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Autoimmune muscle diseases (myositis) comprise a group of complex phenotypes influenced by genetic and environmental factors. To identify genetic risk factors in patients of European ancestry, we conducted a genome-wide association study (GWAS) of the major myositis phenotypes in a total of 1710 cases, which included 705 adult dermatomyositis; 473 juvenile dermatomyositis; 532 polymyositis; and 202 adult dermatomyositis, juvenile dermatomyositis or polymyositis patients with anti-histidyl tRNA synthetase (anti-Jo-1) autoantibodies, and compared them with 4724 controls. Single-nucleotide polymorphisms showing strong associations (P < 5 × 10−8) in GWAS were identified in the major histocompatibility complex (MHC) region for all myositis phenotypes together, as well as for the four clinical and autoantibody phenotypes studied separately. Imputation and regression analyses found that alleles comprising the human leukocyte antigen (HLA) 8.1 ancestral haplotype (AH8.1) defined essentially all the genetic risk in the phenotypes studied. Although the HLA DRB1*03:01 allele showed slightly stronger associations with adult and juvenile dermatomyositis, and HLA B*08:01 with polymyositis and anti-Jo-1 autoantibody-positive myositis, multiple alleles of AH8.1 were required for the full risk effects. Our findings establish that alleles of the AH8.1haplotype comprise the primary genetic risk factors associated with the major myositis phenotypes in geographically diverse Caucasian populations.

Objective. To identify new genetic associations with juvenile and adult dermatomyositis (DM). Methods. We performed a genome-wide association study (GWAS) of adult and juvenile DM patients of European ancestry (n = 1178) and controls (n = 4724). To assess genetic overlap with other autoimmune disorders, we examined whether 141 single nucleotide polymorphisms (SNPs) outside the major histocompatibility complex (MHC) locus, and previously associated with autoimmune diseases, predispose to DM. Results. Compared to controls, patients with DM had a strong signal in the MHC region consisting of GWAS-level significance (P < 5x10(-8) ) at 80 genotyped SNPs. An analysis of 141 non-MHC SNPs previously associated with autoimmune diseases showed that three SNPs linked with three genes were associated with DM, with a false discovery rate (FDR) < 0.05. These genes were phospholipase C like 1 (PLCL1, rs6738825, FDR=0.00089), B lymphoid tyrosine kinase (BLK, rs2736340, FDR=0.00031), and chemokine (C-C motif) ligand 21 (CCL21, rs951005, FDR=0.0076). None of these genes was previously reported to be associated with DM. Conclusion. Our findings confirm the MHC as the major genetic region associated with DM and indicate that DM shares non-MHC genetic features with other autoimmune diseases, suggesting the presence of additional novel risk loci. This first identification of autoimmune disease genetic predispositions shared with DM may lead to enhanced understanding of pathogenesis and novel diagnostic and therapeutic approaches. � 2013 American College of Rheumatology.

Folding and transport of proteins, such as major histocompatibility complex (MHC) class I, through the endoplasmic reticulum (ER) is tightly regulated in all cells, including muscle tissue, where the specialized ER sarcoplasmic reticulum is also critical to muscle fiber function. Overexpression of MHC class I protein is a common feature of many muscle pathologies including idiopathic myositis and can induce ER stress. However, there has been no comparison of the consequences of MHC overexpression in muscle at different ages. We have adapted a transgenic model of myositis induced by overexpression of MHC class I protein in skeletal muscle to investigate the effects of this protein overload on young muscle fibers, as compared with adult tissue. We find a markedly more severe disease phenotype in young mice, with rapid onset of muscle weakness and pathology. Gene expression profiling to compare the two models indicates rapid onset of ER stress in young muscle tissue but also that gene expression of key muscle structural proteins is affected more rapidly in young mice than adults after this insult. This novel model has important implications for our understanding of muscle pathology in dermatomyositis of both adults and children.

