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overweight and obesity accompanied by low levels of physical activity are a widespread and growing global problem. worldwide, the proportion of overweight and obese adults has increased in the last 20 years and continues to increase in children and adolescents in developed and developing countries. in a large us survey, the prevalence of obesity was reported to be 36% in adults. in the uk, childhood obesity is increasing in the world health organization (who) european region, and in slovenia 's capital of ljubljana overweight and obesity in children have become an epidemic in 20 years spanning from 1991 to 2011. in canada, one - quarter of children and youth is overweight or obese, with only 7% attaining the 60 minutes of moderate to vigorous physical activity every day recommended by canadian guidelines. similarly, only 15% of canadian adults attain their recommended level of physical activity, and 61% of canadian men and 44% of canadian women are overweight or obese. lack of physical activity is a risk factor for cardiovascular disease, type 2 diabetes, osteoporosis, depression, anxiety, and some cancers, all chronic diseases that are routinely managed by family physicians. sedentary lifestyle leads to morbidity and mortality for individuals and economic and resource cost for society. although substantial attention has been paid to promoting healthy lifestyles through individual clinical counselling and mass education programs, activity levels have remained low. one of the most effective means of encouraging physical activity is through urban planning and transportation policies. urban planning, such as higher residential density, mixed - use zoning, street connectivity, and increased green space, have all been shown to increase physical activity and decrease body mass index [12, 13 ]. transportation policies, such as increased public transportation usage, reduced car use, and more infrastructure for walking and cycling, can achieve the same results. family physicians are well positioned to educate patients on and advocate for a healthy built environment. given family physicians ' long - standing relationships with patients and their role in chronic disease prevention at the individual level, there may be ethical and financial accountability for physicians to take on a larger health advocacy role. the world most recognized and applied physician competency framework, canmeds, identified that society believes that a fundamental role of physicians is to act as health advocates at the societal level. with family physicians comprising half of all physicians in canada, they and their professional associations have great potential to advocate for health considerations in future planning decisions. family physicians ' knowledge and attitudes about their role in educating patients and advocating for healthy built environments and the perceived barriers for doing so are unclear. therefore, this study sought to measure these constructs in family medicine residents in canada. as recent graduates of medical school, this population provides a good representation of current medical education. an english - language web - based survey of all 353 family medicine residents in postgraduate year one or year two at the university of toronto (u of t) was conducted from january to february 2013. built environment was defined as physical surroundings (i.e., buildings, parks, schools, road systems, and other infrastructures) that we encounter in our daily lives. the survey was designed to address three constructs related to residents educating patients on and advocating for healthy built environment : (1) perceived knowledge, (2) attitudes, and (3) perceived barriers. it was developed based on an extensive literature review of the health impacts of the built environment. questions to assess perceived knowledge and confidence were developed based on well - researched, high impact topics considered as a reasonable level of knowledge for family physicians. the questionnaire consisted of 21 items rated on a five - point likert scale (strongly disagree, disagree, neither agree nor disagree, agree, or strongly agree). it also solicited information on relevant demographic information, preferred ways to learn about built environment, and preferred methods to advocate for healthy built environments and educate patients about this topic. the emails stated the purpose of the study, ensured anonymity, and promised a random drawing for gift certificates as an incentive. using a modified dillman 's method, two reminder emails were sent at one - week intervals. upon the completion of the survey, participants had the opportunity to enter their email address on a separate data collection form for the prize draw. data were entered into spss 19 for windows (ibm corp., armonk, new york). descriptive statistics (mean, median, and standard deviation) were calculated to characterize the demographics of the respondents. to simplify the presentation of results strongly agree and agree into one category, strongly disagree and disagree into a second category, and leaving neither agree nor disagree as a third category. three hundred fifty - three individuals received the online survey ; 151 surveys were initiated and 140 were completed in full, yielding a response rate of 40%. seventy - two percent (n = 110) of completed surveys were by females, close to the 65% in the sample frame. nine percent (n = 14) of respondents were less than 25 years old, 70% (n = 106) were between 25 and 30 years, and 21% were (n = 31) older than 30 years. fifty - four percent (n = 82) of respondents were first year residents, and 46% (n = 69) were in second year. the respondents completed medical school at 14 of the 17 canadian schools. while medical curricula may vary across canada, homogeneity and quality of graduates are guaranteed through independent national organizations responsible for accreditation and certification processes. fifteen percent (n = 23) of respondents were non - canadian medical graduates. residents ' attitudes and perceived knowledge and confidence of educating patients on and advocating for healthy built environment are reported in tables 1 and 2, respectively. briefly, 64.1% (n = 91) and 77.5% (n = 110) of respondents, respectively, indicated that they agreed or strongly agreed with the statements educating patients on the health impact of the built environment is an effective disease prevention activity and advocating for healthy built environments is an effective disease prevention activity. it is part of the family physician 's role to educate patients on the built environment and it is part of the family physician 's role to advocate for healthy built environments (n = 74, 52.2%, and n = 79, 55.7%, resp.). it is not the role of family physicians but rather organizations such as the canadian medical association / ontario medical association (cma / oma) to educate the public on the health impact of the built environment, and 38.0% (n = 54) felt that it is not the role of family physicians but rather organizations such as the cma / oma to advocate for healthy built environments. one - third (n = 51, 35.9%) perceived that educating patients on the impact of the built environment is an effective use of family physicians ' time. at the individual patient level, slightly more than half of the respondents (n = 82, 57.3%) agreed or strongly agreed that they are well trained to consider a patient 's living and work environments as a determinant of their health. at the population level, only 35.0% (n = 50) agreed or strongly agreed that they feel knowledgeable enough to participate in community discussions on how to improve the health of the local population through changes to the built environment. similarly, only one - third (n = 43, 30.1%) perceived they were knowledgeable enough to contribute to a discussion on how urban planning and transportation policies affect health, while half (n = 70, 49.0%) of the respondents disagreed with this statement. residents ' perception of barriers to educate and advocate on this issue by family physicians is reported in table 3. the majority (n = 129, 92.1%) of respondents agreed or strongly agreed that lack of time is a barrier, followed by lack of knowledge (n = 76, 54.3%). most respondents (n = 114, 81.4%) agreed or strongly agreed that they would benefit from additional education on the health impact of the built environment. respondents were asked to select all of their preferred ways to learn about built environment as a determinant of health. listed in order of frequency, they are lecture (n = 94, 67.1%), web module (n = 72, 51.4%), and brochure / publication (n = 64, 45.7%). respondents ' preferred methods to conduct built environment advocacy and deliver patient education in this area, in descending order, are counselling patients (n = 99, 69.7%) ; having pamphlets, posters, or videos in office (n = 95, 66.9%) ; signing a petition (n = 61, 43.0%) ; participating in government public forums (n = 63, 44.4%) ; writing a letter (n = 40, 28.2%) ; and giving a public lecture (n = 34, 23.9%). this study suggests that while residents perceived value in education and advocacy as disease prevention strategies, they did not agree that it is a role of the family physician to advocate for healthy built environments. residents are somewhat knowledgeable but lack specific knowledge to participate in larger system level advocacy work. despite their attitudes, respondents seemed to be open to additional education. there seemed to be a disconnect between respondents ' positive attitudes towards the impact of built environment on health and their lukewarm attitudes towards advocacy as a disease prevention strategy vis - a - vis attitudes towards their role as health advocate (table 1). one hypothesis is that many of the relationships of built environment to health as identified in the literature are intuitive. for example, most physicians would agree that construction of bike lanes could encourage people to bike to work, subsequently resulting in potential health benefits. however, only half thought that family physicians should have a role to educate patients on and advocate for healthy built environments. this finding is not surprising, given the emphasis of medical education on the medical expert role, but is concerning given the influence that physicians ' statements can have on patients ' decisions about their care [18, 19 ]. other studies that have examined medical residents ' attitudes toward health advocacy [20, 21 ] suggest that although it is generally acknowledged to be part of the physician 's social responsibility, residents find few meaningful opportunities to practice advocacy during training, including lack of role models. improving medical education to address this discordance between advocacy endorsement and lack of engagement may encourage physicians to increase their involvement in meaningful and effective advocacy activities related to this determinant of health. social responsibility (e.g., advocating for clearer policy) is an important component of community - oriented primary care and seen to be a key role for family physicians. despite the fact that the role of physicians as health advocate has been long and profusely discussed [14, 2325 ], it is not easy to define this role, which may contribute to residents ' perceptions of their responsibility to educate patients about and advocate for a healthy built environment. interestingly, one - third of survey participants indicated that public education and advocacy for the built environment are roles for medical organizations (table 1). this attitude may reflect family residents ' perceived powerlessness to individually address broader health issues. residents may also be acknowledging the greater power that physicians have when working together as a collective group. when medical associations or specialists support macro level interventions, it is easier to convince politicians to enact broader change. thus, educational strategies on how physicians can support the action of the collective may be beneficial but not a total solution as such organizations may not always recognize a priority. a us study, for example, found that despite a national priority to eliminate health disparities, more than half of national physician organizations were doing little to address this problem with relatively few organizations leading most of the efforts. a perceived knowledge gap was identified related to advocating for healthy built environments at a broader system level (table 2). for example, only one - third of respondents reported that their knowledge level was adequate to participate in community discussions on how to improve the health of the local population through changes to the built environment. most respondents identified that they would benefit from further education on the health impact of the built environment and that this would help them to incorporate this knowledge into their practice (table 3). these results suggest that current medical education in canada on this determinant of health is lacking. other studies also note the knowledge gap and that opportunities for medical trainees to acquire knowledge and skills for health advocacy are lacking [28, 29 ]. medical school curriculum has been largely based on biomedical science to the detriment of knowledge acquisition required to achieve and integrate nonmedical expert roles such as communicator and health advocate. knowledge about the built environment within medical contexts is new for clinicians and educators. yet, to be able to play an active role in educating patients and advocating for healthier communities, physicians must better understand this topic. while advocacy related to the built environment may be less appropriate at the individual patient level in the office setting, broader ecological interventions and advocacy at the system level are highly relevant. as influential and respected members of society, physicians can have a role in participating in strengthened community action and healthy public policies that support physical activity, safe streets and neighbourhoods, affordable housing, and safe air, soil, and water. as this type of role may require physicians to be more political and may take some doctors outside their comfort zone, educational interventions for physicians would help to support them. effective teaching of health advocacy competencies in medical school may support future physicians to make this work a true priority. lecture and web - based learning modules, identified as preferred formats of continuing education, could be distributed online to all canadian family physicians at a low cost. e - learning for physicians and medical students is increasingly popular and may offer cost advantages over traditional pedagogical techniques [31, 32 ]. increasing physician knowledge on built environment impacts (via web modules) to support preventive or chronic disease management plans would be relevant. however, more creative pedagogy may be needed to fully address skills building for system - level advocacy. physicians ' prevention and health promotion efforts have been long - studied, and various studies have highlighted barriers to patient education and advocacy [3336 ]. in our study, inadequate time to educate patients and advocate for healthy built environment was a perceived barrier for nearly all respondents (table 3) and only one - third of residents thought that educating patients was an effective use of their time (table 2). this may reflect a lack of valuing of this issue or lack of clarity around how physician - patient one - on - one encounters will (or will not) create infrastructure or policy changes to support healthy built environments. a broader health advocacy approach may be a more appropriate way to spend time on this issue, and the medical residents may wisely realize this. the fact that residents hold the belief that advocacy or nonclinical work is not appropriately compensated, even before they start earning clinical income, is particularly disconcerting (table 3). residents may have become aware of the barrier of inadequate compensation through conversations with practicing physicians. this barrier may not be specific to advocating for healthy built environment but, rather, may extend to the overall lack of compensation for nonclinical work. in canada, the funding models have remained relatively consistent over time for preventive care activities. this link between remuneration and preventive care has been reported elsewhere in the literature [33, 36, 37 ]. the next step will be to expand this research beyond medical residents to include currently practicing canadian family physicians. furthermore, targeted medical education interventions that foster a sense of responsibility related to health advocacy are indicated. by the time residents become doctors, if they have not already acknowledged the importance of their role in advocacy for a healthy built environment, it is possible that they may never engage in broader advocacy efforts. physician surveys are often characterized by low response rates. while a high response rate is sought after, research evidence indicates that physician surveys are somewhat more resilient to the effects of nonresponse compared with general population surveys. response bias may be less of a concern in this case, as most nonresponse studies have found no or only minimal amounts of response bias [3941 ]. furthermore, the distribution of respondents by sex was similar between the sample population and the respondents, suggesting a satisfactory level of representation. a second limitation is that the survey questions were not validated as to whether they addressed the study constructs. in an effort to reduce this error, finally, response error was minimized by cognitive interviewing and multiple pilot surveys to examine comprehensibility. effective urban planning (i.e., urban design, land use) and transportation policies can impact the health of canadians, and family physicians have the potential to be both educators and advocates for healthy built environments. this survey revealed ambivalent attitudes among residents towards a physician 's role in education and advocacy for a healthy built environment. family medicine residents acknowledged that the built environment significantly impacts health but were divided on whether family physicians have a role in advocacy. they perceived inadequate time, knowledge, and, to a lesser extent, remuneration as barriers to education and advocacy related to the built environment. family medicine residents reported having some knowledge of the built environment but would benefit from further education, particularly on ways to engage in system - level advocacy. some residents wisely acknowledged that professional organizations have a role to play in improvements to the built environment, as such advocacy may be best addressed through collective action. this study supports the idea that implementing physician support and educational interventions to address attitudes and skills deficits related to patient education and system - level advocacy for the built environment would be a step towards effective disease prevention, such as improving physical activity levels. | purpose. to determine family medicine residents ' perceived knowledge and attitudes towards the built environment and their responsibility for health advocacy and to identify their perceived educational needs and barriers to patient education and advocacy. methods. a web - based survey was conducted in canada with university of toronto family medicine residents. data were analyzed descriptively. results. 93% agreed or strongly agreed that built environment significantly impacts health. 64% thought educating patients on built environment is effective disease prevention ; 52% considered this a role of family physicians. 78% reported that advocacy for built environment is effective disease prevention ; 56% perceived this to be the family physician 's role. 59% reported being knowledgeable to discuss how a patient 's environment may affect his / her health ; 35% reported being knowledgeable to participate in community discussions on built environment. 78% thought education would help with integration into practice. inadequate time (92%), knowledge (73%), and remuneration (54%) were barriers. conclusions. while residents perceived value in education and advocacy as disease prevention strategies and acknowledged the importance of a healthy built environment, they did not consider advocacy towards this the family physician 's role. barrier reduction and medical education may contribute to improved advocacy, ultimately improving physical activity levels and patient health outcomes. |
situated between the neuroretina and choroid, it performs several functions that are crucial for supporting sight. among the most important are phagocytosis of shed photoreceptor (pr) outer segments, regeneration of the visual cycle pigment rhodopsin, transportation of glucose and nutrients from the choroid to the distal part of the neuroretina, and transportation of excess fluid in the opposite direction [1, 2 ]. malfunction of the rpe, implying a disrupted ability to perform these tasks, is a direct cause of prevalent retinal diseases like age - related macular degeneration (amd) [3, 4 ] and a consequence of inherited disorders like stargardt disease. a promising approach for treatment of these diseases is the transplantation of tissue engineered rpe [610 ]. however, for the prospect of tissue engineering to become a widespread treatment option, it is necessary to ensure cell availability during short - term storage and transportation of rpe cells. in the process of establishing such a protocol, our research group has demonstrated that storage temperature has a crucial impact on the viability and morphology of cultured rpe cells. arpe-19 cultures stored at 16c displayed the greatest number of viable cells compared to cells stored at eight other temperatures (4c, 8c, 12c, 20c, 24c, 28c, 32c, and 37c) after seven days of storage. having established the potential effect of storage temperature on cell viability, we herein aim to investigate the effect of storage temperature on the gene expression associated with many highly specialized functions of the rpe, using microarray technology. increased knowledge of the effects of storage on cultured arpe-19 cells is imperative for future use of rpe transplantation in treatment of eye diseases affecting millions of people worldwide. adult retinal pigment epithelial cells (arpe-19) were purchased from the american type culture collection (atcc) (manassas, va). dulbecco 's modified eagle 's medium (dmem), nutrient mixture f12, fetal bovine serum (fbs), trypsin - edta, phosphate - buffered saline (pbs), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes), sodium bicarbonate, gentamycin, penicillin, and streptomycin were from sigma - aldrich (st. louis, mo). minimum essential medium (mem) was purchased from invitrogen (carlsbad, ca). nunclon t25 and t75 flasks, pipettes, and other routine plastics were purchased from vwr (west chester, pa). the mirneasy mini kit containing the qiazol lysis reagent was obtained from qiagen (venlo, netherlands). rpe cells from the arpe-19 cell line were cultured under standard conditions in 95% air and 5% co2 at 37c in dmem / f12 medium containing 10% fbs, 50 units / ml penicillin, and 50 g / ml streptomycin. all arpe-19 cells were from passage 4 and lower after acquisition from the vendor. upon reaching confluence, the cells were seeded (5000 cells / cm) in nunclon t25 and t75 flasks. the culture medium was changed after two days, and confluent cultures were obtained on the third day. three cultures were immediately processed for mrna amplification and used as controls, while nine cultures were prepared for storage. the cells were rinsed with pbs, and the culture medium was replaced by storage medium consisting of mem, 25 mm hepes, 22.3 mm sodium bicarbonate, and 50 g / ml gentamycin, hereafter referred to as mem. the cultures were then placed in storage containers maintaining a stable temperature of either 4c, 16c, or 37c and stored for seven days. the configuration and design of the custom - made storage containers have been explained earlier. cultured arpe-19 cells that had been stored for seven days at 4c, 16c, and 37c, as well as control cultures that had not been stored, were rinsed with pbs and directly lysed with qiazol lysis reagent. 150 ng of total rna was subjected to genechip ht one - cycle cdna synthesis kit and genechip ht ivt labeling kit, following the manufacturer 's protocol for whole genome gene expression analysis (affymetrix, santa clara, ca, usa). microarray analyses were performed using the affymetrix genechip human gene 1.0 st arrays (affymetrix, santa clara, ca), which contains approximately 28,000 gene transcripts. signal intensities were detected by hewlett packard gene array scanner 3000 7 g (hewlett packard, palo alto, ca, usa). the scanned images were processed using the agcc (affymetrix genechip command console) software and the cel files were imported into partek genomics suite software (partek, inc. mo, usa). the robust multichip analysis (rma) algorithm was applied for generation of signal values and normalization. gene transcripts with maximal signal values of less than 32 across all arrays were removed to filter for low and nonexpressed genes, reducing the number of gene transcripts to 17,684. for expression comparisons of different groups, gene networks and canonical pathways representing key genes were identified using ingenuity pathways analysis (ipa) (http://www.ingenuity.com/). briefly, the data set containing gene identifiers and corresponding fold changes and p values was uploaded into the web - delivered application and each gene identifier was mapped into its corresponding gene object in the ingenuity pathways knowledge base (ipkb). the functional analysis identified the biological functions and/or diseases that were most significant to the data sets. fisher 's exact test was performed to calculate a p value determining the probability that each biological function and/or disease assigned to the data set was due to chance alone. the data sets were mined for significant pathways with the ipa library of canonical pathways and networks were generated by using ipa as graphical representations of the molecular relationships between genes and gene products. the presentation of the microarray data was divided into two manuscripts : the present and another addressing the genes not presented herein. the differential gene expression data were validated for selected transcripts (tyrp1, dsc1, and glut12) using taqman gene expression assays and the applied biosystems viia 7 real - time pcr system (applied biosystems, life technologies). briefly, 200 ng of total rna was reverse transcribed using qscript cdna super mix (quanta biosciences) following the manufacturer 's instructions. after completion of cdna synthesis, 1/10th of the first strand reaction was used for pcr amplification. a total of 9 l of diluted cdna (diluted in h2o), 1 l of selected primer / probes taqman gene expression assays (life technologies), and 10 l taqman universal master mix (life technologies) were used following the manufacturer 's instructions. transducin - like enhancer of split 1 (tle1) was used as endogenous control due to low coefficient of variation (cv) (0.444) in the affymetrix study. taqman gene expression assays (life technology) used assays detecting tyrp1 (hs00167051_m1), dsc1 (hs00245189_m1), glut12 (hs01547015_m1), and tle1 (hs00270768_m1). p values were calculated using student 's t - test in microsoft excel using delta ct values. normalized target gene expression levels (fc) were calculated using the formula : 2. in order to elucidate the expression patterns of genes critical to important rpe functions, we investigated the expression levels of individual genes associated with distinctive cellular properties (i.e., phagocytosis, pigment synthesis, visual cycle, adherens and tight junctions, and glucose and ion transport). only significantly regulated genes are mentioned, namely, those displaying a p value below 0.05. phagocytosis of photoreceptor (pr) outer segments is a crucial function of the rpe, and the components of its phagocytic machinery have been thoroughly described. compared to control cells, cells that had been stored at 4c and 16c showed no difference in levels of expression for any of the 16 identified genes important for phagocytic functions. cells that had been stored at 37c displayed significant changes in gene regulation of several genes ; however, only the engulfment - related gene pros1 displayed a fold change of more than 1.5. pigment synthesis. production of melanin pigment by the rpe has two important functions in vivo : photoprotection, due to the antioxidant effect of melanin, and prevention of internal reflection of light from the sclera back to the retina. of six identified genes related to pigment synthesis, slc45a2 showed a 1.4-fold increase and mitf a 2.3-fold decrease in expression at 16c compared to controls. the rpe serves a crucial function in the visual cycle by reisomerizing all - trans - retinal to 11-cis - retinal, and defects in key proteins of the cycle can in themselves lead to various retinal diseases [1, 15 ]. most of the identified visual cycle genes were maintained at expression levels similar to controls. rlbp1 and rbp7 expression was decreased 1.3-fold and 1.6-fold, respectively, in cells stored at 37c, while rdh11 was decreased 1.2-fold in cells stored at 4c. in cultures stored at 16c, adherens junctions link actin filaments between epithelial cells and provide a strong mechanical attachment in cellular monolayers. cadherins form homodimers with cadherins of adjacent cells and are pivotal for the integrity of the junction. a total of 13 different cadherins were identified in our data set, and their expression levels were unchanged in cells stored at 4c and 16c compared to the control. at 37c, cadherins 6, 10, and 11 were downregulated 3.0-fold, 1.7-fold, and 2.2-fold, respectively, while dsc1 and cdh13 were upregulated 8.3-fold and 2.0-fold, respectively, compared to control cells. tight junctions of the rpe regulate cell polarity, proliferation, and paracellular diffusion, and they are constituents of the blood - retinal barrier. of the 23 identified genes involved in the tight junction complex, seven were differentially expressed in cells stored at 16c, but only the downregulation of magi1 and magi3 exceeded a fold change of 1.5. a total of 14 genes were differentially expressed at 37c, of which cldn11, f11r, and rab3b were downregulated more than 1.5-fold. the rpe is critical for supplying the inner part of the retina with glucose, and the maintenance and regulation of glut channels are essential for this function [18, 19 ]. in cells that had been stored at 16c, we found an increased expression of three of a total of 11 glucose transporter isoforms identified in our material. glut3 was increased 2-fold, glut8 1.2-fold, and glut14 1.6-fold. in cells stored at 37c, there was a 1.2-fold increase in expression of glut8 and a 2.7-fold decrease of glut12. the na - k - atpase establishes and maintains electrochemical gradients across the plasma membrane, thereby providing the energy for transepithelial transport. of six identified genes involved in the na - k - atpase, atp1a3 and atp1b2 were upregulated 1.4-fold and 1.2-fold at 16c storage, respectively. relative quantification of a few key genes (tyrp1, dsc1, and glut12) was performed with real - time pcr (figure 3). in comparison, the microarray data showed a 3.6-fold upregulation of this gene at 37c. dsc1 expression was significantly and considerably upregulated in the 37c group compared to controls, with a 67.0-fold upregulation. this is higher than the corresponding microarray data, which yielded an 8.3-fold upregulation at this temperature. pcr analysis of glut12 expression showed a similar downregulation compared to microarray results (3.0-fold and 2.7-fold, resp.). however, results were nonsignificant in the pcr group (p value = 0.068). pcr validation showed that expression of tyrp1, dsc1, and glut12 was not significantly regulated in the 4c and 16c culture groups, which is in line with the microarray data. in this study, we investigated the effect of storage temperature on important cellular functions of arpe-19 cells by comparing the expression levels of genes associated with phagocytosis, pigment synthesis, visual cycle, adherens and tight junctions, and glucose and ion transport. the arpe-19 cell line is recognized for displaying significant functional differentiation and forming polarized epithelial monolayers and tight junctions with barrier properties [21, 22 ]. however, the cell line does not mirror all the functions and characteristics of native rpe [2325 ]. some studies have demonstrated a relatively lower expression of some rpe - specific transcripts in arpe-19 cells compared to native rpe cells, while others have not. native rpe exhibits considerable regional variation, and thus any culture models will be inherently heterogeneous [23, 28, 29 ]. cells and cell lines in culture can exceed the normal variation described in rpe in vivo [23, 3032 ]. gene expression by cultured rpe cells is substrate dependent, and arpe-19 grown on plastic displays the phenotype closest to native rpe, capable of yielding a functional profile of differentially expressed genes. the global expression profile of arpe-19 cells can also be directed towards that of primary rpe cells by withdrawing serum. in the present study, cells were cultured and stored on plastic, and the storage medium contained no xenobiotic components. phagocytosis of shed photoreceptor outer segments is vital to photoreceptor repair and represents one of the most critical functions of the rpe [1, 35 ]. we found no changes in expression of phagocytosis - associated genes after storage at 4c and 16c compared to control cells (table 1). two receptor ligand pairs are recognized for exhibiting key roles in the molecular machinery of rpe phagocytosis. these include the receptor tyrosine kinase mertk and its secreted ligands gas6 and protein s, as well as the integrin receptor v5 and its secreted ligand mfg - e8. arpe-19 cells are capable of phagocytosing photoreceptor outer segments [3739 ], but some differences exist compared to primary cultures. both require the integrin receptor v5 for the binding and internalization of outer segments [21, 37 ], the main difference being observed at the level of promoter strength, yielding much higher transcriptional activity in arpe-19. with the exception of protein s, expression of all of these important genes although the differences in expression of the remaining phagocytosis associated genes were modest, these results may indicate a slightly disrupted phagocytic ability in cells stored at 37c. the expression of genes associated with pigment synthesis in the rpe was also evaluated due to its many functions, including protection from oxidative stress [4143 ]. four genes have been described as key contributors in the melanin biosynthesis pathway : tyr, tyrp1, tyrp2, and p gene (oca2). smith - thomas. found that primary human rpe cells failed to express tyrp2 and that a very low percentage of the cells expressed tyrp1, but only if cultured for more than 3 weeks. lu. found that human rpe cultured under standard conditions failed to express any of the four key genes mentioned above. however, we were able to detect both oca2 and tyrp1 in all culture groups, as well as several other genes related to pigment synthesis (table 1). upon transduction of light energy into electrical impulses in the pr, 11-cis - retinal is converted to all - trans - retinal, which is cycled to the rpe for reisomerization. a string of proteins contributes in the visual cycle, and the expression levels of critically important proteins such as cellular retinol binding protein 1 (rbp1, also known as crbp1), lecithin retinol acyltransferase (lrat), cellular retinaldehyde binding protein 1 (rlbp1, also referred to as cralbp), and cellular retinol binding protein 5 (rbp5) were maintained at control levels during storage at all three temperatures. this indicates that the visual cycle can be preserved under the storage conditions used in this study. cell - cell adhesion is important for maintaining the correct rpe phenotype [45, 46 ]. cultures stored at 4c and 16c did not differ from controls in regard to expression of adherens junction genes. cultures stored at 37c, however, showed a differential regulation of five adherens junction genes, among them an 8.3-fold upregulation of dsc1 and a change in expression of several cadherins. this group also showed the largest expression changes of tight junction genes, mostly downregulation. this might indicate a loss of integrity of the intercellular junction in cells stored at 37c compared to control cells. the classic tight junction proteins zo-1 and occludin did not display any changes in expression levels after storage at any of the three temperatures. in a previous study, we demonstrated that the number of viable arpe-19 cells at 4c storage dropped to less than 4% compared to the control group. in the present study first, the cultures stored at 4c contain a large number of dead and dying cells, which have a tendency to detach and be washed away during preparations, thereby not being included in the analysis. second, temperature has a crucial effect on the adhesive abilities of several cell types [4750 ] and adhesion seems to be severely affected during 4c storage, resulting in the loss of otherwise viable and well - functioning cells from the monolayer. unpublished data from our research group demonstrates improved viability following storage at 4c by implementing a radical change in the culture protocol in order to improve cell adhesion. this finding supports our hypothesis that cellular adhesion is severely affected at low storage temperatures. several glut proteins were identified in our material, with glut1 expression being dominant (table 1). this is in line with existing gene expression studies on native rpe [18, 51, 52 ]. given the dominant role of this transporter in rpe cells, the maintenance of its expression in all storage groups indicates a preservation of glucose transport function after storage. in an earlier study by takagi., the addition of fbs to the culture medium was shown to increase the expression of glut1 in human rpe cells. based on this observation, one might anticipate a downregulation of this isoform when replacing the fbs - containing growth medium with a xenobiotic - free storage medium. however, that was not the case in our cultures. expression of glut3 was increased 2-fold after storage at 16c. glut3 is highly effective, displaying both a higher affinity and a fivefold greater transport capacity for glucose than other isoforms including glut1. its expression has been identified in several cell types characterized by very specific and high metabolic demand, such as neurons and placental trophoblasts [5355 ]. its expression in neurons increases in an activity - related manner to meet an increased demand. we speculate whether this strategy is utilized by arpe-19 cells stored at 16c and if it contributes to preserving a larger number of viable cells compared to other temperatures where glut3 expression remains unchanged. active transport of na across the apical membrane of rpe cells creates a high na concentration in the subretinal space, which is crucial for the photoreceptor dark current and for transport of solutes through symporters and antiporters of the rpe. three isoforms of each of the na - k - atpase and subunits were identified, and most were expressed close to control levels in all storage groups. when comparing the expression levels of genes involved in important rpe functions, it is evident that cells stored at 37c display expression changes in a larger number of genes than cells stored at 4c and 16c. in conclusion, the findings of this study show that cells stored at 4c and 16c are capable of maintaining expression levels of genes important for key rpe functions close to the control levels. | purpose. replacement of the diseased retinal pigment epithelium (rpe) with cells capable of performing the specialized functions of the rpe is the aim of cell replacement therapy for treatment of macular degenerative diseases. a storage method for rpe is likely to become a prerequisite for the establishment of such treatment. herein, we analyze the effect of storage temperature on key functions of cultured rpe cells. methods. cultured arpe-19 cells were stored in minimum essential medium at 4c, 16c, and 37c for seven days. total rna was isolated and the gene expression profile was determined using dna microarrays. comparison of the microarray expression values with qrt - pcr analysis of selected genes validated the results. results. expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4c and 16c. cultures stored at 37c displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. conclusion. rpe cultures stored at 4c and 16c for one week are capable of maintaining the expression levels of genes important for key rpe functions close to control levels. |
ewing 's sarcoma (es) is a rare malignant small round cell tumor that primarily affects the skeletal system. it accounts for 4 to 10% of all types of bone cancer, with long bones and pelvis being the most common locations. it affects mainly adolescents and young adults and is rarely seen before the age of 5 and after the age of 30. clinically, this tumor has an aggressive behavior characterized by rapid growth and high probability of micrometastasis at diagnosis. an 11-year - old girl presented to department of oral and maxillofacial surgery, yenepoya dental college and hospital, deralakatte, mangalore, with the complaint of swelling on the left side of the face [figure 1 ]. the swelling started 6 months back which gradually enlarged to present size. on examination, extra orally a diffuse swelling was seen on left side of face which extended superiorly up to ala tragus line, anteriorly from about 2 cm posterior to commisure of mouth to angle of the mandible. intra oral examination revealed mixed dentition and swelling extending from left mandibular canine to left mandibular second molar [figure 2 ]. on palpation, bi - digital palpation revealed expansion of the buccal plate, no perforation of buccal / lingual cortex and no paresthesia of mucosa or lower lip were seen. grade iii mobility was seen on all the teeth from left mandibular canine to left mandibular second molar, which were non tender on percussion. the intra oral periapical radiograph shows radiolucency near the mandibular teeth [figure 3 ]. intra oral periapical radiograph showing radiolucency near the mandibular teeth the ct scan findings revealed the following : an ill - defined expansile cystic lesion was seen in the body of the mandible on left side and also thinning and erosion of buccal cortex was also noticed [figure 4 ]. the results of hematological and biochemical investigations were within normal limits. under local anesthesia, extraction of the mobile teeth left mandibular first molar was performed and the tissue sample obtained through the extraction socket was sent for histopathological examination. extracted tooth specimen for biopsy on microscopic examination, h and e stained sections showed sheets of uniform, small, round cells arranged in diffuse pattern, with indistinct outline, scanty cytoplasm, and well - defined nuclear outline, with round to oval nucleus and inconspicuous nucleoli. a diagnosis of malignant small round cell tumor was made based on the above histopathological findings. histopathological picture a panel of immunohistochemical markers namely cd99, cd3, cd20, chr, mpo, desmin, and syn were used to rule out other small round cell tumors. tissue sections showed positive expression for cd99 (mic2), with characteristic membranous pattern, confirming the diagnosis of ewing 's sarcoma bone scan was also advised which revealed reduced uptake over the body of left mandible without rim of enhanced uptake. a surgical procedure was performed to resect the malignant tissue with generous surgical margins and to reconstruct the resulting defect. intraoperative surgical pictures the postoperative panoramic radiograph revealed a well - aligned reconstruction with intact plating (6 months post operative orthopantamograph) adjuant chemotherapy comprised 20 weeks (3 cycles) of treatment with a four - drug regimen which were vincristine [vc ], dactinomycin [ac ], cyclophosphamide [cp ] and doxorubicin [ad ] after surgery. ewing 's sarcoma (es) is a rare malignant small round cell tumor that primarily affects the skeletal system. it accounts for 4 to 10% of all types of bone cancer, with long bones and pelvis being the most common locations. it affects mainly adolescents and young adults and is rarely seen before the age of 5 and after the age of 30. clinically, this tumor has an aggressive behavior characterized by rapid growth and high probability of micrometastasis at diagnosis. es is a malignant neoplasm that primarily affects long bones of the extremities with nearly 50% of reported cases involving the femur and pelvis. the majority of the patients affected are between the ages 5 and 20, whereas the disease is distinctly uncommon in individuals before age 5 and after age 30 (braz dent). es arising from the bones of the head and neck region is exceedingly uncommon. when it occurs in the jaw, mandible is more frequently affected than the maxilla. modern treatments are based on combined modality of treatment : local therapy (surgery and/or radiotherapy to the main tumor) followed by chemotherapy(for management of micrometastasis). the present case was positive for cd99, vimentin and desmin and negative for other immunomarkers, leading to a diagnosis of es. if root amputation surgery is performed due to an extensive radiolucent lesion, histopathological evaluation should be mandatory. primary bony reconstruction bears the risk of second intervention when dealing with es in the mandible. | ewing 's sarcoma is a malignant tumor of bones that primarily affects children and young adults. the true origin of this small round cell lesion still remains controversial. it was originally described by james ewing in 1921 as arising from undifferentiated osseous mesenchymal cells ; however, recent studies suggest that ewing 's tumor might be neuroectodermally derived from various degrees of differentiation of the primitive neural tissues. this paper reports a rare case of es of the mandible in an 11-year - old girl, which had been previously misdiagnosed and treated as a dental abscess. in the clinical examination, a hard immobile expansive mass of 2 cm diameter was observed on the left side of the mandible. radiographic examination revealed a diffuse radiolucent lesion with ill - defined borders and wide vestibular bone plate destruction. microscopically, the tumor was composed by monotonous small round cells that exhibited immunoreactivity for cd99, vimentin and desmin. surgical resection of mandible followed by mandibular reconstruction was adopted. the patient was subjected to multiagent chemotherapy with vincristine [vc ], dactinomycin [ac ], cyclophosphamide [cp ] and doxorubicin [ad ]). |
true metastases from solid tumors in lung, skin (melanoma), gastrointestinal tract, kidney, testes, and endocrine glands are reported only in 0.2% of all surgical prostatic specimens and 2.9% of all male postmortems. lung is the most common primary site of metastases to the prostate. in all series direct spread of bladder carcinoma clinical context, morphological features, and immunohistochemical localization of prostate specific antigen (psa) are supposed to clarify the differential diagnosis between a secondary and a primary tumor. we report an unusual and rare case of secondary signet ring cell carcinoma (srcc) of prostate in which the clinical data pointed toward the diagnosis of a primary srcc. signet ring cell morphology was also not helpful as incidence of primary srcc was more than that of secondary srcc in prostate. immunohistochemistry (ihc) for psa not only proved the case to be a secondary srcc but also initiated the process for diagnosis of the occult primary malignancy in the patients stomach. a 74-year - old male patient presented with frequency of micturition, dysuria, and features of urinary outlet obstruction developing progressively over a period of last 3 months. he was a smoker, occasional drinker, and complained of no other features of clinical interest except infrequent dyspepsia. transrectal ultrasonography showed grade iii prostatomegaly measuring 5.54.24.8 cm (58 g), with suspicious hypoechoic areas in the peripheral zone and residual urine of 80 ml. serum total psa level was 9.71 ng / ml and free - to - total psa ratio was 10.8. on digital rectal examination the prostate was elastic, slightly hard, painless, and with prominent retroprostatic sulci. cystoscopic examination found no primary abnormality of the bladder and extensive transurethral resection of prostate (turp) was performed under anesthesia with resultant specimen measuring 45 g. histopathology revealed nests and singly infiltrating poorly differentiated, hyperchromatic, pleomorphic adenocarcinoma cells majority of which were of signet ring morphology [figures 13 ]. individual cells and cell nests were seen lying haphazardly among bundles of smooth muscle fibers and also within lymphovascular spaces but no perineural invasion, mucinous fibroplasia or glomerulations were noted. initial diagnosis of signet ring variant of invasive prostatic adenocarcinoma with gleason score 4 + 5=9 was thought of but ihc for psa [figure 3 (inset) ] and carcinoembryonic antigen (cea) were simultaneously performed as primary prostatic signet ring cell adenocarcinoma is rare and it is mandatory to exclude other mucinous tumors of non - prostatic origin based on morphology and immunohistochemistry and if necessary using clinical information. the globoid optically clear cytoplasm of the signet ring cells were stained positively with alcian blue at ph 2.5 confirming the contents as acid mucin [figure 1 (inset) ]. gastric endoscopic biopsy was subsequently performed 2 weeks after turp and histopathology showed infiltrating signet ring cell carcinoma of stomach. (inset : same signet ring cells with positive alcian blue stain at ph 2.5.) signet ring cells lying haphazardly among bundles of smooth muscle fibers. some primary adenocarcinomas of the prostate will have a signet - ring - cell appearance, yet the vacuoles do not contain intracytoplasmic mucin. these vacuolated cells may be present as singly invasive cells, in single glands, and in sheets. only a few cases of prostate cancer have been reported with mucin positive signet ring cells. one should exclude other mucinous tumors of nonprostatic origin based on morphology and immunohistochemistry and if necessary using clinical information. immunohistochemical stain for psa is diagnostically helpful in distinguishing prostatic adenocarcinomas from other neoplasms secondarily involving the prostate and establishing prostatic origin in metastatic carcinomas of unknown primary. a minority of higher grade prostatic adenocarcinomas are psa negative, although some of these tumors have been shown to express psa mrna. some prostatic adenocarcinomas lose psa immunoreactivity following androgen deprivation or radiation therapy. in gastric signet ring cell carcinoma (who classification) more than 50% of the tumor consists of isolated or small groups of malignant cells containing intracytoplasmic acid mucin that stain with alcian blue at ph 2.5. the present case once again underlines the fact that metastatic src carcinoma although rare should be considered as a differential before diagnosing a primary src carcinoma of prostate. a detailed clinical examination and special attention to any prior history of mild symptoms like dyspepsia might lead to the performance of an upper gastrointestinal endoscopy which can help in much earlier detection of a primary gastric tumor. the most important feature for differentiation in this case with an occult primary was psa immunostain supplemented by an alcian blue stain at ph 2.5. dr. tapas moitra, associate professor, department of urology, north bengal medical college, sushrutanagar, darjeeling. | true metastases to prostate from solid tumors are reported only in 0.2% of all surgical prostatic specimens and 2.9% of all male postmortems. clinical context, morphological features, and immunohistochemical localization of prostate specific antigen (psa) are supposed to clarify the differential diagnosis between a secondary and a primary tumor. we report an unusual and rare case of secondary signet ring cell carcinoma (srcc) of prostate in which the clinical data and signet ring cell morphology pointed toward the diagnosis of a primary srcc. immunohistochemistry (ihc) for psa not only proved the case to be a secondary srcc but also initiated the process for diagnosis of the occult primary malignancy in the patients stomach. |
spasm of esophagus is often suggested as the cause of unexplained chest pain or dysphagia. once cardiovascular disease and structural disease have been excluded, esophageal manometry should be performed to detect motility disorders as the cause of symptoms.1 diffuse esophageal spasm (des) is an uncommon motility disorder of unknown etiology in which has been offered as a possible cause for the patient 's dysphagia or chest pain. manometric evaluation of the esophagus is considered to be the gold standard in patients suspected with des.1 the most accepted diagnostic criteria for des is the presence of simultaneous contractions in at least 20% of wet swallows, intermixed with normal peristaltic sequences.2 recently, the development of high - resolution manometry (hrm) and topographical displays has yielded new perspectives and insights into esophageal motor functions.3 - 5 hrm helps to differentiate true esophageal spasm from rapid elevation of the intra - bolus pressure due to focal dysmotility or obstruction. applying this distinction makes the diagnosis of des very rare.4,5 we diagnosed a female patient with dysphagia and chest pain which were triggered by swallowing a large amount of water as des on multiple rapid swallowing testing during hrm. a 52-year old female patient was admitted to our hospital with complaints of intermittent dysphagia and chest pain for 1 year. she had symptoms on the lower substernal area when swallowing a large amount of water, which were accompanied by chest pain. chest pain was a sharp and non - radiating and was relieved by transports of swallowed water to the stomach. these symptoms only occurred when guzzling water and not by taking a sip of water or eating. she was of normal body weight and had no abnormalities on chest and abdominal physical examination. endoscopy of the upper digestive tract was performed in other hospitals, but there were no abnormal mucosal lesions. then she was referred to our hospital for further investigation on dysphagia and chest pain. 24 hour esophageal impedance - ph monitoring was performed and there were no pathologic regurgitation. the patient swallowed 5 ml of water from the first swallow to the seventh swallow, 10 ml from the eighth swallow to the 10th swallow, 15 ml on the 11th swallow, 20 ml on the 12th swallow and 50 ml on the 13th swallow. on the 14th swallow she tried to drink 100 ml water, but could swallow only 25 ml. she did not have symptoms from the first to the 10th swallow, but started to complain chest pain and dysphasia when swallowing more than 15 ml. rapid propagated pressurizations were observed at the second, fourth, sixth and seventh swallows, in which pressurization front velocity was measured as 19.1, 8.4, 42.7 and 32.4 cm / s, respectively. impaired deglutitive inhibition was observed at the 13th swallow, but peristalsis was normal (fig. we diagnosed her with des because more than 20% of total swallows induced symptomatic esophageal spasms. she was advised to drink water little by little and to take diltiazem 30 mg 3 times a day. thereafter, her symptoms have improved. this case was interesting in that the chest pain and dysphagia occurred not by drinking a sip of water, but only by drinking a large amount of water. more interestingly, we were able to prove that esophageal spasms were associated with the symptoms when performing hrm with large amount of water swallowing technique and multiple rapid water swallowing technique. not only were we able to diagnose des with accuracy, we were also able to observe the esophageal spasms developed with onset of symptoms through multiple rapid swallowing test without any other kind of provocation tests. it is noteworthy that we were able to prove the cause of her symptoms during hrm testing. as far as we know, this is the first report of des in which symptoms were reproduced during high - resolution esophageal manometry with multiple rapid swallowing. although many patients with non - obstructive dysphagia and chest pain can be diagnosed accurately by the conventional esophageal manometry, some patients go through difficulties in receiving proper diagnosis. in contrast with conventional esophageal manometry that uses only 3 - 8 pressure sensors with variable spacing positioned within the esophageal lumen to monitor pressure change following water swallows, a novel technique, hrm is a stationary method that uses an increased number of pressure sensors (up to 36) and topography pressure plot can be developed for visualization of the esophageal pressure and peristalsis. hrm analyzed with pressure topography plots provides greater detail regarding the characteristics of individual contractile segments along with the esophagus.3,4,6 the major advantage of hrm with pressure topography compared with conventional manometry is the ability to easily distinguish between the loci of compartmentalized intra - esophageal pressurization (pseudo - spasm) and rapidly propagated esophageal contractions (spasm).4 spastic disorders such as des or vigorous achalasia, are quite rare by hrm, since the majority of patients so classified by conventional criteria actually exhibit a pseudo - spasm pattern.4 however, she had a pfv of more than 8 cm / s in more than 20% of their swallows and met the criteria for a rapidly propagated contraction several studies suggest that the motility disorders such as des, nutcracker esophagus and achalasia share a common pathophysiologic mechanism, which involves the alteration in nitric oxide synthesis / degradation or loss of nitric oxide containing inhibitory neurons in the lower esophageal sphincter (les). the loss of intramural inhibitory neurons leads to the loss of normal peristalsis and inability of the les to relax properly during swallowing.7 - 10 des is associated with incomplete les relaxation, which is likely in a variant case of achalasia.2 the fact that the transition from des to achalasia has been documented and is believed to occur in 3% to 5% of patients supports this observation.11,12 in this patient, hrm findings showed not only intermittent esophageal spasms with some normal peristalsis but also impaired deglutitive relaxation at the 13th swallow which is a typical feature in achalasia.4 since des is possible to progress to achalasia, follow - up studies using hrm will be needed to detect the feasible transition in this patient. this case was notable in that the chest pain and dysphagia develped only after dinking a large amount of water. cold liquid or food ingestion may precipitate episodes of dysphagia and chest pain in patients with spastic esophageal motility disorders.13 - 15 esophageal manometry during food ingestion has been used as a provocation test in patients with unexplained chest pain, because food ingestion compared with water swallowing precipitates symptoms more frequently.16 but this patient did not complain any symptom with eating, and therefore we did not need to test her while swallowing food. a patient showing precipitated esophageal symptoms by the quantity of water swallowed, regardless of the temperature, has not been described before in literature. although we do not know the specific mechanism through which an increase of the quantity of water induces symptoms, increment in the quantity of swallowing water may stimulate secondary peristalsis in esophagus more strongly and esophageal spasm could be provoked more easily. we diagnosed a female patient presenting with dysphagia and chest pain induced by swallowing a large amount of water as des on multiple rapid swallowing testing during hrm. | diffuse esophageal spasm (des) is an uncommon motility disorder of unknown etiology in which the abnormal motility has been offered as a possible cause for the patient 's dysphagia or chest pain. esophageal manometry is the gold standard for the diagnosis of des and the diagnostic hallmark is identification of simultaneous contractions in at least 20% of wet swallows, alternating with normal peristalsis. recently, a new diagnostic technique, high - resolution manometry has been reported to improve the accuracy and detail in describing esophageal function. we report a female patient with intermittent dysphagia and chest pain occurring only when swallowing a large amount of water. on hrm, this patient had esophageal spasms, increased pressurization front velocity attributable to rapid contractile wave front, associated with symptoms, which were provoked by a multiple rapid swallowing test, and thereby was diagnosed with des. |
cases were enrolled when pairs of bilaterally biopsied bone marrow specimens were available. among bone marrow specimens sampled from the right and left iliac crests between january 2013 and july 2014 at gangnam severance hospital, 53 cases were finally included. for the 53 selected cases, 28 right and left bone marrow samples were allocated to the edta (sigma - aldrich, st. louis, mo, usa) protocol and hcl (calci - clear rapid, national diagnostics, atlanta, ga, usa) protocol, respectively. samples from the 25 remaining cases were assigned to the rdo gold (rdo) group (apex engineering products corporation, aurora, il, usa) protocol and hcl protocol. concentration, processing time, and temperature are summarized in table 1. to test dna quality, five cases were randomly selected from each of the three groups (edta, hcl, and rdo). dna was extracted, and the quantity and quality were confirmed using nanodrop nd-2000 spectrophotometer (thermo fisher scientific inc., waltham, ma, usa). the braf pna clamping method (panagene, daejeon, korea) and comparison with ct values of internal controls were used to evaluate the efficacy of polymerase chain reaction (pcr) dna amplification. to evaluate the efficacy of dna in situ hybridization, her2 dual color silver in situ hybridization (ventana, tucson, az, usa) rna in situ hybridization and immunohistochemical studies were performed prospectively. according to the potential differential diagnoses for suspicious lesions observed within bone marrow, appropriate rna probes or protein antibodies were applied. for immunohistochemistry, the following primary antibodies were used and the details are summarized in table 2 : cyclin d1, ki67, bcl2, bcl6, tdt, cd138, cd20, cd79a, cd3, cd5, cd23, cd10, cd30, and myeloperoxidase. to state the process, after deparaffinization and rehydration, the sections were incubated in benchmark xt automated slide stainer (ventana) for 16 minutes at 37c and then counterstained with hematoxylin reagent. two pathologists reviewed the hematoxylin and eosin (h&e), immunohistochemistry, and in situ hybridization slides. the quality of immunohistochemistry and rna in situ hybridization were assessed using a 3-tiered grading scale : good, equivocal, or poor. her2 silver in situ hybridization was assessed by detecting two signals of her2 and cep17 per nucleus from the normal hematopoietic cells of bone marrow. all statistical analyses were carried out by spss ver. 20.0 for windows (ibm co., armonk dna quantity, purity, and ct values of internal controls after real - time pcr in the edta versus hcl group and rdo versus hcl group are depicted in table 3 and fig. 1. although differences were not statistically significant, the dna yield of the edta protocol was about 2 times higher than the hcl protocol. in addition, the ct value of the former protocol was significantly lower than that of the latter (p<.001) with the estimated difference being about 7. furthermore, the ct values of edta processed samples were lower than 30, demonstrating that the amount of intact dna feasible for pcr with the edta protocol is better preserved by a factor of 2 than the hcl protocol. there were no significant differences between dna yield and ct values of rdo and hcl methods. the ct values of both protocols were above 33, indicating that the amount of intact dna feasible for pcr was very small. the morphological comparison and quality assessment of h&e stain, her2/cep17 dual color silver in situ hybridization, kappa / lambda in situ hybridization, and immunohistochemistry studies were analyzed. the rates of high quality staining for each study were compared between the three protocols. there was no difficulty in microtome dissection of 4 m or less in thickness in any of the three methods. the morphological quality of h&e slide was similar in all three protocols, showing well - preserved histological features of the bone marrow (fig. all five cases in the rdo versus hcl group had severe dna breakdown on her2/cep17 dual color silver in situ hybridization, revealing no her2 or cep17 nuclear signal in bone marrow hematopoietic cells. however, in the edta versus hcl group, all 5 cases in the edta protocol showed two her2 and cep17 nuclear signals from almost all of the hematopoietic cells, whereas nearly no nuclear signal was detected in samples from the hcl protocol (fig. bone marrow specimens from the edta and rdo protocols that underwent kappa / lambda rna in situ hybridization showed well preserved rna signal in the nuclei of plasma cells, while those in the hcl group had severe breakdown of rna signals (fig. 4). nuclear proteins such as ki67, cyclin d1, and tdt were relatively better preserved on immunohistochemistry with both edta and rdo protocols, while samples from the hcl protocol showed breakdown and lower quality of nuclear protein staining (fig. immunohistochemistry targeting the cytoplasmic membrane or cytoplasmic cd markers was well preserved in all three protocols (fig. dna quantity, purity, and ct values of internal controls after real - time pcr in the edta versus hcl group and rdo versus hcl group are depicted in table 3 and fig. 1. although differences were not statistically significant, the dna yield of the edta protocol was about 2 times higher than the hcl protocol. in addition, the ct value of the former protocol was significantly lower than that of the latter (p<.001) with the estimated difference being about 7. furthermore, the ct values of edta processed samples were lower than 30, demonstrating that the amount of intact dna feasible for pcr with the edta protocol is better preserved by a factor of 2 than the hcl protocol. there were no significant differences between dna yield and ct values of rdo and hcl methods. the ct values of both protocols were above 33, indicating that the amount of intact dna feasible for pcr was very small. the morphological comparison and quality assessment of h&e stain, her2/cep17 dual color silver in situ hybridization, kappa / lambda in situ hybridization, and immunohistochemistry studies were analyzed. the rates of high quality staining for each study were compared between the three protocols. there was no difficulty in microtome dissection of 4 m or less in thickness in any of the three methods. the morphological quality of h&e slide was similar in all three protocols, showing well - preserved histological features of the bone marrow (fig. all five cases in the rdo versus hcl group had severe dna breakdown on her2/cep17 dual color silver in situ hybridization, revealing no her2 or cep17 nuclear signal in bone marrow hematopoietic cells. however, in the edta versus hcl group, all 5 cases in the edta protocol showed two her2 and cep17 nuclear signals from almost all of the hematopoietic cells, whereas nearly no nuclear signal was detected in samples from the hcl protocol (fig. bone marrow specimens from the edta and rdo protocols that underwent kappa / lambda rna in situ hybridization showed well preserved rna signal in the nuclei of plasma cells, while those in the hcl group had severe breakdown of rna signals (fig. nuclear proteins such as ki67, cyclin d1, and tdt were relatively better preserved on immunohistochemistry with both edta and rdo protocols, while samples from the hcl protocol showed breakdown and lower quality of nuclear protein staining (fig. immunohistochemistry targeting the cytoplasmic membrane or cytoplasmic cd markers was well preserved in all three protocols (fig. some retrospective studies compared the morphology of in situ hybridization or immunohistochemistry by using stored tissues containing bone. other studies extracted nucleic acid from stored tissues containing bone and compared the quantity, purity, and ct value of the dna and/or rna after real - time pcr [1 - 4 ]. we investigated the quality of nucleic acid and protein in decalcified bone marrow tissue employing several types of genetic tools, immunohistochemistry, and morphological assessments. in addition, we compared the effect of decalcification protocol while limiting bias variance, which may be caused by sampling from different patients and cell degeneration from long - term storage. we prospectively investigated pairs of bone marrow biopsy specimens from the same patients sampled for diagnostic purposes in a clinical setting. to our knowledge, this is one of the first studies utilizing clinical samples to assess decalcification protocols. we compared the conventional protocol using hcl, the well - known alternative protocol using edta, and the new protocol using rdo. all three methods had equally good performance with respect to microtome dissection and preservation of cytomorphologic and histomorphologic features. the edta protocol was superior in preserving nucleic acid (dna and rna), allowing for the feasibility of genetic studies, such as real time pcr and in situ hybridization. the edta protocol would be an appropriate option for genetic studies in bone - contained tissues. the present study also confirmed that the hcl protocols are inappropriate for genetic studies due to the severe damage of genetic material. the quality of nucleic acids in the rdo group was equivocal, but this method also did nt seem to be suitable for genetic studies. in immunohistochemistry targeting nuclear protein, edta protocols seem to be the most appropriate in that comprehensive immunohistochemical stains can be applied to the specimens processed by this protocol. the feasibility of immunohistochemistry using specimens processed with the hcl protocol would be limited in many cases, especially when immunostaining for nuclear proteins is necessary. immunohistochemical markers can be more widely applied in samples processed with the rdo protocol than with the hcl protocol, but diagnostic options are still more limited than the edta protocol. in this study, the edta protocol was the most feasible method for several types of genetic studies and immunohistochemistry. such advantages of edta decalcification protocol are already widely known [1,5 - 7 ]. the present study confirms again the superiority of the edta protocol in a wide range of ancillary tests for pathologic diagnosis. the potential of the rdo protocol is higher than the conventional hcl protocol ; however, it is not superior to the edta protocol and thus, it can not be considered an alternative to edta protocols, particularly when genetic studies are needed. when choosing decalcification agents, cost - effectiveness and turn - around time from biopsy to final pathologic diagnosis are important issues. small bone tissues like bone marrow take about three to twenty four hours for decalcification in edta protocol, but most of the other bone specimens need more time when using the same protocol. rdo protocols can shorten decalcification time, but this agent is also expensive. in many cases, bone biopsies are needed for diagnosis of hematologic cancer, metastatic tumors, and primary bone sarcoma ; these types of cancers usually need further gene - based diagnosis. bone marrow biopsy is widely performed in patients with hematologic malignancies and pediatric sarcomas or blastomas. therefore, preserving genetic materials is critical in handling bone marrow tissues, and cost and turn - around time may be less important in such cases. preserving intact nucleic acid, as well as intact proteins, is critical to providing not only accurate pathologic diagnosis but also diverse therapeutic options to the patients. considering this, in this study the edta protocol was the most appropriate method for handling bone marrow specimens. rdo may also be useful in that it requires less decalcification time and it enables more ancillary tests than hcl, but its usefulness is limited by less potential for genetic studies in processed samples than edta. in this era of expanding genetic molecular study, better tissue handling methods are needed. the present study suggests an appropriate approach in handling bone marrow tissues, and this approach should be expanded to other types of tissue specimens. | background : the conventional method for decalcification of bone specimens uses hydrochloric acid (hcl) and is notorious for damaging cellular rna, dna, and proteins, thus complicating molecular and immunohistochemical analyses. a method that can effectively decalcify while preserving genetic material is necessary.methods:pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups : edta versus hcl and rdo gold (rdo) versus hcl. pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the edta versus hcl comparison group, and 25 cases to the rdo versus hcl comparison group. the decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. for molecular analysis, we randomly selected 5 cases from the edta versus hcl and rdo versus hcl groups.results:the decalcification time for appropriate histomorphologic analysis was the longest in the edta method and the shortest in the rdo method. edta was superior to rdo or hcl in dna yield and integrity, assessed via dna extraction, polymerase chain reaction, and silver in situ hybridization using dna probes. the edta method maintained intact nuclear protein staining on immunohistochemistry, while the hcl method produced poor quality images. staining after the rdo method had equivocal results. rna in situ hybridization using kappa and lambda rna probes measured rna integrity ; the edta and rdo method had the best quality, followed by hcl.conclusions:the edta protocol would be the best in preserving genetic material. rdo may be an acceptable alternative when rapid decalcification is necessary. |
the presence and modulation of neuronal responses within specific time and frequency bands has been implicated in the implementation of perception and cognition within the brain. there is also increasing evidence that the most plausible mechanism for the generation of temporally organized network activity is in reciprocally connected neuronal networks containing mixtures of interconnected glutamatergic (excitatory) pyramidal and stellate cells, and gabaergic (inhibitory) interneurons (bartos, 2007 ; traub, 1996). however, the neuropharmacological bases of the spatially summated, population - level, neurophysiological responses (both evoked and induced) that are recorded non - invasively in humans with magnetoencephalographic / electroencephalographic (meg / eeg) are largely unknown. in a recent observational study we found that, across individuals, the frequency of stimulus - induced network gamma oscillations in primary visual cortex was positively correlated with the concentration of gaba measured with edited magnetic resonance spectroscopy (mrs) (muthukumaraswamy, 2009). however, mrs is an indirect measure of synaptic gaba function and could be influenced by a number of anatomical, biochemical / metabolic, or even genetic factors. in this experiment, we wished to test the hypothesis that modulation of endogenous gaba levels would modify either the amplitude or frequency of stimulus - related gamma oscillations in human visual cortex. to do this, we manipulated endogenous gaba levels using the drug tiagabine in a set of healthy control participants in a single - blind crossover design. tiagabine, used in the treatment of epilepsy, binds with high affinity and selectivity to gaba transporter 1 (gat-1 ; borden, 1994), the primary gaba reuptake transporter in the human cerebral cortex (conti, 2004). gat-1 transporters are located on both neurons and glia (minelli, 1995) and their presence at fast - inhibitory gabaa synapses terminates gaba activity and shapes ipsp activity. the effect of tiagabine, measurable by microdialysis, is to elevate the extracellular / synaptic concentration of gaba (dalby, 2000 ; fink - jensen, 1992) and therefore to enhance the actions of endogenous gaba. the electrophysiological effect of tiagabine is to enhance evoked inhibitory postsynaptic current (ipsc) duration without modifying ipsc amplitude (roepstorff and lambert, 1994 ; thompson and gahwiler, 1992). to directly measure neuronal activity in humans, we combined meg recordings with source estimations using an experimental paradigm that we have previously demonstrated to be highly robust to potentially confounding factors such as day, session, or repetition effects (muthukumaraswamy, 2010). meg is an ideal technique to capture higher frequency oscillations as it is more robust to high - frequency artefact sources such as saccades and neck muscle artefacts than eeg (fries, 2008 ; whitham, 2007, 2008 ; yuval - greenberg, 2008, 2009). the principal hypothesis we tested was that modulation of endogenous gaba levels would modify either the amplitude or frequency of stimulus - related gamma oscillations. subsidiary to that hypothesis, we tested for gabaergic modulations of several neurophysiological parameters, including stimulus - induced alpha amplitude decreases and evoked response components, which are also known to occur with these stimuli (muthukumaraswamy, 2010). eighteen right - handed volunteers (14 men and 4 women) participated in the experiment after giving informed consent, with all procedures approved by the uk national research ethics service (south east wales). volunteers were screened and excluded for personal histories of neurological and psychiatric disease, current recreational or prescription drug use, and impaired liver function (by standard liver function tests). early in the data collection, three women volunteers were not able to complete the experiment after becoming too heavily sedated. from this point in the data collection, women were excluded from recruitment, leaving completed data sets from 14 men and 1 woman to analyze (mean age 25.5, range 2032). all results and conclusions are similar whether this one woman was included / excluded from the analysis. participants were scanned on 2 separate days, separated by a minimum 7-day washout period, at approximately the same time - of - day. on each day, an initial pre ' meg recording was obtained then participants orally ingested a capsule containing either placebo or 15 mg of tiagabine (gabitril). this oral dose is similar to the 16 mg used in a pet study by frankle (2009) and identical to several ongoing imaging studies by our group. participants were blinded to the contents of the capsule and placebo / control session order was counterbalanced across participants. following drug / placebo administration, meg recordings were obtained from participants at 1, 3, and 5-h time - points. at the conclusion of every meg recording, participants completed several psychological questionnaires including, the biphasic alcohol effects scale (martin, 1993), the subjective high assessment scale (schuckit, 1980) and asked to qualitatively report their psychological state. during each meg recording, participants sat in a magnetically shielded room and were presented with a visual stimulus consisting of a, vertical, stationary, maximum contrast, three cycles per degree, square - wave grating presented on a mean luminance background. the stimulus was presented in the lower left visual field and subtended 8 both horizontally and vertically. a small red fixation square was located at the top right - hand edge of the stimulus and remained on for the entire stimulation protocol (muthukumaraswamy, 2010 ; swettenham, 2009). the stimulus was presented on a mitsubishi diamond pro 2070 monitor controlled by the psychophysics toolbox (brainard, 1997 ; pelli, 1997). the screen size was 1024 by 768 pixels and the monitor frame rate was 100 hz. the monitor was outside the magnetically shielded room and viewed directly from within, at 2.15 m, through a cut - away portal in the shield. the duration of each stimulus was 11.5 s followed by 1.5 s of fixation square only. participants were instructed to fixate for the entire experiment and, in order to maintain attention, were instructed to responses slower than 750 ms from the termination of the stimulus triggered a visual warning for participants. in each recording session, 120 stimuli were presented and participants responded with their right hand. whole - head meg recordings were made using a ctf 275-channel radial gradiometer system sampled at 1200 hz (0300 hz bandpass). an additional 29 reference channels were recorded for noise cancellation purposes and the primary sensors were analyzed as synthetic third - order gradiometers (vrba and robinson, 2001). three of the 275 channels were turned off because of excessive sensor noise. at the onset of each stimulus presentation participants were fitted with three electromagnetic head coils (nasion and pre - auriculars), which they wore for the entire recording day, and were localized relative to the meg system immediately before and after the recording session. each participant had a 1 mm isotropic mri scan available for source localization analysis. to achieve mri / meg co - registration, the fiduciary markers were placed at fixed distances from anatomical landmarks identifiable in participants ' anatomical mris (tragus, eye centre). offline, data were first epoched from 1 to 1 s around stimulus onset and each trial visually inspected for data quality. data with gross artefacts, such as, head movements and muscle clenching were excluded from further analysis. reaction times were calculated for each trial relative to the (variable) offset of each grating stimulus. any trial where there was no behavioural responses or a response > 750 ms were classified as incorrect responses and excluded from further analysis. for each participant, we ensured that equal number of trials, from each recording was used in the analysis. to achieve this, trials were removed from the end of each recording run so that each participant had an equal number of trials submitted for each condition in the analysis. following these trial removal procedures, the mean number of trials remaining to analyze was 105.5 per participant (range 82117). three source localizations were performed on each data set using synthetic aperture magnetometry (robinson and vrba, 1999), one for gamma (3080 hz), one for alpha (815 hz), and one for evoked responses (samerf ; robinson, 2004). correspondingly, three global covariance matrices were calculated for each data set, one for gamma, one for alpha, and one for samerf (0100 hz). based on these covariance matrices, using the beamformer algorithm (robinson and vrba, 1999), three sets of beamformer weights were computed for the entire brain at 4 mm isotropic voxel resolution. a multiple local - spheres (huang, 1999) volume conductor model was derived by fitting spheres to the brain surface extracted by fsl 's brain extraction tool (smith, 2002). readers unfamiliar with sam are referred to several methodological reviews (hillebrand, 2005 ; vrba and robinson, 2001). for gamma - band sam image reconstruction virtual sensors were constructed for each beamformer voxel and student 's t - images of source power changes computed using a baseline period of 1 to 0 s and an active period of 0 to 1 s, while for alpha - band sam image reconstruction a baseline period of 1 to 0.3 s and an active period of 0.3 to 1 s was used. this latter time window was used to avoid confounding alpha responses with the time - locked evoked response. after generating the source reconstruction images, the voxel with the strongest power increase (in the contralateral occipital lobe) was located for gamma and the voxel with the strongest source power decrease located for alpha. the source analysis and peak voxel identification procedures were carried out separately for each recording as participants were moved in and out of the meg between recordings, which would alter their head position in the meg dewar. to reveal time - frequency responses at these locations, the virtual sensors at these locations were repeatedly band - passed filtered between 1 and 160 hz at 0.5 hz frequency step intervals, filtering with an 8 hz bandpass, third - order butterworth filter (le van quyen, 2001 ; muthukumaraswamy, 2010). the hilbert transform was used to obtain the amplitude envelope and time - frequency spectra were computed as a percentage changed from the pre - stimulus baselines for each frequency band. from these spectra, the peak frequency and amplitude at that frequency for late gamma amplitude increases (3080 hz, 0.31 s) were quantified by collapsing across the time dimension and finding the maximal (or minimal for alpha) frequency. we also checked for confounding effects of using relative change baselines by computing the average absolute values of the source amplitude estimates within the respective time - frequency bins. for samerf, the computed evoked response was passed through the 0100 hz beamformer weights to generate samerf images (robinson, 2004) at 10 ms intervals from 50150 ms. the image window (usually 7080 ms or 8090 ms) with the maximal response in visual cortex was identified and the maximal voxel selected as the peak location for further analysis. for this, the evoked field was computed for this virtual sensor (0.2 to 0-s baseline) and the peak amplitude and latency of the m80 and m150 responses quantified. for the production of grand - average sam maps, individual sam images were first spatially normalized onto the mni (t1) average brain using fmrib 's linear affine registration tool (jenkinson and smith, 2001). this was done by first obtaining a set of warping parameters by registering the participant 's anatomical mri with the average brain and then applying these parameters to the sam source power maps. the grand - average images presented in figure 2 are the grand - average across both participants and conditions. for these analyses, there were main effects of day ', session ', and an interaction term drug ', presented in that order. here, the critically important term is the interaction term, indicative of a tiagabine drug - effect. it should be noted that for f(1, 14) the critical value is 4.6 and for f(3, 42) the critical value is 2.82 (alpha=0.05). eighteen right - handed volunteers (14 men and 4 women) participated in the experiment after giving informed consent, with all procedures approved by the uk national research ethics service (south east wales). volunteers were screened and excluded for personal histories of neurological and psychiatric disease, current recreational or prescription drug use, and impaired liver function (by standard liver function tests). early in the data collection, three women volunteers were not able to complete the experiment after becoming too heavily sedated. from this point in the data collection, women were excluded from recruitment, leaving completed data sets from 14 men and 1 woman to analyze (mean age 25.5, range 2032). all results and conclusions are similar whether this one woman was included / excluded from the analysis. participants were scanned on 2 separate days, separated by a minimum 7-day washout period, at approximately the same time - of - day. on each day, an initial pre ' meg recording was obtained then participants orally ingested a capsule containing either placebo or 15 mg of tiagabine (gabitril). this oral dose is similar to the 16 mg used in a pet study by frankle (2009) and identical to several ongoing imaging studies by our group. participants were blinded to the contents of the capsule and placebo / control session order was counterbalanced across participants. following drug / placebo administration, meg recordings were obtained from participants at 1, 3, and 5-h time - points. at the conclusion of every meg recording, participants completed several psychological questionnaires including, the biphasic alcohol effects scale (martin, 1993), the subjective high assessment scale (schuckit, 1980) and asked to qualitatively report their psychological state. during each meg recording, participants sat in a magnetically shielded room and were presented with a visual stimulus consisting of a, vertical, stationary, maximum contrast, three cycles per degree, square - wave grating presented on a mean luminance background. the stimulus was presented in the lower left visual field and subtended 8 both horizontally and vertically. a small red fixation square was located at the top right - hand edge of the stimulus and remained on for the entire stimulation protocol (muthukumaraswamy, 2010 ; swettenham, 2009). the stimulus was presented on a mitsubishi diamond pro 2070 monitor controlled by the psychophysics toolbox (brainard, 1997 ; pelli, 1997). the screen size was 1024 by 768 pixels and the monitor frame rate was 100 hz. the monitor was outside the magnetically shielded room and viewed directly from within, at 2.15 m, through a cut - away portal in the shield. the duration of each stimulus was 11.5 s followed by 1.5 s of fixation square only. participants were instructed to fixate for the entire experiment and, in order to maintain attention, were instructed to responses slower than 750 ms from the termination of the stimulus triggered a visual warning for participants. in each recording session, 120 stimuli were presented and participants responded with their right hand. whole - head meg recordings were made using a ctf 275-channel radial gradiometer system sampled at 1200 hz (0300 hz bandpass). an additional 29 reference channels were recorded for noise cancellation purposes and the primary sensors were analyzed as synthetic third - order gradiometers (vrba and robinson, 2001). three of the 275 channels were turned off because of excessive sensor noise. at the onset of each stimulus presentation participants were fitted with three electromagnetic head coils (nasion and pre - auriculars), which they wore for the entire recording day, and were localized relative to the meg system immediately before and after the recording session. each participant had a 1 mm isotropic mri scan available for source localization analysis. to achieve mri / meg co - registration, the fiduciary markers were placed at fixed distances from anatomical landmarks identifiable in participants ' anatomical mris (tragus, eye centre). offline, data were first epoched from 1 to 1 s around stimulus onset and each trial visually inspected for data quality. data with gross artefacts, such as, head movements and muscle clenching were excluded from further analysis. reaction times were calculated for each trial relative to the (variable) offset of each grating stimulus. any trial where there was no behavioural responses or a response > 750 ms were classified as incorrect responses and excluded from further analysis. for each participant, we ensured that equal number of trials, from each recording was used in the analysis. to achieve this, trials were removed from the end of each recording run so that each participant had an equal number of trials submitted for each condition in the analysis. following these trial removal procedures, the mean number of trials remaining to analyze was 105.5 per participant (range 82117). three source localizations were performed on each data set using synthetic aperture magnetometry (robinson and vrba, 1999), one for gamma (3080 hz), one for alpha (815 hz), and one for evoked responses (samerf ; robinson, 2004). correspondingly, three global covariance matrices were calculated for each data set, one for gamma, one for alpha, and one for samerf (0100 hz). based on these covariance matrices, using the beamformer algorithm (robinson and vrba, 1999), three sets of beamformer weights were computed for the entire brain at 4 mm isotropic voxel resolution. a multiple local - spheres (huang, 1999) volume conductor model was derived by fitting spheres to the brain surface extracted by fsl 's brain extraction tool (smith, 2002). readers unfamiliar with sam are referred to several methodological reviews (hillebrand, 2005 ; vrba and robinson, 2001). for gamma - band sam image reconstruction virtual sensors were constructed for each beamformer voxel and student 's t - images of source power changes computed using a baseline period of 1 to 0 s and an active period of 0 to 1 s, while for alpha - band sam image reconstruction a baseline period of 1 to 0.3 s and an active period of 0.3 to 1 s was used. this latter time window was used to avoid confounding alpha responses with the time - locked evoked response. after generating the source reconstruction images, the voxel with the strongest power increase (in the contralateral occipital lobe) was located for gamma and the voxel with the strongest source power decrease located for alpha. the source analysis and peak voxel identification procedures were carried out separately for each recording as participants were moved in and out of the meg between recordings, which would alter their head position in the meg dewar. to reveal time - frequency responses at these locations, the virtual sensors at these locations were repeatedly band - passed filtered between 1 and 160 hz at 0.5 hz frequency step intervals, filtering with an 8 hz bandpass, third - order butterworth filter (le van quyen, 2001 ; muthukumaraswamy, 2010). the hilbert transform was used to obtain the amplitude envelope and time - frequency spectra were computed as a percentage changed from the pre - stimulus baselines for each frequency band. from these spectra, the peak frequency and amplitude at that frequency for late gamma amplitude increases (3080 hz, 0.31 s) were quantified by collapsing across the time dimension and finding the maximal (or minimal for alpha) frequency. we also checked for confounding effects of using relative change baselines by computing the average absolute values of the source amplitude estimates within the respective time - frequency bins. for samerf, the computed evoked response was passed through the 0100 hz beamformer weights to generate samerf images (robinson, 2004) at 10 ms intervals from 50150 ms. the image window (usually 7080 ms or 8090 ms) with the maximal response in visual cortex was identified and the maximal voxel selected as the peak location for further analysis. for this, the evoked field was computed for this virtual sensor (0.2 to 0-s baseline) and the peak amplitude and latency of the m80 and m150 responses quantified. for the production of grand - average sam maps, individual sam images were first spatially normalized onto the mni (t1) average brain using fmrib 's linear affine registration tool (jenkinson and smith, 2001). this was done by first obtaining a set of warping parameters by registering the participant 's anatomical mri with the average brain and then applying these parameters to the sam source power maps. the grand - average images presented in figure 2 are the grand - average across both participants and conditions. for these analyses, there were main effects of day ', session ', and an interaction term drug ', presented in that order. here, the critically important term is the interaction term, indicative of a tiagabine drug - effect. it should be noted that for f(1, 14) the critical value is 4.6 and for f(3, 42) the critical value is 2.82 (alpha=0.05). figure 1 shows summed scores from the subjective high assessment scale (schuckit, 1980) that participants completed at the end of each recording session. individual items that showed the largest increase relative to placebo were feeling of intoxication ', dizziness ', and task response accuracy data showed a significant interaction effect (f(1, 14)=3.71, f(3, 42)=2.34, f(3, 42)=2.99, p 30 hz) band (early gamma '), followed by a longer - lasting elevation of gamma - frequency amplitude in a narrower frequency range (late gamma '). in the lower frequencies, there is an initial transient onset response, which is a characteristic signature of the evoked response (clapp, 2006 ; muthukumaraswamy, 2010 ; swettenham, 2009), followed by a sustained alpha amplitude decrease. response components were parameterized (using the optimal virtual sensor locations for each component) for each participant and subjected to statistical analysis (figure 3). neither early gamma frequency (f(1, 14)=0.093, f(3, 42)=2.17, f(3, 42)=0.73) or late gamma frequency (f(1, 14)=1.42, f(3, 42)=1.16, f(3, 42)=1.70) showed any significant effects. similarly, neither early gamma amplitude (f(1, 14)=0.23, f(3, 42)=0.25, f(3, 42)=1.57) or late gamma amplitude (f(1, 14)=0.02, f(3, 42)=2.24, f(3, 42)=1.55) demonstrated any effects. alpha activity showed some non - drug specific effects including, alpha frequency showing a main effect of session but no interaction effect (f(1, 14)=2.32, f(3, 42)=3.58, p<0.03, f(3, 42)=0.53), while alpha amplitude showed a main effect of day effect but no interaction effect (f(1, 14)=7.78, p<0.02, f(3, 42)=0.46, f(3, 42)=0.93). in addition, we performed similar statistical analyses on the individual time - frequency pixels ' to look for subtle time - frequency effects (data not shown) and these demonstrated similar null effects. finally, we checked to see if there were any confounding effects of using relative change baselines by extracting absolute amplitude values (no baselining performed) for the various time - frequency windows. for gamma, no effects were seen for the early (f(1, 14)=3.57, f(3, 42)=0.87, f(3, 42)=0.14), late (f(1, 14)=3.30, f(3, 42)=0.15, f(3, 42)=0.26), or baseline periods (f(1, 14)=1.05, f(3 42)=0.08, f(3, 42)=0.21). for alpha, no effects were seen in the active (f(1, 14)=3.98, f(3, 42)=1.41, f(3, 42)=0.67) or the baseline period (f(1, 14)=0.15, f(3, 42)=0.08, f(3, 42)=0.14). in figure 4 these demonstrate a clear reduction in the amplitude of the first evoked response component that occurs just after 80 ms (labeled m80 ') following tiagabine and an enhanced second component (labeled m150 '). statistical analysis of the m80 amplitudes data showed a main effect of day, session, and critically an interaction effect (f(1, 14)=24.7, p<0.001, f(3, 42)=30.9, p<0.001, f(3, 42)=13.1, p<0.001). m150 amplitudes showed significant effects of day, session, and an interaction effect (f(1, 14)=7.26, p<0.001, f(3, 42)=3.60, p<0.03, f(3, 42)=4.00, p<0.02). the m80 latency showed a day effect but no other effects (f(1, 14)=8.06, p<0.02, f(3, 42)=0.49, f(3, 42)=0.14) and m150 latency showed no significant effects (f(1, 14)=0.14, f(3, 42)=2.34, f(3, 42)=0.35). no correlations were observed between the shas measures or reaction times and the evoked field magnitudes and their respective change from baseline scores. finally, we conducted an analysis to check whether there were any systematic differences in virtual sensor locations that might potentially have confounded the results. the virtual sensor locations for gamma, alpha, and evoked response locations were tested using anova in x, z, and z directions. no significant interaction effects were observed, indicating no systematic changes in response locations with tiagabine. in the current experiment, we elevated endogenous gaba in human participants by administering the gat-1 blocker tiagabine, while measuring evoked and induced visual responses with meg. the data showed a clear modification of visual evoked responses but, contrary to our hypotheses, we found no alteration in stimulus - induced oscillatory responses in either the alpha or gamma - frequency bands. we believe our approach did significantly increase gaba based on previous pet studies using a similar dose of tiagabine that show gaba - related changes in tracer binding (frankle, 2009, 2012 ; myers, 2012). one particularly striking feature of the dissociation between evoked and induced responses is that this occurred in the same temporal segments of meg data. that is, there was a clear reduction in the m80 evoked response but no alteration in the temporally congruent early gamma spike (50100 ms). the early gamma response may reflect early ascending input from the lateral geniculate nucleus (castelo - branco, 1998) and is manifested across all cortical layers, although particularly in layers ii and iii (xing, 2012), whereas early evoked responses are mostly generated in layer iv (kraut, 1985). the pharmacological basis of this selective modulation presumably lies in the complex distribution of gabaa and gabab receptor subunits across the lamina of v1 (hendry, 1994 ; zilles, 2004). in particular, the inhibition provided by gabaa activation is thought to control response gain to stimuli in primary visual cortex (katzner, 2011) and to control the layer - specific spread of excitatory activity caused by visual stimulation (olivas, 2012). reduction of this horizontal spread of excitation (decreased spatial summation) during stimulation onset may be the mechanism underlying the diminished evoked responses we have observed. this selective modulation also suggests other more - simplistic interpretations of the data are incorrect. for example, one might argue that the reduction in m80 amplitude is due to reduced task vigilance, attention, or even fixation control as participants changed psychological state after tiagabine ingestion. however, each of these explanations would also be expected to modulate oscillatory gamma amplitude (hoogenboom, 2010 ; kahlbrock, 2012). similarly, as these dissociable parameters are all based on the same data, they all have the same relative signal - to - noise ratio. the 45% peak reduction we observed in m80 amplitude is significantly larger than other studies examining visual evoked potentials (veps) with benzodiazepenes that affect gabaergic function. in all, 20 mg diazepam administered intravenously (loughnan, 1987) or 10 mg administered orally (bartel, 1988) has no effect on the vep. however, 6 and 12 mg of the benzodiazepine bromazepam has been shown to reduce erp components beginning with the n1 (bauer, 1997) and 10 g / kg of intravenous midazolam caused a relatively small reduction in n75-p100 amplitude (van leeuwen, 1992). in fact, it has been shown that tiagabine, which increases gaba rather than acting via the benzodiazepine receptor, actually increases the amplitude of early somatosensory evoked potential components (restuccia, 2002). the lack of correlation between the subjective measures in the shas and evoked field changes suggest that the changes in evoked field are not simply attributable to the arousal state of participants. another possibility is that the decreased evoked responses we observed might be due to decreased fixation control during the tiagabine - affected conditions. however, loss of fixation control might be expected to alter the response location of both retinotopic evoked responses (di russo, 2002) and also gamma - band responses as these are retinotopically organized (perry, 2011). our analysis of source peak activity locations indicated no shifts in spatial location for either of these response components. further, the amplitude of gamma - band activity is very sensitive to the retinotopic position of the stimulus and falls off more rapidly with eccentricity (swettenham, 2009) than evoked components. nevertheless, measurement of fixation position via either eye - tracking or electrooculography would be a useful addition to future experiments. direct manipulation of gaba levels in this experiment does not appear to have altered gamma frequency, and potential reasons for this are worth considering. it is possible that the lack of effect in the oscillatory domain is due to a lack of statistical power, or perhaps because an inadequate dose of tiagabine was given. however, we do not believe that it would be ethically or feasibly possible to give a much higher dose to control participants and still have them complete the required tasks given the profound behavioural and subjective effects on participants. we also note that overall data quality was very high, with gamma activity identifiable in every subject. further, although there is no guarantee that raising gaba levels with this dose of tiagabine has a direct effect on the synaptic (eg, time constants) properties of neurons in visual cortex, this is unlikely since similar doses have been shown to change 11c - flumazenil and 11c - ro15 4513 binding potentials (frankle, 2009 ; myers, 2012). in addition, tiagabine - induced increases in gaba could potentially modulate gabaergic synapses on all neuronal subtypes, including pyramidal cells and inhibitory interneurons, and receptor subtypes (gabaa (six subtypes) and gabab) with unpredictable effects on oscillatory dynamics until the role of all these gaba receptors is elucidated. in a previous study we found, across individuals, that the frequency of stimulus - induced network gamma oscillations in primary visual cortex was positively correlated with the concentration of gaba measured with mrs. mrs measures the total bulk of gaba in a given area of the brain (usually from a rather large voxel, eg, 27 cm). consistent with invasive evidence, mrs returns concentrations of gaba in the millimolar range (puts and edden, 2012). in the synaptic cleft, gaba concentration can reach the millimolar range (mody, 1994) but this rapidly declines (< 1 ms). although estimates for ambient levels gaba in the extracellular space range from tens of nanomolar to micromolar (farrant and nusser, 2005) these are still orders of magnitude less than the total bulk of gaba. therefore, mrs measures of gaba concentration probably depend on interneuron numbers / inhibitory synapse numbers, and/or the rate constants of glutamic acid decarboxylase, gaba transaminase, and gat-1. in particular, interneuron density / synapse numbers, which are inherently structural parameters (schwarzkopf, 2012), would not be susceptible to short - term pharmacological manipulations, as we have attempted here. although our specific hypothesis was not confirmed, the data here are promising for the relatively unexplored field of pharmaco - meg (hall, 2010). we note another very recent study using the cholinergic agonist phystostigmine found a selective modulation of alpha oscillation amplitude (but not gamma) in response to visual stimuli in humans with meg (bauer, 2012). these studies therefore demonstrate that meg can noninvasively characterize the effect of pharmacologically targeted and physiologically selective agents using alterations in quantitative neuronal biomarkers. future studies systematically investigating the pharmacological sensitivity of meg responses will allow the development of data - driven pharmacological models of these responses. given the wide range of safe and selective interventional agents that can be safely administered to interact with neurotransmitter systems, this bodes well for our future understanding of the neuropharmacological basis of meg / eeg. | the electroencephalographic / magnetoencephalographic (eeg / meg) signal is generated primarily by the summation of the postsynaptic currents of cortical principal cells. at a microcircuit level, these glutamatergic principal cells are reciprocally connected to gabaergic interneurons. here we investigated the relative sensitivity of visual evoked and induced responses to altered levels of endogenous gabaergic inhibition. to do this, we pharmacologically manipulated the gaba system using tiagabine, which blocks the synaptic gaba transporter 1, and so increases endogenous gaba levels. in a single - blinded and placebo - controlled crossover study of 15 healthy participants, we administered either 15 mg of tiagabine or a placebo. we recorded whole - head meg, while participants viewed a visual grating stimulus, before, 1, 3 and 5 h post tiagabine ingestion. using beamformer source localization, we reconstructed responses from early visual cortices. our results showed no change in either stimulus - induced gamma - band amplitude increases or stimulus - induced alpha amplitude decreases. however, the same data showed a 45% reduction in the evoked response component at 80 ms. these data demonstrate that, in early visual cortex the evoked response shows a greater sensitivity compared with induced oscillations to pharmacologically increased endogenous gaba levels. we suggest that previous studies correlating gaba concentrations as measured by magnetic resonance spectroscopy to gamma oscillation frequency may reflect underlying variations such as interneuron / inhibitory synapse density rather than functional synaptic gaba concentrations. |
through the process of natural selection, nature has evolved well - adapted macromolecular structures that interact with biological small molecules. oxidoreductases, for example, rely on the nicotinamide coenzymes to supply them with the redox equivalents required to sustain their catalytic cycles. two forms of natural coenzymes exist : the phosphorylated (nadp / nadph) and nonphosphorylated (nad / nadh) forms (figure 1a). nicotinamide coenzymes essentially contain two structural motifs, the nicotinamide moiety conferring their electrochemical function (i.e., serving as an electron source or sink in the form of a hydride) and the adenosine dinucleotide moiety conferring the separation between anabolic and catabolic pathways. nadp is involved in anabolic redox processes, whereas nad is mostly found in processes dealing with energy metabolism. while this separation between different metabolic pathways is essential for cellular survival, it is irrelevant in chemical applications of redox enzymes. therefore, there is a renewed interest in the design of simple synthetic analogues of the natural nicotinamide coenzymes. the laboratory - based design and synthesis of small molecule biomimetics that can functionally substitute (or even outperform) those available in nature is a major challenge. the development of biomimetics that can be synthesized easily and exploited widely would be a game changer in establishing new manufacturing technologies that would be too expensive using natural biological molecules. the natural coenzymes nad(p)h are prohibitively expensive and chemically too unstable for stoichiometric use in fine and specialty chemicals manufacture. this has prevented their general uptake as a source of reducing equivalents in biocatalytic oxidoreductase - catalyzed reactions and has led to the development of in situ regeneration systems to replenish nad(p)h (e.g., using enzymatic, photochemical, and electrochemical approaches) or the use of hydrogen - borrowing biocatalytic cascades. in turn, the limited stability and expense of natural nicotinamide coenzymes have driven a search for more stable synthetic nicotinamide coenzyme analogues that can interface generally with biological oxidoreductase catalysts. ene reductases (ers) from the old yellow enzyme family (ec 1.3.1.31) are particularly attractive as they are a group of broad specificity biocatalysts that catalyze the asymmetric reduction of activated c = c bonds, forming up to two new stereogenic centers at the expense of the natural nicotinamide coenzyme nad(p)h as an electron source. in particular,,-unsaturated carbonyl compounds (e.g., enals and enones) and nitroalkenes are excellent substrates. in general,,-unsaturated diesters as well as in contrast, the efficient reduction of,-unsaturated monoacids or monoesters requires an additional electron - withdrawing group in the - or -position in order to activate the alkene moiety. the ability to form new stereogenic centers and the wide acceptance of different substrate types are driving the exploitation of ers toward novel applications in redox biocatalysis and implementation in key industrial processes. ers have been studied extensively over the past decade, and there is detailed information known, such as their structure, reaction mechanism, substrate scope, kinetic properties, and biocatalytic approaches. the catalytic cycle of er - catalyzed reactions can be divided into two separated half - reactions : in the reductive half - reaction, a hydride is transferred from nad(p)h to the enzyme - bound flavin (flavin mononucleotide ; fmn). after release of oxidized nad(p), this hydride is then transferred in the oxidative half - reaction from the flavin n5 position to the activated alkene substrate (figure 1b). (a) structure of nad(p)h and synthetic nicotinamide biomimetic mnadhs (15) and (b) the catalytic cycle of er - catalyzed reactions. recently, nicotinamide coenzyme biomimetics (mnadhs) were shown to replace nad(p)h in er - catalyzed reactions. a series of mnadhs (15) has been synthesized to replace the natural coenzymes for various ers (figure 1a). these coenzyme biomimetics show great promise and have sparked renewed interest in redox biocatalysis. in addition, reduction of c = c double bonds and oxyfunctionalization reactions such as epoxidation or hydroxylation using mnadhs have been reported. however, structural mechanistic understanding of biomimetic chemistry in er - catalyzed reactions is lacking, preventing the successful exploitation of these biomimetics. biomimetic complexes and analysis of reaction cycle kinetics, both presented here for the first time for a panel of structurally diverse nicotinamide biomimetics in combination with several ers. these structural mechanistic analyses show how selected biomimetics perform as well and, in some cases, even outperform the natural coenzymes. the chemical stability of the biomimetics, coupled with their enhanced enzymatic reactivity, gives rise to the rare situation where chemical designs surpass those available in the natural world. these biomimetics provide an attractive means of performing redox biotransformations with ers (and mutatis mutandis other redox biocatalysts) under mild reaction conditions and at relatively low cost. we set out to determine the structures of selected complexes formed between er biocatalysts and the biomimetics to ascertain if the biomimetic design features replicate those seen for the natural enzyme the tetrahydro forms of the natural (nadph4) and biomimetic (mnadh4s) ligands were used to prevent hydride transfer in the crystal complexes (figure 2). crystal structures were determined for xena in complex with nadph4 and tetrahydro - biomimetics (1b, 2b, and 3b) at 1.61.8 resolution. clear electron density was observed for the bound nadph4 extending from the nicotinamide moiety and becoming disordered after the diphosphate moieties. nadph4 binds in a stacked arrangement sitting above the isoalloxazine ring of the fmn cofactor, and his181 and his178 are located 2.8 and 2.9, respectively, from the nicotinamide oxygen, with cys25 some 3.6 from the nicotinamide n7 position. each of the three bound biomimetics occupies the same region of the active site as that observed for nadph4 (figure 2), with only very minimal changes observed in the positions of surrounding residues in the active site. however, the reduced bulk of the biomimetics when compared to the dinucleotide portion of nadph4 (figure 2) results in trp302 adopting an alternative conformation that effectively reduces the volume of the active site in this region. this feature is observed with each of the three biomimetics, with the remainder of the active site residues remaining static when compared to the nadph4 complex. the determined structures reveal that the biomimetic designs capture the design features of the natural coenzyme in that the nicotinamide moiety is positioned optimally over the re - face of the enzyme - bound fmn cofactor for hydride transfer from the c4 atom of the biomimetic to the n5 atom of the flavin isoalloxazine. structures cocrystallized in the presence of nadph4 and biomimetic compounds 1b, 2b, and 3b are shown, superimposed based upon secondary structure elements using ssm (coot). residues trp302, his181, and his178 are shown in stick representation colored with white carbon atoms and blue nitrogen atoms. the ordered portion of the nadph4 is shown as semitransparent spheres in all - atom color (carbon, purple ; oxygen, red ; phosphorus, orange) with its associated underlying fmn cofactor shown as gray spheres. three biomimetic compounds are shown in stick representation with their respective trp302 residues colored to match the biomimetic. dual occupancies are present for residue trp302 for each of the three mimics, indicating that occupancy is less than 100% for these compounds. in ers, hydride transfer from the natural coenzyme to the flavin mononucleotide cofactor occurs by quantum mechanical tunnelling. the probability of hydride transfer by quantum mechanical tunnelling is affected by sub - angstrom changes in donor acceptor distance. although the nicotinamide synthetic analogues and the natural coenzyme bind to the er active site with similar geometry, subtle changes in the binding mode will influence the kinetics of hydride transfer. we determined the catalytic parameters for the reductive half - reaction (figure 1b) for four ers, namely, petnr, toye, xena, and tsoye, using anaerobic rapid mixing stopped - flow measurements, employing the natural coenzymes nad(p)h and the mnadh biomimetics (15) (figure 1a). kred, reaction rates [s ] ; kd, dissociation constants [m ] ; n.d., not determined due to poor conversion in biocatalytic reactions. reaction conditions : [er ] = 810 m, buffer 50 mm mops (ph 7.0) + 5 mm cacl2, at 30 c, under anaerobic conditions with constant n2 flow, [coenzyme ] = varied. not possible to measure at lower concentrations of the coenzyme due to non - pseudo - first - order conditions. rate of reduction occurs within the dead time of the stopped - flow instrument at higher concentrations of coenzyme. table 1 reports the reaction rates (kred) and the apparent dissociation constants (kd) for flavin reduction. most ers possess a higher affinity for the natural coenzyme nadph compared to that for nadh. this affinity is evident from the values of the apparent dissociation constants (kd) calculated from stopped - flow studies that differ by about an order of magnitude for all ers investigated. the exception is tsoye, with which the kd values are essentially the same with both coenzymes. furthermore, the values of the kinetic constants for the reductive half - reaction (i.e., hydride transfer ; kred) depend on the type of natural coenzyme employed. the kred values differed from 10- to 36-fold higher when using nadph compared to that using nadh. in particular, petnr shows significantly higher kd values compared to those of the other ers, and it is the only enzyme where kred / kd is 2 orders of magnitude higher for nadph compared to that for nadh. with petnr, the faster rates of flavin reduction with nadph compared to that with nadh have been attributed to promoting vibrations that couple to the reaction coordinate and modulate the donor acceptor distance for hydride tunnelling. the rates of the reductive half - reactions were elevated using the nicotinamide biomimetic 2 compared to that using nadph for each of the four ers investigated. additionally, biomimetic 2 binds with apparent higher affinity to the active site of the ers compared with that of nadph in two cases out of four, namely, with petnr and xena (kd 184 and 8 m, respectively). as a consequence, the kred / kd values for the reductive half - reaction of petnr and xena with the synthetic biomimetic 2 indicate superior catalytic efficiency compared to that of reactions with nadph. the highest kred / kd value of 48 500 mm s was obtained employing biomimetic 2 with xena, and to the best of our knowledge, this is the first report of a nicotinamide biomimetic surpassing the efficiency of natural coenzymes. biomimetic 4 harbors an acid group instead of the amide group in the nicotinamide moiety. the kred values were higher than 500 s with xena and tsoye and reached a maximum of 770 s with toye. further detailed analysis is reported in table 1, which indicates that each biomimetic performed better than nadh in reactions with petnr (i.e., higher reaction rates ; apparent dissociation constants were similar for the biomimetic and nadh). a similar trend was observed in hydride transfer reactions mediated by toye. in this case, the kred values were elevated for all of the biomimetics when compared to those for both natural coenzymes. with that said, the ratio kred / kd was always in favor of the natural coenzymes due to the modest binding affinity of toye for the synthetic biomimetics. xena stands apart from the other ers : xena was the only enzyme capable of catalyzing the reduction of an activated alkene using biomimetic 5 (see enzyme - catalyzed redox reactions using coenzyme biomimetics). biomimetic 5 differs from the others in that it possesses a nitrile instead of carbonyl moiety, and kinetic parameters for the reaction of biomimetic 5 with xena were determined. the kd values for the reduction with 5 and nadph with xena are similar, whereas kred differs by 3 orders of magnitude (i.e., 0.08 and 67 s for 5 and nadph, respectively). in general, the highest reaction rate with xena was obtained using biomimetic 4, whereas three biomimetics (1, 2, and 3) showed a higher apparent affinity compared with that of the natural coenzymes. reactions with tsoye confirmed the general trend, i.e., increased reaction rates with the biomimetics but elevated kd values for the enzyme ers catalyze the reduction of activated alkenes following a ping - pong bibi mechanism. stopped - flow experiments demonstrated that reductive rates are higher for a number of nicotinamide biomimetics than for natural coenzymes, but in order to obtain better insight into the overall catalytic cycle (relevant to exploitation of any er with a coenzyme biomimetic in biocatalysis), steady - state kinetic investigations were undertaken studying three ers, namely, petnr, toye, and xena. in each case, 2-cyclohexen-1-one was used as the second substrate in reactions employing nad(p)h and the biomimetics. for many ers the oxidative half - reaction limits the overall kinetics of the complete catalytic cycle, and the kapp values were similar when using nadph or the synthetic biomimetics. only the use of nadh led to a slight deviation from this trend (table 2). again, biomimetic 2 is of particular interest as it gave the highest ratio of kapp / km in two cases out of three (i.e., petnr, xena), confirming that this synthetic analogue has an improved catalytic efficiency compared to that with nadph. reaction conditions : buffer 50 mm mops (ph 7.0) + 5 mm cacl2, at 30 c, under anaerobic conditions with constant n2 flow. [coenzyme ] = varied, [er ] = 100250 nm ; (mnadhs), see the supporting information. our previous study revealed that biomimetic coenzymes 15 are readily accepted by the ers from thermus scotoductus (tsoye) and bacillus subtilis (yqjm). to evaluate whether this was generally the case for all of the members of the er family, we investigated a more representative selection of ers. we carried out the asymmetric reduction of ketoisophorone 6 to levodione (r)-6a using an extended panel of 12 ers from the oye family (petnr, toye, oye2, oye3, xena, xenb, leopr1, nera, mr, tsoye, droye, and rmoye) and the five biomimetics (15). the results in table 3 show that the biomimetics were overall well - accepted, leading up to quantitative conversions, with the exception of mnadh 5. in fact, xena was the only enzyme that efficiently accepted mnadh 5 as the hydride source. this finding is in line with the kinetic parameters shown in tables 1 and 2. as expected, the source of hydride, whether from the natural or synthetic coenzyme, did not have a significant influence on the enantiomeric excess (table s3, supporting information), which is dependent on the intrinsic stereoselectivity of the enzyme only. the slight deviations in the enantiomeric excess are due to the slow spontaneous racemization of the product in aqueous medium. reaction conditions : = 10 mm, [er ] = 3.05.0 m, [coenzyme ] = 11 mm (unless otherwise specified), mops buffer (50 mm, ph 7.0, 5 mm cacl2), inert atmosphere, 30 c, 4 h ; the mnadhs were directly added as solids. the biocatalytic reduction described in the previous section was carried out at the expense of stoichiometric amounts of nicotinamide biomimetic as hydride donor, which is a limitation in developing inexpensive biocatalytic transformations. the rhodium complex [cprh(bpy)(h2o) ] is capable of reducing biomimetics 1 and 3 using formate as a hydride source. hence, to use the biomimetics in catalytic amounts, we implemented a recycling system for the er - catalyzed reduction of 6 to (r)-6a, as depicted in figure 3a. the reduced form of the rh - catalyst binds the carbonyl moiety of the oxidized form of the biomimetic, followed by hydride transfer from the metal center to the aromatic ring of the biomimetic. after the release of the reduced mimic from the metal center, the organometallic catalyst is reduced back at the expense of formate, which is oxidized to co2. after a preliminary optimization of the reaction conditions (see the supporting information for more details), the reduction of ketoisophorone 6 was performed with tsoye, oxidized biomimetic 1a, and the rhodium complex [cprh(bpy)(h2o) ], using sodium formate as the hydride donor. under these reaction conditions (see the supporting information for more details), 6.6 mm product with an ee of 85% was obtained starting with 1 mm 1a. therefore, an in situ recycling method for mnad1a with an er was demonstrated. (a) investigated recycling system of mnadhs with tsoye and the rhodium complex [cprh(bpy)(h2o) ]. reaction conditions : = 10 mm, [er ] = 3.0 m, [oxidized coenzyme 1a ] = 1 mm, [nahco2 ] = 60 mm, [rh complex ] = 0.25 mm, mops buffer (50 mm, ph 7.0, 5 mm cacl2), inert atmosphere, 50 c, 4 h ; the mnadhs were directly added as solids. (b) proposed mechanism for the reduction of nad(p) by [cprh(bpy)(h2o) ]. the table shows that 66% conversion could be obtained after 4 h reaction time. the structural, kinetic, and biocatalytic studies of the biomimetics with a wide range of er enzymes demonstrate that they faithfully capture, at the functional level, design aspects inferred from the structures of the natural coenzymes and the natural coenzyme er protein complexes. that the biomimetics are also stable and can be recycled in biocatalytic transformations the general utility of these synthetic biomimetics beyond the er family is also important in adopting their use more widely in biocatalytic manufacture. enzymes were chosen to encompass some of the most useful transformations for the manufacture of chemical products such as the monooxygenation of unfunctionalized carbon hydrogen bonds or of ketones to esters, the chemoselective reduction of carboxylic acids to aldehydes and the reverse reaction, the reductive amination of ketones, the reduction of alkenes using non - flavin - dependent enzymes, and the regeneration of the oxidized form of the biomimetics. activity assays using the non - flavin - dependent double - bond reductase from nicotiana tabacum (ntdbr) showed that the biomimetics also support the reduction of cinnamaldehyde to hydro - cinnamaldehyde at the expense of biomimetics 14, with conversion ranging from 5 to 21%. further kinetic data (see the supporting information for more details) showed that ntdbr has the highest preference for nadph as the hydride donor for the reduction of cinnamaldehyde (kcat 40 min, km 91 m). conversely, the observed rate measured at 5 mm substrate concentration revealed a reduced turnover number for nadh (kobs 4 min) as well as biomimetics 1 (kobs 0.15 min) and 3 (kobs 0.08 min). additionally, a nadph oxidase isolated from b. subtilis(53,54) was also capable of efficiently oxidizing biomimetics 14 using molecular oxygen as the only electron acceptor. these are examples that demonstrate the versatility of the cofactor analogues in addition to the previously established system with a styrene monooxygenase, greatly improving the efficiency of electron transfer for the selective epoxidation of styrene derivatives. biocatalyst complexes, similar to that described herein, will guide further design of new biomimetics, extending their reach to other useful transformations. this should extend the biomimetic series beyond compounds 15 and enable widespread application of nicotinamide biomimetics in biocatalytic processes. we have shown that synthetic coenzyme biomimetics can outperform natural coenzymes in biotransformations reactions with widely used redox biocatalysts. that laboratory - based designs can outperform these synthetic coenzymes are inexpensive to manufacture, are stable relative to their biological counterparts, and are operational with a wide range of biocatalysts taken from the er family and a few other oxidoreductases. additionally, the biomimetics can be recycled at the expense of formate and are therefore exploitable using only catalytic amounts. implementation of these synthetic biomimetics as well as the design of more sophisticated analogues capable of operating with a variety of other oxidoreductases will facilitate the use of redox biocatalysts in chemicals production at low cost and thereby transform the use of oxidoreductases more widely in industrial biocatalysis. | the search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. here, we investigate the efficiency of man - made synthetic biomimetics of the natural coenzymes nad(p)h in redox biocatalysis. extensive studies with a range of oxidoreductases belonging to the ene reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase xena with selected biomimetics. in selected cases, these biomimetics outperform the natural coenzymes. better - than - nature biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost. |
when upgrading a material from a low value to a higher value, two main issues need to be considered : the type of potential end - product(s) and the required properties. this study focuses on the extrusion of protein - based plastics for potential use in packaging for two reasons. the present variety of packages is extensive, but the request for renewable and biodegradable low - cost packaging material has increased rapidly in the last decade. this trend seems to be continuing, as most brand owners and legislators are searching for options to create plastics from petroleum. the required material properties for packaging are, in many cases, more demanding than for other plastic products. however the exact criteria differ depending on the type of package, filling / sealing systems, transport, storage, content, appearance, product design, etc. all these parameters should be considered by a packaging developer, but all can not be of the highest priority at once when initiating the development of a new and unexplored material. the properties in focus for this study were the mechanical and barrier performances. extrusion is the processing method of choice for two reasons : extrusion is a common and efficient method for making packaging plastics, and it generally does not involve a solvent, as in solution casting. thus, no drying step is needed at the end of the process. it has shown potential as a packaging plastic in a number of studies. despite this, some challenges remain. crambe abyssinica is an interesting oilseed plant in that it is not a food resource and can be grown in many different agronomic conditions. as with wheat gluten, it is obtained as a defatted crambe meal, with protein as the largest component. it also contains a sizeable amount of nitrogen - free extracts, such as carbohydrates and fiber. the meal has relatively poor cohesive properties and needs to be blended with a material of higher cohesion. in this study, wheat gluten is used as a supportive additive to the crambe meal. to improve the toughness / extensibility of the protein material, a plasticizer is commonly used as an additive as well. in this study, glycerol is used, which is a side product of the plant oil industry (e.g., rape seed methyl ester fuels) and is readily available at a low cost. urea, also renewable, is used as a denaturant in order to give the extrudate the proper cohesion. renewable materials, especially those that are used directly from nature, without purification, modification, or chemical synthesis, are, in most cases, not suitable for high - temperature treatment. the challenge is to find suitable processing parameters and compositions that result in an extrudate with properties that allow it to compete with products from petroleum. this study focuses on the characterization of the mechanical and barrier properties of a new bio - based material produced from crambe meal processed with different additives and at different conditions. the full details of the mechanical and oxygen barrier features are found in rasel.. note : crambe seeds (cultivar galactica) were supplied by the plant research international, wageningen, netherlands. both the crambe meal and the wheat gluten were stored at -18 c until further use. sieving crambe sieve the crambe meal with a round, fine - mesh stainless steel kitchen sieve (pore size : ~1.5 mm, 14 mesh), to remove large fiber fractions and uncrushed seeds. sieve the crambe meal with a round, fine - mesh stainless steel kitchen sieve (pore size : ~1.5 mm, 14 mesh), to remove large fiber fractions and uncrushed seeds. milling crambe to reduce the particle size and make the material more homogeneous, mill the sieved crambe meal in a rotary ball mill.mill 250 g of crambe meal each time in a 7 l jar with 21 - 25 mm diameter ceramic balls using a 53 rpm jar revolution rate and a milling time of 24 hr. to reduce the particle size and make the material more homogeneous, mill the sieved crambe meal in a rotary ball mill. mill 250 g of crambe meal each time in a 7 l jar with 21 - 25 mm diameter ceramic balls using a 53 rpm jar revolution rate and a milling time of 24 hr. conditioning before further processing, condition all the ball - milled crambe meal and wheat gluten powder in open jars for a minimum of 48 hr at 23 c and a relative humidity of 50% in a climate - controlled room. before further processing, condition all the ball - milled crambe meal and wheat gluten powder in open jars for a minimum of 48 hr at 23 c and a relative humidity of 50% in a climate - controlled room. mixing the components grind the urea powder (stored in a closed beaker at ambient conditions) to fine particles with a mortar and pestle.blend urea and glycerol (25.5 g of glycerol and 15 g of urea per 100 g of the final mixture). heat the glycerol to 65 c in a glass flask in an oil bath and add the urea powder slowly.stir the mixture with a magnetic stirrer at 65 c until the urea powder is completely dissolved. grind the urea powder (stored in a closed beaker at ambient conditions) to fine particles with a mortar and pestle. blend urea and glycerol (25.5 g of glycerol and 15 g of urea per 100 g of the final mixture). heat the glycerol to 65 c in a glass flask in an oil bath and add the urea powder slowly.stir the mixture with a magnetic stirrer at 65 c until the urea powder is completely dissolved. heat the glycerol to 65 c in a glass flask in an oil bath and add the urea powder slowly. stir the mixture with a magnetic stirrer at 65 c until the urea powder is completely dissolved. mixing the crambe and wheat gluten blend the crambe meal powder and wheat gluten powder in a kitchen mixing machine for 5 min. for example, for a 60/40 (w / w) crambe / wheat gluten ratio, use 35.7 g of crambe meal and 23.8 g of wheat gluten per 100 g of the final mixture. blend the crambe meal powder and wheat gluten powder in a kitchen mixing machine for 5 min. for example, for a 60/40 (w / w) crambe / wheat gluten ratio, use 35.7 g of crambe meal and 23.8 g of wheat gluten per 100 g of the final mixture. mixing glycerol / urea with crambe / wheat gluten slowly add the glycerol / urea mixture to the crambe / wheat gluten blend in the kitchen mixing machine while stirring the mixture. prepare 500 g of the mixture each time.for the material with 60/40 (w / w) crambe and wheat gluten, use the following relative contents of the respective constituents : 35.7 g of crambe meal, 23.8 g of wheat gluten, 25.5 g of glycerol, and 15 g of urea (per 100 g). for the other two material combinations (i.e., 70/30 and 80/20), change only the crambe and wheat gluten contents. keep the glycerol and urea contents the same as in the 60/40 combination. slowly add the glycerol / urea mixture to the crambe / wheat gluten blend in the kitchen mixing machine while stirring the mixture. prepare 500 g of the mixture each time. for the material with 60/40 (w / w) crambe and wheat gluten, use the following relative contents of the respective constituents : 35.7 g of crambe meal, 23.8 g of wheat gluten, 25.5 g of glycerol, and 15 g of urea (per 100 g). for the other two material combinations (i.e., 70/30 and 80/20), change only the crambe and wheat gluten contents. keep the glycerol and urea contents the same as in the 60/40 combination. low - temperature profile perform the film extrusion in a twin - screw extruder. set zones 1 - 10 (each 80 mm - long) along the extruder barrel at a low temperature profile (subsequently called " low - t profile "), as follows : 75 - 75 - 75 - 80 - 80 - 80 - 80 - 85 - 85 - 85 c. this prevents the wheat gluten from crosslinking in the barrel.use a flat sheet die (45 mm x 0.7 mm) to extrude the films.choose a screw speed between 30 rpm and 200 rpm and record the die pressure.feed the dough manually through the hopper with the help of a wooden pusher to support the material flow towards the screws.at the die, pick up the extrudate with a conveyor belt operating at a speed of 2.0 m / min. place air ventilation cooling (fans) along the belt.run different die temperatures (105 c-(105 c), 110 c-(110 c), 125 c-(115 c), 130 c-(120 c), and 140 c-(125 c)) to select the conditions that give the smoothest extrudate with a minimum amount of voids. note : the values in the brackets correspond to the temperature in zone 11, next to the die. it is adjusted to reach the target temperature in the die.after extrusion, store the extrudates in sealed polyethylene bags until further processing or analysis in order to prevent ageing and atmospheric water absorption. set zones 1 - 10 (each 80 mm - long) along the extruder barrel at a low temperature profile (subsequently called " low - t profile "), as follows : 75 - 75 - 75 - 80 - 80 - 80 - 80 - 85 - 85 - 85 c. use a flat sheet die (45 mm x 0.7 mm) to extrude the films. choose a screw speed between 30 rpm and 200 rpm and record the die pressure. feed the dough manually through the hopper with the help of a wooden pusher to support the material flow towards the screws. at the die, pick up the extrudate with a conveyor belt operating at a speed of 2.0 m / min. place air ventilation cooling (fans) along the belt. run different die temperatures (105 c-(105 c), 110 c-(110 c), 125 c-(115 c), 130 c-(120 c), and 140 c-(125 c)) to select the conditions that give the smoothest extrudate with a minimum amount of voids. note : the values in the brackets correspond to the temperature in zone 11, next to the die. after extrusion, store the extrudates in sealed polyethylene bags until further processing or analysis in order to prevent ageing and atmospheric water absorption. film extrusion using the high - temperature profile extrude films as described in section 2.1, but use a high - temperature profile (subsequently called " high - t profile "), as follows : 85 - 85 - 85 - 100 - 100 - 100 - 110 - 110 - 120 - 120 - 120 c for zones 1 to 11 of the extruder.use die temperatures of 125 c and 130 c. extrude films as described in section 2.1, but use a high - temperature profile (subsequently called " high - t profile "), as follows : 85 - 85 - 85 - 100 - 100 - 100 - 110 - 110 - 120 - 120 - 120 c for zones 1 to 11 of the extruder. film extrusion after pelletization in order to get pellets, extrude the material as continuous strands in the extruder using a two - strand die. use the low - t profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed.use different die-(zone 11) temperatures (130 c-(125c), 125 c-(115 c), 105 c-(100 c), and 85 c-(85 c)) to obtain the strands with the smoothest surfaces. pelletization after passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m / min. film extrusion from the pellets feed the pellets manually into the extruder and extrude films with the low - t profile inside the barrel and with a 125 c-(115 c) flat - sheet die temperature. use a screw rotational speed of 30 rpm. in order to get pellets, extrude the material as continuous strands in the extruder using a two - strand die. use the low - t profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed.use different die-(zone 11) temperatures (130 c-(125c), 125 c-(115 c), 105 c-(100 c), and 85 c-(85 c)) to obtain the strands with the smoothest surfaces. use the low - t profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed. use different die-(zone 11) temperatures (130 c-(125c), 125 c-(115 c), 105 c-(100 c), and 85 c-(85 c)) to obtain the strands with the smoothest surfaces. pelletization after passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m / min. after passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m / min. film extrusion from the pellets feed the pellets manually into the extruder and extrude films with the low - t profile inside the barrel and with a 125 c-(115 c) flat - sheet die temperature. use a screw rotational speed of 30 rpm. feed the pellets manually into the extruder and extrude films with the low - t profile inside the barrel and with a 125 c-(115 c) flat - sheet die temperature. use a screw rotational speed of 30 rpm. film extrusion using volumetric feeding in order to simulate automatic feeding (commonly used in industry), use the pellets previously extruded at 85 c (steps 2.3.1 - 2.3.2.1).connect the feeder to the extruder and choose the volumetric feeder mode of the hopper.use a feeding volume of 35 kg / h and hopper and extruder screw speeds of 16 and 120 rpm, respectively.extrude with the low temperature profile of the barrel and use a die-(zone 11) temperature of 125 c-(115 c). in order to simulate automatic feeding (commonly used in industry), use the pellets previously extruded at 85 c (steps 2.3.1 - 2.3.2.1). connect the feeder to the extruder and choose the volumetric feeder mode of the hopper. use a feeding volume of 35 kg / h and hopper and extruder screw speeds of 16 and 120 rpm, respectively. extrude with the low temperature profile of the barrel and use a die-(zone 11) temperature of 125 c-(115 c). pressing with the frame for the first setup, cut two extrudates into pieces of 4.4 cm x 7.0 cm and 2.6 cm x 7.0 cm. note : this is needed because the frame is broader than the extrudates.place them next to each other in an aluminum rectangular frame (70 x 70 x 0.5 mm).sandwich the frame between two aluminum plates using poly(ethylene terephthalate) (pet) films on both sides to prevent adhesion, and then place them into the press.set the pressure gauge on the press to 200 or 400 bar.for each molding pressure, press the films with a plate temperature of 110, 120, and 130 c for 10 and 20 min.as a reference to the pre - extruded samples, press films from unextruded material. center 7.2 g of fresh material (from section 1.6) in the aluminum frame.press with the same parameter settings as for the pre - extruded films above (steps 3.1.4- 3.1.5). for the first setup, cut two extrudates into pieces of 4.4 cm x 7.0 cm and 2.6 cm x 7.0 cm. place them next to each other in an aluminum rectangular frame (70 x 70 x 0.5 mm). sandwich the frame between two aluminum plates using poly(ethylene terephthalate) (pet) films on both sides to prevent adhesion, and then place them into the press. for each molding pressure, press the films with a plate temperature of 110, 120, and 130 c for 10 and 20 min. as a reference to the pre - extruded samples, press films from unextruded material. center 7.2 g of fresh material (from section 1.6) in the aluminum frame. press with the same parameter settings as for the pre - extruded films above (steps 3.1.4- 3.1.5). pressing without a frame cut out and sandwich rectangular samples (4.4 cm x 4.4 cm) between two aluminum plates using pet films on both sides to prevent adhesion.place them into the press. set the pressure gauge to 50 bar, 75 bar, or 100 bar.for each molding pressure, press films for 5 or 10 min using 110 c, 120 c and 130 c plate temperatures. cut out and sandwich rectangular samples (4.4 cm x 4.4 cm) between two aluminum plates using pet films on both sides to prevent adhesion. for each molding pressure, press films for 5 or 10 min using 110 c, 120 c and 130 c plate temperatures. sieving crambe sieve the crambe meal with a round, fine - mesh stainless steel kitchen sieve (pore size : ~1.5 mm, 14 mesh), to remove large fiber fractions and uncrushed seeds. store the sieved meal at -18 c to prevent material ageing. sieve the crambe meal with a round, fine - mesh stainless steel kitchen sieve (pore size : ~1.5 mm, 14 mesh), to remove large fiber fractions and uncrushed seeds. milling crambe to reduce the particle size and make the material more homogeneous, mill the sieved crambe meal in a rotary ball mill.mill 250 g of crambe meal each time in a 7 l jar with 21 - 25 mm diameter ceramic balls using a 53 rpm jar revolution rate and a milling time of 24 hr. to reduce the particle size and make the material more homogeneous, mill the sieved crambe meal in a rotary ball mill. mill 250 g of crambe meal each time in a 7 l jar with 21 - 25 mm diameter ceramic balls using a 53 rpm jar revolution rate and a milling time of 24 hr. conditioning before further processing, condition all the ball - milled crambe meal and wheat gluten powder in open jars for a minimum of 48 hr at 23 c and a relative humidity of 50% in a climate - controlled room. before further processing, condition all the ball - milled crambe meal and wheat gluten powder in open jars for a minimum of 48 hr at 23 c and a relative humidity of 50% in a climate - controlled room. mixing the components grind the urea powder (stored in a closed beaker at ambient conditions) to fine particles with a mortar and pestle.blend urea and glycerol (25.5 g of glycerol and 15 g of urea per 100 g of the final mixture). heat the glycerol to 65 c in a glass flask in an oil bath and add the urea powder slowly.stir the mixture with a magnetic stirrer at 65 c until the urea powder is completely dissolved. grind the urea powder (stored in a closed beaker at ambient conditions) to fine particles with a mortar and pestle. blend urea and glycerol (25.5 g of glycerol and 15 g of urea per 100 g of the final mixture). heat the glycerol to 65 c in a glass flask in an oil bath and add the urea powder slowly.stir the mixture with a magnetic stirrer at 65 c until the urea powder is completely dissolved. heat the glycerol to 65 c in a glass flask in an oil bath and add the urea powder slowly. stir the mixture with a magnetic stirrer at 65 c until the urea powder is completely dissolved. mixing the crambe and wheat gluten blend the crambe meal powder and wheat gluten powder in a kitchen mixing machine for 5 min. for example, for a 60/40 (w / w) crambe / wheat gluten ratio, use 35.7 g of crambe meal and 23.8 g of wheat gluten per 100 g of the final mixture. blend the crambe meal powder and wheat gluten powder in a kitchen mixing machine for 5 min. for example, for a 60/40 (w / w) crambe / wheat gluten ratio, use 35.7 g of crambe meal and 23.8 g of wheat gluten per 100 g of the final mixture. mixing glycerol / urea with crambe / wheat gluten slowly add the glycerol / urea mixture to the crambe / wheat gluten blend in the kitchen mixing machine while stirring the mixture. prepare 500 g of the mixture each time.for the material with 60/40 (w / w) crambe and wheat gluten, use the following relative contents of the respective constituents : 35.7 g of crambe meal, 23.8 g of wheat gluten, 25.5 g of glycerol, and 15 g of urea (per 100 g). for the other two material combinations (i.e., 70/30 and 80/20), change only the crambe and wheat gluten contents. slowly add the glycerol / urea mixture to the crambe / wheat gluten blend in the kitchen mixing machine while stirring the mixture. for the material with 60/40 (w / w) crambe and wheat gluten, use the following relative contents of the respective constituents : 35.7 g of crambe meal, 23.8 g of wheat gluten, 25.5 g of glycerol, and 15 g of urea (per 100 g). for the other two material combinations (i.e., 70/30 and 80/20), change only the crambe and wheat gluten contents. keep the glycerol and urea contents the same as in the 60/40 combination. low - temperature profile perform the film extrusion in a twin - screw extruder. set zones 1 - 10 (each 80 mm - long) along the extruder barrel at a low temperature profile (subsequently called " low - t profile "), as follows : 75 - 75 - 75 - 80 - 80 - 80 - 80 - 85 - 85 - 85 c. this prevents the wheat gluten from crosslinking in the barrel.use a flat sheet die (45 mm x 0.7 mm) to extrude the films.choose a screw speed between 30 rpm and 200 rpm and record the die pressure.feed the dough manually through the hopper with the help of a wooden pusher to support the material flow towards the screws.at the die, pick up the extrudate with a conveyor belt operating at a speed of 2.0 m / min. place air ventilation cooling (fans) along the belt.run different die temperatures (105 c-(105 c), 110 c-(110 c), 125 c-(115 c), 130 c-(120 c), and 140 c-(125 c)) to select the conditions that give the smoothest extrudate with a minimum amount of voids. note : the values in the brackets correspond to the temperature in zone 11, next to the die. it is adjusted to reach the target temperature in the die.after extrusion, store the extrudates in sealed polyethylene bags until further processing or analysis in order to prevent ageing and atmospheric water absorption. perform the film extrusion in a twin - screw extruder. set zones 1 - 10 (each 80 mm - long) along the extruder barrel at a low temperature profile (subsequently called " low - t profile "), as follows : 75 - 75 - 75 - 80 - 80 - 80 - 80 - 85 - 85 - 85 c. use a flat sheet die (45 mm x 0.7 mm) to extrude the films. choose a screw speed between 30 rpm and 200 rpm and record the die pressure. feed the dough manually through the hopper with the help of a wooden pusher to support the material flow towards the screws. at the die, pick up the extrudate with a conveyor belt operating at a speed of 2.0 m / min. place air ventilation cooling (fans) along the belt. run different die temperatures (105 c-(105 c), 110 c-(110 c), 125 c-(115 c), 130 c-(120 c), and 140 c-(125 c)) to select the conditions that give the smoothest extrudate with a minimum amount of voids. note : the values in the brackets correspond to the temperature in zone 11, next to the die. after extrusion, store the extrudates in sealed polyethylene bags until further processing or analysis in order to prevent ageing and atmospheric water absorption. film extrusion using the high - temperature profile extrude films as described in section 2.1, but use a high - temperature profile (subsequently called " high - t profile "), as follows : 85 - 85 - 85 - 100 - 100 - 100 - 110 - 110 - 120 - 120 - 120 c for zones 1 to 11 of the extruder.use die temperatures of 125 c and 130 c. extrude films as described in section 2.1, but use a high - temperature profile (subsequently called " high - t profile "), as follows : 85 - 85 - 85 - 100 - 100 - 100 - 110 - 110 - 120 - 120 - 120 c for zones 1 to 11 of the extruder. film extrusion after pelletization in order to get pellets, extrude the material as continuous strands in the extruder using a two - strand die. use the low - t profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed.use different die-(zone 11) temperatures (130 c-(125c), 125 c-(115 c), 105 c-(100 c), and 85 c-(85 c)) to obtain the strands with the smoothest surfaces. pelletization after passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m / min. film extrusion from the pellets feed the pellets manually into the extruder and extrude films with the low - t profile inside the barrel and with a 125 c-(115 c) flat - sheet die temperature. use a screw rotational speed of 30 rpm. in order to get pellets, extrude the material as continuous strands in the extruder using a two - strand die. use the low - t profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed.use different die-(zone 11) temperatures (130 c-(125c), 125 c-(115 c), 105 c-(100 c), and 85 c-(85 c)) to obtain the strands with the smoothest surfaces. use the low - t profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed. use different die-(zone 11) temperatures (130 c-(125c), 125 c-(115 c), 105 c-(100 c), and 85 c-(85 c)) to obtain the strands with the smoothest surfaces. pelletization after passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m / min. after passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m / min. film extrusion from the pellets feed the pellets manually into the extruder and extrude films with the low - t profile inside the barrel and with a 125 c-(115 c) flat - sheet die temperature. use a screw rotational speed of 30 rpm. feed the pellets manually into the extruder and extrude films with the low - t profile inside the barrel and with a 125 c-(115 c) flat - sheet die temperature. film extrusion using volumetric feeding in order to simulate automatic feeding (commonly used in industry), use the pellets previously extruded at 85 c (steps 2.3.1 - 2.3.2.1).connect the feeder to the extruder and choose the volumetric feeder mode of the hopper.use a feeding volume of 35 kg / h and hopper and extruder screw speeds of 16 and 120 rpm, respectively.extrude with the low temperature profile of the barrel and use a die-(zone 11) temperature of 125 c-(115 c). in order to simulate automatic feeding (commonly used in industry), use the pellets previously extruded at 85 c (steps 2.3.1 - 2.3.2.1). connect the feeder to the extruder and use a feeding volume of 35 kg / h and hopper and extruder screw speeds of 16 and 120 rpm, respectively. extrude with the low temperature profile of the barrel and use a die-(zone 11) temperature of 125 c-(115 c). pressing with the frame for the first setup, cut two extrudates into pieces of 4.4 cm x 7.0 cm and 2.6 cm x 7.0 cm. note : this is needed because the frame is broader than the extrudates.place them next to each other in an aluminum rectangular frame (70 x 70 x 0.5 mm).sandwich the frame between two aluminum plates using poly(ethylene terephthalate) (pet) films on both sides to prevent adhesion, and then place them into the press.set the pressure gauge on the press to 200 or 400 bar.for each molding pressure, press the films with a plate temperature of 110, 120, and 130 c for 10 and 20 min.as a reference to the pre - extruded samples, press films from unextruded material. center 7.2 g of fresh material (from section 1.6) in the aluminum frame.press with the same parameter settings as for the pre - extruded films above (steps 3.1.4- 3.1.5). for the first setup, cut two extrudates into pieces of 4.4 cm x 7.0 cm and 2.6 cm x 7.0 cm. note place them next to each other in an aluminum rectangular frame (70 x 70 x 0.5 mm). sandwich the frame between two aluminum plates using poly(ethylene terephthalate) (pet) films on both sides to prevent adhesion, and then place them into the press. for each molding pressure, press the films with a plate temperature of 110, 120, and 130 c for 10 and 20 min. as a reference to the pre - extruded samples, press films from unextruded material. center 7.2 g of fresh material (from section 1.6) in the aluminum frame. press with the same parameter settings as for the pre - extruded films above (steps 3.1.4- 3.1.5). pressing without a frame cut out and sandwich rectangular samples (4.4 cm x 4.4 cm) between two aluminum plates using pet films on both sides to prevent adhesion.place them into the press. set the pressure gauge to 50 bar, 75 bar, or 100 bar.for each molding pressure, press films for 5 or 10 min using 110 c, 120 c and 130 c plate temperatures. cut out and sandwich rectangular samples (4.4 cm x 4.4 cm) between two aluminum plates using pet films on both sides to prevent adhesion. for each molding pressure, press films for 5 or 10 min using 110 c, 120 c and 130 c plate temperatures. the blended materials (60 wt% crambe meal and 40 wt% wheat gluten) resulted in a tough dough after the initial mixing procedure. the material was rested for a few minutes before the first extrusion. however, the dough had a too high a viscosity to be able to be fed into the extruder hopper in a regular manner. therefore, it was fed piece - by - piece, directly into the screw. the screws had a constant speed, and the resulting film extrudate was continuous and had a visually smooth surface. the die pressure and temperature were found to be the two most important processing parameters to control in order to obtain homogeneous and smooth film extrudates. too low a die temperature, typically below 110 c, did not result in continuous film extrudates, whereas a temperature above 130 c resulted in the formation of bubbles in the material. the most suitable die temperature to obtain homogeneous and smooth films a two - step process was found to be advantageous, where, in the first step, strands were extruded at a lower temperature (typically 85 c) and pelletized. when the urea content was decreased from 15 to 10 wt%, the cohesion of the dough decreased substantially, resulting in a powder - like material ; no continuous film could be extruded. when the glycerol concentration was decreased (with a retained 15 wt% urea), the dough was found to be more brittle, and the urea did not fully dissolve in the glycerol. also, a considerably higher die pressure was required to get homogeneous films. however, these films were found to be smoother and more homogeneous than those with a higher glycerol content. when increasing the crambe meal powder concentration and decreasing the wheat gluten concentration, the extruded films appeared darker, but also smoother and more homogeneous. the feeding rate could also be increased. the drawback was that the films were only partially continuous, and film breakages appeared a few meters apart. however, when increasing the die temperature to approximately 130 c, continuous films could be prepared, although with some discolorations. compression molding without a frame yielded thin (thickness : 0.1 - 0.2 mm) films that were very flexible and translucent (figure 2). depending on how the extrudates were made and what they contained, the stiffness ranged from 4.9 - 5.6 mpa and the strength from 0.3 - 0.7 mpa, whereas the extensibility ranged from 7 to 16%. the corresponding values for the extrudates after compression molding were 6.4 - 15.0 mpa, 0.3 - 1.1 mpa, and 8 - 19%. 64 mm - long dumbbell specimens were tensile - tested according to astm d882 - 02 at 23 1 c and 50 1% rh, with a crosshead speed of 10 mm / min. figure 3 shows the importance of extruding crambe with the addition of wheat gluten. the strength, and especially the extensibility, decreased with decreasing wheat gluten content. the oxygen permeability ranged from 17 to 39 cm mm/(day m atm), depending on the composition and whether a compression molding step (with a frame) was used or not. figure 1 : extruded material. extruded film using a die temperature of 130 c. it contains 35.7 wt% crambe, 23.8 wt% wheat gluten, 25.5 wt% glycerol, and 15 wt% urea. extruded material compression - molded, without a frame, into thin, translucent films using a 130 c press temperature at 75 bar for 10 min.. maximum stress (filled circles) and strain at maximum stress (open circles) as a function of crambe content in the crambe / wheat gluten mixture. maximum stress was obtained from the maximum force per initial sample cross - section of the narrow part of the dumbbell specimen (narrow section : 16 mm long and 4 mm wide). the low - temperature profile was used with an initial die temperature of 125 c and a zone 11 temperature of 115 c. the screw speed was 30 rpm, and the extrusion was performed without prior pelletization. the reason the die pressure was of such high importance was probably due to the fact that the material needed a certain pressure to avoid bubble formation. however, the different components could phase separate if the pressure was too high. when extruding at too low a temperature, the cohesion was poor, possibly due to a low degree of crosslinking, while too high a temperature resulted in the release of gas (probably moisture together with urea and protein degradation products). the two step extrusion (i.e., where strands were first extruded, pelletized, and then extruded again) resulted in a more homogeneous extrudate because of the more extensive blending that the first extrusion step provided. the poor dough cohesion when decreasing the urea concentration from 15 to 10 wt% was probably due to a lower crosslink density. in analogy to this, a lower glycerol concentration, and thus a lower ability to dissolve the urea, resulted in poorer films unless a higher die pressure was applied. increasing the crambe meal concentration, and thus decreasing the wheat gluten concentration, resulted in a lower degree of aggregation / network formation. this lowered the viscosity of the material in the extrudate, resulting in the need to increase the die temperature to 130 c to raise the viscosity and generate homogeneous films. it is difficult, if not impossible, to extrude plasticized crambe into films of sufficient quality for any use. we show here that this can be overcome by blending crambe with a more readily extrudable protein like wheat gluten. for the best quality, it is shown here that extrusion works on a smaller scale, and upscaling is likely to be more demanding. extrusion, along with injection molding, are the most important commercial methods for producing plastics. in order to replace existing conventional plastics, we show here that it is possible to extrude crambe oilseed meal with the help of wheat gluten. possible applications include packaging and applications for various extruded profiles (e.g., rods and cylinders). we consider the most critical step during the preparation of the samples to be the extrusion step. the final quality of the films depended strongly upon the extrusion parameters and the properties of the material prior to extrusion. | crambe abyssinica is a plant with potential for use in industrial (non - food) plant oil production. the side stream from this oil production is a high - protein crambe meal that has limited value, as it is not fit for food or feed use. however, it contains proteins that could potentially make it a suitable raw material for higher - value products. the purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. the study mainly considered the development of material compositions and methods of preparing and extruding the material. wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. the extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. a denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. the most homogeneous films were made at an extruder die temperature of 125 - 130 c. it is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step. |
neuroblastoma (nb) is a common childhood solid tumor that presents with clinical heterogeneity. the presentation of nb can range from localized tumors to disseminated forms, which have different prognoses and require different treatment. therefore, determining the disease extent and stage at the time of diagnosis is important. a number of genetic and biological markers have been investigated for detection of minimal levels of nb cells. tyrosine hydroxylase (th), which is mainly expressed in sympathetic nervous tissues, has been suggested as a candidate marker for detecting minimal levels of neuroblastoma cells in bone marrow (bm) and peripheral blood (pb). molecular detection of th mrna using reverse transcription quantitative real - time polymerase chain reaction (rt - qpcr) has been proposed as a specific and sensitive method for detection of minimal disease in nb [5 - 8 ]. according to our preliminary reports however, the number of patients was small and the follow - up duration was short. in the current study, we evaluated the significance of th expression in a larger cohort of patients with a longer follow - up period as well as the clinical implications of the level of th expression among th - positive patients. written informed consent for th analysis was obtained at diagnosis from all patients or guardians. this retrospective study was approved by the institutional review board (irb) of samsung medical center and the requirement for informed consent was waived (irb 2014 - 12 - 090). medical records of all patients who were newly diagnosed with nb over a 10-year period from july 2005 to june 2015 were reviewed retrospectively. patients were staged according to the international neuroblastoma staging system and disease extent was evaluated using computerized tomography (ct), magnetic resonance imaging (mri), technetium-99 bone scans, bilateral bm aspirates and biopsy specimens, and i- or i - metaiodobenzylguanidine (mibg) scanning. mycn amplification was determined using competitive polymerase chain reaction (pcr), rt - qpcr, or fluorescence in situ hybridization. tumors were classified as histologically favorable or unfavorable according to the international neuroblastoma pathology classification. measurement of serum lactic dehydrogenase (ldh), ferritin, neuron - specific enolase (nse), and urine vanillylmandelic acid (vma) was also included in routine evaluation at diagnosis. patients were stratified into low - risk, intermediate - risk, and high - risk groups based on age, stage, and mycn status. stage 1, 2, and 4s tumors without mycn amplification were stratified as low - risk tumors whereas stage 4 tumors in patients older than 12 months or any tumors with amplified mycn were stratified as high - risk tumors. otherwise, incisional or percutaneous needle biopsy was performed with definitive surgery after six chemotherapy cycles. alternating cedc (cisplatin+etoposide+doxorubicin+cyclophosphamide) and ice (ifosfamide+carboplatin+etoposide) regimens were used for chemotherapy (table 1). patients with low - risk localized tumors underwent surgery with (stage 2) or without (stage 1) six cycles of preoperative or postoperative chemotherapy. patients with low - risk stage 4s tumors were treated only when there were life - threatening symptoms or the tumor progressed during observation. patients with intermediaterisk tumors received six cycles of preoperative chemotherapy, surgery, three cycles of postoperative chemotherapy, and 12 cycles of 13-cis - retinoic acid (125 mg / m / day for 14 days every 4 weeks). local radiotherapy to the primary site was administered if gross residual tumor remained after surgery. patients with high - risk tumors received tandem high - dose chemotherapy and autologous stem cell transplantation (hdct / auto - sct) after induction treatment (six cycles of preoperative chemotherapy, surgery, and three cycles of postoperative chemotherapy). the second hdct regimen was tm - tbi (thiotepa+melphalan+total body irradiation) for patients diagnosed up to december 2008. for patients diagnosed from january 2009 onwards, tbi was replaced with high - dose i - mibg (12 or 18 mci / kg) to reduce late adverse effects. local radiotherapy was applied to the primary site in all high - risk patients approximately 6 weeks after the second hdct / auto - sct. differentiation therapy involving 13-cis - retinoic acid with immunotherapy using interleukin 2 (210 u / m / day for 5 days every 4 weeks) was administered until 1 year after hdct / auto - sct to reduce relapse from minimal residual tumor cells. pb samples were collected at diagnosis in ethylenediaminetetraacetic acid tubes and mononuclear cells were separated and lysed in trizol reagent (invitrogen corporation, carlsbad, ca). rna was extracted according to the manufacturer s instructions and 1 g of rna was used to generate 20 l of complementary dna using an rna pcr kit (applied biosystems, foster city, ca). rt - qpcr was performed using real - q th quantification kits (biosewoom, seoul, korea) on a lightcycler (roche diagnostics, mannheim, germany). rt - qpcr was performed under the following cycling conditions : denaturation at 95c for 10 minutes, 45 cycles at 95c for 10 seconds, 60c for 10 seconds, and 72c for 30 seconds, followed by a cooling step at 40c for 30 seconds. reactions were performed in a 20-l volume containing pcr reaction mixture, taqman probe (5'-fam agcgcaggaagctgattgctgagtamra-3 '), primer mixture (forward, 5'-tcatcacctggtcaccaagtt-3 ' ; reverse, 5'-ggtcgccgtgcctgtact-3 '), 2 l cdna, and sterile water. if the expression value of abl in the rt - qpcr amplification was less than a copy number of 104, the sample was considered inadequate for rt - qpcr analysis and the rna extraction was repeated. the quantity of th transcripts was normalized to the abl transcript level, and the result was expressed as the ratio of the th to abl transcript copy number. the mann - whitney u test was used for comparison of continuous variables between groups. progression - free survival (pfs) and overall survival (os) rates with standard errors were estimated using the kaplan - meier method. multivariate analyses of prognostic factors for pfs and os were performed using cox regression analysis. medical records of all patients who were newly diagnosed with nb over a 10-year period from july 2005 to june 2015 were reviewed retrospectively. patients were staged according to the international neuroblastoma staging system and disease extent was evaluated using computerized tomography (ct), magnetic resonance imaging (mri), technetium-99 bone scans, bilateral bm aspirates and biopsy specimens, and i- or i - metaiodobenzylguanidine (mibg) scanning. mycn amplification was determined using competitive polymerase chain reaction (pcr), rt - qpcr, or fluorescence in situ hybridization. tumors were classified as histologically favorable or unfavorable according to the international neuroblastoma pathology classification. measurement of serum lactic dehydrogenase (ldh), ferritin, neuron - specific enolase (nse), and urine vanillylmandelic acid (vma) was also included in routine evaluation at diagnosis. patients were stratified into low - risk, intermediate - risk, and high - risk groups based on age, stage, and mycn status. stage 1, 2, and 4s tumors without mycn amplification were stratified as low - risk tumors whereas stage 4 tumors in patients older than 12 months or any tumors with amplified mycn were stratified as high - risk tumors. otherwise, incisional or percutaneous needle biopsy was performed with definitive surgery after six chemotherapy cycles. alternating cedc (cisplatin+etoposide+doxorubicin+cyclophosphamide) and ice (ifosfamide+carboplatin+etoposide) regimens were used for chemotherapy (table 1). patients with low - risk localized tumors underwent surgery with (stage 2) or without (stage 1) six cycles of preoperative or postoperative chemotherapy. patients with low - risk stage 4s tumors were treated only when there were life - threatening symptoms or the tumor progressed during observation. patients with intermediaterisk tumors received six cycles of preoperative chemotherapy, surgery, three cycles of postoperative chemotherapy, and 12 cycles of 13-cis - retinoic acid (125 mg / m / day for 14 days every 4 weeks). local radiotherapy to the primary site was administered if gross residual tumor remained after surgery. patients with high - risk tumors received tandem high - dose chemotherapy and autologous stem cell transplantation (hdct / auto - sct) after induction treatment (six cycles of preoperative chemotherapy, surgery, and three cycles of postoperative chemotherapy). the second hdct regimen was tm - tbi (thiotepa+melphalan+total body irradiation) for patients diagnosed up to december 2008. for patients diagnosed from january 2009 onwards, tbi was replaced with high - dose i - mibg (12 or 18 mci / kg) to reduce late adverse effects. local radiotherapy was applied to the primary site in all high - risk patients approximately 6 weeks after the second hdct / auto - sct. differentiation therapy involving 13-cis - retinoic acid with immunotherapy using interleukin 2 (210 u / m / day for 5 days every 4 weeks) was administered until 1 year after hdct / auto - sct to reduce relapse from minimal residual tumor cells. pb samples were collected at diagnosis in ethylenediaminetetraacetic acid tubes and mononuclear cells were separated and lysed in trizol reagent (invitrogen corporation, carlsbad, ca). rna was extracted according to the manufacturer s instructions and 1 g of rna was used to generate 20 l of complementary dna using an rna pcr kit (applied biosystems, foster city, ca). rt - qpcr was performed using real - q th quantification kits (biosewoom, seoul, korea) on a lightcycler (roche diagnostics, mannheim, germany). rt - qpcr was performed under the following cycling conditions : denaturation at 95c for 10 minutes, 45 cycles at 95c for 10 seconds, 60c for 10 seconds, and 72c for 30 seconds, followed by a cooling step at 40c for 30 seconds. reactions were performed in a 20-l volume containing pcr reaction mixture, taqman probe (5'-fam agcgcaggaagctgattgctgagtamra-3 '), primer mixture (forward, 5'-tcatcacctggtcaccaagtt-3 ' ; reverse, 5'-ggtcgccgtgcctgtact-3 '), 2 l cdna, and sterile water. if the expression value of abl in the rt - qpcr amplification was less than a copy number of 104, the sample was considered inadequate for rt - qpcr analysis and the rna extraction was repeated. the quantity of th transcripts was normalized to the abl transcript level, and the result was expressed as the ratio of the th to abl transcript copy number. the mann - whitney u test was used for comparison of continuous variables between groups. progression - free survival (pfs) and overall survival (os) rates with standard errors were estimated using the kaplan - meier method. multivariate analyses of prognostic factors for pfs and os were performed using cox regression analysis. a total of 210 patients (118 boys and 92 girls) were evaluated for the presence of th mrna in pb at diagnosis. the median age at diagnosis was 22 months (range, 0 to 232 months) and median follow - up duration was 51 months (range, 1 to 122 months). at diagnosis, 134 patients were older than 12 months, 103 patients had metastatic tumors including four stage 4s tumors, 90 had histologically unfavorable tumors, and 34 had mycn amplified tumors. among 89 patients with high - risk tumors, 30 patients used tm - tbi regimen in the second hdct / auto - sct and the remaining 59 patients used i - mibg - tm regimen. th expression was positive in pb at diagnosis in 60 of 210 patients (28.6%). the median expression level (th / abl ratio) of th mrna transcript detected in pb was 1,58010 (range, 210 to 3,711,86410). positive th expression was associated with high - risk features (i.e., advanced stage, older age, unfavorable pathology, and mycn amplification). the levels of serum ldh, ferritin, nse, and urine vma were also higher in th - positive patients (table 2). among 60 th - positive patients, 50 had metastatic tumors and the remaining 10 had localized tumors (stage 1 in three, stage 2 in two, and stage 3 in five patients). among the th - positive patients, a higher th expression level was associated with advanced stage and high - risk tumor, but not with older age (12 months) or mycn amplification (fig. 1a - d). relapse or progression occurred in 19 of 60 th - positive patients (31.7%), compared with only six of 150 th - negative patients (4.0%). lower 5-year probabilities of pfs and os were observed in th - positive patients than in th - negative patients (pfs : 63.86.9% vs. 94.72.1%, p < 0.001 ; os : 70.2 6.7% vs. 92.42.5%, p < 0.001) (fig. 2a). in analysis confined to only high - risk patients, the 5-year probabilities of pfs and os remained lower in th - positive patients (pfs : 55.78.2% vs. 89.65.8%, p=0.001 ; os : 61.48.1% vs. 87.26.1%, p=0.064) (fig. however, in low- and intermediate - risk patients, no difference in the 5-year probabilities of pfs and os was observed between th - positive patients and th - negative patients (pfs : 96.81.9% vs. 90.08.7%, p=0.447 ; os : 100% vs. 94.52.5%, p=0.725). among high - risk patients, there was no difference in the 5-year probabilities of pfs and os according to the hdct regimen (tm - tbi vs. i - mibg - tm) (pfs : 63.38.8% vs. 78.76.6%, p=0.184 ; os : 70.08.4% vs. 76.7 6.4%, p=0.745). in analysis confined to only th - positive high - risk patients, there was also no difference in survival rates according to the hdct regimen (pfs : 50.012.5% vs. 61.2 10.5%, p=0.523 ; os : 56.312.4% vs. 67.09.3%, p=0.923). positive th expression in pb at diagnosis was an independent factor for pfs (hazard ratio, 6.35 ; 95% confidence interval, 2.10 to 19.25 ; p=0.001). the results were similar when the analysis was confined to high - risk patients (hazard ratio, 9.02 ; 95% confidence interval, 2.06 to 39.50 ; p=0.004) (table 4). among th - positive patients, a higher expression level of th was associated with a worse outcome (above median level vs. below median level : 52.9 10.0% vs. 75.78.8%, p=0.075 for 5-year pfs ; 55.99.6% vs. 89.25.9%, p=0.047 for 5-year os) (fig. a total of 210 patients (118 boys and 92 girls) were evaluated for the presence of th mrna in pb at diagnosis. the median age at diagnosis was 22 months (range, 0 to 232 months) and median follow - up duration was 51 months (range, 1 to 122 months). at diagnosis, 134 patients were older than 12 months, 103 patients had metastatic tumors including four stage 4s tumors, 90 had histologically unfavorable tumors, and 34 had mycn amplified tumors. among 89 patients with high - risk tumors, 30 patients used tm - tbi regimen in the second hdct / auto - sct and the remaining 59 patients used i - mibg - tm regimen. th expression was positive in pb at diagnosis in 60 of 210 patients (28.6%). the median expression level (th / abl ratio) of th mrna transcript detected in pb was 1,58010 (range, 210 to 3,711,86410). positive th expression was associated with high - risk features (i.e., advanced stage, older age, unfavorable pathology, and mycn amplification). the levels of serum ldh, ferritin, nse, and urine vma were also higher in th - positive patients (table 2). among 60 th - positive patients, 50 had metastatic tumors and the remaining 10 had localized tumors (stage 1 in three, stage 2 in two, and stage 3 in five patients). among the th - positive patients, a higher th expression level was associated with advanced stage and high - risk tumor, but not with older age (12 months) or mycn amplification (fig. relapse or progression occurred in 19 of 60 th - positive patients (31.7%), compared with only six of 150 th - negative patients (4.0%). lower 5-year probabilities of pfs and os were observed in th - positive patients than in th - negative patients (pfs : 63.86.9% vs. 94.72.1%, p < 0.001 ; os : 70.2 6.7% vs. 92.42.5%, p < 0.001) (fig. 2a). in analysis confined to only high - risk patients, the 5-year probabilities of pfs and os remained lower in th - positive patients (pfs : 55.78.2% vs. 89.65.8%, p=0.001 ; os : 61.48.1% vs. 87.26.1%, p=0.064) (fig. however, in low- and intermediate - risk patients, no difference in the 5-year probabilities of pfs and os was observed between th - positive patients and th - negative patients (pfs : 96.81.9% vs. 90.08.7%, p=0.447 ; os : 100% vs. 94.52.5%, p=0.725). among high - risk patients, there was no difference in the 5-year probabilities of pfs and os according to the hdct regimen (tm - tbi vs. i - mibg - tm) (pfs : 63.38.8% vs. 78.76.6%, p=0.184 ; os : 70.08.4% vs. 76.7 6.4%, p=0.745). in analysis confined to only th - positive high - risk patients, there was also no difference in survival rates according to the hdct regimen (pfs : 50.012.5% vs. 61.2 10.5%, p=0.523 ; os : 56.312.4% vs. 67.09.3%, p=0.923). positive th expression in pb at diagnosis was an independent factor for pfs (hazard ratio, 6.35 ; 95% confidence interval, 2.10 to 19.25 ; p=0.001). the results were similar when the analysis was confined to high - risk patients (hazard ratio, 9.02 ; 95% confidence interval, 2.06 to 39.50 ; p=0.004) (table 4). among th - positive patients, a higher expression level of th was associated with a worse outcome (above median level vs. below median level : 52.9 10.0% vs. 75.78.8%, p=0.075 for 5-year pfs ; 55.99.6% vs. 89.25.9%, p=0.047 for 5-year os) (fig. a number of genetic markers have been investigated for detection of minimal levels of nb cells [13 - 17 ]. viprey. reported that the th, dcx, and phox2b genes are the best candidates for detection of circulating nb cells. corrias., who investigated the advantages of measuring multiple molecular targets over a single molecular target, reported that combinations of multiple targets did not add to the predictive power of th analysis alone. gilbert. also reported that th might be the one of the most useful molecular targets. in this context, in the current study we measured th mrna alone for molecular detection of minimal disease in all patients who were newly diagnosed with nb. positive th expression in pb at diagnosis was associated with high - risk features and worse outcome. more importantly, positive th expression in pb at diagnosis was an independent poor prognostic factor for pfs. current staging systems use conventional diagnostic modalities such as ct, mri, bm biopsy, and various radioisotope scans. patients are stratified into risk groups based on these staging systems and patients within each risk group are treated uniformly. however, our study showed that outcomes within the high - risk group differ according to th expression in pb at diagnosis. previous studies also reported that positive th expression was a poor prognostic indicator in metastatic nb. these findings suggest that th expression at diagnosis might be a significant factor in future methods of risk stratification for nb. some studies examined the clinical implication of positive th expression in non - metastatic nb. yanez. reported an association of positive th expression in pb at diagnosis with a higher probability of relapse in patients with non - metastatic nb. these findings suggest that some patients with localized tumors (by conventional diagnostic modalities) might have disseminated micrometastatic disease and therefore might benefit from upstaging and more aggressive treatment. however, in the current study, because only one of 10 th - positive patients with localized tumor experienced relapse, the significance of th expression in patients with localized tumors was unclear. further studies including more patients are needed to evaluate the prognostic importance of th expression in the pb of patients with localized tumors. among th - positive patients, higher th expression was associated with advanced stage and high - risk tumor, but not with older age or mycn amplification, and a higher th expression level at diagnosis was associated with a worse outcome. yanez. reported that a high level of th mrna in pb was a significant independent predictor of event - free survival and os when detected at diagnosis, post - induction therapy, and at the end of treatment. they proposed inclusion of th mrna monitoring throughout the disease course by rt - qpcr as a standard method of disease evaluation that would be helpful in predicting differences in outcome and identify children for whom current treatment is failing or insufficient. the european hr - nbl1/siopen study defined patients with very high th expression as ultrahigh - risk. these ultrahigh - risk patients showed very poor outcome among the group of high - risk nb patients (pfs, 0%), most of whom underwent single hdct / auto - sct following short induction treatment. the authors suggested that patients with very high th expression are candidates for alternative treatment strategies. in the current study, the prognosis of patients with high th expression was also poorer than for those with low th expression, but was not dismal. the relatively better outcome in our patients with high th expression compared to those in the european hr - nbl1/siopen study might be attributable to the longer induction treatment and more intensive consolidation treatment using tandem hdct / auto - sct. in conclusion, positive th expression in pb at diagnosis of neuroblastoma is associated with high - risk features and is an independent prognostic factor for poor pfs. results of the current study suggest that treatment intensity should be tailored according to th expression in pb at diagnosis. | purposethe purpose of this study is to investigate the clinical significance of tyrosine hydroxylase (th) expression in peripheral blood (pb) at diagnosis in patients with neuroblastoma.materials and methodsth mrna expression in pb was measured by reverse transcription quantitative real - time polymerase chain reaction in 210 patients who were newly diagnosed with neuroblastoma from july 2005 to june 2015 and the clinical significance of th expression in pb at diagnosis was evaluated.resultsth expression was positive in 60 patients (28.6%). fifty of 60 th - positive patients had metastatic tumors and the remaining 10 had localized tumors. th expression was associated with high - risk features (i.e., advanced stage, older age, unfavorable pathology, and mycn amplification) at diagnosis. among th - positive patients, higher th expression level was observed in high - risk patients than in low- or intermediate - risk patients (p=0.035). the probability of 5-year progression - free survival (pfs) was lower in th - positive patients than in th - negative patients (63.86.9% vs. 94.72.1%, p < 0.001). in analysis confined to high - risk patients, the 5-year probability of pfs remained lower in th - positive patients (55.78.2% vs. 89.65.8%, p < 0.001). among th - positive patients, a higher expression level of th was associated with a worse outcome. in multivariate analyses, positive th expression in pb at diagnosis was an independent poor prognostic factor for pfs.conclusionthe treatment intensity should be tailored according to th expression in pb at diagnosis. |
in recent years, the increasing prevalence of infectious diseases resistant to chemotherapy has caused an urgent need to discover and develop new antibiotics. in this context, strain hhs.015, isolated from roots of cucumber plants, belongs to saccharothrix actinomycete [1, 2 ]. the isolate showed a broad antimicrobial spectrum in vitro against plant pathogenic (e.g., valsa ceratosperma and phytophthora capsici) and nonplant pathogenic fungi, for example, candida vulgaris. in field experiments, fermentation supernatant of hhs.015 showed effective biocontrol to tomato leaf mould caused by fulvia fulva and apple tree valsa canker caused by valsa ceratosperma. up to date, many bioactive secondary metabolites have been reported from cultures of saccharothrix. these metabolites are characterised by diverse chemical structures and may be suitable because of their wide range of activity in agriculture to control fungal diseases in cultivated plants and domestic animals. production of secondary metabolites by microorganisms differs qualitatively and quantitatively depending on the strains and species of microorganisms used as well as on their nutritional and cultural conditions. and as fermentation moves into lower - value, higher - volume substrates, it becomes necessary to maximize the efficiency and minimize costs by using waste by - products to complete effectively with traditional high - value, low - volume compounds. in recent years, more and more attention was paid to agricultural pomaces such as apple pomace, wheat / rice bran, corn steep liquor, and peanut meal, for their use as raw materials in the production of high value products [1113 ]. but utilization of these pomaces was limited due to low protein content and poor digestibility. studies on optimization of the production of antimicrobial active compounds using agricultural pomaces have been reported comparatively seldom. the objective of the present study was to produce an alternate culture medium using agricultural waste as medium component and optimize fermentation conditions of the strain hhs.015 in order to greatly reduce the production cost and improve the formation of components with antimicrobial activity. ssy liquid medium consisted of : 20 g soybean powder, 10 g sucrose, 5 g soluble starch, 2 g yeast extract, 2 g protease peptone, 2 g nacl, 1 g caco3, 0.5 g mgso47h2o, 0.5 g kh2po4, and 1 l double distilled (dd) water. ssy solid medium was prepared by adding 10 g agar to the liquid medium. in addition, the following media were used : pda (200 g potato, 20 g dextrose, and 10 g agar in 1 l dd water), and pd (200 g potato and 20 g dextrose in 1 l dd water). all cultural media, plates, and flasks used in the experiments were autoclaved at 121c for 30 minutes. the strain was transferred to ssy solid medium plates and incubated for 7 days in darkness at 28c. the spores produced on the plates were used for inoculation of the liquid fermentation media. two spore cakes (diameter 4 mm) of strain hhs.015 were prepared to inoculate a 250-ml flask containing 50 ml ssy medium. the flasks were incubated at 28c on a rotary shaker at 150 rpm for 48 hours in darkness. for optimization of an alternative culture medium, submerged fermentation was carried out in 250-ml flasks containing 50 ml different test fermentation media. these flasks were inoculated with 10% (v / v) of the spore culture and incubated in darkness at 28c on a rotary shaker at 150 rpm for 5 d. for optimization of fermentation conditions, the experiment was carried out according to the experimental design. after fermentation, all of the test fermentation broths were centrifuged at 11,086 g for 10 minutes ; the supernatants (test samples) were used to evaluate the antimicrobial activity. candida vulgaris as the target microbe was kept on pda medium at 4c and was cultivated in pd liquid culture medium in darkness at 28c on a rotary shaker at 150 rpm for 2 days. then, c. vulgaris culture medium (1010 spores / ml) was kept at 4c for the assay of the antimicrobial activity in the fermentation supernatant produced by hhs.015. in order to investigate the effect of the carbon and nitrogen sources on the antimicrobial activity produced by strain hhs.015, ssy medium was used as a basal medium for the following optimization studies. various single and complex carbon or nitrogen sources (tables 1 and 2) were used to replace the carbon and nitrogen sources in the ssy medium, while all other components were kept constant. in the selection of proper microelements, all of the microelements in ssy medium were replaced by one microelement at different concentrations. the antimicrobial activity was evaluated by measurement of inhibition zones of the target organism after 24 hours of incubation at 30c (see section 2.5). the prices of the different carbon and nitrogen sources are listed in tables 1 and 2. the alternative culture media were optimized according to orthogonal tests. in the orthogonal tests, a total of 16 experiments were carried out simultaneously, each experiment with three replicates. the optimization of the fermentation conditions was carried out according to the central composite tool (ccd) of rsm (response surface methodology) using minitab 14 software (minitab inc., 2003) for selected five environmental factor parameters [15, 16 ]. the crucial factors involved in the study and their concentrations are given in table 4. a total of 32 experiments were carried out simultaneously (table 5), with experiments 2, 4, 6, 8, and 17 as five replicates of the central point. an optimum value of the variables for maximum antimicrobial activity was determined by response optimizer tool of the software. the antibacterial assay was carried out using the oxford cup method in 20-cm - diameter petri dishes. after autoclaving, 100 ml pda medium were cooled to 4050c, and 1% (v / v) of the c. vulgaris suspension (1010 after the medium in the petri dishes solidified, the autoclaved oxford cups were put onto the medium surface and 200 l of the testing samples (supernatants) were added into the cups. the antimicrobial activity was evaluated by measuring the diameter of translucent inhibition zones after incubating the dishes 24 hours at 30c. the data from the orthogonal tests were analyzed by sas software (stat soft, inc. the optimum value of the variables for the maximum antimicrobial activity was obtained by range analysis (table 6). range of every variable can roughly explain the effect of every variable on the antimicrobial activity. the range of every variable was calculated according to the following equation : (1)xij = xijn, n=4, j=1,2,3,4,ri = xijmax xijmin, i=1,2,3,4, where xij is the average value of the j level of one variable in line, i, n is the number of replications of every level of one variable in one line. ri is the difference of the maximum and the minor average value of all of the levels of one variable in line i. the experimental results of rsm were fitted with the response surface regression procedure. in developing the regression equation, the test variables were coded according to the equation : (2)xi=(xixi)xi, i=1,2,3,,k, where xi was the independent variable coded value, xi was the independent variable real value, xi was the independent variable real value on the centre point, and xi was the step change value. the response variable (antimicrobial activity unit) was fitted by a second - order model in order to correlate the response variable to the independent variables. the general form of the second degree of polynomial equation is : y = b0 + ibixi + ijbijxixj + biixi, where y was the measured response, b0 was the intercept term, bi, bij, and bii were the measures of the effects of variables xi, xixj, and xi, respectively. the variable xixj represents the first - order interaction between xi and xj (i < j). the statistical analysis of the model was performed in the form of analysis of variance (anova). this analysis included the fisher 's f - test (overall model significance), its associated probability p(f), correlation coefficient r, and determination coefficient r that measured the goodness of fit of the regression model. the analysis also included the student 's t - value for the estimated coefficients and associated probabilities, p(t). the results indicated that the carbon sources obviously affected the synthesis of substances with antimicrobial activity by strain hhs.015 (table 1). among the various carbon sources tested, strain hhs.015 produced in the presence of glucose (18.65 mm diameter of growth inhibition zones), white sugar (18.33 mm), brown sugar (17.86 mm), apple pomace (17.45 mm), and starch (16.21 mm) greater growth inhibition zones than in the control medium (15.28 mm). statistical analysis indicated that the growth inhibition zones were significantly different from the control for glucose and white sugar, while the growth inhibition zones were not significantly different from glucose and white sugar for apple pomace. significant lower antimicrobial activities were found in the vinegar residue (7.75 mm) as well as in black (7.75 mm) and bran (13.15 mm). taking the costs of the raw materials into consideration, the price of apple pomace (0.6 rmb / kg) was much lower compared with other carbon sources showing similar antimicrobial activity. the results of the nitrogen sources showed that strain hhs.015 could utilize different forms both organic and inorganic nitrogen sources (table 2). rapeseed meal favored production of compounds with antimicrobial activity (19.59 mm diameter of inhibition zones) but not significantly compared to the control. also in media containing urea and nano3, components with low antimicrobial activity were produced ; these sources were not significantly different from the control and other sources (e.g., ammonium phosphate, cotton dregs, ammonium sulfate, and fish meal). thus, considering the price of raw materials and the antimicrobial activity produced, apple pomace and rapeseed meal were used as carbon and nitrogen sources for further experiments. the results showed (figure 1) that the antimicrobial activity produced by strain hhs.015 enhanced in the presence of mgso47h2o concentrations increasing from 0.05% to 0.08% and kh2po4 levels increasing from 0 to 0.01%, but the activity decreased while the concentrations of the other microelements increased, for example, nacl and caco3 (figure 1). the effects of concentrations of the components in the medium on the antimicrobial activity were further investigated by tests of orthogonal design. from the range analysis, the apple pomace as the main variable affected the antimicrobial activity, indicating that the carbon source was essential for the production of compounds with antimicrobial activity by strain hhs.015. from the results of the analysis of variance of orthogonal design, (table 7) showed that each single variable had no significant effect on the antimicrobial activity (f < f0.05), probably because in the experiments the concentration range of every variable g / l, rapeseed meal 4 g / l, kh2po4 0.1 g / l, and mgso47h2o 0.6 g / l were determined according to the range analysis (table 6). the antimicrobial activity produced in the alternative culture medium was not significantly different from the ssy medium (figure 2a, ck), while the price of the materials of 0.1836 rmb / l was drastically lower than the price of the ssy medium, 2.1624 rmb / l ; thus, the price was reduced by 91.5%. applying multiple regression analysis on the experimental data, the results (table 5) of the ccd design fitted with a second - order of polynomial equation : (3)y=15.7443 + 1.4375x11.6625x2 + 3.8792x3 2.0443x122.9743x222.2193x323.9943x42 + 1.5313x1x5 + 1.5313x2x4, where y was the response (the antimicrobial activity units) and x1, x2, x3, x4, and x5 were the coded values of the independent variables, initial ph, medium volume in the flask, rotary speed, temperature, and inoculation volume, respectively. the student 's t - test and p - values were used to check the significance of each coefficient ; the results also indicated interaction strength between each independent variable. the degree of significance (table 8) shows that the linear term regression coefficients of rotary speed were highly significant as demonstrated by the p - values (px3 =.001). initial ph and media volume were also significant at 5% level (px1 =.039, px2 =.020). quadratic coefficients of primary ph, media volume, rotary speed, and temperature were highly significant at 1% level, and showed negative effect on the antimicrobial activity. the few response contour plots of the calculated model for antimicrobial activity were shown in figures 3(a)3(f) by keeping the other three variables constant at their middle level. the analysis of variance of regression for antimicrobial activity was summarized in table 8. in the case of antimicrobial activity, this calculated model was able to explain 93.6% of the results (table 9). the value of the adjusted determination coefficient (adj r = 0.82) was also very high to advocate for a high significance of the model. the coefficient for quadratic effect of ph, medium volume, temperature, and rotary speed may be significant to some extent. the response surfaces 2d contour could be analyzed for determining the optimized value of the variables, but it was difficult to analyze all these simultaneously. finally, the optimum culture condition was obtained as follows : initial ph = 7.0, medium volume = 90.0 ml/250 ml plate, rotary speed = 100 rpm, temperature = 25.0c, and inoculation volume = 15.8%. these values predict a 17.5 mm growth inhibition zone caused by the antimicrobial activity. these optimized values of nutrient parameters were validated in a duplicate flask study and an average of 17.3 0.25 mm growth inhibition zone was obtained. the result proved that the production of ingredients with antimicrobial activity had been improved by 20% when compared with the basal fermentation conditions (14.4 0.15 mm) (figures 2a and b). carbon sources could play an important role in the production of compounds with antimicrobial activity of strain hhs.015. nutrition plays an important role in the onset and intensity of secondary metabolism, not only because of limiting the supply of an essential nutrient is an effective means of restricting growth but also because the choice of limiting nutrients can have specific metabolic and regulatory effects [18, 19 ]. the results of this study showed that production of compounds with antimicrobial activity was drastically reduced when a carbon source such as vinegar residue or no carbon source (black) was present in the culture medium. a similar catabolic repression mechanism for antibiotic production had been described for bacteria, suggesting that the synthesis of the antibiotic was related to phosphoenolpyruvate sugar phosphotransferase system, and controlling the concentration of carbon sources could regulate the synthesis of antibiotics. in the optimization of the culture medium, apple pomace was selected as the carbon source. apple pomace, a by - product from a juice production factory in qianxian, shaanxi province, contained numerous kinds of nutrients, such as proteins, fats, soluble sugars, ca, p, and many other microelements, vitamins, and so forth. for microbe growth and synthesis of the secondary metabolites, microelements (mineral and metal ions) were necessary, which are an important part of enzyme reactive centers or could maintain the structure stability of biomacromolecules and the balance of cell osmotic pressure. for the secondary metabolites in phoma sp. yang. found that mgso4, mgcl2, nacl, kh2po4, kno3, and (nh4)2so4 favored antibiotic production by xenorhabdus spp. d43, but antibiotic production by addition of zn(no3)2 and cuso4 decreased. in this experiment, the results showed that 0.06% mgso47h2o and 0.01% kh2po4 were proper for strain hhs.015 to produce antimicrobial activity. increasing or reducing the concentrations of mgso47h2o and kh2po4, the production of compounds with antimicrobial activity by strain hhs.015 decreased. microelements at different concentrations showed different effects on the microbe physiological activity, so, the suitable concentrations are changing depending on microbe properties and fermentation medium. the apple pomace and rapeseed meal were chosen as two main components in the optimum culture medium for their low market price, 598.4 rmb / t, while soybean in the ssy medium was 3774 rmb / t in the china market. rmb / l in the optimum culture medium ; 91.5% of the production cost were saved compared with the ssy medium, 2.1624 rmb / l, with similar antimicrobial activity. hence, there are a high possibility and feasibility to use the modified cultural media in the scale - up fermentation of strain hhs.015. in the optimization process of fermentation conditions, the approach allowed the determination of the culture conditions that yielded the highest antimicrobial activity by strain hhs.015. in this study, there was a mutual interaction between the medium volume and rotary speed (figure 3(d)) to influence the production of antimicrobial active components. the optimal medium volume in the flask and the rotary speed were around 90 ml and 100 rpm, respectively. our study revealed that rotary speeds, medium volume in the flask are related to the dissolved oxygen (do) in shaken flasks. a proper do level was beneficial to antibiotic production, which is in agreement with those obtained in a 5-l fermentor. lai. found that the shear effect on cell morphology such as mycelium growth or pellet formation was found closely influenced by the do level and antibiotic production was maximized under suboptimal conditions for cell growth. the analysis of significance of each coefficient of the quadratic regression model indicated that initial ph and temperature also have a significant effect on antimicrobial activity of strain hhs.015. it could be observed that the antimicrobial activity increased upon increasing the initial ph from 2.0 to 7.0, but any further increase of ph resulted in decreased production of antimicrobial active components (figures 3(a) and 3(b)). similarly, the optimal temperature was around 25c, lower or higher temperatures reduced the production of compounds with antimicrobial activity (figures 3(c), 3(e), and 3(f)). the results suggested that the broth ph could play a crucial role in the production of secondary metabolites ; this finding is in good agreement with previous studies using for example xenorhabdus species [23, 25, 26 ]. the authors suggested as possible reason that the key enzymes essential for synthesis of antibiotics become inactive at suboptimal ph - values. from the present study, the authors conclude that apple pomace and rapeseed meal were identified as the most suitable medium components for low - cost production of antimicrobial compounds by strain hhs.015. this is the first report about the fermentation of saccharothrix actinomycete using apple pomace as the main medium component. statistical optimization methods for fermentation process could overcome the limitations of classical empirical methods and proved to be a powerful tool for the optimization of conditions of production of antibiotics by strain hhs.015. the optimization of the fermentation process resulted not only in a 91.5% reduction of the costs of raw material compared to the conventional medium but also in a 20% higher antimicrobial activity. furthermore, the information obtained is considered fundamental and useful for developing a cultivation process for efficient production of antibiotics on a large scale. | strain hhs.015 t (saccharothrix yanglingensis sp. nov.), an antagonistic endophytic saccharothrix actinomycete isolated from roots of cucumber plants, exhibited a broad antimicrobial spectrum in vitro and was active as a biocontrol against plant diseases in field trials. the ssy medium was used for production of antimicrobial metabolites by strain hhs.015 t. however, this medium is too expensive for large - scale production. in this study, an alternative culture medium, based on agricultural waste products (e.g., apple pomace), was optimized. the results showed that the alternative medium contained 15 g apple pomace, 4 g rapeseed meal, 0.1 g kh2po4, and 0.6 g mgso47h2o in 1 l distilled water. this medium reduced the material costs by 91.5% compared to ssy medium. response surface methodology (rsm) was used to investigate the influence of environmental variables on production of compounds of antimicrobial metabolites. the optimal conditions achieved were initial ph 7.0, medium volume of 90 ml in 250 ml flasks, rotary speed of 100 rpm, temperature 25c, and inoculation volume of 15.8%. the antimicrobial activity was increased by 20% by optimizing the environmental parameters. the results obtained allow an efficient production of components with antimicrobial activity by strain hhs.015 t on a large scale at low costs. |
it is also known as drumstick tree or horseradish tree ; the leaves are very beneficial and offer important source of beta - carotene, vitamin c, protein, iron, and potassium. the m. oleifera plant is native to the indian subcontinent and has been used by the indians for almost 5000 years. m. oleifera tree can grow well in the humid tropic or hot dry land and it can survive in harsh climatic condition including destitute soil [2, 3 ]. the root, bark, gum, leaf, pods, flowers, seed, and seed oil are used in traditional medicine for treatment of various ailments. the leaves, flowers, roots, gums, fruits, and seeds of m. oleifera are extensively used in the treatment of inflammation, cardiovascular dysfunction, liver disease, and hematological and renal malfunction [57 ]. the leaves of m. oleifera (g / g) have the calcium four times that of milk, vitamin c seven times that of oranges, and potassium three times that of bananas, three times the iron of spinach, four times the vitamin a in carrots, and two times the protein in milk [8, 9 ]. studies have attributed the medicinal benefits of m. oleifera to its anti - inflammatory, antioxidant, and antipathogenic constituents [10, 11 ]. also, the strong antioxidant and scavenging ability of m. oleifera has been linked to chemoprevention of diseases like cancer. research efforts have continued to unearth the medicinal and nutritional potential of m. oleifera in several significant ways. the m. oleifera plant has been used as antispasmodic, stimulant, cough expectorant, and diuretic agent. the use of m. oleifera as diuretic agent is applicable in the treatment of prostatitis, kidney stones, bladder ache, scalding urine, water retention, and obesity. in a separate study, the extract of m. oleifera showed antinephrotoxic effect against dmba - induced renal cancer. in a similar work by owolabi.,, m. oleifera extracts reduced the severity of lead - induced nephrotoxicity. in furtherance of exploring and accentuating the medicinal potential of m. oleifera in protein against heavy metal toxicity, the present study evaluated the nephroprotective capacity of m. oleifera supplemented diets in wistar rats orally administered with nickel sulphate (niso4). commercial reagent kits for the determination of plasma urea, creatinine, potassium, sodium, and protein were either as supplied by randox diagnostic laboratory, crumlin, uk, or the agape laboratory, switzerland. thirty male rats of wistar strain of weight between 190 and 200 g were obtained from the experimental animal farm at the department of biochemistry, university of ilorin, ilorin, nigeria. the wistar rats were housed in plastic animal cages in a well - ventilated experimental room. rats were allowed to acclimatize for a period of 14 days before the commencement of treatments. during the period of acclimatization handling of animals was in accordance with relevant institutional and ethical guidelines as approved for scientific study. the leaves of moringa oleifera were harvested at the landmark university farm, omu - aran, nigeria. bolu ajayi at the herbarium unit, department of plant biology, university of ilorin, ilorin, nigeria. the powdered material was stored at room temperature in cool dry environment until being required for use. protein content, crude fat and fibre, moisture content, carbohydrate, and ash content were determined following established protocols as described by the association of official analytical chemists. thirty male wistar rats of weight between 195 and 200 g were randomly distributed into six experimental groups of five and fed as follows : control received niso4 and fed normal rat chow ; group 2 received niso4 and fed with 5% m. oleifera supplemented diet ; group 3 received niso4 and fed with 10% m. oleifera supplemented diet ; group 4 received niso4 and fed with 15% m. oleifera supplemented diet ; group 5 received normal saline and fed with 15% m. oleifera supplemented diet ; group 6 received normal saline and fed with normal rat chow. control received niso4 and fed normal rat chow ; group 2 received niso4 and fed with 5% m. oleifera supplemented diet ; group 3 received niso4 and fed with 10% m. oleifera supplemented diet ; group 4 received niso4 and fed with 15% m. oleifera supplemented diet ; group 5 received normal saline and fed with 15% m. oleifera supplemented diet ; group 6 received normal saline and fed with normal rat chow. the rats were exposed to daily administration of 20 mg / kg body weight niso4 by oral gavage. 24 hours after the last treatment, the animals were sacrificed under anesthesia in slight diethyl ether. the blood samples were spun at 5000 g for 10 minutes in a refrigerated centrifuge (anke tdl-5000b, shanghai, china) to yield the plasma which was used for the biochemical determinations. the kidneys from each animal were excised into iced physiological solution (ph 7.4), weighed immediately, and fixed in buffered formalin for histopathology examinations. the biochemical indices were determined in rat plasma using a uv / vis spectrophotometer (jenway, staffordshire, uk) where applicable. the levels of rat plasma total protein (tp), creatinine, and urea were determined using randox assay kits (crumlin, uk). the rat plasma electrolytes, sodium, and potassium were determined using reagent assay kits by the agape laboratory (switzerland). the rat kidney was fixed in 10% buffered neutral formalin (bnf) immediately following excision from animals. capture and scoring for morphological changes were done by a pathologist blind to the treatments, the pathology unit, university of ilorin teaching hospital, ilorin, nigeria. data were analysed on graphpad prism 3 (graphpad software inc., san diego, ca) using the one - way analysis of variance (anova). the contents of crude fat, fibre, moisture, and ash in the normal rat chow and m. oleifera supplemented diets showed no significant difference (figure 1). however, the carbohydrate content of supplemented formulated diets decreased with increasing concentration of m. oleifera relative to the normal diet. the various treatments did not evoke any appreciable change in average rat weights over the course of the exposure (figure 2). however, there was 12% decline in rat kidney - to - body weight ratio for the group given niso4 and sustained on normal rat diet. conversely, there were improvements in the kidney weight with restoration to near normal level for groups maintained on m. oleifera supplemented diets. diets containing the 15% m. oleifera caused significant increments in protein level relative to the control group (figure 2). the other treatment groups did not show any appreciable changes. in order to determine whether addition of m. oleifera to diets could reverse or modulate the events of nickel - induced nephrotoxicity, the level of plasma creatinine was determined. for the control group which received niso4 and normal rat diet, the plasma creatinine level was significantly elevated (figure 3). on the contrary, groups given niso4 and sustained on m. oleifera supplemented diets showed significantly lower plasma creatinine level. the restoration or modulation of the rat plasma creatinine to normal level shows correlation with increasing concentration of m. oleifera in the diets given to rats. furthermore, nickel exposure in the control group sustained on normal diet led to increased (p < 0.05) level of plasma urea (figure 3). on the other hand, rats orally exposed to nickel salt and sustained on m. oleifera supplemented diets had significantly reduced level of plasma urea. oral exposure to niso4 and daily sustenance on normal diet in the control group for 21 days lowered (p < 0.05) the level of rat plasma sodium (figure 4). however, for rats given niso4 and sustained on m. oleifera supplemented diets, there were significant elevations in levels of plasma sodium. also, the control group given niso4 and normal diet had elevated (p < 0.05) level of rat plasma potassium compared to groups sustained on m. oleifera supplemented diets in which the rat plasma potassium was appreciably maintained at near normal level. the examination of rat renal sections for morphological changes revealed inimical cellular alterations in control group caused by niso4 exposure. the cellular alterations caused included swollen renal tubules, hyaline change, thickening of glomerular wall, mild nephritis, and necrosis (figures 5, 6, 7, 8, 9, and 10). however, all of the cellular damages caused by niso4 exposure were nonexistent or ameliorated in the groups sustained on the m. oleifera diets. the m. oleifera has shown remarkable potential which could be harnessed for medicinal and nutritional purposes. in recent times, several studies have been able to demonstrate the medicinal significance of m. oleifera [1922 ]. in separate studies, addition of m. oleifera to diet has revealed considerable promises as an adjunct to improving health in a variety of important ways [22, 23 ]. in furtherance of the evidence of medicinal and nutritional benefits of m. oleifera, diets consisting of different concentrations of m. oleifera were compounded and evaluated for potential of attenuating nickel - induced nephrotoxicity in rats. proximate analysis of the diets revealed higher protein content in the m. oleifera supplemented diets. the higher protein content may be due to addition of m. oleifera to the diets. earlier studies have revealed m. oleifera as being rich in protein content [8, 9 ]. to further underscore the nutritional value of m. oleifera diets, the proximate analysis showed reduced carbohydrate content with increasing percentage of m. oleifera. measurement of body weight and/or organ weight may be used to evaluate toxic events due to exposure to a toxicant. alteration in body weight and/or organ to body weight ratio has been shown to imply toxicity arising from exposure to a toxicant [24, 25 ]. no appreciable weight change was recorded for all treatment groups. however, declining kidney - to - body weight ratio in the control group given niso4 and normal diet was improved significantly in groups exposed to niso4 and m. oleifera supplemented diets. this could be an indication that m. oleifera addition to diet was able to attenuate the adverse effect of niso4 administration on rat renal tissues. on the other hand, rat plasma protein levels were not appreciably affected by niso4 exposure. this may be due to the fact that m. oleifera has a high content of protein [8, 9 ]. this is even more plausible if consideration is given to the proximate composition data in which the 15% m. oleifera diet had the highest protein content. plasma levels of creatinine and urea are among the major biochemical indices commonly used to evaluate renal functions [18, 26 ]. creatinine is a by - product of muscle metabolism and under normal physiological condition the amount excreted per day is constant and correlates with body mass. conversely, when creatinine level is elevated due to retention in the blood, it could be used to evaluate glomerular filtration rate. urea, on the other hand, is formed as means to rid body of nitrogenous waste from protein degradation. elevated plasma urea level has been linked to reduced renal function [18, 26 ]. in the present study, the oral exposure of niso4 in rats caused significant elevation to the plasma levels of creatinine and urea in a manner reminiscent of compromised renal integrity. however, the feeding of diets containing different concentrations of m. oleifera to rats prevented the elevation of the indices for renal dysfunction. these findings are in support of previous studies which showed that m. oleifera offered nephro- and hepatoprotection [15, 20, 21, 27 ]. also, in a recent study, the protective effect of m. oleifera against lead - induced nephrotoxicity has been demonstrated. the evidences are demonstrations which strengthen further the belief that addition of m. oleifera to diets could improve health status. to further evaluate nephroprotective effect of diets containing m. oleifera, we determined the level of rat plasma electrolytes. our results revealed significant alteration to levels of rat plasma electrolytes caused by oral exposure to niso4. on the contrary, the significant reductions in levels of plasma sodium and elevations in potassium level caused by niso4 were averted by m. oleifera supplemented diets. perturbation of plasma electrolyte balance may affect the ph, osmolality, and blood volume with adverse impact on the kidney and other related body organs. m. oleifera has been shown to possess diuretic effect [13, 14 ] and this may have contributed to protecting against homeostatic imbalance imposed by nickel exposure. moreover, the maintenance of normal level of plasma sodium and potassium by m. oleifera diet is consistent with reported therapeutic potential including the improvement of the health of renal tissues and general well - being [20, 21 ]. although, at 10 and 15% m. oleifera supplementation, there were increases or decreases in the level of sodium and potassium, respectively, the alterations were found not to be significant when compared to the untreated group given normal saline and 0% m. oleifera diet. the histopathology of renal sections revealed inimical cellular lesions caused by niso4 exposure in rats. the hyaline change, thickening of glomerular wall, mild nephritis, and necrotic areas caused by niso4 were conspicuously absent or attenuated in the groups fed on diets containing m. oleifera. the histopathology presentations support the biochemical findings and confirm further the medicinal protection afforded by m. oleifera against drug - induced tissue damage [15, 16, 19, 21, 27 ]. to our knowledge, this is the first study which revealed that the addition of m. oleifera to diet protected rat kidney against the toxic events caused by niso4 exposure. the data are further scientific demonstration which underpins the medicinal and nutritional potential of m. oleifera. for people working in the mining industries or other related sectors, where the frequency of exposure to heavy metals is high, male wistar rats were daily exposed to oral administration of niso4 and sustained on either normal diets or moringa oleifera supplemented diets.in the control group which was sustained on normal diets, niso4 exposure altered the levels of plasma metabolites including urea, creatinine, and electrolytes in a manner that is reminiscent of impaired renal function.in the other treatment groups sustained on moringa oleifera supplemented diets, there were no alterations to the levels of plasma urea, creatinine, and electrolytes revealing nephroprotective potential of the supplemented diets.oral exposure of niso4 caused damage to renal tubules and glomerular walls. however, moringa oleifera supplemented diets protected against the renal damage.the addition of moringa oleifera to diet protected against nickel - induced nephrotoxicity. male wistar rats were daily exposed to oral administration of niso4 and sustained on either normal diets or moringa oleifera supplemented diets. in the control group which was sustained on normal diets, niso4 exposure altered the levels of plasma metabolites including urea, creatinine, and electrolytes in a manner that is reminiscent of impaired renal function. in the other treatment groups sustained on moringa oleifera supplemented diets, there were no alterations to the levels of plasma urea, creatinine, and electrolytes revealing nephroprotective potential of the supplemented diets. | background. the moringa oleifera plant has been implicated for several therapeutic potentials. objective. to evaluate whether addition of m. oleifera to diet has protective effect against nickel - induced nephrotoxicity in rats. methodology. male wistar rats were assigned into six groups of five. the rats were given oral exposure to 20 mg / kg nickel sulphate (niso4) in normal saline and sustained on either normal diet or diets supplemented with moringa oleifera at different concentrations for 21 days. 24 hours after cessation of treatments, all animals were sacrificed under slight anesthesia. the blood and kidney samples were collected for biochemical and histopathology analyses, respectively. results. niso4 exposure reduced the kidney - to - body weight ratio in rats and caused significant elevation in the levels of plasma creatinine, urea, and potassium. also, the plasma level of sodium was decreased by niso4 exposure. however, addition of m. oleifera to diets averted the nickel - induced alteration to the level of creatinine and urea. the histopathology revealed damaged renal tubules and glomerular walls caused by niso4 exposure. in contrast, the damages were ameliorated by the m. oleifera supplemented diets. conclusion. the addition of m. oleifera to diet afforded significant protection against nickel - induced nephrotoxicity. |
