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Plasma membrane rafts are sphingolipid- and cholesterol-rich patches that function as membrane trafficking and surface signalling regions. Ganglioside GM1 is an integral component of these microdomains, and Escherichia coli enterotoxin B subunit (EtxB) is a pentamer that binds with high affinity to GM1 resulting in GM1 cross-linking. We previously demonstrated that antigen coupled directly to EtxB resulted in enhanced presentation relative to antigen taken up by fluid-phase endocytosis. Here we demonstrate a new role for GM1 in antigen presentation by examining the effects of cross-linking GM1 on the kinetics of presentation and processing of antigen by the B-cell receptor (BCR), fluid-phase endocytosis and GM1-targeted antigen. EtxB bound to B cells does not augment the subsequent kinetics or magnitude of presentation of either BCR-internalized antigen or soluble antigen. Moreover, presentation of GM1-bound antigen is significantly slower than antigen presentation following BCR-mediated uptake. In contrast to the rapid internalization of BCR-bound antigen (which has a half life of 60 min), the majority of EtxB-bound antigen forms a plasma membrane depot detectable for many hours after initial incubation (and with a half life of 12 hr). We conclude that cross-linking of GM1 by EtxB minimally affects the processing and presentation of antigens internalized via other pathways. Nevertheless, binding of antigens to GM1 results in delayed presentation that has important implications for in vivo immunization using GM1-targeted adjuvants.

Successful antigen presentation by antigen-presenting cells is governed by a number of factors including the efficiency of antigen capture by cell-surface receptors, targeting to compartments of antigen processing, surface expression of MHC II-peptide complexes and presence of co-stimulatory signals. Ganglioside GM1 is an important component of membrane glycosphingolipids, and has been implicated in cell differentiation, apoptosis and signal transduction pathways. Using the B subunit of Escherichia coli enterotoxin (EtxB), a potent immunogen that binds GM1 with high affinity, and a non-binding mutant of EtxB, EtxB(G33D), we demonstrate that GM1 is intimately involved in several aspects of antigen presentation. Thus, GM1-mediated presentation of EtxB by B cells and CD11c(+) dendritic cells (DC) significantly enhanced the proliferation and cytokine expression of EtxB-specific CD4(+) T cells. Investigation regarding potential mechanisms revealed that EtxB binding directly augments the expression of MHC class II on B cells, and fractionation of B cells demonstrated that EtxB binding to GM1 results in rapid internalization and targeting to class II-rich compartments. GM1-mediated uptake of antigens and access to class II compartments in B cells can be exploited to significantly enhance the presentation of ovalbumin-conjugated to EtxB. These results demonstrate that GM1 can play an important role in antigen presentation via the MHC II pathway.