Objective To identify interleukin-17 (IL-17)–producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis. Methods Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17–producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)–positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22. Results IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-γ. Conclusion This study is the first to define the frequency and characteristics of “Th17” cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

Juvenile dermatomyositis (JDM) is a childhood autoimmune disease characterised by proximal muscle weakness and cutaneous manifestations. Previous studies have identified an increase in circulating B cells in JDM patients, but their provenance and functional characteristics have not been examined. In this study we investigated whether an immature B cell subset (CD24hiCD38hi), known to be enriched for interleukin (IL)-10 producing regulatory B cells (Breg)1, accounted for the expansion of circulating B cells seen in JDM. The aryl hydrocarbon receptor (AhR) is a ligand based transcription factor, which induces IL-10 expression in T cells2. We investigated the effects of modulating the AhR pathway on IL-10 expression in B cells.

We have studied immune reconstitution in a patient with paediatric-onset polyarteritis nodosa treated with high-dose immunosuppressive agents followed by stem cell rescue. The patient developed several new autoimmune phenomena over the 18 months after immunosuppression and stem cell rescue. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) heteroduplex and isotype-specific RT-PCR analysis of immunoglobulin expression showed that the T- and B-cell repertoires were highly restricted in the first few months after treatment. The dominant T-cell clones seen after reconstitution were persistently expanded, were different from those which could be demonstrated before autologous stem cell transplantation, and were in the CD8(+) population. Our data also show that 12 months after treatment these expanded T-cell clones were within the CD45RA(+) population, suggesting that reversion from the CD45RO(+) to the CD45RA(+) phenotype had occurred in vivo.

While the T-cell receptor (TCR) repertoire of the newborn is highly diverse, a gradual alteration in diversity of the expressed TCR repertoire, in particular the oligoclonality of CD8+ T cells, occurs with increasing age. The timing of the initiation of this process is unknown. These changes are associated with an accumulation of T-cell expansions, thought to be in response to chronic antigen stimulation, frequently by persistent viruses such as Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Using reverse transcription-polymerase chain reaction heteroduplex analysis we have characterized the TCR expression of CD4 and CD8 cells from healthy young children and adults in order to delineate the age range at which these oligoclonal populations appear. We demonstrate that considerable oligoclonality can occur, even in healthy young children, and also that these expanded clonotypes persist. These are shown by heteroduplex to be exclusively within the CD28- subpopulation. The presence of such oligoclonal expansions correlates closely with the percentage of CD8+ cells that have the CD28- phenotype. However, we also show that control of chronic infection with EBV or CMV may coexist with a highly diverse, polyclonal TCR repertoire well into adulthood. These studies suggest that many factors affect the overall regulation of clone size in response to chronic antigens during the development of the immune system.

Juvenile dermatomyositis (JDM) is an autoimmune disease characterized by proximal muscle weakness and cutaneous manifestations, typically heliotrope rash and Gottron's papules. Previous studies have identified an increase in circulating B cells in JDM patients, but their provenance and functional characteristics have not been examined. In this study we investigated whether a recently identified immature B cell subset (CD24hiCD38hi) with known regulatory function (Breg) was enriched in JDM and correlated with disease outcome measures.

The aim of this study was to define normal ranges of histological features in pediatric muscle in comparison with muscle demonstrating inflammatory changes. Sixteen pediatric muscle biopsy samples, considered normal by standard histology, were analyzed for the presence of inflammatory cells, and the expression of neonatal myosin and major histocompatibility complex (MHC) Class 1. Normal findings were defined for each feature. These data will facilitate quantitative analysis of inflammatory changes in pediatric muscle biopsy.

