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Background:Rheumatoid arthritis (RA) is categorized as seropositive or seronegative referring to the presence or absence of IgM rheumatoid factor (IgM-RF) and/ or anti-citrullinated protein antibodies (ACPA).Patients with an autoimmune disease are more likely to develop additional autoimmune conditions than individuals without pre-existing autoimmune disease.Objectives:The aims of this study were to examine patterns of polyautoimmunity in seropositive compared with seronegative patients with recent-onset RA.Methods:The study was register-based and used the nationwide DANBIO register (identification of recent-onset (Table 1.Characteristics, prevalence, and incidence of polyautoimmunity in incident RA patients.SeropositiveSeronegativeN79834534Age in years59.4 (48.6 to 69.1)63.0 (51.5 to 72.3)Women5476 (69 %)2856 (63 %)HAQ-DI0.857 (0.375 to 1.375)0.875 (0.375 to 1.375)DAS28-CRP4.3 (3.3 to 5.2)4.6 (3.6 to 5.5)CRP, mg/mL10 (4 to 23)10 (3 to 26)VAS physician, 0-100mm27 (15 to 45)30 (16 to 47)Treated with methotrexate86 %84 %-other csDMARD, %24 %24 %-Biological DMARD, %5 %6 %Smoking status:Current / previous / never / unknown, %13 / 11 / 17 / 59 %9 / 9 / 23 / 59 %Prevalence of polyautoimmunity / diabetes mellitus type 1 / autoimmune thyroid disease / inflammatory bowel disease549 (10.6 %) / 1.4 % / 6.7 % / 1.4 %349 (12.8 %) / 1.5 % / 7.8 % / 1.5 %Age and sex adjusted odds ratio0.79 (0.71 to 0.89)1 (ref.)Incident cases of polyautoimmunity373 (4.7 %)242 (5.3 %)Adjusted hazard ratio0.86 (0.71 to 1.05)1 (ref.)Continuous variables shown as median with interquartile ranges. Autoimmune conditions included:autoimmune thyroid disease (redeemed prescriptions of ATC=H03A); diabetes mellitus type 1 (ICD-10 diagnosis E10 combined with redeemed prescriptions of insulin, ATC=A10A); pernicious anaemia (D51.0); autoimmune haemolytic anaemia (D59.1); idiopathic thrombocytopenic purpura (D69.3); Autoimmune adrenalitis (E27.1B); multiple sclerosis (G35); neuromyelitis optica (G36.0); Guillain-Barré syndrome (G61.0); myasthenia gravis (G70); inflammatory bowel disease (K50-1); primary biliary cirrhosis (K74.3); primary sclerosing cholangitis (K83.0); autoimmune hepatitis (K75.4); celiac disease (K90.0); pemphigus vulgaris (L10.0); bullous pemphigoid (L12.0); dermatitis herpetiformis (L13.0); alopecia areata (L63); vitiligo (L80); lichen sclerosis (L90.0); chronic interstitial cystitis (N30.1).Using age and sex adjusted logistic regression analysis, the odds ratio (OR) of prevalent polyautoimmunity in seropositive compared with seronegative patients at the time of RA diagnosis was calculated.To estimate the hazard ratio (HR) for developing yet another autoimmune disease in the 5 years after RA diagnosis, adjusted cause-specific Cox regression models were performed. Several sensitivity analyses were carried out including alternative exposure and outcome definitions.Results:In total, 12,517 patients with recent-onset RA were included. The groups were similar in terms of disease characteristics and DMARD treatment, but seropositive patients were younger and included more women, see Table 1. Patients with seropositive RA had an OR of 0.79 (95% CI 0.71-0.89) for baseline presence of polyautoimmunity compared with seronegative patients, whereas the 5-year HR was 0.86 (95% CI 0.71-1.05) for incident polyautoimmunity. The results remained similar in all sensitivity analyses.Conclusion:Patients with seropositive RA had a slightly lower prevalence and incidence of polyautoimmunity compared to seropositive patients. The results were somewhat surprising, yet very robust, and thus raises the question if seronegative RA is in fact “more autoimmune” despite the absence of (identified) autoantibodies.Acknowledgements:We acknowledge all patients and all Danish departments of rheumatology contributing to the DANBIO registry.Disclosure of Interests:Amalie Hagelskjær: None declared, René Cordtz: None declared, Sofie Bliddal: None declared, Anders Sandermann Mortensen: None declared, Salome Kristensen: None declared, Claus Henrik Nielsen: None declared, Ulla Feldt-Rasmussen: None declared, Christian Torp-Pedersen: None declared, Lene Dreyer Grant/research support from: Grants from BMS, Galderma, and Eli Lilly

