) stop - loss. as for nonsynonymous snvs, we found 8595 nonsynonymous snvs in 4901 genes for snu_mm1393_sc, while 8694 nonsynonymous snvs in 4969 genes were found in snu_mm1393_bm. there was overlapping of 8344 nonsynonymous snvs, and 251 nonsynonymous snvs and 350 nonsynonymous snvs were unique for snu_mm1393_sc and snu_mm1393_bm, respectively (figures 1(a)1(c)). the rate of transversion and transition in the coding region was different between the two cell lines. while transversion was dominant event in snu_mm1393_bm cell line, transition was dominant event in snu_mm1393_sc. absolute transversion rate was much higher in snu_mm1393_bm (65.5%) than snu_mm1393_sc (34.0%) (figure 1(d)). after calling of snvs, we compared genomic signatures of snu_mm1393_sc and snu_mm1393_bm with those of tumors in public database. for this comparison, we selected 12 nonsynonymous snvs that is unique for snu_mm1393_bm and 11 nonsynonymous snvs that is unique for snu_mm1393_sc. these snvs were selected according to the criteria below : with the assumption that two cell lines consisted of single cell population, we selected genes with variant allele frequency between 0.4 and 0.6. first, the frequencies of these snvs were investigated in open source data of multiple myeloma (multiple myeloma research consortium) using cbioportal for cancer genomics (http://www.cbioportal.org). around half of snvs found in our cell lines were found with low frequency (0.52%) in open source database of multiple myeloma (table 1). then, the frequencies of these snvs were investigated in open source data of various cancers (http://www.cbioportal.org). when this analysis was performed, snvs which are unique for snu_mm1393_sc were frequently detected in melanoma (52.8%) and uterine cancer (49.0%). on the other hand, snvs those which are unique for snu_mm1393_bm were frequently found in ovarian cancer (74.7%) and bladder cancer (72.8%) (figure 2). using monte carlo simulation (ratio of dns / ds), we examined distribution statistics of snvs found in two cell lines, respectively. it is believed that nonrandomness of snv distribution is related to the functional importance of those snvs. when this analysis was performed, ratio for snu_mm1393_bm was 2.8 (p = 0.14), while it was 1.1 for snu_mm1393_sc (p = 0.07). hence, snv distribution in both cell lines was random with cut - off p value of 0.05. our results indicated that unique nonsynonymous mutations of snu_mm1393_sc seemed biologically more neutral than those of snu_mm1393_bm although they were statistically insignificant. in kegg pathway analysis of unique somatic mutation from both cell lines, chemokine signaling pathway and chemokine - chemokine interaction pathway were showed in two cell lines at the same time. while tight junction and adherens junction were shown to be involved only in snu_mm1393_sc, snu_mm1393_bm cell line showed diverse disorder in pathway such as cell cycle, wnt signaling pathway, and mapk signaling pathway (figure 3). in this report, we analyzed genomic signature of two cell lines derived from single patient. these cell lines have different tropism, and snu_mm1393_sc is plasmacytoma which has tropism for skin. thus, we thought this study would reveal genetic changes that are related to skin tropism and plasmacytoma. in fact, recent study reported the process of segregation of genetic changes in tumor cells during clonal expansion. as far as we know, this is the first wes study comparing the genomics of myeloma cells in bone marrow and plasmacytoma. varscan2 which was used in our study for somatic variant calling is one of the most commonly used tools to detect somatic mutation along with mutect [12, 17 ]. in fact, many papers already published in the field of cancer genomics already used varscan2 [1820 ]. as expected, most of snvs (96.5%) overlapped between the cell lines, suggesting their common originality from a single patient. and the most coding sequence snvs in both snu_mm1393_bm and snu_mm1393_sc were neutral with respect to adaptation and cancer cell growth. this is also supported by the outcome of monte carlo simulation, where distribution of snvs did not show significant nonrandomness. it has been suggested that bone marrow microenvironment strictly regulates the growth of cell via dynamic interplay among hematopoietic cells. and our results indicate that only a small subset of nonsynonymous snvs in cancer are affected by selection, making it possible to interpret snv trends as reflection of underlying mutational processes. the most interesting finding in our data we conjectured in the planning of this study that genetic change unique for snu_mm1393_sc would be related to skin tropism and formation of plasmacytoma. and, as expected, genomic signature that is unique to snu_mm1393_sc had more than 50% of overlapping with melanoma which is a primary skin tumor. this finding highly coincides with our conjecture and snvs such as kiaa1199, fry, ap3b2, and optc may be the very gene related to skin tropism of cancers. these 4 are the genes that are mutated in snu_mm1393_sc and are frequently found in melanoma samples. in fact, it has been known that there are common genetics between melanoma and plasmacytoma such as cdkn2a germline mutation in prenext generation sequencing (ngs) era. one more noticeable finding in our study is that the number of snvs was higher in snu_mm1393_bm than in snu_mm1393_sc and transversion rate was higher in snu_mm1393_bm than in snu_mm1393_sc. also dynamic gene to gene interaction in snu_mm1393_bm was more complex than that in snu_mm1393_sc. along with this phenomenon and from the previous report that the number and pattern of somatic snvs determine pathway underling cancer, we think that it would be biologically simpler to form a tumor in subcutis than to form a tumor in bone marrow in animal xenograft model used in our experiment. we think this is related to the fact that growth and differentiation of cancer cells in bone marrow are strictly regulated by dynamic interplay among various hematopoietic cells, compared to subcutis. in fact, we had difficulties in the analysis of wes data due to the lack of germline reference dna in this study. because we used public germline database as reference in the calling of snvs, the number of snvs was very large compared to the previous multiple myeloma genomic studies. moreover, it is well known that the majority of mutations observed in cancer sequencing studies are believed to be passenger mutations having little impact on the cancer cell. to overcome this issue rather, we analyzed the pattern of genetic changes found in our study and compared them with public database so as to find out genetic clues underlying tropism for skin and plasmacytoma. and snvs for both snu_mm1393_bm and snu_mm1393_sc instead of whole genomic picture of snu_mm1393_bm and snu_mm1393_sc. | background. previously we established two cell lines (snu_mm1393_bm and snu_mm1393_sc) from different tissues (bone marrow and subcutis) of mice which were injected with single patient 's myeloma sample. we tried to define genetic changes specific for each cell line using whole exome sequencing (wes). materials and methods. we extracted dna from snu_mm1393_bm and snu_mm1393_sc and performed wes. for single nucleotide variants (snv) calling, we used varscan2. annotation of mutation was performed using annovar. results. when calling of somatic mutations was performed, 68 genes were nonsynonymously mutated only in snu_mm1393_sc, while 136 genes were nonsynonymously mutated only in snu_mm1393_bm. kiaa1199, fry, ap3b2, and optc were representative genes specifically mutated in snu_mm1393_sc. when comparison analysis was performed using tcga data, mutational pattern of snu_mm1393_sc resembled that of melanoma mostly. pathway analysis using kegg database showed that mutated genes specific of snu_mm1393_bm were related to differentiation, while those of snu_mm1393_sc were related to tumorigenesis. conclusion. we found out genetic changes that underlie tropism of myeloma cells using wes. genetic signature of cutaneous plasmacytoma shares that of melanoma implying common mechanism for skin tropism. kiaa1199, fry, ap3b2, and optc are candidate genes for skin tropism of cancers. |
during the last decades, there have been great advancements in the field of preventive medicine. research has demonstrated that nutrition plays a crucial role in the prevention of chronic diseases. it proposes to consider food not only vital to survive, but also a mean for mental and physical well - being, contributing to the prevention and reduction of risk factors for diseases. however, there is evidence that the concept was believed by ancient physicians as well. rhazes said ; as long as a disease could be treated with food, medicine should be avoided we carried out a review of avicenna s canon of medicine and rhazes books for the definition of food and drug and similar concepts of functional food. rhazes has a book called manafe al - aghziyeh, in which he writes about the medicinal benefits of different nutrition. five concepts (food, drug, medicinal food, nutritional medicine and antidote or poison) were noted in these books. there are many recommendations on food for the prevention and treatment of diseases in tpm books, which can be the basis for novel research studies. | background : during the last decades, there have been great advancements in the field of preventive medicine. research has demonstrated that nutrition plays a crucial role in the prevention of chronic diseases. the concept of functional food was first introduced in japan during the 1980s. it proposes to consider food not only vital to survive, but also a mean for mental and physical well - being, contributing to the prevention and reduction of risk factors for diseases. however, there is evidence that the concept was believed by ancient physicians as well. one of the traditional systems of medicines is traditional persian medicine (tpm). rhazes said ; as long as a disease could be treated with food, medicine should be avoidedmethods : we carried out a review of avicenna s canon of medicine and rhazes books for the definition of food and drug and similar concepts of functional food. we listed the identified concepts along with their examples.results:the classification of food and their therapeutic use were explained in canon of medicine. rhazes has a book called manafe al - aghziyeh, in which he writes about the medicinal benefits of different nutrition. five concepts (food, drug, medicinal food, nutritional medicine and antidote or poison) were noted in these books.conclusion:there are many recommendations on food for the prevention and treatment of diseases in tpm books, which can be the basis for novel research studies. |
the patients have various clinical features depending on the size and deleted region on chromosome 13. the 13q syndrome is characterized into 3 groups : group 1 in which proximal region (q12.2-q31) is involved in deletion and patients tend to appear with minor abnormalities, mild to moderate mental retardation and susceptible to retinoblastoma. group 2 included those with proximal to q32 (q12.2-q32), these patients appear with major malfunctions, moderate to severe mental retardation and microcephaly and growth retardation. group 3 comprised those with q33-q34 ; these patients have severe mental retardation without major malfunctions and growth retardation. a 5-year - old boy was referred for genetic counseling with different phenotypical features [table 1 ]. the patient was the product of a full - term pregnancy and normal vaginal delivery. he had seizure beginning from 2 months ; brain computed tomography (ct) scan displayed agenesis of vermis of the cerebellum [figure 1 ], the clinical features in this case were : clinical features of the patients (a) note to the ptosis, down slanted palpebral fissures, strabismus, and high nasal bridge. (d) agenesis of the vermis cerebellum cleft palate, microcephaly (head circumference of 45 cm [< -2 sd (2%) ]), moderate to severe mental retardation, speech difficulty, asymmetric in left skulp, ptosis, club feet, down slanted palperbal fissures, hypospadias, neurodevelopmental delay, syndactyly in both hands fingers, short metacarp, overriding of the fourth toes, strabismus, left blepharophimosis in the left side, high nasal bridge, thin lips, micrognathia, no ovala, dental decay, small ears, and hypoplastic thumb. a 12-year - old girl was referred with dysmorphic features, mild mental retardation, and neurodevelopmental delay [table 1 ]. the girl was the product of a normal pregnancy and birth weight and height were 3.5 kg and 49 cm, respectively. head circumference was 53 cm normal for the age of 12 years, and she is studying in the elementary school. parents were first cousins once removed with the history of one abortion in the mother. clinical characterizations included [figure 2 ] : generalized weakness of muscle bulk (hypotonia), scapula alata, upper limb girdle weakness, high nasal bridge, wide mandibular angle, high arched palate, thin and long narrow face, down slanted palpebral fissures, hypoplasia of alae nasi, low hairline, webbed neck, overriding toes, and sandal gap. (a) note to the high nasal bridge, wide mandibular angle, thin and long narrow face, down slanted palpebral fissures, web neck. a 5-year - old boy was referred for genetic counseling with different phenotypical features [table 1 ]. the patient was the product of a full - term pregnancy and normal vaginal delivery. he had seizure beginning from 2 months ; brain computed tomography (ct) scan displayed agenesis of vermis of the cerebellum [figure 1 ], the clinical features in this case were : clinical features of the patients (a) note to the ptosis, down slanted palpebral fissures, strabismus, and high nasal bridge. (d) agenesis of the vermis cerebellum cleft palate, microcephaly (head circumference of 45 cm [< -2 sd (2%) ]), moderate to severe mental retardation, speech difficulty, asymmetric in left skulp, ptosis, club feet, down slanted palperbal fissures, hypospadias, neurodevelopmental delay, syndactyly in both hands fingers, short metacarp, overriding of the fourth toes, strabismus, left blepharophimosis in the left side, high nasal bridge, thin lips, micrognathia, no ovala, dental decay, small ears, and hypoplastic thumb. a 12-year - old girl was referred with dysmorphic features, mild mental retardation, and neurodevelopmental delay [table 1 ]. the girl was the product of a normal pregnancy and birth weight and height were 3.5 kg and 49 cm, respectively. head circumference was 53 cm normal for the age of 12 years, and she is studying in the elementary school. parents were first cousins once removed with the history of one abortion in the mother. clinical characterizations included [figure 2 ] : generalized weakness of muscle bulk (hypotonia), scapula alata, upper limb girdle weakness, high nasal bridge, wide mandibular angle, high arched palate, thin and long narrow face, down slanted palpebral fissures, hypoplasia of alae nasi, low hairline, webbed neck, overriding toes, and sandal gap. (a) note to the high nasal bridge, wide mandibular angle, thin and long narrow face, down slanted palpebral fissures, web neck. a 5-year - old boy was referred for genetic counseling with different phenotypical features [table 1 ]. the patient was the product of a full - term pregnancy and normal vaginal delivery. he had seizure beginning from 2 months ; brain computed tomography (ct) scan displayed agenesis of vermis of the cerebellum [figure 1 ], the clinical features in this case were : clinical features of the patients (a) note to the ptosis, down slanted palpebral fissures, strabismus, and high nasal bridge. (d) agenesis of the vermis cerebellum cleft palate, microcephaly (head circumference of 45 cm [< -2 sd (2%) ]), moderate to severe mental retardation, speech difficulty, asymmetric in left skulp, ptosis, club feet, down slanted palperbal fissures, hypospadias, neurodevelopmental delay, syndactyly in both hands fingers, short metacarp, overriding of the fourth toes, strabismus, left blepharophimosis in the left side, high nasal bridge, thin lips, micrognathia, no ovala, dental decay, small ears, and hypoplastic thumb. a 12-year - old girl was referred with dysmorphic features, mild mental retardation, and neurodevelopmental delay [table 1 ]. the girl was the product of a normal pregnancy and birth weight and height were 3.5 kg and 49 cm, respectively. head circumference was 53 cm normal for the age of 12 years, and she is studying in the elementary school. parents were first cousins once removed with the history of one abortion in the mother. clinical characterizations included [figure 2 ] : generalized weakness of muscle bulk (hypotonia), scapula alata, upper limb girdle weakness, high nasal bridge, wide mandibular angle, high arched palate, thin and long narrow face, down slanted palpebral fissures, hypoplasia of alae nasi, low hairline, webbed neck, overriding toes, and sandal gap. (a) note to the high nasal bridge, wide mandibular angle, thin and long narrow face, down slanted palpebral fissures, web neck. (b) scapula alata standard high - resolution gtg banding was carried out in order to investigate the patients chromosomes. case 1, a boy with deletion of q33-q34 [figure 3 ] who had microcephaly, moderate to severe mental retardation, neurodevelopmental delay, and primary diagnosis was moebius syndrome. case 2 was a girl with deletion of q12.3-q14.3 [figure 3 ], with dismorphic features, neurodevelopmental delay and neuropathy, but not microcephaly and seizure ; this case did not have any retinoblastoma. (a) the abnormal chromosome displays deleted region from q33 to q34 which, belongs to group 3. (b) the abnormal chromosome, with deleted region of q12.3-q14.3 belongs to group 1 case 1 related to the group 3 with more severe phenotypical and neurological features and case 2 related to the group 1 with less clinical and neurological characterizations. overall case 1 exhibited more dysmorphic characterizations and more severe mental retardation than case 2. it is recommended that children with seizure, neuropathy, mental retardation, and dismorphic features can be the candidate for chromosomal investigation as well as other tests required for evaluation. | patients with 13q deletion syndrome are characterized with different phenotypical features depending on the size and location of the deleted region on chromosome 13. these patients fall into three groups : in group 1, deleted region is in the proximal and does not extend into q32 ; in group 2, deleted region involves proximal to the q32 and in group 3 q33-q34 is deleted. we present two cases with 13q syndrome with two different deleted region and different severity on clinical features : one case with interstitial deletion belongs to the group 1 with mild mental retardation and minor malformations and the other case with terminal deletion belongs to group 3 with moderate to severe mental retardation and major malformations. |
pentacyclic triterpenoids have generally been utilized as biomarkers to trace genetic sources of organic matter in sedimentary environments, petroleum exploration, or paleoenvironmental reconstructions of biome changes that document climate change [14 ]. the oleananes, ursanes, fernanes, lupanes, and their derivatives, widely distributed mainly as the oxygenated forms in many varieties of higher plant species, belong to this class of compounds [410 ]. their tendencies to also resist biodegradation and occurrence in sediments suggest the potential application as specific higher plant derived biomarkers [9, 10 ]. however, reports of pentacyclic triterpenol methyl ethers (ptmes) in sedimentary environments are limited to lakes [2, 4, 12 ]. other reports have assessed sedimentary input of terrestrial and/or planktonic organic matter with triterpenoid natural products (e.g., [1315 ]) and biomass source tracers to smoke aerosols (e.g., burning of sugar cane). reports of triterpenoid esters in sediments are also limited, because typical extract analyses generally involved saponification as a preparative step. plant wax analyses without hydrolysis do reveal triterpenol esters as part of the wax esters in epicuticular waxes (e.g., [17, 18 ]). under aerobic conditions, the transformation of plant - derived triterpenoids often involves oxidation, dehydration, hydrolysis, decarboxylation, ring opening, and aromatization reactions. for instance, in coal forming environments, higher plant triterpenoids generally undergo aromatization starting from ring a, triggered by the elimination of the oxygenated functionality at c-3 and proceeding to rings d / e [2022 ]. the ptmes, which are natural products, appear to be more resistant to environmental alteration than the triterpenol esters and thus may be good biomarkers. triterpenol esters, on the other hand, may be useful for assessing early diagenesis (i.e., hydrolysis) of terrestrial higher plant detritus during river transport. it is the aim of this paper to report on the characterization, occurrence, and sources of ptmes and triterpenol esters in surface sediments of the cross - river system, nigeria. these are minor compounds occurring with the dominant triterpenoids such as taraxerol, amyrin, and lupeol. the characteristic features of the study area are summarized in table 1 and the sampling locations of surface sediments are shown in figure 1. the cross - river system is one of the largest estuaries located in the eastern edge of the niger delta. the whole cross - river system lies approximately between longitudes 2 03 e and 10 00 e and latitudes 4 00 n and 8 00 n and covers an area of 54,000 km, of which 14,000 km lies in cameroon and 39,000 km lies in nigeria. the river is formed from numerous tributaries arising from the western slopes of the cameroon mountains. it flows southwest into the atlantic ocean with a discharge rate between 879 and 2533 m / sec. the system is exposed to temporal flooding depending on the tides and the season (wet versus dry) and has large fluctuations in hydrographic conditions. the river system is characterized by the interaction of an estuarine and freshwater - seawater frontal system seaward of the river mouth (typical of a deltaic coastal region) with tidal and wind - driven surface currents. previous studies focused mainly on fisheries, ecology, water quality, hydrology of the lower cross - river, and hydrocarbons in sediments [27, 28 ]. sampling stations were chosen to cover the characteristic features of the river environment as summarized in table 1. sediments were collected in january 1999 (dry season) with a van veen grab sampler (0.1 m), wrapped with aluminum foil and stored frozen at 4c until analysis. freeze - dried sediments were grounded in a disc mill and subsequently sieved to pass 230 mesh to obtain the < 63 m fraction. extraction and fractionation of the < 63 m fraction were as previously reported by ekpo.. briefly, 50 g dry samples were extracted in a soxhlet apparatus with dichloromethane and methanol (2 : 1). extracts were concentrated, desulfurized (activated cu), and fractionated by column chromatography on activated silica and alumina. the saturated fraction (f1) was eluted with hexane, the aromatic fraction (f2) with dichloromethane, and the nitrogen - sulfur - oxygen (nso) containing polar fraction (f3) with dichloromethane - methanol. total organic carbon (toc) analyses for all sediment samples were obtained using an leco c - s-444 analyser. gas chromatography - mass spectrometry (gc - ms) analyses of the isolated fractions were performed on a hewlett - packard model 6890 gc coupled to a hewlett - packard model 5973 quadrupole msd. separation was achieved on a db5-ms column (30 m 0.25 mm i.d. temperature holds at 65c for 2 minutes, increases from 65 to 300c at a rate of 6c min, and with final isothermal holds at 300c for 20 minutes. the mass spectrometer was operated in the electron impact mode at 70 ev ionization energy and scanned from 50 to 650 dalton. compounds were identified by comparison with literature data and interpretation of mass spectrometric fragmentation patterns. the percentage of total organic carbon (toc) contents in the sediments ranged between 1.3% and 4.6%, while the extractable organic matter (eom) ranged between 1.1 and 4.1 g / kg dry weight (dw). the total hydrocarbons determined from f1 and f2 fractions showed the lowest concentration range in sediments from the upper river region (range 244 mg / kg dw), a moderate concentration range in sediments from the middle region (range 60148 mg / kg dw), and the highest concentration range in sediments from the lower deltaic region of the estuary (185511 mg / kg dw) (table 1). the data for the upper calabar river and great kwa river are given as ranges and averages (table 1). pentacyclic triterpenol methyl ethers (ptmes) in the bottom sediments were monitored with the m / z 440 key ion (m) in the ms data. examples are shown in figure 2 and the mass spectra of the major ptmes are also given. their concentrations range from 0.02 to 2.4 mg / kg dw (table 1). the fragment ion at m / z 408 (m-32, minor) and m / z 393 (m-15 - 32) indicates loss of the methoxy group as methanol during fragmentation. compound 1 has a base peak at m / z 189 and accompanying ions at m / z 177 and 204, which are characteristic for germanicol (olean-18-en-3-ol). compound 2 has a base peak at m / z 204 which is from the d / e ring of taraxerene after retro - diels - alder rearrangement. the additional ions at m / z 316, 301, 284, 269, 257, 218, and 189 are characteristic for taraxerol methyl ether. compound 3 exhibits a different fragmentation pattern, with significant fragments at m / z 425 (base peak), 393, 273, 241, and 71. the prominent fragment ion at m / z 393 (m-15 - 32) indicates loss of a methyl group followed by methanol, and m / z 273 (m-167) fission of ring c / d and loss of the ring e moiety with the isopropyl group, typical of the fernene type and is assigned as fern-9(11)-en-3-ol methyl ether. the mass spectrum of compound 4 has a base peak at m / z 218 and intense ions at m / z 191 and 203, which, with the m at m / z 440 and the typical fragments at m / z 425, 408, and 393 indicate 3-methoxyolean-12-ene (-amyrin methyl ether or iso - sawamilletin). these mass spectra are also good fits with those reported by jacob.. the mass spectra of the esters are quite simple, reflecting the fragmentation pattern of the triterpane skeleton with minor ions from the additional acid moiety. thus, the key ion for the amyrin esters is m / z 218 and the mass spectrum of -amyrinyl acetate consists of the m at m / z 468, loss of ch3 to m / z 453, and loss of the acetic acid after h transfer to m / z 408 (figures 3(a) and 3(b), resp.). the amyrinyl hexanoates (e.g., figure 3(c) for 3-isomer) have m at m / z 524, followed by loss of ch3 or the acid moiety to m / z 509 and 408, respectively. the mass spectra of lupeyl acetate (figure 3(d)) and lupeyl hexanoate (figure 3(e)) also exhibit the dominant fragmentation pattern of the lupene skeleton and the acid moiety is reflected in the m, m - ch3, and m - acid ions. the mass spectrum of germanicyl acetate (figure 3(f)) has the characteristic fragments for oleana-2,18-diene with significant m at m / z 468, and m - ch3 to m / z 453 and a minor loss of acetic acid to m / z 408. the concentrations of the triterpenol esters range from not detectable to a total of 7.2 mg / kg (table 1). the detection of these ptmes in relatively few sedimentary environments may be linked to variation in environmental conditions such as seasonal and environmental differences that determine the biosynthesis of these compounds in specialized tissues of certain species of higher plants. nevertheless, the ptmes are natural products introduced directly to the river in organic detritus, probably in leaf litter. the primary sources of ptmes in this estuary may be from monocotyledonous plants belonging to the gramineae, on the basis of taxonomic identifications in the vicinity of the study area. according to jacob. plants belonging to the poaceae produce iso - sawamilletin, miliacin, arundoin, and sawamilletin and thus could also contribute to the sources of ptmes in these sediments. for instance, the occurrence of nine ptmes from numerous species of gramineae has been reported (e.g., [30, 34 ]) and arundoin was found in palm trees, elaeis guineensis, and most poaceae reviewed by jacob.. the persistence of these ptmes in this estuary may reflect their relative stability to aerobic degradation. we see no evidence that these ptmes have undergone diagenetic transformations to the 3-ptmes in the sediments. taraxerone also reported for reference (table 1) was detected at almost all the sampling stations. it is a product from the aerobic oxidation of taraxerol from mainly a mangrove origin and a major natural product in most of these samples (table 1) (e.g., [6, 3638 ]). thus, the relative capacity of these ptmes to resist biodegradation may enhance their utility as biomarkers for source correlations of specific higher plant subspecies in environmental samples. the triterpenol esters in these sediments are mainly acetates and lesser amounts of hexanoates (table 1). however, based on previous studies of triterpenoid esters in vegetation and sediments (palmitates, stearates, etc., [17, 18 ]), it is possible that there are even higher molecular weight esters present in these sediments. they are known to elute at much higher gc temperatures and are not detectable by this analytical protocol. the concentrations of the esters are low compared to their parent triterpenols, so a mass balance is not feasible. nevertheless, their presence in certain sediments may indicate a close input source to that locale, because acetates, like the wax esters, are susceptible to hydrolysis during river transport. the concentrations of the acetates are always 2 to 10 times greater than the hexanoates (e.g., figure 3(a), table 1). the highest amounts are observed in the upriver locales and the lowest amounts downriver and in the mangrove bounded estuary. thus, their source is likely in litter and terrestrial detritus from the grasslands and the deciduous forests and not the mangrove stands. pentacyclic triterpenol derivatives, as the methyl ethers (ptmes) and alkanoates, were characterized in the sediments of the cross - river system. the ptmes that were characterized included germanicol methyl ether (miliacin), 3-methoxyfern-9(11)-ene (arundoin), -amyrin methyl ether (iso - sawamilletin), and 3-methoxytaraxer-14-ene (sawamilletin), while the alkanoates consisted mainly of - and -amyrinyl and lupeyl acetates and hexanoates. these distinct biomarkers are readily extractable from river sediments using polar solvent extraction techniques and are identifiable with routine gc - ms analysis by their characteristic mass spectrometric fragmentation pattern. pentacyclic triperpenol natural products, their derivatives, and degradation products are excellent chemical metrics for extrapolating the impacts that the river / estuary environment imparts on higher plant organic matter. | pentacyclic triterpenol methyl ethers (ptmes), germanicol methyl ether (miliacin), 3-methoxyfern-9(11)-ene (arundoin), -amyrin methyl ether (iso - sawamilletin), and 3-methoxytaraxer-14-ene (sawamilletin or crusgallin) were characterized in surface sediments of the cross - river system using gas chromatography - mass spectrometry (gc - ms). triterpenol esters (mainly - and -amyrinyl acetates and hexanoates, and lupeyl acetate and hexanoate) were also found. these distinct compounds are useful for assessing diagenesis that can occur during river transport of organic detritus. poaceae, mainly gramineae and elaeis guineensis higher plant species, are proposed as primary sources for the ptmes and esters in the sediments. ptmes are biomarkers of specific higher plant subspecies, while the triterpenol esters are indicators of early diagenetic alteration of higher plant detritus. |