Background B-cell activating factor of the TNF family (BAFF)is important for B cell maturationand plays a role in (auto)antibodies production. Elevated serum levels in relation to autoantibodies were documented in patients with IIMs. Promoter region of BAFF gene contains several known sites with single nucleotide polymorphism (SNPs). An association between rs9514828 (-871 C/T) SNP and susceptibility to idiopathic thrombocytopenic purpura was shown and possible relations to systemic lupus erythematosus, rheumatoid arthritis or Sjogren’s syndrome were suggested. Objectives To describe four BAFF SNPs located in the BAFF gene promoter in patients with IIMs and analyze relation of particular SNPs to serum BAFF (s-BAFF) levels in autoantibody positive patients. Methods Patients with polymyositis (n=146), dermatomyositis (DM, n=150), juvenile DM (n=11), inclusion body myositis (n=4) and 103 healthy individuals were included. Four SNPs located upstream in the BAFF gene (rs9514827 (-2841 T/C); rs3759467 (-2704 T/C); rs1041569 (-2701 T/A); rs9514828 (-871 C/T)) were analyzed by direct DNA sequencing. Levels of s-BAFF were measured using ELISA. Autoantibodies were detected with immunoprecipitation and Line blot assay. The chi square test for analysis of alleles and genotypes associations and SNPStats software for haplotype frequency studies wereused. Results Significantly higher frequency of -2701T allele was present in patients (18%) compared to healthy controls (12%) (p=0.029; OR 1.68 (CI 95%=1.05-2.7)). Additionally, increased -2841T allele (p=0.09), -2841TT, CT genotype (p=0.07) and -2701TT, AT genotype (p=0.08) frequencies were observed in patients. SNPs were in strong linkage disequilibrium and formed four common haplotypes (TTAC, CTAT, TCAC, TTTT), with significantly different frequency (> 9%) distributions between patients and controls (global P=0.038). Higher frequency of TTTT haplotype was present in patients (16%) compared to controls (9%; OR 1.99 (95% CI 1.15-3.47; P=0.015)) relative to the most frequent haplotype TTAC. Significantly higher s-BAFF levels were detected in patients compared to healthy controls (P Conclusions -2701T allele and TTTT haplotype in the four SNPs located upstream of the BAFF gene were associated with the presence of myositis. Patients with anti-Jo-1 antibodies have higher BAFF levels if they are carriers of -2704T allele. These findings point to differential genetic regulation of BAFF production in patients with IIMs, but need to be validated in the second independent cohort of patients. Acknowledgements Grant support of IGA -Ministry of Health of the Czech Republic NT/12438-4. Disclosure of Interest None Declared

Background Increased serum levels of B cell activating factor (BAFF) have been detected in patients with idiopathic inflammatory myopathies (IIM) [1], which in anti-Jo-1 positive patients correlate with serum autoantibody concentration. The correlation of BAFF expression with disease activity in IIM patients has recently been described. Objectives To investigate serum BAFF levels in anti-PM-Scl positive patients with co-existent myositis and to assess the association with clinical disease activity in a multi-center myositis cohort. Methods Serum BAFF levels of 37 anti-PM-Scl positive IIM patients from two European countries and in 57 healthy controls were measured using ELISA. Nineteen longitudinal serum samples with corresponding clinical data were also obtained. Clinical data were derived from the Euromyositis database (www.euromyositis.eu). Results Serum levels of BAFF in anti-PM-Scl positive PM/DM patients were significantly higher compared to healthy controls (P=0.008) , but there was no statistically significant difference between PM or DM patients (P>0.05) . BAFF levels correlated with both muscular (r=0.509, P=0.002) and extramuscular disease activity (r=0.487, P=0.003). There was a negative correlation with manual muscle test (MMT8) results - increased muscle weakness thus correlated with increased serum BAFF levels (r=-0.423, P=0.025) . The composite disease activity (DA) scores MITAX and MYOACT correlated positively with BAFF levels; MITAX: r=0.374, P=0.027 ; MYOACT: r=0.430, P=0.010. There was a correlation with disease activity (DA) of various systems: Constitutional DA (r=0.382, P=0.023) , Cutaneous DA (r=0.471, P=0.004) , Gastrointestinal DA (r=0.545, P=0.001) and Pulmonary DA (r=0.524, P=0.001) . Furthermore, longitudinal change (∆) in BAFF levels correlated with ∆Physician Global DA (r = 0.6834, P = 0.0035) , ∆Muscular DA (r=0.786, P , and ∆MITAX Score (r=0.672, P=0.002) . Change in BAFF correlated significantly also with ∆MYOACT (r=0.698, P=0.001) and with its components: ∆Cutaneous DA (r=0.491, P=0.033) , ∆Skeletal DA (r=0.588, P=0.008) , ∆Gastrointestinal DA (r=0.548, P=0.015) , ∆Pulmonary DA (r=0.491, P=0.039) and ∆Cardiovascular DA (r=0.471, P=0.042) . Conclusions Serum levels of BAFF are increased in anti-PM-Scl positive patients with IIM, where BAFF levels correlate with clinical disease activity assessments. Furthermore, longitudinal changes in BAFF levels over time also correlate with changes in disease activity measures. These findings indicate that BAFF is likely implicated in the pathogenesis of PM/DM. BAFF is thus a potential biomarker in IIM. References O Krystůfkova, T Vallerskog, S Barbasso Helmers, H Mann, I Půtova, J Bělacek, V Malmstrom, C Trollmo, J Vencovský, I E Lundberg, Increased serum levels of B cell activating factor (BAFF) in subsets of patients with idiopathic inflammatory myopathies, Ann Rheum Dis 2009;68:836-843 doi:10.1136/ard.2008.091405 Acknowledgements IGA -Ministry of Health of the Czech Republic NT/12438-4. Disclosure of Interest None Declared