Background:S100A11 protein, a member of S100 family, has been associated with several autoimmune inflammatory conditions such as rheumatoid arthritis (RA). Although the pathogenesis of autoimmune diseases is not fully understood, the formation of neutrophil extracellular traps (NETs) seems to play a certain role. Recent data indicate that S100A8/A9 is released via NETosis and can further augment inflammatory responses.Objectives:The aim of our study was to examine the association of S100A11 with NETs in RA.Methods:To assess the expression of S100A11 by neutrophils of RA synovial tissue (n=8), immunofluorescence staining of S100A11 and myeloperoxidase (MPO) was performed. The levels of S100A11 and MPO in RA synovial fluid (n=23) were measured by ELISAs (RayBiotech and Abcam), and the activity of peptidyl arginine deiminases (PADs) was measured by an in-house immunoassay. NETosis was induced by adding phorbol 12-myristate 13-acetate (PMA) to neutrophils from RA patients (n=7). Release of NETs was visualised by immunocytochemistry (n=7) and the presence of S100A11 in supernatants was analysed by ELISA (RayBiotech). Neutrophils purified from healthy donors (n=5) were stimulated by S100A11 and the release of cytokines TNF and IL-6 was measured by ELISA (RayBiotech).Results:S100A11 was expressed by synovial tissue neutrophils of the RA patients (n=8). The levels of S100A11 in the synovial fluid of RA patients (n=23) correlated with the levels of a NETosis marker MPO (r=0.562, p=0.005) and with PADs activity (r=0.690, pConclusion:Here we show for the first time that release of S100A11 by neutrophils is dependent on NETosis. Moreover, extracellular S100A11 augments the inflammatory response by inducing TNF and IL-6 secretion in neutrophils.Acknowledgments:Supported by MHCR 023728Disclosure of Interests:Adela Navratilova: None declared, Viktor Becvar: None declared, Jiří Baloun: None declared, Dres Damgaard: None declared, Claus Henrik Nielsen: None declared, Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Jiří Vencovský: None declared, Ladislav Šenolt: None declared, Lucie Andres Cerezo: None declared, David Veigl: None declared

Background:The majority of patients with rheumatoid arthritis (RA) produce autoantibodies against proteins that have undergone post-translational modfication, e.g. citrullination or carbamylation. There is growing evidence of their relevance and their potential utility to improve diagnosis, patient stratification, and prognosis for precision medicine. Investigating new autoantibody patterns may allow further stratification of patients and identifying subsets of patients that benefit from different treatment modalities. Following the discovery of high autoantibody reactivity against multiple modified proteins the interest in native targets decreased. Even though antibodies reacting with native proteins may also have a role in RA pathogenesis, their reactivity patterns are much less studied.Objectives:To identify novel native autoantigens in RA patients and elucidate patterns within autoantibody reactivity against native autoantigens.Methods:We investigated the reactivity of autoantibodies in plasma pools from 15 anti-CCP positive and 10 anti-CCP negative RA patients and 10 healthy donors against more than 1600 human proteins in native configuration using the Immunome high-density protein microarray.Results:We identified 86 native proteins that were recognized by IgG antibodies from anti-CCP positive RA patients and 76 native proteins recognized by IgG antibodies from anti-CCP negative RA patients, but not by antibodies from healthy donors. Examples of proteins recognized by both patient subgroups are