immune checkpoint inhibition with the anti - ctla-4 antibody ipilimumab or the pd-1 antibodies pembrolizumab and nivolumab has become a valuable and effective treatment in metastatic melanoma. all substances have demonstrated significant and durable tumor responses with significant prolonged progress - free and overall survival. side effects of a therapy with ipilimumab are mainly related to the mode of action of the drug resulting in activation of the immune system against autoantigens. immune - mediated adverse events have been reported in 10 - 15% of the patients and primarily manifest as skin rash, diarrhea and colitis, hepatitis and endocrinopathies. however, side effects, particularly endocrinopathies are often difficult to diagnose in early stages because of their lack of specific symptoms. we present the case of a 34-year old female patient with metastatic malignant melanoma undergoing treatment with the anti - ctla-4 antibody ipilimumab. six days after the third infusion, the patient reported a persistent headache since 2 d. she interpreted this symptom as a symptom of premenstrual syndrome as she was waiting for her menstrual period and had already experienced similar headaches. laboratory testing was inconspicuous and showed normal values for routine parameters and for thyroid - stimulating hormone (tsh). a prescription for ibuprofen (administered 2 times daily in a dosage of 400 mg) was given which helped to manage the pain at first. two days later, as the headache did not subside, the patient was examined again. she then presented a light periorbital swelling and was examined from a neurologist and an ophthalmologist who both could not find any cause for the symptoms. a ct scan of the head and brain, a cerebrospinal fluid puncture and a measurement of the intraocular pressure were performed and did not show any abnormalities. pain medication was adapted and the patient left with the requirement to contact us if the headache would get worse. almost a week later, the patient was emergently seen. laboratory results showed thyroid malfunction and a mri scan of the brain confirmed the diagnosis of hypophysitis (fig. 1). in addition, a cellulitis of the periorbital subcutaneous fat tissue was observed (fig. 1). the patient was treated with dexamethasone in a dosage of 4 mg every 6 hours. one day later, the symptoms and swelling declined almost completely, but vitiligo began to develop on the face of the patient. a mri scan of the brain performed 2 d later showed a decrease of inflammation in the periorbital region whereas the signs of hypophysitis had improved only slighly. the vitiligo continued to spread over the next weeks and finally involved more than half of the body surface area. a tumor staging showed progressive disease and treatment with the anti - pd1 antibody nivolumab was initiated. figure 1.gadolinium-enhanced t1-weighted mr images of the brain show increased contrast uptake (red arrows) and enlargement of the right lateral orbital region and pituitary gland on the day of diagnosis (a) and 2 d after the beginning of high dose steroid therapy (b). table 1.course of disease from diagnosis of the primary tumor in 08/2006 until death of the patient due to metastatic disease. 08/2006excision of primary melanoma (amelanotic, tumor thickness > 1,7 mm, clark level iv) right thigh09/2006sentinel lymph node biopsy and lymph node dissection right groin (2/8 nodes positive)10/2006 - 08/2007adjuvant therapy with interferon- (high - dose)2008first pregnancy03/2009birth of a healthy baby girl02/2015second pregnancy, detection of intraabdominal lymph node metastases during ultrasound examination at 19 week of gestation03/2015surgical resection of lymph node metastases (r2)histological examination of tumor tissue : melanoma metastasis. mutation analysis of tumor tissue : wildtype for braf, nras and c - kit03/2015termination of pregnancy04/2015surgical resection of abdominal lymph node metastases (r2)04/2015 - 06/2015therapy with ipilimumab (3 cycles), termination due to side effects07/2015 - 09/2015therapy with nivolumab (3 cycles), termination due to progression of disease10/2015tumordebulking of abdominal metastases12/2015progressive disease with development of multiple brain metastases and pulmonary metastases12/2015whole brain radiation (36 gy)02/2016death of the patient due to further tumor progression gadolinium - enhanced t1-weighted mr images of the brain show increased contrast uptake (red arrows) and enlargement of the right lateral orbital region and pituitary gland on the day of diagnosis (a) and 2 d after the beginning of high dose steroid therapy (b). course of disease from diagnosis of the primary tumor in 08/2006 until death of the patient due to metastatic disease. the onset of these side - effects is often difficult to recognize because of the lack of specific symptoms. in the case presented here, the diagnosis of headache as a sign of hypophysitis was blurred because the patient was waiting for her menstrual period and had already experienced similar headaches as signs of a premenstrual syndrome. other symptoms suspicious for hypophysitis such as nausea or fever were not present at first. furthermore, laboratory testing initially did not show any abnormalities such as disbalanced thyroid hormones. as the periorbital swelling occurred, the clinical presentation mimed a slight case of angioedema. it must remain unclear if the swelling should be interpreted as an independent event of immune - mediated infiltration and inflammation or if it represents a per continuitatem effect due to the inflammation of the pituitary gland. the former scenario seems to be more likely as there are more than 10 similar cases published in the literature so far reporting that ipilimumab therapy may induce orbital myositis and orbital inflammation. in our patient, application of corticosteroids was performed delayed, because brain mri scans had been inconspicuous first. we therefore recommend that every patient should be sensitized to report any symptoms that develop during or after treatment with immunostimulating drugs and that mri of the head and brain should be performed immediately in every patient who presents with an unusual headache. to our knowledge this is the first case in which this combination of common und uncommon immune - mediated adverse reactions occurred in the same patient. whether autoimmunity in patients with melanoma undergoing immunotherapies with i.e. interferon-, interleukin-2, ipilimumab, nivolumab or pembrolizumab is associated with increased response rates and prolonged survival is still discussed controversially. there are reports that the development of leukoderma or vitiligo was associated with a better prognosis. and that immune - related adverse events such as vitiligo and autoimmune thyreoiditis were associated with responses to ipilimumab, at the cost of considerable toxicity. however, there are other studies that report that development of autoimmune diseases whether they were tumor - associated or drug - induced was not associated with a better prognosis. this regrettably applies to our patient who showed no tumor response, neither under ipiliumab nor under nivolumab treatment. | abstractipilimumab is an anti - ctla-4 antibody that is approved for the treatment of metastatic malignant melanoma. side - effects are mostly immune - mediated and in many cases the lack of specific symptoms leads to delayed diagnosis and treatment of adverse events. we present the case of a female patient who experienced an uncommon combination of adverse reactions while undergoing therapy with ipilimumab and where the absence of specificity of the symptoms led to late diagnosis and treatment of side effects. autoimmune disease was neither associated with tumor response nor with prolonged survival. |
thyroid dysfunction results in changes in cardiac contractility, cardiac output, myocardial oxygen consumption, systemic vascular resistance, and blood pressure [1, 2 ]. the relationship between abnormal thyroid function and coronary heart disease (chd) has been recognized for a long time, especially in hypothyroidism status due to the associated hypercholesterolemia and hypertension [3, 4 ]. even subclinical hypothyroidism and subclinical hyperthyroidism have been related to increased risk of chd and mortality, although still controversial [7, 8 ]. results of some cross - sectional studies of patients undergoing coronary angiography suggested that free thyroxine (t4) or free triiodothyronine (t3) level was inversely and thyroid stimulating hormone (tsh) concentration was positively associated with the presence of chd or the severity of coronary atherosclerosis in euthyroid subjects [911 ]. by contrast, one study reported that free t3 level was positively associated with the presence and severity of chd. another study showed that high level tsh in the reference range was not an independent predictor of chd. all these studies were conducted in euthyroid subjects with small samples, and the results were conflicting. the hunt study, a prospective population - based cohort study in norway, found that low thyroid function within the clinically normal range was associated with increased mortality from chd in women during 12-year follow - up. however, they found no association of thyroid function with the risk of being hospitalized with myocardial infarction. therefore, the morbidity finding of the hunt study does not confirm the suggestion that thyroid function in the normal range is associated with the risk of chd. more studies are needed to examine the relationship between thyroid function and chd in euthyroid individuals. from a clinical point of view, the effect of thyroid dysfunction on prevalent chd may be more important than the effect of the thyroid function in the reference range. to the best of our knowledge, the relationship between different thyroid function, including both normal thyroid function and thyroid dysfunction, and the presence of chd and the severity of coronary atherosclerosis in a population undergoing coronary angiography is not determined. using the data from patients who were consecutively admitted to the department of cardiology and underwent coronary angiography, we investigated if free t4 and tsh concentrations were associated with prevalent chd and the severity of coronary atherosclerosis and examined the relationship between thyroid function categories and prevalent chd and the severity of coronary atherosclerosis in the entire study population and in euthyroid individuals. our study enrolled consecutive adults 30 years of age between march 2013 and november 2013 who underwent coronary angiography for suspected chd at the cardiology department of zhongshan hospital in shanghai, which is affiliated to fudan university. these patients had chest pain or dyspnea symptoms and were suspected for chd in primary and secondary hospitals. they were transferred to zhongshan hospital for further diagnosis and were first evaluated for chd at the outpatient department by cardiologists. they underwent routine or dynamic electrocardiogram or coronary computed tomography angiography or exercise treadmill test or stress myocardial perfusion imaging before coronary angiography. if one of these tests was positive, they were hospitalized and had coronary angiography for a definite diagnosis. the exclusion criteria included the following : acute coronary syndrome, using medications (antithyroid medications, thyroid hormone, amiodarone, and glucocorticoid hormone) influencing thyroid function, severe systemic diseases, malignancy, any acute intercurrent illness, and patients with missing data. the study complied with the declaration of helsinki and informed consent was obtained from all patients. free t4 and tsh were measured using the electrochemical luminescence method by modular e170 automatic electrochemiluminescence analyzer (roche diagnostics ltd., the normal range for tsh is 0.274.20 miu / l and the normal range for free t4 is 1222 pmol / l. categories of thyroid function were defined as overt hyperthyroidism (tsh 22 pmol / l), subclinical hyperthyroidism (tsh 4.2 miu / l, normal free t4), and overt hypothyroidism (tsh > 4.2 miu / l and free t4 coronary angiography was performed by using standard judkins techniques or a radial approach. during cardiac catheterization, nitroglycerine or verapamil angiographic findings were reviewed by two experienced cardiologists who were blinded to the study protocol. angiography results were divided into chd (50% stenosis in 1 coronary artery) group and non - chd group. we used the gensini score to assess the severity of stenosis of coronary arteries : it scores it as 1 for 125% narrowing, 2 for 2650%, 4 for 5175%, 8 for 7690%, 16 for 9199%, and 32 for a complete occlusion. this score is then multiplied by a factor, depending on the functional significance of the coronary artery. it is 2.5 for proximal left anterior descending artery (lad) and left circumflex artery (lcx) lesions, 1.5 for a mid - lad lesion, and 1 for distal lad, mid / distal lcx, and right coronary artery lesions. venous blood was drawn in the morning after an overnight fast for at least 12 hours. fasting glucose, 2-hour postprandial glucose, triglyceride, total cholesterol, and high density lipoprotein cholesterol (hdl - c) were determined by enzymatic methods using hitachi 7600 biochemistry autoanalyzer (hitachi high - technologies crop., low density lipoprotein cholesterol (ldl - c) was calculated according to the friedewald formula. glycosylated hemoglobin (hba1c) was measured using high performance liquid ion exchange chromatography by the bio - rad variant hemoglobin testing system (bio - rad laboratories, hercules, ca). systemic arterial hypertension was defined by diagnosis of hypertension made previously by a physician or systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or treatment with antihypertensive medications. diabetes mellitus was defined by diagnosis of diabetes made previously by a physician or fasting plasma glucose 7 mmol / l or 2-hour postprandial glucose 11.1 mmol / l or hba1c 6.5% or use of insulin or oral hypoglycemic agents. a smoking history was defined as current smoking, past smoking, and no smoking ever. continuous variables were expressed as the mean standard error (se), and categorical variables were expressed as percentages. comparisons between groups were performed with t - test and chi - square test for continuous and categorical variables, respectively. the relationship between continuous t4/tsh or thyroid function categories and chd was determined using logistic regression. the thyroid function categories in the entire population were hyperthyroidism, euthyroidism, subclinical hypothyroidism, and overt hypothyroidism. subclinical hyperthyroidism and overt hyperthyroidism were combined as one group hyperthyroidism due to the small numbers., the categories of thyroid function were defined as each category representing one - fourth of the width of the reference range of tsh (tsh 0.271.27, 1.282.28, 2.293.29, and 3.304.20 the group with tsh between 0.27 and 1.27 miu / l was used as the reference. potential confounders were age, sex, bmi, ldl - c, hdl - c, triglyceride, diabetes, hypertension, smoking states, and statin use. the association of continuous t4/tsh with the severity of coronary atherosclerosis which was evaluated by gensini score was assessed by linear regression adjusted for the potential confounders. we used general linear model to determine the association between the categories of thyroid function and the severity of coronary atherosclerosis using the same covariates described above. gensini score was natural log - transformed before analysis due to the obvious deviation from normal distribution. the mean age of the participants was 62.68 0.24 years, and 75.4% were men. the characteristics of the study population by chd and non - chd were presented in table 1. patients with chd were more likely to be male and current and ex - smokers. as expected, patients with chd were older and had a higher proportion of diabetes and hypertension and a higher level of fasting plasma glucose, 2-hour postprandial plasma glucose, hba1c, and systolic blood pressure. the levels of total cholesterol, ldl - c, and triglyceride were similar between chd and non - chd groups, but the hdl - c level was lower in the chd group. the difference of free t4 and tsh levels and thyroid function categories between chd and non - chd groups did not achieve statistical significance. the characteristics of the study population by thyroid function categories were shown in table 2. among the study population, 88.33% of participants were euthyroid (n = 1589), 0.72% had hyperthyroidism (7 subclinical hyperthyroidism patients and 6 overt hyperthyroidism patients), 9.17% had subclinical hypothyroidism (n = 165), and 1.78% had overt hypothyroidism (n = 32). women were more likely to have overt hypothyroidism than men, achieving statistical significance for the comparison between the overt hypothyroidism and euthyroid groups. patients with overt hypothyroidism had higher gensini score compared to the euthyroid patients. in the entire population, free t4 as a continuous variable was significantly associated with decreased odds of chd in the multiple logistic regression model, with each one unit increase in free t4 predicting a 5% decrease in the odds of chd (or = 0.95, 95% ci 0.910.99, p = 0.01) (table 3). the association of tsh with chd was not significant (or = 1.05, 95% ci 0.991.12, p = 0.11) (table 3). ft4 was still associated with chd when ft4 and tsh entered into the model together (or = 0.96, 95% ci 0.920.99, p = 0.04) (table 3). to explore if free t4 and tsh in the reference range were associated with chd, we did analysis in the euthyroid individuals (table 3). neither free t4 nor tsh was found to be associated with chd (or = 0.98, 95% ci 0.921.04, p = 0.49, and or = 1.05, 95% ci 0.901.23, p = 0.51, resp.) (table 3). when comparing to the euthyroid patients, the odds of chd increased gradually across hyperthyroidism, subclinical hypothyroidism, and overt hypothyroidism groups (or = 0.74, 95% ci 0.192.85 ; or = 1.53, 95% ci 0.932.52 ; and or = 1.59, 95% ci 0.584.34, resp.) in the multiple logistic regression model in the entire population, and the trend is borderline significant (p for trend = 0.051) (table 4). to explore if thyroid function in the reference range was associated with chd, we did analysis in the euthyroid individuals by grouping them into four categories defined as each category representing one - fourth of the width of the reference range of tsh (table 4). the thyroid function categories in the reference range were not associated with chd using the group with tsh between 0.27 and 1.27 miu / l as the reference (p for trend = 0.77). in the entire population, free t4 was inversely associated with ln(gensini score) in the multivariate linear regression model (= 0.03, 95% ci 0.050.01, p = 0.005) (table 5). tsh was positively associated with ln(gensini score) in the multivariate linear regression model (= 0.02, 95% ci 0.0040.04, p = 0.02) (table 5). when free t4 and tsh entered into the model together, the association of free t4 with ln(gensini score) was still significant (= 0.02, 95% ci 0.040.004, p = 0.02), and the association between tsh and ln(gensini score) became nonsignificant (= 0.02, 95% ci 0.0020.03, p = 0.08) (table 5). we did analysis in euthyroid individuals to investigate if free t4 and tsh in the reference range were associated with the gensini score (table 5). neither free t4 nor tsh was found to be associated with ln(gensini score) (= 0.01, 95% ci 0.040.02, p = 0.36, and = 0.03, 95% ci 0.040.10, p = 0.36, resp.). when comparing to the euthyroid group, ln(gensini score) was higher in overt hypothyroidism group (p = 0.009) in the general linear model in the entire population (figure 1), although p for trend was not significant (p = 0.08). in the euthyroid group, there was no significant difference of ln(gensini score) between different thyroid function categories (p for trend = 0.49) (figure 1). in the entire study population, we found that free t4 level was inversely associated with prevalent chd and the severity of coronary atherosclerosis, and there was a significant trend of association of thyroid function categories with prevalent chd, with lower thyroid function indicating increased risk of chd. we did not find an association of thyroid function with chd and the severity of coronary atherosclerosis in euthyroid population. thyroid hormone exerts its action on the heart and cardiovascular system through its intranuclear genomic effects and extranuclear nongenomic effects [1, 17 ]. the ability of thyroid hormone to alter vascular smooth muscle cells and endothelial function are very important [1, 17 ]. in hypothyroidism, arterial compliance is reduced, which leads to increased systemic vascular resistance and a rise in diastolic blood pressure [3, 17 ]. thyroid hormone deficiency is accompanied by a reduced number of low density lipoprotein (ldl) receptors in the liver and a decreased ldl receptor activity, which leads to impaired ldl clearance. as a result, overt hypothyroidism is characterized by hypercholesterolemia and a marked increase in ldl - c. the lipid profile changes are reversible with thyroid hormone replacement [19, 20 ]. the dyslipidemia and the diastolic hypertension although direct evidences about the effect of levothyroxine on chd are lacking, clinical studies have shown that levothyroxine treatment of subclinical hypothyroidism may have beneficial effects on endothelial function and carotid artery intima - media thickness, which are early markers of atherosclerosis. as discussed above, it is not surprising that free t4 level was associated with prevalent chd and the severity of coronary atherosclerosis in the current study. we also found that tsh level was positively associated with the severity of coronary atherosclerosis, but this association became insignificant after the adjustment of free t4. some studies investigated the direct action of tsh on lipid metabolism, which is closely linked to the development of atherosclerosis and chd. tian. demonstrated that tsh could upregulate 3-hydroxy-3-methyl - glutaryl coenzyme a reductase in the liver, which indicated a direct role of tsh in the development of hypercholesterolemia. several studies found that tsh concentration was associated with lipids levels independent of thyroid hormones [2325 ]. however, our results do not support the fact that tsh may contribute to the development of atherosclerosis and chd independent of the function of thyroid hormone. circulating tsh reflects the negative feedback effects of t4 and t3 on the pituitary gland and is considered a more sensitive index of thyroid status than free t4. however, tsh is a poor measure for estimating the clinical and metabolic severity of primary hypothyroidism. therefore, it is possible that serum free t4 is a more sensitive index of cardiac thyroid status than tsh, as shown by the current study and previous studies [19, 27 ]. although hypercholesterolemia and increased ldl - c level are one of the important mechanisms underlying the association of hypothyroidism and chd, there was no significant difference of total cholesterol and ldl - c concentrations among different thyroid function categories in the current study. it should be noticed that near 50% of the study subjects use statin treatment in the current study, which may partially explain the similar cholesterol level among different thyroid function categories. we can not determine whether there were different lipid levels among thyroid function categories before the use of statin or the development of chd due to the cross - sectional design of the current study. besides hypercholesterolemia, other mechanisms such as endothelial dysfunction or direct effect on the heart were also very important regarding the relationship between thyroid function and chd. in the current study, we found that thyroid function in the clinically normal range was not associated with chd and the severity of coronary atherosclerosis. as a contrary, several previous studies of patients undergoing coronary angiography demonstrated that free t4 or free t3 level was inversely and tsh concentration was positively associated with the presence of chd or the severity of coronary atherosclerosis in euthyroid subjects [911 ]. first, the difference may have originated from the heterogeneity of the study subjects regarding age and sex distribution and number and characteristics of the selected subjects. some studies recruited both stable angina and acute coronary syndrome, and some did not exclude patients with concomitant diseases and medications which can alter thyroid function. second, the euthyroid population in our study had very high cardiovascular risks. about one - third of the population had diabetes, 70% had hypertension, over 50% used statin, and near 50% were current smokers. as a result, the small effect of variation of thyroid function in the narrow reference range on chd can not be captured in the context of multiple classical cardiovascular risks. third, more prospective cohort studies are needed to determine whether thyroid function in the reference range has an effect on the risk of chd. according to the national health and nutrition examination survey, hypothyroidism is a prevalent condition affecting 4.6% of the general population and about 68% of the 5070-year group. the proportion of hypothyroidism was about 11% in the present study population including 9.17% of subclinical hypothyroidism and 1.78% of overt hypothyroidism, which is relatively higher than the prevalence reported in the epidemiological survey. nonetheless, we can not recommend routine thyroid function evaluation in individuals undergoing coronary angiography. however, our study suggested that thyroid function screening may facilitate risk stratification in individuals with symptoms of chd and could provide additional information for selecting the individuals who would benefit from coronary angiography. the strengths of our study include the large study sample and using coronary angiography to evaluate coronary atherosclerosis. our study was limited by the cross - sectional design, and a causal relationship can not be established. in this study, we excluded 29 patients who received levothyroxine therapy and 3 patients who received antithyroid drugs (2 with methimazole and 1 with propylthiouracil) to exclude the effect of medications on thyroid hormone levels. we inferred that the coronary atherosclerosis of these 29 treated hypothyroid patients should be more serious than that of the euthyroid patients. however, there was no significant difference of gensini score between the levothyroxine treated patients and euthyroid patients. one possibility is that levothyroxine therapy may prevent or slow the development of coronary atherosclerosis and alleviate the degree of coronary lesion. it has been shown by previous study that proinflammatory cytokines decreased and anti - inflammatory cytokines increased after levothyroxine treatment in hypothyroid patients. a decrease in low - grade chronic inflammation may have important clinical relevance due to the known relationship between chronic inflammation, atherosclerosis, and cardiovascular events. cardiovascular risk factors were also improved after levothyroxine treatment, including glucose, lipids, insulin sensitivity, and soluble intercellular adhesion molecule-1 [30, 31 ]. more importantly, many studies have shown that levothyroxine replacement in hypothyroid patients improved endothelial functions and reduced arterial stiffness and carotid intima - media thickness [3134 ]. these studies provide further evidences that hypothyroidism or lower thyroid hormones may be causally important in the development of chd. the mean body weight for these patients was 70 kg, but the mean replacement dose was only 60 g levothyroxine per day and the mean tsh level was 2.59 uiu / ml. so the severity of hypothyroidism in those patients may be close to that of subclinical hypothyroidism. in our study, the gensini score was not significantly different between the euthyroid group and subclinical hypothyroid group. we had no data on thyroid autoimmunity, which may have an important effect on the relationship between thyroid function and chd. it has been shown that thyroid autoimmunity may have some effects on hyperlipidemia and abdominal obesity independent of thyroid function. the association of thyroid peroxidase antibody (tpoab) and endothelium - dependent arterial dilation in euthyroid hashimoto 's thyroiditis patients also has been reported. in addition, we do not know the duration of hypothyroidism of the patients, so the effect of duration of hypothyroidism on coronary atherosclerosis and chd can not be examined. despite the large sample size, the small number of participants with subclinical and overt hyperthyroidism precluded precise estimates for those groups. in conclusion, the present study demonstrated an association of thyroid function with prevalent chd and the severity of coronary atherosclerosis in a population undergoing coronary angiography. our study does not support the fact that thyroid function was associated with prevalent chd and the severity of coronary atherosclerosis in euthyroid individuals. | this study investigated if free t4 and tsh concentrations or thyroid function categories were associated with prevalent chd and the severity of coronary atherosclerosis in a population undergoing coronary angiography. this was a cross - sectional study including 1799 patients who were consecutively admitted and underwent coronary angiography. we evaluated the severity of coronary atherosclerosis using gensini score. in the entire study population, free t4 level was inversely associated with prevalent chd (or = 0.95, 95% ci 0.910.99, p = 0.01) and the natural log - transformed gensini score (ln(gensini score)) (= 0.03, 95% ci 0.050.01, p = 0.005). the odds of chd increased gradually across hyperthyroidism, subclinical hypothyroidism, and overt hypothyroidism groups using the euthyroid group as the reference, and the trend is borderline significant (p for trend = 0.051). when comparing to the euthyroid group, ln(gensini score) of the overt hypothyroidism group was significantly higher (p = 0.009), but the trend was not significant (p for trend = 0.08). a significant association of thyroid function with chd or ln(gensini score) in euthyroid patients was not observed. the present study demonstrated an association of thyroid function with prevalent chd and the severity of coronary atherosclerosis in a population undergoing coronary angiography. however, this association was not observed in euthyroid individuals. |