glycated hemoglobin (hba1c) has been considered as an important marker of long - term glycemic control. in the recent past, the american diabetes association has suggested the use of hba1c as diagnostic tool for prediabetes and diabetes. a value between 5.7% and 6.5% represents prediabetes while a value 6.5% is considered as diabetes mellitus. several factors other than glycemic status can influence hba1c levels, including altered red blood cell (rbc) turnover. we hypothesize that hba1c levels may change due to altered thyroid status, possibly due to changes in rbc turnover. therefore, we measured hba1c levels in hypothyroid and thyrotoxic individuals who do not have diabetes or prediabetes based on fasting and 2 h postglucose plasma glucose criteria. we also followed up some of the patients and measured hba1c again after euthyroidism was achieved. the objective of our study was to determine the effects of altered thyroid status on hba1c levels in individuals without diabetes, and with overt hyper - and hypo - thyroidism. we would, thereafter, follow up the patients and see if such changes in hba1c are reversed after achieving euthyroid state. the objective of our study was to determine the effects of altered thyroid status on hba1c levels in individuals without diabetes, and with overt hyper - and hypo - thyroidism. we would, thereafter, follow up the patients and see if such changes in hba1c are reversed after achieving euthyroid state. the study was carried out in department of endocrinology and metabolism, ipgme and r and sskm hospital, kolkata ; and was approved by the institutional ethics committee. patients from both genders, between 18 and 60 years of age, who were diagnosed to have overt primary hypo- or hyper - thyroidism and were treatment naive, were recruited as cases. overt primary hypothyroidism was defined as free t4 below the lower limit of normal range and thyroid stimulating hormone (tsh) more than 15 /ml. hyperthyroidism was defined as free t4 and/or t3 above the upper limit of normal range with suppressed tsh. patients with diabetes (defined as fasting plasma glucose 126 mg / dl [7 mmol / l ] or 2 h post 75 g glucose plasma glucose 200 mg / dl [11.1 mmol / l ]), impaired glucose tolerance (igt) (defined as 2 h post 75 g glucose plasma glucose between 140 and 199 mg / dl [7.811 mmol / l ]), or impaired fasting glucose (defined as fasting plasma glucose between 100 and 125 mg / dl [5.66.9 mmol / l ]) were excluded from the study. both the fasting and oral glucose tolerance test (ogtt) were done on single day. patients with hemoglobin 1 mg / dl ], reduced albumin [500 mg / day), and pregnant patients, were all excluded. age- and gender - matched healthy euthyroid and euglycemic persons were recruited as controls. at the start of the study, assuming that a difference in hba1c of 0.5% among baseline subgroup would be clinically meaningful and assuming that the standard deviation (sd) would be 0.5% and keeping the power at 80% and p = 0.05 ; the sample size needed was calculated to be 17. the patients thus selected (47 hypothyroid, 34 hyperthyroid and 46 controls) additionally underwent tests for estimation of hba1c and reticulocyte count. hba1c was measured at the baseline using ion - exchange high - performance liquid chromatography method using bio - rad d 10 hba1c program which is certified by national glycohemoglobin standardization program as having documented traceability to the diabetes control and complications trial reference method. the reportable range for hba1c on the d-10 hba1c program is 3.8%18.5% (19179 mmol / mol) (i.e., it shows linearity in this range). serum triglyceride up to 5500 mg / dl (62 mmol / l), bilirubin up to 20 mg / dl (342 mol / l), carbamylated hemoglobin up to 2%, and fetal hemoglobin levels up to 10% have no clinically significant effect on hba1c using this system. the hba1c was compared between hypo- and hyper - thyroid patients with age- and gender - matched healthy control population. we could not follow all the patients due to logistic constraints. moreover, for the before and after analysis for follow - up, the sample size needed was estimated to be 10, keeping the power at 80% and p = 0.05, assuming that a change in hba1c of 0.5% would be clinically meaningful and an expected sd of 0.5%. thus, we decided to follow - up randomly selected 30 overtly hypothyroid and 30 thyrotoxic patients from the aforementioned selected patient population. thus, we decided to follow - up randomly selected 30 overtly hypothyroid and 30 hyperthyroid patients from the aforementioned selected patient population. hba1c was rechecked after establishment and stabilization of euthyroid status for at least 3 months. it was at this point that hba1c was rechecked and median hba1c levels were compared with their own baseline values. fasting plasma glucose, 2-h ogtt, hemoglobin, and reticulocyte count were also rechecked. the baseline variables were compared using the unpaired student 's t - test and mann whitney rank sum test as applicable. the paired student 's t - test and the wilcoxon signed rank test were used to compare variables before and after treatment as applicable. fifty - nine hypothyroid and 58 hyperthyroid patients were initially screened, of whom 12 and 24 patients were excluded, respectively [figure 1 ]. this shows that there was no significant difference in fasting and 2 h post 75 g glucose plasma glucose values between the groups. baseline characteristics hba1c in patients initially selected were found to be significantly higher in hypothyroid group (median interquartile range 5.6 0.07 [38 mmol / mol ] [hypothyroid ] vs. 5.2 0.04 [33 3 mmol / mol ] [controls ] ; p < 0.001) [figure 2 ]. hba1c values in hyperthyroid patients were not significantly different from controls (median interquartile range 5.3 0.5 [34 4 mmol / mol ] [hyperthyroid ] vs. 5.2 0.04 [33 3 mmol / mol ] [controls ] ; p = 0.174) [figure 2 ]. comparison of baseline glycated hemoglobin between groups baseline mean reticulocyte count did not differ significantly between hyperthyroid (mean sd 1.48 0.49) and control population (mean sd 1.33 0.54). mean reticulocyte count was lower in hypothyroid patients (mean sd 1.02 0.5) than controls and the difference was tending toward significance (p = 0.07). moreover, there was a significant inverse correlation of hba1c with reticulocyte count in hypothyroid patients with a correlation coefficient of 0.502 and a p = 0.0003 [figure 3 ]. correlation between glycated hemoglobin and reticulocyte count of hypothyroid patients at baseline in the second part of the study, two patients in hypothyroid group developed igt during follow - up and were excluded from analysis. mean follow - up time was 21.3 3.1 weeks for hypothyroid group and 22.6 3.7 weeks for hyperthyroid group. none of the groups had significantly different fasting and post 75 g ogtt plasma glucose values at baseline and posttreatment. hba1c reduction was significant in hypothyroid group following treatment and achievement of euthyroid state (median interquartile range 5.7 0.75 [39 8 mmol / mol ] [pretreatment ] vs. 5.4 0.75 [36 8 mmol / mol ] [posttreatment ] ; p < 0.001) [figure 4 ]. reticulocyte count increased significantly following treatment in hypothyroid patients (0.9 0.575 vs. 1.9 0.77 ; p < 0.001). glycated hemoglobin change following treatment of hypothyroidism change in hba1c values in the hypothyroid group following achievement of euthyroid state was no longer statistically significant when adjusted for changes in reticulocyte count (5.58 vs. 5.74 [37 vs. 39 mmol / mol ] ; p = 0.37). (median interquartile range 5.35 0.45 [35 4 mmol / mol ] [pretreatment ] vs. 5.35 0.3 [35 3 mmol / mol ] [posttreatment ] ; p = 0.323) [figure 5 ]. the reticulocyte count, however, decreased significantly after treatment in hyperthyroid group, (1.485 0.56 vs. 1.035 0.39 ; p < 0.001). glycated hemoglobin change following treatment of hyperthyroidism at baseline, 23 out of 47 (48.9%) of our hypothyroid patient showed hba1c level of 5.7 (39 mmol / mol) whereas 2 out of 34 (5.9%) patient of hyperthyroid group and 1 out of 46 (2.2%) among controls had hba1c 5.7 (i.e., hba1c criterion for prediabetes). among 27 hypothyroid patients who were followed up six of those 17 patients had values < 5.7 at the end of the study. in our study, median baseline hba1c level was found to be significantly higher in overt hypothyroid patients in comparison to matched control population despite having similar glycemic status. the difference of hba1c between control and hypothyroid patients at baseline was higher in our study than the study by kim. this may be due to the shorter duration of hypothyroidism in their patients (4 weeks postthyroidectomy) than ours (most of whom had possibly longer natural history of disease). a study by anantarapu. also demonstrated false elevation of hba1c values in patients with hypothyroidism which was lowered by thyroid hormone replacement without any change in fasting or ogtt values. the reticulocyte count increased following replacement of thyroid hormone. even though there was a difference in hba1c in hypothyroid patients before and after achievement of stable euthyroidism, this difference was no longer statistically significant when adjusted for reticulocyte count, suggesting that altered rbc turnover is responsible for altered hba1c levels in hypothyroid patients. in contrast, hyperthyroid patients had similar median hba1c value in comparison to controls at the baseline. we propose that this could possibly be due to promotion of glycation by malondialdehyde which is produced by lipid peroxidation induced by excessive thyroid hormones. median hba1c value did not change significantly after treatment of hyperthyroidism despite significant change in reticulocyte count. a limitation of our study was that we could not measure rbc turnover directly with investigation like chromium 51 tagged rbc assay and used reticulocyte count for indirect estimation of rbc turnover. 48.9% of our hypothyroid patient showed discrepant result for diagnosis of prediabetes using plasma glucose (fasting and after 75 g oral glucose load) and hba1c level of 5.7 (39 mmol / mol). in contrast, 5.9% patient of hyperthyroid group and 2.2% among controls showed this discrepancy. in the second part of the study (who were followed up), 17 out of 27 patients in hypothyroid group had hba1c 5.7 at the baseline. six of those 17 patients had values < 5.7 at the end of the study. findings of our study suggest that we should be cautious while interpreting hba1c data in patients with hypothyroidism. baseline hba1c levels were found to be significantly higher in hypothyroid patients compared to control individuals despite similar glucose levels. hba1c reduced significantly with treatment in hypothyroid patients without a significant change in glucose levels. our study suggests that hba1c data should be interpreted with caution in patients with hypothyroidism. | introduction : glycated hemoglobin (hba1c) can be altered in different conditions. we hypothesize that hba1c levels may change due to altered thyroid status, possibly due to changes in red blood cell (rbc) turnover.objectives:the objective of this study was to determine the effects of altered thyroid status on hba1c levels in individuals without diabetes, with overt hyper- and hypo - thyroidism, and if present, whether such changes in hba1c are reversed after achieving euthyroid state.methods:euglycemic individuals with overt hypo- or hyper - thyroidism were selected. age- and sex - matched controls were recruited. baseline hba1c and reticulocyte counts (for estimation of rbc turnover) were estimated in all the patients and compared. thereafter, stable euthyroidism was achieved in a randomly selected subgroup and hba1c and reticulocyte count was reassessed. hba1c values and reticulocyte counts were compared with baseline in both the groups.results:hb a1c in patients initially selected was found to be significantly higher in hypothyroid group. hba1c values in hyperthyroid patients were not significantly different from controls. hba1c reduction and rise in reticulocyte count were significant in hypothyroid group following treatment without significant change in glucose level. hb a1c did not change significantly following treatment in hyperthyroid group. the reticulocyte count, however, decreased significantly.conclusion:baseline hba1c levels were found to be significantly higher in hypothyroid patients, which reduced significantly after achievement of euthyroidism without any change in glucose levels. significant baseline or posttreatment change was not observed in hyperthyroid patients. our study suggests that we should be cautious while interpreting hba1c data in patients with hypothyroidism. |
zinc is an essential trace element required for the growth of humans and other animals.13 prasad described a syndrome characterized by dwarfism, hypogonadism, hepatosplenomegaly and zinc - deficiency. in later years, besides iran, it has been reported from egypt, turkey, portugal, morocco, yugoslavia, and other developing countries.46 these reports represent awareness only in diagnosing overt cases, rather than the actual worldwide incidence of zinc deficiency.7 zinc - deficiency in humans and animals causes a wide variety of symptoms, including impaired growth, alopecia, anemia, dwarfism, impaired sexual development, geophagia, dermatitis, loss of hair, poor appetite, abnormal dark adaptation, delayed wound healing and mental lethargy.17 to our knowledge, although a few of the general symptoms of zinc - deficiency have been reported in the studies published so far,19 its effects on the craniofacial structures have not been thoroughly described.10,11 in this study we attempted to investigate the effects of low levels of zinc intake on the dental, mandibular, maxillary and cranial dimensions of the rats, and to review the related literature. this investigation was carried out at the physiology animal laboratory of medical school (ataturk university, erzurum, turkey). the study protocol was approved by the ethics committee of ataturk university faculty of dentistry. in the study, 14 sprague - dawley rats with cessation of lactation on the 24 day of birth were used. rats were randomly divided into two groups : group i rats were fed with a zinc - deficient diet, and group ii rats (controls) with a zinc - containing diet (table 1).12 the zinc - deficient diet was stored at 4.50.5c in plastic containers and handled with plastic containers, plastic gloves, and appropriate utensils to avoid contamination. the diet was placed in shallow glass food cups with stainless steel follow - through disks to reduce food spills. the rats were kept individually in stainless steel cages and maintained at 2225c with a 12-h light / dark cycle. features of zinc deficiency, including cutaneous lesions, loss of appetite, slowed weight gain, loss of hair, diarrhea, and alopecia were observed in all zinc - deficient rats. at the end of the fourth week on the experimental diet, blood samples were centrifuged at 3000 rpm for five minutes and maintained at 85c until shortly before assay. serum zinc level was measured by atomic absorption spectrophotometry (flame type unicam 929). the skulls and mandibles were freed from soft tissues and divided into two halves midsagittally (figure 1). then, the following 13 measurements were evaluated on the skulls, mandibles and teeth (figure 2). l1 : anterior edge of the glenoid fossa extreme anterior extension of the maxillary bone between the incisors. l2 : anterior edge of the glenoid fossa junction of the mesial surface of the first molar with alveolar bone. l3 : maximum skull length : the intersection of the frontoparietal suture and the interparietal suture on the midsagittal plane - most inferior point of the tympanic process. l4 : posterior edge of the condyle junction of the mesial surface of the first molar with alveolar bone. l5 : posterior edge of the condyle - most anterior extension of the maxillary bone between the incisors. l7 : most superior surface of the condyle line tangential to the inferior border of the mandible. l8 : total skull length : most anterior point of the internasal suture in the midsagittal plane - most posterior and external point of the squama occipitalis. l9 : nasal length : most anterior point of the internasal suture in the midsagittal plane - intersection of the nasofrontal suture and the internasal suture on the midsagittal plane. l10 : interzygomatic width : the distance measured between the right and the left zygion points (most external point of the temporazygomatic suture on the zygomatic arcus). l11 : maximum skull width : the distance measured between the right and the left squamosa temporalis points (most distant point of the squama temporalis from the midsagittal plane). l12 : anterior teeth length of maxilla : the distance between the incisive edges of anterior tooth and the margin of gingiva. l13 : anterior teeth length of mandible : the distance between the incisive edges of anterior tooth and the margin of gingiva. the statistical significance of data for all scores between the groups was determined with the student t - test. the statistical significance of data for all scores between the groups was determined with the student t - test. group i (fed with zinc - deficient diet) consisted of seven rats (five females, two males) ; group ii (fed with zinc - containing diet) contained seven rats (four females, three males). the first observation was growth retardation, poor appetite, loss of hair, diarrhea, and ulcerations of the skin and mucosa in the zinc - deficient rats. these findings were observed at the 1016 days in zinc - deficient rats. although body weight, body length and tail length were retarded in zinc - deficient rats, they were advanced in rats fed with a zinc - containing diet (figure 3). serum zinc level of the zinc - deficient rats (group i) was lower than that of the controls (group ii) (p.05), the decrease of the teeth lengths in the zinc - deficient group was seen in table 3. in those rats fed with a zinc - containing diet, l12, but, the differences in l12, l13 values for group i and group ii were not found to be statistically significant (p>.05). the role of zinc in bone metabolism was shown by its stimulatory effect on bone formation in tissue culture,18 bone growth, and mineralization in weaning rats,19 and potent inhibitory effect in vitro on bone resorption.20 there are few known reports demonstrating what effect changes in zinc intake might exert on osseous tissue during the growth stage. gloub reported that a low - zinc diet during adolescence might slow bone growth, enhancing the risk of osteoporosis in later years. weisman and hoyer22 reported that zinc - deficiency reduced the sensitivity of receptors to growth hormone. elberle compared the counts of various cells active in bone formation in rats fed normal diet with those in rats fed a zinc - deficient diet and reported that the osteoblast count was reduced more significantly in the zinc - deficient group than in the normal diet group. kenshi found the changes in zinc intake might exert an effect on the osseous tissue of the mandible and femur. the zinc - deficient group showed significantly lower trabecular and cortical bone density compared with the control group in the mandible. and also, in an animal study, it was found that the zinc - deficient diet caused a decrease in body weight and long bone (weight and length) growth. growth impairment was significantly greater in zinc deficient rats than in control rats.17 ovesen showed that zinc - deficiency in growing rats induced a reduction of bone strength in the femoral neck, the distal femoral metaphysis, and the femoral diaphysis. the bone abnormalities in zinc - deficient rats are caused not only by changes in the growth plate cartilage, but also by changes in the bone size. the zinc - deficient group showed a significantly lower value in dry skull, mandible and teeth measurements when compared with those of the zinc - containing group. changes in zinc intake might exert an effect on the osseous tissues of the jawbones and teeth during the growth stage. a low - zinc diet during adolescence might slow bone and teeth growth and enhance the risk of oral in later years. | objectivesthe aim of this study was to investigate the effects of low levels of zinc intake on the rat mandible and maxilla during growth and to compare these results with those of zinc - containing rats.methodsthe study was carried out on 14 sprague - dawley rats. the rats were randomly divided into two groups. group i rats were fed with a zn - deficient diet, and group ii rats with a zn - containing diet. at the end of the fourth week on the experimental diet, all the rats were killed and blood samples were taken. serum zn levels were measured by atomic absorption spectrophotometry. then, the skulls and mandibles were freed from soft tissues and measurements were made on the dry skulls, the mandibles, and teeth in both of the two groups.resultsthe zinc - deficient group showed a significantly lower value in dry skull, mandible, and teeth measurements when compared with those of the group ii.conclusionschanges in zinc intake might exert an effect on the growth of craniofacial structures. a low - zinc diet during adolescence might slow bone and teeth growth and enhance the risk of oral, periodontal, and orthodontic problems in later years. |
a longstanding computational theory attributes successful memory to a balance between two complementary functions mediated by the hippocampal dentate gyrus and ca3 regions, which receive input from layer ii neurons of the entorhinal cortex. the model proposes that pattern separation, a function ascribed to the granule cells of the dentate gyrus, reduces mnemonic interference by encoding distinctive representations for similar input patterns, while pattern completion refers to the recovery of a prior representation from partial or degraded input, a function ascribed to the extensive recurrent collaterals of ca3 neurons (mcclelland., 1995 ; norman and o'reilly, 2003 ; o'reilly and mcclelland, 1994 ; treves and rolls, 1994) ; for a review see yassa and stark (2011). it has been proposed that such competing, yet complementary processes would minimize interference while maximizing storage capacity for episodic memories. empirical evidence consistent with this model is supported by studies of the encoding properties of neurons in these brain regions in laboratory animals (alme., 2014 ; lee., 2004 ; leutgeb., 2004 ; neunuebel and knierim, 2014). high - resolution functional magnetic resonance imaging (fmri) has also demonstrated alterations in fmri activation in the dg / ca3 regions consistent with such computational functions in the human brain (bakker., 2008 ; yassa., 2010, 2011a). in elderly human subjects (compared to young adults) and in patients with amci (compared to age - matched controls), increased fmri bold activation was localized to the dg / ca3 region (yassa., 2010, 2011a), using a three - judgment memory task designed to tax pattern separation. in both cases, moreover, this condition was associated with reduced pattern separation and a shift to errors indicative of pattern completion. based on these findings, we conducted a randomized controlled trial (rct) of the functional significance of excess fmri activation and its contribution to cognitive impairment in amci subjects, using levetiracetam, an atypical anti - epileptic. we demonstrated that reduction of dg / ca3 bold overactivity, resulting from levetiracetam treatment, improved performance of the amci subjects on the 3-judgment memory scanning task (bakker., 2012). the full rct enrolled three cohorts of amci patients who were treated with levetiracetam in a range of low doses and evaluated on the 3-judgment memory task designed to assess pattern separation / completion processes. here the study focused on the network components most affected in the animal models, particularly subregions of the hippocampal formation and the entorhinal cortex. consistent with those models, we reliably observed elevated fmri bold activation localized to the dg / ca3 subregion of the hippocampal formation, together with a consistent profile in memory performance showing a shift in bias away from pattern separation in favor of pattern completion across amci cohorts. at doses of levetiracetam that improved memory performance in the scanning task, drug treatment also normalized fmri activation in both the entorhinal cortex and dg / ca3 region, reducing dg / ca3 fmri activity and boosting decreased fmri activation in the entorhinal cortex (ec). those findings are consistent with the close coupling across animal and human data in aging and prodromal alzheimer 's disease, with growing interest in the role of neural hyperactivity as a potential therapeutic target to restore the network properties of circuits that are among the earliest affected in alzheimer 's disease (stargardt., 2015). the design for this study is schematically shown in fig. 1. the entire rct protocol consisted of 4 study visits over an 8-week period. each cohort of amci participants was randomized, double - blind, in a within - subject crossover design, with the order of treatment on drug and placebo counterbalanced within each cohort. age - matched controls were treated single - blind on placebo as further described in the procedures that follow. during the baseline visit all participants completed the dementia rating scale (cdr : morris, 1993), and underwent medical, psychiatric, neurological and neuropsychological evaluations, which included the mini mental status exam (folstein., 1975), the buschke selective reminding test (buschke and fuld, 1974), the verbal paired associated subtest of the wechsler memory scale (wechsler, 1987) and the benton visual retention test (benton, 1974). all amci participants had a global cdr score of 0.5 with a sum of boxes score not exceeding 2.5 and met criteria for amci proposed by petersen (petersen, 2004), which includes impaired memory function on testing and no decline in basic activities of daily living. none of the amci participants or age - matched control subjects met criteria for dementia. other exclusion criteria included major neurological and psychiatric disorders, head trauma with loss of consciousness, history of substance abuse or dependency, and general contraindications to having an mri examination (e.g. cardiac pacemaker, aneurysm coils and claustrophobia) or taking the study medication (e.g. known sensitivity or allergies, or severe renal impairment). participants taking anti - epileptic medications were excluded from participation in the study but use of other neuroactive medications was permitted if the participant was stable on the medication for at least 12 weeks and if the treatment regimen was not altered for the duration of the study. the study protocol was approved by the institutional review board of the johns hopkins medical institutions. all participants provided written informed consent and were paid for their participation in the study. at the baseline evaluation sixty - nine participants with amci and 24 age - matched controls met criteria for enrollment. complete data from 54 participants with amci and 17 control participants were included in the analysis. data from an additional 6 amci participants and 1 control participant were excluded before analysis due to excessive motion or in - scanner task performance that was inadequate for analysis of the fmri data. all participants completed the same study procedures with fmri study visits after each of two treatment phases, separated by a washout period of 4 weeks as shown in fig. 1. control subjects were given placebo during both treatment phases (single - blind) while participants with amci were given placebo during one treatment phase and drug during the other treatment phase, with the order of treatment counterbalanced (randomized, double - blind). a first cohort of amci participants received treatment with 125 mg bid of levetiracetam (keppra, ucb laboratories), as was previously reported (bakker., 2012). based on those initial findings and earlier preclinical data in animals (koh., 2010), two additional doses were selected for two subsequent cohorts of amci participants, receiving treatment with 62.5 mg bid and 250 mg bid of levetiracetam, respectively. all study treatments (drug and placebo) were prepared in identical non - descript capsules by the investigational drug service at johns hopkins hospital. after 2 weeks on treatment, each study visit included a brief medical and psychiatric examination, the neuropsychological assessment, a blood draw and an mri scan during performance of the 3-judgment memory task. at the end of the washout visit, each participant had a blood draw and was provided with the study medication for the second treatment phase of the study. treatment compliance was assessed by participant self - report at the end of each treatment phase, medication diaries, and analysis of levetiracetam blood values at each visit during the study protocol. the study team was blind to the status of the amci participants and levetiracetam blood levels until final group analysis of the data. in addition to providing the study medication the investigational drug service randomized amci participants to the treatment conditions and controlled blinding and unblinding of study data according to standard clinical trial procedures. data safety was monitored by three physicians not related to the study in collaboration with the investigational drug service. the fmri activation paradigm was a 3-alternative forced choice task designed to assess pattern separation and completion processes, as mentioned above (bakker. for this task participants were asked to view a series of stimuli consisting of 768 pictures of common namable objects (see fig. 2). this included 96 pairs of related but not identical pictures of the same object referred to as lures, 96 pairs in which the identical picture was repeated, referred to as repeats and 384 unrelated single pictures of objects used as foils. each stimulus was presented for 2500 ms with a 500 ms inter - stimulus - interval consisting of a blank screen. all trials were presented in pseudo - random order with the limitation that a lure or repeated stimulus was presented within 30 trials of its pair. for each stimulus, stimuli were presented and responses collected using an apple macintosh laptop computer running matlab software (the mathworks, natick, ma), a back - projection screen and an lcd projector located outside of the scan room. responses were made using one button in the left hand and two buttons in the right hand connected to a cedrus rb-610 response box. before each mri session subjects completed a brief practice task consisting of 96 trials outside of the scanner to familiarize themselves with the stimuli and the procedures. imaging sessions were conducted on a 3 tesla philips scanner (philips, eindhoven, the netherlands) equipped with a sense parallel imaging head coil (mri devices, inc., waukesha) and higher order shims to compensate for local field distortions at the f.m. kirby research center for functional brain imaging at the kennedy krieger institute on the johns hopkins medical campus. high - resolution functional images were collected using a t2 -weighted echo planar single shot pulse sequence with an acquisition matrix of 64 64, an echo time of 30 ms, a flip angle of 70, a sense factor of 2, an in plane resolution of 1.5 1.5 mm and a tr of 1.5 s (kirwan., 2007). each volume consisted of 19 oblique 1.5 mm thick axial slices with no gap oriented along the principal axis of the hippocampus and covered the medial temporal lobe bilaterally. four dummy scans were completed at the beginning of each run to allow for stabilization of the mr signal. in addition, a whole brain structural scan was acquired using a magnetization prepared rapid gradient echo (mprage) t1-weighted sequence with 231 oblique slices, 0.65 mm isotropic resolution and a field of view of 240. image data analysis was performed using the analysis of functional neuroimages (afni) software package (cox, 1996). the functional images were first co - registered to correct for slice timing and head motion, using a three - dimensional registration algorithm creating motion vectors to remove trials in which a significant head motion occurred plus and minus one tr from further analysis. of critical interest were the participants ' responses on the lure items used to assess the balance between pattern separation and pattern completion functions in the hippocampal formation. to avoid confusion, throughout the paper we will refer to items subsequently tested with repetitions as subsequent lures. the subsequent items all refer to the 1st presentation trials. during the 2nd presentation, trials will be referred to as targets and lures respectively. following this convention, functional runs were concatenated and 6 vectors were defined to model the different trial types : (1) repeats subsequently called old, (2) lures subsequently called similar, (3) lures subsequently called old, (4) repeats called old, (5) lures called similar and (6) lures called old. all other response types (misses, false alarms, etc.) were modeled but not included in the secondary analyses. the full set of vectors were used to model each individual 's data using a deconvolution approach based on general linear regression treating the single foil presentations that were correctly rejected as a non - zero baseline against which all other conditions were compared. the resulting statistical fit coefficient maps represent the difference in activity between each of the trial types and the baseline for a given time point for a given voxel. the sum of the fit coefficients over the length of the hemodynamic response (~312 s after the onset of the trial) was taken as the model 's estimate of the response to each trial type. the statistical maps were then smoothed using a gaussian kernel of 3 mm to account for variations in individual functional anatomy. methods used for cross - participant alignment in this study increase the power of multi - subject regional fmri studies by focusing the alignment power to the regions of interest using a segmentation of the subject 's anatomical image. initial affine registration was used to transform the subject 's anatomical and functional images to the talairach coordinate system (talairach and tournoux, 1988). subregions of the medial temporal lobe and the hippocampus were manually segmented into three dimensions using the structural scan and methods described previously (bakker., 2008 ; kirwan., 2007 ; yassa and stark, 2009). briefly, the entorhinal cortex, perirhinal cortex, parahippocampal cortex, and temporopolar cortices were defined bilaterally in the coronal plane using methods described by insausti. the ca1, dg / ca3 and subiculum subregions of the hippocampus were also defined in the coronal plane following landmarks described in the atlas of duvernoy (2005). the dg / ca3 region included the ca2/ca3/ca4 and dentate gyrus subregions as these regions can not be reliably separated on mri scans. using both the segmentation label - based information for the point - set expectation (pse) error metric and the grayscale structural image for the pure cross - correlation (pr) error metric, advanced normalization tools (ants) was used to calculate the 3d vector field transformation for each subject needed to align the individual 's rois to a template modal model of the rois based on the entire sample (klein., 2009 ; yushkevich., 2009) equally weighing the segmentations and t1 gray scale data. the 3d vector field for each individual was then applied to the concatenated fit coefficient maps resulting from the functional analysis. age, education and neuropsychological and functional assessment scores between groups were compared using independent sample t - tests. the primary objective of the study was to determine the efficacy of levetiracetam treatment in participants with amci, using within - subject comparisons for each cohort. to facilitate a comparison of the fmri data across the three cohorts of amci participants, independent analyses were first conducted for each cohort using a two - way anova with trial type (6 trial type vectors) and group status (amci on placebo and aged - matched control group) as fixed factors and subject as a random factor nested within group. a liberal voxel threshold of p < 0.07 was used on the main effect of group f - statistic in combination with a spatial extent threshold of 40 voxels to select areas of task - related activation. the resulting areas of activation were then combined with the anatomical segmentations in order to include only voxels within our areas of interest. the hybrid functional / anatomical analysis resulted in clusters of voxels in each amci cohort where activity varied systematically between amci and control cohorts within each of the anatomical regions of interest. voxels within each functional / anatomical region of interest were then collapsed for further analysis. planned comparisons using t - tests were used for comparisons between the control and the amci cohorts on placebo and for the comparisons within each amci cohort comparing the amci participants on placebo and on levetiracetam on the critical trial type. the amci participants in each of the treatment cohorts did not significantly differ in age, education and proportion of males and females from the subjects in the control group. consistent with enrollment criteria, participants with amci scored significantly lower on immediate and delayed recall on tests of verbal and visual memory (table 1). in each of the three amci cohorts, an area localized in the left dg / ca3 subregion of the hippocampus showed significantly increased bold activation during lure trials correctly identified as similar when compared to control subjects (62.5 mg bid, t = 3.074, p = 0.004 ; 125 mg bid, t = 2.636, p = 0.013 and 250 mg bid, t = 2.070, p = 0.047) (fig. the amci participants in each cohort also had a similar profile in the 3-judgment memory task relative to aged - matched controls as assessed by the rates of each response option (old, similar, or new) on the critical lure trials. for those trials, a between - groups anova revealed a significant effect of response type and, importantly, a significant group by response interaction in each cohort. a post - hoc analysis of that interaction by a planned contrast showed that amci participants on placebo incorrectly identified lure items as old more often and gave relatively fewer correct responses of similar compared to control subjects (control vs. amci placebo by old vs. similar 62.5 mg bid, f(1,35) = 4.272, p = 0.046 ; 125 mg bid f(1,32) = 7.687, p = 0.009 ; 250 mg bid f(1,32) = 9.167, p = 0.005) (fig. 3a c). similar responses is consistent with reduced pattern separation and a shift to pattern completion, e.g. more erroneous responses of old in amci. to assess whether low dose levetiracetam treatment effectively reduces hippocampal activation, functional data during the fmri memory task performance in amci participants under placebo treatment the neuroanatomical regions of altered activity obtained from the initial comparison of amci on placebo with the age - matched control group for each of the three treatment cohorts were used to assess the effects of levetiracetam treatment. at the lowest dose, levetiracetam treatment using 62.5 mg bid did not significantly reduce bold activation compared to the placebo condition (t = 1.417, p = 0.1726) during trials correctly identified as similar. however, activation in the dg / ca3 subregion after levetiracetam treatment with 62.5 mg bid no longer differed from activity in that region observed in the age - matched control group. levetiracetam treatment using 125 mg bid significantly reduced bold activation during the correctly identified lure trials relative to the activity during those trials under placebo treatment (t = 2.279, p = 0.037). levetiracetam treatment with 250 mg bid did not change activation during lure trials correctly identified as similar relative to the activity during such trials under placebo treatment (t = 0.326, p = 0.749) (fig. treatment with low dose levetiracetam also improved behavioral performance on the lure trials in treatment groups where levetiracetam treatment normalized increased dg / ca3 activity. a post - hoc analysis of the interaction by planned contrast showed that relative to their performance on placebo, amci participants taking 62.5 mg bid or 125 mg bid of levetiracetam made fewer incorrect responses of old while concomitantly increasing correct judgments of similar (amci drug vs. amci placebo by old vs. similar ; 62.5 mg bid f(1,19) = 4.783, p = 0.041 ; 125 mg bid f(1,16) = 5.028, p = 0.039) and performance under drug treatment in these groups were no longer significantly different from healthy control subjects (amci drug vs. control by old vs. similar ; 62.5 mg bid f(1,35) = 1.823, p = 0.186 ; 125 mg bid f(1,32) = 1.945, p = 0.173) (fig. finally, in addition to not altering activation in the left dg / ca3 during the critical lure trials, treatment with 250 mg bid of levetiracetam also did not improve performance compared to their performance on placebo. relative to their performance on placebo amci participants taking 250 mg bid of levetiracetam did not alter the proportion of old and similar responses to lures (amci drug vs. amci placebo by old vs. similar ; f(1,16) = 1.492, p = 0.2396) and remained significantly different from control subjects (amci drug vs. control by old vs. similar ; f(1,32) = 5.208, p = 0.029) (fig. to confirm the finding that low dose levetiracetam treatment effectively reduces hippocampal activation, a separate analysis was conducted in which voxel selection was based on a one - way anova of trial type only in control subjects. this analysis resulted in an area of task - related activation similarly localized to the left dg / ca3 subregion of the hippocampus. the effect of drug treatment was confirmed by comparing fmri activation in that area of task - related activity in amci participants on placebo and levetiracetam within each of the three amci cohorts. confirming the treatment effects shown in fig. 4 this analysis similarly showed that levetiracetam treatment using 62.5 mg bid lowered but did not significantly reduce bold activation compared to the placebo condition (t = 1.503, p = 0.1492). levetiracetam treatment using 125 mg bid significantly reduced bold activation during the correctly identified lure trials relative to the activity during those trials under placebo treatment (t = 2.192, p = 0.044). levetiracetam treatment with 250 mg bid, in contrast, showed no evidence of reducing activation (t = 0.1773, p = 0.8615). in addition to areas of task related activity in the dg / ca3, analyses of the functional imaging data also revealed an area of reduced task related activity in the left entorhinal cortex (ec) in both the 62.5 mg bid cohort and the 125 mg bid cohort. in the left entorhinal cortex, participants with amci on placebo showed significantly decreased bold activation during those same lure trials compared to control subjects (62.5 mg bid, t = 3.443, p = 0.002 and 125 mg bid, t = 3.278, p = 0.003). after drug treatment bold activation in the left ec was increased and normalized in amci participants during the critical lure trials in comparison to placebo treatment. this increase was statistically significant in the 62.5 mg bid cohort (t = 3.318, p = 0.004) but not statistically significant in the 125 mg bid cohort (t = 1.60, p = 0.129). in both cohorts activity in the left ec after drug treatment was no longer different from activity observed in the left ec in control subjects (fig. 5). these effects on bold activation in the left dg / ca3 and ec in the 62.5 mg bid and 125 mg bid treatment cohorts were obtained with drug doses well below those used clinically for the treatment of epilepsy. drug levels in amci patients were determined to be 2.9 0.29 g / ml (mean sem) for the 62.5 mg bid cohort and 4.4 0.53 g / ml for the 125 mg bid cohort. the ineffective dose of 250 mg bid provided a drug level of 7.91 0.92 g / ml. these levels of drug exposure are well below typical ranges for efficacy of levetiracetam as an anti - epileptic agent where doses of 10003000 mg / day are typical, achieving levels of 1040 g / ml (lyseng - williamson, 2011). (2012) measures of standard neuropsychological test performance were not affected in any of the three treatment cohorts. the primary focus in the current study was to assess the effects of treatment with the atypical anti - epileptic, levetiracetam, on memory performance in the scanning task and fmri signals in amci patients. in the current investigation, low doses of levetiracetam (62.5 and 125 mg bid) significantly improved memory performance in the scanning task with attenuation of overactivity, an effect that was statistically significant at 125 mg bid. in contrast, no difference in either memory task performance or fmri activation was observed at 250 mg bid. the current findings further suggest that in the dose range of 62.5125 mg bid, levetiracetam confers benefit on the network properties of the medial temporal lobe memory system. in addition to excess fmri activation, a region of decreased activation in the entorhinal cortex (ec) was observed during task performance, similar to yassa. while overactivity was attenuated by levetiracetam in amci cohorts treated with low doses (62.5 and 125 mg bid), decreased ec activation was concurrently normalized, with a significant boost in ec activation at the 62.5 mg bid dose compared to placebo. the observed areas of fmri activation, consistently localized to the left dg / ca3 subregion in amci patients relative to age - matched controls, differed somewhat across cohorts in an anterior to posterior location. although differences in anterior posterior localization have been observed in neuroimaging studies using a variety of task - activated fmri paradigms, the functional significance of such localization remains somewhat equivocal (see poppenk. (2010) noted a relatively greater proportion of ca fields (ca1 - 3) in the anterior hippocampal formation with the dg having a proportionately greater representation at the posterior segments of the long axis. overactivity in areas of activation localized anterior to posterior, as reported here and elsewhere (bakker., 2012 ; 2010) in the context of the 3-judgment recognition task could reflect a relatively greater contribution of augmented pattern completion in anterior areas of activation with any excess activation reflecting a loss of pattern separation in dg contributing more in posterior areas of activation. overall, however, these findings suggest that excess fmri activation in the dg / ca3 is associated with a consistent profile of memory task impairment across all three amci cohorts. importantly, no evidence was found in this study to support a beneficial compensatory role in cognition for excess hippocampal activation. instead, the data support the view that hippocampal overactivity contributes to symptomatic impairment in the mci phase of disease (for review see ewers., 2011). because mci patients with hippocampal overactivation also exhibit greater cortical thinning, which is indicative of early alzheimer 's disease (ad) related neurodegeneration (putcha., 2011), and excess hippocampal activation detected by fmri predicts subsequent cognitive decline (miller., 2008), it is hypothesized that hippocampal overactivity contributes to disease progression and neuronal damage if not controlled. the deleterious effect of hippocampal overactivity detected by fmri is further supported by recent evidence of a distinctive gene expression profile underlying excessive hippocampal excitability in mci patients. in a study of autopsy brain samples, (2014) reported that the mrna microarrays from mci brains exhibited a profile that clustered those patients together and segregated them from groups of older controls and patients with a diagnosis of alzheimer 's dementia. in addition, mrna markers for hippocampal excess excitability and aberrant plasticity, which were hallmarks in the mci profile, were correlated with severity of cognitive impairment within the mci cohort. the reliable signature of hippocampal overactivity in the current study for three cohorts of amci patients aligns with such data, as does the detection of overactivity in the ca3/dg subregion reported in a separate study of amci patients (yassa., 2010). studies using neural recordings in rats first focused attention on the computational functions of the dg and ca3 regions as a basis for age - related memory impairment. such studies in aged rats with memory impairment have demonstrated a computational shift away from pattern separation in the encoding properties of hippocampal neurons ; instead of the rapid encoding of new information, neurons retrieve a previously encoded representation (wilson., 2006, 2003). likewise in studies comparing young vs. older adults, the use of lures that are similar, but not identical, in 3-judgment recognition demonstrates that elderly participants make fewer correct responses of similar indicative of pattern separation, while increasing errors that reflect pattern completion, e.g. judging lure items as old repeats (lacy., 2011 ; stark., 2013 ; toner. patients with amci have shown further worsening in memory impairment on lure items compared to age - matched controls (stark., 2013 ; yassa., 2011b), as observed in the current investigation. increased fmri activation in the dg / ca3 subregion in human aging (o'brien., 2011a) and its further augmentation in amci, as reported here and elsewhere (bakker. 2010), may primarily reflect an elevation of ca3 neural activity which has been directly observed in memory - impaired aged rats (wilson., 2005). overactive ca3 neurons and their massive recurrent collaterals are a likely a basis for driving greater pattern completion to shift the balance away from pattern separation. although it is not possible to reliably differentiate dg apart from ca3 in human brain imaging, any greater activation in the dg contributing to the elevated fmri signal in composite dg / ca3 areas of activation could also reflect diminished pattern separation in the dg itself. in addition to elevated activity in the firing rates of ca3 pyramidal neurons, another distinctive signature of aged memory - impaired rats is a loss of integrity affecting the interneurons in the hilus (spiegel., 2013), which normally limits the activation of granule cells receiving perforant path input from the ec (andrews - zwilling., 2010). in a mouse model of genetic risk for alzheimer 's disease, apoe4 has been observed to augment the age - dependent loss of hilar interneuron integrity further reducing inhibitory control of dg granule cells (andrews - zwilling., 2010). although somewhat speculative, if a similar condition occurs in humans, a greater number of granule cells activated by perforant path input could contribute to elevated fmri activation in dg / ca3 reflecting degradation of the sparse encoding by granule cells, a property that is critical for pattern separation. with respect to the ec, the current findings of decreased fmri activation replicate that condition previously reported in amci patients compared to age - matched controls (yassa., 2010). studies with animal models have pointed to the ec as contributing to age - related memory impairment and studies with humans have confirmed the progressive worsening of that impairment in patients with amci. notably, in rats the presence and severity of cognitive impairment in aging are tightly coupled to loss of synaptic integrity for the layer 2 neurons in the ec innervating dg and ca3 (smith., 2000). in humans a further loss of those connections is observed in mci and ad compared to aging, with that synaptic loss correlating with worse delayed - recall memory performance (scheff., 2006). additionally, molecular alterations affecting the ec layer 2 neurons occur in both age - related memory impairment in rodents (stranahan., 2011), as well as in ad animal models (happ mice) and thus the network comprised of the ec together with the hippocampal formation exhibits features underlying age - related cognitive impairment that exhibits a further progression in amci compared to aging. it should be noted, however, that the current methods for detection of decreased fmri activation in the ec were not localized to a specific ec layer. the fmri signature in amci and its restoration by treatment could be due to beneficial effects on the network overall, involving not only ec input to the hippocampus but also hippocampal output to the ec, which innervates deeper ec layers. the relationship between loss of ec input to the dg / ca3 and overactivity in those regions is also not yet clearly defined but could involve the extensive network of interneurons that receive ec and dg input to control excitability of principal neurons in the dg / ca3 region. the current investigation has served to identify a condition detected by brain imaging and its modification by an intervention built on basic research that has increased our understanding of the contribution of circuits involving the ec and its targets in the dg and ca3 regions to memory. the current study leveraged discoveries in cognitive neuroscience based on recordings of the encoding properties of neurons and use of other methods not possible in humans. studies in rodents, both young adults and in a well - characterized model of neurocognitive aging, were especially informative about the properties of the network underlying age - related memory impairment and initial preclinical tests of therapeutic interventions (koh. low doses of the atypical anti - epileptic levetiracetam have shown benefit in conditions of age - related memory impairment in rodents and laboratory models relevant to alzheimer 's disease (koh., 2010 ; rhinn., 2013 ; sanchez., 2012 ; shi., 2013 ; suberbielle., levetiracetam has also been reported to have beneficial effects in circuits throughout the mtl network when treatment improves hippocampal - dependent cognition (sanchez., 2012). moreover, a therapeutic window for levetiracetam treatment similar to that observed in the current clinical investigation has also been reported in animal models (koh. 2013), with a lack of efficacy for levetiracetam dosing in the usual clinical range for treating patients with epilepsy (koh. based on such findings, a translation from animal models to humans with the use of brain imaging represents a promising approach to advance discovery in clinical research. the findings presented here result from a high - resolution approach to defining hippocampal subregions by imaging the medial temporal lobe (mtl). as such the methods do not consider broader network changes that have been observed in aging and mci, for example involving default mode networks in the cortex. further research, incorporating both mtl high - resolution and whole brain acquisitions in the same study population will make possible the assessment of such changes as well as changes in connectivity with regions of task - related fmri in the mtl. | studies of individuals with amnestic mild cognitive impairment (amci) have detected hyperactivity in the hippocampus during task - related functional magnetic resonance imaging (fmri). such elevated activation has been localized to the hippocampal dentate gyrus / ca3 (dg / ca3) during performance of a task designed to detect the computational contributions of those hippocampal circuits to episodic memory. the current investigation was conducted to test the hypothesis that greater hippocampal activation in amci represents a dysfunctional shift in the normal computational balance of the dg / ca3 regions, augmenting ca3-driven pattern completion at the expense of pattern separation mediated by the dentate gyrus. we tested this hypothesis using an intervention based on animal research demonstrating a beneficial effect on cognition by reducing excess hippocampal neural activity with low doses of the atypical anti - epileptic levetiracetam. in a within - subject design we assessed the effects of levetiracetam in three cohorts of amci participants, each receiving a different dose of levetiracetam. elevated activation in the dg / ca3 region, together with impaired task performance, was detected in each amci cohort relative to an aged control group. we observed significant improvement in memory task performance under drug treatment relative to placebo in the amci cohorts at the 62.5 and 125 mg bid doses of levetiracetam. drug treatment in those cohorts increased accuracy dependent on pattern separation processes and reduced errors attributable to an over - riding effect of pattern completion while normalizing fmri activation in the dg / ca3 and entorhinal cortex. similar to findings in animal studies, higher dosing at 250 mg bid had no significant benefit on either task performance or fmri activation. consistent with predictions based on the computational functions of the dg / ca3 elucidated in basic animal research, these data support a dysfunctional encoding mechanism detected by fmri in individuals with amci and therapeutic intervention using fmri to detect target engagement in response to treatment. |
. they control gene expression by targeting the cleavage of cognate mrnas or by inhibiting their translation (13). therefore, when studying the biology of any organism, it is of utmost importance to quantify precisely the expression level of each particular microrna gene during organ development. northern hybridization, microarray analysis, deep sequencing approaches and real - time quantitative pcr (rt qpcr) are standard techniques used to accomplish this task as described previously (47). the rt qpcr is considered a gold standard method in precise quantification of gene transcript levels (8). microrna genes that encode transcripts which are processed to the same or similar mature microrna species are grouped in families. in arabidopsis thaliana, the number of such family members varies between 1 (e.g. mir163, mir173) and 14 (mir169) [mirbase release 17 (9) ]. microrna genes from the same family, although represented by identical or almost identical mature micrornas, differ considerably in gene organization and sequence. in many cases it is only possible to observe the expression of all family members as a group, rather than the individual members due to the large sequence conservation when using northern hybridization or deep sequencing approaches. however, individual members of a given microrna family may be expressed in different developmental stages or in response to various biotic / abiotic stimuli (1012). our resource contains information regarding the expression of 190 a. thaliana microrna genes at the level of primary microrna (pri - mirna) in different developmental stages obtained using a rt qpcr high - throughput platform. many databases that store and allow access to microrna gene expression profiles from variety of organisms already exist [e.g. (1318) ], however, they have various limitations and restrictions. one of the limitations includes the lack of tools and/or data for comparisons between developmental stages and various genes at the same time. with this in mind, we developed the mirex platform as a comprehensive starting point for a comparative investigation of microrna genes expression. our database offers to the scientific community easily accessible data and is of interest to researchers working on the specific microrna function, the expression profile of entire microrna family members during a particular organ / developmental stage, or on microrna biogenesis. additionally, by creating the mirex interface, we hope to propose a new database interface standard for comparative microrna gene expression data mining. in the current release, the mirex database includes expression data of microrna genes from a. thaliana. to collect the data, we prepared a real - time pcr platform for 190 primary microrna sequences. for each microrna, a pair of specific primers amplifying a single product was designed ; 10 micrornas primer sequences were taken from pant. the qpcr reactions for one biological replicate included 12 different controls and standards, were carried out in parallel as described previously (20) using rna template isolated from six developmental stages of a. thaliana columbia-0 wild - type plants. the detailed procedure of running the platform can be found in the mirex database documentation. the plant material used in expression profiling experiments includes : seeds, 10-day - old seedlings, 14-day - old seedlings, 25-day - old plants, 35-day - old plants, 42-day - old plants (rosette leaves and stems) and 53-day - old plants (rosette leaves, stems, inflorescence and siliques). for better visualization of the individual developmental stages our high - throughput platform contains an original and novel set of data on microrna gene expression in dormant seeds. the pp2a (at1g13320) and actin (at3g18780) cdnas were used as an expression reference (22). the mean measurements of three replicas were used to calculate the fold - change value and presented in a form of log10. in a case when the value of correlation coefficient of the three replicas was < 0.995, such data was labeled as low quality and is not shown by default on the graphs, but indicated in gray in the data tables. since the level of expression of most of the primary micrornas is lower than the reference genes, the data shown on the graphs in mirex is rescaled in order to avoid presenting it as a negative value. rescaling shifts the zero value of the graph 's y - axis to the basal expression level of the whole experiment. this allows showing the expression profile in a positive data range, but does not change the actual values. the mirex database interface is designed to be used by a bench scientist on an everyday basis. following a simplicity rule, the interface of mirex has been built on only two types of result windows and a simple two - step querying system. there are two ways to access data in mirex : by searching for a particular microrna or microrna family, or by browsing the database content. a single input window allows searching for an individual or a family of microrna by providing the numerical part of its identification (i d). when entered i d corresponds to a record of a single microrna, the resulting page shows all of the details for this particular sequence (figure 1). in the case of whole family i d, the search results are presented in the form of a line graph, showing expression data for all stages available in the database (figure 2). the table located below the graph contains numerical values for data presented in the graph, including low quality measurements. the ids shown in the table allow quick access to detailed information about particular microrna. this window is divided into distinct sections representing : expression data, sequences and external references. the right panel contains shortcuts to microrna family members ' records, structure of the rna transcript molecule with labeled mature sequences and rt qpcr primers and webform for user comments. the report window contains graphical presentation of the expression levels, as well as actual numerical values presented in tabular format. holding a mouse pointer over any data point allows access to details of expression measurements. the datasets for a particular microrna can be dynamically turned on and off by clicking i d in the legend of the graph. this window is divided into distinct sections representing : expression data, sequences and external references. the right panel contains shortcuts to microrna family members ' records, structure of the rna transcript molecule with labeled mature sequences and rt qpcr primers and webform for user comments. the report window contains graphical presentation of the expression levels, as well as actual numerical values presented in tabular format. holding a mouse pointer over any data point the datasets for a particular microrna can be dynamically turned on and off by clicking i d in the legend of the graph. browsing the first step includes selection of developmental stages, which is followed by the selection of micrornas. in mirex, there are three ways to select micrornas : (i) by typing their ids in the input window, (ii) by uploading a file with a coma - separated list of microrna ids (that can be created in the first step) and (iii) by selecting individual or groups of sequences from the tree - like expandable menu. during the process of entering information using a keyboard it is possible to mix all of the input methods the resulting page will refer to all entered or selected ids. presentation of search / browse results may differ depending on the number of selected developmental stages or microrna genes (see data mining section). by default, the graph of expression profiles for various stages and micrornas shows only high quality data. however, it is a user - defined option in mirex whether the low quality measurements are presented on the graph. the results for a single microrna are presented in a record window in a form that is divided into distinct sections (figure 1). the upper part of the record is dedicated to expression information presented in the form of a graph and two tables. when applicable, the graph contains information about the expression of other members of the microrna family, that can be dynamically turned on and off. additionally, specific shortcuts allow quick access to the records of any member of such family. the two tables, containing either reference - calculated or raw data, can be hidden to reduce the amount of information presented at any given moment on the single record window. each graph or table in the mirex database can be saved for later use by selecting an appropriate button. additionally, below the quantitative expression data, each record contains a web link to a text - based map of publicly available high - throughput next - generation sequencing (ngs) short reads aligned to primary microrna transcript. the information included here contains sequence, location, length and number of the mapping reads. on a separate window, it is also possible to access the original record of the used ngs data. the record window contains a graphical presentation of the structure of microrna transcript with designations of mature molecule(s) and primers used to assess its expression level by rt qpcr. when available, the record data will be modified to include results from northern hybridization with mature micrornas. however, it has to be noted that in most cases, such hybridization represents the level of the whole microrna gene family expression, and will be not specific for an individual microrna gene. moreover, the record window contains a table with sequences relevant to particular microrna and a link to experimental procedures. our aim was to avoid replication of the data available in other databases. for information such as gene structure or available papers, we direct the user to specialized databases, for example tair (23) or pubmed (24). the number of external references will grow in time, when new resources for microrna biology are available, and in response to user requests. to maintain a close relation with users of our database, we provide in the record window a simple tool for entering comments. the comment may be of any nature : concerning the biological aspect of the presented data or any general issue. although the interface is very intuitive and simple, at the main page we provide a video tutorial on all of the capabilities and various ways available to explore the mirex tools and data. a complete road map of the mirex interface is included as supplementary figure s1. the usefulness of the mirex database lies in the ability to compare expression between different developmental stages and various microrna genes. by using the browse button, the user can, in two simple steps, select available developmental stages and a single or sets of micrornas. depending on the number of selected stages, the expression results are presented in the form of a bar graph (for one - stage analysis) or a line - based graph (for many stages). the bar graph presentation allows analysis of all pri - mirnas at once with a zoom - in on an individual gene. the line graph displays by default, data for only 24 micrornas selected from the list. the line graph offers an additional option to dynamically remove and/or add any of the selected microrna genes., the shortcuts represent a new and easy way to explore data stored in the mirex database. they are located in the same area on every page, and their content follows user selections. the use of shortcuts allows quick access to specific sets of data, e.g. the most expressed genes, or records representing micrornas belonging to the same family. periodically, we will modify the shortcuts in response to comments and readapt them according to the most frequently issued queries. the strategy to use primary microrna expression data allows fine exploration of gene activities of members of microrna families. in some families, however, the expression level of the individual pri - mirnas may differ considerably : the fold change in the primary transcript levels within one microrna gene family may range between 10 and 10 (e.g. mir160, mir162, mir164, mir165, mir167, mir168, mir394, mir396, mir398, mir447 families, figure 3a). moreover, a few individual pri - mirnas show profound differences in their expression depending on the developmental stage and/or organ studied (e.g. mir156, mir157, mir158, mir159, mir166, mir169, mir397, mir399, mir404, figure 3b). these differences may reflect either the existence of promoter regulatory elements that are responsive to developmental stimuli, or the various rate of pri - mirna maturation during plant growth and organ formation. microrna families containing a single representative also may exhibit dramatic changes in their expression levels (reaching even 10-fold change) during plant growth and organ development (e.g. mir173, mir774, mir776, mir778, mir780, mir783, figure 3c). conversely, there are also pri - mirnas that show relatively small fluctuations of expression levels during plant growth and organ formation. in conclusion, our data shows that each microrna gene has its own characteristic expression profile reflecting its spatial and temporal regulation that can be followed and comparatively analyzed using tools implemented in the mirex platform. the presented graphs were created with options available in mirex interface and represent (a) mir398 and (b) mir397 gene family (see text for details). (c) example of mir778 showing dramatic changes in expression level during growth and organ development. the presented graphs were created with options available in mirex interface and represent (a) mir398 and (b) mir397 gene family (see text for details). (c) example of mir778 showing dramatic changes in expression level during growth and organ development. in the current release, the mirex database includes expression data of microrna genes from a. thaliana. to collect the data, we prepared a real - time pcr platform for 190 primary microrna sequences. for each microrna, a pair of specific primers amplifying a single product was designed ; 10 micrornas primer sequences were taken from pant. the qpcr reactions for one biological replicate included 12 different controls and standards, were carried out in parallel as described previously (20) using rna template isolated from six developmental stages of a. thaliana columbia-0 wild - type plants. the detailed procedure of running the platform can be found in the mirex database documentation. the plant material used in expression profiling experiments includes : seeds, 10-day - old seedlings, 14-day - old seedlings, 25-day - old plants, 35-day - old plants, 42-day - old plants (rosette leaves and stems) and 53-day - old plants (rosette leaves, stems, inflorescence and siliques). for better visualization of the individual developmental stages our high - throughput platform contains an original and novel set of data on microrna gene expression in dormant seeds. the pp2a (at1g13320) and actin (at3g18780) cdnas were used as an expression reference (22). the mean measurements of three replicas were used to calculate the fold - change value and presented in a form of log10. in a case when the value of correlation coefficient of the three replicas was < 0.995, such data was labeled as low quality and is not shown by default on the graphs, but indicated in gray in the data tables. since the level of expression of most of the primary micrornas is lower than the reference genes, the data shown on the graphs in mirex is rescaled in order to avoid presenting it as a negative value. rescaling shifts the zero value of the graph 's y - axis to the basal expression level of the whole experiment. this allows showing the expression profile in a positive data range, but does not change the actual values. the mirex database interface is designed to be used by a bench scientist on an everyday basis. following a simplicity rule, the interface of mirex has been built on only two types of result windows and a simple two - step querying system. there are two ways to access data in mirex : by searching for a particular microrna or microrna family, or by browsing the database content. a single input window allows searching for an individual or a family of microrna by providing the numerical part of its identification (i d). when entered i d corresponds to a record of a single microrna, the resulting page shows all of the details for this particular sequence (figure 1). in the case of whole family i d, the search results are presented in the form of a line graph, showing expression data for all stages available in the database (figure 2). the table located below the graph contains numerical values for data presented in the graph, including low quality measurements. the ids shown in the table allow quick access to detailed information about particular microrna. this window is divided into distinct sections representing : expression data, sequences and external references. the right panel contains shortcuts to microrna family members ' records, structure of the rna transcript molecule with labeled mature sequences and rt qpcr primers and webform for user comments. the report window contains graphical presentation of the expression levels, as well as actual numerical values presented in tabular format. holding a mouse pointer over any data point allows access to details of expression measurements. the datasets for a particular microrna can be dynamically turned on and off by clicking i d in the legend of the graph. this window is divided into distinct sections representing : expression data, sequences and external references. the right panel contains shortcuts to microrna family members ' records, structure of the rna transcript molecule with labeled mature sequences and rt qpcr primers and webform for user comments. the report window contains graphical presentation of the expression levels, as well as actual numerical values presented in tabular format. holding a mouse pointer over any data point allows access to details of expression measurements. the datasets for a particular microrna can be dynamically turned on and off by clicking i d in the legend of the graph. the first step includes selection of developmental stages, which is followed by the selection of micrornas. in mirex, there are three ways to select micrornas : (i) by typing their ids in the input window, (ii) by uploading a file with a coma - separated list of microrna ids (that can be created in the first step) and (iii) by selecting individual or groups of sequences from the tree - like expandable menu. during the process of entering information using a keyboard, the mirex interface will provide the list of available micrornas. it is possible to mix all of the input methods the resulting page will refer to all entered or selected ids. presentation of search / browse results may differ depending on the number of selected developmental stages or microrna genes (see data mining section). by default, the graph of expression profiles for various stages and micrornas shows only high quality data. however, it is a user - defined option in mirex whether the low quality measurements are presented on the graph. the results for a single microrna are presented in a record window in a form that is divided into distinct sections (figure 1). the upper part of the record is dedicated to expression information presented in the form of a graph and two tables. when applicable, the graph contains information about the expression of other members of the microrna family, that can be dynamically turned on and off. additionally, specific shortcuts allow quick access to the records of any member of such family. the two tables, containing either reference - calculated or raw data, can be hidden to reduce the amount of information presented at any given moment on the single record window. each graph or table in the mirex database can be saved for later use by selecting an appropriate button. additionally, below the quantitative expression data, each record contains a web link to a text - based map of publicly available high - throughput next - generation sequencing (ngs) short reads aligned to primary microrna transcript. the information included here contains sequence, location, length and number of the mapping reads. on a separate window, it is also possible to access the original record of the used ngs data. the record window contains a graphical presentation of the structure of microrna transcript with designations of mature molecule(s) and primers used to assess its expression level by rt qpcr. when available, the record data will be modified to include results from northern hybridization with mature micrornas. however, it has to be noted that in most cases, such hybridization represents the level of the whole microrna gene family expression, and will be not specific for an individual microrna gene. moreover, the record window contains a table with sequences relevant to particular microrna and a link to experimental procedures. our aim was to avoid replication of the data available in other databases. for information such as gene structure or available papers, we direct the user to specialized databases, for example tair (23) or pubmed (24). the number of external references will grow in time, when new resources for microrna biology are available, and in response to user requests. to maintain a close relation with users of our database, we provide in the record window a simple tool for entering comments. the comment may be of any nature : concerning the biological aspect of the presented data or any general issue. although the interface is very intuitive and simple, at the main page we provide a video tutorial on all of the capabilities and various ways available to explore the mirex tools and data. moreover, every window in the mirex interface contains context - specific help guides. a complete road map of the mirex interface is included as supplementary figure s1. the usefulness of the mirex database lies in the ability to compare expression between different developmental stages and various microrna genes. by using the browse button, the user can, in two simple steps, select available developmental stages and a single or sets of micrornas. depending on the number of selected stages, the expression results are presented in the form of a bar graph (for one - stage analysis) or a line - based graph (for many stages). the bar graph presentation allows analysis of all pri - mirnas at once with a zoom - in on an individual gene. the line graph displays by default, data for only 24 micrornas selected from the list. the line graph offers an additional option to dynamically remove and/or add any of the selected microrna genes., the shortcuts represent a new and easy way to explore data stored in the mirex database. they are located in the same area on every page, and their content follows user selections. the use of shortcuts allows quick access to specific sets of data, e.g. the most expressed genes, or records representing micrornas belonging to the same family. periodically, we will modify the shortcuts in response to comments and readapt them according to the most frequently issued queries. the strategy to use primary microrna expression data allows fine exploration of gene activities of members of microrna families. in some families, however, the expression level of the individual pri - mirnas may differ considerably : the fold change in the primary transcript levels within one microrna gene family may range between 10 and 10 (e.g. mir160, mir162, mir164, mir165, mir167, mir168, mir394, mir396, mir398, mir447 families, figure 3a). moreover, a few individual pri - mirnas show profound differences in their expression depending on the developmental stage and/or organ studied (e.g. mir156, mir157, mir158, mir159, mir166, mir169, mir397, mir399, mir404, figure 3b). these differences may reflect either the existence of promoter regulatory elements that are responsive to developmental stimuli, or the various rate of pri - mirna maturation during plant growth and organ formation. microrna families containing a single representative also may exhibit dramatic changes in their expression levels (reaching even 10-fold change) during plant growth and organ development (e.g. mir173, mir774, mir776, mir778, mir780, mir783, figure 3c). conversely, there are also pri - mirnas that show relatively small fluctuations of expression levels during plant growth and organ formation. in conclusion, our data shows that each microrna gene has its own characteristic expression profile reflecting its spatial and temporal regulation that can be followed and comparatively analyzed using tools implemented in the mirex platform. the presented graphs were created with options available in mirex interface and represent (a) mir398 and (b) mir397 gene family (see text for details). (c) example of mir778 showing dramatic changes in expression level during growth and organ development. the presented graphs were created with options available in mirex interface and represent (a) mir398 and (b) mir397 gene family (see text for details). (c) example of mir778 showing dramatic changes in expression level during growth and organ development. by creating the mirex platform, we provide the scientific community with a high quality pri - mirna expression data in seven developmental stages represented by 11 distinct organs of a. thaliana. this is a new and user - friendly platform designed to explore expression data in various developmental stages for a large number of related genes. the querying system has been limited to two simple steps, which allow access to any type of data stored in mirex. additionally, the mirex interface does not require the use of a keyboard the database user interface is fully mouse operated. moreover, graphs presenting expression data are designed to accommodate user selection dynamically, which makes exploration of the mirex content even more efficient. the modular character of the database design makes it possible for further mirex expansion to incorporate new species and datasets. following, we plan to include arabidopsis microrna genes discovered in the future, various mutants and microrna expression profiles from other plant species. incorporation of data from other species will broaden the available tool comparative analyses within and between species. by designing the mirex interface, we would like to propose a new trend in biological databases for simplicity and user - friendliness. additionally, we put special attention to the web browser compatibility issue of the interface by testing all of the most popular tools. careful selection of informatics techniques resulted in the platform that can be accessed even via ios on mobile devices without loosing any of the functionality and interface features. carefully selected links to external databases, prevent the user interface from overloading with data, yet creates the opportunity to easy access - related information from the most significant databases in the field. in every day laboratory work, this approach proved to be very efficient, and made the mirex platform a one - stop information center for arabidopsis microrna data. the polish ministry of science and higher education (grant 3011/b / p01/2009/37) ; the faculty of biology adam mickiewicz university in poznan, poland ; the foundation for polish science (fnp) within the international phd program co - financed from european union regional development fund (mpd 2010/3 to d.b. and j.d.). funding for open access charge : the faculty of biology adam mickiewicz university. | mirex is a comprehensive platform for comparative analysis of primary microrna expression data. rt qpcr - based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user - friendly interface. a new way of accessing gene expression data in mirex includes a simple mouse operated querying system and dynamic graphs for data mining analyses. in contrast to other publicly available databases, the mirex interface allows a simultaneous comparison of expression levels between various microrna genes in diverse organs and developmental stages. currently, mirex integrates information about the expression profile of 190 arabidopsis thaliana pri - mirnas in seven different developmental stages : seeds, seedlings and various organs of mature plants. additionally, by providing rna structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirex can function as a one - stop solution for arabidopsis microrna information. a web - based mirex interface can be accessed at http://bioinfo.amu.edu.pl/mirex. |
[12, 13 ] originally defined sarcopenia as being two standard deviations below the normal appendicular muscle mass divided by height squared, numerous groups have examined the prevalence of low muscle mass. multiple techniques have been used to measure muscle mass, e.g., dual - energy x - ray absorptiometry (dexa), computed tomography, mri, ultrasound, bioelectrical impedance, and anthropometry. on the average, 513 % of older persons over 60 years of age have low muscle mass, with the prevalence increasing to as high as 50 % in persons over the age of 80 years [11, 14 ]. normal aging is associated with approximately a 1 % loss of muscle from 30 years of age, and this loss tends to accelerate after the age of 70 years. in a recent study,. found that about 20 % of community - dwelling persons in italy had low muscle mass. in korea, the prevalence of sarcopenia using the baumgartner criteria was 0.8 % in women and 1.3 % in men over 60 years of age. in barcelona, low lean mass was present in 33 % of elderly women and 10 % of males. in taiwan, low muscle mass was present in 2.5 % of community - dwelling women and 5.4 % of males. two medical conditions, stroke and hip fracture, rapidly lead to an increase in muscle loss [22, 23 ]. this appears to be due to disuse and inflammation, and in the case of stroke, denervation. in addition, persons with diabetes mellitus have accelerated muscle loss [24, 25 ]. persons with muscle loss in combination with excess fat are considered to have obese sarcopenia [2628 ]. using the european working group on sarcopenia in older people (ewgsop) definition, 4.6 % of males and 7.9 % of women in hertfordshire had sarcopenia. in japan, 21.8 % of men and 22.9 % of women aged 65 to 89 years had sarcopenia. in a population of persons 80 years and older, 12.5 % had sarcopenia by the ewgsop definition. in nursing home residents, sarcopenic individuals had a greater than threefold increase in falls [21, 37 ]. sarcopenia has also been shown to prospectively predict mobility and instrumental activities of daily living (iadl) disability. the prevalence of sarcopenia was slightly less common using the international working group on sarcopenia (iwgs) criteria compared to the ewgsop. the sarcopenia, cachexia and wasting disorders (scwd) definition was found to predict adl and iadl difficulties, frailty, and mortality in a longitudinal study. the foundation for the national institutes of health (fnih) criteria were based on developing cutoffs using a variety of large epidemiological studies. these criteria are more restrictive with only 1.3 % of men and 2.3 % of women being defined as having sarcopenia. while there was a strong negative percent agreement with the other definition, the positive percent agreements were low ranging from 5 to 32 %. there is a need to compare the fnih criteria to other definitions to determine which has the best predictive ability. recently, it has been determined that fracture risk can be determined almost as accurately by the frax questions as by measuring bone mineral density. this raises the question of whether or not a simple questionnaire can be used to identify persons with sarcopenia. the sarc - f questionnaire has been validated in two published studies (table 3) [44, 45 ]. found that sarc - f had comparable predictive activity to ewgsop, iwgs, and the asian working group for sarcopenia. like the others in this asian population, it had modest predictive value for 4-year physical limitation. cao. have also provided evidence for validity of the sarc - f. the physical frailty phenotype as originally defined by fried. has been demonstrated to be highly predictive of poor outcomes [47, 48 ]. a simple questionnaire the frail (table 2)has been developed and shown to be equally predictive of poor outcomes [4953 ]. while sarcopenia is clearly a major component of frailty, they are not identical conditions. overlap between sarcopenia and frailty ranges from 50 to 70 %.table 2comparison of the brief screens for sarcopenia and frailty(a) sarc - f(b) frailcomponentquestionscoringstrengthhow much difficulty do you have in lifting and carrying 10 lb?none = 0fatiguesome = 1resistance (can you climb a flight of stairs?)a lot or unable = 2aerobic (can you walk a block?)assistance in walkinghow much difficulty do you have walking across a room?none = 0illness (> 5)some = 1loss of weight (5% in 6 months)(three or more positive answers = frail ; one or two positive answers = prefrail)a lot, use aids, or unable = 2rise from a chairhow much difficulty do you have transferring from a chair or bed?none = 0some = 1a lot or unable without help = 2climb stairshow much difficulty do you have climbing a flight of 10 stairs?none = 0some = 1a lot or unable = 2fallshow many times have you fallen in the past year?none = 013 falls = 14 falls = 2 comparison of the brief screens for sarcopenia and frailty these are listed in table 3. to these needs to be added the measurement of motor unit number index, which can be used to assess the number and the size of the motor units. this is important as loss of motor unit input to muscle is an important cause of sarcopenia in at least half of older persons.table 3biomarkers for sarcopeniabiomarkeragingexercisecommentscreatine kinasealdolase (a)correlates with walking speed in oldcoenzyme qmore strongly correlated with ffmmlc1troponin t?creatine (u)correlates with muscle massmyoglobincreatinine (u) (? /cystatin c)correlates with muscle j - shaped curve with functionn - terminal propeptide iii collagenincreased with testosteronec - terminal agrin fragmentcorrelates with degeneration of the neuromuscular junction biomarkers for sarcopenia 2) [57, 58 ]. disuse coupled with aging is the major underlying cause : poor blood flow to muscle, especially the muscle capillaries due to a decline in nitric oxide production, is another important age - related cause of sarcopenia. aging is associated with an increase in mitochondrial abnormalities leading to damage to the mitochondrial membrane permeability pore and apoptosis.fig. 2the causes of sarcopenia the causes of sarcopenia aging is associated with a physiological anorexia of aging that leads to weight loss [59, 60 ]. weight loss results in a 75 % loss of fat and a 25 % loss of muscle and bone. the increase of fat during weight regain is one of the major causes of sarcopenic obesity. the age - related loss of motor neuron end plates is a major component of sarcopenia. it leads not only to muscle wasting but also to a decrease in muscle function. loss of anabolic hormones, such as testosterone, dhea, growth hormone, and insulin - growth factor 1, occurs with aging. of these, testosterone has been shown to be the hormone that most closely determines the decline in muscle mass and strength. testosterone is important not only for protein synthesis but also for the maintenance of satellite cells [62, 63 ]. insulin resistance, which occurs with aging and obesity, plays an important role in decreasing available glucose and protein for muscle anabolism. obesity and disease lead to an increase in proinflammatory cytokines (e.g., interleukin-6, interleukin-1, and tumor necrosis factor alpha). it is now clear that resistance exercise is the primary therapeutic strategy to prevent and reverse sarcopenia [6670 ]. there is evidence that leucine - enriched essential amino acid supplementation will increase muscle mass and probably function [7275 ]. vitamin d has been shown to enhance muscle function in persons with low muscle function (< 50 nmol) [76, 77 ]. there is a small amount of data suggesting that testosterone will increase muscle mass and strength and may improve function in older frail persons with hypogonadism [7881 ]. selective androgen receptor modulators (sarms) have shown some promise in increasing muscle mass and stair climb. a number of antibodies that modulate myostatin and the activin ii receptor are in clinical trials. ghrelin agonists, which increase food intake and release growth hormone, are also under evaluation. loss of muscle mass is commonly associated with loss of bone, making these individuals at high risk for hip fractures. persons with muscle loss develop accelerated loss of muscle mass and strength when they develop a variety of diseases such as heart failure, chronic obstructive pulmonary disease, and renal failure [8689 ]. like osteoporosis it is time for physicians to screen for sarcopenia and provide treatment for it at a minimum resistance exercise and protein and vitamin d supplementation. | sarcopenia is now defined as a decline in walking speed or grip strength associated with low muscle mass. sarcopenia leads to loss of mobility and function, falls, and mortality. sarcopenia is a major cause of frailty, but either condition can occur without the other being present. sarcopenia is present in about 5 to 10 % of persons over 65 years of age. it has multiple causes including disease, decreased caloric intake, poor blood flow to muscle, mitochondrial dysfunction, a decline in anabolic hormones, and an increase in proinflammatory cytokines. basic therapy includes resistance exercise and protein and vitamin d supplementation. there is now a simple screening test available for sarcopenia sarc - f. all persons 60 years and older should be screened for sarcopenia and treated when appropriate. |
these herbs are relatively cheap and available, and their use depends on ancestral experience [1 - 3 ]. medicinal plants represent a great deal of untapped reservoirs of drugs, and the structural diversity of their components make a valuable source of novel compounds [3, 4 ]. thus, there is a growing interest in the utilization of phytoceuticals and natural product scientists are intensifying efforts towards evaluation of these valuable medicinal plants. one of the various families of herbs that has long been used worldwide in traditional medicine is loranthaceae [5, 6 ], which is formed by three distinct families : loranthaceae (sensu strictu), viscaceae, and eremolepidaceae [7, 8 ]. the members of loranthaceae are about 75 genera, and most of them are globally known as mistletoes [9, 10 ]. historically, the mistletoe, whose name is believed to be derived from the celtic word for all - heal, was used for a variety of treatments. among the recognized therapeutic properties of loranthaceae members include antitumor actions [11, 12 ], cough treatment, headache treatment, uterus tightening following childbirth, and immunomodulatory [15, 16 ], antioxidant [16 - 19 ], antiinflammatory [20, 21 ], antimicrobial [22 - 24 ], antiviral, antidiarrhoeal [16, 26 ], anticancer [23, 27 ], antinociceptive, antipyretic, antihyperglycemic, and antidiabetic [16, 18, 28, 29 ] activities and antihypertensive effects [30 - 32 ]. however, despite its wide applicability, mistletoe is mainly used for its blood pressure (bp) lowering and gastrointestinal treatment properties. chemical and pharmacological studies of some species of the loranthaceae family have identified different kinds of constituents, such as flavonoids [15, 16, 21, 28 - 30, 33 - 35 ], alkaloids, lectins, and polypeptides [11, 12, 16, 36, 37 ], arginine, histamine, polysaccharides [40, 41 ], tannins [16, 28, 29 ], terpenoids and/or steroids [28, 29 ], acidic compounds [16, 27 ], glycosides [16, 28 ], gallic acid, and recently discovered, a new flavocoumarin named loranthin., with the synonym scurrula ferruginea danser, is a member of the loranthaceae family. this herb has a unique way of living, being a hemi parasitic shrub that never grows on land but can partially survive on photosynthesis, and is more dependent on the nutrients and water that can access its roots. these roots have modified structure that attaches the herb onto a host plant which is usually a dicotyledonous tree. the members of the loranthaceae are widely distributed in many countries like malaysia, sumatra, india, singapore, australia and new zealand. particularly in malaysia, l. ferrugineus is locally known as dedalu, dalu - dalu, or dedalu - api where api means fire in the malay language, and probably reflects the rusty appearance of the herb leaves. other reports have demonstrated that an aqueous extract obtained from l. ferrugineus possesses remarkable capabilities to lower bp in vivo. moreover, phytochemical investigation of l. ferrugineus has indicated the presence of components like flavonoids and a high concentration of condensed tannins. three natural flavonol compounds have been isolated from the ethyl acetate fraction of l. ferrugineus : in addition to quercetin and quercitrin, an unusual flavonol glycoside 4-o - acetylquercitrin was also isolated. in addition to its reputable use for treatment of hypertension and gastrointestinal complaints, a decoction of l. ferrugineus is also used as a home remedy for general health maintenance and its gerontological effects, which include the enhancement of memory and well - being in the elderly people. leaves, fruits and flowers are the most common parts of l. ferrugineus used to treat high bp, while the unique roots attaching the herb to the host plant are employed for other therapeutic uses, such as ulcer and cancer treatment. other deep rooted uses for the leaves of this plant are snakebites, wounds, fever, malarial infection or women pouch for after childbirth [45, 46 ]. l. ferrugineus potential antioxidant and anticancer effects represent a relatively unexplored facet of this herb s knowledge database. much research has focused on mistletoe s efficacy as an anticancer treatment [47 - 50 ], and thus spurred a variety of pharmaceutical medicines, such as isorel, eurixor, helixor, and lektinol. however, though there seems to be strong evidence suggesting certain constituents, especially the lectins, exhibit anticancer effects, the value of the whole plant in cancer treatment is not fully accepted due to significant discrepancies in research findings [49, 50, 52 ]. nonetheless, because l. ferrugineus has been shown to contain some chemical constituents which are believed to have anticancer effects [25, 43, 44 ], there is a possibility that l. ferrugineus itself, in addition to its supposed antiviral and cytotoxic activities, may have anticancer effects too. despite l. ferrugineus wide range of medicinal applicability, to date thus, there is a need to review information that can describe this plant and evaluate its traditions, and survey how it has been scientifically and experimentally translated toward evidence based phytomedicine. therefore, this poses a necessity to review and analyze all the relevant scientific publications of l. ferrugineus that can add up to our knowledge and understanding of this least explored herb. furthermore, we aimed to describe the points that remain to be addressed, and thus from this we propose critical future research directions of this herb that could enhance awareness toward evidence based ethnopharmacology. the taxonomical classification of l. ferrugineus was first described by bengal as in table 1. 1) physical and structural appearance can vary slightly, based on the plant s tropical natural habitat and age. branches and twigs of the herb are long, pendulous, and densely clothed with l. ferrugineus down when young, in addition to the underside of the leaves, pedicels, calyxes, and corollas. the rusty colored leaves are opposite in direction, positioned on short petioles, elliptic, obtuse, coriaceous, and glabrous above. these leaves are elliotic, with 4 8 cm long inder surfaces densely covered with reddish scurf. usually, peduncles present as 1 4 together in the axils of the leaves. the flowers of l. ferrugineus are : tetrandrous, 2 6 most commonly, in axillary cymes ; have perianth 1.5 2 cm long ; are drupe clubshaped ; and densely rusty. the flowers are bracteas small, adpressed to the ovaria, and one to each, with a tubular and deeply 4 parted flower corolla. for herbs that are native of the east indies, pulau - penang, singapore and sumatra, the flowers corolla are densely clothed with rusty hairs, 7 lines long. l. ferrugineus berries are yellowish and ovate in shape, and are an orchard pest that is often parasitic on melastoma and many other trees. most traditional productions of l. ferrugineus extract involve a simple water decoction or herbal tea preparation in households, intended for human consumption to treat and alleviate various symptoms. however, this preparation method is not necessarily always implicated for scientific research investigations, because chemically characterizing and isolating constituents from water extracts is laborious. therefore, as in with all natural product chemists, it is necessary to break down herbal material into many different polarity components to ease their identification, enhance yield or mass product, and to occasionally ensure compound stability. based on previous published observations, we summarized a general scheme illustrating how l. ferrugineus extractives are usually produced (fig. we believe that this method can help separate different elements within a plant based on their polarity, potentially purify and optimize yield of active molecules, and most importantly be applied to various bioactive plant constituents. as per the tradition of the most commonly used portions of the herb, fresh aerial parts of l. ferrugineus, including leaves, young stems, twigs, flowers and berries it is important that the plant is collected from the same single host plant to reduce variation between different batches of collection, since chemical composition of the parasitic shrub can be affected by the host plant. only healthy parts of the plant must be taken, while the diseased portions are discarded. this is because, when contaminated by severe microbial infection, diseased portions may affect healthy portions, and thus eventually alter metabolism of the plant : an effect which may contribute to the formation of large amounts of unexpected products [56, 58 ]. the plant material is then cleaned of adulterants, chopped into small pieces, dried in an oven at 42 for 5 days, ground into a fine powder using a milling machine, and the resultant powder successively treated as per fig. 2. as mentioned earlier, various classes of organic compounds described elsewhere have been shown to be present in this herb. these phytochemicals include phenolics, flavonoids, terpenoids, saponins and condensed tannins. the chemical profiling and assays performed thus far on l. ferrugineus and its extractives are summarized in table 2. on the other hand, constituents such as amino acids, peptides, secondary amines, alkaloids, anthraquinones, sterols and coumarins have been reported to be absent at least in part in l. ferrugineus methanol extract (lfme) [2, 59 ]. preliminary thin layer chromatography analysis of lfme and its n - butanol fraction of lfme (nbf - lfme) showed the presence of terpenoids and flavonoids, while ultraviolet visible and fourier transform infrared peak spectroscopy analyses of these two extractives were mostly in favoring terpenoids as the major constituents. few research groups have taken the opportunity to characterize the components of l. ferrugineus ; hence there is limited chemical investigation that would possibly identify or isolate the active components in this plant, and thus elucidation of the mechanism by which its bioactive components may exert its cardiovascular effects is largely unexplored. in addition, there have been no investigations of potential chemical composition changes across seasons. continuing research in this area would considerably enhance our understanding of the natural diversity of this herb, and would definitely open the door for a new era of nutraceuticals. perhaps future studies may employ dna barcoding, which allows the identification of species in samples with short reliable dna regions, or highly degraded dna. dna barcoding has been used in many studies to test regions in plant groups, and provides species identification by using standardized dna regions as tags. in addition, there have been previous studies that have used this method to help identify possible usable dna sequences in plants for the application of dna barcoding. cardiovascular disease is a major public health problem contributing to 17.3 million deaths worldwide, and with over 80% of cardiovascular deaths taking place in the low and middle income countries [62, 63 ]. by 2030, it has been estimated that 23 million people will die annually from cardiovascular complications [63, 64 ]. hypertension is a powerful risk factor for cardiovascular disease and is perhaps one of the most prevalent underlying etiologies of cardiovascular risks. a number of synthetic antihypertensive medications are available in clinical practice ; however, these agents do not always provide optimal control of bp, and in many cases are highly associated with adverse effects. this therefore poses a need for alternative therapies that provide better bp control with minimal side effects. herbal treatments have been successfully utilized to treat patients with congestive heart failure, systolic hypertension, angina pectoris, atherosclerosis, cerebral insufficiency, and arrhythmia [65, 66 ]. specific to hypertension, previous and current evidence suggests that l. ferrugineus possesses promising bp lowering activity. +, present ; lfme, loranthus ferrugineus methanol extract ; cf - lfme, chloroform fraction of loranthus ferrugineus methanol extract ; eaf - lfme, ethyl acetate fraction of loranthus ferrugineus methanol extract ; nbf - lfme, n - butanol fraction of loranthus ferrugineus methanol extract ; wf - lfme, water fraction of loranthus ferrugineus methanol extract ; teac, trolox equivalent antioxidant capacity. earlier preliminary investigations conducted by othman revealed that l. ferrugineus has a remarkable bp lowering activity. here, it was reported that the water extract of l. ferrugineus, along with other water based herbal extracts, produce bp lowering effects in the anesthetized rat when given as intravenous (i.v.) though othman s investigation only involved the use of water based extract, subsequent work involved a series of both in vitro and in vivo ethnopharmacological investigations to explain how l. ferrugineus exerts its effects in hypertension management. it is noteworthy mentioning that characterization of l. ferrugineus potential cardiovascular effects can pave the way to a more useful understanding of natural herb remedies. for example, evidence based research has enabled us to obtain fair knowledge of the antihypertensive therapeutic properties of viscum album [67, 68 ], a mistletoe herb from santalaceae which is of close proximity to l. ferrugineus. early experiments conducted by our laboratory group involved utilization of essential extracts of the herb, as shown in fig. these experiments were conducted by incubation of isolated sprague - dawley (sd) rat aortic ring preparations with increasing concentrations of 0.05% 0.20% (w / v) of petroleum ether extract (lfpee), chloroform extract (lfce), ethyl acetate extract (lfeae), methanol extract (lfme) and water extract (lfwe) in an organ bath chamber, then separately challenging these extracts by cumulative additions of the -adrenergic agonist norepinephrine. the results showed that lfme possesses a remarkable effect in concentration dependently inhibiting norepinephrine induced vasoconstriction. in addition, further examination showed that lfme can modulate norepinephrine induced aortic ring contraction through a reversible noncompetitive antagonism mechanism, suggesting that chemical constituents within lfme modulate vasomotor tone via a non -receptor mediated action and may likely possess bp lowering properties. to this end, a different set of investigations involved the use of i.v. bolus administration of increasing doses of 25 200 mg / kg of l. ferrugineus extracts in in vivo whole animal experimentations. in accordance with in vitro findings, lfme was found to produce the most significant dose dependent yet brief reduction in bp. it was also noted that lfwe had some hypotensive actions ; however, these effects were weaker relative to lfme. although this finding contradicted othman s earlier results, where lfwe was purported to be the most potent extract, this discrepancy between findings may be explained by the fact that the earlier study involved only water based extraction of l. ferrugineus, while subsequent investigations, as illustrated in fig. 2, used increasing polarity solvents for extraction : hence, the active components may be maximally presented in lfme rather than lfwe. collectively, these findings suggest that relative to the other l. ferrugineus extracts, lfme possesses the most significant vasorelaxant and hypotensive activities in rat models. additionally, lfme s promising active constituent(s) is / are of a relatively high polarity, and therefore can justify the herb s use in the management of hypertension in a form of water based decoction. thus, perhaps modification of l. ferrugineus chemical structure may yield compounds that present with more sustained antihypertensive effects. it is well established that bp is a function of cardiac output and total peripheral resistance ; two variables that are regulated by two major branches of the autonomic nervous system (sympathetic and parasympathetic). the autonomic pathways that control the heart and vasculature are primarily sympathetic, targeting both - and -adrenoceptors located within these target organs, and hence altering cardiac output and systemic vascular resistance. parasympathetic autonomic pathways, on the other hand, play an important cardiac effect, modulating both myocardial contractility and heart rate via m2 muscarinic receptors stimulation [72, 73 ]. blood vessels mostly lack parasympathetic innervation, yet m3 muscarinic cholinergic receptor subtype is still expressed within the vasculature and responds to circulating acetylcholine (ach) and other cholinomimetics [73, 74 ]. subsequent studies on l. ferrugineus have therefore focused on investigating the role of active constituents contained within its methanol extract in modulating these key bp regulatory pathways in vivo. thus, the hypotensive mechanisms of l. ferrugineus were characterized by monitoring the extract s effect on mean arterial pressure, following i.v. bolus injections of several antagonists of the adrenergic and cholinergic pathways, using anesthetized normotensive sd rat preparations. possible effects on - and -adrenergic cardiovascular receptors were investigated using respective agonists and antagonists of these receptors. here, the ability of prazosin, an 1-adrenergic blocker, and propranolol, a non selective -adrenoceptor blocker, to alter l. ferrugineus hypotensive effects was investigated with reference to both norepinephrine and isoprenaline, a -adrenoceptor agonist, as positive controls. interestingly, the bp lowering activity of the extract was unaffected by either antagonist, suggesting that l. ferrugineus, unlike extracts obtained from pseuderanthemum palatiferum, artocarpus altilis, solanum torvum and platycapnos spicata, does not exert its hypotensive action via antagonism of cardiovascular - or -adrenergic receptors. indeed, the lack of differences in the hypotensive response to l. ferrugineus following prazosin administration confirmed our previous assertions of a reversible noncompetitive antagonism of norepinephrine mediated vasoconstriction in isolated rat aorta, driven by a non -adrenoceptor mediated action. the possible action of l. ferrugineus on the cholinergic pathways was subsequently studied, with a particular focus on lfme s effects on cardiovascular muscarinic receptors and ach esterase (ache), an enzyme which metabolizes ach. in these experiments, l. ferrugineus mean arterial pressur effects were recorded before and after systemic blockade of muscarinic receptors with atropine or ache inhibition with neostigmine. data driven from these experiments showed that l. ferrugineus retained a behavior almost similar to the positive control ach, as l. ferrugineus hypotensive activity was significantly abolished in the presence of atropine. together, this indicates that l. ferrugineus extract, like extracts derived from tulbaghia violacea, bidens pilosa, moringa oleifera, cinnamomum zeylanicum and chenopodium ambrosioides, possibly possesses some cholinergic properties, and that its bp lowering effect was most likely driven by stimulation of cardiac and/or vascular muscarinic receptors. further exploring the role of the cholinergic pathway neostigmine tended to augment the bp lowering effect of l. ferrugineus and the duration of its action : yet, the effect was not as notably significant when compared to the enhancement of ach action on bp and the action duration. this data suggested that ache may not be the primary enzyme responsible for termination of the lfme effect in plasma and that other esterases, including butyrylcholinesterase or pseudocholinesterase, which is found in great abundance in plasma as compared to ache, is likely to hydrolyze esters of plant sources. importantly, as neostigmine did not block l. ferrugineus action on bp, our study concluded that lfme does not produce its cholinomimetic action through the antagonism of ache, but rather through direct muscarinic receptor stimulation. the cholinergic pathway exerts a powerful antagonistic influence on the heart by modulating cardiac rate (chronotropy), conduction velocity (dromotropy), contraction (inotropy), and relaxation (lusitropy). for instance, the cholinergic hypotensive properties of echinodorus grandiflorus ethanolic extract are associated with powerful reductions in cardiac output and heart rate in spontaneously hypertensive rats. likewise, raphanus sativus (radish) seed crude extract produces muscarinic receptor dependent inhibition of cardiac force and rate of contractions in isolated guinea pig atria ; however, whether l. ferrugineus exhibits similar direct cardiac effects remains unexplored. future investigations should therefore consider exploring cardiac activities of l. ferrugineus, investigating not only chronotropic, inotropic and dromotropic responsiveness of the heart but also changes in ventricular pressure, end diastolic volume and coronary blood flow. these experiments will potentially further our understanding of l. ferrugineus cardiovascular properties and provide comprehensive characterization of its therapeutic benefits. the activity of l. ferrugineus on the autonomic ganglia, which controls cholinergic and preferentially adrenergic cardiovascular targets, were previously studied by monitoring the ability of hexamethonium, a ganglionic blocker, to modulate the hypotensive response evoked by l. ferrugineus extract. it was found that ganglionic blockade did not markedly alter the bp lowering effects of the extract, suggesting l. ferrugineus, unlike dietary soy and gossypium barbadense, does not promote hypotension via ganglionic blockade mechanisms. nitric oxide (no) is probably one of the most important molecules produced by the endothelium and is synthesized by the enzyme no synthase (nos) from the precursor amino acid l - arginine (l - arg). a range of chemical mediators including not only ach but also bradykinin, serotonin, substance p and adenosine diphosphate trigger no release from the endothelium by receptor mediated mechanisms. endothelial derived no has the capacity to maintain vascular tone and to produce vasodilatation through the activation of guanylate cyclase. given the relative similarity between l. ferrugineus and ach pharmacological properties, and the fact that ach mediated activation of vascular muscarinic receptors triggers a cascade of events that ultimately generate no, we predicted that inhibition of nos, as with ach, would perhaps attenuate the bp lowering effects of l. ferrugineus. indeed, n-nitro - l - arginine methyl ester hydrochloride (l - name), a nos inhibitor, was shown to attenuate hypotensive responses to l. ferrugineus in vivo, suggesting that bp lowering activity is not only evoked by activation of cardiovascular cholinergic receptors, but also through promoting no release. it is common practice to subject crude plant extracts to activity guided fractionation to eliminate various types of complex or antagonistic molecules and derive a more purified and more potent form of the extract. subsequent studies on l. ferrugineus have therefore aimed to obtain purer derivatives of the active lfme extract for further in vivo and in vitro testing. in one study, four chemical fractions including chloroform fraction (cf - lfme), ethyl acetate fraction (eaf - lfme), n - butanol fraction (nbf - lfme) and water fraction (wf - lfme) were derived as shown in fig. 2. in vivo data from our laboratory showed that nbf - lfme significantly lowers bp in a dose dependent manner, and has a relatively longer duration of action compared to other fractions. interestingly, it has been further observed that nbf - lfme has more bp lowering potency relative to its mother crude extract lfme, which indicates that fractionation is able to increase the number of bioactive molecules in the extract. in keeping with our in vivo data, in vitro findings showed that, relative to other fractions, nbf - lfme produces a significant concentration dependent inhibition of aortic ring contraction against the -agonist phenylephrine and depolarizing signals of potassium chloride. these in vivo and in vitro results compare very closely to research findings on other plants used in hypertensive treatment, in which bioactivity guided fractionation contributes to a more potent pharmacological action. for example, kane and colleagues investigated an ethanolic extract and fractions of euphorbia thymifolia for diuretic activity, and found that the dose dependent diuretic effect of this herb was more potent in the fractionated extract. in addition, ouedraog performed a bioassay guided fractionation of ethanolic extract of agelanthus dodoneifolius, a plant traditionally used as treatment for hypertension, using rat aorta pre - contracted by norepinephrine to monitor the relaxant activity. they found that the vasorelaxant properties of agelanthus dodoneifolius crude extract were markedly potentiated following column chromatographic separation of its active fractions. as we found previously, lfme exhibits reversible noncompetitive antagonism of norepinephrine induced aortic ring contraction [59, 70 ], and nbf - lfme fraction exhibits the greatest bp lowering effect in vivo relative to other lfme fractions. therefore, we further investigated the effect of the purified nbf - lfme fraction with the aim of finding a more molecular mechanistic explanation to its pharmacological action. the active l. ferrugineus fraction was challenged against vascular endothelium dependent and independent mechanisms, using blockade with various pharmacological interventions including l - name, no - cgmp pathway inhibitor methylene blue, cyclooxygenase inhibitor indomethacin, atp dependent potassium channel blocker glibenclamide, -blocker propanolol and -receptor blocker prazosin. those experiments revealed that nbf - lfme induced vascular relaxation is primarily driven by endothelium dependent mechanisms, stimulating muscarinic receptors, activating the endothelium derived no - cgmp - relaxant pathway, promoting prostacyclin release and lengthening the released no half life through its antioxidant effects. this data therefore provide solid evidence that l. ferrugineus remarkable antihypertensive potentials extend beyond its muscarinic receptor activation, and further involve enhancement of key modulators of vascular function including no and prostacyclin, and possible vascular protection through its antioxidant and free radical scavenging properties. the effectiveness of l. ferrugineus through its enhancement of no or its antioxidant properties is of marked importance, as an imbalance between no and reactive oxygen species production appears to be a common feature of experimental and human hypertension. it is therefore possible that promoting no release and antioxidant action of l. ferrugineus biologically active principles can potentially influence a range of vascular targets to offer diverse protective cardiovascular properties. for instance, ginsenosides and saponins from panax ginseng have been shown to protect against myocardial ischaemia/ reperfusion damage and decrease lipid peroxidation, and that those effects are mediated by release of no from endothelial cells, especially from perivascular nitric oxidergic nerves. crataegus pinnatifida, a plant which has been used as a decoction for antihypertensive treatment for thousands of years in china, is believed to exert its effects through vasorelaxation resulting from no stimulation and antioxidant activity. similarly, other plants such as theobroma cacao, due to their enriched flavonoid constituents and resulting stimulation of no formation, has been shown to improve endothelial function through mechanisms that enhance no synthesis or those that decrease no breakdown. accordingly, further investigation of l. ferrugineus cardiovascular effects and/or targets is warranted in order to fully understand its cardiovascular protective action. herbal medicines could benefit patients suffering from gastrointestinal disorders that can not be treated using conventional drug therapy, including functional dyspepsia, constipation, and postoperative ileus. as reviewed by langmead and rampton, although most indications for the use of such remedies are traditionally derived, controlled research trials suggest some health benefits for ginger in nausea and vomiting, liquorice extracts in peptic ulceration, chinese herbal medicine in irritable bowel syndrome, opium derivatives in diarrhoea and senna, ispaghula and sterculia in constipation. following traditional uses of l. ferrugineus for gastrointestinal complaints, we aimed to investigate the effect of lfme and nb - lfme on the isolated guinea pig ileal preparation in vitro [69, 99 ], to test for gut contractility actions. indeed, this work showed that crude lfme and its nb - lfme retain the ability to dose dependently increase intestinal smooth muscle contractility. importantly, these effects were significantly reduced upon pre - incubation with atropine, enhanced in the presence of neostigmine, and unchanged in the presence of hexamethonium, suggesting a direct cholinomimetic action via muscarinic receptor activation, and that bioactive constituents within l. ferrugineus serve as a substrate for intestinal ache. having previously described a cholinomimetic hypotensive action for l. ferrugineus, our findings in the guinea pig ilium were not counterintuitive. nonetheless, this data has undoubtedly highlighted a new target organ for l. ferrugineus health benefits, confirmed the ability of the extract s active constituents to induce a notable peristalsis, and most importantly provided an immense support for its folklore use. accordingly, l. ferrugineus extracts, like other crude extracts obtained from carissa carandas and fumaria parviflora which possess a cholinergic stimulatory action, can potentially be formulated to treat constipation disorders. muscarinic agonists elicit contraction through the m3 receptor present in smooth muscle ranging from the esophagus to ileum. in the ileum, muscarinic agonists are known to have a dual effect on contraction : a direct m3 mediated contraction and an indirect m2 mediated inhibition of relaxation [102, 103 ]. activation of gastric acid secretion by endogenous ach, on the other hand, is triggered by m3 receptor subtype expressed on gastric parietal cells. despite a cholinomimetic action for l. ferrugineus, it remains undetermined if active constituents within its crude or purified extract would show a muscarinic receptor subtype specificity to elicit their contractile effect, or an ability to modulate acid, mucous or other gastrointestinal secretions. cardiovascular disease is a major public health problem contributing to 17.3 million deaths worldwide, and with over 80% of cardiovascular deaths taking place in the low and middle income countries [62, 63 ]. by 2030, it has been estimated that 23 million people will die annually from cardiovascular complications [63, 64 ]. hypertension is a powerful risk factor for cardiovascular disease and is perhaps one of the most prevalent underlying etiologies of cardiovascular risks. a number of synthetic antihypertensive medications are available in clinical practice ; however, these agents do not always provide optimal control of bp, and in many cases are highly associated with adverse effects. this therefore poses a need for alternative therapies that provide better bp control with minimal side effects. herbal treatments have been successfully utilized to treat patients with congestive heart failure, systolic hypertension, angina pectoris, atherosclerosis, cerebral insufficiency, and arrhythmia [65, 66 ]. specific to hypertension, previous and current evidence suggests that l. ferrugineus possesses promising bp lowering activity. +, present ; lfme, loranthus ferrugineus methanol extract ; cf - lfme, chloroform fraction of loranthus ferrugineus methanol extract ; eaf - lfme, ethyl acetate fraction of loranthus ferrugineus methanol extract ; nbf - lfme, n - butanol fraction of loranthus ferrugineus methanol extract ; wf - lfme, water fraction of loranthus ferrugineus methanol extract ; teac, trolox equivalent antioxidant capacity. earlier preliminary investigations conducted by othman revealed that l. ferrugineus has a remarkable bp lowering activity. here, it was reported that the water extract of l. ferrugineus, along with other water based herbal extracts, produce bp lowering effects in the anesthetized rat when given as intravenous (i.v.) though othman s investigation only involved the use of water based extract, subsequent work involved a series of both in vitro and in vivo ethnopharmacological investigations to explain how l. ferrugineus exerts its effects in hypertension management. it is noteworthy mentioning that characterization of l. ferrugineus potential cardiovascular effects can pave the way to a more useful understanding of natural herb remedies. for example, evidence based research has enabled us to obtain fair knowledge of the antihypertensive therapeutic properties of viscum album [67, 68 ], a mistletoe herb from santalaceae which is of close proximity to l. ferrugineus. early experiments conducted by our laboratory group involved utilization of essential extracts of the herb, as shown in fig. these experiments were conducted by incubation of isolated sprague - dawley (sd) rat aortic ring preparations with increasing concentrations of 0.05% 0.20% (w / v) of petroleum ether extract (lfpee), chloroform extract (lfce), ethyl acetate extract (lfeae), methanol extract (lfme) and water extract (lfwe) in an organ bath chamber, then separately challenging these extracts by cumulative additions of the -adrenergic agonist norepinephrine. the results showed that lfme possesses a remarkable effect in concentration dependently inhibiting norepinephrine induced vasoconstriction. in addition, further examination showed that lfme can modulate norepinephrine induced aortic ring contraction through a reversible noncompetitive antagonism mechanism, suggesting that chemical constituents within lfme modulate vasomotor tone via a non -receptor mediated action and may likely possess bp lowering properties. to this end, a different set of investigations involved the use of i.v. bolus administration of increasing doses of 25 200 mg / kg of l. ferrugineus extracts in in vivo whole animal experimentations. in accordance with in vitro findings, lfme was found to produce the most significant dose dependent yet brief reduction in bp. it was also noted that lfwe had some hypotensive actions ; however, these effects were weaker relative to lfme. although this finding contradicted othman s earlier results, where lfwe was purported to be the most potent extract, this discrepancy between findings may be explained by the fact that the earlier study involved only water based extraction of l. ferrugineus, while subsequent investigations, as illustrated in fig. 2, used increasing polarity solvents for extraction : hence, the active components may be maximally presented in lfme rather than lfwe. collectively, these findings suggest that relative to the other l. ferrugineus extracts, lfme possesses the most significant vasorelaxant and hypotensive activities in rat models. additionally, lfme s promising active constituent(s) is / are of a relatively high polarity, and therefore can justify the herb s use in the management of hypertension in a form of water based decoction. thus, perhaps modification of l. ferrugineus chemical structure may yield compounds that present with more sustained antihypertensive effects. it is well established that bp is a function of cardiac output and total peripheral resistance ; two variables that are regulated by two major branches of the autonomic nervous system (sympathetic and parasympathetic). the autonomic pathways that control the heart and vasculature are primarily sympathetic, targeting both - and -adrenoceptors located within these target organs, and hence altering cardiac output and systemic vascular resistance. parasympathetic autonomic pathways, on the other hand, play an important cardiac effect, modulating both myocardial contractility and heart rate via m2 muscarinic receptors stimulation [72, 73 ]. blood vessels mostly lack parasympathetic innervation, yet m3 muscarinic cholinergic receptor subtype is still expressed within the vasculature and responds to circulating acetylcholine (ach) and other cholinomimetics [73, 74 ]. subsequent studies on l. ferrugineus have therefore focused on investigating the role of active constituents contained within its methanol extract in modulating these key bp regulatory pathways in vivo. thus, the hypotensive mechanisms of l. ferrugineus were characterized by monitoring the extract s effect on mean arterial pressure, following i.v. bolus injections of several antagonists of the adrenergic and cholinergic pathways, using anesthetized normotensive sd rat preparations. possible effects on - and -adrenergic cardiovascular receptors were investigated using respective agonists and antagonists of these receptors. here, the ability of prazosin, an 1-adrenergic blocker, and propranolol, a non selective -adrenoceptor blocker, to alter l. ferrugineus hypotensive effects was investigated with reference to both norepinephrine and isoprenaline, a -adrenoceptor agonist, as positive controls. interestingly, the bp lowering activity of the extract was unaffected by either antagonist, suggesting that l. ferrugineus, unlike extracts obtained from pseuderanthemum palatiferum, artocarpus altilis, solanum torvum and platycapnos spicata, does not exert its hypotensive action via antagonism of cardiovascular - or -adrenergic receptors. indeed, the lack of differences in the hypotensive response to l. ferrugineus following prazosin administration confirmed our previous assertions of a reversible noncompetitive antagonism of norepinephrine mediated vasoconstriction in isolated rat aorta, driven by a non -adrenoceptor mediated action. the possible action of l. ferrugineus on the cholinergic pathways was subsequently studied, with a particular focus on lfme s effects on cardiovascular muscarinic receptors and ach esterase (ache), an enzyme which metabolizes ach. in these experiments, l. ferrugineus mean arterial pressur effects were recorded before and after systemic blockade of muscarinic receptors with atropine or ache inhibition with neostigmine. data driven from these experiments showed that l. ferrugineus retained a behavior almost similar to the positive control ach, as l. ferrugineus hypotensive activity was significantly abolished in the presence of atropine. together, this indicates that l. ferrugineus extract, like extracts derived from tulbaghia violacea, bidens pilosa, moringa oleifera, cinnamomum zeylanicum and chenopodium ambrosioides, possibly possesses some cholinergic properties, and that its bp lowering effect was most likely driven by stimulation of cardiac and/or vascular muscarinic receptors. further exploring the role of the cholinergic pathway neostigmine tended to augment the bp lowering effect of l. ferrugineus and the duration of its action : yet, the effect was not as notably significant when compared to the enhancement of ach action on bp and the action duration. this data suggested that ache may not be the primary enzyme responsible for termination of the lfme effect in plasma and that other esterases, including butyrylcholinesterase or pseudocholinesterase, which is found in great abundance in plasma as compared to ache, is likely to hydrolyze esters of plant sources. importantly, as neostigmine did not block l. ferrugineus action on bp, our study concluded that lfme does not produce its cholinomimetic action through the antagonism of ache, but rather through direct muscarinic receptor stimulation. the cholinergic pathway exerts a powerful antagonistic influence on the heart by modulating cardiac rate (chronotropy), conduction velocity (dromotropy), contraction (inotropy), and relaxation (lusitropy). for instance, the cholinergic hypotensive properties of echinodorus grandiflorus ethanolic extract are associated with powerful reductions in cardiac output and heart rate in spontaneously hypertensive rats. likewise, raphanus sativus (radish) seed crude extract produces muscarinic receptor dependent inhibition of cardiac force and rate of contractions in isolated guinea pig atria ; however, whether l. ferrugineus exhibits similar direct cardiac effects remains unexplored. future investigations should therefore consider exploring cardiac activities of l. ferrugineus, investigating not only chronotropic, inotropic and dromotropic responsiveness of the heart but also changes in ventricular pressure, end diastolic volume and coronary blood flow. these experiments will potentially further our understanding of l. ferrugineus cardiovascular properties and provide comprehensive characterization of its therapeutic benefits. the activity of l. ferrugineus on the autonomic ganglia, which controls cholinergic and preferentially adrenergic cardiovascular targets, were previously studied by monitoring the ability of hexamethonium, a ganglionic blocker, to modulate the hypotensive response evoked by l. ferrugineus extract. it was found that ganglionic blockade did not markedly alter the bp lowering effects of the extract, suggesting l. ferrugineus, unlike dietary soy and gossypium barbadense, does not promote hypotension via ganglionic blockade mechanisms. nitric oxide (no) is probably one of the most important molecules produced by the endothelium and is synthesized by the enzyme no synthase (nos) from the precursor amino acid l - arginine (l - arg). a range of chemical mediators including not only ach but also bradykinin, serotonin, substance p and adenosine diphosphate trigger no release from the endothelium by receptor mediated mechanisms. endothelial derived no has the capacity to maintain vascular tone and to produce vasodilatation through the activation of guanylate cyclase. given the relative similarity between l. ferrugineus and ach pharmacological properties, and the fact that ach mediated activation of vascular muscarinic receptors triggers a cascade of events that ultimately generate no, we predicted that inhibition of nos, as with ach, would perhaps attenuate the bp lowering effects of l. ferrugineus. indeed, n-nitro - l - arginine methyl ester hydrochloride (l - name), a nos inhibitor, was shown to attenuate hypotensive responses to l. ferrugineus in vivo, suggesting that bp lowering activity is not only evoked by activation of cardiovascular cholinergic receptors, but also through promoting no release. it is common practice to subject crude plant extracts to activity guided fractionation to eliminate various types of complex or antagonistic molecules and derive a more purified and more potent form of the extract. subsequent studies on l. ferrugineus have therefore aimed to obtain purer derivatives of the active lfme extract for further in vivo and in vitro testing. in one study, four chemical fractions including chloroform fraction (cf - lfme), ethyl acetate fraction (eaf - lfme), n - butanol fraction (nbf - lfme) and water fraction (wf - lfme) were derived as shown in fig. 2. in vivo data from our laboratory showed that nbf - lfme significantly lowers bp in a dose dependent manner, and has a relatively longer duration of action compared to other fractions. interestingly, it has been further observed that nbf - lfme has more bp lowering potency relative to its mother crude extract lfme, which indicates that fractionation is able to increase the number of bioactive molecules in the extract. in keeping with our in vivo data, in vitro findings showed that, relative to other fractions, nbf - lfme produces a significant concentration dependent inhibition of aortic ring contraction against the -agonist phenylephrine and depolarizing signals of potassium chloride. these in vivo and in vitro results compare very closely to research findings on other plants used in hypertensive treatment, in which bioactivity guided fractionation contributes to a more potent pharmacological action. for example, kane and colleagues investigated an ethanolic extract and fractions of euphorbia thymifolia for diuretic activity, and found that the dose dependent diuretic effect of this herb was more potent in the fractionated extract. in addition, ouedraog performed a bioassay guided fractionation of ethanolic extract of agelanthus dodoneifolius, a plant traditionally used as treatment for hypertension, using rat aorta pre - contracted by norepinephrine to monitor the relaxant activity. they found that the vasorelaxant properties of agelanthus dodoneifolius crude extract were markedly potentiated following column chromatographic separation of its active fractions. as we found previously, lfme exhibits reversible noncompetitive antagonism of norepinephrine induced aortic ring contraction [59, 70 ], and nbf - lfme fraction exhibits the greatest bp lowering effect in vivo relative to other lfme fractions. therefore, we further investigated the effect of the purified nbf - lfme fraction with the aim of finding a more molecular mechanistic explanation to its pharmacological action. the active l. ferrugineus fraction was challenged against vascular endothelium dependent and independent mechanisms, using blockade with various pharmacological interventions including l - name, no - cgmp pathway inhibitor methylene blue, cyclooxygenase inhibitor indomethacin, atp dependent potassium channel blocker glibenclamide, -blocker propanolol and -receptor blocker prazosin. those experiments revealed that nbf - lfme induced vascular relaxation is primarily driven by endothelium dependent mechanisms, stimulating muscarinic receptors, activating the endothelium derived no - cgmp - relaxant pathway, promoting prostacyclin release and lengthening the released no half life through its antioxidant effects. this data therefore provide solid evidence that l. ferrugineus remarkable antihypertensive potentials extend beyond its muscarinic receptor activation, and further involve enhancement of key modulators of vascular function including no and prostacyclin, and possible vascular protection through its antioxidant and free radical scavenging properties. the effectiveness of l. ferrugineus through its enhancement of no or its antioxidant properties is of marked importance, as an imbalance between no and reactive oxygen species production appears to be a common feature of experimental and human hypertension. it is therefore possible that promoting no release and antioxidant action of l. ferrugineus biologically active principles can potentially influence a range of vascular targets to offer diverse protective cardiovascular properties. for instance, ginsenosides and saponins from panax ginseng have been shown to protect against myocardial ischaemia/ reperfusion damage and decrease lipid peroxidation, and that those effects are mediated by release of no from endothelial cells, especially from perivascular nitric oxidergic nerves. crataegus pinnatifida, a plant which has been used as a decoction for antihypertensive treatment for thousands of years in china, is believed to exert its effects through vasorelaxation resulting from no stimulation and antioxidant activity. similarly, other plants such as theobroma cacao, due to their enriched flavonoid constituents and resulting stimulation of no formation, has been shown to improve endothelial function through mechanisms that enhance no synthesis or those that decrease no breakdown. accordingly, further investigation of l. ferrugineus cardiovascular effects and/or targets is warranted in order to fully understand its cardiovascular protective action. herbal medicines could benefit patients suffering from gastrointestinal disorders that can not be treated using conventional drug therapy, including functional dyspepsia, constipation, and postoperative ileus. as reviewed by langmead and rampton, although most indications for the use of such remedies are traditionally derived, controlled research trials suggest some health benefits for ginger in nausea and vomiting, liquorice extracts in peptic ulceration, chinese herbal medicine in irritable bowel syndrome, opium derivatives in diarrhoea and senna, ispaghula and sterculia in constipation. following traditional uses of l. ferrugineus for gastrointestinal complaints, we aimed to investigate the effect of lfme and nb - lfme on the isolated guinea pig ileal preparation in vitro [69, 99 ], to test for gut contractility actions. indeed, this work showed that crude lfme and its nb - lfme retain the ability to dose dependently increase intestinal smooth muscle contractility. importantly, these effects were significantly reduced upon pre - incubation with atropine, enhanced in the presence of neostigmine, and unchanged in the presence of hexamethonium, suggesting a direct cholinomimetic action via muscarinic receptor activation, and that bioactive constituents within l. ferrugineus serve as a substrate for intestinal ache. having previously described a cholinomimetic hypotensive action for l. ferrugineus, our findings in the guinea pig ilium were not counterintuitive. nonetheless, this data has undoubtedly highlighted a new target organ for l. ferrugineus health benefits, confirmed the ability of the extract s active constituents to induce a notable peristalsis, and most importantly provided an immense support for its folklore use. accordingly, l. ferrugineus extracts, like other crude extracts obtained from carissa carandas and fumaria parviflora which possess a cholinergic stimulatory action, can potentially be formulated to treat constipation disorders. muscarinic agonists elicit contraction through the m3 receptor present in smooth muscle ranging from the esophagus to ileum. in the ileum, muscarinic agonists are known to have a dual effect on contraction : a direct m3 mediated contraction and an indirect m2 mediated inhibition of relaxation [102, 103 ]. activation of gastric acid secretion by endogenous ach, on the other hand, is triggered by m3 receptor subtype expressed on gastric parietal cells. despite a cholinomimetic action for l. ferrugineus, it remains undetermined if active constituents within its crude or purified extract would show a muscarinic receptor subtype specificity to elicit their contractile effect, or an ability to modulate acid, mucous or other gastrointestinal secretions. alzheimer s disease is a common neurodegenerative condition that affects the elderly population, primarily resulting in memory loss. the memory dysfunction in alzheimer s disease has been associated with cortical cholinergic deficiency and loss of cholinergic neurons within the brain. furthermore, chronic neuroinflammation and oxidative stress contribute to the neurodegeneration associated with alzheimer s disease and represent targets for therapy. l. ferrugineus antioxidant properties and effects on cholinergic pathways are of particular interest, because cholinergic abnormalities may affect memory or play a role in alzheimer s disease : perhaps l. ferrugineus action on cholinergic pathways may explain its memory enhancement and gerontological effects that have been observed in elderly people. though a variety of medicinal plants and their derivatives have been shown to have cholinomimetic activity in conjunction with improvement of memory and learning functions [108, 109 ], research linking mistletoe and alzheimer s disease is limited [110, 111 ]. hence, we propose this possible relationship between memory effects and l. ferrugineus cholinergic action tentatively, and emphasize that accurate scientific approach is required to test this hypothesis. natural products will continue to be important in three areas of drug discovery : as targets for production by biotechnology ; as sources of new lead compounds of novel chemical structure ; and as the active ingredients of useful treatments derived from traditional systems of medicine. though l. ferrugineus possesses a wide range of medicinal applicability, to date there is limited data available that evaluates this plants traditions, and few studies have been scientifically and experimentally translated toward evidence based phytomedicine. despite the research reviewed, various aspects of l. ferrugineus remain to be addressed : for example, identification of chemical constituents and therapeutic mechanisms. further studies on the isolation and structural elucidation of the pharmacologically active components of l. ferrugineus are likely to yield interesting results. perhaps synthetic or chemical modification of functional groups attached to these constituents could be the base for a more desired pharmacologically active component. for example, the production of a compound that has longer no enhancing properties that would benefit vascular health and serve as a better tool for hypertension control. in addition, to further characterize the bp lowering potential of l. ferrugineus, future investigations may focus on the effects of its extracts both in vivo and in vitro in hypertensive animal models, such as the spontaneously hypertensive rat. the effect of chronic treatment with l. ferrugineus extracts in animal models may also give rise to valuable research outcomes of the herb s effects in high bp treatment. finally, yet another research aspect of l. ferrugineus which remains to be addressed is its toxicology. presently, no current toxicology studies have been performed to investigate its effect on living organisms, despite that toxicology in particular is an important aspect of herbal medicine, since all substances become toxic under certain conditions, whether it be through contaminated growth environments, contamination incurred during collection of the plant materials, or even unfavorable storage conditions. the findings from l. ferrugineus mistletoe are likely to have important implications for understanding the beneficial actions of l. ferrugineus and for developing therapies for clinical problems involving the sites of action of this plant. more in depth research into l. ferrugineus will provide further justification for the employment of this herb in old traditional treatments, as well as provide greater insight into the underlying mechanisms responsible for its therapeutic effectiveness, and thus enhance awareness toward evidence based phytotherapy. | objectives : loranthus ferrugineus (l. ferrugineus) from loranthaceae, a mistletoe, is a medicinal herb used for a variety of human ailments. traditionally, decoctions of this parasitic shrub have been mainly used to treat high blood pressure (bp) and gastrointestinal complaints ; usage which is supported by experimental based pharmacological investigations. nonetheless, there is still limited data available evaluating this plant s traditions, and few studies have been scientifically translated toward evidence based phytomedicine. we therefore provide a concise review of the currently available l. ferrugineus literature and discuss potential directions for future areas of investigation.methods:we surveyed available literature covering ethnopharmacological usage of l. ferrugineus and discussed relevant findings, including important future directions and shortcomings for the medicinal values of this parasitic shrub.results:evidence based pharmacological approaches significantly covered the medicinal application of l. ferrugineus for hypertension and gastrointestinal complaint management, with a particular focus on the active hydrophilic extract of this herb.conclusion:understanding the sites of action of this plant and its beneficial effects will provide justification for its use in old traditional treatments, and potentially lead to the development of therapies. other medicinal applicative areas of this parasitic shrub, such as wound healing, gerontological effects, and antiviral and anticancer activities, are yet to be researched. |
the flexion - relaxation phenomenon (frp) is the myoelectric silencing of a muscle during a full - flexion maneuver1. the frp of the erector spinae (es) muscles shows silencing during upright to full - flexion lumbar movement in asymptomatic subjects. the phenomenon involves the transfer of the role of the extension moment producer to the deep back muscles to achieve spinal stability2. patients with low back pain (lbp) exhibit laxity of passive structures and an altered neuromuscular activation pattern in the back muscles, in which the frp of the es muscles is not present. thus, the frp of es muscles has been used to evaluate lbp and to monitor intervention - related factors after treatment3. previous studies have shown that frp occurs with full lumbar flexion in the sagittal plane. however, asymptomatic and symptomatic subjects can produce asymmetric lumbar flexion by lumbar flexion combined with lateral bending and axial rotation. although most healthy people do not have lbp, they have the potential for asymmetry in the frp in the right and left es muscles due to repetitive movement and poor posture in their work environments or daily activities. ning.4 reported that asymmetric lumbar flexion elicits a loss of the frp in the ipsilateral muscle in asymptomatic subjects. although 90% of non - lbp subjects show frp5, many of them were at risk of lbp, because of asymmetry in the frp of the es muscles. currently, lbp patients perform various deep muscle strengthening exercises for lumbar tissue, with pilates, sling exercises, and lumbar stabilization exercises being the most common6. lumbar stabilization exercises, especially the back bridge and hand - knee position, are the most effective at strengthening the multifidus muscle7. marshall and murphy.3 reported that a 12-week exercise program (side bridge, supine bridge, partial curl - up, bird - dog exercise, swiss ball push - up, single leg hold, and rollouts) decreased self - reported pain and es muscle activity during the relaxation phase of frp tests in patients with chronic non - specific lbp. lumbar stabilization exercises elicit a reappearance of the frp through strengthening of the multifidus muscle, and eventually this could result in symmetry of the frp in non - lbp subjects with an asymmetric frp of the es muscles. the purpose of this study was to evaluate differences in the frp in the right and left es muscles in asymptomatic subjects. in addition, the effects of lumbar stabilization exercises on differences in the frp of the right and left es muscles were investigated. participants (n=30 ; average age, 19.8 1.1 years ; height, 164.5 7.4 cm ; body mass, 57.54 7.20 kg) were recruited from among the healthy student population of silla university. participants were excluded from the study if they had a history of back pain, vertebra surgery, or neuromuscular disorders. all procedures were reviewed and approved by the ethics committee of silla university. to analyze the frp of the es muscles, surface electromyography (emg) (myotrace 400 ; noraxon inc., scottsdale, az, usa) was used with two bipolar surface emg electrodes placed bilaterally 2 cm apart over the right and left l3 area. the data were collected at 1,000 hz and processed using a 20450 hz band - pass filter and a 60-hz notch filter. the root mean square moving average of 300 ms duration of the processed emg data was calculated. the relaxation time was defined as when es activation decreased by two standard deviations (sd) of silence activation in the fully flexed lumbar phase. the absolute value of the difference in relaxation time measured in the right and left es was calculated. the experiment was done in triplicate and the calculations were based on the averages of the three trials. the position of the feet was marked for consistency between trials. in all trials, the move from upright to full - forward flexion (forward flexion phase) was performed over 3s, followed by 3s maintenance of full flexion posture (fully flexed phase), and another 3 s to move from full - forward flexion to the upright posture (re - extension phase)8, 9. participants were assigned randomly to the control or exercise group using a table of random numbers the exercise group performed two exercises that are commonly used in clinical practice and have been used in previous studies (back bridge and hand - knee exercise). these exercises were performed 3 days per week with a trainer and 2 days per week at home for 4 weeks. the exercises were done in three sets with three repetitions per set and a set break of 3 min. instructors provided feedback to ensure that a consistent supine and lower limb posture was maintained during the exercise. for the back bridge exercise, the subjects lay supine on the floor with their feet flat on the ground, their knees bent at 90, their toes facing forward, and their hands on the floor by their sides. with their palms facing down, the subject raised the pelvis to achieve and maintain a neutral hip flexion angle. the subjects were requested to raise the back bridge from the floor (stable condition) and hold the posture for 1 min. after the 1-min hold, the subject was given a 30-s break. for the hand - knee exercise, the subjects started on their hands and knees with a neutral pelvis position and breathing normally. they lifted and held straight the right arm and the left leg before doing the same with the opposite set of limbs. the subjects held each position for 40 s with a 30-s break between positions. 18.0) was used to test the significance of differences between the exercise and control groups and between pre- and post - exercise. a subject factor analysis was used to assess the statistical significance of the frp in the es muscles with a repeated two - way analysis of variance. the paired t - test was used to test the significance of differences between the two groups characteristics. a difference was seen in the frp between the right and left es muscles in asymptomatic subjects (control group 119.2 69.2 ms and exercise group 131.1 85.2 ms ; p>0.05). the exercise group showed a significant decrease post - exercise (50.0 27.0 ms) versus pre - exercise (112.3 41.5 ms) in the right and left frp of the es muscles (p<0.05). in this study, the healthy participants showed a difference in the right and left frp in the es muscles. this lack of balance in the frp is thought to result from repetitive asymmetric posture and activity. previous research has indicated there is an increased risk of back pain when working in asymmetric positions9. motor control deficits and motor dysfunction result in increased passive system loading from repeated stress10. the students who participated in this experiment spent a great deal of time at school in a sitting position. this does not produce lbp, but they exhibited an asymmetric frp in the right and left es muscles. callaghan11 reported a difference in activation between the right and left es in healthy subjects (right 15 and left 17 of 20 participants). this indicates that asymptomatic people can have impairment and imbalance in the frp. when the students exhibited poor posture (e.g., sitting cross - legged or one - leg weighted standing) they experienced no pain, but they demonstrated potential factors for lbp. the bridge exercise is a popular lumbar stabilization exercise that activates weak abdominal muscles and prevents unstable lumbar spine movement via co - activation of the pelvic floor muscles, diaphragm, and deep abdominal muscles12. additionally, the swiss ball exercise activates deep and superficial abdominal muscles by using the characteristics of an unstable environment13. marshall and murphy14 reported that swiss ball exercises increased the frp (from 3.26 3.43 to 6.53 3.34) in lbp patients. in the present study, lumbar stabilization exercises improved muscular balance by reducing asymmetry in frp onset. howarth.15 reported that back muscle instability caused by slumped sitting delayed the onset of the frp. likewise, the intervention used in the present study was meaningful for trunk asymmetry and instability. at the onset of the frp, the external moment generated around the lower back by the upper body s mass is counteracted by the net internal moment produced from the tensile forces developed in elongated passive tissue, instead of the forces generated by the active component of the lower back musculature16. thus, lumbar stabilization exercises can improve the motor control of lumbar spinal movement and lumbar stabilization control. among the various exercises used for lumbar stabilization, the sling and swiss ball exercises show the highest emg activation values17 ; however, this study used the back bridge and hand - knee exercises. these two exercises do not need any special equipment or space, so they are more suited for home - based exercise than sling or ball exercises. additional studies are needed to measure changes in the frp in the es muscles elicited by other lumbar stabilization exercises. while further study is needed, the results of this study suggest that lumbar stabilization exercises can help to correct asymmetry of the frp in the right and left es muscles and this would help prevent lbp in the general population. | [purpose ] this study evaluated the differences in the flexion - relaxation phenomenon (frp) of the right and left erector spinae muscles in asymptomatic subjects and the effect of lumbar stabilization exercises on these differences. [subjects and methods ] twenty - six participants (12 in the exercise group and 14 in the control group) with a difference in the frp in the right and left erector spinae muscles were recruited from among healthy students attending silla university. the exercise group performed two lumbar stabilization exercises (back bridge exercise and hand - knee exercise) for 4 weeks. the control group did not exercise. [results ] no significant group - by - exercise interaction was found. the right and left erector spinae muscles did show a difference in frp between the control and exercise groups (119.2 69.2 and 131.1 85.2 ms, respectively). in addition, the exercise group showed a significant decrease in post - exercise (50.0 27.0 ms) compared to pre - exercise (112.3 41.5 ms) differences in the right and left frp. [conclusion ] these results suggest that lumbar stabilization exercises may counter asymmetry of the frp in the erector spinae muscles, possibly preventing low back pain in the general population. |
it is commonly reported in (1422%) of population studies3, however a recent study found that 25% of employees report fatigue at work4, 5. fatigue is characterized by progressive withdrawal of attention from the environment and demands due to tiredness, dislike of the present activity, and unwillingness to continue, or perform the task at hand6, 7. also, physical resources and the depletion of emotional resources are considered the basic parameters of feelings of being overextended and emotionally exhausted8. it has been reported that fatigued persons show decrements in vigilance when performing a particular task via changes in vigilance, alertness, motivation, and subjective states that occur during this transition9. previous studies have reported that both the sympathetic nervous system (sns) and the hypothalamic - pituitary adrenocortical (hpa) axis are the most vital centers linking stressor exposure to disease10, 11. thus, significant increases in these hormones in line with cortisol are associated with stressful situations, such as caregiving and work strain, which may be diagnosed as adrenal fatigue12,13,14. some studies have reported that the improvement of physical and psychological health is related to physical activity which is considered an effective preventive measure and treatment for stress - related diseases15,16,17. physically active people show higher resistance to physical stressors and nullifying susceptibility to various influences of life stress18, 19, and physical activity has protective effects on non physical stress, such as mental stress20. although the findings are not uniform21, psychological stress protocols show lower responses of the sympathetic nervous system and the hypothalamic - pituitary - adrenal (hpa) axis towards physical activity. a lower cortisol increase was reported in physically active subjects19, as well as lower cardiovascular reactivity19, 22, and more rapid cardiovascular recovery20 to psychological laboratory stressors than less active controls. also, it s more important to notice the variability of stress reactivity which extends to cover all states of physical activity including those who have less distinct differences in activity levels, such as a lower response of heart rate to mental stressors and the faster recovery of elite sportsmen than amateur sportsmen23. these inconsistent results may be related to the differential influence of physical activity on stress reactivity. the relationship between physical activity and stress reactivity may be linked to the personality traits of subjects which may be the most valued stress modulators. depressed persons are physically inactive in daily life and have lower physical work capacity than healthy persons. this indicates that a depressive state is associated with physiological well being and may predispose an individual to diseases related to physical inactivity and limited functional capacity24, 25. the rates of mortality and morbidity for the conditions of most diseases may be related to the negative outcome measures of occupation, income, and education which are characterized collectively as socioeconomic status (ses)26,27,28,29,30,31. these measures are prevalent in countries with state - sponsored health care, suggesting that access to care is not the underlying causal mechanism. it was proposed that persons with lower ses are exposed to conditions with higher levels of psychosocial disruption that increase the risk of disease through continuous provocation of stress eliciting dysregulation of key behavioral and biological systems32, 33. in order to develop or maintain goals towards adequate behavior and performance, it is necessary to increase or minimize both personal competence and willingness34, 35. this strategy was used in this present study, even though the assessment of fatigue has no definite methodology. it was suggested that fatigue can be measured using objective and subjective outcome measures which are significantly different. objective fatigue measures deal with physiological processes, or performance such as reaction time or number of errors36, whereas subjective ways include diary studies, interviews, and questionnaires37, 38. until about 10 years ago, fatigue questionnaires for particular studies were mainly developed on an ad hoc basis, and they were used in large scale studies because of their shortness and self - report format. recently, it was shown that fatigue questionnaires should be developed for specific patient groups, such as patients with cancer39,40,41,42, or ill persons in general43,44,45,46. although there are many benefit outcome measures for both patient and healthy populations, little is known about the suitability of these questionnaires for healthy populations, and only a few questionnaires such as the fatigue scale (fs) have been developed to measure fatigue outcomes among hospital and community populations47, 48. before the start of the 1990s they have studied the effect of five components, general fatigue, physical fatigue, reduction in activity, reduction in motivation, and mental (cognitive) fatigue on depression and fatigue status48,49,50. also, other reviews have documented that multidimensional fatigue scales are seen as more comprehensive and hence as more adequate for providing a complete description of an individual s fatigue experience39, 40. accordingly, this study was designed to assess the effect of physical activity status and environmental factors on the levels of cortisol and acth, as markers of adrenal fatigue status, in a sample of saudi adult volunteers using a questionnaire and biochemical analysis. in the present cross - sectional study, the data were collected in 20122013 by supervised experienced data collectors. the research team collected detailed sociodemographic data of the160 saudi adults aged 1522 years who participated in this study. the present study received prior approval from the ethics committee of the rehabilitation research chair (rrc), king saud university, riyadh, ksa. based on the physical activity of the participants, the sample size was calculated with an estimated difference of 5% between the three groups of mild, moderate, and vigorous exercise for estimating the correlation of cortisol and acth hormones with fatigue and physical activity status. so, in order to investigate cortisol and acth levels in the mild group a sample size of 45 or more was needed for a power of 90%, for the moderate group, a sample size of 37 or more was needed, and for the severe group, a sample size of 60 subjects was required to have the same power. after centrifugation, serum samples of all tests were stored at 80 c until assayed. both cortisol and acth were measured in serum samples using immunoassay techniques following the instructions of the ria - elisa kit (dpc inc., ca, usa), and the sia - elisa kit (md biosciences inc, mn, usa) respectively. a self - administered questionnaire was used to assess living conditions, household income level, employment and marital status, education level, gender, and age. participants chose the most suitable answer from categories of employment status (office worker, independent businessman, professional, public official, student, housewife, part - time worker, not employed), marital status (married, unmarried), living conditions (number of people cohabitating with, living alone), and education level (university, two - year university, vocational college, high school, junior high school, graduate school). the short - term international physical activity questionnaire (ipaq) was used to evaluate the level of physical activity of the participants51, 52. the average number of minutes per day and days per week of physical activity were self - calculated by the participants with reference to the past month. the frequency and duration of walking for all purposes such as work, transport, or recreation, moderate physical activity, vigorous physical activity, and sedentary activity for a usual week were assessed via a self - administered questionnaire. finally, the total numbers of minutes of walking, moderate, and vigorous physical activity per week were computed according to the ipaq scoring manual53, 54. the level of adrenal fatigue status was estimated using the adrenal fatigue test questionnaire55. the adrenal fatigue test assesses adrenal fatigue using a score : a score of 40 suggests no adrenal stress ; 4487 suggests mild adrenal stress ; 88130 suggests moderate adrenal fatigue ; and 130 suggest significant adrenal fatigue problems. a seven - day test - retest reliability was conducted (test - retest), with an interval of 7 days between the two assessments (time 1 and 2). internal consistency was calculated using cronbach s alpha () for seven general adrenal fatigue test items (predisposing factors, key signs and symptoms, energy patterns, frequently observed events, food patterns, aggravating factors, relieving factors). the 7-day test - retest reliability was estimated using pearson s r and spearman s rho statistics. it has been proposed that test - retest reliability coefficients of 0.80 or higher for these statistics are indicative of acceptable test - retest reliability56. the severity index was calculated by simply dividing the total number of points by the total number of questions answered by each participant in the affirmative. it gives an indication of how severely the signs and symptoms are experienced, with 1.0 being normal, 1.11.6 being mild, 1.72.3 being moderate, and 2.4 being severe55. all statistical analyses were performed using the spss statistical package v. 12.0 for windows (spss software, inc., chicago, il, usa). repeated measures anova followed by the bonferroni correction for multiple comparisons was applied for normally distributed parameters, and the wilcoxon test, for nonparametric parameters. exploratory factor analysis of the items of adrenal fatigue and severity scores were conducted to investigate what common components of the scale more effectively respond to physical exercise treatment. data are presented as mean sd. the null hypothesis was rejected at values of p < 0.05, the level of significance. table 1table 1.demographic characteristics of the study sample (n = 160)variablesn, (m sd)%sample size 160100gendermale12075female4025age (yrs)20.22 2.89-height (cm)167.95 13.0-weight (kg)67.32 12.25-bmi (kg / m)23.43 3.76-marital statusunmarried10062.5married6037.5living conditionliving with others11571.9living alone4528.1educational level4-years university or greater65412-years university5031high school or junior high4528employment statusemployed8553not employed7547walking, min / week 91 (260.1 462.3)-moderate - intensity activity, min / week59 (120.6 390.7)-vigorous - intensity activity, min / week10 (75.4 260.6)- shows the characteristics of the participants. the mean age (standard deviation ; sd) of the participants was 20.22 (2.89) years. the percentage of those who were married was 37.5%, that of those living with another person was 71.9%, and that of those who were employed was 53.0%. the mean number of minutes of walking per week (sd) was 260.1 (462.3), for moderate intensity activity excluding walking it was 120.6 (390.7), and for vigorous - intensity activity it was 75.4 (260.6). table 2table 2.test re - test reliability for adrenal fatigue -q measures for participants with an interval of 7 days between the two assessments (times 1 and 2)adrenal fatigue -q measuresitemfull sample (n=160)time 1time 2pearson rspearman s rhocronbach s alphapearson rspearman s rhocronbach s alphapredisposing factors130.810.820.80 (0.640.87)0.890.810.85 (0.600.87)key signs & symptoms310.90.800.81 (0.630.96)0.880.930.88 (0.660.90)energy patterns 130.860.910.87 (0.660.97)0.920.850.95 (0.650.95)frequently observed events220.890.850.82 (0.660.86)0.910.890.88 (0.680.97)food patterns90.870.920.86 (0.550.95)0.850.800.91 (0.820.92)aggravating factors100.960.890.91 (0.660.97)0.980.960.96 (0.860.98)relieving factors50.810.820.81 (0.640.87)0.890.810.89 (0.600.97)overall1030.900.800.85 (0.630.96)0.960.930.96 (0.660.98) shows cronbach s internal consistency for all adrenal fatigue measures. overall, internal consistency was lower at time 1 than at time 2 which remained acceptable at both time points for all participants. internal consistency was consistently higher (= 0.96 for time 2 vs = 0.85 for time 1). also, reliability was excellent for all adrenal fatigue measures (pearson r = 90 ; spearman s rho = 0.80 at time 1 and pearson r = 96 ; spearman s rho = 0.93 at time 2). the results show the questionnaire had good reliability with better results for the investigation of adrenal fatigue stress among the studied participants. table 3table 3.changes in the level of adrenal hormones, social, and environmental factors according to the adrenal fatigue scores of the participants (n = 160)variablesadrenal fatigue scorenormal (40)mild (44 - 87)moderate (88 - 130)severe (130)adrenal hormonescortisol (g / ml)6.2 0.7510.5 1.916.9 3.628.5 3.9acth (pg / ml)16.9 11.727.9 7.336.3 14.242.7 9.5marital status : unmarried19.6 6.345.9 12.192.9 18.3142.3 10.4married10.2 2.758.2 7.498.8 5.7152.2 6.8educational level:4-years university or greater9.1 3.4 51.3 1.2 100.1 3.1 165.3 2.4 2-years university 12.1 2.954.5 4.896.7 7.2175.3 7.2high school or junior high15.3 4348.6 59122.8 9.2181.8 11.6employment status : employed6.1 3.649.3 2.3102.3 2.3170.3 4.5not employed9.7 1.351.7 3.695.4 3.6190.3 5.7living condition : living with others5.8 2.7 49.3 1.5 110.5 3.7 165.8 5.6living alone13.9 5.158.7 6.4108.3 4.3175.5 7.4except where indicated otherwise, values are expressed as the mean. p < 0.001 significant for unmarried versus married status and employed versus not - employed ; p < 0.05 significant for university degree versus 2 year university degree or high school degree and living with others versus living alone for all adrenal fatigue scores. environmental factors and social influences were seen to directly affect the score of adrenal fatigue status in all states, normal, mild, moderate, and severe, of adrenal fatigue. participants who were unmarried (p=0.001), had low educational levels (p=0.05), were not employed (p=0.001), or were living alone (p=0.05) had significantly higher adrenal fatigue scores. these parameters of environmental and social factors had the greatest effect on the status of adrenal fatigue. levels of adrenal hormones such as cortisol and acth were also investigated in this study. there were significant increases in the levels of cortisol (p=0.001) and acth (p=0.001), markers of adrenal fatigue, in participants with moderate to severe adrenal fatigue scores compared to those with normal and mild scores (table 3). the changes in adrenal hormones significantly correlated with the severity of adrenal fatigue. except where indicated otherwise, values are expressed as the mean. p < 0.001 significant for unmarried versus married status and employed versus not - employed ; p < 0.05 significant for university degree versus 2 year university degree or high school degree and living with others versus living alone for all adrenal fatigue scores. based on physical activity, the participants were classified into three groups : mild (n=50), moderate (n=40), and vigorous (n=70). table 4table 4.interaction effect between frequency of adrenal fatigue and physical activity of the participants (n = 160)adrenal fatigue score(m sd)physical activity (ipaq score) (n=160) mild (n= 50)moderate (n= 40)vigorous (n= 70)normal(score, 40) ; n (%) 38.761 3.6 ; 3(6) 22.161 1.9 ; 30 (75)12.8 4.8 ; 41(6)mild(score, 4487) ; n (%) 55.7 5.9 ; 5(10)48.7 2.6 ; 5(11)44.9 3.8 ; 11(16)moderate(score, 88130) ; n (%) 98.75 4.6 ; 13 (26)91.4 3.7 ; 3 (8)88.9 3.8 ; 9(13)severe(score, 130) ; n (%) 148 8.2 ; 29 (58)135.7 2.9 ; 2(5)131.5 1.5 ; 9(13)except where indicated otherwise, values are expressed as the mean sd. improvement in adrenal fatigue status noticed in participants with moderate (p < 0.01) and vigorous activities (p < 0.001) compared to the mild group shows the influence of mild, moderate, and vigorous physical activities on adrenal fatigue severity. participants reporting moderate and vigorous exercise intensities showed significant (p = 0.01 and p = 0.001) decreases in adrenal fatigue severity compared to those reporting mild activities. however, there was significant increase in the ratio (75%) of subjects with a normal fatigue score in the moderate intensity exercise group compared to the vigorous intensity exercise group. the findings of this study suggest that physical activity especially that of moderate intensity may be effective for improving depressive adrenal fatigue. the participants in the moderate and vigorous exercise groups showed significant decreases (p=0.01 and p=0.001) in the severity index score of adrenal fatigue compared to those of mild exercise group (table 5table 5.interaction effect between adrenal fatigue severity index and physical activity status of the participants (n = 160)fatigue severity indexscore(m sd)physical activity (ipaq score) (n=160) mild (n= 50)moderate (n= 40)vigorous (n= 70)normal (score, 1.0) ; n (%) 1.0 0.6 ; 6 (12) 0.86 0.6 ; 25 (62.5)0.65 0.5 ; 46(66)mild(score, 1.1 - 1.6) ; n (%) 1.5 0.75 ; 6(12)1.4 0.6 ; 6(15)1.32 0.8 ; 12(17)moderate(score, 1.7 - 2.3) ; n (%) 2.28 0.92 ; 8 (16)2.2 0.75 ; 5 (12.5)1.9 0.71 ; 9(13)severe (score, 2.4) ; n (%) 5.7 3.2 ; 30(60)3.9 2.9 ; 4(10)131.5 1.5 ; 3 (4)except where indicated otherwise, values are expressed as the mean sd. significant decrease in adrenal fatigue severity noticed in participants with moderate (p < 0.01) and vigorous activities (p < 0.001) compared to the mild group). except where indicated otherwise, values are expressed as the mean sd. improvement in adrenal fatigue status noticed in participants with moderate (p < 0.01) and vigorous activities (p < 0.001) compared to the mild group except where indicated otherwise, values are expressed as the mean sd. significant decrease in adrenal fatigue severity noticed in participants with moderate (p < 0.01) and vigorous activities (p < 0.001) compared to the mild group also, based on biochemical analysis, there were significant correlations between the levels of adrenal hormones and physical activity. significant decreases in the levels of cortisol (p=0.001) and acth (p=0.001) were found in the participants with moderate to high physical activity compared to those with mild or sedentary activity. the decrement in adrenal hormonal levels positively correlated (p=0.01, r= 0.976) with improvements in the adrenal fatigue status and severity index, especially in the physically active participants (table 6table 6.correlation between the level of cortisol and atch adrenal hormones and physical activity status of the participants (n = 160).adrenal hormones(m sd)physical activity (ipaq score) (n=160) mild (n= 50)moderate (n= 40)vigorous (n=70)cortisol (g / ml)28.6 4.8 12.7 3.2 17.3 2.8 acth (pg / ml)34.8 6.8 15.9 6.4 22.7 3.8 except where indicated otherwise, values are expressed as the mean sd. a significant decrease was found in the levels of cortisol and atch as markers of adrenal fatigue status in participants with moderate (p < 0.01) and vigorous activities (p < 0.001) compared to the mild group. the decrement positively correlated with the improvement in adrenal fatigue status and severity index in physically active participants (p=0.01 ; r= 0.976)). except where indicated otherwise, values are expressed as the mean sd. a significant decrease was found in the levels of cortisol and atch as markers of adrenal fatigue status in participants with moderate (p < 0.01) and vigorous activities (p < 0.001) compared to the mild group. the decrement positively correlated with the improvement in adrenal fatigue status and severity index in physically active participants (p=0.01 ; r= 0.976) in recent years, depression has become increasingly more prevalent among adolescent females on a worldwide scale57, 58, and it is considered to be a disease which has a greater worldwide burden than ischaemic heart disease, cerebrovascular disease or tuberculosis59. in this study, the potential influences of environmental and social factors as well as different levels of physical activity on adrenal fatigue stress and reactivity were investigated. this study evaluated the reliability of the adrenal fatigue questionnaire used to investigate the importance of fatigue stress in physical activity using the test - retest reliability method. the results show the questionnaire had good reliability with better results for the investigation of fatigue stress among the studied participants. the present study investigated the environmental and social factors that affect adrenal fatigue stress in males and females aged from 15 to 22 years. environmental and social factors were the most influential factors that directly affected adrenal fatigue, and moderate to severe adrenal fatigue stress was significantly associated with unmarried status, low level education, not in employment, and living alone. this was consistent with a study that reported depression, anxiety, and stress are three common mental health problems worldwide60, and other studies that investigated the psychometric properties of these questionnaires61, 62. it has also been reported that subclinical depression in adolescence is related to depressive episodes, substance abuse, higher levels of neuroticism, academic underachievement, unemployment, and early parenthood63. the ecological perspective suggests that physical activity is influenced by an interaction of demographic, psychological, social, and environmental factors64. in the present study, the relation between physical activity and the status of adrenal fatigue was investigated, and the results show that, pattern of stress responses was related to the level of physical activity. a significant improvement in the stress fatigue status was also shown by participants with moderate physical activity compared to mild and vigorous physical intensity. our findings indicate that higher levels of physical activity are associated with lower stress reactivity suggesting that higher levels of physical activity might be protective against the development of stress related diseases. these results are supported by our measurements of cortisol and acth as markers of adrenal fatigue status. there was significant change in serum adrenal hormones among participants with physical inactivity and higher fatigue scores. there were significant increases in serum cortisol and acth levels among participants with lower physical activity and higher adrenal fatigue scores. however, in participants with moderate to high physical activity, the decrease in the level of cortisol and acth positively correlated with improvement in the severity index and adrenal fatigue score. the results are in agreement with previous studies which focused on the effect of physical activity on depression via the control of adrenal hormones secretion65,66,67,68. another study proposed physical exercise interventions to protect against changes in the adrenal system with ageing69. increased susceptibility to immunosuppression and infection has been linked with extreme levels of exercise70. thus, it may be that physical activity of an intermediate level is the most effective level for stress protection. furthermore, in general, depressive persons are physically sedentary in their daily life and have lower physical work capacity than healthy individuals71. similarly, in the present study, the severity index of fatigue status showed that physical activity plays a pivotal role in reducing the severity of adrenal fatigue status. psychosocial stress increases the risk of developing cardiovascular and mental diseases, such as hypertension or depression72, 73. physical activity is commonly regarded as beneficial for both physical and psychological health, and is seen as an effective preventive measure and treatment for stress - related diseases74. physically active people show reduced reactivity to physical stressors as well as reduced susceptibility to the adverse influences of life stress19. recently, it was reported that the environment has long - term effects on population - based health behavior, and there was a direct relationship between environmental characteristics and physical activity as previously reported in the literature74, 75. interestingly, there are distinct variations in stress reactivity not only between extreme groups of physically active and inactive controls, but also between groups with less distinct differences in activity levels19. finally, the findings of the present study imply that intervention strategies to promote more engagement in physical activity for population based health promotion may be necessary. in conclusion, our data suggest that the level of physical activity and environmental and social factors differentially influence the adrenal fatigue status via changes in adrenal hormone levels. interestingly, the reactivity of adrenal fatigue status was revealed to be sensitive to higher levels of physical activity, unfavorable environmental and social factors. future studies should evaluate whether the level of physical activity might induce an optimal stress protective effect against stress - related diseases. | [purpose ] this research work was performed to examine whether different levels of physical activity and environmental and social factors are associated with changes in adrenal hormones as markers of adrenal fatigue in saudi adult volunteers. [subjects and methods ] a total of 160 saudi adults aged 1522 years were included in this study. the adrenal fatigue score, sociodemographic attributes, and the level of physical activity were evaluated via pre - validated internet - based questionnaire surveys. adrenal hormones such as acth and cortisol were measured using immunoassay techniques. [results ] significant increases in the levels of acth and cortisol biomarkers were found in the participants with moderate to severe fatigue scores, poor environmental factors, and low physical activity. however, in physically active participants, significant decreases in acth and cortisol levels were found with remarkable improvement in adrenal fatigue status. the decrement in adrenal hormonal levels positively correlated (r= 0.976) with the improvement in adrenal fatigue status in the physically active participants. [conclusion ] our results suggest that the level of physical activity and environmental and social factors differentially influence the adrenal fatigue status via changes in the levels of adrenal hormones. also, acth and cortisol biomarkers may be useful as markers measuring the severity of adrenal fatigue. |
bendamustine is an anticancer drug, which has recently evolved as an important agent for a number of lymphoid malignancies in europe and the usa. the drug consists of an alkylating nitrogen mustard group bound to a purine - like benzimidazole ring, and because of this unique bifunctional structure the bendamustine activity profile is significantly different from classical alkylators. although the precise mechanism of action has not been elucidated yet, it is known that bendamustine induces dna cross - linking and dna breaks and induces cell death by apoptosis through intrinsic and extrinsic pathways, which in turn may deregulate the cell cycle and lead to a preclinical studies and clinical observations suggest that bendamustine has limited cross - resistance with other alkylating agents and demonstrates significant synergism with anti - cd20 monoclonal antibody rituximab and purine analogues [2, 3 ]. based on two multicentre randomised studies, bendamustine has received approval for second - line therapy in relapsed / refractory indolent non - hodgkin lymphoma (nhl) [4, 5 ]. a growing body of evidence suggests good efficacy and acceptable tolerability of bendamustine as the first - line option for indolent lymphoma, mantle cell lymphoma (mcl) and selected patients with aggressive lymphoma. this review presents data for bendamustine use in first - line therapy of nhl, taken from all available relevant articles published in the years 20062013, supplemented with abstracts from recent haematology and oncology scientific meetings. a summary of available data, source publications and conference abstracts is presented in tables 1 and 2.table 1bendamustine in first - line treatment for indolent or mantle cell lymphoma prospective studiesstudypatients (n)diagnosisorr % cr % pfsrefbop versus cop prospective phase iiiherold 2006164fl, lpl, mcl66 versus 7622 versus 205 yrs : 59 % versus 46 % 8 br versus chopr prospective phase iiirummel 2013 stil study549fl, mcl, lpl, mzl, sll93 versus 9140 versus 3069.5 versus 31.2 months 9 br versus cvpr or chopr prospective phase iiiflinn 2012 bright study436indolent, mcl94 versus 8431 versus 25 10 br prospective phase iiluminari 201369mzl, sll, lpl84581 yrs : 90 % 14 br + cytarabine prospective phase iivisco 201320mcl100952 yrs : 95 % 19 ribvd (br + bortezomib + dexamethasone) prospective phase iigressin 2013 lysa study76 elderlymcl8760 22 br + lenalidomide prospective phase i iijerkeman 201351mcl9779not reached in 18-month follow - up 21 br + r maintenance prospective phase iirummel 2012162lpl86 9, 12 br prospective phase iisalar 201260malt10098 13 b + ofatumumab prospective phase iifowler 201249indolent9860 17 br + mitoxantrone prospective phase iiboccomini 201276 elderlyfl9675 16 br + bortezomib prospective phase iiflinn 201255indolent8947 15 b + ofatumumab + dexamethasone prospective ii phasemagni 201219 elderlymcl9489 20 b ; bendamustine, br ; bendamustine + rituximab, bop ; bendamustine + vincristine + prednisone, cop ; cyclophosphamide + vincristine + prednisone, cvpr ; cyclophosphamide + vincristine + prednisone + rituximab, chopr ; cyclophosphamide + doxorubicin + vincristine + prednisone + rituximab, ribvd ; rituximab + bendamustine + bortezomib + dexamethasone, fl ; follicular lymphoma, lpl ; lymphoplasmacytic lymphoma, mcl ; mantle cell lymphoma, mzl ; marginal zone lymphoma, sll ; small lymphocytic lymphoma, malt ; mucosa - associated lymphoid tissue, orr ; overall response rate, cr ; complete remission, pfs ; progression - free survival, ref ; reference, yrs ; yearstable 2bendamustine in first - line treatment for aggressive lymphomastudypatients (n), diagnosisorr % cr % pfs monthsos monthsrefbr, retrospectivehorn 201220, dlbcl, elderly55208.319.4 25 br, retrospectivekuntz 201015, dlbcl463314.123.1 27 br, retrospectivehammersen 201321 dlbcl mcl9114824 28 br, retrospectivewalter 201215, dlbcl623869 26 br, prospective phase iiweidmann 201114, dlbcl, mcl elderly69547.77.7 24 br prospective phase iipark 201323, dlbcl, elderly93609.9 29 br ; bendamustine + rituximab, dlbcl ; diffuse large b - cell lymphoma, mcl ; mantle cell lymphoma, orr ; overall response rate, cr ; complete remission, pfs ; progression - free survival, os ; overall survival, ref ; reference bendamustine in first - line treatment for indolent or mantle cell lymphoma prospective studies b ; bendamustine, br ; bendamustine + rituximab, bop ; bendamustine + vincristine + prednisone, cop ; cyclophosphamide + vincristine + prednisone, cvpr ; cyclophosphamide + vincristine + prednisone + rituximab, chopr ; cyclophosphamide + doxorubicin + vincristine + prednisone + rituximab, ribvd ; rituximab + bendamustine + bortezomib + dexamethasone, fl ; follicular lymphoma, lpl ; lymphoplasmacytic lymphoma, mcl ; mantle cell lymphoma, mzl ; marginal zone lymphoma, sll ; small lymphocytic lymphoma, malt ; mucosa - associated lymphoid tissue, orr ; overall response rate, cr ; complete remission, pfs ; progression - free survival, ref ; reference, yrs ; years bendamustine in first - line treatment for aggressive lymphoma br ; bendamustine + rituximab, dlbcl ; diffuse large b - cell lymphoma, mcl ; mantle cell lymphoma, orr ; overall response rate, cr ; complete remission, pfs ; progression - free survival, os ; overall survival, ref ; reference low - grade lymphoma, considered as an incurable disease, represents about 40 % of all nhls. rituximab in combination with chemotherapy, usually a chop (cyclophosphamide, doxorubicin, vincristine, prednisone) regimen, is a standard first - line treatment, especially in follicular lymphoma subset. encouraging results of bendamustine in relapsed or refractory indolent nhl have drawn attention to the first - line use of the drug in this setting. german haematology outpatient centres have reported a continuous increase in the use of bendamustine in combination with rituximab (br) as upfront therapy in patients with indolent nhl, despite no european medicines agency (ema) approval for this indication. the first randomised study evaluating the effectiveness of bendamustine in treatment - naive indolent lymphoma patients was published in 2006. the drug (bendamustine), in combination with vincristine and prednisone (bop), was compared with a cop (cyclophosphamide, vincristine, prednisone) regimen in 164 patients with follicular lymphoma, mcl and lymphoplasmacytic lymphoma (lpl). there were no statistically significant differences between groups with respect to overall response rate (orr) (bop vs. cop : 66 vs. 76 % ; p = 0.1) and overall survival (bop vs. cop : 61 vs. 46 % ; p = 0.2). the median time to progression was significantly longer in bop responders (84 months) than in those who responded to the cop regimen (28 months), p = 0.0369, which translated into the probability of 5-year progression - free survival (pfs) as 59 % versus 46 %, respectively. additionally, both haematological and non - haematological complications were less common and less severe in the bop group than in the cop group (table 3).table 3haematological and non - haematological toxicity of bendamustine - based regimensstudypatients diagnosishaematological toxicity % grade 34non - haematological toxicity % grade 34refbop versus cop prospective phase iiiherold 2006164anaemia10 versus 13alopecia4 versus 48 8 fl, lplthrombocytopenia19 versus 34vomiting0 versus 1mclleucopenia4 versus 1neuropathy1 versus 0br versus chopr prospective phase iiirummel 2013 stil study549anaemia3 versus 5alopecia0 versus 100 9 fl, lplleucopenia37 versus 72neuropathy7 versus 29mzl, sll, mclthrombocytopenia5 versus 6infection37 versus 50br versus cvpr or chopr prospective phase iiiflinn 2012 bright study436anaemia5 versus 5alopecia4 versus 51 10 indolentleucopenia39 versus 87vomiting29 versus 13mclthrombocytopenia10 versus 12neuropathy14 versus 44infection55 versus 57ribvd prospective phase iigressin 201376anaemia2fatigue5 22 mclneutropenia21diarrhoea8elderlythrombocytopenia15neuropathy4 all grades haematological and non - haematological toxicity of bendamustine - based regimens more recently, an important phase iii study comparing bendamustine with rituximab (br) to chopr (cyclophosphamide, doxorubicine, vincristine, prednisone, rituximab) regimen was presented. following the treatment for 549 patients with indolent or mcl, orr was similar for br and chopr groups (93 vs. 91 %), complete remission (cr) was significantly increased in the br group (40 vs. 30 %, p = 0.021) and pfs was significantly longer in the br group (69.5 vs. 31.2 months, p 65 years) with previously untreated mcl grade ii following six cycles of the treatment, the orr was 97 %, cr was 79 % and os after 2 years amounted to 87 %. the results of clinical studies published thus far, assessing the use of bendamustine in upfront treatment for mcl, seem to indicate a risk - stratified approach, based on the mcl international prognostic index (mipi). bendamustine in combination with rituximab could be recommended as a first - line therapy in elderly mcl patients with high or intermediate mipi risk. the combination of rituximab and cyclophosphamide, doxorubicin, vincristine and prednisone is the standard first - line treatment regimen in patients with diffuse large b - cell lymphoma (dlbcl) ; however, the available data do not apply to elderly or frail patients. only a few smaller studies have investigated the role of bendamustine in this setting. a phase ii study demonstrated a cr rate of 54 % in elderly patients treated with br as first - line therapy with a good safety profile. results are acceptable and manageable for toxicity ; however, they seem to be generally unacceptable for pfs and overall survival [2527 ]. defining prognostic factors as gcb - subtype of dlbcl might predict a better outcome in bendamustine treated patients. performance and comorbidity assessment have a great impact on the outcome of frail patients with aggressive lymphoma. the clinical studies published so far have reported fairly low or mild toxicity of bendamustine - containing regimens. the adverse events (ae) observed in lymphoma patients treated with bendamustine are summarised in table 3. in general, bendamustine seems to present a favourable toxicity profile, and the most common complications involve haematological events such as anaemia, leucopenia, neutropenia or thrombocytopenia. despite being reversible, these toxicities are common, with as much as a 94 % occurrence rate for all grade events. the most frequently reported non - haematological toxicities related to bendamustine treatment include nausea, infections, fatigue, constipation, diarrhoea, headache and vomiting. a multicentre phase iii clinical trial comparing first - line br versus chopr revealed a much lower rate of serious ae in the br arm than in chopr treated patients (49 vs. 74). moreover, a granulocyte colony - stimulating factor (g - csf) was used only in 4 % of the patients receiving br and in 20 % of those receiving chopr treatment. similarly, non - haematological aes were of lower grade or incidence in the br group than in the chopr arm. a meta - analysis of randomised, controlled trials comparing the bendamustine - containing regimen to any other regimen demonstrated no effect of bendamustine on the rate of infection when compared to either alkylating agents or fludarabine in haematological, as well as in solid, malignancies despite, remarkably, lymphopenia. a few case studies were published reporting on the safety and effectiveness of the br regimen in dlbcl patients with severe liver impairment. low toxicity of bendamustine, even in these unfavourable settings, makes the drug safe and effective in special patient populations. the exact mechanism behind bendamustine s low toxicity remains not fully understood, and additional basic research aimed at its elucidation is required. it also seems advisable to assess long - term bendamustine toxicity and its potential interactions with other second - line treatments. increasing interest in bendamustine as an upfront therapy has prompted a cost - effectiveness analysis of this drug. when compared to chopr or cvpr regimens, the br was found to be cost - effective, and due to its more favourable toxicity profile it incurred lower costs related to ae management. the remarkably low toxicity profile and high efficacy of bendamustine provide a basis for the use of this drug in the treatment for a variety of lymphoma subsets. recent preclinical data and clinical studies have confirmed the activity of bendamustine in heavily pretreated patients with hodgkin lymphoma or peripheral t - cell lymphoma. the efficacy of bendamustine has been evaluated in treatment for multiple myeloma, with encouraging results [35, 36 ]. these impressive observations may lead to the design of prospective trials evaluating bendamustine in upfront therapy in selected patients in these settings. it was demonstrated that bendamustine provided clinical benefits when combined with other agents, i.e. monoclonal antibodies, purine analogues or more modern drugs such as lenalidomide, bortezomib, ibrutinib or idelalisib. there are a number of clinical trials, expected to be completed in 2014 and 2015, that are assessing the dose - limited toxicity and maximum tolerated dose of bendamustine in combination with those and other drugs. | recently, bendamustine has become an important agent in the treatment for patients with lymphoid malignancies. although the drug has received approval for second - line therapy in indolent lymphoma, a growing body of evidence suggests its efficacy and safety in first - line use. the results of randomised and observational studies with bendamustine as front - line therapy in non - hodgkin lymphoma (nhl) with emphasis on efficacy and toxicity are presented. furthermore, completed and ongoing clinical trials evaluating upfront bendamustine effectiveness in combination with other agents are discussed. the review refers mainly to indolent lymphoma, mantle cell lymphoma and aggressive lymphoma, as the most commonly diagnosed nhl types. finally, we elaborated on the safety profile of bendamustine and the perspectives of using the drug as a first - line therapy. |
tpx2 (targeting protein for the xenopus kinesin xklp2) is a protein essential for spindle assembly and chromosome interaction with microtubules. tpx2 level of expression is critical for spindle function, and altered expression is associated with aneuploidy and cancer. in agreement with a previous report, we show that tpx2 is undetectable in oocytes in prophase and accumulates during maturation to mii (fig. it has been proposed that the absence of tpx2 accumulation in prophase is due to protein degradation through apc / cdh1. indeed, little change in tpx2 mrna translation occurs during the early phases of oocyte maturation, but the late tpx2 accumulation is associated with an increased translation. surprisingly, we found that tpx2 protein accumulation is not only dependent on the stage of the meiotic cell cycle. significant differences in tpx2 protein levels were observed when comparing mii oocytes matured in vivo with those matured in vitro in association with somatic cells, or those matured in vitro after being denuded. this initial finding suggests that tpx2 accumulation is sensitive to the environment in which the oocyte matures. to investigate whether cumulus cells, the somatic cells surrounding the oocyte, play a role in the translation of maternal mrnas and protein synthesis, we developed an in vitro model that preserves the somatic environment in which the oocyte matures. translational reporters were constructed and injected into oocytes still surrounded by cumulus cells (cumulus cell - enclosed oocyte, ceo) (fig. this model enables monitoring translation of selected maternal mrna in oocytes that maintain contact with cumulus cells. furthermore, translation rates in ceos can be compared to those measured in denuded oocytes (dos), which are no longer exposed to somatic signals. reporter constructs with luciferase orfs under the control of 3utrs of tpx2 or dazl, an rna binding protein essential for gametogenesis, were injected into ceos. translation rates of these reporters increased as the oocytes progressed from gv to mii (fig. 2c), consistent with our report of recruitment of the corresponding endogenous transcripts to the polysomes. however, translation in ceo is further increased by supplementing the incubation medium with amphiregulin (areg), an egf - like growth factor that accumulates physiologically in the follicle during ovulation, or egf itself (fig. both ligands signal through egf receptor (egfr) on cumulus cells and are not expressed by oocytes in culture. growth factor - induced effects were not detected when meiotic reentry was prevented with the phosphodiesterase inhibitor milrinone (suppl. fig 1), when a tpx2 reporter with truncated 3utr was injected (suppl. these findings demonstrate that the 3 utr, progression through meiosis, and somatic cells are all required for the growth factor - dependent stimulation. exposure to areg did not increase the stability of the reporter, confirming an effect on translation rate (suppl. consistent with reporter translation, endogenous tpx2 and dazl protein levels increased as oocytes mature (fig. areg further enhanced these protein levels, confirming that increased translation of the reporter reflects translation of the endogenous mrna and accumulation of the encoded protein. when incubated with areg and in contact with somatic cells and areg, a rise in oocyte protein synthesis was independently confirmed by monitoring accumulation of il-7, an oocyte secreted chemokine, in the medium (fig. to conclusively demonstrate that areg does not directly stimulate the oocyte and that the signals are indirect and mediated by somatic cells, we used a genetic mouse model in which egfr is down - regulated only in somatic cells. mice carrying a null egfr allele and a floxed allele (egfr), and expressing a cre recombinase under the control of the granulosa cell - specific cyp19a1 promoter, display a 90% decrease in egfr expression in granulosa cells. although lh - dependent in vivo oocyte maturation is impaired, in vitro spontaneous maturation progresses normally. we used ceo derived from these mice to test whether egfr gene inactivation in somatic cells ablates the areg effects on oocyte translation in vitro. the experiment reported in fig. 2 g demonstrates that areg does not significantly increase translation rate of tpx2 reporter when ceo from egfr : cyp19a1-cre mice are microinjected. to determine whether this somatic effect on oocyte translation occurs also in vivo and whether global translation is affected, genetic mouse models perturbing the egf network were investigated. we have reported that the egf - like growth factors areg, ereg and btc are induced by lh at the time of ovulation and that transactivation of egfr is indispensable for oocyte maturation and ovulation. whereas inactivation of egfr causes a block in oocyte maturation in vivo and defective ovulation, areg mice ovulate and are fertile but litter size 3a). when fertilization rates were assessed in vitro, ceos or denuded oocytes derived from areg mice fertilize at a rate significantly lower than wild type (wt) littermates, suggesting a defect in developmental competence (fig. 3b and c). identical to the areg follicle activated in vivo, isolated ceos from wild type mice cultured in vitro are not exposed to areg. therefore, the role of areg in promoting cytoplasmic maturation was further tested by adding exogenous areg during wt ceo in vitro maturation. this treatment improved their fertilization rate (fig.3d), a finding consistent with other studies demonstrating positive effects of egf network on developmental competence. defective spindle morphology was more frequently detected in mii oocytes derived from areg mice (fig. thus, inactivation of areg in the somatic cells yields oocytes that mature but with signs of reduced developmental competence. our model was then used to test whether the translational program executed during oocyte maturation is affected by the absence of this somatic signal. to capture actively translating mrnas, polysomes were isolated by sucrose density gradient from wt and areg oocytes. maternal transcripts associated with the polysomes were isolated and analyzed by microarray hybridization (fig. consistent with our previous report, 3208 transcripts were recruited or released from the polysomes during oocyte maturation in wild type oocytes (suppl. a similar number of transcripts (3440) moved in and out of the polysome pool also in oocytes from areg mice, confirming that the translation program is qualitatively intact. however, quantitative analysis revealed significant changes in the level of transcripts associated with polysomes in mutant oocytes. when using a cutoff of p<0.05 (fig. 4a), polysome association of approximately 200 - 300 transcripts was altered in the areg oocytes compared to wt (fig. transcripts normally recruited to the polysome during maturation were decreased in the areg oocytes (fig.4c). the affected transcripts included the main functional categories of metabolism, embryonic development, cell cycle and rna regulators (fig. analysis of the 3utrs affected in the areg-/- oocytes did not reveal the presence of a common signature, with the possible exception of two consensus elements enriched in a subset of transcripts (suppl. we confirmed the decrease of selected transcripts in the polysome pool by qpcr (fig. protein levels of dazl and tpx2 in mii oocytes of the areg mice were decreased (fig. 4f, g), which is consistent with the decreased recruitment of these mrnas to the polysome in vivo. polysome recruitment of cyclin b1 and tex19.1, two transcripts known to be highly regulated during oocyte maturation, were not affected in the areg oocyte and were insensitive to areg stimulation in the in vitro translation assay (fig.4h, i, j, k). thus, somatic cell signals affect translation of a subset of maternal mrnas in vivo and in vitro. incubation of ceo with areg caused a rapid increase in akt phosphorylation indicative of activation of the pi3k pathway (fig. surprisingly, we found that the time course of ceo activation is biphasic with a secondary increase in akt phosphorylation detected at 120 - 180 min of incubation with areg (fig. 5a, b). because the delayed increase in akt phosphorylation was reminiscent of the akt phosphorylation detected in the oocyte 2 - 3 hrs after lh / hcg stimulation, we investigated whether this secondary increase takes place in the oocyte. ceos were incubated in the presence of areg ; at the end of the incubation, the oocytes were denuded and used for western blot analysis (fig. a delayed akt phosphorylation was observed in oocytes exposed to areg while still in complex with cumulus cells ; conversely, this delayed phosphorylation could not be detected in denuded oocytes exposed to areg (fig. variable akt phosphorylation was observed in oocytes within the first 30 min whether cultured as ceo or denuded (fig. 5d), a signal independent of areg and likely caused by the mechanical denudation of the oocytes. these data suggest that areg stimulation of ceos causes a delayed transient increase in akt phosphorylation in the oocyte. pharmacological inhibition of pi3k with wortmannin or ly 294002 blocked the akt phosphorylation in oocytes when cultured in complex with cumulus cells in the presence of areg for 2 hrs (fig. the areg - dependent increased translation of the tpx2 reporter was abolished, whereas the cell cycle - dependent increase in reporter translation was not affected (fig. it should be noted that, in the experiments reported above, the pharmacological inhibitors used block the pi3k / akt / mtor pathway both in somatic cells and in the oocyte. to determine whether the pi3k pathway function is required in the oocyte, we used a genetic model to test whether disruption of this pathway exclusively in the oocyte is sufficient to block the areg - dependent increase in translation. to this aim, we used cumulus / oocyte complexes from pten : zp3-cre mice. in this genetic model, the cre recombinase is under the control of an oocyte - specific promoter and the pten gene is ablated only in oocytes but not in cumulus cells. previous data with this model show that pten inactivation causes a constitutive increase in akt phosphorylation. in these oocytes, this latter finding confirms that an intact pi3k pathway in the oocyte is necessary to translate the areg signal from the soma. our findings demonstrate that translation of a subset of oocyte maternal mrnas is under the control of somatic cell inputs acting through the pi3k / akt / mtor pathway. this regulation functions in concert with the translational control by meiotic cell cycle regulators and is involved in establishing the oocyte competence to successfully develop as embryos. our in vivo and in vitro data document that exposure of somatic cell / oocyte complexes to the egf - like growth factor areg causes an increase translation of a subset of maternal mrnas in the oocyte. this activation requires cell - to - cell communication, as it is lost in denuded oocytes. the use of alleles affecting egfr and pi3k signaling in the somatic and germ cell compartments respectively confirms that areg action requires egfr expression in the soma and an intact pi3k signaling in the oocyte. thus, egfr signaling in the somatic cellular compartment is translated into pi3k activation in the contiguous cell compartment, the oocyte. given the critical role of egfr signaling in tumor growth, it is possible that the signaling across cells described here is also necessary for communication of transformed cell with the surrounding cellular environment. growth factor regulation of translation via the pi3k / akt / mtor pathway is established for somatic cells. however, the molecular mechanism underlying the specific regulation of a subset of transcripts in growth factor - target cells remains controversial. through target of rapamycin complex 1 (mtorc) the pi3k pathway converges on regulation of s6 ribosomal protein and eukaryotic initiation factor 4e (eif4e), a key rate - limiting initiation factor for cap - dependent translation. 4e - binding protein 1 (4ebp1), one of the three 4ebp binding proteins expressed in mammals, is phosphorylated by mtorc1 causing the dissociation from eif4e and the formation of an active initiation complex. transcripts sensitive to mtor regulation have signature sequences at the 5 utr, including a polypyrimide (5top) or prte sequence, but it is not clear whether these sequences are indispensable. 5top or prte sequences are not readily discernible in the 5 utr of the prototype transcripts we have investigated in the oocyte. a selective activation is retained when different luciferase reporters are injected in oocytes, even though they have an identical 5 utr sequence. conversely, the 3 utr of these mrnas is essential because tpx2 3utr reporter recapitulates this regulation whereas the 3utr of, for instance, cyclin b does not support this regulation. elements present in the 3 utr are necessary because a truncated tpx2 3utr is not sensitive to somatic cell signals. an additional feature that distinguishes the effects we describe in the oocyte from those in somatic cells is that none of the oocyte targets of regulation identified are ribosomal proteins, whereas many of the growth factor targets in somatic cells are ribosomal proteins. on the contrary, the regulation we describe takes place on a background of widespread destabilization and decreased translation of mrnas coding for ribosomal proteins. the pharmacological and genetic analyses performed indicate that the akt / mtor pathway is required for the areg - depended translational regulation in the oocyte. we propose that mtor signaling in oocytes controls translation via unique mechanisms that target the 3utr (fig. 6). the possible role of the 5utr in this growth factor - dependent regulation remains to be determined. correct mrna translation and accumulation of corresponding proteins in the oocyte is critical for efficient meiotic cell cycle progression, fertilization, and embryo development and necessary to attain full developmental potential. our findings provide a molecular explanation for the widely reported observation that fully grown oocytes dissociated from the surrounding somatic cells may complete nuclear maturation but are defective in fertilization and embryo development. our findings support the idea that components required for maturation, fertilization and embryo development reach optimal levels when oocyte - somatic cell communication is preserved. in the absence of somatic cells, these components accumulate at levels sufficient for cell cycle progression but robustness and stability of the oocyte machinery is compromised, with meiosis becoming prone to errors. this conclusion is supported by our observation that tpx2, a key component in spindle assembly, accumulates significantly less in the absence of somatic input, and spindle defects become more frequent in mii oocytes. in the same vein, decreased accumulation of dazl, which may function upstream of tpx2, likely destabilizes the network of rna binding protein required for maturation and embryo development. thus, correct execution of the program of maternal mrna translation and somatic inputs are essential for oocyte developmental competence. as an extension of this concept, biomarkers monitoring this program, such as secreted proteins, should be of prognostic value for the fitness of an oocyte to develop into an embryo that successfully implants and sustains pregnancy to term. | germ cells divide and differentiate in a unique local microenvironment under the control of somatic cells. signals released in this niche instruct oocyte reentry into the meiotic cell cycle. once initiated, the progression through meiosis and the associated program of maternal mrna translation are thought to be cell - autonomous. here we show that translation of a subset of maternal mrnas critical for embryo development is under the control of somatic cell inputs. translation of specific maternal transcripts increases in oocytes cultured in association with somatic cells and is sensitive to egf - like growth factors that act only on the somatic compartment. in mice deficient in amphiregulin, decreased fecundity and oocyte developmental competence is associated with defective translation of a subset of maternal mrnas. these somatic cell signals that affect translation require activation of the pi3k / akt / mtor pathway. thus, mrna translation depends on somatic cell cues that are essential to reprogram the oocyte for embryo development. |
