Results for serum alpha-fetoprotein, β-human chorionic gonadotropin, and carcinoembryonic antigen were within normal limits. Ultrasonography of the left testis showed increased vascularity and presence of hypoechoic areas containing mottled hyperechogenicity (Wuchereria bancrofti. There was no evidence of malignant cells or fungal elements in the smears examined. The final diagnosis was bancroftian filarial orchitis. The patient was prescribed a 14-day course of diethylcarbamazine.A 37-year-old man from eastern Uttar Pradesh, India, presented with 20 days of a painless swelling of the left testis. There was no history of trauma, fever, infertility, lymphadenitis, lymphangitis, or previous history of testicular tumor. On examination, the left testis measured 5×4 cm, and was found to be slightly enlarged, firm, and nontender on palpation (W. bancrofti.W. bancrofti infection and clinically mimics testicular malignancy or testicular torsion.In India, > 98% cases of filariasis are caused by


GSTO2 is one of the members of this family. GSTO2 (N142D) polymorphism may influence metabolism of immunosuppressive drugs.Acute rejection is the main problem in liver transplantation that occurs in the first days or months of transplantation. It includes histological and cellular rejection. Acute histological rejection is confirmed by biopsy. Glutathione S-transferase family is the most important genes in phase II detoxification working in xenobiotic and drug metabolismGSTO2 polymorphism has association with acute liver rejection.To determine if GSTO2, we used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).The present study included 120 patients with histological-proven acute liver rejection and 182 patients without acute rejection. Both groups were matched for sex and age. To determine variants of GSTO2 genotype and acute liver rejection  and  compared to those with DD geneotype.There was a significant association between the GSTO2 are more likely to develop acute liver rejection compared to those with DD genotype.Recipients with either NN or ND genotype for  Liver transplantation is one the best treatments for patient suffering from end-stage liver disease . DespiteGSTO1 and GSTO2. GSTO2 is on 10q24.3 and has six exons that spans over a 24.5-kbp region that lies 7.5 kbp downstream to GSTO1. GSTO2 has a Cys in its active site that plays an important role in arsenic detoxification. GSTO2 is expressed in whole the body organs, especially liver, kidney, skeletal muscles and prostate [Glutathione-S transferases (GST) are the main enzymes in phase II detoxification that play important role in xenobiotic, and drug metabolism, which catalyze binding of glutathione to these substrates causing them become more water soluble, so that they can be excreted through bile or urine . MutatioGSTO2 (N142D) is one of the polymorphisms of this gene with A>G transition at nucleotide position 424 of exon 5. This change reduces the expression of this enzyme by 20% [GSTO2 (N142D) can affect ALR.e by 20% . Some ste by 20% , gastrice by 20%  and ovare by 20% . There ae by 20% , 11 and e by 20% . We knowStudy GroupsIn the current study, 302 recipients of liver transplants between 2007 and 2011, were studied. All of the patients were Iranian and transplanted at the Transplantation Center of Namazi Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, southern Iran. The study protocol was approved by Ethics Committees of Shiraz University and Shiraz University of Medical Sciences. In the recipients, we investigated the graft outcome and AR episode(s) for at least six months. Acute rejection episodes were identified by an expert gastroenterologist team based on the accepted criteria such as increased serum liver enzymes levels and total serum bilirubin level in the absence of biliary problems, histological findings after biopsy of the liver and clinical and biochemical response to high-doses of steroids, according to criteria for AR described by Banff schema . The rouDNA ExtractionThe buffy coat of the whole blood from liver transplanted patients was available in the sample bank of Transplant Research Center. Patient’s DNA was extracted with DNP kit  according to the manufacturer’s instructions.Genotyping2 (0.45 mM), KCl (50 mM), Tris-HCl , dNTP (0.2 mM), each primer (4 μM), Taq DNA polymerase (1 U)  and DNA sample. Sequences of forward and reveres primers were 5’-AGGCAGAACAGGAACTGGAA-3’ and 3’-GAGGGACCCCTTTTTGTACC-5’, respectively. The PCR program consisted of an initial pretreatment at 95 °C for 5 min, with 35 cycles fallowed by denaturation at 95 °C for 1 min, annealing at 65 °C for 40 sec and extension at72 °C for 1 min. Then, 2.5 μL Tango buffer and 5 U MboI enzyme added to each PCR product tube and incubated at 37 °C for 14 hours. Then, loaded in 2% agarose gel and stained by ethidium bromide and finally illustrated by UV translluminator. When our genotype was GSTO2 NN, we observed one fragment (185 bp) in the gel; in heterozygote case (ND), three fragments  and in DD homozygote two fragments  was observed (Patients’ genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). PCR mixture included MgClobserved .Statistical Analysis® for Windows® ver 17.0  for data analysis. We used Student’s t test for continuous variable. χ2 test was used for categorical variables and logistic binary regression test for risk of liver problems.We use SPSS2 test was performed for GSTO2 polymorphism to determine if the distribution followed the Hardy-Weinberg equilibrium. Regardless of rejection, the studied gene was in Hardy-Weinberg equilibrium (P>0.05). From 302 patients, 120 had AR (mean±SD age of 30.9±18.3 years) and 182 patients did not (mean±SD age of 29.1±19 years). The frequency of D allele was 35% in patients with ALR and 47.3% in those without ALR. There was a significant relationship between GSTO2 (N142D) polymorphism and ALR; increase in D allele reduced the likelihood of ALR; there was a significant association between GSTO2 genotypes and ALR  and also for alleles  (A χp=0.002) . PatientGSTM1 and GSTT1 with ALR [ GSTT1 genotype mismatch (present/null or null/present) is significantly associated with development of acute cellular rejection [GSTO2 and also this polymorphism have so far not been examined. In the current study, we showed that increasing frequency of D alleles decreased the risk ALR. The NN genotype is associated with elimination of immunosuppressive drugs faster than the ND, DD genotypes; ND genotype is faster than DD genotype too. Therefore, the risk of ALR is increased with presence of N allele. In several studies it was shown that concentration of liver enzymes can be useful for predicting ALR [AR is one of the main obstacles after liver transplantation. Several studies show that an increase in D allele frequency decreases the risk of colorectal and gastric cancer , 8. It hwith ALR . Anotherejection , howeverting ALR . We founGSTO2 are more prone to develop hepatic failure. We also observed that males are more prone to develop hepatic failure leading to liver transplantation [In 2013, we showed that patients with D allele of antation .GSTO2 polymorphism N142D is not associated with acute renal rejection [GSTO2 DD genotype in the recipient, results in significant increase in risk of acute rental rejection . This reflects a probable reduction in function of DD genotypes, as anti-oxidant enzymes involved in phase II detoxification, with consequent increase in acute inflammatory processes [A recent study conducted on a group of Iranian people indicated that ejection . An imporocesses .


Chiropractors have been shown to refer for lumbar radiography in clinical scenarios inconsistent with the current clinical guidelines for low back pain. It is unknown whether this is due to lack of adherence with known guidelines or a lack of awareness of relevant guidelines. Therefore, the aim of this study is to determine Australian chiropractors’ awareness of, and reported adherence to, radiographic guidelines for low back pain. Demographic, chiropractic practice and radiographic usage characteristics will be investigated for association with poor guideline adherence.An online survey was distributed to Australian chiropractors from July to September, 2014. Survey questions assessed demographic, chiropractic practice and radiographic usage characteristics, awareness of radiographic guidelines for low back pain and the level of agreement with current guidelines. Results were analysed with descriptive statistics and logistic regression analysis.There were 480 surveys completed online. Only 49.6 % : 44.9, 54.4) reported awareness of radiographic guidelines for low back pain. Chiropractors reported a likelihood of referring for radiographs for low back pain: in new patients ); to confirm biomechanical pathologies ); to perform biomechanical analysis ); or to screen for contraindications ). Chiropractors agreed that radiographs for low back pain could be useful for: acute low back pain ); screening for contraindications ); or to confirm diagnosis and direct treatment ). Poorer adherence to current guidelines was seen if the chiropractor referred to in-house radiographic facilities, practiced a technique other than diversified technique or was unaware or unsure of current radiographic guidelines for low back pain.Only 50 % of Australian chiropractors report awareness of current radiographic guidelines for low back pain. A poorer awareness of guidelines is associated with an increase in the reported likelihood of use, and the perceived usefulness of radiographs for low back pain, in clinical situations that fall outside of current guidelines. Therefore, education strategies may help to increase guideline knowledge and compliance.The online version of this article (doi:10.1186/s12998-016-0118-7) contains supplementary material, which is available to authorized users. The use of lumbar radiography within chiropractic clinical practice is well established as a necessary tool to aid diagnosis and direct appropriate treatment of the low back. The use of radiography is associated with risks to the patient, including risks from ionising radiation; unnecessary diagnosis leading to poorer patient outcomes or potentially unnecessary investigation or treatment; and higher costs . GuideliRadiographic guidelines for low back pain (LBP) have been published by both the medical -4 and chDespite the recommendations made in the published guidelines many chiropractors still believe that radiographs of the lumbar spine are useful in clinical scenarios outside the current guidelines. Surveys conducted by Ammendolia et. al.  and WalkThere is some evidence that educating chiropractors regarding current radiographic guidelines is associated with increased guideline compliance , 12 or aTherefore, the aims of this study were to quantitatively assess whether