Although people with knee and hip osteoarthritis (OA) seek treatment because of pain, many of these individuals have commonly co-occurring symptoms . The purpose of this study was to characterize adults with OA by identifying subgroups with the above comorbid symptoms along with illness burden (a composite measure of somatic symptoms) to begin to examine whether subsets may have differing underlying pain mechanisms.n = 129) participated . Hierarchical agglomerative cluster analysis was used. To determine the relative contribution of each variable in a cluster, multivariate analysis of variance was used.Community-living older adults with symptomatic knee and hip OA (n = 45) had high levels of pain, fatigue, sleep problems, and mood disturbances. Cluster 2 (n = 38) had intermediate degrees of depression and fatigue, but low pain and good sleep. Cluster 3 (n = 42) had the lowest levels of pain, fatigue, and depression, but worse sleep quality than Cluster 2.We found three clusters. Cluster 1  may have relatively stronger central nervous system (CNS) contributions to their symptoms. For such individuals, therapies may need to include centrally-acting components in addition to traditional peripheral approaches. Osteoarthritis (OA) is the leading cause of disability in US adults and its prevalence is expected to double by 2020 . HistoriThere is growing evidence in OA that there is a central component to pain. Recent studies of animal models provide support for central sensitization of nociceptive pathways ,8. In adThe purpose of this study was to identify specific subgroups of individuals who initially presented with symptomatic knee or hip OA. Subgroups would be based upon levels of comorbid centrally-mediated symptomatology, such as fatigue, sleep disturbance, mood, as well as other symptoms, which we refer to as illness burden, known to occur in other centrally-mediated pain states  . We hypoOur primary study was designed to examine the relationship among pain, fatigue, and physical activity in older adults with osteoarthritis. Potential participants were recruited in various ways: through fliers , newspaper advertisements, and online through the University of Michigan's clinical subjects website. All individuals first signed an informed consent document outlining the research project; this was approved by the University of Michigan Institutional Review Board and Ann Arbor Veteran's Affairs Human Subjects Review Board. The individuals underwent a comprehensive screening evaluation with a geriatrics nurse practitioner who was trained by a rheumatologist to ascertain the presence of clinical knee or hip OA using the American College of Rheumatology (ACR) clinical criteria ,34 and tTo recruit people with painful OA, individuals were included if they reported pain in a joint with OA on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scale (Likert version) of ≥ 4 with at least two of the five items on the scale rated as moderate pain or more . This crIndividuals were provided with a wrist-worn accelerometer  that measured physical activity patterns and allowed ecological momentary assessment of pain five times a day over a five-day home monitoring period . Based on our experience in past studies, we chose sampling times that represented each part of the day . After being prompted by the device's audible alarm, participants rated their pain on a scale of 0 to 10 . Pain ratings were then summed over each day and averaged across all days to derive an average pain rating. Because of our interest in multi-focal pain mechanisms, we chose to include this measure of pain rather than the WOMAC pain subscale as we think it better captures global pain experience compared to a disease-specific instrument.Depressive symptomatology was assessed using the Center of Epidemiologic Studies Depression Scale (CES-D) . A scoreThe Brief Fatigue Inventory (BFI) was used to measure total fatigue  calculatIllness burden is a concept that reflects the self-reported symptom load experienced by an individual. For this study, participants were asked whether they experienced any of 41 different somatic symptoms with the sum representing the "illness burden" variable in the analysis. Examples of the types of symptoms include: stomach pains, headaches, cognitive dysfunction, muscle weakness or stiffness, restless sleep, and daytime sleepiness. Individually these symptoms may represent various health problems associated with different body systems, but in aggregate they may be viewed as the amount of burden a given individual experiences stemming from centrally-mediated somatic symptoms, such as fibromyalgia, irritable bowel syndrome, and chronic fatigue syndrome. In other chronic pain conditions, greater endorsement of such symptoms has been associated with greater central pain and dysfunction status -41.Sleep problems were assessed using the Pittsburgh Sleep Quality Index (PSQI) . A totalFunctional status measures were not included in clustering, but were of interest to determine how different symptom clusters were associated with function, included both the short-form WOMAC physical disability scale , and theThe variables selected for analytic clustering were chosen based upon their known association with other central pain conditions  and by virtue of these variables being of centrally-mediated origin rather than being symptoms of peripheral or localized etiology. The symptoms chosen for clustering included the following: pain, fatigue, sleep problems, depressive symptoms, and somatic symptoms . Hierarchical agglomerative cluster analysis utilized Ward's method with Squared Euclidean Distances in the proximities matrix . Visual P = .06) and less physical disability on the short-Form WOMAC scale . These participants had similar levels of fatigue on the BFI  and depression on the CES-D  compared to the sample of 129 people. A total of 79 females and 50 males were included in the current analysis with the majority of the sample identifying themselves as either Caucasian (87%) or African American (9%). Knee OA was the most common diagnosis (69%), followed by both knee and hip OA (11%), and hip OA alone (20%). Scores on the WOMAC indicated a mild-moderate pain experience among participants (8.5 ± 3.2) with overall moderate levels of self-reported physical dysfunction (12.30 ± 4.67). Average pain severity for the sample was 3.06 (1.62) and average fatigue severity was 3.77 (1.67).Characteristics of the sample are shown in Table P < .0001). Subsequent univariate ANOVAs indicated significant differences were observed between clusters for all variables making up the clusters . Post-hoc analyses revealed significant differences between all variables and all pairs of clusters except for pain severity where there was not a significant difference between Clusters 2 and 3 (P = .09). Table Three clusters were identified as being the best fit for these data. Significant separation of the variables contained within the clustering solution was confirmed overall by MANOVA  = 37.69, 36 = 37.6n = 45). This group had the most pathological scores on all clustering variables compared to the other two groups and was characterized by the highest level of depressive symptoms , highest fatigue and average pain (6.2 and 3.9/10 respectively), the worst sleep, and the greatest illness burden (endorsing 30% of somatic symptoms that were queried).Cluster 1 comprised 36% of the sample (n = 38). The group was characterized by subclinical levels of depression and moderate fatigue, but had low pain and lower levels of sleep problems . They had a moderate illness burden (endorsing 25% of somatic symptoms).Cluster 2 comprised 30% of the sample (n = 42). This group had clinically-relevant levels of sleep problems, but mild levels of average pain . The group had the lowest levels of fatigue and depression, and markedly low levels of illness burden compared to the two other clusters .Cluster 3, comprised 34% of the sample (P = .97), and there was no significant relationship between gender and cluster assignment . Similarly, body mass index (BMI) did not differ across clusters, being within the category of obese  on average  = 1.02, P = .36). With regard to OA joints per the ACR clinical criteria, we examined the clusters according to whether they had knee OA alone or whether OA was in both the hip and knee or in the hip joints alone. The most prevalent group in all clusters had knee OA alone ; however, location of OA joint was not significantly different across clusters . Duration of OA pain was not significantly different across the groups and there was a large amount of variability across people  = 2.58, P = .08); although Cluster 2 had the longest pain duration at 177 months (± 180) compared to Cluster 1 and Cluster 3 (124 ± 120 and 105 ± 126).Age did not differ across clusters  = .036, P = .003), specifically between Cluster 1, which had the highest level of stiffness (3.91 ± 1.60), and Cluster 3, which had the lowest level (2.74 ± 1.70). Physical disability on the WOMAC disability scale was also significantly different across clusters  = 16.18, P = .0001). The significant difference in means from post-hoc testing was 14.84 ± 4.50 in Cluster 1 compared to Clusters 2 and 3 (9.84 ± 3.84 and 11.27 ± 3.98 respectively). On the timed up and go test, Cluster 1 had a mean of 13.5 (± 8.9) which is a cut-off score denoting fall risk (43) and was significantly different from Clusters 2 and 3 which were similar in their performance on this test (10.5 ± 2.1 and 10.2 ± 2.3 respectively).Differences in clusters were found in stiffness, physical disability, and objective physical function. Stiffness on the WOMAC scale was significantly different across groups  = 5.95, P < .0001; and for Function 2: χ2 = 59.10, P < .0001). The first function was weighted towards reported fatigue and number of endorsed somatic symptoms, while the second function was weighted toward reported pain and sleep quality. Figure Two discriminant functions  were identified that significantly differentiated the clusters accounting for 71% and 29% of the variance among them  that appeared to have symptoms reminiscent of fibromyalgia, including the highest ratings on both pain and fatigue, as well as the worst ratings on depressive symptoms and sleep. In addition, this group also had the highest illness burden , further supporting that a potential CNS contribution to symptoms.Cluster 2 had intermediate levels of depressive symptoms and modest levels of other somatic symptoms, suggesting these individuals were suffering from mild depression. Cluster 2 appears to be a group of relatively mixed central influence, with perhaps mild depressive symptomatology, but no significant evidence of sensory amplification symptoms that are more classically seen in fibromyalgia and related conditions. Cluster 3 had low levels of all symptoms except difficulties with sleep, where they had slightly greater symptomatology than Cluster 2. Individuals within Cluster 3 may represent those individuals who have been traditionally thought of as having OA confined to the joint. With the exception of sleep problems, these individuals showed minimal evidence of known central factors to be influencing the overall condition.Although our data appear to support the idea that differences in symptom presentation among clusters may be due to differences in pain mechanisms, potential alternative explanations were considered. For example, clusters may have been differentiated due to differences in pain severity rather than differences in pain mechanism. However, because all groups have mild-moderate pain (2.3 to 3.9), this seems unlikely. In addition, it could be argued that the differences among clusters may be due to physical function. Specifically, Cluster 1, the group that was most symptomatic, also had significantly worse scores on the WOMAC physical disability scale and on the timed up and go test than members of the other two clusters. This finding is consistent with known associations between symptoms, depression, and function in chronic pain ,48, and There is a growing body of literature that suggests pain experienced in some individuals with OA could be due to sensitization of both local and central pain pathways ,50. CentThese findings should be considered preliminary given that the clustering solution still requires replication in another sample of OA patients. In addition, this study was a first step into examining pain mechanisms in an OA sample using symptom presentation as a grouping variable. More sophisticated measurement, such as quantitative sensory testing, is needed to more fully investigate the pain mechanisms in these different subgroups. The structural severity of knee and hip OA is not known in this study because the ACR clinical criteria was used and this information would be useful in better understanding these subgroups. Further, generalizability of the study findings are limited to people with symptomatic knee or hip OA and also may be limited by other characteristics of the convenience sample we were able to access for this study. Because a primary goal of the larger research project is to examine fatigue in OA, we excluded people that did not have enough fatigue; however, this was a relatively small portion of the sample (15%). In addition, we included both veterans and non-veterans in this study and this sample comprised about 20% veterans. Veterans are more likely than non-veterans to report chronic joint pain and activity limitations  and a laAmong a group of community-dwelling older adults with symptomatic knee or hip OA, we found evidence of three statistically differentiated subgroups that were characterized by differing symptom presentations which may potentially be due to different pain mechanisms. Although further study and replication of the clusters are needed, the heterogeneity of people with symptomatic OA in this sample highlights the need to tailor treatment strategies for symptom management.ANOVA: analysis of variance; BFI: brief fatigue inventory; CES-D: Center for Epidemiological Studies Depression Scale; CNS: central nervous system; MANOVA: multivariate analysis of variance; OA: osteoarthritis; PSQI: Pittsburgh Sleep Quality Index; WOMAC: Western Ontario and McMaster Universities Osteoarthritis IndexDr. Clauw's financial disclosures are from Pfizer, Lilly, Forest, Cypress Biosciences, Pierre Fabre, UCB, Jazz Pharmaceuticals, and Merck. Dr. Phillips' financial disclosures are from Merck, United Therapeutics, and Actelion. Drs. Murphy and Williams declare that they do not have competing interests.The idea for this manuscript arose from a meeting with all co-authors. SLM and AKL conducted the analysis with specific guidance from DAW. SLM provided an initial draft of the manuscript and all coauthors contributed intellectual content, participating in follow-up meetings and in the writing process. SLM acquired the data and assumes responsibility for the integrity of the data as well as the accuracy of the data analysis. All authors read and approved the final manuscript.