tethered cord syndrome (tcs) is a well - known clinical entity and is characterized by motor and sensorial changes in the legs, back pain, foot deformities, urinary dysfunction and spinal deformity. scoliosis can be a unique and leading sign of tcs.1 in the classic description of tcs by hoffman.,2 scoliosis was a prominent manifestation in 10 of 31 patients. recent reports have suggested that scoliosis is more frequent than thought in tcs and additionally mclone. demonstrated that scoliosis may be the first sign of tcs.1234 pathological entities of the neural tube like meningoceles, lipomyelomeningoceles, myelomeningoceles, split cord malformation, dermal sinus tract, thickened filum and others may be the cause of tethering.5 yamada. showed that longitudinal stretching of the spinal cord interferes with the mitochondrial oxidative metabolism and can result in motor and sensory changes in the lower limbs, incontinence and musculoskeletal deformities including neuromuscular scoliosis.6 in the simplest form, the spinal cord can be tethered by a thickened filum (> 2 mm) with a low conus medullaris (at or below l2l3 level) without other forms of spinal dysraphism. however, the spinal cord can even be tethered by a filum terminale with normal appearance and a normal level conus medullaris.789 it is known that the majority of patients with scoliosis (8090%) are idiopathic because an underlying clinical or radiological cause has not been determined. according to the scoliosis research society, the prevalence of adolescent idiopathic scoliosis is 2% to 3% in the general population.10 tethered cord may be the most important and relatively easily treatable primary disease for associated scoliosis. releasing the tethered spinal cord in these patients may stabilize or even reverse the progression of scoliosis. in addition, untethering aims to improve neuromuscular function even in patients that need surgical correction of scoliosis.11121314 the aim of this study is to evaluate scoliosis due to tcs with and without tethering, findings on magnetic resonance imaging (mri) and also to draw attention to the importance of somatosensorial evoked potentials (ssep) on the differential diagnosis between idiopathic scoliosis and tcs due to filum terminale with normal appearance and normal level conus medullaris. in addition, we also focused to investigate the progression of scoliosis and need for corrective spinal surgery in patients who underwent release of tethering. eleven female and seven male patients with a mean age of 14.72 years (range 342 years) were included in the study [table 1 ]. full spine standing x - rays were obtained for calculating the cobb 's angle on coronal curves. clinical details of patients mri was performed in all patients for the diagnosis of tcs and for probable additional spinal abnormalities. normal conus medullaris level was accepted as between the lumbar 1 and 2 disc spaces and the normal thickness of filum as taken as 10% increase in latency was considered as pathological. all ssep recordings were done at lumbal, thoracal, cervical levels and the somatosensorial cortex simultaneously. urodynamic studies were also performed in all patients to investigate detrussor and sphincter activity evaluations. all cases underwent a standard surgical procedure of a single level lumbar hemilaminectomy at the fifth lumbar vertebrae on the left. after opening the dura mater, we used a nerve stimulator to differentiate the filum terminale from other neural tissue. following the untethering procedure a part of the filum some patients mentioned weak sensorial symptoms such as numbness in the perineum or non radicular pain in the legs. scoliosis was defined as a coronal deformity of up to 10. mean value of cobb angle in the study was 31.6 (range 1845). eleven patients (61.1%) had thoracolumbar scoliosis and 7 (38.9%) had thoracic scoliosis. one patient had a right curved scoliosis and 17 (94.45%) patients had left. besides scoliosis, 10 of 18 patients had thick and/or fatty filum terminale, low level of conus medullaris and developmental abnormalities such as block vertebrae. other eight scoliotic patients (44.45%) had a normal appearance of filum terminale and normal level conus medullaris without different forms of spinal dysraphism on mri. we clinically suspected the tethered cord in scoliotic patients, if there was a late start of the scoliotic curve, in particularly during adolescence. we did not suspect tcs when the spinal deformity progresses in spite of appropriate treatment. on the contrary, progress of spinal deformity after appropriate treatment means untethering did not help to improve or stop the bending. it is for this reason we recommend 6 months of followup period to see the results of untethering. should there be a progress of the curve than we refer the patient to orthopedics for surgical intervention. we found pathological ssep results in 12 of 16 patients (75%) of which there was total block in nine (75%), conduction delay in three (25%). ssep was significant in all cases with normal filum and conus medullaris seen on the mri [figure 1 ]. somatosensorial evoked potentials results, bilaterally lumbar - thoracal total block in urodynamical studies, only four patients had increased detrussor activity with sphincteric dyssynergia defined as neurogenic bladder abnormality. in one patient, there was sensorial delay, both increased bladder capacity and compliance. there were no pathological bladder function findings in the other 13 patients (72.22%). in the histopathological examination of the fila in all patients (including those with normal appearance of the fila on mri), we observed dense collagen fibers and hyaline degeneration in masson 's trichrome staining figures 2 and 3, furthermore there were no elastic fibers seen in the verhoeff 's van gieson staining in these fila [figure 4 ]. histological view of a filum terminale in a scoliosis patient with normal appearance. it is clear that microscopical cut view is not normal with dense collagen tissue and hyaline degeneration. 10 (col : collagen, h : hyaline [masson 's trichrome ]) (a) loose and rich collagen tissue in normal filum terminale. (b) dense collagen (light blue) tissue and large vessel in the middle of a filum of a scoliosis patient. (c) dense collagen and large capillary (c) in the middle of filum terminale of a patient with incontinence (histological sections stained with masson 's tricrome and the pictures a and c are taken from lit. 17) histological view of filum terminale staining with verhoeff 10, seen loose of elastic fibers (black) and dense collagen tissue (red) the cases were followed up postoperatively for a mean of 6 years (range 3204 months). scoliosis is defined as a lateral curvature of the spine on the coronal plane that is cobb 's angle greater than 10. this deformity may include lateral inter - vertebral tilting and rotation of the vertebral bodies across the apex toward the convexity of the curve in the axial plane.1516 neuromuscular scoliosis is a deformity difficult to treat. development of scoliosis may be body 's attempt to minimize the abnormal tension placed on the spinal cord because of the tethering effect of the tight filum. scoliosis and kyphoscoliosis have limited mobility of the spinal column and flexion increases spinal cord traction. the concave side of the spinal canal is shorter than the convex side and rotation is always to the concave side which shortens the distance travelled by the spinal cord. in our series, 10 of 18 patients were in the adolescent group, four patients were under 10 years old and the others were adult. more than half the patients in our series had idiopathic scoliosis with 5 of these 8 adolescent having a normal appearance of the filum terminale and conus medullaris. it 's not always difficult to diagnose tcs if there is an apparent developmental anomaly such as a split cord malformation, dermal sinus tract, neural tube closure defect and the other skin stigmata. in imaging studies too thick, fatty, short filum terminale and low - lying conus medullaris are quite easy to recognize tethered spinal cord for the diagnosis.6915171819202122 problem arises in the patients who have scoliosis without any other pathology or finding to explain the reason of this deformity. after having untethered, the patient is safer for scoliotic curve correction procedure. since 1979, the concept of tethered cord and its effects almost totally changed. it is 2003, when seluki.,9 showed that filum terminale with normal appearance is not normal, for the first time in the literature. these papers have taken their places in the literature and can easily be found elsewhere. protean manifestations of tethered spinal cord by hoffman.,2 was in 1976, and manifestations were pain, incontinence and scoliosis. the prevalence of idiopathic scoliosis has been reported to be 0.912% in the general population and minimum 10% of patients require treatment. weis and moramarco reported that the majority of the patients with scoliosis (8090%) are idiopathic because an underlying cause can not been determined.10 many scoliosis patients who may have spinal cord tethering without mri findings may be included in the idiopathic scoliosis group. conservative treatments like bracing and many others are designed to prevent curve progression during the growing years and is not indicated in patients who are already skeletally mature.23 surgical treatment is generally indicated if the curve reaches 50 at completion of growth. curves < 50 in skeletally immature patients and/or progressive double curves are also indications for corrective surgery.16 the goals of surgery are to correct the deformity producing a balanced spine with a leveled pelvis and a solid spinal fusion to prevent or delay secondary respiratory complications.101213 these corrective spinal surgical interventions are usually difficult, expensive and may carry increased risk of morbidity. limited instrumentation options, persistent anterior spinal growth after posterior fusion and the potential for thoracic insufficiency when a kind of fusion is performed in children or young patients, this may require repetitive spinal surgery. on the other hand, a corrective orthopedic procedure may cause irreversible neurological deficits unless untethering of the spinal cord has been performed previously.1924 since the concept of tethered spinal cord was first proposed, the finding of low - lying conus medullaris has been widely accepted as evidence of cord tethering. hoffman 2 clearly defined radiological criteria on tcs, but seluki.9 showed that normal appearance of the filum terminale does not mean that it really is totally normal. they reported that an abnormal filum terminale with a normal appearance has dense collagen fibers, wide and numerous capillaries and hyaline formation. the high amount of dense collagen fibers and hyalin cause the filum terminale to lose its elastic properties, ultimately leading to conduction of a tethering effect to the conus medullaris [figures 24 ]. somatosensory evoked potentials are not routinely used in spinal dysraphism or idiopathic scoliosis. the sensory stimulus has often been used in clinical evoked potential studies as a way of measuring the spinal cord function.25 sseps are performed by stimulating a peripheral nerve at the wrist or the ankle and recording the responses from the popliteal fossa, cervical spine / brainstem and somatosensory cortex. 8 of 18 patients were with normal appearance of filum terminale and conus medullaris seen in the mri [figure 1 ]. when normal appearance of filum terminale and normal level of conus medullaris are confusing findings, in case of delayed scoliosis then we performed ssep to consider the surgical intervention. we have found that in 12 patients sseps were pathological both in conduction time and amplitude of the wave recorded. while it is not uncommon to see pathological lumbal or thoracic conduction blocks or delays although the ssep results that recorded from cental cerebral lead shows normal waves that can easily be considered as normal, all ssep recordings were done at lumbal, thoracic, cervical levels and at somatosensorial cortex simultaneously. however, contrary to what we expected, ssep results did not match with the urodynamic studies in tethered cord patients with urinary incontinence. in our previous studies we felt that the hyperreflexive, hypertonic bladder is the main indicator of the tethering of the conus medullaris, which is still very important in decision making to untether the spinal cord in patients with urinary incontinence.21 it is interesting that in the majority of scoliosis patients who recorded ssep 's as pathological, the urodynamic studies revealed normal values. most of the patients in the study had normal urodynamic evaluation, and significant part of them had normal mri investigation as far as level of conus medullaris and thickness of filum terminale were concerned. while our impression about the hyperreflexive and hypertonic bladder has not changed so far vis - a - vis patients with bladder and sphincter dysfunction but after seeing these fifteen scoliosis patients with normal urodynamic study scores, we decided that ssep might be a better indicator of tethering of spinal cord especially in normal level conus medullaris and normal thickness filum terminale in scoliosis. after evaluating the results in our series, we noticed that tethered cord induced late onset scoliosis always in the thoracic area with a compensatory curve at the lumbar level. hence, we came to a conclusion whether this left side directed thoracic late onset curve could be a sign of cord tethering. in order to answer this question, we believe accumulation of more data is needed indeed. the tethered cord is vertically tethered spinal cord that is caused by tethering of filum terminale in majority of patients. interestingly, despite the level of the scoliotic curve, untethering of distal spinal cord helps in most of the cases., that untethering, in diagnosed tethered spinal cord cases, also helps in chiari malformation of type i. there is, unfortunately, no clear cut explanation for this. histopathological analysis of the filum terminale with normal appearance revealed either dense collagen tissue or hyaline degeneration. all of the fila with macroscopically normal tissue showed either dense collagen tissue or some hyaline degeneration and markedly wide vessels [figures 2 and 3 ]. these properties of histopathologically abnormal fila terminalia have already been explained in by seluki.914 elastic tissue fibers were seen when stained with verhoff 's van gieson. we did not obtain positive verhoeff elastic fibers staining [figure 4 ] in both normal appearing of fila and others. this histological result may explain stretching of normal level conus medullaris also with normal appearance of filum terminale on mri. in 1982, nachemson. calculated the probability of curve progression before skeletal maturity based on known prognostic factors available at the time 24 [table 2 ].. however, with larger magnitude curvatures, there may be a considerable risk of progression despite maturity.26 probability of progression : magnitude of curve versus age scoliosis surgery is performed by both orthopedic surgeons and neurosurgeons. since tethered cord syndrome is a neurosurgical issue, the orthopedic surgeons must keep in mind this important problem. this is why we wanted to draw attention to this very important point as far as we could. we believe that repetitive impulses (stimulations) on this important issue would be beneficial for true understanding of the intriguing problem. scoliosis may be the first sign of tethered spinal cord. at late onset cases of scoliosis must be assessed for the existence of tcs. in the presence of normal urodynamic studies and mri it has been shown that filum terminale with normal appearance is not histologically normal. in diagnosed tethered cord cases who presented themselves with scoliosis, untethering of the spinal cord after untethering, a followup period of 6 months allows the physician to evaluate the effects of untethering towards the evolution of scoliosis. in spite of improvement or cessation of progression of scoliosis | background : tethered cord syndrome (tcs) is a progressive clinical entity that arises from abnormal spinal cord tension. scoliosis may be a unique symptom in tcs. the aim of this study is to investigate prognosis after releasing the filum terminale in scoliosis due to tcs with / without findings in magnetic resonance imaging (mri) and to draw attention to the importance of somatosensorial evoked potentials (ssep) on the differential diagnosis of idiopathic scoliosis versus scoliosis due to tcs with normal appearance of filum terminale and conus medullaris.materials and methods : eleven female and seven male patients with progressive scoliosis were included in the study. they were evaluated radiologically, ssep and urodynamical studies. preoperative and postoperative anteroposterior full spine x - rays were obtained for measuring the cobb 's angle. mri was performed in all cases for probable additional spinal abnormalities. all patients underwent filum terminale sectioning through a l5 hemilaminectomy. the resected filum terminale were subjected to histopathological examination.results:the mean cobb angle was 31.6 (range 1845). eight patients (44.45%) had a normal appearance of filum terminale and normal level conus medullaris in mri, but conduction delay and/or block was seen on ssep. in the histopathological examination of filum terminale dense collagen fibers, hyaline degeneration and loss of elastic fibers were observed. postoperatively none of the patients showed worsening of the cobb angle. three patients showed improvement of scoliosis.conclusion:in tcs presented with scoliosis, untethering must be performed prior to the corrective spinal surgery. absence of mri findings does not definitely exclude tcs. ssep is an important additional guidance in the diagnosis of tcs. after untethering, a followup period of 6 months is essential to show it untethering helps in stopping the progress of the scoliotic curve. in spite of non progression (curve stopped lesser than 45) or even improvement of scoliosis, there may be no need for major orthopedic surgical intervention. |
medical archives is the oldest medical journal in bosnia and herzegovina (b&h) (founded in 1947.). a total of 104 articles were published in medical archives during 2015. analyzing the type of articles, original articles are present in majority during 201580.7% (in last seven years, 561 (76%) were original out of 738). in last seven years, 651 (88.2%) articles were from the field of clinical medicine (preclinical disciplines, in the last three years are more represented than in previous years). articles written in collaboration of five authors (21.1 %) are found to be predominant. from year to year, most often required time for a decision on acceptance or on the revision prior acceptance is between 50 and 60 days (30% of cases in 2015). during 2015, 47.1% of articles were originally from b&h (eleven countries were represented). h index of medical archive for 2014 was 12, and does not vary during the last decade. in 2015 in b&h about twenty - five journals are issued in the field of biomedical and life sciences in general (six are indexed on medline / pubmed, one is indexed in the science citation index expanded (scie)/web of science base). according to googlescholar the biggest h5 index has bosnian journal of basic medical sciences (bjbms) and medical archives, while the biggest h5 median has bjbms i acta informatica medica. the highest h - index (13) in b&h has izet masic md, phd, enver zerem md, phd and semir vranic md, phd, while highest g - index (22) has enver zerem md, phd (analyzed by software package publish or perish). by comparing the state of medical publishing in b&h with neighboring countries (croatia, serbia, montenegro), we have concluded that b&h is behind croatia and serbia by following parameters : total documents, total cites and h index but in front of montenegro. the oldest medical journal in bosnia and herzegovina (b&h) was jahrbuch des bosnisch - herzegowinischen landesspitales in sarajevo (annual of the national hospital in sarajevo) (figure 1) which was established in 1897 (published in german language) (1). jahrbuch des bosnisch - herzegowinischen landesspitales in sarajevo medical news journal (medicinski vjesnik) in croatia (1877), health news (zdravstveni vestnik) in slovenia (1927), medical archives (medicinski arhiv) in bosnia and herzegovina (1947) and domestic physician (domaci lekar) in serbia (1877), represent a precursor of today s journal in the former yugoslavia (1). in 2015 in b&h about twenty - five journals are issued in the field of biomedical and life sciences in general. systematization is made, 17 of the most relevant journals are selected, and their indexation in scientific databases is presented (table 1). unfortunately, the rest of them are not in the table bellow (lot of them that are not included into account are review journals), due to inefficiency on their web sites, or due to absence of information of the index status on their website. timeliness on their own web sites gives a great view of the seriousness of a journal in contemporary world. representation of the journals in bosnia and herzegovina in the field of medicine in the scientific bases journals that are indexed in medline / pubmed are medical archives (established in 1947), materia socio - medica (established in 1978), acta informatica medica (established in 1993), acta medica academica (established in 2008), bosnian journal of basic medical sciences (bjbms) (established in 2004) and medicinski glasnik (established in 2007). the first three mentioned represent editions of academy of medical sciences of bosnia and herzegovina. acta medica academica is edition of the academy of sciences and arts of bosnia and herzegovina. medicinski glasnik is the official publication of the medical association of zenica - doboj canton (federation of bosnia and herzegovina). bjbms is edition of association of basic medical sciences of the federation of bosnia and herzegovina and is the only one of the above mentioned journals, which is indexed in the science citation index expanded (scie)/web of science base. the journal of health sciences is an open access, periodical journal, with the purpose to publish articles relevant to field of laboratory diagnostics, physical therapy, healthcare and nursing, radiologic technology, health and ecology and related fields, and in this moment has pending status in medline / pubmed base. in last ten years journal is published bi - monthly. till now in medical archives over 5000 articles were published and most of them were original articles from all medical disciplines (1). medical archive is a journal, in which articles in the field of clinical medicine are usually present. considering the number of articles, we can say that it is the most important journal in the field of clinical medicine, because majority of original articles in these areas are published there, compared with all other journals in b&h, which are contained in the medline / pubmed database. journal medical archives, in last ten years is published bi - monthly. medical archives is now indexed in the following databases : pubmed / medline (from 1972, abstracted in this bases more than 5.500 journals) (1), pubmed central (from 2013), and from the year 2009 in databases : excerpta medica / embase, scopus, scirus, ebsco, doaj, index copernicus, ulrich s periodicals directory, geneva foundation for medical education and research - gfmer, hinari, cab abstracts, global health, proquest, newjour, scimago journal and country rank, isc master journals list, crossref, google scholar, genamics journalseek, worldcat, viniti of ras, research gate, catalyst, scopemed, safetylit, bioinfobank library, pubget, getcited, cirrie, kubon and sagner opac. a total of 104 articles were published in medical archives during 2015 (figure 2). although the number of articles was in decrease in the period 2007 - 2010 this number in last three years has stabilized (figure 2). total number of articles during period 2002.-2015. analyzing the type of articles, original articles are present in majority during 2015 (80.7%) (figure 3). by analyzing last seven years, 561 (76%) were original out of 738 (figure 3). type of articles during period 2009 - 2015 during 2015, 90% of publications were related to the field of clinical medicine (27.6% surgery, 25,6% internal medicine, 10.7% dermatovenerology, gynecology 7.4%, 7.4% pediatrics, 6.4% ophthalmology, 5.3% physical therapy, 4.2% ent, 3.2% neuropsychiatry, 1.1% radiology and immunology). analyzing last seven years, 651 (88.2%) preclinical disciplines, in the last three years are more represented than in previous years (in 2015. 10 papers were from preclinical fields, and of these 80% from the field of biochemistry and 20% from the field of microbiology) (figure 5). distribution of articles by fields during period 2009.-2015. preclinical disciplines in medical archives during period 2009 - 2015 (the highest number of articles was in 2014.) collaboration rate (ratio of number of multi - authored articles with the total number of articles) in 2015 was 0,92. articles written in collaboration of five authors (21.1 %) are found to be predominant followed by collaboration of four authors (19,2 %) (figure 6). collaboration of authors during 2015 from year to year, most often required time for a decision on acceptance or on the revision prior acceptance is between 50 and 60 days (30% of cases in 2015) (figure 7). time needed for the decision on acceptance of article in year 2015 during 2015, 47.1% of articles were originally from b&h (eleven countries were represented b&h, iran, kosovo, saudi arabia, albania, turkey, r. macedonia, serbia, jordan, greece, croatia, which is still slightly lower number compared to previous years). growing number of regional collaboration was noticed (figure 8). map of countries from which the articles came from in year 2015 according to scimagojr.com, scimago journal rank (sjr) (average number of weighted citations received in the selected year by the documents published in the selected journal in the three previous years) for 2014 was 0,168 and is increasing (figure 9). sciimago journal rank indicator of medical archives during period 2005 - 2014 analyzing on same portal parameter cites / doc. (2 years) (widely used as impact index), for medical archive was 0.45, and a slight decrease compared to 2013 was observed which was 0,49 in that year (figure 10). (2years) (widely used as impact index) parameter for medical archives during period 2009 - 2014 the sjr indicator measures the scientific influence of the average article in a journal, it expresses how central to the global scientific discussion an average article of the journal is (2). cites per doc. (2y) measures the scientific impact of an average article published in the journal, it is computed using the same formula that journal impact factor (thomson reuters) (2) (figure 11). sjr indicator vs. cites per doc (2 years) for medical archives (2) not every article in a journal is considered primary research and therefore citable figure 12 shows the ratio of a journal s articles including substantial research (research articles, conference papers and reviews) in three year windows (2). ratio of citable and non - citable documents (2) h index (the index that attempts to measure the productivity and impact of published work of scientists) of medical archive for 2014 was 12, and does not vary during the last decade (h index of the medical archive is the highest among journals in bosnia and herzegovina) (figure 13). h index for medical archives during period 2005 - 2014 google corporation in 2005 launched google scholar online service that allows a search for scholarly literature. according to googlescholar, h5 index (h - index for articles published in the last 5 complete years) for medical archives is 14 and h5 median (median number of citations for the articles that make up its h5-index) is 19. the most cited articles by googlescholar are shown in figure 10 (should be interpreted with caution - pubmed shows that articles have higher number of citation) (figure 14). scientometrics is part of scientology (the science of science) that analyzes scientific papers and their citation in the scientific journal selected sample (3). scientometrics is the science of measuring and analyzing science using qualitative, quantitative and computational approaches (4). scientometrics with its various indices is a reliable method for evaluation of scientific development (5). name bibliometrics in the seventies was introduced to denote a quantitative study of the communication process using mathematical and statistical methods to books and other media of communication (6). almost simultaneously, in the countries of the former eastern bloc was introduced scientometrics name derived from the russian language (6). more specifically, in 1969 was introduced the name scientometrics relating to scientific field that deals with the study of science as an information process by applying quantitative (statistical) method, and later tibor braun (who in 1977 established international journal scientometrics), introduces the name scientometrics (7, 8, 9). modern scientometrics is based largely on the work of derek j. de solla price and eugene garfield (garfield founded isi - institute for scientific information is considered to be the father of scientometrics and methods of evaluation of scientific publications) (6). garfield has been striving to mathematical representation developed several factors that allow the assessment value and importance of scientific publications, including the most important impact factor (if) and the h - index (6). if is the number of citations of articles published in the journal during the previous two years divided by the total number of articles published in the journal during the same period (6). factor of influence depends on : the quality of the journal, the language on which it was printed, the area it covers, the magazine distribution system (6). if in the academic journal is a measure that reflects the average number of citations of articles published in the journal (in a given year, if of the journal is the average number of citations received per paper published in that journal during the previous two years) (6). in august 2005, jorge hirsch introduced a new indicator for quantifying the research output of scientists (10, 11). hirsch s so - called h index was proposed as an alternative to other bibliometric indicators - such as the number of publications, the average number of citations and the sum of all citations (12) - and is defined as follows : a scientist has index h if h of his or her np papers have at least h citations each and the other (np - h) papers have h citations each (11). an h - index of 5 means that a scientist has published five papers that each have at least five citations (14). an h - index of 0 does not inevitably indicate that a scientist has been completely inactive : he or she might have already published a number of papers, but if none of the papers was cited at least once, the h - index is 0 (14). h - index is an index that attempts to measure the productivity and impact of published work of scientists (the index is based on the basis of the most cited papers and the number of citations that papers received in other publications) (15). the h - index is given by the largest number h of papers of a researcher which have received at least h citations. one criticism is that the excess citations, i.e., all citations which exceed h for a given publication do not have an effect. therefore egghe (16) proposed the g - index which is given by the largest number g of papers which have received at least g citations on average (it was argued recently that the g - index is a measure of a researcher s specific impact (i.e., impact per paper) as much as it is a measure of overall impact) (17). the main difference between the g - index and the h - index is that the former penalizes consistency of impact whereas the latter rewards such consistency (it is concluded that the h - index is a better bibliometric tool than is the g - index) (17). in 2015 in b&h about twenty - five journals are issued in the field of biomedical and life sciences in general (18). journals that are indexed in medline / pubmed are medical archives, materia socio medica, acta informatica medica, acta medica academica, bosnian journal of basic medical sciences (bjbms) and medicinski glasnik (18). the first three mentioned represent editions of academy of medical sciences of bosnia and herzegovina, acta medica academica is edition of the academy of sciences and arts of bosnia and herzegovina, medicinski glasnik is the official publication of the medical association of zenica - doboj canton (federation of bosnia and herzegovina) (18). bjbms is edition of association of basic medical sciences of the federation of bosnia and herzegovina and is the only one of the above mentioned journals, which is indexed in the science citation index expanded (scie)/web of science base (18 - 22). h index of medical archive for 2014 is 12 (according to scimagojr.com), and is the highest among the journals in b&h (figure 15). h index for b&h journals (according to scimagojr.com) according to scimagojr.com for 2014, bjbms has the biggest scimago journal rank (0,208) (figure 16), while the highest cites / doc. (2years) parameter (widely used as impact index) has acta informatica medica (0,70) (figure 17). (2years) parameter is also differentiated by fields that studies the medicine (figure 18). by analyzing parameter total cites (3 years) (number of citations received in the selected year by a journal to the documents published in the three previous years), it has been shown that medical archives has the highest (figure 19). (2years) parameter for b&h journals (according to scimagojr.com) cites / doc. (2years) parameter vs h index for medicine fields in period 2012 - 13 (2) comparation between medical archives, acta informatica medica and bosnian journal of basic medical sciences by parameter total cites (3 years) (2) according to googlescholar the biggest h5 index (h - index for articles published in the last 5 complete years) has bjbms and medical archives (14) (figure 19), but the biggest h5 median (median number of citations for the articles that make up its h5-index) has bjbms i acta informatica medica (19) (figure 20). the most cited articles from materia socio medica, acta informatica medica and bosnian journal of basic medical sciences are shown on figures 21 - 23. googlescholar information about h5 index and h5 median for b&h journals the most cited articles in materia socio medica during period 2010 - 2015 the most cited articles in acta informatica medica during period 2010 - 2015 the most cited articles in bosnian journal of basic medical sciences during period 2010 - 2015 by comparing the state of medical publishing in b&h with neighboring countries (croatia, serbia, montenegro), we have concluded that b&h is behind croatia and serbia by following parameters : total documents, total cites and h index (figure 24, 25, 26), but in front of montenegro. though interesting is monitoring of international collaboration, where b&h is in front of croatia and serbia, but behind montenegro (figure 27). comparation between b&h, croatia, serbia and montenegro by total documents parameter (2) comparation between b&h, croatia, serbia and montenegro by total cites parameter (2) comparation between b&h, croatia, serbia and montenegro by h index (2) comparation between b&h, croatia, serbia and montenegro by international collaboration parameter (2) ana wil harzing, from melbourne, australia, in 2006 launched a free of charge available software package called publish or perish, which is used for the derivation of various citation analyses, including impact factor (4). we analyzed the most prolific authors of b&h by mentioned software (the highest h - index has izet masic md, phd, enver zerem md, phd and semir vranic md, phd, while highest g index has enver zerem md, phd) (table 2). most profilic authors in b&h analyzed by publish or perish software (based on google scholar database) a total of 104 articles were published in medical archives during 2015. analyzing the type of articles, original articles are present in majority during 201580.7% (in last seven years, 561 (76%) were original out of 738). in last seven years, 651 (88.2%) articles were from the field of clinical medicine (preclinical disciplines, in the last three years are more represented than in previous years). collaboration rate in 2015 was 0,92. from year to year, most often required time for a decision on acceptance or on the revision prior acceptance is between 50 and 60 days (30% of cases in 2015). during 2015, h index of medical archive for 2014 was 12, and does not vary during the last decade. h - index is a better bibliometric tool than is the g - index. in 2015 in b&h about twenty - five journals are issued in the field of biomedical and life sciences in general. journals that are indexed in medline / pubmed are medical archives, materia socio medica, acta informatica medica, acta medica academica, bosnian journal of basic medical sciences (bjbms) and medicinski glasnik (18 - 22). according to scimagojr.com for 2014, bjbms has the biggest scimago journal rank (0,208) while the highest cites / doc. (2years) parameter (widely used as impact index) has acta informatica medica (0,70). according to googlescholar the biggest h5 index has bjbms and medical archives (14), but the biggest h5 median has bjbms i acta informatica medica (18). by comparing the state of medical publishing in b&h with neighboring countries (croatia, serbia, montenegro), we have concluded that b&h is behind croatia and serbia by following parameters : total documents, total cites and h index but in front of montenegro. | introduction : medical archives is the oldest medical journal in bosnia and herzegovina (b&h) (founded in 1947.). a total of 104 articles were published in medical archives during 2015. analyzing the type of articles, original articles are present in majority during 201580.7% (in last seven years, 561 (76%) were original out of 738). in last seven years, 651 (88.2%) articles were from the field of clinical medicine (preclinical disciplines, in the last three years are more represented than in previous years). collaboration rate in 2015 was 0,92. articles written in collaboration of five authors (21.1 %) are found to be predominant. from year to year, most often required time for a decision on acceptance or on the revision prior acceptance is between 50 and 60 days (30% of cases in 2015). during 2015, 47.1% of articles were originally from b&h (eleven countries were represented). h index of medical archive for 2014 was 12, and does not vary during the last decade.findings:in 2015 in b&h about twenty - five journals are issued in the field of biomedical and life sciences in general (six are indexed on medline / pubmed, one is indexed in the science citation index expanded (scie)/web of science base). according to googlescholar the biggest h5 index has bosnian journal of basic medical sciences (bjbms) and medical archives, while the biggest h5 median has bjbms i acta informatica medica. the highest h - index (13) in b&h has izet masic md, phd, enver zerem md, phd and semir vranic md, phd, while highest g - index (22) has enver zerem md, phd (analyzed by software package publish or perish).conclusion : by comparing the state of medical publishing in b&h with neighboring countries (croatia, serbia, montenegro), we have concluded that b&h is behind croatia and serbia by following parameters : total documents, total cites and h index but in front of montenegro. |
cancer of the eyelid accounts for 5 - 10% of all skin cancers with 90% being basal cell carcinoma (bcc). post - operative radiotherapy is usually indicated depending upon the presence of high - risk features for recurrence. positive surgical margins in primary bcc excisions in the head and neck region were reported to be 3 - 20% in the literature and recurrence rates in incompletely excised patients were reported at 26 - 67% as well. among the indian population, squamous and adenocarcinoma involving tarsal glands are more common, incidence ranging between 2.4% to 30.2% of all malignant eyelid tumours in various literatures. they are more aggressive and invasive than basal cell carcinoma, and have a definite metastatic potential. if treatment is delayed, they can invade the orbital and intracranial structures, and hence have a considerable potential for mortality and morbidity. sebaceous carcinoma tends to affect patients at older age, mostly around fifth decade, with a female preponderance. they arise from the meibomian glands of the eyelid and have a local recurrence rate around 30% including invasion of orbital contents and distant metastasis in 20% cases. traditionally, surgical excision has been the mainstay of treatment in this group of patients. moh 's micrographic surgery, cryosurgery, laser excision have been used with 5 year control rates in the range of 90%. radiotherapy, mostly as external beam radiation is added in cases having high chances of local recurrence such as lymph nodal involvement, positive or close margin, perineural invasion, and poorly differentiated tumours [6, 7 ]. the major hindrance in application of post - operative external beam radiotherapy (ebrt) is the close association of vital structures of the globe and the thin skin around the eyelid. radiation - induced ocular morbidity encompasses a spectrum from transient eyelid erythema to complete loss of vision, with or without loss of the globe [810 ]. post - operative adjuvant interstitial brachytherapy (ibrt) has been infrequently used for the treatment of eyelid malignancies. in the presence of high risk factors warranting adjuvant radiotherapy, most of the patients receive ebrt, which has its own shortcomings. post - operative adjuvant high - dose - rate (hdr) brachytherapy can be considered as an effective option in this group of patients for maintaining local control, while preserving cosmesis and function. we present a series of 8 patients who received post - operative hdr brachytherapy (hdr ibrt) in our hospital. this study aims at proposing an effective alternative for preserving function and cosmesis among eyelid cancer patients, warranting adjuvant radiotherapy after primary excision while maintaining disease control. between august 2007 and july 2012, 8 patients of eyelid cancer receiving post - operative hdr ibrt were analysed. there were 5 male and 3 female patients with age between 23 - 82 years. all of them underwent primary surgery in the form of wide excision followed by appropriate reconstruction. the disease was staged by american joint committee on cancer (ajcc) 6 edition of tnm staging. the implant procedure was carried out under general anaesthesia. the tumour volume (post - operative target volume for brachytherapy) the irradiated volume included the primary disease extent with a margin of 3 - 5 mm with modifications if required. plastic tubes were then threaded through these needles and left in place after removal of the stainless steel needles. plastic tubes were then secured, opposing the skin by means of plastic buttons and plastic beads (fig. 1). during treatment, a custom made conformer was fashioned out of dental prosthesis material to prevent additional dose spillage to normal eyes. images were acquired using the ge system light speed ct simulator (ge health care, milwaukee, wi, usa) with patients in supine position with slice thickness of 1 - 1.5 mm. the brachytherapy planning was done using nucletron plato sunrise v 14.3.1 computerized treatment planning system (nucletron, an elekta company, elekta ab, stockholm, sweden). optimization was done along dose points specified at a distance of 2 - 3 mm from the catheters depending on thickness of tumour bed. in the case of single wire implant, dose prescription point of 2 mm was selected as per the american association of physicist in medicine (aapm) task group (tg) report no. the 85 or 100% isodose lines were used as reference isodose based on the adequacy of tumour bed coverage and the dose to normal structures like skin and cornea (fig. postoperative ibrt in eyelid cancer - planning and dosimetry demographics of ibrt patients fractionated high - dose - rate brachytherapy was administered using single high activity ir-192, as radio - active source with predetermined time at various dwell positions which were 2.5 mm apart. total dose was between 21 gy in 7 fractions to 35 gy in 10 fractions, respectively. (twice daily fractionation with gap of 6 - 8 hours between them), local anaesthetic was sprayed over the treatment region, antibiotic ointment dressing was carried out, and patients were kept on oral analgesics and if required intravenous tramadol as well. all the 8 patients were reviewed weekly for one month to document acute radiation reactions, 3 monthly for 2 years, and then 6 monthly up to 5 years, then annually once. one patient was lost to follow up after completion of treatment. on follow - up, patients were examined clinically as well as with ophthalmologic evaluation as warranted. the status of these patients pertaining to disease control was done by clinical examination of both the primary site and neck. at certain intervals it was supplemented by detailed ophthalmologic examination, which also took care of toxicity assessment. any visual deterioration and complications pertaining to skin over eyelid, conjunctiva, and overall cosmesis acute toxicities were graded by rtog acute toxicity scale and ctcae version 3 criteria were used for late complications pertaining to skin, eyelid, and vision, respectively. the indigenous caib scale was designed keeping in mind the different ocular structures and their late effects viz. depigmentation of skin, eyelid dysfunction like ectropion or entropion, dry eye, keratitis, cataract, and glaucoma. if present, they were scored as 1 and 0 if they were absent. on a total score between 0 - 6, 4 groups were made with score 0 being excellent cosmesis, scores 1 - 2 as very good, 3 - 4 as fair, and scores 5 - 6 as poor cosmesis. this scale was specifically devised with responses yes / no to actually document effectiveness of hdr ibrt in terms of cosmetic outcome (table 2). four patients had sebaceous carcinoma, while 3 had squamous cell carcinoma and 1 patient had a nodular basal cell carcinoma. the acute toxicities were graded by rtog acute toxicity criteria. all the 8 patients had grade 1 acute reactions, either faint erythema (6 patients) or mild epiphora (2 patients). the late toxicities that we looked at were the skin of the eyelid in terms of pigmentary changes, eyelid in terms of dysfunction like ectropion or trichiasis, and visual complaints such as cataract, glaucoma and keratitis. only 2 patients had late toxicity in terms of pigmentory changes persisting over skin of right upper eyelid and mild ectropion. one of the patients was lost to follow up and no comment could be made upon late toxicity or cosmesis. there was no incidence of corneal ulcer, conjunctival foreign body sensation, corneal perforation and cataract, which are of significant concern with ebrt. the dosimetric analysis of the organs at risk in vicinity to eyelid cancer like remaining uninvolved eyelid, cornea, lens, and lacrimal gland were carried out. the dmax for uninvolved eyelid was 17.2 gy, for cornea 6.7 gy, lens received 1.9 gy and lacrimal gland on the ipsilateral side 3.4 gy, respectively. dosimetric parameters of eyelid brachytherapy cosmesis was documented as per the objective caib scoring criteria indicated excellent (5 patients) to very good (2 patients) cosmesis. two patients failed at the node but were salvage effectively and remain controlled at subsequent follow - up. the three distinct types of eyelid cancers with their variable local and distant invasion rates coupled with surrounding critical visual apparatus have always posed a challenge towards the effective management. while basal cell cancers are locally recurrent, squamous cell carcinomas are more aggressive and invasive, and sebaceous carcinomas also have high distant failure rates [1416 ]. the therapeutic challenge is to be able to preserve cosmetic and functional results and therefore the goal of therapy would be both tumour control and function preservation [17, 18 ]. a variety of approaches are available for the management of skin cancer, including curettage and electrodessication, mohs micrographic surgery, cryotherapy, surgical resection, and external beam radiotherapy. radiotherapy is used as adjuvant treatment in high - risk lesions, which include positive margins, perineural invasion, and lymph node disease after surgical resection / debulking [6, 7 ]. the indications for postoperative radiation include positive surgical margins, perineural spread, invasion of bone and cartilage, extensive skeletal muscle infiltration and poor differentiation [15, 20 ]. recent literature also suggests pathological stage more than pt2 alone warrants postoperative radiotherapy for better survival in eyelid sebaceous carcinomas. traditional radiotherapy was feared because of acute and late sequelae to the ocular structures. for dry eye to develop, incidence of injury increases from 0% with doses of 30 gy to 100% after doses the eyelid also develops transient erythema at doses of 30 gy, with telangiectasia and loss of eye lashes beginning to occur at 40 gy. late squeal like ectropion and entropion chances of corneal perforation increase when the cornea receives ebrt dose in excess of 60 gy. cataract is a well - known radiation complication presenting as posterior sub capsular opacification. in general, latency and frequency of cataract is a function of radiation dose and conventionally fractionated ebrt dosage exceeding 12 gy is held responsible. the complications in inner eye structures like retina and optic nerve is primarily determined by the total radiation dose. none of the patients receiving a dose of 95%) after almost 8 years median follow up and also acceptable cosmetic outcomes. kowalik. have tried to use individual applicator for different disease sites in the head and neck region, and one of their cases had a recurrence of squamous cell cancer at root of nose close to eyes. they have also showed excellent results in treating these recurrences. to re - emphasise, brachytherapy techniques seem to be the most effective and easy treatment to protect organs located deeper than 2 cm below the surface, and from the perspective of protecting critical healthy organs as well. however, it demands technical expertise in these cases. our study in comparison to available literature also showed an excellent local control with a median follow up of 4 years. the indigenous yet to be validated caib scale, taking into considerations the important late toxicities in the ocular structures was found to be quite useful both in terms of applicability and accuracy. the small sample size and heterogeneous population of eyelid cancer patients actually highlight the rarity of such cases and treatment approach like postoperative hdr ibrt. the caib scale used in our study showed excellent to very good cosmesis in all cases. adjuvant hdr brachytherapy in patients of eyelid cancer undergoing primary surgical excision is safe and efficacious. the small numbers in the series reflect the low incidence of this cancer in our part of the world, but the results prove that effective adjuvant therapy can be provided when necessary with very good disease control and functional outcomes. the indigenous caib scale with future prospective validation studies would definitely streamline management of this rare disease with acceptable cosmetic outcomes. | purposeto analyse feasibility and safety of postoperative interstitial brachytherapy (ibrt) in patients of eyelid cancer treated primarily by surgical excision.material and methodsin this series, 8 patients with eyelid cancer were treated using postoperative interstitial brachytherapy. patients were followed up for local control, cosmetic outcome, and acute and late toxicities. cosmetic outcome was measured using a 6 point indigenous cosmesis after interstitial brachytherapy (caib) scale.resultsthe patients were between 23 - 82 years (median : 71 years). there were 3 females and 5 males, and 3 patients had lesions in upper eyelid. postoperative high - dose - rate brachytherapy was used in all with 2 catheters implanted in most of them (6 out of 8). local control was calculated from end of treatment to last follow - up. at last follow - up, all patients remained locally controlled. two patients had nodal recurrence 6 months after interstitial brachytherapy and were salvaged effectively by external beam radiotherapy. at last follow - up, 7 patients were loco - regionally controlled and one was lost to follow up. all patients had radiation therapy oncology group (rtog) grade 1 acute toxicity and 2 had grade 1 common terminology criteria for adverse events (ctcae) version.3 late toxicities. the cosmesis score for the whole group ranged between 0 - 1 indicating excellent to very good cosmesis.conclusionspostoperative high - dose - rate brachytherapy resulted in excellent disease control and cosmesis without significant acute or late toxicities. it is an effective modality for treatment of eyelid cancers in selected patients. future prospective studies with the validation of caib scale would give us more insight to this effective yet often ignored modality of ibrt. |
the way in which individuals respond to daily stressors is a determinant of reactivity of the sympathetic - adrenal - medullary (sam) and hypothalamic - pituitary - adrenal (hpa) axes and contributes to allostatic load. in order to fully understand the reactivity of these axes it is necessary to observe individuals while they are experiencing stress. naturalistic stressors provide ecologically valid measurement opportunities ; however they can be expensive and lack control and standardisation. alternatively, laboratory stressors allow for the controlled manipulation of stimuli and more specific assessment of the causal factors involved in psychobiological stress responding. a variety of laboratory stressors comprising cognitive challenge, public speaking, emotion induction and interpersonal stress are used for this purpose ; however, these tasks typically serve no function outside of the laboratory (chida and hamer, 2008). to obtain a comprehensive snapshot of how an individual would respond to a stressor encountered in a real - life setting, laboratory stressors should have ecological validity and be representative of experiences in natural settings. such settings rarely involve exposure to a single stressor as modelled in the laboratory, but instead individuals typically deal with multiple sources of stress (chida and hamer, 2008). ecologically valid stressors should therefore comprise multiple stimuli and be representative of the types of situations encountered in everyday life. the multitasking framework (wetherell and sidgreaves, 2005) comprises eight individual cognitive tasks and elicits stress via the manipulation of workload intensity by increasing the difficulty and number of tasks (up to a maximum of four during one presentation) that a user must attend and respond to. although the multitasking framework does not simulate a specific environment, it comprises tasks that are required in many working environments, such as calculations, continuous visual and auditory monitoring, and relevant stimuli identification. moreover, as successful performance requires sustained effort, repeated multitasking does not lead to habituation of responding (wetherell., several studies have demonstrated the efficacy of the multitasking framework as an acute stressor as evidenced by increases in stress, anxiety and fatigue (e.g., haskell., 2010, johnson., 2011, wetherell and carter, 2013) ; cardiovascular reactivity (e.g., kelly - hughes., 2014) ; and mucosal immunity (e.g., wetherell and sidgreaves, 2005). only one study ; however, has reported an increase in cortisol reactivity following multitasking (scholey., 2009). compared with sam responding, the hpa axis has a particularly high threshold for activation and acute increases in cortisol are typically observed in conditions of perceived uncontrollability involving motivated performance tasks accompanied by social evaluative threat (i.e. threats to a valued aspect of self - identity or where the self is at risk of being negatively judged by others ; dickerson and kemeny, 2004). the multitasking framework is a motivated performance task and involves elements of uncontrollability ; but, it does not involve social evaluative threat, and cortisol reactivity would therefore not necessarily be predicted. all of the conditions necessary for reliably inducing a cortisol response are, however, present in other laboratory stressors notably, the trier social stress test (tsst), which involves a preparation period followed by the presentation of free speech and mental arithmetic to a socially evaluative panel whilst being recorded. this paradigm is associated with robust increases in cortisol and has become a standard protocol for stress induction in healthy (e.g., kirschbaum., 1993, kirschbaum., 1995) and clinical (e.g., buske - kirschbaum and hellhammer, 2003) populations of all ages (e.g., kudielka., 2004, jessop and turner - cobb, 2008). there is, however, a need to develop alternative stress protocols that involve other sources of stress and are appropriate for repeated testing (kudielka and wust, 2010). in natural settings, exposure to social evaluation is omnipresent ; for example, giving presentations and being monitored during the performance of tasks in the workplace are commonplace and involve perceived threats to ones abilities, competencies or traits (gruenewald., 2004). a laboratory paradigm that is additionally representative of these settings, cognitive multitasking is analogous to a range of environments requiring attendance and response to multiple stimuli and is associated with cardiovascular and psychological stress reactivity. given that critical social evaluation typically elicits hpa activation, the combination of multitasking and critical evaluation could therefore provide an easily administered acute stressor paradigm representative of everyday stressful situations. the aim of the current study is, therefore, to assess whether a critically evaluated multitasking paradigm elicits activation of psychological, cardiovascular and hpa reactivity. all recruitment and study procedures were granted ethical approval from the faculty ethics committee in line with the regulations of the institution and relevant regulatory bodies. a total of 50 healthy participants (range 1838, mage = 19.6, sd = 2.83 ; females = 34, males = 16) were recruited from an undergraduate population and randomly allocated to either multitasking only (mage = 19.89, sd = 3.93 ; female = 17, male = 8) or multitasking with critical evaluation (mage = 19.32, sd = 0.85 ; female = 17, male = 8). eligibility criteria included : aged 1840 ; resting blood pressure less than 140/90 mmhg ; not pregnant or breastfeeding ; no self - reported anxiety or stress - related disorder. in addition, data were recorded for a number of factors that can alter hpa function ; specifically, body mass index (bmi) ; use of the contraceptive pill (n = 21) ; menstrual cycle stage (first half = 8 ; second half = 14) ; and smoking status (n = 6) were also recorded as appropriate. the multitasking framework (purple research solutions, uk) is a platform for the presentation of performance - driven, cognitively demanding tasks and is analogous to working environments that require attendance and response to simultaneous stimuli. this study used four tasks : auditory monitoring, visual monitoring, number entry, and memory search. all tasks are points drive with points awarded for correct responses and points deducted for missed or incorrect responses. participants are instructed to be as fast and accurate on all of the tasks as possible in order to achieve as high a score as they can. the running total score is displayed in the middles of the screen whilst the tasks are running. a full description of the framework is provided in wetherell and carter (2013). the (10 item) perceived stress scale (pss-10 : cohen., 1983) measured how often in the last month participants felt that life was unpredictable, uncontrollable, and overwhelming. the 16 item bond - lader visual analogue scales (bond and lader, 1974) measured the mood states of alert, content, and calm. the nasa - tlx (hart and staveland (1988) assessed mental, physical and temporal demand, effort, performance and frustration. participants were asked to refrain from eating or drinking (other than water) for 1 h preceding the study. all samples were frozen (20 c) and assayed using the enzyme - linked immunosorbant assay method (salimetrics - europe, uk ; intra and inter - assay coefficients 0.05). the current study assessed psychological, cardiovascular and hpa reactivity in response to critically evaluated multitasking and multitasking alone. multitasking led to increased psychobiological reactivity ; specifically increases in heart rate, diastolic blood pressure, and anxiety and reductions in positive mood states of calmness, contentment and happiness. critically evaluated multitasking increased systolic blood pressure and anxiety, reduced contentment and calmness and led to greater perceived workload on all domains, over and above that observed following multitasking alone. numerous studies (cf dickerson and kemeny, 2004) have demonstrated that critical social evaluation elicits robust increases in cortisol, and alongside the other key stressor components of cognitive challenge and uncontrollability, forms an integral part of laboratory stressors such as the tsst. in this study, however, critically evaluated multitasking did not elicit a cortisol response. the reduction in cortisol observed during the stressor period is likely indicative of the decline associated with the typical diurnal decline of cortisol. physiological processes respond to meet the demands of the environment and terminate that response once the demands are met (gunnar., 2000). furthermore, unlike the sam axis, the hpa axis has a particularly high threshold for activation, and subsequently has longer - lasting effects (shirtcliff., 2012). if the threat associated with a situation is perceived to be low, this will be reflected in the subsequent physiological responses. a situation of low perceived threat may, therefore, involve only brief withdrawal of parasympathetic inhibition or a brief rise in cardiovascular activity to activate the sam - mediated fight - or - flight response. this may account for the observed profile of responding in the current paradigm, that is, cardiovascular responding indicative of activation of the fight - or - flight response, but insufficient perceived threat to activate the hpa axis, despite increased reports of anxiety and demand. an absence of cortisol responding has also been observed in other paradigms involving challenging situations in the presence of others. paradigms that incorporate friendly or inattentive rather than socially evaluative panels do not elicit cortisol reactivity (wiemers., 2013, dickerson., 2008), suggesting that the provision of critical social evaluation, rather than just the presence of others is necessary for hpa activation. the current paradigm incorporated a record of participation (video recording of participant) and critical evaluation of performance, and was perceived as demanding and anxiety provoking ; however, it may not have been perceived as a threat to self. perception of social evaluation was not explicitly measured in this study ; however, the absence of cortisol responding, in an otherwise stressful and demanding paradigm, does suggest a missing stress eliciting component. that is, whilst the current manipulation comprises challenge and critique, it may lack the socially evaluative element that has been previously associated with hpa activation in other paradigms. this may be evidenced by notable differences between the current paradigm and the tsst, which is associated with robust cortisol responding. both paradigms incorporate a motivated performance task and the presence of critical evaluation ; however, they differ in terms of the required response format of the performance task ; and the position of the participant in relation to the evaluator. the tsst requires the participant to verbally perform a free speech task followed by a verbal mental arithmetic task ; in contrast the multitasking framework requires responses via a computer with no verbal component. public speaking is a significant stressor involving the risk of embarrassment and humiliation (garcia - leal., 2014), and as such, the requirement to verbalise responses may represent the salient challenge to self that is necessary for hpa activation. cold pressor, a passive coping task typically associated with minimal hpa activity, leads to significant increases in cortisol when accompanied with social evaluation. specifically, a standard cold pressor procedure whilst being video recorded and evaluated by an experimenter leads to significant increases in cortisol in participants tested alone (schwabe., 2008) and in groups (minkley., 2014), suggesting that social evaluation may represent a salient threat even in the absence of a critique of a motivated performance task or a verbal component. the remaining notable distinction between this paradigm and the tsst relates to the nature of social evaluation. evaluation in the tsst and socially evaluated cold pressor involves being directly observed by an evaluator, and in the case of the tsst involves standing directly in front of a panel. direct social evaluation, even in the absence of critique leads to increased perceived vulnerability and cortisol increases are therefore likely in situations where there is a threat to self - presentation and a greater risk of negative evaluation (schwabe., 2008). although the current paradigm involved the observation and critique of performance, and therefore a potential challenge to one 's self, this challenge was indirect. the participant was therefore able to avoid direct evaluation and focus on the tasks, as evidenced by increases in psychological and cardiovascular responding typical of motivated performance tasks. first, although the sample is small, it was sufficient to observe meaningful differences in variables of interest in line with previous studies that have used the framework (e.g., wetherell and carter, 2013). second, sex differences can impact upon acute stress responding, in particular reactivity of the hpa axis with males typically demonstrating greater reactivity than females (kudielka. although attempts were made to ensure balanced numbers of males and females across conditions, the current sample comprised a greater number of females than males. there were no observed sex differences for any of the psychological or physiological variables ; however, future studies should use this paradigm in larger, more balanced samples. notwithstanding these limitations, the current paradigm affords several advantages and opportunities for stress testing. from a methodological perspective, in response to the call for the development of alternative stress testing protocols (kudielka and wust, 2010) ; the multitasking framework provides an ecologically valid technique for eliciting psychobiological reactivity ; it is appropriate for repeated testing ; and it comprises inherent measures of performance (scholey., 2009). in addition, critically evaluated multitasking offers a paradigm representative of everyday situations requiring attention to multiple stimuli whilst being monitored and evaluated. as the paradigm requires minimal physical and human resource it therefore offers an alternative, economical laboratory stressor. the paradigm can also be used to address a number of research questions relating to the role of social evaluation. the current study reported the effects of additional critical evaluation on psychobiological indices and perceptions of stress and demand ; however, future studies could utilise the inherent performance measures to ascertain the impact of critical evaluation on actual task performance, thus providing a useful tool for modelling work - based performance during evaluation. the absence of cortisol reactivity following critically evaluated multitasking also presents a number of opportunities for assessing the salient social components that are associated with hpa activation in other paradigms. that is, it appears that the levels of interpersonal threat experienced in the tsst are not present in the current paradigm. further manipulations regarding the nature of the critical evaluation received in addition to multitasking, for example, face - to face, rather than over - the - shoulder critical evaluation, or the requirement for verbal responding, would allow for a greater understanding of the role of interpersonal threat in relation to hpa activation. in conclusion, the increases in perceived workload, anxiety and cardiovascular responding following multitasking with critical evaluation demonstrate the stress - inducing effects of this protocol. the current paradigm is an easily administered laboratory analogue of everyday situations involving the performance of multiple tasks whilst being critically evaluated, and therefore provides an ecologically valid paradigm for the assessment of psychological and cardiovascular stress responding. the absence of cortisol reactivity, however, suggests some added subtlety in the factors that elicit hpa responding, that is, not all critically evaluated situations are perceived as a significant threat to self, and direct observation is likely to provide the additional social evaluation that is associated with hpa activation. future developments of this paradigm could therefore assess the importance of in person, face - to - face contact to an evaluative other whilst maintaining the ecologically valid components of the paradigm. | in order to understand psychobiological responses to stress it is necessary to observe how people react to controlled stressors. a range of stressors exist for this purpose ; however, laboratory stressors that are representative of real life situations provide more ecologically valid opportunities for assessing stress responding. the current study assessed psychobiological responses to an ecologically valid laboratory stressor involving multitasking and critical evaluation. the stressor elicited significant increases in psychological and cardiovascular stress reactivity ; however, no cortisol reactivity was observed. other socially evaluative laboratory stressors that lead to cortisol reactivity typically require a participant to perform tasks that involve verbal responses, whilst standing in front of evaluative others. the current protocol contained critical evaluation of cognitive performance ; however, this was delivered from behind a seated participant. the salience of social evaluation may therefore be related to the response format of the task and the method of evaluation. that is, the current protocol did not involve the additional vulnerability associated with in person, face - to - face contact, and verbal delivery. critical evaluation of multitasking provides an ecologically valid technique for inducing laboratory stress and provides an alternative tool for assessing psychological and cardiovascular reactivity. future studies could additionally use this paradigm to investigate those components of social evaluation necessary for eliciting a cortisol response. |
infectious adverse events after transrectal prostate biopsy (tpbx) have been increasing, and even septic and fatal cases have been reported. fluoroquinolones have long been used for prophylactic administration owing to their good efficacy and high concentration in the prostate after dosing. however, especially in urinary tract infections (utis), fluoroquinolone - resistant escherichia coli (fqre) has been rapidly increasing. resistance ratios to levofloxacin (lvfx) have jumped up to 5% to 20%. because tpbx is increasingly used to detect prostate cancer, adverse events are a growing concern. another resistant strain, extended - spectrum beta - lactamase (esbl)-producing e. coli, also poses difficulties for treatment. some kinds of carbapenems are available for this resistant strain, but it has the potential to be an intractable infection. some authors previously reported a case of prostatitis caused by esbl - producing e. coli and it could be treated by imipenem and tazobactam / piperacillin (taz / pipc). beta - lactamase inhibitors have generally been combined with penicillins partly because this combination can bring out the antibiotic activity fully and prevent the emergence of antibiotic - resistant strains. taz / pipc (zosin) is an intravenous antibiotic agent consisting of 4 g of piperacillin and 0.5 g of tazobactam at an 8:1 ratio and is a modification of tazosin, which was used before and consisted of 2 g of piperacillin and 0.5 g of tazobactam. penicillins combined with beta - lactamase inhibitors such as taz / pipc are widely used to treat utis and show good efficacy for e. coli, including fqre and esbl - producing e. coli, and are recommended for tpbx in the japanese uti association guidelines especially in high risk group. in this study, we examined whether taz / pipc plus lvfx could suppress infectious adverse events after tpbx. we performed a study of 201 consecutive men scheduled for tpbx because of an elevated prostate - specific antigen (psa) level, an abnormal digital rectal examination (dre), or abnormal magnetic resonance imaging (mri) or transrectal ultrasound (trus) findings. all biopsies were performed with an 18-gauge bard max core disposable biopsy instrument biopsy needle (c.r. convington, ga, usa) in conjunction with a medical ultrasound console (aloka ssd-2000, aloka co. ltd., acetylsalicylic acid or oral anticoagulant agents were stopped appropriately before tpbx with the approval of the prescribing physician as a rule. no preparatory cleansing enemas were used, and the tpbx procedure was performed with only sacral anesthesia with 1% lidocaine and povidone iodine sterilization just before the tpbx. for prophylactic antibiotic medication, each patient received taz / pipc 4.5 g i.v. and lvfx 100 mg or 500 mg taken orally 30 minutes before tpbx. patients continued to take 100 mg after every meal or 500 mg lvfx once a day for 3 days with or without styptic or anti - plasmin. the dose of lvfx was changed to 500 mg from 300 mg per day from august 2009 in this study. patients were examined for infectious adverse events after tpbx and white blood cell (wbc) counts (/mm) and c - reactive protein (crp) (g / ml) in plasma were measured before and 1 day after tpbx. all patients undergoing tpbx were hospitalized on the day of tpbx and were discharged the next day if there were no adverse events requiring further hospitalization. the day after tpbx, patients ' laboratory data were checked including wbc and crp in serum. if a patient had symptoms from infectious complications such as chills or fever greater than 38.0 after tpbx, he was asked to return to the emergency department. he immediately underwent a urinalysis, urine culture, and blood analysis, and any other complications were recorded. as a rule, if acute prostatitis was diagnosed, the patient was hospitalized and treated with the third- or fourth - generation cephalosporin or carbapenem i.v., and statistical analysis was performed by use of student 's t - test or welch 's t - test or fisher 's exact test with the jstat - java virtual machine statistics monitoring tool (sun microsystems, inc., we performed a study of 201 consecutive men scheduled for tpbx because of an elevated prostate - specific antigen (psa) level, an abnormal digital rectal examination (dre), or abnormal magnetic resonance imaging (mri) or transrectal ultrasound (trus) findings. all biopsies were performed with an 18-gauge bard max core disposable biopsy instrument biopsy needle (c.r. convington, ga, usa) in conjunction with a medical ultrasound console (aloka ssd-2000, aloka co. ltd., acetylsalicylic acid or oral anticoagulant agents were stopped appropriately before tpbx with the approval of the prescribing physician as a rule. no preparatory cleansing enemas were used, and the tpbx procedure was performed with only sacral anesthesia with 1% lidocaine and povidone iodine sterilization just before the tpbx. for prophylactic antibiotic medication, each patient received taz / pipc 4.5 g i.v. and lvfx 100 mg or 500 mg taken orally 30 minutes before tpbx. patients continued to take 100 mg after every meal or 500 mg lvfx once a day for 3 days with or without styptic or anti - plasmin. the dose of lvfx was changed to 500 mg from 300 mg per day from august 2009 in this study. patients were examined for infectious adverse events after tpbx and white blood cell (wbc) counts (/mm) and c - reactive protein (crp) (g / ml) in plasma were measured before and 1 day after tpbx. all patients undergoing tpbx were hospitalized on the day of tpbx and were discharged the next day if there were no adverse events requiring further hospitalization. the day after tpbx, patients ' laboratory data were checked including wbc and crp in serum. if a patient had symptoms from infectious complications such as chills or fever greater than 38.0 after tpbx, he was asked to return to the emergency department. he immediately underwent a urinalysis, urine culture, and blood analysis, and any other complications were recorded. as a rule, if acute prostatitis was diagnosed, the patient was hospitalized and treated with the third- or fourth - generation cephalosporin or carbapenem i.v., and blood and urine culture tests were performed. statistical analysis was performed by use of student 's t - test or welch 's t - test or fisher 's exact test with the jstat - java virtual machine statistics monitoring tool (sun microsystems, inc., one of 201 patients (0.50%) had a febrile complication such as acute prostatitis after tpbx. our data from laboratory tests done the day after tpbx, including serum wbc count and crp, did not demonstrate a significant rise in these variables compared with the pre - tpbx data as shown in table 2 (p>0.05). the serum wbc count before and after tpbx (day 1) was 6,1231,655 and 6,4271,898 (/mm), respectively. serum crp before and after tpbx (day 1) was 0.17020.4020 and 0.26020.4534 (g / ml), respectively (table 2). no patients showed obvious side effects from this regimen (taz / pipc plus lvfx). a case with febrile complication had fever (37.6) from the night of tpbx and his serum wbc and crp rose up (18,100/mm and 4.1 g / ml, respectively) on the next day. doripenem 0.5 g / day and his symptoms and laboratory data reacted very well and improved. in addition, we compared lvfx 500 mg with lvfx 300 mg in the taz / pipc plus lvfx regimen ; however, there were no significant differences between the two groups regarding the febrile complication rate (p>0.05) or change in serum wbc and crp before and after tpbx (table 3). tpbx is a test for the detection of prostate cancer and most patients have cancer - negative results in general ; therefore, severe adverse events must be prevented. recently, the ratio of infectious complications after tpbx has risen and even septic or fatal cases have been reported. one of the main reasons for this unfortunate fact is the increase of antibiotic - resistant enterobacteriaceae. naturally, e. coli is one of the main targets to suppress in this procedure, and infectious adverse events may be partly caused by antibiotic - resistant bacteria. fluoroquinolones are often used for prophylactic medication in tpbx, partly because they are retained at high concentrations in the prostate as mentioned above ; however, fluoroquinolone - resistant e. coli are spreading, especially in uti. a guideline for prophylactic antibiotic medication for tpbx in 2006 and 2007 recommends fluoroquinolones in low - risk groups and taz / pipc in high - risk groups. however, infectious complications and septic cases after tpbx increased after the publication of this guideline. our regimen was set for the following reasons : 1) good efficacy of taz / pipc for e. coli including beta - lactamase - producing bacteria, 2) good intra - prostate concentrations and permeability of lvfx, 3) the data on taz / pipc alone mentioned below, and 4) the higher resistance ratio of e. coli to lvfx in our previous study (data not shown) than before, and 5) the higher ratio of esbl - producing e. coli among all e. coli isolated recently than before in our institution (data not shown). guidelines need to be updated to reflect the current antibiotic susceptibilities of emergent bacterial strains. as reported in the literature, the ratios of febrile infectious complications range from 0.51% to 7.27%, which suggests that our method could be one of the recommendations for this purpose. in addition, our previous method [lvfx plus aminoglycoside (isepamicin) ] has shown good efficacy for this purpose ; however, partly because of the spread of resistant strains, the previous method should be reconsidered and revised by the current data. for instance, kato reported a case of septic shock caused by fluoroquinolone - resistant e. coli after tpbx, and carlson reported multi - drug - resistant e. coli urosepsis cases including a death case following tpbx. in addition, kim reported 923 cases of tpbx and concluded that almost cases of acute prostatitis (2.0%) were caused by fluoroquinolone - resistant e. coli. this may suggest that the trend in infection and resistant strains could change and should be monitored and the guidelines revised if necessary. regarding antibiotic - resistant e. coli, esbl - producing e. coli have been reported as problematic to treat even in utis as mentioned above. lee reported the prevalence of esbl - producing uropathogens in uti patients, and the overall prevalence ratio was 12.6% in their 3-year study. this trend is apparently growing and raises some doubt as to whether we can decrease the dose of taz / pipc or lvfx for future 's study. our laboratory data for serum wbc and crp demonstrate that our regimen offers good efficacy for suppressing inflammation and infectious adverse events after tpbx. our ratio of infectious complications after tpbx was 0.50%, indicating its suitability even for high - risk patients. our next step will, as mentioned above, be to consider reducing the dose of taz / pipc and lvfx to once each on the day of tpbx (just before the procedure) for low - risk patients, while strictly monitoring for resistant strains. some authors report the efficacy of oral antibiotics for this purpose, but it might be necessary to combine intravenous antibiotics for suppression of septic cases. because tpbx detects prostate cancer in only 20% to 30% of cases, and because most of these patients have organ - confined cancer that may not cause early death, serious complications from the diagnostic procedure itself should be regarded as unacceptable as mentioned above. our results of only one febrile complication in 201 consecutive cases and no influence on infection - related laboratory data (wbc and crp) support an updated methodology for prophylactic medication. guidelines need to be updated regularly, because long repetition of a single recommendation may lead to widespread emergence of resistant bacterial strains. in general our data definitely showed a lower infectious complication ratio after tpbx, even though direct comparison could not be done because of the difference in core number, compared with the regimen of taz / pipc alone (13.5 g per day, once daily) as used in kobe university hospital [7/180 : 3.89% in 10 (sextant+2 transitional zone+2 far lateral peripheral zone) cores ; data not shown ]. in addition, the data analyzed to observe an effect of lvfx dosing (300 mg or 500 mg per day as shown in table 3) suggested that our taz / pipc plus lvfx protocol was not affected by lvfx doses. in addition, the tpbx cases from kobe university hospital reported here included low - risk patients because the data were from consecutive tpbx cases. moreover, our previous regimen and study was performed from 2003 to 2008, and the high prevalence of esbl - producing e. coli was reported from the study done from 2007 to 2009. taken together, these data suggest that our current regimen could be considered as one of the recommendations for not only high - risk patients but also non - high - risk patients. even though we showed good efficacy, this study may have limitations and further comparison study with the same patient group, study period, and method may be required. this single - arm study was also performed to confirm not only the effect of this regimen but also the safety for patients without any severe side effects. taz / pipc plus fluoroquinolones can be considered a valuable prophylactic regimen for preventing infectious complications in tpbx. taz / pipc plus fluoroquinolones is one of the options for prophylactic antimicrobial administration in tpbx, especially in high - risk groups. | purposeto investigate the efficacy of tazobactam / piperacillin (taz / pipc) plus levofloxacin (lvfx) as a prophylactic administration in transrectal prostate biopsy (tpbx).materials and methodswe investigated 201 consecutive patients who underwent tpbx in one japanese hospital during the period of 2009 - 2010. the patients received taz / pipc 4.5 g i.v. once just before and 3 hours after tpbx, plus oral lvfx 300 mg or 500 mg daily for 3 days. we examined the infectious adverse events and laboratory data (serum white blood cell [wbc ] count and c - reactive protein [crp ]) before and 1 day after tpbx.resultsonly one patient (0.50%) in 201 cases had febrile complications after tpbx. serum wbc and crp did not rise significantly on the day after tpbx compared with before tpbx (p>0.05). there was no significant difference in the rise of serum wbc and crp before and after tpbx in the comparison of lvfx 500 mg with lvfx 300 mg in the taz / pipc plus lvfx regimen.conclusionstaz/pipc plus lvfx can be considered as a prophylactic regimen for preventing infectious complications in tpbx. |
type 1 diabetes mellitus (t1 dm) is characterized by a loss of beta cell mass due to an autoimmune process. in a phase prior to the onset, immune cells infiltrate the islets creating an inflammatory microenvironment responsible for the -cell - specific toxicity. proinflammatory cytokines, such as interleukin-1 beta (il-1), tumor necrosis factor alpha (tnf-), and interferon gamma (ifn-), secreted by activated lymphocytes and macrophages during insulitis, induce nfkb mediated inos expression which has a key role in -cell apoptosis in the early t1 dm stage. it is produced in tissues by the enzyme nitric oxide synthase (nos) by oxidation of the amino acid l - arginine. there are three different tissue - specific nos isoforms : ca / calmodulin - independent and inducible nos (inos), neuronal nos (nnos), and endothelial nos (enos). these last two isoforms also exhibit constitutive expression and ca / calmodulin - dependent activity. expression of nos isoforms has been studied in pancreatic islets, and the nnos form has been observed in -cell cells, located, mainly, in insulin secretory granules. it exhibits cytochrome c reductase activity in addition to no production activity, and a balance between both functions is essential for a normal insulin secretion in response to glucose stimulation. in contrast, enos expression has not been found in rat islets under basal conditions [2, 3 ]. prooxidant agents formed from no induce damages in dna which activate p53 which induces proapoptotic expression of genes such as bax, fas, noxa, and puma. a p53-independent mechanism has been described in which no acts by inhibiting the sarcoendoplasmic reticulum pump ca atpase 2b (serca2b) protein and, subsequently, by inducing the endoplasmic reticulum stress mechanisms [4, 5 ]. the importance of the mitochondrial pathway has also been highlighted in no - mediated -cell cell apoptosis [6, 7 ]. paradoxically, an antiapoptotic effect of low concentration of no has been reported in several systems, including cells [812 ]. in addition to apoptosis, cell proliferation has been described as a no - regulated process. although certain activating effects have been reported in physiological systems [13, 14 ], the main role of no in cellular proliferation is inhibitory. in the subventricular zone, no induces inhibition of stem cell proliferation by a nitrosylation process. other proposed mechanism for no antiproliferative action is a g1-s inhibition mediated by an induction of the cell cycle inhibitor p21 or cyclins inhibition. recently, no - mediated neogenesis stimulation has been observed in an alloxan - induced murine model of diabetes. a proinflammatory cytokine - mediated inhibition of cultured -cell proliferation in addition to the apoptotic effect has been observed by our group. as described previously, no production is one of the most important events mediated by proinflammatory cytokines. therefore, the aim of this study was to assess the importance of no in the cytokine - mediated antiproliferative effect on cultured cells and to determine the role(s) of different no synthase isoforms present in pancreatic islets. all animal procedures were performed with the approval of the cdiz university school of medicine (cdiz, spain) committee for the ethical use and care of experimental animals. bio - breeding (bb) and wistar rats were kept under conventional conditions in an environment - controlled room (20 - 21c, 12 h light - dark cycle) with water and standard laboratory rat chow available ad libitum and their weight was daily recovered. blood extracted from the tail vein was used in bb rats for weekly random glucose measurements using an automatic glucose monitor (accu - chek optimum, roche diagnostic, basel, switzerland). pancreatic islets were isolated from adult male wistar rats as described previously by mcdaniel.. louis, mo, usa) supplemented with 2 mm l - glutamine (gibco invitrogen, carlsbad, ca, usa), 100 u / ml penicillin, 100 g / ml streptomycin (pen - strep, bio - whittaker europe, verviers, belgium), and 10% fetal bovine serum (fbs, gibco invitrogen, carlsbad, ca, usa). proinflammatory cytokines (peprotech ec ltd, london, uk) used in the experiments were recombinant human il-1 (50 u / ml), recombinant rat ifn- (1000 u / ml), and recombinant rat tnf- (1000 u / ml). these concentrations were selected as being appropriate based on the results of previous published studies [20, 21 ]. no donors, s - nitroso - n - acetyl - dl - penicillamine (snap), and diethylenetriamine / nitric oxide adduct (deta - no), obtained from sigma - aldrich (st. louis, mo, usa) show different no release rates. deta - no is a member of the nonoates family and has a half - life (t1/2) of 20 h at 37c, ph 7.4. the t1/2 of snap is approximately 6 h at 37c and ph 68 although its biological activity is highly influenced by the molecular environment of the parent thiol. inhibitors of nos 7-nitroindazole (7-ni), n-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide, dihydrochloride (1400 w), and n5-(1-iminoethyl)-l - ornithine, dihydrochloride (l - nio), purchased from sigma - aldrich (st. louis, mo, usa) were used to inhibit the activity of neuronal, inducible, and endothelial isoforms of nos, respectively. proliferating cells were detected using 5-bromo-2-deoxyuridine (brdu) 5 mol / l label (sigma - aldrich, st. louis, mo, usa) which was added to the cultures from the start of the assay together with the drugs being tested, when appropriate. following the appropriate culture duration, the islets were recovered and incubated for 15 min with trypsin - edta (0.25% trypsin, 1 mm edta) in hanks ' balanced salt solution without ca and mg (gibco invitrogen, carlsbad, ca, usa) at 37c, and the islets were gently dispersed. after washing with pbs, cells were cytospin on poly - l - lysine - coated slides and fixed in 4% methanol - free formaldehyde. slides were immuno - stained using monoclonal mouse anti - brdu (dako cytomation, denmark) and polyclonal guinea pig anti - insulin (sigma - aldrich, st. cells were permeabilized by incubation for 30 min with 0.1% triton - x100 in pbs and washed twice with 100 mm glycine buffer containing 0.1% triton - x100 and 3% bovine serum albumin. cells were then treated with hcl (2n) in pbs for 30 min, neutralized with borax / borate buffer (0.1 m, ph 8.9) for 30 min, washed, and incubated overnight at 4c with anti - brdu and anti - insulin antibodies. stained cells were revealed using anti - mouse igg antibody (alexa-546 conjugated) and anti - guinea pig igg (alexa-488 conjugated) antibody (molecular probes inc, eugene, or, usa). cell nuclei were stained with 4-6-diamidino-2-phenylindole (dapi). to determine the proliferating fraction, total and insulin - positive / brdu - positive cells were analyzed using a fluorescence microscope in randomized conditions, by a single investigator. for statistical purposes, 48 h cultured islets in presence and absence of cytokines were processed for histology study by histogel and paraffin inclusion. 10 m sections were stained using a polyclonal rabbit anti - inos (ab15323, abcam, cambridge, uk) and a polyclonal guinea pig anti - insulin (sigma - aldrich, st. louis, mo) and examined using a microscope equipped with a digital camera and the image analysis celldsoftware (olympus, hamburg, germany). randomly grouped bb rats daily received an intraperitoneal injection of vehicle (0.1% dmso diluted in injection water) alone or containing l - nmma at a dose of 30 mg / kg, from 4 to 9 weeks of age. animals were sacrificed at 4 (before treatment), 7, and 9 weeks of age. wistar, bb, and thymectomized bb rats were fasted overnight (1618 h) and a blood sample was collected from the tail vein (fasting or 0 min sample). then, an intraperitoneal injection of 40% solution of glucose was administered (2 g / kg), followed by blood sampling at 15, 30, 60, and 120 min after the glucose administration. glycemia was measured with an automatic glucose monitor (accu - chek optimum, roche diagnostic, basel, switzerland). blood samples obtained from fasting and 15 min after glucose administration were also used to plasma extraction and insulin quantification by elisa technique (insulin rat ultrasensitive kit. weeks of age bb rat pancreas were resected, weighed, fixed in bouin 's solution for 6 h, and postfixed in formalin for 12 h. then, they were dehydrated, paraffin embedded, and longitudinal 10 m microtome sections were obtained. insulin was stained in obtained pancreas by immunohistochemical techniques using a mouse anti - rat insulin monoclonal antibody and a peroxidase conjugated goat anti - mouse igg antibody and revealed with dab kit (sigma aldrich, st. insulin - positive areas were quantified in two complete sections of each pancreas using a microscope equipped with a digital camera and the image analysis cell d software (olympus, hamburg, germany). -cell mass values were calculated by multiplying the total insulin - positive area / total pancreatic area ratio by the total pancreas weight. equivalent numbers of islets under the different experimental conditions described previously were lysed in lysis buffer (125 mm tris - hcl buffer ph 68, 2% sds, 1 mm dtt and containing protease and phosphatase inhibitors). protein (40 g) was loaded and electrophoresed on 8% sds - page. proteins were transfer - blotted on to polyvinylidene fluoride (pdfv) membranes and then incubated in blocking buffer (5% nonfat milk in 10 mm tris - hcl, 1.15 m nacl and 0.1% tween-20) for 1 h at room temperature. the blots were then incubated with rabbit polyclonal antibody against enos (santa cruz biotechnology, santa cruz ca, usa) and inos (transduction laboratories bd, nj, usa) overnight according to the manufacturer 's instructions, followed by incubation with peroxidase conjugated anti - rabbit igg for 1 h at room temperature. stained bands were revealed using immun - star western c kit (bio - rad, hercules ca, usa) and quantified by quantity one software version upgrade (bio - rad, hercules, ca, usa). -tubulin expression was used as loading control and enos expression was calculated as ratio of enos : -tubulin densities. histological examination of 7 and 9 weeks of age rat pancreatic islets was performed in harris ' h&e stained pancreas sections using 20 objective lens. the severity of insulitis was graded as a function of the mononuclear cell infiltration of the pancreatic islets : 0 = no infiltrate ; 1 = periductular infiltrate ; 2 = periislet infiltrate ; 3 = intraislet infiltrate ; 4 = intraislet infiltrate associated with -beta cell destruction. thirty islets were examined in each pancreas and the mean score was calculated by dividing the total score by the number of islets examined. results are presented as means sem of measurements performed in at least 3 animals. to determine the role of no donors on -cell proliferation, pancreatic islets were treated with proinflammatory cytokines or different concentrations of no donors snap and deta, alone or in combination with the caspase-3 inhibitor z - vad - fmk. -cell proliferation measured by brdu incorporation showed that the no donors, deta - no (figure 1(a)) and snap (figure 1(b)) exert an antiproliferative action on cells in a dose - dependent manner. this effect of no donors was not modified by addition of z - vad - fmk to the cultures. to determine the contribution of no to the antiproliferative effect of proinflammatory cytokines on pancreatic cells, pancreatic islets were cultured over a 48 h period and treated with proinflammatory cytokines, alone or in the presence of l - nmma (an inhibitor of nitric oxide synthase). inhibition of -cell proliferation induced by proinflammatory cytokines was completely abolished by l - nmma treatment (figure 2). guanylate cyclase is the enzyme through which no exerts the greater part of its effect. to assess the implication of the guanylate cyclase pathway in the no effect on -cell proliferation, cultures of pancreatic islet were treated for 48 h with 8br - cgmp (a guanylate cyclase analogue) and its inhibitor odq, alone or in combination with proinflammatory cytokines. as shown in figure 3, the cgmp analogue exerts no effect on -cell proliferation, and the antiproliferative effect of cytokines is not altered by the cgmp inhibitor odq. to study the role of constitutive and inducible nos isoforms in the antiproliferative action of cytokines, cultured pancreatic islets were treated with proinflammatory cytokines in addition to 1400 w and 7-ni, inhibitors of inducible and constitutive isoforms of nitric oxide synthase, respectively ; the resultant -cell proliferation was quantified. the antiproliferative effect of proinflammatory cytokines underwent no modification when islet cultures were incubated with the constitutive nos inhibitor (figure 4(e)). related to inos, cytokine - dependent induction of protein was observed from 24 h of culture (figure 4(a)). inos expression study by immunohistochemical techniques in control cultured pancreatic islets showed a scarce stain with peripheral profile. however, cultured islet in presence of cytokines showed an increased inos expression localized internally in the islet (figure 4(b)). the specific inhibitor 1400 w partially reverted cytokine - induced decreased proliferation in a dose - dependent manner (figure 4(d)). endothelial isoform of nos expression was initially examined in pancreatic islets cultured under control condition, or after stimulation with proinflammatory cytokines. as shown in figure 4(c), enos isoform was expressed in pancreatic islets and was not altered by exposure to proinflammatory cytokines. to study the role of enos on the antiproliferative effect of cytokines, l - nio (an inhibitor with higher affinity for enos than the other isoforms) a partial inhibition of the antiproliferative effect of proinflammatory cytokines was observed in treated cultures (figure 4(f)) mediated by l - nio. weight and random glycemia monitoring along treatment period show no differences in those parameters between control and l - nmma - treated animals. glucose homeostasis was studied by ipgtt and insulin determination before and 15 min after intraperitoneal glucose administration. as shown in figure 5, ipgtt displays a normal curve at studied times in l - nmma treated as in control animals (figure 6(a)). in addition, insulin quantification also shows no changes between control and treated animals at none of studied times (figure 6(b)). bb rats treated with l - nmma from 4 to 7 or 9 weeks of age were sacrificed and pancreas was removed from each rat to quantify beta cell mass by immunohistological techniques. in figure 7, a beta cell mass loss undergone by bb rats between 7 and 9 weeks of age can be observed. l - nmma reverts this effect at both studied times. to test the effect of l - nmma treatment on infiltration levels, h&e - stained sections obtained from treated and control bb rats were evaluated and infiltration scores were calculated. no changes were observed in infiltration level in response to l - nmma at none of studied times (figure 8). currently, no is considered an important mediator of cell signaling via its classical guanylate cyclase pathway or by exerting posttranslational modifications on an increasing number of proteins and hence regulating their activity. in the present study, we analyzed the role of no in the previously described antiproliferative action of proinflammatory cytokines on pancreatic cells. loss of membrane integrity and cytochrome c release induced by deta - no and snap in insulin producing cell lines has been reported [22, 23 ]. in our system, no donors snap and deta - no induced a decrease in cultured -cell proliferation at concentrations up to 50 m for deta - no and up to 400 m for snap. this effect is dose dependent and similar to that obtained with proinflammatory cytokines (il-1 + tnf- + ifn-) treatment (figure 1). the antiproliferative effect observed in response to no donors is not dependent on the biological t1/2 and suggests that no exerts its biological action in the first hours after exposure. to discard the possibility that the decrease in proliferation was due to a cytokine - induced apoptosis in proliferating cell, we treated pancreatic -cell cultures with the caspase-3 inhibitor zvadfmk, in addition to the no donors. -cell apoptosis inhibition induced by zvadfmk exerts no effect on the antiproliferative effect of no donors suggesting that the two processes are unrelated. although studies in neonatal rat islets indicated that cytokine - induced no production regulates the expression of up to 42 proteins, a considerable number of proteins are regulated in a no - independent manner. thus, the cytokine - induced apoptosis in mouse cells is not caused by the inducible isoform of nos alone. endoplasmic reticulum stress has been described as a possible mechanism by which no induces apoptosis. its presence regulates unfolded protein response (upr) signaling pathway that determines apoptotic response to reticulum stress. in addition to cytokines, there is evidence of no participation in mitochondrial dysfunction and cell death occurring in pc12 cells in response to amyloid. results from our study suggest that, in addition to apoptosis, no can be implicated in the antiproliferative effect of proinflammatory cytokines on pancreatic cell since treatment with nos inhibitor l - nmma is capable of reverting this cytokine - induced effect (figure 2). very few studies have investigated the possible mechanisms underlying the effect of no on cellular proliferation. in pulmonary microvascular smooth muscle cells, exogenous no upregulates p21, a protein which mediates cell cycle arrest. further, no is capable of inhibiting erk 1/2 and akt activation in breast cancer cell lines and pancreatic cells [29, 30 ]. these results are in accord with those observed by our group ; that is, proinflammatory cytokines induced an inhibition of erk 1/2 in islet cultures and in early stages of insulitis. however, no has also been described as an inducer of proliferation, when acting at low concentrations. for example, in mesangial cells, the no donor snap increases proliferation at concentrations of up to 200 m and inhibits proliferation at higher concentrations, in a process implying akt signaling and cox-2 protein. activation of no - sensitive guanylyl cyclase by no binding to the enzyme 's prosthetic heme group has been described as one of the most important no - mediated cellular effects. nevertheless, results observed in our system suggest that this pathway is not involved in the no - mediated antiproliferative effect observed in cells from islets treated with proinflammatory cytokines (figure 4). these results are in accord with those previously reported in tumor cell lines in which the proliferative effect of low no concentration is mediated by the cgmp pathway, but the antiproliferative effect induced by high concentrations of no is independent of this pathway. a proposed cgmp - independent mechanism of no action is its direct interaction with protein and, as such, performing posttranslational modifications such as nitration or s - nitrosylation. for example, s - nitrosylation of egf receptor has been reported in human neuroblastoma nb69 as a mechanism of no - mediated antiproliferative effect. the role of nos isoforms in no production in response to proinflammatory cytokines has not been well defined. our results show that inos, the expression of which is induced in our system after 24 h culture in the presence of proinflammatory cytokines, is implicated while constitutive isoform is not implicated in cytokine - induced antiproliferative response in cells (figure 4). of note is that although inos inhibitor 1400 w inhibited the cytokine - mediated antiproliferative effect, its effect was only partial, and was unlike the total reversion by l - nmma (a general nos inhibitor). it is also interesting the effect of 1400 w increasing beta cell proliferation in absence of cytokines which could be due to a certain level of islet cells inos activation induced by islet isolation process. this partial effect of 1400 w suggests that other nos isoforms are participating in antiproliferative response to cytokines. endothelial nos determination by western blotting was performed in cultured rat islets in the presence of proinflammatory cytokines and, contrary to the results reported by lajoix., enos expression was observed. this expression is maintained in culture and was not affected by the presence of cytokines (figure 4(b)) and could be attributed to endothelial cells in the profuse vascular network present in pancreatic islets. once the presence of enos in islets is confirmed, its implication in -cell antiproliferative response to cytokines was tested in islet cultures treated with l - nio (an inhibitor whose primary target is enos) alone or in combination with cytokines. l - nio induced a partial reversion of cytokine - induced -cell antiproliferation in a similar manner to that induced by the inos inhibitor 1400 w. these results suggest participation of both no isoforms in the antiproliferative response induced by proinflammatory cytokines, possibly by enos producing no in a paracrine manner. in this sense, it has recently reported that inflammatory environment induces changes in enos which may adopt functional features of inos in a ca - independent way. to assess no involvement in the pathophysiology of t1 dm, we treated biobreeding rats, an animal model of autoimmune diabetes, with the general nos inhibitor l - nmma at early stages of the insulitis process. previous observations from our group indicated a halting of -cell proliferation between 4 and 7 weeks of age and which can be reversed by administering neutralizing antibody against ifn-. treatment with l - nmma of bb rats from 5 weeks of age induced a reversal of -cell proliferation loss observed in untreated animals (figure 5). in addition, a relative recovery of -cell area with a regional pattern in treated animals at 7 weeks of age was observed. the effects, either on proliferation of cell or on -cell area, were very much more evident in the head than in the tail of the pancreas. a similar regional pattern of response was reported in earlier studies using a malnutrition model. a plausible explanation for this regional effect could be a different vascularization in the head area compared to that in the tail which precluded equivalent drug availability in all areas of the pancreas volume. further, a different response to environmental factors due to a different islet composition could underlie this observed effect. t1 dm prevention strategies focus, essentially, on early stages of the disease before clinical onset when there is enough -cell mass in the pancreas to maintain its metabolic function. our present study highlights the importance of no as a mediator of the observed impaired -cell proliferative response from the initial stages of insulitis. this impairment in proliferative capacity precludes an appropriate regenerative response of cells to autoimmune damage. our data also highlight the property of l - nmma to maintain -cell proliferative capacity in early insulitis stage and, as such, confirms that nitric oxide might be an important therapeutic target in the strategies of t1 dm prevention. | nitric oxide (no) is involved in several biological processes. in type 1 diabetes mellitus (t1 dm), proinflammatory cytokines activate an inducible isoform of nos (inos) in cells, thus increasing no levels and inducing apoptosis. the aim of the current study is to determine the role of no (1) in the antiproliferative effect of proinflammatory cytokines il-1, ifn-, and tnf- on cultured islet cells and (2) during the insulitis stage prior to diabetes onset using the biobreeding (bb) rat strain as t1 dm model. our results indicate that no donors exert an antiproliferative effect on cell obtained from cultured pancreatic islets, similar to that induced by proinflammatory cytokines. this cytokine - induced antiproliferative effect can be reversed by l - nmma, a general nos inhibitor, and is independent of guanylate cyclase pathway. assays using nos isoform specific inhibitors suggest that the no implicated in the antiproliferative effect of proinflammatory cytokines is produced by inducible nos, although not in an exclusive way. in bb rats, early treatment with l - nmma improves the initial stage of insulitis. we conclude that no is an important mediator of antiproliferative effect induced by proinflammatory cytokines on cultured cell and is implicated in -cell proliferation impairment observed early from initial stage of insulitis. |
recognition of the importance of ion channel regulation by phosphoinositides (pips), especially phosphatidylinositol 4,5-bisphosphate (ptdins(4,5)p2), continues to grow. in earlier studies, the effects of ptdins(4,5)p2 application were examined directly, or a ptdins(4,5)p2 antibody, ptdins(4,5)p2 scavenger (poly - l - lysine) or a pharmacological inhibitor (wortmannin or quercetin) was used to reduce the membrane ptdins(4,5)p2 content. these methods remain solid and are well established, but their utility is limited to assessing steady - state ptdins(4,5)p2 levels. by contrast, methods for using a chemically inducible pip control system and voltage - sensing phosphatase (vsp) are becoming a standardized means of surveying dynamic pip regulation. chemical induction of pips is well described in a recent review article. in this addendum, therefore, we have focused on the advantages of using vsp to study channel regulation. so far vsps from two aquatic species, ciona intestinalis (ci) and danio rerio (dr), have been identified. ci- and drvsp exhibit only small differences, mainly in their voltage - sensitivities and expression levels, and their catalytic activities (pi5-phosphatase) and substrate phosphoinositide (pi) specificities are nearly identical. nonetheless, we think that drvsp may be somewhat better suited for studying pi - mediated regulation of ion channels. this is because gating currents indicate the level of drvsp expression in hek cells to be two to three times higher than that of civsp, and because the voltage - sensitivity of drvsp is shifted rightward by about 50 mv (v12 values from the q - v curves are 94 and 44 mv for drvsp and civsp, respectively). considering that the resting membrane potential is around 40 to 10 mv in hek cells, the enzymatic phosphatase activity driven by the resting potential is more negligible with drvsp. in addition, the rather steep q - v curve for drvsp would be expected to yield more dramatic effects. when we combined drvsp with trpc channels in hek cells, a depolarizing pulse to + 100 mv for 500 ms was sufficient to produce maximum channel inhibition through depletion of ptdins(4,5)p2 (referred to as vmi, vsp - mediated inhibition was evaluated based on the residual current (r) before and after the depolarization). however, such robust depolarizations can have unfavorable effects on channel regulation, e.g., the voltage - dependent inhibition relief observed with high voltage - gated ca channels. to exclude this possibility, we confirmed our results using various voltage - sensing drvsp derivatives [vmi (r)-v curve ], as shown in figure 1a. note that the v - shifted drvsps all inhibited trpc currents to the same degree as wild - type drvsp. these data support our conclusion and further emphasize the advantages of using drvsp to regulate the activities of a variety of ion channels, including voltage - gated channels. (a) top : exemplar of the voltage - dependence of vmi (r) of trpc3/c6/c7 currents observed in hek cells. trpc6 currents were evoked by external application of the dag lipase inhibitor rhc80267 (100 m). bottom : vmi of trpc6 currents plotted against depolarization pulse amplitude applied in the presence of the indicated drvsp mutants (n = more than 4). note that the q - v curves of the mutants are also shifted leftward [v1/2(off ] values for r153q, t156r and i165r are 16, 73 and 60 mv, respectively). (b) top : atypical inhibition trace obtained from hek cells co - transfected with trpc6 and wild - type drvsp. brief depolarizations (+ 100 mv, 500 ms) were applied every 10 sec (protocol displayed in top), and currents were evoked by cch (100 m). middle and bottom : r and -recovery are plotted against stimulus number from the upper trace. the blue dashed line in the middle panel suggests drvsp - available ptdins(4,5)p2 (speculative). the typical trace, averaged r, and averaged -recovery data were shown in ref. vsp has enormous potential for use as a molecular tool with which to clarify the ptdins(4,5)p2 sensitivity and binding kinetics of ion channels, even during periods when ionic currents are flowing. in figure 1b, we present an atypical example of the inhibition of carbachol (cch)-induced trpc6 currents (upper) via vsp activation and subsequent current recovery. in contrast to the typical and averaged data presented in our recent study, in 2 of 11 cells tested, we observed responses like those depicted in the figure [i.e., there was a clear bell- or u - shaped relationship between stimulus number and vmi (r) (middle) and -recovery from the inhibition (bottom) ]. the bell - shaped r curve was closely related to the peak cch - induced current. moreover, -recovery accelerated as the cch - induced current and the value of r became larger. this observation raises the possibility that even during agonist - stimulated macroscopic activity in living cells, vmi magnitude may provide a clue to the level of ptdins(4,5)p2 binding to the channels, as well as to the kinetics of evoked changes in ptdins(4,5)p2 binding. the latter would also reflect to some degree ptdins(4,5)p2 re - synthesis, which would be expected to influence the observed response. in an earlier study, hardie., showed dynamics of living ptdins(4,5)p2 which was accompanied with the light response in drosophila photoreceptors by measuring currents through the ptdins(4,5)p2-sensitive kir2.1 channel. however, we suggest the use of ectopic vsp is an alternative or a more convenient approach because vsp does not itself produce ionic flow other than gating currents. because bioelectric signals are largely attributable to the flow of ions, it is critically important to maintain ion channel activities for appropriate durations. to shorten the duration of ion flow, channels, particularly those contributing to excitation, often possess self - limiting regulatory systems. for instance, figure 2a to c illustrate the mechanisms of self - limiting regulation found in voltage - gated sodium channels, high voltage - gated calcium channels and inotropic atp receptors. the mechanism underlying the self - limiting regulation of trpc3/c6/c7 channels (fig. trpc3/c6/c7 channels are intracellular ligand - gated channels assembled as homo- or heterotetramers, and are activated by dag produced through a reaction catalyzed by g protein- or receptor tyrosine kinase - coupled plc. but when dag is produced from its substrate ptdins(4,5)p2, the resultant reduction in membrane ptdins(4,5)p2 content independently inhibits channel activation. thus both activation and inhibition are simultaneously induced by plc - catalyzed degradation of ptdins(4,5)p2. as a result, the time required for the response to reach the first current peak is more susceptible to modulation than the other modes of self - limiting regulation. (a) : voltage - gated sodium channels (rnav1.2) in vertebrates open (+) and quickly inactivate () in response to depolarization (1 and 2 : difference in the potentials coordinate the v - shaped current trace). (b) : high voltage - gated ca channels (cav1.2) open upon membrane depolarization (1) and inactivate due to negative feedback regulation wherein ca permeating through the channels inactivates the channels (calcium - dependent inactivation). (c) : ligand - gated cys - loop receptors (p2x1) are activated and then desensitized by the concentration - dependent binding of an agonist (a). copyright (1996) national academy of sciences, usa (d) : trpc3/c6/c7 channels are activated by dag, a product of ptdins(4,5)p2, and the resultant reduction in ptdins(4,5)p2 independently inhibits channel opening. strength linkages with g protein - coupled receptors alter the kinetics in trpc6 currents (tight and loose linkages are represented by the red and black traces, respectively). (e) : currents obtained under self - limiting conditions exhibit faster inactivation than those obtained under unlocked conditions induced by application of excessive ptdins(4,5)p2 through the patch - pipette (trpc7 currents obtained from ref. trpc channels are often activated by neurohormones released from autonomic nerves and can be thought of as being downstream of the autonomic nervous system (ans). the ans is important for maintenance of the stable internal physiological conditions often referred to as homeostasis. this makes the self - limiting regulation of trpc channels interesting in part because the resultant regulation of the global effects of the ans appears to arise from the molecular level. enzymatic reactions are often involved in cell signaling through, for example, an increase in the concentration of a product. on the other hand, the functionality of substrate reduction or depletion is easily masked by the reduction in the productivity of the catalytic reaction. consequently, signaling systems mediated by enzymatic reactions might be expected to exhibit physiologically bimodal biochemical responses. the self - limiting regulation linked to plc activity could thus provide profound mechanistic insight of channel modulation as well as rediscovering subtle feature of enzymatic functionality. furthermore, when this regulation is disrupted (which can be seen experimentally in the case of cch - induced currents in the presence of an excess of the substrate analog dic8-ptdins[(4,5)p2 ], the decay phase of the currents is prolonged (fig. 2b), and the ensuing buildup of cytosolic na and/or ca can have significant pathophysiological effects that could underlie the development of such ailments as vascular hypertension, cardiac hypertrophy and renal failure. overall, our results emphasize that trpc3/c6/c7 channels are subject to self - limiting regulation that is related to their close association with the ptdins(4,5)p2-plc - dag cascade and is distinct from the self - limiting mechanisms observed in voltage - gated channels. the specific physiological importance of this type of regulation, as compared with other modes of channel regulation, will be an important area of investigation in the future. | trpc3/c6/c7 channels, a subgroup of classical / canonical trp channels, are activated by diacylglycerol produced via activation of phospholipase c (plc)-coupled receptors. recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. we recently used a membrane - resident danio rerio voltage - sensing phosphatase (drvsp) to study trpc3/c6/c7 regulation and found that the channel activity was controlled by ptdins(4,5)p2-dag signaling in a self - limiting manner (imai y., the journal of physiology, 2012). in this addendum, we present the advantages of using drvsp as a molecular tool to study ptdins(4,5)p2 regulation. drvsp should be readily applicable for studying phosphoinositide metabolism - linked channel regulation as well as lipid dynamics. furthermore, in comparison to other modes of self - limiting ion channel regulation, the regulation of trpc3/c6/c7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. dysfunction of such self - limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists. |
during a 10-month period, 30 non - consecutive patients (6 males and 24 females [age range ; 32 - 80 years, mean age ; 53.7 years ]) with 30 rccs that were detected on ct scan were referred to us for performing us before the masses were histologically confirmed. all of the patients underwent a gray - scale us and an adi scan that were performed by one radiologist (b.k.p.). all the patients gave us an informed written consent for the adi, and this study was approved by the institutional review board of the seoul national university hospital, korea. the rccs measured from 1.5 cm to 10 cm in size (meanstandard deviation ; 4.92.7 cm) and they consisted of 27 clear cell, two papillary and one chromophobe type masses. partial (n=1) or total nephrectomies (n=29) were performed on all the patients. on the pathologic exams, pseudocapsule and intratumoral cyst or necrosis were found in 93% (28/30) and 90% (27/30) of all the rccs. the us contrast agent used in this study was sh u 508a (levovist ; schering ag, berlin, germany) ; it consists of galactose microparticles (99.9%) and palmitic acid (0.1%). four grams of sh u 508 a at a concentration of 300 mg / ml were intravenously administered by a manual bolus injection via an 18-gauge cannula inserted into an antecubital vein. the us exams were performed with a high - resolution us unit (sequoia ; siemens medical solutions, mountainview, ca, usa) that was equipped with adi software. the acoustic power of the adi was adjusted to the default maximal setting (mechanical index, 1.9). a 4c1 convex - array probe only was used for the adi scan. before the adi scan, the gray - scale exam was conducted (a) in order to determine the optimal imaging plane to display the renal masses because the shorter the distance between the probe and the renal masses, the stronger the signal, (b) to compare the diagnostic accuracy between the gray - scale us and the adi. real - time scanning commenced immediately after the intravenous injection of the contrast agent, and the scan time was measured when the contrast agent passed the intravenous cannula. when the first signal of contrast agent appeared in the kidney, we rapidly moved the probe to cover the region including the lesions. rapid sweeping was repeated every 10 - 15 seconds (interval delay scan). for each sweep, we used clip store that can record the continuous images displayed on the monitor during a 6-second period, in a manner similar to video recording, and we also captured still images by employing cine loops. all of the scanned images were automatically stored in the picture archiving communication system (marotech, seoul, korea). h.j.c) working in consensus retrospectively analyzed all of the us images for the enhancement patterns and the presence or absence of intratumoral anechoic areas and pseudocapsule. the enhancement patterns of the tumor were classified as homogeneous, heterogeneous, stippled and peripheral irregular enhancement. homogeneous enhancement was defined as an even signal intensity over the whole tumor with no signal defects. peripheral irregular enhancement was defined as a discontinuous ring of contrast - enhanced peripheral nodules. intratumoral cysts or necrosis were noted depending on whether the contour of the signal defect was smooth or irregular. pseudocapsule was defined as a hypoechoic halo on the gray - scale us and as a peritumoral rim that showed no enhancement in the early phase and delay enhancement in the late phase of adi. we retrospectively compared the diagnostic accuracy for rcc between the gray - scale us and the adi. on the gray - scale us examination, when the lesion had more than two imaging features among intratumoral anechoic areas, pseudocapsule and hyperechogenicity relative to that of the normal renal parenchyma, then we made a diagnosis of rcc. on the adi exam, when the lesion had more than two imaging features among the most common enhancement patterns of rcc, intratumoral anechoic areas and pseudocapsule, then we made a diagnosis of rcc. we then compared the diagnostic accuracy for rcc between the gray - scale us and the adi exam. fisher 's exact test was used to compare the imaging features of rcc that were seen on gray - scale us and adi. the diagnostic accuracy, which included the sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy for rcc on the adi exam, was calculated and then compared with those of the gray - scale us by using the mcnemar test. during a 10-month period, 30 non - consecutive patients (6 males and 24 females [age range ; 32 - 80 years, mean age ; 53.7 years ]) with 30 rccs that were detected on ct scan were referred to us for performing us before the masses were histologically confirmed. all of the patients underwent a gray - scale us and an adi scan that were performed by one radiologist (b.k.p.). all the patients gave us an informed written consent for the adi, and this study was approved by the institutional review board of the seoul national university hospital, korea. the rccs measured from 1.5 cm to 10 cm in size (meanstandard deviation ; 4.92.7 cm) and they consisted of 27 clear cell, two papillary and one chromophobe type masses. partial (n=1) or total nephrectomies (n=29) were performed on all the patients. on the pathologic exams, pseudocapsule and intratumoral cyst or necrosis were found in 93% (28/30) and 90% (27/30) of all the rccs. the us contrast agent used in this study was sh u 508a (levovist ; schering ag, berlin, germany) ; it consists of galactose microparticles (99.9%) and palmitic acid (0.1%). four grams of sh u 508 a at a concentration of 300 mg / ml were intravenously administered by a manual bolus injection via an 18-gauge cannula inserted into an antecubital vein. the us exams were performed with a high - resolution us unit (sequoia ; siemens medical solutions, mountainview, ca, usa) that was equipped with adi software. the acoustic power of the adi was adjusted to the default maximal setting (mechanical index, 1.9). a 4c1 convex - array probe only was used for the adi scan. before the adi scan, the gray - scale exam was conducted (a) in order to determine the optimal imaging plane to display the renal masses because the shorter the distance between the probe and the renal masses, the stronger the signal, (b) to compare the diagnostic accuracy between the gray - scale us and the adi. real - time scanning commenced immediately after the intravenous injection of the contrast agent, and the scan time was measured when the contrast agent passed the intravenous cannula. when the first signal of contrast agent appeared in the kidney, we rapidly moved the probe to cover the region including the lesions. rapid sweeping was repeated every 10 - 15 seconds (interval delay scan). for each sweep, we used clip store that can record the continuous images displayed on the monitor during a 6-second period, in a manner similar to video recording, and we also captured still images by employing cine loops. all of the scanned images were automatically stored in the picture archiving communication system (marotech, seoul, korea). h.j.c) working in consensus retrospectively analyzed all of the us images for the enhancement patterns and the presence or absence of intratumoral anechoic areas and pseudocapsule. the enhancement patterns of the tumor were classified as homogeneous, heterogeneous, stippled and peripheral irregular enhancement. homogeneous enhancement was defined as an even signal intensity over the whole tumor with no signal defects. peripheral irregular enhancement was defined as a discontinuous ring of contrast - enhanced peripheral nodules. intratumoral cysts or necrosis were noted depending on whether the contour of the signal defect was smooth or irregular. pseudocapsule was defined as a hypoechoic halo on the gray - scale us and as a peritumoral rim that showed no enhancement in the early phase and delay enhancement in the late phase of adi. we retrospectively compared the diagnostic accuracy for rcc between the gray - scale us and the adi. on the gray - scale us examination, when the lesion had more than two imaging features among intratumoral anechoic areas, pseudocapsule and hyperechogenicity relative to that of the normal renal parenchyma, then we made a diagnosis of rcc. on the adi exam, when the lesion had more than two imaging features among the most common enhancement patterns of rcc, intratumoral anechoic areas and pseudocapsule, then we made a diagnosis of rcc. we then compared the diagnostic accuracy for rcc between the gray - scale us and the adi exam. fisher 's exact test was used to compare the imaging features of rcc that were seen on gray - scale us and adi. the diagnostic accuracy, which included the sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy for rcc on the adi exam, was calculated and then compared with those of the gray - scale us by using the mcnemar test. on the adi exam, the rccs showed as being heterogeneous in 87% of the patients (26/30), homogeneous in 7% of the patients (2/30), and peripheral irregular enhancement in 7% of the patients (2/30). heterogeneous enhancement was the most common enhancement pattern of the rccs, and thus, it was included into the diagnostic criteria for rcc on the adi exams (figs. 1, 2). the rccs revealed intratumoral anechoic areas in 87% of the cases (26/30), and intratumoral cysts (n=25), necrosis (n=21) or both (n=20) were also noted (figs. 1, 2). on the gray - scale us, the intratumoral anechoic areas were seen in 53% of the cases (16/30), of which ten of the rccs were not able to be classified into cysts or necrosis. the intratumoral anechoic areas were more commonly detected on the adi exam than on the gray - scale us (p < 0.05). pseudocapsule was seen in 77% of the cases (23/30) of rcc on the adi exam, whereas it was seen only in 17% of the cases (5/30) of rcc on the gray - scale us (p < 0.05) (figs. 1, 2). rccs appeared as hyperechoic in 90% of the cases (27/30), and as iso or hypoechoic in 10% of the cases (3/30), relative to the normal renal parenchyma (figs. 1, 2). table summarizes the diagnostic accuracy of rcc with using adi and gray - scale us. when the presence of two or more adi features was noted out of heterogeneous enhancement, intratumoral anechoic areas and pseudocapsule, then this was regarded as positive findings for rcc, and the sensitivity, specificity, and accuracy for rcc were 97%, 93%, and 95%, respectively. however, those for the rcc on the gray - scale us were 70%, 86%, and 78%, respectively and its diagnostic accuracy were significantly different from that of the adi (p < 0.05). agent detection imaging is a microbubble - specific harmonic us modality that is designed for the optimal detection of signals from sh u 508a with using high mechanical index technique (8, 9). it can produce signals from stationary microbubbles as easily as from those that are moving in the vessels, and this unlike conventional us that relies on doppler shift signals. other techniques of the gray - scale contrast - enhanced harmonic imagings such as pulse inversion harmonic imaging (pihi) and coded harmonic imaging (cha), have been developed by various us equipment manufacturers (7, 10, 11). pulse inversion harmonic imaging uses a pair of pulses with opposite polarity, and the nonlinear echoes of the tissue harmonic and the contrast agent responses are detected, while the linear echoes of the fundamental tissue are canceled. coded harmonic imaging uses digital encoding and decoding of the transmitted pulses, and it can separate the contrast from the tissue and reduce the unwanted echoes from the fundamental frequency and the tissue harmonic signals. agent detection imaging uses two pulses with the same polarity and it subtracts the signals from the two pulses. the introduction of us has led to improving the detection of renal tumors in asymptomatic patients. renal cell carcinoma seen on gray - scale us has specific features including intratumoral cyst and pseudocapsule, and aml characteristically shows posterior shadowing (2 - 4). these features are important findings that may help distinguish rcc from aml, but the presence of these features is not sufficient to differentiate rccs from the other solid renal masses that are incidentally detected on gray - scale us. on the power doppler us, the analysis of the vascular distribution has not increased the diagnostic accuracy for small solid renal tumors (4). contrast - enhanced doppler us can increase the detection of intratumoral vascularity compared to color / power doppler us (5, 6). however, its signal intensity hasn't been found to be sufficiently intense for tumor characterization, and it contains artifacts or noises that prevent a correct diagnosis. recently, the development of contrast - enhanced harmonic us imaging has provided for a better assessment of the vascular morphology and the enhancing patterns ; thus, it has improved the detection and characterization of focal liver lesions (7 - 9). to the best of our knowledge, there have been a few reports about the differentiation of rcc with employing contrast - enhanced harmonic us imaging using sh u 508a (10 - 12). however, these studies did not completely evaluate the various enhancement patterns and the intratumoral or peritumoral features of rcc on the adi exam as compared with those features on the gray - scale us and pathologic exams. on the adi exam, heterogeneous enhancement was most commonly seen in the rcc because most of the lesions contain intratumoral signal defects that pathologically corresponded to cystic change, necrosis or both. homogeneous enhancement was shown for the small rccs, and cystic change or necrosis was not found in these tumors on the pathologic exam. intratumoral anechoic areas have been reported to be pathologically cystic change, degeneration or necrosis. on gray - scale us, these were specific for rcc, but their frequency was previously reported as being 12 - 31% (2 - 4). the number of intratumoral cysts in the rccs was larger on the ad scan than on the gray - scale us because the signal of the contrast agent on the adi can be seen in almost all the tissue with vessels, except for the avascular areas such as cysts or necrosis. the pseudocapsule pathologically corresponds to fibrous tissue and renal parenchyma that is compressed by the rcc (13, 14). the preoperative identification of the pseudocapsule was one of the criteria that allow nephronsparing surgery (15). ascenti., reported that the pseudocapsule appeared as a nonenhancing peritumoral rim in the early phase and as a well - enhancing rim in the late phase on the contrast - enhanced harmonic us (12). in their study, the contrast - enhanced second - harmonic us showed a higher sensitivity for the peritumoral pseudocapsule of rcc than did the gray - scale us (12). similarly, the gray - scale us and adi detected the encapsulated rccs in 18% (5/28) and 82% (23/28) of all the cases, respectively.. a larger scale of study will be necessary to support our results for the adi exam. second, it was nearly impossible for us to detect the contrast signals of the rcc situated 10 cm or deeper from the us probe. body habitus, lung or bowel gas, ribs and the location of the lesions also affected the signal intensity of the renal tumors. third, color / power doppler us, which can provide more information on intratumoral vascular distribution, was not performed to evaluate the rcc in this study. it is necessary to compare the diagnostic accuracy between the adi scan and the combination of gray - scale us with power doppler us for the diagnostic accuracy of the solid renal masses. fourth, the diagnostic criteria for rcc with using gray - scale us and adi exams have not been established. we made a diagnosis of rcc when the tumor showed two out of the three most common imaging features on the gray - scale us and adi exams. in conclusion it can improve the diagnostic accuracy of rcc as compared with gray - scale us by allowing better visualization of the intratumoral anechoic areas and the pseudocapsule than can the gray - scale us. | objectivewe wanted to compare the imaging features of renal cell carcinoma (rcc) and their diagnostic accuracy on agent detection imaging (adi) and gray - scale ultrasonography (us).materials and methodsthirty non - consecutive patients (age range ; 32 - 80 years, mean age ; 53.7 years) with 30 rccs were examined with gray - scale us and with adi in conjunction with using sh u 508a. we described the imaging features of the renal tumors obtained from adi according to their enhancement pattern, the intratumoral anechoic areas and the presence of any pseudocapsule. the imaging features and diagnostic accuracy of adi and gray - scale us were then compared.resultson the adi exam, the rccs were shown as being heterogeneous in 87% of the cases (26/30), homogeneous in 7% of the cases (2/30), and there was peripheral irregular enhancement in 7% of the cases (2/30). intratumoral anechoic areas and pseudocapsule were seen in 87% and 77% of the rccs on the adi exam, whereas these features were seen in 53% and 17% of the cases on the gray - scale us, respectively. the diagnostic sensitivity, specificity, and accuracy for rcc with adi were 97%, 93%, and 95%, respectively. however, those for rcc with using gray - scale us were 70%, 86%, and 78%, respectively. there was a significant difference for the diagnostic accuracy of rcc between adi and gray - scale us (p < 0.05).conclusionagent detection imaging can help visualize the enhancement pattern of rcc and improve the diagnostic accuracy of this tumor by better displaying the intratumoral anechoic areas and the pseudocapsule than does the gray - scale us. |
behavioral neuroscience, with the backing of the national institutes of health (nih), has made tremendous progress in establishing powerful tools and techniques that have been used primarily in a small number of model organisms. the nervous system of c. elegans has been poked and prodded in ways previously unimaginable. reproducible and simple rodent behavioral assays have been used to dissect the regional contributions of the brain in behavior. as a result, we now have a tremendous depth of insight in a few, key model systems. in contrast, we have invested relatively few resources toward understanding behavior in other organisms, which has led to two problems. first, in some instances, researchers have tried to force a square block through a round hole by attempting to study specific behaviors in inadequate or inappropriate models. the temptation to do so is understandable given the tremendous wealth of tools available to manipulate these few species. fortunately, though, the diverse animal models of neuroethology provide the solutions to both of these problems. likewise, newer, more broadly applicable technologies that have arisen in behavioral neuroscience will expand the types of questions that can be answered in neuroethological models. adapting cutting edge technologies to dissect the behavioral diversity of lesser studied species represents a novel opportunity to shed new light on human - relevant behaviors and physiologies. for example, it has been proposed that we try to understand facets of human social behavior by applying such tools to model social species, such as songbirds, cichlid fish, social insects, and monogamous and eusocial mammals (robinson., 2008 ; holmes., 2009). neither the tool - rich but organismically limited approach of behavioral neuroscience nor the tool - limited but behaviorally diverse neuroethological approach is right or wrong. a number of examples now exist that demonstrate the power of working at the intersection between neuroethology and behavioral neuroscience, integrating the strengths of both. one of the most well developed lines of research at this intersection focuses on the molecular and neurobiological underpinnings of social cognition and bonding. much of the early work on rodent social affiliation and bonding was performed in rats. among other findings, researchers demonstrated that oxytocin facilitates the onset of maternal behavior in this species (fahrbach., 1984). however, polygamous laboratory rats and mice do not form selective sociosexual bonds with their mates, like humans do. as such, these species are not suitable for investigating the underpinnings of this behavior. instead, the research groups of sue carter and, later, tom insel turned to monogamous prairie voles. building off of the work in rats that suggested that oxytocin modulates maternal affiliation, they demonstrated that oxytocin is also crucial for the formation of selective partner preferences, a proxy for pair bonding, in female prairie voles (carter., 1995). they also found that oxytocin 's partner molecule, vasopressin, which differs from oxytocin by only two of nine amino acids, modulates pair bonding in male prairie voles (wang., 1999). thus, findings in behavioral neuroscience inspired new avenues of research in neuroethology, but this was merely the beginning of the interplay between this two disciplines with respect to our understanding of social affiliation and bonding. oxytocin and vasopressin are exceptionally conserved across species (donaldson and young, 2008), and subsequent comparative work in monogamous and polygamous voles demonstrated that species differences in bonding behaviors are governed by variation in receptor patterns rather than differences in the peptide hormones themselves. monogamous prairie voles have a vasopressin v1a receptor pattern that differs strikingly from that of polygamous meadow voles, and it was hypothesized that these differences underlie the disparate social behaviors of these species (young., 1997). to demonstrate that alteration of receptor patterns can cause differences in social behavior, young. (1999) isolated the prairie vole v1a receptor gene along with a few kilobases of upstream flanking dna and inserted it into the mouse genome. the resulting transgenic mice had a v1a receptor pattern that was reminiscent of that of prairie voles, and when infused with vasopressin, the mice demonstrated higher levels of social affiliation. (2004) altered v1a receptor patterns within the meadow vole brain to make them more prairie - vole - like. lim used a viral vector to increase v1a receptor levels within the ventral pallidum of polygamous meadow voles, a region with high levels of receptors in prairie voles but low levels in meadow voles. subsequently, the meadow voles with the altered receptor distribution demonstrated a social preference for their mating partner, a trait typically exhibited by prairie but not meadow voles. these two experiments demonstrate the causal relationship between central v1a receptor patterns and differences in social behavior, and both merge aspects of behavioral neuroscience and neuroethology. in one instance, a hypothesis generated in a neuroethological model was tested using a model system and tools common to behavioral neuroscience. in the other, a tool developed in behavioral neuroscience research was applied to a neuroethological model. in both cases, human geneticists, psychologists, and psychiatrists have also designed experiments in humans that were inspired by the advances made in rodents with respect to vasopressin, oxytocin, and social behavior (reviewed in donaldson and young, 2008 ; ebstein., 2010). genetic studies have suggested that variation in the 5 flanking region of the human v1a receptor gene is associated with a variety of social traits, predictive of relationship quality, and correlates with differences in the neural processing of social cues. intranasally applied oxytocin has been shown to stimulate trust and other prosocial tendencies, and groups have now begun to explore the possibility of using intranasal oxytocin as a treatment for autism and other neuropsychiatric disorders characterized by deficits in social behavior (bartz., 2008 ; guastella., the trajectory of research on social bonding has continued to make major strides forward through the use of multiple model organisms, both common (mice and rats) and uncommon (prairie voles), and the adoption of the tools of behavioral neuroscience within a neuroethological model. because of these efforts, we are closer to understanding natural variation in human behavior as well as what processes may be disregulated in the human social brain with respect to various psychiatric disorders. social behavior is not the only area in which this approach has proven valuable. although i will not devote much time to other examples, there are currently a number of lines of research that are or could benefit from combining behavioral neuroscience and neuroethological approaches. for instance, the foxp2 gene, which encodes a transcription factor, was found to be mutated in a family with a heritable language impairment (lai., 2001). a foxp2 knockout mouse has highlighted the developmental role of this transcription factor (vernes and fisher, 2009) while work with sirnas targeting this gene in song birds is beginning to shed light on how it may be involved in vocal learning and behaviors (haesler., 2007). these two complimentary approaches will likely each lead to a better understanding of the neurodevelopmental factors important for language - associated traits. finally, since neuroethology owes much to the study of invertebrates, i will outline one last example of interplay between neuroethology and behavioral neuroscience within insects. one of the long - standing neuroethological questions with regard to honeybee social behavior centers around the biological basis of the division of labor within the hive (smith., how does an individual know what job it is supposed to perform within the hive, and likewise, what is the underlying biology of individual job transitions, e.g., from hive - bound nurse early in life to nectar / pollen forager later in life ? within d. melanogaster, regulatory variants in the foraging (for) gene lead to individual differences in foraging behavior ; flies with higher levels of for forage more actively for food than flies with lower transcript levels (sokolowski, 2001). by drawing on this work, ben - shahar. (2002) asked whether the same gene could be involved in different life stages in bees. they demonstrated that upregulation of the foraging (for) gene is associated with the transition from nurse to forager in honey bees and that activation of the pathway regulated by for drives precocious foraging behavior. thus a gene that modulates differences in feeding behavior in a commonly studied model organism yielded insight into the genetic control of social activity in a model social organism (robinson and ben - shahar, 2002). in this instance, behavioral neuroscience in fruit flies inspired a new look at an old neuroethological question in honey bees. one can also imagine that, in the future, hypotheses generated in honey bees will be tested in drosophila. for the time being, genetic manipulation in honeybees is limited, but candidate genes identified in bees may be manipulated within drosophila to better understand their role in behavior. in sum, both neuroethology and behavioral neuroscience have much to gain by capitalizing on each other 's advances. on the one hand, many questions simply can not be answered using the model systems typically employed by behavioral neuroscientists. on the other hand, neuroethologists will be able to explore natural models in new ways by using techniques and approaches pioneered by behavioral neuroscientists. in moving forward, we must seek to identify ways in which the barriers between these groups of scientists can be broken down. pubmed is a database of medline - indexed and other life science articles that plays a dominant role in many neuroscientists literature search habits pubmedding, like googling, has become a verb among scientists. due to its emphasis on biomedical literature and the historical division between biomedical and ecological research, pubmed has had a limited listing of animal behavioral journals. however, a growing interest in behavioral models of human mental health disorders would suggest that animal behavior findings do deserve a seat that the pubmed table. for instance, papers in animal behaviour and the american naturalist are assigned a pubmed i d, but those in behavioral ecology and sociobiology and ethology are not (with the exception of articles deemed to be related to population demographics / reproductive health, which are indexed by medline). this seemingly arbitrary inclusion and exclusion of various behavioral journals does a disservice to the neuroscience community as a whole and limits potential expansion into new animal models. inclusion of more behavioral journals in pubmed will serve to bridge the gap between neuroethology and psychobiology in a number of ways. first, addition of behavioral journals means that anyone searching pubmed for information on a given behavior will have, within their results, relevant references from both neuroethological and behavioral neuroscience perspectives. second, by including journals that routinely publish studies performed in less commonly studied animal models, we will increase the chances that nih reviewers will view such organisms as legitimate biomedical models. finally, scientists will not have to think twice about whether they will get credit for their work, as they do now when they publish in a journal not currently in the pubmed database. a quick pubmed search is often used to identify publications authored by a specific scientist, and when an author publishes in journals outside of pubmed 's current scope, they risk being unfairly seen as less productive than other scientists. thus, simply expanding the number of behavioral journals available in pubmed is a relatively easy first step toward expanding the productive interaction between neuroethology and psychobiology. it also seems to be in line with pubmed 's goal of disseminating life sciences and biomedically relevant findings. the term you are what you eat could just as easily be applied to science. the ideas and approaches we consume during training and at conferences largely dictate the types of experiments that we pursue. thus, attempts to form collaborations and merge neuroethology and behavioral neuroscience will benefit from diverse training opportunities and cross - field conference exposure. programs in neuroscience and psychology within the u.s. typically provide one to two years of classroom - based education. these programs tend to encompass an excellent and many - faceted examination of the brain, but the extent to which neuroethology is included in the curriculum varies across institutions.. we should strive to increase the ubiquitousness of the elective courses in neuroethology, and ensure representation of neuroethological findings in core curricula. it would be considered neglectful to exclude behavioral lessons we have learned from gene knockouts in a graduate level core curriculum and likewise it should be considered ridiculous to exclude key neuroethological findings. a seemless blending of neuroethology and behavioral neuroscience in coursework will stimulate cross - disciplinary approaches, just as new musical genres arise from the fusion of old concepts. in addition, as long as nih relies on the dual inputs of scientists and medical professionals, it would be advantageous to include lectures on diverse animal models in medical schools. medical doctors, and especially clinical scientists, may benefit from thinking outside of the confines of traditional behavioral neuroscience. a few years of graduate training, however, make up only a small portion of most scientific careers. thus, in order to foster communication and the application of new approaches in neuroethology and behavioral neuroscience, we should examine the segregation that occurs at conferences. while it is a tribute to neuroethology 's success that it is recognized as an independent section at the annual society for neuroscience meeting, this partitioning physically separates neuroethologists from behavioral neuroscientists, who typically present their work in other sections of the meeting. simply by serving as a gathering place for both sub - disciplines simultaneously, however, the society for neuroscience meeting serves as a model, for neuroethologists and behavioral neuroscientists often attend different conferences. neuroethologists typically attend the international congress of neuroethology (2010) or the gordon conference on neuroethology (2011), while behavioral neuroscientists attend a smattering of other conferences, including but not limited to the genes, brain, and behavior annual meeting (2010), and the international behavioral neuroscience society (ibns) meeting (2010). one way to begin bridging this conference divide is to invite speakers from the lesser - represented field to each meeting. at this year 's ibns meeting in june, the presidential symposium on the search for effective animal models in behavioral neuroscience will attempt to do this (2010b). however, the symposium 's focus on zebrafish, rats and mice does not delve very deeply beyond the traditional confines of behavioral neuroscience (though one talk about hamsters will be included). likewise, this year 's upcoming international congress on neuroethology (2010c) does a particularly good job of integrating human research into the schedule of presentations but fails to attract speakers from the realm of behavioral neuroscience. providing informative and (hopefully) entertaining cross - field viewpoints at future conferences has the potential to open lines of communication, spur new techniques, and encourage creative scientific avenues. finally, in what may prove to be my most - difficult - to - accomplish suggestion, we must identify ways to bridge the funding divide in the united states. in this sense, america may be able to glean something from other funding models like those of various european countries where a single agency oversees biological research. in the u.s. neuroethological research tends to receive more of its funding from the national science foundation (nsf) than nih. as of february 2010, a grants database search for neuroethology at nsf yields 49 awards with this word in the title or abstract while the same search of the nih report database yields only a single result conversely, the underlying medical motivation of behavioral neuroscience makes it more amenable to nih funding. this divide in the funding of neuroethological and behavioral neuroscience research has more important implications than which governmental organization is represented on one 's curriculum vitae. grants are peer reviewed, and, with some exceptions, nih and nsf often draw on different groups of scientists for their review panels. because the critical review of a grant is limited by the approaches the reviewers are familiar with, such a system fails to encourage the submission of proposals detailing innovative ways to bridge these fields. one way to overcome this difficulty would be to generate a call for proposals specifically geared toward the development of new animal models at nih. another could solicit proposals for technology transfer grants aimed at introducing behavioral neuroscience technologies into less commonly used model species at nsf. ideally, the review panels for such grants would include a mixture of scientists from neuroethological and behavioral neuroscience backgrounds. pubmed is a database of medline - indexed and other life science articles that plays a dominant role in many neuroscientists literature search habits pubmedding, like googling, has become a verb among scientists. due to its emphasis on biomedical literature and the historical division between biomedical and ecological research, pubmed has had a limited listing of animal behavioral journals. however, a growing interest in behavioral models of human mental health disorders would suggest that animal behavior findings do deserve a seat that the pubmed table. for instance, papers in animal behaviour and the american naturalist are assigned a pubmed i d, but those in behavioral ecology and sociobiology and ethology are not (with the exception of articles deemed to be related to population demographics / reproductive health, which are indexed by medline). this seemingly arbitrary inclusion and exclusion of various behavioral journals does a disservice to the neuroscience community as a whole and limits potential expansion into new animal models. inclusion of more behavioral journals in pubmed will serve to bridge the gap between neuroethology and psychobiology in a number of ways. first, addition of behavioral journals means that anyone searching pubmed for information on a given behavior will have, within their results, relevant references from both neuroethological and behavioral neuroscience perspectives. second, by including journals that routinely publish studies performed in less commonly studied animal models, we will increase the chances that nih reviewers will view such organisms as legitimate biomedical models. finally, scientists will not have to think twice about whether they will get credit for their work, as they do now when they publish in a journal not currently in the pubmed database. a quick pubmed search is often used to identify publications authored by a specific scientist, and when an author publishes in journals outside of pubmed 's current scope, they risk being unfairly seen as less productive than other scientists. thus, simply expanding the number of behavioral journals available in pubmed is a relatively easy first step toward expanding the productive interaction between neuroethology and psychobiology. it also seems to be in line with pubmed 's goal of disseminating life sciences and biomedically relevant findings. the term you are what you eat could just as easily be applied to science. the ideas and approaches we consume during training and at conferences largely dictate the types of experiments that we pursue. thus, attempts to form collaborations and merge neuroethology and behavioral neuroscience will benefit from diverse training opportunities and cross - field conference exposure. ph.d. programs in neuroscience and psychology within the u.s. typically provide one to two years of classroom - based education. these programs tend to encompass an excellent and many - faceted examination of the brain, but the extent to which neuroethology is included in the curriculum varies across institutions.. we should strive to increase the ubiquitousness of the elective courses in neuroethology, and ensure representation of neuroethological findings in core curricula. it would be considered neglectful to exclude behavioral lessons we have learned from gene knockouts in a graduate level core curriculum and likewise it should be considered ridiculous to exclude key neuroethological findings. a seemless blending of neuroethology and behavioral neuroscience in coursework will stimulate cross - disciplinary approaches, just as new musical genres arise from the fusion of old concepts. in addition, as long as nih relies on the dual inputs of scientists and medical professionals, it would be advantageous to include lectures on diverse animal models in medical schools. medical doctors, and especially clinical scientists, may benefit from thinking outside of the confines of traditional behavioral neuroscience. a few years of graduate training, however, make up only a small portion of most scientific careers. thus, in order to foster communication and the application of new approaches in neuroethology and behavioral neuroscience, we should examine the segregation that occurs at conferences. while it is a tribute to neuroethology 's success that it is recognized as an independent section at the annual society for neuroscience meeting, this partitioning physically separates neuroethologists from behavioral neuroscientists, who typically present their work in other sections of the meeting. simply by serving as a gathering place for both sub - disciplines simultaneously, however, the society for neuroscience meeting serves as a model, for neuroethologists and behavioral neuroscientists often attend different conferences. neuroethologists typically attend the international congress of neuroethology (2010) or the gordon conference on neuroethology (2011), while behavioral neuroscientists attend a smattering of other conferences, including but not limited to the genes, brain, and behavior annual meeting (2010), and the international behavioral neuroscience society (ibns) meeting (2010). one way to begin bridging this conference divide is to invite speakers from the lesser - represented field to each meeting. at this year 's ibns meeting in june, the presidential symposium on the search for effective animal models in behavioral neuroscience will attempt to do this (2010b). however, the symposium 's focus on zebrafish, rats and mice does not delve very deeply beyond the traditional confines of behavioral neuroscience (though one talk about hamsters will be included). likewise, this year 's upcoming international congress on neuroethology (2010c) does a particularly good job of integrating human research into the schedule of presentations but fails to attract speakers from the realm of behavioral neuroscience. providing informative and (hopefully) entertaining cross - field viewpoints at future conferences has the potential to open lines of communication, spur new techniques, and encourage creative scientific avenues. finally, in what may prove to be my most - difficult - to - accomplish suggestion, we must identify ways to bridge the funding divide in the united states. in this sense, america may be able to glean something from other funding models like those of various european countries where a single agency oversees biological research. in the u.s. neuroethological research tends to receive more of its funding from the national science foundation (nsf) than nih. as of february 2010, a grants database search for neuroethology at nsf yields 49 awards with this word in the title or abstract while the same search of the nih report database yields only a single result conversely, the underlying medical motivation of behavioral neuroscience makes it more amenable to nih funding. this divide in the funding of neuroethological and behavioral neuroscience research has more important implications than which governmental organization is represented on one 's curriculum vitae. grants are peer reviewed, and, with some exceptions, nih and nsf often draw on different groups of scientists for their review panels. because the critical review of a grant is limited by the approaches the reviewers are familiar with, such a system fails to encourage the submission of proposals detailing innovative ways to bridge these fields. one way to overcome this difficulty would be to generate a call for proposals specifically geared toward the development of new animal models at nih. another could solicit proposals for technology transfer grants aimed at introducing behavioral neuroscience technologies into less commonly used model species at nsf. ideally, the review panels for such grants would include a mixture of scientists from neuroethological and behavioral neuroscience backgrounds. the path of science has been, and will continue to be, influenced by the divergent cultures of different sub - disciplines and by disparate funding sources. within the past few decades, especially at nih, there has been a narrowing of focus that emphasizes study of only a few model organisms. as a result, we have been endowed with tremendous depth of understanding and creative techniques for manipulating these organisms, but at the cost of exploring the great diversity of biology. despite this history, there are reasons to believe that the culture of science generally, including, to an extent, the zeitgeist at its major funding sources, may be moving toward the embrace of more integrated and diverse approaches. from a technological perspective, advances in genetic tools have opened up the possibility of performing experiments in a wide range of organisms for the first time. for instance, advances with viral vectors provide a highly efficient means to introduce foreign genes into organisms other than mice or flies. viral - vector mediated gene transfer can be used to modify the properties of a particular neural region or introduce transgenes into the early embryo in order to generate transgenic animals with mutant germlines. to date, scientists have used this and other technologies to generate transgenic prairie voles (donaldson., 2009), zebra finches (agate., 2009), rhesus macaques (chan., 2001), marmosets (sasaki., 2009), cats (yin., 2008), dogs (so gun., 2009), and a variety of agricultural animals (park, 2007 ; whitelaw., 2008). they can also be used to label specific cell types with a fluorescent marker for use in electrophysiology. or they can be used to selectively modulate the firing properties of groups of neurons through alteration of neurotransmitter receptors or to introduce foreign receptors that can be modulated by pharmacological agents or even light (miesenbock, 2009). in short, new technologies now make sophisticated cross - disciplinary studies feasible in less commonly studied organisms. the recent emergence of comparative genomics as enthusiastically embracing krogh 's principle serves as a model for how different fields and groups of scientists are congregating around shared resources and questions. investigation of genomic diversity in humans and animals has applications for studying both human - relevant questions and also exploring hypotheses with respect to evolutionary theory more broadly. as such, genome sequences are the substrate that has brought together groups of scientists that would not normally interact or collaborate. for instance, both human and animal researchers can focus on genomic resources to shed light on mechanisms that generate risk or resilience to cancer as we age. human association studies may identify gene variants associated with predisposition to cancer while investigation of long - living species with low incidence of tumors, such as naked mole rats, can identify novel genetic mechanisms for avoiding cancer (seluanov.,, this model of shared resources may inspire collaboration across other fields, such as neuroethology and behavioral neuroscience. finally, there is reason to believe that the dramatic way in which genomicists are embracing diversity is not the only example of government - funded health science branching out to explore new model organisms. the department of health and human services interagency autism coordinating committee 2009 strategic plan calls for $ 75 million to be allocated to identifying new models (i.e., animal / cellular) relevant to the study and treatment of autism spectrum disorder (2009). hopefully, diverse behavioral models will stimulate new ways to approach the study of complex neurological disorders in humans and, as such, will stimulate a renewed respect and interest in the diverse field of neuroethology. in sum, there are now a number of indicators suggesting that researchers in various groups are recognizing the limits of approaches they are familiar with and are ready to listen to insights from other fields. my intention is not to convince neuroethologists to take on the cause of human health ; nor am i seeking to convince behavioral neuroscientists to take up field studies or abandon the study of mice and rats. i would argue, however, that approaching a question from more than one viewpoint is always useful. the question now is how we can best combine those strengths to facilitate the advancement science, and i hope that this article has provided at least a starting point for discussion. theodore h. bullock, one of the founding fathers of neuroethology (zupanc and zupanc, 2008), has said, with its particularly high demands for expertise in several disciplines, neuroethology is typically a tough challenge, but nothing beats it for satisfaction per square idea ! after all it is time we knew what neuronal systems are used for ! (pg 383, ref hoyle, 1984). understanding how brain function leads to behavior is going to require a creativity that will employ both behavioral neuroscience and neuroethological approaches. the sooner we can realize ways to bridge the ideological and social divides between these scientific groups, the faster this will happen. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | how the brain mediates behavior is a question relevant to a broad range of disciplines including evolutionary biology, basic neuroscience, psychiatry, and population health. experiments in animals have traditionally used two distinct approaches to explore brain behavior relationships ; one uses naturally existing behavioral models while the other focuses on the creation and investigation of medically oriented models using existing laboratory - amenable organisms. scientists using the first approach are often referred to and self identify as neuroethologists, while the second category spans a variety of other sub - disciplines but is often referred to broadly as behavioral neuroscience. despite an overall common scientific goal the elucidation of the neural basis of behavior members of these two groups often come from different scientific lineages, seek different sources of funding, and make their homes in different departments or colleges. the separation of these groups is also fostered by their attendance at different scientific conferences and publication records that reflect different journal preferences. bridging this divide represents an opportunity to explore previously unanswerable questions and foster rapid scientific advances. this article explores the reasons for this divide and proposes measures that could help increase technology transfer and communication between these groups, potentially overcoming both physical and ideological gaps. |
recently, all - ceramic fixed partial dentures (fpds) were introduced ; these are currently in use in clinical dental treatment. due to their superior aesthetics, all - ceramic restorations provide a more desirable approach compared with metal - ceramic combinations for the restoration of natural dentition.1 the application of veneering porcelain over a high - strength core ceramic has resulted in the creation of an aesthetically more acceptable laminate composite system, which has been employed in the construction of all - ceramic crowns.2 however, ceramics are brittle and susceptible to failure beyond a critical stress, which is dependent on the internal and surface flaw distributions,3,4 and their fracture problems have not been solved completely.5 manufacturers have adopted novel engineering processing routes to produce all - ceramic dental core materials with strengths in excess of those achieved using metallic frameworks for metal - ceramic restorations.6 the finite element method (fem) has been shown to be a useful tool when investigating complex systems.7,8 knowledge of stress distribution is important in the understanding of fatigue yielding.9 overall stress distribution within the tooth / restoration complex is determined by geometry and hard tissue / restorative material arrangement.10 it has also been reported that the majority of failures of all - ceramic fpds originate at the gingival side of the connectors, at the interface of the framework and veneer porcelain.11 thus, the framework design of all - ceramic restorations may have an important effect on stress distribution. furthermore, the distribution of stress influences the success of the treatment.1214 in recent years, alternative materials and designs have been developed to suit different clinical situations,15 and fpds must have a design type that satisfies certain structural requirements. an fpd must provide enough strength to resist the forces of occlusion that cause flexure of the framework, producing stress in the restoration, and the abutment. it is known that the arch - type design is the most efficient method of forming a structure with materials that have good compressive strength and low tensile strength.16 however, to develop theories of prosthesis design, the amount of stress likely to be generated in the oral cavity must be quantified.15 thus, this study aimed at evaluating the effects of framework designs on stress distribution at the supporting bone and supporting implants. the null hypothesis of the current study was that the different framework designs associated with all ceramic restorations would not affect stress distribution. the study was conducted using 3d fem and the solidworks 2007 9.0.3 structural analysis program (solidworks corporation, usa). a 3d fem model was constructed to represent a three - unit implant supporting fpds ; this was used to perform the computer simulation (figure 1a). the model contained a three - unit all - ceramic fpd with two implants (iti solid screw implants, 3.8-mm diameter, 10-mm bone sink depth ; straumann ag, waldenburg, switzerland) at each end as abutment. these were supported by alveolar bone structures simulated as spongy bone surrounded by 2 mm of cortical bone. initially, the cross - sections of bone structures included in the mathematical model were hand drawn. they were sketched separately at the front and right planes for each unit in the computer environment. the implant system modeling was conducted using a laser - based 3d scanner (dental wings, montreal, quebec, canada) to reproduce the exact dimensions. the coordinates of the contouring points were then joined to form each structure s volume ; together, these defined the final geometry of the fem model (figure 1b c). the simulated geometries used for analysis were three different framework designs (convex (1), concave (2), and conventional (3) pontic framework designs) (figure 2a c). all - ceramic fpds (ips e - max press ; ivoclar vivadent ag) were simulated as the restorative material. the bottom exterior nodes of the alveolar bone in the fem models were fixed in all directions as the boundary condition (figure 3b). a 300-n static vertical occlusal load17 was applied on the node at the center of occlusal surface of the pontic to calculate the stress distributions (figure 3b). as a second condition, frameworks were directly loaded to clearly demonstrate the effect of framework design (figure 3c). the elastic properties of the materials (young s modulus (e) and poisson s ratio ()) were determined from the literature and are given in table 1.15,18 the fem modeling was accomplished using the solidworks software program, and analyses were run with the cosmosworks software program, which is integrated with solidworks. results are presented by considering the von mises criteria.1924 calculated numerical data were transformed into color graphics to better visualize the mechanical phenomena in the models. both a 3d whole - model view and mesiodistal cross - sectional views were presented for each condition. a narrower range was used for the stress indicator scale to better visualize the differences in stress distribution (0 to 5 mpa). the analysis of the von mises stress values revealed that maximum stress concentrations were located at the loading areas for all models. in addition, high stress values were located at the gingival embrasures of connectors and the cervical regions on the pontic side of abutment implants in all models. the cervical and inter - implantal alveolar bone structure was the other stress concentration area. there were no clear differences in stress distributions among the three design types (figure 4a c). the stress trajectory started from the force application point and continued through the framework to the abutment implants, where it was distributed at the bone structure. the highest stress values observed at the force application areas were 27.3, 29.1, and 26.0 mpa for convex, concave, and conventional designs, respectively. the highest value was observed at the veneering porcelain at the force application area of concave design (29.1 mpa). the stress values observed at the cervical regions of the implants were 1.17, 1.41, and 1.35 mpa for convex, concave, and biconcave designs, respectively. the stress values at the alveolar bone structure between implants were 0.310.76 mpa (figure 5a c). the structures maximum von mises stress values with different models are summarized at table 2. the highest stress values observed at the force application areas of the framework porcelain were 5.12, 5.34, and 5.24 mpa for convex, concave, and biconcave designs, respectively. although the maximum stress concentration values were close, clear stress distribution diversities for example, stress values of 4.65.34 mpa clearly occupied more space in the concave design framework than the other design types. in addition, high stress concentration values at the implant neck occupied more space in the concave design. the maximum von mises stress values observed at the cortical bone structure were 0.81, 1.01, and 0.68 mpa for convex, concave, and biconcave designs, respectively. while stress was directed toward inter - implantal alveolar bone in the concave framework design, at the opposite was the case for the convex and conventional framework designs (figure 7a c). however, the maximum stress value of 0.81 mpa was clearly more widespread for the convex design type than for the conventional framework design. the maximum von mises stress values of structures with different models and a direct loading of the framework are summarized in table 3. there were no clear differences in stress distributions among the three design types (figure 4a c). the stress trajectory started from the force application point and continued through the framework to the abutment implants, where it was distributed at the bone structure. the highest stress values observed at the force application areas were 27.3, 29.1, and 26.0 mpa for convex, concave, and conventional designs, respectively. the highest value was observed at the veneering porcelain at the force application area of concave design (29.1 mpa). the stress values observed at the cervical regions of the implants were 1.17, 1.41, and 1.35 mpa for convex, concave, and biconcave designs, respectively. the stress values at the alveolar bone structure between implants were 0.310.76 mpa (figure 5a c). the structures maximum von mises stress values with different models are summarized at table 2. the highest stress values observed at the force application areas of the framework porcelain were 5.12, 5.34, and 5.24 mpa for convex, concave, and biconcave designs, respectively. although the maximum stress concentration values were close, clear stress distribution diversities for example, stress values of 4.65.34 mpa clearly occupied more space in the concave design framework than the other design types. in addition, high stress concentration values at the implant neck occupied more space in the concave design. the maximum von mises stress values observed at the cortical bone structure were 0.81, 1.01, and 0.68 mpa for convex, concave, and biconcave designs, respectively. while stress was directed toward inter - implantal alveolar bone in the concave framework design, at the opposite was the case for the convex and conventional framework designs (figure 7a c). however, the maximum stress value of 0.81 mpa was clearly more widespread for the convex design type than for the conventional framework design. the maximum von mises stress values of structures with different models and a direct loading of the framework are summarized in table 3. the stress distribution patterns varied with different framework designs. based on these results, the null hypothesis that the framework design would not affect the stress distribution for implant - supported fpds was rejected. in the present study, using the fem technique, the effect of framework design on amount and distribution of stress was evaluated. however, a theoretical and well - known method for calculating stress distribution within complex structures is fem, which allows the investigator to evaluate the influence of model parameter variation once the basic model has been correctly defined.8 the model used in this study involved several assumptions regarding simulated structures. the structures in the model were all assumed to be homogeneous, isotropic, and linearly elastic. the properties of the materials modeled in this study, however, and particularly the living tissues, are different. in addition, it is important to point out that the stress distribution patterns may have been different depending on the materials and properties assigned to each layer of the model, as well as the model used in the experiments.15 furthermore, only a vertical loading condition was used to compare different designs. horizontal or oblique force applications will be used in future studies. von mises stresses depend on the entire stress field and are a widely used indicator of the possibility of damage occurring.20 the value of the occlusal force was selected to be 300 n. however, it is not necessary for this force to match its real value exactly because standardization between conditions has been ensured in the current study and the conditions have been compared qualitatively with one another.15 chen and xu7 emphasized that the value of fem modeling has to do with relative values calculated for distribution patterns. a comparison of stress distributions with different design types revealed lower stress values in frameworks with the convex design. the stress levels for the conventional design type were close to those of the convex design, but the maximum stress value occurred at more places in the framework structure with the conventional type of design. thus, it may be concluded that, biomechanically, the more favorable framework design was the convex one. it was also seen that the veneering porcelain part of the restorations compensated for the mechanical effects of different framework designs. however, more clinical and experimental studies are needed to support the findings of the current study. occlusal loads are transmitted from the point of force application to the point of retainers and, ultimately, travel through the abutments to the bone structure. moreover, the purpose of a restoration is to maintain strength while resisting the forces of occlusion. thus, fixed partial dentures must include materials and a design type that provides enough strength to resist the forces of occlusion. porcelain has an amorphous structure and this produces physical properties typical of a glass.4 when porcelain is stressed in tension, small flaws tend to open up and propagate, resulting in a low tensile strength.4 however, porcelain is much stronger in terms of compression, because compressive stresses tend to close up flaws.4 it is known that when a beam is loaded, the applied loads have a tendency to cause failure of the beam in two main ways : by shearing or by bending the beam.16 arches are commonly used in several structural forms and represent the most efficient method of forming a structure with materials that have good compressive strength and low tensile strength.16 dental porcelains are among such materials because of the abovementioned properties. in order to achieve the most efficient structural solution, the design engineer (in this case, the dentist), can alter the structural configuration to produce an equilibrium condition made up of compression forces.16 thus, the convex framework design can be the most suitable way for inserting ceramic fpds to restore a missing tooth or teeth. in agreement with this information, the results of the current study revealed that von mises stress values decreased with the use of a convex design type. when dealing with implant - supported fpds, modifying the framework design may help to decrease stress concentrations, but the veneering porcelain element may reduce the effect of a framework with recent restorative materials. further studies that better simulate the oral environment and including fatigue loading are recommended. although the clinical implications that can be derived from this study are important, long term clinical evidence is required. within the limitations of this study, the following conclusions were drawn : the use of a concave design for fpd pontic frameworks increases the von mises stress levels on implant abutments and supporting bone structure.the veneering porcelain part reduces the effect of the framework and compensates for design weaknesses. the use of a concave design for fpd pontic frameworks increases the von mises stress levels on implant abutments and supporting bone structure. the veneering porcelain part reduces the effect of the framework and compensates for design weaknesses. | objectives : the biomechanical behavior of the superstructure plays an important role in the functional longevity of dental implants. however, information about the influence of framework design on stresses transmitted to the implants and supporting tissues is limited. the purpose of this study was to evaluate the effects of framework designs on stress distribution at the supporting bone and supporting implants.methods:in this study, the three - dimensional (3d) finite element stress analysis method was used. three types of 3d mathematical models simulating three different framework designs for implant - supported 3-unit posterior fixed partial dentures were prepared with supporting structures. convex (1), concave (2), and conventional (3) pontic framework designs were simulated. a 300-n static vertical occlusal load was applied on the node at the center of occlusal surface of the pontic to calculate the stress distributions. as a second condition, frameworks were directly loaded to evaluate the effect of the framework design clearly. the solidworks / cosmosworks structural analysis programs were used for finite element modeling / analysis.results : the analysis of the von mises stress values revealed that maximum stress concentrations were located at the loading areas for all models. the pontic side marginal edges of restorations and the necks of implants were other stress concentration regions. there was no clear difference among models when the restorations were loaded at occlusal surfaces. when the veneering porcelain was removed, and load was applied directly to the framework, there was a clear increase in stress concentration with a concave design on supporting implants and bone structure.conclusions:the present study showed that the use of a concave design in the pontic frameworks of fixed partial dentures increases the von mises stress levels on implant abutments and supporting bone structure. however, the veneering porcelain element reduces the effect of the framework and compensates for design weaknesses. |
time - dependent density functional theory (td - dft) has been in the limelight of quantum chemistry during the past decade. indeed, paralleling the success of dft for the ground - state (gs), td - dft has now become the most widely applied method to explore the excited - state (es) properties of medium and large compounds, with applications appearing in many applied fields of chemical science. besides the choice of a basis set, two approximations are generally made while running td - dft calculations : the adiabatic formalism is used, inducing the loss of memory effects, and a specific exchange - correlation functional (xcf) is selected to describe the nonclassical component of the electron though these two approximations are certainly not independent of each other, it remains in practice easier to change the xcf than to go beyond the adiabatic approximation, and the vast majority of the benchmarks of td - dft have therefore considered this latter aspect. these benchmarks have been mainly focused on both vertical and 00 transition energies, and to a lesser extended es geometries, and have much less considered the determination of other properties as illustrated by ref (5) reviewing td - dft benchmarks. in this framework, the present investigation is devoted to the es dipole () and traceless quadrupole (q) moments with a focus on medium and large -conjugated molecules. to the best of our knowledge, there are no previous work devoted to the benchmarking of td - dft for es quadrupole calculations, while the previous investigations devoted to the assessment of xcfs in the framework of calculations of the excited - state dipole moments () and/or the excess dipole moment (, the difference between es and gs dipoles) have mainly considered small compounds. the first comparisons probably appeared in 2002 when furche and ahlrichs used experimental values to assess the performances of three generalized gradient approximations (ggas) and two global hybrids for seven tiny molecules. for this set, they found that the errors obtained with the gga (ca. the same year, amos, handy, and co - workers compared td - dft and eom - ccsd dipole moments for numerous excited - states in furan and pyrrole, in studies devoted to the assessment of the hcth and b97 - 1 xcfs. they noted that, contrary to the transition energies, excited - state dipoles are strongly functional dependent, and that this sensitivity is exacerbated for rydberg states. these conclusions were refined by king in 2008, using more accurate reference values. he found that b97 - 1 provides with average deviations of ca. 0.5 d (1.1 d) for a large number of ess in furan (pyrrole). in what is probably the largest study of to date, thiel and co - workers considered their famous set of 20 compact molecules to assess the quality of the dipole moments obtained with bp86, b3lyp, and bhhlyp using their own cas - pt2/tzvp values as references. they concluded that (i) the dft dipole moments tend to be too small ; (ii) the errors are smaller for the gs than the es ; (iii) the average deviations are similar for n and transitions ; (iv) the average td - dft errors are in the 0.60.8 d range and only marginally depend on the functional, though these average deviations are slightly smaller with bhhlyp and b3lyp than with bp86. guido and co - workers reported the determined on five small molecules using six hybrid xcfs and also obtained excess dipole values quite independent of the selected functional, with cases for which increased and decreased when increasing the amount of exact exchange included in the xcf. for small charge - transfer (ct) molecules, tapavicza. found that the cc2 and pbe0 were nicely matching experiment values, whereas pbe led to too small values. more recently, eriksen. compared the cam - b3lyp and eom - ccsd dipoles of p - nitro - aniline in both gas and condensed phases and found that this range - separated hybrid (rsh) xcf considerably undershoots the magnitude of. a few specific investigations also appeared for larger molecules. in 2009, wong. obtained the of the two lowest - lying ess in 12 oligothiophenes and found that the are significantly larger with b3lyp than with a rsh, but no benchmark values were provided to pinpoint the most accurate functional. in 2010, the of a set of donor acceptor benzothiazolium derivatives were determined with b3lyp, cam - b3lyp, and cc2. for the 20 dyes for which direct comparisons are possible, mean absolute deviations of 5.1 and 1.7 d can be determined for b3lyp and cam - b3lyp, respectively, indicating that the latter functional is much more accurate for these ct molecules. in 2013, zaleny and co - workers compared the def2-qzvp gs and es dipole moments computed with cc2 and seven xcfs for five photochromic molecules (four spiro derivatives and one push they found that none of the tested xcfs could deliver accurate in all cases, and, in contrast with the previous studies, that b3lyp and pbe0 were more accurate than rsh for the ct molecule. the same year peluso s group investigated the electro - optical properties of a large azobenzene dye and concluded that while bmk could yield a transition wavelength in very good agreement with cc2, the of the two approaches differed by more than 4 d with the sv(p) atomic basis set. as can be seen from this literature survey, more is needed to obtain a general analysis of the accuracy of the td - dft. indeed, previous works were typically limited to a small number of xcfs, rather compact or specific compounds, and, more importantly, obtained contradictory conclusions, notably regarding both the accuracy of rsh for evaluating the of ct es and the amplitude of the dependency to the selected xcf. this has motivated the present investigation that treats a larger series of real - life dyes and compares a large set of xcfs to cc2 reference results. all dft and td - dft calculations have been performed with the gaussian09.d01 code, using optimal gs geometries determined at the m06 - 2x/6 - 31+g(d) level of theory. the cartesian coordinates of these structures can be found in the si of ref (20). dft and td - dft calculations were performed using tightened dft integration grids and the aug - cc - pvtz atomic basis set. the xcfs benchmarked here are svwn5, blyp, bp86, olyp, m06-l, m11-l, b3lyp, pbe0, m06, bmk, sogga11-x, m06 - 2x, m06-hf, cam - b3lyp, m11, and b97x - d, the six first being pure xcfs (free of exact exchange), the seven next being global hybrid xcfs including a growing fraction of exact exchange, whereas the three last are rsh. note that the es dipole moments reported here correspond to the total es density and do not rely on the so - called 1-particle ci density. the cc2 calculations have been performed on the same geometries with the turbomole program, systematically using the ri scheme, and default convergence parameters that were found to be sufficient for our purposes. note that we report the relaxed cc2 properties in the following and use them as reference values. as for the td - dft calculations, we selected the aug - cc - pvtz atomic basis set and did not apply the frozen - core approximation, both parameters being chosen to obtain accurate reference values. of course, cc2 dipole moments are not error free. nevertheless, by comparing the cc2/tzvp and cas - pt2/tzvp reported by thiel, sauer, and co - workers for their molecular set, a mean absolute error (mae) as small as 0.11 d is obtained. for the, the deviations between cc2 and cas - pt2 values are larger due to the presence of several high - lying es suffering from strong state - mixing. nevertheless, by considering the lowest es of each irreducible representation for the same set of molecules, a mae of 0.43 d is obtained. in subsequent works, thiel, sauer, and co - workers found that if a quite large atomic basis set has to be selected to reach converged cc2 dipole moments, the cas - pt2 and cc2 dipole moments undergo similar variations when increasing the basis set size. in short, this indicates that, despite their inherent limitations, cc2/aug - cc - pvtz dipole moments can be safely used to benchmark td - dft results. in the following the gs and es dipole moments are given as their total values ; e.g., for the gs, 1whereas the were computed as2obviously, as the total dipoles, this quantity is positive irrespective of the relative amplitudes of es and gs dipoles. in the tables below, to give insights into these relative magnitudes, we indicate in italics when 0.95). the value of the latter tends to increase with the amount of exact exchange included in the xcf. we also note that the two tested meta - ggas, namely m06-l and m11-l, yield more accurate than other pure xcfs. for the considered medium - sized molecules, m06 emerges as the most accurate functional for as it delivers the smallest mae (0.33 d) together with a large r (0.99), though several other xcfs, e.g., pbe0 and m06 - 2x, appear to be adequate as well. for the es dipole moments (see r.h.s. of table s-4 in the si), the obtained results are both less accurate and more dependent on the selected functional, and this exacerbated xcf dependence of td - dft compared to dft can be ascribed to the presence of ct es in the set. the mse is positive for all pure xcf and negative for all hybrid xcf, indicating that the former overestimate (to a larger extent than), while the former underestimate, in line of conclusions obtained for small molecules. the maes are significantly larger with the pure xcfs (> 1 d, m06-l and m11-l being again the most accurate of the group) than with hybrids (0.8), though the correlation remains worse than that for the gs property. the smallest mae (0.71 d) is again reached with m06 that also delivers the largest r (0.93) of the series. in a second group, one finds b3lyp, pbe0, and sogga11-x that are slightly less satisfying than m06. for the excess dipole (table 2), we notice a small mse for m11-l, due to the partial error compensation between the overestimation of both and. in contrast, hybrid xcfs that overshoot but undershoot, logically provide a significantly too small with the mse going from 0.45 d for b3lyp to 1.15 d for b97x - d. again, the smallest maes (ca. 1.0 d) are reached with the global hybrid xcfs, range - separated hybrids giving slightly larger deviations (ca. the best correlation between theory and experiment is obtained with m06 - 2x (r = 0.96), and all xcfs including large shares of exact exchange provide large r values, only slightly smaller than the m06 - 2x value. mse, mae, max(+) and max() are the mean signed error, mean absolute error, maximal positive and negative deviations, respectively, whereas r is the linear determination coefficient. the td - dft errors are given with respect to cc2, and are in d (except for r). for both m11-l and pbe0, the problematic data have been removed for the statistics (see footnotes in table 1). for the rest of this work, we have therefore continued with hybrid functionals only and excluded the pure xcfs that obviously deliver less consistent values. the set of larger molecules represented in scheme 2 was next considered. in this set, several states with strong ct character, for example, 17, 21, 25, 28, and 30 are included. the values are listed in table 3 together with the statistical data, whereas the corresponding and values are given in the si. for 17 that presents a huge at the cc2 level (21.8 d), both b3lyp (19.9 d) and pbe0 (19.9 d) provide more accurate estimates than cam - b3lyp (16.1 d) and b97x - d (14.1 d). in contrast, for 30, the b3lyp (10.6 d) and pbe0 (9.7 d) values significantly exceed the cc2 reference (7.2 d) that is reasonably reproduced by cam - b3lyp (6.3 d). this apparent lack of consistency between the ct nature and the adequacy of rsh, already noted above, is discussed in the following section. values in italics indicate that the norm of the total es dipole is smaller than its gs counterpart. at the bottom of the table these states were removed from the statistics. as can be seen in table s-7 in the si, for, the mses are positive for all xcfs except for m06-hf in this set of larger compounds. the maes, in the 0.150.30 d range, are obviously small for all xcfs. very large r are also obtained irrespective of the selected functional, the poorest value being 0.98 with m06-hf. for the, all xcfs underestimate the cc2 reference values with an error that roughly increases with the amount of exact exchange included in the functional, for example, the mse attains 0.25 d with pbe0 but 1.13 d with m06 - 2x. 1.01.5 d with most xcfs, although significantly larger deviations (> 2 d) are reached with both m06-hf and m11. the r values are satisfying, though less impressive than for (see table s-7). in short, the most important trend obtained in section 3.1, that is, the overestimation of by dft and the underestimation of by td - dft is confirmed for the compounds of scheme 2. nevertheless, compared to those in section 3.1, the average deviations tend to be smaller for but larger for. these changes are reflected in the statistical analysis of the given at the bottom of table 3 : the mse is positive for b3lyp and pbe0 but negative with all other xcfs. obviously, the maes are rather large, the most accurate functional being bmk (mae = 1.12 d), the least satisfying being m06-hf (mae = 2.37 d). in figure 2 we provide the mse, mae, and r for both and, considering all 37 cases reported in this work (sections 3.1 and 3.2), the corresponding representations for are given in figure s-1. first, we note that one should expect, on average, an underestimation of and when hybrid xcfs are used, the signed errors being quite large with the three rshs and m06-hf. for both and, the absolute deviations are of the order of 1 d with b3lyp, pbe0, m06, bmk, sogga11-x, and m06 - 2x, but are again larger with the rshs and m06-hf. the r values are in the 0.900.95 d range for and are more dependent on the selected xcfs for. indeed, if b3lyp provides the smallest mse (top of figure 2), it delivers the poorest r for (0.99 for all xcfs) for q and remain rather satisfying though less impressive for q. in other words, as for the dipole moments, td - dft provides less accurate excited - state quadrupole moments than dft does for their ground - state counterparts. the errors that can be expected from td - dft for this property are therefore ca. 0.4 d, though the most accurate xcf of the series for the excess quadrupoles, namely cam - b3lyp, delivers tiny mse, mae, and maximal deviations together with a very large r. several other xcfs, such as pbe0 and sogga11-x, also yield accurate data. interestingly, the m06 - 2x r is comparatively small, mostly due to the presence of an outlier, q3. we have investigated 31 ground - state dipole moments and 37 excited - state dipole moments with td - dft using the large aug - cc - pvtz atomic basis set and considering 16 different exchange - correlation functionals. the results have been compared to reference cc2 (relaxed) values obtained on the same structures with the same basis set. overall, a rather small impact of the selected functional on both and was found, at the notable exception of states presenting a charge - transfer character. on average, dft provides too large, whereas td - dft delivers too small, with a magnitude for the error that is larger for the latter property. consequently, the td - dft excess dipole moments tend to be too small, by ca. 1.01.5 d. gga and meta - gga do not necessarily yield larger deviations than hybrid xcf, but the trends obtained as measured by the correlation with the cc2 values are poorer. this conclusion also holds, to some extent, for hybrid functionals with a small share of exact exchange, e.g., b3lyp. it was known that global hybrid functionals tend to overestimate the electron hole separation in the charge - transfer state leading to too small transition energies. as this exaggerated separation can compensate the generally too small given by td - dft, b3lyp or pbe0 can deliver excess dipole moments matching the cc2 data even for strong ct cases, for which the transition energies obtained with these functionals are obviously far from the reference values. a study of increasingly long push pull systems indicated however that the trends in are much better reproduced by range - separated hybrids. overall, the xcf providing the largest correlation with cc2 for excess dipoles is m06 - 2x, though this functional provides too small by ca cam - b3lyp is another successful xcf, also giving a large r and a relatively small mae. eventually, for the five aromatic hydrocarbon derivatives investigated, a generally nice agreement between the excess quadrupole moments given by td - dft and cc2 is obtained, with average deviations of the order of 0.4 d.. | the accuracies of the excited - state dipole and quadrupole moments obtained by td - dft are assessed by considering 16 different exchange - correlation functionals and more than 30 medium and large molecules. except for excited - state presenting a significant charge - transfer character, a relatively limited dependency on the nature of the functional is found. it also turns out that while dft ground - state dipole moments tend to be too large, the reverse trend is obtained for their excited - state counterparts, at least when hybrid functionals are used. consequently, the td - dft excess dipole moments are often too small, an error that can be fortuitously corrected for charge - transfer transition by selecting a pure or a hybrid functional containing a small share of exact exchange. this error - cancelation phenomena explains the contradictory conclusions obtained in previous investigations. overall, the largest correlation between cc2 and td - dft excess dipoles is obtained with m06 - 2x, but at the price of a nearly systematic underestimation of this property by ca. 1 d. for the excess quadrupole moments, the average errors are of the order of 0.20.6 d for the set of small aromatic systems treated. |