i wish to point out a need for clarification concerning the methodology utilized in the study eye tracking detects disconjugate eye movements associated with structural traumatic brain injury and concussion by samadani., 2015. the authors state that binocular eye movements were recorded using a single - camera infrared - based video - oculography technique (eyelink 1000, sr research, ontario, canada) and that binocular disconjugate characteristics were analyzed without calibration of eye orientation. the authors claim that their variance - based disconjugacy metric was sensitive to the severity of a concussive brain injury and to the status of recovery after the original injury. the eyelink 1000 system is an excellent eye tracker with a single high resolution camera and an infrared light source affixed to the camera. a typical recording setup consists of a computer monitor with which the visual stimuli are presented to the subject and the camera unit (placed in front of the monitor base) with which the eye movement is recorded monocularly or binocularly. the system has an option of easily outputting image - based, uncalibrated eye coordinates with hundreds of units representing 1 of eye rotation. the concern i would raise with samadani.s paper is the unclearness of the relationship between their metric and binocular disconjugacy. logically, for an identical amount of eye rotation, any asymmetry in the spatial relationship that the camera or the infrared light source has with the two fellow eyes would result in different extents of relocation of the images of the pupils or corneal reflections. asymmetries exist because there is a physical separation between the two eyes as well as between the camera and the infrared light source. in addition, although the biometric characteristics of eyes are highly symmetrical within individuals, they are not perfectly symmetrical and a 12% non - conformity in corneal curvature or axial length is not uncommon, which further confounds the relationship between eye rotation and changes in pixel coordinates. moreover, each of the two fellow eyes has its own function that maps pixel movement to the eye rotation, and this mapping is not linear. thus, the arithmetic difference between the uncalibrated coordinates of the two eyes is quite removed from a physical representation of gaze misalignment. beyond the factors associated with the raw data, the analytic methods in the paper it is puzzling why the disconjugacy metric is represented by the variance of the left - right differences after independently averaging for each eye the uncalibrated coordinates over several cycles for a given stimulus position, as opposed to the straightforward variance of the left - right differences at all sample points. furthermore, the ranges of outcome values presented in the series of figures run from 0 to at most 0.25, but how the value 0 could have been obtained is not clear. the question arises because in the two eyes uncalibrated coordinates there must be a constant bias related to the interocular distance. lastly, what the high end of the outcome range represents is not clear. since one unit in eyelink s uncalibrated data output is smaller than 0.01 of eye rotation, being able to report differences in 0.25 square units or less seems implausible. if the raw data were numerically centered or scaled, the procedure should have been noted in the text. however, these points can be directly addressed by implementing a calibration procedure under monocular viewing 3, 4. a comparison between the results from thus calibrated and uncalibrated data, and a demonstration of test - retest reliability could have improved the paper. in summary, the reproducibility of the paper s findings may be challenged simply by the paucity of details in the methodological description. more importantly, however, from the information supplied or cited in the paper it is difficult to evaluate the validity of the potentially interesting conclusion that deficits in conjugacy of eye movements may quantitate physiologic impact of brain injury. | this correspondence points out a need for clarification concerning the methodology utilized in the study eye tracking detects disconjugate eye movements associated with structural traumatic brain injury and concussion, recently published in journal of neurotrauma. the authors of the paper state that binocular eye movements were recorded using a single - camera video - oculography technique and that binocular disconjugate characteristics were analyzed without calibration of eye orientation. it is claimed that a variance - based disconjugacy metric was found to be sensitive to the severity of a concussive brain injury and to the status of recovery after the original injury. however, the reproducibility of the paper s findings may be challenged simply by the paucity of details in the methodological description. more importantly, from the information supplied or cited in the paper, it is difficult to evaluate the validity of the potentially interesting conclusions of the paper. |
complications involving the spleen have been reported with infarction, abscess, subcapsular hematoma, rupture, and hyper - reactive malarial splenomegaly (hms) or tropical splenomegaly syndrome. the latter is commonly understood as an immunological reaction from the infected host and may be confused with lymphoproliferative syndromes. hms is usually described in tropical residents as a result of chronic malarial infection, and is mostly recognized in young and middle - aged adults. a unique case of hms complicated by splenic infarct affecting a caucasian child is reported. a 4-year 11-month - old boy from the united states was staying with his family in a tribal region in indonesia in the preceding five months. he had recurrent febrile illnesses during this period and had been given chloroquine intermittently as prophylaxis, but his general health started to deteriorate about a month before admission. he was first admitted to a local hospital and was diagnosed to have plasmodium vivax infection. he was treated with chloroquine (the exact treatment could not be verified) but the condition did not improve. he was also found to have increasing splenomegaly and severe anemia (lowest hemoglobin recorded, 3.6 g / dl). red cell transfusion was carried out twice before he was evacuated to singapore for further management. the liver and spleen were grossly enlarged at 6 cm and 8 cm below the costal margins, respectively. the first full blood counts showed hemoglobin 9.8 g / dl, white cells 5.48 10/l, platelet 123 10/l. immature trophozoites and gametocytes characteristic of plasmodium falciparum were also seen (figure 1), with a parasite load of 0.1%. treatment with atovaquone 500 mg - proguanil 200 mg for three days was commenced. figure 1photomicrograph of the peripheral blood film with giemsa stain (100) showing an immature trophozoite (t) and a gametocyte (g) characteristic of plasmodium falciparum infection. photomicrograph of the peripheral blood film with giemsa stain (100) showing an immature trophozoite (t) and a gametocyte (g) characteristic of plasmodium falciparum infection. computed tomography revealed enlarged liver and spleen with no signs of thrombosis in the portal venous system. an area of infarction was found in the anterior aspect of the lower pole of the spleen (figure 2). figure 2axial and coronal contrast - enhanced computed tomography showing an infarct in the anterior aspect of the lower pole in the enlarged spleen. axial and coronal contrast - enhanced computed tomography showing an infarct in the anterior aspect of the lower pole in the enlarged spleen. two days later, his hemoglobin dropped to 8.3 g / dl and another red cell transfusion was given. tests for glucose-6-phosphate dehydrogenase deficiency, direct coombs test, and occult blood in stool were negative. serum ferritin (664 mg / ml), bilirubin (15 mol / l), aspartate transferase (37 u / l), and alanine transferase (14 u / l) were normal. serologic tests for hepatitis b, hepatitis c, human immunodeficiency virus, and parvovirus b19 were negative. lymphocyte subsets showed a mild increase in b cells (663/l, normal 65620) only. a week after completion of the anti - malarial treatment, the liver was no longer palpable and the spleen was 4 cm below the costal margin. repeat hemoglobin was 11.7 g / dl, identical to the post - transfusion measurement. parasite load was less than 0.01%, and no more parasites were seen on the blood film a week later. hyper - reactive malarial splenomegaly is believed to be an immunological complication of malarial infection. defective function of suppressive t cells leads to dysregulation of b cells and over - production of igm. the deposition of immune complexes in the reticuloendothelial system results in the enlargement of the liver and spleen even in the absence of significant parasitemia as illustrated in this case. differentiation from chronic lymphoproliferative disorders is important in the adult patient, but this is usually not an issue in childhood, though the condition has rarely been reported in pediatric ages. however, diagnostic confusion may happen as hms may be mistaken as other febrile illnesses in the young child, or when the child presents late after returning from an endemic area. splenic infarct is an unusual complication from malarial infection, but it is often innocuous and no treatment is required. splenic infarct has to be distinguished from abscess which has also been noted after malarial infection. spontaneous rupture is a recognized risk in patients with grossly enlarged spleens with an estimated risk of 2% in those affected by malaria. clinicians evaluating patients returning from the tropics should be aware that diagnostic and therapeutic details from remote healthcare facilities may be difficult to verify. failure to eradicate the parasite may be encountered because of inaccurate parasite identification, re - infection, inadequate drug dosing, drug resistance, or omission of the treatment for the hepatic stage of infection. it is not clear if the malaria parasite had been misidentified or a co - infection with two malarial species had not been found at the initial stage in the reported case. nevertheless, severe splenic complications following malaria should be looked out for in any child returning from the tropics, and they should be followed up for radical cure. | a 4-year - old boy from the united states had been staying in indonesia for five months when he presented with fever, severe lethargy, progressive weight loss, and abdominal distension. he was first diagnosed with plasmodium vivax infection in indonesia and received treatment with chloroquine. however, his condition continued to deteriorate and he required erythrocyte transfusion for severe anemia. three weeks into his illness, he was found to have low parasitemia with plasmodium falciparum with massive hepatosplenomegaly in singapore. a splenic infarct was also documented on computed tomography. treatment with atovaquone - proguanil resulted in stabilization of the hemoglobin level and rapid reduction in splenic size, with clearance of malarial parasites from the bloodstream. although reported typically in adult tropical residents, hyper - reactive malarial splenomegaly may occasionally be found in the pediatric traveler. clinicians receiving children returning from the tropical regions should be aware of this potentially life - threatening complication of partially treated malaria. |
a nosology of psychosis would need to be based on the knowledge of the causes and pathophysiology of these psychotic symptoms. the history of the term will be briefly described, followed by a description of its use in the current classification systems for mental disorders and a discussion on the necessity to deconstruct the term, along with the challenges and future prospects for psychosis research. the term psychosis has been used for about 170 years, and has evolved to reflect the scientific and social contexts of the respective times. it was first used by the austrian medical doctor ernst von feuchtersleben, who used the term in a textbook published in 1845. this reflected the current idea, of the time, of mental disorders being diseases of the mind (geisteskrankheiten or seelenstrungen in german), which von feuchtersleben thought was too narrow and did not convey the idea that it was the interaction between the mind and brain that caused mental disorders. a later position, strengthening the concept of mental disorders as disorders of the brain, was introduced by griesinger in 1845. the term psychosis was soon used by others, and a long and intricate history of its meaning ensued. in the late 19th century, the term was used widely and subdivided as exemplified by wernicke 's distinction between somatopsychoses (affecting the consciousness of one 's own body), autopsychoses (affecting the consciousness of one 's personality), and allopsychoses while such subdistinctions were the first indication that the term psychosis was not a unitary principle, but needed to be deconstructed into its component symptoms, these terms did not gain widespread acceptance. more importantly, kraepelin 's dichotomy of psychosis into dementia praecox and manic - depressive insanity became the rule of the day, and the definition of the several dimensions of psychosis became the center of research in the early and mid-20th century. the concept of jaspers ' layers of mental disorders also comes into play here, in that jaspers hypothesized that neurotic, endogenous, and organic (exogenous) mental disorders reflected three different layers of mental disorders, in which psychotic symptoms could be found on both the endogenous and organic (exogenous) levels. the loss of reality underlying hallucinations and delusions became important, and the term psychosis has been used variably to denote a core syndrome of hallucinations, delusions, and disordered thinking, or in a wider sense, to encompass all severe mental disorders. on the background of such clinical diversity and variability, schneider introduced a ranking of psychotic symptomatology, bringing into the discussion the notion that when diagnosing and classifying mental disorders, some psychotic symptoms may be more important than others. in today 's definition, the characteristic symptoms of psychosis are related to the degree of severity (with psychosis being the severe form of mental disorders), lack of insight, communication disorders, lack of comprehensibility of the symptoms, and reduced social adaptation. classifications of mental disorders and the necessary definitions of the clinical symptoms of mental disorders are mainly based on scientific evidence and aspects of practical utility. while drawing the line between disorder and normality is an important aspect of such classification systems and symptom definitions, questions regarding the validity of the concepts of mental disorders come into play, as well as the quest for defining disease entities. this reflects etiopathological or pathophysiological insights, lending credibility to a concept of psychosis due to valid constructs. in a seminal paper, robins and guze inspired the search for a psychiatric nosology based on etiology and pathophysiology. the rationale is that, while there are some insights into the etiopathoiogy and pathophysiology of psychotic symptoms, we can not yet determine the exact mechanisms that are at work in individual cases of psychotic clinical manifestations. thus, psychosis is still defined by the clinical picture and not by laboratory, genetic, or neuroimaging investigations. the set of symptoms used for a definition should be clearly observable, should be typical of psychosis, and should help to delineate psychotic states from other syndromes and normality. of note, the degree to which these symptoms affect everyday functions should not be a part of the definition of psychosis the presence of the necessary symptoms should suffice to diagnose a psychosis on a level of clinical observation. table i provides an overview of psychotic disorder groups from the american diagnostic and statistical manual of mental disorders (dsm)-5. the introductory text states that psychotic disorders are defined by abnormalities of one, or more, of five domains : delusions, hallucinations, disorganized thinking (speech), grossly disorganized or abnormal motor behavior (including catatonia), and negative symptoms. note that a formal definition of psychosis is not given in the glossary of the dsm-5 ; only psychotic features are defined (features characterized by delusions, hallucinations, and formal thought disorder12) and psychoticism as a feature of personality disorders (exhibiting a wide range of culturally incongruent odd, eccentric or unusual behaviors and cognitions, including both process [eg, perception and dissociation ] and content [eg, beliefs ]). psychoticism is one of the five broad personality trait domains defined in section iii, alternative dsm-5 model for personality disorders. ' at the time of writing, there was only an initial beta version of the international classification of diseases (icd) -11 online, but not the final version. it is evident that the main differences in metastructure occur for the following : (i) brief psychotic disorders, for which dsm-5 has a special category ; (ii) schizotypal disorder, which is classified as a personality disorder in dsm-5 ; and (iii) secondary psychotic disorders, which are grouped together with the primary psychotic disorders in dsm-5, but not icd. both classification systems also include other mental disorders, in which psychosis may occur, like states of delirium or mood disorders with psychotic features. both classification systems keep psychotic syndromes in mood disorders separate from the schizophrenia spectrum (dsm-5 terminology) or the group of schizophrenia and other primary psychotic disorders (icd-11). ostergaard have reviewed the evidence for, and against, separating psychotic depression from the other psychotic disorders, as well as its status compared with the affective disorders, and have made suggestions for the diagnostic criteria of psychotic depression in icd-11 as part of the mood disorders. in the process of developing dsm-5, a working group by the american psychiatric association on psychotic disorders reviewed the available evidence for regrouping the psychotic disorders. the group did acknowledge that the previous dsm - iv grouping had been based on tradition and shared psychopathology, and that the evidence for adding bipolar disorder was, at best, modest, while the evidence for including schizotypal personality disorder was stronger, but that the absence of frank psychosis in schizotypal personality disorder posed a conceptual problem. dsm-5 still uses a categorical classification of psychotic mental disorders since the working group found that the research needed to establish a new nosology of equal or greater validity is lacking. neither dsm-5 nor icd-11 opted to use an attenuated psychosis syndrome as a full diagnostic disease entity. dsm-5 has defined such a syndrome as a clinical condition warranting more research, and the clinical criteria state that it is a syndrome characterized by psychosis - like symptoms below a threshold for full psychosis. this implies two nosological conundrums, in that psychosis - like as compared with psychosis is not defined, and it is unclear how a threshold for full psychosis can be operationalized. in dsm-5, it is suggested to include that the symptoms are less severe and more transient, and insight is relatively maintained. dsm-5 and icd-11 are moving toward harmonization (eg, the course specifiers of the psychotic disorders), but major differences will remain (eg, the time criterion of schizophrenia or the concept of schizoaffective disorder). the composition of psychosis of several symptoms has led to the suggestion of deconstructing the term according to its component symptoms. factor analyses of the symptoms of psychosis in severe mental disorders, like schizophrenia, usually lead to a five - factor solution comprising hallucinations, delusions, disorganization, excitement, and emotional distress. if psychotic symptoms in the general population are taken into account, depressive and manic symptoms also come into play, reflecting the occurrence of the core clinical syndrome of psychosis in affective and other mental disorders. potuzak, after reviewing the available studies on the dimensional structure of psychosis, latent class analyses, and factor analyses, came to the conclusion that there is relatively consistent evidence on appropriate categories and dimensions for characterizing psychosis : the majority of the studies showed that either four or five dimensions describe psychosis, with positive, negative, disorganization, and affective symptom dimensions most frequently reported. similarly, studies showed that the distinction between affective and nonaffective psychotic disorders still has validity and that the symptoms of psychotic disorders are rather stable clinical features when group analyses are carried out over longer observation periods of several years. importantly, in the early stages of disease development (ie, prodromal stages), affective disorders and schizophrenia are similar with dominating affective symptoms, but the occurrence of positive symptoms (eg, hallucinations or delusions) usually sets the mark for differentiation between affective disorders and schizophrenia. a cluster of clinical symptoms encompassing, in a number of possible compositions of symptoms in individual patients, the psychopathologic domains of delusions, hallucinations, and disorganized thinking supplemented by affective domains is the core of psychosis. this notion is supported by the factor analysis results and the finding that these symptoms are characteristic of psychosis across traditional classificatory boundaries. they occur in different mental disorders and there is a considerable overlap between clinical presentations in different mental disorders, although there are symptoms that occur more often in schizophrenia compared with affective disorders with psychotic symptoms, for example. this may indicate that the causes and pathomechanisms of psychotic symptoms in affective disorders are different from schizophrenia and related disorders. however, studies are lacking that address the question about the overlap frequency of symptom domains of the psychosis syndrome (eg, hallucinations, disorganized thinking, or delusions) in individual patients, and about whether these show specific patterns of variation over time. the triad is not necessarily present in all patients, as is shown by disorders like delusional disorders. of note, the clinical psychosis dimensions, such as delusions or hallucinations, need to be subdivided as they are composed of individual symptoms and associated latent factors. attempts are now under way to subdivide the three core psychopathological domains of psychosis even further, indicating that they may be mixed bags of symptoms with different etiopathogenesis, complicating the picture of psychosis even further. another unresolved issue is the question of the temporal variability of the psychotic symptoms in individuals. this leads to a very complex clinical situation : while there is a distinct psychotic syndrome of hallucinations, delusions, and disorganized thinking, the clinical appearance of psychotic symptoms may intraindividually vary greatly over time. this leads to the necessity of group analyses, which by their nature, limit the usefulness for determining the causes and pathophysiology of the symptoms in individual patients. in the future, some major steps remain for the field of psychosis research. first, the causes and etiopathogenesis of the symptoms of psychosis need to be defined. second, a succinct, clinically useful, and internationally harmonized definition of psychosis needs to be provided research into the etiopathogenesis and pathophysiology will benefit from harmonized definitions using research into the essential components of psychosis, which would most likely include delusions and hallucinations. drugs, substances of abuse and their withdrawal, or organic brain disorders (either primary brain disorders or secondary brain disorders that are found in general somatic disorders) may lead to psychosis in any person who may be exposed to these conditions. there has been progress in elucidating the pathophysiology of psychotic symptoms, such as delusions and hallucinations, and one of the new organic aspects is that neuronal autoantibodies have been found to be associated with psychoses. this puts the argument of shared biomarkers into a new light, since there is now a small percentage of persons, among all persons with a psychotic disorder, who carry these autoantibodies. another recent trend in psychosis research relates to the fact that some neurobiological signs are only detectable using sophisticated instrumentation and experimental paradigms in group analyses because the observed alterations of brain circuits are very small and prone to interindividual variation. for example, resting network alterations have been described in schizophrenia, which may help bridge the gap between minor structural brain alterations in patients with schizophrenia, but major disturbances of brain functions such as in perception and thinking. currently, theories are being developed to conceptually link the areas of measurable neurobiological alterations and psychotic phenomenology. neural signatures of psychosis can be expected to be simple and straightforward. on the contrary, changes are manifold and often subtle, they are detectable with sufficient statistical significance based on group analyses, but hardly on an individual level, and they overlap boundaries of traditional icd -10 or dsm-5 mental disorder categories. investigations into the genetic underpinnings of psychotic disorders have also shown a bewildering number of genetic alterations, affecting a wide variety of biological pathways and a rather large overlap of different mental disorders. studying distinct symptom dimensions of psychosis, even in large - scale genetic analyses, did not result in clear associations of specific genes with specific clinical dimensions of psychosis. this genetic research, together with the previously mentioned clinical - course observations in psychotic disorders, supports the notion that psychotic clinical phenomena are spanning traditional classificatory boundaries and may indeed share etiopathology and pathophysiology across diagnostic borders. the national institute of mental health (nimh) research domain criteria (rdoc) initiative is following the path of putting symptoms, syndromes, and neurobiological signatures into the conceptual center of research, thus using the deconstructing approach. conceptual challenges arise because it remains to be seen whether identifying underlying neurocircuits will lead to new nosological definitions, and how the other aspects of the etiopathogenesis (eg, social and environmental factors) will be incorporated. there are probably a vast number of potential individual combinations of relevant factors leading to the clinical picture of psychosis. an important conceptual issue for rdoc to address is how biological predispositions lead to symptoms of psychosis, which is only one of the conceptual and methodological challenges for the rdoc initiative. in the long term, it would be desirable to use additional investigations from the fields of neuroimaging, psychophysiology, and genetics to reclassify psychosis into neurobiologically based subcategories of signs and symptoms. research regarding the association of the symptoms of psychosis with structural or functional brain factors is just beginning (see below), lending some insight into differential associations of some psychotic symptoms with cortical thickness measures, which are, however, not sufficiently distinct on an individual level to provide a novel direction for objectivating and replacing clinical assessments with structural brain measurements. taken together, these findings seem to indicate that the symptoms of psychosis may find neurobiological explanations, but the road to achieving this aim is still long. one of the issues to address is whether a specific bias of reality testing and the resulting reality distortion could be a common denominator of psychosis, with some evidence supporting the notion that impaired reality testing is found in several psychotic disorders and may be further deconstructed into refined neuropsychological dysfunctions. psychological constructs associated with this model would be impaired source monitoring, increased proneness to jumping to conclusions and jumping to perceptions, and aberrant salience of irrelevant information, for which evidence from studies is available. the jumping - to - conclusions mechanism is also associated with other factors in patients with schizophrenia (eg, impairment of working memory), while there is some evidence indicating that alterations of dopamine neurotransmission are involved in the aberrant salience dysfunction. based on these and other findings, recent theories propose that several dysfunctional brain networks interact in schizophrenia, including the salience network, executive network, and default resting state network. such neuropsychological and neurophysiological constructs and other factors (eg, genetic factors), could then be part of the endophenotype assessment battery of psychotic disorders, which could result from such research. endophenotypes are quantitative, heritable, trait - related deficits typically measured with laboratory tests including neuropsychological tests, which could be used to detect