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LA1512

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pubmed-val-1K

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/ a22c / w184a / m185a or p17c / r18l / t19c / w184a / m185a mutations ) were prepared by sequential dialysis of purified protein . crystals were obtained by the sitting - drop vapor diffusion method in tris - buffered precipitant solutions containing polyethylene glycol and sodium iodide . molecular replacement phasing , model building , and crystallographic refinement were performed with the programs molrep , coot , and phenix . the capsid model was built by manual rigid - body docking of the high - resolution structures of the 5-fold symmetric ntd ring ( pdb code 3p05 ) , the 6-fold symmetric ntd ring ( 3h47 ) , and 2-fold symmetric ctd dimers ( 2kod and 1a43 ) into a geometric fullerene cone model .

the mature capsids of human immunodeficiency virus type 1 ( hiv-1 ) and other retroviruses are fullerene shells , composed of the viral ca protein , that enclose the viral genome and facilitate its delivery into new host cells1 . retroviral ca proteins contain independently - folded n - terminal and c - terminal domains ( ntd and ctd ) that are connected by a flexible linker24 . the ntd forms either hexameric or pentameric rings , whereas the ctd forms symmetric homodimers that connect the rings into a hexagonal lattice3,513 . we previously used a disulfide crosslinking strategy to enable isolation and crystallization of soluble hiv-1 ca hexamers11,14 . by the same approach , we have now determined the x - ray structure of the hiv-1 ca pentamer at 2.5 resolution . two mutant ca proteins with engineered disulfides at different positions ( p17c / t19c and n21c / a22c ) converged onto the same quaternary structure , indicating that the disulfide - crosslinked proteins recapitulate the structure of the native pentamer . assembly of the quasi - equivalent hexamers and pentamers requires remarkably subtle rearrangements in subunit interactions , and appears to be controlled by an electrostatic switch that favors hexamers over pentamers . this study completes the gallery of sub - structures describing the components of the hiv-1 capsid and enables atomic level modeling of the complete capsid . rigid - body rotations around two assembly interfaces appear sufficient to generate the full range of continuously varying lattice curvature in the fullerene cone .

METHODS SUMMARY Supplementary Material

spirituality and medical ethics has recently emerged as an important topic in the health care provision and there exists a heated debate surrounding the issue . medical ethics is in the curricula of virtually all medical schools all around the world . on the other hand , spirituality and religion therefore , one could suggest that patients religious ideology and beliefs should be taken into account in any clinical setting ( 1 , 2 ) . spirituality is usually defined as the aspect of humanity that refers to the way an individual seeks to follow in life . it is supposed to add meaning and purpose to life , and facilitate the connection with the moment , self expression , and attitude towards other human beings , nature and the sacred ( 3 ) . spirituality and religious beliefs have been shown to have an enormous impact on how people can cope with serious illness and life threatening stresses ( 4 , 5 ) . spirituality if often claimed to give people a certain feeling of wellbeing , improve the quality of life , and provide social support through spiritual social activities ( 6 , 7 ) . new advances in medical sciences have made it more than obvious that including moral issues in this profession is indispensable . as medical profession deals with individuals bodies and lives , it needs to consider morality and dignity of human beings ( 8) . therefore , medical practice is interlinked with a great deal of responsibility as it deals with human beings physical and mental health ; consequently , it should take all considerations into account including spirituality and religion ( 9 ) . in today s world , medical ethics has been the subject of considerable attention due to the mentioned issues and it is the subject of heated debates . medical ethics include a set of values and codes which aim to build trust and confidence in physicians , patients , and the society as a whole . a physician s competence depends not only on his medical knowledge , clinical decision making , and practical skills , but also , on his ideology and practice of medical ethics ( 10 , 11 ) . as one of the four basic principles of modern ethics is respect to patients choice in clinical practice , physicians should respect patients religious and spiritual choices ( 12 ) . patients are only one side of the interaction in a clinical setting ; physicians too , may have religious ideas and beliefs which may influence their practice . the religious ideology and beliefs of physicians can even more complicate the issue when patients religious choices come into play ( 12 ) . as argued , the concept of morality and medical ethics has become the focus of attention in medical practice , and , its implementation is furiously advocated ( 13 ) . therefore , hospitals need to consider all aspects of patient s rights including the right to be treated based on their religious ideology . in fact , respecting all aspects of patients rights such as their participation in decision making , respect for privacy and dignity , and interpersonal relationships are considered intertwined with several different aspects of health care delivery ( 14 ) . the u.s . legal system has been pioneer in implementing a robust approach to a health care provision system respecting all aspects of patients rights such as their special religious demands , while pursuing excellence in all aspects of health care delivery ( 15 ) . in addition to considering moral issues and respecting religious beliefs of patients , public and governmental hospitals need to promote accountability . respect for patients rights and enhanced communication with patients can pave the way for development of moral competence ( 16 ) . in fact , it is argued that undermining medical and moral accountability in some countries has resulted in patients loosing their confidence in the system regarding moral , legal , monetary , and political affairs ( 17 ) . moral accountability in governmental hospitals is defined as the extent to which they implement and monitor adhering to moral principles by the authorities and staff ( 18 ) . the need for defining a role for religion in human beings everyday tasks is not limited to a specific religion , a certain geographical area , or a particular time period in the history . in fact , it can be argued that man has always felt this need all through the human history including the present time . islam , as both an ideology and a practical way of life , is argued by its followers , to provide solid foundation for different aspects of life ( 19 , 20 ) . the clinical setting and medical affairs seem to be in priority when implementing the religious law is concerned . islam is all about faith , dignity , honesty , and mutual trust , medical ethics is of paramount importance for muslims as it needs to protect them from anything that might endanger the islamic principles ( 21 , 22 ) . in this study , we aimed to conduct a poll to find out what female iranian patients think about the implementation of the same sex health care delivery system ( sshcd ) in accordance with the religious law in a number of teaching general hospitals of tehran , the capital of iran . this plan was first proposed by ministry of health and medical education of the islamic republic of iran due to increasing demand of patients and policy makers , and , consequently , the supreme council of sshcd was established in 1997 . the parliament of the islamic republic of iran passed the bill of this plan in 1998 , and , in the same year , it was communicated to all healthcare organizations in iran . this study is the result of all endeavors to clarify whether the project can be implemented in iranian hospitals or not , and whether the patients would prefer and embrace it . this project is supposed to encompass all aspects of religious law regarding medical practice including the need for female patients to be visited and cared for , exclusively , by health care providers from the same sex ( 24 , 25 ) . the plan of sshcd project also includes a different part which is designed to define medical practice codes based on the religious law . this project is supposed to be followed by all medical practitioners practicing in the islamic republic of iran ( 26 ) . the first step of the project is intended to separate women and men in hospitals ( 21 ) . moreover , physicians should be required to visit patients from the opposite sex only when it is inevitable . this way , any unnecessary contact of the health care provider with the patient from the opposite sex is minimized ( 24 ) . finally , physicians bedside manner and their conduct toward their patients need to be according to the religious law ( 22 ) . this research was a cross - sectional , descriptive analytical study which was conducted at teaching general hospitals in tehran , iran . in this study , we chose four hospitals from different neighborhoods of tehran which were similar in terms of size , number of beds , number of patients admitted during a particular period of time , number of staff , and the health care services delivered . the questionnaires were to be answered anonymously , and , in order to protect their confidentiality , the names of the participating hospitals were not disclosed . the cochran formula was used to calculate the number of female patients as the research cohort . in each hospital , 30 female patients were selected from different wards using cluster sampling method . the assessment tool was a questionnaire comprised of 18 questions and the questions had a focus on demographic information , the preferred gender of the visiting physician , the importance of being visited by a physician from the same sex , and the most important moral issue they faced during their hospitalization . before the commencement of the study , a pilot study was performed to check the reliability of the questionnaire . in order to ensure that , some of the patients were asked to complete the questionnaire randomly two weeks prior to the study . the results obtained from test- retest method were studied and compared with those of the main study . the questionnaires were distributed among 120 women who were admitted to different wards of the hospitals . also , we asked the patients to mention their husbands opinion about their wives being visited exclusively by female physicians and they were asked to express their opinion about the questions . in order to specify the degree of the agreement the three level likert scale was used as agree , disagree and neutral . the statistical analysis , chi - square and pearson tests were used to analyze the data . as it was difficult to analyze patients opinions by direct observation , the attitude was assessed using measurable bits of evidence such as the expression of beliefs and emotions ( 27 ) . most of the patients were 20 40 years old , 89% were married and 79.3% were housewives . 30% of them were illiterate and the remaining had high school diploma , bachelor s degree , master s degree or doctorate degree . 60% of the patients lived in the capitals of the provinces and 40% lived in towns and rural areas . the results indicated that most of the hospitalized women s husbands preferred female physicians to visit their wives . in contrast , 14.8% believed it was not necessary , and 36.5% did not answer this question . almost , half of the patients believed that female physicians were available , 37.2% of them expressed that female physicians were not available , and the rest expressed that they had not noticed it . most of the patients believed that they mostly felt embarrassed when male medical students were present by their bedsides . as it is tabulated in table 1 , more than half of the hospitalized women believed in the competency of the female physicians and staff and preferred to be visited by them even when male physicians were being present . most of the hospitalized women in this study , expressed their concern and dissatisfaction about a number of issues in the questionnaire . moreover , they expressed their feeling embarrassed by being visited by male physicians of their own family members or relatives ( to whom marriage is prohibited by the religious law ) . they also opposed to a number of services delivered by male staff , including nursing care , housekeeping services , changing position , being helped with personal activities , urethral catheterization and intramuscular injections . they also were against presence of male medical students during medical examinations , failure of patient s covering during radiology and laboratory services provided by the technicians or technologists , and the presence of male nurses and staff members of close relationship ( to whom marriage is prohibited ) during physical examinations . in addition , the majority of the hospitalized women were in favor of the separation of men and women s rooms at the hospital wards and the presence of a chaperone during medical examinations . according to table 2 , 3/4 of the hospitalized women were in favor of implementation of the sshcd project in tehran , iran . we analyzed the dependency or independency of the variables based on pearson test , and , it was demonstrated that there was significant relationship between the level of education of the studied women and their opinion about the gender of their physician ( p=0.041 ) and being delivered nursing care by male nurses ( p=0.045 ) . therefore , it can be concluded that more educated patients preferred to be visited by male physicians and received health care by male nurses . also , there was a significant correlation between the age of the participants and their opinion of the presence of male physicians during physical examinations ( p=0.002 ) , being examined by male medical students ( p=0.002 ) , and receiving personal services by male hospital staff ( p=0.019 ) . older patients were in favor of being visited by male physician , being examined by male medical students , and receiving personal services by male hospital staff . moreover , there was a significant correlation between the incidence of hospitalization and the patient s opinion about receiving nursing care provided by male nurses ( p=0.04 ) , and sharing their personal private issues with male physicians and other medical staff ( p=0.01 ) . it was observed that increased incidence of hospitalization was positively correlated with preferring to receive nursing care by male nurses and the other male medical staffs . there was also considerable correlation between the occupation of patients and their attitude towards the presence of male staff during their examinations by male physicians ( p=0.01 ) and the implementation of adjustment plan . there was also correlation between marital status of the hospitalized women and their attitude toward being examined by male medical students ( p=0.025 ) . thus , the hospitalized women with higher job positions preferred to be examined by male physicians ; in other words , woman with higher job positions were not in favor of the implementation of the sshcd project . most interestingly , there was a direct relationship between the opinion of the husbands and the patients , and the patients themselves regarding the preferred gender of physicians as well as being examined by male medical students . firstly , it had a focus on sshcd according to the religious law from a different point of view which is based on medical ethics and patients rights . secondly , this study is one of the few studies conducted on the need for implementation of the sshcd according to the free will and respect to the choice of iranian female patients and their family . thirdly , in this article , the researchers analyzed the demographic characteristics of the hospitalized women ( such as age , education , occupation , the incidence of admission to hospitals , marital status and the opinion of their husbands ) with regard to the implementation of the sshcd . approximately , half of the hospitalized women believed it necessary to be visited by female health care professionals and they considered it as their indisputable right . zare dehabadi has suggested that patient s rights to choose their physician is in accordance with the who declaration and that of the world medical association . this is also considered in patient s rights charters in malaysia , new zealand , and slovakia ( 28 ) . waseem et al . have analyzed several points regarding patients preferences for the gender of their health care deliverer . among the study subjects , 80 percent of the women preferred a female doctor , and when it came to the choice between more experienced and female physicians , none of them chose the doctor who had more experience . they researchers hypothesized some potential underlying basis for the results and suggested the possible implications . however , several studies conducted on hospitalized women s opinion about their preferences for the gender of health care deliverers found that characteristics such as interpersonal communication and clinical skills had priority over physician s gender . the aforementioned results are in congruence with those of the current study ( 29 , 30 ) . the subject of the gender related issues in medical practice and research has been the subject of considerable amount of debate recently . sex and gender related laws and regulations do not only affect the patient , but also include the relationship amongst practitioners themselves . considering the important role it plays in daily activities of medical practitioners and its legal , political , philosophical , and moral consequences , the gender related interpersonal communication issues should be taken into account more seriously ( 31 ) . the most frequently expressed concern of the patients in this study was the issue of a male medical student s being present during physical examinations and ward rounds . the participants also expressed their dissatisfaction with the health care services rendered by male nurses and allied health professionals . moreover , they were strongly in favor of the separation of female patients rooms in hospitals . it is universally acknowledged that patients and their families should have the right to choose their health care providers ( 32 ) . therefore , hospitals bear the responsibility of providing patients with their preferred health care services . these services need to respect patients choice of the clothing , head cover or hijab , and carrying religious relics or other symbolic items , as long as they do not interfere with diagnostic procedures or treatment options . many even argue that such issues need to have priority over medical procedure with more favorable outcomes . privacy , confidentiality of clinical information , the presence of a chaperone during clinical examinations , and having the right to be visited and cared for by health care providers of one s own sex are usually considered as patient indisputable rights ( 33,34 ) . yarsis surakarta hospital in indonesia that is a private healthcare facility with 219 beds and zhejiang woman hospital in china that was founded in 1951 and fit , the local healthcare demand for women and babies , with nearly 1000 staffs , 750 bed , for women and 250 beds for babies , are examples of the world s attention to human rights ( 35 , 36 ) . although the results of our study clearly demonstrated that most of the hospitalized women were in favor of the implementation of the sshcd project according to the religious law , one should consider several different factors when its implementation is concerned . azari argues that one of the most important strategies in this regard is to employ proportionate workforce , especially nursing staff and allied health care professionals ( 37 ) . shojaie and ghofranipour indicated that a conservative and tolerant approach should be considered so that to avoid fierce opposition by those who disagree with this plan , and , also , to prevent extremism . it should be also taken into account that the implementation of the sshcd requires deep and sincere faith of both policy makers and health care providers , and , the strategy of its implementation must be similar to that of the islamic ethics . taking these into consideration , sshcd seems easy to be implemented and requires sincere faith and dedication ( 38 , 39 ) . one of the most important limitations of the present study was that it was conducted over a specific time period . it could be argued that an increased duration of the study might have demonstrated different results . secondly , as all patients were recruited by means of a questionnaire , it is possible that during the information collection process some data are missed . finally , one could suggest that some other factors influencing health care provision for the hospitalized patients that have not been taken into account in our study . considering mentioned limitations further studies with a larger cohort and extending over a longer period of time seems to be of great importance before implementation of the sshcd or any such comprehensive plan at a national scale . it is noteworthy that written informed consent was obtained from all patients and every single aspect of academic honesty has been taken into account to avoid plagiarism , misconduct , data fabrication and / or falsification , and double publication . apparently , although a decade is passed since the proposal of the implementation of the sshcd in iranian hospitals , iranian patients and their families are still in favor of its implementation . in fact , the advocacy of its implementation has increased during the recent decade . moreover , the results of previous studies and those of the present one conclude that not only patients are in favor of the sshcd project , but also a great majority of health care providers advocate it . as a result , we hope that a new common issue about patients rights and medical ethics has opened . therefore , the results of this study , in accordance with those of the previous ones , indicate that iranian health care policy makers need to consider accelerating the implication of the sshcd plan .

as medical ethics indisputably needs to consider patients religious beliefs and spiritual ideas , one can suggest that hospitals are responsible for not only patients rights and dignity , but also for her / his religious concerns and expectations.the current study is designed shed some light on the patients view of the implementation of religious law in iranian hospitals , specifically , the right of patients to be visited and delivered health services by professionals from the same sex . this protocol is proposed by the ministry of health and medical education of the islamic republic of iran as a response to the increasing demand for implementation of the religious law by iranian patients.this research is a cross - sectional study which was conducted at four teaching general hospitals in tehran , iran . the data was collected by the means of a questionnaire distributed to 120 women who were admitted to different wards of the hospitals . these women were asked to express their opinion of the implementation the same sex health care delivery ( sshcd ) system in iranian hospitals . all analyses were performed with the use of spss software , version 16.0.the results indicate that half of the hospitalized women believed that being visited by a physician from the same gender is necessary who advocated the implementation of sshcd in a clinical setting ; and most of their husbands preferred their wives to be visited exclusively by female physicians.this study highlights the view of the iranian patients towards the issue and urges the ministry of health and medical education of the islamic republic of iran to accelerate the implementation of this law.sshcd is what the majority of iranian patients prefer , and , considering patients rights and the medical ethics , it should be implemented by iranian policy makers .

Introduction Materials and Methods Result Discussion Conclusion

acute acalculous cholecystitis ( aac ) is an acute necroinflammatory disease of the gallbladder without gallstones that is typically seen in critically ill patients ( 1,2 ) . the pathology includes endothelial injury , gall bladder ischemia , and stasis and these conditions may cause a concentration of bile salts and gallbladder distension , eventually leading to necrosis of the gallbladder tissue ( 1,2 ) . marked gallbladder wall thickening ( 3.5 mm ) and pericholecystic fluid are the most common imaging features of the disease ( 2 ) . approximately 10% of all acute cholecystitis cases are aac , and the mortality is high in such cases without prompt treatment ( 1,2 ) . aac can be related to trauma , surgery , shock , burns , sepsis , total parenteral nutrition , and/or prolonged fasting ( 2 ) , and it can also be accompanied by autoimmune diseases . macrophage activation syndrome ( mas ) is an aggressive and potentially lethal syndrome of uncontrolled immune activation . in mas , macrophages become activated and overproduce cytokines , thereby causing severe tissue damage and eventually leading to organ failure ( 3 ) . mas is thought to be a form of hemophagocytic lymphohistiocytosis ( hlh ) associated with a rheumatic disease . there are some reports describing the association of aac with systemic lupus erythematosus ( sle ) and other autoimmune diseases ( 4 - 6 ) , but the coexistence of aac and mas has so far only rarely been reported . we herein report a case of aac complicating mas , occurring in a patient with recurrent histiocytic necrotizing lymphadenitis ( kikuchi disease ) and sjgren syndrome . rheumatologists should be aware of this possible association and keep in mind the fact that aac in rheumatic or autoimmune diseases can be successfully treated with early immunosuppressive therapy , although the risk of perforation and the mortality rate is high in aac . a 41-year - old female was admitted to our hospital because of a high fever with lymphadenopathy that had appeared 4 days before admission . kikuchi disease had been diagnosed 5 years earlier by a left cervical lymph node biopsy . sjgren syndrome had also been diagnosed 2 years earlier , but she did not meet the criteria for sle . physical examination revealed a temperature of 38.2 , cervical and axillary lymphadenopathy with tenderness , but no tenderness in the right upper quadrant of the abdomen . laboratory tests showed thrombocytopenia and a coagulation disorder including hypofibrinogenemia ( 143.5 mg / dl ) , suggesting disseminated intravascular coagulation ( dic ) . elevation of hepatobiliary enzymes , c - reactive protein , lactate dehydrogenase , soluble interleukin-2 receptor ( 5,040 u / ml ) , and ferritin ( 9,176.1 ng / ml ) were also noted ( table ) . a histological examination of the lymph node was not performed because of the urgent need for treatment . mas due to the recurrence of kikuchi disease was diagnosed according to the 2004 diagnostic guidelines for hlh ( 7 ) . cervical and axillary lymphadenopathy , hepatosplenomegaly , marked thickening of the gallbladder wall ( > 15 mm ) , pericholecystic fluid , but no signs of intestinal obstruction were detected on computed tomography ( ct ) and ultrasonography ( fig . a gallstone was also found in the fundus , but neither any impaction of the stone nor dilatation of the common bile duct was detected ( fig . so - called acute acalculous cholecystitis with a gallstone secondary to mas was therefore diagnosed clinically and radiologically . aptt : activated partial thromboplastin time , dsdna : double - stranded dna , il-2r : interleukin-2 receptor , mpo - anca : myeloperoxidase - antineutrophil cytoplasmic antibody , pr3-anca : proteinase 3 antineutrophil cytoplasmic antibody , pt / inr : prothrombin time / international normalized ratio imaging performed on admission ( a - f ) and the sixth day of admission ( g , h ) . a , b : ultrasonography showed marked thickening of the gallbladder wall , a gallstone ( a ) , and pericholecystic fluid ( b ; arrow ) , but neither impaction of the stone nor dilatation of the common bile duct was detected . c : cervical lymphadenopathy ( arrows ) and d : axial lymphadenopathy ( arrows ) were detected by contrast - enhanced computed tomography ( ct ) . e , f : marked thickening of the gallbladder wall ( > 15 mm ) and pericholecystic fluid were detected by ct . g , h : follow - up ct performed on the sixth day of the admission showed a significant improvement of the gallbladder wall thickening and pericholecystic fluid . intravenous pulse methylprednisolone ( mpsl ) 1 g / day for 3 days , followed by psl 55 mg / day , and meropenem hydrate ( 1 g / day ) were immediately initiated in addition to recombinant human soluble thrombomodulin ( 19,200 u / day ) for dic treatment . both the fever and lymphadenopathy improved by the next day , as well as thrombocytopenia , coagulation disorder , and hyperferritinemia . on the sixth day of admission , follow - up ct revealed a dramatic improvement of the gallbladder wall thickening and pericholecystic fluid ( fig . she has remained clinically well without any relapse of kikuchi disease or cholecystitis even after the psl was tapered to 6 mg / day . crp : c - reactive protein , ldh : lactate dehydrogenase , mepm : meropenem hydrate , mpsl : methylprednisolone , ne : not examined , plt : platelet count , psl : prednisolone , rhtm : recombinant human soluble thrombomodulin , t - bil : total bilirubin , wbc : white blood cell count aac can develop in patients with mas , but this phenomenon has only rarely been reported . although the association between aac and mas is still not fully understood , endothelial damage caused by the cytokine storm may be primarily involved in the pathogenesis of aac ( 2,3,8 ) . the pathogenesis of mas is characterized by an impaired immune response with the hypersecretion of pro - inflammatory cytokines and the activation of cytotoxic cells ( 8) . this excessive immune reaction results in endothelial injury and increased vascular permeability , possibly leading to local microvascular ischemia of gallbladder wall and bile stasis in aac . endothelial dysfunction has been suggested to play a central role in some forms of drug or infection - associated aac cases ( 9,10 ) . the incidence of aac in mas is not clear , but it may be relatively frequent in children with hlh . schmidt et al . reported that 6 of 9 children with hlh had gallbladder wall thickening on ultrasonographic examination within 1 week of presentation ( 11 ) . reported that all of the 6 children with hlh examined by abdominal ultrasonography revealed thickening of the gallbladder wall , as well as in 14 of 21 children with hlh reported by fitzgerald et al . reported that of 67 children with aac 4 of these children had hlh ( 14 ) . there is only one case report of an adult patient with mas complicated with aac . park et al . reported the case of a 49-year - old female with a 7-year history of adult onset still 's disease ( aosd ) who developed mas and aac ( 15 ) . unlike in children , there is no report evaluating the presence of gallbladder thickening in adult patients with mas . these facts suggest that aac could be more frequently associated with mas in children than that in adults , although the pathogenesis of aac is thought to be similar between children and adults ( 16 ) . the precise mechanisms accounting for this difference are unknown , but one possible explanation is that children in critically ill and febrile conditions are more likely to develop hypovolemia , one of the risk factors for aac ( 2 ) . another explanation could be that the treatment for mas , using high - dose corticosteroids and broad - spectrum antibiotics , is normally initiated before abdominal imaging , which would thus improve aac before it can be identified . aac associated with rheumatic or autoimmune diseases may possibly have a better response to immunosuppressive therapy , compared to that with other causes . in general , surgical intervention is recommended for patients with aac because of the high risk of perforation . it is reported that perforation occurs in 10% and the mortality rate is 30% overall ( 17,18 ) . a delay in treatment could raise the mortality rate up to as high as 75% ( 19 ) . however , in the case reported by park et al . , the patient who developed mas and aac was successfully treated with psl and cyclosporine a without surgical intervention ( 15 ) . it has also been reported that some patients with sle complicated by aac were successfully treated with immunosuppressive therapy ( 4 - 6 ) . our patient recovered immediately after the initiation of intravenous pulse mpsl and the follow - up ct showed significant improvement in the gallbladder wall , resulting in no need for surgical intervention . therefore , early immunosuppressive therapy under careful observation should be carried out for aac cases with rheumatic or autoimmune diseases .

acute acalculous cholecystitis ( aac ) is a severe disease seen in critically ill patients , including those with autoimmune diseases . we herein report the case of a 41-year - old female who developed macrophage activation syndrome ( mas ) accompanied by a recurrence of kikuchi disease . abdominal imaging revealed marked thickening of the gallbladder wall and pericholecystic fluid , typically found in aac . treatment with intravenous pulse methylprednisolone induced in a significant improvement in the gallbladder wall , resulting in no need for surgical intervention . we should consider that patients with mas may therefore sometimes develop aac and that early immunosuppressive therapy can be effective in aac cases associated with rheumatic or autoimmune diseases .

Introduction Case Report Discussion The authors state that they have no Conflict of Interest (COI).

nanochemistry is predominating in major fields of science and technology , specifically in biotechnology and information technology . in the near future , nanochemistry will direct and guide towards nanomedicine and nanodiagnostics . however , obtaining suitable nanoparticles that can be used for diagnostic and medicinal purposes remains a significant challenge . moreover , the effect of these nanoparticles on biological entities such as proteins is considerably significant when it comes to ad . oligomeric aggregates a and tau protein or the protofibrils are considered as precursors for amyloid fibrillation in alzheimer 's disease . recently , the effect of fluorinated magnetic core - shell nanoparticles with the size range of 15.0 2.1 nm has been observed on amyloid model protein insulin ; these fluorinated nanoparticles show inhibition of insulin fibrils [ 3 , 4 ] . furthermore , the effect of various nanoparticles within the dimensions of 6200 nm on another model protein , 2 microglobulin , has been investigated . previously published studies have demonstrated that nanoparticles can act as catalysts for protein fibrillation [ 1 , 5 ] . very recently , li and coworkers have showed an inhibition effect of n - acetyl cysteine - capped cdte qds of size of 35 nm on a ( 140 ) fibrillation . in another case , dual effect of commercial polystyrene nanoparticles with amino modification having various sizes ( 57 , 120 , and 180 nm ) was observed on a ( 140 ) and recombinant a ( 140 ) and a ( 142 ) proteins . furthermore , there is only one recent publication on the effect of nanoparticles on a ( 142 ) fibrils . in this case , it was observed an increase in rate of amyloid fibrillation in presence of tio2 nanoparticles with size of approximately 20 nm . the interaction of nanoparticles with different proteins depends upon various factors such as surface coating of nanoparticles with ligands , surface properties , size , and composition of nanoparticles [ 1 , 5 ] . from the previous studies , [ 1 , 38 ] we can not generalize the concept that different nanoparticles can promote or inhibit the fibril formation for various amyloid model proteins . specifically , the only investigation that explains the effect of tio2 on a ( 142 ) shows that nanoparticles promote the fibrillation process by becoming nucleation centers . we report for the first time in our knowledge that cdse / zns qds of size of 2.5 1.3 nm can inhibit fibrillation of a ( 142 ) . in the present study , we have investigated the effect of the presence of dhla - capped cdse / zns qds either mixed with or conjugated to a ( 142 ) on fibrillation process of a ( 142 ) in aqueous phase . tem and afm studies show that the qds behave uniquely when they are conjugated to a ( 142 ) in comparison to a mixed sample of a ( 142 ) and qds . our study illustrates a considerable difference in morphology of the fibrils and the inhibition of fibrillation process when a ( 142 ) is conjugated to qds versus the mixed system a ( 142 ) and qds . these results are further supported by thioflavin t ( tht ) assay using fluorescence spectroscopy . all chemicals were commercially purchased and used without further purification . cadmium oxide ( cdo ) , selenium ( se ) , trioctylphosphine oxide ( topo ) , trioctylphosphine ( top ) , and hexamethyldisilathiane [ ( tms)2s ] were purchased from sigma - aldrich ( milwaukee , wi ) . the tetradecylphosphonic acid ( tdpa ) was obtained from alfa aesar ( ward hill , ma ) . the diethylzinc ( znet2 , 15 wt% solution in hexane ) was obtained from acros organics ( morrisplains , new jersey ) . dl--lipoic acid , a ( 142 ) , and tht were purchased from mp biomedicals ( solon , oh ) . briefly , cadmium oxide was reacted with a selenium reagent in the presence of a phosphine oxide surfactant at high temperature under argon flow . after the formation of the cdse core , the diethyl zinc and hexamethyldisilathiane in top was added dropwise at 130c . after the synthesis of topo - capped hydrophobic qds , modification to hydrophilic dhla - capped qds was carried out . briefly , first dl--lipoic acid ( 1 g ) was reduced using sodium borohydride ( 2 g ) in methanol / water ( v / v , 1:1 ) solution . after workup product was isolated in chloroform and characterized using h nmr ( 400 mhz , cdcl3 ) : ( ppm ) 1.3 ( d , 1h ) , 1.35 ( t , 1h ) , 1.41.8 ( m , 6h ) , 1.9 ( m , 2h ) , 2.4 ( t , 2h ) , 2.62.8 ( m , 2h ) , 2.9 ( m , 1h ) , and 11 ( s , 1h ) . dhla was used for ligand exchange with topo ; excess of dhla ( 0.5 g ) was added in 5 ml of topo - capped qds in methanol and heated at 60c70c for 4 h. once a homogeneous qds solution was obtained , solution was basified using potassium tert - butoxide and centrifuged to get the precipitates . the water - soluble qds were filtered through 0.2 m filter to get a clear solution . a(142 ) was chemically conjugated to qds by the formation of an amide bond between asp - nh2 end of the polypeptide chain and the cooh end of the dhla ligand using the protocol to conjugate proteins . freshly prepared dhla - capped qds ( 1.51 10 m , 100 l ) were taken in a clean borosilicate glass vial , and 500 l pbs buffer ( ph 7.4 ) was added to the qds . to lyophilize the peptide , 0.5 mg a ( 142 ) was dissolved in hexafluoroisopropanol ( hfip ) to bring the peptide in monomer form and evaporated under gentle flow of n2 . the dried protein was then dissolved in 500 l pbs buffer ( ph 7.4 ) to get the final concentration of 1 mg / ml . freshly prepared a(142 ) solution was then mixed in qds solution . 57 l of freshly prepared 10 mg / ml edc ( 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride ) solution in deionized water was then added to the mixture of peptide and qds , total volume of solution prepared was 1157 l . the solution was stirred for 4 h at the speed of 200 rpm . a ( 142 ) mixed with dhla - capped qds was prepared according to the above - mentioned protocol except for the addition of edc . total volume of the solution was kept at 1157 l . for the induction of fibrillation pure a ( 142 ) solution was prepared similarly , first by lyophilizing the peptide in hfip and evaporating the solvent under gentle nitrogen flow and then redissolving the dried peptide in 1157 l of pbs buffer ( ph 7.4 ) . electrophoresis of qds was performed using a minicuve 8.10 electrophoresis unit ( mp biomedicals , solon , oh ) . hand cast gels were composed of 1% agarose in 1 tbe ( 0.089 m tris base , 0.089 m boric acid , and 0.002 m ethylenediaminetetraaceticacid , ph 8.3 ) . 10 l of each sample was loaded into wells on the agarose gel using a micropipet . the samples were run in 1 tbe buffer on the 1% agarose gel at 84 v for 75 min . for visualization , the gel was placed on a uv transilluminator , and an image was captured with a gel doc xr system ( bio - rad , hercules , ca ) . tem measurements were performed at the center of advanced microscopy , scripps research institute ( la jolla , ca ) and at the center of advanced microscopy , ( cma ) , trinity college dublin ( ireland ) . for the images containing amyloid , briefly , copper grids ( carbon and formvar coated 400 mesh : electron microscopy sciences , hatfield , pa ) were glow discharged and inverted on an 5 l aliquot of sample for 3 min . excess sample was removed and the grids immediately placed briefly on a droplet of double - distilled water . grids requiring the negative stain were then placed on droplets of 2% uranyl acetate solution for 2 min . excess stain was removed and the grid was allowed to dry thoroughly . for unstained grids , the excess water was removed , and the dried grids were examined on a philips cm100 electron microscope ( fei , hillsbrough , or ) at 80 kv and images collected using a megaview iii ccd camera ( olympus soft imaging solutions , lakewood , co ) . grids at ireland were examined on a jeol 2100 electron microscope ( zeiss ) operating at 200 kv and images collected using a ccd camera . analysis of tem images was performed using image j software from nih ( http://rsbweb.nih.gov/ij/ ) . briefly , 4 l aliquots of a solutions were deposited on freshly cleaned and dried silicon wafers ( approximately 1 mm thick ) . after waiting for 10 min , nonadsorbed portions of the samples were washed with deionized water ( 400 l ) . the wet surface of the silicon wafer was then dried using gentle flow of air . the samples were analyzed by atomic force microscopy ( afm , a multimode spm , model no . tapping mode approach was used to acquire the images , which allows intermittent contact of the tip with the sample and minimizes the chances of deformation of the peptide samples . the cantilever force constant was approximately 20100 n / m with the resonance frequency between 200 and 400 khz . the software used for the analysis of fibrils was the nanoscope control , version 5.30 and the histogram analysis was performed using the postanalysis pico image software ( pico view version 1.6.2 ) . uv - vis spectra of solutions were recorded on a perkin - elmer lambda 900 uv / vis / nir spectrometer ( norwalk , ct ) . fluorescence spectra were obtained using spex fluorolog fluorospectrometer ( horiba jobin yvon , edison , nj ) . both uv - vis and fluorescence measurements were obtained using quartz cuvette with1 cm optical path length . 30 l of a ( 142 ) aliquots were extracted at different time periods , and 300 l of tht ( 10 m ) was added to the samples . the tht fluorescence was measured at 482 nm at an excitation wavelength of 440 nm in a semimicro quartz cuvette of an optical pathlength of 1 cm . characterization of dhla - capped qds was carried out using uv - vis and fluorescence spectroscopy ( figure 1(a ) ) . the fluorescein ( qy is 0.94 in 0.1 m naoh ) was employed as reference . the qy was calculated by using the following equation : ( 1)qyq = qyf[afnq2iq()d][aqnf2if()d ] . a is the absorbance at the excitation wavelength , n is the refractive index of the solvent used , i is the emission wavelength - dependent emission intensity , and is the emission wavelength . qy was calculated from the intensity of luminescence and the absorbance in figures 1(b ) and 1(c ) for fluorescein ( 0.1 m naoh as solvent ) and qds ( water as solvent ) , respectively . the qy of the dhla - capped cdse / zns qds was around 25% . to characterize the qds conjugated to a ( 142 ) , it is important to estimate the number of a monomers bound to qds after conjugation . firstly , characterization of samples of pure a ( 142 ) , a ( 142 ) mixed and conjugated to qds was performed using uv - vis and fluorescence spectroscopy ( figure 2 ) . the emission band for the tyrosine moiety in a ( 142 ) was observed at 309 nm at the excitation wavelength of 280 nm , slit width at emission and excitation was set at 5 nm . the absorption band for a ( 142 ) was observed at 275 nm ( figure 2(a ) ) , whereas the emission band for the a ( 142 ) mixed and conjugated to qds was observed at 560 nm at the excitation wavelength of 467 nm . the absorption band for the qds was observed at 547 nm and for the a ( 142 ) a little hump was observed at 275 nm as shown in figure 2(b ) . molar concentration of the qds and a ( 142 ) was calculated from uv - vis spectrum of the solution [ 12 , 13 ] , the optical path length used was 1 cm . the ratio of these concentration values gave the average number of a per quantum dot nanoparticles . the molar extinction coefficient of qds at 547 nm is 105.8 10 m cm and at 275 nm is 2.6 10 m cm . extinction coefficient for a ( 142 ) at 275 nm is 1.4 10 m cm . the calculations to determine the ratio of a ( 142 ) and qds are shown below . qds concentration ( conjugated sample ) is ( 2)abs547547l=0.1561.05 105 m1 cm11 cm=1.5106 m. absorption at 275 nm from qds is ( 3)abs547275547=0.1562637.21.05105=3.9103 . absorption at 275 nm from a ( 142 ) is ( 4)abs275[abs547275547]=2.23.9 103=2.2 . a ( 142 ) concentration in the sample conjugated to qds is ( 5)abs for a at 275 for a at 275l=2.21390 m1cm11 cm=1.6103 m , loading = a concentrationqds concentration=1.6103 m1.5106 m=1.1103 . theoretically , the total number of molecules / particle of a ( 142 ) ( molecular weight 4514.1 g ) present in 0.5 mg of the 1157 l of sample can be calculated by multiplying the number of moles with na ( avogadro 's number ) number of molecules . hence , the total number of molecules of a ( 142 ) present in the sample is 5.8 10 . similarly , the total number of qds particles present in the solution is 9.0 10 . therefore , the ratio of a ( 142 ) molecules per qd particle is 6400 . subtracting the experimental value ( 1100 ) from the theoretical value ( 6400 ) , it can be inferred that there are 5300 molecules of a ( 142 ) that are free in the solution per qd particle that is conjugated to a ( 142 ) . to confirm that the qds were indeed conjugated to a ( 142 ) , we have used the agarose gel electrophoresis for the control dhla - capped cdse / zns qds along with a ( 142 ) mixed and conjugated to the qds ( figure 3 ) . the gel was run in tae buffer ( ph 7.4 ) at 84 v for 75 min , and the volume of the samples in each well was 10 l . figure 3 shows the distance moved by three different samples : dhla - capped qds ( 1 ) , a ( 142 ) mixed with dhla - capped qds ( 3 ) , and a ( 142 ) conjugated with dhla - capped qds ( 5 ) . from the gel electrophoresis , we can clearly distinguish that the distance moved by the qds mixed with a ( 142 ) is the same as for the pure dhla - capped qds . whereas when the qds are conjugated to a ( 142 ) , the distance moved is lower . this shows that when the qds are chemically conjugated to a ( 142 ) , the distance moved is reduced due to the higher molecular weight . moreover , comparison of polyethylene glycol ( peg)-capped qds ( 2 ) , a ( 142 ) mixed peg - capped qds ( 4 ) , and a ( 142 ) conjugated peg - capped qds ( 6 ) shows that peg - capped qds when conjugated travel the least distance . purified fractions of dhla- ( 7 ) and peg - capped ( 8) qds conjugated to peptide show that free a ( 142 ) can be separated from the a ( 142 ) conjugated qds . different fractions of 1 ml each obtained from gel chromatography were checked for the presence of free a ( 142 ) using uv - vis absorption and fluorescence spectroscopy . first evidence on the inhibition in fibrillation comes from tem images ( figure 4 ) taken on the 7th day of incubation at 37c . tem images containing a ( 142 ) are negatively stained using 2% uranyl acetate solution . three different samples , namely , pure a ( 142 ) , a ( 142 ) mixed with dhla - capped cdse / zns qds , and a ( 142 ) conjugated to dhla - capped cdse / zns qds , are shown in figures 4(a ) , 4(c ) , and 4(d ) , respectively . in all the three samples , the concentration of a ( 142 ) is 0.96 10 m and the concentration of qds is 1.4 m . analysis of this image indicates that the length of the fibrils varies from 30 to 1730 nm . the width of the shorter fibrils is 4.0 0.7 nm whereas for the longer fibrils it is 7.5 0.5 nm . figure 4(b ) presents the pure dhla - capped cdse / zns qds , the average particle size is 2.5 1.3 nm ( figure 5(a ) ) . the size of qds is an important parameter for the biodiagnostic studies , since smaller size qds are capable of passing through the blood - brain barrier [ 15 , 16 ] . figure 4(c ) shows the incubated sample of a ( 142 ) in the presence of qds whereas figure 4(d ) shows a ( 142 ) conjugated qds . comparison of the results in the three images containing a ( 142 ) illustrates very interesting pattern . sample containing pure a ( 142 ) , figure 4(a ) , has a large number of fibrils ranging from short fibrils around 30 nm to long fibrils around 2 micron whereas images of samples in presence of qds are very different . figure 4(c ) is the image of a ( 142 ) mixed with qds ; it shows long - length fibrils of around 1 micron . the width of the fibrils for the sample having mixed qds is 7.7 0.7 nm . figure 4(d ) for a ( 142 ) conjugated to qds shows short - length fibrils ranging from 30 to 80 nm . for this sample , the variation in the thickness of fibrils is significant in this case as compared to the other two . one can see a distinguishable inhibition of the fibrillation when a ( 142 ) is conjugated to qds . histogram showing the size of dhla - capped qds is shown in figure 5(a ) . the average size analyzed from the histogram obtained from tem images is 2.5 1.3 nm . furthermore , to consolidate the results , we have performed the statistical analysis on the tem images ( 1318 images per sample ) . the number of short - length fibrils ( 80150 nm ) observed in the sample containing pure a ( 142 ) was extremely high as compared to samples containing qds . statistical analysis showed ( figure 5(b ) ) that the number of fibrils having length 50100 nm dropped to 90% in the case of a ( 142 ) mixed to qds . there were only 26% of short - length fibrils observed for a ( 142 ) conjugated to qds . the total number of fibrils in the samples containing qds mixed or conjugated to a ( 142 ) was similar ( 35 fibrils approximately ) . . figures 6(a ) and 6(b ) show the tem images of unstained samples of a ( 142 ) mixed with qds . contrary to the tem images of stained samples where we can observe only fibrils and no qds , in this case , we were able to observe the qds , as well as fibrils . it could be discerned from the results that qds are enveloping the fibrils and more number of qds are observed at the ends of the fibrils . for the samples containing a ( 142 ) conjugated to qds ( figures 6(c ) and 6(d ) ) , we could observe less number of qds and short - length fibrils . the qds observed in this case are segregated at one end of the fibrils as seen in figure 6(d ) . it could be infered from the results obtained using unstained samples ( figure 6 ) that qds envelop the fibrils and could block the ends to elongate . importance of the c terminus of the a ( 142 ) in controlling the self - assembly of fibrillation was revealed before . the reason for the inhibition of fibrillation in presence of qds could be that the small size of particles could block the c - terminal end of the fibrils ( ~10 nm ) or the protofibrils ( ~5 nm in diameter ) , which is considered as the terminus with lower degree of freedom and accessibility for elongation mechanism . furthermore , binding between the qds and the a ( 142 ) could block the active sites leading to low local protein concentration , hence increasing the lag time for nucleation or disrupting the nucleation process leading to inhibition of the fibrillation process . besides , presence of a ( 142 ) conjugated to qds in the sample containing free a ( 142 ) may perturb the nucleation mechanism with decrease in localised concentration of the a ( 142 ) as thereby inhibiting fibrillation process [ 19 , 20 ] . however , mixed sample of a ( 142 ) and the qds might increase the localised concentration of the polypeptide , thereby increasing the length of fibrils but the number of fibrils still remains low suggesting that there is a perturbation in the mechanism of the fibrillation process . the tem results are supported by the afm images as shown in figure 7 for the 7th day of incubation at 37c . analysis of afm image for a ( 142 ) in absence of qds ( figure 7(a ) ) shows that the length of the longest fibril is 523 nm and the shortest fibril is 30 nm . a bigger number of short length fibrils ( 3080 nm ) are observed as witnessed by the tem images whereas in the sample containing a ( 142 ) mixed to qds ( figure 7(c ) ) the length of the longest fibrils is 849 nm , comparable to the length of fibrils ( 1 m ) found in the tem images . however , when we compare the length of fibrils ( figure 7(d ) ) in the sample containing a ( 142 ) conjugated to qds , very few long fibrils were observed , which correspond to the tem images of the same sample . the length of the longest fibrils found is 468 nm and the shortest fibril is 58 nm , while in the case of the tem images the length of fibrils is between 30 and 80 nm . the height analysis of qds ( figure 7(b ) ) shows that the root mean square height of qds is 2.3 nm , which is comparable to the average height of qds found in tem images ( 2.5 1.3 nm ) . the measurement of z - height of the a aggregates shows that when a ( 142 ) is conjugated to qds the height distribution histogram changes significantly . it is known for the a oligomers that the average height is between 3 and 5 nm , and for the fibrils it varies from 3 to 9 nm [ 21 , 22 ] . from the height distribution curves ( figure 8) one can see that in the case of amyloid fibrils in absence of qds 40% of aggregates have height greater than 8 nm whereas 60% of aggregates have height between 6 and 8 nm . for the sample of a ( 142 ) mixed with qds , 30% of the aggregates have height greater than 7 nm , and 70% of the aggregates have height between 1 and 3 nm . interestingly , when we analyze the height distribution for a ( 142 ) conjugated to qds only 14% of the aggregates have height greater than 7 nm . almost 90% of the aggregates have height between 2 and 4 nm . to further examine the fibrillation process and to support the image analysis , it is known that the fluorescence intensity of the tht dye grows with increasing concentration of fibrils . it has to be pointed out that previous studies confirmed that the fluorescence enhancement of tht depends upon the structure of the aggregated state of the amyloid peptides [ 23 , 24 ] . figure 9(a ) shows the tht assay on the 7th day for the samples incubated at 37c . when the pure solution of 10 m tht in pbs buffer ( ph 7.4 ) is excited at 440 nm , the emission band at 482 nm is observed with a very low intensity . in the presence of amyloid fibrils , the tht emission band at 482 nm is enhanced significantly . for the sample containing qds mixed to the fibrils , the intensity of the emission band at 482 nm is decreased by 66% as compared to the band for pure amyloid fibrils . whereas the amyloid fibrils , conjugated to qds show a decrease in intensity for the emission band by 40% as compared to the qds mixed to the fibrils . the variation of tht intensity yields information regarding the extent of fibrillation . for the sample containing pure a ( 142 ) , a sigmoidal curve is observed , lag phase is between 0 and 24 h , and rapid progress in fibrillation is observed after 50 h of incubation . however , for the sample containing dhla - capped qds mixed to a ( 142 ) , lag time is increased to 48 h and it can be observed with decrease in intensity of fluorescence that the qds are inhibiting the fibril formation . similarly , in the case of sample containing a ( 142 ) conjugated to the dhla - capped qds , a decrease in intensity of fluorescence and completion of saturation in fibrillation are observed at 72 h. these results show , in the presence of qds , that the self - assembly of a ( 142 ) is perturbed . a remarkable diminution in fibrillation process in the presence of qds and a significant change in morphology contrary to the results that have been published previously on the nanoparticles such as tio2 , copolymer particles , cerium oxide , qds , and carbon nanotubes behaving as catalyst for fibrillation [ 5 , 8 ] , we did not observe the same behavior for the dhla - capped cdse / zns qds . moreover , our results are in line with a very recent publication showing the inhibition of a ( 140 ) by cdte nanoparticles which have similar diameter size ( 35 nm ) . major difference from other set of nanoparticles being used could be the composition and the size of the particles . the size range for the particles that have been used for the previous studies varies from 16 to 200 nm whereas the qds used in our work have an average size of 2.5 1.3 nm . to investigate the effect on the tyrosine residue , which is an intrinsic probe of a ( 142 ) , we have examined the tyrosine fluorescence spectra for the three samples on the 7th day of incubation at 37c . there is a notable quenching of the tyrosine fluorescence intensity at 309 nm ( figure 9(c ) ) in the presence of mixed or conjugated qds . this effect could be due to the fact that the tyrosine moiety ( tyr ) interacts with the qds . for example , the three histidine residues ( his , his , and his ) in the vicinity of the tyrosine may interact or form coordination bond with the surface of qds . this phenomenon happens due to the presence of overcoated zns shell offering zn ions , hence rendering tyrosine to interact with qds and consequently decreasing significantly the fluorescence intensity of tyrosine band . another explanation could be the fret mechanism between the donor ( tyrosine moiety ) and the acceptor ( qds ) , since there is an overlap between the absorption spectrum of the acceptor ( qds ) and the emission spectrum of the donor ( tyrosine ) . fret efficiency in case of a ( 142 ) conjugated to qds was 0.84 whereas for a ( 142 ) mixed with qds was 0.94 . it could be interpreted that the forster distance ( ro ) between the a ( 142 ) and qds in aqueous solution was less than 60 which is the critical distance for energy transfer . it means that in both the samples , a ( 142 ) mixed with or conjugated to qds , a ( 142 ) is present very near to the qds . this is an indirect evidence that qds are present very near to fibrils ; that is also observed in the tem images of the unstained samples ( figure 6 ) . furthermore , to examine the inhibition effect of cdse / zns qds , we performed the same set of experiments using polyethylene glycol ( peg ) ( mw 400)-capped cdse / zns qds . no inhibition on fibrillation process is observed when peg - capped qds are mixed or conjugated to a ( 142 ) . figures 10(a ) and 10(b ) show the afm images of peg - capped qds conjugated and mixed to a ( 142 ) , respectively , after 2 days . the length of the fibrils is between 700 nm and 3 m and the height of oligomers is observed between 2.5 and 5.9 nm . increase in intensity of fluorescence at 482 nm for the peg - capped qds mixed or conjugated with a ( 142 ) shows that there is increase in fibrillation in presence of peg - capped qds . experiments were also designed where dhla- or peg - capped qds were purified using gel chromatography to check the inhibition effect . similar results are obtained over a period of 72 h with the purified fractions of dhla - or peg - capped qds , that is , inhibition and absence of inhibition on fibrillation process , respectively . it shows that if we change the ligand of the qds , it changes its behavior towards the fibrillation process . the emission band for the peg - capped qds was observed at 472 nm , when the excitation wavelength was set at 467 nm . the average size for the peg - capped qds obtained using tem analysis was 19.4 4.7 nm ( figure 10 ) . this change in behavior might be due to the fact that peg - capped qds tend to aggregate in buffer solutions , and peg polymer increases the size of the qds . these two factors make the qds nanoparticles less dynamic in solution and less accessible for the a ( 142 ) monomers , where the nanoparticles can block the active sites for extended fibrillation . figures 11(a ) and 11(b ) show the tem images of peg - capped qds . the average size for the peg - capped qds obtained using tem analysis was 19.4 4.7 nm as illustrated in the histogram ( figure 11(c ) ) . qds mixed or conjugated to a ( 142 ) show a decrease in the fibrillation as compared to pure a ( 142 ) , when incubated at 37c for 7 days . longer fibrils ( 2 micron ) are observed in the sample containing a ( 142 ) mixed with qds . pure a ( 142 ) sample contained large number of short- and long - length fibrils ( 301730 nm ) . thicker and shortest length fibrils ( 3080 nm ) are observed in the case of a ( 142 ) conjugated to qds . the height analysis of afm images shows significant decrease in height of aggregates greater than 7 nm ( only 14% ) when qds are conjugated to a ( 142 ) and 30% when qds are mixed to a ( 142 ) as compared to pure a ( 142 ) solution . tht assay for the samples confirmed the inhibition of the fibrillation process when a ( 142 ) is mixed or conjugated to qds . moreover , quenching of tyrosine signal is observed in the presence of qds , which indicates an interaction of the qds with the tyr residue in a ( 142 ) . however , in presence of peg - capped qds mixed or conjugated to a ( 142 ) , an absence of inhibition on fibrillation is observed as revealed by afm images and tht fluorescence . the conclusion of this work is presented in figure 12 that shows diminution in fibrillation in presence of dhla - capped qds , either mixed with or conjugated to a ( 142 ) . to investigate the use of qds in vivo studies is very important part of biomedical applications , there is a recent investigation showing the use of qds for imaging and delivery purposes , where qds carrying snare - tagged rbd were delivered at the synaptic contacts in the cultures from hippocampal neurons obtained from mice . moreover , qds doped with sio2 nanoparticles showed imaging and gene carrier capabilities , it was demonstrated that these qds were internalized by primary cortical neural cells without inducing cell death in vitro and in vivo . point to be noted is cdse quantum dots are toxic and might not be used for medicinal purposes . however , some toxicology studies have shown that the toxicity of qds is size and concentration dependent . for example , cytotoxicity studies of cdse qds on b16 f10 melanoma cells , and c57/bl6 mice showed no detectable toxicity . early studies have shown high toxicity of cdse qds due to the release of toxic cd ions ; however , coating of zns has shown to reduce the toxicity in cell culture to a great extent . nevertheless , extensive studies are required in the field of toxicology . in the light of these studies , it would be important to test a ( 142 ) mixed with or conjugated to qds in the cultures from neurons of mice to investigate the effect of qds in in vivo systems .

nanoparticles have enormous potential in diagnostic and therapeutic studies . we have demonstrated that the amyloid beta mixed with and conjugated to dihydrolipoic acid- ( dhla ) capped cdse / zns quantum dots ( qds ) of size approximately 2.5 nm can be used to reduce the fibrillation process . transmission electron microscopy ( tem ) and atomic force microscopy ( afm ) were used as tools for analysis of fibrillation . there is a significant change in morphology of fibrils when amyloid ( 142 ) ( a ( 142 ) ) is mixed or conjugated to the qds . the length and the width of the fibrils vary under modified conditions . thioflavin t ( tht ) fluorescence supports the decrease in fibril formation in presence of dhla - capped qds .

1. Introduction 2. Experimental Section 3. Results and Discussion 4. Imaging and Analysis 5. Conclusion

statin therapy has been well established as a cornerstone of cardiovascular prevention , and yet despite potent therapies for lowering lowdensity lipoprotein cholesterol ( ldlc ) , substantial residual risk remains.1 , 2 , 3 clinical and epidemiological studies have demonstrated that triglyceride ( tg ) elevation is an independent risk factor for increased cardiovascular ( cv ) events , and therefore may represent one contributive factor of residual cv risk beyond statin therapy.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 more recently , elegant mendelian randomization studies have supported a causal role for tg in the pathogenesis of cardiovascular disease ( cvd ) , showing that elevated tg are not merely a risk marker , but rather a risk factor and thus potentially modifiable.3 despite the available data , an important question that remains is whether treatment of modest degrees of tg elevation would decrease cv events , in particular in patients already receiving ldlc lowering therapy with statins . prior cv outcome studies that administered therapies with tglowering effects ( niacin or fenofibrate ) on top of statin therapy did not reach their primary endpoints . nonetheless , these studies also did not prospectively enroll patients with elevated tg levels despite statin therapy,6 , 12 , 13 , 14 , 15 and subgroup analyses suggested possible benefits to tg lowering in patients with dyslipidemia.5 , 6 outcome studies of relatively low doses of prescription omega3 therapies in japan ( the japan epa lipid intervention study [ jelis])16 and italy ( gruppo italiano per lo studio della sopravvivenza nell'infarto miocardico [ gissi])17 , 18 have suggested that omega3 therapy may provide cv protection . however , these studies were performed in single countries prior to current treatment guidelines , and therefore provide supportive but not conclusive evidence of cv benefit . other more recent omega3 therapy outcome studies conducted in the presence of statins have been less encouraging , but these studies were characterized by evaluating nonhypertriglyceridemic patient populations ( eg , tg < 200 mg / dl ) and administering low doses of longchain omega3 fatty acids ( eg , eicosapentaenoic acid [ epa ] and/or docosoahexaenoic acid [ dha]).19 , 20 , 21 , 22 , 23 , 24 omega3 therapies , including epa , have been postulated to have cardioprotective effects such as beneficial changes to tg and other lipid and lipoprotein parameters ( eg , non highdensity lipoprotein cholesterol [ nonhdlc ] , apolipoprotein ciii ) , as well as other potential benefits beyond plasma lipid modification.19 , 25 , 26 , 27 , 28 , 29 , 30 icosapent ethyl ( vascepa ; amarin pharma inc . , bedminster , nj ) is a highly purified ethyl ester of epa , which has been reported to improve atherogenic dyslipidemia characterized by reductions in tg , tgrich lipoproteins , and factors involved in their metabolism , without raising ldlc.25 , 26 , 27 , 28 , 29 based on trials with tg lowering as the primary endpoint , this prescription therapy is currently approved for use in the united states by the us food and drug administration ( fda ) as an adjunct to diet to reduce tg levels in adult patients with severe hypertriglyceridemia ( 500 mg / dl).25 , 26 in this range of very elevated tg levels , reduction is considered to be clinically necessary to decrease the risk of pancreatitis . in addition to beneficial changes to tgrich lipoproteins and other plasma lipid markers , some clinical studies with higherdose epa also suggest beneficial effects on markers of oxidation and inflammation , coronary plaque characteristics , and major cv events.16 , 25 , 26 , 29 , 31 , 32 , 33 for example , in contrast to the fenofibrate and niacin studies , jelis found a 19% relative risk reduction in cv events in statintreated patients with relatively normal tg but a more pronounced 53% reduction in the subgroup with mixed dyslipidemia , specifically tg 150 mg / dl and hdlc < 40 mg / dl.4 , 16 although confirmation of these results is needed in western populations , the reduction of cv events with epa therapy in a patient population with relatively normal tg levels suggests that epa may have pleiotropic effects beyond plasmalipid modification . it is worth noting that the promising results from jelis occurred with a highpurity epa preparation dosed at 1.8 g / d in a japanese population , for whom the baseline epa levels are higher than in western populations due to greater dietary intake of marine omega3 fatty acids . icosapent ethyl 12week dosing at 4 g / d in a highrisk population similar to that within the reduction of cardiovascular events with icosapent ethyl intervention trial ( reduceit ) who had persistent elevations of tg after treatment with statins resulted in significant reductions in tg and atherogenic lipoproteins,26 , 27 , 28 as well as comparable plasma epa levels as the 1.8 g / d dosing group in jelis.34 therefore , a dose of 4 g / d was selected as the dose for further study . in this context , reduceit was designed to determine if treatment with icosapent ethyl 4 g / d vs placebo would reduce ischemic events in patients at increased cv risk already being treated with statins . reduceit ( nct01492361 ) is a phase 3b , international , multicenter , prospective , randomized , doubleblinded , placebocontrolled , parallelgroup trial of icosapent ethyl 4 g / d ( 2 g twice daily with food ) vs placebo ( figure ) . the main objective is to evaluate whether treatment with icosapent ethyl reduces ischemic events in patients at elevated cv risk concurrently treated with statins . inclusion and exclusion criteria are listed in table 1 and table 2 , respectively . men or women age 45 years with established cvd ( cv risk stratum 1 , table 1 ) or age 50 years with diabetes mellitus in combination with 1 additional risk factor for cvd ( cv risk stratum 2 , table 1 ) were eligible for inclusion . fasting tg levels 150 mg / dl and < 500 mg / dl were required . a study amendment was made during the early part of the trial , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl , to increase enrollment of patients with more significant tg elevations . ldlc levels needed to be > 40 mg / dl and 100 mg / dl , with patients on stable statin therapy ( ezetimibe ) for 4 weeks prior to the ldlc and tg qualifying measurements for randomization . if patients met the inclusion criteria at visit 1 , they were asked to return for the randomization visit ( visit 2 ) and entered the treatment / followup period . patients who were not eligible at visit 1 but who became eligible in the next 28 days ( such as patients whose statin dose changed at visit 1 and/or needed to wash out prohibited medications ) may have returned for an optional second screening visit ( visit 1.1 ) . such patients entered a statin stabilization / medication washout period of 28 days prior to rescreening . patients who were eligible following screening / rescreening entered the treatment / followup period , with followup visits occurring at 4 months , 12 months , and annually thereafter . * a study amendment ( may 2013 ) was made , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl to increase enrollment of patients with tg 200 mg / dl ; it is anticipated that mean and median qualifying tg levels will be > 200 mg / dl . eventdriven design : approximately 1612 primary efficacy events will be required during the study ; study duration will vary accordingly . abbreviations : cv , cardiovascular ; cvd , cardiovascular disease ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; reduceit , reduction of cardiovascular events with icosapent ethyl intervention trial ; t2 dm , type 2 diabetes mellitus ; tg , triglycerides . abbreviations : abi , anklebrachial index ; bp , blood pressure ; cad , coronary artery disease ; cr , creatinine ; crcl , creatinine clearance ; cv , cardiovascular ; cvd , cardiovascular disease ; dm , diabetes mellitus ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highsensitivity creactive protein ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; nsteacs , non stsegment elevation acute coronary syndrome ; pad , peripheral arterial disease ; tg , triglycerides . note : patients with dm and cvd as defined above are eligible based on the cvd requirements and will be counted under cv risk stratum 1 . only patients with dm and no documented cvd as defined above need 1 additional risk factor as listed , and they will be counted under cv risk stratum 2 . a study amendment ( may 2013 ) was made , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl to increase enrollment of patients with tg 200 mg / dl ; it is anticipated that mean and median qualifying tg levels will be > 200 mg / dl . abbreviations : aids , acquired immunodeficiency syndrome ; apocii , apolipoprotein cii ; ck , creatine kinase ; crcl , creatinine clearance ; cvd , cardiovascular disease ; dbp , diastolic blood pressure ; hba1c , glycated hemoglobin ; hf , heart failure ; hiv , human immunodeficiency virus ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; nyha , new york heart association ; om3 , omega3 ; pcsk9 , proprotein convertase subtilisin / kexin type 9 ; sbp , systolic blood pressure ; tg , triglyceride ; uln , upper limit of normal . the primary endpoint is a composite of cv death , nonfatal myocardial infarction ( mi ) , nonfatal stroke , coronary revascularization , or unstable angina . the key secondary endpoint is the composite of cv death , nonfatal mi , or nonfatal stroke . several other secondary , tertiary , and exploratory endpoints are being assessed ( table 3 ) , which were designed to provide additional insights into the potential effects of epa therapy on various outcomes and in distinct highrisk patient populations . abbreviations : apob , apolipoprotein b ; chf , coronary heart failure ; cv , cardiovascular ; dm , diabetes mellitus ; ecg , electrocardiography ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highsensitivity creactive protein ; hstnt , highsensitivity troponin t ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; non hdlc , non highdensity lipoprotein cholesterol ; pvd , peripheral vascular disease ; rlpc , remnant lipoprotein cholesterol ; tc , total cholesterol ; tg , triglycerides ; tia , transient ischemic attack ; ua , unstable angina ; vldlc , very lowdensity lipoprotein cholesterol . the first occurrence of any of these major adverse vascular events during the followup period of the study will be included in the incidence . for the secondary and tertiary endpoints that count a single event , the time from randomization to the first occurrence of this type of event will be counted for each patient . for secondary and tertiary endpoints that are composites of 2 types of events , the time from randomization to the first occurrence of any of the event types included in the composite the time from randomization to occurrence of the first and all recurrent major cv events defined as cv death , nonfatal mi ( including silent mi ) , nonfatal stroke , coronary revascularization , or ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization . including silent mi ; ecg will be performed annually for the detection of silent mi . composite endpoints include : composite of cv death , nonfatal mi ( including silent mi ) , nonfatal stroke , cardiac arrhythmia requiring hospitalization of 24 hours , or cardiac arrest ; composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , or ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization ; composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization , nonfatal stroke , or pvd requiring intervention such as angioplasty , bypass surgery , or aneurysm repair ; and composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization , pvd requiring intervention , or cardiac arrhythmia requiring hospitalization of 24 hours . assessment of the relationship between baseline biomarker values and treatment effects within the primary and key secondary composite endpoints ; assessment of the effect of study drug on each marker ; and assessment of the relationship between postbaseline biomarker values and treatment effects within the primary and key secondary composite endpoints by including postbaseline biomarker values ( for example , at 4 months , or at 1 year ) as a covariate . therefore , 1612 events would be required to have approximately 90% power with a 1sided level of 2.5% and with 2 interim analyses . approximately 70% of randomized patients were to be in cv risk stratum 1 ( established cvd ) and approximately 30% of randomized patients were to be in cv risk stratum 2 ( highrisk primary prevention defined by diabetes mellitus and other risk factors ) . randomization was stratified by cv risk strata , ezetimibe use , and by geographical region . protocol amendment 1 ( may 2013 ) changed the lower limit of tg levels for entry into the trial from 150 mg / dl to 200 mg / dl , as a majority of the steering committee members felt that those were the patients most likely to benefit from tg lowering . protocol amendment 2 ( july 2016 ) designated the composite of hard major adverse cardiovascular events ( cv death , nonfatal mi , nonfatal stroke ) as the key secondary endpoint per suggestions from the fda with steering committee concordance . approximately 8000 patients have been randomized at approximately 470 centers worldwide ( see supporting information , appendix , in the online version of this article ) . follow up will continue in this eventdriven trial until approximately 1612 adjudicated primary efficacy endpoint events have occurred . this study is being conducted in accordance with a special protocol assessment agreement with the fda . despite cv risk reduction through potent ldlc lowering therapies such as statins , substantial residual cv risk remains . epidemiological , biological , and genetic studies have provided robust evidence of a strong association between elevated tg levels and higher rates of cv events.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 furthermore , tg reduction lowers several inflammatory markers associated with cv risk , and subgroup and post hoc analyses of outcome studies suggest possible reductions in major cv events with tglowering therapy.3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 finally , studies administering higherdose epa suggest additional beneficial effects beyond lipidlowering that may be unique to epa relative to other tglowering therapies , such as beneficial changes in coronary plaque characteristics , which may lead to reductions in major cv events.4 , 16 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 however , randomized data from large outcome studies across broad populations regarding pharmacological tg lowering and effect on cv outcomes have been mixed ( table 4).1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 part of the reason may involve differences between the classes of drugs studied , such as fibrates , niacin , and omega3 fatty acids . even among omega3 fatty acid studies , there are marked differences with respect to the relatively low doses of omega3 administered and the ratio of epa to dha.16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 in addition , different tglowering therapies may exert differential effects across lipid profiles . for example , fibrates and dhacontaining omega3 fatty acid mixtures have been shown to increase ldlc , which in turn might adversely influence trial results . among outcome studies administering tglowering agents beyond statin therapy , only the jelis trial using pure epa demonstrated a significant reduction in cv events in patients with relatively normal tg levels.16 the subgroup data ( table 5 ) from the action to control cardiovascular risk in diabetes ( accord ) lipid,6 atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides and impact on global health outcomes ( aimhigh),5 and jelis4 trials further support a prospective study of epa in a broader patient population with hypertriglyceridemia , as exemplified in reduceit , as a potential addon to statin therapy to reduce residual cv risk . that the lipid changes in jelis were relatively modest ( ie , approximately a 5% tg reduction ) raises the possibility that other pleiotropic effects beyond lipid lowering may have also contributed to the reduction in cv risk.4 , 16 finally , any benefits to tglowering therapies may be most pronounced among statintreated patients in the higher range of tg elevation ( ie , 200 mg / dl),4 , 5 , 6 for whom randomized prospectively designed outcome studies have not been previously conducted prior to the reduceit study . cv outcome trials administering therapies that can be used for tg lowering abbreviations : accord , action to control cardiovascular risk in diabetes ; aimhigh , atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides : impact on global health outcomes ; ala , alphalinolenic acid ; alphaomega , study of omega3 fatty acids and coronary mortality ; bip , bezafibrate infarction prevention ; bl , baseline ; chd , coronary heart disease ; ci , confidence interval ; cv , cardiovascular ; cvd , cardiovascular disease ; dha , docosahexaenoic acid ; eos , end of study ; epa , eicosapentaenoic acid ; er , extended release ; feno , fenofibrate ; field , fenofibrate intervention and event lowering in diabetes ; gissip , gruppo italiano per lo studio della sopravvivenza nell'infarto miocardicoprevenzione ; gissihf , gruppo italiano per lo studio della sopravvivenza nell'infarto miocardicoheart failure ; hdlc , highdensity lipoprotein cholesterol ; hf , heart failure ; hps2thrive , heart protection study 2treatment of hdl to reduce the incidence of vascular events ; hr , hazard ratio ; jelis , japan epa lipid intervention study ; mace , major adverse cardiac events ; mi , myocardial infarction ; nyha , new york heart association ; oae , omega acid esters ; omega , effect of omega 3fatty acids on the reduction of sudden cardiac death after myocardial infarction ; or , odds ratio ; origin , outcome reduction with an initial glargine intervention ; pbo , placebo ; rrr , relative risk reduction ; scd , sudden cardiac death ; su.fol.om3 , supplmentation en folates et omega3 ; t2 dm , type 2 diabetes mellitus ; tc , total cholesterol ; tg , triglycerides ; vahit , veterans affairs cooperative studies program highdensity lipoprotein cholesterol intervention trial ; vit e , vitamin e. mean or median values are presented . subgroup analyses of patients with dyslipidemia from cv outcome trials administering tglowering therapies added to statin therapy vs statin monotherapy abbreviations : accord , action to control cardiovascular risk in diabetes ; aimhigh , atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides : impact on global health outcomes ; bl , baseline ; cv , cardiovascular ; epa , eicosapentaenoic acid ; er , extended release ; hdlc , highdensity lipoprotein cholesterol ; hps2thrive , heart protection study 2treatment of hdl to reduce the incidence of vascular events ; jelis , japan epa lipid intervention study ; mace , major adverse cardiac events ; tg , triglycerides . bolded values approached / achieved statistical significance . reduceit is designed to evaluate whether treating atrisk patients with highdose epa will lower the rates of important ischemic events beyond statin therapy . however , this trial alone will not validate whether lowering tg specifically in patients with elevated tg levels will result in lower rates of important ischemic events , because the effects of epa may be broader than tg reduction alone . several trials , including reduceit , are ongoing or planned to determine if different tglowering therapies in patients with elevated tg levels lower the rate of important ischemic events.35 , 36 , 37 , 38 the use of different therapeutic agents across these trials may in aggregate help us better understand the relative importance of tg lowering alone and may also help define which potential effects observed in reduceit might be uniquely attributable to epa therapy . several lines of data , including comparison of the jelis study results to those of fibrate and niacin outcome studies , suggest that epa may be differentiated from other tglowering agents as statin addon therapy , by potentially providing unique pleiotropic cardioprotective benefits in addition to tg lowering . other changes in the lipidlowering field may also affect the interpretation of the ongoing reduceit trial . for example , the proprotein convertase subtilisin / kexin type 9 inhibitors are being tested in large cv outcome trials of patients for whom ldlc control from statin ( ezetimibe ) therapy may be insufficient or poorly tolerated . if these ldlc lowering agents are found to be beneficial , epa therapy could potentially serve as a complementary approach to reduce residual cv risk even further , though this specific combination would not have been studied well in terms of incremental effects on cv events . importantly , residual cv risk remains high in patients with ldlc well controlled by statins , and many of these patients will likely need to be treated from multiple angles . the growing body of tgrelated evidence suggests that tgrich lipoproteins may be a causal factor in such residual risk . consequently , tg lowering represents a target of great interest to optimize further cv risk reduction beyond the ldlc lowering benefits attained with statin use . epaspecific studies suggest that epa may provide unique cv benefits through favorable effects on plasma lipid parameters , as well as on other pleiotropic pathways . a major remaining question is how to achieve cv risk reduction beyond the benefits realized from effective management of ldlc . for patients with persistently high tg levels despite statin therapy , an agent that improves atherogenic dyslipidemia without raising ldlc and provides other potentially pleiotropic benefits may improve cv outcomes . the addition of epa to statin therapy may thus provide additional cv benefit . the reduceit trial with highdose epa is designed to address this longstanding scientific gap and to provide physicians with this muchneeded information to guide clinical care of patients at high cv risk . dr . bhatt has served on advisory boards for cardax , elsevier practice update cardiology , medscape cardiology , and regado biosciences ; has served on the board of directors for boston va research institute and society of cardiovascular patient care ; has been chair of the american heart association quality oversight committee ; has served on data monitoring committees for duke clinical research institute , harvard clinical research institute , mayo clinic , and the population health research institute ; has received honoraria from the american college of cardiology ( senior associate editor , clinical trials and news , for acc.org ) , belvoir publications ( editor in chief , harvard heart letter ) , duke clinical research institute ( clinical trial steering committees ) , harvard clinical research institute ( clinical trial steering committee ) , hmp communications ( editor in chief , journal of invasive cardiology ) , journal of the american college of cardiology ( guest editor , associate editor ) , population health research institute ( clinical trial steering committee ) , slack publications ( chief medical editor , cardiology today 's intervention ) , society of cardiovascular patient care ( secretary / treasurer ) , and webmd ( cme steering committees ) ; has served as deputy editor of clinical cardiology , as chair of the ncdraction registry steering committee , and as chair of the va cart research and publications committee ; has received research funding from amarin ( for his role as chair of the steering committee and principal investigator of reduceit ) , amgen , astrazeneca , bristolmyers squibb , eisai , ethicon , forest laboratories , ischemix , lilly , medtronic , pfizer , roche , sanofiaventis , and the medicines company ; has received royalties from elsevier ( editor , cardiovascular intervention : a companion to braunwald 's heart disease ) ; has served as site coinvestigator for biotronik , boston scientific , and st . jude medical ; has served as a trustee of the american college of cardiology ; and reports unfunded research with flowco , plx pharma , and takeda . dr . steg has received research grants from merck , sanofi , and servier and has received speaking or consulting fees from amarin , amgen , astrazeneca , bayer , boehringer ingelheim , bristolmyers squibb , cslbehring , daiichisankyo , glaxosmithkline , janssen , lilly , merck , novartis , pfizer , regeneron , sanofi , servier , and the medicines company . dr . brinton has received speaking and/or consulting honoraria from alexion , amarin , amgen , aralez , arisaph , astrazeneca , janssen , kastle , kowa , merck , pts diagnostics , regeneron , and sanofiaventis and has received research funding from amarin ( for his role as steering committee member of reduceit ) and kowa ( for his role as steering committee member of prominent ) . jacobson has served as a consultant for amarin , amgen , astrazeneca , merck , regeneron , and sanofi and has done research for amgen and regeneron / sanofi . miller has served as a consultant for amarin , akcea , gemphire , and pfizer . dr . tardif has received research grants from amarin , astrazeneca , dalcor , eli lilly , esperion , merck , pfizer , sanofi , and servier ; has received honoraria from amarin , astrazeneca , dalcor , sanofi , and servier ; and holds equity ( modest position ) in dalcor . ballantyne discloses grant / research support ( all paid to the institution , not individual ) from amarin , amgen , eli lilly , esperion , ionis , novartis , pfizer , regeneron , sanofisynthelabo , the national institutes of health , the american heart association , and the american diabetes association , and has served as a consultant for amarin , amgen , astrazeneca , boehringer ingelheim , eli lilly , esperion , ionis , matinas biopharma inc , merck , novartis , pfizer , regeneron , and sanofisynthelabo .

residual cardiovascular risk persists despite statins , yet outcome studies of lipidtargeted therapies beyond lowdensity lipoprotein cholesterol ( ldlc ) have not demonstrated added benefit . triglyceride elevation is an independent risk factor for cardiovascular events . highdose eicosapentaenoic acid ( epa ) reduces triglyceriderich lipoproteins without raising ldlc . omega3s have postulated pleiotropic cardioprotective benefits beyond triglyceridelowering . to date , no large , multinational , randomized clinical trial has proved that lowering triglycerides on top of statin therapy improves cardiovascular outcomes . the reduction of cardiovascular events with icosapent ethyl intervention trial ( reduceit ; nct01492361 ) is a phase 3b randomized , doubleblinded , placebocontrolled trial of icosapent ethyl , a highly purified ethyl ester of epa , vs placebo . the main objective is to evaluate whether treatment with icosapent ethyl reduces ischemic events in statintreated patients with high triglycerides at elevated cardiovascular risk . reduceit enrolled men or women age 45 years with established cardiovascular disease or age 50 years with diabetes mellitus and 1 additional risk factor . randomization required fasting triglycerides 150 mg / dl and < 500 mg / dl and ldlc > 40 mg / dl and 100 mg / dl with stable statin ( ezetimibe ) 4 weeks prior to qualifying measurements . the primary endpoint is a composite of cardiovascular death , nonfatal myocardial infarction , nonfatal stroke , coronary revascularization , or unstable angina . the key secondary endpoint is the composite of cardiovascular death , nonfatal myocardial infarction , or nonfatal stroke . several secondary , tertiary , and exploratory endpoints will be assessed . approximately 8000 patients have been randomized at approximately 470 centers worldwide . followup will continue in this eventdriven trial until approximately 1612 adjudicated primaryefficacy endpoint events have occurred .

INTRODUCTION METHODS DISCUSSION CONCLUSION Supporting information Conflicts of Interest

the peptide bond linking adjacent amino acids in protein structures can adopt either the cis or the trans conformation . the cis conformation occurs rarely in polypeptides because of the higher intrinsic energy compared to the trans conformation . despite their infrequent occurrence , cis peptide bonds are very important in a variety of biological processes , such as protein folding , regulation , cell signaling and splicing of protein molecules ( 1 ) . recent studies have indicated that prolyl cis / trans isomerization can act as a molecular timer to help control the cellular process , making it a new target for therapeutic interventions ( 2 ) . furthermore , cis peptide bonds , especially the ones between non - proline residues , are located near the active sites of proteins , or have roles in the function of the protein molecules 1 . ( 5 ) trained a support vector machine ( svm ) using only the primary sequence as input in order to discriminate between the two conformations of proline peptide bonds . ( 6 ) predicted the isomerization of proline peptide bonds using multiple sequence alignment profiles and secondary structure as input . the cops algorithm ( 7 ) aimed to predict the peptide bond formation between any two amino acids employing an extension of the chou - fasman parameters . most of the aforementioned studies focus only on the proline residues , ignoring the rare but highly important non - proline cis peptide bonds . here , we make a further distinction of the peptide bonds into four classes , namely cis imide ( cis - pro ) , cis amide ( cis - nonpro ) , trans imide ( trans - pro ) and trans amide ( trans - nonpro ) , by developing the pbond web server . hence , pbond not only predicts the peptide bond conformation between any two amino acids , but also designates potential cis - nonpro formations . furthermore , a reliability index is computed , which represents the confidence assigned to each prediction . a majority voting scheme is also available , which provides consensus prediction of 10 svm classifiers . pbond has been developed using 3,050 high - quality protein sequences with resolution < 2.0 , r - factor < 0.25 and sequence identity < 25% . the pbond web server graphical interface , as shown in figure 1 , consists of six fields . the numbers placed next to every field in the figure follow the same notation as below . 1 . processing : the user may choose to process either a single sequence or upload multiple sequences for batch processing ; there is no upper limit to the number of sequences submitted for batch processing . 2 . upload sequence : amino acid sequences can be provided in fasta format either by pasting them in the text box or uploading them within a text file . more specifically , multiple sequence alignment profiles , in the form of position - specific scoring matrices ( pssms ) , are obtained after running psi - blast ( 8) against one of the provided protein databases ; the choice of the database highly affects the computational time , whereas only slight perturbations are expected in terms of performance . next , the predicted secondary structure of every residue in the query sequence is computed using psipred ( 9 ) ; real valued predictions of solvent accessibility are obtained from rvp - net ( 10 ) ; six widely used physicochemical properties are also employed for every residue ( volume , hydrophobicity , polarity , charge , aromatic and aliphatic character ) . , 11 . , centered at each residue , whose peptide bond with the preceding amino acid we are trying to predict ; outside this range , the influence of the surrounding residues towards the peptide bonds formation decreases . 4 . feature selection : next , the user may choose either to employ the whole feature vector for the prediction or an optimal reduced set of features ( 12 ) identified in our previous study ( 11 ) . voting : the user may choose to invoke either a single svm model or 10 svm models , each one trained with a different dataset . in the latter , each model independently assigns a label ( cis - pro , cis - nonpro , trans - pro , trans - nonpro ) to every residue in the query sequence and then a linear time majority voting algorithm calculates the consensus of the 10 predictions . submit sequence : if a valid e - mail address is supplied , the results are submitted in a compressed file ; otherwise , if the e - mail field is left blank , the prediction results can be downloaded directly from the web page , where they will be available for 10 days . the output of the pbond server consists of a compressed file containing either a single text file , or multiple text files , in case of batch processing . these files contain plain text with the predictions for every sequence uploaded , along with a reliability index for every prediction . the first and last five peptide bonds of every sequence are labeled as n / a since there are not enough residues in the sliding window to make a prediction . due to the scarcity of cis peptide bonds ( both cis - pro and cis - nonpro ) , a severe class imbalance problem emerges , posing a tradeoff between the identification of as many potential cis formations and certain false positive predictions . hence , special attention was given during the training and evaluation of the pbond server so that important cis formations are not neglected . for this purpose , the predictive models of pbond server have been trained using fully balanced datasets , in which all four classes are equally represented . the evaluation of pbond has been performed on fully balanced disjoint data segments coming from the initial unbalanced dataset 13 . , 14 .. table 1 presents the performance achieved using the initial feature vector , with and without performing majority voting . sensitivity and positive predictive value ( ppv ) are also provided for the two general classes ( cis / trans ) . the performance achieved using the initial input vector is in general quite poor , even though voting slightly improves the results . in table 2 , the performance achieved using the optimal reduced set of features is shown , with and without the employment of the majority voting algorithm . it is clear that the feature selection improves the classification outcome to a certain extent ; a further increment in the results is achieved using the consensus prediction of the 10 models . furthermore , the reliability index associated with every prediction can be used for post processing the prediction results . a detailed comparison of the available prediction methods in the literature and pbond is presented in table 3 . based on the physicochemical properties of the 6 surrounding amino acids , frmmel et al . although the reported results are promising , such refined dataset ( 242 proline bonds ) diminishes the credibility of the proposed method . ( 5 ) as well , focused only on the proline residues and employed single sequence information coded in binary form in order to predict the conformation of the peptide bond . the prediction accuracy achieved by this method is 70% and 77% when evaluated with independent datasets and the jackknife test , respectively . ( 6 ) provided multiple sequence alignment profiles coupled with secondary structure information as input to an svm in order to predict the proline cis / trans isomerization . the overall reported accuracy is 71% after performing five - fold cross validation . only pahlke et al . ( 7 ) aimed to predict the peptide bond conformation between any two amino acids , using the secondary structure of amino acid triplets ; however , the reported results ( overall accuracy 66% ) are quite unsatisfactory . this could be attributed to the refined length of the sliding window , as well as to the small number of employed features . the performance of pbond compares well with previously published studies , albeit validated on different datasets and using different evaluation methods . moreover , pbond is able to identify the scarce but highly important non - proline cis peptide bonds . tpe and cp provided valuable comments and suggestions throughout the study and helped in the manuscript preparation . tpe and cp provided valuable comments and suggestions throughout the study and helped in the manuscript preparation .

pbond is a web server that predicts the conformation of the peptide bond between any two amino acids . pbond classifies the peptide bonds into one out of four classes , namely cis imide ( cis - pro ) , cis amide ( cis - nonpro ) , trans imide ( trans - pro ) and trans amide ( trans - nonpro ) . moreover , for every prediction a reliability index is computed . the underlying structure of the server consists of three stages : ( 1 ) feature extraction , ( 2 ) feature selection and ( 3 ) peptide bond classification . pbond can handle both single sequences as well as multiple sequences for batch processing . the predictions can either be directly downloaded from the web site or returned via e - mail . the pbond web server is freely available at http://195.251.198.21/pbond.html .

Introduction Method Evaluation Authors contributions Competing interests

postoperative complications following third molars surgical removal are a remaining topic of discussion with a fallacy of two schools of thought . one school of thought is endorsed by oral and maxillofacial surgeons who contend that most third molars are potentially pathologic and should be removed . the legal system , in which decisions are generally based on norms of practice or local or regional standards of care , credits each school of thought as having equal merit , ignoring the scientific evidence base . a us national institute of health consensus conference meeting in 1979 attempted to establish wide agreement on the indications for third molar surgery . many issues ( including the role of surgical prophylaxis ) were unresolved , although consensus was reached on factors which definitely merited surgery . the british association of oral and maxillofacial surgeons ( baoms ) , the faculty of dental surgery at queen alexandra hospital , portsmouth , uk , and its faculty audit working group have written draft clinical guidelines to guide those involved clinicians . several published papers have raised issues which have implications not only for health service financing but also for the medico legal aspects of professional practice . in the united states more than 11 million patient days of standard discomfort or disabilitypain , swelling , bruising , and malaise result from thirst molars extraction , and more than 11000 people sustained permanent paresthesia numbness lip , numbness tongue , and cheek numbness as a consequence of nerve injury during the surgery . surgical removal of impacted mandibular third molars should be carried out well before the age of 24 years , especially for female patients . a surgeon 's lack of experience could also be a major factor in the development of postoperative complications . the risk factors associated with complicationssuggest that age , tooth location , bone removal , and tooth sectioning appear to be associated with a higher complication rate for third molars removal [ 6 , 7 ] . the extraction of third molars is one of the most common acts in dental practice and certainly the most frequent in stomatology . the indications for general anesthesia concerning this type of intervention are the subject of an ongoing debate , but the choice of the patient is critical and must be framed by clear information . like any surgical procedure , extraction of third molars under general anesthesia is not without complications either related to surgical intervention or to the type of anesthesia . some complications may be subject to litigation and should be reported to the patient before his / her consent . the aim of this work was to study postoperative complications associated with this type of intervention during four years of practice . the safety and comfort of general anesthesia during this period have been assessed in comparison with different studies in this field . particular attention was given to the collection of all data concerning all complications such as those found in the medical record and visit controls . this retrospective monocentric study gathered all observations of patients undergoing an extraction of at least one third molar under general anesthesia over a period of almost four years ( between january 2008 and december 2011 ) . it involved 2112 patients , their ages ranged between 11 and 79 years , with a median age of 19.5 . inclusion criteria : patient programmed for surgical removal of at least one third molar teeth under general anesthesia;patient must have given his / her informed and signed consent;patient must be insured or beneficiary of a health insurance plan;patient is available for 6 months of follow - up;patient under anesthesiologist supervision . exclusion criteria : patient over 18 years old and under judicial protection , under tutorship , patient ( or legal representative ) refuses to sign the consent , the patient is pregnant , parturient , or breastfeeding;addiction or chronic pain treated with morphine;contraindication for regional anesthesia ( e. g. , congenital or acquired coagulopathy , infection , allergy);contraindication to local anesthetics , general anesthetics and analgesics , difficult cooperation and psychiatric disorders that could interfere with assessments;contraindication for all states of sodium and water retention , especially heart failure , edema , and hydroperitoneum associated with cirrhosis.patients were referred by their treating dentist , orthodontist , or directly by the oral surgeon . in less than 1% of cases , patients were referred by an oncologist in order to eliminate an infection in the distribution showed in table 1 . patient programmed for surgical removal of at least one third molar teeth under general anesthesia ; patient must have given his / her informed and signed consent ; patient must be insured or beneficiary of a health insurance plan ; patient is available for 6 months of follow - up ; patient under anesthesiologist supervision . patient over 18 years old and under judicial protection , under tutorship , patient ( or legal representative ) refuses to sign the consent , the patient is pregnant , parturient , or breastfeeding ; addiction or chronic pain treated with morphine ; contraindication for regional anesthesia ( e. g. , congenital or acquired coagulopathy , infection , allergy ) ; contraindication to local anesthetics , general anesthetics and analgesics , difficult cooperation and psychiatric disorders that could interfere with assessments ; contraindication for all states of sodium and water retention , especially heart failure , edema , and hydroperitoneum associated with cirrhosis . the indications in association with multiple extractions involved 11.5% of cases : patients with extensive dental decay problem but also patients candidate for a head and neck radiotherapy , chemotherapy , or bisphosphonates treatment . combination of third molars to maxillary or mandibular cysts was an indication for intervention under general anesthesia for 2% of patients . finally , in less than 1% of cases , the complaint was part of a pain - dysfunction syndrome of the masticatory system . during preoperative consultation , we proposed and explained to all patients the two possible alternatives : intervention under local or locoregional analgesia and intervention under general anesthesia . risks of oroantral fistulae ( oaf ) and labiomental dysesthesia were clearly exposed to patients with a potential higher risk . all surgeries were carried out with the same surgical team , equipment , and technique . surgical technique was classic : patient in supine position , nasal intubation , packing pharyngeal , no periodontal infiltration , sulcular incision , and flap raising as of the first or second molar with subsequent discharge . bone removal , dental cutting , and separation ( cracking ) were carried out using a single bur in tungsten carbide . sutures required the use of braided polyglactin 910 suture ( vicryl rapide 3/0 . ) . in some cases , an absorbable gelatin sponge ( gelfoam ) was placed in alveolar cavity to stop bleeding in the sites of the upper third molars . exceptionally , a sterile hemostatic gauze ( surgicel ) was used . an intraoperative dose of 2 g of amoxicillin or 600 mg of clindamycin , if allergic to penicillin , was administered as well as a dose of 1 mg / kg of methyl prednisolone . antibiotic treatment was continued postoperatively to all patients for a period of 2 to 6 days depending on the operating surgeon , along with an analgesic ( ibuprofen with 1200 mg dosage , divided in 2 or 3 intakes per day for a period of 3 to 5 days ) . 7659 teeth were extracted of which 3731 maxillary teeth ( 1865 and 1866 right to left ) and 3928 mandibular ( 1962 and 1966 right to left ) exist , an average of 3.6 teeth per patient , ranging from a single tooth to six third molars ! the extraction of one third molar under general anesthesia has always been motivated by the need of another dental procedure such as multiple extractions , frenectomy , cyst excision , or surgical exposure of an impacted canine . the duration of the intervention , from incision to the packing retrieval , was around 33 minutes , with extremes going from a range of 10 to 90 minutes . this period also included actions such as associated additional extractions , exposure of canine , frenectomy , or resection of bone or mucosa lesions . a postoperative consultation was planned for all patients between the tenth and the fifteenth postoperative days . out of the 2112 patients examined , 254 patients presented a complication with a minimum follow - up of 6 months ( 12% ) table 1 . postoperative abnormalities information was given to patient for normal and abnormal situations in table 4 . complaints of pain or swelling were identified during the postoperative consultation or an emergency one . pain motivated a consultation for 28 patients ( 1.3% ) without the conclusion of an infection during examination . 38 patients , or 1.8% of cases , showed sensory disturbances of the labiomental area which was transient in all cases . the hemilateral section of the mandibular tooth concerned 20 of these patients with a rate of 52.6% , while in the other 18 cases ( 47.4% ) , there was no dental section . the degree of recovery was judged satisfactory by the patient , between the second and third postoperative months . sensory disturbances of the labiomental area was detected in 9 patients ( 0.4% of all patients ) . in all cases , partial regression was experienced by the patient after 3 months in 6 cases , while in three other cases , it did not seem to improve during the same period . these 9 patients did not attend further consultations ; therefore no other information is available on the recovery process . among other complications , we detected hemorrhage occurring between the first and the eighth days in the case of 6 patients . this required a revision under local analgesia including cleaning of the site and gelfoam or surgicel placement followed by tight suturing . dysfunction of the masticatory system was observed in the case of 19 patients ( 0.9% ) : in 5 cases it concerned a limitation of mouth opening with musculoarticular pain evident in immediate postoperative period and resolved after 3 to 8 weeks . in the case of 14 patients , a pain - dysfunction syndrome occurred during the months following the operation ( between 3 months and 3 years ) . delayed healing after closure of an oroantral fistula ( oaf ) was seen in the case of two patients . spontaneous closure was observed in 3 months in the case of the first patient . the second patient , operated for a maxillary cyst adjacent to third molars , sustained maxillary sinusitis in the postoperative check - up . finally , the accidental mobilization of a sound second molar complicated the gesture in a single case . a following two - month control visit confirmed a grade 1 mobility ( less than 1 mm ) . 95.8% of patients had a one - day hospitalization , and only 4.2% were kept for one night because of their medical history or postoperative state of health . we had to readmit two patients to the hospital : the first one for nausea and vomiting that occurred two days after the surgery and the second one for bleeding that started eight days after surgery without detection of any hematological cause . the cessation of any professional or scholar activities was for an average of a week . infectious complications were the most common outcome of the surgery , reaching 7.15% of patients in this study despite a routine prescription of antibiotics . the reported rate is probably overestimated , since we considered as an infection all persistent or secondary swelling , mucosal inflammation , loosening of sutures , or pain implicating a second antibiotic prescription . the anglo - saxon term inflammatory complications would certainly have been more appropriate . this rate of 7.15% is close to the 8% shown in a french study conducted under the same conditions . according to trost and coworkers infectious complications vary between 2% and 13% in the literature [ 9 , 10 ] , which places our study results in the range of average . antibiotic prophylaxis / therapy for the extraction of third molars is a regular subject of controversy , although double - blinded randomized trials have concluded an important value to the use of it including amoxicillin , amoxicillin / clavulanic acid , or clindamycin [ 1113 ] . the situation is much more delicate when it comes to lingual nerve or inferior alveolar nerve damages . the hemilateral tooth section procedure was not implicated in these cases . a sensory labiomental dysesthesia ( anesthesia , hypoesthesia , or paresthesia ) was reported in 0.4% of cases , always in relation with the hemilateral tooth section . we do not have sufficient data on their long - term evolution , even if partial regression was observed within 3 months after surgery in 6 of the 9 cases . trost and coworkers reported a rate of 1% for transient lingual hypoesthesia and 2% for labiomental hypoesthesia in a study including 180 patients . blondeau and daniel , in a prospective study with a decline of 24 months concerning 550 mandibular molars , reported 1.1% of sensory disorders related to a lesion of the inferior alveolar nerve and did not report any case , even transient , of lingual hypoesthesia . jerjes and coworkers , in a prospective study including 3236 patients , related a sensory disturbance incidence to a lingual nerve damage ( 1.8% ) and mandibular nerve damage ( 1.5% ) a month after the intervention . permanent deficits were estimated at 1.1% to the lingual nerve and 0.6% to the inferior alveolar nerve . the sensitive - sensory deficit is associated with a lingual nerve injury causing more discomfort to the patient and would result in the most frequent medico legal litigation . damage of lingual nerve may inexplicably appear despite a well - controlled technique because of the multiple anatomical variations . therefore , a question surfaces : a random , rare , and unpredictable outcome should be a warning before surgery ? boffano and coworkers disagree with this idea , given the fact of the rarity of this complication and the additional amount of anxiety inflicted on the patients . asked to comment on the legal aspect of iatrogenic disorders , lawton , a legal specialist in the uk , suggested that the most important legal problem facing the majority of practitioners is not negligence but obtaining the informed consent for the treatment . this consent is mandatory as soon as the risk of developing a serious complication or discomfort is considered reasonable . according to lawton , the risk is real if it can affect one in ten patients , probably even if it is a patient in a hundred , but then chances decrease to get distant from reality and approach possibility , but the law is not preoccupied by the possibilities . cited by boffano and coworkers , the american consensus conference on the extraction of third molars under the auspices of the us national institutes of health in 1979 recommended informing the patient of any potential risk higher or equal to 5% if transient and superior to 0.5% if permanent . in the case of our patients , we always try to explain during the preoperative consultation the risks of labiomental sensory disorders and oaf whenever these risks are potentially present . but we judge it as unnecessary , in the light of our experience , to inform our patients on the risk of a lingual nerve disorder because of its rarity , the inability to predict it , and its transient nature in the majority of the cases . we stipulate it though in the postoperative information sheet as a possible postoperative complication . we are not fearing a rejection of the surgery by the patient , but trying to avoid additional stress to often anxious patients . the extraction of third molars under general anesthesia is far from being qualified as an outdated operation . the majority of patients consulting our practice express a request for general anesthesia because of fear or dental phobia . many authors have studied this issue , and it would appear that anxiety concerning dental care is observed worldwide , with variations according to age , sex , and experience [ 1719 ] . this type of anesthesia provides comfort for both patients and practitioners without necessarily increasing the risk , when it is performed in good conditions . the collaboration with the anesthesiologist in the operating room during the intervention ( legal obligation in belgium ) and the standardization of surgical procedure considerably save time . the use of a single tungsten carbide bur to expose and separate the teeth avoids wasting time and therefore a longer exposure to anesthetics . over 95% of our patients were admitted to a one - day care hospital , which significantly helped reducing the cost of care provided in belgium mainly by the national institute of health and disability insurance . the economic cost , taking , for example , the extraction of four third molars under general anesthesia , would be around 1000 euros per patient , including hospitalization and all services related to medical care . on top of that , a social cost occurs since the cessation of professional and scholar activities is around a week . among our 2112 patients , we had to readmit two patients : the first one for nausea and vomiting that occurred two days after surgery and the second one for bleeding starting 8 days following surgery . both incidents compared to the number of patients in our study are an acceptable risk ( < 1/1000 ) ; especially given the benign nature of the complications imposing readmission . safety rules induced prolonged hospitalization when necessary ( unsatisfactory health state , impossibility of returning to the emergency unit in case of serious trouble , and absence of an accompanying person ) . the patients were cleared out by the end of the day after the visit of the surgeon , the anesthesiologist , and the explanation of the different complications that may occur during the recovery period . another matter is the hypersensitivity of the seven ( 37,47 ) ; it seemed in any case a complaint was sent to us as surgeon in this direction . perhaps the complaint was found among dentists . however , some patients had caries ( 37,47 ) like other teeth before avulsion dds , and that required support from their dentist . in this study , we consulted regarded complications after avulsion of dds . no devitalisation has been done , and this entity pains of 7 is not really highlighted . with the ever - growing need for oral health , in orthodontics and aesthetics on one hand , and the public demand for suppression of pain or even discomfort on the other , it is reasonable to assume that surgical extraction of third molars under general anesthesia is an interesting intervention that retains its place in the practice of oral and maxillofacial surgery despite many controversies that this operation rises . this procedure should be framed by clear information , listing all the possible options including surgical abstention helping therefore the patient in making a decision . the gesture is not without complications ; the majorly serious ones are fortunately rare and not dependent on the type of anesthesia , although there are theoretical risks during general anesthesia as well as any local or locoregional anesthesia .

introduction . the aim of this study was to assess the complications resulting from third molar extraction under general anesthesia . material and methods . the retrospective study included all patients who underwent impacted third molars extraction from january 2008 until december 2011 . 7659 third molars were extracted for 2112 patients . postoperative complications were retrieved from medical files . results . no complications were related to general anesthesia . the most frequent postoperative complication was infection ( 7.15% ) . lingual nerve injuries affected 1.8% of the patients . all of them were transient and were not related to tooth section . inferior alveolar nerve injuries were reported in 0.4% of the cases . 95.8% of these patients were admitted for one - day ambulatory care , and only two patients were readmitted after discharge from hospital . discussion . this surgical technique offers comfort for both surgeons and patients . risks are only linked to the surgical procedure as we observed no complication resulting from general anesthesia . one - day hospitalization offers a good balance between comfort , security , and cost . the incidence of complications is in agreement with the literature data , especially regarding pain , edema , and infectious and nervous complications . it is of utmost importance to discuss indications with patients , and to provide them with clear information .

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

the mobile diabetes intervention study was a cluster - randomized clinical trial conducted in primary care practices in four distinct maryland areas . eligible practices included groups of at least three physicians without academic affiliation who provided diabetes care to at least 10% of their patients and were identified from a list of primary care practices in the study geographic areas . data were obtained by abstraction from patients medical charts and primary collection . as shown in fig . 1 , 26 primary care practices were randomized to one of four study groups using a stepped intervention design for groups : group 1 : control usual care ( uc ) , group 2 : coach - only ( co ) , group 3 : coach pcp portal ( cpp ) , and group 4 : coach pcp portal with decision - support ( cpds ) . a total of 2,602 patients were identified by these practices for screening ; 2,103 were determined ineligible , 145 declined participation , 213 were enrolled , and 163 were included in analyses ( uc , n = 56 ; co , n = 23 ; cpp , n = 22 ; and cpds , n = 62 ) . we aimed to identify patients treated in community primary care settings who would benefit from an intensive diabetes intervention . our institutional review board asked us to repeat consent procedures to assure we obtained proper signatures from all parties . we were unable to contact patients not reconsented ; they did not significantly differ ( p > 0.10 ) at baseline from included patients in age , sex , or baseline glycated hemoglobin . participant data were analyzed according to physician practices original randomization treatment assignment ( intention - to - treat analyses ) . patients eligible for recruitment to the study met all inclusion criteria : physician diagnosis of type 2 diabetes for 6 months;glycated hemoglobin 7.5% within 3 months;age 1864 years . physician diagnosis of type 2 diabetes for 6 months ; glycated hemoglobin 7.5% within 3 months ; patients were excluded for any of the following : medicare or medicaid beneficiaries;uninsured;insulin pump users;not currently managed by study physicians;pregnant;active substance , alcohol , or drug abuser ( sober < 1 year);psychotic or schizophrenic under active care;severe hearing or visual impairment ; orno internet or e - mail access . medicare or medicaid beneficiaries ; not currently managed by study physicians ; active substance , alcohol , or drug abuser ( sober < 1 year ) ; psychotic or schizophrenic under active care ; severe hearing or visual impairment ; or no internet or e - mail access . the most common reasons for ineligibility were as follows : glycated hemoglobin < 7.5% ( 72% ) ; patient s diabetes not currently managed by study physician ( 8% ) ; not between the ages of 18 and 64 years ( 5% ) ; uninsured or insured by medicaid or medicare ( 3% ) ; not type 2 diabetes ( 2% ) ; no internet or e - mail access ( 2% ) ; specified medical exclusion ( 2% ) ; and psychiatric exclusion ( 1% ) . patients were excluded if insured by medicare or medicaid or were uninsured because coverage of primary care services differs from patients commercially insured . these patients would be excluded from planned secondary analyses of claims data provided by a commercial insurer . patients on insulin pumps , pregnant , or not meeting other clinical criteria were excluded because their type 2 diabetes required different clinical management . of the patients identified as eligible , 42% were enrolled ( 213 ) and 77% of those enrolled completed the study and were included in the analyses . the intervention was a patient - coaching system and provider clinical decision support ( 13 ) . the patient - coaching system included a mobile diabetes management software application and a web portal . the mobile software allowed patients to enter diabetes self - care data ( blood glucose values , carbohydrate intake , medications , other diabetes management information ) on a mobile phone and receive automated , real - time educational , behavioral , and motivational messaging specific to the entered data . the patient web portal augmented the mobile software application and consisted of a secure messaging center ( for patient - provider communication ) , personal health record with additional diabetes information ( e.g. , laboratory values , eye examinations , foot screenings ) , learning library , and logbook to review historical data . the provider portal had different views of patient data on the basis of study group assignment . the data - only view ( group 3 , cpp ) allowed providers to access unanalyzed patient data . group 4 ( cpds ) providers had access to analyzed patient data linked to standards of care and evidence - based guidelines . patients received a one touch ultra 2 ( lifescan , milpitas , ca ) glucose meter and supplies . patients in the three active treatment groups received identical study materials : mobile phones , 1-year unlimited data and service plan , study mobile diabetes management software , and access to the web - based patient portal . the mobile diabetes management software incorporated over 1,000 automated self - management messages into a feedback algorithm . the algorithm displayed educational and motivational messages to patients after patients self - reported data into the mobile phone application ( supplementary fig . patients in all three treatment groups were allowed to make telephone calls to educators but were encouraged to communicate electronically . on average , < 50% of active patients made or received live phone calls , with an average of one phone call per month . lastly , patients received an electronic action plan every 2.5 months to support improved diabetes self - management and to serve as previsit summaries for physician office visits . providers were not informed of the level of communication to patients but knew whether patients were assigned to an intervention or to the uc group . all providers received the most recent american diabetes association guidelines for diabetes care and were notified when patients enrolled in the study ( 7,8 ) . active treatment providers were informed that their patients received a mobile and web - based patient coaching system . providers in the co group received data from their patients if patients chose to share it . providers in the cpp and cpds groups were trained on accessing the provider internet portal on office compatible computers ( pcs ) , allowing visual access to patients unanalyzed data . providers in the cpds group were trained on accessing the provider internet portal to view patient data on office pcs and also received quarterly reports ( more often if needed ) that summarized patients glycemic and metabolic control , adherence to medication , self - management skills , and relevant evidence - based guidelines . enrolled providers were reimbursed modestly for research effort ( $ 250 per patient enrolled ) . the primary outcome of the study was change in glycated hemoglobin ( % ) comparing uc and maximal treatment ( cpds ) at baseline versus 12 months . medical chart reviews were used to ascertain patient data . for patients without a glycated hemoglobin within 4 months of the desired measurement , a glycated hemoglobin test was offered at no charge at baseline to determine eligibility and at 12 months . at baseline , glycated hemoglobin was measured using one device , the bayer dca 2000 , by trained staff blinded to patient group assignment . at follow - up , if glycated hemoglobin was not ascertained within 14 days of the 12-month time point , reminders were provided to patients and physicians to complete the test . glycated hemoglobin level at intermediate time points ( 3 , 6 , and 9 months ) was collected from patients medical charts . the patient health questionnaire-9 ( phq ) was administered at baseline and at follow - up interviews to assess depressive symptoms ( 15 ) . we used the 9-item version of the self - completion patient outcome instrument to assess patient - reported symptoms associated with diabetes ( 16,17 ) and the 17-item diabetes distress scale ( 18,19 ) . clinical measurement related to diabetes complications ( blood pressure , lipid levels ) hypoglycemic events , hospitalization , and emergency room visits were ascertained through quarterly telephone calls to patients . vital status was ascertained through review of physician charts if we could not contact patients . study data for primary and secondary outcomes were collected by research staff separately from data transmitted through the device . a detailed description of the study design and rationale for primary and secondary outcomes has been reported previously ( 13 ) . the university of maryland baltimore institutional review board approved the study , and a data and safety monitoring board ( dsmb ) was appointed to review study procedures and adverse advents . practices were assigned to treatment groups according to a 1.5:1:1:1.5 ( group 1 , uc : group 2 , co : group 3 , cpp : group 4 , cpds ) ratio using a computer - generated list of random numbers . the ratios were higher in groups 1 and 4 for analyses of the main hypotheses . sample size was determined on the basis of the primary outcome , change in glycated hemoglobin . the comparison of uc , which included 56 patients from nine practices , to cpds , which included 62 patients from seven practices , had 80% power to detect a difference in mean glycated hemoglobin changes of 0.65 sd , corresponding to 1.0% if sd was 1.58% , using a two - sided test with 0.05 type i error after accounting for a within - cluster correlation of 0.10 , similar to a previously reported study ( 20,21 ) . comparisons of the uc to co ( 23 patients from four practices ) and cpp ( 22 patients from six practices ) had 80% power to detect a difference in mean outcome changes of 1.1% ( 0.7 sd difference ) to 1.3% ( 0.8 sd difference ) for glycated hemoglobin . linear mixed - effects models were used to compare mean changes in primary and secondary outcomes between uc and each active intervention . secondary analyses jointly compared 3- , 6- , 9- , and 12-month changes between groups . model fixed effects were treatment group indicators , time indicators , and interactions between treatment group and time . two secondary analyses of glycated hemoglobin were performed as follows : one analysis stratified by baseline glycated hemoglobin ( 9.0 vs. < 9.0 ) ; the other ( prespecified analysis ) adjusted for baseline glycated hemoglobin as a covariate . we performed a sensitivity analysis using weighted estimating equations ( wee ) to address any residual bias from missing data ( 22 ) . the mobile diabetes intervention study was a cluster - randomized clinical trial conducted in primary care practices in four distinct maryland areas . eligible practices included groups of at least three physicians without academic affiliation who provided diabetes care to at least 10% of their patients and were identified from a list of primary care practices in the study geographic areas . data were obtained by abstraction from patients medical charts and primary collection . as shown in fig . 1 , 26 primary care practices were randomized to one of four study groups using a stepped intervention design for groups : group 1 : control usual care ( uc ) , group 2 : coach - only ( co ) , group 3 : coach pcp portal ( cpp ) , and group 4 : coach pcp portal with decision - support ( cpds ) . a total of 2,602 patients were identified by these practices for screening ; 2,103 were determined ineligible , 145 declined participation , 213 were enrolled , and 163 were included in analyses ( uc , n = 56 ; co , n = 23 ; cpp , n = 22 ; and cpds , n = 62 ) . we aimed to identify patients treated in community primary care settings who would benefit from an intensive diabetes intervention . our institutional review board asked us to repeat consent procedures to assure we obtained proper signatures from all parties . we were unable to contact patients not reconsented ; they did not significantly differ ( p > 0.10 ) at baseline from included patients in age , sex , or baseline glycated hemoglobin . participant data were analyzed according to physician practices original randomization treatment assignment ( intention - to - treat analyses ) . patients eligible for recruitment to the study met all inclusion criteria : physician diagnosis of type 2 diabetes for 6 months;glycated hemoglobin 7.5% within 3 months;age 1864 years . physician diagnosis of type 2 diabetes for 6 months ; glycated hemoglobin 7.5% within 3 months ; patients were excluded for any of the following : medicare or medicaid beneficiaries;uninsured;insulin pump users;not currently managed by study physicians;pregnant;active substance , alcohol , or drug abuser ( sober < 1 year);psychotic or schizophrenic under active care;severe hearing or visual impairment ; orno internet or e - mail access . medicare or medicaid beneficiaries ; not currently managed by study physicians ; active substance , alcohol , or drug abuser ( sober < 1 year ) ; psychotic or schizophrenic under active care ; severe hearing or visual impairment ; or no internet or e - mail access . the most common reasons for ineligibility were as follows : glycated hemoglobin < 7.5% ( 72% ) ; patient s diabetes not currently managed by study physician ( 8% ) ; not between the ages of 18 and 64 years ( 5% ) ; uninsured or insured by medicaid or medicare ( 3% ) ; not type 2 diabetes ( 2% ) ; no internet or e - mail access ( 2% ) ; specified medical exclusion ( 2% ) ; and psychiatric exclusion ( 1% ) . patients were excluded if insured by medicare or medicaid or were uninsured because coverage of primary care services differs from patients commercially insured . these patients would be excluded from planned secondary analyses of claims data provided by a commercial insurer . patients on insulin pumps , pregnant , or not meeting other clinical criteria were excluded because their type 2 diabetes required different clinical management . of the patients identified as eligible , 42% were enrolled ( 213 ) and 77% of those enrolled completed the study and were included in the analyses . the intervention was a patient - coaching system and provider clinical decision support ( 13 ) . the patient - coaching system included a mobile diabetes management software application and a web portal . the mobile software allowed patients to enter diabetes self - care data ( blood glucose values , carbohydrate intake , medications , other diabetes management information ) on a mobile phone and receive automated , real - time educational , behavioral , and motivational messaging specific to the entered data . the patient web portal augmented the mobile software application and consisted of a secure messaging center ( for patient - provider communication ) , personal health record with additional diabetes information ( e.g. , laboratory values , eye examinations , foot screenings ) , learning library , and logbook to review historical data . the provider portal had different views of patient data on the basis of study group assignment . the data - only view ( group 3 , cpp ) allowed providers to access unanalyzed patient data . group 4 ( cpds ) providers had access to analyzed patient data linked to standards of care and evidence - based guidelines . patients received a one touch ultra 2 ( lifescan , milpitas , ca ) glucose meter and supplies . patients in the three active treatment groups received identical study materials : mobile phones , 1-year unlimited data and service plan , study mobile diabetes management software , and access to the web - based patient portal . the mobile diabetes management software incorporated over 1,000 automated self - management messages into a feedback algorithm . the algorithm displayed educational and motivational messages to patients after patients self - reported data into the mobile phone application ( supplementary fig . patients in all three treatment groups were allowed to make telephone calls to educators but were encouraged to communicate electronically . on average , < 50% of active patients made or received live phone calls , with an average of one phone call per month . lastly , patients received an electronic action plan every 2.5 months to support improved diabetes self - management and to serve as previsit summaries for physician office visits . providers were not informed of the level of communication to patients but knew whether patients were assigned to an intervention or to the uc group . all providers received the most recent american diabetes association guidelines for diabetes care and were notified when patients enrolled in the study ( 7,8 ) . active treatment providers were informed that their patients received a mobile and web - based patient coaching system . providers in the co group received data from their patients if patients chose to share it . providers in the cpp and cpds groups were trained on accessing the provider internet portal on office compatible computers ( pcs ) , allowing visual access to patients unanalyzed data . providers in the cpds group were trained on accessing the provider internet portal to view patient data on office pcs and also received quarterly reports ( more often if needed ) that summarized patients glycemic and metabolic control , adherence to medication , self - management skills , and relevant evidence - based guidelines . enrolled providers were reimbursed modestly for research effort ( $ 250 per patient enrolled ) . the primary outcome of the study was change in glycated hemoglobin ( % ) comparing uc and maximal treatment ( cpds ) at baseline versus 12 months . medical chart reviews were used to ascertain patient data . for patients without a glycated hemoglobin within 4 months of the desired measurement , a glycated hemoglobin test was offered at no charge at baseline to determine eligibility and at 12 months . at baseline , glycated hemoglobin was measured using one device , the bayer dca 2000 , by trained staff blinded to patient group assignment . at follow - up , if glycated hemoglobin was not ascertained within 14 days of the 12-month time point , reminders were provided to patients and physicians to complete the test . glycated hemoglobin level at intermediate time points ( 3 , 6 , and 9 months ) was collected from patients medical charts . the patient health questionnaire-9 ( phq ) was administered at baseline and at follow - up interviews to assess depressive symptoms ( 15 ) . we used the 9-item version of the self - completion patient outcome instrument to assess patient - reported symptoms associated with diabetes ( 16,17 ) and the 17-item diabetes distress scale ( 18,19 ) . clinical measurement related to diabetes complications ( blood pressure , lipid levels ) hypoglycemic events , hospitalization , and emergency room visits were ascertained through quarterly telephone calls to patients . vital status was ascertained through review of physician charts if we could not contact patients . study data for primary and secondary outcomes were collected by research staff separately from data transmitted through the device . a detailed description of the study design and rationale for primary and secondary outcomes has been reported previously ( 13 ) . the university of maryland baltimore institutional review board approved the study , and a data and safety monitoring board ( dsmb ) was appointed to review study procedures and adverse advents . practices were assigned to treatment groups according to a 1.5:1:1:1.5 ( group 1 , uc : group 2 , co : group 3 , cpp : group 4 , cpds ) ratio using a computer - generated list of random numbers . the ratios were higher in groups 1 and 4 for analyses of the main hypotheses . sample size was determined on the basis of the primary outcome , change in glycated hemoglobin . the comparison of uc , which included 56 patients from nine practices , to cpds , which included 62 patients from seven practices , had 80% power to detect a difference in mean glycated hemoglobin changes of 0.65 sd , corresponding to 1.0% if sd was 1.58% , using a two - sided test with 0.05 type i error after accounting for a within - cluster correlation of 0.10 , similar to a previously reported study ( 20,21 ) . comparisons of the uc to co ( 23 patients from four practices ) and cpp ( 22 patients from six practices ) had 80% power to detect a difference in mean outcome changes of 1.1% ( 0.7 sd difference ) to 1.3% ( 0.8 sd difference ) for glycated hemoglobin . linear mixed - effects models were used to compare mean changes in primary and secondary outcomes between uc and each active intervention . secondary analyses jointly compared 3- , 6- , 9- , and 12-month changes between groups . model fixed effects were treatment group indicators , time indicators , and interactions between treatment group and time . two secondary analyses of glycated hemoglobin were performed as follows : one analysis stratified by baseline glycated hemoglobin ( 9.0 vs. < 9.0 ) ; the other ( prespecified analysis ) adjusted for baseline glycated hemoglobin as a covariate . we performed a sensitivity analysis using weighted estimating equations ( wee ) to address any residual bias from missing data ( 22 ) . the 163 study patients had a mean baseline glycated hemoglobin of 9.4% ( range 7.515.5 ) ( table 1 ) . mean age was 52.8 years , 50.3% were female , 39.3% were african american , and 31.3% were college educated . cpds patients had higher baseline glycated hemoglobin than uc ( 9.9 vs. 9.2% , p = 0.04 ) . primary outcome , glycated hemoglobin change over 12 months ; group 4 ( p < 0.001 ) and group 2 ( p = 0.003 ) have significantly larger changes than group 1 . 2 show that the mean 12-month decrease in cpds glycated hemoglobin was 1.2% more than uc ( 95% ci 0.61.8% ; p < 0.001 ) . furthermore , the cpds patients had a significantly greater decrease in mean glycated hemoglobin than the uc patients when compared at all follow - up time points ( p < 0.001 ) both had greater 12-month glycated hemoglobin reductions than uc ( co , p = 0.02 ; cpp , p = 0.45 ) . co and cpp were similar to cpds over all follow - up time points ( p > 0.05 for both comparisons ) . in a stratified analysis , a greater decline was found with cpds than uc for the stratum with baseline glycated hemoglobin < 9.0% ( difference in decrease 0.7% , 95% ci 0.21.1 , p = 0.003 ) and the stratum with baseline glycated hemoglobin at least 9.0% ( difference in decrease 1.3% , 0.22.4 , p = 0.01 ) ( shown in fig . the test of interaction was not significant ( p = 0.49 ) for baseline glycated hemoglobin stratum and treatment group over time . we obtained the same conclusion whether or not we analyzed the baseline to 12-month changes with intermediate glycated hemoglobin measures . glycated hemoglobin results were unchanged after adjusting for baseline glycated hemoglobin and after performing the wee analysis . although there were mean declines across all groups in lipid values and blood pressure readings , diabetes distress , diabetes symptoms , and phq-9 depression , none of the 12-month changes comparing the uc to any of the active interventions were significantly different ( p > 0.05 ) . hypoglycemic events , hospitalizations , and emergency - room visits were infrequent in all groups . one patient in group 4 ( cpds ) was hospitalized twice for reasons not reported to the study . to our knowledge , this is the first cluster - randomized study of a mobile diabetes - coaching intervention conducted in a community setting over a 1-year treatment period . few previous studies of electronic or mobile communication interventions for diabetes were randomized , included a control group , or covered 1 year ( 13 ) . our study included minority study participants and found clinically meaningful differences and few adverse events , none of which were related to the study or treatment . our study evaluated the intervention for commercially insured patients in primary care settings , where the majority of diabetes care is provided . enrolling and treating study participants according to random assignment of physician practices ( clusters ) reduced the risk of bias in treatment application . we found that a mobile phone based treatment / behavioral coaching intervention improved glycated hemoglobin by 1.9% , compared with 0.7% for uc , a difference of 1.2% this result pertained to people with poorly controlled glycated hemoglobin ( 9.0% ) and people with less severe abnormal initial glycated hemoglobin values ( 7.58.9% ) . first , since cpds and uc had similar mean baseline glycated hemoglobin within strata of baseline a1c ( < 9 vs. 9.0% ) , and the treatment effect is similar in each of the strata , our findings provide evidence of true 12-month treatment differences in glycated hemoglobin , rather than regression to the mean . this stratified analysis is important , showing large changes in a1c by adjusting for baseline a1c . second , the treatment effect in the higher glycated hemoglobin stratum shows this intervention to be suitable to obtain the goals of the more conservative accord approach ( 23 ) . neither accord nor this study collected person - specific data on dietary , physical activity , and pharmacological management adjustments made for individual patients . because of the personalized quality of the mobile phone technology , we expect to be able to make those distinctions in future investigations now that its observed effects on glycated hemoglobin justify their study . third , mobile phone management is efficacious in patients whose glycated hemoglobin levels are clearly above the desired levels as well as patients whose glycated hemoglobin levels are less egregiously elevated . our finding is consistent with the cochrane collaboration review , suggesting the benefit of individual education on glycemic control ( 24 ) . however , we did not see convincing improvements in patient - reported diabetes symptoms , diabetes distress , depression , or other clinical ( e.g. , blood pressure ) or laboratory ( e.g. , lipid ) values . physicians in the community have different experiences with and access to resources , including access to specialists , clinical practice guidelines , and experience or use of electronic communication . we attempted to address these differences by enrolling multiple community physicians to participate in the study and randomization at the practice level . the patient population in the study may also be distinctive because private health care insurance coverage and access to the internet ( either at work or home ) were required . although not all participants provided data at all planned study visits , we addressed missing data in this study in two ways . first , the primary analysis used mixed - effects models , which have the effect of implicitly imputing missing observations ( 25 ) . second , we performed the wee sensitivity analysis that used baseline characteristic data to upweight observations from participants who were most similar to participants with missing data ( 22 ) . as a measure of long - term blood glucose control , change in glycated hemoglobin is an important , commonly used outcome . although low glycated hemoglobin does not imply that diabetes is being well managed , well managed diabetes is characterized by glycated hemoglobin at normal or near - normal levels ( 13 ) . we screened > 2,600 patients ; 72% were ineligible because glycated hemoglobin was lower than eligibility criterion ; many physicians referred patients they thought were not adequately managing their diabetes because of poor control relevant to everyday life , such as blurred vision or pain , self - assessed control of diabetes , or depression ( 13 ) . in this study , we did not observe convincing changes in these indicators . communications as specific for these indicators as ours were for glycated hemoglobin may be able to make a larger difference in future studies . future studies should also consider how mobile communication changes behavior related to blood glucose : medication adherence , treatment intensification , increased physical activity , and number and quality of communications between providers and patients . these may be important mechanisms to explain change in glycated hemoglobin but were not primary or secondary analyses planned for this study . future studies of mobile health should address more specific characterization of patient and provider behaviors that support change in clinical health parameters . mobile phones are ubiquitous more than 2.7 billion people own mobile phones worldwide . in the united states alone , users have increased from 34 million in 1995 to 290 million in 2010 . mobile phone and internet users are increasingly diverse in age and race . the widespread distribution of mobile phones and electronic communication , across socioeconomic , sex , and age - groups , combined with the ability to process and communicate data in real time , make these modalities ideal platforms to create simple , effective , diabetes management programs ( 14 ) . we found mobile phone and web portal communications for diabetes to have a consequential treatment effect when used by patients and their pcps .

objectiveto test whether adding mobile application coaching and patient / provider web portals to community primary care compared with standard diabetes management would reduce glycated hemoglobin levels in patients with type 2 diabetes.research design and methodsa cluster - randomized clinical trial , the mobile diabetes intervention study , randomly assigned 26 primary care practices to one of three stepped treatment groups or a control group ( usual care ) . a total of 163 patients were enrolled and included in analysis . the primary outcome was change in glycated hemoglobin levels over a 1-year treatment period . secondary outcomes were changes in patient - reported diabetes symptoms , diabetes distress , depression , and other clinical ( blood pressure ) and laboratory ( lipid ) values . maximal treatment was a mobile- and web - based self - management patient coaching system and provider decision support . patients received automated , real - time educational and behavioral messaging in response to individually analyzed blood glucose values , diabetes medications , and lifestyle behaviors communicated by mobile phone . providers received quarterly reports summarizing patient s glycemic control , diabetes medication management , lifestyle behaviors , and evidence - based treatment options.resultsthe mean declines in glycated hemoglobin were 1.9% in the maximal treatment group and 0.7% in the usual care group , a difference of 1.2% ( p < 0.001 ) over 12 months . appreciable differences were not observed between groups for patient - reported diabetes distress , depression , diabetes symptoms , or blood pressure and lipid levels ( all p > 0.05).conclusionsthe combination of behavioral mobile coaching with blood glucose data , lifestyle behaviors , and patient self - management data individually analyzed and presented with evidence - based guidelines to providers substantially reduced glycated hemoglobin levels over 1 year .

RESEARCH DESIGN AND METHODS Eligibility and study design Primary outcome Secondary outcomes Study oversight Statistical analysis RESULTS CONCLUSIONS Supplementary Material

cancer detection has always been a major issue for the pathologists and medical practitioners for diagnosis and treatment planning . the manual identification of cancer from microscopic biopsy images is subjective in nature and may vary from expert to expert depending on their expertise and other factors which include lack of specific and accurate quantitative measures to classify the biopsy images as normal or cancerous one . the automated identification of cancerous cells from microscopic biopsy images helps in alleviating the abovementioned issues and provides better results if the biologically interpretable and clinically significant feature based approaches are used for the identification of disease . cancer is one of the common diseases in india which has responsibility to maximum mortality with about 0.3 million deaths per year . the chances of getting affected by this disease are accelerated due to change in habits in the people such as increase in use of tobacco , deterioration of dietary habits , lack of activities , and many more . the possibility of cure from cancer is increased due to recent combined advancement in medicine and engineering . medical professionals use several techniques for detection of cancer . these techniques may include various imaging modalities such as x - ray , computer tomography ( ct ) scan , positron emission tomography ( pet ) , ultrasound , and magnetic resonance imaging ( mri ) and pathological tests such as urine test and blood test . for accurate detection of cancer pathologists use histopathology biopsy images , that is , the examination of microscopic tissue structure of the patient . thus biopsy image analysis is a vital technique for cancer detection [ 2 , 3 ] . histopathology is the study of symptoms and indications of the disease using the microscopic biopsy images . to visualize various parts of the tissue under a microscope , the main goal of staining is to reveal the components at cellular level and counterstains are used to provide color , visibility , and contrast . hematoxylin - eosin ( h&e ) is staining component that has been used by pathologists for over few decades . hematoxylin stains cell nuclei which are blue in color while eosin stains cytoplasm and connective tissues which are of pink color . the histology is related to the study of cells in terms of structure , function , and interpretations of the tissue and cells . microscopic biopsies are most commonly used for both disease screenings because of the less invasive natures . the microscopic biopsy images are easier to analyze specimens compared to histopathology due to absence of noncomplicated structures . the accurate manual identification of cancer from microscopic biopsy images has always been a major issue by the pathologists and medical practitioners observing cell or tissue structure under the microscope . in histopathology , the cancer detection process normally consists of categorizing the image biopsy into cancerous one or noncancerous one . in microscopic biopsy image analysis doctors and pathologists observe many of the abnormalities and categorize the sample based on various characteristics of the cell nuclei such as color , shape , size , and proportion to cytoplasm . for the detection and diagnosis of cancer from microscopic biopsy images , the histopathologists normally look at the specific features in the cells and tissue structures . the various common features used for the detection and diagnosis of cancer from the microscopic biopsy images include shape and size of cells , shape and size of cell nuclei , and distribution of the cells . it has been observed that the overall shape and size of cells in the tissues are mostly normal . the cellular structures of the cancerous cells might be either larger or shorter than normal cells . cancer cells usually do not function in a useful way and their shapes are often not even . ( b ) size and shape of the cell 's nucleus . the shape and size of the nucleus of a cancer cell are often not normal . the image of the cell looks like an omelet , in which the central yolk is the nucleus and the surrounding white is the cytoplasm . the nuclei of cancer cells are larger than the normal cells and deviated from the centre of the mass . the segmentation step mainly focuses on separation of regions of interests ( cells ) from background tissues as well as separation of nuclei from cytoplasm . the function of each tissue depends on the distribution and arrangements of the normal cells . these adjectives of microscopic biopsy images have been included in shape and morphology based features , texture features , color based features , color gray level cooccurrence matrix ( glcm ) , law 's texture energy ( lte ) , tamura 's features , and wavelet features which are more biologically interpretable and clinically significant . the main aim of this paper is to design and develop a framework and a software tool for automated detection and classification of cancer from microscopic biopsy images using the abovementioned clinically significant and biologically interpretable features . this paper focuses on selecting an appropriate method for each design stage of the framework after making a comparative analysis of the various commonly used methods in each category . the various stages involved in the proposed methodology include enhancement of microscopic images , segmentation of background cells , features extraction , and finally the classification . section 2 describes the related works , section 3 presents the methods and models , section 4 describes the results and discussions , and finally section 5 draws the conclusion of the work presented in this paper . in recent years , few works have been reported in the literature for the design and development of tools for automated cancer detection from microscopic biopsy images . kumar and srivastava presented detailed reviews on the computer aided diagnosis ( cad ) for cancer detection from microscopic biopsy images . presented a review on computer aided diagnosis system to detect cancer from microscopic biopsy images using image processing techniques . the quasisupervised learning algorithm operates on two datasets , the first one having normal tissues labeled only indirectly and the second one containing an unlabeled collection of mixed samples of both normal and cancer tissues . this method was applied on the dataset of 22,080 vectors with reduced dimensionality , 119 from 132 . the regions having the cancerous tissues were accurately identified having true positive rate 88% and false positive rate 19% , respectively , by using manual ground truth dataset . used haralick 's textures features , histogram of oriented gradients ( hog ) , and color component based statistical moments ( ccsm ) features selection and extraction approaches to classify the cancerous cells from microscopic biopsy images . the various features used in this paper are contrast , correlation , energy , homogeneity , glcm texture features , rgb , gray level , and hsv . huang and lai presented a methodology for segmentation and classification techniques for histology images based on texture features and by using svm the maximum classification accuracy obtained is 92.8% . presented a method for morphologic characterization of cell neighborhoods in neoplastic and preneoplastic tissue of microscopic biopsy images . in this paper , authors presented watershed transforms to compute the cell and nuclei area and other parameters . the distance measure of the neighborhood value has been used for calculating the neighborhood complexity with reference to the v - cells . the best classification which has been obtained by knn classifier is 83% for dysplastic and neoplastic classes and sinha and ramkrishan extracted some features of microscopic biopsy images which include eccentricity , area ratio , compactness , average values of color components , energy entropy , correlation , and area of cells and nucleus . the classification accuracy obtained by bayesian , k - nearest neighbor , neural networks , and support vector machine was 82.3% , 70.60% , 94.1% , and 94.1% , respectively . extracted the features of microscopic biopsy images including area , perimeter , convex area , solidity , major axis length , orientation filled area , eccentricity , ratio of cell and nucleus area , circularity , and mean intensity of cytoplasm . the knn and neural network classifier are used for classification accuracy 86% and 92% , respectively . in this paper , a framework for automated detection and classification of cancer from microscopic biopsy images using clinically significant and biologically interpretable features is proposed and examined . for segmentation of images the various hybrid features which are extracted from the segmented images include shape and morphological features , glcm texture features , tamura features , law 's texture energy based features , histogram of oriented gradients , wavelet features , and color features . for classification purposes , k - nearest neighbor based method the efficacy of other classifiers such as svm , random forest , and fuzzy k - means is also examined . for testing purposes , 2828 microscopic biopsy images available from histology database it was observed that the proposed method is performing better in comparison to other methods discussed as above . the overall summary and comparison of the proposed method and other methods the detection and classification of cancer from microscopic biopsy images are a challenging task because an image usually contains many clusters and overlapping objects . the various stages involved in the proposed methodology include enhancement of microscopic images , segmentation of background cells , features extraction , and finally the classification . for the enhancement of the microscopic biopsy images , the contrast limited adaptive histogram equalization [ 19 , 20 ] approach is used and for the segmentation of background cells k - means segmentation algorithm is used . in feature extraction phase , various biologically interpretable and clinically significant shape and morphology based features are extracted from the segmented images which include gray level texture features , color based features , color gray level texture features , law 's texture energy ( lte ) based features , tamura 's features , and wavelet features . finally , the k - nearest neighborhood ( knn ) , fuzzy knn , and support vector machine ( svm ) based classifiers are examined for classifying the normal and cancerous biopsy images . these approaches are tested on four fundamental tissues ( connective , epithelial , muscular , and nervous ) of randomly selected 1000 microscopic biopsy images . finally , the performances of the classifiers are evaluated using well known parameters and from results and analysis , it is observed that the fuzzy knn based classifier is performing better for the selected features set . the main purpose of the preprocessing is to remove a specific degradation such as noise reduction and contrast enhancement of region of interests . the biopsy images acquired from microscope may be defective and deficient in some respect such as poor contrast and uneven staining , and they need to be improved through process of image enhancement which increases the contrast between the foreground ( objects of interest ) and background . the contrast limited adaptive histogram equalization ( clahe ) approach is used for enhancement of microscopic biopsy images . several segmentation methods have been adapted for cytoplasm , cell , and nuclei segmentation from microscopic biopsy images like threshold based , region - based , and clustering based algorithms . however the selections of segmentation methods depend on the type of the features to be preserved and extracted . for the segmentation of roi ( region of interest ) , the ground truth ( gt ) of the images is manually cropped and created from histology dataset . the k - means clustering based segmentation algorithms are used because of the preservation of the desired information . from the obtained results through experimentation it is observed that the clustering based algorithms specifically k - means based method are the best suited for microscopic biopsy images . figure 3 shows the original and k - means segmented microscopic biopsy image . for testing and experimentation purpose , twenty ( 20 ) microscopic biopsy images available from histology the visual results of texture based segmentation , fcm segmentation , k - means segmentation , and color based segmentation [ 20 , 2326 ] are presented in figures 3(a)to 3(d ) . thus from the visual results obtained and reported in figures 3(a)to 3(d ) , it is observed that the k - means clustering based segmentation method performs better in most of the cases as compared to other segmentation approaches under consideration for microscopic biopsy image segmentation . finally the roi segmented image of microscopic biopsy is compared to ground truth images for the quantitative evaluation of various segmentation approaches for all 20 sample images from histology dataset . the performance of the various segmentation approaches such as k - means , fuzzy c - means , texture based segmentation , and color based segmentation was evaluated in terms of various popular parameters of segmentation measures . these parameters include accuracy , sensitivity , specificity , false positive rate ( fpr ) , probability random index ( ri ) , global consistency error ( gce ) , and variance of information ( voi ) . the brief description of few of these performance measures used in this paper is as follows . random index between test ( s ) and ground truth ( g ) is estimated by summing the number of pixel pairs with same label and number of pixel pairs having different labels in both s and g and then dividing it by total number of pixel pairs . given a set of ground truth segmentations gk , the pri is estimated using ( 1 ) such that cij is an event that describes a pixel pair ( i , j ) having same or different label in the test image stest(1)pristest , gk=1n/2i , j&i < jcijpij+1cij1pij . the variation of information is a measure of the distance between two clusters ( partitions of elements ) . clustering with clusters is represented by a random variable x , x = { 1 , , k } such that pi = |xi | /n , i x , and n = ixi is the variation of information between two clusters x and y. thus voi(x , y ) is represented using ( 2)voix , y = hx = hy2ix , y , where h(x ) is entropy of x and i(x , y ) is mutual information between x and y. voi(x , y ) measures how much the cluster assignment for an item in clustering x reduces the uncertainty about the item 's cluster in clustering y. ( iii ) global consistency error ( gce ) . the gce is estimated as follows : suppose segments si and gj contain a pixel , say pk , such that s s , g g where s denotes the set of segments that are generated by the segmentation algorithm being evaluated and g denotes the set of reference segments . to begin with , a measure of local refinement error is estimated using ( 3 ) and then it is used to compute local and global consistency errors , where n denotes the set of difference operation and r(x , y ) represents the set of pixels corresponding to region x that includes pixel y. using ( 3 ) the global consistency error ( gce ) is computed using ( 4 ) where n denotes the total number of pixels of the image . gce quantify the amount of error in segmentation ( 0 signifies no error and 1 indicates no agreement):(3)esi , gj , pk = rsi , pkrgj , pkrsi , pk,(4)gces , g=1nminies , g , pi,ies , g , pi . table 2 and figure 4 show the comparison of various segmentation algorithms on the basis of average accuracy , sensitivity , specificity , fpr , pri , gce , and voi for 20 sample images taken from histology dataset . from table 2 and figure 4 , it is observed that k - means , color k - means , fuzzy c - means , and texture based methods are performing better at par in terms of accuracy , specificity , and pri segmentation measures but except for k - means based segmentation methods other methods are not performing better in terms of other important parameters . only the k - means based segmentation algorithm is associated with larger value of accuracy , sensitivity , specificity , and random index ( ri ) and smaller value of fpr , gce , and voi in comparison to other methods and hence it is better in comparison to others . hence , k - means based segmentation is the only method which performs better in terms of all parameters and that is why it is chosen as the segmentation method in the proposed framework for cancer detection from microscopic biopsy images . after segmentation of image features are extracted from the regions of interest to detect and grade potential cancers . the features are extracted at tissue level and cell level of microscopic biopsy images for better predictions . to better capture the shape information , we use both region - based and contour - based methods to extract anticircularity , area irregularity , and contour irregularity of nuclei as the three shape features to reflect the irregularity of nuclei in biopsy images . the cellular level feature focuses on quantifying the properties of individual cells without considering spatial dependency between them . in biopsy images for a single cell , the shape and morphological , textural , histogram of oriented gradients and wavelet features are extracted . the tissue level features quantify the distribution of the cells across the tissue ; for that , it primarily makes use of either the spatial dependency of the cells or the gray level dependency of the pixels . based on these characteristics , some important shape and morphological based features are explained as follows . the nucleus area can be represented by nucleus region containing total number of pixels ; it is shown in ( 5)a=i=1n j=1mbi , j , where a is nucleus area and b is segmented image of i rows and j columns . the average value of intensity of the pixels belonging to the nucleus region is known as nucleus brightness . the largest circle 's diameter circumscribing the nucleus region is known as nucleus longest diameter ; it is shown in ( 6)nld = x1x22+y1y22,where x1 , y1 and x2 , y2 are end points on major axis . it is represented in ( 7)nsd = x2x12+y2y12,where x1 , y1 and x2 , y2 are end points on minor axis . this is represented by the ratio of the shortest diameter to the longest diameter of the nucleus region , shown in ( 8)nucleus elongation = nldperimeter . the length of the perimeter of the nucleus region is represented using ( 9)p = even count+2odd count unit . the ratio of the nucleus area to the area of the circle corresponding to the nucleus longest diameter is known as nucleus compactness , shown in ( 10)=ap=4areap2 . solidity is ratio of actual cell / nucleus area to convex hull area shown in ( 11)solidity = areaconvex area . the ratio of major axis length and minor axis length is known as eccentricity and defined in ( 12)eccentricity = length of mejor axislength of minor axis . there are seven sets of features used for computing the feature vector of microscopic biopsy images explained as follows . [ 3234 ] autocorrelation , contrast , correlation , cluster prominence , cluster shade , difference variance , dissimilarity , energy , entropy , homogeneity , maximum probability , sum of squares , sum average , sum variance , sum entropy , difference entropy , information measure of correlation 1 , information measure of correlation 2 , inverse difference ( inv ) , inverse difference normalized ( inn ) , and inverse difference moment normalized are major texture features which can be calculated using equations of the texture features . ( ii ) morphology and shape feature ( f23f32 ) . in papers [ 35 , the considered shape and morphological features in this paper are area , perimeter , major axis length , minor axis length , equivalent diameter , orientation , convex area , filled area , solidity , and eccentricity . histogram of oriented gradient is one of the good features set to deify the objects . in our observation it will be included for better and accurate identification of objects present in microscopic biopsy images . the wavelets are useful in multiresolution analysis of microscopic biopsy images because they are fast and give better compression as compared to other transforms . the fourier transform converts a signal into a continuous series of sine waves , but the wavelet precedes it in both time and frequency . daubechies wavelets have been used because they have fractal structures and they are useful in the case of microscopic biopsy images . in this paper mean , entropy , energy , contrast homogeneity , and sum of wavelet coefficients are taken into consideration . the components of these models are hue , saturation , and value ( hsv ) . this is represented by the six sided pyramids , the vertical axis behaves as brightness , the horizontal distance from the axis represents the saturation , and the angle represents the hue . here mean and standard deviation of hsv components are taken as features . tamura 's features are computed on the basis of three fundamental texture features : contrast , coarseness , and directionality . coarseness is the measure of granularity of an image ; thus coarseness can be represented using average size of regions that have the same intensity . these features are texture description features which mainly used average gray level , edges , spots , ripples , and wave to generate vectors of the masks . law 's mask is represented by the features of an image without using frequency domain . laws significantly determined that several masks of appropriate sizes were very instructive for discriminating between different kinds of texture features present in the microscopic biopsy images . thus its classified samples are based on expected values of variance - like square measures of these convolutions , called texture energy measures . the lte mask method is based on texture energy transforms applied to the image classification used to estimate the energy within the pass region of filters . table 3 provides the distribution of name of the feature type and the number of features selected for the classification of microscopic biopsy images . the classification of microscopic biopsy images is the most challenging task for automatic detection of cancer from microscopic biopsy images . classification might provide the answer whether microscopic biopsy is benign or malignant . for classification purposes , some commonly used classification methods are artificial neural networks ( ann ) , bayesian classification , k - nearest neighbor classifiers , support vector machine ( svm ) , and random forest ( rf ) . supervised machine learning approaches first step is to prepare the data ( feature set ) , the second step is to choose an appropriate algorithm , the third step is to fit a model , the fourth step is to train the fitted model , and then the final step is to use fitted model for prediction . the k - nearest neighborhood ( knn ) , fuzzy knn and support vector machine ( svm ) , and random forest classifiers are used for classifying the normal and cancerous biopsy images . the proposed methodologies were implemented with matlab 2013b , on dataset of digitized at 5x magnification on pc with 3.4 ghz intel core i7 processor , 2 gb ram , and windows 7 platform . for the testing and experimentation purposes , a total of 2828 histology images from the histology image dataset ( histologyds2828 ) and annotations the image distributions based on the fundamental tissue structures in the histology dataset include connective-484 , epithelial-804 , muscular-514 , and nervous-1026 microscopic biopsy images with magnifications 2.5x , 5x , 10x , 20x , and 40x . although the method is magnification independent , in this work the results are provided on samples digitized at 5x magnification . the features extracted from microscopic biopsy images must be biologically interpretable and clinically significant for better diagnosis of cancer . table 4 provides the brief description of dataset used for identification of cancer from microscopic biopsy images . the proposed methodology for detection and diagnosis of cancer detection from microscopic biopsy images consists of the stages of images enhancement , segmentation , feature extraction , and classification . the contrast limited adaptive histogram equalization ( clahe ) is used for enhancement of microscopic biopsy images , because it has ability to better highlight the regions of interests in the images as tested through experimentation . to better preserve the desired information in microscopic biopsy images during segmentation process , the various clustering and texture based segmentation approaches were examined . for microscopic biopsy images it is required to discover as much as possible the nuclei information in order to make reliable and accurate detection and diagnosis based on cells and nuclei parameters . from results and analysis presented in section 4 , k - means segmentation algorithm was used for segmenting the microscopic biopsy images as it performs better in comparison to other methods . during segmentation process of k - means clustering method , the number of clusters k was set to k = 3 . further , to find the center of the clusters , squared euclidean distance measures are used as similarity measures . in feature extraction phase , various biologically interpretable and clinically significant shape and morphology based features were extracted from the segmented images which include gray level texture features ( f1f22 ) , shape and morphology based features ( f23f32 ) , histogram of oriented gradients ( f33f68 ) , wavelet features ( f69f100 ) , color based features ( f101f106 ) , tamura 's features ( f107f119 ) , and law 's texture energy ( f110f115 ) based features . finally a 2d matrix of 2828 115 feature matrix was formed using all the feature sets , where 2828 are the number of microscopic images in the dataset and 115 are the total number of features extracted . randomly selected 1000 data / samples were used for testing various classification algorithms . the 10-fold cross validation approach was used to partition the data in training and testing sets . thus 900 data / samples were used for training purposes and 100 data / samples were used for testing purposes . the k - nearest neighbor ( knn ) is a simple classifier in which a feature vector is assigned . for knn classification the numbers of nearest neighbor ( k ) were set to 5 , and euclidean distance matrix and the nearest rule to decide how to classify the sample were used . the proposed method was also tested by using support vector machine ( svm ) based classifier for linear kernel function with 10-fold cross validation methods . in svm classification model , the kernel 's parameters and soft margin parameter c play vital role in classification process ; the best combination of c and was selected by a grid search with exponentially growing sequences of c and . each combination of parameter choices was checked using cross validations ( 10-fold ) , and the parameters with best cross validation accuracy were selected . for svm 's linear kernel function , quadratic programming ( qp ) optimization parameter was used to find separating hyperplane . in the case of random the performance of classifiers was calculated using confusion matrix of size 2 2 matrix and the value of tp , tn , fp , and fn was calculated . the performance parameters accuracy , sensitivity , and specificity were calculated using ( 14)(19 ) . the classification accuracy of a technique depends upon the number of correctly classified samples ( i.e. , true negative and true positive ) and is calculated as follows:(14)accuracy = tp+tnn100,where n is the total number of samples present in the microscopic biopsy images it can be calculated using ( 15)sensitivity = tptp+fn , where the value of sensitivity ranges between 0 and 1 , where 0 and 1 , respectively , mean worst and best classification . specificity . the value of sensitivity is calculated using ( 16)specificity = tntn+fp.its value ranges between 0 and 1 , where 0 and 1 , respectively , mean worst and best classification the geometric mean of sensitivity and specificity is considered as balance classification rate [ 43 , 44 ] . it is defined by using ( 18)precision = tptp+fp , recall = tptp+fn , f - measure=2precisionrecallprecision+recall.the value of f - measure ranges between 0 and 1 , where 0 means the worst classification and 1 means the best classification . it can be calculated using the following formula : ( 19)mcc = tptnfpfntp+fntp+fptn+fntn+fp.its value ranges between 1 and + 1 , where 1 , + 1 , and 0 , respectively , correspond to worst , best , at random prediction . table 5 shows classification results of the proposed framework for four different tissues of microscopic biopsy images containing cancer and noncancer cases tested using four popular classifiers like k - nearest neighbor , svm , fuzzy knn , and random forest . from table 5 and figure 5(a ) the following observations are made for sample test cases containing connective tissues.(i)for the identification of cancer from biopsy images of connective tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.921909 , 0.940164 , 0.819922 , 0.880263 , 0.759395 , and 0.717455 , respectively.(ii)for the identification of cancer from biopsy of connective tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.89245 , 0.888438 , 0.948297 , 0.918756 , 0.538314 , and 0.55879 , respectively.(iii)for the identification of cancer from biopsy of connective tissues in the case of fuzzy knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.787879 , 0.867476 , 0.370074 , 0.618789 , 0.356613 , and 0.231013 , respectively.(iv)for the identification of cancer from biopsy of connective tissues , in the case of random forest classifier , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.907245 , 0.993668 , 0.493996 , 0.743832 , 0.647373 , and 0.642137 , respectively . for the identification of cancer from biopsy images of connective tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.921909 , 0.940164 , 0.819922 , 0.880263 , 0.759395 , and 0.717455 , respectively . for the identification of cancer from biopsy of connective tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.89245 , 0.888438 , 0.948297 , 0.918756 , 0.538314 , and 0.55879 , respectively . for the identification of cancer from biopsy of connective tissues in the case of fuzzy knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.787879 , 0.867476 , 0.370074 , 0.618789 , 0.356613 , and 0.231013 , respectively . for the identification of cancer from biopsy of connective tissues , in the case of random forest classifier , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.907245 , 0.993668 , 0.493996 , 0.743832 , 0.647373 , and 0.642137 , respectively . from table 5 and figure 5(b ) the following observations are made for sample test cases containing epithelial tissues.(i)for the identification of cancer from biopsy images of epithelial tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.884727 , 0.916446 , 0.801733 , 0.859435 , 0.795319 , and 0.71626 , respectively.(ii)for the identification of cancer from biopsy of epithelial tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.796998 , 0.7851 , 0.898525 , 0.842279 , 0.472804 , and 0.4587 , respectively.(iii)for the identification of cancer from biopsy of epithelial tissues in the case of fuzzy knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.665834 , 0.76465 , 0.407057 , 0.585984 , 0.401181 , and 0.17053 , respectively.(iv)for the identification of cancer from biopsy of epithelial tissues , in the case of random forest classifier , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.849306 , 0.966243 , 0.555332 , 0.760788 , 0.675868 , and 0.609494 , respectively . for the identification of cancer from biopsy images of epithelial tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.884727 , 0.916446 , 0.801733 , 0.859435 , 0.795319 , and 0.71626 , respectively . for the identification of cancer from biopsy of epithelial tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.796998 , 0.7851 , 0.898525 , 0.842279 , 0.472804 , and 0.4587 , respectively . for the identification of cancer from biopsy of epithelial tissues in the case of fuzzy knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.665834 , 0.76465 , 0.407057 , 0.585984 , 0.401181 , and 0.17053 , respectively . for the identification of cancer from biopsy of epithelial tissues , in the case of random forest classifier , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.849306 , 0.966243 , 0.555332 , 0.760788 , 0.675868 , and 0.609494 , respectively . from table 5 and figure 5(c ) the following observations are made for sample test cases containing muscular tissues.(i)for the identification of cancer from biopsy images of muscular tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.897321 , 0.923277 , 0.650761 , 0.787092 , 0.543009 , and 0.49783 , respectively.(ii)for the identification of cancer from biopsy of muscular tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.884379 , 0.886718 , 0.786303 , 0.83681 , 0.263764 , and 0.320547 , respectively.(iii)for the identification of cancer from biopsy of muscular tissues in the case of fuzzy knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.614958 , 0.672503 , 0.535894 , 0.604364 , 0.538571 , and 0.208941 , respectively.(iv)for the identification of cancer from biopsy of muscular tissues , in the case of random forest classifier , the accuracy , specificity , sensitivity , bcr , f - measure , and mcc are 0.889878 , 0.995023 , 0.193145 , 0.594084 , 0.313309 , and 0.37318 , respectively . for the identification of cancer from biopsy images of muscular tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.897321 , 0.923277 , 0.650761 , 0.787092 , 0.543009 , and 0.49783 , respectively . for the identification of cancer from biopsy of muscular tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.884379 , 0.886718 , 0.786303 , 0.83681 , 0.263764 , and 0.320547 , respectively . for the identification of cancer from biopsy of muscular tissues in the case of fuzzy knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.614958 , 0.672503 , 0.535894 , 0.604364 , 0.538571 , and 0.208941 , respectively . for the identification of cancer from biopsy of muscular tissues , in the case of random forest classifier , the accuracy , specificity , sensitivity , bcr , f - measure , and mcc are 0.889878 , 0.995023 , 0.193145 , 0.594084 , 0.313309 , and 0.37318 , respectively . from table 5 and figure 5(d ) the following observations are made for sample test cases containing nervous tissues.(i)for the identification of cancer from biopsy images of nervous tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.861763 , 0.880866 , 0.835733 , 0.858482 , 0.834116 , and 0.716492 , respectively.(ii)for the identification of cancer from biopsy of nervous tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.769545 , 0.723056 , 0.946068 , 0.834923 , 0.630126 , and 0.552038 , respectively.(iii)for the identification of cancer from biopsy of nervous tissues in the case of fuzzy knn , the accuracy , specificity , sensitivity , bcr , f - measure , and mcc are 0.808453 , 0.882722 , 0.242776 , 0.562835 , 0.225886 , and 0.11837 , respectively.(iv)for the identification of cancer from biopsy of nervous tissues , in the case of random forest classifier , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.843102 , 0.92827 , 0.723262 , 0.825766 , 0.792403 , and 0.676888 , respectively . for the identification of cancer from biopsy images of nervous tissues in the case of knn , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.861763 , 0.880866 , 0.835733 , 0.858482 , 0.834116 , and 0.716492 , respectively . for the identification of cancer from biopsy of nervous tissues in the case of svm , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.769545 , 0.723056 , 0.946068 , 0.834923 , 0.630126 , and 0.552038 , respectively . for the identification of cancer from biopsy of nervous tissues in the case of fuzzy knn , the accuracy , specificity , sensitivity , bcr , f - measure , and mcc are 0.808453 , 0.882722 , 0.242776 , 0.562835 , 0.225886 , and 0.11837 , respectively . for the identification of cancer from biopsy of nervous tissues , in the case of random forest classifier , the average value of accuracy , specificity , sensitivity , bcr , f - measure , and mcc is 0.843102 , 0.92827 , 0.723262 , 0.825766 , 0.792403 , and 0.676888 , respectively . from the above discussions for all four categories of test cases , it is observed that the knn is performing better in comparison to other classifiers for the identification of cancer from biopsy images of nervous tissues . from all above observations , it is concluded that the knn classifier is producing better results in comparison to other methods for the case of biopsy images of connective tissues . the maximum values of the accuracy , sensitivity , and specificity are 0.9552 , 0.9615 , and 0.9543 , respectively . the k - nearest neighbor classifier is also performing better for all cases as well as that was discussed above . table 6 gives a comparative analysis of the proposed framework with other standard methods available in the literature . from table 6 , it can be observed that the proposed method is performing better in comparison to all other methods . an automated detection and classification procedure was presented for detection of cancer from microscopic biopsy images using clinically significant and biologically interpretable set of features . the proposed analysis was based on tissues level microscopic observations of cell and nuclei for cancer detection and classification . for enhancement of microscopic biopsy images contrast after segmentation of images , a total of 115 hybrid sets of features were extracted for 2828 sample histology images taken from histology database . out of 1000 samples of 115 features , 900 samples were selected for training purposes and 100 samples were selected for testing purposes . the various classification approaches tested were k - nearest neighborhood ( knn ) , fuzzy knn , support vector machine ( svm ) , and random forest based classifiers . from table 5 we are in position to conclude that knn is the best suited classification algorithm for detection of noncancerous and cancerous microscopic biopsy images containing all four fundamental tissues . rf classifier provides very low sensitivity and down accuracy rate as compared to knn classifier for this dataset . hence , from experimental results , it was observed that knn classifier is performing better for all categories of test cases present in the selected test data . these categories of test data are connective tissues , epithelial tissues , muscular tissues , and nervous tissues . among all categories of test cases , further it was observed that the proposed method is performing better for connective tissues type sample test cases . the performance measures for connective tissues dataset in terms of the average accuracy , specificity , sensitivity , bcr , f - measure , and mcc are 0.921909 , 0.940164 , 0.819922 , 0.880263 , 0.759395 , and 0.717455 , respectively .

a framework for automated detection and classification of cancer from microscopic biopsy images using clinically significant and biologically interpretable features is proposed and examined . the various stages involved in the proposed methodology include enhancement of microscopic images , segmentation of background cells , features extraction , and finally the classification . an appropriate and efficient method is employed in each of the design steps of the proposed framework after making a comparative analysis of commonly used method in each category . for highlighting the details of the tissue and structures , the contrast limited adaptive histogram equalization approach is used . for the segmentation of background cells , k - means segmentation algorithm is used because it performs better in comparison to other commonly used segmentation methods . in feature extraction phase , it is proposed to extract various biologically interpretable and clinically significant shapes as well as morphology based features from the segmented images . these include gray level texture features , color based features , color gray level texture features , law 's texture energy based features , tamura 's features , and wavelet features . finally , the k - nearest neighborhood method is used for classification of images into normal and cancerous categories because it is performing better in comparison to other commonly used methods for this application . the performance of the proposed framework is evaluated using well - known parameters for four fundamental tissues ( connective , epithelial , muscular , and nervous ) of randomly selected 1000 microscopic biopsy images .

1. Introduction 2. Related Works 3. Methods and Models 4. Results and Discussions 5. Conclusions

workplace - related traumatic injuries ( wris ) account for a significant proportion of injuries that affect mainly the young population . these injuries are a major source of disability and death leading to serious socioeconomic losses to the family of victims . according to the international labor organization ( ilo ) , in addition , around 317 million workers sustain nonfatal wri annually , leading to an average loss of productivity of four days or more per year . an australian report on fatal wri observed 374 deaths , a mortality rate of 1.93 per 100 000 workers , in 2010 - 2011 . of these , 59% were directly job - related , 29% were transport - related , and 12% were bystander fatalities . data from singapore showed that the majority of wri patients were admitted due to fall from height ( ffh ) ( 66% ) and falling of heavy objects ( fho ) ( 21.9% ) at work with a case fatality of 4.7% . international migrants and migrant workers have contributed significantly to the economic growth and rapid development needs of several countries . a review conducted on occupational injury and diseases among immigrant workers found that immigrant workers had a disproportionately higher burden of occupation - related morbidity and mortality in comparison to native workers . other studies reported an increased risk of occupational disability among construction workers compared to those workers involved in less physical labor [ 7 , 8 ] . further , gubran and usel proposed a higher risk of occupational disability in unskilled workers in comparison to the skilled workers . among construction site injuries , ffh is the most frequent cause of injury . in the middle east , the majority of fall - related injuries are associated with the construction and petrochemical industries . the rate of fall - related injuries is markedly increased in rapidly developing middle eastern countries with increasing demands for construction work . there are only few reports from the middle east that describe the pattern of injury and safety measures in those who sustained traumatic injuries purely from fho at the construction sites . in uae , the main causes of such injuries were ffh ( 51% ) and fho ( 15% ) [ 1012 ] . economy and rapid industrialization in qatar attract thousands of skilled and unskilled workers from south asia and other countries . the state of qatar is a rapidly developing , high - income country with a population that is 75% male and is 85% expatriates . its rapid economic growth has been fueled by its vast petrochemical reserves and accompanied by massive infrastructure projects , most notably for the world cup in 2022 , which have necessitated the rapid influx of thousands of migrant workers leading to doubling of its population in the last 7 years . the incidence and mortality from construction that fall in qatar between 2007 and 2008 were reported as 86.7 and 8.44 per 100,000 construction workers . most recently , this worker movement has been accompanied by an amnesty international report that discusses worker rights abuses but does not specify wri as a main issue . herein , we aim to describe the incidence and outcomes of wri in qatar , a rapidly developing country in the middle east between 2010 and 2012 . a descriptive analysis of trauma registry data for occupational injury surveillance was conducted ; this included all wri patients admitted at level i hamad trauma center ( the national trauma referral center and only provider of tertiary trauma care in qatar ) , from january 1 , 2010 , to december 31 , 2012 . labor statistics from the qatar statistics authority were used to compute for the population based incidence and mortality rates from wri in qatar . data analyses included demographics , clinical presentations , mechanism of injury ( moi ) , injury severity score ( iss ) , type of occupation , use of protective measures , hospital length of stay , need for rehabilitation facility , morbidity , and mortality . wris were defined as injuries taking place during the specified working hours , while the employee is engaged in work - related activities . injury burden is the proportion of injuries caused by a specific injury mechanism among the total number of workplace - related injuries . mortality burden is the proportion of deaths caused by a specific injury mechanism among the total number of deaths due to wri . case fatality rate is the number of deaths caused by a specific injury mechanism divided by the total number of workplace - related injuries caused by that specific mechanism of injury . based on a mandate from the supreme council of health in qatar , all emergency medical services ( prehospital and in - hospital services ) are provided free of charge for all qatari nationals and residents , including expatriate workers . moreover , hamad medical corporation is responsible for providing rehabilitation services , free of charge , for all patients until they are discharged home . the majority of migrant workers return to their home countries after serious injuries or disabilities , if they have no family support in qatar . to compute for the iss , each of six anatomical regions was scored with the highest abbreviated injury score ( ais ) using the square of the highest value of the three most severely injured body regions . ethical approval was obtained from the medical research center ( irb number 13186/13 ) at hamad medical corporation , doha , qatar . data were presented as proportions , medians , or mean standard deviation ( sd ) , as appropriate . differences in categorical variables between respective comparison groups were analyzed using chi - square test . data analysis was carried out using the statistical package for the social sciences version 18 ( spss inc . , during the 3-year surveillance , a total of 5152 patients were admitted at the trauma unit , of them 1496 ( 29% ) sustained wri with a mean age of 34 10 years and 97% of them were males . age breakdown revealed that 72% of patients were young ( 20 to 40 years ) ( table 1 ) . the majority of the patients were from south asian countries particularly from nepal ( 29% ) and india ( 21% ) . ffh ( 51% ) , fho ( 18% ) , and motor vehicle crashes ( mvcs ) ( 17% ) were the most common moi . wri patients were mainly laborers involved in industrial work ( 43% ) , transportation ( 18% ) , installation / repair ( 12% ) , carpentry ( 9% ) , and housekeeping ( 3% ) . sixty - four percent of wri patients were not using personal protective equipment ( ppe ) when they were injured . the lower extremities ( 28% ) followed by chest ( 26% ) , upper extremities ( 23% ) , head ( 20.5% ) , and abdomen ( 18% ) were the most frequently injured body regions . there were 308 , 161 , and 92 spinal injuries in terms of lumber , thoracic , and cervical vertebrae , respectively . open reduction and internal fixations ( orif ) ( 27% ) were the most commonly performed surgical procedure followed by exploratory laparotomy ( 6% ) , closed reduction ( 5% ) , and craniotomy ( 3% ) . the study population had median ventilator days of 2 ( 121 ) , icu stay of 3 ( 164 ) , and total hospital stay of 6 ( 1192 ) days . forty - nine percent of the wri patients stayed more than 7 days in the hospital and 13.6% were discharged to long - term rehabilitation facilities . the mean injury severity score ( iss ) was 11.7 8.9 and glasgow coma score was 14.03 2.8 ( table 2 ) . prehospital deaths ( bid ) constituted 40.5% of all mortalities which included mortality either at the workplace or during transportation to the hospital . the main moi of bid was ffh ( 41% ) followed by mvcs ( 23% ) , fho , machinery , burn , and pedestrian injuries ( 9% for each ) . more young workers died but the case fatality was nonsignificantly higher for older ( > 50 years ) compared to young workers ( figure 1 ) . table 3 shows that ffh represents the highest injury ( 51% ) and mortality ( 38% ) burden among the moi , whereas the case fatality was high among burn ( 17 per 100 cases ) and pedestrian ( 12 per 100 cases ) injuries . figure 2 demonstrates the proportion of injuries and case fatality according to type of work . most wri affected construction laborers and workers involved in transport and installers / repairmen job but the highest mortality rate was observed in workers involved in housekeeping jobs . table 4 represents the demographic characteristics , presentation , and outcome of wri patients by moi . laborers were disproportionately highly involved in injuries related to machines , fho and pedestrian injuries , respectively . not surprisingly , almost all wri due to mvc involved transport occupations . installation / repair workers represented around 1216% of patients in all moi except burns ( 31% ) and mvc ( 0% ) . housekeeping workers were involved mainly in injuries due to burns followed by ffh . in comparison to other moi , ffh was more likely associated with lack of ppe use ( p = 0.001 ) and head ( p = 0.002 ) , thoracic ( p = 0.001 ) , and lumbar spine ( p = 0.001 ) injuries . pedestrian injuries were characterized by higher mean iss and more frequently associated with chest , abdomen , pelvic , and lower extremities injuries ( p = 0.001 for all ) . mvcs were more likely to cause cervical spine injuries ( p = 0.02 ) and their victims had higher rate of open reduction / internal fixation ( p = 0.001 ) . workers with burn injuries had significantly higher icu ( p = 0.04 ) and hospital length of stay ( p = 0.004 ) . the aggregate incidence of wri in qatar , from 2010 to 2012 , was 49.27 per 100,000 workers and wri mortality was 1.97 per 100,000 workers per year . this is the first report of occupational injury surveillance data to describe the epidemiology and outcome of serious wri in qatar . the analysis is based on the trauma registry of the only level i trauma center in qatar ; it is an accurate representation of all serious wris that occur in the country because all such cases are transferred to our center . the incidence and death rates of wri in qatar are much lower than the published reports from other similar countries in the region . in particular , developing nations lack reliable information on wri which is mainly attributed to the limited resource allocation , negligence , and underreporting . most of the current literature on traumatic wri is based mainly on reports from the developed countries . according to the international labor organization ( ilo ) , in addition , around 317 million workers sustain nonfatal wri annually , leading to an average loss of productivity of four days or more / year . a high rate of wri ( 1,980 per 100,000 workers per year ) has been reported from oman that has a socioeconomic status similar to qatar . the incidence of wri among developing countries in africa like ghana is also high with an incidence of 1,150 per 100,000 workers per year in the urban areas and 4,490 per 100,000 workers per year in the rural areas . reports from usa showed a similar incidence of wri in the high - risk hispanic workers in massachusetts ( 54.8 per 100,000 workers per year ) and washington state ( 45.5 per 100,000 workers per year ) [ 19 , 20 ] . despite the reports in the popular press questioning worker rights and welfare in qatar , this study reports wri incidence and mortality rates that are much lower than those reported from uae ( 136 per 100,000 workers per year ) , uk ( 99 major injuries per 100,000 workers per year ) , and oman and are equivalent to reports on high - risk hispanic workers in usa [ 19 , 20 ] . the hamad trauma unit admitted about 5152 trauma patients over a 3-year period , of them wri comprises 29% of the total admissions . over a 10-year period , data from washington state trauma registry in usa showed that wri formed only 7.3% of all trauma admissions . this study showed very similar results as the report by barss et al . from the uae , with 30% of all trauma admissions being due to wri . this information should spur the formulation of policies to include initiatives for occupational safety and injury prevention as national priority issues for policy formulation , research , and programs . several epidemiological studies have observed a higher incidence of wri among young males [ 1 , 10 , 17 , 22 , 23 ] . the bureau of labor statistics in usa reported 5,488 workplace - related deaths in 2007 , of which the majority were male workers ( 92% ) . similarly , the majority of workers in this study were males with a mean age of 34 years . in this series , higher proportions of expatriate workers are from nepal and india , who are involved in labor - intensive jobs such as building and road construction and industrial work . in a 2003 report on wri in the neighboring uae , 98% of victims were males , 74% were from south asia , and 69% aged between 25 and 44 years . this is similar to the situation in usa where a large percentage of immigrants work as laborers , of which 47% belong to ethnic minorities and only 15% were white with lower wri rate . a report from singapore showed that the majority of wri occurred among nationals of china ( 53% ) , india ( 23% ) , and bangladesh ( 14% ) . reported the same experience in the uae where 51% of their wri patients were victims of falls from height , 29% of which were from considerable height . . showed that the incidence of fall injuries in qatar was 86.7 per 100,000 workers causing a significant financial burden ( average $ 15,735 per patient ) on the health care system . data from singapore showed that the main moi in the admitted wri patients were ffh ( 66% ) followed by fho at work ( 21.9% ) with an overall mortality rate of 4.7% . the second most common moi was fho ( 18% ) , similar again to the report from uae ( 15% ) . the proportion of wri occurring in laborers and installation / repairmen contrasts with the findings of mock et al . from ghana . they reported that most wri happened in mvc 's ( 12.7% drivers and 19.4% traders ) in urban areas . an australian report on fatal wris observed 374 deaths , a mortality rate of 1.93 per 100 000 workers , in 2010 - 2011 . of these , 59% were directly job - related , 29% were transport - related , and 12% were bystander fatalities . the annual statistics report ( 2010 - 2011 ) in uk found the most dangerous occupations to be construction , agriculture , and waste and recycling which represent the highest mortality rates ( 0.6 per 100 000 workers ) . this belies the marked difference in the most common occupational injury risk exposures between different settings , that is , the extent of construction in qatar and uk . although , work - related burn injuries in our series were only 2% of all wri , 17% of burn victims died and they constituted 9% of mortalities . unfortunately , our database was unable to document the extent , grade , type , associated traumatic injuries , and cause of death among burn - related causalities . analysis based on moi also revealed that among housekeeping workers the percent of injuries due to burns was high and further analysis of the higher rate of deaths among these workers is needed to identify opportunities for improvements in primary , secondary , and/or tertiary prevention of burns - related deaths and injuries . data from the national census of fatal occupational injuries ( 2012 ) showed highest mortality among transportation incidents ( 41% ) , of which 16% involved pedestrians who were struck by vehicles . the other common mechanisms were violence in the workplace ( 17% ) , contact with object or equipment ( 16% ) , fatal falls , slips , or trips ( 15% ) , and fire and explosion ( 3% ) . the present study found the highest number of wri amongst construction laborers ( 42% of all wris ) and transportation workers ( 18% of all wri ) . the overall case fatality was 3.7% , with the most fatalities from falls and the highest lethality being related to burn and pedestrians injuries at work . the variability in the mortality patterns by moi and occupation can be attributable to differential exposures ( i.e. , housekeeping kitchen . the higher incidence of these potentially preventable injuries can be attributed to the lack of stringent safety regulations , consistent enforcement of these regulations , and the proper orientation and training of the expatriate workers . our study confirmed this , as only 36% of wri patients used ppe , while the remaining injured workers were not in compliance with safety regulations in the workplace . also , the mortality was nonsignificantly higher among older workers as compared to other age groups . our findings are supported by an earlier report from centers for disease control and prevention which showed that the mortality rates were higher among workers involved in transportation and material moving . particularly , older workers ( 65 yrs ) constituted a higher percentage ( 23% ) of deaths . over 40% of wri victims were dead on arrival which highlights the importance of primary injury prevention for occupational safety in qatar . in contrast to figures from the national census of fatal occupational injuries , victims of workplace violence did not make up a significant proportion of our patients with wri . interestingly , our study observed an increased need for ventilatory support and prolonged icu stay of wri cases which reflects the severity of injury and financial burden on the healthcare system . there are several limitations and areas for recommended improvement in our study such as underrepresentation of minor wri as the trauma registry only encodes data from patients with severe injury or polytrauma , underreporting of protective measures used , deficient detailed medical history of patients , lack of data about medical insurance issues , work place ergonomics , work site environment , and employers ' details ( both private and public sectors ) . also , language barriers represented a major limitation for medical staff communication with patients and subsequently the accuracy of documentation . further , while our trauma registry data is prospectively encoded , retrospective exploration of this database shows that it still lacks certain wri - specific data elements that are needed to fully inform policies and programs for occupational safety in qatar . the creation of an integrated occupational injury registry with sources of data from the ambulance , emergency , hospital , and rehabilitation services in collaboration with the ministry of labor and insurance companies will mitigate the shortcomings of our present occupational surveillance system . ffh should be the focus of primary prevention efforts based on their high mortality and injury burden . burn injuries in housekeeping workers , mvcs , and pedestrian injuries are other areas for prioritization . future research should focus on improving the quality of data on occupational injuries in qatar . the creation of a dedicated multidisciplinary task force that will prospectively collect all data on risk factors and outcomes for wri and link these with incident investigations from the ministry of labor is the first step needed . the investigation of the relationship between chronic conditions , the incidence and cost of wri among high - risk occupations , workplace violence , and recurrent workplace injuries could be some of the priority issues this dedicated occupational injury registry will address . therefore , occupational health and safety should be specifically tailored for the workers involved in most hazardous occupations and vulnerable groups . strict governance from the ministry of labor is required to ensure compliance with safety measures as well as necessary precautions to avoid health risks from wri in the workplace . there is an urgent need to improve the quality of data regarding wris in the rapidly developing high - income countries of the middle east in order to ensure that progress is sustained but not at the expense of worker health and welfare . we also recommend the need for multiagency review of health provision for migrant workers , which should be based on thorough and independent evaluation for the major causes of mortality among migrant construction workers and identifying key measures to improve health and safety of workers in qatar . in conclusion , although the incidence of wri in qatar is quite substantial , its mortality rate is relatively low in comparison to other countries of similar socioeconomic status . the implementation and enforcement of strict safety regulations and use of ppe should be mandatory in workplaces , particularly in construction industry . wris cause a significant socioeconomic burden as evidenced by their extended length of hospital stay and prevalent need for rehabilitation services . the authors call for better evidence on the risk factors for wri which must be used to formulate and implement focused injury prevention programs in the populations and industries identified to be at the highest risk for wri .

traumatic workplace - related injuries ( wris ) carry a substantial negative impact on the public health worldwide . we aimed to study the incidence and outcomes of wris in qatar . we conducted occupational injury surveillance for all wri patients between 2010 and 2012 . a total of 5152 patients were admitted to the level 1 trauma unit in qatar , of which 1496 ( 29% ) sustained wri with a mean age of 34.3 10.3 . fall from height ( ffh ) ( 51% ) followed by being struck by heavy objects ( fho ) ( 18% ) and motor vehicle crashes ( mvc ) ( 17% ) was the commonest mechanism of injury ( moi ) . wri patients were mainly laborers involved in industrial work ( 43% ) , transportation ( 18% ) , installation / repair ( 12% ) , carpentry ( 9% ) , and housekeeping ( 3% ) . use of protective device was not observed in 64% of cases . the mean iss was 11.7 8.9 , median icu stay was 3 days ( 164 ) , and total hospital stay was 6 days ( 1192 ) . the overall case fatality was 3.7% . although the incidence of wri in qatar is quite substantial , its mortality rate is relatively low in comparison to other countries of similar socioeconomic status . prolonged hospital stay and treatment exert a significant socioeconomic burden on the nation 's and families ' resources . focused and efficient injury prevention strategies are mandatory to prevent future wri .

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

transoral robotic surgery ( tors ) is a technique increasingly used in surgical excision of tongue base neoplasms [ 1 , 2 ] . preoperative evaluation of each patient to determine the ability to achieve adequate exposure becomes critical , especially in light of the increased demand and limited amount of robotic availability . despite the advantages of tors for base of tongue ( bot ) neoplasms , variations of anatomy , tumor characteristics , and surgeon expertise often contribute to the technical difficulty associated with this method and may ultimately result in aborted procedures . although a separate planning endoscopy has been suggested prior to attempting tors , to date , there are no objective measurements utilized to predict the technical challenge of a tors resection . we propose an evaluative process to preoperatively determine the feasibility of robotic surgical access using cephalometric measurements obtained from radiographs . van abel proposed that preoperative evaluation of the tors patient should include an assessment of trismus , tumor volume , tongue , degree of torus on the mandible , and whether the patient has teeth . these parameters provide a global sense of the difficulty of exposure . with the addition of cephalometric measures proposed in this paper , a more informed decision can be made regarding the type of exposure the surgeon will be able to obtain in the operating room . this would minimize the need for separate planning endoscopy , allow for more informed patient counseling and facilitate treatment planning . between 2010 and 2012 , patients who underwent attempted tors resection at one of two tertiary care centers were included in the study . patients met inclusion criteria if they required base of tongue ( bot ) resection ( n = 36 ) . five of these 36 patients were excluded due to poor documentation on ease of exposure and inadequate preoperative imaging . institutional review board approval was obtained to perform a cephalometric analysis on this retrospective cohort . the exposure for each patient was stratified as adequate or restricted by the operative surgeon . twenty cephalometric measurements were obtained from preoperative computed tomographs ( ct ) or magnetic resonance images ( mri ) and compared with the ease of exposure stratification ( fig . 1 ) . these measurements include : a hyoid to gnathion , b hyoid to gonion , c gonion to gnathion , d condyle to gnathion , e posterior pharyngeal wall to hyoid , f posterior pharyngeal wall to soft palate , g tip of tongue to vallecula , h , i two intersecting lines were created in each radiograph , one line from the gonion and to the base of vertebral body of c2 , and another line from the posterior aspect of the hard palate and to the hyoid . the distance from the intersection of these two lines to the gonion and hyoid were subsequently recorded and evaluated independently and as a ratio . l measured involving the sella , nasion anterior nasal spine and suprametale , m measured between the epiglottis and posterior pharyngeal wall , n epiglottis and bot and o epiglottis and larynx . mandibular measurements included , p , q intermandubular distance at two points , r distance between the lower edge of the mandible and the hyoid , s , t anterior and posterior mandibular height various cephalometric measurements obtained from preoperative imaging . these measurements include : a hyoid to gnathion , b hyoid to gonion , c gonion to gnathion , d condyle to gnathion , e posterior pharyngeal wall to hyoid , f posterior pharyngeal wall to soft palate , g tip of tongue to vallecula , h , i two intersecting lines were created in each radiograph , one line from the gonion and to the base of vertebral body of c2 , and another line from the posterior aspect of the hard palate and to the hyoid . the distance from the intersection of these two lines to the gonion and hyoid were subsequently recorded and evaluated independently and as a ratio . l measured involving the sella , nasion anterior nasal spine and suprametale , m measured between the epiglottis and posterior pharyngeal wall , n epiglottis and bot and o epiglottis and larynx . mandibular measurements included , p , q intermandubular distance at two points , r distance between the lower edge of the mandible and the hyoid , s , t anterior and posterior mandibular height the mean , standard deviation , and standard error were calculated for each group and the cephalometric measurements were then compared using the t test . a receiver operating characteristic ( roc ) curve was created for each parameter and those with a fitted roc area greater than 0.8 were considered good predictors of a dichotomous outcome : restricted or adequate exposure . mallampati scores were gathered from anesthesia preoperative records and compared to the surgeon s designation of adequate or restricted exposure using the chi squared test . low scores were considered a grade 1 or 2 mallampati and grade 3 or 4 were classified as high scores . this article does not contain any studies with human or animal subjects performed by any of the authors . all procedures followed were in accordance with the ethical standards of the internal review board ( irb ) and with the helsinki declaration of 1975 . of the 31 patients evaluated , 15 were stratified as adequate exposure and 16 were deemed restricted exposure . table 1 summarizes the patient s pathology , age , operative time and body mass index ( bmi ) . the majority of the bot pathology results ( 17/31 ) were squamous cell carcinomas , with the remainder being lingual hypertrophy . the patient s ages varied 3578 with bmis of 20.736.1 and duration of surgery ranged 40180 min . the duration of surgery for the restricted group , 85 min , was significantly longer than the adequate exposure group , 51 min , ( p = 0.026).table 1patient demographicscharacteristicexposure p valuerestricted ( n = 16)adequate ( n = 15)age ( years ) median62540.40 range33742372body mass index median29260.086 range22352136tumor stage ( no . ) t1640.882 t248 t320 t400 lingual hypertrophy43duration of surgery median85510.026 * range7220212133mallampati score median310.043 * range1413 * significant p values * significant p values upon comparing stratifications of exposure with these measurements , 3 of the 20 measures proved to be statistically significant . the mean distance from the posterior pharyngeal wall to the hyoid in the adequate exposure group was 33.2 5.6 mm , whereas the restricted group had a significantly smaller distance of 26.7 5.4 mm ( p = 0.002 ) . in the restricted group , which has a more posteriorly situated hyoid complex , the adequate exposure group had an angle of 132.9 8.3 compared to 120 19.9 in the restricted group ( p = 0.04 ) . the distance between the most inferior aspect of the soft palate as seen on the sagittal view and posterior pharyngeal wall was significantly larger in the adequate exposure group ( mean of 7.8 3.6 mm ) when compared to the restricted group ( mean of 4.7 1.9 mm ) ( p = 0.006 ) ( fig . . 2posterior pharyngeal wall to hyoid / soft palate distance and epiglottis to vertical laryngeal angle : a , c adequate exposure , and b , d restricted exposure posterior pharyngeal wall to hyoid / soft palate distance and epiglottis to vertical laryngeal angle : a , c adequate exposure , and b , d restricted exposure a roc curve was plotted to determine if the measurements could be used as a tool for selecting the cut off whereby adequacy of exposure could be predicted . when applying this statistical method to the posterior pharyngeal wall to hyoid measurement a distance less than 30 mm was the optimal number to determine if the exposure was restricted based on a plot of sensitivity / specificity vs. distance between the hyoid and posterior pharyngeal wall . the area under the curve ( auc ) for this value is 0.83 with a p < 0.001 ( fig . the auc for distance from the posterior pharyngeal wall to the soft palate is 0.80 ( p = 0.006 ) with a cut off value of less than 8.1 mm and the auc for angle of the epiglottis to larynx is 0.70 ( p = 0.03 ) with a cut off value of less than 130.fig . 3roc curve for posterior pharyngeal wall to hyoid distance roc curve for posterior pharyngeal wall to hyoid distance mallampati scores for the adequate exposure group demonstrated a predominance of lower scores : 12 to 3 ; however , the restricted group had an almost even distribution with seven low scores and nine high scores . the correlation of the mallampati score with difficulty of exposure was statistically significant ( p = 0.045 ) . measurements that assessed retrognathia ( p = 0.46 ) , mandibular height ( p = 0.12 ) and tongue length ( p = 0.63 ) did not significantly differ between the two groups , nor did measurements for sella nasion a ( sna ) ( p = 0.5 ) , sella nasion b ( snb ) ( p = 0.9 ) , or a - nasion - b ( anb ) ( p = 0.5 ) . length of the surgery ( 40180 min ) did correlate with the quality of exposure ( table 1 ) . over the past 4 years the application of transoral robotic surgery has grown extensively , transitioning from the innovative stages to the adaptive stage with use both in academic and private practice settings . the initial reports discussed the issue of safety when considering exposure , such as assessment for retropharyngeal carotid artery and the course of the lingual artery [ 6 , 7 ] . ultimately , one of the main limiting factors in determining success of tors is exposure . the inability to obtain exposure can lead to a cancelled tors approach or aborting the procedure due to poor visualization or decreased confidence in the quality of resection and ultimate margin status . there are numerous reports in the literature regarding access to the infratemporal fossa , anterior skull base , nasopharynx , parapharyngeal space and thyroid [ 2 , 4 ] . the original papers regarding tors radical tonsillectomies and base of tongue resections also focused on feasibility and accessibility . these papers answer the question of possibility , but not of restrictions that may be faced in certain patients . now that we know we can achieve successful operations , these data provide some insight into the limitations of access . the clinical judgment of a restricted verses adequate exposure is subject to the evaluation of the surgeon , which may not be consistent from surgeon to surgeon . to reduce the bias of early experience all three surgeons had at least 20 tors cases to participate in the study . an additional method used to minimize the judgment bias was collecting data from two institutions . perhaps the most critical factor in the accessibility of tors is the location of the tumor . isolated tonsil primaries and some of the high base of tongue primaries can be resected with less difficultly utilizing tors . access becomes critical when tumors extend into the vallecula , glossotonsillar sulcus and supraglottic / hypopharyngeal regions . in order to isolate a homogeneous group for analysis in this study we included only those patients with base of tongue pathology . we can not directly extrapolate these data to supraglottic or hypopharyngeal tumors , but by inference we believe access to these areas would be equally restrictive or open . the patient selection process could be streamlined by clinically and radiographically identifying those patients that may pose an access challenge and schedule them for a staging panendoscopy . those patients that preoperatively appear to have easier access could be directly scheduled for robotic resection . the measurements determined in this study using roc curve and t test analysis would indicate that a posterior pharyngeal wall to hyoid measurement less than 30 mm is considered a risk factor for a restricted view . similarly a distance less than 8.1 mm from the posterior pharyngeal wall to the soft palate and an angle less than 130 between the epiglottis and the larynx would also indicate a possibly challenging exposure . since the hyoid bone is the tethering point of the intrinsic musculature of the tongue , we anticipated finding significance with respect to the length of the tongue and the relationship of the hyoid bone to the mandible . these measurements , however , did not compute out to determine the difficulty of exposure . however , a lower mallampati score was predictive of adequate exposure . this score measures the height of the tongue base in relationship to palate and this measurement has been correlated with the cormack lehane classification system which focuses on the view seen on direct laryngoscopy [ 8 , 9 ] . during the preoperative setting the surgeon can make an informed decision regarding exposure difficulty based on the objective measurement criteria outlined in these data along with , the amount of trismus , presence or absence of teeth or mandibular tori and tumor location . preoperative measurements of radiographic images of the oropharyngeal working space determined that a distance less than 8 mm from the posterior pharyngeal wall to the soft palate and/or 30 mm from the posterior pharyngeal wall to the hyoid , and/or an angle less than 130 between the epiglottis and larynx , may represent restricted exposure for tors resection of the bot . these measures may predict which patients would benefit from a staging endoscopy to determine adequate tors exposure .

transoral robotic surgery ( tors ) is being increasingly used in the treatment of head and neck cancer and we wanted to determine the feasibility of predicting tors access using cephalometric measurements obtained from preoperative imaging . 20 cephalometric measurements were obtained from imaging on 31 tors base of tongue ( bot ) resections and compared to adequacy of exposure . three measurements were found to be significantly different between the restricted and adequate exposure groups . distances from posterior pharyngeal wall ( ppw ) to hyoid , ppw to soft palate and epiglottis to vertical laryngeal angle were all statistically different between the two groups . receiver operating characteristic ( roc ) analysis revealed strong correlation to exposure for all three measurements with cut offs 30 mm between the ppw and the hyoid , 8.1 mm ppw and soft palate and 130 between the epiglottis and vertical plain of the larynx all representing restricted exposure . duration of surgery for the restricted group , 85 min , was significantly longer than the adequate exposure group , 51 min ( p = 0.026 ) . preoperative measurements of radiographic images of the oropharyngeal working space can predict restricted exposure for tors resection of the bot . these measures may be used in conjunction with other subjective assessment parameters to predict which patients could benefit from a staging endoscopy to determine adequate tors exposure .