a number of respiratory viruses including influenza viruses, respiratory syncytial virus (rsv), parainfluenza viruses, adenovirus and the recently described human metapneumovirus (hmpv) play an important role in acute respiratory tract infections (ari) in children. however, in a substantial portion of respiratory infections the aetiological agent is not known. there has been renewed interest in human coronaviruses (hcov) as a cause of some of these infections. coronaviruses are large enveloped singlestranded rna viruses that can infect both humans and a variety of domestic animals causing respiratory and enteric illness. until recently human coronavirus (hcov) 229e and oc43, identified in the 1960s, were the only known coronaviruses to infect humans. although primarily responsible for mild infections including the common cold reports of more severe upper and lower respiratory tract infections associated with hcov229e and hcovoc43 have been documented., the identification of a coronavirus, sarscov, as the causative agent of severe acute respiratory syndrome in 2003 has resulted in an increased interest in this group of viruses. subsequently two new human coronaviruses, hcovnl63, and hcovhku1, have been described. both infect young children, the elderly and immunocompromised and can lead to severe respiratory tract infections requiring hospitalisation. the prevalence and clinical importance of hcovnl63 in the south african hospital setting is not known. in this retrospective study 1055 nasopharyngeal, tracheal aspirate and bronchoalveoloar lavage samples were taken from children (age 13 days to 5 years) hospitalised with respiratory tract infections in 2003 and 2004 in the red cross war memorial children s hospital in cape town. samples had previously been screened by using an indirect immunofluorecence assay (light diagnostics, chemicon international, temecula, ca, usa) for the common respiratory viruses including rsv, influenza viruses a and b, parainfluenza viruses 1, 2, 3, adenovirus and cmv. hmpv was also tested for using reversetranscriptionpcr (rtpcr). in the south african setting, where the prevalence of hiv is high, all infant respiratory samples are routinely screened for cmv as in our setting this virus is a major cause of pneumonia in hivinfected children. for the detection of hcovnl63, rna was extracted from 200 l of sample using the seek viral rna kit according to the manufacturer s instructions (talent, trieste, italy). ten microlitres rna was reverse transcribed into cdna using random primers (roche diagnostics gmbh, penzberg, germany) and the iscript kit (biorad, hercules, ca, usa). pcr amplification of a region of the 1b gene of hcovnl63 was used for screening and positive samples were confirmed by amplification of a portion of the 1a gene. briefly 10 l of cdna was added to a 50 l of pcr mix containing 2 iu supertherm polymerase (jmr holdings, kent, uk), 15 mm mgcl2, 200 m each dntp and 02 m primers. pcr was performed with two sets of outer primers, one set to the 1b gene and one targeting the 1a gene from the study of van der hoek. to improve sensitivity a nested pcr was performed using 25 l outer product and inner primers described by smuts. the 1a amplicons were sequenced directly and the nucleotide sequences were deposited into genbank (eu477762eu477770). a respiratory virus was detected in 401/1055 (380%) samples collected over the 2year period from 2003 to 2004. the detection rate was higher in 2004, 248/559 (444%), compared with 2003, 153/496 (308%). cmv was most frequently found (158/1055 ; 150%) followed by adenovirus (n = 65 ; 62%), rsv (42 ; 40%), parainfluenza 3 (n = 32 ; 30%), hmpv (n = 28 ; 26%), influenza virus a (n = 8 ; 076%), parainfluenza virus 1 (n = 6 ; 057%), parainfluenza virus 2 (n = 4 ; 038%) and influenza virus b (n = 1 ; 009%). in 48 (45%) samples a known respiratory virus was grown in shell vial culture but could not be further identified. of cmvpositive respiratory samples 449% (71/158) were from hivinfected children, 240% (38/158) from hivnegative children and for the remainder the hiv status was unknown. in both hivpositive and hivnegative groups the rates of coinfection with another known respiratory virus were similar, 12/71 (169%) and 7/38 (184%) respectively. hcovnl63 was detected in 4/496 (081%) and 5/559 (089%) samples from 2003 and 2004 respectively. all hcovnl63infected children, with the exception of one aged 30 months, were under 2 years (table 1). the hiv status of only those children from 2004 was known and 4/5 hcovnl63infected children from this year were hivpositive. in two instances in 2003 3/4 positive samples were collected in march (autumn) while in 2004 hcovnl63positive samples were found in march, may, august and september (table 1). a diagnosis of pneumonia or lower respiratory tract infection was made in six (67%) children. two hcovnl63positive infants aged 50 and 71 days respectively, required admission to the intensive care unit ; both were hivpositive. clinical data of hcov nl63infected children nk, not known ; ta, tracheal aspirate ; npa, nasopharyngeal aspirate ; bal, bronchoalveolar lavage ; ltri, lower respiratory tract infection ; hmpv, human metapneumovirus. to determine the role hcovnl63 plays in respiratory illness in infants, respiratory samples that had previously been screened for rsv, influenza viruses a and b, parainfluenza viruses 1, 2, 3, adenovirus, hmpv and cmv were also tested by rtpcr for hcovnl63. due to limited resources, screening for other viruses such as enteroviruses, rhinoviruses and the other hcovs was not undertaken. this may be considered a limitation of the study as there is accumulating evidence that these viruses, in particular rhinoviruses, may play a more significant role in lower respiratory tract infections than previously recognised. the role and clinical significance of the recently identified human bocavirus and polyomaviruses, in respiratory disease is still under investigation. ideally comprehensive screening of respiratory samples for most if not all respiratory viruses and relevant respiratory bacteria should be undertaken in order to obtain greater insight into the epidemiological significance of these pathogens in respiratory disease in the local setting. further this would be beneficial to the clinical management of patients, including the administration of appropriate antiviral drugs and antibiotics. a postmortem study of hivinfected children in kwazulunatal, south africa showed frequent (52%) cmv detection in lung tissue compared with uninfected controls (4%). the significance of cmv detection in respiratory samples from uninfected children is not known but probably represents viral shedding from either a recently acquired primary infection or reactivation. this study is the first to report the presence of hcovnl63 in children hospitalised with respiratory illness in africa. hcovnl63 was found to circulate in infants and young children in both 2003 and 2004 with similar low prevalence rates of 08%. this finding is lower than that reported in previous studies where detection of hcovnl63 ranged from 1% to 73%,,,,,,, although higher prevalences of 88% and 93% have also been reported., most hcovnl63positive children (75%) were under 6 months, indicating that this younger age group may be more susceptible to severe infections requiring hospitalisation ; a finding supported by other studies where 38/76 (50%), 4/5 (80%) and 11/12 (92%) of hcovnl63 infections occurred in this age group respectively.,, further, immunosuppression may also contribute to increased susceptibility to severe hcovnl63 infection. in this study 4/5 all four were < 4 months of age and two required admission to icu. it is not known whether maternal antibodies, if present, would provide protection or reduce the severity of infection. this protection is likely to be most effective in infants under 2 months. in this study this observation is supported by the findings of other studies.,,, in a very recent study by dijkman. these antibodies disappeared within 3 months providing further evidence of their possible protective role in early life. in a recently published study of ambulatory children presenting with acute wheezing at the outpatient s department of the same hospital in 2004, this is significantly different (p = 00186) from the prevalence in the hospitalised group. this indicates that the virus is circulating in the community, probably causing mild symptoms which may trigger a wheezing episode requiring medical attention. in contrast the lower rate of hcovnl63 infection identified in the hospitalised children suggests that the virus rarely causes severe lower respiratory tract infections. however, all hcovinfected children in the ambulatory group were over 12 months of age supporting the possibility that younger children are more susceptible to severe infections requiring hospitalisation. no hcovnl63 infection was detected during the winter months in either 2003 and 2004, a finding also noted in the ambulatory population this pattern differs from that previously reported where hcovnl63 infection was predominantly found during the winter season.,,,,,,, detection during early spring and summer indicates that hcovnl63 may circulate at low levels throughout the year. in this study the clinical symptoms of hcovnl63infected infants were similar to those previously reported with lower respiratory tract infections including pneumonia predominating in this group.,,,,, in this study only specimens from hospitalised children were examined resulting in a bias towards children with more severe respiratory illness. mild hcovnl63 symptoms have also been reported,, indicating that hcovnl63 infections are probably more severe in the very young and immunocompromised. the role of hcovnl63 in enteric disease has not been established but reports of gastroenteritis associated with coronavirus infection have been documented with frequencies ranging from 6% to 33%.,,, in this study one child had a history of gastroenteritis and vomiting. a significant association with hcovnl63 infection and croup has been made but in this study it could not be determined if any of the samples were from children with croup. in conclusion these findings suggest that although hcovnl63 is circulating in the community it plays a minor role in severe respiratory tract infections in young children who require hospitalisation. ethical approval (018/2004) was granted by the research ethics committee of the faculty of health sciences, university of cape town, south africa. | background human coronavirus nl63 (hcovnl63) is a novel respiratory virus which is associated with respiratory tract infections in children. objective to determine the role of hcovnl63 in infants and young children hospitalised with acute respiratory tract infections (ari) in cape town, south africa. methods respiratory specimens were collected from 1055 infants and young children hospitalised with ari in 20032004. samples were screened by rtpcr to detect hcovnl63 and human metapneumovirus (hmpv). standard shell vial culture and immunofluoresence was used to detect the common respiratory viruses including rsv, influenza a and b viruses, parainfluenza viruses 1, 2, 3, adenovirus and cmv. results a respiratory virus was found in 401/1055 (380%) samples. hcovnl63 was detected in 9/1055 (085%) with peak activity during autumn (67%). most patients had a diagnosis of pneumonia or lower respiratory tract infection (6/9 ; 67%). conclusions this is the first report of hcovnl63 infections in hospitalised children in africa. during the 2year period hcovnl63 played a minor role in ari in children. |
laparoscopy in patients with poor cardiac function has been the subject of controversy and is considered by many surgeons a relative contraindication. we report the case of a patient who presented with acute cholecystitis and choledocholithiasis concurrent with unstable angina. our experience in laparoscopic management of patients with calculous biliary disease and severe coronary artery disease is examined. the patient was managed by coronary angioplasty and stenting immediately followed by laparoscopic cholecystectomy and common bile duct exploration under close invasive hemodynamic monitoring and low - pressure pneumoperitoneum. between 1996 and 2001, 39 patients with coronary artery disease and an asa class of iii or iv underwent laparoscopic cholecystectomy. eight of these patients (20.5%) had common bile duct stones necessitating laparoscopic common bile duct exploration. laparoscopic cholecystectomy and common bile duct exploration can be safely performed in patients with severe ischemic cardiac disease under close hemodynamic monitoring and a low - pressure pneumoperitoneum (10 to 12 mm hg). laparoscopy in patients with poor cardiac function has been the subject of controversy and is considered by many surgeons a relative contraindication. the management of patients developing acute cholecystitis following or concurrent with myocardial infarction or unstable angina is particularly challenging. we report here the case of a patient who presented with this scenario and briefly review our experience and management strategies in patients with severe ischemic cardiomyopathy presenting with biliary calculous disease. the patient is a 72-year - old man who presented with right upper quadrant pain radiating to his back. he had experienced multiple similar episodes in the past, but had no fever, chills, nausea, or vomiting. he has a history of systemic hypertension and coronary artery disease with prior coronary angioplasty and stenting. on further questioning, we noted a current worsening of unstable angina pectoris despite a nitroglycerin patch that he had recently been prescribed. in addition, he is on furosemide and quinapril. on physical examination, the patient was afebrile and anicteric. his abdomen was tender in the right upper quadrant with localized guarding and a murphy sign was present. blood workup showed a white blood cell count of 10 400 u / l, and abnormal liver function tests with total bilirubin 1.6 mg / dl, aspartate aminotransferase (ast) 208 iu / l, alanine aminotransferase (alt) 254 iu / l and alkaline phosphatase 191 ultrasonography showed cholelithiasis with a thickened gallbladder wall ; the common bile duct was of normal diameter. the patient was taken directly for a coronary angiogram that revealed severely depressed left ventricular systolic function with an ejection fraction in the range of 20% and severe single - vessel coronary artery disease involving a 90% and 80% sequential mid right coronary artery stenosis ; these lesions were angioplastied and stented. after consultation with the gastroenterologist, we decided to proceed the following day with laparoscopy. an intraoperative cholangiogram was performed and revealed obstruction of the distal common bile duct with multiple filling defects and no flow of contrast in the duodenum. the patient tolerated the surgery very well and was transferred to the surgical intensive care unit extubated and in stable condition. the patient was maintained postoperatively on intravenous hydralazine, enalapril, digoxin, nitroglycerin, milrinone, and furosemide to ensure optimal filling pressure and cardiac output. he was weaned off the milrinone drip by postoperative day 2 at which time the pulmonary artery catheter was removed. at 48 hours postoperatively, a heparin drip was started in view of the stent placement, and the patient was later placed on clopidrogel (plavix). a clear liquid diet was started on the second postoperative day, and the patient was advanced to a low - fat low - sodium diet the following day, at which point the t - tube was clamped then the drain removed. to discharge, an electrophysiologic study with ventricular stimulation was performed followed by implantation of a cardioverter - defibrillator in view of the severe ischemic cardiomyopathy with the increased risk of sudden death. in an epidemiologic study examining the relative risk for hospital admission for acute cholecystitis after admission for myocardial infarction using a statewide hospital discharge database and sequence symmetry analysis, it was noted that hospitalization for myocardial infarction may increase the risk of subsequent hospitalization for acute cholecystitis. in addition, acute cholecystitis may cause a clinical picture similar to that of cardiac ischemia with a syndrome of abdominal fullness, nausea, diaphoresis, chest pain, as well as angina pectoris, arrhythmias, or nonspecific st - t wave changes on the electrocardiogram. a vagally mediated cardiobiliary reflex is the presumed cause of these changes. in patients with known coronary artery disease who present with acute cholecystitis and epigastric or chest pain or a questionable electrocardiogram, the diagnosis of coronary ischemia has to be promptly ruled out with further testing, including stress testing and cardiac catheterization if needed, before proceeding with cholecystectomy ; however, surgeons must keep in mind that undue delay in treatment may result in both cardiac and septic complications. the management of patients presenting with acute cholecystitis immediately following or concurrent with myocardial ischemia is very challenging. anticoagulation or thrombolytic therapy for myocardial infarction (mi) increases the risk of surgical and postoperative bleeding. delaying surgical intervention for 6 months when the risk of recurrent mi has returned to that of the normal population puts the patient at risk for septic as well as other complications of gallstone disease. proceeding to early coronary angiography allows the immediate assessment of the coronary artery anatomy and potentially performing an angioplasty. it is important to start anticoagulation in these patients as soon as deemed safe postoperatively because holding anticoagulation for longer periods puts patients at risk for recurrent mi or stent thrombosis. in cases where the anatomy is not conducive to angioplasty, such as in triple - vessel disease, simultaneous cholecystectomy, and coronary artery bypass grafting, can be performed and has been previously reported. in patients with severely depressed cardiac function secondary to ischemic cardiomyopathy, the intraaortic balloon pump (iabp) has been used successfully to significantly improve cardiac performance so that patients can undergo definitive cholecystectomy (personal communication with franklin me jr.) we believe a definitive biliary procedure following coronary reperfusion is a good alternative to cholecystostomy that has been recommended in high - risk surgical patients. cholecystostomy drains, although occasionally useful in some critically ill ventilator - dependant septic patients, are associated with their own morbidity, have a mortality ranging from 5% to 36%, miss common bile duct stones and ascending cholangitis, are difficult to manage in outpatients with tube occlusion and recurrent symptoms, and usually require readmission for cholecystectomy. laparoscopy has traditionally been relatively contraindicated in patients with severe cardiac disease and cardiac failure in view of its effect on decreasing venous return and significant dropping of left ventricular stroke work, cardiac index, and stroke volume. in addition, releasing pneumoperitoneum may lead to a sudden increase in venous blood return and subsequent congestive heart failure and acute pulmonary edema. studies have shown, however, that laparoscopy may be safely performed in patients with compromised cardiac functions under close hemodynamic monitoring using intraand postoperative pulmonary artery catheters. carroll bj reported their experience with 13 patients with severe cardiac dysfunction undergoing laparoscopic cholecystectomy with appropriate hemodynamic monitoring and adequate perioperative support of cardiac function. although the authors had only 9 successfully completed laparoscopic cholecystectomy procedures and 1 death within 30 days of surgery, they felt that laparoscopic cholecystectomy can be performed safely for acute cholecystitis in patients with severe cardiac disease. a multicenter, prospective, randomized trial comparing two - stage vs one - stage management of patients with gallstone disease and ductal calculi demonstrated equivalent success rates and patient morbidity for the 2 management options but a significantly shorter hospital stay with the one - stage laparoscopic treatment and concluded that in fit patients (american society of anesthesiologists [asa ] class i and ii), one - stage laparoscopic treatment is the better option, and preoperative endoscopic stone extraction should be confined to poor - risk patients, ie, those with cholangitis or severe pancreatitis. in our own experience with laparoscopic common bile duct exploration by choledochotomy, 223 patients have now undergone such a procedure with a 96% success rate in technically clearing the common bile duct. we apply this one - stage approach to less fit patients (asa iii and iv) and feel that these patients would potentially benefit more from a lesser number of interventions, an earlier resumption of anticoagulation, and a shorter hospital stay. our experience with this approach has been extremely encouraging, and the case report presented here only illustrates one of these scenarios. in reviewing our prospectively maintained database, we identified 39 patients between 1996 and 2001 with coronary artery disease and an asa class of iii or iv who have undergone laparoscopic cholecystectomy. eight of these patients (20.5%) had common bile duct stones necessitating laparoscopic common bile duct exploration through a transcystic approach in 2 of them and a choledochotomy in 6. the presence of common bile duct stones was unsuspected in one of these patients based on preoperative ultrasound and liver function tests. laparoscopic cholecystectomy can be performed safely in patients with severe ischemic cardiac disease under close hemodynamic monitoring and a low - pressure pneumoperitoneum (10 to 12 mm hg). this applies as well to patients with unstable angina, following coronary angioplasty. in patients with common bile duct stones and in experienced hands, a laparoscopic common bile duct exploration can be performed in that same setting as a one - stage procedure. | background and objectives : laparoscopy in patients with poor cardiac function has been the subject of controversy and is considered by many surgeons a relative contraindication.methods:we report the case of a patient who presented with acute cholecystitis and choledocholithiasis concurrent with unstable angina. our experience in laparoscopic management of patients with calculous biliary disease and severe coronary artery disease is examined.results:the patient was managed by coronary angioplasty and stenting immediately followed by laparoscopic cholecystectomy and common bile duct exploration under close invasive hemodynamic monitoring and low - pressure pneumoperitoneum. between 1996 and 2001, 39 patients with coronary artery disease and an asa class of iii or iv underwent laparoscopic cholecystectomy. eight of these patients (20.5%) had common bile duct stones necessitating laparoscopic common bile duct exploration. no conversions were necessary, and no major morbidity or mortalities occurred.conclusions:laparoscopic cholecystectomy and common bile duct exploration can be safely performed in patients with severe ischemic cardiac disease under close hemodynamic monitoring and a low - pressure pneumoperitoneum (10 to 12 mm hg). |
inflammatory pseudotumors constitute a heterogenous group of reactive lesions characterized by a mixture of inflammatory cells and a minor component of spindle cells. this benign lesion needs to be distinguished from an inflammatory pseudotumor - like follicular dendritic cell (ipt - like fdc) tumor, a distinctive low - grade malignant neoplasm with consistent epstein - barr virus (ebv) association that has a similar morphology. the ipt - like variant of fdc tumor is usually found in the spleen or liver and appears to be much more indolent compared to conventional fdc sarcoma. splenic ipt - like fdc tumors are extremely rare lesions, and only a few cases have been previously reported. some of the cases referred to as ipt in the literature should be reclassified as ipt - like fdc tumors, especially when spindle cells are positive for ebv and there are no data for the expression of fdc markers within the lesion. we present a case of a splenic ipt - like fdc tumor that was incidentally found in a 61-year - old man and increased gradually in size during a period of 3 months. despite abdominal computed tomography (ct) and magnetic resonance imaging (mri) findings, the definitive diagnosis was established only after immunohistochemical study of the resected spleen. a 61-year - old male was admitted to the emergency surgical department of our hospital due to a car accident. basic laboratory tests of peripheral blood on admission were within normal range, and physical examination was unremarkable. abdominal ultrasonography examination incidentally revealed a well - circumscribed splenic mass 4 cm in diameter. abdominal ct confirmed the presence of a well - circumscribed isodense lesion 4.2 4 cm in size in the splenic hilum with subtle inhomogenous enhancement in the early - phase images (fig. 1). mri of the abdomen with contrast showed inhomogenous enhancement on gd - enhanced images (fig. a follow - up mri scan of the abdomen 3 months later confirmed the above - mentioned findings regarding the same signal characteristics on t1- and t2-weighted images, but the tumor had increased in size and measured 7.5 6 6 cm. two months later, the patient was referred to our surgical department for operative intervention and definite diagnosis. preoperatively, he received vaccination against hemophilus, pneumococcus and meningococcus, and an uncomplicated open splenectomy was performed. on gross examination, the spleen measured 13 11 7 cm and weighed 422 g. the cut surfaces of the tumor revealed a well - circumscribed solid tumor 10 8 7 cm, milky white with a central necrotic area measuring 5 cm. there were no lymph nodes in the splenic hilum. under microscopic examination, the lesion was an admixture of lymphocytes, plasma cells and a smaller percentage of spindle cells (fig. all of the spindle cells were stained with cd35 and a smaller percentage with cd21 and cd23. in situ hybridization analysis for ebv - encoded small rna revealed that most of the spindle cells in the lesion were positive for ebv. the patient remains disease - free with no evidence of recurrence on the last follow - up 1 year postoperatively. fdcs are part of the accessory immune system and are found in the primary and secondary lymphoid follicles serving as antigen - presenting cells for b cells that stimulate b cell proliferation and differentiation. fdc sarcoma is a neoplasmatic proliferation of spindled to ovoid cells showing morphologic and immumophenotypic features of fdcs. it is usually observed as lymphadenopathy, but it can also be present in a variety of extranodal sites and intra - abdominal organs, including the spleen. fdc sarcomas are rare neoplasms with less than 200 cases previously reported in the literature and constitute a heterogenous group of tumors with subtypes showing different histologic appearances and behavior. the ipt - like variant of fdc sarcoma was first classified as a distinct variant of fdc sarcoma by cheuk.. it differs from conventional fdc sarcoma in the following basic aspects : it is characterized by consistent association with ebv, selective localization in intra - abdominal organs and female predominance. the ipt - like variant of fdc tumors is composed of a polymorphic population of inflammatory cells including b - lymphocytes, t - lymphocytes, plasma cells and spindle cells in addition to the expression of fdc markers and ebv expression. clinicopathologic characteristics and radiologic findings of the previously published cases of ipt - like fdc tumors are summarized in table 1. there is a slight female predominance (female / male : 8/7), the median age of patients with ipt - like fdc tumors is 62 years, and the mean tumor diameter 7.2 cm. the maximum tumor diameter ranged from 3.2 to 22 cm [2, 3 ]. the most significant fact is that most of the patients are asymptomatic and without evidence of recurrence during a follow - up period that reaches 4 years or 6.5 years in some studies [3, 12 ]. lewis. proposed that these tumors may arise from a common mesenchymal cell that is capable of differentiating along different pathways. under the stimuli of ebv, some of them would differentiate to a myofibroblastic phenotype with expression of vimentin and sma, while others would acquire fdc characteristics with expression of fdc markers, such as cd21, cd23 or cd35. similarly, shia. proposed that the mesenchymal cells in ipt - like conditions would undergo transformation in fdc tumors under certain oncologic stimuli, such as ebv infection, and eventually become neoplastic. ct and mri findings of ipt - like fdc tumors seem to be similar to the conventional ipt lesions due to their similar morphology and due to the presence of inflammatory cells and fibrotic stroma in both cases. on ct scans, they are usually found as hypodense masses with occasional calcifications and heterogenous slow enhancement. mri depicts a low to isointense mass on t1-weighted images and highly intense areas with surrounding low intensity on t2-weighted images. imaging findings of ipt - like fdc tumors of the spleen are described in only a few studies due to the rarity of the disease. of the 13 previously reported studies of ipt - like fdc tumors of the spleen, 5 describe imaging findings on ct. all of them are characterized by low - density lesions on plain or enhanced ct. mri findings are available in only one case other than ours, and according to this, the lesion had areas of low signal intensity on t2-weighted images and was isointense on t1-weighted images. nevertheless, the degree and distribution of inflammatory cells and proliferating capillaries in ipt and ipt - like fdc lesions influence the staining patterns observed in ct / mri examination, and there is not a single imaging modality that can distinguish ipt from other splenic tumors [7, 9 ]. similar ct / mri findings are observed in primary splenic lymphoma and hamartoma, metastasis and angiosarcoma. therefore, it is difficult to discriminate safely benign lesions as ipt and malignant tumors as lymphoma. the ipt - like variants of fdc tumors found in the spleen are low - grade malignant neoplasms, and their prognosis seems to be excellent. from the previously published data for localized disease, nevertheless, due to the rarity of the disease, the short follow - up and the incompleteness of the published data, it is difficult to assess the need and benefit of adjuvant treatment. this is the first study that demonstrates a significant increase in size during a short period preoperatively and provides both ct and mri imaging findings. | inflammatory pseudotumor of the spleen with expression of follicular dendritic cell markers is an extremely rare lesion with only a few cases reported previously. the present study reports on an inflammatory pseudotumor of the spleen 10 8 7 cm in size that was incidentally found in a 61-year - old man and increased gradually in size during a period of 3 months. abdominal ultrasonography revealed a well - circumscribed splenic mass, and abdominal computed tomography confirmed the presence of a well - circumscribed isodense lesion in the splenic hilum with inhomogenous enhancement in the early - phase images and no enhancement on delayed - phase contrast - enhanced images. magnetic resonance imaging of the abdomen showed a well - defined isodense tumor on t1-weighted images with mildly increased signal intensity on t2-weighted images, and this is only the second study that provides mri findings of this entity. the patient underwent an uncomplicated open splenectomy for definitive histologic diagnosis. under microscopic examination, the lesion was an admixture of lymphocytes, plasma cells and spindle cells. in situ hybridization analysis for epstein - barr virus (ebv) revealed that most of the spindle cells were positive for ebv, and immunochemistry showed the expression of the follicular dendritic cell markers cd21, cd35 and cd23 within the tumor. the diagnosis of inflammatory pseudotumor - like follicular dendritic cell tumor was established. |
according to an estimation of the world health organization (who), more than 366 million people worldwide will have diabetes mellitus in 2030 ; the urban population in developing countries is projected to double between 2000 and 2030. diabetes mellitus (dm) is an endocrine and metabolic disease which affects almost all organs in our body. although the lungs are not thought to be primarily affected by diabetes and lung physiological and structural abnormalities are happening in both type 1 and type 2 diabetes, the lung should be considered as a target organ. chronic hyperglycemia is considered as the main reason for diabetes complications. in the process of prolonged increased glucose concentration, reactive oxygen species (ros) overproduction may be one of the key factors which induce organ injury. decreased antioxidative function and increased oxidative stress were seen in the lung of diabetic rats and rabbits [2, 3 ]. mitochondria dysfunction will cause oxidative stress injury which is one of the underlying factors for neurodegenerative diseases, diabetes, cardiovascular diseases, cancer, and so on. physiological hypoxia triggers the changes of mitochondrial redox and increases superoxide generation at complex iii in alveolar epithelial cells. mitochondrial aldehyde dehydrogenase 2 (aldh2) is a nuclear - encoded mitochondrial enzyme that localizes in mitochondrial matrix ; aldh2 protein had been detected in liver, lung, heart, kidney, testis, esophagus, stomach, colon, and pancreas. our previous study had reported that, in diabetic rats, ethanol (etoh) at low concentration offered myocardial protection through activation of aldh2 expression, and upregulation of aldh2 also plays the protective effect in myocardial ischemia and reperfusion injury, diabetes cardiomyopathy, and kidney and brain injury [912 ] ; meanwhile, some researchers reported the association between aldh2 and lung disease. xu. reported that, in neonatal rat lung, after prolonged hyperoxic exposure, aldh2 was downregulated. in lung epithelial cells, overexpression of aldh2 attenuated hyperoxia - induced cell death through reduction of ros, activation of erk / mapk, and pi3k - akt signaling pathways. but it remains unknown whether activation of aldh2 expression can prevent diabetes induced lung injury. so in this study, we mimic diabetes model by intraperitoneal injection of streptozotocin (stz) to observe the role of etoh at low concentration in diabetes induced lung injury and analyze the related mechanisms. male sprague - dawley (sd) rats (200250 g) obtained from the animal center of bengbu medical college were selected for the study. all animal studies were approved by the animal ethics committee of bengbu medical college and performed in accordance with the ethical standards. streptozotocin (stz) was purchased from sigma (st. louis, mo, usa). malondialdehyde (mda) and superoxide dismutase (sod) assay kits were from nanjing jiancheng bioengineering institute, china. ethanol (etoh, figure 1) was purchased from bengbu new chemical reagent factory, china. the primers used were as follows : for aldh2 forward : 5-gtg ttc gga gac gtc aaa ga-3 and reverse : 5-gca gag ctt ggg aca ggt aa-3 and the product size was 187 bp ; for -actin forward : 5-gat ggt ggg tat ggg tca gaa gga c-3 and reverse : 5-gct cat tgc cga tag tga tga ct-3 and the product size was 630 bp. mouse anti - aldh2 and anti--actin monoclonal antibodies were from santa cruz biotechnology (ca). stz at 55 mg / kg freshly dissolved in 0.1 mol / l sodium citrate buffer (ph 4.5) was injected intraperitoneally to induce diabetic models in overnight fasted rats. the rats in control group were injected with a similar volume of sodium citrate buffer alone. the rats with plasma fasting blood glucose level higher than 16.7 mmol / l 72 h after injection were regarded as diabetic. animals were randomly divided into control, diabetes (dm), and etoh+dm groups, respectively (n = 6). in etoh+dm group, dm rats were fed with 2.5% etoh in their drinking water for one week to initiate drinking and then it was changed to 5% etoh continuous access through the 8 weeks. the plasma fbg level and body weight (bw) were measured at the 8th week. the ratio of lw / bw was calculated. at the end of the experimental period the supernatant was collected after centrifugation for 20 min (2000 rpm). malondialdehyde (mda) content and superoxide dismutase (sod) activity were detected according to the instruction manual. for histological analysis by light microscope, the lung tissue was harvested and fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin and cut into 5 m thick serial sections, and then stained with hematoxylin and eosin (he) for light microscope observation. lung injury degree was evaluated according to mikawa 's scoring standards : (1) alveolar congestion, (2) hemorrhage, (3) infiltration or aggregation of neutrophils in airspace or vessel wall, and (4) thickness of alveolar wall / hyaline membrane formation. each item was scored on a 5-point scale as follows : 0 : minimal damage, 1 : mild damage, 2 : moderate damage, 3 : severe damage, and 4 : maximal damage. the final lung injury score was the summation of the four items. lung tissue was dissected and small pieces were fixed with 2.5% glutaraldehyde in 0.1 mol / l cacodylate buffer for 2 h and postfixed in 1% osmium tetroxide in 0.1 mol / l cacodylate buffer for 1 h. ultrathin sections were cut and contrasted with uranyl acetate followed by lead citrate and observed with jem-1230 transmission electron microscope (jeol, japan). briefly, total rna was extracted with trizol according to the manufacturer 's instructions. a total rna (2 mg) quantification of the result was determined through measuring the optical density of the labeled bands ; the value was normalized to -actin intensity level. anti - aldh2 (1 : 500) antibody was used. mouse anti--actin antibody (1 : 500) was used as an internal control. the immunoblots were exposed to x - ray film and analyzed with a digital image system. statistical comparisons were carried out by one - way variance analysis and the newman - keuls test. in contrast to control group, fbg levels in dm and etoh+dm groups were increased significantly and fbg level in etoh+dm group was lower than in dm group (table 1). compared with control animals, body weight (bw) and lung weight (hw) were significantly decreased in dm and etoh+dm groups, and lw / bw was increased in dm group. in contrast to dm group, bw was increased and lw / bw was decreased in etoh+dm group (table 1). in contrast to control rats, pulmonary sod activity was decreased in dm group while mda content was increased in dm and etoh+dm groups. in etoh+dm group, sod activity was higher while mda content was lower than in dm group (table 2). in control group, alveolar walls in lung tissue had unique shape and size, and hemorrhage and inflammatory infiltration were rare. in dm group, alveolar septum was thickened widely, infiltration of inflammatory cells and hemorrhage appeared, and the lung injury score was higher than in control group. the injury was ameliorated in etoh+dm group than in dm group (figure 2 and table 1). in control group, many regular structure lamellar bodies appeared in type ii alveolar cell cytoplasm. in dm group, the structure of lamellar bodies was collapsed, discontinuous, and vacuolated. in etoh+dm group, the expressions of pulmonary aldh2 mrna and protein were decreased in dm group ; compared with dm group, pulmonary aldh2 mrna and protein expressions were increased in etoh+dm group (figures 4 and 5 and table 3). in the present study, we observed that lung oxidative stress injury occurred in diabetic rats, which was indicated by the increase of pulmonary mda content and decrease of sod activity ; meanwhile, the decrease of pulmonary aldh2 mrna and protein expressions happened accompanying the happening of lung swelling, the destruction of pulmonary tissue, and type ii alveolar cell structure. when the diabetic rats were treated with etoh at low concentration which was reported to activate aldh2 expression [8, 10 ], lung aldh2 mrna and protein expressions were increased, lung oxidative stress injury and swelling degree were attenuated, the destruction of pulmonary tissue and type ii alveolar cell structure was alleviated, suggesting that downregulation of pulmonary aldh2 was likely to be correlated with oxidative stress injury in diabetic rats, and activation of aldh2 with etoh at low concentration attenuated diabetes induced lung injury and oxidative stress overload. in the development of diabetes, oxidative stress induced by chronic hyperglycemia plays a key role in the pathogenesis of diabetes - related complications including lung diseases. diabetes induces impairment of defense system, which is associated with reduced antioxidant capacity, abnormal activity, or expression of antioxidant enzymes. an increase of mda, a lipid peroxidation marker, accompanied by the depressing of sod activity (one of the most important endogenous antioxidase enzymes), is assessed to imply oxidant damage. oxidative stress can destroy the pulmonary structure. in our study, we observed that, in diabetic rat, pulmonary mda content was increased with the decrease of sod activity, and the structures of lung tissue and type ii alveolar cell were destroyed, suggesting the unbalance of lung antioxidative system induced lung injury in diabetes rats. when the diabetic rat was treated with etoh at low concentration, pulmonary mda content was decreased and sod activity was increased accompanying the recovery of lung tissue and type ii alveolar cell structures in contrast to diabetic rat, suggesting that etoh protected the lung tissue through antioxidative stress role. activation of aldh2 with alda-1 alleviated cardiac ischemic and reperfusion injury with the decrease of 4-hne in rodent models. aldh2 overexpression rescued neuronal survival against 4-hne treatment in pc12 cells. in male c57bl/6 mice, activation of aldh2 with etoh prevented renal ischemia and reperfusion injury with suppressed lipid peroxidation and increased sod activity. but few reports focus on the relationship of pulmonary aldh2 and diabetes induced lung injury. aldh2 overexpression attenuated hyperoxia - induced cell death in lung epithelial cells through reduction of ros, activation of erk / mapk, and pi3k - akt signaling pathways. since diabetes induced lung injury accompanied with oxidative stress, while increasing aldh2 expression could decrease oxidative stress, so we investigated whether activating pulmonary aldh2 expression by etoh could decrease lung oxidative stress injury in diabetic rats. the results displayed pulmonary aldh2 mrna and protein expressions were decreased in diabetic rats, while etoh treatment increased aldh2 expression and, meanwhile, decreased oxidative stress injury ; it suggested that, in diabetes induced lung injury, the aggravation of oxidative stress may be derived from the decrease of pulmonary aldh2 expression and improving pulmonary aldh2 expression could be against the happening of hyperglycemia induced oxidative stress. it is worthwhile to note that, in our study, we selected etoh to promote aldh2 expression because there had been many papers reporting that etoh at a suitable concentration could activate aldh2 expression and play the protective effect [8, 10, 12, 18 ]. but in clinic, etoh is difficult to apply for patients because of its toxicity and addiction. so selecting an appropriate drug to activate pulmonary aldh2 expression may be beneficial for diabetic patients who had suffered from lung injury. in conclusion, our results indicated that, in diabetes induced lung injury in rat model, pulmonary aldh2 expression was decreased. treatment with etoh at low concentration can decrease diabetes induced lung injury through activating aldh2 expression. | to observe the changes of lung injury when diabetic rats were treated with low concentration of ethanol (etoh) and analyze the related mechanisms, male sprague - dawley (sd) rats were divided into control, diabetic (dm), and etoh+dm groups. diabetic rat was mimicked by injection of streptozotocin intraperitoneally. fasting blood glucose (fbg) level, lung weight (lw), body weight (bw), and lw / bw were measured. the changes of lung tissue and type ii alveolar cell were detected. pulmonary malondialdehyde (mda) content and superoxide dismutase (sod) activity were measured ; meanwhile, aldh2 mrna and protein expressions were detected by rt - pcr and western blotting, respectively. compared with control group, in dm group, sod activity was decreased ; fbg level, lw / bw, mda content, aldh2 mrna, and protein expressions were decreased. compared with dm group, in etoh+dm group, sod activity, aldh2 mrna, and protein expressions were increased ; lw / bw and mda content were decreased. the structures of lung tissue and lamellar bodies were collapsed in dm group ; the injury was attenuated in etoh+dm group. our findings suggested that, in diabetic rat, pulmonary aldh2 expression was decreased accompanying lung injury. etoh at low concentration decreased diabetes induced lung injury through activating aldh2 expression. |
participants consisted of 108 (66 men and 42 women) older adults with one or more chronic diseases including diabetes, coronary heart disease, chronic pain, and hypertension. the sample was recruited from several retirement clubs such as kholde barin club and tamine ejtemai organization in shiraz - iran, using convenience sampling. inclusion criteria for participation in this study were as follows : (1) age of 55 or older ; (2) having one or more physical disease confirmed by a physician ; and (3) the ability to understand the questionnaire items. demographic characteristics of older adults with chronic disease (n = 108) mhls (9) was used to measure the health locus of control. the mhlc (mhlc - form c) scale consists of three components including internal, chance and powerful others. each of the subscales contains six items, measured on a 6 point - likert scale, ranging from 1 (strongly disagree) to 6 (strongly agree).the god locus of health control (glhc) was added later (6 items) to measure the belief that god is the locus of control of one s health status (15). internal consistency reliability coefficients of 0.66, 0.67, 0.58 and 0.91 has been reported for internal mhloc, powerful others mhloc, chance mhloc and god mhloc, respectively (9, 15). the mhlc has been used in iran and found to have a good reliability and validity. research in iran has reported cronbach s alpha coefficients of 0.72, 0.70 and 0.81 for internal, chance and powerful others health locus of control (16). a score of 0 to 4 on this scale is normal, but a score greater than 5 suggests depression. gds is one of the most widely used scales for screening depression in the old adults and has been used to identify depression in the elderly in iran. the gds has been found to have a good reliability and validity for the elderly population (18). in iran, a cronbach s alpha coefficients of 0.92 have been reported for the gds (19). to analyze the data, descriptive statistics indexes such as mean and furthermore, spearman s correlation coefficient was performed to evaluate the relationship between the components of health locus of control and depression. to determine the role of the component of health locus of control in the prediction of depression, a simultaneous multiple regression analysis was performed. participants consisted of 108 (66 men and 42 women) older adults with one or more chronic diseases including diabetes, coronary heart disease, chronic pain, and hypertension. the sample was recruited from several retirement clubs such as kholde barin club and tamine ejtemai organization in shiraz - iran, using convenience sampling. inclusion criteria for participation in this study were as follows : (1) age of 55 or older ; (2) having one or more physical disease confirmed by a physician ; and (3) the ability to understand the questionnaire items. the mhlc (mhlc - form c) scale consists of three components including internal, chance and powerful others. each of the subscales contains six items, measured on a 6 point - likert scale, ranging from 1 (strongly disagree) to 6 (strongly agree).the god locus of health control (glhc) was added later (6 items) to measure the belief that god is the locus of control of one s health status (15). internal consistency reliability coefficients of 0.66, 0.67, 0.58 and 0.91 has been reported for internal mhloc, powerful others mhloc, chance mhloc and god mhloc, respectively (9, 15). the mhlc has been used in iran and found to have a good reliability and validity. research in iran has reported cronbach s alpha coefficients of 0.72, 0.70 and 0.81 for internal, chance and powerful others health locus of control (16). a score of 0 to 4 on this scale is normal, but a score greater than 5 suggests depression. gds is one of the most widely used scales for screening depression in the old adults and has been used to identify depression in the elderly in iran. the gds has been found to have a good reliability and validity for the elderly population (18). in iran, a cronbach s alpha coefficients of 0.92 have been reported for the gds (19). the mhlc (mhlc - form c) scale consists of three components including internal, chance and powerful others. each of the subscales contains six items, measured on a 6 point - likert scale, ranging from 1 (strongly disagree) to 6 (strongly agree).the god locus of health control (glhc) was added later (6 items) to measure the belief that god is the locus of control of one s health status (15). internal consistency reliability coefficients of 0.66, 0.67, 0.58 and 0.91 has been reported for internal mhloc, powerful others mhloc, chance mhloc and god mhloc, respectively (9, 15). the mhlc has been used in iran and found to have a good reliability and validity. research in iran has reported cronbach s alpha coefficients of 0.72, 0.70 and 0.81 for internal, chance and powerful others health locus of control (16). a score of 0 to 4 on this scale is normal, but a score greater than 5 suggests depression. gds is one of the most widely used scales for screening depression in the old adults and has been used to identify depression in the elderly in iran. the gds has been found to have a good reliability and validity for the elderly population (18). in iran, a cronbach s alpha coefficients of 0.92 have been reported for the gds (19). to analyze the data, descriptive statistics indexes such as mean and standards deviation were used. furthermore, spearman s correlation coefficient was performed to evaluate the relationship between the components of health locus of control and depression. to determine the role of the component of health locus of control in the prediction of depression, the mean age of the sample was 62.34 years old, ranging from 55 to 78. the majority of the participants were married (93.5%) and stheir level of education varied from below high school (58%), to high school (31%) and to undergraduate education (11%). as displayed in table 2, the higher score was on internal locus of control (m = 23.15), followed by god (m = 21.21), powerful others (m = 17.62) and chance (m = 15.25). the mean score on depression scale was in the normal range (m = 4.85 sd =.81). according to the results, about 24 participants (22.7%) [15 women (62.5%), 9 men (37.5%) ] met the criteria for depression. pearson s correlation coefficient was performed to examine the relationship between the components of health locus of control and depression in the sample. as demonstrated in table 3, a significant association was found between the two components of health locus of control including internal (r =.30, p <.01) and god (r =.24, p <.01) with depression. the results did not show any significant correlation of chance and powerful others control with depression. the results revealed that internal hloc (=.32, p <.01) and god hloc (=.20, p <.05) significantly predicted depression. the two other components of health locus of control such as chance and powerful others did not predict depression. findings showed that independents variables explained 26% of the total variance of the model (r2 =.26, p <.001). means and standard deviations on the scales of health locus of control and geriatric depression scale multidimensional health locus of control the relationship between subscales of health locus of control and depression multiple regression analysis for predicting depression b= unstandardized coefficient the purpose of this study was to investigate the prediction of depression based on health locus of control among a group of elderly people with chronic diseases. the results indicated that a great proportion of participants experienced the symptoms of depression (22.7%). this finding is consistent with the majority of research conducted on older people suffering from chronic diseases. these studies have found that elderly people with chronic diseases are more likely to experience the symptoms of depression. for example, in several studies on older adults, the prevalence of depression symptoms was greater among the elderly with chronic diseases compared to those without chronic illnesses (20). older adults with chronic health conditions have higher rates of depression than those who are medically well (21). the results of previous studies on the prevalence of depression among older adults are mixed. some of these studies have reported a higher prevalence and some of them reported lower prevalence of depression compared with the present study (22). the discrepancy between previous research and the results of this study can be due to various demographic characteristics, different types of chronic illnesses and psychosocial factors. the findings also showed that participants obtained higher score on internal control than other dimensions of mhlc. this finding is similar to another research indicating high level of internal control among patients who desired to control their health conditions (23, 24). furthermore, the findings showed that two dimensions of the health locus of control including internal hlc and god hlc predicted depression in the elderly. the older adults with low internal health locus of control and low perceived god control were more likely to have depressive symptoms than others. the findings on the internal locus of control in predicting depression were consistent with a previous research indicating a negative association between internal control and depression (25, 26). the study supports the theory of locus of control that perceiving internal control is an adaptive strategy in coping with illness outcomes (27). these results provide more support for beck s cognitive theory on depression, which emphasizes the significance of adaptive beliefs in emotional state (28). the results of this study on predicting depression based on god control is also in agreement with some other researches that found attributing difficulties to god may reduce depression (29). previous studies have shown that religious beliefs and practices protect the elderly people against depression (2, 30)., religious beliefs may give individuals a form of indirect control over their lives, reducing the need to depend on chance or powerful others (30). one previous research on age has had mixed results. a possible explanation for this may be that the research sample was not diverse on age. these findings lend further support to the importance of internal locus of control in promoting older adults mental health. this study also supports this idea that muslim older adults perceive their health condition as god s will (31). this research contributes to our understanding of the psychological influences on mental health, particularly among the elderly. a small sample was used in this study, so for future studies, it is recommended to include a larger sample size to reach a more reliable results. furthermore, this was a cross - sectional study, thus it could not provide any information on the impact of locus of control on individuals depression over time, so this subject needs to be further investigated in longitudinal studies. finally, this study only investigated the effects of the components of health locus of control on depression. future studies need to survey the impact of other important socio - psychological constructs such as self - efficacy and social support on older adults depression. this study highlighted the importance of the internal locus of control as a protecting factor against depression in older adults suffering from chronic diseases. indeed, older adults who perceived more control on their physical condition were less likely to suffer from depression. moreover, this study suggests that attributing difficult condition such as physical illness to god and destiny helped iranian older adults to have a better mental health. | objective : the purpose of this study was to examine the prediction of depression on a group of iranian older adults based on components of health locus of control.method:sixty-six men and 42 women over the age of 55 were recruited from the retirement clubs in shiraz, using convenience sampling. the participants completed the research questionnaires including the geriatric depression scale (gds) and the multidimensional health locus of control scale (mhlc).results : the findings on health locus of control revealed that the highest score was on internal locus of control followed by god, powerful others and chance. the mean score on depression was on a normal range. multiple regression analysis showed that two independent variables including internal control (=.32, p < 0.01) and god control (=.20, = p < 0.03) significantly predicted depression. the other components of health locus of control such as chance and powerful others as well as age did not predict depression. findings also revealed that the independents variables explained 26% of the total variance of depression (r2 =.26, p < 0.001).conclusion : his study provides more support for the application of theory of health locus of control on depression. |
we recruited patients who were initially diagnosed with ntg at seoul national university hospital glaucoma clinic from september 2007 to march 2009. ntg was diagnosed based on clinical and visual field evaluations as well as glaucomatous optic nerve changes with corresponding visual field defects and a peak iop of less than 22 mmhg. patients diagnosed with ntg were admitted overnight and underwent circadian bp and iop measurements as described below. for unilateral ntg, the affected eye was evaluated ; in bilateral ntg, we evaluated a randomly selected eye. those patients with a previous or current history of anti - glaucoma medication use, severe heart or renal failure causing hemodynamic instability or any other ocular disease that might cause visual field loss and peak iop exceeding 22 mmhg during a hospitalized circadian iop measurement and subsequently lead to the diagnosis of primary open angle glaucoma (poag) were excluded from this study. during the same time period, we recruited a control group of age- and gender - matched patients who were not diagnosed with glaucoma but who had been admitted to kangbuk samsung hospital for cataract surgery. iop was adjusted by 2.5 mmhg for every 50 m that deviated from 525 m. all procedures conformed to the declaration of helsinki, and our study was approved by the institutional review board of the clinical research institute. ntg patients were admitted in the afternoon (between 1 and 2 pm), and they underwent bp and iop measurements at 3, 5, 7, 9, and 11 pm ; 1, 7, 9, and 11 am ; and at 1 pm the following day. systolic blood pressure (sbp) and diastolic blood pressure (dbp) measurements was taken using an automated bp monitor (pa350 ; mediana, wonju, korea) placed on the patient 's upper arm after at least three minutes of rest. iop was subsequently measured using goldmann applanation tonometry three times alternatively for each eye, and the average of the three values was recorded as the iop. control patients were hospitalized in the morning (at 7 am) the day before the cataract surgery and underwent bp and iop measurement in the same manner as our ntg patients every two hours until 11 pm. the 1 am measurement was omitted in the control group to guarantee enough sleep time since most of the patients had cataract surgery scheduled early the next day. all ntg patients had visual field examinations with the 30 - 2 full - threshold humphrey field analyzer (carl zeiss ; meditec inc., dublin, ca, usa) within two weeks after discharge. only those patients with reliable results (false - positive error less than 20%, false - negative error less than 20% and a fixation loss less than 20%) were included in our study. visual field indices for analysis were mean deviation (md) and pattern standard deviation (psd). mean arterial pressure (map) and mean ocular perfusion pressure (mopp) were calculated as follows : map = dbp+1/3(sbp - dbp) ; mopp=2/3map - iop. the fluctuation of each parameter was defined as the difference between the peak and the trough measurement of each parameter. additionally, we measured the degree of bp reduction during the night or morning using the following indices : nocturnal dip index=(diurnal average map - nocturnal lowest map)/diurnal average map100 (%) ; morning dip index=(24-hour average map - morning lowest map)/24-hour average map100 (%). the nocturnal period was defined as from 8 pm to 6 am the following day, and the diurnal period included the remaining times of the day [13, 21 ]. ntg patients were divided into three groups according to the degree of morning bp reduction as follows : non - dipper, morning dip index<5% ; dipper, morning dip index 5% but < 10% ; and over - dipper, morning dip index 10%. spss ver. 15.0 (spss inc., chicago, il, usa) was used for all statistical analyses. patient demographics and hemodynamic and iop parameters were compared between the ntg and control groups using mann - whitney u test and chi - square tests. patient demographics, hemodynamic and iop parameters and visual field indices were compared among three ntg subgroups using kruskal - wallis h test and chi - square tests. we recruited patients who were initially diagnosed with ntg at seoul national university hospital glaucoma clinic from september 2007 to march 2009. ntg was diagnosed based on clinical and visual field evaluations as well as glaucomatous optic nerve changes with corresponding visual field defects and a peak iop of less than 22 mmhg. patients diagnosed with ntg were admitted overnight and underwent circadian bp and iop measurements as described below. for unilateral ntg, the affected eye was evaluated ; in bilateral ntg, we evaluated a randomly selected eye. those patients with a previous or current history of anti - glaucoma medication use, severe heart or renal failure causing hemodynamic instability or any other ocular disease that might cause visual field loss and peak iop exceeding 22 mmhg during a hospitalized circadian iop measurement and subsequently lead to the diagnosis of primary open angle glaucoma (poag) were excluded from this study. during the same time period, we recruited a control group of age- and gender - matched patients who were not diagnosed with glaucoma but who had been admitted to kangbuk samsung hospital for cataract surgery. iop was adjusted by 2.5 mmhg for every 50 m that deviated from 525 m. all procedures conformed to the declaration of helsinki, and our study was approved by the institutional review board of the clinical research institute. ntg patients were admitted in the afternoon (between 1 and 2 pm), and they underwent bp and iop measurements at 3, 5, 7, 9, and 11 pm ; 1, 7, 9, and 11 am ; and at 1 pm the following day. systolic blood pressure (sbp) and diastolic blood pressure (dbp) measurements was taken using an automated bp monitor (pa350 ; mediana, wonju, korea) placed on the patient 's upper arm after at least three minutes of rest. iop was subsequently measured using goldmann applanation tonometry three times alternatively for each eye, and the average of the three values was recorded as the iop. control patients were hospitalized in the morning (at 7 am) the day before the cataract surgery and underwent bp and iop measurement in the same manner as our ntg patients every two hours until 11 pm. the 1 am measurement was omitted in the control group to guarantee enough sleep time since most of the patients had cataract surgery scheduled early the next day. all ntg patients had visual field examinations with the 30 - 2 full - threshold humphrey field analyzer (carl zeiss ; meditec inc., dublin, ca, usa) within two weeks after discharge. only those patients with reliable results (false - positive error less than 20%, false - negative error less than 20% and a fixation loss less than 20%) were included in our study. visual field indices for analysis were mean deviation (md) and pattern standard deviation (psd). mean arterial pressure (map) and mean ocular perfusion pressure (mopp) were calculated as follows : map = dbp+1/3(sbp - dbp) ; mopp=2/3map - iop. the fluctuation of each parameter was defined as the difference between the peak and the trough measurement of each parameter. additionally, we measured the degree of bp reduction during the night or morning using the following indices : nocturnal dip index=(diurnal average map - nocturnal lowest map)/diurnal average map100 (%) ; morning dip index=(24-hour average map - morning lowest map)/24-hour average map100 (%). the nocturnal period was defined as from 8 pm to 6 am the following day, and the diurnal period included the remaining times of the day [13, 21 ]. ntg patients were divided into three groups according to the degree of morning bp reduction as follows : non - dipper, morning dip index<5% ; dipper, morning dip index 5% but < 10% ; and over - dipper, morning dip index 10%. spss ver. 15.0 (spss inc., chicago, il, usa) was used for all statistical analyses. patient demographics and hemodynamic and iop parameters were compared between the ntg and control groups using mann - whitney u test and chi - square tests. patient demographics, hemodynamic and iop parameters and visual field indices were compared among three ntg subgroups using kruskal - wallis h test and chi - square tests. twenty - four patients were included in the ntg group (mean age, 49.813.4 years ; 10 males and 14 females), and 22 patients were included in the control group (mean age, 53.113.7 years ; 15 males and 7 females). two patients admitted for bp and iop measurement after initial diagnosis of ntg revealed peak iop values over 22 mmhg, and these patients were excluded from the study. table 1 summarizes patient demographics and hemodynamic and iop parameters of both ntg patients and normal controls. six of the 24 patients in the ntg group had hypertension and were under medical management for this condition, while none of the control group patients had underlying hypertension. there were no significant differences in the hemodynamic parameters between the two groups in terms of mean sbp, mean dbp, mean map, and map fluctuation (table 1). we found no definite nocturnal bp dip in either group, but the ntg group showed a prominent morning bp dip. therefore, we made a new index, the morning dip index, exhibiting the degree of morning bp dip as represented in our methods section. the ntg group showed a significantly large morning bp dip compared to the control group (7.14.2% vs. 3.83.4%, p=0.022) fig. the ntg group usually showed a higher iop level than the control group, but this difference was not statistically significant (fig. 2 and table 1). in both groups, the mean iop was low during the nighttime and high during the daytime, especially in the early morning. there were no significant differences in peak iop between the two groups, but the ntg group showed a significantly large iop fluctuation compared to the control group (3.91.1 mmhg vs. 3.02.0 mmhg, p=0.040). the ntg group also showed a lower mopp than the control group at all times, and the difference was statistically significant at 9 am and 7 pm (fig.1). however, there were no significant differences in opp between the two groups in terms of mean mopp and mopp fluctuations (table 1). the ntg group was subdivided into three groups according to the degree of morning bp dip : non - dipper, dipper and over - dipper groups. table 2 presents patient demographics, hemodynamic and iop parameters and visual field indices of each subgroup. morning non - dippers were younger than dippers or over - dippers, but this difference did not reach statistical significance. there were no significant differences in mean sbp, mean dbp or mean map among the three groups, while the over - dipper group showed a significantly large map fluctuation. the mean and peak and fluctuation of iop among the subgroups also were not significantly different. while the mean mopp was not significantly different, the mopp fluctuation showed significant differences among the groups. finally, there were no significant differences in md and psd among the groups (table 2). both the ntg and the non - glaucoma control group did not show a significant nocturnal bp dip (2.54.7% in ntg, 3.43.7% in control group) compared to previous studies. investigated circadian bp patterns using ambulatory blood pressure monitoring in 51 ntg patients and 28 age - matched controls, and their study showed that the mean nocturnal arterial dip was 14.16.4% in ntg patients and 15.98.6% in controls, which was higher than our results, although their method of bp measurement differed somewhat from ours. several studies have suggested a correlation between nocturnal hypotension and glaucoma [5, 17, 23, 24 ]. others have reported that there are no significant differences in nocturnal bp dips between glaucoma and control groups [25, 26 ]. in our study, we did not find any significant differences in nocturnal bp dips between the ntg and control groups. however, ntg patients revealed a marked bp dip during the morning (7 am to 11 am). reported that sbp and dbp are low at 7 am and also after lunch (1 pm) in both untreated poag patients and age - matched normal control groups, while mopp showed a significantly different pattern between the two groups. the minimum mopp in the untreated poag group was recorded at 7 am when iop is at its highest. therefore, we evaluated the degree of bp dip in the morning compared to the whole day. the ntg group showed a larger morning bp dip compared to the control group (7.14.2% in ntg and 3.83.4% in controls), although the mean sbp, dbp and map were similar in the two groups. the map fluctuation was larger in the ntg group, but it did not reach statistical significance ; there were also no significant differences in mean mopp and mopp fluctuations between the two groups. therefore, in this study, a larger morning bp dip in ntg patients was not linked to significant map or mopp fluctuations. however, it is possible that a morning bp dip compromises ocular perfusion status along with the increase in morning iop ; 50% of ntg patients experienced peak iop in the morning. when the ntg group was divided into three subgroups according to the degree of morning bp dip, over - dippers showed significantly large map and mopp fluctuations compared to non - dippers or dippers. however, there were no significant differences in visual field indices (md and psd) among the three subgroups (table 2). these results disagree with previous studies that emphasized the correlation between mopp fluctuations and visual field indices or glaucoma severity [18, 19 ]. these discrepancies might be due to our study 's small sample size and short follow - up period. long - term serial follow - up of visual field tests with larger number of cases might reveal the true relationship between mopp or fluctuations between it and visual field indices. in this study, we measured circadian iop and bp profiles without disturbing nocturnal sleep, and we found that there were no significant differences in nocturnal bp dips between the ntg patients and the normal control group. however, ntg patients revealed a significant morning bp dip compared to the control group. our study is the first report that found that ntg patients showed a more prominent morning bp dip than a control group, but it is still inconclusive whether this resulted from undisturbed sleep that is not considered in other studies or if it is just a representation of the hemodynamic instability of ntg patients. further studies with a larger number of patients may clarify the relationship between undisturbed sleep and morning bp dips in ntg patients. previous studies on circadian bp measurement with ambulatory blood pressure monitoring have shown that bp declines during sleep, reaching a trough level roughly between 2 and 4 am. as we did not measure bp during these hours, ntg patients were significantly more likely to have underlying hypertension than members of the control group. we compared iop parameters (including mean and peak and fluctuation of iop) and hemodynamic parameters (including mean sbp, mean dbp, mean map, map fluctuation, and nocturnal and morning dip indices) between those with and without hypertension in ntg patients. we found that there were no significant iop or hemodynamic parameter differences between the two groups (results not shown). therefore, the significant morning bp dip seen in the ntg group could be attributed more to glaucoma than to hypertension. regardless of whether bp dips occur at night or in the morning, ocular perfusion status compromise is possible in ntg patients as both morning and night are well known for having the highest iop level. the morning bp dip might represent the hemodynamic instability of ntg patients and support the vascular theory of glaucoma development. in conclusion a marked morning bp dip was associated with significantly large map or mopp fluctuations, but it was not associated with visual field indices. long - term and serial follow - up of these patients are needed to further investigate the association between hemodynamic changes and the severity and functional aspects of glaucoma. | purposeto investigate and compare the circadian pattern of blood pressure (bp), intraocular pressure (iop) and mean ocular perfusion pressure (mopp) while experiencing undisturbed sleep in normal - tension glaucoma (ntg) and non - glaucoma control patient groups.methodstwenty-four eyes from 24 patients diagnosed with ntg and 22 eyes from 22 control group patients were enrolled. systolic bp, diastolic bp and iop were measured every two hours except for the period of time from 1 am to 7 am in the ntg group and from 11 pm to 7 am in the control group over a one - day period. iop and hemodynamic parameters were then compared between the two groups. ntg patients were subdivided according to the degree of morning bp dip and iop, and hemodynamic parameters and visual field indices (mean deviation and pattern standard deviation) were also compared among these subgroups.resultsthere were no significant differences in mean systolic bp, mean diastolic bp and mean arterial pressure (map) between the ntg and the control groups. the ntg group showed a significantly large morning bp dip compared to the control group (7.14.2% vs. 3.83.4%, p=0.022). however, there were no significant differences in mean or fluctuation of mopp between the two groups. morning over - dippers showed significantly large map and mopp fluctuations compared to non - dippers and dippers, while there were no significant differences in visual field indices among the three subgroups.conclusionsntg patients showed significant morning bp dips compared to the control group. the marked morning bp dip was associated with significantly large map or mopp fluctuations but was not associated with visual field indices. |
iodine deficiency has been well documented in many parts of the developing world and surprisingly it has reemerged in australia despite various programs to reduce its incidence [1, 2 ]. urinary iodine levels, which are used as a measure of dietary iodine intake, have declined over the past decade, and australia is now defined by the world health organization (who) criteria as a mildly iodine - deficient country. the physiological importance of iodine is its central role in maintaining normal thyroid function. inadequate iodine supply to the thyroid gland will result in decreased thyroid hormone synthesis and enlargement of the gland. during pregnancy iodine intake needs to be increased to meet the demands upon the thyroid gland to boost thyroid hormone production by up to 50% more than preconception requirements, to ensure a supply of thyroxine and iodine to the foetus and to compensate for increased renal iodide clearance iodine deficiency in an asymptomatic young female population may have a negative impact on thyroid function and neonatal outcome, as infants born in areas of moderate to - severe iodine deficiency suffer from a variety of neurodevelopmental disorders caused by irreversible brain damage in utero [5, 6 ], but adverse effects from mild maternal iodine - deficiency remain less certain. the incidence of neonatal hypothyroidism is also increased in an iodine deficient population, and there is evidence to suggest that severe iodine deficiency may increase rates of miscarriage and stillbirths. there is an increasing body of information confirming a high prevalence of mild - to - moderate iodine deficiency in pregnant women in the australian state of new south wales (nsw), victoria, and tasmania, as evidenced by reduced median urinary iodine concentrations in a number of sporadic studies. none of these studies in pregnant women have examined thyroid function or thyroid volume so it is unknown if iodine deficiency documented in pregnant australian women has any significant adverse effect on thyroid function. the aim of this study was to assess the iodine nutritional status, and its effect on thyroid function, in pregnant women in a private obstetrical practice in metropolitan sydney. we also wanted to determine if there was an association between urinary iodine concentration, the intake of specific iodine - rich foods, and the use of vitamin and mineral supplements taken by the pregnant women. analysis of supplements was not undertaken to confirm iodine content reported on the label. this study was performed in a private practice setting in north western sydney between november 2007 and february 2009, where 367 new consecutive antenatal patients were reviewed in their first trimester. at the time of initial booking with the private clinic, between 7 and 11 weeks gestation, routine antenatal investigations were performed. at the same time thyroid function tests, thyroid antibodies, and urinary iodine concentrations the patients answered questions in relation to their dietary habits, vitamin and mineral supplementation, and family history of thyroid disease. all women consented to have their blood and urine samples assessed for this study and freely provided the historical information. the study has been approved by the ethics committee of the sydney west area health service. iodine concentrations were measured on spot samples of urine by inductively coupled plasma mass spectrometry (icpms) in laverty pathology laboratories in sydney. serum tsh concentrations were measured by a chemiluminescent immunoassay on the advia centaur platform (bayer health care). serum - free t3 and t4 concentrations were also measured on the advia centaur platform by a chemiluminescent immunoassay method. for serum tsh the detection limit was 0.01 miu / l, and intraassay coefficients of variation (cv) were 2.48 and 2.44% at tsh concentrations of 0.74 miu / l and 5.65 miu / l, respectively. between assay % the intra - assay and inter - assay cvs for ft4 at 13.9 pmol / l were 2.31% and 3.03%, respectively. reference intervals provided by the laboratory for serum tsh, ft4, and ft3 in euthyroid adults were from 0.5 to 4.5 miu / l, 10 to 20 a personal history or family history of thyroid dysfunction was obtained, and dietary questions were asked in relation to servings of milk and dairy products per day and portions of fish per week over the preceding weeks of the pregnancy. the use of iodised or noniodised salt was determined and also the use (if any) of vitamin and mineral supplementation, including the brands used. two - tailed tests with a significance level of 5% were used throughout. chi - squared or fisher 's exact tests, as appropriate, were used to test for association between categorical variables. the mann - whitney test or kruskal - wallis nonparametric analysis of variance were used to test for differences in the distribution of continuous variables by group. spearman rank correlation (r) was used to quantify the degree of association between continuous and ordered categorical variables. the median urinary iodine concentration (uic) for all women (n = 367) was 81 g / l with an interquartile range of 41 to 169 g / l. a histogram illustrates the distribution of uic in figure 1. by conventional who uic standards for a nonpregnant, nonlactating adult population (12), 58% of the women in this study were iodine deficient, comprising 26% with uic 100 g / l, there is no statistically significant evidence of differences in tsh levels or free t4 levels between these uic groups (kruskal wallis nonparametric anova, p = 0.246 and p = 0.586, resp.). similarly, there was no significant evidence of differences in tsh and free t4 levels in those with uic 100 g / l, p = 0.312, and p = 0.514, respectively. however, there was a significant inverse association between serum tsh and free t4 levels (r = 0.490, p < 0.001). there was a high overall prevalence of iodine deficiency of 72%, in our population of pregnant women resident in western sydney, with 32% suffering from moderate - to - severe deficiency. the median urinary iodine level of 81 g / l was similar to the level we found in a pregnant population in sydney approximately 10 years ago indicating that the situation has not improved despite publicity in the medical and lay press about iodine deficiency. similar reports of iodine deficiency have come from victoria and tasmania where the degree of iodine deficiency in pregnant women may be even worse than nsw [9, 11 ]. extrapolating from the urinary iodine excretion to calculate daily intake, it is evident that these pregnant women are only taking, on average, a little more than half (132 g) the recommended daily intake (rdi for pregnancy of 250 g per day. while we are not certain how much reliance one can put on self - reported food and mineral supplement intakes without specific quantitative data, there were some interesting associations between what the participants reported and the measurement of iodine concentrations in their urine. the only food group appearing to influence the urinary iodine level was the intake of milk. it is surprising that other rich sources of iodine such as iodised salt and regular seafood intake did not influence the urinary iodine level. by contrast, there was a highly significant increase in urinary iodine levels in women who were taking a pregnancy supplement. g / l, still well below the cutoff level of 150 g / l, consistent with the rdi for pregnancy of 250 g per day. it is clear that many of the popular pregnancy supplements either do not contain any iodine, or the amounts are less than what is required to optimise iodine intake for pregnant women. in a recent study charlton g / l in a small sample of women attending an antenatal clinic in wollongong. their findings were similar to ours in that the median uic in the women taking a pregnancy supplement was significantly increased to 139 g / l, but still below the recommended level of 150 g / l. as we have not analysed the iodine content of the supplements taken by our patients we have been unable to verify the quality of these products. the shortcomings of the study are firstly that it comprised a relatively homogeneous population of women most of whom were well educated and able to afford private obstetric care. in addition the data obtained by questionnaire regarding food intakes and supplements relied upon recall and could not be independently verified. to our knowledge this is the first study of a pregnant population in australia measuring iodine excretion levels and examining possible negative consequences on thyroid function. as the majority of women were only mildly iodine - deficient significant changes in thyroid function measurement of serum tsh is considered the first line test in the laboratory assessment of thyroid function. while the serum tsh level is normally stable in euthyroid adults, it undergoes dynamic change during pregnancy as the thyroid responds to the challenge of having to dramatically increase thyroid hormone production in the first and second trimesters. unfortunately, tsh reference ranges provided by most laboratories in australia have not been derived from pregnant populations. in a recent study by gilbert. from western australia, examining thyroid function during the first trimester of pregnancy in a large sample (excluding women with positive thyroid autoantibodies), they reported a reference range for serum tsh of 0.4 to 4.0 miu / l and for serum free t4 of 9.0 to 19 pmol / l. they did not assess iodine nutritional status of their population. our data, which does not exclude women with positive thyroid autoantibodies, provide similar results with a 2.5 to 97.5 percentile range for serum tsh and free t4 of 0.03 to 3.4 miu / l, and 10.0 to 20.5 pmol / l, respectively. thus, despite the presence of mild iodine deficiency in our pregnant population, it is reassuring that we could not find any evidence for disturbed thyroid function as a consequence of the iodine deficiency. of course this does not exclude an effect on the thyroid that may be detected by more sensitive measures such as serum thyroglobulin and thyroid volume changes during pregnancy. the fact that 6.5% of the women studied had serum tsh levels greater than 2.5 miu / l identifies these women as suffering from gestational subclinical hypothyroidism in summary, mild - to - moderate iodine deficiency is common in pregnant women attending a private obstetrical practice in western sydney, but there is no evidence of adverse effects in thyroid function from the level of iodine deficiency in the population studied. urinary iodine excretion levels have not changed substantially over the past decade despite attempts to improve iodine nutrition in the population at risk. since this study was completed, it has been mandatory in australia to replace all salt used in bread making with iodised salt. it is estimated that this strategy would increase mean iodine intake by 46 ug / day and reduce the proportion of nonpregnant women with inadequate intakes from 59% to 9%, but most pregnant women will remain iodine deficient. iodine supplementation has been recommended as one of the means to achieve improved iodine nutrition in pregnant australian women. the estimated daily iodine supplement required to provide sufficient iodine intake for pregnant and breastfeeding women has been calculated to be in the range of 100150 ug / day. | aim. the primary objective of the study was to assess the iodine nutritional status, and its effect on thyroid function, of pregnant women in a private obstetrical practice in sydney. methods. it was a cross - sectional study undertaken between november 2007 and march 2009. blood samples were taken from 367 women at their first antenatal visit between 7 and 11 weeks gestation for measurement of thyroid stimulating hormone (tsh) and free thyroxine (ft4) levels and spot urine samples for urinary iodine excretion were taken at the same time as blood collection. results. the median urinary iodine concentration (uic) for all women was 81 g / l (interquartile range 41169 g / l). 71.9% of the women exhibited a uic of < 150 g / l. 26% of the women had a uic < 50 g / l, and 12% had a uic < 20 g / l. the only detectable influences on uic were daily milk intake and pregnancy supplements. there was no statistically significant association between uic and thyroid function and no evidence for an effect of iodine intake on thyroid function. conclusions. there is a high prevalence of mild to moderate iodine deficiency in women in western sydney but no evidence for a significant adverse effect on thyroid function. the 6.5% prevalence of subclinical hypothyroidism is unlikely to be due to iodine deficiency. |
migraine is the most common neurological disorder, affecting 18% of females and 6% of males, with prevalence peaking at age 3040 years, but its pathophysiology is not fully understood.1 this gap in knowledge is especially true in regard to mechanisms for the increased sensory perception for light, sound, and motion in migraine. of the ionic constituents that directly participate in neuronal excitability, evidence is accumulating that sodium homeostasis plays a central role in migraine from both human and animal studies : cerebrospinal fluid (csf) sodium (but not calcium, magnesium, or potassium) increases during migraine in the absence of any change of sodium in blood plasma2 ; the diurnal rhythm of csf sodium is characterized by peaks in early morning and late afternoon,3 corresponding to the most frequent onset times of episodic migraine4, 5, 6, 7, 8 ; intracranial sodium increases in a rat migraine model of nitroglycerintriggered central sensitization9 ; and computergenerated simulation and electrophysiology studies indicate higher extracellular sodium contributes to neuronal hyperexcitability.9 an especially compelling reason to explore dietary sodium is the evidence that higher extracellular sodium has been shown to increase the electrical activity of giant squid axons,10 an effect that we replicated in primary neuronal cultures.11 these results collectively point to a sodium disturbance in migraine. studies have shown that higher dietary sodium increases ventricular csf sodium concentration in rats,12 but sensory evaluation was not reported in these experiments. despite the knowledge that human choroid plexus has an almost identical distribution pattern of water channels and sodium transporters as in the rat,13 we are not aware of prospective human studies focused on the relationship between dietary sodium intake and sodium dysregulation in migraine. the regulation of sodium throughout the body depends on the requirements of different tissues, ranging from the kidneys, skin, and salivary glands to the choroid plexus, but the source of sodium results as a redistribution from elsewhere in the body or from dietary sodium. as sodium has profound effects on neuronal activity, we hypothesized that there would be an association between migraine status (a binary variable) and dietary sodium and used the nhanes database to investigate this possible relationship. we also explored whether an association between migraine status and dietary sodium would vary based on sex and/or body mass index (bmi). nhanes is an ongoing survey conducted by the national center for health statistics at the centers for disease control and prevention (cdc) that collects data on the health and nutritional status of u.s. detailed information on survey design and methodology is available at http://www.cdc.gov/nchs/nhanes.htm.14, 15 nhanes includes a nationally representative sample of approximately 5000 subjects per year. the interview component of nhanes ascertains information on demographic, socioeconomic, and healthrelated factors and includes a 24hour dietary recall assessment. we limited our analysis to adults (ages 20 years and older) who participated in the dietary assessment, whose dietary data were deemed reliable (per the nhanes definition), and who did not take analgesic medications so that respondents who possibly suffered from analgesic overuse headache would be excluded. beginning in 1999, although data are available from 1971 through the present, our analysis includes only the 19992004 cycles due to availability of variables that were of interest to our investigation. the huntington medical research institute institutional review board granted exemption from local review under 45 cfr 46.101(b)(4). data on migraine status were available in the miscellaneous pain section of the nhanes interview. we categorized respondents who answered yes to the survey question : during the past 3 months, did you have severe headaches or migraines ? as probable migraineurs. these selfreports are without the additional parameters necessary for the ihc classification of migraine,16 and alternative diagnoses such as tensiontype or cluster headaches may be erroneously grouped as migraine. nevertheless, it is reasonable to assume most of these respondents had migraine as it is the most common diagnosis for these complaints, an assumption also made by other expert researchers in this field.17 because of this limitation, we excluded respondents who may have had severe headaches due to medication overuse (analgesic users). total grams of daily dietary sodium was an nhanesderived variable from the total nutrient intakes file, produced from respondents ' reporting of individual foods (including drinks) they consumed during the midnighttomidnight 24hour period prior to the inperson dietary interview. in the 20032004 nhanes cycle, a second dietary interview was conducted 310 days later ; to maintain consistency with the 19992002 data, we used data from only the first interview for 20032004. coding of interview data and conversion to total nutrient intake were done by nhanes using the food intake analysis system (fias, university of texas health science center at houston, houston, tx, usa) along with the usda 19941998 survey nutrient database in nhanes cycle 19992000,18 fias in 2001, the usda 's survey net in 2002,19 and the usda 's food and nutrient database for dietary studies in nhanes cycle 20032004.20 final processing of dietary data by nhanes included modifying sodium content for foods for which respondents indicated that they did not use the usual amount of salt in preparation. two sections of the nhanes interview were used to determine use of analgesic medications : analgesic medications, which included a question on whether or not the respondent had ever taken overthecounter or prescription pain medications every day for as long as a month (chronic) ; and prescription medications, which included questions on whether the respondent had taken any prescription medications during the past month and, if so, which medications they reported taking. prescription medications reported by respondents were categorized by nhanes using the multum lexicon therapeutic classification scheme.21 in this scheme, medications are classified by 3 nested levels of therapeutic category, with the first level being the most broad. among medications with firstlevel classification central nervous system agents, analgesics analgesics according to the secondlevel category name ; these included thirdlevel classifications of miscellaneous analgesics, narcotic analgesics, nonsteroidal antiinflammatory agents, salicylates, analgesic combinations, narcotic analgesic combinations, antimigraine agents, and cox2 inhibitors. analgesic users (excluded from our analysis) were defined as respondents who were in either category of analgesic use (chronic or in the last month). this definition did not exclude migraineurs who did not treat their migraines at all, ie, migraineurs who used overthecounter medications were included, as were migraineurs who used prescription medications to treat but not in the most recent month. the primary objective of the analysis was to test for an association between dietary sodium intake and migraine status (a binary variable). secondary objectives were to explore the sodiummigraine relationship within subgroups defined by sex and bmi and to estimate the probability of reporting history of migraine as a function of absolute dietary sodium intake. analyses were done using sas procedures surveyfreq, surveyreg and surveylogistic (sas v9.2, sas institute, cary, nc, usa) to account for the stratified, multistage probability cluster sampling design of nhanes. the nhanes stratification variable (sdmvstra) and primary sampling unit (sdmvpsu) were used as the strata and sampling unit variables, respectively. nhanes provides sampling weights to be used in analyses that account for oversampling of certain subgroups, differences between the sample and the population due to nonresponse, and population sizes, ie, the weights allow each nhanes respondent to represent multiple people from the population from which they were sampled. nhanes provides sampling weights to be used specifically for dietary analyses, which also account for the fact that not all participants completed the dietary interview and that different days of the week were represented in the 24hour periods for which dietary intake was assessed. in general, the nhanes weights are calculated for 2year intervals, but a 4year weight is available for the 19992002 time period. a 2year weight is smaller than a 4year weight because less of the population is included in a sample collected over 2 years compared to a sample collected over 4 years (so each respondent in a 2year sample has to represent more people in the population compared to a 4year sample). because we combined data from three 2year sampling cycles (which included the 19992002 period), per nhanes guidelines we derived a 6year weight equal to 2/3 of the 19992002 weight for respondents sampled during 19992002 or 1/3 of the 20032004 weight for respondents sampled during 20032004. this is possible because the 20032004 2year weights are comparable to the 19992002 4year weights as both sets of weights were based on 2000 census counts.20 total grams of dietary sodium for each respondent was expressed as the respondent 's residual value from the linear regression of total grams of sodium on total calories, ie, the difference between the respondent 's actual grams of dietary sodium and that predicted by his or her total caloric intake. this approach isolates the effect of grams of dietary sodium from factors closely associated with total caloric intake that may be related to migraine (eg, body size, metabolic efficiency) without directly modeling total caloric intake, which is highly correlated with total sodium intake.22 residuals were analyzed both as continuous and categorical variables, with categories based on quartiles of the distribution of residuals. multivariable logistic regression was used to estimate odds ratios (ors) and 95% confidence intervals (cis) in the analysis of the association between dietary sodium (the predictor) and migraine status (the outcome). age, sex, and bmi were included as covariates as these are wellknown to be related to both dietary intake and migraine. interactions were tested between the predictor in its original form and each covariate in their original forms. for stratified analyses, correlations were assessed for each pair of independent variables to check for collinearity. a sensitivity analysis that included analgesic medication users a second sensitivity analysis was done to compare results between the two dietary interviews done on separate days among respondents from the 20032004 nhanes cycle. subgroup analyses were accomplished using the domain statement in the sas procedures surveyfreq, surveyreg, and surveylogistic. nhanes is an ongoing survey conducted by the national center for health statistics at the centers for disease control and prevention (cdc) that collects data on the health and nutritional status of u.s. detailed information on survey design and methodology is available at http://www.cdc.gov/nchs/nhanes.htm.14, 15 nhanes includes a nationally representative sample of approximately 5000 subjects per year. the interview component of nhanes ascertains information on demographic, socioeconomic, and healthrelated factors and includes a 24hour dietary recall assessment. we limited our analysis to adults (ages 20 years and older) who participated in the dietary assessment, whose dietary data were deemed reliable (per the nhanes definition), and who did not take analgesic medications so that respondents who possibly suffered from analgesic overuse headache would be excluded. beginning in 1999, although data are available from 1971 through the present, our analysis includes only the 19992004 cycles due to availability of variables that were of interest to our investigation. the huntington medical research institute institutional review board granted exemption from local review under 45 cfr 46.101(b)(4). data on migraine status were available in the miscellaneous pain section of the nhanes interview. we categorized respondents who answered yes to the survey question : during the past 3 months, did you have severe headaches or migraines ? as probable migraineurs. these selfreports are without the additional parameters necessary for the ihc classification of migraine,16 and alternative diagnoses such as tensiontype or cluster headaches may be erroneously grouped as migraine. nevertheless, it is reasonable to assume most of these respondents had migraine as it is the most common diagnosis for these complaints, an assumption also made by other expert researchers in this field.17 because of this limitation, we excluded respondents who may have had severe headaches due to medication overuse (analgesic users). total grams of daily dietary sodium was an nhanesderived variable from the total nutrient intakes file, produced from respondents ' reporting of individual foods (including drinks) they consumed during the midnighttomidnight 24hour period prior to the inperson dietary interview. in the 20032004 nhanes cycle, a second dietary interview was conducted 310 days later ; to maintain consistency with the 19992002 data, we used data from only the first interview for 20032004. coding of interview data and conversion to total nutrient intake were done by nhanes using the food intake analysis system (fias, university of texas health science center at houston, houston, tx, usa) along with the usda 19941998 survey nutrient database in nhanes cycle 19992000,18 fias in 2001, the usda 's survey net in 2002,19 and the usda 's food and nutrient database for dietary studies in nhanes cycle 20032004.20 final processing of dietary data by nhanes included modifying sodium content for foods for which respondents indicated that they did not use the usual amount of salt in preparation. two sections of the nhanes interview were used to determine use of analgesic medications : analgesic medications, which included a question on whether or not the respondent had ever taken overthecounter or prescription pain medications every day for as long as a month (chronic) ; and prescription medications, which included questions on whether the respondent had taken any prescription medications during the past month and, if so, which medications they reported taking. prescription medications reported by respondents were categorized by nhanes using the multum lexicon therapeutic classification scheme.21 in this scheme, medications are classified by 3 nested levels of therapeutic category, with the first level being the most broad. among medications with firstlevel classification central nervous system agents, analgesics were selected as those classified as analgesics according to the secondlevel category name ; these included thirdlevel classifications of miscellaneous analgesics, narcotic analgesics, nonsteroidal antiinflammatory agents, salicylates, analgesic combinations, narcotic analgesic combinations, antimigraine agents, and cox2 inhibitors. analgesic users (excluded from our analysis) were defined as respondents who were in either category of analgesic use (chronic or in the last month). this definition did not exclude migraineurs who did not treat their migraines at all, ie, migraineurs who used overthecounter medications were included, as were migraineurs who used prescription medications to treat but not in the most recent month. the primary objective of the analysis was to test for an association between dietary sodium intake and migraine status (a binary variable). secondary objectives were to explore the sodiummigraine relationship within subgroups defined by sex and bmi and to estimate the probability of reporting history of migraine as a function of absolute dietary sodium intake. analyses were done using sas procedures surveyfreq, surveyreg and surveylogistic (sas v9.2, sas institute, cary, nc, usa) to account for the stratified, multistage probability cluster sampling design of nhanes. the nhanes stratification variable (sdmvstra) and primary sampling unit (sdmvpsu) were used as the strata and sampling unit variables, respectively. nhanes provides sampling weights to be used in analyses that account for oversampling of certain subgroups, differences between the sample and the population due to nonresponse, and population sizes, ie, the weights allow each nhanes respondent to represent multiple people from the population from which they were sampled. nhanes provides sampling weights to be used specifically for dietary analyses, which also account for the fact that not all participants completed the dietary interview and that different days of the week were represented in the 24hour periods for which dietary intake was assessed. in general, the nhanes weights are calculated for 2year intervals, but a 4year weight is available for the 19992002 time period. a 2year weight is smaller than a 4year weight because less of the population is included in a sample collected over 2 years compared to a sample collected over 4 years (so each respondent in a 2year sample has to represent more people in the population compared to a 4year sample). because we combined data from three 2year sampling cycles (which included the 19992002 period), per nhanes guidelines we derived a 6year weight equal to 2/3 of the 19992002 weight for respondents sampled during 19992002 or 1/3 of the 20032004 weight for respondents sampled during 20032004 this is possible because the 20032004 2year weights are comparable to the 19992002 4year weights as both sets of weights were based on 2000 census counts.20 total grams of dietary sodium for each respondent was expressed as the respondent 's residual value from the linear regression of total grams of sodium on total calories, ie, the difference between the respondent 's actual grams of dietary sodium and that predicted by his or her total caloric intake. this approach isolates the effect of grams of dietary sodium from factors closely associated with total caloric intake that may be related to migraine (eg, body size, metabolic efficiency) without directly modeling total caloric intake, which is highly correlated with total sodium intake.22 residuals were analyzed both as continuous and categorical variables, with categories based on quartiles of the distribution of residuals. multivariable logistic regression was used to estimate odds ratios (ors) and 95% confidence intervals (cis) in the analysis of the association between dietary sodium (the predictor) and migraine status (the outcome). age, sex, and bmi were included as covariates as these are wellknown to be related to both dietary intake and migraine. interactions were tested between the predictor in its original form and each covariate in their original forms. for stratified analyses, correlations were assessed for each pair of independent variables to check for collinearity. a sensitivity analysis that included analgesic medication users a second sensitivity analysis was done to compare results between the two dietary interviews done on separate days among respondents from the 20032004 nhanes cycle. subgroup analyses were accomplished using the domain statement in the sas procedures surveyfreq, surveyreg, and surveylogistic. characteristics of the study population are shown in table 1. of the 15,332 adults who participated in the nhanes 19992004 cycles, 13,424 (88%) had reliable data from the 24hour dietary assessment, and 13,033 of these (97%) had nonmissing dietary total grams of sodium and migraine status. given the relatively low rate of unreliable and/or missing data, per the nhanes guidelines,23 we deemed it unnecessary to account for missing data in the analysis, ie, through imputation or additional weighting. the number of respondents who were not analgesic users was 8819 (68%) ; of these, 4551 (52%) were women. of the 4214 analgesic users, 3098 were because of chronic use (2383 of 10,346 (23%) nonmigraineurs, 715 of 2687 (27%) migraineurs), ie, they took analgesics every day for at least a month at some point in their life. the remaining 1116 exclusions were due to prescription analgesic use during the most recent month (780 of 10,346 (8%) nonmigraineurs, 336 of 2687 (13%) migraineurs). a flow diagram describes the participants : demographic characteristics of analysis population by headache status (nhanes 19992004) based on weighted frequencies. dietary sodium, total caloric intake, analgesic use, age, sex, bmi. # denominator for percentage = respondents with all relevant data nonmissing who did not take analgesics. results from both crude and covariateadjusted analyses of the relationship between dietary sodium and migraine status are shown in table 2. compared to respondents with the lowest dietary sodium residuals, there was a significant trend of decreasing odds of migraine history with increasing levels of dietary sodium residuals (or = 0.93, 95% ci = 0.87, 1.00, p =.0455) ; this relationship remained after covariate adjustment (or = 0.93, 95% ci = 0.87, 1.00, p =.0503). including analgesic users in the analysis had little effect (or = 0.95, 95% ci = 0.89, 1.01, p =.08), and results from the two separate days of dietary interviews among respondents from the nhanes 20032004 cycle were comparable (day 1 : or = 0.88, 95% ci = 0.78, 0.99, p =.04 ; day 2 : or = 0.90, 95% ci = 0.81, 1.01, p =.08). effect of dietary sodium on headache status (respondents who did not use chronic analgesics or prescription analgesics in past month, nhanes 19992004) based on weighted frequencies. we designed our analysis to study those not taking analgesics to avoid the confounding effect of medications ; when we included analgesic users in a post hoc study, a sensitivity analysis suggested little effect from this exclusion. to investigate the effect of sexual dimorphism, we analyzed the relationship between dietary sodium and migraine for men and women separately and observed the same inverse association by sex as was observed for both sexes combined (p for interaction =.99, table 3) ; however, the trend was significant only for women (or = 0.93, 95% ci = 0.86, 1.00, p =.049). in women but not men, bmi was a significant effect modifier of the sodium / migraine relationship (p =.025) ; results by bmi for both sexes are shown in table 4. in women, there was a significant effect of decreasing odds of migraine history with increasing dietary sodium residuals in respondents with lower bmi (or = 0.87, 95% ci = 0.78, 0.96, p =.007). in women with higher bmi, there was no relationship between dietary sodium and migraine history. effect of dietary sodium on headache status by sex (respondents who did not use chronic analgesics or prescription analgesics in past month, nhanes 19992004) adjusted for age and bmi. effect of dietary sodium on headache status by sex and bmi (respondents who did not use chronic analgesics or prescription analgesics in past month, nhanes 19992004) adjusted for age. in women, interaction between sodium residuals and bmi was significant (p =.025). our analysis of nutritional assessment data from nhanes 19992004 provides the first evidence to suggest an inverse relationship between migraine and dietary sodium intake levels independent of age, sex, and bmi ; in women, this observation was limited to those with lower bmi. a comprehensive literature exists that describes the impact that diet can have on migraine occurrence,24 however, few studies have attempted to assess the direct relationship between dietary sodium intake patterns and migraine. the nhanes 19992004 analysis by evans, limited to women, reported that sodium dietary intake was descriptively (but not significantly) lower in migraineurs compared to nonmigraineurs, and in women of normal weight the sodium component of dietary quality indicated less sodium intake in migraineurs compared to nonmigraineurs (p =.04 without adjustment for multiple comparisons).17 thus, their observations are consistent with our analysis of women with lower bmi. we analyzed the nhanes 19992004 data from a strategically different approach ; for example, we included men and women of all ages vs women aged 2050 years, and we analyzed sodium residuals rather than a transformed version of the original sodium variable. this limits a direct comparison between the two studies but, alternatively, lends support to our findings as the two different approaches led to similar conclusions regarding sodium and migraine in women. it is wellknown that higher dietary sodium has been associated with negative outcomes in other fields of medicine and is considered a major health problem worldwide,25 contributing to hypertension and its consequences of stroke and cardiac and renal disease.26 hypertension and hypotension, as well as obesity, are comorbid with migraine,27, 28 and the cardiovascular risk profile is higher in migraineurs.29, 30 sodium intake is also recognized as a sexually dimorphic behavior, controlled by perinatal and adult androgen in rats,31 and includes greater intake by females during acute sodium depletion.32 from these two established correlations for dietary sodium, we expected to find : (1) a higher sodium intake in migraineurs to match their increased cardiovascular risk, and (2) a sexually dimorphic relationship. we found a migraine relationship with dietary sodium and it had sexual dimorphic influences, but the direction of sodium intake lower in migraineurs. first, the inverse relationship between migraine and dietary sodium intake in women was restricted to those with lower bmi, whereas the relationship between hypertension when sodium sensitivity has been reported to be limited to those with higher bmi.33 second, we had previously reported higher csf sodium during migraine2 and kawano found higher dietary sodium increases human csf sodium independent of salt sensitivity / insensitivity34 ; thus it was a surprise that migraineurs would report less dietary sodium intake. third, a post hoc analysis of dietary intervention study of low, medium, and high sodium intake reported a lower risk of headache on a low sodium diet.35 however, the authors suspected that most of the headaches were tension type. further, the controlled high sodium intake in the trial corresponded to the average american intake, whereas the nhanes database used for our analysis was representative of the population distribution of dietary sodium and thus included much higher levels of exposure. extensive studies on the regulation of dietary sodium intake based on salt appetite36, 37 demonstrate the limited understanding of this behavior, and further study is necessary to explain these apparent paradoxes. we previously proposed that brain sodium homeostasis is altered in migraine,38 largely mediated by disturbance of the na, k, atpase, but many other mechanisms have been invoked for migraine. a compelling question is whether disruption of a common activation pathway or component can cause migraine. candidate pathways or components include : cortical spreading depression39, 40, 41 ; neuropeptides42 ; sterile meningeal neuroinflammation with triggering of dural mast cells43, 44, 45 ; central excitatory / inhibitory homeostasis (glutamate / gammaaminobutyric acid)46, 47 ; cortical neuromodulation (serotoninergic, noradrenergic, cholinergic, or dopaminergic)47, 48, 49, 50 ; or channelopathy.49, 51 it will be important to identify if sodium dysregulation is the underlying mechanism for these other pathways and/or is a common mechanism for migraine. it is noteworthy that low or high blood pressures are known comorbidities of migraine,27 and that altered sodium homeostasis and the endogenous na, k, atpase inhibitor, ouabain, have been identified in the regulation of blood pressure28 and depressive disorders.52 we predict that regulators that have already been found for saltsensitive hypertension and depression disorders,52 such as endogenous ouabainlike compounds,28, 53 are worthy of study as modulators of the dietary sodium / migraine relationship. our analysis reported here adds support for the involvement of sodium dysregulation in migraine but is limited due to the retrospective, selfreported, crosssectional nature of nhanes data. this type of data can not demonstrate cause and effect ; ie, one explanation for our observation of lower sodium intake among migraineurs is that respondents who suffered from migraine intentionally lowered their sodium intake as a possible preventive measure. also, the 3month prevalence of headaches captured by nhanes does not allow for an analysis of the effect of dietary sodium on headache frequency. to investigate whether brain sodium alterations and dietary sodium in particular represent a culprit in migraine, it will be necessary to pursue prospective dietary studies with more detailed and valid migraine data, correlated with biochemical parameters. migraine classification was not based on the international headache classification criteria16 and was nonspecific to migraine. another national database, the women 's health initiative, has more conventional migraine classification24 but does not have dietary data comparable to the quality of nhanes 19992004 data. we likely excluded some migraineurs by omitting respondents who had ever used analgesics chronically or who had taken prescription analgesics during the month prior to interview. this latter group, in particular, was more likely to have in included some true migraineurs who were, therefore, not in our migraineur group. however, our goal was to have the most valid migraineur group possible, given the limitations of the data in terms of migraine classification. therefore, we felt it was more important to exclude some true migraineurs than to include as migraineurs those suffering from medicationoveruse headache. ultimately, a sensitivity analysis suggested there was little effect from this exclusion. nutritional assessment was based on 24hour recall, a method with inherent limitations.54 importantly, due to lack of 2 days of dietary recall in 2/3 of our data, we were unable to use more sophisticated methods to account for episodically consumed foods and beverages. despite these limitations, the nhanes 19992004 database and its associated analytical methodology represents best available, stateoftheart practice for survey studies. our analysis of nhanes 19992004 data provides the first evidence of an inverse association between migraine and dietary sodium intake, independent of common migraine covariates. we propose that this dietary sodium intake may impact migraine by modulating the regulation of brain extracellular sodium. our results may stimulate prospective, controlled studies of dietary and body sodium to evaluate whether altered sodium homeostasis is casual in, or an effect of, migraine. category 1 (a) conception and design janice m. pogoda & michael g. harrington (b) acquisition of data janice m. pogoda (c) analysis an interpretation of data janice m. pogoda, noah b. gross, xianghong arakaki, alfred n. fonteh, robert p. cowen, & michael g. harrington (a) conception and design janice m. pogoda & michael g. harrington (b) acquisition of data (c) analysis an interpretation of data janice m. pogoda, noah b. gross, xianghong arakaki, alfred n. fonteh, robert p. cowen, & michael g. harrington category 2 (a) drafting the manuscript janice m. pogoda & michael g. harrington (b) revising it for intellectual content janice m. pogoda, noah b. gross, xianghong arakaki, alfred n. fonteh, robert p. cowen, & michael g. harrington (a) drafting the manuscript janice m. pogoda & michael g. harrington (b) revising it for intellectual content janice m. pogoda, noah b. gross, xianghong arakaki, alfred n. fonteh, robert p. cowen, & michael g. harrington category 3 (a) final approval of the completed manuscript janice m. pogoda, noah b. gross, xianghong arakaki, alfred n. fonteh, robert p. cowen, & michael g. harrington (a) final approval of the completed manuscript janice m. pogoda, noah b. gross, xianghong arakaki, alfred n. fonteh, robert p. cowen, & michael g. harrington | objectivewe investigated whether dietary sodium intake from respondents of a national crosssectional nutritional study differed by history of migraine or severe headaches.backgroundseveral lines of evidence support a disruption of sodium homeostasis in migraine.designour analysis population was 8819 adults in the 19992004 national health and nutrition examination survey (nhanes) with reliable data on diet and headache history. we classified respondents who reported a history of migraine or severe headaches as having probable history of migraine. to reduce the diagnostic conflict from medication overuse headache, we excluded respondents who reported taking analgesic medications. dietary sodium intake was measured using validated estimates of selfreported total grams of daily sodium consumption and was analyzed as the residual value from the linear regression of total grams of sodium on total calories. multivariable logistic regression that accounted for the stratified, multistage probability cluster sampling design of nhanes was used to analyze the relationship between migraine and dietary sodium.resultsodds of probable migraine history decreased with increasing dietary sodium intake (odds ratio = 0.93, 95% confidence interval = 0.87, 1.00, p =.0455). this relationship was maintained after adjusting for age, sex, and body mass index (bmi) with slightly reduced significance (p =.0505). in women, this inverse relationship was limited to those with lower bmi (p =.007), while in men the relationship did not differ by bmi. we likely excluded some migraineurs by omitting frequent analgesic users ; however, a sensitivity analysis suggested little effect from this exclusion.conclusionsthis study is the first evidence of an inverse relationship between migraine and dietary sodium intake. these results are consistent with altered sodium homeostasis in migraine and our hypothesis that dietary sodium may affect brain extracellular fluid sodium concentrations and neuronal excitability. |
neurilemmoma (schwannoma) is a benign neoplasm that arises from the myelinating schwann cells of the nerve sheath. it is a relatively common tumor, comprising approximately 5% of all benign soft tissue tumors, and shows a predilection to affect the sensory nerves. intraosseous neurilemmoma, however, is very rare, accounting for less than 1% of benign bone tumors. the mandible is the most frequently affected site, followed by the sacrum ; this tumor rarely arises in the bones of the extremities. only 2 cases of intraosseous neurilemmoma around the elbow have been described in literature ; these cases arose in the distal humerus. a 21-year - old woman was referred to our orthopedic unit with a 1-month history of pain in the left elbow. the initiating factor, such as trauma, was not clear. on physical examination, the overlying skin was intact, and there was no evidence of warmth, erythema, or induration. no other masses were palpable. the elbows and forearms had a normal range of motion (rom). plain radiographs revealed a well - defined, lytic, and expansile lesion, with thin marginal sclerosis and trabeculation in the proximal ulna (fig. 1). computed tomography (ct) showed considerable destruction of the cortex of the ulna, which connected with a mass of soft tissue. at the edge of 2a), which had invaded into the cortex, and no periosteal reaction was seen (fig. 2b). magnetic resonance imaging (mri) showed the well - defined and lobulated lesion to be isointense to skeletal muscle on t1-weighted images, and heterogeneous and hyperintense on t2-weighted images. the lesion revealed uniform enhancement following gd - dtpa administration. because of uncertainty regarding the histological origin of the tumor, we performed an open biopsy. the pathologic specimen consisted of hypercellular regions with palisading nuclei (antoni type a), and hypocellularity regions with myxoid background (antoni type b). curettage was performed on the bone lesion, and the resulting deficit was grafted with beta - tricalcium phosphate (fig. after 3.5 years of follow up, the patient is clinically asymptomatic, and the radiographs show complete graft incorporation (fig. the patient was informed that the case would be submitted for publication and her consent was obtained. neurilemmomas may involve bone tissue via 3 mechanisms : (1) they may be intramedullary, producing rarefaction of the bone ; (2) they may be located within the nutrient canal, with the formation of a dumbbell - shaped tumor ; or (3) they may be extraosseous, eroding into the bone. with regard to the comparatively high incidence of intraosseous neurilemmoma in the mandible, it has been speculated that the long intraosseous path of the mandibular nerve may predispose to metaplasia of the schwann cells in its nerve sheath. however, some authors have refuted this theory, noting that the nerves innervating the long bones through the nutrient foramina are longer than the mandibular nerve ; nevertheless, the frequency of intraosseous neurilemmoma in the long bones is several times lower than that in the mandible. de la monte and colleagues have suggested that the death of sensory nerve fibers within bone tissue may account for the exceedingly rare occurrence of intraosseous neurilemmoma. we think that the high frequency of neurilemmoma in the mandible is not because of the long intraosseous course, but because the mandibular nerve consists of sensory nerves of the trigeminal nerve origin. the characteristic radiographic features of intraosseous neurilemmoma include the following : (1) a well - defined lytic lesion, (2) sclerotic margins, (3) lobulated or trabeculated contours, (4) cortical expansion, and (5) absence of central calcification. however, the radiographic findings are non - specific. it is difficult to differentiate intraosseous neurilemmoma from other benign neoplasms of such osseous lesions, including solitary bone cyst, aneurysmal bone cyst, giant cell tumor, non - ossifying fibroma, benign fibrous histiocytoma, desmoplastic fibroma, fibrous dysplasia, chondromyxoid fibroma, and enchondroma. on ct, we can confirm the origin of the tumor when there is soft tissue extension of the tumor. if the origin is intraosseous instead of periosteal, no periosteal reaction is seen, and the edge of the destructed cortex will be overhung by a soft tissue mass. young and colleagues pointed out that the aspect of the overhanging edges of cortical bone can assist in distinguishing between an intraosseous desmoplastic fibroma versus a periosteal desmoplastic fibroma. similarly, intraosseous neurilemmoma is considered to originate from intraosseous nerves rather than from periosteal nerves, if ct shows overhanging edges of cortical bone. in our case and in others this may be the characteristic finding of intraosseous neurilemmoma provided the tumor expands within the nutrient canal of the cortex. the final diagnosis of intraosseous neurilemmoma was not made until after histologic examination of tissue obtained during excision. the diffuse immunoreactivity for s-100 protein is indicative of a schwann cell origin, and helps distinguish this tumor from other benign spindle cell lesions of similar histology. the recommended treatment for intraosseous neurilemmoma is conservative resection and bone grafting, as malignant change is extremely rare. recurrence also is rare following complete local excision. in previous reports, after performing only curettage, curettage and bone grafting, or en block resection, local recurrence only occurred in 2 cases. these authors did not refer to the reason for local reference, but recurrence of the tumor is not evidence of malignant transformation. in our case owing to the infrequency of an intraosseous neurilemmoma, the diagnosis is often not even suspected until histologic evaluation after a biopsy. although very rare, intraosseous neurilemmoma should be taken under consideration in the differential diagnosis of painful, radiographically benign - appearing osseous tumor around the elbow. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. case report writing, data collection, and discussion writing were done by munehisa kito and yasuo yoshimura. discussion writing was carried by kenichi isobe, kaoru aoki, takashige momose, and hiroyuki kato. | introductionneurilemmoma is a benign nerve sheath neoplasm commonly located in the soft tissue. intraosseous neurilemmoma is rare, constituting less than 1% of primary bone tumors.presentation of casea 21 year - old woman was presented with left elbow pain of 1-month duration. plain radiographs showed a well - defined, lytic and expansile lesion of the proximal ulna. computed tomography revealed cortical destruction and soft tissue extension. because the tissue of origin for the tumor was uncertain, an open biopsy was performed. the specimens demonstrated a benign spindle cell tumor suggestive of a neurilemmoma, similar to a soft tissue neurilemmoma. the diagnosis of intraosseous neurilemmoma was established. marginal excision of the soft tissue component and curettage of the lesion in the bone were performed. after 3.5 years of follow up, there is no clinical or radiographic finding to suggest any recurrence.discussionthe major site of intraosseous neurilemmoma is the mandible. occurrence in the long bone is particularly rare. only two cases of intraosseous neurilemmoma involving the bones around the elbow have been reported to our knowledge ; these cases arose in the distal humerus. we describe the first case of intraosseous neurilemmoma of the proximal ulna of the left elbow. the recommended treatment is conservative resection and bone grafting, as malignant change is extremely rare.conclusionalthough very rare, intraosseous neurilemmoma should be taken under consideration in the differential diagnosis of painful, radiographically benign - appearing osseous tumor around the elbow. |
carpal tunnel syndrome (cts) is the most frequent entrapment neuropathy, which occurs as a consequence of compression of the median nerve at the wrist. the diagnosis is usually based on characteristic symptoms and signs, and electrophysiological studies4,21). the most reliable method to confirm clinical diagnosis of cts is electrophysiological study18,19) but false negatives can be seen in the variable range of 10 - 20%7). the electrophysiological studies usually show the level of the lesion, but do not provide anatomical information about the nerve or its surroundings. in the last few years, ultrasonography (us) has been shown to be useful diagnostic tools in cts, providing information on the median nerve and surrounding structures4,6,13,23). a few papers have been published in neurosurgery literature regarding diagnostic utility of ultrasonography in carpal tunnel syndrome1,6). to our knowledge, publication has not been available, describing measurements of ultrasonographic parameters for the diagnosis of cts, compared with electrodiagnostic findings as well as clinical severity, assessed by historical - objective scale9). the purpose of the present study is to identify the efficacy of ultrasonographic measurements in a consecutive sample of patients with clinically diagnosed cts and to analyze the correlation of ultrasonographic findings with electrophysiological abnormalities and clinical severity. the clinical diagnostic criterion of cts is based on the american academy of neurology21). one - hundred thirty - five patients who had the symptoms of cts between january 2007 and december 2009 were included in this study. nineteen persons with no clinical symptoms and signs of cts were classified as the control group. patients with diabetes mellitus, collagen disease, thyroid disease, peripheral neuropathy, rheumatic arthritis, traumatic nerve injury were excluded from the study. the patient group contained total of 246 hands, whereas 30 hands were used in the control group. the clinical grade in the patient group was classified into 6 stages according to the historical - objective (hi - ob) clinical scale as follows9) ; stage 0 (no symptoms), stage 1 (paresthesia only at night), stage 2 (paresthesia even for short time in the daytime), stage 3 (hypesthesia in the finger of the median nerve distribution), stage 4 (accompanying weakness or thenar muscle atrophy), and stage 5 (thenar muscle complete atrophy or paralysis). mild cases in hi - ob scale were defined as those patients who reported only symptoms without objective motor deficit of thenar eminence muscles and normal objective sensory function in the median nerve territory of the hand. grades 1 or 2 of a hi - ob scale belong to mild case9). ultrasonographic examination was performed using sonix rp (ultrasonix medical corporation, richmond, canada) by the same experienced examiner (hsn). all wrists were evaluated in the neutral position with the palm up and the fingers semi - extended. the full course of the median nerve in the carpal tunnel was evaluated in both the transverse and sagittal planes. the cross - sectional area (csa) of the median nerve (measured in mm) was measured to proximal carpal tunnel at the level of the pisiform bone. the csa of the median nerve was measured by direct methods as suggested by duncan.7), which was calculated automatically by tracing the inner margin of the epineurium of median nerve, assuming that it has an elliptical shape (fig. the flattening ratio (fr) was defined as the ratio of the nerve 's transverse axis to the antero - posterior axis (fig. the palmar bowing (pb) of the flexor retinaculum is displacement (measured in mm) of the retinaculum from the attachments of a ligament connecting the pisiform bone with the scaphoid bone (fig. the electrophysiological study was performed using a cadwell sierra wave (cadwell laboratories, kennewick, wa, usa). the study consisted of motor and sensory median nerve conduction tests using standard techniques according to the practice parameters for the electrodiagnosis of cts of the american association of electrodiagnostic medicine, the american academy of neurology, and the american academy of physical medicine and rehabilitation3). the electrophysiological abnormalities were classified into three grades according to stevens ' classification as follows23) : mild ; prolonged median sensory distal latency, moderate ; prolonged median sensory and motor distal latency, and severe ; abnormal needle electromyography other than the above two abnormalities, or no response in sensory and motor distal latency. measurements by using us in both the cts patients and control group were compared and the accuracy of diagnostic techniques was calculated. the arithmetic mean, range and the standard deviation of the results were determined and both groups were compared using independent t - tests. sensitivity and specificity of us measurements in cts patients were obtained by determining the cut - off point using the receiver operating characteristics (roc) curve. the correlation between us measurements and hi - ob scale and between the electrophysiological study and us measurements were assessed by using spearman 's test. according to hi - ob scale and electrophysiological severity, accuracy of us was evaluated. chicago, il, usa) was used in all statistical analyses. a p value the clinical diagnostic criterion of cts is based on the american academy of neurology21). one - hundred thirty - five patients who had the symptoms of cts between january 2007 and december 2009 were included in this study. nineteen persons with no clinical symptoms and signs of cts were classified as the control group. patients with diabetes mellitus, collagen disease, thyroid disease, peripheral neuropathy, rheumatic arthritis, traumatic nerve injury were excluded from the study. the patient group contained total of 246 hands, whereas 30 hands were used in the control group. the clinical grade in the patient group was classified into 6 stages according to the historical - objective (hi - ob) clinical scale as follows9) ; stage 0 (no symptoms), stage 1 (paresthesia only at night), stage 2 (paresthesia even for short time in the daytime), stage 3 (hypesthesia in the finger of the median nerve distribution), stage 4 (accompanying weakness or thenar muscle atrophy), and stage 5 (thenar muscle complete atrophy or paralysis). mild cases in hi - ob scale were defined as those patients who reported only symptoms without objective motor deficit of thenar eminence muscles and normal objective sensory function in the median nerve territory of the hand. grades 1 or 2 of a hi - ob scale belong to mild case9). ultrasonographic examination was performed using sonix rp (ultrasonix medical corporation, richmond, canada) by the same experienced examiner (hsn). all wrists were evaluated in the neutral position with the palm up and the fingers semi - extended. the full course of the median nerve in the carpal tunnel was evaluated in both the transverse and sagittal planes. the cross - sectional area (csa) of the median nerve (measured in mm) was measured to proximal carpal tunnel at the level of the pisiform bone. the csa of the median nerve was measured by direct methods as suggested by duncan.7), which was calculated automatically by tracing the inner margin of the epineurium of median nerve, assuming that it has an elliptical shape (fig. the flattening ratio (fr) was defined as the ratio of the nerve 's transverse axis to the antero - posterior axis (fig. the palmar bowing (pb) of the flexor retinaculum is displacement (measured in mm) of the retinaculum from the attachments of a ligament connecting the pisiform bone with the scaphoid bone (fig. the electrophysiological study was performed using a cadwell sierra wave (cadwell laboratories, kennewick, wa, usa). the study consisted of motor and sensory median nerve conduction tests using standard techniques according to the practice parameters for the electrodiagnosis of cts of the american association of electrodiagnostic medicine, the american academy of neurology, and the american academy of physical medicine and rehabilitation3). the electrophysiological abnormalities were classified into three grades according to stevens ' classification as follows23) : mild ; prolonged median sensory distal latency, moderate ; prolonged median sensory and motor distal latency, and severe ; abnormal needle electromyography other than the above two abnormalities, or no response in sensory and motor distal latency. measurements by using us in both the cts patients and control group were compared and the accuracy of diagnostic techniques was calculated. the arithmetic mean, range and the standard deviation of the results were determined and both groups were compared using independent t - tests. sensitivity and specificity of us measurements in cts patients were obtained by determining the cut - off point using the receiver operating characteristics (roc) curve. the correlation between us measurements and hi - ob scale and between the electrophysiological study and us measurements were assessed by using spearman 's test. according to hi - ob scale and electrophysiological severity, accuracy of us was evaluated. of a total of 246 hands in the patient group, 30 hands were male and 216 hands were female. the mean age was 53.0 years. of a total 30 hands in the control group, 27 hands were female and 3 hands were male, and the mean age was 41.7 years. results of this study showed the significant increase in the measures of the csa, fr and pb in the cts, compared with the control group (p<0.05) (table 1). the accuracy of ultrasonographic measurements was evaluated by using cut off points of roc curve. the area under the curve (auc) of csa was 0.95, indicating a sensitivity and specificity of 88.5% and 90.0% respectively, at cut off value of 10.0 mm. the auc of fr was 0.74 at cut off value of 3.4 indicating a sensitivity and specificity of 77.8% and 50.0% respectively. the auc of pb was 0.94 at cut off value of 3.0 mm indicating a sensitivity and specificity of 87.2% and 93.3% respectively (table 2, fig. 2). the csa and pb of us measurements, and hi - ob scale appeared to show a significant positive correlation and as the hi - ob score increased, the csa and pb was increased (r=0.45 : p<0.001, r=0.37 : p<0.001) (table 3). the increase in electrophysiological severity, and the csa and pb of us measurements also showed a significant correlation (r=0.59 : p<0.001, r=0.51 : p<0.001) (table 4). sensitivity of csa and pb for mild patients with less than 3 hi - ob scales were 83.9% and 85.1%, respectively. however, the cases with 3 or more hi - ob scale showed higher sensitivity of csa (92.0%) and pb (89.4%). in relation to electrophysiological study, the mean csa and pb of ultrasonographic measurement usually appeared to be decreased in the mild cases. in mild 65 hands confirmed to the electrophysiological study, 15 hands (22.4%) were found to be negative by csa and 18 hands (26.9%) by pb. however, all of very severe five hands with no response to the electrophysiological study showed positive results in ultrasonographic measurements (table 5). of a total of 246 hands in the patient group, 30 hands were male and 216 hands were female. the mean age was 53.0 years. of a total 30 hands in the control group, 27 hands were female and 3 hands were male, and the mean age was 41.7 years. results of this study showed the significant increase in the measures of the csa, fr and pb in the cts, compared with the control group (p<0.05) (table 1). the accuracy of ultrasonographic measurements was evaluated by using cut off points of roc curve. the area under the curve (auc) of csa was 0.95, indicating a sensitivity and specificity of 88.5% and 90.0% respectively, at cut off value of 10.0 mm. the auc of fr was 0.74 at cut off value of 3.4 indicating a sensitivity and specificity of 77.8% and 50.0% respectively. the auc of pb was 0.94 at cut off value of 3.0 mm indicating a sensitivity and specificity of 87.2% and 93.3% respectively (table 2, fig. the csa and pb of us measurements, and hi - ob scale appeared to show a significant positive correlation and as the hi - ob score increased, the csa and pb was increased (r=0.45 : p<0.001, r=0.37 : p<0.001) (table 3). the increase in electrophysiological severity, and the csa and pb of us measurements also showed a significant correlation (r=0.59 : p<0.001, r=0.51 : p<0.001) (table 4). sensitivity of csa and pb for mild patients with less than 3 hi - ob scales were 83.9% and 85.1%, respectively. however, the cases with 3 or more hi - ob scale showed higher sensitivity of csa (92.0%) and pb (89.4%). in relation to electrophysiological study, the mean csa and pb of ultrasonographic measurement usually appeared to be decreased in the mild cases. in mild 65 hands confirmed to the electrophysiological study, 15 hands (22.4%) were found to be negative by csa and 18 hands (26.9%) by pb. however, all of very severe five hands with no response to the electrophysiological study showed positive results in ultrasonographic measurements (table 5). the diagnosis of cts is made mainly on the basis of the patient 's history and the clinical features10,12). the hi - ob scale is a reliable measurement in cts evaluation, based on clinical history and physical examination9). this scale has no diagnostic purpose but it has a good correlation with the patient - oriented findings and the median nerve electrophysiological impairment9). confirmation of cts is usually evaluated by electrophysiological study3). however, sometimes, it is difficult to diagnose cts using only the electrophysiological study in cases of early and mild cts, even severe cts that show no response to the study, elderly patients and associated peripheral polyneuropathy patients. recently, us techniques came into advancement as a tool to complement the diagnosis of cts. us provide anatomical images of the median nerve, neighboring structures, and space - occupying lesion in the carpal canal as a painless and non - invasive study and can offer dynamic images. however, us is operator dependent that can show high reproducibility after adequate training of the examiners4). in our study, the us measurements used in cts diagnosis are the csa of the median nerve at various levels of the carpal canal, the fr and pb of the flexor retinaculum. in most studies, the increase in the csa at the tunnel inlet demonstrated the highest sensitivity and specificity8,26,27) and moreover, the measurement at this level was easier to perform. some authors considered the proximal edge of the flexor retinaculum, approximately at the level of the distal radioulnar joint, as the tunnel inlet but others considered the level of the pisiform bone as the landmarks11). the sensitivity of the csas ranged from 48% to 89%4,7,8,26,27) and the csa cutoff at which the value was considered abnormal, varied from 9 mm to 15 mm1,2,7,14,18,26). our study showed a sensitivity of 88.5% at cut off value of 10.0 mm for the mean csa. the sensitivities of increased pb of the flexor retinaculum varied from 40% to 81%2,5,14,24), and sensitivities of fr ranged from 37% to 100%5,28). our results showed a sensitivity of 87.2% at cut off value of 3.0 mm for the pb and a sensitivity of 77.8% at cut off value of 3.4 mm for the fr. these data of sensitivity corresponds with the findings reported in earlier studies. in our study, csa and fr, pb of us were significantly increased in the cts group than the control group. among them, csa and pb were evaluated to have a relatively higher accuracy than fr according to the roc curve. therefore, measurement of csa and/or pb can be considered as an alternative modality to distinguish cts patients from asymptomatic controls2,5,14,24,28). many other studies showed good correlation between the hi - ob scale and us, and between electrophysiological grade and us indicating that the nerve swelling detected by calculation of us reflects in itself the degree of nerve damage : the greater the severity of electrophysiological findings or clinical severity, the greater the csa of median nerve13,16,20,29). results of this study showed there is a significant correlation between hi - ob scale and csa as well as pb, respectively. in addition, degree of electrodiagnostic abnormalities is also well correlated with csa and pb, respectively in our study. mild cts can not show abnormal findings on us study. in our data, us could not detect abnormalities in 22.4% of patients with mild cts to electrophysiological study. for patients without abnormal findings on us and electrophysiological study, the diagnosis of cts should be made, based on the patient 's history and the clinical findings. however, our data also demonstrated ultrasonic measurement is helpful to diagnose extremely severe cts patients with no response to electrophysiological studies and these data agree with earlier study14). ultrasonographic measurements of the csa and pb of the median nerve offer comparatively high diagnostic accuracy for cts and ultrasonographic study could be considered as a non - invasive, alternative and complimentary diagnostic modality for the evaluation of cts. in addition to diagnosis of cts, ultrasonographic measurements of the median nerve could also give information about severity of cts. | objectiveto investigate a diagnostic value of ultrasonography in carpal tunnel syndrome (cts) patients and to evaluate a correlation of sonographic measurements with the degree of electrodiagnostic abnormalities and clinical severity.methodstwo-hundred-forty-six symptomatic hands in 135 patients and 30 asymptomatic hands in 19 healthy individuals as control group were included. in ultrasonographic study, we measured the cross - sectional area (csa) and flattening ratio (fr) of the median nerve at the pisiform as well as palmar bowing (pb) of the flexor retinaculum. sensitivity and specificity of ultrasonographic measurements were evaluated and ultrasonographic data from the symptomatic and control hands were compared to the grade of electrodiagnostic and clinical severity.resultsthe mean csa was 13.74.2 mm2 in symptomatic hands and 7.91.3 mm2 in asymptomatic hands. the mean fr was 4.21.0 in symptomatic hands and 3.40.4 in asymptomatic hands. the mean pb was 3.50.5 mm in symptomatic hands and 2.60.3 mm in asymptomatic hands. statistical analysis showed differences of the mean csa, fr and pb between groups were significant. a cut - off value of 10 mm2 for the mean csa was found to be the upper limit for normal value. both the mean csa and pb are correlated with the grade of electrophysiological abnormalities and clinical severity, respectively.conclusionultrasographic measurement of the csa and pb is helpful to diagnose cts as a non - invasive and an alternative modality for the evaluation of cts. in addition, ultrasonography also provides a reliable correlation with the grade of electrodiagnostic abnormalities and clinical severity. |
vascular injury is an uncommon but potentially devastating complication of spine surgery. throughout its course, the aorta remains in close proximity to the thoracic and lumbar spine and is at risk from malpositioned pedicle screws or other hardware.1 despite this risk, the reported rate of vascular injury from spine surgery is low. in one systematic review, papadoulas found the incidence of vascular injury after excision of a herniated lumbar disc via a posterior approach to be less than 0.05%.2 in addition, hicks systematically reviewed 14,570 pedicle screws placed in the thoracic and lumbar spine of 1,666 patients with adolescent idiopathic scoliosis (ais) ; they found a 4.2% overall rate of screw malposition with 6 of 8,147 screws (0.07%) abutting the aorta in the subgroup of studies that reported this finding. there were no reports of major vascular complications.3 despite the low rates of vascular injury identified in these reviews, the literature contains multiple case reports in which pedicle screw instrumentation was associated with early or late aortic injuries.4 5 6 7 8 9 10 in the majority of these reports, treatment consisted of removing of the offending hardware with simultaneous open or endovascular aortic repair (evar). here, we describe the case of an adolescent patient with a malpositioned pedicle screw abutting the thoracic aorta and new complaints of deep - seated thoracic pain. intraoperative aortography demonstrated normal flow in the thoracic aorta, so no graft was placed to avoid the known morbidity of evar. the patient was a 20-year - old man who presented to clinic with 4 months of back pain. at the age of 15 years, he had undergone a t6t8 posterior instrumented fusion with excision of an osteoblastoma from the right t7 pedicle and lamina. at that time, his primary symptom was midthoracic pain ; this pain resolved within 3 months of his index surgery. when he returned to clinic 5 years later, he complained of deep - seated substernal and midthoracic pain., the patient had a well - healed midline incision with no signs of infection. he had normal strength, sensation, and reflexes in his bilateral upper and lower extremities. posteroanterior and lateral radiographs (fig. 1) and a computed tomographic (ct) scan of the thoracic spine (fig. 2) demonstrated that the left t6 screw penetrated the lateral cortex of the t6 pedicle and abutted the posteromedial aspect of the aortic wall. there was no pseudoaneurysm or periaortic inflammation identified to suggest frank penetration into the aortic lumen. however, it was felt that because of ongoing mechanical irritation by the abutting screw and the patient 's new pain symptoms, there was an increased risk of aortic perforation, dissection, or thrombotic complication if the screw were left in place, and that therefore, it was advisable to remove it. posteroanterior and lateral radiographs of the thoracic spine show lateral positioning of the left t6 pedicle screw. an axial computed tomographic scan slice through the t6 pedicle and sagittal reconstruction show the left t6 pedicle screw breaches the lateral cortex and impinges against the posteromedial aorta. after a thorough discussion of the risks and benefits with the patient, his family, and the vascular surgery service, the patient elected to go to the operative theater with plans to obtain endovascular control of the aorta, remove the offending hardware, and stent the aorta if required. in the theater, general anesthesia was induced and a double - lumen endotracheal tube was placed in case open thoracotomy were required. the patient was positioned supine and 7-french sheath was introduced into the right common femoral artery. a catheter was passed via this sheath into the thoracic aorta and an aortogram was performed to confirm appropriate position of the catheter and adequate visualization of the aorta. no evidence of stenosis, contrast extravasation, or intraluminal material was noted. with the right groin catheter in place, the previous midline thoracic incision was reopened and dissection was carried down to the level of the posterior instrumentation. the soft tissues were cleared from around the screws and the end caps removed. once the vascular surgery team indicated their readiness to proceed, the left t6 screw was backed out. the vascular team then performed a second aortogram, which again demonstrated no extravasation of contrast or other abnormal finding. a final aortogram after removal of all hardware showed no evidence of injury or abnormal flow within the thoracic aorta (fig. 3). given the normal aortogram, it was decided no graft placement or further endovascular intervention was necessary. intraoperative aortography demonstrates no extravasation of contrast and normal filling of the descending thoracic aorta. the posterior wound was thoroughly irrigated and closed in layered fashion and the patient was returned to the supine position. estimated blood loss for the procedure was 200 ml. the patient was awakened and found to be neurologically and vascularly intact, unchanged from his preoperative examination. he was admitted to the surgical floor where he progressed appropriately and was discharged home on postoperative day 2. vascular injury has been recognized as a rare potential complication during anterior spine procedures, with a rate of less than 5% reported in a recent systematic review.11 after posteriorly based procedures, reported rates of acute vessel injury in systematic reviews range from 0 to 0.05%.2 3 however, because of the intimate association of the aorta with the thoracic and lumbar spine, it remains at risk of acute or delayed injury from misplaced posterior instrumentation.1 a growing number of reports suggest such injuries are likely an underappreciated complication.4 5 6 7 8 9 10 in cases of frank perforation of the aorta or another major vessel, immediate operative or endovascular intervention is indicated to repair the injury. potential risks of impinging instrumentation include late catastrophic hemorrhage, pseudoaneurysm formation, or thromboembolic complications. the best evidence in favor of observing impinging hardware without intervening comes from foxx who retrospectively reviewed the position of 680 pedicle screws in 107 patients on routine postoperative imaging. of the 680 screws, 33 screws (4.9%) were found to contact a major vessel on imaging, including the aorta in four cases. eight of the nine affected patients were living and asymptomatic at average follow - up of 25 months, with one patient deceased from amyotrophic lateral sclerosis at 16 months after surgery.12 from these data, the authors concluded that asymptomatic, impinging screws may be safely observed with serial imaging. evidence in favor of hardware removal comes primarily from case reports in which pedicle screws impinging against the aorta were diagnosed on a delayed basis and successfully managed with hardware removal and evar.4 5 6 7 8 10 exact details of the procedures varied, but in all cases patients had endovascular stent grafts placed that bridged the affected region of the aorta before or after removal of their hardware. no complications were reported.4 5 6 7 8 10 of note, in several of these reports, patients experienced a symptom - free period after their initial spine surgery and were diagnosed with impinging hardware after returning 4 months to 6 years later with new complaints of pain in the instrumented region ; these complaints resolved after screw removal.6 8 10 we hypothesize that in the setting of impinging hardware, new - onset pain after a symptom - free period may potentially represent a red - flag for surgical intervention. in an in vivo bovine model in which thoracic pedicle screws were left abutting the aorta, faro found histopathologic changes in 96% of aortic specimens at 3, 6, and 12 months. more than half of the specimens showed significant wall thinning, and in aggregate, the impinged aortas demonstrated decreased stress to failure compared with controls. the severity of tissue damage on histology did not correlate with the depth of impingement on ct scans.13 in the context of the limited clinical data, this study raises concerns that screw impingement may compromise of vessel wall integrity even in the absence of frank perforation. the case considered here is unique in that our patient was much younger than those in previous reports (20 years old vs. older than 60 years) and because he was successfully managed without endovascular grafting given his benign findings on intraoperative aortography. as the incidence of pedicle screw instrumentation for ais continues to rise, one expects to see younger patients facing decisions about how to manage impinging hardware.3 on the basis of multiple case reports, placement of an endovascular aortic graft appears to represent an effective, low - morbidity treatment for impinging or perforating pedicle screws in older population. however, available clinical data after evar in the vascular literature suggest a significant incidence of complication and reoperation at intermediate to long - term follow - up that would be more relevant to a younger patient. in one series of 1,066 patients treated with evar for abdominal aortic aneurysm and followed up between 2 and 16 years, 233 patients (22%) required repeat operation, with 131 operations attributed to graft - related complications (12%), and the remainder to progression of vascular disease.14 in another study using a modern endovascular aortic graft exclusively (endurant ; medtronic, minneapolis, minnesota, united states), 16 of 100 grafts showed leakage on ct angiography and 5 of 100 patients required endovascular or operative reintervention within the first year.15 the high rate of complications and reoperation associated with evar is sobering, particularly when considering application in an adolescent patient. here, we describe the safe and successful removal of a pedicle screw impinging on the aorta in a symptomatic 20-year - old patient. on the basis of normal intraoperative aortography, we elected against placement of endovascular graft given the high incidence of associated complications and the expected longevity requirement in a young patient. at more than 2 years after the procedure we would recommend considering similar treatment of other young patients when intraoperative aortography shows no abnormality. neuroscience clinical institute, epworth hospital, melbourne, victoria, australia potter are to be commended for presenting an important yet very uncommon surgical complication of a misplaced thoracic screw requiring revision for aortic impingement.1 the index procedure was an excision of a t7 osteoblastoma 5 years previously. the presentation of the now 20-year - old male patient was midthoracic and retrosternal pain without any evidence of tumor recurrence. a computed tomographic (ct) thoracic scan performed to exclude change in instrumentation or pathology demonstrated a laterally misplaced left t6 screw. interestingly, the ct demonstrated impingement on the thoracic aorta by the screw tip, which was previously unknown. if pain is present, 12% of the patients will report improvement with removal of instrumentation2 ; this also facilitates future imaging for tumor surveillance. the t6 pedicles are among the most technically challenging to cannulate given their narrow width. postoperative ct scans are equally used in many practices, including my own, to exclude neural and/or vascular compromise. delayed aortic rupture is extremely rare.3 however, removal of the t6 screw in this case for vascular impingement is reasonable, especially given the bovine in vivo studies of aortic wall thinning illustrated by faro.4 this is especially true in a young adult. in the setting of luminal or gross mural abnormality, the aorta undergoes constant cyclical movement with cardiac pulsatility being the primary dynamic elastic artery. with each cycle, it undergoes radial and longitudinal distortion. if the point of the screw was in contact with the aorta, it is unlikely that it would be in contact with a single adventitial point promoting erosion over time.4 the aorta is tethered to the vertebral column by multiple radicular branches ; one is the artery of the adamkiewicz, classically thought to arise on the left near the t9 level. although the absence of erosion, thickening, or false aneurysm in the referable area is comforting, having an expedited means for brisk vascular control is vital. combined management with the vascular surgery team involves preparing and positioning the patient for an immediate posterolateral thoracotomy (including a double lung intubation) as detailed by the authors. endoluminal techniques permit control via a compliant balloon that can be prophylactically placed and repair subsequently effected with an appropriately sized stent graft. ideally, imaging the descending thoracic aorta to position a wire and balloon opposite the screw and fix it relative to the sheath allows immediate control to be achieved. a limitation of this case report is extrapolating results and complication rates from abdominal aortic aneurysm endovascular aortic repair (evar) to thoracic endovascular aortic repair (tevar) for trauma. even for aneurysm repair, tevar has lower complication rates5 than the more complicated abdominal evar procedures. furthermore, in this report, the injury is small and the thoracic aortic wall probably normal, and so seal is much more assured, provided proper planning has been performed, making an endoleak very unlikely. there is no aneurysm sac to pressurize in this setting that is the main source of reintervention in evar. evar results are not a surrogate for posttraumatic tevar results. not placing the stent graft in the absence of an aortic abnormality the main concerns with placement of a stent graft in this report is the risk of paraplegia, the potential for devastating (but rare) graft infection, and the uncertainty of the long - term fate of the stent graft in young patients. the post - discovery decision - making described here by the authors and the commenter is very helpful for all of us clinicians. this is a classic scenario for which there is no evidence base as we have no control group ; we have to use our judgment, experience, and the few descriptions offered in the peer - reviewed literature, even if its level of evidence is 4 or 5. this case raises the bigger question : should all patients have some form of routine post - instrumentation advanced imaging to check on segmental hardware placement even in asymptomatic patients ? what do you think ? | study design case report and review of the literature. objective the objective of the article is to report the case of a 20-year - old patient with a threatened aortic injury from pedicle screw instrumentation successfully managed without aortic grafting. methods the patient 's clinical course is retrospectively reviewed. the offending hardware was removed after gaining endovascular control of the aorta. results intraoperative aortography was normal and no graft was placed. the patient remains asymptomatic at 2 years after surgery. conclusions hardware impinging on the aorta can safely be removed by gaining endovascular control of the aorta. in the setting of normal intraoperative aortography in a young patient, we recommend against further intervention to avoid the known morbidity of aortic grafting. |
both wild and captive reptiles are frequently affected by external parasites. of these, acarids (ticks and mites) are the most commonly encountered. the most commonectoparasite in captive snakes is the hematophagous snake mite (ophionyssus natricis) belonging to the macronyssidae family of the suborder mesostigmata (1). unfed females are yellow - brown ; engorged females are dark red, brown or black. 1). the engorged female leaves the host and deposits eggs in cracks and crevices. the eggs hatch in 14 days, developing through larva, protonymph and deutonymph stages to the adult. o. natricis has a worldwide distribution affecting mainly snakes and to a lesser extent lizards. infected snakes often exhibit lethargy, dysecdysis, pruritus, crusting dermatitis (sometimes progressing to abscesses), and behavioral changes (increased bathing time, rubbing against objects). the mite, which thrives in skin crevasses, acts as an irritant to the snake that can cause parasitic dermatitis, but often leads to increased, irregular shedding cycles (3). o. natriciscan also attack humans, causing popular vesiculo - bullous eruption of the skin (4). a 29 years old manworking in sari zoo, sari, mazandaran, iran, presented itchy papullar eruption of the skin (fig. he had noticed small insects fixed on his skin and he had attempted to remove them, also large numbers of these same insects were on a python and its cage in the zoo. department of veterinary parasitology, veterinary faculty, islamic azad university, babol branch was asked to examine the python. the snake was a 2.95 m caramel burmese python snake and insects were collected from the snake and its glass cage in the zoo. the man who feeds and takes care of python with papullar rash was also examined and behavioral changes noticed such as, restlessness, irritation, anorexia, and short cycles of skin shedding in python history. samples were preserved in alcohol, and then made them transparent in lacto phenol in order to observe them under the light microscope. they were identified regarding to their morphological characteristics as o. natricis (geravis, 1844), a snake mite. for diagnosis confirmation, samples were sent to iran veterinary parasitology museum and deposited with accession number of 733. o. natricis is a purely blood sucking parasite of snakes and is of worldwide distribution (5, 6). the diagnosis of snake mite infestation is usually straightforward : the parasites are easily identifiable with the naked eye or with a magnifying glass. snakes are uncommon pets but their popularity is increasing. when patients with unusual pets such as snakes or lizards present at dermatology clinics, o.natricis should be considered as a cause of dermatitis (7). | ophionyssus natricis is a purely blood sucking parasite of snakes and of worldwide distribution. infected snakes often exhibit lethargy, pruritus, crusting dermatitis, and behavioral changes. ophionyssus natricis can also attack humans, causing popular vesiculo - bullous eruption of the skin. a 29 years old man working in zoo, sari, mazandaran, iran, presented itchy papullar eruption of the skin. he had noticed small insects fixed on his skin and large numbers of these same insects on a python and its cage in the zoo. regarding totheir morphological characteristics they were diagnosed as o. natricis (geravis, 1844), a snake mite. it is the first report of o. natricis from iran. |
urinary tract infections in diabetics are a common occurrence, and they may be complicated by the presence of gas producing organisms. emphysematous infection of the kidney is well known, but such infections are rare in the testis and epididymis. these infections are often referred to the surgeon, because of the likelihood of surgical intervention. both emphysematous pyelonephritis and epididymo - orchitis may not be detected clinically and ultrasound or computed tomography (ct) may be needed for the diagnosis. the presentation of epididymo - orchitis with gas bubbles in the scrotum is very rare, and one case that has been reported was secondary to seminal vesicle involvement by diverticulitis of the colon.. however, there are no case reports of emphysematous epididymo - orchitis caused by e. coli with a normal seminal vesicle. a 52-year - old man was brought to emergency with a 3-day history of pain and swelling in the left scrotum with fever [figure 1 ]. clinical examination of the scrotum showed inflamed left scrotum and tender and enlarged left testis and epididymis. per rectal examination showed that the prostate was normal in size and consistency, with its upper limit just reachable. he gave a history of previous urinary tract infection, and the culture had shown e. coli. an ultrasound of the scrotum was done, which showed gas shadows in the left epididymis and testis with inflammation in the skin of the scrotum and fluid in the tunical sacs [figure 2 ]. a ct scan of the abdomen and the pelvis showed the pockets of gas in the scrotum and testis, but the seminal vesicles and the sigmoid colon were normal [figure 2 ]. clinical presentation with acute inflamed scrotum with per operative image showing destroyed testicular tunica and parenchyma radiology of the patient showing in clockwise sequence, gas bubbles in the testis and scrotum seen on ultrasound, ct images showing preserved fat planes around the seminal vesicles, gas bubbles seen in the scrotum and substance of the testis, and increased vascularity of the testis seen on doppler images the patient was taken up for surgery in view of the gas shadows in the testicular substance and poor response to clinical parameters after intravenous antibiotics. there was extensive inflammation in the scrotum with gas bubbles, extending through the tunica into the substance of the testis [figure 1 ]. histopathology of the excised testis showed acute epididymo - orchtis with areas of necrosis and thrombosis in veins. fournier 's gangrene can involve the perineum with gas formation but classically spares the testis and epididymis. an online search revealed one case of emphysematous epididymo - orchitis, secondary to seminal vesicle involvement in diverticulitis of the sigmoid colon. however, our patient had normal outline of seminal vesicles with preserved fat planes and no evidence of diverticulitis on ct scan. pus from the testis cultured e. coli, which has been cultured previously in cases of emphysematous pyelonephritis but has never in emphysematous epididymo - orchitis. there is not much information available about the best modality for the treatment of this condition. initially, most cases merit high - dose broad - spectrum antibiotics till the results of culture sensitivity are available. aspiration of pus with a wide bore needle may be tried in early cases. as in case of emphysematous pyelonephritis diabetes mellitus could have a role in the pathogenesis of the condition as in emphysematous pyelonephritis. the differential diagnosis of a bright, highly reflective tissue interface on ultrasound of the testis, with distal acoustic shadow, can be either emphysematous infections like fournier 's gangrene or emphysematous epididymo - orchitis, or testicular shrapnel, testicular germ cell neoplasm, or testicular microlithiasis. ct scan can be a useful adjunct to the diagnosis. with the availability of ultrasound in most emergency departments, there is a case for applying the ultrasound probe on diabetic patients with an acutely inflamed scrotum to rule out emphysematous infection. | emphysematous epididymo - orchitis is a rare cause of an acute scrotum and is a surgical emergency. diagnosis is clinically difficult, and sonography with a high - frequency probe is useful to pick up gas shadows in the scrotal wall or testicular substance. a diabetic patient presented with fever, urinary tract infection, and an acute scrotal swelling. the patient needed orchidectomy and scrotal debridement. as in emphysematous pyelonephritis, this condition occurs in diabetics, and patients may need surgery. there is a need to perform sonography in all diabetic patients with an acutely inflamed scrotum, because detection of gas shadows makes surgical intervention more likely. |
the genome sequences of gdah16 and gdse16 have been deposited in genbank under the accession numbers ky347898, ky347899. | previously, we identified three porcine circovirus type 2 (pcv2) strains in buffalo meat samples from southern china. in this study, we confirmed the reappearance of those buffalo - origin - like pcv2 strains in swine herds in this region, which supported the possible cross - species infection of pcv2 between buffalos and pigs under field conditions. |
gemella morbillorum is a gram - positive, catalase - negative, facultatively anaerobic coccus that often behaves as commensal bacteria of the mucous membranes of oropharynx, gastrointestinal tract, and female genital tract. however, in some rare cases, this bacterium has been implicated in serious invasive infections, especially endocarditis 1. possible predisposing factors for gemella morbillorum endocarditis include situations in which there is a lack of the gastrointestinal mucosal barrier integrity, as surgical manipulation of the oral cavity and gastrointestinal tract neoplasia 2,3. severe cases of gemella endocarditis have been reported, both cases requiring cardiac valve replacement and cases with fatal outcome 4 ; this fact underscores the potential virulence of this pathogen in the setting of infective endocarditis. we present here a case of cardiogenic shock as a complication of acute mitral valve regurgitation following gemella morbillorum native valve endocarditis in a 72-year - old male. a 72-year - old caucasian male with a history of hypertension, type-2 diabetes mellitus, peripheral arterial disease of the lower limbs, and previous coronary artery bypass graft surgery was admitted to a tertiary referral hospital in southern brazil with dry cough, fever, anorexia, loss of more than 10% of body weight, and dyspnea in the last 30 days. upon arrival to the emergency department, the patient was hypotensive (blood pressure 78/53 mmhg), tachycardic (heart rate 122 bpm), feverish (axillary temperature 38.4c), with jugular turgor at 45, with bibasilar pulmonary rales and with new systolic mitral regurgitation murmur (murmur grade 4). the patient rapidly developed cardiorespiratory collapse requiring mechanical ventilation and continuous infusion of vasopressors ; he also presented oliguria (urine output 2 sec). the initial chest x - ray showed signs of pulmonary congestion and cardiomegaly (fig.1). the complete blood count demonstrated leukocytosis (12 920 white blood cells / mm) and normocytic anemia (hemoglobin level of 11.3 g / dl). the erythrocyte sedimentation rate and the c - reactive protein plasma levels were elevated (91 mm / h and 6.8 mg / dl, respectively). the transesophagic echocardiography at the bedside showed mitral valve thickening suggestive of infectious etiology and large flail of the anterior leaflet ; the mitral regurgitant jet intensity and area were consistent with severe insufficiency (fig.2). the bedside lung ultrasound showed bilateral multiple b - lines in a diffuse pattern, which is also compatible with pulmonary edema. a definitive diagnosis of mitral native valve endocarditis with cardiogenic shock due to severe acute mitral valve regurgitation was made. the initial empirical antimicrobial therapy administered was ceftriaxone plus gentamicin (initiated within 50 min after hospital arrival). the hemodynamic instability was treated with norepinephrine (doses up to 1.0 g / kg / min) and intra - aortic balloon pump counterpulsation implant. the patient was taken to emergency mitral valve replacement surgery ; his preoperative mortality risk according euroscore ii was 55.2%. immediately after valve replacement surgery (bioprosthetic mitral valve), there was progressive improvement in cardiogenic shock with gradual reduction of the vasopressor dose. gemella morbillorum was isolated in two independent samples of blood cultures collected from distinct peripheral veins. although there is no clinical and laboratory standards institute (clsi) standard method and interpretation of susceptibility testing for gemella, susceptibility testing was performed using the clsi method for viridans group streptococci on mueller - hinton agar supplemented with 5% sheep 's blood and incubated at 35c with 5% co2 for 24 h 5 ; the isolated bacteria was sensitive to penicillin, ceftriaxone, and vancomycin. after results of antimicrobial susceptibility tests, the antibiotic regimen was de - escalonated to penicillin g plus gentamicin. during hospitalization, the patient became dependent on mechanical ventilation due to critical illness polyneuropathy and refractory pulmonary congestion (including need for tracheostomy and continuous ultrafiltration in order to negative fluid balance). in the twenty - seventh day of hospitalization, after a brief period of clinical improvement, the patient had a new episode of shock associated with pulmonary edema ; there was lung compliance worsening and need for mechanical ventilation with high pressures and high fraction of inspired oxygen. the patient was taken to emergency surgery for valve replacement again ; however, he ended up dying during the procedure. initial transesophageal echocardiography showing a mass adhered to the ventricular side of the mitral valve. the present case report describes an unusual etiology of infective endocarditis with unfavorable clinical course despite appropriate treatment, gemella morbillorum. although noninvasive infections were reported, the few described cases were generally associated with serious infections, such as endocarditis, bloodstream infections, and central nervous system infections 68. previously, gemella morbillorum was considered to be a viridans streptococcus due to its microbiological similarities with the genus streptococcus ; only in 1988, based on its biomolecular and physiological characteristics, this bacterium was reclassified as part of a distinct genus, gemella 9. however, both the clinical course and the antimicrobial susceptibility patterns of infections associated with this organism seems to be similar to those seen with viridans streptococci 10. the ability of this bacterium to cause destruction of the cardiac valve tissue, contributes to rapid deterioration of patients with endocarditis culminating in cardiogenic shock a very serious condition associated with high mortality rates in the context of infective endocarditis 11. septic shock is another reported complication of gemella morbillorum invasive infections associated with high mortality rates 12. the high virulence profile of gemella morbillorum is not well studied, but is hypothesized that this microorganism may share the same pathogenic mechanisms found in viridans streptococci bacteria, which are often associated with severe infections in both immunocompetent and immunocompromised patients 13,14. in summary, our case report contributes to the limited literature regarding the characteristics of infections by gemella morbillorum and provides further evidence about the high virulence of this pathogen. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review from the editor - in - chief of this journal. | key clinical messagegemella morbillorum is a rare cause of infective endocarditis. consequently, little is known about the natural course of endocarditis secondary to this pathogen. herein, we report a case of cardiogenic shock as a complication of acute mitral valve regurgitation following gemella morbillorum native valve endocarditis. |
in 1960 nowell first described lymphocyte activation by mitogenic lectins to result in " blastic transformation " and subsequent mitosis, which are complex cellular events that require several days to evolve. more than 40 years later, we have advanced considerably in our understanding of the cellular and molecular events responsible for these phenomena. now with the genomic revolution, we are poised on a new threshold of discovery, in that it is finally feasible to begin to identify the genes that are responsible for such complicated cellular processes. however, it is now necessary to develop an experimental approach that will rapidly and accurately facilitate the discovery process. with the description of lymphocyte - derived growth factor activities in the 1960s and 1970s, it was realized that activated lymphocytes secrete soluble factors that are critical for subsequent lymphocyte proliferation. moreover, in 1967 it was demonstrated that monocytes or macrophages serve as necessary accessory cells for the lymphocyte activation process. then, in the early 1970s several reports indicated that mitogen- or endotoxin - stimulated macrophages release soluble lymphocyte activating factors (lafs) that also amplify the activation process. accordingly, lymphokines and monokines, which collectively came to be known as cytokines, became prime research subjects for those interested in lymphocyte activation. however, it was not until the 1980s that the cell surface and soluble molecules responsible for the complex cellular changes of lymphocyte activation were identified. with the isolation of the lymphocyte - derived molecule responsible for t cell growth factor (tcgf) activity, which came to be known as interleukin 2 (il-2), and discovery of il-2 receptors (il-2r), it could be shown that mitogenic lectins and specific antigens triggered an initial activation event, which was analogous to the activation of cell cycle " competence " by serum - stimulated fibroblasts, and resulted in transition from the g0 to the g1 phase of the cell cycle. however, just as in serum - stimulated fibroblasts, this initial activation event did not result in lymphocyte proliferation. instead, it appeared that the initial activation of cellular competence triggered by the mitogenic lectins was amplified by the laf activity, which became known later as il-1, and led to the secretion of the lymphocyte - derived il-2 and the expression of il-2rs. the subsequent interaction of il-2 with the il-2rs then stimulated blastic transformation and progression through g1 and into the s phase of the cell cycle, followed by mitosis. furthermore, detailed studies indicated that successful activation of the il-2r is a quantal (i.e. all - or - none) event that depends on surpassing a threshold of a finite number of il-2-il-2r interactions. this threshold corresponds to surpassing the " restriction (r) point " in the mid to late g1 phase of the cell cycle, first described by pardee for serum - activated fibroblasts. accordingly, activation of lymphocyte proliferation was realized to require at least 3 signals, one delivered to the t cell via the lectin, another supplied as a soluble factor by accessory macrophages, and a third contributed by the il-2/il-2r interaction. with the discovery of the t cell antigen receptor (tcr) in 1983 [20 - 22 ] it was possible to demonstrate that the first signal is delivered via antigenic activation of the tcr. subsequently, discovery of accessory " co - stimulatory " molecule expression by " helper " t cells (th), revealed that activation solely of the tcr itself was insufficient to fully activate il-2 gene expression and t cell proliferation. instead, in the mid ' 80s it was found that the activation of co - stimulatory molecules like cd28 expressed by t cells via ligands such as b7 expressed by antigen presenting cells (apc), deliver additional signals that synergize with those emanating from the tcr, and result in maximal il-2 gene transcription. as well, cd28 activation stabilizes il-2 mrna transcripts, prolonging il-2 mrna life span. thus, in addition to soluble macrophage - derived products, cell surface molecules expressed by apcs also contribute to the 2signal attributed to the apcs. the molecules responsible for the contribution by the soluble macrophage - derived lafs still remain obscure, even today. initially the nomenclature of il-1 and il-2 was created to delineate the sequential action of an assumed single macrophage - derived soluble factor (il-1), which promoted the production of a single lymphocyte - derived factor (il-2). however, the molecular nature of the laf activity was never fully elucidated, even after the isolation and identification of multiple mitogen / endotoxin - stimulated macrophage - derived products, such as il-1, il-6, il-12, il-18, and tumor necrosis factor - alpha (tnf-). it is probable that all of these macrophage - derived soluble factors together make up the activity originally ascribed to laf. accordingly, it is obvious that addition of a polyclonal activating ligand such as phytohemagglutinin to peripheral blood mononuclear cells (pbmcs) leads to the activation of soluble macrophage - derived and lymphocyte - derived activating factors, and the almost simultaneous activation of multiple cell surface receptors, signaling pathways and transcription factors that ultimately all contribute to lymphocyte activation. stimulation of the various receptors expressed by t cells and apcs results in the activation of distinct and also redundant signal transduction pathways, and in the activation of distinct as well as redundant transcription factors. accordingly, it has been assumed that triggering any of these receptors should result in the expression of distinct as well as redundant cellular genes. until recently the identification of large numbers of genes triggered by distinct ligands has been difficult, owing to the cumbersome methods available for gene identification. cochran and stiles were among the first to use differential cloning methods to identify pdgf - regulated genes in fibroblasts. these investigators focused on the expression of immediate / early pdgf - stimulated genes, by activating the cells in the presence of inhibitors of protein synthesis such as cycloheximide, thereby preventing the expression of secondary genes. subsequently, zipfel and co - workers employed subtractive hybridization to isolate and differentially clone immediate / early genes activated via the t cell antigen receptor in the presence of cycloheximide. a total of 60 novel tcr - induced immediate / early genes were isolated, but most were not identified at the time, in that dna cloning and sequencing methods were tedious and time consuming. new differential cloning methods were developed subsequently, including differential display, sulfhydryl labeling and affinity purification of newly - synthesized transcripts, representational difference analysis, and serial analysis of gene expression. each methodological advance has improved both the sensitivity of detection of differentially expressed genes, as well as the speed with which the genes may be identified. however, the development of dna microarray technology, which allows rapid interrogation of thousands of genes and the detection of gene expression at frequencies even less than 1 in 100,000, has now greatly facilitated such experimentation. this communication represents our initial efforts to use dna microarrays in a reductionist approach to begin to identify the genes expressed as a consequence of triggering each individual ligand during lymphocyte activation. because the il-2/il-2r interaction is discrete molecularly, and leads to a very finite, quantal decision to undergo blastic transformation, dna synthesis and mitosis, we have focused our first efforts on identifying il-2-regulated genes expressed by normal human t cells. responses of pbmcs to il-2 are complex, involving direct effects of il-2 on gene expression by t cells, b cells and nk cells, as well as secondary effects mediated by additional cytokines and transcription factors expressed by these cells in response to il-2. to identify the maximal number of il-2-regulated genes, pbmcs comprised of ~80% cd3 + lymphocytes, with smaller fractions of monocytes, b cells and nk cells (table 1) were examined. the cells were pre - activated for 72 hr with anti - cd3 to induce t cell il-2 receptor expression, then washed and rested for 3640 hr, prior to restimulation with il-2 for 4 hr. results are averages se from 5 independent experiments five independent experiments were performed with pbmcs from different donors to identify il-2-regulated genes represented on the affymetrix u95av2 oligonucleotide array. a comparison was made between the pre - activated, rested cells, and those restimulated with il-2 for 4 hr. the expression levels were analyzed using dna - chip analyzer software, with normalization to the average chip signal intensity for each experiment, and a 10percentile value cut - off for ' absent ' genes. approximately 45% of the ~12,600 probe sets were detected above background. using the criterion of p 80-fold (tnf 36296_at). the subtractive differences in fluorescent intensity ranged from 114 20 (c - myb 1474_s_at) to > 1000 (cr7 1779_s_at). four of the genes (c - myb, cr7, flt3 ligand, tnf-) were detected with more than one probe set, and these replicates were in fairly good agreement. it is worthy of note that there is not a correlation between the subtracted difference and the fold change in gene expression levels. this is likely due to the fact that there is a wide range in the abundance classes of rna transcripts derived from different genes, from as few as 1 copy per cell to several thousand copies per cell. the signal intensity of a particular transcript detected on an oligonucleotide array would thus be directly related to this abundance level. therefore, the subtracted change in signal intensity would be profoundly affected by the absolute number of cellular copies of a given transcript. by comparison, expression of known il-2-regulated genes in pbmcs based on the results obtained with known il-2 target genes, the criteria used to identify additional bona fide il-2-regulated genes in these samples were set at 2-fold change, with a subtracted difference in fluorescent intensity 100, and a significance level of the difference between the means of the two treatment groups of p 80-fold (tnf 36296_at). the subtractive differences in fluorescent intensity ranged from 114 20 (c - myb 1474_s_at) to > 1000 (cr7 1779_s_at). four of the genes (c - myb, cr7, flt3 ligand, tnf-) were detected with more than one probe set, and these replicates were in fairly good agreement. it is worthy of note that there is not a correlation between the subtracted difference and the fold change in gene expression levels. this is likely due to the fact that there is a wide range in the abundance classes of rna transcripts derived from different genes, from as few as 1 copy per cell to several thousand copies per cell. the signal intensity of a particular transcript detected on an oligonucleotide array would thus be directly related to this abundance level. therefore, the subtracted change in signal intensity would be profoundly affected by the absolute number of cellular copies of a given transcript. by comparison, based on the results obtained with known il-2 target genes, the criteria used to identify additional bona fide il-2-regulated genes in these samples were set at 2-fold change, with a subtracted difference in fluorescent intensity 100, and a significance level of the difference between the means of the two treatment groups of p < 0.05. of the 316 probe sets that met the standard of p < 0.05, 251 (79%) also met the additional criteria of either fold change 2, or subtracted difference 100, while only 84 probe sets (27%) three criteria. these 84 probe sets represent 72 unique genes (60 il-2-induced genes and 12 il-2-repressed genes) that can be segregated into 8 functional groups. figure 1 shows the fold induction and subtracted differences in expression levels of these probe sets. among the 4 apoptosis - regulating genes, bcl2, caspase 3 and drak2 it is noteworthy that the il-2 induction of bcl2 would be expected to promote cellular survival, while the il-2 regulation of the other three genes would be expected to promote apoptosis. additional il-2-responsive genes include those encoding 6 cell surface proteins, 11 cytokine and chemokine receptors, as well as 15 signal transduction mediators, 6 genes involved in cellular metabolism and biosynthesis, 3 cell cycle regulators and 12 transcription factors. il-2-suppressed genes include lymphocyte antigen 9, inositol 1,4,5-trisphosphate 3-kinase b, and cdc25b, as well as transcriptional regulators max - interacting protein 1, cbp / p300-interacting transactivator, inhibitor of dna binding 3 (id3), glucocorticoid - induced leucine zipper (gilz), and zinc finger protein 36/erf-2. all of the 19 already known il-2-regulated genes (table 2) were also identified in this 72-gene cohort, thereby yielding 53 newly identified il-2-regulated genes expressed by pbmcs after 4 hours of il-2 stimulation. pre - activated, rested human pbmcs were left untreated or restimulated for 4 hr with il-2, and rna probes were prepared for hybridization with affymetrix u95av2 arrays. a total of 84 probe sets were identified that showed fold change 2 (columns, left axis), subtracted difference 100 (diamonds, right axis), and a significance value of the difference between the means of p < 0.05. the probe sets represent 72 unique genes, and can be segregated into 8 functional groups. the pbmc populations used in our initial experiments included nk cells, monocytes and b cells, as well as t cells. in addition, cytokines including tnf, tnf and flt3 ligand were induced by il-2 as detected by the arrays, so that the il-2 effects could be mediated by the action of these intermediary cytokines on other cells in the mixed population, or as well, by the 12 transcriptional targets activated by il-2. therefore to identify the direct il-2 targets in t cells, we used cd56-depleted, cd2-selected t cells, which were 94 1% cd3cells. the pbmcs were pre - activated with anti - cd3 for 72 hr, the t cells purified, and rested for 3640 hr prior to restimulation for 4 hr with 1 nm il-2. in addition, 10 g / ml cycloheximide (chx) was included to block protein synthesis, so that only immediate / early il-2-regulated genes were detected. chx has been shown to both elevate and prolong the expression of many transiently induced transcripts, so that the 4 hr il-2 stimulation in the presence of chx should enable detection of genes induced within minutes as well as hours. samples from 4 independent experiments with pre - activated, rested cells, or cells treated with chx alone were combined for data analysis, and compared to cells treated with il-2 chx. as depicted in figure 2a, using only the criterion of significance level of p < 0.05 for the difference between the means of the two groups, 449 probe sets were identified as il-2-regulated immediate - early targets in the pure t cells. of these probe sets, 110 were also detected in the il-2-stimulated pbmcs. as shown in figure 2b, when a second standard was included, either a fold change 2, or a subtracted difference 100, 161 probe sets (36%) were expressed as immediately / early genes in purified t cells, and of these, 71 were also il-2-regulated in pbmcs. however, when all 3 criteria were applied (figure 2c), a total of only 52 probe sets (12%) were found to be immediate / early, il-2-regulated genes in t cells. twenty - three of these 52 probe sets were among the 84 probe sets identified previously as meeting all 3 criteria in the experiments with pbmcs (figure 3). by comparison, the control genes actin and gapdh showed 1.1 0.1 and 1.3 0.2-fold change, respectively. (a). using the criterion of p < 0.05 for the difference between the means of il-2-treated and untreated groups, a total of 316 il-2-sensitive probe sets were identified in pbmcs (left circle), and 439 in purified t cells (right circle). of these, 110 were identified in both cell populations (overlap between circles). (b). when a second standard of either fold change 2 or subtracted difference 100 was included in the analysis, 251 probe sets were identified in pbmcs, and 161 in pure t cells, with 71 in common. when all three criteria were used, 84 probe sets were identified in pbmcs, 52 in t cells, and 23 of these were common to both populations. within each population, a total of 23 probe sets three expression criteria of fold change 2 (columns, left axis), subtracted difference 100 (diamonds and circles, right axis), and p < 0.05 in both pbmcs and purified t cells. therefore, only ~1/3 of the genes discovered to be il-2-regulated in pbmcs are actually immediate / early direct il-2 targets in t cells. these direct il-2 immediate / early target genes included 4 genes that were suppressed, dsip / gilz, the zinc finger protein 36/erf-2/tis11d, toso, and cdc25b. notably, of the 16 il-2-induced genes, we had already been identified 4 previously as il-2 targets using differential colony hybridization (cr3, cr6, cr7, cr8), while the 3 other known il-2 stimulated target genes included tnf, the il-4r, and the anti - apoptotic gene, bcl2. thus, only 9 genes were newly identified as il-2-induced immediate / early t cell genes ; including the signaling proteins slp76 and rgs1, the transcription factors irf4 and xbp1, and 5 miscellaneous genes (dkfzp586f1323, mitofilin, camp - specific phosphodiesterase 4b, cmp - n - acetylneuraminate monooxygenase, and the aryl hydrocarbon receptor). in addition to the 23 probe sets in common with those identified in pbmcs, 29 others were detected as il-2-regulated immediate / early targets in the purified t cells (figure 2c). these probe sets three of the criteria of 2-fold change, subtracted difference 100, and p < 0.05 in t cells, but were not il-2-regulated in pbmcs. notably, only three of these were induced, while the remaining 24 were all suppressed in response to il-2 + chx. a total of 29 additional probe sets three expression criteria of fold change 2 (columns, left axis), subtracted difference 100 (diamonds and circles, right axis), and p < 0.05 in purified t cells, but did not three criteria in pbmcs. these probe sets represent 27 unique genes. of the remaining 61 probe sets identified in pbmcs (figure 2c), 31 showed changes in expression levels in t cells with p < 0.05, although either the fold change was less than 2.0, or the magnitude of the change was less than 100 (table 3). these 31 probe sets are derived from 27 unique genes, and most likely represent bona fide il-2 targets, since the group includes the genes jab / ssi-1/socs-1, flt3 ligand, il-2rp55, caspase 3, and p78, which have already been reported as il-2-induced genes. if these genes are also included, 47 of the 72 unique genes (65%) identified in pbmcs are il-2-regulated immediate / early genes in pure t cells. t cell expression levels of genes identified in pbmcs similarly, of the 29 probe sets identified as meeting all 3 criteria only in pure t cells, 7 showed differences in expression with p < 0.05 in pbmcs (table 4). these include linker for activation of t cells, t - cell - specific transcription factor 7, stat4, il-7r, pas ser / thr kinase, l - mfa domain - containing protein, and an unknown gene designated dkfzp586d1122. notably, all of the other genes (22) were suppressed by il-2 in the pbmcs, as they were in the t cells, although they did not meet the significance value of p < 0.05 in the pbmcs. lymphocyte activation is initiated by specific antigen / receptor interactions, and aided by accessory molecules. however subsequently, cytokine and cytokine receptor expression ensue, and the cytokines ultimately drive the clonal expansion and functional differentiation of the antigen - selected lymphocyte populations. il-2 was initially identified by virtue of its t cell growth factor activity, and il-2 is now recognized as the pivotal cytokine in promoting mature peripheral t cell proliferation. therefore, to initiate the identification of the genes responsible for lymphocyte activation, we chose first to delineate the genetic mechanisms of il-2 action. using oligonucleotide arrays of known genes to identify il-2-regulated genes in human lymphocytes, a total of 72 unique genes were found to change expression within 4 hr of il-2 stimulation of pbmcs. these genes could be segregated into functional groups, including cell surface proteins, cytokine and chemokine receptors, as well as signal transduction mediators, cytokines, genes involved in cellular metabolism and biosynthesis, cell cycle regulators and transcription factors. in order to define the criteria that we used to sort the microarray data in this series of experiments, we were able to take advantage of previous reports of 19 known il-2-regulated genes. by assessing the characteristics of the changes in expression of these known il-2 targets, we were able to define a range of both fold - induction and subtracted difference that encompassed all of these genes. when applied to the total data set, these criteria were likely to identify additional meaningful il-2-regulated genes. since 19 of these genes had already been reported to be il-2-induced genes, our dna array experiments identified 53 additional il-2-regulated genes expressed by pbmcs. however, only 20 of these genes were found to be il-2-regulated immediate / early t cell genes. it is important to discriminate these results from other analyses of t cell activation that have employed antigen receptor activation, as opposed to il-2. the tcr and il-2 receptors activate some shared signaling pathways, such as the ras - raf - map kinase and pi-3-kinase pathways. however, there are differences, such as the jak - stat pathway, which is unique to the cytokines, and the ca++/calcineurin pathway, which is activated by the tcr. these signaling pathways culminate in the activation of specific transcription factors, so that there must be both shared target genes, as well as genes unique to antigen or il-2 signaling. as tcr signaling induces the expression of il-2, as well as other cytokines and their receptors, the ultimate t cell response is an amalgam of both antigen- and cytokine receptor - triggered events. this point is well illustrated by the report of teague, who used microarrays to identify murine t cell genes induced in vivo by seb stimulation after 8 and 48 hours. again, cytokine and cytokine receptor genes were induced during these time intervals, so that the entire spectrum of genes identified included both antigen- and accessory molecule - activated, as well as cytokine - regulated targets. similarly, ellisen used dna arrays to monitor cona - induced genes in human t lymphocytes, over a time scale ranging from 4 to 48 hours. inducible genes included il-2 and tnf-, as well as the il-1r, il-2r and il-4r. thus, at the time points used in this study, many of the genes detected were likely regulated by these cytokines as well as con a. in a similar vein, herblot used a subtractive approach to identify 66 genes induced by il-2 but not il-4 in a murine t cell line. they further demonstrated that while in vitro, cona could induce expression of the il-2 target genes in t cells from il-2 + /+ mice, the genes were not induced by cona treatment of t cells from il-2-/- mice. thus, cona induces il-2 and il-2r expression, and the cytokine drives expression of additional specific target genes. recently, riley and diehn described genes regulated by the tcr and the costimulatory receptors cd28, icos and ctla-4, over a time course of 148 hours. once again, numerous cytokines, including il-2, are strongly induced within 2 hr, such that a number of the observed regulated genes are likely cytokine - responsive targets. finally, gonzalez have described murine genes induced after 8 hr il-2 stimulation, which likely include direct targets of il-2, as well as genes induced via il-2- regulated cytokines. therefore, to discriminate between the distinct genes induced by each ligand that contributes to the overall response initiated by antigenic stimulation, it is important to use cycloheximide to identify immediate / early genes regulated by the tcr and costimulatory receptors vs. il-2. in addition, the genes activated by tcr, costimulatory receptors and il-2 must be distinguished from those activated by il-1, il-6, il-12, and tnf-, as well as the genes regulated by the toll - like receptors (tlr), all of which have been identified as mediators of lymphocyte activation events that were previously ascribed to laf. moreover, as il-2 itself induces the expression of numerous cytokines and receptors, it is important to discriminate the direct il-2 targets from those induced secondarily through these intermediaries. thus, it is worthy of re - emphasis that of the 72 genes regulated by il-2 in pbmcs, only 20 were found to be bona fide il-2-regulated immediate / early genes in purified t cells. in addition to imparting positive activating signals that result in cell cycle progression, il-2 has also been found to promote negative feedback phenomena. thus, it is especially intriguing that il-2 suppresses the expression of many genes, both among pbmcs in the absence of chx, as well as in purified t cells in the presence of chx. the suppression of the expression of these genes may well be important in mediating the feedback down - regulatory phenomena. the high - affinity il-2 receptor is comprised of three subunits,, and. the chain is specific to il-2, while the chain is shared among il-4, il-7, il-9, il-15 and il-21. the chain of the il-2r is also a component of the il-15 receptor, in conjunction with a specific il-15r chain. the sharing of these receptors by multiple cytokines raises the question of how the ligands can trigger specific responses in the target cells. several signaling events, including the activation of jak kinases 1 and 3 are shared among the ' common chain ' cytokines. other signaling events, such as the activation of shc, gab2, shp-2 and mapk, are shared among il-2 and 15, but not the other common chain cytokine receptors, and tyr 388 of il-2r appears critical for these events. despite these signaling similarities, it is clear that the biological properties of il-2 are unique among the common chain cytokines, as the other cytokines can not compensate for il-2 in the il-2 knockout mouse. in addition, il-15 appears uniquely critical for the development of nk cells, as evidenced by the phenotypes of il-15 and il-15r deficient mice. thus, there must be a means whereby il-2 and il-15, as well as the other common chain cytokines, trigger distinct intracellular signaling pathways, and target genes. therefore, identification of each of the cytokine - specific target genes should shed light on the issue of specificity amongst the common chain cytokines. complete classification of the genes expressed as a consequence of distinct ligand / receptor interactions during lymphocyte activation will also provide a powerful tool for the assessment and treatment of immunological disorders. in addition to defining the target genes of specific cytokines and cell surface ligands, it is possible to further subdivide the genes based on the specific signaling proteins and transcription factors that govern their expression. for example, through the use of stat5-/- mice, ihle have been able to identify those il-2 target genes that require stat5. similar approaches with mutant signaling proteins, knockouts, or over - expression systems enable such dissection of the signal transduction pathways leading to specific target genes. thus, each ligand / receptor pair will ultimately be linked to a specific signature of target genes, derived from the signaling pathways and transcription factors activated. such information will be invaluable in the diagnosis and treatment of disorders involving aberrancies in these pathways. the rapidity with which lymphocyte activation genes may now be identified using the methods described in this report opens the way to a molecular genetic definition of the immune response, based upon the categorization of the genes regulated by distinct ligand receptor pairs. consequently, more precise genetic and molecular diagnoses of immune disorders will soon become available, and hopefully, new treatment regimes may be developed to target the specific molecular nature of each disease. human peripheral blood mononuclear cells (pbmc) were purified from venous blood of healthy donors by density centrifugation over histopaque 1.077 (sigma). cells were cultured in rpmi1640, 10%fcs, at a density of 1 10cells / ml, and activated with anti - cd3 (okt3, 1:500 ; ortho diagnostics). after 72 hr activation, pbmc were washed and replaced in culture without stimulation for 3640 hr prior to restimulation with 1 nm il-2. to purify t lymphocytes, 72 hr okt3-activated pbmc were depleted of cd56, and positively selected for cd2, using antibody - conjugated magnetic beads (anti - cd56 polysciences inc, anti - cd2 dynal) according to manufacturer 's instructions. subsequent to purification, t cells were replaced in culture for 3640 hr prior to restimulation with 1 nm il-2, and/or 10 g / ml cycloheximide. cell populations were stained with anti - cd3-apc, cd14-fitc, cd19-percp and cd56-pe (becton dickinson), and quantitated by flow cytometry (facscalibur, becton dickinson). five independent experiments were performed with pbmcs from different donors, and four independent experiments were carried out with purified t cells. briefly, total cellular rna was prepared with qiagen rneasy reagents, and 5 g samples were used to prepare t7-dt - primed double - stranded cdna (superscript choice, invitrogen). biotin - labeled crna was prepared by in vitro transcription (bioarray high - yield t7, enzo), and hybridization cocktails were prepared with 20 g crna per sample. hybridization, wash, and staining of affymetrix human u95av2 arrays were performed following manufacturer 's instructions. gene expression values were normalized to the average chip signal intensity for each experiment, and a 10percentile value cut - off was used for ' absent ' genes that did not give signals significantly greater than background. to calculate il-2-induced gene expression changes, the subtracted difference values were determined by subtracting the signal intensity of untreated samples from the values of il-2-treated samples. standard errors were derived from 5 independent pbmc experiments, and 4 experiments with purified t cells. the average of the standard errors relative to the mean expression values (se / mean signal intensity)100% was 22% for untreated and 15% for il-2-stimulated pbmcs, and 16% and 19% for the unstimulated and il-2-treated purified t cell samples, respectively. the p - values were determined by testing whether the absolute difference between treatment group means is not zero by unpaired t - test. human peripheral blood mononuclear cells (pbmc) were purified from venous blood of healthy donors by density centrifugation over histopaque 1.077 (sigma). cells were cultured in rpmi1640, 10%fcs, at a density of 1 10cells / ml, and activated with anti - cd3 (okt3, 1:500 ; ortho diagnostics). after 72 hr activation, pbmc were washed and replaced in culture without stimulation for 3640 hr prior to restimulation with 1 nm il-2. to purify t lymphocytes, 72 hr okt3-activated pbmc were depleted of cd56, and positively selected for cd2, using antibody - conjugated magnetic beads (anti - cd56 polysciences inc, anti - cd2 dynal) according to manufacturer 's instructions. subsequent to purification, t cells were replaced in culture for 3640 hr prior to restimulation with 1 nm il-2, and/or 10 g / ml cycloheximide. cell populations were stained with anti - cd3-apc, cd14-fitc, cd19-percp and cd56-pe (becton dickinson), and quantitated by flow cytometry (facscalibur, becton dickinson). five independent experiments were performed with pbmcs from different donors, and four independent experiments were carried out with purified t cells. briefly, total cellular rna was prepared with qiagen rneasy reagents, and 5 g samples were used to prepare t7-dt - primed double - stranded cdna (superscript choice, invitrogen). biotin - labeled crna was prepared by in vitro transcription (bioarray high - yield t7, enzo), and hybridization cocktails were prepared with 20 g crna per sample. hybridization, wash, and staining of affymetrix human u95av2 arrays were performed following manufacturer 's instructions. gene expression values were normalized to the average chip signal intensity for each experiment, and a 10percentile value cut - off was used for ' absent ' genes that did not give signals significantly greater than background. to calculate il-2-induced gene expression changes, the subtracted difference values were determined by subtracting the signal intensity of untreated samples from the values of il-2-treated samples. standard errors were derived from 5 independent pbmc experiments, and 4 experiments with purified t cells. the average of the standard errors relative to the mean expression values (se / mean signal intensity)100% was 22% for untreated and 15% for il-2-stimulated pbmcs, and 16% and 19% for the unstimulated and il-2-treated purified t cell samples, respectively. the p - values were determined by testing whether the absolute difference between treatment group means is not zero by unpaired t - test. the results reported here represent almost 20 years of effort. k.a.s. initiated the search for il-2-regulated genes in 1983, using subtractive hybridization, and reported the first il-2-regulated gene in 1986. extended these initial efforts and reported on the identification of 8 immediate / early il-2-induced genes in 1993. c.b. conceived and performed the experiments contained in this report, and wrote the manuscript. provided advice in experimental design and analysis of the results, and contributed to the revisions of the manuscript that eventually resulted in the final version. il-2 target genes in human pbmcs and t cells this file contains expression data for il-2-regulated probe sets detected on the affymetrix u95av2 array, using samples prepared from human pbmcs and purified t cells. this work was supported by national institutes of health niaid grants ro1 ai 32031 - 24 and ro1 ai 44207 - 02. | backgroundlymphocyte activation culminates in blastogenesis, cell cycle progression, dna replication and mitosis. these complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. until now, the discovery of the genes regulated by each ligand / receptor pair has been hampered by the technologies available.resultsto identify interleukin-2 (il-2)-responsive genes, human peripheral blood mononuclear cells (pbmc) were pre - activated with anti - cd3, rested, and restimulated with il-2 for 4 hr. gene expression was analyzed using affymetrix u95av2 oligonucleotide arrays. to determine the most stringent parameters to score a gene as a bona fide il-2 target, the expression of 19 known il-2-regulated genes was examined first. all were induced at least 2-fold, with a difference in fluorescent intensity of 100 at p < 0.05. an additional 53 unique genes met these criteria. to determine which of these were immediate / early il-2 targets in t cells, purified t cells were stimulated with il-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. of the 72 genes identified in pbmcs, 20 were detected as immediate / early il-2-regulated genes in purified t cells. in addition, 27 unique genes were il-2-regulated in t cells but not in pbmcs.conclusionsfor a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate / early gene expression regulated by each ligand / receptor pair involved. this approach should allow the discovery of genes regulated by all of the ligand / receptor pairs involved in lymphocyte activation. |
direct invasion or metastasis of malignant lymphoma in the middle ear is rare and the clinical features remain unknown [1, 2 ]. we treated a case of non - hodgkin b - cell lymphoma (nhbl) in the middle ear presenting as recalcitrant otitis media and mastoiditis associated with transverse sinus thrombosis. the patient was diagnosed 6 years after the initial symptoms and was treated successfully with surgery and chemotherapy. we report the clinical characteristics of a rare case of nhbl in the middle ear and review the literature. a 57-year - old woman presented with chronic temporal headache and right tinnitus, and the symptoms gradually aggravated. conservative treatment continued at the local otolaryngology clinic based on a diagnosis of otitis media. slight consciousness disturbance, slight fever and auditory disturbance were noted 1 year after the initial symptoms, and the patient was referred to our department. neurological findings showed slight consciousness disturbance, and an audiometric hearing test demonstrated 30 db in the right ear, diagnosed as a mild hearing impairment. magnetic resonance imaging (mri) showed edematous brain swelling in the right temporal lobe and right tentorium and dural enhancement around the transverse sinus (fig. mri showed a heterogeneous mass lesion in the right middle ear and mastoid air cells with dural enhancement around the lesion (fig. she was diagnosed with recalcitrant otitis media invading the mastoid associated with transverse sinus thrombosis and was treated with antibiotics, hyperosmotic fluids, steroids and repeated external drainage of the middle ear. symptoms of temporal headache, auditory disturbance, slight fever and radiological findings of brain swelling did not disappear, and remission and exacerbation of these symptoms occurred repeatedly. consciousness disturbance occurred again, and chronic temporal headache gradually aggravated 5 years after the first examination in our department. mri revealed enlargement of the edematous swelling around the right transverse sinus, and the enhanced lesion was also enlarged (fig. open drainage of the middle ear with mastoidectomy was performed 6 years after the initial symptoms. granulation had invaded the eardrum and stapes, and the auditory tube was also filled with granulation (fig. pathological examination demonstrated diffuse proliferation, predominantly consisting of small to medium - sized lymphoid cells with slightly irregular nuclei (fig. 1f), which were positive for cd20, cd79 and bcl-2, suggesting malignant lymphoma, b - cell type, classified as extranodal marginal zone b - cell lymphoma. computed tomography and blood and bone marrow evaluation did not show other lesions of malignant lymphoma, suggesting that the otitis media lesion was the primary lesion. she was treated with rchop (rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisolone) chemotherapy. malignant lymphoma can spread to all areas of the body, including the head and neck. involvement of the temporal bone as part of generalized lymphoma has been reported ; however, clinical evidence of temporal bone or middle ear involvement is unusual [3, 4, 5, 6 ]. presentation with otoneurological signs prior to systemic involvement of a lymphoproliferative disease is also unusual. to our knowledge, only 10 cases of malignant lymphoma originating around the middle ear have been reported (table 1) [1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ]. in the previous 10 reports, the lesion was located in the middle ear in 3, internal in 3, and external in 4. facial nerve palsy was the most common initial symptom and mastoiditis was associated in about 50% of the cases. there was no direct invasion into the intradural region and no association with sinus thrombosis in 10 cases. our present case included the following marked characteristics : first, it was difficult to diagnose malignant lymphoma under the suspicion of intractable otitis media. second, transverse sinus thrombosis caused by dural invasion of a tumor or spread of otogenic infection manifested neurological symptoms. third, tentorial or dural enhancement adjacent to the transverse sinus did not decrease, and sinus occlusion continued even after remission. fourth, rchop chemotherapy was effective, and the lesion has not recurred for 4 years. infectious symptoms and dural enhancement showed repeated remission and abrogation under treatment for otitis media with antibiotic administration and drainage. there was a possibility that sinus occlusion induced by infection progression might abrogate venous circulation, resulting in brain edema in the early stage, and collateral venous flow might gradually develop during the progression of sinus occlusion. we treated a rare case of b - cell malignant lymphoma presenting with otitis media and mastoiditis associated with transverse sinus thrombosis. the authors report no conflicts of interest concerning the case report presented herein or the findings specified in this paper. | cerebral venous thrombosis as a manifestation of paraneoplastic angitis and otitis media, revealing non - hodgkin b - cell lymphoma (nhbl), is extremely rare. a 57-year - old woman presented with headache, auditory disturbance and recalcitrant otitis media. magnetic resonance imaging showed brain edema in the temporal lobe and transverse sinus thrombosis. external drainage under antibiotic treatment was repeated based on a diagnosis of invasive otitis media and mastoiditis associated with infectious sinus thrombosis, but the condition deteriorated progressively. open surgery for otitis media was performed 6 years after the initial symptoms and after a tumorous lesion had been detected in the middle ear. pathological findings revealed nhbl. we report a rare case of nhbl presenting as otitis media and mastoiditis associated with sinus thrombosis, and a literature review. |