Australian chiropractors reported awareness of current radiographic guidelines for LBP and whether they demonstrated adherence to the key messages in the guidelines. Finally, demographic, chiropractic practice and radiographic usage characteristics were investigated for association with poorer guideline adherence.An online cross-sectional survey was conducted of Australian chiropractors. The survey was designed and implemented using the Qualtrics software. A link to the survey was distributed to Australian chiropractors through two methods: email distribution from two Australian chiropractic associations ; and within the quarterly newsletter from the national registration board for Australian Chiropractors . Only registered chiropractors were invited to partake in the survey. The survey was available from July to September, 2014. No identifying information was requested and participation was voluntary.The survey collected information regarding demographics of the responding chiropractors and characteristics of clinical practice including: year and school of graduation from chiropractic training; years in clinical practice; use of in-house radiographic facilities; further education in radiographic imaging; and the primary manual technique style used in clinical practice. The primary outcome question in the survey asked respondents to state if they felt that they were aware of the current radiographic guidelines for LBP (yes/no/unsure). Respondents were also asked if they were aware (yes/no) of specific guidelines including those put out by both chiropractic  and mediSurvey response rate was calculated as the number of completed surveys divided by the number of chiropractors sent the survey link. The survey link was sent to all registered chiropractors in Australia through the quarterly registration board newsletter, therefore the denominator used was the total number of registered chiropractors in Australia.Descriptive statistics were used to assess the frequency of response to each of the three responses to the primary survey question regarding awareness of radiographic guidelines. Stratification of the responses to the primary research question was performed according to institute of graduation and association was tested for using the chi-squared test. Results for the secondary outcome questions were dichotomised: for those regarding the likelihood of referring for radiographs into those likely to refer for radiographs  compared to the other responses; and for those assessing agreement with indications for referring for radiographs into those agreeing with the statement (agree/strongly agree) compared to all other responses. All of these were calculated as percentages with 95 % confidence intervals (95 % CI).Multivariate logistic regression analysis was used to assess for association and to adjust for possible confounders between six preselected predictor variables and twelve outcome questions: five questions from the respondents’ likelihood for referring for radiographs and seven from their agreement with statements regarding the usefulness of radiographs for LBP ) reported definite awareness of current guidelines whereas 170 ) were unsure of their awareness and 39 ) reported that they were unaware of current guidelines. When questioned regarding specific guidelines only 68 out of 477 respondents ) were aware of the chiropractic guidelines written by Bussieres et. al. [2(8) = 14.2, p = 0.1).Stratification of respondents reported awareness of radiographic guidelines for low back pain by institute of graduation is shown in Fig. The responses for the secondary outcome questions are depicted in Figs. Analysis of associations with outcome questions showing poorer adherence to current radiographic guidelines for LBP are shown in Table The correlation coefficients of the independent variables were assessed. The highest correlation coefficient found was 0.37 between further radiographic education and the year of graduation, indicating that multicollinearity is not significant for this analysis.The primary finding of this study was that 49.6 %  of Australian chiropractors’ report definite awareness of current radiographic guidelines for the low back and only 14.3 %  were aware of published guidelines for the chiropractic profession. This low awareness of current guidelines is consistent with the lack of adherence found in both chiropractors’ reported likelihood of referring for radiographs and their agreement with scenarios where radiographs could be useful. Poorer reported guideline adherence was associated with chiropractors who: refer to in-house radiographic facilities; practice techniques other than diversified; or are unsure or unaware of current radiographic guidelines.The results of this study indicate that adherence to radiographic guidelines for LBP by Australian chiropractors needs to be improved. Current guidelines recommend that radiographs are not useful for the majority of acute LBP cases, including those associated with neurological symptoms; to screen for contraindications; or to analyse for biomechanical change. Radiographs are indicated by the suspicion of trauma or pathology as a cause of LBP or after a period of four to 6 weeks non-response to treatment . AlthougAdherence to radiographic guidelines may be limited by a lack of awareness of the current guidelines. Low awareness of the current guidelines in Australian chiropractors was seen in this study, particularly of those guidelines published for the chiropractic profession specifically . AlthougPoorer guideline adherence was associated with chiropractors referring to in-house radiographic facilities and those practicing techniques other than diversified as their primary manual technique style. Therefore, it may be important to target educational strategies towards these groups of chiropractors to improve adherence to guideline recommendations. Referral to in-house radiographic facilities showed the strongest association with poorer guideline adherence. In chiropractors referring to their own radiographic facilities the odds of being likely to take radiographs of new patients were increased 7.23 times and the odds of agreeing that radiography is useful routinely before spinal manipulative therapy were increased 6.26 times. These findings are of concern since self-referral to practitioner-owned radiographic facilities has the potential to be misused for financial incentives. Strong consideration needs to be given by these practitioners regarding the clinical justification of referring for radiographs, particularly when not in adherence with clinical guidelines. Given their responsibility in owning and operating radiographic facilities and their need to comply with national regulations for ionising radiation, strategies to increase guideline adherence within this group should be prioritised. Diversified technique is the most common spinal manipulative technique practiced among chiropractors -17, howeThe awareness of radiographic guidelines for low back pain within Australian chiropractors has not been previously assessed in the literature. Similarly, no studies assessing awareness of guidelines in other chiropractic populations were found. This information is important as this may indicate the need for education of the key guideline messages to the chiropractic population. Three previous studies have quantitatively assessed the adherence of registered chiropractors to radiographic guidelines for the management of LBP. Two surveys performed in Canada with 26 and 32 responses respectively found that 63 and 59 % would use radiography for acute LBP without indicators of potential pathology and 68 and 66 % thought that radiography was useful in the evaluation of acute LBP . DespiteKey strengths of this study include the primary outcome question and the analysis for potential associations with poorer reported guideline adherence. Previous studies have shown chiropractors’ to demonstrate a lack of adherence to radiographic guidelines for LBP -8, howevThe main limitation of this study is the inability to calculate an accurate response rate. The response rate was calculated as 9.9 % based upon the number of completed surveys compared to the total number of registered chiropractors in Australia. However, this is likely to be an underestimate as the number of people who actually received the survey link could not be accurately determined. The survey was distributed via email distribution to association members and a link in the quarterly newsletter from the Chiropractors’ Board of Australia. Although all registered Australian chiropractors should have received the survey information, it is unknown how many actually received and opened the emails or newsletter and how many followed the link to obtain information about the survey. Calculation of an accurate response rate is necessary to assess non-response bias. As this could not be achieved, assessment of representativeness of the respondent sample was addressed by comparing demographic characteristics from survey respondents to demographic data from the Chiropractic Board of Australia and previous research. Data from the Chiropractors’ Board of Australia  showed aIt is important that Australian chiropractors are aware of current guidelines and demonstrate increased adherence to those guidelines. Implementation of guideline education strategies, both within chiropractic teaching institutes and to chiropractors within the profession, should be undertaken and may be a low-cost strategy to increase awareness and adherence to current guidelines. Further research should be carried out to measure the effectiveness of these educational strategies and whether further intervention is necessary. Targeted interventions may be needed in specific groups within the chiropractic profession, including those with in-house radiographic facilities and those practicing techniques other than diversified technique. Further research may be needed to identify the barriers specific to these populations and design suitable implementation interventions accordingly.This study indicates that only 49.6 % of Australian chiropractors are aware of current radiographic guidelines for LBP. This lack of awareness is reflected in a high percentage of respondents agreeing that radiographs would be useful and that they are likely to refer for radiographs in clinical scenarios that do not adhere to current guidelines. Poorer adherence to current radiographic guidelines was associated with chiropractors who referred to in-house radiographic facilities; practiced techniques other than diversified; or those who reported a lack of awareness of the current guidelines. These findings may be useful in designing education strategies to help improve guideline awareness and guideline adherent behaviour amongst Australian chiropractors.