We examined the independent and combined associations of physical activity and obesity with incident type 2 diabetes in men and women.The InterAct case–cohort study consists of 12,403 incident type 2 diabetes cases and a randomly selected subcohort of 16,154 individuals, drawn from a total cohort of 340,234 participants with 3.99 million person-years of follow-up. Physical activity was assessed by a four-category index. Obesity was measured by BMI and waist circumference (WC). Associations between physical activity, obesity and case-ascertained incident type 2 diabetes were analysed by Cox regression after adjusting for educational level, smoking status, alcohol consumption and energy intake. In combined analyses, individuals were stratified according to physical activity level, BMI and WC.A one-category difference in physical activity  was independently associated with a 13%  and 7%  relative reduction in the risk of type 2 diabetes in men and women, respectively. Lower levels of physical activity were associated with an increased risk of diabetes across all strata of BMI. Comparing inactive with active individuals, the HRs were 1.44  and 1.38  in abdominally lean and obese inactive men, respectively, and 1.57  and 1.19  in abdominally lean and obese inactive women, respectively.Physical activity is associated with a reduction in the risk of developing type 2 diabetes across BMI categories in men and women, as well as in abdominally lean and obese men and women.The online version of this article (doi:10.1007/s00125-012-2532-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Obesity and low levels of physical activity are among the most important modifiable risk factors for type 2 diabetes –6. Moreo–InterAct Study. The EPIC–InterAct Study is a case–cohort study nested within a large pan-European study that involves male and female participants from diverse social and occupational backgrounds, and used a standardised and validated assessment of physical activity at baseline.Meta-analysis of existing published cohort studies, which has been attempted in this context to examine the main association of physical activity , is unli–InterAct project aims to examine: (1) gene × lifestyle interactions in relation to the risk of developing type 2 diabetes; and (2) how knowledge about such interactions may be translated into preventative action . A case–cohort study of incident type 2 diabetes (InterAct Study) was established on the basis of incident type 2 diabetes cases occurring in the EPIC cohorts between 1991 and 2007 at 26 centres from eight  of ten EPIC countries. Due to minor differences in measurements of anthropometry and physical activity between centres in Germany, UK and Sweden, centres from these countries were treated as individual study locations  in all analyses. All participants gave written informed consent, and the study was approved by the local ethics committee in the participating countries and the Internal Review Board of the International Agency for Research on Cancer.The participants, methods, study design and measurements have been described previously . Brieflyn = 5,821) at baseline were excluded.Standard anthropometric data and biological samples were collected from 346,055 of 455,680 individuals from the eight cohorts. Individuals with incident diabetes  were included in the analysis. Due to the random selection, this subcohort also included a random set of 778 individuals who developed incident type 2 diabetes during follow-up .2), overweight (BMI 25–30 kg/m2) and obese (BMI ≥30 kg/m2) groups. We categorised participants as abdominally lean or obese based on the International Diabetes Federation [Body weight (kilograms) and height (centimetres) were measured according to standardised procedures without shoes , except deration  sex-specPhysical activity was assessed at baseline by a validated self-report questionnaire  combininIn one of the centres  a slightly different questionnaire was used to assess physical activity. From this questionnaire, we derived a four-category index that was similar to that derived from all other study locations, but based on two questions on occupational physical activity and LTPA, respectively .p < 0.0001 for trend), with a significant correlation between measured PAEE and the categorical physical activity index being observed in all countries . Further calibration results suggest that the average PAEE across categories of physical activity were: inactive (36 kJ/kg daily); moderately inactive (41 kJ/kg daily); moderately active (46 kJ/kg daily); active (51 kJ/kg daily).We examined the validity of the four-category physical activity index in 1,941 participants of similar age and sex to those in the original EPIC cohort , using cDietary intakes at baseline were measured using country-specific validated food frequency questionnaires and expressed as total daily energy intake .Standard questionnaires were used at baseline to collect information on the participants’ socio-demographic characteristics, smoking status , educational level  and alcohol consumption (grams/day).I2, the percentage of variation in the HRs that is due to heterogeneity between locations, was calculated. The analysis was repeated using LTPA as the exposure. Individuals were also categorised according to levels of (1) BMI (three groups) and physical activity (four groups), and (2) WC (two groups) and physical activity (four groups), and HRs estimated using Prentice-weighted Cox regression, with active as the reference group across strata for BMI and WC. Because in some locations there were very few individuals in some of the groups defined by (1) and (2) above, these latter models were fitted in the full dataset, with adjustment for location. Analyses were performed using Stata version 11.1. .All analyses were performed separately in men and women. Means and SDs for continuous variables, numbers and percentages for categorical variables, and incidence rates of diabetes were calculated across the four physical activity categories. Hazard ratios per category difference in physical activity were estimated separately with data from each location, using Prentice-weighted Cox regression, with age as the underlying timescale and additionally adjusting for educational level, smoking status, alcohol consumption and energy intake. These HRs were then combined across locations using random effects meta-analysis, and The present report includes 11,669 men and 15,695 women, of whom 5,660 and 5,570, respectively, were incident cases of type 2 diabetes. Based on subcohort data, 6.3% of men and 3.9% of women developed type 2 diabetes over a median follow-up time of 12.3 years .Table I2 62.2%, p = 0.005) and WC  was partly explained by the low proportion of men in the obese category in some of the study locations.The significant heterogeneity by study location that was observed in men for the associations including BMI  following adjustment for BMI and the confounders listed above, and 0.94  when analyses were adjusted for WC rather than BMI. In women, the protective effect of LTPA was evident following adjustment for BMI or WC, with the same effect sizes for general and central obesity .We next examined the combined effects of physical activity and BMI on incident diabetes stratified by sex across BMI categories Table . The incOverall, in combined association analyses , the effect of BMI on the risk of diabetes appeared to be more pronounced than the effect of lower levels of physical activity  who were diagnosed with stroke, heart attack, hypertension and hyperlipidaemia at baseline; the conclusions were unchanged (data not shown).In sensitivity analyses, we excluded all participants , and in lean and abdominally obese men and women. However, the protective effect of physical activity appeared to be more pronounced in abdominally obese men than women.While previous studies –8 have eFew previous studies have examined the combined effects of physical activity, and of general and abdominal adiposity on incident diabetes. In those that have addressed this question, strong conclusions have been limited by the use of homogenous samples of men  or womenOne previous study in women  suggesteAs in previous studies –10, we oOur study has some additional potential limitations. Case ascertainment and verification status differed between study locations and screening for blood glucose was not feasible. However, we identified 12,403 incident cases of type 2 diabetes that were verified through multiple sources in each study location. Even if some under-diagnosis may have occurred, the effect on the observed measure of association would be conservative. We only assessed physical activity and anthropometry at baseline, and any change in physical activity and/or obesity between baseline and follow-up might have caused misclassification, therefore attenuating the associations observed. Although we adjusted our analyses for several potential lifestyle-related variables, including dietary intake, alcohol consumption and smoking, residual confounding may have persisted.The association between physical activity and type 2 diabetes independently of obesity is biologically plausible , 22. HowOur results have some potential implications for public health. Results from our calibration study suggest that each increment in physical activity corresponded to approximately 380 and 460 kJ PAEE per day in men and women, respectively. Thus the 19 to 81 % risk reduction observed between extreme categories of physical activity across BMI and abdominal obesity groups could correspond to about 1,200 kJ/day of PAEE. Although these estimates should be interpreted with caution, they were derived from an independent sample of almost 2,000 men and women from all ten EPIC countries, who were measured twice by individually calibrated heart rate- and movement-sensing .Previous lifestyle interventions have been successful in reducing the incidence of type 2 diabetes in those at high risk of developing the disease , 25. HowIn conclusion, physical activity predicts the development of type 2 diabetes independently of general and abdominal adiposity. Higher levels of physical activity are associated with a substantial risk reduction across BMI categories, and in abdominally lean and obese men and women. The promotion of physical activity appears to have beneficial effects on the prevention of type 2 diabetes, regardless of the degree of overall and abdominal adiposity.ESM 1(PDF 30 kb)ESM 2(PDF 29 kb)Below is the link to the electronic supplementary material.