patients aged 12 years with asthma were eligible if they could demonstrate a 12% and 200-ml reversibility of fev1 following albuterol inhalation at screening and had a best evening fev1 of 40% to 85% of the predicted normal value at screening and at randomization. before screening, patients had been taking ics for 12 weeks, with a stable medium dose of ics (fp 250 g bid or equivalent) for 4 weeks. the study was approved by the local ethics review committees (e - table 1) and was conducted in accordance with the declaration of helsinki and good clinical practice guidelines. this phase 3, multicenter, randomized, double - blind, double - dummy, parallel group study was conducted between june 16, 2010, and july 27, 2011. after screening, eligible patients entered a 4-week run - in period during which they replaced their current asthma medication with fp 250 g bid, and their short - acting bronchodilator with albuterol delivered through a metered - dose inhaler. after the run - in period, patients received (1:1) either ff / vi 100/25 g (emitted dose, 92/22 g) once daily in the evening, administered through an ellipta dry powder inhaler (glaxosmithkline), or fp / sal 250/50 g bid (morning and evening) through diskus / accuhaler (glaxosmithkline) for 24 weeks. the central randomization schedule was generated by the sponsor with a validated computerized system (randall ; glaxosmithkline). compliance with study medication was measured by reviewing the dose counter on the dry powder inhaler and diskus / accuhaler. the primary end point was change from baseline in 0- to 24-h serial weighted mean (wm) fev1 after 24 weeks of treatment. secondary end points were individual serial fev1 assessments at week 24 (fev1 at each time point on day 168), time to onset of bronchodilator effect (the time point when fev1 first exceeded the 12% and 200-ml increase over baseline [taken from serial measurements ]) at randomization visit only, 0- to 4-h serial wmfev1 postdose at the randomization visit and at week 24, percentage of patients experiencing a 12% and 200-ml increase from baseline in fev1 at 12 and 24 h at week 24, and change from baseline in clinic visit trough (prebronchodilator and predose) fev1 at week 24. other efficacy end points were scores on the asthma quality of life + 12 questionnaire (aqlq+12), the asthma control test, and the european quality of life-5 dimensions (eq-5d) test, and unscheduled health - care resource utilization. a post hoc analysis of the individual aqlq+12 domains and minimally important difference (0.5-point improvement in aqlq+12 score) was also performed. other safety assessments were 24-h urinary cortisol (uc) excretion at baseline and at the end of the 24-week treatment period (subset of patients) ; vital signs (diastolic and systolic bp, pulse rate) ; incidence of severe asthma exacerbations ; liver safety ; and ecg, clinical chemistry, and hematology screening assessments. patients were withdrawn from the study because of lack of efficacy if they experienced a severe exacerbation or worsening of asthma or at the investigator s discretion. a severe exacerbation was defined as deterioration of asthma requiring treatment with systemic or oral corticosteroids for 3 days or an inpatient hospitalization or ed visit that required systemic corticosteroids. worsening asthma was defined as a requirement for any treatment other than study medication or use of rescue albuterol. severe asthma exacerbations were not recorded as an ae unless they met the definition of a serious adverse event (sae). sample size calculations were based on the primary end point of 0- to 24-h serial wmfev1 at the end of the 24-week treatment period for the treatment comparison of ff / vi 100/25 g once daily vs fp / sal 250/50 g bid. it was estimated that approximately 348 patients with evaluable data per treatment group would provide 90% power to detect a difference of 80 ml between ff / vi 100/25 g and fp / sal 250/50 g ; this assumed an sd of 325 ml. details about the analysis populations and power calculation are provided in e - appendix 4. the primary analysis was performed on the intention - to - treat population with an analysis of covariance model allowing for the effects of baseline fev1, region, sex, age, and treatment group. a two - sided 5% risk (significance level) associated with incorrectly rejecting the null hypothesis was considered acceptable for this study. for the secondary and other efficacy end points, if the statistical test for the primary end point failed to reject the null hypothesis of no treatment difference at the 0.05 significance level, the tests for the secondary and other efficacy end points were to be interpreted as descriptive only. as a consequence of site audits conducted by the study sponsor glaxosmithkline, concerns about the quality of data supplied were identified for two study centers. these centers contributed 57 patients (7% of enrolled patients) to the study. because of these concerns, analysis of the primary end point was repeated, excluding the data from these two centers. the authors and study sponsor are satisfied that the exclusion of data derived from these centers did not materially change the findings of the study ; therefore, we present a complete data set from all the centers. patients aged 12 years with asthma were eligible if they could demonstrate a 12% and 200-ml reversibility of fev1 following albuterol inhalation at screening and had a best evening fev1 of 40% to 85% of the predicted normal value at screening and at randomization. before screening, patients had been taking ics for 12 weeks, with a stable medium dose of ics (fp 250 g bid or equivalent) for 4 weeks. the study was approved by the local ethics review committees (e - table 1) and was conducted in accordance with the declaration of helsinki and good clinical practice guidelines. this phase 3, multicenter, randomized, double - blind, double - dummy, parallel group study was conducted between june 16, 2010, and july 27, 2011. after screening, eligible patients entered a 4-week run - in period during which they replaced their current asthma medication with fp 250 g bid, and their short - acting bronchodilator with albuterol delivered through a metered - dose inhaler. after the run - in period, patients received (1:1) either ff / vi 100/25 g (emitted dose, 92/22 g) once daily in the evening, administered through an ellipta dry powder inhaler (glaxosmithkline), or fp / sal 250/50 g bid (morning and evening) through diskus / accuhaler (glaxosmithkline) for 24 weeks. the central randomization schedule was generated by the sponsor with a validated computerized system (randall ; glaxosmithkline). compliance with study medication was measured by reviewing the dose counter on the dry powder inhaler and diskus / accuhaler. the primary end point was change from baseline in 0- to 24-h serial weighted mean (wm) fev1 after 24 weeks of treatment. secondary end points were individual serial fev1 assessments at week 24 (fev1 at each time point on day 168), time to onset of bronchodilator effect (the time point when fev1 first exceeded the 12% and 200-ml increase over baseline [taken from serial measurements ]) at randomization visit only, 0- to 4-h serial wmfev1 postdose at the randomization visit and at week 24, percentage of patients experiencing a 12% and 200-ml increase from baseline in fev1 at 12 and 24 h at week 24, and change from baseline in clinic visit trough (prebronchodilator and predose) fev1 at week 24. other efficacy end points were scores on the asthma quality of life + 12 questionnaire (aqlq+12), the asthma control test, and the european quality of life-5 dimensions (eq-5d) test, and unscheduled health - care resource utilization. patients were asked to complete questionnaires at baseline and end of treatment. a post hoc analysis of the individual aqlq+12 domains and minimally important difference (0.5-point improvement in aqlq+12 score) was also performed. other safety assessments were 24-h urinary cortisol (uc) excretion at baseline and at the end of the 24-week treatment period (subset of patients) ; vital signs (diastolic and systolic bp, pulse rate) ; incidence of severe asthma exacerbations ; liver safety ; and ecg, clinical chemistry, and hematology screening assessments. patients were withdrawn from the study because of lack of efficacy if they experienced a severe exacerbation or worsening of asthma or at the investigator s discretion. a severe exacerbation was defined as deterioration of asthma requiring treatment with systemic or oral corticosteroids for 3 days or an inpatient hospitalization or ed visit that required systemic corticosteroids. worsening asthma was defined as a requirement for any treatment other than study medication or use of rescue albuterol. severe asthma exacerbations were not recorded as an ae unless they met the definition of a serious adverse event (sae). sample size calculations were based on the primary end point of 0- to 24-h serial wmfev1 at the end of the 24-week treatment period for the treatment comparison of ff / vi 100/25 g once daily vs fp / sal 250/50 g bid. it was estimated that approximately 348 patients with evaluable data per treatment group would provide 90% power to detect a difference of 80 ml between ff / vi 100/25 g and fp / sal 250/50 g ; this assumed an sd of 325 ml. details about the analysis populations and power calculation are provided in e - appendix 4. the primary analysis was performed on the intention - to - treat population with an analysis of covariance model allowing for the effects of baseline fev1, region, sex, age, and treatment group. a two - sided 5% risk (significance level) associated with incorrectly rejecting the null hypothesis was considered acceptable for this study. for the secondary and other efficacy end points, if the statistical test for the primary end point failed to reject the null hypothesis of no treatment difference at the 0.05 significance level, the tests for the secondary and other efficacy end points were to be interpreted as descriptive only. as a consequence of site audits conducted by the study sponsor glaxosmithkline, concerns about the quality of data supplied were identified for two study centers. these centers contributed 57 patients (7% of enrolled patients) to the study. because of these concerns, analysis of the primary end point was repeated, excluding the data from these two centers. the authors and study sponsor are satisfied that the exclusion of data derived from these centers did not materially change the findings of the study ; therefore, we present a complete data set from all the centers. most patients (89%) in both treatment groups completed the 24-week treatment period (fig 1). ae = adverse event ; bd = twice daily ; ff = fluticasone furoate ; fp = fluticasone propionate ; itt = intention to treat ; od = once daily ; sal = salmeterol ; vi = vilanterol. main reason was that patients did not meet inclusion / exclusion criteria (n = 613 [39% ]). main reason was that patients did not meet continuation criteria (n = 103 [7% ]). patient demographics and baseline lung function (intention - to - treat population) data are presented as mean sd or no. ff = fluticasone furoate ; fp = fluticasone propionate ; ics = inhaled corticosteroid ; laba = long - acting 2-agonist ; od = once daily ; sal = salmeterol ; vi = vilanterol. patients needed to have been maintained on a medium dose of ics (eg, fp 250 g bid) for 4 wk before screening. improvements from baseline in 0- to 24-h serial wmfev1 were seen with both ff / vi (341 ml) and fp / sal (377 ml) ; however, the adjusted mean treatment difference was not statistically significant (37 ml ; 95% ci, 88 to 15 ml ; p =.162) (fig 2). there were no differences in key secondary end points, including the change from baseline in individual serial fev1 assessments over time at week 24. these showed sustained 24-h duration of action for both once - daily ff / vi and bid fp / sal at all time points (fig 3). the adjusted mean treatment differences between ff / vi and fp / sal ranged from 2 to 58 ml. no important differences were seen between ff / vi vs fp / sal for the secondary end points of time to onset of bronchodilator effect, 0- to 4-h serial wmfev1 postdose at randomization and at week 24, percentage of patients obtaining a 12% and 200-ml increase from baseline in fev1 at 12 and 24 h at week 24, and change from baseline in clinic visit trough (prebronchodilator and predose) fev1 at week 24 (table 2). change from baseline in predose fev1 over the 24-week treatment period is shown in e - figure 1. adjusted means for 0- to 24-h serial weighted mean fev1 at week 24 (intention - to - treat population). see figure 1 legend for expansion of other abbreviations. adjusted mean change from baseline in fev1 over time at week 24 for ff / vi dosed od in the evening and fp / sal dosed bd (intention - to - treat population). secondary end points data are presented as least squares mean se change from baseline unless otherwise indicated. intention - to - treat population, n = 403 (ff / vi 100/25 g od pm) and n = 403 (fp / sal 250 g bid). hr = hazard ratio ; wm = weighted mean. see table 1 legend for expansion of other abbreviations. results are for all secondary end points other than the end point individual serial fev1 assessments at week 24 (see fig 3). improvements from baseline in the aqlq+12, asthma control test, and eq-5d asthma health outcomes assessments were observed for both ff / vi and fp / sal. health outcomes assessments intention - to - treat population, n = 403 (ff / vi 100/25 g od pm) and n = 403 (fp / sal 250 g bid). act = asthma control test ; aqlq+12 = asthma quality of life + 12 questionnaire ; eq-5d = european quality of life-5 dimensions ; n / a = not applicable ; vas = visual analog scale. aqlq+12, act, and eq-5d vas score, analysis of covariance was used, with covariates of baseline score, country, sex, age, and treatment ; eq-5d, logistic regression analysis was used, with covariates of baseline, region, sex, age, and treatment. the overall incidence of aes and saes reported while on treatment are presented in table 4. the most frequently occurring aes reported by both groups were nasopharyngitis and headache ; treatment - related aes were similar between groups (table 4). nine patients reported on - treatment saes (four receiving ff / vi and five receiving fp / sal) (table 4). saes were single events with the exception of asthma exacerbation and pneumonia (one pretreatment and one on treatment [fp / sal ]). on - treatment aes 3% in any treatment group, all saes, and treatment - related aes (intention - to - treat population) data are presented as no. ae = adverse event ; sae = serious adverse event ; urti = upper respiratory tract infection. there was no statistically significant difference between ff / vi and fp / sal in 24-h uc excretion at week 24 (adjusted treatment ratio, 0.85 ; 95% ci, 0.72 - 1.02 ; p =.075) (fig 4). however, 24-h uc excretion did increase in both groups from baseline to week 24 (ratio to baseline, 1.11 and 1.21 for ff / vi and fp / sal, respectively). adjusted ratios to baseline for 24-h urinary cortisol excretion at week 24 (urinary cortisol population). the incidence of asthma exacerbations was low, and there was no difference between groups (3% vs 2% on fp / sal vs ff / vi, respectively [on - treatment events ]). eight (2%) patients in the ff / vi group and seven (2%) in the fp / sal group withdrew because of their exacerbation. one patient in the ff / vi group and two in the fp / sal group were hospitalized because of their exacerbations. no clinically relevant treatment effects on vital signs (e - appendix 5) or liver function parameters were found, with the exception of one patient in the fp / sal group who met liver stopping criteria of alanine aminotransferase levels more than eight times the upper limit of normal, which was reported as an ae but was not considered treatment related. most patients (89%) in both treatment groups completed the 24-week treatment period (fig 1). ae = adverse event ; bd = twice daily ; ff = fluticasone furoate ; fp = fluticasone propionate ; itt = intention to treat ; od = once daily ; sal = salmeterol ; vi = vilanterol. main reason was that patients did not meet inclusion / exclusion criteria (n = 613 [39% ]). main reason was that patients did not meet continuation criteria (n = 103 [7% ]). patient demographics and baseline lung function (intention - to - treat population) data are presented as mean sd or no. ff = fluticasone furoate ; fp = fluticasone propionate ; ics = inhaled corticosteroid ; laba = long - acting 2-agonist ; od = once daily ; sal = salmeterol ; vi = vilanterol. patients needed to have been maintained on a medium dose of ics (eg, fp 250 g bid) for 4 wk before screening. improvements from baseline in 0- to 24-h serial wmfev1 were seen with both ff / vi (341 ml) and fp / sal (377 ml) ; however, the adjusted mean treatment difference was not statistically significant (37 ml ; 95% ci, 88 to 15 ml ; p =.162) (fig 2). there were no differences in key secondary end points, including the change from baseline in individual serial fev1 assessments over time at week 24. these showed sustained 24-h duration of action for both once - daily ff / vi and bid fp / sal at all time points (fig 3). the adjusted mean treatment differences between ff / vi and fp / sal ranged from 2 to 58 ml. no important differences were seen between ff / vi vs fp / sal for the secondary end points of time to onset of bronchodilator effect, 0- to 4-h serial wmfev1 postdose at randomization and at week 24, percentage of patients obtaining a 12% and 200-ml increase from baseline in fev1 at 12 and 24 h at week 24, and change from baseline in clinic visit trough (prebronchodilator and predose) fev1 at week 24 (table 2). change from baseline in predose fev1 over the 24-week treatment period is shown in e - figure 1. adjusted means for 0- to 24-h serial weighted mean fev1 at week 24 (intention - to - treat population). see figure 1 legend for expansion of other abbreviations. adjusted mean change from baseline in fev1 over time at week 24 for ff / vi dosed od in the evening and fp / sal dosed bd (intention - to - treat population). secondary end points data are presented as least squares mean se change from baseline unless otherwise indicated. n = 403 (ff / vi 100/25 g od pm) and n = 403 (fp / sal 250 g bid). hr = hazard ratio ; wm = weighted mean. see table 1 legend for expansion of other abbreviations. results are for all secondary end points other than the end point individual serial fev1 assessments at week 24 (see fig 3). improvements from baseline in 0- to 24-h serial wmfev1 were seen with both ff / vi (341 ml) and fp / sal (377 ml) ; however, the adjusted mean treatment difference was not statistically significant (37 ml ; 95% ci, 88 to 15 ml ; p =.162) (fig 2). there were no differences in key secondary end points, including the change from baseline in individual serial fev1 assessments over time at week 24. these showed sustained 24-h duration of action for both once - daily ff / vi and bid fp / sal at all time points (fig 3). the adjusted mean treatment differences between ff / vi and fp / sal ranged from 2 to 58 ml. no important differences were seen between ff / vi vs fp / sal for the secondary end points of time to onset of bronchodilator effect, 0- to 4-h serial wmfev1 postdose at randomization and at week 24, percentage of patients obtaining a 12% and 200-ml increase from baseline in fev1 at 12 and 24 h at week 24, and change from baseline in clinic visit trough (prebronchodilator and predose) fev1 at week 24 (table 2). change from baseline in predose fev1 over the 24-week treatment period is shown in e - figure 1. adjusted means for 0- to 24-h serial weighted mean fev1 at week 24 (intention - to - treat population). see figure 1 legend for expansion of other abbreviations. adjusted mean change from baseline in fev1 over time at week 24 for ff / vi dosed od in the evening and fp / sal dosed bd (intention - to - treat population). secondary end points data are presented as least squares mean se change from baseline unless otherwise indicated. n = 403 (ff / vi 100/25 g od pm) and n = 403 (fp / sal 250 g bid). hr = hazard ratio ; wm = weighted mean. see table 1 legend for expansion of other abbreviations. results are for all secondary end points other than the end point individual serial fev1 assessments at week 24 (see fig 3). improvements from baseline in the aqlq+12, asthma control test, and eq-5d asthma health outcomes assessments were observed for both ff / vi and fp / sal. health outcomes assessments intention - to - treat population, n = 403 (ff / vi 100/25 g od pm) and n = 403 (fp / sal 250 g bid). act = asthma control test ; aqlq+12 = asthma quality of life + 12 questionnaire ; eq-5d = european quality of life-5 dimensions ; n / a = not applicable ; vas = visual analog scale. see table 1 legend for expansion of other abbreviations. aqlq+12, act, and eq-5d vas score, analysis of covariance was used, with covariates of baseline score, country, sex, age, and treatment ; eq-5d, logistic regression analysis was used, with covariates of baseline, region, sex, age, and treatment. the overall incidence of aes and saes reported while on treatment are presented in table 4. the most frequently occurring aes reported by both groups were nasopharyngitis and headache ; treatment - related aes were similar between groups (table 4). nine patients reported on - treatment saes (four receiving ff / vi and five receiving fp / sal) (table 4). saes were single events with the exception of asthma exacerbation and pneumonia (one pretreatment and one on treatment [fp / sal ]). on - treatment aes 3% in any treatment group, all saes, and treatment - related aes (intention - to - treat population) data are presented as no. (%). ae = adverse event ; sae = serious adverse event ; urti = upper respiratory tract infection. see table 1 legend for expansion of other abbreviations. there was no statistically significant difference between ff / vi and fp / sal in 24-h uc excretion at week 24 (adjusted treatment ratio, 0.85 ; 95% ci, 0.72 - 1.02 ; p =.075) (fig 4). however, 24-h uc excretion did increase in both groups from baseline to week 24 (ratio to baseline, 1.11 and 1.21 for ff / vi and fp / sal, respectively). adjusted ratios to baseline for 24-h urinary cortisol excretion at week 24 (urinary cortisol population). see figure 1 and 2 legends for expansion of abbreviations. the incidence of asthma exacerbations was low, and there was no difference between groups (3% vs 2% on fp / sal vs ff / vi, respectively [on - treatment events ]). eight (2%) patients in the ff / vi group and seven (2%) in the fp / sal group withdrew because of their exacerbation. one patient in the ff / vi group and two in the fp / sal group were hospitalized because of their exacerbations. no clinically relevant treatment effects on vital signs (e - appendix 5) or liver function parameters were found, with the exception of one patient in the fp / sal group who met liver stopping criteria of alanine aminotransferase levels more than eight times the upper limit of normal, which was reported as an ae but was not considered treatment related. in this study of patients aged 12 years with persistent asthma uncontrolled on a medium dose of ics, ff / vi 100/25 g once daily in the evening was not significantly different from fp / sal 250/50 g bid for the primary end point of 0- to 24-h serial wmfev1 at week 24 or for any of the secondary end points. the ae profiles for both treatments were similar, and those aes reported with fp / sal were consistent with those previously reported with this drug combination. according to current asthma guidelines, these patients would require a step up in their asthma treatment from step 3 to 4. lack of asthma control in the patient population at baseline was evident by an fev1 of approximately 68% of the predicted normal value for both groups and by the fact that 50% of patients had an asthma control test score of 94% in both groups), although the artificial setting of a clinical trial may have encouraged normally nonadherers to take their medication. if poor adherence to the dosing schedule is an important factor in loss of asthma control, then once - daily dosing (as opposed to bid dosing) such as that provided by ff / vi may represent a more - convenient treatment option for patients in a real - world setting. no differences in asthma control test scores or in measures of quality of life (aqlq+12 and eq-5d) were shown in the present study. a difference of 1.4 was observed between groups on the eq-5d test, but this difference was not statistically significant. the outcomes of the post hoc analysis (e - appendix 3) suggest that ff / vi may provide clinically relevant improvements in certain patient - reported quality - of - life measures (environmental domain of the aqlq+12 in the present study), as well as in the percentage of patients who improved by at least the minimally important difference of 0.5, that were not reported with fp / sal. however, this statement is speculative and requires further evaluation in real - world studies outside the environment of a clinical trial. ff / vi and fp / sal were well tolerated, and ff / vi had a similar ae profile to fp / sal. the ae profile with ff / vi reported in the present study is consistent with that seen in the two ff dose - ranging studies that included the 100 g once - daily dose and the two studies of vi 25 g plus concurrent ics. most of the saes reported in the present study were single events, and none were considered treatment related. asthma exacerbations requiring hospitalization occurred in one patient receiving ff / vi and two patients receiving fp / sal. a potential effect of ics treatment is reduction in uc levels as a result of suppression of the hypothalamic - pituitary - adrenal axis, although this usually only occurs at higher doses of ics. in earlier phase dose - ranging studies, once - daily ff 100 or 200 g did not significantly inhibit 24-h uc levels relative to placebo. in the current study, clinically insignificant increases in uc levels from baseline were reported, and no statistically significant differences were observed between the groups at week 24. labas have occasional side effects, such as headache, tremor, and hypertension. in the present study, no clinically relevant effects of ff / vi or fp / sal were found in measures of bp or pulse rate. it may be considered a limitation that the study duration was too short to reliably collect information on important end points such as exacerbations. also, while a once - daily dosing schedule could provide greater convenience to the patient and may therefore increase compliance to treatment, the double - blind, double - dummy nature of the study, together with the very high compliance levels during the study, means that the full benefit of once - daily dosing compared with bid dosing could not be realized in this study. this can only be investigated in a real - world effectiveness study, such as the salford lung study, which is now under way. it is possible that prestudy ics adherence was lower than anticipated by the protocol, as suggested by the high degree of fev1 reversibility seen at screening. once - daily ff / vi had similar treatment effects to bid fp / sal in improving lung function and health status in this double - blind randomized study. it remains to be seen whether it will be superior in a real - world setting. in conclusion, there was no difference in efficacy or safety between ff / vi 100/25 g administered once daily in the evening and fp / sal 250/50 g administered bid. | background : the combination of fluticasone furoate (ff), a novel inhaled corticosteroid (ics), and vilanterol (vi), a long - acting 2 agonist, is under development as a once - daily treatment of asthma and copd. the aim of this study was to compare the efficacy of ff / vi with fluticasone propionate (fp)/salmeterol (sal) in patients with persistent asthma uncontrolled on a medium dose of ics.methods:in a randomized, double - blind, double - dummy, parallel group study, 806 patients received ff / vi (100/25 g, n = 403) once daily in the evening delivered through ellipta (glaxosmithkline) dry powder inhaler, or fp / sal (250/50 g, n = 403) bid through diskus / accuhaler (glaxosmithkline). the primary efficacy measure was 0- to 24-h serial weighted mean (wm) fev1 after 24 weeks of treatment.results:improvements from baseline in 0- to 24-h wmfev1 were observed with both ff / vi (341 ml) and fp / sal (377 ml) ; the adjusted mean treatment difference was not statistically significant (37 ml ; 95% ci, 88 to 15, p = 0.162). there were no differences between 0- to 4-h serial wmfev1, trough fev1, and asthma control and quality - of - life questionnaire scores. there was no difference in reported exacerbations between treatments. both treatments were well tolerated, with no clinically relevant effect on urinary cortisol excretion or vital signs and no treatment - related serious adverse events.conclusions:the efficacy of once - daily ff / vi was similar to bid fp / sal in improving lung function in patients with persistent asthma. no safety issues were identified.trial registry : clinicaltrials.gov ; no. : nct01147848 ; url : www.clinicaltrials.gov |
hyperinsulinemia and elevated circulation angiotensin ii (ang ii) level tend to concomitantly occur in obesity patients and contribute to obesity - related hypertension. in recent years, a number of studies found a cross talk, at multiple levels, between ang ii and insulin [26 ]. several studies showed that ang ii could negatively modulate insulin - mediated actions [24 ]. at the intracellular level, ang ii was found to work on jak-2/irs1-irs2/pi3 kinase, jnk, and erk via ang ii receptor type 1 (at1r), to phosphorylate serine residues of key components of insulin signaling pathway, that is, the insulin receptors, irs1, and the p85 subunit of pi3 kinase, thereby inhibiting pi3 kinase / akt signaling pathway. in addition, by inducing expression of the regulatory protein socs 3, ang ii may inhibit insulin - induced tyrosine phosphorylation of irs1 and irs2 and [ser473 ] phosphorylation of akt, as a consequence, impairing the transduction of insulin signals in the jak2/stat-5b pathway [5, 6 ]. it is generally believed that ang ii acts on insulin predominantly by inhibiting pi3 kinase / akt pathway. nonetheless, researchers do not entirely agree about the role of the map kinase pathway in the cross talk between ang ii and insulin. mayer and colleagues found that, after treatment with ang ii, the phospho - erk1/2 activity at the hypothalamic level was significantly higher in rats pretreated with insulin than in those treated with insulin or ang ii alone. studies by carvalheira. also suggested a direct and positive cross talk between ang ii and insulin in erk pathway in cardiac tissues. on the other hand, other studies revealed a competitive cross talk between ang ii and insulin - mediated erk pathways. in the epithelial cells of renal proximal tubules, insulin - mediated erk activation was found to be suppressed by ang ii, although the two hormones, when working separately, augmented erk1/2-type kinase activity. similarly, a study observed that, in at1ar - ok cells (ok cells that stably express transfected at1ar), insulin suppressed ang ii - mediated erk phosphorylation. the cross talk between ang ii and insulin has been described mainly in cardiovascular cells, hepatocytes, adipocytes, skeletal muscles, and so forth, and, so far, no such cross talk was reported in adrenal. ang ii, insulin, and insulin - like growth factor 1 (igf-1) were shown to play important roles in adrenocortical cells [1113 ], and overexpression of igf-1 receptor was found to be associated with the development of adrenocortical carcinoma [1417 ]. in this study, by employing adrenocortical carcinoma h295r cells [1822 ], we examined the interaction between ang ii and insulin / igf-1 in erk and akt signaling pathways and expression of steroidogenic enzymes in the cells. human recombinant ang ii, insulin, igf-1, and at2 receptor blocker pd123319 were procured from sigma - aldrich (st. louis, mo, usa). pkc inhibitor g6983, pi3k inhibitor ly294202, and mek1/2 inhibitor u0126 were purchased from calbiochem (san diego, ca, usa). at1 receptor blocker candesartan was from astrazeneca. neutralizing anti - igfr1 antibody, mab 391, was bought from r & d systems (minneapolis, mn, usa). dulbecco 's modified eagle 's medium / f12 (dmem / f12) was from life technologies (carlsbad, ca, usa). nu serumtm and its+premix were obtained from bd biosciences (bedford, ma, usa). anti - phospho - erk1/2 (thr202/tyr204), anti - phospho - akt (ser473), anti - erk1/2, and anti - akt antibodies were from cell signaling technology (danvers, ma, usa). secondary antibodies [irdye 800cw conjugated goat (polyclonal) anti - mouse igg and irdye 680 conjugated goat (polyclonal) anti - rabbit igg ] were products of li - cor biosciences (lincoln, ne, usa). nci - h295r cells, a human adrenocortical cell line nci - h295r (atcc, rocksville, md, usa), were cultured in dmem / f12, containing 2.5% nu serum, 1% its plus premix [containing insulin (6.25 g / ml), transferrin (6.25 g / ml), selenium (6.25 ng / ml), bsa (1.25 mg / ml), and linoleic acid (5.35 g / ml) ], 50 units / ml penicillin, and 50 g / ml streptomycin, at 37c in an incubator supplied with humidified air. the cells were passaged by digestion with trypsin - edta (0.25% trypsin and 0.02% edta in pbs without ca and mg). for each experiment, the cells were cultured in six - well plates with the aforementioned growth medium. at 6070% confluence of the cells, the culture medium was replaced by serum - free dmem / f12 at 37c for 24 h before specific stimulation. after the stimulation for indicated times, the media were aspirated, and cells were washed three times with ice - cold pbs and then lysed in 100 l of laemmli sample buffer. after sonication, centrifugation, and heating at 95c for 5 min, the supernatant proteins were analyzed on sds - page (616%) gradient gels and transferred to polyvinylidene difluoride membranes. blots were blocked for 1 h in 5% fat - free milk and incubated overnight at 4c with primary antibodies. after washing three times with pbs containing 0.1% tween 20 for 5 min, blots were incubated with secondary antibodies at room temperature, with gentle shaking, for 1 h, and then washed three times with pbs containing 0.1% tween 20. immunoblots were densitometrically analyzed on an odyssey infrared imager (li - cor biosciences, lincoln, usa). cells were cultured in 24-well plates with growth medium for 1 to 2 days until they became subconfluent. then the medium was replaced with serum - free dmem / f12 and the cells were treated with different agonists. after 24 hours, the cells were lysed with trizol reagent, and total rna was isolated. cdna was synthesized from 1 g total rna by using a high - capacity cdna reverse transcription kit. cyp11b1 and cyp11b2 genes were pcr amplified by using the lightcycler 480 sybr green i master on a lightcycler 480 pcr system. the relative expression of each gene was quantitatively determined by normalizing against the expression of gapdh gene. the primers used were as follows : cyp11b1, 5-aatgcggaactgtcgccagatg-3 (forward) and 5-tcagcaagggaaacaccgtc-3 (reverse) ; cyp11b2, 5-actcgctgggtcgcaatg-3 (forward) and 5-agtgtctccaccaggaagtgc-3 (reverse) ; and gapdh, 5-cggagtcaacggatttggtc-3 (forward) and 5-tgggtggaatcatattggaacat-3 (reverse). all values were expressed as mean sd, and anova with a post hoc test was conducted for data comparison between groups by using spss software package (version 13.0). stimulation of h295r cells with ang ii for 5 min caused a dose - dependent phosphorylation of erk1/2, with the maximum effect occurring at the concentration of 100 nm and 1000 nm (figure 1). both 100 nm insulin and 1000 nm insulin could induce akt phosphorylation whereas only at the highest dose (10 nm) igf-1 induced akt phosphorylation (figures 2(a) and 2(b)). compared to the control, 100 nm ang ii increased phospho - erk1/2 approximately 3-fold. insulin (100 nm) or igf-1 (10 nm) alone raised phospho - erk1/2 1.8- and 1.5-fold, respectively, while, after pretreatment with 100 nm ang ii for 30 min, insulin (100 nm) or igf-1 (10 nm) elevated phospho - erk1/2 level 8- and 7-fold, respectively (figures 2(c) and 2(d)). phospho - erk1/2 was increased much more by ang ii in combination with insulin / igf-1 than by ang ii or insulin / igf-1 alone, indicating that the elevation was caused by more than an additive effect. ang ii marginally suppressed akt activation under the basal condition, while it exerted no effect on phospho - akt induced by insulin / igf-1 (figures 2(c) and 2(d)). the synergistic effect of ang ii and insulin / igf-1 on erk1/2 activation was abolished by candesartan, a selective at1 receptor blocker, but not by pd1233319, a selective at2 receptor blocker, indicating that the action of ang ii was mediated by at1 receptor activation. mab 391, a neutralizing anti - igf-1 receptor antibody, significantly attenuated ang ii- and igf-1-induced phosphorylation of erk1/2 and akt, while it had no effect on the ang ii- and insulin - induced responses (figure 3). ang ii is known to stimulate pkc and mek pathway, while insulin / igf-1 plays its role mainly through pi3k pathway. in h295r cells, both pkc inhibitor and mek1/2 inhibitor fully inhibited ang ii - stimulated phospho - erk1/2 and substantially decreased phospho - erk1/2 stimulated by ang ii plus insulin / igf-1. both erk1/2 inhibitor u0126 and pkc inhibitor g6983 showed a marginal stimulatory effect on phospho - akt, under basal condition or after treatment with agonists (figures 4 and 5). pi3k inhibitor altogether abolished insulin / igf-1 stimulated phospho - akt and markedly decreased phospho - akt costimulated by ang ii and insulin / igf-1 (figure 6). to clarify whether the synergistic effect of ang ii and insulin / igf-1 on erk1/2 activation affects the cellular function, we observed mrna expression of the steroidogenic enzymes stimulated by ang ii and insulin / igf-1, alone or combined. as shown in figure 7, ang ii significantly stimulated mrna expression of cyp11b1 and cyp11b2. insulin receptor (ir) and type 1 igf receptor (igf-1r) serve distinctly different functions. they both possess a highly conserved structure, with a 60% homology between them. each receptor has a homodimeric structure consisting of two and two chains covalently linked in the membrane. these two receptors may engage in synthesis of a hybrid receptor (ir - igf-1r heterodimers). monoclonal antibodies against igf-1r were found to decrease the cell surface expression of igf-1r homodimers and ir - igf-1r heterodimers by causing receptor internalization. the endogenous ligands of ir include insulin, igf-1, and igf-2, but igf-1r is activated only by igf-1 and igf-2. in this study, we found that mab 391, a monoclonal antibody of igf-1r, significantly suppressed phosphorylation of erk1/2 and akt costimulated by ang ii and igf-1 in h295r cells, suggesting that igf-1 exerted its effect predominantly through igf-1r in adrenocortical cells. mab 391 had no effect on the responses induced by ang ii plus insulin, suggesting that insulin acts mainly on ir, and no or hardly any ir - igf-1r heterodimers are present in these cells. although the roles of insulin and igf-1 in adrenocortical cells are well documented, only two studies examined intracellular erk1/2 and akt pathways [25, 26 ]. they reported that igf-1 activated the phosphorylation of both erk1/2 and akt [25, 26 ]. the present study showed that insulin and igf-1 exerted a weak stimulatory effect on erk phosphorylation and a strong stimulatory impact on akt phosphorylation. in adrenocortical cells, after activation of ir and igf-1r, their effects on erk1/2 and akt signaling pathway are similar. we found that, in h295r cells, ang ii suppressed akt activation under the basal condition, while it had no effect on phospho - akt induced by insulin / igf-1. insulin signaling was found to be inhibited by ang ii in several other cells [27, 28 ]. in vascular smooth muscle cells (vsmc), ang ii - elicited erk1/2 activation leads to phosphorylation of irs1 at ser307 and ser616, thereby inhibiting akt activation and ultimately suppressing the insulin - induced glucose uptake. reported that ang ii inhibited insulin - mediated glut4 translocation in rat skeletal muscle cells through at least two pathways : (1) the transient activation of erk1/2 which inhibits irs1/2 and (2) direct inhibitory nitration of akt. the major findings of the present study were that ang ii and insulin / igf-1 exerted a synergistic stimulatory effect on erk1/2 activation in adrenocortical cells. only two in vivo studies showed additive stimulatory effect of these two hormones on erk1/2 activation in hypothalamus and heart tissues [7, 8 ]. activation of erk1/2 by ang ii through at1 receptor may involve three different pathways, that is, gq - pkc, src - ras, and transactivation of the epidermal growth factor receptor, while erk1/2 activation by insulin was found to be mainly through shc - grb2-ras pathway [29, 30 ]. as shown in this study, in adrenocortical cells, pkc was the major pathway involved in ang ii - induced erk1/2 activation. the mechanism underlying the synergistic stimulation is still unknown and might involve other unknown factors. we believe that this synergistic effect can not be explained simply by additive effect of the two hormones. ang ii could stimulate aldosterone and cortisol secretion, which might be mediated by erk1/2 activation [31, 32 ]. we observed that the stimulatory effects of ang ii on the expression of steroidogenic enzymes could be enhanced by cotreatment with insulin / igf-1. the results indicated that the synergistic stimulatory effect of ang ii and insulin / igf-1 on erk1/2 activation might be involved in the enhanced steroid hormone production stimulated by ang ii plus insulin / igf-1. this study, for the first time, demonstrated that the interaction between ang ii and insulin / igf-1 was evidently of synergistic, rather than additive, nature in stimulating erk1/2 activation in h295r cells. such synergistic stimulatory effect on erk1/2 | the cross talk between angiotensin ii (ang ii) and insulin has been described mainly in cardiovascular cells, hepatocytes, adipocytes, and so forth, and to date no such cross talk was reported in adrenal. in this study, we examined the interaction between ang ii and insulin / igf-1 in erk and akt signaling pathways and expression of steroidogenic enzymes in h295r cells. compared to the control, 100 nm ang ii increased phospho - erk1/2 approximately 3-fold. insulin (100 nm) or igf-1 (10 nm) alone raised phospho - erk1/2 1.8- and 1.5-fold, respectively, while, after pretreatment with 100 nm ang ii for 30 min, insulin (100 nm) or igf-1 (10 nm) elevated phospho - erk1/2 level 8- and 7-fold, respectively. the synergistic effect of ang ii and insulin / igf-1 on erk1/2 activation was inhibited by selective at1 receptor blocker, pkc inhibitor, and mek1/2 inhibitor. ang ii marginally suppressed akt activation under the basal condition, while it had no effect on phospho - akt induced by insulin / igf-1. ang ii significantly stimulated mrna expression of cyp11b1 and cyp11b2, and such stimulatory effects were enhanced when cells were cotreated with insulin / igf-1. we are led to conclude that ang ii in combination with insulin / igf-1 had an evident synergistic stimulatory effect on erk1/2 activation in h295r cells and the effect may be responsible for the enhanced steroid hormone production induced by ang ii plus insulin / igf-1. |
polycystic ovary syndrome (pcos) is a heterogeneous and complex endocrine disorder associated with hyperandrogenism and insulin resistance signs and symptoms such as obesity, abnormal glucose tolerance test or type 2 diabetes, abnormal blood lipid profile, high blood pressure and cardiovascular disease (1). this disorder is prevalent and affects 5 - 10% of women of reproductive age (2) and causes infertility in 75% of these women due to chronic anovulation (3). many drugs were used as a treatment for pcos or control its consequences, including metformin (4), simvastatin (5), atorvastatin (6), n - acetyl - cysteine (nac) (7) and flutamide (8). in general, all of these drugs have positive effects in improving hyperandrogenic symptoms and regulation of menstrual cycles, but simvastatin and metformin have been more commonly used than others for treatment and research purposes. in one study, long - term treatment with simvastatin was more effective than metformin for control of hyperandrogenism sign and increased menstrual regulation in women with pco (9). there are several published studies regarding the effectiveness of statins administration in women with pco (10, 11), but in comparison with metformin there is no published research about drug administration before or during icsi cycle. simvastatin a drug from statins group, inhibits hmg - coa reductase and reduces cholesterol production, increases liver ldl receptor sensitivity and hdl level, meanwhile decreases tg level in blood circulation (12). moreover, statins prevent theca - interstitial cell proliferation in ovaries and therefore reduce total androgen production (13). metformin as a biguanid drug, blocks glucose synthesis in liver and enhances peripheral sensitivity to insulin, which finally decreases androgen level in patients with pco and enhances the chance of pregnancy (14). according to latest cochrane published study about metformin treatment before and during icsi cycle, live birth or pregnancy rate was not improved (15) ; the cochrane database announced that although simvastatin improved lipid profile and hyperandrogenism sign, it had no significant effect on pregnancy chance (16), of course in natural cycle. we decided to compare the efficacy of both drugs as a pretreatment regiment before icsi cycle in patients with pco. in this prospective, double blind, randomized clinical trial, 40 women with pcos and a history of infertility referring for first icsi cycle to imam khomeini hospital affiliated with ahvaz jundishapur university of medical sciences (ajums) were selected based on closed enveloped randomization method from december 2010 to november 2012 to receive simvastatin (group a) or metformin (group b). according to the variance of previous studies, the sample size was calculated as 20 patients for each group. in accordance with the guidelines of the declaration of helsinki, after approval of university ethical committee (eth-3251), the study was submitted on iranian health ministry website for clinical trials (www.irrct.ir). pcos considered as presence of two of the following criteria including menstrual irregularities (oligomenorrhea or amenorrhea), clinical and laboratory findings of hyperandrogenism and polycystic ovaries on ultrasound evaluation. besides, inclusion criteria were aged 20 - 35 years, a history of infertility for at least two years (either primary or secondary), normal tsh and prolactin and candidates for the first cycle of icsi. patients with other causes of infertility, including tubal factors, endometriosis and male factor infertility were excluded. patients who used metformin, lipid control drugs, clomiphene, letrozole or doing iui (intrauterine insemination) in the past 3-months before study or having medical problems such as kidney or liver diseases, diabetes or a history of elevated blood lipids were excluded. body mass index (bmi) and the score of hirsutism were assessed according to the ferriman gallwey scoring system (figure 1). in this system, hair growth in a male pattern on a woman shown in four different degrees of severity in 11 different body parts, namely the upper lip, chin, chest, upper back, lower back, upper abdomen, lower abdomen, arm, forearm, thigh and lower leg (17). on the third day of menstruation, all patients were evaluated for total testosterone, lh, fsh, tg, total cholesterol, ldl, hdl, fbs, gtt with 75 grams glucose (blood sugar measured one and two hours after drinking it), prolactin and tsh. after getting the first blood sample for biochemical evaluation, in group a, patients received simvastatin 20 mg daily (osvah pharmaceutical company, iran) plus low dose oral contraceptive pill (ocp) daily for eight weeks. in group b, patients received metformin 500 mg tds (osvah pharmaceutical company, iran) plus low dose ocp daily for eight weeks. then, all patients were again evaluated for previous biochemical parameters (except prolactin and tsh) and bmi measurement and hirsutism scoring. patients were enrolled in the icsi protocol according to the long protocol ; from the day of twenty - one of the second cycle, subcutaneous superfact (0.5 mg daily, triptrolein, hoechst, frankfort, germany) injection was started. after menstruation on the third day of cycle, both drugs (simvastatin and metformin) were discontinued and after vaginal sonography control and reducing superfact to 0.25 mg daily, induction of ovulation with folliotropin alfa (gonal - f, merck, serono. ovarian response was examined by transvaginal sonography six days later and gonal - f dosage adjusted according to the patient response ; when at least three follicles with a size of 18 mm detected, human chorionic gonadotropin (hcg) 10000 units (profasi. switzerland) was injected intramuscularly and ovarian puncture was performed 36 - 40 hours later. after ovarian puncture, the number of mii oocytes and after sperm injection, on the day 3, the number of embryos of grade a was recorded. the incidence of ovarian hyperstimulation syndrome (ohss) was reported in the both groups. then, support of luteal phase was started with vaginal suppository of micronized progesterone 400 mg twice daily. the first ultrasound was performed five weeks after embryo transfer in cases of pregnancy positive test. cases of abortion, ectopic pregnancy (ep), ongoing pregnancy (passing 14 weeks of gestational age) and negative pregnancy were detected. student s t - test for unpaired data and sample t - test for paired information were used. also chi - square and fisher s tests were used to assess frequency distribution and clinical outcome. regression and anova tests were used for comparison between the groups. for gtt result interpretation, p value 30 kg / m) and in group b, 65%. after eight weeks treatment in both groups, reduction of mean hirsutism score was significant, although it was more meaningful in simvastatin group (table 2) ; this reduction in mean bmi level was not significant (table 3). biochemical parameters before the treatment in the both groups were not different (table 4). after eight weeks in group a receiving simvastatin, testosterone, fsh, lh, cholesterol (chol) and ldl significantly decreased ; meanwhile hdl level elevated. in group b, after metformin administration the level of fsh, testosterone, tg and chol reduced meaningfully (table 5). the mean values of three blood sugar measurements (fbs/1 hour/2 hours) before starting the treatment were not different between the two groups (group a : 12208 1658.03 mg / dl vs. group b, 12302 1789.87 after the treatment, roc showed improvement area under curve (auc) in glucose tolerance test in the both groups ; although there were no significant differences between the two groups (p value 0.11) (figure 2). a, before the treatment in metformin ; and c, simvastatin ; b, after the treatment in metformin ; and d, simvastatin. after ovarian puncture, the number of oocytes in phase2 meiosis (m2) in simvastatin treated group was 1.35 1.6, and in metformin treated group 2 3.87 (p value 0.48). on the third day after ovarian puncture, the number of grade a embryos in group a was 1.2 1.3 versus 1.1 1.4 in group b (p value 0.79), which was not significantly different. in the both groups the pregnancy rate in group a was six cases (30%) ongoing and seven cases in group b (35%). to the best of our knowledge, this is the first published study comparing simvastatin and metformin as a pretreatment regiment for icsi cycle in pcos women and there is one study regarding the effectiveness of simvastatin before ivf (18). women with pcos have enlarged ovaries with an increased number of small follicles with more theca cells, which are androgen - producing cells. it is also clear that theca cells of women with pcos produce higher amount of androgens compared with the theca cells of normal women. increased expression of genes involved in androgen production, such as cyp11a and cyp17 has been observed in these cells (9). metformin - induced reduction in testosterone is not only because of decreasing size of the ovarian theca cells, but also due to the direct action of metformin. studies have shown that metformin inhibits the production of testosterone and androstenedione in the cultured theca cells. metformin also decreased expression of steroidogenic acute regulatory (star) protein and 17-hydroxylase/17, 20-desmolase (cyp17) (19). studies have shown that statins reduce cell proliferation, increase apoptosis and inhibit producing testosterone in humans and rat theca cells (10, 13). the positive effects of simvastatin in reducing hirsutism and testosterone have been confirmed by other researchers (5) ; this reduction in testosterone level, but not the hirsutism score after metformin administration has been reported, but in our study bmi decreased after six months therapy (20). in another study, after six months of treatment, simvastatin improved hyperandrogenism parameters like total testosterone, hirsutism score, total cholesterol, ldl and bmi better than metformin (9). lipid profile analysis showed that simvastatin significantly reduced ldl and total cholesterol, and increased hdl. this effect reflects direct competitive prevention of the controlling step of cholesterol production by inhibiting 3-hydroxy3methylglutaryl coenzyme a reductase (21). however, metformin significantly decreased triglyceride and ldl by its anti - lipolytic effects, reducing circulating free fatty acid concentrations and therefor the level of triglycerides (22, 23), although this reduction in ldl level in our study was not significant. in the present study, lh and fsh hormones were significantly lower in simvastatin group after treatment, but in the metformin group only fsh concentrations decreased. in women with pco, reduction in fsh level reflects decreased gnrh frequency secretion due to reduction of hypothalamic dopamine and opioid production and from negative feedback of chronically elevated estrone concentration (increased peripheral aromatization of elevated androstenedione) and decreased progesterone level (chronic anovulation) (24). in contrast to fsh, abnormal lh production is due to increased pulse amplitude and frequency secondary to altered pulsatile gnrh secretion ; this elevated lh led to ovarian hyperandrogenism (25). metformin directly affected gonadotropin - secreting cells and reduced lh production and elevated fsh level by regulating fsh gene expression (26). simvastatin could reduce lh level, but had no effect on fsh (11) ; this impressive effect for normalized gonadotropins level improved in other study (27). simvastatin reduced lh level by decreasing ovarian testosterone production secondary to inhibition of theca - interstitial proliferation. in our study, reduction in lh level was seen in the both groups, although it was more significant in group a ; we could not find any explanation about reduced level of fsh in the both groups. in conclusion, according to only published study about simvastatin administration before ivf cycle (18) and in agreement with cochrane review about co - administration of metformin and icsi cycle (15), the outcome of icsi cycle does not progress significantly, but biochemical parameters, signs and symptom in women with pco improved. these drugs as a pretreatment protocol before icsi cycle could alter the results of cycle if used in more patients, or in longer or higher doses. | background : drugs administration as a pretreatment regiment before icsi cycle in pcos patients could enhance the success rate.objectives:the aim of this study was to compare the effectiveness of metformin with simvastatin in patients with polycystic ovary syndrome (pcos) candidates for intra - cytoplasmic sperm injection (icsi) before starting the cycle.patients and methods : in this prospective, double blind, randomized clinical trial the efficacy of these drugs was evaluated in 40 women with pco syndrome (20 patients in each group ; a : simvastatin and b : metformin) candidates for icsi. in the both groups, metformin and simvastatin administrated for eight weeks before starting the icsi cycle. endocrine, metabolic and clinical parameters were measured before and after drug therapy ; also, the results of icsi cycle evaluated in the both groups.results:both drugs improved hirsutism score significantly, but simvastatin better than metformin (group a, 24.5 3.6 p : 0.0001 vs group b, 22.9 5.9 p : 0.003). the reduction in body mass index (bmi) was not significant in the groups. simvastatin reduced some biochemical parameters such as fsh, lh, testosterone, total cholesterol, ldl and increased hdl level significantly, whereas metformin decreased fsh, tg, testosterone and total cholesterol significantly. overall, respectively 35% and 30% of patients treated with metformin and simvastatin became pregnant. there was no significant difference between the effects of these two drugs on icsi cycle results like oocyte in meiosis2 (m2) phase (1.35 1.6 vs. 2 3.87, p value : 0.4) and the number of grade a, embryo (1.2 1.3 vs. 1.1 1.4, p value : 0.7).conclusions : simvastatin effectively improved hyperandrogenism signs and symptoms in patients with pco, but this effect as a pretreatment regiment was not more expressive than metformin in icsi cycle outcome. |
adhesive bond failure between acrylic teeth and resin material is the most routine type of failure in dental practice. appropriate bonding of acrylic teeth to denture base resin is essential as it increases strength since the teeth becomes an intrinsic part of the prosthesis. the bond strength between acrylic teeth and denture base is multifaceted including mechanical and chemical methods for enhancing the bond between teeth and resin material. many researchers have over the years tried to improve the bond strength by altering the ridge lap area mechanically. (1986) reported that the there is no advantage of vertical retention grooves prepared in the ridge lap area of cross - linked teeth. (1990) contradictated his earlier statement and proved that vertical retention grooves made on cross linked teeth increased bond strength between cross - linked teeth and denture base material. the teeth were divided into three groups : group ano tooth preparation, group ba mesiodistal groove was prepared in the ridge lap area of the tooth, and group ca vertical groove of the same dimensions was prepared at the center of the ridge lap extending halfway up the lingual surface of the tooth. a shear force was applied at an angle of 130 to the lingual surface of the cross - linked teeth until failure occurred. the vertical retention grooves prepared in the ridge lap area of the teeth proved to be beneficial and modified retention to the resin material but there was no benefit of placement of the horizontal grooves. the advantage of mechanical modifications of cross - linked teeth for bond strength between high - impact denture base material and cross - linked teeth is unclear. hence, an attempt has been made to evaluate and compare the bond strength of cross - linked teeth bonded to high - impact denture base material by providing different mechanical modifications of denture teeth. to evaluate bond strength between heat cure resin material and cross - linked teeth without modificationto evaluate bond strength between heat cure resin material and cross - linked teeth modified with round groove, vertical groove, and t - shaped groovecomparative evaluation of different mechanical modifications done on cross - linked teeth for bond strength between high - impact acrylic material and cross - linked teeth. to evaluate bond strength between heat cure resin material and cross - linked teeth without modification to evaluate bond strength between heat cure resin material and cross - linked teeth modified with round groove, vertical groove, and t - shaped groove comparative evaluation of different mechanical modifications done on cross - linked teeth for bond strength between high - impact acrylic material and cross - linked teeth. to evaluate bond strength between heat cure resin material and cross - linked teeth without modificationto evaluate bond strength between heat cure resin material and cross - linked teeth modified with round groove, vertical groove, and t - shaped groovecomparative evaluation of different mechanical modifications done on cross - linked teeth for bond strength between high - impact acrylic material and cross - linked teeth. to evaluate bond strength between heat cure resin material and cross - linked teeth without modification to evaluate bond strength between heat cure resin material and cross - linked teeth modified with round groove, vertical groove, and t - shaped groove comparative evaluation of different mechanical modifications done on cross - linked teeth for bond strength between high - impact acrylic material and cross - linked teeth a total number of 80 cross - linked maxillary left central incisors (21) (acryloc), of the same mold with regard to size and shape were selected to be bonded to lucitone199 resin material. the sample size was calculated using the results of the previous studies by takahashi, chai, takahashi, and habu. the sample size was calculated to be 20 per group keeping a confidence interval of 95% and a power of atleast 80%. the test specimens ' central incisors (21) were divided into four groups. group 1 (control) : denture teeth without any mechanical modification were used as supplied by the manufacturergroup 2 : a round groove 2 mm in diameter was prepared on the ridge lap surfaces of the croos - linked teeth with round burgroup 3 : a vertical groove 2-mm deep and 2-mm wide was prepared into the ridge lap area of the teeth with a straight fissure burgroup 4 : a t - shaped groove 2-mm deep and 2-mm wide was prepared with a straight fissure bur, bisecting the ridge lap surface of the cross - linked teeth. group 1 (control) : denture teeth without any mechanical modification were used as supplied by the manufacturer group 2 : a round groove 2 mm in diameter was prepared on the ridge lap surfaces of the croos - linked teeth with round bur group 3 : a vertical groove 2-mm deep and 2-mm wide was prepared into the ridge lap area of the teeth with a straight fissure bur group 4 : a t - shaped groove 2-mm deep and 2-mm wide was prepared with a straight fissure bur, bisecting the ridge lap surface of the cross - linked teeth. the preparation of the masterpiece was done by aligning the long axis of the central incisor teeth at 45 to the base of a wax block (8 mm 10 mm 30 mm), with ridge lap contacting the base. this mold was used for standardizing the angulation of acrylic tooth with the base of all test specimens. the base of the test specimen was created by placement of acrylic teeth (with and without modification) into the silicone mold, into which molten wax was flown. a total of 80 identical wax models were obtained in this study. these test specimens (wax models) four groups were created by demarcating the specimens, with 20 teeth in each group. thus, each group consists of 20 test specimens and a total of 80 specimens from the four study groups. the prepared wax models were invested in the flask following the manufacturer 's instructions for water powder ratio, mixing time, and setting time. one hour after the stone set, flasks were kept for dewaxing by immersing in boiling water for 5 min. a thin film of alginate separating media was applied on all surfaces of the mold except the saddle portion of teeth with the help of a brush and dried. a combination of polymer and monomer, used in the ratio of 3:1 by volume was proportioned prior to mixing. mixing was done in a porcelain jar, which was kneaded by hand upon achieving the dough consistency to increase its homogenecity and integrity and then packed into mold. after the flasks were clamped, closure was done under force of 20 kn and kept for 30 min. the flasks were then kept at room temperature for 1 h. then the flasks were submerged in water in an acrylizer at room temperature and processing was done as per the manufacturer 's guidelines. after curing of all the specimens all 80 test specimens were prepared by means of these procedures [figure 1 ]. specimen after polishing the specimens created were exposed to load testing by hounsfield universal testing machine (ez20 20 kn, computerised, lloyd company, ametek technologies, uk) [figure 2 ]. bond strength test was performed at the textile department, bapuji institute of engineering and technology (biet), davangere, karnataka, india. all trial specimens were placed securely in a specially created stainless steel jig and were placed in such a way that stress was applied on the palatal region of the sample denture teeth when moved in a downward direction. the bond strength was checked by using a cross - head speed of 5 mm / min. (ibm company, new york, us) and epi - info version 3.0 were used for examining the statistical data. wilk test, one - way analysis of variance (anova), and bonferroni test were done to do statistical investigation. the prepared wax models were invested in the flask following the manufacturer 's instructions for water powder ratio, mixing time, and setting time. one hour after the stone set, flasks were kept for dewaxing by immersing in boiling water for 5 min. a thin film of alginate separating media was applied on all surfaces of the mold except the saddle portion of teeth with the help of a brush and dried. a combination of polymer and monomer, used in the ratio of 3:1 by volume was proportioned prior to mixing. mixing was done in a porcelain jar, which was kneaded by hand upon achieving the dough consistency to increase its homogenecity and integrity and then packed into mold. after the flasks were clamped, closure was done under force of 20 kn and kept for 30 min. the flasks were then kept at room temperature for 1 h. then the flasks were submerged in water in an acrylizer at room temperature and processing was done as per the manufacturer 's guidelines. after curing of all the specimens, the flasks were brought down to room temperature and deflasked. all 80 test specimens were prepared by means of these procedures [figure 1 ]. the specimens created were exposed to load testing by hounsfield universal testing machine (ez20 20 kn, computerised, lloyd company, ametek technologies, uk) [figure 2 ]. bond strength test was performed at the textile department, bapuji institute of engineering and technology (biet), davangere, karnataka, india. all trial specimens were placed securely in a specially created stainless steel jig and were placed in such a way that stress was applied on the palatal region of the sample denture teeth when moved in a downward direction. the bond strength was checked by using a cross - head speed of 5 mm / min. (ibm company, new york, us) and epi - info version 3.0 were used for examining the statistical data. wilk test, one - way analysis of variance (anova), and bonferroni test were done to do statistical investigation. the comparative bond strength values (newton) of different groups created with lucitone 199 are shown in table 1 and graph 1. the relative study of bond strength value among different groups preprared with lucitone 199 proved to be statistically significant. out of all the specimens prepared using lucitone 199, the highest bond strength value (720.6 n) was observed in group 4 followed by group 3 (645.6 n), group 2 (563.4 n), and lastly group 1 (451.8n). comparative bond strength values (newton) of different groups prepared with lucitone 199 heat - cured denture base resin comparative bond strength values (newton) of different groups prepared with lucitone 199 heat - cured denture base resin one of the major issues in prosthodontic practice is failure of tooth - denture base bond. woelfel (1977) listed the principal factors in correct fabrication of acrylic resin dentures : (1) the relationship obtained from the patient and sent to the dental laboratory must be mechanically and physiologically sound ; (2) the wax bulk and contour of the trial denture should be as nearly like that of the final denture as far as possible so that only minimal reduction of the processed denture is necessary ; (3) flasking should be done using entirely dental stone, which is 23 times stronger than plaster ; (4) the boil - out procedure must be timed so that wax is not permitted to soak into the dental stone from prolonged heating but can be removed by lifting it out in one piece, with the wax just softened or melted only around the necks of the teeth and in the ridge undercuts ; (5) the tinfoil substitute should be applied while the flasks are warm and wet ; and (6) during the packing procedures, several things are of utmost importance (a) powder to liquid ratio should be 3:1, (b) on all trial closures, the press closing force must be applied slowly, thus permitting adequate time for the dough to flow and become compressed for optimum density, and (c) the packed flask should be allowed to stand for 3060 min before beginning the processing cycle. studies have calculated the occurrence of various denture repairs and shown tooth separation to be the most common repair for conventional prosthodontics. a survey conducted by darbar, huggett, and harrison (1994) the resultant data showed that 33% of the repairs performed were to correct debonded teeth. acrylic teeth are used for prosthetic rehabilitation as they have the property of good color stability and show perfect aesthetic results. the failure of bond was thought as cohesion failure in the case of remnants of denture base materials, as reported by khalaf, abdulsahib, and abars (2011). yadav, somkumar, mishra, hazari, chitumalla, and pandey (2015) reported that debonding occurs within the body of the tooth rather than tooth acrylic interface ; so, there is no need of surface treatment of ridge lap surface. consani, pucciarelli, mesquita, nogueira, and barao (2014) reported that different polymerization cycles have the same effect on the hardness of heat cure resin material. grando, pacheco, botega, hirakata, and hilgert reorted no significant difference in the wear resistance of different brands ' (trilux, soluut px) teeth. akin, kirmani, tugut, and coskum (2014) worked upon the effects of different surface treatments in the ridge lap area of acrylic resin teeth on shear bond strength to heat polymerized poly (methyl methacrylate) (pmma) denture base resin. they concluded to vanquish tooth debonding, surface treatment should be included as a part of denture fabrication. cardash, liberman, and helft (1986) researched to report the effetiveness of retention grooves of various shapes, cut into the ridge lap surface of acrylic resin teeth in increasing the shear strength bond among heat - cured resin and acrylic teeth. they determined that the force needed for debonding of acrylic resin teeth from denture base was nearly similar to the force needed to fracture the acrylic resin base. a lack of significant improvement was noted on introducing retention grooves of various shapes into the ridge lap portion of acrylic resin teeth in the combined shear compressive strength of bond to heat - cured standard resin and to high - impact denture base resin. cardash, applebaum, baharav, and liberman (1990) checked whether horizontal or vertical retention grooves prepared on the ridge lap area of cross - linked teeth would improve the combined shear compressive strength of the bond to heat - cured resin and to high - impact resin. they interpreted that the presence of vertical retention grooves prepared on ridge lap area of teeth enhanced the retention to acrylic resin. therefore, an effort has been made to check and compare the bond strength of denture teeth bonded to high - impact resin material. group 1 specimens prepared by the lucitone 19 resin material showed significantly lower bond strength value (451.8 n) compared to that of group 2 specimens (round groove) prepared with lucitone 199 resin material (563.4 n). the probable reason is that the round groove on the ridge lap surface of the teeth permits mechanical joint between the tooth and resin material. the control group specimens prepared by the lucitone 199 showed significantly lower bond strength value (451.8 n) compared to that of group 3 specimens (vertical grooves) prepared by the lucitone 199 (645.6 n). these results were similar to the results reported by caradash, applebaum, baharav, and liberman. they stated that vertical retention grooves provide more consistent mechanical joint between cross - linked tooth and the resin material. the control group specimens prepared by lucitone 199 showed significantly lower bond strength value (451.8 newton) compared to that of group 4 specimens (t - shaped groove) prepared by lucitone 199 (720.6 n). the probable reason for these results might be that the t - shaped groove increases the surface area available on the cross - linked teeth to interact with polymerizing denture base resins. group 3 specimens (vertical grooves) prepared by lucitone 199 showed a significantly higher bond strength value (645.6 n) compared to that of group 2 specimens (round groove) prepared by lucitone 199 (563.4 n). the probable reason is that the vertical groove area allows better mechanical bonding between the cross - linked teeth teeth and resin material compared to the bond induced by round groove. the group 4 specimens (t - shaped groove) prepared by lucitone 199 showed significantly higher bond strength value (720.6 n) compared to that of group 3 specimens (vertical grooves) prepared by lucitone 199 heat cure denture base resin (645.6 n). the probable reason for these results might be that the t - shaped groove area being wider area than vertical groove area has more chances of greater flow of acrylic resin denture base material into that area and provided better mechanical retention between the tooth and denture compared to mechanical retention induced by vertical grooves. hence, further studies are required using different types of denture base resins (convetional and high - impact resin materials)we evaluated lone standing denture teeth for bond strength evaluation since adjacent denture teeth also have reinforcing bond strength. further studies are required with denture teeth being present on either side of the the denture tooth being evaluated for the studywe have used only one type of cross - linked denture teeth in our study. there are various composite multilayered denture teeth available in the market, which require further evaluation of bond strength with different mechanical modifications. hence, further studies are required using different types of denture base resins (convetional and high - impact resin materials) we evaluated lone standing denture teeth for bond strength evaluation since adjacent denture teeth also have reinforcing bond strength. further studies are required with denture teeth being present on either side of the the denture tooth being evaluated for the study we have used only one type of cross - linked denture teeth in our study. there are various composite multilayered denture teeth available in the market, which require further evaluation of bond strength with different mechanical modifications. hence, further studies are required using different types of denture base resins (convetional and high - impact resin materials)we evaluated lone standing denture teeth for bond strength evaluation since adjacent denture teeth also have reinforcing bond strength. further studies are required with denture teeth being present on either side of the the denture tooth being evaluated for the studywe have used only one type of cross - linked denture teeth in our study. there are various composite multilayered denture teeth available in the market, which require further evaluation of bond strength with different mechanical modifications. hence, further studies are required using different types of denture base resins (convetional and high - impact resin materials) we evaluated lone standing denture teeth for bond strength evaluation since adjacent denture teeth also have reinforcing bond strength. further studies are required with denture teeth being present on either side of the the denture tooth being evaluated for the study we have used only one type of cross - linked denture teeth in our study. there are various composite multilayered denture teeth available in the market, which require further evaluation of bond strength with different mechanical modifications. the bond strength between lucitone 199 heat - cured denture base material and denture teeth was increased with mechanical modifications of denture teeththe control group specimens without any modification prepared by lucitone 199 heat cure resin showed the lowest bond strength value, whereas the specimens prepared with t - shaped groove packed with lucitone 199 heat cure resin showed the highest bond strength value. therefore, this modification can be a recommended method to secure denture teeth in denture bases. the bond strength between lucitone 199 heat - cured denture base material and denture teeth was increased with mechanical modifications of denture teeth the control group specimens without any modification prepared by lucitone 199 heat cure resin showed the lowest bond strength value, whereas the specimens prepared with t - shaped groove packed with lucitone 199 heat cure resin showed the highest bond strength value. therefore, this modification can be a recommended method to secure denture teeth in denture bases. | aim and objective : acrylic teeth separates from the denture base and remains a major worry in day - to - day routine dental procedure. the present study was conducted to comparatively evaluate different mechanical modifications of acrylic teeth on bond strength between lucitone 199 heat cure resin and cross - linked teeth.materials and methods : the test specimens, central incisors (21) were demarcated into four groups. group 1 was the control group, whereas group 2, group 3, and group 4 were experimental groups modified with round groove, vertical groove, and t - shaped groove, respectively. the preparation of masterpiece was done by aligning the long axis of the central incisor teeth at 45 to the base of a wax block (8 mm 10 mm 30 mm), with ridge lap surface contacting the base. these test specimen (21) was prepared by lucitone 199 heat cure resin. evaluation of bond strength of all the specimens was done using universal tester (materials testing machine). shapiro wilk test, one - way analysis of variance (anova), and bonferroni test were done to do statistical investigation.results:group 1 specimens prepared by lucitone 199 heat cure resin showed the lowest bond strength and group 4 specimens prepared with t - shaped groove packed with lucitone 199 exhibited the highest bond strength.conclusion:the bond strength between lucitone 199 heat cure resin and cross - linked teeth was increased when mechanical modifications was done on denture teeth. the specimens prepared with t - shaped groove packed with lucitone 199 heat cure resin showed the highest bond strength followed by group 3, group 2, and lastly group 1 prepared by lucitone 199 heat cure resin. |
phosphorylation of proteins is the most abundant posttranslational modification used in regulatory mechanisms in eukaryotic cells (khoury., 2011). advanced techniques of phosphoproteomics have led to the identification of tens of thousands of phosphorylated sites in protein kinase targets. a closer look at phosphoproteomes highlights one important feature : a large fraction of protein kinase targets contain multiple phosphorylation sites, and clusters of sites tend to be located within intrinsically disordered regions of the target proteins (holt., 2009 ; tyanova., thus it appears that the mechanism of phosphorylation may hide yet another level of complexity arising from combinatorial patterns of multistep phosphorylation events. however, how this multisite phosphorylation code is to be read and transformed into meaningful signaling information remains unclear because the biochemical details of the signal processing logic of these multisite phosphorylation networks have not been studied until recently. during multisite phosphorylation, phosphate groups are added in either a random or defined order to serine, threonine, or tyrosine residues in kinase substrates. when a crucial set of key sites becomes phosphorylated, the downstream signaling switch will be triggered. our recent studies shed light on a mechanism by which sequential multisite phosphorylation is used for processing of cyclin - dependent kinase 1 (cdk1) signals in cell cycle regulation (koivomagi., here we discuss the main engineering principles of molecular switches based on sequential multisite phosphorylation of cdk1 targets. we also explore a range of possible but as - yet - unobserved properties that multisite phosphorylation networks may provide to the signal processing capacity of cellular systems in general. cdk1-dependent phosphorylation events often lead to the generation of phosphorylated sequence motifs (phosphodegrons) that are recognized by the ubiquitination machinery and thereby marked for destruction (hao., 2007 ;, phosphorylation - dependent destruction of a cdk1 inhibitor protein called sic1 helps to trigger s phase in budding yeast. cdk1, when bound to g1- and s - phase cyclins, phosphorylates sic1 in an ordered sequence at multiple sites, leading to the formation of phosphodegrons that are recognized by the ubiquitin ligase scf - cdc4. the sequential phosphorylation of sic1 and other substrates depends on three important interactions between cdk1 complexes and the disordered substrate chain (figure 1, a and b) : the active site of cdk1 interacts with the consensus phosphorylation site, typically s / t - p or s / t - pxk / r (khoury., 2011) ; specific sites on the cyclin interact with docking motifs on the substrate (holt., 2009) ; and the small adaptor protein cks1 interacts with specific phosphorylated threonines on the substrate (tyanova., 2013). (a) a schematic model of a cyclin - cdk1-cks1 complex containing the catalytic cdk1 subunit, the positive regulatory subunit (cyclin), and an accessory phosphate - binding subunit (cks1). a disordered substrate is shown together with the docking motifs that interact with each of the subunits. these are as follows : the phosphorylated consensus site (s / t - p) interacts with the cdk1 active site (red marks with -oh), a cyclin docking motif interacts with a hydrophobic pocket on the cyclin (blue boxes), and a phosphorylated threonine on the substrate interacts with cks1 (red marks with p). (b) examples of multisite phosphorylation networks in selected cdk1 targets of saccharomyces cerevisiae. l - p rich motifs are specific docking sites for the g1-specific cln2-cdk1 complex (bhaduri and pryciak, 2011 ; koivomagi., 2011b). phosphodegrons are sequence motifs composed of two phosphorylated sites positioned 23 amino acids apart and recognized by the ubiquitin ligase scf - cdc4. (c) graph depicting the ultrasensitive signal - response curves created by multisite phosphorylation. for the model simulation, we created a system of ordinary differential equations describing the phosphorylation of a set of kinase substrates carrying different multisite phosphorylation networks. the differently colored lines indicate simulated phosphorylation output responses at different thresholds of the increasing input signal of the kinase (the diagonal dotted line). the number of phosphorylation sites was varied in each substrate to gain temporal resolution of the response curves (1, 4, 6, 9, and 12 phosphorylation sites in substrates represented by curves from left to right, respectively). in addition, the phosphorylation rate constants of selected steps were varied in some substrates to imitate the effect of different patterning of the sites on the phosphorylation rates on individual steps. the dotted horizontal lines represent the kinase thresholds at which half of the substrate has reached the fully phosphorylated state. analysis of the phosphorylation dynamics of sic1 showed that the phospho - adaptor cks1 is a key factor that controls the dynamics of multisite phosphorylation (figure 1a). cks1 binds to cdk1 and also to phosphorylated sites, thereby enhancing the phosphorylation of neighboring sites positioned c - terminally at optimal distances from the cks1-bound phosphorylated site. this type of event occurs several times, leading to sequential phosphorylation of the entire network of sites and thereby leading to the phosphorylation of output degron sites at the c - terminal end of the network. because clusters of sites are located in disordered segments of the proteins or in entirely disordered cdk1 targets (figure 1b), the cyclin - cdk1-cks1 complex acts as a catalytic scaffold whose net rate of catalysis of multisite phosphorylation through the network is determined by how well the fixed spatial orientation of three docking pockets on the scaffold fits with the linear pattern of phosphorylation sites and docking sites in the substrates. intriguingly, the sequential steps of phosphorylation in cdk1 targets were found to propagate in an n - to - c terminal direction along the disordered polypeptide chain of the substrate. furthermore, the productive interaction of a substrate with the cyclin - cdk1-cks1 scaffold was found to be defined by a set of crucial parameters that control the overall n - to - c terminal flux of multisite phosphorylation : 1) the choice of serine or threonine as the phospho - adaptor residues (only phosphothreonines serve as docking sites for cks1) ; 2) the distance between sites along the disordered peptide chain ; 3) the distance between the cyclin - specific docking sites and the phosphorylation sites ; 4) the consensus binding motif of the phospho - adaptor subunit cks1 ; 5) the overall number of phosphorylation sites ; and 6) the processivity at each phosphorylation step. these findings show that the surrounding consensus specificity of the phosphorylation sites has only limited influence on the phosphorylation of each network. instead, a simple set of rules, or a multisite phosphorylation code, defines the overall pattern and rates of phosphorylation in a multisite cluster, including its key output sites. it has been suggested that multisite phosphorylation of proteins could be the mechanism that processes a graded input signal of a protein kinase into an ultrasensitive, switch - like response (ferrell,1996 ; harvey., 2011 ; trunnell.,, this mode of signal processing requires that the output signal of the switch corresponds to the fully phosphorylated state of the multisite target protein. in the presence of an opposing phosphatase activity, low levels of kinase input signal produce a relatively low fraction of target molecules in which all sites are phosphorylated. at higher input signals, fully phosphorylated molecules begin to accumulate, and the overall signal - response profile shows a more switch - like or digital shape compared with the hyperbolic shape expected from a single - site switch (ferrell 1996 ; harvey., 2011 ; trunnell., this result can be obtained either with a randomly ordered series of phosphorylation sites or when sites are phosphorylated in a specific order, as described for cdk1 targets. in the latter case, a low - level input signal would not produce a sufficient number of phosphorylated priming sites that are required for phospho - adaptor docking and efficient phosphorylation of the c - terminal output sites. at higher levels of the kinase input signal, phosphorylation of the output sites is amplified, and the overall shape of the response begins to resemble a digital step function. ultrasensitive response profiles may provide yet another important quality of the cyclin - cdk1-cks1 scaffold : they could help process different levels of increasing cdk1 signal into discrete, temporally resolved events of the cell cycle. the phosphorylation rates are controlled at each step by the network parameters described here earlier, and different substrates could support networks with different numbers of steps and a different set of rates. the output sites of some substrates are phosphorylated early in the cell cycle at lower cdk1 thresholds (figure 1c). at these low kinase levels, the cdk1 signal is processed much less efficiently through the networks of later substrates, resulting in the output sites being weakly phosphorylated. higher cdk1 levels are required to push a critical amount of the signal through the network of later substrates for a sufficiently phosphorylated output (to use an electronics analogy, the networks of later substrates have more resistance). in this manner, different well - resolved cdk1 thresholds in the cell cycle could be achieved. thus the networks of phosphorylation sites in cdk1 targets may act as timing tags by coding different patterns of sites. intriguingly, our studies of cdk1-dependent multisite phosphorylation revealed that phospho - adaptor mediated docking at primed phosphorylation sites enables cdk1 to phosphorylate residues that are not cdk consensus sites (koivomagi., 2011a). in general, cdk target sites contain either optimal consensus phosphorylation motifs (s / t - p - x - k / r) or suboptimal consensus sites (s / t - p ; songyang. however, docking via optimally positioned and primed phosphorylation sites promotes the efficient phosphorylation of secondary sites that lack the required proline in position + 1 (figure 2a). this phenomenon could prove to be very important in cdk1 signaling because it works as a filter to prevent other proline - directed kinases from erroneously triggering these cdk1-dependent switches. in the case of both sic1 and another cdk1 target far1 (unpublished results), thus the multisite network allows the phosphorylation of the output degron by cdk1 but not by other proline - directed kinases that do not have the cyclin - cdk1-cks1like scaffold structure and thus can not read the multisite phosphorylation code. indeed, many kinases recognize the minimal consensus sequence s / t - p, which highlights the importance of this specificity filter. the output signals of cdk1 targets must be kept very low to prevent both premature and partial signaling via cdk1 switches in g1, as the leaking signals could induce genomic instability (lengronne and schwob, 2002) (a) schematic view of a specificity filter based on sequential phosphorylation of a substrate by cyclin - cdk1-cks1. this system both prevents erroneous firing of the degradation switch by other proline - directed kinases and filters low - level cdk1 signals to ensure that the commitment of the switch occurs only when adequate levels of cdk1 activity are achieved. (b) a three - input and gate created by sequential primed kinases that leads to the degradation of cohesin acetyltransferase eco1 (lyons., 2013). the three kinases recognize motifs primed by previous phosphorylation events (motifs are indicated by red letters on the scheme at right). the resulting ordered sequence of three phosphorylation events leads to the formation of a phosphorylated diphosphodegron. a recent study demonstrates that different kinase inputs can phosphorylate networks of sites in a specific order that is predetermined by the linear pattern of the sites in eco1, an enzyme that performs the acetylation of cohesin in s phase (lyons., 2013). degradation of eco1 at the end of s phase depends on sequential phosphorylation of three sites by three kinase inputs (cdk1, cdc7-dbf4, gsk-3/mck1 ; figure 2b). the resulting phosphorylated configuration corresponds to a diphosphodegron recognized by the scf - cdc4 ubiquitin ligase. by using a sequential priming mechanism such as this, the multisite system acts as a three - branched and gate that ensures that a sufficient level of each input kinase is present before the commitment of the switch. it is highly likely that this principle is widely used in cellular systems and that other logic functions are built by multiple kinase inputs. one additional mechanism for generating complex logic functions could involve phosphorylation sites that negatively affect the phosphorylation of other sites in the same network. indeed, we found evidence for this possibility in our studies of the cdk1 targets and inhibitors far1 and sic1. mitogen - activated protein (map) kinases that mediate external stimuli (osmotic stress, pheromones) influence cdk1 multisite networks by a mechanism we call diversion primed phospho - site diverts the cdk1 complex to an alternative inhibitory network of sites, away from the phosphorylation of sites that are responsible for the cell - cycle - dependent degradation of far1 or sic1. thus the strength of the map kinase input determines the fraction of free cdk1 complex available for generating a signal that triggers entry into the cell cycle and the initiation of s phase. these and other potentially undiscovered logic schemes in multisite phosphorylation networks may provide the cell with the capacity to execute complex computations of responses to external and internal signals. the number of phosphorylation steps in a switch and the complexity of multisite networks can also be increased by combining the phosphorylation of several targets with the assembly of protein complexes. the assembly steps of the complexes could trigger sequences of new phosphorylation events because different subunits in the complex could provide docking interactions for the phosphorylation of other subunits. nature 's ubiquitous use of multisite phosphorylation systems could inspire us to apply this logic to the design of signaling circuits for a diverse range of synthetic biology applications. in the era of affordable dna synthesis and genome - editing tools, creating a toolbox of easily tunable multisite phosphorylation tags may become useful in synthetic systems. the flexibility of multisite networks will enable us to design synthetic protein segments that encode different kinase specificities and output phosphorylation patterns. it is highly likely that future studies will reveal more multisite phosphorylation rules for different kinases and that this knowledge will help us to design additional signal - response modules, including logic gates, oscillators, memory circuits, single - input modules with multiple kinase thresholds, and so on. to facilitate the flexible design of circuits from base components, it is possible to introduce engineered kinase inputs by linking kinase domains (with basophilic, acidophilic, and proline - directed substrate specificity) to various phospho - adaptor modules (cks1, 14 - 3 - 3, polo box, etc.). the resulting chimeric kinases would enable the sequential processing of kinase signals via networks that contain phosphorylation sites with different consensus motifs. using interdependent phosphorylation events within networks of phosphorylation sites in proteins, cellular systems have acquired a vast computational capacity whose diverse possibilities are not well understood. further studies will likely reveal more schemes for encoding logic using multisite networks, and it is also likely that the knowledge gained will offer insights into how this technique could be applied in the design of signaling circuits within synthetic cells. | multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. we recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin - dependent kinase dependent multisite phosphorylation of a crucial substrate is performed sequentially in the n - to - c terminal direction along the disordered protein. the process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. according to our model, linear patterns of phosphorylation along disordered protein segments determine the signal - response function of a multisite phosphorylation switch. here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. we also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications. |
medication - assisted treatment (mat ; sometimes known as opioid substitution therapy or opioid replacement therapy) is widely accepted as one of the most effective interventions for opioid dependence.1 however, variations in treatment access and provision exist across countries. open - access, whereby treatment is predominantly delivered in primary - care settings rather than specialist addiction clinics. this approach has several advantages, including good accessibility, lower costs, reduced stigma, less contact with drug - using peers, and enhanced social integration. methadone was introduced in france in 1995, and 1 year later france became the first country in europe to introduce buprenorphine.2 since then there has been a rapid growth in the number of people receiving mat in france ; an estimated 138,000 people received mat in 2009, of whom 73% received buprenorphine and 27% received methadone.3 a unique feature of the french treatment system is that all registered medical doctors are allowed to prescribe buprenorphine without any special education or licensing ; once prescribed by a doctor, buprenorphine can be dispensed by the pharmacy chosen by the patient and named on the prescription.4,5 regulations allow buprenorphine prescriptions of up to 28 days with seven take - home doses, although physicians have the authority to request daily, supervised dosing by pharmacists or up to 28 days of take - home doses.5 in contrast, treatment with methadone still requires mandatory initiation within a specialized center or health care facility, and management of the patient can only be transferred to a nonspecialist physician once the patient has been stabilized.5 urine testing is compulsory in france when treating with methadone, but is not compulsory with buprenorphine.5 another important feature of the french model is that costs are fully covered by the social security system and psychosocial support is provided free of charge by local authorities.5 in 2008, the french governmental plan to fight drugs and drug addiction (20082011) was launched.6 harm reduction was a central component of this plan, which aimed to reduce deaths by overdose, the spread of infectious diseases, and the social and psychological damage caused by the use of illicit drugs.6 widespread use of mat, primarily buprenorphine, has been associated with a significant decrease in heroin use and improvement in social conditions of those in treatment,79 a decrease in overdose deaths,10 and a decrease in human immunodeficiency virus (hiv) prevalence among injection drug users.10 however, several studies have demonstrated misuse (ie, snorting or injecting) of buprenorphine by patients, which can involve crushing or dissolving their medication. snorting of buprenorphine was reported in 30% (n=33) of 111 stabilized patients receiving office - based buprenorphine in south - eastern france.11 a cross - sectional survey of 404 subjects receiving buprenorphine in various treatment settings found that 47% reported that they previously injected buprenorphine.12 more recently, 9%10% of respondents reported intravenous or inhaled misuse of buprenorphine in the nationwide observation of illicit or misused psychotropic drugs (oppidum) survey.13 diversion (ie, selling or giving away medication) of buprenorphine to the illicit market is also a major concern and has contributed to an extensive black market in some european countries. doctor - shopping, involving patients obtaining prescriptions for the same medication from several physicians at the same time, is thought to be a major route of diversion of buprenorphine to the illicit drug market,14,15 and has recently been estimated to account for 12.5%16.9% of reimbursed buprenorphine in france.13,14,16 the recent introduction of buprenorphine naloxone to france in january 2012 may help to counter these problems as it has reduced abuse liability compared with mono - buprenorphine.17 beyond misuse and diversion, ensuring that treatment is uniformly of a high standard remains an important need. however, national guidelines in france lack clarity, and a lack of standardized mandatory training of physicians in the treatment of drug dependence may lead to variations in the quality of treatment. given the unique history and treatment model for mat in france, collection of real - world data on the status of treatment provision is of potential value to treatment providers and policymakers alike, in seeking to highlight where current policies or treatment approaches could be improved. we report the results of project access france, a national survey of patients in mat, out - of - treatment opioid users, and treating physicians, which was conducted in 2011 and formed part of a larger european initiative, the european quality audit of opioid treatment (equator).18,19 the methodology and questionnaire instruments in this study were adapted from a survey (project [improve ] poverty reduction in europe : social policy and innovation) which was designed to examine the barriers to ost access, entry and retention in germany,20 and to date have been applied in ten national surveys in europe.21 the english versions of the questionnaires have been published previously in full.18 the present survey was conducted between 2011 and 2012. physicians treating opioid - dependent patients were identified by research collaborators or from official lists, and contacted. those who agreed to participate in the study were selected until the number of recruited physicians reached 100. this included 90 general practitioners (gps) in public practice, two specialist physicians in public practice, five physicians practicing at hospitals, and three physicians whose place of work was not clarified. each physician received 70 for participating in the interview and support centers received 20 per recruited physician. physicians were asked to comment on a number of issues including patient awareness / knowledge of treatment options, psychosocial interventions, patient mental and physical health, drug - use outcomes, barriers to treatment entry and retention, and their attitudes and practices. patients in mat and out - of - treatment opioid users were recruited by physicians using a snowball method. physicians (both those who consented to take part in the physician sample and others from the list of treating physicians) were contacted to ask for assistance in recruiting patients. physicians who responded positively were requested to recruit three to four patients in a week and provide them with the self - completion questionnaire. as such both patients and out - of - treatment opioid users received a 40 grocery voucher to take part in the survey. patients currently in mat (n=130) and out - of - treatment opioid users (n=33) were either interviewed or completed questionnaires. briefly, patients and out - of - treatment users answered questions on demographics, details of current mat (for current mat patients only), use of psychosocial counseling, current and past illegal drug use, sharing of injecting equipment, past misuse and diversion of prescribed medications, satisfaction with treatment, previous treatment experience, self - reported stability, past imprisonment, and access to treatment in prison. participation was voluntary and all participants were informed about the study and provided consent prior to participating. a research agency (gfk healthcare), in lyon, france, was responsible for the enrollment of participants and data collection was undertaken in accordance with the european pharmaceutical market research association code of conduct. as part of the wider european (equator) project, as the main objective of this study was to characterize the state of treatment provision, results were analyzed and are presented here using descriptive statistics (means and frequencies). accordingly, formal hypothesis and significance testing was not undertaken or required to meet these study aims. additionally, this decision accounted for the relatively large number of parameters being assessed on a relatively modest sample size, which would have resulted in low statistical power after adjustments (eg, bonferroni) for multiple comparisons. (n=100) were predominantly male (82%) and gps (93%) with a mean duration of 14.7 years experience prescribing mat. patients (n=130) were predominantly male (78%) with a mean age of 34.9 years ; most had not been in mat previously (64% ; mean number of previous mat episodes = 1.5). prior to their current mat episode, 25% of patients had received buprenorphine or buprenorphine naloxone and 12% had received methadone. among surveyed out - of - treatment opioid users (n=33) the mean age was 32.8 years and 73% were male ; most (73%) had never been in a mat program. on a scale of 1 to 10, where 1 was not at all satisfied and 10 was totally satisfied, physicians had an average satisfaction rating with treatment programs in their area of 6.9. lack of education of different treatment options and therapies available (47%), and a lack of awareness of how to get treatment (35%) as barriers to patients entering treatment in their regions. the treatment rules that most physicians considered important for patients to meet to receive treatment were stopping all illegal drug use (60%) and having to attend all appointments (60%) (table 3). most physicians (51%) reported that patients expressly request a specific mat preparation either always or often (table 4) ; 93% of physicians reported that buprenorphine was the most commonly requested mat. most patients (77%) were treated with buprenorphine, whereas 20% were treated with methadone and 3% with buprenorphine naloxone ; 53% of patients receiving buprenorphine (n=88) were receiving daily doses lower than 12 mg, and 44% of patients receiving methadone (n=42) were receiving daily doses 80 mg. most physicians (82%) stated that they were concerned about misuse and diversion of mat medication, and a significant proportion considered that misuse (74%) and diversion (77%) were a what factors most urgently need to be changed in order to encourage more patients to seek mat, physicians selected greater awareness among users that treatment is effective (34%), a greater number of treatment centers (33%), a greater acceptance that addiction is a disease rather than just a criminal act (31%), and key changes that physicians considered were needed to improve patient care were more counseling and behavioral therapeutic intervention (50%), improved education and training of physicians (29%), improved collaboration between treatment services (35%), individualized treatment plans (25%), and easier access to therapy (23%). the majority of patients (n=130) were fairly or very satisfied with their mat (88%) and the success of their mat program (86%). when asked why they decided to begin mat, the most common reasons given by patients were that they wanted to improve their health (61%) and wanted to end their dependence [on illegal drugs ] for good (49%) (figure 1). most patients (78%) reported that it was very easy or fairly easy to find a doctor to provide mat. the treatment rules with most impact on the provision of mat as judged by patients are shown in table 3. stopping all illegal drug use was the treatment rule most frequently cited by patients as a requirement for starting treatment, the hardest rule for them to meet, and the rule with the most impact on daily life once in treatment. greater flexibility in the rules (34%), better availability of treatment (33%), more information about treatment options (31%), and fewer conditions to start treatment (30%) would have encouraged them to start treatment earlier ; furthermore, they reported that greater flexibility (47%), less rules (38%), and more personal responsibility (28%) would have made it easier to stay in treatment. when asked which of the following mat medications had you heard of prior to beginning your therapy?, 82% were aware of buprenorphine, 74% were aware of methadone, and 5% were aware of buprenorphine naloxone ; the timing of the survey was close to the time of introduction of the latter option in france. of patients answering the question, very good impression of buprenorphine compared with methadone (35% for liquid methadone, 19% for methadone tablets, 2% for levomethadone) and 13% for buprenorphine the frequency of patients requesting and receiving specific mat medications (according to patients or physicians) is shown in table 4. when patients were asked if they were currently receiving psychosocial counseling of any kind, 57% reported that they were not. however, of those who had received psychosocial therapy, 74% found it helpful. according to patients, abstinence from all drugs (29%) and long - term mat (19%) were the most common goals set by their physicians during their current treatment. in 19% of cases, patients reported that their physician did not set a goal. most patients were permitted unsupervised dosing, with 15% reporting being allowed take - home doses at weekends and/or holidays and 58% being allowed take - home doses not only at weekends and/or holidays but more often. only a quarter of patients stated that they were required to take every dose under a doctor s supervision or under a pharmacist s supervision. nearly half of patients (48%) reported taking illegal drugs in addition to their mat at least twice a month (figure 2). the most common reasons stated by patients for on - top drug use were because they wanted to get high occasionally (50%) and their drug treatment was not controlling their cravings very well (21%). on - top drug use was reported by 62% of patients on methadone and 42% of patients on buprenorphine. of patients receiving buprenorphine, 7% and 9% reported using cocaine and heroin, respectively, whilst 29% and 26% of patients receiving methadone reported using these drugs. when patients were asked why they took illegal drugs in addition to or instead of their mat, 25% of patients receiving buprenorphine and 15% of patients on methadone said this was because drug treatment was not controlling their cravings very well. past misuse (snorting or injection) of mat medications was reported by 15% of patients, while past diversion (selling or giving away mat medication) was reported by 39% of patients (figure 3). the most common reasons for misuse were to get high occasionally (37%) or because their medication does nt control cravings very well when taken at the prescribed dose (32%). for patients reporting past diversion, 70% said that they diverted medication to help others to treat themselves. over half (60%) of patients judged their physical health as good or very good, whereas a lower proportion of patients (48%) judged their mental health as out - of - treatment opioid users (n=33) reported that they used drugs on a regular basis ; 42% reported using heroin, 21% used benzodiazepines (not prescribed to them), and 18% used crack. some out - of - treatment opioid users reported sharing needles (18%), syringes (21%), spoons (33%), or water (36%). a majority (51%) well informed or very well informed about treatment options, with most being aware of buprenorphine (91%) and methadone (82%) but only 3% being aware of buprenorphine naloxone. the most frequent reasons given by out - of - treatment opioid users for staying out of mat were because they would like to still use drugs sometimes (52%), they did not wish to stop / were happy with their lifestyle (30%), or they did nt like what they heard about treatment programs (30%). almost a third (31%) of out - of - treatment opioid users perceived the rules of going to the dispensing points regularly and always at the same time as a negative aspect of treatment. most (88%) stated that they are registered with a physician and have been in contact with a medical service (59% with gps ; 45% with free clinics) within the last 12 months. despite not currently receiving mat, more than half of out - of - treatment opioid users reported receiving psychological help (58%) or help with reduction of drug consumption (52%). (n=100) were predominantly male (82%) and gps (93%) with a mean duration of 14.7 years experience prescribing mat. patients (n=130) were predominantly male (78%) with a mean age of 34.9 years ; most had not been in mat previously (64% ; mean number of previous mat episodes = 1.5). prior to their current mat episode, 25% of patients had received buprenorphine or buprenorphine naloxone and 12% had received methadone. among surveyed out - of - treatment opioid users (n=33) the mean age was 32.8 years and 73% were male ; most (73%) had never been in a mat program. on a scale of 1 to 10, where 1 was not at all satisfied and 10 was totally satisfied, physicians had an average satisfaction rating with treatment programs in their area of 6.9. lack of education of different treatment options and therapies available (47%), and a lack of awareness of how to get treatment (35%) as barriers to patients entering treatment in their regions. the treatment rules that most physicians considered important for patients to meet to receive treatment were stopping all illegal drug use (60%) and having to attend all appointments (60%) (table 3). most physicians (51%) reported that patients expressly request a specific mat preparation either always or often (table 4) ; 93% of physicians reported that buprenorphine was the most commonly requested mat. most patients (77%) were treated with buprenorphine, whereas 20% were treated with methadone and 3% with buprenorphine naloxone ; 53% of patients receiving buprenorphine (n=88) were receiving daily doses lower than 12 mg, and 44% of patients receiving methadone (n=42) were receiving daily doses 80 mg. most physicians (82%) stated that they were concerned about misuse and diversion of mat medication, and a significant proportion considered that misuse (74%) and diversion (77%) were a what factors most urgently need to be changed in order to encourage more patients to seek mat, physicians selected greater awareness among users that treatment is effective (34%), a greater number of treatment centers (33%), a greater acceptance that addiction is a disease rather than just a criminal act (31%), and key changes that physicians considered were needed to improve patient care were more counseling and behavioral therapeutic intervention (50%), improved education and training of physicians (29%), improved collaboration between treatment services (35%), individualized treatment plans (25%), and easier access to therapy (23%). the majority of patients (n=130) were fairly or very satisfied with their mat (88%) and the success of their mat program (86%). when asked why they decided to begin mat, the most common reasons given by patients were that they wanted to improve their health (61%) and wanted to end their dependence [on illegal drugs ] for good (49%) (figure 1). most patients (78%) reported that it was very easy or fairly easy to find a doctor to provide mat. the treatment rules with most impact on the provision of mat as judged by patients are shown in table 3. stopping all illegal drug use was the treatment rule most frequently cited by patients as a requirement for starting treatment, the hardest rule for them to meet, and the rule with the most impact on daily life once in treatment. patients reported that greater flexibility in the rules (34%), better availability of treatment (33%), more information about treatment options (31%), and fewer conditions to start treatment (30%) would have encouraged them to start treatment earlier ; furthermore, they reported that greater flexibility (47%), less rules (38%), and more personal responsibility (28%) would have made it easier to stay in treatment. well informed or very well informed about treatment options prior to beginning treatment. when asked which of the following mat medications had you heard of prior to beginning your therapy?, 82% were aware of buprenorphine, 74% were aware of methadone, and 5% were aware of buprenorphine naloxone ; the timing of the survey was close to the time of introduction of the latter option in france. of patients answering the question, very good impression of buprenorphine compared with methadone (35% for liquid methadone, 19% for methadone tablets, 2% for levomethadone) and 13% for buprenorphine the frequency of patients requesting and receiving specific mat medications (according to patients or physicians) is shown in table 4. when patients were asked if they were currently receiving psychosocial counseling of any kind, 57% reported that they were not. however, of those who had received psychosocial therapy, 74% found it helpful. according to patients, abstinence from all drugs (29%) and long - term mat (19%) were the most common goals set by their physicians during their current treatment. in 19% of cases, patients reported that their physician did not set a goal. most patients were permitted unsupervised dosing, with 15% reporting being allowed take - home doses at weekends and/or holidays and 58% being allowed take - home doses not only at weekends and/or holidays but more often. only a quarter of patients stated that they were required to take every dose under a doctor s supervision or under a pharmacist s supervision. nearly half of patients (48%) reported taking illegal drugs in addition to their mat at least twice a month (figure 2). the most common reasons stated by patients for on - top drug use were because they wanted to get high occasionally (50%) and their drug treatment was not controlling their cravings very well (21%). on - top drug use was reported by 62% of patients on methadone and 42% of patients on buprenorphine. of patients receiving buprenorphine, 7% and 9% reported using cocaine and heroin, respectively, whilst 29% and 26% of patients receiving methadone reported using these drugs. when patients were asked why they took illegal drugs in addition to or instead of their mat, 25% of patients receiving buprenorphine and 15% of patients on methadone said this was because drug treatment was not controlling their cravings very well. past misuse (snorting or injection) of mat medications was reported by 15% of patients, while past diversion (selling or giving away mat medication) was reported by 39% of patients (figure 3). the most common reasons for misuse were to get high occasionally (37%) or because their medication does nt control cravings very well when taken at the prescribed dose (32%). for patients reporting past diversion, 70% said that they diverted medication to help others to treat themselves. over half (60%) of patients judged their physical health as good or very good, whereas a lower proportion of patients (48%) judged their mental health as out - of - treatment opioid users (n=33) reported that they used drugs on a regular basis ; 42% reported using heroin, 21% used benzodiazepines (not prescribed to them), and 18% used crack. some out - of - treatment opioid users reported sharing needles (18%), syringes (21%), spoons (33%), or water (36%). a majority (51%) well informed or very well informed about treatment options, with most being aware of buprenorphine (91%) and methadone (82%) but only 3% being aware of buprenorphine naloxone. the most frequent reasons given by out - of - treatment opioid users for staying out of mat were because they would like to still use drugs sometimes (52%), they did not wish to stop / were happy with their lifestyle (30%), or they did nt like what they heard about treatment programs (30%). almost a third (31%) of out - of - treatment opioid users perceived the rules of going to the dispensing points regularly and always at the same time as a negative aspect of treatment. most (88%) stated that they are registered with a physician and have been in contact with a medical service (59% with gps ; 45% with free clinics) within the last 12 months. despite not currently receiving mat, more than half of out - of - treatment opioid users reported receiving psychological help (58%) or help with reduction of drug consumption (52%). responding to the acquired immunodeficiency syndrome (aids) epidemic and the significant costs of opioid dependence, france adopted a unique treatment model for opioid dependence based primarily on open access to buprenorphine - based treatment in primary care settings. previous evidence indicates that this approach has been effective in reducing drug - related deaths, blood - borne virus transmission, and other harms associated with illicit opioid abuse.7,9,10 findings from the project access france survey provide further evidence to support the french model for opioid dependence treatment. most patients reported it was easy to access a doctor who could provide treatment and few were still using heroin. in fact, compared with other european union (eu) countries in equator, including some that have invested heavily in specialist clinic - led treatment models, french patients compared favorably on outcomes relating to abstinence from heroin use, employment, avoidance of imprisonment, and number of past treatment episodes.19,22 these findings could translate into lower costs if reflective of real - world outcomes at a national level. notably, unsupervised dosing is also less costly than supervised dosing.23 despite these positives, it is also important to consider the potential for variable, or even suboptimal, treatment provision with this strategy. this includes the involvement of physicians who are often gps, not addiction specialists or psychiatrists, without formal training in prescribing mat. project access france reveals that 38% of patients have diverted their mat medication at some point naloxone has been developed as an effective abuse - deterrent formulation of buprenorphine (the most common form of mat in france)17,25,26 it was not widely used in france at the time of the survey and awareness remained low. notably, where patients reported past misuse of their own medication or continued use of illicit drugs, this was often to get high or because their medication had failed to suppress cravings adequately. just over half of 88 patients receiving buprenorphine were receiving daily doses lower than 12 mg and just under half of 42 patients receiving methadone were receiving daily doses 80 mg. under - dosing may be an important issue in france. in a previous study of gps from south - eastern france (n=345), many untrained gps and a significant minority of trained gps were shown to be prescribing an ineffective dosage of buprenorphine or a potentially dangerous combination of buprenorphine and benzodiazepines.27 a previous eu survey revealed a pattern of under - dosing for both buprenorphine and methadone across four major eu countries, including france.28 in the current study, less than half of patients were participating in psychosocial counseling and similarly less than half rated their mental health as good or very good. moreover, physicians most commonly cited more psychological / behavioral counseling and improved education / training for physicians as steps that would help to improve treatment provision. in addition, they often reported that lack of patient knowledge of different treatment options was a barrier to patients entering treatment. patients in france are often able to make good progress in terms of reducing their drug use and maintaining employment, but further benefits may be possible with improved training of physicians, optimized dosing, and appropriate adjunctive support to target wider health needs. project access france provides new insights regarding the access to and provision of treatment associated with current systems of mat in france from the perspective of physicians, patients, and out - of - treatment opioid users, and differs from other surveys conducted in france. for example, the annual oppidum survey draws respondents from specialty treatment centers.5 these patients tend to be referred from gps and may therefore represent a more challenging or treatment - resistant population.5 similarly, the european monitoring centre for drugs and drug addiction collects data from national monitoring centers in europe but does not collect data at the individual patient level.29 project access france has a number of limitations that may impact on the interpretation of findings. these include the observational, nonrandomized nature of the study and the small sample sizes. these factors expose the study to potential biases and limit the feasibility of more sophisticated statistical analyses. selection bias may have occurred with regard to the convenience sampling of patients, selection of participating sites / regions, and self - selection (participants could decline to take part). the predominantly open - access, gp - based mat model in france is unique in europe and offers the potential to widen access to mat for patients with opioid dependence. however, real - world evidence from physicians, patients, and out - of - treatment opioid users in france suggests that challenges persist with regard to reducing misuse and diversion of mat medications and in appropriate provision of psychosocial support. | aimproject access france was a national survey designed to provide real - world observations on the status of opioid dependence treatment in france.methodsthe views of physicians (n=100), patients (n=130), and out - of - treatment opioid users (n=33) were collected via interviews and questionnaires.resultsphysicians reported being moderately satisfied with treatment programs in their area (rating 6.9 out of 10). most physicians (82%) reported being concerned about misuse and diversion of medication - assisted treatment (mat) medications and 50% identified psychosocial / behavioral counseling as the key change that would most improve patient care. among patients, the mean number of previous mat episodes was low (1.5) ; 78% reported that it was easy to access a doctor to undergo mat ; 14% reported regularly or sometimes using heroin ; misuse and diversion were reported in 15% and 39% of patients, respectively ; and 57% of patients were not receiving psychosocial help. out - of - treatment opioid users reported using drugs on a regular basis (42% regularly used heroin) and cited not wanting to give up drugs completely as the most frequent reason for staying out of mat.conclusionthis survey highlights a number of positive features of the open - access, gp - based treatment model for opioid dependence in france. challenges remain with regard to continued misuse / diversion of mat medications and limited patient access to psychosocial support. |
a thorough understanding of root canal anatomy and morphology is required for achieving high levels of success in endodontic treatment. failure to recognize variations in root or root canal anatomy can result in the unsuccessful endodontic treatment. hence, it is imperative that the clinician be well informed and alerted to the commonest possible variations. hoen and pink in their analysis on teeth requiring re - treatment, found a 42% incidence of missed roots or canals. numerous reports of root canal variations in these teeth have been reported in the literature. vertucci in his series of studies conducted on extracted teeth, reported 2.5% incidence of a second canal. in the case of mandibular first premolars, it is normally a single - rooted tooth. the frequency of occurrence of two roots is 1.8% while three roots are reported to be present in 0.2%of cases. in single - rooted mandibular first premolars, these anatomic abnormalities are additional challenges, which begin at the case assessment and involve all operative stages, including access cavity design, localization, cleaning, and shaping of the root canal system. although preoperative radiography gives a two - dimensional image of a three - dimensional object, precise interpretation can reveal external and anatomic details that suggest the presence of extra canals or roots. the purpose of this clinical report is to describe an anatomic abnormality that was detected during routine root canal treatment in a mandibular first premolar. a 24-year - old male patient came to a private dental clinic with pain in the left lower back tooth. on radiographic examination preoperative radiograph revealed radiolucency involving pulp with respect to 34 [figure 1a ]. diagnosis of acute irreversible pulpitis was made. also iopa revealed 34 having four roots. to confirm the presence of four roots two more radiographs one with distal angulation and the other with mesial angulation access was gained to the pulp chamber after administration of local anesthesia (2% lignocaine with 1:100000 epinephrine), under rubber dam isolation. to gain sufficient access to the canals, the conventional access opening was modified into one that was wider. after careful inspection, four canal orifices were located and patency was ascertained using a small size k - file (kerr, orange, california). working length was established with the use of apex locator (root zx, j. morita inc.) (a) preoperative intraoral periapical radiograph (straight angulation) (b) preoperative intraoral periapical radiograph (mesial angulation) (c) preoperative intraoral periapical radiograph (distal angulation) (a) working length radiograph (b) postobturation radiograph (c) recall radiograph 1-year postoperatively the canals were cleaned and shaped with hand k - files and nickel titanium rotary protaper files (dentsply maillefer, switzerland). the canals were sequentially irrigated using 5.25% sodium hypochlorite and 17% edta during the cleaning and shaping procedure. the canals were thoroughly dried and obturation was done using f2 pro taper gutta - percha and ah plus sealer (dentsply, maillefer, switzerland). the post - endodontic permanent restoration was completed with composite (3 m espe dental products, st paul, mn) [figure 2b ]. a 1-year recall radiograph showed satisfactory healing and was advised to get this tooth crowned [figure 2c ]. the presence of extra roots or canals in mandibular premolars is undoubtedly an endodontic challenge. clearly, these findings are clinically important as in a study at the university of washington assessing the results of endodontic therapy, the mandibular first and second premolars showed failure rates of 11.45% and 4.54%, respectively. conceivably, these findings could be due to the complex root canal anatomy of a large number of these teeth. a wide range of opinions are reported in the literature regarding the number of root canals, but there are very few reports on the variations in the numbers of roots that occur in mandibular premolars. accurate preoperative radiographs, straight and angled, using parallel technique are essential in providing clues as to the number of roots that exist. optimum opening of the access cavity is absolutely necessary. despite the existence of complicated dental anatomy, cautious use of rotary or hand nickel titanium files prepares the canals to a predetermined shape. there are many reports regarding four root canals in mandibular second premolar[1214 ] and five - canaled mandibular second premolar but four canals in mandibular first premolar is hard to find in the published literature. these discussions also validate an important consideration that must not be overlooked, that is, the anatomic position of the mental foramen and the neurovascular structures that pass through it, in close proximity to the apices of the mandibular first and second premolars. there are reports in the literature, of flare - ups in mandibular first and second premolars with associated paresthesia of the inferior alveolar and mental nerves. the failure to recognize the presence of extra root or canals can often lead to acute flare - ups during treatment and subsequent failure of endodontic therapy. successful and predictable endodontic treatment requires knowledge of biology, physiology, and root canal anatomy. the clinician should be astute enough to identify the presence of unusual numbers of roots and their morphology. a thorough knowledge of root canal anatomy and its variations, careful interpretation of the radiograph, close clinical inspection of the floor of the chamber, and proper modification of access opening are essential for a successful treatment outcome. | mandibular premolars have earned the reputation for having aberrant anatomy. the literature is replete with reports of extra canals in mandibular first premolars, but reports about the incidence of extra roots in these teeth are quite rare. this paper attempts at explaining a rare case of successful endodontic management of a four - rooted mandibular first premolar with diagnostic, interoperative and postoperative radiographic records along with a substantial data on the incidence of extra roots in these teeth. the standard method of radiographic appraisal was maintained as the criteria for determining the presence of extra roots. |
it is regarded as the most expensive disorder among all psychiatric disorders in terms of health care expenditure per patient.1 a recent japanese study estimated the annual burden of disease to exceed 3.5 million yen per patient (approximately us$30,000).2 most of the costs can be attributed to the loss of working ability as schizophrenia patients face a higher likelihood of being unemployed. hospitalization has been identified as another significant cost driver not only in japan but also in the united states3,4 and europe.5,6 hence, relapse prevention that helps to reduce inpatient stays is an important element in the treatment of schizophrenia. there is a wide range of antipsychotic medications available ranging from conventional or typical first - generation antipsychotics, atypical second - generation antipsychotics, and conventional and atypical long - acting injectables (lais). what they all have in common is that the efficacy of the drugs is highly dependent on patient adherence.7 however, adherence levels are reported to be low among schizophrenia patients,8,9 and scientific evidence suggests a strong negative relationship between the level of adherence and the probability of hospitalization for patients in the united states1017 and europe;18,19 refer also, higashi for a recent systematic review on this topic. they found lack of insight, medication beliefs, and substance abuse to be key drivers of nonadherence that in turn leads to a greater risk of relapse, hospitalization, and suicide. we are not aware of any study that utilizes japanese data to analyze the relationship between adherence and hospitalization. in our view, it is worth studying the japanese data because stigmatizing attitudes in response to mental disorders are reported to be more severe in japan,21 which some authors attribute to the old japanese character of the word schizophrenia which literally means the disease of disorganized mind.22 in 2002, the japanese name of schizophrenia was changed into togo shitcho sho or integration disorder in an attempt to reduce discrimination and stigmatization.23 stigmatization was such a major issue that only 7% of japanese psychiatrists informed all their patients about the diagnosis of schizophrenia. most of the time, the patients were kept in a state of uncertainty about their disease.24 japanese were also found to be more reluctant to discuss mental disorders with others outside the family.25 moreover, japanese families are believed to think that a family member with schizophrenia should have care provided only by family members, which is argued to be deep - rooted in confucian ideas.26,27 if this was true, we would expect only a weak relationship between poor adherence and hospitalization because the patient would be taken care of by the family when experiencing a mild relapse. the aim of this study is to analyze the relationship between medication adherence and hospitalization in japan using japanese schizophrenia claims data. we address not only underadherence to medication but also the issue of overadherence that has recently been identified as a problem not only for schizophrenia28 but also for other indications.29 within the framework of a multivariate regression analysis, we statistically assess factors that drive the rehospitalization of patients with schizophrenia within 6 months after a schizophrenia - related hospital discharge. the japan medical data center co., ltd database is an employer - based database of health insurance claims with approximately 2.5 million beneficiaries. it covers 40 of approximately 1,500 japanese health insurers and contains detailed electronic records of health insurance claim information on inpatient, outpatient, and prescription drug data at the individual member level over a specified period of time. the database contains data for 12,047 patients who have records for schizophrenia and antipsychotic agents (atc code n05a) between january 1, 2009, and april 30, 2013, in japan. we restricted our analysis to patients between 18 years and 65 years of age who have a schizophrenia diagnosis (icd-10 code f20) and had a hospitalization discharge for schizophrenia within the research timeframe. patients must be prescribed an antipsychotic medicine (n05a) within a month following discharge. the index hospitalization was not counted as a hospitalization ; instead, only hospitalizations that occurred after the index hospitalization were defined as an event in our multivariate regression analysis. only hospital - stays of 7 days or longer were taken into consideration both for the index hospitalization and possible subsequent rehospitalizations because expert interviews indicated that hospital stays shorter than 7 days are probably due to incorrect coding and not schizophrenia related. indeed, a recent investigation of more than 8,000 japanese schizophrenia patients reported an average length of stay of 3,242 days.30 we identified 657 patients in our database who fulfilled our inclusion criteria. we used the medication possession ratio (mpr) that was calculated for antipsychotic agents. the mpr was calculated as the number of days supply of medication divided by the number of days the patient was in the database from their index date (assuming patient should be on medication for the entire period). the average value of the 6-month interval following index hospitalization was used for the analysis. in case of an event (rehospitalization) during the 6-month interval, the average mpr value was taken for the time between index hospitalization and the event. time within a nonpsychiatric hospitalization was included in this analysis as they should be receiving their psychiatric medications and the reason for hospitalization is not related to schizophrenia. we adjusted for the number of days of activity for lais such as risperidone (risperdal consta) or haloperidol decanote. risperdal consta for instance is a 14-day treatment ; every dose was counted as 14 days supply in the mpr calculation. based on the calculated mpr value, we formed three groups : mpr 1.1 (overadherence). this classification is in line with valenstein.14 it should be noted that this adherence measure is based on the number of collected prescriptions. we included the following control variables in the model : if the proposition is true that family members in japan are substantially involved in providing care, we might observe a positive age effect. while parents are probably able to look after their children with schizophrenia up to a certain age, older patients might find it more difficult to receive family support as a substitute for hospitalization. some authors suggest that men suffer more negative symptoms and more severe course of illness than women, which would suggest a higher hospitalization probability of men.3133 most claims data analyses include the insurance type as an explanatory variable ; for instance, refer suh for a korean study.34 in our case, insurance type is either an individual membership or being insured as a family member. for the insurance type, our employer - based database is biased in that it does not cover unemployed persons. the employment rate of japanese patients with schizophrenia is less than 30%,2 and patients suffering from a severe form of schizophrenia are probably underrepresented in the database. note that there is a 100% employment ratio of individual members in our database. on the other hand, there is no such employment bias for family members. hence, we expect family members to be more often hospitalized than individual members, because family membership is not linked to employment status. people with schizophrenia have a wide range of multiple - comorbid physical health problems including alcohol abuse compared with people without schizophrenia.35,36 we expect people with comorbidities to be rehospitalized more often. the length of the index hospitalization captures the disease severity and we also expect a positive impact of this variable. for the statistical analysis, we employed a multivariate cox proportional hazard model.37 for the coefficients, a significance level of 10% was chosen, which is a common threshold within the framework of real - world data analysis.38 if the proposition is true that family members in japan are substantially involved in providing care, we might observe a positive age effect. while parents are probably able to look after their children with schizophrenia up to a certain age, older patients might find it more difficult to receive family support as a substitute for hospitalization. some authors suggest that men suffer more negative symptoms and more severe course of illness than women, which would suggest a higher hospitalization probability of men.3133 most claims data analyses include the insurance type as an explanatory variable ; for instance, refer suh for a korean study.34 in our case, insurance type is either an individual membership or being insured as a family member. for the insurance type individual member, our employer - based database is biased in that it does not cover unemployed persons. the employment rate of japanese patients with schizophrenia is less than 30%,2 and patients suffering from a severe form of schizophrenia are probably underrepresented in the database. note that there is a 100% employment ratio of individual members in our database. on the other hand hence, we expect family members to be more often hospitalized than individual members, because family membership is not linked to employment status. people with schizophrenia have a wide range of multiple - comorbid physical health problems including alcohol abuse compared with people without schizophrenia.35,36 we expect people with comorbidities to be rehospitalized more often. the length of the index hospitalization captures the disease severity and we also expect a positive impact of this variable. for the statistical analysis, we employed a multivariate cox proportional hazard model.37 for the coefficients, a significance level of 10% was chosen, which is a common threshold within the framework of real - world data analysis.38 it can be seen that only a small fraction of 25.9% can be classified as adherent (mpr between 0.8 and 1.1). surprisingly, the majority (51%) of the patients belong to the overadherence group where the number of prescriptions would cover more than 110% of the required time. the regression results of the cox model are displayed in table 2. according to these results, family member type, presence of a bipolar and anxiety disorder, and both poor adherence and overadherence the hazard ratios are 4.7 for the poor - adherence group and 2.0 for the overadherence group. the results suggest that the medication adherence of schizophrenia patients as measured by the mpr is significantly related to the likelihood of rehospitalization. both the poor - adherence group as well as the overadherence group face a higher chance of rehospitalization compared to the good - adherence group. this result is in line with international studies that were undertaken in other cultural environments. valenstein who used the same definition of adherence found that patients with both poor adherence and excess medication fills had higher psychiatric admission rates than patients with good adherence in the united states. we therefore do not observe a specific japan effect as hypothesized in the introduction. nevertheless, this study contributes to the existing evidence in emphasizing the importance of medication adherence in the treatment of schizophrenia patients. one possible option from a clinical point of view is treatment with lais because evidence suggests that lais improve patient s adherence and eventually reduce hospital stays.3943 accordingly, several cost - effectiveness studies demonstrated economic superiority of lais over oral antipsychotics.4448 spill for instance observed patients who switched from an oral treatment to risperidone lai in the framework of a mirror image design study. they report overall savings in drug and institutional - care costs of 21.1% after 1 year.49 on the other hand, waddell and tylor found that lais continue to have an image problem among patients and mental health staff and are primarily used as a last resort for the most stigmatized individuals.50 for this reason, the utilization of lais is still low despite their clinical and economic benefits. the high percentage of patients who are overadherent (51%) is also worth noting. a us claims data analysis found only 7.6% of patients with medication oversupply.26 as we only included patients who have been hospitalized, this gap might be due to differences in the respective patient populations. however, this seems to be only part of the story as a us observational study of patients who are discharged with schizophrenia found only 21% of patients received doses in excess of recommended levels.51 institutional factors of the japanese prescription drug market might serve as an alternative explanation.52 as we only observe the number of prescriptions and not the intake of the medication, the issue of overadherence should be studied in more detail in future research. although our results suggest a correlation between overadherence and hospitalization, we do know little about potential causal relationships. one possible explanation is we could not adequately control for disease severity. if illness severity and oversupply of medication are correlated, the observed increased hospitalization rates would be a consequence of the disease severity and not necessarily of overmedication itself. when it comes to our control variables, we observef that family members are more likely to be rehospitalized than individual members, which can be attributed to the employment bias of the databank we used. patients with comorbidities are more difficult to treat in an outpatient setting or within the family. although the variables age and sex are not significant drivers of rehospitalization, the hazard ratios have the expected values of greater than 1 and less than 1, respectively. both male sex and age are associated with a higher risk. with regard to sex, it is well described in the literature that women with schizophrenia score better both in social53,54 and cognitive functioning,55,56 which probably influences the need for inpatient treatment. the results of this study confirm the notion that good adherence with antipsychotic medication reduces the risk of rehospitalization of schizophrenia patients. health insurers are able to identify poorly adherent insured patients in their data, a timely intervention in collaboration with medical care providers would be desirable. | backgroundthe aim of this study is to analyze if there is a relationship between adherence to antipsychotic medication and rehospitalization for patients diagnosed with schizophrenia in japan.methodsbased on japanese claims data, we constructed three patient groups based on their medication possession ratio (mpr). controlling for potential confounders, a cox proportional hazard model was employed to assess if medication adherence affects the risk of rehospitalization.resultspatients with good adherence (mprs from 0.81.1) had the lowest rates of admission. both poor adherence (mprs 1.1) were associated with a significant higher risk of rehospitalization with hazard ratios of 4.7 and 2.0, respectively.conclusionthe results of this study support the notion that good adherence to antipsychotic medication reduces the risk of rehospitalization of schizophrenia patients. appropriate measures should be taken to improve adherence of schizophrenia patients. |
coats disease first described in 1908,1 is an idiopathic, typically unilateral, retinal vasculopathy that manifests with retinal telangiectasia, exudation, and retinal detachment.24 coats disease shows a male predominance, occurs more often in early childhood, and can lead to vision loss. less commonly, this condition presents in teenagers and young adults often with less aggressive features.4 coats disease is a major simulator of retinoblastoma, a life - threatening pediatric ocular malignancy.14 in a study of angiographic findings of patients with coats disease, we noticed that occlusion of the retinal microvasculature with peripheral nonperfusion and shunt or microshunt formation between arterioles and venules in the retina was a prominent finding.5 this led to a speculation regarding serum factors that could lead to the microocclusions, such as a hypercoagulability state. realizing that such a state would more likely lead to bilateral manifestations, we continued to explore serum features in coats disease. in 2012, we found a significantly high level of anti - cytomegalovirus (cmv) immunoglobulin g (igg) in 92% of coats patients, higher serum proteins c and s, anti - herpes simplex virus (hsv) igg i / ii, alpha-2 globulin, and homocysteinemia in patients with coats disease.6 in the second step of the analysis, it was planned to compare these factors in patients with coats disease with a control group. herein, we evaluated serum hypercoagulable factors and viral and protozoan diseases in patients with coats disease versus normal controls. a prospective, single - center, comparative, consecutive cross - sectional study was conducted for evaluating blood hypercoagulability state and infectious diseases, including cmv, herpes simplex, epstein - barr virus (ebv), toxoplasma and toxocara infections, in all patients with coats disease from february 2011 to december 2013. the research adhered to the tenets of the declaration of helsinki and the study was approved by tehran university of medical sciences institutional review board. each patient or parents were carefully informed about the purpose of the research, and oral consent for laboratory examinations was obtained. each patient or coats disease was defined as unilateral or bilateral retinal vasculopathy characterized by retinal telangiectasia, capillary non - perfusion, multiple aneurysmal formation, exudation, and exudative retinal detachment.19 patients with coats disease were grouped into those aged 10 years or less versus those older than 10 years. clinical factors were compared to a control group, which consisted of patients with cataract undergoing lensectomy for congenital, traumatic, or senile cataract, with no evidence of retinal vascular disease. patients were evaluated with best - corrected visual acuity, indirect ophthalmoscopy for fundus features and color fundus photography, fluorescein angiography, and b - scan echography as needed. fluorescein angiography was performed using a scanning laser ophthalmoscope (hra, heidelberg, germany) or retcam 120 (clarity medical systems, inc., the blood sample for serum studies was obtained from patients immediately before the treatment of coats disease. treatment options included cryotherapy, photocoagulation, and intravitreal antivascular endothelial growth factor and/or subtenon triamcinolone depending on the patient s condition. serum studies included hemoglobinopathies by hemoglobin electrophoresis ; serum protein electrophoresis ; coagulable states by serum protein c, protein s level, antiphospholipid antibody, anticardiolipine antibody, antithrombin iii, homocysteine level, and lipid profile ; and infectious states by anti - hsv igg, anti - cmv igg, anti - epv, anti - toxoplasma and anti - toxocara serum antibodies were measured with a enzyme - linked immunosorbent assay (elisa) technique according to the manufacturer s protocol. statistical analyses were performed using statistical package for social sciences (v 17.0 ; spss, chicago, il, usa). all data are presented as mean standard deviation (sd) or median (range of data). a mann whitney test was used for quantitative or skewed distributed parameters. to assess the relationship between the qualitative variable and coats diagnosis, fisher s exact test was applied. logistic regression models were used to assess the relation of target exposure with coats diseases adjusting for possible confounding factors. statistical analyses were performed using statistical package for social sciences (v 17.0 ; spss, chicago, il, usa). all data are presented as mean standard deviation (sd) or median (range of data). a mann whitney test was used for quantitative or skewed distributed parameters. to assess the relationship between the qualitative variable and coats diagnosis, fisher s exact test was applied. logistic regression models were used to assess the relation of target exposure with coats diseases adjusting for possible confounding factors. a p - value of less than 0.05 was considered to be statistically significant. during the study period, there were 22 patients with coats disease (figure 1) and 19 control patients. of those with coats disease, 13 were 10 years or younger and 9 were older. (table 1). the mean age for coats patients was 14.5 years (median 8 years, range : 2 month to 59 years), and for the control group it was 30 years (median 17 years, range : 282 years) (table 2). for those 22 patients with coats disease, the mean time from the first notice of the disease by patients (or parents) to diagnosis was 7.15.4 months in the younger age group (10 years old) and 7.89.9 months in the older age group (> 10 years old). there were three (13.6%) with bilateral involvement. using the coats disease classification,4 the conditions of the eyes were classified as stage 1 (n=0), stage 2 (n=7), stage 3 (n=12), stage 4 (n=3), and stage 5 (n=0). total retinal detachment was seen in six (28%) cases, whereas macular detachment was seen only in three (14%) cases. in two using fluorescein angiography, avascular non - perfused areas were found in 18 (82%) cases, located in the periphery region in 10 (46%) and in the macular region in 2 (9%) (table 1). the first step of analysis was a comparison of the two age groups of coats disease (10 years vs > 10 years). this revealed no significant difference in the lipid profile, serum antithrombin iii, anticardiolipine and antiphospholipid antibody serum antibody titer as well as polymerase chain reaction test for toxoplasma, toxocara, or ebv in the two groups. there was statistical difference (mann whitney test - skewed deviation) in the two groups with regard to hemoglobin (p=0.02), serum anti - hsv igg (p=0.05), anti - cmv igg (p=0.05), protein c (p=0.07), homocysteine (p 10 years, p=0.05) subgroups compared to controls. applying bonferroni correction did not change the results of serum beta globulin in the group less than 10 years old. logistic regression analysis for the main serologic findings in coats patients (irrespective of age) and control group was performed. after adjustment for age and sex, a significant association persisted for the presence of higher titer of serum beta globulin in coats disease compared to controls (odds ratio [or ] : 1.8, 95% confidence interval [ci ] : 1.03.1, p=0.02) (table 3). anti - hsv showed a mild negative impact (p=0.01, or = 0.94) on the diagnosis of coats disease. after adjusting for age and sex, in those less than 10 years, the serum beta globulin was found to be significantly associated with the diagnosis of coats disease (or : 6.3, 95% ci : 1.232.6, p=0.02). in patients greater than 10 years with coats disease, there was a non - significant association with the diagnosis of coats disease (or : 1.3, 95% ci : 0.82.0, p=0.24). for the older age, anti - hsv antibody was borderline significant (or : 0.98, 95% ci : 0.971.00, p=0.05). the small sample size precluded more conclusive results in the logistic regression analysis, adjusting for all other covariates. in this study, we could not find any association between hypercoagulability state and coats disease (compared to controls). the only factor associated with coats disease was elevated serum beta globulin mainly in the younger age group. we also recognized the negative impact of anti - hsv antibody on coats disease in the older age group. serum protein electrophoresis is a technique to assess the two major fractions of protein in blood, including albumin and globulins.10 albumin is a transport protein that plays a significant role in fat solubility.11 the globulin fraction of blood includes hundreds of serum proteins, including carrier proteins, enzymes, complement, and immunoglobulins. globulins are divided into four groups by electrophoresis : alpha, alpha-2, beta, and gamma, depending on their migratory pattern between the anode and the cathode of the electrophoresis test.11 the beta fraction of serum globulins is known to have two peaks, named as beta 1 and beta 2. beta 1 is composed of mostly transferrin, and beta 2 contains beta - lipoprotein.12 iga, igm, and sometimes igg, along with complement proteins (c3), beta-2 microglobulin, plasminogen, angiostatins, properdin, sex hormone - binding globulin, transferrin, hemopexin, and factor h, can also be identified in the beta fraction.1012 these proteins carry out numerous biological functions in the human body, including iron transport and monitoring immune response.10,11 beta globulin protein can be elevated or depressed in various diseases. beta globulin is found to be elevated in iron deficiency anemia, hypercholesterolemia, pregnancy, estrogen therapy, and also substantially increased in liver disease.9,10,13,14 it is found to be decreased in malnutrition, cirrhosis, and immune deficiency due to decreased synthesis, and in nephrotic syndrome due to protein loss in the kidney.11 in this series of coats disease, the beta globulin fraction was elevated, which is particularly evident in patients lesser than 10 years. this age - related difference could correlate with milder manifestations of coats disease in older patients. the relationship of this fraction with coats disease is unclear, but could correlate to higher c3 (complement protein 3) level or other factors. complement 3 is an important protein in the immune system, which plays a vital role in the acute phase response. it controls several biological processes, including cell lysis, chemotaxis, anaphylaxis, vascular permeability, and cellular membrane adhesion. this protein is categorized as a beta globulin and might represent the elevation of beta globulin in coats disease in this report.12,1518 chronic low - grade inflammation has been correlated with elevated c - reactive protein, as well as complement c3 plasma levels, and these can be predictive of arterial thrombotic events.1620 elevated c3 is also associated with prolonged fibrinolysis.18,2123 both of these roles could relate to the vascular pathology of coats disease. the discrepancy found in the younger and older age groups could suggest a difference in the main pathophysiological basis of coats disease in children and adults. there are limitations of this study, including the small sample size and unclear relevance of beta - globulin relationship to the condition studied. this relationship could be further explored with larger cohort and more precise fractionation of the beta - globulin subgroup to better understand the exact protein(s) that contribute to this finding and its relationship to coats disease. this analysis demonstrated that children with coats disease showed elevated serum levels of beta globulin. we speculate that this finding could reflect the known fluorescein angiographic - evident ischemic component and possibly now a low - grade inflammatory component. | purposethe purpose of this study was to investigate the serum hypercoagulability state and common viral and protozoan infections in coats disease versus a normal control group.materials and methodsin this comparative case series, 22 consecutive patients with coats disease and 19 non - coats patients undergoing lensectomy for congenital, traumatic, or senile cataract between january 2011 and june 2014 were included. laboratory data for hypercoagulability states and common viral and protozoan infections were investigated.resultsthe mean age for the coats group was 14.5 years (median 8 years, range : 2 months to 59 years), and for the control group it was 30.6 years (median 17 years, range : 282 years). in patients aged 10 years or younger, anticytomegalovirus immunoglobulin g (igg) (p0.01), homocysteine (p=0.03), and serum beta globulin (p<0.001) were associated with coats disease. in those older than 10 years, higher serum protein s (p=0.04), beta globulin (p=0.05), and gamma globulin (p=0.04) were related to coats diagnosis. after adjusting for sex and age as confounding factors, only beta globulin was found to be associated with coats disease in logistic regression analysis (odds ratio : 1.8, 95% confidence interval : 1.03.1, p=0.02).conclusionserum beta globulin levels appear to be elevated in patients with coats disease. |
juvenile idiopathic arthritis (jia) is the most common chronic rheumatic disease of childhood. it is an heterogeneous disease group of unknown aetiology with distinct presentation, clinical features, and genetic background. jia is a complex genetic disease caused by the effects of environmental factors and multiple genes that act in concert to predispose the host to the development of jia and to determine the different disease phenotypes. cytokines, a group of modulatory proteins or glycoproteins produced by a wide range of cells in response to a variety of stimuli, are important mediators and regulators of synovial inflammation. basal and cell - stimulated cytokine levels differ between individuals ; both genetic and environmental influences have been shown to play a role in their variability. genetic variation that produces altered structure or expression of a cytokine can have evident pathological consequences, as chronic diseases, increased risk of infection, and altered outcome of acute disorders. variations in dna include single nucleotide polymorphisms (snps), microsatellites, and insertion / deletion polymorphisms. a large number of polymorphisms within the coding and noncoding regions of cytokines genes have been identified, and several thousand disease - association studies have been carried out using these variants. defining cytokine gene polymorphisms in jia is linked to the necessity of understanding the aetiology, discovering possible markers of severity, and identifying targets for therapeutic intervention. one of the most important molecule is tnf-, several studies have demonstrated high tnf- levels in both sera and synovial fluid of children with chronic arthritis [810 ]. several snps have been identified within the promoter, exonic, intronic, and 3-untranslated region of the tnf- gene, but conflicting results have been reported [11, 12 ]. although many snps have been reported, only a very small minority of the genetic variants published have proven functional consequences and have been associated with susceptibility to jia. furthermore, associations between tnf- polymorphisms and subtypes of jia have been found [1316 ]., biologic drugs have been introduced in the treatment of jia. among these drugs tnf- antagonists (etanercept, infliximab, and adalimumab) play a primary role. etanercept has proven highly efficacious in children with polyarticular jia and is a food and drug administration (fda) approved drug for these patients. unfortunately, there is a group of patients, defined as nonresponders, that has no benefit from tnf- blockade and has a worst prognosis. defining the gene responsible for the phenotype might allow the identification of subjects who could benefit from a specific therapeutic intervention. at this regard, most medical literature data are based on studies performed in adult rheumatoid arthritis (ra) patients and few studies in jia subjects [15, 2023 ]. the aim of this study is to collect the present knowledge about the principal tnf- gene polymorphisms in jia, their possible role in the pathogenesis, the severity, and the response to drugs. in this work we reviewed the current knowledge regarding the role of tnf- polymorphisms in jia with focus on the evidence for pathogenesis, phenotypes, prognosis, and therapeutic response to anti - tnf- drugs. using pubmed from the international library of medicine, relevant literature on the role of tnf- polymorphisms in jia from january 1990 to november 2010 five researches were performed for the keywords : tumor necrosis factor alpha polymorphism, juvenile arthritis and tumor necrosis factor alpha, juvenile arthritis and tumor necrosis factor alpha polymorphism, tumor necrosis factor alpha and arthritis therapy, and tumor necrosis factor alpha polymorphism and arthritis therapy. expert opinion and case - report works were not included. using the first keyword, tumor necrosis factor alpha polymorphism, we found 2794 articles and selected 88 of these ; for the second research we used the keyword juvenile arthritis and tumor necrosis factor alpha and found 327 works and of these 54 articles were considered. for the third keyword, juvenile arthritis and tumor necrosis factor alpha polymorphism, we detected 22 articles and considered 14 works. tumor necrosis factor alpha and arthritis therapy detected 4864 and selected 81 of these. the last search tumor necrosis factor alpha polymorphism and arthritis therapy found 69 works and selected 33 of them. finally, a further review of all selected articles led us to choose a total of 105 works. juvenile idiopathic arthritis (jia) is a group of chronic arthritides of unknown aetiology occurring in children under the age of 16 years. in the united states, the international league of association for rheumatology (ilar) defines seven subtypes of jia that exhibit differences in age at onset, clinical features, prognosis, and genetic background. the oligoarticular type is characterized by the involvement of one to four joints (monoarticular or oligoarticular) ; the polyarticular type involves five or more joints ; the systemic arthritis is characterized by the presence of fever and systemic involvement at onset. the diagnosis of juvenile psoriatic arthritis need the presence of arthritis and a typical psoriatic rash or a family history of psoriasis. may involve the sacroiliac and spinal joints thus producing the clinical picture of ankylosing spondylitis [2, 4 ]. all jia subtypes are characterized by persistent joint swelling caused by accumulation of synovial fluid and thickening of the synovial lining. the aetiology of jia is still poorly understood ; the heterogeneity of this disease implies that different factors probably contribute to the pathogenesis. significantly elevation of sera and synovial levels of proinflammatory cytokines and the presence of autoantibodies in jia subjects suggest the involvement of the immune system [2628 ]. the results of genome - wide association studies (gwas) in children and sibling recurrence risk in family studies support the assumption that multiple genes probably contribute to jia susceptibility : it is accepted that an environmental factor (i.e., infections) triggers the disease in genetically predisposed subjects [3, 4, 24, 29 ]. first genetic investigations focused on the human leucocyte antigens (hla) within the major histocompatibility complex (mhc) loci on the sixth chromosome. mhc genes are highly conserved sequences of genome that present a certain variability which cause an individual response to various stimuli. for this reason a variable mechanism of starting and maintenance of the inflammatory response exists in different individuals. the strongest association has been observed in the oligoarthritis subgroup, in particular with the alleles hla - drb1 01, hla - drb1 11 (dr5), hla - drb1 08 (dr8), hla - drb1 13 (dr6), and hla - dpb02. positive rheumatoid factor polyarthritis has been associated with hla - dr4 and hla - drb1 11 ; negative rheumatoid factor polyarthritis has been demonstrated to be associated with hla - drb1 08 and hla - dpb1 03 [30, 31 ]. the hla - drb1 04 has been linked to systemic arthritis [3, 32 ] and the hlab27 have long been recognised as a contributing factor to the development of enthesitis related arthritis ; moreover, this allele seems to be related to axial inflammation with hip involvement and subsequent juvenile ankylosing spondylitis [3, 4, 26, 33, 34 ]. several other genes are probably involved in jia pathogenesis ; indeed elevated synovial and sera levels of tnf- and other inflammatory cytokines as interleukin-1 (il-1), interleukin-6 (il-6), and interferon - gamma (inf-) in jia subjects are likely related to abnormal expression of genes for cytokine production and regulation [26, 33 ]. several studies have focused blood and synovial cytokine polymorphisms ; a major involved cytokine in jia is the tnf- ; the polymorphisms of its gene have been evaluated in different studies to establish its role in the pathogenesis and in the therapeutic response to anti - tnf- drugs [4, 6, 7, 35, 36 ]. tnf- is a proinflammatory cytokine produced as a membrane - bound 26 kda molecule from which the soluble 17 kda active tnf- molecule is released by the tnf- converting enzyme (tace). tnf- is involved in several biologic processes such as tissue remodelling, epithelial cell barrier permeability, macrophage activation, recruitment of inflammatory cells, effectiveness of the local and systemic inflammation, and amplification of other proinflammatory cytokine actions [3941 ]. the biological functions of tnf- have been demonstrated to be related to the concentration and the duration of exposure to tnf- molecule. in the acute situation, local production of tnf- have a clear positive action increasing the expression of adhesion molecules on the vascular endothelium to allow immune cells, as macrophages and neutrophils, to reach the sites of tissue damage or infection. furthermore, tnf- activates phagocytes to clear infectious agents and cellular debris [37, 41 ]. the upregulation of tnf- gene expression has been involved in the pathogenesis of several autoimmune inflammatory illnesses, such as systemic lupus, rheumatoid arthritis, and inflammatory bowel disease [4245 ]. the tnf- acts by binding to tnf cellular receptor (tnfr), present on all cells in the body. the family of tnfrs has many members : the first two discovered were the tnfr1 and tnfr2. tnfr2 has a higher affinity for tnf-, especially at lower molecule concentrations, and causes the proliferation of t - lymphocytes and other proinflammatory responses. both the tnfrs are released also in a soluble form that neutralises the tnf- action competing with the cell - bound receptors. at the same time, generally, higher tnf- levels are related to the severity of inflammatory response, although it is not clear if greater tnf- production causes more severe inflammation or, conversely, if more severe inflammation elicits greater tnf- synthesis. tnf- levels seem to vary too on an individual basis and increase in circulating tnf- levels is much greater in some patients than in others. the tnf- gene is located on human chromosome 6p21.3 within the major histocompatibility complex (mhc) (figure 1). it lies in the so called class i region, between the genes encoding the mhc class human leukocyte antigen (hla) class ii cell surface molecules (hla - dp, dq, and dr) and the mhc class i antigen (hla - a, b, and c). the 5 flanking region of the tnf- gene contains multiple potential regulatory sites that seems to be responsive to inflammatory stimuli [12, 48 ]. genetic factors may affect tnf- levels as showed by in vitro and in vivo studies. differences in cytokine production may be partly attributed to the presence of single nucleotide polymorphisms (snp) within its corresponding gene. at least 12 snp have been identified in the tnf- locus, some of which have also been shown to influence the rate of transcription and production of tnf- cytokine [49, 50 ]. the most commonly studied tnf- polymorphism is the 308a / g, also known as tnf 1/2 (rs1800629 [maf : ceu 0.22 hcb 0.03, jpt 0.02, yri 0.06 ]). the function of this snp has been suggested by conflicting disease association studies rather than in vivo / vitro analysis. the presence of the less common 308a allelic form has been found to be correlated with enhanced spontaneous or stimulated tnf- production. several studies suggested that the protein preferentially binding to the 308a is likely to be a transcriptional activator, although it has yet to be characterized [50, 5355 ]. the less common 308a allele is strongly associated with the mhc haplotype hla - a1-b8 and dr3, which is in turn associated with high tnf- production and autoimmune disease. this genetic propensity to produce elevated tnf- levels, due to the presence of the 308a polymorphism, may alter the course of an immune response [44, 55, 56 ]. in vitro studies using different techniques (transfection with two variant construct cell lines, allele specific tnf- transcript quantification, 308 tagging snp within the tnf- primary mrna transcript) failed to demonstrate function in vitro for the 308 tnf- snp [15, 54, 57 ]. in vivo studies have demonstrated that the 308a tnf- allele had higher transcriptional activity compared with the 308 g allele [45, 58 ]. however, this association has not been found by other authors, probably due to the linkage disequilibrium of truly functional polymorphism with the 308 position and to the variable inclusion of these functional polymorphism in the gene construct [59, 60 ]. other differences may be related to the type of cells and of stimuli used in the studies. another possible functional promoter snp is the 238g / a (rs361525 [maf : ceu 0.07 hcb 0.04, jpt 0.00, yri 0.01 ]) that is located within the tnf- repressor site, but it has shown contradicting function. some works demonstrated that the 238a allele is associated with higher tnf- production with respect to the 238 g allele, but this data is not confirmed by other studies [12, 53, 6164 ]. moreover, brinkman. demonstrated a faster radiological damage in gg patients with respect to the ga genotype. the rare 376g / a (rs1800750 [maf : ceu 0.01 hcb 0.00, jpt 0.00, yri 0.01 ]) is a binding site for the transcriptional factor oct-1. on the other hand, the promoter containing the 376a allele demonstrated a promoter activity superior of 35% compared to the g allele (p = 0.002) in a monocyte cell line [12, 66 ]. studies regarding the 863c / a (rs1 800630 [maf : ceu 0.16 hcb 0.18, jpt 0.14, yri 0.12 ]) and 857c / t genotypes (rs1199724 [maf : ceu 0.05 hcb 0.18, jpt 0.11, yri 0.03 ]) showed that the rarer a and t alleles provide increased promoter activity and high production of tnf- [6, 32 ]. many other promoter variants have been described, as + 489, + 386, 1301, 857, 419, 376, and 244, but these snps are rare, particularly in caucasian, with conflicting and inconsistent data [6, 19, 32, 53 ]. the involvement of tnf- protein and its receptors in the pathogenesis of jia has been suggested by many studies [4, 23, 33 ]. tnf- plays a key function in the initial and prolonged inflammation and in joint destruction, controlling the production of interleukin 1 (il-1) and other proinflammatory cytokines including interleukin-6 (il-6) and interleukin-8 (il-8). tnf- mediates joint inflammation and destruction by inducing the synthesis and release of inflammatory metalloproteinases, prostaglandins, and nitric oxide in a variety of cell types, as well as inhibiting the production of matrix components. the role of tnf- in jia is suggested by the findings of high tnf- levels in the synovial fluids of these patients, from studies on transgenic mice overexpressing tnf- and developing a polyarthritis, and from the observation of a positive response to anti - tnf- biologic drugs in arthritis patients [7173 ]. in order to better understand the genetic background of jia and the role of cytokine snps in this disease, some authors found that the a allele was significantly more frequent in jia subjects with respect to controls and was related to a higher disease activity and a poor prognosis [43, 74 ]. showed that the tnf 308a allele is more frequently found in rheumatoid factor positive juvenile polyarthritis and is associated with a more severe disease, while the more common tnf 308 g allele may be protective. modesto. found no relationship between genotypes and juvenile arthritis, but 308a was more frequent in systemic jia subgroup. other authors found no association between 308a / g genotype and juvenile arthritis [23, 75 ]. ozen. found that the 308g / a polymorphism was significantly associated with a poor outcome in the turkish group of jia patients (p = 0.005) but not in the czech jia subjects ; the authors suggested a possible ethnic allele distribution. besides, in both jia cohorts, the distribution of genotypes was not significantly different among different jia subsets. so, most jia studies are in accord with adult ra results, demonstrating a direct involvement of this polymorphism in the severity of arthritis [42, 7678 ]. some authors demonstrated that the 238g / a allele has a significant association with jia, particularly with persistent oligoarthritis subtype ; these data were not confirmed by others [23, 43, 63, 74 ] demonstrated that the 863a, 1013c, and 857 t alleles were significantly higher in systemic jia patients with respect to healthy controls. moreover, the 857 t allele seems to enhance the effect of drb1 0405/dqb1 0401 haplotype in predisposing the development of systemic jia. studies in adult patients have recently demonstrated that 857 t allele is an independent risk allele for psoriatic arthritis [79, 80 ] but similar data were not founded in children. examined the association of multiple tnf snps (1031, 863, 857, 376, + 489a, + 851, + 1304) with juvenile oligoarthritis by constructing and analyzing snp - tagged tnf haplotype in 144 simplex families consisting of parents and affected children. no relationship was found for the other snps investigated [13, 23, 74 ]. the + 489 polymorphism was found positively associated with radiographic bone damage in studies on adult patients ; oen. studied the radiographic joint damage and 308a / g snp in patients with juvenile arthritis but they did n't find any association, although the + 489 snp was not investigated in this study. the central role of tnf- in the inflammatory process makes this cytokine an excellent therapeutic target. knowledge about genetic variants may help to predict drug response or optimal dose in the individual patient [82, 83 ]. biologic drugs have been demonstrated effective in the treatment of progressive jia [18, 84 ] ; however, approximately 2040% of children affected, especially with the polyarticular and the systemic onset subtypes, have been defined as nonresponders and still have a poor prognosis [37, 85 ]. as several snps have been noted in the tnf- promoter and some reports have shown that production of tnf- is influenced by these snps, an association has been suggested between some tnf- promoter snps, jia subtypes, and clinical response to biologic therapy [13, 14, 50 ]. etanercept, a fusion protein of extracellular domain of the tnf- receptor combined with the fc portion of the human immunoglobulin molecule, is the first tnf- antagonist approved for the use in jia and to date it has proven to be highly efficacious in children with polyarticular jia. other important biological agents in jia are adalimumab, a fully humanized monoclonal antibody, approved for treatment of moderate to severe polyarticular jia, and infliximab, a chimeric human - murine monoclonal anti - tnf- antibody, not formally approved for jia patients but commonly used in selected cases. three studies are actually detectable regarding the influence of tnf- snps on the anti tnf- effects in paediatric arthritis population. to test the influence of tnf- polymorphisms in the etanercept therapy response, schmeling and horneff (see table 2) studied 137 children and founded 308gg genotype in 101, 308aa genotype in 3, and heterozygous in 33 patients. patients with the 308gg genotype more frequently reached a response to etanercept therapy than patients leading the a allele ; the response was most pronounced and significant in patients with rheumatoid factor negative polyarthritis. in contrast, a recent study by cimaz. considered 107 children with different juvenile arthritis subtypes nonresponders to other first line drugs, treated with etanercept (34 patients), infliximab (71 children), and adalimumab (2 subjects). in these patients the authors were not able to find a link between the two tnf- snps considered (238a / g and 308a / g) and clinical response to anti - tnf-. more recently a serbian group detected the influence of 308a / g tnf- snp on the metalloproteinase-9 (mmp-9) levels and on the clinical response to etanercept in 66 polyarticular jia children. they found that patients with the 308gg genotype achieved a clinical response more significant than those with the 308aa genotype (p = 0.035) and that mmp-9 levels in patients with the genotype 308gg were significantly decreased after 1 year of treatment with etanercept. major information can be obtained from studies performed in adults affected by rheumatoid arthritis (ra). it is known that ra and jia are two distinct entities whereas they have several common characteristics. works in ra patients investigating response to several anti - tnf- therapy showed some important evidence (see table 3). a report analysed whether polymorphisms of several cytokine genes are associated with the responsiveness to etanercept treatment in 123 ra patients. no association between 308a / g snp and therapy response was found also by ongaro and colleagues ; they found that the 676tt genotypes is related to a better response to anti - tnf- drugs with respect to 676tg. besides, a certain combination of alleles (308gg) was associated with good responsiveness to etanercept (p > 0.05) [20, 9092 ]. another korean study showed that 70 ra patients with the t allele of tnf promoter snp 857 responded better to 12 weeks etanercept therapy than homozygous for the c allele. similar results were found in patients treated with other anti - tnf- drugs. in adult cohorts, mugnier. tested if the 308g / a tnf- snp influences the response to infliximab therapy in ra patients. according to these authors, patients with 308gg genotype were better infliximab responders and they concluded that this genetic evaluation can be useful for predicting infliximab therapy response. considered the influence of tnf- gene 308g / a polymorphism on therapeutic efficacy of infliximab in patients with ra and crohn 's disease. other studies showed that patients carrying the 308g / g allele responded to infliximab treatment better than 308a / g subjects [91, 96, 97 ]. furthermore, marotte. found no association between the 308 snp and response to infliximab, but the level of circulating tnf- bioactivity resulted was higher in 308a / a or a / g patients than in g / g subjects. another study in ra patients considered 152 patients subdivided in 3 groups of treatment : adalimumab plus methotrexate, adalimumab plus other dmards, and adalimumab alone. the authors studied 3 tnf- snps : 308a / g, 238a / g e, 857c / t. at evaluation after 12 weeks of therapy no association between the three tnf- snps and clinical response was noted. however, the ggc haplotype (308 g, 238 g e, 857c) in a homozygous form presented significant association with lower clinical response in patients on adalimumab plus methotrexate treatment. in the biologic era, several studies reported an elevated rate of malignancy in jia patient treated with tnf- inhibitors and in 2009 the us food and drug administration (fda) placed a black box warning for these drugs, as result of the identification of 48 malignancies cases occurring in children exposed to anti tnf- biologic drugs. studies in children exposed to biologic drugs reported an increased risk of lymphoma and other cancers with respect to healthy population [105107 ]. indeed, two paediatric studies in jia patient never treated with biologic drugs reported a 2- to 3-fold increased risk of cancer, and in particular 4-fold increased risk of lymphoproliferative disease [108, 109 ]. also the work of beukelman and colleagues showed that jia children had an increased rate of incident malignancy compared to children with asthma and attention deficit hyperactivity disorders, but the authors showed that specific therapies as methotrexate and tnf inhibitors did not alter this rate. a very recent work of nordstrom. considering a cohort of biologics - nave patients diagnosed with jia between 1998 and 2007, matched with a non jia cohort, found that the jia incidence rates of cancer were significantly higher in jia with respect controls (67.0 cases/100,000 person - years for jia and 23.2 cases/100,000 for non - jia). however, they found a nearly 3-fold increased risk of cancer in biologics - nave jia patients. in these studies, it is difficult to evaluate the real risk of cancer because of the potential risk of malignancy associated with underlying illnesses and the use of concomitant immunosuppressants ; a clear causal relationship could not be established and the findings suggest an elevated underlying risk of cancer in this disease population independently from biologic therapy. wolfe and michaud found about 3-fold increased risk for lymphoma in 18.572 ra biologic drug treated patients with respect to controls. however, the authors referred that increased lymphoma rates observed with anti - tnf therapy may reflect channeling bias, whereby patients with the highest risk of lymphoma preferentially receive anti - tnf therapy and consequently the data are insufficient to establish a causal relationship between ra treatments and the development of lymphoma. the same authors in a following work, during 89,710 person - years of followup of ra from 1998 to 2005, did not observe evidence for an increase in the incidence of lymphoma among patients who received anti - tnf therapy. this work considered 1114 ra patients treated with anti - tnf agents after failing to respond to traditional dmards, over an average observational period of 23.32 months. comparison with the general population showed that the overall cancer risk was similar, but the risk of lymphoma was about five times higher in the ra patients treated with a biological agent. however, even in this study there is the same bias : patients treated with biologic drugs are the same subjects with severe disease that did not respond to several previous therapies. the reported studies seem to indicate that not the biologic drug use but the prolonged inflammatory state itself due to the autoimmune disease that can be responsible for the increased risk of cancer [117, 118 ]. indeed a recent sponsored swedish nationwide cohort study found that ra patient anti - tnf- biologic drugs nave have a significantly higher risk of malignancy compared with general population. at this regard, several studies showed an association between elevated circulating tnf- levels and cancer development [120, 121 ] stimulating cell proliferation, causing dna damage and promoting angiogenesis. this data was not confirmed by other studies that reported a direct cytotoxic effect of tnf- on tumoral cell, an indirect action on tumor vessels and a synergic action with conventional antineoplastic agents [127, 128 ]. moreover, a relationship was found between some tnf- polymorphisms and development of different types of cancer. the + 488a and the 857 t polymorphisms have been associated to bladder cancer. the + 488ga genotype seems to be related to development of renal cell carcinoma and prostate carcinoma [130, 131 ]. the 238ga snp have been also correlated to renal carcinoma and the + 857 t snp to leukemia and lymphoma. the 308 g have been found associated with gastric, breast, and liver cancer [133136 ] and represents a negative prognostic factors in pediatric leukemia. although some biologics seem to have a high association with certain cancer compared to control, there is no consistency of data. the actual knowledge regarding the role of tnf- gene polymorphisms in the pathogenesis of jia is still incomplete. numerous studies have focused on understanding the contribution of tnf- polymorphisms in the ra and jia pathogenesis. the tnf- polymorphisms have shown an association with higher or lower levels of circulating tnf-, aggressive or mild disease and poor or good prognosis related to the response to anti tnf- treatment. the most studied tnf- snp is located at 308 position, where the presence of rare a allele was associated with a major gene expression, high level of tnf- expression, and more aggressive jia phenotypes [36, 43, 74 ] such as systemic juvenile arthritis and rheumatoid factor positive juvenile polyarticular arthritis [13, 74 ]. several data suggest an association of this polymorphism with systemic manifestations, radiological progression, work disability, and joint surgeries. the presence of 308a allele has been moreover linked to increased susceptibility and severity of a variety of other autoimmune disorders including systemic lupus erythematosus [43, 138 ], dermatomyositis [139, 140 ], inflammatory bowel disease [141, 142 ], and asthma. this 308 snp was found mostly in caucasian population and represents an important risk factor for jia appearance in this population. also the 238a tnf- gene polymorphism has been associated with higher tnf- production and more aggressive jia phenotypes [59, 74, 75 ]. moreover these data were not confirmed by others [12, 23, 42, 54, 6165, 74 ]. several other polymorphisms have been identified but their frequency, their pathogenic role, and their influence on biologic drug response have been poorly characterized. this is likely related to several elements : the complex pathogenesis of jia, involving different cytokine genes and non hla genes, largely still not well defined, and also environmental factors that are, in the major part actually unknown. anyway tnf- blocking agents are among the most effective therapies for jia but unfortunately not all patients have a good response. actually, the reasons for the interindividual variability in the response to anti - tnf- therapy are unclear, although it is supposed that the genetic background might play a role. considering the increasingly wide range of biologicas available for aig and the cost of these therapies, there is an increasing need to predict responsiveness to identify patients more suitable to the therapy, to define the timing of treatment, and to avoid complications. results of studies in ra patients indicate tnf- as candidate genes potentially involved in the modulation of clinical response to anti tnf- blocking agents. many studies demonstrated that patients with the 308gg genotype are better responders to anti tnf- therapy. indeed only some authors showed that the 308g / g genotype is associated with a better anti - tnf- treatment response also in jia [83, 85 ]. other authors did not confirm this data, therefore adjunctive information are necessary. to conclude, data gathered so far indicate a possible influence of the 308 snp promoter position on the production of tnf- and consequently on the severity of jia and the response to anti - tnf- treatment. further and larger studies are needed to investigate the influence of tnf- polymorphisms on the treatment response to individualize the management of the disease. | whether tumor necrosis factor alpha (tnf-) gene polymorphisms (snps) influence disease susceptibility and treatment of patients with juvenile idiopathic arthritis (jia) is presently uncertain. tnf- is one of the most important cytokine involved in jia pathogenesis. several single nucleotide polymorphisms (snps) have been identified within the region of the tnf- gene but only a very small minority have proven functional consequences and have been associated with susceptibility to jia. an association between some tnf- snps and adult rheumatoid arthritis (ra) susceptibility, severity and clinical response to anti - tnf- treatment has been reported. the most frenquetly studied tnf- snp is located at 308 position, where a substitution of the g allele with the rare a allele has been found. the presence of the allele 308a is associated to jia and to a poor prognosis. besides, the 308 g genotype has been associated with a better response to anti - tnf- therapy in jia patients, confirming adult data. psoriatic and oligoarticular arthritis are significantly associated to the 238 snp only in some works. studies considering other snps are conflicting and inconclusive. large scale studies are required to define the contribution of tnf- gene products to disease pathogenesis and anti - tnf- therapeutic efficacy in jia. |
the myocyte enhancer factor 2 (mef2) proteins belong to the mads (mcm-1-agamous - deficiens - serum response factor) box evolutionarily conserved family of transcription factors (tf). four vertebrate genes encode for distinct isoforms (a - d) of the mef2 family, whose expression is detected in a wide range of tissues, but the proteins are most abundant in muscle and brain. in the cns, mef2 isoforms exhibit distinct but overlapping patterns throughout the developing brain through adulthood, indicating that they are tightly regulated in multiple processes. in the mature brain, mef2 proteins are expressed in brain regions involved in memory formation, including amygdala, hippocampus, cortex and striatum. each isoform is characterized by a highly conserved n - terminal region including the mads box, a dna binding domain that recognizes a / t - rich motif in target genes, and the mef2 domain that mediates homo- and hetero - dimerization. the c - terminal region of mef2 is characterized by a divergent transactivation domain that mediates the interaction with numerous co - factors, including co - activators, such as the acetyl - transferases p300 and cbp, or co - repressor, such as class ii histone deacetylases (hdacs) and ncor / smrt co - repressor complex. mef2 factors function as bivalent transcriptional regulators. in the absence of stimulating inputs, mef2 proteins form a complex with co - repressor complexes to keep their target genes in a repressed state. upon stimulus, the interaction with hdacs is disrupted to permit mef2 proteins to recruit co - activator complexes that promote transcription of target genes. the transcriptional activity of mef2 proteins is tightly regulated by post - translational modifications that include phosphorylation, sumoylation, acetylation, and nitrosylation. in the central nervous system, neuronal activity induced by depolarization, neurotrophins or synaptic stimuli, such as reelin at glutamatergic synapses, triggers calcium signaling cascades involving calmodulin - dependent protein kinases (camk) activation that results in phosphorylation of class iia hdacs. these phosphorylation events lead to hdacs nuclear export and dismissal from mef2 target genes, permitting recruitment of co - activators. furthermore, protein phosphatase 2b (pp2b) or calcineurin, a serine / threonine phosphatase, directly dephosphorylates mef2 influencing the affinity to dna target sequences and promoting transcriptional activity. in neuronal cells, mef2 target genes regulate different aspects of synaptic function such as presynaptic vesicle release, excitatory and inhibitory postsynaptic strengthening ; with many target genes linked to increased genetic susceptibility to neurological disorders, including autism, epilepsy and intellectual disabilities. recent efforts in elucidating the distinct function of mef2 family members using in vitro and in vivo systems strongly suggest important roles for specific mef2 isoforms in brain plasticity. while mef2c specific ko clearly impairs hippocampal - dependent learning and memory by increasing synapses number and potentiating synaptic transmission, manipulation of mef2a and mef2d results in deficits in motor coordination and enhanced hippocampal short - term synaptic plasticity without impairments in learning and memory behaviors. consistently, an increase in mef2a suppresses synapses number and inhibits dendritic spine growth in vitro. collectively, these findings indicate the sophisticated and complex regulation of diverse neuronal processes by distinct mef2 isoforms that is likely affected by other factors, such as environmental cues or other signaling pathways. however, the precise transcriptional programs regulated by individual mef2 isoforms remain to be defined. here, we report the genome - wide epigenetic analysis of mef2a and mef2c cistromes in primary cortical neurons by chip - seq, which identifies the unique and overlapping genomic loci occupied by each isoform. our analysis reveals a widespread localization of mef2 transcription factors to enhancer regulatory elements in the genome of neuronal cells, suggesting a function of these factors in directing neuronal lineage specification. both transcription factors orchestrate overlapping but unique programs correlated to a variety of neuronal functions, such as glutamatergic synaptic transmission, drug addiction and mapk signaling pathway. although, both isoforms orchestrate a similar epigenomic program, the motif discovery reveals some key differences indicating that the combinatorial action of different transcription factors might determine the regulation of distinct enhancer - driven transcriptional programs. the finding that mef2a / d restrain memory formation, whereas mef2c promotes associative learning and memory, raises the question of whether their divergent actions are mediated via regulation of distinctive or common cohorts of target genes. to identify the direct target genes of different isoforms of the mef2 family, we used genome - wide readout of mef2a and mef2c binding sites by chromatin immuno - precipitation coupled with deep sequencing (chip - seq) in mouse primary cortical neurons. as previously reported, mef2a and mef2c are the most abundant isoforms expressed in cultured neurons. using homer chip - seq analysis program (http://homer.salk.edu/homer/motif/), we identified a total of 75,072 mef2a or 35,710 mef2c binding sites with fdr threshold 90% of identified peaks for both mef2a and mef2c (fig. the median distance from the tss is 56 kb for mef2a and 55 kb for mef2c enriched regions. as expected the most represented sequence element by de novo motif analysis was the mef2 motif for both factors, which was enriched in roughly 40% of chip - seq peaks compared to the background sequences (fig., peaks were considered co - bound when they were found within 200bp of each other. we observed that the majority (66.7%) of mef2c binding regions were co - bound by mef2a, while only a minor fraction of mef2a were detected as common regions (fig. (a) pie charts showing the genomic distribution of mef2a binding sites in the genome. (b) pie charts showing the genomic distribution of mef2c binding sites in the genome. (c) de novo motif analysis of mef2a or mef2c - bound genomic regions showing the top enriched sequence motifs. (a) pie charts showing the genomic distribution of mef2a binding sites in the genome. (b) pie charts showing the genomic distribution of mef2c binding sites in the genome. (c) de novo motif analysis of mef2a or mef2c - bound genomic regions showing the top enriched sequence motifs. p - values and frequencies of motifs are indicated. to gain further insight in the putative functional role of the genomic regions bound by mef2 transcriptional regulators, we used histone 3 lysine 27 acetylation (h3k27ac) profiling by chip - seq, which marks both promoters and enhancers cis - regulatory elements of the mammalian genome. enhancer elements are defined as dna sequences that function far away from the promoter or tss of target genes independently of their orientation, and have the capability to promote basal transcriptional activity in a cell - type specific manner and in response to external stimuli. a large body of evidence has shown that active enhancers are characterized by defined properties of the chromatin, including a nucleosomal free region flanked by nucleosomes containing histones with specific posttranslational modifications. the histone 3 lysine 27 acetylation (h3k27ac) mark has been extensively reported to mark cell - type specific and active enhancers. therefore, we used available h3k27ac chip - seq data in cortical neurons as an enhancer mark. first, we overlapped the h3k27ac chip - seq peaks with those identified for each transcription factor, and we identified 17,290 and 9,314 co - bound peaks for mef2a and mef2c, respectively. we observed that h3k27ac mark labels 3035% distal regions for mef2a or mef2c chip - seqs. only the distal peaks away from the tss (> 1, 000 bp) were considered as enhancers (14,593 for mef2a and 8,518 for mef2c) (fig. scatter plots of the tag counts around peak coordinates of tfs enable comparative analysis of mef2a- and mef2c - dependent enhancer programs. based on this analysis, we identified 6,554 common h3k27ac - enhancers (fig. 2b). to examine functional pathways associated to the genes in proximity of mef2 enhancers, we used gene ontology analysis for 3 datasets of mef2-enhancers (mef2a - specific, mef2c - specific and mef2a / c - common). based on the kegg pathway analysis, the most enriched functional annotations were nearly identical for the common or isoform - specific subsets of genes and they included signaling pathways known to be regulated by mef2 factors, such as glutamatergic synaptic transmission, drug addiction, axon guidance, and mapk signaling pathways (fig. 2c). this result suggests that mef2a and mef2c regulate common or distinct transcriptional targets that are involved in similar neuronal processes. to explore whether specific sequence determinants may account for distinct mef2 binding patterns, we examined the mef2-enhancer subsets for enrichment of distinctive sequence elements by de novo motif analysis (fig. we observed that in the top 10 most enriched sequences each subset of enhancers contains a core combination of consensus motifs, including the mef2, ap1 and neurod1. we believe that mef2 tfs together with neurod1 and ap1 constitute the factors that determine neuronal - lineage specific enhancers. however, each class is associated with different transcription factors, such as srf for the common enhancers, creb / atf for the mef2a enhancers, cebp / a and forkhead box factors for mef2c. how different combinations of transcription factors cooperate to activate cis - active regulatory elements regulated by mef2 isoforms remains an interesting question to address in the future. we speculate that the activation of diverse signaling pathways might differentially regulate specific isoforms of mef2 family by posttranslational modifications or activation of other transcription factors. recently, we have shown that the secreted protein reelin triggers a synapse - to - nucleus pathway involving the -secretase - dependent cleavage of its receptor lrp8 that is required for proper hippocampal - dependent associative learning. this signaling pathway relies on the induction of transcriptional programs activated by a specific subset of enhancers, known as lrp8-reelin - regulated neuronal enhancers (lrn). these enhancers are characterized by the occupancy of both mef2a and mef2c factors before stimulation, but only mef2c isoform exhibits an increased recruitment upon reelin treatment, suggesting a specific role for mef2c in mediating learning and memory processes. other synaptic inputs might be involved in the fine - tuned regulation of subsets of enhancers bound by mef2 factors. characterizing the mef2 transcriptional programs has the potential not only to shed light on underlying mechanisms of transcriptional regulation of memory, but also might provide key insights into the disease mechanisms involved in the development of many neurological disorders linked to dysfunction of mef2 transcription factors and to the misregulation of their specific target genes. (a) venn diagram showing h3k27ac - mef2a or h3k27ac - mef2c overlapping peaks identified using a false discovery threshold of 0.001. peaks were considered co - bounded when they were found within 200 bp of each other. (b) on the right, the scatter plot of mef2a and mef2c chip - seq peaks in cortical neurons is represented by log2 of normalized chip - seq tag counts ; on the left, the venn diagram shows the fractions of common or isoform - specific h3k27ac - enhancers. (c) functional gene ontology annotations associated with common or isoform - specific h3k27ac - enhancers. (d) de novo motif analysis of common or isoform - specific h3k27ac - enhancers showing the top enriched sequence motifs, with associated p values as indicated. (a) venn diagram showing h3k27ac - mef2a or h3k27ac - mef2c overlapping peaks identified using a false discovery threshold of 0.001. peaks were considered co - bounded when they were found within 200 bp of each other. (b) on the right, the scatter plot of mef2a and mef2c chip - seq peaks in cortical neurons is represented by log2 of normalized chip - seq tag counts ; on the left, the venn diagram shows the fractions of common or isoform - specific h3k27ac - enhancers. (c) functional gene ontology annotations associated with common or isoform - specific h3k27ac - enhancers. (d) de novo motif analysis of common or isoform - specific h3k27ac - enhancers showing the top enriched sequence motifs, with associated p values as indicated. the finding that mef2a / d restrain memory formation, whereas mef2c promotes associative learning and memory, raises the question of whether their divergent actions are mediated via regulation of distinctive or common cohorts of target genes. to identify the direct target genes of different isoforms of the mef2 family, we used genome - wide readout of mef2a and mef2c binding sites by chromatin immuno - precipitation coupled with deep sequencing (chip - seq) in mouse primary cortical neurons. as previously reported, mef2a and mef2c are the most abundant isoforms expressed in cultured neurons. using homer chip - seq analysis program (http://homer.salk.edu/homer/motif/), we identified a total of 75,072 mef2a or 35,710 mef2c binding sites with fdr threshold 90% of identified peaks for both mef2a and mef2c (fig. the median distance from the tss is 56 kb for mef2a and 55 kb for mef2c enriched regions. as expected the most represented sequence element by de novo motif analysis was the mef2 motif for both factors, which was enriched in roughly 40% of chip - seq peaks compared to the background sequences (fig., peaks were considered co - bound when they were found within 200bp of each other. we observed that the majority (66.7%) of mef2c binding regions were co - bound by mef2a, while only a minor fraction of mef2a were detected as common regions (fig. (a) pie charts showing the genomic distribution of mef2a binding sites in the genome. (b) pie charts showing the genomic distribution of mef2c binding sites in the genome. (c) de novo motif analysis of mef2a or mef2c - bound genomic regions showing the top enriched sequence motifs. (a) pie charts showing the genomic distribution of mef2a binding sites in the genome. (b) pie charts showing the genomic distribution of mef2c binding sites in the genome. (c) de novo motif analysis of mef2a or mef2c - bound genomic regions showing the top enriched sequence motifs. to gain further insight in the putative functional role of the genomic regions bound by mef2 transcriptional regulators, we used histone 3 lysine 27 acetylation (h3k27ac) profiling by chip - seq, which marks both promoters and enhancers cis - regulatory elements of the mammalian genome. enhancer elements are defined as dna sequences that function far away from the promoter or tss of target genes independently of their orientation, and have the capability to promote basal transcriptional activity in a cell - type specific manner and in response to external stimuli. a large body of evidence has shown that active enhancers are characterized by defined properties of the chromatin, including a nucleosomal free region flanked by nucleosomes containing histones with specific posttranslational modifications. the histone 3 lysine 27 acetylation (h3k27ac) mark has been extensively reported to mark cell - type specific and active enhancers. therefore, we used available h3k27ac chip - seq data in cortical neurons as an enhancer mark. first, we overlapped the h3k27ac chip - seq peaks with those identified for each transcription factor, and we identified 17,290 and 9,314 co - bound peaks for mef2a and mef2c, respectively. we observed that h3k27ac mark labels 3035% distal regions for mef2a or mef2c chip - seqs. only the distal peaks away from the tss (> 1, 000 bp) were considered as enhancers (14,593 for mef2a and 8,518 for mef2c) (fig. scatter plots of the tag counts around peak coordinates of tfs enable comparative analysis of mef2a- and mef2c - dependent enhancer programs. based on this analysis, we identified 6,554 common h3k27ac - enhancers (fig. to examine functional pathways associated to the genes in proximity of mef2 enhancers, we used gene ontology analysis for 3 datasets of mef2-enhancers (mef2a - specific, mef2c - specific and mef2a / c - common). based on the kegg pathway analysis, the most enriched functional annotations were nearly identical for the common or isoform - specific subsets of genes and they included signaling pathways known to be regulated by mef2 factors, such as glutamatergic synaptic transmission, drug addiction, axon guidance, and mapk signaling pathways (fig. 2c). this result suggests that mef2a and mef2c regulate common or distinct transcriptional targets that are involved in similar neuronal processes. to explore whether specific sequence determinants may account for distinct mef2 binding patterns, we examined the mef2-enhancer subsets for enrichment of distinctive sequence elements by de novo motif analysis (fig. we observed that in the top 10 most enriched sequences each subset of enhancers contains a core combination of consensus motifs, including the mef2, ap1 and neurod1. we believe that mef2 tfs together with neurod1 and ap1 constitute the factors that determine neuronal - lineage specific enhancers. however, each class is associated with different transcription factors, such as srf for the common enhancers, creb / atf for the mef2a enhancers, cebp / a and forkhead box factors for mef2c. how different combinations of transcription factors cooperate to activate cis - active regulatory elements regulated by mef2 isoforms we speculate that the activation of diverse signaling pathways might differentially regulate specific isoforms of mef2 family by posttranslational modifications or activation of other transcription factors. recently, we have shown that the secreted protein reelin triggers a synapse - to - nucleus pathway involving the -secretase - dependent cleavage of its receptor lrp8 that is required for proper hippocampal - dependent associative learning. this signaling pathway relies on the induction of transcriptional programs activated by a specific subset of enhancers, known as lrp8-reelin - regulated neuronal enhancers (lrn). these enhancers are characterized by the occupancy of both mef2a and mef2c factors before stimulation, but only mef2c isoform exhibits an increased recruitment upon reelin treatment, suggesting a specific role for mef2c in mediating learning and memory processes. other synaptic inputs might be involved in the fine - tuned regulation of subsets of enhancers bound by mef2 factors. characterizing the mef2 transcriptional programs has the potential not only to shed light on underlying mechanisms of transcriptional regulation of memory, but also might provide key insights into the disease mechanisms involved in the development of many neurological disorders linked to dysfunction of mef2 transcription factors and to the misregulation of their specific target genes. (a) venn diagram showing h3k27ac - mef2a or h3k27ac - mef2c overlapping peaks identified using a false discovery threshold of 0.001. peaks were considered co - bounded when they were found within 200 bp of each other. (b) on the right, the scatter plot of mef2a and mef2c chip - seq peaks in cortical neurons is represented by log2 of normalized chip - seq tag counts ; on the left, the venn diagram shows the fractions of common or isoform - specific h3k27ac - enhancers. (c) functional gene ontology annotations associated with common or isoform - specific h3k27ac - enhancers. (d) de novo motif analysis of common or isoform - specific h3k27ac - enhancers showing the top enriched sequence motifs, with associated p values as indicated. (a) venn diagram showing h3k27ac - mef2a or h3k27ac - mef2c overlapping peaks identified using a false discovery threshold of 0.001. peaks were considered co - bounded when they were found within 200 bp of each other. (b) on the right, the scatter plot of mef2a and mef2c chip - seq peaks in cortical neurons is represented by log2 of normalized chip - seq tag counts ; on the left, the venn diagram shows the fractions of common or isoform - specific h3k27ac - enhancers. (c) functional gene ontology annotations associated with common or isoform - specific h3k27ac - enhancers. (d) de novo motif analysis of common or isoform - specific h3k27ac - enhancers showing the top enriched sequence motifs, with associated p values as indicated. for chip - seq, the 50 bp sequence tag was aligned to the mm9 assembly by using bowtie2. during alignment to the reference genome the total number of mapped reads was normalized to 10 ^ 7 for each experiment presented in this study. using homer and applying the same criteria as in our previously published methods (telese et. al, 2015), we identified the mef2a / mef2c or h3k27ac chip - seq peaks. first, we used input sequencing run as a control to filter out false positive peaks. second, we used different parameters to define the width of peaks by applying a distinct sliding window for searching the enrichment regions, which is 200 bp for transcription factors and 500bp for histone modifications. third, redundant peaks detection was applied by merging peaks when the distance between each other was less than 500 bp. furthermore, to reduce clonal amplification only single tags per each genomic position are used for the identification of chip - seq peaks. finally, a threshold of false discovery rate (fdr) < 0.001 was applied to call peaks by comparing randomized tag positions with an effective genomic size of 210 bp. all the peaks identified as described above are assigned to genes by cross - referencing the ncbi reference sequence database (refseq). peaks from individual experiments were considered overlapping if they were located within 200 bp from each other. the motif finding and enrichment p - values were computed by using homer algorithm as described before (heinz, 2010). the scatter plots were then generated by plotting as (x, y) coordinates the normalized tag counts (log2) within 1000bp around the center of the peaks identified for each experiments as describe above. the ncbi gene expression omnibus accession number for the sequencing data is reported in previously published work. for chip - seq, the 50 bp sequence tag was aligned to the mm9 assembly by using bowtie2. during alignment to the reference genome the total number of mapped reads was normalized to 10 ^ 7 for each experiment presented in this study. using homer and applying the same criteria as in our previously published methods (telese et. al, 2015), we identified the mef2a / mef2c or h3k27ac chip - seq peaks. first, we used input sequencing run as a control to filter out false positive peaks. second, we used different parameters to define the width of peaks by applying a distinct sliding window for searching the enrichment regions, which is 200 bp for transcription factors and 500bp for histone modifications. third, redundant peaks detection was applied by merging peaks when the distance between each other was less than 500 bp. furthermore, to reduce clonal amplification only single tags per each genomic position are used for the identification of chip - seq peaks. finally, a threshold of false discovery rate (fdr) < 0.001 was applied to call peaks by comparing randomized tag positions with an effective genomic size of 210 bp. all the peaks identified as described above are assigned to genes by cross - referencing the ncbi reference sequence database (refseq). peaks from individual experiments were considered overlapping if they were located within 200 bp from each other. the motif finding and enrichment p - values were computed by using homer algorithm as described before (heinz, 2010). the scatter plots were then generated by plotting as (x, y) coordinates the normalized tag counts (log2) within 1000bp around the center of the peaks identified for each experiments as describe above. the ncbi gene expression omnibus accession number for the sequencing data is reported in previously published work. we thank janet hightower for assistance with figure preparation and rachel pardee for assistance with editing of the manuscript. | the transcription factors of the myocyte enhancer factor 2 family (mef2 a - d) are highly expressed in the brain and play a key role in neuronal survival / apoptosis, differentiation and synaptic plasticity. however, the precise genome - wide mapping of different members of the family has not yet been fully elucidated. here, we report the comparative analysis of mef2a and mef2c genome - wide mapping in mouse cortical neurons by chip - seq, a powerful approach to elucidate the genomic functions of transcription factors and to identify their transcriptional targets. our analysis reveals that mef2a and mef2c each orchestrate similar epigenomic programs mainly through the binding of enhancer regulatory elements in proximity of target genes involved in neuronal plasticity and calcium signaling. we highlight the differences in the enhancer networks and molecular pathways regulated by mef2a and mef2c, which might be determined by the combinatorial action of different transcription factors. |