ObjectivesTo study muscle biopsy tissue from patients with juvenile dermatomyositis (JDM) in order to test the reliability of a score tool designed to quantify the severity of histological abnormalities when applied to biceps humeri in addition to quadriceps femoris. Additionally, to evaluate whether elements of the tool correlate with clinical measures of disease severity.Methods55 patients with JDM with muscle biopsy tissue and clinical data available were included. Biopsy samples (33 quadriceps, 22 biceps) were prepared and stained using standardised protocols. A Latin square design was used by the International Juvenile Dermatomyositis Biopsy Consensus Group to score cases using our previously published score tool. Reliability was assessed by intraclass correlation coefficient (ICC) and scorer agreement (α) by assessing variation in scorers’ ratings. Scores from the most reliable tool items correlated with clinical measures of disease activity at the time of biopsy.ResultsInter- and intraobserver agreement was good or high for many tool items, including overall assessment of severity using a Visual Analogue Scale. The tool functioned equally well on biceps and quadriceps samples. A modified tool using the most reliable score items showed good correlation with measures of disease activity.ConclusionsThe JDM biopsy score tool has high inter- and intraobserver agreement and can be used on both biceps and quadriceps muscle tissue. Importantly, the modified tool correlates well with clinical measures of disease activity. We propose that standardised assessment of muscle biopsy tissue should be considered in diagnostic investigation and clinical trials in JDM.

Pediatric normal brachial biceps (14 specimens) and quadriceps muscles (14 specimens) were studied by immunohistochemistry to quantify fiber-type, diameter and distribution, capillary density, presence of inflammatory cells (CD3, CD20, CD68) and expression of neonatal myosin and MHC class 1 proteins. Brachial biceps showed more fast-twitch fibers and lower capillary/fiber ratio than quadriceps. The mean diameter of both fiber types was smaller in biceps than quadriceps. Fast-fibers were smaller than slow-fibers, and capillary/fiber ratio was

International audience; IntroductionThe aetiopathogenesis of juvenile dermatomyositis (JDM) remains poorly understood. In particular the contribution of monocytes or macrophages, which are frequently observed to be an infiltrate within muscle tissue very early in the disease process, is unknown. We hypothesised that these cells secrete the pro-inflammatory myeloid related protein (MRP) 8/14 which may then contribute to muscle pathology in JDM.MethodsIn this study of 56 JDM patients, serum MRP8/14 levels were compared with clinical measures of disease activity. Muscle biopsies taken early in disease were assessed by immunohistochemistry to determine the frequency and identity of MRP-expressing cells. The effects of MRP stimulation and endoplasmic reticulum (ER) stress on muscle were tested in vitro. Serum or supernatant levels of cytokines were analyzed by multiplex immunoassay.ResultsSerum MRP8/14 correlated with physician's global assessment of disease activity in JDM (R = 0.65, p = 0.0003) and muscle strength/endurance, childhood myositis assessment score (CMAS, R = −0.55, p = 0.004). MRP8/14 was widely expressed by CD68+ macrophages in JDM muscle tissue. When cultured with human myoblasts, MRP8 led to the secretion of MCP-1 and IL-6, which was enhanced by ER stress. Both inflammatory mediators were detected in significantly higher levels in the serum of JDM patients compared to healthy controls.ConclusionsThis study is the first to identify serum MRP8/14 as a potential biomarker for disease activity in JDM. We propose that tissue infiltrating macrophages secreting MRP8/14 may contribute to myositis, by driving the local production of cytokines directly from muscle.

Objective. Recent advances in molecular techniques have revealed that there is bi-directional transfer of cells between mother and child during pregnancy, and the presence of a mother's cells in her child has been termed maternal microchimerism (MMc). There is the potential for maternal cells to provoke inappropriate immune responses in the child, which could be a factor in autoimmunity including JDM. The aim of this study was to determine whether maternal (female) cells could be detected in frozen muscle sections from seven males (age range 3–13 years) with JDM participating in the Juvenile Dermatomyositis National (UK and Ireland) Cohort Biomarker Study and Repository for Idiopathic Inflammatory Myopathies and sections of muscle controls (age range 2–12 years). Methods. At least 1000 cells from each section underwent FISH and confocal imaging through each nucleus. Concomitant IF for CD45 was used to determine whether MMc in muscle were lymphocytes. A non-parametric Mann–Whitney U-test was used to detect statistical differences. Results. The frequency of MMc was higher in JDM muscle (0.42–1.14%) than in controls (0.08–0.42%) P = 0.01. No CD45+ MMc were observed. Conclusion. These data confirm an increased frequency of MMc in JDM. More detailed characterization of MMc is required, particularly using phenotypic markers, to explain the role of these cells in JDM.