The human leukocyte antigen (HLA) system is widely used as a strategy in the search for the etiology of renal function impairment.This study was carried out to detect the most common HLA alleles’ distribution in kidney transplant in both donors and recipients, and clarify the association between HLA alleles and renal dysfunction immediately after transplantation.HLA-class I and II alleles typing by PCR-SSOP was performed on a total of 874 recipients aged 40.7 ± 13.8  and 874 donor aged 27.5 ± 5.3 , between 2006 and 2009 in Baqiyatallah, hospital, Tehran, Iran. In this retrospective, cross sectional study, data were obtained from personal files. Donors aged 40.9 ± 13.6 years and male/female 390/195, while recipients had a mean age 27.5 ± 5.3 and male/female 523/83. Renal dysfunction defined as acute rejection, acute tubular necrosis and Delay graft function.In this study common alleles at each of the loci for the human leukocyte antigen (HLA) class I  and class II (DR and DQ) were A2 , Bw6 , Cw4 , DR52 , DQ3  for donors; while A2 , BW6 , Cw6 , DR511 , DQ1  for recipients. We detected a total of 139 case of renal dysfunction among RTRs. By the way only cold ischemic time (P = 0.03) and severe anemia (P = 0.000) were significantly associated with renal dysfunction early post kidney transplantation.We can predict high risk groups before kidney transplantation and try to establish a screening program for the detection of genetic susceptibility to renal function impairment. HLA typing of the donors and recipients might influence the development of new treatment strategy. Human leukocyte antigen (HLA) molecules are expressed on almost all nucleated cells . The HLADue to the high polymorphism in the HLA system and since some of the HLA antigens appear to be more immunogenic, this study was designed to detect the most common distribution of HLA alleles in Iranian kidney transplant donors and recipients. The understanding of HLA subtype frequency has also permitted a better ABDR matching of donor and recipient and better clinical results. HLA-class I and II alleles typing by PCR-SSOP was performed on a total of 874 recipients aged 40.7 ± 13.8  with median dialysis duration time 11 (0-438) months, and 874 donor aged 27.5 ± 5.3 , between 2006 and 2009 in Baqiyatallah, hospital, Tehran, Iran. In this retrospective, cross-sectional study, allele frequencies, history of renal dysfunction, and other participants’ data were obtained from personal files by two trained medical students. Polymerase chain reaction method was used for DNA replication and HLA typing. All data were extracted from personal file.In order to determine allele frequencies, participants’ data files were reviewed by two trained medical students. Polymerase chain reaction method was used for DNA replication and HLA typing. Two or more alleles that express the similar antigen were defined as a single allele.The definition of acute rejection was based on the conventional histological and clinical criteria of the reporting centers. Acute tubular necrosis (ATN) was defined clinically and histologically.2).Delay graft function (DGF) means dialysis requirement during the first week after kidney transplantation. World Health Organization criteria, has defined a hemoglobin (Hb) level less than 11 g/dL for men and less than 10 g/dL for women as severe anemia. Body mass index (BMI) was defined in weight (kg)/height . Quantitative variables were expressed as mean ± standard deviation, whereas qualitative variables were shown as number and percentage. Continuous data were compared by Student’s t-test, and categorized data were analyzed using the Chi-square or Fisher’s exact test.In the current study, common alleles at each loci for the human leukocyte antigen (HLA) class I  and class II (DR and DQ) are summarized in The most common alleles in diabetes mellitus, hypertension and autosomal dominant poly cystic kidney disease were summarized in In the univariate analysis, only long cold ischemic time  and severe anemia  were associated with renal dysfunction. ATN  and DGF  were frequent in long cold ischemic time. Association between ATN, DGF and acute rejection with other variables such as recipients and donors’ age, gender, BMI, donor source and warm ischemic time were not significant. We revealed demographic differences and HLA distribution among ATN, DGF and acute rejection in HLA antigens expressed on the cell membrane encompass an important role in the immune response to grafted tissue. In addition, distribution of HLA allelic subtypes vary in different populations so, information about the distribution of HLA genotypes in different populations may be important because probability of incompatibility at the allele level will be affected by different origins of recipients and donors  and theoIn transplantation, frequencies of HLA genotypes can be used to predict the chance of detecting a highly HLA match donor for an exacting recipient and to determine the effect of HLA mismatching on different renal transplant complications . In the Conclusion: HLA typing of the donors and recipient might influence the development of new treatment strategy; additionally it provides useful data for HLA matching in transplantation. We can predict high risk group before kidney transplantation and try to establish a screening program for the detection of genetic susceptibility to renal function impairment. HLA typing of the donors and recipient might influence the development of new treatment strategy.


Few studies have evaluated short-term functional outcome and its improvement with time. We determined the time course of functional outcome as evaluated by the knee injury and osteoarthritis outcome score (KOOS) over the first 2 years after Oxford medial UKR. In a prospective multicenter study, we included 99 unselected knees  years, 57 women) operated with Oxford medial UKR at 3 hospitals in the southeast of Norway between November 2003 and October 2006. Data were collected by independent investigators preoperatively and at 6 weeks, 3 months, 6 months, 1 year, and 2 years postoperatively. KOOS and range of motion (ROM) were determined at all follow-ups. Mean KOOS values for pain and activities of daily living were improved already after 6 weeks, and increased between each time point up to 2 years postoperatively. However, no statistically significant improvements were seen after 6 months. Mean active and passive ROM gradually improved up to 2 years after UKR, and were then better than before surgery. Most of the expected improvements in pain and function after UKR are achieved within 6 months of surgery. Only minimal improvement can be expected beyond this time. Unicompartmental knee replacement (UKR) has regained popularity in recent years. Studies comparing UKR to total knee replacement (TKR) have shown faster recovery, shorter hospital stay, more normal kinematics, better range of motion, less blood loss, fewer thromboembolic incidents, and fewer surgical site infections . SurvivaWith a possible higher revision risk for UKR than for TKR , more inWe determined (1) the time course of patient-relevant functional outcome evaluated by the KOOS, and (2) the time course of range of motion (ROM) during the first 2 years following UKR using the Oxford medial unicompartmental knee prosthesis. Improvements in patients' self-reported pain and daily function during the study period were of particular interest.96 unselected patients (99 knees) to be operated with medial unicompartmental knee arthroplasty at Martina Hansens Hospital, at Akershus University Hospital, and at Baerum Hospital between November 2003 and October 2006 were included in a prospective study. The inclusion criteria were: medial knee osteoarthritis, age 50–80 years, no rheumatoid arthritis or previous knee infection, a correctable varus deformity of less than 10°, a fixed flexion deformity of less than 10°, intact anterior and posterior cruciate ligaments, absence of osteoarthritis in the lateral compartment in standing radiographs, absence of lateral compartment tenderness, and no more than minimal patellofemoral symptoms or radiological abnormalities. Informed consent was obtained from all patients. All patients were operated with the Oxford phase III medial unicompartmental knee . The operation was performed in the hanging-leg position under spinal/epidural anesthesia, using a minimally invasive approach . Mean opData were collected by independent investigators (physiotherapists) preoperatively and at 6 weeks, 3 months, 6 months, 1 year, and 2 years postoperatively. The primary outcome measures were the KOOS subscales for pain and for ADL. Knee pain and knee daily function are among the most important clinical considerations when deciding whether or not to have a knee replacement; thus, these variables were assumed to be the 2 most important. The secondary outcome measures were the KOOS subscales for symptoms, knee-related quality of life (QoL), and sport and recreational function (see below); the Oxford score; the UCLA activity scale; and active and passive ROM.Patients completed the KOOS —the NorwPatients completed the Oxford knee score   with the patient in supine position in order to allow free hip flexion. A goniometer was used.Patients were asked to report any complications or adverse events at all follow-ups.The minimal perceptible clinical improvement (MPCI) of the KOOS is suggested to be approximately 10 score units . PreviouThe data were analyzed by fitting separate linear mixed models for the KOOS subscales, for the Oxford score, for the UCLA score, and for the ROM measurements, choosing an unstructured  covariance matrix for the repeated measurements. Missing data were assumed to be missing at random. The mean of each outcome was estimated along with its corresponding 95% confidence interval (CI) at all observation times. Post hoc comparisons between the main effects of all pairs of points in time  were performed separately for each model, corresponding to the KOOS subscales, the Oxford score, the UCLA activity scale, and the measurements of active and passive ROM. Bonferroni adjustments, including all pairwise comparisons within a specific model, were applied to the respective confidence intervals and p-values to account for multiple testing. The level of statistical significance was set to p < 0.05. Statistical analysis was performed using SPSS software version 18.Data from all 96 patients (99 knees) initially included in the study were available for analysis.Of 594 evaluations planned, 38 (6%) were missing for 27 patients. 21 patients failed to attend 1 or more of the postoperative evaluations, 5 patients were revised to have a total knee replacement, and 1 patient died of gastrointestinal cancer shortly after the operation. The mean age, sex distribution, and KOOS values of these 27 patients were similar to those of the 69 patients with complete data.5 knees (5 patients) were revised to a total knee replacement during the study period: 2 due to pain, 2 because of aseptic loosening, and 1 because of a deep infection. 1 patient developed a stiff knee because of arthrofibrosis, and was treated with manipulation under anesthesia with a satisfactory result (ROM was 110 degrees at 2 years). 