today there is a great debate about the role of inflammation in control and regulation of tumor microenvironment directly influencing the development of neoplasms. inflammation plays a fundamental role in tumor dynamic and is accepted as one of the hallmarks of cancer [1, 2 ]. currently there are two marked ways by which inflammation is associated with cancer development, an intrinsic pathway characterized by the gene expression and an extrinsic pathway, characterized by the formation of an inflammatory microenvironment. within this microenvironment, macrophages are the major cell populations involved in the inflammatory process associated with the development of neoplasms. considered highly plastic cells, they are able to respond to stimuli and produce numerous pro- and anti - inflammatory factors and when related to cancer, they are termed tumor - associated macrophages (tams). it is believed that tams play a key role in promoting and coordination phenomena of tumor growth due to their ability to modulate the angiogenesis and lymphangiogenesis that are processes involved in the progression of neoplasms. tams are activated by different stimuli, such as growth factors, nutrients, and cytokines, produced in the tumor microenvironment that are responsible for inducing differentiation in functionally distinct populations. in populations of classically activated macrophages (m1), there is a high production of proinflammatory cytokines displaying an immune - stimulatory function. on the other hand, the alternative activation of macrophages (m2) is based on the release of anti - inflammatory cytokines supporting an immunosuppressive environment [79 ]. this environment, formed by cells, extracellular matrix, growth factors, nutrients, and cytokines, may be responsible for the macrophages differentiation and behavior of tumor cells. however, like a feedback loop, these cells alter the environment in reply to stimuli., the role of tams polarization in m1 and m2 profiles might have clinical and pathological importance and appears to be associated with angiogenesis, proliferation, aggressiveness of tumor, and apoptosis. moreover, the debate appears to be controversial about the relation between inhibitory or promoter ability of m1 or m2 on tumor. recently, one of the ways suggested for activation of macrophages subpopulations involves the signaling akt / mtor pathway. a change in regulation of this metabolic pathway plays a crucial role in activation controlling and acquisition of macrophages effector activity, depending on the context in which they are, as well as the tumor microenvironment. with this view of the tumor stroma and its influence on the progression of neoplasms, the study about the role of macrophage polarization in the tumor pathogenesis may emerge as a new therapeutic approach. in this review, we discuss the role of tumor microenvironment and macrophages in cancer ; m1 and m2 differentiation and the role of mtor pathway ; and m1 and m2 macrophages as possible tumor markers. the monocyte - derived macrophages are cells of the myeloid lineage that have functional plasticity [12, 13 ]. they are important cells of the innate immune system and can act in different tissues as phagocytes, antigen presenting cells, and tissue remodeling [14, 15 ]. the functional plasticity associated with phenotypic and metabolic differences led to the characterization of two macrophage subtypes, m1 and m2. these macrophages subtypes were primarily defined in murine models, but actually the m1 and m2 terms are widely used for humans and others mammalians. however, clearly, there are not only two functional subtypes of macrophages and the polarization process has a large spectrum of subpopulations with phenotypic differences showing the plasticity of macrophages [15, 16 ]. m1 macrophages are always related to the inflammatory response by presenting a large phagocytic and microbicidal capacity, increased expression of mhc ii molecules, cd80, and cd86 and also secreting cytokines such as tnf - alpha. this m1 profile is induced by the presence of pamps that bind mainly to tlr2 and tlr4 and th1 cytokines such as ifn - gamma that activate the transcription factors stat1 and irf5 [12, 14, 15 ]. m2 macrophages, in turn, are typically activated in the presence of th2 cytokines such as il-4 and il-13 that improve the stat6 and irf4 interaction. the m2 profile is characterized by low phagocytic and microbicidal activity, and it is linked to the presence of helminthes or to the tissue remodeling processes. these m2 macrophages, unlike the m1, do not activate inos and therefore do not produce nitric oxide (no) ; however, they have great arginase activity that is important in wound healing and tissue remodeling process [14, 16 ]. the differences between these profiles are also observed in their metabolism, m1 have preferably glycolytic pathways while m2 has increased beta - oxidation [11, 12 ]. tams are a major stromal component and exhibit, mostly, m2 similar profile. however, according to sica and mantovani (2012) macrophages change during tumor development. tams show m1-like phenotype characterized by high il-12 releases that cause tumor cell disruption, when in tumoral early - stage. despite this, in late - stage of tumor progression, tams exhibit an m2-like phenotype accompanied to reduced antitumoral activity. the antitumoral m1-dependent response is related to inflammatory response and the activation of specific lymphocytes in an attempt to eliminate tumor cells. on the other hand, m2 participation in tumor environment provides angiogenesis and survival, supporting metastasis and tumor growth [11, 18 ]. thus, tams play a crucial role in the modulation of tumor microenvironment producing a large amount of growth factors and inflammatory mediators that stimulate proliferation, angiogenesis, deposition, or degradation of extracellular matrix [19, 20 ] the tumor cells present an unusual metabolic activity and consume a large amount of nutrients and oxygen ; in addition, these cells need to remove the waste metabolic products such as carbon dioxide. thus, the new vascularization associated with tumor, generated by the angiogenesis process, allows these conditions. thus, without a pathological neovascularization the tumors grow slower decreasing their capacity to promote metastasis. at the same time, ineffective blood supply does not allow the action of anticancer drugs in effective amounts in tumor regions. in some cancers the first occurs directly by oncogenes such as ras and myc that can increase the expression of angiogenic factors in cancer cells, while, in second, the angiogenic signals are indirectly produced by inflammatory cells, specially tams that play a crucial role in tumor promotion and progression, contributing to neovascularization and tumor cell proliferation and metastasis [2427 ]. the epidermal growth factor (egf) ; family members of fibroblast growth factor (fgf) ; vegf ; matrix metalloproteinases (mmps) ; heparanase ; plasmin, activator of the urokinase - type plasminogen (upa) ; the upa receptor ; and cytokines and chemokines as tgf-, which enlarge the inflammatory state, are among the key related factors secreted by tams in the tumor microenvironment [21, 25, 28 ]. proangiogenic factors produced by tams are critical to regulate tumor development and have ability to maintain a favorable environment to encourage and maintain the remodeling and a complex vascular network within the lesion. vegf and matrix metalloproteinase-9 zymogen (prommp-9) are considered key factors involved in this increased organization of tumor vasculature. 2013, demonstrated that the angiogenic capacity of m2 macrophages critically depends on production of the readily activatable proenzyme prommp-9 and only m2 macrophages approach the high angiogenic capability of neutrophils. in addition to angiogenesis researchers have also studied a new approach in vascular biology the lymphangiogenesis. lymphangiogenesis is described as the development of a new lymphatic vessels, which in normal condition occurs in embryonic development being absent in adults. however, this process can be induced by pathological condition such as chronic inflammation, wound healing, and several neoplastic lesions [31, 32 ]. as angiogenesis, the process of lymphangiogenesis can be stimulated by cytokines and growth factors ; the vascular endothelial growth factor family (vegfs) plays a pivotal role in this mechanism. related to lymphangiogenesis, vegf - c and vegf - d present an exclusive involvement and recent data has also described vegf - a, a well known angiogenic growth factor, as an important lymphangiogenic growth factor [33, 34 ]. the expression of lymphangiogenic growth factor and related protein was revised by ran and montgomery (2012) and, together with vegf, the authors listed pdgf, adrenomedullin, hgf, cox-2, fgf, tnf - alpha, mmps, heparanase, and angiopoietin-2 as factors that can also provide lymphangiogenesis. (2012) describes the vegf production not only by tumor cells but also by tams and this starts the lymphangiogenic process through vegf - c and vegf - d via vegfr3 results presented on cancers of the cervix postulate the hypothesis of peritumoral lymphangiogenesis process mediated by tams. in first step, thus, activated tams serve as a major producer of vegf - c and vegf - d that cause proliferation of lymphatic microvessels and this new dense vessel network could favor the lymphatic metastases formation. once recruited into neoplastic microenvironment tams may also disrupt lymphangiogenesis and lymphatic tumoral spread in pancreatic cancer ; kurahara and colleagues (2011) reported that this tumor presented a high density of tams correlated with lymph node metastasis. additionally, these authors also analyzed the correlation between cd163 + densities with clinic - pathological data, and, as a result, the the evidences that macrophages can cooperate with lymphangiogenesis also came from studies on bladder cancer, breast, lungs, and melanoma. the research data presented above suggest that macrophages induce the formation of both angiogenic and lymphangiogenic microenvironment. in contrast, these cells can also express antiangiogenic molecules and damage the integrity of the blood vessels. this paradoxical role of m1 and m2 in cancer is explained by their functional plasticity, which results in the expression of pro- or antitumor functions [24, 42, 43 ]. the treatment induced an antitumor effect by subsequent reduction of m2 cd206 + macrophages and a simultaneous increase in m1 macrophages expressing class ii mhc and cd86 +. this change was preceded by an increase in il-12 production within the tumor and a reduction in its neovascularization. takeuchi. (2016) to correlate the prevalence of m2 macrophages, angiogenesis, and clinic - pathological features in bladder cancer showed higher density of m2 macrophages in invasive bladder cancer in relation to non - muscle invasive cancers. moreover, the higher the distribution of microvessels, the greater the degree of severity of cancer, and the predominance of m2 macrophage correlated to the amount of microvessels in bladder cancer and can have prognostic value for this type of cancer. although high concentration of m2 promotes tumor angiogenesis, the mechanism that promotes the differentiation of monocytes in tams and their phenotypes is still unclear. thus, it was proposed that the mtor pathway could be a critical element in the regulation of tams differentiation. the molecular control of polarization and activation of m1 and m2 has been the subject of many investigations and has well established the participation of stat1 and stat6, respectively [13, 16 ]. in addition to these canonical pathways, molecules of other routes are also being related to differentiation of macrophages [11, 46 ]. mammalian target of rapamycin (mtor), a serine - threonine kinase that is highly conserved, is a component of the pathway cell survival phosphatidylinositol 3-kinase (pi3k) that monitors the availability of nutrients, mitogenic signals, cellular energy, and oxygen levels and is therefore insignificant in the regulation of cell growth and proliferation [11, 47, 48 ]. mtor is regulated by nutrients and growth factor and regulates cell growth by controlling many biological process such as autophagia, mrna translation, and energetic metabolism [guertin and sabatini, 2007 ]. the mtor acts both upstream and downstream of the akt, operating as a connection with pi3k pathway and forming two different multiprotein complexes, mtorc1 (mtor complex 1) and mtorc2 (mtor complex 2), which regulate protein synthesis required for cell growth and proliferation [48, 50 ]. the stimulation of tlrs by pamps leads to the recruitment of pi3k that activates mtorc1 and mtorc2 in macrophages. the mtorc1 may be inhibited physiologically by the action of tsc1 (tuberous sclerosis complex 1) and tsc2 (tuberous sclerosis complex 2) and by rapamycin due to the presence of raptor (regulatory associated protein of mtor) that is sensitive to the action of rapamycin [48, 5053 ]. mutations in tsc1 or tsc2 genes lead to tuberous sclerosis syndrome with the appearance of various benign tumors in skin, kidney, brain, heart, and lung [48, 50, 54 ]. mtorc1 is described as a regulator of the inflammatory activity of macrophages and other innate cells because its activation is related to reduction of the nfb activity and increase of il-10, tgf-, and pdl1 expression. mtorc2, on the other hand, is insensitive to rapamycin and rictor (rapamycin insensitive companion of mtor) and sin1 are two specific proteins for this complex. moreover, the mtorc2 can be activated by specific growth factors in macrophages and phosphorylates akt. the specific mtorc2 function in macrophages remains unclear, but recent studies showed that since the lack mtorc2, the inflammatory response is augmented in dendritic cells and macrophages. in this study with murine macrophages lacking rictor, the authors demonstrated the overresponse to tlrs ligands and the increase of the levels of m1 related - genes and lower expression of m2 markers. these data suggest that mtorc2 plays key roles in the macrophage polarization and in the regulation of the inflammatory response. in 2013, a group of researchers using rapamycin in human monocytes showed that monocytes had a cytokine profile after stimulation similar to that expected for m1. in the same year, byles and colleagues showed that the tsc1-mtor pathway regulated the polarization of human macrophages. they showed that, in the absence of tsc1, the macrophages did not differ to m2 even in the presence of il-4 and suggested that activation of mtorc1 would lead to downregulation of akt interfering with the expression of characteristic m2 genes. in addition, recently, huang and collaborators (2015) observed that glycolytic metabolism, specific of m2 profile, requires both mtorc2 and stat6 pathway activation in il-4 and csf1-stimulated macrophages. lin. (2002) highlighted the csf-1 as one of the major regulators to mononuclear cells and its expression in breast carcinomas is correlated with poor prognosis. in 2014, zhu and colleagues also related the macrophages polarization to tsc1 and mtor. in this case, the authors used tsc1 knockout mice and observed resistance to m2 profile polarization and m1 macrophages increased. however, they concluded that m2 inhibition occurred in mtor - dependent manner and the m1 increase was mtor - independent and erk - dependent. additionally, mtor pathway is also responsible for the control of cellular metabolism, which appears differently between the poles. in the tumor context chen. (2012) demonstrated that, in human peripheral monocytes stimulated by lipopolysaccharide, the mtor was inhibited by rapamycin or activated by rna interference - mediated knockdown of the mtor repressor tuberous sclerosis complex 2 (tsc2). thus, rapamycin took to differentiation of monocytes into macrophages m1 releasing more il-12 and less il-10 while tsc2 knockdown led to the differentiation of monocytes into macrophages m2 releasing less il-12 and more il-10. moreover, angiogenic properties were stimulated from endothelial cells from human umbilical vein cocultured with tsc2 deficient monocytes or reduced in the group treated with rapamycin. the tumor angiogenesis and growth in murine xenografts were held after infusion of tsc2 deficient monocytes or reduced with monocytes overexpressing tsc2. finally, in vivo depletion of macrophages was sufficient to block the antiangiogenic effects of rapamycin in tumors. these results define the tsc2-mtor pathway as a determinant in the differentiation of monocytes in tam m2 phenotype that promote angiogenesis.. (2016) demonstrated that macrophages, associated with the microenvironment of the renal cell carcinoma, favor tumor metastasis by activation of akt / mtor pathway. in addition, as for the mtor signaling to be related to macrophages differentiation and consequently in angiogenesis, studies show its role in proliferation, migration and cell survival [59, 61, 62 ]. these data together reinforce that the mtor pathway can be used as a therapeutic target in an attempt to modulate macrophage response in the tumor environment or the raised immune response so that there is an antitumor effect. not only macrophages but also cancer cells present mtor pathway activation that modulates different molecules that contribute to the tumoral microenvironment. in turn, this environment can induce the tam polarization, demonstrating the close and complex relationship between cancer cell, macrophages, and tumor progression. gene mutations such as oncogenes and tumor suppressor genes confer constitutive activation of the mtor pathway in conditions of nutrient depletion increasing its expression in various types of cancer, such as akt in breast and ovarian cancer, pi3k in ovarian, gastrointestinal, breast, and prostate cancer, tsc1/tsc2 in the hamartomas formation and lymphangioleiomyomatosis development. however, the inhibition by rapamycin or nutrient depletion induces the activation of autophagy by pi3 kinase, akt, and mtor pathways, which allows the cancerous cells survival under conditions of a microenvironment with low amount of nutrients [59, 63 ]. in autophagy, the release of amino acids arising from autophagic degradation generates reactivation of mtorc1 and the restoration of lysosomal cell population and promotes cell growth. mtor pathway plays a critical role in the tumoral neovascularization providing an environment where cells grow. initially, tumor microenvironment modifications can induce mtor to increase the translation of hypoxia - inducible factor 1 (hif-1)/hypoxia - inducible factor 2 (hif-2), which drive the expression of genes for hypoxic stress response, including angiogenic growth factors such as vegf, pdgf-, and tgf-. interestingly, vegf has also presented the direct capability to activate the mammalian target of rapamycin complex 1 (mtorc1) pathway through the cross talk with phospholipase c gamma (plc). as mtorc1 is a metabolic sensor, metabolic signals may be integrated with signals from vegf in the regulation of angiogenesis leading to a positive feedback loop to enhance angiogenic responses. furthermore, the lymphangiogenesis can be also controlled by mtor pathway modulation. to illustrate the relation between mtor and lymphangiogenesis ekshyyan. the authors observed that rapamycin (mtor inhibitor) significantly inhibited tumor progression and lymphangiogenesis when compared to control, and in conclusion ekshyyan. (2013) describes mtor inhibitor impairing the autocrine and paracrine vegf - c / vegfr-3 axis. (2013) conclusion could be linked to tmas population ; mtor inhibitor can affect macrophages changing their expression profile. in last decades significant improvement of overall survival has been observed in patients with neoplastic disease. some factors (locoregional recurrences, distant metastases, and a second primary tumor) may influence the prognosis reducing overall survival rates. in recent years, a better understanding of tumor - associated macrophages associated with these tumors microenvironment might help to find adjuvant therapies to improve patients ' overall survival. we need to remember that tams may present an m2-like (immunosuppressive) phenotype that leads to enhancing the tumor development. therefore, the tams phenotype may play a dual role in tumoral progression and disease overall survival. an animal model of lung cancer demonstrated that the immunomodulation of m2 tams population was able to reduce the tams population and tumor size and enhances overall survival rates. (2011) aimed at analyzing the impact of tumoral microenvironment on survival outcomes showed that patients diagnosed with human gastric cancer that have high level of tams presented a significantly lower overall survival when compared to patients presenting low level of tams. in a retrospective study comparing tams population in pancreatic ductal adenocarcinoma chen. (2015) described that tumor presenting a high level of tams infiltration has a significantly worse relation to prognostic factors and lower survival rates. as a first analysis the authors observed distribution of cd68 and cd163 (m2-like macrophages), and focused on m2 phenotype the authors observed a positive relation to histological grade and n stage. 2015) have also demonstrated the relation between tams and survival ; neoplasm with high m2-like macrophage infiltration presented a worse survival rate when compared to lesion presenting a low m2-like infiltration. corroborating the idea of worse prognoses of m2-infiltrating tams a study evaluating the correlation between cd163 + macrophages and survival rates in melanomas showed that lesions presenting a high population of cd163 + macrophages have significantly poor overall and melanoma - specific survival. in the same way, breast cancer has also demonstrated a worse prognosis related to tams infiltration, and tsutsui. (2005) described that patients with high tams population have a reduced disease - free survival. however, this behavior (relation between tams and worse prognoses) is not rule for all neoplastic entities. to exemplify the correlation between tams infiltration and better prognosis, edin. (2012) studying colorectal cancer demonstrated a significant association between improved prognosis and cd163 + tams. to explain this relation the authors demonstrate the parallel existence of a nos2 + tams population (m1-like macrophage), and edin. (2012) believe that patient 's outcome was determined by m1 and m2 phenotypes balance. the tumor - associated macrophages have emerged as a main component effector and regulator of the innate immune response to neoplasms. biologically, the cancer microenvironment has provided important signals to macrophages plasticity. in this context, the mtor signaling pathway, a key factor in m1 and m2 polarization, can be, in the near future, considered as possible target in anticancer therapy. | since 2000, written with elegance and accuracy, hanahan and weinberg have proposed six major hallmarks of cancer and, together, they provide great advances to the understanding of tumoral biology. our knowledge about tumor behavior has improved and the investigators have now recognized that inflammatory microenvironment may be a new feature for the tumor entities. macrophages are considered as an important component of tumoral microenvironment. biologically, two forms of activated macrophages can be observed : classically activated macrophages (m1) and alternative activated macrophages (m2). despite the canonical pathways that control this puzzle of macrophages polarization, recently, mtor signaling pathway has been implicated as an important piece in determining the metabolic and functional differentiation of m1 and m2 profiles. currently, it is believed that macrophages related to tumoral microenvironment present an m2-like feature promoting an immunosuppressive microenvironment enhancing tumoral angiogenesis, growth, and metastasis. in the present review we discuss the role of macrophages in the tumor microenvironment and the role of mtor pathway in m1 and m2 differentiation. we also discuss the recent findings in m1 and m2 polarization as a possible target in the cancer therapy. |
the meiotic cell cycle in mammalian oocytes proceeds up to the diplotene or dictiate stage of the first prophase and stay at this phase before lh surge (hubbard & terranova, 1982 ; dutta., 2016). during growth phase of the follicle, the oocyte remains the arrested condition at prophase i. at this time it is incompetent to complete meiosis. the germinal vesicle intact oocytes in cumulus - oocyte - complex removed from mature antral follicles spontaneously progress to the metaphase ii stage by culture without gonadotropin (pincus & enzmann, 1935 ; erickson & sorrenson, 1974). on the other hand, the oocytes enclosed in an antral follicle, do not enter meiosis even though these follicles are removed from their environment. by the adding of gonadotropin or releasing from follicle therefore, some low molecular weight substances in the follicular fluid have been suggested as a mediator for meiotic arrest (eppig. cyclic adenosine monophosphate (camp) is a key signaling mediator in oocyte for meiotic prophase arrest in most species studied. elevated camp concentration in the oocyte has been demonstrated to maintain meiotic arrest and a transient decrease in camp levels in the oocytes is thought to be a primary trigger for resumption of meiosis. camp works through protein kinase a (pka)-mediated phosphorylation of maturation promoting factor (jones, 2008). the levels of camp can be modulated by the activity of camp phosphodiesterase, pde3a in the oocyte (hambleton., 2005). on the other hand, camp can be transported from the surrounding cumulus cells (norris., 2009) or generated by oocyte itself through the activation of heterotrimeric g protein (gs)-linked receptor, gpr3 or grp12 g (mehlmann, 2005). in the case of cyclic gmp, it has been shown to increase in the whole ovaries in hamsters until the lh surge when camp rises and cgmp falls (hubbard, 1980). cgmp works in maintaining of the meiotic arrest in oocyte and that this inhibition is dose and time dependent and expressed only in oocytes with intact cumulus (jankowski., 1997 ; zhang., 2010 ; robinson., 2012 ; egbert., 2014 ; shuhaibar., it appears that the inhibitory action of cgmp on oocyte maturation is mediated by the cumulus oophorous while camp acts directly on the oocyte (hubbard & terranova, 1982). however, so far, the essentiality of cumulus cells for cgmp in oocyte is controversy. therefore, in the present study, we investigate whether the cgmp - mediated meiotic arrest can be possible without cumulus cells during in vitro culture. cd1 mice were maintained on a 10 hr light and 14 hr dark cycle under standard vivarium condition and were supplied with food and water ad libitum. immature female mice (20 - 24 days old) were injected with 5 iu of pregnant mares serum gonadotropin (pmsg ; i.p.) to enhance multiple follicular development. oocytes were collected by ovarian follicular puncture with a needle under a dissecting microscope. to get cumulus free gv oocyte, the cumulus mass were removed with a fine - bore pipetted. the entire procedure did within 20 min after puncture and only the healthy oocytes and cumulus - enclosed oocytes were chosen for examination. cumulus - oocyte complexes and oocytes were transferred into bww medium containing 0.4% bsa and cultured. oocytes (8 - 10 oocytes / drop) were cultured in 10 l drops of bww in mineral oil (sigma) or 15 hr at 37 in a humidified atmosphere conditioning 5% co2. the progression of meiotic maturation was scored by observing the breakdown of germinal vesicles (gvb) and the subsequent appearance of polar bodies with an inverted microscope. cell permeable cgmp analogs, 8-bromoguanosine 3,5-cyclic monophosphate (8-br - cgmp ; 1 mm or 1 m), rp-8-bromo - cgmps (1 mm or 1 m), rp - pcpt-8-bromo - cgmp (1 mm or 1 m) were treated into the medium and gv intact oocytes were cultured. the progression of meiotic maturation was scored by observing the breakdown of germinal vesicles (gvb) and the subsequent appearance of polar bodies with an inverted microscope. chi - squared test was used in statistical analysis, and a p value less than 0.05 was considered significant. to know the difference of cgmp sensitivity to meiotic arrest by the existence of cumulus, cell permeable cgmp analog, 8-bromoguanosine 3,5-cyclic monophosphate (8-br - cgmp) was treated to gv intact oocytes as mentioned in materials and methods. gvb was inhibited concentration dependently by the 8-br - cgmp in the cumulus - enclosed oocyte (fig., it also inhibited gvb in denuded oocytes with concentration dependent manner (fig. at 6 hr of incubation, the gv intact percentage of cumulus - enclosed oocytes was 0% at control, 21% at 1 mm 8-br - cgmp, and 41% at 1 m 8-br - cgmp. on the other hand, in the case of denuded oocyte, the gv intact percentage was 0% at control, 1.4% at 1 mm 8-br - cgmp, and 95% at 1 m 8-br - cgmp. at 15 hr of incubation, the intact percentage was 0% at control, 0% at 1 mm 8-br - cgmp, and 11% at 1 m 8-br - cgmp. in the case of denuded oocyte, gv intact percentage was 0% both at control and 1 mm 8-br - cgmp, and 85% at 1 m 8-br - cgmp. the inhibitory effect of 8-br - cgmp was more severe in denuded oocytes than cumulus - enclosed oocytes. at 1 m 8-br - cgmp, the gvb inhibition was significantly higher in denuded oocytes than cumulus - enclosed oocytes at all time points (fig. gv oocytes were cultivated in medium containing 0.001 mm or 1 mm 8-br - cgmps for 15 hr and nuclear status were determined under the investigated microscope (nomarski dic modulated) at 0 hr, 1.5 hr, 3 hr, 6 hr, 9 hr and 15 hr. the cell permeable cgmp analog rp-8-br - cgmp and rp - pcpt-8-br - cgmp but have a different function from 8-br - cgmp were treated to the gv intact oocytes. rp-8-br - cgmp and rp - pcpt-8-br - cgmp have antagonistic effects in cgmp target cellular mediator, a cgmp - dependent protein kinase. in cumulus - enclosed oocyte 2a). in denuded oocyte, it also did not block the gvb (fig. at 6 hr of culture, gv intact percentages in cumulus - enclosed oocytes were 0% both at control and 1 m rp-8-br - cgmp, and 48% at 1 mm rp-8-br - cgmp. in denuded oocytes, the gv intact percentages were 4% at control, 70% at 1 mm rp-8-br - cgmp, and 20% at 1 m rp-8-br - cgmp. at 15 hr after culture, the gv arrest percentages in cumulus - enclosed oocytes were 0% both at control and 1 m rp-8-br - cgmp, and 10% at 1 mm rp-8-br - cgmp. in denuded oocytes, the gv arrest percentages were 0% at control, 10 % at 1 mm rp-8-br - cgmp, and 2.5% at 1 m rp-8-br - cgmp (fig. 2). compared with the results of 8-br - cgmp in denuded oocytes, gv oocytes were treated with 0.001 mm or 1 mm rp-8-br - cgmp combined with 750 m guanosine for 15 hr and nuclear status were determined under the investigated microscope (nomarski dic modulated) at 0 hr, 1.5 hr, 3 hr, 6 hr, 9 hr and 15 hr. : p<0.05 versus control. in the case of rp - pcpt-8-br - cgmp, its effect on gv arrest was stronger than rp-8-br - cgmp (fig. 2 and 3). in cumulus - enclosed oocytes which were cultured in rp - pcpt-8-br - cgmp containing media, the gv arrest percenttages were 0% at control, 43% at 1 mm rp - pcpt-8- br - cgmp, and 9.5% at 1 m rp - pcpt-8-br - cgmp after 6 hr of culture (fig. the gv arrest percentages were 0% both control and 1 m rp - pcpt-8-br - cgmp, and 23% at 1 mm rp - pcpt-8-br - cgmp. in cumulus - enclosed oocytes, the gv arrest effect is similar between rp-8-br - cgmp and rp - pcpt-8-br - cgmp. in denuded oocyte, the gv arrest percentages after 6 hr of culture were 40% at control and 73% both in 1 mm and 1 m rp - pcpt-8-br - cgmp. at 15 hr of culture, the gv arrest percentages were 0% at control, 57% at 1 mm rp - pcpt-8-br - cgmp, and 60% at 1 m rp - pcpt-8-br - cgmp. when compared with the results of cumulus - enclosed oocytes and denuded oocytes, the inhibitory effects of rp - pcpt-8-br - cgmp severe in denuded oocytes (fig. gv oocytes were treated with 0.001 mm or 1 mm rp - pcpt-8-br - cgmp combined with 750 m guanosine for 15 hr and nuclear status were determined under the investigated microscope (nomarski dic modulated) at 0 hr, 1.5 hr, 3 hr, 6 hr, 9 hr and 15 hr. gv ; germinal vesicle, cont ; control. : p<0.05 versus control. fertility of female is depending on the quantity and quality of follicles, and the getting a competence for development in the oocyte is critical for oncogenesis. oocytes have to stay until ovulation at gv intact status, prophase i of meiosis. cotreatment of fsh and adenosine markedly inhibits the gvb of ceo in a dose - dependent manner (mille & behrman, 1986). recently, we reported about the possible roles of adenosine and adenosine receptor - mediated calcium signaling in gv arrest of oocytes enclosed with cumulus (hwang & cheon, 2013). it supports the role of calcium in camp action (powers & paleos, 1982 ; preston., 1987). on the other hand, cyclic gmp also is also suggested as a mediator for camp regulation in oocyte. cgmp inhibit camp hydrolysis through pde3a (norris., 2009 ; vaccari., 2009). the cgmp levels in follicle are uniformly high (about 2 - 4 m). the level of cgmp in oocyte is also decreased uniformly to about 100 nm and this decrease causes of meiotic restart (shuhaibar., 2015). in follicle cgmp producing guanylyl cyclase natriuretic peptide receptor 2 (npr2, guanylyl cyclase b) is present in all of the granulosa cells, but not in the oocyte (jankowski., 1997 ; zhang., 2010 ; robinson., 2012 ; egbert., the cell permeable cgmp analog, 8-br - cgmp inhibits gvb with concentration - dependent manners both in cumulus - enclosed oocytes and denuded oocyte. the effects of 8-br - cgmp were stronger in cumulus - enclosed oocytes than denuded oocytes. such a responsibility of oocyte to cgmp suggest that cumulus may modulate the cgmp effects on gvb arrest. the antagonists of cgmp in cellular signaling, rp-8-br - cgmp and rp - pcpt-8-br - cgmp were showed opposite effect to 8-br - cgmp. however, its effects were specific to the concentration of rp-8-br - cgmp in cumulus - enclosed oocytes but not in denuded oocytes. in denuded oocytes, the spontaneous maturation processed dependently to the concentration of rp-8-br - cgmp. in cumulus - enclosed oocytes, the effects of rp - pcpt-8-br - cgmp also had a similar pattern with the rp-8-br - cgmp concentration. in denuded oocytes, rp - pcpt-8-br - cgmp was significantly inhibited gvb in all groups compared with control, though its gvb inhibitor effect were less than 8-br - cgmp. the responsibility to the rp - pcpt-8-br - cgmpthe ministry of education, science and technology and rp-8-br - cgmp however, interestingly rp - pcpt-8-br - cgmp inhibited severely more than rp-8-br - cgmp at 1 mm but not 1 m. such differences between antagonists for cgmp may be originated from the difference of the affinity to the target molecule. based on them, it means that the sensitivity for gv arrest to cgmp may be modified by cumulus cells. the cgmp transported from granulosa cells to oocyte through gap junction has been suggested as a key factor for gv arrest. based on the results, we know that the responsibility to agonist or antagonist is modified by the cumulus. the gv arrest was maintained in denuded oocyte by 8-br - cgmp significantly compared with rp-8-br - cgmp or rp - pcpt-8-br - cgmp. put together, it suggests that the role of cgmp for spontaneous gvb is modulated by cumulus. | abstractintact germinal vesicle (gv) arrest and release are essential for maintaining the fertility of mammals inducing human. intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. cyclic guanosine monophosphate (cgmp) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. in this study we examine the effects of cgmp on germinal vesicle breakdown in cumulus - enclosed oocytes and denuded oocytes. spontaneous maturation was inhibited by a cgmp agonist, 8-br - cgmp with concentration dependent manners both in cumulus - enclosed oocytes and denuded oocytes. the inhibitory effect was more severe in denuded oocytes than cumulus - enclosed oocytes. the rp-8-br - cgmp and rp - pcpt-8-br - cgmp did not severely block gvb compared to 8-br - cgmp. the spontaneous gvb inhibitory effects were different by the existence of cumulus. based on them it is suggested that the cumulus modulates the role of cgmp in gv arrest. |
cardiomyopathies are difficult and complicated diseases of heart muscle associated with heart failure and/or sudden cardiac death, which are classified by the world health organization (who) in 1995 into four main forms ; hypertrophic cardiomyopathy (hcm), dilated cardiomyopathy (dcm), restrictive cardiomyopathy (rcm), and arrhythmogenic right ventricular cardiomyopathy (arvc). hcm is characterized by ventricular muscle hypertrophy, which especially involves the increased thickness of interventricular septum, leading to a marked decrease in left ventricular (lv) chamber volume. hcm has impaired lv diastolic function probably because of hypertrophy itself, interstitial fibrosis and/or myocyte disarray, while it usually has normal, or slightly attenuated lv systolic function., the prevalence of hcm is about 1/500, in which more than 70% are familial cases. dcm is characterized by abnormal enlargement of lv or both ventricular chambers with impaired systolic function, affecting about 36.5 in 100,000 people, of whom about 25%30% are familial cases. its prognosis is poor due to high frequency of arrhythmias and sudden death, and there are no effective therapeutic drugs at end stage and heart transplantation is the most effective treatment for survival. rcm, which is sometimes familial, is a rare form of cardiomyopathy, characterized by impaired ventricular filling with normal or decreased diastolic volume of either, or both ventricles and normal, or near normal, systolic function and wall thickness., arvc is characterized by progressive fibrofatty replacement of right ventricular myocardium, with palpitations, syncope, and sudden death. its prevalence is between 1/1000 and 1/5000, with 10% of deaths occurring before age 19 and 50% before age 35. reported the first hcm - causing mutation of -myosin heavy chain (-myhc) gene in 1990, a large number of mutations in sarcomeric protein genes that encode -myhc, cardiac troponin t (ctnt), cardiac troponin i (ctni), cardiac troponin c (ctnc), -tropomyosin (-tm), cardiac myosin binding protein c (mybp - c), ventricular myosin light chains 1 and 2 (lc1, lc2), actin and titin / connection, etc.,, as well as cytoskeletal and nuclear membrane protein genes that encode dystrophin, desmin, tafazzin, -sarcoglycan, lamin a / c, etc., have been identified as a cause of hcm, dcm and rcm. in contrast, no sarcomeric protein genes have been shown to be responsible for arvc. molecular genetic testing has so far indicated that arvc is associated with more than eight genes which encode the transforming growth factor -3 gene (tgfb3), ryanodine receptor 2, desmoplakin, transmembrane protein 43, junction plakoglobin (also known as gamma catenin), etc., many studies have been made on the structural and/or functional consequences of mutations, but the molecular mechanisms by which mutations found in the above genes lead to the pathogenesis of various forms of cardiomyopathy are not entirely clear. sarcomere, a structural unit of the contractile apparatus myofibril in cardiac muscle, has thick and thin filaments, which are composed of myosin and actin / tropomyosin / troponin complex, respectively. the molecular mechanism of ca regulation of muscle contraction has extensively been investigated since dr., it is now common knowledge that troponin consists of three subunits with distinct function and structure, in which tnt anchors tn complex to tm, tni inhibits the actin - myosin contractile interaction, and tnc removes the inhibitory action of tni upon binding ca. until now, there have been over 100 mutations found in three ctn subunits causing hcm, dcm or rcm. this article focuses on the molecular mechanisms underlying the pathogenesis of cardiomyopathies caused by these mutations. cardiac muscle contraction is generated by the interaction between myosin head and thin filament, upon activated actomyosin mg - atpase. actin molecules polymerize into a double helical filament, which forms the backbone of the thin filament. tm is an -helical coiled - coil fibrous protein interacting with adjacent tm molecules in a head to tail manner and is located along polymerized actin together with tn complex at a actin : tm : tn ratio of 7 : 1 : 1., tn consists of three subunits : tnt, which anchors tn complex to tm, also interacts directly with actin ; tni, which is involved in the inhibition of actomyosin mg - atpase and inhibits actin - myosin interactions at diastolic levels of intracellular ca ; tnc, which is a ca - binding subunit that removes tni inhibition upon ca binding. tn plays a key role in the transition from diastole to systole of cardiac muscle. when the cytoplasmic ca concentration of cardiac myocytes is low around 2 10 mol / l, the contractile interaction of actin with myosin head is inhibited by tn - tm. when cytoplasmic ca concentration is raised by electrochemical coupling involving a membrane controlled release of ca into sarcoplasm and ca mobilization known as calcium - induced calcium release, ca binds to tnc and triggers a series of conformational changes of protein - protein interactions in the thin filament, which relieves the inhibition by tn - tm and thus promotes the contractile interaction between actin and myosin. tnt has two functionally and structurally distinct domains named t1 and t2., t1 (n - terminal region of tnt) anchors tn complex to the thin filament through its strongly binding to tm. on the other hand, t2 (c - terminal region of tnt) interacts with other tn subunits (tni and tnc) and tm / actin. the molecular weight of tnt is 3136 ku, with 250300 amino acid residues. ctnt is a peptide of 288 amino acid residues with approximate 35 ku of molecular weight. in human heart, there are four ctnt isoforms (ctnt1-ctnt4) produced by alternative splicing of ctnt transcripts at exon 4 and exon 5., it is suggested that the alternative splicing of ctnt may contribute to altered myofilament response to ca in cardiac muscle contraction. ctnt is a substrate for protein kinase c (pkc) containing up to four potential phosphorylation sites. sumandea,. reported that phosphorylation at thr-206 significantly reduces maximum force generation, ca - sensitivity and cross - bridge cycling rate, suggesting that pkc- mediated phosphorylation of ctnt may play a role in the regulation of cardiac contraction. studies on inherited cardiomyopathies caused by ctnt mutations have revealed an important function of this protein in the ca regulation of muscle contraction. since thierfelder,. found two missense mutations (ile79asn and arg92gln) and a splice donor site (intron 16g1a) mutation in ctnt in inherited hcm patients in 1994 and until only recently, a new mutation p80s was reported in a japanese family, 36 hcm - linked mutations, 13 dcm - linked mutations and 4 rcm - linked mutations dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. mutations in ctnt account for approximately 15%30% of all hcm case. however, they usually lead to poor prognosis and high incidence of sudden cardiac death ; e.g., i79n, r92w, r92q and e160, etc., induce a malignant clinical phenotype with the average life of patients being no more than middle age.,,, the functional consequences of these mutations have been studied by many groups., we studied the functional effects of these hcm mutations using rabbit left ventricular skinned fibers and myofibrils with a ctnt - exchange technique., we found most of these hcm - linked ctnt mutations, such as i79n, r92q, r92l, r92w, r94l, a104v, r130c, e160, e163r, e163k and e244d, etc., increased the ca sensitivity of force generation in skinned fibers and shifted the force - pca relationship leftwards, but did not significantly affect the maximum force generating capability or atpase activity and ca - cooperativity. on the other hand, r278c, r278p, tnt28(+7) and tnt14 decreased ca - cooperativity in force generation in skinned fibers in addition to a ca - sensitizing effect. almost all hcm - causing mutations occur in the tm - anchoring region (residues 79 - 170 and 272 - 288) in ctnt, strongly suggesting that these mutations impair the interaction of ctnt and tm leading to an increase in the ca - sensitivity by decreasing the inhibitory action of ctni on the thin filament. recently, manning,. suggested that the ctnt hcm - related mutations in, or flanking, the n - tail t1 domain (residues 79 - 170) directly interacting with overlapping tm, may alter the flexibility of t1, which is inversely proportional to ca - cooperativity. in 2000, a deletion mutation k210 of ctnt was reported as the first dcm - causing mutation of sarcomeric proteins. since then, this mutation has extensively been studied both in vitro and in vivo. with the ctnt - exchange technique, we have shown that this mutation confers a lower ca - sensitivity on the force generation of skinned cardiac muscle fibers and atpase activity of cardiac myofibrils compared with wild - type, while having no effects on the maximum force, or atpase activity, and ca - cooperativity. other groups also reported similar results. almost all dcm - causing mutations reported in ctnt to date, e.g., r131w, r141w, r205, k210, r205l, and d270n, etc., have demonstrated a ca - desensitizing effect on skinned fiber force generation and myofibrillar or actomyosin atpase activity., these results strongly suggest that ca desensitization of cardiac muscle contraction is a primary mechanism for the pathogenesis of dcm caused by ctnt mutation, in contrast to hcm where ca sensitization is a primary mechanism for the pathogenesis. however, it is still not completely clarified how one amino acid mutation can increase or decrease the ca - sensitivity of force generation of cardiac muscle in the respective form of inherited cardiomyopathy. we demonstrated that the dcm - causing mutation of ctnt r141w increased the affinity of ctnt for tm, by using a quartz - crystal microbalance. this result strongly suggests that r141w mutation in the strong tm - binding region in ctnt strengthens the integrity of ctni in the thin filament by stabilizing the interaction between ctnt and tm, which might allow ctni to inhibit the thin filament more effectively, leading to ca desensitization. rcm - causing mutations, unlike hcm- and dcm - causing mutations, are rare and recently found in ctnt (i79n, e96, and e136k)., pinto,., reported that e96 showed a large increase in the ca - sensitivity and impaired abilities to inhibit atpase and to relax skinned fibers, which could contribute to the severe diastolic dysfunction in rcm. more recently, pinto,., found a novel double deletion in ctnt of two highly conserved amino acids (n100 and e101) in a rcm pediatric patient, which also conferred a large increase in the ca - sensitivity. in addition, this double deletion mutation decreased the ca - cooperativity of force development, suggesting alterations in intra - filament protein - protein interactions. ctni binds to a specific region of each tm molecule with a 38 nm periodicity along the tm / actin filament., ctni is the inhibitory subunit of ctn complex, which is responsible for inhibition of actomyosin atpase activity. in the absence of ca, ctni inhibits contraction through interacting with actin ; the inhibitory effect of ctni is relieved during muscle contraction through interacting with ctnc upon ca binding to ctnc. hence, ctni is a key regulatory protein in cardiac muscle contraction and relaxation cycle. ctni consists of several functional domains, a cardiac specific n - terminal extension region (residues 1 - 32), an it - arm region, the inhibitory region (residues 128 - 147), the switch region (residues 147 - 163), and the c - terminal mobile domain., phosphorylation of ctni by several different kinases plays important roles in the regulation of cardiac muscle contraction under physiological or pathological conditions. protein kinase a (pka)-mediated phosphorylation of ctni at ser-23/ser-24 reduces myofilament ca - sensitivity, increases the rate of ca dissociation from ctnc, increases crossbridge cycling rate, and enhances unloaded shortening velocity. pkc may phosphorylate ctni at ser-23/ser-24, ser-43/ser-45 and thr-144 ; phosphorylation at ser-43/ser-45 decreases maximum ca - activated force generation in skinned fibers and maximal sliding velocity in motility assays., we have recently found that ctni is phosphorylated by pkc at ser-23/ser-24 and thr-144, which leads to the depressed cooperative activation of the thin filament. phosphorylation of ctni at different sites by different protein kinases may have distinct effects in vivo, although they are poorly understood. the effects of ctni phosphorylation could be affected by some mutations that cause inherited cardiomyopathies. thirty, four, and nine mutations of ctni have so far been found in hcm, dcm, and rcm, respectively,,, most of which are located in the c - terminal region (figure 2). in 1997, kimura,. reported six mutations in ctni (r145 g, r145q, r162w, k183, g203s, and k206q) associated with hcm in an autosomal dominant manner. studies on the functional consequences of these mutations at the level of skinned cardiac muscle force generation and myofibrillar atpase activity revealed that they have a ca - sensitizing effect on cardiac muscle contraction as in the hcm - linked mutations in ctnt. studies of the ca affinity of reconstituted cardiac thin filament with a fluorescence (iaans) labeling technique revealed that r145 g, which is located in the inhibitory region of ctni, increases the basal level of atpase activity and increases the ca binding affinity of the regulatory site of ctnc in the thin filament. mutations of r145 g, r145q, r162w, and g203s show increased minimum force in skinned cardiac muscle fibers with no significant changes in the maximum force. a deletion mutation k183 increases the ca - sensitivity with no effects on either maximum or minimum force generation of skinned cardiac muscle. dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. until 2009, only one mutation a2v was reported to cause dcm in an autosomal recessive mannar, which impairs the interaction of ctni with ctnt. in 2009, carballo,. reported that the mutations of k36q and n185k of ctni cause dcm in an autosomal dominant manner. k36q has been shown to mediate the movement of the n - terminal region in ctni upon phosphorylation of s22/23 by camp- dependent protein kinase. functional studies of k36q and n185k have revealed that these dcm mutations of ctni decrease the maximum activity and ca - sensitivity of actin - myosin s1 atpase and significantly reduce the ca binding affinity of the regulatory site of ctnc in the thin filament. more recently, murakami,. reported a new missense mutation p16 t in dcm patients. in 2003, published the first report of ctni mutations in rcm patients (l144q, r145w, a171 t, k178e, d190 g, and r192h). we examined the functional and structural consequences of these six mutations by using in vitro assays of skinned cardiac muscle fibers and nmr and revealed that they cause dramatic ca - sensitization of force generation in cardiac muscle with a subtle structural perturbation within ctni molecule. ca - sensitizing effects of these rcm mutations are much greater than those of hcm mutations in ctni. however, d190 g mutation with the smallest ca - sensitizing effect was reported to cause both rcm and hcm in a single large family, suggesting that rcm and hcm are caused by the same mechanism through ca - sensitization with greater sensitization leading to rcm. in addition, all six mutations elevate the resting force of skinned cardiac muscle fibers at low ca concentrations, and four mutations, l144q, r145w, a171 t, and r192h decrease the maximum level of force generation at high ca concentrations. troponin c is a member of the ef - hand family of ca binding proteins and consists of two globular lobes at n- and c - terminus, which are connected with an -helix. each globular lobe contains a pair of ca - binding sites, hence there are four ca - binding sites named i through iv from the n - terminal of vertebrate skeletal tnc (stnc). in contrast to stnc, site i of ctnc dose not bind ca under physiological conditions. analysis of equilibrium ca binding of ctnc demonstrates that the dissociation constant (kd) of site ii is approximate 1.2 10 m, and plays an important ca - regulatory role in cardiac muscle contraction. on the other hand, the dissociation constant (kd) of sites iii and iv are approximate 7.4 x 10 m. sites iii and iv also bind mg with kd at 0.9 10 m, play a structural role to keep the c lobe tightly bound to ctni and to stabilize the interaction between ctnc and ctnt. compared with ctnt and ctni, much fewer mutations have so far been found in ctnc, with only six mutations both in hcm and dcm. c84y and d145e increase the ca - sensitivity of force generation in skinned cardiac muscle fibers. a8v and d145e also increase the force recovery. in contrast to these mutations, e134d shows no changes in ca - sensitivity and force recovery. fluorescence studies using iaans - labeled ctnc suggested that l29q increases the ca binding affinity of site ii of ctnc. functional studies indicated that this mutation increased the ca - sensitivity with no effect on the maximal force generation, and increased the ca affinity of ctnc isolated or incorporated into troponin complex and thin filament. in contrast to the dcm - linked mutations found in ctnt and ctni, g159d mutation in ctnc does not significantly decrease the ca - sensitivity of force generation of skinned fibers, but it induces a decrease in atpase activity of reconstituted myofilaments as well as filament sliding velocity. interestingly, biesiadecki,. provide evidence that g159d may alter myocardial functional responses to -adrenergic stimulation by blunting the ca - desensitizing effect of ctni phosphorylation at ser-23/ser-24. afterward, two novel missense mutations localized in the regulatory ca - binding site ii of ctnc, e59d and d75y, were identified in a dcm patient. these mutations showed a decrease in the myofilament ca - sensitivity and ca binding affinity in the force - pca relationship measurements and fluorescence spectroscopy, respectively. d75y disrupts molecular motions critical for ca binding and cardiomyocyte contractility, whereas functional defect caused by e59d is benign. most recently, pinto,. reported y5h, m103i, and i148v mutations in ctnc in dcm patients. functional studies showed that y5h and m103i decrease the ca2-sensitivity of force generation and that the effects of pka phosphorylation of ctni on the myofilament ca - sensitivity is abolished by m103i but diminished by y5h and i148v. t1 (n - terminal region of tnt) anchors tn complex to the thin filament through its strongly binding to tm. on the other hand, t2 (c - terminal region of tnt) interacts with other tn subunits (tni and tnc) and tm / actin. the molecular weight of tnt is 3136 ku, with 250300 amino acid residues. ctnt is a peptide of 288 amino acid residues with approximate 35 ku of molecular weight. in human heart, there are four ctnt isoforms (ctnt1-ctnt4) produced by alternative splicing of ctnt transcripts at exon 4 and exon 5., it is suggested that the alternative splicing of ctnt may contribute to altered myofilament response to ca in cardiac muscle contraction. ctnt is a substrate for protein kinase c (pkc) containing up to four potential phosphorylation sites. sumandea,. reported that phosphorylation at thr-206 significantly reduces maximum force generation, ca - sensitivity and cross - bridge cycling rate, suggesting that pkc- mediated phosphorylation of ctnt may play a role in the regulation of cardiac contraction. studies on inherited cardiomyopathies caused by ctnt mutations have revealed an important function of this protein in the ca regulation of muscle contraction. since thierfelder,. found two missense mutations (ile79asn and arg92gln) and a splice donor site (intron 16g1a) mutation in ctnt in inherited hcm patients in 1994 and until only recently, a new mutation p80s was reported in a japanese family, 36 hcm - linked mutations, 13 dcm - linked mutations and 4 rcm - linked mutations dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. mutations in ctnt account for approximately 15%30% of all hcm case. however, they usually lead to poor prognosis and high incidence of sudden cardiac death ; e.g., i79n, r92w, r92q and e160, etc., induce a malignant clinical phenotype with the average life of patients being no more than middle age.,,, the functional consequences of these mutations have been studied by many groups., we studied the functional effects of these hcm mutations using rabbit left ventricular skinned fibers and myofibrils with a ctnt - exchange technique., we found most of these hcm - linked ctnt mutations, such as i79n, r92q, r92l, r92w, r94l, a104v, r130c, e160, e163r, e163k and e244d, etc., increased the ca sensitivity of force generation in skinned fibers and shifted the force - pca relationship leftwards, but did not significantly affect the maximum force generating capability or atpase activity and ca - cooperativity. on the other hand, r278c, r278p, tnt28(+7) and tnt14 decreased ca - cooperativity in force generation in skinned fibers in addition to a ca - sensitizing effect. almost all hcm - causing mutations occur in the tm - anchoring region (residues 79 - 170 and 272 - 288) in ctnt, strongly suggesting that these mutations impair the interaction of ctnt and tm leading to an increase in the ca - sensitivity by decreasing the inhibitory action of ctni on the thin filament. suggested that the ctnt hcm - related mutations in, or flanking, the n - tail t1 domain (residues 79 - 170) directly interacting with overlapping tm, may alter the flexibility of t1, which is inversely proportional to ca - cooperativity. in 2000, a deletion mutation k210 of ctnt was reported as the first dcm - causing mutation of sarcomeric proteins. since then, this mutation has extensively been studied both in vitro and in vivo. with the ctnt - exchange technique, we have shown that this mutation confers a lower ca - sensitivity on the force generation of skinned cardiac muscle fibers and atpase activity of cardiac myofibrils compared with wild - type, while having no effects on the maximum force, or atpase activity, and ca - cooperativity. other groups also reported similar results. almost all dcm - causing mutations reported in ctnt to date, e.g., r131w, r141w, r205, k210, r205l, and d270n, etc., have demonstrated a ca - desensitizing effect on skinned fiber force generation and myofibrillar or actomyosin atpase activity., these results strongly suggest that ca desensitization of cardiac muscle contraction is a primary mechanism for the pathogenesis of dcm caused by ctnt mutation, in contrast to hcm where ca sensitization is a primary mechanism for the pathogenesis. however, it is still not completely clarified how one amino acid mutation can increase or decrease the ca - sensitivity of force generation of cardiac muscle in the respective form of inherited cardiomyopathy. we demonstrated that the dcm - causing mutation of ctnt r141w increased the affinity of ctnt for tm, by using a quartz - crystal microbalance. this result strongly suggests that r141w mutation in the strong tm - binding region in ctnt strengthens the integrity of ctni in the thin filament by stabilizing the interaction between ctnt and tm, which might allow ctni to inhibit the thin filament more effectively, leading to ca desensitization. rcm - causing mutations, unlike hcm- and dcm - causing mutations, are rare and recently found in ctnt (i79n, e96, and e136k)., reported that e96 showed a large increase in the ca - sensitivity and impaired abilities to inhibit atpase and to relax skinned fibers, which could contribute to the severe diastolic dysfunction in rcm. more recently, pinto,., found a novel double deletion in ctnt of two highly conserved amino acids (n100 and e101) in a rcm pediatric patient, which also conferred a large increase in the ca - sensitivity. in addition, this double deletion mutation decreased the ca - cooperativity of force development, suggesting alterations in intra - filament protein - protein interactions. ctni binds to a specific region of each tm molecule with a 38 nm periodicity along the tm / actin filament., ctni is the inhibitory subunit of ctn complex, which is responsible for inhibition of actomyosin atpase activity. in the absence of ca, ctni inhibits contraction through interacting with actin ; the inhibitory effect of ctni is relieved during muscle contraction through interacting with ctnc upon ca binding to ctnc. hence, ctni is a key regulatory protein in cardiac muscle contraction and relaxation cycle. ctni consists of several functional domains, a cardiac specific n - terminal extension region (residues 1 - 32), an it - arm region, the inhibitory region (residues 128 - 147), the switch region (residues 147 - 163), and the c - terminal mobile domain., phosphorylation of ctni by several different kinases plays important roles in the regulation of cardiac muscle contraction under physiological or pathological conditions. protein kinase a (pka)-mediated phosphorylation of ctni at ser-23/ser-24 reduces myofilament ca - sensitivity, increases the rate of ca dissociation from ctnc, increases crossbridge cycling rate, and enhances unloaded shortening velocity. pkc may phosphorylate ctni at ser-23/ser-24, ser-43/ser-45 and thr-144 ; phosphorylation at ser-43/ser-45 decreases maximum ca - activated force generation in skinned fibers and maximal sliding velocity in motility assays., we have recently found that ctni is phosphorylated by pkc at ser-23/ser-24 and thr-144, which leads to the depressed cooperative activation of the thin filament. phosphorylation of ctni at different sites by different protein kinases may have distinct effects in vivo, although they are poorly understood. the effects of ctni phosphorylation could be affected by some mutations that cause inherited cardiomyopathies. thirty, four, and nine mutations of ctni have so far been found in hcm, dcm, and rcm, respectively,,, most of which are located in the c - terminal region (figure 2). in 1997, kimura,. reported six mutations in ctni (r145 g, r145q, r162w, k183, g203s, and k206q) associated with hcm in an autosomal dominant manner. studies on the functional consequences of these mutations at the level of skinned cardiac muscle force generation and myofibrillar atpase activity revealed that they have a ca - sensitizing effect on cardiac muscle contraction as in the hcm - linked mutations in ctnt. studies of the ca affinity of reconstituted cardiac thin filament with a fluorescence (iaans) labeling technique revealed that r145 g, which is located in the inhibitory region of ctni, increases the basal level of atpase activity and increases the ca binding affinity of the regulatory site of ctnc in the thin filament. mutations of r145 g, r145q, r162w, and g203s show increased minimum force in skinned cardiac muscle fibers with no significant changes in the maximum force. a deletion mutation k183 increases the ca - sensitivity with no effects on either maximum or minimum force generation of skinned cardiac muscle. dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. until 2009, only one mutation a2v was reported to cause dcm in an autosomal recessive mannar, which impairs the interaction of ctni with ctnt. in 2009, carballo,. reported that the mutations of k36q and n185k of ctni cause dcm in an autosomal dominant manner. k36q has been shown to mediate the movement of the n - terminal region in ctni upon phosphorylation of s22/23 by camp- dependent protein kinase. functional studies of k36q and n185k have revealed that these dcm mutations of ctni decrease the maximum activity and ca - sensitivity of actin - myosin s1 atpase and significantly reduce the ca binding affinity of the regulatory site of ctnc in the thin filament. more recently, murakami,. reported a new missense mutation p16 t in dcm patients. in 2003, published the first report of ctni mutations in rcm patients (l144q, r145w, a171 t, k178e, d190 g, and r192h). we examined the functional and structural consequences of these six mutations by using in vitro assays of skinned cardiac muscle fibers and nmr and revealed that they cause dramatic ca - sensitization of force generation in cardiac muscle with a subtle structural perturbation within ctni molecule. ca - sensitizing effects of these rcm mutations are much greater than those of hcm mutations in ctni. however, d190 g mutation with the smallest ca - sensitizing effect was reported to cause both rcm and hcm in a single large family, suggesting that rcm and hcm are caused by the same mechanism through ca - sensitization with greater sensitization leading to rcm. in addition, all six mutations elevate the resting force of skinned cardiac muscle fibers at low ca concentrations, and four mutations, l144q, r145w, a171 t, and r192h decrease the maximum level of force generation at high ca concentrations. troponin c is a member of the ef - hand family of ca binding proteins and consists of two globular lobes at n- and c - terminus, which are connected with an -helix. each globular lobe contains a pair of ca - binding sites, hence there are four ca - binding sites named i through iv from the n - terminal of vertebrate skeletal tnc (stnc). in contrast to stnc, site i of ctnc dose not bind ca under physiological conditions. analysis of equilibrium ca binding of ctnc demonstrates that the dissociation constant (kd) of site ii is approximate 1.2 10 m, and plays an important ca - regulatory role in cardiac muscle contraction. on the other hand, the dissociation constant (kd) of sites iii and iv are approximate 7.4 x 10 m. sites iii and iv also bind mg with kd at 0.9 10 m, play a structural role to keep the c lobe tightly bound to ctni and to stabilize the interaction between ctnc and ctnt. compared with ctnt and ctni, much fewer mutations have so far been found in ctnc, with only six mutations both in hcm and dcm. c84y and d145e increase the ca - sensitivity of force generation in skinned cardiac muscle fibers. a8v and d145e also increase the force recovery. in contrast to these mutations, e134d shows no changes in ca - sensitivity and force recovery. fluorescence studies using iaans - labeled ctnc suggested that l29q increases the ca binding affinity of site ii of ctnc. functional studies indicated that this mutation increased the ca - sensitivity with no effect on the maximal force generation, and increased the ca affinity of ctnc isolated or incorporated into troponin complex and thin filament. in contrast to the dcm - linked mutations found in ctnt and ctni, g159d mutation in ctnc does not significantly decrease the ca - sensitivity of force generation of skinned fibers, but it induces a decrease in atpase activity of reconstituted myofilaments as well as filament sliding velocity. provide evidence that g159d may alter myocardial functional responses to -adrenergic stimulation by blunting the ca - desensitizing effect of ctni phosphorylation at ser-23/ser-24. afterward, two novel missense mutations localized in the regulatory ca - binding site ii of ctnc, e59d and d75y, were identified in a dcm patient. these mutations showed a decrease in the myofilament ca - sensitivity and ca binding affinity in the force - pca relationship measurements and fluorescence spectroscopy, respectively. d75y disrupts molecular motions critical for ca binding and cardiomyocyte contractility, whereas functional defect caused by e59d is benign. most recently, pinto,. reported y5h, m103i, and i148v mutations in ctnc in dcm patients. functional studies showed that y5h and m103i decrease the ca2-sensitivity of force generation and that the effects of pka phosphorylation of ctni on the myofilament ca - sensitivity is abolished by m103i but diminished by y5h and i148v. in order to determine the significance of the in vitro findings concerning inherited cardiomyopathies, especially to validate whether the shift in cardiac myofilament ca- sensitivity is the most important and a common cause of cardiomyopathies associated with tn mutations in vivo, it is necessary and important to produce animal models. we have succeeded to create a knock - in mouse model in which a deletion of three base pairs coding for k210 in ctnt found in dcm patients was introduced into mouse endogenous ctnt gene using gene - targeting technology. this mouse model showed a particularly high incidence of sudden death in their growth periods from one to three months old, and enlarged hearts and heart failure, closely recapitulating the phenotypes of human dcm patients., surface ecg showed that mutant mice commonly had an electrophysiological abnormality in the heart with long qt, which might be involved in their frequent sudden death. telemetric ecg recordings showed that mice died by abruptly developing repetitive torsade de pointes several hours before death, which ultimately degenerated into ventricular fibrillation. consistent with the results of in vitro studies, skinned cardiac muscle fibers prepared from mutant mice showed significantly lower ca - sensitivity in force generation. ca transient analysis using fura-2 in cardiomyocytes showed a significant increase in the peak amplitude in mutant mice, suggesting that ca transient is augmented to compensate for decreased myofilament ca - sensitivity. an increased intracellular camp level might be responsible for the augmented ca transient in mutant mice. skinned fibers prepared from f110i - ctnt transgenic mice showed an increased ca - sensitivity of force and atpase activity, and a much increased energy cost. in contrast, no changes in force or the atpase- pca dependencies were observed in transgenic r278c- ctnt skinned fibers. the increased ca - sensitivity and higher energy cost in f110i - ctnt hearts may be responsible for the severe phenotype compared with r278c - ctnt. this result supports the hypothesis that a greater shift in ca - sensitivity of force development results in more severe clinical phenotype and prognosis., a knock - in mouse model of hcm caused by ctni mutation of r21c also showed an increased ca - sensitivity of force development, consistent with the findings in mouse models of hcm caused by ctnt mutations. heterozygous and homozygous mutant mice both developed a remarkable degree of cardiac hypertrophy and fibrosis. r21c is the only mutation identified within the unique n - terminus of ctni and is located in the region close to -adrenergic- activated pka - mediated phosphorylation sites. heterozygous mice reduced and homozygous mice abolished the well - known decrease in the ca sensitivity of force generation by the phosphorylation of ctni at ser23/ser24 with pka, suggesting that the impaired regulation of myofilament ca sensitivity by pka phosphorylation in ctni may also play a role in pathogenesis of hcm caused by this mutation. although studies using knock - in and transgenic mouse models have provided much important and useful information, we have to note that mouse models are different from human in ca handling during contraction / relaxation and in alterations in ca flux during heart failure., made a hcm - causing r146g - ctni transgenic rabbit model that reflects the human system more accurately. this rabbit model also showed an increased ca - sensitivity of skinned fibers prepared from ventricular papillary muscle, as well as cardiomyocyte disarray, fibrosis, and altered connexin43 organization, which recapitulate the human hcm phenotype. human patients should have different contractile dynamics in cardiac muscle compared with mouse models, because mouse heart mainly expresses the -myosin heavy chain (mhc) isoform, whereas human heart mainly expresses the -mhc isoform with much lower atpase activity., recently created a mouse model expressing hcm - related mutation r92l ctnt and -mhc by crossing the transgenic mouse strain expressing r92l ctnt with the transgenic mouse strain predominantly expressing the -mhc isoform. they found that the length - dependence of contractile activation and myofilament ca - sensitivity was blunted in these mice, suggesting that the mhc isoform has an important effect on the outcome of ctnt mutations. many functional consequences of ctn mutations in inherited cariomyopathies have been revealed, such as changes in regulatory protein - protein interaction, alterations of crossbridge cycling rate or unloaded shortening velocity, and alterations of atpase activity or phosphorylation level of key regulatory proteins like ctni., however, it should be noted that almost all mutations in ctn shift the ca - sensitivity of cardiac muscle contraction. recently, liu,., demonstrated by fluorescence studies that cardiomyopathy - related cardiac tn mutations alter the ca association and dissociation rates of ctnc in the thin filament, also implying that the alteration of steady state ca - sensitivity of the thin filament by the mutations in ctn may be a primary cause for cardiomyopathies. changes in ca - sensitivity and subsequent alteration of ca homeostasis could be a common, and the most important underlying mechanisms that trigger the arrhythmias leading to cardiac sudden death or the development of congestive heart failure.,,, to fully understand the pathogenic mechanism of inherited cardiomyopathies and their therapies, it is important to generate animal models that closely recapitulate human pathological features. we attempted to treat the dcm - causing knock - in k210-ctnt mice with a ca sensitizer pimobendan, which was expected to be beneficial for the mice with decreased cardiac myofilament ca - sensitivity. this result strongly supports the hypothesis that ca desensitization of cardiac muscle contraction is a primary mechanism for the pathogenesis of dcm caused by this ctnt mutation, which has been led by our in vitro studies, and suggests that ca sensitizers might be beneficial for the treatment of dcm patients associated with the sarcomeric regulatory protein mutations that cause myofilament ca desensitization, including k210 ctnt mutation. on the other hand, ca desensitizers might be beneficial for the treatment of hcm, in which increased ca - sensitivity could be a primary pathogenic mechanism. further studies using animal models closely recapitulating human disease phenotypes are important to develop therapeutic drugs for inherited cardiomyopathy patients. | genetic investigations of cardiomyopathy in the recent two decades have revealed a large number of mutations in the genes encoding sarcomeric proteins as a cause of inherited hypertrophic cardiomyopathy (hcm), dilated cardiomyopathy (dcm), or restrictive cardiomyopathy (rcm). most functional analyses of the effects of mutations on cardiac muscle contraction have revealed significant changes in the ca2 + -regulatory mechanism, in which cardiac troponin (ctn) plays important structural and functional roles as a key regulatory protein. over a hundred mutations have been identified in all three subunits of ctn, i.e., cardiac troponins t, i, and c. recent studies on ctn mutations have provided plenty of evidence that hcm- and rcm - linked mutations increase cardiac myofilament ca2 + sensitivity, while dcm - linked mutations decrease it. this review focuses on the functional consequences of mutations found in ctn in terms of cardiac myofilament ca2 + sensitivity, atpase activity, force generation, and cardiac troponin i phosphorylation, to understand potential molecular and cellular pathogenic mechanisms of the three types of inherited cardiomyopathy. |
in tissue engineering research, it is necessary to develop effective culture substrates for cells to facilitate regeneration of deficient tissue. it has been shown that the environment surrounding cells has a significant effect on cell behavior. the physical conditions of any scaffold to which cells adhere are an important determinant of cell growth [16 ], along with biochemical signals. cells receive various types of information from the culture substrate, such as surface stiffness [14 ] or roughness [5, 6 ]. therefore, a good understanding of the relationship between cellular behavior and the physical characteristics of culture substrates is critical for development of effective scaffolds that allow suitable cell adhesion, proliferation, and differentiation. in a previous study, we demonstrated that ferric - ion - cross - linked alginate (fe - alginate) was an efficient material for use as scaffold, allowing good cell adhesion and proliferation. alginates are a material widely used in various biomedical fields, and are composed of 1,4-linked -d - mannuronic acid (m) and -l - guluronic acid (g) residues, forming gels with certain multivalent metal ions [8, 9 ]. calcium ions are used most frequently as a cross - linking agent for alginates, but calcium - ion - cross - linked alginate (ca - alginate) is known to be an unfavorable culture substrate for cells. fe - alginate is able to overcome the deficiencies of ca - alginate, such as poor protein adsorptive capacity, and has been established as an effective cell culture substrate. alginates are commonly derived from seaweed, and have widely varying proportions and sequences of two the m and g monomers depending on the source from which they are derived [8, 10 ]. it is known that the m / g ratio influences the mechanical properties of ca - alginate gels : those with a high g - content have higher mechanical strength than those with a high m - content. it has also been reported that high - g ca - alginate forms more porous gels, and that the cross - linkages tend to be more stable over a longer period, than is the case for high - m alginate. in the case of fe - alginate, it is considered that the alginate composition can also influence the condition of the gel. however, the effects of fe - alginate substrate composition on gel properties and the growth of cultured cells have not been clarified. the aim of this study was to define the relationship between fe - alginate composition and the growth of cells cultured on fe - alginate films. we evaluated the efficiency of cell adhesion and proliferation on fe - alginate films with differing m / g ratios, using normal human dermal fibroblasts (nhdf) as model cells. we also focused on the degree of change in the condition of the fe - alginate gel during cell culture. here we report that favorable cell proliferation was observed on high - g, rather than on high - m fe - alginate gels, and that the stability of fe - alginate gel with firm cross - linkage between alginate and ferric ions seems to be a key supportive element for good cell proliferation. eagle 's minimum essential medium (e - mem) was obtained from nissui pharmaceutical (tokyo, japan), fetal bovine serum (fbs) was from tissue culture biologicals (tulare, ca, usa), and trypsin was from becton dickinson (sparks, md, usa). anti - vitronectin polyclonal antibody was purchased from abd serotec (oxford, uk). horseradish peroxidase (hrp)-conjugated anti - rabbit igg was from santa cruz biotechnology (santa cruz, ca, usa). all other chemicals were purchased from wako pure chemical co. (tokyo, japan). two types of alginate were used in this study : a high g - content alginate (m : g ratio 34 : 66) and a high m - content alginate (m : g ratio 57 : 43). the viscosity (1% (w / v) solution at 20c) of both alginates, purchased from kimica corporation (tokyo, japan), was about 100200 mpas. the procedures for preparation of the fe - alginate gel films have been described previously. briefly, 3 ml of 1% (w / v) alginate solution was added to the wells of 6-well tissue culture plates along with cellulosic sheets (bemcot, ozu corporation, tokyo, japan) as frames, and then dried at 60c overnight. after drying, 3 ml of 500 mm or 50 mm fecl3 was added to each well to induce cross - linkage of the alginate at 25c for 30 min. the resulting gel films were washed with deionized water to remove unreacted ions on the surface, and sterilized with 70% (v / v) ethanol. before seeding of cells on the films, the films were stabilized by immersion in e - mem at 37c under a 5% co2 atmosphere for 72 h, and the medium was replaced every 24 h. nhdf were cultured in e - mem with 10% (v / v) fbs at 37c under a 5% co2 atmosphere. to detach cells by trypsinization, the cells were incubated with 0.25% (w / v) trypsin and 0.02% (w / v) edta in ca-, mg - free phosphate - buffered saline (pbs) at 37c for 10 min. e - mem containing 10% (v / v) fbs was subsequently added to terminate the enzyme reaction. the cell suspension was centrifuged at 1000 g for 5 min and resuspended in e - mem containing 10% (v / v) fbs. the cells were counted with a coulter counter (beckman coulter corporation, fl, usa) and seeded onto fe - alginate films in 6-well tissue culture plates at a density of 4000 cells / cm in 3 ml of culture medium. samples were incubated at 37c under a 5% co2 atmosphere and the medium was replaced every day. after 1, 5, and 8 days, the cells were observed using a phase - contrast microscope and photographed, and then cell counting was performed to quantify the degree of cell proliferation. to count the attached cells, the cells were detached by trypsinization at 37c for 10 min, and then resuspended in e - mem containing 10% (v / v) fbs. cells were also cultured on high m - content fe - alginate films after immersion in e - mem at 37c under a 5% co2 atmosphere for 8 days to stabilize the gel condition. alginate films were immersed in fbs (0.2 ml / cm) in a 6-well tissue culture plate at 37c for 2 h. after incubation, the films were washed with deionized water, and transferred to new tissue culture plates. then 1% triton x-100 (0.1 ml / cm) was added to desorb the proteins on the film surfaces at 37c for 2 h. protein concentrations of the desorption fraction were determined using a bicinchoninic acid (bca) protein assay reagent kit (thermo fisher scientific pierce biotechnology, il, usa) in accordance with the manufacturer 's instructions. the solutions were also collected to separate the proteins in the solutions using sds - page on 10% polyacrylamide gel. protein bands in the gel were transferred to immobilon membranes (millipore, bedford, ma, usa) for immunoblot analysis. the blotted membranes were treated with 5% (w / v) non - fat dried milk in pbs for blocking, and then incubated with anti - vitronectin antibody diluted with the blocking solution at room temperature for 60 min. after washing with 0.05% (v / v) tween 20 in pbs, the membranes were incubated with hrp - conjugated secondary antibodies at room temperature for 60 min, and the immunospecific bands on the membranes were visualized by the dab color reaction method. quantitative analysis of adsorbed vitronectin was performed using image j 1.40 g (http://rsb.info.nih.gov/ij). alteration of alginate gel conditions during the culture period was evaluated by measuring the degree of swelling and polymer content of the gels. the assessments were conducted at 1, 5, 8, and 16 days after the start of incubation. to investigate the degree of gel swelling, the thickness of the alginate films was determined using a dial thickness gauge (g-7c ; ozaki mfg. co., ltd.). for estimation of polymer content, dry weight was measured after cultivation by weighing the gel that had been dried at 60c for 2 h. statistical analysis was performed using kaleidagraph version 4.0 software. the adequacy of high g- and m - content fe - alginate films as culture substrates was investigated by cultivation of nhdf on the respective gels. in the initial stage of culture, the number of attached cells and cell morphology showed little difference between the two fe - alginates (figures 1 and 2). however, as culture time progressed, the number of attached cells on the high - g fe - alginate gel was notably higher than that on the high - m gels (figure 1). cells on the high - g fe - alginate film showed good spread morphology throughout the culture period (figures 2(a)2(c)). conversely, on the high - g fe - alginate film, many cells became detached from the substrate, while maintaining cell - cell association, after 8 days of culture (figure 2(f)). the protein adsorption ability of high - g and high - m alginate was investigated. quantitative analysis demonstrated that both types of gel had an equivalent ability to adsorb serum proteins (figure 3(a)). moreover, immunoblot analysis revealed the same degree of adsorption of vitronectin, the major cellular adhesion - related serum protein, on both alginate gels (figure 3(b)). changes in the conditions of fe - alginate gel in the culture medium were evaluated by measurement of gel thickness and dry weight (figure 4). gel thickness gave an indication of the degree of swelling, whereas dry weight reflected the degree of gel disintegration. the conditions of the high - g fe - alginate gel showed no significant change even after 16 days. in contrast, the high - m fe - alginate gel became thicker, and the dry weight decreased with time. the rate of change in the properties of the high - m fe - alginate gel was rapid until around 8 days, but became more stable thereafter. fe - alginate gel was stabilized by immersion in culture medium for 8 days to prevent any further changes in gel condition. good cell proliferation was confirmed on stable high - m fe - alginate films at the same level as that on high - g fe - alginate films (figure 5). to investigate whether the amount of cross - linked ferric ions has a decisive influence on the properties of the two types of alginate, we studied cell growth on fe - alginate films with a low concentration of cross - linked ions. the concentration of ferric ions for cross - linkage of alginate employed under ordinary circumstances (500 mm fecl3) was determined previously to obtain sufficient film strength and to avoid excessive inhibition of cell growth, and the minimum required concentration of fecl3 solution to maintain the film strength needed for cell culture using our methodology was 50 mm. in both types of alginate, cell growth on gels with a low concentration of cross - linked ferric ions was comparable to that on gels with a normal iron concentration (figure 6(a)). the same tendency was revealed with regard to gel condition (figures 6(b) and 6(c)), although the thickness of both types of alginate gel increased as the concentration of cross - linked ferric ions decreased (figure 6(b)). to establish fe - alginate as an effective material for biomedical applications in research and therapeutics, an understanding of how the composition of fe - alginate influences its suitability as a culture substrate for encouraging good cell growth is of considerable significance. in this study, we observed that strong cross - linkage with ferric ions was necessary to ensure good cell proliferation during culture, and that alginate with a high g rather than a high m content showed better conservation of ferric ion cross - linkages. in the case of ca - alginate gels, it is reported that the m / g ratio influences cell compatibility. for instance, murine insulinoma cells encapsulated in high - m rather than high - g ca - alginate showed rapid growth and good metabolic activities. conversely, in the case of encapsulated neural stem cells, high - g ca - alginate microcapsules allowed better release of neurotrophic factor. studies of ca - alginate as a scaffold for cells have revealed that a high g content promptes good cell growth [14, 15 ]. our present results demonstrated superior nhdf cell growth on high - g rather than high - m fe - alginate films (figure 1). additionally, we demonstrated that good cell growth occurred on high - m fe - alginate films under conditions that tended not to result in gel swelling and disintegration (figure 5). it is known that cells recognize proteins that are adsorbed onto the surfaces of substrate materials [1618 ]. in particular, vitronectin is a major factor that mediates initial adhesion of nhdf onto fe - alginate films in the presence of serum. in the present study, there was little difference in the ability to adsorb serum proteins, including vitronectin, between the two types of fe - alginate gel (figure 3). high - m and high - g alginate gels showed comparable efficacy to promote initial cell adhesion (figures 1 and 2), being consistent with their similar protein adsorption capacities. the interaction between cells and high - m fe - alginate appeared to be weakened in response to alterations in the condition of the gel. a reduced degree of cell - substratum interaction probably caused cell detachment, although cell - cell interaction was maintained. swelling and dissolution of high - m fe - alginate observed in this study may have been caused by loss of cross - linked ferric ions from the gels. under physiological conditions, disengagement of cross - linking ions from alginate gels occurs in the presence of chelators, monovalent ions, and non - cross - linking divalent ions [19, 20 ]. it is also reported that high - m ca - alginate shows loss of mechanical strength during culture. calcium ions are known to bind to g- and mg - blocks of alginate. however, since interactions between calcium ions and the hydroxyl groups of mg are prevented by the intrinsic configurational characteristics of the mg building blocks, mg sequences in ca - alginate have a more flexible structure than g sequences. therefore the structures formed by mg junctions are likely to disassociate in response to the presence of competing ions or application of stress. although the blocks of alginate involved in binding to ferric ions are unknown, the present study suggested that the binding of ferric ions to alginate might occur through a mechanism similar to that for calcium ions. it can be inferred that ferric ions combine with not only g - blocks but also mg- and/or m - blocks in alginate, because gel swelling and breakage were observed in high - m rather than high - g alginate under physiological conditions (figure 4). further clarification of the interaction between ferric ions and alginate may lead to better understanding of the properties of fe - alginate affecting cell culture. it has been reported that high - m ca - alginate shows no further changes in material properties after 7 days of exposure to nacl. in the present study, high - m fe - alginate gels immersed in culture solution for 8 days also showed little change in gel conditions, such as swelling or polymer content (figure 4). on high - m alginate gels under the same conditions, nhdf were able to proliferate at the same level as that on high - g alginate gels (figure 5). there is some question as to whether cell growth on high - m alginate films is inhibited by elution of substances from the gels. however, no harmful effects on cell growth were evident when cells were cultured on the surface of tissue culture plates in medium together with high - m alginate gel film (data not shown), thus suggesting that the eluted materials did not influence cell growth appreciably. reduction of the concentration of ferric ions cross - linked with alginate did not affect the growth of nhdf seeded on both types of alginate gel (figure 6). in fact, cells proliferated well on highly swollen high - g alginate with a decreased amount of cross - linked ferric ions. thus, swelling of fe - alginate gels was not directly linked to scanty growth of cells cultured on gel surfaces. in the case of high - m alginate, gel conditions were changed during immersion in culture medium, even though the concentration of cross - linked ferric ions was low. it is considered that the percentage of firm cross - linkage in high - m alginate gels is unrelated to the amount of cross - linked ferric ions. in the case of high - m alginate gels under physiological conditions, strong cross - linkage may be maintained, while weak cross - linkage may occur as a result of exchange of cross - linked ferric ions for other competing ions. in this study, we have revealed for the first time the effects of fe - alginate composition on the properties of the gels and their suitability as a culture substrate for cells. fe - alginate gel with a high g content showed stable cross - linkage with ferric ions, and supported good cell proliferation. in the case of high - m alginate, although partial weak cross - linkage was observed during culture and the cell growth rate was suboptimal, the effectiveness of the gel for supporting cell growth was recognized after it had been stabilized by immersion in culture medium for an appropriate period. in fact, the protein adsorption abilities of the two types of fe - alginate gels ware similar. therefore, it is considered that the major factor influencing cell growth on two types of fe - alginate films is the stability of the gel during culture, and that the mode of cross - linkage between ferric ions and alginate differs according to alginate composition. these results suggest that the composition of fe - alginate gels could be altered according to their intended purpose. it is anticipated that our findings will be useful for extending the application of fe - alginate to diverse biomedical fields. | we investigated the suitability of ferric - ion - cross - linked alginates (fe - alginate) with various proportions of l - guluronic acid (g) and d - mannuronic acid (m) residues as a culture substrate for human dermal fibroblasts. high - g and high - m fe - alginate gels showed comparable efficacy in promoting initial cell adhesion and similar protein adsorption capacities, but superior cell proliferation was observed on high - g than on high - m fe - alginate as culture time progressed. during immersion in culture medium, high - g fe - alginate showed little change in gel properties in terms of swelling and polymer content, but the properties of high - m fe - alginate gel were altered due to loss of ion cross - linking. however, the degree of cell proliferation on high - m fe - alginate gel was improved after it had been stabilized by immersion in culture medium until no further changes occurred. these results suggest that the mode of cross - linkage between ferric ions and alginate differs depending on alginate composition and that the major factor giving rise to differences in cell growth on the two types of fe - alginate films is gel stability during culture, rather than swelling of the original gel, polymer content, or protein adsorption ability. our findings may be useful for extending the application of fe - alginate to diverse biomedical fields. |
glucose-6-phosphate dehydrogenase (g6pd) deficiency is one of the most common hereditary hemolytic disorders in human, affecting around 400 million people worldwide. the majority of deficient individuals live in tropical and subtropical regions where malaria is still endemic or has been eradicated only recently. in fact, g6pd deficient individuals have been protected from severe forms of malaria in these areas. although most of individuals with g6pd deficiency are usually asymptomatic, deficient subjects may suffer from neonatal jaundice, severe non - spherocytic hemolytic anemia or acute hemolytic anemia induced by infection, ingestion of fava beans or some drugs. the severity of these serious consequences of g6pd varies based on different gene mutations which cause different levels of residual enzyme activity. therefore to prevent the above complications, it is important to investigate their molecular bases. this gene is located on the xq28 region with a length of 18.5 kb, which contains 13 exons and 12 introns. since g6pd deficiency is an x - linked recessive disorder, it is more frequent in males than females. glucose-6-phosphate dehydrogenase enzyme, the product of g6pd gene, catalyzes the first step of the pentose phosphate pathway (ppp), which provides cells with pentoses and reduction power in the form of nicotinamide adenine dinucleotide phosphate (nadph). nicotinamide adenine dinucleotide phosphate cofactor is required for various redox reactions, and protects cells against oxidative stress via glutathione and catalase. glucose-6-phosphate dehydrogenase is the only source of nadph in erythrocytes, so any oxidative stress in g6pd deficient red blood cells may results in hemolytic anemia. approximately 140 mutations and 400 biochemical variants have been reported for this enzyme till now. the g6pd cosenza mutation was described for the first time in the of, southern. previous investigations have revealed that g6pd cosenza (g1376c), which is a common g6pd mutation in some parts of iran, has a variable frequency ranging from 0% to 12.33%. given the variability and high frequency of g6pd cosenza in iran, in the present study we have characterized g6pd cosenza among deficient individuals in the province of khuzestan, which is located in the southwest of the country bordering iraq and the persian gulf with a population of about five million mostly iranian arabs. screening study was performed on 1064 randomly selected blood samples from volunteer male donors referring to ahvaz blood transfusion center from february to april 2008. screening test for the diagnosis of g6pd deficiency was done by fluorescent spot method (sigma). eighty - one (7.6%) of them were found to be severely g6pd deficient. however blood sample were taken only from 79 deficient male blood donors for next studies. in order to identify g6pd molecular characterization, 231 g6pd deficient blood samples were collected from 79 screened male blood donors and 152 individuals (116 males and 36 females) who were referred to hospitals of khuzestan province with a history of favism, acute anemia or neonatal jaundice. all samples were collected into 0.5 m edta (becton dickinson,) solution, carried on ice and kept at -70 c for dna extraction. because g6pd mediterranean (c563 t) is reported as the most common mutation in middle east and some provinces of iran, at first we analyzed all samples for this mutation. finally 64 (55 males and 9 females) out of 231 samples were recognized without mediterranean mutation, which were then analyzed to identify cosenza mutation.genomic dna was extracted from leukocytes by using " picopure " dna extraction kit from molecular devices (san diego, ca). mutation site is located in exon 12 of g6pd gene. for detection of the cosenza mutation, exon 11 - 13 of g6pd gene was selectively amplified by pcr method using f - cos (5-gca gcc agt ggc atc agc aag-3) and r - cos (5-ggg aag gag ggt ggc cgt gg-3) primers. polymerase chain reaction (pcr) assay was performed in final volume of 25 l. pcr reaction mix contained 10x pcr buffer, 10 mm of each deoxynucleotide triphosphate, 25 pmol of each primer, 0.5 g genomic dna, 2 u / ml of taq dna polymerase and 50 mm mgcl2. the pcr reaction was carried out for 30 cycles as follows : 10 cycles (94 c for 30 seconds, 68 c for 1 min and 72 c for 30 seconds) and 20 cycles (95 c, 65 c, 72 c each temperature for 1 min). in order to certify the fidelity of pcr, amplified segments since the mutation creates an eco81i recognition site (figure 2), this endonuclease was used to perform restriction fragment length polymorphism (rflp) analysis. cozenza pcr products were digested by eco81i enzyme (fermentas gmbh,) at 37 c, overnight. among the 231 g6pd deficient individuals (a total of 267 alleles), 195 (84.1%) were males and 36 were females. only 64 samples (55 males and 9 females) out of 231 deficient subjects did not have g6pd mediterranean. cosenza pcr product was a 548 bp fragment, which appeared as two fragments with 232 bp and 316 bp lengths after digestion by eco81i on 2% agarose gel in mutant subjects (figure 3). therefore the mutation relative rate and allele frequency in khuzestanian deficient subjects are 2.6% and 0.023, respectively. polymerase chain reaction (pcr) products related to glucose-6-phosphate dehydrogenase cosenza mutation on 1.5 % agarose gel. lane 1 : size marker 1 kbp, lane 2 : negative control, lanes 3, 4, 5, 6, 7, 8 and 9 : cosenza pcr products with 548 bp length oligonucleotide primers f - cos and r - cos amplify a 548pb fragment across exon 11 and 13 of the glucose-6-phosphate dehydrogenase gene that after digestion by eco81i appeared as two fragments with 232 bp and 316 bp restriction digestion analysis of polymerase chain reaction (pcr) products related to glucose-6-phosphate dehydrogenase cosenza mutation with eco81i enzyme on 2% agarose gel. lane 1 : size marker 100 bp, lane 2 : pcr product, lanes 3, 4, 5, 6, 7, 8 and 9 : samples without cosenza mutation, lane 10 : hemizygote male with cosenza mutation g6pd deficiency is a very prevalent disorder in africa, southern europe, south east asia, oceania and middle east especially neighboring persian gulf countries including iran, kuwait, united arab emirates, iraq, bahrain, and oman, with a prevalence of 11.55, 5.51, 8.7, 6.1, 26.45 and 26 - 29%, respectively. khuzestan province is located in the south west iran, bordering iraq and the persian gulf. the prevalence of g6pd deficiency among male khuzestanian blood donors was reported to be 7.6%. this prevalence is obviously higher than the 6 % reported earlier for male blood donors in fars province of southern iran. however, one other recent study showed that the overall prevalence of g6pd deficiency among male and female children in the city of shiraz (fars province) was 11%. since g6pd deficiency is so frequent in khuzestan, it is very important and desirable to fully identify the molecular basis of this disorder. our previous study revealed that g6pd mediterranean (c563 t, ser188phe) was the most common mutation in khuzestan, like the other provinces of iran. to pursue our investigation cosenza mutation, which was initially described in the north of calbaria, southern italy, by frequency of 1.9%, is caused by 1376 g c (459 arg pro) substitution. its phenotype is associated with a severe enzyme deficiency (enzyme activity less than10 %). thus far, g6pd cosenza has been identified in some parts of italy, daltamation region of south croatia, and some parts of iran including mazandaran, kermanshah, and hormozgan. however, it hasnt been found in gilan, golestan, sistan and balochestan, khorasan, and fars provinces. the highest incidence (37.5%) of g6pd cosenza it not known whether this distribution of g6pd cosenza is the result of a common ancestry or an independent origin in mediterranean basin and middle east. we have detected cosenza mutation in 6 of 231 samples, resulting in a prevalence of 2.6% and allele frequency of 0.023. therefore the incidence of mutation in khuzestan is higher than that in, but lower than those in hormozgan and mazandaran. kermanshah, hormozgan and mazandaran are provinces that are respectively located in the western, southern and northern parts of (table 1). the great difference between the incidences of g6pd cosenza in some parts of could be explained by immigration issues, which might have induced a flow of gene from countries. or alternatively, it might be due to the origin of ethnic groups, which may be clarified by studying additional markers in populations. the variation in the prevalence on cosenza mutation may be partially comparable to that of thalassemia in the south of the country. we also studied the association of mediterranean and cosenza mutation with (1311ct) haplotype. the haplotype analysis revealed that unlike mediterranean g6pd, mutation was associated with (1311c) haplotype. fifty eight samples, which have not mediterranean and mutations, are kept for identification of other g6pd mutations. the findings of the present study indicate that g6pd cosenza is a common mutation in khuzestanian g6pd deficient individuals. | glucose-6-phosphate dehydrogenase (g6pd) deficiency is one of the most common hereditary enzymatic disorders in human, increases the vulnerability of erythrocytes to oxidative stress. it is also characterized by remarkable molecular and biochemical heterogeneity. according to previous investigations, g6pd cosenza (g1376c) is a common g6pd mutation in some parts of. therefore in the present study we have characterized mutation among g6pd deficient individuals in khuzestan province. in order to identify g6pd cosenza, we analyzed the g6pd gene in 64 samples out of 231 deficient individuals who had not g6pd mediterranean mutation, using pcr- restriction fragment length polymorphism (rflp) method. g6pd cosenza mutation was found in 6 males of 231 samples, resulting in the relative rate of 2.6% and allele frequency of 0.023 among khuzestanian g6pd deficient subjects. a comparison of these results with previous findings in some parts of suggests that g6pd cosenza is a common mutation in khuzestanian g6pd deficient individuals. |
two days after the onset of fever, vomiting, and diarrhea, a 10-month - old previously healthy boy was admitted with generalized tonic - clonic seizure. brain ct, performed elsewhere, revealed the presence of symmetric low - density thalamic lesions. on admission conventional brain mr images obtained using a 1.5-t system on the second day of hospitalization depicted symmetric distribution of t1- and t2-prolonged areas in the thalami (fig. -weighted gradient - echo images (tr / te = 800/30, flip angle = 20) demonstrated low signal intensities within the thalamic lesions, suggesting acute hemorrhage (fig. after the intravenous administration of gadolinium - diethylene triamine penta - acetic acid, the lesions showed no abnormal enhancement. diffusion - weighted mr imaging (b value=1000 sec / mm) demonstrated high signal intensity in all the lesions, though this was absent in the central portion of thalamic lesions, and other than in this same area, apparent diffusion coefficient (adc) mapping revealed low signal intensity (fig. localized proton mr spectroscopy of the thalami using a stimulated echo - acquisition mode sequence (tr / te=3000/30, 96 acquisitions, volume of interest=7 ml) showed that compared with an age - matched control subject, peak intensities were higher, occurring at 2.0 - 2.5 and 0.8 - 1.5 ppm (figs. laboratory findings on admission showed increased serum aspartate and alanine aminotransferase levels, though those of blood ammonium and lactate were normal. polymerase chain reaction analysis of the csf was negative for deoxynucleic acid (dna) of herpes simplex virus and enterovirus, and similar analysis of peripheral blood was negative for major mutations in mitochondrial dna, which would indicate mitochondrial encephalopathy with lactic acidosis and stroke - like episode (melas) syndrome. his mental state improved, and on the fourth day of hospitalization he was almost alert. follow - up brain mr imaging performed three months later revealed residual atrophic change in the previously observed lesions ; both t1- and t2-weighted images depicted small areas of high signal intensity at the center of the thalami, indicating residual subacute hemorrhage. four days after the development of fever, cough, and rhinorrhea, a 6-month - old, previously healthy girl was admitted with generalized tonic - clonic seizure and mental change, as well as increased rigidity of the extremities. ct images of the brain, obtained at another hospital, depicted symmetric low - density lesions in the thalami and external capsules. conventional brain mr imaging performed on the second day of hospitalization indicated that symmetric t1- and t2-prolonged areas were present in the thalami and external capsules. t2 -weighted gradient - echo images clearly showed that within the thalamic lesions, acute hemorrhage had occurred. after the intravenous administration of gadolinium - diethylene triamine penta - acetic acid, the lesions showed no abnormal enhancement and adc mapping revealed areas of low signal intensity within them (fig. localized proton mr spectroscopy using a stimulated echo - acquisition mode sequence (tr / te=3000/30, 96 acquisitions, volume of interest=7 ml) showed a small doublet at 1.33 ppm (fig. laboratory findings indicated slightly increased levels of serum aspartate aminotransferase, lactate, and ammonia, though the lactate level rapidly returned to normal. polymerase chain reaction analysis of the csf was negative for dna of herpes simplex virus and enterovirus, and similar analysis of peripheral blood was negative for major mutations in mitochondrial dna. the patient was thought to be suffering from acute necrotizing encephalopathy, and was treated with mannitol, acyclovir, and steroid. her mental state improved and on the second day of hospitalization her level of alertness was almost normal. the extremities gradually became less rigid, and follow - up brain mr imaging and mr spectroscopy performed one week later revealed marked improvement of the initial lesions and disappearance of the doublet at 1.33 ppm (fig. two days after the onset of fever, vomiting, and diarrhea, a 10-month - old previously healthy boy was admitted with generalized tonic - clonic seizure. brain ct, performed elsewhere, revealed the presence of symmetric low - density thalamic lesions. on admission conventional brain mr images obtained using a 1.5-t system on the second day of hospitalization depicted symmetric distribution of t1- and t2-prolonged areas in the thalami (fig. -weighted gradient - echo images (tr / te = 800/30, flip angle = 20) demonstrated low signal intensities within the thalamic lesions, suggesting acute hemorrhage (fig. after the intravenous administration of gadolinium - diethylene triamine penta - acetic acid, the lesions showed no abnormal enhancement. diffusion - weighted mr imaging (b value=1000 sec / mm) demonstrated high signal intensity in all the lesions, though this was absent in the central portion of thalamic lesions, and other than in this same area, apparent diffusion coefficient (adc) mapping revealed low signal intensity (fig. localized proton mr spectroscopy of the thalami using a stimulated echo - acquisition mode sequence (tr / te=3000/30, 96 acquisitions, volume of interest=7 ml) showed that compared with an age - matched control subject, peak intensities were higher, occurring at 2.0 - 2.5 and 0.8 - 1.5 ppm (figs. laboratory findings on admission showed increased serum aspartate and alanine aminotransferase levels, though those of blood ammonium and lactate were normal. polymerase chain reaction analysis of the csf was negative for deoxynucleic acid (dna) of herpes simplex virus and enterovirus, and similar analysis of peripheral blood was negative for major mutations in mitochondrial dna, which would indicate mitochondrial encephalopathy with lactic acidosis and stroke - like episode (melas) syndrome. his mental state improved, and on the fourth day of hospitalization he was almost alert. follow - up brain mr imaging performed three months later revealed residual atrophic change in the previously observed lesions ; both t1- and t2-weighted images depicted small areas of high signal intensity at the center of the thalami, indicating residual subacute hemorrhage. four days after the development of fever, cough, and rhinorrhea, a 6-month - old, previously healthy girl was admitted with generalized tonic - clonic seizure and mental change, as well as increased rigidity of the extremities. ct images of the brain, obtained at another hospital, depicted symmetric low - density lesions in the thalami and external capsules. conventional brain mr imaging performed on the second day of hospitalization indicated that symmetric t1- and t2-prolonged areas were present in the thalami and external capsules. t2 -weighted gradient - echo images clearly showed that within the thalamic lesions, acute hemorrhage had occurred. after the intravenous administration of gadolinium - diethylene triamine penta - acetic acid, the lesions showed no abnormal enhancement and adc mapping revealed areas of low signal intensity within them (fig. localized proton mr spectroscopy using a stimulated echo - acquisition mode sequence (tr / te=3000/30, 96 acquisitions, volume of interest=7 ml) showed a small doublet at 1.33 ppm (fig. laboratory findings indicated slightly increased levels of serum aspartate aminotransferase, lactate, and ammonia, though the lactate level rapidly returned to normal. polymerase chain reaction analysis of the csf was negative for dna of herpes simplex virus and enterovirus, and similar analysis of peripheral blood was negative for major mutations in mitochondrial dna. the patient was thought to be suffering from acute necrotizing encephalopathy, and was treated with mannitol, acyclovir, and steroid. her mental state improved and on the second day of hospitalization her level of alertness was almost normal. the extremities gradually became less rigid, and follow - up brain mr imaging and mr spectroscopy performed one week later revealed marked improvement of the initial lesions and disappearance of the doublet at 1.33 ppm (fig. acute necrotizing encephalopathy of childhood is a distinct form of acute encephalopathy recently reported from japan (2 - 4). since then, similar cases in other countries have been described (5 - 7). the clinico - radiological features of our cases closely correspond to the diagnostic criteria suggested by mizuguchi (1), namely clinical findings of acute encephalopathy following febrile prodromata, rapid deterioration in the level of consciousness, and convulsions ; laboratory findings which indicate an absence of csf pleocytosis, together with frequently increased csf protein and serum aminotransferase levels but no increase in blood ammonia ; characteristic neuroimaging findings ; and the exclusion of similar disease. among the characteristic neuroimaging findings, symmetric lesions in the thalami were most constant, being observed in all reported cases (1 - 8). bilateral tegmental involvement of the brain stem was the second most common finding, present in about 60% of cases. neuropathologically, the lesions occurring in acute necrotizing encephalopathy have been described as having a concentric or laminar structure (9). perivascular petechial hemorrhage and necrosis of neurons and glial cells occurred in the center of lesions, and in the outer portion, all vessels were congested and acutely swollen oligodendrocytes were observed. along the edge of lesions, the pathologic findings were extravasation mainly around the arteries and myelin pallor. the diffusion mr imaging findings of acute necrotizing encephalopathy have previously been reported (8). the adc value was found to be initially lower than normal, indicating decreased water diffusibility, a very useful finding in differentiating acute necrotizing encephalopathy from acute demyelinating encephalomyelitis ; this low adc value seen during the acute phase slowly increased during follow - up studies. in our cases, a low adc was observed, except in the central portion of thalamic lesions, where adc was high. the causes of adc decrease during the acute phase of acute necrotizing encephalopathy are not clear, though it is most likely due to swollen neuroglial cells within the lesions, as in acute ischemic infarctions, or to the swelling of myelin. supportive evidence for this is the presence of swollen oligodendrocytes or myelin pallor, revealed by pathologic examination. another possible cause is the presence of acute petechial hemorrhage within a lesion, particularly a thalamic lesion. it is well known that diffusion mr imaging reveals the lower than normal adc caused by acute hemorrhage (10), but the adc decrease seen in non - hemorrhagic lesions is difficult to explain. we believe, therefore, that the cytotoxic edema occurring in neuroglial cells and possible swelling of myelin is the dominant cause of adc decrease in acute necrotizing encephalopathy. in the brain, decreased adc is observed most frequently in cases involving hyperacute infarction, but rarely in status epilepticus, hemiplegic migraine or venous infarction. acute necrotizing encephalopathy is a further example of a brain disorder in which a lower adc is found in the involved areas, and in most clinical situations this decrease involves significant neuroglial damage. we speculate that the degree and extent of adc decrease can be used to predict brain damage or the clinical outcome in patients with acute necrotizing encephalopathy, and our experience, though limited to only two cases, supports this possibility. in the first patient, in whom the adc of thalamic lesions showed a greater decrease, there was more significant morphologic damage and a poorer clinical outcome, as shown at follow - up, than in the second, whose adc of thalamic lesions was only slightly decreased. in both our cases, the central high adc area surrounded by a thick, rim - like area of low adc in the thalami correlated closely with the pathologic findings of lesions with a concentric or laminar structure, as mentioned above. in severe cases of acute necrotizing encephalopathy, rapid neuroglial cell death or necrosis may occur in the central portion of a lesion, surrounded by a less severely damaged zone, pathological change which can be clearly demonstrated by diffusion mr imaging. in milder cases, as in our second patient, brain damage may be less severe and the pathological change occurring may be seen at diffusion mr imaging as a slight decrease in adc. the proton mr spectroscopic findings of acute necrotizing encephalopathy have previously been reported (5, 8). follow - up studies showed that a small lactate peak seen at initial mr spectroscopy had rapidly returned to within the normal range (8), and we also observed this change in our second patient. in our first case, we noted that peak intensities were higher at 2.0 - 2.5 ppm, a finding not reported in previous proton mr spectroscopic studies of this disease. the peak is usually caused by the glutamate / glutamine complex within the brain, and it is well known that cerebral glutamine is increased in the presence of hyperammonemia (11). because of the absence of hyperammonemia in our first case, it is less likely that the peak was due to increased cerebral glutamine, and we speculate that it may, instead, have been due mainly to increased cerebral glutamate. glutamate is a well - known excitatory neurotransmitter but can cause excitotoxic neuronal damage if excessive amounts are released into the synaptic cleft. it is, therefore, tempting to explain the neuronal damage occurring in acute necrotizing encephalopathy in terms of glutamate - mediated excitotoxicity. an increase in glutamate was not, however, observed in our second case or in those previously reported, suggesting that it may be transient or depend on the severity of the disease. in our first case, another interesting mr spectroscopic finding was the broad peak occurring at 0.8 - 1.5 ppm. as the increased peak intensities were broad rather than a narrow doublet at 1.33 ppm, we believe that most are due to mobile lipids, the increase of which may result from cell membrane damage or disintegration ; pathological evidence which supports this hypothesis is the neuroglial cell necrosis occurring at the center of the lesions. this was not, however, observed in the second case, in which diffusion mri demonstrated a slightly decreased adc and the clinical outcome was good. we consider that the combined increase in the glutamate / glutamine complex and mobile lipids is a spectroscopic sign suggesting significant neuroglial damage in the investigated regions. at follow - up t1-weighted imaging, subacute perivascular petechial hemorrhage has been detected (1 - 3, 6, 7), and in our cases, t2 -weighted gradient - echo imaging detected acute petechial hemorrhage in the center of the thalamic lesions ; t2-weighted images obtained during the acute phase showed homogeneous high signal intensity and the presence of hemorrhage was not detected. small petechial hemorrhage may, however, be obscured within the lesions on t2-weighted images, and t2 -weighted gradient - echo imaging may help characterize the lesions by identifying central hemorrhage during the acute phase. in acute necrotizing encephalopathy, the infectious organisms identified are almost always viruses, and so an antiviral agent such as acyclovir is used for treatment. another widely used agent is steroid, which has an anticytokine effect, and since the effect of hypothermia is similar, it too has recently been used in the treatment of patients with acute necrotizing encephalopathy (12). such anticytokine therapies are based on the observation that in the csf and serum of patients with this disease, proinflammatory cytokine levels, including those of interleukin-6 and tumor necrosis factor-, increase. in this report, we have described two cases of acute necrotizing encephalopathy examined by diffusion mr imaging and proton mr spectroscopy, as well as routine brain mr imaging. diffusion mr imaging revealed a more prominent decrease of adc in the first patient, who had more severe brain damage and a poorer clinical outcome than the second. proton mr spectroscopy demonstrated increased glutamate / glutamine complex and mobile lipids in the first case and only a small increase of lactate in the second, less severe, case. we conclude that diffusion mr imaging and proton mr spectroscopy may provide useful information not only for diagnosis, but also for estimating the severity and clinical outcome of acute necrotizing encephalopathy. | in this report, we describe the findings of diffusion mr imaging and proton mr spectroscopy in two infants with acute necrotizing encephalopathy in which there was characteristic symmetrical involvement of the thalami. diffusion mr images of the lesions showed that the observed apparent diffusion coefficient (adc) decrease was more prominent in the first patient, who had more severe brain damage and a poorer clinical outcome, than in the second. proton mr spectroscopy detected an increase in the glutamate / glutamine complex and mobile lipids in the first case but only a small increase of lactate in the second. diffusion mr imaging and proton mr spectroscopy may provide useful information not only for diagnosis but also for estimating the severity and clinical outcome of acute necrotizing encephalopathy. |
in past three decades, minimal access and minimally invasive techniques have been firmly established in the field of surgery. though, initially it was restricted to abdominal and gynaecological surgeries, minimal access techniques have been employed in sub specialities in the latter period. similarly, thyroidectomy is not an exclusion to these minimally invasive approaches. various endoscopic thyroidectomy (et) techniques ranging from totally endoscopic, video- assisted, trans - axillary, chest wall approaches have been reported. the gas dependent techniques also lead to complications such as hypercarbia, subcutaneous emphysema, mediastinal emphysema, cardiac arrythmias etc. in few cases. in this context, we evaluated our technique of a safer et through trans - axillary route for its feasibility and safety. this is a retrospective study conducted in endocrine and metabolic surgery department of a tertiary care teaching hospital in southern india. this study complied with the international ethical norms according to helsinki declaration - ethical principles for medical research involving human subjects. the details of operative technique were documented and recorded in all the cases. only cases with benign thyroid nodules were included. exclusion criteria were diffuse toxic goitres (graves disease), thyroid cancer, > 6 cm nodules, recurrent goitres and patients with shoulder joint pathology. all the cases were performed by the operative team -the primary operating surgeon (os) and two assisting surgeons (as). the ipsilateral arm was abducted to 90 and elbow flexed such that hand stays besides the head. the surface markings of relevant anatomical landmarks such as clavicle, anterior axillary line (aal), sternocleidomastoid (scm) and jugular notch were made [figure 1 ]. surface markings for surgical procedure - axillary incision (vertical arrow) ; anatomical landmarks (trasverse arrows) a 4 - 5 cm long skin incision was used 1 cm posterior and parallel to aal. skin incision was deepened up to pectoral fascia. pectoral fascia was incised in the same line and sub - fascial dissection was performed by blunt digital and sharp instrumental dissection to create operative space. after initial dissection, subsequent dissection was done under telescopic guidance placed through the same incision and held by camera assistant (ca). thus, dissection for operative space was done both under synergistic view of naked eye and monitor. the operating surgeon (os) and as stand on ipsilateral side of goitre to visualize the operative field through the axillary incision. thyroid bed was entered through the distal avascular area between the two heads of scm [figure 2 ]. the sternal head of scm and strap muscles are retracted by as to the opposite side. while managing the clavicular head of scm, os retracts carotid sheath and superior vascular pedicle is ligated with conventional knotting or clipping individual vessels [figure 3 ]. further medial mobilization of helps in visualizing and securing the recurrent laryngeal nerve (rln) and superior parathyroid gland (ptg). ipsilateral goitre was resected after incising the isthmus, which was sutured in conventional way. a 14 fr suction catheter is placed in to thyroid bed and operative space through a separate stab wound in axilla. it was retained in situ till the drain output was less than 20 ml on two consecutive days with no erythema or bogginess in dissected area. intraoperative view i - approach to thyroid through sternomastoid muscle - sternal head (white arrow) ; clavicular head (black arrow) intraoperative view ii - clipping of superior pole vessels (white arrow) ; superior parathyroid (black arrow) ; retracted strap muscles (hollow arrow over goiter) intraoperative view iii - recurrent laryngeal nerve (black arrow) ; superior parathyroid (lower end of double headed arrow) ; suture being threaded to tie a branch of inferior thyroid artery (hollow arrow) post - operative period was uneventful in all the cases. the ipsilateral arm was abducted to 90 and elbow flexed such that hand stays besides the head. the surface markings of relevant anatomical landmarks such as clavicle, anterior axillary line (aal), sternocleidomastoid (scm) and jugular notch were made [figure 1 ]. surface markings for surgical procedure - axillary incision (vertical arrow) ; anatomical landmarks (trasverse arrows) a 4 - 5 cm long skin incision was used 1 cm posterior and parallel to aal. skin incision was deepened up to pectoral fascia. pectoral fascia was incised in the same line and sub - fascial dissection was performed by blunt digital and sharp instrumental dissection to create operative space. after initial dissection, subsequent dissection was done under telescopic guidance placed through the same incision and held by camera assistant (ca). thus, dissection for operative space was done both under synergistic view of naked eye and monitor. the operating surgeon (os) and as stand on ipsilateral side of goitre to visualize the operative field through the axillary incision. thyroid bed was entered through the distal avascular area between the two heads of scm [figure 2 ]. the sternal head of scm and strap muscles are retracted by as to the opposite side. while managing the clavicular head of scm, os retracts carotid sheath and superior vascular pedicle is ligated with conventional knotting or clipping individual vessels [figure 3 ]. further medial mobilization of helps in visualizing and securing the recurrent laryngeal nerve (rln) and superior parathyroid gland (ptg). a 14 fr suction catheter is placed in to thyroid bed and operative space through a separate stab wound in axilla. it was retained in situ till the drain output was less than 20 ml on two consecutive days with no erythema or bogginess in dissected area. intraoperative view i - approach to thyroid through sternomastoid muscle - sternal head (white arrow) ; clavicular head (black arrow) intraoperative view ii - clipping of superior pole vessels (white arrow) ; superior parathyroid (black arrow) ; retracted strap muscles (hollow arrow over goiter) intraoperative view iii - recurrent laryngeal nerve (black arrow) ; superior parathyroid (lower end of double headed arrow) ; suture being threaded to tie a branch of inferior thyroid artery (hollow arrow) post - operative period was uneventful in all the cases. 6/15 patients had compensated comorbidities such as diabetes, hypertension, chronic airway disease. at the time of surgery, all the patients were rendered euthyroid. demographic and clinical profile operative, postoperative morbidity and histopathology details are shown in table 2. the only specific complications were induration in the infraclavicular area between axillary incision and thyroid region, prolonged wound drainage and stiffness in lower 3 of sternocleidomastoid. induration of infraclavicular area resolved at a mean of 20.7 (15 - 35) days and sternocleidomastoid induration at a mean of 36.5 (22 - 50) days in all the cases. all the patients had excellent cosmetic outcome with a hidden surgical scar in axilla [figure 5 ]. surgical and post - operative details 12th post - operative day clinical photograph - no cervical scar (white arrow) ; axillary incision to be hidden by clothes (lower black arrow) ; induration in ipsilateral dissected area (vertical arrow) a glance at the history of thyroidectomy reveals how it was developed by pioneer endocrine and thyroid surgeons through various stages till the present era of minimal access surgery. though, open conventional thyroidectomy is still the favored approach worldwide, a plethora of endoscopic thyroidectomy (et) techniques ranging from totally endoscopic, video - assisted, trans - axillary, chest - wall approaches have been already employed. even novel approaches such as thoracoscopic, robotic, trans - oral, post - auricular routes have been explored. though none of them are validated universally, chest - wall and trans - axillary approaches are favoured by the majority. the objectives of any et technique are better cosmesis, lesser pain, lower morbidity and feasibility in patients with co - morbidities. most of available et techniques fulfill cosmetic and pain aspects, but fail to address the latter two aspects. based on use of gas (co2), et may be broadly divided in to gas dependent and gasless techniques. the gas dependent techniques can cause serious hypercarbia associated complications such as subcutaneous emphysema, mediastinal emphysema, cardiac arrhythmias and even death in few cases. especially, patients with comorbid conditions such as hypertension, diabetes and chronic obstructive airway disease are susceptible to the complications. these complications are related to co2 insufflation pressures, length of surgery, susceptibility and comorbid conditions of the patient. lower co2 insufflation pressures, intensive end tidal co2 monitoring (etco2), intra - operative arterial blood gas analysis (abg), intermittent desufflation, shorter operative times, proper patient selection have been recommended to counter these gas related complications. on the contrary, gasless techniques are very safe as it nullifies the whole gamut of gas related morbidity. thus, even patients with well to moderately controlled comorbidities can be taken up for surgery and allows longer operative times with extra safety. it also reduces the cost of intensive etco2 and abg monitoring used for gas dependent techniques. the only practical disadvantage of gasless over gas dependent technique is inadequate operative space, which can be compensated by proper retraction by as and synergistic naked eye and camera vision of thyroid area. in conventional open approach, the gland is reached through a range of variably sized collar neck incisions in the neck, which can result in scar with hyperesthesia, paraesthesia and increased patient self - awareness. furthermore, keloid or hypertrophic scar worsens the cosmetic outcome, especially in dark skinned individuals. total et has reduced the level of surgical invasiveness with extra - cervical short incisions with superior cosmetic outcome, but is a gas dependent technique with its associated risk. the trans - axillary approach utilizes an access through the axilla, thus avoiding a cervical or chest wall incision. this is especially applicable in modern times, due to increasing use of low neck tops exposing the upper anterior chest wall, which is the surgical access area for et through chest wall. the position of the patient has great importance, as abduction of the arm and flexion of the elbow reduces the distance between the aal and the thyroid bed by almost 4 - 6 cm, ensuring easier accessibility of thyroid through axilla. pre - operative surface marking was very much useful in limiting dissection during creation of the space. the plane of dissection was confined below the pectoral fascia, which reduces the chances of skin burns due to electro - cautery and subcutaneous ecchymosis. the development of this natural plane was easier compared to traumatic dissection of subcutaneous space and beyond clavicle ; it was continued in to sub - platysmal plane. the use of telescope with magnified view on monitor with synergistic naked eye vision has a practical advantage (vide infra). both steps are complementary, especially for identification and preservation of critical structures like rln and ptg. the entry in to the thyroid bed was through the two heads of scm which is a relatively avascular plane in the lower 1/3. further, it reduces the morbidity related to strap muscle division. throughout the procedure it requires efficient retraction of the sternal head of scm along with strap muscles the as. thus it does not require co2 gas to maintain the working space for dissection and thus precludes its related morbidity. the dissection performed utilizes both open as well as laparoscopic instruments and the combines the advantages of both the techniques. maneuvering in the depth to ligate the superior pole by open knotting technique is one of the disadvantages for the surgeons with shorter fingers. in such situations, all the haemostatic techniques such as direct knotting, diathermy, clips can be used complementarily and interchangeably as per surgeon 's experience. the sternohyoid muscle to create a good visual space even for the contralateral lobe dissection has been described, but has disadvantage of prolonged operative time and post - operative neck pain with discomfort while swallowing. to counterbalance this situation, we employed a similar procedure on contralateral side through its corresponding axilla for total thyroidectomy. this bilateral axillary approach for total thyroidectomy was not associated with any extra morbidity compared to unilateral approach and ensures better identification of rln and ptg on both sides. kang., described a similar technique, but employed a subcutaneous route with an additional 5 mm chest wall port. holsinger. and landry., described a robotic technique with similar approach, but are feasible in very few centers in near future. though, it increases the area of dissection in infra - clavicular region, it compensates with better post - operative cosmetic result. comparision between various studies of trans - axillary thyroidectomy the three notable morbidities, we observed with this technique are prolonged drainage (mean : 5.4 days), induration of lower part of scm and induration of infraclavicular area. infraclavicular induration is evidently due to larger area of dissection compared to conventional thyroidectomy, through neck and lymphatic plexuses in subpectoral mammary area. in our experience, with conventional thyroidectomy though, we did not perform formal analysis of this variable in the present study, this observation is significant enough to investigate further as it prolongs hospital stay. the increased seroma rates in other studies might be due to earlier drain removal or understatement, as we found wound drainage lasted for longer duration than the hospital stay in those studies. induration of lower 1/3 of scm is probably due to splitting and dissection between its two heads. though, it was self - limiting, it appears to be a unique complication specific to gstat. induration of ipsilateral infraclavicular area corresponded to the dissected area between axillary incision and thyroid bed. similar to scm induration, this was self - limiting and unique to gstat, although of shorter duration than the former. in an effort to obviate the adverse aesthetic and morbid effects of conventional open surgical technique and gas dependent et techniques, we employed this gasless single incision trans - axillary thyroidectomy (g - stat). the basic sequence of steps in the procedure is nearly identical to a conventional open thyroidectomy. an endoscope provides excellent lighting and magnification, thus the entire surgical team can see the structures and procedure better than in a conventional open procedure. thus, this technique combines the magnification of endoscopy and tactile sensation of open surgery. this study demonstrates the feasibility and safety of gasless trans - axillary endoscopic thyroidectomy technique for nodular goitres with usual general surgical risk. | introduction : a range of minimally invasive endoscopic techniques (gas dependent and gasless) have been attempted for thyroidectomy in the past two decades. in this context, we evaluated the feasibility and safety of our technique of a gasless trans - axillary thyroidectomy.material and methods : this retrospective study from the department of endocrine and metabolic surgery in southern india included 15 cases. the details of operative technique, intra and postoperative data were documented in all the cases. only cases with benign thyroid nodules were included. exclusion criteria were diffuse toxic goiters (graves disease), thyroid cancer, > 6 cm nodules, recurrent goitres and patients with shoulder joint pathology. statistical analysis was done with spss software 12.0 version.results:f:m -14:2. mean age of the patient group was 26.4 years (15 - 52). mean operative time was 123.4 (82 - 206) minutes. the only specific complications were induration in the infraclavicular area between axillary incision and thyroid region, prolonged wound drainage and stiffness in lower 3rd of sternocleidomastoid. wound drainage lasted for 5.4 days (4 -8). there was no significant operative morbidity.conclusion:this gasless trans - axillary technique for nodular goitres is safe and effective in the hands of an experienced thyroid surgeon. |
different cystic lesions may occur both in the brain parenchyma and in the ventricular system, alone or in association to hydrocephalus. these include porencephalic cysts, ependymal cysts, cysts arising from a defect in the hemispheric cleavage and from the choroid plexus (1). they are typically small and asymptomatic and are discovered incidentally in older patients, usually in the trigone (also known as atrium) of the lateral ventricle. symptomatic cpcs (usually exceptionally large, 2 - 8 cm) are rare (2). symptomatic cpcs were treated to date either by direct surgical fenestration or excision through a craniotomy or by a cyst - peritoneal shunt. the development of endoscopic techniques has provided a new modality of surgical treatment of these cysts, which allows one to avoid further treatment in most cases (3). we report on a case of a large symptomatic cpc, located in the trigone of the right lateral ventricle resulting in an blockage of the right foramen of monro in a 26-yr - old man who underwent endoscopic removal. a 26-yr - old man, with no other medical history, complained of progressive headache and vomiting for 3 months. computed tomographic (ct) scans demonstrated only asymmetry of the lateral ventricles, with enlargement of the right lateral ventricle (fig. 1). subsequent magnetic resonance imaging revealed a large, cystic, nonenhancing lesion with a thin wall in the right lateral ventricle. the cyst contents had the same signal intensity as cerebrospinal fluid (csf) on mri (fig. the patient was positioned supine with head in neutral position and the thorax elevated 15 degree. a linear incision on the right frontal scalp, parallel to the sagittal suture, was applied to the pericranium. a 15-mm burr hole was placed 3 cm from the midline and just behind the hair line. a 12-french introducer sheath was inserted into the frontal horn of the right lateral ventricle under freehand - guidance. a 2.7 mm rigid telescope was introduced, and the cyst wall obstructing the right foramen of monro was identified (fig. an avascular area in the midportion of the cyst wall above the foramen of monro was chosen for fenestration. the cyst wall was punctured by gentle pushing the tip of the probe. with microscissors and forceps,, subsequent intraventricular hemorrhage was identified, so continuous irrigation of lactated ringer 's solution was done because the bleeding focus could not be found. after identifying no active bleeding, a small piece of gelfoam was placed in the cortical chimney, and the wound was closed in a routine fashion. external ventricular drainage catheter was changed due to catheter infection at 8 days after surgery and it was removed at 14 days after surgery. a ct scan performed 14 days after endoscopic removal of the cyst showing no intraventricular hemorrhage (fig. 6). he was discharged home at 37 days after the first surgery with no neurologic deficit. netsky and shuangshoti have reported cysts of the choroid plexus in 38% of the telencephalic choroid plexuses in their 124 autopsy cases (4). small cpcs are usually asymptomatic and are usually incidentally encountered at autopsy (5). their size usually does not extend beyond 1.5 - 2 cm in diameter at the most (4, 5). they are most frequently found in children with a preponderance for the male sex and account for approximately 3% of all pediatric cerebral pathological entities (6). they can be uni- or bilateral, single or multiple. in the lateral ventricle, the presence of cpc on prenatal screening has been associated with trisomy 18 and possibly trisomy 21, but the majority of cysts are asymptomatic incidental findings. when present, symptoms are secondary to the obstruction of csf, particularly at the foramen of monro. patients frequently present with intermittent worsening of their symptoms, which implies a mobile cyst with a ball - and - chain configuration. patients with unilateral lesions above the third ventricle can present with asymmetrical dilatation of the lateral ventricle. other possible clinical manifestations of large cysts may range from increased pressure to epilepsy (7). histologically, cpcs are consist of a fibrous outer membrane and an inner layer of cuboidal choroid plexus epithelium, often with ciliated cells (6, 8). most authors believe the origin of these lesions is the primitive neuroepithelium that lines the neural tube. the most unifying theory about their pathogenesis suggests that a neuroepithelial tube or a small cyst is formed by a folding of the neuroepithelium into the choroid 's matrix and of stroma into the ventricle (evagination and invagination). the neck of the folded epithelial sacs occasionally may be pinched off and become separated from the ventricle. the accumulation of secretions from the secretory activity of these epithelial cells in the congenitally formed cyst and the fluid activity transported from the exterior will produce a clear csf - like substance (1). computerized tomography most often reveals a focal enlargement of the trigone with a well - defined low density mass in the lateral ventricle of similar density to csf. the cyst does not enhance after administration of contrast material, whereas the enhancing choroid plexus is usually displaced. the cyst wall can be easily identified, even though it is very thin and closely applied to the ventricular lining. the content of the cyst has a signal on both t1- and t2-weighted mr images, corresponding to csf (2). the differential diagnosis of intraventricular cysts includes colloid cysts, ependymal cysts, arachnoid cysts, rathke cyst, cysticercosis, and histocytosis x. immunohistochemical studies are useful in differentiating between these different types of cysts. the epithelium of choroid and ependymal cysts shows immunoreactivity for markers of normal neuroepithelial structures of the cns. choroid plexus epithelium has a positive result with transhyretin, and ependymal epithelium has a positive result with glial fibrillary acidic protein. on the contrary, the epithelial walls of colloid cysts and rathke cleft cysts express epithelial markers, such as mucin (mucicarmin). these markers support the theory of a neuroepithelial origin of a cpc or ependymal cyst and an ectodermal origin of a colloid cyst or rathke cleft cyst (9). regarding treatment, it should be emphasized that the majority of these cysts are asymptomatic and only require periodic observation. there are several surgical options for treating symptomatic cpcs : 1) creation of communication of the cyst cavity with the ventricle, either by open surgery or through ventriculoscopy ; 2) placement of ventriculocystoperitoneal shunt ; 3) stereotactic evacuation of the cyst content ; and 4) direct surgical approach with total or partial resection of the lesion (6). in fact, the endoscopic approach to the cyst is a scarcely invasive technique, which allows us to avoid the direct approach by craniotomy or the insertion of a shunt in most cases. however, the surgical approaches and the technique of endoscopic treatment are rather variable, according to the location of the cyst, and are today debated (3). cpcs develop near the foramen of monro, thus producing monoventricular hydrocephalus (10). the endoscopic approach to the cpcs should be different, according to the size and the residual lateral ventricle. the burr hole must be performed at the level of the more superficial point of the cyst and according to an easy and direct trajectory, as defined by mri sequence. large and multiple fenestrations are necessary to avoid closure of the stomy and recurrence of the cyst (3). in our case, burr hole was placed 3 cm ipsilateral from the midline and 5 cm anterior to the coronal suture. however, gangemi. advised to approach large choroid plexus cysts through a contralateral occipital burr hole and to perform the fenestration from the contralateral not enlarged ventricle to the cyst, because when the cyst is large and occupies almost completely the enlarged lateral ventricle, the cannulation of the ipsilateral ventricle is difficult or even impossible (3). total resection of these lesions was recommended when the cyst can be easily separated from the ependymal walls like our case. when the cyst is firmly adherent to surrounding brain close to the choroid plexus, it is better to leave a strip of the cyst wall undisrupted rather than risk of injury to important fine vessels (6). there must be a caution in dissecting cyst from the ventricular wall because of such a narrow surgical field like endoscopic approach. symptomatic large cpc is very rare. the endoscopic surgery plays a very important role in the treatment of symptomatic cpc and the endoscopic fenestration rather than excision should be considered as the first surgical procedure because the goal of treatment is shrinkage of the cyst until normal csf flow is restored. | choroid plexus cysts (cpcs) are the most commom neuroepithelial cysts, occuring in more than 50% of some autopsy series. they are typically small and asymptomatic and are discovered incidentally in older patients, usually in the trigone of the lateral ventricle. symptomatic cpcs (usually exceptionally large, 2 - 8 cm) are rare. the authors report a case of large symptomatic choroid plexus cyst, located in the trigone of the right lateral ventricle in a 26-yr - old man who presented with headache and vomiting. the patient underwent endoscopic removal through a burr hole placed 3 cm from the midline and just behind the hair line. the histological examination of the cyst wall was consistent with choroid epithelium. despite of postoperative intraventricular hemorrhage and catheter infection, he discharged home without neurologic deficits. the endoscopic fenestration rather than excision should be considered as the first surgical procedure because the goal of treatment is shrinkage of the cyst until normal cerebrospinal fluid flow is restored. |
pulmonary tuberculosis is divided into primary and post - primary types according to pathogenic and radiological features. pleural involvement is generally seen in the form of a pleural effusion, which can be a component of both primary and post - primary tuberculosis. empyema, residual pleural thickening and calcification are other forms of pleural tuberculosis involvement and are generally seen in association with parenchymal disease. in approximately two - thirds of cases, there is no associated parenchymal lesion on the chest radiograph ; yet tubercle bacilli are frequently found in biopsy specimens of pleural tissue. we report an unusual form of pleural tuberculosis demonstrated by pleuroscopy and computed tomography (ct) presenting with multiple pleural nodules and masses with large hemorrhagic pleural effusion and without any evidence of either parenchymal or lymphatic disease. a 68-year - old male was admitted with complaints of gradually progressive exertional dyspnea and dry cough of three months duration. he had pleuritic chest pain, low grade fever and weight loss for one month. his routine hemogram, urine examination, renal and liver function tests and coagulation profile were normal except raised erythrocyte sedimentation rate. chest x - ray was suggestive of large right - sided pleural effusion. at thoracentesis, it was a lymphocytic exudate (total protein 3.4 g / dl, sugar 76 mg / dl, cholesterol 67 mg / dl, lactate dehydrogenase 253 iu / l, adenosine deaminase 9 polymorphs : 5%, lymphocytes : 92%, cytology negative for malignant cells, gram stain and afb smear negative). post - aspiration chest x - ray showed multiple irregular pleural masses [figure 1 ]. ct of the chest revealed multiple different size masses involving the parietal as well as visceral pleura [figure 2 ]. post taping chest x - ray shows multiple irregular pleural masses computed tomography (ct) chest shows multiple different size masses on parietal pleura as well as on visceral pleura a medical pleuroscopy was performed using a flexible bronchoscope placed through the chest tube. multiple pleural - based (parietal as well as visceral) masses of varying sizes were observed. histopathological examination of the pleural biopsy from a parietal pleural mass revealed epitheloid cell granulomas indicative of tuberculosis [figure 3 ]. the major differential diagnostic considerations in a patient with multiple pleural nodules and masses are malignant mesothelioma, pleural lymphoma, hematogenous pleural metastases, malignant thymoma and on rare occasion sarcoidosis.[38 ] although pleural tb infection is thought to result from the rupture of a subpleural caseous focus within the lung into the pleural space, it may occasionally be as a result of hematogenous dissemination or contamination from adjacent infected lymph nodes. pleural infection in tuberculosis is usually characterized by an effusion in early stages, or by diffuse pleural thickening and adhesions often with calcification in long standing cases. pleural thickening may also result from chronic inflammatory conditions, for example, hemothorax, empyema, tubercular effusion, or recurrent pneumothorax, often with calcification. in our case the provisional diagnosis was a pleural neoplasm (primary or secondary) based on clinical, radiological and pleuroscopic findings. to our knowledge, pleural tuberculosis presenting with multiple pleural nodules and masses without parenchymal involvement or lymphadenopathy have been reported rarely in the standard english medical literature,[1113 ] but pleural tuberculosis presenting as multiple pleural masses with large hemorrhagic pleural effusion has not been reported. it should be considered in the differential diagnosis of pleural masses without radiographically visible parenchymal lung disease even when there is large hemorrhagic effusion. confirmation of the diagnosis with sputum smears, culture, or histology is mandatory since treatment is highly successful. | tuberculous involvement of the pleura usually presents as pleural effusion, empyema or pleural thickening. pleural tuberculosis presenting with multiple masses without parenchymal involvement or lymphadenopathy has been reported rarely in the english literature. we report a case of a 68-year - old male with unilateral pleural tuberculosis presenting as multiple different sized nodular masses evident on computed tomography (ct) of the chest with a large hemorrhagic pleural effusion, which initially raised a possibility of mesothelioma. pleuroscopy also revealed multiple pleural masses. pathological examination of the biopsy specimen showed multiple epitheloid cell granulomas confirming the diagnosis. |
age - related macular degeneration (amd) is a progressive disease characterized by the degeneration of retinal pigment epithelium (rpe) and photoreceptor atrophy in the macula [1, 2 ]. recently, the inflammasome, a multimeric protein consisting of nod - like receptor (nlr), apoptosis - associated speck - like domain contains a caspase - recruitment domain (asc), and pro - caspase-1 plays a central role in innate immunity and has been implicated in the pathogenesis of amd [4, 5 ]. activation of the nlrp3 inflammasome results in caspase-1 cleaving pro - il-1 and pro - il-18 into their mature proinflammatory forms in macrophages and rpe cells [5, 6 ]. however, the direct effect of il-1 and il-18 on other retinal cells has not been well studied. in combination with il-23, il-1 or il-18 can induce interleukin-17a (il-17a) production by th17 cells, t cells, and inkt cells [710 ]. higher levels of il-17a are found in the serum and macular tissues of the amd patients when compared to age - matched controls [11, 12 ]. in vitro, il-17a is cytotoxic to arpe-19 cells, characterized by the accumulation of cytoplasmic lipids, autophagosome formation, and the presence of cleaved caspase-9 and cleaved caspase-3. il-17rc, a member of il-17r family and the primary receptor for il-17a, is highly expressed in amd macular tissues and in arpe-19 cells. in a study of twins with discordant amd status, hypomethylation of the il-17rc promoter this finding was correlated with elevated expression of il-17rc in peripheral blood cells as well as the macular tissue of amd patients. however, the direct effect of il-17a on other cell types remains to be explored. to test the hypothesis that il-18 and il-1 could stimulate il-17a secretion in retinal cells, we used a mouse - derived multipotent retinal stem cell line (rscs) as a model. rscs are cultured stem cells from the mouse retina and can be efficiently differentiated into photoreceptor cells and all major cell types of neural retina under optimized differentiation conditions. subretinal injection of these differentiated photoreceptors into slowly degenerating rd7 mouse eyes can form new synapses with resident retinal neurons ; in fast degenerating rd1 mouse eyes, injection of these cells can restore light response. these findings suggest that human retinal or neuronal stem cells could be useful for treating retinal degeneration in amd. we stimulated rscs with il-1, il-18, or il-17a and characterized the inflammatory and cytotoxic responses. the rsc line was obtained from primary culture of adult cd-1 mouse neuroretina and cultured as described previously. briefly, rscs were cultured in medium for retinal stem cells (rcm) composed of dmem / f12 (1 : 1, sigma, st louis, mo, usa), insulin - transferrin - selenium - a supplement (invitrogen, eugene, or, usa), 1.0 g / l bovine serum albumin (bsa, sigma), 1.0 g / l glucose (sigma), 1.0 g / l lactose (sigma), 0.045 g / l proline (sigma), 11.25 g / ml linoleic acid (sigma), 5 mm glutamine (invitrogen), 2 mm nicotinamide (sigma), 5% knockout serum replacement (life technologies, ny, usa), 20 ng / ml epidermal growth factor (egf, millipore, billerica, ma, usa), and 20 ng / ml basic fibroblast growth factor (bfgf, r&d systems, minneapolis, mn, usa). rscs grown to 70%80% confluence were treated with 1100 ng / ml recombinant mouse il-1 (r&d systems), recombinant mouse il-18 (mbl, woods hole, ma, usa), or recombinant mouse il-17 (r&d systems) for 24 hours. all procedures using animals adhered to the association for research in vision and ophthalmology statement for the use of animals and the nei 's institutional animal care and use committee approved protocols. mouse rpe was isolated from retinas of c57/b6j mice at 68 weeks of age as described previously. the globes were washed with pbs containing 1% penicillin - streptomycin (sigma) and then were dissected free of periocular connective tissue. then, the globe was placed on 2% dispase ii (neutral protease, grade ii, roche, indianapolis, in, usa) and incubated at 37c for 40 min. the globe was transferred to dmem / f12 media, the anterior segment was removed, and the retina containing the rpe layer was dissected free. the loosely adherent rpe cell layer was gently separated from the retina and transferred to a 15 ml tube containing dmem / f12, 20% fbs, and 1% l - glutamine - penicillin - streptomycin. the medium was changed after 5 - 6 days and every 2 - 3 days thereafter. the rpe cells between two and three passages were stimulated with 100 ng / ml recombinant mouse il-1 (r&d systems), 10 ng / ml recombinant mouse il-18 (mbl), or 10 ng / ml recombinant mouse il-17 (r&d systems) for 24 hours. the assessment of cell viability was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) assay in rscs as described previously. after stimulation with il-1, il-18, or il-17a for 24 hours, cells were washed with pbs and incubated with 20 l of 5 mg / ml mtt solution (sigma) for 4 h at 37c. cell viability was determined by measuring the optical density at 570 nm using an elisa plate reader (biotek, burlington, vt, usa). cell viability represented the optical density ratio of stimulated cells relative to that of unstimulated cells. for transmission electron microscopy (tem), cells were fixed in glutaraldehyde (2.5%, pbs buffered) for 24 hours, then suspended in warm low - melting point agarose (1.5%), pelleted down, and refrigerated overnight at 4c ; solidified pellets were rinsed with pbs three times, doubly - fixed with osmium tetroxide, rinsed again three times with pbs, dehydrated in ethanol, and embedded in spurr 's epoxy resin. ultrathin sections (100 nm) were mounted on 200 lines / inch copper grids, double - stained with uranyl acetate and lead citrate, and viewed with a jeol jem-1010 transmission electron microscope. the cells were seeded into 2-well chamber slides, and stimulation was performed at 70% confluence. after stimulation, cells were fixed with acetone, blocked with 1% bsa, and incubated overnight with the following primary antibodies : rabbit anti - mouse fasl (1 : 100, santa cruz, dallas, texas, usa) ; rabbit anti - mouse fas (1 : 100, santa cruz) ; rabbit anti - mouse cleaved caspase-3 (1 : 200, cell signaling technology, danvers, ma, usa) ; rabbit anti - mouse cleaved caspase-9 (1 : 200 cell signaling technology). after washing with pbs, secondary antibodies conjugated to either alexa-488 or alexa-555 (1 : 500, invitrogen) were added and incubated for 1 h. after rinsing with pbs, cells were counterstained with 40, 6-diamidino-2-phenylindole dihydrochloride (dapi, 1 : 1000, invitrogen) for 5 min. total rna was extracted from rscs by using an rneasy mini kit (qiagen, hilden, germany), and equal amounts of rna were synthesized to cdna with superscript ii rnase h reverse transcriptase (invitrogen) according to the manufacturer 's instructions. quantitative rt - pcr (qrt - pcr) was performed using rt sybr green rox qpcr mastermix (qiagen). cdna was amplified with primers -actin, il-6, il-17rc, or il-17a (qiagen) separately for 50 cycles. statistical analyses were performed using spss version 17.0 (spss, chicago, il, usa). unpaired t - tests or analysis of variance (anova) rscs cultured in rcm medium maintained spindle - shaped morphology (figure 1(a)). because the inflammatory response in rscs has not yet been characterized, we evaluated expression of il-17rc, which has been implicated in amd pathogenesis previously [12, 13 ]. indeed, il-17rc mrna expression was significantly increased in a dose - dependent fashion after stimulation with each cytokine (figure 1(b)). further, increased expression of il-17rc protein was detected after treatment with 100 ng / ml il-1, 10 ng / ml il-18, or 10 ng / ml il-17a, respectively (figure 1(c)). interestingly, il-17rc mrna expression was also significantly increased in primary cultured mouse rpe cells after stimulation with each cytokine (figure 1(d)). in order to test whether il-1, il-18, or il-17a could induce apoptosis in rscs, cleaved caspase-3, cleaved caspase-9, fas, and fasl were evaluated by immunohistochemistry. il-1 (100 ng / ml) or il-18 (10 ng / ml) induced the expression of all the tested proapoptotic proteins when compared to the untreated cells (figure 2) ; however, il-17a had minimal effect on the cells. accordingly, the mtt assay results demonstrated lower rsc viability in a dose - dependent manner after the cells were treated with il-1 and il-18. interestingly, rscs were also less viable after treatment with il-17a for 24 hours despite little increase in expression of any proapoptotic proteins (figure 3). to further elucidate the subcellular features of rscs after treatment with il-1, 100 ng / ml) or il-18 (10 ng / ml), the rscs showed autophagosome formation, mitochondrial degeneration, cytoplasmic vacuoles, and glycogen accumulation (figure 4). the average number of autophagosomes per cell increased from 1.3 in untreated controls to 9.8, 14.3, and 11 when rscs were stimulated with il-1, il-18, and il-17, respectively. a few necroptotic and pyroptotic cells with degradation of cytoplasmic contents and chromatin condensations were also noted. il-17a (10 ng / ml) had a similar effect as il-18, but to a lesser extent and without necroptosis (figure 4). surprisingly, only the highest concentration of il-1 (100 ng / ml) induced significantly higher expression of il-6 transcripts in rscs (figure 5(a)). both il-18 and il-17a induced high il-6 transcriptsin a dose - dependent manner (figures 5(b)-5(c)). consistent with these findings, il-1 (100 ng / ml), il-18 (10 ng / ml), and il-17a (10 ng / ml) could induce higher expression of il-6 mrna transcripts in primary cultured mouse rpe cells (figure 5(d)). however, neither il-1 nor il-18 could induce detectable il-17a expression from the rscs (data not shown). rscs can be differentiated into many types of retinal cells, including ganglion cells, bipolar cells, and photoreceptor cells. differentiated photoreceptors from this stem cell line could effectively integrate into rd1 or rd7 mouse retinas, improving vision. recently, the potential for stem cell therapy in amd has been highlighted [16, 17 ]. however, no extensive studies on the inflammatory response of rscs have been performed previously. in our study our tem finding of necroptosis and pyroptosis in the cells stimulated by the cytokines is unique. in contrast to apoptosis, necroptosis requires the function of ripk3 [18, 19 ], which regulates the nlrp3 inflammasome [20, 21 ]. pyroptosis is a caspase - dependent form of programmed cell death that differs from apoptosis. nlrp3, asc, and pro - caspase-1 induce caspase-1 activation and can lead to maturation and secretion of il-1 and il-18. this suggests a link between these two cytokines and pyroptosis / necroptosis, which could be novel pathways for cell death in amd in addition to apoptosis. our findings of releasing proinflammatory cytokines are in parallel with previous studies [4, 12, 24 ]. we found that il-1 could induce expression of il-6 and il-8 at both the transcript and the protein level in arpe-19 and human rpe cells, yet this treatment had no effect on cell viability. in our study, il-1 could also induce il-6 expression in primary cultured mouse rpe cells and rscs. however, il-1 upregulated proapoptotic protein expression and decreased cell viability in rscs, suggesting that il-1 may be more destructive to these cells than to rpe cells. tarallo and colleagues found that intravitreal injection of recombinant il-18 could induce rpe degeneration in mice, and il-18 neutralization protected against palu - induced rpe degeneration ; however, doyle and her group reported that il-18 has a protective role in laser induced choroid neovascularization (cnv), as intravitreally injected il-18-neutralizing antibodies resulted in increased cnv development in mice. these two seemingly conflicting studies may point to diverging roles of il-18 in rpe versus the myeloid cells and vascular endothelium. supporting the hypothesis that il-18 is damaging to the neuroretina, we found that il-18 decreased cell viability, induced necroptosis / pyroptosis by ultrastructure (figure 4), and induced proinflammatory response (il-6 production) in rscs. interestingly, it was found that there are increased level of nlrp3 protein, il-1 and il-18 mrna in the rpe of donor eyes from individuals with geographic atrophy and neovascular amd [4, 25 ]. combined with our findings that both il-1 and il-18 could induce rscs death in vitro, this mechanism may to some extent explain neuroretinal (photoreceptor) atrophy in amd patients. il-17rc serves as an essential subunit of the il-17 receptor complex and mediates the signal transduction and proinflammatory activities of il-17a and il-17f, which have been implicated in autoimmune and neurodegenerative diseases [2729 ]. recent research has also implicated the il-17a / il-17rc pathway in the pathogenesis of amd [13, 30 ] ; however, the exact role of il-17a still remains elusive. in a previous study silencing of il-17rc could prevent upregulation of cleaved caspase-3 and cleaved caspase-9 and was protective against il-17a - mediated cell death. in rscs this may imply that il-17a - induced effect in rscs proceeds through pathways other than apoptosis. one of the most notable roles of il-17 is its involvement in inducing and mediating proinflammatory response. in synoviocytes, interestingly, we found that not only il-17a but also il-1 and il-18 could induce il-6 expression in rscs and rpe cells. generally, il-6 is an important proinflammatory cytokine and has been associated with incidence of early amd. furthermore, elevated plasma il-6 was found in amd patients with the cc variant in the cfh y402h polymorphism, indicating a potential role for il-6 in inflammation - related damage in amd pathogenesis. it has also been shown that il-6 can contribute to th17 cell differentiation from nave t cells. il-1 combined with il-23 can promote il-17 production in nave and memory t cells [36, 37 ]. thus, il-6 secretion by rscs or rpe cells could result in a positive feedback loop through which th17 and cells are locally induced. although neither il-1 nor il-18 led to increased expression of il-17a in rscs, both could independently induce il-17rc expression, which may amplify the effect of il-17a. interestingly, primary cultured rpe cells could also express notably higher il-17rc and il-6 under the stimulation of il-1, il-18, or il-17a. these findings may account for a potential mechanism of il-17a - induced pathogenesis in amd via il-6 production. first, we only explored the response of rscs, not the differentiated mature neuroretinal cells to il-1, il-18, and il-17a ; in future studies, we plan to differentiate the rscs to photoreceptor cells and explore their response to these cytokines, which will be a better model for photoreceptor changes in amd. additionally, this study only evaluates the in vitro effects of il-1, il-18, and il-17a ; in the future, we hope to explore the effect of these cytokines in vivo. in conclusion, we demonstrated that il-1, il-18, and il-17a have cytotoxic (necroptosis, pyroptosis, and apoptosis) effect and induce proinflammatory response in rscs. though il-1, il-18 alone could not induce il-17a expression in rscs, they all induce il-17rc expression, which may mediate the effect of il-17a. | purpose. to investigate how multipotent retinal stem cells (rscs) isolated from mice respond to the proinflammatory signaling molecules, il-1, il-18, and il-17a. materials and methods. rscs were cultured in a specific culture medium and were treated with these cytokines. cell viability was detected by mtt assay ; ultrastructure was evaluated by transmission electron microscopy ; expression of il-17rc and proapoptotic proteins was detected by immunocytochemistry and expression of il-6 and il-17a was detected by quantitative rt - pcr. as a comparison, primary mouse retinal pigment epithelium (rpe) cells were also treated with il-1, il-18, or il-17a and analyzed for the expression of il-6 and il-17rc. results. treatment with il-1, il-18, or il-17a decreased rsc viability in a dose - dependent fashion and led to damage in cellular ultrastructure including pyroptotic and/or necroptotic cells. il-1 and il-18 could induce proapoptotic protein expression. all treatments induced significantly higher expression of il-6 and il-17rc in both cells. however, neither il-1 nor il-18 could induce il-17a expression in rscs. conclusions. il-1, il-18, and il-17a induce retinal cell death via pyroptosis / necroptosis and apoptosis. they also provoke proinflammatory responses in rscs. though il-1 and il-18 could not induce il-17a expression in rscs, they both increase il-17rc expression, which may mediate the effect of il-17a. |
it is generally whitish, yellowish, or greenish vaginal discharge in females that might be normal or a symptom of infection. it is almost mucus discharge, which exhibit exfoliation of vaginal epithelial cells due to estrogen influence on the vaginal mucosa. leucorrhea is a well - known disease in iranian traditional medicine (itm). in their manuscripts, the word sayalan - e rahem was used by avicenna and some other iranian traditional practitioners to describe this condition. ancient practitioners believed that excessive residue (kesrate fozool) and weakness of digestion (zaafe hazm) were the main causes of leucorrhea, for which herbal therapy was the main proposed treatment. in the present study, in this research, six iranian traditional textbooks including canon of medicine (avicena 980 - 1037 ad), a - hawi (razes 865 - 925 ad), tuhfat ul - momineen (momen tonekaboni, 17th century), makhzan - ul - adwiah (aghili 18th century), ikhtiarat badii (ansari 1329 - 1404 ad), and al - jmi li - mufradt al - adwiyawa al - aghdhiy (ibn al - baitar 1197 ad) were studied and searched for anti - leucorrhea medicines. then the herbal medicines were selected and scored depending on their frequency in the above - mentioned textbooks. this study introduced many materia medica with anti - leucorrhea activity and among them seven herbs including rubus fruticosus l., rhus coriaria l., phoenix dactylifera l., pimpinella anisum l., rumex acetosa l., olea europaea l. and quercus lusitanica lam. showed the most repetition in itm prescriptions these herbs can be introduced as new anti - leucorrhea herbal medicines for clinical research. | background : leucorrhea or vaginal discharge is a conventional complaint. it is generally whitish, yellowish, or greenish vaginal discharge in females that might be normal or a symptom of infection. it is almost mucus discharge, which exhibit exfoliation of vaginal epithelial cells due to estrogen influence on the vaginal mucosa. it is important to identify the differences between physiologic and pathologic discharges. leucorrhea is a well - known disease in iranian traditional medicine (itm). in their manuscripts, the word sayalan - e rahem was used by avicenna and some other iranian traditional practitioners to describe this condition. ancient practitioners believed that excessive residue (kesrate fozool) and weakness of digestion (zaafe hazm) were the main causes of leucorrhea, for which herbal therapy was the main proposed treatment. in the present study, medicinal plants used in itm for leucorrhea are introduced.methods:in this research, six iranian traditional textbooks including canon of medicine (avicena 980 - 1037 ad), a - hawi (razes 865 - 925 ad), tuhfat ul - momineen (momen tonekaboni, 17th century), makhzan - ul - adwiah (aghili 18th century), ikhtiarat badii (ansari 1329 - 1404 ad), and al - jmi li - mufradt al - adwiyawa al - aghdhiy (ibn al - baitar 1197 ad) were studied and searched for anti - leucorrhea medicines. then the herbal medicines were selected and scored depending on their frequency in the above - mentioned textbooks. additional attention was paid to provide the most suitable scientific name for each plant.results:this study introduced many materia medica with anti - leucorrhea activity and among them seven herbs including rubus fruticosus l., rhus coriaria l., phoenix dactylifera l., pimpinella anisum l., rumex acetosa l., olea europaea l. and quercus lusitanica lam. showed the most repetition in itm prescriptions.conclusion:these herbs can be introduced as new anti - leucorrhea herbal medicines for clinical research. |
the babydiab study examined the natural history of islet autoimmunity in children of patients with type 1 diabetes (8). families were eligible if one or both parents had type 1 diabetes. venous blood samples were obtained from children at study visits scheduled at age 9 months and 2, 5, 8, 11, 14, 17, and 20 years. if children had a positive autoantibody result, visit frequencies and islet autoantibody measurements were subsequently performed at 6- to 12-month intervals. the study was coordinated centrally from munich and conducted from this site by directly contacting the participating families and their family pediatrician. the babydiab cohort contains 1,650 offspring followed from birth to last sample for a median of 8.8 years (range 0.7520.1). families were asked to report the occurrence of symptoms of diabetes. in children with islet autoantibodies, diabetes onset was defined according to american diabetes association criteria, which include unequivocal hyperglycemia with acute metabolic decompensation or the observation (on at least two occasions) of a 2-h plasma glucose > 200 mg / dl after an oral glucose challenge, or a random blood glucose > 200 mg / dl if accompanied by unequivocal symptoms. since 1997, fasting blood glucose > 126 mg / dl on two occasions was added to the diabetes diagnosis criteria. insulin autoantibodies (iaas), gad autoantibodies (gadas), ia-2a autoantibodies (ia-2as), and znt8 autoantibodies (znt8as) were measured by radiobinding assays as previously described (8,9). the upper limits of normal were determined using qq plots and corresponded to the 99th percentile of control children. performances in the diabetes autoantibody standardization program are shown as laboratory 121 in published reports (10,11). offspring were considered islet autoantibody positive when two consecutive samples collected after birth were positive. thyroid peroxidase antibodies (tpoas) were measured by radiobinding assay according to the manufacturer s instructions (centak anti - tpo ; medipan, dahlewitz / berlin, germany) as previously described (12). samples were tpoa positive if levels were > 50 units / ml, as suggested by the manufacturer and confirmed using qq plot analysis. iga antibodies to tissue transglutaminase autoantibodies (ttgas) were measured by elisa according to the manufacturer s instructions (eurospital, trieste, italy) and by radiobinding assay with [s]methionine - labeled in vitro transcribed / translated recombinant human tissue transglutaminase as previously described (13). positive thresholds were determined using qq plots and corresponded to the 95th percentile of control children without diabetes or celiac disease for the elisa and the 99th percentile of control samples for the radiobinding assay. hla - drb1, hla - dqa1, and hla - dqb1 alleles were typed using pcr - amplified dna and nonradioactive sequence - specific oligonucleotide probes as described previously (5). classification into high - risk hla genotypes was based on the environmental determinants of diabetes in the young (teddy) study inclusion genotypes for first - degree relatives (14) : dr4-dqa1 030x - dqb1 0302/dr3-dqa1 0501-dqb1 0201 ; dr4-dqa1 030x - dqb1 0302/dr4-dqa1 030x - dqb1 0302 ; dr4-dqa1 030x - dqb1 0302/dr8- dqa1 0401-dqb1 0402, dr3-dqa1 0501-dqb1 0201/dr3-dqa1 0501-dqb1 0201 ; dr4-dqa1 030x - dqb1 0302/dr4-dqa1 030x - dqb1 020x ; dr4-dqa1 030x - dqb1 0302/dr1-dqa1 0101-dqb1 0501 ; dr4-dqa1 030x - dqb1 0302/dr13-dqa1 0102-dqb1 0604, dr4-dqa1 030x - dqb1 0302/dr4-dqa1 030x - dqb1 0304, dr4-dqa1 030x - dqb1 0302/dr9-dqa1 030-dqb1 0303 ; dr3-dqa1 0501-dqb1 0201/dr9-dqa1 030x - dqb1 0303, where includes dqb1 0302 and 0304. tested were the originally described ptpn2 rs1893217and cd25 rs11594656 snps (1) and, to facilitate typing in the multiplex method, the proxy snps ptpn22 rs6679677, erbb3 rs705704, kiaa0350 rs12935413, and ifih1 rs2111485. snp genotyping was performed with the massarray system using iplex chemistry (sequenom, san diego, ca) as previously described (15). reproducibility was assessed by duplicate genotyping in 16.3% of samples (discordance rate 200 mg / dl after an oral glucose challenge, or a random blood glucose > 200 mg / dl if accompanied by unequivocal symptoms. since 1997, fasting blood glucose > 126 mg / dl on two occasions was added to the diabetes diagnosis criteria. insulin autoantibodies (iaas), gad autoantibodies (gadas), ia-2a autoantibodies (ia-2as), and znt8 autoantibodies (znt8as) were measured by radiobinding assays as previously described (8,9). the upper limits of normal were determined using qq plots and corresponded to the 99th percentile of control children. performances in the diabetes autoantibody standardization program are shown as laboratory 121 in published reports (10,11). offspring were considered islet autoantibody positive when two consecutive samples collected after birth were positive. thyroid peroxidase antibodies (tpoas) were measured by radiobinding assay according to the manufacturer s instructions (centak anti - tpo ; medipan, dahlewitz / berlin, germany) as previously described (12). samples were tpoa positive if levels were > 50 units / ml, as suggested by the manufacturer and confirmed using qq plot analysis. iga antibodies to tissue transglutaminase autoantibodies (ttgas) were measured by elisa according to the manufacturer s instructions (eurospital, trieste, italy) and by radiobinding assay with [s]methionine - labeled in vitro transcribed / translated recombinant human tissue transglutaminase as previously described (13). positive thresholds were determined using qq plots and corresponded to the 95th percentile of control children without diabetes or celiac disease for the elisa and the 99th percentile of control samples for the radiobinding assay. hla - drb1, hla - dqa1, and hla - dqb1 alleles were typed using pcr - amplified dna and nonradioactive sequence - specific oligonucleotide probes as described previously (5). classification into high - risk hla genotypes was based on the environmental determinants of diabetes in the young (teddy) study inclusion genotypes for first - degree relatives (14) : dr4-dqa1 030x - dqb1 0302/dr3-dqa1 0501-dqb1 0201 ; dr4-dqa1 030x - dqb1 0302/dr4-dqa1 030x - dqb1 0302 ; dr4-dqa1 030x - dqb1 0302/dr8- dqa1 0401-dqb1 0402, dr3-dqa1 0501-dqb1 0201/dr3-dqa1 0501-dqb1 0201 ; dr4-dqa1 030x - dqb1 0302/dr4-dqa1 030x - dqb1 020x ; dr4-dqa1 030x - dqb1 0302/dr1-dqa1 0101-dqb1 0501 ; dr4-dqa1 030x - dqb1 0302/dr13-dqa1 0102-dqb1 0604, dr4-dqa1 030x - dqb1 0302/dr4-dqa1 030x - dqb1 0304, dr4-dqa1 030x - dqb1 0302/dr9-dqa1 030-dqb1 0303 ; dr3-dqa1 0501-dqb1 0201/dr9-dqa1 030x - dqb1 0303, where includes dqb1 0302 and 0304. tested were the originally described ptpn2 rs1893217and cd25 rs11594656 snps (1) and, to facilitate typing in the multiplex method, the proxy snps ptpn22 rs6679677, erbb3 rs705704, kiaa0350 rs12935413, and ifih1 rs2111485. snp genotyping was performed with the massarray system using iplex chemistry (sequenom, san diego, ca) as previously described (15). reproducibility was assessed by duplicate genotyping in 16.3% of samples (discordance rate < 0.5%). snps were tested for deviation from hardy - weinberg equilibrium by or fisher exact test. the study design was a priori established to examine overall genotype association with diabetes and subsequently examine relationships to islet autoantibodies and progression to diabetes only in diabetes - associated genes. hazards ratios (hrs) were determined using cox proportional hazards model (with and without adjustment for hla risk genotype). within islet autoantibody positive children, kaplan - meier analysis was used to calculate the probability of progression to diabetes where follow - up time was calculated from the age when autoantibodies were first detected to the age of type 1 diabetes diagnosis, or last contact. statistical analysis was performed using the statistical package for the social sciences (spss 18.0 ; spss, chicago, il). ifih1 snp rs2111485 was associated with diabetes development in the babydiab cohort (hr 2.08 ; 95% ci 1.163.74 ; p = 0.014 ; table 1). the probability of type 1 diabetes was 5% (95% ci 3.26.8) by age 15 years for children with gg genotypes and 2% (95% ci 0.83.2) for children with ga or aa genotypes (p = 0.004 ; fig. the association remained when adjusted for hla genotypes (hr 1.98 ; 95% ci 1.013.56 ; p = 0.023 ; table 1), and ifih1 genotypes were able to stratify diabetes risk in children with high - risk hla genotypes (fig. no significant association with diabetes development could be observed for snps in the other five gene regions (table 1). gene associations with development of type 1 diabetes in the babydiab cohort hrs (95% ci) are shown for the homozygous expected susceptible genotype vs. other genotypes ; p value is for diabetes development using cox proportional hazards model across all genotypes ; p values shown in parentheses are adjusted for hla drb1-dqb1 risk or other genotype on the basis of teddy risk genotypes (14). a : children are grouped with respect to ifih1 snp rs2111485 genotype into those carrying gg genotype (solid line) and the ga or aa genotype (dashed line). b : children are grouped by ifih1 genotypes after stratification of hla genotypes (solid and dashed lines are children with teddy hla risk genotypes, and the dotted and dot - dashed lines represent children with low - risk hla genotypes). p values are provided for comparison of ifih1 gg vs. ga and aa genotypes in the total cohort (a) and for high - risk (p = 0.03) and low - risk (p = 0.06) genotypes. numbers below the x - axis indicate the number of diabetes - free children remaining on follow - up. to determine whether the association with diabetes observed in the cohort was at the stage of autoimmunity development or the progression to clinical diabetes, we examined ifih1 associations with islet autoantibody development. no ifih1-associated difference in the development of autoantibodies was observed (p = 0.80 ; fig. autoantibody appearance curves were similar between susceptible and protective ifih1 genotypes for iaas (p = 0.44), gadas (p = 0.24), and znt8as (p = 0.20) and slightly, but not significantly, higher for susceptible genotypes for the later marker ia-2a (p = 0.06). moreover, no significant difference between ifih1 genotypes was found for the probability of developing autoantibodies to thyroid autoantigens and transglutaminase antigen (p = 0.55 and 0.66 ; fig. 2f and g). cumulative risk is shown for at least one islet autoantibody (a), iaas (b), gadas (c), ia-2as (d), znt8as (e), tpoas (f), and ttgas (g) by ifih1 genotypes. children are grouped with respect to ifih1 snp rs2111485 genotype into those carrying the gg genotype (solid line) and the ga or aa genotype (dashed line). numbers below the x - axis indicate the number of autoantibody - negative children remaining on follow - up with respect to age. among 137 islet autoantibody positive children, 47 developed diabetes (median, 4.66 years after their first islet autoantibody positive sample). in contrast to the lack of association with islet autoantibody development, a significant association of the ifih1 gg genotype with progression from islet autoantibody positivity to diabetes was observed (31 vs. 11% within 5 years ; p = 0.006 ; fig. this remained significant (hr 1.9 ; p = 0.05) after adjustment for islet autoantibody status of the child as single or multiple and hla genotype. cumulative risk for the progression from islet autoimmunity to type 1 diabetes by ifih1 genotypes. islet autoantibody positive children are grouped with respect to ifih1 snp rs2111485 genotype into those carrying the gg genotype (solid line) and the ga or aa genotype (dashed line). follow - up (x - axis) is from the age of the first islet autoantibody positive sample. numbers below the x - axis indicate the number of diabetes - free children remaining on follow - up. understanding the mode of action of genes influencing the development of type 1 diabetes requires knowledge as to whether genes influence the development of islet autoimmunity and/or progression from autoimmunity to diabetes. here we have examined association in a cohort of genetically at - risk children who were followed from birth for both development of islet autoantibodies and diabetes. an association of the ifih1 gene with diabetes development in this cohort allowed us to determine at what stage the gene is likely to influence diabetes development. unlike hla class ii genes, which strongly influence the risk for developing islet autoantibodies (57), association of the ifih1 gene was restricted to the progression to diabetes after development of islet autoimmunity. in view of the involvement of the ifih1 gene in responses to virus infection (16,17), the findings are consistent with a role of infection in determining the progression to diabetes after islet autoimmunity has been initiated. the findings are from a unique cohort characterized by a family history of type 1 diabetes, perspective follow - up from birth with relatively frequent testing for islet autoantibody development, monitoring for diabetes development, and testing and monitoring for the development of thyroid- and celiac disease associated autoimmunity up to age 20 years. to minimize the number of comparisons, we chose association with diabetes as the outcome and selected genes that showed association both alone and together with hla genotypes in a multiple cox proportional hazards model. the disadvantage of this approach is that modest numbers developed diabetes, allowing us to identify only moderate to strong genetic associations. thus, the findings from our study are not informative for snps in the five gene regions, where we found no association with type 1 diabetes. a potential caveat is that not all islet autoantibodies may be specific for type 1 diabetes (18). the findings were, however, significant for the ifih1 snp after adjustment for multiple islet autoantibodies, which is a specific characteristic of type 1 diabetes. finally, because our study is in subjects with a type 1 diabetes family history, we can not make conclusions for case subjects without a family history. analyses in the finnish diabetes prediction and prevention (dipp) study (6) and the multicenter teddy study (14) are informative in this respect. the ifih1 gene encodes helicase c domain 1, which mediates induction of the interferon response to viral rna (16,17). the association of ifih1 polymorphisms including rare variants with type 1 diabetes (19,20) has been noteworthy because of their link to the inflammatory response caused by infectious agents, including enteroviruses. viral infection is hypothesized to cause islet autoimmunity and/or influence progression to diabetes (2123). data in humans are inconclusive, whereas data from the murine models lean toward effects at the progression stage (24). first, a common polymorphism of the ifih1 gene had an odds ratio for diabetes development of around 2 in our cohort, potentially implying relatively strong effects in children with a type 1 diabetes family history. second, we found no association with the initiation of autoimmunity, as defined by the development of islet autoantibodies, and the development of thyroid- or celiac disease associated autoantibodies. examining individual islet autoantibodies found no association with the development of iaas or gadas, which appear early in the disease process, and borderline association with ia-2as, which in our cohort appear later than iaas or gadas. of interest, the development of all these autoantibodies is strongly influenced by the hla class ii genotype (57,10,11), whereas progression to diabetes after the appearance of islet autoantibodies is only minimally associated with hla class ii (6,25). in contrast, we observed a strong effect of the ifih1 genotype on the rate of progression to disease from first islet autoantibody detection. with respect to pathogenesis, our findings would be consistent, with infection playing a more prominent role in diabetes development after the onset of islet autoimmunity than during initiation of autoimmunity. they suggest that intervention in autoantibody - positive children to delay or prevent the onset of type 1 diabetes is possible and furthermore suggest that interference with host response to infection may be a means to achieve successful intervention. | objectivegenome - wide association studies have identified gene regions associated with the development of type 1 diabetes. the aim of this study was to determine whether these associations are with the development of autoimmunity and/or progression to diabetes.research design and methodschildren (n = 1,650) of parents with type 1 diabetes were prospectively followed from birth (median follow - up 10.20 years) for the development of islet autoantibodies, thyroid peroxidase antibodies, tissue transglutaminase antibodies, and diabetes. genotyping for single - nucleotide polymorphisms of the ptpn22, erbb3, ptpn2, kiaa0350, cd25, and ifih1 genes was performed using the massarray system with iplex chemistry.resultsislet autoantibodies developed in 137 children and diabetes developed in 47 children. type 1 diabetes risk was associated with the ifih1 rs2111485 single - nucleotide polymorphism (hazard ratio 2.08 ; 95% ci 1.163.74 ; p = 0.014). none of the other genes were significantly associated with diabetes development in this cohort. ifih1 genotypes did not associate with the development of islet autoantibodies (p = 0.80) or autoantibodies against thyroid peroxidase (p = 0.55) and tissue transglutaminase (p = 0.66). islet autoantibody positive children with the ifih1 rs2111485 gg genotype had a faster progression to diabetes (31% within 5 years) than children with the type 1 diabetes protective ga or aa genotypes (11% within 5 years ; p = 0.006).conclusionsthe findings indicate that ifih1 genotypes influence progression from autoimmunity to diabetes development, consistent with the notion that protective genotypes downregulate responses to environmental insults after initiation of autoimmunity. |
cystic fibrosis (cf) or mucoviscidosis is a multisystem inflammatory disorder where the primary cause of morbidity and mortality is pulmonary destruction, characterized by chronic inflammation, bacterial colonization and frequent exacerbations (1, 2). cf is an autosomal - recesive disease caused by mutations in the cf transmembrane conductance regulator (cftr) protein at the chromosome 7q31.2 (cftrf508). the mutation is a deletion of the three nucleotides that comprise the codon for phenylalanine at position 508. mutations of the cftr gene affecting chloride ion channel function lead to dysregulation of epithelial fluid transport in the lung resulting in cystic fibrosis (3 -7). in the united states approximately 30, 000 individuals have cf, and in canada 4,000 (8). in the european union, 1 in 2000 - 3000 newborns is found to be affected by cf (5, 6, 7). cf occurs in one of every 3,200 live white births, and in 1 of every 15,000 live african american births, 1 of every 31,000 live asian americans births (2, 8). in normal conditions, cftr regulates the normal absorption of sodium ions (na) from the lumen of the channel gland, by controlling the function of neighboring epithelial sodium channel (enac). in cf this control is missing, which leads to excessive absorption of na in the epithelial cells. inhibit secretion of cl and na hyperabsorption lead to the withdrawal of water from the lumen of the secretory canals of glands and secretion in them remains dehydrated and viscous and difficult to be removed by normal mucociliary mechanisms. this leads to obstruction of the airway lumen and a suitable basis for the development of pathogenic microorganisms (1, 2, 9). in the thick layer of dense and tenacious secretions airway gradually create anaerobic conditions, which encourages bacteria to adjustment, especially pseudomonas, with the formation of alginate and mucous form of growth (10). cystic fibrosis is a chronic progressive disease that affects multiple organ systems and whose manifestations related primarily to the development of chronic lung disease and malnutrition. common cf symptoms are : salty - testing skin, slow weight gain, abnormal bowel movements, wheezing, cough and increased lung mucus, pneumonia, nasal polyps, clubbing, severe constipation, meconium ileus, rectal prolapse, pancreatic insufficiency, multifocal biliary cirrhosis, male infertility, etc (1, 2, 9). diagnosis is made by finding elevated levels of chloride in the sweat test.. they can be performed at any time from 1 week of age, but adequate sweat volumes may not be obtained before 6 weeks of age. if there is any clinical suspicious of cf, a sweat test should be arranged, even if the patient has had newborn screening, since approximately 5% of cf cases are not detected by newborn screening. sweat electrolyte values in adults are often higher than children and need to be interpreted by an expert. the sweat glands are made to produce sweat with a mild chemical called pilocarpine and a little electricity (pilocarpine iontophoresis) (11, 12). sweat - testing involves application of a medication that stimulates sweating (pilocarpine). to deliver the medication through the skin, iontophoresis is used to, whereby one electrode is placed onto the applied medication and an electric current is passed to a separate electrode on the skin. the resultant sweat is then collected on filter paper or in a capillary tube and analyzed for abnormal amounts of sodium and chloride. people with cf have increased amounts of sodium and chloride in their sweat (11). the reference ranges of sweat chloride that are currently accepted for the diagnosis of cf in infants up to and including 6 months of age are : equal to or less then 29 mmol / l means cf is very unlikely, 30 - 59 mmol / l means that cf is possible, greater than or equal to 60 mmol / l means cf is likely to be diagnosed. the reference ranges of sweat chloride that are currently accepted for the diagnosis of cf in people older than 6 months of age are : equal to or less then 39 mmol / l means cf is very unlikely, 40 - 59 mmol / l means that cf is possible, greater than or equal to 60 mmol / l means cf is likely to be diagnosed (1, 11). the test is carried out by collecting sweat from pilocarpine iontophoresis in two or more occasions and chemical determination of the concentration of chloride. the conditions other than cystic fibrosis, which are associated with elevated concentrations of sweat electrolytes include malnutrition, adrenal insufficiency, glycogen storage diseases, anorexia, celiac disease, hypothyroidism, hypoparathyroidism, nephrogenic diabetes inspidus, ectodermal dysplasia, atopic eczema, and congenital metabolic disease. most of errors are caused by using unreliable methodology, irregular way of collecting sweat, technical errors and misinterpretation of results (1, 2, 11). we retrospectively analyzed all hospital records at the pediatric clinic of university clinical center of sarajevo between march 2003 and december 2014. we included all resident patients of the federation of bosnia and herzegovina, consisting of canton sarajevo and 9 other cantons. we selected all patients with positive sweat test, and also recorded age, gender, date of initial diagnosis, amount of chloride in the sweat test, the number of patients in federation of bosnia and herzegovina. statistical analysis was performed using spss 20.0 (spss, chicago, il, usa). in the analysis of the difference between the observed group was used pearson s test, chi - square test, ficher exact test, yates chi - square test with correction for small samples (with tables in which the number of cells in less than 5), and student s t - test and one - way analysis of variance (anova), depending on the type of data. the results of these statistical tests were considered statistical significance at p < 0.05 or at the level of 95%. the analysis was performed using the statistical package spss statistics ibm v21.0 (chicago, illinois, usa). statistical analysis was performed using spss 20.0 (spss, chicago, il, usa). in the analysis of the difference between the observed group was used pearson s test, chi - square test, ficher exact test, yates chi - square test with correction for small samples (with tables in which the number of cells in less than 5), and student s t - test and one - way analysis of variance (anova), depending on the type of data. the results of these statistical tests were considered statistical significance at p < 0.05 or at the level of 95%. the analysis was performed using the statistical package spss statistics ibm v21.0 (chicago, illinois, usa). the median age in all patients (n=40) with cystic fibrosis was 3,223,85 years (interquartile range 0 to 15 years). the median age for male children (19/40) was 2.153.11 years, and for female children (21/40) 4.194.26 years. there was no significant age difference (t=2.912 ; df=39 ; p=0.096) between male and female children. the total number of sick children (male and female) from march 2003 to december 2014 was 40, 19 male children and 21 female children. analysis of the frequency of gender according to the year of diagnosis of cystic fibrosis showed that the largest number of children fell ill in 2011, seven. the minimum number of detected children was in 2010, two children (table 1). there was no statistically significant differences in male compared to female children by the year of discovery cf, (2 = 9.801 ; df = 22 ; p = 0.458). sweat test is a method used to measure an average amount of chloride in the sweat of children. an average amount of chloride in the sweat in male children (19/40) was 103.0521.26 mg / l, (interquartile range 60 to 131 mmol / l chloride) ; and in the female children 89.7122.95 mmol / l (interquartile range 60 to 128 mmol / l chloride).. the total average amount of chloride in sweat at all affected children was 96.0528.89 mmol / l (interquartile range 60 to 131 mmol / l chloride). the lowest measured amount of chloride, which was enough to confirm the diagnosis of cystic fibrosis in male and female children was 60mmol / l in sweat, while the maximum amount of chloride in male children in sweat was 131 mmol / l, in the female children 128 mmol / l. according to the analysis there was no statistically significant difference (t=3.612 ; df=39 ; p=0.046) between the average amount of chloride by gender (figure 1). an average amount of chloride in sweat by gender children from the federation of bosnia and herzegovina consisting of canton sarajevo and other cantons, are tested at the pulmonary department of the pediatric clinic at the university clinical center sarajevo. the total number of tested children in the federation was 625 for the period 2003 - 2014 years, while the number of children tested just in the sarajevo canton was 351, and from others cantons 272 children (figure 2). the demographic distribution of patients with cf in the federation of bosnia and herzegovina pseudomonas aeruginosa is a common cause of respiratory infections in chronically sick children with cf, and there is a colonization with pseudomonas aeruginosa. the data are from 2008 to 2014, and pseudomonas was isolated in 13 children 36 times, mostly two and more time per each child (table 2). the evidence of infected children with p.aeruginosa (2008 - 2014) this study shows for the first time the number of children suffering from cf in the federation of bosnia and herzegovina from march 2003 to december 2014. advanced diagnostics led to the diagnosis in the early stages of the disease which greatly facilitates the further treatment of patients and seventy years ago patients died in the first year of life most of malabsorption and diarrhea. today, patients experience the second and third decade, and life expectancy is determined by duration of pulmonary disease. survival of patients with cf has dramatically improved since the disease was discovered, 70 years ago. progressive pulmonary disease is leading to morbidity and pulmonary arrest, and the average life expectancy of people with 37.4 years, 37 years for women and 40 years for men (13, 14). progress in the length of life of patients is the result of many new improvement in treating disease. progress has been made in the diagnosis of cf and the introduction of neonatal screening. it is possible to act on intracellular events and encourage, correct or potentiate the synthesis of mutated cftr - a (ivacafor, lumocaftor, ataluran), (15, 16, 17). in the final stage lung disease is available and lung transplantation, individuals with cf must have both lungs replaced because the remaining lung might contain bacteria that could infect the transplanted lung (18). although the lifespan moved from childhood to adult age, patients are still faced with an abundance of therapeutic needs, and their doctors with a new pathology. the challenge is identifying new manifestations and complications of the disease as well as the actual carrying out of treatment. a special, more a problem in the treatment of patients with cf becomes a problem of economic sustainability of therapy. respiratory tract infections and prevent colonization of p. aeruginosa is one of the fundamental problems in the treatment of cf. in this sense, nowadays there are many therapeutic strategies that must be counted and the possibility of active immunization against bacterial and viral infections (19). by 18 years of age, 80% of patients with classic cf harbor p. aeruginosa, and 3.5% harbor b. cepacia (20). in cystic fibrosis (cf) lung occupy a special place, because it in most patients with cf are determined by the quality and length of life. the lung is an organ that before birth is not affected by the changes, but soon after birth becomes a place of chronic infection and inflammation. the main goal of therapy in cf is focused primarily on the lungs, and that means delay and reduce the damage and thereby contribute to the quality and duration of life expectancy of cf patients. therapy of cf today are not causal, or untreated primary disorder, but is based on the treatment of a number of consequences that illness affects many organ systems. children born with cf today have a greater chance of living longer, thanks to early diagnosis and multidisciplinary approach to treatment, where children may benefit from treatment with mitigation damaged ionic transport with medications such as inhalers, mucolytics, antibiotics, anti - inflammatory drugs, and methods of cleaning the airways. male children with cf tend to live longer than female children with cf, and there is no gender (sex gap). socioeconomic status may affect the length of life of the sick and environmental factors, exposure to cigarette smoke, air pollutants. the most difficult patients in the post - war period were from the central bosnia canton, while lately the number of children from canton sarajevo with manifestations of harder lung obstruction has risen but without growth deformation and changes in radiogram like in post - war children. whether this can be explained by new mutations of children with cf or not p.aeruginosa is the most common bacterial species involved in respiratory tract infection in cf, while burkholderia have also been identified as causing infection of cf patients. | aim : the aim of this study is to present the first total number of tested children in the federation of bosnia and herzegovina and the number of children with positive sweat test. during the study we determined the number of ill children, the median age of children with cystic fibrosis, date of initial diagnosis, an average amount of chloride in the sweat.material and methods : the study was a retrospective, conducted at the department of pulmonology pediatric clinic of university clinical center of sarajevo.results:in the period from march 2003 to december 2014, we have tested 625 children. 351 child were from sarajevo canton and 272 children from other cantons. female children were more affected then male children, in the ratio of 1 : 1,105. an average age of female children was 4.194.26 years, and the male 2.153.11 years. the median concentration of chloride in the sweat measured by sweat test was for male children 103.0521.29 mmol / l, and for the female children 96.0528.85 mmol / l.conclusion : most of children in federation of bosnia and herzegovina have f508 gene mutation. in the post - war period we started to use a sweat test. male children tend to live longer than female children with cf. |
dna and rna nucleobases contain the genetic information of all living cells and play a vital role in the development and functioning of all known living organisms. because of their importance in maintaining the genome integrity during cell replication, increasing attention continues to be devoted to understanding the specific mechanisms (particularly ionization and electron - impact radiation damage) that can dramatically alter their electronic and structural states. an important first step in understanding the reactivity and damage mechanisms in dna and rna is the ability to both efficiently and accurately predict electronic properties such as ionization energies and electron affinities. while numerous theoretical studies using high - level wave - function - based techniques have been used to accurately predict the electronic properties of nucleobases, (see ref (1) and references within), their immense computational costs prevent their routine use for complex biological environments or for large geometries (i.e., a fully periodic dna / rna strand is currently not possible with wave - function - based methods). because of its favorable balance between accuracy and efficiency, density functional theory (dft) has become the most widely used quantum mechanical method for obtaining electronic structure information of molecules and solids. however, selecting the best exchange - correlation functional continues to be a difficult task due to the large number of functionals available. herein, we assess the accuracy of a nonempirically tuned range - separated (long - range corrected) density functional theory (lc - dft) method for predicting both the quasiparticle properties (homo lumo fundamental gaps) and the excitation energies in dna and rna nucleobases. the fundamental gap is rigorously defined as the difference in energy between the first ionization potential (ip) and the first electron affinity (ea), whereas the excitation / optical gap is the difference in energy between the lowest dipole - allowed excited state and the ground state. in general, the true optical excitation energy is smaller than the fundamental gap due to excitonic effects that arise from the coulombic attraction between the excited electron and hole within the molecule (see figure 7 in ref (5) for a clear pictorial example of the fundamental gap and exciton binding energy). as discussed extensively by izmaylov and scuseria, semilocal functionals are incapable of accurately predicting excitonic effects whereas exchange - correlation kernels that include a portion of nonlocal long - range exact exchange (such as the lc - dft kernels used in this work) give rise to a nonzero exciton binding energy, resulting in an accurate prediction of the optical gap. in this study, we investigate the quasiparticle properties for guanine, adenine, cytosine, thymine, and uracil (molecular structures shown in figure 1), and we evaluate the performance of the nonempirically tuned lc - dft approach against both wave - function - based and conventional dft methods. finally, using the same nonempirical tuning procedure, we assess the accuracy of this approach and discuss the implications for predicting the excited - state energies and properties in these systems. over the past few years, the use of range - separated functionals for both dft and time - dependent dft (tddft) applications has significantly grown in popularity. in particular, we and other researchers have shown that these functionals show a dramatic improvement for strong charge - transfer systems and also surprisingly show improved accuracy even for relatively simple valence excitations. in contrast to conventional hybrid functionals which incorporate a constant fraction of hartree fock exchange, the range - separated formalism mixes exchange densities nonuniformly by partitioning the electron repulsion operator into short - range (first term) and long - range (second term) contributions as1the erf term denotes the standard error function, r12 is the interelectronic distance between electrons 1 and 2, and is the range - separation parameter in units of bohr. for a pure density functional (i.e., blyp or pbe) which does not already include a fraction of nonlocal hartree fock exchange, the exchange - correlation energy according to the lc formalism is2where ec, dft is the dft correlation functional, ex, dftsr is the short - range dft exchange functional, and ex, hflr is the hartree fock contribution to exchange computed with the long - range part of the coulomb operator. the modified (nonlocal) ex, hflr term can be analytically evaluated with gaussian basis functions, and the short - range ex, dftsr contribution is computed with a modified exchange kernel specific for each generalized gradient approximation. recently, baer, kronik, and others and srebro and autschbach have demonstrated that the range - separation parameter in these exchange - correlation kernels is highly system dependent but can be nonempirically tuned for a given system. this tuning process ensures that the negative of the homo energy is equal to the ionization potential (ip) of the n electron system, which is a fundamental condition within the kohn this condition would naturally be satisfied if the exact exchange - correlation functional were known ; as a result, tuning in a self - consistent manner to satisfy this fundamental constraint is both intuitive and theoretically justified. more rigorously, the ip of a given system can be determined from the difference between the ground - state energy of the n electron and the n 1 electron systems. this energy difference corresponds to the energy required to remove an electron from the system and, according to janak s theorem, should be equal to the negative of the homo energy. as mentioned, this condition can be satisfied (or approximately satisfied) with an optimal range - separation parameter that can be obtained by minimizing the following function:3the second term in this function takes into consideration the n + 1 system to indirectly tune the lumo of the n electron system. the lumo can not be directly incorporated in eq 3 as there is no formal equivalent of janak s theorem for the lumo ; that is, janak s theorem does not explicitly relate the electron affinity to the negative of the lumo. as we will see, the tuning of, besides being theoretically rigorous, significantly improves the ability to predict homo and lumo levels, fundamental gaps, and even excitation energies. in order to maintain a consistent comparison across previously published benchmark calculations, identical molecular geometries obtained from ref (1) were used for this work. optimal values were determined for guanine, adenine, cytosine, thymine, and uracil with the long - range corrected blyp functional (lc - blyp) using a polarized triple- basis set (cc - pvtz). we also investigated the effect of including diffuse functions using a larger aug - cc - pvtz basis but found that the use of larger or more diffuse basis sets did not significantly change our overall findings (see results and discussion section for further details). the optimal range - separation values were determined by varying from 0.05 0.9 in increments of 0.05 (increments of 0.01 were used from 0.2 0.4). in order to determine j, single - point calculations were carried out for the n and n 1 states for each value. figure 2 graphically illustrates j as a function of for the different nucleobases. the minimum (optimal) of each curve was obtained by spline interpolation, and these optimal values are reported in table 1. these optimal range - separation parameters were used for all subsequent lc - blyp calculations. all calculations were carried out with the gaussian 09 package using default scf convergence criteria (density matrix converged to at least 10) and the default dft integration grid (75 radial and 302 angular quadrature points). j (eq 3) as a function of for the different dna / rna nucleobases as determined using the lc - blyp functional and cc - pvtz basis. the inset shows a magnified view of j in the 0.2 2.0 ev). again, this example demonstrates that traditional / popular density functionals can fail significantly for predicting ionization energies (homo) ; however, significant improvements can result from the use of nonempirically tuned lc - dft functionals. ionization energies (ev) of the five nucleobases considered determined by the negative of the homo orbital at different levels of theory compared to the experimental range. finally, in addition to our detailed study of (ground - state) quasiparticle properties, we also analyzed the accuracy of nonempirically tuned range - separated methods for predicting optical excitation energies using time - dependent density functional theory (tddft). as benchmarks for assessing the quality of the various tddft methods, we calculated equation - of - motion couple - cluster (eom - ccsd) excitation energies with the same geometries and cc - pvtz basis set used previously in our tddft calculations. it is important to mention that the lowest excited states in these nucleobases are largely dominated by valence excitations and have been shown by the krylov group to be well - described with the cc - pvtz basis set. for both the tddft and eom - ccsd calculations, we also compute the oscillator strength since this provides another stringent benchmark test for assessing excited - state properties. table 3 compares both the lowest excitation energy and oscillator strengths between b3lyp, lc - blyp, and eom - ccsd for all five nucleobases. overall, we find that the nonempirically tuned lc - blyp results are in significantly better agreement with the eom - ccsd benchmark calculations in all cases. specifically, table 3 shows that the mae is reduced by more than half for both the lc - blyp excitation energies and oscillator strengths. in particular, we draw specific attention to the excitation energies of adenine, thymine, and uracil, which are all significantly underestimated (by almost 0.5 ev) using b3lyp. in order to understand the possible cause of these dramatic errors, we calculated tozer s lambda () diagnostic at the b3lyp / cc - pvtz level of theory for all five nucleobases. this test numerically quantifies the spatial overlap between the occupied and virtual orbitals involved in an excitation. by construction, the diagnostic metric is bounded between 0 and 1, with small values signifying a long - range excitation and large values indicating a localized, short - range transition. on the basis of their extensive benchmarks, if is less than 0.3, indicating little overlap and significant long - range charge transfer character, hybrid functionals are predicted to yield inaccurate results. as shown in the last column of table 3, none of the values are less than 0.3 ; however, the results for adenine, thymine, and uracil are nearly borderline cases with smaller diagnostic values (0.4). as a result, b3lyp exhibits larger errors for these particular systems when compared to our benchmark eom - ccsd results. in contrast, it is remarkable to note that the nonempirically tuned lc - blyp method demonstrates significant accuracy for these excited states even though they have only been tuned to satisfy kohn sham ground - state constraints (eq 3) without relying on any a priori knowledge of excited - state properties. the lambda () diagnostic values were determined at the b3lyp / cc - pvtz level of theory. in conclusion, we have shown that nonempirically tuned range - separated dft methods represent a significant improvement over traditional functionals for predicting both the quasiparticle properties (homo lumo fundamental gaps) and excitation energies in dna and rna nucleobases. we have demonstrated that the nonempirically tuned lc - blyp method accurately reproduces experimental ionization potentials for the various nucleobases considered ; in addition, our results demonstrate an excellent agreement with the more computationally intensive gw and caspt2 methods. furthermore, even though this nonempirical tuning procedure has been used to satisfy kohn sham ground - state constraints, we have shown that this methodology also leads to significantly improved excited - state energies and properties in nucleobases, compared to conventional dft methods. we believe that these types of functionals are at the forefront of density functional theory and will continue to grow in popularity because of their improved accuracy, computational efficiency, and theoretical rigorousness. | using a nonempirically tuned range - separated dft approach, we study both the quasiparticle properties (homo lumo fundamental gaps) and excitation energies of dna and rna nucleobases (adenine, thymine, cytosine, guanine, and uracil). our calculations demonstrate that a physically motivated, first - principles tuned dft approach accurately reproduces results from both experimental benchmarks and more computationally intensive techniques such as many - body gw theory. furthermore, in the same set of nucleobases, we show that the nonempirical range - separated procedure also leads to significantly improved results for excitation energies compared to conventional dft methods. the present results emphasize the importance of a nonempirically tuned range - separation approach for accurately predicting both fundamental and excitation gaps in dna and rna nucleobases. |