calcium/calmodulin-dependent protein kinase type II subunits, histone deacetylases, keratin, and vimentin. Reactivity against the ribonucleic protein SSB was observed in anti-CCP negative RA patients only.Conclusion:Several human proteins in their native conformation are recognized by autoantibodies from anti-CCP positive as well as anti-CCP negative RA patients. In general, anti-CCP positive patients had higher autoantibody activity than anti-CCP negative patients and healthy donors.References:[1] Konig, M.F., Giles, J.T., Nigrovic, P.A., Andrade, F., 2016. Antibodies to native and citrullinated RA33 (hnRNP A2/B1) challenge citrullination as the inciting principle underlying loss of tolerance in rheumatoid arthritis. Ann. Rheum. Dis. 75, 2022–2028.[2] Zheng, Z., Mergaert, A.M., Fahmy, L.M., Bawadekar, M., Holmes, C.L., Ong, I.M., Bridges, A.J., Newton, M.A., Shelef, M.A., 2019. Disordered Antigens and Epitope Overlap Between Anti-Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis. Arthritis Rheumatol. art.41074.[3] Sirotti, S., Generali, E., Ceribelli, A., Isailovic, N., De Santis, M., Selmi, C., 2017. Personalized medicine in rheumatology: the paradigm of serum autoantibodies. Autoimmun. Highlights 8.Acknowledgments :The Department of Clinical Immunology at Rigshospitalet Copenhagen is acknowledged for providing the healthy donor blood. The study is part of the PROCIT study financed by the Danish Council for Independent Research (grant no. DFF - 7016-00233). Moreover, the Obelske Family Foundation, the Svend Andersen Foundation, the Spar Nord Foundation and the Danish National Mass Spectrometry Platform for Functional Proteomics (PRO-MS; grant no. 5072-00007B) are acknowledged for grants to the analytical platform are acknowledged for the funding to enabling parts of this study.Disclosure of Interests:Thomas B.G. Poulsen: None declared, Dres Damgaard: None declared, Malene Møller Jørgensen: None declared, Ladislav Senolt: None declared, Jonathan Blackburn Shareholder of: Sengenics Corporation, Consultant of: Director of Sengenics Corporation, Employee of: Director of Sengenics Corporation, Claus Henrik Nielsen: None declared, Allan Stensballe: None declared

Background:Inflammatory and autoimmune diseases include multifactual pathomechanisms and systemic responses. The etiology of the joint diseases rheumatoid arthritis (RA), spondyloarthritis (SpA) in relation to osteoarthritis (OA) remain incomplete and establishing the correct diagnose remains nontrivial. Advances in high-throughput molecular technologies have increased investigations into the utility of transcriptomic, proteomic and high-density protein arrays approaches as diagnostic tools and companion diagnostics for precision medicine. To increase our understanding of the molecular pathogenesis, we extracted synovial fluid from the joints from multiple patient groups and characterized the protein composition in relation to plasma. Basic blood tests include inflammatory markers and autoantibodies, however, now analysis speed and robustness allow more readily clinical insight biofluids.Objectives:We present recent Omics concepts and studies investigating inflammatory state and treatment outcome in different biofluids from plasma to synovial fluid accessing causalities leading to inflammation and pain. Additionally, the aim was to investigate in any proteomic findings in synovial fluid can be correlated to proteomic changes in patient plasma and can be used as biomarkers for treatment effect.Methods:Plasma and synovial fluid were investigated in multiple pathologies before and after treatment in patients (biologics; MTX; intraarticular gold). Deep proteome, PTM and EV profiling were accomplished using quantitative proteomics approaches using quantitative mass spectrometry-based analysis by DIA/PASEF followed by deep datamining. All biological samples were digested according to a Filter Aided Sample Preparation (FASP) protocol before analysis with tandem mass spectrometry (MS/MS). PTM profiling were evaluated by 4D CCS based feature finding.Results:Mass spectrometry based profiling allowed quantitative profiling of up to 480 proteins in matched synovial fluid and plasma. Complementary analysis by Olink proteomics, cytokine profiling and cell-free DNA. Multiple acute inflammatory proteins were more abundant in the RA synovial fluid, including proteins originating from neutrophil granulocytes, whereas SpA patients had a higher concentration of haptoglobin. Complementary analysis by Olink immunoassay identified significantly downregulated inflammation markers out of 96 tested in relation to antiinflammatory treatment.Conclusion:Discovery of biomarkers and/or inflammatory signatures through integration of multi-omic data allowed stratify patients for improved treatment and prognosis. Firstly, our data using next generation proteomics approaches alleviates many pitfalls of missing values and poor proteome coverage including unbiased PTM profiling without enrichment strategies.Disclosure of Interests:None declared

Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes. These features may be relevant for clearance of MPs in SLE pathogenesis. Methods: Attached C3 fragments (C3b, iC3b, C3d), membrane integrity and cell surface markers of MPs from 18 patients with SLE and 11 HCs were measured by adding specific antibodies, 7-aminoactinomycin D (7AAD) and annexin V. MPs from all subjects were labelled with carboxyfluorescein diacetate succinimidyl ester and allowed to bind to autologous phagocytes and erythrocytes in the presence of autologous serum, and the binding to individual cell populations was assessed by flow cytometry. Results: The proportion of MPs bearing C3 fragments was higher in patients with SLE than in HCs (p=0.026), but the amount of opsonising C3b/iC3b molecules was lower ( p=0.004). The C3b/iC3b level correlated with the concentration of circulating C3 (rs=0.53, p=0.036). Phagocytes and erythrocytes from patients and HCs bound autologous MPs, and granulocytes from patients bound 13% more MPs than those from HCs (p=0.043). The presence of erythrocytes inhibited the MP binding to granulocytes by approximately 50%. Conclusions: Our demonstration of altered composition of C3 fragments on MPs from patients with SLE, including decreased numbers of opsonising C3 fragments, and competitive binding of MPs to circulating phagocytes and erythrocytes corroborates the hypothesis of defective clearance of apoptotic material in SLE, and indicates that differences in both MP opsonisation and binding of MPs to cells are important in the pathogenesis of SLE.

Background According to national guidelines issued in 2015, a non-medical switch from originator infliximab (IFX) to biosimilar IFX was conducted in all Danish patients with inflammatory rheumatic diseases treated in routine care. Objectives To investigate effects of the switch on serum (s) IFX and anti-drug antibodies (ADA) in patients with rheumatoid arthritis (RA), axial spondyloarthrits (SpA) and psoriatic arthritis (PsA). Methods We included originator infliximab treated patients, who switched to and were treated with biosimiilar infliximab for >2 months with 2 available trough level serum samples (baseline immediately before switching/follow-up 2–4 months after switching time of 2nd–4th infusion). Patients stopping treatment earlier were not included. Trough sIFX 30AU/l median-high. sIFX and ADA were analyzed using automated in-house assays at OUS-Radiumhospitalet. Characteristics and outcomes were registered in the DANBIO registry. Remission was defined by disease activity indices (DAS28 Results 96 pts (192 samples) from 4 departments (49 RA, 27 SpA, 10 PsA, 10 other) were included (age 52 (43–62) years, 47% women). Previous IFX treatment duration was 7.5 (5.1–10.3) years. Follow-up was after 81 (71–90) days. 58 patients (60%) received concomitant methotrexate (15 (10–20) mg/wk). Baseline IFX dose was 3.1 (3.0–4.8) mg/kg every 7 (6–9) weeks. At