1 patient had a meniscal bearing dislocation caused by a hyperflexion trauma, and was reoperated with exchange of the meniscal bearing.The linear mixed-models analysis revealed significant differences in KOOS scoring between the 6 time points of measurement. This was true for all subscales of the KOOS (p < 0.001).Mean KOOS score values for ADL and for pain continued to improve until 1 year after surgery. Minimal changes were seen after 6 months . Using tPatients with complete KOOS data at 2 years (n = 84) were divided into groups according to the magnitude of the difference between mean preoperative values and mean 2-year values in the KOOS subscales pain and ADL . 75 patiMean KOOS score values for Symptoms, QoL, and Sport/rec also continued to improve until 2 years after surgery. Only small changes were observed after 6 months, however .The linear mixed-models analysis revealed significant differences in Oxford scoring between the 6 times of measurement (p < 0.001). Oxford mean score values continued to improve until 2 years after surgery, but only small changes were seen after 6 months .The linear mixed-models analysis revealed that there were significant differences in UCLA scoring between the 6 times of measurement (p < 0.001). UCLA mean score values continued to improve until 1 year after surgery, with only minimal changes observed after 6 months .The linear mixed-models analysis revealed significant differences in both active and passive ROM between the 7 times of measurement (p < 0.001). Mean values for active and passive ROM continued to improve until 2 years after surgery .Patients were divided into groups according to their preoperative passive ROM, and mean preoperative ROM was compared to mean 2-year ROM for each group. Patients with a preoperative ROM of 80–99 degrees (n = 6) improved in mean ROM , patients with a preoperative ROM of 100–119 degrees (n = 26) improved in mean ROM , patients with a preoperative ROM of 120–129 degrees (n = 43) improved in mean ROM , and patients with a ROM of 130–149 degrees preoperatively (n = 24) ended up with approximately the same mean ROM  2 years postoperatively.To our knowledge, only 2 studies have used the KOOS to evaluate functional recovery after UKR , and ourWe found improvement in pain and function as early as 6 weeks postoperatively; more than 50% of the improvement in KOOS Pain and KOOS ADL had already been achieved at this point. Improvement in KOOS Pain and KOOS ADL mean values continued until 2 years postoperatively, although the differences between the 6-month and 2-year values were not statistically significant. The minimum perceptible clinical improvement (MPCI) in the KOOS is suggested to be approximately 10 score units . The meaParadowski et al. (2006) collected population-based reference data for the KOOS in different age groups. The reference data for KOOS ADL in the age group 55–74 years were 86 for men and 77 for women. For KOOS Pain in the age group 55–74 years, the reference data were 88 for men and 78 for women. Our results for KOOS ADL were actually somewhat better than the reference data whereas our results for KOOS Pain were similar. Studies on total knee replacement indicate that the most important factor determining postoperative ROM is the preoperative ROM . We founDuring the last decade, several authors have emphasized the importance of measuring the patient's own experience of disability using self-reported questionnaires . DiffereThe present study has some limitations. One weakness is that we had some missing data. However, when designing the study, we decided to use linear mixed-models statistics. This method makes allowances for some missing data, and all the patients who had been included could still be kept in the study. The analysis of missing data revealed only small differences between patients with missing data and patients with complete data sets. Another objection might be the method of measuring ROM—clinically with a goniometer. A study of functional results after UKR should preferably include both a patient-relevant self-evaluation score and more demanding objective tests. Kleijn et al. (2007) studied functional results at multiple time points after UKR using the clinician-based KSS score and the more objective Dynaport test. They found that the KSS score showed no statistically significant improvement after 6 months, whereas the Dynaport knee test showed improvement up to 2 years in one subscore, indicating that the more objective Dynaport knee test is more discriminative than the KSS score. ROM was the only objective parameter we studied.In summary, we found that functional results and recovery after UKR are time-dependent. There was improvement in pain and function as early as 6 weeks postoperatively, and most of the expected improvements were achieved at 6 months. ROM gradually improved up to 2 years after UKR, and was then better than before surgery.


Chronic wasting disease (CWD), the only known prion disease endemic in wildlife, is a persistent problem in both wild and captive North American cervid populations. This disease continues to spread and cases are found in new areas each year. Indirect transmission can occur via the environment and is thought to occur by the oral and/or intranasal route. Oral transmission has been experimentally demonstrated and although intranasal transmission has been postulated, it has not been tested in a natural host until recently. Prions have been shown to adsorb strongly to clay particles and upon oral inoculation the prion/clay combination exhibits increased infectivity in rodent models. Deer and elk undoubtedly and chronically inhale dust particles routinely while living in the landscape while foraging and rutting. We therefore hypothesized that dust represents a viable vehicle for intranasal CWD prion exposure. To test this hypothesis, CWD-positive brain homogenate was mixed with montmorillonite clay (Mte), lyophilized, pulverized and inoculated intranasally into white-tailed deer once a week for 6 weeks. Deer were euthanized at 95, 105, 120 and 175 days post final inoculation and tissues examined for CWD-associated prion proteins by immunohistochemistry. Our results demonstrate that CWD can be efficiently transmitted utilizing Mte particles as a prion carrier and intranasal exposure. C) to a pathologic, misfolded form (PrPCWD) causes CWD. This conversion process, according to the prion hypothesis, allows aberrantly folded prions to replicate without a genome Chronic wasting disease (CWD) is a naturally occurring transmissible spongiform encephalopathy (TSE) of deer, elk and moose that affects captive as well as wild populations. Currently, 15 states, 2 Canadian provinces and South Korea have reported cases of CWD Intranasal (IN) infection has been validated in rodent models by placing liquid inoculum into or in front of the nasal cavity All procedures involving animals were performed to minimize suffering and were approved by the Institutional Animal Care and Use Committee at Colorado State University in accordance with the USDA Animal Welfare Act Regulation. CFR, title 9, chapter 1, subchapter A, parts 1–4.Odocoileus virginianus) fawns of mixed sex and PRNP genotype (codon 96) were purchased from a CWD-free private deer facility  in June 2011 and transported to the USDA National Wildlife Research Center (NWRC) in Fort Collins, CO. At the time of transport fawns ranged from two to fourteen days of age. Fawns were bottle-fed and were weaned by 16 weeks. One fawn was euthanized at eight weeks of age due to an encephalitis of unknown origin.Fifteen white-tailed deer (At twelve weeks of age 12 deer were transported to a Colorado State University (CSU) BSL-2 facility for CWD-inoculation. The remaining two deer served as controls and remained at the USDA National Wildlife Research Center (NWRC) facility.PRNP. All animals were homozygous for the wild type amino acid at residue 95-(glutamine Q). There were three genotypes present for codon 96: 5 were homozygous for the wild type allele encoding glycine (GG), four were heterozygous for G and the alternative allele encoding serine (GS) and five were homozygous SS. To assess any effects of genotype on study results, deer from each genetic group were distributed as evenly as possible among the euthanasia time point groups.Blood was drawn from fawns upon arrival and sent to the USDA Agricultural Research laboratory in Pullman, WA for genetic analysis of the open reading frame of A 20% homogenate of white-tailed deer brain from experimentally inoculated, CWD-terminal or CWD-negative deer was generated in 0.5× phosphate buffered saline (PBS) and glass beads utilizing a Blue Bullet homogenizer  as previously described 2+ and Ca2+. Homogenate was passed through 18-, 23-, 26- and 28-gauge needles 15 times each, and stored at −80°C until used. A rabbit kidney epithelial (RK13) cell line  engineered to express deer prion protein, referred to as Deer5E9-S1, which is highly sensitive to the infection with deer CWD prions (manuscript in preparation) was utilized in this assay Twenty thousand cells per well were plated in 96-well plates. One day later, cells were exposed to three-fold serial dilutions of deer CWD+ brain homogenate prepared in cell culture medium, ranging from 10−2 to 10−5, in a volume of 100 µl per well. Cells that were not exposed to CWD prions served as a background control.We quantified the infectivity of our CWD+ inoculum with the cervid prion cell assay (CPCA) as described previously Cells were passaged three times at four day intervals at 1∶4 and 1∶7 split ratios. When cells reached confluence at the third passage, 20,000 cells per well were filtered onto Multiscreen IP 96-well 0.45-µm filter plates . Plates were dried at 50°C and cells were digested for 90 min at 37°C in 60 µl of lysis buffer containing 5 µg/ml proteinase K (PK) then terminated with phenylmethanesulfonylfluoride (PMSF) (2 mM). To expose the epitope of PrP27-30, cells were incubated in 120 µl 3 M guanidinium thiocyanate in 10 mM Tris-HCl (pH 8.0) for 10 min at room temperature then rinsed four times with 160 µl PBS. For immunodetection, wells were filled with 120 µl of filtered 5% superblock  and incubated for one hr at room temperature. The solution was removed by vacuum, and wells were incubated with 60 µl of 6H4 mAb, diluted 1: 5000 in TBST for one hr at RT or overnight at 4°C. Wells were rinsed four times with 160 µl of TBST then incubated with 60 µl AP-α-Mouse IgG , diluted 1: 5000 in TBST, after one hr at RT, the wells were rinsed four times with 160 µl TBST, followed by a final wash with PBS. Plates were allowed to dry completely. Visualization was done by adding 60 µl of AP conjugate substrate kit  at RT and rinsing twice with 160 µl water and allowed to completely dry. Images were scanned with a ImmunoSpot S6-V analyzer , and spot numbers were determined using ImmunoSpot5 software . Statistical analyses were performed using GraphPad Prism 5.0 for Mac OS X software. Prion titer was calculated in spot-forming CPCA units per gram of wet brain homogenate.The deer were bottle-fed and hand-raised so they were easily manually restrained for the intranasal inoculations. The plastic transfer pipette containing the powdered inoculum