Background Hydroxy-methyl-glutaryl Coenzyme-A reductase (HMGCR) inhibitors or statins are a well-acknowledged cause of a spectrum of myopathic consequences including an asymptomatic increase of creatine kinase (CK), myalgias, myositis and rhabdomyolysis. A unique autoantibody with specificity against 200-kd and 100-kd proteins has been recently associated with statin induced myositis (1) based on identification of cases with features of necrotizing myopathy. The 100kd autoantigen has been identified as HMGCR (2). We sought to further characterize cases of clinically suspected statin-induced myositis using clinical, serological and pathological data. Objectives The aim was to characterize statin induced myositis patients in a clinical setting and test the utility of the newly identified anti-200/100 autoantibody. Methods Patients with a statin induced myositis were identified from a large tertiary myositis referral centre in Salford, UK. Case records of all patients were reviewed with selected patients’ blood samples tested for anti-200/100 autoantibody. Clinical features, concomitant medications, co-morbidities, immunology, electromyography (EMG), magnetic resonance imaging (MRI) and muscle biopsies for each patient were reviewed. Results 10 patients were identified with a clinical diagnosis of statin induced myositis. Mean age of diagnosis was 59 years (70% male). Median CK level detected at onset was 1,750 IU/litre (range 28-11,984). 9/10 patients were on a lipophilic statin (simvastatin/atorvastatin) with 1 patient on a hydrophilic statin (rosuvastatin); mean daily dose of 39mg/day prior to symptom onset. 5/10 patients were on a concomitant medication known to interact with a statin (amiodarone, proton pump inhibitors, high dose vitamin D). 3/10 patients were anti-nuclear-antibody positive (2 had low titres, 1/100; 1 positive 1/1000), whilst 2 patients were anti-Ro antibody positive. 6 patients demonstrated some evidence of myositis on MRI, of which 3 patients exhibited myopathic changes on EMG. 8 patients had evidence of myopathic muscle on biopsy, 2 patients with type II fibre atrophy. Of the 7 patients tested for anti-HMGCR by Western blotting using a recombinant source, 6 tested positive. 2 patients who tested strongly 200/100 positive required immunosuppression and had biopsy features of necrosis, phagocytosis and regeneration. 2 further 200/100 positive patients (1 strong and 1 weak positive) had occasional necrotic fibres with little regenerative activity on biopsy and did not require further treatment. The remaining 2 200/100 positive patients had no features of necrosis on biopsy, one of whom required immunosuppression without corticosteroids. Conclusions EMG and MRI were non-specific in identifying patients with a statin induced myositis, whilst CK levels varied significantly in this group. A strongly positive anti-200/100 antibody in combination with a necrotizing myopathy on muscle biopsy could be useful clinically to identify which patients have an autoimmune element to their disease, hence may potentially benefit from immunosuppression. References Christopher-Stine L et al. Arthritis Rheum 2010;62(9):2757–66. Mammen AL et al. Arthritis Rheum. 2011 Mar;63(3):713-21. Acknowledgements MJ is a MRC Clinical Training Fellow funded by the North West England Medical Research Council Fellowship Scheme in Clinical Pharmacology and Therapeutics. Disclosure of Interest None Declared