the underlying impairments of reality testing in psychotic disorders. delineating and defining assessments will be part of the rdoc approach, as was recently shown for hallucinations. pending the results of such sophisticated analyses and ensuing revelations of putative highly intricate etiopathogenetic mechanisms, psychosis will remain a clinical description of a set of core symptoms, which can be detected by psychopathoiogical investigations. notably, this concept should be regarded philosophically as a realistic concept, which entails that the conceptual scheme mirrors the real world. this means that psychosis is not a social or theoretical construct, but that psychosis is observable in the world outside theories and concepts. as malmgren put it, our concepts are formed while we are interacting with these natural phenomena. the border toward normality and questions about the early detection of psychosis emerge as essential critical issues, which are, however, an issue for all mental disorders and not just psychosis. as to the early detection of psychotic disorders, it is currently clear that many psychotic disorders have a long period in which symptoms occur and in which psychosocial interventions may be helpful to prevent the progression toward schizophrenia, for example. another aspect is that even after frank psychotic symptoms have occurred, the duration until appropriate treatment is initiated is very long, but a long duration of untreated psychosis implies a less favorable prognosis, although other factors (eg, involvement of cognition) are also important for predicting functional outcomes. another aspect is that there are symptoms of psychosis (or psychosis - like experiences), mostly of a fleeting nature in the population, leading to the question of the border toward normality. over the lifespan of an individual affected by such symptoms, these are sometimes followed by progression to a mental disorder, but the symptoms usually subside spontaneously. the transition from psychosis - like experiences in otherwise healthy adolescents to psychosis is at a low rate of approximately 0.56% per year in persons with such psychosis - like experiences, which is, however, greatly increased compared with persons without such experiences (0.16% per year). also, such periods may be due to identifiable and treatable or preventable clinical situations, such as sleep problems, sensory deprivation, intoxicating effects of drugs or substances of abuse, states of withdrawal from drugs or substances of abuse, or they may be associated with somatic disorders including brain disorders (see van gastel on the association with cannabis use). such psychotic symptoms and psychosis - like experiences may signal hitherto unidentified mental disorders. in the unselected, general population, as many as 17% endorse having had lifetime psychotic symptoms (as defined by the composite international diagnostic interview [cidi ]), but only 2% to 5% have ever had a diagnosis of a psychotic disorder. in such studies, there are some associations (eg, for delusions and female sex, and hallucinations and male sex), but there is considerable overlap between associated factors and symptom profiles. also, in such studies, a range of mental disorders (eg, substance addiction or affective disorders), emerge as psychosis - associated, besides the primary research in adolescents who are at risk of psychosis indicate that the overlap of these symptom groups and the ensuing pattern of psychotic symptoms may become an indicator of progression to psychotic disorders, although research into this question is still in its infancy. psychotic - like experiences, unusual subjective experiences, and similar experiences, and what is their prospective value for predicting the future occurrence of mental disorders in general and psychotic disorders in particular ? obviously, further research is necessary to delineate the experiences of psychotic symptoms from those of a psychosis - like nature, and such psychopathoiogical research is just beginning. given the high frequency of such experiences in the general population, and the impairments and suffering associated with them if they progress, there is a clear clinical need to address these questions, which may have consequences for the nosological status of mild or attenuated psychotic experiences in the general population. first, there is now stereoelectroencephalographic evidence derived from studies with intracranial electrodes during epilepsy surgery showing that some symptoms of psychosis may result from stimulation in different brain areas, and that complex brain networks are obviously involved. interestingly, there seems to be considerable overlap in the pathophysiology of hallucinations and delusions using such technologies, which has prompted a debate on whether the psychiatric distinction between hallucinations and delusions was warranted. a second technique is the use of neuroimaging methods to identify areas of the brain involved in the pathophysiology of hallucinations and delusions. while this research is ongoing, it seems clear that there are no single brain regions that are more decisive, but that complex network disturbances occur in the context of these phenomena and that many combinations of functional alterations may be detectable. the question is whether the symptoms of primary (or endogenous) psychotic disorders will prove to have similar pathophysiology compared with those in other brain disorders (eg, alzheimer 's disease). the genetics of psychotic manifestations in alzheimer 's disease show some overlap with schizophrenia genetics, but both fields of research have so far yielded a bewildering array of associations with a multitude of genes. a third approach utilizes novel methods of brain network analyses (connectome) and the results are preliminary and complex, and have not yet provided distinguishing landmarks for analysis suitable for clinical practice. however, research advances in the brain network analysis of the symptoms of psychosis (eg, relevant neurotransmitter systems including -aminobutyric acid [gaba ] and glutamate) as well as proteomic approaches combined with genomic approaches are beginning to reshape the concept and therapeutic approaches of psychotic disorders. new concepts of psychosis are emerging as the result of the neurobiological research progress, including the theory of dopamine hypersensitivity caused by a range of pathological insults that may be a common denominator, with the concept also taking into account brain reactions in the different dopamine pathways (both intracellular and intercellular) and the counterreactions by the same pathways or due to altered interactions among each other. today, alterations of the dopamine system and their interactions with other neurotransmitter systems are viewed not as the causes, but rather as the consequences of a cascade of events in the etiopathogenesis of psychosis. they seem to represent a common final pathway, amenable to treatment with antipsychotic drugs across traditional diagnostic boundaries of mental disorders. gene - environment interactions are another important aspect here as it appears highly likely that not only do endogenous processes play a role, but also exogenous factors (eg, environmental factors) codetermine the timing, type, and course of psychosis. however, large - scale epidemiological studies indicate that the gene - environment - symptom pathway is complex and highly variable between individuals, leading to weak associations between these factors, even in large - scale studies. now there are attempts to associate specific developmental insults with specific symptoms or specific disease trajectories of psychotic disorders, but this research is just beginning. ' once psychotic disorders have developed, studies in patients with treatment - refractory vs nonrefractory psychotic disorders show that the old these classes of mental disorders are associated with differential treatment responses in specific clinical domains of psychosis, and they emerge in latent class analyses in cohorts of patients with mental disorders and healthy controls. these findings indicate that there may be differential pathways into these specific symptoms, which need to be unraveled by future research in more detail. while this research has a rather long - time perspective between the insult and ensuing symptoms, research is now also addressing fluctuations of psychotic symptoms on a micro - timescale of hours. initial results indicate that such assessments are feasible, may lend insight into the complex array of environmental or affective factors influencing the clinical presentation of psychosis, and indicate that such momentary states are the basic units of psychosis. the expected progress in these areas of (neuro) systems - oriented research will clearly bear on the concept of psychosis and the methodology of elucidating the etiopathogenesis of mental disorders in general. taken together, the etiopathogenesis of the symptoms of psychosis is complex and involves a number of environmental and endogenous factors (eg, genetic and neurodevelopmental factors), which interact in an intricate manner leading to a range of structural and functional adaptive or maladaptive responses of the brain. novel research approaches based on the deconstruction of the psychosis syndrome into its symptoms hold promise for unraveling the causes and pathophysiology of these symptoms in the future. psychosis is a clinical syndrome composed of several symptoms. it is not a nosological entity. symptoms of psychosis occur in a wide range of mental disorders and show a high degree of interindividual variability between persons with different mental disorders, and a high degree of intraindividual variability over time. symptoms of psychosis are usually embedded in the wider clinical picture of the mental disorder, which may include symptoms of mania and depression. the elucidation of the symptoms of psychosis by drugs or brain disorders indicates that every person may experience symptoms of psychosis. while the concept and definition of psychosis are characterized by the core clinical symptoms of delusions, hallucinations, and disorganized thinking, it is most likely that these symptoms are common final outcomes of a range of different causes and etiopathogenetic pathways, which may all lead to a similar clinical picture. as kraepelin put it, the human brain only has a limited number of reaction types (a concept relating to bonhoeffer 's reaction types) in the face of etiopathogenic insults. the clinical efficacy of antipsychotic drugs against the symptoms of psychosis, irrespective of the mental disorder, indicates that such final common pathways play a role. the symptoms of psychosis are a common clinical result of a number of causes and pathomechanisms. therefore, the need arises to deconstruct the construct into its clinical dimensions with a view to identifying the causes and pathomechanisms of each of the symptoms of psychosis. there may be shared causes and pathomechanisms, since the symptoms of psychosis commonly occur together, which seem to converge on some common final pathways of the brain, leading to the similar efficacy of antipsychotic drugs in different mental disorders where these symptoms occur. future challenges are to identify the causative and pathophysiological components, and their interplay in individual cases. | the concept of psychosis has been shaped by traditions in the concepts of mental disorders during the last 170 years. the term psychosis still lacks a unified definition, but denotes a clinical construct composed of several symptoms. delusions, hallucinations, and thought disorders are the core clinical features. the search for a common denominator of psychotic symptoms points toward combinations of neuropsychological mechanisms resulting in reality distortion. to advance the elucidation of the causes and the pathophysiology of the symptoms of psychosis, a deconstruction of the term into its component symptoms is therefore warranted. current research is dealing with the delineation from normality, the genetic underpinnings, and the causes and pathophysiology of the symptoms of psychosis. |
metabolic syndrome (ms) is a cluster of cardiovascular risk factors such as diabetes mellitus (dm), pre - diabetes, central obesity, dyslipidemia, and high blood pressure (bp).13 it is estimated that approximately one quarter of the world s population have the syndrome and people with this condition are likely to have a heart attack or stroke compared with people without it.2 even more alarming is the fact that the risk of developing dm is fivefold greater in people with this syndrome.1 in 1976, wellborn and wearne4 established the link between type 2 diabetes mellitus (t2 dm) and cardiovascular disease (cvd). pyorala5 showed strong links between glucose intolerance, hyperinsulinemia, and coronary heart disease (chd) and this link was extended in the 1980s by modan to include obesity, hypertension, and cvd. although the pathogenesis is not fully understood, it has been postulated that genetically determined insulin resistance in a setting of suitable environmental factors is the pivotal pathogenic mechanism underlying ms.7 lipoprotein lipase deficiency largely accounts for the lipid abnormalities seen in ms,8 while hypertension is due to enhanced sympathetic activities, salt sensitivity, and increased transmembrane cation transport.9,10 the role of tnf in obesity and insulin resistance has also been described.11 developing nations are witnessing rapid industrialization, urbanization, and increased economic prosperity and the resulting acquisition of the western lifestyle characterized by calorie excess and physical inactivity would provide a suitable milieu for the development of ms in genetically predisposed individuals. the prevalence of ms is known to vary across the world : age and ethnicity of the populations being studied are also key issues influencing the prevalence.12 cvd is unanimously recognized as the major burden in t2 dm in terms of mortality and morbidity.13,14 unfortunately, myocardial infarction, stroke, and non - ischemic cvd are the causes of death in up to 80% of patients with t2dm.15 it is worthy to note that the constellation of metabolic derangements often seen in people with insulin resistance and t2 dm is individually associated with an increased risk of a cvd.16 the relationship between pre - diabetes and a family history of dm and cardiovascular risk has been increasingly emphasized. several hypotheses have been generated in order to explain such relationship between family history of diabetes and cardiovascular risk profile of individuals.1724 family history of diabetes increases per se the risk of chd even in non - diabetic subjects.21 this may be due to an increased prevalence of abdominal fat content in such subjects,22 elevated systolic bp (sbp), higher triglyceride and cholesterol plasma concentration,23 and higher pai-1 activity.24 all these conditions could increase the cardiovascular risk profile of individuals and lead to them being considered as an at risk population even considering their apparently healthy general clinical condition. family history of dm is not only related to a pure increase in metabolic alterations predisposing to the onset of overt diabetes but it seems to overcome the endocrinology alterations and to provoke early lesions even in the vascular walls. although this is not supported by strong scientific evidences, many observational studies revealed its relationship with increased cardiovascular risk. although literature is poor about human protocols demonstrating the effects of pre - diabetes and diabetes on direct evidence of cardiac myocyte hypertrophy, some evidences can be outlined.2527 among turkish people, a coronary heart disease study revealed that two out of every three cases originate from ms28 and according to the world health organization (who) ; chd is developing in developing countries it is a plague to avoid.29 chd was thought to be uncommon in nigeria but the prevalence appears to be rising in keeping with urbanization, westernized diet, and increasing levels of physical inactivity. though the current prevalence is not known, it seems reasonable to expect an increase in its frequency as the lifespan of the population increases, particularly with the increasing prevalence of t2dm.30 in eastern nigeria, dagogo - jack and odia31 reported six cases of myocardial infarction in port - harcourt in 1990 and four out of the six cases had t2 dm. out of three cases of myocardial infarction reported by danbauchi and onyemelukwe in zaria, northern nigeria, two of them had t2dm.32 thus it can be said that the cardiovascular complications of dm (which are also a leading cause of blindness, amputation, and renal failure) account for much of the social and financial burden of the disease.1 with ms driving the twin global epidemics of t2 dm and cvd, there is an overwhelming moral, medical, and economic imperative to identify people with this syndrome early so they can benefit from lifestyle interventions and treatment that may alter the course of the disease. diagnosing ms will help in identifying individuals at increased risk of cvds : a similar risk is known to exist in individuals with t2 dm. the question this study will also attempt to answer is to what extent is cvd risk altered in t2 dm persons with ms ? the united kingdom prospective diabetic study risk engine (ukpds - re) is a computer based estimation of cardiovascular risk in patients with t2 dm : it will be used to estimate the cardiovascular risk in t2 dm. the effect of ms on the estimated cardiovascular risk in these subjects will also be analyzed. this is the first study in benin city that has assessed the impact of t2 dm on cardiovascular risk in subjects with ms using the ukpds - re. to the best of our knowledge this study sets out to evaluate the impact of the coexistence of ms and t2 dm on the estimated cardiovascular risk as calculated using the ukpds - re and also to determine the impact of the coexistence of ms and t2 dm on the 10-year risk of developing chd and stroke. this study sets out to evaluate the impact of the coexistence of ms and t2 dm on the estimated cardiovascular risk as calculated using the ukpds - re and also to determine the impact of the coexistence of ms and t2 dm on the 10-year risk of developing chd and stroke. ethical approval was obtained from the ethics and research committee of the university of benin teaching hospital (ubth) before the commencement of the study and consent was obtained from study subjects. this was a cross - sectional study carried out at the diabetes clinic of the ubth, a 500-bed federal government tertiary hospital in benin city, edo state in the south - south geopolitical region of nigeria. the ubth receives referral cases from edo state and neighboring states like delta, ondo, ekiti, and kogi states and the federal capital territory, abuja. a total of 124 subjects were recruited from the diabetes clinic of the ubth and the inclusion criteria includes : t2 dm patients presenting to ubth within the last 24 months using the 1999 who criteria,29 people aged 30 years and above, on treatment with oral hypoglycemic drugs plus or minus non - pharmacological therapy and not requiring insulin for survival, and finally subjects who consented to participate in the study. the exclusion criteria included subjects diagnosed with other types of dm, with t2 dm and age < 30 years, and those who declined. ninety - six control subjects were recruited from among the staff of ubth and healthy relatives of non - diabetic patients. the inclusion criteria includes : non - diabetic age - and sex - matched adult with normal fasting blood sugar less than 110 mg / dl while the exclusion criteria includes : non - diabetics less than 30 years of age, first degree relative of type 2 diabetic, non - diabetics who declined being a part of the study. a convenience sampling technique was used to recruit 124 subjects with t2 dm and 96 controls using a questionnaire administered technique. the following were assessed : anthropometric indices, bp, serum lipid profile, fasting blood sugar, proteinuria, and microalbuminuria.30,31 the who criterion was used in this study to define ms (table 1). in order to determine the sensitivity and specificity of these diagnostic criteria in persons living with t2 dm, the ukpds - re was used to identify persons with increased risk for stroke and those with increased risk for chd. individuals with stroke risk < 15% (in the green zone) were considered normal (figure 1). those with stroke risk 15% (outside the green zone) were considered as having an increased risk for stroke. similarly, persons with risk of < 15% for chd were considered normal while those with chd risk 15% were considered as having an increased risk for a coronary event. anthropometric measurements of weight, height, waist circumference (wc), and hip circumference (hc) were measured for each subject as follows : the weight was measured with subjects in light clothing, without shoes using a weighing scale and recorded in kilograms measured to the nearest 0.1 kg. the height was measured, without shoes and the subject standing upright and looking straight ahead (along the coronal plane) using a stadiometer and was recorded to the nearest 0.1 m. the body mass index (bmi) was calculated by the formula : bmi = weight (kg)/height2 (m). the wc was taken at the point midway between the inferior margin of the rib cage and the iliac crest to the nearest 0.1 cm using measuring tape. the hc was measured at the level of the maximal gluteal circumference (along the greater trochanter) to the nearest 0.1 cm with subjects standing erect, hands at the sides, and feet together. the waist - hip ratio (whr) the bp was measured to the nearest 2 mmhg using a standard mercury sphygmomanometer with subjects in the sitting position and the arms resting on the arms of a chair and the sphygmomanometer at the level of the heart using the first and fourth korotkoff sounds for the sbp and diastolic bp (dbp) respectively. all subjects were instructed to observe an overnight fast for 810 hours before the day of sample collection. approximately 20 ml of blood was collected from the ante - cubital vein using sterile disposable needles and syringes, for the following investigations : fasting blood sugar : 2 ml of blood was collected in fluoride oxalate bottles and then analyzed for plasma glucose within 1 hour by the glucose oxidase method.serum lipid profile : blood was collected in plain bottles, allowed to clot and the serum separated and stored at 20c until analyzed. the assay was done by enzymatic method using randox kit.approximately 1 ml of blood was collected for glycated hemoglobin assay.urinalysis testing for proteinuria using combi 10 test strips and microalbuminuria using micral test kit. fasting blood sugar : 2 ml of blood was collected in fluoride oxalate bottles and then analyzed for plasma glucose within 1 hour by the glucose oxidase method. serum lipid profile : blood was collected in plain bottles, allowed to clot and the serum separated and stored at 20c until analyzed. urinalysis testing for proteinuria using combi 10 test strips and microalbuminuria using micral test kit. the ukpds - re is a type 2 diabetes specific risk calculator based on 53,000 patient years of data from the uk prospective diabetes study, which also provides an approximate margin of error for each estimate. first release of the ukpds - re program and excel spreadsheet was on march 26, 2002. it provides risk estimates and 95% confidence intervals, in individuals with type 2 diabetes not known to have heart disease, for : non - fatal and fatal chd, fatal chd, non - fatal and fatal stroke, fatal stroke. these can be calculated for any given duration of type 2 diabetes based on current age, sex, ethnicity, smoking status, presence or absence of atrial fibrillation, and levels of hba1c, sbp, total cholesterol, and high - density lipoprotein (hdl) cholesterol. ethical approval was obtained from the ethics and research committee of the university of benin teaching hospital (ubth) before the commencement of the study and consent was obtained from study subjects. this was a cross - sectional study carried out at the diabetes clinic of the ubth, a 500-bed federal government tertiary hospital in benin city, edo state in the south - south geopolitical region of nigeria. the ubth receives referral cases from edo state and neighboring states like delta, ondo, ekiti, and kogi states and the federal capital territory, abuja. a total of 124 subjects were recruited from the diabetes clinic of the ubth and the inclusion criteria includes : t2 dm patients presenting to ubth within the last 24 months using the 1999 who criteria,29 people aged 30 years and above, on treatment with oral hypoglycemic drugs plus or minus non - pharmacological therapy and not requiring insulin for survival, and finally subjects who consented to participate in the study. the exclusion criteria included subjects diagnosed with other types of dm, with t2 dm and age < 30 years, and those who declined. ninety - six control subjects were recruited from among the staff of ubth and healthy relatives of non - diabetic patients. the inclusion criteria includes : non - diabetic age - and sex - matched adult with normal fasting blood sugar less than 110 mg / dl while the exclusion criteria includes : non - diabetics less than 30 years of age, first degree relative of type 2 diabetic, non - diabetics who declined being a part of the study. a convenience sampling technique was used to recruit 124 subjects with t2 dm and 96 controls using a questionnaire administered technique. the following were assessed : anthropometric indices, bp, serum lipid profile, fasting blood sugar, proteinuria, and microalbuminuria.30,31 the who criterion was used in this study to define ms (table 1). in order to determine the sensitivity and specificity of these diagnostic criteria in persons living with t2 dm, the ukpds - re was used to identify persons with increased risk for stroke and those with increased risk for chd. individuals with stroke risk < 15% (in the green zone) were considered normal (figure 1). those with stroke risk 15% (outside the green zone) were considered as having an increased risk for stroke. similarly, persons with risk of < 15% for chd were considered normal while those with chd risk 15% were considered as having an increased risk for a coronary event. anthropometric measurements of weight, height, waist circumference (wc), and hip circumference (hc) were measured for each subject as follows : the weight was measured with subjects in light clothing, without shoes using a weighing scale and recorded in kilograms measured to the nearest 0.1 kg. the height was measured, without shoes and the subject standing upright and looking straight ahead (along the coronal plane) using a stadiometer and was recorded to the nearest 0.1 m. the body mass index (bmi) was calculated by the formula : bmi = weight (kg)/height2 (m). the wc was taken at the point midway between the inferior margin of the rib cage and the iliac crest to the nearest 0.1 cm using measuring tape. the hc was measured at the level of the maximal gluteal circumference (along the greater trochanter) to the nearest 0.1 cm with subjects standing erect, hands at the sides, and feet together. the waist - hip ratio (whr) was thereafter determined as the wc divided by the hc. the bp was measured to the nearest 2 mmhg using a standard mercury sphygmomanometer with subjects in the sitting position and the arms resting on the arms of a chair and the sphygmomanometer at the level of the heart using the first and fourth korotkoff sounds for the sbp and diastolic bp (dbp) respectively. all subjects were instructed to observe an overnight fast for 810 hours before the day of sample collection. approximately 20 ml of blood was collected from the ante - cubital vein using sterile disposable needles and syringes, for the following investigations : fasting blood sugar : 2 ml of blood was collected in fluoride oxalate bottles and then analyzed for plasma glucose within 1 hour by the glucose oxidase method.serum lipid profile : blood was collected in plain bottles, allowed to clot and the serum separated and stored at 20c until analyzed. the assay was done by enzymatic method using randox kit.approximately 1 ml of blood was collected for glycated hemoglobin assay.urinalysis testing for proteinuria using combi 10 test strips and microalbuminuria using micral test kit. fasting blood sugar : 2 ml of blood was collected in fluoride oxalate bottles and then analyzed for plasma glucose within 1 hour by the glucose oxidase method. serum lipid profile : blood was collected in plain bottles, allowed to clot and the serum separated and stored at 20c until analyzed. urinalysis testing for proteinuria using combi 10 test strips and microalbuminuria using micral test kit. the ukpds - re is a type 2 diabetes specific risk calculator based on 53,000 patient years of data from the uk prospective diabetes study, which also provides an approximate margin of error for each estimate. first release of the ukpds - re program and excel spreadsheet was on march 26, 2002. it provides risk estimates and 95% confidence intervals, in individuals with type 2 diabetes not known to have heart disease, for : non - fatal and fatal chd, fatal chd, non - fatal and fatal stroke, fatal stroke. these can be calculated for any given duration of type 2 diabetes based on current age, sex, ethnicity, smoking status, presence or absence of atrial fibrillation, and levels of hba1c, sbp, total cholesterol, and high - density lipoprotein (hdl) cholesterol. two hundred and twenty - three subjects were enrolled : 125 subjects living with t2 dm, 98 non - diabetic control subjects (without a history of dm in a first degree relative). out of the 125 subject living with t2 dm, the only subject excluded had an incomplete result and an incomplete questionnaire. out of the 98 non - diabetic control subjects, one hundred and twenty - four subjects with t2 dm and 96 controls who the requirements were included. therefore, a total number of 220 subjects participated in this study. of the 96 controls, 38 (39.6%) were males while 58 were females (60.4%) and their mean age was 58.611.2 years while the age range was 3183 years. the majority (over 60%) of the control subjects were within the age range of 5069 years (table 2). the mean age (standard deviation) of the subjects with t2 dm was 58.611.2 years. no significant age and sex difference was observed in these groups and this is suggestive of a study with subjects well matched for age and sex. for subjects with t2 dm, the statistical analysis comparing the age group distribution yielded