Multiple Sclerosis (MuS) is a complex multifactorial neuropathology, resulting in heterogeneous clinical presentation. A very active MuS research field concerns the discovery of biomarkers helpful to make an early and definite diagnosis. The sphingomyelin pathway has emerged as a molecular mechanism involved in MuS, since high levels of ceramides in cerebrospinal fluid (CSF) were related to axonal damage and neuronal dysfunction. Ceramides are the hydrolysis products of sphingomyelins through a reaction catalyzed by a family of enzymes named sphingomyelinases, which were recently related to myelin repair in MuS. Here, using a lipidomic approach, we observed low levels of several sphingomyelins in CSF of MuS patients compared to other inflammatory and non-inflammatory, central or peripheral neurological diseases. Starting by this result, we investigated the sphingomyelinase activity in CSF, showing a significantly higher enzyme activity in MuS. In support of these results we found high number of total exosomes in CSF of MuS patients and a high number of acid sphingomyelinase-enriched exosomes correlated to enzymatic activity and to disease severity. These data are of diagnostic relevance and show, for the first time, high number of acid sphingomyelinase-enriched exosomes in MuS, opening a new window for therapeutic approaches/targets in the treatment of MuS. A very active MuS research field concerns the discovery of new biomarkers helpful to understand its etiology, and to assist in early diagnosis as well as prognosis3. CSF is an excellent source of biological information regarding the CNS, since it is in close contact and accurately reflects alterations of this system. Since the brain is the organ with one of the highest lipid concentration, lipid metabolism become of specific interest in understanding the molecular mechanism underlined to different neurological disease4, including MuS5. Sphingolipids are integral components of biomembranes, which are involved in many cellular functions, including cell proliferation, signaling cascades and apoptosis6. Sphingolipids include sphingomyelins (SMs), ceramides and sphingosines, involved in the same molecular cascade, in which one component emerges from the other by sequential enzymatic reactions7. The interest in the study of the SMs pathway increased with the use of FTY720 as an oral drug in MuS treatment. Indeed, the drug is an analogue of Sphingosine1-P, that represents the last product of SMs cascade8. In addition, higher phospholipid and lower sphingolipid content were found in white and gray matter from MuS patients compared to controls9, while sphingosine content is increased in MuS white matter10. On the other hand, ceramide levels increase in regions surrounding plaques11, and it was recently demonstrated, through a lipidomics approach, that patients with MuS had increased CSF levels of ceramide C16:0 and C24:0. Moreover, the presence of these ceramide species are sufficient to induce neuronal mitochondrial dysfunction and axonal damage12. Ceramides are the hydrolysis product of SMs through a reaction catalyzed by sphingomyelinase (SMase), a family of enzymes that have recently become of interest in MuS. Neutral SMase (nSMase) was also identified as a possible biomarker of MuS13, while, Acid SMase (ASMase) deficiency seems to enhance myelin repair after acute and chronic demyelination14. The exact mechanism of SMase release and activity (strongly dependent on the pH of environment) are still unclear, although the constitutive extracellular release, through exocytosis mechanism, was described and seems to be linked to exosomes and microvesicles (MVs) formation16. However, the role of exosomes and microvesicles in the release of secretory SMase and their involvement in MuS remain unclear17, even if some evidence is beginning to emerge18. Moreover, the link between SMs and vesicles was already demonstrated since the FTY720 significantly reduced the amount of MVs in the CSF of EAE-treated mice19. In our previous studies, we reported lipidomics profiling of serum20 and CSF21 from MuS patients. In the present work, we applied a lipidomics approach for investigating CSF phospholipid profile, with particular focus on SMs, in patients with MuS compared with CSF from patients suffering from Other Central and Peripheral Neurological Disease . Moreover, the ASMase activity in CSF of MuS patients and patients with C_OND and P_OND was measured and correlated to the number of total exosomes in CSF. Finally, CSF exosomes carrying ASMase were identified, gated and counted in the enrolled patients.Multiple Sclerosis (MuS) is a highly complex CNS disease characterized by a multifactorial pathogenesis and a great heterogeneity in clinical presentation22 focusing on the acquisition of phosphatidylcholines (PCs) and SMs profile. 421 signals of PCs and SMs species were obtained following the data processing described in method section, and their relative intensities were subjected to different explorative statistical approaches with the aim of highlighting specific altered lipid patterns in MuS. Figure CSF lipids from 20 MuS patients and 17 OND patients were analyzed by our already reported methodFigure Finally, we used a more stringent univariate statistical analysis comparing all the 421 lipid signals taken individually. Figure In order to evaluate the diagnostic predictive power of the selected lipids, we performed a combined Receiver Operating Characteristic (ROC) curve in a training set of patients (80%), as well as for external validation purpose, we predict diagnosis for new samples . In Fig. 14. These studies are in agreement with our lipidomics results where the low levels of several SMs in CSF of MuS patients means an altered sphingolipid metabolism. To elucidate the potential involvement of ASMase in MuS, we analyzed the expression and the activity of secretory isoforms16 of ASMase in CSF. Figure 23 for measuring ASMase activity in human CSF (of the same pooled samples) to determine if the overexpression of the protein was also correlated to an increase of enzymatic activity. As shown in Fig. ASMase is a key enzyme in sphingolipid metabolism and it is involved in the regulation of cell fate and signaling via hydrolysis of sphingomyelin to ceramide. Recently, an involvement of this enzyme in MuS and in particular in demyelination mechanism was establishedIn order to investigate whether the measurement of ASMase activity may represent a potential diagnostic tool for MuS, we analyzed 87 MuS CSF and 57 OND patients divided into 34 C_OND and 23 P_OND. Figure 2 = 0.03) as reported in Fig. Similar results were achieved by comparing MuS with a sub-group of inflammatory neurological diseases, suggesting that ASMase behavior in CSF is independent of unspecific inflammatory condition Fig. . A pears16. To evaluate the potential relationship between the number of released exosomes in CSF and ASMase activity in MuS, a flow cytometry investigation was carried out. In Fig. ASMase is described to be matured in secretory isoform by Golgi and translocated into extracellular compartment by a still unclear mechanismThe high number of exosomes/µL was able to reclassify MuS from C_OND whit 83,33% sensitivity and 64,29% specificity, resulting in AUC = 0.74 (p < 0.0001) setting a cut off = 45 exosomes/µL Fig. . By compTo better understand the relationship between exosomes and ASMase activity in CSF, we labeled the enzyme on these nano-vesicles with a fluorescent antibody. Figure 6. Between sphingolipids, the SMs are the most abundant structural component of myelin and demonstrated that in adrenoleukodystrophy a depletion of SMs can be observed in demyelination areas, in accordance with our data24. The SMs are hydrolysed to ceramide by SMases, a family of enzymes strongly dependent on environmental pH. Ceramide serves as a potential lipid second messenger or mediator molecule modulating diverse cellular signal pathways e.g. apoptosis. Recently, patients with MuS were found to have increased CSF levels of ceramide C16:0 and C24:0 and these ceramide species were sufficient to induce neuronal mitochondrial dysfunction and axonal damage12. This evidence may be correlated with high degradation of SMs consistent with our data. Between these SMs, we found low level of C16:0 precursor (SM (d18:1/16:0)) in MuS in respect to OND.MuS is presumed to be an autoimmune disease directed against the lipid-rich myelin sheath. We showed, through lipidomics approach, decreased SMs content in MuS CSF able to discriminate disease with good statistical performances. Moreover, our data suggest as level of SMs may serve as potential biomarkers. Further investigation demonstrated that low levels of SMs are due to the overexpression of active ASMase carried by specific exosomes. Actually, SMs are widely distributed in the nervous tissue and they are integral component of bio membranes, involved in many cellular function, including cell proliferation, signalling cascades and apoptosis25. Therefore, CD3926 and CD16127 have direct interactions with ASMase, which mediates downstream signals including STAT3 and mTOR and thus defines human Th17 cells.To explain these results, we found overexpression of ASMase accompanied by high enzymatic activity in MuS CSF. Recently, ASMase was proposed as a novel target for the therapy of human autoimmune diseases, since it shows a pivotal role in regulation of human CD4 + T-cell activation and responses. In fact, several studies have shown that upon stimulation of CD3 and/or CD28, ASM-dependent ceramide signalling mediates intracellular downstream signal cascades of CD3 and CD28, and regulates CD4 + T-cell activation and proliferation28. In addition, it has been demonstrated that the activation of ASMase is associated with MV release from glial cells, opening new strategies for the treatment of neuro-inflammatory disease29, even if we did not find data on ASMase as exosomal cargo. The number of exosomes was already characterized in serum of MuS patients30 and recently, it was demonstrated that they carry transcriptome biomarkers31. In this work, we show that the number of CSF exosomes is significantly higher in MuS patients compared to patients with OND, and that this number is correlated to CSF ASMase activity, highlighting an implication of exosomes in release and safe transport of ASMase in extracellular environment. Interestingly, we found that ASMase is located in CSF exosomes by labelling the enzyme with fluorescent antibody, showing that the ASMase abundance per exosome is strongly correlated to its activity. ASMase activity was known to act only close to membrane cell domain16, which allows the correct enzymatic condition. Our data show, for the first time, a high number of released exosomes carrying a cargo of active ASMase, that probably acts far from the original cells, inducing neuronal mitochondrial dysfunction and axonal damage through the conversion of SMs in ceramides . Futures studies may consider comparison with other, less common diseases, like Neuromylitis Optica with antiacquaporin antibodies or Neurosarcoidosis, where the inflammatory burden could be similar to MuS Patterns.The study design was made following the guidelines for the local Ethics Committee that approved the study , and conducted according to Declaration of Helsinki . All patients were informed about the procedures and provided written informed consent to participate