Background Idiopathic inflammatory myopathies (IIM) are frequently accompanied by presence of autoantibodies. A correlation of anti-Jo-1 and anti-SRP antibody levels with disease activity and creatine kinase levels has been described. Recently a new anti-PM1α quantitative ELISA test detecting antibodies to the main epitope of PM-Scl-100 has become available. Objectives To investigate the prevalence of anti-PM1α autoantibodies and association with clinical symptoms, clinical disease activity and serum CK in a multicenter myositis cohort. Methods Two hundred and thirty eight consecutive patients with established IIM (103 DM, 99 PM, 8 JDM, 3 IBM and 25 IIM/CTD-overlap syndrome) were tested for the presence of anti PM-Scl autoantibodies by immunoblot and by immunoprecipitation. Eight additional anti-PM-Scl positive sera were also obtained. Serum levels of anti-PM1α autoantibodies were detected by ELISA kit (Dr. FOOKE Laboratorien GmbH, Neuss, DE). Clinical data were derived from the Euromyositis database (www.euromyositis.eu). Global and organ clinical disease activities were assessed by Myositis Activity Assessment Tool (MYOACT) 10 cm visual analogue scales. Results 19/238 (8%) patients (11 DM, 2 PM and 6 IIM/CTD-overlap syndrome (3 DM + SSc, 2 PM + SSc, 1 PM + SjS)) were positive for anti-PM-Scl in immunoblot, immunoprecipitation and anti-PM1α ELISA. 19 PM-Scl+ were 16/181 women and 3/57 men, average age 50.5 years (22.0 – 74.2) and average disease duration 3.0 years (0 – 11.6 years). Good agreement between detection tests was found. The most common clinical features among anti-PM-Scl positive patients were muscle weakness (96%), interstitial lung disease (77%), Raynaud’s phenomenon (69%) and mechanic’s hands (65%). One patient had a cancer. Anti-PM1α levels correlated with serum CK activity in the DM group (p=0.041, r=0.533) and with constitutional disease activity and seriousness of dysphagia in the PM group (p=0.048, r=0.768 and p=0.007, r=0.927). Furthermore longitudinal change in anti-PM1α levels correlated with HAQ scores in the whole group (p=0.016, r=0.730) and with global disease activity score in DM group (p=0.023, r=0.685), but not with other MYOACT parameters. Conclusions Anti-PM-Scl autoantibodies are detected in 8% of patients with myositis. They are more frequent in those with DM and overlap syndromes. Presence of these autoantibodies is associated with muscle weakness, ILD, Raynaud’s phenomenon and mechanic’s hands. Anti-PM1α levels correlate with serum CK levels in DM and their change is correlated with change of HAQ scores in the whole group and with MYOACT scores in DM group. This suggests that the quantitative measurement of anti-PM-1α may be a useful tool in the assessment of patients with this syndrome. Disclosure of Interest None Declared