no significant difference (= 2.107, degrees of freedom [df ] = 8, and p=0.97). similarly, no significant sex difference was observed (= 0.017, df = 8, and p=0.99). table 3 shows the prevalence of ms using the who diagnostic criteria. using the who as the gold standard criteria for diagnosis, the prevalence of ms in the control and t2 dm groups were 13.5% and 87.1% respectively. a group by group comparison of proportions of subjects with ms showed a statistically significant higher proportion of persons with ms in the t2 dm group than in the control (=1.183, df = 1, and p=0.000). a statistically significant difference in the means of wc (98.1011.94 cm and 84.8112.43 cm), bmi (27.295.04 kg / m and 23.102.26 kg / m), sbp and dbp (137.8517.10 mmhg/126.569.42 mmhg and 86.609.92 mmhg/78.016.24 mmhg) respectively of t2 dm subjects with ms and those without ms was observed (p=0.01, 0.01, 0.01, 0.01 respectively). there was no significant difference observed in the mean whr of t2 dm subjects with or without ms even though the mean whr was higher in subjects with ms. there was a statistically significant difference in the mean of the triglycerides, sbp, dbp, fbs, and hba1c in t2 dm subjects with ms compared to t2 dm subjects without ms (p=0.02, 0.01, 0.01, 0.01, 0.01 respectively). however, no significant difference in total cholesterol levels, hdl, and low - density lipoprotein (ldl) levels was observed (table 4). of the 124 subjects with t2 dm, 15 (12.10%) were identified by the ukpds - re version 2.0 as having an increased risk for stroke while ten (8.06%) were identified as having an increased risk for a coronary event. seven subjects (5.65%) had increased risk for both conditions. the ability of the who criteria to identify these individuals with t2 dm who have significant cardiovascular risk was assessed and odds of developing a stroke and that of developing a coronary event was calculated to determine the possible impact of ms in the lives of t2 dm subjects with ms. this implies that the odds of a t2 dm subject with ms developing a stroke compared with that of a t2 dm subject without ms developing a stroke are equal (0.95791). table 5 shows the odds associated with developing an increased risk for stroke while having ms. this implies that the risk of a coronary event occurring in a t2 dm subject with ms is not the same as that of a t2 dm subject without ms. persons with t2 dm and ms have a threefold risk of having a coronary event when compared with persons with t2 dm without ms. table 5 also shows the odds associated with developing an increased risk for coronary as a result of having ms. table 3 shows the prevalence of ms using the who diagnostic criteria. using the who as the gold standard criteria for diagnosis, the prevalence of ms in the control and t2 dm groups were 13.5% and 87.1% respectively. a group by group comparison of proportions of subjects with ms showed a statistically significant higher proportion of persons with ms in the t2 dm group than in the control (=1.183, df = 1, and p=0.000). a statistically significant difference in the means of wc (98.1011.94 cm and 84.8112.43 cm), bmi (27.295.04 kg / m and 23.102.26 kg / m), sbp and dbp (137.8517.10 mmhg/126.569.42 mmhg and 86.609.92 mmhg/78.016.24 mmhg) respectively of t2 dm subjects with ms and those without ms was observed (p=0.01, 0.01, 0.01, 0.01 respectively). there was no significant difference observed in the mean whr of t2 dm subjects with or without ms even though the mean whr was higher in subjects with ms. there was a statistically significant difference in the mean of the triglycerides, sbp, dbp, fbs, and hba1c in t2 dm subjects with ms compared to t2 dm subjects without ms (p=0.02, 0.01, 0.01, 0.01, 0.01 respectively). however, no significant difference in total cholesterol levels, hdl, and low - density lipoprotein (ldl) levels was observed (table 4). of the 124 subjects with t2 dm, 15 (12.10%) were identified by the ukpds - re version 2.0 as having an increased risk for stroke while ten (8.06%) were identified as having an increased risk for a coronary event. seven subjects (5.65%) had increased risk for both conditions. the ability of the who criteria to identify these individuals with t2 dm who have significant cardiovascular risk was assessed and odds of developing a stroke and that of developing a coronary event was calculated to determine the possible impact of ms in the lives of t2 dm subjects with ms. this implies that the odds of a t2 dm subject with ms developing a stroke compared with that of a t2 dm subject without ms developing a stroke are equal (0.95791). table 5 shows the odds associated with developing an increased risk for stroke while having ms. this implies that the risk of a coronary event occurring in a t2 dm subject with ms is not the same as that of a t2 dm subject without ms. persons with t2 dm and ms have a threefold risk of having a coronary event when compared with persons with t2 dm without ms. table 5 also shows the odds associated with developing an increased risk for coronary as a result of having ms. the mean age of t2 dm subjects in this study was (58.611.2 years) and there were more females (59.7%) than males (40.3%) with t2 dm. a comparative analysis of clinical and biochemical variables between t2 dm patients with and without ms revealed that the mean bmi, sbp, dbp, fbs, hba1c, total cholesterol, ldl cholesterol, and triglycerides were higher in t2 dm subjects with ms ; the mean bmi, sbp, dbp, fbs, hba1c, and triglycerides were significant. on the other hand, the mean hdl cholesterol was higher in t2 dm subjects without ms though this was not significant. this is despite the fact that the use of medications for both hypertension and hyperlipidemia was much more common in t2 dm patients with ms. this finding was similar to that reported in japan by hirohito who observed that bp and serum triglycerides were significantly higher and hdl cholesterol was significantly lower in ms patients. similarly, carole reported that patients with ms were more likely to have higher hba1c, total cholesterol, and ldl cholesterol. diabetic patients are known to be at greater risk for cvd than non - diabetic subjects,35 and it has been suggested that ms is responsible for the increased prevalence of chd seen in diabetic patients.36 to the best of our knowledge, there have been few cohort studies specifically targeting diabetic patients to determine the relative risk of ms on the incidence of cvd3638 and mortality due to cvd.39 the ukpds - re is a risk model used only for diabetes patients. van der heijden reported that the ukpds risk function was more accurate in predicting chd in a hoorn study cohort of newly diagnosed t2 dm patients than were the framingham and score methods. the ukpds - re version 2.0 was used to identify t2 dm persons at increased risk for stroke and coronary events. fifteen subjects were identified in our study as having an increased 10-year risk for stroke and ten subjects had an increased risk for a coronary event. using the who criteria to screen for persons with ms who are likely to present with these events, the odds of a t2 dm person with ms having an increased risk for stroke compared with that of a t2 dm person without ms was 0.95791. this implies that a t2 dm person with ms and a t2 dm person without ms have equal risk of developing an increased risk for stroke. thus, ms from this study does not appear to cause an additional increase in the risk of stroke in persons with t2 dm, however dm is clearly one of the most important risk factors for stroke.41 commonly, the relative risk of stroke in diabetic patients is higher than non - diabetic, but the rate of relative risk is wide and varies in different studies.42 in black diabetic americans, the peak rate of risk for stroke (eight to ten times higher) is at the age of 3445 years, but in the same age range, whites have a 2.65.3 relative risk for stroke.42 the highest risk for stroke for white diabetic americans is at the age of 4564 years. approximately 37%42% of all incidences of stroke are associated with diabetes alone or in combination with hypertension43 and one - third of all acute stroke patients may have diabetes. for patients presenting with post - stroke hyperglycemia, impaired glucose tolerance or diabetes is present in two - thirds of survivors.44 japanese with diabetes have two to five times the risk for stroke compared to non - diabetics.45 the risk of stroke in japanese men in hawaii increased with age for diabetics and non - diabetics. the risk was substantially higher among diabetic compared with non - diabetic individuals at almost all ages.45,46 women with diabetes and no history of cvd have a threefold increased fatal stroke risk compared with non - diabetic women without cvd.47 the odds of a t2 dm subject with ms developing an increased risk for a coronary event compared with that of a t2 dm subject without ms was 3.4513. this implies that the risk of having an increased risk for a coronary event in a t2 dm subject with ms is not equal to the risk of a t2 dm subject developing a coronary event and it appears that t2 dm subjects with ms are more likely to suffer a coronary event than t2 dm subjects without ms. it has been shown that ms can increase (by three to four times) the risk of death due to ischemic heart disease by the kuopio ischaemic disease risk factor study in finland.48 qiao reported in finnish and swedish cohorts that ms and its components predicted the incidence of stroke and chd equally well. bruno in the casale monferrato study, reported that diabetic subjects with even only one component of ms have more than twofold higher risk of cardiovascular mortality than subjects with diabetes only, independent of age, sex, smoking, total cholesterol level, chd, and cumulative hba1c. similarly guzder reported that ms at baseline is associated with an increased risk of cvd incident in the 5 years following diagnosis of t2dm.49 the cvd - free survival rates declined incrementally as the presence of ms features increased. though there are few studies that have compared cardiovascular mortality amongst t2 dm patients with and without ms, there are several studies that have looked at the cardiovascular outcomes in patients with t2 dm. in a study by seon the 10-year chd risk and 10-year stroke risk were 14.92% and 4.03%, respectively, calculated using the ukpds - re. the 10-year chd risk was relatively high, and the risk of chd tended to increase after onset of t2 dm : although they did not study patients with ms like in our study, it is worthy to note that patient outcome was similar to what we reported. the ukpds - re has recently become controversial in its reliability to predict chd or stroke. bannister noted that the relatively poor performance of the ukpds - re may be explained, at least in part by the differences in the baseline profiles of the ukpds and clinical practice research datalink populations. these plausible explanations include the epidemiological setting, changes in life expectancy, changes in smoking habits, the presence or absence of comorbidities, temporal changes in diabetes management, and changes in the general quality of care. other plausible explanations include the possible harm of overly aggressive treatment with sulfonylureas and insulin in the early stages of the disease. in the external validation of the ukpds - re in patients with t2 dm study by van dieren ;52 they observed that their mean follow - up time was 8 years ; therefore, they could not validate 10-year cvd and chd risks. however, the ukpds - re is in principle, designed for all risk periods including periods shorter than 10 years. secondly, their population consisted of all diabetes cases, not just individuals newly diagnosed with diabetes. therefore, they could only validate the use of the ukpds - re for patients who have been diagnosed with diabetes for some time. finally, they had some missing values in the baseline factors. there are several explanations for the poor to moderate performance of the ukpds - re to predict chd and cvd risk in this population. first, the ukpds - re was developed from a cohort that started including patients in 1977. treatment of t2 dm and prevention of cvd has improved since 1977 and the risk of developing cvd has declined with better treatment of t2 dm. also, as diabetes is now detected at an earlier stage, therapeutic intervention can be initiated earlier, reducing cvd risk even further. altogether, this may likely explain the large differences in predicted and observed absolute risks that have led to poor calibration. a major strength of this study is that it is the first study in benin city that assessed the impact of t2 dm on cardiovascular risk in subjects with ms using the ukpds - re. to the best of our knowledge this was a small study with notable limitations which include : 1) the original ukpds model tended to overestimate event rates across studies, therefore a modified ukpds model which includes adjustments for prior cardiovascular history has the potential for use as a tool for benchmarking and may be useful for predicting cardiovascular rates in clinical studies. this modification could be further evaluated, recalibrated, and validated using patient - level information derived from prospective clinical studies to yield greater predictability. 2) we did not consider medication use in the diagnosis of ms in this study. 3) mortality was not analyzed because we did not have sufficient occurrences at this stage of the study. a larger and specifically designed study is needed to evaluate the effects or impact of the ms on the cardiovascular status of nigerians with t2 dm, as cardiovascular events are known to be a major cause of mortality in this group of persons worldwide. in conclusion, the increased risk of cvd at the onset of type 2 diabetes observed in this study is consistent with previous studies. this study has demonstrated that the ms appears to increase the risk of a coronary event in a person with t2 dm by threefold. | backgroundthe aim of this study is to evaluate the impact of coexistence of metabolic syndrome (ms) and type 2 diabetes mellitus (t2 dm) on the estimated cardiovascular risk as calculated using the united kingdom prospective diabetic study risk engine (ukpds - re) and also to determine the impact of the coexistence of ms and t2 dm on the 10-year risk of developing coronary heart disease and stroke.methodologythis is a cross - sectional study in which convenience sampling technique was used to recruit 124 consecutive persons with t2 dm and 96 controls using a questionnaire administered technique. the world health organization (who) criterion was used to define ms and the ukpds - re was used to identify persons with increased risk for stroke and those with increased risk for coronary heart disease. the data obtained were analyzed using spss version 16. statistical comparisons were made with chi - square for comparison of proportions. a p - value of less than 0.05 was taken as statistically significant.resultsfifteen subjects were identified as having an increased 10-year risk for stroke and ten as having an increased risk for a coronary event. the odds of a t2 dm subject with ms having an increased risk for stroke compared with a t2 dm subject without ms was 0.95791 while the odds of a t2 dm subject with ms developing an increased risk for a coronary event compared with a t2 dm subject without ms was = 3.4513.conclusionms was more common in subjects with t2 dm compared with controls (irrespective of the diagnostic criteria used) and ms appears to increase the risk of a coronary event in subjects with t2 dm by threefold. also from this study, ms did not appear to cause an additional increase in the risk of stroke in subjects with t2 dm. |
the sample consisted of 51 right - handed non - demented patients between 40 and 80 years, diagnosed with ipd. they were presenting at least three cardinal signs of the disorder (bradykinesia, rest tremor and rigidity). there were 42 males and 9 females with mean (sd) age of 53 years (9.58). their educational level ranged from being literate to post graduation. among them, 33 were employed and 18 were retired. the sample was taken from the movement disorder clinic, department of neurology, at bangur institute of neuroscience and psychiatry, kolkata, india. those with evidence of parkinsonism plus syndrome (pps), or secondary or drug - induced parkinson 's disease were excluded. other exclusion criteria were ipd of less than 1 year duration, lack of response to levodopa, vertical gaze abnormality, early dementia, ataxia and autonomic neuropathy as evident from postural dizziness, erectile impotency and sweating disturbances, clinical depression and severe impairment of daily activities as evaluated from the information battery. cognitive information battery by das. for obtaining demographic and behavioral information relevant to cognitive impairment.kolkata cognitive screening battery (kcsb) initially used by ganguly. and validated among urban population in bengali and hindi speaking population. field - testing of kcsb was done to evaluate intra- and inter - rater reliability and the values for different sub - tests ranged from 0.77 to 0.99. the final battery consisted of 10 sub - tests among which the following 8 sub - tests have been used in this study : verbal fluency tests for categories, naming tests, bengali validated mental status examination, calculation, word list memory task, delayed word list memory task, delayed recognition word task, and visuoconstructional ability. the subscales of depression and activities of daily life were not used in the present study as the subjects with notable depression and severe impairment of everyday functioning were excluded.wechsler adult intelligence scale (wais) : four verbal sub - tests from wais were used to assess higher mental functioning. these sub - tests were information, comprehension, similarities, and arithmetic.hoehn and yahr staging of disease : this scale was developed by hoehn and yahr in 1967. the patients are evaluated on a 7-point scale by the clinicians.0 = no sign of disease1 = unilateral disease1.5 = unilateral plus axial involvement2 = bilateral disease, without impairment or balance2.5 = mild bilateral disease, with recovery on pull test3 = mild to moderate bilateral disease, some postural instability, physically independent4 = severe disability ; still able to work or stand unassisted cognitive information battery by das. for obtaining demographic and behavioral information relevant to cognitive impairment. kolkata cognitive screening battery (kcsb) initially used by ganguly. and validated among urban population in bengali and hindi speaking population. field - testing of kcsb was done to evaluate intra- and inter - rater reliability and the values for different sub - tests ranged from 0.77 to 0.99. the final battery consisted of 10 sub - tests among which the following 8 sub - tests have been used in this study : verbal fluency tests for categories, naming tests, bengali validated mental status examination, calculation, word list memory task, delayed word list memory task, delayed recognition word task, and visuoconstructional ability. the subscales of depression and activities of daily life were not used in the present study as the subjects with notable depression and severe impairment of everyday functioning were excluded. wechsler adult intelligence scale (wais) : four verbal sub - tests from wais were used to assess higher mental functioning. hoehn and yahr staging of disease : this scale was developed by hoehn and yahr in 1967. the patients are evaluated on a 7-point scale by the clinicians. 0 = no sign of disease 1 = unilateral disease 1.5 = unilateral plus axial involvement 2 = bilateral disease, without impairment or balance 2.5 = mild bilateral disease, with recovery on pull test 3 = mild to moderate bilateral disease, some postural instability, physically independent 4 = severe disability ; still able to work or stand unassisted data were collected from a specific hospital in kolkata. the patients were diagnosed with ipd without dementia and identified as non - depressed by the attending neurologists according to dsm iv criteria. then, with the help of the attending neurologist, the hoehn and yahr staging of disease, kcsb, and the four verbal sub - tests from wais were applied. approximately 90 min were required for each patient to complete the whole protocol. to determine the effects of age, stage, and education, the sample was divided into subgroups. three age groups were identified, namely below 50 years, 5059 years, and 60 years and above. three groups in terms of hoehn and yahr (hy) staging were identified, namely early (rated as 1 or 1.5 on hy scale), middle (rated as 2 or 2.5 on hy scale), and advanced (rated as 3 or 4 on hy scale). these were lower (below class v), middle (class v to class x), and upper (above class x). statistical analyses were done using descriptive statistics, z tests, and multivariate analyses of variance. since the number of participants was small, the interaction of age, stage, and education was not calculated, but separate analysis of the effects of each of these variables on the cognitive functioning was done. the sample consisted of 51 right - handed non - demented patients between 40 and 80 years, diagnosed with ipd. they were presenting at least three cardinal signs of the disorder (bradykinesia, rest tremor and rigidity). there were 42 males and 9 females with mean (sd) age of 53 years (9.58). their educational level ranged from being literate to post graduation. among them, 33 were employed and 18 were retired. the sample was taken from the movement disorder clinic, department of neurology, at bangur institute of neuroscience and psychiatry, kolkata, india. those with evidence of parkinsonism plus syndrome (pps), or secondary or drug - induced parkinson 's disease were excluded. other exclusion criteria were ipd of less than 1 year duration, lack of response to levodopa, vertical gaze abnormality, early dementia, ataxia and autonomic neuropathy as evident from postural dizziness, erectile impotency and sweating disturbances, clinical depression and severe impairment of daily activities as evaluated from the information battery. cognitive information battery by das. for obtaining demographic and behavioral information relevant to cognitive impairment.kolkata cognitive screening battery (kcsb) initially used by ganguly. and field - testing of kcsb was done to evaluate intra- and inter - rater reliability and the values for different sub - tests ranged from 0.77 to 0.99. the final battery consisted of 10 sub - tests among which the following 8 sub - tests have been used in this study : verbal fluency tests for categories, naming tests, bengali validated mental status examination, calculation, word list memory task, delayed word list memory task, delayed recognition word task, and visuoconstructional ability. the subscales of depression and activities of daily life were not used in the present study as the subjects with notable depression and severe impairment of everyday functioning were excluded.wechsler adult intelligence scale (wais) : four verbal sub - tests from wais were used to assess higher mental functioning. these sub - tests were information, comprehension, similarities, and arithmetic.hoehn and yahr staging of disease : this scale was developed by hoehn and yahr in 1967. the patients are evaluated on a 7-point scale by the clinicians.0 = no sign of disease1 = unilateral disease1.5 = unilateral plus axial involvement2 = bilateral disease, without impairment or balance2.5 = mild bilateral disease, with recovery on pull test3 = mild to moderate bilateral disease, some postural instability, physically independent4 = severe disability ; still able to work or stand unassisted cognitive information battery by das. for obtaining demographic and behavioral information relevant to cognitive impairment. kolkata cognitive screening battery (kcsb) initially used by ganguly. and validated among urban population in bengali and hindi speaking population. field - testing of kcsb was done to evaluate intra- and inter - rater reliability and the values for different sub - tests ranged from 0.77 to 0.99. the final battery consisted of 10 sub - tests among which the following 8 sub - tests have been used in this study : verbal fluency tests for categories, naming tests, bengali validated mental status examination, calculation, word list memory task, delayed word list memory task, delayed recognition word task, and visuoconstructional ability. the subscales of depression and activities of daily life were not used in the present study as the subjects with notable depression and severe impairment of everyday functioning were excluded. wechsler adult intelligence scale (wais) : four verbal sub - tests from wais were used to assess higher mental functioning. these sub - tests were information, comprehension, similarities, and arithmetic. hoehn and yahr staging of disease : this scale was developed by hoehn and yahr in 1967. the patients are evaluated on a 7-point scale by the clinicians. 0 = no sign of disease 1 = unilateral disease 1.5 = unilateral plus axial involvement 2 = bilateral disease, without impairment or balance 2.5 = mild bilateral disease, with recovery on pull test 3 = mild to moderate bilateral disease, some postural instability, physically independent 4 = severe disability ; still able to work or stand unassisted the patients were diagnosed with ipd without dementia and identified as non - depressed by the attending neurologists according to dsm iv criteria. then, with the help of the attending neurologist, the hoehn and yahr staging of disease, kcsb, and the four verbal sub - tests from wais were applied. to determine the effects of age, stage, and education, the sample was divided into subgroups. three age groups were identified, namely below 50 years, 5059 years, and 60 years and above. three groups in terms of hoehn and yahr (hy) staging were identified, namely early (rated as 1 or 1.5 on hy scale), middle (rated as 2 or 2.5 on hy scale), and advanced (rated as 3 or 4 on hy scale). these were lower (below class v), middle (class v to class x), and upper (above class x). statistical analyses were done using descriptive statistics, z tests, and multivariate analyses of variance. since the number of participants was small, the interaction of age, stage, and education was not calculated, but separate analysis of the effects of each of these variables on the cognitive functioning was done. the means and standard deviations of the eight sub - tests of kcsb were calculated. these were compared with the normative values obtained in the original article by das. the z tests were done to determine whether the obtained mean values for the functions differed significantly from the mean values of the population [table 1 ]. z values for testing the significance of difference between the normal and ipd groups in different areas of functioning subsequently, the effects of age were considered. the means and standard deviations of the three age groups (4049 years, 5059 years, and 60 years and above) were calculated. multivariate analysis of variance (manova) was conducted with age as an independent variable and the cognitive functions as a dependent variable. table 2 indicates that there is no significant effect of age categories on the dependent variables taken in combination. this implies that at least for the age range taken in this study, age may not be a highly significant factor for cognitive decline in general. however, there remains a possibility that some of the specific variables may be affected by age. results imply that age turned out to be a significant variable only for delayed recall and delayed recognition [table 2 ]. the means and sds of the three age groups (4049 years, 5059 years, and 60 years) for the cognitive functions and results of manova and anovas subsequently, multiple comparisons between each age group in terms of these two dependent variables were done with tukey 's post hoc tests. the results revealed that for both delayed recall and recognition memory, the mean differences between the 60 years age group was obtained. for delayed memory, for the delayed recognition memory test, the difference of 4.77 was significant at 0.008 level. the means and standard deviations of the three groups with differing stages of disease (early, middle, and advanced) are presented in table 3. subsequently, manova was conducted with stage of disease as an independent variable and the cognitive functions as dependent variables. the means and sds of the three stage groups (early, middle, and advanced) for the cognitive functions and results of manova and anovas table 3 indicates that there is no significant effect of stage categories on the dependent variables taken together. subsequently, univariate anovas were conducted separately for each cognitive variable [table 3 ]. results imply that stage of disease turned out to be a significant variable for verbal fluency, findings from mini mental status examination (mmse), delayed recall memory, and fund of information. subsequently, multiple comparisons between each stage group in terms of the dependent variable were done with tukey 's post hoc tests. the results revealed that for verbal fluency, mmse, delayed recognition, and fund of information, the mean differences between the early and middle stages or middle and advanced stages were not statistically significant. however, significant differences between early and advanced stages were obtained. for verbal fluency, the difference of 6.24 between the early and advanced stages was significant at 0.025 level. for mmse, the difference of 4.97 between the early and advanced stages was significant at 0.021 level. for delayed recall, the difference between early and advanced stages of 2.02 was significant at 0.024 level. for fund of information, the difference of 6.80 between the early and advanced stages was significant at 0.027 level. the means and standard deviations of the three groups with differing educational levels (below class v, classes v x, and above class x) are presented in table 4. subsequently, manova was conducted with educational level as an independent variable and the cognitive functions as dependent variables. the means and sds of the three educational level groups (class x) for the cognitive functions and results of manova and anovas table 4 indicates that there is significant effect of educational levels on the dependent variables taken together. this implies that educational level is a highly significant factor for cognitive decline when all the variables are taken in combination. subsequently, univariate anovas were conducted separately for each cognitive variable [table 4 ]. results imply that educational level was a