was warmed to room temperature, inserted approximately 3 cm into the left nostril and inoculum puffed into the nasal cavity once a week for six weeks, exposing each deer to a total of 1.2 g (wet weight) of brain. Two mock-infected control deer were inoculated in the same fashion using CWD-negative brain/clay inoculum. Three deer were euthanized at each of three time points after the final inoculation: 95, 105, 120 days post inoculation (DPI); and two deer at 175 DPI. Because of the presence of genetic variation at codon 96, a GG, GS and SS deer was placed in each of the four time groups with the exception of the 105 DPI group, which contained one GG and two SS genotypes. The control deer were composed of one GG and one GS.Two tracking methods were employed to determine the distribution of the CWD inoculum within nasal the cavity, both grossly and microscopically, after inoculation. To grossly determine the distribution of the clay inoculum, 500 µl of green fluorescent dye  and 500 µl of deionized water were added to 250 mg of Mte clay and mixed thoroughly. The mixture was prepared and inoculated IN as described for brain homogenates. The ink was visible with both ultraviolet and visible light. One control deer was manually restrained and inoculated IN with GFD/Mte into the left nostril. Forty-five minutes later the deer was euthanized and necropsy conducted.2O then floated in the water bath of a 3000MP sonicator  at 37°C and sonicated for 30 sec at 70% maximum power. Sonicated, fluorescent prions were mixed with an additional 450 µl of DI H2O then with 250 mg of Mte clay in a weigh boat and lyophilized. Prion/Mte dust was prepared and inoculated as the brain inocula described above. The two deer from the final 175 DPI euthanasia time point were manually restrained and given the prion/Mte inoculum in the left nostril at either 60 or 45 min prior to euthanasia.To track the uptake of the CWD/Mte material within the nasal mucosa, a lyophilized, fluorescently-labeled CWD prion was strategy employed. Purified deer CWD prion rods were isolated as previously described Deer were sedated with Xylazine  then euthanized via intravenous injection of Beuthanasia-D . Deer were immediately transported to the Colorado State University Veterinary Diagnostic Laboratory for a complete post mortem examination.At necropsy of the GFD/Mte tracer control deer, the nasal cavity, throat, and head lymph nodes were examined for the presence of GFD/Mte using a hand-held UV light source . Nasal turbinates were serially dissected and photographed. To detect microscopic-sized dyed particles, a fluorescent microscope was used with a UV excitation source and UV emission filter.18 and 100 ng/mL DAPI for 20 minutes, then mounted using ProLong Gold fluorescent mounting medium . Five to twelve 5 µm thick, paraffin-embedded tissue sections from nasal turbinates, retropharyngeal, submandibular and parotid lymph nodes and tonsils were viewed and photographed using an Olympus BX60 microscope  equipped with a cooled charge-coupled diode camera.To visualize fluorescent prions, sections were counterstained with 1 µg/mL of the Carbocyanin lipophilic tracer DiOCCWD, a biomarker for CWD, followed by incubation with alkaline phosphatase-conjugated anti-mouse IgG secondary antibody and visualized using an automated immnuostainer and an alkaline phosphatase red kit . Slides were counterstained with hematoxylin for four minutes at 37°C. Positive and negative control slides containing lymphoid tissues and brain were stained with each run. PrPCWD was visualized as granular red staining showing protease-resistant deposits within lymphoid follicles. All follicles were counted for each section present on each slide, totaling between 1600–3069 follicles for each deer.Retropharyngeal, submandibular, parotid, pre-scapular, pre-femoral, mesenteric, and illiocecalcolic junction lymph nodes as well as tonsils, Peyer's patches, rectum, brain, and nasal tissues  were collected from the deer at necropsy and placed in 10% neutral buffered formalin for one week. The nasal tissues were then placed in 10% formic acid for three to five days for decalcification, trimmed, and embedded in paraffin blocks. Other tissues were trimmed and two to four sections of each were placed in plastic cassettes and allowed to fix for an additional two days. Slides were prepared as previously described for visualization and evaluation +/− mouse-passaged CWD prion isolate 012-09442 were calculated as 106.1 and 106.6 cervid prion cell units per gram of brain homogenate, (CPCA units/g), respectively. The results of the titer assay revealed that the CWD prion inoculum titer was approximately three times lower than that of a transgenic mouse strain commonly used to generate disease in a mouse bioassay  prior to inoculation. Control brain inoculum remained negative after six 24 hr rounds of PMCA amplification and western blot visualization. Positive brain inoculum was positive on western blot visualization. The deer CWD prion infectivity titer was determined by CPCA using a highly sensitive Deer 5E9-S1 cell line . The pribioassay . All deeCWD by IHC in the obex, thalamus, hypothalamus, basal ganglia, olfactory bulb, or hippocampus of the brain in any of the deer at any of the time points. We next investigated subclinical infection by searching for PrPCWD in lymph nodes that drain or are proximal to the nasal cavity; retropharyngeal, submandibular, parotid, and palatine tonsil, as well as distal lymph nodes, pre-scapular, pre-femoral, mesenteric, Peyer's patches and the recto-anal mucosa associated lymphoid tissue (RAMALT). PrPCWD was not detected in either of the two mock-infected control deer (0/2 at 175 DPI), but was detected in lymph nodes of 10/11 of the CWD-inoculated deer by IHC  in the GG animals as compared to SS deer. Follicle number in the palatine tonsils and the rectum were significantly greater (Student's one-tailed T-test p = <0.05) than in GS deer , all three genotypes  were represented at codon 96. We therefore sampled deer from each genotype at each time point. We found that the genotype at codon 96 affected PrPd system . Overall GS deer . Deer ha GS deer . We founph nodes . Genotypph nodes .To determine if the deposition of the initial inocula could influence later replication sites and to understand the deposition and initial contact/entry sites for prions and the Mte within the nasal cavity, we sought to track the location of inocula immediately after exposure. We first puffed GFD/Mte dust  into theWe detected no prions associated with blood or lymphatic vessels in the lamina propria, or in any of the lymph nodes examined, including the retropharyngeal, submandibular and parotid lymph nodes, and palatine tonsils at these very early time points.CWD in the brain as late as 175 days after IN PrPCWD dust exposure. All brain regions analyzed were negative, demonstrating that although PrPCWD staining could be detected in the lymph nodes of the head in all genotypes and in more distal nodes in the GG genotype, observable PrPCWD accumulation had not yet progressed to the brain.Indirect environmental CWD transmission has been demonstrated and several infectious tissues and excreta have been identified that could contaminate forage, soil and water RES deposition has been documented in the olfactory bulb and nasal mucosa of terminally ill, infected animals C in mice CWD by IHC, demonstrating that the PrPCWD did not travel along these neurons to enter the brain by 175 DPI. Our results are in agreement with rodent studies that show prion diseases are unable to make use of this direct connection to the brain and that olfactory bulb and nasal mucosa deposition occurs later in the disease, moving retrograde from the brain to the olfactory bulb and then down the OSNs to the nasal mucosa The olfactory bulb was of particular interest in this study. There are millions of olfactory sensory neurons (OSN) present in the nasal mucosa that connect directly to the olfactory bulb CWD in the lymph nodes and tonsils of the head  in all genotypes. The tonsil findings are in agreement with those of a concurrent study using aerosolization of liquid inoculum CWD in mesenteric lymph nodes, Peyer's patches and in lymphoid follicles at the ileocecalcolic junction of the gut and rectum only in deer expressing the GG PRNP genotype. Despite detecting original inoculum deep within the nasal turbinates on OE and mucus-secreting Bowman's glands, IHC analysis of the nasal turbinates did not reveal the presence of CWD prions later in infection. Taken together, these data suggest that after IN exposure, CWD infection begins in lymphoid tissues proximal to the nasal cavity and later progresses to the brain.In contrast to the brain, we found substantial PrPDust can be an environmentally realistic, chronic, low dose mode of intranasal CWD exposure for deer and elk in the wild, particularly in dry western states such as Colorado and Wyoming. The size of dust particles is directly proportional to how far the particles are able to travel into the respiratory tract, with particles ≤1 µm able to travel into the deepest regions of the lung, 1–5 µm into the bronchiolar region, 5 µm into the upper lung and >10 µm caught in the nose and oral pharynx CWD traffics from the nasal mucosa to draining lymph nodes via immune cells, or autonomously through lymphatic drainage, or both We detected fluorescent prions on and within the olfactory epithelium and associated with nerve fibers and serous cells of the Bowman's glands in the lamina propria within one hr of exposure. We hypothesize that PrP3 of nasal epithelium CWD from the olfactory epithelium and lamina propria and transported them to regional draining lymph nodes. However, we cannot eliminate paracellular or cell autonomous movement of prions, as has previously been shown Peyer's patches in the gut and the nasal mucosa contain dendritic cells (DC) and specialized membranous (M) cells CWD deposition in the Peyer's patches in one GS deer. These data suggest that GS and SS deer have slower CWD progression in the peripheral lymphoid tissues of the body, which may explain the delayed progression of CWD observed in GS and SS mule deer by Miller et. alThis study provided a unique opportunity to observe the effect of the dimorphism at codon 96 on IN infection of deer with Mte-adsorbed prion particulate. Several studies examining the prevalence of codon 96 polymorphisms have been done on wild populations with the GG polymorphism being the most common (72%), followed by GS (13–25%), and SS (3%) The results of this study confirm that CWD can be successfully transmitted IN as a lyophilized prion particulate adsorbed to Mte and that genotype at codon 96 affects the lymphoid distribution of CWD within the body. Additionally, two novel intranasal tracking methods were employed that provided insight into CWD translocation within the nasal cavity. The data collected in this study may also shed light on why there is a higher prevalence of CWD in males, as males participate in more behaviors that generate dust. We propose chronic, long-term exposure to CWD prions adsorbed to dust particles to be a natural CWD infection route in addition to chronic oral and nasal contact exposure.