in the study. In order to protect human subject identity a number code was employed for specimen identification.Table 21 and in Supplementary methods.A training set of samples (20 MuS and 17 OND) was used for the untargeted lipidomics approach. The extraction procedure was carried out on 200 µL of CSF as reported in our previous work22. The differential lipid signals were tentatively identified by Lipidmaps Database and HMDB database, by the retention time and by using the mass fragmentation data.An HPLC Alliance HT 2795 Separations Module coupled to Quattro Ultima Pt ESI tandem quadrupole mass spectrometer  was used for lipidomics study. The total lipid extract was injected (20 µL) and analyzed following our already reported method with few modifications concerning the profile of biological phospholipids analyzed that was focused on the acquisition of SMs and Phosphatidylcholines (PCs)15 CSF from MuS patients and 15 CSF from OND subjects were used for SDS-PAGE and WB analysis  for HPLC measurement of BODIPY-FL-C12-SM as substrate and BODIPY-FL-C12-CERAMIDE as product of the enzymatic activity. A Kinetex 5 µ XB-C18 100 Å 50 × 2.10 mm  column fitted with guard column C18 4 × 2.0 mm  was used, kept at 45 °C. Analytes were monitored at emission wavelength 520 nm . The enzyme activity was determined as a ratio of peak area of BODIPY-FL-C12-CERAMIDE (product) and the sum of peak area of the product and the BODIPY-FL-C12-SM (substrate). Data were normalized on the total proteins content for each CSF sample. Examples of chromatograms are reported in Fig. CSF from 87 MuS patients and CSF from 57 OND subjects were studied for ASMase and nSMase activity experiments by using the fluorescent substrate BODIPY-FL-C12-SM  sphingosyl-Phosphocholine (ThermoFisher scientific). 6 μL of CSF were incubated following the protocols suggested by Mühle 32. Briefly, CSF samples were stained using CD81-FITC , CD63-PE , CD9-BV450 . After 30 min of staining (4 °C in the dark), 200 µL of PBS 1× were added to each tube and 1 × 106 events/sample were acquired by flow cytometry . In a separate tube, ASMase antibody  was added, using its specific secondary antibody FITC-conjugated  after a dilution step (50 µl/test). Amplifier settings for forward scatter (FSC) and side scatter (SSC) as well as for any fluorescence channel were set in height (H) and in logarithmic mode. Exosomes scatter properties were established by running Megamix-Plus SSC beads  at the same photomultiplier (PMT) voltages used for exosome detection. Each antibody/reagent was titrated (8 point titration) under assay conditions; dilutions were established based on achieving the highest signal  for the positive population and the lowest signal for the negative population, representing the optimal signal to noise ratio33, and stain indexes were calculated34. The immune complex formation and unspecific background linked to antibody aggregation was prevented by spinning the antibody stock solution before use, at 21,000 g for 12 minutes.From each enrolled subject, 100 µL of CSF were processed by a common flow cytometry no-lyse and no-wash method35. Compensation was assessed using CompBeads and FACSuite FC Beads (BD Biosciences) and single stained fluorescent samples. Data were analyzed using FACSuite v 1.0.5 (BD Biosciences) and FCS Express 5.01 software . Exosome numbers were obtained by volumetric count.Instrument performances, data reproducibility and fluorescence calibrations were sustained and checked by the Cytometer Setup & Tracking Beads (BD Biosciences). In order to evaluate non-specific fluorescence, Fluorescence Minus One (FMO) controls were used36. Aggregates of exosomes were monitored by plotting both FSC-Area (FSC-A) signals versus FSC-High (FSC-H) signals, or SSC-Area (SSC-A) signals versus SSC-High (SSC-H). As evidenced in the representative dot-plots reported in Fig. A morphological gate was placed on a SSC-H/FSC-H dot-plot on the basis of the area containing the exosome compartment established by running MegMix-Plus SSC Beads . The exosome compartment was analyzed for the expression of CD9, CD63 and CD81 and Annexin V (in a sub-group of patients) on the basis of the respective FMO control, while the ASMase compartment was identified on the basis of the respective negative control (unspecific bound of the specific secondary antibody on the exosome gate). MFI Ratio for the ASMase was calculated by dividing the MFI of the positive events by the MFI of the secondary antibody aloneExosomes were separated, by instrumental cell sorting , on the basis of the above described flow cytometry method and gating strategy. Pure samples (92–97% of purity) were analyzed for their size by Dynamic Light Scattering.k is the fluid viscosity, D is the translational diffusion coefficient]. Data have been analyzed in terms of both scattering intensity and particles number by using either a monomodal or a multimodal size distribution software depending on the specific sample. The polydispersity index has been used as a measure of the homogeneity of the exosomal population.Pure exosomes dimensions have been determined through Dynamic laser Light Scattering by using a 90Plus/BI-MAS Zeta Plus multiangle particle size analyzer (Brookhaven Instruments Corp.) and showed in Table Mass spectra obtained by LC-MS/MS phospholipids analysis were processed using MarkerLynx software . After the exclusion of lipid signals absent in 75% of samples, that resulted the optimum compromise to eliminate most of the useless variables, data matrix was subjected to different explorative statistical approaches.The first multivariate statistical analysis was performed using Neural Designer software (version 2.9.5) by neural network implementation on data matrix. Partial least squares Discriminant Analysis (PLS-DA), was also performed on the same data matrix, by using SIMCA-P + 11.0 software . Lastly, the univariate statistical analysis visualized as Volcano-plot using Metaboanalyst 3.0 software, was carried out. The significant lipid signals resulted by comparing the above mentioned statistical approaches, were used to perform a combined ROC curve, an internal and external diagnostic model validations and a logistic regression model, using Metaboanalyst 3.0 software. Unpaired t test with Welch’s correction or Kruskal-Wallis test with Dunn’s Multiple Comparison post-test were performed for comparisons between clinical groups, using GraphPad Prism . The ROC curve analysis based on single variable, and Rank correlation between variables were performed using MedCalc 7.6 , by using a Spearman non-parametric correlation test. The values of p < 0.05 were considered significant. 95% of confidence interval was assumed for each test. ChemBioDraw Ultra 13.0 software was used for the cartoon making of the proposed mechanism reported in Fig. Supplementary Materials


Despite having higher sensitivity as compared to conventional troponins,sensitive troponins have lower specificity, mainly in patients with renalfailure.Study aimed at assessing the sensitive troponin I levels in patients withchest pain, and relating them to the existence of significant coronarylesions.® TnI-Ultra assay  was used. The ROC curve analysis was performed toidentify the sensitivity and specificity of the best cutoff point oftroponin as a discriminator of the probability of significant coronarylesion. The associations were considered significant when p < 0.05.Retrospective, single-center, observational. This study included 991 patientsdivided into two groups: with (N = 681) and without (N = 310) significantcoronary lesion. For posterior analysis, the patients were divided into twoother groups: with (N = 184) and without (N = 807) chronic renal failure.The commercial ADVIA CentaurThe median age was 63 years, and 52% of the patients were of the male sex.The area under the ROC curve between the troponin levels and significantcoronary lesions was 0.685 (95% CI: 0.65 - 0.72). In patients with orwithout renal failure, the areas under the ROC curve were 0.703 (95% CI:0.66 - 0.74) and 0.608 (95% CI: 0.52 - 0.70), respectively. The best cutoffpoints to discriminate the presence of significant coronary lesion were: inthe general population, 0.605 ng/dL ;in patients without renal failure, 0.605 ng/dL ; and in patients with chronic renal failure, 0.515 ng/dL.In patients with chest pain, sensitive troponin I showed a good correlationwith significant coronary lesions when its level was greater than 0.605ng/dL. In patients with chronic renal failure, a significant decrease inspecificity was observed in the correlation of troponin levels and severecoronary lesions. The coronarylesion was considered significant when ≥ 70% on coronary angiography.Chronic renal failure was defined as a creatinine level > 1.5 mg/dL.The patients were divided into two groups: with (N = 681) and without (N = 310)significant coronary lesion. For Receiver Operating Characteristic (ROC) curveanalysis, the patients were divided into two other groups: with (N = 184) andwithout (N = 807) chronic renal failure.® TnI-Ultra assay  was used for current sensitivetroponin with a 99th percentile value of 0.04 ng/mL. The flowchart ofthe management of all patients with chest pain met the criteria established bythe last American Heart Association guideline.9Non-ST-elevation acute coronary syndrome was defined as presence of chest painassociated with electrocardiographic changes or troponin elevation/drop onadmission or, in the lack thereof, clinical findings and risk factors compatiblewith unstable angina . The highesttroponin level during hospitalization before coronary angiography was consideredfor analysis, following the every 6-hour marker collection protocol of theinstitution.The commercial ADVIA CentaurThe following data were obtained: age, sex, presence of diabetes mellitus,systemic arterial hypertension, smoking habit, dyslipidemia, family history ofearly coronary artery disease, chronic coronary artery disease, previous acutemyocardial infarction, creatinine, ST-segment depression or T-wave inversion onthe electrocardiogram.This study was submitted to the Ethics Committee in Research and approved by it.All patients provided written informed consent.The ROC curve analysis was performed to identify the sensitivity and specificityof the best cutoff point of troponin as a discriminator of the probability ofsignificant coronary lesion, and 95% confidence interval (CI) was used. Thatanalysis was performed for the general population and separately for patientswith and without chronic renal failure.th percentile of the method and the best cutoff point found inthis study) were entered into the univariate analysis. Comparison betweenpatients with versus without significant coronary lesion wasperformed.Descriptive analysis of the categorical variables was performed by use ofpercentages. Continuous variables with non-normal distribution were expressed asmedians and interquartile intervals, and those with normal distribution, asmeans and standard deviations. The comparison between groups was performed byuse of the chi-square test for categorical variables. The continuous variables,when the Kolmogorov-Smirnov test showed normal distribution, were assessed byusing the unpaired T test, and when the distribution was not normal, theMann-Whitney U test was used. Both troponin cutoff points analyzed (the99th percentile of the method and the bestcutoff point found in this