congenital lip pits are malformations that occur on the paramedial portion of the vermilion border of the lip. if it occurs along with cleft lip and palate, it is termed as vander woude 's syndrome. the other anomalies which may or may not be associated with syndrome are hypodontia, hypoplasia, ankyloglossia, high arched palate, limb anomalies, congenital heart defects, etc. most cases have been associated with a deletion of chromosome 1q32-q41, but an extra chromosomal locus at 1p34 has been identified. lip pits may result due to notching of the lip at an early stage of development with fixation of the tissues at the base of the notch or it may result from a failure of complete union of embryonic lateral sulci of the lip. the surface opening of the lip may present as a circular or transverse slit or be located at the apex of nipple - like elevations. its diameter may be upto 3 mm and its depth can range from 1 to 15 mm. the problem associated with lip pits is exudation of mucous on the lower labial skin which is a source of embarrassment for the patient. other than the lip pits, the other intraoral features should also be properly looked for and treated simultaneously. the cleft lip and palate patients have various dental anomalies like hypoplastic, malformed, supernumerary, congenitally missing, and peg - shaped teeth. it is also important to identify and treat these anomalies at an early stage thus improving the esthetics and function of the patient. a 7-year - old male child reported to the department of pedodontics and preventive dentistry with a chief complaint of carious teeth. natal history revealed that he was delivered through a caesarean section. maternal history relieved no medications or illness during pregnancy., it was seen that the patient was a treated case of unilateral cleft lip and palate, and two lip pits were present on the lower lip [figures 1 and 2 ]. when the lip was compressed there was mucous secretion. intraoral examination revealed carious teeth with 16, 26, 36, and 46 and root stumps with 55, 64, and 65 [figure 3 ]. extraoral examination of the patient lip pits present on the lower lip intraoral examination of the patient (preoperative) : maxillary arch intraoral examination of the patient : anterior bite radiographic examination of the patient : opg the treatment planned was restoration of carious teeth followed by extraction of root stumps [figure 6 ]. the patient was also advised to undergo orthodontic treatment and a cosmetic lip correction for the lip pits. it is important to diagnose these syndromes from oro facial digital syndrome and popliteal syndrome which manifests similar clinical features. these entities are allelic variants of the same condition. the other features which help to differentiate are as follows : orofacial digital syndrome shows cleft lobular tongue, digital malformation and mental retardation, whereas popliteal syndrome shows syndactly, digital reduction, and syngathia. because the symptoms of these children are limited and because the affected individuals have normal intelligence, this disorder should not be confused with non - syndromic cleft lip and palate. the treatment of such cases should be surgical excision of the labial and commissural pits if esthetics is a major concern. it should include total removal of the minor salivary glands that exude secretions at the base of pits to prevent formations of cysts and mucoceles. the treatment should be carried out in collaboration with plastic surgery, oral maxillofacial surgeons, and orthodontists. other treatments like cross bite corrections, maxillary expansions, restorations, and extractions should also be carried out. a multidisciplinary approach is very necessary to carry out the treatment thus improving the self - esteem of the patient at an early age. an early and proper diagnosis followed by a multidisciplinary approach is necessary to improve the self - esteem of the patient. | one of the most common developmental defects seen in south india is cleft lip and palate. among them a few are associated with lip pits and termed as vander woude 's syndrome. the early diagnosis of this rare syndrome is very necessary followed by a multidisciplinary approach. it is also necessary to differentiate this syndrome from the other syndromes which may present similar features. a case report of the same is presented here requiring a multidisciplinary approach for a functional and esthetically pleasing outcome. |
prostate cancer (pca), the most frequently diagnosed malignancy, has become the second leading cause of cancer - related deaths among men in western countries [1, 2 ]. endocrine therapies which aimed at inhibiting the androgen receptor (ar) function was the mainstay of treatment for advanced prostate cancer based on that the androgen signaling will promote the proliferation of prostate cancer cell. unfortunately, most of treated patients progressed toward castration - resistant prostate cancer (crpc) from castration - dependent prostate cancer. and crpc characterized by aggressive growth and ability to colonize distal organs, which made crpc still incurable and the median survival time for patients with crpc was only 12 months. the status of ar was highly predictive of prostate cancer patients that will benefit from endocrine therapy but was not correlated with a better clinical outcome [4, 5 ]. prostate - specific antigen (psa) is a protein produced by the prostate gland cells. although many published studies have assessed the performance of candidate biomarkers in predicting time to relapse of prostate cancer following radical prostatectomy [6, 7 ], no molecular markers suitable for routine clinical practice that can identify those prostate cancer patients with a high risk of early clinical progression or prostate cancer - specific mortality have been found. g - proteins are composed of,, and subunits and subunit are classified into 4 families : gs, gi / o, gq/11, and g12/13. each of them has multiple members with different expression specificity [8, 9 ]. although gs is the most extensively characterized and clinically relevant, literature is not unanimous on the role of gs in different types of cancers. in lung cancer, choi. found that gs could augment cisplatin - induced apoptosis of lung cancer cells through upregulating bak expression by increasing transcription and by decreasing the rate of protein degradation and the efficacy of radiotherapy of lung cancer may be improved by modulating gs signaling pathway. but in cervical cancer or intrahepatic cholangiocarcinoma (icc), the situation was just opposite. cho. found gs inhibited cisplatin - induced apoptosis by increasing transcription of x - linked inhibitor of apoptosis protein (xiap) and by decreasing degradation of xiap protein in hela cervical cancer cells. in icc, schmitz. also found a significant association of both unfavorable disease - specific overall survival and recurrence - free survival with the homozygous tt genotype of the gnas1 gene which encoded gs protein [13, 14 ] however, they also reported that t393c polymorphism in the gene gnas1 was significantly associated with favorable clinical outcome of patients suffering from bladder cancer, chronic lymphocytic leukemia, and renal cell carcinoma. there were studies reported no association was found between gnas t393 t genotype and prostate cancer [17, 18 ]. but liu. identified that membrane caveolae - associated gas was involved in androgen receptor (ar) transactivation by modulating the activities of different pi3k isoforms. more importantly, it had been reported that the expression of gs and gi decreased 30% to 40% after neoplastic transformation. and the levels of gs and gi subunits correlated inversely with serum prostate specific antigen in patients with prostate cancer, which indicated an important regulatory role of gs and gi for cell proliferation and neoplastic transformation in human prostate cancer and they may have prognostic value. therefore, more in - depth investigations are necessary to address this controversy and identify the role of gs in prostate cancer. thus, we assessed the potential of gs as a prognostic marker by determining the level of gs protein expression in a series of 347 postradical prostatectomy prostate cancer tissue microarrays (tma) which include 56 metastatic pcas and 291 localized pcas and 67 benign prostatic hyperplasia (bph) as controls using immunohistochemistry (ihc). in the present study, we found that expression of gs protein was decreased in high grade and metastatic pcas. and low gs protein levels were strongly associated with adverse clinicopathologic features and poor clinical outcomes in metastatic and localized pca patients. multivariate cox regression analysis showed that low expression of gs was an independent predictor of prostate cancer recurrence and cancer - specific death in metastatic and localized pca. to the best of our knowledge, this is the first study to identify the independent predictive role of gs in patient with prostate cancer. in order to study gs expression in prostate cancer by immunohistochemistry, a total of 347 formalin - fixed, paraffin - embedded prostate tissues between 1994 and 1997 were retrieved from the archives of the first affiliated hospital, college of medicine, and xi'an jiao tong university, and a tissue microarray (tma) was constructed. in addition, 67 benign prostatic hyperplasia (bph) samples were collected as control. this research project was approved by the ethical committee of the xi'an jiao tong university, and all the patients had been given their fully written informed consent. data were collected on patients with disease baseline and clinicopathologic characteristics as well as 2 treatment outcomes : time to progression and prostate cancer - specific mortality (pcsm). prostate cancers were graded based on the gleason system by 2 independent pathologists at the first affiliated hospital, college of medicine, and xi'an jiao tong university in a blind and consecutive manner to ensure adequate diagnosis and grade. the tnm staging system was used to describe the extent of prostate cancer in patients (based on the ajcc cancer staging manual, seventh edition, 2010, springer, new york, inc.). briefly, slides were deparaffinized in xylene and rehydrated in a graded alcohol series before endogenous peroxidase activity was blocked with 3% h2o2 in methanol. after nonspecific protein binding was blocked, the primary antibody diluted into recommended concentration for gs, which was purchased from abcam (ab58810), was applied overnight in a humidity chamber at 4c. biotinylated secondary antibody was applied for 30 min at room temperature after washing with pbs for 3 times. negative control was conducted by replacing the primary antibody with preimmune rabbit serum. to evaluate gs expression, we used the immunoreactive score (irs) as previously implemented by tischler. the intensity of staining was arbitrarily graded as absent (0), weak (1 +), moderate (2 +), and strong (3 +). the percentage of stained cells was use d to quantify the react ion as negative (0% of positive cells), 1 + (80% of positive cells). the final value of the analysis of each tissue sample was then expressed as an absolute value through the obtained score by multiplying the two individual scores (i.e., intensity of staining score times the percentage of stained cells score) then generates a final score ranging from (no expression) to + (weak expression), + + (moderate expression), or + + + (strong expression). and we identified and + as negative for gs expression and + + and + + + as positive gs expression. examples of scoring according to staining intensity and the percentage of stained cells are shown in figure 1. spss version 13.0 (spss, chicago, il, usa) was used for statistical analyses. psa progression - free and overall survival curves were constructed by the kaplan - meier method and compared using the log - rank test. to evaluate the role of prognostic variables, a series of cox proportional hazards models since psa was a continuous estimate, with the median psa level for the entire cohort of patients (n = 347) as 34.9 ng / ml, we divided the cohort into those with psa levels 35 ng / ml and > 35 ng / ml. the following parameters were included : psa levels (35 ng / ml, > 35 ng / ml) ; extraprostatic extension (yes, no) ; involvement of surgical margins (no, yes) ; involvement of seminal vesicles (no, yes) ; involvement of pelvic nodes (n0, n+) ; gleason scores (26, 7, 810). 145 patients (41.8%) presented gleason score of 26, 127 (36.6%) patients presented gleason score of 7, and the remaining 75 cases (21.6%) presented gleason score between 8 and 10. 49 patients (11.5%) presented tnm stage i ; 125 (36.0%) patients presented stage ii ; 117 (33.7%) presented stage iii ; and 56 (16.1%) patients presented tnm stage iv. psa progression was observed in 229 (66.0%) patients at a median interval of 123.5 month (range 7167). other clinicopathological features are summarized in table 2. moreover, prostate cancer patients who had higher gleason scores (p < 0.001 and p < 0.001, resp.), higher tnm stages (p < 0.001 and p < 0.001, resp.), higher preoperative psa level (p < 0.001 and p < 0.001, resp.), positive surgical margin (p = 0.009 and p < 0.001, resp.), angiolymphatic invasion (p = 0.004 and p = 0.032, resp.), extraprostatic extension (p = 0.031 and p < 0.001, resp.), and seminal vesicle invasion (p = 0.046 and p = 0.007, resp.) present shorter overall survival and psa progression - free survival (tables 5 and 6). psa progression and overall survival time correlated with tnm stage, gleason score, extraprostatic extension, positive surgical margins, and seminal vesicle invasion demonstrate the representability of study group. the number of patients with positive lymph node involvement (n = 34) was too small to find any significant correlation with psa progression - free survival and overall survival. to determine the prevalence and clinical significance of gs in prostate cancer tissues, we determined the expression of gs protein by immunohistochemistry in a retrospective cohort of 347 tumor tissue samples from prostate cancer patients and 67 samples from patients who were diagnosed with benign prostatic hyperplasia (bph) after tumor resection. among the 347 patients, 114 patients had not expression of gs () ; 73 patients were weak expression (+) ; 86 patients were moderate expression (+ +), and 74 patients were strong expression (+ + +) (as shown in figure 1). thus, as we described in the methods section, there were 160 (46.1%) samples positive for gs expression and 187 (53.9%) samples negative for gs expression in our pca cohort. we also found that positive ratio of gs expression was downregulated in metastatic pca compared to localized pca and bph (p = 0.012 and p < 0.001, respectively, as shown in table 1). in patients with bph, there were 42 (62.7%) samples positive for gs expression and 25 (37.3%) samples negative for gs expression. in the patients with localized pca, there were 151 (51.2%) samples positive for gs expression and 140 (48.1%) samples negative for gs expression. whereas in the patients with metastatic pca, there were 9 (16.1%) samples positive for gs expression and 47 (83.9%) samples negative for gs expression. ihc staining for gs was sharp and reliable, no background or nonspecific staining was observed. then, we further evaluated the relationship between gs expression and clinical features of prostate cancer patients by pearson chi - square test or fisher 's exact test. we found that the positive ratio of gs expression was decreased as the level of gleason score or preoperative psa increased. these result showed that there was inverse correlations between gs expression and preoperative psa and gleason score and tnm stage at diagnosis (p = 0.030, p < 0.001, and p < 0.001, respectively, table 2). but we found no specific correlation between gs expression and the rest of pathological parameters (age ; angiolymphatic invasion ; extraprostatic extension ; positive margin ; seminal vesicle invasion ; positive lymph node) that we evaluate in the present analysis. in the localized pca specimens, the expression of gs was correlated with preoperative psa level, gleason score, and tnm stage (p = 0.028, p = 0.016, and p = 0.011, respectively, table 3). however, in metastatic pca specimens, the expression of gs was only associated with preoperative psa level and gleason score much more significantly (p < 0.001, and p < 0.001, resp., table 4). in our retrospective cohort with 347 patients, we got the detailed follow - up information of 15 years. at the time of our analysis, 121 patients died and 256 patients progressed. median time to psa progression for the whole cohort was 123.5 months (range 7167 months), while the median time to death was 123.5 months (range 3179 months). we found that patients with negative expression of gs had a higher ratio of psa progression than those with positive gs expression (table 2). more importantly, negative gs expression was associated with psa progression - free survival and overall survival. the group of patients with negative expression of gs showed significantly shorter overall survival than patients with positive expression of gs (p = 0.001, figure 2(a)). these patients also showed a trend for shorter psa - free survival time (p < 0.001, figure 2(d)). in localized pca specimens, negative gs expression was also associated with better psa progression - free and overall survival rate (p < 0.001, figure 2(c) ; p < 0.001, figure 2(f)). in metastatic pca specimens, a similar trend was found between negative gs expression and psa progression - free / overall survival time (p = 0.0003, figure 2(e) ; p = 0.0146, figure 2(b)). as expected, at the univariate level, gleason scores, tnm stages, preoperative psa, positive margin, angiolymphatic invasion, extraprostatic extension, and seminal vesicle invasion were associated with psa progression - free and overall survival. negative expression of gs protein was a prognostic predictor of psa progression - free and overall survival in pca patients at univariate level (tables 5 and 6). we further conducted a multivariate cox regression analysis to assess whether gs was a prognostic predictor of survival independent of age, gleason scores, tnm stages, preoperative psa, positive margin, angiolymphatic invasion, extraprostatic extension, seminal vesicle invasion, and positive lymph node. multivariate analysis showed that negative gs expression was a strong independent predictor of outcome providing survival information (both psa progression - free or overall survival) above other independent prognostic features (tnm stage, gleason score), with a hazard ratio of 4.328 and 3.904 and a 95% confidence interval of 1.8768.432, p < 0.001 (negative gs group versus positive gs group) and 1.2785.873, p < 0.001 (negative gs group versus positive gs group). in our cohorts, psa, positive margin, angiolymphatic invasion, extraprostatic extension, and seminal vesicle invasion it has been reported that the expression of gs correlated inversely with serum prostate specific antigen in patients with prostate cancer and the expression of gs decreased 30% to 40% after neoplastic transformation. but there was no study concerning the role of gs protein in the prognosis of prostate cancer patients. in the present study, we characterized the expression pattern of gs protein in a large number of tissues derived from prostate cancer patients, consisting of localized and metastatic pca, and assessed the utility of gs as a prognostic marker in these patients. in agreement with previous reports, we confirmed that gs expression was localized in nuclear and cytoplasm in neoplastic cells. moreover, we found that expression of gs was downregulated in metastatic pca compared to localized pca and bph. and gs was inversely associated with psa level and gleason scores both in localized and metastatic pca. at the univariate level, gs, gleason scores, tnm stages, preoperative psa level, positive margin, angiolymphatic invasion, extraprostatic extension, and seminal vesicle invasion but in multivariable cox regression analysis, only high gs expression and gleason scores were independent predictors of both psa progression - free and overall survival. these findings support the potential clinical utility of incorporating gs into clinical nomograms to help determine the risk of psa progression and death. the prognostic significance of gs as biomarker for prostate cancer is likely to its biological functions. gs is a member of gtp - binding protein superfamiliy and could independently regulate a variety of effectors including adenylate cyclases, phospholipase c, and ion channels [22, 23 ]. however, gs and t393c polymorphism in the gene gnas1 which is encoded by gs plays distinct roles in different cancers. gs could augment cisplatin - induced apoptosis of lung cancer cells, but it inhibited cisplatin - induced apoptosis in cervical cancer or intrahepatic cholangiocarcinoma (icc). t393c polymorphism in the gene gnas1 was significantly associated with favorable clinical outcome of patients suffering from bladder cancer, chronic lymphocytic leukemia, and renal cell carcinoma, and significantly associated with unfavorable clinical outcome of patients suffering from icc and breast cancer [13, 14 ]. in prostate cancer, previous study reported that low expression level of gs was found in t2 stage pca compared to high levels in normal controls. more importantly, the expression of gs was found downregulated in hormone refractory c4 - 2b and pc3 cell lines compared to hormone sensitive lncap and rwpe-1 cell lines. all these studies indicating the functionality and expression of gs are selectively modified in human prostate adenocarcinoma and downregulated gs levels may play an important regulatory role for cell proliferation and neoplastic transformation in prostate cancer. thus, we did our efforts to investigate and validate whether gs functioned as an efficient prognostic biomarker to predict the outcome of pca patients. consistent with previous studies, we found that the expression of gs was much lower in metastatic pca than it is in localized pca. furthermore, the patients who had the low expression of gs tend to have shorter progression - free survival and overall survival time, nonetheless, metastatic or localized pca. more importantly, multivariable cox regression analysis proved that gs was an independent predictor of prognosis in prostate cancer. though the answer to the discrepancy of gs function in different cancers was still unclear, and the role of gs in prostate cancer was also in dispute, our results were relatively easy to understand for gs had a close relationship with egfr. egfr belongs to erbb oncogene family which also includes erbb-2, 3, and 4 and is comprehensively expressed in epithelial cells including prostate cancer cells. egfr are known to regulate cell proliferation, differentiation, angiogenesis, and survival. in prostate cancer, it has been reported that egfr was highly expressed in du145 and pc3 cell lines which were hormone - independent human prostate cancer cell lines and responsive to egf stimulation [2527 ]. it was also found that prostate cancer bone metastases express significantly higher level of egfr. more importantly, egfr expression increased as prostate cancer progressed from an androgen - dependent to an androgen - independent stage. di lorenzo., found 41%, 76%, and 100% egfr expression in radical prostate ectomy hormone - sensitive and hormone - refractory metastatic patients in a cohort consisting of 76 patients with androgen - dependent and androgen - independent prostate cancer, respectively. many studies confirmed that overexpression of egfr contributes significantly to the progression of prostate cancer [3134 ]. zheng. reported that overexpression of the stimulatory gs promotes ligand - dependent degradation of epidermal growth factor (egf) receptors and texas red egf, and knock - down of gs expression by rna interference (rnai) delays receptor degradation. recently, they demonstrated that egf - induced, proliferative signaling occurs from eea1 endosomes and was regulated by the gs through interaction with the signal transducing protein giv (also known as girdin). when gs or giv was depleted, activated egfr and its adaptors accumulate in eea1 endosomes. then egfr signaling was prolonged and egfr downregulation was delayed, which made cell proliferation greatly enhanced. basing on our finding that gs was downregulated in advanced pca and the previous studies concerning the function of egfr in pca, we hypothesise that downexpression of gs inhibit the degradation of egfr, then androgen receptors which are activated by egfr were prolonged and cell proliferation increased, eventually causing tumor progression and hormone - resistant in prostate cancer. that maybe the underlying mechanism account for our results in which the expression of gs was downregulated in metastatic pca and inversely associated with psa level and gleason scores. but experiments would be desirable to further clarify the relationship between gs and egfr and identify their function in the progression of prostate cancer. however, such functional studies were beyond the scope of this study. in summary, we discovered that gs is a promising biomarker of prostate cancer patients. to our knowledge, this is the first study to describe the predictive role of gs in prostate cancer. we found that the expression of gs was downregulated in metastatic prostate cancer compared to localized prostate cancer. and although our results are promising, gs expression needs to be validated in relationship to outcome in the context carefully controlled clinical trials. all in all, targeting gs could be a promising therapeutic strategy for enhancing the therapy effect of patients. | background. t393c polymorphism in the gene gnas1, which encodes the g - protein alpha s subunit (gs) of heterotrimeric g protein, is significantly associated with the clinical outcome of patients suffering from several cancers. however, studies on the role and protein expression of gs subunit in prostate cancer were still unavailable. methods. the immunohistochemical staining was used to assess gs expression through tissue microarray procedure of 56 metastatic pcas, 291 localized pcas, and 67 benign hyperplasia (bph). gs expression was semiquantitatively scored and evaluated the correlation with pathologic parameters and biochemical recurrence of prostate - specific antigen (psa). results. gs expression was localized in nuclear and cytoplasm in prostate cancer cells and downregulated in metastatic pca compared to localized pca and bph (p < 0.001). gs was inversely associated with psa level and gleason scores ; patients with low expression of gs had adverse clincopathological features. in multivariable cox regression analysis, high gs expression and gleason scores were independent predictors of both psa progression - free and overall survival. conclusions. gs down - expression is associated with adverse pathologic features and clinical psa biochemical recurrence of prostate cancer. gs is an independent predictor to help determine the risk of psa progression and death. |
ultra - high magnification endoscopy, also known as endocytoscopy (ec) or endomicroscopy, enables assessment of the muscularis propria (mp) or myenteric plexus in vivo through a submucosal tunnel 1 2 3. this technique is expected to be applicable for clinical use for the treatment of neuromuscular diseases because the nidus of such diseases is under the mucosa, a location that is difficult to assess using conventional endoscopy. achalasia is an idiopathic esophageal motility disorder characterized by a lack of peristalsis in the esophageal body and absent or incomplete relaxation of the lower esophageal sphincter (les) 4 5. the novel procedure, peroral endoscopic myotomy (poem), has become one of the best treatment options for achalasia because it is safe, offers long - lasting symptom control, and is less invasive than surgery 6 7 8. moreover, the submucosal tunnel created during poem allows insertion of the endoscope under the mucosa and access to the mp. during poem, thick muscle at the middle to lower esophagus, as well as thin muscle at the les area, can be identified (fig. 1), although there are variations among patients with achalasia 9 10. during peroral endoscopic myotomy, thick muscle is observed at the mid- to lower- esophagus (a), whereas thin muscle is observed at the les (b). the histopathology of achalasia has been insufficiently investigated because of the low prevalence of this condition, and because the mainstream treatment for achalasia has been balloon dilatation, which does not allow tissue sampling. surgical myotomy may allow access to the mp at the les but, because of the limited treatment window (difficult to pull the mid - esophagus into the abdominal cavity), using surgical myotomy to assess the mid - esophagus is technically difficult. the approach to and examination of the esophageal mp was difficult until the introduction of poem. the purpose of the current study, therefore, is to determine the feasibility of ec for real - time histopathologic examination of the mp in a clinical setting, and to provide more knowledge about the histopathologic features of esophageal tissues features of esophageal tissues in achalasia. this study was performed at showa university northern yokohama hospital, a tertiary referral center in japan. enrollment began in december 2013 and patients with consecutive achalasia who were candidates for poem were recruited for this study. patients younger than 18 years and those whose health was not favorable for enrollment because of severe comorbidity in organs such as the heart or lungs were excluded from this study. in instances where full - thickness myotomy was performed, ec examination was not performed, and these patients were also excluded from the analysis. the current study was approved by the institutional review board (no. 1311 - 05). written informed consent was obtained from all participants, and the study was conducted according to the declaration of helsinki., esophageal contents were cleared by endoscopic suction using a 3.7-mm channel endoscope (gif-1t240 ; olympus co., tokyo, japan) under intravenous anesthesia to prevent aspiration caused by intubation. under general anesthesia with positive pressure ventilation, after submucosal injection at the level of the mid- esophagus, a 2-cm longitudinal mucosal incision was created as the point of entry. a one - third circumferential submucosal tunnel was created from entry down to the les and into approximately 2 3 cm of the gastric side. dissection of the circular muscle (from 2 cm distal to the mucosal entry) was performed at the center of the submucosal tunnel. after myotomy, a totally relaxed c (egj) was confirmed by inserting an endoscope into the natural lumen down to the gastric side, and by a retroflex view from the gastric side to egj (fig. 2). the mucosal entry was closed with hemostatic clips. as compared to pre - poem (a), a totally relaxed esophagogastric junction is seen on retroflex view from the gastric side with poem (b). all ec examinations were performed using the integrated - type endocytoscope gif - y0002 (prototypes from olympus co.). gif - y0002 has one lens that can consecutively increase the magnification from the conventional endoscopy level to 380 magnification (tissue field of view, 700 600 m) using a hand lever. the gif - y0002 allows gradual magnification at the center of the monitor, thus ensuring accurate localization of the area being viewed. for ec, a mixture of 0.1 % methylene blue was used to stain the tissues and to obtain images similar to those obtained in conventional pathology examination. ec examination was performed after poem and prior to closure of the mucosal entry site. after staining with 2 ml of methylene blue, the ec examination was started from the proximal 2 cm of circular muscle through remnant circular muscle into the gastric side. two biopsy specimens were obtained from the proximal circular muscle and at the les area (high - pressure zone) for histopathological assessment as confirmation of in vivo diagnosis. ec findings were assessed according to the previous in vivo report and histopathology reports 1 11 12. ec findings of the mp were classified as : atrophy, normal, or hypertrophy. this study was performed at showa university northern yokohama hospital, a tertiary referral center in japan. enrollment began in december 2013 and patients with consecutive achalasia who were candidates for poem were recruited for this study. patients younger than 18 years and those whose health was not favorable for enrollment because of severe comorbidity in organs such as the heart or lungs were excluded from this study. in instances where full - thickness myotomy was performed, ec examination was not performed, and these patients were also excluded from the analysis. the current study was approved by the institutional review board (no. 1311 - 05). written informed consent was obtained from all participants, and the study was conducted according to the declaration of helsinki. this study was performed at showa university northern yokohama hospital, a tertiary referral center in japan. enrollment began in december 2013 and patients with consecutive achalasia who were candidates for poem were recruited for this study. patients younger than 18 years and those whose health was not favorable for enrollment because of severe comorbidity in organs such as the heart or lungs were excluded from this study. in instances where full - thickness myotomy was performed, ec examination was not performed, and these patients were also excluded from the analysis. the current study was approved by the institutional review board (no. 1311 - 05). written informed consent was obtained from all participants, and the study was conducted according to the declaration of helsinki. esophageal contents were cleared by endoscopic suction using a 3.7-mm channel endoscope (gif-1t240 ; olympus co., tokyo, japan) under intravenous anesthesia to prevent aspiration caused by intubation. under general anesthesia with positive pressure ventilation, after submucosal injection at the level of the mid- esophagus, a 2-cm longitudinal mucosal incision was created as the point of entry. a one - third circumferential submucosal tunnel was created from entry down to the les and into approximately 2 3 cm of the gastric side. dissection of the circular muscle (from 2 cm distal to the mucosal entry) was performed at the center of the submucosal tunnel. after myotomy, a totally relaxed c (egj) was confirmed by inserting an endoscope into the natural lumen down to the gastric side, and by a retroflex view from the gastric side to egj (fig. 2). the mucosal entry was closed with hemostatic clips. as compared to pre - poem (a), a totally relaxed esophagogastric junction is seen on retroflex view from the gastric side with poem (b). all ec examinations were performed using the integrated - type endocytoscope gif - y0002 (prototypes from olympus co.). gif - y0002 has one lens that can consecutively increase the magnification from the conventional endoscopy level to 380 magnification (tissue field of view, 700 600 m) using a hand lever. the gif - y0002 allows gradual magnification at the center of the monitor, thus ensuring accurate localization of the area being viewed. for ec, a mixture of 0.1 % methylene blue was used to stain the tissues and to obtain images similar to those obtained in conventional pathology examination. ec examination was performed after poem and prior to closure of the mucosal entry site. after staining with 2 ml of methylene blue, the ec examination was started from the proximal 2 cm of circular muscle through remnant circular muscle into the gastric side. two biopsy specimens were obtained from the proximal circular muscle and at the les area (high - pressure zone) for histopathological assessment as confirmation of in vivo diagnosis. ec findings were assessed according to the previous in vivo report and histopathology reports 1 11 12. ec findings of the mp were classified as : atrophy, normal, or hypertrophy. seven patients with achalasia who underwent poem were enrolled in this study and underwent concomitant ec examination. the mean (sd) age of the participants was 35.0 18.1 years, and the study population consisted of four male and three female patients. the duration of achalasia symptoms was 5.5 7.5 years, and ranged from 3 to 20 years. according to chicago classification criteria 13 14, manometric findings were classified as type i in one patient and type ii in four patients ; in two patients, manometry was not completed because of difficulty in inserting the catheter through the les. the total length of the endoscopic myotomy was 12 2.2 cm with a mean of 10 2.0 cm in the esophagus and 3.0 0.69 cm in the stomach (table 1). nothing particular manometry : manometric findings according to chicago classification criteria 13 14 : manometry was not completed endocytoscopy (ec) images showed no evidence of atrophy or hypertrophy, which was consistent with the diagnosis established by histopathology. favorable ec images were obtained, and spindle - shaped smooth muscle cells were observed. a strong staining nucleus, surrounding cell bodies, and vessels in the muscular layer were visualized. however, none of these structures showed particularly notable changes, that is, non - atrophy and non - hypertrophy of the mp (fig. 4). in other words, changing patterns such as atrophy or hypertrophy were not identified on the biopsy specimens. on postoperative day 2, a liquid diet was started, and on day 4, all the patients were discharged on a normal diet. in vivo images of the muscularis propria of circular and longitudinal muscle in achalasia obtained by endocytoscopy. spindle - shaped smooth muscle cells, including the nucleus and cell bodies, are clearly identified. conventional microscopic analysis of the muscularis propria shows the smooth muscle layer from one of the achalasia patients with well - preserved spindle - shaped, smooth muscle cells. to our knowledge, this is the first study to report in vivo histopathology of the mp in patients with achalasia. first, ec can be used for in vivo real - time assessment not only for intestinal mucosa but also for mp via a submucosal tunnel created after poem. in poem for achalasia, real - time histopathological assessment of mp may be helpful to identify the morbid lesion preoperatively, although no atrophy or hypertrophy was identified in our small cohort. the intermittent and time - consuming biopsy method, which also includes adverse event risks such as bleeding, can not substitute for the continuous and real - time ec assessment. furthermore, if ec can be performed by resecting the mucosal / submucosal layer using endoscopic mucosal resection / endoscopic submucosal dissection, ec is potentially effective for the diagnosis of other disorders with a nidus located in the mp. second, the endocytoscopic morphology of the mp showed no evidence of hypertrophy or atrophy of smooth muscle cells despite a thick or thin appearance on a conventional endoscopic view, and the ec findings were confirmed by histology. this result suggests that the thick or thin appearance of mp in achalasia is caused by the changing histopathology rather than hypertrophy or atrophy, or that such change requires long - term symptoms. previous studies have examined muscle biopsies from patients with achalasia treated at an earlier stage of disease. these studies have detected intact mp in these patients who had a shorter duration of symptoms and a non - dilated esophagus 15 16 17, findings that are almost consistent with our results. first, the staining technique used in this series allowed the evaluation of cell structure, cytoplasm, and nuclei alone. the nerve plexuses were not visualized in this setting because of a lack of neuron - specific fluorescent stain available for safe use in humans. development of stains and methods to evaluate stain toxicity and long - term effects are essential. in the current study, the nerve plexus was not identified by biopsy, probably because the tissue sampling should be nonconsecutive, and with a small specimen. in addition, achalasia and the other esophageal motility disorders have multidimensional etiology and pathogenesis. the most familiar histomorphologic change is a loss of ganglion cells in the auerbach plexus of smooth muscle ; however, controversy exists regarding the trigger, extent, and degree of the change 10 18 19. further studies involving a larger series, particularly including participants with a longer duration of symptoms, is warranted. however, the application of this technique should remain limited and performed only in select centers so that poem and ec can be performed safely. in conclusion, in vivo real - time assessment of the mp is possible using ec | background : the histopathology of the muscularis propria (mp) is unknown in patients with achalasia. endocytoscopy (ec) was developed as an ultra - high magnification endoscopy, and the submucosal tunnel created during peroral endoscopic myotomy (poem) not only provides access to the mp but also enables subsequent endoscopic assessment of the mp. patients and methods : in seven patients with achalasia (mean sd ; 35 18.1 years ; men : women, 4:3) who underwent poem (myotomy length : 12 2.2 cm), subsequent ec examination was performed from the mid - esophagus to the gastric side. ec images were compared to the results of histopathologic examination (two biopsies from the mid - esophagus and lower esophageal sphincter), which was the standard. results : in all patients, favorable ec images were obtained, and spindle - shaped smooth muscle cells were detected. in our series, we observed no notable features such as atrophy or hypertrophy of smooth muscle cells. in addition, the ec assessment was consistent with the results of biopsy. no complications were encountered during any of the procedures. conclusion : in a clinical setting, real - time assessment of the mp using ec is feasible. this technique may play an important role in determining the pathology of achalasia and other diseases that affect gastrointestinal function. |
cardiovascular diseases (cvds) are the leading cause of death worldwide composed of heart and blood vessel diseases. in the recent years, the incidence of cvds has been increasing at a sharp rate globally. according to the world health report 2010, cvds contributed to 17.5 million deaths and these numbers are estimated to increase to 23.3 million by 2030 [1, 2 ]. fibroblast growth factor (fgf) is a cytokine superfamily with pleiotropic biological functions including regulating cell growth, differentiation, development, and metabolism [37 ]. human fgfs contain 22 members which can be divided into 7 subfamilies based on phylogeny and sequence [810 ]. due to the lack of a heparin binding domain, fgf19 subfamily members (fgf19, fgf21, and fgf23) function in an endocrine manner rather than an autocrine manner as other subfamily members of fgfs. among them, fgf21 is a polypeptide with 209/210 (human / rodent) amino acid residues that is primarily produced and secreted by the liver, adipose tissue, and thymus. fgf21 expression is mainly regulated by peroxisome proliferator - activated receptor (ppar) in the liver and ppar in adipocytes [13, 14 ]. fgf21 was firstly cloned in 2000 and received global attention in recent years due to its outstanding ability on regulating carbohydrate and lipid metabolism including improving insulin sensitivity, lowering blood glucose, reducing hepatic / plasma triglycerides, inducing weight loss by increasing energy expenditure, and reducing fat mass [1518 ]. further studies indicated that fgf21 functions by binding to (fgfr)1c and (fgfr)2c in the presence of coreceptor -klotho and activation of downstream signaling pathway [19, 20 ]. although fgf21 and other members of fgfs share the same fgf receptors, the coreceptors are different (-klotho for fgf21 and heparin for others) which determined that they have different bioactivity due to activation of various pathways [21, 22 ]. the possible explanation is that fgf21 induces physiological role in healthy condition and pharmacological role under unhealthy condition [23, 24 ]. additionally, fgf21 does not lead to carcinogenic event due to lack of mitogenic function which makes it possible to be administrated in vivo in clinics. therefore, fgf21 may hold promise as a clinically therapeutic option due to the abovementioned characters and advantages. in recent clinical and preclinical studies, cvds have been closely associated with serum fgf21 which increased in the patients with atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy [2527 ]. therefore fgf21 has the potential to be considered as a biomarker for the above cvds. whether the increased serum fgf21 level is the basis for cvd pathogenesis or is induced to protect the heart form cvds is still under discussion. however, growing evidence indicated that administration of exogenous fgf21 induces preventive effects on most of the above cvds, suggesting that fgf21 not only is a simple marker of cardiovascular risk but also induces a protective effect on the cardiovascular system contributing to a reduction in risk (table 1). in clinics, serum fgf21 levels were increased in patients with obesity or type 2 diabetes which was associated with high risk of cvds. the paradoxical phenomenon was supposed to be explained by a compensatory response to induce cardiac protection or resistance to fgf21 which impaired its bioactivity [28, 29 ]. in animal study, we found that at the early - stage of diabetes serum fgf21 level of mice was sharply increased compared with nondiabetic mice (c57bl/6j), while it was dramatically decreased at the late - stage of diabetes which further confirmed that early - stage increase of serum fgf21 was a compensatory response and induced beneficial effect on the heart ; late - stage decrease may be the cause of diabetes - induced cardiac damage, since the above cvds are always attributed to lipid metabolic disorder. mechanistic studies indicated that fgf21-induced cardiac protection in cvds is possibly attributed to the suppression of lipotoxicity since the above cvds are always the consequences of lipotoxicity. this review tries to illuminate the underlying relationship between fgf21 and cvds and the possible mechanisms. atherosclerosis is a chronic, inflammatory disorder characterized by the deposition of excess lipids in the arterial intima. the accrued evidence indicated that lipid - lowering therapy limits the progression of atherosclerosis and reduces cad events. since fgf21 plays an important role in the regulation of lipid metabolism, the effect of fgf21 in atherosclerosis is of interest. clinical studies showed that increased circulating fgf21 levels were discovered in atherosclerotic patients or the individuals with high risk of developing atherosclerosis [33, 34 ]. additionally, an in vivo study demonstrated that increased serum fgf21 was observed in aortas of apoe mice (c57bl/6j background). strong evidence identified that administration of exogenous fgf21 significantly improved lipid metabolic disorders and reduced atherosclerotic plaque areas in these animals. moreover, lin. also reported that fgf21 deficiency enhanced atherosclerotic deterioration and mortality in apoe mice (c57bl/6j background), implying that increased serum fgf21 in patients with atherosclerosis described previously induces beneficial effect rather than the basic for atherosclerotic pathogenesis. mechanistic study indicated that fgf21-induced prevention of atherosclerosis was associated with suppression of endoplasmic reticulum stress - mediated apoptosis in apoe mice (c57bl/6j background). further mechanistic studies revealed that prevention of atherosclerosis by fgf21 was attributed to the fine - tuning of multiorgan cross talk among the liver, adipose tissue, and blood vessels, characterized by suppression of hepatic sterol regulatory element - binding protein-2 and induction of adiponectin in mice with atherosclerosis. although fgf21 functions in an endocrine manner, whether fgf21 can also induce a direct protection to the blood vessels remains unclear. for decades, lowering levels of low - density lipoprotein (ldl) cholesterol and increasing level of high - density lipoprotein (hdl) have formed the cornerstone of management of patients with atherosclerotic cardiovascular disease. strong evidence demonstrated that fgf-21 dramatically improved the condition of atherosclerosis in wistar rats by decreasing serum ldl levels and increasing serum hdl levels. moreover, fgf-21-induced antioxidative function is also involved in its therapeutic effect in atherosclerotic wistar rat characterized by increased levels of superoxide dismutase, reduced glutathione, and reduced malondialdehyde. along with the development of atherosclerosis, the artery 's lining becomes hardened, stiffened, and swollen with all sorts of gunge, including fatty deposits and abnormal inflammatory cells, to form a plaque and then eventually deteriorate into coronary heart disease [3941 ]. strong evidence indicated that cardiac endothelial cell dysfunction may be an early initiating factor for atherosclerosis which facilitates the development of coronary heart disease. oxidized ldl (ox - ldl) is a proatherogenic lipoprotein that accumulates in the vascular wall and contributes to vascular dysfunction at the early - stage of atherosclerosis development [4353 ]. enhanced serum ox - ldl and antibodies against its epitopes are predictive for endothelial dysfunction and subsequent coronary heart disease. previous in vitro study indicated that both fgf21 mrna and protein expressions were increased in response to ox - ldl treatment in cardiac endothelial cells and this was protective against apoptosis caused by ox - ldl. also, fgf21 has been reported to prevent high glucose induced cell damage and endothelial nitric oxide synthase dysfunction through an amp - activated protein kinase- (ampk-) dependent pathway in endothelial cells. shen. reported that serum fgf21 level was positively associated with coronary heart disease in clinics [56, 57 ]. our previous work confirmed that serum levels of fgf-21 are increased in patients with coronary heart disease independently associated with adverse lipid profiles. in contrast, another study indicated that serum fgf21 has been associated with hypertriglyceridemia, hyperinsulinemia, and pericardial fat accumulation but not associated with coronary heart disease. this paradox may be explained by decreased body mass index of healthy controls compared to patients with coronary heart diseases. myocardial ischemia, a disorder causing cardiomyocytes injury and myocardial infarction and malfunction, activates adaptive responses enhancing myocardial tolerance to ischemia. liu. indicated that, in response to myocardial ischemia in the c57bl/6j mouse, liver- and adipocytes - derived fgf21 was upregulated and secreted into the circulation. after interacting with fgfr1 in cardiomyocytes in the presence of -klotho, fgf21 activates its downstream kinases and proteins including phosphatidylinositol 3-kinase (pi3k), protein kinase b (pkb / akt), and bcl2 antagonist of cell death (bad), thereby reducing myocardial ischemia - induced apoptosis characterized by reduction of caspase-3 activity. reversely, myocardial ischemic size was significantly smaller in fgf21 transgenic mice than that in wild type mice, suggesting that upregulated endogenous fgf21 derived from the liver and adipose tissue in response to myocardial injury induced cardiac protection mediated by activation of fgfr1/-klotho - pi3k - akt1-bad signaling pathway. although various growth factors and cytokines were upregulated during myocardial ischemia, the expression and secretion of cardiac fgf21 had no alteration, implying fgf21 induces cardiac protection against myocardial ischemia in an endocrine rather than an autocrine manner [59, 60 ]. to date, a question of whether administration of exogenous fgf21 can also induce cardiac protection during myocardial ischemia and if so whether the protection of exogenous fgf21 against myocardial ischemia can be direct to the heart or cardiomyocytes appears. they found that administration of exogenous fgf21 induced significant cardioprotection and restored cardiac function following global ischemia in langendorff perfused rat hearts. further study revealed that inhibition of akt, extracellular signal - regulated kinase (erk1/2), and ampk impaired fgf21-induced antimyocardial ischemia effect in the hearts of obese wistar rats, suggesting that the above kinases are involved in this cardioprotection of fgf21. our previous in vitro study also confirmed that administration of exogenous fgf-21 attenuated ischemia - reperfusion induced damage in h9c2 cells characterized by inhibition of oxidative stress and apoptosis. the mechanistic study revealed that fgf21-induced protection against ischemia - reperfusion injury in cardiac cells mainly depended on the activation of akt - gsk-3-caspase-3 signaling pathway by preventing oxidative stress and recovery of the energy supply. hypertrophic remodeling characterized by enlarged cardiomyocytes is an adaptive response of the heart to certain stresses. and it is also the leading cause of multiple cardiovascular problems including hypertension, myocardial ischemia, valvular disease, and cardiomyopathy [6769 ]. mature cardiomyocytes are considered to be terminally differentiated cells with no regenerative ability [7072 ]. under stresses, cardiac hypertrophy is characterized by cardiomyocytes enlargement, rather than cells division [73, 74 ], and this phenomenon is accompanied by the increase of extracellular matrix and fibroblasts inside the heart [75, 76 ]. recently, cardiac hypertrophy was reported to induce fgf21 gene expression in the cardiomyocytes of mouse, and this was subjected to transcriptional regulation of the hepatic silent mating type information regulation 2 homolog 1/ppar pathway. in turn, fgf21 knockout mice had greater heart weights and more severe cardiac dysfunction in response to isoproterenol infusion along with induction of hypertrophic inflammatory markers. however, administration of recombinant fgf21 significantly prevented isoproterenol - induced cardiac hypertrophy damage in mice. mechanistic studies indicated that fgf21 prevented cardiac hypertrophy by activating mitogen - activated protein kinase (mapk) signaling via activation of fgfr1c/-klotho [64, 77 ]. additionally, fgf21 prevented cardiac hypertrophy by promoting multiple antioxidant genes expressions (e.g., uncoupling proteins 2 and 3, also superoxide dismutase-2) and inhibiting the formation of reactive oxygen species in an autocrine manner. diabetic patients develop the diabetic cardiomyopathy independent of coronary artery disease and hypertension [78, 79 ]. diabetic cardiomyopathy is attributed to multiple pathogenic factors, including hyperglycemia, hyperlipidemia, and inflammation [8082 ]. cardiomyopathy is a late consequence of diabetes - induced early cardiac responses especially the myocardial apoptosis [83, 84 ]. recently, we reported that cardiac fgf21 mrna expression was positively associated with the development of diabetes in the type 1 diabetic mice, suggesting that the increased cardiac fgf21 expression may be beneficial to the heart in this regard. in the study we also observed cardiac apoptosis in early diabetic mice, which was remarkably prevented by administration of recombinant fgf21. similar protection by fgf21 was observed in mice with cardiac lipotoxicity induced by fatty - acid. mechanistic studies indicated that fgf21-induced antiapoptotic effects in vitro and in vivo were mediated by erk1/2-p38-mapk - ampk signaling pathway. thus, fgf21-induced cardioprotection in diabetic mice is mainly attributed to prevention of lipotoxicity by fgf21. also, long - term treatment of fgf21 prevented diabetic - induced cardiac dysfunction and fibrosis mediated by the same signaling pathway as above. our work also revealed that fgf21 deletion - aggravated cardiac lipid accumulation is likely mediated by cardiac nrf2-driven cd36 upregulation in type 1 diabetic mice, which contributes to increased cardiac oxidative stress and remodeling, and eventual development of diabetic cardiomyopathy. cvd includes atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy which are all closely associated with severe lipid metabolic disorders [8587 ]. fgf21, a metabolic regulator of carbohydrates and lipids, has been shown to improve insulin sensitivity and glucose uptake and suppress lipogenesis and lipid oxidation [1518 ]. clinical studies indicated that serum fgf21 changes were positively associated with the development of atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy, which implies that upregulated endogenous fgf21 may improve cvds. specifically, fgf21 prevented atherosclerosis and subsequent coronary heart disease was attributed to multiorgan cross talk among the liver, adipose tissue, and blood vessels and was characterized by suppression of lipid accumulation and increased lipid oxidation. similarly, fgf21 prevented stress - induced ch via enhancing lipid oxidation mediated by the erk1/2-creb - pgc-1 signaling pathway. fgf21 also prevented myocardial ischemia and diabetic cardiomyopathy via akt- or ampk - mediated signaling pathways which regulate lipid and glucose metabolisms (figure 1). since serum fgf21 increases in several kinds of cvds, serum fgf21 levels might be regarded as a potential biomarker not only for diagnosis of metabolic disorders but also for diagnosis of cvd in clinics. and supplementation of exogenous fgf21 might also induce beneficial effect in patients with cvd based on the conclusion of preclinical studies. | cardiovascular disease (cvd) is one of the most severe diseases in clinics. fibroblast growth factor 21 (fgf21) is regarded as an important metabolic regulator playing a therapeutic role in diabetes and its complications. the heart is a key target as well as a source of fgf21 which is involved in heart development and also induces beneficial effects in cvds. our review is to clarify the roles of fgf21 in cvds. strong evidence showed that the development of cvds including atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy is associated with serum fgf21 levels increase which was regarded as a compensatory response to induced cardiac protection. furthermore, administration of fgf21 suppressed the above cvds. mechanistic studies revealed that fgf21 induced cardiac protection likely by preventing cardiac lipotoxicity and the associated oxidative stress, inflammation, and apoptosis. normally, fgf21 induced therapeutic effects against cvds via activation of the above kinases - mediated pathways by directly binding to the fgf receptors of the heart in the presence of -klotho. however, recently, growing evidence showed that fgf21 induced beneficial effects on peripheral organs through an indirect way mediated by adiponectin. therefore whether adiponectin is also involved in fgf21-induced cardiac protection still needs further investigation. |
strabismus, also referred to as squint, presents as a misalignment of the eyes. strabismus is also a feature of several syndromes, such as congenital fibrosis of extraocular muscles, duane retraction syndrome, and chronic progressive external ophthalmoplegia. population - based studies show that the familial clustering of strabismus has been observed, and the incidence of specific types of strabismus differs, in different racial groups, which suggests that the etiology of strabismus has genetic factors [38 ]. michaelides. studied the genetics of strabismus and found that it was related to mitochondrial cytopathies and cranial nerve misrouting, which indicated that strabismus has a significant genetic component. however, significant genetic heterogeneity makes it difficult to determine the mode of inheritance and the relevant causative genes. in a large family with nonsyndromic strabismus, parikh. found a presumptive strabismus susceptibility locus to chromosome 7p22.1 with a multipoint lod score of 4.51 under a model of recessive inheritance. several studies have identified the causative gene mutations associated with strabismus - related disorders, such as congenital stationary night blindness, intellectual disability, and kaufman oculocerebrofacial syndrome. however, the identity of the strabismus susceptibility genes has been enigmatic and the genetic mechanisms involved in the pathogenesis of strabismus remain poorly understood. mutation screening of candidate genes may be necessary to localize genes that predispose humans to strabismus. exome sequencing is a powerful and cost - effective tool for dissecting the genetic basis of diseases [1618 ]. in the present study, we investigated a chinese strabismus pedigree with the parents unaffected and 2 offspring affected. by whole - exome sequencing, we identified 2 mutations c.a3257 g (p.e1086 g) in ahi1 and c.a914 g in neb as the most likely causative mutations of the disorder. of interest, biallelic pathogenic variant in ahi1 gene can cause joubert syndrome - related disorders with oculomotor apraxia characteristics. eye examinations focused on the measurement of visual acuity (unaided and best - corrected) and refractive status. his unaided visual acuity was 1.0 in the right eye and 1.0 in the left eye. the proband s sister was also diagnosed with comitant exotropia (ii2) (figure 1a). moreover, neither of them had any other symptoms such as nystagmus or joubert - like symptoms or any sign of hypotonia or vision / retinal. additionally, their parents, grandparents, and maternal grandparents were unaffected. based on these findings, we considered that the etiology of strabismus in this family had a genetic factor. therefore, the proband and his parents were enrolled for exome sequencing screening (figure 1a). for the purpose of biological investigations, the study was approved by the ethics committee of the affiliated hospital of qingdao university (2015 - 012) and complied with the declaration of helsinki principles. venous blood was drawn from the affected individual (ii-1) and 2 unaffected individuals (i1 and i2) within the examined strabismus pedigree, then stored in tubes containing edta. all dna samples were prepared from the venous blood of the available patients by standard procedures. the qualified genomic dna was used to perform exome target enrichment using the agilent sureselect human all exon 50 mb exon kit (agilent technologies, santa clara, ca, usa). the captured whole exomes were sequenced on the platform of illumina hiseq 2500 sequencer (illumina, san diego, ca, usa). for each sample, sequencing reads with paired - end 125-bp long and mean coverage of 100x was generated. after filtering the adapter, contaminating reads and low - quality reads, the clean paired reads were then mapped to reference human genome sequence (hg19) using the burrows - wheeler alignment tool (bwa), generating the sequence alignment / map (sam) file. pcr duplicate reads were further marked and removed using the picard software program (version 1.07). genome analysis toolkit (gatk) and mutect software were used to detect the single - nucleotide variants (snvs), insertions, and deletions within genome - wide regions. to obtain the important candidate genes, the program annovar (http://www.openbioinformatics.org/annovar/) was used to annotate the variants of the information from various genetic variation databases. based on the reported variant frequencies, the common variants were first excluded with the minor allele frequency (maf) greater than 0.01 represented in the 1000 genomes project. according to the variant location within genes, the variants in the coding region were given higher priority, and the variants that altered the coding sequence (nonsynonymous) were selected. the deleteriousness of selected variants was subsequently predicted by various bioinformatics programs (sift, polyphen2, lrt, mutationtaster, mutationassessor, fathmm, and radialsvm, lr), and the variants were retained whose changes to the protein were damaging. according to the autosomal recessive inheritance pattern, only the variants which were homozygous in the affected individual and heterozygous in the unaffected members were screened. moreover, we required at least 5x sequence coverage at any given position for genotype calling. genomic dna was prepared from peripheral blood samples of the original 3 individuals who underwent exome sequencing and 5 additional sporadically affected individuals. primer - premier 5.0 (premier biosoft international, palo alto, ca, usa) was used to design oligonucleotide primer sets for the variants from whole - exome sequencing. pcr reactions and sanger sequencing were performed by majorbio company (shanghai, china). conservation of affected residue was analyzed using the ucsc genome browser (http://genome-asia.ucsc.edu/index.html) and visualized using mega7.0. the protein conserved domains were further identified using the conserved domain search service (http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi). eye examinations focused on the measurement of visual acuity (unaided and best - corrected) and refractive status. his unaided visual acuity was 1.0 in the right eye and 1.0 in the left eye. the proband s sister was also diagnosed with comitant exotropia (ii2) (figure 1a). moreover, neither of them had any other symptoms such as nystagmus or joubert - like symptoms or any sign of hypotonia or vision / retinal. additionally, their parents, grandparents, and maternal grandparents were unaffected. based on these findings, we considered that the etiology of strabismus in this family had a genetic factor. therefore, the proband and his parents were enrolled for exome sequencing screening (figure 1a). for the purpose of biological investigations, the study was approved by the ethics committee of the affiliated hospital of qingdao university (2015 - 012) and complied with the declaration of helsinki principles. venous blood was drawn from the affected individual (ii-1) and 2 unaffected individuals (i1 and i2) within the examined strabismus pedigree, then stored in tubes containing edta. all dna samples were prepared from the venous blood of the available patients by standard procedures. the qualified genomic dna was used to perform exome target enrichment using the agilent sureselect human all exon 50 mb exon kit (agilent technologies, santa clara, ca, usa). the captured whole exomes were sequenced on the platform of illumina hiseq 2500 sequencer (illumina, san diego, ca, usa). for each sample, sequencing reads with paired - end 125-bp long and mean coverage of 100x was generated. after filtering the adapter, contaminating reads and low - quality reads, the clean paired reads were then mapped to reference human genome sequence (hg19) using the burrows - wheeler alignment tool (bwa), generating the sequence alignment / map (sam) file. pcr duplicate reads were further marked and removed using the picard software program (version 1.07). genome analysis toolkit (gatk) and mutect software were used to detect the single - nucleotide variants (snvs), insertions, and deletions within genome - wide regions. to obtain the important candidate genes, the resulting variants were annotated and filtered systematically. the program annovar (http://www.openbioinformatics.org/annovar/) was used to annotate the variants of the information from various genetic variation databases. based on the reported variant frequencies, the common variants were first excluded with the minor allele frequency (maf) greater than 0.01 represented in the 1000 genomes project. according to the variant location within genes, the variants in the coding region were given higher priority, and the variants that altered the coding sequence (nonsynonymous) were selected. the deleteriousness of selected variants was subsequently predicted by various bioinformatics programs (sift, polyphen2, lrt, mutationtaster, mutationassessor, fathmm, and radialsvm, lr), and the variants were retained whose changes to the protein were damaging. according to the autosomal recessive inheritance pattern, only the variants which were homozygous in the affected individual and heterozygous in the unaffected members were screened. moreover, we required at least 5x sequence coverage at any given position for genotype calling. genomic dna was prepared from peripheral blood samples of the original 3 individuals who underwent exome sequencing and 5 additional sporadically affected individuals. primer - premier 5.0 (premier biosoft international, palo alto, ca, usa) was used to design oligonucleotide primer sets for the variants from whole - exome sequencing. pcr reactions and sanger sequencing were performed by majorbio company (shanghai, china). conservation of affected residue was analyzed using the ucsc genome browser (http://genome-asia.ucsc.edu/index.html) and visualized using mega7.0. the protein conserved domains were further identified using the conserved domain search service (http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi). genomic dna from i : 1, i : 2 and ii : 2 were analyzed by whole - exome sequencing (wes). for each sample, an average of 9.67 gb raw data were generated with paired - end 125-bp length. above 99% clean reads were then aligned to the human reference sequence (hg19) (table 1). a total of 567 705 single - nucleotide variations (snvs) were identified by variant detection with the genome analysis toolkit (gatk) and mutect software. among the variants identified by wes, we first removed variants with minor allele frequency > 0.01 in the 1000 genomes project, resulting in 118 542 variants, and 3191 variants were located in the exonic region. further filtering of synonymous variants led to 1851 loss - of - function variants, which altered the coding sequence and were more likely associated with the disease. based on the recessive - inherited mode of the strabismus pedigree, only 2 snvs fulfilled our filtering criteria : c. 914a - g mutation in neb (chr2 : 151485334.. 151734487) and c. 3257a - g mutation in ahi1 (chr6 : 135283972.. 135497775). the proband of ii : 1 was homozygous for these mutations, and his clinically unaffected parents were heterozygous for these variants. variant frequencies were 0.0228 for mutation in neb and 0.0357 for mutation in ahi1 in the population of east asian ancestry from the 1000 genomes project. the c.3257a - g mutation in ahi1 resulted in p.1086e - g change, which was predicted to be damaging by polyphen2. we searched the literature and found that biallelic pathogenic variants in ahi1 gene were identified in individuals with joubert syndrome - related disorders, which are characterized by cerebellar ataxia, oculomotor apraxia, hypotonia, neonatal breathing abnormalities, and psychomotor delay. it is reported that the expression of neb is remarkably decreased in strabismic extraocular muscles compared to normal muscles. therefore, we considered c.3257a - g mutation in ahi1 and c.914a - g mutation in neb as the most likely causal variants in this strabismus pedigree. to further confirm the variants of c.3257a - g mutation in ahi1 in strabismus, sanger sequencing was performed in 10 individuals with the basic type of strabismus and in 3 healthy individuals. the results showed that the mutation was not observed in other individuals with strabismus, indicating the genetic heterogeneity of strabismus. however, the mutation was found to be a homozygous mutation in his sister, which was consistent with the proband. the protein in 1086 locus of ahi1 protein sequence is highly conserved across species (human, rhesus monkey, mouse, dog, and elephant). however, we found the rare mutation of c.3257a - g in ahi1 (p.e1086 g) (figure 1b) in this study. searching for conserved domains within the ahi1 protein indicated that the altered amino acid residue (p.e1086 g) was located in the conserved src homology 3 (sh3) domain of ahi1 (figure 1c). sh3 domains are protein interaction domains that bind to proline - rich ligands with moderate affinity and selectivity, and bind preferentially to pxxp motifs. they play versatile and diverse roles in the cell, including the regulation of enzymes, changing the subcellular localization of signaling pathway components, and mediating the formation of a multiprotein complex assembly. genomic dna from i : 1, i : 2 and ii : 2 were analyzed by whole - exome sequencing (wes). for each sample, an average of 9.67 gb raw data were generated with paired - end 125-bp length. above 99% clean reads were then aligned to the human reference sequence (hg19) (table 1). a total of 567 705 single - nucleotide variations (snvs) were identified by variant detection with the genome analysis toolkit (gatk) and mutect software. among the variants identified by wes, we first removed variants with minor allele frequency > 0.01 in the 1000 genomes project, resulting in 118 542 variants, and 3191 variants were located in the exonic region. further filtering of synonymous variants led to 1851 loss - of - function variants, which altered the coding sequence and were more likely associated with the disease. based on the recessive - inherited mode of the strabismus pedigree, only 2 snvs fulfilled our filtering criteria : c. 914a - g mutation in neb (chr2 : 151485334.. 151734487) and c. 3257a - g mutation in ahi1 (chr6 : 135283972.. 135497775). the proband of ii : 1 was homozygous for these mutations, and his clinically unaffected parents were heterozygous for these variants. variant frequencies were 0.0228 for mutation in neb and 0.0357 for mutation in ahi1 in the population of east asian ancestry from the 1000 genomes project. the c.3257a - g mutation in ahi1 resulted in p.1086e - g change, which was predicted to be damaging by polyphen2. we searched the literature and found that biallelic pathogenic variants in ahi1 gene were identified in individuals with joubert syndrome - related disorders, which are characterized by cerebellar ataxia, oculomotor apraxia, hypotonia, neonatal breathing abnormalities, and psychomotor delay. it is reported that the expression of neb is remarkably decreased in strabismic extraocular muscles compared to normal muscles. therefore, we considered c.3257a - g mutation in ahi1 and c.914a - g mutation in neb as the most likely causal variants in this strabismus pedigree. to further confirm the variants of c.3257a - g mutation in ahi1 in strabismus, sanger sequencing was performed in 10 individuals with the basic type of strabismus and in 3 healthy individuals. the results showed that the mutation was not observed in other individuals with strabismus, indicating the genetic heterogeneity of strabismus. however, the mutation was found to be a homozygous mutation in his sister, which was consistent with the proband. the protein in 1086 locus of ahi1 protein sequence is highly conserved across species (human, rhesus monkey, mouse, dog, and elephant). however, we found the rare mutation of c.3257a - g in ahi1 (p.e1086 g) (figure 1b) in this study. searching for conserved domains within the ahi1 protein indicated that the altered amino acid residue (p.e1086 g) was located in the conserved src homology 3 (sh3) domain of ahi1 (figure 1c). sh3 domains are protein interaction domains that bind to proline - rich ligands with moderate affinity and selectivity, and bind preferentially to pxxp motifs. they play versatile and diverse roles in the cell, including the regulation of enzymes, changing the subcellular localization of signaling pathway components, and mediating the formation of a multiprotein complex assembly. strabismus is characterized as a misalignment of the eyes, which is also a feature of several syndromes such as congenital fibrosis of extraocular muscles. family studies suggest that there is a strong genetic component to the etiology of strabismus [1,68 ] the advent of whole - exome sequencing technologies is a well - justified strategy for discovering candidate causative genes underlying genetic disease phenotypes. in this study, we aimed our efforts in this direction, and whole - exome sequencing was performed on the affected individual and his parents in a chinese strabismus pedigree, identifying 2 mutations in the genes of ahi1 and neb, which may be associated with strabismus. additionally, we detected the ahi1 homozygous mutation in the affected individual in this study. the inheritance patterns of strabismus are complex and are associated with complex sensory and motor pathways in the retina, thalamus, visual cortex, and brainstem [2527 ]. herein, genetic analysis was conducted on a chinese strabismus pedigree, and a mutation in ahi1 was identified. the structure of the ahi1 gene contains at least 33 exons and spans 213.7 kb. genomewide screening of 5 pedigrees with autosomal recessive joubert syndrome-3 identified 3 independent mutations in the ahi1 gene. using next - generation sequencing, najmabadi also identified homozygosity for a nonsense and a missense mutation in the ahi1 gene in affected members of 2 families with joubert syndrome-3. pathogenic variants in ahi1 were identified in 7.3% of 137 persons with joubert syndrome. moreover, about 80% of them had retinal dystrophy. ahi1 expression is required for the development of cerebellar and cortical structure, and its missense and frameshift mutations were identified in families with joubert syndrome plus cortical polymicrogyria. additionally, joubert syndrome has been reported to be genetically heterogeneous with mutations in ahi1, suggesting that this genotypic variant of joubert syndrome may be related to functional abnormalities of the cerebellum and brain stem. the ahi1 protein includes a sh3 domain, a coiled - coil domain, and 6 wd40 repeats. the ahi1 mutations occurred in a phenotype - specific group of joubert plus retinal abnormalities, and most of the mutations abolished all of the sh3 domain. consistent with that, we also found that the altered amino acid residue (p.e1086 g) was located in the conserved sh3 domain of ahi1, suggesting its potential roles in strabismus. previous studies reported that approximately 1520% of strabismus was associated with global central nervous system (cns) defects rather than with ocular disorders. it has been suggested that the ipsilateral non - decussating projection from the temporal retina is suppressed in strabismus, indicating that the cns and retina play an important role in the pathology of strabismus. functional studies on mice showed that ahi1-null mice had defects of retinal outer segment morphogenesis and mislocalization of opsin in photoreceptors, which contributed to the loss of photoreceptors. the protein encoded by ahi1 (also termed jouberin) is involved in canonical wnt signaling. lancaster found that ahi1-mutant mice showed cerebellar hypoplasia and the wnt reporter activity in the developing cerebellum was decreased. these findings suggest that our variant in ahi1 may contribute to strabismus phenotype in this chinese pedigree. in this study neb encodes nebulin, which accounts for 3% to 4% of the total myofibrilla protein and regulates the length of actin filaments and contraction strength [4446 ]. it has been reported that neb has significantly down - regulated expression in strabismic extraocular muscles compared to normal muscles, which further demonstrates the relationship between neb and strabismus. our results defined 2 relevant genes, ahi1 and neb, that may predispose to strabismus, possibly through an additive effect. our finding revealed the high degree of genetic heterogeneity inherent in the strabismus phenotype, as demonstrated by parikh., who discovered that the presumptive strabismus susceptibility locus to chromosome 7p22.1 was not presented in 6 other multiplex families. we conclude that this chinese strabismus pedigree best fits a model of autosomal recessive disease transmission. | backgroundthe etiology of strabismus has a genetic component. our study aimed to localize the candidate causative gene mutant in a chinese family with strabismus and to describe its underlying etiology.material/methodsgenomic dna was extracted from the affected individual and his parents in a chinese pedigree with strabismus. the resulting exomes were sequenced by whole - exome sequencing. after variant calling and filtering, the candidate causative gene mutations were selected for the rarity and predicted damaging effect, which complied with the model of recessive disease transmission.resultswe examined a chinese strabismus pedigree with the parents unaffected and 2 offspring affected. whole - exome sequencing and bioinformatics filtering identified 2 variants including abelson helper integration site 1 (ahi1) gene and nebulin (neb) gene. the variant in the ahi1 gene, c.a3257 g (p.e1086 g), and the altered amino acid had a damaging effect on the encoded protein predicted by polyphen2. moreover, this change was located in the conserved sh3 domain of ahi1. biallelic pathogenic variant in ahi1 gene can cause joubert syndrome - related disorders with oculomotor apraxia characteristics. additionally, c.a914 g mutation was found in nebulin (neb) gene. therefore, we concluded that ahi1 c.3257a > g and neb c.914 a > g were potential causal variants in this strabismus pedigree.conclusionswe detected an ahi1 homozygous mutation in the affected individual. whole - exome sequencing is a powerful way to identify causally relevant genes, improving the understanding of this disorder. |
chronic hepatitis c virus (hcv) infection is a major worldwide cause of liver - related deaths, including those caused by cirrhosis and hepatocellular carcinoma. the eradication of hcv using interferon - based treatment improves liver - related mortality and reduces the risk of hepatocellular carcinoma development. recently, interferon - free direct - acting antiviral therapy was used, which showed an excellent rate of hcv eradication among patients with chronic hcv genotype 1b, including those with prior interferon - based treatment failure. however, interferon - based therapy is still a treatment option, and negativity for hcv rna at the end of antiviral treatment (end - of - treatment response ; etr) is essential to achieve the viral clearance of chronic infection. although delayed viral clearance after the completion of 2448 weeks of interferon - based therapy is rarely reported, such a situation has not been observed among patients with the early discontinuation of therapy. here, we report a case of hcv clearance at 7 months after a 6-week treatment with peginterferon (pegifn) plus ribavirin (rbv). a 76-year - old japanese male (height, 161 cm ; weight, 63 kg) with chronic hcv genotype 1b infection had initiated treatment with pegifn -2b plus rbv. previously, he had been followed up for inactive chronic hepatitis for 15 years, with alanine aminotransferase (alt) levels < 40 iu / l, which had fluctuated from 40 to 140 iu / l for a year before the initiation of the latest treatment regimen. his medical history included hypertension, hyperuricemia, and resistant gastroesophageal reflux disease, which were being treated with nifedipine, candesartan cilexetil, allopurinol, and lansoprazole for more than 5 years. laboratory examinations (table 1) revealed the following biochemical levels : alt, 129 iu / l ; albumin, 4.3 g / dl ; -glutamyl transpeptidase, 72 iu / l ; platelet count, 23.6 10/l, and -fetoprotein, 4.9 ng / ml. hepatitis c viral load was 5.3 log iu / ml. genetic studies revealed tt of il28b (rs8099917) and human leukocyte antigen (hla)-drb1 01 : 01/08 : 03. after the initiation of therapy, he reported a loss of appetite at week 2 and complained of anxiety, irritability, insomnia, and poor concentration at week 3. although daily etizolam before sleep was started, he experienced poor appetite, taste disorder, and weight loss of 2 kg from the initiation of therapy. therefore, sulpiride was added, and the rbv dose was reduced from 800 to 600 mg per day because of anemia. however, symptoms did not subside with a further weight loss of 1 kg and the progression of anemia ; thus, the rbv dose was reduced to 400 mg / day. nonetheless, the symptoms persisted, and he reported depression at week 6, which prompted treatment discontinuation. following this, the symptoms slowly but gradually subsided, and he recovered after 5 weeks. meanwhile, the hcv rna load decreased to weakly positive, with measured values of less than 1.2 log iu / ml at week 4, which persisted at this level until 12 days after the discontinuation of treatment (fig. 1). then, the load gradually increased to 2.6 log iu / ml and 4.3 log iu / ml at 710 and 19 weeks after discontinuation, respectively. concurrently, serum alt levels, which had persisted at levels of less than 34 a posttreatment ultrasound exam revealed a 1.0-cm bladder tumor, which was diagnosed by urinary cytology as class v, and the patient was treated by transurethral resection of the bladder tumor (tur - bt) at week 26 of discontinuation. iu / l) at 1 week before tur - bt, and hcv rna was found to be negative at 11 days after surgery (28 weeks after the discontinuation of antiviral therapy), which has been maintained along with low levels (< 25 this patient showed an unusual and unique clinical course of delayed viral clearance despite only 6 weeks of pegifn plus rbv therapy, without the achievement of hcv rna negativity. there are relatively few reports of delayed viral clearance among patients with non - etr and those with relapse after pegifn plus rbv therapy ; all these patients had completed 2448 weeks of therapy, and almost all had genotype 2 or 3 and were aged < 60 years [4, 5, 6 ]. compared with those cases, our patient was unique in terms of being infected with hcv genotype 1b, older, and treated for only 6 weeks without achieving etr. although the mechanism underlying delayed viral clearance remains unknown, we speculate two possibilities for this patient. one is an immunological modification due to surgical stress caused by tur - bt, and the other is a delayed effect of pegifn plus rbv therapy. it has been reported that surgical stress or invasive treatment can sometimes induce the clearance of hcv rna [7, 8 ], but this is not applicable to this case because it is quite unlikely that hcv rna with 4.3 log copies / ml was eliminated within 11 days after tur - bt. furthermore, although hcv rna was not examined, the normalization of serum alt had already occurred before tur - bt. chronic infection with hcv is characterized by hcv - specific t cell dysfunction, and t cell function is considered to play an essential role in the elimination of the virus. although it is controversial whether pegifn plus rbv therapy restores t cell function, the marked restoration of hcv - specific cd8 t cells was observed from 1 month after treatment in patients with a sustained viral response, and a correlation between the restoration of hcv - specific cd8 + t - cell function and early / sustained viral response has been reported. in this case, hcv rna rapidly decreased after treatment, although the levels remained positive at < 1.2 log iu / ml at week 4, and the low levels of viral load persisted for at least 10 weeks after the discontinuation of therapy, suggesting immunological recovery from hcv infection. furthermore, the mild elevation of alt at 19 weeks could be considered as the t cell response against increased hcv rna. this phenomenon is well known in patients with chronic hepatitis b virus (hbv) infection, in whom alt flare occurs with a preceding increase in hbv dna, often resulting in continuous virus suppression. the immunological response observed in this phenomenon is also well understood. in our patient, although the elevation of alt was mild, a similar mechanism was presumed. this patient had a genetic background of hla - b1 01 : 01 and tt of il28b rs8099917. the cellular immune responses coordinated by cd4 and cd8 t cells are genetically predetermined by the hosts hla molecules. recently, several studies have revealed that the class ii drb1 01 : 01 hla allele and the c and t alleles of il28b (rs12979860 and rs8099917, respectively) are associated with the spontaneous clearance of hcv [13, 14, 15 ]. furthermore, hla - drb1 01 is suspected to be associated with delayed viral clearance after pegifn plus rbv therapy. therefore, the genetic background of this patient may be related to the viral clearance after treatment discontinuation. in conclusion, interferon - based therapy remains a viable treatment option for chronic hcv infection, and virological follow - up is necessary even for patients with non - etr, particularly for those with persistent low viral loads after treatment. | following interferon - based therapy for chronic hepatitis c, the negativity of hepatitis c virus rna is essential to achieve viral clearance at the end of treatment. we report a case of clearance of chronic hepatitis c virus infection following early discontinuation (at 6 weeks) of peginterferon plus ribavirin therapy, without negativity for hepatitis c virus rna during the treatment period. the patient was a 76-year - old japanese male infected with hepatitis c virus genotype 1b and tt of il28b rs8099917. hepatitis c virus rna remained positive at persistently low levels for more than 2 months after the cessation of therapy and became negative at 7 months after the discontinuation of therapy. spontaneous clearance of hepatitis c virus rna can occur following antiviral failure in patients with persistently low viral loads, and virological follow - up is therefore necessary in chronic hepatitis c virus infection, even after antiviral failure. |
nondopaminergic and nonmotor symptoms of parkinson 's disease (pd), which include psychiatric, autonomic, and gastrointestinal symptoms, are sometimes present before a diagnosis of pd and almost inevitably emerge with the disease progression. in contrast to motor symptoms of pd, nonmotor symptoms have been poorly recognized and inadequately treated. however, methods for recognizing and quantifying the nonmotor symptoms of pd have become a new focus of research that will form the basis for improved treatments. among the nonmotor symptoms, cognitive impairment adversely influences their activities of daily living and quality of life and worsens caregiver burden. a community - based study found that within a group of patients with a diagnosis of pd of at least 20 years, 83% had experienced dementia, and a similar number of patients had experienced frequent falls (87%), moderate dysarthria (81%), visual hallucinations (74%), or urinary incontinence (71%). in pd dementia (pd - d) patients, -synuclein pathology can be widespread in the cerebral cortex. in addition, there is less acetylcholinesterase activity in the cerebral cortex of pd - d patients than of alzheimer 's disease patients. moreover, clinical studies have reported on the effectiveness of cholinesterase inhibitors for treating cognitive impairment in pd - d patients. the prevalence of cognitive dysfunction among pd patients has led to the recognition that a diagnostic criterion is needed for pd - d. as a result, the movement disorder society task force published diagnostic criteria for pd - d in 2007 and simultaneously proposed a practical diagnostic procedure for pd - d with the aim of its widespread international application. level i testing includes a simple and short algorithm based on current cognitive tools that can be used in an office or at the bedside. it is considered a screening tool for diagnosing pd - d, whereas level ii testing provides a more detailed assessment that characterizes the components of pd - d and monitors the elements that may be responsive to intervention. level i testing was designed as a screening tool for use in the clinic and in clinical trials. although it has been used in practice, no study has yet verified its validity or utility in a large sample of pd patients. in this study, we used level i testing on 304 pd patients with the objective of determining which patients would be diagnosed with pd - d and what impairments would be diagnosed among the 4 cognitive domains tested. the subjects included in our study were japanese patients with idiopathic pd who were clinically diagnosed and treated by board - certified neurologists at 1 of the 13 participating institutions between july and september 2011. a diagnosis of pd was made according to the uk parkinson 's disease society brain bank criteria as well as a differential diagnosis. the study design and protocol were approved by the ethics committees for human research of the keio university school of medicine and all the participating institutions. this was a cross - sectional study which examined the clinical and neurological features of these pd patients, including sex, age at the assessment, education, disease duration, and the hoehn and yahr (h&y) stage. their cognitive function was evaluated with the japanese versions of the mini - mental state examination (mmse) and montreal cognitive assessment (moca). the mmse consists of 10 subtests scored to a total of 30 points : pentagon copying (1 point), serial 7 subtraction (5 points), immediate (3 points) and delayed 3-word recall (3 points), naming (2 points), repeating a sentence (1 point), 3-stage commands (3 point), reading a sentence (1 point), writing a sentence (1 point), and orientation to time and place (10 points). the moca consists of 12 subtests also scored to a total of 30 points : cube copying (1 point), clock drawing (3 points), trail making (1 point), phonemic fluency (1 point), verbal abstraction (2 points), digit span (2 points), serial 7 subtraction (3 points), target tapping (1 point), delayed 5-word recall (5 points), naming (3 points), repeating 2 sentences (2 points), and orientation to time and place (6 points). we used the algorithm for level i testing to examine the present patient population (table 1). in this algorithm, the diagnosis of pd - d is based on the following 5 criteria : (1) diagnosis of pd, (2) pd developed prior to the onset of dementia, (3) a decreased global cognitive efficiency, (4) a cognitive deficiency that impairs daily life, and (5) impairment in > 1 cognitive domain. in this study, however, we did not investigate whether cognitive deficits were severe enough to affect daily living (item 4, typically assessed with a caregiver interview or the pill questionnaire). for item 5, the results of the following subtests of the moca and mmse were used : serial 7 subtraction (seven backward in table 1), phonemic fluency (lexical fluency), and clock drawing from the moca, and pentagon copying (mmse pentagons) and delayed 3-word recall from the mmse. memory for delayed 3-word recall (table 1). in the months reversed test, the patient must name the months in reverse order. however, japanese months are named by number, which is different from their naming in english ; thus, the months reversed test was excluded from this study. according to the level i testing procedures, test results that indicated impairment were : 2 incorrect responses on the seven backward test, a score of 9 words in the lexical fluency test, an inability to correctly draw the clock face or clock hands pointing to the correct time in the clock drawing test, an inability to correctly draw 2 pentagons that overlap in the pentagon copying test, and 1 word missing in the 3-word recall test. the jmp software version 8.0 (sas institute, tokyo, japan) was used for all statistical analyses. the level of statistical significance was defined as p 1 cognitive domain. in this study, however, we did not investigate whether cognitive deficits were severe enough to affect daily living (item 4, typically assessed with a caregiver interview or the pill questionnaire). for item 5, the results of the following subtests of the moca and mmse were used : serial 7 subtraction (seven backward in table 1), phonemic fluency (lexical fluency), and clock drawing from the moca, and pentagon copying (mmse pentagons) and delayed 3-word recall from the mmse. however, japanese months are named by number, which is different from their naming in english ; thus, the months reversed test was excluded from this study. according to the level i testing procedures, test results that indicated impairment were : 2 incorrect responses on the seven backward test, a score of 9 words in the lexical fluency test, an inability to correctly draw the clock face or clock hands pointing to the correct time in the clock drawing test, an inability to correctly draw 2 pentagons that overlap in the pentagon copying test, and 1 word missing in the 3-word recall test. the jmp software version 8.0 (sas institute, tokyo, japan) was used for all statistical analyses. the level of statistical significance was defined as p 80%) even in the low - moca - score group. in contrast, sentence repeating, phonemic fluency, and delayed recall scores decreased remarkably (90%) in the high - mmse - score group, indicating that no mmse subtest was significative for these pd patients (fig. a significant decrease was observed only for the serial 7 subtraction and delayed recall subtests in the low - mmse - score group (1 cognitive domain), we observed that 53 subjects (17.4%) showed no impairment in any cognitive domain, whereas the remaining subjects showed impairments in 1 (n = 74 ; 24.3%), 2 (n = 87 ; 28.6%), 3 (n = 74 ; 24.3%), or 4 (n = 16 ; 5.3%) cognitive domains. therefore, 177 subjects (58.2%) showed impairments in 2 cognitive domains. among these 177 subjects, 99 also had mmse scores 80%) even in the low - moca - score group. in contrast, sentence repeating, phonemic fluency, and delayed recall scores decreased remarkably (90%) in the high - mmse - score group, indicating that no mmse subtest was significative for these pd patients (fig. a significant decrease was observed only for the serial 7 subtraction and delayed recall subtests in the low - mmse - score group (1 cognitive domain), we observed that 53 subjects (17.4%) showed no impairment in any cognitive domain, whereas the remaining subjects showed impairments in 1 (n = 74 ; 24.3%), 2 (n = 87 ; 28.6%), 3 (n = 74 ; 24.3%), or 4 (n = 16 ; 5.3%) cognitive domains. therefore, 177 subjects (58.2%) showed impairments in 2 cognitive domains. among these 177 subjects, 99 also had mmse scores < 26 (32.6% of all subjects ; 94.3% of the total group with mmse scores < 26) and therefore met the diagnostic criteria for pd - d. only 6 subjects (5.7% of 105 subjects) with mmse scores < 26 did not meet the diagnostic criteria for pd - d, because they had only 1 impaired cognitive domain. of the 105 subjects with mmse scores < 26, most showed impairments in 3 cognitive domains (n = 62 ; 59.0%). figure 3 shows the number of subjects with mmse scores < 26 or 26, along with the number of impaired domains. no subject with an mmse score < 26 was without impairment in any domain. among the subjects with mmse scores 26 (n = 199), we observed 1 (n = 68 ; 34.2%), 2 (n = 65 ; 32.7%), 3 (n = 12 ; 6.0%), and 4 (n = 1 ; 0.5%) impaired domains ; 53 (26.6%) had no impairment in any cognitive domain. figure 4 shows the proportions of subjects with mmse scores < 26 or 26, along with the number of impaired domains. subjects with mmse scores 26 accounted for the majority of subjects with 1 impaired domain, and they accounted for 74.7% of subjects with 2 impaired domains. in contrast, subjects with mmse scores < 26 accounted for the majority of subjects with 3 or 4 impaired domains. this finding was as expected because patients with lower mmse scores should have an increased likelihood of being affected in more cognitive domains. figure 5a shows the numbers of subjects with impairments for each of the 4 cognitive domains. the most common impairment was executive dysfunction (224 subjects ; 73.7%), followed by memory (n = 161 ; 53.0%) and attention (n = 119 ; 39.1%) impairments. visuospatial functional impairments were the least common and were apparent in only 30 subjects (9.9%). of the 224 subjects who showed executive dysfunction, most exhibited impairment in lexical fluency (180 subjects ; 80.4%) or clock drawing (n = 133 ; 59.4%), whereas 89 subjects (39.7%) displayed impairments in both (fig. the most common combinations in the group with mmse scores 26 were executive dysfunction alone (49 subjects ; 24.6%) and executive dysfunction with memory impairment (n = 47 ; 23.6%), followed by executive dysfunction with attention impairment (n = 16 ; 9.5%) and memory impairment alone (n = 15 ; 7.5%). the most common combination of impaired cognitive domains in the group with mmse scores < 26 was executive dysfunction with both memory and attention impairments (n = 54 ; 51.4%), followed by impairments in all 4 domains (n = 15 ; 14.3%). interestingly, visuospatial impairment was always accompanied by executive dysfunction ; however, visuospatial impairment alone, or in combination with attention, memory, or both attention and memory impairments, did not occur in any of the subjects. for the purpose of screening pd patients for dementia, it is necessary to use a test in which achievement does not decline in pd patients who have normal global cognitive efficiency (as measured by the total mmse or moca score) but declines in those patients with impaired global cognitive efficiency indicative of dementia. with regard to the hypothesis that an appropriate test is one in which the high- and middle - total - score groups by the mmse or the moca maintain a score 70% while the low - total - score group scores < 70%, the copy cube, draw clock, verbal abstraction, digit span, target tapping, and serial 7 subtraction tests of the moca (marked # in fig. 2a) and the serial 7 subtraction and 3-word delayed recall tests of the mmse (marked # in fig. in level i testing, this includes the clock drawing, serial 7 subtraction, and delayed 3-word recall subtests, but not the phonemic fluency or pentagon copying subtests. as regards evaluating executive function, scores for the phonemic fluency test declined markedly to approximately 50% in the high - moca - score group, whose scores (24 - 30) were consistent with pd without dementia. the other subtest that evaluates executive function in level i testing is the clock drawing test. scores for this test declined to < 70% only in the low - moca - score group. this suggests the clock drawing test may be more appropriate than the phonemic fluency test for screening for a decline in executive function in pd - d. on the other hand, when evaluating visuospatial function, scores for the pentagon copying test stayed at 70% even in the low - mmse - score group, whose scores (12 - 25) were below the 26-point cutoff for pd - d. the authors of the original moca test indicated that the cube copying and clock drawing tests are concerned with visuospatial function, but in level i testing, the clock drawing test is used for executive function. for the cube copying test, the authors stated that if any of the following criteria are not met, the abnormal result was assigned : (1) drawing must be 3 dimensional, (2) all lines are drawn, (3) no line is added, (4) lines are relatively parallel and their length is similar. applied to the 304 subjects in the present study, 101 were classified as abnormal according to the cube copying test, including 43 subjects in the group with mmse scores 26 and 58 subjects in the group with mmse scores < 26. these are far more than the 30 subjects with abnormal results for the pentagon copying test, and almost as many as the 119 subjects who were categorized as abnormal in attention by the serial 7 subtraction test. some previous studies have examined the screening properties of level i testing for pd - d. have demonstrated that level i testing is more sensitive for the detection of pd - d, detecting up to 22% more cases than the dsm - iv. di battista. validated level i testing in comparison with the dsm - iv criteria for pd - d and revealed its relatively lower sensitivity (78%) but high specificity (95.5%) with a 12% prevalence of pd - d in 76 pd patients. the authors raised their doubts about the sensitivity of an mmse cutoff value of 26, stating this to be the main cause of false negatives, and also pointed out that more sensitive cognitive domain - related psychometric tests would maximize the screening property of level i testing. compared level i testing with full neuropsychological testing for detecting pd - d in 91 pd patients and found that 7.7% of the subjects met the level i testing criteria, while 16.5% of the subjects were classified as pd - d by full neuropsychological testing. they revealed that level i testing showed 100% specificity but only 46.7% sensitivity for diagnosing pd - d compared with the full neuropsychological assessment. they showed that the low sensitivity is largely due to the total mmse cutoff values for decreased global cognitive efficiency and the 15-item geriatric depression scale score for excluding depression. we found that 34.5% of all pd patients examined in the present study had mmse scores < 26. most of these patients (94.3%) were impaired in 2 cognitive domains and therefore were cases of probable pd - d. many of these patients (59%) showed impairment in 3 cognitive domains, and patients rarely showed impairment in a single cognitive domain. at least 1 of these 4 cognitive domains was always impaired in pd patients with mmse scores < 26. even among pd patients with mmse scores 26, many had impairments in 1 or 2 cognitive domains, and the proportion of those with no impairment in any cognitive domain was 26.6%. subjects with mmse scores 26 accounted for 75% of pd patients who had impairments in 2 cognitive domains, which was far more than in the group with mmse scores < 26. among the cognitive domains, executive function, which was tested by the phonemic fluency and clock drawing subtests, was most frequently impaired ; visuospatial function, which was tested by the pentagon copying test, was least frequently impaired. no patient showed visuospatial impairment alone, and an impaired visuospatial function was always accompanied by executive dysfunction. among the subjects in the group with mmse scores 26, the most common combinations were executive dysfunction alone or executive dysfunction with memory impairment, each of which accounted for approximately 25% of these subjects. among the subjects in the group with mmse scores < 26, the combination of executive dysfunction with both attention and memory impairment was most frequent, accounting for approximately 50% of these subjects. the inclusion of impairments in 2 cognitive domains as an item in level i testing stems from the conventional thinking behind the diagnostic criteria for dementia resulting from research on alzheimer 's disease. level i testing includes 4 cognitive domains that may often be impaired in pd patients. we showed that for the mmse and moca subtests, some test results were abnormal even among pd patients with mmse scores 26. this suggests that for dementia screening tools such as level i testing, it may be appropriate to use tests in which abnormalities are rare in patients with mmse scores 26 but tend to be common in those scoring < 26. thus, once tests that tend to yield abnormal scores even for pd patients with mmse scores 26 are included in a screening tool, its sensitivity for pd - d diagnosis will increase, even if its specificity will decrease. a total of 32.6% of the pd patients had mmse scores < 26 and impairments in 2 cognitive domains, and may therefore have met the diagnostic criteria for probable pd - d by level i testing. this point prevalence of pd - d in pd is similar to the mean value of 31.1% (95% ci : 20.1 - 42.1) reported in a systematic and critical review of previous studies, thus indicating the validity of level i testing. the fact that 1 of the 5 tests concerning the 4 cognitive domains included in these diagnostic tests showed abnormal scores for the group of patients with mmse scores < 26 indicates the validity of these tests as a whole for pd - d screening. however, the results of this study show that the moca clock drawing subtest might be more appropriate than the phonemic fluency test for revealing executive dysfunction when screening for pd - d. the frequency of abnormal results for the mmse pentagon copying subtest was < 10%, and thus was markedly lower than for the other tests. this is inconsistent with previous reports which found that visuospatial function tends to be impaired in pd - d, and it may be necessary to replace this test with another that also examines visuospatial function. item 4 (activities of daily living) in level i testing was not evaluated and no extensive neuropsychological assessment was done, because of which dementia was not accurately diagnosed for each subject. therefore, this study could not address the validity, sensitivity, or specificity of level i testing regarding clinically diagnosed pd - d. the months reversed test was not used as there was no japanese equivalent to this english subtest in level i testing ; therefore, we could not examine its validity. by using the mmse and moca, level i testing for pd - d can evaluate both decreased global cognitive efficiency and impairments in cognitive domains that are vulnerable in pd - d. level i testing is therefore regarded as a practical and useful tool for screening dementia in pd patients. a total of 32.6% of the pd patients met the diagnostic criteria for probable pd - d. among the subjects with mmse scores < 26, the combination of impaired attention, executive dysfunction, and memory impairment was the most common (51.4%). the months reversed test can not be used in japanese as a test for attention owing to linguistic nonequivalence. the moca clock drawing test may be more appropriate than the phonemic fluency test as a measure of executive function. perhaps consideration should be given to replacing the mmse pentagon copying test with a different test of visuospatial function, such as the moca cube copying test, to improve sensitivity when screening for pd - d. | backgrounddementia is a new focus of research on improved treatment for parkinson 's disease (pd). in 2007, a screening tool for pd dementia (pd - d) was developed by the movement disorder society (level i testing), which still requires verification by a large population study.methodswe conducted a cross - sectional and multicenter study including 13 institutions administering the mini - mental state examination (mmse) and montreal cognitive assessment (moca) to 304 pd patients (mean age : 70.6 8.3 years ; mean hoehn and yahr stage : 2.7 0.7).resultsin all, 34.5% of the patients had mmse scores < 26 ; 94.3% of these patients had impairments in 2 cognitive domains and met the criteria for probable pd - d by level i testing. executive dysfunction combined with attention and memory impairment was most common (51.4%). in the level i subtests of executive function, the score for phonemic fluency declined by < 50% in patients with high moca scores (24 - 30 points) and lacked specificity for pd - d. no patient had visuospatial impairment (measured by the pentagon copying subtest) alone, and the score for pentagon copying stayed at 70% even in patients with low mmse scores (12 - 25 points), therefore lacking sensitivity for pd-d.conclusionslevel i testing with administration of the mmse and moca is a practical and efficient screening tool for pd - d. however, the phonemic fluency and pentagon copying tests should be replaced by more specific / sensitive ones when screening for pd - d. |
we conducted a cross - sectional study among poultry farmers and cullers from 1 district in each of the 5 provinces (chachoengsao, kanchanaburi, khon kaen, sukhothai, and suphanburi) where outbreaks of avian influenza (h5n1) among poultry and human infections had been confirmed since january 2004 (figure). with the assistance of provincial human and animal health authorities informed consent was obtained, and a brief interview was conducted. because the precise timing of potential exposures could not be determined, a single serum sample was collected from each patient and stored at 20c until tested under biosafety level 3 (bsl-3) conditions. (4), at the department of microbiology, faculty of medicine, siriraj hospital, mahidol university by microneutralization assay (micro - nt) for antibody to h5n1 viruses. before this study, senior laboratory staff from siriraj hospital received 2 weeks of on - site training by a visiting scientist from the us centers for disease control and prevention who had expertise with this assay. the world health organization (who) defines a positive test result as a microneutralization antibody titer for influenza virus (h5n1) of > 80 with a confirmatory elisa or western blot assay (3,4) (www.who.int/csr/disease/avian_influenza/guidelines/case_definition2006_08_29/en/index.html). serum samples from persons > 50 years of age were excluded from laboratory analysis because the microneutralization assay for antibodies against subtype h5n1 has been reported to be less specific for older persons (5). gray shading indicates provinces with confirmed avian influenza outbreaks ; black outlines indicate provinces included in this study. of 350 farmers asked to participate, 322 (92%) enrolled in the study, of which 167 (52%) the mean age of participants was 34 years (range 550 years) (table). among participants, 188 (58%) reported handling sick or dying poultry, 107 (33%) were involved in culling operations of apparently well poultry in outbreak areas, and 27 (9%) reported only contact with well poultry in the context of routine farming practices. although no study participant had an anti - h5n1 antibody titer of > 80, 7 (2.2%) farmers had lower reactive antibody titers. of these, 4 had titers of 10, 2 had titers of 20, and 1 had a titer of 40. the small number of study participants with anti - h5 antibody titers precluded statistical comparisons to those without reactive antibodies. poultry farmers and cullers are at increased occupational risk for exposure to avian influenza viruses. however, since 2004, infections have been less commonly reported in cullers, while poultry farmers have made up a large proportion of cases worldwide. a study in hong kong special administrative region, people s republic of china, examined influenza virus (h5n1) transmission and risk factors among poultry workers and government workers involved in culling during the 199798 outbreak (3). the study concluded that although no hospitalized poultry workers were identified among the 18 patients in that outbreak, 3% of 293 cullers and 10% of 1,525 poultry workers had antibody titers against influenza (h5n1) of > 80, which suggested that a substantial number of mild or asymptomatic infections had occurred in this occupationally exposed population. in contrast, we found that no poultry workers had microneutralization titers > 80, whereas 7 (2%) had lower titers that did not meet the who definition for seropositivity. the lower titers may have resulted from cross - reactivity with circulating antibodies after previous human influenza virus infections (5,6). these low titers could be the result of mild or asymptomatic influenza (h5n1) infections because not all influenza virus infections invariably result in marked antibody responses (7). likewise, these results could reflect the decay of antibody titers over time (8). finally, the micro - nt assay is a highly specific and strain - sensitive test. although we used the same virus that was circulating in thailand at that time, these lower titers could be attributable to infections with another virus variant. most human influenza (h5n1) infections have occurred in persons who had had direct contact with sick or dying poultry (911). while human infections with avian influenza (h5n1) continue to be reported, growing evidence indicates that this virus is not easily transmitted from poultry to humans and that mild or asymptomatic infections in humans are not common. a seroepidemiologic investigation in rural cambodia surveyed 351 participants from 93 households in an area where influenza (h5n1) infections in poultry and a single fatal human case had been documented (10). despite frequent, direct contact with poultry suspected of having influenza (h5n1) infection, a similar study in nigeria found that all of 295 poultry workers had negative test results for influenza (h5n1) neutralizing antibodies (12). studies of healthcare workers suggest that transmission of influenza virus (h5n1) to hospital staff who cared for infected patients also appears to be uncommon (1315). our study provides additional evidence to suggest that influenza virus (h5n1) is not easily transmitted to humans. however, the wide geographic distribution of this subtype, ubiquitous exposures, and the high case - fatality ratio from the infection underscore the importance of adherence to poultry - handling practices recommended by the food and agriculture organization and who (www.wpro.who.int/nr/rdonlyres/7693baf7-13e7-42db-b92b-004cf5d517e7/0/whointerimrecommendation26012004.pdf, www.fao.org/ag/againfo/subjects/en/health/diseases-cards/avian_qa.html#8). molecular surveillance indicates that the avian influenza virus (h5n1) continues to evolve rapidly (www.who.int/csr/disease/avian_influenza/guidelines/recommendationvaccine.pdf). additional seroepidemiologic studies are warranted to monitor for changes in transmissibility and the spectrum of clinical illness. | in thai provinces where avian influenza outbreaks in poultry had been confirmed in the preceding 6 months, serum from 322 poultry farmers was tested for antibodies to avian influenza virus subtype h5n1 by microneutralization assay. no study participant met the world health organization serologic criteria for confirmed infection. |
dental surfaces with pits and fissures are susceptible for dental caries. at the same time, the usual prevention measures against caries (topical and systemic fluoride therapy) have the lowest effects in these areas. this advantage is enhanced through sealant strength in penetrating to the depth of the fissures and retention in the porosity of the etched enamel surfaces. the better the resin and the etched enamel contact, the more increased will be the adhesion of s sealant to the enamel. therefore, the cleaner the teeth surface and the better etching, rinsing and drying, the better resin can wet the etched enamel and penetrate into deeper depth. the remains of the etching products will cause in lesser resin penetration in the etched enamel and causes lesser retention ; therefore, the total eradication of dissolved calcium phosphate salts will result into improvement of sealant bond to the tooth. the purpose of the present study is to assess the effect of different rinsing angles of the etched enamel to the bond strength between the tooth and the sealant. in the present study, 60 first - maxillary premolars, which had been extracted for orthodontic purposes were collected. all teeth were free of any caries, breakage or enamel problems at buccal or lingual surfaces. following rinsing with water, the teeth depending on the rinsing angle, the teeth were divided into six groups as follows : group i (with a rinsing angle of 90) ; group ii (with a rinsing angle of 75) ; group iii (with a rinsing angle of 60) ; group iv (with a rinsing angle of 45) ; group v (with a rinsing angle of 30) ; and group vi (with a rinsing angle of 15). following this division, the teeth in each of the groups were sub - divided into two groups as follows : buccal (five teeth) and palatal (five teeth) randomly. next, the teeth roots were mounted in plaster blocks (vel - mix stone, kerr, italy) with a diameter of 2.5 cm and a height of 2 cm. they were mounted in such a way that the height of contour of the surfaces were almost in vertical positions. tow fissures, one vertically and one horizontally were cut by a high speed hand piece to indicate the location, which was etched and rinsed. by means of a low speed hand piece, the examination sites on the teeth were cleaned or 10 sec. in this step, the teeth were microscopically tested for a second time to find any enamel problems. in order to clarify the rinsing angle exactly, a syringe on a moving protractor in a distance of 2 cm of the tooth surface a barometer, on the water pouring system, helped to uniform the water pressure, pouring on all samples. after 20 sec of etching (by scotch bond, 3 m, st paul, mn, usa), the teeth surfaces were rinsed for 20 sec at each of the angles mentioned above. the teeth were dried in such a way that the chalky - white enamel surface was visible. to put the sealants (concise, 3 m, espe, usa) on the etched surfaces, transparent plastic pipes with an internal diameter of 3.6 mm and a height of 4 mm were used. finally, following sealant placing in the plastic cylinders and curing them, the plastic matrix was dissected by the scalpel blade and finally separated. a cylinder with a diameter of 3.5 and a height of 4 mm was remained on the buccal or palatal surface of the teeth. the roots were dissected from 2 cm below the c.e.j. by a diamond discus (d and z diamond, germany) and separated. all samples were kept in distilled water for 24 h, before being prepared for micro tensile bond test. a water syringe mounted onto a protractor to determine the exact rinsing angle the teeth were sectioned in x and y directions to obtain bonded sticks with a cross - sectional area of approximately 1.0 mm, measured to the nearest 0.01 mm with a digital caliper (mitutoyo corp, japan). the samples were then fixed on the plate of the micro tensile test machine (bisco inc., usa) conforming to the midline of the plates. in this machine, a pulling strength of 200 n with a speed of 0.5 mm / min was exerted to the samples. the load (n) and the bonding surface area of the specimens in mm were noted and the microtensile bond strength calculated in mpa. the type of failure in the samples was determined at a magnification of 90 by stereo microscope (szx olympus, japan). the failure mode was classified in three forms as follows : adhesive ; failure between the enamel and the sealantcohesive ; failure inside the sealanta mixed form of adhesive and cohesive. adhesive ; failure between the enamel and the sealant cohesive ; failure inside the sealant a mixed form of adhesive and cohesive. in the case of a mixed failure mode, the amount of the remaining composite on the surface was calculated by a grid lens and expressed in percentage. the normal distributions of bond strength and sealant remnant were verified using the kolmogrov - smirnov test and an all pair - wise multiple comparison post - hoc tukey test used to determine the statistical significance between the groups. the normal distributions of bond strength and sealant remnant were verified using the kolmogrov - smirnov test and an all pair - wise multiple comparison post - hoc tukey test used to determine the statistical significance between the groups. the results concerning the tensile bond strength of the samples mpa (mean sd) are shown in table 1. tukey honestly significant difference test showed that the tensile bond strength in the group i was significantly higher than those in groups v and vi (p < 0.05). kolmogroy - smirnov test results on the amount of tensile bond strength in different rinsing angles the percentage values of different failures at various rinsing angles and in total are demonstrated in table 2. the most prevalent failure in the present study was a furthermore, an increase in the rinsing angle from 15 to 90, is associated with a reduction in the number of specimens with adhesive failures and an increase in mixed and cohesive failures [figure 2 ]. based on table 3, the highest average of residual composite percentage was at 90 while the lowest was at 15. percentage of different failures at various rinsing angles comparison of failure modes in different rinsing angles kolmogrov - smirnov test results on the % of the residual composite at different rinsing angles the intention of this study was to improve caries preventive effect of sealants by improving their adhesion to the underlying enamel. a move in this direction is needed as the interest of the dental profession in preventive dentistry is increasing. considerable research has been undertaken to optimize the bond strength between resin based dental materials and dental enamel. the ability of the resin to penetrate between the enamel crystallites and rods is a determinant of the bond strength between the resin and the etched enamel. to enhance the enamel surface free - energy and resin penetration,, we showed that a general trend exists between reduction in bond strength and the rinsing angle from 90 to 15 [table 1 ]. it seems that the increased tensile bond strengths, associated with the more thorough washing, were due to the increased rinsing angle. as shown in [figure 3 ], at 90, the complete evacuation of the etching products can be achieve while the persistence of deposit remnants on enamel surface might possibly be a consequence of reduction in the rinsing angle. these data ; therefore support the hypothesis that failure to remove the etched enamel surface deposits markedly reduces bonding between the sealant and the enamel. indeed, scanning electron microscopy of etched enamel surfaces has shown the presence of such deposits after inadequate washing. a : 90, b : 75, c : 60, d : 45, e : 30, f : 15. increasing the angle from 15 to 90 results in discharging of more precipitates from the resin tags incomplete cleansing obscures the fine structure of the etched prism by a generalized precipitate layer so that the surface does not lend itself to mechanical bonding. the efficacy of sealants and resins has been evaluated by numerous mechanical testing methods, such as shear bond strength, tensile bond strength and micro - leakage. in this study, we used the micro tensile technique, a recent but widely employed test to evaluate the bond strength of adhesive materials to dental tissues. compared to other debonding tests, this method tends to increase adhesive failures. because of the small size of the specimens in the micro tensile test, stress distributions are improved and failures occur in materials with true ultimate strength closer to the applied load. thus, the mean micro tensile bond strength varied between 23 mpa and 24.98 mpa [table 1 ]. in spite of such high bond strength this behavior may be attributed to the micro tensile test characteristics, which have been shown to produce a more homogeneous stress distribution and to the prismatic orientation of the intact enamel surface, which is mostly perpendicular to the enamel periphery and parallel to the applied stress. these will result in higher bond strength and fewer cohesive failures. a possible explanation for the cohesive failures occurred in groups i and ii [figure 2 ] may be the higher bond strength in these groups, which was probably stronger than the ultimate tensile bond strength of the adhesive, leading to failures in sealant material. a reduction in the rinsing angle was coupled with more adhesive failures, which would be attributable to the lower bond strength observed in the acute angles. light stereo microscopy of the specimens revealed that the presence of more sealant remnants at the failure site when a rinsing angle of 90 was used. this indicates that bond failures in acute angles occur more frequently at the enamel adhesive interface consistent with the other findings in this study, showing that rinsing at 90 results in improved adhesion of sealant to the enamel surface. it should be further noted that in view of the fact that many researchers consider a clinically successful bond as being between 15 mpa and 35 mpa, it seems that there was acceptable bond strength in the acutest rinsing angles. since many variables encountered in the clinical cases can not be controlled for in - vitro, we are unable to make direct inferences from the information obtained in this study to the clinical cases. therefore, future clinical trials that assess the longevity of sealants placed subsequent to washing with different rinsing angles are necessary. within the limitations of this in - vitro study, the following conclusions can be drawn : rinsing the etched enamel surface at an acute angle (15 and 30) reduces the bond strengtha direct relationship existed between the rinsing angle and percent values of sealant remnants on the tooth surface after failure, contributing to the bond strength of each grouphowever, there will be acceptable bond strengths in the acutest rinsing angle. rinsing the etched enamel surface at an acute angle (15 and 30) reduces the bond strength a direct relationship existed between the rinsing angle and percent values of sealant remnants on the tooth surface after failure, contributing to the bond strength of each group however, there will be acceptable bond strengths in the acutest rinsing angle. | background and objectives : attempts to enhance bond strength of the sealant have been among the most important sides of dental research. the purpose of the present study was to evaluate the effect of using different rinsing angles on the micro - tensile bond strength of the sealant to the etched enamel.materials and methods : sixty first - premolars were randomly assigned to six groups based on the rinsing angle applied (15, 30, 45, 60, 75 and 90). following etching and rinsing, a 4-mm height build - up of sealant material was created. bonded specimens were sectioned into sticks (1 mm 1 mm), which were subjected to micro - tensile bond strength, testing at a cross head speed of 0.5 mm / min.statistical analysis used : the data were analyzed by kolmogorov - smirnov and post - hoc tukey test.results:the tensile bond strength in specimens rinsed at 90 were statistically higher compared to those rinsed at 15 and 30 (p < 0.05) and increasing the angle from 15 to 90 was correlated with a reduction in the number of specimens with adhesive failures.conclusions:rinsing the conditioned enamel surface at 90 may improve the bond strength and retention of the sealant. |
urine has now become one of the most attractive biological fluids in clinical proteomics [1, 2 ]. a number of urinary proteomic studies have been conducted and have revealed urinary biomarker candidates for renal systemic diseases and malignancies of urinary tract [35 ]. proteomic analysis of urines can be applied to biomarker search in nonrenal diseases as well [68 ]. although urinary proteome analyses have been conducted by various gel - based and gel - free techniques, comprehensive urinary proteome analysis is not an easy task because the urine has very diluted protein concentration with high levels of salts. sample preparation, processing, and storage for urinary proteomics have been reviewed [1013 ]. more recently, an optimized quantitative proteomic strategy for urine biomarker discovery was described. in any event, maximal protein recovery from urine is essential for detecting trace quantities of proteins present in urine for potential biomarker discovery. for this purpose, one possible factor responsible for loss of trace amounts of urinary proteins could be adsorption to sample tubes, but this issue has not been well addressed so far to our knowledge. many attempts have been made to prevent the adsorption of plasma proteins and to improve blood compatibility by surface modification [15, 16 ]. ishihara and coworkers reported on rapid development of hydrophilicity and protein adsorption resistance by polymer surfaces bearing poly(2-methacryloyloxyethyl phosphorylcholine (mpc)-co - n - butyl methacrylate) (poly(mpc - co - bma)) [17, 18 ]. we took advantage of this coating method in the present study and assessed whether conventionally used tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(mpc - co - bma) can minimize the adsorption. poly(mpc - co - bma) was obtained from ai bio - chips co., ltd (tokyo, japan). a total of 6 different types of conventional urine collection tubes were used in this study. tubes made from polystyrene (ps) (cat # 10200), polypropylene(pp) cat # 72200, polyethylene terephthalate(pet) (cat # 23540), and styrene - butadiene copolymers / methyl methacrylate - styrene (sbc / ms) (cat # 17300) were purchased from toyo kagaku kizai co., ltd. tubes made from acrylonitrile - styrene (as) copolymers (cat # 479511373) were from nittobo medical co., ltd., japan and those made from styrene - butadiene copolymers (sbc) (cat # 3324a000a-10) were from asiakizai co., ltd. the conventional tubes made by as were coated by poly(mpc - co - bma) as described by futamura.. an aliquot of the samples was taken for routine urinalysis, and the rest of the samples were centrifuged (700 g, 5 min at room temperature), and the supernatant was subjected to assess protein adsorption on test tubes as described below. one ml of two different levels of pooled proteinuric urines (equivalent to 15 mg / dl and 50 mg / dl, resp.) obtained from 10 patients with renal disease were put into poly(mpc - co - bma)-coated and noncoated urine correction tubes and were kept at room temperature for 15 min. after aspiration of the urines, the tubes were washed with 200 l of pbs three times. after the third wash and pbs being aspirated, 100 l of page sample buffer (electrophoresis) (50 mm tris - hcl, ph 6.8 containing 50 mm dithiothreitol, 0.5% sds, and 10% glycerol) or 1% tca aqueous solution (maldi - tof ms analysis) was added and the tubes were vortexed for 30 sec to dissolve possibly adsorbed proteins. the solution which contained proteins possibly adsorbed on the urine tubes was then analyzed using sds - page (perfect nt gel w, 1020% acrylamide, 20 wells ; drc co., ltd., the gel was stained with cbb (phastgel blue r ; ge healthcare, little chalfont, uk). the proteins separated by sds - page were identified by in - gel tryptic digestion of the proteins followed by ms. in - gel molar quantities of recovered peptide fragments were estimated from the staining intensity of the sds - page bands that were digested in - gel with trypsin. digested peptides roughly equivalent up to 1 pmol of protein were injected into a trap column : 0.3 5 mm l - trap column (chemicals evaluation and research institute, saitama, japan), and an analytical column : 0.1 50 mm monolith column (amr, tokyo, japan), which was attached to a hplc system (nanospace si-2 ; shiseido fine chemicals, tokyo, japan). the solvent composition of the mobile phase was programmed to change in 35 min cycles with varying mixing ratios of solvent a (2% v / v ch3cn and 0.1% v / v hcooh) to solvent b (90% v / v ch3cn and 0.1% v / v hcooh) : 550% b 20 min, 5095% b 1 min, 95% b 3 min, 955% b 1 min, 5% b 10 min. purified peptides were introduced from hplc to an ltq - xl (thermo scientific, calif, usa), an ion trap mass spectrometer (itms), via an attached pico tip (new objective, mass, usa). the ms and ms / ms peptide spectra were measured in a data - dependent manner according to the manufacturer 's operating specifications. the mascot search engine (matrix science, london, uk) was used to identify proteins from the mass and tandem mass spectra of peptides. peptide mass data were matched by searching the human international protein index database (ipi, july 2008, 72079 entries, european bioinformatics institute) using the mascot engine. the minimum criterion of the probability - based mascot / mowse score was set with 5% as the significant threshold level. for 2-de analysis, we used the method described by oh - ishi. and kawashima. briefly, one ml aliquots of urine samples kept at room temperature for 15 min in poly(mpc - co - bma)-coated and noncoated urine correction tube were concentrated up to 20-fold by bjp concentrator (prochem, ma, usa) to 50 l and lyophilized. the lyophilizate was resuspended by 200 l of immobiline reagent (7 m urea, 2 m thiourea, 4% chaps, 2% dtt, 2% pharmalyte, broad range ph 310). finally, 50 l of the 5-fold urine sample was applied to the ief agarose gel. the agarose gel was then transferred to the perfect nt gels w (1020% gradient of polyacrylamide gel ; drc. the protein spots were detected, quantified, and matched with the 2-de gel view analysis software, progenesis samespots (nonlinear dynamics ltd. the protein spots were excised from the gel and identified, as we previously described. one ml aliquots of urine samples (containing 50 mg / ml protein) were kept at room temperature for 15 min in poly(mpc - co - bma)-coated and noncoated urine collection tube. proteins possibly adsorbed on the tubes were collected as described above for the gel - based method and were analyzed by the maldi - tof ms. to obtain quantitative data of the possibly adsorbed proteins, we used stable isotope - labeled 5.9 kda fibrinogen alpha c chain fragment (fic 5.9) as an internal standard as described by sogawa.. we obtained the stable isotope - labeled synthetic fic 5.9 from the anygen co., ltd. (the underlined amino acids were synthesized with c, n uniformly labeled fmoc amino acids.). in urine analysis, ten microliters of sid (stable isotope - labeled) -fic 5.9 solution (0.5 pmol/l sid - fic 5.9, mb - wcx binding solution) and 5 l of urine sample were transferred to a 200 l pcr tube (thermo fisher scientific k.k., ten microliters of sid - fic 5.9 solution (0.025 pmol/l sid - fic 5.9, mb - wcx binding solution) and 5 l of extracted samples (urine samples kept in tube) were transferred to a 200 l pcr tube. a 10 l homogenous magnetic particle solution was added, mixed with the other solutions, and allowed to sit for 5 min. the pcr tubes were placed in a magnetic bead separator (mbs ; bruker daltonics gmbh) for 30 s for magnetic fixation of the mb - wcx particles. we added 100 l of the washing solution and carefully mixed it with the magnetic beads. we then replaced the tube into the mbs device and moved it back and forth between adjacent wells on each side of the device 's magnetic bar. after fixation of the magnetic beads for 30 s, the supernatant was aspirated. after the final wash, we eluted the bound molecules by incubating them for 1 min with 5 l mb - wcx elution solution and then used the mbs device to collect the eluate. for the final step, we added 5 l of the mb - wcx stabilization solution to the eluate. we then mixed 1 l of the eluate with 5 l of a matrix solution (0.3 g / l a - cyano-4-hydroxycinnamic acid in ethanol : acetone, 2 : 1). we spotted 1 l of the mixture onto an anchorchip target plate (bruker daltonics gmbh) and allowed it to dry. protein calibration standard (protein calibration standard 1, bruker daltonics gmbh) was dissolved in 125 1 l. we applied 0.5 1 l of the solution to target spots in proximity to the urine samples for external calibration. we placed the anchorchip target plate into the autoflex ii tof / tof mass spectrometer (bruker daltonics gmbh) and into the ultraflex iii tof / tof mass spectrometer (bruker daltonics gmbh), which is controlled by flexcontrol software 3.0 (bruker daltonics gmbh). spectra were acquired in a positive linear mode in the mass range of 600 to 10,000 da. the concentration of the proteins adsorbed to urine collection tube was estimated from the ratio of the peak intensity of adsorbed proteins to the peak intensity of sid - fic 5.9. for identification of peptides as we previously described, the anchorchip target plate was also placed in an ultraflextreme tof / tof mass spectrometer (bruker daltonics) and the maldi - tof / tof ms / ms spectrum was recorded in lift mode. the maldi - tof / tof ms / ms spectrum was subjected to a database search using the mascot (matrix science, london, uk) database search engine. the search parameters were as follows : no enzyme specificity, 25 ppm mass tolerance for the parent mass, and 0.2 da for fragment masses. one hundred urine samples requested for urinalysis on routine basis at the division of laboratory medicine and clinical genetics, chiba university hospital were used. the urine samples were aliquoted (10 ml) to poly(mpc - co - bma)-coated and noncoated collection tubes and were kept at room temperature for 15 min before use. nine different quantitative urinalysis such as protein, glucose, creatinine, microalbumin, beta-2-microglobulin, amylase, n - acetyl--d - glucosaminidase, urea nitrogen, uric acid, and six kinds of electrolytes were conducted using biomajesty jca - bm6010 (jeol ltd. ten different parameters are assessed : specific gravity (sg, measured via a built - in refractometer), erythrocytes, leukocytes, nitrite, ph, protein, glucose, ketones, bilirubin, and urobilinogen. this equipment includes digital imaging and auto - particle recognition (apr) (chatsworth, ca, usa) software to classify urine particles and quantitatively report results. in this study, 4 categories red blood cells (rbc), white blood cells (wbc), squamous epithelial cells (sec), and casts were classified by the apr software. we evaluated the statistical significance using ibm spss statistics 18 software (spss inc., il, usa). a p < 0.05 was considered significant using the mann - whitney u test. proteins adsorbed on the poly(mpc - co - bma)-coated and noncoated tubes were analyzed by sds - page. as shown in figure 1(a), a few distinct protein bands (60 kda, 66 kda and 100 kda) were noted in samples obtained from noncoated as tubes. no clear bands were visible in samples obtained from poly(mpc - co - bma)-coated as tubes under these experimental conditions. lc - ms analysis of trypsin digests of these bands identified 11 proteins as listed in table 1. protein adsorption on the tubes was observed in 6 different types of conventionally used urine collection tubes as shown in figure 1(b). urine specimens kept in poly(mpc - co - bma)-coated and noncoated tubes were subjected to the agarose 2-de as described in the methods section. nine protein spots the intensities of which were significantly greater (p < 0.05) in samples kept at poly(mpc - co - bma)-coated tubes compared with those kept at noncoated tubes were selected based on the results obtained in seven different experiments. these differences were most likely as the results of more protein adsorption on noncoated tubes. these spots were excised and subjected to in - gel trypsin digestion followed by lc - ms. proteins and/or peptides adsorbed on the conventional urine tubes were also detectable by the maldi - tof ms. the intensities of the two peaks (2556 m / z and 2654 m / z) were notably greater in samples obtained from poly(mpc - co - bma) noncoated tubes. similar results were obtained in 7 different experiments ; the expression levels of the two peaks expressed as the ratio to the internal standard, sid - fic 5.9, were significantly greater (p < 0.001) in poly(mpc - co - bma) noncoated tubes than in coated tubes. using maldi - tof / tof ms / ms technology, we successfully identified the two peaks (2556 m / z and 2654 m / z) as internal sequences of the fibrinogen alpha c chain fragment. the peptide sequences of the 2556 m / z and 2654 m / z were deagseadhegthstkrghaksrp and deagseadhegthstkrghaksrpv, respectively. the mean value of the ratio of m / z 2654 to sid - fic5.9 was 5.70 (figure 2(a) right panel) in poly(mpc - co - bma) noncoated urine collection tube. it is 0.85 g / ml when converting it into the protein concentration. figure 2(b) shows representative view of the spectrum of urine samples kept in urine tubes. the relative intensities of the two peaks (2556 m / z and 2654 m / z) were greater in the samples kept in poly(mpc - co - bma)-coated tubes than those kept in noncoated tubes. similar results were obtained in 7 other experiments ; the differences quantified using the sid - fic 5.9 were statistically significant (p < 0.007 for 2556 m / z and p the quantitative values of urinalysis parameters in samples kept in poly(mpc - co - bma)-coated and noncoated collection tubes are presented in table 3. they were all comparable between the two groups in linear regression equation, slope of linearity, correlation coefficients ranged from 0.997 to 1.014. the results of dipstick urinalysis and urinary sediment determinations were also comparable between the two groups. the issue of preanalytical factors affecting sample integrity is often overlooked and yet is critically important. although preanalytical factors for serum or plasma proteome analysis have been extensively studied, the impact of adsorption of proteins and peptides on urine tubes on biomarker discovery using urinary proteomics is not well investigated. the results of this study indicate that conventionally used urine collection tubes adsorb proteins and/or peptides and that the surface coating of the tubes by poly(mpc - co - bma) can minimize the adsorption without any significant effects on routine chemical determinations. in this study, urine samples were kept in poly(mpc - co - bma)-coated and noncoated tubes for 15 min. this is because it is generally known that proteins adsorb onto a surface within a few minutes when the material contacts body fluids such as blood, plasma, and tears [2426 ]. protein adsorption is one of the most important phenomena in determination of the biocompatibility of materials [16, 18 ]. polymers composed of mpc and hydrophobic alkylmethacrylate units have been extensively used in many medical devices as coating materials to improve the blood compatibility of these devices [1518 ]. however, this coating requires a long wetting pretreatment time to achieve equilibrium hydration by the reorientation of the phosphorylcholine groups [16, 27 ]. in this study, urinary proteins were found to be adsorbed on poly(mpc - co - bma) noncoated urine collection tubes made from six different types of materials. recently, futamura. succeeded in rapid development of hydrophilicity and protein adsorption resistance poly(ethylene terephthalate) (pet) surfaces bearing poly(mpc - co-2-vinylnaphthalene(vn)) (pmvn). it should be considered, however, that coating effects on the plastic tubes appear to be dependent on the initial properties of the plastic tubes. we took advantage of this coating method and showed that protein adsorption can be reduced in urine samples as well. most of the proteins listed in table 1 are representative protein in urine and have theoretical isoelectric points between 4.7 and 7.0, suggesting that proteins with isoelectric point of this range are likely to be adsorbed on the conventional urine tubes employed in this study. since the ph of the urine samples kept in poly(mpc - co - bma)-coated and noncoated tube was similar, it is unlikely that the differences obtained in this study were due to ph difference. it has been reported that the factor responsible for protein adsorption to the plastic tube might depend on the relation of sample ph and protein pi. three proteins (alpha-1-antitrypsin, igkv1 - 5 protein, prostaglandin - h2 d - isomerase) were detected in common for two different comparisons. ceruloplasmin, one of the proteins listed in table 1, is a biomarker of uranium nephrotoxicity. the use of the coated tubes did not have any impact on the urine analysis of routine parameters. since there were no significant differences in the quantitative data of abundant urinary proteins including albumin and beta-2-microglobulin, the effects of adsorption on abundant proteins may be minimal. but, in searching for urinary protein biomarkers with low concentration, possible adsorption on conventional urine tubes should be considered. since the material used in the conventional and noncoated tubes employed in the present study is widely used around the world, possible adsorption of trance amount of proteins to urine collection tubes should be considered in proteome analyses of urine samples. urine is one of the attractive biofluids in clinical proteomics. in chasing very low abundance urinary proteins and peptides, however, loss of biomarker candidates by adsorption on urine tubes should be considered. in this study, we found that protein adsorption on the conventionally used urine collection tubes is not negligible, and that the adsorption can be reduced by using a tube coated by hydrophilic polymers without any effects on routine urinalysis. i believe that these findings should be shared by those who are interested in urinary proteomic study. | one possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. this issue, however, has not been well addressed so far. recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (mpc)-co - n - butyl methacrylate (bma)) (poly(mpc - co - bma)) has been developed mainly to prevent the adsorption of plasma proteins. we assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(mpc - co - bma) can minimize the adsorption. proteinuric urine samples were kept in poly(mpc - co - bma)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by sds - page, 2-de, and the maldi - tof ms. it was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(mpc - co - bma) can minimize the adsorption without any significant effects on routine urinalysis test results. although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine. |
cutis laxa is a rare connective tissue disorder, which may be inherited or acquired, and is characterized by progressive looseness of skin, associated with abnormalities of other organs and structures such as lung, vasculature or gastrointestinal tract as they contain elastic tissues. the hereditary form of cutis laxa may be inherited as either autosomal dominant, x - linked recessive or autosomal recessive trait. the acquired form is even rarer than the inherited form and is often associated with some form of preceding or accompanying cutaneous eruptions like eczema, urticaria, erythema multiforme, sweet 's syndrome or multiple myeloma. the clinical appearance is characterized by inelastic, loose and pendulous skin that produces a wrinkled, prematurely aged appearance. the primary change is in the elastic fiber, which is decreased or nearly absent in the dermis. here an itchy eruption had developed on the anterior neck 5 years ago, and it lasted for about a month. several months later, she noticed that the skin of that area was becoming wrinkled and loose. the wrinkling and looseness progressed for 4 to 5 months after which it became stable. she was otherwise well and had no history of dyspnoea, chest pain, palpitation or visual problems. family history of similar skin lesion was absent. physical examination revealed a circumscribed palm sized area of lax, extensible, wrinkled skin localized to the anterior part of the neck [figure 1a ]. lesion on the neck of a 14-year - old female lesion on the neck showing stretching of the lax skin a 40-year - old muslim male presented to us with a complaint of loose skin all over his body for last 10 years. he was well till the age of 30, when he noticed that his skin was gradually becoming wrinkled and loose. it started from forehead and progressed slowly downwards over a couple of years after that the process stabilized. at the time of presentation the skin of his forehead [figure 2a ], neck, abdomen and back [figure 2b ] was diffusely wrinkled and it hung in folds. lesion on the scalp of a 40-year - old male lesion on the back of a 40-year - old male in both the cases investigations revealed normal values for complete blood count, erythrocyte sedimentation rate, routine urine analysis, blood biochemistry, hepatic enzymes and serum electrophoresis. biopsy specimens taken from the involved area were stained with hematoxylin - eosin (h and e), von kossa and verhoeff - van gieson stain. h and e staining revealed normal epidermis with diminished elastic fibres and mild non - specific chronic lymphohistiocytic infiltrate in upper dermis [figure 3a ]. the elastic fibres present were fragmented, stained uneven and showed granular appearance, as evidenced in verhoeff - van gieson stain [figure 3b ]. histopathology of skin lesion of case 1 showing normal epidermis with diminished elastic fibres and mild non - specific chronic lymphohistiocytic infiltrate in upper dermis (h and e, original magnification 10) histopathology of skin lesion of case 2 showing fragmented and unevenly stained elastic fibres with granular appearance, (verhoeff - van gieson stain, original magnification 10) an itchy eruption had developed on the anterior neck 5 years ago, and it lasted for about a month. several months later, she noticed that the skin of that area was becoming wrinkled and loose. the wrinkling and looseness progressed for 4 to 5 months after which it became stable. she was otherwise well and had no history of dyspnoea, chest pain, palpitation or visual problems. family history of similar skin lesion was absent. physical examination revealed a circumscribed palm sized area of lax, extensible, wrinkled skin localized to the anterior part of the neck [figure 1a ]. lesion on the neck of a 14-year - old female lesion on the neck showing stretching of the lax skin a 40-year - old muslim male presented to us with a complaint of loose skin all over his body for last 10 years. he was well till the age of 30, when he noticed that his skin was gradually becoming wrinkled and loose. it started from forehead and progressed slowly downwards over a couple of years after that the process stabilized. at the time of presentation the skin of his forehead [figure 2a ], neck, abdomen and back [figure 2b ] was diffusely wrinkled and it hung in folds. lesion on the scalp of a 40-year - old male lesion on the back of a 40-year - old male in both the cases investigations revealed normal values for complete blood count, erythrocyte sedimentation rate, routine urine analysis, blood biochemistry, hepatic enzymes and serum electrophoresis. biopsy specimens taken from the involved area were stained with hematoxylin - eosin (h and e), von kossa and verhoeff - van gieson stain. h and e staining revealed normal epidermis with diminished elastic fibres and mild non - specific chronic lymphohistiocytic infiltrate in upper dermis [figure 3a ]. the elastic fibres present were fragmented, stained uneven and showed granular appearance, as evidenced in verhoeff - van gieson stain [figure 3b ]. histopathology of skin lesion of case 1 showing normal epidermis with diminished elastic fibres and mild non - specific chronic lymphohistiocytic infiltrate in upper dermis (h and e, original magnification 10) histopathology of skin lesion of case 2 showing fragmented and unevenly stained elastic fibres with granular appearance, (verhoeff - van gieson stain, original magnification 10) acquired cutis laxa usually begins in adulthood with insidious development of loose skin often starting at face and neck. the first case with localized lesion described above could be differentiated from pseudo xanthoma elasticum (pxe) by the absence of the characteristic yellow color of pxe and lack of calcification with von kossa stain. the second case could be differentiated from elher danlos syndrome, as the skin was lax and recoiled slowly, and there was no joint involvement. the girl (case 1) was referred to a reconstructive surgeon for treatment, and after successful excision of the loose skin only a fine linear scar was visible. but not much could be done for the older patient (case 2). he was advised to take one vitamin e capsule (400 mg) daily for 2 months. there was not much improvement after 2 months and the patient was lost to follow up. this option may be a less invasive way to improve facial defects in these patients. the cause of acquired cutis laxa is largely unknown, though various hypotheses such as altered copper metabolism, excess elastase, and immune - mediated mechanisms have been proposed. is that elastolysis may result from release of granulocytic elastase because neutrophils contain large amounts of elastase, and is also found in macrophages and platelets. up to 100-fold increase in the serum elastase level has been demonstrated in a patient with autosomal recessive cutis laxa. a suggested mechanism of cutis laxa is that, unknown causes stimulate mast cells to release mediators of inflammation, such as histamine, leucotrienes, neutrophil and eosinophil chemotactic factors. consequently, there is a progressive infiltration of neutrophils and eosinophils around the blood vessels in the papillary dermis and between the collagen fibers in the mid- and reticular dermis. these neutrophils release elastase, and elastolysis first begins in areas with neutrophilic infiltration around the blood vessels and between collagen fibers, and then it gradually spreads to the entire dermis. these changes could be localized, as in the first case or they could be generalized leading to universal involvement, as in the second case. | cutis laxa is a rare disease that may be either inherited or acquired. the acquired form is rarer than the inherited form. pathogenesis of this disease is largely unknown. two cases of acquired cutis laxa are reported here and neither of them had any systemic involvement or any history of drug intake. one of them had localized disease with history of preceding cutaneous inflammation. the other patient with generalized lesion lacked any history of preceding illness. the patient with localized lesion was treated satisfactorily by reconstructive surgery. the other patient had generalized involvement, for which no satisfactory treatment could be offered. |
pierre robin sequence (prs) is the triad of glossoptosis, micrognathia, and cleft palate1. varying degrees of airway obstruction are a major problem that may be encountered in these patients. if left untreated, these airway obstructions can lead to acute and chronic hypoxia, apnea, cyanosis, aspirations, respiratory tract infections, feeding difficulty, malnutrition and failure to thrive. the treatment protocol for prs starts with conservative management, which mostly consists of positional adjustments (prone positioning) and nasopharyngeal airway placement2. surgical options include tongue - lip adhesion (tla), glossopexy, tracheostomy and early mandibular lengthening by distraction osteogenesis34. in 1946, douglas5 described the tla technique for the management of pierre robin patients. tla is a simple procedure in which the tongue is attached anteriorly to the lower lip. glossoptosis and microretrognathia were managed conservatively with oxygen via nasal cannula and prone positioning during the first few months of life. moreover, this child 's presentation was complicated by poor weight gain, hence was referred to plastic surgery department and tla was performed. following the procedure, patient showed clinical improvements such as weight gain, reduced episodes of respiratory infection and improvement in oxygen saturation. the central surface of the tongue was pulled anteriorly to make contact with the lower lip mucosa in order to estimate the necessary raw area. b) an inferiorly based 21-cm flap was raised from the lower lip, and a superiorly based flap of the same size was raised from the ventral surface of the tongue.(fig. 3) written permission had been obtained from the patient to report and publish the identifying photographs prior itself. based on a study by sher.6, in which flexible fiberoptic nasopharyngoscopy was used in 53 infants with prs, four types of obstruction have been described. in type i obstruction, the most common, obstruction is caused by posterior movement of the tongue against the posterior pharyngeal wall. type ii obstruction is due to posterior and superior displacement of the tongue, promoting contact between the tongue, the velum and the pharyngeal wall in the superior oropharynx. type iv is due to constriction of the pharynx in a circular manner by movement of the tongue and the lateral pharyngeal walls. infants with prs requiring surgical intervention for airway management should be treated with procedures offering the lowest morbidity. nasaopharyngeal tubes are an unstable mode of therapy, especially for long - term use7. in a recent survey, 91% of pediatric otolaryngology fellowship programs cited tracheostomy as the safest and most reliable surgical method for long - term airway management in infants with prs3. however, complications related to tracheostomy (tracheal stenosis, granuloma formation, tracheal fistula formation), cannula obstruction and accidental decannulation have been reported in 19% to 49% of patients, and tracheostomy - related mortality ranges from 2% to 8.5%3. due to the longer associated hospital stays, difficulty in obtaining consent, demand for home care and greater morbidity, especially in newborns, most surgeons avoid tracheostomy. in tracheostomy, an additional deterrent is that fact that infants who have undergone tracheostomy may demonstrate significant delays in speech production and language development. unlike with tracheostomy, infants treated with tla suffer minimal or no adverse effects on their speech development 3. however, tla is only an appropriate treatment option in infants with type i obstruction without any associated intubaanomalies. in type ii, iii and iv obstructions the concept of tla for obstructive apnoea relief in prs was first popularised by douglas in 19463. this procedure involved excising a rectangular area of mucosa on the undersurface of the tongue and extending the area onto the floor of the mouth, alveolus and lower lip. the tongue was brought forward, and the lateral edges of the mucosal incision were sutured to one another, creating broad - based adhesion of the tongue to the lip, alveolus, and floor of the mouth. the procedure was associated with complications like dehiscence, tongue laceration, injury to wharton 's duct and cicatricial ankyloglossia. in 1960, routledge8 reported a modification that avoided many of these complications. then, in 1977, randall 9 described moving the retention suture to the posterior aspect of the tongue, providing better support for the tongue base during healing. in 1992, argamaso10 later described the use of an internal retention suture looped around the mandible and sutured to the tongue 's muscularis propria. he stressed the importance of genioglossus release from its mandibular insertion to allow improved anterior displacement of the tongue11 serial procedures such as division of tla (performed after 1 year) and mandibular distraction osteogenesis are performed for cosmetic reasons after facial skeletal maturity has been attained. in the case of a failed tla, repeat surgery, tracheostomy or early mandibular distraction osteogenesis mandibular distraction osteogenesis has multiple complications, such as damage to tooth buds, inferior alveolar nerve injury, and unsightly facial scars. thus, mandibular distraction osteogenesis should be reserved for the small minority of infants who demonstrate persistent apnea despite tla. we perform tla in infants who do not benefit significantly from non - surgical techniques such as prone positioning, supplemental oxygen, oral airway, nasopharyngeal airway, continuous positive airway pressure or endotracheal intubation. serial procedures such as division of tla (performed after 1 year) and mandibular distraction osteogenesis are performed for cosmetic reasons after facial skeletal maturity has been attained. in the case of a failed tla, repeat surgery, tracheostomy, or early mandibular distraction osteogenesis prs infants may have associated cleft palates. if cleft palate is present, a repair is required at 1.5 to 2 years of age as was done in this child. in conclusion, the surgical management of airway obstruction in neonates with pierre robin anomaly is dependent on adequate assessment of the site, the mechanism of airway collapse and associated congenital anomalies. tla can safely be employed with less morbidity in comparison with tracheostomy and mandibular distraction osteogenesis and therefore plays an important role in relieving airway obstruction refractory to positioning and cannulation in neonates. | patients with pierre robin sequence exhibit varying degrees of airway obstruction and feeding difficulty. in some patients, airway obstruction may be profound, warranting surgical intervention to maintain a patent airway. the purpose of this article is to highlight the advantages of the tongue - lip adhesion procedure for the management of airway obstruction in such patients compared to the currently available options. |
the research sample consisted of 40 individuals with asd who referred to forensic medicine centers of mazandaran province and 40 individuals with war - related ptsd following displacement after iran - iraq war who referred to clinical centers of mazandaran province. then, 40 individuals who had the experience of trauma but were not diagnosed as patients with acute stress disorder wereas normal individuals for the study. to ensure the correctness of psychiatrist 's diagnosis, the composite international diagnostic interview was utilized. age over 25 years, minimum education level of grade 9, being married, being informed, and voluntary consent were considered as the entrance criteria. data were analyzed using multivariate analysis of variance (manova) and multivariate regression analysis. instruments utilized for data collection were as follows : the composite international diagnostic interview (cidi) : the original version of cidi has been edited by world health organization (1997). this interview is of good validity in diagnosing mental disorders. in terms of validity of cidi, the specificity for most psychiatric disorders is high (over 0.85), but the sensitivity for diagnoses is to some extent lower, especially for severe psychiatric disorders (like schizophrenia). the instrument 's reliability assessment through test - retest method showed that the reliability of diagnosis for most psychiatric disorders is within an acceptable range kappa over 0.4 ; however, for schizophrenia, this figure was smaller. results obtained from the two diagnostic systems of dsm - iv and icd were similar to a large extent (20). stroop color - word test (scwt) : stroop test is a classic scale for attention processing and cognitive set shifting ability. the test in its original form consists of four cards : word reading (first card), color naming (second card), reading words regardless of the color (third card), and telling the color of words regardless of what is written (forth card). each card displays 25 stimuli ordered in 5 lines and 5 columns (21). the impact of event scale revised (ies - r) : the impact of event scale revised (7) has 22 items graded on a 5-point scale. emmerik. came to a range of 0.76 to 0.84 for cronbach 's alpha coefficient of these scales (22). in study of abdi, moradi&akramian (2008), abolghasemi, bakhshian and narimani(2013) showed that the impact of event scale revised was correlated with the thought control questionnaire (r= 0.54, p < 0.01) (24). posttraumatic cognitions questionnaire (pcq) : this questionnaire was developed by foa. the posttraumatic cognitions questionnaire consists of 33 items to which the subjects should answer on a 7-point scale ranging from fully disagree (1) to fully agree (7). cronbach 's alpha coefficient for this test is 0.97. in a study by louise field, cronbach 's alpha coefficients for this test were reported as 0.83, 0.83, and 0.70 in subscales of negative self - appraisal, negative world appraisal, and self - blame respectively (15). in study of abolghasemi, bakhshian and narimani(2013) cronbach 's alpha coefficient for this inventory was 0.96. bakhshian (2011) showed that this questionnaire was correlated with the thought control questionnaire (r = 46, p < 0.01) (24). first, individuals encountering damaging events who were diagnosed as having acute stress disorder (in legal medical center of sari) and posttraumatic stress disorder (veterans of iran - iraq war) were identified. at the next stage, to ensure the correctness of psychiatrist 's diagnosis, subjects were administered the composite international diagnostic interview for asd and ptsd. after diagnosis the research sample consisted of 40 individuals with asd who referred to forensic medicine centers of mazandaran province and 40 individuals with war - related ptsd following displacement after iran - iraq war who referred to clinical centers of mazandaran province. then, 40 individuals who had the experience of trauma but were not diagnosed as patients with acute stress disorder wereas normal individuals for the study. to ensure the correctness of psychiatrist 's diagnosis, the composite international diagnostic interview was utilized. age over 25 years, minimum education level of grade 9, being married, being informed, and voluntary consent were considered as the entrance criteria. data were analyzed using multivariate analysis of variance (manova) and multivariate regression analysis. instruments utilized for data collection were as follows : the composite international diagnostic interview (cidi) : the original version of cidi has been edited by world health organization (1997). this interview is of good validity in diagnosing mental disorders. in terms of validity of cidi, the specificity for most psychiatric disorders is high (over 0.85), but the sensitivity for diagnoses is to some extent lower, especially for severe psychiatric disorders (like schizophrenia). the instrument 's reliability assessment through test - retest method showed that the reliability of diagnosis for most psychiatric disorders is within an acceptable range kappa over 0.4 ; however, for schizophrenia, this figure was smaller. results obtained from the two diagnostic systems of dsm - iv and icd were similar to a large extent (20). stroop color - word test (scwt) : stroop test is a classic scale for attention processing and cognitive set shifting ability. the test in its original form consists of four cards : word reading (first card), color naming (second card), reading words regardless of the color (third card), and telling the color of words regardless of what is written (forth card). each card displays 25 stimuli ordered in 5 lines and 5 columns (21). the impact of event scale revised (ies - r) : the impact of event scale revised (7) has 22 items graded on a 5-point scale. emmerik. came to a range of 0.76 to 0.84 for cronbach 's alpha coefficient of these scales (22). in study of abdi, moradi&akramian (2008), abolghasemi, bakhshian and narimani(2013) showed that the impact of event scale revised was correlated with the thought control questionnaire (r= 0.54, p < 0.01) (24). posttraumatic cognitions questionnaire (pcq) : this questionnaire was developed by foa. (1999) to assess thoughts and ideas associated with trauma (16). the posttraumatic cognitions questionnaire consists of 33 items to which the subjects should answer on a 7-point scale ranging from fully disagree (1) to fully agree (7). cronbach 's alpha coefficient for this test is 0.97. in a study by louise field, cronbach 's alpha coefficients for this test were reported as 0.83, 0.83, and 0.70 in subscales of negative self - appraisal, negative world appraisal, and self - blame respectively (15). in study of abolghasemi, bakhshian and narimani(2013) cronbach 's alpha coefficient for this inventory was 0.96. bakhshian (2011) showed that this questionnaire was correlated with the thought control questionnaire (r = 46, p < 0.01) (24). first, individuals encountering damaging events who were diagnosed as having acute stress disorder (in legal medical center of sari) and posttraumatic stress disorder (veterans of iran - iraq war) were identified. at the next stage, to ensure the correctness of psychiatrist 's diagnosis, subjects were administered the composite international diagnostic interview for asd and ptsd. after diagnosis in asd group, 57.5% of subjects were male and 42.5% were female ; in ptsd group, 62.5% of the subjects were male and 37.5% were female ; and in normal group, 50% of the subjects were male and 50% were female. educational status of the subjects in asd group was as follows : 10% with grade 9 certificate, 37.5% with high school diploma, 25% with technician 's degree, and 7.5% with a bachelor 's degree and higher. the mean age of the subjects was 45.20 (sd = 9.29) for the ptsd group, 37.05 (sd = 7.67) for the asd group, and 39.60 (sd = 7.73) for the normal group with a range of 25 - 40 years. as table 1 indicates, manova test results revealed a significant difference between individuals with asd and those with ptsd considering at least one of the dependent variables (wilks lambda = 0.748, f = 30.39, p = 0.001). significant differences among the mean response inhibition scores (f = 82.82) and negative self - appraisal (f = 230.98) in three groups of asd, ptsd, and normal (p < 0.001). there were also significant differences among the mean scores obtained for the constituents of and self - blame(f = 22.46) and post - trauma symptoms (f = 264.45) in the three groups of subjects (p < 0.001). comparing mean of response inhibition and cognitive appraisal scores in patients with asd and ptsd, and normal subjects acute stress disorder(asd) posttraumatic stress disorder (ptsd) as observed in table 2, the results of lsd test on comparison of the mean response time scores in stroop test show that the mean response time in patients with asd is significantly more than that of those with ptsd, and so it is about those with asd compared to normal subjects, and about those with ptsd compared to normal subjects (p < 0.01). also, it is revealed that the mean scores for the number of errors (stroop cards) in patients with asd are significantly more compared to patients with ptsd, and it is the same for patients with asd compared to normal subjects and for patients with ptsd compared to normal subjects (p < 0.01). moreover, the mean cognitive appraisal is significantly more in patients with asd than in patients with ptsd and normal subjects, and similarly, it is significantly more in patients with ptsd compared to normal subjects (p < 0.001). to determine the role of each variable, response time, number of errors, and cognitive appraisal were analyzed as predictive variables, and post - trauma symptoms were analyzed as criterion variable in regression equation.the observed f value in the table is significant and 61.4% of the variance of asd symptoms is explained by the variables of response time, number of errors, and cognitive appraisal (adjusted r2 = 0.745, f= 42.25, p < 0.001). considering beta values, response time (= 0.706), number of errors (= 0.159), and cognitive appraisal (= 0.730) can significantly predict the changes relevant to post trauma symptoms in subjects with ptsd (table 3). also, 86.3% of the variance in the symptoms of acute stress disorder is explained by the variables of response time, number of errors, and cognitive appraisal (adjusted r2 = 0.376, f= 39.71, p < 0.001). considering beta values, response time (= 0.409) and cognitive appraisal (= 0.503) can significantly predict the changes related to post - trauma symptoms in subjects with asd (table 3). mean of response inhibition and cognitive appraisal scores in subjects with asd, ptsd, and normal individuals with lsd test acute stress disorder(asd) posttraumatic stress disorder (ptsd), normal(n) regression results for predictor of post trauma symptoms in ptsd and asd acute stress disorder (asd) posttraumatic stress disorder(ptsd) results showed that the mean of response inhibition(response time and error rate) scores is significantly higher in patients with asd compared to patients with ptsd ; similarly, it is higher in both groups of patients compared to the normal group (p < 0.01). the mentioned studies showed that individuals with ptsd have longer reaction times in responding to stroop test compared to normal people. it can be said that when an intrusive thought occurs, it is associated with emotional disturbance, physiological arousal, and interference in concentration or task completion which can last for a long time. therefore, the presence of intrusive thoughts causes interference in concentration which entails occurrence of more errors and longer reaction time (25). cognitive theories emphasize that manifestations of negative emotions, like anxiety, cause attentional biases in information processing which results in occupation of the individual 's mind and response to the perceived danger (26). thus, belief system of the individual or cognitive schemata may be an important predisposing factor in developing posttraumatic disorders. specifically, the occurrence of intrusive and unacceptable thoughts is part of normal experience, but individuals who develop posttraumatic disorders have inflexible beliefs about personal meaning of these thoughts and their catastrophic implication. occupation of the mind with such beliefs and thoughts causes interference in individual 's concentration, and this entails psychomotor retardation which is followed by increased number of errors and longer time responding to tasks. therefore, it seems that the presence of bothering and intrusive thoughts produces interference in the process of individual 's attention and concentration on tasks ; and the effort to cope with these thoughts causes occupation of the mind and influences the individual 's responding to the tasks. thus, individuals act more slowly in responding to the tasks and display more errors. to explain the obtained results, it can be said that individuals with acute stress disorder who are in a closer time interval to the traumatic event experience more frequent and intense disturbance and intrusive thoughts. results also showed that the mean of impaired cognitive appraisal scores(negative self - appraisal, negative world appraisal and self - blame) are significantly higher in asd patients compared to ptsd persons, and this is also the case with the two groups of patients compared to normal individuals. this result suggests that although both groups of patients have impaired cognitive appraisals, the problem is more serious in patients with asd. 's (15) studies, which showed that individuals have more negative cognitive self - appraisals after traumatic events. recent cognitive theories (13) state that negative appraisal of an event and its consequences leads to the development of a sense of ongoing threat which causes the disorder to remain. negative self - appraisals following trauma can focus on durable negative interpretations about oneself (e.g. i will never improve). according to ehlers & clark 's model of posttraumatic disorders, these negative appraisals determine individual 's emotional response to the trauma and base cognitive and behavioral strategies (e.g. thought suppression, behavioral avoidance form fearful situations, social seclusion, etc) which later on contribute to maintenance of disorder. despite the discrepancies observed in the results, there is a consensus that self - opinion, specifically negative self - appraisal, has important roles in development and maintenance of posttraumatic reactions, and that such a negative self - view may have a central role in hindering one 's natural health improvement following traumatic events. they believe that one of the sources of one 's intense and constant fear is the appraisals they make of the trauma and/or its sequels based on their own traits. negative appraisal of the trauma consists of negative appraisal of emotions appeared during a shocking event and negative appraisal of feelings and activities occurred during a shocking event, negative perception of others responses, and perception of a permanent change in the life after the accident (26). in the present research, the significance of self - blame deference in the groups under study may be a reflection of variant nature of traumatic events that the individuals under study have experienced. in traumas like heart attack that the individual 's life style, e.g. physical activity, diet, smoking, and alcohol consumption, plays an important role in the creation of trauma or in road accidents that the individual plays a role in its happening, they blame themselves more following the trauma (13). however, in traumas like war that the individual has no part in its occurrence, the amount of self - blame is smaller. since the acute stress disorder group in this study included individuals who were selected to be studied following road accidents, intrusion, rape, and violence, and the ptsd group included war veterans results of showed that response inhibition and cognitive appraisal explain 86.3% of the variance symptoms in ptsd. this result indicates that response inhibition and cognitive appraisal significantly predict changes associated with ptsd symptoms in the persons involved (p < 0.01). ehlers & clark (13) suggest that negative cognitions probably lead to different cognitive - behavioral coping strategies and end up developing or intensifying ptsd symptoms. these strategies can contribute to the development and maintenance of ptsd symptoms by directly producing ptsd symptoms, preventing negative appraisals of trauma and its sequels from alteration, and preventing the nature of incidental memory from alteration (13). research evidence also shows that thought suppression and selective attention are maladaptive cognitive strategies that directly predict the symptoms of ptsd (26). in addition, a number of studies have reported the power of these dysfunctional coping strategies to predict symptoms of ptsd (26, 27). also, results revealed that variables of response inhibition and cognitive appraisal explain 61.4% of the variance of symptoms in asd patients. this result indicates that response inhibition and cognitive appraisal significantly predict changes associated with the symptoms of acute stress disorder in the persons involved (p < 0.01). research evidence (28, 29, 30) suggests that the experience of traumatic event is in contrast with individual 's previous schemata, and the disruption of individual 's beliefs about the self and the world leads to the development of acute stress symptoms. this evidence predicts that experience of traumatic event leads the individual to challenge with one or more constant beliefs. consequently, the individual 's system of integrated and intact self would be under pressure and disturbed resulting in acute stress symptoms. the oldness of stroop color - word test may have affected the results of this study. the cross - sectional study design restricts our understanding of the relationships between the variables. furthermore, longitudinal designs in particular would be able to determine the role of response inhibition and cognitive appraisal on the subsequent development of psychopathology. collecting data on the basis of self - report scales can bring about the distortion of information because of unconscious defenses, bias in responding, and personal introducing styles. conducting the research with adult groups restricts the generalizability of the results to different age groups. moreover, it is recommended that tools which are not self - reporting be applied to collect the research data. these results have important implications for pathology, prevention, and treatment for ptsd patients. these directions may provide important information about the influence of these variables on individual 's perceptions and evaluations of inappropriate behaviors and symptoms which may inform our conceptualization and treatment of psychopathology. to sum, the results of this study showed that response inhibition and cognitive appraisal is significantly higher in patients with asd than in patients with ptsd. also, the research results indicated that response inhibition and cognitive appraisal significantly predict changes associated with ptsd symptoms in the persons involved (p < 0.01). in conclusion, | objectivethe purpose of the present study was to compare response inhibition and cognitive appraisal in clients with acute stress disorder, clients with posttraumatic stress disorder, and normal individuals.methodthis was a comparative study. the sample consisted of 40 clients with acute stress disorder, 40 patients with posttraumatic stress disorder, and 40 normal individuals from mazandaran province selected through convenience sampling method. data were collected using composite international diagnostic interview, stroop color - word test, posttraumatic cognitions inventory, and the impact of event scale.resultsresults showed that individuals with acute stress disorder are less able to inhibit inappropriate responses and have more impaired cognitive appraisals compared to those with posttraumatic stress disorder. moreover, results showed that response inhibition and cognitive appraisal explain 75% of the variance in posttraumatic stress disorder symptoms and 38% of the variance in posttraumatic stress disorder symptoms.conclusionthe findings suggest that response inhibition and cognitive appraisal are two variables that influence the severity of posttraumatic stress disorder and acute stress disorder symptoms. also, these results have important implications for pathology, prevention, and treatment of posttraumatic stress disorder and acute stress disorder. |
cord blood is currently used as a source of hematopoietic stem cells (hscs) to treat a variety of malignant and nonmalignant hematologic disorders [15 ]. its utilization has expanded beyond the pediatric field thanks to several favorable factors, including ease of collection, prompt availability, absence of risk to donors, and reduced risk of adverse effects such as graft versus host disease (gvhd) [68 ]. in adult patients, the therapeutic potential of this procedure is still limited by the low number of nucleated cells contained in each unit and by the consequent risk of delayed or inadequate recovery of neutrophils (pmn) and platelets (plt) [914 ]. direct cord blood cells intrabone transplantation (ib - cbt) has been proposed as a tool to improve the hscs engraftment and thus their therapeutic effectiveness [914 ]. however, the mechanisms underlying this potential benefit have not been fully defined so far. local hscs engraftment might be assumed after ib - cbt, yet a recent experimental study documented that, in rats, approximately 5% of hscs remain within the injected bone site after administration. it, however, favorably compares with the 10% seeding efficiency of conventional protocols implying intravenous administration [16, 17 ]. in fact, should the same homing features characterize transplanted hscs present in the circulating blood, the extra local engraftment produced by ib - cbt might play a relevant role in accelerating the reconstitution of recipient hematopoiesis. in order to test this hypothesis, we verified whether the metabolic activity in the site of injection predicts the subsequent reconstitution of recipient hematopoiesis in adult patients treated with ib - cbt. to this purpose, we exploited the high sensitivity of f - fluorodeoxiglucose (fdg) pet - ct imaging. this method has been proved to accurately estimate bone marrow (bm) mitotic activity as well as its hematopoietic response to chemotherapy in a large number of conditions. in this setting, this technique provided us with the advantage to simultaneously evaluate bm metabolism, as an index of proliferating activity, within the injected bone as well as in remote sites. the subsequent monitoring of peripheral blood cell reconstitution confirmed the main role of injected sites in the early phases of hematopoietic recovery. the study group included 21 subjects (15 males, age 43 11 years, range 2266) who were submitted to ib - cbt according to the indications reported in table 1. in all patients, cord blood was used as a source of hscs due to the absence of human - leucocyte - antigen-(hla-) matched donors in a clinically useful time frame. treatment plan was reviewed and approved by the local ethical committee and written informed consent was obtained from all patients before any treatment. as requested by our standard operative treatments, all patients received fractionated 1012 gy total body irradiation on days 6, 5, and 4 before transplantation (subdivided into one fraction per day or fractions of 2 gy twice a day). drug treatment included the administration of thiotepa (8 mg / kg) on day 8, treosulfan (1012 g / m daily) on days 7, 6, and 5, fludarabine (40 mg / m per day) from day 7 to day 3, and cyclophosphamide (60 mg / kg per day) on days 2 and 1. all patients received a combination of cyclosporine, mycophenolate mofetil, and antithymocyte globulin as gvhd prophylaxis. intravenous cyclosporin was started on day 7 at a daily dose of 1 mg / kg and, in the absence of gvhd, was tapered until discontinuation from day + 90 to day + 180 after transplantation. the dose of cyclosporin was adjusted in order to maintain serum through concentration within the 150300 mg / l range. mycophenolate mofetil was given at a dose of 15 mg / kg orally twice a day in the first 28 days after ib - cbt. antithymocyte globulin was given at a dose of 3 mg / kg per day on days 3 and 2. all patients received granulocyte colony stimulating factor. to verify the dependence of fdg uptake upon the history of bone trauma within the ib - cbt site, we evaluated a group of 18 patients (14 males, mean age 47 12 years, range 1965) as control. they were submitted to pet - ct scanning 2540 days after bm aspiration biopsy from the iliac crests. these evaluations were performed in the course of followup, two months after the last chemotherapy application for lymphoma. cord - blood units were obtained from several cord - blood banks. before ib - cbt, surface expression of cd34 antigen was determined after thawing with a facsaria flow cytometer using a single platform protocol (ishage). total number of nucleated cells was in all cases greater than 1 10 per kilogram of recipient body weight, as determined before freezing. cord - blood units were thawed in a 37c water bath, according to the procedure described by rubinstein.. to remove dimethyl - sulphoxide, the same solution was used to prepare a 20 ml cell suspension that was divided into four 5-ml syringes for administration. before the procedure, patients were sedated with intravenous propofol and positioned in the flank posture. a standard needle for bm aspiration (14 gauge) was inserted into a superior - posterior iliac crest ; an aspiration of about 1 ml was then executed in order to ascertain that the needle was securely inserted into bm cavity. subsequently, each cord - blood - cell suspension was gently infused into the intrabone space. this procedure was then repeated for all the remaining aliquots at a distance of about 35 cm from each other. in all cases, an uneven distribution of injections was used, randomly selecting the side subjected to three or one administration. peripheral blood cell counts was estimated daily for the first month after ib - cbt and twice weekly thereafter. for the purposes of the present study, the last time point was set at 100 days. plt counts, obtained using standard methods, were thus considered as marker of hematopoiesis reconstitution. growth of colony forming cells (cfc) was assessed by use of a complete semisolid medium (methocult, stem cell technologies inc., vancouver, bc, canada) under standard conditions at day + 30 and + 100 after bm transplant. after 6 hours fasting, serum glucose level was measured before intravenous injection of 4.85.2 mbq of 18f - fdg per kilogram of body weight. all these procedures were performed in a quiet room with the patient recumbent in supine position and instructed not to move. fdg - pet imaging, from vertex to toes, started 60 to 90 minutes after tracer administration and was performed using an integrated pet / ct scanner (hirez ; siemens medical solutions, knoxville tn, usa). pet raw data were reconstructed by means of ordered subset expectation maximization (osem) and attenuation correction was performed by means of ct. the entire ct dataset was coregistered with the 3-dimensional pet data using an integrated software interface (syngo image fusion ; siemens erlangen, germany) to combine anatomical and functional images in all body districts. fdg uptake was semiquantitatively evaluated in the injection sites as well as in remote bm. to this purpose, volumetric regions of interest (vrois) were manually drawn on the anatomic ct images in order to identify the trabecular space within each iliac crest as well as in the soma of all five lumbar vertebrae (figure 1). maximal standardized fdg uptake values (suvs) were measured in these three districts to estimate the metabolic activity of both injection sites and remote bm. the extension of the metabolic effect produced by ib - cbt in both iliac crests was also evaluated. to this purpose, we applied a criterion previously described by kidd and grigsby to measure the total mass of metabolically active solid cancer. this method, implemented in syngo software, provides an estimation of the total volume included in the iliac crest vroi presenting fdg uptake values scoring at least 40% of maximum suv. once defined in each iliac crest, this volume was multiplied for the corresponding average suv providing an integrated index of both extension and metabolic activity of engrafted bm (the so - called metabolic volumetric product). unpaired or paired t - test was used, as appropriate, to compare the same variables at the three different time points. cord blood unit was mismatched with the recipient for 2/6hla antigen and the disparity was in class i at the antigenic level. infused cell dose was 3.2 0.68 (range 1.95.1) 10 cells / kg. no immediate side effects, such as pain, hemorrhage, or local infections, were recorded. three out of 21 (14%) patients died within 50 days from the procedure due to multiorgan failure. in two further patients, plt counts at day 100 the remaining patients achieved complete hematological recovery as documented by the monitoring of peripheral blood cells counts and remained in remission thereafter. in fact, pmn concentration was 0.019 0.009 10/l at the time of bm transplantation (day 0), it increased to 3.9 2 10/l (p < 0.0001 versus baseline) at day 50, and remained relatively stable thereafter until day 100 (4.6 1.7 10/l, p = ns versus day 50). despite the needed transfusion before ib - cbt, plt counts showed a different pattern, progressively rising from day 0 (3.1 1.1 10/l) to 82 50 10/l at day 50 (p < 0.01 versus day 0), up to 122 46 10/l at day 100 (p < 0.01 versus both previous temporal milestones). pmn and plt recovery was considered established when these elements reached for five consecutive days and in the absence of transfusions a steady and consistent concentration greater than 0.5 10/l and 20 10/l, respectively. as expected, this event occurred earlier for pmn than for plt (22.6 4.5 versus 34 8.6 days, p < 0.001). among surviving patients, acute gvhd incidence and severity were graded at day 100 as 0 (n = 6), 1 (n = 8), 2 (n = 3), or 3 (n = 1) according to conventional criteria. no patient showed areas of abnormal fdg uptake diagnostic for neoplastic localization within the skeleton or in the rest of the body. however, fdg uptake in the iliac crests was clearly visible and it was most often higher than in the rest of bm (figure 2). in particular, with respect to the soma of the five lumbar vertebrae, injected bone districts showed significantly higher maximal suvs (4.1 1.7 versus 3.2 0.7, resp. in fact, in control group, suvs presented the opposite pattern being lower within iliac crests with respect to lumbar vertebrae (1.8 0.6 versus 4.2 1.5, resp., p < 0.001 ; figure 1). this finding was also confirmed by the fact that suvs were significantly higher within the injected bone of transplanted patients than in the corresponding bm segments of controls (4.1 1.7 versus 1.8 0.6, resp. conversely, lumbar bm metabolic activity showed the opposite, though less evident, difference being lower in ib - cbt patients than in controls (3.2 0.7 versus 4.2 1.5, resp. furthermore, iliac crests submitted to three injections showed higher suvs than those treated with only one (4.3 1.1 versus 3.3 0.9, resp., p < 0.01). this could suggest the existence of a link between the number of injected cells and local metabolic increase. this concept is further confirmed by the fact that a trend toward the correlation was observed between metabolic activity in the injected bone and number of locally injected cd34 cells (r = 0.35, p = 0.12). to verify whether bm metabolic activity was related to the speed of hematopoietic recovery, we divided the population into two groups according to the median interval between ib - cbt and recovery of cell counts. dividing the population according to the median interval needed for pmn recovery did not show any significant different maximal suvs, in neither iliac crest nor in remote bm between the two groups. on the contrary, when plt renewal was considered, patients who presented faster repopulation (recovery occurring before the median day 35) displayed higher maximal suvs than the slow - recovery group. this metabolic increase was evident within the injected site (4.43 1.7 versus 2.9 0.8, resp., p < 0.05), but it was not significant within lumbar vertebrae (3.5 0.7 versus 3.1 0.8, resp., intensity and extension of metabolic activity in both injected sites predicted the reconstitution of recipient hematopoiesis at day 100. in fact, a close direct correlation was observed between maximal suvs in the site of injection and in remote bm (r = 0.70, p < 0.001) (figure 3). metabolic activity in both iliac crests was unrelated to plt counts both at time of transplant (r = 0.32, p = ns) and at day 50 (r = 0.26, p = ns) ; however, it robustly predicted plt counts at day 100 (r = 0.72, p < 0.001 ; figure 4). in fact, maximal suv in lumbar vertebrae did not correlate with plt counts neither at day 0 (r = 0.06, p = ns) nor at day 50 (r = 0.28, p = ns), while it approached the statistical significance threshold at day 100 (r = 0.31, p = 0.06) (figure 5). this finding was confirmed and corroborated by data related to the volumetric extension of hscs engraftment in the site of injection. in fact, the volume encompassing voxels with suvs 40% of the maximum was 2.5 0.5 ml and correlated with late follow - up plt counts (r = 0.64, p < 0.01). similarly, the metabolic volumetric product in the sites of injection showed an even closer correlation with plt counts at the end of the study (r = 0.75, p < 0.01). the present study aimed to verify whether hematopoiesis recovery after ib - cbt is at least partially explained by the local increase in bm metabolism, as an index of hscs proliferating activity, within the injection site. fdg uptake was higher in iliac crests than in remote bm and it was dependent upon the number of locally administered cord blood cells. the degree of glucose consumption in the site of injection was strictly correlated with the metabolic pattern in lumbar vertebrae, suggesting a role for locally engrafted cells in determining cell density and metabolism in remote bm. plt counts recovery at day 100 was accurately predicted by degree and extension of metabolic activity in the injected bone and only loosely by fdg uptake in remote bm. although the mechanisms underlying this potential benefit are far beyond the scope of the present study, these data suggest that ib - cbt could induce a local engraftment store capable to transiently accelerate the hematopoietic recovery and thus to bridge the traditional gap that limited the clinical potential for cord blood transplantation in adult patients. the potential role of fdg imaging in evaluating hematopoietic function has been documented by a number of studies reporting a biphasic response of bm suv to chemotherapy with a reduction during cytotoxic treatment followed by an increase during the proliferating reaction in the subsequent recovery phase [2123 ]. in hematologic patients, the presence of residual disease might partially limit the interpretation of tracer uptake as an index of hscs metabolism. however, several considerations suggest that this possibility should have played a relatively minor role in the present study. in fact, all patients were exposed to a complex regimen encompassing chemotherapy and total body irradiation before ib - cbt. moreover, the focal hot spots typical of residual disease did never occur in our patients who rather displayed a homogeneous and diffuse increase in bm fdg uptake, with a relatively higher tracer retention selectively involving the injected iliac crests. accordingly, although a small number of diseased cells might have been present in some patients, their dispersed nature should have been not recognizable due to the limited spatial resolution of pet / ct imaging. in agreement with these considerations, the high sensitivity of whole - body fdg imaging permitted to simultaneously evaluate the metabolic activity in the injected bone and in the remote bm. the comparison with a control group of patients studied 2040 days after iliac crest biopsy rules out the possible interference on bm metabolism caused by the bone trauma following injection. in fact, these patients not only showed lower suvs in iliac crests with respect to subjects treated with ib - cbt, rather they also displayed an opposite tracer distribution, presenting highest uptake values in lumbar vertebrae bodies. accordingly, both observations strongly and concordantly indicate that the enhancement in iliac crests metabolism in transplanted patients is the consequence of local hscs administration rather than a possible metabolic consequence of previous bone trauma or residual disease. this concept is further supported by the observation that fdg uptake in each injected bone was dependent on the corresponding number of locally administered cells. one month after ib - cbt corroborates the concept that the increased glucose consumption represents both an index of a high cell density and a marker of increased proliferating activity within the site of injection. the volume of metabolically active hscs within the iliac crests was estimated using a method validated for studies dealing with bm metabolism and cancer evaluation [19, 24 ] and appears too small to explain the restored production of circulating blood cells. on the other hand, fdg uptake in the injection site was directly correlated with the corresponding value in lumbar vertebrae. moreover, metabolic pattern in iliac crests closely predicted both the day of plt normalization as well as plt concentration 100 days after transplant. these findings suggest that metabolic rate of locally engrafted cells profoundly contributes to remote bm colonization, although this process is not yet completed at one month after transplant when fdg uptake in lumbar vertebrae only loosely correlates with the subsequent hematopoietic recovery. in line with the notion that megakaryopoiesis is a more accurate indicator for the quality of hscs function than granulopoiesis [13, 2527 ], however, neutrophil counts display a slower recovery with respect to plt [19, 27 ] and a large variability due to the interference of numerous confounding factors. both these features hamper the analysis of a possible correlation between hscs metabolism and granulocyte counts in a small population sample. thus, independently from the extremely relevance of leukocyte reconstitution in determining patients survival, plt count was more suited for the purpose of the present study. present data indicate that direct injection of cord blood cells into bm spaces facilitates their in situ proliferating activity. the initial benefit is probably followed by an improved migration of stem cells to remote uninjected bm sites. altogether, these effects at least partially explain the accelerated hematopoietic recovery observed after ib - cbt. the present study does not provide any insight into the clinical potential and effectiveness of ib - cbt that has been already reported in the literature. nevertheless, the robustness of our findings and their agreement with previous experimental and clinical evidence indicate that ib - cbt might represent a useful model to elucidate the biology of donor hscs in the recipient. | background. despite advancements in comprehension of molecular mechanisms governing bone marrow (bm) homing of hematopoietic stem cells, cord blood transplant (cbt) suffers from a slow rate of hematopoietic recovery. intrabone (ib) injection has been proposed as a method able to improve speed of bm engraftment with respect to conventional iv protocols. however, the mechanisms underlying this benefit are largely unknown. aim. to verify whether ib - cbt determines a local engraftment able to predict the reconstitution of recipient hematopoiesis. design and methods. twenty - one patients with hematologic malignancies received ib injection into both iliac crests of 3.2 0.68 107/kg cord blood cells. one month following ib - cbt, pet - ct imaging was performed. maximal standardized uptake values (suvs) were assessed in bm of both iliac crests and in all lumbar vertebrae. results. maximal suv within iliac crests was higher than in lumbar vertebrae (4.1 1.7 versus 3.2 0.7, resp., p = 0.01). however, metabolic activity in these two different bm districts was significantly correlated (r = 0.7, p < 0.001). moreover, fdg uptake values within the injection site closely predicted platelet recovery 100 days after ib - cbt (r = 0.72, p < 0.01). conclusions. the metabolic activity of injected bm predicts the subsequent rate of hematopoietic recovery after ib - cbt, suggesting a pivotal role of the local engraftment in the reconstitution of recipient hematopoiesis. |