Introduction Methods Results Discussion Conclusions

hepatosplenic t - cell lymphoma ( hstcl ) is a rare and aggressive extranodal t - cell lymphoma that comprises < 5% of peripheral t - cell lymphomas.1 it is characterized by extranodal infiltration of malignant mature post - thymic t - lymphocytes in the sinusoids of the liver , spleen , and bone marrow.1 the majority of cases harbor the t - cell receptor ( tcr ) , but recently , a few cases have been shown to express the tcr . since the first report of an hepatosplenic t - cell lymphoma ( hstcl ) in 2000,2 < 30 cases have been reported in the literature . as the majority of early cases were of the subtype , hepatosplenic gamma - delta t - cell lymphoma in the revised european american lymphoma ( real ) classification.3 with increasing reports of new cases with the subtype , the term hstcl is preferred in the current world health organization ( who ) classification.4 comparison of the two subtypes5 shows similar clinicopathologic and cytogenetic features ; however , due to the paucity of reported cases , it is not clear whether they are prognostically distinct entities . diagnosis proved challenging due to an unusual blastoid morphology with the absence of typical intrasinusoidal distribution of tumor cells in the bone marrow . this unique case adds to the growing list of this rare subtype of t - cell lymphomas , which warrant urgent attention due to the lack of effective treatment options and dismal prognosis . a male in his 20s from nepal with no chronic illness presented at our hospital with a one - month history of high - grade fever , skin rash , weight loss , symptoms of anemia , and abdominal pain . he was in fair general condition with normal vital signs except for fever ( 39 c ) . physical examination revealed a purpuric rash over his trunk and extremities , hepatomegaly ( 4 cm ) , and splenomegaly ( 10 cm ) below the costal margin . initial investigations showed bicytopenia with a platelet count of 36 10/l and hemoglobin 7.0 g / dl with normal mean corpuscular volume of 90 fl ( elevated reticulocytes at 233 10/l ; reticulocyte index 4.5% , suggesting adequate marrow response ) . a peripheral blood smear showed a leukoerythroblastic picture with several blast - like cells with fine chromatin and one or more prominent nucleoli ( fig . 1 ) . there were also features of hemolysis , including spherocytosis , significant polychromasia , and nucleated red blood cells . iu / l ; normal up to 225 ) and haptoglobin was low ( <3 mg / dl ) . serologies and polymerase chain reaction ( pcr ) tests for hepatitis b and c virus , human immunodeficiency virus , cytomegalovirus , epstein barr virus , leishmania , and human t - lymphotropic virus type 1 were negative . scan showed marked splenomegaly with a span of 21.8 cm associated with arterial hypoperfusion ( perhaps explaining the abdominal pain ) . enlarged axillary , porta hepatis , mesenteric , and para - aortic lymph nodes were also noted ( the largest lymph node was 19 13 mm in the abdomen ) . a whole - body fdg pet / ct scan showed moderate uptake in the left axillary and upper retroperitoneal lymph nodes , suggesting lymphoma activity with spleen and bone marrow involvement . the aspirate showed hypercellular marrow with erythroid hyperplasia ( mixed normoblastic and megaloblastoid ) , hypoplastic granulopoiesis , and reduced megakaryocytes . there was marrow infiltration with 18% abnormal pleomorphic cells with patchy distribution , mostly showing blast morphology with primitive chromatin , prominent nucleoli , and irregular nuclear contour . 2 ) . flow cytometric analysis ( navios , beckman coulter ) of the bone marrow aspirate ( fig . 3 ) revealed a population of abnormal t - cells ( 18% ) of high side scatter expressing cd2 , cd3 , cd8 , and tcr with brighter cd45 and dimmer cd7 than the normal t - lymphocytes with partial loss of cd5 . these cells were negative for cd4 , cd56 , cd16 , cd57 , cd34 , cd1a , and tdt . flow cytometry analysis of the tcr v ( vb ) repertoire revealed that the abnormal cells failed to express any of the tcr vb regions , suggesting monoclonality . the immunostains confirmed that the remarkably increased t - cells were positive for cd2 , cd3 , cd5 , and cd8 with loss of cd7 expression ( fig . 4 ) and negative for cd34 , tdt , cd99 , granzyme b , and alk1 . cytogenetic analysis of the bone marrow sample revealed normal karyotype , and tcr gene rearrangement performed using peripheral blood showed no evidence of clonality . based on the peripheral blood and bone marrow findings , a diagnosis of mature t - cell lymphoma was made . after further multidisciplinary discussion , a diagnosis of hstcl stage iv ( subtype ) was confirmed . the hyper - cvad protocol was initiated after discussion at a multidisciplinary conference , and the patient subsequently received the first cycle of treatment , which was complicated by febrile neutropenia and hypotension . he was managed with intravenous antibiotics but continued to have lymphoma - related fevers with persistent massive splenomegaly and no changes in his complete blood count findings . at this point magnetic resonance imaging of the brain showed a small lesion ( 7 9 9 mm ) in the right temporal lobe , and the possibility of lymphomatous deposition was considered as a likely etiology . a lumbar puncture was not pursued because of refractory thrombocytopenia ( < 50,000/l ) . repeated bone marrow examinations after the first cycle of treatment showed increasing abnormal t - cells ( 53% ) with blast morphology scattered in many cohesive clusters . flow cytometry findings were comparable to those at presentation except for loss of cd5 expression . a second bone marrow aspirate revealed abnormal karyotype with 47,x ,- y , i(7 ) ( q10),+8,+8,add(13)(p11.1 ) /46,x ,- y , i(7)(q10),+8,add(13 ) ( p11.1 ) /46,xy . interval fluorescence in situ hybridization analysis revealed additional signals at 7q31 ( d7s486 probe ) on chromosome 7 in 15% of the analyzed cells . at this time after further discussion , a second cycle of hyper - cvad was initiated , which was complicated by enterobacter cloacae sepsis that was treated with meropenem . after two cycles of hyper - cvad therapy , bone marrow evaluation showed 17% neoplastic t - cells ( similar to 18% at initial diagnosis ) and flow cytometry showed the same immunophenotype ( as at initial diagnosis ) except for persistent loss of cd5 . interval pet - ct imaging showed no significant change compared to baseline , suggesting the absence of response to hyper - cvad therapy . given the poor prognosis of the disease , the patient was offered salvage therapy with regimens containing high - dose cytarabine and platinum analogs ( dhap or esha ) , followed by autologous stem cell transplantation . the poor prognosis of the disease and its refractoriness to chemotherapy along with the salvage therapy options were discussed with the patient . this case of hstcl confirms several of the previous findings of this rare disease5,6 and also illustrates some atypical features that make it a diagnostic challenge , especially given its extremely low incidence . the peak incidence of hstcl is in adolescents and young adults with a median age at presentation of 20 years and with a gender ratio of 9:1 ( male : female ) . the largest case series of hstcl ( 14 cases ) was reported by macon et al.5 who showed no significant clinicopathologic and cytogenetic difference between the and subtypes , except for female preponderance , older median age , and variable age distribution in the former . up to 20% of hstcl cases arise in the background of chronic immune suppression , particularly solid organ transplantation,79 a finding denied by nagai et al for the subtype.6 it is worth mentioning that our patient had no significant past medical history . in this context hstcl usually presents with marked splenomegaly and hepatomegaly , but without lymphadenopathy.1 interestingly , our patient had no palpable lymphadenopathy , but ct and pet imaging unveiled axillary , porta hepatis , and retroperitoneal lymphadenopathy . patients also commonly have b symptoms , such as fever , fatigue , and weight loss.1 abnormalities in liver function tests may be seen in half of the patients.1 thrombocytopenia is also a common feature of this disease with associated leukopenia and anemia . autoimmune hemolytic anemia ( warm type and cold agglutinin disease)10,11 and immune thrombocytopenic purpura12 have been reported as initial manifestations of the disease in rare cases . cytopenias may result from hypersplenism , bone marrow failure , cytokine - mediated hemophagocytic histiocytosis,13 or a combination of these mechanisms . the pathogenesis of autoimmune hemolytic anemia in hstcl is unknown , although neoplastic t - cells as mediators of autoimmune disease have been proposed as a potential explanation.14 our patient initially presented with a picture of coombs-negative hemolytic anemia . it is also worth noting that 5%10% of autoimmune hemolytic anemias are coombs negative,15 and several causes for this phenomenon have been identified , which include hemolysis by natural killer ( nk ) cells independent of antibody , small number of subthreshold immunoglobulin ( ig ) g attached to red blood cells , low - affinity autoantibodies , iga autoantibodies , and igm autoantibodies.14 coombs-negative hemolytic anemia was previously reported in one case of hstcl.16 to the best of our knowledge , this is the first reported case of hstcl with such a presentation . morphologically , hstcl is characterized by a monotonous neoplastic infiltrate consisting of medium - sized lymphocytes with a moderate cytoplasm and irregular cellular borders . the splenic red pulp is expanded with infiltration of cords and sinuses associated with atrophy of the white pulp . liver biopsy also shows an intrasinusoidal infiltrative pattern.1 the bone marrow is frequently involved in the disease.1 trephine biopsy typically shows a hypercellular marrow with characteristic selective infiltration and expansion of the sinuses , which is a useful diagnostic criterion.17,18 however , with progression , the pattern of bone marrow involvement becomes increasingly interstitial,16 which was likely the case in our patient , thereby posing a diagnostic challenge . furthermore , morphology of the tumor cells was predominantly blastoid , another atypical feature of hstcl that is typically associated with disease progression.16,17,19 cohesive clustering of lymphoma cells was another unusual finding . these atypical findings , along with the possibility of false - negative initial karyotype and tcr rearrangement studies , delayed the definitive diagnosis . the false - negative result is possibly related to sample dilution , leading to loss of signal below the detection limit . given the blastoid morphology of the cells , acute lymphoblastic leukemia / lymphoma was considered in the differential diagnosis . however , the negativity for precursor markers ( cd34 , tdt , and cd99 ) excluded this possibility . other differential diagnoses considered included splenic marginal zone lymphoma , t - cell large granular lymphocytic leukemia , and aggressive nk / t - cell leukemia , which were excluded because of the inappropriate morphology and immunophenotype . hstcl typically has the following phenotype : cd2 + , cd3 + , cd4 , cd5 , cd8 , cd16 , cd56+/ , and tcr/+.4,20 frequently , loss of cd3 , cd5 , and/or cd7 ( cd5 lost in this case ) occurs with disease progression . the lymphoid infiltrate in our case was cd8 + , which is reported in a minority of cases.4,20 interestingly , macon et al.5 showed that the type was more likely to be cd8 + when compared to the type . nk cell markers may also be usually expressed with an inactive cytotoxic profile ( tia+ , granzyme , and perforin ) ; however , our case did not show any . most hstcls are associated with the presence of an isochromosome 7q ( i[q10 ] ) with associated abnormalities such as trisomy 8 and loss of chromosome y. the isochromosome 7q has also been demonstrated in hstcl cases.5,11,21 all the abovementioned karyotypic abnormalities were observed in our patient . the biological significance of the isochromosome 7q has not been worked out to date and is not entirely specific to this disease . the prognosis of hstcl , or type , is extremely poor with a median survival of less than one year.5 according to the international peripheral t - cell and natural killer / t - cell lymphoma study,22 the five - year overall survival was only 7% and five - year failure - free survival was 0% , this being the worst among all subtypes of t - cell lymphomas . the majority of patients respond to chop or chop - like regimens but quickly relapse with very few long - term survivors.19 therapies that are quite effective in curing a substantial number of patients with other aggressive lymphoid malignancies such as diffuse large b - cell lymphoma have been shown to be ineffective for hstcl . several case reports have studied the efficacy of new treatments , such as bortezomib , alemtuzumab , and allogeneic bone marrow transplantation,2326 but more efforts are still needed to treat this rare but devastating disease .

hepatosplenic t - cell lymphoma ( hstcl ) is a rare and aggressive extranodal t - cell lymphoma that comprises < 5% of peripheral t - cell lymphomas . the majority of cases harbor the t - cell receptor ( tcr ) , but recently , a few cases have been shown to express the tcr . comparison of these two subtypes ( and ) shows similar clinicopathologic and cytogenetic features ; however , due to the paucity of reported cases , it is not clear whether they are prognostically distinct entities . we report a case of hstcl with a rather unusual presentation of coombs-negative hemolytic anemia . diagnosis proved challenging due to an unusual blastoid morphology with the absence of typical intrasinusoidal distribution of tumor cells in the bone marrow . this unique case adds to the growing list of this rare subtype of t - cell lymphomas , which warrant urgent attention due to the lack of effective treatment options and dismal prognosis .

Introduction Case Report Discussion

ischaemic heart disease is the leading cause of morbidity and mortality in the western world . it was recently estimated that 935 000 new cases of acute coronary occlusion are identified each year in the usa . several experimental studies have demonstrated that the region supplied by the occluded coronary artery , also known as myocardium at risk ( mar ) , is a major independent variable that determines the final infarct size , which in turn is closely related to the clinical outcome . the difference between mar and infarct size is used to calculate the myocardial salvage index , which is a measurement of the effectiveness of acute interventions aimed at reducing the extent of myocardial infarction . thus , a feasible and accurate method for determination of mar is of significant value in clinical studies evaluating the efficiency of cardioprotective therapies . cardiovascular magnetic resonance ( cmr ) imaging is currently considered the reference standard for in vivo assessment of myocardial infarction using late gadolinium - enhanced ( lge ) imaging . recently , cmr has also been introduced as a promising method for assessing mar using t2-weighted imaging up to 1 week after the acute event . thus , cmr can be used to calculate the myocardial salvage index by a single cmr imaging session several days after the acute event in a stable clinical setting . even though t2-weighted imaging is promising for the assessment of mar , this technique still has certain limitations such as difficulties in distinguishing blood pool from the myocardium , especially in the apical parts of the left ventricle ( lv ) where hypokinesia and trabeculation cause stagnant blood flow . more recently , a new cmr method for assessment of mar , referred to as contrast - enhanced steady - state free precession ( ce - ssfp ) , has been introduced . this technique is based on acquisition of time - resolved steady - state free precession images after injection of the gadolinium - based contrast agent and was recently validated against myocardial perfusion single - photon emission computed tomography ( spect ) . still , ce - ssfp has not been compared head - to - head with t2-weighted imaging . therefore , the aim of the present study was to explore the relationship between ce - ssfp and t2-weighted imaging with regard to mar in patients with first - time reperfused acute myocardial infarction . the protocol and procedures were approved by the regional research ethics committee and all patients gave their written consent . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . all patients were treated with primary percutaneous coronary intervention with coronary stenting , resulting in timi grade iii flow in the culprit artery . one week after admission , cmr was performed using a 1.5 t philips intera cv ( philips , best , the netherlands ) . images were obtained using a five - element chest array surface coil with two anterior and three posterior elements . all subjects were placed in supine position and images were acquired at end - expiratory breath hold with vectrocardiographic gating . initial scout images were acquired to locate the heart , and a dark - blood t2-weighted triple inversion turbo spin - echo sequence ( t2-stir ) was employed to depict the mar . t2-weighted images were acquired in the short - axis view , covering the lv from the base to the apex . imaging parameters for the t2-weighted sequence were : echo time , 100 ms ; repetition time , 2 heart beats ; echo train length , 33 ; number of averages , 2 ; inversion time , 180 ms ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed , but surface coil intensity correction was performed to minimize the signal inhomogeneities due to differences in receiver coil sensitivity . acquisition of short - axis , retrospectively gated ssfp cine images , covering the lv from the base to the apex , were initiated within 8 min after the administration of 0.2 mmol / kg extracellular gadolinium - based contrast agent ( gadoteric acid , gd - dota ; guerbet , gothia medical ab , billdal , sweden ) . these images were referred to as ce - ssfp images ( see supplementary data online , movie s1 ) . typical image parameters were : echo time , 1.4 ms ; repetition time , 2.9 ms ; flip angle , 60 ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed to maximize the signal - to - noise ratio ( snr ) . long- and short - axis lge images covering the lv were then acquired 15 min after injection of gadolinium . the lge images were acquired with an inversion - recovery sequence with following image parameters : slice thickness , 8 mm ; field of view , 340 mm ; flip angle , 15 ; repetition time , 4.2 ms ; echo time , 1.3 ms ; image resolution , 1.4 1.4 8 mm ; slice gap , 0 mm ; parallel imaging with a sense factor of 2 . all cmr images were analysed using the freely available software segment v1.8 ( http://segment.heiberg.se ) . the mar derived from t2-weighted imaging was assessed according to a previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices , followed by manual delineation of the hyperintense regions by two blinded observers . the mar was then defined as the total amount of hyperintense myocardium in all short - axis slices and expressed as percentage of lv . if present , hypo - intense myocardium within the area of increased signal intensity ( microvascular obstruction ) was included in the mar . the mar derived from ce - ssfp was also assessed according to previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices in end - diastole and end - systole , followed by manual delineation of the hyperintense regions in both end - diastole and end - systole , by two observers blinded to both the t2-weighted and lge images . the values of mar in end - diastole and end - systole were averaged and expressed as a percentage of the lv . the contrast ratio ( cr ) for the ce - ssfp images was determined for each patient as the mean signal intensity in the mar divided by the mean signal intensity in remote myocardium . a slice - by - slice comparison of mar between t2-weighted imaging and ce - ssfp was also performed for corresponding slice positions . the infarcted myocardium was automatically quantified from the short - axis lge images according to a previously described method . in short , the lge myocardium was then defined using a computer algorithm that takes into consideration partial volume effects within the infarcted region . if present , a hypointense signal within the area of lge ( microvascular obstruction ) was included in the analysis as 100% infarction . finally , the myocardial infarct size was expressed as a percentage of the lv . the myocardial salvage index was defined as : 100*[(mar infarct size)/mar ] , where mar was assessed by both t2-weighted imaging and ce - ssfp . the snr ratio , contrast - to - noise ( cnr ) ratio and contrast ratio ( cr ) were determined for t2-weighted , ce - ssfp , and lge images , respectively . the snr was calculated as the mean signal intensity within the affected region ( mar or infarcted myocardium ) divided by the standard deviation of signal intensities within a background region of interest . the cnr was calculated as the snr in the affected region ( mar or infarcted myocardium ) snr within remote myocardium and cr was calculated as the mean signal intensity in the affected region ( mar or infarcted myocardium ) divided by the mean signal intensity in remote myocardium . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . spss version 17.0 software package ( chicago , il , usa ) was used for analysis . the protocol and procedures were approved by the regional research ethics committee and all patients gave their written consent . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . all patients were treated with primary percutaneous coronary intervention with coronary stenting , resulting in timi grade iii flow in the culprit artery . one week after admission , cmr was performed using a 1.5 t philips intera cv ( philips , best , the netherlands ) . images were obtained using a five - element chest array surface coil with two anterior and three posterior elements . all subjects were placed in supine position and images were acquired at end - expiratory breath hold with vectrocardiographic gating . initial scout images were acquired to locate the heart , and a dark - blood t2-weighted triple inversion turbo spin - echo sequence ( t2-stir ) was employed to depict the mar . t2-weighted images were acquired in the short - axis view , covering the lv from the base to the apex . imaging parameters for the t2-weighted sequence were : echo time , 100 ms ; repetition time , 2 heart beats ; echo train length , 33 ; number of averages , 2 ; inversion time , 180 ms ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed , but surface coil intensity correction was performed to minimize the signal inhomogeneities due to differences in receiver coil sensitivity . acquisition of short - axis , retrospectively gated ssfp cine images , covering the lv from the base to the apex , were initiated within 8 min after the administration of 0.2 mmol / kg extracellular gadolinium - based contrast agent ( gadoteric acid , gd - dota ; guerbet , gothia medical ab , billdal , sweden ) . these images were referred to as ce - ssfp images ( see supplementary data online , movie s1 ) . typical image parameters were : echo time , 1.4 ms ; repetition time , 2.9 ms ; flip angle , 60 ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed to maximize the signal - to - noise ratio ( snr ) . long- and short - axis lge images covering the lv were then acquired 15 min after injection of gadolinium . the lge images were acquired with an inversion - recovery sequence with following image parameters : slice thickness , 8 mm ; field of view , 340 mm ; flip angle , 15 ; repetition time , 4.2 ms ; echo time , 1.3 ms ; image resolution , 1.4 1.4 8 mm ; slice gap , 0 mm ; parallel imaging with a sense factor of 2 . all cmr images were analysed using the freely available software segment v1.8 ( http://segment.heiberg.se ) . the mar derived from t2-weighted imaging was assessed according to a previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices , followed by manual delineation of the hyperintense regions by two blinded observers . the mar was then defined as the total amount of hyperintense myocardium in all short - axis slices and expressed as percentage of lv . if present , hypo - intense myocardium within the area of increased signal intensity ( microvascular obstruction ) was included in the mar . the mar derived from ce - ssfp was also assessed according to previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices in end - diastole and end - systole , followed by manual delineation of the hyperintense regions in both end - diastole and end - systole , by two observers blinded to both the t2-weighted and lge images . the values of mar in end - diastole and end - systole were averaged and expressed as a percentage of the lv . the contrast ratio ( cr ) for the ce - ssfp images was determined for each patient as the mean signal intensity in the mar divided by the mean signal intensity in remote myocardium . a slice - by - slice comparison of mar between t2-weighted imaging and ce - ssfp was also performed for corresponding slice positions . the infarcted myocardium was automatically quantified from the short - axis lge images according to a previously described method . in short , the lge myocardium was then defined using a computer algorithm that takes into consideration partial volume effects within the infarcted region . manual adjustments were made when image artefacts caused misinterpretation by the computer algorithm . if present , a hypointense signal within the area of lge ( microvascular obstruction ) was included in the analysis as 100% infarction . the myocardial salvage index was defined as : 100*[(mar infarct size)/mar ] , where mar was assessed by both t2-weighted imaging and ce - ssfp . the snr ratio , contrast - to - noise ( cnr ) ratio and contrast ratio ( cr ) were determined for t2-weighted , ce - ssfp , and lge images , respectively . the snr was calculated as the mean signal intensity within the affected region ( mar or infarcted myocardium ) divided by the standard deviation of signal intensities within a background region of interest . the cnr was calculated as the snr in the affected region ( mar or infarcted myocardium ) snr within remote myocardium and cr was calculated as the mean signal intensity in the affected region ( mar or infarcted myocardium ) divided by the mean signal intensity in remote myocardium . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . spss version 17.0 software package ( chicago , il , usa ) was used for analysis . in 59% ( 12 of 21 ) of the patients , the right coronary artery ( rca ) was the culprit vessel and in 29% ( 6 of 21 ) of the patients , the left anterior descending coronary artery ( lad ) was the culprit vessel . furthermore , 2 patients presented with an occlusion of the left circumflex coronary artery and 1 patient had a left main occlusion . in all patients , t2-weighted imaging and ce - ssfp identified the mar in the same perfusion territory and in concordance with angiography . figure 1a shows an example of multi - slice short - axis images from the base to the apex demonstrating mar by t2-weighted imaging and ce - ssfp as well as myocardial infarction by lge , in one patient with an occlusion in the left anterior descending coronary artery . for one patient , myocardial infarct size another patient had a clinical history of a prior infarction in a coronary territory different from that supplied by the current culprit vessel . this patient had no signs of infarction by lge imaging in the part of the myocardium previously reported to be infarcted . figure 1the myocardium at risk by t2-weighted imaging and ce - ssfp , and the infarct size by late gadolinium enhancement . ( a ) t2-weighted , ce - ssfp , and late gadolinium - enhanced short - axis images at the corresponding lv levels in a patient with a reperfused left anterior descending coronary artery occlusion . ( b ) single corresponding mid - ventricular short - axis images from a patient with an occlusion in the left anterior descending coronary artery ( lad ) , left circumflex coronary artery ( lcx ) , and the right coronary artery ( rca ) , respectively . the hyperenhanced regions constituting the myocardium at risk ( dashed arrows ) and the infarcted myocardium ( solid arrows ) are traced in white . note the similarity in location and the extent of the affected region between t2-weighted imaging and ce - ssfp . also note the significantly smaller infarction compared with the myocardium at risk indicating a significant myocardial salvage accomplished by the acute reperfusion therapy . the myocardium at risk by t2-weighted imaging and ce - ssfp , and the infarct size by late gadolinium enhancement . ( a ) t2-weighted , ce - ssfp , and late gadolinium - enhanced short - axis images at the corresponding lv levels in a patient with a reperfused left anterior descending coronary artery occlusion . ( b ) single corresponding mid - ventricular short - axis images from a patient with an occlusion in the left anterior descending coronary artery ( lad ) , left circumflex coronary artery ( lcx ) , and the right coronary artery ( rca ) , respectively . the hyperenhanced regions constituting the myocardium at risk ( dashed arrows ) and the infarcted myocardium ( solid arrows ) are traced in white . note the similarity in location and the extent of the affected region between t2-weighted imaging and ce - ssfp . also note the significantly smaller infarction compared with the myocardium at risk indicating a significant myocardial salvage accomplished by the acute reperfusion therapy . ce - ssfp , contrast - enhanced steady - state free precession . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . infarct size could not be assessed due to poor image quality related to frequent arrhythmias . figure 1b shows three examples of the agreement of the mar by t2-weighted imaging and ce - ssfp . another example including the cine ce - ssfp can be seen in supplementary data online , movie s1 . figure 2a shows a scatter plot indicating the relationship between t2-weighted imaging and ce - ssfp ( mean of two observers ) . there was a strong correlation between the two methods ( r = 0.89 , p < 0.01 ) . figure 2b shows the limits of agreement between t2-weighted imaging and ce - ssfp , demonstrating a bias of 3.0 4.0% of the lv ( p < 0.01 ) . for the slice - by - slice comparison , r was 0.69 ( p < 0.01 ; y = 0.98x + 0.61 ) with a bias of 1.8 16% per slice . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . the myocardium at risk by t2-weighted imaging and ce - ssfp . ( a ) mar by t2-weighted imaging ( t2w ) vs. ce - ssfp . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . the ce - ssfp imaging lasted for 24 min and the latest image was acquired 12 min after contrast agent administration . there was no change in the relationship between the two techniques as a function of time after contrast agent administration . this is illustrated in a patient with an lad occlusion in figure 3b . figure 3the mar by ce - ssfp as a function of time after gd administration . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) an example of a patient with an infarction within the lad territory . it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . in six patients , cine ssfp short - axis imaging was also performed prior to contrast agent administration . in five of six patients , for one patient , a slight hyperenhancement with indistinct borders was found within the mar . snr within the mar was 156 7 and 132 10 for the t2-weighted imaging and ce - ssfp , respectively ( mean sem ) . the cnr was 58 3 and 27 6 for the t2-weighted imaging and ce - ssfp , respectively ( mean sem ) . the contrast ratio between mar and remote myocardium for t2-weighted imaging was 1.7 0.3 compared with 1.5 0.4 for ce - ssfp , which was not statistically significant different ( p > 0.05 ) . the interobserver variability was 5.0 5.4% of the lv for t2-weighted imaging and 0.1 6.2% of the lv for ce - ssfp . the mean infarct size by lge was 14 11% ( range 149 ) of the lv . the infarct size was smaller in all patients when compared with mar assessed by t2-weighted imaging ( p < 0.01 ) and ce - ssfp ( p < 0.01 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . the non - coloured part of the myocardium at risk defines the salvaged myocardium . for this patient , ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . the non - coloured part of the myocardium at risk defines the salvaged myocardium . for this patient , ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . for the lge images , the snr within the infarction was 115 12 and the cnr was 73 8 . note that the lge images were acquired using parallel imaging increasing the noise levels in these images . the contrast ratio between remote and infarcted myocardium was 2.9 0.8 , which was significantly higher than for t2-weighted imaging and ce - ssfp ( p < 0.001 ) . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . infarct size could not be assessed due to poor image quality related to frequent arrhythmias . figure 1b shows three examples of the agreement of the mar by t2-weighted imaging and ce - ssfp . another example including the cine ce - ssfp can be seen in supplementary data online , movie s1 . figure 2a shows a scatter plot indicating the relationship between t2-weighted imaging and ce - ssfp ( mean of two observers ) . there was a strong correlation between the two methods ( r = 0.89 , p < 0.01 ) . figure 2b shows the limits of agreement between t2-weighted imaging and ce - ssfp , demonstrating a bias of 3.0 4.0% of the lv ( p < 0.01 ) . for the slice - by - slice comparison , r was 0.69 ( p < 0.01 ; y = 0.98x + 0.61 ) with a bias of 1.8 16% per slice . figure 2the myocardium at risk by t2-weighted imaging and ce - ssfp . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . the ce - ssfp imaging lasted for 24 min and the latest image was acquired 12 min after contrast agent administration . there was no change in the relationship between the two techniques as a function of time after contrast agent administration . this is illustrated in a patient with an lad occlusion in figure 3b . figure 3the mar by ce - ssfp as a function of time after gd administration . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) an example of a patient with an infarction within the lad territory . it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) an example of a patient with an infarction within the lad territory . it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . in six patients , cine ssfp short - axis imaging was also performed prior to contrast agent administration . in five of six patients , for one patient , a slight hyperenhancement with indistinct borders was found within the mar . snr within the mar was 156 7 and 132 10 for the t2-weighted imaging and ce - ssfp , respectively ( mean sem ) . the cnr was 58 3 and 27 6 for the t2-weighted imaging and ce - ssfp , respectively the contrast ratio between mar and remote myocardium for t2-weighted imaging was 1.7 0.3 compared with 1.5 0.4 for ce - ssfp , which was not statistically significant different ( p > 0.05 ) . the interobserver variability was 5.0 5.4% of the lv for t2-weighted imaging and 0.1 6.2% of the lv for ce - ssfp . the mean infarct size by lge was 14 11% ( range 149 ) of the lv . the infarct size was smaller in all patients when compared with mar assessed by t2-weighted imaging ( p < 0.01 ) and ce - ssfp ( p < 0.01 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . the non - coloured part of the myocardium at risk defines the salvaged myocardium . for this patient , t2-weighted imaging and ce - ssfp showed 52 and 55% myocardial salvage , respectively . ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . for this patient , t2-weighted imaging and ce - ssfp showed 52 and 55% myocardial salvage , respectively . ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . for the lge images , the snr within the infarction was 115 12 and the cnr was 73 8 . note that the lge images were acquired using parallel imaging increasing the noise levels in these images . the contrast ratio between remote and infarcted myocardium was 2.9 0.8 , which was significantly higher than for t2-weighted imaging and ce - ssfp ( p < 0.001 ) . this study explored the agreement between mar assessed by t2-weighted imaging and mar assessed by ce - ssfp . the agreement was shown to be good , both for the assessment of mar and myocardial salvage index . both t2-weighted imaging and ce - ssfp have previously been validated against mar assessed by myocardial perfusion spect , with a mean difference of 2.3 5.7 and 0.5 5.1% of the lv , respectively . in accordance with these results , the present study found a small bias of 3.0 4.0% when t2-weighted imaging was compared with ce - ssfp . this is , however , the first time these two techniques have been compared head - to - head which is important in order to ensure that both techniques can be used interchangeably in clinical cardioprotection trials using myocardial salvage as endpoint . the correlation between mar by t2-weighted imaging and ce - ssfp was not as strong for the slice - by - slice comparison as for the global measure of mar normalized to the entire lv . as the heart rate varies , the timing within the cardiac cycle at which the t2-weighted image is acquired varies resulting in difficulties with local registration of the myocardium when comparing the two techniques . even though the slice position was approximately the same for the two acquisitions , the part of the myocardium depicted may differ due to av - plane movement during the cardiac cycle . this is not a problem when comparing global measures of mar normalized to the entire lv . more recently , bright - blood t2-weighted sequences have been developed to increase the diagnostic performance of t2 cmr for oedema depiction . in a recent study by payne et al . , dark - blood t2-stir was shown to underestimate the mar with 9% units compared with the recently introduced bright - blood t2-weighted sequence ( acut2e ) . it was concluded that acut2e was more accurate for the determination of mar and myocardial salvage than was dark - blood t2-stir . thus , ce - ssfp might be more accurate than dark - blood t2 in the present study since ce - ssfp showed larger mar and , previously , showed no bias compared with myocardial perfusion spect . the pathophysiological basis for the enhanced myocardium observed using t2-weighted imaging is still not completely understood . following acute coronary occlusion , the ischaemic myocardium shifts from aerobic metabolism to anaerobic glycolysis and ceases to contract . this failure of the energy - regulated membrane channels results in swelling of the myocytes due to influx of water and sodium . furthermore , reperfusion leads to inflammatory - like response , increasing the amount of extracellular fluid . this increased water content in the affected myocardium is likely to explain the increased signal intensity compared with the non - affected myocardium as seen by t2-weighted imaging . the ischaemic episode causes post - ischaemic stunning , associated with a decreased contractility in the previously ischaemic myocardium . this decreased contractility is likely associated with a decreased lymphatic drainage from this part of the myocardium , which may also contribute to residual increased water content 1 week after the acute event . the mechanisms behind the enhanced myocardium observed in ce - ssfp are not completely known either . the contrast in ssfp images is dependent on the t2/t1 ratio . in the presence of paramagnetic gadolinium this is utilized for infarct visualization in t1-weighted inversion - recovery lge imaging , where the concentration of an extracellular gadolinium - based contrast agent is increased due to an increased distribution volume in ischaemically injured myocardium . it has been shown that even reversibly injured myocardium within the mar has an increased distribution volume in the acute phase after an ischaemic episode . hence , the t2/t1 ratio in the entire mar , including both reversible and irreversible injured myocardium , is affected by the presence of gadolinium . this might explain the increased signal intensity in the mar seen by ce - ssfp . furthermore , it has been shown that the relationship between the change in t1-relaxation rates before and after contrast agent administration ( r1 ) in different parts of the myocardium ( remote , salvaged , and infarcted ) in relation to r1 in blood ( r1 ratios ) remains constant from 4 to 29 min after contrast agent administration in acute myocardial infarction . these earlier findings indicate that the rate of exchange of contrast agent between the myocardium ( normal and injured ) and the blood pool is constant and much faster than the clearance rate in the kidneys during the first 30 min after contrast agent administration . . recently showed similar findings supporting this concept using t1 mapping before and after contrast administration in experimentally induced infarction . thus , this can explain why the relationship between mar assessed by t2-weighted imaging and ce - ssfp did not change with time after contrast agent administration in the present study ( figure 3 ) and why the timing of ce - ssfp after contrast agent administration is not critical . therefore , ce - ssfp could potentially be added to clinical protocols so that cine imaging is acquired after contrast agent administration for the assessment of lv function and mar . one advantage with ce - ssfp imaging is that this technique is based on a multi - phase acquisition throughout the cardiac cycle . this enables tracking of the mar and myocardial borders in multiple time frames ( see supplementary data online , movie s1 ) , making delineation of both mar and myocardial borders more robust . in the present protocol , mar in the ce - ssfp images were traced in both end - systole and end - diastole and averaged . another situation where ce - ssfp could be advantageous is when limited time for scanning is available , due to heavy clinical load or an unstable patient . in such a situation , gadolinium can be injected prior to the examination and the imaging protocol can be shortened since lv dimensions / function and mar can be assessed from the same set of images . on the other hand , t2-weighted imaging for the determination of mar can be performed in those patients where administration of a gadolinium - based contrast agent is contraindicated . thus , there are several advantages of having access to more than one method for determination of mar by cmr and we recommend both t2-weighted imaging and ce - ssfp to be implemented in the imaging protocol . owing to the relatively small differences in signal intensity between mar and remote myocardium , we recommend not to use parallel imaging when acquiring ce - ssfp images as this decreases the snr . no significant difference in the contrast ratio between t2-weighted imaging and ce - ssfp imaging was found in the present study where no parallel imaging was performed . the present study was performed on a limited number of stemi patients , all undergoing successful reperfusion . thus , how this would translate to a non - stemi population or to patients treated with thrombolytic therapy is not known . since only a few female patients where included , gender perspective can not be evaluated . no semi - quantitative method was used to determine the mar by both t2-weighted imaging and ce - ssfp , since the signal intensities of the mar and the remote myocardium varied between slices and between patients , making it difficult to choose a fixed standard deviation of signal intensities to differentiate mar from remote myocardium . there is a good agreement between mar assessed by t2-weighted imaging and mar assessed by ce - ssfp in patients with reperfused acute myocardial infarction 1 week after the acute event . thus , both methods can be used to determine mar and subsequently myocardial salvage at this point in time . this study was funded by the swedish research council ( 521 - 2008 - 2461 ) , the swedish heart and lung foundation , the medical faculty at lund university , sweden , european commission fp7 consortium cardiocell , region of scania , sweden , the stockholm county council and karolinska institutet / stockholm county council strategic cardiovascular program .

aimsto determine the myocardial salvage index , the extent of infarction needs to be related to the myocardium at risk ( mar ) . thus , the ability to assess both infarct size and mar is of central clinical and scientific importance . the aim of the present study was to explore the relationship between t2-weighted cardiac magnetic resonance ( cmr ) and contrast - enhanced steady - state free precession ( ce - ssfp ) cmr for the determination of mar in patients with acute myocardial infarction.methods and resultstwenty - one prospectively included patients with first - time st - elevation myocardial infarction underwent cmr 1 week after primary percutaneous coronary intervention . for the assessment of mar , t2-weighted images were acquired before and ce - ssfp images were acquired after the injection of a gadolinium - based contrast agent . for the assessment of infarct size , late gadolinium enhancement images were acquired . the mar by t2-weighted imaging and ce - ssfp was 29 11 and 32 12% of the left ventricle , respectively . thus , the mar with t2-weighted imaging was slightly smaller than that by ce - ssfp ( 3.0 4.0% ; p < 0.01 ) . there was a significant correlation between the two mar measures ( r2= 0.89 , p < 0.01 ) , independent of the time after contrast agent administration at which the ce - ssfp was commenced ( 28 min).conclusionthere is a good agreement between the mar assessed by t2-weighted imaging and that assessed by ce - ssfp in patients with reperfused acute myocardial infarction 1 week after the acute event . thus , both methods can be used to determine mar and myocardial salvage at this point in time .

Introduction Methods Study population Cardiovascular magnetic resonance Image analysis Statistics Results Myocardium at risk Myocardial salvage index Discussion Study limitations Conclusions Supplementary data Funding Supplementary Material

many studies investigating the relationships between skin aging and environmental factors have been reported.14 these studies have indicated that environmental factors , including ultraviolet irradiation and cigarette smoking , markedly influence skin aging . other environmental factors , such as body mass index ( bmi ) , alcohol consumption , and marital status have also been suggested to be associated with skin aging.2,5,6 twin studies , especially those involving monozygotic ( mz ) twins , provide unique opportunities to control for age , gender , and genetic effects in the analysis of the effects of environmental factors on skin aging.5,7 in the present study , we conducted medical interviews and facial photographic assessments of 67 pairs of adult , japanese , mz twins between 2010 and 2013 . this study , conducted at the osaka twin research center , evaluated the influence of environmental factors on skin aging . using a complexion analysis system , the subjects included 67 pairs of adult , mz twins visiting the osaka twin research center . the research center was set up in 2010 at osaka university , to provide comprehensive medical examinations to japanese mz and dizygotic adult twins . volunteers for the twin study were recruited from the general community through various media advertisements . the study was approved by the osaka university ethics committee , and all of the participants provided written , informed consent . the mz twins included 27 pairs of men and 40 pairs of women , aged 4087 years . the subjects participated in medical interviews and photographic assessments designed to analyze the important visible features of facial skin aging . the medical interviews were conducted using a structured questionnaire to collect information regarding : age , alcohol consumption , smoking history , marital status , hormone replacement therapy , medical history , sun exposure history , bmi , and sunscreen or foundation use . photographic assessments were performed using the visia complexion analysis system ( canfield scientific . , fairfield , nj , usa ; fig . 1).810 the visia system , with a configurable head support , ensured consistent positioning of each subject 's head . the subjects cleaned their skin with a gentle facial makeup remover before the image was obtained . the photographic images were captured with standard , cross - polarized , parallel polarized , and ultraviolet light . images were taken in two different close - up views ( right lateral 37 , left lateral 37 ) for each subject to quantify the scores and percentiles of the cheek skin , including spots , wrinkles , pores , texture , and erythema . the percentiles enabled the evaluation of the subject 's complexion analysis results through a comparison between the individual 's scores and those of people of the same sex , generation , and skin type in the database . the scores provided a comprehensive measurement of the impact that each feature had on the client 's complexion . the scores factored in the total size and area , as well as the intensity of the feature being analyzed . skin with a lower score for each factor was considered to be more youthful in appearance ( values ranged from 0 to 100 ) . in the present study , we used the scores to more objectively assess skin condition . skin texture represents the degree of uniformity of the surface of cheek skin . a subject with shallow sulcus cutis and closely shaped crista cutis skin wrinkle indicated to folds resulting from dermal connective tissues including elastic and collagen fibers . the average scores for the right and left cheeks were calculated , and the differences between individuals in each pair of twins were recorded and chronologically compared . in addition , the results of the pairs of twins who provided answers to the medical interviews were compared with the visia data to explore the effects of environmental factors on their facial features . the twin pairs who differed by more than 4 bmi points from each other were also compared . all calculations were performed with the jmp statistical software package ( sas , cary , nc , usa ) . the data were analyzed using pearson 's correlation coefficient test and the wilcoxon signed - rank test ; a p - value of < 0.05 was considered statistically significant . of the 67 pairs of mz twins , two male pairs were excluded from the analysis due to the interference of their mustaches , with the photographic assessments . therefore , 25 pairs of men and 40 pairs of women were evaluated in this study . the characteristics of the evaluated subjects , based on their medical interviews , are presented in table 1 . the subjects ranged in age from 40 to 87 years and represented all the regions in japan . alcohol consumption , smoking histories , and outdoor work were more frequent among men than women , although sunscreen or foundation use was more frequent in women . in 11 twin pairs , the individuals differed from each other by more than 4 bmi points . baseline characteristics of subjects by using the visia system , differences in facial feature scores ( facial spots , wrinkles , pores , texture , and erythema ) in each pair of twins were calculated and chronologically compared , by comparing the results for twins of various ages . 2 . as the ages of the twins increased , the intrapair differences in facial texture scores also increased ( p = 0.03 ) . however , significant correlations between increasing ages and intrapair differences in facial spots , wrinkles , pores , and erythema were not observed ( p = 0.27 , 0.08 , 0.63 , and 0.23 , respectively ) . the differences in the visia scores for spots , wrinkles , pores , texture , and erythema in twin pairs . as the age of the twin increased , the intrapair differences in texture scores significantly increased as well ( p = 0.03 ) . alcohol consumption , smoking history , hormone replacement therapy , sun exposure history , sunscreen or foundation use , marital status , medical history , and bmi were also investigated for their effects on facial skin aging . comparing the visia scores of each factor in the twins of each pair , the twins who smoked showed significantly higher facial texture scores compared with the twins who were nonsmokers ( p = 0.04 ) . the twins who did not use sunscreen or foundation also showed significantly higher facial wrinkle scores compared with the twins who used these products ( p = 0.03 ) ( table 2 ) . there were no significant intrapair differences in the other environmental factors , namely alcohol consumption , hormone replacement therapy , sun exposure history , marital status , medical history , or bmi . comparison of the visia scores of each factor in the twins of each pair who provided answers to the medical interviews the numbers are the average scores of each twin . the bmi ( large ) was > 4 bmi points of the bmi ( small ) . the characteristics of the evaluated subjects , based on their medical interviews , are presented in table 1 . the subjects ranged in age from 40 to 87 years and represented all the regions in japan . alcohol consumption , smoking histories , and outdoor work were more frequent among men than women , although sunscreen or foundation use was more frequent in women . in 11 twin pairs , the individuals differed from each other by more than 4 bmi points . by using the visia system , differences in facial feature scores ( facial spots , wrinkles , pores , texture , and erythema ) in each pair of twins were calculated and chronologically compared , by comparing the results for twins of various ages . 2 . as the ages of the twins increased , the intrapair differences in facial texture scores also increased ( p = 0.03 ) . however , significant correlations between increasing ages and intrapair differences in facial spots , wrinkles , pores , and erythema were not observed ( p = 0.27 , 0.08 , 0.63 , and 0.23 , respectively ) . the differences in the visia scores for spots , wrinkles , pores , texture , and erythema in twin pairs . as the age of the twin increased , the intrapair differences in texture scores significantly increased as well ( p = 0.03 ) . alcohol consumption , smoking history , hormone replacement therapy , sun exposure history , sunscreen or foundation use , marital status , medical history , and bmi were also investigated for their effects on facial skin aging . comparing the visia scores of each factor in the twins of each pair , the twins who smoked showed significantly higher facial texture scores compared with the twins who were nonsmokers ( p = 0.04 ) . the twins who did not use sunscreen or foundation also showed significantly higher facial wrinkle scores compared with the twins who used these products ( p = 0.03 ) ( table 2 ) . there were no significant intrapair differences in the other environmental factors , namely alcohol consumption , hormone replacement therapy , sun exposure history , marital status , medical history , or bmi . comparison of the visia scores of each factor in the twins of each pair who provided answers to the medical interviews the numbers are the average scores of each twin . marital status ( + ) twin showed the twins who were married . the bmi ( large ) was > 4 bmi points of the bmi ( small ) . skin aging is influenced by the interaction of both intrinsic and extrinsic factors.11,12 intrinsic aging is an inevitable process that is regulated by a genetically programmed pattern , characterized by cellular senescence , decreased proliferative capacity , decreased cellular dna repair capacity , oxidative stress , and gene mutations . extrinsic aging is regulated by various external factors such as ultraviolet irradiation , cigarette smoking , alcohol consumption , marital status , and medical history.2,5,6,13 skin aging is manifested as spots , wrinkles , pores , coarseness , and telangiectasia , which are significantly correlated with the appearance of elderly individuals.14,15 therefore , accurate assessment of facial features is of great importance for esthetic surgeries and may also provide important information about several skin disorders . there have been many reports regarding the relationship between skin aging and environmental factors.14,16 up to 40% of the changes that contribute to an aged appearance are reported to be due to nongenetic factors.2,15 the first proposed correlation between smoking and skin aging was made by solly in 1856.17 sun exposure has also been associated with an older appearance and has been suggested to accelerate with age . similarly , a history of outdoor activities and failure to use sunscreen has been correlated with an older appearance.5,16 guyuron et al.6 reported that the use of hormone replacements helped to preserve a younger appearance . the use of antidepressants , various diseases ( diabetes , asthma , and cardiovascular disease ) , marital status , alcohol consumption , and bmi have also been suggested to be associated with skin aging.2,5,6 despite the number of studies on skin aging , the effects of various environmental factors are still not conclusively understood . the study of twins , especially mz twins , provides a unique opportunity to control for age , gender , and genetic susceptibility to aging in order to analyze environmental influences on aging . gunn et al . performed heritability analyses of skin aging features in the twin population and demonstrated that 4160% of the variations in sun damage , skin wrinkling , wrinkle depth , and pigmented age spot measurements were explained by genetic factors.12 shekar et al.18 concatenated the variations in epidermal reticular patterning into genetic and environmental influences and estimated the concomitant effects of sun exposure and skin color in mz twins . the objective evaluation of the characteristic features of the facial skin , however , has rarely been reported . the visia complexion analysis system enables the procurement of high - quality digital facial photographs with standardized lighting and configurable head positioning . the images obtained allow the quantitation of the physical properties of the cheek skin , such as spots , wrinkles , pores , texture , and erythema . differences in these properties between individual mz twins were objectively analyzed , in this study , by the visia system . the results indicated that as the age of the twins increased , the intrapair differences in facial texture scores also increased significantly ( p = 0.03 ) , suggesting that facial texture tends to be influenced by environmental factors . environmental factors , including smoking and failure to use sunscreen or foundation , resulted in significantly higher facial texture and wrinkle scores ( p = 0.04 and 0.03 , respectively ) . alcohol consumption , hormone replacement use , sun exposure history , marital status , medical history , and bmi did not demonstrate a significant correlation with facial skin aging . these results did not completely support previous reports , possibly due to the more objective assessments achieved using the visia system . if the sample sizes for most factors were sufficiently large , the intrapair differences of most environmental factors would be expected to be more significant . moreover , if more male subjects were included , the impact of additional environmental factors might be more apparent . further investigations may help clarify the relationship between other environmental factors and skin aging in mz twins . as the age of the twins increased , facial texture scores were less similar between individual twins , within a pair . this suggests that facial texture is influenced by environmental factors rather than solely by genetic factors . moreover , smoking and the failure to use skin protection resulted in significantly higher facial texture and wrinkle scores .

twin studies , especially those involving monozygotic ( mz ) twins , facilitate the analysis of factors affecting skin aging while controlling for age , gender , and genetic susceptibility . the purpose of this study was to objectively assess various features of facial skin and analyze the effects of environmental factors on these features in mz twins . at the osaka twin research center , 67 pairs of mz twins underwent medical interviews and photographic assessments , using the visia complexion analysis system . first , the average scores of the right and left cheek skin spots , wrinkles , pores , texture , and erythema were calculated ; the differences between the scores were then compared in each pair of twins . next , using the results of medical interviews and visia data , we investigated the effects of environmental factors on skin aging . the data were analyzed using pearson 's correlation coefficient test and the wilcoxon signed - rank test . the intrapair differences in facial texture scores significantly increased as the age of the twins increased ( p = 0.03 ) . among the twin pairs who provided answers to the questions regarding history differences in medical interviews , the twins who smoked or did not use skin protection showed significantly higher facial texture or wrinkle scores compared with the twins not exposed to cigarettes or protectants ( p = 0.04 and 0.03 , respectively ) . the study demonstrated that skin aging among japanese mz twins , especially in terms of facial texture , was significantly influenced by environmental factors . in addition , smoking and skin protectant use were important environmental factors influencing skin aging .

Introduction Patients and methods Results Subject's baseline characteristics VISIA complexion analysis system assessments Environmental factors Discussion

all animal procedures were performed in a facility accredited by the association for assessment and accreditation of laboratory animal care international in accordance with protocols approved by ut austin ( # 2012 - 00084 , # 13080701 ) and the university of oklahoma health sciences center ( # 14 - 072-i ) animal care and use committee and the principles outlined in the guide for the care and use of laboratory animals . a total of 15 baboons between six and nine months old were infected intra - nasally and intra - tracheally with 1010 cfu b. pertussis strain d420 on day 0 . of these , eight animals were treated with human anti - ptx antibodies on day 2/3 ( denoted t1-t8 ) , while the remaining seven were untreated controls ( denoted c1-c7 ) . serial dilutions of nasopharyngeal washes were plated on selective regan - lowe agar plates supplemented with cephalexin ( 40 g / ml ) . the specific details of procedures performed at the oklahoma baboon research resource at the university of oklahoma health sciences center have been described previously . microbial identification was performed by idexx ( westbrook , me , u.s.a . ) using maldi - tof for a preliminary identification based largely on 16s ribosomal rna followed by a panel of standard microbiological tests , including gram stain , lactose fermentation on macconkey agar , urease and oxidase activity and the triple sugar iron test . elisa was used to detect and quantify antibodies binding fha , los and ptx antigens from b. pertussis ( list labs , campbell , ca , u.s.a . ) as described . curves were fit using a 4-parameter logistic model and compared to a reference monoclonal human antibody or high titer baboon serum run on each plate . the reference was standardized by comparison to the who reference reagent pertussis antiserum 06/142 ( nibsc , potters bar , hertfordshire , u.k . ) , and serum antibody concentrations converted to iu / ml . day 0 serum anti - fha titers were log - transformed , and the values for baboons in the typical and atypical groups were found to be statistically different with a p - value of < 0.001 in a t - test . for los elisas , plates were coated with 5 g / ml los , with results reported as raw absorbance values in the dose - response regime at a serum dilution of 1:625 . all elisas were performed in duplicate with standard deviations of the measurements reported as error . all animal procedures were performed in a facility accredited by the association for assessment and accreditation of laboratory animal care international in accordance with protocols approved by ut austin ( # 2012 - 00084 , # 13080701 ) and the university of oklahoma health sciences center ( # 14 - 072-i ) animal care and use committee and the principles outlined in the guide for the care and use of laboratory animals . a total of 15 baboons between six and nine months old were infected intra - nasally and intra - tracheally with 1010 cfu b. pertussis strain d420 on day 0 . of these , eight animals were treated with human anti - ptx antibodies on day 2/3 ( denoted t1-t8 ) , while the remaining seven were untreated controls ( denoted c1-c7 ) . serial dilutions of nasopharyngeal washes were plated on selective regan - lowe agar plates supplemented with cephalexin ( 40 g / ml ) . the specific details of procedures performed at the oklahoma baboon research resource at the university of oklahoma health sciences center have been described previously . microbial identification was performed by idexx ( westbrook , me , u.s.a . ) using maldi - tof for a preliminary identification based largely on 16s ribosomal rna followed by a panel of standard microbiological tests , including gram stain , lactose fermentation on macconkey agar , urease and oxidase activity and the triple sugar iron test . elisa was used to detect and quantify antibodies binding fha , los and ptx antigens from b. pertussis ( list labs , campbell , ca , u.s.a . ) as described . curves were fit using a 4-parameter logistic model and compared to a reference monoclonal human antibody or high titer baboon serum run on each plate . the reference was standardized by comparison to the who reference reagent pertussis antiserum 06/142 ( nibsc , potters bar , hertfordshire , u.k . ) , and serum antibody concentrations converted to iu / ml . day 0 serum anti - fha titers were log - transformed , and the values for baboons in the typical and atypical groups were found to be statistically different with a p - value of < 0.001 in a t - test . for los elisas , plates were coated with 5 g / ml los , with results reported as raw absorbance values in the dose - response regime at a serum dilution of 1:625 . all elisas were performed in duplicate with standard deviations of the measurements reported as error . during a series of studies designed to assess the potential for passive immunization to treat pertussis , several baboons had unexpectedly variable wbc and bacterial colonization levels 2-to-3 days after inoculation with b. pertussis ( fig . 1afig . 1.serum antibody levels indicate susceptibility to pertussis infection . a : wbc and b. pertussis colonization levels were measured on day 2 or 3 after experimental infection . levels typical of the baboon model on day 2/3 are shown for wbc ( > 13,000/l ; solid line ) and colonization ( > 10/ml , dashed line ) . baboons exhibiting expected symptoms fell into the upper right quadrant , while baboons that appeared to be protected from disease fell into the lower left quadrant . b : the day 9/10 los elisa absorbance values correlated with the log - transformed day 9/10 fha antibody responses with a pearson correlation coefficient of 0.90 . animals exhibiting low values in both assays were classified as nave , while animals exhibiting high values in both assays were classified as previously exposed . c : anti - fha antibody sera concentrations collected on day 0 correlated with typical and atypical model responses ; * * * p<0.001 in a t - test of the log - transformed concentration values . typical responses include high levels of colonization , a marked wbc rise and lack of a secondary antibody response . shown are responses for animals without b. bronchiseptica co - infection ; bars indicate geometric means . ) . six of 15 animals exhibited typical responses for this model : namely , a rapidly rising wbc and high bacterial colonization level on day 2/3 . however , six animals had low wbc and colonization , one was heavily colonized but had a low wbc count , and two were not heavily colonized but had a two - to - four - fold wbc rise . additionally , nasopharyngeal wash samples from four baboons ( c2 , c3 , c4 and t5 ) yielded rapidly growing colonies apparent one day after plating on regan - lowe media selective for bordetella species and overgrew bordetella pertussis colonies appearing one to three days later . serum antibody levels indicate susceptibility to pertussis infection . a : wbc and b. pertussis colonization levels were measured on day 2 or 3 after experimental infection . levels typical of the baboon model on day 2/3 are shown for wbc ( > 13,000/l ; solid line ) and colonization ( > 10/ml , dashed line ) . baboons exhibiting expected symptoms fell into the upper right quadrant , while baboons that appeared to be protected from disease fell into the lower left quadrant . b : the day 9/10 los elisa absorbance values correlated with the log - transformed day 9/10 fha antibody responses with a pearson correlation coefficient of 0.90 . animals exhibiting low values in both assays were classified as nave , while animals exhibiting high values in both assays were classified as previously exposed . c : anti - fha antibody sera concentrations collected on day 0 correlated with typical and atypical model responses ; * * * p<0.001 in a t - test of the log - transformed concentration values . typical responses include high levels of colonization , a marked wbc rise and lack of a secondary antibody response . shown are responses for animals without b. bronchiseptica co - infection ; bars indicate geometric means . cultures of the rapidly growing bacteria from animal c4 were positively identified as b. bronchiseptica . maldi - tof was used to make a preliminary species identification , followed by a panel of biochemical tests to confirm the bordetella assignment and discriminate between b. bronchiseptica and b. pertussis . the results showed that the cultured organism was gram negative , did not ferment lactose on macconkey agar , did not ferment lactose , sucrose or dextrose but catabolized peptone in the triple sugar iron test ( k / k result ) and was positive for oxidase and urease . notably , b. bronchiseptica but not b. pertussis is urease - positive , and only b. bronchiseptica has flagella and is motile . while not definitively confirmed , the other three animals were strongly suspected of b. bronchiseptica coinfection by virtue of similar culture characteristics . in all , nine of 15 baboons behaved aberrantly in this model ( table 1table 1.baboon responses to experimental pertussis infectionbaboonactive b. br infection anti - fha titer>100 iu / mlanti - los absorbance > 0.1 wbc < 13,000/lcfu < 10/mlresponse to pertussis infection c1 - ----typicalc2yes----excludedc3yes -- yes - excludedc4yes*yesyesyesyesexcludedc5-yesyesyesyesatypicalc6 - ----typicalc7 - ----typicalt1-yesyesyesyesatypicalt2-yesyesyesyesatypicalt3-yesyesyesyesatypicalt4-yesyesyesyesatypicalt5yesyesyes - yesexcludedt6-yesyes - yesatypicalt7 - ----typicalt8 - ----typicala ) b. br = b. bronchiseptica ; -animal did not exhibit this behavior ; * = b . b ) anti - fha and anti - los titers were measured in serum samples collected on day 9/10 after experimental pertussis infection . d ) typical baboon responses to experimental pertussis infection are defined by wbc > 13,000 /l , cfu > 106/ml on day 2/3 and low fha and los serum titers ; animals with known or suspected b. bronchiseptica infection were excluded from this classification . , supplementary table 1 ) . a ) b. br = b. bronchiseptica ; -animal did not exhibit this behavior ; * = b . b ) anti - fha and anti - los titers were measured in serum samples collected on day 9/10 after experimental pertussis infection . d ) typical baboon responses to experimental pertussis infection are defined by wbc > 13,000 /l , cfu > 106/ml on day 2/3 and low fha and los serum titers ; animals with known or suspected b. bronchiseptica infection were excluded from this classification . with compelling evidence of an active b. bronchiseptica infection in several animals , we sought to determine whether any other baboons in this study had been previously exposed to the organism . this question was addressed by examining antibody titers to fha and los prior to infection and the kinetics by which those titers rose after infection . specifically , high titers before pertussis infection and a rapid rise after infection indicate a secondary immune response and strongly suggest prior b. bronchiseptica exposure . prior experiments indicated that nave baboons do not exhibit primary responses to fha until after day 12 , while secondary responses are detectable on day five [ 9 , 13 ] . thus , a high antibody titer on day 9/10 to fha indicates prior exposure to any bordetella species , while a similar response against los indicates prior exposure to either b. bronchiseptica or b. pertussis . detection of antibodies against ptx at day 9/10 was used to identify prior exposure to b. pertussis as opposed to b. bronchiseptica , which does not express this antigen due to mutations in the promoter . accordingly , serum titers against fha and los on day 9/10 were determined for each baboon serum sample . comparison of the resulting anti - fha and anti - los serum titers revealed two distinct groups : seven baboons with low anti - fha and low anti - los titers and eight baboons with high titers for both bordetella antigens ( pearson correlation 0.90 ; fig . next , anti - fha titers were determined for all time points , as these responses were stronger than those recognizing los . animals with high anti - fha titers on day 9/10 also exhibited high titers early in the experiment , and these remained high for the ~21 day experiment ( supplementary fig . six of the seven baboons with no apparent secondary response had rising anti - fha titers starting around day 16 or later , indicative of a primary response to the experimental infection . one baboon , c2 , had no response at day 9/10 , but a very strong response starting at day 13 . rapidly growing bordetella colonies were recovered from this animal on day 9 , suggestive of b. bronchiseptica co - infection . only the seven untreated baboons could be analyzed for the presence of anti - ptx igg on day 9/10 due to interference from humanized anti - ptx antibodies administered to the treated baboons . one baboon , c5 , was positive for anti - ptx antibodies as well as anti - fha and anti - bp los antibodies on day 9/10 ( supplementary table 1 , supplementary fig . c5 had a colony count of 5 cfu / ml on day 2/3 and no wbc rise , indicating near complete protection from b. pertussis colonization . while we can not exclude the possibility that this baboon was exposed to b. bronchiseptica as well , this result indicates that c5 had likely been previously exposed to b. pertussis . we next aimed to develop a quantitative description of typical versus atypical baboon responses to pertussis infection . published data [ 14 , 16 ] indicate typical responses on day 2/3 include bacterial colonization > 10 cfu / ml , an increase in wbc count to > 13,000/l and the absence of secondary immune responses to bordetella antigens . atypical responses do not meet one of more of these metrics ( table 1 ) . baboons with confirmed or suspected active b. bronchiseptica infection ( c2 , c3 , c4 and t5 ) were excluded from this analysis to avoid potential confounding factors , such as competition with b. pertussis , unpredictable wbc rise and unpredictable timing of the secondary antibody response . for future studies , a predictive test to exclude baboons likely to exhibit atypical responses to b. pertussis infection thus , the presence of serum anti - fha antibodies on day 0 was evaluated as a potential screening tool . when baboons with active b. bronchiseptica infections were removed from analysis , anti - fha antibody presence on day 0 was significantly predictive of the baboon s subsequent responses ( p<0.001 ; fig . the baboon model of pertussis brings substantial improvements over previous models and has generated considerable enthusiasm due to its similarity to human disease . unfortunately , the majority of baboons ( nine of 15 ) in this study did not develop symptoms as expected ( fig . 1a ) . in an effort to improve the utility of this model , we aimed to understand why some baboons varied in their response to b. pertussis infection . three baboons ( c3 , c4 and t5 ) were likely infected with b. bronchiseptica when the study began and were partially protected against b. pertussis infection . prior studies in mice have shown that exposure to an engineered b. pertussis strain can protect against subsequent b. bronchiseptica infection ; conversely , an engineered b. bronchiseptica strain can protect against b. pertussis infection . there are numerous overlapping antigens between these subspecies which are likely responsible for cross - protection . specifically , anti - los and anti - pertactin antibodies are often bactericidal and importantly , they would be expected to have the substantial impact on subsequent b. pertussis infection levels and symptoms caused by infection observed in this study . additionally , baboon c2 was cohoused with b. bronchiseptica infected baboon c3 and appears to have been infected during the study , as this organism was not observed on plates until day 9 . an additional six animals were suspected of prior b. bronchiseptica or b. pertussis exposure , based on antibody profiles indicative of secondary responses after experimental b. pertussis infection ( fig . these animals all exhibited lower than expected colonization levels either with or without a suppressed wbc rise . while informative retrospectively , these data can not be used to screen baboons prior to initiating an experiment . to this end , day 0 anti - fha antibody concentrations were predictive of the baboons subsequent response to infection , although these exhibited greater variability than day 9/10 titers ( fig . because fha is a shared antigen present in b. pertussis , b. parapertussis and b. bronchiseptica , it is particularly useful as a simple screen to rule out confounding prior exposures for studies involving b. pertussis infection . while prior bordetella exposure complicates scientific studies , it further supports the baboon model s relevance to human disease . in humans , whooping cough in humans can be difficult to diagnose , because of widely varying infection severity and clinical manifestations which depend on the patient s prior exposures to b. pertussis and other microbes with overlapping antigens and timing of that exposure , exact age and level of general health . these factors may play a similar role in the diversity of the baboon responses seen here and must be carefully controlled . in comparison to other model animals used for pertussis , baboons are typically housed with access to the outdoors where small rodents , such as rabbits or mice , for whom b. bronchiseptica is endemic could pass disease to the research animals . in our hands , commercial baboon serum ( sigma ) had a relatively high anti - fha antibody concentration of ~90 iu / ml ( supplementary table 1 ) , indicating that bordetella infections are likely common in baboon colonies . the well - documented , highly communicable nature of both b. pertussis infection in baboons and b. bronchiseptica infection in other mammals requires extremely careful handling , containment and observation of baboons involved in pertussis studies . the university of oklahoma health science center is home to the only specific pathogen free indoor baboon colony that was the source of baboons c7 , t7 and t8 . these three specific pathogen free baboons responded to infection with b. pertussis as expected and were typical of the model . in our ongoing work , neonatal baboons from the specific pathogen free colony are pre - screened for anti - fha titer and consistently fall below 5 iu / ml at the time of infection and exhibit high colonization as expected ( data not shown ) . to maximize the impact of pertussis experiments in baboons , we recommend the following baboon exclusion criteria : ( 1 ) clinical evidence of illness , ( 2 ) b. bronchiseptica - culture positive nasopharyngeal wash and ( 3 ) anti - fha titers > 5 iu / ml indicating prior exposure to bordetella sp . with close attention to these factors , the baboon model of pertussis is poised to be an important new tool in development of next - generation pertussis vaccines and therapeutics . funding . this work was supported by national institutes of health [ ai066239 and ai122753 to j.a.m . and p40od010431 and p40od010988 to r.f.w . ] and synthetic biologics [ j.a.m . ] . conflicts of interest . this work was supported in part by funding from synthetic biologics .

the baboon model of bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date . however , among the 15 experimentally infected baboons in this study , a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count . moreover , cultures of nasopharyngeal wash samples from several baboons suggested b. bronchiseptica coinfection . analysis of serum antibodies recognizing filamentous hemagglutinin , pertussis toxin and b. pertussis lipo - oligosaccharide indicated that several baboons had likely been previously exposed to bordetella species and that prior exposure correlated with partial protection from b. pertussis infection . notably , all animals with a baseline fha titer of 5 iu / ml or below exhibited symptoms typical of the model , suggesting this value can be used as inclusion criteria for animals prior to study enrollment . while b. pertussis infection is endemic to human populations and b. bronchiseptica is common in wild small mammals , this study illustrates that baboons can readily harbor both organisms . awareness of bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis .

MATERIALS AND METHODS Ethics statement Baboon challenge study Detection of serum antibodies RESULTS DISCUSSION Supplementary

traditionally , post bronchodilator forced expiratory volume in 1 second ( pb fev1 ) is used to assess the severity of chronic obstructive pulmonary disease ( copd ) . however , it has now been shown that functional exercise capacity is a strong predictor of survival following pulmonary rehabilitation and so assessment of functional status is very important for proper prescription of medical therapy and rehabilitation programs . van stel et al . suggested that 6 min walk test ( 6mwt ) was an easy to perform procedure and was able to predict functional status in all patients with chronic respiratory disease ; however , pinto - plata et al . argued that walking by itself might not reflect the true functional status in these patients as a person has to do several other activities in addition to walking . a sit - to - stand test ( stst ) was proposed as a better alternative to 6mwt , but it lacks universal application , more specifically in countries like india where people resort to squat ( sit on the floor ) rather than sitting on chair . a more practical approach for them will be to assess the functional ability to stand from the squatting position , i.e. , squat - to - stand test ( sqtst ) . therefore , a study was undertaken to compare sqtst with pb fev1 , 6mwt , and stst and to assess whether it is a feasible and effective tool in evaluating the functional status of the copd patients . all patients attending the outpatient department of respiratory medicine , national institute of medical sciences , jaipur , with clinical history , consistent with copd , were recruited . twenty age- and sex - matched , healthy and nonsmoking attendants / hospital staff were also recruited to serve as controls . was also taken from all the patients and controls after duly explaining the study protocol . all the recruited patients were evaluated in detail , including present and past clinical history , physical examination , complete blood counts , random blood sugar , renal and liver function tests , two sputum smears for acid - fast bacillus using ziehl neelsen staining , skiagram chest posteroanterior view , and a standard electrocardiogram . patients showing obvious pulmonary or other system abnormalities such as active pulmonary tuberculosis , malignancy , diabetes mellitus , coronary artery disease , stroke , and renal or hepatic disease were excluded . the remaining patients were subjected to spirometry including the reversibility test as per the american thoracic society guidelines using an rms helios spirometer . three attempts were made , and the best was selected and recorded to obtain the cases of forced vital capacity ( fvc ) , fev1 , and fev1/fvc ratio . a repeat spirometry was performed 20 min after inhaling 200 g of salbutamol to obtain pb fvc , fev1 , and fev1/fvc ratio . all patients with fev1/fvc ratio < 70% and fixed airway obstruction on spirometry ( pb improvement in fev1 of < 200 ml or in fev1/fvc of < 12% ) were included in the study , but patients with history of wheeze , chest tightness , eye allergy , nasal allergy , or skin allergy , suggesting bronchial asthma , those suffering from osteoarthritis and oxygen saturation < 90% , were also excluded from the study . the body mass index ( bmi ) of the study patients was calculated as body weight in kg / height in meters square and was classified as follows : underweight < 18.5 kg / m , normal 18.525.0 kg / m or overweight > 25 kg / m as per the world health organization criteria . pack years of smoking was calculated as : number of bidi packs smoked / day number of years smoked ( where a bidi pack was calculated as number of bidies/20 ) . the study patients were further subjected to combined risk assessment , which included the modified british medical research council questionnaire , history of exacerbations in the past 2 years , and history of exacerbations needing hospitalization in the past 2 years and categorized as risk category a , b , c , or d as per gold guidelines . all the patients and controls were then subjected to 6mwt , stst , and sqtst ( a modified form of stst , used for the first time for rural patients ) as follows : the participants were instructed by the command start for them to stand from their squatting position and they then asked to go to their squatting position without any delay , repeating these steps as many times as possible in 1 min at a self - selected speed which was felt safe and comfortable by them or until asked to stop . in case a patient was unable to stand unaided , support of a wide block of one feet height was allowed . the patient 's functional status was recorded as ( a ) unable to stand even with support , ( b ) able to stand with support only , and ( c ) number of times he was able to stand in 1 min without support . since normal ranges of 6mwt in meters ( m ) , stst , and sqtst are not available for universal use , the mean of the respective parameters minus twice the standard deviation in normal controls was used as cutoff for normal values for these parameters . data so obtained were tabulated and assessed for statistical significance using student 's t - test , anova test / test , and fisher 's exact test , as and when applicable . ninety stable copd patients and twenty controls could be studied , between july 2014 and january 2016 . the basic parameters of the study patients and the controls are shown in table 1 . the mean age of the patients in the four categories was similar to that of the controls ( p = 0.0059 ) . the mean age was higher in category b patients , but the differences from other categories were statistically insignificant ( p = 0.058 ) . males outnumbered the females in all the risk categories but the sex - wise distribution was also fair ( p = 1.000 ) . the duration of illness was higher in category a patients as compared to the rest , but the differences from other categories were statistically insignificant ( p = 0.823 ) . sixty - eight patients were current smokers and the rest 22 were reformed or ex - smokers . there were no differences in the copd categories with regard to the smoking status ( p = 0.913 ) , but the mean pack years of smoking was lower in category a patients as compared to the rest ( p = 0.914 ) . the mean bmi and pb fev1% were significantly lower in category d patients as compared to the rest ( p = 0.000 ) . basic parameters of the patients and controls the mean of 6mwt , stst , and sqtst in controls were 244 + 30.15 m , 27.05 + 7.89 , and 17.8 + 5.38 , respectively , so the respective cutoff values were derived as 184 m , 11 , and 07 . the mean distance walked by category d patients was lower as compared to the rest ( f = 1.47 , p = 0.109 ) , but the difference was statistically insignificant . on the contrary , the stst and sqtst values were significantly lower in category d patients as compared to the rest ( p = 0.000 ) . eight patients desaturated ( sao2 < 90% ) at the end of sqtst in group d as compared to 3 and 2 in group c and b , respectively , but all recovered of their own in due course . none of the patients in group a or the controls desaturated . the study of functional status is very vital in assessment of severity of copd as it has been shown to be a strong predictor of survival following pulmonary rehabilitation . 6mwt and more recently stst have been used in functional assessment of copd , but for rural population , squatting is also an important routine activity . this study was , therefore , undertaken to assess the feasibility and efficacy of sqtst in evaluation of the functional status of the copd patients residing in rural areas . data of this study were found to be valid for statistical comparisons as the controls in the study were age and sex matched , and the distribution of the patients in various risk categories of copd was fair . the mean age of the patients , sex , duration of illness , smoking status , and pack years of smoking did not correlate to severity of copd in this study ( p > 0.5 ) . have reported more dyspnea , lower exercise tolerance , and higher incidence of severe exacerbations in the elderly ( p < 0.05 ) . movahed and milne could observe a direct correlation between amount of expectoration / cough and the duration or amount of smoking . some of these observations , made at variance from the current study , can be explained on the basis of differences in the disease progression in copd in individual patients of different studies . it has been observed that smoking patterns of the cases , such as depth of smoking and the holding time , may play a more crucial role in pathogenesis of copd than the duration or pack years of smoking . further , it was significantly lower in category d patients as compared to the rest of the risk categories in this study ( p = 0.000 ) . harik - khan et al . reasoned that the nutritional abnormality and weight loss in copd are caused due to decreased caloric intake and increased basal metabolic rate . stated that copd produced malnutrition due to loss of fat as well as fat - free components . the mean distance covered in 6 min ( 6mwt ) was significantly lower in copd patients as compared to controls ( p = 0.003 ) . further , it progressively declined with progression of the severity of the disease , but at the cutoff distance of 184 meters , there was a significant overlap in different categories and the correlation was rather poor ( p = 0.109 ) . suggested stst as a superior alternative to 6mwt , the former being less stressful , easier to apply , and more sensitive for the patient 's clinical status , compared to the latter . in the present study , further , at a cutoff of 11 , all patients in category a were classed as normal and all patients in category d as abnormal . thus , stst was found to be superior to 6mwt in the present study also . in this study , cutoff , none of the category a patients had abnormal sqtst and none in category d was classed as normal . further , the mean sqtst value was highest in category a and lowest in category d as compared to rest ( p 0.000 ) . thus , a strong correlation was observed between sqtst and disease severity ( p = 0.000 ) . further , a strong correlation was also observed between sqtst and stst ( p = 0.000 ) , but its correlation with 6mwt was moderate ( p = 0.004 ) and to that with fev1% , was poor ( p = 0.091 ) . the major limitations of the study are the small number of patients and exclusion of patients with evident hypoxia due to fear of further drop in oxygen saturation . in spite of these limitations , sqtst was found to be an easy , feasible , and effective tool to study the functional status in stable , rural copd patients , as the test is very close to their routine daily activities . whether the findings of this study are applicable to other population groups who routinely resort to squat or not needs to be studied further . sqtst is an easy , feasible , and effective tool to study the functional status in stable copd patients of rural india as it is physiologically more close to their routine daily activities . further , bmi of the patients was observed to be an important surrogate marker of the functional status in these patients .

background : assessment of functional status is vital for proper therapy and rehabilitation programs in chronic obstructive pulmonary disease ( copd ) patients . traditionally , 6 min walk test ( 6mwt ) has been used but it by itself may not reflect the total functional status in these patients as it poorly correlates to the disease severity . a sit - to - stand test ( stst ) has been proposed as a better alternative to 6mwt but to test their ability to stand from the squatting position , i.e. , a squat - to - stand test ( sqtst ) will be more appropriate in rural patients.material and methods : all patients , diagnosed to have stable copd , after exclusion of asthma and other respiratory or nonrespiratory diseases were evaluated by applying 6mwt , stst , and sqtst in them . data so obtained were statistically analyzed.results:ninety patients and twenty healthy controls were studied . sixteen , 24 , 23 , and 27 of the patients were classed as copd risk category a , b , c , and d , respectively . the mean post bronchodilator forced expiratory volume in 1st second ( pb fev1 ) and body mass index ( bmi ) were significantly lower in category d as compared to the rest . the cutoff values for 6mwt , stst , and sqtst were derived as 184 m , 11 , and 07 , respectively . 6mwt correlates poorly to disease severity ( p = 0.109 ) but there was a strong correlation between disease severity and sqtst and stst ( p = 0.000).conclusion : sqtst is a feasible and effective tool to assess functional status of copd patients residing in rural areas . bmi is an important surrogate marker of the functional status in these patients .

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

apart from an esthetic concern , excessive gingival exposure with lip incompetence is considered an instigation factor for gingivitis in anterior maxillary region.1,2 osteotomy surgery combined with orthodontic therapy is considered the standard treatment in the management of maxillary excess . but this treatment modality has got its own limitations , especially when the patient does not have suitable posterior teeth to support the treatment , the teeth under consideration are periodontally weak or the patient is apprehensive in undergoing a complex surgical procedure.3 - 5 still the prime consideration of treatment was orthognathic surgery , provided the periodontal condition become favourable . a proper periodontal management by itself changed the clinical presentation of the case , and few well - designed crowns on four upper incisors could completely manage the case avoiding the need of surgery . an 18-year - old girl presented with a complaint of excessive gum visibility while smiling as well as in resting lip position . the central portion of her upper lip was raised exposing good amount of gingiva , and her lower lips were hiding upper front teeth while smiling ( figure 1 ) . she had severe anterior deep bite and several posterior teeth had severe attrition and were malposition . gingiva in the anterior sextant showed reduced stippling , rolled out crest with flat architecture . she had undergone orthodontic therapy with myofunctional and fixed appliances for 2 years to upright her inclined teeth . the orthodontic treatment had to be called off in between because of poor response to treatment . she had then consulted for the possibilities of orthognathic surgery to correct the gingival appearance and deep bite , but lack of proper posterior teeth occlusion , compound gingival pockets and high expectation of treatment outcome challenged the option of maxillary osteotomy surgery . the surgical option that would require removal of two premolars was not preferred in the present condition anyway , she was referred for periodontal management of deep pockets before its further planning . on examination of gingiva , pockets ranging from 3 to 5 mm were found on all aspects of teeth without attachment loss indicating the unusual anatomical crown length of anterior teeth . trans gingival probing indicated the presence of alveolar bone at very higher level than the present gingival margin ( figure 2 ) . gingivectomy , to expose anterior teeth till the cementoenamel junction ( cej ) and to obtain more physiologic gingival contour was proposed to eliminate the gingival pocket . after trans gingival probing the level of alveolar crest is marked with a sharp explorer . because she had undergone orthodontic therapy for 2 years period to upright the front teeth without much improvement and the treatment had to be stopped in between , a thorough periodontal assessment was crucial.6 clinical and radiographical assessment of the concerned area thorough oral prophylaxis was performed to make the pocket area and the gingival margin firm . since there was no attachment loss of gingiva and the future gingival level has to be positioned in cej , gingivectomy surgery was preferred over flap surgery.1,7 owing to the extended bevelling required to shape the gingival margin and the thickness of gingiva under consideration , scalpel surgery was decided.7 - 9 gingivectomy surgery bevelling the gingiva upto a level 3 mm coronal to the alveolar bone crest was done , preserving adequate biologic width of dento gingival unit.10,11 the cej was barely visible and sufficient exposure of crown was achieved to rectify the unaesthetic gingival exposure in resting lip position . the case was reviewed after 1 month ; though the gingival visibility became normal , and gingival margin was healthy , the inversely inclined position of teeth was more visible ( figure 3 ) . a treatment plan of prosthetic crowns on four incisor teeth with a straightened profile was suggested to correct the inclined profile of teeth , and she was ready for the procedure . since the crown preparation was planned in an esthetic zone and subgingival restorations were to be placed , patient was recalled after 6 months for prosthetic treatment.12 one month post - operative view ; after healing of gingivectomy area . the four upper incisors were prepared with labial angulations to receive crowns following the principles of subgingival crown preparation to favour gingival health ( figure 4).13 porcelain fused to metal crowns was fabricated with required lip fullness and the labial inclination towards incisor tip . the crowns were tried for its marginal fit , aesthetics and patient judgment , and upon finding completely satisfied , they were permanently cemented ( figure 5 ) . crown preparation to favour the required labial inclination of crowns . the marginal gingiva - crown relationship was found satisfactory , and crown contours were favourable for gingival maintenance . her complaint of gingival visibility during resting lip position and the inclined teeth profile were well attended with absolute satisfaction ( figure 6 ) . the patient was given post treatment instructions highlighting the importance of maintaining gingival health and the importance of periodic review . she was reviewed after a month , and 3 years follow - up of the case has been completed , and she is found satisfied with the prosthesis and also maintains good gingival health . vertical maxillary excess with open bite is a common condition whereas the condition described here is relatively rare with maxillary excess being visible in resting lip position and the upper anterior teeth hidden under lower lip while smiling.1,14,15 the condition presented here is commonly referred to as altered passive eruption . altered passive eruption is a condition wherein the gingiva does not recede apically as the teeth erupt to reach its final position . this gives rise to gingival pockets that may lead to complaints ranging from unaesthetic gingival position to gingival inflammation.1,2,15 the commonly seen causes of excessive gingival exposure are short upper lip , hyper - mobile lip , maxillary skeletal excess and altered passive eruption sequence resulting in unattached gingival coverage over teeth crown.15,16 the objectives of its treatment would be to enhance facial appearance , chewing ability , speech articulation , favor nasal air flow and obtain needed lip closure.2,17,18 the conventional method for treating vertical maxillary excess is orthognathic surgery . orthodontic treatment is usually combined with the surgery to gain optimum result.3 the present case was treated orthodontically for 2 years , but the anterior teeth resisted any movement and also developed gingival inflammation in relation to untreated gingival pockets . the possibilities of non - conventional techniques like botox therapy and lip repositioning surgery were considered and were found not appropriate for the present case.19,20 the case was further referred for orthognathic surgical management , but the planning of surgery required elimination of gingival pockets . the multiple deep gingival pockets in the upper anterior teeth dragged the attention of the treatment plan to attempt gingivectomy procedure as the prime treatment in managing the case . upon attaining a complete correction of gingival excess visibility after the gingivectomy procedure , an attempt to upright its appearance through prosthetic treatment of four incisors were found more suitable compared to orthognathic surgery . orthognathic surgery of the present case demanded extraction of upper 2 premolars and also the possibility of pre - maxilla being positioned without lip support.4,5 the patient had limited occlusion in posterior teeth and premolars were crucial for her , moreover achieving lip support was one of the objectives of treatment . this lead to a more favorable and limited treatment plan of giving crowns on upper for incisors . prosthetic interrelationship always remain a challenge unless the marginal gingival relationship with crown margin is in harmony and the dentogingival unit area is spared in crown preparation.21,22 considering these two factors , enough time for new healthy sulcus formation was given before crown preparation . as supported by literature in this regards , a healing period of 6 months were allowed following the periodontal surgical procedure for the final maturation and location of the free gingival margins.12 a proper maintenance protocol was also scheduled to ensure optimum gingival health . dentistry is a multi speciality science practice with a variety of treatment modality available for similar clinical situations . an understanding of the real aetiology behind the presenting disease , various treatment techniques available in its management , the feasibility and prognosis of each of these techniques , all combined with the need and acceptability of the patient should be the guideline in framing a tailor made treatment plan of each case . the present case could have been approached in many ways , but short listing various treatment possibilities and limiting it to patient s absolute demand was given the importance . this case report point at situations where conventional treatment modalities fall short in meeting the patient s demand , but combining relatively simple techniques of two different disciplines of same science could derive an alternative approach to address it .

excessive gingival visibility during smile is a common esthetic complaint in dentistry , but excessive gingival visibility in resting lip position is relatively uncommon condition . several exclusive radical to conservative surgical treatment or its combination with orthodontic therapy to treat this condition are within the reach of every patient nowadays . a case report of an excessive gingival visibility because of altered passive eruption along with inversely inclined maxillary anterior teeth is presented here . a relatively simple treatment approach combining periodontal and restorative therapy is used here to achieve the desirable aesthetic results . the gingival exposure resulting from altered passive eruption was completely managed by a gingivectomy , but the inclined appearance of teeth was still an aesthetic complaint . after complete healing of gingivectomy surgery , appropriately designed crowns were given to rectify the inversely inclined appearance . a complete rehabilitation of patient s smile could be achieved , the case was followed up for 3 years and the condition was found satisfactory .

Introduction Case Report Discussion Conclusion

the german decision maker federal joint committee ( fjc ) commissioned the institute for quality and efficiency in health care ( iqwig ) to perform a scientific evaluation on the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures . on may 20 , 2010 the fjc decided , on the basis of the present systematic review , that routine care can not be made dependent upon whether the opposing arch is provided with fixed or removable partial dentures and has therefore revoked its previous directive on fixed allowances for health care services . in the fourth german dental oral health study ( dms - iv ) it was estimated that , without taking wisdom teeth into account , in persons aged between 35 and 44 years , on average about 2.7 teeth were missing ; 48.5% of missing teeth had been replaced . in persons aged between 65 and 74 years , 14.2 teeth on average were missing and 31.3% had an edentulous upper arch ; 88.7% of missing teeth had been replaced . with the exception of a shortened dental arch , every tooth gap requires the earliest possible prosthetic restoration in order to avoid secondary damage . when deciding upon the type of denture most suitable for the respective patient , besides the nature of the opposing dentition or the type of prosthetic restoration in the opposing arch , the following factors play a crucial role : ( 1 ) size of gap ( number of missing teeth ) , type of gap ( free - end or interdental ) , and gap localization ; ( 2 ) age of patient ; ( 3 ) specific factors in the patient 's lifestyle , including in particular the quality of the patient 's oral hygiene as well as his or her tobacco consumption . the subsequent specifications define the aim of the investigation , which is as follows : assessment of the functionality of fixed and removable partial dentures as test interventions in relation to variations in the opposing dentition and their prosthetic restoration . following patient - relevant outcomes were evaluated : ( 1 ) denture longevity , ( 2 ) change in dietary habits , ( 3 ) oral health - related quality of life to include patient satisfaction and phonetic rehabilitation , which are parameters relevant to quality of life , and ( 4 ) denture cleansability and aftercare required . the test intervention assessed was the treatment of residual dentition by means of fixed partial dentures depending on the nature of the opposing dentition . the comparator intervention assessed was the treatment of residual dentition by means of removable partial dentures or combined fixed / removable partial dentures , again depending on the nature of the opposing dentition . implant - supported dentures ( fixed or a combination of fixed / removable partial dentures ) were also considered in the assessment of the test and comparator interventions . adult patients with residual dentition who had an indication for restoration with partial dentures were included in the investigation . residual dentition was classified as interdental ( kennedy class iii+iv ) as well as free - end gaps ( kennedy class i+ii ) comprising at least the first two molars . all patients with the following conditions were excluded from the investigation : posttraumatic status , status after carcinoma resection , craniofacial deformities and syndromes , and oligodontia . systematic literature searches were carried out in the following databases : central , medline , embase , biosis , scisearch , ccmed , dare , and hta ( search period from 1982 to 2009 , inclusive ) . in addition , a manual search was carried out in german dental journals and the search was extended topic specifically to the following databases : cdsr , cdmr , cdms , nhs eed , cinahl , amed , cab abstracts , global health , istpb + istp / isshp , medikat , and the publisher databases of karger ( secondary search ) , kluwer , springer , thieme , and hogrefe ( secondary search ) . finally , the opportunity to cite additional topic - relevant studies for the timespan until middle of 2008 was provided in july 2008 when interested parties were invited to submit written comments on a preliminary version of the report . randomized controlled trials ( rcts ) together with prospective and retrospective studies without control groups were included for the patient - relevant outcomes , provided the patients were included in the study consecutively and the confounding variables were adequately monitored . also included in the assessment were case reports and case series with a sample size of at least ten and of adequate biometric quality to avoid any bias in selection . the minimum observation time for all studies was a follow - up period of six months . the literature screening was carried out by two reviewers independently of each other . after assessing the biometric quality of the studies , the results from the individual studies were collated according to therapy goals and outcomes , compared , and described . iqwig 's preliminary benefit assessment , the preliminary report , was published on the internet in german language and interested parties invited to submit written comments ( https://www.iqwig.de/download/n05-02_vorbericht_v_1_0_relevanz_der_beschaffenheit_der_gegenbezahnung.pdf ) . in addition to data consistency within the publication , particular attention was paid to the homogeneity of the follow - up period . in prospective studies , this is equivalent to homogeneity in the age of the prostheses fitted . this factor was also examined in the case of retrospective analyses , that is , whether the age of the prostheses deviated considerably from each other or at least whether their age was adequately documented in the studies . in studies using questionnaires , particular focus was placed on the methodology of patient recruitment , paying special attention to the application of a consecutive procedure . the complexity of the topic lies in the great variability of very different dental patterns resulting from the lack of one or more of the total of 32 teeth in the human dentition . to enable subgroup analyses of patients with the numerous teeth patterns , documentation of gap classification according to kennedy was a major factor in the usability of the individual results for the present systematic review . finally , taking the above - mentioned aspects into account , the study and publication quality was assessed by means of a biometric quality tool comprising four grades ( no detectable flaws , minor flaws , major flaws , and unclear ) . exist if it is assumed that their correction would not substantially influence the results and therefore the overall conclusions of the study . in the case of major flaws the overall conclusions of the study would be called in question , as correction of the flaws might lead to different conclusions . initially , a total of twenty five papers were identified that met the inclusion criteria . after more detailed screening , eight studies had to be excluded from the assessment , as the data provided could not be broken up on the basis of variations in the opposing dentition ( figure 1 ) . in five out of 17 papers definitely included , there were prepublications with no additional relevant information . in eight of the 17 studies , there was information about the denture longevity outcome , in five studies information on the change in dietary habits outcome , in four studies information on the oral health - related quality of life and patient satisfaction outcome , and in nine studies information on the denture cleansability and aftercare required outcome , whereby 11 studies reported on one outcome , three studies on two , and three studies on three outcomes . five publications reported only on fixed partial dentures , three publications only on removable partial dentures , one publication on fixed and removable partial dentures , one publication on fixed partial dentures and complete dentures , three publications on removable partial dentures and complete dentures , one publication on removable partial dentures and fully dentulous patients , and three publications on removable partial dentures and complete dentures as well as fully dentulous patients . there were control interventions in nine papers ; however , in eight cases they represented interventions that did not meet the inclusion criteria ( i.e. , complete dentures or fully dentulous patients ) ( see tables 1 and 2 ) . this ultimately resulted in indirect comparisons being carried out on the test interventions of fixed versus removable partial dentures in relation to variations in the opposing dentition . the overall study and publication quality of the relevant studies was for the most part inadequate ( table 3 ) . there was only one prospective trial on the topic under investigation that could be described as randomized controlled , yet it provided no information on the randomization technique used . the six prospective studies [ 1318 ] identified revealed unequal periods of observation and flaws in how study discontinuations were dealt with . the three retrospective studies [ 1921 ] also revealed considerable flaws in the quality of studies and publication . the findings were similar for the seven prevalence studies identified [ 2228 ] , although here it was mainly the selection methods of the patient population that were inadequately described . outcome , one had no detectable flaws , two had minor , and five had major flaws in the biometric quality of the studies and publication . a comparison between the longevity of fixed and removable partial dentures was only possible for one opposing dentition variant ( complete dentures in the opposing arch ) ( tables 4 and 5 ) . only one trial , described as randomized controlled , contains data on the longevity of fixed and removable partial dentures in combination with a complete denture in the opposing arch . the validity of this trial is reduced through the following biometric flaws : ( 1 ) no data on the kennedy classes of the intervention arches ; ( 2 ) incomplete data on prognostic factors and comorbidity ; ( 3 ) inhomogeneous size of tooth gaps in the group with fixed partial denture : 44.4% teeth gaps of two to three teeth , 25.9% gaps of four to five teeth , and 29.7% gaps of nine to 11 teeth ; ( 4 ) no data on teeth gaps for the group with removable partial denture ; ( 5 ) drop - out rate of 18.9% over a follow - up period of five years ; ( 6 ) detailed description of randomization procedure is missing ( only mentioned as a term ) , so it should rather be classed as a nonrandomized controlled trial ; ( 7 ) no significance level given ( p value ) in the subgroup analysis . consequently , a significant difference can not be considered as detected in the 5-year survival rate of fixed partial dentures ( 95.2% ) and removable partial dentures ( 100% ) with a complete denture in the opposing arch . data on the longevity of fixed tooth - borne partial dentures with differing variations in the opposing dentition were found in two studies : in one publication , the 4-year survival rate with natural opposing dentition is given as 86,7% ; in the above - mentioned rct , the 5-year survival rate with complete denture in the opposing arch is given as 95.2% . apart from other differences in the study design and setting , a direct comparison between these two trials is limited by the different follow - up periods , since one of the publications gives no survival rates for shorter periods . data on the longevity of fixed implant - supported partial dentures with differing variations in the opposing dentition were found in two additional trials : the 3-year survival rate in case of natural opposing dentition is given as 97.8% in one publication ; the survival rate for fixed implant - supported partial dentures in the opposing arch after an average followup of 44.5 months is given as 100% in the other publication . apart from other differences in the study design and setting , a direct comparison of these data does not appear to have much value , since the follow - up period in one of the studies does not indicate survival rates for shorter periods . no conclusions can be drawn on the basis of the existing data regarding whether variations in the opposing dentition influence the longevity of fixed or removable partial dentures ( table 6 ) . ( a ) the first trend suggests increased longevity of removable partial dentures , compared to fixed partial dentures , with a fully edentulous opposing arch fitted with a removable prosthesis . ( b ) the second weaker trend suggests increased longevity of implant - supported partial dentures , compared to conventionally fixed partial dentures , with natural opposing dentition or with a removable partial denture in the opposing arch . there were major flaws in the quality of the biometric studies and publication in all 5 studies that contained data on the change in dietary habits in fixed and removable partial dentures in relation to variations in the opposing dentition . as no evaluable data were found on dietary habits in patients fitted with a fixed partial denture , it was not possible to compare with dietary habits in patients fitted with a removable partial denture ( tables 7 and 8) . data on the relevance of opposing dentition for removable partial dentures could only be drawn from one trial . the validity of this trial is reduced due to the following biometric flaws : ( 1 ) inhomogeneous residual dentition in the intervention / opposing arch : on average 17.4 teeth in the opposing natural dentition group , 11.8 teeth in the opposing removable partial denture group , and 5 teeth in the opposing complete denture group ; ( 2 ) no data on the kennedy classes in the intervention arches ; ( 3 ) no data on prognostic factors or on comorbidity ; ( 4 ) only male patients between 67 and 68 years of age ; ( 5 ) inhomogeneous age of prostheses : 35% less than 2 years old , 48% between 2 and 9 years old , and 17% over 10 years old ; ( 6 ) data collection instrument based on 6 hard and 6 soft meals not validated ; ( 7 ) inappropriate percentage analysis of restriction in dietary habits . in another trial , all patients interviewed featured natural dentition in the opposing arch , so that different dentition patterns could not be compared . the existing data does not allow any conclusion to be drawn on whether variations in the opposing dentition influence dietary habits when a fixed or removable partial denture is fitted . the data in another trial show that , in the case of removable partial dentures only , no or little difference can be determined in relation to the opposing dentition when eating soft or hard food ( table 9 ) . out of the four identified trials , one displayed minor flaws and three [ 20 , 22 , 24 ] major flaws in the biometric quality of the studies . it was only possible to compare the satisfaction between a fixed and removable partial denture with one opposing dentition variant ( complete denture in opposing arch ) . one trial described as randomized controlled contained data on patient satisfaction for fixed and removable partial dentures in combination with a complete denture in the opposing arch . due to the biometric flaws already described in the results for the denture longevity a significant difference ( p < 0.05 ) is indicated in patient satisfaction regarding stability in general and during chewing with fixed ( 77.8% and 85.2% of patients , resp . , were satisfied ) and with removable partial denture ( 61.5% and 53.9% , resp . ) . however , the fact that fixed prostheses have greater stability than removable ones appears reasonable . data on general patient satisfaction with removable partial dentures and differing variations in the opposing dentition were found in one trial : the percentage of satisfied patients with removable partial denture in the opposing arch was 37% ( n = 102 ) , and 65% for those with complete denture in the opposing arch ( n = 147 ) . however , due to the variable sampling size and the unequal age of prosthesis ( one to 15 years ) , a comparison of these data would not appear to be worthwhile . based on the existing data , it is not possible to draw any conclusions on whether variations in the opposing dentition influence patient satisfaction when fitting fixed or removable partial dentures . data on denture cleansability and aftercare required when fitting fixed and removable partial dentures were included in nine publications . of these trials , two showed minor [ 12 , 13 ] and seven major flaws [ 2024 , 27 , 28 ] in the biometric quality of studies and publication . it was only possible to compare the maintenance requirements of fixed and removable partial dentures for one opposing dentition variant ( complete denture in the opposing arch ) . as no analysable data were found on prosthesis cleansability or aftercare required in fixed partial dentures , it was not possible to make a comparison with the prosthesis cleansability or aftercare required in removable partial dentures . only one rct contained data on the maintenance requirements of fixed and removable partial dentures when the opposing arch was fitted with a complete denture . however , as already mentioned above , it contained biometric flaws that reduced its validity . consequently , a significant difference could not be proven in the level of repair required for fixed partial dentures ( 22.2% of prostheses ) and removable partial dentures ( 23% ) in combination with a complete denture in the opposing arch . in another trial , data were found on the level of repair required for removable partial dentures with differing variations in the opposing dentition : the number of repairs required within a period of 16 months was 72 for natural opposing dentition , eight for removable partial dentures in the opposing arch , and 18 for removable complete dentures in the opposing arch . however , due to the nondocumented sample size of the individual subgroups and the inhomogeneous age of prosthesis ( one to six years ) , a comparison of these data would not appear to be worthwhile . no conclusions could be drawn on the basis of the existing data as to whether variations in the opposing dentition influence denture cleansability and aftercare in case of a fixed or removable partial denture . after a comprehensive literature search and an assessment of the evidence base on the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures , no robust conclusions could be drawn . this is due both to the low quantity and the methodological weaknesses of the few studies identified . only one of the 17 studies compared the two denture types investigated within the context of a controlled prospective study , even though this study can not be regarded as an rct either , due to its inadequately documented randomization procedure . all the remaining data extracted originated from prospectively or retrospectively planned studies without a control group ( one - arm studies ) or from one - off data collections by way of questionnaires . it should be noted , however , that all the studies included were not per se concerned with the question of the relevance of opposing dentition but addressed this question mostly in the form of subgroup analyses . for this reason the literature search for this report was particularly time intensive , as it could mostly be decided only on the basis of full - text screening whether a subgroup analysis considering opposing dentition had been performed . within the framework of the quality assessment for the present systematic review , the serious biometric flaws in study and publication quality identified in nearly all studies refer to the research question investigated here , that is , the relevance of opposing dentition . this quality assessment is not an evaluation of the informative value of the individual studies with regard to their original research questions . the assessment of denture longevity , that is , the survival or success rate of dentures , should follow a clear definition . it must be quite evident what medical , functional , or even aesthetic requirements the dentures must fulfill in order to be classed as functioning or successful . in the present systematic review all irreparable damage was viewed as a failure ( end of denture longevity ) and all repairable damage was allocated to the outcome denture aftercare required . functional and medical reasons ( medical failure = tooth loss ) account for 69.5% and 28.5% of fixed partial dentures failures , respectively ; removable dentures fail nearly exclusively for medical reasons . implant - supported fixed dentures can fail due to prosthesis failure but also through failure of the implants themselves . in most of the studies included , no definition was offered as to what criteria were applied for success of the dentures , nor was the status of the opposing dentition over the course of the entire study period clearly reported . first , some differentiations should be made between the objective chewing performance ( i.e. , the instrumentally measured ability to break down a specific test meal into pieces ) and subjective chewing efficiency ( i.e. , chewing capacity as experienced by the patient ) , depending on the status of the opposing dentition . some objective tests indicate that by means of fixed and removable dentures the chewing function can be equally restored , whereas others claim a better chewing capacity for hard food with fixed prostheses . however , it is still controversial whether the objective effect measured correlates with the patient 's subjective perception . objectively measured improvements in chewing capacity through adaptation of the prosthesis have not been perceived by patients in previous studies . a study in which patients with only one partial prosthesis that estimated their chewing ability to be the same as that of patients with two complete prostheses shows that the parameter subjectively reported chewing efficiency is potentially flawed . independent of the choice of measurement methods , changes in dietary habits should always be compared intraindividually . a further patient - relevant aspect is the change in the perception of taste through dentures . studies were able to show that dissatisfaction due to a changed perception in taste was evident only for prostheses covering the entire palate , that is , for complete prostheses . this aspect plays no role for the removable partial prostheses assessed in this systematic review . oral health - related quality of life was not analyzed in the studies identified ; we therefore extended the outcomes investigated and also included patient satisfaction so as to be able , at least , to draw conclusions on this quality - of - life relevant parameter . every patient should first be provided with removable dentures and then , after assessment of satisfaction , with fixed ones . this approach is more costly than an interindividual assessment , but reduces uncertainty and contradictions in patients ' statements . as an inverse approach is not feasible on practical grounds , an intraindividual approach would , however , have to dispense with randomization with regard to the sequence of the interventions , as the study would not have a cross - over design in the proper sense of the term . particularly in view of the variety of patterns regarding the status of teeth and opposing dentition , this study design represents the most reliable comparison between fixed and removable dentures for the evaluation of patient satisfaction . independently of the nature of the opposing dentition , patient satisfaction has been found to be 96% for fixed dentures and 90% for removable ones . it has also been shown that patient satisfaction diminishes significantly with the number of teeth remaining . patients with at least 25 intact teeth are significantly more satisfied with any type of prosthesis than patients with 1 to 24 intact teeth . above all , however , patient - related factors are of importance : satisfaction with dentures is not only significantly dependent on personality type but also on acceptance of tooth loss . . in the studies included , patient satisfaction was exclusively assessed by means of nonvalidated questionnaires that enquired about patients ' subjective satisfaction . the oral health - related quality of life ( ohqol ) instrument is a validated tool . ohqol also diminishes considerably with the number of intact teeth ( p < 0.001 ) and the condition of those remaining . in patients with less than 20 intact teeth above all , the lack of the front teeth even if these are replaced by a prosthesis substantially lowers the ohqol ( or = 21.5 ) . however , demographic factors such as ethnicity or immigrant status have a stronger influence on the ohqol than the status of the teeth . current studies even come to the conclusion that the ohqol is not meaningful as an indicator for patient satisfaction or the disease burden of patients with tooth gaps . the outcome denture cleansability and aftercare required covers four different patient - related aspects : ( 1 ) effort involved in the cleaning of the remaining teeth , as well as ( 2 ) effort involved in the cleaning of the denture ( the third and fourth aspects are described further below ) . the first and second aspects cover all measures demanded of the patient with regard to oral or prosthesis hygiene . fixed and removable dentures differ fundamentally here , as the cleansability of the remaining teeth is considerably facilitated after the removable dentures are taken out , while extra effort is required for prosthesis hygiene . fixed dentures are to be cleansed within the context required for routine denture care ; not only the demands in time but also the dexterity required by the patient is greater , however , in comparison to the care of natural teeth . in patients no longer possessing sufficient motor capacities due to age or illness , fixed dentures are for this reason often contraindicated . the question as to whether and what influence the nature of the opposing dentition has on oral and prosthesis hygiene could not be answered in the present analysis . in principle it can , however , be assumed that for every denture , independently of the opposing arch , certain efforts are necessary for hygiene , efforts which are doubled , for example , if the opposing arch is equipped with a similar denture . without taking opposing teeth into account , numerous studies have shown that , assuming adequate care , patients with fixed as well as those with removable dentures bear no increased risk of caries [ 5052 ] or periodontitis [ 29 , 53 , 54 ] . for both fixed and removable dentures , adherence to specific regulations on design is essential in the production of the devices in order to keep plaque formation as low as possible and so ensure the hygienic qualities of the dentures . in addition , it is the dentist 's responsibility to inform the patient not only of the necessity and possibilities of oral and prosthesis hygiene , but also to motivate him or her on a regular basis . the reality , however , is that 83% of all denture wearers are not properly informed and 12% of all wearers show no adequate behaviour regarding denture care ; in hospitalized patients the percentage is even 45% . further aspects of the outcome denture cleansability and aftercare required are ( 3 ) the level of repair required for dentures and ( 4 ) the effort involved in denture aftercare . for denture repairs , not only the potential costs incurred but also the often necessary deprivation of the denture during repair in a dental laboratory are relevant issues for patients . the following points were noted in the evaluation of the studies included.to be able to draw robust conclusions , a randomized controlled design of the study is essential . stratified randomization should be used , at least with regard to the different opposing dentition groups . the problem of randomized allocation of patients to the fixed or removable denture group arises from the fact that fixed dentures are more expensive to produce . study design used in numerous denture studies , that is , the intraindividual comparison between the left- and right - hand halves of the jaw of the same patient , is only suitable for the assessment of fixed dentures , not removable dentures , which extend over the whole jaw for one - sided tooth gaps . in the case of non - rcts , the intervention arches should be comparable . as an intact jaw has at least 14 teeth , not less than 2 = 16,384 variations in tooth gaps are possible . to limit the number of subgroups , classification according to type , localization , and size of the gap , as practiced in this report , would be sensible , that is , specification of kennedy class ( iiv ) of the jaw , as well as gap width ( number of missing teeth ) . by this classification these data should be supplemented by the number of intact teeth remaining , which should be similar between groups . in the description of the opposing dentition , at least the five groups distinguished in this systematic review ( nd , rpd , cd , fpd , and ifp ) should also be assessed separately . in many publications , these data are only reported within the framework of the demographic description of the patient population , but the results are not presented separately for groups according to opposing dentition . in part , for example , the natural opposing dentition is treated as equal to a fixed denture in the opposing arch . in the case of a follow - up period of ten years or longer , it should be noted that , over time , the condition of the opposing dentition may change . due to the loss of teeth in the opposing arch , natural opposing dentition may turn into residual dentition and can eventually lead to a fully edentulous arch . it should further be considered whether only patients with a constant status of their opposing dentition should be included in the assessment in terms of a per - protocol ( pp ) analysis or whether , parallel to that , patients with changes in this status should be evaluated during the follow - up period by way of an intention - to - treat ( itt ) analysis . as a relevant comorbidity , the periodontal status of the remaining teeth should primarily be considered . every type of denture with the exception of implants results in additional strain on the remaining teeth . if their stability is compromised by periodontal disease , this has an effect on the expected longevity of the denture . the presence of parafunctions such as bruxism is a second important factor , as dentures in people with such a condition are overstressed . prognostic factors include smoking habits and oral hygiene . as these are changeable risk factors , smokers and patients with inadequate oral hygiene need not necessarily be excluded from the study , but could receive counseling for smoking cessation or be given instructions in oral hygiene . as the compliance of study participants is important in every long - term study , this would also represent a preselection with regard to less cooperative patients . many of the studies identified compared newly manufactured prostheses and others that had in part been used for over ten years . in prospective study designs , the age of the prostheses is equivalent to the follow - up period , which should not be subject to deviations . furthermore , attention should be paid to a standardized manufacturing process of the prostheses . for fixed dentures this means the same preparation technique , the same molding material , the same alloy and ceramic material , and if possible the same dental laboratory . for removable dentures it would in principle be important to focus on a uniform , systematic design of the prosthesis , on the same materials , and on a standardized manufacturing process . comparison of the level of repair required for differently fabricated dentures must be regarded as a methodological error in the study design . in retrospectively planned studies it can be assumed that the prostheses were not manufactured under standardized conditions . as a matter of principle , only dental indications should be compared in which both fixed and removable dentures are considered as treatment options . for one- or two - sided free - end gaps ( kennedy class i+ii ) with a gap width of one tooth , a fixed denture in the form of an extension bridge is possible , but due to the greater strain arising through the leverage effect , the longevity or level of repair of such bridges is , for example , not comparable to that of conventional bridges in patients with kennedy class iii gaps . regarding the longevity of implants it should be noted that their survival rate depends , among other factors , on their position , length , diameter , and surface . if the jawbone material available is insufficient to fit an implant , then bone augmentation , that is , an increase in the local bone material , must first be performed . the survival rate of implants in augmented areas is again lower than that of those set in original , local bone material . although restrictions such as cost aspects , the impossibility of blinding , and the lack of randomization due to patient preferences play an important role in the conduct of dental care studies , some approximation to the state - of - the - art standards implemented in other areas of medical care would be desirable . overcoming these restrictions ultimately , the conduct of informative studies is urgently recommended in order to successfully clarify the present research questions . to be able to draw robust conclusions , stratified randomization should be used , at least with regard to the different opposing dentition groups . the problem of randomized allocation of patients to the fixed or removable denture group arises from the fact that fixed dentures are more expensive to produce . study design used in numerous denture studies , that is , the intraindividual comparison between the left- and right - hand halves of the jaw of the same patient , is only suitable for the assessment of fixed dentures , not removable dentures , which extend over the whole jaw for one - sided tooth gaps . in the case of non - rcts , the intervention arches should be comparable . as an intact jaw has at least 14 teeth , not less than 2 = 16,384 variations in tooth gaps are possible . to limit the number of subgroups , classification according to type , localization , and size of the gap , as practiced in this report , would be sensible , that is , specification of kennedy class ( iiv ) of the jaw , as well as gap width ( number of missing teeth ) . by this classification the number of subgroups is reduced . these data should be supplemented by the number of intact teeth remaining , which should be similar between groups . in the description of the opposing dentition , at least the five groups distinguished in this systematic review ( nd , rpd , cd , fpd , and ifp ) should also be assessed separately . in many publications , these data are only reported within the framework of the demographic description of the patient population , but the results are not presented separately for groups according to opposing dentition . in part , for example , the natural opposing dentition is treated as equal to a fixed denture in the opposing arch . in the case of a follow - up period of ten years or longer , it should be noted that , over time , the condition of the opposing dentition may change . due to the loss of teeth in the opposing arch , natural opposing dentition may turn into residual dentition and can eventually lead to a fully edentulous arch . it should further be considered whether only patients with a constant status of their opposing dentition should be included in the assessment in terms of a per - protocol ( pp ) analysis or whether , parallel to that , patients with changes in this status should be evaluated during the follow - up period by way of an intention - to - treat ( itt ) analysis . as a relevant comorbidity , the periodontal status of the remaining teeth should primarily be considered . every type of denture with the exception of implants results in additional strain on the remaining teeth . if their stability is compromised by periodontal disease , this has an effect on the expected longevity of the denture . the presence of parafunctions such as bruxism is a second important factor , as dentures in people with such a condition are overstressed . prognostic factors include smoking habits and oral hygiene . as these are changeable risk factors , smokers and patients with inadequate oral hygiene need not necessarily be excluded from the study , but could receive counseling for smoking cessation or be given instructions in oral hygiene . as the compliance of study participants is important in every long - term study , this would also represent a preselection with regard to less cooperative patients . suitable variables as indicators of patients ' oral hygiene are plaque and bleeding indices . many of the studies identified compared newly manufactured prostheses and others that had in part been used for over ten years . in prospective study designs , the age of the prostheses is equivalent to the follow - up period , which should not be subject to deviations . furthermore , attention should be paid to a standardized manufacturing process of the prostheses . for fixed dentures this means the same preparation technique , the same molding material , the same alloy and ceramic material , and if possible the same dental laboratory . for removable dentures it would in principle be important to focus on a uniform , systematic design of the prosthesis , on the same materials , and on a standardized manufacturing process . comparison of the level of repair required for differently fabricated dentures must be regarded as a methodological error in the study design . in retrospectively planned studies it can be assumed that the prostheses were not manufactured under standardized conditions . as a matter of principle , only dental indications should be compared in which both fixed and removable dentures are considered as treatment options . for one- or two - sided free - end gaps ( kennedy class i+ii ) with a gap width of one tooth , a fixed denture in the form of an extension bridge is possible , but due to the greater strain arising through the leverage effect , the longevity or level of repair of such bridges is , for example , not comparable to that of conventional bridges in patients with kennedy class iii gaps . regarding the longevity of implants it should be noted that their survival rate depends , among other factors , on their position , length , diameter , and surface . if the jawbone material available is insufficient to fit an implant , then bone augmentation , that is , an increase in the local bone material , must first be performed . the survival rate of implants in augmented areas is again lower than that of those set in original , local bone material . the assessment of outcomes must follow a standardized procedure . although restrictions such as cost aspects , the impossibility of blinding , and the lack of randomization due to patient preferences play an important role in the conduct of dental care studies , some approximation to the state - of - the - art standards implemented in other areas of medical care would be desirable . overcoming these restrictions represents a challenge that evidence - based dental medicine should successfully meet . ultimately , the conduct of informative studies is urgently recommended in order to successfully clarify the present research questions . this systematic review assesses the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures . there is currently no proof of sufficient certainty regarding the relevance of opposing dentition in removable and fixed partial dentures for any of the following patient - relevant outcomes : denture longevity , change in dietary habits , oral health - related qol , condensed into patient satisfaction , and denture cleansability and aftercare required . no evidence - based statements could be generated as to whether , and if so how , variations in the opposing dentition have a bearing on the decision to fit a partially edentulous arch with a fixed or removable partial denture . there were only few indications of increased patient satisfaction in favor of the fixed partial denture in combination with the opposing dentition variant of complete denture in the opposing arch . however , these indications are based on a small number of methodologically weak studies , and this is characteristic of the field of prosthetic dentistry , as the systematic review shows .

the aim of this systematic review was to evaluate the functionality of fixed and removable partial dentures as test interventions in relation to variations in the opposing dentition and their prosthetic restoration . the abstracts identified in the respective databases were screened independently by two investigators . rcts and uncontrolled studies were considered , provided the patients were included consecutively and the confounding variables were adequately monitored . seventeen papers were included . the study and publication quality was assessed using a biometric quality tool showing an overall poor quality . the reported outcomes , such as survival rates , were in each case obtained from a single study . two possible trends could be deduced for the endpoint longevity : ( a ) the first trend in favor of removable partial dentures , compared to fixed partial dentures , with a fully edentulous opposing arch fitted with a removable prosthesis ; ( b ) the second trend in favor of implant - supported partial dentures , compared to conventionally fixed partial dentures , with natural opposing dentition or with a removable partial denture in the opposing arch . no evidence could be generated as to whether , and if so how , variations in the opposing dentition have a bearing on the decision to fit a partially edentulous arch with a fixed or removable partial denture .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions

nonalcoholic fatty liver disease ( nafld ) is the most prevalent cause of chronic liver diseases . this disorder occurs because of the pathologic accumulation of fat ( mainly triglycerides ) in the liver . it is estimated that in iran , 7% of children and 35% of adults are affected . until now results of these studies have sometimes caused the researchers to suggest different nutritional reasons for this disease such that some of studies suggest that high - calorie food patterns , which lead to obesity , can also increase the risk of accumulation of lipids in liver and incidence of steatosis . on the other hand , high carbohydrate level diets can affect nafld by influencing on de novo synthesis of fatty acids and increasing blood triglycerides . results of some other studies indicates a relation between type of lipid intake and developing this disease . such that it seems increase in receiving omega 6 to omega 3 ratio is in relation to advanced stages of the disease by activating inflammatory pathways . moreover , it seems that saturated fatty acids can damage the structure of hepatocytes by inducing apoptosis and thus advancing nafld . in some studies , such that it seems patients with nafld have a lower level intake of vitamin d and antioxidant vitamins ( e and c ) than healthy individuals . moreover , results from some of these studies indicated that calcium and zinc intake in patients with nafld was less than healthy individuals . despite these results , the relation between dietary intake and nafld , in some other studies , no relation has been observed . these inconsistent results might be because of difference in studies design and aims , participant groups , and small sample size in some studies . because of some limitations in previous studies and also according to the fact that nutrition has an essential role in etiology of nafld , further investigation of relationship between diets components and this disease can help us to prevent and control it . the present study is a case - control study that was approved by the tehran university committee and ethics advisory committee , and written consent was obtained from all participants . patients with nafld who admitted isfahan fatty liver research center were chosen as the case group . in this study , according to sample size , estimation of 140 people for case and control group : and on the basis of expectancy for over- and under - reporting of dietary intakes while completing the 24-h dietary recall , 170 people were selected for each group . after calculating participant 's energy intake , who receiving under 800 kcal and over 4200 kcal of energy each day inclusion criteria for case group included the following : individuals between 20 and 60 years old who were diagnosed with nafld after giving blood tests and performing ultrasonography , by a radiology specialist ( the device used for ultrasonography was esaot medica , which is equipped with a convex 3.5 mhz probe ) . inclusion criteria for control group included the following : volunteers who were the neighbors of case group and were same as them in age and sex . blood tests and ultrasonography were performed on them and their wellbeing in case of nafld was approved ( not suffering from any stages of hepatic steatosis ) . exclusion criteria included the following : alcohol consumption , following special diets 2 months before the study , regular use ( at least continues for 1 week ) of any nutritional supplements in the last 6 months , pregnant or breastfeeding women , individuals with type b or c hepatitis and diagnosed wilson 's disease , use of drugs effective on liver , and biochemical biomarkers . data about age and sex were recorded by researcher by means of general information questionnaire . physical activity rate was estimated by short form of international physical activity questionnaire . based on instructions of this questionnaire , low - level physical activity category : if the individual does not meet the criteria for intermediate- or high - level categories . intermediate - level physical activity category : if the individual meets one of these criteria : three days or more , each day at least 20 min of intense physical activity or five days or more , each day at least 30 min of intermediate physical activity or walking . high - level physical activity category : if the individual meets one of these criteria : having at least three days of intense physical activity which reaches the minimum of 1500 met - min / week or seven days a week , any combination of walking , intermediate , or intense activity , if the total score of physical activity reaches a minimum of 3000 met - min / week . for determining the nutritional intake , then , foods were converted into their ingredients , and their amounts were calculated into grams and were encoded . then , the amounts of energy , macronutrients , and micronutrients were calculated by entering the data to nut4 software nutritionist 4 ( version7 ; n - squared computing , or usa ) which was modified for iranian items were used . after collecting quantitative and qualitative data were presented as mean standard deviation ( sd ) and frequency and percentages , respectively . the relationship of the disease status with qualitative variables was assessed by chi - square and comparing the quantitative data between groups was conducted using independent t - test and analysis of covariance as appropriate . the present study is a case - control study that was approved by the tehran university committee and ethics advisory committee , and written consent was obtained from all participants . patients with nafld who admitted isfahan fatty liver research center were chosen as the case group . in this study , according to sample size , estimation of 140 people for case and control group : and on the basis of expectancy for over- and under - reporting of dietary intakes while completing the 24-h dietary recall , 170 people were selected for each group . after calculating participant 's energy intake , who receiving under 800 kcal and over 4200 kcal of energy each day inclusion criteria for case group included the following : individuals between 20 and 60 years old who were diagnosed with nafld after giving blood tests and performing ultrasonography , by a radiology specialist ( the device used for ultrasonography was esaot medica , which is equipped with a convex 3.5 mhz probe ) . inclusion criteria for control group included the following : volunteers who were the neighbors of case group and were same as them in age and sex . blood tests and ultrasonography were performed on them and their wellbeing in case of nafld was approved ( not suffering from any stages of hepatic steatosis ) . exclusion criteria included the following : alcohol consumption , following special diets 2 months before the study , regular use ( at least continues for 1 week ) of any nutritional supplements in the last 6 months , pregnant or breastfeeding women , individuals with type b or c hepatitis and diagnosed wilson 's disease , use of drugs effective on liver , and biochemical biomarkers . data about age and sex were recorded by researcher by means of general information questionnaire . physical activity rate was estimated by short form of international physical activity questionnaire . based on instructions of this questionnaire , low - level physical activity category : if the individual does not meet the criteria for intermediate- or high - level categories . intermediate - level physical activity category : if the individual meets one of these criteria : three days or more , each day at least 20 min of intense physical activity or five days or more , each day at least 30 min of intermediate physical activity or walking . high - level physical activity category : if the individual meets one of these criteria : having at least three days of intense physical activity which reaches the minimum of 1500 met - min / week or seven days a week , any combination of walking , intermediate , or intense activity , if the total score of physical activity reaches a minimum of 3000 met - min / week . for determining the nutritional intake , a 24 h dietary recall questionnaire was filled for every individual . then , foods were converted into their ingredients , and their amounts were calculated into grams and were encoded . then , the amounts of energy , macronutrients , and micronutrients were calculated by entering the data to nut4 software nutritionist 4 ( version7 ; n - squared computing , or usa ) which was modified for iranian items were used . after collecting , data were analyzed using spss software version 16 ( spss inc . , quantitative and qualitative data were presented as mean standard deviation ( sd ) and frequency and percentages , respectively . the relationship of the disease status with qualitative variables was assessed by chi - square and comparing the quantitative data between groups was conducted using independent t - test and analysis of covariance as appropriate . there is no difference between two groups in frequency of two genders , average of age , height , and weight ( p > 0.05 ) . however , the body mass index ( bmi ) was higher in the case group ( p < 0.05 ) . physical activity comparisons show that patients are less active than healthy individuals ( p < 0.05 ) . comparison of demographic , anthropometric , and physical activity data between nonalcoholic fatty liver disease and control groups mean sd of total energy and macronutrients intake between case and control groups is shown in table 2 . no difference in these variables was observed between two groups ( p > 0.05 ) . intake of saturated fat in patients with nafld was more than healthy individuals ( p < 0.05 ) . other dietary lipids intake in case and control groups had no difference ( p > 0.05 ) . comparison of energy and macronutrients intake between nonalcoholic fatty liver disease and control groups sugar intake had a statistically significant difference between two groups such that it was denoted that sugar consumption in patients with nafld is more than healthy individuals ( p < 0.05 ) . mean sd of total dietary fiber ( tdf ) in healthy individuals was more than patients with nafld ( p < 0.05 ) , but no difference was observed in soluble dietary fiber and insoluble dietary fiber ( p > 0.05 ) [ table 3 ] . comparison of dietary carbohydrates and dietary fiber intake between nonalcoholic fatty liver disease and control groups in table 4 , intake of vitamins and minerals after adjustment of energy intake between two groups are shown . mean sd of dietary intake of folic acid and vitamin d in healthy individuals are more than patients with nafld ( p < 0.05 ) . moreover , it was declared that intake of potassium and zinc in healthy individuals is higher than patients with nafld ( p < 0.05 ) . no other difference was observed in intake of other vitamins and minerals in two groups ( p > 0.05 ) . there is no difference between two groups in frequency of two genders , average of age , height , and weight ( p > 0.05 ) . however , the body mass index ( bmi ) was higher in the case group ( p < 0.05 ) . physical activity comparisons show that patients are less active than healthy individuals ( p < 0.05 ) . comparison of demographic , anthropometric , and physical activity data between nonalcoholic fatty liver disease and control groups mean sd of total energy and macronutrients intake between case and control groups is shown in table 2 . no difference in these variables was observed between two groups ( p > 0.05 ) . intake of saturated fat in patients with nafld was more than healthy individuals ( p < 0.05 ) . other dietary lipids intake in case and control groups had no difference ( p > 0.05 ) . comparison of energy and macronutrients intake between nonalcoholic fatty liver disease and control groups sugar intake had a statistically significant difference between two groups such that it was denoted that sugar consumption in patients with nafld is more than healthy individuals ( p < 0.05 ) . mean sd of total dietary fiber ( tdf ) in healthy individuals was more than patients with nafld ( p < 0.05 ) , but no difference was observed in soluble dietary fiber and insoluble dietary fiber ( p > 0.05 ) [ table 3 ] . comparison of dietary carbohydrates and dietary fiber intake between nonalcoholic fatty liver disease and control groups in table 4 , intake of vitamins and minerals after adjustment of energy intake between two groups are shown . mean sd of dietary intake of folic acid and vitamin d in healthy individuals are more than patients with nafld ( p < 0.05 ) . moreover , it was declared that intake of potassium and zinc in healthy individuals is higher than patients with nafld ( p < 0.05 ) . no other difference was observed in intake of other vitamins and minerals in two groups ( p > 0.05 ) . our study results indicated that waistline , hipline , and bmi in the case group are higher than control group . the results of hashemi kani et al . study indicate that patients with nafld have higher average of weight , waistline , and bmi than healthy individuals . moreover , capristo et al . ( by using dual - energy x - ray ) found that weight and body fat percentage of patients with nafld are more than healthy individuals . as the results suggest , physical activity level in healthy participants is more than nafld patients . an overview of the epidemiological evidence reported lower chance of diagnosis with nafld in individuals who exercise two times per week compared to sedentary people . according to the present study , consumption of saturated fatty acids in patients is more than healthy individuals . in studies conducted by musso et al . and zelber - sagi et al . , it was shown that consumption of saturated fat sources such as red meat has a direct relation with nafld . glucose - dependent insulinotropic polypeptide has a role in lipid metabolism as an intermediate polypeptide . intake of sugar in patients with nafld is higher than healthy individuals but no difference in other carbohydrates was observed . until now , a few studies have surveyed type of consuming carbohydrate in nafld patients . in the study of yoshari et al . showed direct relation between weekly intake of fructose - rich sources and nafld . in two other studies conducted by zelber - sagi et al . and toshimitsu et al . , it was indicated that the consumption of food sources containing simple carbohydrates is in direct relation with developing this disease . according to the conducted studies using high levels of sugar can lead to advance in steatosis by increasing de novo synthesis of free fatty acids and lipid accumulation in hepatic tissue . in our study , tdf intake in healthy individuals is higher than patients with nafld . in two studies conducted by cortez - pinto et al . and , it was found that healthy people use more fiber - rich foods such as vegetables than patients with nafld . it seems intake of dietary fiber can be a protective factor against nafld because by increasing dietary fiber intake , blood ldl , fat accumulation in body , and resistance to insulin are decreased . results of our study indicated that dietary intake of vitamin d was higher in control group than nafld . also in a case - control study conducted by targher , the results indicated that vitamin d intake in patients with nafld was much lower than healthy individuals . it is declared that deficiency vitamin d has a relation with lipid accumulation outside of hepatocytes and development of nafld . results of the presents study show that healthy individuals receive more folic acid , potassium , and zinc than patients with nafld . in study of toshimitsu , it was found that decrease in intake of zinc has a direct relation with the intensity of stages of this disease . studies indicate that some antioxidants in diet ( such as zinc ) can regulate hepatic aminotransferases and also prevent lipid accumulation in the liver of patients with nafld . on the basis of our information from studies conducted until now limitations of this study are using ultrasonography for diagnosing nafld because this method has much lower accuracy than fibro - scan or liver biopsy methods . also using only one 24 h strength , however , is using case - control nature of the study that is better than cross - sectional designs and provides stronger evidence regarding risk factors . also comparing all of the dietary macronutrients and micronutrients between two groups of patients with nafld and control group which were matched for age and sex in total , it seems that in etiology of nafld what is more important than essential macronutrients intake ratio , is the type of intake sources . such that according to our study results , individuals with nafld consume more amounts of sugar and saturated fatty acids and less amount of sources containing fiber ( soluble and insoluble ) in comparison to healthy individuals . also it seems that low intake of vitamin d , folic acid , zinc , and potassium are associated with the development of this disease .

background : nonalcoholic fatty liver disease ( nafld ) is the most common cause of chronic liver disease in the world . although some studies have been conducted about dietary intakes of these patients , but the results are inconsistent . the aim of this study was to survey all macronutrients and micronutrients included in dietary intake of these patients for better understanding the factors influencing this disease.methods:the present study is a case - control conducted in isfahan city , iran . the cases were recently diagnosed patients with nafld who identified by ultrasonography . the case ( 159 ) and control ( 158 ) individuals were matched in age and gender . data of general characteristics and physical activity of individuals were collected through questionnaire . dietary intake was also collected using 24 h dietary recall questionnaire.results:waistline and body mass index for the case group were more than the control group ( p < 0.05 ) . physical activity level in healthy individuals was more than patients with nafld . dietary intake of saturated fatty acids and sugar in patients with nafld was more than healthy individuals ( p < 0.05 ) . intake of total dietary fiber , folic acid , vitamin d , zinc , and potassium in healthy individuals was more than patients with nafld ( p < 0.05).conclusions : in total , it seems the type of dietary intake source is associated with nafld . increasing saturated fatty acids and sugar and decreasing fiber , folic acid , vitamin d , zinc , and potassium intake might play a role in the progression of this disease .

INTRODUCTION METHODS Research participants Nutritional intake assessments Statistical data analysis RESULTS Demographic variables and physical activity Energy and macronutrients intake Vitamins and minerals intake DISCUSSION CONCLUSIONS Financial support and sponsorship Conflicts of interest

the main objectives of endodontic therapy are to eliminate bacteria from the root canal and to prevent the regrowth of residual microorganisms . antimicrobial agents are recommended for intracanal antisepsis , to prevent the growth of microorganisms between appointments . e. faecalis plays a major role in the etiology of persistent periradicular lesions after root canal treatment . its prevalence in asymptomatic , persistent endodontic infection is about 77% . c. albicans is the most common fungus isolated from failed endodontic cases . however , e.faecalis and c.albicans have been reported to be resistant to the antimicrobial effect of calcium hydroxide as a result of their ability to penetrate the dentinal tubules and adapt to the changing environment . therefore , a search for a better alternative has led to various formulations of calcium hydroxide , using different vehicles and newer antimicrobial agents such as chlorhexidine . bhat and walkevar , in 1975 , demonstrated the strong antibacterial action of propyleneglycol against the common microorganisms found in infected root canals and suggested its wider application in endodontics as a gentle vehicle for intracanal medicaments . its hygroscopic nature permits the absorption of water , which ensures a good sustained release of calcium hydroxide for long periods . calcium hydroxide with iodoform and silicone oil as a vehicle ( vitapex , neo dental chemical products co. ltd , tokyo , japan ) is very popular . it is composed of calcium hydroxide ( 30.3% ) , iodoform ( 40.4% ) , silicone oil ( 22.4% ) , and inert substances ( 6.9% ) . chlorhexidine has a reasonably wide range of activity against aerobic and anaerobic organisms as well as the candida species . to date , no study has compared the antimicrobial efficacy of calcium hydroxide with two different vehicles ( propylene glycol and iodoform with silicone oil ) , with chlorhexidine , against e.faecalis and c.albicans . the aim of this study is to determine the antimicrobial efficacy of two different formulations of calcium hydroxide and compare it with that of chlorhexidine , using an in vitro model of e.faecalis and c.albicans dentinal tubule infection . the model proposed by haapasalo and orstavik was modified for this study , similar to the one that was used by krithikadatta et al . a rotary diamond disk was used to decoronate 80 freshly extracted , single - rooted human teeth , 5 mm below the cementoenamel junction . the remaining root was sectioned such that 6 mm of the middle third of the root was obtained . the cementum was removed from the root surface to standardize the external diameter to approximately 4 mm , using a cylindrical diamond bur ( mani inc . , japan ) . the internal diameter was standardized to gates glidden drill number 3 ( mani inc . , the organic and inorganic debris was removed by treating the blocks with 17% ethylenediaminetetraacetic acid ( dent wash ; prime dental products pvt . gg-41 ) for five minutes , followed by 5% sodium hypochlorite ( prime dental products pvt . ltd . ; b.no 188777 - 0 ) for five minutes . next they were dipped in distilled water for five minutes and sterilized in an autoclave at 121c . half the number of blocks was subjected to a second cycle of sterilization , with the blocks immersed in 1 ml of tryptone soya ( ts ) broth in individual microcentrifuge tubes , and the other half in sabourauds dextrose ( sd ) broth . a rotary diamond disk was used to decoronate 80 freshly extracted , single - rooted human teeth , 5 mm below the cementoenamel junction . the remaining root was sectioned such that 6 mm of the middle third of the root was obtained . the cementum was removed from the root surface to standardize the external diameter to approximately 4 mm , using a cylindrical diamond bur ( mani inc . , the internal diameter was standardized to gates glidden drill number 3 ( mani inc . , the organic and inorganic debris was removed by treating the blocks with 17% ethylenediaminetetraacetic acid ( dent wash ; prime dental products pvt . gg-41 ) for five minutes , followed by 5% sodium hypochlorite ( prime dental products pvt . ltd . ; b.no 188777 - 0 ) for five minutes . next they were dipped in distilled water for five minutes and sterilized in an autoclave at 121c . half the number of blocks was subjected to a second cycle of sterilization , with the blocks immersed in 1 ml of tryptone soya ( ts ) broth in individual microcentrifuge tubes , and the other half in sabourauds dextrose ( sd ) broth . twenty - four hour colonies of pure culture of e.faecalis ( atcc 29212 ) and c. albicans ( 90028 ) grown on tryptone soya agar and sabourauds dextrose agar , respectively , were suspended in 5 ml of ts broth for e.faecalis and sabourauds infusion broth for c. albicans , and incubated for 24 hours at 37c . the culture suspensions were adjusted to match the turbidity equivalent of a 0.5 mcfarland standard . fifty microliters of inocula were transferred to presterilized individual microcentrifuge tubes containing 1 ml of respective broths and dentin block . the dentin blocks were transferred to a fresh broth containing microorganisms , every second day . the purity of the culture was checked by subculturing 5 l of the broth from the incubated dentin blocks in the respective broths , on agar plates . the blocks were irrigated with 5 ml of sterile saline , to remove the incubation broth . the dentin blocks were assigned to the following groups ( n = 5 ) for each day . group ii : calcium hydroxide ( deepashree products , india , b.no.051 ) + propylene glycol ( hi media ) [ 150 mg of calcium hydroxide powder was mixed with 0.15 ml of the vehicle , i.e. , distilled water ] . group iii : calcium hydroxide ( 30.3% ) , iodoform ( 40.4% ) , silicone oil ( 22.4% ) , and inert substances ( 6.9% ) , ( vitapex ; neo dental chemical products co. ltd . , tokyo , japan ) . group iv : two percent chlorhexidine gel ( 20% chx digluconate solution ; sigma aldrich , bangalore , c9394 ) [ 20% solution was first diluted to 2% and then the 2% chx gel was prepared by mixing it with polyethylene glycol as the vehicle and methyl cellulose as the thickening agent ] . all the blocks , after medication , were sealed above and below with paraffin wax and incubated in an aerobic environment , at 37 c. an antimicrobial assessment was performed at the end of two and seven days , with five blocks from each group , for every time interval . the blocks were washed with 5 ml of sterile saline combined with ultrasonics to remove the medicament . dentin debris were harvested at depths of 200 m and 400 m by using gg drills ( mani inc . , japan ) nos . 4 and 5 , respectively , collected in 1 ml of sterile broths , and incubated in aerobic environment at 37c for 24 hours . after the incubation period , the content of each microcentrifuge tube was evaluated by measuring the optical densities using the multiskan spectrum ( thermo scientific ) microplate spectrophotometer , at 620 nm . the data were analyzed with one - way analysis of variance followed by tukey multiple comparison test , to check the differences in microbial inhibition between the groups ( p < 0.05 = * , p < 0.01 = * * , p < 0.001 = * * * ) . the paired t test was used to check the differences in growth at different time intervals within the groups and for differences at the two depths at ( p < 0.05 = * , p < 0.01 = * * , p < 0.001 = * * * ) . after the incubation period , the blocks were irrigated with 5 ml of sterile saline , to remove the incubation broth . the dentin blocks were assigned to the following groups ( n = 5 ) for each day . group ii : calcium hydroxide ( deepashree products , india , b.no.051 ) + propylene glycol ( hi media ) [ 150 mg of calcium hydroxide powder was mixed with 0.15 ml of the vehicle , i.e. , distilled water ] . group iii : calcium hydroxide ( 30.3% ) , iodoform ( 40.4% ) , silicone oil ( 22.4% ) , and inert substances ( 6.9% ) , ( vitapex ; neo dental chemical products co. ltd . , group iv : two percent chlorhexidine gel ( 20% chx digluconate solution ; sigma aldrich , bangalore , c9394 ) [ 20% solution was first diluted to 2% and then the 2% chx gel was prepared by mixing it with polyethylene glycol as the vehicle and methyl cellulose as the thickening agent ] . all the blocks , after medication , were sealed above and below with paraffin wax and incubated in an aerobic environment , at 37 c. an antimicrobial assessment was performed at the end of two and seven days , with five blocks from each group , for every time interval . the blocks were washed with 5 ml of sterile saline combined with ultrasonics to remove the medicament . dentin debris were harvested at depths of 200 m and 400 m by using gg drills ( mani inc . , japan ) nos . 4 and 5 , respectively , collected in 1 ml of sterile broths , and incubated in aerobic environment at 37c for 24 hours . after the incubation period , the content of each microcentrifuge tube was evaluated by measuring the optical densities using the multiskan spectrum ( thermo scientific ) microplate spectrophotometer , at 620 nm . the data were analyzed with one - way analysis of variance followed by tukey multiple comparison test , to check the differences in microbial inhibition between the groups ( p < 0.05 = * , p < 0.01 = * * , p < 0.001 = * * * ) . the paired t test was used to check the differences in growth at different time intervals within the groups and for differences at the two depths at ( p < 0.05 = * , p < 0.01 = * * , p < 0.001 = * * * ) . there was no statistical difference between group ii and group iv and between 200 m and 400 m . for all the three medicaments the bacterial inhibition was significantly more on the seventh day as compared to the second day . the inhibition of growth in all the groups was statistically significant in comparison to group i [ figure 1 ] . comparison of the mean od values for the four groups at 200 and 400 micrometers depth on the second and seventh days for e.faecalis all the three medicaments used in this study exerted antimicrobial activity . no statistical difference was seen between group iii and group iv and between 200 m and 400 m . for all the three medicaments the fungal inhibition was significantly more on the seventh day as compared to the second day . the inhibition of growth in all the groups was statistically significant in comparison to group i [ figure 2 ] . comparison of mean od values for the four groups at 200 and 400 micrometers depth on the second and seventh days for c.albicans there was no statistical difference between group ii and group iv and between 200 m and 400 m . for all the three medicaments the bacterial inhibition was significantly more on the seventh day as compared to the second day . the inhibition of growth in all the groups was statistically significant in comparison to group i [ figure 1 ] . comparison of the mean od values for the four groups at 200 and 400 micrometers depth on the second and seventh days for e.faecalis no statistical difference was seen between group iii and group iv and between 200 m and 400 m . for all the three medicaments the fungal inhibition was significantly more on the seventh day as compared to the second day . the inhibition of growth in all the groups was statistically significant in comparison to group i [ figure 2 ] . comparison of mean od values for the four groups at 200 and 400 micrometers depth on the second and seventh days for c.albicans e.faecalis is a normal inhabitant of the oral cavity and is associated with different forms of periradicular disease . c. albicans is the fungal species most commonly detected in the oral cavity . therefore , different vehicles have been added to calcium hydroxide in an attempt to enhance its antimicrobial activity against the two tested microorganisms . the model proposed by haapasalo and orstavik was modified for this study , by adapting it to extracted human teeth rather than the previously used bovine incisors . this modification was considered appropriate because of the marked difference in diameter between the canals of bovine and human teeth . in addition , studies with human dentin blocks would definitely be more suitable to simulate the clinical scenario . the presence of the cementum affected the ability of the e.faecalis cells to infect the dentinal tubules , therefore , the cementum was removed from the specimens and a period of 21 days for microbial inoculation was taken for this study . the effective antimicrobial action of the tested intracanal medicaments decreased after 48 hours , therefore , one of the time periods chosen to evaluate the antimicrobial assessment was after the second day . calcium hydroxide requires a minimum period of seven days as an intracanal dressing , therefore , in half of the specimens the medications were kept for seven days . the direct contact test method using spectrophotometry was selected for this study as the optical density of the broth ( containing dentinal debris ) , which was directly related to the number of microorganisms present , and therefore , could be used to examine the antimicrobial efficacy of a medication . this method was especially helpful when a large number of specimens were taken . according to the results of this study calcium hydroxide with propylene glycol ( group ii ) and 2% chlorhexidine gel ( group iv ) performed the best for e.faecalis,and the differences between their antibacterial activities were not statistically significant . the possible reason for the antimicrobial action of calcium hydroxide with propylene glycol might be due to the mechanism of action of the antimicrobial activity of propylene glycol , as given in the book antimicrobials in food, by michael davidson ; which was related to the tendency of its molecules to orient themselves between the lipid and the water phases of the microbial membrane , which possibly altered the permeability of the cell membrane function . as the fungal cell wall mainly contained chitin ( polysaccharide ) and the cell membrane of sterols , propylene glycol might be more effective for e.faecalis , which had lipoteichoic acid as a major component of the cell membrane . the results of this study showed that for c.albicans , group iii ( calcium hydroxide with iodoform ) and group iv ( 2%chlorhexidine gel ) had the highest antifungal activity , and the differences between their antifungal activities were not statistically significant . the antimicrobial action of calcium hydroxide with iodoform against c. albicans might be due to the role of the calcium ions in the regulation of c. albicans morphogenesis . the antimicrobial effect of calcium hydroxide , due to the release of hydroxyl ions , might be enhanced due to the inhibition of c.albicans growth by calcium ions . this might be the possible cause for the better performance of group iii , for c.albicans . chlorhexidine has a reasonably wide range of activity against aerobic and anaerobic organisms as well as the candida species the inhibition of the growth of e.faecalis and c.albicans at 200 m and 400 m was uniform , with no statistical difference . the antimicrobial action of all the three medicaments increased on the seventh day as compared to that on the second day , for both the organisms tested in the study . propylene glycol was hygroscopic in nature , and therefore , there was a sustained release of hydroxyl ions from calcium hydroxide . this might be the reason for the increased antimicrobial activity of group ii on the seventh day . as oily vehicles have the lowest solubility and diffusion of the paste within the tissues , it might have resulted in the better antimicrobial action of group iii on the seventh day . for chlorhexidine , it had the property of substantivity , which might have been responsible for its higher antimicrobial activity in group iv on the seventh day as compared to the second day . results obtained from the in vitro laboratory experimental study can not be directly extended to the clinical situation ; however , they do provide reproducible and reliable means for comparing and testing the antimicrobial efficacy of various intracanal medicaments . from the results of this study it was concluded that : two percent chlorhexidine gel was an effective antibacterial and antifungal intracanal medicament against e.faecalis and c.albicans , respectively.the type of vehicle used would alter the antimicrobial property of calcium hydroxide as an intracanal medicament.addition of propylene glycol to calcium hydroxide as an intracanal medicament made calcium hydroxide an effective antibacterial agent against e.faecalis.addition of iodoform with silicone oil to calcium hydroxide as an intracanal medicament made calcium hydroxide an effective antifungal agent against c.albicans . two percent chlorhexidine gel was an effective antibacterial and antifungal intracanal medicament against e.faecalis and c.albicans , respectively . the type of vehicle used would alter the antimicrobial property of calcium hydroxide as an intracanal medicament . addition of propylene glycol to calcium hydroxide as an intracanal medicament made calcium hydroxide an effective antibacterial agent against e.faecalis . addition of iodoform with silicone oil to calcium hydroxide as an intracanal medicament made calcium hydroxide an effective antifungal agent against c.albicans .

aim : to evaluate the disinfection of dentinal tubules using calcium hydroxide with propylene glycol and calcium hydroxide with iodoform in silicone oil , as compared to 2% chlorhexidine gel.materials and methods : the antimicrobial efficacy of the medicaments against e.faecalis and c.albicans were assessed in vitro , using a dentinal tubule model at depths of 200 m and 400 m in extracted single rooted teeth . saline was taken as the negative control ( group i)results : all three medicaments used in this study exerted antibacterial and antifungal activity . group ii ( calcium hydroxide with propylene glycol ) and group iv ( 2% chlorhexidine gel ) had the highest antimicrobial activity and the differences between their antibacterial activities were not statistically significant . group iii ( calcium hydroxide with iodoform in silicone oil ) and group iv had the highest antifungal activity and the differences between their antifungal activities were not statistically significant . the inhibition of growth at 200 m and 400 m was uniform , with no statistical difference.conclusions:two percent chlorhexidine gel was effective against both e.faecalis and c.albicans . calcium hydroxide with propylene glycol was the most effective intracanal medicament along with 2% chlorhexidine against e.faecalis , whereas , calcium hydroxide with iodoform in silicone oil was the most effective intracanal medicament along with 2% chlorhexidine against c.albicans .

INTRODUCTION MATERIALS AND METHODS Dentin block preparation INFECTION OF THE BLOCKS Antimicrobial assessment Statistical analysis RESULTS For For DISCUSSION CONCLUSIONS

although the importance of diabetes mellitus ( dm ) as a major risk factor for macrovascular and microvascular disease is well recognized , the recent increase in the prevalence of dm , especially in younger individuals , has raised alarm . it is probable that the increased incidence of dm will slow the age - adjusted decline in coronary heart disease ( chd ) and will even raise the possibility of a significant increase in the risk of cardiovascular ( cv ) disease during the next several decades . recent data from the center for disease control showed that , although there was an increase in the prevalence of dm across all ethnic groups and all ages , the most striking increase ( by 70% ) was observed in people aged between 30 and 39 years . these data suggest that there is not only an increase in the prevalence of dm , but that the disease is occurring at younger ages and that such trends , if uninterrupted , can potentially lead to a new epidemic of cv disease during this millennium . cv complications are the leading cause of death and disability in patients with type ii dm . the magnitude of cv complications in dm is best illustrated by a recent report demonstrating that the risk of myocardial infarction in diabetic patients with no prior myocardial infarction was as high as that in nondiabetic patients with a history of prior myocardial infarction . although it has been known for years that dm is a major coronary risk factor , the precise mechanism for the increased risk of chd in patients with dm has not been well defined [ 3 - 6 ] . several studies have evaluated the role of potential factors that could increase the risk of atherosclerosis in association with dm [ 7 - 10 ] . the results of these studies demonstrate that there are a number of pathways by which dm increases the risk of chd . most diabetic patients frequently have one or more of the well - established risk factors , including hypertension , dyslipidemia , and obesity . although these are well - established , independent risk factors in nondiabetic patients , they can only account for approximately one - half of the excess chd risk associated with dm . the additional risk of chd must therefore be partly accounted for by the predominant metabolic abnormalities in dm , which are hyperglycemia and hyperinsulinemia due to insulin resistance . a number of recent studies have documented a relationship between the risk of chd events and hyperglycemia , increased levels of glycated hemoglobin ( hga1c ) , and hyperinsulinemia [ 2 - 10 ] . whether these metabolic abnormalities play a direct role in the pathogenesis of atherosclerosis or increase the risk of chd due to other associated risk factors is currently being investigated . based on the available information , it appears that hyperinsulinemia secondary to insulin resistance is an important and independent risk factor for chd [ 5 - 12 ] . the strongest evidence to support the association between hyperinsulinemia and chd events comes from the prospective study of men from quebec , which demonstrated a fourfold to fivefold increase in the risk of chd in individuals with the highest levels of fasting plasma insulin . the precise pathogenetic mechanism by which insulin increases the risk of atherosclerosis and chd has not been established . insulin might contribute to the risk of hypertension by stimulating sympatho - adrenal axes through modulation of cation transport and vascular remodeling by stimulating hypertrophy of vascular smooth cells . insulin not only enhances the effects of platelet - derived growth factor , but also directly stimulates vascular smooth cell hypertrophy . hyperinsulinemia can also produce dyslipidemia ( e.g. increase triglyceride and decrease high - density lipoprotein cholesterol by increasing catecholamine levels and by increased synthesis of very low - density lipoprotein . in addition , the increased levels of glucose and insulin promote secretion of plasminogen activator inhibitor-1 , which increases the risk of thrombosis by producing a prothrombotic state . diabetic patients also have increased levels of serum fibrinogen and von willebrand factor , which further enhance thrombosis . it is now well recognized that endothelial dysfunction and injury are key initial steps in the process of atherosclerosis . many of the factors associated with insulin resistance can also lead to endothelial dysfunction directly or indirectly . although in the normal physiologic state insulin stimulates production of nitric oxide by endothelial cells , recent studies have shown that in a hyperinsulinemic state endothelial response to insulin is impaired . the presence of endothelial dysfunction has also been demonstrated in association with hyperglycemia and in patients with type ii dm . in addition , the increased oxidative stress in association with diabetes can further impair endothelial function . it is thus clear that diabetes and the associated insulin resistance have damaging effects on the endothelium that , together with the cluster of associated abnormalities , increase the risk of atherosclerosis and chd . it has been suggested that as many as 50% of newly diagnosed patients with type ii dm might already have evidence of chd [ 3 - 6 ] . it is therefore conceivable that the insulin resistance that often precedes the development of clinical signs and symptoms of type ii dm is responsible for the increased risk of chd [ 7 - 11 ] . several studies have demonstrated that insulin resistance and hyperinsulinemia often coexist with other metabolic abnormalities that increase the risk of chd ( table 1 ) [ 7 - 11 ] . although insulin resistance syndrome has been recognized for some time as syndrome x or reaven 's syndrome , this was recently redefined as cardiovascular dysmetabolic syndrome ( cds ) . this newly coined term appears to be more logical because it includes all of the important elements of the syndrome and it highlights the involvement of the cv system in this dysmetabolic state . frequently observed metabolic , hemostatic , and other abnormalities in cardiovascular dysmetabolic syndrome , increased ; , decreased cds has four critical elements : dyslipidemia , insulin resistance , obesity , and high blood pressure ( easily remembered by the mnemonic drop ) ( table 2 ) . a diagnosis of cds requires the presence of at least two of the first three components : dyslipidemia , insulin resistance , and obesity . all components of cds are risk factors for macrovascular disease ( coronary disease , carotid disease , cerebral disease , and peripheral vascular disease ) . the presence of more than one component confers additive risk , although some components ( e.g. diabetes or insulin resistance ) confer more risk than others . it is important to note that , whereas the presence of dm doubles the risk of macrovascular disease in men , this risk is increased by fourfold to fivefold in women . women with diabetes thus lose the gender protection and are at equally high risk of chd as men of the same age and risk profile . although it is difficult to define insulin resistance ( the critical element of cds ) in the clinical setting , it has been suggested that fasting plasma glucose 110 mg / dl identifies most individuals with an insulin - resistant state [ 13 - 15 ] . the other elements of cds , such as obesity and hypertension , require only good physical examination and measurement of weight , waist circumference , and blood pressure . those with an increased waist - hip ratio ( > 0.9 ) and/or truncal obesity can be suspected to have an insulin - resistant state . a simple , the new criteria of the american diabetes association have also lowered the cut - off point that defines dm to fasting glucose 126 mg / dl , in recognition of the microvascular disease that is often seen with glucose values above that level . fasting glucose 126 mg / dl corresponds to a postprandial glucose 200 mg / dl , 2 hours after a 75 g glucose load , and either one of these values denotes the presence of dm . it has also been recommended that any casual nonfasting blood glucose 200 mg / dl indicates the presence of dm . it is important to note that , although good control of glucose levels decreases the risk of microvascular disease , patients with well - controlled glucose continue to be at increased risk of macrovascular disease due to persistence of insulin resistance , hyperinsulinemia , and associated metabolic and hemodynamic abnormalities . although routine screening for dm should be carried out at 3-year intervals starting at 45 years of age , earlier and more frequent screening is recommended for individuals who have one or more elements of cds , for those who have a family history of dm , and for members of high - risk ethnic groups ( e.g. hispanics , native americans , or migrant southeast asians ) . there are numerous nonpharmacologic and pharmacologic interventions that can have beneficial effects in individuals with cds and/or dm . although weight reduction by as little as 10% of body weight can improve insulin sensitivity , it is generally desirable to reduce weight to the ideal level , achieving body mass index < 25 kg / m . regular aerobic physical exercise can substantially improve insulin sensitivity and , in many cases , improve many of the abnormalities seen in cds . it has been suggested that even low levels of exercise , such as brisk walking for 3045 min three to five times a week , are useful in improving insulin sensitivity . there are several pharmacologic modalities that can be used for patients with dm and cds . in patients with dm , although exogenous insulin supplements endogenous production by the pancreas , it does not improve insulin resistance and it often causes hunger and additional weight gain . sulfonylureas work by stimulating production of insulin and would have the same metabolic drawbacks as exogenous insulin . alpha - glucosidase inhibitors , such as acarbose , delay carbohydrate absorption and decrease the stimulation for endogenous insulin secretion . biguanides , such as metformin , primarily decrease hepatic glucose production and , to a limited extent , reduce insulin resistance . although rare , metformin can cause fatal lactic acidosis , particularly in patients with renal or hepatic dysfunction . however , it can improve insulin sensitivity and can help reduce weight in some individuals . the thiazolidinediones , such as pioglitazone or rosiglitazone , reduce insulin resistance in muscle and , to a lesser extent , reduce hepatic glucose production . the thiazolidinediones can decrease blood pressure by reducing peripheral vascular resistance due to improved endothelial function . some of these agents can increase high - density lipoprotein cholesterol and decrease triglyceride levels . low - density lipoprotein cholesterol might increase without an increase in apolipoprotein b in some cases , indicating an increase in low - density lipoprotein particle size without an increase in number of particles , which may have an anti - atherogenic effect . a number of ongoing studies are now evaluating the cv benefits of treatment with thiazolidinediones ( pioglitazone and rosiglitazone ) in patients with type ii dm . although improvement in insulin sensitivity with these agents might be beneficial in the short term , we need to evaluate their long - term safety and efficacy , especially in light of the serious adverse experience observed with troglitazone . in summary , treatment of type ii dm should emphasize improvement in insulin sensitivity , a decrease in hepatic glucose production , and a decrease in stimulation of endogenous insulin production , rather than increased endogenous insulin production or exogenous administration of insulin . future drug development in this area should focus on drugs that can improve several of these abnormalities . it is only through development of such therapy , as well as public health measures directed to reducing obesity in the population at large , that we will be able to control this emerging epidemic of the 21st century . recent data show that the prevalence of type ii diabetes is rapidly increasing in the world . people are also developing dm at a younger age , and this trend is likely to continue with increased prevalence of obesity in the adolescent and youth years . dm has clearly become a major public health issue and it is likely to result in a new epidemic of premature coronary artery disease in the new millennium . early identification by regular evaluation for cds in susceptible individuals should help to identify the high - risk individuals who will benefit from aggressive nonpharmacologic as well as pharmacologic measures ( when necessary ) directed toward decreasing their risk for developing dm and cv disease . cds = cardiovascular dysmetabolic syndrome ; chd = coronary heart disease ; cv = cardiovascular ; dm = diabetes mellitus .

although diabetes mellitus is predominantly a metabolic disorder , recent data suggest that it is as much a vascular disorder . cardiovascular complications are the leading cause of death and disability in patients with diabetes mellitus . a number of recent reports have emphasized that many patients already have atherosclerosis in progression by the time they are diagnosed with clinical evidence of diabetes mellitus . the increased risk of atherosclerosis and cardiovascular complications in diabetic patients is related to the frequently associated dyslipidemia , hypertension , hyperglycemia , hyperinsulinemia , and endothelial dysfunction . the evolving knowledge regarding the variety of metabolic , hormonal , and hemodynamic abnormalities in patients with diabetes mellitus has led to efforts designed for early identification of individuals at risk of subsequent disease . it has been suggested that insulin resistance , the key abnormality in type ii diabetes , often precedes clinical features of diabetes by 56 years . careful attention to the criteria described for the cardiovascular dysmetabolic syndrome should help identify those at risk at an early stage . the application of nonpharmacologic as well as newer emerging pharmacologic therapies can have beneficial effects in individuals with cardiovascular dysmetabolic syndrome and/or diabetes mellitus by improving insulin sensitivity and related abnormalities . early identification and implementation of appropriate therapeutic strategies would be necessary to contain the emerging new epidemic of cardiovascular disease related to diabetes .

None Clinical relevance of cardiovascular dysmetabolic syndrome When and how to screen for CDS Therapeutic strategy in CDS and DM Conclusion Competing interests Abbreviations

celiac disease is a prevalent autoimmune disorder , with documented presence in populations of north and south america , europe , north africa , south and west asia , and australia . the symptoms of the disease are triggered in genetically susceptible individuals by ingestion of wheat and related cereal proteins of rye and barley . the ensuing innate and adaptive immune responses to the ingested proteins are responsible for inflammation , villous atrophy , and crypt hyperplasia in the small intestine , as well as the production of autoantibodies against the transglutaminase 2 ( tg2 ) enzyme . in addition to intestinal symptoms , celiac disease may involve extra - intestinal complications . dermatitis herpetiformis is the skin manifestation of celiac disease , affecting about 1020% of celiac disease patients and is characterized by papulovesicular lesions and presence of granular deposits of iga in the dermal papillae . elimination of the offending cereals from diet is currently the only effective mode of treatment for celiac disease . willem karel dicke was the first to recognize the importance of the removal of wheat and related cereals from diets of celiac disease patients in 1950 . shortly after that , the alcohol - soluble subfraction containing the gliadin proteins was determined to contain the main toxic factor in the offending grains . gliadin proteins were found to trigger b and t cell - mediated immune responses , which were thought to play a significant role in the inflammatory cascade in celiac disease . antibodies against gliadin proteins were demonstrated to be closely associated with celiac disease and were widely utilized as serologic markers of the condition prior to the discovery of anti - tg2 autoantibodies . antibodies against specific deamidated sequences of gliadin were eventually found to be more specifically associated with celiac disease than those against unmodified native gliadin . in addition to the gliadins , glutenin proteins of wheat , initially thought to be harmless , were later reported to trigger antibody and t cell immune responses in celiac disease patients . together , gliadins and glutenins comprise approximately 70 different proteins , collectively referred to as gluten . they are the major storage proteins of wheat and related cereals , representing about 75% of the total protein content of wheat grain . the nongluten proteins comprise the remainder of the wheat proteome and include several minor storage proteins , -amylase / protease inhibitors , and a variety of other enzymes . these proteins are generally much more soluble in water or aqueous salt solutions than gluten proteins and have been historically referred to as albumins / globulins . several nongluten proteins , including -amylase / protease inhibitor , thiol reductase , serine protease inhibitor ( serpin ) , and -amylase have been identified as potent allergens in ige - mediated wheat allergy and/or baker s asthma . however , the nongluten proteins of wheat and related cereals generally have been considered to be nontoxic and to lack immunogenic potential in the context of celiac disease . a few studies on small numbers of patients have examined immune reactivity to crude albumin / globulin extracts in celiac disease , with divergent results . the earliest of these investigated antibody reactivities in 24 untreated celiac disease patients ( but no healthy controls ) by an immunodiffusion assay , reporting antibodies to gliadin in six and to a crude pbs extract of wheat flour in seven patients . a subsequently published article reported elevated igg reactivity to both gliadin and albumin / globulin protein extracts in 15 untreated celiac disease patients when compared with three normal controls , although the patients exhibited significantly lower titers of antibody to the albumin / globulin extract than to gliadin . another report on three patients with celiac disease described increased antibodies to an extract of albumins / globulins in comparison to two controls , but the utilized extract appeared to contain gluten contamination . a study of six children with celiac disease found increased antibodies to albumin and globulin fractions in comparison to six unaffected controls , although information about the relative purity of the antigenic mixtures used in the immunoassays was not provided . in contrast , the latest of these studies with 10 celiac disease patients found no significant cellular or humoral response to the total extracts of albumins or globulins in comparison to 10 unaffected controls . the authors of this study attributed the contradiction between their and others observations to the presence of extensive gluten contamination in the earlier studies . a recently published report suggested that the nongluten -amylase / protease inhibitor proteins play a role in triggering the inflammatory response associated with celiac disease as innate immune activators through the engagement of the tlr4-md2-cd14 complex , but probably without concomitant b or t cell involvement . apart from these specific studies , the nongluten proteins of wheat and related cereals have not been examined in the context of celiac disease , and their potential for immunogenicity remains unclear . in this study , we use high - resolution two - dimensional gel electrophoresis , immunoblotting , proteome mapping , and tandem mass spectrometry ( ms / ms ) to show that specific nongluten proteins of wheat trigger a robust humoral immune response in patients with celiac disease or dermatitis herpetiformis . serum samples were from 120 individuals , including 50 patients with celiac disease ( 18 male , 46 white race , mean [ sd ] age 44.6 [ 17.5 ] years ) , 20 patients with dermatitis herpetiformis ( 11 male , 20 white race , mean age 43.1 [ 20.8 ] years ) , and 50 unaffected controls ( 20 male , 46 white race , mean age 37.1 [ 11.3 ] years ) . all cases of celiac disease were biopsy - proven and diagnosed according to previously described criteria . all patients with dermatitis herpetiformis had biopsies demonstrating classic histology , as well as the characteristic immunofluorescence pattern showing clear granular iga deposits in the dermal papillae . the celiac disease and dermatitis herpetiformis patients were on a gluten - containing diet . serum samples were obtained from patients and healthy controls under institutional review board - approved protocols at columbia university and at university of utah . this study was approved by the institutional review board of columbia university medical center . hard red spring wheat triticum aestivum butte 86 flour was suspended in 1 ml of 40% ethanol and mixed for 30 min at room temperature . the supernate was removed , chilled at 4 c for 1 h , combined with 1.9 ml of 1.5 m nacl , and stored at 4 c overnight . the precipitate was removed by centrifugation , rinsed with h2o , and dissolved in 0.2 ml of 0.1 m glacial acetic acid . the solution , containing gluten proteins , was lyophilized and stored at 20 c . fifty milligrams of flour was suspended in 200 l of buffer ( 50 mm tris - hcl , 100 mm kcl , 5 mm edta , ph 7.8 ) at 4 c and incubated for 5 min with intermittent vortex mixing . samples were centrifuged at 4 c for 15 min at 14,500 g. the supernate was collected , and proteins were precipitated by the addition of 4 volumes of cold ( 20 c ) acetone . following incubation overnight at 20 c , samples were centrifuged at 14,000 g for 15 min at 4 c . the pellet was rinsed with cold acetone , air - dried , and stored at 20 c . all patients and controls were tested for the currently recommended full panel of the most sensitive and specific serologic markers of celiac disease , including iga antibody to tg2 , igg antibody to deamidated gliadin , and iga antibody to deamidated gliadin . iga antibody to recombinant human tg2 was measured by elisa , according to the manufacturer s protocol ( euroimmun ag , luebeck , germany ) . igg and iga antibody reactivities to deamidated gliadin , as represented by a previously described glutamine glutamate substituted trimer of a fusion peptide containing the sequences plqpeqpfp and peqlpqfee , were measured by separate elisas , according to the manufacturer s protocols ( euroimmun ag ) . serum igg and iga antibodies to the gluten and nongluten protein extracts were measured separately by elisa as previously described , with some modifications . prior to the elisa analyses , the protein profile of each extract was assessed by sds - page , using the xcell surelock mini - cell electrophoresis system , 412% nupage bis - tris precast gels , and 2-(n - morpholino)ethanesulfonic acid ( mes ) buffer ( life technologies , carlsbad , calif . ) . a 2 mg / ml stock solution of the gluten extract in 70% ethanol or the nongluten protein extract in pbs was prepared . wells of 96-well maxisorp round - bottom polystyrene plates ( nunc , roskilde , denmark ) were coated with 50 l / well of a 0.01 mg / ml solution of protein extract in 0.1 m carbonate buffer ( ph 9.6 ) or left uncoated to serve as controls . after incubation at 37 c for 1 h , all wells were washed and blocked by incubation with 1% bovine serum albumin ( bsa ) in pbs containing 0.05% tween-20 ( pbst ) for 1.5 h at room temperature . serum samples were diluted at 1:200 for iga and at 1:800 for igg measurement , added at 50 l / well in duplicate , and incubated for 1 h. each plate contained a positive control sample from a patient with biopsy - proven celiac disease and elevated igg and iga antibodies to each protein extract . after washing , the wells were incubated with hrp - conjugated antihuman igg ( ge healthcare , piscataway , n.j . ) or iga ( mp biomedicals , santa ana , calif . ) the plates were washed and 50 l of developing solution , containing 27 mm citric acid , 50 mm na2hpo4 , 5.5 mm o - phenylenediamine , and 0.01% h2o2 ( ph 5 ) , was added to each well . after incubating the plates at room temperature for 20 min , absorbance was measured at 450 nm . absorbance values were corrected for nonspecific binding by subtraction of the mean absorbance of the associated uncoated wells . the corrected values were first normalized according to the mean value of the positive control duplicate on each plate . the mean antibody level for the unrelated healthy control cohort was then set as 1.0 au , and all other results were normalized accordingly . the kcl - soluble proteins were separated by two - dimensional electrophoresis as previously described . mg / ml in a solution containing 9 m urea , 4% nonidet p-40 , 1% dithiothreitol , and 2% servalyt 3 - 10 iso - dalt the first dimension capillary tube gels contained 9.2 m urea , 4% ( total monomer ) acrylamide / bis , 2% nonidet p-40 , 2% servalyt 3 - 10 iso - dalt , 0.015% ammonium persulfate , and 0.125% temed . isoelectric focusing was performed using a mini protean ii tube cell ( bio - rad , hercules , calif . ) . eighteen micrograms of protein was loaded for gels that were to be coomassie - stained for visualization of the separated proteins , while 3.6 g of protein was loaded for gels that were to be used for subsequent transfer and immunoblotting . proteins were separated in the second dimension by sds polyacrylamide gel electrophoresis , using the xcell surelock mini - cell electrophoresis system , 412% nupage bis - tris precast gels , and mes buffer . protein transfer onto nitrocellulose membranes was carried out with the iblot dry blotting system ( life technologies ) . the membranes were incubated for 1 h in a blocking solution made of 5% milk and 0.5% bsa in a solution of tris - buffered saline containing 0.05% tween-20 ( tbst ) . incubation with patient and control serum specimens ( 1:2000 for iga and 1:4000 for igg determination in dilution buffer containing 10% blocking solution and 10% fetal bovine serum in tbst ) was done for 1 h. serum samples from celiac disease ( n = 14 ) and dermatitis herpetiformis ( n = 6 ) patients with elevated iga and/or igg antibody reactivity to nongluten proteins , in addition to 5 healthy controls , were included . detection of bound antibodies was by the ecl system ( millipore , billerica , mass . ) and autoradiography film ( crystalgen , commack , n.y . ) . following immunodetection , bound antibodies were removed from the nitrocellulose membranes with restore western blot stripping buffer ( thermo scientific , rockford , ill . ) , and the membrane proteins were visualized using colloidal gold stain ( bio - rad ) . each immunoblot was aligned to its corresponding colloidal gold - stained membrane using the samespots software ( version 4.5 ) ( totallab ltd . proteins in the two - dimensional electrophoresis spots that were the main targets of the antibody response were identified initially by comparison to a previously generated proteomic map of butte 86 flour . spots were excised from gels and placed in wells of a 96-well reaction plate , leaving a blank well between each sample . proteins in each sample - well were reduced , alkylated , and then digested with trypsin using a digestpro instrument ( intavis , koeln , germany ) according to the manufacturer s instructions . the resulting tryptic peptides were eluted into a collection tray that was then placed into the autosampler compartment of an easy - nlc ii ( thermo scientific , waltham , mass . ) that was interfaced by a nanoelectrospray source to an orbitrap elite mass spectrometer ( thermo scientific ) . four microliter fractions were loaded by the autosampler onto an integrafrit trap column ( 100 m 200 mm , with 5 m , 120 , reprosil - pur c18 aq packing ) ( new objective , woburn , mass . ) , washed with 20 l of solvent a to remove salts , then switched in - line with the analytical column ( 75 m 10 cm of 3 m , 120 , reprosil - pur c18 aq packing ) ( new objective ) and eluted with a gradient of 100% solvent a to 30% solvent b over 30 min into the orbitrap elite mass spectrometer . solvent a was water and solvent b was acetonitrile , both optima lc / ms grade , containing 0.1% formic acid ( thermo scientific ) . peptides were detected in the orbitrap with the ft survey scan set to scan a range from 300 to 2000 m / z at a resolution of 60,000 . the 10 most intense peaks were automatically subjected to collision - induced dissociation ( cid ) . the mass range for the cid scans was set to high and the minimal signal threshold to 10,000 . dynamic exclusion with a repeat count of 1 was enabled for a duration of 10 s. normalized collision energy was set to 35 . processing of raw files to mascot generic format ( mgf ) files was carried out using msconvert from the proteowizard open - source project [ http://proteowizard.sourceforge.net/downloads.shtml ] . a first pass search of the ms / ms spectra was conducted against the superwheat database ( version # 140828 ) . for this study , the superwheat database ( version # 100211 ) described in dupont et al . was updated with ncbi triticeae ( taxid : 147389 ) protein sequences ( download date : 08/28/2014 ) as well as with the translated sequences of 137 ests , contigs , or pcr products from butte 86 ( supplemental file 1 ) . mascot version 2.3 [ www.matrixscience.com ] and x ! tandem version 2010.12.01.1 [ http://www.thegpm.org/tandem/ ] were used to match instrument - generated spectra to fasta sequences in the superwheat database . for the first and second pass search , the parent mass tolerance was set to 15 ppm and the fragment error to 0.4 da . charge state screening allowed + 1 , + 2 , and + 3 charge states to be selected , and one missed cleavage was allowed . analysis , validation , and display of the data were carried out using scaffold version 4.3.2 [ http://www.proteomesoftware.com ] . the output results files from the two different search engines were combined in individual folders and analyzed as separate mudpit experiments . a second database of fasta files was generated from the first pass search by exporting from scaffold all proteins that contained one peptide hit and had a protein probability of 20% and peptide probability of 0% . a reverse - concatenated database was created from these sequences . the resulting subset database , containing 14,776 sequences , was used for the second pass search . criteria for protein acceptance in the second pass search was set in the scaffold validation software package to a protein probability of 99% and a requirement for four matching peptides having a parent peptide mass accuracy of 2 ppm and a calculated 95% probability . the list of proteins identified in each spot is shown in supplemental file 2 . the protein with the greatest number of exclusive unique spectra sequence coverages determined by scaffold were adjusted for the presence of the signal peptides as predicted by signalp 4.1 [ http://www.cbs.dtu.dk/services/signalp/ ] . the data files associated with this study were archived at ucsd center for computational mass spectrometry in the massive data set project . the raw mass spectrum files , the sequence database files , and the scaffold results report can be downloaded from ftp://msv000078887:a@massive.ucsd.edu/. in order to visualize scaffold report results , a free viewer can be downloaded from http://www.proteomesoftware.com/products/free-viewer . to visualize the instrument raw files , the cdnas encoding the identified serpin protein in spot 1a ( gi : 224589270 ) and the purinin protein in spot 2a ( bu_purinin#3 ) ( table 2 ) were synthesized ( after codon optimization for an e. coli expression system ) based on amino acid sequences shown in supplemental file 4 . for the purinin , a potential signal peptide cleavage site was detected at position 19 and amino acids 119 were therefore excluded . the synthesized genes were inserted between the restriction sites ndei and hindiii of the tag - free vector e3 to create the e. coli expression vectors e3 - 224589270 and e3-bu_purinin#3 . linearized vectors were transformed into e. coli , and protein expression was induced by adding 100 mm isopropyl -d-1-thiogalactopyranoside to the culture for 4 h at 37 c . cells were lysed in lysis buffer ( 50 mm tris , 1 mm pmsf , ph 8.0 ) , and inclusion bodies , containing insoluble proteins , were collected for purification . after washing ( 50 mm tris , 1% triton x-100 , 300 mm nacl , 2 mm dtt , 1 mm edta , ph 8.0 ) , pellets were dissolved in 8 m urea . the purified proteins were suspended in refolding buffer ( 50 mm tris , 10% glycerol , 150 mm nacl , ph 8.0 ) . the molecular weight and relative purity of the proteins were assessed by sds - page , following the above protocol . the identity of each protein was confirmed by mass spectrometry - assisted peptide mass mapping , as previously explained . antibody reactivity to the generated recombinant serpin and purinin proteins was assessed by immunoblotting . recombinant proteins ( 0.2 g ) were separated by sds - page and transferred onto nitrocellulose membrane . detection of bound hrp - conjugated secondary antibodies was by ecl and the fluorochem m imaging system ( protein simple , santa clara , calif . ) . differences between groups were analyzed by parametric or nonparametric one - way analysis of variance ( anova ) with posthoc testing for multiple comparisons . statistical analyses were performed with prism 6 ( graphpad , san diego , calif . ) . iga antibody to tg2 and igg / iga antibodies to deamidated gliadin , which are considered to be highly specific and sensitive for celiac disease , were measured in all patients and controls . celiac disease and dermatitis herpetiformis groups displayed significantly higher mean levels of iga anti - tg2 antibody ( figure 1a ) , iga antideamidated gliadin antibody ( figure 1b ) , and igg antideamidated gliadin antibody ( figure 1b ) than those of healthy controls ( p < 0.0001 for all comparisons ) . mean levels of antibody to ( a ) human tg2 ( iga ) and ( b ) deamidated gliadin fusion peptide ( iga and igg ) in patients with celiac disease and dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . the gel electrophoresis profiles of the gluten and nongluten protein extracts used for the elisa analyses are shown in figure 2a . levels of iga and igg class antibodies to the extracted gluten and nongluten protein fractions were measured in all patients and controls . compared with healthy controls , the two patient groups displayed significantly higher mean levels of iga ( p < 0.0001 for celiac disease ; p < 0.001 for dermatitis herpetiformis ) and igg ( p < 0.0001 for both ) antibody to gluten proteins ( figure 2b ) . in addition , compared with healthy controls , the celiac disease and dermatitis herpetiformis patient cohorts exhibited significantly increased serum iga ( p < 0.0001 for both ) and igg ( p < 0.0001 for both ) antibody reactivity to the proteins of the nongluten extract ( figure 2c ) . ( a ) gel electrophoresis profile of the butte 86 protein preparations used for the antibody assays : ( 1 ) gluten extract ( 5 g ) and ( 2 ) nongluten protein extract ( 5 g ) . molecular weight markers , shown to the left of the panel , are in kda . ( b , c ) comparison of mean levels of iga and igg antibodies to gluten ( b ) and nongluten ( c ) proteins in patients with celiac disease or dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . error bars represent the standard error of the mean . in order to identify the molecular targets of the detected increased antibody response to nongluten proteins in celiac disease and dermatitis herpetiformis patients , a randomly selected subset of antibody - positive sera was further analyzed . antibody reactivity was characterized by immunoblotting following two - dimensional separation of the nongluten proteins of the wheat cultivar butte 86 . the two - dimensional electrophoresis pattern of the nongluten proteins following coomassie staining is shown in figure 3a . the immunoblotting analyses demonstrated antibody reactivity to specific nongluten proteins of wheat in 19 of 20 analyzed patient sera . these included 10 of 11 celiac disease and 2 of 3 dermatitis herpetiformis samples tested for iga reactivity , and 9 of 9 celiac disease and 6 of 6 dermatitis herpetiformis samples tested for igg reactivity . none of the control sera exhibited antibody binding to any proteins at the dilutions and exposures tested . ( a ) pattern of proteins of the nongluten extract after separation by two - dimensional gel electrophoresis and coomassie staining . proteins in five groups , shown in boxes , were found to be the main nongluten antigen targets of the antibody response : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . ( b e ) immunoblots showing serum antibody reactivity to the two - dimensionally separated proteins in representative patients and healthy controls : ( b ) igg reactivity to serpins , purinins , -amylase / protease inhibitors , globulins , and farinins in a patient with celiac disease ; ( c ) igg reactivity to serpins , purinins , and -amylase / protease inhibitors in a patient with celiac disease ; ( d ) igg reactivity to serpins and globulins in a patient with dermatitis herpetiformis ; ( e ) iga reactivity to serpins and farinins in a patient with dermatitis herpetiformis ; ( f ) igg and ( g ) iga reactivity in healthy controls , indicating lack of binding to nongluten proteins . molecular weight markers ( in kda ) proteins within five regions of the two - dimensional gels reacted with patient sera ( figure 3 ) . comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour suggested that the reactive proteins belonged to the following distinct protein groups : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins ( figure 3 and table 1 ) . identities of proteins in individual spots that reacted with sera were verified by ms / ms using an orbitrap elite mass spectrometer and subsequent database search . peptide data for the predominant protein in each spot are displayed in supplemental file 3 , along with the sequence coverage of each protein . supplemental file 4 contains the amino acid sequences of the identified target proteins from table 2 . because of the sensitivity of the instrument , the analysis indicated that most spots contained multiple proteins ( supplemental file 2 ) . however , the majority of the spectra obtained from each spot corresponded to a specific protein that was deemed the predominant protein in the spot ( table 2 and supplemental files 2 and 3 ) . the ms / ms data confirmed the presence of the five protein types that had been initially identified through comparison of spot positions with the proteomic map of butte 86 flour . for spots 2c and 2d , the ms / ms data indicated that the spots contained purinin proteins ( bu_purinin#1 and bu_purinin#2 ) ( supplemental files 2 and 3 and table 2 ) , even though nonpurinin type proteins in those spots had the highest exclusive unique spectrum count . five distinct protein groups were identified as the main targets of the antibody response through comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . numbers refer to the specific immunoreactive protein groups , as marked in figure 3a . number of sera found to have igg and/or iga antibody reactivity to spots in each protein type group , as described in the materials and methods section . protein in each spot for which the highest number of exclusive unique spectra were obtained . protein sequences derived from the translation of butte 86 ests or contigs are indicated by protein sequences derived from the translation of contigs from harvest 1.14 est assembly [ http://harvest.ucr.edu/ ] are indicated by sequences of all identified proteins in the table are provided in supplemental file 4 . number of sera found to have igg and/or iga antibody reactivity to each protein spot , as described in the materials and methods section . ms / ms data indicate that the spot also contains a purinin , bu_purinin # 1 ( supplemental files 2 and 3 ) . ms / ms data indicate that the spot also contains a purinin , bu_purinin # 2 ( supplemental files 2 and 3 ) . in this spot , a similar number of exclusive unique spectra were obtained for globulin-2 , bu_globulin-2_contig_18428 , but a greater number of total spectra were obtained for bu_purinin # 3 ( supplemental files 2 and 3 ) . description of results according to patient group is presented in tables 1 and 2 . patients were most frequently reactive to serpins ( 75% of tested celiac disease and dermatitis herpetiformis patient samples ) , followed by purinins ( 65% ) , -amylase / protease inhibitors ( 60% ) , globulins ( 40% ) , and farinins ( 35% ) . the most frequently reactive individual protein spot was identified as a serpin ( spot 1a in figure 3a ; gi : 224589270 ) ( table 2 ) . fifteen of the 20 patients ( 75% ) and none of the controls exhibited antibody reactivity to this protein spot . within the purinin group , the most reactive spot ( 2a ) was identified as bu_purinin # 3 ; 10 of 20 patients ( 50% ) displayed antibody reactivity to it . frequencies of reactivity to the identified proteins did not appear to be substantially different between the celiac disease and dermatitis herpetiformis groups . in order to further confirm the presence of antibody reactivity to selected nongluten antigens identified in this study , we generated proteins by recombinant expression based on the available amino acid sequences for the serpin in spot 1a and the purinin in spot 2a . immunoblotting with the recombinant proteins confirmed the presence of igg and/or iga antibody reactivity to the specific serpin and purinin proteins in patients with celiac disease or dermatitis herpetiformis who had been found to be positive for antibodies to spot 1a and/or spot 2a ( figure 4 ) . healthy controls did not display any reactivity at the serum dilutions and image acquisition exposures used . serum antibody reactivity to a recombinant serpin , representing spot 1a ( gi : 224589270 ) , and a recombinant purinin representing spot 2a ( bu_purinin # 3 ) . ( a ) sds - page profile of the purified recombinant serpin ( lane 1 ) and purinin ( lane 2 ) proteins , following coomassie staining . ( b e ) immunoblotting with the recombinant proteins , demonstrating iga ( b , d ) and igg ( c , e ) antibody reactivity to serpin ( b , c ) and purinin ( d , e ) . lanes 1 and 2 correspond to sera from two representative patients , while lanes 3 and 4 represent sera from two healthy controls . molecular weight markers are shown to the left of each panel ( in kda ) . heightened adaptive immune response to ingested gluten proteins of wheat and related cereals is a hallmark of celiac disease . a few earlier studies , carried out on small numbers of celiac disease patients , had attempted to assess immune reactivity to nongluten proteins of wheat . however , the results were inconsistent , and the purity of the antigenic mixture in the crude nongluten protein extracts used for detecting immune reactivity was later questioned . here , we have demonstrated the presence of a robust humoral response to specific nongluten proteins of wheat in patients with celiac disease and dermatitis herpetiformis . the detailed analysis of antibody reactivity through two - dimensional immunoblotting and ms / ms identification of target proteins provides unequivocal confirmation that the main targets of the antibody response are , in fact , specific proteins that have not been categorized as gluten or previously recognized as triggers of humoral response in celiac disease . wheat serpin proteins belong to the large family of serine protease inhibitors that are present in many organisms , most of them acting as suicide substrate inhibitors of chymotrypsin - like proteases . other nongluten target antigens of wheat included purinin , -amylase / protease inhibitor , globulin , and farinin proteins . the mechanism responsible for generating antibodies against proteins that are generally thought of as nontoxic in the context of celiac disease is not clear . the elevated antibody reactivity to nongluten proteins may be driven by the enhanced inflammatory environment in the gut , brought on by the villous damage and ensuing epithelial barrier dysfunction that are triggered by gluten in celiac disease . however , it should be noted that we did not detect antibodies against a broad array of nongluten proteins . for example , no antibody reactivity to triticin proteins , which are present at similar or higher concentration in wheat flour as serpins , globulins , farinins , and purinins , was found . also , serpins make up a substantially lower percentage of total protein content of butte 86 flour than the -amylase / protease inhibitors but were a more frequent target of patient antibody response . therefore , the elevated antibody reactivity to nongluten proteins appears to be directed at a rather specific set of antigens . another possibility is that the peripheral antibody response to gluten proteins in celiac disease may cross - react with specific nongluten antigens that contain similar epitopes , thus contributing to the detected levels of antibodies against nongluten proteins . a homology analysis indicates that the newly named purinin proteins are close in sequence to -gliadins . in addition , there are short sequences in the identified -amylase / protease inhibitors and another newly characterized group of proteins , the farinins , which are similar to those in certain -gliadin and low molecular weight glutenin proteins . in addition , the reactive centers of some of the identified serpin antigens resemble the glutamine - rich repeats in gluten proteins . whether these particular shared sequences are actually immunogenic and capable of contributing to the presence of cross - reactive antibodies the observed increase in iga and igg antibody responses to specific nongluten proteins brings up the obvious question of whether the identified immune response might be relevant to the pathogenic mechanism of celiac disease . similar to gluten proteins , serpins and -amylase / protease inhibitors are resistant to proteolytic digestion and are therefore likely to be present in the form of long stretches of incompletely digested , and potentially immunogenic , sequences in the small intestine . the observed iga , along with igg , reactivity to the identified nongluten proteins points to a mucosal origin for the immune - triggering event . however , it remains to be seen whether the mucosal b cell response is accompanied by a corresponding intestinal cd4 t cell reactivity to the nongluten proteins . presence of lamina propria cd4 t cells with specificity for sequences of the identified nongluten proteins may contribute to the celiac disease - associated pathways in the gut , for example , by providing additional help to the autoreactive cd8 intraepithelial cytotoxic t cells that drive epithelial cell damage . it is also possible that the detected antibodies to nongluten proteins would themselves contribute to the mucosal lesion . similar to the effect shown for antigluten antibodies , the antibodies against the nongluten proteins may be involved in inducing local complement activation and mucosal damage . they may also contribute to recruitment of various leukocytes that lead to antibody - dependent cell - mediated cytotoxicity , a process that has been previously demonstrated for antibodies to gluten . however , the observed immune response to nongluten proteins , as was mentioned earlier , may be the result and a bystander marker of intestinal barrier damage and inflammation , without playing a role in the pathogenic mechanism of celiac disease . in conclusion , the results of this study clearly demonstrate that the humoral response to wheat in celiac disease is not limited to gluten antigens but is also directed at specific nongluten proteins . while direct conclusions can not be drawn about the pathogenic effects of the identified nongluten proteins , these findings should prompt further research into their potential role in contributing to the inflammatory processes that result in mucosal damage in patients with celiac disease . the possibility of such a role for these proteins is worthy of attention , especially as therapies other than gluten exclusion from the diet are under development . for example , proteolytic enzymes with specificity for the toxic fragments of gluten , may be insufficiently active against other immunogenic proteins . in addition , further investigation of the utility of the identified antibodies as potential biomarkers in celiac disease or other gluten - related disorders may be warranted .

while the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched , the immune response to nongluten proteins of wheat has not been characterized . we aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease . serum samples from patients and controls were screened for igg and iga antibody reactivity to a nongluten protein extract from the wheat cultivar triticum aestivum butte 86 . antibodies were further analyzed for reactivity to specific nongluten proteins by two - dimensional gel electrophoresis and immunoblotting . immunoreactive molecules were identified by tandem mass spectrometry . compared with healthy controls , patients exhibited significantly higher levels of antibody reactivity to nongluten proteins . the main immunoreactive nongluten antibody target proteins were identified as serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens . the results demonstrate that , in addition to the well - recognized immune reaction to gluten , celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat .

Introduction Materials and Methods Results Discussion

the term gossypiboma , derived from the latin word gossypium ( cotton ) and the swahilli boma the condition may manifest as an exudative inflammatory reaction with formation of abscess or aseptic fibrotic reaction with formation of a mass ( 2 ) . the patient may present early with features of sepsis or may remain asymptomatic or may present with non - specific symptoms months or years after the operation . although early recognition of this problem will ensure institution of appropriate treatment , every measure should be taken to prevent this complication . herein , we report a case of gossypiboma presented as an abdominal lump 7 years after open cholecystectomy . a 54-year - old woman presented with a 2-years history of dull aching pain in the upper abdomen and 1 year history of an abdominal swelling . she developed fever and purulent discharge from the wound in the immediate postoperative period which was treated conservatively . on examination a large non - tender mass with restricted side to side mobility was found in the right hypochondrium extending up - to the right lumber region . blood investigations revealed hemoglobin of 8.5gm / dl , leukocyte count of 9600/mm , serum bilirubin of 2.9 mg / dl and alkaline phosphatase of 507 u / l . the plain abdominal radiography did not show any sign of a radio - opaque marker in the abdomen . however , contrast enhanced abdominal computerized tomography revealed a 1512x10 cm round well - defined soft - tissue mass with multiple septae . ct scan abdomen ( both oral and intravenous contrast ) showed a mass in right upper quadrant with displacement of gut loops towards the left . the mass showed contrast enhanced internal septa ( white arrow ) a radio - opaque material was seen in the lesion raising the suspicion of a foreign body . common bile duct was dilated and contained a stone which was removed endoscopically . at exploration , a large globular lesion was found which was densely adhered to the adjacent structures ( anterior abdominal wall , omentum , transverse colon , and antrum of the stomach ) . en - bloc resection of the mass along with the adhered transverse colon was done ( figure-2 ) . cut section of the mass showed a large surgical sponge and a foreign body within it ( figure-3).the patient had an uneventful recovery and she was well at 19-month follow - up . operative photograph showing a large mass ( white arrow ) with adherent transverse colon ( black arrow ) . cut section of the mass showing a large sponge ( white arrow ) and a radio - opaque marker ( black arrow ) within it . gossypiboma or a mass of cotton that is retained in the body following surgery is rarely seen in daily clinical practice . although the real incidence is unknown , it has been reported as 1 in 100 to 3000 for all surgical interventions and 1 in 1000 to 1500 for intraabdominal operations ( 3,4 ) the clinical presentation of gossypiboma varies and depends on the location of the sponge and the type of reaction . there are 2 types of foreign body reaction in pathology : an exudates reaction leading to abscess formation or chronic internal or external fistula formation , and an aseptic fibrinous reaction resulting in adhesion , encapsulation , and eventual formation of granuloma of various size . they usually remain asymptomatic or present with pseudotumor syndrome , as in our case . because , the symptoms of gossypiboma are usually nonspecific and may appear years after surgery , the diagnosis of gossypiboma usually comes from imaging studies and a high index of suspicion . the most impressive imaging finding of gossypiboma is the curved or banded radio - opaque lines on plain radiograph . the ultrasound feature is usually a well - defined mass containing wavy internal echogenic focus with a hypoechoic rim and a strong posterior shadow . on ct scan , a gossypiboma may manifest as a cystic lesion with internal spongiform appearance with mottled shadows as bubbles , hyperdense capsule , concentric layering , or mottled mural calcifications ( 6 ) . when no radio - opaque marker is seen on x - ray or ct , the characteristic internal structure of the gauze granuloma is best visualized on magnetic resonance imaging . it may appear as a low - signal - intensity lesion on t2-weighted images with wavy , striped or spotted appearance ( 7 ) . nosher and siegel ( 8) described six patients in whom percutaneous extraction was successfully performed for removal of foreign bodies . besides diagnostic and therapeutic difficulties the presence of a foreign body inside the patient can be easily proved and the patient may litigate the responsible surgeon because this is an avoidable problem . moreover , gossypiboma may be misdiagnosed as a malignant tumor and lead to unnecessary invasive diagnostic procedures or extensive extirpative surgery ( as in our case ) which may result in further complications ( 9 ) . avoidance of leaving foreign bodies inside the patients could be possible by implementation of three measures : meticulous count of all surgical materials , thorough exploration of the surgical site at the conclusion of the procedures and routine use of surgical textile materials impregnated with a radio - opaque marker . gawande et al ( 3 ) published an article about risk factors of retained foreign bodies ( rfbs ) . of the 8 risk factors , the authors identified [ emergency operation , unexpected change in operation , more than one surgical team involved , change in nursing staff during procedure , body mass index ( bmi ) , volume of blood loss , female sex , and surgical counts ] only 3 were found to be statistically significant by multivariate logistic regression . the 3 significant risk factors were emergency surgery , unplanned change in the operation , and bmi . the counting of sponges and instruments was not a significant predictor in the multivariate model . although all 3 factors were significant , the 9-fold increase in risk associated with emergency surgery was impressive . in addition , in 88% of the cases where there was a rfb and counts were performed , the counts were falsely called correct . the authors recommended radiographic screening at the end of high risk cases as a possible adjunct to improve detection of rfb . although human errors can not be completely avoided , continuous medical training and strict adherence to rules of the operation room should reduce the incidence of gossypiboma to a minimum .

gossypiboma is very rare in clinical practice . despite its clinical importance , it carries some medico legal implications . we report a case of gossypiboma in a 54 years old female who presented with pain and a slowly growing lump in the abdomen 7 years after open cholecystectomy . computed tomography ( ct ) scan of the abdomen showed a mass with enhanced internal septae and a radio - opaque marker within it , raising the suspicion of a foreign body . she underwent excision of the mass along with a segment of densely adherent transverse colon . post - operative recovery was uneventful and the patient was well at 19-month follow up .

INTRODUCTION CASE REPORT DISCUSSION

it is well known that early diagnosis and management of neonatal infection is mandatory for outcome . clinical difficulties in identifying early the infection in neonates and also in discriminating between bacterial and viral aetiology have led to the evaluation of several biomarkers including hematologic parameters , acute phase proteins , chemokines , cytokines , and cell - surface antigens [ 1 , 2 ] . however , none of the biomarkers of neonatal infection or sepsis assessed up to date has been shown to be ideal . the urokinase - type plasminogen activator receptor ( upar ) , a membrane - linked receptor with extracellular protease activity that transduces intracellular signaling pathways , is expressed on the surface of several inflammatory cells , including neutrophils , lymphocytes , and monocytes , as well as on endothelial cells . it is the cognate receptor for urokinase - type plasminogen activator which is activated upon binding and involved in the conversion of plasminogen to the active fibrinolytic enzyme plasmin . after cleavage and release from the cell membrane , increased supar levels have been reported in several biological fluids including blood , urine , and saliva , as well as in cerebrospinal , pleural , pericardial , and peritoneal fluid [ 35 ] , in response to a variety of infections in adults [ 68 ] , and they correlate positively with the activation level of the immune system . in neonates , the properties of supar as a biomarker of infection are not known ; to the best of our knowledge , the only information regarding supar in neonatal infections derives from one study aimed to identify by multiplexed immunoassay arrays differentially expressed serum proteins in clinically infected and noninfected preterm infants . the objective of the present study was to evaluate the clinical value of supar in the detection of neonatal infection or sepsis , discrimination between bacterial and viral infections , and monitoring the responsiveness to treatment . criteria for eligibility in the study included : ( 1 ) term infant , ( 2 ) postnatal age between 4 and 28 days of life , ( 3 ) bacterial ( confirmed by positive blood , urine , and/or csf culture ) or viral infection ( confirmed by detection of virus in biological fluids or highly probable viral infection suggested by accompanying symptoms , well - appearing , negative bacterial cultures , normal for age total leukocyte count / differentiation , and serum c - reactive protein [ crp ] values less than 10 mg / l ) . exclusion criteria were as follows : ( 1 ) perinatal asphyxia , ( 2 ) antibiotic treatment in the last 10 days before admission , ( 3 ) surgery , and ( 4 ) positive culture(s ) for staphylococcus epidermidis . on admission to our special care unit , all infected neonates underwent blood , csf , and suprapubic urine sampling for analyses and cultures ; a small quantity of plasma ( 0.2 ml ) was isolated within 30 min and stored at 80c for measurement of supar levels . additional biological specimens including synovial fluid , stools , throat swabs , or nasopharyngeal secretions were also obtained if required according to the presenting symptoms . following admission , blood samples were drawn from all infected infants at 24 hours , 48 hours , 35 days , and 710 days for routine blood tests ( full blood count , renal and liver function , and serum crp levels ) , as well as for 0.2 ml plasma isolation and storing at 80c for supar levels determination . plasma supar concentrations were finally measured in 47 infected neonates of mean sd gestational age 39.0 1.0 weeks , birth weight 3 , 135 351 g , postnatal age 18 6 days , and male / female ratio 29/18 , and in 18 healthy term infants who had similar gestational age ( 38.6 1.0 weeks ) , birth weight ( 3 , 216 406 g ) , postnatal age ( 17 6 days ) , and gender distribution ( 11 males , 7 females ) to those of infected infants , as controls . five serial supar measurements were performed in each patient during the course of the disease , whereas supar plasma levels were determined only once in the control group along with full blood count , renal and liver function tests , and serum crp levels . in all infants studied , associations of supar concentrations on admission with clinical and laboratory parameters ( leukocyte count and differentiation , platelet count , serum crp levels , renal and liver function ) were assessed . nineteen out of 47 infected neonates suffered from bacterial infections confirmed only by blood ( n = 4 ) , urine ( n = 8) , or csf ( n = 1 ) cultures , or by both blood / urine ( n = 4 ) , blood / csf ( n = 1 ) , and blood / synovial the remaining 28 neonates had definite ( n = 12 ) or probable ( n = 16 ) viral infections ( table 1 ) with no evidence of bacterial super - infection . the definite diagnosis of viral infections was performed by polymerase chain reaction ( enterovirus , adenovirus ) , immunochromatographic tests ( adenovirus or rotavirus in faeces ) or direct immunofluorence in nasopharyngeal secretions ( respiratory syncytial virus ) . sepsis was defined according to the criteria established by the international pediatric sepsis consensus conference ; evidence of systemic inflammatory response syndrome ( sirs ) in the presence of or as a result of suspected or proven infection was required . the children 's hospital ethics committee approved the study and informed parental consent was also obtained . hematologic parameters were measured using a siemens - advia 120 whole blood autoanalyzer ( siemens healthcare diagnostics , tarrytown , ny , usa ) . blood chemistry for renal and liver function was performed using the siemens advia 1800 clinical chemistry system ( siemens healthcare diagnostics , tarrytown , ny , usa ) , whereas crp concentrations were determined with immunonephelometry using a beckman coulter immage immunochemistry system ( beckman coulter , inc . , the intra- and interassay coefficients of variation ( cvs ) were 3.5% and 7.0% , respectively . for supar determination , plasma was isolated from edta - k3 anticoagulated blood samples of neonates following centrifugation at 3000 g for 10 minutes and stored at 80c until analysis . plasma supar levels were determined using a commercial enzyme - linked immunosorbent assay ( elisa ) ( suparnostic standard kit ; virogates a / s , birkerd , denmark ) . this assay utilizes a double monoclonal antibody that measures all circulating supar , including full - length and cleaved forms of the receptor . according to the standards provided by the manufacturer in ng / ml , a curve was constructed by us and the results were expressed as ng / ml , in a range between 0.6 and 20.0 ng / ml . the intra- and interassay cvs ranged from 1.3% to 4.7% and 1.7% to 5.1% , respectively , whereas the sensitivity limit was 0.1 ng / ml . the sample size of the study was calculated using our preliminary results of plasma supar concentrations in infected and healthy neonates . assuming an alpha risk of 0.05 , a power of 0.80 , and a bilateral test , it was estimated that 15 neonates was the minimum number of infants needed in each group to detect a significant difference of one sd in mean supar levels between infected and healthy infants . values of supar were not normally distributed overall ( kolmogorov - smirnov test ) , but the distribution was normal for neonates with infections ( bacterial or viral ) and controls separately . comparisons of quantitative variables between more than two groups were conducted by anova with bonferroni post hoc analysis or by kruskal - wallis and mann - whitney u test , as appropriate . a one - way repeated measures anova was used to compare the values of supar with baseline ( on admission ) within the infected group , in bacterial and viral infections separately . correlation ( pearson 's or spearman 's correlation test , as appropriate ) and regression analyses were used to examine relations among the variables of interest . the diagnostic properties of supar concentrations on admission were assessed by receiver operating characteristic ( roc ) analysis . all statistical analyses were performed using the spss statistical package ( spss , version 19.0 , chicago , il ) . demographic , clinical characteristics , and laboratory findings including white blood cells ( wbc ) , absolute neutrophil count ( anc ) , immature neutrophil and platelet count , liver and renal biochemistry , serum crp , and plasma supar levels in infants with bacterial and viral infections , separately , on admission to the unit , as well as in controls , are shown in table 2 . infants with bacterial infections did not differ significantly than infants with viral infections in gestational and postnatal age , birth weight , gender , duration of fever before admission , and maximum body temperature ( table 2 ) . the time for fever recession following admission was less than 36 hours in equal percentage ( approximately 75% ) of infants in the bacterial and viral infection group , but it was more than 72 hours in 3 out of 28 infants with viral infections ( 10.7% ) and in none infants with bacterial infection ; however , the difference between groups was not statistically significant ( table 2 ) . as expected , the wbc , anc , and serum crp values were significantly higher in neonates with bacterial infections than those with viral infections and controls ; immature neutrophil count was also higher , whereas platelet count was lower in neonates with bacterial infections but the difference between groups was not significant ( table 2 ) . plasma supar levels were significantly higher ( p < 0.001 ) in the entire group of infected neonates ( 5.01 1.52 ng / ml ) , as well as in neonates with bacterial or viral infections separately ( p < 0.001 and p = 0.02 , resp . ) in comparison with controls ( 3.61 0.74 ng / ml ) . no difference was recorded in supar levels between neonates with bacterial infections and those with viral infections ( table 2 ) . furthermore , in the group of neonates with viral infections , supar levels did not differ significantly between neonates with confirmed and those with probable infections . in the 13 out of 47 infected neonates who fulfilled the criteria for sepsis ( 11 with bacterial and 2 with viral infections ) , baseline mean s.d . plasma supar levels ( 6.05 1.96 ng / ml ) were significantly higher than levels in the remaining 34 nonseptic neonates ( 4.58 1.08 ng / ml ; p = 0.002 ) and controls ( p < 0.001 ) . the difference in supar levels between nonseptic neonates and controls was also significant ( p = 0.02 ) ( figure 1 ) . in the entire study population , supar levels on admission correlated positively with postnatal age ( rs = 0.29 , p = 0.01 ) , wbc ( rs = 0.26 , p = 0.03 ) , serum urea ( rs = 0.28 , p = 0.03 ) , and crp levels ( rs = 0.42 , p = 0.001 ) . the positive correlation between supar and crp concentrations was driven by the group of neonates with bacterial infections ( rs = 0.52 , p = 0.02 ) ( figure 2 ) . moreover , supar levels correlated negatively with platelet number in neonates with bacterial infections ( r = 0.55 , p = 0.01 ) , whereas a positive influence of male gender on supar levels was recorded in controls ( regression coefficient = 0.516 , p = 0.02 ) . receiver operating characteristic ( roc ) analysis of supar values on admission to the unit resulted in significant areas under the curve ( auc ) for detecting either infected ( auc = 0.801 ( 95% ci : 0.6870.916 ) , p < 0.001 ) or septic neonates ( auc = 0.788 ( 95% ci : 0.6530.923 ) , p = 0.001 ) ( figures 3(a ) and 3(b ) , resp . ) . however , the diagnostic performance of supar was not superior compared to crp either for detecting infection ( auc = 0.905 ( 95% ci : 0.8340.976 ) , p < 0.001 ) or sepsis ( auc = 0.858 ( 95% ci : 0.7141.000 ) , p < 0.001 ) , whereas it was almost equal compared to wbc and anc for detecting infection ( auc = 0.787 ( 95% ci : 0.6340.940 ) , p = 0.001 , and auc = 0.820 ( 95% ci : 0.6760.963 ) , p < 0.001 , resp . ) , but better than wbc and anc for detecting sepsis ( auc = 0.633 ( 95% ci : 0.4130.853 ) , p = 0.14 , and auc = 0.675 ( 95% ci : 0.4610.888 ) , p = 0.05 , resp . , figure 3 ) . in our study population , supar values higher than 4.07 ng / ml on admission could detect infection with sensitivity 74.5% and specificity 78% , whereas values greater than 4.79 ng / ml could detect sepsis with sensitivity 61.5% but specificity 89% . however , supar values on admission could not discriminate between bacterial and viral cause of infection ( auc = 0.515 ( 95% ci : 0.3310.699 ) , p = 0.86 ) . within the entire group of infected neonates , the time points at which repeat blood samples were obtained had a significant effect on plasma supar levels ( f = 10.5 , p < 0.001 ) by one - way repeated measures anova , whereas the effect of the infection type ( bacterial or viral ) was not significant . in comparison with values on admission ( baseline ) , supar concentrations decreased significantly ( p < 0.001 ) in the entire population of infants with bacterial and viral infections ( figure 4(a ) ) ; however , at 710 days following admission , supar levels were still significantly higher in comparison with controls ( p = 0.01 ) ( figure 4(a ) ) . plasma supar levels also declined significantly during the course of the disease in the group of neonates with bacterial ( p = 0.004 ) and viral infections ( p < 0.001 ) , separately ( figure 4(b ) ) , whereas no difference was recorded between the two groups . in neonates with bacterial infections , supar concentrations decreased significantly from baseline to 24 hours ( p = 0.01 ) , as well as from 24 to 48 hours ( p = 0.04 ) , whereas the alteration in supar levels from 48 hours to 35 days or from 35 days to 710 days following admission was not statistically significant . in neonates with viral infections , a significant decrease in supar levels was firstly observed between 24 and 48 hours ( p = 0.007 ) ; supar levels also decreased from 48 hours to 35 days ( p = 0.05 ) , but they did not alter significantly from 35 days to 710 days following admission ( figure 4(b ) ) . in the septic group of infants , separately , supar concentrations decreased significantly from 24 hours onwards in comparison with baseline ( p < 0.001 ) ( figure 4(c ) ) , but remained higher than in controls at 710 days following admission ( p = 0.05 ) . according to the results of this study , circulating supar levels are increased in term neonates with acute infections in comparison with healthy controls . moreover , supar levels in septic neonates are higher than levels in infected neonates who do not fulfill sepsis criteria . increased systemic supar levels were previously reported in critically ill adults with or without sirs , or sepsis ; a gradual increase in levels of supar was shown from adults who did not fulfill sirs criteria to patients with sirs and patients with sepsis [ 8 , 9 ] . supar levels were almost two times higher in septic adults than levels in our septic neonates , whereas levels in healthy adults were similar to those in our control group . the difference in supar levels between septic neonates and septic adults can possibly be attributed to the less severity of sepsis in our neonates ; all studied infants were hospitalized in a special , but not intensive , care unit and in none of them the course of disease was complicated or fulminant . supar levels in our study population were similar to those in older children with acute malaria infection , community - acquired pneumonia , and urinary tract infection . there is only one published study of supar levels in neonates ( 10 ) ; supar has been recognized as one among eight proteins increased in clinically infected preterm infants , but neither its association with bacterial or viral cause of infection , or with the course of disease , was studied . despite the increased circulating supar levels , the diagnostic value of supar for identifying infection or sepsis in adults is questioned as there are reports showing that it is good , uncertain , or poor [ 7 , 8 , 15 , 16 ] . it was previously shown that in comparison with other , frequently used in clinical practice , biomarkers of sepsis including crp and procalcitonin , supar was not a better diagnostic marker [ 7 , 15 ] . in our study , the diagnostic value of supar for neonatal infection was good , whereas it was much better for neonatal sepsis . however , similarly to studies in adults [ 7 , 16 ] , supar was less accurate than crp as diagnostic biomarker and also did not have any value in discriminating bacterial from viral infections . furthermore , in neonates with bacterial infections , supar levels did not seem to correlate with bacteria species ( data not shown ) . whereas the diagnostic utility of supar is controversial , several studies have shown that supar is a valuable prognostic biomarker [ 6 , 1619 ] . circulating supar levels , but not crp levels [ 20 , 21 ] , were reported to be higher on admission in nonsurvivor adults compared to patients who survived sepsis [ 6 , 1618 ] . the prognostic value of supar for mortality could not be evaluated in our study as all septic neonates survived . however , the higher supar levels on admission in septic neonates compared to infected , but nonseptic , ones and the negative correlation between supar levels and platelet number in the bacterial infant group indicate that supar is indeed associated with the severity of infection . the positive correlation between supar and crp levels in our study population is in accordance with the fact that circulating supar levels reflect the degree of immune activation and systemic inflammation . there is also some evidence that individual factors , such as age and possibly female gender , are positively associated with systemic supar levels [ 9 , 13 , 22 , 23 ] . in our study , supar levels correlated positively with postnatal age , whereas a positive influence of male , but not female , gender was observed in controls . we wonder whether supar could be used as a biomarker of clinical recovery or response to treatment in patients with infection or sepsis . changes in supar levels after initiation of antimicrobial therapy have been studied only in adults [ 8 , 16 , 19 , 24 ] ; it was shown that supar may remain elevated for days ( even for weeks ) after initiation of treatment . in our study , supar levels declined significantly in infected neonates ; however , 7 to 10 days following admission , although all infected neonates had fully recovered , supar levels were higher than in healthy controls . given that supar levels were not halved even at 7 to 10 days following recovery , we assume that either the half - life of supar , being unknown so far , is long or the production / release of supar is continued for a period of time following clinical improvement . supar participates in a number of immunological functions , including cell adhesion , migration , chemotaxis , proteolysis , immune activation , tissue remodeling , invasion , and signal transduction . however , its precise role in infection and sepsis is still not clear . knockout mice lacking upar have shown impaired host defense against sepsis , including reduced neutrophil migration towards the primary site of infection and markedly impaired phagocytosis in comparison with wild - type mice [ 25 , 26 ] . on the other hand , upar and circulating supar have been implicated in the promotion of disease progression [ 25 , 27 ] . further studies are needed to elucidate whether supar is merely an epiphenomenon of the increased activation level of the immune system , or either this receptor possesses a protective role in neonatal sepsis or promotes inflammation / tissue damage and should , therefore , become a potential therapeutic agent or target in the future .

background . supar , the soluble form of the urokinase - type plasminogen activator receptor , has been identified as a biomarker of infection in adults but its properties in neonatal infection are not known . methods . plasma supar levels were determined by elisa in 47 term neonates with infection ( 19 bacterial and 28 viral ) and in 18 healthy neonates as controls . thirteen out of 47 infected neonates were septic . in all infected neonates , supar levels were repeated at 24 hours , 48 hours , 35 days , and 710 days following admission . results . plasma supar levels were significantly increased in infected neonates upon admission , whereas they were highest in septic neonates , in comparison with controls ( p < 0.001 ) and correlated positively with serum crp levels ( p = 0.001 ) . at infection subsidence , supar concentrations decreased significantly in comparison with baseline ( p < 0.001 ) but remained higher than in controls ( p = 0.01 ) . receiver operating characteristic analysis resulted in significant areas under the curve for detecting either infected or septic neonates , but not for discriminating between bacterial and viral cause of infection . conclusions . supar is a diagnostic biomarker of infection or sepsis in term neonates ; however , it can not discriminate bacterial from viral infections and also its utility for monitoring the response to treatment is questioned .

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

a 30-year - old man with no significant past medical history was scheduled for tibial rod removal and possible bone graft of the ankle . the patient received a single injection femoral and a continuous popliteal sciatic nerve block in the holding area for postoperative pain management . the patient was positioned prone , and an axial ultrasound ( us ) image of the sciatic nerve was obtained 8 cm superior to the popliteal crease . the needle guidance system by philips ultrasound system hd-11 with 12 - 3 linear probe ( philips medical systems ; andover , ma ) was used to measure the distance from the needle holder to the nerve . this system highlights the needle trajectory on the us image with superimposed dotted lines and secures a precise in - plane view of the needle . when the biopsy guide is activated on the hd-11 us system , it produces a thick blue crosshair . the track ball can control the needle length crosshair , displaying the distance from the top of the needle guide to the target . this system highlights the needle trajectory on the us image with superimposed dotted lines and secures a precise inplane view of the needle ( figure 2 ) . the advancement of the 17 g plexus block needle has centimeter markings that concur with the markings on the us system and distance to the target nerve can be continuously calculated with needle advancement . the patient was prepped , draped , and a sterile us probe cover was applied . the guidance system is a disposable , sterile , plastic attachment for the us probe that contains a needle holder in line with the center of the us beam . the arrow international ( reading , pa ) 17 g 3.5 inch insulated tuohy needle under simultaneous real - time us visualization and nerve stimulation of 1.0 milliamp ( ma ) observed tracking exactly along the dotted guide line that was displayed on the screen ( figure 3 ) . in addition to maintaining an in - plane view , the needle and probe function as one unit , helping to secure the us probe placement . the needle position was maintained solely by holding the us probe ( figure 4 ) . then the needle stylet was removed and an arrow stimcath ( reading , pa ) catheter was advanced with continuous visualization and nerve stimulation . after securing the catheter in place , 30 ml of 0.5% the patient was then repositioned supine , and a single injection femoral nerve block was performed . a 2 inch , 22 g b - braun ( bethlehem , pa ) stimuplex ptfe insulated needle was percutaneously advanced 1 cm lateral to the femoral artery with electrostimulation . patellar snap was evident to 0.40 ma , and the practitioner incrementally injected 20 ml of 0.5% ropivacaine . ten minutes after completion of the femoral nerve block , testing revealed absence of sensation in both the femoral and sciatic distribution . the patient received laryngeal mask general anesthesia for the procedure , and the pain scores in the recovery room were 0/10 . the patient was discharged to home with an i - flow on - q on demand ( lake forest , ca ) disposable infusion device . daily telephone contact revealed excellent analgesia requiring one dose of po narcotics on postoperative day 1 . the patient was maintained on acetaminophen after discontinuation of the peripheral nerve catheter on postoperative day 5 . recent opinion on us guidance has revealed that it may not eliminate complications of nerve blocks . in fact , there have been several case reports of intraneural and intravascular injection during blocks , despite the adjunct use of us.24 although complications have been reported , researchers have also revealed that use of us can improve block placement times as well as block onset times.58 ultrasound difficulties have been well delineated . these include clinician expertise , maintenance of an exact in - plane view , artifacts , and pitfall errors . of these , one of the most troublesome is keeping the needle tip in view.1 one solution may be to devise user - friendly needle guides that secure the needle track in line with the us beam . though in - plane visualization is widely accepted as the safest way to conduct us blocks , it can be very difficult to obtain a precise in - plane view and to ensure a safe location of the needle tip . physicians have tried a variety of methods from cameras to lasers in order to combat this very problem.911 radiography literature reveals good success with needle guides , especially when targets are small or conventional methods have failed.12 an additional advantage of the needle guide is that the imaging system and the delivery system become one unit . the practitioner fastens the needle guide , containing the track needle , securely onto the ultrasound probe . the practitioner can hold the probe with one hand and control the needle with the second hand . instead of requiring a third hand for the catheter , one practitioner can release the needle because the needle guide holds it in a stable relationship with both the tissue planes and the us beam . a common practice today is to guide the needle to the nerve with us , but set the probe aside and blindly advance the catheter . the arrow stim catheter has a metal inner coil and tip that is echogenic and enhances visualization of catheter advancement . the sciatic nerve is a fairly mobile structure in which the pressure applied from the us probe during scanning will displace the nerve . when the needle is positioned adjacent to the target , it is not uncommon to observe the nerve move several centimeters with removal of the us probe for catheter advancement , which may explain secondary block failure . we have performed over 20 successful popliteal nerve catheters using the us needle guidance system . we found it easy to use ; the needle tracking was precise and the target depth was accurate . the limitation of the needle guide is that once the practitioner initiates a trajectory , he or she can only make minor adjustments . in addition , there are only a few trajectories preprogrammed into the us machine ; therefore , one must be more adept at acquiring an us image . in other words , not only must the target nerve appear in the image , but it must also be in direct alignment with a path predetermined by the needle holder . in this case report , the needle guide only had one available trajectory , but other models allow a greater selection of four trajectory lines . these other models allow clinicians to change the trajectory to match the target in the us image instead of adjusting the us image to follow a different path . aside from the trajectory restrictions second , in order to overlay the trajectory on a live image , the practitioner must synchronize the needle guide with the us machine software . also , increasing the size of the probe by adding an additional fixture is likely to hamper efforts in block areas that are already compromised by an unusual probe angle or a small anatomic space . despite the aforementioned weaknesses , the needle guide remains a simple adjunct to increasing block success and has a long , proven history in other medical specialties . the role of us in peripheral nerve blocks continues to evolve rapidly , but in each case , success and safety partly depends on needle tip identification within tissue . the needle guidance systems are one way to improve that visibility ; especially with novice clinicians .

while ultrasound ( us)-guided placement of peripheral nerve blocks is rapidly gaining popularity , expert practitioners agree that two of the most significant barriers to safety and efficacy are keeping the needle tip within the image and unintentional probe movement during the procedure.1 in addition , placing a nerve catheter past the needle tip under direct us observation requires two practitioners : one to hold the us probe and needle and another to advance the catheter . we present a case of a needle guidance system that attaches to the ultrasound probe and facilitates in - plane imaging . it enables a single practitioner to successfully execute a popliteal sciatic nerve block and visualize catheter placement . therefore , a needle guidance system may represent an additional modification to ultrasound imaging that increases both time efficiency as well as safety .

Case report Discussion

the online version of this article ( doi:10.1007/s00249 - 009 - 0412 - 6 ) contains supplementary material , which is available to authorized users . analytical ultracentrifugation is an analytical absolute technique applying centrifugal force to fractionate the sample and optical detection systems to detect the concentration distribution of the sample inside the ultracentrifuge cell . these experiments yield a number of important physico - chemical quantities of the sample like size , shape , density , molar mass , sedimentation and diffusion coefficient , interaction constants and stoichiometry etc . and many of them in form of distributions . however , often the available information depends on the applied optical detection system . optical systems that have been constructed are uv / vis absorption , rayleigh interference , schlieren and the lavrenko optics ( lavrenko et al . development of new optical detection systems can expand the possible use of auc for different types of samples . size distributions can be determined with turbidity optics ( mchtle 1992 ; mller 1989 ; scholtan and lange 1972 ) . fluorescence optics has expanded the use for extremely diluted labeled samples even in presence of other solutes at a much higher concentration ( macgregor et al . the only commercially available machine at the moment is the beckman xl - i which is equipped with a uv / vis absorption optics and rayleigh interference optics ( giebeler 1992 ) . recently , the uv / vis absorption detection was significantly improved by the development of a multiwavelength uv / vis absorption detector ( mwl - auc ) . by essentially replacing the monochromator with a spectrograph , the mwl - auc delivers an entire uv / vis spectrum for each radial point instead of a single wavelength reading ( bhattacharyya et al . this technology has a number of advantages over the commercial absorption optics detecting at a single wavelength with time - consuming wavelength scanning , thus excluding the study of all fast processes , obtaining full uv / vis spectra at each point in the ultracentrifuge cell rather than a radial concentration profile at a single wavelength can give much more structural information about the sample , allows for averaging and can even decrease the experimental time when modern fast ccd based spectrometers are used ( bhattacharyya et al . successful optical , mechanical , radial scan , linearity and noise tests of the mwl detector have been published recently ( strauss et al . 2008 ) . combined with the fractionating power of the auc , application of the mwl detector with its additional structural and/or compositional information on light absorbing samples can yield distributions of the individual components in complex mixtures with respect to composition and size / density related to different chromophores . this can start with relatively straightforward issues like sample homogeneity and purity but can then get increasingly complex in case of composite and/or interacting samples . especially for such complex samples mwl - auc has a huge potential , as spectral discrimination can synergistically enhance the hydrodynamic resolution ( balbo et al . this is also an important issue for any colored industrial product composed of at least two components which at least slightly differ in their uv / vis spectra . in this work , we will show the capabilities of mwl - auc for the analysis of an industrial composite sample of -carotene and gelatin . this system was investigated before with x - ray scattering , uv / vis absorption spectroscopy , foqels ( fiber - optic quasi - elastic light scattering ) , microelectrophoresis and on basis of these results , a core - shell structure was presented ( auweter et al . the core structure with 120 nm diameter consists of partially crystallized , partially amorphous -carotene as active ingredient . this hybrid structure self - assembles in a carefully tuned co - precipitation of gelatin ( from an aqueous solution ) and the active ingredient ( from a lipophilic solvent ) . such particulate formulations can transport an active ingredient that is not water soluble across an aqueous phase with high bioavailability , in this case provitamin a. these particles are not persistent , but disassemble and get digested quickly in biological media . -carotenes can precipitate as h - aggregate or j - aggregate ; the two morphologies do not interconvert and are regarded to be kinetically stable over years . the h - aggregate is observed in precipitation of dilute solutions ( 0.3 weight% ) whereas the j - aggregate is observed at higher concentration ( 1.0 weight% ) . ( 1999 ) calculated a 40 nm hypsochromic shift observed for an h - aggregate and a bathochromic shift in j - aggregates . this results in a significant color change from yellow to red of the product depending on the precipitation conditions and hybrid particle size ( figs . 1 , 2 ) . this color change is the basis for the industrial application of the -carotenes as pigments for food applications.fig . 1dashed line uv / vis spectrum of the core - shell -carotene / gelatin sample with 0.05 g / l concentration ; solid line uv / vis spectrum of gelatin at 1 g / lfig . 1999 ) , right side color change of -carotene / gelatin microparticles due to particle size and structure dashed line uv / vis spectrum of the core - shell -carotene / gelatin sample with 0.05 g / l concentration ; solid line uv / vis spectrum of gelatin at 1 g / l left side assumed structure of the -carotene microparticle system ( auweter et al . 1999 ) , right side color change of -carotene / gelatin microparticles due to particle size and structure not only the purity of the sample concerning the color characteristics ( brilliance of color due to steep absorbance profiles ) or sample homogeneity ( different species or unbound gelatin ) is of interest for the industrial application but furthermore any possible transitions between different structures . this is a problem which can be advantageously solved in a single mwl - auc experiment , which we will describe in this work . the -carotene product was obtained in powder form as a laboratory sample from basf se , ludwigshafen . an aqueous dispersion in water the uv / vis spectrum of the dispersion and of the free gelatin is shown in fig . 1 . in contrast to conventional sedimentation velocity experiments , where the sample between boundary and bottom of the cell is only diluted by radial dilution , the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . the reservoir is filled with a small amount of concentrated sample . column and sample sectors are filled with d2o with a density which is higher than that of the sample solution and lower than the density of the dispersed solute . we have prepared a 20 g / l solution and deposited 15 l of the solution into the cell reservoir . after preliminary experiments , this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with od s < 1.4 in the experiment without too much dilution which causes noisy data . after cell assembly , the auc was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the d2o column . forty scans were taken with a time interval of 90 s and a radial step size of 50 m to observe the full sedimentation of the sample . the selected wavelength range was 250750 nm . in the prototype setup , we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 od ( see fig . 3i ) after the experiment , while cleaning the cell , we saw some precipitate in the cell reservoir . thus , not all particles were transferred to the sample column , but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.fig . 3three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) each of the 40 scans produces a 3-dimensional graph . we have radial position as x - dimension , wavelength as y - dimension and absorbance as z - dimension . in the present contribution , we will perform a semi - quantitative evaluation based on simple model - free transformations of the data without any prior knowledge . for evaluation , we have converted the radial position ( r ) to the sedimentation coefficient s by using eq , rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{s } } = \frac{{{\text{ln}}\,(r / r_{\text{m } } ) } } { { \omega^{2 } t } } $ $ \end{document } the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . a multiwavelength auc as described in bhattacharyya et al . ( 2006 ) and strauss et al . in contrast to conventional sedimentation velocity experiments , where the sample between boundary and bottom of the cell is only diluted by radial dilution , the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . the reservoir is filled with a small amount of concentrated sample . column and sample sectors are filled with d2o with a density which is higher than that of the sample solution and lower than the density of the dispersed solute . we have prepared a 20 g / l solution and deposited 15 l of the solution into the cell reservoir . after preliminary experiments , this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with od s < 1.4 in the experiment without too much dilution which causes noisy data . after cell assembly , the auc was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the d2o column . forty scans were taken with a time interval of 90 s and a radial step size of 50 m to observe the full sedimentation of the sample . the selected wavelength range was 250750 nm . in the prototype setup , we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 od ( see fig . 3i ) after the experiment , while cleaning the cell , we saw some precipitate in the cell reservoir . thus , not all particles were transferred to the sample column , but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.fig . 3three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) each of the 40 scans produces a 3-dimensional graph . we have radial position as x - dimension , wavelength as y - dimension and absorbance as z - dimension . in the present contribution , we will perform a semi - quantitative evaluation based on simple model - free transformations of the data without any prior knowledge . for evaluation , we have converted the radial position ( r ) to the sedimentation coefficient s by using eq . 1 . in eq . 1 , rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{s } } = \frac{{{\text{ln}}\,(r / r_{\text{m } } ) } } { { \omega^{2 } t } } $ $ \end{document } the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . in principle , the entire dataset can be evaluated globally , and efforts are underway to incorporate such routine into the ultrascan evaluation software package ( demeler 2005 ) . however , even then we are confronted with a confounded polydispersity of both optical and colloidal / hydrodynamic properties . in the left side of fig . 2 , the assumed core - shell structure of a -carotene microparticle is shown ( auweter et al . 1999 ) . such a complex hybrid particle exhibits several levels of polydispersity , which impact the distribution of sedimentation coefficients observed in an auc . oil content , of the inner core;concentration of the adsorbed protection colloid ( gelatin);degree of swelling of the gelatin . oil content , of the inner core ; concentration of the adsorbed protection colloid ( gelatin ) ; degree of swelling of the gelatin . parameters 1 and 2 determine the optical properties and bioavailability that are decisive for the commercial application profile . for smallest particle sizes , -carotene is an h - aggregate while for the biggest particle sizes -carotene forms j - aggregates . intermediate particle sizes are assumed to integrate h- and j - aggregates in differing ratio ( auweter et al . parameters 3 and 4 determine the thickness of the protection colloid layer , which is typically 40 nm in pure water . the buoyant density of gelatin is rather high ( above 1.3 g / cm ) , and can not be matched with a non - interfering solvent such as heavy water . all parameters 14 enter into the calculation of the effective density and the hydrodynamic diameter of the hybrid particle . the frictional force under sedimentation depends on the ion concentration and ph because the gelatin may collapse or swell thus changing the effective frictional forces ( and thus changing the observable sedimentation constant ) although the chemical composition and buoyant density , which in principle could be measured in a krattky gauge or density gradient , stay the same . the swelling of the gelatin corona alone impedes an exact conversion from measured sedimentation constants to hydrodynamic diameters . considering that also parameters 1 and 2 contribute to the polydispersity in the observable distribution of sedimentation coefficients , we decided to limit ourselves to a conservative evaluation on the level of sedimentation fractions , not sizes . we now discuss the optical properties that result from the specific colloidal microstructures as discussed above . due to different preparation conditions , the morphology of the -carotene core changes . h- and j - aggregates have different uv / vis spectra , shown as visual impression on the right side of fig . 2 . figure 1 dot line curve shows the uv / vis spectrum of 0.05 g / l product without any ultracentrifugation . four peaks at 288 , 449 , 478 and 518 nm can be seen . the three peaks in the visible can be attributed to the 1ag ( s0)1bu+ ( s2 ) transition with the vibrational progression 20 , 10 , 00 of the c c stretch vibration along the alternatingly double bonded electronically conjugated backbone of the carotenoid ( polivka and sundstrom 2004).the uv peak partially can be attributed also to the carotenoid transition 1ag1ag+ , which is forbidden by symmetry , but becomes allowed in the crystalline assembly . the spectrum of the composite particle indicates the -carotene j - aggregate ( auweter et al . 1999 ) . it can be seen that gelatin only contributes to the uv region of the spectra below 280 nm . however , the contribution of gelatin is vanishing compared to the three times stronger absorption of the composite sample at 20 times lower overall concentration . another component that presumably contributes to the uv absorption is the ascorbylpalmitat dispersant that is added during the co - precipitation . if we put all these 40 scans in sequence , we can form a 3d movie of the sedimentation process . figure 3.1 shows scan 1 where particles just have been transferred from the reservoir to the sample column . the baseline offset is 0.05 ( purple ) due to the absorption calculation with an empty cell as reference . , there is an overlay of two peaks , one is the uv peak of -carotene ( see fig . 2 ) and the other is the uv signal of gelatin . after 15 min of sedimentation , fractionation of the sample is obvious and the first sedimentation fraction proceeds to the bottom of the cell . scan 10 ( 15 min ) is the last scan where the entire particle range can be seen before the first particles reach the bottom . if we compare the height of the peak in the uv and visible region at different radial positions , the ratio changes . this is the first important result , demonstrating that the sample is not homogenous . instead the observed effect can be explained by a higher content of stabilizing agent that induces smaller particle sizes . 1 ) does not exactly match the gelatin absorption and that the expected contribution of gelatin is weak at the applied concentrations , hinting at a combined action of both gelatin and the ascorbylpalmitat added during the co - precipitation in particle synthesis . the third part of fig . , the fastest particles have sedimented already . in the fourth part of fig . 3 , we see the last fraction that remained after 60 min of sedimentation , which is mainly composed of gelatin . however , some -carotene absorption is still visible , which seems to be solubilized in small amount by the excess gelatin or excess ascorbylpalmitat . we do not detect free gelatin in the analysis . in an independent experiment we measured the characteristic sedimentation behavior of gelatin with the interference optics of the beckman xli auc at 44,000 rpm . this confirms our assignment that the last fraction can not be pure gelatin . to summarize the global evaluation , fig . we can differentiate particles , observe the full uv / vis spectra of the particles and can already draw conclusions about the different components in the complex sample mixture without any further evaluation , as the y - axis shows the full uv / vis wavelength range . we can also use projections of the data onto individual axes and proceed thus to a more quantitative evaluation . in order to calculate the full s - distribution of all particles , we have selected scan 10 for all further evaluation as this scan shows fractionation of the mixture while no particles are yet lost due to complete sedimentation . more information is potentially available with a global evaluation of the entire dataset . in fig . 4 , the s - distribution of the particles is shown for 5 different representative wavelengths out of 330 ( 250750 nm with a wavelength resolution of 1.5 nm ) . we have selected the wavelengths according to the peaks of the -carotene microparticles : 260 , 280 , 450 , 480 and 520 nm in fig . 4sedimentation coefficient distributions at different wavelengths sedimentation coefficient distributions at different wavelengths the s - distribution is obviously very broad . due to the chemical heterogeneity of the particles and the resulting density distribution , however , all important sample characteristics can be discussed for the s - distributions . from fig . 4 , we conclude that there are at least three fractions in the sample : small hybrid particles with s < 25 s , a main fraction around 100 s and larger particles around 200 s. their absorption spectra ( and chemical composition ) are clearly different as can be seen in fig . 5.fig . 5top normalized uv / vis spectra of particles with different sedimentation coefficients , bottom zoom the range around 450 nm and peak positions of 10.6 s ( 448 nm ) up to 232 s ( 439 nm ) top normalized uv / vis spectra of particles with different sedimentation coefficients , bottom zoom the range around 450 nm and peak positions of 10.6 s ( 448 nm ) up to 232 s ( 439 nm ) in fig . 5 , seven representative uv / vis spectra are shown . the spectra agree well with those of pure h - aggregates ( auweter et al . however , the original sample contained j - aggregates too ( fig . 1 , dashed line ) . we believe that the j - aggregates were already precipitated before the first scan in the auc cell was taken . indeed precipitation of particulate material inside the reservoir of the vinograd cell was observed visually after cell disassembly following the experiment . however , irrespective of the actual nature of the particulate material that remained in the vinograd cell reservoir , the result stands for itself that the coloristic polydispersity ( fig . 1 ) is not due to an intra - particle but inter - particle distribution of morphologies ( fig . this result is contrary to the previous assumption that is sketched in fig . 2 , where h / j - aggregates would coexist in the particles.fig . 6ad hoc structural model of the -carotene microparticle system on basis of the presented auc results . the different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before ( fig . 2 ) ( auweter et al . 1999 ) but instead separate particles contain pure h- or j - aggregates and the concentration ratio between these particles determines the colour of the final sample . note the difference to fig . 2 ad hoc structural model of the -carotene microparticle system on basis of the presented auc results . the different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before ( fig . 2 ) ( auweter et al . 1999 ) but instead separate particles contain pure h- or j - aggregates and the concentration ratio between these particles determines the colour of the final sample . 2 the peak around 520 nm slightly shifts to lower wavelength with increasing sedimentation coefficient and the peak height also decreases . therefore , this excitation of -carotene microparticles decreases with increasing sedimentation coefficient . for the peak at 450 nm , only the spectral shift to lower wavelength is observed with increasing sedimentation coefficient . we suspect that the displacement of the electronic potential energy surfaces changes , such that the frank factors change for the vibrational progression , due to the changing incorporation of the chromophore into the partially crystalline assembly . as discussed above , an overlay of the signal from -carotene , gelatin and the ascorbylpalmitat a shift of the peak maximum to higher wavelength with increasing sedimentation coefficient is detected . in addition , a drastic decrease of the peak height relative to the 450 nm peak is observed with increasing sedimentation coefficient . the particles that sediment slower show stronger uv absorption , which we attribute to a higher content of ascorbylpalmitat , and hence smaller particle diameters due to the formation process in co - precipitation auweter et al . 1999 ) . to our knowledge , it is the first time that relatively small steps of spectral shift among an h - aggregate are shown for composite particles . although the spectral changes appear to be continuous , that does not exclude defined and different spectra for different particle populations as the detected raw signals are those of a sample band , which is broadened by polydispersity in size , composition and diffusional broadening . industrial -carotene gelatin composite particles are a highly heterogeneous system both in particle size and chemical composition . fractionation of this mixture in mwl - auc allows to resolve individual components in the mixture and detect compositional changes in an experiment , which takes only 1 h. this proves the power of mwl - auc as a direct technique that can differentiate particles with respect to size and uv / vis spectra . although our current analysis does not allow to unambiguously assign the spectra to defined particles as the particle density , swelling , composition and size maybe varying simultaneously , the presented multiwavelength analysis allows insights into this complex system , which were not possible before by other techniques . we do not further evaluate the directly experimentally determined sedimentation coefficients as for our sample , in addition to the above mentioned polydispersity , ph effects as well as charge interactions between the colloids also have to be taken into account . despite these restrictions , we have shown the existence of h - aggregates inside a sample that was previously known as j - aggregate and have detected spectral changes of different h - aggregate populations as well as changes in the electronic potential energy surfaces of different hybrid particles . we restricted ourselves to a semi - quantitative evaluation based on simple model - free transformations of the data of 1 out of 40 scans without any prior knowledge . clearly , even richer phenomena can potentially be discovered with a global evaluation of the entire dataset . below is the link to the electronic supplementary material . supplementary information ( avi 10.4 mb ) .

a multiwavelength uv / vis detector for the analytical ultracentrifuge ( mwl - auc ) has been developed recently . in this work , -carotene gelatin composite particles are investigated with mwl - auc . band centrifugation with a vinograd cell is used to ensure maximum sample separation . spectral changes of the system are observed in dependence of the sedimentation coefficient and are attributed to a previously unknown inhomogeneity of the -carotene chemical composition with both h- and j - aggregates coexisting in a mixture . in addition , our data suggest that pure h- and j - aggregates exist in a particle while their relative concentrations in a mixture determine the color characteristics of the sample . the unique abilities and properties of mwl - auc include sedimentation coefficient distributions for all possible wavelengths , full uv / vis spectra of each different species in the mixture and 3d movies of the sedimentation process . these properties significantly extend the scope of the analytical ultracentrifuge technique and show that complex biopolymer multicomponent mixtures can be resolved into their individual species.electronic supplementary materialthe online version of this article ( doi:10.1007/s00249 - 009 - 0412 - 6 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Materials and methods Multi-wavelength analytical ultracentrifugation (MWL-AUC) Results and discussion Conclusion Electronic supplementary material

in benin , 64% of the population has consumed once rabbit meat at least , and 95% of them appreciated it positively . thus , to supply the market with white meat , rabbits ' farms are installed in periurban areas . to promote optimal growth performance of rabbits , appropriate feeding strategy is necessary . fibres are one of the main components of rabbits ' diets ; because they play a key role in rabbit feeding by contributing to caecum activity for efficient digestion . moreover , demonstrated that particle size is a major factor of fibre digestibility in rabbits . crude fibre level in growing rabbits ' diet varies from 14 to 16% , whereas in reproductive rabbits ' diets that level is between 12 and 13% . unfortunately , the availability of forages , main sources of fibre is low in peri - urban areas , and farmers have difficulties to provide rabbits with grass . the processing of complete diets with optimal level of fibres is therefore suitable to efficiently feed rabbits . palm - press fibres contain about 86.2% of dry mater , 4% of crude proteins , 21% of fat , 0.31% of calcium , 0.13% of phosphate and 36.4% of crude fibre . palm - press fibres can be dried and pelleted to overcome the problems of poor keeping quality and bulkiness . palm - press fibres can be therefore included in complete diets of rabbits , rodents and ruminants . crude proteins and crude fibre digestibility decreases when the level of palm - press fibres exceeds 2530% in ruminants diet . the marketing of palm - press fibres for animal feeding will provide additional revenues to palm oil producers in developing countries . the objective of this study was to contribute to the valorization of palm - press fibres in growing rabbits feeding by evaluating its optimal level in diets . a total of 64 rabbits weaned at 35 days old were divided in 16 groups of 4 rabbits each . at the beginning of the experiment , the average live body weight of these rabbits of local breed reared in benin was 857.2 g. each group of 4 rabbits ( 2 males and 2 females ) was housed in a cage ( length width height : 80 50 30 cm ) . they contained , respectively , 0% ( f0 , control diet ) , 5% ( f5 ) , 10% ( f10 ) , and 15% ( f15 ) of palm press fibres ( table 1 ) . two cages of rabbits were randomly selected in each row . at the beginning of the experiment , similar ( p > 0.05 ) average live body weights of rabbits were recorded in f0 ( 858.1 g ) , f5 ( 836.3 g ) , f10 ( 880.0 g ) , and f15 ( 854.4 g ) dietary treatments . the experiment took six weeks and finished when rabbits were thirteen weeks old . at six weeks old , four starved rabbits ( 2 males and 2 female ) were slaughtered per diet for carcass study . the performances of rabbits were compared using each cage of four rabbits ( two males and two females ) as repetition . repetition effect and interaction between diets and repetitions were not significant ( p > 0.05 ) . thus , analyses were performed according to the model as follows : ( 1)yi=+fi+i , where yi is the observation for dependent variables ; is the general mean ; fi is the fixed effect of the feed ; i is the residual error . the daily feed intakes of rabbits fed with the four diets ( figure 1 ) were similar during the experiment ( p > 0.05 ) . on average , daily feed intakes were , respectively , 78.4 10.7 g ( f0 ) , 79.5 10.2 g ( f5 ) , 79.7 9.7 g ( f10 ) and 79.94 11.0 g ( f15 ) . in the sixth week of experiment ( 13 week old ) the feed intake decreased in all dietary treatments . the decrease of feed intake was more important when the level of crude fibre in diet is low as it was the case in f0 and f5 diets ( table 2 ) . the daily body weight gains ( table 3 ) were similar between diets ( p > 0.05 ) . thus , at the end of the experiment ( 77 days - old ) , the live body weights were not significantly different ( p > 0.05 ) between rabbits fed f0 ( 1788.5 g ) , f5 ( 1805.0 g ) , f10 ( 1718.5 g ) and f15 ( 1801.3 g ) . the lowest feed conversion ratio and feeding cost were recorded in rabbits fed f15 diet ( table 3 ) . the economic feed efficiency that reports the revenue from the live body weight gain comparatively to the feeding cost , was not significantly affected by the dietary treatment ( p > 0.05 ) . thus , included , up to 15% in balanced diet , palm - press fibres had no significant effect on the survivability of growing rabbits . the carcass yields of rabbits were similar ( p > 0.05 ) between dietary treatments ( table 4 ) . additionally , the relative weight of digestive tract and abdominal fat evaluated comparatively to the carcass weight were not significantly affected by the level of palm - press fibres in diets ( p > 0.05 ) . however , compared to the control diet ( f0 ) , the proportion of liver was significantly lower in rabbit fed f5 and f15 dietary treatments ( p < 0.05 ) . the feed intakes recorded in all dietary treatments were close to 78.7 g / day ; but higher than 53.360.4 g / day and lower than 136.3 and 149.9 g / j . the light increase of feed intake of rabbits fed with palm press fibres - based diets ( f5 , f10 and f15 ) confirms the statements of [ 4 , 12 ] according to which , the feed intake is positively correlated with the level of fibre in diet . in hot and humid climate , the keeping of an optimal level of feed intake is a challenge in livestock . the use of palm press fibres - based diets can therefore , reduce the requirement of forages to feed rabbits and allow them to develop their intestinal microflora that is necessary for efficient digestion . the similar growth between rabbits fed different level of palm press fibres - based diets indicates the efficiency of that feedstuff in rabbit feeding . the daily weight gains of the experimental rabbits ( 19.65 to 23.07 g ) were higher than the 7.48 to 10.91 g reported in nigeria and 16.7 g found in benin with similar rabbit breed . compared to the control diet f0 , f5 and f15 diets improved lightly the growth performance of rabbits without any adverse effect on their survivability . the final live body weights of rabbit ( 1718 to 1805 g ) are close to 1746 g found at the same age in local population of rabbit in tunisia . the body weights of rabbits are also in the range of 1638.9 to 1862.5 g . the low performance of rabbits fed f10 diet might be due to a health or digestive problem . the decrease of feed conversion ratio and feeding cost in f5 and f15 diets confirms the efficiency of these diets and the profitability of palm press fibres inclusion in growing rabbits ' diets . feed conversion ratios ( 3.78 to 4.64 g feed / g body weight gain ) were lower than 5.81 to 8.11 feed / g weight gain , found in nigeria . however , feeding costs ( 760 to 816 fcfa feed / kg body weight gain ) were higher than about 596.4 to 685.1 fcfa feed / kg body weight gain . this economic result can be explained by the feed price ( 189 to 192 fcfa / kg feed ) that was higher than about 85.03 to 128.8 fcfa / kg feed reported by these authors . the result indicates in certain instance , the difference in feeds market between benin and nigeria . in monogastric livestock , compared to broiler , growing rabbits fed palm fibres - based diets produce therefore , about equally profitable white meat in benin . the carcass yields ( 60.8 to 62.9% ) are higher than 57.1% ; but lower than the 64.8 to 66.6% reported in rabbit fed pellet diets and fodder . the palm press fibres - based diets did not affect significantly the relative weight of digestive tract of rabbits . the abdominal fat in rabbits carcass increase lightly in f5 and f15 diets compared to the control diet ( f0 ) . these results show a similar effect of the experimental diets on rabbit meat productivity and carcass quality . moreover , the diets kept rabbits in apparently good health conditions as demonstrated the significantly low liver relative weight of rabbits fed f5 and f15 . this experiment suggests that growing rabbits can be fed exclusively with balanced palm press fibres - based diets . in such feeding strategy , up to 15% of palm press fibres can be included efficiently in the diets without any significant adverse effect on rabbits ' survivability and their abdominal carcass fat . to reduce grass and forage need in peri - urban rabbitry

an experiment was carried out to define the optimal rate of palm - press fibres in growing rabbits ' diet . in total , 64 weaned rabbits ( 35 days old ) of beninese breed were divided in 16 groups of 4 rabbits ( 2 males and 2 females ) each . during six weeks , rabbits were fed with 4 complete diets containing 0% ( f0 , control ) , 5% ( f5 ) , 10% ( f10 ) , and 15% ( f15 ) of fibres from a palm oil industry . results demonstrated that up to 15 of palm - press fibres can be included efficiently in growing rabbits ' diet . the daily feed intake was not significantly affected by the diet ( p > 0.05 ) . at 13 weeks old , the average live weights of rabbits were 1788.5 g , 1805.0 g , 1718.5 g , and 1801.3 g in respectively , f0 , f5 , f10 and f15 groups . no mortality of rabbits was recorded . compared to f0 , the feed conversion ratio and feeding cost decreased in the group of rabbits fed f15 diet . the carcass yield was similar between diets .

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

when reconstructing an anorectal malformation ( arm ) with posterior sagittal anorectoplasty ( psarp ) , the dissection of the rectourethral fistula is one of the challenges . the psarp procedure includes opening the rectum posteriorly to clarify the anatomy before dividing the rectourethral fistula.1 the aim of this report is to describe an approach including placing of a guidewire passed through the endoscope in the colostomy , through the fistula to the urethra to facilitate the localization and dissection of the rectourethral fistula early in the operative intervention . a boy was born in the 38th gestational week , with a birth weight of 2,980 g. on examination , he was found to have an anal atresia without fistula to the perineum . the boy was provided with a left loop divided colostomy by a laparoscopic approach . at the age of 6 weeks preoperative colon x - ray examination disclosed the open rectourethral fistula in the distal end of the rectum . before the psarp operation , an endoscope was inserted through the distal stoma and the fistula to the urethra was identified at the distal end of the rectum . a guidewire was passed through the endoscope into the fistula and out through the urethra ( figs . 1 and 2 ) . a catheter was placed into the urinary bladder in the traditional way through the urethra . video endoscope of 6.5 mm , a guidewire of 2.5 mm , and bladder catheter of 6 charrire were used . through the videoendoscope the rectourethral fistula is easily identified and a guidewire is passed through the videoendoscope and through the rectourethral fistula . through the endoscope of 9 mm , a guidewire of 2.5 the fistula could initially be shown and dissected without opening the rectum and the guidewire was visualized on opening the distal end of the rectum . surgical damage to surrounding tissues and intestine was thereby avoided by using this artificial benchmark of the fistula . no dissection in search of the rectourethral fistula was needed . during the psarp operation , the fistula was divided and used for reconstructing the new anus , while the opening to the urinary tract was left without suture.2 the postoperative course was uneventful . an x - ray of the urinary bladder and urethra , voiding cystourethrogram , performed 6 weeks later showed a normal urinary tract without any fistula to the bowel . this case report demonstrates the use of an endoscopy - aided placement of a rectourethral guidewire to facilitate the reconstruction of anus in patients born with arm . the placement of a guidewire from the distal end of the rectum to the urethra was found to be easy . a guidewire through the fistula facilitates the dissection and thereby helps avoiding any unnecessary dissection searching for the rectourethral fistula . the anatomy becomes clearer , and with the wire in place , opening of the intestine during the initial part of the dissection is prevented . by using the guidewire , any suspicion of nonexisting fistula could be disregarded through the view from the endoscope . with the video endoscope introduced through the distal stoma , it is usually easy to identify the rectourethral fistula preoperatively . the risk of damage to the urethra may be reduced and that might lead to fewer problems with urethral stricture or urethral diverticula . with more exact dissection , it is easier to save the tissues in the fistula for reconstruction of the anus . furthermore , the air in the bowel introduced through the endoscope facilitates the dissection of the blind - ending bowel . endoscopy - assisted laparoscopic excision of rectourethral fistula in a male patient with imperforate anus has been described without the use of any guidewire.3 by using the method described here , laparoscopy / laparotomy intervention in the abdomen is facilitated . to summarize since the first time this operative intervention described here was performed , we have used the method in several children with arm with rectourethral fistula , including a child with cloaca . when performing these interventions , we suggest that both options for placing a guidewire through a cystoscopy from the urethra or from a video endoscope from the bowel should be available .

the aim of this report is to present a technical detail of use in the management of an anorectal malformation ( arm ) . a boy with arm and a fistula to the urethra was operated on with a posterior sagittal anorectoplasty ( psarp ) . before the psarp operation , a videoendoscopy was performed through the distal stoma down to the distal end of the rectum . the rectourethral fistula was identified and a guidewire was passed through the endoscope , through the fistula , and out through the urethra . the endoscopically placed rectourethral guidewire was used as a landmark and facilitated the psarp operation .

Introduction Case Report Discussion

this is achieved by fine - tuning of all the processes governing gene expression , including transcription , splicing , mrna transport , rna stability , translation , protein stability and posttranslational modification . all the steps within this cascade of events are subjected to their own specific regulation , and contribute to generate a different composition of the proteome by modifying not only the levels but also the identity of the proteins present in the cell under specific conditions . the components that participate in these regulatory events are often engaged in the formation of macromolecular complexes . protein - protein as well as rna - protein interactions allow a compartmentalization of the factors needed to control gene expression . translation initiation can modify the proteome by altering the efficiency of translation as well as by enabling the synthesis of different forms of a protein from specific genes . the process of rna translation includes a series of sequential steps , known as initiation , elongation , termination and ribosome recycling . most of translational control is exerted at the initiation step , assisted by specific proteins designated translation initiation factors ( eifs ) . translation initiation of most eukaryotic mrnas commences with 5 end - dependent recruitment of the 43s complex ( that is composed of a 40s subunit bound to eif2-gtp / met - trnai , eif1a , eif1 and eif3 ) by eif4f recognition of the mgpppn ( cap ) at the 5 end of the mrna . in turn , the eif4f complex comprises eif4e that physically binds to the cap , eif4a that unwinds secondary structures in the 5 untranslated region ( 5utr ) and eif4 g , a scaffold protein that interacts with eif4e , eif4a and eif3 . aided by the helicase activity of eif4a and its cofactor eif4b , the 43s pre - initiation complex scans in 5 to 3 direction until an appropriate initiation codon is encountered . auxiliary factors , eif1 , eif2 and eif5 , help to identify the correct aug start codon , resulting in the formation of the 48s complex . eif5 induces hydrolysis of eif2-bound gtp , which is recycled to the active form by eif2b ( guanine nucleotide exchange factor ) . the poly(a ) tail present in the 3utr of most mrnas synergistically stimulates the efficiency of translation through recruitment of poly(a)-binding protein ( pabp ) , enabling its interaction with eif4f located at the mrna 5 end . finally , eif5b mediates joining of the 60s and 40s subunits , generating the 80s complex competent for protein synthesis . translation initiation , particularly in viral mrnas , can occur by an alternative mechanism driven by internal ribosome entry site ( ires ) elements , discovered near 20 years ago in two picornaviruses , encephalomyocarditis virus ( emcv ) and poliovirus ( pv ) [ 2 , 3 ] . these elements are cis - acting sequences that form secondary and tertiary rna structures and recruit the translation machinery to an internal position in the mrna , bypassing a large number of stable structural elements in the viral 5utr . hence , picornavirus ires - driven initiation is 5 end - independent and does not require eif4e to recruit the 40s subunit , in contrast to the cap - dependent initiation mechanism . the subsequent discovery of an ires element in hepatitis c virus ( hcv ) rna that was able to recruit 40s ribosomal subunits in the absence of eif4 g represented a major breakthrough in the translation field [ 5 , 6 ] . this finding opened the question of how iress differing in primary sequence , rna structure , and factor requirements , perform the same function . over the last two decades , the process of internal initiation has been found to be more general than originally thought , that is , it operates in other rna and dna viruses as well as in cellular mrnas . in all cases , iress direct translation of a subset of proteins when the excellent performance of iress , together with the fact that they are active in genetically engineered constructs , has been exploited to study how these diverse rna elements perform the same function . with the exception of one or more polypyrimidine tracts , no primary sequence conservation neither overall structural similarity is detected between picornavirus and hcv iress , strongly suggesting that different strategies may be used to recruit the ribosomal subunits . these strategies could be under the control of a distinct group of proteins specifically interacting with each of these regulatory elements . in this review understanding the role played by these ires trans - acting factors ( itafs ) may help to unravel the strategies employed by mrnas to capture the translation machinery internally . translation initiation of all picornavirus rnas is dependent on the ires located in the long 5utr ( figure 1(a ) ) . various structural elements in the 5utr region , which differ among picornavirus genera , control the viral replication cycle in concerted action with the 3utr [ 811 ] . the ires region spans about 450 nucleotides immediately upstream of the functional translation start codon of the polyprotein [ 2 , 3 , 12 , 13 ] . although less than 50% of primary sequence is conserved between different members of the picornavirus family , the similarity of their secondary structure allows their classification into four types , i to iv . type i includes enterovirus ( ev , pv , hrv ) , type ii , cardiovirus ( emcv ) and aphthovirus ( foot - and - mouth disease virus , fmdv ) , type iii , is used for hepatitis a virus ( hav ) , and the hcv - like ires conforms group iv . the acquisition of a proper structural organization is a key determinant of internal translation initiation driven by picornavirus ires [ 14 , 15 ] , and all viral ires in general [ 16 , 17 ] . this feature is well illustrated by mutational and structural studies conducted on the central domain ( termed 3 or i ) of type ii ires ( figure 1(a ) ) . this region is organized as a cruciform structure with phylogenetically conserved structural motifs that are essential for ires activity [ 18 , 19 ] and determine the tertiary structure of this region [ 2023 ] . picornavirus ires - driven translation initiation depends on the recognition of the ires by specific cellular proteins . iress belonging to types i and ii require eif4 g , eif4a , eif2 , eif3 and atp , but no eif4e , eif1 or eif1a to assemble 48s complexes in a reconstitution assay with purified components [ 13 , 24 , 25 ] . specific structural motifs in the stem - loops j - k - l ( or 4 - 5 ) provide the preferential binding site for eif4 g , eif4b and eif3 ( figure 1(a ) ) [ 2628 ] . however , interaction of these eifs is necessary but not sufficient to promote ires activity , demonstrating the essential function of domains 2 and 3 in ires function . the contribution of domains 2 and 3 to ires activity may consist in the acquisition of a proper rna structural organization , assisted by auxiliary proteins . along this line , while the requirement for eifs is well established , the itafs involved in picornavirus ires activity are still under study . here we review the rna - binding proteins ( rbps ) that can form ribonucleoprotein ( rnp ) complexes with iress modulating their efficiency of translation . picornavirus rnas differ on their ability to operate in the cell - free rabbit reticulocyte lysates ( rrl ) . early studies conducted on the pv rna , which was inactive in rrl , evidenced that its translation efficiency was greatly enhanced upon supplementation of the lysates with hela cell extracts . this difference was related to the requirement of factors that were missing or present in limiting amounts in rrl . hence , addition of hela cells soluble extract resulted in reconstitution of pv rna translation . in due course , these observations led to the discovery of auxiliary proteins behaving as ires trans - acting factors . most itafs described so far are well characterized rbps that contain rna - binding motifs organized in a modular structure [ 31 , 32 ] , as it also occur in proteins involved in rna processing and transport . however , modulation of ires activity by itafs is not well understood , and at least in part it is a controversial issue because , depending on the ires element , some proteins show a more stringent requirement than others do [ 33 , 34 ] . over the last decade , the interaction of rbps with picornavirus ires ( table 1 ) has been analyzed taking advantage of riboproteomic affinity methods . of interest , and confirming the validity of this methodology , proteins previously know to interact with iress by other methods this is the case of eif4b and eif3 , which were specifically identified bound to fmdv or hcv ires by mass spectrometry following rna affinity purification [ 3538 ] . soon after the discovery of picornavirus iress , a direct interaction of the polypyrimidine tract - binding protein ( ptb ) with emcv and fmdv iress was shown by uv - crosslinking [ 3941 ] , and later by rna foot - printing and hydroxyl radical probing . ptb is a multifunctional rbp with four rna recognition motifs ( rrm ) that belongs to the heterogeneous nuclear ribonucleoprotein ( hnrnp ) family . the rrm domains of ptb recognize u - rich loops on short stems and in general , u / c - rich sequences . however , it also performs critical roles in cellular processes pertaining rna metabolism , including polyadenylation , mrna stability and translation initiation . regarding its function as itaf , ptb stimulates picornavirus and retrovirus iress , but it represses translation initiation driven by the bip ires . as a consequence of their role as regulator of ires activity , itafs can mediate cell type specificity , and hence , determine viral spread . this property was brought about by the effects of the neural form of ptb ( nptb ) , that determines the neurovirulence of theiler 's murine encephalitis virus ( tmev ) , and by the double - stranded rna - binding protein 76 ( drbp76 , also termed nf90/nfar-1 ) , that forms a heterodimer with nf45 ( nuclear factor of activated t cells ) . the drbp76:nf45 heterodimer binds to the hrv2 ires and differs in subcellular distribution in neuronal and non - neuronal malignant cells , arresting hrv translation in neuronal cells but not in glioma [ 48 , 49 ] . picornavirus iress often contain more than one polypyrimidine tract located in distant domains at each end of the ires region . recent studies have shown that a single ptb molecule binds in a unique orientation to the emcv ires , with rrm1 - 2 contacting the 3 end , and rrm3 contacting the 5 end of the ires , thereby constraining the ires structure in a unique orientation . however , studies carried out on the fmdv ires raised the conclusion that rrm3 - 4 of ptb were bound in an oriented way to domain 2 and the ires 3 region , respectively . although the rrms involved in the ires - ptb interactions are significantly different between these two studies , both are consistent with a role of ptb in stabilizing the ires structure , thereby acting as a chaperone . itafs , as it is the case of ptb , do not act alone but in combination with various factors presumably contributing to explain the opposite effects on ires activity . hence , rbps interacting with different targets may result in different effects depending on the target and the other partners of the complex . for instance , two iress such as emcv and fmdv with apparent similar secondary structure but different primary sequence , exhibit different requirements in terms of functional rna - protein association . one example is ebp1 protein ( erbb-3-binding protein 1 ) , also known as proliferation - associated factor pa2g4 and itaf45 , identified interacting with domain 3 of fmdv ires in proteomic analysis . ebp1 cooperates with ptb to stimulate fmdv ires activity in reconstitution studies [ 13 , 51 ] , but its depletion does not produce any effect on emcv ires activity . this protein is expressed in proliferating cells during the s phase but not during cell cycle arrest consistent with the fact that fmdv ires is active in proliferating tissues . another example of a factor that mediates ires activity is unr ( upstream of n - ras ) , a cold - shock rbp that interacts with pabp1 and stimulates hrv and pv translation through its interaction with two distinct ires domains [ 53 , 54 ] . in support of the specific role of unr in internal initiation , ires activity of c - myc , apaf-1 , unr and pitslre cellular mrnas is differentially regulated depending on the unr - partners , hnrnp k / poly r(c)-binding protein pcbp1 - 2 , nptb , or hnrnp c1 - 2 , respectively [ 5658 ] . other example of rbp identified with iress is the constitutive heat shock protein hsc70 , although the possibility of an indirect binding can not be discarded . hsc70 forms part of rnp complexes that interact with au - rich elements in the 3utr of specific mrnas , enhancing their stability . in addition to ptb , several proteins implicated in rna splicing can function as itafs , suggesting the existence of a network of interactions between different gene expression processes . an illustrative example is the splicing factor srp20 that up - regulates pv ires - mediated translation via its interaction with pcbp2 . another example of an itaf involved in a different gene expression process is gemin5 that binds directly to fmdv and hcv ires , acting as a downregulator of translation . not surprisingly , gemin5 is associated with other factors in rnp complexes that perform rather different roles during gene expression control . gemin5 is the rna - binding factor of the survival of motor neurons ( smn ) complex , which assembles sm proteins on snrnas playing a critical role in the biogenesis of key components of the mrna splicing machinery , the small nuclear ribonucleoproteins ( snrnps ) . gemin5 is a nuclear protein , but it is predominantly located in the cell cytoplasm and it also appears to be present in p bodies . together , the conclusions derived from the study of multifunctional proteins such as ptb , pcbp2 , gemin5 and other itafs , suggest a novel mechanism for the coordinated regulation of translation initiation of a subset of mrnas bound by shuttling proteins such as hnrnps or splicing factors . in support of this , the splicing factor sf2/asf mediates post - splicing activities promoting translation initiation by suppressing the activity of 4e - bp and modulating the internal initiation of cellular mrnas . in fact , it has been suggested that some rbps might exert its function in translation control by binding to the ires of specific cellular mrnas during splicing complex assembly before nuclear export . this could be an additional layer of posttranscriptional regulation for proteins whose functions are important when cap - dependent translation is compromised . hnrnps are a family of proteins ( named from hnrnp a1 to hnrnp u ) with rna - binding and protein - protein binding motifs [ 31 , 66 ] . they have a nuclear localization associated with nascent rna polymerase ii transcripts and shuttle with the rna to the cytoplasm . the rgg rna - binding motif that mediates the interaction with rna as well as with other hnrnps was first described in hnrnp u , one of the factors identified by mass spectrometry interacting with the fmdv ires . both hnrnp u and gemin5 form part of a complex with eif4e , that may explain the down - regulatory role of gemin5 in cap - dependent translation by virtue of eif4e sequestration or its localization to p bodies . several members of the hnrnp family , hnrnp k , pcbp1 ( hnrnp e1 ) and pcbp2 ( hnrnp e2 ) , have been identified associated with various iress ( table 1 ) . these proteins have in common the kh rna binding domain first described in hnrnp k and , subsequently , in pcbp1 , 2 , 3 and 4 . hnrnp k is the most abundant member of the family of proteins that recognize poly(rc ) regions , and regulates transcription , rna turnover and translation [ 69 , 70 ] . proteins hnrnp k , pcbp1 and pcbp2 , together with daz-1 , have been identified associated to domain 3 of the fmdv ires . daz1 is a 3utr - binding protein that has been found bound to polysomes and stimulates translation initiation of polyadenylated mrna . pcbp2 interacts with a c - rich loop in stem - loop iv of pv , cvb3 and hrv iress and specifically stimulates their activity [ 7275 ] . in contrast , the activity of emcv and fmdv iress that also interact with pcbp2 was not modified by the addition of recombinant pcbp2 protein to depleted - rrl lysates , in agreement with the lack of effect of nucleotide substitutions in the c - rich loop of fmdv ires . pcbp2 performs a dual role in translation initiation and rna replication of the poliovirus genome through its interaction with different targets in the viral 5utr . furthermore , consistent with its relevance in ires function , pv ires competes out with the hav ires for pcbp2 binding . the balance between translation initiation and silencing depends on the cellular response to stress . indeed , many viruses regulate the assembly or disassembly of stress granules ( sgs ) modifying translation of host and virus - encoded mrnas . consistent with this observation , some rbps have been localized in sgs , as pabp1 , or cytoplasmic processing bodies ( pbs ) , as pcbp2 . thus , in response to stress signals including viral infection , these multifunctional proteins may perform distinct roles depending on their localization . the signaling factor ras - gtpase - activating protein ( g3bp ) that was identified interacting with the fmdv ires , belongs to a new family of rbps that link tyrosin kinase - mediated signals with rna metabolism . these cytoplasmic aggregates contain stalled translation preinitiation complexes thought to serve as sites of mrna storage during the cell stress response . g3bp has been found associated to the 3utr of hcv rna , and its depletion induced a reduction of both hcv rna and proteins , supporting the idea that it might be a component of hcv replicating complexes . interestingly , g3bp interacts with the transcriptional regulator gp1-anchored membrane protein ( gpiap1 ) also identified as an ires - binding protein . many itafs are predominantly nuclear proteins that localize to the cytoplasm in picornavirus - infected cells . nucleolin is a protein involved in rdna transcription , rrna maturation , ribosome assembly and nucleo - cytoplasmic transport , and is translocated into the cytoplasm following infection of cells with poliovirus . nucleolin interacts with hrv , fmdv and pv ires and stimulates pv ires - mediated translation in transfected cells overexpressing the full - length protein . during enterovirus ev71 infection , the nuclear protein fbp2 ( far upstream element ( fuse ) binding protein 2 ) was enriched in the cytoplasm where viral replication occurs , whereas in mock - infected cells fbp2 was localized in the nucleus . fbp2 is a kh protein that negatively regulates ev71 ires activity presumably through its capacity to compete with ptb binding . together with hnrnps , a group of proteins that are involved in gene silencing , transport , and stabilization ( eif2c , rna helicases ) have been identified in riboproteomic approaches bound to different iress ( table 1 ) . the recurrent identification of a subset of factors with different rna targets [ 91 , 92 ] points to the existence of a network of rnps with the potential for multiple levels of regulation . moreover , the modular structure of rbps that is at the basis of their capacity to recognize a large number of targets raises the possibility that binding to any particular rna could facilitate different sorts of regulation depending on the other protein partners and the cellular environment . initiation of protein synthesis in the positive - strand rna genome of hcv is also driven by an ires . the ires region spans 340 nucleotides and differs from picornavirus iress in rna structural organization and factor requirement . the hcv ires is organized in three conserved structural domains , termed ii , iii and iv ( figure 1(b ) ) that adopt a tertiary fold whose integrity is required for efficient protein synthesis . domain ii , which consists of a hairpin with basal and apical loops , is essential for hcv ires activity . this domain promotes eif5-induced gtp hydrolysis during 80s ribosome assembly and eif2/gdp release from the initiation complex . the basal portion of domain iii forms the core of the high - affinity interaction with the 40s subunit including a small stem - loop ( iiie ) and a pseudoknot ( iiif ) . in the absence of eifs , the hcv ires can establish a high - affinity interaction with ribosomal 40s subunits through the binding surface between subdomains iiiabc , iiief and iiid . despite the capacity to form binary complexes , localization of the met - trnai on the surface of the 40s subunit by eif2 is essential for translation , and eif3 significantly enhances formation of the 48s initiation complex interacting with the junction of subdomains iiiabc [ 5 , 99 ] . interaction of eif3 subunits with hcv ires has been analyzed by cryo - electron microscopy of the binary ires - eif3 complex and by mass spectrometry of ires - bound protein complexes [ 36 , 37 ] . however , under conditions of inactivation of eif2 by phosphorylation , the hcv ires can form a preinitiation complex in the presence of eif3 and eif5b , which is reminiscent of the bacterial - like initiation mode . the ribosomal proteins that participate in ires-40s interaction have been identified by different approaches as well , such as cross - linking studies [ 102104 ] and mass spectrometry . besides ribosomal proteins and eif3 subunits , the non - canonical factors rack1 and nucleolin were identified in native and ires-40s ribosomal complexes . rack1 functions as the receptor for activated protein kinase c , and regulates translation initiation by recruiting protein kinase c to the 40s subunit . it forms a stable complex with the 40s subunit , exposing the wd - repeats as a platform for interactions with other proteins to the ribosome . another non - canonical host factor , the insulin - like growth factor ii mrna - binding protein 1 ( igf2bp1 ) has been reported to associate with both the ires and the 3utr of hcv , and remarkably , to coimmunoprecipitate with eif3 and the 40s subunit . this result suggests that this factor may enhance hcv ires - dependent translation by recruiting the ribosomal subunits to a pseudo - circularized rna . in agreement with this possibility , a subset of the identified proteins , nf90/nf45 , also interact with the ends of the viral rna contributing to enhance viral rna replication . moreover , in support of the role of the 35 interactions in the control of gene expression in positive - strand rna viruses , stimulation of ires activity by the homologous 3utr has been shown in fmdv and hrv [ 10 , 11 ] , presumably mediated by functional bridges that bring together the rna ends by long - range rna - rna interactions . despite some controversy regarding the effect of ptb , most of the identified itafs regulate hcv ires activity in a positive manner [ 110 , 111 ] . la binds to pv , emcv and hcv ires stimulating translation [ 113115 ] , but it suppresses hav ires activity . nsap1 protein has a dual function in hcv life cycle participating in rna replication and enhancing ires - dependent translation through its binding to a - rich sequences in the core coding region . similar to nsap1 , hnrnp l interacts with the 3 border of the hcv ires in the core - coding sequence and it is required for ires - mediated translation . this protein is necessary for efficient translation of the cat-1 arginine / lysine transporter mrna during amino acid starvation . other hcv - interacting protein is hnrnp d that binds to the stem - loop ii and promotes translation . proteins of this family , hnrnp a / b 38 , have been identified interacting with diii of hcv ires . hnrnp a1 binds to the 5utr of ev71 and sindbis rna and is required for viral rna replication . this protein also mediates internal initiation of fgf-2 and apaf-1 mrnas [ 123 , 124 ] . in addition to direct rna - protein interactions , protein - protein association between rbps , such as hnrnp u or hnrnp a / b [ 125 , 126 ] during mrna transport can explain the identification of proteins belonging to the cytoskeleton machinery with fmdv and hcv ires [ 36 , 38 , 55 ] . protein - protein interactions may also explain the identification of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) with hav ires . this protein competes with ptb for binding to stem - loop iiia , suppressing the ability of the hav 5utr to direct cap - independent translation . gapdh forms a macromolecular complex that binds to u - rich sequences in the 3utr of a selective group of cellular mrnas controlling their translation . however , as already mentioned for some factors , indirect interactions may be behind the identification of very abundant rbps , such as yb-1 , in riboproteomic studies . thus , the functional involvement of each factor as well as whether the binding is direct or mediated by another partner in the rnp complex , needs to be verified individually . a few proteins identified by mass spectrometry with a discrete domain of the hcv ires have been also identified in similar approaches interacting with the entire hcv ires , giving additional information about the binding site of the protein . this could be the case of rna helicase deah - box polypeptide 9 ( dhx9 ) or dead - box polypeptide 1 ( ddx1 ) . the ddx / dhx family of proteins play important roles in nucleic acid metabolism , including pre - mrna processing , ribosome biogenesis , rna turnover , rna export , translation , and association / dissociation of large rnp complexes . dhx9 recognizes a complex structure at the 5-utr of retrovirus mrna precursors , facilitating its association to polyribosomes . ddx1 , a dual interactor of hnrnp k and poly(a)-mrna , has been also identified bound to the 3utr of hcv suggesting a role for this protein in viral rna replication . in general , itafs are rna - binding proteins that shuttle between the nucleus and the cytoplasm . thus , a network of rna - protein and protein - protein interactions may assist to recruit the ires to the ribosome and possibly , to other cytoplasmic structures . rbps are key cellular components that control the temporal , spatial and functional dynamics of rna within the competitive cell environment . indeed , changes in the expression of rbps have profound implications for cellular physiology , affecting rna processes from pre - mrna splicing to protein translation . thereby , the composition of rnp complexes bound to the rna in a particular situation will determine the fate of the rna ( e.g. , stability , translatability , compartmentalization ) . in other words , binding of proteins , even those considered to be promiscuous , to a given rna could mediate specific regulation . in agreement with this , recent mass spectrometry identification of the proteins associated with argonaute proteins , the protein complex responsible for gene - silencing pathways guided by small rnas , revealed a common group of helicases , hnrnps and other rbps which are shared with rnp complexes involved in other cellular processes such as mrna transport , stabilization and translation . the observation that proteins with the potential for multiple levels of regulation can recognize various rna targets raises the possibility that protein - binding to specific rnas could facilitate different sorts of regulation depending on the other partners and the cellular environment . thus , elucidating the function of itafs will require a deep understanding of their rna targets , their protein partners , and their potential modifications . concerning the first issue , the recent advances in cross - linking immunoprecipitation and high - throughput sequencing appears to be a promising technique to help in this task . implementation of new proteomic approaches will continue to help in the second and third tasks . finally , regarding the modification of rbps in infected cells , understanding the effect of proteolytic cleavage of factors such as pcbp2 , ptb , pabp or g3bp [ 78 , 133 , 134 ] will need to be extended to newly identified itafs . all together , this will help to understand the integrated action of itafs on mrna targets . the rbps modulating picornavirus and hcv ires activity offer promising targets to combat these infectious pathogens . indeed , iress are ideal candidates to interfere with virus replication through direct ires - targeting or through itaf - targeting . in the first case , antiviral agents based on rna molecules aimed to disrupt the ires structure have been partially successful [ 135138 ] . in the second case , knowledge of the structural organization of itafs provided the basis to design antiviral therapy , as shown by a synthetic peptide derived from the rrm2 of la which acts as a dominant negative inhibitor of hcv rna translation . in the coming years , elucidation of the structural determinant of peptides derived from different itafs , interfering with the capacity of these proteins to generate protein - protein and rna - protein networks , will provide the basis for developing small peptidomimetic structures as potent anti - viral therapeutics .

translation initiation is a highly regulated process that exerts a strong influence on the posttranscriptional control of gene expression . two alternative mechanisms govern translation initiation in eukaryotic mrnas , the cap - dependent initiation mechanism operating in most mrnas , and the internal ribosome entry site ( ires)-dependent mechanism , first discovered in picornaviruses . ires elements are highly structured rna sequences that , in most instances , require specific proteins for recruitment of the translation machinery . some of these proteins are eukaryotic initiation factors . in addition , rna - binding proteins ( rbps ) play a key role in internal initiation control . rbps are pivotal regulators of gene expression in response to numerous stresses , including virus infection . this review discusses recent advances on riboproteomic approaches to identify ires transacting factors ( itafs ) and the relationship between rna - protein interaction and ires activity , highlighting the most relevant features on picornavirus and hepatitis c virus iress .

1. Translational Control of Gene Expression 2. Internal Initiation of Translation in Picornavirus RNAs 3. Internal Initiation of Translation in HCV RNA 4. Concluding Remarks

rheumatoid arthritis ( ra ) and systemic lupus erythematosus ( sle ) are common rheumatologic diseases , and considered to be separate diseases . rhupus syndrome is a rare clinical condition and there are only a small number of well - documented cases in the literature . the prevalence of rhupus syndrome was found to be far less than expected chance of concurrence ( 0.09% versus calculated 1.2% ) . rhupus syndrome has clinical manifestations of both ra and sle but the organ damages found due to sle are usually mild . we are reporting a young lady with bilateral acute lupus pneumonitis in rhupus syndrome , a rare condition with only few cases reported worldwide . a 23-year - old female , diagnosed as ra five years back and on treatment with analgesics , hydroxychloroquine and methotrexate , had developed high grade intermittent fever for seven days , joint pain with swelling involving bilateral elbow and wrist joints , dyspnea and cough with scanty mucoid expectoration for four days . she was transferred to our respiratory care unit as she was not responding to parenteral antibiotics ( intravenous cefepime 2 gm twice daily and levofloxacin 500 mg daily for 10 days ) . on general survey , following were the findings : oral temperature 104f , moderate pallor , blood pressure 110/72 mmhg , pulse 110/min , regular , respiratory rate 32/min with accessory muscles working , alopecia , oral ulcer , swan neck deformities of both hands with ulnar deviation of wrist joints . there was no skin nodule , skin rash , lymphadenopathy , hepato - splenomegaly , puffy fingers , sclerodactyly , or she did not give history of raynaud 's phenomenon , dysphagia , hemoptysis or any bleeding episodes , muscle weakness , seizures or abnormal behavior . chest examination revealed impaired percussion notes in both infrascapular areas with decreased vesicular breath sound and bilateral basal crepitations . investigations showed normochomic normocytic anemia with hemoglobin of 9.1 gm / dl , total white blood cell ( wbc ) count of 1,800/mm with neutrophil 83% , lymphocyte 17% ; platelet count of 225,000/mm , erythrocyte sedimentation rate at 110 mm in the1st hour and fasting blood sugar at 78 mg / dl . x - ray of both hands showed juxta articular osteopenia , loss of carpometacarpal and metacarpophalangeal joint spaces [ figure 1 ] . her x - ray chest postero - anterior view showed bilateral lower zone consolidation with bilateral pleural effusion [ figure 2 ] . high resolution computed tomography ( hrct ) thorax showed bilateral , multiple areas of consolidation in lower lobes with bilateral small pleural effusion [ figure 3 ] . electrocardiography showed sinus tachycardia and echocardiography showed mild pericardial effusion with slightly thickened pericardium and mild pulmonary arterial hypertension . arterial blood gas analysis ( abg ) showed hypoxia ( pao2- 54 mm hg with 4 litre of oxygen / min ) with respiratory alkalosis ( ph- 7.48 , paco2- 32 mm hg ) . pleural fluid examination showed cell count 540 cells / mm with lymphocyte 65% , neutrophil 30% , eosinophil 05% ; sugar 89 mg / dl ; protein 3.39 mg / dl ; lactate dehydrogenase 2,783 units / liter and adenosine deaminase 51.3 units / liter ; ziehl - neelsen stain negative ; gram stain and culture negative and mycobacterium dna polymerase chain reaction was negative . consecutive 3 days sputum examinations revealed no acid fast bacilli ( afb ) or organisms and one time bronchiolo - alveolar lavage ( bal ) examination from the affected areas was also negative for organism , afb and hemosiderin - laden macrophage . special investigation showed ra factor was positive ( 1:480 ) ; antinuclear antibody ( ana ) was 3 + ( 1:320 , homogenous ) and anti - dsdna with the immunofluorescence technique ( ift ) using crithidia luciliae as a substrate was positive . x - ray of both hands showing juxta articular osteopenia , loss of carpo - metacarpal and meta - carpophalangeal joint spaces chest x - ray postero - anterior view shows bilateral lower zone consolidation with bilateral pleural effusion hrct scan thorax showing bilateral lower lobe consolidation we had diagnosed the case as bilateral lupus pneumonitis and started treatment with methyl prednisolone intravenous 1,000mg / day for three days , followed by oral prednisolone 40 mg / day for two weeks then tapered over two months . on follow - up , x - ray chest postero - anterior view after one month showed there was complete clearance of consolidations [ figure 4 ] . abg findings were normal , on pulse - oximetery spo2 was 96% in room air , six minute walking distance was 340 meters and on spirometry fev1/fvc ratio was 84% , fev1 and fvc were 82% and 86% of predicted values , respectively . follow - up x - ray chest postero - anterior view after one month showed complete clearance of consolidations the diagnostic criteria for rhupus was suggested as inflammatory symmetrical erosive polyarthritis , positive ra factor , clinical features suggestive of sle and positive anti - dsdna or anti - smith auto - antibodies . a retrospective study in china observed that the mean age of rhupus was 36.8 years ; sle developed about 7.7 years after initial presentation and sle associated severe organ damages other than hematopoietic abnormalities were less frequent . features of ra are dominated by erosive polyarthritis in all cases and rheumatoid nodules in around 40% of the cases . sle is usually manifested by cutaneous ( butterfly skin rash , photosensitivity and alopecia ) , hematological ( leukopenia and thrombocytopenia ) , serosal ( pleural and pericardial effusion ) and mucosal involvement . we diagnosed the case as rhupus by clinical manifestations of ra and sle with positive ra factor , ana and ds - dna . we excluded the possibilities of other differential diagnosis of rhupus like ra patients with extrarticular manifestations , sle patients with arthritis and mixed connective tissue disease . major organ involvements are rare , though there are scattered reports of lupus encephalitis in rhupus . lupus pneumonitis is an unusual and life threatening complication of sle ; usually occurs during flare - up or rarely as a presentation . the usual radiological signs of lupus pneumonitis are consolidations in one or more areas usually basal and bilateral and often associated with pleural effusion and pulmonary arterial hypertension . the mortality of lupus pneumonitis is around 50% , rest of them responds dramatically to steroids and some require cyclophosphamide . the diagnosis of lupus pneumonitis is essentially by exclusion of other causes of lung infiltration such as infective pneumonia ( bacterial , mycobacterial , fungal and viral ) , organizing pneumonia ( op ) , alveolar hemorrhage , pulmonary embolism , etc . we excluded infective pneumonia by repeated sputum and single bal fluid examination ; alveolar hemorrhage by the absence of hemoptysis and hemosiderin - laden macrophage in bal fluid and the syndrome of acute reversible hypoxemia by the presence of abnormal chest x - ray and hrct - scan of thorax . organizing pneumonia , a rare complication of ra that may simulate lupus pneumonitis , is usually not life threatening , subacute in onset , the mean age of onset around 50 - 60 years and begins with a mild flu - like illness with fever , cough , malaise , mild dyspnoea , anorexia and weight loss . the radiographical pattern in op secondary to connective tissue disease ( ctd ) is reticularnodular , and thus , indistinguishable from idiopathic pulmonary fibrosis while that of idiopathic op is usually patchy alveolar infiltration . ct findings are commonly accompanied by ground glass opacity , nodules ( indicating bronchiolitis ) , and the findings of non - specific / usual interstitial pneumonia ( in ctd - associated op ) . relapses are common upon stopping or reduction of corticosteroids to < 20mg / day , thus often leading to prolonged treatment . we excluded the possibility of ctd associated op by absence of typical clinic - radiological picture and good response to treatment without relapse . the diagnosis of acute lupus pneumonitis was established by the typical clinical manifestations , hrct findings and dramatic response to corticosteroids .

rhupus syndrome , the overlap of rheumatoid arthritis ( ra ) and systemic lupus erythematosus ( sle ) , is an extremely uncommon condition . organ damages found due to sle are usually mild in rhupus . lupus pneumonitis in rhupus syndrome has not been reported worldwide . we are reporting a 23-year - old female with bilateral symmetric erosive arthritis , oral ulcer , alopecia , polyserositis , anemia , leucopenia , positive ra - factor , anti nuclear antibody ( ana ) and anti ds - dna . she presented with acute onset dyspnea , high fever , chest pain , tachycardia , tachypnea , hypoxia and respiratory alkalosis . high resolution computed tomography ( hrct)-thorax showed bilateral , basal consolidation with air bronchogram . repeated sputum and single broncho alveolar lavage ( bal ) fluid examination revealed no organism or hemosiderin - laden macrophage . the diagnosis of rhupus was confirmed by combined manifestations of ra and sle , and the diagnosis of acute lupus pneumonitis was established by clinico - radiological picture and by excluding other possibilities .

INTRODUCTION CASE REPORT DISCUSSION

one of the most contentious issues related to lyme disease has been whether infection with b. burgdorferi can be easily eradicated by conventional antimicrobial treatment or whether it is a persistent , recurrent and difficult to eradicate infection . post - treatment lyme disease syndrome refers to patients with persistent , non - specific symptoms , such as arthralgia , fatigue or perceived cognitive impairment 6 months after completion of treatment for lyme disease . many of those who believe in a condition termed chronic lyme disease argue that documenting that the bacteria can survive a standard course of antimicrobial treatment will prove that chronic lyme disease and post - treatment lyme disease syndrome are indeed a consequence of persistent infection with b. burgdorferi . this belief persists , despite a mountain of scientific evidence that chronic lyme disease , a label without a case definition that is applied to patients with non - specific symptoms more properly designated medically unexplained symptoms , does not exist [ 13 ] . several studies in which investigators have claimed to have documented persistence of b. burgdorferi in humans , despite treatment with antimicrobials , have not been reproduced or have been shown to be due to laboratory contaminants [ 48 ] . some studies in which models of the infection in animals ( usually in either mice or non - human primates ) have been performed have lent support to the argument that b. burgdorferi can persist in tissues despite antimicrobial treatment . however , there are numerous problems with the animal models that have led many to question either the reliability of the results of these studies or the generalizability of the results to disease in humans . among the many problems with animal models is the difficulty of mimicking the route and infectious dose of b. burgdorferi in human infection , differences in the pharmacokinetic and pharmacodynamic parameters of antimicrobial treatment in the animals vs. humans , and differences in immune responses of different animal species ( for example , mice are reservoirs for b. burgdorferi in nature ) . in addition , even in the rare instances in which b. burgdorferi has been identified in treated animals , it has not been shown to be viable ( that is , able to replicate in culture media ) . moreover , whether it is producing symptomatic disease in these animals is speculative . several recent studies have shed additional light on this topic and have added to the evidence that viable b. burgdorferi do not persist after antimicrobial treatment of humans . it is well recognized that subsequent episodes of erythema migrans are not unusual in patients with an initial episode who receive prompt antimicrobial treatment . if b. burgdorferi persist despite antimicrobial treatment , it would make sense that subsequent episodes of erythema migrans might be due to the originally infecting strain of the organism . in a study to assess whether such episodes were new infections or recurrences , strains of b. burgdorferi from 17 patients with two or more episodes of culture - proved erythema migrans had 22 paired episodes with organisms available for analysis ( first and second episodes , and , for those with additional episodes , third and fourth episodes ) . strains were compared by amplifying their dna with polymerase chain reaction ( pcr ) assay of each strain , followed by genotyping of outer surface protein c of each strain . in all instances , the strains in each of the paired episodes were different , indicating that each of the subsequent episodes of erythema migrans was due to a new infection ( presumably transmitted from a new tick bite ) rather than recrudescence of a persistent infection . moreover , investigators used statistical simulation based on these data to conclude that patients treated for early lyme disease develop protective immunity that is strain specific and that lasts for at least 6 years . proponents of the existence of chronic lyme disease postulate that the organism often persists despite conventional treatment with an antimicrobial , and that very prolonged treatment with antimicrobials is necessary to eliminate the organism . however , other than a few isolated claims ( that could not be replicated ) of recovery of b. burgdorferi from patients with chronic symptoms , there is no evidence that viable organisms persist as a cause of chronic symptoms in humans , despite conventional treatment with antimicrobials [ 48 ] . in infected patients , b. burgdorferi is present only in low concentrations in blood and spinal fluid and can be difficult to culture . xenodiagnosis is a method of documenting the presence of a microorganism in tissue by allowing a vector to feed on potentially infected tissue and then examining the vector for the presence of the microorganism it may have ingested . in the past , it was used extensively as a method of diagnosing chagas disease . some say it may be the most sensitive test in mammals because of evolutionary adaptations of the vector and the microorganism . for example , there is evidence that a chemoattractant is secreted by ixodes scapularis that can enhance migration of b. burgdorferi to its mouthparts while feeding on a host . the first use of xenodiagnosis to try to detect b. burgdorferi in humans was reported recently . in this study , 36 human subjects ( median age : 55 years ) had 2530 laboratory - raised , pathogen - free , larval i. scapularis ticks placed on them under a retention dressing and were then collected after feeding for 3 to 7 days . the 36 subjects included 10 patients who had been treated for lyme disease and still had high concentrations of antibodies against the c6 peptide of the membrane protein of b. burgdorferi , 10 patients treated for lyme disease who had post - treatment lyme disease syndrome with continuing symptoms severe enough to impair their normal activities , 5 patients who recently completed antibiotic therapy for erythema migrans , 1 patient early in the course of antibiotic therapy for erythema migrans , and 10 healthy controls . the patients recently treated for erythema migrans were meant potentially to be positive controls , since it was felt to be unethical to delay treatment of infected patients , but it was thought that these patients had the best chance of testing positive by xenodiagnosis . biopsies of skin at the site where ticks fed and homogenates of the ticks were cultured for b. burgdorferi . ticks were also tested for dna of b. burgdorferi by pcr assay and/or by isothermal amplification followed by pcr and electrospray ionization mass spectroscopy . in addition , attempts were made to infect immunodeficient mice by having the potentially infected ticks feed on them or by inoculation of homogenates of fed ticks into the mice . no viable organisms were recovered from ticks or from skin biopsies from any of the patients . one was the patient who was early on in the course of antibiotic treatment for erythema migrans . most would say that only a positive culture ( recovery of viable organisms ) would constitute positive xenodiagnosis . it is well recognized that fragments of dna of b. burgdorferi can persist for a very long period after successful antibiotic treatment and killing of viable organisms . a positive result of a highly sensitive pcr assay of a tick from a patient with post - treatment lyme disease syndrome certainly is provocative . can persistence of antigens of organisms killed after antimicrobial treatment lead to prolonged non - specific symptoms ? perhaps persistent ( non - viable ) fragments of organisms can persist and provoke inflammation that leads to symptoms . certainly the results of this study do not provide evidence for the suppositions that viable b. burgdorferi persist after conventional antimicrobial treatment , or suggest that additional clinical trials of prolonged antimicrobial treatment of patients with only non - specific symptoms after lyme disease are warranted . it is well recognized that subsequent episodes of erythema migrans are not unusual in patients with an initial episode who receive prompt antimicrobial treatment . if b. burgdorferi persist despite antimicrobial treatment , it would make sense that subsequent episodes of erythema migrans might be due to the originally infecting strain of the organism . in a study to assess whether such episodes were new infections or recurrences , strains of b. burgdorferi from 17 patients with two or more episodes of culture - proved erythema migrans had 22 paired episodes with organisms available for analysis ( first and second episodes , and , for those with additional episodes , third and fourth episodes ) . strains were compared by amplifying their dna with polymerase chain reaction ( pcr ) assay of each strain , followed by genotyping of outer surface protein c of each strain . in all instances , the strains in each of the paired episodes were different , indicating that each of the subsequent episodes of erythema migrans was due to a new infection ( presumably transmitted from a new tick bite ) rather than recrudescence of a persistent infection . moreover , investigators used statistical simulation based on these data to conclude that patients treated for early lyme disease develop protective immunity that is strain specific and that lasts for at least 6 years . proponents of the existence of chronic lyme disease postulate that the organism often persists despite conventional treatment with an antimicrobial , and that very prolonged treatment with antimicrobials is necessary to eliminate the organism . however , other than a few isolated claims ( that could not be replicated ) of recovery of b. burgdorferi from patients with chronic symptoms , there is no evidence that viable organisms persist as a cause of chronic symptoms in humans , despite conventional treatment with antimicrobials [ 48 ] . in infected patients , b. burgdorferi is present only in low concentrations in blood and spinal fluid and can be difficult to culture . xenodiagnosis is a method of documenting the presence of a microorganism in tissue by allowing a vector to feed on potentially infected tissue and then examining the vector for the presence of the microorganism it may have ingested . in the past , it was used extensively as a method of diagnosing chagas disease . some say it may be the most sensitive test in mammals because of evolutionary adaptations of the vector and the microorganism . for example , there is evidence that a chemoattractant is secreted by ixodes scapularis that can enhance migration of b. burgdorferi to its mouthparts while feeding on a host . the first use of xenodiagnosis to try to detect b. burgdorferi in humans was reported recently . in this study , 36 human subjects ( median age : 55 years ) had 2530 laboratory - raised , pathogen - free , larval i. scapularis ticks placed on them under a retention dressing and were then collected after feeding for 3 to 7 days . the 36 subjects included 10 patients who had been treated for lyme disease and still had high concentrations of antibodies against the c6 peptide of the membrane protein of b. burgdorferi , 10 patients treated for lyme disease who had post - treatment lyme disease syndrome with continuing symptoms severe enough to impair their normal activities , 5 patients who recently completed antibiotic therapy for erythema migrans , 1 patient early in the course of antibiotic therapy for erythema migrans , and 10 healthy controls . the patients recently treated for erythema migrans were meant potentially to be positive controls , since it was felt to be unethical to delay treatment of infected patients , but it was thought that these patients had the best chance of testing positive by xenodiagnosis . biopsies of skin at the site where ticks fed and homogenates of the ticks were cultured for b. burgdorferi . ticks were also tested for dna of b. burgdorferi by pcr assay and/or by isothermal amplification followed by pcr and electrospray ionization mass spectroscopy . in addition , attempts were made to infect immunodeficient mice by having the potentially infected ticks feed on them or by inoculation of homogenates of fed ticks into the mice . no viable organisms were recovered from ticks or from skin biopsies from any of the patients . one was the patient who was early on in the course of antibiotic treatment for erythema migrans . most would say that only a positive culture ( recovery of viable organisms ) would constitute positive xenodiagnosis . it is well recognized that fragments of dna of b. burgdorferi can persist for a very long period after successful antibiotic treatment and killing of viable organisms . a positive result of a highly sensitive pcr assay of a tick from a patient with post - treatment lyme disease syndrome certainly is provocative . can persistence of antigens of organisms killed after antimicrobial treatment lead to prolonged non - specific symptoms ? perhaps persistent ( non - viable ) fragments of organisms can persist and provoke inflammation that leads to symptoms . certainly the results of this study do not provide evidence for the suppositions that viable b. burgdorferi persist after conventional antimicrobial treatment , or suggest that additional clinical trials of prolonged antimicrobial treatment of patients with only non - specific symptoms after lyme disease are warranted .

whether or not borrelia burgdorferi can persist after conventional treatment with antimicrobials has been a very controversial issue . two recent studies took different approaches to try to answer this question . in one , investigators showed that , in each of 22 instances in 17 patients with two consecutive episodes of culture - proved erythema migrans , the strains of b. burgdorferi were different based on their genotypes . this indicated that the repeat episodes were due to new infections rather than recrudescence of the original infection . in another study , in which persistence of b. burgdorferi was assessed by using xenodiagnosis , no viable b. burgdorferi were cultured from ticks fed on any of the patients . there continues to be no evidence that viable b. burgdorferi persist in humans after conventional treatment with antimicrobials .

Introduction Recent studies Repeat episodes of erythema migrans Xenodiagnosis for B. Burgdorferi Disclosures

we have known for over 60 years that a dark - adapted human observer can reliably identify a flash of light activating only a small subset of the photoreceptor cells . a requirement for such exquisite sensitivity is that the individual rod photoreceptors must be able to produce a reliable signal in the ganglion cells , the cells that provide the output from the retina . many of the details of how the single - photon response is generated in the rods and how it is processed by retinal interneurons have remained in question . over the past decade , several important advances have identified and characterized the mechanisms that preserve the single - photon responses amid intrinsic noise in the receptor cells and retinal circuitry . in response to an absorbed photon , rod photoreceptors generate small graded hyperpolarizations on the order of approximately 1 mv . the challenge for retinal processing is to convey this small signal to higher visual centers in the presence of significant receptor noise . efficient signal transmission through the retina is accomplished by means of a conserved and highly convergent pathway in the mammalian retina called the rod bipolar pathway . on bipolar cell and a depolarizing ( aii ) amacrine cell to boost the single - photon response amplitude and suppress noise , before feeding this signal back into the cone circuitry . the rod signals are then relayed to spiking ganglion cells , which in turn convey this information to the lateral geniculate nucleus and higher visual areas . rod phototransduction has received intense study over the past 30 years , but our understanding has deepened considerably with recent advances in mouse genetics that have facilitated the creation of mouse lines with selective perturbations in the signaling cascade . we are now beginning to see in detail how components of the signaling cascade contribute to properties of the single - photon response . these components include : ( a ) the role of ca feedback in the synthesis of cyclic guanosine monophosphate ( cgmp ) , which limits rod noise and speeds the time course of the single - photon response ; ( b ) the role played by rhodopsin phosphorylation and arrestin binding in the shutoff of the single - photon response [ 9 - 11 ] ; and ( c ) the role played by regulators of g - protein signaling ( rgss ) proteins in accelerating the gtpase activity of the g - protein transducin . advances in our understanding of rod synaptic transmission have also revealed specializations for the transmission of the single - photon response . rod photoreceptors expend considerable energy to maintain a depolarized membrane potential in darkness which sets synaptic ca channels near the steepest point in the relationship between membrane potential and ca influx . proteins like calcium - binding protein 4 ( cabp4 ) have been implicated in the further fine - tuning of this voltage dependence to allow small changes in membrane potential to produce proportionally larger changes in ca concentration in the presynaptic terminal , which in turn results in a larger change in glutamate release . while the factors that control the size and time course of the single - photon response and of glutamate release have been studied in detail , light detection will ultimately depend on how the system is able to discriminate the single - photon response from noise . studies of phototransduction have identified two major sources of noise that limit the detection of single photons : the thermal activation of rhodopsin , which generates discrete noise events , and the spontaneous activity of the cgmp phosphodiesterase ( pde ) , which generates continuous noise . either of these forms of noise can produce fluctuations in cgmp that resemble the single - photon response . the key to light detection is therefore the ability of our visual system to discriminate light - driven signals from these two forms of noise . the rod - to - rod bipolar synapse plays a key role in this process . in particular , a threshold - like nonlinearity at this synapse is positioned to make an optimum separation of the single - photon response from continuous noise . as shown in figure 1 , this threshold - like nonlinearity acts at every rod - to - rod bipolar synapse to allow only signals that exceed a criterion amplitude to pass through the synapse , so that only the largest responses from the rods can be reliably detected by the bipolars . rod bipolars are thus able to sum responses from rod photoreceptors in a manner that allows them to preserve the larger single - photon responses while rejecting much of the continuous noise component . such an operation is believed to improve the signal - to - noise ratio of the single - photon response in rod bipolar cells by more than 300-fold over what we would expect from a linear combination of rod signals . mechanistically , this threshold - like nonlinearity is generated by postsynaptic saturation at the rod - to - rod bipolar synapse . this saturation appears not to be attributable to saturation of binding to metabotropic glutamate ( mglur6 ) receptors on the rod bipolar dendrites but seems instead to occur somewhere within the g - protein cascade that couples mglur6 to cation channels . ( a ) a rod bipolar cell pools inputs from many rods , but near absolute visual threshold , only one rod may absorb a photon ( red ) while the remaining rods are generating electrical noise ( blue ) . a threshold - like nonlinearity ( dashed ) improves photon detection at this synapse by retaining responses that exceed threshold ( red ) in rods most likely absorbing a photon , while discarding responses that do not exceed threshold and that are more likely noise in the remaining rods . ( b ) nonlinear signal processing can improve the fidelity of rod signals . if rod outputs from ( a ) are simply summed , the resulting trace is noisy , but when summed after applying the threshold - like nonlinearity for each rod in ( a ) , the response is easier to discern . r , rod ; rb , rod bipolar cells . reprinted with permission from physiology . copyright 2007 , the american physiological society . understanding how the nonlinear threshold is set by rod bipolar cells will ultimately require the elucidation of the mglur6 signaling pathway in the bipolar dendrites . as shown in figure 2 , glutamate released from rods in darkness is sensed postsynaptically by mglur6 receptors , which in turn activate a go-containing g - protein . through a series of unknown steps , the activity of this pathway leads to the closure of transduction channels , part of which may consist of trpm1 , a member of the trpm family of transient receptor potential ( trp ) ion channels . recent physiological evidence indicates that the properties of these channels are similar to those of other trp channels they can , for example , be gated by trpv1 agonists . since postsynaptic saturation sets the position of the nonlinear threshold , elucidating the remaining transduction components will be key to identifying the components that set the level of synaptic saturation . ( a ) the rod spherule is a specialized invaginating structure where the dendrites of horizontal ( h ) and rod bipolar ( b ) cells are apposed to glutamate release sites controlled by a synaptic ribbon . ca channels ( cav1.4 ) located near the active zone ( az ) allow the continuous release of glutamatergic vesicles in darkness . in on rod bipolar cells , glutamate is sensed by metabotropic glutamate receptor type 6 ( mglur6 ) receptors located near the mouth of the invagination . inset : release of glutamate is dependent on ca influx through ca channels , which is graded by voltage over the physiological range . mglur6 activation leads to the activation of go , which through unknown mechanisms leads to the closure of nonselective cation channels , which may be in part trpm1 ( transient receptor potential cation channel , melastatin family , member 1 ) . the light - evoked reduction in glutamate release relieves activity in this cascade and opens cation channels leading to membrane depolarization . while the properties of signaling in the rod photoreceptors and rod bipolar cells have received much attention , other mechanisms downstream also play key roles in improving the fidelity of the single - photon response . for example , signals in aii amacrine cells are boosted by multivesicular release at the rod bipolar cell - to - aii amacrine cell synapse , increasing the discrimination of the single - photon responses from synaptic noise . furthermore , aii amacrine cells create an electrically coupled network that may suppress synaptic noise . this network is formed by gap junctions consisting of connexin 36 and is critical for the transmission of high - sensitivity signals to ganglion cells by means of efficient signal transmission to cone bipolar cells . over the past decade , significant advances have been made that have helped us understand how signals are preserved and noise is eliminated in the rod circuitry in order to maximize the detection of the single - photon response . is it the discrete noise due to thermal activation of rhodopsin , or the continuous noise produced by spontaneous pde activation ? in principle , either discrete or continuous noise could limit light detection by generating false - positive signals . mechanisms such as the threshold - like nonlinearity at the rod - to - rod bipolar synapse may eliminate much of the rod noise , but the identification of the molecular mechanism that is responsible will require the elucidation of the mglur6 signaling cascade in the rod bipolar dendrites . one other area of future investigation will be the identification and characterization of the retinal pathways that carry rod signals to ganglion cells . while the rod bipolar pathway may function predominantly near absolute threshold , it saturates at light levels where the rods are not saturated . under these conditions , these include the rod - cone pathway and the rod off pathway , which are believed to relay signals for mesopic vision . the threshold and dynamic range of each pathway for relaying rod signals to ganglion cells remain to be determined .

the performance of sensory systems in many cases is limited by the physical nature of the stimulus . for vision , the quantal nature of light limits detection by dark - adapted observers ; only now are we beginning to be aware of the subtleties in the biophysical mechanisms underlying this exquisite sensitivity .

Introduction and context Major recent advances Future directions Competing interests

gait disturbance is one of the most disabling sequelae of stroke1 , 2 , with a reported prevalence of 5480%3 , 4 . gait disturbance often leads to loss of independence , which has profound effects on both the patients and the caregivers and results in higher health - care costs4,5,6,7 . several traditional gait rehabilitation techniques have been developed to improve gait disturbance in stroke patients , including neurophysiological techniques ( e.g. , the bobath , brunnstrm , proprioceptive neuromuscular facilitation , vojta , and rood methods)8 and motor learning techniques ( e.g. , the perfetti , carr and shepherd , conductive education , and sensory integration methods)9 . however , these traditional methods are very physically intensive for the therapists , making it difficult for these practitioners to continuously provide gait training for stroke patients10 . robotic technology has progressed rapidly in recent years , and several gait - training robots have now been developed , including the lokomat ( hocoma inc . , volketswil , switzerland ) and g - eo ( rha technologies , olten , switzerland ) . these devices use a powered exoskeleton with a treadmill ( lokomat ) or powered footplates with programmable trajectories , or are analogous to a robotic elliptical trainer ( g - eo)11 . these devices have alleviated the physical workload for the therapists and have thus gained popularity . several studies have also suggested that gait - training robots can improve locomotor recovery in stroke patients12 . to aid in the further development and refinement of gait - training robots , it is important to survey the views of the physiatrists and physical therapists who might use these machines , in terms of merit , indication , type , price , treatment duration , and design . in our current study , these parameters were evaluated by means of a questionnaire survey . a questionnaire was developed to collect the opinions of physiatrists and physical therapists on robot - assisted gait - training therapy . the questionnaire was divided into six sections : merit of robot - assisted gait training , indications for gait - training robots , robot type , treatment duration , price , and design of gait - training robots . on the basis of these six categories , most questions were either closed ended ( multiple choice ) , or based on a 10-point likert scale that gauges the level of agreement with or feelings on the importance of an item . a total of 100 physiatrists and 100 physical therapists from 38 hospitals participated in the survey from november 2013 to may 2014 . a self - written survey method was used , either face - to - face or by mail . a software was used to randomly enroll participants from the registry of physiatrists and physical therapists at the korean academy of rehabilitation medicine . this study was exempted from institutional review board ( irb ) review requirements based on the guidelines of asan medical center ( irb no . the spss version 18.0.0 software package was used for statistical analyses , and p - values < 0.05 were considered to indicate statistically significant differences . questions such as perspectives on the use of gait - training robots for stroke patients according to rehabilitation experience parameters were analyzed by using pearson s chi - square test and anova . a total of 106 physiatrists and 107 physical therapists were invited to complete our study survey . six physiatrists and seven physical therapists could not complete the survey for various reasons , resulting in a 94% participation rate . for 62 ( 31% ) participants , the remaining participants ( 69% ) completed the survey by mail . concerning the experience of the study participants in providing rehabilitation to stroke survivors , 33% had worked with stroke survivors for > 5 years , whereas 67% of these practitioners had worked with such patients for 15 years . sixty - seven ( 33.5% ) participants had a previous experience with using a rehabilitation robot . the survey questions asked about the perception of the treatment effects of gait - training robots by using a 10-point likert scale for effectiveness . participants with previous experience with a gait - training robot selected a higher score in this regard . the average score of this experienced group was 7.63 , and that of the nonexperienced group was 6.93 ( on a 10-point likert scale , p < 0.001 ; table 1table 1.perception of the treatment effects of a gait - training robotexperiencednonexperiencedtreatment effect7.636.93values are on a 10-point likert scale . a more detailed description of these statistics is provided in table 2table 2.baseline characteristics of the study participantsn ( % ) gendermale137 ( 68.5)female63 ( 31.5)occupationphysiatrist100 ( 50)physical therapist100 ( 50)careermore than 5 years56 ( 28)less than 5 years144 ( 72)experienced in gait rehabilitation with a gait - training robotexperienced67 ( 33.5)nonexperienced133 ( 66.5)institution of employmenttertiary hospital63 ( 31.5)general hospital40 ( 20)rehabilitation hospital72 ( 36)care hospital25 ( 12.5 ) . p < 0.05 by using fisher s exact test the survey included questions on the merit of robot - assisted gait training for stroke patients . ( 17% ) , and elevation of a patient s self - esteem the survey also queried the appropriate period to use a gait - training robot . at 13 months after stroke onset ( 47.3% ) was the most frequently selected answer , followed by 36 months after stroke onset ( 27.4% ) , within 1 month after stroke onset ( 14.4% ) , 612 months after stroke onset ( 8% ) , and 12 months after stroke onset ( 3% ) . the survey participants were also asked about the appropriate locomotor status of a patient to warrant the application of a gait - training robot . patient locomotor status was rated on the basis of a functional ambulation classification ( fac ) score from 0 to 5 . the most selected response for the optimal patient locomotor status was fac 1 ( 27% ) , followed by fac 2 ( 24% ) , fac 0 ( 23% ) , fac 3 ( 11% ) , fac 4 ( 8% ) , and ( 2% ) . the extent of agreement with the application of a gait - training robot at each locomotor status by using a 10-point likert scale was also surveyed . the average scores for each locomotor status classification are reported in table 3table 3.average scores for each locomotor statusmeansdfac 06.342.37fac 16.891.94fac 26.252.10fac 34.932.41fac fac : functional ambulation classification , and the mean scores for each locomotor status score showed an identical pattern . fac : functional ambulation classification the most useful type of gait - training robot for stroke patients was also surveyed . ( 40% ) , foot plate - based gait trainer ( 11.5% ) , and fixed - gait trainer the survey also asked about the appropriate treatment duration for robot - assisted gait therapy . the 3045 min period was selected most often ( 47% ) , followed by ( 18% ) , 60 min ( 5% ) , and when queried about the most suitable price for a gait - training robot for stroke patients , the survey participants most frequently selected from $ 50,000 to $ 100,000 ( 38.3% ) , followed by from $ 100,000 to $ 200,000 ( 37.8% ) , below $ 50,000 ( 14.4% ) , and from $ 200,000 to $ 500,000 ( 8.5% ) . most of the respondents selected stability ( 49% ) , followed by comfort ( 28% ) , cutting edge ( 13% ) , friendliness ( 9% ) , and other ( 1% ) . when asked about the most important consideration for the production of gait - training robots , fitting sense ( 23.9% ) , light weight ( 23.2% ) , miniaturization ( 10.2% ) , and aesthetics ( 3% ) . the most frequently selected response concerning the intention detector sensors for gait - training robots was emg ( electromyography ) ( 25% ) , followed by foot pressure ( 22% ) , joint torque when asked about the ideal graph type for training results , bar graph ( 42.3% ) was selected most often , followed by line graph ( 27.9% ) , radiant graph ( 21.9% ) , and pie graph ( 8.0% ) . in terms of the most preferred materials for direct contact areas , most respondents to the survey selected silicon ( 60% ) , followed by leather ( 21% ) , cloth ( 13% ) , wood ( 4% ) , and others ( 2% ) . in our present study , the views of physiatrists and physical therapists on the use of gait - training robots were surveyed , specifically their views on the merits of robot - assisted gait training ; the indications for using these devices ; the optimal treatment duration of robot - assisted gait training ; and the type , price , and design of gait - training robots . the largest group of participants selected treatment effect of gait training as the principal reason that would merit robot - assisted gait training , followed by standardized treatment . this finding is consistent with those of previous reports showing that robot - assisted gait training exhibited equivalent or better treatment effects compared with conventional physical therapy11 , 13 , 14 . in conventional gait training by physical therapists , the protocols and intensities of by contrast , robot - assisted gait training can provide reproducible symmetrical gait kinematic patterns of leg movements15 , and thus enable standardized treatment to be continuously provided to stroke patients16 . in the survey of experts , the most appropriate patient locomotor status for the application of gait - training robots was indicated to be an fac score ranging from 0 to 2 , which corresponds to patients who can not walk without assistance17 . conventional gait training thus imposes a significant physical burden on therapists when treating patients with fac scores of 0218 , and this approach could not therefore be readily applied on a large scale for such cases . importantly , this limitation could be overcome by the application of robot - assisted gait training19 . our current study survey indicated that the most appropriate period to apply gait - training robots in stroke patients is at 13 months after the disease onset . this result concurs with previous reports of a significant improvement in walking speed and functional outcomes when these devices are used during the acute post - stroke phase ( 2.514 weeks)20,21,22,23 . additionally , several clinical studies have reported that recovery from stroke mainly occurs during the first 3 months after its onset24,25,26 . treadmill gait - training robots were the most selected type in our survey , followed by overground walking - type robots . this finding was consistent with those of preliminary studies that suggested that the use of treadmill gait training leads to a better recovery of ambulation , with beneficial effects on overground walking speed and endurance , and a reduction in the physical assistance required to walk27,28,29 . conversely , fewer of our survey participants selected a foot plate based gait trainer . this result was not surprising , as foot plate - based gait trainers are considered to have several drawbacks compared with other types of gait - training robots . first , foot plate based gait trainers lack a true swing phase during the gait cycle30 . additionally , they can not control spasticity of the lower extremities11 and would be difficult to use for patients with a lower locomotor status ( fac 02 ) . the appropriate duration of a robot - assisted gait therapy session was considered to be 3045 min by most of our survey respondents . a previous study has suggested that > 30 min of gait training is effective in improving the locomotor function of stroke patients31 . moreover , > 30 min of aerobic exercise is required for an improved cardiovascular function32 . however , a prolonged treatment duration can cause patients to experience physical fatigue and to lose interest in continuing the therapy33 , 34 . concerning the suitable price for a gait - training robot , the most commonly selected response was the $ 50,000$100,000 range , followed by $ 100,000$200,000 . the most important requirements to consider in the design of a gait - training robot were considered by our survey participants to be ( in order of importance ) : stability , comfort , cutting - edge features , and friendliness . bar graphs were the most preferred graphical representations of real - time training states and training results , perhaps because these data are easier to comprehend . an emg sensor was considered to be the most appropriate type for the detection of intended gait initiation . from these responses , we contend that gait - training robot producers should consider the results of our survey when designing and then manufacturing gait - training robots . to the best of our knowledge , only one previous study has investigated the views of therapists on the optimal practices and design requirements for the development of an upper - limb stroke rehabilitation robot35 . however , no study to date has surveyed the views of practitioners on their use of gait - training robots . our current study is the first to investigate the actual user demands for gait - training robots , and to evaluate the perspectives on the use of these devices to help in the rehabilitation of stroke patients . a notable limitation of our current investigation was that only one - third of the participants had experience with robot - assisted gait training .

[ purpose ] gait - training robots have been developed for stroke patients with gait disturbance . it is important to survey the views of physiatrists and physical therapists on the characteristics of these devices during their development . [ subjects and methods ] a total of 100 physiatrists and 100 physical therapists from 38 hospitals participated in our questionnaire survey . [ results ] the most common answers about the merits of gait - training robots concern improving the treatment effects ( 28.5% ) , followed by standardizing treatment ( 19% ) , motivating patients about treatment ( 17% ) , and improving patients self - esteem ( 14% ) . the subacute period ( 13 months post - stroke onset ) was most often chosen as the ideal period ( 47.3% ) for the use of these devices , and a functional ambulation classification of 02 was the most selected response for the optimal patient status ( 27% ) . the preferred model was the treadmill type ( 47.5% ) over the overground walking type ( 40% ) . the most favored commercial price was $ 50,000$100,000 ( 38.3% ) . the most selected optimal duration for robot - assisted gait therapy was 3045 min ( 47% ) , followed by 1530 min ( 29% ) , 4560 min ( 18% ) , 60 min ( 5% ) , and < 15 min ( 1% ) . [ conclusion ] our study findings could guide the future designs of more effective gait - training robots for stroke patients .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

in this pilot , multicenter , prospective , open - label , randomized study , we enrolled 90 adult patients admitted to general medicine and surgery services . recruited patients had a known history of t2d with a blood glucose ( bg ) prior to randomization of between 140 and 400 mg / dl and a known history of t2d for > 3 months , were between 18 and 80 years of age , and were treated at home with diet alone , any combination of oral antidiabetic agents , or low - dose insulin therapy at a daily dose 0.4 units / kg prior to admission . on admission , we stopped oral antidiabetic agents and insulin therapy , and bg was measured before meals and bedtime . we excluded patients with any bg between admission and randomization of > 400 mg / dl or with a prior history of hyperglycemic crises ; patients with hyperglycemia but without a known history of diabetes ; patients admitted to or expected to require icu admission or cardiac surgery ; patients with a history of pancreatitis or active gallbladder disease , corticosteroid therapy , clinically relevant hepatic disease , or impaired renal function ( glomerular filtration rate [ gfr ] < 30 ml / min or serum creatinine 3.0 mg / dl ) ; and patients with a history of diabetic ketoacidosis ( 24 ) , pregnancy , or any mental condition rendering the subject unable to give informed consent . patients were randomized according to a 1:1:1 ratio into three regimens : sitagliptin once daily , sitagliptin and basal insulin ( glargine lantus ; sanofi ) once daily , and basal bolus insulin with glargine once daily and lispro before meals ( humalog ; eli lilly and company ) . patients treated with sitagliptin received a single dose of 100 mg / day ( at any time of day ) if gfr > 50 ml / min or 50 mg / day if gfr was between 30 and 50 ml / min . patients in the sitagliptin and basal group received a starting total daily dose ( tdd ) of glargine of 0.25 units / kg / day , except for those patients 70 years of age and/or with a serum creatinine 2.0 mg / dl who received a starting tdd of 0.15 units / kg . patients in the basal bolus group were started at a tdd of 0.5 units / kg divided half as insulin glargine once daily and half as insulin lispro before meals . in patients 70 years of age and/or with a serum creatinine 2.0 mg / dl , the starting tdd in the basal bolus group was reduced to 0.3 units / kg in the basal bolus group . patients in all three groups received supplemental ( correction ) doses of insulin lispro before meals and bedtime for bg > 140 mg / dl . the goal of therapy was to maintain a fasting and premeal glucose concentration between 100 and 140 mg / dl . the doses of insulin were adjusted daily according to protocol ( included in supplementary table 1 ) . treatment failure was arbitrarily defined as an average daily bg > 240 mg / dl or two consecutive values bg > 240 mg / dl ( 11,14 ) . if this occurred , patients in the sitagliptin and sitagliptin plus glargine groups were switched to basal bolus regimen starting at a tdd of 0.5 units / kg . bg was measured before each meal and at bedtime ( or every 6 h if a patient was not eating ) using a point - of - care glucose meter ( accu - check ; roche , indianapolis , in ) . in addition , bg was measured at any time if a patient experienced symptoms of hypoglycemia or if requested by the treating physician . the results of bg values are presented as premeal glucose , bedtime glucose , and mean daily bg during the hospital stay after day 1 . this study was conducted at grady memorial hospital ( atlanta , ga ) , emory university hospital , and university of michigan health system . the study protocol and consent form were approved by the institutional review board at each participating institution . a research pharmacist at each institution according to a computer - generated randomization table coordinated the randomization and treatment assignment . all patients were managed for medical and surgical problem(s ) by their primary care team who received a copy of the assigned treatment protocol . the primary outcome of the study was to determine differences in glycemic control as measured by mean daily bg concentration among treatment groups . secondary outcomes included differences between treatment groups in any of the following measures : number of bg values within range , number of hypoglycemic events ( bg < 70 and < 40 mg / dl ) , number of episodes of hyperglycemia ( bg > 200 mg / dl ) after the first day of treatment , ttd of insulin , length of hospital stay , hospital complications , and differences in glycemic control between medicine and surgery patients . this was a noninferiority study design based on the hypothesis that the difference in mean daily bg between basal plus sitagliptin and basal bolus regimens would be no greater than 18 mg / dl ( 1 mmol / l ) ( 11,14 ) . we compared baseline and clinical characteristics and outcomes , such as mean daily bg after day 1 , occurrence of hypoglycemia , and occurrence of complications , among treatment groups and between medical and surgical patients . the comparisons were made with the use of one - way anova for continuous variables and tests ( or fisher exact test ) for discrete variables . the data were generally presented as mean sd for continuous variables and count ( percentage ) for discrete variables . the primary outcome of the study was to determine differences in glycemic control as measured by mean daily bg concentration among treatment groups . secondary outcomes included differences between treatment groups in any of the following measures : number of bg values within range , number of hypoglycemic events ( bg < 70 and < 40 mg / dl ) , number of episodes of hyperglycemia ( bg > 200 mg / dl ) after the first day of treatment , ttd of insulin , length of hospital stay , hospital complications , and differences in glycemic control between medicine and surgery patients . this was a noninferiority study design based on the hypothesis that the difference in mean daily bg between basal plus sitagliptin and basal bolus regimens would be no greater than 18 mg / dl ( 1 mmol / l ) ( 11,14 ) . we compared baseline and clinical characteristics and outcomes , such as mean daily bg after day 1 , occurrence of hypoglycemia , and occurrence of complications , among treatment groups and between medical and surgical patients . the comparisons were made with the use of one - way anova for continuous variables and tests ( or fisher exact test ) for discrete variables . the data were generally presented as mean sd for continuous variables and count ( percentage ) for discrete variables . a total of 90 patients with t2d were consented ( 55 medicine and 35 surgery ) ; 8 patients were excluded from further analysis because they received < 24 h of insulin treatment , were transferred to the icu , or received corticosteroid therapy . a total of 27 patients in the sitagliptin alone group , 29 patients in the sitagliptin and glargine group , and 26 in the basal bolus group were included in the final analysis . there were no significant differences in the mean age , racial distribution , bmi , duration of diabetes , type of treatment prior to admission , or mean hospital length of stay ( los ) among groups . the most common admitting diagnoses in medicine patients were cardiovascular ( 14% ) , infectious ( 28% ) , and pulmonary ( 22% ) disorders , whereas the most common types of surgery were orthopedic ( 28% ) , urologic ( 19% ) , thoracic ( 16% ) , and abdominal ( 9% ) procedures . clinical characteristics of study patients the admission bg , hba1c concentration , and changes in glycemic control during the hospital stay are shown in table 2 . the mean admission glucose for the entire cohort was 211.9 63 mg / dl and the mean hba1c was 8.2 2% . all treatment regimens resulted in prompt and similar improvement in mean daily bg concentration after the 1st day of therapy ( fig . 1 ) . the percentages of glucose readings within target range between 70 and 140 mg / dl were slightly higher in the sitagliptin and glargine ( 43% ) and basal bolus ( 43% ) regimens compared with sitagliptin ( 36% ) , but results were not statistically significant ( p = 0.53 ) ( table 2 ) . similarly , there were fewer bg readings > 200 mg / dl in the sitagliptin and glargine group compared with basal bolus and sitagliptin alone ( 13 , 21 , and 21% , respectively ) ; however , the difference was not statistically significant ( p = 0.23 ) . in addition , there were no differences in the number of treatment failures ( 8 vs. 11 vs. 10% , respectively , p > 0.99 ) . glycemic control , insulin therapy , and hypoglycemic events in patients treated with sitagliptin alone or in combination with basal insulin and basal bolus regimen differences in glycemic control in medicine and surgery patients with t2d treated with sitagliptin alone or in combination with basal insulin and basal bolus regimen . a : mean daily glucose levels in patients treated with sitagliptin alone or in combination with basal ( glargine ) insulin and basal bolus ( glargine + lispro ) insulin regimens . all groups received supplemental ( correction ) doses of lispro before meals and bedtime for bg > 140 mg / dl . b : mean bg levels before meals and bedtime during the hospital stay in patients treated with sitagliptin alone or in combination with basal insulin and basal bolus insulin regimens . the tdd of insulin ( units / day ) was higher in the basal bolus group ( 39.8 22 units / day ) than in the glargine plus sitagliptin ( 28.2 12 units / day ) and sitagliptin ( 11.5 7 units / day ) groups ( p < 0.001 ) . there were no differences in the total dose of basal insulin between basal bolus ( 17 9 units / day ) and sitagliptin and glargine ( 20 9 units / day ) groups , but patients in the basal bolus group received three times the amount of lispro ( 22.4 15 units / day ) before meals compared with the sitagliptin and glargine ( 7.9 6 units / day ) and sitagliptin ( 11.5 7 units / day ) groups ( p < 0.001 ) ( table 2 ) . most patients received insulin supplements for correction of hyperglycemia during treatment with basal bolus 96% , glargine and sitagliptin 93% , and sitagliptin 100% ( p = 0.65 ) . in addition , patients in the basal bolus group received a higher number of insulin injections per day ( 2.4 0.8 ) than patients in the sitagliptin and glargine and sitagliptin groups ( 1.8 0.9 and 1.8 1.1 , respectively , p < 0.01 ) ( table 2 ) . a bg < 70 mg / dl was reported in one patient in the sitagliptin group ( 4% ) , two patients ( 8% ) in the basal bolus group , and two patients ( 7% ) in the sitagliptin and glargine group ( p = 0.86 ) . there were no patients with severe hypoglycemia ( < 40 mg / dl ) . in all cases , hypoglycemia was corrected with oral dextrose , and none of these episodes were associated with adverse outcomes . the level of glucose at admission or at randomization was found to be a good predictor of glycemic control and treatment response during the hospital stay . compared with patients with glucose 180 mg / dl , those with a bg > 180 mg / dl had significantly higher mean daily glucose levels after the 1st day of therapy ( p < 0.001 ) . there were no differences in mean daily bg concentration or in the number of treatment failures among different treatment groups in patients with a randomization bg < 180 mg / dl ( supplementary fig . 2b ) ; however , patients with a randomization bg > 180 mg / dl treated with sitagliptin alone had higher mean daily bg ( 182.7 30 mg / dl ) compared with patients treated with basal bolus ( 168.1 31 mg / dl ) and sitagliptin plus glargine ( 161.8 31 mg / dl ) ( p = 0.08 ) . this pilot , multicenter , randomized clinical trial compared the efficacy and safety of a daily dose of sitagliptin alone or in combination with glargine insulin to a standard basal bolus regimen in general medicine and surgery patients with t2d . we observed similar improvements in glycemic control in all treatment groups with no differences in the mean daily bg , number of bg readings within target , number of treatment failures , hospital los , or number of hypoglycemic events . in addition , the total daily insulin dose and number of insulin injections were significantly less in the sitagliptin groups compared with the basal bolus regimen . the result of this preliminary study suggests that treatment with sitagliptin alone or in combination with basal insulin is safe and effective for the management of general medicine and surgery patients with t2d . the association between hyperglycemia and increased risk of hospital complications is well established in icu and non - icu patients ( 36,2527 ) . recent guidelines from professional organizations ( 1820 ) recommend the use of subcutaneous insulin as the preferred therapy for glycemic control in hospitalized patients in a non - icu setting . the two most common subcutaneous insulin regimens for inpatient glycemic management are sliding scale regular insulin ( ssi ) and basal bolus insulin therapy in combination with correction insulin scale . the use of basal bolus regimen is preferred as it improves glycemic control and reduces the rate of hospital complications ( 12,13 ) . the rabbit 2 medicine trial ( 11 ) reported that a bg target of < 140 mg / dl was achieved in two - thirds of patients treated with basal bolus regimen , whereas only one - third of those treated with ssi achieved target glycemia . the rabbit surgery trial also reported a higher percentage of glucose readings < 140 mg / dl with basal bolus compared with ssi treatment ( 53 30 vs. 31 28% ) ( 14 ) . in this study , we report that sitagliptin alone or in combination with basal ( glargine ) insulin resulted in similar improvements in glycemic control compared with basal bolus regimen . in agreement with recent reports , the level of glucose at admission or at randomization was found to be a good predictor of glycemic control and treatment response during the hospital stay ( 25 ) . compared with patients with glucose > 180 mg / dl , those with a bg 180 mg / dl had a lower mean daily glucose and less treatment failures , independent of treatment regimen . in patients with an admission or randomization bg 180 mg / dl , we observed no differences in mean daily bg concentration or in the number of treatment failures among patients treated with sitagliptin plus supplements compared with patients treated with sitagliptin and glargine or basal bolus regimens ( p = 0.63 ) . patients with a randomization bg > 180 mg / dl treated with sitagliptin alone had higher mean daily bg compared with sitagliptin and glargine or basal bolus regimens ( p = 0.08 ) . this observation indicates that sitagliptin plus rapid - acting supplements ( correction ) before meals is useful in patients with mild - to - moderate hyperglycemia , whereas treatment with sitagliptin plus basal insulin or basal bolus regimens should be considered in those with more severe hyperglycemia . as previously reported ( 11,14 ) , we show that the use of basal insulin as part of a basal bolus regimen or in combination with sitagliptin is well tolerated with a low rate of hypoglycemia . in the rabbit medicine trial , 3% of patients in the basal bolus group had a bg < 60 mg / dl and no patients had a value < 40 mg / dl ( 11 ) . in the rabbit surgery trial , 12% of patients treated with basal bolus had a bg < 60 mg / dl and 4% had a value < 40 mg / dl ( 14 ) . in the current study , a bg < 70 mg / dl was reported in 7% of patients treated with sitagliptin and glargine and in no patients treated with basal bolus or sitagliptin alone . minimizing hypoglycemic events is of major importance in hospitalized patients because it has been shown to be an independent risk factor of poor outcome ( 26,27 ) . we recruited a relatively small number of patients in this pilot study and excluded a large number of patients , which included those admitted to the icu , with clinically relevant hepatic disease , with pancreatitis , with serum creatinine 3.0 mg / dl or gfr < 30 ml / min , with severe hyperglycemia ( bg > 400 mg / dl ) , and receiving a total dose of insulin > 0.4 units / kg / day prior to admission . in such patients , a standard basal bolus approach may be the preferred approach in achieving glycemic control . in addition , our study was not powered to determine differences in hospital complications across the three groups . a large , prospective , randomized , multicenter trial of glycemic control comparing sitagliptin alone or in combination with basal insulin with the basal bolus approach is needed to address these important issues . in summary , these preliminary results indicate that the inpatient use of sitagliptin alone or in combination with basal insulin resulted in a similar improvement in glycemic control compared with a standard basal bolus insulin regimen . these results indicate that sitagliptin alone or in combination with basal insulin is an effective alternative to the basal bolus insulin regimen for general medicine and surgery patients with t2d .

objectivethis study investigated the safety and efficacy of sitagliptin ( januvia ) for the inpatient management of type 2 diabetes ( t2d ) in general medicine and surgery patients.research design and methodsin this pilot , multicenter , open - label , randomized study , patients ( n = 90 ) with a known history of t2d treated with diet , oral antidiabetic agents , or low total daily dose of insulin ( 0.4 units / kg / day ) were randomized to receive sitagliptin alone or in combination with glargine insulin ( glargine ) or to a basal bolus insulin regimen ( glargine and lispro ) plus supplemental ( correction ) doses of lispro . major study outcomes included differences in daily blood glucose ( bg ) , frequency of treatment failures ( defined as three or more consecutive bg > 240 mg / dl or a mean daily bg > 240 mg / dl ) , and hypoglycemia between groups.resultsglycemic control improved similarly in all treatment groups . there were no differences in the mean daily bg after the 1st day of treatment ( p = 0.23 ) , number of readings within a bg target of 70 and 140 mg / dl ( p = 0.53 ) , number of bg readings > 200 mg / dl ( p = 0.23 ) , and number of treatment failures ( p > 0.99 ) . the total daily insulin dose and number of insulin injections were significantly less in the sitagliptin groups compared with the basal bolus group ( both p < 0.001 ) . there were no differences in length of hospital stay ( p = 0.78 ) or in the number of hypoglycemic events between groups ( p = 0.86).conclusionsresults of this pilot indicate that treatment with sitagliptin alone or in combination with basal insulin is safe and effective for the management of hyperglycemia in general medicine and surgery patients with t2d .

RESEARCH DESIGN AND METHODS Outcome measures Statistical analysis RESULTS CONCLUSIONS Supplementary Material

increasing threat of communicable as well as non - communicable disease epidemics coupled with acute shortage of healthcare professionals is a real challenge in developing countries . the problem can be solved by a rapidly growing mobile communications as mobile phones penetrate deeper into developing countries than any other information and communication technology ( itc ) and health infrastructures.(1 ) in the current era of technological innovations- mobile phones , therefore , have occupied a special space due to their rapid spread from richest to the poorest in developing countries and faster penetration in low- and middle - income countries . the global observatory for ehealth ( goe ) by who ( 2011 ) has defined mobile - health or m - health as the provision of health services and information via mobile technologies such as mobile phones and personal digital assistants ( pdas).(2 ) mobile - health field is one such emerging area in use of mobile phones , which can not only improve the well - being of people in developing counties but also it can improve health outcomes in long run . mobile - health consequently has expanded in numbers and type of initiatives , and it is expected to become a multi - billion dollar industry by 2017 . mobile - health approaches are currently emerging as an upcoming promising tool in providing greater access to healthcare to populations in developing countries , as well as creating cost - efficiencies and improving the capacity of health systems to provide quality healthcare.(2 ) a multitude of m - health solutions have emerged recently in countries such as ethiopia , kenya , nigeria , and south africa i.e. leaders in using mobile - health services , as per the global observatory report for e - health by who ( 2011).(2 ) mobile phones can be an appropriate and promising tool for disease control interventions in developing countries , for diseases such as hiv / aids , and this approach has been found to be well accepted.(3 ) moreover , mobile phones as a research tool in public health can also be used for household surveys , clinical trials , surveillance , and spatial / geographical data collection.(4 ) with this problem in mind , authors have chosen this research area and systematically reviewed the capacity of mobile - health in improving public health system of developing countries in this paper briefly . the global observatory for ehealth ( goe ) by who ( 2011 ) has defined mobile - health or m - health as the provision of health services and information via mobile technologies such as mobile phones and personal digital assistants ( pdas).(2 ) mobile - health field is one such emerging area in use of mobile phones , which can not only improve the well - being of people in developing counties but also it can improve health outcomes in long run . mobile - health consequently has expanded in numbers and type of initiatives , and it is expected to become a multi - billion dollar industry by 2017 . mobile - health approaches are currently emerging as an upcoming promising tool in providing greater access to healthcare to populations in developing countries , as well as creating cost - efficiencies and improving the capacity of health systems to provide quality healthcare.(2 ) a multitude of m - health solutions have emerged recently in countries such as ethiopia , kenya , nigeria , and south africa i.e. leaders in using mobile - health services , as per the global observatory report for e - health by who ( 2011).(2 ) mobile phones can be an appropriate and promising tool for disease control interventions in developing countries , for diseases such as hiv / aids , and this approach has been found to be well accepted.(3 ) moreover , mobile phones as a research tool in public health can also be used for household surveys , clinical trials , surveillance , and spatial / geographical data collection.(4 ) with this problem in mind , authors have chosen this research area and systematically reviewed the capacity of mobile - health in improving public health system of developing countries in this paper briefly . a systematic review of studies in literature as per prisma ( 2009 ) guidelines published till 31 october 2013 of last 10 years on key search word : capacity of mobile - health in improving health system of developing countries was done from medical search engines abstracting databases such as pub - med , who , cochrane database , google scholar , and bio - med central . both types of studies elucidating utility and no benefit of mobile - health in developing countries were included as main criteria for deciding the capacity of mobile - health approach in health system of developing countries . m - health studies on areas of impact , effectiveness , and evaluation and previous reviews , conferences data , and exploratory studies were the main study designs incorporated . studies on m - health in developed world , indian studies as well data from thesis or dissertation were excluded . a systematic review of studies in literature as per prisma ( 2009 ) guidelines published till 31 october 2013 of last 10 years on key search word : capacity of mobile - health in improving health system of developing countries was done from medical search engines abstracting databases such as pub - med , who , cochrane database , google scholar , and bio - med central . out of 40 articles searched , 16 met the inclusion criteria . both types of studies elucidating utility and no benefit of mobile - health in developing countries were included as main criteria for deciding the capacity of mobile - health approach in health system of developing countries . m - health studies on areas of impact , effectiveness , and evaluation and previous reviews , conferences data , and exploratory studies were the main study designs incorporated . studies on m - health in developed world , indian studies as well data from thesis or dissertation were excluded . the result of the systematic review is summarized in table 1 , to show the qualitative synthesizing process of key issues and concepts emerged from systematic review of the literature as per prisma ( 2009 ) guidelines . to achieve the mdgs and to maintain better maternal and neonatal health beyond 2015 , there is an emerging need of searching new ways of using existing resources . expanding mobile phone penetration and network coverage is one such solution , which can remove traditional geographic and economic barriers to health care in developing world . combining mobile technologies with existing health system resources offers significant potential to provide women and newborns with adequate and appropriate care . the combined effect of communicable disease and chronic non - communicable disease is described as an epidemiological burden in developing countries , and efforts to reduce the dual burden of disease can improve quality of life for millions . ( 2010 ) reveal the fact that mobile technologies are widely available and can play an important role in health care at the regional , community , and individual levels.(5 ) the integrated nature of mobile communication systems provides unique opportunities for m - health in large geographic areas . at the community level , social networking can be used to exchange information about the local health system . and at the individual levels , mobile - health offers improved communication , access to diagnostic tools , and ability to store and access personal medical data in central repositories.(5 ) mobile technology also promises a wide range of functions through the use of intelligent handsets called smart - phones . the functions which smart - phones can play include remote diagnostic monitoring , data collection , information dissemination , and public education and alerts systems as revealed by m - health running projects in developing countries in studies found from literature as shown in table 2.(567 ) currently running m - health projects in developing countries(567 ) the mobile communication technologies are currently considered essential for improving the access and quality of health services , and overall health outcomes , by providing informational support and regular care as found by many authors in literature ( klasnja and pratt , 2012 ; noordam , kuepper , stekelenburg , and milen , 2011 ; tezcan , von rege , henkson , and oteng - ntim , 2011 ; kreps and neuhauser , 2010 ; krishna , boren , and balas , 2009).(8 ) in 2011 , a global survey of 114 nations done by the world health organization ( who ) has also found that m - health initiatives have been established in many countries , but there is variation in adoption levels , and the most common activity noted was the creation of health call centers , which respond to patient inquiries.(9 ) remote monitoring occupies two - thirds of the m - health market as doctors and patients use these devices to manage chronic illnesses . good example of efforts in this direction is china where qualcomm 's wireless reach in partnership with life care networks and the community health association of china had deployed an electrocardiogram sensing handset , which can record 30 seconds of heart data and transmits that information electronically to the 24-hour life care networks center in beijing.(10 ) 3 mobile for development ( m4d ) conference held in 2012 also reveal similar key issues and areas in m - health remote monitoring and mobile telemedicine approaches by few researchers such as haque et al . ; hoefman et al . and littman - quinn et al . on new areas , for example : sms and medical sensors , mobile oral telemedicine , mobile tele - radiology , mobile cervical cancer screening , mobile tele - dermatology , andmobile tele - mentoring.(5810 ) sms and medical sensors , mobile oral telemedicine , mobile tele - radiology , mobile cervical cancer screening , mobile tele - dermatology , and mobile tele - mentoring.(5810 ) m - health studies currently are revealing mixed results in terms of demonstrating the potential that fixed - line and mobile phones can serve as a support for more effective delivery of healthcare services ( kaplan , 2006).(11 ) with the ubiquity of mobile devices both in developing and developed countries , there have been innovations in awareness , prevention , diagnosis , and treatment . yang , and kahn ( 2010 ) have emphasized the steady growth of disease in developing countries and that using m - health to combat this growth shows promise.(12 ) the potential impact of m - health in health delivery in developing countries is largely untapped due to technical as well as socio - economic , cultural , and regulatory barriers ( kaplan , 2006).(11 ) kaplan ( 2006)(11 ) et al . from his study suggest that functional and structural properties of mobile phones like low start - up cost , text messaging , and flexible payment plans make them attractive to use as a healthcare intervention . with the development of standardized health - related software applications , mobile phones can provide real - time feedback and pre - programmed portable automated services that enable support to increasingly decentralized health systems as advocated also by lacal , ( 2003 ) and mechael ( 2006).(31314 ) most m - health studies are not guided by any conceptual framework , neither the research questions are instigated by existing theories , and research in this domain has not focused on the impact of the interventions on the various stakeholders . there are also a number of impending challenges such as cost , both at the macro systems level ( istepanian and lacal , 2003),(1013 ) as well as at the level of the individual citizen and healthcare provider ( mechael , 2006).(14 ) although most qualitative and quantitative results are generally consistent , and overall , there is enthusiasm for m - health technology , there are a number of potential inhibiting factors found also in study by chang et al . ( 2013 ) in their mixed methods formative research approach on m - health area in kampala ( uganda).(15 ) the primary barriers in m - health field identified currently are language , timing of messages , mobile network fluctuations , lack of financial incentives , data privacy , and mobile phone turnover affecting effective health care delivery through m - health approach despite their immense potential ( motamarri , s 2012).(16 ) the combined effect of communicable disease and chronic non - communicable disease is described as an epidemiological burden in developing countries , and efforts to reduce the dual burden of disease ( 2010 ) reveal the fact that mobile technologies are widely available and can play an important role in health care at the regional , community , and individual levels.(5 ) the integrated nature of mobile communication systems provides unique opportunities for m - health in large geographic areas . at the community level , social networking can be used to exchange information about the local health system . and at the individual levels , mobile - health offers improved communication , access to diagnostic tools , and ability to store and access personal medical data in central repositories.(5 ) mobile technology also promises a wide range of functions through the use of intelligent handsets called smart - phones . the functions which smart - phones can play include remote diagnostic monitoring , data collection , information dissemination , and public education and alerts systems as revealed by m - health running projects in developing countries in studies found from literature as shown in table 2.(567 ) currently running m - health projects in developing countries(567 ) the mobile communication technologies are currently considered essential for improving the access and quality of health services , and overall health outcomes , by providing informational support and regular care as found by many authors in literature ( klasnja and pratt , 2012 ; noordam , kuepper , stekelenburg , and milen , 2011 ; tezcan , von rege , henkson , and oteng - ntim , 2011 ; kreps and neuhauser , 2010 ; krishna , boren , and balas , 2009).(8 ) in 2011 , a global survey of 114 nations done by the world health organization ( who ) has also found that m - health initiatives have been established in many countries , but there is variation in adoption levels , and the most common activity noted was the creation of health call centers , which respond to patient inquiries.(9 ) remote monitoring occupies two - thirds of the m - health market as doctors and patients use these devices to manage chronic illnesses . good example of efforts in this direction is china where qualcomm 's wireless reach in partnership with life care networks and the community health association of china had deployed an electrocardiogram sensing handset , which can record 30 seconds of heart data and transmits that information electronically to the 24-hour life care networks center in beijing.(10 ) 3 mobile for development ( m4d ) conference held in 2012 also reveal similar key issues and areas in m - health remote monitoring and mobile telemedicine approaches by few researchers such as haque et al . ; hoefman et al . and littman - quinn et al . on new areas , for example : sms and medical sensors , mobile oral telemedicine , mobile tele - radiology , mobile cervical cancer screening , mobile tele - dermatology , andmobile tele - mentoring.(5810 ) sms and medical sensors , mobile oral telemedicine , mobile tele - radiology , mobile cervical cancer screening , mobile tele - dermatology , and mobile tele - mentoring.(5810 ) m - health studies currently are revealing mixed results in terms of demonstrating the potential that fixed - line and mobile phones can serve as a support for more effective delivery of healthcare services ( kaplan , 2006).(11 ) with the ubiquity of mobile devices both in developing and developed countries , there have been innovations in awareness , prevention , diagnosis , and treatment . yang , and kahn ( 2010 ) have emphasized the steady growth of disease in developing countries and that using m - health to combat this growth shows promise.(12 ) the potential impact of m - health in health delivery in developing countries is largely untapped due to technical as well as socio - economic , cultural , and regulatory barriers ( kaplan , 2006).(11 ) kaplan ( 2006)(11 ) et al . from his study suggest that functional and structural properties of mobile phones like low start - up cost , text messaging , and flexible payment plans make them attractive to use as a healthcare intervention . with the development of standardized health - related software applications , mobile phones can provide real - time feedback and pre - programmed portable automated services that enable support to increasingly decentralized health systems as advocated also by lacal , ( 2003 ) and mechael ( 2006).(31314 ) most m - health studies are not guided by any conceptual framework , neither the research questions are instigated by existing theories , and research in this domain has not focused on the impact of the interventions on the various stakeholders . there are also a number of impending challenges such as cost , both at the macro systems level ( istepanian and lacal , 2003),(1013 ) as well as at the level of the individual citizen and healthcare provider ( mechael , 2006).(14 ) although most qualitative and quantitative results are generally consistent , and overall , there is enthusiasm for m - health technology , there are a number of potential inhibiting factors found also in study by chang et al . ( 2013 ) in their mixed methods formative research approach on m - health area in kampala ( uganda).(15 ) the primary barriers in m - health field identified currently are language , timing of messages , mobile network fluctuations , lack of financial incentives , data privacy , and mobile phone turnover affecting effective health care delivery through m - health approach despite their immense potential ( motamarri , s 2012).(16 ) mobile technology - supported health care undoubtedly has many potentials in improving health outcomes in the long run in developing world provided m - health technological solutions are designed according to local realities and local needs . there is also an emerging need to evaluate the benefits and pitfalls of this technology application urgently ; therefore , further research in this field is suggested by authors such as more in - depth qualitative as well as quantitative studies in future , if we need to use this technology for better public health status in developing world . m - health : mobile healthwho : world health organizationhiv / aids : human immunodeficiency virus / acquired immuno deficiency syndromemdgs : millennium development goalsprisma : preferred reporting items for systematic review and meta - analysis m - health : mobile health who : world health organization hiv / aids : human immunodeficiency virus / acquired immuno deficiency syndrome mdgs : millennium development goals prisma : preferred reporting items for systematic review and meta - analysis

background : the mobile - health approach is currently knocking the doors of public health to make use of this rapidly advancing technology in developing countries ; therefore , it needs a critical look on its capacity in improving health system of developing countries.materials and methods : a systematic review of studies in literature published till 31st october 2013 of last 10 years on key search word : capacity of mobile - health in improving health system of developing countries was done from medical search engines abstracting databases such as pub - med , who , cochrane database , google scholar , and bio - med central . both types of studies elucidating utility and no benefit of mobile - health in developing countries were included as main criteria for deciding the capacity of mobile - health approach in health system of developing countries . m - health studies on areas of impact , effectiveness , and evaluation and previous reviews , conferences data , and exploratory studies were the main study designs incorporated . studies on m - health in developed world , indian studies as well data from thesis or dissertation were excluded in this review.discussion:multi-faceted mobile - health applications , strategies , and approaches currently lack proper regulation and standardization from health care authorities , and currently their results also vary from good to no beneficial effects as found in this review.conclusion:umbrella of mobile - health approaches must be used intelligently , keeping in mind the fact that , it can provide a greater access and quality health care to larger segments of a rural population and its potential to improve the capacity of health system in developing countries .

Background Emergence and growth of mobile-health technologies in developing world Materials and Methods Search strategy Inclusion criteria Exclusion criteria Results Discussion Emergence and growth of m-health projects, applications, strategies, and approaches at different levels Expansion of m-Health activity in developing world Remote monitoring in chronic diseases via m-Health approach Current m-Health activities and its future role in developing world Promises from m-Health approach Pitfalls in adopting m-Health approach Conclusion List of abbreviations used

protein deacetylases counter the action of protein acetyl transferases ( verdin and ott , 2015 ) by removing acetyl groups added to lysine residues by the latter . this large family of enzymes is often referred to as histone deacetylases ( hdacs ) , but histones are certainly not their only substrates . in mammals , hdacs are broadly divided into four classes ( i iv ) ( yang and seto , 2008 ) . class i hdacs ( hdacs 1 , 2 , 3 and 8) are homologous with the yeast reduced potassium deficiency 3 ( rpd3 ) , are ubiquitously expressed , and are primarily localized to the nucleus . class ii hdacs ( namely hdacs 4 , 5 , 6 , 7 , 9 and 10 ) , which are homologues of yeast histone deacetylase 1 ( hda1 ) , shuttle between the nucleus and the cytoplasm , and are more tissue specific in terms of expression . hdac11 has only fairly low homology to both rpd3 and hda1 , and has been proposed to constitute a separate class iv ( gregoretti et al . , 2004 ) sirtuins are a family of proteins with homologies to the silence information regulator 2 ( sir2 ) gene of the yeast s. cerevisiae ( brachmann et al . , 1995 ) , and are not structurally homologous to the other hdacs . lysine deacetylation by sirtuins is coupled to the cleavage of a key intermediate of cellular energy metabolism , nicotinamide adenine dinucleotide ( nad ) , into nicotinamide and 1-o - acetyl - adp - ribose ( tanner et al . , 2000 ) or 2- and 3-o - acetyl - adp - ribose ( jackson and denu , 2002 ) . the activities of sirtuins are thus obligatorily dependent on cellular nad , inhibited by the reaction product nicotinamide , and effectively influenced by cellular metabolic and redox states . homologues of yeast sir2 exist in lower model organisms , such as the nematode c. elegans sir-2.1 and the fruit fly d. melanogaster sir2 . the mammalian genome has seven sirtuin paralogues ( sirt1 - 7 ) , with sirt1 having the highest homology to yeast sir2 ( haigis and guarente , 2006 ; haigis and sinclair , 2010 ) . in yeast , sir2p regulates chromatin silencing , and reduces the accumulation of rdna circles during yeast replicative aging ( sinclair and guarente , 1997 ) . sir2 and its metazoan orthologues have been prominently associated with lifespan extension ( howitz et al . , 2003 ; tissenbaum and guarente , 2001 ) , in particular that induced by caloric or dietary restriction ( cr / dr ) ( rogina and helfand , 2004 ; wood et al . , 2004 ) . a large number of studies have connected sirt1 expression and/or activation by compounds such as resveratrol , with increase health span and lifespan ( guarente , 2011 ) . however , the notion that sirt1 is a mediator of cr effects and a longevity factor in mammals and humans has been controversial in two major ways . there were doubts about the specificity and effectiveness of natural and synthetic sirt1 activators such as resveratrol and srt1720 ( kaeberlein et al . sirt1 , at least in certain contexts ( longo , 2009 ; tang , 2006 ) , may also promote aging . sirt1 s role in longevity has been more extensively investigated in yeast and lower invertebrate models , and although the notion of sirt1 as a longevity gene has a great deal of support , there were also controversies and evidence to the contrary ( burnett et al . , 2011 ) . sirt1 s knockout phenotype was difficult to interpret , as the mice often died early due largely to developmental defects ( cheng et al . , 2003 ; mcburney et al . , 2003 earlier work on transgenic over - expression of sirt1 in mice did not result in a significant increase in lifespan ( herranz et al . , 2010 ) . more recent findings have , however , provided some reconciliation of the contrasting results and opinions . the mode of action of sirtuin - activating compounds ( stacs ) , possibly via an allosteric mechanism , has been better clarified ( hubbard et al . , 2013 ) . additionally , both the transgenic expression of sirt1 ( satoh et al . , 2013 ) and the specific activator srt1720 ( mitchell et al . , 2014 ) have eventually been shown to increase lifespan in mice . indeed , dissection of sirt1 s role in cr and aging is difficult because sirt1 has multiple physiological functions as well as wide - ranging roles in pathological settings . this is due to its astonishingly large repertoire of targets , many of which are key transcription factors associated with important aspects of cell survival and metabolism . prominent amongst these are the tumor suppressor tp53 ( vaziri et al . , 2001 ) , the forkhead box class o ( foxo ) family members ( brunet et al . , 2004 ) , and nuclear factor b ( nf-b ) ( yeung et al . , 2004 ) , all key regulators of cell death and survival . sirt1 represses a key metabolic regulator , peroxisome proliferator - activated receptor gamma ( ppar ) ( picard et al . , 2004 ; sugden et al . , 2010 ) , via deacetylation of ppar coactivator-1 ( pgc-1 ) ( gerhart - hines et al . , 2007 , 2005 ; rodgers et al . , 2005 ) , which regulates energy metabolism in skeletal muscle , adipose tissues and the liver . sirt1 has also been implicated in cellular metabolism through its activation of amp - dependent kinase ( ampk ) via liver kinase b1 ( lkb1 ) ( hou et al . , 2008 ; lan et al . , 2008 ; zu et al . , 2010 ) , as well as suppression of the mammalian target of rapamycin ( mtor ) pathway ( ghosh et al . , 2010 ; hong et al . of the seven mammalian sirtuins , three ( sirt3 , sirt4 and sirt5 ) have a primary mitochondrial localization ( michishita et al . , 2005 ) . sirt1 , on the other hand , is primarily nuclear , but its activities have large bearings on mitochondrial biogenesis and turnover . in the ensuing paragraphs we first look at some evidence for the predominantly nuclear sirt1 being physically localized at the mitochondria . sirt1 is primarily found in the nucleus of most cell types ( michishita et al . , 2005 ) , and it is not just a bona fide histone deacetylase ( vaquero et al . , 2006 ) but also deacetylates histone modifying enzymes such as the histone methyl - transferase suv39h1 ( vaquero et al . , 2007 ) . both sirt1 and suv39h1 are required for the epigenetic control of the rdna locus for rrna synthesis ( murayama et al . , 2008 ) , and sirt1 befitting its nuclear localization , a majority of sirt1 s known function is associated with deacetylation of transcription factors , as mentioned in the section above . as per all nuclear proteins sirt1 , however , appears to possess both nuclear localization signals and nuclear export signals , and could shuttle between the cytoplasm and the nucleus ( tanno et al . , 2007 ) . sirt1 s cytoplasmic appearance has been associated with enhanced apoptotic cell death ( jin et al . , 2007 ) , and also shown to be due to an increased protein stability via oncogenic signaling through the insulin - like growth factor-1-phosphoinositide 3-kinase ( igf-1/pi3 kinase ) axis in cancer cells ( byles et al . , 2010 ) . on the other hand , sirt1 does have cytosolic substrates whose deacetylation by sirt1 results in distinct physiological or functional consequences . for instance , the cytoplasmic acetyl - coa synthetase ( hallows et al . , 2006 ) and the actin binding protein cortactin ( zhang et al . , 2009 ) are both deacetylated by sirt1 , and these influence cytosolic fatty acid synthesis and cytoskeletal changes during cell migration , respectively . sirt1 and its substrate , pgc-1 , regulate aspects of energy metabolism through the mitochondria , but this was initially thought to occur through their influence on nuclear localized transcription . a rather interesting aspect of sirt1 s activity and function that is less well defined is its extranuclear localization , particularly at the mitochondria ( aquilano et al . , 2012 ) . this was first shown by aquilano and colleagues using both confocal imaging and subcellular fractionation analysis ( aquilano et al . , 2010 ) . both sirt1 and the nuclear transcription factor pgc-1 could be found in the mitochondria of human cell lines and platelets , as well as in various mouse organs . within the mitochondria , both deacetylase and its substrate are associated with the mitochondrial dna ( mtdna ) nucleoids ( bogenhagen , 2012 ) , as well as with the mitochondrial transcription factor a ( tfam ) , a key mitochondrial gene transcription factor and regulator of mtdna copy number ( campbell et al . , 2012 ) . these findings are fascinating , as they suggest that sirt1 and pgc-1 may also directly affect mitochondrial transcription . however , the degree of mitochondrial sirt1 and pgc-1 s functionality in this regard is still uncertain . in the sections that follow , we shall outline findings that point to how sirt1 mediates aspects of mitochondrial biogenesis and turnover , as well as the controversies associated with these findings . mitochondria are dynamic organelles , and inheritance occurs by partitioning existing mitochondria between daughter cells in dividing cells ( mishra and chan , 2014 ) . however , within all cells , either dividing or terminally differentiated , the mitochondria population is constantly renewed , and the steady state number of this organelle represents an equilibrium between mitochondrial biogenesis and eventual degradation through the process of mitophagy ( stotland and gottlieb , 2015 ; vega et al . , 2015 ) . mitochondrial biogenesis is orchestrated by the peroxisome proliferator - activated receptor gamma coactivator-1 ( pgc-1 ) family of transcriptional coactivators ( austin and st - pierre , 2012 ) . the pgc-1 family consists of three members , namely , pgc-1 , pgc-1 and the pgc related coactivator ( prc ) . pgc-1 , in particular , is often cited as a master regulator of this process . pgc-1 co - activates the transcription of nuclear respiratory factor ( nrf ) 1 and 2 , which , in turn , regulate the transcription of tfam . tfam translocates to mitochondrial matrix where it stimulates mitochondrial dna replication and mitochondrial gene expression . pgc-1 undergoes several modes of post - translational modification , which includes acetylation and phosphorylation . acetylation of pgc-1 occurs at several of its lysine residues and is catalyzed by the ubiquitous histone acetyl transferase general control of amino acid synthesis 5 ( gcn5 ) ( dominy et al . conversely , deacetylation of pgc-1 was shown by several studies to be dependent on sirt1 activity ( amat et al . , 2009 ; dominy et al . , 2010 ; gerhart - hines et al . , 2007 ; gurd , 2011 ; , 2006 ; nemoto et al . , 2005 ; olmos et al . , 2013 ; rodgers et al . , 2005 ) , which increases pgc-1 s transcriptional activity . pgc-1 is phosphorylated by both mitogen - activated protein kinase p38 ( barger et al . , 2001 ) , and the amp - dependent kinase ( ampk ) in skeletal muscle ( leick et al . , 2010 ) , which also stabilizes the protein and increases its activity . on the other hand , phosphorylation of pgc-1 by akt / protein kinase b ( pkb ) , downstream of insulin signaling in the liver , is known to decrease its stability and transcriptional activity ( li et al . , 2007 ) . given the importance of its acetylation status to pgc-1 activity , the connection between sirt1 and pgc-1 has been a focus of investigations into metabolic regulation and mitochondrial biogenesis . finkel s group has provided clear biochemical evidence that sirt1 physically and functionally interacts with pgc-1 ( nemoto et al . , 2005 ) , and this interaction could be distinguished by a single amino acid mutation in the putative adp - ribosyltransferase domain of sirt1 . this mutation abolishes sirt1 s interaction with pgc-1 , but not with two other major substrates , p53 and foxo3a . in what contexts a rather simplified view in this regard is that sirt1 acts a metabolic and redox sensor of changes in nutrient and energy status , such as those occurring during cr ( cant and auwerx , 2009 ) . cr has been shown to significantly increase mitochondrial biogenesis in multiple tissues in mice ( nisoli et al . , 2005 ) and early work from puigserver s group showed that sirt1 is induced during fasting , and induced sirt1 interacts with pgc-1 to increase the expression of hepatic gluconeogenic genes ( rodgers et al . , 2005 ) . the group also showed that fasting induced pgc-1 deacetylation by sirt1 in skeletal muscle , which is required for the activation of mitochondrial fatty acid oxidation genes ( gerhart - hines et al . , 2007 ) . auwerx s group , on the other hand , showed that resveratrol treatment of mice increased running time and oxygen consumption by the skeletal muscles , which is accompanied by the induction of genes for oxidative phosphorylation and mitochondrial biogenesis ( lagouge et al . , 2006 ) . the authors attributed these changes to activation of sirt1 , and a consequential decrease in pgc-1 acetylation and activity . the authors also showed that resveratrol treatment protected mice against diet - induced - obesity and insulin resistance . a more specific sirt1 activator , srt1720 , likewise enhanced muscle endurance and protected mice from diet - induced obesity and insulin resistance by enhancing oxidative metabolism in skeletal muscle , liver , and brown adipose tissue ( feige et al . , 2008 ) . auwerx and colleagues also found that deletion of the poly(adp - ribose ) polymerase-1(parp-1 ) , a major nad - consuming enzyme , resulted in increased sirt1 activity . parp-1 knockout mice have a higher mitochondrial content , an increase in energy expenditure , and were protected against metabolic disease ( bai et al . , 2011 ) . the work of anderson and colleagues ( anderson et al . , 2008 ) provided dynamical insights as to how sirt1 may regulate pgc-1 action . the latter s nuclear transcription activity is enhanced by sirt1-dependent nuclear accumulation and is counter regulated by glycogen synthase kinase beta ( gsk3 ) , which targets pgc-1 for intranuclear proteasomal degradation . despite the apparently convincing functional connection between sirt1 and pgc-1 , whether sirt1 is critically or directly involved in mitochondrial biogenesis has been controversial . gurd and colleagues ( gurd et al . , 2009 ) examined the relationship between sirt1 levels and activity , pgc-1 and markers of mitochondrial density ( such as cytochrome c oxidase subunit iv , coxiv ) in skeletal and heart muscles , and surprisingly found a largely negative correlation between sirt1 levels and the rest of the parameters . in fact , overexpression of sirt1 in muscle downregulated pgc-1 and mttfa levels , and while muscle stimulation and the ampk activator aicar upregulated pgc-1 and cox iv , sirt1 level was downregulated . the group further showed that while nuclear sirt1 activity correlated with indices of mitochondrial density , nuclear sirt1 protein levels were negatively correlated in skeletal and heart muscles ( gurd et al . , 2011 ) . while training did not alter muscle or nuclear sirt1 protein content in human muscle , it did increase muscle and nuclear pgc-1 and sirt1 activity . thus , it is an increased nuclear sirt1 activity that may actually contribute to exercise - stimulated mitochondrial biogenesis in muscle . from a more fundamental perspective , hancock and colleagues ( hancock et al . , 2011 ) have reexamined the claim that cr resulted in dramatic increases in mitochondrial biogenesis in multiple mouse tissues ( nisoli et al . , 2005 ) . in contrast to previous work , the authors found no elevation in pgc-1 levels after cr , and no evidence of any significant elevation in mitochondrial biogenesis markers in heart , brain , skeletal muscle , liver and adipose tissues . although the authors did not report any measurement of sirt1 level / activity status or pgc-1 s acetylation status , the study does challenge the reproducibility of the cr - induced mitochondrial phenomenon . another study directly questioned the role of sirt1 in exercise - induced mitochondrial biogenesis through pgc-1 decetylation ( philp et al . , 2011 ) . floxed and crossed with cre driven by muscle creatine kinase promoter ) , and found that skeletal muscle endurance , mitochondrial oxidative metabolism , and mitochondrial biogenesis were all not impaired . moreover , mice deficient in muscle sirt1 are similar to wild - type mice with regard to pgc-1 expression , nuclear translocation , activity , and deacetylation status . the authors further suggest that it is really the changes in gcn5 acetyltransferase activity that are important in regulating pgc-1 activity after exercise , and sirt1 may not be relevant . another recent study added to the controversy by showing that resveratrol feeding had no effect on mitochondrial proteins in muscle ( higashida et al . , 2013 ) , while high levels of resveratrol activated ampk in c2c12 myotubes in culture , which did increase mitochondrial protein levels . the group further showed that sirt1 silencing or a dominant negative mutant did increase pgc-1 acetylation , and over - expression of sirt1 reduced it . however , the latter actually suppressing , instead of enhanced pgc-1 s co - activator activity , with a consequential reduction in mitochondrial proteins . the discussion above highlights a particularly controversial point pertaining to the effect of resveratrol on pgc-1 and mitochondrial biogenesis , as well as the mode of involvement of the key cellular nutrient and energy status sensor ampk ( hardie , 2011 ) . using an inducible knockout model , sinclair s group has shown that intact sirt1 in mice is required for resveratrol s activity on mitochondrial biogenesis and function ( price et al . , 2012 ) . the authors also showed that with moderate levels of resveratrol , ampk activation in the system was sirt1-dependent . although higher levels of resveratrol could activate ampk in a sirt1-independent manner , improvement in mitochondrial function in both cases of moderate and high dosages of resveratrol was not observed in the absence of sirt1 . that sirt1 may act upstream of ampk has support in a previous report , which showed that sirt1 could potentially deacetylate and activate the major ampk activating kinase , serine / threonine kinase 11 ( stk11 ) ( or liver kinase b1 , lkb1 ) ( lan et al . , 2008 ) . resveratrol s pharmacological effect is multifaceted , and amongst its known effects is the direct activation of ampk ( dasgupta and milbrandt , 2007 ) and inhibition of camp - degrading phosphodiesterases ( park et al . , 2012 ) . the work of dasgupta and milbrandt indicates that resveratrol could directly activate ampk , at least in certain cell types , in a manner that is completely independent of sirt1 activity ( dasgupta and milbrandt , 2007 ) . another report has also shown that ampk deficient mice are fairly resistant to the metabolic improvement effect of resveratrol ( um et al . , 2010 ) . a role for ampk in mitochondrial biogenesis in skeletal muscle and other tissues kukidome et al . , 2006 ; reznick and shulman , 2006 ; reznick et al . , 2007 ; zong et al . , 2002 ) . in skeletal muscle , ampk has been shown to directly phosphorylate pgc-1 ( jger et al . , 2007 ) , and the phosphorylation of thr-177 and ser-538 is required for the pgc-1-dependent induction of its own promoter . it was also demonstrated that a single administration of the ampk activator aicar increased the mrna expression of all pgc-1 isoforms ( tadaishi et al . it is therefore clear that any compound or physiological regimes that elevate ampk levels may also influence pgc-1 levels / activity and consequent mitochondrial biogenesis , in a manner that may be largely independent of sirt1 . while some results , such as those of price et al . ( 2012 ) are in support of sirt1 acting upstream of ampk , others have demonstrated just the opposite . auwerx s group has shown that ampk could at least exert an influence on sirt1 activity by increasing cellular nad levels ( cant et al . , 2009 ; 2010 ) . the work of park et al . ( 2012 ) indicated that resveratrol inhibits camp phosphodiesterases , thus leading to elevated camp levels and the activation of ampk and camp - inducible factors such as the rap exchange factor directly activated by camp 1 ( epac1 ) . the authors showed that epac1 is required for resveratrol - mediated increases in nad levels , which were abolished by silencing of epac1 . what could be discerned from these remarkably different reports is that the connection between sirt1 , ampk and pgc-1 is nonlinear , is definitely cell / tissue - type dependent and is influenced by energy and nutrient status . given sirt1 s role in mitochondrial biogenesis discussed above , it might appear paradoxical that sirt1 has also been recently implicated in the opposite process , namely , the destruction of damaged or aged mitochondria via mitophagy ( yoshii and mizushima , 2015 ) . a detailed examination of mitophagy and its mechanism is beyond the scope of this review , and the reader is referred to several excellent recent review articles on this topic ( durcan and fon , 2015 ; eiyama and okamoto , 2015 ; hamacher - brady and brady , 2015 ; yoshii and mizushima , 2015 ) . mitophagy is critically dependent on two factors - the pten - induced putative kinase 1 ( pink1 ) and the e3 ubiquitin ligase parkin . these proteins act in sensing the functional and health statuses of mitochondria , and mark damaged mitochondria for autophagic disposal ( eiyama and okamoto , 2015 ) . sirt1 s regulation of general macroautophagy is well known ( ng and tang , 2013 ) and is broadly viewed as a cellular protective mechanism against stress and death insults ( ou et al . , 2014 ; suzuki and bartlett , 2014 ) . a connection between sirt1 and mitophagy was first shown by hwang s group , when the authors found that nicotinamide treatment of primary human fibroblasts extended their replicative lifespan apparently by accelerating autophagic degradation of mitochondria ( kang and hwang , 2009 ) . nicotinamide decreased mitochondrial mass and increased mitochondrial membrane potential , with elevations of the autophagosome marker light chain 3 ( lc3)-ii and levels of proteins that regulate mitochondrial fusion and fission . a subsequent paper from the hwang lab indeed suggested that the mitophagic effect of nicotinamide is mediated through an increase of the nad / nadh ratio and sirt1 activation ( jang et al . , 2012 ) , and mitochondrial dysfunction in xeroderma pigmentosum group a ( xpa ) , a nucleotide excision dna repair disorder , is characterized by defective mitophagy ( fang et al . , 2014 ) . this is apparently due to hyperactivation of the dna damage sensor parp-1 , a major nad consuming enzyme , and the consequent lowering of sirt1 activity . another recent report indicated that the sirt1 activator resveratrol acts through a signaling axis that involves sirt1-sirt3 and pink1/parkin - mediated mitophagy , resulting in cardio - protection ( das et al . , 2014 ) . furthermore , loss of sirt1 in the luminal epithelium in human prostate cancer delayed parkin translocation to the mitochondria and reduced mitophagy ( di sante et al . , 2015 ) . a particularly interesting aspect of the findings discussed above is sirt1 s known role in protection against neurodegenerative disorders ( ng et al . , 2015 ; tang , 2009 ; zhang et al . , 2011 ) . over the years , sirt1 activity has been extensively associated with neuroprotection , and autophagy ( or more specifically , mitophagy ) induction could be part of its protective repertoire . sirt1 activity is known to attenuate -synuclein aggregation and toxicity , a hallmark of parkinson s disease ( pd ) ( albani et al . , 2009 ; donmez et al . , 2012 ; sampaio - marques et al . , 2012 ) . the mitophagy regulators pink1 and parkin are both encoded by pd susceptibility genes that are mutated in patients with juvenile or early - onset parkinsonism ( mullin and schapira , 2015 ) . interestingly , a genetic screen in drosophila found that ectopic expression of the fly sir2 rescued mitochondrial defects in pink1-null mutants ( but not parkin mutants ) ( koh et al . , 2012 ) . a better known connection between sirt1 and neurodegenerative diseases is not a mitophagy factor , but rather pgc-1 ( rna - vrs and weydt , 2010 ; tsunemi and la spada , 2012 ) . pgc-1 suppression promotes -synuclein accumulation in cellular models of pd ( ebrahim et al . , 2010 ) . the zn - finger protein parkin - interacting substrate ( paris , or znf746 ) accumulates in cellular models of parkin inactivation and in the human pd brain ( shin et al . , 2011 ) . paris represses the expression of pgc-1 and nrf-1 , and transgenic overexpression of paris leads to the selective loss of dopaminergic neurons in the substantia nigra , the disease - susceptible neuron group in pd . another report indicated that loss of parkin in cells from patients with early - onset pd increased pgc-1 , but the elevated pgc-1 appeared transcriptionally non - functional ( pacelli et al . , 2011 ) . meta - analysis of gene expression profiles and molecular pathways clearly implicated pgc-1-responsive genes amongst those downregulated in pd co - transduction of adenovirus carrying human pgc-1 in rat e17 mid - brain neuron cultures attenuated dopaminergic neuron loss induced by either mutant -synuclein , or the mitochondrial respiratory chain complex i - inhibitor rotenone . transgenic mice that over - express pgc-1 in substantia nigra s dopaminergic neurons are resistant to degeneration induced by the neurotoxin mptp ( mud et al . , 2012 ) . loss of pgc-1 is also a well - known feature in huntington s disease ( hd ) , a hereditary disorder where aggregation of mutant huntingtin protein with an extended polyglutamine tract causes striatal neuron loss ( ross and tabrizi , 2011 ) . nuclear accumulation of mutant huntingtin repressed pgc-1 gene transcription by associating with its promoter ( cui et al . , 2006 ) , and over - expression of pgc-1 could partially reverse the toxicity of mutant huntingtin in cultured striatal neurons . interestingly , nicotinamide could upregulate pgc-1 and the neuroprotective brain - derived neurotrophic factor ( bdnf ) in a transgenic mouse model of hd , and improve motor deficits ( hathorn et al . , 2011 ) . another study with inducible pgc-1 expression in hd mice not only rescued the hd phenotype and spared neurons , but could result in significant elimination of mutant huntingtin aggregates ( la spada , 2012 ) . given pgc-1 s beneficial effect on a multitude of neurodegenerative pathologies , it is likely that the neuroprotective effect of sirt1 may , at least in part , be exerted through activation of pgc-1. while all of the above results appear to attest to the promise of pgc-1 as a drug target in pd and hd , one must avoid generalizing pgc-1 s potential beneficial effects on other neurodegenerative diseases . in particular , a recent report has shown that crossing of an alzheimer s disease ( ad ) mouse model ( tg19959 mice ) with transgenic strains over - expressing human pgc-1 exacerbated amyloid and tau accumulation ( dumont et al . , sirt1 s protective activity against the detriments of mitochondrial dysfunction involves other sirt1 substrates , particularly transcription factors that influence mitochondrial activity . one such group of factors are members of the forkhead box o ( foxo ) transcription factor family ( webb and brunet , 2014 ) . sirt1 is known to deacetylate members of the foxo family , particularly in response to stress ( brunet et al . , 2004 ) . drosophila sir2 expression prevented the demise of fly dopaminergic neurons in pink1-null mutants in a dfoxo - dependent manner ( koh et al . , 2012 ) . activation of foxo3a , in particular , has been shown to inhibit mitochondrial gene expression , causing a reduction in mitochondrial dna copy number and respiratory activity ( ferber et al . , 2012 ) . in fact , a declined in nuclear nad , and a consequentially diminished sirt1 activity , was also found to underlie a specific loss of mitochondrial - encoded subunits of the oxidative phosphorylation system ( gomes et al . , 2013 ) . interestingly , foxo3a could translocate into the mitochondria where it could potentially be a substrate of mitochondrial sirt3 ( jacobs et al . , foxo3a mitochondrial translocation was also shown to occur during gtp - induced erythroid differentiation ( meshkini and yazdanparast , 2012 ) . recent work in c. elegans has indicated that worm sir2.1 mediates longevity via the mitochondrial unfolded protein response ( upr ) and the activation of the worm foxo orthologue daf-16 ( mouchiroud et al . , 2013 ) . it was further shown that inhibition of the mitochondrial chaperone trap1 generates a foxo - dependent retrograde cell protective signal from the mitochondria to the nucleus ( kim et al . , 2015 ) . sirt1 s modulation of foxo activity could therefore influence mitochondria function via gene expression in the nucleus , as well as affecting retrograde signaling from the mitochondria to the nucleus . a few other sirt1-modified transcription factors could also have a role , either directly or indirectly , in mitochondrial activity and function . sirt1 deacetylates and represses the activity of the hypoxia inducible factor 1-alpha ( hif-1 ) ( lim et al . , 2010 ) . hif-1 is known to repress mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 ( pdk1 ) ( papandreou et al . , 2006 ) , and sirt1 activity may thus influence mitochondrial respiratory function via the former . one prominent transcription factor substrate of sirt1 is the tumor suppressor tp53 ( vaziri et al . , 2001 ) , and sirt1 deacetylation of p53 suppresses its transactivation capacity and promotes cell survival ( luo et al . , 2001 ) interestingly , it has been shown that mouse embryonic stem ( mes ) cells produces endogenous reactive oxygen species ( ros ) , which causes p53 translocation into the mitochondria in the presence of sirt1 , but induces p53 nuclear translocation in sirt1-null mes cells ( han et al . , 2008 ) . sirt1 activity may therefore influence mitochondria - mediated apoptosis by influencing mitochondrial translocation of factors such as p53 . another transcription factor that is deacetylated by sirt1 which has known roles in mitochondrial physiology is the nuclear factor erythroid 2-related factor 2 ( nrf2 ) ( kawai et al . , 2011 ) . nrf2 and its inhibitor , the kelch - like ech associated protein 1 ( keap1 ) , are master regulators of the cellular anti - oxidative response , and have key roles in cellular bioenergetics ( dinkova - kostova et al . , 2015 ) . it is clear from these examples that sirt1 activity exerts a complex and multi - effector mediated influence on mitochondrial functions . in this review , i discussed evidence for sirt1 s regulation of mitochondrial biogenesis and turnover , particularly in relation to pgc-1 ( see fig . the emerging view in the field is that sirt1 activity could exert important influence on mitochondrial function , but the degree of influence is likely cell type- and physiological context - dependent . sirt1 activation could promote mitochondrial biogenesis in conditions of energy deficiency associated with disease and injury . on the other hand , it could also have an important role in triggering the demise or turnover of damaged mitochondria . should one pathway occur preferentially over the other , which pathway will predominate would likely depend on the nature of disease / injury , the relevant sirt1 substrates available , and the cell s energetic and metabolic status , such as the availability of nad . for example , factors that result in direct mitochondrial damage may induce a higher rate of mitophagy . in terminally differentiated cells with acute energy requirements such as neurons , sustaining a steady supply of mitochondria and salvaging whatever that remains functional could be important to ensure survival . in dividing cells , a quick mitophagic clearance of damaged mitochondria may be more urgent so that these are not passed down to daughter cells during division . on the whole , sirt1 s regulation of mitochondrial biogenesis and mitophagy could thus act in concert for mitochondria quality maintenance . the large regulatory repertoire of sirt1 could potentially be exploited for therapeutics , but a better understanding of the molecular factors it influences and their dynamic interactions would be necessary .

sirt1 is the most prominent and extensively studied member of sirtuins , the family of mammalian class iii histone deacetylases heavily implicated in health span and longevity . although primarily a nuclear protein , sirt1 s deacetylation of peroxisome proliferator - activated receptor gamma coactivator-1 ( pgc-1 ) has been extensively implicated in metabolic control and mitochondrial biogenesis , which was proposed to partially underlie sirt1 s role in caloric restriction and impacts on longevity . the notion of sirt1 s regulation of pgc-1 activity and its role in mitochondrial biogenesis has , however , been controversial . interestingly , sirt1 also appears to be important for the turnover of defective mitochondria by mitophagy . i discuss here evidences for sirt1 s regulation of mitochondrial biogenesis and turnover , in relation to pgc-1 deacetylation and various aspects of cellular physiology and disease .