study).Multivariate analysis was performed with logistic regression, p < 0.05 beingthe significance level adopted. All baseline characteristics listed in The calculations were performed with the SPSS software, version 10.0.th percentile) showedsensitivity of 93.7% and specificity of 23%. For patients with chronic renal failureto reach a specificity of 67% , an elevation in thetroponin level to 1.58 ng/dL was necessary.The median age was 63 years, and 52% of the patients were of the male sex. The areaunder the ROC curve between the troponin levels and significant coronary lesions was0.685 (95% CI: 0.65 - 0.72). In patients with or without renal failure, the areasunder the ROC curve were 0.703 (95% CI: 0.66 - 0.74) and 0.608 (95% CI: 0.52 -0.70), respectively. The best cutoff points to discriminate the presence ofsignificant coronary lesion were: in the general population, 0.605 ng/dL; in patientswithout renal failure, 0.605 ng/dL ; and in patients with chronic renal failure, 0.515ng/dL  . In the Troponin was negative in 143 patients, and, in 40.6% of them, significant lesionswere observed on coronary angiography. In addition, 10.5% of those patients withnegative troponin showed ST-segment depression/T-wave inversion onelectrocardiogram. Using the gold-standard procedure of cardiac catheterization, theacute coronary syndrome diagnosis was confirmed in 68.7% of the patients admitteddue to chest pain. In 9.1% of those without significant coronary lesion on coronaryangiography and with positive troponin, the acute coronary syndrome diagnosis wasconfirmed by cardiac magnetic resonance. The baseline characteristics of thepopulation studied and the univariate analysis between the groups are shown in th percentile of the method,there were significant differences between the groups with and without coronarylesion regarding smoking habit , ST-segment depression/T-waveinversion  and troponin positivity , respectively. However, when considering the best troponin cutoff pointfound in this study, there were significant differences between the groups with andwithout coronary lesion regarding the male sex , smoking habit, ST-segment depression/T-wave inversion  and troponin positivity , respectively. Themultivariate analysis results are shown in In multivariate analysis, considering the 99th percentile of the method. When assessingthe subgroup with renal failure, that level is even higher, hindering its correctinterpretation.The results of this study in the Brazilian population are in accordance with those ofrecently published literature. Troponin positivity without association with coronaryangiographic findings was observed in 31.3% of the patients. In addition, betterspecificity values were only achieved with a troponin cutoff point of 0.605 ng/dL,approximately 15 times the 9912 That prevalence of troponin positivity not relatedto acute coronary artery disease is similar to that found in our study, although weassessed specifically patients with chest pain.In a study published in 2012 derived from the Scottish Heart Health Extended Cohort,blood samples were collected and high-sensitivity troponin I levels were measured.The results showed that, in a population of 15340 individuals, 31.7% of the men and18.1% of the women had high high-sensitivity troponin with no clinical manifestationat the time of blood collection, highlighting the problem of the specificity of themethod. Positivity and worse prognosis were correlated in the long run (p <0.0001), as reported in other studies.13Likewise, a prospective cohort study of 6304 patients with chest pain presenting tothe emergency department has reported positive high-sensitivity troponin T in 39% ofthe cases diagnosed as non-coronary.14 have conducted anobservational multicenter study with 1181 patients hospitalized because ofnon-cardiac causes, 15% of whom had positive high-sensitivity troponin T. Of themajor factors related to that unexpected elevation, the presence of kidneydysfunction was identified as a significantly influencing factor. In addition, onceagain, patients with elevated troponin were at higher risk for death .14Irfan et al.15That specificity value can be greater than ours found in the general population,probably because of the inclusion of more patients with other heart diseases,because we belong to a referral tertiary cardiology center.In individuals older than 75 years, high-sensitivity troponin T was assessed in thecontext of chest pain, being measured at baseline and 3-4 hours. Approximately 27%of the patients were classified as having acute coronary syndrome. The sensitivityand specificity found in that population were 88% and 38%, respectively. The greaterthe initial level or the increase (mainly absolute) in the subsequent measures, thehigher the specificity found.1The concept of variation in the levels of sensitive troponin and high-sensitivitytroponin in different measurements has been studied, and establishing a correlationbetween the amplitude of variability and the probability of coronary artery diseasehas been consecutively attempted. In addition, amplitude can be relative (expressedas percentages) or absolute, with possible implications and distinctinterpretations.16A retrospective study published in 2014, including 1054 patients with chest pain,assessed the variability related to high-sensitivity troponin T. Approximately 40%of the patients showed alteration in at least one measurement. Even with a variationgreater than 20% as compared to the initial level, the specificity did not exceed70%.17 publisheda study comparing current sensitive troponin I versushigh-sensitivity troponin I in 381 patients with chest pain at the emergencydepartment. Those authors found sensitivity values for the two assays of 94% and97%, and negative predictive values of 98% and 99%, respectively, with nosignificant difference.17 Anothersimilar study of 1807 patients with non-ST-segment elevation acute coronary syndromehas shown no significant difference regarding prognosis when comparing thepositivity of current sensitive troponin I versus high-sensitivitytroponin I.18 Differently from thefindings of those studies and using the same assay, ours showed lower sensitivityand specificity of 23% when using the 99th percentile of the method. Thatshows the importance of assessing each center’s population, respecting theirspecific individualities.Assessing specifically the same current sensitive troponin assay used in this study,in 2013 Bonaca et al.2In alignment with that, the meta-analysis published in 2014 with 17 studies and 8644patients with chest pain compared the use of high-sensitivity troponin with that ofconventional troponin. There were differences regarding sensitivity  and specificity , respectively.Despite that increase in sensitivity with high-sensitivity troponin, the number ofpatients with the final diagnosis of myocardial infarction and the need foradditional tests for ischemia did not differ between the groups, showing noadditional clinical advantage with the use of high-sensitivity troponin.20 The studyconducted in 2015 compared seven assays of current sensitive troponins andhigh-sensitivity troponin in 2813 patients with chest pain, and with (16%) orwithout kidney dysfunction. Of the patients with nephropathy, in only 45-80% ofthose with positive troponin, the final diagnosis was myocardial infarction. Theoptimal cutoff point varied from 1.9 to 3.4 times that of the general population todetect acute coronary artery disease. Assessing only the same current sensitivetroponin assay used in this study, in 27% of those with positive troponin, the finaldiagnosis of myocardial infarction was ruled out. The area under the curve ofaccuracy of that assay decreased from 0.92 to 0.87 (p = 0.013), comparing thegeneral population with the patients with kidney dysfunction.19 That cutoff point elevation is inaccordance with our findings, showing a clear specificity reduction in the group ofpatients with nephropathy.Finally, some studies have validated the new troponin assays.Despite the large case series, this is a retrospective  single-center study, with a much higher number of patients withoutchronic renal failure than with it. In addition, we used only one troponinassay, and most patients were of the male sex.In the study population of patients with chest pain, sensitive troponin I showed agood correlation with significant coronary lesions when its level was greater than0.605 ng/dL. In patients with chronic renal failure, a significant decrease inspecificity was observed in the correlation of troponin levels and severe coronarylesions.


GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A Spondyloarthritis is an inflammatory disease with Th17 cells implicated in the pathogenesis. Here the authors show that patients with spondyloarthritis have increased numbers of GM-CSF-secreting blood and synovial lymphocytes, Th17 or not, that carry a unique transcriptional profile including enhanced GPR65 expression. The prevalence of spondyloarthritis is at least 0.5% in European and US populations3. Only one third of patients achieve disease remission with TNF inhibitor therapies, therefore a significant unmet clinical need remains7.Spondyloarthritis encompasses a group of inflammatory diseases with common pathologic and genetic features. These disorders include ankylosing spondyltis (AS), reactive arthritis (ReA), psoriatic arthritis (PsA) and enteropathic arthritis (EA)10. Within the CD4 T cell compartment, Th17 cells can be pleotropic and, in addition to the classical “type 17” cytokines IL-17A, IL-17F and IL-2212, can also produce IFN-γ13 and GM-CSF14. Murine models of inflammation suggest that co-expression of GM-CSF and IL-17A by CD4 T cells marks out a pathogenic subset of Th17 cells15, and neutralisation of GM-CSF in the Sagakuchi model of spondyloarthritis causes improvement in joint inflammation15. Noster et al.16 have provided evidence that GM-CSF and IL-17A are antagonistically regulated, but it is unclear if the CD4/GM-CSF axis is acting within the context of Th17 immunity or independently. Additional pathogenic markers of murine Th17 cells include GPR6517, a receptor implicated in the pathogenesis of spondyloarthritis by genome-wide association studies18 .Genetic, functional and therapeutic evidence implicates IL-17-producing cells in the pathogenesis of spondyloarthritis19, γδ T-cells20, innate lymphoid cells21 (ILC) and B cells22 have also been shown to be producers of IL-17A and/or GM-CSF. ILC are broadly divided into three groups; ILC1s express T-bet, ILC2 express GATA3 and ILC3 express RORγt and are thought to be involved in type 17 immunity23. ILCs are present in the gut24, lung25 and skin26, and have been described in the joint in psoriatic arthritis27. The contribution of ILCs to pathogenic immune responses is still being actively investigated.In addition to classical CD4 Th17s, CD161-expressing CD8 T cellsGPR65, and we identify a function for this receptor in CD4 lymphocyte GM-CSF production. Therapeutic targeting of GPR65 may potentially attenuate pathogenic T cells while preserving physiological type 17 immunity.Here we show that multiple lymphoid populations (including ILCs) express more GM-CSF in patients with spondyloarthritis. Increased lymphocyte GM-CSF production occurs both in the context of a polyfunctional type 17 response, but also independently of IL-17A. Primary human GM-CSF-producing CD4 cells, isolated using a