significant variable for visuoconstructional ability, fund of information, comprehension, similarities, and arithmetic. subsequently, multiple comparisons between each educational level group in terms of these dependent variables were done with tukey 's post hoc tests. the results revealed that for visuoconstructional ability, the mean difference of 3.867 between the highest and the lowest educational levels was significant at 0.01 level. the mean difference of 6.13 between the middle and the lowest levels in case of fund of information was significant at 0.039 level. mean difference of 11.20 between the highest and the lowest levels in case of fund of information was significant at 0.000 level. for comprehension, the mean difference of 5.39 between the middle and the lowest levels was significant at 0.041 level. mean difference of 10.17 between the highest and the lowest level in case of comprehension was significant at 0.000 level. the mean difference of 4.77 between the highest and middle levels of comprehension was significant at 0.037 level. for similarities, the mean difference of 6.10 between the highest and the lowest educational levels was significant at 0.021 level. for arithmetic, the obtained mean difference of 3.13 between the highest and the lowest educational levels was significant at 0.04 level. subsequently, the effects of age were considered. the means and standard deviations of the three age groups (4049 years, 5059 years, and 60 years and above) multivariate analysis of variance (manova) was conducted with age as an independent variable and the cognitive functions as a dependent variable. table 2 indicates that there is no significant effect of age categories on the dependent variables taken in combination. this implies that at least for the age range taken in this study, age may not be a highly significant factor for cognitive decline in general. however, there remains a possibility that some of the specific variables may be affected by age. results imply that age turned out to be a significant variable only for delayed recall and delayed recognition [table 2 ]. the means and sds of the three age groups (4049 years, 5059 years, and 60 years) for the cognitive functions and results of manova and anovas subsequently, multiple comparisons between each age group in terms of these two dependent variables were done with tukey 's post hoc tests. the results revealed that for both delayed recall and recognition memory, the mean differences between the 60 years age group was obtained. for delayed memory, for the delayed recognition memory test, the difference of 4.77 was significant at 0.008 level. effects of stage of disease were subsequently scrutinized. the means and standard deviations of the three groups with differing stages of disease (early, middle, and advanced) subsequently, manova was conducted with stage of disease as an independent variable and the cognitive functions as dependent variables. the means and sds of the three stage groups (early, middle, and advanced) for the cognitive functions and results of manova and anovas table 3 indicates that there is no significant effect of stage categories on the dependent variables taken together. subsequently, univariate anovas were conducted separately for each cognitive variable [table 3 ]. results imply that stage of disease turned out to be a significant variable for verbal fluency, findings from mini mental status examination (mmse), delayed recall memory, and fund of information. subsequently, multiple comparisons between each stage group in terms of the dependent variable were done with tukey 's post hoc tests. the results revealed that for verbal fluency, mmse, delayed recognition, and fund of information, the mean differences between the early and middle stages or middle and advanced stages were not statistically significant. however, significant differences between early and advanced stages were obtained. for verbal fluency, the difference of 6.24 between the early and advanced stages was significant at 0.025 level. for mmse, the difference of 4.97 between the early and advanced stages was significant at 0.021 level. for delayed recall, the difference between early and advanced stages of 2.02 was significant at 0.024 level. for fund of information, the difference of 6.80 between the early and advanced stages was significant at 0.027 level. the effects of education were analyzed in the last section. the means and standard deviations of the three groups with differing educational levels (below class v, classes v x, and above class x) are presented in table 4. subsequently, manova was conducted with educational level as an independent variable and the cognitive functions as dependent variables. the means and sds of the three educational level groups (class x) for the cognitive functions and results of manova and anovas table 4 indicates that there is significant effect of educational levels on the dependent variables taken together. this implies that educational level is a highly significant factor for cognitive decline when all the variables are taken in combination. subsequently, univariate anovas were conducted separately for each cognitive variable [table 4 ]. results imply that educational level was a significant variable for visuoconstructional ability, fund of information, comprehension, similarities, and arithmetic. subsequently, multiple comparisons between each educational level group in terms of these dependent variables were done with tukey 's post hoc tests. the results revealed that for visuoconstructional ability, the mean difference of 3.867 between the highest and the lowest educational levels was significant at 0.01 level. the mean difference of 6.13 between the middle and the lowest levels in case of fund of information was significant at 0.039 level. mean difference of 11.20 between the highest and the lowest levels in case of fund of information was significant at 0.000 level. for comprehension, the mean difference of 5.39 between the middle and the lowest levels was significant at 0.041 level. mean difference of 10.17 between the highest and the lowest level in case of comprehension was significant at 0.000 level. the mean difference of 4.77 between the highest and middle levels of comprehension was significant at 0.037 level. for similarities, the mean difference of 6.10 between the highest and the lowest educational levels was significant at 0.021 level. for arithmetic, the obtained mean difference of 3.13 between the highest and the lowest educational levels was significant at 0.04 level. the results suggested that most of the functions with the exception of information were significantly poorer among the ipd group in comparison to the normative non - demented scores, indicating substantial impairment in cognitive functions in ipd. table 2 reveals that age came out to be a significant factor only for delayed memory. in the interpretation of age - related changes in this study, it needs to be remembered that the number of subjects was small and majority of the subjects were below 70 years. similar findings have been obtained earlier. in a recent study, kim. observed that among ipd patients, age was associated with mild cognitive impairment around the age of 64 years. it is possible that cognitive impairment becomes more pronounced for a higher age group than was taken in this study. in table 3 it was found that stage was significant only between the early and the advanced stages and in case of selected cognitive functions, namely, verbal fluency, mmse, delayed recall memory, and fund of information. the impairment of various cognitive areas in advanced ipd patients supports the models describing parkinsonism - related changes in dorsolateral prefrontal regions participating in cognitive basal ganglia and thalamocortical circuits. impairment was also more frequent in conditions of delayed recall than in conditions of delayed recognition, where cues guide retrieval. results from table 4 suggest that the level of education was more significant for overall impairment of the cognitive faculties taken together in comparison to age and stage of disease. lower educational level was associated with greater impairment. specifically, it affected visuoconstructional ability and the higher cognitive functions. the present findings reveal that as expected, cognitive functions are impaired in patients with ipd in comparison to normative data. the study also suggests that cognitive functions deteriorate along with increasing age, advanced stage of disease, and educational level. among these three factors this has important implication in management, as cognitive practice seems to deter the impairment rate. the non - demented ipd remains a relatively ignored area where management strategies and rehabilitation efforts are concerned. while the present study has significant implications in understanding the nature of cognitive impairment associated with ipd, it is limited by its small sample size and the lack of any internal control group, which would have enhanced its value as evidence. it may be extended along these lines in future research and a charter of cognitive training and practice may be prepared for ready use of the caregiver. | background : parkinsonism is known to be associated with clinically significant impairments on an array of cognitive deficits. the degree of impairment is dependent not only on the course of the disease, but also on other bio - social factors. the objective of the present study was to examine the cognitive dysfunction in non - demented idiopathic parkinson 's disease (ipd) in relation to age, stage of disease, and educational level in a sample in kolkata, india.materials and methods : the sample consisted of 51 (42 males, 9 females) right - handed patients suffering from non - demented ipd, of age between 40 and 82 years. data were collected during on - phase medication by using the kolkata cognitive screening battery. data were analyzed using means, standard deviations, and multivariate analyses of variance (manova).results and conclusion : the patients with ipd were impaired in comparison to the available normative data in almost all aspects of cognitive functioning and higher order mental processes. with increasing age, the patients showed greater impairment in delayed memory and recognition task. patients of more severe stage showed greater impairment in mmse, delayed recall, and information. those with lower education had more impaired visuoconstructional ability, information, comprehension, similarities, and arithmetic. |
subsets of 270 participants who visited our hospital for knee pain and met the american college of rheumatology criteria for symptomatic oa of the knee19) from october 2005 to september 2013 were evaluated. we limited the subsets to women for avoiding the confounding variables and bias, because there were only a few male patients (n = 8). all of the patients had a k - l grade 1 in the affected knee and dxa scan was taken of the ipsilateral proximal femur (femoral neck, trochanter, intertrochanter, total hip - neck, trochanter, and ward 's triangle) and lumbar spine (l1 - 4) within 6 months of the first visit (mean interval, 2.7 months). to avoid confounding in the statistical analysis, patients with valgus alignment of knees (3 valgus about the mechanical axis), and other comorbidities (e.g., rheumatoid arthritis, septic arthritis) that could also be the causes of knee pain and change in bone structure were excluded. subjects who had unsatisfactory medial tibial plateau alignment in the radiographs were also excluded from the final analysis. weight was measured to the nearest 0.1 kg using a single pair of electronic scales. height was measured to the nearest 0.1 cm using a stadiometer. from these data, body mass index (bmi ; weight / height, kg / m) was calculated. mechanical axis in the scanogram, jsn in the anteroposterior (ap) view and the rosenberg view, and k - l grade were measured to assess the severity of oa. thus, the relationship between the measured values of bmd and radiographic severity of osteoarthritic changes was analyzed considering the confounding variables. standing ap films of the knee in full extension were obtained with the horizontal x - ray beam centered at the level of the superior patellae. views were standardized with the back of the knees in contact with the cassette, the patella centralized over the lower end of the femur, and the beam centered 2.5 cm below the apex of the patella, with a tube - to - film distance of 100 cm. also, the rosenberg view of the knee in 45 of flexion was obtained for accurate detection of jsn in the state of maximal load weight - bearing. the rosenberg view is a 45 flexion, posteroanterior, weight - bearing view of the knee with the patellae touching the image receptor. the x - ray tube is 100 cm away from the image receptor, centered at the patellae, and pointing caudad 10. the mechanical axis of the knee was measured from the angle formed by a line drawn from the center of the femoral head to the medial tibial spine and a line drawn from the medial tibial spine to the center of the ankle joint. as mentioned above, patients who having valgus alignment of knees were excluded.20) the radiographic determinations of knee alignment were made by two examiners who had adequate knowledge about the measurement techniques and the k - l grading scale. all of the radiographs were digitized by the picture archiving and communication system software (piviewstar, infinit technology, seoul, korea) in our hospital, and all of the distances and angles were measured by using the calipers and goniometer provided in the software. the software enables straight - line measurements with an accuracy of 0.01 mm and 0.01. jsn was defined as the narrowest interbone distance between the medial femoral condyle and the tibial plateau. k - l grades were assessed using a standard atlas.21) the inter - observer reproducibility between two examiners was assessed in a randomly chosen subset of 30 radiographs. icc for inter - observer variability was more than 0.94 and icc for intra - observer variability was more than 0.97 in all of the measurements. partial correlation analysis was performed to analyze the relationship between the bmds (femoral neck, femoral trochanter, femoral intertrochanter, total hip and spine) and joint space distance in the ap and the rosenberg views, between the bmds and mechanical axis of the knee adjusted for demographic variables (age, bmi). analysis of covariance (ancova) was used to analyze the difference in bmd in the k - l grade category groups, which was also adjusted for covariates (age, bmi). we determined the sample size after conducting a pilot study with 50 cases. in the pilot study, we set the level of significance () and power to 0.05 and 0.8, respectively ; therefore, the result of sample size calculation was 193 and we ensured that a sufficient number of patients were included in the study. subsets of 270 participants who visited our hospital for knee pain and met the american college of rheumatology criteria for symptomatic oa of the knee19) from october 2005 to september 2013 were evaluated. we limited the subsets to women for avoiding the confounding variables and bias, because there were only a few male patients (n = 8). all of the patients had a k - l grade 1 in the affected knee and dxa scan was taken of the ipsilateral proximal femur (femoral neck, trochanter, intertrochanter, total hip - neck, trochanter, and ward 's triangle) and lumbar spine (l1 - 4) within 6 months of the first visit (mean interval, 2.7 months). to avoid confounding in the statistical analysis, patients with valgus alignment of knees (3 valgus about the mechanical axis), and other comorbidities (e.g., rheumatoid arthritis, septic arthritis) that could also be the causes of knee pain and change in bone structure were excluded. subjects who had unsatisfactory medial tibial plateau alignment in the radiographs were also excluded from the final analysis. weight was measured to the nearest 0.1 kg using a single pair of electronic scales. height was measured to the nearest 0.1 cm using a stadiometer. from these data, body mass index (bmi ; weight / height, kg / m) was calculated. mechanical axis in the scanogram, jsn in the anteroposterior (ap) view and the rosenberg view, and k - l grade were measured to assess the severity of oa. thus, the relationship between the measured values of bmd and radiographic severity of osteoarthritic changes was analyzed considering the confounding variables. standing ap films of the knee in full extension were obtained with the horizontal x - ray beam centered at the level of the superior patellae. views were standardized with the back of the knees in contact with the cassette, the patella centralized over the lower end of the femur, and the beam centered 2.5 cm below the apex of the patella, with a tube - to - film distance of 100 cm. also, the rosenberg view of the knee in 45 of flexion was obtained for accurate detection of jsn in the state of maximal load weight - bearing. the rosenberg view is a 45 flexion, posteroanterior, weight - bearing view of the knee with the patellae touching the image receptor. the x - ray tube is 100 cm away from the image receptor, centered at the patellae, and pointing caudad 10. the mechanical axis of the knee was measured from the angle formed by a line drawn from the center of the femoral head to the medial tibial spine and a line drawn from the medial tibial spine to the center of the ankle joint. as mentioned above, patients who having valgus alignment of knees were excluded.20) the radiographic determinations of knee alignment were made by two examiners who had adequate knowledge about the measurement techniques and the k - l grading scale. all of the radiographs were digitized by the picture archiving and communication system software (piviewstar, infinit technology, seoul, korea) in our hospital, and all of the distances and angles were measured by using the calipers and goniometer provided in the software. the software enables straight - line measurements with an accuracy of 0.01 mm and 0.01. jsn was defined as the narrowest interbone distance between the medial femoral condyle and the tibial plateau. the inter - observer reproducibility between two examiners was assessed in a randomly chosen subset of 30 radiographs. icc for inter - observer variability was more than 0.94 and icc for intra - observer variability was more than 0.97 in all of the measurements. 12 (spss inc., chicago, il, usa) was used for the statistical analysis. partial correlation analysis was performed to analyze the relationship between the bmds (femoral neck, femoral trochanter, femoral intertrochanter, total hip and spine) and joint space distance in the ap and the rosenberg views, between the bmds and mechanical axis of the knee adjusted for demographic variables (age, bmi). analysis of covariance (ancova) was used to analyze the difference in bmd in the k - l grade category groups, which was also adjusted for covariates (age, bmi). we determined the sample size after conducting a pilot study with 50 cases. in the pilot study, we set the level of significance () and power to 0.05 and 0.8, respectively ; therefore, the result of sample size calculation was 193 and we ensured that a sufficient number of patients were included in the study. the study population included 195 females who had radiographic features of knee oa (k - l grade 1). the mean age of study participants was 68.5 years, with a range of 38 to 83 years. the mean height, weight, and bmi were 151.58 cm (range, 139.40 to 177.90 cm), 60.25 kg (range, 39.10 to 96.00 kg), and 26.17 kg / m (range, 18.39 to 39.33 kg / m), respectively. mean jsn in the ap view was 2.00 mm, and mean jsn in the rosenberg view was 1.48 mm. thirty participants were classified into k - l grade 1, 22 into k - l grade 2, 51 into k - l grade 3, and 92 into k - l grade 4. there was a statistically significant relationship between mechanical axis of the knee and femoral neck, trochanter, intertrochanter, and total hip bmd (p < 0.05). less severity of varus alignment was positively associated with the bmd of the proximal femur. there was also a statistically significant difference in spine bmd in terms of the mechanical axis. in terms of the joint space distance, there were statistically significant relationships between bmd of the proximal femur (femoral neck, trochanter, intertrochanter, and total hip) and joint space distance in the ap view (p < 0.05). there were also statistically significant differences between bmd of the proximal femur and joint space distance in the rosenberg view (p < 0.05). the bmd values of the proximal femur were positively associated with increased joint space distance. joint space distance increased with greater total hip bmd both in the ap view and the rosenberg view (in the ap view : femoral neck, r = 0.147, p = 0.041 ; femoral trochanter, r = 0.251, p = 0.000 ; femoral intertrochanter, r = 0.224, p = 0.002 ; total hip, r = 0.233, p = 0.001 and in the rosenberg view : femoral neck, r = 0.226, p = 0.002 ; femoral trochanter, r = 0.289, p = 0.000 ; femoral intertrochanter, r = 0.295, p = 0.000 ; total hip, r = 0.307, p = 0.000). spine bmd, however, was not significantly associated with joint space distance (table 2). the mean values of bmd of the proximal femur had a tendency to decrease with increasing k - l grades, and there were significant differences in the bmd of the proximal femur (femoral neck, trochanter, intertrochanter, total hip) according to the k - l grade groups (p = 0.028, p = 0.004, p = 0.002, and p = 0.003, respectively). the contrast test was also conducted, and the k - l grade 1 group showed a statistically significant difference in the bmd of the proximal femur compared to k - l grade 2, 3, and 4 groups, and k - l grade 1, 2, and 3 groups showed statistically significant differences in the bmd of the proximal femur compared to k - l grade 4 group. there was no significant difference in spine bmd among the k - l grade groups (p = 0.072) (table 3). in this study, we focused on the relationship between the bmd of the proximal femur and severity of radiological knee oa in the ipsilateral side based on the cross - sectional data in a korean population especially among women, while studying the relationship between spine bmd and the severity of radiological knee oa at the same time. the overall results demonstrated that after adjusting for demographic variables (age, bmi), low bmd of the proximal femur (femoral neck, trochanter, intertrochanter and total hip) was positively correlated with joint space width. also, higher k - l grade was associated with lower bmd of the proximal femur. on the other hand, a number of studies about the relationship between bmd and oa have been reported and the inverse relationship between op and oa has been shown in the cross - sectional data.1,2,3,4,5,6) in the framingham study, mean femoral bmd at the 3 proximal femur sites was 5%-9% higher in patients with either grade 1, grade 2, or grade 3 knee oa compared with those with no knee oa.3) another cohort study reported that higher bone density was associated with an increased prevalence of knee oa and an increased risk of incident knee oa, but higher bmd was associated with a decreased risk of progressive knee oa.11) other cross - sectional studies showed that oa subjects had a 6%- 9% increase in lumbar spine bmd4) and patients who had hip oa were found to have 8%-10% higher spine and hip bmd.6) our data are obtained from patients who had both symptomatic and radiological knee oa. also, we made an effort to minimize the difference in the time between the assessment of knee oa and the bmd measurement (6 months) for collecting the cross - sectional data. development of oa is associated with changes in the subchondral bone22,23) and crosstalk between the subchondral bone and the cartilage contributes to progression towards different stages of oa.24) as the initiation of oa precedes the appearance of radiographic features,23) it may be more difficult to analyze the actual relationship between bmd and oa. severity of radiological oa, as demonstrated by a narrower joint space or a higher k - l grade, had a negative correlation with the bmd of the ipsilateral proximal femur. also, contrary to the result of a previous study,25) there was no correlation between the spine bmd and knee oa (in terms of joint space distance and the k - l grade). pain and disuse of the affected limb may have caused a local decrease in bmd of the proximal femur on the ipsilateral side, while spine bmd was relatively unaffected by these effects. so in our setting, it can be suggested that local factors affected the interaction between oa and bone density in addition to systemic factors. our data, obtained from a korean population, especially from women, may reflect a different lifestyle compared with that of caucasian population3,6,9,10,11) as the patients with pain due to oa mostly avoid physical activities and exercises. therefore, we think that these factors led to the obtainment of results, which are the different from those in previous studies. as a corollary, while studying the relationship between oa and op, various factors should be taken into consideration. only females were included in the study population ; hence, it did not accurately reflect the korean population. although we made an effort to adjust for physical variables, we were not able to control for individual lifestyle and physical activity of knee oa patients. also, we only included symptomatic patients with knee oa ; therefore, we could not assess the relationship between the bmd and radiographic knee oa in healthy groups and could not analyze the association between bmd and the incidence of knee oa. however, we believe that this report provides useful information about the relationship between oa and op in asian patients. | backgroundthe relationship between osteoarthritis (oa) and osteoporosis (op) is complicated and it may differ according to the site or stage of disease. the purpose of this cross - sectional study is to examine the relationship between the severity of radiological knee oa and the degree of op in the ipsilateral proximal femur as denoted by bone mineral density (bmd) in a korean population, especially among women.methods one hundred ninety - five female patients who had knee pain and radiological knee oa were investigated with respect to the relationship of knee oa severity with bmd. the bmd of the proximal femur and spine was measured by dual energy x - ray absorptiometry, and the severity of knee oa was evaluated based on kellgren - lawrence (k - l) radiographic criteria, joint space narrowing (jsn) and mechanical axis of knee alignment. partial correlation analysis and ancova adjusted for confounding factors (age and body mass index) were performed to assess the relationship.resultsthere was a statistically significant relationship between the bmd of the proximal femur and jsn, and the bmd of the proximal femur was positively associated with increased joint space width. there was a lack of association between the spine bmd and jsn. the bmd of the proximal femur was also significantly lower in patients who had a higher k - l grade.conclusionsthe radiographic finding of severe oa in the knee is associated with decreased bmd of the ipsilateral proximal femur including the femoral neck, trochanter, intertrochanter, and region of the entire hip (neck, trochanter, and ward 's triangle). |