infection is one of the major complications of total knee arthroplasty (tka) that can be best treated with two - stage revision arthroplasty1,2). however, the use of non - articulating spacers, such as bead spacers and static block spacers, in two - stage revision has been associated with undesirable side effects : the difficulty of joint exposure at the time of revision arthroplasty due to scarring and shortening of the joint capsule and quadriceps, additional bone loss, and impaired postoperative rehabilitation and functional recovery due to restriction on weight - bearing between the two stages3). the introduction of articulating prosthesis with antibiotic - loaded acrylic cement (prostalac ; depuy orthopaedics inc., warsaw, in, usa) has contributed to reduced infection rates and increased range of motion (rom). unfortunately, it is neither affordable nor applicable to all patients4,5). thus, some studies suggested using articulating prostheses that are manufactured intraoperatively by the surgeon6,7). the custom - made spacers are described as facilitating the procedure, allowing for early ambulation and minimal soft tissue contractures, and yielding favorable outcomes in terms of preservation of the muscle strength and rom. still, prolonged use of such prostheses has been associated with quadriceps contractures and joint capsule adhesion in two - stage revision. hence, efforts have been focused on inventing temporary articulating spacers for two - stage revision arthroplasty. in this article, we present our experience of two - stage revision tka using a modified articulating spacer that was effective for facilitating joint mobility and eradicating infection with a review of the literature. the surgery consisted of reinsertion of the autoclaved femoral component and implantation of a new polyethylene with antibiotic - loaded cement in the proximal tibia. of the patients who were treated for infected tka between january 2006 and december 2011, 20 patients (20 knees) who underwent two - stage revision arthroplasty using a modified articulating spacer were included in this study. fifteen patients had undergone primary tka at our institution and the remaining 5 patients were transferred from other institutions after having been diagnosed with infection after tka. infection was diagnosed based on blood test results including white blood cell (wbc) count, erythrocyte sedimentation rate (esr), and c - reactive protein (crp) level, radiographic findings (fig. there were 5 males and 15 females and their mean age at the time of primary tka was 61.5 years (range, 36 to 75 years). the mean follow - up period was 22.3 months (range, 14 to 60 months). the indications for surgery were degenerative arthritis in 16 patients and rheumatoid arthritis in 4 patients. a skin incision was made over the previous incision line created during the primary tka and the knee joint was exposed using a medial parapatellar approach. inflammatory tissues obtained during this procedure were sent for gram staining, aerobic and anaerobic bacterial culture, fungal culture, and acid fast bacilli staining and culture. the removed femoral component was cleaned of bone cement and debris, washed, and sterilized in an autoclave for 7 minutes at 137 in the operating room. meanwhile, joint irrigation, removal of the remaining bone cement and necrotic debris, and synovectomy were performed. for bone cementing, if the preoperative culture was negative, 4 g of vancomycin was mixed with cmw 3 (blue cross, depuy orthopaedics inc.) bone cement containing 1 g of gentamycin sulphate per 40 g packet of cement. otherwise, a mixture of cephalosporin antibiotics sensitive to the cultured organism was used. in the tibia, the antibiotic - loaded bone cement was applied without molding and a new polyethylene insert was placed not to cause shift of the joint line from primary tka. the new polyethylene insert was thicker than that used in the primary surgery to compensate for the removal of infected bone tissues and was mobile bearing for implantation with bone cement in the tibia. joint exercises using a continuous passive motion machine were initiated within one to two days after the first stage procedure. from the fifth to sixth postoperative day, partial weight - bearing with a walker was started. the lenox hill derotation orthosis was prescribed to wear for patients complaining of discomfort during walking (15 patients, 75%). the intravenous antibiotic treatment was discontinued if the blood test performed once per week shows normal wbc count, esr, and crp level, joint aspirate culture is negative at the 6th postoperative week, and symptoms of inflammation, such as swelling, heat sensation, and tenderness, are not present. subsequently, four weeks of oral antibiotic treatment was started and joint aspiration was performed at the end of the treatment. patients were followed without further therapy for the following 2 weeks until another joint aspiration was done. the blood test results were normal and the aspirate culture was negative in all patients during the period when the antibiotics were withdrawn. since the intraoperative frozen section examination revealed less than five wbcs per high power field in all patients, the second stage procedure was performed as planned (fig. 3). the postoperative rom, knee society knee score (ksks), knee society function score (ksfs), hospital for special surgery (hss) score, and recurrence of infection were assessed. a paired t - test was used for all statistical analysis with p<0.05 considered statistically significant. of the patients who were treated for infected tka between january 2006 and december 2011, 20 patients (20 knees) who underwent two - stage revision arthroplasty using a modified articulating spacer were included in this study. fifteen patients had undergone primary tka at our institution and the remaining 5 patients were transferred from other institutions after having been diagnosed with infection after tka. infection was diagnosed based on blood test results including white blood cell (wbc) count, erythrocyte sedimentation rate (esr), and c - reactive protein (crp) level, radiographic findings (fig. there were 5 males and 15 females and their mean age at the time of primary tka was 61.5 years (range, 36 to 75 years). the mean follow - up period was 22.3 months (range, 14 to 60 months). the indications for surgery were degenerative arthritis in 16 patients and rheumatoid arthritis in 4 patients. a skin incision was made over the previous incision line created during the primary tka and the knee joint was exposed using a medial parapatellar approach. inflammatory tissues obtained during this procedure were sent for gram staining, aerobic and anaerobic bacterial culture, fungal culture, and acid fast bacilli staining and culture. the removed femoral component was cleaned of bone cement and debris, washed, and sterilized in an autoclave for 7 minutes at 137 in the operating room. meanwhile, joint irrigation, removal of the remaining bone cement and necrotic debris, and synovectomy were performed. for bone cementing, if the preoperative culture was negative, 4 g of vancomycin was mixed with cmw 3 (blue cross, depuy orthopaedics inc.) bone cement containing 1 g of gentamycin sulphate per 40 g packet of cement. otherwise, a mixture of cephalosporin antibiotics sensitive to the cultured organism was used. in the tibia, the antibiotic - loaded bone cement was applied without molding and a new polyethylene insert was placed not to cause shift of the joint line from primary tka. the new polyethylene insert was thicker than that used in the primary surgery to compensate for the removal of infected bone tissues and was mobile bearing for implantation with bone cement in the tibia. joint exercises using a continuous passive motion machine were initiated within one to two days after the first stage procedure. from the fifth to sixth postoperative day, partial weight - bearing with a walker was started. the lenox hill derotation orthosis was prescribed to wear for patients complaining of discomfort during walking (15 patients, 75%). the intravenous antibiotic treatment was discontinued if the blood test performed once per week shows normal wbc count, esr, and crp level, joint aspirate culture is negative at the 6th postoperative week, and symptoms of inflammation, such as swelling, heat sensation, and tenderness, are not present. subsequently, four weeks of oral antibiotic treatment was started and joint aspiration was performed at the end of the treatment. patients were followed without further therapy for the following 2 weeks until another joint aspiration was done. the blood test results were normal and the aspirate culture was negative in all patients during the period when the antibiotics were withdrawn. since the intraoperative frozen section examination revealed less than five wbcs per high power field in all patients, the second stage procedure was performed as planned (fig. the postoperative rom, knee society knee score (ksks), knee society function score (ksfs), hospital for special surgery (hss) score, and recurrence of infection were assessed. a paired t - test was used for all statistical analysis with p<0.05 considered statistically significant. of the 20 patients, the culture was positive in 16 patients (80%) and negative in 4 patients (20%). the organisms identified from preoperative and intraoperative cultures were methicillin - resistant staphylococcus epidermidis in 7 patients, methicillin - resistant staphylococcus aureus in 4 patients, methicillin - sensitive staphylococcus aureus in 3 patients, and enterococcus feacalis (e. feacalis) in 2 patients (table 1). the mean wbc count decreased from 14,580/l (range, 10,400 to 24,240 l) prior to first stage surgery to 7.830/l (range, 4,300 to 8,540 l) prior to second stage surgery. the mean esr decreased from 54.11 mm / hr (range, 48 to 82 mm / hr) prior to first stage surgery to 23.41 mm / hr (range, 12 to 28 mm / hr) prior to second stage surgery. the mean crp level decreased from 6.98 mg / dl (range, 1.31 to 14 mg / dl) prior to first stage surgery to 0.39 mg / dl (range, 0.05 to 0.48 mg / dl) prior to second stage surgery. the mean interval from the onset of symptoms of infection following tak to first stage surgery was 39 months (range, 24 to 77 months) and the mean interval from first stage surgery to second stage surgery was 3.3 months (range, 3 to 4 months). the 16 patients with positive cultures after the first stage surgery were treated with intravenous antibiotics sensitive to the cultured organisms. in the remaining 4 patients with negative cultures, cephalosporin class antibiotics were administered intravenously and orally. the mean rom was increased from 69.8 (range, 50 to 100) prior to first stage surgery to 98 (range, 70 to 115) prior to second stage surgery, and then to 102.8 (range, 80 to 130) following second stage surgery (p<0.001). the mean ksks increased from 33.8 points (range, 28 to 52 points) prior to first stage surgery to 85.3 points (range, 77 to 94 points) following second stage surgery (p<0.001). the mean ksfs increased from 35.0 points (range, 20 to 55 points) prior to first stage surgery to 87.5 points (range, 70 to 100 points) following second stage surgery (p<0.001). the mean hss score increased from 57.6 points (range, 27 to 80 points) prior to first stage surgery to 82.6 points (range, 56 to 100 points) following second stage surgery (p<0.001). partial weight - bearing was permitted at a mean of 5.6 days (range, 5 to 6 days) after first stage surgery. there was no sign of infection recurrence in 18 patients until the last follow - up based on the clinical examination and blood test results ; however, extended period of antibiotic therapy and revision surgery using an articulating cement spacer were required in 2 patients (10%) due to recurrent infection. although diabetes mellitus was present in the latter 2 patients, 9 of the former 18 patients (50%) without recurrent infection were also diagnosed with the disease, indicating there is no statistically significant relationship between the disease and relapse of infection (p=0.494). complications, such as medial collateral ligament tears and periprosthetic fractures, were not observed in any of the patients. of the 20 patients, the culture was positive in 16 patients (80%) and negative in 4 patients (20%). the organisms identified from preoperative and intraoperative cultures were methicillin - resistant staphylococcus epidermidis in 7 patients, methicillin - resistant staphylococcus aureus in 4 patients, methicillin - sensitive staphylococcus aureus in 3 patients, and enterococcus feacalis (e. feacalis) in 2 patients (table 1). the mean wbc count decreased from 14,580/l (range, 10,400 to 24,240 l) prior to first stage surgery to 7.830/l (range, 4,300 to 8,540 l) prior to second stage surgery. the mean esr decreased from 54.11 mm / hr (range, 48 to 82 mm / hr) prior to first stage surgery to 23.41 mm / hr (range, 12 to 28 mm / hr) prior to second stage surgery. the mean crp level decreased from 6.98 mg / dl (range, 1.31 to 14 mg / dl) prior to first stage surgery to 0.39 mg / dl (range, 0.05 to 0.48 mg / dl) prior to second stage surgery. the mean interval from the onset of symptoms of infection following tak to first stage surgery was 39 months (range, 24 to 77 months) and the mean interval from first stage surgery to second stage surgery was 3.3 months (range, 3 to 4 months). the 16 patients with positive cultures after the first stage surgery were treated with intravenous antibiotics sensitive to the cultured organisms. in the remaining 4 patients with negative cultures, cephalosporin class antibiotics were administered intravenously and orally. the mean rom was increased from 69.8 (range, 50 to 100) prior to first stage surgery to 98 (range, 70 to 115) prior to second stage surgery, and then to 102.8 (range, 80 to 130) following second stage surgery (p<0.001). the mean ksks increased from 33.8 points (range, 28 to 52 points) prior to first stage surgery to 85.3 points (range, 77 to 94 points) following second stage surgery (p<0.001). the mean ksfs increased from 35.0 points (range, 20 to 55 points) prior to first stage surgery to 87.5 points (range, 70 to 100 points) following second stage surgery (p<0.001). the mean hss score increased from 57.6 points (range, 27 to 80 points) prior to first stage surgery to 82.6 points (range, 56 to 100 points) following second stage surgery (p<0.001). partial weight - bearing was permitted at a mean of 5.6 days (range, 5 to 6 days) after first stage surgery. there was no sign of infection recurrence in 18 patients until the last follow - up based on the clinical examination and blood test results ; however, extended period of antibiotic therapy and revision surgery using an articulating cement spacer were required in 2 patients (10%) due to recurrent infection. although diabetes mellitus was present in the latter 2 patients, 9 of the former 18 patients (50%) without recurrent infection were also diagnosed with the disease, indicating there is no statistically significant relationship between the disease and relapse of infection (p=0.494). complications, such as medial collateral ligament tears and periprosthetic fractures, were not observed in any of the patients. treatment of infection following tka has been the focus of numerous clinical studies that suggest treatment options, such as antibiotic therapy, debridement, resection arthroplasty, knee arthrodesis, revision arthroplasty, and limb amputation1,3,7 - 11). currently, two - stage revision arthroplasty is widely performed for the treatment of infected tka. the two - stage procedure consists of removal of knee components followed by intravenous antibiotic treatment and use of antibiotic - loaded bone cement to increase the concentration of antibiotics at the infection site3,10,12,13). in particular, articulating cement spacers are preferred in two - stage revisions for prevention of joint contractures and retention of rom. compared to non - articulating ones, the articulating spacers provide better functional outcomes by enabling early rehabilitation and rom exercises between the two stages and make soft tissue resection easier during the second stage procedure14). although implants designed for two - stage revision arthroplasty, such as the prostalac system (depuy orthopaedics inc.), have been introduced, they are neither affordable nor applicable to all patients2,3). hence, the procedure is mostly performed by using an articulating spacer custom - made by the surgeon during surgery. techniques for manufacturing personalized articulating cement spacers for two - stage tka are well described by hoffmann.14), fehring.2), and goldstein.15) and satisfactory results of two - stage revision using custom - made articulating cement spacers have been documented in some domestic studies by bae and yim3), kim.16), and ha and awe17). the technique we used among our patients is similar to that described by kim.16), but it can be differentiated in terms of the use of a new polyethylene insert. a failure to maintain an appropriate joint space between the two stages due to bone defect and collateral ligament insufficiency in the infected knee increases the risk of dislocation or instability of the implanted antibiotic - loaded cement spacer18). thus, we took care to lower the risk of joint instability by maintaining the thickness of the previous tibial component with use of antibiotic - loaded cement and inserting a thicker polyethylene than that used in the primary tka. the advantages of our technique include 1) reduced cost due to the reuse of the femoral component after autoclaving, 2) prevention of autoclaving - related changes in the morphological and functional properties of the polyethylene insert, and 3) customization of the polyethylene insert size according to the extent of bone loss. the instability and flexion / extension of the knee were assessed with the polyethylene insert in place. the relatively delicate procedure for manufacturing a femoral component could be avoided by reusing the component that had been placed in the primary surgery after autoclaving, which resulted in reduced operating time. the timing of reimplantation is crucial to treatment success of two - stage revision19). insall.20) suggested that a second stage surgery should be performed when soft tissue is in good condition and joint aspiration shows no evidence of infection. mont.21) advocated the importance of preoperative evaluation of cultures and sensitivity through biopsy and joint aspiration. regarding the period of antibiotic therapy, mahmud.22) proposed at least 6 weeks of intravenous antibiotic injection prior to reimplantation. in our study, we assessed clinical symptoms and soft tissue condition before proceeding with the second stage surgery. joint aspiration was performed after 6 weeks of intravenous antibiotic injection following the first stage surgery. the antibiotic treatment was withdrawn for the subsequent 2 weeks and three separate joint aspirations were performed. if the aspirate cultures were negative, it was determined to proceed with reimplantation. reimplantation was performed taking care to restore the closest to the preoperative joint condition to facilitate postoperative ambulation and rehabilitation. the mean interval between the first stage surgery and initiation of partial weight - bearing was 5.6 days (range, 5 to 6 days), indicating relatively early ambulation was possible. early ambulation increased patient satisfaction in performing daily living activities, such as using the bathroom without help and walking without aid, and thus patient compliance was high until the second stage surgery. the mean rom was significantly improved from 69.8 preoperatively to 98 prior to second stage surgery, and then to 102.8 after reimplantation, indicating that the two - stage revision using a modified articulating cement spacer was effective for improving rom and functional recovery (p<0.001). statistically significant improvements were also noted in the mean ksks (from 33.8 prior to first stage surgery to 85.3 after reimplantation, p<0.001) and the mean ksfs (from 35.0 prior to first stage surgery to 87.5 after reimplantation, p<0.001). we believe these results demonstrate the efficacy of the two - stage revision arthroplasty using a modified articulating spacer. infection recurred in two of the 20 patients. during the follow - up after reimplantation, the two patients presented with pain, localized heat, and increased wbc count, esr, and crp level, which were considered as evidence of infection, and thus repetitive debridement and intravenous antibiotic injection were performed. in spite of this, the symptoms did not subside, which led us to remove the femoral component and polyethylene insert with cement that were implanted in the first stage surgery and perform another second stage surgery to manage the infection. the two - stage revision arthroplasty using a modified articulating cement spacer provided successful results in 18 of the 20 patients. however, the limitations of our study include that there was no comparison with tkas using non - articulating spacers and the number of cases was small. two - stage revision arthroplasty using a modified articulating cement spacer can be a reliable and effective procedure for the treatment of infected tka. the procedure improved patient function and compliance with the treatment and facilitated recovery of rom and joint function by providing joint stability and mobility during the interval treatment period and yielded a high infection eradication rate. two - stage revision arthroplasty using a modified articulating cement spacer can be useful for the treatment of infected tka in terms of infection eradication and recovery of joint mobility and function. | purposeto evaluate clinical results of two - stage revision using a modified articulating spacer for treatment of infected total knee arthroplasty (tka).materials and methodswe retrospectively reviewed 20 cases treated by two - stage revision arthroplasty using a modified articulating spacer under the diagnosis of infected tka from january 2006 to december 2011. the mean follow - up period was 22.3 months. the first operation consisted of debridement after removal of the prosthesis, reinsertion of the femoral component after autoclaving, and implantation of antibiotic - loaded cement with a new polyethylene in the proximal tibia.resultsthe mean period between the primary tka and the first stage operation was 39 months and between the first stage operation and the revision arthroplasty was 3.3 months. the average range of motion (rom) increased from 69.8 preoperatively to 102.8 postoperatively (p<0.001). the mean knee society knee score increased from 33.8 points to 85.3 points (p<0.001). the mean knee society function score increased from 35 points to 87.5 points (p<0.001). the mean hospital for special surgery score increased from 57.6 points preoperatively to 82.6 points postoperatively (p<0.001). two cases (10%) were re - infected after the revision arthroplasty.conclusionstwo-stage revision arthroplasty using an articulating cement spacer can be an effective therapy not only for the treatment of an infected tka but also for recovery of knee rom and function. |
the purpose of this article is to report on the opening plenary session of the association of chiropractic colleges educational conference research agenda conference (acc - rac) 2014, aiming for effective change : leadership in chiropractic education, research and clinical practice. speakers with extensive backgrounds with implementing substantial change on a broad level shared personal examples from their experiences in education, research, political organizations, and clinical practice. they described efforts, challenges, and opportunities that are encountered in order to implement effective change and shared their personal thoughts on leadership. each of the speakers shared their diverse, unique insights and personal experiences to convey the process and meaning of leadership. leadership may be defined by one s actions and ability to direct others towards a common goal. successful leaders establish direction and vision, communicate goals, and are able to develop coalitions and alliances. although leadership seems to be an elusive concept, it is one that most people agree is necessary to promote effective change. the world has seen leaders, such as adolph hitler or jim jones, who were able to motivate people into action ; but their actions were devastating. as well, just because someone is popular enough to be voted into a position of leadership does not make that person a good leader. we must look beyond the title or position and understand the qualities that are necessary to implement change and, through good leadership, for us to improve the chiropractic profession. leader position but is focused on himself, group efforts aim to satisfy his self - aggrandizement instead of what is best for the group. unfortunately, a position of power is a magnet to those who use others for their own gain. to be an effective leader, a good leader focuses on moving the group towards a better future and is willing to endure difficult times to get there. by focusing on the best interest of the group, as history has shown, sometimes it is not until years have passed that we can truly distinguish the good from bad leaders. fortunately, each of us can make the choice as to what type of leaders we follow or what type of leaders we wish to be. i have participated in a variety of research, educational, leadership, and agenda conferences over the past 20 years. from these meetings, dedicated people have met and developed lists of action steps, great ideas, and potential plans ; but few of these are fully carried out. once the meeting has concluded and the documents have been published, somehow the group expects that the leaders will magically implement them. certainly we have limited resources, but the lack of follow through may be caused by not engaging others in the call to take action. a good friend once told me that after the meeting someone must do the work and that is where leadership begins. i often hear that the chiropractic profession needs to have better, stronger, or more effective leaders. i hear, they need to do something about the problems that our profession is facing. have we forgotten that each of us is a participant in the profession and it is our responsibility, not the responsibility of others, to move our profession forward ? each of us must commit to being a part of the solution if the chiropractic profession is going to thrive. we need others to see what we are seeing in order to create the path to achieve a better future. we need to reach out and expand our leadership potential and inspire all doctors of chiropractic to lead from where they currently are. this may mean that each of us begins with leading ourselves, and then we can move on to leading others by expanding our circles of influence. we need to encourage our chiropractic practitioners, educators, and researchers, who are supportive in spirit, to engage and be part of the solution through personal leadership. we have accomplished much in the past 120 years, and yet we still have a long way to go. even though we see problems that we think someone else should solve, we must remind ourselves that each one of us has the potential to be a part of the solution. therefore, the purpose of this session was to assemble a panel of good and effective leaders to discuss main issues in leadership from their point of view, describe what it takes to make effective change, and discuss what can be done to develop new leaders, especially for the chiropractic profession. in preparation for the association of chiropractic colleges educational conference research agenda conference (accrac) 2014, we sought speakers who had been influential in various areas of leadership. we considered those with positions of power ; those with national and international influence ; and those with academic, business, and research backgrounds. the speakers were selected based upon their breadth and depth of experience and being change agents in multiple areas over many years. much of the change that they have been responsible for has been done without others being aware of their actions, which is one of the signs of a selfless leader. they have sought to make changes for the better : not to improve themselves, but to help others. when invited to speak on leadership, each one responded in a very similar manner : humble and contrite. it is not often that we have the opportunity to listen to wisdom from 1, 4, outstanding leaders. therefore, we asked their permission to audiotape the session so that others may gain from this presentation. the following is a transcription of the opening plenary session, learning from a lifetime of leading effective change, of the accrac 2014. the audio recordings were transcribed, and the text has been edited for length and clarity purposes. this raises the question, does chiropractic need leadership ? at last year s accrac, futurist clem bezold challenged us by asking, where are we going as a profession ? what will become of chiropractic s future ? although we may think we know the answer, one thing is for certain, we can not get to wherever we are going without leadership., leaders can leave their dazzling picture in our mind s eye. and, although they may no longer be with us, we continue to be inspired by them. like rainbows, leaders are often unaware of themselves, their glory, or their greatness. before you is a panel of speakers that represents decades of experience. each of these distinguished leaders has challenged and inspired thousands of people over the years. each speaker will make a short presentation, and mr jim hunter will follow with his comments. dr clum is a 1973 palmer graduate, founding faculty member of life university, and first president of life chiropractic college west, holding that office for 30 years. dr clum served on the board of directors or as an officer of the association of chiropractic colleges, the council on chiropractic education, the international chiropractors association, the foundation for chiropractic progress, the chiropractic summit, and the world federation of chiropractic. he presently serves on the executive committee and board of directors of the foundation for chiropractic progress as treasurer and serves a member of the board of directors of the integrated healthcare policy consortium. i would like to begin by thanking life university for sponsoring my participation this morning and for the opportunity to be here with you. i have to tell you that i have approached this challenge, or this opportunity, with a great deal of trepidation and a great deal of anticipation as well but when you speak about leadership, to me it is similar to judge potter stewart s definition of pornography : i do not know how to define leadership, but i do know it when i see it. i would also like to thank the accrac for the remarkable timing of this presentation. it is at the same hour that fortune magazine released its top 50 leaders in the world. and, i would like to ask you to think about 4 of them that have applicability to us in our circumstance. i rejected that tradition as a teenager for all sorts of reasons that i held as valid and true until a few months ago, when pope francis was asked about the behavior of a vatican monsignor who was alleged to be involved in a sexual relationship with a priest. the pope s response was who am i to judge ? in that one phrase, it changed decades of antipathy, anger, and angst that i had toward the faith that i was raised in. in that one moment of giving up the power and asserting the authority that he did in that statement, it changed the deal for me in that relationship. the second leader for us to consider is the fifth person on the list : president clinton. and i am thrilled with him as a past president. to be pilloried to a level that few of us can imagine, withstand and to continue on ; this is an unbelievable perspective in that consideration. the third person, the ninth on the list, is the dalai lama. if you have had the good fortune to read his book beyond religion : ethics for a whole world, you have the leader of the entire buddhist community worldwide suggesting that what we need to do is take a step back from our faith and into our humanity. he put forward in his concept of secular ethics ; that secular ethics, based upon our humanness first, becomes consistent with every religious tradition there is. but in reverse, we have problems assimilating those perspectives depending upon faith and where we want to go. i put him on our list because i do not know how the hell he got there. this is a really good illustration that one person s leader is not the next person s leader. and where the pope s perspective and authority may have value to me, derek jeeter s does not matter much to me. and in this room, and in our profession, we have that reality. i was brought up in chiropractic in an era where one was a student of dr joseph janse or a student of dr ernest napolitano or a student of dr sid williams our current reality, in terms of our leadership as a discipline, is that we are going through a very difficult phase where we have transitioned from a personality - driven profession to a profession that is content driven. however, i am not sure we know what content we are driven by at this moment. but we are not being driven by the personalities, the great personalities, that were so instrumental at a very important time in our history. what we have right now is the opportunity for us to do something very different than our predecessors have done. when the keynote speaker, mr jim hunter, talked about influence, i thought about archimedes. archimedes said that if you give me a fulcrum and a lever long enough, i can move the world. our fulcrum in this moment is our values the lever is the commitment to those values. to the degree that we can commit to more powerful values, we increase the size of the fulcrum, we increase the length of the lever, and we increase the ability to move the world. our original goal as chiropractors was to change the whole health care system, to change the whole thing by turning it upside down, to completely reverse it, to make it be about health rather than disease. i think the moment in time is here for us to come closer to realizing this potential than ever before in our history. the need for leadership in health care, the need for leadership in health politics, and the need for leadership in health education have never been greater. one of the things that we were asked to address was the relevance, the action, and the development of leadership. my start in chiropractic was under dr sid williams who founded life university, life chiropractic college at the time. leadership development in me came very simply from somebody looking at me, my mentor looking at me, and saying that i can do it. i had such faith and such confidence in him that if he said i could, then i could. i do not know that there are many of us, myself included, that have dealt with people within our institutions and our organizations and our groups with the generosity that i was given by him as a young man. i am embarrassed that there has not been more effort and action on my part committed to that in others. there may be a person on your campus or someone you are going to cross paths with today or tomorrow. it could be our next einstein or a mother theresa, the person that leads us into the future if given a moment of encouragement, inspiration, and support in the process. the opportunity that we all have as leaders and as followers is to use that influence for good. sid taught me that the ethic we wanted to conduct our institution and our lives by was to love, to serve, to give, and to do so out of abundance. i am thrilled to have had the opportunity to be exposed to that ethic and perspective throughout my entire career. he has served as a university chief financial officer and served as president of 3 institutions. most recently, he served as chancellor of the dallas county community college, a system consisting of 7 colleges that enrolled over 105,000 students and is the largest community college system in texas. he is a trustee of dallas baptist university and is a past chairman of the board. he is currently serving as the chairman of the board of trustees of parker university and chiropractic college in dallas. he is an ordained baptist minister and has written 11 books, including the friday messages : food for thought : perspectives on leadership from a leading educator and the power of thought : a series of messages celebrating the life of dr martin luther king, jr. when i was asked to be a part of this panel, i did not know that i would follow jim hunter. as you heard, i served as the chancellor of the dallas county community college for 28 years. the board of trustees asked me to write a little paper entitled the academic administrator as a leader ", including what might be some critical principals, roles, and responsibilities. when you hear these, you are going to think that i am jim hunter, jr, because much of what i am going to say, he said. what is leadership ? to answer this, perhaps it can be expressed this way. they are found in the poor, the rich, the humbled, the proud, the common man, and the brilliant thinker. trust provides the confidence that the one who leads will act in the best interest of those who follow. this is the assurance that he or she will serve the group without sacrificing the rights of the individual. knowing when to advance, when to pause, when to criticize, when to praise, and how to encourage others to excel. from the leader s reserves of energy and optimism, followers draw their strength. drawing from a leader 's determination and his or her self - confidence, followers will find inspiration. this command by conscience asserts itself more from commitment and example than by directive. at the level of practice and action if we had to pick out 5 or 6 qualities that are essential to good leadership, they would be the following : firstly, you must have a vision, have a dream. faith to try means that when you walk to the edge all of the light that we have and we take that step into the darkness of the unknown, we must believe that 1 of 2 things will happen. one, there will be something solid for us to stand on ; or 2, god will teach us how to fly. other qualities include enthusiasm, energy to work, skill to motivate, and determination. someone asked if they were to write a profile of an outstanding leader what it would look like. i would start by saying leadership is an elusive quality. that it is an asset with great potential for stimulating productivity in higher education. the outstanding academic leader is unusually competent, dynamic, and very confident, and somehow seems to have it all together. one, the outstanding academic leader sets a particularly positive example as a person. that individual brings out the best in people. that individual demonstrates great skill in directing day - to - day activities and takes a dynamic approach to activities. you heard reference made to jim collins by jim hunter about why some companies make the leap and others do not. if you are going to be a good academic leader, you want to strive very hard to become the best at something. and becoming the best at something is not about being a charismatic leader. collins calls that first who, then what. and in a disciplined culture like the chiropractic profession, i suggest that the stop doing lists may be more important than the to do lists. if anything, higher education is an industry that is infected with the view of all things to all people, which leads to trouble. great organizations with academic leaders use technology to advance their plan, their strategy, and their momentum where they do not create it. and finally, i was asked to talk about what might be some of the dos and donts of effective leadership. far too many leaders believe that what they do and why they do it must be obvious to everybody in the organization. too many people believe that when they announce things as a leader, everybody understands. and these are the ideas that i have and now i need your ideas. and the first duty of a leader in an academic arena is to have and value followers. leadership is always accountable for our results, and you need a team to accomplish that. i had the privilege of teaching at the army command and general staff college and the army war college. a good leader makes sound discipline decisions on time. and finally, today is my birthday. the book is entitled the 10 best decisions a leader can make. and these are some of those 10 things. one, even the most fundamental element of society, the family, has leaders in the husband and the wife. every local expression of god s love and truth is guided by the church leaders. half the books in the bible use the word leader somewhere in it. everyone in the church is called to remember your leaders as it is in our colleges, our universities : those leaders who speak the words of truth. and so i say to you as my other colleagues would, welcome to the challenge and the privilege of leadership. dr phillips graduated from national college of chiropractic in 1973, followed with a masters in science in community medicine and a phd in sociology both from the university of utah. he was president of the los angeles college of chiropractic, which he transitioned into the multidisciplinary southern california university of health sciences. he has served as president of the acc, the council on chiropractic education (cce), and cce - international. he serves on 6 editorial boards, has served as the editor - in - chief of the journal for chiropractic education, and currently serves as the executive editor for american chiropractic association (aca) publications. he was instrumental in the inclusion of chiropractic in the veterans affairs (va) and military through his roles as the chair of the chiropractic advisory committee for the veteran s health affairs and participation on the oversight advisory committee for the department of defense demonstration project. dr phillips has published numerous articles ; and as a social historian, he has published 4 books on various aspects of chiropractic history. currently, he serves as the executive director of the ncmic foundation and the executive director for the councils on chiropractic education international. i want to share with you an experience that will help demonstrate some examples of leadership rather than giving an academic lecture. i look to mickey mouse as one of the great leaders of the world for 2 reasons : he is always smiling, and he has influenced this world for good more than most people. i would like to thank southern california university of health sciences for sponsoring my travel to this conference. this committee was organized to advise anthony joseph principi, secretary of veterans affairs, how best to incorporate chiropractic services into theva health care facilities. so it was not an issue of whether it was going to happen or not ; it was an issue of how best to do it. as a profession, we are continually faced with a series of great opportunities that are disguised as insoluble problems. this committee had been dubbed, not in print but off the record, as a committee that was designed to fail. the medical side of the committee included paul shekelle, md, phd, a well - known name amongst chiropractors for the work he did with rand in the 1990s. warren jones, md, a medical doctor in the navy and then retired into family medicine, chaired the department of family practice at the university of mississippi. he and dr jones served on the department of defense committee as well as the va committee. brian murphy, pt, was a physical therapist from the va facility at salt lake city, ut. michael orourke was a physician s assistant but was an active lobbyist for the veterans of foreign wars and a representative of several other va organizations. if ever there was a leader that was key to our success, it was sarah mcvikar. on the chiropractic side of the committee, but my good friend terry yochum, dc, dacbr, leaned on me quite heavily. i started giving him a list of people who could do a better job than i could ; and he stopped me and said, what would dr janse have done ? rick mcmichael, dc, who later served as president of the aca, was an aca representative. leona fisher, dc, represented the world chiropractic association ; and charles duvall, jr, dc, represented the national association of chiropractic medicine. this was quite a disparate group of chiropractors. at the end of our service, jay cook, dc, a practicing chiropractor from california, joined the committee. not on the list and not a member of the committee but in constant attendance in giving service to the committee was acc executive director mr david obryon. someone suggested it was the luck of the irish that brought us through to a successful completion, meeting the requirement of a federal advisory committee, that is, to have a representation of all interested parties who have a stake in the purpose of the committee. the charge of this committee was to meet the requirement of public law 107 : to provide direct assistance and advice to the secretary in the development and implementation of chiropractic health program in the va health care system. we had our mission set for us ; we did not have to come up with one. we started in 2002 meeting 3 or 4 times each year for a period of 3 years. at the end the only one we did not all agree on was related to the scope of practice. those familiar with the va system said that would never work in the va system. that is not to suggest we did not have discussions. over the course of 3 years, we had a lot of disagreement. however, we eventually came to accept the recommendation unanimously except for one. over the years i have asked myself, how did success like this happen ? was it my leadership as chair ? was it the makeup of the committee ? reflecting back, i think there was a commonality shared by all : a desire to provide a service for our veterans, those men and women who had put their life on the line to preserve our freedom, and our love for our country and what it represents. we all had different opinions on what chiropractic was, how it could be applied, and how it could be inserted into the system. but those 2 driving forces, service and love, held us together and allowed us to reach success. every time we gathered, we would have our formal meetings and then we would have our informal meetings during breaks, when we went to lunch and when we met after hours. instead, interactions integrated across that barrier as well as across differences of opinions within both sides of the group. so i attribute the success to what this committee accomplished to the members of the committee and to the diligence, the work, and the effort they put forth to achieve our mission and to fulfill our goal. as a leader, my job was to suggest let s go this way a little bit ; let s go that way a little bit. but if i did not have the quality of people on that committee on both sides of the aisle, we would have never achieved what we accomplished. when we finished our recommendations, the committee was sunset and we disbanded., success is not measured in achievement of goals, but in the stress and strain of meeting those goals. it was a highlight of my career, and i thank all of you for the opportunity to make this presentation. the last speaker on the panel before dr hunter has a chance to give us his comments is dr lou sportelli. dr lou sportelli received his doctor of chiropractic degree from palmer in december of 1962 and has been in private practice since 1963 in palmerton, pa. he served as chairman of the board of the aca and as president of the world federation of chiropractic. dr sportelli currently serves as president of the ncmic group, an insurance and financial services company. ncmic insurance company is the largest chiropractic malpractice insurance company in the nation, insuring more than 40,000 doctors of chiropractic in the united states. ladies and gentlemen, each of us at one time or another will have the opportunity to accept or decline the potential to rise to leadership roles in our lifetime. many times that opportunity to be a leader is not revealed until long after the occasion has passed. and only later when the opportunity to reflect on what transpired in the previous experience is manifest in some other circumstance are the lessons learned and the skills developed in the previous challenge used again, only this time with less effort and sort of a knowing of the value of the prior lessons truly realized. today we talk about the era of the information age ; and interestingly enough, if you notice, it is not called the knowledge age because there is a huge difference between information and knowledge. the biggest issue in defining leadership is mistaking a position or a title to automatically transfer leadership ability. before i go any further, the way a leader handles failure reveals more about the leader than how the leader handles success. i have been blessed in my 50-plus years in chiropractic to have had a mentor very early on in my career. you need to remember that when i went to chiropractic school, students could go right out of high school ; and that is exactly what i did. i had to wait until i was 21 years old to get my license in pennsylvania. unfortunately, i did not realize it at the time ; but upon reflection, its significance has become crystal clear. he is 89 years old, and i visit him every year since he moved away from my area. i owe him an awful lot for what i have become today, largely because of his early mentoring. his keen insight into the weaknesses of a young, enthusiastic, but novice chiropractor armed with nothing to fight the opposition with but a gun filled with philosophical bullets, which i did not know at the time were only lethal to me as the carrier of a questionable message regarding a new health profession. the message about chiropractic and the lack of scientific evidence has in the 21st century been exposed ; but back in the 1960s, the profession had little to use as evidence to support the professional positions we took and the public messages we espoused. another important mentor was a public relations consultant, mr irv davis, who painfully taught me the skill sets necessary to successfully and diplomatically convey our message with confidence and credibility. for his tutoring, ask him or her to be your mentor. or simply borrow them without their knowledge if you are shy about asking. and then, when valuable lessons are learned be a mentor to others the first opportunity you have. my early involvement in chiropractic advocacy and the world of media controversy i owe, strangely enough, to a medical doctor, a gentleman by the name of stephen barrett, who still to this day operates and manipulates a web site called quack busters. i met him in 1969 and through his actions launched my career because he subversively formed a group called the lehigh valley committee against health fraud. so, i showed up to attend the initial meeting of this group, but they would not let me in. so obviously, he angered me to a point which launched my advocacy career ; and the rest, as they say, is history. before i knew it, things evolved rapidly and soon we were faced with the wilk v american medical association (ama) suit. i was practicing in 1963 which was long before i or anybody in my era knew that there was the ama committee on quackery. this clandestine activity went on long before we knew the ama had devised a plan to contain and eliminate the chiropractic profession. so that experience of being denied access to a group opposed to health fraud catapulted me into a leadership role whether i wanted it or not. i became my state association president ; lobbyist ; media spokesperson ; and, for those of you in this largely academic room, a black lung coordinator. you heard me right. what could chiropractic do for pneumoconiosis, known as black lung disease ? it was a question that i was asked by the governor of the state of pennsylvania, and this very serendipitous path led me to the ability and the opportunity to have a state - funded black lung conference in the mid-1970s. funded by the state ! our state chiropractic association did not have enough money to buy dinner, however the state of pennsylvania wanted to fund a black lung conference ! the interesting part about the black lung conference was the names of some people who you may be familiar with when i name them. he did not at any time ever say to me, are you nuts ? energetic and supportive individuals like scott haldeman, jim winterstein, and al adams were invited. other giants such as joseph howe, earnest napolitano, and many whom i have forgotten had the opportunity to ask me if i was nuts. and of course, there were others who did not share in my enthusiasm you can be sure ; but they were always negative. it was the selfless desire in everyone who did participate to do something to support that conference and advance the entire profession. i am glad today that i got to know the researchers and, as we clinicians like to say, those pointy - headed academics early on in my career ; they helped mold and expand my broad view of the chiropractic world we as clinicians are forced to function in. unquestionably, the world of chiropractic politics, education, and research was indeed blurry. the black lung conference was a success ; but more importantly, that was a unique opportunity given to our profession when we had limited resources. many of those individuals who participated had never met each other. and because of the black lung conference, we had the opportunity to get to meet each other long before another very serendipitous event occurred in our profession called the national institute of neurological and communicative disorders and stroke, otherwise known as the nincds conference. those of you with gray hair may remember that seminal event back in 1975 ; look at the roster of participants, and it will look uncannily similar.. some of you may know that i traveled as state association president with a myriad of challenges, to then become chair of the aca with challenges, then to be president of the world federation of chiropractic with challenges, and then to be president of ncmic with challenges. all of these unexpected and unforeseen challenges were essential to mold my thinking of today. fundamentally, as you evolve in your career and life, not much is really different.. each challenge may be on a different day, with different players ; but fundamentally, the leadership issues are the same. there will always be similar problems no matter where or what stage of development you are in. in each of these roles, i had the good fortune to have a mentor who guided me. i do not want to embarrass the tall irishman in the room ; but mr patrick mcnerney, who is the ceo of ncmic organization, has turned ncmic from a mom and pop organization into an incredibly viable corporate entity in this profession, one that is needed so badly. he has also been a mentor to me, teaching me the nuances of business and finance ; but more importantly, he has created an incredibly viable and successful organization in ncmic that has benefitted the entire chiropractic profession in ways that our profession will never be able to measure ; only history will reveal its true relevance. i learned early on from my dear friend charlie tremendous jones. in his famous line when he ended all his talks, you 're the same today as you 'll be in 5 years except for 2 things : the people you meet and the books you read. he also said, readers are not always leaders, but leaders are always readers. and from those hundreds and hundreds of books that i read since first meeting charlie jones back in 1969, several pearls of wisdom have emerged. do not waste time on your weaknesses. always surround yourself with people brighter than yourself. you do not have to know everything ; you just have to know where to get the information. brains are the cheapest commodity in the world to buy. however, applying what that intelligence tells you to do is leadership. a lot can get done when you are not concerned about who gets the credit. take time to thank those who help you. and, if possible, learn the lessons that keith ferrazzi writes about in his book never eat alone (and have dinner without your iphone). when the stakes are high and the risks are great, when the outcome is in jeopardy, that is when you have to earn the right to be an exceptional leader. in conclusion, just remember, only 1 of the hundred us companies in the fortune 500 from 1900 is still in business today. so leadership is an ongoing process ; and in the words of futurist eric hoffer, in a time of drastic change it is the learners who inherit the future. the learned usually find themselves equipped to live in a world that no longer exists. leaders can help us see that vision. mr jim hunter, the keynote speaker who you heard earlier, is the author of 2 internationally best - selling books : the servant : a simple story about the true essence of leadership and the world s most powerful leadership principle : how to become a servant leader. his books are the texts used in many mba and other higher - education curricula, have been translated into 2 dozen languages, and have sold over 4.5 million copies worldwide. mr hunter speaks on servant leadership to audiences around the world and has personally coached over 2300 executives in developing the skills of servant leadership. as each panelist spoke, one of things that captured me was how different each person is. one can go to the bookstore and find a lot of books on leadership style, but i do not think they are worth the paper they are written on. you look at wwii and you look at a general patton versus a general eisenhower. how more different styles could 2 men possibly have ? a john wooden or a bobby knight, a lee iacocca or a mary kay ; each has a completely different style. some of the great leaders i know are introverted, which allows them to listen well. extroverts have to work at some other things, listening and thinking through their decisions. hence, the term maturity. moral maturity. doing the right thing even when you do not feel like it. as i look at the speakers, i see very different people ; and it is just a wonderful, wonderful thing the diversity in god s creation. so do not think you have to be a certain type of person to be a leader. he said in terms of style, in terms of personality, they were very different. he said some were tall, some were short, some were heavy, some were thin, some were dressed for success, and some were dressed for failure. some were articulate, but many were inarticulate. and he said a couple were charismatic. he said the quality they all had in common is what we just saw in these 4 speakers. they did not lay awake at night wondering when they were going to get a corner office or get some perk out of serving on a committee. do my people have everything they need to win ? are my people being served ? that is what kept them up at night. the second quality collins found in all the great leaders was a strong will to do the right thing for their people and for their organization. we have an old - fashioned word for that ; it is called character. doing the right thing even when you do not feel like it. very different people, but each one has character ; each stepped up at a time when someone needed to step up and fill a need. perhaps you will be inspired to look within yourself and recognize these traits and offer your own personal leadership to help move the chiropractic profession forward (figure 1). cost for transcription was funded by the national university of health sciences. no funding sources or conflicts of interest | objectivethe purpose of this article is to report on the opening plenary session of the association of chiropractic colleges educational conference research agenda conference (acc - rac) 2014, aiming for effective change : leadership in chiropractic education, research and clinical practice.discussionspeakers with extensive backgrounds with implementing substantial change on a broad level shared personal examples from their experiences in education, research, political organizations, and clinical practice. they described efforts, challenges, and opportunities that are encountered in order to implement effective change and shared their personal thoughts on leadership.conclusioneach of the speakers shared their diverse, unique insights and personal experiences to convey the process and meaning of leadership. |
transurethral resection of the prostate (turp) is considered the best option for treating benign prostatic enlargement (bpe) ; however, it is associated with significant bleeding. in recent years, we have seen a gradual increase in life expectancy and, particularly in more developed countries, an increased incidence of cardiovascular diseases, metabolic syndrome, and cerebrovascular stroke, all of which are conditions that require oral antiplatelet or anticoagulation (oap / oac) therapy. owing to the severe bleeding risk in these patients, turp is contraindicated. furthermore, suspending oap / oac therapy is also not recommended. for example, the use of laser technology has advantages in patients receiving oap / oac therapy who have severe comorbidities and bleeding disorders. thulium vaporesection of the prostate (thuvarp) with a 2-m continuous - wave (cw) thulium - doped yttrium - aluminum - garnet (tm : yag) laser has been established as a new approach for bpe treatment. this laser has a peak absorption spectrum closer to that of water than the holmium laser, which causes increased tissue vaporization and results in reduced penetration depth. however, because the thulium laser operates in cw mode, it permits smaller and more precise cutting and consequently decreased risk of bleeding. the aim of this study was to demonstrate the efficacy and safety of vaporesection with the 120-w tm : yag laser (revolix duo) in patients with bpe receiving systemic oap / oac therapy and to evaluate medium - term functional outcomes. between october 2011 and december 2013, a total of 103 patients with lower urinary tract symptoms due to bpe were enrolled in the study, and data were retrospectively analyzed. a, oap / oac therapy was interrupted for 10 days before the operation and patients were switched to low molecular weight heparin (lmwh) until 2 weeks after the procedure. in group group b patients were also divided into subgroups according to the oap / oac therapy that they were using : warfarin, acetyl salicylic acid (asa), clopidogrel, ticlopidine, or asa+clopidogrel. according to the standard protocol of the university hospital in which the study was carried out, each patient was informed of the procedure upon admission and signed an informed consent allowing data collection for research purposes. the study design was in accordance with the helsinki declaration, conformed to the committee on publication ethics guidelines, and was approved by the institutional review board of the department of human pathology, section of urology, university of messina, messina, italy in which it was performed. all the design, analysis, data interpretation, drafting, and revisions followed the strobe (strengthening the reporting of observational studies in epidemiology) statement as well as the guidelines for reporting observational studies, available through the equator (enhancing the quality and transparency of health research) network. all operations were performed by a single surgeon (cm) with extensive experience in laser prostatectomy and prostate resection. the thuvarp technique used in this study was the same described in our previous publication. the procedure was performed by using the 120-w 2-m cw tm : yag laser (revolix duo). the revolix duo has 2 laser generators : (1) a 120-w cw mode for soft tissue use and (2) a 20-w pulsed head to holmium for lithotripsy. a 550-m core nude - ended fiber (rigifib ; ohg, lisa laser, katlenburg - lindau, germany) was used in combination with a 26-f continuous - flow laser resectoscope (karl storz gmbh & co. kg, tuttlingen, germany). incisions were made on the bladder neck at the 5 and 7 o'clock positions and vaporesection was performed on the median lobe. finally, vaporesection of the lateral lobe was performed until the prostatic capsule was reached. by performing this technique, small tissue chips are obtained, which were evacuated through the resectoscope sheath. at the end of every procedure, a 20-f foley catheter was placed and bladder irrigation was performed in the case of hematuria. the catheter was routinely removed 48 to 72 hours after the procedure regardless of preoperative prostate size when the urine was clear. cefazolin (2 g) was administered as a short protocol 1 hour before the procedure. data collected included operative time, hospital stay, catheterization time, and hemoglobin values pre- and postoperatively. in addition, maximal flow rate (qmax), postmicturition residual urine (pmr), international prostate symptom score (ipss), and quality of life (qol) scores were assessed immediately before surgery (baseline) and at 3, 6, 12, 18, and 24 months after treatment. prostate - specific antigen (psa) values were assessed preoperatively and at 12 and 24 months. baseline variables were described by using mean and standard deviation or percentages, as appropriate. mann - whitney u - tests were used to evaluate the difference between quantitative measurements that had nonparametric distributions. a total of 103 patients who underwent laser prostatectomy were included in the study. in group a, anticoagulant therapy was interrupted and switched for lmwh in 47 patients, whereas in group b, 56 patients underwent surgery while still on their anticoagulant therapy regimen. the mean ages of groups a and b were 68.56 and 69.66.8 years, respectively. the mean operation, hospitalization, and catheterization durations are given in table 1 for each group. the mean preoperative prostate volumes were 66.814.1 and 65.39.5 ml for groups a and b, respectively. the mean amounts of resected prostate tissue were 43.810.9 and 43.16.7 ml for groups a and b, respectively. in group b, 7 patients were on warfarin, 23 patients were on asa, 14 were on ticlopidine, 8 were on clopidogrel, and 4 patients were on asa + clopidogrel combination treatment. the mean preoperative hemoglobin level of patients in group a was 13.71 and that of patients in group b was 13.51.5. the mean postoperative hemoglobin level of patients in group a was 131 and that of patients in group b was 13.21.5. neither pre- nor postoperative hemoglobin values were statistically significantly different between groups a and b. however, the drop in hemoglobin values in the pre- and postoperative periods was significantly higher in group a : 0.70.5 and 0.30.5 in groups a and b, respectively (p<0.005). when subgroup analyses were performed within group b, the mean drop in hemoglobin was significantly lower in patients who were on warfarin and ticlopidine compared with patients in group a (p<0.005, p<0.005, respectively). the mean pre- and postoperative hemoglobin values along with the mean drop in hemoglobin are given for each group in fig. when the ipss values were compared between groups a and b, group a had significantly lower ipss values at 3, 12, 18, and 24 months after surgery (p<0.005, p<0.05, p<0.005, p<0.001, respectively). however, in the preoperative period, the ipss was 15.9 in group a and 18.6 in group b and the difference was not statistically significant. no statistically significant differences were found between groups a and b or subgroups of group b in qol scores, qmax values, or pmr volumes (ml). during the whole follow - up period, the mean ipss, qmax, and pmr values along with qol scores for groups a and b and the subgroups of group b are given in table 2 and fig. the mean psa values in the preoperative period were 3.11.5 and 3.11.9 in groups a and b, respectively. at 12 months after the operation, the values were 2.30.9 and 2.41.2, and at 24 months, the values were 2.20.8 and 2.30.9 for groups a, 38 patients had no complications, whereas 4 had transient urge incontinence, 4 had urinary tract infection (uti), and 1 patient needed recatheterization for slight hematuria for 5 days which resolved without further intervention. no complications occurred in 41 patients in group b ; however, in 6 patients, transient urge incontinence occurred ; 7 patients had a uti ; 1 patient needed recatheterization for 4 days due to hematuria ; and 1 patient needed transfusion due to intraoperative bleeding. according to the latest guidelines of the european association of urology, turp is the best treatment option for patients with nonneurogenic lower urinary tract symptoms affected by bpe. however, morbidity with turp remains fairly high, with associated bleeding risk and electrolytic disorders. in particular, patients receiving oap / oac treatment carry a major risk for hemorrhage, and discontinuation of these treatments carries the risk of thromboembolic events. this may be considered a double - edged sword : the management of bpe is problematic for the urologist owing to the increased use of oap / oac for primary or secondary prevention of cardiovascular complications. in their study, capodanno. expressed that this commonly used bridging practice is potentially harmful in patients undergoing noncardiac surgeries and can result in worse ischemic outcomes, especially in the first 30 days, along with significant risk of bleeding.. pointed out the importance of these risks and proposed an immediate change in daily practice to abandon bridging therapy in most of these patients. to clarify this issue for urological procedures, several studies were conducted on turp in patients receiving oap / oac therapy regimens. analyzed 163 cases and found a higher rate of perioperative bleeding in patients who continued oap / oac therapy than in those who were not on an oap / oac therapy. also, cerebrovascular and cardiovascular events occurred in patients who had discontinued their oap / oac treatments. this study demonstrates the importance of studies in which patients are evaluated while they are still receiving oap / oac treatments. to question the safety of bridging therapy with lmwh and the discontinuation of prophylactic anticoagulants, dotan. compared the bleeding rates of patients who were on chronic oap / oac treatment and switched to lmwh perioperatively with those of non warfarin - treated patients. they found a bleeding rate of 20% in patients who switched to perioperative lmwh, and this situation is presumed to result in longer hospitalization duration. high rates of bleeding were also reported by parr. in a cohort of 12 patients with more than 30% of them having transfusions. however, they reported that several such patients who switched from warfarin to heparin before surgery presented to the emergency department with major thromboembolic events and some even required open - heart surgery. thus, they concluded that there is no need for bridging therapy and that bleeding problems can be easier to manage than thromboembolic events. ong. reported that in a population of 293 patients on chronic oap / oac therapies who underwent turp, bleeding complications occurred in 10% of patients, of whom 8% required prolonged bladder irrigation, 2% required re - catheterization, 2% required reoperation, and 2% needed blood transfusion. although turp has been the gold standard for the surgical management of bpe for the past several decades, in recent years, a new series of lasers and techniques have become well established as an alternative treatment for bpe. in particular, holmium and thulium lasers have shown safety and efficacy in numerous studies throughout the past decade. studies reporting the safety of laser prostatectomy in patients on oap / oac therapies are also becoming more common. in their study in 2007, ruszat. reported that in 116 patients on oap / oac therapy who were compared with a group of 92 control cases, none of the patients required transfusions. owing to slight hematuria, 17% of these patients required prolonged bladder irrigation ; however, this frequency was 5% among the control cases. in our patient groups, one patient in each group was catheterized as a result of bleeding after the retrieval of the urinary catheter and required prolonged irrigation. reported no statistically significant differences among the 2 groups in terms of ipss, qol scores, or qmax. these findings agree with our study. in our patient cohort, although these variables all improved after surgery, there were no statistically significant differences between the groups showing that bridging oap / oac with lmwh or continuing oap / oac affected functional outcomes. in various studies, for example, fu. compared thuvarp and monopolar turp and stated that the efficacy of the two modalities was similar, although thuvarp resulted in less blood loss and a shorter hospital stay. bach. compared the 120-w and 70-w tm : yag lasers in terms of hemostatic properties. the device that we used, the 120-w tm : yag laser, provided better ablation rates and fewer bleeding problems compared with the 70-w device. the 120-w thulium laser has a wavelength of 2013 nm, which is close to the absorption peak of water. thus, the effects are independent of tissue vascularization, which provides good hemostasis, comparable to that of the holmium : yag laser. additionally, the cw laser beam in tm : yag lasers provides precise incisions and sufficient vaporization. in the study by bach. in 2010 and the study by fu. in 2010 about thuvarp, the authors did not indicate the vaporized prostate volume. in our study written by netsch. in 2015 comparing thuvarp and thuvep, the amount of vaporized tissue was 42.5 ml. the amount of vaporized tissue in our current study was 43.1 and 43.8 ml in the different groups. psa is a tool that can be used to predict prostate volume and prostate growth. additionally, according to the pless (proscar long - term efficacy and safety) study, psa is valuable in predicting changes in symptoms, qol, and qmax. in our study, psa values were lower in the postoperative period compared with preoperative values in both groups a and b. this drop was due to tissue vaporization and the decrease in prostate volume, which also supports the amelioration of symptoms and improved qol after surgery. as expressed earlier, the bleeding complications during turp in patients receiving oap / oac therapies are high. with technological advancements and new laser devices, the bleeding rate is expected to decrease as a result of the high ablative and coagulative power of these modalities. in our study, apart from showing low bleeding rates similar to those reported in fu. 's study, we also compared patients on continuous oap / oac therapy and patients who underwent lmwh bridging. although the subgroup comparisons showed statistically significant differences, the drop in hemoglobin levels was similar and the differences were not clinically relevant. these results show that bridging therapy with lmwh has a small impact on bleeding complications that have clinical relevance. although no cardiopulmonary adverse events occurred in our patient cohort, the high level of evidence in the literature about the seriousness of these events draws great attention to patients undergoing bridging therapy with lmwh. when the low risk due to bleeding complications in our patient group is combined with the high risk of potential cardiopulmonary adverse events in the case of lmwh bridging, continuing oap / oac therapy during thuvarp seems to be the better way of patient management. apart from bleeding complications, we did not encounter any bladder neck contracture or urethral stricture in the long - term follow - up. fu. reported one case of urethral stricture and no cases of bladder neck contracture in their study. in a recent study by yu., the authors reported a uti rate of 10%. in our study, 4 of 47 patients in group a (8.5%) and 7 of 56 patients in group b (12.5%) had uti. in fu. 's study, the rate of uti in thuvarp group was 6.9%. another minor limitation was the lack of comparison with a group of patients undergoing turp, which is considered the gold standard operation for bpe. however, in the absence of good prospective trials, the results obtained from well - analyzed retrospective studies can play significant roles in clinical practice and can set up new ideas for future randomized prospective studies. continuing oap / oac therapy regimens for certain endoscopic procedures has been strongly encouraged in recent years owing to the high risk of cardiopulmonary thromboembolic events in these patients. our study showed the safety profile of continuing different oap / oac regimens in terms of bleeding problems in patients undergoing thuvarp. we strongly recommend abandoning lmwh bridging and maintaining the patient 's current oap / oac therapy regimen. a larger cohort of patients and a longer follow - up period are needed to add more valuable contributions to our results. | purposethulium vaporesection of the prostate (thuvarp) is a new and safe approach for patients receiving anticoagulant therapy in whom transurethral resection of the prostate (turp) may possess a high bleeding risk. we aimed to demonstrate the efficacy and safety of thuvarp in patients receiving oral antiplatelet / anticoagulant (oap / oac) therapy.materials and methodsa total of 103 patients who underwent thuvarp between 2011 and 2013 were enrolled in the study. patients were divided into 2 groups. group a consisted of 47 patients who underwent low molecular weight heparin (lmwh) bridging and group b consisted of 56 patients who were operated on while receiving oap / oac therapy.resultsthe drop in hemoglobin levels in the pre- and postoperative periods was significantly higher in group a than in group b. when subgroups were analyzed, the mean drop in hemoglobin was significantly lower in the warfarin and ticlopidine subgroups of group b than in group a. international prostate symptom scores were significantly lower 3, 12, 18, and 24 months after surgery in group a than in group b. quality of life scores, maximal flow rate values, and postmicturition residual urine volumes (ml) were similar between the 2 groups. a total of 38 and 41 patients in groups a and b, respectively, had no complications.conclusionsour study showed the safety profile of continuing different oap / oac therapies in terms of bleeding problems in patients undergoing thuvarp. we strongly recommend abandoning lmwh bridging and maintaining the oap / oac regimen patients are already receiving. |
catecholaminergic polymorphic ventricular tachycardia (cpvt) is an inherited arrhythmogenic disease that can cause syncope or sudden cardiac death during emotional or physical stress in the absence of detectable structural heart disease and a prolonged qt interval on an electrocardiogram (ecg). mutations in two genes, the cardiac ryanodine receptor gene (ryr2) and calsequestrin 2 gene (casq2), have been identified in patients with cpvt. these mutations lead to an increase in intracellular ca concentration, resulting in life - threatening ventricular arrhythmias, possibly via delayed depolarizations. a 16-year - old male, who had suffered from recurrent episodes of syncope triggered by physical exertion was referred to our hospital after undergoing defibrillation at a local emergency department (ed). he had lost consciousness at school during a volleyball test that was also emotionally stressful. an initial ecg at a local ed revealed ventricular fibrillation. although his arrhythmia was converted to sinus rhythm after defibrillation, the patient was stuporous and had repeated episodes of nonsustained ventricular tachycardia (vt). after referral to our hospital, ecg monitoring in the ed and intensive care unit showed intermittent nonsustained vt and bidirectional vt (fig. when the patient became agitated, his heart rate increased, and the ecg showed nonsustained polymorphic vt despite intravenous administration of a -blocker. he was sedated and placed on mechanical ventilation. two days later, his heart rate was regular, and no additional episodes of vt were observed. his ecg showed a sinus rhythm with a corrected qt interval of 423 msec (fig. 2). routine laboratory findings, including electrolytes and two - dimensional echocardiography, were unremarkable. a detailed history of the patient included several episodes of syncope since the age of 8 years associated with physical activities, such as sprinting, playing football, and fighting with a sibling. his father also experienced an episode of syncope associated with sprinting at the age of 9 years. the patient and his family underwent a genetic analysis, and his father and mother each had one different de novo missense mutation in ryr2 (exon 97 c.14009t > a p.l4670h in the father and exon 37 c.5428g > c p.v1810l in the mother). as his episodes of syncope were associated with sympathetic activation, an exercise ecg test and epinephrine infusion test (intravenous bolus of 0.1 g / kg followed by continuous infusion at rates of 0.1 g / kg per minute) were performed. however, only premature ventricular complex (pvc) bigeminy was induced with no episodes of syncope during the exercise ecg test (fig. 3) and epinephrine only increased his heart rate without an arrhythmia. with these clinical features, we made the diagnosis of cpvt. atenolol (37.5 mg bid ; 1.9 mg / kg / day) was prescribed, and a follow - up exercise ecg test while on the -blocker was performed 3 days later. during the follow - up exercise ecg test the patient remains on 50 mg bid atenolol (2.3 mg / kg / day) and is being followed. to date catecholaminergic polymorphic ventricular tachycardia, arrhythmogenic right ventricular cardiomyopathy, and long qt syndrome are well known causes of exercise - induced syncope that commonly manifest normal baseline ecgs.1)2) therefore, many provocation tests, including vt induction, an exercise ecg test, and epinephrine infusion tests, have been used to diagnose exercise - induced syncope.1)2) exercise - induced bidirectional vt has been considered the hallmark of cpvt for many years. however, recent studies1)2) have shown that ventricular bigeminy is the most common exercise - induced arrhythmia among patients with cpvt, and serious arrhythmias such as bidirectional vt and polymorphic vt are rarely induced. therefore, exercise - induced ventricular bigeminy no longer should be considered an innocent arrhythmia, at least in suspicious cases of cpvt. catecholaminergic polymorphic ventricular tachycardia may have both an autosomal dominant and an autosomal recessive pattern of inheritance. the more common type 1 autosomal dominant cpvt is caused by mutations in ryr2 on chromosome 1q42-q42 and results from defective calcium release from the sarcoplasmic reticulum, which is required for myocardial contraction.3) a mutation in ryr2 can increase calcium release and cause life - threatening ventricular arrhythmias. patients with ryr2 mutations become symptomatic at an early age, and men are at higher risk for cardiac events.4) in the autosomal recessive variant, type 2 cpvt, the causative gene is casq2, located on chromosome 1p13 - 21.5) casq2 encodes calsequestrin, a calcium buffering protein in the sarcoplasmic reticulum, which binds a large amount of calcium. while the mechanism by which this mutation causes ventricular arrhythmias has not been clearly established, the mutated protein may be associated with the following : loss of polymerization of casq monomers, loss of calcium buffering capability, or indirect destabilization of the ryanodine receptor channel.6)7) patients with cpvt, in which many forms of exercise are associated with catecholamine release that triggers vt, should be cautioned against virtually all forms of vigorous and/or competitive physical activity.8) since the early reports on cpvt, -blockers have been used as primary therapy for cpvt,9)10) and they are indicated for both chronic treatment and acute therapy for sustained vt. other antiarrhythmic drugs such as amiodarone and class i drugs have proved to be ineffective.11) however, these issues were addressed in a study that evaluated both the molecular mechanisms and clinical efficacy of flecainide in mice and humans.12) implantable cardioverter defibrillators can be considered for primary prevention despite the potential drawbacks in young patients and concerns about provoking arrhythmic storms. left cardiac sympathetic denervation has been reported effective in patients whose symptoms were not adequately controlled by -blockers.13) this approach may also be considered for individuals with intractable arrhythmic storms to reduce the number of implantable cardioverter - defibrillator shocks. | a 16-year - old male with a prior history of recurrent syncope was referred to our hospital after being resuscitated from cardiac arrest developed while playing volleyball. his electrocardiogram (ecg) demonstrated ventricular fibrillation at a local emergency department. after referral, an ecg showed bidirectional ventricular tachycardia (vt) and nonsustained torsade de pointes. two days later, his heart rate became regular, and no additional episodes of vt were observed. his ecg showed sinus rhythm with a corrected qt interval of 423 msec, and two - dimensional echocardiography was unremarkable. we made the diagnosis of a catecholaminergic polymorphic vt. however, only premature ventricular complex bigeminy was induced on exercise ecg and epinephrine infusion tests, and the patient showed no episodes of syncope. his father and mother had different missense mutations in the cardiac ryanodine receptor on genetic testing. the proband had both mutations in different alleles and was symptomatic. it was recommended that the patient avoid competitive physical activities, and a -blocker was prescribed. |
in molecular biology, the comparative analysis of organisms exhibiting different phenotypes is still mostly performed on the level of genes. however, many genes encode proteins that are involved in complex metabolic pathways, whose functions result from the interplay of all involved enzymes. so to understand the phenotype of any living system, it is essential to not only investigate single genes in isolation, but also the metabolic pathway variant of the particular organisms under study. interesting questions to study in this context are what are the commonalities and differences within a group of organisms or more elaborately, if you subdivide the group of organisms of interest into subsets, what are the features all organisms of one set share while all members of another set completely lack these functions? for example, consider analyzing a set of pathogens versus a set of non - pathogens for identifying reactions associated with pathogenicity. sets of organisms instead of single representatives are used for comparison, because it is not the goal to find special innovations present in one species only, but rather more general differences that could be interpreted as principles of pathogenicity. the set of reactions or respectively their catalyzing enzymes resulting from answering the above questions may serve as candidate set for finding new drug targets. throughout the rest of this article we will use the term differential reaction content to describe the set of reactions that do not occur in all organisms under study. (1) have published an algebraic method for comparing networks that can be used to find the metabolic innovations in a set of organisms as compared to a second set of organisms. they use this method to find those reactions that occur in at least one organism out of a predefined set of organisms and are missing in all organisms of another predefined set. however, it is of interest to also detect all reactions that occur in exactly all organisms of the first set, while missing in precisely all organisms of the second set (which is a subset of the above mentioned metabolic innovations and can also be computed using the algebraic method) and of course vice versa. these reactions will in the following be called unique reaction content. a method for pairwise protein interaction network alignment there also exists a web server (3) allowing queries of a short protein interaction path against a target protein protein interaction network selected from a network database. the metacyc / biocyc collection of pathway / genome databases (4) relies on a program called pathway tools that permits comparative metabolic pathway analysis for two or more organisms. in the web version results are presented as lists, whereas the gui version not only contains improved comparative methods, but also displays the results visually on pathway maps. however, this version needs to be installed on a local machine along with the organism databases, which requires large amounts of disk space and computational power. yet, in order to enable easy access to results, it is essential to visualize the detected reaction content in a graphical way. it should become apparent at one glance whether a reaction occurs in all organisms or only in organisms of one of the sets and in the latter case whether it occurs in all members of this set or only in a subset. a crucial point in detecting the differential reaction content is the choice of which organisms to put into the two sets to be compared. if not chosen appropriately, reaction content that is unique for one of the sets and thus possibly worth to be further analyzed might not be found (e.g. a reaction shared by all organisms of set 1 and one organism of set 2). in order to identify a good subdivision, one has to find sets of organisms with high similarity in terms of their reaction content within each set and low similarity across the sets. furthermore, our analyses showed that the metabolic reaction content of a group of organisms of interest often does not allow for a grouping that makes it possible to detect unique reaction content. when comparing the whole network at once, differences in subnetworks may cancel each other out and thus may lead to groupings with less discriminative power. in this article, it aims at finding unique metabolic reaction content that is worth to be further analyzed. the website provides a visualization engine for displaying the differential metabolic reaction content resulting from comparing two sets of organisms. furthermore, the server provides an automated approach based on clustering techniques for finding an appropriate grouping. in order to find sets that are well suited for detecting unique reaction content, we use a subdivision of the overall metabolic network into smaller subnetworks and separately apply the clustering on each of them. we have designed and built a web server that supports researchers in comparative metabolic network analysis. it is based on the metabolic reaction annotation provided by the kegg database (5) and thus currently comprises 155 eukaryotes, 569 bacteria and 49 archaea (30 december 2007). in order to keep the contents up to date, users can additionally upload their own reaction annotation data to include it in their analysis. currently, two file formats are supported : text files containing one kegg reaction identifier (e.g. r00001) per line, and files in embl format (6) containing ec number annotation. one feature of our web server is a visualization engine called reaction content visualizer that like kegg permits the display of the reaction content of organisms on static metabolic pathway maps by coloring the respective enzymes. unlike the kegg website, our server allows the user to choose several organisms, and displays simultaneously their reaction content using a user - specified color for each organism (figure 1). visualizing the reactions within their pathway context and not as plain lists facilitates assessing the functional relevance of missing a certain reaction. using maps of individual pathways for visualization, instead of one map of the whole metabolic network, allows for easy visual inspection. figure 1.reaction content visualizer showing the reaction content in the biotin metabolism (kegg) for several corynebacteria simultaneously. reaction content visualizer showing the reaction content in the biotin metabolism (kegg) for several corynebacteria simultaneously. a second visualization engine called differential reaction content visualizer allows the user to choose two sets of organisms and a kegg pathway (figure 2). pressing the display pathway button invokes calculating the differential reaction content, which then will be displayed on the well - known kegg pathway maps. here, each reaction is colored according to whether it occurs in both sets of organisms or only one and for the latter case whether it occurs in all organisms of the respective set or only in a subset (figure 3). figure 2.differential reaction content visualizer : select boxes allow the user to choose pathway, organisms in set one and organisms in set two (from top to buttom). organisms already selected for the first set are not allowed to be included in the second set and vice versa. figure 3.differential reaction content visualizer applied to the biotin metabolism for the corynebacteria c. jeikeium (cjk) and c. diphtheriae (cdi) in the first set compared against c. glutamicum (kyowa hakko) (cgl), c. efficiens (cef) and c. glutamicum (bielefeld) (cgb) in the second set. for each box containing an ec number a tooltip lists all kegg reactions associated to the respective ec number and all organisms this reaction is annotated for. differential reaction content visualizer : select boxes allow the user to choose pathway, organisms in set one and organisms in set two (from top to buttom). organisms already selected for the first set are not allowed to be included in the second set and vice versa. differential reaction content visualizer applied to the biotin metabolism for the corynebacteria c. jeikeium (cjk) and c. diphtheriae (cdi) in the first set compared against c. glutamicum (kyowa hakko) (cgl), c. efficiens (cef) and c. glutamicum (bielefeld) (cgb) in the second set. for each box containing an ec number a tooltip lists all kegg reactions associated to the respective ec number and all organisms this reaction is annotated for. as already indicated, a crucial point for analyzing the differential reaction content is the choice of which organisms to put into the two sets to be compared. to support the user in making this decision, we provide a number of hierarchical clustering methods that automatically group organisms with similar variants of their metabolic network together. thus, the user can either rely on preliminary knowledge or use the clustering dendrograms to find an adequate grouping of organisms. unexpected groupings and the corresponding unique reaction content are likely to be detected using the latter. our analyses showed that for different parts of the metabolic network in most cases different groupings would be a good choice. in other words, once you fix the grouping, you find some of the discrepancies, but will miss others, which you would only find using another grouping. however, the second grouping will in turn miss the findings enabled by the first grouping. to avoid this, we subdivide the overall metabolic network into smaller networks and apply the clustering analysis separately on each of them. on our web server we use the subdivision provided by the kegg pathway maps : all reactions on one of these maps constitute one pathway. additionally, users can define their own pathway by uploading a list of kegg reaction identifiers. because in this case the resulting differential reaction content can not be displayed on kegg pathway maps, results are provided in tabular form as well as displayed on pathway maps generated by the graph visualization software graphviz (http://www.graphviz.org/). the start page for the clustering allows the user to choose the organisms of interest and pathways to analyze. furthermore, users can upload their own annotation data as well as define their own pathways. then the user can decide which distance measure to use for calculating the similarity between the pathway variants in two different organisms. two choices are provided : a reaction edit distance on metabolic network graphs and the normalized network distance defined in forst. once calculations are finished, results are displayed on a new page (figure 4). on the top of the page the user can select one pathway from the analysis set. after pressing the display results button, clustering dendrograms of average linkage (7), complete linkage (7) and ward (8) hierarchical clustering are displayed for this pathway. below each of these images, preselected lists of the automatically detected groupings are presented to the user who can directly invoke the visualization of the differential reaction content based on this grouping. additionally, the user can make his own selection after visual inspection of the dendrograms. this is useful, since the automatic detection can never be guaranteed to find the optimal solution. hyperlinks are provided next to each grouping to start the differential reaction content visualizer using the respective grouping and pathway. on the very top of this page the user can follow a link to an overview page listing all pathways and the respective differential reaction content. the pathways are sorted according to the differential reaction content so that most different pathways appear at the top of the list. this is helpful, because it is often not known which pathway yields interesting results. cjk : c. jeikeium ; cdi : c. diphtheriae ; cgb : c. glutamicum (bielefeld) ; cef : c. efficiens ; cgl : c. glutamicum (kyowa hakko). cjk : c. jeikeium ; cdi : c. diphtheriae ; cgb : c. glutamicum (bielefeld) ; cef : c. efficiens ; cgl : c. glutamicum (kyowa hakko). we calculate a distance measure to assess how close two organisms are to each other in terms of their metabolic reaction content. we calculate the pairwise distances for each single pathway and then cluster the organisms with average linkage, single linkage and ward hierarchical clustering techniques. these groupings are those that maximize the cophenetic correlation coefficient (9), which is a measure for cluster quality. one feature of our web server is a visualization engine called reaction content visualizer that like kegg permits the display of the reaction content of organisms on static metabolic pathway maps by coloring the respective enzymes. unlike the kegg website, our server allows the user to choose several organisms, and displays simultaneously their reaction content using a user - specified color for each organism (figure 1). visualizing the reactions within their pathway context and not as plain lists facilitates assessing the functional relevance of missing a certain reaction. using maps of individual pathways for visualization, instead of one map of the whole metabolic network, allows for easy visual inspection. figure 1.reaction content visualizer showing the reaction content in the biotin metabolism (kegg) for several corynebacteria simultaneously. reaction content visualizer showing the reaction content in the biotin metabolism (kegg) for several corynebacteria simultaneously. a second visualization engine called differential reaction content visualizer allows the user to choose two sets of organisms and a kegg pathway (figure 2). pressing the display pathway button invokes calculating the differential reaction content, which then will be displayed on the well - known kegg pathway maps. here, each reaction is colored according to whether it occurs in both sets of organisms or only one and for the latter case whether it occurs in all organisms of the respective set or only in a subset (figure 3). figure 2.differential reaction content visualizer : select boxes allow the user to choose pathway, organisms in set one and organisms in set two (from top to buttom). organisms already selected for the first set are not allowed to be included in the second set and vice versa. figure 3.differential reaction content visualizer applied to the biotin metabolism for the corynebacteria c. jeikeium (cjk) and c. diphtheriae (cdi) in the first set compared against c. glutamicum (kyowa hakko) (cgl), c. efficiens (cef) and c. glutamicum (bielefeld) (cgb) in the second set. for each box containing an ec number a tooltip lists all kegg reactions associated to the respective ec number and all organisms this reaction is annotated for. differential reaction content visualizer : select boxes allow the user to choose pathway, organisms in set one and organisms in set two (from top to buttom). organisms already selected for the first set are not allowed to be included in the second set and vice versa. differential reaction content visualizer applied to the biotin metabolism for the corynebacteria c. jeikeium (cjk) and c. diphtheriae (cdi) in the first set compared against c. glutamicum (kyowa hakko) (cgl), c. efficiens (cef) and c. glutamicum (bielefeld) (cgb) in the second set. for each box containing an ec number a tooltip lists all kegg reactions associated to the respective ec number and all organisms this reaction is annotated for. as already indicated, a crucial point for analyzing the differential reaction content is the choice of which organisms to put into the two sets to be compared. to support the user in making this decision, we provide a number of hierarchical clustering methods that automatically group organisms with similar variants of their metabolic network together. thus, the user can either rely on preliminary knowledge or use the clustering dendrograms to find an adequate grouping of organisms. unexpected groupings and the corresponding unique reaction content are likely to be detected using the latter. our analyses showed that for different parts of the metabolic network in most cases different groupings would be a good choice. in other words, once you fix the grouping, you find some of the discrepancies, but will miss others, which you would only find using another grouping. however, the second grouping will in turn miss the findings enabled by the first grouping. to avoid this, we subdivide the overall metabolic network into smaller networks and apply the clustering analysis separately on each of them. on our web server we use the subdivision provided by the kegg pathway maps : all reactions on one of these maps constitute one pathway. additionally, users can define their own pathway by uploading a list of kegg reaction identifiers. because in this case the resulting differential reaction content can not be displayed on kegg pathway maps, results are provided in tabular form as well as displayed on pathway maps generated by the graph visualization software graphviz (http://www.graphviz.org/). the start page for the clustering allows the user to choose the organisms of interest and pathways to analyze. furthermore, users can upload their own annotation data as well as define their own pathways. then the user can decide which distance measure to use for calculating the similarity between the pathway variants in two different organisms. two choices are provided : a reaction edit distance on metabolic network graphs and the normalized network distance defined in forst. once calculations are finished, results are displayed on a new page (figure 4). on the top of the page the user can select one pathway from the analysis set. after pressing the display results button, clustering dendrograms of average linkage (7), complete linkage (7) and ward (8) hierarchical clustering are displayed for this pathway. below each of these images, preselected lists of the automatically detected groupings are presented to the user who can directly invoke the visualization of the differential reaction content based on this grouping. additionally, the user can make his own selection after visual inspection of the dendrograms. this is useful, since the automatic detection can never be guaranteed to find the optimal solution. hyperlinks are provided next to each grouping to start the differential reaction content visualizer using the respective grouping and pathway. on the very top of this page the user can follow a link to an overview page listing all pathways and the respective differential reaction content. the pathways are sorted according to the differential reaction content so that most different pathways appear at the top of the list. this is helpful, because it is often not known which pathway yields interesting results. cjk : c. jeikeium ; cdi : c. diphtheriae ; cgb : c. glutamicum (bielefeld) ; cef : c. efficiens ; cgl : c. glutamicum (kyowa hakko). cjk : c. jeikeium ; cdi : c. diphtheriae ; cgb : c. glutamicum (bielefeld) ; cef : c. efficiens ; cgl : c. glutamicum (kyowa hakko). we calculate a distance measure to assess how close two organisms are to each other in terms of their metabolic reaction content. we calculate the pairwise distances for each single pathway and then cluster the organisms with average linkage, single linkage and ward hierarchical clustering techniques. these groupings are those that maximize the cophenetic correlation coefficient (9), which is a measure for cluster quality. as an application case we analyze biotin metabolism (10) in different corynebacterium species, with the goal of detecting which species show similar (annotated) reaction content with respect to this pathway. for this task we choose the cpa clustering website, select pathway and organisms, and start the calculations. the resulting clustering dendrograms and suggested groupings are displayed on another web page. in this case, the clustering dendrograms for different clustering techniques are almost identical ; they only differ in the height, at which different groups are merged (figure 4). corynebacterium efficiens (cef) and c. glutamicum (kyowa hakko) (cgl) have no differences, c. glutamicum (bielefeld) (cgb) is close to them, though not identical, c. diphtheriae (cdi) differs and c. jeikeium (cgk) is different from both c. diphtheriae and the set of the other three organisms. however, we decide to put c. jeikeium and c. diphtheriae into a single set and compare this set against the set of the other three. we select the respective checkboxes for group 1 and group 2 on the website and click visualize. now the differential reaction content is calculated and displayed using the differential reaction content visualizer (figure 3). one immediately notices that reactions in the upper part of the map are not shared among all organisms. kegg reaction r03182 (ec 6.3.3.3) and r03231 (ec 2.6.1.62) occur in all organisms of set 1 and c. diphtheriae, whereas r03209 (ec 6.2.1.14) and r03210 (ec 2.3.1.47) are only annotated for c. diphtheriae. although this image reflects current knowledge, it has not been proven yet in the wet - lab, whether c. diphtheriae actually uses the additional path from pimelate to 8-amino-7-oxononanoate for synthesizing biotin. note, that the automatic clustering for this pathway correctly suggested not to put the two pathogens among the analyzed organisms, c. diphtheriae and c. jeikeium, into the same set, as an unexperienced user might have done. only the green colored reactions are annotated for c. jeikeium, which clearly makes it the outsider in this analysis. another application case is the comparison of metabolic reaction content of pathogenic bacteria against human. host comparison can be the identification of reactions that occur only in bacteria but not in human. bacterial enzymes coding for these reactions may serve as potential drug targets, if they are specific to the bacteria and essential for their survival. using the comparative pathway analyzer, we apply the clustering aproach to pseudomonas aeruginosa pa7, pseudomonas aeruginosa pao1 and homo sapiens. when screening the results on the overview page, all pathways are of interest for that any reactions are reported to be present in the pathogens only. one example is the kegg alanine and aspartate metabolism : the detected reactions are the kegg reactions r00401, r00490 and r00357 (supplementary figure 1). r00401, which in p. aeruginosa is catalyzed by the alanine racemase, has already been shown by perumal. (11) to be a potential drug target in their comprehensive analysis of metabolic enyzmes in p. aeruginosa. the conversion between l- and d - alanine is essential for p. aeruginosa pao1 due to the fact that d - alanine is a necessary component of the bacterial cell wall (12). whether or not the other reactions may serve as potential drug targets remains to be proven. we presented cpa, a web server that supports researchers in analyzing the metabolic reaction content of organisms. analyses are based on the annotation provided by the kegg database and optionally by additional data sets the user may upload. it also permits finding and visualizing the differential reaction content of two sets of organisms. clustering methods can be invoked for finding appropriate sets of organisms as a basis for detecting the differential reaction content. visualization and pathway definition are by default based on kegg pathway maps, but we also offer this functionality for user defined pathways. note that results of the analyses presented here are not only sensitive to true differences, but are also strongly influenced by the quality of annotation of the organisms under investigation. | in order to understand the phenotype of any living system, it is essential to not only investigate its genes, but also the specific metabolic pathway variant of the organism of interest, ideally in comparison with other organisms. the comparative pathway analyzer, cpa, calculates and displays the differences in metabolic reaction content between two sets of organisms. because results are highly dependent on the distribution of organisms into these two sets and the appropriate definition of these sets often is not easy, we provide hierarchical clustering methods for the identification of significant groupings. cpa also visualizes the reaction content of several organisms simultaneously allowing easy comparison. reaction annotation data and maps for visualizing the results are taken from the kegg database. additionally, users can upload their own annotation data. this website is free and open to all users and there is no login requirement. it is available at https://www.cebitec.uni-bielefeld.de/groups/brf/software/cpa/index.html. |
diabetes mellitus is a common metabolic disease characterized by hyperglycemia which often leads to nerve injury probably through the induction of systemic and neuronal oxidative stress (1, 2). it has been shown that high glucose (hg) induces oxidative stress and cell death via nadph oxidase dependent generation of reactive oxygen species and no production (3, 4). during cerebrovascular accidents, ischemia induced metabolic deficiency appears to promote a cascade of events leading to neuronal cell death via both necrosis and apoptosis (-). in other words, ischemia induced glucose and/or oxygen deficiencies can cause both cellular and functional damage to the brain (8, 9). also, neuronal cells deprived of means of production of energy appear to be highly susceptible to excitotoxicity (8, -). although glucose appears to be essential in normal function of the nervous system (14), there is conflicting evidence regarding the effects of extracellular glucose concentration on various forms of ischemic injury (9). some studies have found that hyperglycemia worsens the effects of ischemic insult, probably due to enhanced lactate generation which results in tissue acidosis and leads to generation of reactive oxygen species (9, -). in contrast, other studies have found that elevated glucose levels before hypoxic insult reduced neuronal damage (9, -), while other studies have demonstrated that hyperglycemia can decrease glutamate levels released during ischemia (9, 15, -). corroborating evidence of neuroprotective effects of glucose was provided by dave kr, pileggi a and raval ap () who showed that exposure of hippocampal slices to hypoglycemia prior to ischemic insult exacerbated ischemia induced damage. research on noscapine, a non - addictive antitussive alkaloid of opium, as a neuroprotective agent in our laboratory had previously shown it efficacy in attenuating brain injury after hypoxia in neonatal rats (33). also, in a limited clinical trial, it was shown that noscapine reduced sharply the mortality rate of stroke patients (34). in order to delineate the mechanism of the neuroprotective effects of noscapine, in this study, we investigated if noscapine could protect primary murine fetal cortical neurons exposed to oxygen - glucose deprivation/24 h. recovery during high glucose condition. the intracellular free calcium concentration and no production were measured as two important factors during ischemic insult. chemicals and reagents noscapine was obtained from temad - d pharmaco - chemical company (tehran, iran). the stock solution was prepared at 1 mol / l in dimethyl sulfoxide (dmso) and stored at 20 c. rpmi-1640, glutamine - free dulbecco 's modified eagle 's medium (dmem), foetal bovine serum (fbs), horse serum, penicillin- streptomycin, cytosine arabinoside, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl - tetrazolium bromide (mtt), d - glucose, fura-2 am, poly - l - lysine, n nitro - l - arginine methyl ester (l - name), mannitol, sodium - azide (nan3) and modified griess reagent were purchased from sigma chemical co. (st. all other chemicals used were of the purest grade available from regular commercial sources. murine neuronal cell cultures primary cultures of cerebral cortex neurons were obtained from mouse embryos (e1518) as described previously (35, 36). briefly, timed - pregnant mice were anaesthetised with chloroform and killed by cervical dislocation. skull cap were removed, cerebral cortex was isolated and the cortex was cut into small pieces. the resulting homogenate was centrifuged at 300g and 20 c for 5 min and plated on poly - l - lysine - coated 12-well culture dishes at a density of 610 cells in rpmi-1640 medium supplemented with 10% fbs, 5% horse serum, 100 iu / ml penicillin and 100 g/ ml streptomycin. poly - l - lysine (150,000300,000 mw) was dissolved in deionised water, and the plate bases were filled with this solution (20 g/ ml) 24 h. before the experiment. after 5 min standing at room temperature, the solution was aspirated, and the plates were left to dry in a laminar flow bench overnight. the culture dishes were kept at 37 c in humidified 95% air and 5% co2. after 2448 h. 10 m cytosine arabinoside (cytosine 1--d - arabinofuranoside) was added to the culture medium to inhibit the proliferation of non - neuronal cells. the culture medium was changed twice weekly, and the neurons were used for experiments after 1316 days of incubation. all experiments were approved by the institute of animal care committee at the iran university of medical sciences. oxygen glucose deprivation and drug exposure procedures for oxygen glucose deprivation (ogd) were performed as described previously (37). neuronal cells were incubated for 24 h. in rpmi-1640 containing 25, 100 mm d - glucose in presence or absence of noscapine. the culture medium was replaced with glucose / glutamine - free dmem, and cells were exposed to ogd for 30 min in a small anaerobic chamber previously filled with 95 % (v / v) n2 and 5 % (v / v) co2 at 37 c. to terminate ogd, the chamber was opened, the medium was replaced with rpmi-1640 and the cultures were then placed in an incubator with 5 % co2 for 24 h. to examine the effects of noscapine, the drug (0.5 - 2 m) was added to cultures in media containing 25 mm or 100 mm d - glucose 24 h. before ogd. the concentrations of noscapine were chosen based on the results of preliminary experiments to identify nontoxic levels (data not shown). analysis of neuronal cell viability neuronal cell viability was measured using the colorimetric mtt assay, as previously described by mosmann t (). briefly, cells were incubated with 0.5mg / ml mtt in rpmi at 37 c under 5 % co2 for 3 h. the blue formazan reduction product, which is generated by the action of the succinate dehydrogenase on the dye only in living cells, was dissolved in 100l dmso, and its absorbance was read at 570 nm using a dynex mmx microplate reader (dynex, richfield, mn, usa). the data were expressed as the percentage of viable cells in oxygen glucose deprivation - exposed plates compared with control normoxic plates. measurement of nitrite levels the level of nitrite as an indicator of no production in the culture medium was measured using modified griess reagent. in brief, after the experiment, the medium in each well was removed and centrifuged at 10,000 g for 10 min at 20 c. then, 100 l of the supernatant was mixed with an equal volume of griess reagent at room temperature for 10 min, and the absorbance was measured at 540 nm using a microplate reader. plates not exposed to ogd were used as external control, and plates exposed to ogd without the addition of any drug were used as an internal control. measurement of intracellular free calcium measurement of intracellular free calcium concentration was performed using the ca - sensitive indicator fura-2 bya olympus ix-71 inverted microscope and ccd camera equipped with an automated filter changer. cortical cell cultures grown on a coverslips were loaded with 4 m fura-2/am at room temperature for 30 min. cells were washed three times with a salt solution containing (in mm) : 140 nacl, 3 kcl, 2.5 cacl2, 1.2 mgcl2, 7.7 glucose and 10 hepes, ph 7.4. the fura-2 uorescent images (ex 340/380 nm ; ratio metric analyses were carried out using image j software (nih). statistical analysis the effects of noscapine in high glucose conditions on primary cortical neurons exposed to 30 minutes ogd we examined the effect of d - glucose in cell viability as measured by the mtt assay. our results demonstrated that after 24 h. of exposure to 25 or 100 mm d - glucose neuronal cell viability was reduced by 73.51.04 and 53.150.47 respectively. as shown in figure 1 the decrease in cell viability induced by hg media was concentration dependent and statistically significant when compared to the control cultures which were incubated in media with normal glucose concentration (4.5 mm). we explored the effects of high osmolarity on neuronal cell death by pre - treating neuronal culture with 100 mm mannitol (negative control for osmolarity). our results indicated that there was no significant difference in cell viability between neuronal cultures in control group (100 %) versus 100 mm mannitol (98.670.1). thus any observed change in cell viability is due, directly, to the effect of glucose on neuronal cells. effects of normal and high glucose on cell viability for 24 h. primary cultured murine cortical neurons incubated in low glucose (4.5 mm), high glucose (25 and 100 mm), or mannitol (100 mm) containing medium. mannitol (100 mm) was used as an osmotic control for the high - glucose concentration. exposure to 30 min of ogd decreased viability of neuronal cells which had been kept in media with normal glucose to 55.581.09. neuronal cells pretreated for 24 h. with 25 or 100 mm glucose prior to ogd exposure showed an increase in cell viability (71.470.79 and 80.560.66 respectively) when compared with normal glucose medium (figure 2a - b). the effect of noscapine (nos) in the presence of 25 mm (a) or 100 mm (b) d - glucose (glu) on primary cultured murine cortical neurons subjected to a 30 min oxygen - glucose deprivation/24 h. recovery - induced cell injury. 0.001 vs. internal control group (normal glu). external control : the plates not exposed to ogd and without addition any drugs in order to determine if noscapine (0.5 - 2 m) could affect neuronal cells viability in the presence of 25 and 100 mm d - glucose prior to 30 min ogd, cultures were pretreated with noscapine and hg. our results showed that in the presence of noscapine protective effect of glucose was potentiated significantly compared to that of d - glucose alone (figure2a - b). the increase in cell viability was 95.671.02 and 98.910.66 for 2 m noscapine, the most effective concentration, in the presence of 25 and 100 mm d - glucose. the effects of noscapine in high glucose conditions on no production in primary cortical neurons exposed to 30 minutes ogd we examined the effects of noscapine in hg conditions on no production in primary cortical neurons exposed to 30 min ogd. as shown in figure. 3a - b, under normal glycemic pre - treatment, neuronal cells exposed to 30 min ogd, produced significantly more nitrite compared to groups which were not exposed to ogd (p<0.001). cortical neurons pre - treated with 25 or 100 mm d - glucose prior to exposure to 30 min ogd produced significantly less no 1.420.09 m and 1.270.08 m respectively. pre - treating neurons with noscapine (0.5 - 2 m) during hg condition (25 or 100 mm) induced a further decrease in no production as compared with hg alone (p<0.001). the neuronal no production induced by 30 min of ogd was attenuated by 100 m l - name, a nitric oxide synthase inhibitor (figure 3a - b). the effect of noscapine (nos) on no production in the presence of 25 mm (a) or 100 mm (b) d - glucose (glu) on murine primary cultured cortical neurons during a 30 min oxygen - glucose deprivation/24 h. recovery - induced cell injury. p < 0.001 vs. internal control group (normal glu). the plates not exposed to ogd and without addition any drugs the effects of noscapine in high glucose conditions on intracellular calcium levels against chemical ogd to determine the intracellular calcium levels during ischemia, the neuronal cells were exposed to 30 min sodium - azide (4 mm) as a model of chemical ogd in the absence of glucose. 4 mm nan3 was able to induce a significant increase in (ca)i levels in cortical neurons bathed in ca - containing medium (169.874.73) (figure.4 - 5). exposure of cortical neurons to sodium azide and 25 mm or 100 mm d - glucose after 30 min of ogd, decreased (ca)i levels by 161.234.29 and 143.332.23 respectively. furthermore, the ischemia - evoked increase in (ca)i levels was decreased by 150.822.7 and 124.121.87 in the presence of 2 m noscapine with 25 or 100 mm d - glucose respectively (figure 4 - 5). changes in (ca)i levels induced by chemical ogd (4 mm nan3) in the presence and absence of 2 m noscapine during 25 mm d - glucose in primary cortical neurons. (a) time course of nan3-induced (ca)i rise in presence and absence of 2 m noscapine during 25 mm d - glucose. nan3 (4 mm) stimulated (ca)i levels increase. 25 2 m noscapine added to 25 mm d - glucose during ischemia reduced the (ca)i levels than that of 25 mm d - glucose alone. (b) bar graph of mean change in (ca)i levels acquired in response to chemical ischemia in presence and absence of 2 m noscapine during 25 mm d - glucose. significant difference from control group (baseline) and chemical ischemic groups p < 0.001 changes in (ca)i levels induced by chemical ogd (4 mm nan3) in the presence and absence of 2 m noscapine during 100 mm d - glucose in primary cortical neurons. (a) time course of nan3-induced (ca)i rise in presence and absence of 2 m noscapine during 100 mm d - glucose. 100 mm d - glucose could decrease (ca)i levels during chemical ischemia. 2 m noscapine added to 100 mm d - glucose during ischemia reduced the (ca)i levels than that 0f 100 mm d - glucose alone. (b) bar graph of mean change in (ca)i levels acquired in response to chemical ischemia in presence and absence of 2 m noscapine during 100 mm d - glucose. significant difference from control group (baseline) and chemical ischemic groups p < 0.001 hyperglycemia appears to be an important factor in the development of diabetic neuropathy (2). it induces neuronal damage and dysfunction probably via the generation of reactive oxygen species (2, 39). as such, many studies have shown that hg does indeed cause neuronal cell death (-). however, there is conflicting evidence regarding the role of extracellular glucose during brain ischemic insults (9). it is generally held that glucose supplies the essential energy needs of the brain and alteration of glucose metabolism may result in dysfunction of the cns (8, 14). a number of studies have obtained evidence for the beneficial effects of enhanced glucose entry into neurons during ischemia (14, 40, 41). these works suggest a protective role for glucose against excitotoxicity and hypoxic ischemia. in the current study, we aimed to show the effects of hg on intracellular ca levels, no production, and general cell viability in the absence and presence of noscapine which we had previously shown to have neuroprotective effects of its own (42). we examined the effect of d - glucose (25 or 100 mm) on neuronal cell death. cortical cultures were exposed, for 24 h. to 4.5 mm (as normal glucose control), 25 and 100 mm glucose (as high glucose). our results showed that 25 and 100 mm d - glucose could significantly reduce cell viability in a concentration dependent fashion as compared with control. in fact, neuronal cell viability treated with 100 mm glucose was markedly reduced as compared to 25 mm d - glucose. this was consistent with previous studies demonstrating that high concentrations of glucose induced neuronal injury in an in - vitro model (2, 4, 43). for example, koshimura k, tanaka j, murakami y and kato y () showed that hg increased no production and induced cell death in differentiated pc12 cells. also, vincent am, mclean ll, backus c and feldman el () demonstrated that in neurons, hg caused oxidative stress, induced mitochondrial dysfunction and programmed cell death. thus, the data obtained by us and other workers, confirmed exposure to hg for 24 h. or more, to be detrimental to neuronal cells. however, there is data to suggest a neuroprotective role for short period exposure to hg after ischemia (40). here toping up of intracellular glucose stores has been proposed to be the reason for this effect (9, 44). thus, to determine whether a short exposure to hg could protect cortical neuronal cells from ogd insult, cells were pre - incubated for 24 h. in rpmi-1640 containing 25 or 100 mm glucose prior to 30 min ogd. this effect was more visible for 100 mm d - glucose than that of 25 mm d - glucose. other workers have shown hg to be protective against the effects of excitotoxins (8, 9, 41). seo sy, kim ey, kim h and gwag bj () showed that although pretreatment with 25 mm glucose did not induce great protection against nmda evoked neuronal death, 100 mm glucose pretreatment enhanced cell survival after treatment with nmda. it has been demonstrated that ogd induces cell death by enhancing the release of extracellular glutamate which in turn increases activation of nmda receptors leading to increased intracellular ca levels and augmented no production (-). thus the data obtained by us is in part agreement with the work of many other researchers (8, 9, 14, 40, 41). all this suggests an important role for intracellular ca and no in both excitotoxicity and ogd induced cell death. in a previous study carried out in this lab, effects of noscapine on ogd induced death in primary cortical neurons had been studied (42). noscapine appeared to impart protection against ischemia by attenuating ogd induced no production. in this study we investigated the possibility that noscapine could potentiate the protective effects of hg from ogd in primary cortical neurons. when neuronal cells pre - treated with noscapine (0.5 - 2 m) subsequent the addition of hg (25 or 100 mm) and then subjected to 30 min ogd, the cell viability increased as compared with the cell pre - treated with high - glucose alone. in order to find out the neuroprotective effect of noscapine in hg condition, we set out to measure the change in ca and no after ogd in cells pretreated with hg. sodium azide / glucose deprivation (chemical ogd) model was used to create in - vitro ischemic conditions. this model has been shown to elicit electrophysiological and neurochemical changes that are similar to the ogd model of ischemia. while this model having the advantage of producing neurochemical changes more rapidly and reproducibly (49, 50), thus facilitating the recording of changes in (ca)i (51). our results showed that during chemical ogd the (ca)i was significantly increased compared to control condition. this is consistent with previous studies indicating that nan3 could increase neuronal (ca)i, as a consequence of energy depletion (52). we next examined the (ca)i level in neuronal cells exposed to 25 or 100 mm d - glucose after 30 min of chemical ogd treatment. the results suggested that exposure to 25 or 100 mm d - glucose was able to reverse the effects of chemical ogd on intracellular ca levels in cultured cortical neurons. also, 100 mm glucose was more effective in decreasing (ca)i levels than 25 mm glucose. chan j and greenberg da () showed that in pc12 cells, increasing glucose concentration in the culture medium by a factor of 4, attenuated ischemic injury by reducing the dihydropyridine - sensitive and depolarization - induced increase in ca. these results suggested that hg might decrease neuronal cell death by reducing (ca)i levels in neuronal cells. finally, our findings demonstrated that cells exposed to d - glucose together with 2 m noscapine following chemical ogd had significantly lower (ca)i compared to d - glucose treatment alone. this data suggests that noscapine reinforced the protective effect of hg via reduction in intracellular ca levels. also, noscapine potentiated the effects of 100 mm glucose more than it decreased the effects of 25 mm glucose. however, because 100 mm d - glucose could considerably reduce cell death during ischemic insult, the neuroprotective effect of 2 m noscapine in presence of 100 mm d - glucose also could better act on neuronal cells to reduce (ca)i following chemical ogd. many studies have been shown that no is involved in both physiological and pathological events, and is believed to play different roles in both lethal and sub - lethal events (47, 54). several studies have found evidence indicating an involvement of no synthase (nos) in glutamate - induced excitotoxicity and there were some corroborating data that no production increases during ischemia (35, 45, 47, -). for example, it has been shown that during ogd, calcium enters the neurons through nmda receptor and/or l - type voltage - gated calcium channel activation and activates nos which leads to no production (45). zhang m, ning g - m, hong d - h, yang y, kutor j and zheng x - x () had shown that ca - free medium markedly inhibited the ogd - induced increase in no, suggesting that the inhibition of ca might cause the decrease of no synthesis. our results confirmed that in neuronal cells exposed to 30 min ogd, no production was signicantly increased, supporting a role for no in the cascade of events leading to ogd - induced cell death (45). however, the exposure of neuronal cells to 25 or 100 mm d - glucose could decrease the no production during 30 min ogd. after that neuronal cells were pre - treated with noscapine (0.5 - 2 m) in the presence of the 25 or 100 mm d - glucose and then subjected to 30 min ogd. the results indicated that the no production was significantly reduced as compared with 25 or 100 mm d - glucose alone. in addition, when the cultures were pre - treated with the nos inhibitor, l - name, no production was signicantly decreased and returned to the level of the external control. suggesting that nos inhibitors could markedly decrease the no production after nmda receptor activation, and decreased excitotoxic injury in cultured neurons (45, 58, 59) in conclusion, noscapine potentiated the neuroprotective effect of hg (25 and 100 mm) treatment prior to ogd. the enhanced cell viability after treatment with both noscapine and hg was mirrored by a dampening of ogd induced intracellular caand no rise. these observations support the hypothesis that calcium homeostasis and no production have crucial roles in excitotoxicity process. | in the present work we set out to investigate the neuroprotective effects of noscapine (0.5 - 2 m) in presence of d - glucose on primary murine foetal cortical neurons after oxygen glucose deprivation/24 h. recovery. cell viability, nitric oxide production and intracellular calcium ((ca2+)i) levels were evaluated by mtt assay, the modified griess method and fura-2 respectively. 25 and 100 mm d - glucose could, in a concentration dependent manner, improve cell viability and decrease no production and (ca2+)i level in neuronal cells after ischemic insult. moreover, pre - incubation of cells with noscapine, noticeably enhanced protective effects of 25 and 100 mm d - glucose compared to similar conditions without noscapine pre - treatment. in fact, noscapine attenuated no production in a dose - dependent fashion, after 30 minutes (min) ogd, during high - glucose (hg) condition in cortical neurons. pretreatment with 2 m noscapine and 25 or 100 mm d - glucose, was shown to decrease the rise in (ca2+)i induced by sodium azide / glucose deprivation (chemical ogd) model. these effects were more pronounced than that of 25 or 100 mm d - glucose alone.the present study demonstrated that the neuroprotective effects of hg before an ischemic insult were augmented by pre - treatment with noscapine. our results also suggested that the neuroprotection offered by both hg and noscapine involve attenuation of no production and (ca2+)i levels stimulated by the experimental ischemia in cortical neurons. |
there is ample evidence showing that 711% of hcv - infected children and adult individuals have thyroid dysfunction, mainly subclinical hypothyroidism, prior to the initiation of treatment [1, 2 ]. the association between autoimmune thyroiditis in hcv - infected patients, in particular in subjects with hcv - related mixed cryoglobulinemia, has also been confirmed. once combined pegifnalpha2b + ribavirin therapy is initiated, the percentage of patients with thyroid dysfunction goes up to 1520%. immunological mechanisms at the origin of this thyroid dysfunction are presumed but the precise mechanism still is unknown. it is involved in some organ - specific autoimmune diseases such as rheumatoid arthritis, asthma, inflammatory bowel disease, multiple sclerosis, and even organ allograft rejection [69 ], among many others. the main function of il-17-secreting t cells is to mediate inflammation, by stimulating production of inflammatory cytokines, such as tnf - a, il-1b, il-6, and inflammatory chemokines that promote the recruitment of neutrophils and macrophages. initial evidence suggested that human th17 cells were regulated by cd4 + 25+foxp3 + regulatory t cells, but recent evidence suggests an alternative regulatory mechanism mediated by il-10 and tgf - b [11, 12 ]. ag - specific th17 cells are induced in hcv - infected patients, but the hcv - ns4 protein also induces the secretion of il-10 and tgf - b by monocytes. neutralization of both anti - inflammatory cytokines significantly enhances ns4-specific il-17 and ifn - gamma production by t cells from hcv - infected donors. although this may represent a novel immune subversion mechanism by the virus to evade host protective immune responses, it could also be responsible for the thyroid dysfunction an initial evaluation of the thyroid function in chronically infected hcv patients referred to the gastroenterology service at mexico 's h.r. issste, showed that a high percentage of patients had subclinical hypothyroidism prior to the initiation of combined treatment. therefore, our aim was to evaluate the serological status of il-10, tgf - b and il-17 in chronically non - interferon treated hcv - infected patients with or without subclinical hypothyroidism. all patients tested positive for antibodies to hcv using an enzyme immunoassay (abbott diagnostics). they were all infected as determined by consistently positive hcv rna serum detection results using a qualitative rt - pcr (amplicor ; roche diagnostic systems). none of the patients (25 males and 62 females) had ever received treatment with pegylated interferon and/or ribavirin. all the patients were catalogued as a class a child - pugh score and were selected to initiate pegylated interferon treatment. the control group was conformed by 40 individuals (20 males, 20 females ; with a mean age of 48 years old) with no history of liver disease, negative to hcv antibodies and normal liver function tests. blood samples, freshly obtained from each patient one week before treatment was initiated, stood for 5 minutes at room temperature before being centrifuged for 10 minutes at 1800 rpm at 4c. plasma and sera was obtained from each patient and aliquots of 0.1 ml were kept at 70c until use. plasma levels of il-2, il-10, il-12, il-17, and tgf - b were determined using commercially available kits purchased from genzyme and r & d systems (quantikine) carefully following the manufacturer instructions. the r value for the linear regression of the standard curve had to be 0.95 or higher, a lower value was considered inadequate and the data obtained of such plates was eliminated. in order to activate latent tgf - b to immunoreactive tgf - b platelet - poor plasma samples was incubated for 10 min at room temperature with 1 n hcl and then the sample was neutralized with a solution made of 1.2 n naoh/0.5 m hepes for another 10 minutes before the determination was performed. commercially available kits, also from r & d systems were used to determine antithyroglobulin (tg), antithyroid peroxidase (tpo), anticardiolipin, antiglomerular basement membrane (gbm) and anticyclic citrullinated peptide (ccp) antibodies. the plates were read in a multiskan ascent (thermolab systems, maltham, ma, usa) microplate reader using a 450 and 490 nm filters. hormone determination (tsh, t3, and free t4) and liver function tests (alt, ast, albumin, immunoglobulin) were performed in the laboratory of the hospital. subclinical hypothyroidism was defined as a tsh value of 4 pg / ml with normal t3 and t4 values. the results of all these evaluations in the control group were within the established normal values determined in our laboratory (0.254.0 ui / ml for tsh ; 0.61.9 ng / ml for t3 ; 0.71.8 ng / dl for free t4 ; 240 ui / ml for ast and 240 ui / ml for alt). the results obtained in the control samples for the different cytokines were 6.8 2.7 pg / ml for il-2 ; 4.9 1.4 pg / ml for il-10 ; 52.7 8.1 pg / ml for il-12 ; 6.75 1.14 pg / ml for il-17, and 398 271 pg / ml for tgf - b1). the normal value for anti - tg is 034 iu / ml, 0 - 12 iu / ml for anti - tpo, > 10 iu / ml for anti - cardiolipin, and 07 iu / ml for anti - gbm and anti - ccp antibodies. differences between groups were evaluated by student 's t test, confidence interval at the 95% value, tukey test and kruskal - wallis one way analysis of variance on ranks, and holm sidak method. the mean age of male patients was 45.7 years old whereas that of the females was 51.8 years old. the predominant hcv genotype was 1b (42 patients) followed by 1a (18 patients), 2b (17 patients), 2a (9 patients), and one patient with genotype 4. seventy percent of the patients with elevated tsh (6.45 2.21 iu / ml) (group a) were females ; the percentage of female patients in the group with normal tsh values (2.58 0.95 ui / ml) (group b) was similar (71%). the difference in tsh values between both groups was highly significant (p <.0001) as well as between group a and control group (p <.001 ; according to the all pairwise multiple comparison procedures - holm - sidak method). there were no statistical differences in tsh values related to gender (5.82 1.73 ui / ml for males and 6.71 2.40 ui / ml for females in group a versus 2.26 0.76 ui / ml for males and 2.68 0.99 ui / ml for females, in group b). similarly there were no statistical significant differences between both groups in relation to viral load. although the values of the following markers were statistically different from those of the control group, there were no statistical significant differences between group a and group b results in relation to ast, alt, t3, and free t4. one patient with normal anti - tg, anti - tpo, and anti - ccp values had elevated anti - gbm and anti - cardiolipin antibodies. this patient had consistently low platelet counts but had no history of miscarriage or deep vein thrombosis. none of the anti - tpo nor anti - tg positive patients in group a had signs or symptoms of hashimoto 's thyroiditis. the percentage of autoantibodies in group b was 33% for anti - tg antibodies, and 7% for anti - tpo. none of the patients in group b had elevated values of anti - gbm or anti - ccp, and only one had elevated anti - cardiolipin antibodies. interestingly, the latter patient was also positive for anti - tpo antibodies and had deep vein thrombosis of the lower extremities. in relation to the cytokines it was interesting to observe that although the concentrations in hcv+ patients were significantly higher than those found in the control group for all the cytokines, the intergroup differences (group a individuals versus group b) for il-10 (14.83 2.98 pg / ml versus 15.27 1.70 pg / ml) and il-12 (271.59 196.43 pg / ml versus 221.13 101.60 the cytokines that showed a highly statistically significant difference between group a and group b patients were : il-2, il-17, and tgf - b (table 1). the kruskal - wallis analysis of variance at the 0.05 level of significance for il-2 showed a median value of 611 pg / ml for group a, 195 pg / ml for group b, and 7.36 pg / ml for the control group (p <.001) ; the significance of these differences between groups a and b with the control was confirmed by the tukey test (p <.05). as far as il-17 was concerned, the kruskal - wallis analysis of variance showed a median value of 19.4 pg / ml for group a, 18.5 pg / ml for group b, and 8.9 pg / ml for the control group (p <.001) ; similarly to il-2, il-17 results were also confirmed by the tukey test. there were no differences in il-17 concentration in both groups in relation to the viral genotype. finally, tgf - b values passed the normality test and the one way analysis of variance demonstrated a significant difference between groups a and b with the control group (p <.001) but not between group a and group b. it was interesting to observe that tgf - b concentration in group a patients was 38% below that of group b patients. hcv infection has been related to various autoimmune disorders including thyroid dysfunction [14 ] and papillary thyroid cancer. the presence of hypothyroidism in chronically hcv - infected individuals, either under treatment or not, has been well documented [1, 16, 17 ]. one interesting observation was the high percentage of hcv - infected patients having hypothyroidism (23%) versus an average 12% cited in the literature ; this could be secondary to the significantly higher frequency of thyroid microsomal antibodies in mexican women in comparison with caucasian or black women thus representing a higher predisposition to autoimmunity against thyroid gland. we found autoimmune thyroid involvement as judged from serum auto - antibodies and tsh values in 20 of the 87 patients that we analyzed. the percentage of individuals in the subclinical hypothyroidism group with autoantibodies was significantly higher than the percentage found in normothyroid hcv+ patients and control. none of our patients had manifestations of hashimoto 's disease and only one of the female patients in the hypothyroid group, had a pattern compatible with an antiphospholipids syndrome, an association found in 20% of hcv - infected patients. the percentage of females with autoimmune disease in our study (70%) is identical to that found in a multicenter international study [20, 21 ]. nevertheless, it has recently been suggested that the presence of autoantibodies in hcv - infected patients do not appear to be of clinical importance. one patient in each group had elevated anticardiolipin antibodies but none of them suffered stroke, although the patient with deep vein thrombosis is a strong candidate. the thyroid dysfunction in hcv - infected patients has been associated to interleukin 2 [2426 ], a cytokine secreted primarily by th1 cells. patients with non - hcv - related diseases being treated with high doses of il-2 also develop thyroid dysfunction [27, 28 ]. the increased interleukin 2 serum concentration determined in our non - il-2 or ifn - treated hcv+ hypothyroid patients, corroborates the above mentioned findings. interleukin 2 is known to expand th17 cells in some autoimmune diseases. a key cytokine for il-2 production and th1 cell differentiation is il-12 [30, 31 ]. although the core protein of hcv seems to have a suppressive action on il-12 production at the transcriptional level, our results showed that the concentration of il-12 was similar in both of our hcv - infected groups, but considerably higher than the control group. this suggests that hypothyroidism was not related to interleukin-2 despite the fact that transgenic mice with il-12 serum concentrations in the 150 pg / ml range develop a moderate primary hypothyroidism. the th17 response is characterized by a serum increase in interleukin-17a, commonly known as il-17. this cytokine, secreted by the cd4 + t cells known as th17, is also secreted by some cd8 + t cells, nkt, alpha - beta or gamma - delta t cells, eosinophils, neutrophils, and monocytes [3537 ]. th17 cells are mainly linked to autoimmune diseases, namely, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, lyme disease, psoriasis, and uveitis [29, 38 ]. of the five different il-17 receptors (a to e), il-17 only bind to il-17ra and c [35, 39 ]. the pituitary gland and the thyroid, the regulation of th17 cells is basically mediated by cd4+cd25+foxp3 + t reg cells which are capable of inducing anergy towards self- and alloantigens, thus playing an important role in autoimmunity [4143 ]. nevertheless, a new regulatory mechanism mediated by il-10 and tgf - b, both of which suppress il-17 production [11, 12 ], has been suggested. our hypothyroid patients had similar amounts of il-10 than the normo thyroid group but lower tgf - b serum concentration. therefore, the only abnormality that could explain the high il-17 serum concentration and the thyroid dysfunction was related to alterations in the foxp3 + treg cells, probably through enhanced stat3 phosphorylation [36, 45 ]. nevertheless, recent evidence shows that tgf - beta orchestrates th17 cell differentiation in a concentration - dependent manner. low tgf - b concentrations favors th17 cell differentiation whereas high tgf - b concentrations favors foxp3 + treg cell differentiation. the former is accompanied by high levels of il-17, as we observed in our hypothyroid patients, whereas the latter is accompanied by a tighter regulation of il-17 secretion, as we observed in our normothyroid patients. despite tgf - b apparent importance it has recently been shown that the proportion of peripheral th17 cells in patients with autoimmune thyroid disease is higher than in control subjects. although there is evidence showing that il-2 can inhibit human th17 cell differentiation through a stat-5 mediated pathway and enhanced tgf - b - induced foxp3 expression [48, 49 ], a recent report by deknuydt. show that cd4+cd25 + foxp3 + t regulatory cells in the presence of il-2, in an inflammatory microenvironment, can be converted into proinflammatory th17 cells. this could explain the link between inflammation, high il-2 and il-17 concentrations, and autoimmunity in our patients. interleukin 17 is also involved in human alcoholic liver disease, an entity that shares some features with autoimmune diseases. the viral load in our patients could be definitively considered low and such viral loads have been linked with the development of auto antibodies due to the lowering of the b cell activation threshold or by inducing self - reactivity through a mechanism of molecular mimicry [52, 53 ]. we believe that our results point towards an abnormality in the t regulatory cells, induced by the hepatitis c virus. the fact that not all the hcv - infected patients develop an full blown autoimmune disease suggests that there are many individual variations that go beyond the viral load, viral genotype, presence of auto antibodies or liver biochemical alterations. we must not forget that the hcv does replicate within the thyroid tissue and that the evolution of the hcv infection is related to distinct immune cell cytokine expression profiles. recently it has been postulated that a th1cell mediated immune response underpins the association of chronic hcv infection with endocrine disease, our results suggest that a th17 immune response should also be considered. | hcv - ag - specific th17 cells secrete il17, a cytokine involved in autoimmune diseases and regulated by il10 and tgf - b. 512% of patients with chronic hcv infection have hypothyroidism. we evaluated the role of these cytokines in this patients by determining serum concentration of tsh, t3, free t4, il2, il10, il12, il17, tgf - b, anti - tg, tpo, ccp, gbm, and cardiolipin antibodies in 87 chronically noninterferon treated hcv - infected patients. 20 patients (group a) had elevated tsh values (> 5 ui / ml) whereas the remaining 67 (group b) had normal values. the percentage of anti - tpo, tg, gbm, and cardiolipin antibodies in group a patients (33%, 41%, 5% and 5%, resp.) as well as il17, il2 and tgf - b concentrations (25 23 pg / ml, 643 572 pg / ml, and 618 221 pg / ml, resp.) were significantly higher than group b. abnormal th17 regulation mediated by il-2 and low tgf - b concentrations is associated with hypothyroidism in chronically - infected hcv patients. |
eight adult cats weighing between 2.9 and 4.4 (mean, 3.9) kg were used in this study. each animal was anesthetized with 5% halothane administered through an endotracheal tube by a mechanical ventilator (mini-7 ; royal medical, seoul, korea). pancuronium bromide (panslan ; reyon pharmaceutical, seoul, korea) (0.6 mg / kg) was used for skeletal muscle relaxation. to monitor blood pressure and allow pulse - gated mr imaging, a femoral artery was cannulated, and for the administration of drugs and contrast agents, this procedure was applied to a femoral vein. after a left lateral thoracotomy along the fifth intercostal space, pericardiotomy was performed by means of a midline incision, and a pericardial cradle was prepared by attaching the margins of the dissected pericardium to the adjacent thoracic wall. the left anterior descending (lad) coronary artery was isolated distal to the first diagonal branch, and a snare loop was made with 4 - 0 silk placed in a slender plastic tube. occlusion or reperfusion of the lad artery was achieved simply by fastening or releasing the snare loop. obstruction of this artery was confirmed by observing changes in the color of the myocardium at risk during a preliminary test occlusion. in each cat the lad artery was occluded for 90 minutes, and this was followed by 90 minutes of reperfusion. gd - dtpa has been widely used as a clinical mr contrast agent. after injection, it is rapidly distributed from the vascular compartment to the extracellular fluid space. in rats, the plasma half - life is approximately 12 minutes (8), and due to a rapidly changing blood concentration resulting from the large volume distributed, the utility of gd - dtpa for the mr diagnosis of acute myocardial ischemia is therefore limited (9, 10, 11). gadomer-17, provided by schering, is designed for prolonged intravascular retention and reduced transcapillary diffusion and has a molecular weight (mw) of 17453 g / mol. noh. demonstrated that following the administration of gadomer-17, the signal intensity of the enhanced area seen on t1-weighted images increased rapidly, and maximum enhancement was detected during a 40 - 60 minute period (7). gadophrin-2, a necrosis - avid contrast material, consists of mesoporphyrins linked to gadolinium, and its mw is 1697.25 g / mol. the methods by which it is synthesized, and its chemical structure, physiochemical properties and imaging behaviors have been previously described in detail (12). mr imaging was performed on a 1.5 t magnetom vision system (siemens, erlangen, germany). for signal reception, a circularly polarized head array coil 27 cm in diameter (siemens, erlangen, germany) was used. during the imaging procedure electrocardiography - triggered breath - hold turbo spin - echo t2-weighted mr images were obtained along the short axis of the heart prior to the injection of contrast agents, and in order to obtain additional information as to myocardial status, images in the sagittal plane were also acquired. the acquisition parameters for t2-weighted mr images were as follows : repetition time msec/ echo time msec of 400 - 600 (according to heart rate) / 82, echo train length of 33, acquisition time of 9 - 10 seconds, matrix size of 132256, field of view of 210280 mm, and section thickness of 5 mm. after acquiring the baseline image and intravenously injecting contrast medium, gadomer-17-enhanced t1-weighted mr images were obtained dynamically for one hour. after visual comfirmation of complete washout of gadomer-17 (3 hours after its injection), gadophrin-2 was administered, and contrast - enhanced t1-weighted mr images were obtained for the next hour. electrocardiography - triggered multisection t1-weighted spin - echo imaging was performed with the following imaging parameters : repetition time msec/ echo time msec of 300 - 500 (according to heart rate)/ 25, section thickness of 5 mm, field of view of 210280 mm, and one signal acquisition. all images were obtained along the short axis of the heart, and to provide additional confirmation of the signal enhancement of irreversibly damaged myocardium, images in the sagittal plane were also occasionally acquired. after mr imaging studies were completed, each cat was sacrificed by intravenous injection of kcl solution. the heart was excised and cut into five or six consecutive slices, 5 mm thick, in the same planes in which the mr images were obtained. the specimens were immersed in 1.5% ttc solution at 36 and stained for 15 minutes, and were then stored in 10% formalin solution for 12 hours.. photographs of ttc - stained specimens in the same planes, for which mr images were obtained along the short axis of the heart, were scanned into a computer (macintosh ; apple computer inc., u.s.a.) to measure the size of the infarct area and of the total left ventricle mass using public domain image processing software (nih image 1.55 ; national institutes of health, bethesda, md., u.s.a.). all mr images were independently analyzed by two experienced radiologists, and discrepancies were resolved by consensus. the size of the infarct area in the myocardium of the left ventricle was measured by outlining the high signal area seen on t2-weighted images and enhanced areas on gadomer-17- and gadophrin-2-enhanced t1-weighted images. the size of the infarct area was expressed as a percentage of the size of the total left ventricle, as revealed by mr imaging and ttc histochemical staining. the size of the enhanced areas seen on gadomer-17 and gadophrin-2-enhanced t1-weighted images, and of the high signal area on t2-weighted images, was compared with that of the infarct area disclosed by ttc histochemical staining. to determine statistical significance (defined as p < 0.05), a paired student t test was used. electron microscopic examinations were performed on tissue taken from three areas. that from the infarct area was sampled from the centers of ttc - unstained areas ; for the lateral border zone, tissue from the ttc - stained peripheral region adjacent to the ttc - unstained area (2 mm apart from that area) was sampled ; and for normal myocardium, tissue was taken from the center of the ttc - stained area of the posterior wall. after collection, tissues were cut into 1-mm cubes and fixed in a 2.5% buffered glutaraldehyde solution for 12 to 16 hours followed by additional fixation in a solution of osmotic acid at 5 for 2 hours. the cubes were then dehydrated in graded alcohol at room temperature, passed through propylene oxide, and placed in a 1:1 mixture of propylene oxide and epon 812 (polyscience inc.) for 12 to 16 hours. sections approximately 0.5 m thick were cut on an lkb ultramicrotome (bromma, sweden) using a diamond knife, and were mounted on a copper grid and stained with 4% aqueous uranyl acetate and lead citrate for examination with a transmission electron microscope (jem-1200 ex ii, tokyo, japan). the electron microscopic criteria for irreversibly damaged myocardium were that all mitochondria were swollen, had disorganized cristae, and contained electron - dense deposits and contraction bands in addition to disrupted sarcolemmas. on the other hand, the ultrastructures of reversibly damaged myocardium showed mild edematous myocytes, increased sarcoplasmic space, a prominent i - band, and mild peripheral aggregation of nuclear chromatin without the features of irreversibly damaged myocardium (13). eight adult cats weighing between 2.9 and 4.4 (mean, 3.9) kg were used in this study. each animal was anesthetized with 5% halothane administered through an endotracheal tube by a mechanical ventilator (mini-7 ; royal medical, seoul, korea). pancuronium bromide (panslan ; reyon pharmaceutical, seoul, korea) (0.6 mg / kg) was used for skeletal muscle relaxation. to monitor blood pressure and allow pulse - gated mr imaging, a femoral artery was cannulated, and for the administration of drugs and contrast agents, this procedure was applied to a femoral vein. after a left lateral thoracotomy along the fifth intercostal space, pericardiotomy was performed by means of a midline incision, and a pericardial cradle was prepared by attaching the margins of the dissected pericardium to the adjacent thoracic wall. the left anterior descending (lad) coronary artery was isolated distal to the first diagonal branch, and a snare loop was made with 4 - 0 silk placed in a slender plastic tube. occlusion or reperfusion of the lad artery was achieved simply by fastening or releasing the snare loop. obstruction of this artery was confirmed by observing changes in the color of the myocardium at risk during a preliminary test occlusion. in each cat the lad artery was occluded for 90 minutes, and this was followed by 90 minutes of reperfusion. after injection, it is rapidly distributed from the vascular compartment to the extracellular fluid space. in rats, the plasma half - life is approximately 12 minutes (8), and due to a rapidly changing blood concentration resulting from the large volume distributed, the utility of gd - dtpa for the mr diagnosis of acute myocardial ischemia is therefore limited (9, 10, 11). gadomer-17, provided by schering, is designed for prolonged intravascular retention and reduced transcapillary diffusion and has a molecular weight (mw) of 17453 g / mol. noh. demonstrated that following the administration of gadomer-17, the signal intensity of the enhanced area seen on t1-weighted images increased rapidly, and maximum enhancement was detected during a 40 - 60 minute period (7). gadophrin-2, a necrosis - avid contrast material, consists of mesoporphyrins linked to gadolinium, and its mw is 1697.25 g / mol. the methods by which it is synthesized, and its chemical structure, physiochemical properties and imaging behaviors have been previously described in detail (12). mr imaging was performed on a 1.5 t magnetom vision system (siemens, erlangen, germany). for signal reception, a circularly polarized head array coil 27 cm in diameter (siemens, erlangen, germany) was used. during the imaging procedure electrocardiography - triggered breath - hold turbo spin - echo t2-weighted mr images were obtained along the short axis of the heart prior to the injection of contrast agents, and in order to obtain additional information as to myocardial status, images in the sagittal plane were also acquired. the acquisition parameters for t2-weighted mr images were as follows : repetition time msec/ echo time msec of 400 - 600 (according to heart rate) / 82, echo train length of 33, acquisition time of 9 - 10 seconds, matrix size of 132256, field of view of 210280 mm, and section thickness of 5 mm. after acquiring the baseline image and intravenously injecting contrast medium, gadomer-17-enhanced t1-weighted mr images were obtained dynamically for one hour. after visual comfirmation of complete washout of gadomer-17 (3 hours after its injection), gadophrin-2 was administered, and contrast - enhanced t1-weighted mr images were obtained for the next hour. electrocardiography - triggered multisection t1-weighted spin - echo imaging was performed with the following imaging parameters : repetition time msec/ echo time msec of 300 - 500 (according to heart rate)/ 25, section thickness of 5 mm, field of view of 210280 mm, and one signal acquisition. all images were obtained along the short axis of the heart, and to provide additional confirmation of the signal enhancement of irreversibly damaged myocardium, images in the sagittal plane were also occasionally acquired. after mr imaging studies were completed, each cat was sacrificed by intravenous injection of kcl solution. the heart was excised and cut into five or six consecutive slices, 5 mm thick, in the same planes in which the mr images were obtained. the specimens were immersed in 1.5% ttc solution at 36 and stained for 15 minutes, and were then stored in 10% formalin solution for 12 hours. photographs of ttc - stained specimens in the same planes, for which mr images were obtained along the short axis of the heart, were scanned into a computer (macintosh ; apple computer inc., to measure the size of the infarct area and of the total left ventricle mass using public domain image processing software (nih image 1.55 ; national institutes of health, bethesda, md. all mr images were independently analyzed by two experienced radiologists, and discrepancies were resolved by consensus. the size of the infarct area in the myocardium of the left ventricle was measured by outlining the high signal area seen on t2-weighted images and enhanced areas on gadomer-17- and gadophrin-2-enhanced t1-weighted images. the size of the infarct area was expressed as a percentage of the size of the total left ventricle, as revealed by mr imaging and ttc histochemical staining. the size of the enhanced areas seen on gadomer-17 and gadophrin-2-enhanced t1-weighted images, and of the high signal area on t2-weighted images, was compared with that of the infarct area disclosed by ttc histochemical staining. to determine statistical significance (defined as p < 0.05), a paired student t test was used. electron microscopic examinations were performed on tissue taken from three areas. that from the infarct area was sampled from the centers of ttc - unstained areas ; for the lateral border zone, tissue from the ttc - stained peripheral region adjacent to the ttc - unstained area (2 mm apart from that area) was sampled ; and for normal myocardium, tissue was taken from the center of the ttc - stained area of the posterior wall. after collection, tissues were cut into 1-mm cubes and fixed in a 2.5% buffered glutaraldehyde solution for 12 to 16 hours followed by additional fixation in a solution of osmotic acid at 5 for 2 hours. the cubes were then dehydrated in graded alcohol at room temperature, passed through propylene oxide, and placed in a 1:1 mixture of propylene oxide and epon 812 (polyscience inc.) for 12 to 16 hours. sections approximately 0.5 m thick were cut on an lkb ultramicrotome (bromma, sweden) using a diamond knife, and were mounted on a copper grid and stained with 4% aqueous uranyl acetate and lead citrate for examination with a transmission electron microscope (jem-1200 ex ii, tokyo, japan). the electron microscopic criteria for irreversibly damaged myocardium were that all mitochondria were swollen, had disorganized cristae, and contained electron - dense deposits and contraction bands in addition to disrupted sarcolemmas. on the other hand, the ultrastructures of reversibly damaged myocardium showed mild edematous myocytes, increased sarcoplasmic space, a prominent i - band, and mild peripheral aggregation of nuclear chromatin without the features of irreversibly damaged myocardium (13). the high signal area seen on t2-weighted images and the enhanced area on gadomer-17-enhanced t1-weighted images were larger than the enhanced area on gadophrin-2-enhanced t1-weighted images and the infarct area disclosed by ttc histochemical staining (t2= 39.2 % ; gadomer-17 = 37.25 % vs gadophrin-2 = 29.6 % ; ttc staining = 28.2 % ; p < 0.05). the size of the high signal area seen on t2-weighted images correlated closely with that of the enhanced area on gadomer-17-enhanced t1-weighted images, and the size of the enhanced area on gadophrin-2-enhanced t1-weighted images showed close correlation with that of the infarct area revealed by ttc histochemical staining (figs. 1, 2). electron microscopic examination of tissue taken from the three areas showed virtually the same results in each cat. ultrastructural changes in the infarct area indicated irreversibly damaged myocardium ; in the lateral border zone, the features of reversibly damaged myocardium were observed but findings of irreversibly damaged myocardium were absent (fig. in this study, we found that the high signal area seen on t2-weighted images and the enhanced area on gadomer-17-enhanced t1-weighted images were larger than the enhanced area on gadophrin-2-enhanced t1-weighted images and the infarct area revealed by ttc histochemical staining, with statistical significance. electron microscopic examination showed that tissue taken from the ttc - stained peripheral region adjacent to the ttc - unstained area exhibited the features of reversibly damaged myocardium. in a cat model of reperfused myocardial infarction, identification of the lateral border zone is therefore possible, and by means of mr imaging we were able to determine the size and distribution of this zone. it has been well documented that the coronary artery is the major source of ischemic heart disease : thrombotic occlusion of an epicardial coronary artery is usually the cause of acute myocardial infarction (14). the myocardium initiates anaerobic glycolysis within 10 seconds of occlusion of the coronary artery, with the accumulation of lactate and other metabolites (15). acute myocardial infarction first begins in the subendocardium within 20 - 40 minutes of occlusion and spreads toward the subepicardium. this concept of infarct progression has been previously described and termed the wavefront of myocardial necrosis by reimer at al. a part of the transmural progression of injury is related to the transmural gradient of collateral blood flow. previous clinical and experimental studies have indicated that collateral blood flow following coronary occlusion is extremely poor in the subendocardial region and blood is shunted preferentially to the subepicardial zone. furthermore, two other factors, greater systolic wall stress and oxygen consumption, can contribute to subendocardial ischemia. previous studies involving dogs and humans have shown that the evolution of infarction is usually completed within six hours of coronary occlusion ; if reperfusion therapy is instituted while viable myocardium is present, the infarct can therefore be confined to a smaller area. another study has suggested, however, that further myocardial cell death due to reperfusion may occur after prolonged ischemia (reperfusion injury) (18). the reduction of infarct size by reperfusion results primarily from the salvage of myocardium in the subepicardial region of the ischemia. in cases associated with coronary artery stenosis, coronary collateral flow plays an important role in maintaining the viability of the myocardium and in the smaller and confined infarct area (19, 20). the lateral border zone (peri - infarct area) is defined as the lateral area of reversibly injured myocardium adjacent to the core of the infarct and within the area supplied by the occluded artery. studies of acute myocardial infarction (21, 22) have determined whether salvageable myocardium existed along the lateral border zone as well as in the transmural direction. they found that intermediate levels of collateral blood flow and biochemical derangement, as well as intermediate functional impairment of myocardium, occur in the lateral margins of an ischemic region. such data have been interpreted to imply the existence of a lateral border zone where salvage of the myocardium may be possible. however, some studies have argued that the lateral border zone of acute myocardial infarction is limited to a narrow zone or does not exist as a quantitatively significant region (23, 24). this controversy over the existence of such a zone of intermediate injury may have arisen because of a difference in research methods. in their analysis of biochemical and flow gradient, yellon. reported that a quantitatively significant and spatially identifiable " border zone " region did not exist, a conclusion probably due to the tissue sampling technique they employed and differences in the ligation time of the coronary artery (23). on the other hand, others have reported that if necrosis - avid contrast agents were used, a lateral border zone was demonstrated by cardiac mr imaging. various mr sequences and techniques have been developed for the assessment of ischemic heart disease. previous reports have indicated that non - enhanced t1-weighted mri fails to distinguish between myocardial infarction and normal myocardium. (25) reported that breath - hold turbo spin - echo t2-weighted mri can successfully detect acute myocardial infarction, providing excellent tissue contrast and high spatial resolution in a reasonably short scan time. in that study, segmental analysis of acute myocardial infarction showed a diagnostic concordance rate between t2-weighted mri and rest thallium - spect of 95%. however, even optimal t2-weighted images revealed both reversibly and irreversibly injured myocardium (26, 27). many attempts have been made to use contrast - enhanced t1-weighted mri for the evaluation of acute myocardial infarction. 28) reported that if infarct size was estimated on the basis of gd - dtpa - enhanced spin - echo images, the effect of reperfusion therapy on infarct size could be accurately assessed. other researchers, however, have reported that the use of a contrast agent such as gd - dtpa overestimated the extent of myocardial infarction by approximately 10 - 20 % (29 - 31). using gd - dtpa polylysine - enhanced mri in a cat model of reperfused myocardial infarction, choi. (32) investigated changes in the size and degree of signal enhancement during the evolution of myocardial infarction over a six - day period. after observing that during those six days the enhanced area became smaller, they concluded that the highly enhancing area seen during the acute stage of reperfused myocardial infarction included both an irreversibly damaged necrotic area and a reversibly damaged peri - infarct zone. a necrosis - avid mr contrast agent, bis - gadolinium mesoporphyrins (gadophrin-2 ; schering, berlin, germany), has recently become available and this shows a marked affinity for non - viable tissue components. the mechanism of signal enhancement by the contrast material in irreversibly damaged myocardium is still not well understood, but it can be assumed to result from some kind of binding of the compound to the sites of denatured tissue components by means of reperfused coronary flow and progressive extravascular diffusion. further studies to elucidate the mechanism of accumulation are needed. in an animal model with occlusive and reperfused myocardial infarction, gadophrin-2-enhanced mr imaging has proved capable of distinguishing between irreversibly and reversibly injured myocardium, with strong, persistent signal enhancement of the infarct area (33, 34). in addition, another intravascular contrast agent, gadomer-17 (schering, berlin, germany), has recently been introduced. according to studies performed at this institution, the enhanced area seen on gadomer-17-enhanced t1-weighted images was similar to the high signal area on t2-weighted images, and statistical analysis showed no significant difference between them. we therefore believe that the enhanced area seen on gadomer-17-enhanced t1-weighted images probably included both infarct and peri - infarct areas. consequently, direct comparison of the enhanced area revealed by contrast - enhanced mri when gadophrin-2 and gadomer-17 are used may provide the means of distinguishing between irreversibly and reversibly injured myocardium. for revascularization therapy to be successful, it is imperative to distinguish between viable and non - viable myocardium : only the former is likely to benefit from this therapy. in this current study, the statistical difference in size between the abnormal signal areas on mr images and the infarct areas revealed by ttc histochemical staining may represent the lateral border zone and thus suggest the extent of reversibly damaged myocardium. first of all, we did not consider the in - vivo interaction between gadophrin-2 and gadomer-17. a previous study (34) reported that maximal enhancement took place 40 - 60 minutes after the administration of gadomer-17 but 1 - 3 hours after the administration of gadophrin-2, and that with both agents gradual washout then occurred ; we therefore expected that since the maximal enhancement time of these two agents differed, the effect would be minor or non - existent. second, we did not quantitatively analyze the lateral border zone seen on mr images which suggested reversibly damaged myocardium. thus, in order to better understand the clinical usefulness of gadomer-17 and gadophrin-2, and the interaction between them, and to quantitatively analyze the lateral border zone seen on mr images, further study may be required. in conclusion, by means of mr imaging and pathologic correlation we were able to identify the lateral border zone in reperfused myocardial infarction in a cat model, and it may therefore be assumed that both the lateral and transmural border zone contain potentially salvageable myocardium. contrast - enhanced mr imaging using gadophrin-2 and gadomer-17 is potentially useful for determining the size and distribution of the lateral border zone. | objectiveto identify and evaluate the lateral border zone by comparing the size and distribution of the abnormal signal area demonstrated by mr imaging with the infarct area revealed by pathological examination in a reperfused myocardial infarction cat model.materials and methodsin eight cats, the left anterior descending coronary artery was occluded for 90 minutes, and this was followed by 90 minutes of reperfusion. ecg - triggered breath - hold turbo spin - echo t2-weighted mr images were initially obtained along the short axis of the heart before the administration of contrast media. after the injection of gadomer-17 and gadophrin-2, contrast - enhanced t1-weighted mr images were obtained for three hours. the size of the abnormal signal area seen on each image was compared with that of the infarct area after ttc staining. to assess ultrastructural changes in the myocardium at the infarct area, lateral border zone and normal myocardium, electron microscopic examination was performed.resultsthe high signal area seen on t2-weighted images and the enhanced area seen on gadomer-17-enhanced t1wi were larger than the enhanced area on gadophrin-2-enhanced t1wi and the infarct area revealed by ttc staining ; the difference was expressed as a percentage of the size of the total left ventricle mass (t2= 39.2% ; gadomer-17 = 37.25% vs gadophrin-2 = 29.6% ; ttc staining = 28.2% ; p < 0.05). the ultrastructural changes seen at the lateral border zone were compatible with reversible myocardial damage.conclusionin a reperfused myocardial infarction cat model, the presence and size of the lateral border zone can be determined by means of gadomer-17- and gadophrin-2-enhanced mr imaging. |
enhancing the coupling of light and matter at the level of a few photons is one of the challenges inevitable in fundamental research in quantum optics. it is likewise important in applications related to quantum information processing and quantum communication. without any advanced measures the coupling of light and matter especially single atoms is inefficient under standard laboratory conditions, where optics with low numerical apertures (na 4/3, i.e. for more than half of the solid angle, and zero phase shift for 4/(3). this is equivalent to rsc > 2, i.e. the power scattered into the full solid angle is more than twice the incident power. this condition can only be met for focusing optics covering more than half the solid angle. calculated phase shift induced by a single atom for illumination with a dipole - like radiation pattern from full solid angle (solid line), using a deep parabolic mirror (dashed line), a lens with na = 0.68 (dotted line) and a lens with na = 0.55 (dash - dotted line). the above solid angle and na values correspond to values of /(8/3) = 1, 0.94, 0.18 and 0.11, respectively. (the color version of this figure is included in the online version of the journal.) in cavity qed, one usually speaks of strong coupling when the coupling constant g is larger than the cavity field 's decay rate and larger than the spontaneous emission rate associated with the transition of the atom inside the cavity. here one has to keep in mind that the spontaneous emission rate of an atom inside a cavity may differ substantially from the free space value. now the question is whether the cavity qed criteria for strong coupling can be met in free space. the relation g > does not seem relevant at first sight, since trivially is not defined in free space. however, the effective interaction time between the atom and any wave packet propagating inside the optical cavity is determined by independent of the wave packet 's shape which includes continuous waves. similarly, the interaction time in free space is given by the temporal width of a wave packet impinging onto an atom. arguing along this line, one may just reach g = in free space atom - light interactions. we now check the condition g >. the coupling constant g is given by with being the matrix element of the transition dipole moment, the frequency of the atomic transition and v the quantization volume. in cavity qed v is the volume of the cavity mode. for the free space case, where we tightly focus an incident light pulse onto the atom, we make the following ansatz which is the area of the focus with radius rfocus times the spatial length of the incident pulse. the latter is given by c0t with the speed of light c0 and the pulse duration t. we express rfocus in terms of = 2c0/ : rfocus =. likewise, we write the pulse duration as t = /, i.e. in units of the atom 's excited state lifetime. relating g to we insert equation (7) with the above definitions into equation (6) and use the definition of the free space spontaneous emission rate = /(30c0). this yields as outlined in section 2, one example of efficient free space interaction is the absorption of an exponentially shaped single photon pulse by a single atom. such a pulse has an effective length of suggesting = 1. for determining the value of we recall that light - matter interaction in free space the transverse area of a focused dipole wave that is generated by, e.g. an infinitely deep parabolic mirror can be determined with simulations of the intensity in the focal region based on a generalization of the method by richards and wolf. as a radiation pattern incident onto the parabolic mirror we use the one for a linear dipole transition with the quantization axis parallel to the optical axis of the mirror. for comparison also the case of a finite parabolic mirror covering roughly 94% of the solid angle is shown, a mirror geometry matching the one used in refs.. choosing the area at half maximum height as the spot size yields 0.2 in both cases. this results in g 0.97, i.e. the coupling constant would be almost equal to the spontaneous emission rate. another ansatz that does not rely on the definition of the quantization volume is the following : the coupling constant g is also called the single photon rabi frequency. in general, for a field of amplitude e that is polarized parallel to the atomic transition dipole moment the rabi frequency is defined as we use equation (3) to determine e and calculate the power p of the incident radiation by taking the energy of a single photon pulse and dividing it by the pulse duration t =, i.e. p = /. inserting these values into equation (9) and expressing through yields radial intensity distribution in the focus of a parabolic mirror covering the full solid angle (solid line) and a mirror covering 93.65% of the solid angle when weighted with the emission pattern of a linear dipole oriented along the mirror axis (dashed line). (the color version of this figure is included in the online version of the journal.) hence, for a single photon dipole wave pulse whose effective length is the excited state lifetime (= 1) the rabi frequency is twice the spontaneous emission rate. the electric field based estimate for the rabi frequency, however, should be taken with a grain of salt, because the average electric field amplitude is zero for pure fock states. nevertheless it might be tempting to increase the rabi frequency (or equivalently the coupling constant g) of a single photon pulse by reducing the pulse duration t. this, however, has two consequences : (i)the spectrum of the pulse broadens proportional to 1/t.(ii)the rabi frequency increases proportional to the spectrum of the pulse broadens proportional to 1/t. the rabi frequency increases proportional to for t t as can be inferred from the discussion of the free space equivalent to at the beginning of this section this underlines the similarities of the figures of merit for free space and cavity systems. also for monochromatic continuous wave excitation the condition for the vacuum rabi frequency r > may be misleading. under this condition the saturation parameter s = 2r is much larger than one and strong saturation results in power broadening and hence in a reduction of the atomic response on resonance. likewise, strong saturation results in a decrease of the index of refraction, i.e. the real part of the atomic susceptibility. therefore, both the phase shift and the extinction imposed onto a cw light mode by a single atom will decrease, a scenario treated in refs. [comparison of the possible effects of a single atom on the light field in free space and in cavity qed. mechanism : destructive interference for all the reasons discussed above the inequalities g, r > are not the most useful criteria for indicating efficient coupling of light and single atoms in free space. we therefore suggest that the measure of the efficiency of light - matter interaction in free space is not the rabi frequency or coupling constant in comparison to the spontaneous emission rate, but rather the efficiency of converting the power of an incident light mode into a high intensity of the field e at the location of the atom. with the intensity of a sinusoidally oscillating field given by i = 0c0e/2 we find with the help of equation (3) since the maximum effect is obtained for = 8/3 and = 1, we normalize i / p to this case and obtain the free space coupling efficiency this free space coupling efficiency can be related to another quantity used in cavity qed, the cooperativity parameter c = g/(2). the condition c 1 is a necessary (although not sufficient) condition for strong coupling in cavity qed. as outlined in ref., c can be interpreted as a geometric quantity that is the product of the solid angle fraction /(8/3) covered by the cavity mode times the average number n of round trips of a photon wave packet inside the cavity. assuming = 1 for the cavity mode, one can write the cooperativity parameter as c = g n, i.e. as the free space coupling efficiency set by the cavity geometry times the number of cavity round trips. the conclusion of this paper may be summarized as follows : (a)the typical condition for strong coupling in cavity qed, g >, can be just reached in free space (g =,), while(b)only in cavity qed is g, possible. the typical condition for strong coupling in cavity qed, g >, can be just reached in free space (g =,), while only in cavity qed is g, possible. it follows from (a) that there should be several phenomena which can be observed in both coupling schemes. and indeed, the absorption of a single photon by a single atom and the effects a single atom can have on cw beams (or coherent states) via elastic scattering belong to this class of phenomena (cf. a single atom may absorb a single photon pulse, it may switch the transmission of a laser beam and it may impose a large phase shift. corresponding experiments in cavity qed yielded phase shifts on the order of 30 and absorption probabilities of 17%, respectively, for weak coherent state pulses and for true single photon pulses produced in another cavity. extinction of a transmitted beam on resonance with the cavity and the atom has been observed as well. however, the mechanism differs substantially from the free space case. in the strong coupling regime there, extinction is due to destructive interference (see the previous section) without any modification of the components of the transmission spectrum. in cavity qed the plethora of beautiful effects includes collapse and revival of the atom 's excited state population as well as rabi frequency induced nonlinearities. on the other hand, fundamental studies involving the possibly structured continuum of modes are only possible in free space. in either scheme the limits of the effects possible in both coupling schemes have yet to be reached. atom is used to denote various kinds of quantum emitters or quantum targets, such as single ions, quantum dots, molecules and, of course, neutral atoms. | we review recent experimental advances in the field of efficient coupling of single atoms and light in free space. furthermore, a comparison of efficient free space coupling and strong coupling in cavity quantum electrodynamics (qed) is given. free space coupling does not allow for observing oscillatory exchange between the light field and the atom which is the characteristic feature of strong coupling in cavity qed. like cavity qed, free space qed does, however, offer full switching of the light field, a 180 phase shift conditional on the presence of a single atom as well as 100% absorption probability of a single photon by a single atom. furthermore, free space cavity qed comprises the interaction with a continuum of modes. |
deep vein thrombosis, ischemic stroke, and pulmonary embolism are manifestations of the same disease process, summed up over 100 years ago by rudolph virchow.1 his hypothesis that thrombosis was the result of the interaction of the three factors stasis of blood flow, hypercoagulability of the blood, and damage to the vascular endothelium has become the basis of risk - association diagnosis in patients who have developed venous thrombosis embolism. atrial fibrillation (af) is the most common tachyarrhythmia with prevalence of over 10% in older patients (> 70 years). af is the leading cause of ischemic stroke, and stroke due to af is one of the leading causes of death and adult disability.2 besides rate and rhythm control, stroke prevention is the key management strategy for patients with nonvalvular atrial fibrillation and one or more additional risk factors for stroke.3 thrombosis risk can be quantified using the chads2 or recently quantified cha2ds2-vasc scores (documenting risk factors for stroke : history of congestive heart failure, hypertension history ; age 75 [or age 65 years associated with one of the following : diabetes mellitus, coronary artery disease, or hypertension ] ; diabetes mellitus ; stroke or transient ischemic attack or thromboembolism history ; vascular disease history ; sex) (see also table 1).46 by considering these additional risk factors the score is calculated to determine whether antithrombotic therapy is required or not. anticoagulation with vitamin k antagonists (vka), ever since their introduction in the 1950s, has been an enduring gold standard for stroke prevention in af as well as for the prophylaxis and long - term treatment of venous thromboembolism.7,8 vkas such as phenprocoumon (marcumar ; meda pharma gmbh & co. kgaa, bad homburg, germany) or warfarin (coumadin ; bristol - myers squibb gmbh & co. kgaa, munich, germany) prevent hepatic synthesis of coagulation factors ii, vii, ix, and x by inhibiting vitamin k - dependent -carboxylation. due to the wide spectrum of food and drug interactions of vkas, several pathological conditions, and the unpredictability of genetically determined interindividual differences in drug metabolism, treatment with vka requires more or less frequent monitoring of the anticoagulant effect with dose adjustment.9 regarding the problems and disadvantages of these drugs with respect to efficacy, safety, and quality of life, many efforts have been undertaken to develop new anticoagulants targeting only single factors of the coagulation cascade. the licensed drugs rivaroxaban (xarelto ; bayer pharma ag, leverkusen, germany), dabigatran (pradaxa ; boehringer ingelheim gmbh, ingelheim, germany), and apixaban (eliquis ; bristol - myers squibb gmbh & co. kgaa ; pfizer pharma gmbh ; munich, germany) are already available for clinical use in many countries for stroke prevention in af. other new substances targeting factor xa such as edoxaban (lixiana ; daiichi sankyo company, limited, tokyo, japan) are in final stages of clinical studies. the predictability of these new oral direct anticoagulants is based on their pharmacodynamic and pharmacokinetic profiles. unlike vkas, multiple food and drug interactions are not seen with noac and, thus, routine monitoring with laboratory tests is not recommended. rivaroxaban as the first direct oral factor xa inhibitor is a small molecule (molecular weight 436 g / mol) that is almost insoluble in water and exhibits high plasma protein binding (92%95%) in humans, with serum albumin being the main binding component. the absolute bioavailability of rivaroxaban is high (80%100%) and is not affected by food intake. in patients with nonvalvular atrial fibrillation receiving xarelto 20 mg once daily, median maximal concentration (cmax) at steady state reaches approximately 290 g / l (5th95th percentile : 195420 ng / ml) and a trough concentration (ctrough) of approximately 32 g / l (5th95th percentile : 587 ng / ml). rivaroxaban has a dual mode of excretion with the renal route accounting for one third of the overall elimination of unchanged active drug.1012 apixaban as second direct oral factor xa inhibitor with good bioavailability and a half - life of approximately 12 hours has high affinity for factor xa similar to rivaroxaban, and inhibits free factor xa, factor xa in the prothrombinase complex, and factor xa bound to platelets. following oral administration, peak plasma concentrations are observed at about 34 hours post dosing. apixaban is eliminated predominantly via the fecal route (56%), with 25%29% of the recovered dose eliminated via urinary excretion. in patients receiving eliquis 5 mg twice daily, mean ctrough and cmax reaches approximately 19162 ng / ml.12 edoxaban is another oral reversible direct factor xa inhibitor with 62% oral bioavailability. it achieves maximum concentrations within 12 hours, its mean terminal elimination half - life is 8.7510.4 hours. edoxaban is primarily eliminated unchanged through multiple pathways, with approximately 50% of systemically absorbed drug eliminated via renal excretion. the most abundant metabolites are formed through hydrolysis with minor contribution from cytochrome p450 - 3a.13 dabigatran etexilate is a potent synthetic nonpeptide competitive rapidly acting oral direct thrombin inhibitor. dabigatran is taken orally as a prodrug in its inactive precursor form, dabigatran etexilate, which is converted after absorption by nonspecific esterases to the active substance that inhibits thrombin directly. in the treatment of atrial fibrillation, for which dabigatran was approved by the food and drug administration and the european medicine agency (in 2011) pradaxa is prescribed as having a cmax at steady state of 25470.5 ng / ml (mean standard deviation) and a ctrough after 12 hours of 80.318.7 ng / ml in elderly subjects.14,15 as seen in table 2, the three noac, dabigatran, rivaroxaban, and apixaban, differ in mode of action (factor iia and factor xa inhibition), pharmacology, pharmacokinetic and pharmacodynamic parameters, drug interactions, and side effects. dabigatran (pradaxa) was the first new oral anticoagulant to be approved based on the results of the randomized evaluation of long - term anticoagulation therapy (re - ly) trial for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.16 in the re - ly study, 18,113 patients with nonvalvular atrial fibrillation and at least one additional risk factor for stroke or systemic thromboembolism were randomized into two different treatment arms : dabigatran 150 mg or 110 mg twice daily or conventional therapy with the vka warfarin in therapeutic international normalized ratio (inr)-targets of 2.03.0. both dabigatran treatment regimes were statistically noninferior to the vka in terms of the rate of primary efficacy endpoints (stroke and systemic embolism) as well as with respect to the primary safety endpoint (severe bleeding complications). the higher dabigatran dosing (150 mg twice daily) was significantly superior to the tested vka in reducing the primary efficacy endpoints, without increasing the risk of bleeding. the lower dosing (110 mg twice daily) showed a comparable effectiveness in terms of preventing stroke and thromboembolism, while the risk of bleeding complications was significantly lower. interestingly, both dabigatran dosage regimes were associated with a significantly lower risk of intracranial bleeding compared to the vka. clinically relevant side effects in gastrointestinal complaints (dyspepsia) and gastrointestinal bleeding occurred more frequently among dabigatran treated patients. rivaroxaban (xarelto) was the first oral factor xa inhibitor to have gained approval for the prophylaxis of thromboembolism in nonvalvular atrial fibrillation. in the double - blind randomized pivotal trial rivaroxaban once - daily, oral, direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation (rocket - af), a high risk population of approximately 14,000 patients with nonvalvular atrial fibrillation was included ; patients had at least two risk factors or had history of stroke, transitory ischemic attack, or systemic embolism (average chads2 score : 3.5).17 rivaroxaban 20 mg once daily was compared with the vka warfarin at inr target of 2.03.0. patients with impaired renal function (creatinine clearance of 3049 ml / minute) were treated with a reduced rivaroxaban dose of 15 mg once daily. in the statistical analyses, a noninferiority of rivaroxaban compared to the conventional treatment with warfarin could be demonstrated for preventing thromboembolic complications and safety. in the on - treatment analysis, rivaroxaban resulted in a significant reduction in the primary efficacy endpoint consisting of stroke and systemic embolism compared to with the vka. the primary safety endpoint of the study consisted of all bleeding complications (severe and nonsevere clinically significant bleeding) and was not significantly different in both study treatment arms. rivaroxaban was associated with a significantly lower risk for intracranial bleeding and fatal bleeding complications. apixaban (eliquis) as second oral factor xa inhibitor was examined in two randomized, multicenter studies in patients with atrial fibrillation. in the apixaban versus acetylsalicylic acid to prevent strokes (averroes) trial, 5,599 patients with atrial fibrillation and at least one additional risk factor were double - blind randomized.18 patients who were not suitable for treatment with vka and, were either treated with acetylsalicylic acid (asa) at doses of 81324 mg once daily or apixaban at a dosing of 5 mg twice daily. it has long been known that asa has only a limited antithrombotic effect in atrial fibrillation ; therefore, apixaban could demonstrate its superior antithrombotic activity compared to asa. remarkably, the bleeding rates among apixaban were not significantly increased in comparison to asa. due to the superior benefit it was finally investigated in the apixaban for reduction in stroke and other thromboembolic events in atrial fibrillation (aristotle)-trial whether apixaban in the dosing of 5 mg twice daily is non - inferior to warfarin at target - inr of 23.19 the study design was double - blind and randomized, and included patients with nonvalvular af and one additional risk factor (average chads2 score : 2.1). patients with increased risk of bleeding and the presence of two risk markers (age > 80 years, body weight less than 60 kg, creatinine > 1.5 mg / dl) were treated with the lower dose of apixaban 2.5 mg twice daily. both the risk of stroke and systemic embolism and the risk of major bleeding complications were statistically significantly lower with apixaban than with warfarin. moreover, apixaban had an impact on the mortality of the patients that was significantly reduced in the statistical analyzes in comparison to warfarin. edoxaban (lixiana), another oral factor xa inhibitor, was studied in a large multicenter phase iii trial versus warfarin in subjects with af : engage af - timi 48.20 with more than 20,000 patients, this study was the largest and longest single comparative clinical trial performed for prevention of embolic events in nonvalvular af. two edoxaban regimens (30 or 60 mg once daily) were tested for noninferiority in comparison to warfarin during the treatment period of 2.8 years. the primary efficacy endpoint was stroke or systemic embolism. both once - daily regimens of edoxaban were noninferior to warfarin with respect to the prevention of stroke or systemic embolism and were associated with significantly lower rates of bleeding and death from cardiovascular causes. all studies, which compared the efficacy and safety of the new direct oral anticoagulants, used dose - adjusted warfarin as standard of control and showed noninferiority or superiority for the efficacy outcome of ischemic stroke and systemic embolism. furthermore, the rates of major bleeding complications were similar or even reduced for the new drugs in comparison with warfarin.21 table 3 summarizes the results for efficacy and safety data of dabigatran, rivaroxaban, apixaban, and edoxaban based on the re - ly, rocket af, aristotle, and engage af - timi 48 trials. no clinical comparisons regarding efficacy and safety outcomes between the new direct oral drugs have been performed with patient cohorts, and it is highly unlikely that such a comparison would be performed in the near future due to the expense and risks to the manufacturer of such an undertaking. the vka such as warfarin, acenocoumarol, and phenprocoumon require drug monitoring mainly due to their narrow therapeutic window and numerous food and drug interactions. the relationship between monitoring vkas and their efficacy / safety balance is proven.22,23 the exponential increase in studies evaluating health - related quality of life as an important outcome in anticoagulated patients has shown that monitoring these patients leads to more anticoagulation stability, lower incidence of bleeding, and less ischemic events.2430 however, active changes in lifestyle can be potentially troublesome for many patients. qualitative studies have confirmed that frequent monitoring of blood tests and visits to the clinic, anxiety related to adverse events, patient autonomy, dietary restrictions, and the impact of anticoagulant medication on physical activities have a negative impact on the health - related quality of life.24 moreover, it is often intended that the oral anticoagulation should be maintained over the long term, sometimes for the remainder of patient s life. advantages of noac include the wide therapeutic window, low inter- and intraindividual variability of the dose effect relation, and only few known drug interactions or genetic determinations of the metabolism ; thus, their clinical use is easy to handle, and frequent monitoring and dose adjustments have not been considered necessary. however, there are several situations in which it may be of assistance to assess the anticoagulant effects, even of noac such as rivaroxaban, apixaban, or dabigatran.31,32 in patients with severe renal impairment (creatinine clearance 120 kg) did not result in loss of drug efficacy.35 dose adjustment based on low body weight may also not be warranted ; however, combination of additional risk factors for bleeding (age 80 years, body weight 60 kg, and serum creatinine 1.5 mg / dl) might lead to dose adjustment. as substrates of cyp3a4 and p - glycoprotein, rivaroxaban and apixaban are not recommended for concomitant use with strong inhibitors of both pathways, eg, most azole antimycotics and protease inhibitors. since dabigatran is eliminated primarily by renal excretion, the use of low dosage is recommended at a reduced kidney function and also at an increased risk of bleeding in patients, as well as in the elderly (75 years).3639 a corresponding control value of the renal function before starting treatment and subsequently is required at regular intervals. in severe renal insufficiency (creatinine clearance 1 or equivalent ], a renal creatinine clearance of > 50 ml / minutes, and no previous contraindications to anticoagulant therapy). for a baseline 65-year - old patient with af and chads2 > 1 or equivalent in a time range over 20 years, the cost - utility analysis for warfarin ranged from 7,622 to 9,069 and for noac from 19,537 to 20,048.65 self - management entails the measurement of the inr by the patient using point - of - care devices and, when necessary, self - adjustment of the vka dosage.2730 in the review of regier,66 start - up cost in canada for self - managing treated patients was estimated to be $ 1,567 per patient, and the annual cost of physician management and self - management was estimated to be $ 357 and $ 352, respectively, per patient. self - management for noac - treated patients has not been considered because the novel drugs are established without drug monitoring, and no point - of - care device is now available for monitoring noac - therapy. after a major event, patients with mild disability underwent 1 year of rehabilitation at a cost of $ 2,176. for those with permanent disability, however, data of efficacy from large randomized trials exists for noac,1620 and they showed the reduction of major adverse bleeding events with noac. this is likely to lower the overall health care costs in comparison to following the vka strategy, though it is hard to get a real - world cost estimate for this. dabigatran, rivaroxaban, and apixaban are approved for stroke prevention in patients with nonvalvular af. among the factors of oral anticoagulation that induce dissatisfaction, vka as current standard therapy used for prolonged stroke management have a narrow therapeutic window, interindividual variability in dose response, and numerous drug drug and drug food interactions. however, special care is indicated in patients with increased risk of bleeding or with renal or hepatic insufficiency. older patients represent a particularly difficult cohort of patients in which both the thromboembolic risk and the risk of bleeding are increased. in these patient cohorts monitoring anticoagulation therapy methods for accurate and effective monitoring of direct oral anticoagulants are available. in order to accurately estimate the cost, the cost differences of the various anticoagulant drugs have to be calculated, but also the costs of monitoring vka versus measuring noac - concentrations in special clinical situations, the cost of bleeding events, and the costs of stroke have to be considered. uncertainties in clinical practice, that may be solved in the near future, arise in particular from : the lack of availability of specific antidotes for rapid antagonism of the anticoagulant effect of direct thrombin or factor xa inhibitors ; the lack of validated point - of - care tests to monitor and review the quality of anticoagulation in certain emergency situations (for example, in acute bleeding and chronic renal insufficiency) ; and from the absence of clinical data on the management of bleeding under direct oral anticoagulants or perioperative switching and bridging (timely discontinuation and resumption in operations). there are also no data on possible interactions with other drugs in the presence of much comorbidity. in figure 1 therefore, it has to be carefully considered if using fixed doses without laboratory - guided dose adjustment is a real benefit for patients quality of life and adherence. to conclude, individual patients satisfaction profiles are influenced by many factors, and have to be adjusted with physicians satisfaction profiles. | atrial fibrillation (af) continues to be a leading cause of cerebrovascular morbidity and mortality resulting from cardioembolic stroke. oral anticoagulation therapy has been shown to decrease the incidence of cardioembolic stroke in patients with af by more than 50%. appropriate use of anticoagulation with vitamin k antagonists requires precise adherence and monitoring. a number of factors that potentially induce patients dissatisfaction reduce quality of patient life. new direct oral anticoagulants, such as the direct factor xa inhibitors rivaroxaban, apixaban, edoxaban, and the thrombin inhibitor dabigatran, were developed to overcome the limitations of the conventional anticoagulant drugs. however, models to optimize the benefit of therapy and to ensure that therapy can be safely continued are missing for the new oral anticoagulants. this review will briefly describe the new oral anticoagulants dabigatran, rivaroxaban, apixaban, and edoxaban with focus on their use for prevention of embolic events in af. moreover, it will discuss the safety, efficacy, cost data, and benefit for patients quality of life and adherence. |
grip was a prospective, nonrandomized, open - label phase iv study performed in 55 centers in 9 countries (germany [18 centers ], italy, spain, austria, switzerland, canada, australia, south korea, and thailand). the primary objective was to assess the magnitude of potential risk of developing nsf after gadobutrol administration in patients with moderate to severe renal impairment. the study population consisted of patients with moderate to severe renal impairment scheduled for gadobutrol - enhanced mri within the approved indications and dose. the study was conducted in accordance with all international and local guidelines and laws stated by the involved institutional review boards. the classification of cohorts based on the degree of renal impairment was already described by lauenstein in a study on gadoxetate disodium. the definitions in brief are as follows : severe renal impairment was defined by an egfr of 59 and 65 ml / min per 1.73 m at baseline) and (2) a mild renal impairment cohort (presenting an egfr 59 ml / min per 1.73 m at screening, but an egfr > 65 ml / min per 1.73 m at baseline) (table 1). classification of study cohorts all patients received a single intravenous bolus injection of 0.1 mmol / kg (0.1 ml / kg) bw gadobutrol followed by a 20 ml saline flush in the framework of the clinical routine diagnostic workup. gadobutrol is marketed in all participating countries and was provided by the local hospital pharmacies. the primary target variable was defined as the number of patients with moderate to severe renal impairment who developed nsf during the 2-year follow - up period. secondary target variables included (1) number of patients without biopsy but who later developed nsf - like symptoms based solely on the clinical score by girardi ; and (2) number and characteristics of adverse events reported in association with the administration of gadobutrol. our focus was to establish a standardized diagnostic workup and data collection for potential nsf cases in the daily clinical routine. blood sampling to assess egfr was performed twice first during screening within 6 weeks before the mri examination at the local laboratory and second at baseline (ie, 48 hours before gadobutrol administration) at a central laboratory. after gadobutrol administration, patients were followed for clinical examination and review of source documents after 12 and 24 months. in addition, telephone interviews were performed 1, 3, 6, and 18 months after gadobutrol administration by health care professionals who were trained on the study protocol and specifically for identification of clinical signs of nsf. any skin finding with the faintest suspicion of nsf was clinically assessed. in cases where a biopsy was taken, a histopathological assessment had to be performed. here, the girardi criteria had to be applied. this strategy ensured that the risk to miss a potential case of nsf was minimized. descriptive statistics including sample size, mean, standard deviation, minimum, and maximum were calculated for quantitative variables. summaries are presented by renal status cohort (mild, extended moderate, moderate, and severe renal impairment) and overall study population. the joint meeting of the cardiovascular and renal drugs and drug safety and risk management advisory committee of the fda (december 8, 2009) stipulated a 2-year observational study for all gbcas approved in the united states to assess the likelihood of nsf development. the fda proposed a sample size of 1000 patients, consisting of 600 patients with moderate and 400 patients with severe renal impairment. the fda 's sample size suggestion was based on a retrospective study of 370 patients with severe renal insufficiency who received gadodiamide. on june 2, 2011, the fda released the pharmaceutical companies manufacturing gbcas from completing study enrollment but the full 2-year follow - up was required for all patients already enrolled. we continued the grip study in accordance with the study protocol and concluded enrollment by december 31, 2012, with 927 of the 1000 originally planned patients enrolled. grip was a prospective, nonrandomized, open - label phase iv study performed in 55 centers in 9 countries (germany [18 centers ], italy, spain, austria, switzerland, canada, australia, south korea, and thailand). the primary objective was to assess the magnitude of potential risk of developing nsf after gadobutrol administration in patients with moderate to severe renal impairment. the study population consisted of patients with moderate to severe renal impairment scheduled for gadobutrol - enhanced mri within the approved indications and dose. the study was conducted in accordance with all international and local guidelines and laws stated by the involved institutional review boards. the classification of cohorts based on the degree of renal impairment was already described by lauenstein in a study on gadoxetate disodium. the definitions in brief are as follows : severe renal impairment was defined by an egfr of 59 and 65 ml / min per 1.73 m at baseline) and (2) a mild renal impairment cohort (presenting an egfr 59 ml / min per 1.73 m at screening, but an egfr > 65 ml / min per 1.73 m at baseline) (table 1). all patients received a single intravenous bolus injection of 0.1 mmol / kg (0.1 ml / kg) bw gadobutrol followed by a 20 ml saline flush in the framework of the clinical routine diagnostic workup. gadobutrol is marketed in all participating countries and was provided by the local hospital pharmacies. the primary target variable was defined as the number of patients with moderate to severe renal impairment who developed nsf during the 2-year follow - up period. secondary target variables included (1) number of patients without biopsy but who later developed nsf - like symptoms based solely on the clinical score by girardi ; and (2) number and characteristics of adverse events reported in association with the administration of gadobutrol. the study procedures were identical to those described by lauenstein. our focus was to establish a standardized diagnostic workup and data collection for potential nsf cases in the daily clinical routine. blood sampling to assess egfr was performed twice first during screening within 6 weeks before the mri examination at the local laboratory and second at baseline (ie, 48 hours before gadobutrol administration) at a central laboratory. after gadobutrol administration, patients were followed for clinical examination and review of source documents after 12 and 24 months. in addition, telephone interviews were performed 1, 3, 6, and 18 months after gadobutrol administration by health care professionals who were trained on the study protocol and specifically for identification of clinical signs of nsf. any skin finding with the faintest suspicion of nsf was clinically assessed. in cases where a biopsy was taken, a histopathological assessment had to be performed. here, the girardi criteria had to be applied. this strategy ensured that the risk to miss a potential case of nsf was minimized. descriptive statistics including sample size, mean, standard deviation, minimum, and maximum were calculated for quantitative variables. summaries are presented by renal status cohort (mild, extended moderate, moderate, and severe renal impairment) and overall study population. the joint meeting of the cardiovascular and renal drugs and drug safety and risk management advisory committee of the fda (december 8, 2009) stipulated a 2-year observational study for all gbcas approved in the united states to assess the likelihood of nsf development. the fda proposed a sample size of 1000 patients, consisting of 600 patients with moderate and 400 patients with severe renal impairment. the fda 's sample size suggestion was based on a retrospective study of 370 patients with severe renal insufficiency who received gadodiamide. on june 2, 2011, the fda released the pharmaceutical companies manufacturing gbcas from completing study enrollment but the full 2-year follow - up was required for all patients already enrolled. we continued the grip study in accordance with the study protocol and concluded enrollment by december 31, 2012, with 927 of the 1000 originally planned patients enrolled. nine hundred eight patients received gadobutrol and completed the mri examination and were included in the analysis : 284 with severe, 540 with moderate, 46 with extended moderate, and 38 with mild renal impairment (table 2). a total of 581 patients (64.0%) completed the 24 months ' follow - up. demographic and baseline characteristics by degree of renal impairment (fas) the mean age was 66.7 years (range, 1994 years) with 63.4% of patients being 65 years or older. there were no apparent differences in the degree of renal impairment among the various demographic subgroups (table 2). the period from initial diagnosis of renal disease increased with the severity of renal impairment and ranged from 1.83 years in the extended moderate renal impairment cohort to 5.49 years in the severe renal impairment cohort (overall range less than 0.1 to 60.4 years). hypertension and diabetes were the most frequently reported causes of renal disease, with incidence rates of 58.5% and 31.9%, respectively. eighty - three patients (9.1%) were dependent on dialysis and thus assigned to the severe renal impairment cohort. vascular injuries were reported by 386 patients (42.5%), with the majority in the cohort with moderate (43.0%) and severe (46.1%) renal impairment. seventy patients (7.7%) had a history of organ transplant surgery, all in the moderate and severe renal impairment cohort (table 3). history of renal disease by degree of renal impairment (fas) overall, 228 patients (25.1%) underwent contrast - enhanced mri with another gbca within 12 months before the inclusion in the study start or in the follow - up period. ninety - seven patients (10.7%) of the 908 were exposed to gadobutrol before the inclusion in the study, and 184 patients (20.3%) received additional different gbcas during follow - up, that is, after gadobutrol administration at baseline. the number of gbca administrations ranged from 1 (114 patients, 12.6%) to > 5 (14 patients, 1.5%) (table 4). two of the patients with more than 5 additional injections of gbcas had received these injections at baseline, and 6 patients received more than 5 additional injections during follow - up. patients with and number of gbca injections from 12 months before study start (gadobutrol administration) and during follow - up (fas) during the period immediately after gadobutrol administration and before leaving the mri facility, 3 patients (0.3%) experienced drug - related adverse events (aes) : urticaria, retching, and rash. none of these aes were considered serious or life threatening. during the 24-month follow - up, ae reporting focused on skin - related findings and other findings suggestive of nsf. patients with mild renal impairment were not included in the follow - up. during this follow - up, four patients (0.4%) (3 in the moderate, 1 in the severe renal impairment cohort) suffered a rash, but no patient developed symptoms indicative of nsf. no serious adverse events occurred between the date of informed consent and the date the patient left the mri facility on the day of gadobutrol injection or during the follow - up. the frequency of deaths was related to the severity of renal impairment, with 10.9%, 18.1%, and 22.2% of patients in the cohorts with extended moderate, moderate, and severe renal impairment, respectively. following the fda 's postmarketing requirement for all us - approved gbcas, we voluntarily initiated this study on gadobutrol adhering to the fda 's proposed study design. this was a preemptive measure as we expected gadobutrol to be approved in the united states shortly. no case of nsf was detected in our study. to the best of our knowledge, this is the first publication of prospective data on the incidence of nsf after gadobutrol administration. as of may 2016, another recent study by amet investigated the risk of gadoteric acid in 255 patients on dialysis with no findings of nsf. in addition, soulez reported 2 prospective 2-year studies in 534 patients with either stage 3 chronic kidney disease (ckd) or stage 4 to 5 ckd. no signs or symptoms of nsf were reported after administration of gadobenate dimeglumine or gadoteridol. smorodinsky retrospectively evaluated 1167 patients with chronic liver disease where 72% also had some degree of renal insufficiency. the gbcas applied in that study were gadobenate dimeglumine, gadoversetamide, gadopentetate dimeglumine, gadodiamide, and gadoteridol. a total of 284 patients had severe, and 586 had moderate renal impairment, consistent with the target population requested by the fda. in a number of patients, the 2 egfr determinations (one at screening and one immediately before the mri examination at baseline) resulted in different patient classifications based on the cutoff values for mild, moderate, or severe renal impairment. some patients who originally fulfilled the criterion for moderate renal impairment at the time of screening actually showed improved renal function at the time of contrast injection. to reflect a worst - case scenario, we subsumed those patients still in the category of moderate renal impairment and performed a 2-year follow - up. however, to be fully transparent, we reported these patients in a subgroup as extended moderate for the 38 patients in the mild renal disease group an elevated nsf risk has not been established, so follow - up was waived according to protocol. a total of 228 patients (25.1%) received other, additional gbca administrations 12 months before study start and/or during the 2-year follow - up. being fully aware that this might confound / harm our primary objective, we still decided to include all these patients in our analysis. thus, we chose the most conservative approach and report the full - analysis data set that includes all patients who have received at least 1 dose of gadobutrol. therefore, we feel confident that our study population reflects the clinical reality of patients with renal impairment. in addition, as the increasing number and dose of gbca administrations is postulated to increase the likelihood of triggering nsf development, it appears reassuring that even in patients with exposure to multiple gbcas no case of nsf was observed. in our study cohort, most patients had renal disease caused by hypertension (58.5%) or diabetes (31.9%), both being very common in the modern western world. no serious adverse events occurred between the date of informed consent and the date the patient left the mri facility or on the day of gadobutrol injection or during the follow - up period. our results are in accordance with other reports on gadobutrol use in patients with renal impairment. even in patients with chronic renal impairment, including hemodialysis, gadobutrol can safely be applied at doses up to 0.3 mmol / kg, providing evidence that gadobutrol is safe in patients with renal impairment. today, gadobutrol is approved in more than 100 countries worldwide, including the european union, switzerland, australia, united states, canada, japan, and china. since its introduction to the market in february 1998 until december 2015, the cumulative patient exposure is estimated to be more than 29 million patients. as of may 2016, the pharmacovigilance department of bayer pharma ag has received 3 single - agent reports, so - called unconfounded reports, for gadobutrol consistent with the clinicohistopathological definition of nsf. this classification was rigorously performed after a most stringent and conservative approach according to the criteria by girardi. in case clinicopathological criteria or laboratory parameters bayer continues to follow a policy of total transparency regarding nsf, with expedited case reporting to health authorities all over the world. finally, it is important to note that the fda and the european medicines agency have defined risk categories for gbcas. gadobutrol, as well as the 2 other macrocyclic gbcas, gadoterate meglumine and gadoteridol, belong to the class with the lowest risk for nsf development. also, the european society of urogenital radiology (esur) followed those categories in their recommendations. the esur classified macrocyclic agents as low risk but still recommends that they should be used with caution in patients with ckd 4 and 5 (gfr < 30 ml / min per 1.73 m), and there should be at least 7 days between 2 injections. furthermore, pregnant women should only be imaged with contrast - enhanced mri if the expected diagnostic information is essential. however, laboratory testing of renal function (egfr) is not mandatory, and the esur contrast medium safety committee guidelines state that a questionnaire on renal function should be sufficient. on may 23, 2007, there are several limitations to our study that need to be taken into account when interpreting the results. although we followed the fda 's stipulations, the sample size must be considered as a major limitation. in a letter dated june 2, 2011, the fda stated that the estimate of the incidence of nsf in patients with renal insufficiency, based on postmarketing surveillance reports, is lower than the original literature - based estimate. therefore, the trial 's sample size became inadequate to assess the magnitude of nsf risk of gbcas in patients with renal insufficiency. we stopped enrollment on december 31, 2012, but still conducted the 2-year follow - up. the second limitation is related to the concomitant application of other gbcas during the course of the study. however, we consider it reassuring that none of these patients developed signs of nsf. because the fda asked all manufacturers of gbcas marketed in the united states to run similar studies, further reports on other gbcas once all results are available, we suggest that a final assessment of the impact of gbca administration on nsf development should be performed. gadobutrol in patients with moderate to severe renal impairment did not raise any clinically significant safety signals. | objectivethe aim of this study was to assess the potential risk of gadobutrol - enhanced magnetic resonance imaging (mri) in patients with moderate to severe renal impairment for the development of nephrogenic systemic fibrosis (nsf).materials and methodswe performed a prospective, international, multicenter, open - label study in 55 centers. patients with moderate to severe renal impairment scheduled for any gadobutrol - enhanced mri were included. all patients received a single intravenous bolus injection of gadobutrol at a dose of 0.1 mmol / kg body weight. the primary target variable was the number of patients who develop nsf within a 2-year follow - up period.resultsa total of 908 patients were enrolled, including 586 with moderate and 284 with severe renal impairment who are at highest risk for developing nsf. the mean time since renal disease diagnosis was 1.83 and 5.49 years in the moderate and severe renal impairment cohort, respectively. overall, 184 patients (20.3%) underwent further contrast - enhanced mri with other gadolinium - based contrast agents within the 2-year follow - up. no patient developed symptoms conclusive of nsf.conclusionsno safety concerns with gadobutrol in patients with moderate to severe renal impairment were identified. there were no nsf cases. |
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