Microviridae, a group of viruses infecting bacteria. In this study we used metagenomic analysis, genome assembly, and amplicon sequencing of purified ssDNA to show that bacteriophages belonging to the subfamily Gokushovirinae within the Microviridae are genetically diverse and widespread members of marine microbial communities. Metagenomic analysis of coastal samples from the Gulf of Mexico (GOM) and British Columbia, Canada, revealed numerous sequences belonging to gokushoviruses and allowed the assembly of five putative genomes with an organization similar to chlamydiamicroviruses. Fragment recruitment to these genomes from different metagenomic data sets is consistent with gokushovirus genotypes being restricted to specific oceanic regions. Conservation among the assembled genomes allowed the design of degenerate primers that target an 800 bp fragment from the gene encoding the major capsid protein. Sequences could be amplified from coastal temperate and subtropical waters, but not from samples collected from the Arctic Ocean, or freshwater lakes. Phylogenetic analysis revealed that most sequences were distantly related to those from cultured representatives. Moreover, the sequences fell into at least seven distinct evolutionary groups, most of which were represented by one of the assembled metagenomes. Our results greatly expand the known sequence space for gokushoviruses, and reveal biogeographic separation and new evolutionary lineages of gokushoviruses in the oceans.Much remains to be learned about single-stranded (ss) DNA viruses in natural systems, and the evolutionary relationships among them. One of the eight recognized families of ssDNA viruses is the  For each sample, the viruses were concentrated ~10–100-fold  using ultrafiltration  collected from different locations and at different times within a geographic region were combined into a single mix. Except for the SI and freshwater samples, these mixes corresponded to those used by Angly et al.  in which2O for pyrosequencing or Tris-HCl for PCR amplification.As described in Labonté and Suttle , ssDNA wMetagenomic libraries were constructed from WGA ssDNA from SI, SOG, and GOM ssDNA. The purified WGA DNA was resuspended in 100 μL of RNAse- and DNAse-free water (Invitrogen) and concentrated using a Millipore YM-30 Microcon centrifugal filter to a final volume of ~50 μL; 3–5 ug of DNA from each sample was sent for pyrosequencing (Roche 454 FLX instrumentation with Titanium chemistry) at Genome Québec, McGill University (SOG) and the Broad Institute at the Massachusetts Institute of Technology (GOM and SI).Microviridae using the tBLASTx algorithm with an e-value cut-off of 10−5. Reads with significant similarity to gokushoviruses were aligned onto the assembled contigs using the add454Reads.perl script and were reassembled into new contigs using the phredPhrap.perl script of the Consed package  using the Newbler Assembler (Roche). The individual reads and assembled sequences were compared to a database of all available genomes in GenBank (as of February 2010) from viruses belonging to the  Gordon, . AdditioGokushovirinae in the Microviridae.Two forward  and two reverse  primers with respective degeneracies of 256, 215, 256, and 256 were designed from alignments of the inferred amino acid sequences of the major capsid protein (VP1) of the chlamydiaphages Chp1 (accession number NC_001741.1), Chp2 (NC_002194.1), Chp3 (NC_008355.1), Chp4 (NC_007461.1), phiCPAR39 (NC_002180.1), and phiCPG1 (NC_001998.1), Spiroplasmaphage Sp4 (NC_003438.1), bdellovibriophage phiMH2K (NC_002643.1), and the Sargasso Sea Chp1-like assembled genome . Negative controls contained all reagents except DNA template. The samples were denatured at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 50 s, with a final elongation step of 72°C for 5 min.Prior to use in PCR reactions, the purified WGA DNA was resuspended in 100 μL of TE, and 10 μL was used as a template in each PCR reaction mixture consisting of Taq DNA polymerase assay buffer , 1.5 mM MgClE. coli Top10 cells. For each sample, 30 clones were checked by colony PCR to verify that they contained an insert of the correct size. Restriction fragment length polymorphism (RFLP) analysis was then performed on 20 positive clones. For each RFLP reaction, 15 μL of colony PCR product was digested with AluI (New England BioLabs) in a reaction containing 1 U/μg of DNA and 1× NEBuffer 4  by incubating at 37°C for 16 h, followed by heat inactivation at 65°C for 20 min. RFLP products were separated on a 2% agarose gel in 0.5× TBE  running at 110 V for ~2 h. Sequencing of representative clones confirmed that each unique restriction pattern could be considered as an operational taxonomic unit (OTU). Forward and reverse sequences (~800 bp) were obtained for each RFLP pattern using Big-Dye Terminator Cycle Sequencing (Applied Biosystems) and ABI 373 Stretch or ABI Prism 377 sequencers .PCR amplicons were purified with a MinElute PCR purification kit (Qiagen), ligated into pCR2.1-Topo (Invitrogen), and used to transform chemically competent For the whole genome phylogeny, non-coding sequences were removed and the five major open reading frames were ordered. The sequences were aligned using MAFFT .VP1 from the previously sequenced isolates, environmental sequences, and the degenerate PCR products from this study were trimmed to the PCR-product length (~800 bp) and aligned using MAFFT . The genome sizes varied from 4062 to 5386 bp, and were uniformly shorter than those from previously sequenced isolates  that has interspaced conserved and variable regions. For the assembled genomes, the phylogeny of VP1 is congruent with the whole genome; thus, the phylogeny of VP1 can be used to infer viral phylogeny. PCR amplification was performed on samples from the SOG (4 mixes), GOM (4 mixes), SI (9 samples), and Arctic  (Table S1). No products were amplified from the ARC, LA, SOG-Low Salinity, or Eastern GOM mixes. This means that gokushoviruses were absent or at low concentrations in these samples, or that they are too divergent to be amplified by the primers.AluI to reveal 77 different RFLP patterns. Representative clones from each restriction pattern were sequenced (data not shown). Of these, 43 sequences were at least 98% different at the nucleotide level, and thus identified as unique gokushovirus VP1 sequences. Some sequences occurred in more than one sample from the same geographic region; for example, the sequence SI-07 was sequenced from multiple dates in SI , but no sequences occurred in more than one location  using labeled VP1 sequences to probe natural microbial communities. Another approach that has been used to visualize phage-infected gammaproteobacterial cells is phageFISH (Allers et al., Discovering the hosts of marine gokushoviruses is a high priority in order to understand the roles that these viruses play in ecosystems. Given the challenges in culturing marine microbes, culture-independent techniques will likely be needed to determine the hosts for most of these viruses. One approach that we have tried with some success is to use fluorescence Chlamydia and Bdellovibrio. Phylogenetic analysis of the major capsid protein, combined with fragment recruitment of environmental metagenomic sequences shows that the distribution of some evolutionary groups of gokushoviruses is very environment dependent, whereas others are more cosmopolitan. The high-burst size, rapid replication rates and likely lytic nature of these viruses suggests that they may play an important role as mortality agents in marine systems.This manuscript presents a new set of degenerate primers that have been used to reveal at least five new evolutionary groups of gokushoviruses, and clearly show they share a common evolutionary history with viruses that infect the obligate intracellular parasitic bacteria The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A few studies are available actually comparing the clinical efficacy of intravenous acetaminophen with other medications such as dexamethasone to inhibit postoperative adverse events in children.This randomized blinded controlled trial was designed to compare controlling status of postoperative events in children after tonsillectomy randomized to receive either intravenous acetaminophen or dexamethasone.Eighty four children aged between 4 to 13 undergoing tonsillectomy were randomized using a computer-generated schedule to double-blind treatment with intravenous acetaminophen (15 mg/kg) or intravenous dexamethasone (0.1 mg/kg). Children were post-operatively assessed for swallowing pain, pain while opening mouth, ear pain, and postoperative sore throat in recovery room (within one hour after surgery), at the time of admission to the ward, as well as at 12 and 24 hours after surgery, assessed by the objective pain scoring system .There were no significant differences between the two groups with regard to the severity of postoperative pain due to swallowing or opening mouth measured at the different study time points from postoperative recovery to 24 hours after the surgery. There was no difference in ear pain severity at the time of postoperative recovery, at the admission time to ward and also at 12 hours after surgery; however mean score of ear pain severity was significantly higher in those who administered acetaminophen 24 hours after operation. Also, the mean score severity of sore throat was significantly higher in the acetaminophen compared with the dexamethasone group within 12 hours of surgery. Postoperative vomiting and bleeding were similarly observed between the two study groups. The severity of swallowing pain, pain while opening mouth, ear pain, as well as postoperative sore throat as gradually assuaged within 24 hours of tonsillectomy in both groups, however no between-group differences were observed in the trend of the severity of these events.The dexamethasone-based regimen may have more advantage over the intravenous acetaminophen regimen for inhibiting pain and PONV following tonsillectomy in children. Tonsillectomy is one of the most frequent surgical procedures carried out in children which is commonly associated with increased risk of some complications such as severe pain while swallowing and postoperative nausea and vomiting (PONV) . Poorly Dexamethasone is the other drug used for diminishing postoperative pain and PONV in various operations. The effects of the dexamethasone on decreased edema and fibrosis have been clearly substantiated. The anti-inflammatory mechanism of corticosteroids is considered to be a result of the inhibition of phospholipase A2, the crucial membrane enzyme regulating the cascade for the production of hyperalgesic leukotrienes and prostaglandins from arachidonic acid in response to inflammatory stimuli -14, the There are few studies truely comparing the clinical efficacy of intravenous acetaminophen with other medications such as dexamethasone. This randomized blinded controlled trial was designed to compare controlling status of postoperative events in children after tonsillectomy randomized to receive either intravenous acetaminophen or dexamethasone.This randomized blinded controlled trial was designed to compare controlling status of postoperative events in children after tonsillectomy randomized to receive either intravenous acetaminophen or dexamethasone.This study was a randomized controlled trial with blinded assessment of postoperative pain severity and nausea and vomiting (PONV). Children were eligible if they were undergoing tonsillectomy, aged between 4 and 13 years and of ASA physical status I to II. Exclusion criteria were any renal or hepatic impairment, neurological disorder impairing accurate pain assessment, sensitivity to any trial drug, requirement for premedication and other analgesics administered on the day of surgery. After institutional Ethics Committee approval and informed consent from a parent in each case, 84 patients were randomized using a computer-generated schedule to double-blind treatment with intravenous acetaminophen 15 mg/kg (Acetaminophen 1gr/6.7mL - UNIPHARMA S.A manufactured in E.U) or intravenous dexamethasone 0.1 mg/kg (8mg/2mL – DAROU PAKHSH IRAN). Drugs were administered in 50 ml normal saline while using micro set and infused over 30 minutes. Study drugs were marked only with coded number labels and administered within the last 30 minutes of the surgical