multiple cytokine capture approach, show a specific transcriptional profile that includes upregulation of 28, 14 patients with rheumatoid arthritis (RA) matched for CRP, and 17 sex and age matched healthy donors (Supplementary Table + CD4 cells (Th17 cells) were increased in spondyloarthritis compared to both control groups  and RA inflammatory disease controls . IL-17A+GM-CSF+ cells were also increased in CD8, γδ and CD56+ lymphoid subsets in spondyloarthritis  from 38 patients with axial spondyloarthritis fulfilling the assessment of spondyloarthritis (ASAS) criteriaups Fig. . We thenups Fig. . We obsetis Fig. .Fig. 1GM+IL-17A− cells in both CD4 and CD8 cells in spondyloarthritis compared to the two control populations   Fig. . We usedls) Fig. . In all + events and performed an unbiased clustering analysis of all parameters using viSNE29  from the inflamed joints of 5 patients with peripheral spondyloarthritis  in human joints has recently been described in psoriatic arthritis, a form of spondyloarthritision Fig. . The preset Fig. . Figure set Fig. .Fig. 3Ty+CD4+ may constitute a distinct functional subset, we next asked if GM-CSF+ CD4 T cells have a distinct transcription signature compared to classical IFN-γ+ Th1 cells and IL-17A+ Th17 cells. In the absence of known surface markers that define these cells we developed a triple-cytokine capture assay  showed GM-CSF single positive cells to have a gene expression profile with both unique  might have be potentially pathogenic, integrating our RNA sequencing data with eQTL and GWAS data sets  in the percentage of IL-22+ cells but no difference in IFN-γ+, IL-17A+ and IL-17A+GM-CSF+ cells. However, culture in the presence of IL-7 did not increase expression of GPR65 . This overlaps with an expansion of “type 17” cytokine production previously reported for CD431, and have recently been shown to produce GM-CSF in the gut24. We show that ILC1s are also present in the joint and show a downregulation of IFN-γ relative to blood. Thus, our data suggest that GM-CSF may be an important mediator in both innate and adaptive immune responses in spondyloarthritis.We also show increased numbers of lineage-negative IL-7R-positive type 3 innate lymphoid cells (ILC3) in inflamed joints, and further show that ILC3 cells derived from the joint abundantly produce GM-CSF upon in vitro stimulation, albeit after in vitro culture. C-Kit-expressing ILC3 are thought to contribute to the cytokine environment through the production of IL-17A and IL-22 in response to tissue stress15 and in EAE models of neuro-inflamation16, with a T cell specific-GM-CSF knockout shown to be protected from disease32. GM-CSF-mediated immunity has been implicated in several human diseases. Elevated GM-CSF-producing T cells have been shown in the joints of patients with Juvenile Inflammatory Arthritis33. Moreover an increased percentage of GM-CSF+ CD4 T cells have been found in the cerebrospinal fluid of patients with multiple sclerosis (MS) compared to non-MS controls16, and CD4 and CD8 T cells expressing GM-CSF have been shown to be elevated in the peripheral blood of patients with MS34.Several lines of evidence have suggested that T cell-derived GM-CSF may be pathogenic in murine and human inflammatory diseases. Neutralization of GM-CSF has been shown to be effective in the Sagakuchi mouse model of spondyloarthritis+GM-CSF+ double-positive cells is greatly increased in the peripheral blood of patients with spondyloarthritis, with further increases in the ex vivo synovial fluid. Th17 cells co-expressing GM-CSF are thought to be particularly pathogenic in animal models of inflammatory disease36. Lee et al.35 showed pathogenic Th17 cells to be dependent on IL-23 and downstream TGF-β3 signalling, with Csf2 (GM-CSF) one of the key upregulated genes in murine pathogenic Th17 cells. Csf2 was also shown recently to be upregulated in pathogenic Th17 cells isolated from the central nervous system of diseased experimental autoimmune encephalitis (EAE) mice17.In this study, we observed GM-CSF production both independently and in combination with Th17 subsets. Importantly, we here show that the percentage of IL-17A+ CD4 T cells have a distinct phenotypic and transcriptional profile. Several reports have shown GM-CSF production to be more aligned with a STAT5 rather than a STAT3 programme16, with IL-7 implicated as a driver of STAT5-mediated GM-CSF-driven inflammation in the mouse EAE model37. Moreover, it has been shown that GM-CSF production by CD4 T cells is influenced by IL-1β39, with GM-CSF in turn acting via monocytes to boost IL-1β production40. We recently showed that RORγt inhibition of human Th17 cells in vitro led to suppression of IL-17A+ but not GM-CSF+ CD4 T cells in vitro, suggesting GM-CSF can act independently of RORγt41. In the absence of clear surface phenotypic markers to identify GM-CSF+ CD4 T cells and IL-17A+GM-CSF+ double-positive CD4 T cells, our RNA sequencing data represent an important insight into the transcriptional regulation of these cells. We show GM-CSF+ cells to have a transcriptional profile that is distinct from IFN-γ+ and IL-17A+ cells, supporting the concept that GM-CSF+ T cells may be a distinct T-cell phenotype. Gene clustering analysis identified distinct, as well as overlapping groups of differentially expressed genes for both GM-CSF+ cells and IL-17A+GM-CSF+ double-positive cells.Our flow cytometry, CyTOF and gene expression data suggest that GM-CSF+ CD4 and CD8 T cells in spondyloarthritis to be significantly higher than rheumatoid arthritis (RA), as well as healthy controls. Interestingly, the percentage of GM-CSF+ CD4 cells was especially high in patients receiving anti-TNF therapy, suggesting that this pathway is downstream of TNF. This increase in GM-CSF may be due to the fact that anti-TNF treated patients represent a subset with more aggressive disease requiring the need for biological therapies. Alternatively, the effects of TNF blockade may lead to a loss of negative feedback leading to an increase of GM-CSF. An increase in Th17 numbers after anti-TNF has been previously reported in RA42 and the two mechanisms may be similar.In this study, we observe the percentage of GM-CSF44. Even though we did not observe an increase in circulating GM-CSF+ lymphocytes in RA, the efficacy of the anti-GM-CSF antibody in this disease may be predominantly due to the local effects of GM-CSF in synovial tissue, where increased numbers of cells expressing the GM-CSF receptor have been described in both RA and psoriatic arthritis45. Additionally, in RA, GM-CSF has been shown to play a function in inflammatory dendritic cell differentiation46.Our data would support development of clinical trials of anti-GM-CSF in spondyloarthritis, particularly in view of the safety and efficacy already reported in RA+ Th17 cells are characterized by high levels of GPR65 expression. GPR65 is G-protein coupled receptor with an extracellular proton sensing domain47 associated with ankylosing spondyloarthritis by GWAS18. Its function in immune cells remains unclear. We show culture of primary human CD4 cells in an acidic environment significantly upregulates GM-CSF while silencing of GPR65 reduced GM-CSF. Notably, a low pH environment has been reported in the inflamed synovium48 . Of interest, Gpr65 expression was upregulated in pathogenic murine Th17 cells, with knockout protecting against EAE17. The upregulation of GPR65 in ex vivo sorted spondyloarthritis Th17 cells further validates a function for this receptor in driving pathogenic T cell responses in human disease. Since silencing of GPR65 in human CD4 cells leads to a significant decrease in GM-CSF, inhibition of GPR65 using small molecules might constitute a promising therapeutic approach, in combination with or as an alternative to current therapies, although the potential human in vivo side effect profile of such agents remains to be determined. Such an approach may potentially preserve the protective function of non GM-CSF secreting Th17 cells49.We here show that both human GM-CSF-producing CD4 T cells, and GM-CSFGPR65. Our data provide a strong scientific rationale for clinical trials targeting GM-CSF in spondyloarthritis and additionally identify further targets including GPR65 involved in this likely ‘pathogenic’ module.Taken together, our data suggest an important function in human spondyloarthritis for GM-CSF production by multiple lymphocyte populations, including CD4, CD8 and γδ T cells and ILCs, within the context of globally enhanced type 17 immunity. GM-CSF-producing CD4 cells have a distinct transcriptional profile, which includes expression of 28. Age and sex matched healthy donors and rheumatoid arthritis (RA) patients meeting American college of rheumatology/European league against rheumatism 2010 criteria50 matched for C-reactive protein were recruited. Patient and control characteristics are shown in Supplementary Table 51. A minimum sample size of 11 was needed for each group for power of 0.8 and type 1 error rate of 5%.Peripheral blood samples were recruited, following informed written consent, from patients attending the Oxford University Hospitals National Health Service Foundation Trust . All spondyloarthritis patients met assessment of spondyloarthritis international society (ASAS) criteria for axial spondyloarthritis28 or psoriatic arthritis as defined by classification criteria for psoriatic arthritis (CASPAR) criteria52 were also recruited under the same ethics as the peripheral blood cohort with informed written consent. Additional synovial tissue samples from spondyloarthritis28 and RA50 patients were studied . Patient characteristics are shown in Supplementary Table Synovial fluid from patients with peripheral spondyloarthritis as defined by ASAS criteria2. Staining antibodies and dilutions shown in Supplementary Table The cells were stimulated with phorbol myristate acetate  and ionomycin , in the presence of Golgiplug (Brefeldin A) and Golgistop (Monensin) (both from BD Biosciences diluted according to manufacturer’s instructions) and incubated for four hours at 37 °C in 5% COA volume of 8 μl of 103Rh intercalator (Fluidigm) was added to cells and incubated for a 15 min at 37 °C. Cells were resuspended in 50 μl staining buffer containing surface antibody master mix and incubated for 20 min at room temperature, washed, then re-suspended in 500 μl of fix buffer (Fluidigm-Maxpar) and incubated at room temperature for 20 min. Cells were permeabilised using 2 ml of perm-S-buffer (Fluidigm-Maxpar) and intracellular staining was performed for 30 min at room temperature. All antibodies used are listed in Supplementary Table Fresh lymphocyte cones were obtained from the NHS blood and transplant service and PBMCs obtained by density centrifugation. CD4 T cells were isolated by negative selection (Miltenyi). The next day cells were stimulated with phorbol myristate acetate  and ionomycin  for 2 h. Cells were stained on ice for 5 min with IL-17A, IFN-γ and GM-CSF capture antibodies  and incubated at 37 °C for 45 min. At 5 min intervals the mixture was resuspended. Cells were then placed on ice to stop cytokine secretion and stained with MACS detection antibodies  all at a concentration of 1:25 μl. In addition, cells were also stained with CD45RA-PerCpCy5.5, CD3 BV605, CD4 BV421 and ef780 viability dye followed by with anti-biotin APC secondary. Sorting was carried out using an automated cell sorter