Objective: Treatment-resistant muscle wasting is an increasingly recognized problem in idiopathic inflammatory myopathy (IIM). TNF-α is thought to induce muscle catabolism via activation of nuclear factor-kappa B (NF-κB). Several genes share homology with the NF-κB family of proteins. This study investigated the role of NF-κB-related genes in disease susceptibility in UK Caucasian IIM. Methods: Data from 362 IIM cases [274 adults, 49 (±14.0) years, 72% female; 88 juveniles, 6 (±3.6) years, 73% female) were compared with 307 randomly selected Caucasian controls. DNA was genotyped for 63 single nucleotide polymorphisms (SNPs) from NF-κB-related genes. Data were stratified by IIM subgroup/serotype. Results: A significant allele association was observed in the overall IIM group vs controls for the IKBL-62T allele (rs2071592, odds ratio 1.5, 95% CI 1.21, 1.89, corrected P = 0.0086), which strengthened after stratification by anti-Jo-1 or -PM-Scl antibodies. Genotype analysis revealed an increase for the AT genotype in cases under a dominant model. No other SNP was associated in the overall IIM group. Strong pairwise linkage disequilibrium was noted between IKBL-62T, TNF-308A and HLA-B*08 (D' = 1). Using multivariate regression, the IKBL-62T IIM association was lost after adjustment for TNF-308A or HLA-B*08. Conclusion: An association was noted between IKBL-62T and IIM, with increased risk noted in anti-Jo-1- and -PM-Scl antibody-positive patients. However, the IKBL-62T association is dependent on TNF-308A and HLA-B*08, due to strong shared linkage disequilibrium between these alleles. After adjustment of the 8.1 HLA haplotype, NF-κB genes therefore do not independently confer susceptibility in IIM. © The Author(s) 2011. Published by Oxford University Press on behalf of The British Society for Rheumatology.

Background Methotrexate (MTX) is the first line of therapy for patients with juvenile idiopathic arthritis (JIA) and juvenile dermatomyositis (JDM). The mechanisms of action of MTX in childhood autoimmunity are not fully elucidated. The primary target sites of inflammation of JIA and JDM i.e. joint and muscle, respectively do not represent sites from which samples can be easily obtained to monitor inflammatory markers. In contrast peripheral blood is accessible and can be obtained at time of routine clinical screen. We hypothesized that changes in peripheral blood of patients would be informative in monitoring disease and understanding inflammatory pathways that are modulated by MTX.

Results Overall, Treg number and phenotype of JDM patients was similar to age-matched controls. While JDM patients had increased numbers of Treg during active disease compared to remission, this increase was only found in JDM patients treated with high doses of corticosteroids. Treg of remitting JDM patients, but not of all active JDM patients were able to suppress effector T cell activation in vitro. Furthermore, in inflamed muscle tissue we detected high numbers of CD4 T cells, of which a large proportion expressed FOXP3.

The aim of this study was to define normal ranges of histological features in pediatric muscle in comparison with muscle demonstrating inflammatory changes. Sixteen pediatric muscle biopsy samples, considered normal by standard histology, were analyzed for the presence of inflammatory cells, and the expression of neonatal myosin and major histocompatibility complex (MHC) Class 1. Normal findings were defined for each feature. These data will facilitate quantitative analysis of inflammatory changes in pediatric muscle biopsy.

Objective To devise and test a system with which to evaluate abnormalities on muscle biopsy samples obtained from children diagnosed with juvenile dermatomyositis (DM). Methods We established an International Consensus Group on Juvenile DM Biopsy and carried out 2 phases of consensus process and scoring workshops. Biopsy sections (n = 33) were stained by standard methods. The scoring tool was based on 4 domains of change: inflammatory, vascular, muscle fiber, and connective tissue. Using a Latin square design, biopsy samples were scored by 11 experts for items in each domain, and for a global abnormality measure using a 10-cm visual analog score (VAS 0–10). The tool's reliability was assessed using an intraclass correlation coefficient (ICC) and scorer agreement (α) by determining variation in scorers' ratings. Results There was good agreement in many items of the tool, and several items refined between the meetings improved in reliability and/or agreement. The inflammatory and muscle fiber domains had the highest reliability and agreement. The overall VAS score for abnormality had high agreement and reliability, reaching an ICC of 0.863 at the second consensus meeting. Conclusion We propose a provisional scoring system to measure abnormalities on muscle biopsy samples obtained from children with juvenile DM. This system needs to be validated, and then could be used in prospective studies to test which features of muscle pathology are prognostic of disease course or outcome. We suggest that the process we used could be a template for developing similar systems in other forms of myositis.