Background and Objectives B-cell activating factor of the TNF family (BAFF) is important for B cell maturation and plays a role in (auto)antibodies production. Elevated serum levels in relation to autoantibodies were documented in patients with IIM. Promoter region of BAFF gene contains several known sites with single nucleotide polymorphism (SNPs). An association between Rs9514828 (–871 C/T) SNP and susceptibility to idiopathic trombocytopaenic purpura was shown and possible relations to systemic lupus erythematosus, rheumatoid arthritis or Sjogren’s syndrome were suggested but in IIM have not been studied yet. Here, we analysed relation of four BAFF SNPs located in the BAFF gene promoter with the development of IIM. Materials and Methods 146 patients with polymyositis (PM), 150 with dermatomyositis (DM), 11 patients with juvenile DM and 4 patients with inclusion body myositis and 103 healthy individuals were included. Four SNPs located upstream in the BAFF gene (Rs9514827 (–2841 T/C); Rs3759467 (–2704 T/C); Rs1041569 (–2701 T/A); Rs9514828 (–871 C/T)) were analysed by direct DNA sequencing. Serum levels of BAFF (s-BAFF) were evaluated using ELISA. Autoantibodies were detected with immunoprecipitation. The chi square test for analysis of alleles and genotypes associations and SNPStats software for haplotype frequency studies were used. Results Significantly higher frequency of –2701T allele was present in patients (18%) compared to healthy controls (12%) (P = 0.029; OR 1.684 (CI 95% = 1.050 – 2.699)). Additionally, increased –2841T allele (P = 0.086), –2841TT, CT genotype (P = 0.066) and –2701TT, AT genotype (P = 0.079) frequencies were observed in patients. SNPs were in strong linkage disequilibrium and formed four common haplotypes (TTAC, CTAT, TCAC, TTTT), with significantly different frequency (>9%) distributions between patients and controls (global P-value Conclusions We describe significant association of SNP (Rs1041569) with myositis and a relation of SNP (Rs3759467) to presence of anti-Jo-1 autoantibodies and s-BAFF levels. These results should be confirmed in an independent cohort of patients and possible relations of s-BAFF levels with disease activity and treatment should be considered. Acknowledgement IGA -Ministry of Health of the Czech Republic NT/12438–4.

Background and Objectives The idiopathic inflammatory myopathies (IIM) are systemic connective tissue diseases in which autoimmune pathology is responsible for promotion of chronic muscle inflammation and weakness. As in many other autoimmune diseases, the development of IIM is also associated with genes of HLA complex. The aim of this study was to determine the basic relation between alleles of HLA genes and IIM. Materials and Methods We have performed low to high resolution genotyping to characterise the allelic profiles of HLA-DRB1, -DQB1 and -DQA1 loci in a large group of single centre cohort of patients suffering from IIM (n = 269). The genomic DNA was prepared by standard DNA extraction methods and the HLA typing was done using the commercial LABType® SSO kit (One Lambda, USA). Statistical evaluation of results was done with chi-2 test and Fisher exact test. Autoantibody profiles were analysed with radioactive immunoprecipitation. Results The frequencies of HLA-DRB1*03:01 and -DRB1*16:01 alleles were increased in IIM patients and the difference reached statistical significance when compared to healthy controls (P Presence of HLA-DRB1*03:01 allele was associated with anti-Jo-1, anti-Ro52, or anti-Pm-Scl positivity in all IIM patients (P The DRB1*16:01 allele was associated with negativity of all studied autoantibodies, particularly in subgroup of DM patients (P Conclusions This study identifies different genetic background between patients with dermatomyositis and polymyositis in a homogenous population of patients from a single centre. Acknowledgement This work is supported by Internal Grant Agency of Ministry of Health of the Czech Republic NT/12438–4 and NT/13699.

Autoantibodies targeting intracellular proteins involved in key processes are detected in patients with idiopathic inflammatory myopathies. These myositis-specific autoantibodies have been increasingly demonstrated to correlate with distinct clinical phenotypes within the myositis spectrum. This review highlights the clinical associations of the myositis-specific autoantibodies, with particular attention to the recently identified and characterized novel myositis autoantibodies: p155/140, p140 (MJ), CADM-140 (MDA5), SAE, and 200/100.

Myositis specific autoantibodies (MSA), exclusively found in patients with Idiopathic Inflammatory Myopathies can be detected in approximately 60% of children with JDM. Anti-MDA5 antibodies, a subgroup of MSA, appear to be associated with clinically amyopathic myositis, rapidly progressive interstitial lung disease (RP-ILD) and a poor prognosis in adult East Asian dermatomyositis patients. Small studies in Japanese children with JDM have suggested similar disease phenotype. This contrasts dramatically with findings in predominantly Caucasian US adult populations where anti-MDA5 has been associated with a distinct cutaneous phenotype and no association with RP-ILD has been found.