baseline, 60% of pts had low sIFX and 29% very low and at follow-up it was 57% and 29%, respectively. At baseline, 14 pts (15%) had medium-high ADA, at follow up 16%. Median sIFX was lower at baseline vs. follow-up (1.8 (0.8–5.8) vs 2.4 (0.8–6.2) mg/l, p=0.006) whereas ADA were similar (p=0.7). Six of 58 pts with low baseline sIFX had high sIFX at follow-up, and 3 of 38 pts with high baseline sIFX had low sIFX at follow-up. Similar numbers for low vs. medium–high ADA at baseline and follow-up were 2/81 and 3/15. For the rest, sIFX and ADA remained stable between baseline and follow-up in 87/96 (91%) and 91/96 (95%), respectively (Figure). MTX use was not associated with sIFX or ADA (both p>0.05). Patients with low sIFX received lower IFX doses than pts with sIFX ≥3mg/l (255 (200–320) mg vs. 300 (250–400) mg, p=0.02) and with longer intervals (8 (6–10) weeks vs. 6 (6–6)weeks, p 0.05). Conclusions In this highly selected group of patients treated with originator infliximab for >5 years, non-medical switch to biosimilar IFX had no negative impact on sIFX or ADA 2–4 months following switch. At baseline, 60% of patients had low sIFX but few patients had ADA, perhaps indicating low immunogenicity in these patients. Disclosure of Interest None declared

Background There is increasing evidence for a pathogenetic role of DNA methylation changes in RA. Monozygotic (MZ) twins provide a unique opportunity to study the epigenetic effects in complex diseases like RA. Recent estimates on DNA methylation in twins showed a mean genome-wide heritability of 18%. Recent studies indicate that DNA methylation may be an intermediary factor of genetic risk in RA. 1 In addition, tobacco smoking, which has emerged as the single most important environmental risk factor for RA, leads to extensive Genome-Wide changes in DNA methylation. 2 Objectives To identify differentially methylated positions (DMPs) associated with RA and smoking. Methods Blood-derived DNA from 28 monozygotic RA discordant twin pairs was analyzed for epigenome wide methylation (EWAS) using Illumina HumanMethylation450 array. The twins were characterized with regard to smoking, DMARD-treatment, anti-CCP, and the pair specific genetic variables shared epitope and PTPN22 as well as age and sex. We used a mixed model designed for EWAS on discordant twins taking these covariates as well as batch effects into consideration. Results A total of 407 CpG sites were differentially methylated in RA patients related to smoking with p Conclusions These observations indicate that tobacco smoking elicits profound epigenetic modifications in RA when adjusting other RA associated covariates. References Liu Y, Aryee MJ, Padyukov L, Fallin MD, Hesselberg E, Runarsson A, et al. Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis. Nat Biotech 2013; 31(2): 142-7. Zeilinger S, Kuhnel B, Klopp N, Baurecht H, Kleinschmidt A, Gieger C, et al. Tobacco smoking leads to extensive genome-wide changes in DNA methylation. PLoS One 2013; 8(5): e63812. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2461

The possible adverse consequences of biological therapies in inflammatory bowel diseases (IBDs) were recently highlighted in this journal by D’Haens ( Gut 2007; 56 :725–32). We here describe a hitherto unappreciated adverse effect to treatment with the B lymphocyte (B cell) depleting agent rituximab (RTX),1 namely the occurrence of ulcerative colitis and arthritis shortly after treatment with RTX. Our patient, a 45-year-old Caucasian female, had had mild irritable bowel symptoms since 1992 at which time rigid sigmoidoscopy and bowel x ray were normal. She had never received any therapy, had never previously had joint pain, and was not predisposed to IBD. In April 2005 she was diagnosed with Graves’ disease, and after uncomplicated standard methimazole therapy she received four weekly doses of 375 mg/m2 RTX from day 1 to 22 as part of a clinical trial.2 The trial was approved by the local ethics committee …