BD Aria. Cells were sorted at 4 °C into tubes containing sterile PBS.Freshly obtained surgical material was re-suspended in 25 ml of sterile RPRMI immediately. The sample was placed in a large petri dish and cut into 3–4 mm pieces by hand. 20–30 pieces of the sample were placed in a 6-well plate containing 5 ml of warm of RPMI supplemented with 10% human serum containing 100 IU/ml IL-2 (Peprotech) + IL-7 10 ng/ml (Peprotech). Volume of 2.5 ml of media was aspirated every 72 h and replaced with the same volume containing IL-2 100 IU/ml + IL-7 10 ng/ml. After 14 days non-adherent cells were washed off the plate and filtered through a 70 μm filter. Cells were counted, rested overnight and stimulated in the morning for intracellular cytokine staining.The AllPrep DNA/RNA/miRNA kit  was used for RNA extraction. FACS sorted cells were spun down and re-suspended in 350 μl of RLTplus buffer and transferred to 2 ml tubes. Samples were then stored at −80 °C for batched RNA extraction. Homogenisation of the sample was carried out using the QIAshredder (Qiagen). DNase I was used during the extraction protocol to minimise DNA contamination. RNA was eluted into 35 μl of RNase-free water. The RNA amount was quantified by nano-drop and the RNA samples stored at −80 °C for storage until ready for sequencing.RNA sequencing was carried out at the Wellcome Trust Centre for Human Genetics core facility. RNA underwent quality control testing using a bioanalyser followed by cDNA library preparation. Paired end sequencing was performed at 100 base pairs on each side of the DNA fragment on the HiSeq 4000 platform. Total of 20–80 million fragments were sequenced per sample.53, deduplicated using samtools v.1.254. Gene counts were retrieved using htseq-cout55 and the Ensembl gene annotation. DESeq256, an R package, was used for differential gene expression analysis.Prism version 6 was used for statistical analysis. Bioinformatic analysis of RNA sequencing samples was carried out using validated packages in R. Reads were mapped to human genome reference sequence GRCh37 with tophat2+GM-CSF+ and GM-CSF+ were defined if they, as compared to naive CD45RA+ cells, were differentially expressed (FDR <5%) in both IL-17A+GM-CSF+ cells and GM-CSF+ cells but not in IL-17A+ cells. Pathway analysis was conducted using the XGR software to identify enriched pathways . Network analysis was performed using the dnet package to identify a gene network that contains groups of interconnected genes. In brief, the dNetPipeline function in the package took as inputs a list of common genes with the significance level  and output a gene network (FDR <1%) from a human gene interaction data .Genes common to IL-17A57 to generate evidence scores prioritising key genes involved in spondyloarthritis. We utilised GWAS summary data  for susceptibility in spondyloarthritis59 and eQTL summary data61 to prioritise the 1643 genes that were differentially expressed in GM-CSF+ CD4 cells from our data set. We prioritised genes based upon the relative weight of evidence of GWAS association and the eQTL significance level linking a SNP to a gene.We applied an in-house prioritisation system using random forest plot modellingt-test was used for statistical analysis. When comparing two unpaired groups with data that was not normally distributed a Mann–Whitney test was used. For paired data, a paired t-test and Wilcoxon matched-pairs signed-rank tests were used for parametric and non-parametric data, respectively. When comparing more than two unpaired groups, a one-way analysis of variance (ANOVA) with Bonferroni’s correction was used for parametric data. Where the data were not normally distributed a Kruskal–Wallis test with Dunn’s multiple comparison analysis was used for statistical analysis. For matched data with more than two groups, a one-way ANOVA with Greenhouse-Geisser correction was performed in parametric data sets. In non-parametric data a Friedman test, a Dunn’s multiple comparison analysis was performed. Significance was defined as p ≤ 0.05.Prism version 6 was used for statistical analysis. When comparing two unpaired groups with normal distribution of data a two-tailed unpaired RNA sequencing data have been deposited in the National Centre for Biotechnology Information gene expression omnibus under the accession number GSE103930. The remainder of the data that support the findings of this study are available on request from the corresponding author. The patient data are not publicly available due to them containing information that could compromise research participant privacy.Supplementary InformationPeer Review File


The troponin complex, composed of Troponin-I, Troponin-T and Troponin-C, is an essential mediator of the contraction of striated muscle downstream of calcium signaling in almost all bilaterians. However, in echinoderms and hemichordates, collectively termed Ambulacraria, the components of the troponin complex have never been isolated, thus suggesting that these organisms lost the troponin system during evolution. Here, by analyzing genomic information from sea urchins, we identify the troponin-I gene and isolate its complete mRNA sequence. Using this information, we reveal that the larval muscles express this gene and its translated product and that the protein is definitely a functional molecule expressed in sea urchin larvae by showing that Troponin-I morphants are unable to swallow algae. We conclude that muscular contraction in all bilaterians universally depends on a regulatory system mediated by Troponin-I, which emerged in the common ancestor of bilaterians. The force in each contractile unit depends on the sliding actin-myosin assembly, which is highly conserved among eumetazoans1S. purpuratus (http://www.echinobase.org)9Hemicentrotus pulcherrimus , a candidate TnI gene was found in H. pulcherrimus (LC187281). The complete cDNA was isolated, and it encodes a clear troponin motif in the C-terminal region. These findings are supported by phylogenic analysis of the troponin motif  using MUSCLE alignmentBy analyzing the genomes and transcriptomes of 2+  might be an alternative to TnC, because the Ca2+-binding EF-hands of TnC and CaM are well conservedS. purpuratus, the TnT gene has been manually annotated (SPU_006532)8H. pulcherrimus is awaited, and biochemical analysis of HpTnI and HpTnT with and without CaM on muscular components would indicate how members of the sea urchin group use the troponin complex in regulating muscle contraction via Ca2+.The primary structure of HpTnI and the alignment with fly and zebrafish TnI showed characteristics specific to HpTnI. One such characteristic is the long N-terminal extension, and another is the absence of a Troponin-C (TnC) binding-switch region, which is essential for initiating actin-myosin interaction in the presence of CaS. purpuratus, a temporal microarray analysis suggested that the incompletely annotated TnI gene (SPU_013183) is mainly expressed after gastrulation during embryogenesisIn situ hybridization indicated that HpTnI-mRNA is expressed in the esophagus, the esophageal muscles and the pyloric and anal sphincters  with 50 μg/ml of kanamycin. To examine the food intake ability, we fed 5.0 μl of Sunculture  to 20–100 individuals of 72-h larvae in 3.0 ml FSW for 30 min. The number of larvae whose stomach and intestine were filled with algae was counted, and the ratio was calculated from three independent batches . For each sample, the number of swallowing behaviors was counted under the microscope for one minute. Criteria for evaluating swallowing were based on the contraction of the esophagus.Adults of the sea urchins, TnI gene of Mesocentrotus nudus was partially cloned (LC187280), and the protein equivalent to amino acids 307–422 in H. pulcherrimus TnI (Escherichia coli (strain BL21 Rosetta2 (DE3); Merck-Millipore, Darmstadt, Germany). The protein was purified with Toyopearl CM-650 M  and hydroxyapatite  columns under conditions of 6 M urea, and it was then immunized to rabbits. The serum was collected and the specific antibody was purified with a TnI-conjugated Toyopearl AF-formyl-650 M (Tosoh) column. All experimental procedures using rabbits were performed according to the guidelines of and approved by the animal care committee of Hokkaido University (ID: 14–0073)The These methods were described in detail previouslyHpTnI-MO1 (0.5 mM): 5′-CTGCTTCATAGTCGTCACCCATAGT-3′,HpTnI-MO2 (0.5 mM): 5′-TACCCTTCCAATTCAGGCCCTACAC-3′,Nodal-MO (0.3 mM): 5′-AGATCCGATGAACGATGCATGGTTA-3′,Lefty-MO (0.4 mM): 5′-AGCACCGAGTGATAATTCCATATTG-3′.μg/μl in 24% glycerol in needles.Nodal mRNA was synthesized from linearized plasmids using the mMessage mMachine kit (Thermo Fisher Scientific) and injected at 0.2 μl final concentration) against the whole coding region of HpTnI was used for in situ hybridization. The sequence information of full-length HpTnI was provided by transcriptome analysis of H. pulcherrimus, which will be published elsewhere. The coding region was confirmed and isolated by the polymerase chain reaction using a cDNA pool of embryonic stages with KOD-FX (TOYOBO) and the following primers complementary to the 5′- and 3′-untranslated regions:Dig-labeled RNA probe  at 4 °C. The samples were fixed with formaldehyde and washed with PBS (three 10-min washes) before treating rhodamine-phalloidine for 30 min to visualize the fibrous actin.μl, separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition according to the methods of Porzio and Pearsonet al.Four-day larvae were stored at −80 °C until use. Each sample was dissolved in sodium dodecylsulfate (SDS)-sample buffer, approximately 10 larvae/How to cite this article: Yaguchi, S. et al. Troponin-I is present as an essential component of muscles in echinoderm larvae. Sci. Rep.7, 43563; doi: 10.1038/srep43563 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We utilized a survival modification of the cecal ligation and puncture (CLP) model of sepsis in humanized mice. Animals were sacrificed 28 days after CLP (tCLP+28d). Untouched (CONTR) or sham-operated (SHAM) animals served as controls. Some animals received rescue from stem cells originally used for grafting 2 weeks after CLP. We found profound decrease of MO→iDC in the humanized mice 28 days after sepsis, demonstrated by depressed expression of CD1a, CD83, and CD209, diminished production of IL-12p70, and depressed ability to stimulate T cells in mice after CLP as compared to SHAM or CONTR. In vitro defect in MO→iDC was accompanied by in vivo decrease of BDCA-3+ endogenous circulating DC. Interestingly, post-CLP MO had persistent activation of M-CSF pathway, shown by exaggerated secretion of M-CSF, activation of PU.1, and demethylation of SPII. Neutralization of the M-CSF in vitro reversed the post-CLP MO→iDC aberration. Furthermore, transplantation of naïve, autologous stem cell-derived MO restored CLP-deteriorated ability of MO to become DC, measured as recovery of CD1a expression, enhanced production of IL-12p70, and ability of IL-4 and GM-CSF MO to stimulate allogeneic T cells. Our results suggest the role of epigenetic mediated M-CSF aberration in mediating post-sepsis immune system recovery.The duration of post-sepsis long-term immune suppression is poorly understood. Here, we focused on the role of monocytes (MO) as the pivotal cells for long-term regulation of post-sepsis milieu. Lost ability of MO to adapt is seen in several acute conditions, but it is