INTRODUCTION SIRT1S MITOCHONDRIAL LOCALIZATION SIRT1S DEACETYLATION OF PGC - 1 - METABOLIC REGULATION AND MITOCHONDRIAL BIOGENESIS THE CONNECTION BETWEEN SIRT1, AMPK AND PGC-1 IN REGULATING MITOCHONDRIAL FUNCTION SIRT1 AND MITOPHAGY SIRT1, PGC-1 AND NEURODEGENERATIVE DISEASES OTHER SIRT1-REGULATED FACTORS/SUBSTRATES IN MITOCHONDRIAL FUNCTION CONCLUSION

etoposide is a cytotoxic agent ( anticancer drug ) which has been used in the treatment of a variety of malignant conditions . etoposide is an essential component of chemotherapy in patients with rare gynecological malignancies such as germ cell tumors and gestational trophoblastic disease . hypersensitivity reactions ( hsr ) to etoposide have been reported in 13% patients . in most patients , reactions arise within 10 min and are treated by discontinuing the infusion and administering antiallergic pharmacologic agents . there are no suitable substitutes for etoposide in regimens such as emaco ( etoposide , methotrexate , actinomycin d , cyclophosphamide , vincristine ) . etoposide should only be discontinued when there is severe patient intolerance during a course of treatment , necessitating second - line therapy . a 39-year - old patient with a diagnosed recurrence of choriocarcionma in the site of vaginal scar tissue and distant metastases in the lungs and brain was admitted for treatment with a standard emaco regimen . two years earlier , she was diagnosed with choriocarcinoma with lung metastasis and was successfully treated with an emaco regimen ( 8 cycles ) and hysterectomy . this time , the council for trophoblastic disease prescribed 8 cycles of chemotherapy . on admission , she had severe anemia [ red blood cells 3.89 10/l ( 4.105.10 ) and hemoglobin 73.5 g / l ( 115165 ) ] , which required blood transfusion . during the second dose of blood transfusion , the administration of etoposide was commenced ( 200 mg in 500 ml of sodium chloride 0.9% over 60 min ) . after 20 drops , the patient complained of generalized heat , tightness in the throat , shortness of breath and palpitations . she was found to be hypotensive ( blood pressure = 100/60 mm hg ) , tachycardic ( pulse rate = 120 beats / min ) and desaturated to 84% on pulse oximetry . in addition , the anesthesiologist noticed a discrete rush on the hand opposite to that used for transfusion . the patient was supplemented with oxygen and given fluids , methylprednisolone and diphenhydramine intravenously , which resolved her symptoms rapidly within 45 min . the following day , the patient received premedication with methylprednisolone ( 60 mg ) , ranitidine ( 50 mg ) and diphenhydramine ( 50 mg ) intravenously . then , she was given an infusion with sodium chloride 0.9% over 10 min ( placebo ) . after a few drops of infusion , the patient developed acute dyspnea and chest tightness . the following day the patient received an equivalent dose of etoposide phosphate intravenously , a water - soluble ester of etoposide , after the same premedication described above . the etiology and the exact mechanism are unknown , but they are believed to be of nonimmunogenic origin . nevertheless , there are some hypotheses that the concentration of the drug and the rate of infusion could explain hsr . another possible way of hsr is that the additive used in the preparation to dissolve etoposide ( benzyl alcohol , polysorbate 80 ) may cause hsr . in animal models , it is a type i reaction and antibody mediated . in our case , it is believed that the patient experienced the same type of reaction since the acute symptoms occurred immediately , as a result of releasing vasoactive substances such as histamine . it was reported that the substitution of etoposide phosphate in patients with etoposide hsr seems to be appropriate . in the reported case , we have completed the treatment without any complications after changing to another pharmaceutical form of etoposide . etoposide phosphate is a water - soluble prodrug of etoposide , and this variant does not require polysorbate 80 to enter solution . to date , hsr to etoposide phosphate has not been reported [ 3 , 7 ] . in our case , it is likely that polysorbate 80 is a causative factor in the hsr to etoposide . however , another alternative to avoid any possibility of hsr is the use of oral instead of intravenous etoposide because this has a lower incidence of hsr . also , a case of successful treatment of choriocarcinoma by diluting the etoposide concentration to half and reducing the drug administration rate by half to avoid hsr is described in the literature . in situations where a patient expresses hsr to etoposide and where subsequent doses of etoposide are deemed necessary , replacement with etoposide phosphate is a reasonable and viable alternative with antihistamine and corticosteroid premedication .

etoposide is commonly used in the treatment of a variety of neoplasms . hypersensitivity reactions to etoposide are infrequently reported and include hypotension , hypertension , flushing , diaphoresis , chest discomfort , dyspnea , bronchospasm and loss of consciousness . we report the case of a 39-year - old woman who experienced acute bronchospasm , tachycardia , hypoxia and hypotension . the symptoms resolved within an hour after administration of intravenous fluids , methylprednisolone , diphenhydramine and oxygen . subsequently , the patient was given etoposide phosphate without incident .

Introduction Case Report Discussion Conclusion

adolescent reproductive health ( arh ) outcomes in africa are the worst in the world.1 for this reason , the world health organization ( who ) has strongly promoted youth - friendly reproductive health services for over a decade ; however , targets for service access , uptake and funding remain unmet.2 3 infrastructural limitations including health system failures,4 5 extreme lack of human resources , irregular medicine supply,6 inaccessibility due to long distances,7 and inflexible work routines combine with poor attitudes of government health workers8 to limit the effectiveness of public sector arh service provision . against this background , close - to - community providers such as private drug vendors and traditional healers are often the first port of call for adolescents with reproductive health needs.9 such close - to - community providers are perceived to be more convenient than government service providers such as clinical officers based in dispensaries and health centres.10 11 for this reason , health planners have increasingly explored different strategies for community - based and lay reproductive and maternal health service provision.1215 however , it is unclear what capacity close - to - community agents have to provide effective reproductive health services for adolescents . we report the results of a study examining the experience , attitudes and capacity of a range of close - to - community providers of such services , as well as their readiness and ability to integrate with the mainstream health sector . for the purpose of this study , we define a close - to - community provider as any service provider at the community level offering one or more of a range of arh services including contraception , antenatal care , obstetric services , sexually transmitted infection ( sti ) and hiv services , counselling and support . we adopt the who definition of adolescence , namely anyone who is 1019 years of age.16 we aimed to determine which cadres of close - to - community providers were providing reproductive services to adolescents in nine communities in two districts ( magu and sengerema ) in mwanza region on the northwest shore of lake victoria ; what services they offered ; their skills and capacity to provide them ; their attitudes towards arh ; and their attitudes to cooperation with the mainstream health sector , especially referral of their clients to formal health facilities . these providers were selected from communities that were stratified into rural , urban and high - risk clusters . we also included formal health service providers from government dispensaries and health centres to triangulate views on community referral and integration . cadre - specific focus group discussions ( fgds ) with 814 participants were conducted in order to capture the consensus view on the respective experiences and attitudes17 18 to arh . fgds were facilitated by senior researchers from the tanzania national institute for medical research ( nimr ) mwanza . the fgds were conducted in february 2011 at central locations ( e.g. schools , health centres or village offices ) . village executive officers invited eight people per cadre to participate in planned fgds ; however , word - of - mouth spread of information about the study led to greater numbers of participants than expected . in view of the distance they had travelled ethical approvals were obtained from the liverpool school of tropical medicine research ethics committee and the tanzania medical research coordinating committee . permission was obtained from administrative leaders at the regional , district and ward levels , and informed written consent was sought from all participants . fgd guides were prepared , pretested in the field and revised to incorporate the views of pre - test participants . discussion focused on skills , attitudes and practices in family planning and contraception , antenatal care and maternal delivery , hiv and sti prevention and treatment as well as post - abortion care . after each round of discussion the guide was further tuned to key themes based on reflection on the previous fgds . the discussions were digitally recorded , transcribed in kiswahili language and translated into english . using nvivo 9 software ( qsr international , doncaster , victoria , australia ) , the transcripts were analysed using a thematic framework based on nodes that were deductively drawn from predefined themes in the discussion guide.19 we aimed to determine which cadres of close - to - community providers were providing reproductive services to adolescents in nine communities in two districts ( magu and sengerema ) in mwanza region on the northwest shore of lake victoria ; what services they offered ; their skills and capacity to provide them ; their attitudes towards arh ; and their attitudes to cooperation with the mainstream health sector , especially referral of their clients to formal health facilities . these providers were selected from communities that were stratified into rural , urban and high - risk clusters . we also included formal health service providers from government dispensaries and health centres to triangulate views on community referral and integration . cadre - specific focus group discussions ( fgds ) with 814 participants were conducted in order to capture the consensus view on the respective experiences and attitudes17 18 to arh . fgds were facilitated by senior researchers from the tanzania national institute for medical research ( nimr ) mwanza . the fgds were conducted in february 2011 at central locations ( e.g. schools , health centres or village offices ) . village executive officers invited eight people per cadre to participate in planned fgds ; however , word - of - mouth spread of information about the study led to greater numbers of participants than expected . in view of the distance they had travelled , additional participants could not be turned away . ethical approvals were obtained from the liverpool school of tropical medicine research ethics committee and the tanzania medical research coordinating committee . permission was obtained from administrative leaders at the regional , district and ward levels , and informed written consent was sought from all participants . fgd guides were prepared , pretested in the field and revised to incorporate the views of pre - test participants . discussion focused on skills , attitudes and practices in family planning and contraception , antenatal care and maternal delivery , hiv and sti prevention and treatment as well as post - abortion care . after each round of discussion the guide was further tuned to key themes based on reflection on the previous fgds . the discussions were digitally recorded , transcribed in kiswahili language and translated into english . using nvivo 9 software ( qsr international , doncaster , victoria , australia ) , the transcripts were analysed using a thematic framework based on nodes that were deductively drawn from predefined themes in the discussion guide.19 we conducted 35 fgds with a total of 323 close - to - community providers distributed among nine cadres as shown in table 1 . six key themes were identified as follows : knowledge , skills and attitudes towards arharh services offered by close - to - community providersarh needs and close - to - community providers ' ability to provide themclose - to - community providers ' trainingcommunity referralpreferred referral service . knowledge , skills and attitudes towards arh arh services offered by close - to - community providers arh needs and close - to - community providers ' ability to provide them close - to - community providers ' training preferred referral service . summary of close - to - community providers views within the emerging themes arh , adolescent reproductive health ; art , antiretroviral therapy ; arv , antiretroviral ; fp , family planning ; phc , primary health care ; rh , reproductive health ; sti , sexually transmitted infection ; vct , voluntary counselling and testing . most close - to - community providers were subject to a number of serious misconceptions and negative attitudes about arh . specifically , they had negative attitudes towards provision of reproductive health services to adolescents and expressed hostility to arh rights ; dismissing the right of adolescents to choose when and where to seek reproductive health services or when to use contraceptives as nonsense . some maintained the belief that condoms were impregnated with hiv , while others believed that condoms could cause cancer.there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] such sentiments were also linked to doubts on the general purpose of family planning interventions : people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . [ fgd # 17 , traditional healers ] people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . [ fgd # 17 , traditional healers ] drug shops mainly sell medicines . however , whilst they are only licensed to sell non - prescription drugs and items such as analgesics and condoms , they reported treating stis and selling antibiotics . home - based care volunteers and social workers reported their main services to be palliative care , education and counselling . they felt that provision of guidance on hiv prevention including condom use , education and informal referral to health facilities in the community was their responsibility ; however , clinical officers from dispensaries did not corroborate that these referrals take place . in addition , village aids committee members stated that if consulted by adolescents on reproductive health issues they would disclose the discussion to parents . village health workers ( also known as community health workers ) reported providing counselling , distribution of condoms and referral to health facilities . traditional birth attendants conducted home births and provided advice to pregnant mothers . in one community , tbas worked 1 day per week at dispensaries helping with deliveries . traditional healers , the largest group of close - to - community providers , were the least likely to have had any form of training , despite the range of services that they claim to provide ; including infertility treatment , abortions , home births and treatment of stis using herbal remedies and charms . all close - to - community providers reported that the main reproductive health need of the adolescents was condoms . the next most requested service was information on hiv prevention , pregnancy and family planning . the close - to - community providers and dispensary clinicians also highlighted a number of specific reproductive health demands that they were not able to meet . for example , dispensaries could not offer complex family planning services such as sterilisation , and although they could not provide abortion due to its illegality in tanzania , its need amongst adolescents was reported to be on the rise . youth - friendly service provision was also difficult for some due to lack of space.to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] some drug sellers complained of being consulted for sti treatment but unable to provide it , as they are not licensed to do so:we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] close - to - community providers also felt that the demand for information and other reproductive health services could not be met as they lack the ability or resources . most close - to - community providers did not have any training except drugstore attendants and dispensary clinical officers who said they had certificates in drug dispensing and diplomas in clinical medicine , respectively . community health workers , home - based care volunteers and social workers reported receiving orientations from various civil society organisations promoting hiv prevention in the community , although the level and quality of such orientations to provide arh services was unclear . the other close - to - community providers had received no training or orientation , and all except dispensary staff felt that they had limited knowledge on arh and requested further training . unfortunately verification of the actual level of training attained by the close - to - community providers was beyond the scope of our study . our fgds explored the acceptability of establishing community referral mechanisms from close - to - community providers to formal health facilities ( i.e. dispensaries and health centres ) . we established that referral is done informally between close - to - community providers , for example , from village health workers to tbas . referrals from drug shops to dispensaries were also reported:patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] participants discussed ideal community referral interventions to increase adolescents access to reproductive health services . they wanted linkages and connections with formal health facilities as well as recognition and integration into the health sector . dispensaries were also willing to work with drug shops saying:we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] drug sellers also stated that linkages to dispensaries would be beneficial to them:we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] table 1 summarises the close - to - community providers views within the themes discussed above . most close - to - community providers were subject to a number of serious misconceptions and negative attitudes about arh . specifically , they had negative attitudes towards provision of reproductive health services to adolescents and expressed hostility to arh rights ; dismissing the right of adolescents to choose when and where to seek reproductive health services or when to use contraceptives as nonsense . some maintained the belief that condoms were impregnated with hiv , while others believed that condoms could cause cancer.there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] such sentiments were also linked to doubts on the general purpose of family planning interventions : people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . [ fgd # 17 , traditional healers ] people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . drug shops mainly sell medicines . however , whilst they are only licensed to sell non - prescription drugs and items such as analgesics and condoms , they reported treating stis and selling antibiotics . home - based care volunteers and social workers reported their main services to be palliative care , education and counselling . they felt that provision of guidance on hiv prevention including condom use , education and informal referral to health facilities in the community was their responsibility ; however , clinical officers from dispensaries did not corroborate that these referrals take place . in addition , village aids committee members stated that if consulted by adolescents on reproductive health issues they would disclose the discussion to parents . village health workers ( also known as community health workers ) reported providing counselling , distribution of condoms and referral to health facilities . traditional birth attendants conducted home births and provided advice to pregnant mothers . in one community , tbas worked 1 day per week at dispensaries helping with deliveries . traditional healers , the largest group of close - to - community providers , were the least likely to have had any form of training , despite the range of services that they claim to provide ; including infertility treatment , abortions , home births and treatment of stis using herbal remedies and charms . all close - to - community providers reported that the main reproductive health need of the adolescents was condoms . the next most requested service was information on hiv prevention , pregnancy and family planning . the close - to - community providers and dispensary clinicians also highlighted a number of specific reproductive health demands that they were not able to meet . for example , dispensaries could not offer complex family planning services such as sterilisation , and although they could not provide abortion due to its illegality in tanzania , its need amongst adolescents was reported to be on the rise . youth - friendly service provision was also difficult for some due to lack of space.to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] some drug sellers complained of being consulted for sti treatment but unable to provide it , as they are not licensed to do so:we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] close - to - community providers also felt that the demand for information and other reproductive health services could not be met as they lack the ability or resources . most close - to - community providers did not have any training except drugstore attendants and dispensary clinical officers who said they had certificates in drug dispensing and diplomas in clinical medicine , respectively . community health workers , home - based care volunteers and social workers reported receiving orientations from various civil society organisations promoting hiv prevention in the community , although the level and quality of such orientations to provide arh services was unclear . the other close - to - community providers had received no training or orientation , and all except dispensary staff felt that they had limited knowledge on arh and requested further training . unfortunately verification of the actual level of training attained by the close - to - community providers was beyond the scope of our study . our fgds explored the acceptability of establishing community referral mechanisms from close - to - community providers to formal health facilities ( i.e. dispensaries and health centres ) . we established that referral is done informally between close - to - community providers , for example , from village health workers to tbas . referrals from drug shops to dispensaries were also reported:patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] they wanted linkages and connections with formal health facilities as well as recognition and integration into the health sector . dispensaries were also willing to work with drug shops saying:we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] drug sellers also stated that linkages to dispensaries would be beneficial to them:we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] table 1 summarises the close - to - community providers views within the themes discussed above . improved integration of close - to - community providers into formal health services has been identified as a strategy to strengthen health service provision.20 training and involvement of close - to - community provider cadres is included in the tanzania national strategic plan 20092015.21 however , our findings suggest that despite activities by the government of tanzania ministry of health and social welfare to engage this segment of the health sector22 the capacity of community - based providers to offer effective adolescent reproductive health services is poor . our study systematically explored views about arh across 10 cadres of close - to - community providers in nine communities in two districts in tanzania . the large number of fgds utilised in this study and the stratification of communities to include rural , urban and high - risk increases the representativeness of our findings , and makes this one of the largest studies of its kind.23 24 however , although large , representative and inclusive , this study is not without limitations . first , while we emphasised the need for confidentiality and set clear ground rules during each session , fgds are a communal activity and disclosure is dependent on the degree of trust generated within the discussion group . it is therefore possible that some close - to - community providers did not fully express their views out of embarrassment or fear of retribution . the use of the village executive officers as an entry point may also have led to unknown biases arising from local and interpersonal relationships ; however , we hope that the large number of participants and geographical breadth of the study has offset some of this bias . there was no gender or age separation within the cadre - specific groups as we believed service provision to be a topic that both male and female participants within a cadre could discuss openly ; however , the presence of older male participants could well have inhibited younger or female participants from speaking their minds extensively . whilst this did not appear to be the case from a review of field reports overall , our results suggest that levels of knowledge about arh among close - to - community providers remains poor and attitudes negative . misconceptions and mistrust are especially demonstrated through observed perceptions that hiv has been put into condoms to harm the public . these misconceptions date back to the very early days of the hiv epidemic and it is disappointing that they persist despite the substantial and varied community information campaigns that have been implemented over the past two decades.23 25 strongly held close - to - community provider views about adolescent sexual behaviour are based on cultural norms , which are difficult to change . there is a need to develop and model rights - based approaches to reduce judgemental views on adolescents sexuality . the perceived increase in demand for condoms among adolescents beyond the available supply could suggest that the number of adolescents seeking protection during sex is increasing . other hiv - prevention interventions have reported increases in this demand;26 27 however , such trends are difficult to interpret . whilst it may mean that decades of health promotion strategies are finally translating into increased condom use , it could also represent increasing numbers of sexual encounters among adolescents . however , our findings suggest that abortions are being sought by adolescents , potentially leading to increased numbers of unsafe abortions being carried out . whilst reported data of this kind are a good indicator of perceptions , they must be interpreted with caution in the absence of quantitative documentation of actual condoms dispensed or abortions conducted . we also found that close - to - community providers services are largely fragmented and under - resourced . the close - to - community providers are juggling hiv / sti , family planning and maternal health in large populations with very little capacity . they promote themselves as providers of these services , even though they are not in a position to offer them effectively . notwithstanding this , research shows that some close - to - community providers such as drug shops are more popular than mainstream health services.24 28 it means that close - to - community providers have the potential to powerfully influence health outcomes in the communities . a systematic review of evidence on health care workforce task - shifting demonstrated that issues identified in our study such as training and integration were key to improving health outcomes in communities where largely untrained and unsupported human resources are to be deployed.29 for example , dispensaries with staff who have diagnostic and prescribing skills often lack supplies of medicines,30 31 whereas drug shops where such skills are lacking may have stocks of these medicines . at the time of writing the ministry of health was upgrading drug stores into accredited drug distribution outlets ( addo ) with a licence to sell controlled medicines , and further development of the addo programme to include linkages with local dispensaries could significantly improve accessibility of skilled reproductive health services and effective treatment . our findings suggest that tbas may be one of the effective cadres in the community . in our fgds , tbas reported weekly participation in facility - based , in addition to house - to - house , visits . although evidence on population - level benefits of tba involvement in maternal health is equivocal,32 a recent study in southern tanzania reported that only 46.7% ( n=974 ) of women delivered in a health facility.33 we argue that the popularity of tbas at the community level can not be ignored . the regular use of their services , even at the institutional level as demonstrated in the present study , indicates a continued need for their role . improving the capability of tbas to provide arh services is an option worth considering in the context of tanzania . close - to - community providers potential to provide effective services is limited by their low capacity and skills , negative attitudes and lack of resources . other researchers have argued that training , integration and provision of resources to close - to - community providers is important if the health system achievements they contribute to are to be realised.20 our findings suggest that currently this training is lacking and that there have been few integration efforts to include the close - to - community providers in the health system or to address their lack of capacity and resources . close - to - community providers , known to be the first port of call for many adolescents seeking health care in tanzania , continue to have negative attitudes towards the provision of arh services , family planning and condom use . although they lack training , skills or resources to provide effective arh care , they are open to integration with the formal health sector . potentially therefore they could provide an entry point for expanded access to reproductive health services , but only with significant skills building and training to address negative attitudes .

objectivesyoung people in tanzania are known to access reproductive health services from a range of close - to - community providers outside formal health settings such as drug stores , village aids committees , traditional healers and traditional birth attendants ( tbas ) . however , questions remain about the quality of services such agents provide . this study investigated their capacity to provide adolescent reproductive health ( arh ) services and explored their readiness and ability to integrate with the mainstream health sector through community referral interventions.methodsthirty-five focus group discussions exploring close - to - community provider experiences and attitudes to arh service provision were carried out in two districts in northern tanzania . discussions were conducted in kiswahili , digitally recorded , verbatim - transcribed , translated and back - translated from swahili to english . a thematic analysis was conducted using nvivo 9.resultsthe major close - to - community cadres providing reproductive health services were drug stores , traditional healers , tbas and village health workers . they reported being the first port of call for adolescents seeking reproductive health services , but their knowledge of arh needs was poor . they had negative attitudes to , and lacked the necessary resources for , the provision of such services for adolescents . some were particularly unwilling to provide condom services and were prejudiced against adolescents using them . there was poor integration between the close - to - community providers and the formal health sector , further limiting their ability to provide adequate services.conclusionsalthough close - to - community providers are considered a key resource in the community , most have limited capacity to provide arh services . without capacity - building investments such as training and cooperation with the mainstream health sector , their contribution to positive reproductive health outcomes is limited , or could indeed lead to adverse outcomes .

Introduction Methods Study setting Study type and participant selection Ethical considerations Data collection and analysis Results Theme 1: Knowledge, skills and attitudes towards ARH Theme 2: ARH services offered by close-to-community providers Theme 3: ARH needs and close-to-community providers ability to meet them Theme 4: Close-to-community providers training Theme 5: Community referral Theme 6: Preferred referral service Discussion Conclusions

cough is a common symptom for which medical attention is sought by patients . in the united states , cough was the third principal reason for office visits after progress visit and general medical examination ; there were 31 million visits for cough in 2012 ( 1 ) . in uk general practice , cough was the most common symptom in more than half of all new referrals ( 2 ) . in japan , cough is also a common symptom among patients . among the 35,768 patients who visited the department of general medicine of juntendo university hospital in 2012 , 4,543 had a cough , which was the second most common symptom following a fever ( 3 ) . various guidelines for the diagnosis and management of cough have been published ( 4 - 9 ) . pertussis is one of the important causes of cough , especially chronic cough ( 10 ) . pertussis remains one of the top 10 causes of childhood death worldwide , mainly in unvaccinated children , and it causes nearly 300,000 deaths in children every year ( 11 ) . pertussis is the most dangerous in infants , where the cough can be minimal or absent but can induce apnea , which leads to death . in unvaccinated children , clinical suspicion of pertussis is made in patients with a cough - associated illness lasting at least two weeks with one of the following : paroxysms of coughing , inspiratory whoop , and post - tussive vomiting . in the initial catarrhal stage , the differentiation of pertussis from a common cold is difficult . the paroxysmal whooping cough sets in after one to two weeks and can persist for four weeks or longer . adults and older children are less likely to have typical symptoms , instead showing only mild , non - specific symptoms like prolonged cough ; therefore , the diagnosis of pertussis is difficult when it is based on symptoms alone . all studies assessing the relative proportion of pertussis in adults with longer - lasting coughs agree that a significant proportion of them have pertussis ( 12 ) . the best way to prevent pertussis is to get vaccinated . in japan , the pertussis vaccine has been administered since 1950 , and the number of patients with pertussis decreased from more than 100,000 in 1950 to 206 in 1971 . the first vaccine against pertussis included whole - cell killed bordetella pertussis ( b. pertussis ) bacteria and contained pertussis endotoxin ( surface lipooligosaccharide ) , which had side effects . severe neurologic disorders led to the suspension of pertussis vaccination in 1975 , and the number of patients increased to about 13,000 in 1979 in japan . new acellular pertussis vaccines , which include only a few selected pertussis antigens , were introduced in 1981 ( 13 ) . outbreaks occurred in university students in 2007 , and it was suspected that many young people did not have immunity against b. pertussis , although recent studies have suggested another mechanism : asymptomatic transmission from individuals who were vaccinated with the currently used acellular b. pertussis vaccines ( 14 ) . identification of the pathogen by culturing nasopharyngeal secretions has been considered the gold standard for diagnosis , but this method is unsuitable in clinical practice for young and adult patients , as b. pertussis loads are very low in these populations , and the isolation and identification of bacteria is very difficult , especially in previously vaccinated persons ( 15,16 ) . compared with the culture method , nucleic acid amplification tests such as the is481-based real - time polymerase chain reaction ( pcr ) and b. pertussis - specific loop - mediated isothermal amplification ( lamp ) are highly sensitive for the detection of b. pertussis dna ( 17 - 19 ) . therefore , these nucleic acid amplification tests have been recommended for the accurate diagnosis of pertussis ( 20 ) . nevertheless , the pcr and lamp are virtually impossible to perform in clinical practice due to their cost and the fact that they are not covered by public health insurance in japan . the most common pertussis serologic assays use enzyme - linked immunosorbent assay ( elisas ) with highly purified pertussis toxin ( pt ) antigen ( 21,22 ) . pt antigen is expressed only by b. pertussis , and no cross - reacting antigens have been described ; furthermore , igg responses occur in most patients with b. pertussis infection ( 23,24 ) . a two- to four - fold increase in the igg titers against pertussis toxin antigen ( pt - igg ) in paired acute and convalescent sera a review reported that a single measurement of pt - igg can be indicative of current pertussis infection , with a sensitivity of 76% and a specificity of 99% for the diagnosis of acute pertussis ( 11 ) . some reports have proposed cut - off values for pt - igg to indicate recent contact ( 25 ) . in the japan respiratory society guidelines for cough , a cut - off value of 100 eu / ml was used for a definitive diagnosis of pertussis , and pt - igg levels < 10 eu / ml were used to indicate non - immunized status ( 26 ) . it was reported that concentrations of pt - igg fall below the defined cut - off about 4.5 months after infection on average , and in most patients ( 82% ) within 1 year ( 27 ) . we herein investigated the effect of previous vaccination for pertussis on the levels of pt - igg . we also examined the time course of the levels of pt - igg in the japanese population . we retrospectively identified subjects who had visited ikebukuro otani clinic between december 2012 and october 2014 . in japan , about half of the patients were given a final diagnosis of bronchial asthma or cough - variant asthma , and one third were diagnosed with acute bronchitis by viral or bacterial infection . all patients who had their pt - igg levels measured at multiple time points as of february 2015 were recruited for the present study , as well as those with measurements at a single time point ( fig . 1 ) . information on sex , date of birth , and pt - igg levels with the sampling date were collected from the medical records of each patient . the patients were followed up for one year , and their data were updated in february 2016 . we conducted epidemiologic studies of the age distribution for natural and vaccine - induced immunity against b. pertussis to evaluate the correlation between the age and pt - igg levels . to study the natural time course of the pt - igg levels , the average change in the logarithm of the pt - igg level versus elapsed time was analyzed for patients who had their pt - igg levels measured at multiple time points . we excluded the subjects who were deemed inappropriate for the natural time course analysis . to exclude subjects with active b. pertussis infection during the observation period , the data from subjects with more than a two - fold increase of pt - igg level were excluded from the analysis . pt - igg levels exceeding 160 eu / ml were reported as pt - igg > 160 , and we were unable to obtain the exact values ; therefore , these data were also excluded from the change rate analysis . the levels of pt - igg antibody were measured using commercially available enzyme immunoassays for pertussis ( denka seiken co. , ltd . , briefly , a plate coated with the pertussis toxin antigen was incubated with standard or test sera , washed , incubated with goat anti - human igg conjugated with horseradish peroxidase , and then washed again . the pt - igg titers were determined using calibration curves obtained from standard serum jnih-10 ( 28 ) from the national institute of infectious diseases ( niid ) in japan . the measurements were conducted at the laboratories of the showa medical science corporation in accordance with the manual . pt - igg levels exceeding 160 eu / ml were reported as pt - igg > 160 ; serum was not diluted , and no reexamination was performed to obtain the exact values . the robustness of our results was assessed using the graphpad prism 6 software program for mac os x ( graphpad software , inc . , la jolla , usa ) . the correlation coefficients between the age and pt - igg levels were calculated by spearman 's correlation , a method that makes no assumptions about gaussian - like distributions . differences in the pt - igg levels between groups were estimated using the kruskal - wallis test . this study was approved by the ethics committee of the tokyo medical and dental university ( approved number 1957 ) . given the retrospective nature of the study , informed consent from the participating patients was waived by the institutional ethics committee and not obtained , but the public was notified of the study by posters . a total of 205 ( men , 66 ; mean age , 40.913.8 ; women , 139 ; mean age , 39.811.4 ) patients were recruited who had pt - igg levels measured at multiple time points ( fig . 201 ( men , 88 ; mean age , 42.412.8 ; women , 113 ; mean age , 39.714.1 ) first - visit patients between march 2014 and may 2014 who had pt - igg levels measured once were recruited . thus , the age distribution of pt - igg levels in a total of 406 patients was determined ( fig . 2 ) . we found no correlation between the age and pt - igg level ( r=0.06 , p=0.20 ) . 3 shows the distribution of the pt - igg levels in the three groups by date of birth . only a small portion of babies who were born between 1975 and 1985 were vaccinated for pertussis . after more than 30 years , there was little difference in the levels between those born in or before 1974 ( 51.949.5 eu / ml ) , those born between 1975 and 1984 ( 44.343.6 eu / ml ) , and those born in or after 1985 ( 43.046.7 eu / ml ) ( p=0.15 ) . the distribution of pt - igg levels in the three groups by date of birth . pertussis vaccination was suspended in 1975 , and new pertussis vaccines were introduced in 1981 , but only a small number of babies were vaccinated . more than 18% ( 37 out of 205 ) of recruited patients had pt - igg levels exceeding 100 eu / ml . because patients with a high pt - igg level tend to be examined multiple times , we analyzed first - visit patients who had single measurements . among the first - visit patients with single measurements , 15% ( 29 out of 201 ) had a pt - igg level exceeding 100 eu / ml . the pt - igg levels of very few subjects had diminished over a long period of time , and the average decay rate of pt - igg levels per year was 0.97-fold . in the 37 patients with an initial pt - igg level exceeding 100 eu / ml , only 3 had a pt - igg level that had dropped below 100 eu / ml during follow - up . most patients continued to show levels exceeding 100 eu / ml for at least several months ( fig . the temporal changes in the pt - igg levels in patients with an initial pt - igg level exceeding 100 eu / ml . the average annual decay rate of the pt - igg levels was 0.97-fold . igg - pt levels exceeding 160 eu / ml ( upper shaded area ) were not reported as exact values . only a small portion of patients showed pt - igg levels that had decreased to below 100 eu / ml ( lower shaded area ) . in patients with pt - igg levels between 10 and 100 eu / ml , more than 6% ( 9 out of 135 ) showed a more than 2-fold increase in the pt - igg level during long - term follow - up and were therefore regarded as having b. pertussis infection . except for these subjects , the average change rate for the pt - igg levels per year was 1.11-fold ( fig . the temporal changes in the pt - igg levels in patients with an initial pt - igg level between 50 and 100 eu / ml . the average annual change rate of the pt - igg levels was 1.18-fold . the temporal changes in the pt - igg levels in patients with an initial pt - igg level between 20 and 50 eu / ml . the temporal changes in the pt - igg levels in patients with an initial pt - igg level between 10 and 20 eu / ml . the average annual change rate of the pt - igg levels was 1.20-fold . in the patients with pt - igg levels below 10 eu / ml , 9% ( 3 out of 32 ) showed a more than 2-fold increase in the pt - igg level during long - term follow - up , resulting in a level exceeding 10 eu / ml during follow - up . except for these subjects , the average change rate for the pt - igg levels per year was 1.15-fold ( fig . the temporal changes in the pt - igg levels in patients with an initial pt - igg level below 10 eu / ml . we found no correlation between the age and pt - igg levels . because only a small portion of babies who were born between 1975 and 1984 had been vaccinated for pertussis , we stratified subjects according to the date of birth , and the results showed that , after more than 30 years , there was little difference in the levels between the age groups . using a cut - off value of < 10 eu / ml as an indication of non - immunization for pertussis ( 26 ) , the proportion of subjects without immunity for b. pertussis was 14% ( 55 out of 406 ) , and 9% ( 3 out of 32 ) of these subjects were regarded as having b. pertussis infection subsequently . the patients with pt - igg levels between 10 and 100 eu / ml are usually regarded as having immunity against pertussis . in the present study , more than 5% of subjects the relation between antibody and protection is not straightforward , with no particular serological correlate of protection ( 11 ) . we showed that the values of pt - igg changed very little over a long period of time . it is unlikely that concentrations of pt - igg had naturally fallen over time and increased again after another infection by b. pertussis . because there is a very low possibility that many subjects would show almost equal concentrations of pt - igg before naturally falling over time and then rising after b. pertussis reinfection . because patients with a high pt - igg level tend to be examined multiple times , we analyzed the patients who only had single measurements . among them , 15% had a pt - igg level exceeding 100 eu / ml . in other words , more than 15% of subjects could be definitively diagnosed with pertussis based on a single measurement of pt - igg , according to the japan respiratory society however , only a small portion of them were clinically given a diagnosis of active pertussis . one reason for this is that the pt - igg levels of very few subjects had diminished over a long period of time , and many subjects continued to have a value that exceeded the cut - off after infection or immunization . our finding that the pt - igg levels of only a few subjects diminished over a long period of time was inconsistent with the results from previous reports ( 27 ) . this discrepancy in findings may be due to differences in race / ethnicity or epidemic strains of b. pertussis in japan . in the present study , the levels of pt - igg antibody were measured using a commercially available enzyme immunoassay for pertussis ( denka seiken co. , ltd . ) , which is currently the only way to measure pt - igg level in japan . this technique is considered to allow the comparison of pt - igg titer data obtained in the iu with values from other countries , as the iu value was approximately 70 - 80% of the eu value in this assay ( 28 ) . since serum antibodies subtle differences in the measurement methods for pt - igg levels may also explain these discrepancies in findings . the purities of antigens , purification methods , strains , composition of buffer solutions , etc . used in the test differ among manufacturers . in the present study , we showed that a pt - igg level exceeding 100 eu / ml might simply indicate past infection or immunization , at least in japan . a single measurement of pt - igg using cut - off values might therefore lead to the overdiagnosis of pertussis . commercially available measurement levels of pt - igg range from 0 to 160 eu / ml in japan . exact values for subjects with a pt - igg exceeding 160 eu / ml might be needed for detailed analyses . the main limitation associated with this study was that the subjects were retrospectively identified at a single institute that specialized in treating patients with cough . furthermore , we did not use culture , pcr , or lamp methods to identify the pathogen . while we did perform culture and nucleic acid amplification tests , an accurate diagnosis of pertussis was impossible , as b. pertussis loads are very low in adults ( 15,16 ) , creating the possibility of false - negative results . only a small number of subjects were given a clinical diagnosis of active pertussis , although more than 15% of recruited patients had pt - igg levels exceeding the cut - off value . about half of the patients were given a final diagnosis of bronchial asthma or cough variant asthma , and one third were diagnosed with acute bronchitis by viral or bacterial infection . in fact , only one subject was diagnosed with acute pertussis , and he was indistinguishable from the others because he did not present with typical symptoms such as paroxysmal coughing , inspiratory whoop , or post - tussive vomiting . based on paired serum titers showing a more than 2-fold increase in igg antibodies , about 7% of subjects were regarded as having b. pertussis infection in the long - term follow - up , which does not necessarily indicate an infection at the time of study entry . in summary , a single measurement of pt - igg level for the diagnosis of pertussis is insufficient , and using cut - off values for the diagnosis may run the risk of overdiagnosis . the guidelines ( 26 ) have three problems associated with diagnosing pertussis : ( a ) patients with a pt - igg level exceeding 100 eu / ml can be definitively diagnosed with pertussis ; ( b ) patients with a pt - igg level exceeding 10 eu / ml who have no history of immunization can be definitively diagnosed with pertussis ; and ( c ) patients with a pt - igg level exceeding 10 eu / ml who previously had a pt - igg level less than 10 eu / ml can be definitively diagnosed with pertussis . with respect to ( a ) , in actual clinical practice in japan , the pt - igg level rarely falls below the defined cut - off value ( i.e. 100 ) ( fig . 4 ) . with respect to ( b ) , pertussis vaccinations were stopped from 1975 to 1981 , and therefore , subjects born between 1975 and 1984 are at high risk of not having been vaccinated for pertussis . in this group , 86% ( 128 out of 149 ) of subjects showed a pt - igg level exceeding 10 eu / ml ( fig . 3 ) and therefore should have been diagnosed with acute pertussis , according to the guideline ( 26 ) . with respect to ( c ) , 3 . orange line ) observed in the present study are within normal limits and not caused by pertussis . to make a definitive diagnosis , we recommend the use of the b. pertussis - specific lamp or the confirmation of a two- to four - fold increase in the pt - igg values in paired sera .

objective adult patients with pertussis rarely show typical symptoms , such as paroxysmal coughing , inspiratory whoop , or post - tussive vomiting . while a culture is regarded as the gold standard for diagnosis , the sensitivity is very low . therefore , the diagnosis of pertussis in adults in clinical practice is mostly based on single - sample serology using an enzyme - linked immunosorbent assay ( elisa ) with the pertussis toxin antigen . various cut - off values for the anti - pertussis toxin igg ( pt - igg ) have been proposed . it has been reported that concentrations of pt - igg fall below the defined cut - off about 4.5 months after infection on average , and within 1 year in most patients . we investigated the distribution and time course of the pt - igg levels . methods the data were collected from the medical records . patients the study retrospectively identified subjects who had visited ikebukuro otani clinic , which is a specialized clinic for patients with cough . we retrospectively reviewed 406 patients with pt - igg measurements to investigate the age distribution of pt - igg levels . the changes in pt - igg levels over time were assessed in the 205 patients who had more than one pt - igg measurement . results pt - igg levels were 100 eu / ml in more than 15% of subjects . the pt - igg levels of a few subjects had diminished over a long period of time . conclusion a pt - igg level greater than the defined cut - off value simply indicates past infection or immunization in most subjects . as such , a single measurement of pt - igg using the cut - off values might lead to overdiagnosis of pertussis . further data collection and analysis are required .

Introduction Materials and Methods Results Discussion The authors state that they have no Conflict of Interest (COI).

cardiovascular diseases ( cvd ) are the main cause of diabetes - related morbidity and mortality [ 1 , 2 ] . they include myocardial infarction , which is due to premature atherosclerosis , and diabetic cardiomyopathy , both leading to heart failure . patients with diabetes have a higher prevalence of cardiovascular morbidity and mortality as compared to the general population , such that diabetes is considered not only as an independent cardiovascular risk factor but also as a cardiovascular event equivalent , meaning that patients with diabetes have a risk of cardiovascular complications equal to that of patients with a prior myocardial infarction . this excess cardiovascular risk in comparison to the general population is explained only partly by conventional cardiovascular risk factors , such as hyperglycemia , dyslipidemia , hypertension , and cigarette smoking . one of the links between diabetes and such a high prevalence of cvd is renin - angiotensin - aldosterone system ( raas ) activation . it has been shown that the raas plays a major role in the development of diabetic cardiovascular complications , as it promotes atherosclerosis [ 6 , 7 ] , cardiomyocyte loss , and extensive myocardial fibrosis [ 8 , 9 ] . consistent with this view , ace inhibitors and angiotensin ii receptor blockers represent the first line therapy for primary and secondary cvd prevention in patients with diabetes . recent research has uncovered new dimensions of the raas and , therefore , new potential therapeutic targets against diabetic cvd . here we describe the timeline of paradigm shifts in raas understanding , how diabetes modifies the raas , and what new parts of the raas pathway could be targeted in order to achieve raas modulation against diabetic cvd . the renin - angiotensin - aldosterone system ( raas ) consists of a group of enzymes and peptides whose main function is to control blood pressure by regulating vasoconstriction , sodium reabsorption , and body fluid homeostasis . the modern view of the raas began with the concept that this was a life - saving system , which raised blood pressure by approximately 30 mmhg in case of an acute hemorrhage . classically , the process whereby the raas raises blood pressure usually starts within the kidney , where a blood pressure fall stimulates renin release into the bloodstream [ 12 , 13 ] . then , circulating renin cleaves hepatic angiotensinogen and generates angiotensin ( ang ) i , which is converted to ang ii by pulmonary angiotensin - converting enzyme ( ace ) , as represented in figure 1 [ 1416 ] . soon after its generation , ang ii causes smooth muscle cell vasoconstriction , stimulates the sympathetic nervous system , and promotes renal retention of salt and water by binding to its specific receptors [ 17 , 18 ] . moreover , in the adrenal glands , ang ii stimulates the release of aldosterone , which enhances tubular sodium reabsorption in the kidney and increases the effective circulating plasma volume . ang ii has two main receptors : ang ii type 1 receptor ( at1r ) and ang ii type 2 receptor ( at2r ) , as represented in figure 2 . studies in mice lacking at1r showed that the hemodynamic actions of ang ii depended on at1r [ 2022 ] . on the other hand , at2r , which was found highly expressed in differentiated mesenchymes during fetal life and decreased rapidly after birth , seemed to regulate fetal development [ 18 , 23 ] . nevertheless , at2r is still detectable in adulthood in different organs , such as the heart , kidney , adrenal glands , brain , ovaries and uterus , and the vessels , where its main function is to counterbalance at1r . for instance , at2r - knockout mice exhibit high blood pressure levels and increased vascular sensitivity to ang ii [ 25 , 26 ] , suggesting that at2r antagonizes angii - at1r peripheral effects . such an action could be achieved not only by vasodilator effects that are independent of at1r [ 27 , 28 ] but also by at1r downregulation and/or direct inhibition of at1r signaling [ 30 , 31 ] , as represented in figure 2 . although the raas was a life - saving system , the first aspect that became clear was that if the ang ii cascade was activated inappropriately , it could lead to hypertension and cvd . therefore , ace inhibitors and ang ii type 1 receptor blockers ( arb ) were designed as antihypertensive therapies [ 33 , 34 ] . a paradigm shift in raas understanding occurred when the consensus and the solvd trials showed that the ace inhibitor enalapril reduced overall mortality by 27% and 16% in patients with heart failure [ 35 , 36 ] . enalapril 's benefit suggested that the raas had significant nonhemodynamic actions that were associated with cardiovascular pathology . these trials led to mechanistic studies demonstrating that ang ii promoted ventricular hypertrophy [ 3739 ] , myocardial infarction , and atherosclerosis [ 4143 ] , independent of blood pressure values . in particular , by binding to at1r , ang ii was able to induce reactive oxygen species generation , tissue inflammation and fibrosis , and the regulation of cell growth and differentiation as well as apoptosis and survival as summarized in table 1 [ 4163 ] . the observation that many tissues were capable of synthesizing the raas key components led to another paradigm shift in the timeline of raas understanding : the raas was not anymore only a circulating hormonal system but also a tissue system widespread in cardiovascular organs [ 6468 ] . ace and ang ii receptors were identified in all the relevant target tissues including the heart , kidney , blood vessels , and adrenal glands [ 6971 ] , where they have not only endocrine but also paracrine and autocrine effects . interestingly , circulating and local systems can sometimes behave in opposite ways , like in cases of high salt intake , which generally downregulates circulating and upregulates local raas . renin , angiotensinogen , ace , and ang ii receptors were all present in the heart , where they were found upregulated in models of cardiac injury , such as volume overload , myocardial infarction , and heart failure [ 7678 ] . for example , one of the first experimental observations was that ace increased in heart failure , its cardiac induction was tissue specific , and it correlated with the size of myocardial infarction . interestingly , ace was expressed not only in the cardiomyocytes adjacent to the infarct size , but also in fibroblasts , macrophages , and endothelial cells . further studies demonstrated that the increase in cardiac ace was functionally significant , as it was associated with an increased intracardiac conversion of ang i to ang ii , which could potentially impair ventricular function and induce ventricular arrhythmias , eventually promoting heart failure . almost a decade after the consensus and the solvd studies , the hope trial extended to high - risk patients , such as those with diabetes , the concept that raas blockade protected against cardiovascular morbidity and mortality . in that trial , ramipril significantly reduced the rates of death , myocardial infarction , and stroke in patients with vascular disease or diabetes . likewise , the life trial showed that losartan was more effective than atenolol in reducing cardiovascular morbidity and mortality in patients with hypertension , diabetes , and ventricular hypertrophy . also the europa and advance trials provided evidence that an ace inhibitor treatment improved survival and reduced the risk of major cardiovascular events in patients with diabetes . consistent with these results , experimental studies demonstrated that ang ii blockade significantly reduced cardiac damage [ 8 , 9 ] and atherosclerotic plaque accumulation [ 6 , 7 ] in the context of diabetes . more recently , the ontarget trial has evaluated whether the combination of an ace inhibitor with an arb was better than the full dose of either drug . in the end , there was no superiority of the ace inhibitor versus the arb in terms of cardiovascular events . moreover , dual blockade did not confer greater cardiovascular protection and it actually put risk of more adverse events . at the same time the rales and ephesus trials showed that aldosterone antagonism on top of ang ii blockade provided a major additive benefit as it reduced significantly overall mortality and the rate of death from cardiovascular causes among patients with severe heart failure or after acute myocardial infarction . these results suggested that ang ii blockade could not totally suppress the production of aldosterone and that other factors in addition to ang ii were important in the production of this hormone . moreover , they reinforced the notion that aldosterone promoted organ damage by a series of cellular and tissue effects , elicited by binding predominantly to the mineralocorticoid receptors , which are reported in table 1 [ 86103 ] . more recently , eplerenone benefits have been extended to diabetic patients , where this treatment reduced adverse cv events in diabetic patients with heart failure following myocardial infarction . a randomized study evaluating the effects of two aldosterone antagonists , finerenone versus eplerenone , in diabetic patients with heart failure is still ongoing . nevertheless , ace2 discovery represents perhaps the last fundamental paradigm shift in raas understanding [ 106 , 107 ] . ace2 is a carboxypeptidase whose main function is to degrade ang ii to generate ang 17 , as represented in figure 3 . although ace2 can also degrade ang i to generate ang 19 , its catalytic efficiency is 400-fold higher with ang ii , and therefore its main effect is the degradation of ang ii to ang 17 [ 108 , 109 ] . ang 17 is a biologically active peptide , which exerts opposite peripheral actions to those of ang ii by binding predominantly to the mas1 receptor ( mas1r ) . cellular and tissue effects of ang 17/mas1r are summarized in table 1 [ 112128 ] . moreover , ang 17 , like all ang ii breakdown products , has the potential to act as an endogenous ligand of at2r , further promoting organ protection ( figure 3 ) . the identification of ace2 provided evidence that the raas had two pathways with opposite effects : the classic ace / ang ii / at1r and the new ace2/ang 17/mas1r ( and at2r ) pathway . accordingly , the current scientific opinion is that what is critical in cvd development is an imbalance in ace - ang ii and ace2-ang 17 . consistent with this view , ace2 is regarded as the central regulator of the raas , as its changes can affect not only ang ii detrimental actions but also ang 17 protective effects . for instance , a decrease in ace2 results in activation of the ang ii / at1r axis with a parallel reduction of ang 17 [ 51 , 132 ] . nevertheless , ace2 protective effects can be attributed not only to the degradation of ang ii and the generation of ang 17 or to the degradation of ang i and the generation of ang 19 , as represented in figure 3 , but also to the local increase of anp . having said that , the final entry in the raas is the ( pro)renin receptor [ ( p)rr ] , which is a specific receptor for both renin and its inactive precursor prorenin , collectively called ( pro)renin ( figure 3 ) . when ( pro)renin binds to ( p)rr it can degrade angiotensinogen to ang i , but it can also trigger intracellular signaling pathways , which are independent of ang ii generation . interestingly , the ability of prorenin to activate the raas depends on the fact that its binding induces a conformational change that exposes prorenin active site , which becomes catalytically active and generates ang i. the biology of ( p)rr is very complex and only partially understood . the association between ( p)rr gene polymorphism and ambulatory bp in japanese men has suggested that ( p)rr was related to cardiovascular disease leading to experimental studies supporting this hypothesis . the cellular and tissue effects of ( pro)renin are reported in table 1 [ 136145 ] . nevertheless , transgenic animals overexpressing ( pro)renin developed hypertension that was sensitive to ace inhibition , indicating that this effect was due to ang ii generation , which led to arguing against an independent role of ( p)rr in cvd [ 146 , 147 ] . on the other hand , batenburg and danser have suggested that ( pro)renin overexpression led to a 400-fold increase of plasma ( pro)renin levels , which is far from the 1000-fold100.000-fold increase that is required to observe ( pro)renin effects in vitro . certainly , ( p)rr has essential functions that are independent of the raas and are related to the vacuolar h - proton adenosine triphosphatase ( v - atpase ) . in particular , ( p)rr is a cofactor of the v - atpase and is required for central nervous system development [ 151 , 152 ] . ( p)rr is also an essential partner in wnt receptor complex signaling , which regulates normal pattering of the embryo , and in adults , cell proliferation , migration , polarity , and tissue repair . these findings explain not only why ( p)rr deletion yielded embryos that died before the end of embryogenesis [ 153 , 154 ] but also why mice with specific atp6ap2/(p)rr knockout in cardiomyocytes died of severe heart failure within 3 weeks of age . the current picture of the raas is that of an extremely complex pathway whose overstimulation triggers a chain of events contributing to cardiovascular disease . the raas is found not only in the circulation , where it has hemodynamic effects , but also at a tissue level , where it has nonhemodynamic effects . it is activated in the hypertrophied , ischemic , and failed heart , and activation of the ace / angii / at1r axis leads to both accelerated atherosclerosis and direct cardiac injury , as shown by infusing ang ii or by the studies where raas blockade was able to attenuate or prevent cardiac damage . patients with diabetes have a higher prevalence of cardiovascular morbidity and mortality as compared to the general population . on one hand , diabetes is associated with accelerated atherosclerosis affecting the coronaries , which increases the risk for myocardial infarction and heart failure . on the other hand , it can cause diabetic cardiomyopathy , which corresponds to a cardiac dysfunction independent of coronary artery disease , hypertension , and valvular complications . miller and colleagues demonstrated that during the early stages of diabetes there was an increase in plasma renin activity , mean arterial pressure , and renal vascular resistance . moreover , the fact that losartan lowered blood pressure more in hyperglycemic than in euglycemic conditions , and that captopril and eprosartan caused a greater renal vasodilator response during hyperglycemia , suggested that glucose levels were associated with an activation of the raas , which made diabetic patients more sensitive to raas antagonism . consistent with this view , direct renin inhibition with aliskiren led to significant improvement of left ventricular hypertrophy and end - systolic volume only in groups of patients with diabetes . the importance of raas antagonism in the prevention / reduction of cardiovascular complications has clearly demonstrated that diabetes - induced raas activation contributes substantially to diabetic cvd [ 5 , 8082 ] . activation of angii / at1r pathway in the setting of diabetes can in fact promote cell growth and proliferation , apoptosis , oxidative stress generation , inflammation , and fibrosis , which are all leading to cardiac remodeling and atherosclerosis , that can be reversed / reduced by raas blockade [ 79 ] . firstly , hyperglycemia directly stimulates local ang ii production in cardiomyocytes , cardiac fibroblasts , and endothelial cells as well as in murine and human diabetic heart tissues [ 164 , 166 ] . for example , fiordaliso and colleagues demonstrated that glucose increased ang ii in cardiac myocytes , which made them more susceptible to undergo apoptosis , consistent with the view that the raas promotes the development of diabetic cardiomyopathy [ 162 , 166 ] . studies on cardiac myocytes suggest that the mechanism whereby hyperglycemia increases local ang ii in the heart is the generation of intracellular ang ii by intracellular chymase and/or internalized prorenin . then , intracellular ang ii could directly produce oxidative stress and cellular apoptosis and/or enhance raas components expression through a positive feedback mechanism . the second mechanism underlying angii / at1r activation in diabetes is that high glucose concentrations can enhance the tissue response to ang ii and vice versa . while ang ii can increase the activation of glucose - induced transcription factors in vascular smooth muscle cells , hyperglycemia can increase the contractile aortic response to ang ii . this additive effect could be ascribed to the fact that diabetes induces at1r expression in the heart [ 171 , 172 ] and the vasculature . interestingly , several works have demonstrated that hyperglycemia can also stimulate aldosterone secretion by increasing local ang ii . xue and siragy observed an upregulation of renal aldosterone synthase in diabetic rats , which was significantly reduced by at1r blockade . locally , aldosterone triggers a vicious cycle that promotes angii / at1r effects as it mediates part of ang ii effects , increases tissue ace and at1r , and reduces tissue ace2 [ 178 , 179 ] . moreover , together with ang ii , aldosterone promotes oxidative stress , fibrosis , and apoptosis . therefore , many studies are now supporting at least a synergistic role for aldosterone in the pathogenesis of diabetic cardiovascular complications . the third way whereby diabetes promotes ang ii tissue actions is through the several metabolic abnormalities associated with hyperglycemia . these include advanced glycation end products , which form after prolonged hyperglycemia and oxidative stress , dyslipidemia , and low - grade inflammation . all of them can in fact stimulate the angii / at1r pathway by upregulating at1r expression [ 181184 ] . an interesting aspect that should be considered is that , in the heart , the conversion of ang i to ang ii relies not only on ace activity but also on other enzymes such as tissue chymase [ 185 , 186 ] , which are found primarily in mast cells . given this localization , another intriguing mechanism whereby diabetes enhances angii / at1r actions is ace2 downregulation , which does not only promote ang ii actions but also reduce local ang 17 leading to an imbalance of the raas . this concept is supported by the works of tikellis and colleagues , who showed that the induction of diabetes was associated with a significant reduction of ace2 expression and ace2 activity in the heart and the vasculature together with a significant increase in circulating ang ii and a significant reduction of ang 17 levels . similar changes were reported in the kidney of diabetic mice [ 132 , 188 ] . it has been argued that ace2 may be more important than ace in regulating cardiac levels of ang ii and ang 17 and therefore more important for balancing raas activation . this concept relies on the experimental evidence that while ace2 deficiency results in increased cardiac levels of ang ii and reduced levels of ang 17 , ace deficiency does not modify cardiac ang ii , which could be generated by non - ace pathways , including chymases . at2r expression was found increased in the hearts of diabetic rats [ 171 , 191 ] , as well as in their kidneys . similar results were obtained in kidney biopsies of patients with type 2 diabetes ; this is in line with the report that glucose induced at2r renal expression both in vivo [ 194 , 195 ] and in vitro through the expression of the transcriptor factor irf-1 . lastly , diabetic patients have higher prorenin levels than normal subjects , and their prorenin levels are 10-fold higher than their renin levels . given that the binding of prorenin to ( p)rr can generate ang i and also stimulate ( p)rr intracellular signaling , it has been argued that plasma prorenin could contribute more substantially than renin to the pathogenesis of end - organ damage . this is supported by the finding that diabetic patients with high prorenin levels had a higher risk of microvascular complications , whose development could be predicted by the same prorenin levels . consistent with these circulating changes , connelly and colleagues demonstrated that , also at a tissue level , diabetic cardiomyopathy was associated with a 3-fold increase in both ( p)rr gene and protein expression , which were reduced by the renin inhibitor aliskiren . although the increased expression of ( p)rr in the diabetic heart was interpreted as a beneficial response to limit intracellular acidosis , due to its sequence identity with the v - atpase , it was argued that ( p)rr abundance could still modulate the activity of the local raas and expose cardiac myocytes to ang ii increased concentrations . ace inhibition , at1r antagonism , and mr blockade are some of the most classic therapeutic strategies against cvd . another classic therapeutic strategy against heart failure is to increase natriuretic peptide levels , as they are natriuretic , diuretic , vasodilating , and able to inhibit pathologic growth in heart failure . however , although the idea to boost their activity in patients with heart failure seemed theoretically infallible , the approaches used for years have produced only partial if not disappointing effects . these approaches included short - term intravenous infusions of natriuretic peptides or inhibition of neprilysin , which is the enzyme that degrades natriuretic peptides , together with bradykinin , adrenomedullin , and possibly other substrates such as ang ii . so , the most likely explanation underlying the disappointing effect of the oral inhibitor of neprilysin was that neprilysin degraded also ang ii , and therefore inhibiting neprilysin would increase not only natriuretic peptides but also ang ii actions . eventually , the combination of an ace and a neprilysin inhibitor solved this problem and turned out to be superior to either approach in terms of cardiovascular benefits [ 201 , 202 ] . unfortunately , in clinical trials this combination was associated with angioedema [ 203 , 204 ] . in order to overcome this issue , angiotensin receptor - neprilysin inhibitors ( arnis ) have been developed , such as lcz696 , which is an association of the arb valsartan with the neprilysin inhibitor sacubitril ( figure 4 ) . in a recent trial this association was found superior to enalapril in reducing the risk of death and hospitalization in patients with heart failure , including those with diabetes . this is consistent with the observation that the administration of lcz696 in an experimental model of diabetes significantly reduced cardiac hypertrophy and fibrosis and improved ejection fraction after myocardial reperfusion injury . another classic target against cvd is aldosterone , due to the growing appreciation of its contribution to cvd development and progression . the drugs that are available for blocking aldosterone actions are spironolactone and eplerenone , which are both mineralocorticoid - receptor antagonists . nevertheless , spironolactone and eplerenone have still limited clinical use , due to the poor selectivity of spironolactone , the low potency of eplerenone , and the fact that only mr - dependent effects of aldosterone can be inhibited . an alternative approach to antagonize aldosterone the compounds targeting cyp11b2 that have been studied so far are fad286 , which has been used mostly in experimental settings , and lci699 , which has been developed for human use in 2010 . a few works have demonstrated that fad286 reduced mortality , improved cardiac remodeling [ 209 , 210 ] , and reduced atherosclerosis . nevertheless , both drugs have exhibited clear side effects due to the inhibition of cyp11b1 , which synthesizes glucocorticoids . moreover , another concern prior to their use is that aldosterone synthase inhibitors could allow the activation of unprotected mr by glucocorticoids , and this needs further research . this can be achieved with the renin inhibitor aliskiren , which attenuates ang ii generation and reduces blood pressure comparably to ace inhibitors and arb . however , similarl to what was reported in the ontarget trial , the altitude study has demonstrated that the simultaneous administration of aliskiren with an ace inhibitor or an arb should be avoided . a critical concern on the use of aliskiren is that it is associated with high renin levels that might bind to ( p)rr [ 214 , 215 ] . therefore , specific blockade of ( p)rr could provide more benefits as compared to renin inhibition alone , as it could not only reduce ( pro)renin enzymatic activity but also prevent some angii - independent effects of ( pro)renin . so far , a ( p)rr antagonist , called hrp ( handle region peptide ) , has been designed . in particular , this molecule mimics the handle region of prorenin , which allows its binding to ( p)rr and its subsequent nonproteolytic activation . studies in animal models of diabetes show promising results as reviewed in , possibly because hrp exerts its effects only in diseases associated with high prorenin and low renin levels , such as diabetes . a promising therapeutic strategy in cardiovascular medicine , raas modulation combines ace / angii / at1r blockade with the stimulation of ace2/ang 1 - 7/mas1r and at2r . the latter can be achieved by a series of new therapies that includes ace2 replenishing strategies , ang 17 administration , and at2r agonists . however , also ace inhibitors and at1r blockers should be considered raas modulating agents , as they can increase ace2 expression and ang 17 levels severalfold , suggesting that part of their beneficial effects is due to ace2/ang 17 effects [ 216 , 217 ] . having said that , crakower and colleagues showed that ace2 is an essential regulator of cardiac function , by demonstrating that ace2 deficiency was associated with reduced systolic function . similar findings have been reported in animal models of type 1 and type 2 diabetes [ 219 , 220 ] . in addition to that , we have demonstrated that ace2 deficiency was associated with accelerated atherosclerosis . it has also to be noted that ace2 deficiency might in fact influence not only the risk of developing cvd but also the response to the treatment , particularly in the setting of diabetes where ace2 expression is constitutively low . these findings emphasize the potential utility of ace2 repletion as a strategy to reduce cvd . current therapeutic tools that modulate ace2 levels or activity , which are still under investigation , include adenoviral ace2 gene transfer , recombinant human ace2 ( rhace2 ) , ace2 activators , oral ace2 , and ang 17 bioencapsulated in plant cells . so far , both ace2 gene transfer and the administration of an ace2 activator have ameliorated diabetic cardiomyopathy [ 221 , 222 ] . moreover , the reports that rhace2 administered intravenously to healthy human subjects was well tolerated and that it resulted in sustained reduction in plasma ang ii levels and elevation in ang 17 levels are encouraging , as they are shortening the gap between bench and bedside with respect to the possibility of using rhace2 in clinical practice . based on the protective cardiovascular effects of ang 17 , several experimental studies have tested the hypothesis that ang 17 infusion could ameliorate diabetic cardiomyopathy . in all these works , ang 17 improved all the structural hallmarks of diabetic cardiomyopathy , which is characterized by left ventricle hypertrophy and left and right ventricle fibrosis and dysfunction [ 224226 ] . in db / db mice with early and advanced disease , ang 17 reversed diabetes - induced changes . in particular , in the early disease model , ang 17 increased cardiac output and cardiac index and decreased cardiomyocyte hypertrophy , heart fibrosis , and inflammation . vascularization was also improved , which correlated with increased numbers of bone marrow residing in and circulating endothelial and mesenchymal stem cells . here , mas1r seemed to be the main receptor mediating ang 17 effects , which explains why ave0991 , which is a mas1r agonist , had cardioprotective effects in diabetic rats . in addition , in the advanced disease model , ang 17 ameliorated diastolic function and decreased not only cardiac hypertrophy and fibrosis but also triacylglycerol accumulation . moreover , consistent with the report that ang 17 preserved cardiac function , coronary perfusion , and aortic endothelial function in a rat model of heart failure , ang 17 improved cardiac recovery from ischemia / reperfusion and restored the normal reactivity to constrictor and dilator stimuli in the vasculature . recently , we have shown that ang 17 administration significantly reduced ex vivo leukocyte recruitment in an animal model of diabetes . this was associated with a reduction of glucose - induced molecule adhesion expression and leukocyte adhesion to endothelial cells in vitro . these effects were completely blocked by the mas1r antagonist , suggesting that mas1r was the main receptor mediating ang 17 effects on endothelial cells . at2r activation has been currently achieved by compound 21 ( c21 ) , which is a nonapeptide that acts as a highly selective at2r agonist and stimulates at2 receptors . however , those that have been carried out in diabetic conditions have explored its actions mostly against renal fibrosis . in this setting , c21 was able to significantly reduce renal fibrosis in experimental models of both type 1 and type 2 diabetes . in one of these studies , c21 was able to significantly reduce also the expression of several inflammatory mediators . this finding is consistent with the vascular effects of this drug , which prevented endothelial inflammation and reduced leukocyte adhesion both in vivo and in vitro . in addition , recently , c21 was found to be able to reduce diabetes - associated atherosclerosis . nevertheless , the current picture of the raas is that of an extremely complex pathway , which has not been fully characterized yet and might hold in store new aspects that have still to be discovered . this is particularly true in diabetic conditions , where ang ii / at1r pathway is activated , while the ang 17/mas1r is not . therefore , the aim of new therapies is not only to block ang ii harmful effects but also to augment the activity and actions of potentially beneficial pathways , by ace2 replenishing strategies , ang 17 administration , and at2r agonists . here is another paradigm shift : treatment of diabetic cardiovascular disease should move from raas inhibition to raas modulation .

since the advent of insulin , the improvements in diabetes detection and the therapies to treat hyperglycemia have reduced the mortality of acute metabolic emergencies , such that today chronic complications are the major cause of morbidity and mortality among diabetic patients . more than half of the mortality that is seen in the diabetic population can be ascribed to cardiovascular disease ( cvd ) , which includes not only myocardial infarction due to premature atherosclerosis but also diabetic cardiomyopathy . the importance of renin - angiotensin - aldosterone system ( raas ) antagonism in the prevention of diabetic cvd has demonstrated the key role that the raas plays in diabetic cvd onset and development . today , ace inhibitors and angiotensin ii receptor blockers represent the first line therapy for primary and secondary cvd prevention in patients with diabetes . recent research has uncovered new dimensions of the raas and , therefore , new potential therapeutic targets against diabetic cvd . here we describe the timeline of paradigm shifts in raas understanding , how diabetes modifies the raas , and what new parts of the raas pathway could be targeted in order to achieve raas modulation against diabetic cvd .

1. Introduction 2. Paradigm Shifts in the Renin-Angiotensin-Aldosterone System Understanding 3. Diabetes, the Activation and Imbalance of the Renin-Angiotensin-Aldosterone System, and Cardiovascular Disease 4. New Potential Therapies against Diabetic Cardiovascular Complications 5. Conclusions

although endovascular aneurysm repair ( evar ) has been established as a standard intervention for abdominal aortic aneurysm ( aaa ) ( 1 , 2 ) , it is not ideal or feasible for the patients who have challenging anatomic features that can not be overcome by the design of currently available devices ( 3 - 6 ) . the estimated proportion of aaa patients suitable for evar varies and has been reported as high as 80% ( 7 - 11 ) . however , most of the reports came from western countries and there have been only a few data from asian population who have smaller body size and subsequently may have different anatomic features ( 12 , 13 ) . with rapid aging of the population and economical development in east asia , the incidence of newly diagnosed aaa is rapidly increasing ( 14 , 15 ) . so , it would be pertinent to answer the following questions ; 1 ) how large proportion of patients with aaa can be effectively treated with evar in the region , 2 ) are the suitability rate and anatomic features different from those of western patients , 3 ) is there need for modification of device design specific to asian patients ? this study aimed to answer those questions by retrospective analysis of the aortoiliac morphology in a single - center korean patient cohort . from the database linked to the electronic medical records of the hospital , the patients who had aaa as one of their diagnoses were sought . among them , only the patients who underwent contrast - enhanced spiral computed tomography ( ct ) and whose maximal aneurysm diameter was 40 mm or larger were included for this study . in a 7-yr period from july 2003 through august 2010 , a total of 191 patients were identified ( table 1 ) . for these patients , suitability of the aneurysm morphology for evar was retrospectively investigated by review of their ct images . morphologic characteristics of the aneurysm such as the diameters and the lengths of the proximal aortic neck and both common iliac arteries were measured with digital calipers on a pacs workstation . all the diameters were measured in the perpendicular plane to the vessels ' long axes on the sagittal or coronal images . infrarenal neck angulation ( in degrees ) was determined with the digital goniometer . for the common iliac artery , , the narrowest diameter was determined along the external iliac and common femoral arteries at both sides . suitability of evar was determined according to the criteria recommended by the manufacturers ( table 2 ) of the four fda - approved devices which are available in korea ; aneurx ( medtronic cardiovascular , santa rosa , ca , usa ) , talent ( medtronic cardiovascular ) , zenith ( cook medical . , bloomington , in , usa ) , and excluder ( w. l. gore & associates , flagstaff , az , usa ) . the aneurysm was considered suitable for evar when the morphologic characteristics met the manufacturer 's recommendation of any of the four devices . in determining the suitability , factors prone to subjective variation such as wall calcification , luminal thrombus or atheroma , shape of the neck ( straight vs conical ) , and iliac artery tortuosity were not taken into consideration . comparison between groups were performed for statistical significance with student t - test , one - way anova , pearson 's correlation , or fisher 's exact tests according to the type of the variables . those tests were performed using spss version 15.0 ( spss inc , chicago , il , usa ) . b-1011 - 116 - 106 ) and waived the need of consent from individual patients . b-1011 - 116 - 106 ) and waived the need of consent from individual patients . endovascular repair of the abdominal aortic aneurysm would have been feasible in 89 patients ( 46.6% ) if the manufacturer 's guidelines had been strictly followed without compromise of the criteria . the most frequent causes for unsuitability were aneurysmal dilatation of the common iliac artery and excessive angulation of the aneurysm neck ( table 3 ) . especially , common iliac artery dilatation was the most frequently found problem ( 63/191 , 33.0% ) and bilateral dilatation was present in almost the same number of patients as unilateral dilatation . the problem of short proximal landing zone was found in a relatively minor proportion of the patients ( 17/191 , 8.9% ) . other factors that may be expected to be present in asian population whose body size is small were present also in relatively small numbers ; small neck diameter in 12 patients ( 6.3% ) , small common iliac artery in 5 patients ( 2.6% ) , and small external iliac / femoral arteries in 9 patients ( 4.7% ) . the problems making evar unsuitable were present in the neck and the common iliac arteries with almost equal incidences . in addition , both the neck and common iliac arteries had morphological characteristics unsuitable for evar in 46 patients ( 24% of entire patients and 45.1% of unsuitable patients , table 3 ) . when the characteristics of the aortoiliac morphology in this series were compared with previous reports from other countries ( 12 , 13 , 16 , 17 ) , there was no difference in the diameter and length of the proximal neck and the lengths of the common iliac arteries . the only remarkable difference was the larger average diameter of the common iliac arteries in our patients ( table 4 ) . between the patients who had aneurysm suitable for evar and those who did not , there was no significant difference in the age ( 72.2 8.3 vs 73.6 7.6 yr ) , height ( 165.5 7.4 vs 163.9 8.8 cm ) , or body weight ( 62.2 10.0 vs 59.2 10.8 kg ) . there was also no significant difference in evar suitability between genders ( 47.4% in male vs 42.9% in female ) . while female patients had more chance of small access ( external iliac and common femoral artery diameter < 8 mm ) than male patients ( 8.6% vs 3.8% ) , the difference was not statistically significant . although the height ( 160.6 8.1 cm vs 164.9 8.2 cm ) and body weight ( 54.5 13.4 kg vs 60.9 13.4 kg ) were smaller in the patients who had small access problem , the difference was not statistically significant , either . meanwhile , suitability of evar was significantly correlated with the maximal diameter of aneurysm sac . more than 60% of aneurysms with the maximal diameter 6 cm or less were suitable for evar without compromise of the manufacturer 's guidelines . on the contrary , the suitability decreased markedly as the aneurysm sac size increased larger than 6 cm ( p < 0.05 , fig . 1 ) . in addition , the larger the aneurysm became , the more was the chance of having problems in both of the proximal and the distal landing zones . almost 60% of the aneurysms with the maximal diameter 8 cm or larger had unfavorable morphology in the neck and common iliac arteries at the same time . considering the aging of the large population along with economic growth and advance of health care system in asia , there will be a rapid increase in the incidence of aortic aneurysm in the region . the question of whether the suitability for evar is the same between different races would be pertinent to modifying the stent - graft design targeting the large potential market . however , we could find only two articles addressing this issue in previous literature ( 12 , 13 ) . while the former two studies investigated the anatomical features only in the patients who underwent endovascular repair , our study included all the patients who had abdominal aortic aneurysm with the maximal diameter 4 cm or larger . as a result , many patients who declined any intervention or underwent conventional surgery were subject to analysis ( table 1 ) . many of our surgical patients could have been treated with endovascular procedure if the fda - approved commercial devices had been available ( they became available in korea since 2007 ) . this fact makes our study helpful for giving insight into the suitability rate for evar among the entire aaa population and reasons for unsuitability . according to our results , morphological characteristics ' ideal ' for stent - graft implantation were found in about a half of korean aaa patients . if the criterion of common iliac artery diameter is compromised by placement of the iliac limb of stent - graft in the external iliac artery and sacrificing one or both internal iliac arteries , the proportion of ' feasible ' patients can increase to 65% . however , the actual proportion of ' ideal ' or ' feasible ' patients would have been less if we had taken into consideration other factors prone to subjective variation such as wall calcification , luminal thrombus or atheroma , shape of the neck , and iliac artery tortuosity . considering the all of above factors , we think that the overall ' feasibility ' rate for evar may be almost the same or a little lower in korean patients compared with western population . although the estimates regarding the rate of suitability or feasibility for evar vary between different reports , recent western studies reported 48%-66% rate with current commercial devices ( 7 , 8) . while the overall feasibility rate is not thought much different , we could find differences between our data and previous reports regarding the anatomical features making evar unsuitable . in a report from the netherlands ( 7 ) , unsuitability was more frequently caused by inadequate proximal neck , especially inadequate length of the neck , than by inadequate iliac anatomy . on the contrary , in our series , neck features and iliac anatomy were the reasons for unsuitability in almost the same frequency and excessive angulation was the most frequent features making the neck unstable for evar . reported that both the neck and common iliac arteries of chinese patients were larger and shorter compared with caucasians ( 12 ) . however , the only difference between our patients and previous western series ( and the only common finding between ours and the chinese report ) was that the common iliac artery was significantly larger in asian patients . another difference from cheng 's report was that our patients had significantly higher incidence of severe neck angulation . it could be explained by the difference of the study material because cheng 's report included only the patients who underwent evar while our study examined all the patients with aaa . we have to admit that our measurement of the neck length may be inaccurate in some patients , because it was made in sagittal or coronal view ( instead of center - line program ) and sometimes with 4-mm - thick slices that is too large for a short neck . however , considering that the difference of average neck lengths was just 5 mm between ours and the chinese series that included only the evar cases , we do not think the possible measurement error had a significant impact in interpretation of our data . in a study of hawaiian patients , masuda et al . ( 13 ) reported that the external iliac artery diameter was significantly smaller in asians than in other races ( 8.2 vs 9.5 mm ) and this factor was related with higher incidences of access - related and device - related complications after aortoiliac endografting . however , the mean external iliac artery diameter of our patients was well above 9 mm and small access problem was found in only 9 patients ( 4.7% ) . the difference may be related to the fact that the patients in hawaiian study were significantly older than ours . although their older patients might have smaller body size and subsequently smaller iliofemoral arteries than ours , their data did not mention about the body size of the patients . as both of chinese series and ours show that the common iliac arteries are larger in aaa patients , common iliac artery aneurysm may be the most frequent problem that makes evar more complicated in asian patients . among western literature , we could find only one report addressing the issue of common iliac artery aneurysm in aaa patients . armon et al . ( 18 ) reported that the mean common iliac artery diameter was significantly larger in aaa patients compared with general population . they suggested that common iliac artery aneurysm in aaa patients should be defined as those greater than 2.4 cm in diameter . with their definition , the incidences of unilateral and bilateral common iliac artery aneurysm were 16% and 12% , respectively . our series shows the similar incidences ( 14.1% and 15.2% ) and internal iliac artery coverage would have been necessary in 34% of our patients if evar had been performed . ( 12 ) reported that internal iliac artery coverage was necessary in 51% of chinese patients undergoing evar . correlation between evar feasibility with gender , age , and maximal aneurysm diameter has been controversial in the previous studies ( 7 , 8 , 19 , 20 ) . in our series , feasibility for evar was not different according to the patient 's age or gender . however , there was a strong correlation between the size of the aneurysm and evar suitability . as the maximal aneurysm diameter increases larger than 6 cm , the suitability decreased significantly . in addition , it is worth mentioning that unsuitability was caused by multiple anatomic features rather than a single factor as the aneurysm size increases . our result is concordant with the previous studies which showed that larger aneurysms were less suitable for evar and had higher incidence of complications or unsatisfactory outcome after evar ( 20 - 22 ) . in conclusion , the overall ' feasibility ' rate for endovascular repair of abdominal aortic aneurysm in korean patients is not thought to be much different compared with western patients . however , considering that the most frequent causes for unsuitability were different from those in western patients , future modification of stent - graft design should address excessive neck angulation and more attention has to be paid to common iliac artery aneurysm to provide endovascular treatment for more of korean patients especially those who have large aneurysm . suitability of endovascular repair with current stent grafts for abdominal aortic aneurysm in korean patients kay - hyun park , cheong lim , jae hang lee and jae suk yoo in 191 patients with abdominal aortic aneurysm ( aaa ) , aortoiliac morphologic characteristics in ct images were reviewed to determine suitability for evar . the frequent causes for unsuitability were common iliac artery ( cia ) aneurysm ( 61.8% ) and excessive neck angulation ( 52.9% ) . larger aneurysms were more frequently unsuitable for evar and had more chance of having multiple features making evar unsuitable . in conclusion , however , the difference in the major causes of unsuitability requires further consideration in designing stent - graft for evar .

suitability rate of endovascular aneurysm repair ( evar ) and the anatomic features causing unsuitability have not been well determined in asian patients who have abdominal aortic aneurysm ( aaa ) . in a single korean center , a total of 191 patients with abdominal aortic aneurysm ( maximal diameter 4 cm ) were identified . aortoiliac morphologic characteristics in contrast - enhanced computed tomography images were retrospectively reviewed to determine suitability for evar with four fda - approved stent - grafts . aaa was considered ideally suitable for evar in 46.6% of patients . the most frequent causes for unsuitability were common iliac artery ( cia ) aneurysm ( 61.8% ) and excessive neck angulation ( 52.9% ) . problems such as small and/or short neck and small access were found in minor incidences . if cia aneurysm is dealt by overstenting with sacrifice of internal iliac artery , suitability rate can increase to 65% . larger aneurysms were more frequently unsuitable for evar and had more chance of having multiple unfavorable features . in conclusion , the overall feasibility rate for evar in korean patients was not different from that in western patients . however , considering the difference in the major causes of unsuitability , more attention has to be paid to neck angulation and cia aneurysm to provide evar for more korean patients especially who have large aneurysm .

INTRODUCTION MATERIALS AND METHODS Ethics statement RESULTS DISCUSSION AUTHOR SUMMARY

parkinson 's disease ( pd ) is neurodegenerative disorder characterized by progressive loss of substantia nigra pars compacta ( snpc ) neurons1,4,5,20 ) . the progressive loss of cells leads to motor dysfunctions including tremor , rigidity , and bradykinesia6,10,28 ) . neural tissue transplantation has been a promising strategy for pd because the transplanted tissues can produce dopamine in the striatum , which has a therapeutic effect in pd patients2,15 ) . however , tissue grafts have the disadvantages of low rates of cell survival and/or development of dyskinesia9 ) . stem - cell transplantation was recently tried , but the effects were controversial due to possible tumorigenesis8 ) , poor differentiation into dopaminergic cells , or low survival rates19 ) . the transplantation of spheroids , or cell aggregations , has been proposed to overcome these problems , because the cell aggregates can extend their axons into the host brain and establish synaptic connections with host neurons following transplantation into specific brain areas13 ) . in our previous study , human brain - derived neural stem cells ( hb - nsc ) which were injected into the striatum of a pd rat model as single - cell suspensions showed partial differentiation to dopaminergic cells , but without therapeutic effect30 ) . we hypothesized that the injected single hb - nsc cell suspensions could partially differentiate into dopaminergic cells , which produced dopamine within the cell , but were unable to secrete it . therefore , in the present study , we transplanted hb - nsc as cell aggregates form into the striatum of pd rats to determine their efficacy compared to single - cell transplantations . we expected that the cell aggregates have the advantage of a pre - established cell network which reported in transplantation of spheroid form13 ) that might promote high rates of survival and differentiation . after the transplantation , we evaluated the results by behavioral tests , pet study and histological study . all animals were housed individually in cages , with free access to food and water in a climate - controlled room . this study was approved by the institutional animal care and use committee of the asan institute for life science . at the beginning of the study , each animal was anesthetized with zoletil 50 ( virbac s.a , carros , france ) and rompun ( bayer , leverkusen , germany ) at doses of 37.5 mg / kg and 6 mg / kg , respectively , using an intraperitoneal ( i.p . ) injection . a total of 22 rats were administered unilateral injections of 14 g 6-ohda hydrochloride ( sigma , st . louis , mo , usa ) in 4 l of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm , lateral + 1.2 mm relative to the bregma and ventral -7.8 mm from the dura by paxinos 's rat atlas , with the tooth bar set at -2.4 mm . the injections were delivered at a rate of 1 l / min using a hamilton syringe ( 33-gauge ) and an automated microsyringe pump ( harvard apparatus , holliston , ma , usa ) . once the injection was finished , the needle was left in place for 5 minutes to prevent backflow of the solution . among 22 rats , 18 rats which showed apomorphine - induced rotation exceeded 6 rotations per min after 4 weeks of 6-ohda injection ( success rate 81.8% ) were selected for this study . then , hb - nsc cells were injected in 9 rats ( hb - nsc group ) and pbs in 9 rats ( control group ) . however , 2 rats were dead during pet study , so the final numbers of experimental animals became 9 in hb - nsc group and 7 in control group . hb - nsc ( san pedro , ca , usa ) were expanded in brain stem - cell growth medium according to the manufacturer 's instructions . for formation of homogenous aggregates , polyethylene glycol ( peg ) hydrogel microwell arrays with a diameter of 500 m were produced using micro - fabrication procedures , as reported previously13 ) . the expanded hb - nsc were allowed to form aggregations within the peg hydrogel microwell arrays . briefly , hb - nsc at an approximate cell density of 110 cells/200 l were deposited into each microwell array , and then placed in a 5% co2 humidified incubator for 30 min to allow the cells to settle . non - adherent cells were carefully removed by washing with pbs , and the adherent cells were allowed to form aggregations within each microwell . the cell aggregations were retrieved from the peg hydrogel after 5 days of culture in the brain stem - cell growth medium ( celprogen , san pedro , ca , usa ) . the cell aggregates are visible with the naked eyes and the diameter of each aggregate is approximately 200 m . and then , the cell aggregation were administered in sterilized pbs at a concentration of 810 cells/8 l . we could evaluate the concentration of cell aggregation by cell counting with trypan blue viability staining which was partially harvested from cell aggregation solution and transformed into cell suspension . the cell aggregations transplanted into the right striatum of the pd rat model ( hb - nsc group , n=9 ) using a 22 g hamilton syringe and a stereotaxic microinjection device ( coordinates : ap + 0.6 mm from the bregma , ml + 3.5 mm from the midline , dv -4.5 mm , nose bar + 2.3 mm ) 4 weeks after the 6-ohda injection . the control group ( n=7 ) received 8 l of pbs ( ph 7.4 ) injected at the same coordinates . all injections used a rate of 1 l / min , and the needle was held in place for 5 min after the injection , followed by withdrawal at a rate of 1 mm / min . postoperatively , all rats received daily cyclosporine a ( chong kun dang pharm , seoul , korea ) at 10 mg / kg ( i.p . ) until the animals were sacrificed . the stepping tests were performed six times : prior to any treatment , 1 week after 6-ohda injection , and 2 , 4 , 6 , and 8 weeks after cell transplantation , as previously described24 ) , with slight modifications . briefly , both hind limbs were firmly held in one hand of the experimenter , while the other hand was used to hold one of the forelimbs . the anterior end of the animal was lowered onto a treadmill ( jeung do bio & plant co. , seoul , korea ) , which was set at a rate of 0.9 m/5 s. the rat 's body remained stationary while the unilateral forelimb was allowed to touch the moving treadmill track for > 5 seconds while the treadmill was operating . all of the experiments were video recorded to count the number of adjusted steps taken in the backward direction . every rat performed the stepping test twice during every session , and the data were averaged . the data are expressed as the percentage of contralateral forelimb steps versus ipsilateral forelimb steps . the rotation tests were conducted 1 week after 6-ohda injection , and 4 and 8 weeks after cell transplantation , as previously described25 ) , with slight modifications . briefly , as soon as 0.5 mg / kg apomorphine ( sigma ; in sterile water , injected subcutaneously ) was administered , the rat was harnessed to an automated rotometer ( panlab , barcelona , spain ) and its rotational behavior was assessed over 45 minutes . data are expressed as the net average rotations ( contralateral - ipsilateral turns ) per minute . 6-ohda - lesioned rats that demonstrated > 6 rotations per minute were selected for inclusion in the present study . all animals were scanned using the micropet focus 120 system ( siemens medical solutions , inc . , erlagen , germany ) , which includes a 1212 array of lutetium oxyorthosilicate and 96 detector blocks . glucose metabolic activity was assessed in the control ( n=3 ) and hb - nsc groups ( n=5 ) using [ f]-fdg at 10 weeks posttransplantation . after fasting for at least 12 h , the rats were injected with 1 mci [ f]-fdg in 0.2 ml of sterile saline via the tail vein . one hour after the injection , the animals were scanned for 30 min under isoflurane anesthesia . dopamine transporter function was assessed with the same animals in the control ( n=3 ) and hb - nsc group ( n=5 ) using [ f]-fp - cit , which binds to the dopamine transporter with high affinity , at 1 or 2 weeks after 6-ohda injection ( pd status before cell transplantation ) and at 5 and 10 weeks after cell transplantation . rats were injected with 1 mci [ f]-fp - cit in 0.4 ml sterile saline via the tail vein , immediately followed by dynamic scanning for 90 minutes . the [ f]-fdg pet data were reconstructed using a 3-dimensional ( 3d ) maximum a posteriori ( map ) algorithm , and the [ f]-fp - cit data were reconstructed using a 2d ordered - subset expectation maximization16 ) . pet images were analyzed using the asipro software ( siemens preclinical solutions , knoxville , tn , usa ) to generate a 3d volume of interest that consisted of three regions of interest ( roi ) . to produce 3d volume of interest , we selected a coronal image which showed highest uptake of striatum , and then , three consecutive rois in axial images which show striatum were taken on the selected coronal image base . data are expressed as the percentage of ipsilateral standardized uptake values based on the mean uptake in the 3d volume of interest against the contralateral standardized uptake values . the equation of standardized uptake values is [ tissue concentration ( mbq / cc)]/[injected dose ( mbq)/body weight ( g ) ] . rats were transcardially and sequentially perfused with 0.9% saline containing 10000 iu heparin ( hanlim pharm , seoul , korea ) and 4% paraformaldehyde in pbs . extracted brain tissues were postfixed overnight in the same fixative , followed by incubation and dehydration in 30% sucrose until they sank . serial sections from frozen tissue , including the striatum ( anteroposterior + 2.5 to 0.0 mm ) , were produced and the brain tissue was cut into 40 m sections on a sliding microtome ( hm450 ; sliding electric microtome , thermo scientific ) . for ihc , striatal sections were incubated overnight with primary antibodies ( anti - th monoclonal 1 : 5000 , sigma aldrich , st . louis , usa ) diluted in pbs ( ph 7.4 ) containing 0.5% bsa ( bioworld , dublin , usa ) . these sections were subsequently incubated for 2 h at room temperature with biotinylated secondary antibodies produced in mouse ( vector laboratories , burlingame , ca , usa ) and diluted 1 : 200 in pbs containing 0.5% bsa . th staining was detected by adding diaminobenzidine ( r&d system , minneapolis , mn , usa ) . diaminobenzidine - treated tissues were dehydrated in a series of alcohol and clearing agents , and a coverslip was applied . th - positive cells were observed in the coronal sections of the striatum using a nikon 80i microscope ( nikon , tokyo , japan ) at 100 magnification using the nis - elements f3.0 software ( nikon , tokyo , japan ) . for if , tissue sections were incubated with blocking solution containing 1% bsa , 0.2% triton x-100 ( sigma aldrich , st . louis , usa ) and 0.05% sodium azide , rinsed in 0.5% bsa in pbs twice and then incubated with anti - th monoclonal ( 1 : 5000 , sigma aldrich , st . louis , usa ) and anti - human 2 microglobulin polyclonal ( 1 : 800 , dako , seoul , korea ) antibodies for 1 h at room temperature . the sections were washed three times and incubated for 90 min with alexa 546-labeled anti - mouse igg ( 1 : 200 , invitrogen , grand island , ny , usa ) and alexa 488-labeled anti - rabbit igg ( 1 : 200 , invitrogen grand island , ny , usa ) . after washing twice , the sections were mounted onto gelatin - coated slides , dried at 50 for 30 min , and cover - slipped with vector mounting ( dako , seoul , korea ) medium for fluorescence . fluorescent images were obtained using a confocal microscope ( tcs - st2 ; leica , wetzlar , germany ) . behavioral parameters were analyzed using mann - whitney u test , and pet data were analyzed by the paired sample t - test for working out the relative importance of each data which was mat - ched one - on - one . all animals were housed individually in cages , with free access to food and water in a climate - controlled room . this study was approved by the institutional animal care and use committee of the asan institute for life science . at the beginning of the study , each animal was anesthetized with zoletil 50 ( virbac s.a , carros , france ) and rompun ( bayer , leverkusen , germany ) at doses of 37.5 mg / kg and 6 mg / kg , respectively , using an intraperitoneal ( i.p . ) injection . a total of 22 rats were administered unilateral injections of 14 g 6-ohda hydrochloride ( sigma , st . louis , mo , usa ) in 4 l of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm , lateral + 1.2 mm relative to the bregma and ventral -7.8 mm from the dura by paxinos 's rat atlas , with the tooth bar set at -2.4 mm . the injections were delivered at a rate of 1 l / min using a hamilton syringe ( 33-gauge ) and an automated microsyringe pump ( harvard apparatus , holliston , ma , usa ) . once the injection was finished , the needle was left in place for 5 minutes to prevent backflow of the solution . among 22 rats , 18 rats which showed apomorphine - induced rotation exceeded 6 rotations per min after 4 weeks of 6-ohda injection ( success rate 81.8% ) were selected for this study . then , hb - nsc cells were injected in 9 rats ( hb - nsc group ) and pbs in 9 rats ( control group ) . however , 2 rats were dead during pet study , so the final numbers of experimental animals became 9 in hb - nsc group and 7 in control group . hb - nsc ( san pedro , ca , usa ) were expanded in brain stem - cell growth medium according to the manufacturer 's instructions . for formation of homogenous aggregates , polyethylene glycol ( peg ) hydrogel microwell arrays with a diameter of 500 m were produced using micro - fabrication procedures , as reported previously13 ) . the expanded hb - nsc were allowed to form aggregations within the peg hydrogel microwell arrays . briefly , hb - nsc at an approximate cell density of 110 cells/200 l were deposited into each microwell array , and then placed in a 5% co2 humidified incubator for 30 min to allow the cells to settle . non - adherent cells were carefully removed by washing with pbs , and the adherent cells were allowed to form aggregations within each microwell . the cell aggregations were retrieved from the peg hydrogel after 5 days of culture in the brain stem - cell growth medium ( celprogen , san pedro , ca , usa ) . the cell aggregates are visible with the naked eyes and the diameter of each aggregate is approximately 200 m . and then , the cell aggregation were administered in sterilized pbs at a concentration of 810 cells/8 l . we could evaluate the concentration of cell aggregation by cell counting with trypan blue viability staining which was partially harvested from cell aggregation solution and transformed into cell suspension . the cell aggregations transplanted into the right striatum of the pd rat model ( hb - nsc group , n=9 ) using a 22 g hamilton syringe and a stereotaxic microinjection device ( coordinates : ap + 0.6 mm from the bregma , ml + 3.5 mm from the midline , dv -4.5 mm , nose bar + 2.3 mm ) 4 weeks after the 6-ohda injection . the control group ( n=7 ) received 8 l of pbs ( ph 7.4 ) injected at the same coordinates . all injections used a rate of 1 l / min , and the needle was held in place for 5 min after the injection , followed by withdrawal at a rate of 1 mm / min . postoperatively , all rats received daily cyclosporine a ( chong kun dang pharm , seoul , korea ) at 10 mg / kg ( i.p . ) until the animals were sacrificed . the stepping tests were performed six times : prior to any treatment , 1 week after 6-ohda injection , and 2 , 4 , 6 , and 8 weeks after cell transplantation , as previously described24 ) , with slight modifications . briefly , both hind limbs were firmly held in one hand of the experimenter , while the other hand was used to hold one of the forelimbs . the anterior end of the animal was lowered onto a treadmill ( jeung do bio & plant co. , seoul , korea ) , which was set at a rate of 0.9 m/5 s. the rat 's body remained stationary while the unilateral forelimb was allowed to touch the moving treadmill track for > 5 seconds while the treadmill was operating . all of the experiments were video recorded to count the number of adjusted steps taken in the backward direction . every rat performed the stepping test twice during every session , and the data were averaged . the data are expressed as the percentage of contralateral forelimb steps versus ipsilateral forelimb steps . the rotation tests were conducted 1 week after 6-ohda injection , and 4 and 8 weeks after cell transplantation , as previously described25 ) , with slight modifications . briefly , as soon as 0.5 mg / kg apomorphine ( sigma ; in sterile water , injected subcutaneously ) was administered , the rat was harnessed to an automated rotometer ( panlab , barcelona , spain ) and its rotational behavior was assessed over 45 minutes . data are expressed as the net average rotations ( contralateral - ipsilateral turns ) per minute . 6-ohda - lesioned rats that demonstrated > 6 rotations per minute were selected for inclusion in the present study . all animals were scanned using the micropet focus 120 system ( siemens medical solutions , inc . , erlagen , germany ) , which includes a 1212 array of lutetium oxyorthosilicate and 96 detector blocks . glucose metabolic activity was assessed in the control ( n=3 ) and hb - nsc groups ( n=5 ) using [ f]-fdg at 10 weeks posttransplantation . after fasting for at least 12 h , the rats were injected with 1 mci [ f]-fdg in 0.2 ml of sterile saline via the tail vein . one hour after the injection , the animals were scanned for 30 min under isoflurane anesthesia . dopamine transporter function was assessed with the same animals in the control ( n=3 ) and hb - nsc group ( n=5 ) using [ f]-fp - cit , which binds to the dopamine transporter with high affinity , at 1 or 2 weeks after 6-ohda injection ( pd status before cell transplantation ) and at 5 and 10 weeks after cell transplantation . rats were injected with 1 mci [ f]-fp - cit in 0.4 ml sterile saline via the tail vein , immediately followed by dynamic scanning for 90 minutes . the [ f]-fdg pet data were reconstructed using a 3-dimensional ( 3d ) maximum a posteriori ( map ) algorithm , and the [ f]-fp - cit data were reconstructed using a 2d ordered - subset expectation maximization16 ) . pet images were analyzed using the asipro software ( siemens preclinical solutions , knoxville , tn , usa ) to generate a 3d volume of interest that consisted of three regions of interest ( roi ) . to produce 3d volume of interest , we selected a coronal image which showed highest uptake of striatum , and then , three consecutive rois in axial images which show striatum were taken on the selected coronal image base . data are expressed as the percentage of ipsilateral standardized uptake values based on the mean uptake in the 3d volume of interest against the contralateral standardized uptake values . the equation of standardized uptake values is [ tissue concentration ( mbq / cc)]/[injected dose ( mbq)/body weight ( g ) ] . rats were transcardially and sequentially perfused with 0.9% saline containing 10000 iu heparin ( hanlim pharm , seoul , korea ) and 4% paraformaldehyde in pbs . extracted brain tissues were postfixed overnight in the same fixative , followed by incubation and dehydration in 30% sucrose until they sank . serial sections from frozen tissue , including the striatum ( anteroposterior + 2.5 to 0.0 mm ) , were produced and the brain tissue was cut into 40 m sections on a sliding microtome ( hm450 ; sliding electric microtome , thermo scientific ) . , striatal sections were incubated overnight with primary antibodies ( anti - th monoclonal 1 : 5000 , sigma aldrich , st . louis , usa ) diluted in pbs ( ph 7.4 ) containing 0.5% bsa ( bioworld , dublin , usa ) . these sections were subsequently incubated for 2 h at room temperature with biotinylated secondary antibodies produced in mouse ( vector laboratories , burlingame , ca , usa ) and diluted 1 : 200 in pbs containing 0.5% bsa . th staining was detected by adding diaminobenzidine ( r&d system , minneapolis , mn , usa ) . diaminobenzidine - treated tissues were dehydrated in a series of alcohol and clearing agents , and a coverslip was applied . th - positive cells were observed in the coronal sections of the striatum using a nikon 80i microscope ( nikon , tokyo , japan ) at 100 magnification using the nis - elements f3.0 software ( nikon , tokyo , japan ) . for if , tissue sections were incubated with blocking solution containing 1% bsa , 0.2% triton x-100 ( sigma aldrich , st . louis , usa ) and 0.05% sodium azide , rinsed in 0.5% bsa in pbs twice and then incubated with anti - th monoclonal ( 1 : 5000 , sigma aldrich , st . louis , usa ) and anti - human 2 microglobulin polyclonal ( 1 : 800 , dako , seoul , korea ) antibodies for 1 h at room temperature . the sections were washed three times and incubated for 90 min with alexa 546-labeled anti - mouse igg ( 1 : 200 , invitrogen , grand island , ny , usa ) and alexa 488-labeled anti - rabbit igg ( 1 : 200 , invitrogen grand island , ny , usa ) . after washing twice , the sections were mounted onto gelatin - coated slides , dried at 50 for 30 min , and cover - slipped with vector mounting ( dako , seoul , korea ) medium for fluorescence . fluorescent images were obtained using a confocal microscope ( tcs - st2 ; leica , wetzlar , germany ) . behavioral parameters were analyzed using mann - whitney u test , and pet data were analyzed by the paired sample t - test for working out the relative importance of each data which was mat - ched one - on - one . the stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation ; however , at 6 weeks posttransplantation , a significant improvement of 52.9% was measured . at 8 weeks posttransplantation , the increase was 48.9% and this improvement was maintained through to 10 weeks , at which time the increase was 64.2% in the hb - nsc group compared to the control group ( fig . 1a ) . in apomorphine - induced rotation tests , the hb - nsc group showed a slight decrease in contralateral rotations at week 8 , compared to week 4 ; however , the difference was not statistically significant ( fig . there was no significant decrease in uptake of [ f]-fdg in either the control or hb - nsc transplantation group , indicating that glucose metabolism was normal , as is characteristic of pd ( fig . 2a ) . using [ f]-fp - cit to quantify dopamine transporter activity , a gradual increase in radioactivity in the lesion - side striatum was detected in the hb - nsc group and , in particular , there was a statistically significant increase in dopamine trans - porter activity by 10 weeks posttransplantation compared to the pretransplantation state ( 48.53.9% vs. 41.02.6% , respectively ) ( fig . these micropet data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward . ihc and if analysis revealed the presence of tyrosine hydroxylase ( th)-positive cells in the ipsilateral striatum of the hb - nsc transplantation group ( fig . high - magnification images showed that the th - positive cells corresponded to the cells positive for the human 2 microglobulin marker ( fig . the th - positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation ( fig . these results indicate that the implanted hb - nsc aggregates successfully differentiated into dopaminergic cells within the host striatal tissue . this finding differs from our previous study using single - cell suspensions for injection which demonstrated abnormal parallel staining of dendrites ( fig . dendrites of the stained cells extended in various directions , suggesting network formation with the neighboring cells ( fig . the stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation ; however , at 6 weeks posttransplantation , a significant improvement of 52.9% was measured . at 8 weeks posttransplantation , the increase was 48.9% and this improvement was maintained through to 10 weeks , at which time the increase was 64.2% in the hb - nsc group compared to the control group ( fig . 1a ) . in apomorphine - induced rotation tests , the hb - nsc group showed a slight decrease in contralateral rotations at week 8 , compared to week 4 ; however , the difference was not statistically significant ( fig . there was no significant decrease in uptake of [ f]-fdg in either the control or hb - nsc transplantation group , indicating that glucose metabolism was normal , as is characteristic of pd ( fig . 2a ) . using [ f]-fp - cit to quantify dopamine transporter activity , a gradual increase in radioactivity in the lesion - side striatum was detected in the hb - nsc group and , in particular , there was a statistically significant increase in dopamine trans - porter activity by 10 weeks posttransplantation compared to the pretransplantation state ( 48.53.9% vs. 41.02.6% , respectively ) ( fig . these micropet data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward . ihc and if analysis revealed the presence of tyrosine hydroxylase ( th)-positive cells in the ipsilateral striatum of the hb - nsc transplantation group ( fig . high - magnification images showed that the th - positive cells corresponded to the cells positive for the human 2 microglobulin marker ( fig . the th - positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation ( fig . these results indicate that the implanted hb - nsc aggregates successfully differentiated into dopaminergic cells within the host striatal tissue . this finding differs from our previous study using single - cell suspensions for injection which demonstrated abnormal parallel staining of dendrites ( fig . dendrites of the stained cells extended in various directions , suggesting network formation with the neighboring cells ( fig . in previous studies , neural stem cell transplants in pd had difficulty for surviving and differentiating in the host striatum19 ) . over the past decade , clinical trials of neural stem cell transplantation in pd patients as well as animal experiments with pd model used intrastriatal grafts because intrastriatal dopaminergic cell grafts not only are capable of reinnervate with the host striatal tissue but also replace the functions of dopaminergic cells of substantia nigra21,23,26,27 ) . on the contrary , fetal dopaminergic neurons transplanted into the substantia nigra have been reported to regrow axons into the striatum in one report22 ) , but another literature showed failure of extending axons into the striatum12 ) . in this regard , we think the transplantation into substantia nigra does not seem to be effective and appropriate compared to striatal injection and also the injection of enough volume of cells might be difficult in substantia nigra compared to the striatal injection . our previous study on transplantation of hb - nsc as a suspension of single cells showed that the injected cells survived and were demonstrated as positive in th staining , but behavioral tests and [ f]-fp - cit scans showed no improvement30 ) . thus , in this study , aggregation form of hb - nsc were transplanted into the striatum of 6-ohda rat models and the results were evaluated using behavioral tests , multitracer - micropet scans , ihc , and if tissue staining . in the behavioral stepping test , the hb - nsc group showed insignificant increases in contralateral forelimb touches up to 4 weeks after transplantation , but then drastically improved from 6 to 10 weeks posttransplantation . in apomorphine - induced rotation tests , the hb - nsc group exhibited a slight decrease in contralateral rotations by week 8 compared to week 4 posttransplantation , although the difference was not statistically significant . in recent cell transplantation studies , apomorphine - induced rotation tests did not fully reflect the pd status because of apomorphine sensitization18 ) . the significant improvement in the stepping test in the hb - nsc group was not found in the previous single - cell suspension transplant study30 ) . in the stepping test , the drama - tic improvement at 6 weeks posttransplantation suggests an increase in transplanted cell function , previously described as small - world network development by 4 weeks posttransplantation7 ) . the network formed among transplanted hb - nsc or between hb - nsc and host striatal cells could affect cell survival as well as provide therapeutic effects . imaging of dopaminergic neurons by techniques such as [ f]-fp - cit pet scans has been used to diagnose pd3,14 ) . striatal uptake of [ f]-fp - cit , a ligand of the dopamine transporter , decreases in pd . [ f]-fp - cit micropet images were useful and consistent indicators of movement disorders in a rat model in our previous reports29,30 ) . in this study , there was a significant increase in [ f]-fp - cit uptake by 10 weeks posttransplantation compared to the pretransplantation state . these results suggest that the improvements in the behavioral stepping test are the result of dopamine secretion from the transplanted hb - nsc . however , as we described in the methods section , 2 rats in control group were dead during micropet study and comparison between hb - nsc group and control group could not be performed which is a limitation of this study . [ f]-fdg pet scan was performed to evaluate glucose metabolism in the striatum . in typical pd model made by 6-ohda injection into medial forebrain bundle , glucose metabolism of striatum does not decrease . therefore , we studied [ f]-fdg pet at 10 week posttransplantation , and the uptake of bilateral striatum did not show significant differences in control group and hb - nsc group , which suggest successful pd models were made . transplantation of both single - cell suspensions , reported in our previous study30 ) , and aggregates of hb - nsc results in the appearance of th - positive cells in striatum . however , improvements in the stepping tests and increasing uptake of [ f]-fp - cit were demonstrated only after transplantation of hb - nsc aggregates , indicating that dopamine secretion at therapeutic levels only resulted from the trans - plantation of cell aggregates . although single - cell suspensions transplantation resulted in a wide range of th - positive cells in the striatum , the transplanted cells showed abnormal parallel staining which considered to be dendrites of partially differentiated and poorly connected dopaminergic cells ( fig . , no change in [ f]-fp - cit scans in single - cell suspensions transplanted into the striatum suggests that dopamine secretion from the transplanted cells is absent as a result of incomplete differentiation30 ) . by contrast , cell ag - gregate transplantations showed th - positive cells only in a small area in the striatum ( fig . 3b , d ) , but dendrites of the stained cells extended in various directions ( fig . 4d ) suggesting the formation of a network with the neighboring cells . from these data , we hypothesize that the cell aggregates adapt well and efficiently secrete neurotransmitter within the host tissue . the number of cells per transplant in this study was less than 10% of the single - cell suspensions used in the previous study ( 810 vs. 1 10 ) , and this difference in injected cell numbers may explain the small area of th - positive cells compared to previous study . this study has the limitation of showing the advantage of pre - established cell network in cell transplantation because we used cell aggregates not spheroid form which is currently difficult to make and transplant deep into the brain . in addition , the phenomenon of marginal th positive staining in the injection site of cell aggregates could not be explained and quantitative comparison between cell aggregation form and single - cell suspensions was not carried out since the cell numbers of two studies were not similar . in conclusion , this study showed that cell aggregates of hb - nsc transplanted into the striatum not only survive well , but also exert a therapeutic effect in a rat pd model by secreting dopamine . such success was not achieved in the transplantation of single - cell suspensions of hb - nsc . thus , this result suggests that the form of cell transplantation is critical for successful therapy .

objectiveneural tissue transplantation has been a promising strategy for the treatment of parkinson 's disease ( pd ) . however , transplantation has the disadvantages of low - cell survival and/or development of dyskinesia . transplantation of cell aggregates has the potential to overcome these problems , because the cells can extend their axons into the host brain and establish synaptic connections with host neurons . in this present study , aggregates of human brain - derived neural stem cells ( hb - nsc ) were transplanted into a pd animal model and compared to previous report on transplantation of single - cell suspensions.methodsrats received an injection of 6-ohda into the right medial forebrain bundle to generate the pd model and followed by injections of pbs only , or hb - nsc aggregates in pbs into the ipsilateral striatum . behavioral tests , multitracer ( 2-deoxy-2-[18f]-fluoro - d - glucose ( [ 18f]-fdg ) and [ 18f]-n-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ( [ 18f]-fp - cit ) micropet scans , as well as immunohistochemical ( ihc ) and immunofluorescent ( if ) staining were conducted to evaluate the results.resultsthe stepping test showed significant improvement of contralateral forelimb control in the hb - nsc group from 6 - 10 weeks compared to the control group ( p<0.05 ) . [ 18f]-fp - cit micropet at 10 weeks posttransplantation demonstrated a significant increase in uptake in the hb - nsc group compared to pretransplantation ( p<0.05 ) . in ihc and if staining , tyrosine hydroxylase and human 2 microglobulin ( a human cell marker ) positive cells were visualized at the transplant site.conclusionthese results suggest that the hb - nsc aggregates can survive in the striatum and exert therapeutic effects in a pd model by secreting dopamine .

INTRODUCTION MATERIALS AND METHODS Animal and 6-OHDA lesion procedures Transplantation of aggregates of human brain-derived neural stem cells Behavioral tests Multitracer [ Immunohistochemical and immunofluorescent tissue staining Statistical analysis RESULTS Behavioral tests Multitracer [ Immunohistochemistry and immunofluorescence DISCUSSION CONCLUSION

childbirth , even though a normal physiological process has been associated with a number of risks , which may , in extreme cases , lead to loss of life . this concern was underscored by the millennium development goal 5 ( mdg ) which focused on improving maternal health services ( mhs ) to reduce morbidity , disability , and mortality due to pregnancy and delivery . worldwide , it has been estimated that 289,000 cases of maternal deaths were recorded in 2013 , indicating a raising trend by 2000 deaths compared to the figure obtained in 2011 , with 99 % of these deaths occurring in the developing countries . although it is the wealthiest nation in the central african sub - region , with an impressive economic growth since the end of the country s civil war in 2002 , the republic of angola has a higher life time risk of 1 in 39 women dying as a result of pregnancy and its related complications compared to neighboring republic of namibia with a life time risk of 1 in 160 . the current maternal mortality ratio ( mmr ) for angola was reported to be 460 per 100,000 live births in 2013 , which is still far above the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . the high mmr ( 460 per 100,000 live births ) is partly due to low utilization of maternal health services . for instance , between 2006 - 2013 , only 47% and 49% of pregnant women had at least four ante natal care visits as recommended by the world health organization ( who ) and delivered under the supervision of a skilled health worker respectively . more disturbing is the fact that only 32% of rural pregnant women were reported to have had their delivery supervised by a skilled health worker compared to 82% of their urban cohort . this scenario is further worsened by the fact that many pregnancies end up as stillbirths or neonatal deaths . one of the major reasons for sub - saharan african countries having the highest life time risk of dying due to pregnancy related issues and poor pregnancy outcomes such as still births and neonatal deaths is lack of systematic search for the root causes of these deaths as being done in the industrialized countries where confidential inquiries is the norm rather than an exception . unfortunately , the findings of these confidential inquiries in the developed countries are used to set out downstream public health intervention strategies in the developing nations notwithstanding their small sample sizes ( pregnancy related deaths are rare event in europe and north america ) and differences in terms of socio - economic , cultural and political systems including health care financing mechanisms between the developed and the underdeveloped countries . concerns about the lack of use of area - specific data have been referred to as inverse information and care law . this concern is legitimate in terms of the need for the utilization of local information in order to come up with the root causes that lead to poor utilization of maternal and child care services , which will subsequently guide local interventions at provincial / state and district levels . for instance , of the 2,500 articles on maternal mortality reviewed by gil - gonzlez and colleagues , and another 5,575 articles on maternal health services utilization and pregnancy outcomes reviewed by say and raine , and 54 articles on the effectiveness of interventions on maternal mortality in low income countries reviewed by burchette and mayhew , there was no single article from the republic of angola despite the country s high mmr of 460 per 100,000 live birth , and a life time risk of a woman dying from pregnancy and its related complications over 1000 times higher when compared to canada and scandinavian countries . this dearth of data on mhs utilization and maternal mortality in angola and many other high burden countries was further reported to have neither timely nor complete data on maternal deaths surveillance system . surveillance for maternal mortality was launched in 2004 with the aim not just to estimate the burden of the problem but , also to provide better insights on the why , how and where these deaths occur . nearly , a third of all districts in sub - saharan africa have not integrated maternal mortality among the immediate notifiable events / diseases in their integrated disease surveillance and response ( idsr ) program . furthermore , a review of the status of maternal mortality surveillance in 2012 showed that data on maternal deaths are lacking or incomplete in 48.9% of the 180 countries reviewed including angola . a recent review on status of implementation of maternal death surveillance and response among african countries has shown that the republic of angola was among the countries with no available data on maternal death surveillance and response . the country still relies on traditional non - electric surveillance information tools that focus on clinical causes of maternal deaths such as hemorrhage , ruptured uterus , sepsis , eclampsia etc . , which does not provide insights on what transpired at home , on the way to health facility and appropriateness of treatment received . these issues indicate the need for area specific studies in order to complement the weak surveillance system and also to guide interventions that are in context of the local issues which forms the objective of this study . independent autonomous decision making on reproductive health desires of women in many developing countries is defined not only by a woman s level of education or personal income but also by the norms in her community as dictated by cultural and religious beliefs . in order to identify the underlying root causes of utilization behavior and how they impact pregnancy outcomes , the anderson medical care utilization model was used as the theoretical framework of this study . the model is made up of three constructs namely : 1 ) the predisposing characteristics of women such as biological , cultural , social , and economic ; 2 ) the enabling characteristics such as health system and health care financing mechanisms ; and 3 ) the need characteristics which is the individual pregnant woman s perceived need to use modern health services and the perceptions of the health care worker . the constructs of the model are highly adaptable attested by its application in numerous research studies . this unique characteristic of the anderson s model was underscored by its use in various health.[18 - 20 ] and other social issues.[21 - 23 ] we examined the associations between women s bio - socio demographic and health system characteristic ( independent variables ) and pregnancy outcome ( mother is dead or alive ) as the outcome / dependent variables . this study will bridge the existing current gap of evidence - based information noted above and highlight areas that require further research in order to come up with interventions that are specific to local context in angola and in other sub - saharan african countries . kuando kubango is one of the 18 provinces of angola , situated in the southern part of the country . it has a common international boundary with the republic of namibia to the south and the republic of zambia to the southeast . it also shares local boundaries with moxico province to the north east , bie to the north , huila and cunene provinces to the west . it has an estimated population of 510 , 369 with 60% living in urban areas based on the 2014 census . it has a total of 102 health facilities out of which 10 provided obstetric services . the population of women in the reproductive age group is 104,342 with an estimated annual number of pregnancies of 24,843 based on the 2014 census . this study was conducted in the main regional referral center due to the availability of better skilled workers and diagnostic services in this center . all hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . subsistence farming , petty trading , fishing , and hunting are the main occupations of the indigenes . the target population of this study was women that received obstetric services ( antenatal , delivery or post - natal ) in the provincial main referral maternity hospital menongue , kuando kubango province of angola , between 2010 and 2014 . it is basically the analysis of data on pregnancy , labor and puerperium from patient case notes and the delivery register for a period of five years ( 2010 - 2014 ) . only maternal deaths that meet the world health organization s ( who ) definition of maternal mortality were included in the study . according to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy , from any cause related to or aggravated by pregnancy or its management , irrespective of the duration and site of the pregnancy , but not from accidental or incidental causes . the data collection instruments were case files kept in the medical records department , labor ward ( delivery ) register , and a form designed to keep records for data extracted from each case file . the tool documented data on personal , and clinical information such as age , tribe , religion , marital status , place of domicile , parity , occupation of husband , occupation of cases , mode of admission ( self or referred ) , booking status , indication for admission , date of admission , duration of labor , interval between onset of labor and admission to hospital , place of delivery ( as indicated in the referral letter or case notes ) , previous obstetric operations , interval between admission and maternal death , cause of maternal death and date of death . maternal mortality ratio was calculated for each of the five years ( 2010 - 2014 ) and for the overall study period . the causes of death were examined under two heading viz : direct obstetric causes and indirect non obstetric medical causes . cross tabulations of variables , the chi square test of association and fisher exact test were conducted with the level of statistical significance set at p<0.05 at 95% confidence interval . kuando kubango is one of the 18 provinces of angola , situated in the southern part of the country . it has a common international boundary with the republic of namibia to the south and the republic of zambia to the southeast . it also shares local boundaries with moxico province to the north east , bie to the north , huila and cunene provinces to the west . it has an estimated population of 510 , 369 with 60% living in urban areas based on the 2014 census . it has a total of 102 health facilities out of which 10 provided obstetric services . the population of women in the reproductive age group is 104,342 with an estimated annual number of pregnancies of 24,843 based on the 2014 census . this study was conducted in the main regional referral center due to the availability of better skilled workers and diagnostic services in this center . all hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . subsistence farming , petty trading , fishing , and hunting are the main occupations of the indigenes . the target population of this study was women that received obstetric services ( antenatal , delivery or post - natal ) in the provincial main referral maternity hospital menongue , kuando kubango province of angola , between 2010 and 2014 . it is basically the analysis of data on pregnancy , labor and puerperium from patient case notes and the delivery register for a period of five years ( 2010 - 2014 ) . only maternal deaths that meet the world health organization s ( who ) definition of maternal mortality were included in the study . according to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy , from any cause related to or aggravated by pregnancy or its management , irrespective of the duration and site of the pregnancy , but not from accidental or incidental causes . the data collection instruments were case files kept in the medical records department , labor ward ( delivery ) register , and a form designed to keep records for data extracted from each case file . the tool documented data on personal , and clinical information such as age , tribe , religion , marital status , place of domicile , parity , occupation of husband , occupation of cases , mode of admission ( self or referred ) , booking status , indication for admission , date of admission , duration of labor , interval between onset of labor and admission to hospital , place of delivery ( as indicated in the referral letter or case notes ) , previous obstetric operations , interval between admission and maternal death , cause of maternal death and date of death . maternal mortality ratio was calculated for each of the five years ( 2010 - 2014 ) and for the overall study period . the causes of death were examined under two heading viz : direct obstetric causes and indirect non obstetric medical causes . cross tabulations of variables , the chi square test of association and fisher exact test were conducted with the level of statistical significance set at p<0.05 at 95% confidence interval . during the period under study ( 2010 - 2014 ) , 131 maternal deaths were recorded , out of 7,158 live births , giving a maternal mortality ratio ( mmr ) of 1830 per 100,000 deliveries . the annual and overall maternal mortality ratio the denominator of the former ( ratio ) is live births while the later ( rate ) is women in the reproductive age group 15 - 49 years . in this study we used live births as the denominator to calculate the annual and overall mortality ratio is shown in table 1 . annual distribution of maternal mortality ratio the peak incidence of mmr was recorded in 2012 with mmr of 2,487 per 100,000 live births ( figure 1 ) . the overall trend although decreasing between 2013 and 2014 , however , the 2014 mmr of 1,757 per 100,000 live births is still higher than the figure of 1,360 per 100,000 live births recorded in 2010 ( figure 1 ) . trend of mmr per 100,000 live births , 2010 - 2014 , menongue , kuando kubango province out of the 131 maternal deaths , 31 ( 24% ) of these cases had inadequate documentation on working diagnosis , presenting complain , history of presenting complain and or management outline and therefore were not included in analysis of direct and indirect causes of maternal death . of the remaining 100 deaths that had information on diagnoses , 51 ( 51% ) and 49 ( 49% ) were as a result of direct and indirect causes respectively ( table 2 ) . the three leading direct causes of maternal deaths are hemorrhage ( 15% ) , puerperal sepsis ( 13% ) and eclampsia ( 11% ) which combined accounted for 39% of all deaths ( table 2 ) . proportion of direct obstetric and indirect non - obstetric causes of deaths , 2010 - 2014 , kuando kubango , angola indirect non - obstetric medical causes , accounted for 49% of all maternal deaths . malaria in pregnancy ( 14% ) , anemic heart failure ( 13% ) and pneumonia ( 5% ) were the leading cause of indirect non obstetric medical causes of maternal death , accounting for 32% of all deaths ( table 2 ) . the age distribution of maternal deaths indicated that more than half of all deaths were accounted by women between the ages of 15 - 19 and those > 35 years with age specific mmr ( asmmr ) of 1,058 and 1,354 per 100,000 live births respectively . women between the ages of 20 - 34 years had the lowest asmmr of 876 per 100,000 live births . however , the difference is not statistically significant ( =4.572 ; df 2 ; p>0.05 ) ( table 3 ) . pregnancy outcomes in relation to some bio - socio - demographic factors among 12,573 deliveries , 2010 - 2014 , kuando kubango , angola fisher exact test with yates correction one hundred and seventeen out of the 131 cases of deaths were from comuna sede of menongue district accounting for 89% of all deaths recorded during the review period . additionally , caiundo and missombo comunas of menongue district accounted for another 2.7% indicating that about 93% of all deaths were from menongue district . the districts of cuito cuanavale and cuchi accounted for only 3.8% and 1.5% of all deaths respectively . women living in rural areas accounted for 96.2% of all deaths and the difference in the place of domicile ( rural versus urban ) was significantly associated with maternal deaths ( p < 0.0001 ) ( table 3 ) . distance to the nearest health facility that provides maternal health services ( mhs ) was significantly associated maternal mortality ( p < 0.0001 ) , with women who responded that distance is not an obstacle accounting for only 30% of all maternal deaths during the period under review ( table 3 ) . out of the 131 maternal deaths , 52 ( 40% ) occurred within the first 24 hours of been admitted in the hospital , 29 ( 22% ) deaths occurred 24 - 48 hours after admission , while 47 ( 35.6% ) occurred between 2 to 11 days after admission . the maternity hospital has two medical doctors in 2010 which increased to four in 2014 . with an estimated 62 , 736 women in the reproductive age group and 14,937 annual pregnancies for menongue district where the hospital is located , the ratio of midwife per 1000 women in the reproductive age group for menongue district is 1 per 2,134 pregnancies per annum . the study hospital has no ultrasound services that could aid in gynecological and obstetric diagnoses . the mean mmr of 1830 per 100,000 live births recorded in this study is five times higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 , and four times higher than the who estimate for angola . as earlier pointed out above , there is a general lack of published journal articles on maternal mortality ( hospital or community based ) in angola . the official report by the health authorities of cabinda province of angola indicated in 2012 the mmr was 234.3/100,000 live births . however , a review of records in all the 10 health facilities that provide obstetric care in cabinda province by rodrigues ( 2013 ) concluded that the provincial official mmr is an under estimation and that the magnitude of the problem is unknown because data is generally incomplete or not available in all the 10 health facilities . however , when compared with published studies ( 2010 - 2015 ) from central africa region to which angola belongs , the mmr of this study ( 1,830 per 100,000 ) was higher than other hospital based study in cameroon ( 287.57100 , 000 ) . the huge difference in mmr between our finding and the cameroonian study may partially be due to the fact that more than half of all the cases of maternal mortality in our study area was among women who were less than 20 years or more than 35 years and presented with complicated pregnancies that resulted in maternal deaths moreover , the difference in mmr could also be as a result of a general lack of high skilled medical doctors who have not yet specialized in obstetrics and the lack of basic obstetric diagnostic equipment s like ultrasound machine which might have limit the quality of case management in our study area compared to the cameroonian study that was conducted in a tertiary , research and training hospital and therefore more capable to deal with high risk pregnancies . when compared with hospital based studies that were conducted between 2010 - 2015 from other regions of africa , a high level of mmr was also reported from nigeria , west africa , ranging from 866 to 1,791 per 100 , 0000 live births in the southern and northern parts of nigeria respectively . however , lower figures of between 124 and 492 per 100,000 live births was reported east african countries - kenya and tanzania respectively . it is important to note that since 2010 , tanzania and kenya were reported to have made significant progress in the implementation of emergency obstetric care and maternal death surveillance and response compared to angola and therefore may partly account for their lower mmr . similar disparity was observed in hospital based studies among south east asian countries with nepal and pakistan both having lower mmr of 357 and 1007 per 100,000 live births respectively compared to the 1,830 per 100,000 live births reported in this study . however , when compared with the mmr from northern europe and north america , the figure obtained in this study is 167 times higher and hence , reinforces the huge disparity in population health outcome between developed and developing countries . this is driven , in part , by differences in socioeconomic , cultural and political development of these developed countries compared to under developed countries like angola . overall , the mmr obtained in this series is far higher than angola s 2014 estimated mmr of 460 per 100,000 live births by the who . this might not be unrelated to the fact that , the data used in this study were largely incomplete with more than a third of all cases of maternal deaths lacking information on the working diagnosis or clinical management and hence could be a marker for possible under estimation . the possibility of under estimation is further reinforced by the reports on the status of implementation of maternal death surveillance and response which rated it to be low in angola . the dearth of published information on maternal deaths in angola , which could have provided additional information on the state of maternal mortality , is also a big challenge . for instance , extensive systematic reviews of over 7000 articles on maternal mortality , reproductive health and female autonomy , there was no single article from angola , despite having a higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . although , hospital based studies are likely to limited by misrepresentation or selection bias , we believe that the likelihood of under estimation can not be easily dismissed without conducting large scale community based quantitative and qualitative studies . the available data for this study were characterized by the lack of basic information on some or all of the following variables such as parity , birth order , booking status and number of ante natal care visits in majority of the cases . this was further compounded by none of the cases have information on well - established drivers of utilization of maternal health services such as the level of education , income , religion , ethnicity , and occupation as was reported by several studies.[39 - 41 ] the lack of information on these variables makes it difficult to estimate the risk of dying from pregnancy and its related complications based on parity , education , income , and religion , which invariably compromises the appropriateness and quality of any population based public health intervention that is currently being implemented in the province . furthermore , although a maternal mortality review committee was established , however , during the period under review ( 2010 - 2014 ) , not a single review meeting was held . this further reinforces the likelihood of the current plans directed to address maternal deaths are not data driven and could be contributory to the high mmr recorded in this study . the causes of direct obstetric death ( table 2 ) in this study were the same to those reported from various regions of the developing countries . however , although , literature on direct obstetric causes of maternal death was reported to account for 80% of all maternal deaths in the developing countries , however , a much lower figure ( 51% ) was recorded in this study . the reason been largely attributable to the poor documentation with at least a quarter of cases had no working diagnosis . nevertheless , hemorrhage , puerperal sepsis , eclampsia , and ruptured uterus were the major causes of direct deaths as was similarly reported from several developing countries.[42 - 44 ] the high proportion of these causes in this study may be because many of the cases were teenagers ( 37.8% ) and had delivered at home ( 27.2% ) before seeking help in a health facility . thus , teenage pregnancy , lack of prenatal care and delay in seeking early medical intervention increase the risk of ruptured uterus due cephalo - pelvic disproportion / malpresentation , and undiagnosed preeclampsia that resulted in eclampsia , hemorrhage and sepsis due to unhygienic condition associated with home delivery . first , this study is essentially hospital based and therefore , estimates calculated , tend to be very high . thus , women who died or deliver in this hospital are not the true representation of the entire country . second , patients referred to this hospital , are high risk women who had presented themselves for prenatal care at other health institutions , but were referred here for delivery . this means that , among the women giving birth in these hospitals , there will be a disproportionate number of women with obstetric complication and women who die here which may partly explain the high mmr recorded . third , a large proportion of women who died might have been admitted as an emergency and in moribund condition ; but whose deaths will swell the number of total hospital deaths . first , this study is essentially hospital based and therefore , estimates calculated , tend to be very high . thus , women who died or deliver in this hospital are not the true representation of the entire country . second , patients referred to this hospital , are high risk women who had presented themselves for prenatal care at other health institutions , but were referred here for delivery . this means that , among the women giving birth in these hospitals , there will be a disproportionate number of women with obstetric complication and women who die here which may partly explain the high mmr recorded . third , a large proportion of women who died might have been admitted as an emergency and in moribund condition ; but whose deaths will swell the number of total hospital deaths . with an mmr of 1,830 per 100,000 live births , the scourge of pregnancy and childbearing are enormous in this environment . this study demonstrated that , mmr is higher than the estimates by who and the province is not likely to achieve the desired mdg 5 target of mmr 300 per 100,000 live births . although , hospital based studies suffer from berksonian bias , however , the lack of periodic reviews or community based surveys made this study useful by highlighting the lack of documentation of basic information that constituted the who framework on social determinants of disparities in population health , and documentation is the starting point of evidence based planning . hence , the result of the study is an indictment on the policy standard operating procedures for maternal health services and the likelihood of using plans that are not data driven . the fact that the hospital operates without an ultrasound machine to enhance appropriate diagnosis and management of cases , further underscores the likelihood of minimal impact of clinical intervention to avert maternal deaths . this study also sheds more light on the need for operational and community based quantitative and qualitative studies , in order to provide better insights on what operates in the community from the time when danger signs of pregnancy are recognized at household level to the time when appropriate management has commence in a health facility . such approach will provide basis for the development of holistic multi - prong evidence based intervention that will improve the quality of service with subsequent reduction in mmr . based on the findings above , the following recommendations were made : measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound . there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . based on the findings above , the following recommendations were made : measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound . there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death .

background : increasing global health efforts have focused on preventing pregnancy - related maternal deaths , but the factors that contribute to maternal deaths in specific high - burden nations are poorly understood . the aim of this study was to identify factors that influence the occurrence of maternal deaths in a regional maternity hospital in kuando kubango province of angola.methods:the study was a retrospective cross - sectional analysis of case notes of all maternal deaths and deliveries that were recorded from 2010 to 2014 . the information collected included data on pregnancy , labor and post - natal period retrieved from case notes and the delivery register.results:during the period under study , a total of 7,158 live births were conducted out of which 131 resulted in maternal death with an overall maternal mortality ratio of 1,830 per 100,000 live births . the causes of death and their importance was relatively similar over the period reviewed . the direct obstetric causes accounted for 51% of all deaths . the major causes were hemorrhage ( 15% ) , puerperal sepsis ( 13% ) , eclampsia ( 11% ) and ruptured uterus ( 10% ) . in addition , indirect non - obstetric medical causes such as malaria , anemia , hepatitis , aids and cardiovascular diseases accounted for 49% of all maternal deaths . there is poor documentation of personal data and clinical case management of cases . the factors of mutual instability of statistical significance associated with maternal death are : place of domicile ( p=0.0001 ) and distance to the hospital ( p=0.0001).conclusion and global health implication : the study demonstrated that the mmr in maternity hospital is very high and is higher than the who 2014 estimates and the province is yet to achieve the desired mdg 5 target by the end of 2015 . a reversal of the present state requires data driven planning in order to improve access and use of maternal health services ( mhs ) and ultimately lower the number of pregnancy - related maternal deaths .

Introduction Methodology Study area Study population Study design Data analysis Results Discussion Limitation of the study Conclusions and Global Health Implication Recommendations

abiotrophia defectiva is part of the normal human microbiota , colonizing the oral , genitourinary , and intestinal tracts . it is a rare , yet important , cause of infective endocarditis , and is estimated to cause approximately 5 - 6% of all cases of infective endocarditis , including being a major cause of blood culture - negative infective endocarditis . it affects diseased valves in 90% of cases and it is implicated in embolic complications and valvular destruction , despite being sensitive to antibiotics . previous studies have shown mortality and relapse rates as high as 17% despite antibiotic treatment , and this makes accurate and quick identification important . herein we report the first case of infective endocarditis caused by a. defectiva in korea . a 62-year - old female was admitted to the emergency department ( ed ) after two consecutive episodes of syncope . the patient was diagnosed with severe rheumatic mitral stenosis , and underwent a mitral valve replacement ( mvr ) . two months previously , the patient had undergone a simple extraction of her # 16 tooth due to secondary dental caries and had taken prophylactic antibiotics ( amoxicillin 2,000 mg ) . upon admission to the ed the patient was alert and had a body temperature of 38.6c , a pulse rate of 66 beats / min , and blood pressure of 149/51 mmhg . upon physical examination , no cardiac murmur was auscultated and no other evidence , such as clubbed fingers , laboratory studies showed a white blood cell count of 14,260/mm(neutrophil 79.6% ) , erythrocyte sediment rate of 92 mm / h and c - reactive protein of 111.1 mg / l . chest x - ray revealed mild cardiomegaly and electrocardiography showed a newly developed complete atrioventricular block . chest ct and abdominal - pelvic ct was performed to evaluate fever focus , which showed no significant finding . a transthoracic echocardiogram ( tte ) showed that the mechanical prosthetic mitral valve functioned well , and there was no visible vegetation , however , infective endocarditis could not be completely ruled out because of the patient s previous mvr . thus , empirical antibiotics ( vancomycin 1 g intravenous q12hr , gentamicin 60 mg intravenous q8hr and rifampin 600 mg per oral q24hr ) were administered , and a transesophageal echocardiogram ( tee ) performed the day after admission showed mitral valve vegetation and mild transvalvular mitral regurgitation ( fig . 1 ) , leading to a conclusive diagnosis of infective endocarditis according to duke criteria ( one major and three minor criteria ) . ( a ) long - axis view ( 135 ) of a transesophageal echocardiography showing two examples of vegetation attached to the prosthetic mitral valve . the larger one measures 0.7 0.5 cm ( large arrow head ) and the smaller one measures 0.5 0.3 cm ( small arrow head ) . ( b ) two - chamber view ( 59 ) of a transesophageal echocardiography ( zoomed view ) showing vegetation attached to the prosthetic mitral valve ( arrowhead ) . a color doppler shows the jet flow with mild transvalvular mitral regurgitation in systole ( arrow ) . three sets of blood cultures taken on admission showed tiny , non - hemolytic colonies on a blood agar plate ( asan pharmaceutical , hwaseong , korea ) , and pleomorphic gram - positive cocci from smear preparation of the blood agar plate ( fig . a. defectiva was identified by maldi - tof - ms ( matrix - assisted laser desorption / ionization time - of - flight mass spectrometry , bruker daltonics inc . , ma , usa ) , not identified in vitek 2 ( biomrieux , marcy l'etoile , france ) systems . however , because the amount of bacteria was insufficient , cultures were sub - cultured on a medium containing vitamin b6 to assess antibiotic susceptibility . tiny , non - hemolytic colonies on a blood agar plate ( asan pharmaceutical , hwaseong , korea ) after 48 hour of incubation at 35c with 5% co2 ( left ) , and pleomorphic gram - positive cocci from smear preparation of the blood agar plate ( gram stain , 1,000 , right ) . susceptibility to cefotaxime , penicillin g and vancomycin was tested by the e - test method ; the isolate tested cefotaxime - sensitive ( mic 0.75 g / ml ) , penicillin g - intermediate ( mic 1.0 g / ml ) and vancomycin - sensitive ( mic 0.38 g / ml ) . an adjusted antibiotic regimen of vancomycin 1 g q12hr and gentamicin 60 mg q8hr was initiated , after which the patient s fever subsided . vancomycin therapeutic drug monitoring was performed , and vancomycin dosage was adjusted to 400 mg q12hr . on day 12 of admission , the patient became feverish once more and in the subsequent tee the mitral valve vegetation on the medial side seemed to have resolved , but there was an increase in the size of the vegetation on the lateral annulus ( 0.5 0.3 cm 0.8 0.3 cm ) which indicated a perivalvular infection ( fig . operation finding showed small vegetation in prosthetic mv and annulus , which was removed , and mvr was conducted using 23 mm ats medical open pivot heart valve ( ats medical , inc . , pathology finding showed myxoid degeneration , acute and chronic inflammation , fibrosis and calcification , and tissue culture showed no growth of organism ( it was on the 19 day on antibiotics , and blood culture was negatively converted at this time ) . on day 23 of admission , the patient was treated with antibiotics for 5 weeks and was discharged on day 36 . the patient was followed - up at an out - patient clinic for 3 months after discharge with no significant complications . organisms of the genus abiotrophia were first classified as nutritionally variant streptococci ( nvs ) in 1961 . bouvet et al . identified two species of nvs ; streptococcus defectivus and streptococcus adjacens . in 1995 , kawamura et al . nvs are part of the normal flora of the mouth and urogenital and intestinal tracts . recently , an increasing number of cases have been described of a. defectiva isolates recovered from invasive and non - invasive infections following dental procedures . has fastidious culturing and the unspecific colony morphology that they presents on primary detection , such strains have caused major diagnostic difficulties . thus , it has been supposed that many culture - negative endocarditis could have been caused by these species , which could have lead to an underestimation as pathogens of infective endocarditis . in this case report maldi - tof - ms is a new technology for routine identification of bacteria in clinical microbiology laboratories . much of the work using maldi - tof - ms for microbial identification has focused on demonstrating that reproducible mass spectra can be obtained using intact cells and developing algorithms for interpretation and comparison of these spectra . by testing colonies , it takes only a few minutes to have a correct identification which makes not only possible to identify the microorganisms at the species levels but sometimes at the sub - species and strains levels , allowing the detection of epidemic lineages . a. defectiva can cause serious infections such as bacteremia , osteomyelitis , brain abscess , pancreatic abscess , septic arthritis , crystalline keratopathy and in rare cases , infective endocarditis . endocarditis caused by nvs is implicated in 56% of all streptococcal endocarditis cases , and < 1% of all endocarditis cases are caused by a. defective . however , a. defectiva has a higher affinity for the endocardium because of its ability secrete exopolysaccharide , enabling it to adhere to fibronectin in the extracellular matrix . of all the nvs - induced endocarditis patients , 90% suffered from heart disease , and in most cases , bacteremia associated with the pre - existing valvular heart disease led to the development of endocarditis with subacute prognoses . infective endocarditis caused by a. defectiva and other nvs had higher mortality , morbidity and complication rates than those caused by other viridans streptococci . most deaths were due to refractory congestive cardiac failure or major systemic emboli . a mortality rate of up to 17% has been reported , which is higher compared to endocarditis caused by viridians streptococci ( 012% ) . previous studies have shown a treatment failure rate as high as 41% , despite the use of appropriate antibiotics . the antibiotic regimen to combat a. defectiva endocarditis includes penicillin or ampicillin , plus an aminoglycoside or vancomycin for cases of antimicrobial resistance , taken for 46 weeks . infective endocarditis due to a. defectiva progresses slowly , but despite its sensitivity to antimicrobials ~50% of cases need surgical management . surgical treatment combined with concurrent antimicrobial therapy result in better prognoses , and the main indications for surgery are persistent sepsis and vegetation , severe congestive heart failure and recurrent embolism . in korea , 2 cases of infective endocarditis caused by s. adjacens were first reported in 1996 . since then , a case of infective endocarditis by grnulicatella adjacens was reported in 2010 . after classified as a. defectiva , this is the first report as the pathogen of infective endocarditis in korea . the case presented here is an example of the successful treatment of infective endocarditis caused by a. defectiva following a tooth extraction in a post - mvr patient , and represents the first reported case of infective endocarditis caused by a. defectiva in korea . this case shows that a. defectiva could be considered as a causative organism of infective endocarditis in korea .

abiotrophia defectiva , a nutritionally variant streptococci can cause bacteremia , brain abscess , septic arthritis and in rare cases , infective endocarditis , which accounts for 5 - 6% of all cases . a. defectiva is characteristically difficult to diagnose and the mortality , morbidity and complication rates are high . here , we discuss a case of infective endocarditis caused by a. defectiva . a 62-year - old female had previously undergone prosthetic valve replacement 6 years prior to admission . she developed infective endocarditis after tooth extraction . her endocarditis was successfully treated with antimicrobial therapy and mitral valve replacement surgery . this is the first case of infective endocarditis caused by a. defectiva reported in korea . this case shows that a. defectiva could be considered as a causative organism of infective endocarditis in korea .

Introduction Case report Discussion

one of the most common complaints of the gait disorders in infants and children is an intoeing gait , which is caused by excessive anteversion of the proximal femur , internal tibial torsion , and/or metatarsal adductus [ 15 ] . a careful evaluation of the children is essential to rule out serious pathological conditions such as cerebral palsy , infantile blount 's disease , metabolic bone diseases , and skeletal dysplasias . an intoeing gait in the majority of patients without these pathological conditions is a minor problem and is observed to spontaneously improve with time . the parents or grandparents are concerned that the child will have a permanent disability or that the condition will interfere with the child 's physical performance ; thus they sometimes hope for some treatments . a shoe modification with wedges or arch supports is one of the tolerable nonsurgical treatments for children to correct an intoeing gait and modify the gait pattern . the sensomotor insole ( smi ) , which had been introduced by the german shoemaker jarhling , was originally developed to improve abnormal gait patterns in children with spastic gait [ 6 , 7 ] . it was expected to change the muscle tone of the lower limbs by stimulating the proprioceptors of the sole . jahrling and rockenfeller demonstrated improvement of the gait patterns in cerebral palsy patients with flexible equinus or equinovarus deformities using smi . this information prompted us to try the use of smi for a pediatric intoeing gait , since the patients usually have more flexible feet than the cerebral palsy patients . in the present study , the effects of smi on a pediatric intoeing gait were evaluated using the three - dimensional ( 3d ) gait analysis with a vicon motion capture system . the parents of patients treated with smi for their intoeing gait at our institution between january 2009 and december 2010 were invited to have their child participate in this prospective study , which was approved by an institutional review board . patients who had rigid deformities of the feet with an ankle range of motion less than 30 degrees or forefoot abduction less than 10 degrees were excluded from this study . there were five boys and five girls , and the average age at testing was 5.6 years ( range , 39 years ) . bilateral intoeing gait was observed in eight , right side in one , and left side in one . among six congenital clubfeet patients who had initially been treated with serial casting and braces , the torsional profile was assessed with the patient prone on the examining table , as described by staheli et al . , so that the examiner can determine the amount of internal and external rotation of the hip as an indication of the amount of femoral anteversion , measure the thigh - foot angle in order to estimate tibial torsion , and examine the shape of the lateral border of the foot to assess the presence of metatarsal adductus . smi comprises of five exclusive bars , including medial and lateral heel bars , a retro bar , a toe bar , and a lateral wedge ( figure 1 ) . before first application of the insole , patients were individually ground to size velcro trial elements that were attached to a base sole by the orthopaedic shoe technician . the trial elements can be readjusted repeatedly until the desired gait pattern has been achieved . once the final position of the bars has been determined , the individual smi was made up in the workshop using the care set . the computerized motion analysis tests were performed with a six - camera vicon 3d motion system ( vicon mx system , oxford , uk ) during walking at a self - selected speed along a five - meter walkway using 16 passive retroreflective markers attached to specific bony landmarks of the lower extremities and the pelvis . patients underwent gait analysis in the same shoes with and without smi . walking speed , stride length , and cadences were calculated using the vicon plug - in gait ( vicon mx system , oxford , uk ) , and the 3d joint kinematic data were obtained at 100 hz for the hips , knees and ankles in the sagittal plane and the hips and knees in the coronal and transverse planes from bilateral lower extremities . comparisons in walking speed , step length , cadences , and joint kinematic data of the lower extremities were made between the conditions with and without smi . the data were analyzed using the vicon nexus plug - in gait and the custom - made software ; the vicon normalizer ver . gait cycle events that were defined by perry were adopted for descriptions in this study . paired t - test was conducted to determine any difference among the demographics of the two groups ( with and without smi ) . analysis was performed with spss version 16.0 ( spss inc , chicago , il , usa ) . all patients with clubfeet showed residual metatarsal adductus without apparent hindfoot deformities . in four patients with idiopathic intoeing gait , excessive anteversion of the proximal femur was seen in three , and internal tibial torsion was found in two ( one of the four patients had both deformities ) ( table 1 ) . improvement of the gait pattern was clinically recognized after a couple of steps on wearing smi in most patients . there were significant differences in transverse plane motions of the hip and knee joints between the patients with and without smi . smi decreased internal rotation of the proximal femur relative to the pelvis in loading response phase ( 18.3 28.1 versus 21.6 28.0 , p = 0.009 ) and terminal swing phase ( 16.3 27.4 versus 19.0 26.4 , p = 0.047 ) ( table 2 , figure 2 ) . smi also decreased internal rotation of the tibia relative to the femur in mid stance phase ( 0.7 12.5 versus 2.0 14.9 , p = 0.030 ) and terminal stance phase ( 1.4 11.9 versus 2.3 14.5 , p = 0.042 ) ( table 3 , figure 3 ) . there were no significant differences of the hip and knee kinematics in the sagittal plane between the patients with and without smi . ankle dorsiflexion was increased in loading response phase and terminal swing phase and decreased in terminal stance phase and terminal swing phase on wearing smi . in regard to cadence parameters , smi significantly increased the walking speed ( 67.9 m / min versus 64.9 m / min , p < 0.001 ) and the stride length ( 500 mm versus 477 mm , p < 0.001 ) , although cadence did not differ between the two groups ( 137.6 steps / min versus 136.7 steps / min , p = 0.89 ) ( table 4 ) . li and leong emphasized the importance of a correct diagnosis and understanding the causes and natural course of the condition in the management of a pediatric intoeing gait . thackeray and beeson pointed out that identification of the level of torsional deformities ( excessive femoral anteversion , internal tibial torsion , or metatarsal adductus ) that produced an intoeing gait should be essential for planning of the specific intervention . the present study , however , demonstrated that smi improved abnormal gait patterns not only children with idiopathic intoeing gait but also those with congenital clubfeet , who had deformities of different levels in lower limbs . although smi can not correct dynamic and structural abnormalities of the lower extremities of feet and its long - term effectiveness is unknown , it is one of the easy and effective treatment options in the management of a pediatric intoeing gait . smi inhibited internal rotation of the leg in loading response phase , which is usually observed in normal gait . nakajima reported that the foot progression angle , which is the angle formed by the direction of gait progression and the foot axis at mid stance , was reduced by using a lateral wedged sole , but it recovered in the use of a lateral wedge and an arch support that resembles smi . the present study revealed that the decreased internal rotation of the lower limb by smi was shown to be due to decreased internal rotation of the proximal femur through the end of the swing phase and the beginning of the stance phase and decreased internal rotation of the tibia during the mid and terminal stance phases . although the precise biomechanical effect of this specific insole on the hip and knee joints remains unknown , it is recognized that the leg - ankle - foot alignment is affected by the varus - valgus of the calcaneus . we speculated that the calcaneus stabilized in neutral position between the medial and the lateral heel bar at the initial contact may provide favorable effects on entire lower limbs by suppressing pronation of the subtalar joint which is accompanied by internal rotation of the leg ( figure 4 ) . in addition to changing the rotational profile of the lower limb , smi accelerated children 's walking speed and increased stride lengths . the increase of the walking speed depended on the increase of the stride length because cadence did not show significant differences between children with and without smi . smi increased external rotation of the femur relative to the pelvis during the end of the swing phase , which reflected the internal rotation of the pelvis relative to the femur . the increment of stride lengths could be due to internally rotated pelvis toward the direction of progression in walking . young children were tested under a nonthreatening environment with parents present and distractions such as toys available in the present study , but the likelihood of not walking in their normal fashion while undergoing instrumented gait analysis is questionable . the foot progression angle , which is the most important variable to be measured , was not addressed in the present study because we unfortunately did not possess a force plate instrument that is indispensable to show the accurate direction of gait progression . the measurements of children without smi may not reflect the actual gait situation because the shoes , which were adjusted on wearing smi , would be a little large when children put them on without smi . finally , the number of patients examined in this study was limited because of difficulties in performing the gait analysis for toddlers or naughty children . in conclusion , smi improved an intoeing gait pattern in patients with congenital clubfeet and idiopathic intoeing by externally rotating the proximal femur through the end of the swing phase and the beginning of the stance phases and externally rotating the tibia during the mid and terminal stance phase , resulting in acceleration of the walking speed and increase in the stride lengths .

background . the sensomotor insole ( smi ) has clinically been shown to be successful in treating an intoeing gait . we investigated the biomechanical effect of smi on a pediatric intoeing gait by using three - dimensional gait analysis . methods . six patients with congenital clubfeet and four patients with idiopathic intoeing gait were included . there were five boys and five girls with the average age at testing of 5.6 years . the torsional profile of the lower limb was assessed clinically . three - dimensional gait analysis was performed in the same shoes with and without smi . results . all clubfeet patients exhibited metatarsal adductus , while excessive femoral anteversion and/or internal tibial torsion was found in patients with idiopathic intoeing gait . smi showed significant decreased internal rotation of the proximal femur in terminal swing phase and loading response phase . the internal rotation of the tibia was significantly smaller in mid stance phase and terminal stance phase by smi . in addition , smi significantly increased the walking speed and the step length . conclusions . smi improved abnormal gait patterns of pediatric intoeing gait by decreasing femoral internal rotation through the end of the swing phase and the beginning of the stance phase and by decreasing tibial internal rotation during the stance phase .

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

her2 ( erbb2/neu ) is a member of the erbb family of transmembrane receptor tyrosine kinases ( rtks ) , which also includes the epidermal growth factor receptor ( egfr , erbb1 ) , her3 ( erbb3 ) , and her4 ( erbb4 ) . ligand binding to the ectodomains of egfr , erbb3 , and erbb4 results in the formation of catalytically active homo- and heterodimers to which her2 is recruited as a preferred partner . although her2 can not bind any erbb ligand directly , its catalytic activity can potently amplify signaling by erbb - containing heterodimers via increasing ligand binding affinity and/or receptor recycling and stability [ 25 ] . activation of the erbb network leads to receptor autophosphorylation of c - terminal tyrosines and recruitment to these sites of cytoplasmic signal transducers that regulate cellular processes such as proliferation , differentiation , motility , adhesion , protection from apoptosis , and transformation . cytoplasmic signal transducers activated by this network include plc-1 , ras - raf - mek - mapks , pi3k - akt - ribosomal s6 kinase , src , the stress - activated protein kinases ( sapks ) , pak - jnkk - jnk , and the signal transducers and activators of transcription ( stat ) . several rtks , including the erbb family members , fibroblast growth factor receptors , insulin receptor , and vascular endothelium growth factor receptor flk1/kdr , are known to migrate to the nucleus and act as transcription factors for certain target genes . nuclear her2 has been found to associate with multiple genomic targets in vivo , including the cyclooxygenase enzyme cox-2 gene promoter , and stimulate gene transcription . her2 gene amplification is reported in 20% of metastatic breast cancers , where it is associated with poor patient outcome . studies of her2-overexpressing breast cancer cell lines and human tumors have shown constitutive her2 phosphorylation [ 9 , 10 ] . overexpression of her2 is associated with mammary epithelial cell transformation [ 11 , 12 ] and shorter survival in breast cancer patients [ 8 , 13 ] . trastuzumab ( herceptin ) , a humanized igg1 that binds to her2 ectodomain , is an approved therapy for treating her2-overexpressing breast cancers [ 14 , 15 ] . trastuzumab has been shown to induce tumor regressions in 12%~35% of heavily pretreated metastatic breast cancers with her2 overexpression [ 1618 ] . meanwhile , most metastatic breast tumors with her2 gene amplification and/or very high levels of her2 protein do not respond to trastuzumab , and the majority of those that initially respond relapse later , suggesting de novo and acquired mechanisms of therapeutic resistance however , recent reports suggest that overexpression of the igf - i receptor or activated egfr as well as aberrant pi3k / akt signaling or pten deficiency may all result in acquired resistance to trastuzumab . lately , intragenic somatic mutations in the her2 gene were reported in < 10% of non - small - cell lung cancers ( nsclcs ) [ 23 , 24 ] . these involve in - frame duplications / insertions in a small stretch within exon 20 that correspond to the identical nine - codon region in exon 20 of the egfr gene , where duplications / insertions have also been reported . because of the location of these insertions at the c - terminal end of the c - helix in the tyrosine kinase domain , it has been postulated that they result in a conformational change and shift in the helical axis , thus narrowing the atp - binding cleft and increasing kinase activity over that in wild - type receptors . her2 kinase domain mutations within exons 1822 are identified in 5% of gastric carcinomas , 3% of colorectal carcinomas , and < 5% of breast carcinomas from asian patients . mechanistic studies indicate that the mutant her2 induces constitutive transphosphorylation of egfr and activation of the downstream signal transducers in a ligand - independent manner , resulting in increased tumorigenicity and decreased sensitivity to trastuzumab and egfr inhibitors in cells carrying these mutations . the tgf- ligands are a family of multitasking cytokines that play important roles in cell proliferation , lineage determination , extracellular matrix production , cell motility , apoptosis , and modulation of immune function . these ligands bind to a heteromeric complex of transmembrane serine / threonine kinases , the type i and type ii receptors ( tri and trii ) . upon ligand binding to trii this allows for the constitutively active trii kinase to transphosphorylate and activate the tri kinase which subsequently phosphorylates the transcription factors smad2 and smad3 . smad2/3 then associate with a common mediator smad , smad4 , and translocate to the nucleus where as a heteromeric complex , they regulate gene transcription . in addition to smads , other signaling pathways have been implicated in tgf- actions in the recent studies . these include the extracellular signal - regulated kinase ( erk , mapk ) , c - jun nh2-terminal kinase ( jnk ) , p38mapk , phosphatidylinositol-3 kinase ( pi3k ) , and rho gtpases ( reviewed in [ 34 , 35 ] ) . the critical role of these non - smad pathways on mediating the cellular effects of tgf- remains to be fully characterized . subsequent studies indicated that tgf- is a potent inhibitor of cell proliferation and therefore , a tumor suppressor [ 37 , 38 ] . consistent with its tumor suppressor role , many cancers lose or attenuate tgf--mediated antimitogenic action by mutational inactivation of tgf- receptors or their signal transducer smads [ 3944 ] . studies using transgenic mice with conditional knockout of trii indicate that loss of trii in the context of polyomavirus middle t antigen ( pyvmt ) expression results in a shortened median tumor latency and an increased formation of pulmonary metastases . on the other hand , increasing evidence shows that excess production and/or activation of tgf- in tumors can accelerate cancer progression by a combination of autocrine and paracrine mechanisms , resulting in enhancement of tumor cell motility and survival , increase in tumor angiogenesis and production of extracellular matrix and peritumoral proteases , and the inhibition of immune surveillance mechanisms in the cancer host ( reviewed in [ 34 , 35 , 45 ] ) . for example , overexpression of active tgf-1 or active mutant of tri ( alk5 ) in the mammary gland of bigenic mice also expressing mouse mammary tumor virus ( mmtv)/neu ( erbb2 ) accelerates metastases from neu - induced mammary cancers [ 4648 ] . in transgenic mice bearing pyvmt - expressing mammary tumors , inhibition of tgf- with the soluble fusion protein trii : fc results in increased apoptosis of tumor cells and a reduction in both circulating tumor cells and lung metastases . in the same transgenic model , conditional induction of active tgf-1 in mice bearing established mammary cancers increases lung metastases by > 10 folds without a detectable effect on mammary tumor proliferation or size . mice expressing soluble trii under the regulation of the mmtv / ltr promoter exhibit high levels of the tgf- antagonist in the circulation which suppress metastases from neu - induced mammary tumors as well as metastases resulting from injected b16 melanoma cells . in breast cancer models , exogenous as well as transduced tgf- confer motility and invasiveness to mcf10a nontransformed human mammary epithelial cells ( hmecs ) stably expressing transfected her2 [ 52 , 53 ] . indeed , a genetic modifier screen in these cells identified tgf-1 and tgf-3 as molecules that cooperate with her2 in inducing cell motility and invasion [ 37 , 52 ] . taken together , these data suggest that oncogenic signals , such as overexpression of her2 , are permissive for tgf--induced signals associated with tumor cell motility and , potentially , metastatic progression . inhibition of her2 with trastuzumab blocks the promigratory effect of tgf- on her2-overexpressing hmec , suggesting that oncogene function is required for the transforming effect of tgf-. our recent studies demonstrate that tgf- and her2 cooperate at various levels , including ( 1 ) transcriptional regulation of the smad target genes and pathways ; ( 2 ) activation of the pi3k / akt pathway in a smad - independent manner ; ( 3 ) modification of the tumor microenvironment by inducing the secretion of tgf- , erbb ligands , and angiogenic mediators . we have utilized a cell culture model overexpressing her2 ( mcf10a / her2 ) or empty vector ( mcf10a / vec ) to investigate synergy of her2 overexpression and tgf- signaling . a chromatin immunoprecipitation-(chip- ) based screen was carried out to identify chromatin smad targets ( chsts ) in tgf--treated mcf10a / her2 cells . the regulatory regions of several potential tgf- target genes are identified from the chst dna pool established in this study . these genes include the receptor - type phosphatase ( ptprk ) , serine / threonine kinase 24 ( stk24 ) , integrin 9 ( itga9 ) , and vimentin - similar genes . interestingly , tgf- induces binding of smads to some of these gene promoters only in mcf10a / her2 but not in mcf10a / vec cells . this suggests that cofactors regulated by her2 signaling modulate smad - mediated transcription and , thereby , the biological functions of tgf- in her2-overexpressing cells . further investigation on ptprk , a smad target gene indentified in this study , indicates that while tgf- upregulates ptprk expression in both tumor and nontumor mammary cells , her2 overexpression downregulates ptprk . rna interference of ptprk accelerates cell cycle progression , enhances response to egf , and abrogates tgf--mediated antimitogenesis , suggesting a tumor - suppressive role of ptprk . therefore , by suppressing ptprk expression , her2 abrogates the ability of tgf- to induce antimitotic factors . another example of altered regulation of smad target genes is the muts homolog 2 ( msh2 ) , a tumor suppressor and central component of the dna mismatch repair ( mmr ) system . tgf- upregulates msh2 expression in non - tumor cells through promoter activation mediated by smads and p53 . however , overexpression of her2 impairs p53 function and increases the level of mir-21 , a microrna that targets and downregulates msh2 transcripts . as a result , in her2-transformed cells , tgf- fails to activate msh2 promoter but decreases msh2 expression by further stimulating mir-21 . this downregulation of msh2 by tgf- also contributes to resistance to dna - damaging chemotherapy agents in cancer cells , as msh2 is required for the recognition of drug - induced dna damages , which triggers apoptosis . in another study , we show that addition of exogenous tgf- or expression of constitutively active tri ( alk5 ) induces motility of mcf10a / her2 cells but not mcf10a / vec cells . this is mediated by pi3k activation and involves her2 translocation to cell membrane protrusions , where it colocalizes with vav2 , rac1 , pak1 , and actin skeleton , resulting in prolonged rac1 activation and enhanced cell survival and invasiveness . by anchoring her2 to actin skeleton , tgf- also induces clustering of her2 and integrin 6 , 1 and 4 , which is mediated by focal adhesion kinase ( fak ) and required for tgf--induced motility and oncogenic signaling of her2 in breast cancer cells ( figure 1 ) . we further investigated the mechanism through which tgf- activates pi3k in her2-overexpressing cells and found that treatment with tgf- or expression of alk5 induces phosphorylation of the tace / adam17 sheddase and its translocation to cell surface , resulting in increased secretion of tgf- , amphiregulin , and heregulin . in turn , these ligands enhance association of pi3k p85 subunit with erbb3 and activate pi3k / akt ( figure 1 ) . in addition , activation of tgf- signaling in her2-overexpressing breast cancer cells also reduces their sensitivity to trastuzumab , as a result of pi3k activation . while tgf- induces shedding of erbb ligands into the microenvironment , her2 signaling also induces the expression and secretion of tgf-1 and tgf-3 through a mechanism involving rac1 activation and jnk - ap1-dependent transcription . vascular endothelial growth factor ( vegf ) , a target of the tgf--smad transcriptional regulation , is synergistically induced by her2 and tgf- . thus , the crosstalk between her2 and tgf- not only alters intracellular signaling in cancer cells but also influences other components of the tumor microenvironment through inducing several proinvasive growth factors , which may serve as extracellular targets of novel therapeutic strategies directed at both cancer - driving oncogenes and the modified tumor microenvironment . to understand the clinical relevance of the her2:tgf- crosstalk , we mapped an alk5-induced gene expression signature to a previously published 295-array data set by van de vijver et al . and chang et al . . the tumors with a positive correlation with the active tri signature are mostly her2 positive , basal - like , and some luminal b tumors while the tumors with a negative correlation are predominantly luminal a and normal - like tumors ( figure 2(a ) ) . cancers with a positive correlation with the alk5 signature show a worse recurrence - free survival ( rfs ) and overall survival ( os ) compared to tumors with a negative correlation ( figure 2(b ) ) . we further explored possible correlation of the alk5 signature with resistance to trastuzumab by mapping this gene expression signature to an array data set reported by harris et al . obtained from 22 patients with her2-overexpressing breast cancer treated with neoadjuvant trastuzumab and vinorelbine . hierarchical clustering analysis shows that all 3 patients who achieved pathological complete response do not share similar expression with the tgf- signature ( figures 2(c ) and 2(d ) ) , which supports a role of tgf- in inducing clinical resistance to trastuzumab . as indicated by the studies reviewed herein , the cell readouts of the multifunctional tgf- signaling is context dependent and largely edited by the overexpression of her2 , which is one major dysregulation in breast cancer . in her2-transformed cells , tgf- , in turn , further stimulates her2 signaling to promote malignancy and induces resistance to anti - her2 therapy . documented evidence suggest that blockage of her2:tgf- crosstalk may significantly enhance the efficiency of conventional therapies in breast cancer patients with her2 overexpression .

accumulating evidence indicates a functional crosstalk between the her2 ( erbb2 ) tyrosine kinase and the tgf- signaling mediated by its serine / threonine kinase receptors . in her2-overexpressing breast cancer , this crosstalk results in increased cancer cell proliferation , survival and invasion , accelerated cancer progression and metastasis in animal models , and resistance to chemotherapy and her2-targeted therapy . the transformed cellular context with constitutively active her2 signaling , as a consequence of her2 gene amplification or overexpression , converts tgf- from a tumor suppressor to a malignancy - promoting factor . tgf- , in turn , potentiates oncogenic her2 signaling by inducing shedding of the erbb ligands and clustering of her2 with integrins . in addition , tgf- is associated with resistance to trastuzumab , an anti - her2 therapeutic antibody . recent mechanistic studies indicate that tgf- and her2 cooperate through both smad - dependent and independent mechanisms . blockade of her2:tgf- crosstalk may significantly enhance the efficiency of conventional therapies in breast cancer patients with her2 overexpression .

1. HER2 Is a Proto-Oncogene and a Therapeutic Target in Breast Cancer 2. HER2 Converts TGF- 3. The Crosstalk between HER2 and TGF- 4. Clinical Relevance of the Crosstalk between HER2 and TGF-

obesity is a growing problem in the united states ( flegal et al . , 1998 ; kuczmarski et al . , 1994 ; mokdad et al . among many determinants of obesity , frequency of meals , snacking , and eating - out have attracted research and practical interests because of their direct applicability to everyday life . fabry and co - workers reported that eating small frequent meals ( nibbling ) was strongly associated with lower body weight than eating large few meals ( gorging ) ( fabry et al . , 1964 , 1966 ; hejda & fabry , 1964 ) . but , the inverse relationship between meal frequency and body weight has not been consistently found in recent other studies in population studies ( charzewska et al . , 1981 ; dreon et al . , 1988 ; edelstein et al . , 1992 ; metzner et al . , 1977 ; summerbell , 1996 ) . nibbling did not produce better results in weight loss than gorging in clinical studies , when the amount of total energy was restricted ( finkelstein & fryer , 1971 ) . ( 1997 ) concluded in their review paper that nibbling does not have energy metabolic advantages in terms of energy utilization over gorging . it has also been hypothesized that eating frequent meals may result in higher amount of energy than eating infrequent meals , however , data on this hypothesis is relatively limited . kant ( 1995 ) reported that energy intake was positively associated with meal frequency in the national health and nutrition examination and survey i epidemiologic follow - up study . however , energy intake levels in the dataset were below the predicted minimal energy needs ( 1.4 * bmr ) ( goldberg et al . , 1991 ) . with limited data availability and imprecision of dietary assessment methods , snacking has been discussed in relation to obesity in similar ways to meal frequency has . snacking has sometime been studied in the context of meal frequency , because definitions of meals or snacking have not been fully established ( gatenby , 1997 ) . booth ( 1988 ) proposed that snacking in addition to meals may lead to higher energy intake . high - fat , high - sugar and high - energy foods are often associated with snacking , possibly resulting in consuming " empty calories ( drummond et al . , 1996 ) . " studies on whether frequent meals or snacking leads to higher energy consumption reported mixed results ( kirk , 2000 ; lioret et al . , 2008 ) . eating - out is one of the significant phenomna in the industrialized and modernized society ( finklestein , 1989 ) . in the u.s . , about half of the food expenditures of adults are spent eating away from home ( blisard et al . , 2002 ; clausen , 2000 ) . in the u.k . , eating - out became much more commonplace at the end of the twentieth century ( warde & martens , 2000 ) . eating - out is thought to be associated with obesity because foods eaten away home tend to be in larger portion size and higher fat and energy content ( ma et al . , 2003 ; young & nestle , 2002 ) . if and how frequent eating - out is related to body weight , however , has not been fully studied . frequency of meals , snacking , and eating - out are determined by many factors . immigrants and their children provide unique information on relationships among meal frequency , snacking , eating - out , and body weight , because people acculturate in different speed and patterns . acculturation refers to a process of overall adaptation on both individual and group levels , including cultural , psychological , social , economic , and political aspects ( berry , 1997 ) . since current international migration flows from undeveloped or developing countries to developed countries , immigrants and their children may be in different stages of showing changes from industrialization and modernization . this study examined whether acculturation was associated with meal frequency , snacking , and eating - out and whether meal frequency , snacking and eating - out were related to weight status using korean americans as an example . specifically , we hypothesized that : 1 ) higher acculturation is related to lower meal frequency , more frequent snacking , and more frequent eating - out and 2 ) lower meal frequency , more frequent snacking , and more frequent eating - out are associated with higher prevalence of overweight . data for this study were collected by a cross - sectional mail survey of a national sample of korean americans in the continental u.s . adult ( 17 years old or older ) korean americans were randomly sampled from u.s . korean surnames were used to target ethnicity and the listing was screened to make sure that only people with korean surnames were included . the sample was stratified by four variables : 1 ) region ( northeastern , midwestern , southern , and western following the national health and nutrition examination and survey ( u.s . department of health and human services . 1981 ) ) , 2 ) urban - rural area ( standard metropolitan statistical area ( smsa ) and non - smsa ) , 3 ) age as a proxy for generation ( under 35 years and over 35 years ) , and 4 ) gender ( male and female ) . the pre - tested questionnaire was mailed to a total of 1113 people with korean surnames in the continental u.s . a personally addressed cover letter in both english and korean explaining the purpose of the study was sent along with two questionnaires ( in english and korean ) . out of the 1113 questionnaires mailed out , 259 ( 33% ) were returned as undeliverable . of the 854 deliverable questionnaires , 470 ( 55% of the total deliverable sample ) responded , with 105 who responded that they were not korean americans and two who refused to participate . after careful screening , 16 questionnaires were determined to be unusable . this analysis used a total of 347 questionnaires , which was 42% of the total deliverable sample . the respondents did not show significantly different patterns from the sampling list distribution by region , area , or gender . there are no widely accepted acculturation scales for korean americans . in this study , a bidimensional model ( berry , 1997 ) provided a framework and gordon 's seven dimensions ( gordon , 1964 ) were used to identify variables to measure overall acculturation in korean americans . each dimension was independently measured in two axes : relation to american society and retention of korean ethnic society . cluster analysis on acculturation resulted in three distinct groups : acculturated , bicultural , and traditional ( lee et al . , 2000 ; 2003 ) . acculturated korean americans were either born in the u.s . or immigrated to the u.s . when they were very young . generally , they were most comfortable with american society compared to the other acculturation groups . the largest , traditional group was situated on the opposite end from the acculturated group . they were more likely to have limited english proficiency and felt most comfortable with korean culture and environments . the bicultural group appeared to be in between the acculturated and the traditional group , however , it was notable that the bicultural group had a wider social network and social participation with american mainstream than the acculturated group . more detailed information on acculturation scale and acculturation groups can be found elsewhere ( lee et al . , 2000 ) . frequency of meal was estimated by asking how often respondents eat breakfast , lunch , and dinner . response categories were never , once a week or less , 2~3 times a week , 4~6 times a week , and everyday . response categories were re - coded because of low frequencies in some of the categories . " never " and " once a week or less " category for regularity of lunch were combined together , leaving " once a week or less , " " 2~3 times a week , " " 4~6 times a week , " and " everyday . " response categories for regularity of dinner were re - coded into three response categories with the first three categories ( " never , " " once a week or less , " and " 2~3 times a week " ) collapsed . frequency of snacking was also asked using the same response categories . with responses for frequency of breakfast , lunch , and dinner , a new variable ( daily frequency of meals ) was created . respondents who reported eating each meal everyday were given a score of 1 for each meal , while others received a score of 0 . combining the scores for three meals produced a value for frequency of meals , ranging from 0 to 3 ; a value of 0 would mean a respondent did not have regularly established meal frequency , while a value of 3 would mean a respondent had a regularly established meal frequency of three . there were few respondents with a value of 0 , therefore , they were combined with those with a value of 1 . eating - out behaviors was determined by asking how often respondents eat out or take out from restaurants : never or rarely , 1~2 times a month , about once a week , 2~3 times a week , and almost everyday . weight status was determined by calculating body mass index ( bmi ) with self - reported height and weight . bmi is a good indicator of adiposity and self reported height and weight have adequate validity for population studies ( bowman & delucia , 1992 ) . respondents were grouped by bmi into four groups : underweight ( bmi<18.5 ) , normal weight ( 18.5bmi<25 ) , overweight ( 25bmi<30 ) , and obese ( bmi30 ) ( who , 1998 ) . in multiple logistic regressions , overweight individuals ( bmi25 ) were compared to non - overweight individuals . control variables included sex , age , size of place of residence , education , income , working status , and marital status . size of place of residence was divided into smsa ( 0 ) vs non - smsa ( 1 ) according to zip code of residence . income categories were re - coded to six categories because of low frequency in some of the categories . working status was collapsed into two categories : currently working ( 1 ) and not working ( 0 ) . married people were treated as a reference category and all other categories were collapsed into unmarried . chi - squares and analysis of variance with scheff test were used to examine bivariate relationships . logistic regression ( proportional odds model ) was applied to simultaneously investigate the relationships of all study variables in multi - variable models . adult ( 17 years old or older ) korean americans were randomly sampled from u.s . korean surnames were used to target ethnicity and the listing was screened to make sure that only people with korean surnames were included . the sample was stratified by four variables : 1 ) region ( northeastern , midwestern , southern , and western following the national health and nutrition examination and survey ( u.s . department of health and human services . 1981 ) ) , 2 ) urban - rural area ( standard metropolitan statistical area ( smsa ) and non - smsa ) , 3 ) age as a proxy for generation ( under 35 years and over 35 years ) , and 4 ) gender ( male and female ) . the pre - tested questionnaire was mailed to a total of 1113 people with korean surnames in the continental u.s . a personally addressed cover letter in both english and korean explaining the purpose of the study was sent along with two questionnaires ( in english and korean ) . out of the 1113 questionnaires mailed out , 259 ( 33% ) were returned as undeliverable . of the 854 deliverable questionnaires , 470 ( 55% of the total deliverable sample ) responded , with 105 who responded that they were not korean americans and two who refused to participate . after careful screening , 16 questionnaires were determined to be unusable . this analysis used a total of 347 questionnaires , which was 42% of the total deliverable sample . the respondents did not show significantly different patterns from the sampling list distribution by region , area , or gender . there are no widely accepted acculturation scales for korean americans . in this study , a bidimensional model ( berry , 1997 ) provided a framework and gordon 's seven dimensions ( gordon , 1964 ) were used to identify variables to measure overall acculturation in korean americans . each dimension was independently measured in two axes : relation to american society and retention of korean ethnic society . cluster analysis on acculturation resulted in three distinct groups : acculturated , bicultural , and traditional ( lee et al . acculturated korean americans were either born in the u.s . or immigrated to the u.s . when they were very young . generally , they were most comfortable with american society compared to the other acculturation groups . the largest , traditional group was situated on the opposite end from the acculturated group . they were more likely to have limited english proficiency and felt most comfortable with korean culture and environments . the bicultural group appeared to be in between the acculturated and the traditional group , however , it was notable that the bicultural group had a wider social network and social participation with american mainstream than the acculturated group . more detailed information on acculturation scale and acculturation groups can be found elsewhere ( lee et al . , 2000 ) . frequency of meal was estimated by asking how often respondents eat breakfast , lunch , and dinner . response categories were never , once a week or less , 2~3 times a week , 4~6 times a week , and everyday . response categories were re - coded because of low frequencies in some of the categories . " never " and " once a week or less " category for regularity of lunch were combined together , leaving " once a week or less , " " 2~3 times a week , " " 4~6 times a week , " and " everyday . " response categories for regularity of dinner were re - coded into three response categories with the first three categories ( " never , " " once a week or less , " and " 2~3 times a week " ) collapsed . frequency of snacking was also asked using the same response categories . with responses for frequency of breakfast , lunch , and dinner , a new variable ( daily frequency of meals ) was created . respondents who reported eating each meal everyday were given a score of 1 for each meal , while others received a score of 0 . combining the scores for three meals produced a value for frequency of meals , ranging from 0 to 3 ; a value of 0 would mean a respondent did not have regularly established meal frequency , while a value of 3 would mean a respondent had a regularly established meal frequency of three . there were few respondents with a value of 0 , therefore , they were combined with those with a value of 1 . eating - out behaviors was determined by asking how often respondents eat out or take out from restaurants : never or rarely , 1~2 times a month , about once a week , 2~3 times a week , and almost everyday . weight status was determined by calculating body mass index ( bmi ) with self - reported height and weight . bmi is a good indicator of adiposity and self reported height and weight have adequate validity for population studies ( bowman & delucia , 1992 ) . respondents were grouped by bmi into four groups : underweight ( bmi<18.5 ) , normal weight ( 18.5bmi<25 ) , overweight ( 25bmi<30 ) , and obese ( bmi30 ) ( who , 1998 ) . in multiple logistic regressions , overweight individuals ( bmi25 ) were compared to non - overweight individuals . control variables included sex , age , size of place of residence , education , income , working status , and marital status . size of place of residence was divided into smsa ( 0 ) vs non - smsa ( 1 ) according to zip code of residence . income categories were re - coded to six categories because of low frequency in some of the categories . working status was collapsed into two categories : currently working ( 1 ) and not working ( 0 ) . married people were treated as a reference category and all other categories were collapsed into unmarried . chi - squares and analysis of variance with scheff test were used to examine bivariate relationships . logistic regression ( proportional odds model ) was applied to simultaneously investigate the relationships of all study variables in multi - variable models . about half of the sample 's acculturation status was traditional , a third was bicultural and a sixth could be classified as acculturated . the respondents reported average age of 40 years , average education of 16 years , and average income of approximately $ 35,000 . the majority of them were working , while men were more likely working than women . korean american men had significantly higher bmi than korean american women . while more korean american men ( 30% ) were overweight than korean american women ( 8% ) , more korean american women were underweight ( 9% ) than korean american men ( 4% ) . only about a third reported that they ate three meals a day everyday ( table 2 ) . breakfast was the least frequent meal of the day with less than half reporting eating breakfast everyday . dinner was the most frequent meal of the day with 86% eating dinner everyday . about a quarter of the respondents reported having a snack everyday , and women tended to snack more often than men ( =11.025 , p<0.05 ) . more than half ( 58% ) reported that they usually eat out or get take - out food at least once a week . since there were few significant and meaningful sex differences among acculturation , socioeconomic , meal frequency variables , further analyses were carried out with combined male and female respondents . in the case of analyses with weight status , the low prevalence of overweight and obesity among women forced the analyses done only in men . relationships between acculturation and independent variables ( frequency of meals , frequency of snack , and frequency of eating - out ) were examined ( table 3 ) . controlling for sociodemographic variables ( age , sex , size of place of residence , income , education , working status , and marital status ) , relationships between acculturation and frequency of snacking and eating - out were significant . acculturated korean americans were more likely to eat snacks more frequently than traditional korean americans ; however , significant relationships were not found for any other acculturation group comparisons . higher frequency of eating - out was also significantly associated with being men , younger age , higher income , and unmarried status . after controlling for age , size of place of residence , income , education , working status , and marital status , bicultural men were more than twice as likely to be overweight than traditional men ( table 3 ) . when frequency of each meal , snacking , and eating - out were entered into the model , only frequency of snacking was significant . korean american men who snack more often were 1.3 times more likely to be overweight . when daily frequency of meals , rather than frequency of each meal , were entered to the model , only acculturation remained significant . this study examined how acculturation was related to frequency of meals , snacking , and eating - out behaviors in korean americans and whether frequency of meals , snacking , and eating - out were associated with body weight . acculturation was not significantly associated with frequency of meals , but significantly associated with frequency of snacking and eating - out . multiple regression models showed that only frequency of snacking was significantly related to the likelihood of being overweight in men . acculturated korean americans tended to more frequently eat snacks and eat out than traditional korean americans . this relationship between acculturation and snacking and eat - out was also reported in japanese americans . third - generation japanese americans were engaged in more frequent snacking and eating - out than second - generation japanese americans ( kudo et al . , 2000 ) . these results seem to support that higher acculturation may lead to changes in daily eating patterns , which includes more eating occasions outside of traditional meals at home . it is worth mentioning that frequency of meals was not significantly related to most sociodemographic variables . older korean americans were more likely to eat breakfast and korean americans living in non - smsa areas to eat lunch . none of the variables examined in this study were significantly associated with frequency of dinner . these results seem to indicate that dinner is the most constant and stable meal a day . in contrast , frequency of eating - out was significantly associated with several sociodemographic variables as well as acculturation . korean americans were more likely to eat out if they were acculturated , unmarried , educated men with higher income . . both married and unmarried individuals appeared to engage in similar daily eating patterns , suggesting that culture and hunger structured the timing and frequency of consumption . however , the source of food differed substantially by marital status , with unmarried people much more often seeking food out of their household than those who are married . marriage provides a commensal partner for eating occasions , and norms of marriage encourage eating at home ( bove et al . , 2003 ) . many people are reluctant to or not able to prepare foods just for themselves , and this may lead many unmarried people to use commercially prepared foods and eat in restaurants or bring takeout food home . this study was limited in making causal inference because of the cross - sectional study design . we used the traditional and colloquial definition of meals ( breakfast , lunch , and dinner ) and snacking , therefore , information from people who do not fit into the traditional meal patterns may not have been fully obtained . since meals and snacks are defined by respondents , results may have been affected by personal bias . however , since this study asked each meal separately then combined meals into one variable , personal bias on a definition of " meals " would have been less than asking " how many meals do you eat a day ? " definition of " meals " warrants more attention because consistent use of a well - defined " meal " variable will help clarify possible relationships and mechanisms between meal frequency and body weight ( chiva , 1997 ; gatenby , 1997 ) . this study did not examine mechanisms of the relationships between meal frequency , eating - out and body weight . one speculated mechanism on the relationship between meal frequency and body weight is that meal frequency affects energy utilization . studies appear to indicate that there are no significant relationships between meal frequency and energy utilization , when energy intake is controlled ( bellisle et al . , 1997 ) . therefore , meal frequency would not affect body weight in short terms , if isocaloric diets are consumed ( bellisle et al . however , it is not conclusive if the same results will be found with free - living individuals where energy intake will not be controlled . although it has been speculated that frequent meals may result in higher energy intake ( booth , 1988 ) , issues in dietary assessment ( under - and over - reporting ) have hindered to reach meaningful conclusion ( bellisle et al . , 1997 ) . whether frequent meals and/or snacking lead to over consumption needs to be further studied with combination of dietary assessment and metabolic assessment . in addition to the relationship between frequency of meal and obesity , there is growing evidence on possible advantages of frequent meals on other health conditions such as maintaining better control of blood glucose ( jenkins et al . , 1994 ) , although some studies ( franceschi et al . , 1992 ; gerhardsson de verdier & longnecker , 1992 ; young & wolf , 1990 ) reported that frequent meals may be related to colorectal cancer . future research should examine relative risk and benefit of having frequent meals in order to make a recommendation to healthy and free - living population .

consuming regular meals has been studied in relation to better health , while higher regularity of eating - out has been linked to obesity . this study examined whether acculturation was associated with regularity of meals , eating - out , and overweight in korean americans . pre - tested questionnaires were mailed to a u.s . national sample with korean american surnames , and 55% of the deliverable sample responded , producing 356 usable questionnaires . acculturation was measured using a two - culture matrix model and gordon 's theoretical work , and showed there were three distinct groups ( acculturated , bicultural , and traditional ) . only 36% reported that they regularly ate three meals a day . breakfast was the least frequent meal of the day with 43% reporting eating breakfast everyday . more than half ( 58% ) reported that they usually eat out or get take - out food at least once a week . after controlling for age , sex , income , education , and working status , higher acculturation was related to greater regularity of eating - out , but not meal regularity . a total of 28% of men and 6% of women were overweight ( bmi>25 ) , and there were significant and positive relationships between body weight status and acculturation in men but not women . however , no significant relationships between frequency of meals and eating - out and overweight status were present . this study did not find significant relationships of meal regularity and eating - out with body weight , however , given the positive relationship between acculturation and eating - out among the subjects and the well - established relationship between eating - out and obesity , nutrition education about skipping meals and eating - for korean americans may be useful to prevent such relationships from developing .