This study focuses upon three chemokines, namely CCL5, CXCL10 and CCL3, which are potential novel therapeutic targets in arthritis. The aim of the study was to analyse the expression and production of these three chemokines within the joints of children with juvenile idiopathic arthritis (JIA) of the oligoarticular and polyarticular subtypes. All three of these chemokines are highly expressed at the level of mRNA, with the most significant increase in mRNA levels being demonstrated for CCL5 when compared with matched peripheral blood samples and controls. We show that high levels of all three chemokines are present in synovial fluid of children with JIA. We investigate the major source of CCL5 from inflammatory synovial cells, which we show to be CD8+ T cells. This CD8+ synovial T cell population has an unexpected phenotype that has not been described previously, being CCR7- yet predominantly CD28+ and CD45RA-. These cells contain high levels of stored intracellular CCL5, and rapid release of CCL5 takes place on T cell stimulation, without requiring new protein synthesis. In addition, we demonstrate that CCL5 is present in synovial biopsies from these patients, in particular on the endothelium of small and medium sized vessels. We believe this to be the first in depth analysis of these mediators of inflammation in JIA.

To assess muscle expression of MHC Class I complexes (heavy chain and beta2-microglobulin) and to analyze the composition of infiltrating mononuclear cells, specifically cells that bear receptors for class I MHC molecules, in the muscles of children with early juvenile dermatomyositis (JDM).Light microscopic and immunohistochemical analysis of muscle biopsies from 10 patients with JDM and 3 controls. The mean duration from initial weakness was 2.8 months. At the time of biopsy, 9 patients had not received steroid treatment or immunomodulatory drugs.MHC Class I over-expression was evident on muscle fibers in all 10 JDM samples, even in a biopsy reported as normal by conventional histology. MHC class I heavy chain and beta2-microglobulin were over-expressed in an identical distribution. Variable infiltration of T cells and macrophages was seen in the JDM biopsies, with minimal lymphocytic and monocytic infiltration in 4 cases, and none in one. Only very occasional natural killer lymphocytes were identified. Neuronal cell adhesion molecule (NCAM, CD56) staining of regenerating muscle fibers was seen in all samples and these cells were confirmed as being of muscle origin by co-staining for dystrophin.MHC Class I over-expression is an early event in JDM, and may occur in the absence of lymphocytic infiltration and muscle damage. Immunostaining for MHC Class I could be used routinely in the assessment of muscle histology in juvenile dermatomyositis.

Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4+CD25 +FOXP3+ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne's Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne's muscular dystrophy. Both in JDM and Duchenne's muscular dystrophy the proportion of FOXP3+ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.

Clinically distinct forms of childhood arthritis are associated with different risk alleles of polymorphic loci within the MHC, which code for the antigen-presenting class I or class II molecules. We have compared the TCR diversity of synovial T cells from children with enthesitis-related (HLA-B27(+)) arthritis and oligoarticular arthritis (with class II MHC risk allele associations) in parallel with peripheral blood T cells from each child, using a high-resolution heteroduplex TCR analysis. We demonstrate that multiple clonal T cell expansions are present and persistent within the joint in both groups, but that there is disease-specific divergence in the dominant T cell subset containing these expansions. Thus, the largest clonotypes within the inflamed joints of children with class II-associated arthritis are within the CD4(+) synovial T cell population, while the dominant clones from children with enthesitis-related arthritis (associated with a class I allele) are within the CD8(+) synovial T cell population. These data provide powerful data to support the concept that recognition of MHC-peptide complexes by T cells plays a role in the pathogenesis of juvenile arthritis.