procedure.At operation room and after setting ECG monitoring, non-invasive blood pressure, capnography, and pulse oxymetry, intravenous line was secured with 22G i.v canula and ringer lactate infusion (8 to 10 cc/kg) was started. All patients were uniformly premedicated with Midazolam 0.05 mg/kg, and Fentanyl 2 µg/kg IV. After preoxygenation with 100% O2, anesthesia was induced with sodium thiopental (5 mg/kg) and Cisatracurium (0.15 mg/kg) and maintained with Sevofluran 2.5%. After the three minutes, patients were intubated endotracheal tube without cuff but tailored to the patients’ age and weight. All intubations were performed by an experienced anesthesiologist. After surgery, all patients were transferred to the recovery room. The mean duration of surgery was 45 minutes.Study Parameters Measurement: Children were postoperatively assessed for swallowing pain, pain when opening mouth, ear pain, and postoperative sore throat in recovery room (within one hour after surgery), on the time of admission to the ward, as well as at 12 and 24 hours after surgery, assessed by the Objective pain scoring system . The incidence of PONV and postoperative bleeding was also assessed postoperatively. Our study outcome measure was the incidence of postoperative pain severity as well as PONV and bleeding. After surgery and in the ward, children with OPS ≥ 5 medicated with meperidine PRN (0.3 mg/kg). In the cases with the appearance of vomiting, ondansetron PRN (0.1 mg/kg) was administered. Also, after establishment of oral feeding, Ibuprofen syrup (6 mg/kg/q8h) was administered.A sample size calculation was performed using a change in OPS mean values of 2.0 with a SD of 1.5. Calculations were performed to determine the number of patients required in this study to have 80% power to detect as statistically significant (P < 0.05). By accepting a type I error of 0.05 and a type II error of 0.20, the study size needed was 40 patients per group. Results were reported as mean ± standard deviation (SD) for the quantitative variables and percentages for the categorical variables. The groups were compared using the Student’s t-test or the Mann-Whitney U test for the continuous variables and the chi-square test (or Fisher’s exact test if required) for the categorical variables. Trend of the changes in continuous variables was compared between the groups using the repeated measure for ANOVA test. This study was done with the power of 80%. P values of 0.05 or less were considered statistically significant. All the statistical analyses were performed using SPSS version 16.0 .From a total of 84 eligible patients that were invited to participate, all of them completed the trial (42 patients in the Acetaminophen group and 42 cases in the dexamethasone group). Average age of all participants was 7.07 ± 2.22 years, median 7.00 years, ranged 4-13 years). The two interventional groups received acetaminophen or dexamethasone were matched in terms of mean age  and frequency of male gender . As summarized in Considering trend of the changes in the severity of pointed postoperative events showed that the severity of swallowing pain, pain when opening mouth, ear pain, as well as postoperative sore throat as gradually reduced within 24 hours of tonsillectomy in both groups, but no between-group differences were observed in the trend of the severity of these events . RegardiIn a total of seven patients in the dexamethasone groups and five cases of the acetaminophen groups, Amp mepridine 0.3 mg/kg PRN were used once in order to reduce pain during 24 hours after the surgery however, it didn’t cause any significant difference. . And a case of the acetaminophen group had vomited three times during 24 hours (Amp ondansetron 0.1 mg/kg PRN was used once) in which the P value = 0.314 was not significant.Postoperative pain and PONV remains a significant problem for children undergoing tonsillectomy, and is the commonest reason for readmission after procedures. The present study compared two different analgesic regimens including intravenous acetaminophen and dexamethasone. Although we could demonstrate a significant deceasing trend of pain severity within 24 hours of surgery in both groups, but between-group difference was not observed regarding trend of the changes in swallowing pain and pain when opening mouth. Also, more pain removing effect of dexamethasone was observed in dexamethasone group compared with acetaminophen group at 24 hours after surgery. Moreover, those who administered dexamethasone experienced severer postoperative sore throat compared to another group within 12 hours after surgery. In fact, our study emphasizes more efficacy of dexamethasone administration than intravenous acetaminophen with respect to controlling postoperative complications in children undergoing tonsillectomy. Much discomfort after tonsillectomy is potentially due to local inflammation, edema and nerve irritation. Dexamethasone is one of the most potent glucocorticoids available that can effectively suppress a basic inflammatory response to tissue injury , 16. DexMalde  studied In our study, although the appearance of PONV was scarce after tonsillectomy, but its rate was more observed in the group which received dexamethasone in comparison with acetaminophen administration. It has been shown that in patients with a high risk of postoperative PONV, a single prophylactic dose of dexamethasone can effective compared with placebo, without evidence of any clinically relevant toxicity , 27. AltThere has been limitations in our study, for instance IV acetaminophen was given once and the half-life is 2-4 hours and duration effect 4-6 hours; we did not control for other medication use that impacts post-op pain and PONV ; even though there is a statistical difference in post op sore throat at the first three time points, one wonders if this is really clinically significant.


Resistin is an adipocytokine, associated with obesity and inflammation. Its exact role in insulin resistance and diabetes in the general population is still controversial. The relation between resistin plasma levels, insulin resistance and risk of impaired glucose metabolism in OSA patients has not been investigated.Plasma levels of resistin were measured in 67 patients with OSA and impaired glucose metabolism. 34,7% (23/67) had diabetes; 40% (27/67) patients had impаired glucose tolerance(IGT); 25,3%(17/67) had normal glucose metabolism (NGM). The association between resistin, BMI, obesity, markers of insulin resistance, oxidative stress and sleep study characteristics was analysed. The different groups of patients were compared in regards to glucometabolic parameters and biomarkers of oxidative stress – isoprostanes and insulin resistance – free fatty acids (FFA).Plasma levels of resistin were higher in patients with diabetes , compared to those with IGT  and NGM . Resistin did not differ between patients with IGT and NGM . In OSA patients with BMI>40 resistin plasma levels correlated neither to the clinical parameters , nor to the biomarkers of oxidative stress and insulin resistance. Free fatty acids  were higher in diabetics in comparison to NGM.Plasma resistin levels in OSA patients with BMI>40 are independent of insulin resistance and are not associated with the parameters, characterising the oxidative stress or severity of OSA. Resistin could be used in a multiple panel of clinical and biomarkers to discern patients with diabetes from those with IGT; in OSA patients with BMI >40 resistin together with HbA1C could discern patients with diabetes from those with NGM. In OSA patients with BMI >40 FFA and HbA1C are useful clinical markers in assessing the risk of dysglycaemia among patients with normal and IGT. Obstructive sleep apnea (OSA) is closely related to the metabolic syndrome. Systemic inflammation and oxidative stress have been thoroughly described as the main pathological triggers, provoking both metabolic and cardiovascular derangements in OSA patients. The inflammatory milieu is associated with the development of vascular injury in OSA . Indeed,OSA is also closely related to insulin resistance which itself is a cardiovascular risk factor. Insulin resistance, oxidative stress and chronic inflammation coexist in OSA leaving the patients under increased risk for cardiovascular diseases.In turn, the presence of OSA has emerged as an independent risk factor contributing to insulin resistance -6 and thIt is now clear that the routine biochemistry and the indexes, related to OSA severity are not the best indicators neither for the cardiovascular derangements, nor for the metabolic disruptions. This leads the door open for future research that is mostly clinically oriented providing a good study design as well as reasonable and pathophysiologically related biomarkers.Resistin is an adipocyte-derived cytokine (adipokine) that may contribute to the development of obesity -12, insuFree fatty acids have been used as predisposing factors for insulin resistance for a long time as well as a cardioavascular risk factor. In addition in OSA patients they are associated with sympathetic activity. Their role in insulin resistance in OSA patients is still elusive ,21. In cThe design of the study was conducted so that to find the role of resistin in the generation of insulin resistance, including subjects with newly found OSA. The primary aim was to investigate whether there is an incremental change of resistin from healthy to diabetic patients, analyzing its association with the clinical indicators – IRI, HOMA index; whether it refelects the oxidative stress - evaluated by the level of urinary isoprostanes, or is more closely related to insulin resistance and immunoreactive insulin. The secondary aim was to determine whether OSA severity, from normal to severe affects resistin levels.We further evaluated the associations between resistin and lipid profiles and free fatty acids and tried to use them in a multipanel diagnostic model for the early detection of glucose impairment in OSA patients.The protocol of the study was approved by the Ethics Committee of the University Hospital “Alexandrovska” and all the participants gave informed consent. We consecutively recruited 67 patients with newly defined OSA between June - December 2011. None of them had undergone any treatment for OSA. The definition of OSA was based on a combination of clinical symptoms (i.e. daytime excessive sleepiness) and a standard polysomnography.2).After polysomnography, fasting blood samples were taken between 7 and 8 a.m. to examine plasma levels of resistin, and free fatty acids. A sample of overnight urine was taken to define the levels of urinary isoprostanes. Тhe biochemical tests were performed in the laboratory of synthesis and analysis of compounds, Department of Biochemistry, University Hospital “Alexandrovska”. Following admission, smoking habits , medical history and regular medication use were recorded. Anthropometric data (height and weight) were recorded and BMI was then calculated as body weight (kg) divided by height squared  x serum insulin( U/ml)/22,5 .An oral glucose tolerant test was performed to define the patients with impairments in glucose metabolism. The test was performed as described by WHO  – subjecExclusion criteria were as follows: 1) age > 80 years; 2) current prescription of anti-inflammatory drugs, statins, steroids; 3) the presence of any of the following medical conditions: chronic kidney diseases, chronic respiratory failure, primary heart diseases, endocrine disorders or neoplasm.Polysomnographic parameters were continuously recorded via a computerized data acquisition system Compumedics, E-series. The following parameters were documented:sleep stage , electrocardiogram, nasal airflow, thoracic and abdominal movements and oxyhemoglobin saturation (SpO2). Apnea was defined as complete cessation of nasal airflow for at least 10s. Hypopnea was defined as a > 50% reduction in nasal airflow lasting at least 10s, with oxygen desaturation > 3%. The AHI was calculated as the average number of apnea plus hypopnea events per hour of sleep. The degree of nocturnal hypoxia was assessed as: 1) the percentage of the total sleep time during which SpO2 decreased below 90% (time SpO 2 < 90%).Blood samples were centrifuged immediately after collection and isolated plasma was stored in vials at −80°C until assayed. Resistin and free fatty acids were measured