with the increasing elderly population, dementia has become one of the most common social problems1. because memory impairment is the core symptom of dementia, the majority of early interventions aim to slow cognitive decline. in recent years however, studies into their use for dementia patients have found them to have limited effectiveness3. recent studies, however, have focused on non - pharmacological cognitive interventions to alleviate the cognitive deficits related to dementia4, 5. non - pharmacological cognitive interventions for dementia include re - learning strategies that promote the management ability to reduce cognitive impairment3, 6. cognitive interventions differ from pharmacological interventions in that they consider the interaction between the patients and therapist, as well as the needs of the patient7. cognitive interventions have been shown to temporarily slow the rate of cognitive decline and the loss of functional abilities. on the other hand, they can fail because cognitive rehabilitation interventions that are beneficial for the patients with dementia require significant effort and attention. spaced retrieval training (srt) is a good candidate strategy for cognitive intervention in dementia because it does not require considerable cognitive effort8. srt is an expanded interval timer with independent data - tracking and timing of up to 3 targets4. srt recalling targets over increasingly longer periods of time has been proven to result in improved learning in people with mild to moderate dementia. this training regime was developed based on the expanding rehearsal technique which was originally developed for improving memory in cognitively intact persons9. errorless learning (el) is a teaching technique that prevents the making of mistakes while subjects are learning a new skill or acquiring new information, and it is conducted together with srt or the vanishing cues technique. srt with el is a technique for training the memory which uses an expanded interval that increases with correct responses and decreases with incorrect ones10. kinsella.11 suggested that srt would be beneficial for individuals with dementia because it utilizes the relatively unimpaired automatic implicit memory system. srt can be used with el because it minimizes the possibility of error during the acquisition phase of learning by ensuring mistakes. in addition, srt has a spacing effect that is retrieved more effectively when trials are distributed over time. srt appears to be attracting attention within the field of dementia treatment because of these advantages12, 13. non - pharmacological interventions for dementia, such as music, art, physical activity, and gardening, are often performed, but cognitive intervention of srt with el has rarely been conducted in clinical settings. in particular, studies aimed at observing the effects of srt in a clinical environment are difficult to perform. therefore, the purpose of this study was to examine effects of srt with errorless learning (el) for the rehabilitation of patients with dementia. twenty - nine participants with vascular dementia (vd) and alzheimer s disease (ad) participated in the present study. the study participants were enrolled from among inpatients in a rehabilitation - care hospital of korea. all the subjects provided their written informed consent to participation in the experiment in accordance with the ethical principles of the declaration of helsinki. subjects diagnosed with dementia including major depressive disorder by the dsm - iv criteria and those with serious medical and neurological problems that could have affected their mental function were excluded. table 1table 1.general characteristics of the subjects (n=29)characteristicsvd (n=22)ad (n=7)gender (male / female)13/92/5age (years)58.99.978.14.4education (years)7.95.01.72.9mmse - kc15.75.19.23.2gds - k18.85.615.53.4vd : vascular dementia ; ad : alzheimer dementia ; mmse - kc : korean version of mini mental status evaluation in the cerad - k assessment packet, gds - k : korean version of geriatric depression scale lists the general characteristics of the 29 participants. the ad group had a higher average age than the vd group, and had lower mean values in the korean version of mini - mental status evaluation (mmse - kc), and the korean version of geriatric depression scale (gds - k), as well as lower education levels than the vd group. vd : vascular dementia ; ad : alzheimer dementia ; mmse - kc : korean version of mini mental status evaluation in the cerad - k assessment packet, gds - k : korean version of geriatric depression scale mmse - kc was used to provide an index of the current cognitive status14. the maximum score on mmse - this measure consists of 30 items with each item scoring 1 point, and higher total scores indicate a higher level of depression. the cognitive functions were investigated using the korean version of the consortium to establish a registry for the alzheimer s disease (cerad - k) battery, which consists of 8 subtests16. the independence of activities of daily living (adl) was examined using the modified barthel index which consists of 10 subtests. the srt with el was administered by first providing the patients with a prompt question and associated target response. when successful at recalling the response, the patient was required to recall the target information at longer inter - trial intervals. if at any time the patient was unable to recall the response, they were provided with the information and asked to repeat it. the training was considered successful if the patient correctly responded to the prompt question at the beginning of each session (45, 90, 180, 360, and 720 sec.). all words were composed of 100 different words presented as high frequency words by the national institute of the korean language. if a subject repeatedly failed to remember a word for 45 seconds, the subject was excluded. if the subject remembered the word in two sessions (720 sec. 2 session), the number of words added ranged from one to five. during each interval, the subject was asked to perform inter - retrieval activities in order to prevent them from rehearsing the given set of words. the inter - retrieval activities consisted of simple physical activities that did not require much memory function (e.g., jenga, puzzle, pegboards). spss for windows version 18.0 was used for the statistical analysis, and p values less than 0.05 were considered significant. wilcoxon s matched - pairs signed - rank test was used to compare the pre- and post - test neuropsychological, depression, and adl performances of each group. as listed in table 2table 2.comparison of the effect of srt on cerad - k of dementia patientsvd group (n=22)ad group (n=7)beforeafterbeforeafterverbal fluency test5.822.747.183.13 3.291.604.292.56boston naming test7.413.208.143.113.712.143.572.30mmse - kc15.735.1717.274.659.293.2510.004.32word list memory8.823.8410.774.46 2.573.552.863.80constructional praxis6.002.916.143.315.711.985.712.06word list recall2.451.743.361.870.861.570.861.57word list recognition4.363.035.683.47 1.861.951.292.21constructional recall2.412.523.642.360.431.130.431.13srt : spaced retrieval training ; cerad - k : korean version of consortium to establish a registry of alzheimer s disease ; vd : vascular dementia ; ad : alzheimer dementia ; values are expressed as mean sd. significant difference from pre - test, p<0.05, p<0.01, all items of the cerad - k score in the vd group except for constructional praxis increased significantly after the srt with el intervention, but their values were similar to those of the ad group. significant differences were observed in the gain of the verbal fluency test, the boston naming test, mmse - kc, word list memory, word list recall, word list recognition, and constructional recall in the vd group between before and after the intervention. there were no significant differences in the subtests in the ad group between before and after the intervention. srt : spaced retrieval training ; cerad - k : korean version of consortium to establish a registry of alzheimer s disease ; vd : vascular dementia ; ad : alzheimer dementia ; values are expressed as mean sd. significant difference from pre - test, p<0.05, p<0.01 table 3table 3.comparison of the changes in gds - k and mbi between before and after the interventionvd group (n=22)ad group (n=7)beforeafterbeforeaftergds - k18.85.617.26.1 15.53.417.84.5mbi57.216.557.316.791.24.190.84.0gds - k : korean version of the geriatric depression scale ; mbi : modified barthel index ; values are expressed as mean sd. significant difference from pre - test, p<0.05 compares the effects of the srt on gds - k and mbi of the dementia groups. gds - k : korean version of the geriatric depression scale ; mbi : modified barthel index ; values are expressed as mean sd. the subjects with dementia could perform the srt with el. in particular, the srt with el intervention was beneficial for the vd patients. this suggests that the cognitive ability of dementia patients may be improved by srt with el. previous studies have shown that dementia patients have decreased communication skills and voluntary activity because of their reduced cognitive function. therefore, it is more difficult for dementia patients to have interpersonal relationships and engage in social participation17. cognitive interventions are essential to maintain the motivation for community participation. in addition, they are required for enhancing memory in cognitive interventions. considerable research has focused on the efficacy of cognitive intervention in rehabilitation. the in - session maximum retention span was maintained at 720 seconds during srt for the vd and ad groups. in addition, the intervention time was set at 30 minutes, considering the clinical environment and the disease characteristics. srt was previously administered in an in - session retention span of 1,440 seconds for mild dementia, but 720 seconds was used in the present study because of the various characteristics of dementia. srt with el had a significant effect on the performance of the vd group in the cerad - k except for the item of constructional praxis. after the srt with el, the vd group showed superior cognitive performance compared to the post - test values of the ad group. findings similar to those of the vd group have been reported by previous studies19. although not statistically significant, srt with el as a cognitive approach elicited improvements in the ad group. moreover, increasing the number of target words during the srt intervention may enhance the memory function in the early stages of ad, and might improve the acquisition of new information by patients with dementia20. it is our opinion that this is due to the difficulty in accurately calculating individual effect sizes for the adls. mbi has limited ability to compare the changes in the performance of patients with dementia after a short training period. a limitation of the present study is that the changes were not compared with another dementia group. while there were before and after changes in the vd and ad groups, it was difficult to identify differences between the groups. therefore, additional research will be needed to determine the effects of srt on ad patients according to the method of intervention. future research using sufficient sample sizes will be needed to obtain strong evidence for its efficacy not only in pre- and post - test results, but also between groups. | [purpose ] among the non - pharmacological interventions for dementia, spaced retrieval training (srt) is a good method for rehabilitating cognition. the purpose of this study was to examine effects of srt with errorless learning (el) in the rehabilitation of patients with dementia. [subjects and methods ] twenty - nine participants with vascular dementia (vd) and alzheimer s disease (ad) participated in the present study. the korean version of the consortium to establish a registry for alzheimer s disease (cerad - k) and modified barthel index (mbi) were performed to assess the changes in the neuropsychological performance and the independent activities of daily living after srt with el. all tests were administered both before and after srt with el. each srt with el intervention was performed for 30 minutes per day for 5 weeks. spss for windows version 18.0 was used for statistical analysis. [results ] all items of the cerad - k score of the vd group except for constructional praxis increased significantly after the srt with el intervention, but no significant differences from the ad group were found. the korean version of the geriatric depression scale (gds - k) of the vd group increased significantly after the srt with el intervention. the mean mbi scores of each group showed no significant difference after the intervention. [conclusion ] srt with el is an effective intervention for memory training of patients with dementia. future research using sufficient sample sizes will be needed to obtain strong evidence for comparing not only the before and after intervention data but also between the groups. |
globally, colorectal cancer (crc) is the fourth most common cancer in men and the third most common cancer in women1). although most crc occurs sporadically, genetic predisposition related to its pathogenesis has been reported2). one of the most investigated genotypic subtypes of crc is the aberrancy of the mismatch repair pathway, usually found in combination with microsatellite instability (msi)3). lynch syndrome, also called hereditary nonpolypsosis colorectal cancer (hnpcc), is caused by germline mutation of mismatch repair (mmr) genes4). individuals affected by lynch syndrome by heterozygous mutation of mmr usually present crc in the fourth or fifth decade ; however. k - ras, a critical oncogene and its product playing a key role in the kinase signaling growth pathway, plays a critical role in the pathogenesis of crc6). the revised bethesda guidelines recommendations were established for a better understanding and identifying individuals with hnpcc ; however, diagnosis of hnpcc at a young age, less than second decades, is difficult as clinician does not suspect it due to its rarity. we recently cared for a female patient with lynch syndrome, carrying heterozygous mlh1 germline mutation and k - ras missense somatic mutation ; she had crc at a very early age, 13-years old, and congenital heart disease (transposition of great arteries). a 13-year - old korean female was born without complications. soon after delivery, she was diagnosed with transposition of great arteries and corrective surgery was performed at 3 months of age. she grew up without any specific health problem until she reached 12 years of age. at 13 years of age she visited the hospital for weight loss of 10 kg over 2 months and a pale appearance. she was diagnosed as having helicobacter pylori gastritis by endoscopy and referred to our hospital for further h. pylori eradication and iron supplementation for management of iron deficiency anemia (hemoglobin, 7.1 g / dl ; serum iron, 13 g / dl ; total iron - binding capacity, 381 g / dl ; ferritin, 6.50 ng / dl). she did not have any skin lesions such as caf - au - lait spots and physical finding was not remarkable. after treatment, she was free from h. pylori gastritis ; however, despite taking a sufficient iron supplement for 1 month, iron deficiency persisted without improvement and palpable mass was noticed. for further evaluation of resistant anemia and palpable abdominal mass, colonoscopy was performed and revealed a huge irregular multilobular mass located on the rectum approximately 6 cm from the anal verge. biopsy was performed at that site and revealed adenocarcinoma of the rectum. computed tomography of the abdomen, pelvis, and chest showed a polypoid mass (2.6 cm2.5 cm) in the rectum and a huge mass (6.7 cm5.1 cm) in the transverse colon and ascending colon with lymph node enlargement. total proctocolectomy with ileal pouch anal anastomosis was performed and stage of cancer was t4n2m0. 1). genetic study of the pathologic specimen revealed a k - ras gene mutation at codon 12 as gly12asp (c. 35g > a) and msi was found in cancer cells by polymerase chain reaction amplification and fragment analysis by gene analyzer. germline mutation study of mmr genes using patient 's blood revealed a splicing mutation at mlh1 as c. 678 - 1g > c while other findings were unremarkable (fig. 2). genetic study of her parents was performed and revealed that mother had a same mlh1 mutation of patient ; however father had none. in addition, 2 cases of crc were detected on her maternal relatives (fig. 3). after informed consent was obtained, 12 cycles of chemotherapy consisting of oxaliplatin (85 mg / m on day 1), leucovorin (200 mg / m on days 1, 2), and 5-fluorouracil (1,500 mg / m on days 1, 2) (folfox) were completed without any specific problems, with the exception of myelosuppression. diagnostic work - up for recurrence was followed and a suspicious recurrent lesion was noticed at the duodenal second portion and pancreas head portion after completion of the eighth cycle of chemotherapy. after completion of 12 cycles of chemotherapy, imaging study revealed increased mass at the duodenal portion. nonetheless, some cancers arise as the consequence of inherited genetic mutations (germline mutations). lynch syndrome, caused by germline mutation of mmr genes, such as mlh1, msh2, msh6, and pms2, is an inherited cancer syndrome789). lynch syndrome - related tumors, such as crc and endometrium tumor in females, and a tumor spectrum comprised of hematological malignancies, development of crc by heterozygous mutation of mmr usually occurs during the fourth or fifth decade. however, in recent years, early onset of crc has been reported in children with either compound heterozygosity or homozygosity for the mmr gene defect101112). in our case crc by heterozygous germline mutation of mmr gene and a point somatic mutation of the k - ras developed in teenage without compound heterozygosity or homozygosity for the mmr gene defect. somatic mutation of k - ras gene, mainly missense mutation at codons 12 and 13, is found less frequently in crc with lynch syndrome than sporadic crc13). the effect of k - ras gene mutation on the time of occurrence and prognosis of crc is uncertain14). however, it could be considered that the mutation on the growth factor signaling pathway (k - ras) and mmr gene mutation could contribute synergistically to occurrence of crc at a very early age. some studies have also shown that age at diagnosis of crc decreases in successive generations of lynch families15). early diagnosis and management of crc in teenagers with lynch syndrome is difficult, since occurrence of crc is very unusual in this period. in our case, diagnosis was delayed and crc was managed improperly. the necessity of early screening for detection of crc in subjects with a family history of early onset crc, even before two decades, should be emphasized. | lynch syndrome is the most common inherited colon cancer syndrome. patients with lynch syndrome develop a range of cancers including colorectal cancer (crc) and carry a mutation on one of the mismatched repair (mmr) genes. although crc usually occurs after the fourth decade in patients with lynch syndrome harboring a heterozygous mmr gene mutation, it can occur in children with lynch syndrome who have a compound heterozygous or homozygous mmr gene mutation. we report a case of crc in a 13-year - old patient with lynch syndrome and congenital heart disease. this patient had a heterozygous mutation in mlh1 (an mmr gene), but no compound mmr gene defects, and a k - ras somatic mutation in the cancer cells. |
pylephlebitis is defined as septic thrombophlebitis of the portal vein or one of its tributaries, usually secondary to suppuration either in the region drained by the portal venous system or in structures contiguous to the portal vein (e.g., the common bile duct). pylephlebitis, that previously well - characterized complication of appendicitis, is rare now, owing to core expeditious surgical management of intra - abdominal infections and the advent of antibiotic therapy. despite this rarity, mortality remains high and the number of case reports dealing with this entity seems to have increased over the past 15 years. the current widespread use of abdominal ultrasonography and ct may, in part, explain the recent increase in reported cases. the 37-year - old man had a 5-days duration of fever, chills, small amount of watery diarrhea and yellowish skin discoloration. his past medical and surgical history was unremarkable, but he recalled an episode of diffuse abdominal pain, nausea and vomiting more than 3 weeks earlier. bowel sounds were slightly increased and minimal right upper and lower quadrant tenderness was presented. peripheral white blood cell count was 11,300/mm (segment neutrophil : 66%, metamyelocyte : 20%). iu / l, gamma - gpt 68 iu / l, alkaline phosphatase 152 iu / l, total - bilirubin 16.79 mg / dl, direct bilirubin 14.30 mg / dl, protein 5.54 g / dl and albumin 2.81 g / dl. abdominal ultrasonography revealed a thrombus in the upper smv, lower extrahepatic portal vein and dilated smv. doppler studies showed antegrade flow in the portal vein and space - occupying lesions in the liver. a contrast - enhanced ct scan confirmed the portal vein thrombosis and also demonstrated superior mesenteric vein thrombosis(fig. dirty fat sign, with streaky infiltration and enlarged lymph nodes, were noted in the right lower abdomen, adjacent to the cecum. an inflammatory condition around the cecum, such as chronic perforated appendicitis, was highly suggested(fig. a small amount of abnormal fluid was seen around the liver, right paracolic gutter and in the pelvis(fig. treatment was done with imipenem alone 0.5g / iv q 6hours. on the 7th hospital day, the patient s condition continued to improve and he was discharged 4 weeks later. follow - up ct scan showed minor residual pericolonic inflammation, resolution of the multiple liver abscess and improvement of the portal vein thrombosis. after 14 days from treatment with imipenem, his bilirubin level was lowered to near - normal level (total - bilirubin 2.5 mg / dl, direct - bilirubin 1.3 mg / dl). pylephlebitis or septic thrombophlebitis of the portal vein, a precursor of liver abscess, is an extremely rare and frequently fatal complication of diverticulitis. in 1846, waller documented autopsy findings in a patient with appendicitis, including multiple liver abscesses and pylephlebitis. he concluded that appendicitis was the direct cause of pylephlebitis and liver abscess. by the turn of the century, the incidence of pylephlebitis following appendicitis gradually diminished with the advent of antibiotics and early surgical treatment. the true incidence of pylephlebitis is difficult to estimate, however, since the diagnosis may be obscured by either the inciting focus of infection or the liver abscesses resulting from portal pyemia, such as acute diverticulitis, intestinal perforation, uterine infection, suppurative pancreatitis and pancreatic abscess formation, splenic abscess, perirectal abscess, hemorrhoid, epididymitis, omphalitis and inflammatory bowel disease. in our case, furthermore, the high morbidity and mortality of suppurative pylephlebitis and intrahepatic abscesses are, at least in part, attributable to the nonspecificity of the symptoms and signs and the limitations of available diagnostic modalities. a clinician must be cognizant of both the nonspecificity of the clinical presentation and the limitations of the sensitivity of these diagnostic modalities. nothing has superseded the necessity for a high index of clinical suspicion regarding these diagnoses. diagnoses are based on clinical features, laboratory findings, blood culture and radiological examinations, or postmortem examination. clinical feature includes fever, right upper quadrant pain, diarrhea, jaundice and hepatomegaly. the clinical pearl that jaundice is rare in pylephlebitis, except in cases complicated by multiple liver abscesses, appears to be valid. jaundice developed late (no earlier than 5 days after the onset of symptoms) since the relative absences of jaundice early in the illness helps distinguish pylephlebitis from ascending escherichia coli, followed by proteus mirabilis, klesiella pneumoniae, enterobacer species, pseudomonas species and gram - positive cocci (staphylococcus aureus, streptococcus species). more recently, coloenteric anaerobic bacteria (clostridia, bacteroides, fusobacterium and anaerobic streptococcus species) have been isolated with increasing frequency, as a consequence of both improved culture techniques and selective pressure by the prevalent use of antibiotics. in our case, modern radiological imaging techniques provide supportive diagnostic evidence. with regard to diagnostic imaging in pylephlebitis, the current series suggests that ultrasonography is a useful modality for demonstrating portal vein thrombosis. ct scanning also shows promise in this regard, and may be less operator - dependent than ultrasonography. in the setting of probable intra - abdominal infection, ct scanning may be the most reasonable initial choice for imaging, given its proven ability to detect not only thrombi but pericolonic abscesses as well. early in the disease process, hepatic infection and inflammation are microscopic, and contrast ct may demonstrate patchy areas of attenuated hepatic parenchyma. later in the clinical course, parenchymal hepatic infection may coalesce to form multiple microabscess, or a single macroabscess amenable to percutaneous ct - guided drainage. also in our case, inhomogenous patchy infiltration in the hepatic parenchyme was observed at the time of diagnosis, and resolution of these lesions were observed in follow - up film. although modern imaging techniques have allowed the early diagnosis of pylephlebitis, the patient s history and physical examination still provide clues to the diagnosis. on the basis of treatments, it appears that empirical antibiotic therapy for a patient with suspected pylephlebitis should include broad coverage for enteric facultative gram - negative bacilli and agents active against anaerobes, especially b. fragilis, and coverage for aerobic streptococcus species. the ideal duration of antibiotic therapy for pylephlebitis is unclear. given the frequency of liver abscess as a complication of pylephlebitis, however, a minimum 4 weeks of therapy seems prudent since developing abscesses may not be visualized on ct scans. patients with demonstrated macroscopic liver abscess complicating pylephlebitis should probably receive at least 6 weeks of antibiotic therapy, with or without drainage. in our case, imipenem (2.0g / d) monotherapy was performed for 2 weeks and resulted in complete resolution of hepatic lesions. no further treatment was performed. the role of anticoagulation in the treatment of pylephlebitis is controversial. in prior writings, all of these opinions appear to have been based on limited data and personal experience, since no formal study of anticoagulation in pylephlebitis has ever been done. despite such evidence that heparin may not be critical for the survival of patients with pylephlebitis, the possibility exists that anticoagulation might benefit some patients by decreasing the chance of septic embolization to the liver from infected portal thrombi and pulmonary emboli. surgical intervention for pylephlebitis typically involves opening and drainage of the focus of infection, separation of large vessels of the portal system from the focus of infection (i.e., pericolonic or hepatic abscess) rather than surgery on the infected vessels themselves. in our patient, a previously healthy patient presented with nonspecific symptoms and fever. bacteroides fragilis was grown from blood, which clearly suggested an intra - abdominal origin. appendicitis was the most likely cause of sepsis, confirmed by abdominal ct, with seeding to the portal vein and liver. no surgical intervention was performed, since the intra - abdominal phlegmonous process responded to antibiotic therapy. outcome depends on multiple factors, including age greater than 70 years, multiple liver abscess, combination of hepatic abscess and pylephlebitis, concomitant intrahepatic and extrahepatic abscesses, usually subphrenic, deterioration of liver function due to intrahepatic infection, particularly jaundice, and hypoalbuminemia. although radiological imaging studies (e.g. contrast - enhanced abdominopelvic ct scan) facilitates diagnoses, early suspicion and prompt antibiotic therapy can lead to resolution of portal vein thrombosis and multiple liver abscesses thus resulting in recovery. in conclusion, pylephlebitis is an uncommon complication of suppurative infections (most often diverticulitis) in the portal drainage area. although rare, pylephlebitis is a treatable but often lethal complication of intra - abdominal sepsis, pylephlebitis should be suspected in patients with intra - abdominal sepsis with liver function abnormalities. the timely administration of broad - spectrum antibiotics appears to be the most critical component of therapy for pylephlebitis. | pylephlebitis usually occurs secondary to infection in the region drained by the portal venous system. a most common antesecent focus of infection is diverticulitis and the most common blood isolate is e. coli (54%), followed by proteus mirabilis (23%). overall mortality is 32% and most of the patients who had died had severe sepsis prior to the initiation of antibiotic therapy. we describe a case of pylephlebitis which had appendicitis and consequent septic thrombosis of the portal vein and its branches, with dissemination of infection to the liver. the patient had recovered due to timely antibiotic treatment alone and resulted in complete resolution. early diagnosis and treatment are basic to a favorable clinical course. |