Introduction Subjects and Methods Sample Data collection procedure Measures Analysis Results Discussion

scleromyxedema is a rare , chronic idiopathic disorder characterized by the appearance of numerous papules and areas of induration that are due to dermal mucin deposition in association with an increase in dermal collagen . this entity must be distinguished from localized variants of lichen myxedematosus where skin is the sole site of involvement . there are , however , occasional patients who have an atypical constellation of findings and fall between scleromyxedema and localized lichen gentleman who had findings of scleromyxedema and associated peripheral eosinophilia , but no monoclonal gammopathy . a 59-year - old gentleman from punjab , india , presented with severe generalized pruritus and closely studded papules all over the body for 7 years with an exacerbation for 15 days . the rash initially began as erythematous papules over the face , then progressed to involve the trunk and limbs . general physical examination revealed cervical and axillary lymphadenopathy with 1 cm - sized , mobile , firm and multiple lymph nodes . systemic examination was normal , apart from a palpable liver ; 6 cm below the costal margin . cutaneous examination revealed numerous , 3 - 4 mm sized , closely spaced papules , which were firm and waxy , arranged in a widespread symmetrically distributed pattern all over the body including the scalp , and sparing only the mucosae and genitalia . the glabella was typically involved with deep longitudinal furrowing and madarosis , giving the appearance of leonine facies . skin over the flexures was thrown into folds , however mobility was not restricted . overlying the proximal interphalangeal joints , a doughnut sign was visible as a central depression surrounded by an elevated rim of infiltration due to skin thickening [ figure 1 ] . infiltration of the face resulting in leonine facies , coalescing sheets of papules all over the body with thedoughnut sign visible on the fingers and the skin of the flexures thrown into folds abnormal investigations included a high total leukocyte count of 36,800/mm , with an absolute eosinophil count of 31,000/mm . high - resolution computed tomography ( hrct ) of chest showed mediastinal and axillary lymphadenopathy . the upper and mid dermis showed a moderately dense chronic inflammatory cell infiltrate of lymphocytes , plasma cells and histiocytes . in the mid dermis , collagen bundles showed fragmentation and splitting surrounded by loose myxoid stroma and plump fibroblastic cells , consistent with the diagnosis of scleromyxedema [ figure 2 ] . collagen bundles with fragmentation and splitting surrounded by loose myxoid stroma and plump fibroblastic cells all over the mid - dermis , hematoxylin and eosin 10 scleromyxedema , also known as arndt gottron syndrome or generalized lichen myxedematosus , is a rare disease , etiology of which is not yet known . it is a chronic and progressive disease , in which response to therapy has been inconsistent . diagnostic criteria of scleromyxedema include a generalized papular eruption and sclerodermoid features , histological features of mucin deposition , fibroblast proliferation and fibrosis , monoclonal gammopathy on serum protein electrophoresis and the absence of thyroid disease . accentuation of facial folds and creases may distort the features giving the appearance of leonine facies . as the condition progresses , skin stiffening , sclerodactyly and decreased mobility of joints is seen . nail fold and mat - like telangiectasias , and calcinosis , as seen in systemic sclerosis are absent . the skin in scleromyxedema moves over the subcutaneous tissue , unlike in scleroderma . the monoclonal gammopathy is usually igg with lambda light chains seen in 80% patients . the paraproteinemia is said to either stimulate the production of hyaluronic acid and prostaglandin e by fibroblasts or represents an immunologic response to dermal mucin . the patients who do not have paraproteinemia should be followed up for its presence for years . patients of scleromyxedema can have a number of internal manifestations , in particular- muscular , neurologic , rheumatologic , pulmonary , renal and cardiovascular involvement . staging of the disease has been proposed , with stage i being limited cutaneous papular mucinosis , stage ii is generalized cutaneous mucinosis and/or extracutaneous manifestation(s ) and stage iii generalized cutaneous mucinosis and/or extracutaneous manifestation(s ) and disease related karnofsky ps < 50% . the characteristic histopathologic triad includes diffuse dermal mucin deposition , increased collagen and numerous irregularly shaped fibroblasts . treatment is difficult ; melphalan is the most commonly used therapeutic agent ; high doses have been recommended for stage ii and iii patients . other therapies include steroids , methotrexate , cyclophosphamide , thalidomide , cyclosporine , oral retinoids , extracorporeal photopheresis and plasmapheresis . iv immunoglobulins can be considered as a well tolerated and efficacious second line therapy and also as first line therapy in cases of acutely worsening neurologic function . high - dose melphalan along with autologous stem cell therapy was found to be effective . beam regimen ( bcnu , etoposide , cytarabine and melphalan ) has been recommended as the conditioning prior to autologous stem cell transplantation ( asct ) for prolonged remission . the level of evidence ascribed to all these therapies is level iii , as this is a rare disease . scleromyxedema can also present with severe pruritus , scalp involvement , unrestricted mobility and associated eosinophilia without paraproteinemia .

scleromyxedema is a rare , chronic and persistent idiopathic disorder characterized by a generalized papular eruption due to dermal mucin deposition with an increase in dermal collagen . patients usually have associated paraproteinemia . we describe the case of a 59-year - old gentleman with features of scleromyxedema , who had severe pruritus , scalp involvement , unrestricted mobility and associated peripheral eosinophilia , but no monoclonal gammopathy .

Introduction Case Report Discussion

osteopetrosis is a rare bone disease , with an estimated prevalence of 1 case in every 20,000 people ( 1 ) . this disease , caused by metabolic imbalances resulting from genetic mutations , was first reported in 1904 by the german radiologist heinrich albers - schnberg , as reviewed by bollerslev ( 2 ) and garca et al ( 3 ) . within one of a limited number of reports on this disease , garca et al ( 3 ) demonstrated osteomyelitis of the mandible in a patient with osteopetrosis . the pathophysiological mechanism of the disease involves an osteoclastic dysfunction that results in an impaired bone resorption ability ; as a result , bone modeling and remodeling are inhibited . the cortical bone mass is therefore able to increase in density and develop into a hard , marble consistency when the medullary bone is remodelled . two types of osteopetrosis exist : malignant and benign , categorized based on mortality rate , and disease severity is highly heritable ( 1 ) . malignant osteopetrosis , or autosomal recessive osteopetrosis has a poor prognosis associated with fatality in infancy . patients with aro demonstrate a survival rate of 7% , even subsequent to curative treatment such as allogenic hemopoietic stem cell transplantation ( 4 ) . currently , it is understood that autosomal dominant osteopetrosis ( ado ) is a relatively benign disorder caused by a missense mutation in the clcn7 gene ( 5 ) . benign osteopetrosis typically has a late onset in children and adults , the symptoms are milder and prognosis is better than in malignant osteopetrosis . however , curative care is not available for patients with the benign form , thus only supportive care is provided . osteopetrosis is difficult to treat and can cause complications , such as chronic osteomyelitis and bone fractures , as a result of poor blood supply and iatrogenic fractures . as there is no cure , the current study presents a case of chronic mandibular osteomyelitis and a maxillary sinusitis - generated fistula of benign osteopetrosis . the current recommended treatment options include high - dose systemic antibiotics , debridement of the necrotic region and hyperbaric oxygen ( hbo ) therapy ( 3 ) . however , the current report describes treatment without the use of high - dose antibiotics or hbo therapy , a justified approach due to the insufficiency of the blood supply to the bone . treatment of the debrided fistula , which included repeated flushing and cleaning using nasal endoscopy every 2 weeks in the acute infected period , was successful . therefore , the present study investigated whether the debridement and drainage of pus from fistulas generated by maxillary sinusitis and chronic osteomyelitis is an effective treatment method . in december 2014 , a 50-year - old chinese female was admitted to the xiamen chang gung hospital ( xiamen , china ) for the evaluation and treatment of chronic purulent nasal discharge in the left side of the nose . the patient reported that the discharge appeared 3 years previously , following a tooth extraction from the left side of the mouth . intraoral examination revealed exposed necrotising bone in the left maxillary region , sixth , seventh and eighth tooth agenesis in the upper left side and fistula formation . the pus had accumulated in the mouth and the left side of the nose in the fistula , generated as a result of maxillary sinusitis . previous medical history revealed that the patient was diagnosed with osteopetrosis at the age of 19 years old , with six rib , femoral and thoracic vertebra bone fractures ( fig . 1 ) . the patient did not accept any treatment for the osteopetrosis , but was fitted with a plaster cast for 6 weeks . however , the patient developed splenomegaly due to secondary bone marrow suppression , subsequent to which the spleen was resected . examination of the patient 's family history revealed that her older sibling was diagnosed with osteopetrosis at the age of 23 years old , but the patient 's child had not developed the disease . non - contrast computed tomography scans ( ge healthcare life sciences , chalfont , uk ) displayed evidence of low bone density in the posterior left side of the mandible with destruction of the vestibular cortex that was suggestive of a fistula , resulting from chronic osteomyelitis with maxillary sinusitis ( fig . a bone marrow puncture , hematoxylin and eosin staining and brightfield imaging of these structures were performed . the medullary cavity was obliterated and the bone marrow was reported to be suppressed , determined by hematoxylin and eosin staining using an olympus bx51 brightfield microscope ( fig . the sequestra were removed and debridement of the necrotic region was performed via nasal endoscopy , without antibiotics or any other form of treatment a month later , intravenous low - strength antibiotic therapy ( 1 g cefazolin , every 8 h ; mosinter group , ltd . , ningbo , china ) was administered for a week , and the fistula was flushed and drained of pus via nasal endoscopy every 2 weeks . after a week , the infection had been successfully treated without the necessity of additional antibiotic treatment , chronic osteomyelitis was resolved and the fistula was replaced with fresh granulation after 2 months ( fig . osteopetrosis is a rare hereditary disease caused by an imbalance in bone metabolism , resulting from decreased osteoclast activity ( 6 ) . this leads to a lack of bone remodeling , which can result in a high mortality rate in children within the first 10 years of their life ( 6 ) . early onset osteopetrosis is known as severe infantile malignant autosomal recessive disease , or intermediate mild autosomal recessive disease ( 6 ) . this condition is typically fatal as a result of anemia with congestive heart failure or sepsis due to bone overgrowing in the bone marrow space . in adults , the same disease is known as benign ado with a late onset , and has a lower mortality rate ( 3 ) . at present , the curative treatment for malignant osteopetrosis is allogenic hemopoietic stem cell transplantation ( 4 ) ; however , only supportive care is available for benign osteopetrosis ( 7 ) . therefore , symptom management of benign osteopetrosis is important to increase the patient survival rates . the present study reported the case of a patient diagnosed with benign osteopetrosis at the age of 19 years , with fractures identified based on presentation and radiology . in benign osteopetrosis , the most common complications are chronic osteomyelitis , occurring in 10% of cases involving the mandible ( 3 ) , and bone fractures , in 78% of cases ( 8) . the patient in the current study presented with chronic purulent nasal discharge in the left side of the nose , following tooth extraction from the left side of the mouth 3 years earlier associated with chronic osteomyelitis . since the healing process in the bone is slow , it is difficult to fight and prevent infection in osteopetrosis ( 1 ) . in ado , care must be taken when removing the sequestra , since it is easy to create iatrogenic fractures due to dense bone , which are difficult to repair once the bone is fractured ( 9 ) . currently , administering a high - dose of systemic antibiotics with debridement of the necrotic region alongside hbo therapy is recommended ( 3 ) . however , as the patient in the current report presented with insufficient blood supply to the bone due to osteopetrosis , the use of high - dose antibiotics was not suitable . instead , pus from the fistula was flushed and drained via nasal endoscopy every 2 weeks for 1 month , subsequent to careful removal of the sequestra and debridement of the necrotic region . low dose and intensity antibiotics were administered following debridement , and the infection was cleared . to the best of our knowledge , this is the first case report indicating that debridement with low doses of antibiotics may be effective in the treatment of osteopetrosis . notably , patients with osteopetrosis should be aware of good dental care and oral hygiene . decayed teeth should be endodontically treated and tooth extraction should be avoided , if possible , on the account of necrosis development . care must therefore be taken when removing the necrotic bone to prevent iatrogenic fractures , which may occur due to inexperience of the surgeon ( 9 ) . the findings of the present study demonstrated that debridement and drainage of pus from fistulas are important , and possibly a more effective strategy compared with high - dose antibiotic usage alone in osteopetrosis . in conclusion , the complications of osteopetrosis include bone fractures and chronic osteomyelitis , which are difficult to treat . in a patient with osteopetrosis currently , there is no cure for osteopetrosis and , therefore , symptom management is important to increase the patient survival rates . in the present study , it was demonstrated that debridement and drainage of pus from fistulas may be more effective than high - dose antibiotic usage in treating osteopetrosis .

osteopetrosis is a rare bone disease caused by metabolic imbalances as a result of genetic mutations . for instance , autosomal dominant osteopetrosis is caused by a missense mutation of the c1cn7 gene . this was first reported in 1904 and is thought to be caused by osteoclastic dysfunction and an impaired bone resorption ability . an accumulation of cortical bone mass during the remodeling of the medullary bone may increase the bone density and give rise to a hard marble consistency . osteopetrosis can be divided into benign and malignant forms ; however , no curative treatment exists for benign osteopetrosis . the management of complications , such as chronic osteomyelitis and fractures , serves a key role in influencing the patient survival rates . previous studies have demonstrated that a combined treatment of hyperbaric oxygen ( hbo ) lavage for debridement of the necrotic region and high - dose systemic antibiotics may be effective in the management of osteopetrosis . the present study reported a case of chronic mandible osteomyelitis and fistula occurring in association with maxillary sinusitis , who was successfully treated by through nasal endoscopy , using repeated flushing and cleaning every 2 weeks as a form of debridement , in the absence of high - dose antibiotics and hbo .

Introduction Case report Discussion

systemic local anesthetic toxicity is a rare but potentially fatal complication that is intractable to conventional cardiopulmonary resuscitation . systemic local anesthetic toxicity also has a risk of progressing to " recurrent systemic local anesthetic toxicity after successful resuscitation " . intravenous infusion of les reverses intractable cardiac toxicity in an animal model , and les are effective for treating local anesthetic - induced cardiac toxicity . therefore , various studies associated with les have been actively conducted to understand the mechanism of lipid rescue and improve treatment regimens . intralipid 20% , which contains only 100% long - chain triglycerides , is commonly used to treat local anesthetic - induced systemic toxicity , whereas lipofundin mct / lct 20% which is composed of 50% long - chain triglycerides and 50% mediumchain triglycerides , is occasionally used to treat local anestheticinduced systemic toxicity . however , both types of le affect hemodynamics in an ex vivo model but the associated cellular mechanism remains unknown . therefore , we investigated the inotropic effect of two les ( intralipid or lipofundin mct / lct ) on a hanging heart model mounted in a langendorff perfusion system to elucidate the mechanism associated with changes in intracellular calcium level in myocardial cells . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . if the tail moved ( i.e. a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . the perfusion solution was maintained at 37 0.2 using thermostatically controlled water circulating system ( water bath ntt-2200 ; tokyo rikakikai co. ltd . , perfusion pressure was maintained at 70 mmhg with a 95 cm high fluid column and an overflow pump . the left atrium was removed and a 2f millar catheter ( model spr-407 ; millar instruments inc . , houston , tx , usa ) was inserted into the left ventricle . a pressure transducer ( model ml 118 ; adinstruments pty . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . baseline measurements were taken on quiescent cells for 34 min prior to the application of le . fluorescent images were collected every 0.6 sec for fast signals and analyzed by fluoview image processing software ver . twenty signal curves were taken from one view for each and averaged after each le treatment ( n = 4 ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . all analyses were performed using spss statistical software ver . 18.0 for windows ( spss inc . , experimental outcomes were analyzed using one - way analysis of variance and the bonferroni post - hoc test . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . if the tail moved ( i.e. a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . the perfusion solution was maintained at 37 0.2 using thermostatically controlled water circulating system ( water bath ntt-2200 ; tokyo rikakikai co. ltd . , perfusion pressure was maintained at 70 mmhg with a 95 cm high fluid column and an overflow pump . the left atrium was removed and a 2f millar catheter ( model spr-407 ; millar instruments inc . , houston , tx , usa ) was inserted into the left ventricle . a pressure transducer ( model ml 118 ; adinstruments pty . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . baseline measurements were taken on quiescent cells for 34 min prior to the application of le . fluorescent images were collected every 0.6 sec for fast signals and analyzed by fluoview image processing software ver . twenty signal curves were taken from one view for each and averaged after each le treatment ( n = 4 ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . if the tail moved ( i.e. a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . the perfusion solution was maintained at 37 0.2 using thermostatically controlled water circulating system ( water bath ntt-2200 ; tokyo rikakikai co. ltd . , perfusion pressure was maintained at 70 mmhg with a 95 cm high fluid column and an overflow pump . the left atrium was removed and a 2f millar catheter ( model spr-407 ; millar instruments inc . , houston , tx , usa ) was inserted into the left ventricle . a pressure transducer ( model ml 118 ; adinstruments pty . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . baseline measurements were taken on quiescent cells for 34 min prior to the application of le . fluorescent images were collected every 0.6 sec for fast signals and analyzed by fluoview image processing software ver . twenty signal curves were taken from one view for each and averaged after each le treatment ( n = 4 ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . all analyses were performed using spss statistical software ver . 18.0 for windows ( spss inc . , chicago , il , usa ) . experimental outcomes were analyzed using one - way analysis of variance and the bonferroni post - hoc test . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the ratio of + dp / dtmax ( control , 98.5% 6.9% ; il1 , 103.2% 7.2% ; mct1 , 108.7% 15.7% ) , dp / dtmax ( control , 97.6% 10.0% ; il1 , 103.9% 6.0% ; mct1 , 102.1% 9.6% ) , and hr ( control , 93.2% 7.8% ; il1 , 96.6% 1.5% ; mct1 , 94.0% 5.0% ) were not significant between the groups . the change of intracellular calcium level was estimated by comparing the cellular fluorescence ratio with the basal fluorescence ratio using the fluo-3/am total calcium imaging technique . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the ratio of + dp / dtmax ( control , 98.5% 6.9% ; il1 , 103.2% 7.2% ; mct1 , 108.7% 15.7% ) , dp / dtmax ( control , 97.6% 10.0% ; il1 , 103.9% 6.0% ; mct1 , 102.1% 9.6% ) , and hr ( control , 93.2% 7.8% ; il1 , 96.6% 1.5% ; mct1 , 94.0% 5.0% ) were not significant between the groups . the change of intracellular calcium level was estimated by comparing the cellular fluorescence ratio with the basal fluorescence ratio using the fluo-3/am total calcium imaging technique . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the major findings of our study are : 1 ) the lipofundin mct / lct infusion significantly increased lvsp in hanging hearts , 2 ) lipofundin mct / lct has had more of a positive inotropic effect than that of intralipid , and 3 ) the le treatments increased the intracellular calcium levels in h9c2 cells . lipofundin mct / lct increased intracellular calcium levels more than that of intralipid. although there are some reports indicate that long - chain triglycerides has have virtually no effect on hemodynamics , it is a generally accepted that le has a positive hemodynamic effect . however , a mixture of medium - chain triglycerides , long - chain triglycerides , and omega-3 polyunsaturated fatty acid emulsions increase mean aortic blood pressure . thus , we investigated the effect of commercially available les ( intralipid 20% and lipofundin mct / lct 20% ) on hemodynamics in an ex vivo heart model . as results , lipofundin mct / lct had a more positive inotropic effect than that of intralipid. therefore , careful observations of hemodynamic changes are needed during bolus infusion of les . in particular , lipofundin mct / lct should be used with caution as a total parenteral nutrition component in critically ill patients with compromised cardiovascular function . the second aim of this study was to elucidate the mechanism responsible for the le - induced increase in lvsp and intracellular calcium levels in myocardial cells . long chain fatty acids directly activate calcium channels at some lipid sites near the channels or on the channel protein itself , as assessed by the standard whole cell voltage clamp technique in ventricular myocytes . therefore , the positive inotropic effect of lipofundin mct / lct shown on the hearts connected to the langendorff perfusion system may be associated with cardiac myocyte calcium concentration . however , one study showed that 10% intralipid exerts a modest positive inotropic effect , although the intracellular calcium remains unchanged by intralipid . thus , we measured the change in calcium current after adding les to rat cardiac myoblastic h9c2 cells to clarify their effect on calcium current . most of the signal transduction pathways that stimulate inotropy ultimately involve calcium either by increasing calcium influx , by increasing release of calcium from the sarcoplasmic reticulum , or by sensitizing troponin - c to calcium . in this study , we found that les increased intracellular calcium level more than three times in h9c2 cells , and that lipofundin mct / lct increased intracellular calcium level more than that of intralipid , suggesting that lipofundin mct / lct has a more positive inotropic effect than that of intralipid. les are used for various clinical purposes and have become an integral part of parenteral nutrition , effective treatment for local anesthetic - induced cardiovascular collapse , and treatment for lipophilic drug toxicity . the mechanism of action of les for treating local anesthetic - induced cardiac toxicity is not completely understood . however , local anesthetic - induced systemic toxicity is caused by inhibiting inotropic and metabotropic cell signal systems and possibly mitochondrial metabolism . the " lipid sink theory " ( reduced tissue binding by re - establishing equilibrium in the plasma lipid phase ) and the " energetic - metabolic effect " ( reversal of inhibited mitochondrial fatty acid transport ) are the most widely known theories for the mechanism of action of les for treating local anesthetic - induced systemic toxicity . in addition , a " positive inotropic effect " is proposed as another mechanism . in this regard , lipofundin mct / lct seems to be a more effective le for treating this condition than intralipid. our study had some limitations . however , we did not verify the source of the increase or the channel involved . thus , further research regarding the effect of calcium channel blockers on le - induced increase in calcium is needed to elucidate the calcium source . third , we showed previously that 3 ml / kg intralipid increases lvsp in an in vivo rat model . however , as this large volume of le could change the hemodynamics in an ex vivo heart model , we chose 1 ml / kg in this study . we assume that this may have affected the limited increase in lvsp after the intralipid treatment despite the increased intracellular calcium level . in conclusion particularly , lipofundin mct / lct had more of a positive inotropic effect than that of intralipid. the inotropic effect of les may be related to increased intracellular calcium levels in the heart .

backgroundlipid emulsions have been used to treat various drug toxicities and for total parenteral nutrition therapy . their usefulness has also been confirmed in patients with local anesthetic - induced cardiac toxicity . the purpose of this study was to measure the hemodynamic and composition effects of lipid emulsions and to elucidate the mechanism associated with changes in intracellular calcium levels in myocardiocytes.methodswe measured hemodynamic effects using a digital analysis system after intralipid and lipofundin mct / lct were infused into hearts hanging in a langendorff perfusion system . we measured the effects of the lipid emulsions on intracellular calcium levels in h9c2 cells by confocal microscopy.resultsinfusion of lipofundin mct / lct 20% ( 1 ml / kg ) resulted in a significant increase in left ventricular systolic pressure compared to that after infusing modified krebs - henseleit solution ( 1 ml / kg ) ( p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) . lipofundin mct / lct 20% had a more positive inotropic effect than that of intralipid 20% ( p = 0.009 , 95% ci , 1.411.6 ) . both lipid emulsion treatments increased intracellular calcium levels . lipofundin mct / lct ( 0.01% ) increased intracellular calcium level more than that of 0.01% intralipid ( p < 0.05 , 95% ci , 0.01.9).conclusionsthese two lipid emulsions had different inotropic effects depending on their triglyceride component . the inotropic effect of lipid emulsions could be related with intracellular calcium level .

Introduction Materials and Methods Experimental protocols Experiment 1 Experiment 2 Statistical analysis Results Effect of LE on hemodynamic functions of hanging hearts in a Langendorff perfusion system Effects of the lipid emulsions on intracellular calcium in H9c2 cells Discussion

cholangiocarcinoma ( cca ) is a devastating complication of primary sclerosing cholangitis ( psc ) that often results in removal from the liver transplant list and death . in half of cases the malignancy is found within a year of psc diagnosis.1 early detection of cca may lead select psc patients to be included in specialized protocols where chemoradiation precedes and may follow orthotopic liver transplantation ( olt ) . these protocols have been modestly successful and are reportedly available in about a third of liver transplant centers across the united states.2 this emerging potential for cure highlights the importance of identifying risk factors for or warning signs of concurrent or impending cca in this patient population . the aim of this study is to examine the association between increased biochemical liver dysfunction , as evidenced by bilirubinemia and coagulopathy , and the presence of synchronous or subsequently developing cca in patients with psc . institutional board review ( irb ) approval was sought and secured prior to the initiation of the project . we did a chart review of all patients diagnosed and treated for psc at the outpatient hepatology clinic of the johns hopkins hospital from 1998 to 2012 . primary end points included diagnosis of cca or olt with no evidence of malignancy in the explanted liver . first clinic visit was designated as day 0 and age , gender , race , alcohol and tobacco consumption , and co - existing diagnosis of inflammatory bowel disease ( ibd ) were recorded for each patient . patients with known concurrent viral hepatitis were excluded from the study , as were those with documented biliary instrumentation prior to or fever and leukocytosis on day 0 . labs obtained on day 0 included aspartate and alanine aminotransferases ( ast , alt ) , total bilirubin ( tbil ) , alkaline phosphatase ( alp ) , international normalized ratio ( inr ) , and serum ca 19 - 9 antigen levels . patients that developed cca beyond 18 months of follow - up were excluded from the study . included in the control group were only those patients that underwent olt with no sign of cca in the explanted liver , the rationale being that explant pathology examination would exclude underlying , subclinical cca in a psc patient with reasonable confidence . the final study population consisted of patients who developed cca within 18 months of day 0 ( cca group ) and the control group ( ncca group ) . mean lab values were calculated for each group and student s t - test was implemented to determine the statistical significance of the differences between means . chi square test was utilized to detect association of smoking , alcohol , and presence of ibd with eventual development of cca . software used for statistical analysis was stata ( by statacorp lp , college station , tx , usa ) . the records of 43 patients with psc were retrieved from our electronic database . sixteen patients were diagnosed with cca at some point during their follow - up ; half of these patients ( 8) developed cca within 18 months of day 0 , while the other half developed malignancy at a later time in their course , thus excluding them from the study . twenty - seven patients remained cca - free as of their last clinic visit , and 21 of them underwent olt with no evidence of cca in their explant pathology , over a mean period of 52.9 months . five of these 21 patients were excluded due to missing laboratory values on day 0 . patients that met all our criteria formed a final sample size of 24 ( eight in the cca group , 16 in the ncca group ) . demographic characteristics included predominance of male sex ( n = 6 in the cca " , n = 13 in the ncca group ) and caucasian race ( n = 7 in the cca , n = 24 in the ncca group ) . patients in the cca group were of significantly older age ( 48 vs. 36 years , p = 0.017 ) than those in the ncca group . tobacco and alcohol use were not quantified for each patient , but their presence was identical in the two groups ( 37.5% and 25% for smoking and alcohol , respectively ) . mean follow - up from day 0 to cca detection was 7 months for the cca group ; mean follow - up until the time of olt for the ncca group was 60 months ( p = 0.000 ) . prevalence of ibd ( irrespective of subtype ) was documented in five out of eight ( 62.5% ) patients in the cca group and in 12 out of 16 ( 75% ) patients in the ncca group ; this difference was not significant ( p = 0.52 ) . none of the patients of the cca group experienced variceal bleeding , while four of the ncca group had at least one ( 0% vs 25% , p = 0.121 ) . in terms of liver biochemistry , patients of the cca group on day 0 were found to have significantly more elevated levels of ast ( 283.7 vs. 87.7 , u / l , p = 0.034 , fig . 1 . ) , tbil ( 8.0 vs. 3.2 , mg / dl , p = 0.042 , fig . 2 ) , and inr ( 1.17 vs. 1.01 , p = 0.026 ) than the ncca group . on the other hand , levels of alt ( 185.5 vs. 105.6 , u / l , p = 0.104 ) , alp ( 334.0 vs. 491.3 , u / l , p = 0.136 ) or serum ca 19 - 9 ( 347.0 vs. 116.6 , u / ml , p = 0.126 ) did not differ significantly between the two groups . alp , alkaline phosphatase ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; ibd , irritable bowel syndrome ; inr , international normalized ratio . the incidence of cca in psc has been estimated to be up to 1.5% per year.3 identifying psc patients susceptible to developing cca would allow for implementation of timely interventions . to that end , an increase in the frequency of tp53 , bcl-2 , k - ras , and p16-ink4a mutations in cca tissue as well as in the bile of psc patients with the malignancy have been demonstrated,46 but mutations such as these characterize the tumorigenesis in a variety of human cancers . melum et al.7 found single nuclear polymorphisms in the natural killer group 2 member d ( nkg2d ) cell receptor gene associated with an increased risk for cca in psc ; the authors noted , however , that the detection of the predisposing alleles as a means to isolate the high - risk patients would yield too many false positives . elevated serum levels of leucine - rich a2 glucoprotein 1 ( lrg1 ) , alone or in combination with interleukin 6 ( il-6 ) and ca 19 - 9 , were shown to significantly aid in the discrimination between malignant and benign biliary strictures.8 bile9 and urine10 proteomic analyses and most recently the detection of extracellular vesicles in the bile and specific arrays of micrornas within those vesicles11 show promise in distinguishing underlying malignancy from a psc flare . the degree of biochemical deterioration , serum ca 19 - 9 levels , presence and duration of ibd , alcohol consumption , and smoking have all been suggested as risk factors for the development of cca in this population , along with a history of colonic dysplasia , cancer , proctocolectomy , and variceal bleeding . in the largest study of its kind,1 the european study group of primary sclerosing cholangitis retrospectively examined the records of 394 consecutive psc patients , 48 of whom ( 12.2% ) developed cca , half of them within a year of psc diagnosis . the latter subgroup was found to contribute to significantly higher levels of serum bilirubin and coagulopathy compared to the majority of psc patients that did not develop cca . in an older , but almost equally sizeable swedish study of 305 psc patients,12 serum bilirubin at diagnosis of psc thus , the available data from high - powered studies confirm our own finding of significantly higher bilirubin levels in the cca group . the aminotransferase levels , however , did not significantly differ between cca and noncca psc patients in those studies , which is in contrast to our finding of significantly higher ast ( but not alt ) levels in the cca group . a very recent study from the university of hiroshima13 examining the biochemical and cholangiographic characteristics of cca in psc and featuring an almost equal sample size ( n = 23 ) corroborated our findings of both significantly higher bilirubinemia and ast transaminitis in cca patients . elevated ca 19 - 9 certainly raises suspicion of cca and often triggers further evaluation with non - invasive ( ultrasound , magnetic resonance cholangiopancreatography ) and invasive ( endoscopic retrograde cholangiopancreatography with biliary brushings , cholangioscopy ) modalities . however , it can also occur in acute / recurrent bacterial cholangitis , benign stricturing of the biliary tree , or for other reasons unknown . some authors consider serum ca 19 - 9 a screening tool that is in itself unreliable and only to be used in combination with imaging and/or cytology.14 sinakos et al.15 showed that patients subsequently proven to have benign strictures presented with initial serum ca 19 - 9 values of up to 2000 u / ml ( although not exceeding that ) , and more than a third of all psc patients with significantly elevated ca 19 - 9 did not in fact have cca . serum ca 19 - 9 levels were found to be significantly elevated in the cca group compared to the controls in the population studied by ishii et al.13 ( 47 u / ml vs 7 u / ml , p = 0.017 ) , whereas in our study , the difference in marker levels between the two groups , albeit marked , did not attain statistical significance ( 347 u / ml in the cca vs 116.6 u / ml in the ncca group , p = 0.126 ) . the prevalence of ibd did not differ significantly between psc patients who did and did not develop cca in our study or the study by boberg et al.1 the latter , however , did demonstrate that the duration of ibd for the cca group was significantly longer . an earlier study by bergquist et al.,16 albeit one of more limited power , showed no association between duration of ibd and the development of cca but significant frequency of smoking in the psc / cca group . chalasani et al.17 also found no association between duration of ibd and cca but none between smoking and cca either , implicating alcohol consumption instead . in a multivariate analysis by the mayo clinic,3 only history of variceal bleeding proved to be a significant risk factor for the development of cca in psc patients . in contrast , our population exhibited identical prevalence of tobacco and alcohol use in the two groups . small size , the comparatively very short follow - up time until the primary end point ( cca ) , and the poor group survival rates may explain the absence of episodes of variceal bleeding among our cca patients . the most significant limitation of the present study is the relatively small sample size ; which may have contributed to the lack of statistical significance of the difference in mean serum ca 19 - 9 between the two groups and the lack of clinically meaningful , albeit statistically significant , difference in coagulopathy , as evidenced by the two groups mean inrs . a study with a greater sample size may demonstrate a wider gap , thereby linking more strongly biochemical dysfunction and cholangiocarcinoma . in summation , we have demonstrated and confirmed the finding of prior large - scale retrospective studies , which found that psc patients with co - existing or impending cca showed greater biochemical liver dysfunction , as demonstrated by significantly more increased total bilirubin and inr levels compared to those who do not develop the malignancy . levels of ast also appeared to be more elevated in patients who develop cca , possibly due to a greater degree of cholestasis - mediated liver injury . amidst the current effort to identify sensitive , specific , accurate , and practical markers to detect cca in this challenging population and until such markers are found and become widely available , we recommend that psc patients who present with significant bilirubinemia and coagulopathy are intensely evaluated with all available means to exclude the presence of cca .

background and aim : patients with primary sclerosing cholangitis ( psc ) who develop cholangiocarcinoma ( cca ) have a median survival of less than 6 months . in half of cases , psc and cca will be diagnosed either concurrently or within a year of one another . the aim of the present study is to demonstrate that the degree of biochemical liver dysfunction is associated with concomitant or impending cca . methods : we did a chart review of patients diagnosed with psc and cca up to 18 months from presentation ( cca group ) as well as patients with psc that underwent transplantation with no sign of cca in their explanted liver ( ncca group ) . along with demographic data and follow - up length , we recorded their presenting liver function tests , including alanine and aspartate aminotransferases ( alt , ast ) , total bilirubin ( tbil ) , alkaline phosphatase ( alp ) , international normalization ratio ( inr ) , and serum ca 19 - 9 levels . differences between mean values of the two groups were analyzed with a student s t - test . results : twenty - four patients were included . the cca group consisted of eight patients , and the non - cca group had 16 patients . there was no significant difference between the two groups in their presenting values of alt , alp , or serum ca 19 - 9 . however , the cca group had significantly higher levels of ast , tbil , and inr . conclusion : patients with psc and concurrent or impending cca appear to exhibit significantly greater biochemical liver dysfunction than those who do not develop cca . therefore , newly - diagnosed psc patients presenting with these findings may warrant more rigorous evaluation .

Introduction Methods Results Discussion Conclusion

forensic odontology / forensic dentistry is defined as that branch of forensic medicine which in the interest of justice deals with the proper handling and examination of dental evidence and with the proper evaluation and presentation of the dental findings . however , tasks of forensic odontologists have broadened in recent years to cover issues related to child abuse and domestic violence , human rights protection , insurance claims , and professional ethics . for all the above tasks , records have to be maintained through casts , radiographs etc . the storage of physical dental records like dental casts and radiographs is fraught with difficulties of space and is also very expensive . this has led to increasing dependence on digital photography and digital radiology for preservation and documentation of ante - mortem and post - mortem dental records . the very nature of digital imaging makes it very easy for the operator to adjust or modify digital image files . many such manipulations , however , constitute inappropriate changes to original data , and making such changes can be classified as scientific misconduct . consequently , to protect the integrity of research , the scientific community takes strong action against perceived scientific misconduct . in the current definition provided by the u.s . research misconduct is defined as fabrication , falsification , or plagiarism in proposing , performing , or reviewing research , or in reporting research results . at the outset , it is imperative to define what constitutes digital image fraud . any alteration of the original image in part or full with malicious intent of altering its implication constitutes digital fraud . it includes : ( a)additions,(b)removal or masking,(c)modifications of specific characteristics of image ( color / contrast etc.),(d)splicing , blending of multiple images into a single composite , and(e)combination of any or all of the above . being accused of misconduct initiates a painful process that can disrupt one 's research and career . to avoid such a situation , it is important to understand where the ethical lines are drawn between acceptable and unacceptable image adjustment . modifications of specific characteristics of image ( color / contrast etc . ) , splicing , blending of multiple images into a single composite , and combination of any or all of the above . being accused of misconduct initiates a painful process that can disrupt one 's research and career . to avoid such a situation , it is important to understand where the ethical lines are drawn between acceptable and unacceptable image adjustment . in this article , we highlight some universal guidelines for the apposite handling of digital image data and provide some examples to exemplify pitfalls and inapt practices . this article also discusses anti - tampering techniques and methods of detecting and preventing manipulation of digital images . digital image records of forensic data ( casts , lip prints , bite marks ) were manipulated using freely available softwares . softwares used were adobe photoshop ( adobe systems inc , san jose , ca , usa ) , corel draw ( corel corporation , ottawa , ontario , canada ) , picasa 3.9.1.535 version . to record digital images of casts , lip prints , bite marks , the tools employed on images for manipulation are listed together with their function in table 1 . it was quite evident that ostentatious digital images can be created either by merging two or more digital images or by altering an existing image . figure 1 shows use of merge tool on a bite mark photographfigure 2 shows tsuchihashis classificationfigure 3 shows original image of a lip print manipulated to another varietyfigure 4 shows digital photographic records of palatal rugae on castsfigure 5 shows multiplication of data in figure 4 using clone stamp tool . figure 1 shows use of merge tool on a bite mark photograph figure 2 shows tsuchihashis classification figure 3 shows original image of a lip print manipulated to another variety figure 4 shows digital photographic records of palatal rugae on casts figure 5 shows multiplication of data in figure 4 using clone stamp tool . usage of merge tool on a bite mark photograph tsuchihashi 's classification original image of a lip print manipulated to another variety digital photographic records of palatal rugae on casts modification of figure 4 showing how the same data was multiplied the old adage of publish or perish is very pertinent in the present scenario and it is not unexpected for sloppiness , plagiarism , and even fraud to find their way too easily in today 's intense research atmosphere . by far and away , the most significant problem is that scientists do not understand complex data - acquisition tools and occasionally seem to be duped by the ease of use of image - processing programs to manipulate data in a manner that amounts to misrepresentation . procedural hindrances to objectionable image manipulation have been greatly reduced resulting in an ease of bringing about any change to an image with just the click of a mouse . over the last 10 - 15 years , there have been a few highly public instances of falsified images ( abbott 1997 ; aldhous and reich 2009 ; weissmann 2006 ; xin 2006 ; young 2008 ) , but most of the problem lies with the lack of a basic understanding of how to properly handle image data . digital image modification is done at a pixel level and makes it to a large extent undetectable when viewed with a naked eye . several measures , such as few described below , help in detection of altered digital images : look for evident anomalies in the photo . this can comprise items not in the correct perspective , conflicting angles of reflection or unexpected discontinuities . any inconsistencies may divulge the photo to be forged . with the assumption that tampering disturbs certain underlying statistical properties of an image air - brushing or re - touching can be detected by measuring deviations of the underlying color filter array correlations . a digital composite of two people can be detected by measuring differences in the direction to the illuminating light sources from their faces and body . the presence of identical and spatially coherent regions in an image can be used as an evidence of tampering . if there is doubt about integrity of images and previous steps did n't detect the same , then the images can be submitted to a proficient image processing lab . detection of altered images has also been made easier by forensic tools that scrutinize scientific images ( available through the us department of health and human services office of research integrity ) . these tools have been used by some journals , and their routine use is currently being considered by many editors and publishers . the accuracy and reliability of digital dental records is very important when such records are considered as forensic evidence . the knack of image fakery has a long history , and in today 's digital age , it is possible to very easily change the information represented by an image without leaving any apparent traces of tampering . no method yet exists , which accomplishes efficiently and precisely the image forge recognition mission . considering that prevention is better than cure , we propose that the person making a digital record should validate his data by taking certain steps to prevent the manipulation of the data by any third party for any spurious purpose . a few points for prevention of fraudulent use of digital images can be stated as : it is attaching of information in the form of data to digital image . it 's like a miniature text file appended to files only adding a few bytes to the total file size so as to discourage manipulation at an amateurish scale . most of the images are stored in exchangeable image file ( exif ) format , so that you can view it in any organizer that can speak exif format . example for image metadata a digital watermark is a super impose on a digital photo ( consisting of text / logo / copyright notice ) . the purpose of a watermark is to recognize the work and discourage its unauthorized usage . though a watermark ca n't thwart unauthorized use of digital image , it makes tampering more difficult and offer shield from manipulation . it can differentiate between malicious and non - malicious changes to a greater or lesser extent . figure 7 shows a superimposed water mark on previously used image [ figure 4 ] of casts . this can comprise items not in the correct perspective , conflicting angles of reflection or unexpected discontinuities . check for inconsistent gloom or features that seem slightly bigger or smaller than standard . with the assumption that tampering disturbs certain underlying statistical properties of an image , these forensic techniques can detect specific forms of tampering . air - brushing or re - touching can be detected by measuring deviations of the underlying color filter array correlations . a digital composite of two people can be detected by measuring differences in the direction to the illuminating light sources from their faces and body . the presence of identical and spatially coherent regions in an image can be used as an evidence of tampering . if there is doubt about integrity of images and previous steps did n't detect the same , then the images can be submitted to a proficient image processing lab . detection of altered images has also been made easier by forensic tools that scrutinize scientific images ( available through the us department of health and human services office of research integrity ) . these tools have been used by some journals , and their routine use is currently being considered by many editors and publishers . the accuracy and reliability of digital dental records is very important when such records are considered as forensic evidence . the knack of image fakery has a long history , and in today 's digital age , it is possible to very easily change the information represented by an image without leaving any apparent traces of tampering . no method yet exists , which accomplishes efficiently and precisely the image forge recognition mission . considering that prevention is better than cure , we propose that the person making a digital record should validate his data by taking certain steps to prevent the manipulation of the data by any third party for any spurious purpose . a few points for prevention of fraudulent use of digital images can be stated as : it 's like a miniature text file appended to files only adding a few bytes to the total file size so as to discourage manipulation at an amateurish scale . most of the images are stored in exchangeable image file ( exif ) format , so that you can view it in any organizer that can speak exif format . a digital watermark is a super impose on a digital photo ( consisting of text / logo / copyright notice ) . the purpose of a watermark is to recognize the work and discourage its unauthorized usage . though a watermark ca n't thwart unauthorized use of digital image , it makes tampering more difficult and offer shield from manipulation . it can differentiate between malicious and non - malicious changes to a greater or lesser extent . figure 7 shows a superimposed water mark on previously used image [ figure 4 ] of casts . digital imaging has provided scientists with new prospects to obtain and manipulate data using techniques that were difficult or impossible to employ in the past . there is a possible use of retouched images for fraudulent purposes even in forensic investigations . computer alertness , especially about imaging software 's , should be promoted among forensic odontologist / dental professionals . anticipatory measures such as attached metadata and preferably water marking of digital images should be done in order to avert their malicious reuse . until there is an integrated response from the research community as to what constitutes appropriate image manipulation

introduction : in present - day times , freely available software allows dentists to tweak their digital records as never before . but , there is a fine line between acceptable enhancements and scientific delinquency.aims and objective : to manipulate digital images ( used in forensic dentistry ) of casts , lip prints , and bite marks in order to highlight tampering techniques and methods of detecting and preventing manipulation of digital images.materials and methods : digital image records of forensic data ( casts , lip prints , and bite marks photographed using samsung techwin l77 digital camera ) were manipulated using freely available software.results:fake digital images can be created either by merging two or more digital images , or by altering an existing image.discussion and conclusion : retouched digital images can be used for fraudulent purposes in forensic investigations . however , tools are available to detect such digital frauds , which are extremely difficult to assess visually . thus , all digital content should mandatorily have attached metadata and preferably watermarking in order to avert their malicious re - use . also , computer alertness , especially about imaging software 's , should be promoted among forensic odontologists / dental professionals .

Introduction Aims and objectives Materials and Methods Results and Observations Discussion Cautious skepticism Scrutinize Image analyzing software, tools, and Laboratory Investigation Meta data (data about data) Digital watermarking Summary and Conclusion

a successful orthodontic movement is directly related to the ability of orthodontic wires to slide through brackets slots and tubes , and it is well known that the sliding resistance between the bracket slot and archwire can drastically influence the tooth movement . the sliding mechanism is important not only to close the space , but also in the initial phase of the treatment in which leveling and alignment of the teeth occur . in an attempt to fulfill an increasingly aesthetic demand , aesthetic accessories consisting of different materials have been developed , among which are the aesthetic ceramic and polycarbonate brackets . therefore , these accessories alleviate the aesthetic problem despite the limitations of their use , such as fracture of the brackets , abrasion of antagonist teeth and , mainly , the increased friction resulting from the mechanical sliding . friction is defined as the force opposing the movement of two objects in direct contact to each other , and its direction is tangent to a common interface between both surfaces . the intensity of this force is closely related to the surface characteristics and properties of the materials involved . therefore , knowing the influence of different materials on the sliding mechanism , the aim of this study was to assess the frictional force existing between conventional and aesthetic brackets in association with stainless steel ( ss ) and titanium - molybdenum alloy ( tma ) wires by correlating the mean roughness values of wires and bracket slots . conventional right upper canine brackets ( b ) made of pure polycarbonate ( pb ; american orthodontics , sheboygan , wi , usa ) , ceramic ( cb ; american orthodontics , sheboygan , wi , usa ) , and ss ( ssb ; 3 m unitek , monrovia , ca , usa ) with slots of 0.022x0.030-in were used for study . ss and tma wires ( american orthodontics ) , both measuring 0.019x0.025-in , were evaluated ( figure 1 ) . systems assessed and their respective abbreviations a device for mechanical test was developed in order to simulate the sliding movement of the wire through the bracket slots as seen during the orthodontic treatment . holes were perforated in this glass support to fit high - rotation penholders ( dabi atlante , ribeiro preto , sp , brazil ) into which the brackets were inserted ( figure 2a ) . the penholders were filled with self - curing composite ( 3 m unitek , so paulo , sp , brazil ) , thus creating a base for bonding the brackets . the brackets were then positioned and bonded to their respective penholders by using cyanoacrylate ester ( super bonder ; loctite , so paulo , sp , brazil ) ( figure 2b ) . this set was hold within the glass box through lateral rods made from glass lamina in order to keep the system stable enough during the traction test . the glass box was designed to keep the system immersed in artificial saliva , thus providing a better simulation of the oral cavity . also , a heater with thermostat was added to this system so that the inner temperature was maintained at 37c during the whole experiment . a ) device made for stabilizing the combinations of bracket and wire during mechanical test . b ) detail of the bracket moving through the wire and angulation close to 0 a rectified 8-cm segment from both orthodontic wires was attached to the universal testing machine ( emic dl 10.000 ; so jos dos pinhais , pr , brazil ) , mounted onto the bonded bracket with no active torque and then tied to it with an elastic thread ( 3 m unitek , usa ) by using a ligature elastic applicator ( morelli , sorocaba , sp , brazil ) . the universal testing machine simulated the sliding movement of the bracket through the orthodontic wire during the retraction movement of the canines , which yielded a total dislocation of 8 mm that corresponds to the mean width of a premolar . a personal computer connected to the equipment recorded graphic results showing values of maximum load ( n ) generated by a load cell . such a test was repeated 5 times for each of the 3 combinations of bracket / wire of each group . the wings of the brackets were removed with a steel diamond discs ( 22-mm diameter , 0.15-mm thickness , ref . 7016 , kg sorensen , so paulo , sp , brazil ) at low speed on a handpiece ( dabi atlante ) for reading through a profilometer ( surf test sj , 201 , mitutoyo co. , kawasaki , honshu , japan ) . three milliliters of slots of the 3 types of brackets were analyzed , yielding three readings for each bracket . similarly , 5-mm segments of each wire submitted to mechanical traction test were sectioned , and 3 mm of them were analyzed , also yielding three readings for each wire . the orthodontic brackets and wires were randomly selected , and sectioned for analysis of their surface with a jeol scanning electron microscope ( 2000 fx , tokyo , japan ) . next , the orthodontic bracket and wires were positioned on a double - faced adhesive tape whose sequence was carefully recorded . the samples were then placed in the sample chamber of the microscope for visualization of the surfaces of the bracket slots and wires . the data obtained were submitted to simple anova with tukey s test for post - hoc multiple comparison tests between the systems . conventional right upper canine brackets ( b ) made of pure polycarbonate ( pb ; american orthodontics , sheboygan , wi , usa ) , ceramic ( cb ; american orthodontics , sheboygan , wi , usa ) , and ss ( ssb ; 3 m unitek , monrovia , ca , usa ) with slots of 0.022x0.030-in were used for study . ss and tma wires ( american orthodontics ) , both measuring 0.019x0.025-in , were evaluated ( figure 1 ) . a device for mechanical test was developed in order to simulate the sliding movement of the wire through the bracket slots as seen during the orthodontic treatment . holes were perforated in this glass support to fit high - rotation penholders ( dabi atlante , ribeiro preto , sp , brazil ) into which the brackets were inserted ( figure 2a ) . the penholders were filled with self - curing composite ( 3 m unitek , so paulo , sp , brazil ) , thus creating a base for bonding the brackets . the brackets were then positioned and bonded to their respective penholders by using cyanoacrylate ester ( super bonder ; loctite , so paulo , sp , brazil ) ( figure 2b ) . this set was hold within the glass box through lateral rods made from glass lamina in order to keep the system stable enough during the traction test . the glass box was designed to keep the system immersed in artificial saliva , thus providing a better simulation of the oral cavity . also , a heater with thermostat was added to this system so that the inner temperature was maintained at 37c during the whole experiment . a ) device made for stabilizing the combinations of bracket and wire during mechanical test . b ) detail of the bracket moving through the wire and angulation close to 0 a rectified 8-cm segment from both orthodontic wires was attached to the universal testing machine ( emic dl 10.000 ; so jos dos pinhais , pr , brazil ) , mounted onto the bonded bracket with no active torque and then tied to it with an elastic thread ( 3 m unitek , usa ) by using a ligature elastic applicator ( morelli , sorocaba , sp , brazil ) . the universal testing machine simulated the sliding movement of the bracket through the orthodontic wire during the retraction movement of the canines , which yielded a total dislocation of 8 mm that corresponds to the mean width of a premolar . a personal computer connected to the equipment recorded graphic results showing values of maximum load ( n ) generated by a load cell . such a test was repeated 5 times for each of the 3 combinations of bracket / wire of each group . the wings of the brackets were removed with a steel diamond discs ( 22-mm diameter , 0.15-mm thickness , ref . 7016 , kg sorensen , so paulo , sp , brazil ) at low speed on a handpiece ( dabi atlante ) for reading through a profilometer ( surf test sj , 201 , mitutoyo co. , kawasaki , honshu , japan ) . three milliliters of slots of the 3 types of brackets were analyzed , yielding three readings for each bracket . similarly , 5-mm segments of each wire submitted to mechanical traction test were sectioned , and 3 mm of them were analyzed , also yielding three readings for each wire . the orthodontic brackets and wires were randomly selected , and sectioned for analysis of their surface with a jeol scanning electron microscope ( 2000 fx , tokyo , japan ) . next , the orthodontic bracket and wires were positioned on a double - faced adhesive tape whose sequence was carefully recorded . the samples were then placed in the sample chamber of the microscope for visualization of the surfaces of the bracket slots and wires . the data obtained were submitted to simple anova with tukey s test for post - hoc multiple comparison tests between the systems . ss wire had a lower mean value of roughness value compared to tma wire . on the other hand , the polycarbonate bracket had a greater roughness compared to the ceramic and metallic ones ( table 1 ) . the results regarding roughness of brackets and wires after be submitted to mechanical traction test demonstrated a reduction in the roughness in all materials studied , with the metallic brackets having the most significant reduction ( 61.7% ) and the polycarbonate brackets having the lowest ( 47.6% ) . reading regarding 3-mm segment of each sample comparing the maximum mean friction values for the bracket systems using ss wire ( table 2 ) , no statistically significant differences were found between metallic ( ssb - ss ) and polycarbonate ( pbss ) brackets . there were significant differences between the systems of ceramic ( cb - ss ) and metallic brackets using ss wire ( ssb - ss ) ( p<0.001 * ) as well comparing the systems cb - ss and pb - ss ( p=0.001 ) . statistical data on friction coefficient of the systems of bracket and wire ( n=15 ) significance level of 95% ( * p<0.05 ; anova and tukey 's post hoc test ) . sd = standard deviation with regard to systems of brackets using tma wire , no statistically significant differences were found between metallic ( ssb - tma ) and polycarbonate ( pb - tma ) brackets or between metallic ( ssb - tma ) and ceramic ( cb - tma ) brackets . however , statistically significant differences were observed between the pb - tma and cb - tma systems ( p=0.038 ) . the ssb - ss system had the lowest friction coefficient , differing significantly from the ssb - tma ( p=0.012 ) . however , when the systems pb - ss and cb - ss were compared to the systems pb - tma and cb - tma , respectively , no statistical differences were found . the ssb - ss and cb - ss systems had the lowest and highest friction coefficients , respectively . figure 3a shows that the friction coefficient tends to be even higher in the cb - ss group . on the other hand , the pb - tma system had the lowest friction coefficient , followed by the ssb - ss and cb - tma systems , with the highest friction coefficient among all systems ( figure 3b ) . box plot showing maximum frictional coefficients between metal , polycarbonate , and ceramic brackets in association with different wires : a ) stainless steel wire ; b ) titanium - molybdenum alloy ( tma ) wire . simple analysis of variance ( anova ) and tukey s test as post hoc multiple comparison test at confidence interval of 95% ( * p<0.05 ) the tma wire ( figure 5a , b ) has less smooth surfaces compared to ss wire ( figure 4a , b ) . the conventional metallic bracket ( figure 6a , b ) also exhibits a smoother surface than the polycarbonate ( figure 7a , b polymers homogeneously distributed ) and ceramic ( figure 8a , b the most irregular surface among all ) brackets . scanning electron microscopy micrograph showing surface topography of tma wire ( associated with mb ) before ( a ) and after ( b ) mechanical traction scanning electron microscopy micrograph showing surface topography of ss wire ( associated with mb ) before ( a ) and after ( b ) mechanical traction scanning electron microscopy micrograph showing surface topography of mb slot ( associated with ss wire ) before ( a ) and after ( b ) mechanical traction scanning electron microscopy micrograph showing surface topography of pb slot ( associated with ss wire ) before ( a ) and after ( b ) mechanical traction scanning electron microscopy micrograph showing surface topography of cb slot ( associated with ss wire ) before ( a ) and after ( b ) mechanical traction after the mechanical traction test , the surfaces of all materials studied were more polished , which was more evident in the polycarbonate brackets and less evident in the ceramic brackets . the friction on a given surface is closely related to both material involved and surface characteristics . with regard to roughness , the ss wire exhibited less irregular surface compared to that of tma wire , thus corroborating other findings in the literatureand confirming that the greater the roughness , the higher the friction coefficient . in the present study , the systems using tma wire had a higher friction coefficient regardless of the type of bracket used . in addition , tma has been shown to present lower modulus of elasticity , springback greater than that of steel . with regard to the brackets , the slots of metallic brackets exhibited less roughness however , the polycarbonate bracket showed a friction coefficient lower than that of ceramic bracket either with ss wire ( p=0.01 ) or tma wire ( p=0.038 ) , both statistically significant . this may be explained by the fact that the profilometer could not detect small irregularities which were easily observed in the sem images , and the ceramic bracket showed more irregularities than the polycarbonate accessory , thus justifying the similarity of roughness between ss and tma wires . the authors developed an apparatus that was immersed in artificial saliva during mechanical traction test , which not only reduces the friction but also simulates the sliding mechanism as seen in vivo and control an important variable , the lubrication . furthermore , studies demonstrated that lubrication reduces significantly the friction between bracket and wire . also , a device was developed to correct the small vertical angulations so that the angle formed between wire and bracket was close to 0 , thus helping to reduce the friction coefficient . according to the literature , friction coefficient increases as the angulation between bracket and wire increases . generally , when extraction is indicated during orthodontic treatment , closing of extraction spaces can be performed mainly by canine retraction through distal movement . wire was attached to the bracket with elastic ligatures because it promotes an additional force in comparison to wire ligature . in addition , attachment to the bracket with elastic guarantees a standard force in comparison to the wire ligature . the different combinations comparing the ssbss and pb - ss systems showed that both yielded the lowest friction coefficients without statistically significant differences . this fact is explained by the use of brackets and wires made from the same material , which reduces friction as surfaces from the same material tend to have less friction compared to surfaces from different materials , in addition to their low roughness compared to other groups . with regard to the pb - ss system , despite using different materials , the low friction coefficient observed may be explained by the fact that the polymeric chains ( macromolecules consisting of simple molecules ) are small spheres homogeneously distributed on the surface of the polycarbonate bracket , being partially responsible for its roughness . these factors may explain the lack of statistically significant difference in the low friction coefficient between these both systems . on the other hand , the cb - ss system had the highest friction coefficient , being statistically significant different from the ssb - ss system , which is also in agreement with the literature . this can be explained by the higher surface roughness of the ceramic brackets and also by the different wire material ( ss ) , thus making the sliding movement of the bracket through the wire difficult . in addition , the ceramic bracket showed higher friction coefficient compared to the polycarbonate bracket , since the former consists of crystal grains that are clearly larger than the monomers of the polycarbonate . as can be seen in the sem images , the ceramic surface also exhibits more irregularities , thus increasing the friction . similarly , no statistically significant difference between polycarbonate and metallic brackets was found when they were used in combination with tma wire , with both systems having a lower friction coefficient . significant difference was observed in relation to the ceramic brackets ( p=0.038 ) , but not between metallic and ceramic brackets . this is due to the fact that both ceramic bracket and tma wire have higher friction coefficients because they have a rougher topography compared to other systems . however , no statistically significant difference was observed between polycarbonate and metallic brackets regarding the friction coefficient because of the increased friction observed in relation to the ss wire . however , even in the final phases of the treatment in which the long - range movements are not the main goal , tma wire should be carefully used as the results of the present study have shown that its friction is significantly higher than that of ss wire for metallic brackets , a finding also corroborated elsewhere . however , when polycarbonate and ceramic brackets are used in combination with tma wire , the friction coefficient decreases compared to systems using ss wire . it is suggested that despite the brackets being of different materials and tma wire having a rougher surface compared to ss wires , it is possible that the flexibility of the tma wire might have produced less friction , reducing the frictional resistance for small inclination angles , although the brackets were in a rotating platform . therefore , no statistically significant difference between metallic and polycarbonate brackets was observed , whereas significant difference was found between the polycarbonate and ceramic brackets . this latter system also showed higher friction coefficient compared to the other systems , but with no statistically significant difference . one can observe that the ss wire has a lower friction coefficient when used with metallic bracket compared to the tma wire , which is well described in the literature . as the materials composing brackets and wires of each system are different , no significant differences between the systems using polycarbonate and ceramic brackets in association with ss and tma wires were observed . the orthodontic brackets and wire submitted to mechanical traction tests were then evaluated by using a profilometer in order to verify changes on the surface . the results showed a decrease in the roughness of all materials tested : ss wire ( 52.2% ) , tma wire ( 84.1% ) , polycarbonate bracket ( 47.6% ) , ceramic bracket ( 60.1% ) , and metallic bracket ( 61.7% ) . based on the data obtained the present study , it is important to emphasize that in sliding mechanics the effective force to be clinically applied to metallic brackets should be increased when using tma wire instead of ss wire in order to maintain a satisfactory speed of the tooth movement . on the other hand , some undesirable consequences such as loss of anchorage , tooth inclination , root absorption , and intense pain may occur , among other complications that may cause short- and long - term irreversible damages . future studies are suggested to evaluate how human salivary components act on the friction between bracket and wire during orthodontic treatment . the following conclusions can be drawn : the system formed by metallic bracket and ss wire exhibited less roughness and lower friction . the use of ceramic brackets with tma wire should be judiciously used as this system was found to have a higher friction coefficient .

objectivethe objective this work was to assess the friction coefficient between brackets and wires of different materials under conditions simulating the oral environment.material and methodsstainless steel ( ss ) and titanium - molybdenum alloy ( tma ) wires of 0.019x0.025-in diameter ( american orthodontics ) and polycarbonate bracket ( american orthodontics ) , ceramic bracket ( american orthodontics ) , and metal bracket ( 3 m unitek ) with slots of 0.022x0.030-in were used . the friction coefficient was assessed by means of mechanical traction with the system immersed in artificial saliva . the mean roughness of both wire surface and bracket slots was evaluated by using a surface profilometer.resultsthe system using tma wire and polycarbonate bracket had the highest roughness ( p<0.05 ) . ss wire with ceramic bracket had the highest friction coefficient , whereas the use of metallic bracket yielded the lowest ( p<0.05 ) . however , it was observed a statistically significant difference in the system using tma wire and ceramic bracket compared to that using tma wire and polycarbonate bracket ( p=0.038).conclusionceramic brackets in association with ss wire should be judiciously used , since this system showed a high friction coefficient .

INTRODUCTION MATERIAL AND METHODS Materials Mechanical Traction Test Surface Roughness Scanning Electronic Microscopy (SEM) Statistical Analysis RESULTS DISCUSSION CONCLUSIONS

it has a profound impact on every aspect of quality of life and is thus the dominant cause of total suffering for cancer patients even though pain relief is achievable in more than 90% of the cases . inadequate pain relief in practice is well documented and can involve up to 40% of patients . nurses have a key role in effective pain management ; therefore , nurses knowledge is of critical importance in the care of patients with cancer pain.[46 ] the nurse 's accurate assessment , prompt intervention , and adequate evaluation of pain relief measures are necessary for better clinical outcomes . however , nurses may not be prepared to assume a critical role because their lack of knowledge and individual attitudes on pain can interfere with their ability to effectively manage pain . the most common explanation is that nurses receive too little pain management education in their nursing curricula . the majority of studies determined that nurses answered less than 70% of questions correctly , indicating that many nurses continue to lack sufficient knowledge regarding appropriate pain management . one of the most important deficiencies of nurses about cancer pain assessment is that their assessment often focuses on patients behaviors rather than pain intensity and other descriptive characteristics of pain . as a result , most nurses have overestimated the percentage of patients who over - report their pain . it has been reported that opioid knowledge of nurses who cared for patients with cancer was generally weak . nurses also were unaware that a common side effect of opioid analgesics , constipation , does not decrease over time . in addition , many nurses do not know that the incidence of psychologic dependence in patients with cancer is less than 1 in 1000 patients . many studies have also demonstrated that negative nursing attitudes create barriers to effective cancer pain management . howell et al . , found that nurses believed that their patients should experience pain first before being given pain medication . in their study affirmed that nurses have poor knowledge and negative attitudes toward the approach and treatment of patients in pain . the health belief model ( hbm ) is a psychological model that attempts to explain and predict health behaviors . the hbm was spelled out in terms of four constructs representing the perceived threat and net benefits : perceived susceptibility , perceived severity , perceived benefits , and perceived barriers . the purpose of this study was to identify the level of knowledge , attitude , and hbm constructs among aalzahra hospital nurses regarding the management of cancer pain . the health belief model ( hbm ) is a psychological model that attempts to explain and predict health behaviors . the hbm was spelled out in terms of four constructs representing the perceived threat and net benefits : perceived susceptibility , perceived severity , perceived benefits , and perceived barriers . the purpose of this study was to identify the level of knowledge , attitude , and hbm constructs among aalzahra hospital nurses regarding the management of cancer pain . in this cross - sectional study , 98 randomly selected nurses , who were employed in alzahra educational hospital in isfahan , iran , were enrolled in the study . all nurses agreed to participate in the study and therefore they did sign the consent form . content and construct validity of the questionnaire was revised by a group of specialists in the field of health education , anesthesiology , and oncology . reliability analysis was conducted for testing the reliability of knowledge and hbm constructs . internal consistency of these sections of the questionnaire was calculated using cronbach 's alpha technique ( 0.82 for knowledge , 0.79 for attitude , 0.75 for perceive benefits , 0.68 for perceived barriers , 0.72 for perceived threat , 0.74 for self - efficacy , and 0.85 for cues to action ) . the questionnaire contained two sections : ( a ) socio - demographic characteristics of nurses ( such as sex , education , marital status ) and ( b ) knowledge and hbm constructs . to assess the knowledge of nurses regarding cancer pain management each correct answer was given a score of 1 and each wrong answer a score of 0 . hbm constructs section consisted of attitude ( 10 items ) , perceived benefits ( 5 items ) , perceived barriers ( 5 items ) , perceived threat ( 5 items ) , self - efficacy ( 5 items ) , and cues to action ( 5 items ) . for scoring this section of questionnaire , 5-point likert scale ( 0=strongly disagree , 1=disagree , 2=no opinion , 3=agree , and 4=strongly agree ) was used . for negative questions , after completion of the questionnaire by the nurses , the obtained data were analyzed by spss ( version 11.5 , chicago , il , usa ) using statistical tests ( descriptive , pearson correlation , independent t - test ) at the significance level of =0.05 . the demographic characteristics of the 98 nurses who responded to the questionnaire are presented in table 1 . as shown in the table , of the 98 nurses participating in the study , 81.6% were females , 67.3% were married , 80.6% had bachelors degree , and 68.4% had moderate income . the majority of nurses had insufficient knowledge and attitude regarding cancer pain management . among the hbm constructs , perceived barriers and perceived threat were in low range . the nurses also reported high self - efficacy in cancer pain management [ table 2 ] . pearson correlation test showed that the knowledge of the nurses had a direct relationship with their attitude , perceived benefits , self - efficacy , and cues to action . but knowledge had reverse relationship with perceived barriers and perceived threat . the same test ( pearson correlation ) also indicated that nurses attitude regarding cancer pain management had a direct relationship with perceived benefits , self - efficacy , and cues to action , but had reverse relationship with perceived barriers and perceived threat [ table 3 ] . independent t - test showed that the mean scores of model variables in the two sexes had no significant difference ( p>0.05 ) . in other words , no relationship was observed between the two sexes and the mean score of hbm variables . pearson correlation indicated that there was no significant relationship of age and work experience with the score of model variables ( p>0.05 ) . demographic characteristics of the nurses participating in the study mean ( sd ) of the knowledge , attitude , and hbm constructs of nurses regarding cancer pain management pearson correlation coefficient between knowledge , attitude and hbm constructs this study provides important information about the level of nurses pain knowledge and attitude in isfahan , iran . the findings showed that on average , the nurses included in this study showed low scores on knowledge and attitudes regarding cancer pain management . in other words , these results demonstrated that the nurses knowledge and attitude about cancer pain management is far from optimal . regarding knowledge section , inadequacy of health care professionals knowledge and education has been proposed as one of the major factors contributing to inadequate pain relief in cancer patients worldwide . it is also well documented that there is a lack of professional health care education about pain management , which has resulted in less than optimal pain management for cancer patients . an earlier survey has shown that an addiction to opioid is observed in less than 1% of cancer patients . in this study , only 12.4% of nurses correctly identified that less than 1% of patients who receive opioids for pain relief will develop addiction and 87.6% erroneously believed that addiction will occur in patients . compared with the nurses in western countries , more nurses in this study our study 's results revealed that among the hbm constructs , the highest mean score was related to self - efficacy . in other words , the participating nurses believed that they had enough ability to manage cancer pain issues such as emotional relationship with patients , to use the needed dose of opioid , and to give relevant information to the patient and his / her caregivers . 's study which had reported low self - efficacy among nurses regarding cancer pain management . we considered that , this difference may arise from work experience of our study subjects who have long - term clinical experiences . regarding cues to action , results of this study indicate that 78.5% of nurses agreed with the important role of seminars and workshops to increase their knowledge toward cancer pain management . in mamishi 's study , the most important source of information from nurses viewpoint was university curriculum . in our study , there was direct correlation between knowledge and other hbm constructs except for perceived barriers and perceived threat . it means with increasing knowledge of nurses , their perceived barriers and perceived threat regarding cancer pain management have been decreased . there was also a reverse correlation between nurse 's self - efficacy and their perceived barriers and perceived threat . this finding is in accordance with earlier findings in this regard.[2931 ] the present study provides important information about nurse 's knowledge and attitude deficits in pain management of cancer patients . when major medical and nursing textbooks published worldwide are examined , it is seen that pain makes up a small part of the content . in a study by ferrell et al . in 2000 , it was observed that pain - related issues and pain management were addressed in only 284 of 45,683 pages in nurses textbooks . additionally , it is reported that baccalaureate nursing programs allocate little time to pain management . first , the sample size was relatively small and may not represent all nurses in isfahan . additionally , the study sample was taken from a university hospital , so it can not necessarily be generalized to other staff populations . first , the sample size was relatively small and may not represent all nurses in isfahan . additionally , the study sample was taken from a university hospital , so it can not necessarily be generalized to other staff populations . the deficits in knowledge and attitude identified in this survey are a significant barrier to the effective management of pain in cancer patients . we believe that pain management issues in master nursing programs in iran are insufficient as well . national and local course programs about pain management are also insufficient , although remedial interventions such as rearing palliative care nurses to improve the standard of clinical practice are currently being implemented and the impact on knowledge and outcomes of these interventions will be the subject of future studies in a countrywide continual quality improvement project .

background : effective cancer pain management requires accurate knowledge , attitudes , and assessment skills . the purpose of this study was to obtain information about the knowledge and attitudes of nurses concerning cancer pain management with the use health belief model ( hbm ) as conceptual framework.materials and methods : the study was a descriptive survey and included 98 randomly selected nurses from alzahra hospital , isfahan , iran . a self - administered questionnaire which was designed on the basis of hbm was used to collect the data . knowledge , attitudes , and hbm constructs regarding cancer pain were the main research variables . the obtained data were analyzed by spss ( version11.5 ) using descriptive statistics , independent t test , and pearson correlation at the significant level of =0.05.results : ninety - eight nurses aged 38.7 7.04 years were studied in this survey . from the 10 pain knowledge questions assessed , the mean number of correctly answered question was 61.2 ( sd=16.5 ) , with a range of 30100 . there was a direct correlation between knowledge and attitude of nurses with hbm constructs except for perceived barriers and perceived threat . among the hbm constructs , the highest score was related to self - efficacy with mean score of 87.2 ( sd=16.4).conclusions : the findings support the concern of inadequate knowledge and attitudes in relation to cancer pain management . we believe that basic and continuing education programs may improve the knowledge level of nursing about pain management .

INTRODUCTION Health belief model MATERIALS AND METHODS RESULTS DISCUSSION Limitations CONCLUSION

orthodontists traditionally consider restored oral health , function , and aesthetics as the principal therapeutic goals.1 however , improved aesthetics and its positive psychosocial impact are increasingly being accepted as important benefits of treatment.1,2 occlusal or orthodontic treatment need indices are available to classify the anatomical and aesthetic aspects of malocclusion . the best - known and most - used tools are the dental aesthetic index ( dai ) and index of orthodontic treatment need ( iotn).3,4 however , they do not account for the influence of malocclusion on the patient 's quality of life.5 tools that attempt to assess the oral health - related quality of life ( ohrqol ) offer information on the patient 's perception of their welfare in relation to a particular oral condition.6 several authors have employed ohrqol assessment questionnaires together with normative indices to study the impact of malocclusion on the patient 's quality of life.7,8 in the past , ohrqol research focused on adults with periodontal disease , tooth loss , or inadequate dentures.9 recently , however , the ohrqol of children and adolescents has aroused considerable interest,10 to some extent because adolescents usually show great concern about their appearance , which plays an important role in their psychosocial welfare.11,12 the psychosocial impact of dental aesthetics questionnaire ( pidaq ) is a valuable tool that provides information on one aspect of the ohrqol . this self - rating instrument was designed to assess the psychosocial impact of dental aesthetics in young adults.6 brazilian , chinese , and spanish versions of the pidaq have been published recently,13 - 15 so its global use is increasing . the aims of the present study were to evaluate the psychosocial impact of malocclusion , determine its relationship with the severity of malocclusion , and assess the influence of gender and social class on this relationship in adolescents . the study sample was composed of 627 adolescents aged 12 - 15 years in 42 schools chosen randomly from 1,200 subjects of all the schools in valencia , spain . the fieldwork was carried out in november and december 2010 . in each of the selected schools , 15 - 20 adolescents underwent intraoral examinations by three examiners , who were calibrated against a gold standard in the use of the iotn ( intraexaminer and interexaminer kappa > 0.85 ) . adolescents with visible lesions on their anterior teeth due to caries , traumatic injury , or hypoplasia / fluorosis or those who wore orthodontic appliances were excluded . the study was approved by the human research ethical committee of the university of valencia ( approval number h1352114553202 ) and complied with the recommendations of the declaration of helsinki . written informed consent for the intraoral examinations and survey was obtained from the parents . the pidaq , a psychometric instrument containing 23 items , is composed of four subscales , representing four areas , one positive and three negative : aesthetic concern ( ac , three items ) , psychological impact ( pi , six items ) , social impact ( si , eight items ) , and dental self - confidence ( dsc , six items ) . a five - point likert scale is used for each item . the response options are as follows : 0 = not at all ; 1 = a little ; 2 = somewhat ; 3 = strongly ; and 4 = very strongly.5 each subscale score can be calculated separately and is obtained by summing the item scores.14 to calculate the total pidaq score , the dsc items were re - coded to align them with the other subscales . this index is composed of 2 parts : the dental health component ( dhc ) and the aesthetic component ( ac).3,15 the iotn - dhc is assessed by the examiner and classified into five grades according to the therapeutic need : grade 1 = none ( normal occlusion ) ; grade 2 = little ( minor malocclusion ) ; grade 3 = borderline ( moderate malocclusion ) ; grade 4 = great ( severe malocclusion ) ; and grade 5 = very great ( very severe malocclusion ) . the iotn - ac is assessed by the patient using 10 photographs that show the degrees of malocclusion ranging from the least severe to the most severe . the 10 iotn - ac grades are combined into three groups : grades 1 - 4 , grades 5 - 7 , and grades 8 - 10.3 the uk registrar - general 's social class scale was employed . this scale groups the population into the following five categories : i = professionals and higher managerial and technical occupations ; ii = lower managerial and technical occupations , trade ; iii = intermediate supervisory and clerical occupations ; iva = skilled manual workers ; ivb = partly skilled manual workers ; and v = unskilled workers.16 in this study , categories i and ii were considered the high social class , category iii was considered the middle social class , and the remaining categories were considered the low social class . univariate descriptive statistics were used to calculate the means of the quantitative variables , proportions of the categorical variables , and confidence intervals of both . the means were compared by using student 's t - test and analysis of variance ( p < 0.05 ) . stepwise linear regression models were employed to study the linear relationship between the pidaq data as the dependant variable and the iotn components , gender , and social class as the independent predictive variables . the study sample was composed of 627 adolescents aged 12 - 15 years in 42 schools chosen randomly from 1,200 subjects of all the schools in valencia , spain . the fieldwork was carried out in november and december 2010 . in each of the selected schools , 15 - 20 adolescents underwent intraoral examinations by three examiners , who were calibrated against a gold standard in the use of the iotn ( intraexaminer and interexaminer kappa > 0.85 ) . adolescents with visible lesions on their anterior teeth due to caries , traumatic injury , or hypoplasia / fluorosis or those who wore orthodontic appliances were excluded . the study was approved by the human research ethical committee of the university of valencia ( approval number h1352114553202 ) and complied with the recommendations of the declaration of helsinki . written informed consent for the intraoral examinations and survey was obtained from the parents . the pidaq , a psychometric instrument containing 23 items , is composed of four subscales , representing four areas , one positive and three negative : aesthetic concern ( ac , three items ) , psychological impact ( pi , six items ) , social impact ( si , eight items ) , and dental self - confidence ( dsc , six items ) . a five - point likert scale is used for each item . the response options are as follows : 0 = not at all ; 1 = a little ; 2 = somewhat ; 3 = strongly ; and 4 = very strongly.5 each subscale score can be calculated separately and is obtained by summing the item scores.14 to calculate the total pidaq score , the dsc items were re - coded to align them with the other subscales . this index is composed of 2 parts : the dental health component ( dhc ) and the aesthetic component ( ac).3,15 the iotn - dhc is assessed by the examiner and classified into five grades according to the therapeutic need : grade 1 = none ( normal occlusion ) ; grade 2 = little ( minor malocclusion ) ; grade 3 = borderline ( moderate malocclusion ) ; grade 4 = great ( severe malocclusion ) ; and grade 5 = very great ( very severe malocclusion ) . the iotn - ac is assessed by the patient using 10 photographs that show the degrees of malocclusion ranging from the least severe to the most severe . the 10 iotn - ac grades are combined into three groups : grades 1 - 4 , grades 5 - 7 , and grades 8 - 10.3 the uk registrar - general 's social class scale was employed . this scale groups the population into the following five categories : i = professionals and higher managerial and technical occupations ; ii = lower managerial and technical occupations , trade ; iii = intermediate supervisory and clerical occupations ; iva = skilled manual workers ; ivb = partly skilled manual workers ; and v = unskilled workers.16 in this study , categories i and ii were considered the high social class , category iii was considered the middle social class , and the remaining categories were considered the low social class . the pidaq , a psychometric instrument containing 23 items , is composed of four subscales , representing four areas , one positive and three negative : aesthetic concern ( ac , three items ) , psychological impact ( pi , six items ) , social impact ( si , eight items ) , and dental self - confidence ( dsc , six items ) . a five - point likert scale is used for each item . the response options are as follows : 0 = not at all ; 1 = a little ; 2 = somewhat ; 3 = strongly ; and 4 = very strongly.5 each subscale score can be calculated separately and is obtained by summing the item scores.14 to calculate the total pidaq score , the dsc items were re - coded to align them with the other subscales . this index is composed of 2 parts : the dental health component ( dhc ) and the aesthetic component ( ac).3,15 the iotn - dhc is assessed by the examiner and classified into five grades according to the therapeutic need : grade 1 = none ( normal occlusion ) ; grade 2 = little ( minor malocclusion ) ; grade 3 = borderline ( moderate malocclusion ) ; grade 4 = great ( severe malocclusion ) ; and grade 5 = very great ( very severe malocclusion ) . the iotn - ac is assessed by the patient using 10 photographs that show the degrees of malocclusion ranging from the least severe to the most severe . the 10 iotn - ac grades are combined into three groups : grades 1 - 4 , grades 5 - 7 , and grades 8 - 10.3 this scale groups the population into the following five categories : i = professionals and higher managerial and technical occupations ; ii = lower managerial and technical occupations , trade ; iii = intermediate supervisory and clerical occupations ; iva = skilled manual workers ; ivb = partly skilled manual workers ; and v = unskilled workers.16 in this study , categories i and ii were considered the high social class , category iii was considered the middle social class , and the remaining categories were considered the low social class . univariate descriptive statistics were used to calculate the means of the quantitative variables , proportions of the categorical variables , and confidence intervals of both . the means were compared by using student 's t - test and analysis of variance ( p < 0.05 ) . stepwise linear regression models were employed to study the linear relationship between the pidaq data as the dependant variable and the iotn components , gender , and social class as the independent predictive variables . by gender , 47.8% and 52.2% of the sample were boys and girls , respectively . in terms of social class , 49.3% , 38.7% , and 12.0% belonged to the low , middle , and high social classes , respectively . the mean total pidaq score was 32.2 ( 95% confidence interval = 31.1 to 33.3 ) . the mean dsc score was 11.3 ( 10.8 to 11.7 ) , si score was 6.1 ( 5.6 to 6.5 ) , pi score was 5.9 ( 5.6 to 6.3 ) , and ac score was 7.44 ( 7.2 to 7.7 ) . in the iotn - dhc , 54.4% of the adolescents had grades 1 and 2 , 27.4% had grade 3 , and 18.2% had grades 4 and 5 . the iotn - ac distribution was 90.3% for grades 1 - 4 , 6.4% for grades 5 - 7 , and 3.3% for grades 8 - 10 . table 1 shows the relationships of the pidaq and iotn data with gender and social class . the total and subscale pidaq scores showed no significant differences by gender , except the pi subscale , where girls had higher scores ( p = 0.02 ) . no significant differences by social class were noted . the iotn - ac presented significant differences by gender , with a greater number of girls having grades between 5 and 10 ( p = 0.02 ) . the total pidaq score and the si , pi , and ac scores presented a significant positive linear relationship and the dsc score presented a significant negative linear relationship with the iotn - dhc grades ( table 2 ) . table 3 shows that the iotn - dhc was a predictive variable of the total and subscale pidaq scores . neither gender nor social class was an independent predictive variable of the relationship between the pidaq scores and the iotn - dhc grades , except the pi subscale , where gender was a predictive variable in the linear model . the total pidaq and si and pi scores showed a significant positive linear relationship and the dsc score presented a significant negative linear relationship with the iotn - ac grades . the pidaq - ac scores did not exhibit significant differences in the iotn - ac grades ( table 4 ) . the iotn - ac was a predictive variable of the total pidaq and dsc , si , and pi scores . a linear relationship between the pidaq - ac scores and the iotn - ac grades could not be established ( table 5 ) . table 6 shows a descriptive analysis of the main occlusal conditions requiring orthodontic treatment ( iotn - dhc grades 3 - 5 ) and increasing the pidaq scores : increased overjet , impeded tooth eruption or submerged deciduous teeth , tooth displacement , and increased overbite . the present study included a randomized and representative sample , good reliability of the examiners , and a validated questionnaire to measure the psychosocial impact of malocclusion in adolescents . however , considering the age of the subjects , comparisons with other studies should be made cautiously . many authors consider it more advisable to analyze the psychosocial impact of dental aesthetics in adults , who are emotionally stable and have a realistic view of dentofacial aesthetics , than in adolescents.17,18 cooper et al.19 observed that the perception of dental aesthetics changes and even improves with age . tuominem et al.20 also concluded that the perceived orthodontic treatment need seems to lessen with age even if the patient does not undergo orthodontic treatment . several authors agree that the iotn - dhc measures the severity of malocclusion,21 but the reliability of the iotn - ac has been questioned.22,23 indeed , many studies have shown that results differ considerably according to whether the treatment need is measured objectively with the iotn - dhc or subjectively with the iotn - ac.22,24,25 nevertheless , the present study showed a significant linear association between the pidaq scores and the grades of both the iotn components . the pidaq - ac was the only subscale that did not show a linear correlation with the iotn - ac . although the pidaq - dsc showed a linear relationship with the iotn - dhc , this association was not much stronger than that of the other subscales . in agreement with other studies,8,17,26 this study showed that the psychosocial impact of dental aesthetics increased with the severity of malocclusion , so the pidaq has considerable validity . this finding confirms that malocclusion has a psychosocial impact in adolescents , which can considerably influence their self - confidence and social life.6,27 increased overjet , tooth displacement , and increased overbite were the occlusal conditions that had a higher psychosocial impact . dahong et al.28 obtained similar results ; they considered that the overjet might induce a protrusive or retrusive profile and further influence the psychology of patients . in this study , only angle 's classification or the incisor classification was used . only sardenberg et al.13 and lin et al.14 have analyzed the psychosocial impact of malocclusion , but neither study was conducted in adolescents using the pidaq . we related the pidaq with a normative index ( iotn ) that has been used by many authors . sardenberg et al.13 used the dai as a normative index , but lin et al.14 did not use any objective index . although some studies have shown a relationship between the aesthetic impact of malocclusion and social class,8 the present study showed no such association , as previously reported.29 noteworthily , the methods employed to obtain socioeconomic information differ among the relevant studies , so more research should be conducted with this aspect in mind . other authors such as doan et al.30 also concluded that socioeconomic factors are unrelated to the perception of malocclusion . although it did not influence three pidaq subscales ( dsc , si , and ac ) , it affected the pi subscale . de oliveira and sheiham31 also found that the psychosocial impact of malocclusion is significantly greater in women than in men and affects their quality of life . similarly , the present study showed that the girls ' perception of their dental aesthetics was worse than that of the boys . other studies have also indicated that men tend to be more satisfied with their dental aesthetics.22 we have observed lower punctuation than the brazilian version on the pidaq dsc subscale . malocclusion has a psychological impact in adolescents and this impact increases with the severity of malocclusion . social class may not influence this association , but gender has some effect , because the psychological impact is greater in girls .

objectiveto evaluate the psychosocial impact of malocclusion , determine its relationship with the severity of malocclusion , and assess the influence of gender and social class on this relationship in adolescents.methodsa random sample of 627 spanish adolescents aged 12 - 15 years underwent intraoral examinations by 3 calibrated examiners ( intraexaminer and interexaminer kappa > 0.85 ) at their schools . psychosocial impact was measured through a self - rated psychosocial impact of dental aesthetics questionnaire ( pidaq ) . the severity of malocclusion was measured by the index of orthodontic treatment need ( iotn ) . gender and social class were also recorded.resultsthe total pidaq score and those of its 4 subscales , social impact , psychological impact , aesthetic concern , and dental self - confidence , presented significant differences ( p 0.05 by analysis of variance ) and linear relationships with the iotn grades ( p 0.05 by linear regression ) . stepwise linear regression models showed that the iotn dental health component was a predictive variable of the total and subscale pidaq scores . neither gender nor social class was an independent predictive variable of this relationship , except the linear model for psychological impact , where gender was a predictive variable . the occlusal conditions responsible for higher pidaq scores were increased overjet , impeded eruption , tooth displacement , and increased overbite.conclusionsmalocclusion has a psychological impact in adolescents and this impact increases with the severity of malocclusion . social class may not influence this association , but the psychological impact seems to be greater among girls .

INTRODUCTION MATERIALS AND METHODS Subjects Data collection Psychosocial impact of dental aesthetics Severity of malocclusion Social class Statistical analysis RESULTS DISCUSSION CONCLUSION

breast cancer is a multifaceted , heterogeneous disease whose treatment is evolving as genetic profiles shed more light on potential targets . 's 2000 publication detailing the classification of breast cancer based on gene - expression assays . among this classification was the basal - like subtype , described as frequently ( but not always ) being er , pr , and her-2 deficient while also expressing basal cytokeratins 5/6 and 17 and epidermal growth factor ( egfr ) [ 1 , 2 ] . these basal - like breast cancers make up 17 to 37% of all breast cancers [ 24 ] . having genetic profiles outlining the inherent differences in breast cancer has allowed for new research paths attempting to develop novel therapeutics that are subtype dependent . the definition of triple - negative breast cancer is based on clinical observations ; the tumor must lack estrogen receptors ( ers ) , progesterone receptors ( prs ) , and hormone epidermal growth factor receptor type 2 ( her-2 ) expression . these tumors are particularly vexing for physicians because there are no known endocrine targets nor are there specific receptors to block . women diagnosed with tnbc tend to be younger and are more likely to present with poorly differentiated tumors . although tnbc is responsive to chemotherapy and features a higher pathologic complete response ( pcr ) rate compared to other breast cancer types ( in the presence of neoadjuvant therapy ) , the prognosis for tnbc patients is still poor [ 7 , 8 ] . there are many similarities between tnbc and basal - like breast cancer , but the two terms are not synonymous ( figure 1 ) . they share demographic characteristics such as age of first menarche and increased incidence in the african - american and hispanic female population . it has been noted that roughly 80% of tnbc tumors are basal - like breast cancers . however , immunohistochemical studies have shown that 1740% of basal - like breast cancers do not have a triple - negative phenotype . up to 20% of basal - like breast cancers actually express er or her-2 to some extent . one important similarity between tnbc and basal - like breast cancer is the incidence of mutations in the breast cancer susceptibility gene 1 and 2 ( brca1 and 2 ) . brca mutations are only 2 - 3% of all breast cancers but signify an increased lifetime risk of breast and ovarian cancer . it is estimated that methylation of the brca1 promoter can be found in 1114% of sporadic breast cancers [ 1517 ] . brca1 is a key player in mammary gland development , and both brca1 and brca2 are connected with dna repair . a majority of tumors in women with brca mutations feature similar expression patterns as basal - like tumors [ 1820 ] , clouding the picture of where brca - mutated cancers , basal - like breast cancers , and tnbc originate ( figure 1 ) . researchers have found the links between tnbc , basal - like breast cancer , and brca mutations to be a potential source of directed therapy . this is a strategy to target and kill specific cell types , without collateral damage . it is achieved by locating a gene that , when inhibited , will kill cancerous cells that contain a specific genetic signature . the design and exploration of poly(adp - ribose ) polymerase ( parp ) inhibitors have emerged as a potential target to cause synthetic lethality in cancerous cells while sparing normal mammary tissue . the aim of this paper is to discuss the molecular basis behind parp inhibitors and an update on their current status in several clinical trials . poly(adp - ribose ) polymerase ( parp ) is a nuclear protein that is activated in the presence of dna damage . while several parp proteins have been detected , parp1 and parp2 have been associated with dna stability . when single strand dna ( ssdna ) damage occurs , it is identified and repaired by a cellular process that includes parp and base excision repair . if ssdna breaks are not repaired ( e.g. , parp inhibition ) , the breaks build up and are converted at the replication fork to double - strand dna ( dsdna ) breaks [ 2325 ] . at this point , homologous recombination or nonhomologous end joining repairs the double - stranded breaks in dna [ 23 , 25 ] . homologous recombination is mediated by several factors , including brca1 , brca2 , and rad51 [ 2628 ] . cells deficient in functioning homologous recombination , such as ones with defective brca1 and/or brca2 genes , are forced into less precise repair pathways that make them more susceptible to cell death when overwhelmed with defects to repair . the incorrect pairing of ends of dna then possibly leads to genomic instability , ultimately ending in apoptosis ( figure 2 ) . interestingly , parp is also involved in dsdna repair in combination with nonhomologous end joining , so parp inhibition also hinders the cell 's other repair routes . parp1 inhibitors are being investigated as pharmacologic interventions for metastatic tnbc due to a theory of selectivity : if only brca - defective genes are terminated , then other cells that maintain a normal , functioning brca allele will not be killed by a parp inhibitor . this synthetic lethality is being developed to create a new class of drugs that aim to efficiently kill cancer cells . several parp1 inhibitors are being studied at the clinical trial level , and this paper will focus specifically on iniparib , olaparib , and veliparib ( table 1 , http://www.clinicaltrials.gov/ ) . results of an open - label phase ii trial for iniparib ( bsi-201 , sanofi - aventis ) combined with chemotherapy on metastatic tnbc patients were recently published . this trial compared the use of gemcitabine and carboplatin alone versus those two agents and iniparib . the median progression - free survival increased when iniparib was added , from 3.6 to 5.9 months . the median overall survival was also significantly increased in the iniparib group , up to 12.3 months from 7.7 months . a complete or partial response was seen in 56% of patients receiving iniparib , while only 34% exhibited such a response in the gemcitabine / carboplatin arm . common side effects seen amongst the 116 patients were nausea , fatigue , anemia , and neutropenia . it is notable that these side effects did not increase when iniparib was added to the regimen , suggesting that the side effects originate from gemcitabine and/or carboplatin . a notable component of this study is that brca1/2 status was not assessed on the patients . domagala et al . have claimed that 18% of brca1-associated cancers have low or no nuclear expression of parp1 and low parp1 expression in 21% of triple - negative brca1-associated breast cancers . when looking at cytoplasmic and nuclear parp , another group has observed its presence in all intrinsic types of breast cancer , albeit with different frequencies . clearly , the expression pattern and full mechanism of parp1 needs to be investigated to better understand if it will be an effective target for tnbc . at this year 's meeting of the american society of clinical oncology , o'shaughnessy and colleagues presented their results of the phase iii iniparib trial this trial enrolled 519 women and again looked at gemcitabine and carboplatin versus the same regimen with added iniparib . the results did find an increase in progression - free survival amongst the iniparib / gemcitabine / carboplatin arm ( 5.1 versus 4.1 , p = 0.027 ) , but this did not achieve the prespecified criteria for significance ( p = 0.01 ) . a possible explanation behind the change in results from phase ii to phase iii is that the heterogenous nature of tnbc will continue to make finding a single agent problematic in treating all comers . by not stratifying the patients based on brca status or tnbc subtype , it leaves questions as to which patients will truly benefit from this drug and which have a genetic makeup that is not conducive to iniparib . iniparib is continuing to be studied in other phase iii clinical trials , including its effects on nonsmall cell lung cancer and ovarian cancer . iniparib evidently is not being discontinued completely from breast cancer research ; rather , the drug maker has continued with phase ii trials analyzing different doses , schedules , and chemotherapy combinations . olaparib ( azd2281 , astrazeneca ) is another parp1 inhibitor that is being tested on various cancers , including breast . preclinical models showed an increased selective potency for this compound . the subsequent phase i trial revealed 400 mg twice daily to be the maximum dose . with a brca1- or brca2-defective cohort of 22 patients , results of a phase ii trial detailed how olaparib is effective in breast cancer patients with a brca1 or brca2 mutation and advanced disease . while admittedly not a flawless design , such as lacking randomization , the results showed promise . all patients in the study had locally advanced breast cancer ( labc ) or metastatic breast cancer . for the tnbc and brca1/2 carrier patients in this cohort , twice daily 400 mg dosages of olaparib were more effective than twice daily 100 mg dosages when analyzing objective response ( 54% versus 25% ) and progressive disease ( 15% versus 31% ) . these data were observed , but it must be noted that this trial was not designed or powered for this comparison . when looking at all of the women in the trial , 41% of the brca1- or brca2-mutated breast cancer patients had an objective response when assigned 400 mg twice - daily olaparib . despite these encouraging results , london - based drug maker astrazeneca has decided to suspend olaparib prior to a phase iii trial . astrazeneca has shifted its olaparib focus to ovarian cancer and currently has a phase ii trial to study its effects on that cancer type . veliparib ( abt-888 , abbot laboratories ) has been investigated as a single agent and also has been shown to improve laboratory outcomes when paired with platinum agents and radiotherapy . were able to show that 5 and 25 mg / kg / d of veliparib combined with cisplatin were significant in tumor regression of murine models compared to cisplatin alone . 10 mg / kg / d of veliparib was also shown to be effective in combination with carboplatin when compared to carboplatin alone . in addition to improving the effectiveness of platinum agents on murine models of breast cancer , veliparib has shown to assist in radiation therapy . in mice , 3 gy with added veliparib was significantly more effective in inducing early cellular senescence than just the radiation alone . a phase ii trial recently studied the effects of veliparib combined with temozolomide on metastatic breast cancer and included tnbc patients . progression - free survival was 5.5 months in the brca - mutated group versus 1.8 months for patients without a brca mutation . tnbc is a clinical term used to describe women whose tumors lack expression of er , pr , and her-2 . this subset of breast cancer partially fits into a molecular subtype known as basal - like breast cancer . regardless of whether one looks at data through a tnbc or basal - like spectrum , the prognosis is worse compared to other subtypes . while there is no specific treatment regimen for tnbc patients , neoadjuvant therapy has been effective in achieving complete pathologic response ( pcr ) that subsequently correlates to improved outcome [ 7 , 44 ] . tnbc patients who achieve pcr had similar overall survival rates to non - tnbc patients who achieved pcr . however , tnbc patients that did not reach pcr had a worse outcome compared to non - tnbc patients that did not reach pcr . therapeutic options for tnbc have the potential to drastically increase in the near future . combinations of platinum compounds for neoadjuvant therapy are being tested in various clinical trials . epidermal growth factor receptors ( egfrs ) are noted in 4570% of tnbc [ 45 , 46 ] , resulting in egfr antagonists such as cetuximab ( merck serono ) to be explored . noted vegf to be increased in their tnbc patients compared to non - tnbc , and the antiangiogenic agent bevacizumab is being studied in combination with several chemotherapy agents in clinical trials . still other emerging avenues for treatment include mammalian target of rapamycin ( mtor ) inhibitors and src tyrosine kinase inhibitors . many potential therapeutic agents are in the pipeline in laboratories worldwide , but parp inhibitors have the potential to alter the outcome of tnbc patients . in addition to iniparib , olaparib , and veliparib , there are more being constructed . these include cep-9722 ( cephalon ) , ino-1001 ( genentech ) , pf-01367338 ( clovis / pfizer ) , and mk-4827 ( merck ) . most notably is the fact that recent trial data have landed huge blows to the momentum of parp inhibitors for breast cancer . at the 2011 asoc , it was announced that iniparib did not perform at its expected effectiveness in a phase iii trial with metastatic tnbc patients . astrazeneca has maintained an interest in parp inhibitors , but is doing so through further trials in other organs , such as ovarian . yet another complication that has emerged is resistance to parp inhibitors that is being observed in the laboratory . recently reported to observe cell lines with brca1/2 restoration mutations exhibiting resistance to platinum therapy in patients with hereditary ovarian cancer . they also observed these restoration mutations to predict resistance to parp inhibitors , but did not have a large sample size . more research must be done on these compounds to prepare for these and other , unknown , complications . it will be imperative to continue exploring the pathway connecting tnbc , basal - like breast cancer , and brca . there appears to be more questions to explore and compounds to test in the tnbc population with these therapeutics . also , further testing is necessary to identify the optimal doses of not only the parp inhibitor but also any combined chemotherapy . these key components of parp inhibitor development will hopefully improve the quality of this class of cancer - fighting drugs and provide hope for patients currently facing such bleak diagnoses .

triple - negative breast cancer ( tnbc ) is an aggressive type of breast cancer that is clinically defined as lacking estrogen and progesterone receptors , as well as being erbb2 ( her-2 ) negative . without specific therapeutic targets , tnbc carries a worse prognosis than other types of breast cancer in the absence of therapy . research has now further differentiated breast cancer into subtypes based on genetic expression patterns . one of these subtypes , basal - like , frequently overlaps with the clinical picture of tnbc . additionally , both tnbc and basal - like breast cancer link to brca mutations . recent pharmaceutical advances have created a class of drugs , poly(adp - ribose ) polymerase ( parp ) inhibitors , which are showing potential to effectively treat these patients . the aim of this paper is to summarize the basis behind parp inhibitors and update the current status of their development in clinical trials for the treatment of tnbc .

1. Introduction 2. PARP1 Inhibitors 3. Current Therapeutic Strategy 4. Conclusion

a review of the long history of orthodontic bonding adhesives shows that many evolutional developments have occurred since the first chemically - cured composite resins to the most recently introduced light - cured color - change adhesives ( ccas ) . while chemically - cured composite resins have some shortcomings , such as incorporation of air bubbles during mixing and limitation in controlling the curing time accurately , light - cured composite resins provide controlled polymerization time , more accurate bracket placement , and easier removal of excess adhesive prior to curing . however , there are still two major concerns about light - cured adhesives : improper removal of excess adhesive material from the tooth surface during bracket placement , which has been considered a potential risk for subsequent gingival irritation and white spot lesions on enamel surface;restoring enamel , as closely as possible , to an intact and flawless surface after bracket removal . improper removal of excess adhesive material from the tooth surface during bracket placement , which has been considered a potential risk for subsequent gingival irritation and white spot lesions on enamel surface ; restoring enamel , as closely as possible , to an intact and flawless surface after bracket removal . to this end , all the adhesive material must be removed from the enamel surface , taking care to prevent removal of enamel . similarity in shade and color makes it difficult to clearly delineate the enamel - adhesive interface , which may subsequently result in incomplete removal of the adhesive or loss of enamel during flash clean - up . accordingly , a range of 5 - 150 m has been reported in various studies for enamel loss . color - change adhesives are a recent development in the world of orthodontic adhesives , with the purpose of facilitating the discrimination between the adhesive material and enamel . they provide a distinct color and contrast and can be easily differentiated from enamel during both bracket placement and adhesive removal . related studies have mostly demonstrated high bond strengths for ccas under various conditions , suggesting that they can be used efficiently in orthodontic practice . although a minimum of bond strength is required for any orthodontic bonding adhesive , a high bond strength may increase the risk of enamel fracture and pulp damage upon debonding . a range of 6 - 8 mpa has been reported as the adequate bond strength for orthodontic purposes . enamel cracks appear when bond strength exceeds 13.5 mpa or 14 mpa and become more frequent as bond strength increases . since most of the studies on ccas have reported the shear bond strength ( sbs ) more than the safe upper limit , there is a demand to find ways to decrease it . some researchers have evaluated the effect of light - curing time on the sbs of orthodontic adhesives and concluded that the bond strength significantly decreases by reducing the curing time from 40 to 20 s. however , the available literature lacks information about the effect of reducing light - curing time on bond strength of ccas . thus , the aim of this study was to evaluate the effect of different light - curing times on the sbs of two commonly used ccas , i.e. , greengloo and transbond plus . in this in vitro study , 72 recently extracted sound human upper premolar teeth without any caries , restorations or former root canal therapy were included . after removing residual periodontal tissues and debris , all the teeth were immersed in 5.25% naocl for 2 h and then stored in normal saline . enamel surfaces were assessed under a stereomicroscope ( dm143 , motic digital microscope ) at 4.5 magnification to detect cracks , calcifications or any other defects . nonfluoridated pumice and rubber cups were used to clean the tooth surfaces . after mounting the specimens in auto - polymerizing acrylic resin , they were randomly assigned into 6 groups of 12 teeth each . in groups i and ii , resilience composite resin ( orthotechnology , fl , usa ) was used as the adhesive material and cured for 20 and 40 s , respectively . in groups iii and iv , greengloo composite resin ( ormco , glendora , ca , usa ) was used as the bonding adhesive and cured for 20 and 40 s , respectively . in groups v and vi , transbond plus composite resin ( 3 m , unitek , monrova , ca , usa ) was applied as the adhesive with the curing time of 20 and 40 s , respectively . bonding procedure commenced as follows : in groups i - iv , the buccal surfaces of the specimens were etched with 37% phosphoric acid gel ( ultradent , usa ) for 30 s , rinsed with water , and dried with moisture - free air until a frosty white etched area was observed . in groups i and ii a thin layer of resilience sealant resin ( orthotechnology , fl , usa ) and in groups iii and iv ortho solo primer ( ormco , glendora , ca , usa ) were applied to the buccal surface and thinned by a gentle current of air . in groups v and vi , transbond plus self - etching primer ( 3 m , unitek , monrovia , ca , usa ) was applied by an applicator to the buccal surface , and after 10 s thinned by a gentle current of air . for each specimen , the orthodontic adhesive was applied to the base of a 0.018 " standard edgewise metal bracket ( dentaurum , ispringen , germany ) . the adhesive was cured by a visible light - curing unit ( demetron lc , sds kerr , usa ) at a light intensity of 800 mw / cm from mesial and distal directions . subsequent to the completion of bonding procedure , all the specimens were stored in distilled water at 37c for 24 h in a dark environment . then , the specimens were mounted in the jig of a universal testing machine ( testometric , m350 - 10ct , england ) with the long axis of the specimen parallel to the direction of the shear load . in order to apply the shear load , a blade measuring 0.3 mm in thickness was advanced at a rate of 1 mm / min until debonding occurred [ figure 1 ] . measurement of shear bond strength of a specimen in a universal testing machine . in this study , debonding was defined as the point at which a sharp and instantaneous drop > 25% of the applied load occurred . the load at debonding ( measured in newton ) was recorded , and in order to calculate the sbs in mpa , it was divided by the surface area of the bracket base in millimeters . the remnant adhesive material on the tooth surface was evaluated under a stereomicroscope at 10 magnification and scored according to the modified adhesive remnant index ( ari ) . in this five - point scale , score 1 represents the entire composite resin with an impression of the bracket base remaining on the tooth ; score 2 represents more than 90% of composite resin remaining on the tooth ; score 3 represents 10 - 90% of composite resin remaining on the tooth ; score 4 represents < 10% of composite resin remaining on the tooth ; and score 5 represents no composite resin remaining on the tooth . finally , the tooth surface was scrutinized under 10 magnification for any postexperiment enamel fracture . one - way analysis of variance and tukey honestly significant difference were used to compare the mean sbss of the groups . multiple comparison test was used to determine the significance of the differences in the ari scores . all statistical analyses were performed using spss software ( version 11 ; spss , chicago , il , usa ) . one - way analysis of variance and tukey honestly significant difference were used to compare the mean sbss of the groups . multiple comparison test was used to determine the significance of the differences in the ari scores . all statistical analyses were performed using spss software ( version 11 ; spss , chicago , il , usa ) . tables 1 and 2 present the mean sbss of all the study groups and the significance of their differences . in all the three adhesives tested in this study , the mean sbs was lower when the specimens were light - cured for 20 s compared with the situation in which they were cured for 40 s ; however , only the difference in transbond plus adhesive ( groups v and vi ) was statistically significant ( p = 0.003 ) . the highest sbs values were obtained in group iv , in which greengloo composite resin was cured for 40 s ; and the lowest values were observed in group v , in which transbond plus composite resin was cured for 20 s [ tables 1 and 2 ] . sbs values of all the study groups ( mpa ) tukey hsd comparison of the mean sbss among study groups the remnant adhesive and the location of bond failure were scored according to ari and the results are shown in table 3 . in none of the groups , debonding occurred at the bracket adhesive interface . totally , 88.8% of bond failures occurred in the adhesive layer ( scores 2 , 3 and 4 ) and 11.1% of debondings occurred at the enamel - adhesive interface with no adhesive remaining on the tooth . the most frequent bond failures at enamel - adhesive interface occurred in groups v and vi with 25% and 33% , respectively [ table 3 ] . ari scores of all the study groups multiple comparison test indicated significant differences between ari scores of transbond plus and other groups . based on the results of this test , the ari scores of group v were significantly higher than those of group i. the ari scores in groups v and vi were significantly higher than those in group ii as well . stereomicroscopic evaluation revealed six postexperiment enamel fractures , 1 in group ii , 2 in group iii , and 3 in group iv . the main purpose of these studies is to obtain optimal bond strength for orthodontic brackets , in which the bond is strong enough to prevent bracket debonding , and simultaneously weak enough to avoid enamel damage upon debonding . bond strengths higher than 13.5 mpa or 14 mpa may result in enamel damage . in this study , the sbs of the test groups ranged from 14.05 4.24 mpa to 31.25 2.43 mpa . it was shown that reducing the light - curing time from 40 to 20 s decreased the sbs in all the adhesives and the decrease was statistically significant in transbond plus adhesive ; however , it seems that this decrease is not sufficient yet , since the mean sbss were still more than the safe limit . thus , it may be of interest to evaluate lower light - curing times in future studies . measured the mean sbss of four adhesives in the range of 16.0 - 22.1 mpa . like the present study , they measured the highest sbs among ccas in greengloo and the lowest in transbond plus . unlike the present study , the difference between the sbs of these ccas was not statistically significant , while in the present study the sbs in greengloo was significantly more than that in transbond plus . according to the manufacturer , higher bond strength of greengloo is due to the sealant used in this adhesive , ortho solo , a fluoride - releasing universal sealant . this sealant has a unique glass filler , which acts as a shock absorber and enhances bond strength by reducing the incidence of cracks that can lead to bond failure . chemical affinity of greengloo for some metal brackets has also been mentioned as a useful factor in improving bond strength . however , there are conflicting results about the bond - enhancing role of ortho solo . a mean sbs of 6.44 mpa for greengloo and 7.69 mpa for transbond plus adhesive at 24 h was reported by duers et al . lower amounts of sbs in their study compared to the present study may be attributed to the use of bovine teeth instead of human teeth , or the use of ground enamel surface instead of natural intact enamel surface . they used transbond etching gel and transbond primer for all the study groups and cured all the adhesives for 20 s. ekhlassi et al . used transbond plus self - etching primer and 20 s of light - curing time for all the groups and reported a mean sbs of 14.5 2.8 mpa for transbond plus at 24 h , which is very close to the results of the present study ( 14.05 4.24 mpa after 20 s of light - curing ) . on the contrary , they reported a mean sbs of 11.3 2.8 mpa for greengloo at 24 h , which is obviously different from 27.55 3.47 mpa in the present study . this difference might be due to the different primers applied , i.e. , transbond plus primer and ortho solo . conflicting results for the same adhesive type and curing time imply that other factors such as the type of primer may be responsible for differences in sbs . reported that etch - and - rinse primer systems that use acid etchants provided higher surface roughness and subsequently higher sbs than self - etch primer systems . in the above - mentioned study , since all the groups had a sbs well above minimally acceptable level , the authors suggested the use of self - etching primers instead of etch - and - rinse systems as a step toward conservative treatment in an ideal clinical situation like perfect isolation and manipulation . similarly , in the present study , the sbs with transbond plus adhesive which was used with a self - etch primer was significantly lower than that with greengloo adhesive which was used with an etch - and - rinse primer system . however , the authors of an in vitro study concluded that the bond strengths obtained by self - etch and etch - and - rinse systems are not statistically different . maintaining a sound and intact enamel surface is an important objective in orthodontic treatments . to this end , bond failures within the adhesive layer or at the bracket - adhesive interface are preferred to those at the enamel - adhesive interface . on the other hand , thus , there is a tradeoff between conservancy of orthodontic adhesives and convenience of the procedure for clinicians . ccas are an impressive step forward toward faster and facilitated adhesive removal . in the present study , ari scoring revealed that most of the debondings ( 88.8% ) occurred in the adhesive layer . the ari scores of greengloo groups ( iii and iv ) and transbond plus groups ( v and vi ) did not exhibit a statistically significant difference ; however , transbond plus groups showed significantly higher scores of ari compared to the controls . mavropoulos et al . in their study have reported that ari scores were not significantly different with 20 and 40 s of light - curing times , consistent with the results of the present study . in their study as well as some others , most of the debondings occurred at the bracket - adhesive interface or in the adhesive layer . in the present study , the majority of bond failures in greengloo adhesive occurred within the adhesive layer , which is consistent with the results of a study by trkkahraman et al . based on the results of the present study , regarding the highest amounts of sbs in greengloo adhesive and the safe region of bond failure within the adhesive layer , this cca may be a good candidate in cases where a high sbs is needed , such as rebonding of debonded brackets or bonding brackets to mutilated enamel . in the present study as well as in the study by trkkahraman et al . , the majority of debondings at enamel - adhesive interface occurred with transbond plus adhesive , suggesting a potential risk of enamel damage with the use of this adhesive . however , the question is to what extent bond failures at enamel - adhesive interface contribute to enamel damage . although , most of the debondings at enamel - adhesive interface ( ari score of 5 ) were observed with transbond plus adhesive , enamel fractures were predominantly observed in greengloo adhesive ( five out of six ) . however , due to the limited number of fractures for each adhesive , drawing a conclusion based on the current data is not feasible . have reported , based on scanning electron microscope evaluation , that etch - and - rinse primer systems caused dramatic changes in the enamel surface due to enamel demineralization , resulting in higher bond strength . it was demonstrated that higher amounts of sbs lead to a higher frequency of enamel fracture , consistent with the results of the present study . therefore , the extent to which ari scoring is predictive of enamel damage remains an important question . an ideal adhesive will show minimal amount of remnant adhesive and minimal change in enamel surface , while providing sufficient bond strength . according to the limitations of this study , precise comparison between studies in the literatures requires matching of factors such as brand of light cure device , composites , their manufacturer propositions , power , intensity , area , wavelength of light , distance and etc . in addition , the findings of this study were obtained under in vitro conditions that may differ from intraoral conditions . some factors such as enamel composition , saliva contamination and the difference between the universal testing machine forces and intraoral forces may affect the results in clinical situations . paucity of data indicates the demand for further clinical studies on the bond strength , risk of enamel damage , and measures to minimize this risk when color - change orthodontic adhesives are applied . reducing the light - curing time from 40 to 20 s decreased the sbs in all the adhesives , and the difference between the sbss of transbond plus groups was statistically significant ; however , the mean sbss of all the groups were still more than the safe limit . among the two ccas studied , greengloo adhesive showed significantly higher sbs than transbond plus adhesive . ari scores in transbond plus adhesive were significantly higher than the controls ; however , ari assessment did not yield significantly different results in transbond plus and greengloo adhesives .

background : the purpose of this study was to evaluate the effect of light - curing time on the shear bond strength ( sbs ) of two orthodontic color - change adhesives ( ccas).materials and methods : a total of 72 extracted premolars were randomly assigned into 6 groups of 12 teeth each . subsequent to primer application , a metal bracket was bonded to the buccal surface using an orthodontic adhesive . two ccas ( greengloo and transbond plus ) were tested and one conventional light - cured adhesive ( resilience ) served as control . for each adhesive , the specimens were light - cured for two different times of 20 and 40 s. all the specimens underwent mechanical testing using a universal testing machine to measure the sbs . adhesive remnant index ( ari ) was used to assess the remnant adhesive material on the tooth surface . all statistical analyses were performed using spss software . the significance level for all statistical tests was set at p 0.05.results:the sbss of the tested groups were in the range of 14.05 - 31.25 mpa . greengloo adhesive showed the highest sbs values when light - cured for 40 s , and transbond plus adhesive showed the lowest values when light - cured for 20 s. ari scores of transbond plus adhesive were significantly higher than those of controls , while other differences in ari values were not significant.conclusion:within the limitations of his study , decreasing the light - curing time from 40 to 20 s decreased the sbs of the tested adhesives ; however , this decline in sbs was statistically significant only in transbond plus adhesive

INTRODUCTION MATERIALS AND METHODS Statistical analysis RESULTS DISCUSSION CONCLUSION

the genomes of the two mimivirus strains , the mamavirus and apmv , are highly similar but show characteristic divergence in the terminal regions . the mamavirus genome is the largest available virus genome , in part due to the presence of a 13-kb unique 5-terminal region that apparently evolved by duplication of internal genomic sequences , possibly combined with the acquisition of a dna fragment from an unknown source . a comprehensive comparative sequence analysis of the mamavirus and apmv proteins led to a substantial amendment of the functional annotation of the mimivirus genome and revealed several unique predicted proteins in the mamavirus . supplementary methods and files 13 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .

the genome sequence of the mamavirus , a new acanthamoeba polyphaga mimivirus strain , is reported . with 1,191,693 nt in length and 1,023 predicted protein - coding genes , the mamavirus has the largest genome among the known viruses . the genomes of the mamavirus and the previously described mimivirus are highly similar in both the protein - coding genes and the intergenic regions . however , the mamavirus contains an extra 5-terminal segment that encompasses primarily disrupted duplicates of genes present elsewhere in the genome . the mamavirus also has several unique genes including a small regulatory polya polymerase subunit that is shared with poxviruses . detailed analysis of the protein sequences of the two mimiviruses led to a substantial amendment of the functional annotation of the viral genomes .

Conclusions Supplementary Material

however , in a proportion of the patients with major depression , despite the use of adequate antidepressant doses for the adequate duration , clinical remission is not achieved . although there is no consensus , but in general it is accepted that those patients with major depression who do not respond to 2 - 3 adequate trials of antidepressants are considered to have treatment - resistant depression ( trd ) . some of the authors have suggested staging for trd and based on the level of nonresponse the patient is allocated to different stages of trd . it is suggested that whenever a patient present 's with trd , a thorough evaluation needs to be done to evaluate the underlying organic and psychosocial causes . we here , report a case of recurrent depressive disorder , current episode severe depressive episode without psychotic symptoms , who did not respond to adequate trials of antidepressants and showed minimal response to electroconvulsive therapy ( ect ) . in view of the lack of remission , on investigation she was managed with ketoconazole 400 mg / day along with the continuation of antidepressants with which she achieved remission . mrs . a , 40-year - old , known case of recurrent depressive disorder , with first episode occurring at the age of 36 years , with two episodes in the past which responded to antidepressant treatment , presented with severe depressive episode without psychotic symptoms of 18 months duration . for the current episode , the onset was insidious with the evolution of symptoms over the period of 1-month , without any precipitating event and the course was continuous for the current episode . her clinical presentation was characterized by persistent sadness of mood with morning worsening , poor interaction , anhedonia , lethargy , psychomotor retardation , sleep disturbance in the form of difficulty in falling asleep with frequent midnight awakenings , reduced appetite associated with weight loss of 3 kg , reduced libido , ideas of guilt , suicidal ideations , suicidal planning with one unsuccessful attempt and off and on anxiety symptoms . her treatment history revealed that during the current episode she was treated with tablet paroxetine 12.5 - 37.5 mg / day for 4 months , tablet mirtazapine 15 - 30 mg / day for 3 months , tablet imipramine up to 175 mg / day for 5 months , c. venlafaxine up to 300 mg / day for 2 months with no response . later she was treated with c. venlafaxine 300 mg / day along with thyroxine 75 g / day ( for 2 months ) and c. venlafaxine 300 mg / day and lithium 600 mg / day for a period of 2 months but with minimal improvement . on mental status examination , she had sadness of mood , psychomotor retardation , ideas of hopelessness , worthlessness , guilt , and suicidal ideas . investigations in the form of hemogram , liver function test , renal function test , serum electrolytes , thyroid function test , serum vitamin b12 levels were did not reveal any abnormality . her magnetic resonance imaging ( mri ) scan of the brain did not show any abnormality . her psychosocial history did not reveal any evidence of chronic stressors and her family was very supportive . there was no history suggestive of mania , psychotic symptoms , alcohol or drug abuse , seizure , head injury , and cognitive decline . she was continued on c. venlafaxine 300 mg / day along with tablet lithium carbonate 300 mg / day ( with serum levels in the therapeutic range ) . in addition , due to lack of response to adequate doses of antidepressants she was treated with 14 sessions of modified ect over the period of 6 weeks with minimal improvement ( hdrs score reduced to 32 ) . in view of the lack of response to ect , further investigations were done for cushing 's syndrome although her physical examination was not suggestive of the same . workup for cushing 's syndrome revealed raised plasma cortisol level ( 722.7 nmol / l [ normal range 193 - 634 mri scan of the abdomen revealed small homogenous , well - defined lesion measuring 2 cm in the adrenal cortex with clear margins suggestive of an adrenal adenoma . as a result , she was started on tablet ketoconazole 200 mg / day and increased to 400 mg / day over next 15 days along with the continuation of c. venlafaxine 300 mg / day . patient improvement was monitored clinically and using ham - d score . over a period of next 4 weeks , the patient showed significant improvement in her depressive symptoms with no associated side effects . she has been maintaining well on tablet ketoconazole 400 mg / day and of c. venlafaxine 225 mg / day for the last 4 years . her adrenal mass has been monitored with no increase in the size of the tumor . according to the staging of trd by thase and rush , the index case can be considered as stage-5 trd , that is , patient who has not responded to antidepressants of two different classes , tricyclic antidepressants and ect . in addition , the patient had also not responded to augmentation with thyroxine and lithium . it is suggested that whenever a patient presents with trd , first there is a need to evaluate the patient for pseudo - resistance . the factors that contribute to pseudo - resistance include poor compliance , inadequate dosing , and discontinuation of antidepressant before adequate duration . the history of the index case did not reveal the same . in view of the stage-5 nonresponse , she was empirically evaluated for cushing 's syndrome and was found to have positive evidence for the same . due to the role of stress and involvement of cortisol in understanding the etiopathogenesis of depression , researchers have used antiglucocorticoid drugs such as metyrapone , aminoglutethimide , ketoconazole , and mifepristone in the management of trd . in a review , which included 11 studies , authors reported that 67 - 77% of the patients show at least a partial antidepressant response and largest two series documenting response rates of 70 - 73% . our case highlights the fact that while dealing with patients with trd , psychiatrists should look into all possible medical causes for depression . further , our case suggests that antiglucocorticoid medications can be considered in patients with trd who do not respond to conventional treatments .

treatment resistant depression ( trd ) is a common clinical occurrence among patients treated for major depressive disorder . a significant proportion of patients remain significantly depressed in spite of aggressive pharmacological and psychotherapeutic approaches . management of patient with treatment resistant depression requires thorough evaluation for physical causes . we report a case of recurrent depressive disorder , who presented with severe depressive episode without psychotic symptoms , not responding to multiple adequate trials of antidepressants , who on investigation was found to have cushing 's syndrome and responded well to ketoconazole .

INTRODUCTION CASE REPORT DISCUSSION

a 30-year - old woman visited our clinic with pain on the right inguinal area and lateral aspect of the thigh , which spontaneously started 3 months ago and had become aggravated a week before . the pain intensity in the visual analogue scale ( vas ) was about 80 of 100 . she was experiencing right groin and thigh pain precipitated by walking , which made it difficult for her to walk normally . lumbar magnetic resonance imaging ( mri ) done at a previous clinic showed otherwise normal ( fig . , lumbar epidural steroid injection was done twice in combination with medications such as nonsteroidal anti - inflammatory drugs ( nsaids ) and muscle relaxants for 2 months ; however , these failed to improve her symptoms . she has even had a gynecological examination to rule out the disorder such as pelvic inflammatory disease , yield to normal . on physical examination , she complained of tenderness over the right buttock and the anterolateral aspect of the thigh , as well as pain with flexion and internal rotation of the right hip joint . both the straight - leg - raising test and the flip test were negative , as concordant with previous mri showing non - specific finding . her body temperature was 36.3 , and laboratory findings such as complete blood count , erythrocyte sedimentation rate , c - reactive protein , and rheumatoid factor were normal . we checked the hip radiograph and found a small amorphous calcification near the right anterior inferior iliac spine ( aiis)-the attachment site of the rectus tendon - suggesting calcific tendinitis of rectus femoris . additionally , it was more visible in frog leg lateral views around the bony structure ( fig . , calcification was noted in the right rectus femoris tendon , showing thickening with a small amount of fluid collection and edematous change of peripheral soft tissue ( fig . , the transducer was positioned over the hip area to find the calcification . around the calcified lesion , we injected 5 ml of 1% of mepivacaine with 20 mg triamcinolone acetate , by using a 25 g 38-mm needle ( bd precisionglide needle ; becton dickinson medical , singapore ) with the short - axis in - plane technique . at 1 week intervals , the pain intensity started to decrease 2 days after the first injection and decreased to 0 - 10 of 100 in the vas 3 days after the second injection . she returned to work in a pain - free state and after 6 weeks of follow - up , us image demonstrated decreased echogenicity of surrounding fat and also decreased amount of fluid collection with resolved symptom ( fig . painful periarticular calcification is an acute inflammatory reaction , with pain , exquisite tenderness , local swelling , and redness . it is most commonly seen within the rotator cuffs of the shoulder , although it can develop around the wrist , elbow , hip , knee , foot , and , rarely , in the neck . in the hip area , since the report of king and vanderpool in 1967 , it has been rarely reported in the literature . the exact etiology of the origin and resorption of the calcium deposits are not clearly understood , although several hypothetical mechanisms have been proposed . the only disorders reported to be associated with the disease are chronic renal failure with persistent hemodialysis and diabetes mellitus . however , historically , it was assumed that these deposits might have resulted from trauma around the involved joints , although no specific mechanism was elucidated . as repetitive trauma has been cited as a factor , especially in the shoulder region , tendinous tears of hip abductors , the patient 's history of overuse of the hip abductors , in relation to her job that involves walking for 6 - 8 h a day , supported the above by describing repetitive trauma hypothesis . it is known that periarticular calcific tendinitis can be classified as acute or chronic . in acute disease , most attacks last 2 weeks and the calcification disappears from radiographs at 4 weeks to 8 months post - onset . despite its self - limiting nature , if mild or moderate pain persists for 2 - 24 months after disease onset , the disease can be classified as chronic . in the hip , it is known that calcification occurs at three sites , namely the tendon of the gluteus medius , the bursa between the tendon of the gluteus medius and the greater trochanter , and the undersurface of the gluteus medius but not connected to the trochanter . all locations can cause pain in the posterolateral aspect of the thigh that can simulate radicular pain from a herniated intervertebral disc . hence , misdiagnosis is common and leads to a delay in treatment , as in our case ; this case was primarily managed under the diagnosis of a herniated lumbar disc , and the patient 's condition failed to improve . the diagnosis is usually based on clinical suspicion and typical findings of plain radiography of the hip , locating calcific depositions in the tendon or bursa . however , plain radiography can lead to misdiagnosis without additional diagnostic tools , as the bone , adjacent soft tissue , ligaments , and tendons around hip are arranged three - dimensionally . furthermore , if the calcification is very thin and small , and located in the posterior aspect , it is even more difficult to detect with only plain radiography , sometimes even in retrospective examinations . the use of the " frog - leg position " while obtaining the plain x - ray image also supports this three - dimensional anatomical characteristic as it was also shown in our case . in the diagnosis of calcific tendinitis , computed tomography ( ct ) allows the best visualization and localization of proper lesions , as well as facilitates accurate injection , when required . mri can be a useful tool to rule out other conditions , especially when the lesions involve soft tissue areas , and to show the severity of inflammation . the real - time nature of ultrasonography has led to its use in both diagnostic and therapeutic procedures , as we did in our case . ultrasonography is reliable for the detection and localization of calcifications ; however , it is unable to classify the pathophysiologic phase . therefore , clinical correlations and other radiographs are necessary to evaluate the disease . the differential diagnoses of calcifications in the rectus femoris include osacetabuli , avulsion fractures , sesamoid bones in the rectus femoris , myositis ossificans , and malignancies , usually based on the size , characteristic ovoid shape , bony trabeculation , and painlessness of the ossicle . the comet - tail appearance of the calcific deposit suggests that it lies within a tendon . periarticular calcific tendinitis is widely accepted as a self - limited disease ; however , some refractory patients need treatment because the duration of symptoms varies from months to years . the treatment options include conservative management such as rest , nsaids , local applications of heat and cold , arthroscopy , local surgical excision , and local steroid injection . surgical excision is usually considered in case of long - lasting symptoms that are refractory to conservative treatment . among the conservative modalities , local steroid and anesthetic injections provide rapid and long - lasting pain relief and shorten the clinical course . however , their mechanisms of action are still unclear . there are several literature reports in which a prompt diagnosis and early treatment with a local injection of corticosteroid under ct or fluoroscopy guidance were performed . evaluating the general condition by using ultrasonography alone has some limitations ; however , it has the benefits of convenience and the lack of radiation exposure compared with ct or fluoroscopy . there are insufficient cases reported in the literature in which ultrasonography was mainly used to treat calcific tendinitis in the hip area as this case , compared with that in the rotator cuff of the shoulder . we performed local injection under ultrasound guidance because it has the advantage of real - time image acquisition , enabling fast and accurate diagnosis and treatment . especially in a hip lesion , although a tiny calcification can cause severe pain , a misdiagnosis is easily made , causing the disease to progress into the chronic state . it is difficult to detect the calcification initially because of its rarity and the complex three - dimensional anatomic structure of the hip . it could be more easily found in conjugation with valid radiographs of the patient 's pain area . physicians should consider acute calcific tendinitis of the rectus femoris when making a differential diagnosis of hip pain , although it is a rare disease entity , as misdiagnosis leads to inappropriate treatment and delays in recovery . we also recommend ultrasonography in the evaluation of hip pain , as it can be used as a rapid and effective diagnostic and therapeutic tool .

painful periarticular calcification most commonly occurs within the rotator cuff of the shoulder and rarely around the elbow , hip , foot , and neck . as acute inflammatory reaction develops , severe pain , exquisite tenderness , local swelling , and limitation of motion with pain occur . in case of calcific tendinitis of the shoulder , it can be easily diagnosed according to the symptoms and with x - ray . however , in lesions of the hip , as it is a rare location and usually involves pain in the posterolateral aspect of the thigh , which can simulate radicular pain from a lumbar intervertebral disc , it could be difficult to diagnose . hence , physicians usually focus on lumbar lesions ; therefore , misdiagnosis is common and leads to a delayed management . here , we report the case of a 30-year - old female patient with calcific tendinitis of the rectus femoris that was successfully managed with ultrasound - guided steroid injection . this study offers knowledge about the rectus femoris calcification .

CASE REPORT DISCUSSION

breast cancer , which has the highest incidence and mortality rate of women cancers globally , imposes a significant disease burden in developing countries . among breast cancer risk factors , dense breasts found on mammography due to breast epithelium and stroma are known to be a potent risk factor , raising breast cancer risk by four to six times [ 2 - 4 ] according to previous systematic reviews ( srs ) [ 5 - 9 ] . however , these srs have mostly been performed on studies of white women in the west . asian women , whose breast cancer incidence rate is lower than that of western white women , have been reported to have dense breasts on mammography more frequently [ 11 - 14 ] . that is , asian women have denser breasts on mammography , but lower breast cancer incidence than white women . these facts make us doubt the proposal that breast density may be a risk factor in asian women . in particular , the incidence curves of breast cancer in accordance with age are significantly different in asian women , including koreans , than in western women [ 18 - 20 ] . thus , we can infer that the risk factor of breast density works differently in asian women than in western women . some studies have reported that breast density on mammography was a risk factor for breast cancer in asian women [ 21 - 23 ] . however , the risks varied depending on the density measurement index , statistical significance varied for each density interval , and a dose - response relationship has not been shown . as of the end of december 2015 , no sr was found that evaluated the association between breast density on mammography and breast cancer risk in asian women . the final selection criteria for the meta - analysis included analytical epidemiology studies that evaluated the association between breast density levels determined using mammography and breast cancer risk in asian women . article selection was conducted in accordance with the preferred reporting items proposed for srs and meta - analyses , including three stages : searching , screening using titles and abstracts , and evaluating articles . the databases searched in the first stage were pubmed ( http://www.ncbi.nlm.nih.gov/pubmed ) and scopus ( www.scopus.com ) , and the following search formula was applied : [ ( breast ) or ( mammary ) ] and [ ( cancer ) or ( neoplasm ) ] and [ ( density ) or ( index ) ] and [ ( asia ) or ( women ) ] . furthermore , a list was made while performing a hand search to investigate whether an sr had already been published evaluating the same hypothesis . the lists from the three search sources were combined to remove duplicates . for the 2-stage screening process , the following exclusion criteria were applied based on the titles and abstracts to the summarized list : ( 1 ) studies not related to breast cancer , ( 2 ) laboratory studies , ( 3 ) expert or systematic reviews , and ( 4 ) descriptive epidemiological studies including cross - sectional prevalence studies . after the 2-stage screening process and exclusion , the third - stage evaluation was conducted for the remaining articles . for this purpose , the content was evaluated using the full text of each article , and articles that fell into the following categories were excluded sequentially : ( 5 ) analytical epidemiology studies that did not provide the information necessary for a meta - analysis , and ( 6 ) duplicate studies . judgment of duplicates was made when the study subjects were selected from the same institution during the same recruiting period . among duplicates , the following information was extracted from each article : the nationality of the subjects , the recruiting institution , data sources for cohort construction , menopausal status , number of cancer cases and controls , type of breast density measurement index , and adjusted odds ratios ( aors ) or relative risks and their 95% confidence intervals ( cis ) for potential confounders at each density level . the measurement indices included the wolfe classification ( wolfe ) , percent density ( pd , % ) , volumetric density grade ( % ) , density area ( cm ) , total breast area ( tba , cm ) , absolute dense area ( cm ) , and mean dense area ( mda , cm ) . an article showing aors divided according to menopausal status and several measurement indices was considered to provide independent datasets for each stratum . the effect size ( es ) of each dataset to be used in the meta - analysis was calculated using the interval collapsing method ( icm ) rather than highest vs. lowest intake method ( hlm ) , because the icm increases the statistical precision more than the hlm . icm application adopts the es and its 95% ci calculated by performing a meta - analysis with a random effect model ( rem ) on the aor and its 95% ci presented for each density level within a dataset as the es for each dataset . after dividing the data into subgroups according to menopausal status and measurement indices , a rem meta - analysis was performed again using the es for each dataset to calculate a summary effect size ( ses ) and its 95% ci . meta - analysis was performed only in cases in which two or more datasets were found in the subgroup analyses . in the meta - analysis , furthermore , to investigate the dose - response relationship in breast cancer risk in accordance with the density level presented in breast density indices , a random effects dose - response meta - regression ( drmr ) was conducted . the median values within the interval were used for dosage determination , and the lower limit was set at zero when the lowest interval was open . when the highest interval was open , the median interval of the adjacent interval was used . if the density index was pd , the dosage unit was determined to be 25% . the statistical significance level was set at 5% , and stata version 14.0 ( statacorp , college station , tx , usa ) was used . the final selection criteria for the meta - analysis included analytical epidemiology studies that evaluated the association between breast density levels determined using mammography and breast cancer risk in asian women . article selection was conducted in accordance with the preferred reporting items proposed for srs and meta - analyses , including three stages : searching , screening using titles and abstracts , and evaluating articles . the databases searched in the first stage were pubmed ( http://www.ncbi.nlm.nih.gov/pubmed ) and scopus ( www.scopus.com ) , and the following search formula was applied : [ ( breast ) or ( mammary ) ] and [ ( cancer ) or ( neoplasm ) ] and [ ( density ) or ( index ) ] and [ ( asia ) or ( women ) ] . furthermore , a list was made while performing a hand search to investigate whether an sr had already been published evaluating the same hypothesis . the lists from the three search sources were combined to remove duplicates . for the 2-stage screening process , the following exclusion criteria were applied based on the titles and abstracts to the summarized list : ( 1 ) studies not related to breast cancer , ( 2 ) laboratory studies , ( 3 ) expert or systematic reviews , and ( 4 ) descriptive epidemiological studies including cross - sectional prevalence studies . after the 2-stage screening process and exclusion , the third - stage evaluation was conducted for the remaining articles . for this purpose , the content was evaluated using the full text of each article , and articles that fell into the following categories were excluded sequentially : ( 5 ) analytical epidemiology studies that did not provide the information necessary for a meta - analysis , and ( 6 ) duplicate studies . judgment of duplicates was made when the study subjects were selected from the same institution during the same recruiting period . among duplicates , the following information was extracted from each article : the nationality of the subjects , the recruiting institution , data sources for cohort construction , menopausal status , number of cancer cases and controls , type of breast density measurement index , and adjusted odds ratios ( aors ) or relative risks and their 95% confidence intervals ( cis ) for potential confounders at each density level . the measurement indices included the wolfe classification ( wolfe ) , percent density ( pd , % ) , volumetric density grade ( % ) , density area ( cm ) , total breast area ( tba , cm ) , absolute dense area ( cm ) , and mean dense area ( mda , cm ) . an article showing aors divided according to menopausal status and several measurement indices was considered to provide independent datasets for each stratum . the effect size ( es ) of each dataset to be used in the meta - analysis was calculated using the interval collapsing method ( icm ) rather than highest vs. lowest intake method ( hlm ) , because the icm increases the statistical precision more than the hlm . icm application adopts the es and its 95% ci calculated by performing a meta - analysis with a random effect model ( rem ) on the aor and its 95% ci presented for each density level within a dataset as the es for each dataset . after dividing the data into subgroups according to menopausal status and measurement indices , a rem meta - analysis was performed again using the es for each dataset to calculate a summary effect size ( ses ) and its 95% ci . meta - analysis was performed only in cases in which two or more datasets were found in the subgroup analyses . in the meta - analysis , furthermore , to investigate the dose - response relationship in breast cancer risk in accordance with the density level presented in breast density indices , a random effects dose - response meta - regression ( drmr ) was conducted . the median values within the interval were used for dosage determination , and the lower limit was set at zero when the lowest interval was open . when the highest interval was open , the median interval of the adjacent interval was used . if the density index was pd , the dosage unit was determined to be 25% . the statistical significance level was set at 5% , and stata version 14.0 ( statacorp , college station , tx , usa ) was used . figure 1 shows a flow diagram illustrating a series of processes from the searching , screening , and evaluation stages to the final selection of articles to include in the analysis . from the two databases , pubmed and scopus , eight articles acquired from the hand - searching process were added to the list , and then 197 duplicates were removed , leaving a list of 1374 articles . from this list , the texts of the 53 remaining articles were obtained and the content was evaluated to remove 47 articles , leaving the final six articles for meta - analysis [ 20 - 22,28 - 30 ] . the exclusion criteria during the selection process were the following : ( 1 ) 766 studies were not related to breast cancer ; ( 2 ) 48 articles were laboratory studies ; ( 3 ) 97 studies were expert or srs ; ( 4 ) 410 articles were descriptive epidemiological studies , including cross - sectional prevalence studies ; ( 5 ) 40 analytical epidemiology studies did not provide sufficient information for a meta - analysis ; and ( 6 ) seven articles were duplicate studies . the study subjects of nagata et al . were patients at gifu city hospital , and four duplicates were removed [ 31 - 34 ] . the study subjects of lee et al . were participants of the singapore breast cancer screening programme ( sbcsp ) , and two duplicates were removed . the study subjects of kim et al . were patients at the samsung medical cancer in korea , and one duplicate was removed . table 1 shows a summary of the final six articles with 17 datasets based on type of breast density index and menopausal status . categorized by country , the six articles included three japanese studies , two korean studies , and one singaporean study . seven datasets were of premenopausal women and eight were of postmenopausal women . in terms of the breast density index , five used pd , four used tba , and two or fewer datasets used the remaining indices . since the findings varied in accordance with the index type and menopausal status , subgroup analysis was performed rather than calculating the ses values of the 17 datasets in order to control for potential heterogeneity . in other words , the datasets were divided according to menopausal status , and meta - analysis was performed only in cases with two or more datasets for each density index ( table 2 , figure 2 ) . in premenopausal women , the pd index was significantly associated with elevated breast cancer risk ( ses , 3.23 ; 95% ci , 2.23 to 4.66 ; i=0.0% ) , whereas the tba index did not show a statistically significant association . in the group of postmenopausal women , the tba index did not show a statistically significant association , whereas the pd index was associated with a significant increase in breast cancer risk ( ses , 1.62 ; 95% ci , 1.13 to 2.32 ; i=0.0% ) . for premenopausal and postmenopausal women , meta - analysis was performed if two or more datasets were present for each density index , and the results showed significantly elevated breast cancer risks for the pd , density area , and volumetric density indices . among the six articles selected , three provided the information necessary for drmr analysis . three datasets ( 2 , 5 , and 8 in table 1 ) were obtained using the pd index , and homogeneity was detected between two datasets ( 5 and 8 in table 1 ) of postmenopausal women ( p=0.35 ) , showing a risk increase of 1.73 times for each 25% increase in pd in postmenopausal women ( 95% ci , 1.20 to 2.47 ) . in this study , the first sr of breast density and breast cancer risk in asian women , breast cancer risk was found to increase as the pd value increased . although the tba index did not show statistical significance , the risk increased by 73% for each 25% increase in pd in postmenopausal women , which indicates that higher breast cancer risk is associated with higher pd values in women in asian countries . however , the risk calculated for pd in premenopausal and postmenopausal women was estimated to be 2.21 times that of baseline ( 95% ci , 1.52 to 3.21 ) , which is lower than the risk elevation of four to six times that has been confirmed in western women ( two to four ) . first , breast density itself is not a risk factor , but a phenomenon determined by other risk factors [ 37 - 40 ] . density can be affected by obesity , family history , genotype as well as obstetrical history . second , in asian women , the positive predictive value in breast cancer diagnosis decreases with decreased sensitivity in mammography of denser breasts , resulting in underestimation of cancer occurrence . third , risk levels were found to change in accordance with the type of density measurement index [ 43 - 45 ] , suggesting that different measurement indices have been used by different researchers , and that different indices may be appropriate depending on race . fourth , this study analyzed limited data , meaning that the conclusions may be tentative . further studies evaluating breast density , as measured using several indices , and the risk of breast cancer in asian women are needed . first , an overall es reflecting information from all 6 articles was not calculated , not only because the number of articles related to asian women was small , but also because the breast density index varied across articles . however , the study performed by wong et al . , which was excluded because its participants overlapped with the sbcsp participants , presented breast cancer risks adjusted for menopausal status and the pd index . when this was added to the five data - sets using pd in the meta - analysis , as shown in table 2 , the ses increased from 2.12 to 2.30 ( 95% ci , 1.67 to 3.16 ; i=40.5% , not shown in table 2 ) . when two groups ( premenopausal and postmenopausal women ) were differentiated in a single article , it was possible that the ses values were determined by calculations within the subgroup analysis . however , the studies of nagao et al . and lee et al . could not be used in the subgroup analysis because the results were not divided according to menopausal status . furthermore , in cases where various indices were used for the same subjects , these results could not be incorporated without omission , resulting in a selective analysis within subgroups as well . applying drmr to the density indices was possible because three datasets were established from the total of two articles on japanese women , while only one was possible for premenopausal women . the aforementioned three limitations could be overcome by creating a single database for a pooled analysis of the selected articles . first , the subjects included only women who were born and lived in asia . in other words , this decision was based on studies reporting that immigration as an environmental change affects breast cancer risk . in the future , studies will be needed to investigate how breast density affects cancer risk among people of the same race depending on emigration . second , in the five case - control studies , the most recent mammography results before breast cancer diagnosis were used as breast density values . this design does not reflect the fact that breast density changes with age in individual women [ 46 - 48 ] . in the future , cohort studies that investigate breast cancer risk according to individual changes in breast density will be needed . in conclusion , regardless of menopausal status , breast cancer risk in asian women increased with breast density measured using pd . in particular , postmenopausal women with a high pd index had an elevated risk of breast cancer . as this sr suggests that the pd index represents breast cancer risks well in asian women , we propose the further development of a breast - cancer risk prediction model involving pd for asian women . to investigate the risks more precisely , a pooled analysis is proposed , along with the application of pd in breast cancer prediction models in asian women

objectivesthe established theory that breast density is an independent predictor of breast cancer risk is based on studies targeting white women in the west . more asian women than western women have dense breasts , but the incidence of breast cancer is lower among asian women . this meta - analysis investigated the association between breast density in mammography and breast cancer risk in asian women.methodspubmed and scopus were searched , and the final date of publication was set as december 31 , 2015 . the effect size in each article was calculated using the interval - collapse method . summary effect sizes ( sess ) and 95% confidence intervals ( cis ) were calculated by conducting a meta - analysis applying a random effect model . to investigate the dose - response relationship , random effect dose - response meta - regression ( re - drmr ) was conducted.resultssix analytical epidemiology studies in total were selected , including one cohort study and five case - control studies . a total of 17 datasets were constructed by type of breast density index and menopausal status . in analyzing the subgroups of premenopausal vs. postmenopausal women , the percent density ( pd ) index was confirmed to be associated with a significantly elevated risk for breast cancer ( ses , 2.21 ; 95% ci , 1.52 to 3.21 ; i2=50.0% ) . the re - drmr results showed that the risk of breast cancer increased 1.73 times for each 25% increase in pd in postmenopausal women ( 95% ci , 1.20 to 2.47).conclusionsin asian women , breast cancer risk increased with breast density measured using the pd index , regardless of menopausal status . we propose the further development of a breast cancer risk prediction model based on the application of pd in asian women .

INTRODUCTION METHODS Search and Selection of Related Articles Statistical Analysis RESULTS DISCUSSION CONCLUSION

osteoarthritis ( oa ) is a major cause of disability affecting millions of people worldwide . in the united states alone , to date there are no proven therapies for the prevention or treatment of oa . pain relief and visco - supplementation are prescribed to attenuate the symptoms of oa until disease progression significantly impairs joint function and joint replacements are required . the lack of disease modifying oa drugs ( dmoads ) may be a function of incongruence between in vitro models of oa and the pathogenesis in vivo , and between disease mechanisms in humans and model animals . to overcome these issues , there is increasing momentum to develop human cell - based organotypic models in vitro that functionally represent the osteochondral tissue directly affected by oa . the development of physiologically relevant models requires an understanding of the tissue architecture , physiology , and pathophysiological responses to biochemical ( or biophysical ) insults . this is especially the case for the osteochondral complex , where the main tissues , cartilage and bone , differ so substantially . cartilage is composed of a collagen type ii / aggrecan - rich , highly hydrated , viscoelastic , anisotropic matrix that encapsulates chondrocytes within biochemically distinct chondrons . in contrast , bone is composed of a collagen type i - rich , laminated or woven calcified structure that is much stiffer and encapsulates osteocytes , osteoblasts , and osteoclasts , blood vessels , and nerves . the cartilage and bone are intimately connected at the osteochondral junction ( ocj ) , a highly organized structure that represents a significant challenge to mimic in vitro by tissue engineering . it is composed of distinct , interacting layers that include ( epi - to - diaphyseally ) deep zone cartilage , a basophilic tidemark , calcified cartilage , the cement line , and the subchondral bone plate . interestingly , there is growing evidence of significant biochemical communication between cartilage and bone across the ocj . in the pathogenesis of oa , changes in the physical linkage between cartilage and bone at the ocj are critical components of disease progression . these include remodeling of the tidemark , microcracks , and fissures in both tissues and ingrowth from the underlying bone of blood vessels and nerves , all of which may enhance the cartilage - bone crosstalk allowing a better passage of growth factors , cytokines , and signaling molecules . these ocj changes accelerate cartilage degeneration and are associated with joint pain and disease morbidity , pointing to the need of a better understanding of the complex network of interactions between bone and cartilage in oa . cartilage and bone exist not only in a different matrix but also in very different biophysical environments . in vivo , there is a steep oxygen gradient from bone ( essentially normoxic ) to cartilage ( extremely hypoxic ) . these differences are reflected in the in vitro culture systems often used to maintain chondrocytes and osteoblasts . starved environment : low glucose , serum - free medium supplemented with pyruvate and abundant matrix precursors or -enhancing molecules ( proline and ascorbate ) in hypoxic conditions . however , osteoblasts are maintained in high glucose , serum - containing medium supplemented with -glycerol phosphate and vitamin d3 in normoxic conditions . with these fundamental environmental differences between chondrocytes and osteoblasts , it is not surprising that oa elicits specific responses from each tissue . oa disease progression is most frequently characterized by a net loss of cartilage matrix that results from an imbalance between cartilage matrix degradation and synthesis by chondrocytes in the cartilage . progressive chronic destruction of articular cartilage is the most obvious characteristic of oa , and the etiology of the disease is believed to be at the intersection of genetics and abnormal mechanical forces . therefore , the primary locus of the disease is traditionally presumed to be the cartilage , and as a result , most in vitro oa models focus exclusively on cartilage to study oa disease mechanisms and therapeutic intervention . however , there is increasing evidence from in vivo and clinical studies that subchondral bone lesions may precede cartilage degeneration , implying that oa is an osteochondral disease and possibly bone dependent . in addition , it has been often reported that the health of mature articular cartilage in vitro is positively impacted by the presence of subchondral bone . despite these observations , most in vitro oa research has not taken into account the effects of bone - cartilage interactions , focusing primarily on cartilage alone . this may account for the dearth of new therapeutics for the prevention and treatment of oa . we theorize that the development of a model system of osteochondral tissue using human cells in a physiologically relevant environment that can accurately replicate in vivo osteochondral tissue homeostasis and pathophysiology will lead to greater predictive power in the development of dmoads . the challenges in developing such a system include : ( 1 ) mimicking or inducing production of appropriate extracellular matrix critical to the function of cartilage and bone , ( 2 ) replicating the tissue architecture , ( 3 ) reconciling the different growth and maintenance conditions of bone and cartilage while promoting their interaction with each other , and ( 4 ) replicating the biomechanical environment known to be essential to cartilage and bone health . current in vitro models to investigate bone - cartilage interactions are mostly limited to cell co - culture systems in which bone and cartilage cells are both exposed to the same medium , arguably a very distant condition from the in vivo environment . here , we report the development of a bioreactor designed to accommodate the biphasic nature of an osteochondral plug by creating two separate compartments for the chondral and osseous microenvironments . these are separated only by the tissue itself and are supplied by a microfluidic system . the two microenvironments can be independently controlled and regulated via introductions of bioactive agents or candidate effecter cells , and the medium can be individually sampled for compositional assays . the central hypothesis of the study is that a gradient of tissue specific nutrients and conditions is required for the formation and maintenance of the osteochondral tissue . furthermore , we hypothesize that induction of an oa - like condition in the engineered osseous or the engineered chondral component alone will induce a corresponding oa - like response in the other component . to test these hypotheses , we have generated distinct chondral and osseous zones within the same construct by controlling the different media exposures within the bioreactor . then , we induced an oa - like response by exposing the osseous or chondral compartments to the pro - inflammatory cytokine ( il-1 ) and assayed the intervening changes in expression and secretion from both the engineered chondral and osseous components . all chemicals used in this study were purchased from sigma - aldrich ( st . louis , mo ) unless stated otherwise . the 3d structures of the bioreactor ( figure 1 ) were modeled using magics 14 ( materialise , belgium ) . the chamber and insert were fabricated using a stereolithography apparatus ( envisiontec , germany ) employing e - shell 300 as the resin . ( a ) an individual bioreactor composed of the removable insert ( dark gray ) within a chamber ( light gray ) of the microfluidic plate ( b ) and fixed in place with two o - rings . the osteochondral construct within the insert creates the final separation between the upper and lower medium conduits . opposing gradients of chondrogenic and osteogenic factors and stimulants will aid in forming an interface . ( b ) a single bioreactor formed by the inset and lid in the context of a 24-well plate . red circles indicate the o - rings that seal the joint space between lid / insert and chamber . hbmscs were isolated from the femoral heads of patients undergoing total joint arthroplasty with irb approval ( university of pittsburgh ) , cultured and expanded as previously described ( caterson , 2002 ; song , 2004 ) . briefly , bone marrow was flushed out from the trabecular bone of the femoral neck and head using an 18-gauge needle and resuspend in dulbecco s minimal essential medium ( dmem ) . the suspension was filtered through a 40 m strainer and the flow - through was centrifuged at 300 g for 5 min . after the supernatant was discarded , the pellets were suspended using growth medium ( gm , -mem containing 10% fetal bovine serum ( fbs , invitrogen ) , 1% antibiotics - antimycotic , and 1.5 ng / ml fgf-2 ( raybiotech , norcross , ga ) ) , and then plated into 150 cm tissue culture flasks at a density of 20,00040,000 nucleated cells / cm , and medium was changed every 3 to 4 days . the colony formation and trilineage mesenchymal differentiation capacity of hbmscs was validated before use ( data not shown ) . all experiments were performed with passage 3 ( p3 ) hbmscs from 3 patients ( 3 female patients 44 , 52 , and 72 years old ) , which were pooled for use in this study . the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate ( lap ) was synthesized as described by fairbanks et al . mgl was synthesized by reacting gelatin with methacrylic anhydride ( ma ) in water according to a procedure previously described . mha was prepared as previously reported using sodium hyaluronate powder ( research grade , mw 66 kda , lifecore ) . both mgl and mha were lyophilized and stored in a desiccator for future use . to test medium leakage between ( 1 ) the chamber wall and the insert and ( 2 ) the insert and scaffold material , the insert was filled with 10% mgl/0.15% lap in hbss and alexa fluor 488-conjugated soybean trypsin inhibitor ( ti488 , 21kd , molecular probes , ca ) and alexa fluor 555-conjugated albumin from bovine serum ( bsa ) ( bsa555 , 65kd , molecular probes ) were diluted in hbss individually at 10 g / ml and then perfused through the top and bottom of bioreactor , respectively , at 1 l / min . at different time points , effluent from the upper and lower medium conduits was collected and the fluorescence intensity at both wavelengths measured using a microplate reader ( synergy ht , biotek , winooski , vt ) . leaking between top and bottom conduits was estimated by the ratio of ti488 ( bottom)/ti488 ( top ) and bsa555(top)/bsa555 ( bottom ) . because of the permeable nature of gelatin scaffold used as the scaffold model , leaking was assayed for 24 h only . p3 hbmscs were pelleted and drained completely in order to prevent the unwanted dilution of polymers . chondrogenic cell suspension : hbmscs were resuspended in 10% mgl/1%mha/0.15% lap ( w / v ) hbss solution ( ph adjusted to 7.4 ) at a final density of 20 10/ml ( chondrogenic suspension ) . hbmscs were resuspended in the 10% mgl/1% hydroxyapatite/0.15% lap ( w / v ) hbss solution ( ph was adjusted to 7.4 ) at a final density of 20 10/ml . osteochondral construct preparation : first , the insert was placed within a hollow cylindrical well to prevent suspension leaking from the pores in the insert . second , 60 l of osteogenic suspension was pipetted into the insert and cross - linked using the uv light source . after 2 min of uv light exposure , the inset with photopolymerized osseous construct was removed from the chamber . third , 30 l of chondral suspension was added on the top of osseous construct within the same insert and cured for another 2 min . previous studies have shown cell viability within the scaffold > 90% after photo - cross - linking ( data not shown ) . the second round of cross - linking had the added benefit of bonding the osseous and chondral layers together as well , and the fabrication of the osteochondral construct within the insert created a tight seal . the inserts with nave osteochondral constructs were placed into the microfluidic plate as shown in figure 1 . chondrogenic medium ( cm ) was supplied through the upper conduit , while osteogenic medium ( om ) through the bottom conduit at a flow rate of 1 l / s . the following formulas were used for the differentiation media : om ( gm supplemented with 10 ng / ml bmp-2 ( peprotech , rocky hill , nj ) , 1% l - alanyl - l - glutamine ( glutamax ) , 10 nm dexamethasone ( dex ) , 0.1 mm l - ascorbic acid 2-phosphate ( asa2-p ) , and 10 mm beta - glycerophosphate ( -gp ) ; cm ( dmem supplemented with 10 ng / ml tgf-3 ( peprotech ) , 1% its , 50 m asa2-p , 55 m sodium pyruvate , and 23 m l - proline ) . the perfusion rate was 1 l / min , and used syringes were replaced with syringes and new medium every 3 days . after 4 weeks of differentiation , engineered osteochondral tissues were collected for validation using real - time pcr and histological analysis , or treated with il1 - 1. chondral and osseous constructs were collected separately . to avoid the potential contamination , total rna was extracted using trizol ( invitrogen ) following the standard protocol and purified with the rneasy plus mini kit ( qiagen , hilden , germany ) . superscript iii kit ( invitrogen ) was utilized with random hexamer primers to complete the reverse transcription . real - time rt - pcr was performed using the steponeplus thermocycler ( applied biosystems , foster city , ca ) and sybr green reaction mix ( applied biosystems ) . sox 9 , aggrecan ( acn ) , collagen type ii ( col2a1 ) , runx2 , osteocalcin ( ocn ) , and bone sialoprotein ( bsp ii ) expression were analyzed , and primer sequences are listed in table 1 . monolayers of hbmscs cultured in gm on 2d tissue culture plastic were used as negative controls . transcript level of 18s rrna was used as endogenous control , and gene expression folder changes were calculated using the comparative ct ( ct ) method . aggrecan ( acn ) , collagen type ii ( col2 ) , runt - related transcription factor 2 ( runx2 ) , osteocalcin ( ocn ) , bone sialoprotein ii ( bsp ii ) , and 18s rrna ( 18s ) . intact engineered osteochondral tissues were fixed in 10% neutral buffered formalin ( fisher scientific , pittsburgh , pa ) for 7 days , dehydrated , embedded in paraffin with 10 m sections cut from each sample . safranin o / fast green and alizarin red staining were used to detect the gag and calcium deposition , respectively . after 4 weeks of differentiation , engineered osteochondral constructs were treated with il-1 ( 10 ng / ml , r&d ) on the chondral or osseous sides only to investigate the cell / neo - tissue response to pro - inflammatory cytokines and possible communication through the osteochondral construct . the media used in this test were cm without tgf-3 ( chondral ) and om without bmp-2 ( osseous ) , both supplemented with 10 ng / ml il-1. there were three experimental groups : ( 1 ) cm / om , ( 2 ) cm + il1 - 1/om , and ( 3 ) cm / om + il1 - 1. the treatment lasted 7 days , with effluent medium collected and frozen at 1 and 7 days for elisa . after 7 days , the osteochondral constructs were bisected into the chondral and osseous halves and processed for gene expression analysis as described before . in addition to tissue specific gene expression , matrix metalloproteinase 1 , 3 , and 13 were also analyzed . media was collected separately from chondral and osseous constructs , cleared of cell debris via centrifugation ( 1000 g ) , and analyzed via il-1 ( abcam , cambridge , ma ) , mmp-1 ( r&d ) , mmp-3 ( abcam , cambridge , ma ) , and mmp-13 ( abcam ) elisas according to the manufacturers instructions . significant differences were determined with anova followed by a bonferroni post hoc analysis for multiple group comparisons using spss statistics 21 ( ibm , armonk , ny ) . significance was determined at p < 0.05 ( * ) and p < 0.01 ( * * ) . the robustness of the microbioreactor was tested by assessing the extent of leakage of two molecules perfused independently in the upper and lower medium conduits : ( 1 ) trypsin inhibitor ( 21 kda ) , with a molecular weight similar to the two commonly used osteoinductive ( bmp-2 , 26kd ) and chondroinductive ( tgf3 , 25kd ) factors , and ( 2 ) bsa ( 65kd ) , the most abundant protein in serum . as shown in figure 2a , after 24 h of perfusion , the extent of mixing between top and bottom was < 1% , indicating there was minimal medium exchange through the interfaces between the chamber wall and the inset and between the inset and scaffold - only construct . these results were further confirmed by elisa assay for il-1 in both medium conduits during the il-1 test ( figure 2b ) . ( a ) trypsin inhibitor-488 and bsa-555 were simultaneously perfused through the top and bottom space of bioreactor , respectively , and the percent leakage at different time was estimated based on the bottom / top ratio of 488 nm fluorescence readings ( bottom / top 488 ) and top / bottom 555 nm fluorescence readings ( top / bottom 555 ) . ( b ) il-1 was included in top or bottom stream and perfused for 24 h. its concentration in top or bottom medium was then measured . the naive hbmscs seeded within tissue - specific scaffolds in the freshly fabricated osteochondral construct were induced to differentiate using cm in the top stream and om in the bottom stream . we anticipated chondrogenesis in the upper , chondral half of the construct and osteogenesis in the bottom , osseous half . after 4 weeks of differentiation , biphasic osteochondral constructs were produced ( figure 4c ) . as shown in figure 3 , cells in the chondral half showed enhanced expression of chondrogenic genes , including sox 9 , aggrecan , and collagen type ii as compared to those in the osseous half , while the cells in the osseous half had higher expression of osteogenic genes , including runx2 , osteocalcin , and bsp ii . monolayers of hbmscs cultured in gm on 2d tissue culture plastic were used as negative controls . histological staining with alcian blue / alizarin red revealed high matrix gag content in the upper chondral half than the osseous half ( figure 4b ) ; although the amount of calcium deposition in the osseous half was not detectable . taken together , these results strongly indicate a spatially defined , biphasic differentiation of these engineered osteochondral constructs , with the chondral component undergoing more characteristic differentiation . in addition , h&e staining revealed a distinct , < 100 m wide basophilic band in the interface between the chondral and osseous halves , potentially indicative of a developing tidemark ( figure 4a ) . after 4 weeks of culture in the bioreactor , osteochondral constructs were separated into chondral ( cartilage ) and osseous ( bone ) components , and each were analyzed for expression of cartilage ( sox9 , col2 , and aggrecan ) or bone ( runx2 , osteocalcin , bspii ) markers . expression levels are normalized to 18s rrna and then to corresponding 2d control expression levels . expression of cartilage markers was found only in the chondral component , and bone markers in the osseous compartment . * p < 0.05 ; * * p < 0.01 ) . top , chondral component ( cc ) ; bottom , osseous component ( oc ) . ( a ) alizarin red staining ; ( b ) safranin o / fast green staining . dashed lines indicate the border between cc and oc . as described above , the microtissue bioreactor presented here , with its two separate medium flow systems and biphasic construct compartments , has the capability for targeted treatment of one ( or both ) tissue construct(s ) with soluble factors . osseous and chondral components were separately treated with the pro - inflammatory cytokine il-1 ( 10 ng / ml ) for 7 days ( control conditions consisted of untreated osteochondral constructs ) , and the responses of each of the two components were separately analyzed . media samples were collected from chondral and osseous components streams at days 1 and 7 , and after day 7 , the osteochondral constructs were separated into osseous and chondral components , and each was separately analyzed for gene expression of catabolic genes ( mmp-1 , mmp-3 , and mmp-13 ) and either cartilage markers ( sox9 , col2 , and aggrecan ) ( figure 5 ) or bone markers ( runx2 , osteocalcin , and bspii ) ( figure 6 ) . media samples from the chondral and osseous components stream collected at days 1 and 7 were analyzed via mmp-1 , mmp-3 , and mmp-13 elisas ( figure 7 ) . effects of il-1 treatment on cartilage gene expression in the engineered osteochondral microtissue . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 7 days , cartilage components were analyzed for the expression of cartilage markers and mmps . expression levels were normalized to 18s rrna expression and then to corresponding gene expression under control conditions . * p < 0.05 ; * * p < 0.01 . effects of il-1 treatment on bone gene expression in the engineered osteochondral microtissue . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 7 days , bone components were analyzed for expression of bone markers and mmps . expression levels were normalized to 18s rrna expression and then to corresponding gene expression under control conditions . effects of il-1 treatment on osseous and chondral mmp secretion in the engineered osteochondral microtissue . after treatment of either osseous ( bone ) or chondral ( cartilage ) component with 10 ng / ml il-1 for 1 or 7 days , medium samples collected from the bone or cartilage medium compartment were analyzed by elisa for the levels of secreted mmp-1 , mmp-3 , and mmp-13 . values were normalized to those measured under control conditions , which involved untreated osteochondral constructs . treatment of chondral constructs with il-1 caused decreases in expression of cartilage genes sox9 , col2 , and aggrecan , consistent with physiological outcomes of damaged or stressed cartilage ( figure 5 ) . chondral construct expression of these genes also decreased in response to il-1 treatment of osseous constructs , suggesting signaling between the osseous and chondral components . evidence of this osseous - to - chondral communication was even more apparent in results concerning expression of catabolic genes ; expression of mmp-1 , mmp-3 , and mmp-13 of the chondral constructs increased substantially in response to il-1 treatment of the osseous component . crosstalk between the two components was also detected in the case of chondral - to - osseous communication ( figure 6 ) . il-1 treatment of the chondral construct caused decreases in expression of the bone genes osteocalcin and bspii and increases in mmps production in the osseous construct , particularly mmp-13 , which is one of the most important mediators of oa cartilage degradation . elisa analysis of mmps secreted by the chondral and osseous components at different time points allowed for observations on the rate of signal propagation between the two components ( figure 7 ) . for example , the chondral construct responded to il-1 treatment of the osseous component with increases in mmp-1 , mmp-3 , and mmp-13 secretion . the chondral mmp-13 response occurred quickly , within 1 day , while the chondral mmp-1 response took 7 days . the chondral mmp-3 response time was intermediate between those of mmp-13 and mmp-1 . again , these results are interesting considering the central role mmp-13 plays in cartilage degeneration . the osseous construct response to treatment of chondral component with il-1 , however , was quick yet increased further over time , and by day 7 was overall stronger than the chondral responses to the osseous component treatment . it is worth noting that gene expression and protein levels of mmps should not be expected to be necessarily consistent . this stems from differences in the ways in which elisa and real - time pcr samples were collected and measured . elisa samples consisted of culture media conditioned by cells for 24 h and were collected at day 1 or day 7 for each experiment . the proteins contained in day 1 samples were secreted between days 0 and 1 , and day 7 samples contained proteins secreted between days 6 and 7 . pcr samples , however , were collected after 7 days of cultures and represent the expressional activities taking place at the moment of collection . in other words , the mrna levels analyzed by pcr at day 7 are not necessarily totally reflective of the protein levels analyzed by elisa in day 7 conditioned media samples . this disconnect between pcr and elisa measurements may be more pronounced in mmps , which need to be translated , secreted , and then diffuse out of the 3d construct before they are detected by elisa . furthermore , differences between pcr and elisa values also arise from differences in normalization . pcr results are normalized to 18s rrna expression , thereby taking into account cell number . elisa results are instead a representation of the entire culture and normalized to control conditions . thus , any experimental treatment that may affect cell number would have a larger impact on elisa results than pcr results . since chondrocytes are particularly sensitive to il-1 , this may explain why results concerning cells of the chondral component exhibit the greatest degree of inconsistency between pcr and elisa measurements when chondral constructs are directly stimulated by il-1. in this study , we have developed a novel bioreactor system for the engineering of osteochondral tissue . rt - pcr and histological analyses showed that hbmscs - derived nave constructs have been successfully differentiated into cartilage - like tissue on the top and bone - like tissue on the bottom , using separated culture medium for 4 weeks . a transition layer between 2 tissues is also observed . our results show that il-1 exposure decreases the ecm anabolic gene expression but greatly enhances the levels of mmp expression and secreted amount into the medium . interestingly , the il-1 insulted osseous construct induces a catabolic gene expression response into the untreated chondral component , which is not due to leakage of il-1 , suggesting active osseous chondral interaction and the likely importance of bone injury in oa development . in this study , a dual - chamber bioreactor has been developed to generate and maintain osteochondral constructs derived from human hbmscs . the design parameters included individual compartments to separate the chondral and osseous microenvironments that are individually accessible for the introduction of bioactive agents and/or candidate effector cells , tissue and medium sampling , and compositional assays , including noninvasive imaging techniques . furthermore , the total dimension and geometry of the bioreactor matches that of a multiwell culture plate chamber for the development of medium- to high - throughput analysis . validation of the system included successful , simultaneous differentiation of osseous and chondral constructs from hbmscs from the same source ( pooling of three donors ) and subsequent application of il-1 , a potent inflammatory mediator implicated in oa pathophysiology , to test the physiological response of the osteochondral construct . we have shown that in the course of 6 weeks , hbmscs undergo tissue - specific differentiation in response to the tissue specific growth media and hydrogel composition provided . the differentiating bbmscs expressed tissue - specific transcription factors and ecm molecules , as shown by rt - pcr and histological staining . most impressively , there was an indication of a basophilic , tidemark - like zone separating the chondral and osseous components . the recreation of the a tidemark - containing biphasic tissue is vital to drug testing using an osteochondral organotypic culture since changes in the ocj are mechanistically involved in oa progression and likely to be a target of toxicants and dmoads . we subsequently tested the response of the msc - based osteochondral tissue to il-1. the test served two purposes : ( 1 ) to validate the utility of the bioreactor in osteochondral studies and ( 2 ) to assess the physiological replication of the oc tissue by the mscs in this bioreactor . il-1 is almost ubiquitous in inflammatory diseases , is prominent in advanced oa in both the cartilage and synovial lining , and is frequently employed as a pathogenic initiator in in vitro models of oa . application of il-1 to both osseous and chondral components results in clear matrix degeneration and phenotypic changes in the resident cells , similar to what has been observed in monocultures of chondrocytes and osteoblasts . while chronic degeneration of the articular surface is most prevalent in oa , it is not clear whether alterations in the subchondral bone or articular cartilage is the primary trigger in oa . using the bioreactor in this study , we are able to study interactions between cartilage and bone that may contribute to oa progression clear osseous and chondral tissue interactions are observed when il-1 is applied to the osseous component , which results in low levels of anabolic gene expression ( sox9 , col2 , and aggrecan ) but robust expression of mmps in the cartilage component . conversely , application of il-1 to the cartilage induces in bone low levels of anabolic bone gene expression ( runx2 , opn , and bspii ) but robust expression of mmps . this apparently contradictory simultaneous induction of anabolic and catabolic processes within a tissue is entirely in keeping with the hypothesis that oa begins initially with a shift in the balance between anabolic and catabolic activities , followed by phenotypic changes in the cells in response to the modified environment . to some degree , inflammation can have beneficial effects on tissues , but at higher concentrations , inflammatory mediators induce tissue remodeling / destruction . focusing on the response of chondral component to il-1 treatment of the osseous component , it is interesting that direct application of il-1 to the chondral component has a less impressive catabolic response than indirect exposure via the osseous component . this result implies that the affected osteoblasts in the osseous component are producing bioactive factors , in addition to il-1 , that are causing greater catabolic responses than il-1 itself and vice versa . the formation of an ocj between regions of engineered cartilage and bone is often reported , but generally is not analyzed beyond histological identification . in our study , we have employed opposing chondrogenic and osteogenic nutrient gradients to stimulate ocj formation by nave , differentiating mscs . it is thus difficult to relate the potential tidemark development in our construct with frequently reported constructs that combine solid , porous polymeric sponges and hydrogels for osteochondral engineering in vitro or in vivo because of the great disparity in tissue architecture and scaffold biochemistry . tidemark development similar to what is seen here has been reported in studies employing microparticle - mediated spatially restricted growth factor release , microbead - encapsulated msc - derived chondrocytes and osteocytes , mscs encapsulated within scaffold material gradients , and mscs stimulated by growth factor gradients . in all cases , the basophilic tidemark is indistinct and broad , particularly in models without loading . we expect that appropriate mechanical loading and enhancement of cell differentiation , e.g. , with an oxygen gradient , may enhance the collection of metabolites to form the tidemark at the deep zone / calcified cartilage interface , particularly if differentiation is enhanced with an oxygen gradient and/or the addition of hydroxyapatite . while formation of ocj has been reported in in vivo implanted cell - seeded scaffold , there have been relatively few studies using a controlled bioreactor as reported here . the features of our bioreactor design , including separate compartments for the chondral and osseous microenvironments supplied by independent tissue - specific media that can be controlled and regulated via introductions of bioactive agents or candidate effecter cells , and capability of individual sampling of the different compartments , are thus of potential value in allowing more individual manipulations . specifically , we envision its application for the assessment of drug and environmental factor toxicity . we have postulated that catabolic insults to one tissue component comprising the osteochondral unit would influence the other in a manner reminiscent of tissue degeneration in oa . of particular interest is to investigate the communication of biomechanical signals / forces . to our knowledge , we are the first to provide evidence of communication between different compartments of an osteochondral construct in response to catabolic cues ( il-1 ) . the tissue responses reported here reflect a subset of pathophysiological conditions reported in in vitro and in vivo models of oa . il-1 is utilized in models of both rheumatoid and oa , with the effect of causing cartilage matrix breakdown and down regulation of cartilage matrix gene expression as shown here . in contrast , il-1 has been reported to induce increased bone matrix deposition , although it is a matrix of inferior quality , which may explain the response of the osseous component to direct exposure to il-1 in our model . the fact that catabolic gene expression in the osseous component is more enhanced by exposure of the overlying chondral component to il-1 suggests that the chondral construct is producing additional signals and catabolic factors that travel to and affect the osseous component below , possibly constituting a form of intercellular communication not previously reported . in summary , we have fabricated a new microfluidic - based , multichamber bioreactor for osteochondral differentiation and toxicity testing . we demonstrated clear biphasic tissue differentiation in response to opposing chondrogenic and osteogenic gradients produced by tissue - specific differentiation factors supplied by independent medium streams to the chondral and osseous components of the construct . finally we have shown that msc - based chondral and osseous tissues are capable of responding to il-1 in a relevant manner and that changes in one tissue compartment are communicated , and perhaps amplified , to the other along the osteochondral axis . this bioreactor / organotypic osteochondral culture combination will enable us to focus on the relationship of cartilage and bone in growth and degeneration and perhaps help to elucidate the roles of each tissue in oa . finally , with minimal modification and appropriate coupling , the bioreactor reported here can be adapted as a tissue - specific component of an interacting multitissue bioreactor platform to study systemic multitissue interactions .

osteoarthritis ( oa ) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes . an engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of oa and testing the efficacy of potential disease - modifying oa drugs ( dmoads ) . in this study , a multichamber bioreactor was fabricated and fitted into a microfluidic base . when the osteochondral construct is inserted , two chambers are formed on either side of the construct ( top , chondral ; bottom , osseous ) that is supplied by different medium streams . these medium conduits are critical to create tissue - specific microenvironments in which chondral and osseous tissues will develop and mature . human bone marrow stem cell ( hbmscs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue - specific media . we observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark . introduction of interleukin-1 ( il-1 ) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type . for example , il-1 treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase ( mmp ) expression and activity , and tissue - specific gene expression . this induction was greater than that seen with il-1 application to the chondral component directly , indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of oa . the microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of oa and serving as a high - throughput platform to test potential dmoads .

Introduction Experimental Section Results Discussion

post - translational modifications of the n - terminal tails of core histones , including methylation , acetylation , ubiquitylation and phosphorylation , influence chromatin configuration , which can modulate accessibility of transcription factors and transcriptional activity of nearby genes 1 . four lysine residues in the n - terminal tail of histone h3 , k4 , k9 , k27 and k36 , are primary targets of specific histone methyltransferases and demethylases . methylated h3k27 is abundant , and three different statues ( mono- , di- or tri - methylation ) exist . for example , based on quantitative mass spectrometry 50% of h3k27 in embryonic stem ( es ) cells is dimethylated , 15% trimethylated and 15% monomethylated 2 . di- and tri - methylation are characteristic of pcg ( polycomb group ) target genes and are associated with transcriptional repression 3 . prc1 catalyzes the monoubiquitylation of histone h2a and prc2 contributes to the methylation of h3k27 4 . prc2 is composed of several proteins , including eed , suz12 , rbap46/48 and either ezh1 or ezh2 . ezh1 and 2 are characterized by a set domain , which functions as a histone - lysine n - methyltransferases 5 - 8 . while prc2 complexes containing ezh2 are primarily responsible for h3k27 methylation , prc2 complexes containing ezh1 can also catalyze h3k27 methylation 9 , or compact chromatin through mechanisms other than histone methylation . experiments with cell - specific ezh2 knockout mice have demonstrated that ezh2 influences proliferation and differentiation of stem cells and progenitor cells in three distinct cell types , adipocytes , keratinocytes , and neurons 11 - 13 . experimental evidence from several systems suggests that h3k27 methylation is mainly achieved by ezh2 and to a lesser extent by ezh1 . the critical importance of ezh2 is reflected by the fact that ezh2-null mice die in utero , while ezh1-null mice have no overt defects , possibly due to compensatory functions of ezh2 . however , ezh1 can also compensate to some extent for ezh2 as loss of both ezh1 and 2 evoke more severe consequences than loss of ezh2 alone 14 . in addition , contribution to h3k27 monomethylation by g9a , a well - known h3k9 methyltransferase , was observed in vitro and in vivo 15 , 16 . although no changes in h3k27 di- and tri - methylation were observed between wild type and g9a es cells , the extent of monomethylation was significantly decreased in g9a es cells . it is possible that g9a introduces a monomethyl group on h3k27 in the absence of ezh2 and that this is sufficient the binding by embryonic ectoderm development ( eed ) , a component of the prc2 complex , which contains the methyl - lysine histone - binding domain wd40 . jmjd3 ( kdm6b , [ jumonji - domain containing protein 3 ] ) and utx ( kdm6a [ ubiquitously transcribed tetratricopeptide repeat gene on the x chromosome ] ) are demethylases acting on h3k27 ( figure 1b ) . they remove the gene - inactivating h3k27 di - methyl and tri - methyl marks and thereby presumably contribute to the maintenance of gene expression . abberant of utx and jmjd3 levels have been associated with cancer and defective differentiation programs 17 . excessive ezh2 concentrations have been reported as a marker of aggressive breast cancer 18 , 19 and associated with invasion and cancer progression . chinnaiyan and colleagues tested ezh2 protein levels in 280 breast cancer patients using high - density tissue microarray 20 . ezh2 levels were elevated in patients with invasive breast carcinoma relative to normal or atypical hyperplasia . notably , increased expression was already observed in ductal carcinoma in situ ( dcis ) , a precursor of invasive carcinoma 21 . elevated ezh2 concentration has been associated with increased tumor cell proliferation in various cancers including breast , prostate and melanoma 22 . the two - layered regulatory loop includes transcriptional regulation of the ezh2 gene and ensuing consequences of associated h3k27me3 ( figure 2 ) . e2f , a target of the retinoblastoma protein ( prb ) , plays a critical role in regulating cell cycle progression through activating genes that control entry into the s phase and genes associated with dna replication 23 . upon phosphorylation of prb , activated e2f binds to promoter regions of ezh2 and eed , and controls their expression . however , helin and colleagues confirmed that the interrupt of ezh2 and eed expression was not enough to increase expression of negative regulators of cell cycle including p14arf , but positive regulators of cell proliferation , cyclind1 ( ccnd1 ) , cycline1 ( ccne1 ) , cyclina2 ( ccna2 ) and cyclinb1 ( ccnb1 ) , significantly decreased in ezh2 lacking cells . they concluded that the prc complex , including ezh2 , is required for the activation or maintenance of the activated state of certain genes in proliferating cells through h3k27 methylation 24 . ezh2 expression is also controlled by hypoxia through hif response elements ( hre ) in the ezh2 gene promoter 25 . in hypoxic microenvironment , lastly , mek / erk / elk pathway can also leads to ezh2 overexpression in erbb2-overexpressing breast cancer cell lines 26 . a set of genes associated with cell proliferation and invasion is regulated by ezh2 . among them rad51 , runx3 , and cdkn1c ( p57 ) regulate cell proliferation , and foxc1 and cdh1 ( e - cadherin ) have been linked to metastasis . the runt - related ( runx ) family is associated with normal development and neoplasia and runx3 is an established tumor suppressor gene in gastric cancer 46 . inactivation of runx3 expression through dna hypermethylation has been reported in various cancers , including those of prostate , lung and pancreas . notably , upon reducing ezh2 expression in the mcf-7 breast cancer cell line , runx3 expression was recovered . it has been suggested that runx3 down - regulation is controlled by h3k27me3 through ezh2 and histone deacetylation through hdac1 . even though dna methylation affects repression of runx3 , h3k27me3 by ezh2 has a critical role in down - regulation of runx3 . runx3 reduction leads to the decrease of the cyclin - dependent kinase inhibitor p21expression , which results in the induction of cell proliferation in breast cancer 28 . foxc1 , a member of the forkhead box transcription factor family , plays an important role in differentiation . increased ezh2 influences transcriptional repression of foxc1 through the accumulation of h3k27me3 and diminished acetylation of h3/h4 in mda - mb-231 cell lines . elevated foxc1 concentrations lead to reduced migration and invasion in vitro and in vivo 29 . the cyclin - dependent kinase ( cdk ) inhibitor , cdkn1c ( p57 ) encodes the tumor suppressor p57 protein . reduced cdkn1c expression in various breast cancer cell lines was associated with ezh2 overexpression and increased h3k27me3 30 . upon treatment with the histone methylation inhibitor 3-deazaneplanocin a ( dznep ) , recovery of cdkn1c expression was observed suggesting a direct link between h3k27me3 and transcriptional suppression . the transmembrane receptor e - cadherin maintains epithelial cellular adhesion and integrity and it has been proposed that its down - regulation leads to enhanced invasion in cancer . in ezh2-overexpressing breast epithelial cells , reduced cdh1 ( e - cadherin ) expression correlated with h3k27me3 levels in the promoter . however , elevated expression of ezh2 was not sufficient to increase enzymatic activity for h3k27me3 at the cdh1 promoter , which also required hdac activity . increased hdac activity leads to the removal of acetyl groups from h3k27 , a prerequisite for ezh2-controlled histone methylation . increased ezh2 induces reduction of cdh1 expression , and it leads to metastasis and invasion in breast cancer as well as pancreatic cancer and ovarian cancer 31 - 33 . ezh2 also appears to regulate breast tumor initiating cells ( btics ) through suppressing rad51 expression . pho and gaga motifs , which are targets for prc2 complexes , are located in the rad51 promoter and h3k27me3 coincides with these sites . reduction of rad51 affects chromosome breaks , deletion and translocation as well as increase of centrosome number and genome instability through activated raf / perk/ - catenin signaling 25 . mutations in brca1 are a cause of basal like breast carcinomas that are er , pr and her2-neu negative ( triple negative ) and increased ezh2 concentrations were found mainly in basal type breast carcinomas 34 . germline brca1 mutations predispose women to develop breast cancer , and patients have a poor prognosis , in part because brca1 's role in dna repair and genomic stability 35 . even though the majority of basal breast cancers do not carry brca1 germline mutations , reduced concentration of brca1 protein and mrna levels were reported in these patients 36 . however , the mechanisms causing decreased brca1 expression in basal like carcinomas remains to be established . ezh2 is frequently overexpressed in er - negative breast cancer cell lines , and a reduction of ezh2 results in suppressed cell proliferation . biological effects of ezh2 knockdowns were observed in vivo and in vitro system using xenograft transplantations . reduced tumor growth was confirmed in xenograft mouse models injected with ezh2 knockdown cells 37 . ezh2 overexpression inhibits brca1 phosphorylation ( ser1423 ) and thereby promotes an increase of cdc25c , an essential player for g2/m checkpoint control . once cdc25c is activated , it induces the activity of the cdc2/cyclinb1 complex . in summary , elevated ezh2 concentrations in er - negative breast cancer cell lines confer increased cell proliferation in part possibly through the inhibition of brca1 phosphorylation 37 . brca1 is a nuclear protein involved in numerous activities , including dna repair and estrogen receptor modulation . it has been proposed that akt-1 ( ser473 ) , but not akt-2 and 3 , are phosphorylated and activated by ezh2 in the ezh2 overexpressing breast cancer cell line cal51 , and nuclear location of brca1 protein is suppressed in these cells by a yet to be determined mechanism ( figure 3 ) . this in turn results in aberrant mitoses with extra centrosomes , and genomic instability 38 . elevated ezh2 concentrations were observed in mouse mammary epithelium from which the brca1 gene had been deleted . as a result gene expression related to differentiation was suppressed and excessive cell proliferation was promoted . it was confirmed that brca1-deficient cells are more sensitive to dznep , a potential treatment , when compared with brca1-proficient cells 39 . genome - wide approaches have been applied to identify genomic localizations and target genes of prc complexes in human and mouse es cells . this system has been explored using a combination of chromatin immunoprecipitation ( chip ) , chip on chip and microarray analyses 40 - 43 . transcription factors related with developmental regulation , including homeobox protein ( hox ) , gata binding protein ( gata ) , and runt related transcription factor ( runx ) are regulated by the prc2 complex in human es cells . these include genes encoding the transcription factor families fox , sox , gata and tbx 40 . moreover , enriched h3k27me3 was found in proximal promoter regions of these genes , which were also derepressed in suz12- and eed - null cells 40 , 42 supporting the notion that their suppression is dependent on the prc complex . chip - seq analyses with antibodies against ezh2 , ring1b , h3k4me3 and h3k27me3 have been performed to identify the conservation of chromatin states in orthologous genomic loci in human and mouse es cells 44 . bivalent promoters , which carry h3k4me3 and h3k27me3 marks , are highly conserved between mouse and human es cells . in addition to prc2 , prc1 binding was identified in the above genes . in general , h3k27me3 retention was most pronounced in regions that were occupied by both prc1 and 2 , while poor retention was detected on sequences occupied by prc2 only . these findings suggest that prc2 catalyzes and regulates h3k27 methylation but is not sufficient to maintain the methylation status of respective target genes . genome - wide approaches have been used to predict the importance of the prc2 complex in the regulation of developmental transcription through h3k27 methylation . however , it is not clear what concentration of ezh2 is required to regulate h3k27 methylation , and whether excess ezh2 has other functions besides its methyltransferase activity . the suggestion that excess ezh2 , and thereby altered histone modifications , leads to a changed epigenetic genomic landscape , and thereby contributes to cancer progression , is mainly based on correlative studies and cell culture investigations . although biochemical studies are capable of linking elevated ezh2 concentrations with altered genetic programs in cancer , clear genetic evidence is scarce . it will be necessary to conduct decisive genetic experiments in mice to link ezh2 to the h3k27 methylation status and subsequent genetic programs in relevant breast cancer models , such as mice carrying mutant brca1 45 . it will be necessary to explore breast cancer initiation and progression in relevant mouse models in which h3k27 methylation is disrupted . by ablating the ezh1 and ezh2 genes in mammary epithelium the mammary genome will lack h3k27me3 marks and upon deletion of the utx and jmjd3 genes a persistent h3k27me3 should be expected . such experiments , although labor intensive and time - consuming , shall provide definitive answers on the role of h3k27 methylation in development and the etiology of cancer .

histone modifications are thought to control the regulation of genetic programs in normal physiology and cancer . methylation ( mono- , di- , and tri - methylation ) on histone h3 lysine ( k ) 27 induces transcriptional repression , and thereby participates in controlling gene expression patterns . enhancer of zeste ( ezh ) 2 , a methyltransferase and component of the polycomb repressive complex 2 ( prc2 ) , plays an essential role in the epigenetic maintenance of the h3k27me3 repressive chromatin mark . abnormal ezh2 expression has been associated with various cancers including breast cancer . here , we discuss the contribution of ezh2 and the prc2 complex in controlling the h3k27 methylation status and subsequent consequences on genomic instability and the cell cycle in breast cancer cells . we also discuss distinct molecular mechanisms used by ezh2 to suppress brca1 functions .