Objective To study the expression of chemokine receptors CCR5 and CXCR3 and the Th1/Th2 cytokine balance in children with oligoarticular or polyarticular juvenile idiopathic arthritis (JIA). Methods Using 3-color immunofluorescence, we studied the expression of CCR5 and CXCR3 on, and T cell cytokine production by, paired samples of synovial fluid (SF) and peripheral blood (PB) T cells from 20 patients with oligoarticular- or polyarticular-onset JIA. Chemokine and cytokine phenotypes were also compared within the CD45RO+,CD3+ subsets. CCR5 genotypes were confirmed by polymerase chain reaction typing and sequencing. Results In the majority of samples, the number of T cells that were CCR5+ and CXCR3+ was higher in SF than in PB, and this difference was significant. One child was homozygous for the null Δ32 CCR5 allele; 4 others had lower expression of CXCR3 in SF than in blood. All samples showed strongly Th1-type cytokine production by synovial T cells compared with that by PB T cells. Both features were also markedly polarized within the synovial CD45RO+ subset compared with PB CD45RO+ T cells. Conclusion The high expression of CCR5 and CXCR3 and high interferon-γ:interleukin-4 ratios suggest a type 1 phenotype of SF T cells in JIA. The difference between CD45RO+ T cells from SF and from PB suggests that specific activation events have occurred in synovial T cells. We suggest that the highly activated, Th1-type phenotype of T cells within the chronically inflamed joints of children with JIA may reflect specific recruitment events that contribute to the polarization of these cells.

This study focuses upon three chemokines, namely CCL5, CXCL10 and CCL3, which are potential novel therapeutic targets in arthritis. The aim of the study was to analyse the expression and production of these three chemokines within the joints of children with juvenile idiopathic arthritis (JIA) of the oligoarticular and polyarticular subtypes. All three of these chemokines are highly expressed at the level of mRNA, with the most significant increase in mRNA levels being demonstrated for CCL5 when compared with matched peripheral blood samples and controls. We show that high levels of all three chemokines are present in synovial fluid of children with JIA. We investigate the major source of CCL5 from inflammatory synovial cells, which we show to be CD8+ T cells. This CD8+ synovial T cell population has an unexpected phenotype that has not been described previously, being CCR7- yet predominantly CD28+ and CD45RA-. These cells contain high levels of stored intracellular CCL5, and rapid release of CCL5 takes place on T cell stimulation, without requiring new protein synthesis. In addition, we demonstrate that CCL5 is present in synovial biopsies from these patients, in particular on the endothelium of small and medium sized vessels. We believe this to be the first in depth analysis of these mediators of inflammation in JIA.

Predicting disease course in Juvenile Idiopathic Arthritis (JIA) is difficult. During the first 6 months of oligoarticular JIA (O-JIA) disease presents with 4 or fewer affected joints, however after 6 or more months severity might increase with more than 4 joints involved (extended O-JIA), or persist with 4 or fewer joints affected (persistent O-JIA). However patients do not show any clinical differences at onset making a decision on treatment strategy difficult before extension occurs. It has previously been shown that the CD4/CD8 T cell ratio in extended-to-be patients (samples before extension has occurred) is lower compared to those who persist (Hunter et al 2010), and thus might be a useful biomarker in predicting the course of disease. However measuring this cell ratio currently requires specialized expertise. To translate the measurement of the CD4/8 synovial T cell ratio as a biomarker a method is needed, which could be used in a wide range of hospital facilities, hence a real-time quantitative PCR (qPCR) method was tested.

This study investigates the role of CD4+CD25+ regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4+CD25bright T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4+CD25bright T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4+CD25int T cells and therefore of CD4+CD25total in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4+CD25bright T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4+CD25int T cell population. The CD4+CD25bright cells of both patient groups and the CD4+CD25int cells of pers-OA JIA patients were able to suppress responses of CD25neg cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4+CD25bright T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4+CD25+ Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4+CD25bright T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4+CD25bright T cells. These data suggest that CD4+CD25bright Treg cells play a role in determining the patient’s fate toward either a favorable or unfavorable clinical course of disease.