prostate cancer is the most commonly diagnosed cancer in men in the united states and the second most commonly diagnosed malignancy worldwide. although many prostate cancer patients have localized disease at the time of diagnosis, some present with evidence of metastasis. in the latter cases, androgen deprivation therapy (adt) although this therapy is very effective initially, the disease eventually progresses in all patients and becomes resistant to treatment, which is also known as castration - resistant prostate cancer (crpc). 3-hydroxy-3-methyl - glutaryl - coa reductase inhibitors, commonly known as statins, are highly effective in lowering cholesterol levels and reducing the risk of cardiovascular disease. however, statins can also modify the cholesterol levels needed for signal transduction and have shown an effect on prostate cancer cells. statins are thought to modulate androgen receptor expression and activity, which may reduce the proliferation of prostate cancer cells and induce apoptosis. statins may also reduce the levels of prostate - specific antigen (psa) released by prostate cancer cells. a number of epidemiological studies have shown a relationship between statin use and lower cancer risk and mortality, including in prostate cancer. in many studies, statin use has shown an antitumor effect in prostate cancer, decreasing the risk of recurrence and prostate cancer mortality. in patients who have undergone radical in addition, statins are associated with reduced mortality rates in prostate cancer patients who have been treated with radiation therapy. the aim of our current study was to determine whether statins have an effect on prostate cancer patients who can not be treated with standard definitive therapies owing to the extent of disease. we investigated whether statin use delays the development of crpc in metastatic prostate cancer patients who had been treated with adt. this study was performed with the approval and oversight of the institutional review board (irb) of asan medical center (irb no. a database of prostate cancer patients who already had metastasis and were treated with adt between january 1997 and december 2013 at asan medical center was retrospectively analyzed. because of the retrospective nature of this analysis, the requirement for informed consent was waived by the irb. a total of 196 patients who had metastatic prostate adenocarcinoma at the time of diagnosis and had been treated with adt and eventually progressed to crpc were selected for analysis. the patient characteristics we assessed included age at diagnosis, diabetes mellitus, hypertension, body mass index (bmi), gleason score for a prostate biopsy, and initial psa level before adt. clinicopathological characteristics were compared between patients exposed or not to statins by use of the chi - square test and student t - test. cox proportional hazards models were used to estimate hazard ratios (hrs) with a 95% confidence interval (ci) for crpc occurrence to determine the effect of statin use on the time to crpc onset. this study was performed with the approval and oversight of the institutional review board (irb) of asan medical center (irb no. a database of prostate cancer patients who already had metastasis and were treated with adt between january 1997 and december 2013 at asan medical center was retrospectively analyzed. because of the retrospective nature of this analysis, the requirement for informed consent was waived by the irb. a total of 196 patients who had metastatic prostate adenocarcinoma at the time of diagnosis and had been treated with adt and eventually progressed to crpc were selected for analysis. the patient characteristics we assessed included age at diagnosis, diabetes mellitus, hypertension, body mass index (bmi), gleason score for a prostate biopsy, and initial psa level before adt. clinicopathological characteristics were compared between patients exposed or not to statins by use of the chi - square test and student t - test. cox proportional hazards models were used to estimate hazard ratios (hrs) with a 95% confidence interval (ci) for crpc occurrence to determine the effect of statin use on the time to crpc onset. data were analyzed by using the ibm spss statistics ver. 21.0 (ibm co., armonk, ny, usa). the clinicopathologic characteristics of all the prostate cancer subjects according to statin use are summarized in table 1. the patients ' mean age at diagnosis was 67.1 years (standard deviation, 9.1 years), with a mean follow - up time of 52 months. of the 171 patients evaluated, 132 died of prostate cancer, with a mean survival time after diagnosis of 48.6 months. there were no statistically significant differences in the prevalence of comorbidities such as diabetes mellitus or hypertension between the groups. among patients with diabetes mellitus, 15 patients (48%) had metformin in their medication : 5 patients (10%) in the statin group and 10 patients (8%) in the non statin group. cross - tabulation analysis showed no statistical difference in the use of metformin between the two groups (p=0.879). among patients with hypertension, an angiotensin converting enzyme (ace) inhibitor was included in three patients and a beta - blocker was included in eight patients ; one patient used both an ace inhibitor and a beta - blocker. cross - tabulation was also done and showed no statistical difference between the two groups for the above medications (p=0.393). furthermore, there were no significant differences between the study groups in terms of the gleason score for prostate cancer or the psa level before treatment (p=0.547, p=0.681). also, there was a significant difference in the time to development of crpc between statin users and nonusers with a median time of 30.5 and 22.7 months, respectively (p=0.016). a total of 23 patients (13%) did not receive chemotherapy and 33 patients had other treatments such as mitoxantrone, enzalutamide, and cyclophosphamide vincristine dexamethasone combination therapy. docetaxel was used in 69% of the patients in the statin group and in 67% of the patients in the nonstatin use group. cross - tabulation analysis showed no significant difference in treatment method after crpc between the two groups (p=0.701). the results of our primary analysis using univariate and multivariate hr models with cox regression analysis are listed in tables 3, 4. in addition, the presence of diabetes mellitus was associated with adt duration before crpc (hr, 1.573 ; 95% ci, 1.02 - 2.43 ; p=0.041). using statins was associated with a longer adt duration (hr, 0.61 ; 95% ci, 0.41 - 0.91 ; p=0.015), with a difference in the crpc - free survival curve as shown in fig. 1 (p=0.044). similar to adt duration, the gleason score, diabetes mellitus, and statin use were associated with cancer - specific survival. a higher gleason score was associated with increased risk of cancer - specific mortality (p=0.002). diabetes mellitus was also associated with increased risk (hr, 1.641 ; 95% ci, 1.03 - 2.61 ; p=0.037), and statin users showed a lower risk of cancer - specific mortality than did nonusers (hr, 0.405 ; 95% ci, 0.25 - 0.66 ; p<0.001) and also showed a significant difference in the survival curve as shown in fig. 2 (p=0.001). in addition, a longer duration of adt use was associated with a lower cancer - specific mortality rate (hr, 0.909 ; 95% ci, 0.89 - 0.93 ; p<0.001). the antitumor effects of statins in prostate cancer patients have been reported in many previous studies. in our current study, we evaluated the association between statin use and prognosis of metastatic prostate cancer because statins are known to reduce the risk of advanced prostate cancer. previous studies have reported that the effects of statins on prostate cancer are linked to several pathways related to cancer development and growth. the results of our current study of metastatic prostate cancer patients indicate that the use of statins is associated with delay in the development of crpc, even though all patients eventually developed crpc. one explanation for this result may be that statin users may have more favorable prostate cancer characteristics and better cancer outcomes. in our present analysis the possibility of healthy - user bias is another important factor that should be considered. statin users may be more focused on their health and thus may have better lifestyle habits, which could have an effect on prostate cancer survival. also, we noted a reduced hr of 59.5% associated with statin use, which suggests that factors other than lifestyle habits play a role in the effects of statins. although other confounding factors may have been present in our study cohort, statin users had a higher bmi and higher cholesterol levels, which may be associated with poorer survival and may weaken the effects of the statins. nevertheless, our current results show that statin use may decrease prostate cancer mortality and delay the development of crpc. statins are also known to lower serum psa levels, which may influence the development and metastasis of prostate cancer. however, in our current study, we saw no significant difference in psa levels between the groups : 515 ng / ml and 559 ng / ml in the nonstatin user group and the statin user group, respectively. the discrepancy among studies in this regard may be due to differences in the patient populations. our present patients all had metastatic prostate cancer at the time of diagnosis, and therefore the burden of prostate cancer may have been higher in this group than in previously reported cohorts. statins inhibit 3-hydroxy-3-methylglutaryl (hmg) coenzyme a reductase and limit cholesterol biosynthesis by affecting the pathway that converts hmg coenzyme a to mevalonate, eventually reducing the level of mevalonate. the lower level of cholesterol in the blood may limit cholesterol use in cells and modulate the signal pathway associated with cancer apoptosis, such as akt signaling in prostate cancer cells. reduced mevalonate levels lead to deprivation of isoprenoids and thus limit the activation of proteins such as ras and rho in the cell membrane, eventually decreasing their growth promotion and survival activities. according to this hypothesis, statins may have an effect in patients without hypercholesterolemia. in our present study, however, we did not assess any biochemical markers that could be used to support this mechanism. the duration of adt was also found to be associated with prostate cancer survival, with a longer duration of adt associated with a lower risk of cancer - specific mortality. a linear correlation was observed between the duration of adt and the survival time after the initial diagnosis of prostate cancer, as shown by simple correlation analysis (fig. 3). this may be because patients who received adt for a longer duration may have had better pathologic features. however, the duration of adt also showed statistically significant associations with factors that influenced cancer - specific survival in our multivariate analysis. diabetes was found to be a statistically significant risk factor for both crpc and cancer - specific survival. diabetic patients usually have a higher level of morbidity than do nondiabetic patients, and this may also have an effect on survival ; however, the difference in the duration of adt owing to diabetic status is likely to be attributable to more than just lifestyle differences. several studies have shown that diabetes is a risk factor for cancer, including prostate cancer, while other studies have reported that diabetes is a protective factor for prostate cancer. the use of glucose - lowering agents such as metformin may also affect prostate cancer development and prognosis. the relationship between diabetes and prostate cancer is not yet clear, and further studies on this subject are currently underway although all of our study patients presented with bone metastasis, some patients also had visceral metastasis at the time of diagnosis. the patients who presented with visceral metastasis were expected to have a poorer prognosis and shorter time to crpc onset, likely as a result of a higher tumor burden than in patients who presented with only a bone metastasis. surprisingly, however, we found no statistically significant difference in the risk of crpc and cancer - specific survival in patients with an additional visceral metastasis. initial psa levels before treatment also did not significantly differ between patients with only bone metastasis and patients with a visceral metastasis also. this may indicate that a combination of visceral and bone metastases does not correlate with a higher tumor burden and a poorer prognosis. there were some limitations of note to our study, mainly due to the retrospective nature of the analyses. for example, there may have been gaps in our data, including comorbidities and statin use, owing to an incomplete reporting of medical histories. records of any existing medications were obtained by querying the patients at the time of admission. the duration of medication use before admission was calculated by using the date provided in the medical records, but the degree of patient compliance with those medication regimens is unknown, which may have biased our calculations. also, when this study was conducted, only 19 patients (30%) in the statin user group were alive to respond to phone calls about duration of statin use. therefore, data on total statin duration and the accumulative dose of statins were not appropriately obtained or analyzed. there may also have been data gaps for statin use if any patients failed to report their use. however, such a bias would result in statin users being included in the nonstatin user group, which would weaken the measured effects of statins on prostate cancer. the use of statins is associated with a delayed development of crpc and also with lower prostate cancer - specific survival. although statin use may also be associated with a decreased risk of prostate cancer mortality, additional prospective evaluations of the impact of statins are needed. | purposeto determine whether statin use delays the development of castration - resistant prostate cancer (crpc) in patients with metastatic prostate cancer treated with androgen deprivation therapy (adt).materials and methodsa total of 171 patients with metastatic prostate cancer at the time of diagnosis who were treated with adt between january 1997 and december 2013 were retrospectively analyzed. the patients were classified into two groups : the nonstatin use group (a group) and the statin use group (b group). multivariate analysis was performed on statin use and other factors considered likely to have an effect on the time to progression to crpc.resultsthe mean patient age was 67.19.1 years, and the mean follow - up period was 52 months. the mean initial prostate - specific antigen (psa) level was 537 ng / ml. of the 171 patients, 125 (73%) were in group a and 46 (27%) were in group b. the time to progression to crpc was 22.7 months in group a and 30.5 months in group b, and this difference was significant (p=0.032). blood cholesterol and initial psa levels did not differ significantly according to the time to progression to crpc (p=0.288, p=0.198). multivariate analysis using the cox regression method showed that not having diabetes (p=0.037) and using a statin (p=0.045) significantly increased the odds ratio of a longer progression to crpc.conclusionsstatin use in metastatic prostate cancer patients appears to delay the progression to crpc. large - scale, long - term follow - up studies are needed to validate this finding. |
sarcoidosis is an idiopathic multisystem granulomatous disease that commonly involves the lungs, eyes, lymph nodes and skin. the disease usually begins at around 40 years of age and nearly two - thirds of the cases are females. sarcoidosis confined to skin is quite uncommon as reported in literature, contrary to the latest scenario. because lesions assume a vast array of morphologies, cutaneous sarcoidosis is known as one of the great imitators in dermatology. we report three cases of cutaneous sarcoidosis without systemic involvement with varied presentation with regard to age and morphologies. a 28-year - old female presented with a complaint of itchy skin lesions over the face and extremities. initially, the lesions started on the elbow and leg, which was diagnosed as lichen planus and treated with topical steroid. cutaneous examination revealed multiple, discrete, erythematous papules [figure 1 ] along with flat - topped, violaceous papules and plaques, morphologically classical of lichen planus [figure 2 ] lesions over the face, forearm, abdomen and leg. case 1 erythematous papules of sarcoidosis over face case 1 violaceous lichenoid papules of sarcoidosis over elbow skin biopsy from the face and elbow lesion revealed classical noncaseating epithelioid cell granulomas of sarcoidal type involving the entire dermis [figure 3 ]. serum calcium, liver function tests (lft), renal function tests (rft), ultrasonogram (usg) abdomen and chest x - ray were normal case 1 h and e low power showing noncaseating granuloma extending till basal cell layer ; (inset) high power showing epithelioid granuloma consisting of langhan 's giant cell case 1 reticulum stain showing intact black reticulin fibers the patient was treated with hydroxy - chloroquine 200 mg bid for 3 months. the erythematous lesions started resolving but there was an increase in the number and size of the flat - topped, violaceous colored skin lesions. the repeat skin biopsy of these lesions revealed the classical histopathologic features of lichen planus, enabling us to come to the final diagnosis of cutaneous sarcoidosis and lichen planus overlap. lesions responded to oral prednisolone with a starting dose of 1 mg per kg body weight, tapered over a period of 5 weeks, with complete resolution without any recurrence for 6 months post - treatment. a 52-year - old female presented with multiple reddish skin lesions over the face and extremities of 1 year duration, which increase in size and number. there was no history of itching or sudden exacerbation and remission with trauma, sunlight, stress, infection or medication. the case was treated as psoriasis with emollients and topical steroid by a few dermatologists and the patient presented to us since the lesions did not resolve. on examination, multiple, well - defined, discrete and confluent erythematous papules and plaques with a few polycyclic lesions were seen over the extensor aspects of limbs, face, neck and scalp [figures 5 and 6 ]. the morphology of the lesions was classical of psoriatic lesions, but auspitz sign was negative. the skin biopsy revealed epithelioid cell, noncaseating, naked granulomas, mainly in the mid and lower dermis, with multinucleate giant cells and sparse lymphocytic infiltrate [figure 7 ]. case 2 erythematous psoriasiform lesion of sarcoidosis over forearms case 2 erythematous papules and plaques with scales of sarcoidosis over neck, back and scalp case 2 h and e low power showing noncaseating granuloma ; (inset) high power showing epithelioid granuloma consisting of langhans giant cell, epithelioid cell and sparse lymphocytes case 2 reticulum stain showing intact black reticulin fibers routine blood and urine tests along with serum calcium, ace levels, usg abdomen and chest x - ray were normal. a final diagnosis of psoriasiform cutaneous sarcoidosis was made. the patient was treated with hydroxyl - chloroquin 200 mg orally bid, combined with a short course of low - dose oral prednisolone initially for the first 3 weeks only. the lesions started resolving after 1 month of initiating the treatment ; however, the patient was lost for follow - up later. a 15 year - old boy from kolar gold fields, karnataka, presented with multiple asymptomatic lesions of 6 months duration over the face. there was no history of fever, joint pain, cough weight loss, joint pains, eye complaints or any other systemic complaints. dermatologic examination revealed multiple skin colored to reddish - brown, translucent papules, measuring 2 - 5 mm, on the cheeks, nose, upper lip, chin and forehead [figure 9 ]. lesions were non - tender. palms, soles, hair, nail and mucosal areas were uninvolved. differential diagnosis of acne, histoid hansen 's disease, and trichoepithelioma were considered and investigated further. papular lesions on face of case 3 ; (inset) grouped papules over chin investigations revealed raised serum ace (118 u / l) level, negative mantoux test, and lft, serum calcium, serum creatinine and blood urea were normal. venereal disease research laboratory test (vdrl) was nonreactive ; skin slit smear for acid fast bacilli was negative. radiologic examination of chest, hands and feet, ultrasound abdomen and lung function tests revealed no abnormality. the granulomas predominantly composed of epithelioid cells, with few langhan 's giant cells and sparse lymphocytic infiltrate (naked granulomas) [figure 10 ]. special stains for acid fast bacilli (afb), ld bodies, fungi and calcium were negative. case 3 h and e low power showing noncaseating granuloma ; (inset) high power showing epithelioid granuloma consisting of giant cell, epithelioid cell and sparse lymphocytes case 3 special stain low power showing intact black reticulin fibers the patient was treated with hydroxychloroquine 200 mg od and topical fluticasone. the patient responded with near- complete resolution at the end of 4 months treatment [figure 12 ]. a 28-year - old female presented with a complaint of itchy skin lesions over the face and extremities. initially, the lesions started on the elbow and leg, which was diagnosed as lichen planus and treated with topical steroid. cutaneous examination revealed multiple, discrete, erythematous papules [figure 1 ] along with flat - topped, violaceous papules and plaques, morphologically classical of lichen planus [figure 2 ] lesions over the face, forearm, abdomen and leg. case 1 erythematous papules of sarcoidosis over face case 1 violaceous lichenoid papules of sarcoidosis over elbow skin biopsy from the face and elbow lesion revealed classical noncaseating epithelioid cell granulomas of sarcoidal type involving the entire dermis [figure 3 ]. serum calcium, liver function tests (lft), renal function tests (rft), ultrasonogram (usg) abdomen and chest x - ray were normal case 1 h and e low power showing noncaseating granuloma extending till basal cell layer ; (inset) high power showing epithelioid granuloma consisting of langhan 's giant cell case 1 reticulum stain showing intact black reticulin fibers the patient was treated with hydroxy - chloroquine 200 mg bid for 3 months. the erythematous lesions started resolving but there was an increase in the number and size of the flat - topped, violaceous colored skin lesions. the repeat skin biopsy of these lesions revealed the classical histopathologic features of lichen planus, enabling us to come to the final diagnosis of cutaneous sarcoidosis and lichen planus overlap. lesions responded to oral prednisolone with a starting dose of 1 mg per kg body weight, tapered over a period of 5 weeks, with complete resolution without any recurrence for 6 months post - treatment. a 52-year - old female presented with multiple reddish skin lesions over the face and extremities of 1 year duration, which increase in size and number. there was no history of itching or sudden exacerbation and remission with trauma, sunlight, stress, infection or medication. the case was treated as psoriasis with emollients and topical steroid by a few dermatologists and the patient presented to us since the lesions did not resolve. on examination, multiple, well - defined, discrete and confluent erythematous papules and plaques with a few polycyclic lesions were seen over the extensor aspects of limbs, face, neck and scalp [figures 5 and 6 ]. the morphology of the lesions was classical of psoriatic lesions, but auspitz sign was negative. the skin biopsy revealed epithelioid cell, noncaseating, naked granulomas, mainly in the mid and lower dermis, with multinucleate giant cells and sparse lymphocytic infiltrate [figure 7 ]. case 2 erythematous psoriasiform lesion of sarcoidosis over forearms case 2 erythematous papules and plaques with scales of sarcoidosis over neck, back and scalp case 2 h and e low power showing noncaseating granuloma ; (inset) high power showing epithelioid granuloma consisting of langhans giant cell, epithelioid cell and sparse lymphocytes case 2 reticulum stain showing intact black reticulin fibers routine blood and urine tests along with serum calcium, ace levels, usg abdomen and chest x - ray were normal. a final diagnosis of psoriasiform cutaneous sarcoidosis was made. the patient was treated with hydroxyl - chloroquin 200 mg orally bid, combined with a short course of low - dose oral prednisolone initially for the first 3 weeks only. the lesions started resolving after 1 month of initiating the treatment ; however, the patient was lost for follow - up later. a 15 year - old boy from kolar gold fields, karnataka, presented with multiple asymptomatic lesions of 6 months duration over the face. there was no history of fever, joint pain, cough weight loss, joint pains, eye complaints or any other systemic complaints. dermatologic examination revealed multiple skin colored to reddish - brown, translucent papules, measuring 2 - 5 mm, on the cheeks, nose, upper lip, chin and forehead [figure 9 ]. lesions were non - tender. palms, soles, hair, nail and mucosal areas were uninvolved. differential diagnosis of acne, histoid hansen 's disease, and trichoepithelioma were considered and investigated further. papular lesions on face of case 3 ; (inset) grouped papules over chin investigations revealed raised serum ace (118 u / l) level, negative mantoux test, and lft, serum calcium, serum creatinine and blood urea were normal. venereal disease research laboratory test (vdrl) was nonreactive ; skin slit smear for acid fast bacilli was negative. radiologic examination of chest, hands and feet, ultrasound abdomen and lung function tests revealed no abnormality. the granulomas predominantly composed of epithelioid cells, with few langhan 's giant cells and sparse lymphocytic infiltrate (naked granulomas) [figure 10 ]. special stains for acid fast bacilli (afb), ld bodies, fungi and calcium were negative. case 3 h and e low power showing noncaseating granuloma ; (inset) high power showing epithelioid granuloma consisting of giant cell, epithelioid cell and sparse lymphocytes case 3 special stain low power showing intact black reticulin fibers the patient was treated with hydroxychloroquine 200 mg od and topical fluticasone. the patient responded with near- complete resolution at the end of 4 months treatment [figure 12 ]. a 28-year - old female presented with a complaint of itchy skin lesions over the face and extremities. initially, the lesions started on the elbow and leg, which was diagnosed as lichen planus and treated with topical steroid. cutaneous examination revealed multiple, discrete, erythematous papules [figure 1 ] along with flat - topped, violaceous papules and plaques, morphologically classical of lichen planus [figure 2 ] lesions over the face, forearm, abdomen and leg. case 1 erythematous papules of sarcoidosis over face case 1 violaceous lichenoid papules of sarcoidosis over elbow skin biopsy from the face and elbow lesion revealed classical noncaseating epithelioid cell granulomas of sarcoidal type involving the entire dermis [figure 3 ]. serum calcium, liver function tests (lft), renal function tests (rft), ultrasonogram (usg) abdomen and chest x - ray were normal case 1 h and e low power showing noncaseating granuloma extending till basal cell layer ; (inset) high power showing epithelioid granuloma consisting of langhan 's giant cell case 1 reticulum stain showing intact black reticulin fibers the patient was treated with hydroxy - chloroquine 200 mg bid for 3 months. the erythematous lesions started resolving but there was an increase in the number and size of the flat - topped, violaceous colored skin lesions. the repeat skin biopsy of these lesions revealed the classical histopathologic features of lichen planus, enabling us to come to the final diagnosis of cutaneous sarcoidosis and lichen planus overlap. lesions responded to oral prednisolone with a starting dose of 1 mg per kg body weight, tapered over a period of 5 weeks, with complete resolution without any recurrence for 6 months post - treatment. a 52-year - old female presented with multiple reddish skin lesions over the face and extremities of 1 year duration, which increase in size and number. there was no history of itching or sudden exacerbation and remission with trauma, sunlight, stress, infection or medication. the case was treated as psoriasis with emollients and topical steroid by a few dermatologists and the patient presented to us since the lesions did not resolve. on examination, multiple, well - defined, discrete and confluent erythematous papules and plaques with a few polycyclic lesions were seen over the extensor aspects of limbs, face, neck and scalp [figures 5 and 6 ]. the morphology of the lesions was classical of psoriatic lesions, but auspitz sign was negative. the skin biopsy revealed epithelioid cell, noncaseating, naked granulomas, mainly in the mid and lower dermis, with multinucleate giant cells and sparse lymphocytic infiltrate [figure 7 ]. case 2 erythematous psoriasiform lesion of sarcoidosis over forearms case 2 erythematous papules and plaques with scales of sarcoidosis over neck, back and scalp case 2 h and e low power showing noncaseating granuloma ; (inset) high power showing epithelioid granuloma consisting of langhans giant cell, epithelioid cell and sparse lymphocytes case 2 reticulum stain showing intact black reticulin fibers routine blood and urine tests along with serum calcium, ace levels, usg abdomen and chest x - ray were normal. the patient was treated with hydroxyl - chloroquin 200 mg orally bid, combined with a short course of low - dose oral prednisolone initially for the first 3 weeks only. the lesions started resolving after 1 month of initiating the treatment ; however, the patient was lost for follow - up later. a 15 year - old boy from kolar gold fields, karnataka, presented with multiple asymptomatic lesions of 6 months duration over the face. there was no history of fever, joint pain, cough weight loss, joint pains, eye complaints or any other systemic complaints. dermatologic examination revealed multiple skin colored to reddish - brown, translucent papules, measuring 2 - 5 mm, on the cheeks, nose, upper lip, chin and forehead [figure 9 ]. differential diagnosis of acne, histoid hansen 's disease, and trichoepithelioma were considered and investigated further. papular lesions on face of case 3 ; (inset) grouped papules over chin investigations revealed raised serum ace (118 u / l) level, negative mantoux test, and lft, serum calcium, serum creatinine and blood urea were normal. venereal disease research laboratory test (vdrl) was nonreactive ; skin slit smear for acid fast bacilli was negative. radiologic examination of chest, hands and feet, ultrasound abdomen and lung function tests revealed no abnormality. the granulomas predominantly composed of epithelioid cells, with few langhan 's giant cells and sparse lymphocytic infiltrate (naked granulomas) [figure 10 ]. special stains for acid fast bacilli (afb), ld bodies, fungi and calcium were negative. case 3 h and e low power showing noncaseating granuloma ; (inset) high power showing epithelioid granuloma consisting of giant cell, epithelioid cell and sparse lymphocytes case 3 special stain low power showing intact black reticulin fibers the patient was treated with hydroxychloroquine 200 mg od and topical fluticasone. the patient responded with near- complete resolution at the end of 4 months treatment [figure 12 ]. sarcoidosis commonly affects the lungs, lymph nodes, liver, eyes, bones and skin. hutchinson recorded the first case in 1865, but first unequivocal case of sarcoidosis in english literature was reported by boeck in 1899. it occurs worldwide with the highest prevalence in scandinavia and the prevalence was 20/100,000 in 1975 but recent report says it to be 64/100,000. only 300 cases of sarcoidosis were reported in our country till 1986 but a recent report says there is an increased prevalence. the authors are of the strong opinion that this increase in incidence is because of increased awareness about the not so uncommon occurrence of sarcoidosis in india in the recent times. missed diagnosis due to lack of clinical suspicion, better understanding and newer investigative modalities have also contributed. in patients with systemic disease, however ; cutaneous lesions are the sole manifestation of sarcoidosis in approximately 10% of patients. among the numerous reported morphologic presentations are papules, micropapules, plaques, subcutaneous nodules, scar sarcoidosis, lupus pernio, erythema nodosum, ulcer and alopecia. multiple other cutaneous morphologies are possible ; many are quite rare, although the incidence is difficult to determine. papules of sarcoidosis may be of various colors, including red, reddish - brown, violaceous, translucent, or hyperpigmented. because of this varied color, it mimics many common conditions including violaceous color of lichen planus. case 1 is rare example where lesions exhibited violaceous color which was associated with pruritus and the histopathology of the differently colored lesions was different (lichen planus and sarcoidosis). rare presentations like follicular, verrucous, ichthyosiform, hypomelanotic, and annular lesions have also been described. the disease usually begins at around 40 years of age and nearly two - thirds of the cases are females. cutaneous sarcoidosis has been reported rarely from india and incidence in children is much lesser. also, in all these reports, lesions of skin were associated with some forms of systemic involvement. the age of onset in case 3 of this report is 14 years, which is rare as per the existing reports. sarcoidosis is a great imitator of other dermatologic diseases because the cutaneous manifestations are quite variable and occur in both localized as well as generalized forms. greater awareness about the not so rare occurrence of sarcoidosis and its varied manifestations mimicking the most common dermatologic conditions like psoriasis (as in case 2) and its association with common conditions like lichen planus (as in case 1) | it is a known fact that cutaneous sarcoidosis is a great imitator in dermatology. we report three cases of cutaneous sarcoidosis without systemic involvement and with varied dermatologic presentation with regard to age and morphology. lesions mimicked various common dermatologic conditions, causing great confusion for the diagnosis and posing problems for management. awareness of these varied morphologic presentations is essential for the early diagnosis and management of the master mimicker - cutaneous sarcoidosis. |
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