unclear for how long MO aberrancy post-sepsis can persist. Interestingly, the positive feedback loop sustaining secretion of macrophage-colony stimulation factor (M-CSF) can persist even after resolution of sepsis and significantly alters performance of MO. Here, we investigated the activation of M-CSF, and it as critical regulator of PU.1 in mice surviving 28 days after sepsis. Our primary readout was the ability of MO to differentiate into dendritic cells   During sepsis, an early, innate response transforms into acquired immunity-driven processes followed by a resolution , 2. It win vitro. Its secretion can be sustained by a positive feedback loop even when initial stimulus has resolved. Prolonged stimulation with M-CSF has been linked to negative outcomes in trauma, burn, heart failure, and surgical recovery.Numerous mechanisms are involved in the homeostasis of the immune system , 2, 6. AHere, we hypothesize that sepsis results in prolonged aberration in the plasticity and function of the MO well beyond the acute phase of sepsis. Prior work led us to formulate a more focused hypothesis suggesting that the sustained production of M-CSF is potentially responsible by virtue of preventing an optimal emergence of regulatory DC, long after sepsis resolution –21. Henc+ cells  as previously described  and similar to other researchers  in small pilot run. Also, no difference in peripheral blood leukocytes was seen in post-CLP animals re-transplanted with naïve stem cells .A permit for study was approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Animals were care in accordance in the USDA Animal welfare Act and all efforts were made in order to minimize number of enrolled animals and their suffering. Sublethally irradiated (3 Gy/mouse) NSG mice were transplanted with human umbilical cord blood CD34escribed . Human iearchers , 23. No ad libidum. Day/night cycle was maintained throughout. At 28 days after CLP, the animals were sacrificed. Technical and clinical details of this model are published in another manuscript (under review).The CLP model was derived from pre-existing literature and modified for survival –26. Brie5 cell/animal) originally used to graft the animals 2 weeks after CLP or sham procedure.Some animals after CLP were reinjected intravenously with stem cells (2 × 10(bright) and 80% for CD14(+). Isolated MO were incubated in X-VIVO 10/15™ media with gentamycin and phenol red , supplemented with human IL-4  at 500 IU/ml and human GM-CSF  at 1,000 IU/ml at 37°C and 5% CO2. On day 3, 50% of the supernatant was collected and replenished with fresh media and cytokines. After 5 days, the supernatants and cells were collected using an ice-cold 10 mM EDTA buffer , followed by a washout with PBS without Ca2+ or Mg2+   . An addiNY, USA) .5 cells were incubated with a flow cytometry media  enriched with human TrueStainFcX™  for 15 min at 4°C in the dark. Cells were then incubated with the antibodies for 30 min at 4°C in the dark, with additional mixing at 15 min. Cells were washed twice in FACS media and re-suspended in 100 µl of 1% FlowFix . The following antibodies were employed: CD1a , CD14 , CD83 , CD115 , CD124 , and CD126 .A total of 10The frequency of naturally occurring DC was analyzed with a BDCA enumeration kit  , 28. Enu4 were collected. Data were expressed as a frequency of cells while mean fluorescent intensity (MFI) was a measure of receptor density. MFI is presented as relative to non-specific binding after exposing cells to non-specific antibodies.Cells were analyzed with an LSR™  or a FACSCalibur™ . Non-specific antibodies were used as a negative control. At least 10 × 104 harvested cells (IL-4 and GM-CSF differentiated MO) were added to 2 × 105 allogeneic T cells and incubated for 24 h. Twenty microliters of Alamar Blue  were subsequently added. After 18 h of incubation, the absorbance at 570 nm (with a reference filter at 630 nm) was measured to assess T cell proliferation using the Opsys MR  with Revelation software   .4 IL-4 and GM-CSF stimulated myeloid cells were added to 200 µl of X-VIVO 10/15™ media with gentamycin (10 µg/ml) and phenol red  supplemented with lipopolysaccharide (LPS)  (10 ng/ml)  . After 2The level of cytokines  in supernatants were measured using a magnetic multiplex kit  as per the manufacturer’s protocol and analyzed on the BioRad™ platform .SPI1 was conducted. Using the CpG Island Finder program (http://dbcat.cgm.ntu.edu.tw), we analyzed 3 kb fragment of the SPI1 gene encoding the PU.1 transcription factor. The analyzed fragment was located from 1.5 kb upstream to 1.5 kb downstream of the position corresponding to the first base of the major gene transcript . Within the 750-bp long fragment indicated as CpG island, a 375-bp fragment including potential transcription start sites of the major and two other gene transcripts, containing 17 CpG dinucleotides, was selected for further analysis. The methylation status of this fragment was analyzed using the OneStep qMethyl Kit  following the manufacturer’s protocol. The real-time PCR reaction was performed using LightCycler 480 II . The primers used were: forward 5′ATGTCACCCCAAGGGGACTA3′ and reverse 5′GGAAACCCTGACTTCCCACT3′. The PCR conditions were: initial denaturation for 10 min at 95°C, then 45 cycles of 30 s at 95°C, 30 s at 63°C, 30 s at 72°C, and then one melting curve cycle.In order to assess epigenetic regulation of PU.1, an analysis of methylation of in vitro using several indices of this process . At least six independent animals were used from each study group for statistical contrast based on power analysis for primary outcome. The parametric nature of the data was confirmed by Levene’s and Shapiro–Wilk tests. Parametric data were presented as average (X) ± SD while non-parametric variables were denoted as median (Me) with 25–75% interquartile range (IQ). t-Tests and Wilcoxon matched pair tests were conducted depending on the characteristics of the data. ANOVA was conducted for multiple group comparisons with a Bonferroni test for post hoc analysis. The data were flagged as significant if the two-tailed hypothesis test was <0.05, unless otherwise specified. Statistica v8.0  was used.The study was designed as a cohort study of the ability of MO to become professional iDC under the influence of IL-4 and GM-CSF p = 0.42)  . Finally, IL-4&GM-CSF-differentiated MO stimulated with LPS secreted significantly less IL-12p70   , 30. The = 0.03) , 12, 27.+ was significantly lower in CLP survivors as compared to sham-operated animals at 28 days after surgery (%BDCA-p = 0.03), while other types of endogenous DC remained the same or tended to slightly increase (%BDCA-p = ns) (%BDCA-p = ns). When recalculated for absolute number of BDCA-positive cells, we detected similar tendencies since there was no difference in absolute number of leukocytes per milliliter of blood (data not shown).We assessed the frequency of naturally occurring DC using specific markers , 28. Thein vitro (R (CD116) was elevated in the CLP group in terms of frequency of positive cells (%) (p = 0.0019) and receptor density (MFI) (p = 0.0013)  expression was not different between the three studied groups  (MFI CD12p = ns). However, the expression of M-CSFR (CD115) was significantly elevated in terms of average receptor density (p < 0.000051)  and secretion of M-CSF are elevated up to 28 days after CLP, we focused on the M-CSF pathway as a potential culprit for depressed MO→DC (p = 0.000183) . Finally, we added a neutralizing antibody for M-CSF (FN M-CSF) to IL-4&GM-CSF-differentiated MO obtained from CLP animals and SHAM. We observe at least partial restoration of MO→iDC differentiation demonstrated by acquisition of DC markers  if the MO obtained from CLP animals were differentiated in the presence of FN-M-CSF . In contrast, FN-M-CSF was ineffective in further boosting CD1a level on IL-4&GM-CSF differentiation MO obtained from SHAM (data not shown).Considering that both expression of M-CSFed MO→DC , 31, 32.p = 0.0003  , 34. Furp = 0.0058) on the surface of IL-4&GM-CSF-stimulated myeloid cells, while the expression of a monocyte/macrophage marker, CD14, was diminished  and ability of T cells to stimulate was assessed . We also assessed the frequency of endogenous DC and found that the frequency of BDCA-3+ cells was significantly higher than in animals after CLP .In the last set of experiments, we injected the mice 2 weeks after CLP with the naïve stem cells originally used to graft the animals. When the animals were sacrificed 28 days after initial insult, we observed a partial restoration of MO’s ability to become DC, as evidenced by the increased expression of CD1a p = 0.008 on the M-CSF after LPS = 406.1 ± 134.7 vs CLPM-CSF after LPS = 4,319.3 ± 2,042.4; CLP + Stem Cell TxM-CSF after LPS = 1,118.1 ± 93.4; p = 0.0001). The same trend was seen during in vitro IL-4&GM-GM-CSF differentiation in respect to M-CSF secretion . Methylation of PU.1 also partially normalized .Interestingly, animals after stem cells transplant had secretion of M-CSF in response to LPS and comparable CONTR animals  DC in vivo. An elevated level of M-CSF production in response to LPS as well as during differentiation of MO into DC was accompanied by upregulation in PU.1 and demethylation of SPI1. Finally, the diminished capacity of MO to become DC was reversed by neutralization of M-CSF in vitro or by transplanting autogenic, naïve stem cells in vivo. Our study provides an explanation as to how the immunological milieu may be altered by long-term sepsis leading to the emergence of comorbidities in some individuals  MO population but no increase in frequency of CD16+/CD33+ leukocytes were observed (data not shown). The preservation of CD14, a markers not present on the surface of any DC, suggest that MO obtained from humanized mice are MO arrested in a pre-DC process and are unable to acquire antigen-presenting features (in vitro MO→DC differentiation defect was accompanied by diminished expression of BDCA-3(+) endogenous circulating DC. In contrast to tissue-residing DC , circulating DC are critical for several immune functions. Interestingly, we observed a diminished frequency of BDCA-3 that are pivotal for T cell stimulation and resemble the mouse equivalent of MO-derived DC . These strongly suggested the paracrine effect of M-CSF and observed alteration. One has to keep in mind that M-CSF is critical for MO emergence from bone marrow and their survival in the circulation. Therefore, the techniques almost totally abrogating its production result in increased MO apoptosis or suboptimal function  cells suggests bone marrow defect. Elevated level of PU.1, a critical factor in regulation of not only M-CSF but also other inflammatory and regulatory pathways, suggests that M-CSF can be one of the plausible effects of CLP , 49–51. ironment , 35, 36.ironment . Naïve sde facto a xenotransplant model  approved this study.KL—study design, experiment, data collection, data analysis, and manuscript preparation. MZ—experiment, data collection, data analysis, and manuscript preparation. NL—data collection, analysis, and manuscript preparation. GW—study design, data analysis, and manuscript preparation. GD-D—study design, experiment, data collection, and manuscript preparation. JP—experiment, data analysis, and manuscript preparation. MP-K—experiment design, data analysis, and manuscript preparation.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.