Transcriptional regulation modulated by the H3K27 methylation status EZH2 and human breast cancer Dynamic signaling regulated by EZH2 A BRCA1-EZH2 connection Genome-wide approaches to explore PRC Moving forward using mouse genetics

alkaptonuria is a rare autosomal recessive disease , resulting from deficiency of the enzyme homogentisic acid oxidase , which is involved in the metabolism of phenylalanine and tyrosine aminoacids . due to partial or total deficiency of homogentisic acid oxidase in the liver and kidneys , homogentisic acid accumulates in blood , part being excreted in the urine . on exposure to the atmosphere it is also reported to be deposited in the skin , tympanic membrane , heart valves , sclera , cartilages of nose and ears , kidneys , pancreas , and large vessels . due to accumulation of homogentisic acid , cartilages get a dark discoloration and become brittle and more vulnerable to mechanical stress . the development of degenerative arthritis with accompanying brownish black pigmentation of the tissues and dark discoloration of the urine is called ochronosis . clinical diagnosis is made on the basis of skeletal manifestations including lower back pain , stiffness , and degenerative arthropathy of the large joints , ochronotic pigmentation of the tissues and blueish black discoloration of urine . the definitive diagnosis of the alkaptonuria may be confirmed with several biochemical tests for the quantitative presence of homogentisic acid in urine . since there is no effective medical treatment for alkaptonuria , treatment at present is symptomatic by local heat , physiotherapy , analgesics , external support and with surgical treatment in the form of synovectomy , arthroscopic debridement , fusion , or arthroplasty as indicated regarding the affected joint . we present a patient who was diagnosed to have alkaptonuria during further laboratory tests after observing ochronotic brownish black pigmentation of the menisci and femoral cartilage during arthroscopy of the knee . a 30-year - old woman presented with complaints of pain in the left knee along with occasional episodes of locking for 1 year . her physical examination revealed tenderness on the medial joint line and positive mcmurray 's test . ordinary x - ray images of the knees were found to be normal . magnetic resonance imaging ( mri ) of the left knee revealed bucket handle tear of medial meniscus along with partial tear of anterior cruciate ligament . she was planned for arthroscopic partial meniscectomy in view of her complaints and mri findings . during the arthroscopic examination of her left knee joint there was mild synovial hypertrophy and a bucket handle tear was found in the medial meniscus that appeared to be brownish black in color [ figures 1 , 2 ] . meniscectomy for bucket handle tear of medial meniscus was done and specimen was sent for histopathological examination . excised medial meniscus showing brownish black discoloration excised medial meniscus ( under surface ) further investigations were planned for the diagnosis of ochronosis considering discoloration of the joint cartilage and menisci . the patient 's freshly passed urine turned dark brown after addition of sodium hydroxide to the sample to facilitate oxidation . histopathological examination of the excised tissue showed pigmentation and degenerative changes [ figure 3 ] . no pathological findings were detected with the detailed examination of the ear , sclera , skin , and cardiovascular system . further treatment was done in the form of physiotherapy and by administering vitamin c and analgesics . after the medical and surgical treatment , the patient 's complaints were alleviated and no complaints were registered , during the next follow - up . few reports of arthroscopic diagnosis and treatment of ochronosis have been found in the literature . chen et al . reported the arthroscopic findings of a 50-year - old male patient who was complaining of pain in both knees . reported the arthroscopic findings of a 40-year - old man who had arthroscopy of the knee and shoulder and kural et al . presented a 50-year - old woman in whom arthroscopic examination of the right knee revealed brownish black discoloration of the articular cartilage and menisci leading to the diagnosis of alkaptonuria by further laboratory examinations . generalized synovial hypertrophy accompanying brownish black discoloration of articular cartilage and menisci were our common arthroscopic findings with other reported cases in the literature . generalized cartilage degeneration was not present in our case probably due to comparatively younger age of our patient . in our case there was an additional meniscus tear unlike in that of thacker 's case and similar with chen 's , kural 's and raaijmaaker 's case . similar to raaijmaaker 's case arthroscopy was a diagnostic and treatment tool to remind us of ochronosis and direct us for further research . during arthroscopy , brownish black discoloration of the articular cartilage and menisci warned us to have further research for ochronosis . macroscopic examination of the urine following alkalization and positive ferric chloride test further established our diagnosis . but we were not able to perform the laboratory tests that determine the level of homogentisic acid in urine and blood due to higher cost involved in these tests . approximately 50% of the alkaptonuric patients manifest one clinical form of ochronosis after the fourth decade . diagnosis can be made during childhood by observing dark coloration of diapers or urine when left standing . our patient 's mother had noticed the dark color of her urine in early childhood . ochronotic discoloration of the cartilage of the ears and nose may be observed at the age of 30 . other less common extra articular manifestations are ochronosis of the heart with valve stenosis and urinary tract obstruction by ochronotic calculi . treatment with high dose of ascorbic acid proved to lower the levels of homogentisic acid in excreted urine , but does not prevent the occurrence of arthropathy . nitisinone is shown to have significantly altered the excretion of homogentisic acid in urine in both human and rat studies . nitisinone is suggested to be used for treatment of the tyrosinemia type 1 but its use in ochronotic patients has yet to be proved . since there is no effective treatment to replace the deficient enzyme , symptomatic treatment is initiated with nsaids , intra - articular steroid injection and ascorbic acid . arthroscopic debridement , synovectomy , partial , or total joint arthroplasties and fusion are the surgical treatment options . . raaijmaakers et al . reported to provide symptomatic relief with administering 400 mg / day chondroitin sulfate and 1500 mg / day glucosamine for 4 months . finally , ochronosis is a rare metabolic disorder affecting peripheral joints as well as spine and the soft tissues and arthroscopy has great importance in suspecting and diagnosing ochronosis .

ochronotic arthropathy is a rare condition found in patients with alkaptonuria , which is a hereditary metabolic disease associated with deposition the of homogentisic acid derivatives in various connective tissues of the body . we present the case of a 30-year - old woman in whom arthroscopic examination of the left knee prior to meniscectomy for bucket handle tear of medial meniscus revealed brown - black discoloration of the articular cartilage and menisci leading to the diagnosis of alkaptonuria by further laboratory evaluation . after medical and surgical treatment , patient 's complaints were alleviated and no further complaints were registered , during the next follow - up .

INTRODUCTION CASE REPORT DISCUSSION

tsc is an autosomal dominant disorder caused by the inactivation of either of 2 tumor suppressor genes , hamartin ( tsc1 ) or tuberin ( tsc2 ) . in the normal state , the hamartin - tuberin complex activates the protein ras homolog enriched in brain ( rheb ) , which inhibits mammalian target of rapamycin ( mtor ) . if a tsc mutation is present , mtor is constitutively activated , leading to abnormal cellular proliferation , ribosome biogenesis , and mrna translation . as a consequence , tsc is characterized clinically by the growth of benign tumors in multiple organs , including the brain , the heart , the kidneys , the lungs , and the skin1 ) . the severity of the disease is highly variable , ranging from mild skin manifestations to intractable epilepsy , mental retardation , and autism3 ) . rapamycin ( also called sirolimus ) is an immunosuppressive drug that has recently been shown to extend lifespan in multiple species , including mammals4 ) . this anti - aging property is presumably related to the mtor - inhibiting properties of rapamycin . the mtor pathway is crucial for the coordination of growth in response to energy status , stress , and nutrient availability5 ) . the potential anti - aging properties of rapamycin and other mtor inhibitors , such as rad001 ( everolimus ) , and cci-779 ( temsirolimus ) are of great interest . unfortunately , the side effects associated with these drugs preclude research trials to study their impacts on aging in healthy individuals . in view of this obstacle , experts in the field of aging have suggested that these potential anti - aging drugs should be introduced in clinical trials for the treatment of particular diseases , and then , if appropriate , be approved for prevention of all age - related diseases in healthy individuals6 ) . mtor is a 290-kda serine / threonine protein kinase that is highly conserved among mammals and also has closely related analogs in lower eukaryotes , such as drosophila and yeast7 ) . mtor has been implicated in numerous cellular functions , many of which are related to the fundamental processes of cell growth , survival , and homeostasis8 ) . a variety of upstream signaling pathways can regulate mtor activity in response to different extracellular stimuli or intracellular signals , including nutrient and energy status , growth factors , and stress9 ) . in turn , mtor responds to these upstream signals by modulating multiple downstream pathways , which mediate cellular growth , proliferation , metabolism , and survival , usually due to direct changes in the translation of relevant proteins10 ) . thus , during anabolic states in the presence of nutrients , growth factors , or insulin , signaling through specific upstream pathways , such as the phosphatidylinositol-3 kinase ( pi3k)/akt ( protein kinase b ) pathway , activates mtor , leading to increased protein synthesis , cellular growth , and proliferation11 ) . in catabolic states with nutrient / energy or oxygen deprivation , other upstream regulators , such as amp - kinase , inhibit mtor activity , thus decreasing protein translation and cellular growth , proliferation , and metabolism9 ) . activation or inhibition of mtor by upstream pathways is generally accomplished through opposing effects on the tuberous sclerosis gene products , hamartin and tuberin , and on the small gtpase protein , rheb . the cell signaling pathway involving mtor is further complicated by poorly defined intermediate steps , multiple feedback loops , and the formation of mtor complex 1 ( mtorc1 ) and mtor complex 2 ( mtorc2 ) . mtorc1 and mtorc2 are functional complexes of mtor bound to the regulatory proteins raptor and rictor respectively , which differ in their sensitivity to the mtor inhibitor , rapamycin12 ) . in addition to its functions in cellular growth and proliferation , mtor has other important and complex roles in regulating cell survival and cell death , especially in relation to the processes of autophagy , apoptosis , and immune regulation . autophagy involves the degradation and recycling of proteins and other macromolecules , and normally promotes cell survival under conditions of bioenergetic stress or in catabolic states where resources are limited . however , in some situations , autophagy may also mediate an alternative ( non - apoptotic , autophagic ) form of programmed cell death ( type ii pcd ) , thus revealing a dual role of autophagy in promoting both cell survival and death , depending on the cellular context13 ) . in anabolic states , in addition to stimulating protein synthesis , mtor generally inhibits autophagy and thus reduces the degradation of proteins . conversely , mtor inhibitors , such as rapamycin , usually stimulate autophagy , with a resultant neuroprotective effect in various models of brain injury14 ) . finally , mtor plays a critical role in immune responses via regulation of antigen - presenting cells and t - cells , and rapamycin is used clinically as a potent immunosuppressant drug . while the effects of rapamycin on autophagy , apoptosis , and immune regulation may most directly translate into neuromodulatory and neuroprotective properties , these features may also have anti - epileptogenic effects . the clinical and therapeutic importance of mtor is wide - reaching and continues to expand . abnormal mtor activity , leading to excessive cellular growth and proliferation , has been implicated in the pathophysiology of numerous human cancers , including both sporadic , isolated organ - specific and multiorgan tumors , genetic tumor syndromes . in many of these cases , specific mutations of some component of the mtor signaling pathway has been documented , resulting in hyperactivation of mtor or its downstream effectors . on the basis of the physiological and pathophysiological properties of mtor , it is reasonable to hypothesize that mtor signaling could be involved in mechanisms of epileptogenesis15 ) . the current main clinical complication related to tsc for which treatment with mtor inhibitors is indicated is subependymal giant cell astrocytoma ( sega ) . this complication affects approximately 15% of patients with tsc and it occurs in the pediatric age group16 ) . the traditional management approach is to monitor segas with periodic neuroimaging , and to resect those that exhibit growth and/or are associated with clinical signs of intracranial hypertension . this approach is being challenged by recent observations that suggest that mtor inhibitors such as rapamycin ( sirolimus ) and rad001 can induce partial regression of segas17,18 ) . the first report showing clear regression of segas in 5 patients with the use of rapamycin recently , a phase ii trial18 ) using everolimus to treat segas in 28 patients with tsc showed sega reduction of at least 30% in 21 patients ( 75% ) and at least 50% in 9 patients ( 32% ) . everolimus was well tolerated , as only single cases of grade 3 treatment - related sinusitis , pneumonia , viral bronchitis , tooth infection , stomatitis , and leukopenia were reported . these observations suggest that treatment with mtor inhibitors could serve as an acceptable alternative to sega surgery . renal angiomyolipomas and lymphangioleimyomatosis are other tsc manifestations against which mtor inhibitors have proven potential efficacy19 ) . in addition , animal models of tsc have suggested that mtor inhibitors could have beneficial effects on cognitive deficits20 ) and on epileptogenesis15 ) . whether similar benefits would be observed in humans with tsc there is clear evidence that segas grow back after administration of the mtor inhibitor is stopped17 ) . this cohort of patients , who will experience prolonged exposure to mtor inhibitors , should be carefully followed longitudinally to better document long - term side effects , but also to compare their longevity with that of similar patients receiving tscs . these patients represent a unique opportunity to study the potential anti - aging properties of mtor inhibitors in humans . in animal models , mtor inhibitors showed that mtorc1 blockade alone and pi3k - mtor blockade lead to suppression of tumor development and longer survival of the treated animals21 ) . rapamycin , the first mtor inhibitor used in individuals with tsc - associated lesions , was able to stimulate regression of subependymal giant cell tumors ( sgcts)17 ) . subsequent studies have confirmed its efficacy in sgcts , but also in other lesions such as angiomyolipomas22 ) . the effects of mtor inhibitors on the mtor pathway result in decreased protein synthesis and cell - cycle arrest , as well as decreased angiogenesis . more recently , a new mtor inhibitor , rad001 , has been used in the treatment of 28 patients with tsc - associated brain lesions but with no symptoms of increased intracranial pressure18 ) . in particular , this study reports a reduction in tumor size of at least 30% in 75% of patients and at least 50% in 32% of treated individuals . varying degrees of reduction of sgct size have been observed in all the 38 patients treated with mtor inhibitors ( sirolimus or everolimus ) to date . most sgct reductions occur in the first 3 months of mtor inhibitor treatment , after which the rate of reduction slows . in recent case reports , a similar anti - tumor efficacy was achieved , even with lower serum levels of everolimus23 ) . none of the patients treated with mtor inhibitors required surgery or developed new sgcts while receiving treatment17,18 ) . the treatment was also associated with a clinically relevant reduction in the overall frequency of seizures and an improvement in quality of life . unfortunately , regrowth of sgcts occurred a few months after drug discontinuation in all but one of the reported patients24 ) . therefore , mtor inhibition may need to be continuous for the benefits to persist , and the benefits and hazards of long - term treatment with low - dosage mtor inhibitors should be evaluated . an early diagnosis of sgct in neurologically asymptomatic children with tsc may allow prompt surgical removal of the tumor before the appearance of signs of increased intracranial pressure , and this approach is being progressively adopted to lessen the morbidity / mortality rate . however , the dramatic response of tsc - associated sgcts to mtor inhibitors suggests that these drugs could be a potential alternative to surgery in many cases . mtor inhibitors could be recommended when an asymptomatic sgct shows growth in 2 consecutive magnetic resonance imaging evaluations following diagnosis . mtor inhibitors could also be used as an initial treatment to facilitate subsequent surgery in individuals with bilateral lesions . medical therapy may also have a role when sgcts present in an atypical location or exhibit aggressive growth . furthermore , in case of regrowth after a first resection , considering the higher risk of further surgery , pharmacotherapy could provide an alternative method to keep lesion size under control . little is known about the long - term efficacy and safety of low dosage use of mtor inhibitors and whether regrowth could be prevented by a more prolonged treatment course . in animal models , rapamycin dosing comparison studies indicated that the duration of rapamycin treatment is more important than dose intensity in terms of efficacy ; prolonged treatment with low doses of mtor inhibitors resulted in more complete and durable tumor responses25,26 ) . our current understanding of the effects of continuous mtor inactivation in individuals with tsc is still poor . mtor inhibitors may also activate pathways that should not be activated , and this issue will need to be taken into account when a long - term treatment is proposed . the feasibility and timeline for discontinuation of mtor inhibitor - based pharmacotherapy also remains unclear , and further studies are required to explore the optimal duration of treatment . since it is known that the growth of sgcts tends to slow in early adulthood , mtor inhibitor treatment should theoretically be undertaken until the patient reaches around 20 years of age . strategies for future clinical trials with mtor inhibitors may include the investigation of longer treatment durations with minimum dosage . when choosing between surgical and/or medical intervention , clinicians should take the risks and benefits of each option into account . there are several issues to be considered , and every decision should be discussed thoroughly with the parents and tailored to the individual case . depending on the age of the patient , one option may be more valid than the other . for example , pharmacotherapy might be preferred when a growing sgct is discovered in adolescents , as the therapy may only be required for a few years . on the other hand , in childhood the positive effect that mtor inhibitors have on several manifestations of tsc is an important factor in favor of pharmacotherapy , and should be considered in patients presenting with problems such as renal angiomyolipomas , pulmonary lymphangioleiomyomatosis , and/or intractable epilepsy , in addition to sgcts . since the activation of the mtor pathway has been implicated in epileptogenesis , mtor inhibition could have antiepileptic effects in patients with tsc18,27 ) . inhibition of the mtor pathway may provide a biologically targeted therapy that has the potential to change current clinical practice regarding management of sgcts . currently , it is still unclear whether pharmacotherapy is able to prevent or merely delay the need for surgical resection of sgcts . in the coming years , medical treatment will certainly play a larger role in the management of children with tsc , as our understanding of the pathogenesis of this disorder at the molecular level improves . in conclusion , a new treatment era has begun in the field of tsc since the discovery of the potential beneficial effects of mtor inhibitors . although the use of mtor inhibitors is becoming increasingly accepted , especially for the treatment of segas in tsc , questions remain concerning the duration of treatment and long - term side effects . whether mtor inhibitors will have a significant impact on longevity in tsc is unknown , but warrants attention , as mtor inhibitors are increasingly recognized as anti - aging drugs in animal models . long - term prospective studies in patients with tsc will provide information on the potential anti - aging properties of mtor inhibitors in humans .

tuberous sclerosis complex ( tsc ) is a genetic multisystem disorder that results from mutations in the tsc1 or tsc2 genes , and is associated with hamartomas in several organs , including subependymal giant cell tumors . the neurological manifestations of tsc are particularly challenging and include infantile spasms , intractable epilepsy , cognitive disabilities , and autism . the tsc1- and tsc2-encoded proteins modulate cell function via the mammalian target of rapamycin ( mtor ) signaling cascade , and are key factors in the regulation of cell growth and proliferation . the mtor pathway provides an intersection for an intricate network of protein cascades that respond to cellular nutrition , energy levels , and growth factor stimulation . in the brain , tsc1 and tsc2 have been implicated in cell body size , dendritic arborization , axonal outgrowth and targeting , neuronal migration , cortical lamination , and spine formation . the mtor pathway represents a logical candidate for drug targeting , because mtor regulates multiple cellular functions that may contribute to epileptogenesis , including protein synthesis , cell growth and proliferation , and synaptic plasticity . antagonism of the mtor pathway with rapamycin and related compounds may provide new therapeutic options for tsc patients .

Introduction mTOR mTOR inhibitors and TSC Conclusions and future perspectives

a plausible prebiotic synthesis of the canonical nucleotides has long been a major goal in origins of life research . the oligomerization of hydrogen cyanide one of the major products of spark discharge in atmospheres containing methane and nitrogen to furnish the hcn tetramer diaminomaleonitrile ( 1 ) , its near - quantitative intramolecular photochemical rearrangement to 5-aminoimidazole-4-carbonitrile ( aicn , 2 ) , and the subsequent hydrolysis of aicn 2 to 5-aminoimidazole-4-carboxamide ( aica , 3 ) were demonstrated over 50 years ago.(4 ) however , the further elaboration of aminoimidazoles 2 and 3 to give the purine nucleobases and , after ribosylation and phosphorylation , the purine nucleotides is both very low yielding and , most importantly , not selective for the canonical nucleotides over isomers thereof . for example , adenine and hypoxanthine have been condensed under dry conditions at high temperatures with an excess of ribose in the presence of a high concentration of magnesium salts to yield 3 and 15% of the respective canonical n9-linked -d - ribofuranonucleosides in complex mixtures with many other isomers.(5 ) thus , irrespective of the prebiotic availability of ribose , the formation of adenosine ( and inosine ) is in low yield , alongside a multitude of regioisomers and pyranosyl isomers . furthermore , guanine , cytosine , and uracil do not give any of their respective nucleosides under similar conditions.(6 ) recently , a stepwise route from simple abiotic molecules considered to be prebiotically available to activated pyrimidine nucleotides was demonstrated ( scheme 1b).(1 ) however , this route does not solve the problem of purine synthesis . ideally , further development of this chemistry would also lead to a stepwise synthetic route for purine nucleotide assembly , thus providing a plausible pathway by which the four nucleotides required for an information - rich rna coding system would be prebiotically available . the key to effective pyrimidine synthesis was the stepwise increase in molecular complexity from simple aldehydes and cyanamide 4 through in situ formation of 2-aminooxazole ( 2ao , 5 ) as a hybrid sugar and nucleobase synthon . therefore , we considered ways in which 5 could additionally participate in the synthesis of purines . the diversity of chemical species likely to have been present under prebiotic conditions and the proven utility of multicomponent reactions ( mcrs ) to yield biologically relevant structures suggested that the development of novel mcrs could lead to the rapid buildup of molecular complexity and , in particular , to the structures that are central to molecular biology . the formation of 1 is catalyzed by simple aldehydes via the formation of cyanohydrins during hydrogen cyanide oligomerization ( scheme 1a).(9 ) it therefore seemed important to assess the chemistry of hydrogen cyanide tetramers with simple aldehydes in aqueous solution toward the prebiotic synthesis of purine ribo - nucleotides.(10 ) specifically , we proposed that the addition of 2 or 3 to the reaction of 5 with aldehydes might yield regiospecifically tethered aminoimidazole nucleotide intermediates . the close relationship of aminoimidazole tethering and pyrimidine synthesis is depicted in scheme 1b , wherein we propose that divergent access to purines and pyrimidines may be achieved by the addition of 5 to an imine or an aldehyde , respectively . as the five carbon atoms of ribose are contiguous , the formation of the canonical purine ribonucleotides from simple aldehydes should be considered both in the context of one c2 and one c3 fragment , and of one c1 and two c2 fragments . we first assessed the efficiency of forming an aminoimidazole - tethered masked c3-aldehyde upon reaction of 2ao 5 with formaldehyde and aicn 2 or aica 3 , which could ultimately translate into a c5-tethered aminoimidazole . upon mixing formaldehyde , 5 , and 2 or 3 in water at room temperature and near neutral ph , the rapid , efficient synthesis of rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines 6 or 7 was observed ( scheme 2 ) . synthesis of 6 and 7 demonstrates the selective sequestration of 2 and 3 through sequential formation of iminium ion 8 at equilibrium , followed by intermolecular carboncarbon bond formation to furnish 9 and 6-exo - trig intramolecular imidazoleiminium trapping ( scheme 3 ) . these reactions were found to be scalable to multigram levels , and the products crystallized from concentrated aqueous solution , giving 6 and 7 in 89 and 80% isolated yield , respectively . the -fused tricyclic structures were proven by x - ray diffraction analysis of a single crystal of 7 crystallized at ph 6.0 and a single crystal of the hemi - hydrochloride salt of 6 crystallized at ph 5.0 . pyrimidine nucleotide precursors result from two - component chemistry ( 2cr),(1 ) while three - component chemistry ( 3cr ) leads to potential purine nucleotide precursors . to further explore the generality of the mcr the reaction was shown to tolerate acetaldehyde to yield 11 and 12 as a 2:1 ( erythro : threo ( e : t ) ) mixture of diastereomers . the minor rac-[2s,3r]-isomer 12 was then isolated by precipitation and crystallization , which allowed the structure to be proven unambiguously by single - crystal x - ray diffraction . to explore this reaction outside of the prebiotic context , an aromatic aldehyde , 13 , was shown to undergo reaction in the presence of dmso as a co - solvent , giving a 3:2 e : t ratio of products 14 and 15 ( scheme 4 ) . to continue our investigation into the potential of this mcr for prebiotic nucleotide assembly the reaction of these -hydroxyaldehydes with aicn 2 or aica 3 and 2ao 5 was very clean and high yielding for the expected c4 ( c2 + c2 ) and c5 ( c2 + c3 ) products.(11 ) ph - dependent reactivity was observed , with three - component chemistry dominating at lower ph values and two - component chemistry dominating a higher ph values ( vide infra ) . notably , the products were uncontaminated with homo - aldolization or homo - mannich byproducts , and the three - component products exhibited a high diastereoselectivity for rac-[3r,4r]-products important for realization of a -ribo - nucleotide synthesis by c3-stereochemical inversion ( schemes 5 and 6 ) . proposed routes for rna synthesis must be continually evaluated for their potential to yield alternative structures capable of watsoncrick base - paring . glycolaldehyde , as a c2 sugar synthon , has the potential to furnish precursors of threose nucleic acid ( tna ) upon reaction with 5.(12 ) at pd 5.0 , the reaction of glycolaldehyde , aicn 2 , and 5 gave a 4:1 mixture of diastereoisomers 16 and 17 , alongside 10% two - component tetrose aminooxazoline products 18 and 19 . at pd 4.0 , the reaction gave a near - quantitative conversion to three - component products ; however , 21% of these products were 20 and 21 , which result from 5-exo - trig ring closure of intermediate 9.(13 ) an improvement in diastereoselectivity accrued upon reaction with aica 3 in lieu of aicn 2 . at pd 5.0 , a 7:1 mixture of diastereoisomers 22 and 23 ( 95% yield as determined by h nmr integration ) was observed , with complete 6-exo - trig selectivity . compound 22 was isolated in 74% yield and crystallized to allow the [ 2r,3r]-stereochemical relationship to be proven by x - ray crystallography . interestingly , 22 and 16 are related to tna via c3-stereochemical inversion.(12 ) glyceraldehyde is a c3 sugar synthon ; therefore , its reaction with the c2 sugar synthon 2ao 5 can yield precursors of pentose nucleic acids ( e.g. , rna).(1 ) however , a high - yielding synthesis of ribo - purinosides via c3-tethered aminoimidazoles would require high [ 3r,4r]-diastereoselectivity ( lyxo / xylo ) , a reversal of the facial selectivity of glyceraldehyde upon bimolecular reaction with 5.(14 ) at pd 5.0 , the reaction of glyceraldehyde , 5 , and aica 3 furnished lyxo - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine ( 24 ) in 60% yield ( by h nmr analysis ; 46% isolated yield ) , alongside three other diastereoisomers , 2527 , in 11 , 5 , and 8% yields , respectively ( by h nmr analysis ) . at pd 4.0 , the reaction of aicn 2 , 5 , and glyceraldehyde was highly selective for three - component products ( 89% by h nmr analysis ) . however , likely due to an increased thorpeingold effect in 9 ( where r = ch(oh)ch2oh relative to r = ch2oh ) and the lower ph required for selective three - component reaction of 2 relative to 3 , competitive 6-exo - trig imidazole cyclization and 5-exo - trig hydroxyl cyclization resulted in a 1:1 mixture of rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines 2831 and rac-3-aminoimidazole-2-aminooxazolines 3235 . the three - component reaction of glyceraldehyde with 2 or 3 and 5 is highly lyxo - selective , yielding 66% of 28/32 or 60% of 24 , respectively , whereas the bimolecular reaction of glyceraldehyde and 5 is highly ribo / arabino - selective ( ribo36 = 44% and arabino37 = 30%).(14 ) the high lyxo - selectivity of the three - component reaction establishes the required c1 , c3 , and c4 stereochemistry for the proposed ribo - nucleotide synthesis by c3 stereochemical inversion , while the ribo / arabino - selectivity of the two - component reaction establishes the required c3 and c4 stereochemistry for pyrimidine synthesis . the stereochemical preference for lyxo / xylo three - component products or ribo / arabino two - component products likely derives from the control of facial selectivity of the imine or aldehyde component , respectively,(14 ) coupled with the obligatory z - geometry of the nucleophilic c = c bond of 5 ( see supporting information , figure s36 ) , though calculations to support this conjecture have not been carried out . interestingly , with regard to the furanosyl selectivity of ribo - nucleotide synthesis , isolated 32 is observed to equilibrate with 28 , but not with detectable amounts of the pyranosyl isomer 40,(16 ) though lyxo - furanosyl aminooxazoline 39-f equilibrates to give significant amounts of the pyranosyl isomer ( 39-p/39-f , 5:1 ) ( scheme 7).(17 ) equilibration of 28 and 32 is proposed to occur via intermediate 9 and to be controlled by imidazole protonation ; therefore , at low ph , protonation of the imidazole moiety results in complete furanosyl selectivity ( see supporting information , figure s27 , for h nmr spectra of 28/32 at pd 112 ) . although efficient three - component reactivity is observed at ph values lower than 6.0 , the reaction profile of -hydroxyaldehydes with aicn 2 or aica 3 and 2ao 5 was observed to shift to furnish only bimolecular aminooxazoline products at higher ph s , even in the presence of a large excess of 2 or 3 . the reaction of glycolaldehyde and 5 with 2 or 3 , above pd 6.5 and 7.0 , respectively , was found to predominantly yield tetrose aminooxazolines 18 and 19 in a 1.2:1 [ 2r,3r]:[2s,3r ] ratio.(14 ) similarly , the reaction of glyceraldehyde , 2 or 3 , and 5 at pd 7.0 yields a mixture of pentose aminooxazolines 3639 in a 1.5:1 [ 2r,3r]:[2s,3r ] ratio.(14 ) the more or less complete switch between two- and three - component reactivity is observed to occur over 2 ph units at an aminoimidazole pka - dependent ph . however , surprisingly , at pd 7.0 , formaldehyde and 2 or 3 were observed to react selectively with 5 as imine / iminium 8 , resulting in the synthesis of 6 or 7 ( schemes 2 and 3 ) . even at elevated ph , and in the absence of 2 and 3 , no hydroxymethylation of 5 to give 41 was observed a standard sample of this latter compound being prepared by the reaction of glyceraldehyde with cyanamide 4 ( scheme 8).(18 ) it is possible that , due to the stability of the hemiaminal 42 formed between the amino group of 5 and formaldehyde , there is insufficient free aldehyde form of formaldehyde to react with free 5 at an observable rate.(19 ) the selective mcr of formaldehyde , and the absence of hydroxymethylation of 5 , may have interesting implications for the selectivity of prebiotic nucleotide synthesis . in effect , glycolaldehyde may be sequestered as 5 by the action of cyanamide 4 even in the presence of other aldehydes , such as formaldehyde , that can not undergo oxazole or aminooxazoline formation . as an additional point , aldehydes are known to accelerate the rate of hydrolysis of syn - disposed -aminonitriles at elevated ph values , but no concurrent nitrile hydrolysis was observed under the conditions of tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine formation . as the nitrile of derivatives of 2 retains the potential to give divergent access to adenine and guanine nucleotides , it is of note that no significant nitrile hydrolysis was observed in our experiments . selective cyanovinylation of the aminooxazoline moiety of the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines is one of several routes that may allow for the subsequent removal of those atoms derived from cyanamide 4 ( scheme 3 ) through photoactivated hydrolytic ( or otherwise ) removal of the resultant cytosine moiety , thereby affording products constitutionally closer to purine nucleotides . to investigate this potential for pyrimidine nucleobase elaboration and potential subsequent loss , a series of cyanovinylation experiments was undertaken to show that the aminooxazoline moiety of the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine structure could be selectively cyanovinylated . because there are several potentially nucleophilic heteroatoms in the generic tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidine structure that could react with an alkylating agent , we were initially unsure as to the likely regioselectivity of cyanovinylation upon treatment with aqueous cyanoacetylene . nonetheless , with regard to the nucleophilicty of n1 , we were encouraged by the observation that 6 crystallized as the hemihydrochloride salt at ph 5.0 , wherein two molecules of 6 were hydrogen - bonded via a common proton between n1 ( see supporting information , figure s3 ) . furthermore , we thought that the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines were likely to be intrinsically protected from c3nh cyanovinylation by delocalization of the nitrogen lone pair into the imidazole ring . interestingly , and very pleasingly , highly selective cyanovinylation was observed upon treatment of 6 , 17 , 21 , 24 , and 30 with cyanoacetylene . even in the presence of 5 equiv of cyanoacetylene , clean conversion of 6 , 17 , 21 , 24 , and 30 to 4-amino-1-(3-hydroxy-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidin-4-yl)pyrimidin-2-(h)-ones 4347 was observed over the period of 13 days at 60 c in unbuffered aqueous solution in 8095% yield.(21 ) this range of products demonstrates the efficacy this cyanovinyl in both tetrose and pentose series for both 2,3-cis- and 2,3-trans - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines . additionally , compounds 43 and 44 were isolated and crystallized from d2o , providing samples for x - ray diffraction to unambiguously prove the novel structural motif ( scheme 9 ) . experiments to investigate the propensity for cytosine loss from these compounds under a variety of conditions are now underway . we have described a concise and high - yielding route to the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines . on the basis of these key intermediates cytidines are known to undergo both pyrimidine loss and c2-epimerization upon uv - irradiation , possibly due to the photochemical generation of nucleobaseiminium ions.(22 ) these intermediates can undergo dissociative hydrolytic processes that are similar to those observed during the acid - catalyzed hydrolysis of -d - uridine,(23 ) but at near - neutrality . it is of note that the mcr described provides a high yield of [ 3r,4r]-products ( 70% lyxo / xylo ) , reversing the facial selectivity of glyceraldehyde observed in the biomolecular reaction with 2ao 5 ( 70% ribo / arabino ) , with the lyxo and xylo products being stereochemically related via c2-epimerization.(24 ) upon cyanovinylation and dissociative cytosine cleavage , compounds 27 , 31 , and 34 should , via intramolecular ring closure , furnish 5,3-anhydro - aicn - riboside 49 ( where r = ch2oh ) , which has an obvious relationship to aica - riboside 50 , an intermediate in the de novo biosynthesis of purines ( scheme 10 ) . furthermore , those compounds with trans-2-hydroxyl-3-anhydronucleoside relationships , such as 49 , have the clear potential to allow 2,3-cyclic phosphate formation under urea - mediated phosphoryl - transfer conditions,(1 ) thus generating 51 . access to 49 should allow further study of the selectivity issues involved in synthesizing the canonical nucleotides as opposed to closely related chemical structures capable of watsoncrick base - pairing interactions . it is of note that the proposed phosphorylation of the 2-hydroxyl group of 49 could lead to purines via a c3 stereochemical inversion , which would be directly analogous to the c2 inversion previously described to give access to the pyrimidine nucleotides . center : comparison to aica - riboside 50 , an intermediate in the de novo stepwise biosynthesis of canonical nucleotides . in summary , we have described additional predisposed complex structures that can be accessed through the participation of the key intermediate 2-aminooxazole 5 in a novel aqueous multicomponent reaction system . this mcr has proved to be scalable and high - yielding , and it generates a ph - dependent product distribution . further , the intrinsically controlled cyanovinylation of the low - ph mcr products generates potential precursors of the purine nucleotides . these reactions help to define the chemical structural space and chemical distribution likely to be accessible under plausible prebiotic chemical scenarios . in particular , we have demonstrated the concurrent synthesis of 37 and the tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines in the ph range in which activated pyrimidine synthesis can occur ( ph < 6.5 , where hydrolysis of 53 is prevented),(1 ) suggesting that both the pyrimidine and the purine ribonucelotides could be made together at the same time , in the same place , and under the same conditions.(29 ) the reactions may therefore be of direct relevance to the problem of abiogenesis of a full set of canonical ribonucleotides , and hence the chemical origins of molecular biology . reagents and solvents were purchased from sigma - aldrich , tci america , frontier scientific , or cambridge isotope laboratories . flash column chromatography was carried out using merck 9385 silica gel 60 ( 230400 mesh ) . nmr spectroscopy was carried out on a varian nmr spectrometer ( oxford as-400 ) operating at 20 c probe temperature ( unless specified ) . where possible , the chemical shift of the corresponding solvent was used as a reference . high - resolution mass spectrometry was carried out on a waters q - tof micro lc / ms / ms system . infrared spectra were recorded as manually pressed potassium bromide ( kbr ) discs on a perkinelmer spectrum 100 series ft - ir spectrometer . single - crystal x - ray crystallography was carried out with a bruker apex ii ccd diffractometer ( mo k radiation , = 0.71073 ) , equipped with an oxford cryosystems nitrogen flow apparatus , at 100 k. data integration down to 0.76 resolution was carried out using saint v7.46 a ( bruker diffractometer , 2009 ) with reflection spot size optimization . the structure was solved by the direct methods procedure and refined by least - squares methods against f using shelxs-97 and shelxl-97 ( sheldrick , 2008 ) . non - hydrogen atoms were refined anisotropically . method a : a solution of aminoimidazole 2 or 3 ( 1 equiv ) and 2-aminooxazole 5 ( 1 equiv ) in d2o was adjusted to the desired pd . a solution of aldehyde ( 1 equiv ) in d2o and , if required , dmso - d6 was added . the pd was adjusted as necessary by addition of dcl or naod , and the reaction was volumetrically adjusted to the required concentration and then incubated at the specified temperature . method b : a solution of aminoimidazole 2 or 3 ( 1 equiv ) and 2-aminooxazole 5 ( 1 equiv ) in h2o was adjusted to the desired ph . a solution of aldehyde ( 1 equiv ) in h2o was added . the ph was adjusted as necessary by addition of hcl or naoh , and the reaction was volumetrically adjusted to the required concentration and then incubated at the specified temperature . method c : an aqueous solution of cyanoacetylene ( 0.98 m , 35 equiv ) was added to tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines ( 20 mm ) , and the solution was then heated at 60 c for 13 d with stirring . method a : 5-aminoimidazole-4-carbonitrile 2 ( 24.8 mg , 0.23 mmol ) and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 1.0 ml ) at pd 5.0 , 6.0 , or 7.0 . formaldehyde ( 37% , 18.6 mg , 0.23 mmol ) was added in d2o ( 0.5 ml ) , the pd was rechecked , and the reaction volume was adjusted to 2 ml by the addition of d2o . the reactions were incubated at room temperature , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s4 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . 6 was then crystallized by cooling the reaction at pd 5.0 to 4 c for 2 d. 6 was isolated by filtration and washed with ice - cold water , and a single crystal was removed for x - ray diffraction . method b : 5-aminoimidazole-4-carbonitrile 2 ( 1.29 g , 11.9 mmol ) and 2-aminooxazole 5 ( 1.00 g , 11.9 mmol ) were dissolved in h2o ( 10 ml ) at ph 5.0 . formaldehyde ( 37% , 0.96 g , 11.9 mmol ) was added , the ph was rechecked , and the reaction volume was adjusted to 20 ml with h2o . after 20 min a white precipitate had formed in the reaction . the reaction was mechanically stirred for a further 15 h. the solids present were isolated by filtration , and the filtrate was lyophilized . slowly cooling to room temperature gave 1.68 g ( 69% ) of 6 as a white solid . recrystallization of the lyophilized filtrate afforded a further 0.5 g ( 20% ) of 6 . ir ( kbr , cm ) : 3467 ( nh ) , 3380 ( oh ) , 3115 , 2986 , 2938 ( ch ) , 2212 ( cn ) 1698 , 1610 ( n = c ) . h nmr ( 400 mhz , d2o ) : h 7.39 ( s , 1h , h-(c2 ) ) ; 6.08 ( d , j = 7.8 hz , 1h , h-(c1 ) ) ; 5.30 ( ddd , j = 7.8 , 2.0 , 1.8 hz , 1h , h-(c2 ) ) ; 3.54 ( abx , j = 14.2 , 1.8 hz , 1h , ha-(c3 ) ) ; 3.37 ( abx , j = 14.2 , 2.0 hz , 1h , hb-(c3 ) ) [ peak partially obscured by hod peak in dmso ; see supporting information , figure s2 ] . c nmr ( 101 mhz , dmso - d6 ) : c 166.6 ( c = o ) ; 164.0 ( c2 ) ; 142.1 ( c5 ) ; 129.6 ( c2 ) ; 112.7 ( c4 ) ; 76.4 ( c2 ) ; 72.8 ( c1 ) ; 42.3 ( c3 ) . m / z ) : 205 ( 100% , [ m + h ] ) , 227 ( 10% , [ m + na ] ) . hrms ( m / z ) : [ m ] calcd for c8h9n6o , 205.0832 ; found , 205.0831 . method a : 5-aminoimidazole-4-carboxamide 3 ( 29.0 mg , 0.23 mmol ) and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2.0 ml ) at pd 5.0 , 6.0 , or 7.0 . formaldehyde ( 37% , 18.6 mg , 0.23 mmol ) was added in d2o ( 0.5 ml ) , the pd was rechecked , and the reaction volume was adjusted to 2 ml with d2o . the reactions were incubated for 15 h , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s4 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . 7 was then crystallized by cooling the reaction solution to 4 c for 1 d. 7 was isolated by filtration and washed with ice - cold water . method b : 5-aminoimidazole-4-carboxamide 3 ( 1.50 g , 11.9 mmol ) and 2-aminooxazole 5 ( 1.00 g , 11.9 mmol ) were dissolved in h2o ( 10 ml ) at ph 5.0 . formaldehyde ( 37% , 0.96 g , 11.9 mmol ) was added , the ph was rechecked , and the reaction volume was adjusted to 20 ml with h2o . the reaction was mechanically stirred for 15 h. the solids present were isolated by filtration , and the filtrate was lyophilized . the combined solids were redissolved in water by heating , agitated , and then allowed to cool to give 1.95 g of 7 as a yellow solid . the solids were dissolved in hot water ( 4 ml ) , and the product was allowed to crystallize upon cooling . 7 was isolated by filtration , washed with methanol ( 10 ml ) , and air - dried to give 1.30 g ( 49% ) of 7 as a white solid . a further portion of methanol was added to the filtrate , and the solution was then cooled to give a second batch ( 0.82 g , 31% ) of 7 as an off - white powder . ir ( kbr , cm ) : 3365 ( nh ) , 3096 , 2979 , 2765 , 2660 ( ch ) , 1676 ( n = c ) , 1640 ( c = o ) . h nmr ( 400 mhz , d2o ) : h 7.21 ( s , 1h , h-(c2 ) ) ; 5.83 ( d , j = 7.8 hz , 1h , h-(c1 ) ) ; 5.03 ( dt , j = 7.8 , 1.8 hz , 1h , h-(c2 ) ) ; 3.53 ( abx , j = 13.8 , 1.8 hz , 1h , ha-(c3 ) ) ; 3.18 ( abx , j = 13.8 , 1.8 hz , 1h , hb-(c3 ) ) . c nmr ( 101 mhz , d2o ) : c 166.2 ( c = o ) ; 164.1 ( c2 ) ; 144.3 ( c5 ) ; 130.0 ( c2 ) ; 110.4 ( c4 ) ; 78.4 ( c2 ) ; 66.1 ( c1 ) ; 43.4 ( c3 ) . m / z ) : 223 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] x - ray diffraction structure solved ; deposited as ccdc 784244 . method a : acetaldehyde ( 10 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 86.9 mg , 0.69 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 6.0 . the reaction was incubated for 15 h , and crystals ( colorless needles ) were observed to form . the supernatant was removed , and two products were observed by nmr spectroscopy ( see supporting information , figure s7 , for h nmr spectrum ) . the crystals were dissolved in the supernatant by warming , and nmr spectroscopy showed a 2:1 ratio of 11:12 . the crystals were isolated by filtration ( 18 mg ) , and an analytical sample was dissolved in dmso - d6 ( 0.75 ml ) for nmr spectroscopy . the remaining crystals were recrystallized from slowly cooling h2o to provide a sample for x - ray diffraction . h nmr ( 400 mhz , d2o ) : h 7.18 ( s , 1h , har ) ) ; 5.81 ( d , j = 7.6 hz , 1h , h-(c1 ) ) ; 4.74 ( dd , j = 7.6 , 2.9 hz , 1h , h-(c2 ) ) ; 3.65 ( dq , j = 6.9 , 2.9 hz , 1h , h-(c3 ) ) ; 0.90 ( d , j = 6.9 hz , 3h , me ) . m / z ) : 237 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c9h13n6o2 , 237.1095 ; found , 237.1099 . 12 . ir ( kbr , cm ) : 3413 ( nh ) , 3178 , 3087 , 2960 ( ch ) , 1684 , 1663 ( n = c ) , 1616 ( c = o ) . h nmr ( 400 mhz , d2o ) : h 7.15 ( s , 1h , h-(c2 ) ) ; 5.81 ( d , j = 7.7 hz , 1h , h-(c1 ) ) ; 4.83 ( dd , j = 7.7 , 2.0 hz , 1h , h-(c2 ) ) ; 3.32 ( dq , j = 6.6 , 2.0 hz , 1h , h-(c3 ) ) ; 1.16 ( d , j = 6.6 hz , 3h , me ) . h nmr ( 400 mhz , dmso - d6 ) : h 7.16 ( s , 1h , h - ar ) ; 6.68 ( br d , 2h , nh2 ) ; 6.40 ( br s , 2h , nh2 ) ; 5.79 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 5.59 ( br s , 1h , nh ) ; 4.70 ( dd , j = 7.4 , 0.5 hz , 1h , h-(c2 ) ) ; 3.41 ( dd , j = 6.0 , 0.5 hz , 1h , h-(c3 ) ) ; 1.29 ( d , j = 6.0 hz , 3h , me ) . c nmr ( 101 mhz , dmso - d6 ) : c 166.7 ( c = o ) ; 166.7 ( c2 ) ; 142.0 ( c5 ) ; 129.5 ( c2 ) ; 112.5 ( c4 ) ; 79.7 ( c2 ) ; 73.6 ( c1 ) ; 47.9 ( c3 ) ; 17.9 ( me ) . es - ms ( pos . m / z ) : 237 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c9h13n6o2 , 237.1095 ; found , 237.1099 . x - ray diffraction structure solved ; deposited as ccdc 784245 . method a : 2-pyridine carboxaldehyde 13 ( 24.6 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 58 mg , 0.42 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o : dmso - d6 ( 10:1 , 2 ml ) at pd 6.0 . the reaction was incubated for 15 h , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s9 , for h nmr spectrum ) . the formation of two products , 14/15 , was observed in a 3:2 ratio by h nmr spectroscopy . compound 14 was observed to crystallize from the solution over 3 d and was isolated by centrifugation , washed with methanol , and dried to give 15 mg ( 20% ) of 14 . an analytical sample of crystalline 14 was then recrystallized from ethanol three times for single - crystal x - ray diffraction . ir ( kbr , cm ) : 3412 ( nh ) , 2937 , 2528 , 2458 ( ch ) , 1693 ( n = c ) , 1669 , 1637 ( c = o ) 1591 . h nmr ( 400 mhz , dmso - d6 ) : h 9.1 ( s , nh ) ; 8.43 ( dd , j = 4.8 , 0.8 hz , 1h , ar ) ; 7.75 ( td , j = 7.8 , 1.8 hz , 1h , ar ) ; 7.36 ( d , j = 7.8 hz , 1h , ar ) ; 7.24 ( ddd , j = 7.8 , 4.8 , 1.8 hz , 1h , ar ) ; 5.78 ( d , j = 7.8 hz , 1h , h-(c1 ) ) ; 5.40 ( dd , j = 7.6 , 2.5 hz , 1h , h-(c2 ) ) ; 4.88 ( d , j = 2.5 hz , 1h , h-(c3 ) ) . c nmr ( 101 mhz , dmso - d6 ) : c 166.6 ( c = o ) ; 163.7 ( c2 ) ; 159.0 ( ar ) ; 149.6 ( c5 ) ; 139.9 ( arh ) ; 137.9 ( arh ) ; 129.8 ( c2 ) ; 123.6 ( arh ) ; 122.2 ( arh ) ; 112.7 ( c4 ) ; 78.6 ( c2 ) ; 72.3 ( c1 ) ; 56.2 ( c3 ) . m / z ) : 300 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c13h14n7o2 , 300.1203 ; found , 300.1205 . x - ray diffraction structure solved ; deposited as ccdc 784252 . method a : glycolaldehyde ( 14.2 mg , 0.23 mmol ) , 5-aminoimidazole-4-carbonitrile 2 ( 24.8 mg , 0.23 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 4.0 , 5.0 , 5.5 , or 6.0 . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see table 1 and supporting information , figure s11 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . the formation of 16 ( 60% ) , 17 ( 16% ) , 20 ( 14% ) , and 21 ( 7% ) was observed at pd 4.0 ( see supporting information , figure s16 , for h , hh cosy , and 2d noesy spectra ) . 17 was isolated by precipitation from warm methanol and recrystallized four times from ethanol and once by vapor diffusion ( chloroform into methanol ) to provide a sample for x - ray crystallography . 17 was studied by 2d homo- and heteronuclear nmr spectroscopy ( see supporting information , figures s13s15 ) . the formation of 18 ( 60% ) and 19 ( 40% ) was observed at pd 7.0 . noe intensity volumes are given as strong ( s ) , medium ( m ) , weak ( w ) , or not observed ( ) relative to the volume of h(c1)h(c2 ) of the same compound which is defined as strong . h nmr ( 400 mhz , d2o ) : h 7.22 ( s , 1h , h-(c2 ) ) ; 5.81 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 4.99 ( dd , j = 7.4 , 2.7 hz , 1h , h-(c2 ) ) ; 3.74 ( dt , j = 5.7 , 2.7 hz , 1h , h-(c3 ) ) ; 3.50 ( t , j = 5.7 hz , 2h , h-(c4 ) ) . m / z ) : 235 ( 100% , [ m + h ] ) , 257 ( 10% , [ m + na ] ) , 273 ( 3% , [ m + k ] ) . hrms ( m / z ) : [ m ] calcd for c9h11n6o2 , 235.0938 ; found , 235.0944 . ir ( kbr , cm ) : 3467 ( nh ) , 3380 ( oh ) , 3115 , 2986 , 2938 ( ch ) , 2212 ( cn ) 1698 , 1610 ( n = c ) . h nmr ( 400 mhz , d2o ) : h 7.16 ( s , 1h , h - ar ) ) ; 5.78 ( d , j = 7.7 hz , 1h , h-(c1 ) ) ; 4.8 ( dd , j = 7.7 , 1.9 hz , 1h , h-(c2 ) ) ; 3.63 ( abx , j = 11.4 , 6.6 hz , 1h , ha-(c4 ) ) ; 3.56 ( abx , j = 11.4 , 6.8 hz , 1h , hb-(c4 ) ) ; 3.56 ( app td , j = 6.7 , 1.9 hz , 1h , h-(c3 ) ) . h nmr ( 400 mhz , dmso - d6 , 25 c ) : h 7.35 ( s , 1h , h - ar ) ; 6.83 ( s , 1h , hn ) ; 6.55 ( s , 2h , nh2 ) ; 5.87 ( d , j = 7.5 hz , 1h , h-(c1 ) ) ; 5.08 ( t , j = 5.85 hz , 1h , oh ) ; 4.92 ( d , j = 7.5 hz , 1h , h-(c2 ) ) ; 3.74 ( abx , j = 10.5 , 6.0 hz , 1h , ha-(c4 ) ) ; 3.60 ( abx , j = 10.5 , 6.7 hz , 1h , hb-(c4 ) ) ; 3.42 ( m , 1h , h-(c3 ) ) . c nmr ( 101 mhz , dmso - d6 , 25 c ) : c 163.6 ( c2 ) ; 145.4 ( c5 ) ; 131.9 ( c2 ) ; 116.9 ( cn ) ; 88.8 ( c4 ) ; 75.5 ( c2 ) ; 73.5 ( c1 ) ; 60.3 ( c4 ) ; 53.6 ( c3 ) . m / z ) : 235 ( 100% , [ m + h ] ) , 257 ( 10% , [ m + na ] ) . m / z ) : 233 ( 70% , [ m h ] ) . hrms ( m / z ) : [ m ] calcd for c9h11n6o2 , 235.0938 ; found , 235.0944 . x - ray diffraction structure solved ; deposited as ccdc 784246 . method a : glycolaldehyde ( 14.3 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 29.0 mg , 0.23 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 5.0 , 6.0 , or 7.0 . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s17 , for h nmr spectra at pd 5.0 , 6.0 , and 7.0 ) . the formation of 22 ( 78% ) and 23 ( 11% ) was observed at pd 5.0 . 22 was isolated by precipitation with methanol and studied by 2d homo- and heteronuclear nmr spectroscopy ( see supporting information , figures s19s21 ) . the formation of 18 ( 60% ) and 19 ( 40% ) was observed at pd 7.0 . method b : glycolaldehyde ( 357 mg , 5.9 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 743 mg , 5.9 mmol ) , and 2-aminooxazole 5 ( 500 mg , 5.9 mmol ) were dissolved in h2o ( 20 ml ) at ph 6.0 . the reaction was incubated for 15 h , after which a white precipitate had formed . the precipitate was isolated by filtration and washed with ice - cold water to give 1.1 g ( 74% ) of 22 . an analytical sample of 22 was then recrystallized five times from aqueous ethanol for x - ray diffraction analysis . ir ( kbr , cm ) : 3315 ( oh ) , 3188 ( nh ) , 2922 , 2850 ( ch ) , 1711 ( n = c ) , 1659 ( c = o ) , 1614 , 1575 ( n = c ) . h nmr ( 400 mhz , dmso - d6 ) : h 7.14 ( s , 1h , h-(c2 ) ) ; 6.65 ( bd , 2h , nh2 ) ) ; 6.45 ( s , 2h , nh2 ) ) ; 6.18 ( s , 1h , nh2 ) ) ; 5.74 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 5.04 ( t , j = 5.12 hz , 1h , ho ) ) ; 4.78 ( dd , j = 7.4 , 2.9 hz , 1h , h-(c2 ) ) ; 3.49 ( m , 1h , h-(c3 ) ) ; 3.44 ( abx , j = 11.0 , 5.0 hz , 1h , ha-(c4 ) ) ; 3.38 ( abx , j = 11.0 , 5.3 hz , 1h , hb-(c4 ) ) . c nmr ( 101 mhz , dmso - d6 ) : c 166.7 ( c = o ) ; 163.4 ( c = n ) ; 140.4 ( c5 ) ; 129.2 ( c2 ) ; 112.2 ( c4 ) ; 76.3 ( c1 ) ; 72.5 ( c2 ) ; 62.4 ( c3 ) ; 54.2 ( c4 ) . m / z ) : 253 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c9h13n6o3 , 253.1044 ; found , 253.1049 . glyceraldehyde ( 20.7 mg , 0.23 mmol ) , 5-aminoimidazole-4-carboxamide 3 ( 29.0 mg , 0.23 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 5.0 , 6.0 , 6.5 , or 7.0 . the reaction was incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see supporting information , figure s23 , for h nmr spectra at pd 5.0 , 6.0 , 6.5 , and 7.0 ) . the predominant formation of four tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines ( 84% ) 24 ( 60% ) , 25 ( 11% ) , 26 ( 5% ) and 27 ( 8% ) was observed at pd 5.0 . the predominant formation of pentose aminoxaolines 36 ( 44% ) , 37 ( 30% ) , 38 ( 8% ) , 35-f ( 2% ) , and 35-p ( 10% ) was observed at pd 7.0 . the reaction initiated at pd 5.0 was lyophilized and dissolved in methanol ( 5 ml ) . 24 was isolated by precipitation with anhydrous diethyl ether ( 10 ml ) and crystallized from methanol to give 29 mg ( 46% ) of 24 as colorless crystals . an analytical sample of 24 was crystallized three times from aqueous ethanol and then recrystallized from methanol : ch2cl2 ( 1.5:1 ) by evaporation . ir ( kbr , cm ) : 3443 ( nh ) , 3305 ( oh ) , 2866 , 2807 , 2662 ( ch ) , 1678 ( c = o ) 1652 , 1577 ( n = c ) . h nmr ( 400 mhz , d2o ) : h 7.16 ( s , 1h , har ) ; 5.72 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 4.93 ( dd , j = 7.4 , 2.4 hz , 1h , h-(c2 ) ) ; 3.72 ( dd , j = 4.2 , 2.4 hz , 1h , h-(c3 ) ) ; 3.63 ( app q , j = 5.2 hz , 1h , h-(c4 ) ) ; 3.50 ( abx , j = 11.7 , 5.0 hz , 1h , ha-(c5 ) ) ; 3.42 ( abx , j = 11.7 , 6.2 hz , 1h , hb-(c5 ) ) . c nmr ( 101 mhz , d2o ) : c 168.1 ( c = o ) ; 165.1 ( c2 ) ; 141.3 ( c5 ) ; 131.0 ( c2 ) ; 110.4 ( c4 ) ; 78.4 ( c2 ) ; 71.3 ( c1 ) ; 62.5 ( c4 ) ; 53.3 ( c5 ) ; 48.9 ( c3 ) . m / z ) : 283 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c10h15n6o4 , 283.1149 ; found , 283.1154 . x - ray diffraction structure solved ; deposited as ccdc 784247 . method a : glyceraldehyde ( 20.7 mg , 0.23 mmol ) , 5-aminoimidazole-4-carbonitrile 2 ( 49.6 mg , 0.46 mmol ) , and 2-aminooxazole 5 ( 20 mg , 0.23 mmol ) were dissolved in d2o ( 2 ml ) at pd 4.0 , 4.5 , 5.0 , 6.0 , or 7.0 . the reactions were incubated for 5 d , and the progress of the reaction was assessed by h nmr spectroscopy ( see table 2 and supporting information , figure s26 , for h nmr spectra at pd 4.0 , 4.5 , 5.0 , 6.0 , and 7.0 ) . at pd 4.0 , the formation of four rac - tetrahydroimidazo[1,3]-2-aminooxazolo[1,2]-pyrimidines , 2831 ( 46% , 13:2.2:1 ratio ) , and four 3-imidazoloaminooxazolines , 3235 ( 43% , 16:2.2:1 ratio ) , was observed by h nmr spectroscopy ( see supporting information , page s17 , for partial nmr characterization of compounds 28 , 29 , 31 , 33 , 34 , and 35 ) . the predominant formation of pentose aminoxaolines 36 ( 44% ) , 37 ( 30% ) , 38 ( 8% ) , 35-f ( 2% ) , and 35-p ( 10% ) was observed at pd 7.0 . noe intensity volumes are given as strong ( s ) , medium ( m ) , weak ( w ) , or not observed ( ) relative to the volume of h(c1)h(c2 ) of the same compound which is defined as strong . method b : glyceraldehyde ( 207 mg , 2.3 mmol ) , 5-aminoimidazole-4-carboxamide 2 ( 496 mg , 2.3 mmol ) , and 2-aminooxazole 5 ( 200 mg , 2.3 mmol ) were dissolved in h2o ( 10 ml ) at ph 4.0 . the reaction was incubated for 2 d , lyophilized , and dissolved in meoh ( 10 ml ) . sio2 ( 2 g ) was added , the mixture was concentrated to a fine , free - flowing powder in vacuo , and the compounds were eluted with 1:4 to 1:3 methanol : ch2cl2 . the solids were dissolved in aqueous methanol ( h2o : meoh 0.5:10 ml ) , and etoac ( 6 ml ) was added to partially precipitate the mixture . the solids were filtered and washed with methanol to give 98 mg of 32 ( 16% ) . those fractions eluted from silica gel that predominately contained 2831 were concentrated in vacuo to a white foam . the foam was dissolved in d2o and studied by h , hh cosy , and hh noesy nmr . the solution was lyophilized , and the solid was dissolved in warm aqueous ethanol . upon cooling , white crystalline solids formed ; these were isolated by centrifugation and then dissolved in d2o . repeated recrystallization of the crystalline solids from h2o afforded 20 mg of 30 as fine , clear needles ( see supporting information , figure s25 ) . ir ( kbr , cm ) : 3440 ( nh ) , 3323 ( oh ) , 2873 , 2800 , 2658 ( ch ) , 2216 ( cn ) , 1579 ( n = c ) . h nmr ( 400 mhz , d2o ) : h 7.40 ( s , 1h , h-(c2 ) ) ; 6.00 ( d , j = 7.4 hz , 1h , h-(c1 ) ) ; 5.22 ( dd , j = 7.4 , 1.9 hz , 1h , h-(c2 ) ) ; 3.93 ( ddd , j = 8.1 , 5.6 , 3.9 hz , 1h , h-(c4 ) ) ; 3.83 ( abx , j = 12.1 , 3.9 hz , 1h , ha-(c5 ) ) ; 3.73 ( abx , j = 12.1 , 5.6 hz , 1h , hb-(c5 ) ) ; 3.55 ( dd , j = 8.1 , 1.9 hz , 1h , h-(c3 ) ) . es - ms ( pos . m / z ) : 265 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c10h12n6o3 , 265.144 ; found , 265.1050 . h nmr ( 400 mhz , d2o , pd 1.0 ) : 8.21 ( s , 1h , h-(c2 ) ) ; 5.97 ( d , j = 5.4 hz , 1h , h-(c1 ) ) ; 5.45 ( t , j = 5.4 hz , 1h , h-(c2 ) ) ; 4.17 ( dd , j = 10.0 , 5.4 hz , 1h , h-(c3 ) ) ; 3.87 ( ddd , j = 10.0 , 3.9 , 2.0 hz , 1h , h-(c4 ) ) ; 3.77 ( abx , j = 12.5 , 2.0 hz , 1h , ha-(c5 ) ) ; 3.55 ( abx , j = 12.5 , 3.9 hz , 1h , hb-(c5 ) ) . c nmr ( 400 mhz , d2o ) : 162.7 ( c2 ) ; 151.4 ( c5 ) ; 135.5 ( c2 ) ; 114.9 ( cn ) ; 88.2 ( c1 ) ; 84.9 ( c2 ) ; 84.0 ( c4 ) ; 78.0 ( c4 ) ; 59.1 ( c5 ) ; 55.5 ( c3 ) . m / z ) : 265 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c10h12n6o3 , 265.144 ; found , 265.1051 . glyceraldehyde ( 1.8 g , 20 mmol ) and cyanamide 4 ( 840 mg , 20 mmol ) were dissolved in h2o ( 10 ml ) at ph 10 and heated at 60 c for 2 h. the solution was then cooled , diluted with h2o ( 10 ml ) , and extracted in etoac ( 5 20 ml ) . the residue was dissolved in etoac and recrystallized by the addition of heptane to give 1.5 g ( 66% ) of 5-(hydroxymethyl)-2-aminooxazole 41 as a white solid . h nmr ( 400 mhz , meod ) : h 6.58 ( s , 1h , h-(c4 ) ) ; 4.40 ( s , 2h , ch2 ) . c nmr ( 400 mhz , meod ) : c 163.9 ( c2 ) ; 146.2 ( c5 ) ; 124.5 ( c4 ) ; 55.2 ( ch2 ) . m / z ) : 115 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c4h7n2o2 , 115.0502 ; found , 115.0507 . method c:7 ( 100 mg , 0.49 mmol ) and cyanoacetylene ( 0.98 m in h2o , 1.5 ml ) were incubated at 60 c for 72 h. the product was then purified by silica gel chromatography ( eluting with 1:5 to 1:1 ch2cl2:meoh ) , and the fractions containing the product ( rf 0.3 ; 1:5 ch2cl2:meoh ) were concentrated in vacuo . the product was crystallized from aqueous ethanol to give 120 mg of 43 ( 85% ) as clear , colorless crystals . h nmr ( 400 mhz , d2o ) : h 7.25 ( d , j = 7.6 hz , 1h , h-(c6 ) ) ; 7.08 ( s , 1h , h - ar ) ; 6.75 ( d , j = 3.8 hz , 1h , h-(c1 ) ) ; 5.93 ( d , j = 3.8 hz , 1h , h-(c5 ) ) ; 4.37 ( ddd , j = 5.3 , 3.8 , 2.2 hz , 1h , h-(c2 ) ) ; 3.53 ( abx , j = 13.0 , 2.2 hz , 1h , h-(c3 ) ) ; 3.41 ( abx , j = 11.7 , 5.0 hz , 1h , h-(c3 ) ) . c nmr ( 101 mhz , d2o ) : c 168.2 ( c = o ) ; 166.3 ( c2 ) ; 157.8 ( c4 ) ; 144.0 ( c6 ) ; 142.7 ( c5 ) ; 130.6 ( c2 ) ; 109.5 ( c5 ) ; 96.6 ( c4 ) ; 64.8 ( c2 ) ; 62.4 ( c1 ) ; 43.5 ( c3 ) . m / z ) : 292 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c11h12n7o4 , 292.1153 ; found , 292.1146 . method c:17 ( 117 mg , 0.50 mmol ) and cyanoacetylene ( 0.98 m in h2o , 2.0 ml ) were incubated at 60 c for 48 h. ( 2r,3s,4s)-3-hydroxy-2-(hydroxymethyl)-4-(2-imino-6-oxo-2h - pyrimido[1,6-a]-pyrimidin-7(6h)-yl)-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidine-8-carbonitrile ( 48 ) was observed to precipitate from solution over 4 h and was removed by filtration to give 2 mg of 48 as a yellow solid . ( 2r,3s,4s)-4-(4-amino-2-oxopyrimidin-1(2h)-yl)-3-hydroxy-2-(hydroxymethyl)-1,2,3,4-tetrahydroimidazo[1,5-a]-pyrimidine-8-carbonitrile ( 44 ) was observed to crystallize from aqueous solution over 48 h. the large colorless crystals were used directly for x - ray diffraction . h nmr ( 400 mhz , d2o ) : h 7.28 ( d , j = 7.4 hz , 1h , h-(c6 ) ) ; 7.13 ( s , 1h , h-(c2 ) ) ; 6.76 ( d , j = 2.7 hz , 1h , h-(c1 ) ) ; 5.96 ( d , j = 7.4 hz , 1h , h-(c5 ) ) ; 4.27 ( d , j = 2.7 hz , 1h , h-(c2 ) ) ; 3.773.87 ( m , 1h , ha-(c4 ) ) ; 3.673.77 ( m , 2h , h-(c3 ) , hb-(c4 ) ) . c nmr ( 101 mhz , d2o ) : c 166.2 ( c2 ) ; 157.4 ( c4 ) ; 147.2 ( c5 ) ; 142.7 ( c6 ) ; 131.1 ( c2 ) ; 116.5 ( cn ) ; 96.3 ( c5 ) ; 88.6 ( c4 ) ; 66.0 ( c2 ) ; 62.2 ( c1 ) ; 60.4 ( c4 ) ; 55.2 ( c3 ) . m / z ) : 304 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c12h14n7o3 , 304.1152 ; found , 304.1119 . h nmr ( 400 mhz , d2o ) : h 8.34 ( s , 1h , h-(c2 ) ) ; 7.62 ( d , j = 7.5 hz , 1h , h-(c6 ) ) ; 7.33 ( d , j = 9.4 hz , 1h , h-(c7 ) ) ; 6.10 ( d , j = 3.5 hz , 1h , h-(c1 ) ) ; 5.96 ( d , j = 7.5 hz , 1h , h-(c5 ) ) ; 5.55 ( d , j = 9.4 hz , 1h , h-(c8 ) ) ; 4.48 ( dd , j = 3.5 , 2.0 hz , 1h , h-(c2 ) ) ; 4.43 ( dd , j = 9.8 , 4.0 hz , 1h , ha-(c4 ) ) ; 4.26 ( app dt , j = 3.0 , 1.9 hz , 1h , h-(c3 ) ) ; 4.0 ( dd , j = 9.8 , 1.9 hz , 1h , hb-(c4 ) ) . uvvis : max 252 nm ( 9760 ) , 320 ( 6400 ) . es - ms ( pos . m / z ) : 355 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c15h15n8o3 , 355.1261 ; found , 355.1261 . method c:21 ( 50 mg , 0.20 mmol ) and cyanoacetylene ( 0.98 m in h2o , 0.5 ml ) were incubated at 60 c for 24 h. the formation of 45 was observed in 80% yield by h nmr integration . h nmr ( 400 mhz , d2o ) : h 7.28 ( d , j = 7.4 hz , 1h , h-(c6 ) ) ; 7.13 ( s , 1h , h-(c2 ) ) ; 6.76 ( d , j = 2.7 hz , 1h , h-(c1 ) ) ; 5.96 ( d , j = 7.4 hz , 1h , h-(c5 ) ) ; 4.27 ( d , j = 2.7 hz , 1h , h-(c2 ) ) ; 3.773.87 ( m , 1h , h-(c4 ) ) ; 3.673.77 ( m , 2h , h-(c3 ) , h-(c4 ) ) . c nmr ( 101 mhz , d2o ) : c 167.6 ( c = o ) ; 165.6 ( c2 ) ; 157.3 ( c4 ) ; 142.4 ( c5 ) ; 142.0 ( c6 ) ; 129.5 ( c2 ) ; 108.4 ( c4 ) ; 96.2 ( c5 ) ; 63.3 ( c1 ) ; 62.9 ( c2 ) ; 60.9 ( c4 ) ; 54.1 ( c3 ) . m / z ) : 304 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c12h16n7o4 , 322.1258 ; found , 322.1284 . method c:30 ( 5 mg , 0.019 mmol ) and cyanoacetylene ( 0.98 m in h2o , 50 l ) were incubated at 60 c for 24 h. the formation of 46 was observed in 82% yield by h nmr integration ( see supporting information , figure s34 , for crude h nmr ) . h nmr ( 400 mhz , d2o ) : 7.16 ( d , j = 7.6 hz , 1h , h-(c6 ) ) ; 7.03 ( s , 1h , h-(c2 ) ) ; 6.64 ( d , j = 3.1 hz , 1h , h-(c1 ) ) ; 5.84 ( d , j = 7.6 hz , 1h , h-(c5 ) ) ; 4.30 ( d , j = 3.1 , 1.0 hz , 1h , h-(c2 ) ) ; 3.663.77 ( m , 2h , h-(c4 ) , ha-(c5 ) ) ; 3.58 ( abx , j = 12.1 , 5.8 hz , 1h , hb-(c5 ) ) ; 3.51 ( app d , j = 8.6 hz , 1h , h-(c3 ) ) . c nmr ( 101 mhz , d2o ) : c 165.6 ( c2 ) ; 157.5 ( c4 ) ; 147.2 ( c5 ) ; 142.0 ( c6 ) ; 130.8 ( c2 ) ; 116 ( cn ) ; 96.0 ( c4 ) ; 96.0 ( c5 ) ; 65.9 ( c1 ) ; 61.7 ( c2 ) ; 69.0 ( c4 ) ; 62.3 ( c5 ) ; 55.2 ( c3 ) . m / z ) : 334 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c13h16n7o4 , 334.1258 ; found , 334.1251 . method c:24 ( 20 mg , 0.07 mmol ) and cyanoacetylene ( 0.98 m in h2o , 0.35 ml ) were incubated at 60 c for 24 h. the formation of 47 was observed in 87% yield by h nmr integration ( see supporting information , figure s34 , for crude h nmr ) . h nmr ( 400 mhz , d2o ) : h 7.16 ( d , j = 7.5 hz , 1h , h-(c6 ) ) ; 7.00 ( s , 1h , h-(c2 ) ) ; 6.74 ( d , j = 4.3 hz , 1h , h-(c1 ) ) ; 5.81 ( d , j = 7.5 hz , 1h , h-(c5 ) ) ; 4.22 ( dd , j = 5.8 , 4.3 hz , 1h , h-(c2 ) ) ; 3.68 ( ddd , j = 5.6 , 4.8 , 2.7 hz , 1h , h-(c4 ) ) ; 3.60 ( abx , j = 11.8 , 4.8 hz , 1h , h-(c5 ) ) ; 3.53 ( abx , j = 11.8 , 5.6 hz , 1h , h-(c5 ) ) ; 3.50 ( dd , j = 5.8 , 2.7 hz , 1h , h-(c3 ) ) . c nmr ( 101 mhz , d2o ) : c 168.3 ( c = o ) ; 166.1 ( c4 ) ; 157.8 ( c2 ) ; 143.2 ( c5 ) ; 142.6 ( c6 ) ; 129.8 ( c2 ) ; 109.0 ( c4 ) ; 96.7 ( c5 ) ; 69.7 ( c4 ) ; 64.4 ( c2 ) ; 63.7 ( c1 ) ; 63.6 ( c5 ) ; 55.5 ( c3 ) . m / z ) : 352 ( 100% , [ m + h ] ) . hrms ( m / z ) : [ m ] calcd for c13h16n7o4 , 352.1364 ; found , 352.1363

the recent development of a sequential , high - yielding route to activated pyrimidine nucleotides , under conditions thought to be prebiotic , is an encouraging step toward the greater goal of a plausible prebiotic pathway to rna and the potential for an rna world . however , this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides . to address this problem , and further explore prebiotically accessible chemical systems , we have developed a high - yielding , aqueous , one - pot , multicomponent reaction that tethers masked - sugar moieties to prebiotically plausible purine precursors . a ph - dependent three - component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles , which allows the first divergent purine / pyrimidine synthesis to be proposed . due to regiospecific aminoimidazole tethering , the pathway allows n9 purination only , thus suggesting the first prebiotically plausible mechanism for regiospecific n9 purination .

Introduction Results and Discussion Outlook and Summary Experimental Section

we discuss through a new case and a review of the literature the pathological pattern and the management of this uncommon entity . we report the case of a 58 year - old man who presented with a macroscopic hematuria . transurethral bladder resection was consistent with the diagnostic of a poorly differentiated carcinoma infiltrating the bladder 's muscle . lymphoepithelioma - like carcinoma of the bladder is a rare bladder cancer that is important to recognize since it has a favourable prognosis . a 58 year - old north african man presented with a macroscopic hematuria of two weeks duration . pathologic examination of the transurethral bladder resection was consistent with the diagnostic of poorly differentiated carcinoma infiltrating the muscle of the bladder and a radical cysto - prostatectomy was performed . grossly , there was a well demarcated tumour in the dome that measured 2 3 1 cm . tumour cells were characterized by a syncytial growth pattern in a dense lymphoid stroma ( fig . 1 , 2 ) . malignant cells having a syncytial appearance in a lymphoid stroma ( hematoxylin - eosin , original magnification 100 ) . anaplastic cells with vesicular nuclei and prominent nucleoli ( hematoxylin - eosin , original magnification 400 ) . immunohistochemically , most of the tumour cells were positive for epithelial membrane antigen ( ema ) and cytokeratin . lymphoepithelioma like carcinoma ( lelc ) of the bladder is an extremely rare tumour that was first reported by zukerberg et al in 1991(1 ) and since that time , about 50 cases have been reported in the english literature(2 ) . lelc of the bladder is characterized by a syncytial growth pattern of the malignant epithelial cells , indistinct cytoplasmic borders and a non - neoplastic lymphocytic infiltration . according to the proportional expression of lymphoepithelial elements in the tumour mass , tumours are categorized as pure ( all lymph epithelial elements ) , predominant ( more than a half ) or focal ( less than a half)(3 ) . the origin of bladder lelc is unknown ; they probably represent modified urothelial basal cells(4 ) . unlike undifferentiated nasopharyngeal carcinomas , the lelc of the bladder were uniformly epstein - barrvirus ( ebv ) negative in western and asian patients(56 ) , and also in our case . histologically , the lelc of the bladder must be distinguished from large cell lymphomas which are usually metastatic(7 ) , but the immunohistochemical findings using cytokeratin and ema are invaluable to confirm their epithelial origin(7 ) . moreover , identification of a polyclonal lymphoid population is controversial in the diagnosis of lymphoma(7 ) ; immunohistochemistry can be used to detect the malignant epithelial cells in the inflammatory elements and to exclude the diagnosis of chronic cystitis(7 ) . the lelc has a relatively favourable prognosis(347 ) which is attributed to the intense immunological response of the host against the neoplasm(3 ) ; moreover , the inflammatory infiltration causes early symptoms like macroscopic hematuria alerting the patient promptly(4 ) . the combination of transurethral resection of the bladder and adjuvant chemotherapy is probably effective against lelc , particularly in the pure form and focal forms , avoiding radical cystectomy(134 ) . in the present case

context : lymphoepithelioma like carcinoma of the bladder is an extremely rare tumour . we discuss through a new case and a review of the literature the pathological pattern and the management of this uncommon entity.case report : we report the case of a 58 year - old man who presented with a macroscopic hematuria . transurethral bladder resection was consistent with the diagnostic of a poorly differentiated carcinoma infiltrating the bladder 's muscle . a radical cysto - prostatectomy was performed . the pathological examination revealed an ebv negative lymphoepithelioma - like carcinoma of the bladder.conclusion:lymphoepithelioma-like carcinoma of the bladder is a rare bladder cancer that is important to recognize since it has a favourable prognosis .

Context: Case report: Conclusion: Introduction Case Report Discussion

the global incidence of malignant diseases has significantly increased during the last decades in parallel with the rapid industrialization of most countries . in iran , as the third cause of death and the second prevalent chronic diseases , cancers pose a significant health burden . malignancies are considered primarily as environmental diseases with 90 - 95% of cases attributed to environmental factors and 5 - 10% to genetic factors . common environmental factors that are attributable to cancer death include tobacco use ( 25 - 30% ) , dietary habits and obesity ( 30 - 35% ) , infections ( 15 - 20% ) , radiation ( ionizing and non - ionizing , up to 10% ) , tensions , and environmental pollutants . environmental pollution is considered as one of the main causes for some types of cancers . for instance , each year nearly one million ton of lead ( pb ) is added to the soil at global level . this contains large quantities of atmospheric dust , scattering ash , chemical fertilizers used in agriculture , and industrial and urban wastes . although inorganic contaminants and extensive distribution of metals are considered as one of the most likely potential exposure ways for populations , still enough attention is not paid to the environmental factors affecting health . isfahan , a province with an area of about 107,045 square kilometers , equivalent to 6.3% of the total area of iran , is located between 30 degrees 43 minutes and 34 degrees 27 minutes north latitude and 49 degrees 38 minutes and 55 degrees 32 minutes east of the greenwich meridian . this province is also considered as one of the major agricultural poles of the country and the usage of chemical fertilizers in agriculture has been added plenty of pb into the province 's soils . pb is up taken by the plants from contaminated land , thus agricultural products are the most important way to enter this element in the food chain . pb is not biodegradable , does not undergo dissociation , and can not be metabolized in human body . therefore , after entering the body , pb will be deposited and accumulated in blood , skin , organs , and tissues such as fat , muscles , bones , and joints . this study aimed to map pb distribution and the spatial distribution of the ten most common types of cancers in the province . the ten most common cancers in isfahan were determined by using data recorded from 2007 to 2009 by the isfahan cancer registry program of the isfahan provincial health center . we obtained the distribution map of pb in the province of isfahan during 2007 to 2009 from the mineral exploration organization , and the information on soil pb concentration from the isfahan agricultural jihad organization . the pb concentration was documented in three fields of agricultural , non - agricultural , and urban and industrial land . the geographic mapping of cancers and soil pb was then incorporated using geographic information system ( gis ) software . after producing the spatial distribution model , overall 10,142 medical records of patients with documented pathology report of cancer in 2007 to 2009 were recruited from the cancer registry data . most prevalent cancers in the province of isfahan during 2007 - 2009 spatial distribution of pb in 2007 - 2009 is depicted in figure 1 . it shows that pb was more abundant in south , west , center , north , and east of the province . skin cancer was more prevalent in cities of isfahan , najaf abad , kashan , naeen , ardestan , and natanz ( figure 2 ) . the rate of skin cancer was correlated with pb distribution in the province , i.e. it was lower in areas away from pb - contaminated regions . soil lead distribution map in the province of isfahan , iran ( values are presented in km . ) spatial distribution of skin cancer in the province of isfahan , iran ( values are expressed as number of persons . ) spatial distribution of breast cancer in the province of isfahan , iran ( values are expressed as number of persons . ) spatial distribution of hematological malignancies in the province of isfahan , iran ( values are expressed as number of persons . ) spatial distribution of bladder cancer in the province of isfahan , iran ( values are expressed as number of persons . ) spatial distribution of prostate cancer in the province of isfahan , iran ( values are expressed as number of persons . ) as shown in figure3 , the prevalence of breast cancer was also directly related with the soil pb concentration . the cities of isfahan , najaf abad , kashan , borkhar , meymeh , naeen , ardestan , and natanz had the highest prevalence of breast cancer ( figure3 ) . the spatial distribution of hematological malignancies was consistent with the distribution of soil pb throughout the province ( figure4 ) . mapping of bladder and prostate cancers were not associated with soil pb distribution ( figures 5 and 6 ) . plotting the spatial distribution of the most common types of malignant diseases and soil pb concentration showed significant associations between mapping for skin , breast , and hematological malignancies and pb distribution in the province of isfahan . this finding may suggest that soil contamination by pb , and possibly other industrial metals , may contribute in the incidence of cancers . we should acknowledge that in addition to the possible association of soil pb with malignancies , the higher prevalence of diagnosed cancers might have been a result of better medical facilities and health centers in larger cities of the province . however , our data comprised the records from the whole province including smaller cities for three years , and earlier diagnosis of malignancies in larger cities would not affect our findings . although pb naturally exists in the environment , most pb concentrations found in the environment are accumulated from human activities . the industrial pollutants , fertilizers , and other agricultural items are the main sources of pb added to the environment by humans . pb distribution in the areas where the population and human activities are higher is more prominent . the main reason can be contamination by industrial pollution , pb - acid batteries , electronic components , cable sheathing , ammunition , glass , ceramics , pb pipes , paints , alloys , connections , and joints in roofs used for protection against rain . the larger pb particles drop to the ground straightaway and will pollute soils or surface waters , whereas the smaller particles will be transported long distances through air and may remain in the atmosphere . this pb - cycle caused by human production is more extended than the natural pb - cycle . although western lifestyle , obesity , smoking , and epidemiologic transition have crucial roles in the development of non - communicable diseases , including cancers , they can not solely explain the worldwide rapid increase in the incidence of cancers . environmental pollution , including soil contamination by toxic metals during industrialization may be an important contributing factor.[810 ] in 1980 , barrett depicted the spatial distribution as the possible etiology of disease , and proposed the importance of medical geography on human health . he suggested for the first time that a geographic area represents the complex physical , biological , and cultural processes . by analyzing the elements and patterns , it is possible to determine and track the spatial distribution of various diseases . a large body of evidence supports the health hazards of environmental pb exposure.[1217 ] it is estimated that reducing the concentrations of environmental pb can significantly decrease the standardized death rate . a study in taiwan revealed that living in high pb - polluted areas can result in an increased incidence rate of brain cancer . an ecologic study conducted in 2000 - 2007 among chinese population living in nine agricultural villages with high environmental pollution showed significant associations between exposure to multiple heavy metals , including pb , and cancer mortality . our findings on the probable association of pb exposure with breast cancer are consistent with some previous studies.[2123 ] in addition to ecological studies , such association has also been documented in animal models . it is suggested that pb may accelerate tumor growth rates , and may interact with some protective factors against breast cancer development . a growing body of evidence proposed that environmental metal exposure may be associated with changes in epigenetic factors . this association may lead to a possible link between heritable changes in gene expression and disease susceptibility and development.[2527 ] to solve the global problem , soil resources should be tested and evaluated periodically . preventive measures , such as using filters , lime , and other things to prevent excessive entry of sewage into rivers and freshwater resources and soil , should be considered . cultivation of plants like borage or oxtongue plant that absorb toxic elements may be beneficial for reducing pb levels . however , products of such plants should not be consumed in areas with pb - contaminated soil . an intersectoral collaboration is necessary for reducing environmental pb production . on the other hand , increasing public knowledge on how to prevent exposure is another important strategy in this regard . health professionals have a distinctive capacity to increase the knowledge of the whole population through educating their patients . according to the results of this study , not all diseases are caused by inheritance or genetic factors . in fact , environmental factors could also be responsible for some diseases such as malignant . control pb producing industries to improve work - related environmental health and increasing the knowledge of health professionals and the general population in this regard are also of high importance . programs aiming at lowering the cancer risk will thus have to consider effective measures to reduce the production of and exposures to pb and other industrial metals that are currently contaminating the environment .

background : malignancies are primarily environmental diseases mostly attributed to environmental factors . by plotting the prevalence and spatial distribution maps , important differences can be observed in detail . this study aimed to determine the association between map distribution of malignancies and the geological phenomena of lead ( pb ) accumulation in soil in the province of isfahan , iran.methods:spatial distribution maps of malignant diseases were plotted by using data recorded during 2007 to 2009 in the isfahan cancer registry program . data on pb accumulation in soil was obtained from the national geological survey and mineral exploration . pb concentrations were documented in three parts of agricultural , non - agricultural , urban , and industrial land . the geographical mapping of cancers and soil pb were then incorporated into a geographic information system ( gis ) to create a spatial distribution model.results:the spatial distributions of ten common malignant diseases in the province , i.e. skin cancers , hematological malignancies , and breast cancers , followed by other malignancies were scattered based on pb distribution . in fact , common cancers were more prevalent in the parts of the province where soil pb was more abundant.conclusion:the findings of this study underscore the importance of preventing pb exposure and controlling industrial production of pb . the data is also important to establish further effects modeling for cancers . moreover , physicians and health professionals should consider the impact of environmental factors on their patients health .

INTRODUCTION METHODS RESULTS DISCUSION CONCLUSION

one hundred and fifty million people are infected by hepatitis c virus ( hcv ) worldwide , and chronic hepatitis c is now the leading indication for liver transplantation in the us . transmitted principally by blood , hcv is passed by transfusion of inadequately screened blood and blood products , and by intravenous drug use ; sexual and vertical transmission also occur , but at substantially lower rates than in other blood - borne viral infections . unsterile dental equipment , accidental needle punctures in medical facilities , and tattooing are also linked to transmission . following acquisition of infection , 70 to 85% of patients will develop persistent viremia , usually for the duration of their lives . not all of these will develop liver failure , however - infection is often indolent for long periods . nonetheless , two decades after infection about 20% of hcv - infected subjects will have developed end - stage liver disease . hepatitis c is now a more common cause of cirrhosis than alcoholism . though chronic hepatitis is the major result of hcv infection , there are other manifestations of the disease that stem from chronic inflammation and associated immune cell stimulation with cytokine release . these include arthritis , antibody - mediated thrombocytopenia , itching , porphyria cutanea tarda , dermatitis , glomerulonephritis and cryoglobulinemia . hcv is a small ( 50 nm ) single - stranded rna member of the flaviviridae family . the rna has one open reading frame , preceded by a ribosome - binding site within a utr ( untranslated region ) . the open reading frame encodes a 3,011 amino acid protein that is cleaved into several different proteins by proteases of viral and cellular origins . the amino acid sequences include nucleocapsid , envelope , protease , helicase , transmembrane , and rna polymerase proteins . the hcv rna polymerase is highly error prone , and the sequences of hcv genomes display enormous amounts of variation . they differ from one another at nearly one - third of their nucleotide positions , but in practice are defined by mutations in the 5'utr . these six genotypes are stable , heritable variants that differ in their geographic distribution . superimposed on this already substantial variation is the fact that large numbers of mutations occur in any given individual 's hcv isolate over time - that is , as in hiv , each new infection gives rise to a cloud of sequence variants descended from the original infection ; this cloud is sometimes referred to as a ' quasispecies ' . this extraordinary sequence diversity renders the virus very resistant to standard immune responses and inhibits the production of a useful preventive vaccine . the two coat proteins of the virus are heavily glycosylated and recognize receptors on the hepatocyte . though not entirely proven , these receptors are thought to be cd81 , claudin-1 and occludin [ 3 - 5 ] . virions replicate at an enormous rate in hepatocytes , from which they are released into the blood ( they may invade monocytes and b cells as well , but this is uncertain ) . the titer of virus in the blood may therefore provide a fairly accurate index of the hepatic load of virus . recently a relatively well - conserved sequence of the non - coding region has been identified , permitting the development of an inexpensive and highly accurate diagnostic polymerase chain reaction ( pcr)-based blood test for the virus and its titer . this protein activates the janus kinase / signal transducers and activators of transcription ( jak / stat ) signaling pathway to induce the transcription of interleukins and caspases that kill viral loaded cells , but the toxicity of the thrice - weekly treatment was considerable and the original remission rate was only 10 to 15% . particularly poor results were seen in those infected with viral genotype 1 , which accounts for 65% of us cases of hepatitis c - including the bulk of caucasian and the vast majority of american black patients . the addition of ribavirin brought the remission rate up to 20 to 25% , and a change to weekly pegylated interferon alfa further increased the response rate . but the treatment remains difficult , with side - effects that resemble the cytokine - releasing consequences of cancer chemotherapy , and the hemolytic anemia induced by ribavirin weakens patients still further . american patients with genotype 1 continue to respond less well than those with genotypes 2 and 3 , but nonetheless a significant subset of genotype 1 carriers do benefit from treatment . given the toxicities of interferon / ribavirin , it would be very useful to be able to predict which patients might fall into the responder subclass . connected to this , a startling advance has recently been made by ge , thomas and their co - workers , who have shown that a polymorphism in close proximity to the il28b gene that encodes interferon lambda 3 predicts both spontaneous clearance of hcv and response to interferon and ribavirin treatment in genotype 1 hepatitis c infection . clearance and response to therapy are not affected by the polymorphism in those with genotype 2 and 3 infections . the reasons for these disparities are not at all evident , since the genotypes are not associated with any known protein differences . finally , the treatment of hepatitis c - induced chronic liver failure with liver transplantation illustrates the dilemma posed by the growth of technology in medical care . indeed , all such liver transplants become re - infected by persistent virus , and in some patients the march to secondary cirrhosis is accelerated , necessitating a second transplant . ironically , alcoholic cirrhotics are routinely denied transplants in the current donor - liver shortage because it is thought that their bibulous habits can not be broken . yet patients with hepatitis c receive transplants routinely , even though their livers are doomed to be re - infected and cirrhosis induced at a far faster rate than that caused by alcohol . since the natural progression of the disease takes two or more decades , and since most patients come to transplant in their 50 s or 60 s , most do not require a second transplant but the costs of these decisions are massive . hcv : hepatitis c virus ; jak : janus kinase ; pcr : polymerase chain reaction ; stat : signal transducers and activators of transcription ; utr : untranslated region . the authors are grateful to maureen jonas md and chinwe ukomadu md for their valuable help in the preparation of this discussion , and particularly express their appreciation to don ganem md for his careful review of the manuscript and his important suggestions .

hepatitis c is a viral disease transmitted principally by blood , which affects millions of people worldwide . a significant proportion of those affected develop severe liver disease as a result . only a fraction of patients are responsive to interferon treatment , highlighting the need for further research into genetic factors involved in response to therapy in order to optimize treatment . the only current approach for end - stage disease is liver transplant , which ironically does not cure the condition , and thus poses a clinical dilemma in the face of liver - donor shortage .

None Abbreviations Acknowledgements

the patient was seen in 1996 at the age of 32 , with blurred vision in the right eye and headache . there was mild epithelial edema of the cornea with a few keratic precipitates and no cells seen on slit - lamp examination of the right eye . he had five episodes of pss crises over the next 14 years , with a highest iop of 60 mmhg . anterior segment inflammation and elevated iop were controlled after few weeks of treatment with antiglaucoma medication and topical corticosteroids . this was his treatment course after each pss event . at his first medical examination in 1996 , his cup / disc ratio was noted to be 0.4 in the right eye and 0.4 in the left eye . 1a and 1b ) . after his fifth attack , in 2010 , his uncorrected visual acuity was 1.0 in both eyes and a relative afferent papillary defect of right eye was observed . his right optic disc was pale and the field loss in the right eye had progressed to a superior paracentral scotoma ( fig . there was p100 delay of the right eye in the visual evoked potential test ( fig . 4 ) . there were no abnormal findings on brain magnetic resonance image ( fig . pss is a syndrome associated with recurrent anterior segment inflammation and elevated iop . after an attack , iop and facility of aqueous outflow return to normal . the etiology of pss is unknown , although abnormal vascular process , autonomic defect , allergic conditions , variation of developmental glaucoma , association of hla - bw54 , cytomegalovirus , and herpes simplex virus have been suggested [ 6 - 8 ] . the presumed mechanism causing naion was decreased perfusion to the optic disc secondary to the rise in intraocular pressure . risk factors for naion such as old age , small cup to disc ratio , and hypertension were considered when treating pss patients . this is case of optic disc pallor in a patient with recurrent anterior segment inflammation and high intraocular pressure . optic disc perfusion is directly proportional to mean arterial pressure and inversely proportional to iop . we had not found something that explained optic disc pallor excluding high iop . decreased optic nerve perfusion by recurrent high iop might induce optic disc pallor . in conclusion , pss could cause optic disc changes such as glaucomatous change and ischemic change . thoughtful attention and aggressive iop control are required especially in pss patients with prolonged periods of high intraocular pressure and recurrent attacks .

a 32-year - old man with blurred vision in the right eye and headache presented with anterior uveitis , an intraocular pressure ( iop ) of 60 mmhg , an open angle , no visual field defects , and normal optic nerve . he had a history of five previous similar attacks . in each of the previous instances , his anterior uveitis and high iop were controlled with antiglaucoma medications and topical steroids . however , at the fifth attack , his optic disc was pale and a superior paracentral visual field defect was shown . brain magnetic resonance image studies were normal . this case represents that a recurrent posner - schlossman syndrome ( pss)-induced optic disc atrophy likely due to ocular ischemia caused by a recurrent , high iop . although pss is a self - limiting syndrome , we should manage high iop and prevent ischemia of the optic nerve head by treating with ocular antihypertensive medications .

Case Report Discussion

obtaining a satisfying long - term survival of thas in patients younger than 40 years remains a challenge . young patients must function longer with their tha than the typical patient who has a tha , and they also engage in a higher level of activity , which is associated with higher revision rates [ 19 , 27 ] . therefore , this population is more dependent on durable implants with excellent long - term survival . although stem survival is acceptable in most studies , in general , cup survival is the weakest link in patients younger than 40 years [ 5 , 8 , 9 , 15 , 18 , 20 , 25 ] . the difference in reported survival rates between the cup and stem varies from 1% ( 97% [ stem ] versus 96% [ cup ] ) to 11% ( 98.3% [ stem ] versus 87.6% [ cup ] ) . despite attempts to improve cup designs and using new materials in tha one popular option is to implant uncemented acetabular cups in young patients as part of a total uncemented tha or hybrid tha ( uncemented cup , cemented stem ) . although cement in young patients commonly is not used [ 1 , 24 , 35 ] , we always have implanted cemented cups in patients of all ages , but with one substantial modification : in all patients with substantial acetabular bone stock deficiencies , we have reconstructed this bone stock loss using impaction bone grafting with a cemented cup . secondary osteoarthritis resulting from underlying diseases in these young patients often is seen with associated loss of acetabular bone stock ( for example , in developmental dysplasia of the hips and juvenile rheumatoid arthritis ) . with this approach using cemented cups in young patients for many years , we asked whether there were any differences between cemented cups in young patients ( younger than 40 years ) with and without reconstruction with impaction grafting concerning ( 1 ) clinical scores , ( 2 ) revisions , ( 3 ) complications , ( 4 ) radiographic appearances , ( 5 ) polyethylene wear , and ( 6 ) survival . we retrospectively reviewed prospectively collected data of all 130 patients ( 175 hips ) who had a primary tha in our department between january 1988 and july 2004 and who were younger than 40 years at the time of index surgery . we used a cemented femoral stem and cemented acetabular polyethylene cup in all patients . in patients with acetabular bone deficiencies , these deficiencies were reconstructed with the impaction grafting technique . the decision to use bone impaction grafting a trial cup was placed on the transverse ligament ; in the case of a protrusion hip or a superolateral rim defect , a reconstruction was performed . eighty - four hips ( 48% ) had impaction grafting whereas 91 ( 52% ) did not have impaction grafting . because a cemented tha was our only treatment technique , patients with all diagnoses were included ( table 1 ) . the majority ( 62% ) of the patients had developmental dysplasia of the hips , rheumatoid arthritis , or corticosteroid - induced avascular necrosis . fifty - five ( 42% ) patients were males and 75 ( 58% ) were females . eighty - nine ( 51% ) thas were on the left side and 86 ( 49% ) were on the right . the average age of the patients at index surgery was 31.3 years ( range , 1639 years ) . the mean body mass index was 25.5 ( range , 17.936.3 ) . according to the classification of charnley , 46 hips were in category a , 71 in b , and 58 in c. we followed all patients in this prospective cohort on a regular basis and the minimum followup was 2 years ( average , 8.1 years ; range , 2.018.5 years ) after surgery . during followup , six patients ( eight hips ) died of causes not related to the hip or hip surgery . all patients who died were followed on a regular basis and their data included ; none had revision surgery . of the original group of 175 cups , the data of only one patient were incomplete . based on a telephone interview , the prosthesis of this patient functioned well ; however , a recent radiograph was missing.table 1indications for primary tha with and without reconstruction with bone impaction graftingindicationnumber of hipswithout bone impaction graftingwith bone impaction graftingtotaldevelopmental dysplasia of the hip103242rheumatoid arthritis171027perthes disease448avascular necrosis of unknown cause628epiphyseal dysplasia527posttraumatic osteoarthritis246bechterew s disease325posttraumatic avascular necrosis415morquio s disease134epiphysiolysis134septic coxitis213protrusio acetabuli033osteomyelitis033spontaneous fusion of the hip of unknown cause112osteogenesis imperfecta022polycystic disease of unknown cause202psoriatic arthritis011gigantism of unknown cause011pseudohypoparathyroidism101monoarthritis of unknown cause011alcohol - induced avascular necrosis101corticosteroid - induced avascular necrosis31839 systemic lupus erythematosus9 kidney transplantation / nephropathy7 subarachnoid hemorrhage4 non - hodgkin s lymphoma3 crohn s disease3 cerebral aneurysm2 head trauma2 thrombocytopenia2 hypothalamus hormone substitution1 germ cell tumor1 aplastic anemia1 pituitary adenoma1 wegener s disease1 acute lymphatic leukemia1 meduloblastoma1total9184175 indications for primary tha with and without reconstruction with bone impaction grafting we categorized acetabular defects in accordance with the classification system of the american academy of orthopaedic surgeons . type i segmental deficiencies occurred in 16 hips , type ii cavitary defects in 39 hips , and type iii combined deficiencies in 29 hips . one patient ( two hips ) had ankylosis of the hips , a type v deficiency . using impaction grafting , we reconstructed all deficiencies , including mild cavitary defects ; however most were larger defects . differences between the two groups ( with and without impaction grafting ) were analyzed regarding diagnosis and gender ( chi square test , both p = 0.001 ) . in the group with an acetabular reconstruction , a larger proportion was female and was diagnosed with developmental dysplasia of the hips compared with the group without reconstruction . there were no differences regarding age at surgery , side , bilateral thas , followup , type of cup used , cup inner diameter , and body mass index between the two groups . two - thirds of the operations ( 67% ) were performed by or under the supervision of two senior faculty orthopaedic surgeons ( bws , jwmg ) . a posterolateral approach without trochanteric osteotomy was used in all hips , with the exception of two . intraoperatively , in one patient , a preplanned sugioka procedure was converted to a tha ; however , a trochanteric osteotomy already had been performed . in the other patient , a trochanteric osteotomy was performed in a technically demanding hip with a short femoral neck . in one patient , all acetabular deficiencies were reconstructed ( with the exception of one case ) with impaction grafting using autografts and/or allografts in 84 hips ( 48% ) ; this technique has been described in detail [ 2830 ] . segmental bone defects first were reconstructed with wire meshes before the morselized bone graft was impacted and a conventional full polyethylene cup was cemented . in one patient , we reconstructed a lateral rim deficiency without impaction grafting using a solid autograft fixed with two screws . in one of the ankylosed hips ( type v deficiency ) we used allografts only with impaction grafting in four hips ( 4.8% ) , autografts only in 72 hips ( 85.7% ) , and combined allografts and autografts in eight hips ( 9.5% ) . allografts were used when the original femoral head was not large enough to reconstruct the defect or in cases with pathologic femoral heads ( for example , avascular necrosis of the femoral head ) . in three cases , instead of a solitary metal mesh , a solid fragment was used in combination with impaction grafting . in two of these cases , a minor segmental defect in the medial wall was closed using a cortical - trabecular fragment of a femoral head . a wire mesh was placed medial on top of the fragment and the acetabulum was reconstructed with impaction grafting . in the third case , a cortical head fragment was used to support the anterior rim together with a rim mesh in a reconstruction . the number of femoral heads used as grafts varied from one to four . in 40 hips ( 48% ) , metal wire mesh was used for acetabular reconstruction with impaction grafting ( 10 medial wall meshes , 39 rim meshes ) . in nine early cases , we placed a mesh on top of the bone graft just before cementation , but this mesh was not part of a segmental defect reconstruction . however , after we realized this mesh did not add any stability to the reconstruction and there were no signs of damaging of the graft or graft healing by direct contact with cement , we abandoned the use of a mesh for this purpose . we used 79 ( 45% ) exeter contemporary cups with an inner diameter of 28 mm ( n = 75 ) and 22.225 mm ( n = 5 ) ( stryker howmedica , newbury , uk ) , 71 ( 41% ) charnley elite cups with an inner diameter of 22.225 mm ( n = 6 ) or 28 mm ( n = 65 ) ( depuy , leeds , uk ) , and 25 ( 14% ) mller / allopro cups with an inner diameter of 32 mm ( n = 19 ) , 28 mm ( n = 2 ) , or 22.225 mm ( n = 4 ) ( sulzer , winterthur , switzerland ) . for the femoral component , we used an exeter stem in 111 cases , a charnley elite stem in 48 cases , and a mller stem in 16 cases . all femoral heads used were made of a cobalt - chrome alloy ; no ceramic implants were used . we cemented acetabular components with a third - generation cementing technique . in the directly cemented cups , after reaming , multiple small drill holes were made with a 2.6-mm drill . after using pulse lavage , vacuum - mixed cement was injected directly from the cement gun and the cement was pressurized by a seal . in cases of reconstruction with bone grafts , we reamed the acetabulum , made multiple drill holes in sclerotic areas , and irrigated the acetabulum . again , vacuum - mixed cement was injected and pressurized and the cup was inserted . before 1989 , we used palacos bone cement ( merck , darmstadt , germany ) ; however , since 1989 , we have used surgical simplex ( stryker howmedica ) . in 165 cases ( 94% ) , all patients received antibiotic prophylaxis consisting of 2 g cefazolin intravenously just before surgery . other precautionary measures to prevent infections were use of an operating theater with laminar airflow and use of two pairs of sterile gloves . postoperatively , all patients received thrombosis prophylaxis with low - molecular - weight heparin for 6 weeks , or before 1999 , with acenocoumarol ( the individual dosage regimens regulated with regular coagulation tests ) for 3 months . to prevent heterotopic ossification , we used nonsteroidal antiinflammatory drugs ( nsaids ) for 7 days . in six patients in whom nsaids were contraindicated , we administered one dose ( 7 gy ) of radiotherapy 1 day postoperatively . patients without acetabular reconstruction were mobilized under supervision of a physiotherapist after 1 or 2 days . full weightbearing was increased in 2 to 6 weeks with the aid of one or two crutches . the patients who underwent impaction grafting were mobilized according to a modified protocol ; in the first 6 weeks , only 10% weightbearing was allowed and then 6 to 12 weeks of 50% weightbearing using two crutches was allowed . thirty - one hips had such an extensive reconstruction of major defects that several weeks of bed rest were maintained ranging from 1 to 6 weeks . routine followups were scheduled at 6 weeks ; 3 , 6 , and 12 months ; and yearly or biannually thereafter . at our outpatient clinic , student researchers not participating in the treatment performed clinical analysis using the harris hip score , the oxford hip questionnaire score ( since 1998 ) , and visual analog scales for pain during rest and physical activity on a scale from 0 ( no pain ) to 100 ( unbearable pain ) . we report the clinical scores of all patients excluding the 21 patients whose hips were revised during followup . all anteroposterior pelvis and lateral radiographs of all hips were analyzed on a consensus basis by two of the authors ( dcjdk , bws ) . radiographic evaluation included assessment of cup position , loosening of the acetabular component , polyethylene wear , presence of osteolysis , structural quality of the bone graft , application and position of the meshes , migration , heterotopic ossification , and fracture of the cement , mesh , or prosthesis . radiolucent lines and osteolysis were recorded according to the three acetabular zones as described by delee and charnley . radiographic loosening was defined as 2 mm or greater demarcation in two or three zones around the acetabular component , progressive demarcation , 3 mm or greater component migration , 5 or greater component tilting , and/or cement or prosthesis fracture . we determined cup migration ( > 3-mm shift in any direction or > 5 tilting ) in relation to the interteardrop line instead of the kohler line . graft incorporation was defined as the presence of the crossing of trabecular bone on the bone - graft interface on the radiographs . clinical failure was defined as the need for revision of the acetabular component for any reason . we calculated kaplan - meier curves to study the survival ( time to revision ) . the end points were ( 1 ) cup revision for any reason , ( 2 ) cup revision for any reason excluding infections , ( 3 ) cup revision for aseptic loosening , and ( 4 ) radiographic signs of cup loosening . with an average followup of 8.1 years , 30% of all patients had a followup longer than 10 years . the log - rank test was used to test the differences in survival between cups with and without impaction grafting . differences in outcomes between the groups were determined with the student s t - test ( continuous variables after checking for normal distribution ) or chi square test ( nominal variables ) . the outcome of the harris hip score and the oxford hip questionnaire score improved ( p < 0.0001 ) after surgery for both groups ; there were no differences in preoperative and postoperative clinical outcomes between the cups with and without acetabular reconstruction ( table 2 ) . the postoperative experienced pain score was low.table 2outcome of clinical questionnairesquestionnairepreoperative scorepostoperative scorewith bone impaction graftingwithout bone impaction graftingindependent t test p valuewith bone impaction graftingwithout bone impaction graftingindependent t test p valueharris hip score48 ( 1581 ) ( n = 46)50 ( 2882 ) ( n = 50)0.67292 ( 35100 ) ( n = 72)96 ( 12100 ) ( n = 73)0.546oxford hip questionnaire score39 ( 3052 ) ( n = 9)38 ( 1252 ) ( n = 15)0.10017 ( 1245 ) ( n = 70)15 ( 1241 ) ( n = 72)0.151vas pain at restnana0 ( 075 ) ( n = 70)0 ( 070 ) ( n = 71)0.260vas pain during physical activitynana10 ( 090 ) ( n = 70)0 ( 0100 ) ( n = 71)0.267values are expressed as median , with range in parentheses ; vas = visual analog scale ; na = not available . outcome of clinical questionnaires values are expressed as median , with range in parentheses ; vas = visual analog scale ; na = not available . the number of revisions in the groups with and without bone grafts was not different ( p = 0.152 ) . at last followup , 21 of the 175 cups ( 12% ) had been revised , seven of which had reconstruction with impaction grafting ( table 3 ) . reasons for revision were infection ( eight ) , recurrent dislocations ( two ) , traumatic loosening ( one ) , and aseptic loosening ( 10 ) . the cup only was revised in eight cases and the cup and stem were revised in two cases . four of the 10 revised cups had reconstruction with impaction grafting and six cups were implanted with standard techniques without any graft . the failed cups reconstructed with impaction grafting were revised after 4.1 , 9.8 , 16.2 , and 16.8 years ( average , 11.7 years ) . the six directly cemented cups were revised after an average of 4.0 years ( range , 1.110.0 years ) . the time to revision for aseptic loosening was longer ( p = 0.032 ) for the reconstructed cups with impaction grafting than for the cups implanted with standard techniques . the eight infected hips ( 4.6% ) all had revision because of culture - proven infection of the implant . the average time to revision for septic loosening was 5.3 years ( range , 2.28.1 years ) . staphylococcus epidermidis was isolated in three , staphylococcus aureus in two , proprioni in two , pseudomonas aeruginosa in one , and streptococcus oralis in one . as a result of recurrent dislocations , two cups ( 1.1% ) one implant ( 0.6% ) was radiographically and clinically loose after trauma and needed revision of both components.table 3overview of the revised cups ( n = 21)patientyears to revisioncausepart revisedbone impaction graftingindicationyears to radiographic looseningprevious hip operations 57.3infectionthanocorticosteroids ( systemic lupus erythematosus)no206.1infectionthayesavascular necrosis of unknown causeyes505.7infectionthanocorticosteroids ( crohn s disease)2.6yes688.1infectionthanorheumatoid arthritisno875.3infectionthanodevelopmental dysplasia of the hip5.2yes1044infectionthanocorticosteroids ( subarachnoid bleeding)no1133.4infectionthayescorticosteroids ( pituitary adenoma)yes1232.2infectionthanomedial column fracture0.5yes1118.6recurrent dislocationscupnoposttraumatic coxarthrosisno1603.5recurrent dislocationscupyescorticosteroids ( cerebral aneurysm)yes8410.3traumatic looseningthanocorticosteroids ( head trauma)9.9no294.1aseptic looseningcupyesrheumatoid arthritis4no412.3aseptic looseningcupnocorticosteroids ( systemic lupus erythematosus)2.2yes453.1aseptic looseningcupnospontaneous fusion of unknown cause0.3no491.1aseptic looseningcupnocorticosteroids ( kidney transplantation)4.2no7716.8aseptic looseningcupyesposttraumatic coxarthrosis16.6yes789.8aseptic looseningcupyescoxarthritis9.8yes7916.2aseptic looseningthayesdevelopmental dysplasia of the hip16.2no8210aseptic looseningcupnodevelopmental dysplasia of the hip5.2no906.4aseptic looseningthanodevelopmental dysplasia of the hipno1531.1aseptic looseningcupnoepiphysiolysisyes overview of the revised cups ( n = 21 ) we observed similar ( p = 0.959 ) numbers of overall complications in the groups with and without bone grafts . however , dislocations were more common ( p = 0.045 ) in the group without bone grafts than in the group with bone grafts ( 15 versus 5 , respectively ) . patients without reconstruction with impaction grafting had an increased dislocation chance of 1:2.9 . during followup , there were nine intraoperative complications and 30 postoperative complications ( table 4 ) . one additional stem was revised because of aseptic loosening and two femoral heads were exchanged because of recurrent dislocations . seven hips underwent additional surgery because of postoperative complications ( table 4).table 4overview of complicationstype of complicationnumberintraoperative complications ( n = 9 ) entrapment of sciatic nerve during reposition , permanent damage1 false route femur1 incomplete femoral fracture2 malposition cup1 malposition stem1 instrument failure1 suspicion of breakthrough of sterility2postoperative complications ( n = 30 ) superficial wound infection3 single dislocation9 recurrent dislocations6 sensory nerve palsy4 sensory and motor nerve palsy1 hematoma6 bleeding after 4 months1heterotopic ossifications ( n = 44 ) brooker class i15 brooker class ii19 brooker class iii10postoperative complications leading to revision ( no cup revision ) ( n = 3 ) stem revision for aseptic loosening1 head exchange because of recurrent dislocations2postoperative complications requiring surgical intervention ( no revision ) ( n = 7 ) deep wound infection4 heterotopic ossifications1 traumatic dislocation1 persistent motor and sensory nerve palsy1 overview of complications there were no differences between the cups with and without acetabular reconstruction concerning the occurrence of cup migration , radiographic loosening , or the presence of osteolysis , cysts , and abnormal cup position ( table 5 ) . cups with impaction grafting had fewer radiolucent lines ( p = 0.02 ) and fewer lines in zone i ( p = 0.001 ) ( table 5 ) . all lines , except two , were on the bone - cement interface . in 28 ( 48% ) of the 58 cups with radiolucent lines , the lines were progressive . of the 175 hips , 160 were radiographically stable ( fig . 1 ) . fifteen cups were difficult to evaluate because of overlap of the metal mesh ( 11 zone i ; four zones i + ii ) . we observed graft osteolysis in only one patient with impaction grafting ; the hip revised because of traumatic loosening had a fracture in zone ii of the acetabulum ; no other fractures were seen . fifteen ( 8.6% ) cups were radiographically loose , three had cup migration ( after 1.8 , 9.8 , and 11.2 years postoperatively ) , and 12 had evident radiolucent lines in all zones and/or severe osteolysis ; 12 of these cups were revised ( table 3).table 5radiographic findings of all cups*radiographic findingallwith bone impaction graftingwithout bone impaction graftingp value ( where appropriate)radiographic loosening155100.608cup migration3120.234radiolucent lines5818400.02 zone i183150.001 zone ii202 zone iii1798 zones i + ii422 zones ii + iii523 zones i + iii514 zones i + ii + iii716osteolysis11560.861 zone i725 zone ii110 zone iii320cysts1000.033 zone i100 zone ii000 zone iii000cup position neutral position ( 3555)1607783 abnormal position15780.914 vertical ( > 55)1266 horizontal ( < 35)312polyethylene wear mean nonrevised cups ( mm / year)0.0800.0760.0840.539 mean revised cups ( mm / year)0.2140.1820.2300.525 * total cups ( n = 175 ) ; cups with ( n = 84 ) or without ( n = 91 ) reconstruction with bone impaction grafting.fig . 1a cthe radiographs illustrate reconstruction of the acetabuli in a 34-year - old woman with bilateral ddh ( crowe grade 3 ) . ( b ) an anteroposterior radiograph taken immediately postoperatively shows the thas with the acetabuli reconstructed with impaction grafting . ( c ) an anteroposterior radiograph taken 12 years postoperatively shows the thas remain radiographically stable , but brooker classes iii ( left ) and i ( right ) heterotopic ossifications are visible . radiographic findings of all cups * * total cups ( n = 175 ) ; cups with ( n = 84 ) or without ( n = 91 ) reconstruction with bone impaction grafting . the radiographs illustrate reconstruction of the acetabuli in a 34-year - old woman with bilateral ddh ( crowe grade 3 ) . ( b ) an anteroposterior radiograph taken immediately postoperatively shows the thas with the acetabuli reconstructed with impaction grafting . ( c ) an anteroposterior radiograph taken 12 years postoperatively shows the thas remain radiographically stable , but brooker classes iii ( left ) and i ( right ) heterotopic ossifications are visible . there was no difference in polyethylene wear rates between the cups with and without impaction grafting ( p = 0.539 in 154 unrevised cups and p = 0.525 in the 21 revised cups ) ( table 5 ) . when looking at all cups ( with and without acetabular reconstruction ) , the revised and radiographically loose cups had more wear compared with the cups that were not revised ( both p < 0.0001 ) . patients with an abnormal position of the cup had similar ( p = 0.196 ) polyethylene wear rates to those who had a normal position . analysis of polyethylene wear rates of cups with different inner diameters showed no differences ( independent t test , 22 versus 28 mm : p = 0.135 , 22 versus 32 mm : p = 0.484 , 28 versus 32 mm : p = 0.620 ) . there were no differences in survival after 10 years between the groups with and without bone impaction grafting ( table 6 ) . the midterm survival rates of all cemented polyethylene cups varied from 85% to 92% at 10 years with four end points ( table 6 ; figs . 2 , 3 ) . cup survival with an end point of radiographic loosening was 89% ( 95% confidence interval , 83%95%).table 6the 10-year survival rates*end pointall cupswithout bone impaction graftingwith bone impaction graftinglog - rank p valuerevision for any reason 85% ( 78%92%)79% ( 68%90%)91% ( 82%99%)0.21revision for any reason excluding infections 91% ( 85%97%)87% ( 78%99%)94% ( 87%100%)0.56revision for aseptic loosening92% ( 87%98%)90% ( 81%99%)95% ( 89%100%)0.73 * kaplan - meier estimates ; 95% confidence interval in parentheses.fig . 2a bkaplan - meier survival curves with 95% confidence intervals ( broken lines ) of all cups with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . 3a bkaplan - meier survival curves of cups without impaction grafting ( thick broken line , 95% confidence intervals in thin broken lines ) and cups with impaction grafting ( thick solid line , 95% confidence intervals in thin solid lines ) with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . the 10-year survival rates * * kaplan - meier estimates ; 95% confidence interval in parentheses . kaplan - meier survival curves with 95% confidence intervals ( broken lines ) of all cups with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . kaplan - meier survival curves of cups without impaction grafting ( thick broken line , 95% confidence intervals in thin broken lines ) and cups with impaction grafting ( thick solid line , 95% confidence intervals in thin solid lines ) with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . the use of cemented tha in young patients is not very popular and most surgeons will use uncemented or will resurface hips in these patients . however , we have continued to use only cemented implants in tha even in young patients . in our view , the real challenge in tha in these young patients is to manage the commonly seen acetabular deficiencies . in cases of acetabular defects , we reconstruct these deficiencies with impaction bone grafting . we questioned whether there was a difference in clinical outcome , revisions , complications , radiographic appearances , polyethylene wear , and survival between the cups implanted with an acetabular reconstruction with impaction grafting and those implanted with standard cementing techniques . our study has several limitations : short followup , lack of assessment of activity levels , clinical interobserver variability , heterogeneous group , no comparison with other reconstruction techniques , and different types of implants used . with no patients lost to followup , our followup is representative and reliable for the midterm results , and long - term followup ( > 15 years ) was not available at the time of this review . our results can be biased by an important factor we did not evaluate : the level of activity . theoretically , with restoration of the affected hip(s ) into well - functioning artificial joints , most patients will increase their level of activity . however , young patients undergoing tha with acetabular deficiencies and therefore more complex reconstructions could still have a lower level of activity after surgery relative to primary cemented cups . however , the average wear of the cups with impaction grafting was the same as the cups without impaction grafting ( both 0.08 mm / year ) . provided that activity is a major cause of polyethylene wear , this might imply the level of activity is similar in these two groups . several studies suggest the revision and polyethylene wear rates are correlated to level of activity [ 2 , 19 , 27 , 31 , 38 ] . additional research on level of activity and impaction grafting in young patients is necessary to confirm this hypothesis . the clinical questionnaires were obtained by student researchers who did not participate in the treatment . multiple researchers were involved in the data collection and interobserver variability has not been tested ; however , all researchers were trained and supervised to obtain these questionnaires correctly . the clinical scores were comparable between the two groups and comparable to published scores ( table 7 ) . although the cups reconstructed with bone impaction grafting were the more demanding procedures , no clinical differences were seen.table 7reported outcomes of the harris hip score in patients < 40 years for primary thastudyquestionnairepreoperative scorepostoperative scorepaired t - test p valuechiu et al . harris hip score44 ( 26 - 74 ) 88 ( 74 - 99 ) < 0.001duffy et al . harris hip score51 92 < 0.001current study with bone impaction graftingharris hip score48 ( 1581 ) 92 ( 35100 ) < 0.001 without bone impaction graftingharris hip score50 ( 2882)96 ( 12100 ) < 0.001values are expressed as median ( current study ) or means ( other studies ) , with range in parentheses . reported outcomes of the harris hip score in patients < 40 years for primary tha values are expressed as median ( current study ) or means ( other studies ) , with range in parentheses . although revision rates in both groups were comparable , the time to revision was longer in the cups reconstructed with bone impaction grafting . we have no clear explanation for this observation ; possibly the cement - bone interface was better in cups with bone impaction grafting with better interdigitation of the cement into the bone . this also may explain the lower incidence of radiolucent lines in the cups reconstructed with bone impaction grafting . the number of revisions for septic loosening was relatively high during this midterm followup study ( 4.6% ) . only one septic loosening likely was related to the surgery ; we considered all other infections acute hematogenous infections of previously well - functioning prostheses . the use of corticosteroids and newer rheumatic disease - modifying drugs , which were used in most of the infection cases , can explain this higher risk of infection [ 3 , 4 ] . sochart and porter had only two infections in their study , but both were in patients with rheumatoid arthritis . still , our revision rate for septic loosening of 4.6% is relatively high in contrast to other studies , such as that of joshi et al . , with an infection rate of 1.3% . remarkably , many septic loosenings occurred late ( > 2 years postoperatively ) . the cups reconstructed with impaction grafting showed fewer complications by having fewer dislocations than the cups implanted by standard techniques . this might be attributed to the different mobilization protocol for the patients who received cups with impaction grafting . however , subluxation rates in young patients having tha have been reported to be as much as 18.2% . the overall complication rate of 17% ( 30 postoperative complications ) is also relatively high . reported a complication rate of 11.5% in cemented hips and duffy et al . reported a complication rate of 12% during the perioperative period . this is consistent with previous reports showing wear particles are associated with osteolysis in tha [ 27 , 33 , 37 ] . the average wear rate of the cups of 0.08 mm / year is within the normal limits , keeping in mind that wear in younger patients can be 33% to 40% higher than wear in older patients . in a large study of 226 hips in patients younger than 40 years with a charnley prosthesis , sochart and porter reported an average wear rate of 0.08 to 0.10 mm / year in the nonrevised cups , which is comparable to our results . . found a correlation between inclination of the cup and higher / lower wear rates . however , we did not observe higher wear with abnormal position or inner cup diameter . the observed overall midterm survival of cemented polyethylene cups in patients younger than 40 years in our study was acceptable . especially in these young patients , there is a need for total hip implants with proven long - term survival . although the use of uncemented prostheses in these young patients is very popular , literature regarding long - term outcome of tha in patients younger than 40 years concerns mainly studies of cemented implants and less about uncemented implants ( table 8) [ 5 , 8 , 9 , 15 , 18 , 20 , 25 , 3234 ] . a limitation of the reported midterm or long - term results of uncemented cups is the fact that in these studies first - generation uncemented cups were used . the only report of uncemented cups at 15 years after surgery with an end point of revision for any reason showed a survival rate of 54% . this is less favorable than the results of cemented cups at that time ( table 8) . sochart and porter had survival rates of 71% and 68% at 20 and 25 years , respectively , for cemented charnley cups . the survival of the acetabular uncemented cups with an end point of revision for aseptic loosening in patients younger than 40 years reported in one study was 85% , in contrast to a survival rate of 96% after 10 years of the charnley cups in the study by joshi et al . . we found a survival rate with cemented cups of 92% at 10 years with an end point of revision for aseptic loosening.table 8long - term acetabular cup survival rates in patients younger than 40 yearsstudynumber of hipsage ( years)*followup ( years)*type of cupsurvival5 years7 years10 years15 years20 years25 years30 yearsuncementedbizot et al . 8732.3 ( 1740)7.7 ( 019)screw - in alumina insert94.7 ( 68.599.2)88.8 ( 62.097.1)88.8 ( 62.097.1)cerapress alumina10095.1 ( 69.899.3)cerafit alumina10094.3 ( 66.399.2)duffy et al . 8232 ( 1739)10.3 ( 1014)pca , osteonics , h - g porousaseptic : 84.6 ( 7693)excl inf : 81.8 ( 7391)mcauley et al . 25633.2 ( 1640)7.3 ( 019)duraloc , aml , arthropor , triloc , h - g , solution97.4 ( se 2.2)87.6 ( se 6.0)53.8 ( se 13.9)odent et al . 6218.3 ( 11.831)6 ( 313)zweymuller90.1 ( se 7.1)cementedbizot et al . 4132.3 ( 1740)7.7 ( 019)plain alumina97.3 ( 81.999.6)94.1 ( 78.198.5)90.4 ( 73.096.8)78.9 ( 54.791.1)chiu et al . 4728.8 ( 1739)14.9 ( 721)charnley polyethylene86.3 ( 75.597.1)27.0 ( 10.743.3)chmell et al . 6619.9 ( 1129)15.1 ( 1122)trapezoidal , aufranc - turner , custom - madeaseptic : 98 ( se 1.6)aseptic : 97 ( se 2.8)aseptic : 84.5 ( se 4.7)aseptic : 70 ( se 7.3)aseptic : 40 ( se 15.6)joshi et al . 21832 ( 1739)16 ( 1024)charnley polyethyleneaseptic : 99 ( se 0.5)aseptic : 96 ( se 1.4)aseptic : 91 ( se 2.3)aseptic : 84 ( se 4.6)sochart and porter 22631.7 ( 1739)19.7 ( 230)charnley polyethylene93 ( 9096)71 ( 6577)68 ( 6175)sochart and porter 8324.9 ( 1729)20 ( 5.230)charnley92 ( 8598)70 ( 6081)68 ( 5779)sochart and porter 4328.8 ( 1939)22.4 ( 0.130.3)charnley73 ( 6184)70 ( 5783)current study17531.0 ( 1639)8.1 ( 2.018.5)exeter , charnley , mller / allopro85 ( 7892)aseptic : 92 ( 8798)with impacted bone grafts91 ( 8399)aseptic : 95 ( 89100)studies published through 2007 ; * values are expressed as mean , with range in parentheses ; values are expressed as percentage , with 95% confidence interval in parentheses ; deceased and revised excluded ; lost to followup , deceased excluded , only chinese patients ; aml = anatomic medullary locking ; pca = porous - coated anatomic ; excl inf = excluding infections ; h - g = harris - galante ; se = standard error . long - term acetabular cup survival rates in patients younger than 40 years studies published through 2007 ; * values are expressed as mean , with range in parentheses ; values are expressed as percentage , with 95% confidence interval in parentheses ; deceased and revised excluded ; lost to followup , deceased excluded , only chinese patients ; aml = anatomic medullary locking ; pca = porous - coated anatomic ; excl inf = excluding infections ; h - g = harris - galante ; se = standard error . a remarkable finding of our study was the survival of cups with acetabular reconstructions with impaction grafting was at least comparable to the survival of standard cemented cups , especially considering the more difficult hips of our study population needed reconstruction with impaction grafting . our data on the cemented cups with impaction grafting showed similar survival , where rather lower survival rates would be expected . the outcome of these cups reconstructed with impaction grafting even fulfilled the nice criteria ( a survival of > 90% after 10 years ) , with a survival rate of 91% at 10 years with an end point of revision for any reason . the survival rates of the cemented cups in our study are comparable to those reported for cemented cups [ 8 , 18 , 34 ] . although cemented cups are not commonly used in young patients , our data suggest cemented conventional polyethylene cups are still a good option in tha in young patients . even reconstruction of ( severe ) acetabular deficiencies with impaction grafting and a cemented conventional polyethylene cup produced very acceptable survival rates , comparable to the rates of cemented cups implanted in acetabuli without deficiencies with standard cementing techniques .

although uncemented cup implants frequently are used in young patients , we believe long - term survival rates of cups in these patients are somewhat disappointing , and therefore we have continued to use cemented cups in primary tha , even in young patients . however , in cases of acetabular bone stock defects , we also use bone impaction grafting . we prospectively followed 130 patients with 175 cemented cups ; no patients were lost to followup . the mean age of the patients at surgery was 31 years ( range , 1639 years ) . an acetabular reconstruction with bone impaction grafting was performed in 84 hips ( 48% ) . the minimum followup was 2 years ( average , 8.1 years ; range , 2.018.5 years ) . twenty - one of the 175 cups ( 12% ) were revised at an average of 8.1 years ( range , 2.018.5 years ) . reasons for revision were infection ( one early , seven late ) , recurrent dislocations ( two ) , traumatic loosening ( one ) , and aseptic loosening ( 10 ) . the 10-year survival rate of all cemented cups with end point of revision for any cause was 85% . survival with end point of aseptic loosening of all cups was 92% . survival with end point of revision for aseptic loosening was 90% for the cups without impaction grafting and 95% for the cups with impaction grafting . we believe cemented acetabular cups in young patients have acceptable midterm survival ; however , in the case of acetabular bone defects , we recommend reconstruction with impaction grafting.level of evidence : level iii , therapeutic study . see the guidelines for authors for a complete description of levels of evidence .

Introduction Materials and Methods Results Discussion

more than 90% of the increasing radiative forcing imbalance is reflected as global ocean warming [ alexander et al . , the deceleration of the upper ocean heat storage since the mid2000s [ levitus et al . cheng and zhu , 2015 ] has instigated an active search for the missing heat in the deep ocean [ meehl et al . , 2011 ; trenberth and fasullo , 2010 ; katsman and van oldenborgh , 2011 ; balmaseda et al . , 2013 ; kosaka and xie , 2013 ] . the enhanced downward heat transfer necessary for this vertical heat redistribution has been proposed to be related to the hiatus in global warming , although the hiatus started earlier and lasted for the 15 years period 19982013 [ trenberth et al . two natural mechanisms have been proposed to explain the deceleration of the rise in global mean surface temperature and the efficient transfer of heat to the deep ocean : ( i ) the intensification of zonal winds causing la nialike conditions in the tropical pacific [ meehl et al . , 2011 ; trenberth and fasullo , 2010 ; katsman and van oldenborgh , 2011 ; balmaseda et al . , 2013 ; kosaka and xie , 2013 ] and ( ii ) decadal variations in the strength of deep convection and currents in the north atlantic [ meehl et al . , 2011 ; katsman and van oldenborgh , 2011 ; chen and tung , 2014 ; drijfhout et al . , modeling simulations show that la nia surface cooling in the eastern and central tropical pacific can decrease the rate of increase in atmospheric temperature ( although the similarity of observed and simulated deceleration is still debated [ trenberth , 2015 ; karl et al . , 2015 ] ) and induce an anomalous heat flux into the ocean [ kosaka and xie , 2013 ; england et al . , 2014 ; balmaseda et al . , associated to this anomalous heat flux into the ocean , subsurface heat uptake in the western tropical pacific and indian oceans occurs [ goddard , 2014 ; england et al . , 2014 ; meehl et al . , 2013 ; lee et al . , 2015 ] mostly above 300 m ( see figure s1 in the supporting information ) [ chen and tung , 2014 , figures 1 and 3 ; nieves et al . , 2015 ] , thus not largely involving transfer of heat into the deep ocean . regarding the second mechanism , vertical heat inventories of the north atlantic show notable heat transfer from the upper ( 0300 m ) to intermediate ( 300700 m ) and deep ( > 700 m ) layers since the mid2000s ( see figure s1 and corresponding comments ) [ alexander et al . , 2013 , figure 3.1 ; chen and tung , 2014 , figure 6 ; williams et al . such recent downward heat transfer follows decades of sustained surface warming and increasing ocean heat content in the basin [ drijfhout et al . , 2014 ; however , concomitant with the downward heat export after the mid2000s , the increase in ocean heat content in the upper 2000 m of the north atlantic paused [ chen and tung , 2014 , figures 1 and 3 ; hkkinen et al . , 2015 , figure 1 ] , making the bulk rate of change of ocean heat content per unit area in the basin almost zero . bulk rates of change in ocean heat content per unit area are considered an estimation of the ocean heat uptake ( q , w m ) at ocean basin scales [ e.g. , mauritzen et al . , 2012 ; guemas et al . , 2013 ; chen and tung , 2014 ] . changes in ocean circulation can generate ocean heat content anomalies of opposite signs between different ocean layers and regions ( e.g. , subtropical versus subpolar gyre ) , but the anomalies compensate when integrated at ocean basin scales and decadal timescales [ lozier et al . , 2008 ; mauritzen et al . , therefore , one can conclude that the heat redistribution in the north atlantic since the mid2000s as a whole has apparently occurred without extra heat uptake from the atmosphere . overall , during the past decade , different basins have played contrasting roles on the earth 's atmosphereocean heat budget distribution . in particular , the north atlantic would not have contributed notably as in previous decades to atmospheric heat uptake , in contrast to that observed in the tropical pacific and southern oceans [ roemmich et al . , 2015 ] , but to the quick transfer of heat accumulated during the previous decades in the upper layers to the deep ocean . what are the causes for this shift ? deep convection in the subpolar north atlantic under highsalinity conditions might account for the quick transfer of heat to the deep ocean [ chen and tung , 2014 ] . according to these authors , recent highsalinity conditions may be caused by a strengthening of the atlantic meridional overturning circulation ( amoc ) . agreeing that high salinity in deep convection regions favors heat transfer to the deep ocean [ mauritzen et al . , first , available observational records do not show a strengthening of the amoc [ kanzow et al . second , deep convection in the subpolar north atlantic is restricted to the labrador and irminger seas ( see figure 1 ) , but far from these prominent deep ocean ventilation sites , ocean observations seem to show the largest deep ocean heat uptake at midlatitudes in the eastern north atlantic ( figure 1 ) [ desbruyres et al . , warming pattern in the north atlantic at middepth . temperature differences ( t in color , c ) in the 300700 m layer between the 20072012 and the 19972002 periods ( i.e. , average values are estimated for each time range ) . figure 1 is the zoom into the north atlantic in figure s1 ocean reanalysis system 4 ( oras4 ) ( d ) ( see figure s1 procedure description for details ) . arrows represent ( in red ) warm and salty upper water currents , ( in dark blue ) cold and fresh deep water currents , ( in black , dotted ) the subpolar gyre , and ( in black , dashed ) the subtropical gyre . deep convection sites in the irminger and greenland seas as well as the position of the santander standard section in the bay of biscay are marked by green squares . midlatitudes in the eastern north atlantic ( mlena ) , considered as the oceanic area including the mode water formation area of the eastern north atlantic central water ( enacw ) and the oceanic waters of the bay of biscay and surrounding the iberian peninsula to north africa as in somavilla et al . we reexamine the mechanism responsible for the quick transfer of ocean heat from upper to deeper layers in the north atlantic focusing on the role of ena modal waters . highquality hydrographic data with global coverage are required for providing observational evidence to the mid2000s shift in the north atlantic . these data are obtained from regularly observed hydrographic sections , which are affected by gaps in coverage both in time and space . to overcome this issue , we combined standard hydrographic observations ( salinity and temperature profiles ) at oceanographic sections regularly maintained in the bay of biscay and the greenland sea ( see figure 1 ) with other data sets that offer more global coverage such as argo floats , globalgridded ocean reanalysis ( european centre for mediumrange weather forecasts ocean reanalysis system 4 , oras4 ) and objective analysis ( met office hadley centre monthly objective analysis , en4 , and world ocean database 2001 , woa01 ) . along the work , temperature and salinity time series from oceanographic sections are systematically presented together with those constructed from argo floats and reanalysis data . in this manner , we prove that the changes detected at oceanographic sections are representative of larger oceanic areas ( e.g. , figures 2 and 5 ) , and it serves as an important validation for both argo floats and gridded data sets affected by factors such as changes in observation coverage around 2005 [ roemmich et al . , 2015 ] , contamination from model errors in ocean reanalysis data [ balmaseda et al . , 2013 ] , or the gapfilling strategy in climatological data [ cheng and zhu , 2015 ] . with a similar purpose of increasing the reliability of individual data sets , warming / cooling and saltening / freshening maps obtained from different gridded data are compared ( figures s1 and s2 ) thus , we increase the confidence of the enhanced warming at midlatitudes in the eastern north atlantic as shown in figure 1 , since similar warming distributions showing enhanced heat uptake at midlatitudes in the ena and surrounding the subpolar north atlantic margins are observed in other gridded products ( figure s1 based on en4 ; figure 3c in chen and tung based on ishii data ; figure 4 to 6 in hkkinen et al . based on noaa / national oceanographic data center database ; figure 3.1 on the intergovernmental panel on climate change report [ alexander et al . , 2013 ] constructed from an update of the annual analysis of levitus et al . ) . a complete description of the data sets , methods , and further details are included in the supporting information . strong mixinginduced deep ocean heat injection at mlena . ( a ) mean temperature ( white line ) between the shallower winter mixed layer depth achieved in warm winters and the enacw upper bound ( 100300 m ) and ( black line ) in the depth range of the enacw core ( 300550 m ) in the enacw formation area ( 4348n , 1020w ) obtained from oras4 reanalysis data set . ( b and c ) same as figure 2a but for temperature and salinity at =27.227.3 kg m ( corresponding to the + 0.1 kg m 2005 denser enacw core ) . the blue circles indicate temperature and salinity at =27.227.3 kg m as recorded at station 7 of the santander standard section ( figure 1 ) . the magenta line in figure 2b shows the background average warming trend ( + 0.1c decade ) at the intermediate depth ( 300700 m ) for the global ocean ( table s1 ) . the winter of 2005 was exceptionally cold and dry in southwestern europe [ shein , 2006 ; somavilla et al . , , the ocean surface at midlatitudes in the eastern north atlantic ( mlena , see figure 1 for geographical reference ) experienced the highest densification ( buoyancy loss ) since the 1960s , causing the winter mixed layer to reach very deep levels likely unprecedented for decades [ somavilla et al . the induced deep convective mixing resulted in homogeneization of properties of the water column from the surface to density levels of =27.227.3 kg m , at depth ranges of 350500 m , well beyond the normal airsea interaction extent at the core of enacw ( =27.127.2 kg m ) . this mixing of ( warmer and saltier ) upper water and ( colder and fresher ) modal waters made the upper waters colder ( 0.4c ) , creating a denser ( + 0.1 kg m ) , warmer ( + 0.23c ) , and more saline ( + 0.015 ) type of enacw ( figures 2 and s1 ) [ somavilla et al . , 2009 ] . such temperature increase at the enacw core ( + 0.23c ) resulted in a sudden heat content increase of 2.410 j m for the 350500 m layer ( 3.9 0.610 j m for the 300700 m layer , table s1 ) . this suggests that 70% of the estimated 5.810 j m of heat gained slowly but persistently between 1994 and 2005 in the shallower levels ( 0.7c in the 100300 m layer , table s1 and figure 2a ) was transferred into a denser variety of mode waters through the extreme winter mixing . in addition , considering that such heat injection took place during winter 2005 , the heat uptake ( q ) at intermediate depths would be 30 w m ( 5 w m for the 300700 m layer ) , between 65 and 100 times faster than the average ocean heat uptake at such intermediate depths ( 300700 m ) between 2000 and 2010 ( see table s1 ) . note that the heat uptake at intermediate depths originates from the heat transfer from the upper layers and not from the atmosphere . forced by atmospheric cooling , the convective mixing would have released heat to the atmosphere ( see comments in table s1 for additional information ) . most of the enhanced warming observed at intermediate depths at mlena shown in figure 1 ( + 0.33c gained between 1997 and 2012 ) is explained by the increase in the modal waters during the winter of 2005 ( + 0.23c ) . the estimated overall heat content increase in the region is 1.2 10 j and accounts for 44% of the total heat content increase at intermediate depths in the north atlantic from 36 to 65n from 1997 to 2012 ( table s2 ) . evidence suggests that this extreme winter mixing episode may have contributed to the deep ocean heat injection at higher latitudes by inducing ocean circulation changes that enabled northward propagation of the relatively salty enacw . mlena climatological circulation charts and specific studies indicate slow southward flow west of the bay of biscay and iberia [ pollard et al . , 1996 ; pingree , 1993 ; reid , 1994 ] ( figure 3a ) . the 27.15 isopycnal became shallower from the midatlantic eastward and peaked at about 18w , beyond which it deepened slowly . after winter 2005 , the isopycnals became shallower all the way against the european margin ( figures 3b3d ) . the associated zonal dynamic height sections were thus altered , and the zonal dynamic height slope eastward of 22w changed from almost flat or negative to positive ( figures 3e3 g ) . hence , the associated meridional flow shifts from southward to northward . estimated as the geostrophic meridional current between 22 and 12w ( v g=/f l , where is the difference between the dynamic height at 12w and 22w , f the coriolis parameter , and l the distance between the two longitudes ) , the meridional current after 2005 estimated from the mean filtered dynamic height from argo floats is + 0.7 cm s ( northward ) with respect to 0.4 cm s ( southward ) estimated from woa01 climatological dynamic height ( with reference level at 1500 m both for argo floats and woa01 data ) . oras4 meridional current data show a comparable northward reversal of the flow centered at 18w from 2005 to 2010 ( mean value = + 1 cm s , maximum value = + 3 cm s ) with respect to its southward direction ( mean value = 0.5 cm s , maximum value = 3 cm s ) from 1990 to 2005 ( see figure s3 ) . the inversion of the zonal dynamic height slope is a robust feature ( e.g. , with reference level at 1000 m , the meridional current after 2005 estimated from argo floats is + 0.6 cm s ( northward ) with respect to 0.35 cm s ( southward ) estimated from woa01 climatological dynamic height ) as also observed in oras4 meridional current ( figure s3 ) . a similar flow reversal was predicted by a sensitivity experiment in a global ocean circulation model , and anomalous northward advection of salt was obtained as a result of surface cooling at 45n in the ena [ svellec et al . , ( a ) map of the northeast atlantic showing the location of the nwse , we , and swne sections across the enacw formation area for which the data are shown in figures 3b3 g . ( b d ) location of the = 27.15 at the nwse , ew , and swne sections marked in figure 3a based on woa01 climatological data ( grey ) and available argo floats in the corresponding section between 2005 and 2010 ( cyan ) . the blue line indicates the mean filtered value of all the argo floats each 2 of longitude . ( e g ) idem for the dynamic height between 300 and 1500 m ( see section 2.3 in the supporting information for details ) . black arrows in figure 3a indicate the southward flow west of the bay of biscay based on pollard et al . , inferred in figures 3e3 g from the climatological data ( grey dots ) ; and the red arrow indicates the reversed flow after 2005 , shown in figures 3e3 g by argo floats ( blue line ) . it is worth noting that the maximum water exchange between the subtropical and the subpolar gyres occurs at a density level of =27.227.4 kg m [ brambilla and talley , 2006 ] . thus , the + 0.1 kg m denser core of enacw ( =27.227.3 kg m after 2005 ) might easily get incorporated to the major branch of the north atlantic current flowing northward . the anomalous northward flow of salty enacw is also inferred directly from largescale hydrography ( see figure 4b ) as will be described in detail in the next section . increasing salinities at middepths after 2005 . salinity difference ( s in color ) in the 300700 m layer between ( a ) the 20022005 and the 19972002 periods and ( b ) the 20072012 and the 19972002 periods based on en4 data set ( i.e. , average values are estimated for each time range ) . figure 4 is the zoom into the north atlantic in figure s2 en4 ( c and d ) ( see figure s2 procedure description for details ) . due to the weak circulation and dominance of mesoscale activity in the mlena region , finding direct evidence of a flow reversal as the one inferred from dynamic height and reanalysis current data is difficult . argobased deep displacements [ ollitrault and rannou , 2013 ] are an available source , and luckily , a number of floats drifting at enacw levels were active in the region ( see figure s4 ) . the outcome from the analysis of their lagrangian displacements upper water temperature and salinity in the subpolar gyre and the nordic seas increased since early the 2000s ; this tendency is attributed to the arrival of warmer and saltier western subtropical waters caused by the contraction of the subpolar gyre [ htn et al . , 2005 ; woollings , 2011 ] . though the higher salinities peaking between 2007 and 2010 in these areas ( see figure 4b ) [ holliday et al . , 2008 ; beszczynskamller and dye , 2013 ; http://ocean.ices.dk/iroc/ ] would be consistent with a higher transport of western subtropical waters , that higher transport does not account for the drop in nutrient concentration in the eastern subpolar north atlantic ( rockall trough , figure 1 ) observed from 2007 onward [ johnson et al . , 2013 ] . this drop in nutrient concentration is explained by a higher contribution of enacw in the area [ johnson et al . , 2013 ] , thus adding further evidence in support of the circulation reversal previously described . the enhanced northward flow in the ena would have reinforced the northwestward retreat of the subpolar front [ hkkinen et al . , 2013 ] , broadening the passage for both enacw and western subtropical waters toward the north ( see figure 4b ) . both the eastern and western north atlantic central water types have the potential to increase temperature and salinity at higher latitudes . however , enacw are saltier than western waters arriving at the eastern subpolar north atlantic [ johnson et al . , 2013 ; hkkinen et al . , 2013 ] , providing an alternative explanation for the highestsalinity anomalies observed between 2007 and 2010 in the subpolar north atlantic and nordic seas . assuming mean anomaly propagation speeds of 2 to 3 cm s [ larsen et al . 2008 ] , a lag of 2 to 5 years is expected for the salinity anomaly ( from the mlena ) to reach first the eastern subpolar north atlantic and then the western subpolar gyre and northern nordic seas . in these areas , the arrival of salty southern waters due to the circulation shift in the mlena would have contributed to the highsalinity conditions required for deep ocean heat uptake through density compensated anomalies [ mauritzen et al . , 2012 ; chen and tung , 2014 ] without requiring an increase of the amoc as suggested by chen and tung . such circulation shift and enhanced northward flow in the mlena do not seem to contradict the observed weakening of amoc [ srokosz and bryden , 2015 ] . indeed , a strong northward flow east of 22w and south of 48n in the a priori recirculation area at the ' ovide ' section crossing the north atlantic from portugal to greenland was observed in 2006 , coincident with the lowest amoc measured through the section between 1997 and 2010 [ mercier et al . , consistent with the aforementioned spreading timescale and presence of the saltiest upper waters after 2007 in these areas , the heat and salt uptake below 700 m in the subpolar gyre and the nordic seas increased after 2007 ( figures s1 and s2 ) , coinciding with more intensive deep convection activity in both the subpolar gyre ( irminger and labrador seas , [ yashayaev and loder , 2009 ; kieke and yashayaev , 2015 ; de jong et al . , 2012 ; piron et al . , 2015 ] ) and nordic seas ( greenland sea , [ beszczynskamller and dye , 2013 ] ) . after years of sustained warming and increasing salinity in the upper layers , the first deep convection events at each of these sites resemble the enacw transformation in 2005 . in the deep water formation region of the greenland sea , a strong mixinginduced deep ocean heat injection event occurred through density compensated anomalies in 2008 ( figure 5 ) . upper ocean salinity in this region had been increasing since the mid1990s , and the 2008 winter cooling made the surface waters dense enough to trigger deep convection with temperature and salinity higher than typical values ( figure s5 ) . as a consequence of the overturning , the heat and salt gained slowly over time by the upper layers were directly injected to great depths , with a resulting sudden temperature and salinity increase of 0.16c and 0.01 , respectively ( figures 5c and 5d ) . with a less stratified water column , convection occurred at greater depths in the subsequent years as compared to previous decades in the greenland sea [ beszczynskamller and dye , 2013 ] . coincident with the arrival of highly saline waters in the upper layers of the subpolar gyre , a strong winter mixing occurred in the irminger sea in 2007 [ piron et al . , figures 5a and 5b show sudden increases in temperature ( 0.15c ) and salinity ( 0.01 ) at depth , related to this event . as in the greenland sea , subsequent deep convection events followed in later years ( 2008 , 2009 , and 2012 ) [ de jong et al . , 2012 ; piron et al . , 2015 ] . finally , coinciding with the arrival of highly saline waters in the upper levels , deep convection has reached levels deeper than 1700 m in the labrador sea in 2008 after more than a decade of weak convection , being repeated in 2012 and 2014 [ kieke and yashayaev , 2015 ] . as in the greenland and irminger seas , the first deep convection events appear to be associated with downward transfer of heat and salt [ see yashayaev and loder , 2009 , figure 2a ; kieke and yashayaev , 2015 , figure 4b ] . in general , the steplike temperature increases at depth at deep convection sites caused by the strong mixinginduced heat injection events are equivalent to heat uptake pulses between 16 and 25 w m , which is between 50 and 100 times faster than the average heat uptake below 700 m in the global ocean ( see table s1 ) . as in the mlena , concurrent with the deep ocean heat injection , the deep convective mixing events would have released heat to the atmosphere , favoring deceleration of the north atlantic heat uptake from the atmosphere , as apparently observed since the mid2000s [ chen and tung , 2014 ; hkkinen et al . , deep ( a ) temperature and ( b ) salinity ( 14001600 dbar , =27.7827.8 kg m ) in the irminger sea ( 6062n ; 3540w , green square in figure grey dots indicate vertically averaged argo temperature and salinity values in the pressure range of interest from individual profiles . deep ( c ) temperature and ( d ) salinity ( 15001800 dbar , =28.05828.062 kg m ) in the greenland sea ( 7476n ; 06w , see figure 1 ) . blue circles indicate vertically and spatially averaged conductivitytemperaturedepth ( ctd ) casts in the area and pressure range of interest and grey dots vertically averaged temperature and salinity of individual argo floats . the magenta line in figures 5a and 5c shows the background global ocean average warming trend below 700 m ( + 0.015c decade ) ( table s1 ) . blue lines highlight the years 2007 ( in figures 5a and 5b ) and 2008 ( in figures 5c and 5d ) . the primary cause of the extremely cold winter in 2005 in the mlena was the occurrence of atmospheric blocking anomalies in the ena . in addition to its effects on ocean circulation described in section 2 ( i.e. , regional enhanced mixing altering the largescale density structure ) , blocking anomalies in the ena generate a dipole of temperature anomalies between europe ( cold ) and greenland ( warm ) associated with a strong northward meridional atmospheric flow [ barriopedro et al . , 2008 ] with a potential to reinforce largescale ocean circulation changes . blocking anomalies over the ena occurred previously in the late 1950s and 1960s and extreme winter mixing events ( similar to that in 2005 ) enhanced northward flow of enacw may have occurred and contributed ( along with western subtropical waters ) to the intrusion of warmer and saltier waters into the eastern subpolar north atlantic and nordic seas during the 1960s [ holliday et al . , 2005 ] , as well as enhanced heat uptake by the subpolar gyre through densitycompensated temperature anomalies [ mauritzen et al . , 2012 ; chen and tung , 2014 ; hkkinen et al . , 2013 ; williams et al . , the heat uptake in the 1960s had smaller amplitude compared to the 2005 event , and this may be related to the sustained warming trend in the global upper ocean in the few decades prior to 2005 . the ultimate driver for the occurrence of blocking anomalies at midlatitudes is under debate [ kintisch , 2014 ] . both warmer arctic and tropical pacific have been associated with a wavier jet stream responsible for an increase in blocking events in the north atlantic and higher frequency of extreme weather events in general [ francis and vavrus , 2012 ; trenberth et al . , the blocking pattern developed in winter 2005 in the ena region is not inconsistent with natural climate variability [ barnes et al . however , overall global warming is expected to affect the development of such atmospheric features [ alexander et al . , 2013 ] as well as the subsequent ocean response . estimating the global significance of ocean heat transfer from upper to deeper levels by the mechanism described here requires a better model representation of the frequency and duration of wintertime blocking events , which have been underestimated in most global climate models thus far [ hov et al . , since the mid2000s , in contrast to the enhanced ocean heat uptake observed in other ocean basins , the north atlantic deep ocean heat content has significantly increased without apparently intervening ocean heat uptake from the atmosphere . about 40% of the increase in deep ocean heat content increase below 300 m is centered at midlatitudes in the eastern north atlantic and occurred relatively quickly around the mid2000s far from prominent deep ocean ventilation sites . we suggest that a sequence of connected strong mixinginduced heat injection events originating from this region triggered large transfer of heat from upper to deeper ocean layers , implying a deceleration or halt of the north atlantic heat uptake from the atmosphere . a large transformation of modal water masses in the eastern north atlantic in the winter of 2005 played a major role in the observed quick transfer of heat from upper to deep ocean layers but also aided a reverse of the southward circulation of saltier waters from the eastern side of the temperate atlantic with far reaching consequences ; the northward flow of saltier waters into the subpolar north atlantic and nordic seas favored additional heat injection through deep convection events in later years . thus , we agree that existing highsalinity conditions in the late 2000s in areas of deep convection have aided heat transfer to the deep ocean , but the suggestion that an increase of the amoc is behind such salinity increase is not supported by observations . a greater influence of the saltier enacw and broadened passage for both enacw and western subtropical waters toward the north appears to be a more plausible explanation . anomalous atmospheric patterns such as the one behind this shift are not unique to the last decade , although they may have been exacerbated under global warming .

abstractprior to the 2000s , the north atlantic was the basin showing the greatest warming . however , since the mid2000s during the socalled global warming hiatus , large amounts of heat were transferred in this basin from upper to deeper levels while the dominance in terms of atmospheric heat capture moved into the indopacific . here we show that a large transformation of modal waters in the eastern north atlantic ( ena ) played a crucial role in such contrasting behavior . first , strong winter mixing in 2005 transformed ena modal waters into a much saltier , warmer , and denser variety , transferring upper ocean heat and salt gained slowly over time to deeper layers . the new denser waters also altered the zonal dynamic height gradient reversing the southward regional flow and enhancing the access of saltier southern waters to higher latitudes . then , the excess salinity in northern regions favored additional heat injection through deep convection events in later years .

Introduction Data Sets Transformation of NorthEast Atlantic Modal Waters in 2005 and Implications for BasinScale Circulation Increasing Salinities and Deep Ocean Heat Injection at Higher Latitudes Further Remarks Conclusions Supporting information

dementias due to neurodegenerative diseases are frequent in the elderly population but at the same time , vascular pathology is prevalent in the same age groups , too . vascular factors contribute to ad pathology and perivascular a amyloid deposits have been observed in both ad and vascular dementia ( vad ) . the correct identification of patients with dementia might be hampered by the possible overlap with stroke events . an ischemic event will significantly lead into the aggravation of the clinical symptoms or might even trigger clinically yet unapparent disease , which might unmask after a strategic stroke . these interactions are not well understood and only limited information is available in the literature how csf biomarkers which are used in dementia diagnosis might be influenced by ischemic events . if so , a careful exclusion of acute ischemic events will be necessary to support the common dementia diagnosis in elderly persons , and clinicians should take into account the possibility of a silent stroke . we identified a series of patients with acute ischemic events as detected by mri in combination with a poor performance in neuropsychological tests . in this study , we performed an analysis to see if csf biomarkers might be altered in the same way in dementia and after stroke . patients having different forms of neurological diseases who underwent lumbar puncture for diagnostic purposes which included csf dementia marker profile , were analyzed . we selected data from those with a clinical diagnosis of ad ( n = 47 ) , mild cognitive impairment ( mci ) ( n = 22 ) , vad ( n = 44 ) and stroke ( n = 18 , including three patients with previously diagnosed ad and a recent stroke ) for further analysis ( table 1 ) . for statistical analysis we considered all 18 patients with stroke , including three patients with ad and stroke.table 1clinical characteristics of patients included in the studydiagnosis n age in years**gender m : fduration in months***severity****control * arithmetic mean2258.910:11(n = 9)84.8 min479 max72240mci arithmetic mean2269.814:8(n = 11)22.50 = 21 = 192 = 1 min553 max8660ad arithmetic mean4770.511:36(n = 32)25.10 = 11 = 182 = 203 = 8 min321 max8784vad arithmetic mean4473.921:23(n = 22)26.71 = 262 = 133 = 5 min532 max86108stroke arithmetic mean1872.610:8(n = 12)1.41 = 62 = 73 = 44 = 1 min510 max8630 not determined , 1 still independent , 2 need of care , 3 high need of care , 4 exitus * controls = patients with depression and other non - neurological diseases * * age = age at csf analysis * * * corresponds to the period passed by since the first diagnosis of disease * * * * number of patients dedicated to a certain degree of self - dependence determined by mmse , demtect and their symptoms clinical characteristics of patients included in the study 0 not determined , 1 still independent , 2 need of care , 3 high need of care , 4 exitus * controls = patients with depression and other non - neurological diseases * * age = age at csf analysis * * * corresponds to the period passed by since the first diagnosis of disease * * * * number of patients dedicated to a certain degree of self - dependence determined by mmse , demtect and their symptoms all tests were performed in the neurochemistry laboratory at the department of neurology , university medical school , gttingen . csf was examined for standard parameters such as cell count , proteins and immunoglobulins , and tau , phosphorylated tau and amyloid-142 according to established protocols . csf tau protein was quantitatively analysed using a commercially available elisa kit according to manufacturer s instruction ( innotest htau ag , innogenetics ) . human tau , phosphorylated at thr181 ( phosphorylated tau ) was measured quantitatively with a commercially available elisa kit [ innotest phospho - tau(181p ) , innogenetics ] . a pathological elevated phosphorylated tau level was considered at > 61 pg / ml according to manufacturer s instruction , aimed at the diagnosis of alzheimer s disease . amyloid 142 was detected with a commercially available elisa kit [ innotest - amyloid(142 ) innogenetics ] for quantitative analysis . a pathological decreased amyloid 142 assay was considered at < 450 pg / ml according to manufacturer s instruction . amyloid 140 was detected by elisa ( genetics company , schlieren , switzerland ) . the diagnoses of the patients were based on the following criteria : adthe diagnosis was based on recent criteria icd-10 definition for alzheimer s disease ( f.00 g.30)mcithe diagnosis was based on neuropsychological evaluation ( decline in mmse)strokethe diagnosis was based on clinical syndrome and neuroimaging ( ct , mri)vadthe diagnosis was based on icd 10 definition ( f 01 ) the diagnosis was based on recent criteria icd-10 definition for alzheimer s disease ( f.00 g.30 ) the diagnosis was based on neuropsychological evaluation ( decline in mmse ) the diagnosis was based on clinical syndrome and neuroimaging ( ct , mri ) the diagnosis was based on icd 10 definition ( f 01 ) the anova test ( levene , bonferroni and tamhane t2 ) and kruskal wallis test were used to compare the values patients having different forms of neurological diseases who underwent lumbar puncture for diagnostic purposes which included csf dementia marker profile , were analyzed . we selected data from those with a clinical diagnosis of ad ( n = 47 ) , mild cognitive impairment ( mci ) ( n = 22 ) , vad ( n = 44 ) and stroke ( n = 18 , including three patients with previously diagnosed ad and a recent stroke ) for further analysis ( table 1 ) . for statistical analysis we considered all 18 patients with stroke , including three patients with ad and stroke.table 1clinical characteristics of patients included in the studydiagnosis n age in years**gender m : fduration in months***severity****control * arithmetic mean2258.910:11(n = 9)84.8 min479 max72240mci arithmetic mean2269.814:8(n = 11)22.50 = 21 = 192 = 1 min553 max8660ad arithmetic mean4770.511:36(n = 32)25.10 = 11 = 182 = 203 = 8 min321 max8784vad arithmetic mean4473.921:23(n = 22)26.71 = 262 = 133 = 5 min532 max86108stroke arithmetic mean1872.610:8(n = 12)1.41 = 62 = 73 = 44 = 1 min510 max8630 not determined , 1 still independent , 2 need of care , 3 high need of care , 4 exitus * controls = patients with depression and other non - neurological diseases * * age = age at csf analysis * * * corresponds to the period passed by since the first diagnosis of disease * * * * number of patients dedicated to a certain degree of self - dependence determined by mmse , demtect and their symptoms clinical characteristics of patients included in the study 0 not determined , 1 still independent , 2 need of care , 3 high need of care , 4 exitus * controls = patients with depression and other non - neurological diseases * * age = age at csf analysis * * * corresponds to the period passed by since the first diagnosis of disease * * * * number of patients dedicated to a certain degree of self - dependence determined by mmse , demtect and their symptoms all tests were performed in the neurochemistry laboratory at the department of neurology , university medical school , gttingen . csf was examined for standard parameters such as cell count , proteins and immunoglobulins , and tau , phosphorylated tau and amyloid-142 according to established protocols . csf tau protein was quantitatively analysed using a commercially available elisa kit according to manufacturer s instruction ( innotest htau ag , innogenetics ) . human tau , phosphorylated at thr181 ( phosphorylated tau ) was measured quantitatively with a commercially available elisa kit [ innotest phospho - tau(181p ) , innogenetics ] . a pathological elevated phosphorylated tau level was considered at > 61 pg / ml according to manufacturer s instruction , aimed at the diagnosis of alzheimer s disease . amyloid 142 was detected with a commercially available elisa kit [ innotest - amyloid(142 ) innogenetics ] for quantitative analysis . a pathological decreased amyloid 142 assay was considered at < 450 pg / ml according to manufacturer s instruction . amyloid 140 was detected by elisa ( genetics company , schlieren , switzerland ) . the diagnoses of the patients were based on the following criteria : adthe diagnosis was based on recent criteria icd-10 definition for alzheimer s disease ( f.00 g.30)mcithe diagnosis was based on neuropsychological evaluation ( decline in mmse)strokethe diagnosis was based on clinical syndrome and neuroimaging ( ct , mri)vadthe diagnosis was based on icd 10 definition ( f 01 ) the diagnosis was based on recent criteria icd-10 definition for alzheimer s disease ( f.00 g.30 ) the diagnosis was based on neuropsychological evaluation ( decline in mmse ) the diagnosis was based on clinical syndrome and neuroimaging ( ct , mri ) the diagnosis was based on icd 10 definition ( f 01 ) the anova test ( levene , bonferroni and tamhane t2 ) and kruskal wallis test were used to compare the values . we report similarities in the csf profiles of those with ad and those who underwent lumbar puncture after stroke . apart from their different sizes and the distribution of gender , both groups are especially comparable concerning age ( ad arithmetic mean of 70.5 years , stroke 72.6 years ) ( tables 1 and 2 ) . increased tau and decreased a42 were similar in patients with ad and in those after stroke . the highest total - tau levels were found in the stroke group ( arithmetic mean 516 pg / ml , median 468 pg / ml ) . the highest p - tau levels were seen in ad patients , followed by mci , stroke and vd . also a40 levels were low in ad and stroke patients ( table 2 ; fig . 1).table 2cerebrospinal fluid levels of tau , p - tau , a142 and a140 stratified by disease typecsfcontrols*mciadvad**strokepg / mlmean ( sd)mean ( sd)mean ( sd)mean ( sd)mean ( sd)medianmedianmedianmedianmedian(min max ) n ( min max ) n ( min max ) n ( min max ) n ( min max ) n tau131 ( 58)311 ( 243)391 ( 232)302 ( 252)516 ( 317)126200.5339258468(75253)(751,080)(75910)(751,145)(1211,300)2122474418p - tau33 ( 15)65 ( 35)74 ( 44)58 ( 34)62 ( 36)3057.5705556(252)(16156)(20243)(21148)(15111)101431217a42927 ( 164)856 ( 362)580 ( 211)701 ( 341)553 ( 245)942825.5537.5608443(4761,685)(2561,650)(2461,026)(2511,775)(2431,002)2122464417a406,421 ( 2,045)9,437 ( 3,081)7,062 ( 1,890)9,964 ( 5,639)6,258 ( 2,328)5,885.59,830.56,8438,2765,846(4,28811,031)(3,32113,729)(3,60411,257)(4,49329,786)(3,3569,599)121224205 * controls : patients with depression * * vad without recent strokefig . 1 a [ t - tau ] csf levels across the groups b [ p - tau ] csf levels across the groups c [ a42 ] csf levels across the groups cerebrospinal fluid levels of tau , p - tau , a142 and a140 stratified by disease type * controls : patients with depression * * vad without recent stroke a [ t - tau ] csf levels across the groups b [ p - tau ] csf levels across the groups c [ a42 ] csf levels across the groups t - tau levels of ad - patients ( arithmetic mean 391 pg / ml ) showed less variation than in stroke ( arithmetic mean 516 pg / ml ) . in vad , t - tau levels were significantly lower than in stroke patients ( arithmetic mean 302 pg / ml ) ( p = 0.018 ) . there were no significant differences between stroke ( arithmetic mean 62 pg / ml ) and ad ( arithmetic mean 74 pg / ml ) , p = 0.465 , ad and mci ( arithmetic mean 65 pg / ml ) , p = 1.0 and ad and vad ( arithmetic mean 58 pg / ml ) , p = 0.579 . the highest values of p - tau were found in ad , followed by mci , similar to stroke and finally vad ( table 2 ) . as a biomarker , p - tau was not useful to distinguish between any of the groups , although higher levels are observed in ad . for a42 , there was a significant difference between ad ( arithmetic mean 580 pg / ml ) and mci ( arithmetic mean 856 pg / ml ) , p = 0.015 , as well as between mci and stroke ( arithmetic mean 553 pg / ml ) , p = 0.022 . no difference was observed for ad and stroke ( p = 0.999 ) , which points out the similarity between csf a42 levels in both . similar findings were obtained for a42 in stroke and vad ( arithmetic mean 701 pg / ml ) , p = 0.341 . a42 levels in ad and vad were in the same range ( p = 0.247 ) . no significant differences between the groups were found when we calculated the a ratio [ p - values between 0.757 and 1.0 , proof for homogeneity of variances among the single groups ( data not shown ) ] . in this study because the time of the lumbar puncture varied from the day of ischemic event up to several weeks after and because the sizes and localization of the lesion generally affects the csf protein profile ( middle brain , thalamus , capsula interna and media total infarction ) , the tau values vary to a great extend ( fig . 2 ) . the tau levels were even still increased within the period over 1 month , reaching values of 1,300 2tau - values in pg / ml after stroke defined by the size of damage and time point after the event tau - values in pg / ml after stroke defined by the size of damage and time point after the event we stratified the single patients to different subgroups considering the infarction size , time of lumbar puncture related to the time of stroke . the lowest levels were observed in stroke patients with an acute and limited brain volume damage , such as pons ischemia ( n = 2 , tau 144 and 121 pg / ml ) , while the highest were observed in a patient 1 month after a media infarction ( tau 1,300 pg / ml ) . as final result , we found highest tau levels in patients with large stroke areas and subacute stage ( media total infarction and multiple strokes , n = 3 , arithmetic mean 986 pg / ml ) , followed by those patients with similar location but in acute stage ( n = 4 , arithmetic mean 510 pg / ml ) , followed by those with only limited infarction size ( n = 2 , arithmetic mean 132.5 pg / ml ) . of importance , even small infarct areas led into increased csf tau levels , especially several days after the stroke . the csf analysis offers an excellent opportunity to detect early signs of neuronal degeneration and this has been widely used for ad and other dementia disorders . thus , we studied csf alterations in csf marker profiles of commonly used ad biomarkers in patients with ad , vad and stroke . increased values of tau and a decrease of a42 were detected in ad in a similar manner as in vad and acute ischemic events . with special regard to total tau , which is known as an indicator of neuronal damage and discussed to be specifically altered in ad , an increase after stroke was not expected to this degree . in our literature research , we found only limited data dealing with csf biomarkers after stroke , all of them well in line with results obtained here [ 25 ] . tau was described to be significantly increased at day 23 ( 179 % ) , showing a peak after 1 week ( 257 % ) and after 3 weeks ( 425 % ) , normalizing not until 35 month ( 140 % ) . no significant alteration of p - tau was reported . for the distinction between vad and ad , csf proteins were found to be more altered in ad , such as a42 decrease and tau increase , especially phosphorylated forms ( table 2 ) [ 68 ] . according to the literature , to differentiate ad from vad , a combination of all three is recommended . with special one important aspect is mci and its progression to ad , which can potentially be predicted with the help of csf profile [ 5 , 10 , 11 ] . apparently those mci patients with a more ad - like csf profile progress to ad in a shorter period of time , mostly showing low a42 and high tau levels [ 12 , 13 ] . because csf biomarkers are already altered very early , there is the possibility to detect ad patients at risk or at very early stages . more precisely , amyloid - peptide burden and changes in app metabolism are altered at first up to 10 years before clinical symptoms . csf a42 values are supposed to predict disease progression in ad , but can be altered in vad , too . up to now studies have mostly focused either on ad or on stroke and directly comparative analysis is rarely given . similar to ad , imbalances concerning the total cleavage of app , with tendencies towards accumulation to amyloid plaques , was reported in vad . analogies between cognitive decline in patients after stroke with or without cerebrovascular pathologies prior to this can be seen in comparison to amyloid impact in ad . total tau might reflect the degree of neuronal damage after ischemia [ 3 , 4 ] . csf samples taken immediately after stroke , some days up to several weeks after stroke document an increase of t - tau with a peak after 1 week and a re - normalization 35 months later . the level of tau alteration depends on stroke size as well as on time passed since the event . neuronal apoptosis caused by ischemia is supposed to lead into hyperphosphorylation of tau , and p - tau is not increased directly after stroke . moreover , as a biomarker it is more useful for ad diagnosis but can be altered in the context of a chronic process like vad . whether it can be considered as a new therapeutic target that should be regulated after ischemia and reperfusion process , remains to be determined . some research has been done using serum in patients after stroke and recently also in ad . blood samples are of special research interests , because they are much easier to obtain and the analyses can be done sequentially . increased serum tau some other biomarkers were supposed to reflect processes of oxidative stress and inflammation followed by blood a larger review concentrated on processes after ischemic brain injury , inflammatory processes contributing to neurodegeneration or by the ischemic event itself . amyloid balance is supposed to be hampered because of the down - regulation of -secretase resulting into the non - amyloidogenic pathway of total cleaving of app , but accumulating soluble neurotoxic amyloid peptides via striking the second pathway using - and -secretases . this explains the finding of ad - like pattern in rodents , starting some days after ischemia an increase of 200 % of app in the penumbra on the seventh day post - stroke is described up to 1 year after the event . it also raises the question of the cause of progressive cognitive decline following stroke , inflammatory or degenerative processes . a striking overlap is found in vad and ad , such as hypertension , hyperlipidemia , diabetes mellitus and white matter changes subsequently lead into vad on one hand ; on the other hand patients with ad showed more often large vessel abnormalities , like carotid artery stenosis , carotid intimal - medial thinning and bilateral present carotid plaques . prevalence of cardiovascular risk factors , atrial fibrillation , vessel stiffness and microinfarction increase with age and as result cognitive impairment is not distinctly due to ad or microvascular brain damage alone , rather than a mixture of both . as a drawback of our study the deviation of our control group concerning age has to be mentioned ( arithmetic mean 58.9 years ) . being around one decade younger than ad or stroke patients with less developed age related changes of the vascular system or degenerative processes , their csf analysis is expected to show less affected values of the proteins a priori . a control group with more similar age in comparison with the rest would have been more significant ; however , since white matter lesions are common in the elderly , such a cohort will be difficult to obtain . there is a clear importance to keep in mind the possibility of stroke and vascular degenerative processes , which might lead to increased tau and decreased a42 in csf in a similar manner as in ad . based on csf biomarker analysis alone , we could not distinguish stroke from ad patients , since csf alterations of t - tau , p - tau and a42 levels did not differ across groups . because of its high incidence in elderly population , ischemic events have to be considered in the interpretation of given pathologic csf profile with increased csf tau , p - tau and decreased a42 levels . our conclusions based on our observations reported here are : ischemic events detected in neuroimaging can cause a pathological csf profile which resembles those obtained in neurodegenerative dementia / ada follow - up examination of the biomarkers after several months might be necessary to exclude underlying dementia via normalized csf profilepatients suffering from stroke show distinct alterations of pathological csf profile , especially an increase of t - tauin differential diagnosis of cognitive decline after stroke , the pathological biomarker profile can be caused by the ischemic event itself and not by the neurodegeneration alone ischemic events detected in neuroimaging can cause a pathological csf profile which resembles those obtained in neurodegenerative dementia / ad a follow - up examination of the biomarkers after several months might be necessary to exclude underlying dementia via normalized csf profile patients suffering from stroke show distinct alterations of pathological csf profile , especially an increase of t - tau in differential diagnosis of cognitive decline after stroke , the pathological biomarker profile can be caused by the ischemic event itself and not by the neurodegeneration alone

vascular factors are thought to contribute to the development of disease pathology in neurodegenerative dementia such as alzheimer s disease ( ad ) . another entity , called vascular dementia ( vad ) , comprises a less defined group of dementia patients having various vascular diseases that especially emerge in the elderly population and require valid options for examination and differential diagnosis . in the context of a retrospective study , we analyzed the cerebrospinal fluid ( csf ) biomarkers t - tau , p - tau and a42 of a total of 131 patients with ad ( n = 47 ) , mild cognitive impairment ( mci ) ( n = 22 ) , vad ( n = 44 ) and stroke ( n = 18 ) . we found a remarkable alteration in csf biomarker profile in ad , vad and in acute ischemic events . csf profile in ad patients was altered in a very similar way as in stroke patients , without statistical differences . in stroke , increase depend largely on size and duration after the initial event . total tau levels were useful to differ between vad and stroke . a42 decreased in a similar way in ad , vad and stroke and had a trend to lower levels in mci but not in controls .