by commercial kits, following the procedure protocol.Resistin was determined by an ELISA kit (RayBio_ Human Resistin ELISA Kit Protocol (Cat#:ELH-Resistin-001) The intra- and interassay coefficients of variation in this assay kit ranged from 10 to 12%. Plasma resistin levels were measured in ng/ml.The concentration of free fatty acids was determined by an enzymatic colorimetric method assay for the quantification of non-estserified free fatty acids – (WAKO NEFA-HR2). Results are given in mmol/l.The levels of 8-isoprostane in urine samples were determined by HRAM (high resolution accurate mass) mass spectrometry on LTQ Orbitrap® Discovery  mass spectrometer, equipped with Surveyor® Plus HPLC system and IonMax® electrospray ionization module. The analyses were carried out by stable isotope dilution method in negative ionization mode using HESI II (heated electrospray ionization) source type.2O) and 110 μl affinity sorbent. The sample was incubated for 45 min at room temperature. After that the sample was centrifuged for 15 min at 4°C, 13500 rmp and supernatant was carefully removed by decanting. The sample was washed once with 1 ml of phosphate buffer , centrifuged for 15 min at 4°C, 13500 rmp and the supernatant was carefully removed. After that the same washing step was repeated with 1 ml of ultra pure water. The 8-isoprostane was eluted from the affinity sorbent by 2 x 0.5 ml of 95% ethanol. The samples were evaporated to dryness under vacuum and reconstructed in 150 μl mixture of water-acetonitrile .The concentration and purification of 8-isoprostane from urine samples was processed by affinity sorbent , following the producer’s protocol with some modification. Briefly, approximately 1 ml of urine sample was centrifuged for 10 min at 4°C and 13500 rmp. Then, to 300 μl of supernatant was added 700 μl of phosphate buffer , 10 μl internal standard  using as eluents: A-0.1% Formic acid in water; B - 0.1% Formic acid in acetonitrile at flow rate of 250 μl/min. The followed binary gradient was used: 30–90% B for 10 min; 90–30% B for 1 min; and 30% B for 4 min.The HRAM spectra in range of m/z 200–700 were acquired on Orbitrap® mass analyzer set to operates at 30 000 resolution. The all Orbitrap MS parameters were optimized for sensitivity to the target analyte using the instrument control software program. The below presented parameters were used: spray voltage 3.2 kV; Spray current 100 μA; Sheath gas flow rate 40; Auxiliary gas flow rate 10; Sweep gas flow rate 0; Capillary voltage −37 V, Capillary temperature 280°C; Vaporizer temperature 250°C and Tube lens −99.07 V. All data acquisition and processing were carried out with XCalibur® software package . Results are given in pg/mkmol/creatinine.Statistical analysis was performed using SPSS  A p<0,05 was considered of statistical significance. Continuous variables are expressed as means ±SD. Kolmogorov-Smirnov was used to find if normal distribution existed. Comparison between the three groups of OSA patients  was made using one way analysis of variance (ANOVA) if normal distribution or Kruskall-Wallis if non-parametric data was found. Mann-Whithney test was performed to compare two groups of patients according to different variables. Correlations between paired variables were performed using Pearson  or Spearman (nonparametric data) correlation analysis.Patients were divided into three groups according to their glucose metabolism – patients with already known diabetes – 23; patients with impaired glucose tolerance – 27; patients with normal glucose metabolism – 17. Anthropometric, glucometabolic, sleep study and biological characteristics are given in Table Regarding the anthropometric and sleep study characteristics no large discrepancies existed between groups. They were similar according to the age, BMI, smoking status, gender distribution. No difference was established, when analyzing their sleep study parameters. The lipid profiles did not differ in regards to any of the fractions investigated.The analysis of the indicators related to the glucose metabolism showed a statistically significant difference between the plasma levels of the immunoreactive insulin in patients with IGT in comparison to those with diabetes – 31,06±28,05 vs.18,92±16,57 (p −0.006). The HOMA – index did not differ much between the groups. The glycated haemoglobin however was statistically higher in patients with IGT - 6,41±1,06 vs. those with normal glucose metabolism 5,63±0,44 p-0.003. The same trend could be found when the diabetics and the patients with normal glucose metabolism were compared - 6,69±1,11 vs 5,63±0,44 . Resistin plasma levels were significantly higher in patients with diabetes - 6.12±5.93 in comparison to those with normal glucose metabolism - 3.77±3.23, p-0.043. Similar are the results when diabetics and patients with impaired glucose metabolism are compared – p −0.021. The significant difference remains even after adjustment for age, BMI and HOMA-I.Based on the AHI, OSA patients were subdivided into mild-to-moderate (AHI = 5–30 events/h) OSA group – 19; and severe (AHI > 30 events/h) OSA group - 48. Patients’ characteristics are given in Table The analysis of the relationship between resistin plasma resistin levels and anthropometric, clinical, biological and sleep apnea parameters, cigarette consumption is shown in Table ROC analysis was performed to analyse the clinical utility of the biomarkers that differed significantly between groups. The ROC analysis of FFA, resistin and HbA1C among diabetic and healthy patients is presented in Table The ROC analysis shows that resistin plasma levels of 3,45ng/ml had sensitivity - 68% and specificity – 69% in distinguishing diabetic from healthy patients; FFA at a cut-off of 0,211 mmol/l has sensitivity 61% and specificity – 67%; HbA1c – 5,7% seems to be with the best sensitivity (78%) and specificity (75%) as a diagnostic marker. Similar are the results when comparing patients with impaired glucose tolerance and healthy ones. ROC analysis could be performed in regards to FFA and HbA1c. Data is presented in Table ROC analysis shows that in OSA diabetic patients with high grade obesity the validated by the American Diabetes Association diagnostic value of HbA1c – 6,4% is less specific and sensitive, compared to 5,7%. Similar are the results regarding OSA patients with impaired glucose tolerance and high grade obesity - HbA1c – 5,7% is better than the range 5,7-6,4%.The only biomarker that differed between diabetic and impaired glucose tolerance patients was resistin At plasma levels of 4,03ng/ml it shows sensitivity - 64% and specificity – 67% in distinguishing diabetic from impaired glucose tolerance patients Table Patients with sleep apnea commonly develop the metabolic syndrome, the hallmark of which is insulin resistance. It appears to be one of the major risk factors for atherosclerotic and cardiovascular diseases . The mecAdipose tissue dysfunction that is characterized by the imbalanced secretion of different adipokines has been largely disputed in clinical studies. Adiponectin and leptin are adipose-specific hormones that improve glucose tolerance. Both have been thoroughly investigated in different studies. Several other adipose-derived hormones oppose insulin action, and they circulate at increased levels in obesity, thus also possibly contributing to insulin resistance. Prominent among them are tumor necrosis factor-α and interleukin-6, two cytokines that are also produced at high levels in macrophages and were first identified as inflammatory mediators ,28. ResuResistin is a 12-kDa polypeptide that was initially linked to insulin resistance in animal models . Early rA number of studies have examined plasma resistin levels or adipose resistin expression, and have found variable associations with insulin resistance ,33-35. AAn investigation of resistin in OSA will therefore provide a better understanding of the complex interrelation between metabolic disorders, inflammation and cardiovascular involvement.According to the results of our study there is not a significant difference between the plasma levels of resistin in patients with impaired and normal glucose tolerance. The two groups were comparable regarding the age, BMI, waist circumference and the severity of OSA. The duration of sleep, the average desaturation index and the time spent under SpO2 <90% also did not differ much between the groups.The analysis of resistin plasma levels on the other hand showed correlation neither to the anthropometric , nor to the sleep study or glucometabolic characteristics. The other biomarkers, indicative of insulin resistance and oxidative stress also turned to play no role on resistin plasma levels. Assuming this data it seems that resistin alone is not a trigger of insulin resistance in obese OSA patients. It more likely plays a secondary role in the complex adipokine signaling, accompanying adipose tissue dysfunction.Our results support those of Yamamoto et al. , who finHaving this in mind and considering that each of the three groups we studied consisted of extremely obese patients with an average BMI >40 (only 10% have BMI 30–35) we can speculate that the secretion of resistin could be blunted. If true this makes it rather difficult to detect the slight difference in resistin plasma levels between OSA patients with IGT and NGM if any exists.Wysoczka et al. , describWe found no association between high levels of insulin and HOMA-I and resistin neither in patients with normal, nor in those with IGT. This is in controversy to what is reported in obese patients (BMI-33) without diabetes . Again iIn human adipose tissue, resistin seems to be produced mainly by infiltrating macrophages .Recent studies have shown the causative association between resistin and systemic inflammation , especiaIn our study we find a tendency for an increase of resistin with the severity of OSA that is not statistically significant. The plasma levels of the marker in the group with moderate apnea, compared to those with severe one are respectively – 3,92vs4,96ng/ml, not reaching statistical significance. A reason for this can be that our patients were extremely obese – the average BMI >40. In the study of Yamamoto et al. , and LeeThe limitations of our study are that: First patients were of similar age and BMI, but extremely obese, which complicates the applicability of data to the general OSA population; Second patients were predominantly men as a gender dimorphism is present in plasma resistin ,43 the rLast but not least we performed a cross-sectional study, encompassing a relatively small number of subjects with various impairments of glucose metabolism, which does not allow the establishment of any causality, but only the propositions of certain hypothesis about the role of resistin in the metabolic derangements in OSA.In conclusion according to the results of our study resistin plasma levels in OSA patients with BMI>40 do not correlate to any of the clinical parameters - anthropometric, glucometabolic, lipid or sleep study characteristics of the patients. Resistin plasma levels were higher in patients with diabetes, compared to those with IGT and NGM. In OSA patients with BMI>40 resistin did not differ between patients with IGT and NGM and did not change significantly with the severity of OSA. This shows that resistin alone could not be an indicator of insulin resistance in OSA patients, at least not in extremely obese one. It could however be a part of a multiple diagnostic panel. In OSA diabetic patients with high grade obesity HbA1c – 5,7% is of better diagnostic value than the validated – 6,4%.OSA: Obstructive Sleep Apnea; AHI: Apnea+Hypopnea Index; BiPAP: Bilevel Positive Airway Pressure; BMI: Body Mass Index; HOMA-I: Homeostasis Model Assessment Index; IRI: Immuno Reactive Insulin; OGTT: Oral Glucose Tolerance Test; RR: Riva Rocchi; FFA: Free Fatty Acids; IGT: Impaired Glucose Tolerance; NGM: Normal Glucose Metabolism.The authors declare that they have no competing interests.RCh, OG, VM and DP participated in the design of the study and in writing the manuscript. RCh recruited the patients and collected the data. TM,OG performed the PSG studies. SSR and VL performed the HRAM analysis and free fatty acids measurements. AC performed the ELISA for resistin and the enzymatic determination of urinary creatinine levels. All reviewed and approved the final version of the manuscript.