Introduction Methods Patients data collection and analysis Statistical evaluation Results Discussion Conclusions

motor neuron diseases ( mnd ) are a heterogeneous group of disorders which result in death of motor neurons amyotrophic lateral sclerosis ( als ) is the most common form of mnd in adults , affecting both anterior horn cells and corticospinal tracts . amyotrophic refers to the muscle atrophy , weakness , and fasciculation that signify disease of the motor neurons . fifty percent of patients die within three years of onset of symptoms , and 90% die within five years . the incidence of als is from approximately 2 per 100,000 per year and may be increasing . the majority of als cases which have been reported are sporadic ( sals ) ; 10% are familial ( fals ) , some of which arise from mutations in superoxide dismutase-1 ( sod1 ) , tar dna - binding protein ( tdp43 ) [ 5 , 6 ] and fused in sarcoma / translated in liposarcoma ( fus / tls ) [ 7 , 8 ] , als2 [ 9 , 10 ] , dynactin , and senataxin . genomic studies suggest the existence of at least eleven additional loci for fals , but the genetic defects remain to be identified . using a genome - wide association study ( gwas ) approach , it has been recently reported that a locus on chromosome 9p21 accounted for 40% of familial als and nearly 1 fourth of all als cases in a sample of 405 finnish patients . this association signal had previously been reported by van es et al . as related to als , and a meta - analysis amongst many studies showed that this was indeed the major signal for this disease . similarly , recent gwas for frontotemporal dementia ( ftd ) with tdp-43 pathology had also been identified on this locus . linkage analysis of patients affected with multiple cases of als , ftd , and ftd - als with type 2 tdp-43 pathology had suggested that there was an important locus for the disease on chromosome 9p [ 1821 ] , but it is not clear whether the linkage and association signals related to a single locus or whether the different studies are reporting the same alleles at that locus . some more studies have shown that there is a large hexanucleotide ( ggggcc ) repeat expansion in the first intron of c9orf72 on the affected haplotype , and a common mendelian genetic lesion in c9orf72 is implicated in many cases of sporadic and familial als and ftd . recently one of them is mutations in ubqln2 , which encodes a ubiquitin - like protein , ubiquilin 2 , cause dominantly inherited chromosome x - linked als and als / dementia . ubiquilin 2 is a member of the ubiquilin family ( ubiquilins ) , which regulate the degradation of ubiquitinated proteins and found in both inherited and sporadic form of als . functional analysis showed that mutations in ubqln2 lead to an impairment of protein degradation , abnormal protein aggregation and neurodegeneration . another is mutation in sqstm1 gene , which encodes the ubiquitin - binding protein p62 ( also known as sequestosome-1 ) . these findings provide evidence of a direct genetic role of p62 in als pathogenesis as it is supposed to be involved in protein - recycling system by regulating the protein degradation pathways , and thus , p62 , related to ubqln2 may represent an important therapeutic target in als . the key role these mutations play in als points towards their probable contribution in als metabolomics , which needs to be explored in future research studies . although the root cause of als is not clearly understood , but multiple mechanisms have been found to be associated in the pathogenesis of motor neuron death in als . these include oxidative stress , glutamate - mediated excitotoxicity , protein aggregation , mitochondrial dysfunction , and transition - metal - induced toxicity [ 2628 ] . metabolomics is the comprehensive study of the repertoire of small molecules present in cells , tissues , or other biological samples . the lack of biological tools to detect als together with the nonspecificity and heterogeneity of clinical symptoms leads to difficulty in diagnosing the disease in its early stages . metabolomic analysis is a universally applicable strategy for defining the metabolite composition of cells and tissues . ideally metabolomic analyses are rapid , unbiased , and comprehensive , and , to some extent mass spectrometry , h1 nuclear magnetic resonance ( h1 nmr ) spectroscopy and ir spectroscopy are progressively considered as metabolomic techniques . metabolomic approach evolves to study most or all implicated pathways , thereby revealing a biochemical signature for the disease and providing valuable new insights into disease mechanisms . metabolomics should be seen as a complementary technique to genomics , transcriptomics , and proteomics . additionally , it will be of great interest to integrate metabolomic , transcriptomic , and proteomic data into a system biology approach to understand global changes in als biological states . the understanding of regulatory metabolic processes of a complex living organism at the system level requires the assessment of spatiotemporal interorgan metabolic crosstalks through the analysis of biofluids . this challenge can be addressed by studying the metabolic correspondence among tissue and biofluid metabolic profiles . when associated with a well - defined physiological condition , metabolic profiles provide a snapshot of a functional phenotype , or metabotypes , as a result of multiple interactions between metabolic pathways under the influence of environment , lifestyle , genetics , and microbial factors [ 3032 ] . a specific metabolic profile of a systemic biofluid , such as blood or serum / plasma , reflects the overall metabolic status of an individual as the result of highly complex metabolic exchanges between diverse biological compartments , including organs , biofluids , and microbial symbionts . although h1-nmr is a well - established approach for multicompartmental metabolic profiling of intact tissue and biofluid samples , gc / tofms has the advantage to discover changes in metabolites present at lower concentrations due to the increased sensitivity ; hence , nmr and mass spectrometry should be regarded as complementary techniques . the potential use of blood ( serum or plasma ) and cerebrospinal fluid ( csf ) biomarkers associated with als have been studied by many authors , and these target molecules are supposed to be linked to als pathogenesis [ 3436 ] . recently , high - throughput techniques have been used to evaluate a combination of markers in patients with neurological diseases which is performed via different analytical methods such as high - performance liquid chromatography followed by electrochemical detection . nmr spectroscopy is a noninvasive technique , needs little sample preparation , and gives an overview of the principal metabolic pathways . recently , kumar et al . have analyzed blood ( serum ) metabolic profile to study 13 metabolites by h1 nmr ( figure 1 ) and found a significant elevation of the metabolite glutamate in serum sample of als patients . these higher glutamate signals were consistent with the hypothesis of glutamate excitotoxicity in als pathogenesis . plaitakis proposed the hypothesis of glutamatergic dysfunction in als , that is , imbalance between brain and blood glutamate levels . babu et al . also observed that blood glutamate levels were significantly higher in als patients . furthermore , kumar et al . found that , with increased glutamate concentration , glutamine concentration decreased in als , which might represent the imbalance between glutamate - glutamine conversion cycle that occurs in postsynaptic buttons and astrocytes during excitotoxicity . earlier study of pioro also showed in vivo evidence of abnormal glutamate metabolism in the cns parenchyma of als patients . there were previous studies by authors , who measured glutamate and found decreased glutamate concentrations in cns tissue and increased concentrations in the serum and csf of als patients , and hence , proposed a hypothesis suggesting an imbalance in the intracellular versus extracellular glutamatergic neurotransmitter system [ 43 , 44 ] . further some more important metabolites related to energy and oxidant and antioxidants have been studied , and it has been hypothesized that glutamate excitotoxicity exacerbates the formation of ros , which may be responsible for the oxidant - antioxidant imbalance in serum of als patients . for example , histidine metabolite , which is considered to be powerful antioxidant , and n - acetyl derivative ( n - acetyl - x ) concentrations significantly decreased in serum of als patients with increase concentration of bhbt , acetone , and glutamate ( figure 1 ) . unfortunately due to space limitations only a small number of promising article findings related to blood metabolites can be discussed in this section , and readers are directed to other excellent research articles for some other changes in metabolic profile in blood of als patients [ 4547 ] . csf is known as good source for the study of biomarkers because of its direct contact with the brain , its accessibility , and its dynamic changes with the cerebral environment . reduced levels of cystatin c and increased levels of neurofilaments have been proposed as csf biomarkers for als [ 48 , 49 ] . a recent study has found a relationship between the levels of galectin-3 to be indicative of the onset of als symptoms in mice , and the result was found transferrable to als . measured 17 metabolites in als csf and compared csf metabolic profiles between als and non - als patients . his univariate analysis showed higher concentrations of csf acetone , pyruvate , and ascorbate in als patients , while acetate was found decreased in als compared to controls . in this interesting study , the authors concluded that perturbations in energy metabolism and ketone metabolism were associated with als . however , other earlier studies of ascorbate metabolism have shown contradictory results [ 52 , 53 ] . the elevated levels of this antioxidant molecule ( ascorbate ) are compatible with oxidative stress previously described in als and could also be linked to ascorbic acid release from astrocytes after glutamate stimulation [ 54 , 55 ] . in earlier studies performed over the past twenty years and using different methodologies and usually only a small number of patient samples , glutamic acid has been reported as being elevated , normal , or reduced in the csf of als patients [ 5658 ] . the studies reporting an increase in csf glutamic acid content have been taken as support for the theory of glutamic acid excitotoxicity as a cause of als and could result in elevated levels of ascorbic acid . ascorbic acid might , by itself , modulate neuronal metabolism through the inhibition of glucose consumption during episodes of glutamatergic synaptic activity and by stimulating lactate uptake in neurons . in contrast to the interesting study by blasco et al . , wuolikainen et al . found that the content of ascorbic acid to be nonsignificantly different between als and controls . the sources of the compound , creatine , and creatine phosphate , are important for the energy metabolism and are present at high levels in the cns . the major formation of creatinine in the body , however , takes place in the skeletal muscles . this suggests that amyotrophy is the most likely explanation to the lower csf creatinine levels in als . studied the csf metabolome by using gc - tofms platform in als patients with six different mutations in the sod1 gene and compared it with als - patients without such mutations and found that patients with a sod1 mutation have a distinct metabolic profile in the csf . in particular , the patients who are homozygous for the d90a sod1 mutation showed a distinctively different signature when modeled against als patients with other sod1 mutations and sporadic and familial als patients without a sod1 gene mutation . among the metabolites that contributed most to the csf signature were arginine , lysine , ornithine , serine , threonine , and pyroglutamic acid , all found to be reduced in patients carrying a d90a sod1 mutation . analysis of the neurofilament light chain in the csf suggests that patients with sod1 gene mutations constitute a distinct subgroup within the als syndrome , and more so patients homozygous for the d90a sod1 mutation . wuolikainen et al . also reported that patients with sals have a heterogeneous metabolite signature in the csf . the metabolome of the csf of als patients with a sod1 gene mutation was found to form a separate homogeneous group . analysis of metabolites revealed that glutamate and glutamine were reduced , particularly in patients with a familial predisposition . there are significant differences in the metabolite profile and composition among patients with fals , sals , and patients carrying a mutation in the sod1 gene suggesting that the neurodegenerative process in different subtypes of als may be partially dissimilar . kynurenine pathway ( kp ) plays a very important role in the pathogenesis of als . the kn is a major route for the conversion of l - tryptophan ( trp ) to nicotinamide adenine dinucleotide ( nad ) [ 62 , 63 ] , initiated by either tryptophan 2,3-dioxygenase ( tdo ) or indoleamine 2,3-dioxygenase ( ido ) [ 64 , 65 ] . along the kynurenine ( kn ) , they include the free radical generator 3-hydroxykynurenine ( 3-hk ) , the excitotoxic n - methyl - d - aspartate ( nmda ) receptor agonist , quinolinic acid ( quin ) , the neuroprotective nmda receptor antagonist , kynurenic acid ( kyna ) [ 68 , 69 ] , and the neuroprotective picolinic acid ( pic ) ( figure 2 ) [ 70 , 71 ] . with respect to the pathogenesis of als , there is increasing evidence suggesting the involvement of the kp , especially that of the neurotoxin quin . in familial sod1 als , the formation of covalent aggregates of sod1 is tryptophan dependent . kynurenic acid ( kyna ) , produced from l - kynurenine , is an endogenous antagonist of ionotropic excitatory amino acid receptors acting preferentially at the glycine site associated with the nmda receptor complex . demonstrated the highest kyna levels in the caudate nucleus , lower levels in the thalamus , globus pallidus , hippocampus , parietal cortex and frontal cortex , and the lowest level in the cerebellum . it has been suggested that the reduced concentration of kyna may cause neurodegeneration . on the other hand , this endogenous neuroprotectant could have beneficial effect on several animal models of neurological diseases [ 62 , 63 ] . . demonstrated that kyna concentration was significantly higher in the csf of als patients with bulbar onset of als compared to control group , whereas serum kyna concentration was significantly lower in als patients with severe clinical status than in controls . these findings suggest that in als patients csf kyna concentration does not depend on its production in the periphery . but recent study by chen et al . in als patients has shown twofold increase in csf trp , serum trp , and serum kyn , a tenfold increase in csf kyn and a fourfold increase in intracellular csf ido activity in als compared to controls . although kyn can be transported across the blood brain barrier , the tenfold increase in csf kyn , compared to only a twofold increase in serum kyn , the elevated levels of serum and csf trp are speculated to reflect a dysfunction in protein - binding and l - neutral amino acid transporters , respectively , or perhaps , an over - compensatory response , whereby the increase in csf ido activity results in a decrease in csf trp , which encourages the dissociation of trp from albumin to facilitate transport of trp across the blood brain barrier ( bbb ) . a recently published study with glutamate has found that abnormal neurons exhibit an upregulation in a particular form of glutamate - aspartate transporter , which may make them more vulnerable to glutamate - mediated excitotoxicity . as such , a similar event is hypothesized to occur between quin and motor neurons . pathological concentrations of quin could result in a myriad of unfavourable consequences that could exacerbate and accelerate the condition of als . as an endogenous nmda agonist the main source of quin is immune cells rather than astrocytes or neurons [ 79 , 80 ] . however , high levels of kyn produced by astrocytes can be taken up by surrounding activated microglia for quin production . once quin exceeds the safety threshold ( < 150 nm ) , its adverse effect is exerted via the generation of reactive oxygen species , augmentation of the excitotoxic impact through disruption in the glutamatergic transport system , apoptosis , mitochondrial dysfunction , and the production of cytokines and chemokines , all of which are putatively thought to be contributory factors in als ( figure 1 ) . chen et al . showed a significantly increased microglial activation in als motor cortex and spinal cord which indicate a heightened immune response in als cns . it has been also shown that synthesis of kyna in the brain takes place predominantly in astrocytes . in als , an intensive astrocytosis has been found . reported reactive astrogliosis in the ventral horns , dorsal horns , and at the transition between gray matter and anterior and lateral funiculi in the spinal cords of als patients . feeney et al . showed that motor neuron loss and reactive astrocytosis were correlated with the progression of the disease in sod 1 transgenic mouse model of als . observed that the loss of glutamate transporter subtype 1 ( glt-1 ) in als transgenic sod1 mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis . thus , it seems that astrocytosis occurring in als may cause an increase of kyna production and observed elevation of kyna in csf . als is a neurodegenerative disease characterized by progressive degeneration of both upper motor neurons ( umn ) in the primary motor cortex ( pmc ) and lower motor neurons ( lmn ) in the brain stem and spinal cord anterior horns . despite the identification of pathologies in the pmc and corticospinal tracts ( cst ) of autopsy tissue samples with als [ 8487 ] , there are no biomarkers identified to date that reliably indicate presence of such pathologies in the brain of patients with als . in an effort to find neuroimaging biomarkers indicative of umn degeneration , advanced mr techniques ( e.g. , magnetic resonance spectroscopy , diffusion tensor imaging , and functional mri ) have been explored [ 88 , 89 ] . it is hypothesized that the neuropathologically proven motor neuron and cst degeneration in als might occur gradually with no apparent manifestation of macroscopic tissue structural changes in the early stage which are detectable by conventional mri methods . in contrast , neurochemicals indicative of metabolic processes responsible for degeneration of the motor neurons and cst in patients with als can be accessed from the disease onset stage using proton mr spectroscopic ( 1h mrs ) methods . several previous cross - sectional and longitudinal studies have demonstrated the value of proton mrs for the evaluation of metabolite alterations in the pmc [ 90100 ] and cst [ 95 , 101 ] of patients with als using single voxel svs and 2d mr spectroscopic imaging [ 101103 ] methods . in general , most of these studies have reported significantly altered metabolite concentrations or ratios in the motor pathways of patients with als . 1h mrs of the human brain reveals important information about compounds such as n - acetylaspartate ( naa ) which has been suggested to be a marker for neuron viability and axonal density in the brain , creatine ( cr ) as a putative marker of gliosis [ 105 , 106 ] , and choline ( cho ) which is associated with membrane phospholipids . several 1h mrs studies in patients with definite als and clear signs of umn involvement showed a reduction of naa in the motor cortex [ 90 , 108110 ] . in another study , chemical shift - imaging follow - up measurements in als patients after a three - month period demonstrated a decline in concentrations of naa , cr , and cho in the most affected motor cortex areas of als in comparison with healthy controls . . found significant changes of naa concentrations and the naa/(cr + cho ) ratio of the investigated grey matter areas . it has been observed that the ratio of naa and naa/(cr + cho ) as a biomarker of neuronal loss or neuronal metabolism in als patients is significantly apparent . this was supported by significantly lower naa values in the more affected hemispheres at the time of study inclusion . also reported significantly decreased naa / myo inositol in als patients compared to healthy controls . today als is thought to be to some degree a multisystem disorder and mitochondrial abnormalities that have been observed not just in motor neurons but even in skeletal muscle cells of als patients . recent work in msod1 mice has shown that motor neuron death is not cell autonomous and involves defects in other cell types than neurons [ 112 , 113 ] . consistent with this notion , recent evidence showed that the pathophysiology of als includes widely systemic defects in both patients and animal models . in particular , energy homeostasis is strikingly abnormal in als patients since both sals and fals patients present with increased energy expenditure ( hypermetabolism ) and hyperlipidemia [ 114 , 115 ] . further it is also reported that , a severe deficiency of nicotinamide adenine dinucleotide to coq oxidoreductase ( naoh : coq ) in skeletal muscle biopsies of patients with sals was observed using saponin - permeabilized muscle fibers . this muscular mitochondrial defect had a heterogeneous distribution and was correlated in some patients with multiple mitochondrial dna deletions , decreased mitochondrial dna levels , and low levels of membrane - associated manganese sod . these results suggest that some sals patients may have muscular mitochondrial damage that may contribute to disease pathogenesis . it has been found that metabolism is higher and bodyweight and fat mass are lower in mutant sod1 mice than in wild - type mice . loss of tdp-43 or its overexpression [ 121 , 122 ] both lead to growth retardation and thus impaired energy homoeostasis . furthermore , adult loss of tdp-43 led to massive decreases in adipose tissue , probably through muscle hypermetabolism , and tdp-43 overexpression led to the formation of morphologically abnormal mitochondria [ 121 , 122 ] . tdp-43-als , like mutant sod1-als , therefore , seems to be associated with impaired energy homoeostasis in transgenic animals . abnormalities in muscle energy metabolism have been suggested as the direct cause of energy deficit and hypermetabolism in mutant sod1 mice . cellular levels of atp are decreased [ 122124 ] and expression of mitochondrial uncoupling proteins and concentrations of markers of lipid and carbohydrate use are increased [ 120 , 125 ] . several reports have indicated the existence of mitochondrial defects in the muscle tissue of patients with als that develops as the disease progresses [ 126128 ] . low - level mitochondrial defects might , however , be present earlier , as increased sensitivity of als myoblasts to oxidative stress has been seen in some patients . the extent and origin of such mitochondrial dysfunction remain controversial , however , and the impairment of energy metabolism seen in patients with als and mutant sod1 mice might be due at least partly to dysfunctional regulation of metabolic pathways . numerous studies in als patients have shown collagen abnormalities of skin , such as decreased amount of collagen , alteration of cross - linking of collagen , and increased solubility of collagen . recently , it has been demonstrated that the basement membrane of skin in als patients is weakly positive for type iv collagen compared with controls , and also serum type iv collagen levels in als patients are lower than controls . it showed that the absolute and relative concentrations of two collagen metabolites , glucosylgalactosyl hydroxylysine ( glu - gal hyl ) , and galactosyl hydroxylysine ( gal hyl ) , excreted in urine , indicate the tissue origin of the collagen metabolites and the rate of degradation of collagen [ 135137 ] . glu - gal hyl accounts for 61% of the hydroxylysine glycosides in skin collagen , and gal hyl constitutes approximately 75% of the glycosides in bone collagen [ 135 , 138 ] . measurement of glu - gal hyl and gal hyl may indicate whether bone or skin collagen is preferentially being degraded . the study demonstrated that large increase in degradation of skin collagen produces a large increase in urinary excretion of glu - gal hyl . it has also been reported that , in the case of bone collagen , patients with bone complications tend to have elevated urinary gal hyl excretion . a high ratio of glu - gal hyl to gal hyl increases the probability that the patient will develop skin complications , and a low ratio is associated with bone - related complications . therefore , these two hydroxylysines , that is , glu - gal hyl and gal hyl , offer more reliable parameters of collagen breakdown than hydroxyproline . the present study has indicated that the urinary excretion of glu - gal hyl is markedly decreased in als and is more pronounced with the duration of illness . [ 141 , 142 ] reported markedly decreased contents of collagen and collagen - associated amino acids in the lateral corticospinal tract and the anterior horn in the spinal cord of patients with als , suggesting that these data could be associated with the degeneration of the upper and lower motor neurons in patients with als . riluzole was the first drug approved by the fda ( usa ) in 1995 for the treatment of als , but its mechanisms of action in slowing the progression of this disease remain obscure . in initial study , to evaluate the efficacy and safety of the antiglutamate agent riluzole , bensimon et al . conducted a prospective , double - blind , placebocontrolled trial in 155 als patients and found that the antiglutamate agent riluzole appears to slow the progression of als disease , and it may improve survival in patients with bulbar onset of disease . further , lacomblez et al . also carried out a double - blind , placebocontrolled at multicentre and found that the 100 mg dose of riluzole has the best benefit - to - risk ratio , and it decreased mortality and slowed muscle - strength deterioration in als patients . it has been reported that the drug riluzole induces a strong metabolic signature which seems not to derive chemically from the drug , but may reflect modified metabolic processes . elucidation of the structures of these molecules could identify biochemical pathways that are subject to perturbations by the drug , some of which could contribute to its efficacy and some to its side effects . though the precise mechanism of action for riluzole remains unclear , it appears to interfere with excitatory amino acid signalling , possibly through the inhibition of glutamate release [ 145147 ] , blockade of inactivated sodium channels , and interaction with guanosine triphosphate - binding proteins . it is a tetracycline with anti - inflammatory action coupled with an independent antimicrobial property . minocycline has proved to be a promising neuroprotective agent [ 151154 ] when studied in mouse models of cerebral ischemia , spinal cord injury , and parkinson 's disease ( pd ) . as an anti - inflammatory , minocycline inhibits apoptosis ( programmed cell death ) via the attenuation of tnf- and downregulating proinflammatory cytokine output . this effect is mediated by a direct action of minocycline on the activated t cells and on microglia , which results in the decreased ability of t cells to contact microglia which impairs cytokine production in t - cell - microglia signal transduction . it has been also found that minocycline inhibits mitochondrial permeability - transition - mediated cytochrome - c release . a pilot study on als patients showed that it could be safe to take minocycline together with riluzole with no significant side effects . in a recently completed multicentre randomized placebocontrolled phase iii trial , however , the positive outcome seen in als mouse models was not reproduced in the human study . it has been reported that minocycline , which is known as an antibiotic and inhibitor of microglial activation , riluzole , which works as an inhibitor of glutamate release , and nimodipine , which is voltage - gated calcium channel blocker , is designed to target the different pathways leading to neuronal death . formerly , minocycline and riluzole have each alone been effective in delaying progression of als [ 143 , 144 , 156 , 160 , 161 ] . when the three drugs were tested in combination on sod1 mice , it resulted in strikingly improved conditions , delaying disease onset by 4 weeks , increasing survival by 6 weeks , and attenuating neurodegeneration by reducing cyclin - dependent kinase 5 mislocalization , caspase-3 activation , astrocytosis , and microgliosis . it has been found that in rat model it has the capability to increase glt1 expression by threefold . it has been also reported that ceftriaxone considerably reduced motor neuron loss and hypercellular gliosis , delaying muscle strength and body weight loss and improving longevity in sod1g93a mice . various antioxidants have been used as potential therapeutic agents in transgenic mice expressing the mutated human sod-1 enzyme . polyamine or putrescine - modified catalase , an antioxidant enzyme that removes hydrogen peroxide and has good permeability at the bbb , increases the survival of transgenic mice bearing the human msod-1g93a [ 147 , 148 , 163 , 164 ] . moreover , the copper chelator and thiol compound penicillamine , the copper chelator trientine , carboxyfullerenes , vitamin e , and n - acetylcysteine have been reported to increase the survival time in this mouse model and/or delay the onset of the disease to a small extent [ 165 , 166 ] . in als , weight loss is frequently observed and can occur early or later during the course of disease . all patients lose some weight due to the unavoidable reduction in skeletal muscle mass that results from denervation and decreased physical activity . in many cases , however , weight loss also has a nutritional component , with loss of fat mass and fat - free mass ( ffm ) attributable to malnutrition , the principal cause of which is decreased dietary intake [ 168 , 169 ] . swallowing disorders and a reduction of energy intake is associated with increased weight loss and the degree of dysphagia . anorexia , digestive disorders , and upper extremity motor difficulties also contribute to low intake . reports that resting energy expenditure ( ree ) may be increased in this context [ 171 , 172 ] appear paradoxical because ffm , the main determinant of ree , decreases in als . many research studies point towards significant role of mitochondria in als pathogenesis and thus may provide a cause of malnutrition in als patients . it has been found that prevalence of substantial mitochondrial degeneration in motor neurons of g93a sod1 mutant mice at the onset of the disease , when electron transfer chain activity and atp synthesis appear severely dysfunctional . furthermore , reduction in the activity of cytochrome - c oxidase , encoded by the mitochondrial genome , in motor neurons of sals patients has also been reported . there is a direct correlation between the amount of mitochondrial dna in the spinal cord of als patients with decrease in the activities of citrate synthase and the respiratory chain complexes i , ii , iii , and iv . not only nervous system but other tissues , including liver , lymphocytes , and muscle , are also affected by mitochondrial dysfunction . found a deficiency of nadh : coq oxidoreductase in muscles of sals and also noticed that decreased activities of nadh : coq oxidoreductase and cytochrome c oxidase are associated with dna abnormalities and reduced levels of the mitochondrial mn - superoxide dismutase . reported the early increase in the mrna levels of the mitochondrial uncoupling protein-3 in skeletal muscles of als - linked g86r sod1 mutant mice . in addition , isolated mitochondria from g86r muscle tissue exhibited a reduced respiratory control ratio , which is in line with the existence of mitochondrial uncoupling . supporting the mitochondrial dysfunction hypothesis , and administration of creatine , an intracellular energy shuttle between mitochondria and sites of energy consumption that is known to ameliorate muscle function , increased the life expectancy of g93a mice . although clinical trials failed to show a beneficial effect of creatine in als patients , the findings in g93a mice invoke the existence of a characteristic energetic imbalance . currently , hypermetabolism in als remains unexplained . the suggestion that hypermetabolism can be explained by an increase in the work of respiratory muscles lacks credibility because there is no correlation between ree and pulmonary function . the evaluation of a patient 's nutritional status and elucidation of the causes of any malnutrition are essential to provision of adequate nutritional care . in fact , malnutrition is common in als , with a frequency reported to be between 15 and 55% , depending on the stage of disease and the definition of malnutrition adopted . moreover , malnutrition is an independent prognostic factor for survival [ 171 , 172 ] . . indicated a chronic hypermetabolism in a subset of sporadic als patients , which complements a decrease in fat - free mass frequently observed in these patients . reported that fat - enriched high - energy diet to g86r mice resulted in an increase in body mass and adipose tissue accumulation , thus showing that increasing energy intake is sufficient to reduce the energetic deficit . . also showed that the number of cells in the ventral horns of the lumbar spinal cord , which is significant lost in large sized cells , most probably representing motor neurons , in symptomatic g86r , mice was prevented in high - fat diet - fed animals of the same age . it is worth emphasizing , however , that at least a subset of als patients showed characteristic hypermetabolic phenotype reminiscent of that observed in mice . from clinical perspective , the nutritional status is a prognostic factor for survival in als , and more evidence suggests that the proper customized nutritional management of patients may constitute a primary symptomatic treatment for the disease . nutritional intervention may prove to be a very significant aspect in als and can be explored further . the kp is a major route for the conversion of trp to nicotinamide adenine dinucleotide [ 62 , 63 ] generating neuroactive intermediates in the process . targeting the kp could offer a new therapeutic option to improve als treatment . in order to proceed further in this regard , two possible approaches can be taken , either to develop analogues of the neuroprotective kynurenines or to inhibit the synthesis of the neurotoxic quin . some of the below - mentioned drugs may show some promising therapeutic approach towards als . ( a ) intracerebral and intraperitoneal administration of 4-chlorokynurenine , which is a precursor of 7-chlorokynurenate , with quin , showed successful enzymatic transamination into the active 7-chlorokynurenate , conferring neuroprotection [ 188 , 189 ] by preventing quin neurotoxicity . ( b ) laquinimod , which is a novel synthetic quinoline , inhibited disease progression and infiltration of cd4 + t - cells and macrophages into the central nervous system ( cns ) . ( c ) an immunosuppressive and anti - inflammatory prodrug , leflunomide , gets converted to its active open - ring metabolite , teriflunomide , an inhibitor of mitochondrial dihydroorotate dehydrogenase , and known to successfully reduce the development of active lesions in patients with relapsing multiple sclerosis . ( d ) the synthesis of quin can also be blocked by inhibiting either kynureninase or kynurenine hydroxylase activity , thus , diverting the kp towards the synthesis of kyna . in immune activated mice , meta - nitronemzoylalanine also significantly reduced the formation of quin in the blood and brain . ( e ) ro61 - 8048 is another potent kynurenine-3-mono - oxygenase ( kmo ) inhibitor . it also reduces glutamate concentrations in the extracellular spaces of the basal ganglia in rats without impairing the learning or memory process typically associated with glutamate receptor antagonists and significantly reduces the neurotoxic levels of 3-hk and quin in the cns . ( f ) clioquinol is a quinoline metal chelator that binds selectively to zinc and copper ions with a hydrophobic nature that allows it to pass easily across the bbb . recent research with clioquinol in neurological disorders involving an imbalance in metal ions has led to promising results , presenting the possibility of a new therapeutic strategy . the wide range of survival time in als patients suggests that multiple prognostic factors are involved ; only some have been clearly identified . a specific biochemical marker for early diagnosing and for monitoring disease progression in als will have important clinical applications . a minority of patients carries mutations in the cu / zn - superoxide dismutase ( sod1 ) gene , but the disease mechanism remains unknown for all types of als . as the glutamate modulator , riluzole , is the only drug currently approved for als treatment , however , combination therapies that target other pathogenic mechanisms may be more effective in slowing disease progression and prolonging survival . the most recent finding in the als genetics suggests a very important role of mutation in sqstm1 and ubqln2 genes , and it is suggested that als patients should be monitored in a broad spectrum to study the altered effect on metabolic pathways [ 24 , 25 ] . the d90a sod1 mutation findings suggest that metabolomic profiling using gc - tofms and multivariate data analysis may be a future tool for diagnosing and monitoring disease progression and may cast light on the disease mechanisms in als . for this , the use of metabolomics as the link to pathway information in genome - wide association studies could be of great interest for mapping and interpreting the effects of different mutations and even combinations of mutations in relation to als subtypes . global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as csf . exploration of metabolomics by the use of small molecules derived from biofluids provides a strong platform to understand the metabolic characters of the living system and plays a significant role in the detection of diagnostic biomarkers . future clinical and experimental studies , therefore , need to concentrate on the complex relations between metabolism and als , and in this way may answer the question of whether targeting defective metabolism in als is an efficient way to alter disease progression . though a single technique is not sufficient to study the entire metabolomic profile , there is a need to converge some state of the art techniques namely , gc / ms , lc / ms , and nmr to realize the full worth of metabolomics . there is definite need to further elaborate on the sensitive and specific metabolic therapeutic signatures by advance analytical means and bioinformatics application which will help us to elucidate the structures of more signature molecules in als disease and provide us insight into aberrant biochemical pathways and may prove to be helpful in building up diagnostic markers and targets for drug design .

amyotrophic lateral sclerosis ( als ) is one of the most common motor neurodegenerative disorders , primarily affecting upper and lower motor neurons in the brain , brainstem , and spinal cord , resulting in paralysis due to muscle weakness and atrophy . the majority of patients die within 35 years of symptom onset as a consequence of respiratory failure . due to relatively fast progression of the disease , early diagnosis is essential . metabolomics offer a unique opportunity to understand the spatiotemporal metabolic crosstalks through the assessment of body fluids and tissue . so far , one of the most challenging issues related to als is to understand the variation of metabolites in body fluids and cns with the progression of disease . in this paper we will review the changes in metabolic profile in response to disease progression condition and also see the therapeutic implication of various drugs in als patients .

1. Introduction 2. Metabolomics 3. ALS and Peripheral Biomarkers 4. Brain Metabolites 5. Muscle Metabolite 6. Urinary Collagen Metabolite 7. Therapeutic Consequences 8. Conclusion

being overweight or obese is considered to be one of the biggest risk factors of poor health among school children worldwide1 . previous studies have shown a significant increase in obesity over a 12 year period , in all age groups of children and adolescents which was likely to result in them become overweight adults2 , 3 . studies have proposed the inclusion of daily physical activity as an essential intervention to prevent obesity among school populations4,5,6,7 . recently , leisure time has become more sedentary because of activities such as watching television ( tv ) , playing video game , and surfing the internet , and more people , especially in industrialized countries are expending less daily energy3 . a sedentary lifestyle has been associated with increased risk of obesity and related diseases ( e.g. , diabetes)8,9,10 . the average time spent watching tv in the us , europe , and australia has been reported to be 5 hours , 3.5 hours and 4 hours , respectively7 . during leisure time , the proportion of time spent watching tv while sitting and using a computer has increased from 26% in 1975 to 43% in 200511 . physical activity has been shown to lower the genetic predisposition to increased body mass index ( bmi)12 , 13 . it has been widely reported that an active lifestyle during childhood and adolescence optimizes growth and development14,15,16 . the body mass index has also been found to be related to total percentage of body fat in both boys and girls which can be influenced by genetic predisposition and environmental factors17,18,19,20,21,22,23 . different instruments have been used to measure daily physical activity including physiological ( e.g. , oxygen consumption , heart rate ) and behavioral tools ( e.g. , questionnaires , interviews , diaries)24 , 25 . it is also important to investigate the amount of time spent performing sedentary activities daily ( e.g. , television , electronic games , computers ) , which may contribute to increased weight , body fat , blood pressure , and serum lipids26 , 27 . because of the increased prevalence of overweight and obese youth and the risk of subsequent chronic disease in adulthood , it is important to determine the correlates of obesity in youth28 , 29 . however , to the best of our knowledge , there have been no studies that have examined the relationships between physical activity and sedentary behaviors ( such as tv viewing , computer usage , and passive commuting ) among obese and non - obese saudi children and adolescents . therefore , understanding the effect of sitting time associated with media consumption on lifestyle and physical activity patterns is important for the development of health - promoting interventions among schoolchildren who exhibit these behaviors . accordingly , this study was performed to assess the effect of daily sitting time associated with media consumption on physical fitness , total energy expenditure , and body composition indices of saudi schoolchildren . a total of 214 healthy saudi school students ( 818 years ; mean sd age = 14.40 2.36 years ) from different saudi schools were recruited for this study during the period of 20132014 . the study lasted three months and the participants were recruited through electoral roll randomized selection . out of these , only 180 healthy participants ( 90 boys , 90 girls ) were included in the study . none of the school children included in this study reported any kind of disability such as cerebral palsy , muscle weakness , or paralysis . all the participants provided informed consent before participation and this study was approved by the ethical committee of rehabilitation research , chair of king saud university . the demographics and baseline characteristics of the participants are shown in table 1table 1.demographic data of the school children participating in the study ( mean sd ; n=180)boys ( n=90)girls ( n=90)total ( n=180)age ( y)14.69 2.3914.01 2.2614.40 2.36height ( cm)169.6 7.4165.6 6.9167.8 7.2weight ( kg)69.72 7.466.1 7.9767.9 8.1bmi ( kg / m)23.1 3.3**24.11 2.124.6 4.1wc ( cm)100.70 18.67**111.27 20.15108.5 18.7whtr0.49 0.09 * * 0.43 0.050.48 0.08bmr ( kcal / day ) 1,749 124.6**1,518 94.91,635 109.8tee ( kcal / day ) 2,662 328.7**2,261 417.52,463 375.67-d activity ( counts /min)476 98.7**436 68.3462 88.7television viewing ( h / d)2.52 0.93**2.72 1.02.64 1.2computer usage ( h / d)2.93 0.85**2.56 0.872.750.86data expressed as mean sd ; p < 0.05 , * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) . data expressed as mean sd ; p < 0.05 , * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) . wc : waist circumference ( cm ) children or their parents were asked to assess the average number of minutes per day ( weekdays and weekend days combined ) that the children spent with screen media ( tv , video , computer , and video game usage)30 . tv viewing time was defined as the time spent watching tv , videotapes , or dvds , while computer time was defined as the time spent on a home computer or playing video games . body mass was measured to the nearest 0.1 kg using a portable digital metric scale , that was calibrated using standard weights . standing height was measured to the nearest 5 mm using a wall - mounted height board and bmi was subsequently calculated as body mass / height ( kgm-2 ) . waist circumference ( wc ) was measured as the minimum circumference between the iliac crest and the rib cage31 and whtr was calculated as wc divided by height . the participants pattern of physical activity was measured with the short form international physical activity questionnaire ( ipaq)32 , 33 . the participants were asked to report the average number of days per week and minutes per day during which they participated in physical activities . the total number of weekly minutes of walking as well as moderate and vigorous physical activity was computed according to the ipaq scoring manual34 , 35 . the participants were classified according to physical activity level ; mild ( 500 mets - min / week ) , moderate ( 5002500 mets - min / week ) , or active ( 2500 mets - min / week ) . basal metabolic rate ( bmr ) and total daily energy expenditure ( tdee ) were estimated from body mass , height , age , sex , and physical activity according to the harris and benedict equation36 . statistical analyses were performed using spss version 13.0 for windows ( spss inc . , chicago ) and data are presented as the mean sd . the normality of the data distribution was assessed using the kolmogorov - smirnov test and since body mass values were skewed , a non - parametric ( mann - whitney u ) test was adopted to compare normal and overweight children . associations between bmi , physical activity level , and other variables were calculated using correlation coefficients ( r & ) tests . overall , 180 saudi school students participated in this study and table 1 shows the characteristic baseline values of the participants . boys had significantly higher ( p < 0.01 ) whtrs and lower ( p < 0.01 ) wcs and bmis than girls . however , girls reported significantly lower ( p < 0.01 ) physical activity , bmr , tee , and computer usage values along with significantly higher ( p < 0.01 ) tv watching times than boys . based on physical activity , the students were classified into three groups ; mild ( n=40 ) , moderate ( n=35 ) , and active ( n=105 ) . table 2table 2.association between body composition indices , energy expenditure , total sitting time during media consumption and physical activity status of school children ( n = 180)mild ( n=40 ; 22.2%)( 500 mets - min/ week)moderate ( n=35 ; 19.4%)(5002500 mets - min/ week)active ( n=105 ; 58.3% ) ( 2,500 mets - min/ week)boys ( n=10)girls ( n=30)boys ( n=15)girls ( n=20)boys ( n=65)girls ( n=40)bmi ( kg / m)30.4 4.226.9 6.425.3 2.6**24.1 2.4**21.8 1.3**22.6 2.4**waist ( cm)112 7.9102 27.985 18.5 * * 98 5.8**65 16.5**81 4.5**hips ( cm)98 7.588.5 8.9100 9.1**81.5 5.4 * * 85 6.3**78.6 2.3**whtr0.59 0.090.62 0.050.49 0.06**0.48 0.03**0.44 0.08**0.45 0.06**bmr ( kcal / day)1,410 45.31,542 38.51,761 76 * * 1,545 76.2**1,735 88.5**1486 75.8**tee ( kcal / day)1,765 120.81,786 98.52,560 252**2,320 314.5**2,850 314.5**2690 180.5**television viewing ( h / d)2.9 1.33.6 0.962.6 0.96**3.1 0.85**1.9 0.73**2.6 0.81**computer usage ( h / d)3.5 0.893.4 0.852.8 0.88**3.0 0.83**1.85 0.83**2.3 0.75**tmst ( h / d)3.1 1.53.9 1.62.6 2.7**3.2 0.56**1.66 1.3**2.7 1.6**data expressed as mean sd ; p < 0.05 , * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) ; tmst : total media sitting time shows the effect of these scores on body composition indices , energy expenditure , and total time spent sitting during media consumption . the exercise levels were found to directly affect the body composition indices of the participants . the students in the moderate and active physical activity groups had significantly lower ( p=0.01 ) bmi , wc hips circumference , and whtr values than those of the low or sedentary physical activity group students with longer media sitting times ( table 2 ) . also , the values of significantly higher bmr and tee and significantly lower sitting time were greater in the physically active boys than in the physically active girls ( table 2 ) . the findings of this study suggest that physical activity , especially at moderate and active intensities , may be effective at reducing high body weights and obesity caused by sitting while consuming media for long periods . data expressed as mean sd ; p < 0.05 , * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) ; tmst : total media sitting time tables 3table 3.correlation coefficients ( r and ) between body composition indices , energy expenditure , and physical fitness score of school children derived from partial correlation and multiple regression analysisbody composition and tee ratephysical fitness scorerbmi 0.415 * * * 0.015 * * wc ( cm)0.522 * * * 0.025 * * * whtr0.396 * * * 0.012 * * * tee ( kcal / day)0.275 * * * 0 . 48 * * * * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) ; tmst : total media sitting time ; bmi : body mass index , wc : waist circumference , whtr : waist height ratio and 4table 4.correlation coefficients ( r & ) between body composition indices , energy expenditure , physical fitness score , and total sitting time during media consumption of school children derived from partial correlation and multiple regression analysisbody compositiontotal media time ( h / d)tv ( h / d)computer usage ( h / d)rrrbmi 0.64**0.011**0.98**0.035**0.74**0.042**wc ( cm)0.53**0.021**0.64**0.028**0.39**0.031**whtr0.91**0.029**0.49**0.027**0.23**0.036**physical fitness score0.245**0.009**0.123**0.013**0.250**0.048**tee ( kcal / day)0.115**0.012**0.145**0.019**0.265**0.057 * * * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) ; tmst : total media sitting time ; bmi : body mass index , wc : waist circumference , whtr : waist height ratio present the partial correlation coefficients and the results of the multiple regression analysis ( r and ) of physical activity , media time , and body composition indices . the students in the moderate and active physical activity groups showed significant inverse correlations with the body composition indices , bmi ( p < 0.001 ) , wc ( p < 0.001 ) , and whtr ( p < 0.001 ) , and a positive association with tee ( p < 0.001 ) ( table 3 ) . * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) ; tmst : total media sitting time ; bmi : body mass index , wc : waist circumference , whtr : waist height ratio * * p < 0.01 ; * * * p < 0.001 . bmr : basal metabolic rate ( kcal / day ) ; tee : total energy expenditure ( kcal / day ) ; tmst : total media sitting time ; bmi : body mass index , wc : waist circumference , whtr : waist height ratio finally , for girls , there were significant positive correlations between total media time and bmi ( p < 0.001 ) , wc ( p < 0.001 ) , and whtr ( p < 0.001 ) . in boys , computer usage was the only variable that was significantly associated with body composition ( p < 0.001 ) . physical activity scores and tee correlated negatively with total media time , tv viewing , and computer usage in all participants ( table 4 ) . although genetics , nutrients , and hormones are major determinants of the normal maturation and growth of children , the level of habitual physical activity is another factor that affects several biological traits , including body composition37 , 38 . recently , studies have proposed that the higher rates of low physical activity among young persons may be related to changes in social and environmental factors that initiate sedentary behaviors39 , 40 . these changes may affect the body composition indices of children and adolescents leading to childhood obesity that can extend into adulthood with consequent health issues such as cardiovascular and metabolic diseases41 . in this study , the potential effects of media time and computer usage on physical activity , total energy expenditure , and body composition indices were investigated . the girls reported longer average daily hours of watching tv than boys of the same age . these findings agree well with previous studies that have also reported females have longer average of media times than to males42 , 43 . previous research has reported that young adults ( 1830 years ) spend almost twice as much of their leisure time using a computer than older adults44 . our present data is also in agreement with studies that concluded gender was associated with computer time and tv viewing45 ; gender may be a determinant of screen - viewing time in young adults . in this school - based study , the negative effects of tv viewing and computer usage on body composition although there were significant increases in bmi , wc , and whtr in girls who reported more screen - viewing time , computer usage was the only variable that was significantly correlated with body composition in boys . partial correlation and regression analysis revealed significant positive correlations between body composition parameters and total sitting time , tv viewing , and computer usage . this result is supported by other studies that showed an increase in bmi in response to a greater number of hours watching tv in childhood and adolescence , which may extend into adulthood24 , 25 . an increase in tv viewing has been associated with bmi in both children and adults9 , 46 ; however , this relationship is stronger among young children27 . these results support the recommendation that children and adolescents should only spend two hours per day watching tv47 , 48 . the present study investigated the effect of tv viewing and computer usage on tee and revealed that girls had lower values of tee than boys . there was an inverse correlation between total screen viewing ( tv watching and computer usage ) and these results agree with a previous study that addressed the link between tv viewing and energy expenditure in older children49 . the reduction in tee may be related to the fact that a considerable amount of girls daily energy intake is spent while watching tv50 . also , tv watching increases the consumption of food , consequently resulting in a higher energy intake in children51 , 52 . furthermore , a decrease in tv viewing has been shown to have a positive effect on bmi by reducing energy intake53 . tv viewing and computer usage are habits of a sedentary lifestyle that is linked with lower total energy expenditure ( tee ) ; thus , it should be monitored in children to overcome obesity54 . most studies have shown that during tv viewing , tee may be slightly higher ( 18% ) than the resting metabolic rate ( rmr ) in adolescents and adults55 , 56 . physical inactivity promoted by a sedentary lifestyle may play a role in increasing the number of overweight and obese children57 , 58 . therefore , being able to estimate the potential correlation between physical activity , a sedentary lifestyle , and body composition in young children and adolescents may be useful for understanding the etiology of childhood obesity . the present study examined physical activity levels in relation to body composition as parameters of physical fitness in saudi schoolchildren . the participants were classified into three activity level groups : mild ( 22.2% ) , moderate ( 19.4% ) , and active ( 58.3% ) . it was found that physical activity significantly correlated with better physical fitness as measured by body composition and tee levels in boys but not girls . boys with higher physical activity levels had better bmis , wcs , and whtrs than those in the mild or sedentary activity level groups . in physically active children , of both genders , physical fitness status showed significant inverse correlations with bmi ( p < 0.001 ) , wc ( p < 0.001 ) , and whtr ( p < the data presented here are in agreement with previous studies in different adolescent populations worldwide that reported that boys had better fitness than girls40 , 59 , 60 . these differences in physical fitness between the genders may be due to variances in hematological parameters and ventricular chamber sizes61 . similarly , a positive relationship was observed between overall obesity in both genders and physical inactivity during the transformation from adolescence to adulthood , and it was linked with abdominal obesity in females at the age of 31 years62 . other research has also discussed the link between obesity and physical inactivity ( i.e. , lower fitness ) in adolescents and children , and proposed that low physical activity is one of the biggest factors connecting obesity and impaired physical fitness63,64,65,66 . in the study presented here , physically active participants had significant negative relationships between physical fitness and total time spent watching tv media and computer usage compared to participants in the mild activity level group . in the female sample , those who spent more time in front of a screen showed lower levels of physical activity than boys . these data are in agreement with others the results of other researchers who have reported statistically significant relationships between tv viewing times and body fat percentages in children and youth27 . it has been reported that patterns of physical activity and behaviors of a sedentary lifestyle play a pivotal role in weight gain67 and that media - based screen time such as tv viewing and computer usage interfere with the time that should be spent being physically active68 . most studies have reported that children watching tv for fewer hours have lower obesity rates than those who watch tv for four hours or more69 . thus , reducing sedentary behaviors in children should be considered as one of the most important interventions for reducing and treating obesity26 . finally , the data show that physically inactive students with sedentary behaviors such as time spent in front of screen may be at risk of overweight and obesity syndromes . in conclusion , the data suggest that poor physical fitness , lower tee , and more time spent sitting while consuming media may differentially influence normal body composition indices among schoolchildren and may lead to diseases associated with being overweight or obese . thus , decreasing sitting time while consuming media and enhancing physical activity , may have an important role in preventing obesity in young children .

[ purpose ] this study was performed to assess the effect of daily sitting time during media consumption on physical fitness , total energy expenditure ( tee ) , and body composition indices of saudi school children . [ subjects and methods ] a total of 180 healthy saudi school students ( 818 years ) were included in this study . sitting time , total energy expenditure , and levels of physical activity were evaluated with pre - validated internet based questionnaires . body composition indices were evaluated using anthropometric analysis . [ results ] out of the studied participants , only 22.2% of students were physically inactive . children with moderate and active physical scores demonstrated less sedentary behavior ( tv viewing and computer usage ) , lower body composition values ( bmi , wc , whtr ) , and higher tee than sedentary or mild activity level participants . boys showed higher fitness scores and less sedentary behavior than girls . media sitting time among the studied subjects correlated negatively with physical scores and positively with body composition . [ conclusion ] the data presented here suggests that poor physical fitness , lower tee , and longer sitting times differentially influence normal body composition indices among school children which may lead to overweight or obese individuals . thus , decreasing sitting time during media consumption and enhancing physical activity may play a pivotal role in preventing obesity in young children .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

the term oligodontia is generally used to describe the absence of more than six teeth , excluding third molars , and complete failure of dentition to develop is called anodontia . it has been reported that in 20% to 30% of the population , at least one of the third molars is absent . the prevalence of agenesis of other permanent teeth have been reported in different races and countries . after third molars , agenesis of lower second premolars and upper lateral incisors are the most common types of hypodontia . the reported prevalence of hypodontia in orthodontic patients have been different between studies , ranging from 2.7% to 11.3% . the etiology of hypodontia can be a combination of genetic and environmental factors , and it can occur as an isolated condition ( non - syndromic hypodontia ) or can be associated with a systemic condition or syndrome ( syndromic hypodontia ) . the agenesis of permanent teeth represents clinical problems and requires a multidisciplinary approach to cover the needs of the patients . aesthetic problems , periodontal damage , malocclusion , and alveolar bone deficiency are some of complications following hypodontia . for example , agenesis of maxillary lateral incisor impairs dental aesthetics and function from a very young age . hence , investigating the prevalence of tooth agenesis is of significant clinical value in terms of early diagnosis and effective treatment planning . although the prevalence of hypodontia has been studied in other countries , it has not been well documented in studies of iranian population . therefore , the aim of this study was to investigate the prevalence , characteristics , and sex distribution of hypodontia as well as its association with different malocclusions among orthodontic patients in shiraz , iran . in this descriptive retrospective cross - sectional study , a total of 494 records of patients ( 162 males , 332 females ) including dental casts , intraoral photographs , panoramic and lateral cephalometric radiographs were evaluated . patients with any syndrome or cleft lip / palate , those with previous loss of teeth due to caries , periodontal diseases , trauma or orthodontic extraction were excluded from the study . two expert clinicians evaluated all radiographs , and the radiographic findings were checked with recorded information of the patients to assure that the tooth had not been extracted . pertinent information including age , gender , clinical findings written in the file , the type of malocclusion , number and type of missing tooth , the site of the agenesis ( maxilla or mandible , right or left side ) , being unilateral or bilateral was determined . to diagnose and categorize the type of malocclusion anb angle between 2 and 4 degrees were categorized as class i occlusion / malocclusion . accordingly , higher and lower anb angles were categorized as class ii and class iii malocclusions , respectively . the findings from the anb angle were checked to be consistent with photographs and clinical findings documented in the patient file , and if there was any doubt about the accuracy of the anb angle , wit 's appraisal measurement was used to accurately categorizing . wit 's appraisal of 0 to 1 were classified as class i , those with positive wit 's measurements were classified as class ii and negative wit 's measurements more than 1 as class iii . all comparative statistical analyses were performed using the spss software ( version 12.0 , spss inc . , chicago , il , usa ) . to compare the difference between different malocclusions , chi - square test and to evaluate the difference between male and female patients , all comparative statistical analyses were performed using the spss software ( version 12.0 , spss inc . , chicago , il , usa ) . to compare the difference between different malocclusions , chi - square test and to evaluate the difference between male and female patients , the distribution of tooth agenesis according to the gender in different malocclusion groups is shown in table 1 . from the total cases congenital absence of one or more teeth was observed in 38 out of 494 patients , with a frequency of 7.66% . also , from all of the 38 patients with hypodontia , 63.15% had class ii , 23.7% were class i , and 13.15% had class iii malocclusions [ table 1 ] . regard , class iii patients showed the highest rate of hypodontia ( 9.6% ) followed by class ii and class i patients ( 8.3% and 5.84 , respectively ) . distribution of different malocclusions in our study sample also , from all of the patients with missing teeth , 22 ( 58% ) were females and 16 ( 42% ) were males . however , this difference was not statistically significant ( fisher 's exact , p = 0.552 ) [ table 2 ] . distribution of hypodontia according to gender there were a total of 93 missing teeth in our examined patients . of all 93 missing teeth , the most ( 50.54% ) was observed in class ii group , and the least amount belonged to class i group ( 22.58% ) . however , there was no statistical significant difference between different malocclusions in the number of missing teeth . the most and the least affected teeth were upper lateral incisor ( 27.95% ) and upper second molar ( 1.07% ) , respectively . fifty four percent of all missing upper lateral incisors and 50% of all missing lower second premolars were found in class ii patients . thus , the most prevalent missing tooth types were found more in class ii patients , and interestingly , tooth types that were absent less frequently , were found more in class iii patients . most of all missing teeth were found in the maxilla , and only 40.87% of absent teeth belonged to mandible . this higher frequency of hypodontia in the upper arch was observed in all types of malocclusions [ table 3 ] . prevalence of various tooth type agenesis in different mallocclusions and genders there was a higher frequency of missing teeth in the left side in all types of malocclusions ( 55.91 ) . prevalence of missing teeth in different types of malocclusions with respect to the affected jaw and side among 38 patients with missing teeth , only 7 patients ( 18.4% ) had missing of more than one tooth type , and 73.68% had 1 or 2 missing teeth , and 21 patients ( 55% ) had more than one missing tooth . as tooth agenesis is the most common anomaly in the development of the human dentition , many studies are found on the prevalence of hypodontia in different ethnic groups . the etiology consists of environmental and genetic factors including infection , trauma , and medicines , as well as genes associated with certain syndromes . early diagnosis and suitable intervention seems to be necessary and can play an important role on prevention of its serious aesthetical , physiological , functional , and even emotional complications . therefore , detection of missing teeth before age 10 might cause false and unreliable results . disruption of the findings in terms of the most frequently missing teeth can also affects the results due to delayed development of premolars . that 's why higher prevalence of premolar missing has been reported in some studies . considering this matter , results of a meta analysis showed that the prevalence for both sexes was higher in europe and australia than for north american caucasians . present study carried out among orthodontic patients in southern iran , shiraz , and showed 7.66% prevalence for hypodontia . in a previous study , prevalence of hypodontia was reported to be 9.1% in an iranian orthodontic population that is higher than that of our study . prevalence of hypodontia in our study is comparable to the results of sisman et al . ( 6.2% ) in orthodontic populations , and is higher than that reported in a pediatric orthodontic population in venezuela ( 4% ) . however , prevalence of hypodontia in our population is within range of asian populations reported in different studies . the prevalence of tooth agenesis in females was also reported to be 1.37 times higher than in males in north american caucasians . it has been mentioned that this higher prevalence of hypodontia in females might be associated with biological differences and makes a greater orthodontic treatment need , that increases their seeking for treatment due to aesthetical concerns . however , several investigations found higher prevalence in males and some reported no difference between gender . nevertheless , according to the our results , dental agenesis was more common in males.this study as well as some others did not find a statistically significant difference between hypodontia prevalence in males and females . in our study , 9.6% of class iii patients had hypodontia ; while 8.3% of class ii patients and 5.84% of class i patients showed this anomaly . this higher prevalence of hypodontia ( although not significant ) in class iii patients is in accordance with some of previous studies . although , celikoglu et al . , found more prevalence of hypodontia in their class i study sample . also , the prevalence of hypodontia in the maxillary arch was higher than in mandibular arch , that is consistent with most of the previous studies . however , there are studies which had found a greater missing rate in the mandible . this higher prevalence of hypodontia in the maxillary arch and in class iii patients is an interesting finding that might demonstrate an association between these two findings and may be considered as etiologic factor . consistent with the findings of several studies , the most prevalent missing teeth types in our study were upper lateral incisor ( 27.95% ) followed by lower second premolar ( 21.25% ) and maxillary first and second premolar . in most previous studies , either upper lateral incisor or lower second premolar has been reported as the most prevalent . nevertheless , in the results of some other investigations the mandibular second premolar was introduced as the most prevalent absent tooth . another interesting finding of our results was that the most prevalent missing tooth types ( upper lateral incisor and lower second premolar ) were more found in class ii patients , while other missing tooth types were observed more in class iii patients . found that patients with maxillary lateral incisor agenesis had a significantly increased prevalence rate of permanent tooth agenesis . they observed the frequencies of maxillary second premolar agenesis , mandibular second premolar agenesis , microdontia of maxillary lateral incisors , and distoangulation of mandibular second premolars were significantly increased . the missing teeth were more often absent bilaterally ( 73.11% ) , and only 26.89% were unilaterally missed , with a tendency of higher prevalence in the left side . silva meza also reported that missing teeth are mostly found bilaterally , whereas fekonja observed more prevalence of missing teeth in right side . from all of the patients with tooth agenesis , only 18.4% had missing of more than one tooth type . thus , finding shows that most of the hypodontia patients had only one missing tooth type . by early detection of tooth agenesis , a multidisciplinary team approach can be performed in order to establish an esthetic and functional dentition and to minimize the complications of hypodontia in the future . the prevalence of hypodontia in our orthodontic patients was 7.66% that showed no significant difference between males and females . higher prevalence of hypodontia in the maxillary arch and in class iii patients may be considered as an etiologic factor .

objectives : to evaluate the pattern and prevalence of hypodontia and its association with different malocclusions among orthodontic patients in southern iran.materials and methods : in this study , a total of 494 records of orthodontic patients ( 162 males , 332 females ) were evaluated . to investigate percent of hypodontia pertinent information of patients was evaluated . chi - square and fisher 's exact tests were used for statistical analysis.results:the prevalence of tooth agenesis was 7.66% . thirty - eight patients were found to have at least one tooth missing . number of patients having hypodontia was the most in class iii patients , whereas the number of missing teeth was more in class ii sample . however , it was not statistically significant ( p = 0.569 ) . upper lateral incisor ( 27.95% ) , lower second premolar ( 21.51% ) , and upper first premolar ( 12.9% ) were respectively the most frequently absent teeth . there was not statistically significant difference between sexes ( p = 0.580 ) . hypodontia observed to be more bilaterally , in the upper arch and in the left side.conclusion:the pattern and prevalence of hypodontia is different among races and ethnic groups . in our study population , hypodontia was found to be 7.66% . higher prevalence of hypodontia in the maxillary arch and in class iii patients may be considered as an etiologic factor .

INTRODUCTION MATERIALS AND METHODS Statistical analysis RESULTS DISCUSSION CONCLUSION

an ongoing issue is whether similar or different mechanisms contribute to the pathogenesis of different ms subtypes . current opinion implicates peripheral t cells in an autoimmune response directed against cns antigens as the pathogenetic mechanism in rrms , but not in ppms or in spms [ 1 , 2 ] . instead , some claim that cns injury in progressive ms is due to neurodegeneration , or as suggested recently , to a neuroinflammatory process confined within the blood brain barrier . the basis of autoimmunity in ms is a subject of considerable interest . over the past twenty years , work in my laboratory investigated the possibility that a thymic abnormality contributes to the pathogenesis of ms . the results of our studies of the t - cell receptor ( tcr ) repertoire ( see below ) led us to question whether rrms patients have altered t - cell homeostasis . we also considered the possibility that ppms patients share t - cell homeostatic alterations with rrms patients . the thymus has been shown to have an important role in the regulation of experimental autoimmune encephalomyelitis ( eae ) . peripheral homeostatic t - cell responses to lymphopenia have a central role in the development of experimental autoimmune diabetes and in patients after islet cell transplants . in rheumatoid arthritis , one group of investigators reported homeostatic peripheral t - cell alterations , possibly secondary to reduced thymic output although direct evidence of a thymic alteration was not obtained . as summarized below , we have direct evidence of reduced thymic output not only in rrms but also in ppms . this reduced thymic output leads to peripheral homeostatic responses and t - cell activation in both ms subtypes . it is proposed here , analogous to lymphopenia - induced autoimmunity , that reduced thymic output results in a homeostatic response that contributes to the pathogenesis of both rrms and ppms . in 1993 , utz et al . reported that antigen - stimulated t - cells from rrms patients have major shifts in their overall tcr diversity , that is , tcr repertoires . this report led us to question whether tcr repertoire shifts occur in unstimulated t - cells from rrms patients and whether these shifts could contribute to the pathogenesis of rrms . in order to address this possibility , we analyzed freshly isolated naive cd4 t - cells , which by definition have not been antigen - stimulated . we reasoned that changes in these t cells would be independent of the immune response in rrms and precede the onset of rrms . we studied identical twins discordant for rrms . in comparisons with healthy identical twin pairs , both the affected twins and their healthy cotwins had tcr repertoire shifts , specifically in the complementarity - determining 3 ( cdr3 ) regions of several tcr v beta gene segments . since we analyzed nave t - cells , the findings suggested that these shifts precede rrms and likely predispose to the development of rrms . importantly , it seemed unlikely that these shifts were sufficient to initiate ms by themselves , as both the healthy and the affected members of the discordant twin pairs had tcr repertoire shifts . firstly , we excluded genetic differences between twin pairs , including hla differences , as explanations for the tcr repertoire shifts ( see for a detailed discussion ) . in this regard , a genetic influence on cdr3 repertoires is highly unlikely ; 90% of tcr diversity is randomly generated by nucleotide additions within the cdr3 region during thymopoiesis and these additions are independent of genetic background . secondly , a stochastic basis is improbable , since both members of the discordant ms twin pairs shared the tcr repertoire shifts . thirdly , and a more likely explanation is that unknown environmental factors affected both members of each discordant twin pair so as to alter tcr repertoire formation in the thymus ; we have no direct evidence to support this explanation . since the thymus has a central role in nave t - cell generation and t - cell homeostasis ( reviewed in ) , we hypothesized that ms is a disorder of t - cell homeostasis . homeostasis is the capacity of a biological system to maintain its equilibrium by physiological mechanisms in response to change . t - cell homeostasis is tightly regulated and includes a balance between thymic output , which decreases exponentially with increasing age , delivery of death and survival signals , nave t - cell differentiation into memory cells , and peripheral t - cell proliferation . this t - cell proliferation is an important contributor to t - cell homeostasis in healthy individuals , as it helps maintain t - cell numbers in the face of thymic involution . signal joint t - cell receptor excision circles ( sjtrecs ) are often quantified as a measure of thymic output [ 1426 ] . these sjtrecs are cytoplasmic circular dna fragments ( episomes ) and can not replicate during mitosis [ 14 , 15 ] . consequently , reduced sjtrec frequencies , that is , reduced sjtrecs in constant cell numbers ( e.g. , sjtrecs/10 cells ) , could be due either to reduced thymic output or to increased peripheral t - cell proliferation [ 14 , 15 ] . in rrms , various investigators report reduced sjtrec frequencies in peripheral blood mononuclear cells ( pbmc ) or in t subsets . as one might expect from the difficulty in interpreting sjtrec frequencies , some claim reduced thymic output whereas others raise the possibility of increased peripheral t cell proliferation in rrms [ 1822 ] . an important reason for the different interpretations is that none of these studies included a direct measure of thymic output , which excludes the contribution of peripheral t cell proliferation . nave t cells contain the vast majority of sjtrecs whereas memory t cells contain very few sjtrecs . memory t cell numbers increase with age and might also vary with the magnitude of the chronic immune response in ms . consequently , any demonstration of patient - control differences in the sjtrec content of pbmc or total t cells could reflect , in part , differences in memory t - cell numbers between patients and controls . in order to avoid these difficulties in interpreting sjtrecs , we isolated nave t subsets and studied their sjtrec content . in order to exclude possible treatment influences on sjtrecs , we only studied rrms patients in remission ; none had been treated with corticosteroids , immunomodulatory agents in the year prior to the study , and none of the patients had ever received cytotoxic agents . since most rrms patients receive some form of treatment , we could not identify sufficient patients to control for disease duration . in one study , we found that rrms patients under , but not over , age 40 have significantly reduced sjtrec frequencies compared to age - matched controls . in a second study of both rrms and ppms patients , we observed that the sjtrec frequencies of naive cd4 and nave cd8 t cells decrease exponentially with age in healthy controls , as shown in figures 1(a ) , and 1(b ) . this finding is consistent with several reports indicating that thymic output decreases with increasing age [ 14 , 15 ] . in contrast to controls , naive t - cell sjtrec frequencies did not show a significant decrease with age in rrms or in ppms . instead , the younger rrms and ppms patients had lower sjtrec frequencies than controls of a similar age , and these frequencies remained at low levels with increasing age of the patients . since sjtrecs are influenced not only by thymic output but also by t cell division [ 14 , 15 ] , we sought direct evidence of reduced thymic output in ms patients . total sjtrec numbers in the nave t cell pool , for example , sjtrecs / ml of peripheral blood , are unaffected by peripheral t - cell proliferation and consequently provide a superior measure of thymic output . total naive cd4 t - cell sjtrecs were significantly reduced in rrms and ppms patients compared to controls ( see figure 1(c ) ) , indicating reduced thymic output in both ms subtypes . to extend this work , we exploited a mathematical model , which provides the first reported quantitative measure of thymic export ( t - cells / day ) . this model takes into account nave cd4 t - cell sjtrec content and the absolute number of nave cd4 t - cells and identifies and excludes the contribution of t - cell proliferation , measured by expression of the proliferation marker ki-67 , to nave cd4 t - cell sjtrec content . application of this model demonstrated that both rrms and ppms patients have reduced thymic export of nave cd4 t cells / day compared to controls , and that thymic export is low in young patients from both patient groups and remains low with increasing age ( see figure 1(d ) ) . surprisingly , thymic export of nave cd4 t - cells / day was lower in ppms than rrms . taken together , the quantitative data indicate that both rrms and ppms patients have a major thymic alteration with early - onset thymic involution and resulting reduced thymic output [ 23 , 24 ] . the long - term maintenance of function and survival of peripheral nave tcells depends upon tcr signalling via self - peptide - mhc class ii molecules and upon il-7 receptor ( cd127 ) signalling via il-7 , which in turn induces expression of the anti - apoptotic molecule bcl-2 . , we found apparently increased expression of bcl-2 by cd31-negative ( cd31neg ) cells from rrms patients versus controls ; cd31-positive nave cd4 t cells include cd4 recent thymic emigrants whereas cd31neg cells are distant in origin from the thymus [ 16 , 28 ] . we found increased expression of bcl-2 by both nave cd4 and cd8 t cells from ppms patients compared to controls . these findings suggest increased delivery of survival signal in both patient groups , which may partly compensate for the reduced thymic output in these patients , that is , helps maintain the size of the peripheral naive t - cell pools . increased bcl-2 expression may also have relevance for ms susceptibility ; overexpression of -arrestin 1 , a positive regulator of bcl-2 expression , increases susceptibility to eae , and cd4 t cells in ms have increased expression of -arrestin 1 . the magnitude of proliferation of early ( cd3-cd4-cd8- ) thymocytes is the main determinant of thymic output . proliferation of early thymocytes decreases progressively with age , which explains the progressive age - associated decreasing thymic output that occurs in healthy individuals [ 30 , 31 ] . we used two strategies to assess peripheral homeostatic proliferation ; in all studies , we analyzed nave t - cell expression of the proliferation marker ki-67 , which is expressed in all cells in the g1 , s , g2 , and m phases of the cell cycle ; in one study , we quantified nave cd4 t - cell trecs , which are a byproduct of early thymocyte proliferation . in an early study , we reported increased ki-67 proliferation levels in nave cd8 t cells from rrms patients versus controls . in a later study , we reported progressive age - associated reduction of nave cd4 t - cell trecs in rrms patients versus controls . this finding suggested that rrms patients have increased nave cd4 t - cell homeostatic proliferation ; an increase in thymocyte proliferation seems an unlikely explanation of the reduced trecs , as such proliferation is inconsistent with thymic involutional processes [ 30 , 31 ] . we also attempted to use a more direct measure of thymocyte proliferation , the sj/trec ratio ; to obtain this ratio some quantify either 6 or 10 trecs , extrapolate to 13 trecs , and then calculate the sj/trec ratio [ 31 , 33 ] . after quantifying six trecs in cohorts of rrms patients and age - matched controls we concluded in our laboratory that calculations of sj/trec ratios would not provide accurate , direct measures of thymocyte proliferation . detailed comparisons of ki-67 expression levels in nave cd4 t - subsets from rrms patients and controls did show , however , increased nave cd4 t - cell proliferation in rrms , predominantly in the cd31-positive cells having the highest levels of cd31 expression ( cd31hi cells or cd4 recent thymic emigrants for details , see ) but also in cd31neg cells . our proliferation data suggest that increased peripheral t - cell proliferation may partly contribute to reduced sjtrecs in rrms , as suggested in other reports [ 18 , 22 ] , but , as noted in section 4 , reduced sjtrecs in rrms are mainly due to reduced thymic output . several reports emphasize that tcr signalling of cd31-positive naive cd4 t cells induces loss of cd31 expression , which is accompanied by increased naive cd4 t - cell proliferation [ 28 , 34 ] . we analyzed the proportion of naive cd4 t - cells expressing cd31 as a potential indicator of increased proliferation in ppms , in comparisons with rrms and controls . an age - associated decrease in this proportion only in ppms ( see figure 2 ) initially suggested increased naive cd4 t - cell proliferation in ppms compared to the other two groups . also , if increased proliferation had occurred in ppms , nave cd4 t - cell sjtrec frequencies should also have decreased with age , but that did not occur . a recent study has showed that tcr signalling does not induce total loss of cd31 from the t - cell surface but rather cleavage and shedding of a portion of cd31 containing the epitope recognized by the commonly used anti - cd31 antibody . accordingly , our data indicate increased tcr signalling of nave cd4 t cells in ppms without increased proliferation . the direct evidence for early - onset thymic involution and reduced thymic output in rrms , described above , is supported by the indirect evidence of reduced thymic output from other laboratories [ 1922 ] . to my knowledge , one limitation to our studies is that we have no definite evidence as to the basis of the reduced thymic output in ms although our tcr repertoire studies raise the possibility that some unknown environmental factor / factors target(s ) the thymus in rrms ( see above ) . direct analysis of thymic export suggests that thymic alterations occur early in life in rrms and ppms , either at the time of ms onset or before and that the thymic defect persists throughout life , as indicated by low but constant levels of thymic export with increasing age . in other words , . one study of immunoablation / autologous stem cell transplantation as a treatment of ms showed that the post transplant ms patients rebooted their immune system such that the thymus produced new t cells having a diverse tcr repertoire . this finding is not inconsistent with our results ; rrms patients over age 40 continue to generate recent thymic emigrants , which contain the majority of nave t cell tcr diversity although this generation is at significantly lower levels than age - matched controls . a critical question is whether reduced thymic output has relevance to the pathogenesis of ms . both rrms and ppms patients have increased naive cd4 t - cell expression of bcl-2 [ 23 , 24 ] . increased expression of this survival signal may partly compensate for reduced thymic output in rrms and ppms by helping to maintain the size of the nave t - cell pool . as noted in section 5 , increased expression of this anti - apoptotic molecule may also increase susceptibility to ms . homeostatic proliferation induced by self - peptide mhc constricts the tcr repertoire and inevitably expands autoreactive t cells , particularly autoreactive cd31neg naive cd4 t cells [ 28 , 34 ] . the increased homeostatic proliferation in rrms is analogous to the homeostatic proliferation that initiates autoimmune diabetes in a mouse model , that is , it seems logical to implicate this process in the pathogenesis of rrms . this shedding abrogates the activity of cd31-associated immunotyrosine - based inhibitory motifs ( itims ) . in ppms , increased tcr signalling of naive cd4 t cells , presumably by self - peptides , could activate self - reactive naive cd4 t cells and initiate autoimmune responses . since naive cd4 t cells have a central role in initiating immune responses , including autoimmune responses in rrms , the peripheral tcr signalling / proliferative responses to reduced thymic output likely initiate autoreactivity in ppms and rrms . if this view is correct , it remains unresolved as to why ppms patients show minimal responses to anti - inflammatory and immunosuppressive agents and why ppms patients have a paucity of new mri lesions . obviously , peripheral homeostatic naive t - cell responses to early - onset thymic involution will persist throughout life in ppms and rrms in order to maintain the naive t - cell pool . natural regulatory t cells ( ntregs ) are included within the naive cd4 t subset and show disturbed development and function in spms . one possibility in ppms is that early - onset thymic involution alters ntregs , either quantitatively or qualitatively , so that cns injury is continuous without relapses , as suggested by one study of t - cell regulation in ppms . further analysis of regulatory t - cell function in ppms is essential to address this issue .

uncertainty exists as to whether similar or different mechanisms contribute to the pathogenesis of different subtypes of multiple sclerosis ( ms ) . detailed analysis of naive t cell homeostasis shows that patients with relapsing - remitting ms ( rrms ) and with primary progressive ms ( ppms ) have early - onset thymic involution that causes reduced thymic output . the reduced thymic output leads to secondary peripheral homeostatic alterations in nave cd4 t - cells , which closely mimic t - cell alterations observed in an experimental animal model of diabetes mellitus . homeostatic t - cell receptor ( tcr ) signalling and proliferation of nave t cells are induced by self - peptides . consequently , the findings of increased tcr signalling of nave cd4 t - cells , without increased proliferation , in ppms , and the increased homeostatic proliferation of nave cd4 t - cells in rrms favour the development of autoimmunity . thus , it seems highly likely that peripheral t - cell alterations secondary to a thymic abnormality contribute to the pathogenesis of both ms subtypes .

1. Introduction 2. Studies of the TCR Repertoire in RRMS 3. T-Cell Homeostasis: A Brief Summary 4. Reduced Thymic Output in RRMS and in PPMS 5. Increased Nave CD4 T-Cell Survival Signals in MS 6. Increased Naive T-Cell Proliferation and TCR Signalling in MS 7. Conclusions

corneal endothelium dysfunction , as well as the resulting reduced transparency due to corneal edema , remains a major indication for corneal transplantation . until 1998 , the only known technique for exchanging the corneal endothelium was a full - thickness corneal transplantation penetrating keratoplasty , even though this type of disease affects only a thin inner layer of cells the endothelium . in 1998 , its main advantages compared to conventional penetrating keratoplasty are a rapid improvement in visual functions , lower incidence of serious postoperative complications , a sutureless technique and significantly higher comfort for the patient [ 1 , 2 ] . the disadvantages of these surgeries are the relatively high technical difficulty involved and the high loss of transplanted endothelium cells during the procedure in the early postoperative period [ 3 , 4 ] . therefore , many eye surgeons have focused on this issue in an attempt to simplify the procedure and improve the long - term results of lamellar keratoplasty . the optimal procedure has not yet been clearly established and there are many variations of the operation . the first type of lamella is composed of endothelium , descemet membrane ( dm ) , and a part of the deeper stroma , which works as a structural support to enable feasible manipulation of the lamella and to prevent massive damage of the fragile endothelium . such techniques are called descemet 's stripping endothelial keratoplasty ( dsek ) , if the lamella is created manually , or descemet 's stripping automated endothelial keratoplasty ( dsaek ) , if the lamella is made using a microkeratome or femtosecond laser [ 57 ] . the main advantages of these techniques are relatively easy manipulation , less stress on the endothelial cells , and good recognition of the stromal and endothelial side of the lamella into the anterior chamber of the recipient 's eye . a lamella with stroma keeps a convex shape , and it is also possible to mark the stromal side . the main disadvantages are the plus of the stromal tissue and possible interlamellar problems , so the best corrected visual acuity is very often slightly decreased [ 3 , 6 , 810 ] . the second type of lamella consists of dm with endothelium , while the stromal support is absent . the donor discs are prepared from the corneoscleral donor button by stripping a circular portion of tissue ; the transplantation technique has been referred to as descemet membrane endothelial keratoplasty ( dmek ) [ 11 , 12 ] . the preparation of the recipient corneal bed is the same as in a dsek lamella . a donor button ( 9.5 mm ) is trephined from the endothelial side and subsequently stripped from the posterior stroma . in the storage medium , the endothelium - dm roll may be evaluated after treatment with trypan blue and sucrose . the preparation technique is standardized , and its basic parameters , as well as the postoperative follow - up , have been repeatedly published . these techniques are hypothetically optimal , because the surgeon replaces the involved endothelium and dm with the same portion of tissue . in this approach , there are no problems with interlamellar opacities , and the vision after surgery is very often excellent ; unfortunately , however , manipulating such a thin lamella is difficult ; in particular , the unwrapping of the lamella at the insertion may be quite stressful on the endothelium . the third type of lamella has a thicker periphery , similar to the lamella in dsek or dsaek , and a central part that consists of bare dm and endothelium , as in the dmek lamella . the techniques by which this lamella is prepared can be called hybrid techniques , because they combine the advantages of both the previous techniques . the central part without stroma insures excellent optical results after surgery , comparable with those of successful dmek patients ( figure 1 ) . the stromal rim fixes the thin , fragile central part , helping to maintain its shape and preventing the scrolling of the dm . moreover , the stromal part allows the anterior - posterior orientation of the lamella to be marked . this enables the surgeon to know exactly on which side the endothelium is located when manipulating the lamella , as well as its orientation in the anterior chamber . we called this special type of surgery dmek - s ( descemet membrane endothelial keratoplasty with peripheral stromal support ) . this paper describes the surgical procedure , implantation technique , and visual outcomes in each group of patients after dmek - s surgery . seventy - one eyes of 55 patients enrolled in a single - center study underwent posterior lamellar keratoplasty with a hybrid lamella dmek - s implantated using a solution implantation technique , owing to endothelial dysfunction . the study was held in the ophthalmology department of kralovske vinohrady teaching hospital and the 3rd medical faculty of charles university , prague , from 2009 through 2011 . the mean age of the patients was 71.25 12.6 years ( range 4094 years ) . the predominant indications for posterior lamellar keratoplasty were pseudophakic or aphakic bullous keratopathy ( 27 eyes ) , fuchs ' endothelial dystrophy ( 15 eyes ) , and posterior polymorphous corneal dystrophy ( 7 eyes ) . in 22 eyes , preoperative medication routinely consisted of antibiotic - steroid combination drops applied 5 times daily , 2 days preoperatively . patients applied tobradex ( alcon ) , which contains tobramycin and dexamethasone . if the patient was using antiglaucomatous medication , he continued using it until the day of surgery . the surgery was performed under an inpatient department regimen ; the duration of hospitalization was about 4 to 7 days . the proper preoperative preparation on the day of surgery was the same as in other anterior segment surgeries . to induce preoperative pupil dilation , the surgeries were performed with topical anesthesia using local anesthetic drops ( bupivacaine , lidocaine ) . in the case of single - procedure dmek - s and cataract surgery , topical anesthesia was combined perioperatively with intracameral anesthetics ( lidocaine ) according to need . in the operating theatre , the standard procedure for disinfection was followed , as was periocular and eyelid skin disinfection , and conjunctival sac and corneal surface irrigation , using a 5% povidone iodine solution ( betadine , egis pharmaceuticals ) for 3 minutes . we obtained the tissue used for donor lamella preparation before the dmek - s surgery from the international eye tissue bank otb01 in prague , czech republic . the 17 mm corneoscleral buttons were held in an eusol medium with an expiry date 14 days after the death of the donor . we consider 2500 cells / mm to be the minimum required endothelial cell density prior to the preparation of lamellae . all lamellae were prepared in the operating room under sterile conditions just prior to implantation . we placed the corneoscleral disc endothelium side up in the barron artificial chamber ( katena ) and separated the descemet membrane from the stroma using the big bubble technique . we injected an insulin needle into the corneal stroma in the outer periphery of the donor disc to minimize damage to the transplanted endothelium . air was subsequently injected into the stroma that caused the whitish appearance of the cornea , after which a central air bubble was formed , which separated the descemet membrane from the stroma . given that the corneal endothelial side was at this stage placed upwards , the forming bubble could be monitored visually . we covered the endothelial surface with a fine layer of dispersive viscomaterial ( viscoat , alcon ) , turned the donor cornea , and placed it securely in the artificial chamber , endothelium downwards . we completely removed the stroma from the central 6 mm zone dissecting the big bubble and left the deep stromal portion in the periphery of the donor cornea using a crescent knife . we used a color marker ( skin marker , kendall ) to mark the lamella in the peripheral area with retained deep stromal layers , enabling the next phase of the operation , which was to identify the stromal and endothelial sides of the disc . for this purpose , we chose either laterally asymmetric letters ( s , f ) or two different consecutive symbols ( one point , two points ) . after this , we used an 8.5 mm barron punch for trephination of the lamella . the preparation of the lamella usually took about 1015 minutes ( see the video in supplementary material available online at http://dx.doi.org/10.1155/2013/254383 ) . in four cases , we observed rupturing in the descemet membrane caused by rapid big bubble formation during preparation . in these cases , there was no detachment of the descemet membrane and no big bubbles were created . in these cases , we reached the descemet membrane without rupturing it , but in three cases , a microperforation of the descemet membrane occurred ; therefore no other preparation was performed , and the lamella was left with deep stromal layers and implanted into the eye in this condition . we performed a 2.75 mm corneal tunnel incision at 12 o'clock in the limbus using a single - use knife ; we then made 2 side ports at 3 and 9 o'clock , respectively , in the limbus using a paracentesis knife . if there was any significant corneal edema preventing sufficient visualization of the anterior chamber structures , we performed epithelial abrasion . this procedure was chosen for 14 patients . in these cases , we covered the cornea with a bandage contact lens at the end of the surgery , and it was left in the eye until the epithelium healed . we used the technique of continual descemetorhexis to remove the patient 's descemet membrane and damaged endothelial cells in a central area of approximately 9 mm . to maintain the anterior chamber during this procedure , we used an irrigation cannula from the phacoemulsification machine ( katena ) , introduced through the left paracentesis and connected to the infusion of a ringer solution ( fresenius kabi ) . we implanted the donor lamella in the eye so prepared . in patients where corneal transplantation was combined with cataract surgery , we filled the anterior chamber with a cohesive viscoelastic material ( provisc , alcon ) after making an incision . using capsular forceps , we performed continuous circular capsulorhexis . subsequently , we performed the descemetorhexis and followed to completion the standard phacoemulsification cataract surgery technique and posterior chamber iol implantation . the anterior chamber was properly flushed after the complete of the cataract surgery using irrigation and aspiration cannulas to completely remove the residual viscoelastic material . we implanted the lamella using our own technique utilising balanced salt solution ( bss ) flow and a plastic cartridge , which we named solution implantation technique ( sit ) . our aim was to speed up and simplify this part of the surgery and to minimize the burden on endothelial cells during implantation . the endothelial side of the lamella is covered with a small amount of dispersive viscoelastic solution ( viscoat , alcon ) and folded approximately 50 : 50 , endothelial side inwards . to transport the tissue into the open entrance of the cartridge , we held the peripheral stromal part of the lamella with forceps . we pulled the tissue into the closed part of the cartridge using a small amount of pressure with a cyclospatula on the stromal rim . we then closed and connected the cartridge to the end of an infusion tube set with a syringe filled with bss . the irrigation pressure has to be low so as not to push the lamella out of the eye . the surgeon held the irrigation cannula in his nondominant hand ( left in our case ) and inserted it through the paracentesis ; the surgeon held the cartridge containing the rolled lamella and connected with the syringe in his dominant hand . the opening of the cartridge was inserted into the main incision ( 3.0 mm . ) . at this moment it is very important to switch off the irrigation by releasing the pedal , so as not to overpress the lamella deeper into the cartridge . with a small amount of pressure on the syringe we removed the implantation device and the irrigation cannula , and with repeated amounts of small pressure on the cornea , using a cannula for instance , we could centrate the lamella . after this centration we left the anterior chamber completely filled with the air bubble for 60 minutes , with the patient lying in the supine position in the operating theatre . afterwards , we replaced part of the air using bss , so the remaining bubble was still covering the edges of the lamella ( see supplementary video ) . we checked the water tightness of the corneal wounds and performed hydration of the supplement paracentesis . at the end of the operation , we applied combined antibiotic and corticosteroid eye drops ( tobradex , alcon ) . we covered the eye with a transparent plastic cover ; the patient was transferred to the inpatient department and advised to maintain the correct , supine position , that is , looking up at the ceiling . the air bubble in the anterior chamber was left to its spontaneous absorption , that is , about 2 - 3 days . in the postoperative period , we biomicroscopically observed the position and attachment of the lamella . if the lamella was detached after complete air bubble absorption from the anterior chamber , we performed rebubbling . this was usually done 7 days after the principal surgery in the operating theatre under sterile conditions and an outpatient regimen . the anterior chamber was completely filled with the air bubble and the patient was kept for 60 minutes in the same position on his back . after one hour , the air was partially replaced with ringer solution so that the edge of the air bubble covered the borders of the lamella . if , despite this procedure , the lamella was still detached , rebubbling was repeated again after one week . if the lamella was not attached even after repeated air - bubble - filling ( after a maximum of 3 times ) , the surgery was assessed to be unsuccessful and the patient was indicated for retransplantation , but only after a period of at least three months after principal surgery . in the postoperative period , patients applied topical antibiotic ( tobramycin ) and steroid ( dexamethasone ) combined drops ( tobradex , alcon ) 5 times daily for two weeks after the surgery . two weeks postoperativly , the patients started to apply only the local steroid drops ( dexamethasone , wzf polfa ) 3 times daily and continued this for a 6-month period . after six months the steroid drops were replaced with nonsteroid antiphlogistic indomethacin ( indocollyre , laboratoire chauvin s.a . ) , which was applied 3 times daily . when faced with an increase in intraocular pressure , local medication was combined with antiglaucomatous drops ( timolol , dorsolamide ) . we recorded the number of peroperative and postoperative complications . in each patient , we noted the number of further air injections into the anterior chamber needed to properly attach the donor lamella ( figure 2 ) . in the postoperative period , we monitored the value of intraocular pressure using noncontact tonometry , corrected and uncorrected visual acuity of the snellen optotypes 1 month , 3 , 6 , 12 , 18 , and 24 months after surgery , and corneal endothelial density using a noncontact specular endothelial microscope ( topcon sp 3000 ) at 6 , 12 , and 24 months . since the endothelial cell replacement is the main objective of this type of corneal transplantation , the endothelial cell density ( ecd ) data best indicates the surgical success rate of this surgery . we evaluated endothelial cell loss caused by the surgery itself by comparing the ecd data , declared by the eye tissue bank in the document accompanying the transplanted tissue , with postoperative ecd in patients with transplanted endothelium . the corrected and uncorrected visual acuity indicated the clinical success rate of this surgery and , primarily , the benefit that it brings to the patient . we evaluated the statistical significance of the results using both a student 's paired t - test and a multidimensional test ( bonferroni test ) . the study involved 71 eyes demonstrating endothelial dysfunction of various etiologies , but without other ocular pathologies that could affect proper surgery or postoperative results of visual acuity . in our group of patients , we realized primary lamella attachment in 27 eyes ; in 31 eyes we , performed one rebubbling ; in 9 eyes we repeated the air - bubble filling twice in 3 cases 3 times and in 1 case 4 times ( figure 3 ) . in cases involving total detachment of the lamella we , performed only 3 rebubblings according to our internal criteria . we had to replace the lamella due to its failure or malfunction in 17 patients ( 23.9% ) . therefore , we supposed that endothelial loss was one of the causes of unsuccessful surgery . we decided to exchange the lamella in 10 patients within 3 months following primary surgery , in 5 patients within 6 months , and in the remaining 2 patients within a year . in these cases , the repeated surgery was successful , with comparable visual and endothelial outcomes as those presented in the main group of patients . the average preoperative endothelial cell density of the donor graft was 2907 51.9 cells / mm . we analyzed the ecd in a 1-month postoperative follow - up , and the average endothelium cell count was 1273 82.7 cells / mm , which meant an endothelial cell loss of 43.8% . during further follow - ups in months 3 , 6 , 12 , 18 , and 24 , we observed a stabilization of postoperative findings or at most a very low rate of corneal endothelial cell loss . the mean ecd was 1177 78.6 in the 3-month follow - up , 1108 72 in the 6-month follow - up , 1005 79.9 in the one - year follow - up , 1163 145.9 in the 18-month follow - up , and 989 195.7 in the two - year follow - up . we compared the preoperative and postoperative ecd results and found a statistically significant difference ( p 0.05 ) . we also found a statistically significant decrease ( p = 0.044 ) in the endothelium cell count between the postoperative follow - ups in the sixth and twelfth months . we did not find statistically significant differences among ecd results when compared with results of all postoperative follow - ups . our results show that the most stressful times for the endothelium are the surgery itself and the early postoperative period . during further follow - ups , we observed endothelial cell density stabilization and only a small decrease in ecd , which was not statistically significant . another value that we evaluated over the follow - up period was uncorrected visual acuity ( ucdva ) . in our group of patients , the mean ucdva was 1.0 0.02 logmar postoperatively , 0.44 0.04 logmar at one month postoperatively , 0.35 0.04 logmar at three months postoperatively , 0.34 0.03 logmar at six months postoperatively , 0.29 0.04 logmar at twelve months postoperatively , 0.18 0.08 logmar at eighteen months postoperatively , and 0.15 0.10 logmar at twenty - four months postoperatively . we observed a statistically significant improvement in ucdva when compared with preoperative ucdva and all further follow - up data . we also observed a statistically significant improvement in visual acuity when comparing the uncorrected at the one - month follow - up with that of the 18- and 24-month follow - ups ( figure 4 ) . the best corrected visual acuity was 0.86 0.02 logmar postoperatively , 0.28 0.04 logmar at 1 month postoperatively , 0.18 0.04 logmar at 3 months postoperatively,0.18 0.04 logmar at 6 months postoperatively , 0.15 0.04 logmar at 12 months postoperatively , 0.04 0.02 logmar at 18 months postoperatively , and 0.03 0.01 logmar at 24 months postoperatively . we observed a statistically significant improvement in bcdva when comparing preoperative bcdva with all further follow - up data . we also observed a statistically significant improvement when comparing bcdva the first month after surgery and at follow - up checks 18 and 24 months after surgery ( figure 5 ) . plk is becoming a gold - standard technique in the treatment of corneal endothelial diseases , including endothelial dystrophies , pseudophakic bullous keratopathy and endothelial graft failures . three main types of posterior corneal lamellae ( pcl ) are currently used in plk according to their histological structure - dsek / dsaek , dmek and hybrid technique dmek - s . the main disadvantages of these operations are either their technical demands ( dmek ) or the decrease in postoperative visual function due to the presence of interlamellar opacities ( dsek / dsaek ) [ 12 , 15 ] . for this reason , the hybrid technique dmek - s seems to be an ideal compromise , eliminating both disadvantages of the techniques mentioned previously . in comparison with that of other published data , endothelial cell loss immediately after surgery is higher [ 9 , 11 , 12 , 16 ] . theoretically , the viscomaterial used during the preparation of the lamella could be a cause of the higher rate of lamella detachment . despite this , we suppose that its influence is very low , because we used only a thin layer of the viscomaterial exclusively in the central bending of the lamella . our explanation of the higher rebubbling rate is that the transition between the central bare descemet membrane and the stromal rim in the periphery of the lamella is not completely smooth . there is always a small amount of fluid that can contribute to an increased incidence of lamella detachment . in order to adopt this new technique in other practices consequently , it may become the method of choice for many surgeons because of the ease of handling the lamella and the excellent visual results . the preparation of the lamella on the premises of the eye tissue bank , avoiding the risk of destroying the cornea in the operating room , contributes to this being a method of choice . in summary , dmek - s combines the advantages of dsek / dsaek and dmek . the central zone of bare descemet membrane and endothelium allows for very good visual outcomes , and the peripheral rim allows for better manipulation of the lamella during implantation . it is an effective method of treating the endothelial dysfunction of various etiologies , but the high complication rate needs to be addressed before widespread implementation of the technique in the future .

purpose : to evaluate the outcomes of the hybrid technique of posterior lamellar keratoplasty ( dmek - s ) . materials and methods : 71 eyes of 55 patients enrolled in a single - center study underwent posterior lamellar keratoplasty with a hybrid lamella dmek - s implanted using a solution implantation technique , owing to endothelial dysfunction . the outcome measures studied were visual acuity and endothelial cell density . results : the rate of endothelial cell loss caused by surgery was 43.8% . during followups , we observed the stabilization of postoperative findings , or at minimum a very low rate of corneal endothelial cell loss . the ucdva and bcdva dramatically improved postoperatively . the rebubbling rate in our group of patients was 61.9% . we replaced the lamella due to its failure or malfunction in 17 patients ( 23.9% ) . conclusion : in summary , dmek - s combines the advantages of dsek / dsaek and dmek . the central zone of bare descemet 's membrane and endothelium allows for very good visual outcomes , and the peripheral rim allows for better manipulation of the lamella during implantation . it is an effective method of treating the endothelial dysfunction of various etiologies , but the high complication rate needs to be addressed before widespread implementation of the technique in the future .

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

oral submucous fibrosis ( osmf ) is a well - known precancerous condition , which clinically presents with reduced mouth opening , decreased salivary flow rate , blanching of oral mucosa and burning sensations . submucous fibrosis is an insidious , chronic disease affecting any part of the oral cavity and sometimes the pharynx , occasionally , it is preceded by and/or associated with vesicle formation and is always associated with a juxta - epithelial inflammatory reaction followed by progressive hyalinization of the lamina propria . the later subepithelial and submucosal fibrosis leads to stiffness of the oral mucosa and deeper tissues with progressive limitation in opening of the mouth and protrusion of the tongue , thus causing difficulty in eating , swallowing and phonation . the name candida is derived from a custom in ancient rome for a candidatus , a candidate for public office , to dress in white . albico means to be white , so the name candida albicans is redundant . among more important pathogenic species , c.albicans , candida tropicalis , candida parapsilosis , candida krusei , candida lusitaniae and candida glabrata , c. the presence of candida in the mouth together with epithelial changes may predispose individuals to candidal infection . epithelial changes of the oral mucosa , such as atrophy , hyperplasia and dysplasia , may compromise the mucosal barrier and facilitate candidal invasion , especially in the event of epithelial atrophy . these opportunistic fungal pathogens may colonize , invade and induce lesions in any part of the oral cavity in both immunocompetent and immunocompromised individuals . however , those who are immunocompromised are affected at a significantly higher frequency than healthy individuals . according to shinozaki et al . , the salivary flow rate is negatively correlated with candidal carriage . hence , the present study was undertaken to compare and assess the salivary flow rate and salivary candidal carriage in the osmf patients and healthy individuals . the present study comprised sixty patients , 30 osmf patients of different grades ( nine patients of grade i osmf , 14 patients each of grade ii and iii , respectively and two patients of grade iv osmf ) and control group comprised 30 healthy individuals who were not having any relevant medical , dental and habit history . there was no history of any treatment such as antibiotic or steroid usage in any of these patients . all the osmf patients were divided into four groups according to severity , following the criteria from a study done by ranganathan et al . the criteria considered was mouth opening that is graded as follows : grade i : only symptoms , with no demonstrable restriction in mouth openinggrade ii : limited mouth opening , twenty millimeters and abovegrade iii : mouth opening < 20 mmgrade iv : osmf advanced with limited mouth opening . grade i : only symptoms , with no demonstrable restriction in mouth opening grade ii : limited mouth opening , twenty millimeters and above grade iii : mouth opening < 20 mm grade iv : osmf advanced with limited mouth opening . salivary flow was estimated using preweighed cotton rolls placed at the openings of major salivary gland duct for 5 min . the cotton rolls were then removed from the oral cavity and weighed again . the difference in weight was recorded . salivary samples were collected by the oral rinse technique and cultured on sabouraud dextrose agar ( sda ) medium . the tests performed for the identification of candida species were gram stain , periodic acid - schiff ( pas ) technique and germ tube test . one hundred gram of sda was mixed in distil water to obtain a uniform suspension . it was heated and boiled and then sterilized in an autoclave at a temperature of about 121123c for 15 min . a slant is prepared in the test tube for the growth . according to chin and saat ever since c. albicans was reported to form germ tubes on incubation in serum for 3 h at 37c , the germ tube test has been widely used to identify and differentiate c. albicans from other species of candida . according to saigal et al . , human serum from patient 's venous blood was collected and kept vertically to form clot ; then it was centrifuged in centrifuge machine for about 3000 rpm for 10 min . about 0.30.5 ml of human serum obtained from this method was used for the detection of germ tube . a small inoculum from an isolated candidal colony was picked up by sterile inoculating loop and mixed it with the human serum in a disposable sterile container . the mixture was incubated at 42c for 23 h. a drop of obtained mixture is kept on a clean glass slide and covered with a coverslip . the subjects in both groups were asked to rinse their mouth with 10 ml of phosphate buffered saline for 2 min and then expectorate in a sterile container . the samples were incubated at 37c for 48 h. the growth appeared in 48 h as cream / white colored , smooth and pasty colonies [ figure 1 ] . candidal colony in sabouraud dextrose agar media student t - test and chi - square test were done to compare between two groups . one hundred gram of sda was mixed in distil water to obtain a uniform suspension . it was heated and boiled and then sterilized in an autoclave at a temperature of about 121123c for 15 min . a slant is prepared in the test tube for the growth . according to chin and saat ever since c. albicans was reported to form germ tubes on incubation in serum for 3 h at 37c , the germ tube test has been widely used to identify and differentiate c. albicans from other species of candida . according to saigal et al . , human serum from patient 's venous blood was collected and kept vertically to form clot ; then it was centrifuged in centrifuge machine for about 3000 rpm for 10 min . about 0.30.5 ml of human serum obtained from this method was used for the detection of germ tube . a small inoculum from an isolated candidal colony was picked up by sterile inoculating loop and mixed it with the human serum in a disposable sterile container . the mixture was incubated at 42c for 23 h. a drop of obtained mixture is kept on a clean glass slide and covered with a coverslip . the subjects in both groups were asked to rinse their mouth with 10 ml of phosphate buffered saline for 2 min and then expectorate in a sterile container . the samples were incubated at 37c for 48 h. the growth appeared in 48 h as cream / white colored , smooth and pasty colonies [ figure 1 ] . one hundred gram of sda was mixed in distil water to obtain a uniform suspension . it was heated and boiled and then sterilized in an autoclave at a temperature of about 121123c for 15 min . according to chin and saat ever since c. albicans was reported to form germ tubes on incubation in serum for 3 h at 37c , the germ tube test has been widely used to identify and differentiate c. albicans from other species of candida . according to saigal et al . , human serum from patient 's venous blood was collected and kept vertically to form clot ; then it was centrifuged in centrifuge machine for about 3000 rpm for 10 min . about 0.30.5 ml of human serum obtained from this method was used for the detection of germ tube . a small inoculum from an isolated candidal colony was picked up by sterile inoculating loop and mixed it with the human serum in a disposable sterile container . the mixture was incubated at 42c for 23 h. a drop of obtained mixture is kept on a clean glass slide and covered with a coverslip . the subjects in both groups were asked to rinse their mouth with 10 ml of phosphate buffered saline for 2 min and then expectorate in a sterile container . the samples were incubated at 37c for 48 h. the growth appeared in 48 h as cream / white colored , smooth and pasty colonies [ figure 1 ] . student t - test and chi - square test were done to compare between two groups . three tests were performed for the identification of candida species . they were gram stain , pas technique and germ tube test . gram stain- gram staining , showing candidal spores [ figure 2 ] pas technique - showing candidal yeast cells was recorded as a positive finding [ figure 3 ] germ tube test- a germ tube was seen in the wet smear preparation , obtained from human serum [ figure 4 ] which confirmed the presence of fungal hyphae in the wet smear [ figure 5 ] ( according to saigal et al . and chin and saat ) . gram stain- gram staining , showing candidal spores [ figure 2 ] pas technique - showing candidal yeast cells was recorded as a positive finding [ figure 3 ] germ tube test- a germ tube was seen in the wet smear preparation , obtained from human serum [ figure 4 ] which confirmed the presence of fungal hyphae in the wet smear [ figure 5 ] ( according to saigal et al . and chin and saat ) . ( gram stain , x100 ) periodic acid - schiff technique showing candidal yeast cells recorded as a positive finding . ( pas stain , x100 ) human serum containing small inoculum from an isolated candidal colony germ tube formation in human serum . ( wet smear , x400 ) when comparing saliva flow rate between osmf patients and control group , there was a statistically significant ( p < 0.001 ) decrease in the salivary flow rate in osmf individuals as compared to the control [ graph 1 ] . the results also showed that there was a constant decrease in the salivary flow rate among different grades of osmf patients [ graph 2 ] which was statistically significant ( p < 0.001 ) . there were absence of candidal carriage in healthy individuals ( control ) as well as in grade i and grade iv osmf patients as compared to grade ii and grade iii osmf patients ( grade iii , iv ) which were also statistically significant ( p < 0.001 ) [ graph 3 and 4 ] . there was a statistically significant difference in the salivary flow rate and candidal carriage in the osmf patients as compared to the healthy individual / control group ( p < 0.001 ) [ graph 5 ] . salivary flow rate was constantly reduced with different grades of osmf patients , although candidal carriage was seen in grade ii and grade iii osmf patients . there was statistically significant ( p < 0.001 ) decreased salivary flow rate in the oral submucous fibrosis individuals as compared to the control./changes in the salivary flow rate between control and oral submucous fibrosis as a single group changes in the salivary flow rate between the groups of oral submucous fibrosis comparison of candidal isolation between control and oral submucous fibrosis as a single group comparison of candidal isolation between control and different grades of oral submucous fibrosis patients association / relation or comparison between salivary flow rate and candida carriage the predominant species is c. albicans , which has the potential to infect any tissue within the body under immunocompromised conditions . an association between candida and various precancerous and cancerous lesions has been reported in the literature . a delicate balance clearly exists between the potentially damaging effects of candida virulence factors and the nature of the immune response elicited by the host . frequently , it is changes in the host factors that lead to candida seemingly changing from a commensal to pathogenic existence . however , given the often reported heterogeneity in morphological and biochemical factors that exist between candida species and indeed strains of c. albicans , it may also be the fact that colonizing strains differ in the way they exploit the resources , to allow persistence at mucosal surfaces and as a consequence this too may affect the way candida interacts with epithelial cells . results of our study suggest an inverse relationship between salivary flow and candidal colony , which was in accordance to the study of torres et al . factors such as poor oral hygiene , denture wearing , oral epithelial dysplasia , smoking habits , orthodontic appliances , mouth breathing and chronic irritants have been found to be the local factors that predispose to candidiasis . compared osmf and healthy individual with xerostomia and found significantly decreased salivary flow rate , increased rate of oral mucosal symptoms and higher numbers of candida in osmf patients than a healthy individual . salivary flow rate was negatively correlated with the number of candida , which is in agreement with the findings of our study . our result was in accordance with the result of saigal et al . , which found no fungal growth in the normal control group and 3 out of 15 cases of osmf showed fungal growth . the results of our study were similar to that of kamat et al . , who suggested that osmf favors the colonization of candida . field et al . suggested that c. albicans plays an important role in the development of oral cancer by means of endogenous nitrosamine , oligosaccharides and lectin - like component production . our results confirmed the findings of ariyawardana et al . , who showed higher candidal carriage in osmf patients , using the oral rinse technique . as oral mucosa is compromised in osmf patients , it can be argued that candida may predispose the individual to candidal infection and invasion . a study done by anila et al . suggested a higher incidence of candidal carriage in osmf patients and increased salivary flow in osmf patients , which contradicts our study . on the contrary , reichart et al . reported that there was no significant difference in the candidal carriage rate between the osmf patients and control group . at present , the results of our study suggest that a higher candidal carriage in grade ii and grade iii osmf patients could be related to decreased salivary flow rate . candidal carriage was not seen in grade iv osmf patients ; the reason might be because of small sample size of this group , as this study was done on random number of patients . a higher incidence of candida was observed in the osmf patients when compared to the healthy individuals . also , the study showed that there is a constant decrease in the salivary flow rate among the different grades of osmf patients from grade i to grade iv . absence of candidal carriage was seen in the control group as compared to the osmf patients . prospective studies with larger sample size of osmf patients and different culture media to identify various species of candida along with long - term follow - up will help in assessing the role of increased candidal carriage in the coarse and malignant transformation of osmf .

background : oral submucous fibrosis ( osmf ) is a high - risk precancerous condition that predominantly affects indians due to prevalent gutka chewing . changes in the salivary flow rate and its effect on candidal carriage in patients suffering from osmf have not been extensively explored.aim:the aim of this study is to compare the salivary flow rate and salivary candidal carriage in osmf patients and healthy individuals.materials and methods : this pilot study included a total of 30 osmf patients and 30 healthy individuals . salivary flow was estimated using preweighed cotton rolls placed at the openings of major salivary duct for 5 min . the cotton rolls were then removed from the oral cavity and weighed again . the difference in weight was recorded . salivary samples were collected by the oral rinse technique and cultured on sabouraud agar medium . the cultured yeast colonies were identified based on gram 's staining , periodic acid - schiff ( pas ) and germ tube formation.result:there was statistically significant ( p < 0.001 ) decreased salivary flow rate in osmf individuals as compared to the control . salivary flow rates constantly reduced with different grades of osmf patients , although candidal carriage was seen in grade ii and grade iii osmf patients.conclusion:a higher incidence of candida was observed in osmf patients when compared to the healthy individuals . the results of our study suggest that a higher candidal carriage in grade ii and grade iii osmf patients could be related to decreased salivary flow rate .

INTRODUCTION MATERIALS AND METHODS Preparation of culture media For fungus growth For germ tube test Saliva culture technique for candida Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

adenocarcinoma is the most common malignant gastric tumor , while gastrointestinal stromal tumors ( gists ) are relatively rare . from all gastrointestinal locations , gists occur most frequently in the stomach ( 60 - 70% ) . however , a combination of an adenocarcinoma and a gist is a very rare finding and an actual collision of these tumors even more so . this case report is , to our best knowledge , the second report in literature . laboratory data demonstrated an esr of 15 mm / h ( normal 1 - 7 mm / h ) , a hemoglobin level of 6.5 mmol / l ( normal 8.5 - 11.0 mmol / l ) , thrombocytes 341 10 ( normal 150 - 400 10 ) , leukocytes 8.9 10 ( normal 4.0 - 10.0 10 ) . endosonography showed an irregular course of the gastric wall where it was adjacent to the liver , not excluding possible invasive growth into the liver and thus suggesting the possibility of a t4 carcinoma . sonography of the neck region and the abdomen showed no lymph node involvement or signs of metastasis to other organs . primary surgery consisting of a total gastrectomy with roux - en - y esophagojejunostomy was performed . except from a postoperative septic period as a result of a spondylodiscitis , which was successfully treated with antibiotics , two and a half years after the total gastrectomy the patient developed pleural effusion due to adenocarcinoma metastases and died shortly thereafter . the resected specimen consisted of a totally resected stomach with en bloc part of the distal oesophagus . alongside the lesser curvature on cut section the tumor invaded through the muscularis propria into the fat of the omentum minus . microscopically the tumor consisted of both a conventional adenocarcinoma and tumor nodules composed of cells with another phenotype . most of the adenocarcinoma was found in the submucosa , but focally the adenocarcinoma invaded all layers of the gastric wall into the outer serosal surface or perigastric fat . this tumor was composed of a combination of intestinal , gland - forming growth and a diffuse , poorly adhesive , often signet - ring morphology . it consisted of nodules up to 6 mm composed of spindle cells with little cytonuclear polymorphism and with no significant amount of mitosis ( < 5/50 hpf ) ( fig . 1b ) and cd34 , while other stromal cell markers like s100 , smooth muscle actin and desmin were not expressed . laboratory data demonstrated an esr of 15 mm / h ( normal 1 - 7 mm / h ) , a hemoglobin level of 6.5 mmol / l ( normal 8.5 - 11.0 mmol / l ) , thrombocytes 341 10 ( normal 150 - 400 10 ) , leukocytes 8.9 10 ( normal 4.0 - 10.0 10 ) . endosonography showed an irregular course of the gastric wall where it was adjacent to the liver , not excluding possible invasive growth into the liver and thus suggesting the possibility of a t4 carcinoma . sonography of the neck region and the abdomen showed no lymph node involvement or signs of metastasis to other organs . primary surgery consisting of a total gastrectomy with roux - en - y esophagojejunostomy was performed . except from a postoperative septic period as a result of a spondylodiscitis , which was successfully treated with antibiotics , two and a half years after the total gastrectomy the patient developed pleural effusion due to adenocarcinoma metastases and died shortly thereafter . the resected specimen consisted of a totally resected stomach with en bloc part of the distal oesophagus . alongside the lesser curvature on cut section the tumor invaded through the muscularis propria into the fat of the omentum minus . microscopically the tumor consisted of both a conventional adenocarcinoma and tumor nodules composed of cells with another phenotype . most of the adenocarcinoma was found in the submucosa , but focally the adenocarcinoma invaded all layers of the gastric wall into the outer serosal surface or perigastric fat . this tumor was composed of a combination of intestinal , gland - forming growth and a diffuse , poorly adhesive , often signet - ring morphology . it consisted of nodules up to 6 mm composed of spindle cells with little cytonuclear polymorphism and with no significant amount of mitosis ( < 5/50 hpf ) ( fig . 1b ) and cd34 , while other stromal cell markers like s100 , smooth muscle actin and desmin were not expressed . these tumors are the most common mesenchymal tumors in the digestive tract and are thought to originate either from the stem cells that differentiate towards interstitial cells of cajal , or directly from interstitial cells of cajal [ 3 , 4 ] . small gists are often detected incidentally during surgery for other reasons , or are detected during gastroscopy . in general , gists can be differentiated from other tumors made up from spindle cells and/or epitheloid cells by expressing cd117 ( c - kit ) and cd34 in 50 - 80% of cases [ 3 , 4 ] . the kit gene codes for c - kit protein which activates a tyrosine kinase after binding with a stem cell factor . 90% of gists have a mutation in the kit oncogene , but 10% miss this mutation . these sometimes cd117-negative tumors often have a mutation in the platelet - derived growth factor receptor a ( pdgfra ) . nowadays , the prognosis and biological behaviour of gist depends on a combination of two criteria : the size of the tumor and the mitotic index . approximately 70 - 85% of patients have a complete resection , but overall five - year survival is only 50% , because of the tendency of the gist to metastasize or to recur . since 2002 a new therapy , imatinib mesylate , is developed to treat this kind of malignancies when metastasized or irresectable . this is a tyrosine kinase inhibitor and demonstrates dramatic effects in the majority of patients . possibly the kind of kit gene mutation has an effect on the development of resistance and the tumor response on imatinib . the development of gastric collision tumors is very rare . in the english literature we found one case report describing the occurrence of an adenocarcinoma mixed with a gist . several cases describing various other combinations of tumor have been reported [ 7 , 9 , 10 , 11 ] . they considered whether or not such an association was incidental or whether the two lesions were connected by a causal relationship . some articles suggest that gene mutations or a single carcinogenic agent might interact with two neighboring tissues , inducing the development of tumors of different histiotypes in the same organ , but no evidence of this last hypothesis has yet been found [ 8 , 10 ] . both tumors have different precursor cells and molecular make - up , and if there were a single carcinogen , these types of tumor would probably be diagnosed more often . in this case the diagnosis of gist was made based on the tumor phenotype in combination with immunoreactivity to cd117 and cd34 . the gist was presumed to be benign due to the low mitotic count , size and to a lesser extent the low degree of cellularity and cellular polymorphism . although multicentricity is often considered a sign of malignancy , in this case , only very small nodules with no histological signs of malignant clinical behavior were present . therefore it was expected that in this case the prognosis would most probably be determined by the adenocarcinoma component as this tumor penetrated the entire gastric wall into the serosa , had invaded blood vessels and showed lymph node metastasis . we present a patient suffering from a collision tumor consisting of an adenocarcinoma originating from the gastric mucosa and a gist that was presumed to behave in a benign fashion . as shown by the adenocarcinoma metastasis in the pleural effusion and death two and a half years after surgery , the patient 's prognosis was determined by the adenocarcinoma component . . however , an actual collision of both an epithelial adenocarcinoma and a gist is exceedingly rare and as far as we know now , both with different and unknown etiology .

a collision tumor of the stomach is a rare event . we report the case of a collision tumor of the stomach consisting of an adenocarcinoma and a gastrointestinal stromal tumor ( gist ) . this is , to our knowledge , the second report in the literature of such a case . a 71-year - old man with abdominal discomfort underwent an esophagogastroduodenoscopy which revealed a tumor of the oesophagogastric junction . a total gastrectomy was performed . histologic examination showed a mixed tumor consisting of a primary adenocarcinoma and multiple nodules of gist . the adenocarcinoma showed both diffuse and intestinal growth , angio - invasion and metastasis to lymph nodes . the gist tumor cells were strongly immunoreactive to cd117 and cd34 . based on mitotic index , size and cytonuclear details , the biological behavior of this gist tumor was supposed to be benign . this case reports the rare finding of a collision tumor consisting of an adenocarcinoma and a gist with an unknown etiology .

Introduction Case Report Clinical Presentation Pathologic Findings Discussion Conclusion

chronic low - grade inflammation and aberrant regulation of the stress response system are two prominent hallmarks of obesity , a major risk factor for the development of insulin resistance , type 2 diabetes , metabolic syndrome , and cardiovascular diseases [ 1 , 2 ] . sedentary lifestyles and excessive food intake are considered as key contributors to this chronic condition . the white adipose tissue has been identified as the predominant site of obesity - associated inflammatory reactions due to its infiltration by immune inflammatory cells such as monocytes , macrophages , th1 t cells , and dendritic cells [ 24 ] . these immune cells , together with adipocytes and stromal vascular cells , constitute a cellular network that produces various inflammatory mediators . obesity - induced inflammatory response impairs insulin signaling in insulin - responsive organs and causes systemic insulin resistance , which leads to a perturbation of glucose homeostasis and ultimately type-2 diabetes [ 5 , 6 ] . studies on mice indicated that obesity also alters the balance between pro- and anti - inflammatory activities in adipose tissue by promoting the phenotypic switch from m2 anti - inflammatory macrophages to m1 proinflammatory macrophages and thereby perpetuating further the inflammatory response and insulin resistance [ 5 , 7 ] . regulated upon activation normal t cells expressed and secreted ( rantes or ccl5 ) is a powerful proinflammatory mediator of the cc chemokine family that regulates the mobilization and , in certain cases , promotes survival of immune inflammatory cells from the bloodstream into tissues and other areas of injury and infection [ 3 , 8 , 9 ] . although the chemotactic activity of rantes on immune cells to injured and infected areas is beneficial , sustained production of rantes is associated with several detrimental effects such as atherosclerosis [ 10 , 11 ] , arthritis rheumatoid , liver disease [ 13 , 14 ] , and viral infection that share in common chronic inflammatory response . consistent with its critical role in the pathophysiology of these chronic inflammatory - related diseases , treatments that interfere with rantes signaling such as neutralizing antibody [ 16 , 17 ] , peptide mimetics [ 18 , 19 ] , and pharmacological inhibitors [ 20 , 21 ] are associated with improved outcomes . rantes orchestrates its effects through binding to one of its cognate receptors including ccr1 , ccr3 , and ccr5 [ 22 , 23 ] . the crucial role of ccr5 in mediating the inflammatory response in adipose tissue was demonstrated recently by using ccr5 knockout mice that showed a dominant shift from m1-phenotype to m2-phenotype , which contributed to attenuated inflammatory response and improved impaired glucose tolerance and insulin sensitivity in response to diet - induced obesity . physical exercise is an important component of healthy lifestyle that is widely advocated as a first line for the treatment and management of obesity , insulin resistance , diabetes , and cardiovascular diseases [ 2527 ] . previous studies linked the protective effect of physical exercise to the improvement of the inflammatory and stress responses [ 2830 ] . the mechanisms by which physical exercise mediates its anti - inflammatory effect are multiples and they include a reduction of visceral fat mass with subsequent decrease in the production of proinflammatory adipokines and a reduction in the circulating number of proinflammatory monocytes in favor of an increase in the circulating number of regulatory t cells [ 31 , 32 ] . other studies revealed also that physical exercise prevents monocytes and macrophages from infiltrating into adipose tissue and induces a phenotypic switching from m1-phenotype macrophages to m2-macrophages within the adipose tissue . other beneficial effects of physical exercise include attenuated activation of c - jun nh2-terminal kinase together with improvement of lipid , glucose , and oxidative homeostasis [ 3537 ] . in the current study , we investigated the effect of a 3-month physical exercise on the expression and release of rantes in obese subjects . given the critical role of rantes and its ccr5 receptor in promoting obesity - induced metabolic inflammation , we hypothesized that physical exercise may mediates its anti - inflammatory effect in part , by impairing rantes signaling pathway via downregulation of rantes and/or its ccr5 receptor . we report here for the first time a decrease in the expression of both rantes and ccr5 receptor by physical exercise in the adipose tissue of obese humans . the study was conducted on adult male and female nondiabetic subjects consisting of 17 lean ( bmi = 2024.9 kg / m ) and 40 obese ( bmi = 3040 kg / m ) . all subjects gave written informed consent and the study was approved by the ethical review board of dasman diabetes institute ( reference number : ra-2010 - 003 ) . participants that followed any physical exercise within the last 6 months prior to this study as well as those with prior major illness or intake of medications or supplements known to influence the body composition or bone mass were excluded from the study . none of the participants that were enrolled in this study reported any cerebrovascular complication , kidney disease , asthma , and food and drug allergies and chronic viral infection . according to the medical questionnaire , furthermore , no specific diet or nutritional change was prescribed to our subject during the 3 months of physical exercise protocol as our aim was mainly to investigate the sole effect of physical exercise on those subjects . all eligible subjects were enrolled to a supervised exercise program at the fitness and rehabilitation center ( frc ) of dasman diabetes institute . prior to exercise prescription , each individual underwent a symptom - limited maximal incremental cardiopulmonary exercise test as well as a muscle strength and endurance test . it consisted of a combination of both moderate intensity of aerobic exercise and resistance training using either treadmill or cycling . each exercise session included 10 minutes of warming - up and cooling down steps at 5060% of max hr , along with 40 minutes of the prescribed exercise program at 6580% of max hr . for the duration of the 3-month period , participants exercised 3 to 5 times per week and they were instructed to reach and maintain the recommended heart rate range . strength training was performed 2 to 3 times a week according to the program plan . all trainings were supervised by qualified fitness professionals from frc . to assess the effectiveness of the exercise , the same physical stress and fitness tests were performed for all subjects at the end of the exercise program . venous peripheral blood and subcutaneous adipose tissue biopsies were obtained prior to the start of the 3-month protocol and within 2 to 3 days after its completion . plasma and serum were prepared using vacutainer tubes and then aliquoted and stored at 80c . subcutaneous superficial adipose tissue biopsies ( ~0.5 g ) were obtained from the periumbilical area by surgical biopsy after a local anesthesia . once removed , the biopsy was rinsed in cold pbs , divided into 4 pieces , and stored appropriately until assayed . glucose and lipid profiles were measured on the siemens dimension rxl chemistry analyzer ( diamond diagnostics , holliston , ma ) . hemoglobin a1c ( hba1c ) was determined using the variant device ( biorad , hercules , ca ) . plasma levels of inflammatory and metabolic markers were measured using the bio - plex 27-plex human cytokine and 12 bio - plex human diabetes kits , respectively ( biorad , hercules , ca ) . data were analyzed with bio - plex manager software version 6 ( biorad , hercules , ca ) . lipid peroxidation was assessed by measuring plasma levels of malonaldehyde , using tbars assay kit ( cayman chemical company , ann arbor , mi ) . serum levels of ros were determined using the oxiselect ros assay kit ( cell biolabs inc , san diego , ca ) . total rna was extracted from frozen adipose tissue and pbmcs using rneasy lipid tissue mini kit and allprep rna / protein kit , respectively ( qiagen , inc . , the cdna was synthesized from total rna sample using high capacity cdna reverse transcription kits ( applied biosystems , foster city , ca ) . qrt - pcr was performed on rotor - disc 100 system using sybr green normalized to gapdh ( qiagen , inc . , cell lysates were prepared from pbmcs by the addition of ripa buffer ( 50 mm tris - hcl ph 7.5 , 150 mm nacl , 1% triton x100 , 1 mm edta , 0.5% sodium deoxycholate , and 0.1% sds ) . protein concentration was determined by bradford method using globulin as a standard and 20 g of proteins was resolved on 10% sds - page gels . proteins were transferred onto pvdf membranes and probed with primary and secondary antibodies using standard protocols . protein bands were visualized by chemiluminescence and the images were captured by using the versadoc 5000 system ( biorad , hercules , ca ) . the primary antibodies used in this study are raised against rantes ( abcam , cambridge , ma ) and actin ( santa cruz biotechnology , santa cruz , ca ) . for densitometric analysis , the intensity of the bands was determined using quantity one software ( biorad , hercules , ca ) . formalin - fixed and paraffin - embedded adipose tissue sections were deparaffinized and rehydrated prior to antigen retrieval by boiling in the unmasking solution ( dako , denmark ) . sections were blocked with 5% fat - free milk for 60 min at rt followed by 1% bsa for another 60 min and then incubated at 4c for overnight with primary antibodies against rantes ( abcam , cambridge , ma ) , tnf - a ( abcam , cambridge , ma ) , il-6 ( novus biologicals , littleton , co ) , and phospho - jnk ( staining with horseradish conjugated secondary antibody ( dako , denmark ) was done for 60 minutes at rt . colors were developed using dab kit ( dako , denmark ) and sections were counterstained with hematoxylin ( sigma aldrich , st . quantification of the immunohistochemical data was done using aperio software version 6.3 ( molecular devices , downingtown , pa ) with an established arbitrary threshold . approximately 5 10 of frozen pbmcs were thawed and washed with rpmi medium supplemented with 10% fbs , antibiotics , and l - glutamine ( complete media ) . for rantes staining , cells were cultured overnight in complete media at 37c and then washed with pbs containing 2% fbs followed by facs buffer ( bd biosciences , san jose , ca ) and then costained with anti - human apc - conjugated rantes ( r&d systems ) , fitc - conjugated cd3 ( mca463f , abd serotec , raleigh , nc ) , and pe - conjugated cd14 ( fab3832p , r&d systems , minneapolis , mn ) antibodies for 45 minutes on ice . cells were then washed twice with permeabilization buffer ( ebioscience , san diego , ca ) and resuspended in 300 l facs buffer for analysis . regarding ccr5 staining , cells were allowed to recover for 1 - 2 hours in complete media . the cells were then cultured in complete media in presence of 2 m monensin ( ebioscience , san diego , ca ) at 37c and 5% co2 for 16 hours . pbmcs were then washed with fb and surface stained simultaneously with fitc - conjugated cd3 ( mca463f ) and pe - conjugated cd14 ( fab3832p ) antibodies . after 45 minutes of incubation on ice in the dark , cells were washed twice and fixed with fixation buffer ( ebioscience , san diego , ca ) for 20 minutes . cells were then washed twice with permeabilization buffer ( ebioscience , san diego , ca ) according to manufacturer 's recommendation followed by staining with ccr5-apc antibody ( fab1802a , r&d systems , minneapolis , mn ) for 20 minutes in the dark . acquisition and analysis were carried out on a facs canto ii flow cytometer using facsdiva software ( bd biosciences , san jose , ca ) . after forward scatter ( fsc ) and side scatter ( ssc ) gating on either lymphocytes or monocytes the rantes expression level was determined in the cd3 as well as in cd14 subsets . statistical analyses were performed with sas version 9.2 ( sas institute inc , cary , nc ) . unless otherwise stated , all descriptive statistics for the variables in the study were reported as means standard error . nonparametric mann - whitney test was used to determine significance of difference in means between the two groups as indicated in the figure legends . the physical characteristics of the two groups , namely , lean ( n = 17 ) and obese ( n = 40 ) , enrolled in this study are displayed in table 1 . as expected , body mass index , percent body fat , and waist and hip circumferences were significantly higher in obese group compared to lean group ( p < 0.0001 ) . obese subjects had also higher systolic blood pressure and higher levels of triglycerides ( p < 0.05 ) ; however , there was no significant difference in the cardiopulmonary parameters between the two groups . obese subjects had higher levels of glucose and hba1c ( p < 0.05 ) in spite of not being diabetic . compared to lean group , the metabolic and inflammatory profiles are dysregulated in obese group as indicated by increased levels of leptin ( p = 0.0006 ) , resistin ( p = 0.04 ) , ip-10 ( p = 0.0008 ) , and rantes chemokines ( p = 0.023 ) . no significant difference was observed in the other metabolic and inflammatory mediators as well as the oxidative stress markers ( table 1 and data not shown ) . in agreement with our recent investigation , obese subjects had higher levels of rantes in the circulation that correlated negatively with il-1ra ( r = 0.42 ; p = 0.001 ) and positively with ip-10 ( r = 0.31 ; p = 0.02 ) and tbars levels ( r = 0.29 ; p = 0.03 ) ( figure 1(a ) ) . to investigate the endogenous expression of rantes between the two groups at the protein level , we determined its expression in pbmcs and adipose tissue by western blot , flow cytometry , and immunohistochemistry ( ihc ) . as shown in figure 2(a ) , there was no difference in the expression of rantes protein in pbmcs between lean and obese subjects ( n = 3 for each group ) . consistent with this finding , flow cytometry indicated that rantes levels were comparable between lean and obese subjects ( n = 3 for each group ) in both monocytes and lymphocytes ( figure 2(b ) ) . by contrast to pbmcs , the expression of rantes protein in the adipose tissue as monitored by ihc studies was significantly higher in obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.01 ; figure 2(c ) ) . the differential expression of rantes between pbmcs and adipose tissue in lean and obese subjects was also confirmed at the mrna level by using quantitative real - time pcr ( qrt - pcr ) and the results are displayed in figures 2(d ) and 2(e ) . accordingly , there was no change in rantes mrna levels in pbmcs from lean and obese subjects ( n = 10 for both lean and obese , figure 2(d ) ) , whereas the levels of rantes mrna in the adipose tissue were significantly higher in obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.02 , figure 2(e ) ) . under the same conditions , the endogenous expression of the two key inflammatory markers tnf- and il-6 in the adipose tissue were significantly increased in obese subjects both at the protein level ( p < 0.05 , figure 2(f ) ) and mrna level ( p < 0.05 , figure 2(g ) ) . we next examined the expression profile of ccr5 receptor between lean and obese subjects using pbmcs and adipose tissue . indeed , there was a clear upregulation of ccr5 mrna in the adipose tissue of obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.03 , figure 3(b ) ) . by contrast , ccr5 mrna was significantly downregulated in pbmcs of obese subjects compared to lean subjects ( n = 10 for both lean and obese ) ( p = 0.04 , figure 3(a ) ) . on the other hand , flow cytometry analysis carried out on pbmcs from lean and obese ( n = 5 for each group ) revealed a significant and unique increase of ccr5 in cd14 + monocyte subset from obese subjects ( p = 0.02 , figure 3(c ) ) . the specific mean fluorescence intensity for ccr5 in obese individuals revealed a higher signal for cd14 + monocytes compared to cd14++ monocytes ( p = 0.001 , figure 3(c ) ) . we previously reported the effectiveness of our physical exercise protocol on improving the physical , clinical , and metabolic parameters on obese subjects . accordingly , there was a significant reduction of pbf and sbp and increase in vo2max along with a decrease in tbars levels and a reduction of inflammatory markers tnf- and il-6 in the circulation . however , physical exercise did not reduce the levels of rantes in the circulation . to investigate whether physical exercise has an impact on the endogenous expression of rantes and ccr5 , qrt - pcr and ihc were carried out on adipose tissue from obese subjects before and after the exercise program . as shown in figure 4(a ) , ihc carried out on adipose tissue from obese subjects before ( n = 11 ) and after ( n = 7 ) the exercise program indicated a significant decrease in the expression of rantes by physical exercise ( p = 0.003 ) . qrt - pcr performed on obese before and after the exercise program ( n = 10 for each group ) confirmed the reduction of rantes mrna expression by physical exercise ( p = 0.01 , figure 4(b ) ) . likewise , ccr5 mrna was significantly reduced by physical exercise in the adipose tissue ( p = 0.02 , figure 4(b ) ) . using the same samples , we observed a decrease in the endogenous expression of tnf- and il-6 both at the mrna level ( p < 0.05 , figure 4(b ) ) and protein level ( p < 0.05 , figure 4(c ) ) . since obesity is known to induce the phosphorylation of jnk protein , we measured the levels of phosphorylated jnk in adipose tissue by ihc before ( n = 11 ) and after ( n = 7 ) the physical exercise program . data shown on figure 4(c ) indicated that physical exercise decreases significantly the levels of phosphorylated jnk ( p = 0.002 ) in obese in a manner that was concomitant with a decrease of ccr5 and its ligand rantes . no effect was observed for total jnk before and after exercise in obese subjects ( data not shown ) . taken together , these data suggest that exercise is interfering with obesity - mediated expression of rantes and ccr5 . chronic low - grade inflammation state is a key feature of obesity and it is caused mainly by infiltration of macrophages and other inflammatory cells into the adipose tissue . the present study explored the effect of physical exercise on the expression of rantes and its main receptor ccr5 in obese humans . our focus on ccr5 receptor was based on a recent study in which ccr5 knockout mice were protected from obesity - induced adipose tissue inflammation and insulin resistance . in addition , it has been previously shown that obesity triggers a modest change in the expression of ccr3 and no change in that of ccr1 . the main findings of our current investigation are as follows : ( 1 ) the expression of both rantes and ccr5 was significantly higher in the subcutaneous adipose tissue of obese subjects , ( 2 ) by contrast to the adipose tissue , ccr5 was downregulated in pbmcs of obese subjects compared to lean subjects but there was no significant difference in the expression of rantes between the two groups , and ( 3 ) physical exercise corrected the dysregulated expression of both rantes and ccr5 in the subcutaneous adipose tissue but has a marginal effect on circulating levels of rantes . while our findings showing increased expression of rantes and ccr5 in the adipose tissue are consistent with previous clinical and animal studies [ 17 , 40 ] , the downregulation of their expression by physical exercise is novel . based on the crucial role of rantes / ccr5 signaling pathway in the pathology of obesity and its associated complications , our findings add further evidence that physical exercise might be one of nonpharmacologic approaches that can attenuate rantes / ccr5 signaling pathway and thereby mitigating the inflammatory and metabolic stress triggered by obesity . rantes is a powerful proinflammatory chemokine that controls the trafficking of immune inflammatory cells such as monocytes , macrophages , th1 t cells , and dendritic cells from the circulation into various tissues including adipose tissue [ 3 , 9 ] . previous studies carried out on obese humans and rodents reported high levels of rantes as well as increased frequency of macrophages in the adipose tissue that were concomitant with increased inflammatory response , insulin resistance , and type 2 diabetes [ 5 , 17 , 4144 ] . our findings showing high levels of rantes in the adipose tissue of obese subjects at both mrna and protein levels corroborate these pioneer studies that associated rantes with obesity . unlike the adipose tissue , the levels of rantes were however comparable between lean and obese subjects as monitored by western blotting , flow cytometry , and qrt - pcr . the mechanism underlying this differential expression of rantes between pbmcs and adipose tissue remains to be investigated but there is evidence for depot - specific differences that dictate the levels of rantes released by various adipose tissues in humans . one of the key characteristics of macrophages that infiltrate the adipose tissue is the heterogeneity of their phenotype and depending on their polarization status ; they can be proinflammatory macrophages ( m1-phenotype ) secreting various inflammatory mediators such as tnf- , il-6 , and inos [ 45 , 46 ] or anti - inflammatory macrophages ( m2-phenotype ) that secrete anti - inflammatory cytokines such as il-10 [ 41 , 47 ] . in obesity , the differentiation of m2 into m1 macrophages is considered as a major event that sustains chronic inflammation . indeed , studies on mice indicated that obesity induces a shift in macrophage balance towards the m1-phenotype macrophages that perpetuate further the inflammatory response and insulin resistance [ 5 , 46 ] . in our study population , we did not evaluate the status of m1- and m2-phenotypes , which may represent a limitation to this investigation . however , in our study the high levels of circulating rantes correlated negatively with the anti - inflammatory il-1ra and positively with the levels of proinflammatory ip-10 chemokine and tbars supporting its role in mediating inflammation . in the current study , we have also investigated the expression pattern of ccr5 in obese humans and similar to our findings with rantes , both mrna and protein levels of ccr5 were increased in the adipose tissue of obese compared to lean group . the observed increase is consistent with recent studies both on humans and rodents [ 24 , 40 ] . the direct role of ccr5 in the regulation of obesity - induced adipose tissue inflammation and development of insulin resistance was recently demonstrated using ccr5 knockout mice . a dominant shift occurred from proinflammatory m1 to anti - inflammatory m2 macrophage phenotypes , which contributed to an improved impaired glucose tolerance and insulin sensitivity in response to diet - induced obesity in these animals . unexpectedly , our data demonstrated that , unlike the adipose tissue , the expression of ccr5 mrna was significantly downregulated in pbmcs from obese individuals . to the best of our knowledge , our data is the first to report the downregulation of ccr5 mrna in the pbmcs of obese individuals . this observed downregulation is intriguing and might suggest a complex regulation process of its transcription . the altered expression of ccr5 in pbmcs has been described in rheumatoid arthritis and in women with systemic lupus erythematosus . it is possible that other receptors for rantes such as ccr1 and ccr3 may be compensating for the reduced expression of ccr5 in obese subjects . interestingly , ccr5 expression on cd14 + monocyte subset appears to be upregulation in comparison with cd14++ subset in obese individuals . cd14 + cells are known to exhibit a macrophage - like phenotype with enhanced antigen - presenting capacities , higher endothelial affinity , and they are potent producers of proinflammatory cytokines ; thus , they may contribute to the development of chronic inflammation . of particular interest , our study emphasized more on the role of physical exercise in regulating the expression of rantes and its receptor ccr5 . it is well established that exercise reduces the risk of chronic metabolic and cardiorespiratory diseases by modulating the inflammatory and stress responses [ 51 , 52 ] . our data showed for the first time that physical exercise significantly reduced the expression of both rantes and ccr5 receptor in the adipose tissue in a manner that was concomitant with decreased pbf and improved cardiopulmonary performance as monitored by decreased sbp and increased vo2max. the reduced expression of rantes and ccr5 by physical exercise was also consistent with attenuated inflammatory response as indicated by decreased tnf- and il-6 in the circulation and reduced levels of tbars . our finding revealed also that exercise - mediated decrease in the expression of rantes and ccr5 in the adipose tissue was not translated to a decrease in the circulating levels of rantes . the lack of parallel changes between systemic and endogenous levels of rantes after exercise raises the question regarding the role of rantes in the circulation . earlier study indicated that systemic levels of rantes are 100-fold higher than those being released from any of the adipose tissue depots . therefore , it is sensible to suggest that while adipose tissue is able to produce this chemokine , its contribution to systemic levels may be less significant . physical exercise is also known to inhibit metabolic stress in part by dephosphorylating and thus inactivating jnk stress kinase [ 34 , 53 , 54 ] . in agreement with these findings , our data indicate that there was a significant increase in the levels of phosphorylated jnk along with high levels of rantes and ccr5 in obese subjects that were all reduced by physical exercise ( figure 4(c ) ) . whether there is a direct crosstalk between rantes / ccr5 signaling pathway and jnk signaling or not remains to be investigated , despite the fact that ccr5 knockout mice displayed attenuated signaling via nf-b and jnk . in conclusion , the dysregulation of rantes and its negative correlation with the metabolic and stress responses are supporting the pathological role of rantes in obesity . our results also provide strong evidence that physical exercise downregulates the expression of both rantes and ccr5 in the adipose tissue . these findings give strong support to the concept that physical exercise can be used as a nonpharmacologic approach to mitigate the complications associated with obesity , particularly inflammation , in part by attenuating rantes / ccr5 signaling in the adipose tissue .

rantes and its ccr5 receptor trigger inflammation and its progression to insulin resistance in obese . in the present study , we investigated for the first time the effect of physical exercise on the expression of rantes and ccr5 in obese humans . fifty - seven adult nondiabetic subjects ( 17 lean and 40 obese ) were enrolled in a 3-month supervised physical exercise . rantes and ccr5 expressions were measured in pbmcs and subcutaneous adipose tissue before and after exercise . circulating plasma levels of rantes were also investigated . there was a significant increase in rantes and ccr5 expression in the subcutaneous adipose tissue of obese compared to lean . in pbmcs , however , while the levels of rantes mrna and protein were comparable between both groups , ccr5 mrna was downregulated in obese subjects ( p < 0.05 ) . physical exercise significantly reduced the expression of both rantes and ccr5 ( p < 0.05 ) in the adipose tissue of obese individuals with a concomitant decrease in the levels of the inflammatory markers tnf- , il-6 , and p - jnk . circulating rantes correlated negatively with anti - inflammatory il-1ra ( p = 0.001 ) and positively with proinflammatory ip-10 and tbars levels ( p < 0.05 ) . therefore , physical exercise may provide an effective approach for combating the deleterious effects associated with obesity through rantes signaling in the adipose tissue .

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

the term adenoid cystic carcinoma ( acc ) was first used by spies in 1930 . it is more common in the minor salivary glands ( > 25% ) rather than in the major salivary glands ( about 5% ) . the tumor is characterized by slow and relentless growth with a tendency for extensive local invasion . a particular feature of acc is the tumor 's propensity for perineural invasion that may result in the spread along the involved nerves . a few point mutations in known cancer genes , including kras , braf and tp53 have been reported in accs . recently , recurrent translocation t ( 6;9)(q22 - 23;p23 - 24 ) was identified resulting in the fusion of the v - myb myeloblastosis viral oncogene homolog ( myb ) gene on chromosome 6 to the nuclear factor i / b gene on chromosome 9 in a major number of cases . however , the extent to which other genes contribute to the disease is not well understood . most commonly followed the method of treatment for acc seems to be surgical resection combined with radiotherapy . a 64-year - old man presented to the department of oral medicine and radiology with a 3-month history of swelling in the right anterior floor of the mouth . the patient had noted reduced salivation for 2 months along with a little discomfort in speech and mastication due to the size of the tumor . extra - oral examination revealed a palpable right submandibular lymph node measuring around 2 cm 2 cm in size , tender and soft in consistency suggestive of an inflammatory nature of the lesion . intra - oral examination revealed a tender , solitary , soft , and sessile swelling in the right anterior floor of the mouth measuring about 3 cm 2 cm extending mesiodistally from central incisor to premolar region and superioinferiorly from the depth of the lingual vestibule to the level of occlusion . examination also revealed an ulcer over the swelling [ figure 1 ] . considering the history of reduced salivation and discomfort in mastication , provisional diagnosis of submandibular salivary sailolith and ranula was considered . figure 164-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . intraoral photograph shows smooth surface with well - defined borders ( solid arrow ) . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . acc may require ultrasonography ( us ) , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) for evaluating perineural , vascular , or skull base infiltration . the limitation of us includes its inability to evaluate the deep lesions . because ct is less accurate than mri for detecting the extent of disease , mri is preferred over ct . in case of bone pain , acc is best evaluated by using t1-weighted and fat - suppressed t2-weighted and gadolinium enhanced mri . the lesion exhibits homogenous hypointense signals with ill - defined margins on t1-weighted mri and after injecting gadolinium intravenously acc shows continuous rapid or gradual enhancement pattern . on t2-weighted mri , low signal intensity corresponds to the solid type with poor prognosis and high signal intensity corresponds to either the cribriform or tubular subtypes with better prognoses . mandibular cross - sectional occlusal radiograph of the 64-year - old patient showed no identifiable sailoliths [ figure 2 ] . the patient 's socioeconomic status did not allow use of the more expensive diagnostic imaging modalities . with a provisional diagnosis of ranula , surgical excision of the swelling figure 264-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . the swelling was found to begin from the sublingual duct and to obstruct the submandibular duct [ figures 3 and 4 ] . histological investigation of the excised tissue stained with hematoxylin and eosin revealed basaloid epithelial cells arranged in cyst like spaces ( duct like appearance ) , with many of the spaces presenting with hyalinized and mucoid appearance . the cells appeared small and uniform with deeply basophilic nuclei and a predominently cribriform architecture suggestive of acc arising from the right sublingual gland [ figure 5 ] . considering the patient 's age and socioeconomic status , no further investigations or radiotherapy was done . patient was advised to return for a follow - up examination once in 3 months because of the relentless growth of acc which has a tendency for extensive local invasion . figure 364-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . figure 464-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . figure 564-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . photomicrograph of the hematoxylin and eosin stained tissue ( 10 ) shows multiple basaloid epithelial cells with isomorphic and basophilic nuclei arranged in cribriform architecture ( solid arrow ) . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . 64-year - old - male with swelling in the right anterior floor of the mouth diagnosed with adenoid cystic carcinoma . photomicrograph of the hematoxylin and eosin stained tissue ( 10 ) shows multiple basaloid epithelial cells with isomorphic and basophilic nuclei arranged in cribriform architecture ( solid arrow ) . accs may show any one of the following patterns : solid , trabecular , tubular , and cribriform . among these cribriform type is the most common variant . cribriform form shows the presence of extensive sheets , uniform bands and is composed of small , darkly stained , slightly separated basal cells with duct like structures , which may contain secretory products . the present case showed the most common cribriform pattern of acc . according to many authors histological subtype influence the prognosis of the disease . histological subtypes of tubular and cribriform acc have a better prognosis than lesions with trabecular and solid patterns . acc of the sublingual salivary gland causing obstruction of the submandibular duct is exceedingly rare : only three cases have been reported in the literature . the peak incidence is seen during the fifth and sixth decades of life and shows a female predominance . most of the patients with acc present with a mass , which has been there for months or years . pain is felt by a few patients and is more often associated with advanced or recurrent tumors . in the present case , a 64-year - old - male patient presented with swelling of only 3-months duration and experienced mild pain , which may be associated with traumatic ulceration on the swelling rather than due to the tumor itself . possible treatment modalities of acc include surgical therapy , radiotherapy , chemotherapy , and combined therapy . post - operative radiotherapy is indicated for tumors associated with perineural spread , local recurrence , and distant metastases . according to avery et al . alcedo et al . , opened up a new possibility in the pharmacological treatment of this tumor by demonstrating its response to imatinib mesylate , a potent inhibitor of kit tyrosine kinase , an enzyme involved in the pathogenesis of this tumor . several genomic investigations have been carried out in accs to identify biological markers of therapeutic potential . these efforts , however , have been largely unrewarding and more investigations of new targets are needed . in the present case , only surgical therapy was preferred and further management was not done due to patient 's socio - economic status . in spite of only surgical management , 9-month follow - up showed no recurrence . this may be due to better prognosis associated with the less aggressive cribriform type of the lesion . further studies are required to validate treatment with only surgery in cases of tubular and cribriform forms of accs . acc of sublingual glands is rare and tumors causing obstruction to submandibular duct are extremely rare . review of the literature shows that until date , only three cases have been reported . us , ct , and mri are helpful tools in the diagnosis of accs . the diagnosis can be confirmed and the subtype identified by histological investigations . most common method of treatment for acc seems to be surgical resection combined with radiotherapy . the present case was managed only with surgical modality and 9-month follow - up showed no recurrence . long - term follow - up and newer biological markers of therapeutic potential are necessary to validate use of only surgery to manage tubular and cribriform accs .

sublingual salivary gland malignancies are extremely rare and account for only 0.3 - 1% of all epithelial salivary gland tumors . here , we report a case of adenoid cystic carcinoma ( acc ) of the sublingual salivary gland that presented as a swelling in the right anterior floor of the mouth obstructing the submandibular duct . sublingual salivary gland acc obstructing the submandibular duct is rare and only three cases have been reported in the literature until date . we discuss the different patterns of acc seen during the pathologic investigations and its radiologic features .

INTRODUCTION RADIOLOGIC FEATURES PATHOLOGIC FEATURES DISCUSSION CONCLUSION

ureteric injuries and strictures can occur iatrogenically from pelvic surgery or result from urolithiasis , repeated ureteroscopic procedures and congenital vesicoureteric reflux . the available options include ureteroscopic stenting and ureteric reimplantation by open , laparoscopic or robotic approaches . minimally invasive techniques are preferred in patients in whom open surgery is a high risk . they have been shown to be feasible , and short - term outcomes are comparable to open surgery . preserving renal function in a solitary kidney is vital , and in the current case , the patient was unsuccessfully treated with repeated ureteric stenting for a ureteric stricture . in the current report , laparoscopic reimplantation of ureter in a complex case of ureteric stricture secondary to urolithiasis and repeated ureteroscopy is described . a 56-year - old caucasian female who has a history of recurrent pyelonephritis , obstructive uropathy and stage 4 chronic kidney disease initially presented to the infectious disease team with bacterial endocarditis . a ct demonstrated bilateral renal obstruction : right secondary to two 4-mm distal ureteric stones , and left secondary to long - standing pelviureteric junction ( fig . the patient recovered well and outpatient investigations revealed the following : ( 1 ) a 5 mm distal right ureteric calculus and a further 7-mm calculus more distally on repeat ct , ( 2 ) kidney split function of the left kidney 17% , and of the right 83% on the dmsa renogram . the patient desired an improved quality of life and wanted to be free of repeated urosepsis and free of the insertion of nephrostomy . in view of this , an open left nephrectomy was performed 13 months later , following a period of haemodialysis . a nephrostogram demonstrated a right renal obstruction due to an ureteric stricture , showing inflammatory changes secondary to the patient 's infected stones . ureteroscopic laser fragmentation of the right ureteric calculi and stent insertion was performed ; however , a repeat nephrostogram showed a long stricture in the lower third of the right ureter . the stent expelled spontaneously , and a new antegrade ureteric stent and nephrostomy was inserted , and a post - procedure ct confirmed a satisfactory nephrostomy and stent position ; but , in addition , a persistent 7-mm right distal ureteric calculus was shown . the stent expelled again spontaneously , and the right kidney was found to be grossly hydronephrotic on the ultrasound scan ( creatinine 476 mol / l , egfr 8 ml / min/1.73 m ) , and a right nephrostomy was inserted temporarily . another nephrostogram showed a persistent 4-cm - long stricture within the distal right ureter ( fig . 2 ) . in view of her chronic ureteric stricture and repeated spontaneous passing of stents , laparoscopic right ureteric reimplantation into the bladder dome was performed on her single urinary tract system . routine pre - operative assessment and optimisation was employed with no specific mechanical bowel preparation . the ascending colon was mobilised , the right ureter was identified and dissected off of iliac vessels to scar tissue . the bladder was mobilised , the umbilical artery obliterated and the superior vesical pedicle clipped . the bladder was hitched to the psoas muscle , opened , and the ureter spatulated . 3 ) performed at 6 weeks revealed free urine flow into the bladder , and the nephrostomy was subsequently removed . sixteen months post - operatively , the patient is well , nephrostomy - free , and her renal function has improved with a creatinine of 291mol / l ( egfr = 14 ml / min/1.73 m ) . routine pre - operative assessment and optimisation was employed with no specific mechanical bowel preparation . the ascending colon was mobilised , the right ureter was identified and dissected off of iliac vessels to scar tissue . the bladder was mobilised , the umbilical artery obliterated and the superior vesical pedicle clipped . the bladder was hitched to the psoas muscle , opened , and the ureter spatulated . 3 ) performed at 6 weeks revealed free urine flow into the bladder , and the nephrostomy was subsequently removed . sixteen months post - operatively , the patient is well , nephrostomy - free , and her renal function has improved with a creatinine of 291mol / l ( egfr = 14 ml / min/1.73 m ) . laparoscopic ureteric re - implantation was initially performed on animal models for vesicoureteric reflux in 1993 and was introduced in humans in 1994 shortly after . since then , refinements , including laparoscopic boari flap procedures , have been described to enable the repair of more complex distal ureteric strictures . ( no symptoms , renal deterioration , radiological evidence of residual obstruction , or need for subsequent procedures ) [ 5 , 6 , 7 ] . rassweiler et al . performed a retrospective comparison of laparoscopic ( n = 10 ) and open ( n = 10 ) techniques and concluded that the laparoscopic approach is feasible , provides comparable functional outcomes , but offers the advantages of less post - operative analgesic requirement , shorter hospital stays and convalescence . the patient in the current case developed ureteric stricture in a solitary kidney secondary to repeated instrumentation . preserving renal function in a single system is vital . the patient had repeated stent insertion ; however , these expelled spontaneously on a number of occasions , and no clear causes were identified . in patients who experience repeated spontaneous passing of stents , titanium - nickel memokath stents can be offered ; however , these are not permanent and may occlude , and multiple further procedures are likely to be required in the future . laparoscopic reimplantation offers treatment of the stricture and avoidance of stent complications and the need for further ureteroscopy or nephrostomies . the current report presents a successful case of laparoscopic ureteric reimplantation in a solitary kidney to preserve renal function and to avoid repeated endoscopic procedures and nephrostomies .

ureteric strictures can be caused by traumatic pelvic surgery , urolithiasis and instrumentation . there are various treatment options for ureteric stricture , including laparoscopic ureteric reimplantation . a 56-year - old female with a history of chronic left pelviureteric junction obstruction presented with urosepsis secondary to right - sided urolithiasis . the patient had a left nephrectomy and developed right - sided ureteric stricture following repeated ureteroscopy to manage her stone disease . the treatment with ureteric stenting was unsuccessful . here we present a case on the feasibility of laparoscopic reimplantation for ureteric stricture in a solitary kidney to preserve renal function and avoid further ureteroscopy or nephrostomies .

Introduction Case Presentation Before Surgery Procedure After Surgery Discussion Conclusion Disclosure Statement

following elimination of virus , the virus - specific t cell population progressively contracts , and a small population of memory t cells is preserved . however , after infection with cytomegalovirus ( cmv ) , a ubiquitous -herpesvirus , the virus - specific cd8 t cell population is maintained , and it accumulates at a high frequency , acquiring a phenotype of effector memory cells ( 1234 ) . although primary cmv infection is usually asymptomatic in healthy individuals , its resolution does not result in complete elimination of the virus from the host . instead , cmv persists latently in the host , and the latent virus is reactivated according to the immune status of the host ( 5 ) . consequently , the cmv - specific cd8 t cell population is boosted by repeated reactivation of cmv over a lifetime , and its size becomes extraordinarily large , often reaching 20% of total cd8 t cells ( 678 ) . most of these t cells are functional , and their populations are maintained throughout the life of the host . this phenomenon of gradual increase in cmv - specific cd8 t cell population over time is known as memory inflation ( 12910 ) . although inflationary cd4 t cells have also been observed , their responses are less exaggerated ( 11 ) . this peculiar nature of the cmv - specific cd8 t cell population in the elderly has been considered to be related to senescence in the t cell population . here , we review the general features of cmv - specific memory t cell inflation . in particular , we focus on the factors that contribute to memory t cell inflation in cmv infection and its potential adverse effects in the elderly . inflationary memory cd8 t cells are characterized by an effector memory phenotype in mice , humans , and other primates , whereas non - inflationary memory t cells have a typical phenotype of central memory during the latent phase of the infection ( 1234 ) . inflationary memory cd8 t cells generally express low levels of lymph node homing markers , such as cd62l and ccr7 , and accumulate in non - lymphoid peripheral organs . they also display reduced expression of co - stimulatory receptors such as cd28 and cd27 ( 13 ) , and express large amounts of inhibitory receptors such as klrg-1 and cd85j in mice and humans , respectively ( 1012 ) , but they do not express pd-1 ( 313 ) . despite the chronic latent nature of cmv infection , inflationary cd8 t cells are proliferative and are capable of producing effector cytokines such as ifn- and tnf- , and maintain cytotoxicity . these functional features are contrary to those of the exhausted t cells observed in other chronic persistent viral infections ( 314 ) . although inflationary memory t cells possess a terminally differentiated phenotype , they can constantly turnover and respond to viral reactivation ( 15 ) . there was an issue of whether inflationary cmv - specific cd8 t cells are a polyclonal or an oligoclonal population with restricted t cell receptor ( tcr ) usage . mhc class i tetramer - positive populations using v8.1 or v 8.2 tcr chain increased in latent murine cmv ( mcmv ) infection ( 10 ) . similarly , in humans , cmv - specific cd8 t cells are restricted in tcr diversity ( 16 ) . in this regard , it is considered that t cells with high affinity for cmv epitopes are preferentially selected during acute infection , and they continuously retain their clonal dominance ( 17 ) . one of the main causative factors that originated the notion of memory t cell inflation is the presence of repetitive antigen exposure , which was primarily investigated using murine models ( 1819 ) . it was demonstrated that mcmv - driven t cells that were transferred to nave mice failed to divide , suggesting the requirement of antigen stimulation for memory inflation ( 3 ) . inflationary t cells are short - lived , with a half - life of approximately 40~50 days , and exhibit a slow turnover rate through antigen exposure . cells that are primed early in infection may proliferate and maintain the inflationary t cell population . however , it was reported that nave precursor cells could also be primed when adoptively transferred to latently infected mice and recruited to the inflationary t cell pool ( 3 ) . overall , it is concluded that both nave precursor cells and memory cells may contribute to the inflating memory cell population over time . recently , a study showed that low dose infection of mcmv severely hampered the accumulation of inflationary memory t cells , suggesting that the dose of viral inoculum influences the magnitude of memory inflation ( 20 ) . for memory inflation cmv is well known for its ability to evade cd8 t cell responses by interfering with antigen processing and presentation of mhc class i pathways in the infected cells . however , robust cd8 t cell responses are observed in cmv - infected hosts ( 212223 ) . this suggests that cmv - specific cd8 t cells are primed by cross - presentation of cmv antigens by non - infected professional antigen - presenting cells ( 24 ) . interestingly , it was observed that the inflationary response was triggered through antigen presentation by non - hematopoietic cells in lymph nodes at a latent stage of mcmv infection ( 25 ) . following expansion in lymph nodes , cd8 t cells move to non - lymphoid peripheral tissues , where they undergo terminal differentiation and accumulate . however , it was recently reported that inflationary t cell populations are primarily generated by exposure to antigens in blood circulation , suggesting a novel mechanism of mcmv - driven memory inflation ( 15 ) . in addition to tcr stimulation , co - stimulatory molecules and inflammatory cytokines are profoundly involved in effector and memory cd8 t cell responses ( 26 ) . however , terminally differentiated cd8 t cells lose their ability to express cd28 . as a result , cd28 does not participate as a t cell co - stimulatory receptor during chronic latent infection ( 27 ) . instead , other co - stimulatory receptors such as 4 - 1bb and ox40 are expressed on t cells after antigen - driven t cell activation , and they contribute to mcmv - specific t cell inflation ( 2829 ) . furthermore , use of a bone marrow chimeric mouse model demonstrates that the cmv - specific inflationary t cell response depends on il-2 . in addition , secondary expansion is engendered by cd8 t cell - derived autocrine il-2 but not by cd4 t cell- or dendritic cell - generated il-2 ( 3031 ) . il-7 and il-15 , which are important cytokines for homeostatic proliferation of memory cd8 t cells , may affect the proliferation of hcmv - specific t cells ( 32 ) , but their pivotal role in memory t cell inflation remains to be elucidated . il-10 was shown to clearly inhibit the inflation of t cells in the chronic phase of mcmv infection , but the specific molecular mechanism is still unknown ( 33 ) . one of the main causative factors that originated the notion of memory t cell inflation is the presence of repetitive antigen exposure , which was primarily investigated using murine models ( 1819 ) . it was demonstrated that mcmv - driven t cells that were transferred to nave mice failed to divide , suggesting the requirement of antigen stimulation for memory inflation ( 3 ) . inflationary t cells are short - lived , with a half - life of approximately 40~50 days , and exhibit a slow turnover rate through antigen exposure . cells that are primed early in infection may proliferate and maintain the inflationary t cell population . however , it was reported that nave precursor cells could also be primed when adoptively transferred to latently infected mice and recruited to the inflationary t cell pool ( 3 ) . overall , it is concluded that both nave precursor cells and memory cells may contribute to the inflating memory cell population over time . recently , a study showed that low dose infection of mcmv severely hampered the accumulation of inflationary memory t cells , suggesting that the dose of viral inoculum influences the magnitude of memory inflation ( 20 ) . for memory inflation , the manner of antigen processing and presentation might be also important . cmv is well known for its ability to evade cd8 t cell responses by interfering with antigen processing and presentation of mhc class i pathways in the infected cells . however , robust cd8 t cell responses are observed in cmv - infected hosts ( 212223 ) . this suggests that cmv - specific cd8 t cells are primed by cross - presentation of cmv antigens by non - infected professional antigen - presenting cells ( 24 ) . interestingly , it was observed that the inflationary response was triggered through antigen presentation by non - hematopoietic cells in lymph nodes at a latent stage of mcmv infection ( 25 ) . following expansion in lymph nodes , cd8 t cells move to non - lymphoid peripheral tissues , where they undergo terminal differentiation and accumulate . however , it was recently reported that inflationary t cell populations are primarily generated by exposure to antigens in blood circulation , suggesting a novel mechanism of mcmv - driven memory inflation ( 15 ) . in addition to tcr stimulation , co - stimulatory molecules and inflammatory cytokines are profoundly involved in effector and memory cd8 t cell responses ( 26 ) . however , terminally differentiated cd8 t cells lose their ability to express cd28 . as a result , cd28 does not participate as a t cell co - stimulatory receptor during chronic latent infection ( 27 ) . instead , other co - stimulatory receptors such as 4 - 1bb and ox40 are expressed on t cells after antigen - driven t cell activation , and they contribute to mcmv - specific t cell inflation ( 2829 ) . furthermore , use of a bone marrow chimeric mouse model demonstrates that the cmv - specific inflationary t cell response depends on il-2 . in addition , secondary expansion is engendered by cd8 t cell - derived autocrine il-2 but not by cd4 t cell- or dendritic cell - generated il-2 ( 3031 ) . il-7 and il-15 , which are important cytokines for homeostatic proliferation of memory cd8 t cells , may affect the proliferation of hcmv - specific t cells ( 32 ) , but their pivotal role in memory t cell inflation remains to be elucidated . il-10 was shown to clearly inhibit the inflation of t cells in the chronic phase of mcmv infection , but the specific molecular mechanism is still unknown ( 33 ) . asymptomatic human cmv ( hcmv ) infection is present in most of the human population , and t cell response to hcmv is sustained and amplified over time . consequently , the hcmv - specific t cell population acquires dominance in the peripheral blood of healthy elderly hosts . among the total cd8 t cell population in elderly individuals , the expansion of hcmv - specific t cells in the elderly has been implicated in relation to immunosenescence . the concept of immunosenescence was first used to describe the age - associated deterioration of immune functions , characterized by functional and phenotypic changes at both cellular and systemic levels ( 3435 ) . although it is unclear whether hcmv is the main causative agent of systemic immunosenescence , hcmv is one of the most important antigens that induce repetitive t cell stimulation , which is a powerful driving factor of immunosenescence ( 3637 ) . in addition , hcmv - specific inflationary t cells possess rather similar phenotypic and functional features to those of replicative senescent t cells . in the elderly , a majority of hcmv - specific cd8 t cells are cd45ra effector memory cells , representing the typical phenotypic feature of terminally differentiated , replicative senescent t cells that gain cd57 and lose cd28 , ccr7 , and il-7r expression ( 383940 ) . the cd45ra effector memory cell population gradually expands with age and comprises more than half of the total memory cd8 t cell population , even in healthy elderly individuals ( 38 ) . considering the oligoclonality of these cells along with shortened telomere length due to extensive cell division , frequently encountered antigens , such as latent or persistent chronic viruses , are considered as major contributors to the expansion of cd45ra effector memory t cells ( 36 ) . among these viruses the clinical significance of hcmv - specific t cell inflation with ageing is supported only by circumstantial evidence . cmv seropositivity and the clonal expansion of cmv - specific cd8 t cells were identified as the major immune risk phenotype ( irp ) in a longitudinal study in the elderly ( 44 ) . in addition , latent hcmv infection has been associated with reduction in life span ( 745 ) , acceleration of tumor growth ( 4647 ) and occurrence of cardiovascular diseases ( 48 ) . further studies are necessary to understand the role of memory t cell inflation in health and diseases , particularly in the senior population . ' memory inflation ' is a term describing progressive expansion of virus - specific cd8 t cells , particularly in the context of chronic latent cmv infection . the expanded cmv - specific cd8 t cell population is extraordinarily large and is more prominent in the elderly . inflationary cd8 t cells possess rather similar phenotypic and functional features to those of replicative senescent t cells . until now , the detailed nature of cmv - specific t cell responses has been explored mainly using mouse models . however , there is a concern for the discrepancies of longevity and genetic backgrounds between mice and humans , which result in an incomplete picture for elucidating cmv - specific t cell responses and immunosenescence . therefore , non - human primates may be a more suitable alternative as cmv animal models to understand memory t cell inflation ( 49 ) . moreover , it is necessary to dissect the roles and characteristics of inflationary t cells induced by hcmv infection . the mechanism by which inflationary t cells contribute to the pathogenesis of geriatric diseases requires further investigation . ' memory inflation ' is a term describing progressive expansion of virus - specific cd8 t cells , particularly in the context of chronic latent cmv infection . the expanded cmv - specific cd8 t cell population is extraordinarily large and is more prominent in the elderly . inflationary cd8 t cells possess rather similar phenotypic and functional features to those of replicative senescent t cells . until now , the detailed nature of cmv - specific t cell responses has been explored mainly using mouse models . however , there is a concern for the discrepancies of longevity and genetic backgrounds between mice and humans , which result in an incomplete picture for elucidating cmv - specific t cell responses and immunosenescence . therefore , non - human primates may be a more suitable alternative as cmv animal models to understand memory t cell inflation ( 49 ) . moreover , it is necessary to dissect the roles and characteristics of inflationary t cells induced by hcmv infection . the mechanism by which inflationary t cells contribute to the pathogenesis of geriatric diseases requires further investigation .

cytomegalovirus ( cmv ) infection in healthy individuals is usually asymptomatic and results in latent infection . cmv reactivation occasionally occurs in healthy individuals according to their immune status over time . t cell responses to cmv are restricted to a limited number of immunodominant epitopes , as compared to responses to other chronic or persistent viruses . this response results in progressive , prolonged expansion of cmv - specific cd8 + t cells , termed ' memory inflation ' . the expanded cmv - specific cd8 + t cell population is extraordinarily large and is more prominent in the elderly . cmv - specific cd8 + t cells possess rather similar phenotypic and functional features to those of replicative senescent t cells . in this review , we discuss the general features of cmv - specific inflationary memory t cells and the factors involved in memory inflation .

INTRODUCTION CHARACTERISTICS OF INFLATIONARY MEMORY T CELLS FACTORS INVOLVED IN MEMORY T CELL INFLATION Repetitive antigen exposure Antigen presentation Co-stimulation and cytokines CMV, MEMORY INFLATION AND T CELL SENESCENCE Conclusions and prospects

the reactant is resorcinol , a highly activated aromatic molecule that reacts quickly and efficiently by electrophilic aromatic substitution under acidic conditions . each student prepares a solution of resorcinol ( 0.60 mmol ) in d2o ( 1 ml ) containing h2so4 ( 20 l ) . one reaction solution is analyzed by h nmr spectroscopy , and the other solution is used in the iodination reaction ( as the reaction mixture ) . students prepare another solution of resorcinol in acidic d2o that is not refluxed as a ( nondeuterated ) control . students conduct iodination of resorcinol at room temperature by adding a dilute solution of molecular iodine and potassium iodide in ethanol simultaneously to two vials , one containing the reaction mixture and the other containing the ( nondeuterated ) control . the reaction progress is monitored visually via the disappearance of iodine color in the reaction solutions due to the formation of 2-iodoresorcinol and 4-iodoresorcinol . the kinetic isotope effect of the iodination reactions is quantified by analysis of uv vis spectroscopic data provided to students . a solution of sulfuric acid in d2o can be prepared in advance for students to use . no safety information is available for resorcinol - d5 , but similar safety concerns are reasonably expected . the solution of iodine / iodide in ethanol may be corrosive and should be handled with care . the iodination products of resorcinol , including 2-iodoresorcinol , are harmful if swallowed , inhaled , or absorbed through the skin . students should wear proper personal protective equipment at all times when performing this experiment , including gloves , a lab coat , and safety goggles . chemical manipulations are recommended to be done in an appropriate fume hood if possible . this experiment has been completed once in a laboratory session of 3 h by a total of 15 first - semester undergraduate organic chemistry students . h bonds in resorcinol did not undergo significant h d exchange in refluxing d2o in a time frame reasonable for use in a classroom setting . however , when d2o and sulfuric acid were mixed together , the concentration of d3o in solution and subsequent reactivity were greatly increased . to conduct the reaction , a microscale reflux apparatus , consisting of a small vial containing a spin vane ( a boiling chip would be sufficient ) with a reflux condenser , was heated on a hot plate . the reaction typically reached equilibrium within 30 min of heating , after which no further net deuteration would occur . the appearance of the reaction mixture did not change over the course of the experiment . d exchange at the 2- , 4- , and 6-positions on the ring ( scheme 1 ) . this result was in accord with a typical eas mechanism involving slow addition of d+ to the aromatic ring to form an intermediate arenium ion followed by rapid elimination of the proton . though there was a negligible kinetic preference for elimination of the proton over the deuteron , the high concentration of deuterium in solution resulted in preferential formation of the trideuterated species . the alcohol protons were also exchanged , but this had no effect on the outcome of the experiment . the extent of deuteration was determined by integrating the remaining proton at the 5-position and comparing it to any remaining proton signals ( figure 1 ) . this proton was meta to the activating hydroxyl groups on the ring and thus not activated toward electrophilic exchange . as a result , deuterium incorporation at this position was negligible during the course of the experiment . the nmr signal associated with this proton collapsed from a triplet into a broad singlet , providing strong evidence that neighboring protons were exchanged with deuterium . d coupling in the spectrum , despite deuterium being nmr active ( i = 1 ) , typified important differences between h h and h d spin spin coupling and can provide an introduction to more advanced nmr concepts such as the magnetogyric ratio . the spectrum was used as an effective explanation of electrophilic aromatic substitution trends due to the clear distinction between near complete exchange at the positions ortho and para to the activating hydroxyl groups and very minor exchange at the meta position . h nmr spectra of resorcinol prior to ( left ) and after ( right ) h d exchange . by adapting the procedure from a previous work , students visualized the kinetic isotope effect by adding a dilute ethanolic solution of iodine and sodium iodide to the reaction mixture containing deuterated resorcinol and a control mixture of resorcinol and sulfuric acid in d2o at ambient temperature ( scheme 2 ) . before addition of the iodine solution , the deuterated mixture and control ( nondeuterated ) mixture were clear , nearly colorless , and visually indistinguishable . after initial addition of the iodine solution , both the deuterated mixture and control mixture were translucent and dark orange - red in color . the color faded , and the solutions clarified as the iodine reacted with the resorcinol . in each case , after completion of the reaction , the solution was clear and very light yellow in color , indicating that a trace amount of unreacted iodine may remain . in the control experiment , the iodine color of the solution faded much more rapidly ( i.e. , a change was apparent within 5 min ) compared to the deuterated example , and the reaction was nearly complete after approximately half an hour . comparatively , the isotopically labeled resorcinol - d5 reacted much more slowly , and the reaction remained incomplete after well over an hour . the concentration of iodine remained consistently higher in the vial containing deuterated resorcinol throughout the duration of the experiment , providing a qualitative visual representation of the comparative reactivities of each substrate . the effect was best observed when the experimental and control reactions were conducted side - by - side and a student could observe both results simultaneously . while reaction time and the concentration of iodine varied somewhat from student to student , the trend was very clear in each case and the effect was easily visualized . the kinetic isotope effect kh / kd of the iodination reaction was also determined quantitatively the reaction of iodine with resorcinol under the aforementioned conditions was found to be second - order overall , providing an excellent opportunity to quantify the kie by monitoring the uv vis absorption spectrum of iodine as it reacted . however , because a significant amount of time was required to collect sufficient uv vis data and the outcome appeared to be somewhat sensitive to minor variances in preparation , previously obtained data were given to students for interpretation . when 1/[i2 ] versus time was plotted for the control and experimental conditions and the slopes of these plots were compared , a kh / kd of approximately 3.4 was calculated . representative uv vis absorbance data for this experiment are in the supporting information . a key aspect of this experimental methodology is its adaptability to fit the target audience . this experiment was primarily utilized as part of the lab curriculum for first - semester undergraduate organic chemistry students . prior to conducting the experiment , most students only had a cursory understanding of isotopes and their effect on chemical reactivity . students were introduced to the concepts of electrophilic aromatic substitution , isotopic exchange , and the kinetic isotope effect as part of the required reading before the experiment . by performing the experiment , students were able to comprehend better eas and isotopic exchange . using h nmr spectroscopy , they were able to familiarize themselves with the instrumentation and its utility in characterizing such isotopic exchange reactions . additionally , they interpreted uv vis absorbance data to quantify the kinetic isotope effect of the iodination reaction and rationalize its rate and mechanism . after the experiment , post - lab write - ups indicated that all students had a significantly greater understanding of eas and the kinetic isotope effect , and many were interested in the potential applications of the chemistry , including the production of deuterated drugs . the materials given to students , including introductory material as well as pre - lab and post - lab questions , are provided in the supporting information . as a secondary project , the deuteration and iodination reactions were successfully performed and the results interpreted by ambitious high school students looking to gain experience with undergraduate - level chemistry . these students had previous experience with chemistry courses but were not familiar with isotopic substitution reactions or their effects . while some of the more complicated aspects of the transformation were omitted , the fundamental aspects of isotopic exchange could be understood and demonstrated experimentally . isotope effects were explained by comparing the physical properties of d2o and h2o ( slightly different molar masses and densities ) , as well as by monitoring the iodination reaction visually . in each case , students saw firsthand that isotopically substituted compounds often have very similar physical properties but can be differentiated through experimentation . this methodology is amenable to a diverse audience of chemistry students and educators , with the complexity of the material highly adaptable to the desired outcome .

an efficient laboratory experiment has been developed for undergraduate students to conduct hydrogen deuterium ( h d ) exchange of resorcinol by electrophilic aromatic substitution using d2o and a catalytic amount of h2so4 . the resulting labeled product is characterized by 1h nmr . students also visualize a significant kinetic isotope effect ( kh / kd 3 to 4 ) by adding iodine tincture to solutions of unlabeled resorcinol and the h d exchange product . this method is highly adaptable to fit a target audience and has been successfully implemented in a pedagogical capacity with second - year introductory organic chemistry students as part of their laboratory curriculum . it was also adapted for students at the advanced high school level .

Experimental Overview Hazards Results and Discussion Student Experiences

naturally produced biopolymers in living organisms play crucial roles in materials discovery and development . they have inspired scientists to synthesize novel biomaterials through mimicking mother nature , or they can further serve as templates and building blocks to prepare new generations of biocompatible , bioregenerative , or biodegradable materials for biomedical applications . for instance , dna has been used to rationally design plasmonic nanostructures , to build nanoscaffolds for incorporating multiple - affinity ligands , and to self - assemble into numerous prescribed 3d shapes . cellular membranes have also been widely imitated by phospholipids and polysaccharides to form liposome or micelles for drug and imaging agent delivery . leukocyte membranes have also been used to coat silicon nanoparticles ( nps ) to yield hybrid nps that achieve cell - like functions , including avoiding clearance by the immune system . multimodal imaging combines different modalities together to provide complementary information and achieve synergistic advantages over any single modality alone . it has emerged as a very promising strategy for preclinical research and clinical applications . one major challenge of multimodal imaging is to develop an efficient platform to load various components with individual contrast properties together while maintaining compact size , good biocompatibility and targeting capability . exogenous inorganic nps - based reporters have attracted considerable interests , such as iron oxide nps for magnetic resonance imaging ( mri ) and quantum dots for fluorescence imaging . compared with inorganic nps , organic nps generally exhibit good biocompatibilities , biodistribution and clearance , although most of them only appear to possess optical imaging properties . some biomolecules based nps such as liposomes have been widely used for loading contrast agents and drugs . therefore , such biomolecules need complicated and time - consuming processes to prebuild various contrast properties or require chemical modifications to integrate different reporting moieties into one entity , which we term as a passive platform . for example , organic ligands are generally incorporated into a nanoplatform before chelating to radioactive or magnetic metal ions for positron emission tomography ( pet ) and magnetic resonance imaging ( mri ) . melanin , an amorphous , irregular functional biopolymer and a ubiquitous natural pigment that presents in many organisms including human skin , is a typical biomarker for disease imaging including melanoma detection and parkinson diseases diagnosis . in this study , we report the successful transferring of this biomarker into an imaging nanoplatform . by mimicking natural melanin , water - soluble melanin nanoparticle ( mnp ) has been synthesized and used as the active platform for multimodal imaging of tumors . we demonstrate that mnp can not only offer its native optical properties for photoacoustic imaging ( pai ) , but also actively chelate to metal ions ( cu , fe ) for pet and mri with a high loading capacity and stability utilizing its intrinsic chelating function . furthermore , ultrasmall size mnps ( 4.5 nm ) can be easily prepared and surface - modified . overall , these unique properties significantly simplify the process of preparation of multimodal imaging probes and make mnp a highly promising nanomaterial for biomedical applications . figure 1 schematically illustrates the procedure to prepare ultrasmall water - soluble mnp with multimodal imaging properties . to change the intrinsic poor water - solubility of melanin , pristine melanin granule was first dissolved in a 0.1 n naoh and then neutralized under the assistance of sonication to decrease interchain aggregation . ultrasmall mnp in high water monodispersity and homogeneity with a size of 4.5 0.5 nm , which was termed as plain water - soluble mnp ( pws - mnp ) , were successfully obtained ( figure 2a , b and figure s1a , supporting information ) . pws - mnp exhibited excellent water - solubility of 40 mg / ml and stability , which can be attributed to the highly negative potential of approximately 22.2 mv on the np surface that efficiently blocks the np aggregation through electrostatic repulsion ( figure s1b ) . furthermore , pws - mnp can be stored as lyophilized powder for over six months and effectively redissolved in water allowing long - term usage ( figure 2a ) . the ft - ir spectra of pristine melanin granule and pws - mnp were similar to each other , indicating no significant change of molecular structure ( figure s2a ) . the h nmr spectrum of pws - mnp in d2o showed no obvious signal belonging to the hydrogen atom on the arylene groups , suggesting most of the conjugated backbones were buried in the np ( figure s2b ) . the molecular weight of a pws - mnp was calculated from the nanoparticle size and its density ( 1.3 g / cm ) , which is about 40 kda . the melanin granules were first dissolved in 0.1 n naoh aqueous solution , and then neutralized under sonication to obtain melanin nanoparticles in high water monodispersity and homogeneity . after peg surface - modification , rgd was further attached to the mnp for tumor targeting . then fe and/or cu were chelated to the obtained mnps for pai / mri / pet multimodal imaging . characterization of physical properties of mnps . ( a ) from left to right : pictures of ( 1 ) pristine melanin granule in h2o , ( 2 ) melanin neutralized without sonication in h2o , ( 3 ) freeze - dried pws - mnp , ( 4 ) freeze - dried pws - mnp redissolved in pbs ( ph = 7.4 ) , ( 5 ) freeze - dried peg - mnp , ( 6 ) freeze - dried peg - mnp redissolved in pbs ( ph = 7.4 ) . ( b ) tem of pws - mnp ( left ) and peg - mnp ( right ) , scale bar = 20 nm . ( c ) the plot of the relationship between the number of metal ions attached on one mnp with feed ratio ( wions : wmnp ) . ( d ) stability study of metal ion - chelated mnps in pbs ( ph = 7.4 ) . in vitro and in vivo study of pai of mnps . ( a ) the photoacoustic signal produced by peg - mnps at concentrations of 0.625 , 1.25 , 2.5 , 5.0 , 10 , and 20 m , and it was observed to be linearly dependent on its concentration ( r = 0.995 ) . mice were injected subcutaneously ( region enveloped by blue dotted line ) with peg - mnp at concentrations of 0 , 5 , 10 ( from left to right in top row ) , and 20 , 40 , 80 ( from left to right in bottom row ) m . one vertical slice in the photoacoustic image ( red ) was overlaid on the corresponding slice in the ultrasound image ( gray ) . the linear regression is calculated on the five most concentrated inclusions ( r = 0.998 ) . ( d ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imagings of u87 mg tumor ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) and their subtraction imagings . ( e ) quantitative analysis of enhanced pa signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. to retain the water - solubility of pws - mnps for further biomodification and metal ion - chelating , polyethylene glycol ( peg ) chains were introduced to the mnp . nh2-peg5000-nh2 was used because the amine groups can react with dihydroxyindole / indolequinone groups in melanin . the number of peg chains per mnp was determined to be 19 ( figure s3a and s3b ) . the diameter of the peg - functionalized mnp ( peg - mnp ) became large and reached 7.0 nm ( figure 2b and figure s1a ) . moreover , the surface potential of peg - mnp decreased to 6.1 mv ( figure s1b ) because of introduction of peg and positive nh2 groups on the mnp surface . the similar absorption spectrum of peg - mnp to pws - mnp demonstrated that the peg - modification did not influence the absorption properties of melanin ( figure s3c ) . lastly , for demonstrating that mnp can be used as a platform for biomodification , peg - mnp was further modified with biomolecules such as cyclic arg - gly - asp - d - phe - cys [ c(rgdfc ) ] peptide ( abbreviated as rgd ) , which can target tumor v3 integrin . the number of rgd attached to the mnp was calculated to be about 8 per mnp and the size of rgd - functionalized peg - mnp ( rgd - peg - mnp ) increased a little to 9.6 nm ( figure s4 ) . in vitro and in vivo study of mri of fe - chelated mnps . ( a ) t1 relaxation rates ( 1/t1 , s ) as a function of fe - rgd - peg - mnp ( mm ) in agarose gel ( 1.0 t , 25 c ) . ( b ) mri detection of fe - rgd - peg - mnps in living mice . mice were injected subcutaneously ( region enveloped by red dotted line ) with fe - rgd - peg - mnps at concentrations of 0 , 1.25 , 2.5 ( from left to right in upper layer ) , and 5 , 10 , 20 ( from left to right in bottom layer ) m . ( c ) quantitative analysis of enhanced mr signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h , compared with at 0 h. ( d ) mri images of u87 mg tumors ( region enveloped by yellow dotted line ) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice ( n = 3 ) ( tr : 700 ms , te : 5.2 ms ) . top row shows black and white images , and bottom row shows the pseudocolored images . to investigate the possibility of mnp as a platform for pet and mri , its chelating properties to cu ( cu for pet ) and fe ( for mri ) were studied . after adding metal ions ( 0.2 ml of 10 mm fecl3 or cucl2 ) into mnp aqueous solutions ( 1 ml of 20 m for pws - mnp and peg - mnp ) , the precipitation of pws - mnp quickly appeared , while peg - mnp maintained good water - solubility ( figure s5 ) . the fe or cu - chelated mnp ( fe - peg - mnp , fe - rgd - peg - mnp , cu - peg - mnp and cu - rgd - peg - mnp ) exhibited high loading capacities . the maximum quantities of one mnp to chelate to cu and fe are about 100 and 90 ions , respectively , no matter whether rgd is attached to the mnp or not ( figure 2c ) . after fe - chelating , the mnp sizes increased to 8.9 nm and 10.7 nm for fe - peg - mnp and fe - rgd - peg - mnp respectively and their zeta - potential remained in the neutral region ( figure s4 and table s1 ) . the optical stabilities of peg - mnp and rgd - peg - mnp under increasing durations of light exposure were further tested . compared with those reported dyes for pai , which exhibit significant reduced absorption ( > 30% ) under light exposure , peg - mnp and rgd - peg - mnp showed intriguing photostability ( only 3% reduced absorption ) ( figure s6 ) , indicating their high capability for pai . further stability assay of fe or cu - chelated mnps in pbs solution showed that only about 3% cu and 7% fe were released from those mnps at the first 2 h , and there was no further release at longer incubation time points , indicating the high stability of the chelating platform ( figure 2d ) . the first 2 h released metal ions may derive from those that were absorbed on the mnps through weak electrostatic interaction . furthermore , the high viability of nih3t3 and u87 mg cells ( about 90110% as compared to the nontoxic control ) after 24 h of incubation with peg - functionalized mnps was found , indicating high biocompatibility and low cytotoxic effect of peg - functionalized mnps ( figure s7 ) . to investigate the possibility of mnps to be used as a photoacoustic agent , we first studied the detection sensitivity of peg - mnp in aqueous solution at increasing concentrations from 0.625 to 20 m . the peg - mnp with 0.625 m was detected , and the photoacoustic signals increased linearly with the increase of peg - mnp concentrations ( r = 0.995 ) ( figure 3a ) . the detection sensitivity of mnp in living body was further tested by subcutaneous injection of peg - mnp on the lower back of mice ( n = 3 ) at increasing concentrations of 5 to 80 m ( figure 3b ) . a linear correlation ( r = 0.998 ) between the mnp concentration and the corresponding photoacoustic signal 2.5 m of peg - mnp was found to give the equivalent photoacuoustic signal strength as the tissue background . to further investigate the in vivo pai properties , one group of u87 mg tumor mice were tail - vein injected with 250 l of rgd - peg - mnp at a concentration of 200 m . mice showed obvious increase of photoacoustic signal in tumors after injection with rgd - peg - mnp at 4 h than that of prescan ( figure 3d ) . the increased photoacoustic signal of rgd - peg - mnp ( figure 3e ) could be attributed to the enhanced permeability and retention ( epr ) effect and the tumor targeting ability of rgd - peg - mnp to v3 integrin . furthermore , instead of vevo lazr pai system , using inveon research workplace ( nexus 128 ) was able to obtain 3d pa imaging , which provided the more clearly enhanced blood vessel signals in tumor after mnp injection ( figure s8 ) . in vitro and in vivo study of pet of cu - labeled mnps . ( a ) uptake of cu - rgd - peg - mnp with and without blocking in u87 mg cells at 37 c for 1 , 2 and 4 h incubation . all results , expressed as percentage of cellular uptake , are mean of triplicate measurements sd . ( b ) representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal pet images ( left three images ) and the overlaying of ct ( gray ) and pet ( color ) images ( right three images ) of u87 mg tumors ( region enveloped by yellow dotted line ) acquired at 2 , 4 , and 24 h after tail vein injection of cu - rgd - peg - mnp . ( c ) biodistribution of cu - rgd - peg - mnp in mice ( n = 3 ) at 2 , 4 , and 24 h after injection . the radioactive signal from each organ was calculated using a region of interest drawn over the whole organ region . to study whether fe ( t1 contrast agent ) retains mr signal - enhancing property after loading into mnps , t1-weighted mri images of various concentrations of fe - rgd - peg - mnp in agarose gel was investigated ( figure s9a ) . with the increase of np concentration , mr signal was significantly enhanced , suggesting fe - rgd - peg - mnp generate a high magnetic field gradient on their surface . r1 value of fe - rgd - peg - mnp ( the slope of the fitted curve in figure 4a , using gd as standard ) was calculated to be 1.2 mm s. the magnetic sensitivity in living mice was then tested by subcutaneous injection of fe - rgd - peg - mnp on the lower back of mice ( n = 3 ) at increasing concentrations of 1.25 to 20 m . it was extrapolated that 1.25 m of fe - rgd - peg - mnp produced the equivalent mri signal intensity as the tissue background ( figure 4b ) . to demonstrate the use of mnp as the platform for mri of tumors , t1-weighted images were obtained from mice bearing u87 mg tumors ( n = 3 ) . u87 mg tumors displayed increased signals after 4 h mnp injection ( figure 4d ) . the relative mr signal intensity of tumor at 4 h increased 30% compared with at 0 h , demonstrating that mnp can be used as a platform for mri ( figure 4c ) . furthermore , further optimizing the mr imaging conditions can provide more clearly enhanced mri signal and mnp accumulation in tumor ( figure s9b ) . to investigate the pet imaging properties of mnp , cu was selected as a pet radiolabel for mnp because it can be readily chelated by melanin and the intermediate half - life of cu ( 12.7 h ) makes it suitable for radiolabeling of biomolecules and imaging . simple mixing of rgd - peg - mnp and peg - mnp with cu allowed successfully labeling the nps in the yield of 80% . the resulting mnps , cu - rgd - peg - mnp and cu - peg - mnp , displayed excellent stability in pbs solution ( figure s10 ) . similar to cu - chelated mnps , only 3% cu released from the mnps after 24 h of incubation . thus , cu - labeled mnps were easily and reliably produced and exhibited reasonable stability in vitro . in vivo multimodality imaging of tumor ( region enveloped by yellow dotted line ) bearing mice with pai and mri / pet respectively . ( b ) the overlaying of ultrasonic ( gray ) and photoacoustic ( red ) imaging of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp ( 200 l of 10 m ) in living mice and their subtraction imaging . ( c ) the overlaying of representative decay - corrected coronal ( top ) and transaxial ( bottom ) small animal ct ( gray ) and pet ( color ) images of u87 mg tumors acquired at 2 , 4 , and 24 h after tail vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) . ( d ) mri images of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp ( 250 l of 200 m ) in living mouse . top row shows black and white images , and bottom row shows the pseudocolored images . uptake of cu - rgd - peg - mnp by u87 mg cells with or without blocking agent rgd at 1 , 2 , and 4 h are shown in figure 5a . cu - rgd - peg - mnp exhibited higher uptakes than blocking group at all the incubation time , with a value of 3.32 0.37% , 5.32 0.43% and 7.18 0.33% for cu - rgd - peg - mnp at 1 , 2 , and 4 h. in comparison , for cu - rgd - peg - mnp blocking group , much lower uptake of cu - rgd - peg - mnp was observed with a value of 2.00 0.15% , 3.16 0.20% and 3.48 0.29% at 1 , 2 , and 4 h , respectively , indicating the successful biomodification of mnps with rgd peptide and the specific targeting ability of rgd contribute to the uptake of cu - rgd - peg - mnp by u87 mg cells . the in vivo pet of mnps was performed in u87mg - tumor - bearing mice . cu - rgd - peg - mnp showed tumor accumulation and clear tumor contrast after 2 h postinjection ( figure 5b ) . quantification analysis revealed that the tumor uptake values of cu - rgd - peg - mnp gradually increased with time to 24 h , and they were 4.75 0.63 , 5.87 0.87 , and 5.93 0.89% id / g at 2 , 4 , and 24 h , respectively ( figure 5c ) . in addition to the tumor , moderate activity accumulation was observed in the liver ( e.g. , 15.78 2.55% id / g at 24 h for all mnps ) , and relative lower activity accumulation was also found in the kidneys ( e.g. , 5.34 0.62% id / g at 24 h for all mnps ) . these data indicated the mnp was cleared mainly through hepatobiliary system . to further investigate the possible targeting property of mnps , the cu radiolabeled rgd - peg - mnp and control cyclic arg - ala - asp - d - phe - cys [ c(radfc ) ] peptide ( abbreviated as rad , with nontargeting property for tumor v3 integrin ) functionalized peg - mnp ( cu - rad - peg - mnp ) for u87 mg tumor pet imaging the obvious stronger pet signal of cu - rgd - peg - mnp can be found in tumor at 4 h than that of cu - rad - peg - mnp ( p < 0.05 ) , indicating the good and specific targeting property of rgd - peg - mnp . to investigate the possibility of using mnp platform for multimodality imaging , mnp were mixed with fe and cu in sequence to form the multifunctional probes fe - rgd - peg - mnp and cu - fe - rgd - peg - mnp ( the amount of fe per mnp is 56 ) for pet / pai / mri . pet of mice bearing u87 mg tumors were then obtained first at 2 , 4 , and 24 h postinjection of cu - fe - rgd - peg - mnp . after 48 h , t1-weighted mri and pai of mice bearing u87 mg tumors were then obtained respectively at 4 h after another injection of large dose of fe - rgd - peg - mnp . in figure 6 , cu - fe - rgd - peg - mnp showed very similar pet , mri and pai properties on u87 mg tumor , compared with the corresponding single modality imaging from cu - rgd - peg - mnp , fe - rgd - peg - mnp , and rgd - peg - mnp respectively . these results showed that using mnp as the active platform to load cu and fe together can efficiently combine its native photoacoustic properties with radioactive and magnetic properties together for multimodality imaging . to change the lack of contrast properties of biomolecule - based nanoplatform for multimodality imaging , recently porphyrin were successfully introduced into phospholipid to provide the platform with desirable optical properties , while it still requires complicated and time - consuming chemical modifications and other reporting molecules to achieve multimodality imaging ability . we herein developed the functional biomarker , melanin , as a novel nanoplatform with its native optical property and multifunctions , which can simply and actively collecting optical , magnetic and radioactive properties together for multimodality imaging . melanin , the oxidation products of tyrosine , plays an important role in living organism . accompanied with the development of molecular imaging probes in the past decade , melanin has been used as an effective molecular target as well as endogenous contrast agent for pai because of its strong light absorption properties . besides , melanin has intrinsic strong chelating properties to many metal ions including cu and fe , which can be used to nuclear imaging and mri . consequently melanotic melanomas show hyperintensity on t1-weighted mri images . considering the attractive properties of the biomarker melanin , we and others have engineered cancer cells to biologically produce melanin for multimodality imaging ( pai / mri / pet ) of cancer . however , this method requires genetic modification of cells , which is time - consuming and may have limited clinical value . thus , water - soluble mnps are more appropriate to behave as a natural active platform to simplify the preparation procedure for multimodal applications . considering only trace amount of cu ions utilized for pet and its final decay to zn ions , which is necessary for life process , and the abundant amount of fe ions in living body , cu and fe ions used in our system are expected to be metabolized in living subjects . in comparison , although gd ion has higher t1 mri effect than fe ion , its potential toxicity is still a problem . concomitantly , the traditional nanoparticle - based platform needs complicated functionalization of ligand to chelate gd for mri and cu for pet . more interestingly , the new mnp system can serve as an active nanoplatform and easily bind with metal ions without the traditional needs of surface modification and introducing chelating groups , which significantly simplifies the preparation process and reduces the heterogeneity of the resulting multimodal nps . furthermore , the mnp is an organic and biodegradable material and showed relatively good tumor imaging properties . all of these properties make mnps highly promising for potential clinical translation . despite its important functions , developing melanin for molecular imaging therefore , preparing mnps is desired while still a challenge for well - dispersing in water , especially for those with size around 10 nm that can provide not only appropriate blood circulation time , but also high surface - to - volume ratio to chelate enough metal ions for efficient bioimaging . although the formation mechanism of polymeric melanin is not clear , its molecular structure is generally considered to be composed of dihydroxyindole / indolequinone segments with hydrophobic conjugated main chain having strong interaction and hydrophilic hydroxyl groups on the benzene rings . therefore , to realize melanin water - soluble at neutral environments , decreasing the interchain aggregation of conjugated main chains and lowering the formed melanin particle size to expose more hydrophilic hydroxy groups on the surface of melanin is a promising way . in our work , we first realized the synthesis of ultrasmall mnps in water with high monodispersity and homogeneity under the assistance of sonication . another problem that should be resolved is the metal ion - initiated cross - linking and the formation of mnp precipitation . recent reports showed that fe is a strong cross - linker for catechol groups , which is one of the components in melanin molecular structure . in our work , peg encapsulation is found can not only enhance the biocompatibility and the water - solubility , but also efficiently prevent the formation of metal ion - initiated precipitation . investigation on the in vivo subcutaneous phantom showed that the signals between mri and pai had good linear relationship ( figure s12 ) . in addition , the slope of enhanced pa signal with different concentrations was higher than that of mri signal , indicating the pai imaging of mnp was more sensitive than the mri imaging . moreover , because of their easy conjugation with targeting groups , the mnps modified with rgd exhibited the tumor targeting ability to the v3 integrin overexpressed on the surface of tumor vasculatures and the u87 mg tumor cells ( from pet data ) , which afforded rgd - conjugated mnps higher accumulation capability in tumor than those rad - modified mnps through enhanced permeability and retention ( epr ) effect . this further indicated that mnps can conveniently function as a good nanoplatform for targeted imaging . though it is difficult to compare the imaging effects of our platform with other type of nanoplatforms ( considering the lack of the triplemodality analogues ) , we demonstrated the unique ability of mnp nanoplatform to combine pet , photoacoustic and mr imaging modalities together to get complementary information for tumor imaging . for example , pet efficiently provides the in vivo pharmacokinetics and biodistribution of the nanoprobes and can also provide physiological information on disease with whole body imaging capability , but it can not image tissues at high spatial resolution ; pai provides functional and molecular information on the tumor with high spatial resolution . it is a cheap and convenient way to image tumor in real time but suffers from limited tissue penetration ability , which can be compensated by pet . mri provides high spatial resolution image and anatomical information on disease but generally lacks of molecular imaging capability . combination of pet / pai / mri thus allows us to image diseases at different depths with molecular and anatomical information . recent developments showed that multimodality imaging is promising not only for accurate tumor imaging but also for guiding tumor resection . for example , pet / mri , which combines the exquisite anatomical information by mri with the extreme sensitivity of pet , can be used for whole - body imaging and deep tumor localization . mri / fluorescence imaging ( fi ) is appropriate for guiding superficial tumor resection . in comparison , combining pet / mri / pai together is anticipated to help for guiding both superficial and deep tumor surgery . to our knowledge , our triple - modality mnp system can be first used for pet and mri to obtain the detailed information on tumor for surgical planning in presurgery . pai can then be used to localize the superficial and relatively deep tumors in surgery for helping the tumor resection . overall , a reliable method for preparation of water - soluble mnp has been developed in our work , which lays down a foundation for its future biomedical applications . it can be easily envisioned that mnp can serve as a nanoplatform not only for molecular imaging but also for theranostics . considering the abundant functionalities of melanin , such as binding drugs , mnp - based platform used for drug delivery and therapy are now being investigated . in conclusion , we report mnp as the natural biomarker - transferred active platform for multimodality imaging . mnp is of particular interest because such an endogenous agent with native photoacoustic signals and strong chelating properties with metal ions can act as an active platform to simplify the assembling of different imaging moieties . mnp can be easily modified with biomolecules for targeted tumor multimodality imaging , and it showed good in vivo tumor imaging properties . we expect this work will stimulate further studies of multifunctional endogenous material as nanoplatforms for potential imaging and therapeutic applications . the following reagents were acquired and used as received : melanin ( sigma - aldrich ) , sodium hydroxide ( sigma - aldrich ) , hydrochloric acid ( 37 wt % , sigma - aldrich ) , nh4oh solution ( 28 wt % , sigma - aldrich ) , amine - peg5000-amine ( nh2-peg5000-nh2 , 5 kda , laysan bio ) , dimethylthiazolyl - diphenyltetrazolium ( mtt ; biotium ) , phosphate buffered saline ( pbs , gibco ) , and agarose ( invitrogen ) . tyrosine - derived synthetic melanin ( 20 mg ) was first dissolved in 10 ml of 0.1 n naoh aqueous solution under vigorous stirring . after dissolving , hcl aqueous solution ( 0.1 n ) was swiftly dropped into the obtained basic melanin solution to adjust the ph to 7.0 under sonication with output power = 10 w for 1 min . the neutralized solution was further centrifuged with a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and washed with deionized water and repeated several times to remove the produced nacl . the aqueous solvent was removed by freeze - drying to obtain 15 mg black solid of pws - mnp . nh4oh solution ( 28 wt % ) was added to 5 ml of pws - mnp aqueous solution ( 1 mg / ml of water ) to adjust the ph of the solution to 9 . this mixed solution was added dropwise into nh2-peg5000-nh2 ( 5 , 10 , 25 , and 50 mg ) aqueous solution with ph = 9 . after vigorous stirring for 12 h , peg - modified mnp was retrieved by centrifugation with a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and washed with deionized water several times by redispersion / centrifugation processes to remove the unreacted nh2-peg5000-nh2 . the aqueous solvent was removed by freeze - drying and the obtained peg - mnp was weighed to preliminary calculate the quantity of the peg attached on mnps . because of the existence of one nh2 group per peg chain on the surface of mnp , we then accurately determined the nh2 group on mnp with fluorescamine by spectrofluorometer to calculate the amount of peg on the nanoparticles ( using ethamine as the standard ) . the cross - linker solution was prepared freshly . the 4-(n - maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo - n - hydroxysuccinimide ester sodium salt ( sulfo - smcc ) ( 1.2 mg ) the water - soluble peg - mnp [ 1 mg in 1 ml of pbs ( ph = 7.2 ) ] was incubated with the above cross - linker solution for 2 h at room temperature . the resultant thiol - active mnp ran through a pd-10 column prewashed with pbs ( ph = 7.2 , 10 mm ) to remove the excessive sulfo - smcc and byproducts . the purified mnp was concentrated to the final volume of 0.5 ml with a centrifugal - filter ( mwco = 30 kda ) . the crgdfc stock solution ( 120 l of 5 mm in the degassed water ) was added to the above mnp solution with stirring . the uncoupled rgd peptide was removed through a pd-10 column and collected to analyze its quantity through hplc . the resultant product , rgd - peg - mnp , were concentrated by a centrifugal - filter ( mwco = 30 kda ) and stored at 4 c for one month without losing targeting activity . the final rgd - peg - mnp were reconstituted in pbs and filtered through a 0.22 m filter for cell and animal experiments . the mnp ( 1 mg in 1 ml h2o ) was labeled with fe or cu by addition of 20 l of fresh fecl3 ( 10 mg / ml ) in pbs ( ph = 7.4 ) or 20 l of cucl2 ( 10 mg / ml ) in buffer solution of ph = 5.5 followed by a 1 h incubation at 40 c ( the chelating mechanisms of mnps were shown in figure s13 ) . the products were washed out by pbs and passed through a 0.22-m millipore filter into a sterile vial for in vitro and animal experiments . the fe and cu concentrations of mnps were measured by inductively coupled plasma - mass spectrometry ( icp - ms ) analysis . the stability of metal ion - chelated mnps were studied by incubating those mnps in pbs ( ph = 7.4 ) at 37 c . those mnps were placed in dialysis tube ( mwco 10k ) with magnetic stirring , dialysis against 10 ml pbs . at a certain time , dialysate was removed for icp - ms analysis and replaced with fresh pbs solution . for icp - ms analysis , 100 l of the detected sample was first heated to evaporate the water solvent and then digested with 0.5 ml of concentrated nitric acid ( 70% w / w ) under heating . after the solvent was evaporated , the residue was then dissolved in 7 ml of dilute nitric acid ( 2% w / w ) for final icp - ms analysis . ft - ir spectra were measured in a transmission mode on a bio - rad ft - ir spectrophotometer ( model fts135 ) under ambient conditions . samples of pristine melanin granules and functionalized mnps were ground with kbr and then compressed into pellets . transmission electron microscopy ( tem ) images were recorded on a jeol 2010 transmission electron microscope at an accelerating voltage of 100 kv . the tem specimens were made by placing a drop of the nanoparticle aqueous solution on a carbon - coated copper grid . the hydrodynamic sizes of the mnps were determined by dynamic light scattering ( dls ) using a 90 plus particle size analyzer ( malvern , zetasizer nano zs90 ) . zeta potentials were measured using a zeta potential analyzer ( malvern , zetasizer nano zs90 ) . the h nmr spectra were recorded at 20 c on a 400 mhz nmr spectrometer ( bruker ) , using d2o as solvent . the mnps with or without fe were further radiolabeled with cu by addition of 11.5 mci of cucl2 in 0.1 n naoac ( ph 5.5 ) buffer followed by 1 h incubation at 40 c . the radiolabeled mnps were then purified by a pd-10 column ( ge healthcare , piscataway , nj , usa ) . the product was washed out by pbs and passed through a 0.22-m millipore filter into a sterile vial for in vitro and animal experiments . the investigation of the radiolabeling stability of mnps is similar to the metal ion - chelated mnps except that the detector icp - ms was replaced by perkinelmer 1470 automatic gamma - counter for counting radioactivity . in vitro cytotoxicity of mnps was determined in nih-3t3 and u87 mg cells by the mtt assay . nih-3t3 and u87 mg cells were incubated on 96-well plate in dmem medium containing 10% fbs and 1% penicillin / streptomycin at 37 c in 5% co2 humidified atmosphere for 24 h and 0.5 10 cells were seeded per well . cells were then cultured in the medium supplemented with indicated doses of different mnps for 24 h. the final concentrations of mnps in the culture medium were fixed at 3.125 , 6.25 , 12.5 , and 25 m in the experiment . addition of 10 l of mtt ( 0.5 mg / ml ) solution to each well and incubation for 3 h at 37 c was followed to produce formazan crystals . then , the supernatant was removed and the products were lysed with 200 l of dmso . the absorbance value was recorded at 590 nm using a microplate reader . the absorbance of the untreated cells was used as a control and its absorbance was as the reference value for calculating 100% cellular viability . u87 mg cells ( 1 10 per well ) were seeded in 12-well tissue culture plates and allowed to attach overnight . the cells were washed twice with serum - free dmem and incubated with the cu - labeled mnps ( 2 ci per well , final concentration approximately 6 nm ) in 400 l of serum - free dmem at 37 c . the specific binding of the probes with u87 mg cells was determined by coincubation with rgd ( 30 g per well ) . after 1 , 2 , and 4 h , the cells were washed three times with cold pbs and lysed with the addition of 200 l of 0.2 m naoh . the radioactivity of all fractions was counted using a perkinelmer 1470 automatic gamma - counter . the uptake ( counts per minute ) was expressed as the percentage of added radioactivity . for studying the pai properties of mnps , different concentrations of mnps aqueous solutions ranging from 0.625 to 20 m were filled into polyethylene capillaries and then the capillaries were laid on the surface of solidified agarose gel . the capillaries were further covered with thin 1% agarose gel to make the surface smooth . for the particle s sensitivity in living body , mnps aqueous solutions with different concentrations from 5 m to 80 m were mixed with matrigel at 0 c and then subcutaneously injected on the lower back of mice . toronto , canada ) with a laser at excitation wavelength of 680 nm and a focal depth of 10 mm was used to acquire photoacoustic and ultrasound images . mri experiments were performed at 25 c in a magnetic resonance ( mr ) scanner ( siemens 1.0 t ) . to simulate the biological environment , agarose gel , prepared in 300 l of the pcr tube using secondary distilled water as the solvent for dissolving the agarose , was used to demonstrate the magnetic signal . the bottom of the tube was first covered with a layer of 1% agarose gel . when agarose gel was cooled , the mixtures of mnps and aqueous solution of agarose ( ratio 1:1 by volume ) with iron concentrations at 62.5 , 125 , 250 , 500 , and 1000 m fe ( amount to 1.25 , 2.5 , 5 , 10 , 20 m mnp ) , were then filled into the intermediate portion of the pcr tube respectively while the sample was hot . after cooling , another 1% agarose gel was covered on the top layer of the cube . the tubes were placed into the mr scanner and a number of mr sequences were run , spin echo for r1 determination ( tr : 503000 ms ; te : 5.5 ms , flip angle 30 ; fov : 3.5 cm , matrix : 256 256 ; slice thickness : 1 mm ) . the luminance values of the resulting image were obtained through the imagej software processing , thereby calculating the r1 value . for measurement the mnps detection sensitivity in living subject , fe - chelated mnps aqueous solution with different concentrations from 1.25 m to 20 m were mixed with matrigel at 0 c and then subcutaneous injected on the lower back of mice . tr : 700 ms , te : 5.2 ms ; fov : 3.5 cm , matrix : 256 256 ; slice thickness : 1 mm . all animal experiments were performed in compliance with the guidelines for the care and use of research animals established by the stanford university animal studies committee . female athymic nude mice ( nu / nu ) in 46 weeks old were obtained from the charles river laboratories ( boston , ma , usa ) and kept under sterile conditions . u87 mg cells suspended in 100 l of pbs were inoculated subcutaneously in the shoulder of nude mice . when the tumors reached 0.50.8 cm in diameter , the tumor bearing mice were subjected to in vivo multimodality imaging ( pai , mri and pet ) and biodistribution studies . mice bearing tumor ( u87 mg ) were anesthetized with 2% isoflurane in oxygen and placed with lateral position . mri was performed using the same instrument , protocols and conditions as in the phantom mri study . pai was carried out using the same vevo lazr pai system as the in vitro study . quantification analysis of pa and mr signals was performed on the pai and mri images . because the enhanced signals of mri and pai in tumors were dispersed heterogeneously , the enhanced signal regions in tumors on the pai and mri images were used as rois to analysis the signal change with injection time ( comparing the signal intensity at 4 h injection with 0 h injection ) . small animal pet imaging of tumor - bearing mice was performed on a siemens inveon micropet - ct . mice bearing u87-mg tumors were injected with cu - labeled mnps ( 110.0 5.0 ci ) via the tail vein . at different times after injection ( 2 , 4 , and 24 h ) , the mice were anesthetized with 2% isoflurane and placed prone near the center of the fov of the scanner . all the small animal pet images were reconstructed using irw4.0 software by two - dimensional ordered - subsets expectation maximization ( osem ) algorithm . the radioactivity uptake in the tumor and normal tissues was calculated using a region of interest ( roi ) drawn over the whole organ region and expressed as a percentage of the injected radioactive dose per gram of tissue ( % id / g ) .

developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging . here , we report the successful transfer of an important molecular target , melanin , into a novel multimodality imaging nanoplatform . melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging . in our work , the multifunctional biopolymer nanoplatform based on ultrasmall ( < 10 nm ) water - soluble melanin nanoparticle ( mnp ) was developed and showed unique photoacoustic property and natural binding ability with metal ions ( for example , 64cu2 + , fe3 + ) . therefore , mnp can serve not only as a photoacoustic contrast agent , but also as a nanoplatform for positron emission tomography ( pet ) and magnetic resonance imaging ( mri ) . traditional passive nanoplatforms require complicated and time - consuming processes for prebuilding reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity . in comparison , utilizing functional biomarker melanin can greatly simplify the building process . we further conjugated v3 integrins , cyclic c(rgdfc ) peptide , to mnps to allow for u87 mg tumor accumulation due to its targeting property combined with the enhanced permeability and retention ( epr ) effect . the multimodal properties of mnps demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation .

Introduction Results Discussion Conclusion Materials and Methods

male landrace pigs ( 19 to 26 kg ) were pre - dosed orally with aspirin and clopidogrel 24 h before surgery , and they were maintained on this dual antiplatelet therapy throughout the study to reduce the risk of in - stent thrombosis . vascular access was obtained by femoral artery cutdown and the insertion of a 6-f transradial sheath ( arrow international , reading , pennsylvania ) . coronary angiography was performed before the deployment of either bms ( gazelle , biosensors europe sa , morges , switzerland ) or biolimus a9 eluting stents ( biomatrix flex , biosensors europe sa ) to achieve a target ratio of stent to artery diameter of 1.2:1 . animals were euthanized after 7 or 28 days . in the murine model , a stainless steel stent ( 5-cell , 2.5 mm 0.8 mm ; strut thickness 0.06 mm ; cambus medical , galway , ireland ) was crimped onto a 1.20-mm 8-mm mini - trek balloon angioplasty catheter ( abbott vascular , abbott park , illinois ) and deployed ( 10 atm for 30 s ) into the thoracic aorta before engraftment into a recipient mouse . mice were allowed to recover in heated chambers for 24 h and were returned to normal housing conditions , where they were maintained on aspirin - supplemented water and a normal chow diet for another 28 days before being killed . murine aortas were harvested from male mice 8 to 12 weeks of age , and vascular smcs were isolated and cultured . bone marrow derived macrophages ( bmdm ) were generated and bone marrow cells isolated from femurs and tibiae of wild - type ( wt ) and mir-21 knockout ( ko ) mice . flow cytometry was performed by using a bd facscanto ii or lsr ii ( bd biosciences , san jose , california ) . global profiling for mirnas in the control unstented arteries and stented porcine coronary arteries was performed with mirna quantification by using real - time polymerase chain reaction . for analysis , fold changes for each mirna were normalized to u6 because this mirna was the most suitable endogenous mirna in porcine tissue . data analysis was performed by using sds software version 2.3 ( applied biosystems , carlsbad , california ) , and the baseline and threshold were automatically set . data were normalized and then analyzed to identify mirnas differentially expressed between the control ( unstented ) arteries and arteries subjected to stenting for 7 or 28 days . visual assessment was used to check for any lack of normality ; because there was no evidence of this , 1-way analysis of variance followed by a tukey multiple comparison test ( for comparison of > 2 groups ) or student t test ( for comparison of 2 groups ) were conducted . for all the quantitative polymerase chain reaction experiments , values are expressed as fold - change . all statistical analyses were conducted by using prism version 4 ( graphpad software , san diego , california ) . comparisons of in vitro smc proliferation and migration were performed by using 2-way analysis of variance and bonferroni post - hoc tests . all animal procedures were performed in accordance with the united kingdom home office guidance on the operation of the animals ( scientific procedures ) act 1986 and institutional ethical approval . the effects of bms and des on isr were assessed in pig coronary arteries by using optical coherence tomography imaging ( figures 1a to 1d ) . the des reduced neointima formation by 33% compared with bms at 28 days ( figure 1a ) , leading to a significantly larger luminal area ( figure 1b ) with no difference in stent expansion ( figure 1c ) . 116 mirnas were differentially regulated in the bms group , with 23 mirnas remaining dysregulated at 28 days . at 7 days , multiple mirnas associated with inflammatory cell infiltration and activation were altered ( online table 1 , online figure 1 ) . of note , mir-21 - 3p was substantially up - regulated in both bms- and des - treated arteries at 7 days , suggesting that the mir-21 stem loop ( i.e. , both lead and passenger strands ) may be important post - stenting . the expression profile of mir-21 - 3p and mir-21 - 5p were validated , and this revealed that mir-21 - 5p was significantly up - regulated in stented arteries at 7 days regardless of stent type and remained up - regulated at 28 days compared with unstented control arteries ( figure 2a ) . mir-21 - 3p was also up - regulated at 7 and 28 days post - stenting in both groups ( figure 2b ) . we then identified the localization of expression of the mir-21 axis in the vessel wall after stenting , focusing on mir-21 - 5p ( because the absolute levels of mir-21 - 3p are lower and below the detection threshold for in situ hybridization ) . in control coronary arteries , mir-21 - 5p was detected within the media ( figure 2c ) . in injured vessels 28 days post - stenting , increased signal intensity was observed in the medial layer and developing neointima , particularly around stent struts . to determine if this expression pattern is maintained in the clinical setting , in situ hybridization was performed in human coronary arteries 14 days post - stenting from tissue obtained from a heart transplant patient . in concordance with the staining pattern in porcine arteries , abundant mir-21 expression was observed in the developing neointima and infiltrating inflammatory cells ( figure 2d , online figure 2 ) . taken together , these findings suggest that mir-21 - 5p and mir-21 - 3p may be important in the development of post - stent pathophysiological responses to injury . hybridization confirmed abundant mir-21 - 5p staining around the stent struts and developing neointima ( figure 3 ) , consistent with the porcine data . morphometric analysis found that mir-21 ko mice stent grafts had a reduced neointimal area , neointimal thickness , and neointima - to - medial ratio compared with wt controls ( 1.37 0.18 [ n = 8 ] vs. 2.11 0.17 [ n = 9 ] ; p < 0.05 ) ( figures 4a and 4b , online table 2 ) . measurements of strut depth were concordant with greater strut depth in wt - stented grafts than in ko - stented grafts . a significant reduction in luminal area in wt mice compared with mir-21 ko mice was also observed ( figures 4c and 4d , online table 2 ) . there were no apparent differences in luminal , media , or total vessel area between wt and mir-21 ko mice at baseline ( before stent injury ) , although an increased sample size would be needed to confirm this observation ( online table 3 ) . furthermore , the total vessel area did not differ significantly at 28 days ( figure 4e ) , and no differences were observed in unstented vessels at baseline ( online table 3 ) . the neointimal lesions from mir-21 ko mice contained significantly less -actin positive smcs ( 28 2.4% [ n = 8 ] vs. 14 3.7% [ n = 9 ] ; p < lesions in wt mice contained 50% more elastin than in ko mice ( figures 5c and 5d , online table 2 ) . no difference in cell proliferation was observed after quantification of cells in the neointima ( figures 5e and 5f , online table 2 ) . we assessed vessel reendothelialization and , importantly , found no significant difference between mir-21 ko and wt mice ( 91.0 3.9% vs. 88.7 2.9% , respectively ) ( figures 5 g and 5h ) . no differences were observed at baseline ( i.e. , unstented vessels ) ( online table 3 ) . to explore the mechanisms responsible for the reduction in neointima formation in mir-21 ko mice , proliferation and wound healing assays were performed . the proliferative response of mir-21 ko smc was dramatically attenuated in response to platelet - derived growth factor ( pdgf ) ( figure 6a ) , as was migration in response to pdgf ( figures 6b and 6c ) . to identify potential targets responsible for these effects , we stimulated aortic smc from mir-21 wt and ko mice with pdgf ( n = 5 ) to elevate mir-21 levels ( 3.03 0.23-fold vs. controls ; p < the messenger rna levels of known mir-21 targets with defined roles in smc proliferation and migration were profiled . the expression of programmed cell death protein 4 ( pdcd4 ) and signal transducer and activator of transcription 3 were significantly reduced in aortic smc from wt mice after pdgf stimulation . these changes were not observed in mir-21 ko cells stimulated with pdgf ( figures 6e and 6f ) ; however , further experiments are required to determine whether repression of these genes is direct or indirect via mir-21 regulation in this setting . mir-21 ko mice contained greater numbers of galactin-3 + ( mac-2 ) macrophages in the neointima compared with wt mice ( 0.79 0.23% vs. 2.54 0.75% ; p < 0.05 ) ( figures 7a and 7b ) and enhanced levels of ym-1positive macrophages , a validated murine marker of the alternatively activated ( m2 ) macrophage ( 1.93 0.54% vs. 0.50 0.21% ; p < 0.01 ) ( figures 7c and 7d ) . thus , loss of mir-21 results in altered inflammatory cell phenotype within injured vessels . to investigate whether these effects were derived from any hematological defect , before cell recruitment to the vessel wall , the populations of immune cells in both bone marrow and blood of wt and mir-21 ko mice were examined ( 16 ) . in bone marrow , the percentage of cluster of differentiation ( cd ) 3 + t cells was significantly reduced in mir-21 ko mice , but neutrophils , monocytes , b cells , cd4 + , and cd8 + t cells did not differ ( online figures 3a and 3b ) . in blood , mir-21 ko mice exhibited a significantly reduced percentage of circulating ly6c+hi monocytes and cd3 + t cells ( figures 7e and 7f ) , potentially indicating a reduced capacity to develop proinflammatory responses . to investigate whether the absence of mir-21 leads to altered macrophage differentiation , bmdms from wt and mir-21 ko mice were generated and treated with either lipopolysaccharide ( lps ) or interleukin ( il)-4 in vitro to induce m1 and m2 polarization , respectively . both lps and il-4 significantly up - regulated the expression of mir-21 - 3p and mir-21 - 5p in wt macrophages ( 3.00 0.23-fold , 4.38 0.91-fold , 4.74 0.13-fold , and 4.88 0.7-fold , respectively ; p < 0.05 ) , indicating that inflammatory mediators can modulate the expression of mir-21 ( figure 8a ) . at baseline , unstimulated ( m0 , nonpolarized ) mir-21 ko macrophages had significantly higher levels of peroxisome proliferator activated receptor ( ppar)- expression ( m2 polarization marker ) than wt cells ( p < 0.01 ) ( figure 8b ) . after activation with lps , expression of nitric oxide synthase ( an m1 marker ) was significantly reduced in mir-21 ko versus wt macrophages ( figure 8c ) . in addition , the ratio of messenger rna expression of nitric oxide synthase , compared with arginase 1 ( an m2 marker ) , was significantly higher in wt than mir-21 ko macrophages treated with lps ( 20.6 6.52-fold vs. 2.7 1.24-fold ; p < 0.001 ) ( figure 8d ) . flow cytometric analysis demonstrated that after activation with lps , cd69 was significantly reduced in mir-21 ko versus wt lps - treated macrophages ( 1.8-fold ; p < 0.05 ) ( figure 8e ) . however , all other markers examined by using flow cytometry ( major histocompatibility complex - ii , cd11c , cd86 , cd206 , and toll - like receptor-2 ) were not significantly different in either lps- or il-4treated wt or mir-21 ko bmdm ( online figure 4 ) . the levels of several proinflammatory mediators were significantly reduced from lps - stimulated macrophages of mir-21 ko compared with wt mice : il-1 , il-1 , il-6 , il-12 , tumor necrosis factor- , and macrophage inflammatory protein-1 ( figure 8f ) . in addition , the il-12/il-10 ratio for lps - stimulated mir-21 macrophages was almost double that of ko macrophages ( 24 vs. 13 ) . to address whether mir-21 ko macrophages had a reduced capacity to infiltrate and migrate into vascular lesions after injury , we studied bmdm migration through matrigel - coated transwell inserts containing 8-m pores ( sigma , united kingdom ) . these experiments showed that both bmdm isolated from wt and mir-21 ko mice had the same capacity to migrate through a matrix membrane in response to monocyte chemoattractant protein-1 ( figure 8 g ) . we report for the first time mirna patterns associated with delayed arterial healing and neointima formation after stenting ( central illustration ) . of particular interest , the stem loop of mir-21 , including lead ( mir-21 - 5p ) and passenger ( mir-21 - 3p ) strands , was up - regulated in both the bms and des groups compared with controls . further experiments highlighted that loss of the mir-21 stem loop in ko mice blocked neointimal formation through effects on smc proliferation and migration , macrophage polarization , and inflammatory activation . a causal network analysis was performed of mir-21 - 3p and mir-21 - 5p targets to determine whether these mirnas affect the inflammatory networks associated with vascular injury . despite a relatively small overlap of predicted genes , pathways associated with tumor necrosis factor- and il-1 were 1 of the top - ranking regulatory networks ( online figure 5 ) ; this finding may be relevant because both of these cytokines play prominent roles in neointima formation ( 17,18 ) . mir-21 ko mice exhibited reduced smc deposition , neointima formation , and an altered inflammatory phenotype , resulting in enhanced levels of anti - inflammatory m2 macrophages in response to vascular injury and stenting , with no effect on endothelial regeneration . subsequent profiling of immune cell populations in the blood of mir-21 ko mice demonstrated reduced numbers of ly6c+hi cells , a cell type that can differentiate into m1 macrophages after tissue infiltration . this finding suggests that mir-21 ko mice have a reduced capacity to develop an m1 inflammatory phenotype in response to injury . we also found reduced total cd3 + t - cell counts in the bone marrow and blood of the mir-21 ko . recently , it has been reported that mir-21 can modulate t - cell responses , including alterations in cytokine production and apoptosis rates ( 1921 ) . t cells are already known to contribute to the inflammatory response after coronary artery stenting ( 22 ) ; thus , the absence of mir-21 in t cells likely contributes to the altered inflammatory responses and reduced in - stent stenosis seen in mir-21 ko . in support of the altered inflammatory responses in vivo , our bmdm experiments revealed that mir-21 ko macrophages contain enhanced baseline levels of ppar- , a well - characterized m2 macrophage marker , and a reduced ratio of nitric oxide synthase / arginase 1 ( m1/m2 markers ) . these findings are particularly relevant in the setting of inflammatory vascular disease because numerous studies suggest that ppar- activation can curtail the inflammatory response . activation of ppar- in human macrophages reduces matrix metallopeptidase 9 activity and inhibits expression of il-1 , il-6 , and tumor necrosis factor- ( 2325 ) . furthermore , several groups have reported that ppar- agonists inhibit atherosclerosis development and reduce inflammatory markers in apolipoprotein e ko mice ( 26,27 ) . furthermore , mir-21 ko macrophages exhibited a reduced capacity to secrete proinflammatory mediators such as il-1 , tumor necrosis factor- , macrophage inflammatory protein-1 , il-6 , and il-12 in response to lps , a substance known to stimulate the inflammatory m1 macrophage phenotype . however , a defective response to lps does not necessarily mean altered polarization and may simply reflect , for example , defective cd14 expression . further phenotyping of surface receptors from bmdm also showed reduced expression of cd69 in mir-21 ko mice . a cd69 deficiency may contribute to reduced inflammatory cytokine secretion , as previous studies have reported that cd69 activation mediates numerous inflammatory processes such as nitric oxide production and release of tumor necrosis factor- from murine macrophages and t cells ( 28,29 ) . thus , loss of mir-21 may accelerate wound healing and resolution of the inflammatory response after vascular injury and stenting , events that could reduce the incidence of late stent thrombosis . it is important to note that we used human arrays in the porcine samples to identify mirnas that would extrapolate to the pathology in the clinical setting . it is possible that a proportion of mirnas may be underrepresented due to the sequence variation or chromosomal locations between pig and human . we identified an almost 20-fold up - regulation of mir-21 - 3p after stenting with bms and des . our subsequent experiments in bmdm demonstrated that both mir-21 - 3p and mir-21 - 5p were up - regulated in response to lps , which induces classical macrophage polarization ( m1 ) . in combination with reduced inflammatory cytokine release from mir-21 ko macrophages , these results suggest that mir-21 - 3p and mir-21 - 5p may both play a pathological role in macrophage activation in response to inflammatory stimuli . we observed that mir-21 plays a prominent role in smc proliferation and migration in response to vascular injury , consistent with other studies ( 30,31 ) . aortic smc isolated from mir-21 ko mice had a reduced capacity to migrate and proliferate in response to pdgf , an important observation because novel des must retain the antiproliferative effect on smc accumulation to maintain their clinical efficacy . these findings are consistent with previous reports demonstrating that pharmacological or genetic knockdown of mir-21 reduces smc proliferation and neointima formation after balloon injury or vein grafting ( 3033 ) . several of these reports suggest that mir-21 mediates a beneficial effect on smc proliferation and neointima , at least in part , via inhibition of phosphatase and tensin homolog . we also found that the levels of pdcd4 were suppressed after pdgf exposure in wt mice . importantly , the levels of pdcd4 were increased in mir-21 ko mice , suggesting that pdcd4 is modulated after exposure to pdgf and plays a role in smc proliferation and migration , effects repressed in mir-21 ko smcs . in concordance with these findings , previous studies suggest that pdcd4 is down - regulated after vascular injury , and overexpression of pdcd4 with adenoviral vectors increases apoptosis and reduces smc proliferation ( 34 ) . although the present study demonstrates that mir-21 play a prominent role in the pathology of in - stent restenosis , it is important to note that these studies are based on data from pre - clinical animal models of restenosis . although our results in mir-21 knockout mice demonstrate that loss of mir-21 reduces in - stent restenosis and inflammatory cell function , it is important to note that these defects are present before vascular injury , these deficiencies may alter the response to injury in these mice . in order to demonstrate that these finding have efficacy in the clinic , further studies are required to demonstrate that pharmacological knockdown of mir-21 or mir-21 targets can inhibit neointimal formation and vessel inflammation from current drug - eluting stent platforms . furthermore , detailed pharmacokinetic profiling would be needed to demonstrate an effect elution profile of antimir-21 therapy , without any off - target effect . although the present study demonstrates that mir-21 play a prominent role in the pathology of in - stent restenosis , it is important to note that these studies are based on data from pre - clinical animal models of restenosis . although our results in mir-21 knockout mice demonstrate that loss of mir-21 reduces in - stent restenosis and inflammatory cell function , it is important to note that these defects are present before vascular injury , these deficiencies may alter the response to injury in these mice . in order to demonstrate that these finding have efficacy in the clinic , further studies are required to demonstrate that pharmacological knockdown of mir-21 or mir-21 targets can inhibit neointimal formation and vessel inflammation from current drug - eluting stent platforms . furthermore , detailed pharmacokinetic profiling would be needed to demonstrate an effect elution profile of antimir-21 therapy , without any off - target effect . the mir-21 stem loop plays an important role in smc and macrophage activation after vascular injury . our findings in the murine model of isr revealed that loss of mir-21 attenuates neointima formation and macrophage activation , resulting in a less inflammatory phenotype . these findings suggest that mir-21 modulation could enhance wound healing and resolve the inflammatory response , effects that could improve the clinical efficacy of currently available des . the mir-21 axis warrants further investigation as a therapeutic target in the setting of stent - induced inflammatory vascular disease . lineage - restricted knockouts maybe required to unravel the complex role of mir-21 in this setting.perspectivescompetency in patient care : des reduce in - stent restenosis after percutaneous coronary intervention but are associated with a greater risk of stent thrombosis due to delayed arterial healing that is characterized histologically by incomplete reendothelialization and persistent fibrin and inflammatory cell deposition . an array of microrna molecules are involved in the inflammatory processes driving the cellular response to vascular injury , and genetic ko of the mir-21 stem loop attenuates neointimal formation after arterial stenting in mice.translational outlook : future investigations should seek to determine whether local antimir to knockdown mir-21 levels delivered directly from drug - eluting stents could reduce vessel inflammation and neointima formation and reduce the risk of both restenosis and stent thrombosis . competency in patient care : des reduce in - stent restenosis after percutaneous coronary intervention but are associated with a greater risk of stent thrombosis due to delayed arterial healing that is characterized histologically by incomplete reendothelialization and persistent fibrin and inflammatory cell deposition . an array of microrna molecules are involved in the inflammatory processes driving the cellular response to vascular injury , and genetic ko of the mir-21 stem loop attenuates neointimal formation after arterial stenting in mice . translational outlook : future investigations should seek to determine whether local antimir to knockdown mir-21 levels delivered directly from drug - eluting stents could reduce vessel inflammation and neointima formation and reduce the risk of both restenosis and stent thrombosis .

backgrounddrug - eluting stents reduce the incidence of in - stent restenosis , but they result in delayed arterial healing and are associated with a chronic inflammatory response and hypersensitivity reactions . identifying novel interventions to enhance wound healing and reduce the inflammatory response may improve long - term clinical outcomes . micro ribonucleic acids ( mirnas ) are noncoding small ribonucleic acids that play a prominent role in the initiation and resolution of inflammation after vascular injury.objectivesthis study sought to identify mirna regulation and function after implantation of bare - metal and drug - eluting stents.methodspig , mouse , and in vitro models were used to investigate the role of mirna in in - stent restenosis.resultswe documented a subset of inflammatory mirnas activated after stenting in pigs , including the mir-21 stem loop mirnas . genetic ablation of the mir-21 stem loop attenuated neointimal formation in mice post - stenting . this occurred via enhanced levels of anti - inflammatory m2 macrophages coupled with an impaired sensitivity of smooth muscle cells to respond to vascular activation.conclusionsmir-21 plays a prominent role in promoting vascular inflammation and remodeling after stent injury . mirna - mediated modulation of the inflammatory response post - stenting may have therapeutic potential to accelerate wound healing and enhance the clinical efficacy of stenting .

Methods Results Discussion Study limitations Conclusions Appendix

atypical central neurocytomas are rare central nervous system tumors in the pediatric population [ 1 , 2 ] . they are treated unsuccessfully with gross total resection and require consolidative radiation therapy or chemotherapy to prevent recurrence [ 35 ] . the atypical nature and aggressive behavior of the central neurocytoma we describe in our patient along with her young age led to the use of systemic chemotherapy . the use of chemotherapy in the treatment of typical and atypical central neurocytoma is reported in adults and children [ 616 ] . this is the second report describing the use of high dose chemotherapy followed by autologous stem cell rescue in a child with atypical central neurocytoma . we describe a 14 month old female with episodic agitation upon awakening followed by a very intense episode of agitation with associated respiratory compromise . she was transported to a local hospital where ct imaging demonstrated a large hemorrhagic lesion in the right frontal lobe extending into the right lateral ventricle ( see fig . a gross total resection of this hemorrhagic lesion was completed as demonstrated by ct imaging ( see fig . 2a d ) . the histology was that of a mildly pleomorphic round cell tumor with diffuse , strong immunolabeling for synaptophysin . atypical features were noted including the presence of mitoses , glomeruloid vascular proliferation , and necrosis . the ki-67 labeling index was 10% overall though focally was higher . a decision to avoid consolidative radiation therapy 1a preoperative post contrast coronal ct images demonstrating a hemorrhagic lesion in the right frontal lobe extending into the right lateral ventricle . b postoperative post contrast coronal ct images demonstrating a gross total resection of the right frontal lesionfig . 2a microscopic evaluation showed an atypical neurocytic tumor composed of cells with round to oval nuclei . d the ki-67 labeling index was approximately 10% overall ( 400 ) a preoperative post contrast coronal ct images demonstrating a hemorrhagic lesion in the right frontal lobe extending into the right lateral ventricle . b postoperative post contrast coronal ct images demonstrating a gross total resection of the right frontal lesion a microscopic evaluation showed an atypical neurocytic tumor composed of cells with round to oval nuclei . d the ki-67 labeling index was approximately 10% overall ( 400 ) two months after her resection she developed a recurrence with obstructive hydrocephalus and midline shift requiring placement of a ventriculo - peritoneal shunt . imaging demonstrated diffuse meningeal enhancement as well as interval development of an area of local enhancement concerning for recurrence ( see fig . she subsequently received 3 cylces of chemotherapy including vincristine ( 0.05 mg / kg / dose , days 1 & 8) , cisplatin ( 3.5 mg / kg / dose , day 2 ) , and etoposide ( 2.5 mg / kg / dose , days 13 ) . imaging demonstrated a partial response with decreased leptomeningeal and local enhancement in the resection cavity . 3post contrast coronal and axial t1-weighted images show nodular enhancement ( black arrow ) along the margin of the right posterior frontal resection cavity , suggesting recurrent tumor post contrast coronal and axial t1-weighted images show nodular enhancement ( black arrow ) along the margin of the right posterior frontal resection cavity , suggesting recurrent tumor at 24 months she was noted to have development of a cystic lesion in the resection cavity and increased leptomeningeal enhancement . as a result mg / kg / dose , days 12 ) and mesna ; however , the second cycle was complicated by hyponatremia and seizures . she was then placed back on the chemotherapy regimen consisting of vincristine , cisplatin , and etoposide . repeat imaging demonstrated stable disease . at 28 months of age prior to the completion of additional chemotherapy she underwent gcsf mobilized peripheral blood stem cell collection . a total of 12 10 cd34 + cells / kg were collected in anticipation of high dose chemotherapy and autologous stem cell rescue . additional chemotherapy was provided with 3 cycles of ifosfamide ( 1800 mg / m / dose days 15 ) , carboplatin ( 400 mg / m / dose days 12 ) , and etoposide ( 100 mg / m / dose days 15 ) . repeat imaging was completed demonstrating stable disease . due to a lack of significant clinical response and nephrotoxicity ( hypomagnesaemia , hypokalemia , and academia ) she she received one cycle of therapy followed by imaging demonstrating interval progression of her leptomeningeal disease . at 34 months of age , high dose chemotherapy per the consolidation phase of children s cancer group protocol 99703 this chemotherapy regimen consisted of carboplatin ( 17 mg / kg , days 01 ) and thiotepa ( 10 mg / kg , days 01 ) . this was followed by a day of rest , stem cell infusion , and then gcsf . this phase was repeated every 4 weeks for 3 cycles with the infusion of 2.4 10 cd34 + cells / kg , 3.68 10 cd34 + cells / kg , and 3.86 10 cd34 + cells / kg , respectively . toxicity included fever and neutropenia . at 40 months of age she was disease free by imaging criteria and was started on isotretinoin ( 8 mg / kg / day , 14 days / month ) and oral etoposide ( 3 mg / kg / day , 21 days / month ) for 8 months until the development of neutropenia . unfortunately , 6 months later abnormal enhancement in the cervical , thoracic , or lumbar spine was noted on mri , suggesting recurrent disease ( see fig . following her most recent relapse , a decision was made to begin therapy with temodar ( 5 days per week every 2 weeks ) and irinotecan ( every 2 weeks ) . 4post contrast sagittal t1 and axial t1-weighted images show enhancement of the surface of the cord ( white arrow ) , suggesting recurrent disease post contrast sagittal t1 and axial t1-weighted images show enhancement of the surface of the cord ( white arrow ) , suggesting recurrent disease central neurocytoma , first described in 1982 , is a rare central nervous system ( cns ) tumor that accounts for 0.10.5% of cns tumors [ 1 , 2 ] . this tumor typically affects young adults in their third decade of life [ 1 , 2 ] . neurocytomas are believed to arise from subependymal plate of lateral ventricles and are believed to be neuronal or neuroglial in origin [ 1 , 2 ] . histologically they are divided into typical and atypical central neurocytomas [ 1 , 2 ] . microscopically they appear as small round cells with round nuclei and scant cytoplasm and will often stain positive for synaptophisin . typical central neurocytomas are well differentiated and benign appearing ; however , atypical central neurocytomas are less well differentiated and malignant appearing . the presence of necrosis , increased mitotic activity , and vascular proliferation is not uncommon . treatment of atypical central neurocytoma in children relies upon surgical therapy and radiation therapy [ 35 ] . of these 438 cases 73 patients were 18 years of younger at the time of initial surgery . typical central neurocytomas comprised 62 individuals in the sample and atypical central neurocytomas only 11 individuals . those individuals that had a complete resection with or without radiation therapy had an excellent 5 year overall survival rate . those individuals that had an incomplete resection followed by radiation therapy achieved improved local control rates , but did not benefit from improved overall survival . this data suggests that durable cures are likely in individuals that achieve a complete resection ; however , this data did not parse out responses of atypical central neurocytomas from typical neurocytomas . based upon the extant literature present in table 1 , there is little experience with the use of conventional chemotherapy for atypical central neurocytoma and high dose chemotherapy followed by autologous stem cell transplant for atypical central neurocytoma [ 617 ] . our patient was disease free by imaging criteria following the completion of high dose chemotherapy and autologous stem cell rescue which is a notable difference when looking at the cases described previously ; however , the duration of this remission was brief . myeloablative chemotherapy followed by autologous stem cell rescue has been reported as an effective consolidative therapy for patients with malignant brain tumors and those individuals that are not candidates for radiation therapy [ 1821 ] . avoidance or delay of radiation therapy use may also help limit some of the potential long - term complications of craniospinal irradiation including increased risk of future second cancers , neuro - cognitive dysfunction , and endocrinologic deficits [ 2224].table 1summary of reported cases of neurocytoma treated with chemotherapystudyage / genderclinical history / therapychemotherapyoutcomeamini et al . 5 years old / male(1 ) resected and treated with adjuvant chemotherapy(1 ) adjuvant therapy : vcr and cisplatin monthly alternating with cyclophosphamide . he completed 5 cyclesstable disease with no evidence of recurrence or progression(2 ) local recurrence one year later which was reselected(2 ) no chemotherapy(3 ) disseminated recurrence 5 months later(3 ) gleevec ( 200 mg / m twice daily ) with a lack of response followed by local radation and temozolamide ( 75 mg / m / day ) followed by maintenance ( 175 mg / m / day ) for 5 days every 4 w aeks , complete response(4 ) local recurrence 9 months later(4 ) it liposomal ara - c ( 2 courses ) and radiosurgery followed by temodar maintenance , complet(5 ) spinal metastasis treated with induction chemotherapybrandes et al . 43 years old / femalestereotactic radiotherapy led to a complete response recurrence was noted six years later and was treated with chemotherapyetoposide ( 40 mg / m / day , days 14 ) , cisplatin ( 25 mg / m / day , days 14 ) , and cytoxan ( 1000 mg / m , day 4 ) cycles were repeated every 4 weeks . she completed 5 cyclespartial repsonse for a follow - up of 15 monthsbrandes et al . 61 years old / malelimited field radiation was completed with a partial response noted and stable disease for 5 years recurrent disease developed and was treated with chemotherapy.etoposide ( 40 mg / m / day , days 1 - 4 ) , cisplatin ( 25 mg / m / day , days 14 ) , and cytoxan ( 1000 mg / m , day 4 ) cycles were repeated every 4 weeks . he completed 5 cyclesstable disease for a follow - up of 18 monthsbrandes et al . 22 years old / femaletotal resection was completed recurrent disease 3 years later with ventrcular disease and a spinaletoposide ( 40 mg /m / day , days cisplatin ( 25 mg / m / day , days 14 ) , and cytoxan ( 1000 mg / m , day 4 ) cycles were repeated every 4 weeks she completed 3 cyclescomplete response of the spinal lesion and stable ventricular disease craniospinal radiation led to a complete response for a follow - up of 36 monthscoelho et al . 6 years old / malerecurrent ventrile and thalamus and peritoneal dissemination 3 5 yrs after subtotal resection vp shunt for hydrocephalusetoposide , carboplatin , doxorubicin , cyclophosphamidedied 3 days after diagnosis of disemminationdodds et al . 15 years old / malesubtotal resection completed along with placement of a vp shunt treated with chemotherapycarboplatin ( 500 mg / m , day 12 ) , etoposide ( 100 mg / m , days 13 ) , and ifosfamide ( 3 g / m , days 13 ) the etoposide and ifosfamide are repeated at week . 3 he completed 4 cyclesresponse with tumor shrinkage for 22 months until symptoms and tumor growth treated with subtotal resection and radiation therapy stable disease with a follow - up period of 6 years . 22 years old / femalesubtotal resection completed along with placement of a vp shunt a local recurrence occurred 20 months later and a second subtotal resection was done . a ventricular recurrence and leptomenningeal enhancement were noted 14 months later and treated with chemotherapy.cyclophosphamide , etoposide , and cisplatinnot reportedkulkarni et al . 21 years old / maleradiation therapy and placement of bilateral vp shunts and chemotherapylomustine ( 7 doses)partial response with follow - up for 60 months followed by deathkulkarni et al . 21 years old / femaleradiation therapy and chemotherapylomustine ( 9 doses)subependymal spread with increased size of primary lost to follow - up at 15 monthskulkarni et al . 14 years old / femaleradiation therapy and chemotherapylomustine ( 7 doses)no change in tumor with follow - up of 108 months underwent shunt surgerykulkarni et al . 45 years old / maleradiation therapy and placement of vp shunt and chemotherapylomustine ( 8 doses)no change in tumor with follow - up of 90 monthskulkarni et al . 38 years old / maleradiation therapy and chemotherapylomustine ( 9 doses)no change in tumor with follow - up of 114 monthskulkarni et al . 27 years old / femaleradiation therapy and placement of bilateral vp shunts and chemotherapylomustine ( 7 doses)no change in tumor with follow - up of 96 monthsleenstra et al . 17 years old / femaleradiation therapy followed by 1st recurrence treated with total resection at 45 months second , third , and fourth recurrences with local and drop metastases treated with chemotherapy at 150 , 172 , and 185 months , respectivelyetoposide ( 60 mg / m days 13 , cisplatin ( 20 mg / m days 15 ) , cytoxan ( 500 mg / m day 1 every 4 weeks she completed 7 cylces with second recurrence four more cycles were given for third recurence then given carboplatin ( 250 mg /m day 1 and ifosfamide 1 35 g / m days 13 ) with mesna for fourth recurrencepartial responses with 4 relapses as described had seizures after 1 st cycle of chemotherapy and stroke after 4th cycle died 28 months after completion of initial salvage chemotherapyleenstra et al . 20 years old / maletotal resection with second total resection at 3 months following local recurrence . second relapse at 6 months treated with chemotherapycisplatin ( 75 mg / m / day , day 1 ) , vincristine ( 15 mg / m days 7 and 14 ) , cytoxan ( 1000mg / m , days 21 and 22 ) with mesna . he completed 4 cyclesalive with disease at 12 months of follow - upleenstra et al . 25 years old / malesubtotal resection , radiation therapy and chemotherapycisplatin ( 50 mg / m / day , days 13 ) , and ranimustine ( 50 mg / m d1)stable disease , no recurrence , no progression alive 3.7 yearsleenstra et al . 8 years old / maletotal resection , radiation therapy and chemotherapycisplatin , vincristine , and prednisone every 8 weeksno disesase or recurrence alive at 10.8 years cognitive deficitsleenstra et al . 23 years old / femaletotal resection , radiation therapy and chemotherapylomustine ( 200 mg / m / day , days 1 ) , and carmustine ( 200 mg / m d1 ) , every 6 weeks . 17 years old / malesubtotal resection , radiation therapy and chemotherapycyclophosphamide and cisplatinalive and well at 14 months . 26 years old / femalesubtotal resection , radiation therapy and chemotherapycyclophosphamide and cisplatinalive and well at 11 months . she completed 3 cyclesparital response with eventual dissemination and death one year after radiation therapyogawa et al . 71 years old / femaleradiation therapy followed by chemotherapyacnudeveloped disseminated disease and died 1.5 years after diagnosissgourous et al . 19 years old / femaleresection followed by recurrence at 12 months second resection followed by radiation response noted , but developed progressive disease treated with chemotherapycarboplatinpartial repsonse for a 58 years old / maleradiation therapy and chemotherapy followed by progression 5 months later treated with subtotal resectiontemozolamide ( 175 mg/ 2/day days 15 of 28 ) he completed 3 cyclesstable disease on chemotherapy . developed symptoms of progression 2.5 months later followed by subtotal resection no residual or recurrent tumor at 28 months of follow - upvon koch et al . 15 years old / femaleseveral subtotal resections over a 3 year period followed by tumor progression treated with chemotherapyprocarbazine ( 60 mg / m , days 821 ) , ccnu ( 110 mg / m , day 1 ) , and vcr ( 14 mg / m , days 8 and 29 ) . she completed 6 cyclesdurable response for at least 16 months of follow - up summary of reported cases of neurocytoma treated with chemotherapy moreover , the use of novel therapeutic agents should be considered in these patients as we described above . as an example , the use of retinoids in the care of children with brain tumors is under investigation [ 25 , 26 ] . their potent anti - tumor effect which occurs by induction of neuronal differentiation , growth arrest , and apoptosis has been demonstrated in childhood cancers with neuronal differentiation such as neuroblastoma . isotretinoin and other novel retinoids are currently under investigation as therapeutic adjuncts in a host of childhood cancers including brain tumors . several recent articles have successfully utilized metronomic combinations of agents such as oral etoposide , isotretinoin , thalidomide , cyclophosphamide , and celecoxib in children and adults with brain tumors [ 28 , 29 ] . the mechanism of metronomic therapy is believed to be due in part to anti - angiogenic effects although further research is needed to fully understand the complete biology of this approach . a recent publication described the use of metronomic therapy for ten pediatric patients under 5 years old with malignant brain tumors following high - dose chemotherapy and autologous stem cell rescue . similar to our case , it was tolerated well and had encouraging activity suggesting that this approach may warrant further evaluation in larger clinical trials . surgical resection and radiation therapy serves as the mainstay of therapy for atypical central neurocytoma . the toxicity of radiation therapy in young children poses a risk to the use of this therapy in the treatment of atypical central neurocytomas including examples such as neuro - cognitive deficits and second cancers . those young patients with atypical neurocytomas that behave aggressively may also be candidates for the use of systemic chemotherapy . limited experience with the use of chemotherapy and myeloablative chemotherapy followed by autologous stem cell rescue in central neurocytoma is described . we add to the literature a case describing the use of intensive chemotherapy followed by autologous stem cell rescue . further exploration of this potentially effective modality of therapy in combination with other novel adjunctive therapeutic agents and approaches is needed in patients with atypical central neurocytoma that is refractory to traditional curative therapy .

the authors describe a 9 month old female with recurrent atypical central neurocytoma and leptomeningeal spread treated with high dose chemotherapy , autologous stem cell rescue , and adjuvant therapy . she had a complete response to therapy and was disease free at 4 years of age until a recurrence 6 months later . the use of intensive chemotherapy followed by autologous stem cell rescue for atypical neurocytoma may be considered as an adjunct to surgical therapy in young patients with atypical neurocytoma not amenable to radiation therapy .

Introduction Case report Discussion Conclusion

atrial - esophageal fistula ( aef ) is a rare severe disease , which may be associated with radiofrequency catheter ablation ( rfca ) of atrial fibrillation ( af ) or intraoperative radiofrequency ablation of atrial fibrillation ( iraaf ) . we presented a case of a 67-year - old man with aef following rfca treated with esophageal stenting and surgical repair , and then reviewed literatures about aef to explore the appropriate treatment options . a 67-year - old man , who had coronary heart disease , af , and heart failure , was admitted because of dyspnea and rapid af . . then rfca of rf was to perform wide area circumferential ablations to achieve pulmonary vein isolation using a 3.5 mm tip electrode on a d - curved thermocool catheter through agelis sheath . the lesions were applied for 30 to 60 s with 30 to 30 walter . 18 days after the procedure , the patient was admitted to local hospital with complaint of chest discomfort and fever for 1 day . his temperature was 41c , white blood count ( wbc ) 5.2 10/l , and high sensitivity c , physical examination revealed that his temperature was 37.9c , blood pressure was 99/73 mm hg , and pulse rate was 78 beats / min . the initial laboratory studies showed n - terminal pro brain natriuretic peptide ( nt - probnp ) 1550.00 pg / ml , alanine aminotransferase 21 u / l , cardiac troponin i 3.95 g / l , wbc 10.58 10/l , neutrophil 97.2% , hscrp 26.0 mg / l , and fecal occult blood 3 + . contrast - enhanced ct ( cect ) showed suspicious thrombus in left atrial appendage ( fig . contrast - enhanced computed tomography showed suspicious thrombus in left atrial appendage ( indicated by red arrow ) . patient was asked to fast with gastrointestinal ( gi ) decompression , and used antibiotic , imipenem / cilastatin and linezolid , to control infections . he regained consciousness from the next day after admitting our institution , but he still had repeated epileptic seizures . cerebral magnetic resonance imaging ( mri ) was performed and showed lacuna infarctions ( fig . emergency esophagogastroduodenoscopy ( egd ) showed a small and deep ulcer at 30 cm from incisors . the esophagus was stented with self - expanding , polyester - covered , length 100 mm , width 18 mm ( nanjing micro - tech , nanjing , china ) ( fig . 3 ) . in the following days , wbc counts and hscrp level decreased , epileptic seizures went away . but the symptom of acid reflux occurred from the 5th day after esophageal stenting when he ate fluid food . temperature and cardiac cect showed esophageal - mediastinum fistula with encapsulated effusion and pneumatosis behind left atrium ( la ) , and post wall of la depression . emergency esophagogastroduodenoscopy showed a small and deep ulcer at 30 cm from incisors ( left ) . right thoracotomy incision through the right 5th intercostal space was made under general anesthesia . during the surgery , we noticed the membranaceous and funicular adhesions between the right lung , diaphragm and mediastinum , white lamellar scars under azygos vein beside left atrial . after opening mediastinal pleura , tight adhesion between esophagus and pericardium was found and separated carefully , then a 6 mm diameter fistula in the anterior wall of esophagus was discovered ( fig . tight adhesion between la and pericardium , combined with bleeding at the posterior wall of la , was found during exploratory pericardiotomy . rid - muscle flap pedicled with vessels was made to repair la posterior wall and fistula between esophagus and la ( fig . pleuroclysis using povidone iodine and nature saline was performed before sternal closure . a 6 mm diameter fistula ( indicated by blue arrow ) in the anterior wall of esophagus . rid - muscle flap pedicled with vessels ( indicated by blue arrow ) was used to repair la posterior wall and fistula between esophagus and la . , the patient was transferred to intensive care unit , and then out - of - control sepsis and infectious shock , which showed repeated fever and chills , metabolic acidosis , hypotension , tachycardia and oliguria , attacked the patient , and caused his death . aef , a rarely complication of rfca of af , was first reported in 2004 . some surveys revealed that the incidence was 0.03% to 0.04% in rfca of af and about 1% in iraaf , and its mortality ranging was from 71% to 83%.24 as the number of rfca of af cases grows , more aef cases could be found in recent years . it was considered that the ablation of the posterior wall of la by the high temperature might be an important risk of aef . elevated esophageal temperatures during la ablation may occur in more than 50% of patients.5 6 esophageal stenting and surgical repair are the commonest treatment for aef . to date , we still did not know which one is the best treatment for aef . fifty - seven relevant articles , including 86 aef patients ( 60 males ) , were analyzed.1 661 seventy - eight patients were secondary to rfca of af , and 8 patients were secondary to iraaf . the incubation period of aef symptoms was from 2 to 60 ( average : 24.8 ) days . . the symptoms of neurological symptoms , including hemiplegia and epilepsy , consciousness disorder , gi bleeding , chest pain , were relatively common . the other uncommon symptoms included : nausea and vomiting , dysphagia , dyspnea , cough , arrhythmia , headache , blindness , syncope , abdominal pain , and petechiae ( fig . chest ct and cect were the most common noninvasive diagnosis methods with high sensitive for aef , which could show the air bubbles or vegetation at the site near the esophagus of la . transthoracic echocardiography , transesophageal echocardiography , cardiac mri , and contrast esophagram could discover aef ( fig . 7 ) . abnormal cerebral ct findings , such as cerebral air embolism and multifocal cerebral ischemia , revealed in most of patient on admission . cect = contrast enhanced computed tomography , ctpa = computed tomography pulmonary angiogram , egd = esophagogastroduodenoscopy , et = exploratory thoracotomy , tee = transesophageal echocardiography , tte = transthoracic echocardiography . hemorrhage from aef , sepsis , air embolism , and cerebrovascular accidents are the major reasons for a fatal outcome.2 6 9 12 23 57 esophageal stenting and surgical repair were the major therapies of aef . different therapeutic strategy might influence outcome of patients . twenty - eight patients without esophageal stenting or surgical repair had higher mortality , only 1 patient who performed endoscopic ligation survived . but relatively , surgical repair decreased the mortality of the patients significantly , only 12 patients died in 43 patients who only performed surgical . three patients implanted esophageal stenting after performing emergency atrial wall repair with thoracotomy , 2 patients survived . two patients , who received esophageal stenting and subsequently performed surgical , were all survived ( table 2 ) . it is known to all that aef is a life - threatening complication of ablation of af . according to the literature review of aef , we found that conservative treatment or esophageal stenting alone seemed to be a poor clinical outcomes , and surgical repair combined with or without esophageal stenting may be the preferred option for the patients . early surgical treatment have lower mortality than delayed strategy.8 19 23 26 37 though there were many disputes about the opportunity to put esophageal stent , stenting alone was not recommended for treating aef.26 37 the clinical outcomes of combination therapy of esophageal stenting and surgical repair were not clear because of few cases . in our case , the patient underwent early esophageal stenting with temporary improvement of the symptoms in the first few days . but the conditions of the patient were worse , and eventually caused death despite surgical repair was performed . early resuming oral intake and delayed surgical intervention may be the important factors of relapse . make a definition diagnosis in time with early primary surgical repair may save their lives . conservative treatment or esophageal stenting alone may not be a better choice for aef patients .

abstractintroduction : atrial - esophageal fistula ( aef ) is a rare severe disease , which may be associated with radiofrequency catheter ablation ( rfca ) of atrial fibrillation ( af ) or intraoperative radiofrequency ablation of atrial fibrillation ( iraaf).clinical findings : we reported a case of a 67-year - old man with aef following rfca of af , who treated with esophageal stenting and surgical repair.outcomes:he was attacked by out - of - control sepsis and infectious shock after surgery and died.literature review : we analyzed 57 relevant articles about aef from 2003 to 2015 by searching pubmed database . according literatures , the most common symptoms were fever , rigor , sepsis , and neurologic symptoms . chest computer tomography ( ct ) and contrast enhanced ct may be the reliable noninvasive diagnosis methods because of high sensitive for aef.conclusion:make a definition diagnosis in time with early primary surgical repair may save their lives . conservative treatment or esophageal stenting alone may not be a better choice for aef patients .

Introduction Case presentation Discussion Conclusion

we reviewed respiratory infection outbreaks registered with the public health laboratory ( phl ) , ontario agency for health protection and promotion dating back to october 2007 ( table 1 ) . molecular detection methods were used for a subset of outbreaks registered during october 1 , 2008april 19 , 2009 . after emergence of severe respiratory illness clusters in mexico in early april , intensified tracking of respiratory infection outbreaks in ontario was undertaken . consequently , more information was available on outbreaks registered during the spring ( april 20 to june 12 , 2009 ) ; these data comprise the bulk of the study . * influenza season is delineated as october 1april 19 ; spring season is delineated as april 20june 12 . greater toronto area includes submissions by peel , york , and toronto public health units . specimens from 78 of the 101 outbreaks were tested by the luminex xtag respiratory viral panel ( luminex molecular diagnostics , toronto , ontario , canada ) . respiratory infection outbreaks in ltcfs were defined as any of the following : 2 cases of acute respiratory tract illness , 1 of which was laboratory - confirmed ; 3 cases of acute respiratory tract illness within 48 hours in a geographic area ( e.g. , unit , floor ) ; and > 2 units having a case of acute respiratory illness within a 48-hour period . influenza - like - illness was defined as acute onset of respiratory illness with fever and cough with > 1 of the following : sore throat , arthralgia , myalgia , or prostration . from april 20 through june 12 , 2009 , a total of 112 respiratory infection outbreaks were registered . molecular testing was not used in 29 outbreaks ( e.g. , insufficient / inappropriate sample ) . most of the remaining 83 outbreaks submitted for molecular testing originated from ltcfs ( 91% ) ; hospitals ( 2% ) , child care centers ( 2% ) , and psychiatric care facilities ( 1% ) comprised the remainder . mean age of persons tested as part of an outbreak investigation was 82 years ( sd 13.96 years ) and median age was 85 years ; 95% were > 57 years of age . testing on the 589 specimens received from 161 outbreaks registered from october 1 , 2008 through june 12 , 2009 was performed by real - time reverse transcription pcr ( rt - pcr ) for the influenza a virus matrix gene and the luminex respiratory viral panel ( rvp ) ( luminex molecular diagnostics , toronto , ontario , canada ) for other respiratory viruses . an etiologic agent was identified in 89% of the 161 outbreaks tested by molecular methods . two and 3 different pathogens were identified in 24 ( 15% ) and 6 ( 4% ) outbreaks , respectively . no etiologic agent was identified in 18 ( 11% ) of the outbreaks tested by molecular methods , which includes 1 specimen in which the result was indeterminate for coronavirus oc43 . a wide range of causative etiologic agents were detected for outbreaks by the rvp assay ( table 2 ) . specimens from most patients were positive for enterovirus / rhinovirus ( 114 patients ) followed by metapneumovirus ( 85 ) , parainfluenza virus type 3 ( 55 ) , and human influenza virus a ( h3 ) ( 41 ) . no virus was identified in 186 patients . * luminex molecular diagnostics , toronto , ontario , canada . spring season is delineated as april 20june 12 ; influenza season is delineated as october 1april 19 . seven of the 15 outbreaks were confirmed as rhinovirus by the seeplex rv12 detection kit ( seegene , inc . , co - infections were noted in 22 of the patients tested by the rvp assay . in 1 ltcf outbreak , 2 patients had co - infection of an untypeable influenza a and enterovirus / rhinovirus on testing by rvp . an influenza a real - time rt - pcr result was negative in both patients ; 1 patient had a co - infection with respiratory syncytial virus b and enterovirus / rhinovirus . co - infections with coronavirus subtypes 229e and nl63 were the most common , observed in 10 of the 22 patients ( 45% ) infected with multiple pathogens . isolates from 1 patient were positive for 3 viruses ( coronavirus subtypes 229e and nl63 and enterovirus / rhinovirus ) . one of the 2 outbreaks identified as caused by pandemic ( h1n1 ) 2009 originated from a ltcf was not observed until june 3 , 2009 , six weeks into the evolving pandemic , despite widespread community prevalence . the second pandemic ( h1n1 ) 2009 outbreak , registered on june 11 , 2009 , originated from a hospital treating patients with influenza - like illness . seasonal influenza ( h1n1 and h3n2 ) or pandemic ( h1n1 2009 ) was detected in 2,966 ( 25.5% ) , and pandemic ( h1n1 ) 2009 in 2,203 ( 19% ) of 11,612 persons tested at phl for influenza a by real - time rt - pcr during april 20june 12 , 2009 . seasonal influenza a ( h3n2 ) was only identified in 273 specimens ( 11.0% ) of the 2,476 influenza a positive samples subtyped . however , it was the strain responsible for 15 ( 88% ) of the typeable influenza a outbreaks at the same time . seasonal influenza a ( h1n1 ) was absent from institutional outbreaks and only detected in 41 ( 2% ) of subtyped influenza a positive samples from the general population . persons with laboratory - confirmed pandemic ( h1n1 ) 2009 infection tested at the phl , ontario agency for health protection and promotion , were younger than those tested as part of outbreak investigations . mean and median ages were 21.5 and 16 years , respectively ; only 10% were > 46 years of age . the number of respiratory infection outbreaks in institutions submitted to phl may reflect disease impact caused by respiratory viruses during the influenza season . respiratory viruses during the 200708 season may have been more active than those of the 200809 season because the number of outbreaks registered with phl decreased from 1 year to the next . declaration of pandemic status for the novel ( h1n1 ) virus has not influenced the reporting of respiratory infection outbreaks from institutions in ontario because submission rates for the corresponding period in 200708 and 200809 are similar . variation would not be expected because reporting is required by ontario law ( 4 ) . respiratory viruses detected in outbreaks in institutions reflect those known to be major causes of acute respiratory disease in the community ; prevalence varies based on geographic location , season , and detection methods ( 57 ) . free access to such institutions by members of the community ( staff or visitors ) , in conjunction with communal close quarters of residents , creates an ideal environment for propagation of viral respiratory outbreaks ( 8) . current guidelines for isolation during viral respiratory outbreaks are not tailored for the specific virus . as shown in this study , multiplex molecular testing makes it possible to identify the virus causing most ltcf respiratory infection outbreaks . infection control guidelines for a specific outbreak could be modified based on the incubation period and duration of viral shedding for the identified virus ( 9 ) . clinicians should be reminded that rhinovirus can cause severe lower respiratory tract infection , including death , as documented in several ltcf outbreaks ( 10,11 ) . these data highlight the need for molecular capacity to diagnose rhinovirus infection because detection is otherwise limited to less sensitive viral culture systems . this review of outbreaks predominantly involving elderly persons in ltcfs highlights the sparing of older persons by pandemic ( h1n1 ) 2009 . possible explanations include cross - protective antibodies from previous exposure to influenza a ( h1n1 ) strains circulating before the antigenic shift of influenza a to subtype h2n2 in 1957 or minimal contact with those most likely to have imported the pandemic strain into canada ( young travelers ) ( 12 ) . in addition , older persons may have less contact with the age group ( children 1019 years of age ) , with most cases being in ontario . our findings support centers for disease control and prevention guidelines for vaccination with monovalent pandemic ( h1n1 ) 2009 virus vaccine . these guidelines have not placed older persons in a high priority group for vaccination because increased rates of hospitalization and severe disease caused by pandemic ( h1n1 ) 2009 have not been observed ( 13,14 ) . investment in multiplex technologies to investigate respiratory outbreaks in ltcfs shortens time for pathogen detection , helps guide infection control and vaccination policies , and can potentially save resources spent on other investigations .

outbreaks of respiratory infection in institutions in ontario , canada were studied from april 20 to june 12 , 2009 , during the early stages of the emergence of influenza a pandemic ( h1n1 ) 2009 . despite widespread presence of influenza in the general population , only 2 of 83 outbreaks evaluated by molecular methods were associated with pandemic ( h1n1 ) 2009 .

The Study Conclusions

this case report seeks to describe a novel technique for treating post - operative pancreatic fistulas . conventional non - surgical treatments include nothing by mouth ( npo ) , total parenteral nutrition ( tpn ) and octreotide administration . when such a treatment fails , re - operation with complete pancreatectomy or entero - fistulotomy may be required . we present a case of post - operative pancreatic fistula treated with pancreatic duct embolization via a percutaneous approach as a new technique , which to the best of our knowledge has not been previously reported . a 55-year - old caucasian male with a history of morbid obesity , sleep apnea , chronic obstructive pulmonary disease and kidney stones was evaluated for acute onset of right upper quadrant abdominal pain , nausea and vomiting , unintentional 19-pound weight loss , and jaundice 3 weeks was found to have an apple - core lesion involving the duodenum at the level of the ampulla causing gastric outlet obstruction and biliary obstruction . there was no evidence of metastatic disease and tumor markers were found to be normal . the patient underwent a whipple procedure , and was found to have periampullary adenocarcinoma . of note his postoperative course was complicated with respiratory failure requiring prolonged intubation , multiple abdominal abscesses , candidemia , wound infections , and post - operative bleeding requiring re - exploration . one month after the procedure , high output from the right corner of his incision persisted . a ct scan of the abdomen and pelvis demonstrated a fistulous communication from the pancreatico - jejunostomy in the retroperitoneum to the skin ( fig . this was managed by insertion of a percutaneous drainage catheter along the fistulous track to the pancreatico - jejunostomy . the drain was left in place for 2 months in an attempt to conservatively treat the fistula in conjunction with npo , tpn and octreotide , but the fistula persisted . despite multiple attempts to percutaneously embolize the endoscopic retrograde cholangiopancreatography ( ercp ) was performed and demonstrated that the pancreatic duct anastomosis had closed , thus the fistula was the only egress of pancreatic secretions . prior to completion pancreatectomy it was decided to proceed with percutaneous embolization of pancreatic duct via the fisutlous tract . the percutaneous drain was exchanged over a guidewire and replaced with an 8 french vascular sheath ( terumo medical corp . ) , through which a kumpe catheter ( cook medical ) was advanced to the central aspect of the fistula . a 2.4 french progreat microcatheter ( terumo medical corp . ) was advanced over a 016 fathom guidewire ( boston scientific ) through the kumpe catheter . using this catheter and guidewire combination , the pancreatic duct was successfully cannulated . a 3 - 1 mixture of n - bca trufill glue was injected under fluoroscopic supervision completely filling the pancreatic duct , side - branches and the fistulous tract . the sheath , base catheter and microcatheter were removed and replaced with a 12 french ultrathane percutaneous drain ( cook medical ) ( fig . the patient developed moderate abdominal pain within 20 min of embolization , which was controlled with a morphine patient - controlled analgesia ( pca ) . he was admitted to the hospital for 3 days post embolization for pain control and tpn . his lipase peaked at 538 within 12 h but returned to normal in 5 days . his pain resolved and his drain output decreased to less than 10 ml per day . the drain was removed one week later and the patient has not experienced leakage since . follow up ct three weeks after embolization demonstrated no evidence of pancreatitis , fluid collection or abscess formation . in post - pancreaticoduodenectomy pancreatic fistulas , the severity of the leakage dictates the aggressiveness of treatment . the overarching goal is to control the leak by the least invasive means possible while preventing sepsis and maintaining adequate nutrition in the post - operative patient . minimally invasive catheter drainage has been described as a choice of treatment for pancreatic duct leakage . they , however , used a sclerosing agent , prolamine , as opposed to embolic glue . in the approach discussed in , the sclerosant was delivered via the percutaneous drain that was positioned adjacent to the pancreas , not in the pancreatic duct as in our case . moreover , hirota discussed two different techniques depending on the situation . they performed two procedures in their post - surgical patients , resulting in trans - hepatic catheterization of the pancreatic duct to great an enteric fistula . for the patients with disconnected duct syndrome , they also use prolamine to sclerose the duct and their drainage catheter remains at the margin of the duct and does not cannulate the duct . pancreatic duct embolization has been previously reported including pancreatic duct remnant during pancreaticoduodenectomy or as an alternative to complete pancreaticoduodenectomy in patients with chronic pancreatitis to block exocrine function . previous methods of duct embolization have used fibrin sealants ; however , this process is not always successful and often necessitates repeat injections to seal the fistula . this method was attempted in our patient , but was unsuccessful , producing similar results to the above studies . the use of n - butyl-2-cyanoacrylate has been used before to occlude pancreatic fistulas in several cases . recent studies have determined that endoscopic sealing of pancreatic fistulas have been performed successfully , resulting in complete occlusion of the duct with leakage no longer occurring in these patients . this approach was not possible in our patient as the surgically created anastomosis had closed . in this case report , we present a novel approach to treat pancreatic fistulas that occur as a complication of pancreaticoduodenectomy . although n - butyl-2-cyanoacrylate has been used to embolize the pancreatic duct via ercp , it has not been previously reported to treat pancreatico - cutaneous fistula in the post - whipple patient . if percutaneous access to the pancreatic duct can be achieved , embolization is possible and can useful as a minimally invasive treatment prior to more complex interventions such as completion pancreatectomy .

highlightsnovel approach for treating pancreatic - cutaneous fistulas developed post-pancreaticoduodenectomy.use of liquid embolic agent to treat a high - output pancreatic - cutaneous fistula after a whipple procedure , which to the best of our knowledge , has not been performed before.minimally invasive treatment for a difficult problem that often results in additional surgery or prolonged drainage and tpn .

Introduction Case presentation Discussion Conflict of interest Funding Ethical approval Author's contribution

addiction is an acquired , chronic relapsing disorder that begins with some experimentation and pleasurable responses and , for subgroups of individuals , is followed by preoccupation , escalation , tolerance , denial , and a series of medical , psychological , and social consequences that relate directly to the continued addictive behaviors . discoveries over the last 20 years have clearly defined a relationship between obesity and systemic inflammation . the association of numerous diseases including diabetes mellitus , heart disease , common forms of cancer , as well as depression and alzheimer s disease with chronic low - grade systemic inflammation due to abdominal obesity has raised the possibility that obesity - associated inflammation affecting the brain may promote addictive behaviors leading to a self - perpetuating cycle that may affect not only foods but addictions to drugs , alcohol , and gambling . a key element of chronic addictions is continued addictive behavior in the face of clear knowledge of the expected consequences . many obese individuals identify specific foods that promote continued consumption in the face of the known consequences of obesity . . while for many individuals , these are simply bad habits that can be changed ; there is increasing evidence suggesting that genes implicated in other addictive behaviors may also be associated with food addiction , most commonly to sweet and fat foods . specific associations of genetic polymorphisms of the dopamine , opiomelanocortin , and leptin receptor have been hypothesized to increase the risk of food addiction . exercise and regular physical activity can entrain some of the same reward pathways and its efficacy in maintaining body weight after weight loss may be due to the activation of neural pathways involved in the initiation of overeating . insights into the genetics of addictive behaviors associated with obesity may both lead to improved therapies of obesity and at same time may provide insights into adjunctive therapies useful in treating drug addiction , alcoholism , and other addictions . humans eat even when they are not physically hungry as psychological , environmental , and hedonic influences affect food urges . the peripheral and central pathways which regulate food intake are geared toward the storage of calories when food is eaten in excess of metabolic needs , leading to obesity in situations of high fat and sugar food availability coupled with sedentary lifestyles . dopamine increases the motivation for food intake in animals and humans as demonstrated by imaging studies carried out using food images . it has also been demonstrated that obese individuals have reduced levels of dopamine d2 receptors and that these are inversely related to body weight . presentation of desirable foods increases metabolism in the orbitofrontal cortex ( ofc ) , the area of the brain that programs chewing and salivation , in proportion to the subjective perception of hunger and the desire to eat . obese subjects studied with functional magnetic resonance imaging ( fmri ) , which detects changes in blood flow , had evidence of activated striatum and ofc as well as the insula while viewing pictures of high - caloric food . wang and co - workers used positron emission tomography imaging which detects uptake of 2-deoxy-2[f]fluoro - d - glucose ( fdg ) to assess the ability of men and women to inhibit the activation of brain metabolism following presentation of images of favorite foods . in men food stimulation with inhibition significantly decreased activation in amygdala , hippocampus , insula , orbitofrontal cortex , and striatum , but this did not occur in women . the regions studied are integral to emotional regulation , conditioning , and motivation of food intake and in men suppressed activation of the orbitofrontal cortex with inhibition was associated with decreased self - reports of hunger . these observations suggest that hormonal mechanisms may underlie a gender difference in the ability to suppress hunger , and may contribute to the greater incidence of obesity in women compared to men . the repeated stimulation of da ( dopamine ) reward pathways has been proposed by volkow to trigger adaptations in other neurotransmitters and in brain reward circuitry that may lead to increases in compulsive behaviors affecting both food and drug intake . moreover , exposure to drugs that elicit dependence alters gene expression profiles throughout the reward circuitry of the brain . drugs of abuse induce repeated profound dopamine stimulation which , with chronic use , may induce changes in neuronal plasticity resulting in increased emotional reactions to drugs and reduced ability to inhibit drug consumption leading to compulsive chronic drug abuse . sugar - induced dependence in rats has been shown to promote increased consumption of ethanol , suggesting that food - related dependence may influence drug addiction . as reviewed above , repeated exposure to favorite foods , especially those that are calorie dense due to excess sugar and fat could lead to binge eating disorder , and poor inhibitory control of food intake . the increased usage of high fructose corn syrup in the american diet has been further suggested to reduce satiety that would have normally been satisfied with consumption of natural sugars . studies of rats show that excessive binging on sugar followed by deprivation creates a state of anxiety and suggested opiate - like withdrawal . while both pathways have similarities , that is , whether addictive drugs affect the emotional reaction to foods and vice versa . such an interaction could have significance for efforts to combat drug abuse as well as obesity . a possible mechanism for such an interaction is the systemic inflammation associated with obesity which could affect the brain and promote changes that make individuals more susceptible to dopamine - related rewards . consumption of favorite foods increases dopamine concentrations in the nucleus accumbens , activating the mesocorticolimbic dopaminergic reward pathways of the brain , resulting in the reinforcing effects of euphoria or pleasure . these discoveries have been the result of advances in brain imaging including positron emission tomography scan and fmri that enables visualization of brain activation on simply seeing images of favorite foods . since consuming food is critical to survival , it is perfectly reasonable that this activity would be highly valued within our inherited biological control mechanisms that evolved over eons of food scarcity and insecurity . only in recent times has there been a surplus of calories available to all americans including those in the lower socioeconomic strata . while these foods are inexpensive , they also have limited nutritional value and excess amounts of sugar and fat . pleasurable responses to sugar and fat linked to activation of reward circuits in the brain activated by dopamine and other neurotransmitters can lead to food addiction and obesity in susceptible individuals as has been documented for drugs , alcohol , tobacco , sexual activity , and gambling . the d2 dopamine receptor ( drd2 ) gene has most commonly been linked to alcoholism , although the gene has been associated with other addictive behaviors such as smoking , illicit drug use , gambling , and overeating . the taq1a polymorphism , the most frequently studied polymorphism , is located more than 10 kilobase - pairs downstream from the coding region of the drd2 gene at chromosome 11q23 . recent studies have further localized the coding region of taq1a within the coding region of a neighboring gene , ankk1 . the d2 dopamine receptor , a g - protein - coupled receptor located on postsynaptic dopaminergic neurons , is centrally involved in reward - mediating and reward - deficiency pathways . the drd2 gene encodes two molecularly distinct isoforms with distinct functions . signaling through dopamine d2 receptors governs physiologic functions related to locomotion , hormone production , and drug abuse . d2 receptors are also known targets of antipsychotic drugs that are used to treat neuropsychiatric disorders such as schizophrenia . by a mechanism of alternative splicing , d2l differs from d2s by the presence of an additional 29 amino acids within the third intracellular loop . usiello et al . demonstrated that these receptors have distinct functions in vivo ; d2l acts mainly at postsynaptic sites and d2s serves presynaptic autoreceptor functions . there is evidence of a direct association in taq1a a1 allele carriers with increased body fat and co - morbid substance use disorders . a clinical study found increased prevalence of taq1a a1 allele in obese patients with other drug dependencies compared to non - abusing obese patients . among taq1a a1 allele carriers , individualized custom formulations of neutraceuticals have been proposed to treat individuals with known genetic deficiencies such as taq1a a1 allele carriers . consumption of sweet or fat foods increases dopamine concentrations in the nucleus accumbens , activating the mesocorticolimbic dopaminergic reward pathways of the brain , resulting in the reinforcing effects of euphoria or pleasure . the a1 allele of the taq1a polymorphism ( rs1800497 ) , located 10 kb downstream of the drd2 gene has been investigated for possible associations with habitual use of alcohol [ 21 , 22 ] , cocaine , nicotine [ 2427 ] , and opioids . evidence indicates that individuals carrying this allele have reduced brain d2 receptor density compared with other individuals [ 2932 ] . adolescent girls shown with pictures of palatable food had weaker activation of the frontal operculum , lateral orbitofrontal cortex , and striatum if they are carriers of the drd2 taq1a a1 allele or the drd4 - 7r allele . persistent substance abuse in individuals with the a1 allele may be a form of self - stimulatory behavior that compensates for insufficient dopamine activity . the a1 allele has also been observed to be associated with obesity [ 3537 ] . more research is needed to determine whether the taq1a genetic polymorphism interacts with other well - known candidate genes for obesity including polymorphisms of the leptin receptor gene and others , and whether these interactive genetic effects could promote the development of obesity - associated inflammation . in turn , systemic inflammation could affect the brain leading to mild depression or other effects which promote continued addictive behaviors . other candidate genes for appetite control may interact with the drd2 a1 allele to increase the likelihood of overeating and obesity . the leptin receptor gene has polymorphisms that affect binding of leptin to the leptin receptor . the leptin receptor gene ( lep - r , formerly ob - r ) identified in 1995 , is expressed in the hypothalamus and other organs , binds to free - circulating leptin released from adipose tissue , and plays a major role in metabolism and appetite signaling . genetic variants potentially affecting receptor structure , function , and binding to leptin include three single - nucleotide polymorphisms ( snps ) that result in amino acid substitutions ( lys109arg ( k109r ) rs1137100 ; gln223arg ( q223r ) rs1137101 ; and lys656asn ( k656n ) rs8179187 ) [ 4042 ] located at highly conserved positions in the extra - cellular domain of the receptor protein . research from our group demonstrated an association between percent body fat and the lep - r variant lys109arg . other studies reported linkage of lys109arg with fat mass , association with serum leptin levels in young dutch men , an interaction between lys109arg and menopause with body mass index ( bmi ) , and fat mass loss in a caloric restriction intervention . while the drd2 gene increases the likelihood of food addiction and obesity [ 36 , 37 ] , the lep - r gene influences appetite control and has been associated with elevated levels of body fat and body composition [ 4347 ] . the joint influence of drd2 and lep - r could therefore predispose individuals to weight gain and obesity if risk alleles are carried from both genes . evidence for potential drd2 and lep - r interaction has been demonstrated in mice lacking d2 receptors . d2 receptor knock - out mice demonstrated leptin sensitivity , while wild - type mice had heavier body weights and were leptin resistant . further studies are needed to characterize the interplay between genes that encode for reward / pleasure neurotransmitters and energy balance regulation . the mu - opioid receptor gene ( oprm1 , located on chromosome 6q24 ) is one of four genes whose protein products bind to endogenous opioids . oprm1 is a member of the large family of transmembrane g - protein - coupled receptors . the mu - opioid receptor is the primary site of action for the most commonly used opioids , including morphine , heroin , fentanyl , and methadone . by sequencing , dna from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol dependence , five different snps in the coding region of the mu - opioid receptor gene were identified . the most prevalent snp is a nucleotide substitution at position 118 ( a118 g ) , predicting an amino acid change at a putative n - glycosylation site . significant differences in allele distribution were observed among ethnic groups studied . in a targeted study of food addiction , the functional a118 g polymorphism of the mu - opioid receptor gene over the last 200 years , changes in the human diet and lifestyle have outstripped the ability of the genome to adjust to a changing environment and billions of people around the world are now overfat as a result [ 5258 ] . while studies demonstrate that hunter gatherers ate over 800 varieties of plant foods , americans eat only about three servings per day which fail to provide the naturally occurring antioxidants , phytochemicals , vitamins , and minerals essential to good health . highly processed foods with refined starches , sugars , fats , and oils often fail to contain essential nutrients found in nutrient - dense foods . at the same time , labor - saving devices of all kinds and advances in transportation have made a highly sedentary lifestyle possible in just the last 30 years . a global epidemic of obesity and chronic disease that is underestimated using the bmi is sweeping the globe as industrialization and improved standards of living are spreading to urban centers . certain ethnic groups including african americans , asians , asian indians , latinos , native americans , and eastern europeans are genetically susceptible to metabolic syndrome resulting from the accumulation of visceral fat with resulting inflammation . it is estimated that in the next 10 years , the majority of all heart disease will be associated with type 2 diabetes mellitus . the metabolic syndrome has emerged as an important cluster of risk factors for atherosclerotic disease . common features are central ( abdominal ) obesity , insulin resistance , hypertension , and dyslipidemia , with high triglycerides and low high - density lipoprotein cholesterol . according to the clinical criteria developed by atp iii , it has been estimated that about one of every four adults living in the usa merits the diagnosis . chronic inflammation may represent a triggering factor in the origin of the metabolic syndrome : stimuli such as overnutrition , physical inactivity , and aging would result in cytokine hypersecretion and eventually lead to insulin resistance and diabetes in genetically or metabolically predisposed individuals . alternatively , resistance to the anti - inflammatory actions of insulin would result in enhanced circulating levels of proinflammatory cytokines resulting in persistent low - grade inflammation . a generally enhanced adipose tissue - derived cytokine expression may be another plausible mechanism for the inflammation / metabolic syndrome relationship . the role of adipose tissue as an endocrine organ capable of secreting a number of adipose tissue - specific or enriched hormones , known as adipokines , may play a pathogenic role in metabolic syndrome . although the precise role of adipokines in the metabolic syndrome is still debated , an imbalance between increased inflammatory stimuli and decreased anti - inflammatory mechanisms may be an intriguing working hypothesis . the chronic inflammatory state that accompanies the metabolic syndrome associates with both insulin resistance and endothelial dysfunction , providing a connection between inflammation and metabolic processes which is highly deleterious for vascular function . however , small amounts of weight loss which relieve the pressure for abdominal adipocytes to proliferate have been shown to reduce inflammation in patients with type 2 diabetes , and in two large trials to reduce the incidence of new cases of diabetes over 5 years by 58% in individuals with glucose intolerance and insulin resistance . our brains are programmed to activate the pleasure centers of the brain on visualizing food and then eating it , to remember the food that was eaten in specialized memory neurons , and to activate our digestive processes including chewing and hormone secretion in preparation for digesting a delicious meal . eating food is a life - giving event in the wild and so it is perfectly reasonable that this activity would be highly valued within our inherited biological control mechanisms . however , when the stimuli are linked in susceptible individuals to increases in reward circuitry activity as detected in changes in dopamine and glucose metabolism , then neuronal plasticity could be altered in ways that both increase the tendency to eating disorders and obesity as well as drug addiction . whether these pathways interact in susceptible individuals is unknown but has potential significance for efforts to prevent or treat chronic drug abuse through changes in diet and lifestyle including the treatment of obesity which may reduce brain inflammation .

there is increasing evidence that the same brain reward circuits involved in perpetuating drug abuse are involved in the hedonic urges and food cravings observed clinically in overweight and obese subjects . a polymorphism of the d2 dopamine receptor which renders it less sensitive to dopamine stimulation has been proposed to promote self - stimulatory behavior such as consuming alcohol , abusing drugs , or binging on foods . it is important to determine how this polymorphism may interact with other well - known candidate genes for obesity including polymorphisms of the leptin receptor gene and the opiomelanocortin gene . leptin is a proinflammatory cytokine as well as a long - term signal maintaining body fat . upper - body obesity stimulates systemic inflammation through the action of multiple cytokines including leptin throughout many organs including the brain . the association of numerous diseases including diabetes mellitus , heart disease , as well as depression with chronic low - grade inflammation due to abdominal obesity has raised the possibility that obesity - associated inflammation affecting the brain may promote addictive behaviors leading to a self - perpetuating cycle that may affect not only foods but addictions to drugs , alcohol , and gambling . this new area of interdisciplinary research holds the promise of developing new approaches to treating drug abuse and obesity .

Introduction Food Urges, Addiction, and Eating Behavior Genetic Polymorphisms Associated with Obesity and Food Addiction Dopamine Receptor Leptin Receptor Mu-Opiod Receptor Gene ( Human Diets, Obesity, and Inflammation Conclusion

transgenic mice expressing ib.dn or traf2.dn were described previously ( 5 , 12 ) . in vitro jnk assays were done as described previously ( 5 ) . in brief , 25 10 cells were treated with medium alone or rmu tnf- ( r&d systems , inc . , minneapolis , mn ) and lysed . for the kinase reaction , 1.53.0 g of purified glutathione s - transferase ( gst)c - jun(1 - 79 ) ( a gift from dr . h. hanafusa , the rockefeller university ) , 0.5 ci of [ -p ] atp and atp ( 20 m ) was incubated with the immunoprecipitated jnk in a total volume of 30 l of jnk reaction buffer for 20 min at 30c . the reactions were stopped with 2 loading buffer , boiled for 5 min , and run on a 12% sds - page gel . to measure nf-b activation , nuclear extracts prepared from thymocyte lysates stimulated with medium alone or tnf were used for gel - shift assays as outlined previously ( 5 ) . for thymocyte apoptosis assays , freshly isolated thymocytes ( 2 10 cells / well ) were treated with the indicated amounts of rmu tnf- ( r&d systems , inc . ) , or anti mu - fas mab ( jo2 ; pharmingen , san diego , ca ) for 22 h in the presence or absence of 30 g / ml cycloheximide ( chx ; sigma chemical co. , st . louis , mo ) , with anti - cd3 ( 10 g / ml ) and anti - cd28 antibody ( 10 g / ml ) , or with the indicated amounts of daunorubicin . viable cells were quantified for 30 s at a constant rate by flow cytometry after staining with 5 g / ml propidium iodide ( pi ) as described previously ( 5 , 13 ) . specific cell death ( % ) was determined from the percentages of viable ( pi - negative ) cells and was calculated as 100 ( 1 no . of viable cells in a treated sample / no . of viable cells in the control ) . transgenic mice expressing ib.dn or traf2.dn were described previously ( 5 , 12 ) . in vitro jnk assays were done as described previously ( 5 ) . in brief , 25 10 cells were treated with medium alone or rmu tnf- ( r&d systems , inc . , minneapolis , mn ) and lysed . for the kinase reaction , 1.53.0 g of purified glutathione s - transferase ( gst)c - jun(1 - 79 ) ( a gift from dr . h. hanafusa , the rockefeller university ) , 0.5 ci of [ -p ] atp and atp ( 20 m ) was incubated with the immunoprecipitated jnk in a total volume of 30 l of jnk reaction buffer for 20 min at 30c . the reactions were stopped with 2 loading buffer , boiled for 5 min , and run on a 12% sds - page gel . to measure nf-b activation , nuclear extracts prepared from thymocyte lysates stimulated with medium alone or tnf were used for gel - shift assays as outlined previously ( 5 ) . for thymocyte apoptosis assays , freshly isolated thymocytes ( 2 10 cells / well ) were treated with the indicated amounts of rmu tnf- ( r&d systems , inc . ) , or anti mu - fas mab ( jo2 ; pharmingen , san diego , ca ) for 22 h in the presence or absence of 30 g / ml cycloheximide ( chx ; sigma chemical co. , st . louis , mo ) , with anti - cd3 ( 10 g / ml ) and anti - cd28 antibody ( 10 g / ml ) , or with the indicated amounts of daunorubicin . viable cells were quantified for 30 s at a constant rate by flow cytometry after staining with 5 g / ml propidium iodide ( pi ) as described previously ( 5 , 13 ) . specific cell death ( % ) was determined from the percentages of viable ( pi - negative ) cells and was calculated as 100 ( 1 no . of viable cells in a treated sample / no . of viable cells in the control ) . to determine the relationship between traf2- and nf-b dependent antiapoptotic signaling pathways during tnf stimulation of normal cells , we have tested the role of these proteins in the tnf - induced apoptosis of thymocytes from transgenic mice expressing trans - dominant repressors of ib ( ib.dn ) and/or traf2 ( traf2.dn ) . expression of ib.dn , which lacks sequences required for signal - dependent degradation and functions as a constitutive repressor of multiple nf-b / rel proteins ( 14 ) , abolished the activation of nf-b , but not jnk , in thymocytes upon tnf stimulation ( fig . 1 ) . as reported previously ( 5 ) , expression of traf2.dn , which lacks the nh2-terminal ring and zinc finger domains , impaired the activation of jnk by tnf , but not that of nf-b . these results show that the activation of jnk and nf-b by tnf in thymocytes is mediated by two discrete signaling pathways ( fig . 1 ) . when both ib.dn and traf2.dn were expressed , the activation of nf-b and jnk by tnf were both severely impaired ( fig . 1 ) . to determine the relationship between the nf-b and traf2 pathways in normal cells , we measured tnf-induced death using thymocytes . cells from control mice were relatively resistant to tnf - induced apoptosis , which could be enhanced to a modest degree by treatment with chx to prevent new protein synthesis ( fig . inhibition of traf2 by traf2.dn expression potentiated the tnf - induced apoptosis of thymocytes , and this level of killing could be further increased by chx treatment , suggesting that tnf retained the ability to induce synthesis of antiapoptotic proteins despite the observed impairment of jnk activation ( fig . 2 , a and b ) . to determine whether nf-b activation suppresses the tnf - induced apoptosis of normal t lymphoid cells , thymocytes from ib.dn mice were treated with increasing concentrations of tnf . inhibition of nf-b activation by overexpression of ib.dn significantly enhanced tnf - induced apoptosis of thymocytes ( fig . chx treatment of thymocytes from ib.dn mice led to no further enhancement in their tnf - induced apoptosis , suggesting that the effect of chx on tnf - induced apoptosis is mediated mostly through inhibition of the synthesis of nf-b dependent antiapoptotic proteins ( fig . dn double - transgenic ( tg ) mice were treated with increasing concentrations of tnf- , they were at least 1,000 times more sensitive to tnf - induced apoptosis than cells from normal mice , and at least 100 times more sensitive than those from either ib.dn or traf2.dn tg mice ( fig . these results indicate that nf-b and traf2-dependent antiapoptotic signals synergistically protect cells from tnf - induced apoptosis . moreover , treatment of thymocytes from ib.dn traf2.dn double - tg mice with tnf- induced a level of apoptosis similar to that induced by tnf in chx - treated thymocytes from traf2.dn single - tg mice ( fig . this finding further supports the conclusion that the principal effect of chx is to inhibit de novo synthesis of antiapoptotic proteins that are regulated by the state of nf-b activation . fas death domain associated protein ( fadd ) , an effector of tnf - induced apoptosis recruited by heterotypic death domain interactions with tradd , is also an effector of apoptosis induced by the fas receptor ( 3 , 15 , 16 ) . moreover , nf-b signaling occurs in thymocytes in situ ( 17 , 18 ) , suggesting that the activation state of nf-b could influence fas - induced apoptosis . to investigate this possibility , thymocytes from the four sets of mice were cultured in the presence of increasing concentrations of a cross - linking anti - fas antibody ( fig . 3 a ) . compared with the dramatic increase in tnf - induced apoptosis caused by simultaneous inhibition of traf2 and nf-b activation , increased sensitivity to fas - induced apoptosis was minimal . in addition , when cd4cd8 thymocytes were activated by anti - cd3 and anti - cd28 antibodies , physiological stimuli involved in thymocyte selection , the induction of apoptosis in these cells was not affected by ib.dn and traf2.dn expression ( fig . tradd and the nf-b signaling pathway have also been implicated in the apoptosis induced by interactions between tnf - related apoptosis inducing ligand ( trail ) and death receptors ( dr4 , dr5 [ 19 , 20 ] ) . however , trail did not induce any significant cell death in wild - type thymocytes or in thymocytes from transgenic mice expressing traf2.dn and ib.dn ( data not shown ) . finally , nf-b activation has been suggested to play a role in the induction of apoptosis of some tumor cells by cancer chemotherapeutic compounds such as daunorubicin ( 11 ) , a finding offering the hope of potential therapeutic benefits . the therapeutic window for such effects would be more favorable if they were restricted to cancer cells and did not affect primary cells . importantly , then , neither traf2 nor nf-b activation played any role during daunorubicin - induced apoptosis of thymocytes ( fig . therefore , these results suggest that in nonneoplastic cells , the antiapoptotic role of traf2 and nf-b may be restricted to the context of tnf signaling . as shown in this study and in previous reports ( 5 , 6 , 8) , tnf - induced jnk and nf-b activation appear to be triggered by independent signaling pathways , with the former but not the latter mediated by traf2 . in addition , nf-b mediated antiapoptosis required de novo protein synthesis , whereas traf2-mediated antiapoptosis did not . therefore , it was surprising to see that traf2 and nf-b played synergistic roles in preventing tnf - induced apoptosis . one possibility is that traf2- and nf-b mediated pathways reconverge at some point to effect antiapoptosis during tnf stimulation ( fig . 4 a ) ; for instance , traf2 may be responsible for recruitment of an antiapoptotic protein whose basal level of expression is induced by tnf- in an nf-b dependent , jnk - independent manner . alternatively , traf2/jnk and nf-b activation could lead to the independent inhibition of discrete apoptotic effectors whose synergistic action is required for efficient induction of apoptosis ( fig . 4 b ) . although tnf was identified by its ability to kill various tumor cells , most cells do not undergo apoptosis in response to tnf . our results indicate that two distinct antiapoptotic signals are generated by tnf stimulation , and that removal of either one is insufficient to allow efficient apoptosis induction by tnf . thus , the combined inhibition of both antiapoptotic pathways during tnf stimulation may enhance the efficacy of tnf - based antineoplastic therapies . tnf - induced jnk and nf-b activation are mediated by distinct signal transduction pathways . ( top ) cell lysates prepared from thymocytes unstimulated ( ) or stimulated ( + ) with tnf ( 10 ng / ml ) for 2.5 min were used to measure jnk activity by immunocomplex kinase assay with gst c - jun(1 - 79 ) as a substrate . ( bottom ) nuclear extracts ( 4 g ) prepared from thymocytes unstimulated ( ) or stimulated ( + ) with tnf for 15 min were used in gel - shift assays of nf-b / rel binding activity . ( a ) thymocytes ( 2 10/ well ) from the indicated mice ( 68 wk old ) were treated for 22 h with increasing amounts of murine tnf- as indicated , and the percentages of cell death are shown as mean sd of triplicate samples from each group . similar results were also obtained with human tnf- ( data not shown ) , which is specific for murine tnfr1 . of note , the number and distribution of the major thymic subsets were normal , with the exception of a previously reported decrease in single positive cells in ib.dn-expressing mice and in double - tg mice expressing both traf2.dn and ib.dn ( data not shown ) ; however , this subset represented < 1% of thymocytes in wild - type littermates . ( b ) thymocytes from the indicated mice were treated overnight with tnf ( 33 ng / ml ) , with or without chx ( 30 g / ml ) , as indicated . mean ( sd ) data from one of three experiments , all of which yielded similar results , are shown . inhibition of traf2 and nf-b activity does not affect the sensitivity of thymocytes to apoptosis induced by other stimuli . thymocytes ( 2 10/well ) from the indicated mice were treated for 22 h with ( a ) anti - fas antibody ( jo2 ) ; ( b ) anti - cd3 and anti - cd28 antibodies ; or ( c ) daunorubicin . mean ( sd ) data from one of three experiments with similar results are shown .

tumor necrosis factor ( tnf ) signaling leads to pleiotropic responses in a wide range of cell types , in part by activating antiapoptotic and proapoptotic signaling pathways . thus , although tnf can cause apoptosis and may prove useful in the treatment of malignancies , most cells are resistant to tnf - induced cell death unless de novo protein synthesis is inhibited . previous studies suggested that tnf activation of the nuclear factor ( nf)-b transcription factor family antagonizes the proapoptotic signals initiated by tnf-. tnf receptor associated factor ( traf)2 has also been shown to mediate crucial antiapoptotic signals during tnf stimulation , yet is not essential in activation of nf-b under physiologic conditions , thus raising questions about the relationship between these antiapoptotic pathways . we report here that inhibition of traf2 and nf-b function in primary cells , by coexpression of a constitutive repressor of multiple nf-b / rel proteins ( ib.dn ) and a dominant negative form of traf2 ( traf2.dn ) , synergistically enhanced tnf - induced apoptosis . the effects were stimulus dependent , such that neither inhibitory molecule affected fas- and daunorubicin - induced apoptosis to the same degree as tnf - induced death . these findings indicate that the nf-b and traf2 pathways activate independent antiapoptotic mechanisms which act in concert to suppress the proapoptotic signals induced by tnf-.

Materials and Methods Transgenic Mice. Kinase and NF-B Assays. Cell Killing Assay. Results and Discussion Figures and Tables

identification of a myocardial pseudoaneurysm has a high morbidity and mortality and this case how special manipulation of the echocardiographic transducer not only identified the presence of an abnormal color doppler signal , but also prompted the use of agitated saline and contrast injections to confirm the presence of a pseudoaneurysm . we report a case of a 61-year - old caucasian female who presents to a community hospital with chest pain , shortness of breath , and electrocardiographic changes suggestive of an acute inferior myocardial infarction . upon arrival to our institution , cardiac catheterization was emergently performed in view of the persistent st - segment elevations in the inferior leads ii , iii , and avf with elevation in serum troponin levels and showed extensive disease of a dominant right coronary artery ( rca ) with an 8090% lesion in the midsegment followed by a long 6070% more distal stenosis as well as a proximal 70% lesion of the left anterior descending artery . right heart hemodynamics showed evidence of right ventricular infarction and multiple taxus stents were used to treat the lesions within the rca . a transthoracic echocardiogram was then performed in the intensive care unit for evaluation of left ventricular function showing an inferior wall aneurysm with overall preserved systolic function . three days later , another transthoracic echocardiogram was performed for a new murmur , which showed an abnormal color flow signal suggestive of a ventricular septal defect in the short axis view as seen in figure 1 . from the apical 4-chamber view , a cystic structure was seen lateral to the right ventricular wall [ figure 2 ] . inferior angulation of the transducer from the 4-chamber view , as seen in figure 3 , shows a long neck of the pseudoaneurysm connecting the inferior aneurysm to the cystic structure with color doppler confirming blood flow within this structure . a parasternal short axis view at the chordal level shows an abnormal color flow signal traversing the myocardium from the left ventricle , suggestive of a ventricular septal defect located in the posterior aspect of the inferior septum at the junction with the right ventricle . lv : left ventricle , rv : right ventricle four - chamber apical window view showing a cystic structure lateral to the right ventricular wall which appears to be within the ventricle shown by the white arrow . lv : left ventricle , rv : right ventricle ( a ) inferior angulation of the transducer from the 4 chamber view showed a long neck of a pseudoaneurysm connecting the inferior aneurysm to the cystic structure with ( b ) color doppler confirming blood flow within this structure . lv : left ventricle , rv : right ventricle , pseudo : pseudoaneurysm injection of agitated saline failed to opacify the cystic structure [ figure 4 ] . however , opacification of this cystic structure occurred after the intravenous administration of definity [ figure 5 ] . furthermore , a better definition of the contained rupture that compressed the basal right ventricular free wall was obtained with this maneuver [ figure 6 ] . injection of agitated saline was done and ruled out the presence of a ventricular septal defect or any other intracardiac shunt as the agitated bubbles stayed within the right ventricular chambers and this cystic structure was never opacified during this maneuver . rv : right ventricle , lv : left ventricle , la : left atria , pseudo : pseudoaneurysm opacification of this cystic structure occurs after the left ventricle was opacified with the intravenous administration of definity . lv : left ventricle , rv : right ventricle , la : left atria , pseudo : pseudoaneurysm furthermore , a better definition of the contained rupture or pseudoaneurysm that compressed the basal right ventricular free wall was obtained with this maneuver . lv : left ventricle , rv : right ventricle , ra : right atria , pseudo : pseudoaneurysm a pseudoaneurysm , or false aneurysm , develops after an acute myocardial infarction , which is complicated by a ventricular free wall rupture that is contained by localized pericardial adhesions . the diagnosis needs to be established early since these aneurysms are prone to rupture . in the most extensive review of the literature , frances et al . reported that 253 patients with a pseudoaneurysm in whom the cause was reported , 55% were related to an inferior wall myocardial infarction , as reported in our case . in this review , the authors also reported that the second most common cause of pseudoaneurysm formation was surgery , which was responsible for 33% of cases followed by trauma that accounted for 7% of cases . the site of pseudoaneurysm varies with etiology and in a review of 52 patients seen at the mayo clinic pseudoaneurysms were primarily seen in the inferior or posterolateral wall after a myocardial infarction , in the right ventricular outflow tract after congenital heart surgery , in the posterior subannular region of the mitral valve after mitral valve replacement , and in the subaortic region after aortic valve replacement . sudden cardiac death was the presenting manifestation in about 3% , whereas 12% of patients were asymptomatic . the most reliable method for diagnosis of a pseudoaneurysm has been angiography , which demonstrates a narrow orifice leading to a saccular aneurysm and lack of surrounding coronary arteries . transthoracic echocardiography has been considered a reasonable first step , but definitive diagnosis of pseudoaneurysm has only been reported in up 26% of patients . this case report presents this very unusual case showing how an abnormal color flow signal that traverses a myocardial wall is not always a ventricular septal defect . as described above , further echocardiographic manipulation utilizing modified views along with sequential injections of both agitated saline and definity proved very useful to identify the correct pathologic process and by itself was sufficient to provide definitive therapy . there was no further need for any other diagnostic test , and the patient was treated surgically , undergoing successful repair of the pseudoaneurysm as well as coronary artery bypass grafting of the left coronary artery .

a case of a patient presenting with an acute myocardial infarction is presented . a transthoracic echocardiographic examination revealed an abnormal color flow signal that traversed the myocardial wall from a large inferior aneurysm and initially considered to be a ventricular septal defect . however , further echocardiographic manipulation utilizing modified views along with sequential injections of both agitated saline and definity proved very useful to identify a pseudoaneurysm . there was no further need for any other diagnostic test , and the patient was treated surgically , undergoing successful repair of the pseudoaneurysm as well as coronary artery bypass grafting of the left coronary artery .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

image bioinformatics as a field is characterized by an odd mixture of tremendous advances in its computational statistics aspects while suffering from a persistent resistance to their use by biomedical domain experts . the image analysis platform with the deepest domain penetration in the research community is probably national institutes of health ( nih 's ) imagej in large part because of its free open source and pluggable implementation . over the years , an extensive community of image analysis developers and researchers has developed a comprehensive environment for image analysis software development using java . the development cycle can be roughly described as follows : when a promising new algorithm is identified , it might be first prototyped in a specialized environment such as mathwork 's matlab , but to reach a broader non - programming audience , it is then laboriously ported as an imagej plugin . illustrating the domain penetration challenge , a regular stream of new and updated plugins feeds a comprehensive repository that now has several hundred entries ( http://rsbweb.nih.gov/ij/plugins/ ) . however , a visit to the laboratory will show that those who could benefit from an add - on are typically not aware of them . even more striking is the situation that the few researchers using image analysis , who know how to find and install plugins , often also need image analysis applications that are slight modifications or combinations of existing functionalities . paradoxically , the computational statisticians with the quantitative expertise to develop those modifications often do not have the substantial software development skills needed to deliver the novel application as a stand - alone application or as a plugin module . a common solution to for example , the quantitative image analysis of estrogen receptor , progesterone receptor , and more generically , the ki-67 immunohistochemistry staining results in breast cancer tissue sections can be conveniently performed by submitting the image to a public web service . users benefit from improvements in the code automatically and no application needs to installed , therefore overcoming concerns with compromising privacy or degrading the performance of existing applications in the client machine . submitting to a remote service exposes the image to a third party outside the reach of institutional it ; updates in the web service may cause results not to be reproducible and the server side application is typically not exposed to public scrutiny , limiting the opportunities for other quantitative researchers to advance or even fully understand the underlying algorithms ; bioinformatics web services are also notorious for relatively short lifetimes , reflecting a sustainability model that can only be met in the long term by institutional commitment or by commercial products . a third solution might be to develop web services that deliver the software rather than process the data , the software - as - a - service ( saas ) model . the imagejs webapp ecosystem described in this report advances along those exact lines without crossing the lines set by requirements of privacy , scalability , reproducibility , and sustainable deployment associated with the biomedical environment . it also pays particular attention to the need for producing and delivering reproducible research , a topic that is taking center stage as bioinformatics increasingly seeks to deliver data - intensive solutions with translational usage . specifically , if an image analysis result can be retrieved directly from the published image , with both the data and the analytical environment that generated the result , then one could indeed claim to be reporting reproducible research . accordingly , imagejs seeks to deliver image analysis results meeting the gold standard in that report 's description of the spectrum of reproducibility [ figure 1 ] . imagejs migrates through script tag loading to the browser ( by pointing a script.src to http://imagejs.github.com/imagejs.js ) , where it creates an object with a method , imagejs . loadmodule , that will import and register additional components onto imagejs.modules , while keeping data meant to be shared between modules in imagejs.data . the corresponding uml representation is appended to the lower left side of the browser box . the solid lines represent programmatic exchanges and dashed line represents an exchange that requires user intervention , such as the loading or saving an image / analysis the possibility of approaching software development as an open organic process , which is explored by the imagejs webapp environment described here , has been proposed before as a requirement for medicine 2.0 . as discussed in that report , such an approach would blur the separation between software development and software deployment , turning coding into a conduit for workflow assembly that is more akin to social networking mechanisms . this is a departure from conventional bioinformatics infrastructure that can also be construed as a response to the mounting knowledge re - engineering bottleneck . it would provide a beyond the data deluge solution to image analysis where the computation is pushed to where the data is , rather than the customary approach of shuffling the data among a myriad of analytical services . imagejs was entirely developed in javascript ( ecmascript 5th ed . ) , the assembler language of the web . to illustrate the distributed and collaborative nature of the proposed webapp ecosystem , its component modules were developed in multiple versioned code hosting services , github ( http://github.com ) and google code ( http://googlecode.com ) . it can also be started directly from http://imagejs.github.com/ , which serves code hosted at https://github.com/imagejs/imagejs.github.com . the segmentation , feature extraction , and filtering modules were developed , and are hosted at http://imagejs.googlecode.com/. the list of modules can be accessed directly through http://module.imagejs.googlecode.com/git/ , and the development history can be inspected at http://code.google.com/p/imagejs/source/list?repo=module . in some examples , a web read - write resource is used to provide persistent image storage as a web service . a number of alternatives exist ; in this report , we used webrw http - rest api , which is supported by two independent implementations , both open source : one is written in nodejs ( https://github.com/jonasalmeida/webrw ) and another in php ( https://github.com/ebadedude/webrw ) . important note : although the core components and most modules will work in different web browsers , this study makes use of recent html5 features such as the file api ( http://www.w3.org/tr/fileapi/ ) that are still being adopted . to avoid problems with the syntax or support of recent developments in html5 , the reader is advised to use the latest version of google chrome ( version 18 at the time of this reporting ) . the imagejs browser based computational ecosystem is loaded by pointing it ( preferably using google chrome , see availability in materials and methods ) to http://imagejs.github.com . this action will cause imagejs 's document object , imagejs , to migrate to the web browser . a quick inspection of this object will reveal that it includes only a handful of methods , including the one that will load external code , loadmodule . a closer inspection of this method will show that a ) it imports external code by pointing a browser 's document object model ( dom ) script tag to the url of the external code ( module ) and b ) it registers the imported module in imagejs.module(url ) . as is the rest of this section , this inspection is also provided as a webcast video , publicly available at http://www.youtube.com/watch?v=qbkbgb4eche . the features of the imagejs environment as a vehicle to deliver computation were studied by developing and deploying an image analysis workflow that included standard modules for feature segmentation , analysis , and filtering . each of these modules was configured not only to deliver a desirable image analysis functionality and in the process test if the browser is a suitable platform to do so but also to test different models of dependency between modules . the goal of testing different models of articulation between analytical modules is to explore the balance between flexibility and robustness of browser - based computational ecosystems as platforms for collaborative software development . each module represents an independent development exercise , hosted and versioned in its own externally hosted service . as stated in the materials and methods section , to reinforce the perception of independent development , the multiple modules and imagejs core code are scattered between distinct versioned hosting services , namely , github and google code . examples of interface triggers for loading of modules illustrated in this section : menu driven , dependency driven , and programmatically associated with action by another module . the analytical workflow can therefore be assembled by loading modules in response to actions by the user or it can be initiated by a particular result of the analysis the image analysis procedure developed starts with the selection by the user of a chromomarker . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . for example , the n m distance matrix , d , defined by equation 1 , can be resolved efficiently by nesting two map operations on the image rgb intensities , i : d = i.map(function(x , i){return x.map(function(y , j){returni[r , g , b](i , j ) } ) } ) ( 3 ) the efficiency of this implementation in imagejs relies on the javascript compiler mapping the explicit parallelization of the code to local opportunities for distribution . for example , by possibly using the machine 's graphics processing units ( gpus ) . as can be verified by operating imagejs ( or by viewing the accompanying webcast at http://www.youtube.com/watch?v=qbkbgb4eche ) , chromomarkers will display the edges of segmented image superimposed on the original image . the edge detection is performed by another mapping operation which is worth inspecting in the native javascript syntax to recognize the nested mapping pattern used in equation 3 , and is highlighted below in the code for equation 4 . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . feature extraction represents a tougher challenge . here , the goal is to identify which sets of segmented pixels define a contiguous feature . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . the description of this feature extraction procedure provides the opportunity to demonstrate how code can be invoked in a publication without concern that subsequent updates or renaming will cause this information not to be available . for example , at the time of this writing , the code for feature extraction can be found in line # 171 of version 4ec7931d17ccc17d27e724d51ae0 of jmat.js , which can be following that link will not only return the code used , but also expose comments made by co - developers . it is again worth noting that although the imagejs core is hosted and versioned at imagejs.github.com , this procedure happens to be hosted and versioned by a distinct environment , this time at google . both hosting services , which is to say , the hosting services resolved by the links , are likely to last longer ( forever ) than the interest or even the memory of its authors . the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . in the previous section , the feature extraction procedure was described by reference to the specific version and line of code deployment at the time of this writing . the ability to reference specific versions of the code therefore extends to the ability to execute them as well . this feature is enhanced by imagejs by allowing the submission of modules by passing them as arguments to the url call . for example , http://imagejs.github.com/?https://raw.github.com/imagejs/imagejs.github.com/27b596ed08d41e79c6c96e86f0318694ca98cc9e/mainmenu.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.chromomarkers.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.countshapes.js specifies a very particular combination of versions of the multiple modules involved in the assembly of imagejs . this is of course not a convenient url to exchange , so a shorter reference could be generated , for example , http://bit.ly/h3hvbz . it is worth repeating the note that none of the resources used to enable the assembly of a specific version of the imagejs tool depends on the commitment of the authors of this report to maintain those resources . the imagejs computational environment will natively allow for three mechanisms to incorporate code generated by others . the easiest to distribute is to simply add the address of the modules to the url of imagejs core application , as illustrated above . a more interactive possibility is to use an input box as exemplified by the main menu / load option . the filtering of segmented images offers an illustration of the usefulness of this mechanism in facilitating the interaction with researchers focused on the computational statistics aspects of the analysis . this module , mathbiol.filtershapes , is strictly dedicated to specifying a number of matrix manipulation procedures erode and round at the time of this writing . the association of these procedures with the user interface buttons that trigger them is dealt by the feature extraction module . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . figure 2 summarizes the orchestration of modules described here , highlighting the salient features and dependencies they were designed to illustrate . the conventional approach of manually loading images from the local file system , by file select or by drag and drop , is supported . manually saving the image and its analysis results back to the files system is also supported and consists of saving the contents of imagejs.data [ figure 1 ] as a json structure . this behavior relies on a relatively new development in web computing standards , the file api ( http://www.w3.org/tr/fileapi ) . another possibility that is particularly useful in collaborative and distributed environments is to rely on the read - write capabilities anticipated for web 3.0 apps . in a nutshell , this is a cloud computing option that treats web services as both remote storage and remote devices , allowing for exchange of data simply by reference . one of the modules , countshapes , illustrates this functionality by using the webrw service described in the materials and methods section . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . accordingly , the same module loading mechanism described in the previous sections will now be used to deal with and saving and loading of data . as an example , see either https://bitly.com/withdata or point a qr reader to the qr code in figure 3 http://bit.ly/withdata.qrcode ; both link to the same data with analysis , as depicted in figures 3 and 4 . quick response ( qr ) code , generated automatically by http://bit.ly/withdata.qrcode for the link http://bit.ly/withdata , which in turn points to http://imagejs.github.com/?http://module . js and http://165.225.128.64/?doc=uid5716226333752275 and http:// module.imagejs.googlecode.com/git/mathbiol.showdata.js , which corresponds to a particular image analysis result that is assembled , re - analyzed , reproducing the display depicted in figure 4 image analysis corresponding to http://bit.ly/withdata . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . as proposed , this was pursued by building not only on top of the imagejs abstraction [ figure 1 ] but also integrated with one of the generic modules described earlier , chromomarkers . as a consequence , this exercise was conveniently reduced to the writing of a few image analysis filters that rely on the chromomarkers segmentation utilities to select segmentation thresholds . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . to address the vagaries of manual estimation of proliferation from such images , the ki67 imagejs module was designed to assist with the calculation and , more importantly , to report the final value with the graphic display of the corresponding segmentations . a snapshot of this application is presented in figure 5 , and can be invoked by a short link such as http://bit.ly/ki67js or http://uab.mathbiol.org/ki67 in a modern web browser , preferably google chrome . snapshot of the result of using imagejs 's ki67 module to determine proliferation index in a labeled glioblastoma sample imaged by transmission microscopy . a webcast illustrating its usage is available at http://www.youtube.com/watch?v=ncpngrxwwdq and is also provided with the help option of this imagejs application the image analysis procedure developed starts with the selection by the user of a chromomarker . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . for example , the n m distance matrix , d , defined by equation 1 , can be resolved efficiently by nesting two map operations on the image rgb intensities , i : d = i.map(function(x , i){return x.map(function(y , j){returni[r , g , b](i , j ) } ) } ) ( 3 ) the efficiency of this implementation in imagejs relies on the javascript compiler mapping the explicit parallelization of the code to local opportunities for distribution . for example , by possibly using the machine 's graphics processing units ( gpus ) . as can be verified by operating imagejs ( or by viewing the accompanying webcast at http://www.youtube.com/watch?v=qbkbgb4eche ) , chromomarkers will display the edges of segmented image superimposed on the original image . the edge detection is performed by another mapping operation which is worth inspecting in the native javascript syntax to recognize the nested mapping pattern used in equation 3 , and is highlighted below in the code for equation 4 . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . feature extraction represents a tougher challenge . here , the goal is to identify which sets of segmented pixels define a contiguous feature . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . the description of this feature extraction procedure provides the opportunity to demonstrate how code can be invoked in a publication without concern that subsequent updates or renaming will cause this information not to be available . for example , at the time of this writing , the code for feature extraction can be found in line # 171 of version 4ec7931d17ccc17d27e724d51ae0 of jmat.js , which can be forever available at http://code.google.com/p/jmat/source/browse/jmat.js?r=4ec7931d17ccc17d27e724d51ae0342b871bc98a#171 . following that link will not only return the code used , but also expose comments made by co - developers . it is again worth noting that although the imagejs core is hosted and versioned at imagejs.github.com , this procedure happens to be hosted and versioned by a distinct environment , this time at google . both hosting services , which is to say , the hosting services resolved by the links , are likely to last longer ( forever ) than the interest or even the memory of its authors the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . in the previous section , the feature extraction procedure was described by reference to the specific version and line of code deployment at the time of this writing . the ability to reference specific versions of the code therefore extends to the ability to execute them as well . this feature is enhanced by imagejs by allowing the submission of modules by passing them as arguments to the url call . for example , http://imagejs.github.com/?https://raw.github.com/imagejs/imagejs.github.com/27b596ed08d41e79c6c96e86f0318694ca98cc9e/mainmenu.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.chromomarkers.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.countshapes.js specifies a very particular combination of versions of the multiple modules involved in the assembly of imagejs . this is of course not a convenient url to exchange , so a shorter reference could be generated , for example , http://bit.ly/h3hvbz . it is worth repeating the note that none of the resources used to enable the assembly of a specific version of the imagejs tool depends on the commitment of the authors of this report to maintain those resources . the imagejs computational environment will natively allow for three mechanisms to incorporate code generated by others . the easiest to distribute is to simply add the address of the modules to the url of imagejs core application , as illustrated above . a more interactive possibility is to use an input box as exemplified by the main menu / load option . the filtering of segmented images offers an illustration of the usefulness of this mechanism in facilitating the interaction with researchers focused on the computational statistics aspects of the analysis . this module , mathbiol.filtershapes , is strictly dedicated to specifying a number of matrix manipulation procedures erode and round at the time of this writing . the association of these procedures with the user interface buttons that trigger them is dealt by the feature extraction module . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . figure 2 summarizes the orchestration of modules described here , highlighting the salient features and dependencies they were designed to illustrate . the conventional approach of manually loading images from the local file system , by file select or by drag and drop , is supported . manually saving the image and its analysis results back to the files system is also supported and consists of saving the contents of imagejs.data [ figure 1 ] as a json structure . this behavior relies on a relatively new development in web computing standards , the file api ( http://www.w3.org/tr/fileapi ) . another possibility that is particularly useful in collaborative and distributed environments is to rely on the read - write capabilities anticipated for web 3.0 apps . in a nutshell , this is a cloud computing option that treats web services as both remote storage and remote devices , allowing for exchange of data simply by reference . one of the modules , countshapes , illustrates this functionality by using the webrw service described in the materials and methods section . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . accordingly , the same module loading mechanism described in the previous sections will now be used to deal with and saving and loading of data . as an example , see either https://bitly.com/withdata or point a qr reader to the qr code in figure 3 http://bit.ly/withdata.qrcode ; both link to the same data with analysis , as depicted in figures 3 and 4 . quick response ( qr ) code , generated automatically by http://bit.ly/withdata.qrcode for the link http://bit.ly/withdata , which in turn points to http://imagejs.github.com/?http://module . js and http://165.225.128.64/?doc=uid5716226333752275 and http:// module.imagejs.googlecode.com/git/mathbiol.showdata.js , which corresponds to a particular image analysis result that is assembled , re - analyzed , reproducing the display depicted in figure 4 image analysis corresponding to http://bit.ly/withdata . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . as proposed , this was pursued by building not only on top of the imagejs abstraction [ figure 1 ] but also integrated with one of the generic modules described earlier , chromomarkers . as a consequence , this exercise was conveniently reduced to the writing of a few image analysis filters that rely on the chromomarkers segmentation utilities to select segmentation thresholds . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . to address the vagaries of manual estimation of proliferation from such images , the ki67 imagejs module was designed to assist with the calculation and , more importantly , to report the final value with the graphic display of the corresponding segmentations . a snapshot of this application is presented in figure 5 , and can be invoked by a short link such as http://bit.ly/ki67js or http://uab.mathbiol.org/ki67 in a modern web browser , preferably google chrome . snapshot of the result of using imagejs 's ki67 module to determine proliferation index in a labeled glioblastoma sample imaged by transmission microscopy . a webcast illustrating its usage is available at http://www.youtube.com/watch?v=ncpngrxwwdq and is also provided with the help option of this imagejs application because of the increasing volume and heterogeneity of data , the organic development of software that delivers computational solutions to translational environments is not only a challenge of increasing proportions , but also one with increasing rewards . accordingly , advances in biomolecular methodology , coupled with the benefits of an integrated environment in which data science in a biomedical context may be applied , are increasingly seen as a key new frontier for pathology . the opportunity to develop encompassing integrative infrastructure for image analysis has not been missed by the research community , with very comprehensive initiatives such as omero advancing open source software that pathologists with access to informatics research resources and partnerships can use . the advantages of these partnerships are also increasingly compelling as the amount of annotated image data in the public domain increases . furthermore , these are also increasingly object of large - scale modeling exercises such as the recent morphological analysis of the cancer genome atlas ( tcga ) slide images to identify classifiers of tumor subtypes . these advances make the prospect of pushing image analysis to the lab medicine or to the point of care all the more enticing and the inability to do so all the more frustrating . in principle , applications such as nih imagej provide a good solution to the last mile problem of delivering computational solutions to the environments and machines that have access to the image data that need to be analyzed . the use of the java language ( the j in imagej ) to deliver such a solution is in line with its 1990s motto of write once , run anywhere . it was then widely anticipated that this would be the language of the web and that not only would applications be delivered to the browser as applets ( http://java.sun.com/applets/ ) but also eventually the browser would mature to such a high level of integration with the java language ( it would have a native jvm ) that even individual procedures would be delivered into a communal computational pool where an ecosystem of workflows might emerge in reaction to the data and user interaction context . in a nutshell , the web browser was , correctly , anticipated to become the ultimate saas platform endowed with efficient processors and with a protective sandbox such that the machine 's own file system would not be exposed to the foreign code injection . this should allow for the promise of personalizing the analysis of an image in that private file system to benefit from the wealth of reference data being annotated elsewhere . in fact , this promise has since been delivered , not by object - oriented java which still requires a virtual machine to be first loaded by the browser before being interpreted , but by its little functional helper , javascript , which went on to provide the basis for the second generation of web technologies a decade later . another key variation on the original prediction was the emergence of cloud computing infrastructure as a logical extension of the machine 's file system . this is the modern scenario that the imagejs prototype , described in the results section , explores : we now have all the features needed for pervasive image analysis that we can port all the way to the point of care . specifically , a ) we have an efficient computational environment and b ) we can deliver saas in a manner that is both safe and reproducible . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . initially , it was not clear that this environment is sufficiently performant for image analysis . the world wide web consortium ( w3c ) has recognized the need for metrics for different computational operations , but has only recently standardized their acquisition . we have also recently found out when functionally decomposing sequence analysis into mapreduce operations that these improvements may actually justify rather than deter the use of the browser to deploy advanced computational statistics procedures . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . this argument will be on even more solid ground if efficiency refers to power consumption , since that is the very argument why web browsers in mobile devices are increasingly not supporting graphics rendering using adobe 's flash , microsoft 's silverlight , and oracle - sun 's jvm . the argument that the browser offers a safer delivery of applications is the same that leads it departments to disable the installation of applications by researchers and clinicians in biomedical facilities . there certainly are opportunities to make webapps unsafe , but the fact that they are executed within the browser 's sandbox raises effective barriers to accessing the file system or devices attached to the machine where the webapp is being executed . the argument for a more reproducible delivery of image analysis solutions is also not an absolute claim . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . as illustrated in figure 4 , the reproduction in the client machine can even include the assembly of the analytical environment where alternative analysis can be attempted . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . initially , it was not clear that this environment is sufficiently performant for image analysis . the world wide web consortium ( w3c ) has recognized the need for metrics for different computational operations , but has only recently standardized their acquisition . we have also recently found out when functionally decomposing sequence analysis into mapreduce operations that these improvements may actually justify rather than deter the use of the browser to deploy advanced computational statistics procedures . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . this argument will be on even more solid ground if efficiency refers to power consumption , since that is the very argument why web browsers in mobile devices are increasingly not supporting graphics rendering using adobe 's flash , microsoft 's silverlight , and oracle - sun 's jvm . the argument that the browser offers a safer delivery of applications is the same that leads it departments to disable the installation of applications by researchers and clinicians in biomedical facilities . there certainly are opportunities to make webapps unsafe , but the fact that they are executed within the browser 's sandbox raises effective barriers to accessing the file system or devices attached to the machine where the webapp is being executed . the argument for a more reproducible delivery of image analysis solutions is also not an absolute claim . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . as illustrated in figure 4 , the reproduction in the client machine can even include the assembly of the analytical environment where alternative analysis can be attempted . imagejs provides a pervasive mechanism to deliver image analysis workflows and interactive interfaces to where the data are . it includes a core object model equipped with data - specific attributes and methods , such as a module loader that registers the source urls of the components loaded . the ability to define workflows by chaining the addresses of the modules with imagejs 's own the use of versioned code hosting as a mechanism for deployment is also a stark departure from conventional bioinformatics infrastructure , where the ensuing code injection is normally avoided rather than promoted . however compelling this mechanism for distributed development and deployment may be , and no matter how convenient and scalable the delivery of computation to image repositories may be , imagejs webapp ecosystem is primarily an experiment in informatics . the ability to gather data about the population of this computational webapp ecosystem by others and about collaborative data processing to inform further development of distributed image analysis infrastructure may well be its most valuable purpose .

background : image bioinformatics infrastructure typically relies on a combination of server - side high - performance computing and client desktop applications tailored for graphic rendering . on the server side , matrix manipulation environments are often used as the back - end where deployment of specialized analytical workflows takes place . however , neither the server - side nor the client - side desktop solution , by themselves or combined , is conducive to the emergence of open , collaborative , computational ecosystems for image analysis that are both self - sustained and user driven.materials and methods : imagejs was developed as a browser - based webapp , untethered from a server - side backend , by making use of recent advances in the modern web browser such as a very efficient compiler , high - end graphical rendering capabilities , and i / o tailored for code migration.results:multiple versioned code hosting services were used to develop distinct imagejs modules to illustrate its amenability to collaborative deployment without compromise of reproducibility or provenance . the illustrative examples include modules for image segmentation , feature extraction , and filtering . the deployment of image analysis by code migration is in sharp contrast with the more conventional , heavier , and less safe reliance on data transfer . accordingly , code and data are loaded into the browser by exactly the same script tag loading mechanism , which offers a number of interesting applications that would be hard to attain with more conventional platforms , such as nih 's popular imagej application.conclusions:the modern web browser was found to be advantageous for image bioinformatics in both the research and clinical environments . this conclusion reflects advantages in deployment scalability and analysis reproducibility , as well as the critical ability to deliver advanced computational statistical procedures machines where access to sensitive data is controlled , that is , without local download and installation .

INTRODUCTION MATERIALS AND METHODS RESULTS Dissimilarity Metric Module Module Modular Orchestration Data Management A Pathology Application: KI67 Labeling for Cellular Proliferation DISCUSSION The Web Browser for Computational Efficient Image Analysis Safe and Reproducible SaaS? CONCLUSION

intestinal parasitic infections are widely distributed throughout the world causing substantial intimidation to the public health , economy , and physical and cognitive development particularly among children in developing countries like ethiopia . the poor personal hygiene , poor environmental hygiene , and poor health system commonly observed in developing countries make the prevalence to be highest among these populations [ 1 , 2 ] . the consumption of fruits and vegetables helps in protecting human body from a number of diseases by providing nutrients , vitamins , minerals , protein , and fibers . it could also have a positive impact on body - weight regulation and related conditions , including diabetes and hypertension . however , fruits and vegetables , especially , those that are consumed raw and or not properly washed , have been the major way for the transmission of human pathogens [ 35 ] . intestinal parasitic infection may be acquired in different ways like by consumption of contaminated fruits , vegetables , other food stuff , and water . eating unclean , raw , or undercooked fruits and vegetables is one of the means by which the transmission of intestinal parasitic infections is propagated . fruits and vegetables act as vehicles for the transmission of parasitic infections when contaminated as a result of various associated factors related to planting , such as while they are still on the field , harvesting , transportation , storage , market chain , and even at home [ 5 , 8 ] . despite the fact that intestinal parasitosis is common in jimma town [ 9 , 10 ] , there are no studies conducted to assess the level of contamination of fruits and vegetables with parasites of medical and zoonotic importance . if our target is to control the intestinal parasitic diseases , it is not enough to depend merely on the chemotherapeutic intervention of identified cases , but need the concerted effort to reduce and eliminate the potential sources of infection . to our knowledge there is no published document to attest the level of parasitological contamination of fruits and vegetables in jimma town . therefore , this study was designed to determine the level of parasitic contamination of selected fruits and vegetables and associated factors in jimma town . the study was conducted in jimma town , which is located at south west of ethiopia , about 352 km from addis ababa , the capital of ethiopia . a cross - sectional study was conducted to determine the level of parasitic contamination of fruits and vegetables sold in selected local markets in jimma town from april 22 to may 14 , 2013 . eight types of fruits and vegetables including lettuce , cabbage , carrot , tomato , green pepper , banana , mango , and salad were purchased from four conveniently selected local markets , namely , bishishe , hirmata merkato , kochi , and agip found in jimma town . equal numbers of samples ( 45 each , totally 360 samples ) were collected from the selected markets . the samples were collected , put in plastic bags , properly labeled , and brought to the medical parasitology laboratory of jimma university , for parasitological analysis . a portion ( 200 g ) of each fruit and vegetable was washed separately in 500 ml of normal saline for detaching the parasitic stages ( ova , larvae , cysts , and oocysts ) of helminths and protozoan parasites commonly assumed to be associated with vegetable contamination . after overnight sedimentation of the washing solution , 15 ml of the sediment was then transferred to a centrifuge tube using sieve , to remove undesirable matters . for concentrating the parasitic stages , finally , the sediment was examined under a light microscope using 10 and 40 objectives . modified zeihl - neelsen staining technique was also used for identification of oocysts of cryptosporidium and cyclospora spp as described elsewhere . data were entered into , cleaned , and analyzed using spss for windows version 16.0 . the difference between prevalence of intestinal parasites among different categories was compared using pearson chi - square test . univariate and multivariate logistic regression was used to identify factors associated with parasitic contamination of the fruits and vegetables data collection using questionnaire was done after the purpose of the study was explained to the respondents ( vendors of fruits and vegetables ) and verbal consent was obtained . a total of 360 samples of fruits and vegetables were collected from the local markets and examined for parasitological contamination . the results of the study showed that 208 samples were identified to be contaminated with at least one type of parasite , which gave rise to the overall contamination rate of 57.8% . these include 53% of green pepper , 68.9% of cabbage , 55.6% of lettuce , 77.8% of salad , 62.2% of carrot , 46.7% of tomato , 51.1% of banana , and 46.7% of mango ( table 1 ) . the stages and species of parasites detected include larvae of strongyloides like parasite , ova of ascaris lumbricoides , toxocara spp , hymenolepis nana , and hymenolepis diminuta , oocysts of cyclospora spp and cryptosporidium spp , and cysts of giardia lamblia , and entamoeba histolytica / dispar . table 2 shows that strongyloides like parasite ( 21.9% ) was the most frequently detected parasitic contaminant followed by toxocara spp ( 14.7% ) , cryptosporidium spp ( 12.8% ) , h. nana ( 8.3% ) , g. lamblia ( 7.5% ) , a. lumbricoides ( 6.7% ) , e. histolytica / dispar ( 5.3% ) , cyclospora spp ( 5.0% ) , and h. diminuta ( 1.4% ) . the highest frequency of strongyloides like parasite was detected in samples of salad and the least frequency from samples of carrot and tomato . ova of toxocara spp was detected most frequently from cabbage samples but not recovered from mango samples . ova of a. lumbricoides was detected from salad samples with highest frequency and not detected from samples of green pepper . 37.5% of the total samples were contaminated with two species of parasites , while 6.25% of the samples with three species of parasites and quadruple parasitic contamination were observed in two samples ( table 1 ) . strongyloides like parasite and toxocara spp combination was the most frequently encountered with 35.48% of the multiple contamination . the parasitic contamination rate among the different fruits and vegetables was significantly different ( p = 0.027 ) table 3 . the highest prevalence of intestinal parasites was recorded in salad ( 16.8% ) followed by cabbage ( 14.9% ) , carrot ( 13.5% ) , lettuce ( 12% ) , green pepper ( 11.5% ) , banana ( 11.1% ) , and tomato and mango each 10.1% . further analysis with a binary logistic regression showed that , as - compared to mango , salad was significantly contaminated ( aor = 4.5 , 95% ci ( 1.7 , 11.9 ) ) ; see table 4 . in addition to the parasitological investigations , factors associated with contamination of fruits and vegetables were also assessed . these factors were assessed by interviewing the vendors of fruits and vegetables in the selected markets of jimma town . the educational status of the vendors was ascertained and the majority ( 52% ) of the vendors had no formal education , while 36% of the vendors had primary education and only 12% had secondary education . there was no significant association between education level of vendors and parasitic contamination rate of the produces they were selling ( p = 0.845 ) , table 3 . bishishe ( 28.4% ) , agip ( 22.1% ) , and kochi ( 19.7% ) markets . the percentage contamination rate was significantly different among samples collected from the different markets ( p = 0.003 ) , table 3 . samples from groceries contributed for 15.4% of the positive samples while 84.6% of the contamination was contributed by open markets with no statistically significant difference ( p = 0.811 ) , table 3 . another factor associated with parasitic contamination of fruits and vegetables is the act of washing the produces before displaying for sale . according to this study , majority ( 73.1% ) of the produces were not washed before display for selling while only 26.9% of the produces were washed before displaying for sale . 82.2% of the unwashed produces were contaminated with one or more parasites , while 17.8% of the washed produces were contaminated with intestinal parasites . the cross tabulation of washing the produces before display for sale and result of parasitological analysis showed a significant difference in contamination rate among washed and unwashed produces ( p = 0.000 ) , table 3 . as compared to the washed produces , the odds of parasitic contamination for unwashed ones was 3.33 times ( aor = 3.3 , 95% ci ( 1.9 , 5.6 ) ) , table 4 . the sources of water used for washing the produces among the vendors include pipe water ( 62.5% ) , well water ( 30.2% ) , and river water ( 7.3% ) . well water , and river water were contaminated with at least one parasitic species , respectively . there was no significant difference in contamination rate among produces washed by water from different sources ( p = 0.412 ) , table 3 . the means of display for selling is also another factor assessed for association with parasitic contamination of fruits and vegetables . various means of display were observed among the vendors as follows : 67.5% of the produces are displayed on the floor by the road sides while 23.1% on tables by the road sides . the detection of intestinal parasitic stages from fruits and vegetables is an indicative of the fecal contamination from human and or animal origin . as in many tropical countries , intestinal parasites are widely distributed in ethiopia not only due to the favorable climatic conditions for the survival and dissemination of the parasites but also due to the unsanitary conditions that facilitate fecal pollution of water , food stuffs , and soil . the present study has attempted to assess the level of contamination and prevalence of different intestinal parasites from different fruits and vegetables sold in selected markets of jimma town . the overall parasitic contamination rate was found to be 57.8% , which is in agreement with the findings reported elsewhere [ 3 , 5 ] . however , it is higher than what was reported in similar studies from other areas [ 8 , 1217 ] . on the other hand , it is lower when compared with the findings of some studies [ 18 , 19 ] . the discrepancy between the present study and previous studies might be as a result of the variations in geographical locations , climatic and environmental conditions , the kind of sample and sample size examined , the sampling techniques , methods used for detection of the intestinal parasites , and socioeconomic status . so long as these factors differ , consequently the discrepancy of the results would be expected . salad ( 77.8% ) was found to be the most frequently contaminated produce followed by cabbage ( 68.9% ) , carrot ( 62.2% ) , lettuce ( 55.6% ) , green pepper ( 53.3% ) , and banana ( 51.1% ) . this variation among the produces might be due to the fact that salad , cabbage , carrot , and lettuce have uneven surfaces which make the parasitic stages attach more easily to the surface of these vegetables . the smooth surface of green pepper , tomato , and mango might reduce the rate of parasitic attachment hence had lower contamination rate . in this study , larvae of strongyloides like parasite was the most frequently detected parasite with a prevalence of 21.9% . this might be due to the fact that the parasite has a free living state and does not require a host for its proliferation , in addition to its parasitic mode of life . the predominance of strongyloides like parasite is similar with similar studies conducted elsewhere [ 5 , 8 , 12 , 17 ] . however , the finding is in contrast with what was reported by other investigators where ascaris lumbricoides , cryptosporidium spp , e. histolytica / dispar , and toxocara spp were the predominant parasites detected [ 1316 ] . ova of toxocara spp was the second most prevalent contaminant next to strongyloides like parasite . this dominance might be attributed to the high fertility of toxocara female adult producing up to 10,000 eggs daily and the resistant nature of the eggs , which may survive for up to ten years resisting harsh conditions in the environment . cryptosporidium spp was the third most frequently detected parasite in this study with a prevalence of 12.8% . however , majority of researches did not report the parasite as vegetable contaminant [ 3 , 5 , 12 , 13 , 2022 ] . this might be due to differences in methods used between this study and the previous ones . in the present study , modified acid fast stain was used for detection of the coccidian oocysts , while the majority of the previous studies did not . no ova of hookworm species were detected from the samples examined in the present study . this is in agreement with other studies conducted elsewhere [ 3 , 14 , 16 , 20 , 23 ] . this might be due to the fact that hookworms have very short life span in the soil . however others have reported the contamination of vegetables with hookworm species [ 5 , 6 , 25 ] . the differences might be attributed to differences in geographical locations , climate conditions , and the type of soil . this might indicate the possibility of high level contamination of the fruits and vegetables , which perhaps results in multiple parasitic infections in human . the contamination rate was significantly different for the samples collected from the different markets in which samples collected from this might be associated with the act of washing of the produces before display ; 77.8% of the samples collected from the market were not washed . majority ( 69% ) of the produces were displayed for sale on the floor where it is exposed to dusts and flies . it is well established that the flies can act as vectors for a number of pathogenic microorganisms including parasites like cryptosporidium parvum and toxoplasma gondii . besides , there might be bacterial and viral contamination of the produces during display for sale on the floor . the habit of eating raw vegetables like salad and tomato is commonly practiced in the study area . hence , the findings of the present study are of public health importance , requiring an appropriate intervention to prevent transmission of parasitic diseases that can be acquired through consumption of contaminated fruits and vegetables . however , this study did not address the effect of seasonal variation on the contamination of the fruits and vegetables . the findings of this study could not underscore the infectivity of the parasitic stages detected as viability study was not conducted . in conclusion , this study highlighted the importance of raw fruits and vegetables as the potential source of transmission for intestinal parasites to humans . the fruits and vegetables contamination with the pathogenic parasites poses health risk to the consumers if consumed without proper cleaning and or cooking . a comprehensive health education should be given to vendors and farmers of fruits and vegetables and to the general population on the health risks associated with consumption of contaminated fruits and vegetables . the consumers should always observe the basic principle of food and personal hygiene , that is , thorough washing of the fruits and vegetables before eating and washing hands before meal . the vendors of fruits and vegetables should avoid the contact of the produces with soil while display for selling . further studies should be conducted on the viability of parasitic contaminants of fruits and vegetables . also , other researches must be done to evaluate the level of parasitic contamination of farm produces , water , and soil in which fruits and vegetables are cultivated .

background . a study aimed at determining the prevalence and predictors of parasitic contamination of fruits and vegetables collected from local markets in jimma town , ethiopia , was conducted between april and may 2013 . methods . a total of 360 samples of fruits and vegetables were examined by sedimentation concentration after washing using normal saline . results . the overall prevalence of parasitic contamination was 57.8% . strongyloides like parasite ( 21.9% ) was the most frequent parasitic contaminant followed by toxocara spp ( 14.7% ) , cryptosporidium spp ( 12.8% ) , h. nana ( 8.3% ) , g. lamblia ( 7.5% ) , a. lumbricoides ( 6.7% ) , e. histolytica / dispar ( 5.3% ) , cyclospora spp ( 5.0% ) , and h. diminuta ( 1.4% ) . washing of the fruits and vegetables before display for selling was significantly associated with decreased parasitic contamination ( p < 0.001 ) . conclusion . since fruits and vegetables are potential sources of transmission for intestinal parasites in the study area , consumers should always avoid acquiring parasitic infection from contaminated fruits and vegetables supplied in jimma town through proper cleaning and cooking .

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

ulcerative colitis ( uc ) is a chronic disease characterized by diffuse mucosal inflammation of the colon . it involves the rectum and may extend proximally in a contiguous pattern to cause procto - sigmoiditis , left - sided colitis , or pancolitis . uc is initially treated 5-aminosalicylate and oral glucocorticoids or immunosuppressive agents for refractory patients , and proctocolectomy in advanced cases . fluorine-18 fluorodeoxyglucose positron emission tomography / computed tomography ( fdg pet / ct ) can be used to identify regions of active inflammation in uc . here present a patient with pan - uc with multiple strictures , in whom fdg pet / ct was used to noninvasively evaluate the extent and severity of the disease . a 60-year - old male patient with a history of uc for 2 years presented with fever for 3 weeks duration while on treatment with standard immunosuppressive drugs . fdg pet / ct was performed to look for any inflammation in the large bowel [ figure 1a c ] . a few fdg avid retroperitoneal lymph nodes were detected [ arrows in figure 1 ] , with no definite abnormality in the large intestine . the entire large intestine showed healthy mucosa during colonoscopy , and he reported relief of symptoms with conservative management . one year later , he reported increased frequency of bowel movements ( > 6/day ) and more than 10% weight loss . sigmoidoscopy showed loss of vascular pattern with granularity , ulcerations , and pseudopolyps in the rectosigmoid . biopsy from this region showed changes suggestive of acute colitis . with his symptoms persisting for more than 6 months , fdg pet / ct [ figure 2a c ] was repeated 18 months after the first scan to rule out malignant transformation in the sigmoid colon , after stopping oral steroids for 15 days . diffusely increased fdg uptake was seen in homogeneous mural thickening involving the entire large intestine . the scan showed intensely fdg avid multiple segmental strictures [ arrows in figure 2 ] located closely in the ascending colon and approximately every 5 cm in the transverse colon , descending colon and sigmoid colon , the longest measuring 7 cm . since the patient was not responding to medical treatment , total proctocolectomy with ileo - pouch anal anastomosis ( tpiaa ) and loop ileostomy was performed . maximum intensity projection , ( a ) coronal fused ( b ) and axial fused ( c ) images of initial fluorodeoxyglucose positron emission tomography / computed tomography show a few fluorodeoxyglucose - avid retroperitoneal lymph nodes ( arrows ) , with no definite abnormality in the large intestine maximum intensity projection , ( a ) coronal fused ( b ) and axial fused ( c ) images of second fluorodeoxyglucose positron emission tomography / computed tomography show diffusely increased fluorodeoxyglucose uptake in homogeneous mural thickening involving the entire large intestine and intensely fluorodeoxyglucose - avid multiple segmental strictures ( arrows ) . no abnormal extra - intestinal fluorodeoxyglucose uptake is seen in the maximum intensity projection image it involves the rectum and may extend proximally in a contiguous pattern to cause proctosigmoiditis , left - sided colitis , or pancolitis . increased frequency of bowel movements ( > 6/day ) with more than 10% weight loss constitutes a severe form of the disease . uc is initially treated with a combination of rectal and oral 5-aminosalicylate and oral glucocorticoids or immunosuppressive agents for refractory patients , followed by maintenance therapy . this patient initially had nonactive left - sided colitis that progressed to active pancolitis over a period of 2 years as demonstrated noninvasively by fdg pet / ct . diagnosis with colonoscopy would have been difficult due to nonnegotiable strictures in the sigmoid colon . fdg pet / ct can be used to identify regions of active inflammation in uc . this uncommon case demonstrates that , in patients with multiple segmental strictures involving the entire colon , this investigation can be used to noninvasively evaluate the extent and activity of the disease to guide further management .

ulcerative colitis ( uc ) is an inflammatory bowel disease characterized by waxing and waning inflammation that changes in severity and extent and may progress to neoplasia , especially in the presence of strictures . when patients have nonnegotiable strictures or severe inflammation with ulcers , colonoscopy is difficult and carries the risk of perforation . the authors present a patient with pan - uc with multiple strictures , in whom fluorodeoxyglucose positron emission tomography / computed tomography was used to noninvasively evaluate the extent and severity of the disease .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

ductal carcinoma in situ ( dcis ) is characterized by proliferation of malignant cells confined by the basement membranes of ductal structures , without evidence of extraductal invasion . dcis comprises different subtypes with heterogeneous proliferative disease processes and varies in architecture , imaging features , and clinical outcome . although the progression of dcis is not fully understood , it is known that untreated dcis is likely to progress to invasive cancer within 10 years of diagnosis . therefore , dcis is considered a preinvasive form of invasive breast cancer that requires immediate treatment [ 2 , 3 ] . with the widespread use of screening mammography , dcis now accounts for 15%20% of all newly detected breast cancer , with the trend still increasing . in mammography , although mammography has been the mainstay for diagnosis of dcis , it has limitations in defining the extension and the margin , especially in patients without microcalcifications or in those with dense breasts or breast implants . furthermore , there is a tendency to underestimate the tumor size of dcis on mammography [ 57 ] . positive surgical margin is known as an independent risk factor for local recurrence [ 810 ] . dynamic contrast - enhanced mri ( dce - mri ) has been proven more sensitive than mammography for detecting breast cancer in young women with a high risk of developing breast cancer [ 1113 ] . in many instances , dce - mri can reveal early stage breast cancer , including dcis and dcis with small invasive carcinomas , which are mammographically , sonographically , and clinically occult . in addition to screening , breast mr can also be used for staging purposes to better characterize the disease extent and the presence of multifocal multicentric lesions , which can impact on treatment planning and the subsequent management . imaging characterization of patients with mixed cohort of pure dcis and dcis with invasive components [ 1417 ] and patients with merely pure dcis [ 1825 ] on dce - mri has been reported before . in a study of 167 patients , kuhl et al . found that mammography only diagnosed 56% pure dcis preoperatively , while mri achieved a diagnostic rate of 92% . for 89 high - grade lesions , mri detected 87 lesions ( 98% ) while mammography only diagnosed 52% of lesions . nevertheless , consensus regarding how mri can be used to aid in management of pure dcis has not been reached yet . given the recent evidence that additional diseases diagnosed by preoperative mri often led to more aggressive treatment yet did not improve the treatment outcome [ 26 , 27 ] , continuing exploration of the role of breast mri for the diagnosis of dcis is needed . the purpose of this retrospective study was to characterize the morphology and enhancement kinetic pattern of pure dcis on dce - mri . since high - grade dcis is associated with a greater risk of local recurrence and progression to invasive breast cancer [ 2830 ] , it would be very helpful if the high - grade and low - grade dcis could be differentiated on imaging . in this paper , the mri features of dcis between high - grade ( grade iii ) and non - high - grade ( including low grade i and intermediate grade ii ) dcis based on the van nuys classification were compared . a total of 34 consecutive patients with histological - proven pure dcis ( without any microinvasion ) were identified from our breast mri database collected from 2002 to 2006 . these patients came to our center to participate in a breast mri research study due to suspicious lesions found in mammography , sonography , or physical examination . the medical records , breast mri images , and mammogram reports ( if available ) were reviewed retrospectively . among these 34 patients , three patients had received surgical excision biopsy before mr imaging , and they were excluded in the analysis . the final diagnosis of pure dcis was obtained from pathological examination of mastectomy ( n = 21 ) and lumpectomy ( n = 10 ) specimens . this study was approved by the institutional review board , and informed consents were obtained from all patients . the mri study was performed using a 1.5 t phillips eclipse mr scanner with a standard bilateral breast coil ( philips medical systems , cleveland , ohio ) . the imaging protocol consisted of high - resolution precontrast imaging and dynamic contrast - enhanced imaging . after setting the iv line , then a sagittal view unilateral t1-weighted precontrast images were acquired from the breast of concern , using a spin echo pulse sequence with tr = 1000 ms , te = 12 ms , and fov = 20 cm , and matrix size = 256 256 . following this , a 3d spgr ( rf - fast ) pulse sequence with 16 frames ( repetitions ) was prescribed for bilateral dynamic imaging . thirty - two axial slices with 4 mm thickness were used to cover both breasts . the imaging parameters were tr = 8.1 ms , te = 4.0 ms , flip angle = 20 , matrix size = 256 128 , fov = 3238 cm . the sequence was repeated 16 times for dynamic acquisitions , four precontrast , and 12 postcontrast sets . the contrast agent ( omniscan , 1 cc/10 lbs body weight ) was manually injected at the beginning of the 5th acquisition and was timed to finish in 12 seconds to make the bolus length consistent for all patients . immediately following the contrast , 10 cc saline the subtraction images at 1-minute postinjection were generated by subtracting the precontrast images acquired in frame number 3 from the postcontrast - enhanced images acquired in frame number 6 . the maximum intensity projections ( mips ) were also generated from the subtraction images to help identify the lesion . the enhancement kinetic was analyzed from manually selected roi ( region of interest ) based on the subtraction images at 1-minute post - injection . the enhanced tumor area was outlined on each imaging slice covering the lesion by an experienced breast radiologist ; then a mean signal intensity time course from all 16 time frames was obtained . the percent enhancement time course was calculated by first subtracting the mean precontrast signal intensity ( mean of first 4 frames ) from each of the subsequent 12 postcontrast signal intensities and then normalized by the mean precontrast signal intensity 100% . morphological appearances and enhancement kinetic features of lesions shown on mri were categorized according to the acr breast imaging reporting and data system ( bi - rads ) breast mri lexicon . the features were evaluated by 2 radiologists ( with 4 and 5 years of experience in reading breast mri ) separately . for cases with different results , based on the lesion size , the mass type can be further separated into single focus / multiple foci ( < 5 mm ) and mass ( 5 mm ) . then for mass lesions other characteristics such as shape , margin , and internal enhancement patterns were evaluated . the nonmass - like lesions were described by the type of enhancements ( diffuse , regional , segmental , focal , ductal , linear ) and the internal enhancement patterns ( punctate / stippled , clumped , and heterogeneous ) . the tumor size was measured as the longest diameter on the maximum intensity projection ( mip ) of subtraction image . the enhancement kinetics was divided into two phases : the initial enhancement phase , defined as enhancement patterns within the first 2 minutes or before the curve starts to change , and the delayed phase , defined as enhancement pattern after 2 minutes or after the curve starts to change . when the enhancement kinetics showed a rapid initial enhancement followed by washout or reaching to a plateau in the delayed phase , it was determined as suspicious of malignancy . all cases are pure dcis without presence of microinvasion . the pathological diagnosis was evaluated based on the van nuys system . dcis was classified according to the nuclear grade ( non - high grade versus high grade ) and the morphologic subtype ( comedo and noncomedo ) . grade - i ( low grade ) and grade - ii ( intermediate grade ) were categorized as non - high grade , and grade - iii was the high - grade . for statistical analysis , fisher 's exact test was used to examine the significant difference between non - high grade dcis and high grade dcis . according to the nuclear grading system , 2 patients had grade - i , 16 patients had grade - ii , and 13 patients had grade - iii , all together 18 non - high grade and 13 high grade . the mean age for non - high grade dcis was 52 years old and 57 years old for high - grade dcis ( p = .27 ) . the comedo type was noted in 12 patients ( 7 high grade and 5 non - high grade ) , and the noncomedo type was noted in 19 patients ( 6 high grade and 13 non - high grade ) ( table 1 ) . there was no significant difference in the histological comedo or noncomedo subtype between the non - high grade and high grade dcis ( p > .05 ) . two cases of high - grade dcis with noncomedo morphology were not detected by mri and were false negative diagnosis . the imaging features of these 29 cases are summarized in table 2 , and 5 case examples are shown in figures 15 . among these 29 cases , mass lesions were seen in 15 cases ( 52% ) including 12 masses and 3 focus / foci lesions and nonmass - like lesions in 14 cases ( 48% ) . two cases were multifoci ( figures 2 and 5 ) , and the size was not measured . for the remaining cases , the size ranged from 0.4 to 5 cm with the mean of 1.9 1.2 cm . the mean size was 1.6 1.2 cm for non - high grade dcis and 2.2 1.3 cm for high - grade dcis , not significantly different ( p = .21 ) . for the two false negative cases , in mammography , one case was occult , and the other was undetermined ( i.e. , needed additional imaging evaluation ) . the mammographically occult case received ultrasound exam and found a suspicious hypoechoic mass in the left breast . after needle biopsy confirming malignancy , the patient received lumpectomy , and a pure dcis of 1.4 cm was found . this patient was diagnosed with dcis in the right breast 2 years ago and had already received right mastectomy . she decided to receive left mastectomy , and a pure high grade dcis of 1.1 cm was found . among the 15 mass lesions , 9 cases were non - high grade , and 6 were high grade . for the 14 nonmass - like lesions , 9 lesions were non - high grade , and 5 cases were high grade ( p = .26 ) . the shape , margin , and internal enhancement patterns were evaluated in 12 mass lesions that were 5 mm . the most frequently seen features were irregular shape ( 50% ) , spiculated / irregular margin ( 92% ) , and heterogeneous enhancement ( 67% ) ( table 3 ) . there was no significant difference in the mr morphology pattern between the non - high - grade and high - grade mass type dcis lesions . three case examples are shown in figure 1 ( low grade ) , figure 3 ( intermediate grade ) , and figure 4 ( high grade ) . of 14 nonmass lesions , 4 lesions showed regional enhancement ( 28% ) , 3 showed ductal ( 22% ) , and 3 showed focal ( 22% ) enhancements . internal enhancement pattern was dominated by the clumped pattern ( figure 5 , high grade ) ( 9/14 , 64% ) followed by the heterogeneous enhancement pattern ( 4/14 , 29% ) ( table 4 ) . two grade - ii lesions had severe motion artifacts due to patient 's movement during the delayed phase , and the enhancement kinetic curves could not be reliably measured . the early postcontrast subtraction images had good quality , and the morphological features of these two lesions could be evaluated . twenty - one lesions ( 21/27 , 78% ) showed the suspicious malignant type enhancement kinetics with a rapid initial enhancement followed by plateau ( figures 1 and 5 ) or washout ( figures 24 ) , including 1/2 grade - i lesion , 12/14 grade - ii lesions , and 8/11 grade - iii lesions . therefore , no significant differences were found between enhancement kinetics of high - grade and non - high - grade dcis ( p = .38 ) . among the other 8 enhanced lesions that showed benign type enhancement kinetic curves or undetermined kinetic curves , 6 lesions showed malignant morphological features . therefore , of the total 31 cases , mri diagnosed 27 as suspicious of malignancy , with the sensitivity of 87% ( 27/31 ) . correlation between screening mammogram and mr imaging was performed in 22 patients whose mammography reports were available . sixteen lesions ( 16/22 , 73% ) were classified as bi - rads 4 or 5 , as suspicious or five of 22 patients were classified as bi - rads category 1 ( normal mammogram ) and one patient as bi - rads category-0 ( need additional imaging evaluation ) . on mri , four of 5 patients with normal mammogram were correctly diagnosed as suspicious of malignancy , showing malignant type enhancement kinetics ( rapid enhancement followed by washout or plateau ) and/or the morphological appearance ( irregular shape and spiculated / irregular margin ) . mammography may miss dcis that does not present certain suspicious patterns of microcalcifications , and mri may have a complementary role in diagnosis of these lesions . most of the mr imaging studies of dcis analyzed mixed patient cohort of pure dcis and dcis with invasive components . several studies reported the mr kinetic and morphologic appearance of pure dcis correlated with histopathology findings [ 1825 ] . in a study by kuhl et al . , only 56% pure dcis was detected by mammography , while mri could detect more than 90% . in another study of 33 pure dcis patients by vag et al . the sensitivity of mammography and mri was 64% and 88% , respectively . in this study , we detected 29/31 ( 94% ) enhancing lesions and diagnosed 27/31 ( 87% ) as suspicious of malignancy . of the 22 patients whose mammography reports were available , the sensitivity of mammography was 74% ( 16/22 ) . the result was comparable to other published studies [ 15 , 17 , 19 ] . it was found that most false - negative diagnosis of dcis by mri appeared to be non - high - grade , noncomedo - type lesions [ 23 , 32 , 33 ] . in this study , we had two false negative cases of high - grade dcis with noncomedo morphology . one case showed strong background tissue enhancements in both breasts , and the 1.4 cm lesion could not be identified . the other case showed clumped enhancement in the affected breast , and the 1.1 cm dcis could not be identified . nuclear grade has been consistently associated with poor prognosis and local recurrence in dcis [ 26 , 3436 ] , and the combination of nuclear grade and comedo necrosis was reported to correlate with the risk of local recurrence after breast conserving surgery [ 34 , 35 ] . found that high - grade pure dcis missed by mammography can be diagnosed by mri alone . the diagnostic yield of mri for non - high - grade pure dcis is also significantly higher than mammography . in the series reported by vag et al . , all 12 mammographically occult dcis lesions ( three low grade , four intermediate grade , five high grade ) were correctly diagnosed by mri . in our series all these 4 lesions were high grade . with the prognostic significance of early detection for high - grade dcis , this result further strengthens the recommendation of mri for diagnosis of dcis . pure dcis lesions often appear as nonmass clumped enhancement in a regional , ductal , or linear distribution [ 16 , 18 , 21 , 22 ] . reported a high specificity of focal branching enhanced pattern on mri for dcis . as the spatial resolution of mri improves , it is anticipated that the ductal pattern as linear or linear branching may be seen more often , which can be a signature feature on mri suggesting dcis . in our study , the typical morphologic appearance of pure dcis on mri was a mass- or a nonmass - like lesion with heterogeneous or clumped enhancement . although our study showed a higher percentage of mass lesions ( 52% ) ( figures 3 and 4 ) compared to previous publications [ 18 , 22 ] , the classification of mass and nonmass lesions is subjective , and this may lead to variations in the determined percentages . in the study by viehweg et al . , of the 48 enhancing pure dcis lesions , 35 lesions ( 35/48 , 73% ) showed either well - defined ( n = 12 ) or ill - defined focal mass lesions ( n = 23 ) . the percentage will change if some ill - defined mass lesions are reclassified as nonmass lesions . , we did not find significant differences between morphologic patterns of high - grade and non - high - grade dcis . pure dcis does not always exhibit the typical malignant washout kinetic curves and could show persistent and plateau curve types [ 16 , 18 , 21 , 22 ] . reported that the peak enhancement ratio at one minute ( e1 ) for pure dcis was less than invasive cancers and more than benign lesions . studies on the enhancement kinetic characteristics among the different nuclear grades of pure dcis did not show consistent results . some reports have indicated that the kinetic characteristics of low - grade pure dcis lesions are different from those of intermediate- and high - grade lesions [ 18 , 37 ] , whereas other studies have revealed no difference [ 22 , 34 ] . we did not find significant difference between the enhancement kinetics of high - grade and non - high - grade groups . for the size measurement of dcis , different local recurrence rates were found between dcis lesions smaller and larger than 10 mm , thus an accurate size measurement is important . . showed a 8% deviation of mri measured tumor size relative to histopathologic size , compared with 29% for mammography . despite that in general the size measurement is more accurate on mri , false positive findings and tumor size overestimation may occur on mri . reported that cases in which mri overestimated dcis were mostly non - high - grade and noncomedo - type lesions . the false - positive enhancement in most cases is due to coexistence of benign proliferative processes such as fibrocystic changes or atypical ductal hyperplasia [ 16 , 40 ] . several limitations existed in our study , including small case number and retrospective case review . nevertheless , the reported cases were from consecutive patients referred to participate in a breast mri research study , with diagnosis of pure dcis ; therefore , allowing objective analysis as presented in this study . the research study was designed to analyze the dce kinetics , so a protocol with a relatively high temporal resolution ( 42 seconds compared to 2 minutes used in a clinical setting ) was chosen . this could only be accomplished by using a compromised spatial resolution , which would inevitably affect the quality of the image . however , since the tumor size of our subjects was relatively large ( 1.6 1.2 cm for mass lesions and 2.2 1.3 cm for nonmass lesions ) , we believe that the relatively low spatial resolution did not affect our findings on the analysis of lesion morphology . the morphological features of the three focal lesions < 5 mm were not evaluated . the feature about spiculated margin might be affected by the low spatial resolution , and the percentage of lesions with spiculated margin could be higher than the reported 42% ( 5/12 cases ) . therefore , the cases with spiculated and irregular margin were reported together . with improved spatial resolution and reduced slice thickness , it is possible that more subtle morphological features , such as linear or linear branching rather than regional enhancements , can be revealed for improved diagnosis of dcis . in this retrospective analysis , the majority ( 94% ) of pure dcis lesions showed contrast enhancement . mri was more sensitive in detecting pure dcis compared to mammography , hence reinforcing the role of mri in detection and preoperative management of pure dcis . however , we did not observe any significant difference in mr morphologic or kinetic features between non - high - grade and high - grade pure dcis . therefore , our results did not provide strong evidence to support the use of mri in staging of dcis for treatment planning .

to characterize imaging features of pure dcis on dynamic contrast - enhanced mr imaging ( dce - mri ) , 31 consecutive patients ( 37 - 81 years old , mean 56 ) , including 2 grade i , 16 grade ii , and 13 grade iii , were studied . mr images were reviewed retrospectively and the morphological appearances and kinetic features of breast lesions were categorized according to the acr bi - rads breast mri lexicon . dce - mri was a sensitive imaging modality in detecting pure dcis . mr imaging showed enhancing lesions in 29/31 ( 94% ) cases . pure dcis appeared as mass type or non - mass lesions on mri with nearly equal frequency . the 29 mr detected lesions include 15 mass lesions ( 52% ) , and 14 lesions showing non - mass - like lesions ( 48% ) . for the mass lesions , the most frequent presentations were irregular shape ( 50% ) , irregular margin ( 50% ) and heterogeneous enhancement ( 67% ) . for the non - mass - like lesions , the clumped internal enhancement pattern was the dominate feature , seen in 9/14 cases ( 64% ) . regarding enhancement kinetic curve , 21/29 ( 78% ) lesions showed suspicious malignant type kinetics . no significant difference was found in morphology ( p > .05 ) , tumor size ( p = 0.21 ) , and kinetic characteristics ( p = .38 ) between non - high grade ( i+ii ) and high - grade ( iii ) pure dcis .

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

bullous systemic lupus erythematosus ( sle ) is a clinicopathologically distinctive immunobullous eruption occurring during the course of sle . circulating antibodies to type vii collagen ( nc1 domain ) subepidermal blister formation in the course of severe systemic lupus erythematosus ( sle ) can occur due to extensive interface inflammation and basal cell vacuolation , presenting as polycyclic erosions with advancing blistering border predominantly on sun - exposed areas . in contrast , bullous systemic lupus erythematosus ( bsle ) is a distinctive bullous eruption occurring with sle , presenting with typical clinical and pathological findings including circulating antibodies to type vii collagen ( nc1 [ noncollagenous domain 1 ] domain ) . occasionally other antigens such as laminin 5 , laminin 6 , and bp230 ( bullous pemphigoid antigen ) have also been described . a 39-year - old lady , a homemaker and mother of three , presented with largely asymptomatic vesicobullous lesions ( 2 months duration ) , involving head and neck , upper limbs , and trunk over both photo - exposed and unexposed areas . the blisters would rupture over 2 - 3 days ; yielding clear ( sometimes hemorrhagic ) fluid ; heal over 2 - 3 weeks ; leaving behind pigmentation and occasional scarring . concurrent painful oral ulcers were reported ; though , other mucosae were uninvolved . a 6-month history suggestive of raynaud 's phenomenon ; arthralgias involving small joints of hands and feet ; the patient reported three previous , spontaneous abortions , ( 5months gestation ) the last one being 6 years back . on examination , there were multiple , tense , clear as well as hemorrhagic , vesicles and bullae , arising over an erythematous base and predominantly involving upper body [ figure 1 ] . in addition , superficial erosions , areas of postinflammatory hyperpigmentation , and small superficial scars were noted . superficial erosions were also noted over the buccal mucosa , hard palate , and lips . presence of tense vesicles ( marked with an arrow ) filled with cleat fluid along with superficial erosions with polycyclic margins . lesions are present on covered as well as exposed areas a provisional diagnosis of bsle was considered and she was thoroughly investigated . she had a significant anemia ( 8.8 gm / dl ) with evidence of iron deficiency on peripheral smear and iron studies . extensive serological workup revealed antinuclear antibody positivity of 1:640 ( using hep-2 cells ) ; positive anti - dsdna , anti - ro , and anti - la antibodies ; low levels of c3 and c4 with a normal ch50 component ; negative antiphospholipid antibody , rheumatoid factor , and c - reactive protein . x - rays of chest and involved joints were normal . a lesional biopsy revealed subepidermal split with neutrophilic infiltrate in the papillary dermis [ figure 2 ] . direct immunofluorescence ( both perilesional and distant - covered skin ) revealed igm and c3 deposition along the basement membrane zone ( bmz ) as well as perivascular location . due to resource constraints , indirect immunofluorescence or salt - split skin based on clinical , histopathological , and serological evidence , a diagnosis of bsle was confirmed . biopsy taken from vesicular lesion revealed a subepidermal split , containing numerous red blood cells . a marked neutrophilic infiltrate is seen in the papillary dermis ( h and e , 250 ) . inset shows a positive lupus band ( igg ) at the basement membrane zone ) with a normal glucose 6-phosphate dehydrogenase ( g6pd ) activity , we planned to start her on dapsone ; however , her low hemoglobin prevented this . she was started on prednisolone ( 1 mg / kg / day as a single morning dose ) ; hydroxychloroquine ( 200 mg twice a day ) ; along with complete sunprotection and topical care . there was only partial improvement with new lesions developing even after 4 weeks ; wherein , she was started on dapsone . she responded quite favorably with no new lesions and satisfactory healing of pre - existing ones [ figure 3 ] . over the next 6 months , steroids were completely withdrawn , and dapsone and hydroxychloroquine continued for further 1 year . the patient has remained in remission without any systemic manifestations as well , over the last 1 year . cutaneous lesions are reported during the course of sle in 76% patients ; however , vesicobullous lesions account for less than 1% of these . it generally affects young adult females and involves both sun - exposed and nonexposed areas . clinically , it is characterized by subepidermal blisters ; appearing on erythematous or urticated base ; with a predilection for face , upper trunk , and proximal extremities . lesions can mimic bullous pemphigoid ( bp ) , inflammatory variant of epidermolysis bullosa acquisita ( eba ) , and linear iga disease . our understanding of the pathology of this interesting and unique dermatologic condition has evolved with improved diagnostic criteria and clinicohistologic profiling . gammon and briggaman suggested six diagnostic criteria : fulfillment of american rheumatism association ( ara ) criteria for sle ; acquired vesicobullous eruption on ( but not limited to ) sun - exposed skin ; histologic evidence of subepidermal blister with neutrophilic infiltrate ; presence of igg , igm , iga , and c3 at the bmz ; evidence of antibodies to type vii collagen ; and codistribution of immunoglobulin deposits with anchoring fibrils / type vii collagen by immunoelectron microscopy . on the basis of these , bsle can be of two distinctive types : type i fulfilling all six criteria , while type ii ( undetermined antigen or dermal antigen other than type vii collagen ) satisfies criteria 1 - 4 only . as seen in our case , bsle developing with or prior to features of sle can present a diagnostic dilemma ; hence , other subepidermal vesicobullous disorders need to be ruled out . histopathologically , a predominant neutrophilic infiltrate may suggest dermatitis herpetiformis ( dh ) and dramatic response to dapsone is also common to both diseases . eba is histopathologically and immunopathologically identical , as it is also mediated by antibodies to type vii collagen ; however , a dramatic response to dapsone , as in our case , helps differentiate the two as eba is notoriously resistant to treatment . the major antigenic epitopes for both bsle and eba reside within the fibronectin type iii homology region ( fn3 ) of nc1 domain of type vii collagen . this region is important in mediating the interaction between anchoring fibrils and other matrix proteins . blister formation occurs as a result of antibody - induced interference with normal interactions between type vii collagen and its extracellular matrix ligands ; destabilization of anchoring fibrils due to impaired antiparallel dimer formation of type vii collagen ; or complement mediated tissue damage . whether or not bsle is associated with systemic activity is a hotly debated topic . some authors argue against this ; though , others have reported a clinical and/or serological flare with the eruption . in our patient , symptoms of sle developed shortly before the eruption , though no significant systemic activity was recorded ; however , a meticulous search for systemic involvement is highly suggested . most reported cases have dramatically responded to dapsone , with cessation of new lesions in 1 - 2 days and healing within several days . the same was seen in our case with dapsone ( 2 mg / kg / day ) . though , it may be difficult to introduce the drug initially , as in our case ; every attempt should be made to use it , as relatively low doses ( 25 - 50 mg ) have also been shown to be efficacious . a relative resistance to steroids and immunosuppressives our patient showed only modest improvement on steroids and antimalarials , complete response being seen only with dapsone . the case is presented for its typical and distinctive features which should prompt an astute physician to suspect and investigate underlying systemic manifestations of sle . also , the dramatic response to dapsone is highlighted which remains the drug of choice in the management of this disorder . dapsone should be considered the drug of choice for bullous systemic lupus erythematosus as treatment response to other options is suboptimal . even if one is not able to initiate dapsone due to relative contraindications , as in our case , every effort should be made to use it as early as possible . the course of the disease is benign with remission reported in most of the cases within a year , including ours .

bullous systemic lupus erythematosus ( bsle ) is a rare manifestation of systemic lupus erythematosus ( sle ) , characterized histologically by subepidermal bullae with predominantly neutrophilic infiltrate and immunoglobulin ( igg , iga , igm ) and c3 deposition at the basement membrane zone . often associated is a heterogeneous profile of autoimmunity to components of type vii collagen . it needs to be differentiated from other subepidermal bullous diseases in view of potentially associated systemic manifestations of sle . we report a 39-year - old lady who presented with an extensive vesicobullous eruption on face , neck , trunk , and mucosa . she was also found to have photosensitivity , significant anemia , and high antibody titers confirming sle . routine histopathology and immunoflorescence studies suggested bsle and she responded favorably to dapsone . the importance of clinical and histopathological findings in confirming a diagnosis of bsle along with the very encouraging response to dapsone therapy is highlighted .

Introduction Case Report Discussion

colorectal cancer is a common disease . according to the ministry of health , labour and welfare , cancer has been the most common cause of death in japan since 2013 ; colorectal cancer is the most frequent cause of cancer - related death in women . colonoscopy plays a critical role in the early detection and treatment of colorectal cancer 1 2 . oral polyethylene glycol ( peg ) is the most popular means of preparing the bowel for colonoscopy in japan , but magnesium citrate ( mc ) and sodium phosphate ( sodium dihydrogen phosphate monohydrate / sodium dihydrogen phosphate anhydrous ) are also widely used . oral bowel preparation for colonoscopy is associated with potential adverse events 3 . many studies have reported adverse events ( aes ) associated with colonoscopy , but most have focused on those caused by the conduct of colonoscopy or by endoscopic treatment of the abnormalities detected rather than those caused by bowel preparation for colonoscopy 4 . the most severe aes associated with oral bowel preparation are large bowel obstruction ( lbo ) and perforation 5 . acute , complete lbo requires emergency abdominal surgery , and is associated with high morbidity and mortality rates if left untreated . in september 2003 , an emergency safety bulletin was issued by the ministry of health , labour and welfare in japan highlighting the risks of lbo and perforation caused by oral bowel preparation for colonoscopy 6 . according to this bulletin , lbo occurred in 7 patients between july 1993 and september 2003 ( 1of whom died [ 14.3 % ] ) and perforation occurred in 11 patients ( 5 of whom died [ 45.5 % ] ) . when a patient had severe constipation or standard preparation was suboptimal , we always used additional preparation . nevertheless , the risk of lbo or perforation may be elevated by the use of additional bowel preparation drugs . consequently , clinicians must have a high index of suspicion that complications might arise in patients with severe constipation , or in whom bowel preparation is inadequate with standard techniques . but it contained no scientific analysis of the likely pathogenesis of the adverse events documented . consequently , the risk factors for lbo and perforation during bowel preparation for colonoscopy are not fully understood . we undertook a retrospective analysis of patients who had developed lbo or a bowel perforation associated with oral preparation drugs , to inform the development of a suite of preventive measures for patients undergoing colonoscopy . we undertook a retrospective analysis of patients clinical records at showa university northern yokohama hospital ( sunyh ) , japan . the subjects of the study were patients of sunyh treated between april 2001 and december 2015 , who were diagnosed with lbo or perforation caused by oral drugs for bowel preparation . we recorded the following for each patient : age , sex , indications for colonoscopy , preparation drug or drugs used , location of lbo / perforation , pathogenesis , requirement for surgery , requirement for emergency surgery , requirement for colostomy and mortality . from these data , we analyzed the pathogenesis of lbo and perforation , and we propose prophylactic measures for the prevention of these aes . we identified 20 patients ( 13 men and 7 women ) diagnosed with lbo or perforation associated with oral bowel preparation drugs , who were treated at sunyh in the study period . fourteen cases underwent colonoscopy at sunyh , while six occurred at other hospitals and were referred to sunyh for treatment . the frequency of lbo or perforation associated with oral bowel preparation at sunyh was 0.016 % ( 14 out of 86,463 colonoscopies ) . among the 14 patients of sunyh , 12 were diagnosed with lbo and two were diagnosed with perforation . a representative case of bowel perforation is illustrated in fig . 1 . a representative case of a patient with large bowel perforation . the patient drank 2800 ml polyethylene glycol and defecated 7 times , and subsequently reported sudden - onset abdominal pain and cold sweats . we performed computed tomography and confirmed the presence of free air , ascites and rectal cancer . emergency surgery was undertaken and at laparotomy , a large amount of intraperitoneal stool was present . the mean duration of symptoms before colonoscopy was undertaken was 70.7 92.4 days ; 8 patients had presented with constipation , 6 with abdominal pain and 2 with anorexia ; 3 were asymptomatic . mc , magnesium citrate ; lbo , obstruction ; p , perforation ; t , tumor ( uicc t classification ) ; r , rectal ; s / c , sigmoid colon ; t / c , transverse colon ; a / c , ascending colon ; lit , long intestinal tube ; sunyh , showa university northern yokohama hospital ; peg , polyethylene glycol . there were three types of cancer : 1 s / c and 2 r cancer . the drug used for bowel preparation was peg in 15 cases and mc in 1 case ; 1 patient was given both , 2 were given sodium picosulfate hydrate ( sph ) and 1 patient was given sph and mc the day before colonoscopy . the cause of the ae was judged to be advanced cancer in 18 patients , transverse colon stenosis resulting from inflammation that had spread from an inflamed gallbladder in 1 patient , and bowel obstruction by feces in 1 patient . for patients with cancer , the mean maximum tumor diameter was 55.6 21.8 mm . the tumor was situated in the sigmoid colon in 10 patients , the rectum in 4 patients , the transverse colon in 2 patients and the ascending colon in 2 patients . patients with lbo were treated in 1 of 3 ways : urgent surgery within 3 days ( 8 patients ) ; elective surgery ( 3 patients ) ; or decompression by placement of a long intestinal tube ( lit ) or self - expanding metallic stent ( sems ) followed by surgery ( 7 patients ; 5 had a lit and 2 a sems ) . we have used a strategic protocol for colonoscopy preparation since 2004 ( fig . 2 ) , to prevent lbo and perforation . a detailed history of defecation status during preparation taken by specialist endoscopy nurses can contribute to early prevention and detection of aes . after we started using the protocol , the incidence of aes was reduced from 0.044 % ( 2001 2003 , 4 out of 9,175 patients ) to 0.013 % ( 2004 this difference was not statistically significant ( p = 0.053 ) , but might suggest the presence of a trend towards fewer aes since introduction of the protocol ( p < 0.05 was considered significant ) . strategy for colonoscopy preparation at the showa university northern yokohama hospital for preventing bowel obstruction and perforation . peg , polyethylene glycol ; ct , computed tomography ; ge , glycerin enema ; lit , long intestinal tube ; sems , self - expandable metallic stent . large bowel obstruction and perforation associated with oral preparation for colonoscopy are rare , but they can be fatal and , thus , require early recognition and prompt treatment . aes that have been reported include : mallory - weiss syndrome and esophageal perforation caused by vomiting 7 8 9 10 ; acute respiratory distress syndrome caused by aspiration pneumonia 11 ; allergy and anaphylactic shock 12 13 14 ; hyperphosphatemia 15 16 and ischemic colitis caused by peroral preparation drugs 17 18 19 . there have also been a few reports of lbo and perforation caused by bowel preparation . 5 a previous report of bowel perforation in 2 patients ( 1 of whom died from colon perforation caused by colorectal cancer , the other underwent emergency colostomy ) suggested that use of magnesium sulfate , an osmotic laxative that creates hypertonic pressure in the intestine after oral administration , might have contributed . 5 magnesium sulfate prevents water reabsorption , mechanically stimulates intestinal peristalsis and facilitates bowel movement , thereby softening the stool and cleansing the intestinal tract . in this case , it was judged that magnesium sulfate had increased the intraluminal pressure of the obstructed colon until it ruptured . ours was a retrospective review of patients with lbo or perforation . in both cases of perforation , although peg is excreted in the feces without disturbing serum and urinary electrolyte concentrations or urine volume , it nonetheless increases intraluminal pressure in the intestinal tract . as with the reported cases with magnesium sulfate , we judge increased pressure within the intestine and the presence of a vulnerable lesion to have been the cause of bowel perforation on our cohort . the preparation strategy outlined in fig . prompt use of imaging , administration of enemas and adjustment of drug doses can mitigate against precipitous increases in intestinal pressure , and contributes to the early detection and management of lbo and perforation . we should pay attention to the cases if the existence of malignant tumor is known or strongly suspected by previous doctor 's introduction , computed tomography ( ct ) or high tumor marker . in lbo and perforation caused by cancer cases , 11 cases ( 61 % ) about lbo or perforation cases , even though it is difficult to predict lbo or perforation , it may have been possible to suspect the presence of a tumor in more than half the cases . we also judge exacerbation of constipation to be a risk factor for bowel perforation , given that 7 patients who subsequently developed lbo had initially presented with disturbance of bowel movement . indeed , a total of 12 patients had reported abnormal bowel movement if stool narrowing and diarrhea were included , both of which may accompany severe constipation . both patients with perforation presented with stool narrowing , not constipation . strict confirmation of the defecation state before bowel preparation may have the potential to avert aes . in routine clinical practice , we attempt to diagnose aes as promptly as possible using imaging tests such as x - ray and ct if patients report lack of defecation and abdominal symptoms after bowel preparation has been administered . nevertheless , it is not possible to prevent all aes , as some occur with sudden onset and some patients are asymptomatic when the decision to undertake colonoscopy is made . an appreciation of the risks of oral preparation for colonoscopy and a high index of suspicion among healthcare professionals can ensure that aes are detected and treated promptly to avoid the need for surgery and to prevent deaths . in 8 patients in our cohort large tumors had been identified on ct before colon preparation . we suggest that non - oral bowel preparation should be used ( for example , glycerin enema ) when massive tumors are detected before colonoscopy . patients medical history and serum albumin concentrations are presented in table 2 . hypoalbuminemia ( serum albumin concentration < 3.8 g / dl ) was evident in 11 cases ( 61.1 % ) . mean body mass index ( bmi ) was 21.0 kg / m , and was below 18.5 kg / m in 4 cases ( 22.2 % ) . we judge that nutritional condition , colonic diverticular disease and bmi did not strongly influence our findings . the number of control groups was too large , it was difficult to evaluate the risk factor of this study , and it became a one arm evaluation . in future , larger , prospective observational or retrospective case - controlled studies may help to illuminate the risk factors for lbo and perforation . in conclusion , lbo and perforation associated with an oral preparation for colonoscopy are rare , but can have severe consequences . lob or perforation can not accurately be predicted from bowel habit and abdominal findings alone . all staff working in endoscopy units should have an appreciation of the adverse events associated with preparation for colonoscopy .

background and study aims patients undergoing bowel preparation for colonoscopy are at risk of potentially severe adverse events such as large - bowel obstruction ( lbo ) and perforation . these patients usually need emergency surgery and the consequences may be fatal . little is known about the risk factors for lbo and perforation in these circumstances . we sought to establish the natural history of lbo and perforation caused by oral preparation for colonoscopy . patients and methods we retrospectively analyzed data from 20 patients with lbo or perforation associated with oral preparation for colonoscopy . all patients were treated at the showa university northern yokohama hospital ( sunyh ) between april 2001 and december 2015 . drugs used for bowel preparation , age , sex , indication for colonoscopy , pathogenesis and treatment were recorded . results eighteen of the patients had lbo and 2 had perforation . fourteen events occurred at sunyh , which accounted for 0.016 % of patients who underwent bowel preparation during this period . seventeen patients were symptomatic when the decision to undertake colonoscopy was made ( including 7 who complained of constipation and 4 who complained of abdominal pain ; 3e were asymptomatic ) . nineteen patients ultimately required surgery , 13 within 3 days of presentation . eleven patients ultimately required colostomy . there was no perioperative mortality in our cases . conclusion large bowel obstruction and perforation are rare events associated with oral preparation for colonoscopy , but frequently require surgery . exacerbation of constipation might be a risk factor for lbo or perforation . potentially catastrophic situations can be avoided by early detection and treatment .

Introduction Patients and methods Results Discussion Conclusion