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BackgroundIn rheumatoid arthritis (RA) around two-thirds of patients are autoantibody-positive for rheumatoid factor, anti-citrullinated protein antibodies (ACPA) and/or anti-carbamylated protein antibodies (anti-CarP). The remaining seronegative subgroup of RA is clinically heterogeneous and thus far, biomarkers predicting the disease course in these patients are lacking. Therefore, we set out to investigate the value of a new/different autoantibody in rheumatoid arthritis directed against advanced glycation end-product (AGE) modified proteins (anti-AGE). AGEs are a marker of oxidative stress, and anti-AGE have been described in multiple diseases including diabetes, and hypertension.ObjectivesTo investigate the prevalence of anti-AGE in RA and non-RA arthritis patients, as well as their association with clinical parameters and disease outcome in RA.MethodsIn 648 RA patients and 538 non-RA arthritis patients from the Leiden Early Arthritis Clinic anti-AGE IgG antibody levels were measured using an in-house fetal calf serum (FCS) ELISA based assay using native FCS as control. The cutoff for positivity was set as the mean optical density plus two times the standard deviation of 80 healthy controls. Radiological progression was measured with the with Sharp van der Heijde score (SHS) on yearly basis and the association with anti-AGE was assessed with a multivariate normal regression model.ResultsAnti-AGE was found in 299 (46%) of RA patients versus 163 (30%) of non-RA arthritis patients. Interestingly, 67 (34%) of completely seronegative (RF-, ACPA- and anti-CarP-negative) RA patients were positive for anti-AGE. Within RA, anti-AGE-positive patients had significantly higher ESR (median anti-AGE-positive: 38, anti-AGE-negative: 32, pConclusionAlmost half of the RA patients are anti-AGE positive including a substantial part of otherwise completely seronegative RA patients. Anti-AGE antibodies are associated with inflammatory parameters and radiologic progression in seronegative RA patients. Therefore, although these autoantibodies are not specific for RA, anti-AGE could potentially identify patients with a more inflammatory phenotype and more severe disease outcome in “classic” autoantibody-negative RA patients.Disclosure of InterestsNone declared.

BackgroundANCA-associated vasculitides (AAV) are characterized by recurrent, chronic small vessel inflammation and deleterious organ damage. The main targets of ANCA are myeloperoxidase (MPO) and proteinase 3 (PR3). ANCA, B cells and the complement system are crucial to AAV pathogenesis, as evidenced by the clinical benefit of B cell depletion with rituximab and, more recently, the C5a receptor antagonist avacopan. While ANCA in serum have been studied extensively, phenotypic and functional characteristics of the underlying B cell responses remain largely unknown.ObjectivesTo develop a flow cytometry-based technique for identifying MPO-specific B cells in the circulation of MPO-positive AAV patients in order to characterize this B cell response and its potential contribution to disease pathogenesis.MethodsHuman neutrophil-derived MPO was conjugated to two different fluorochromes and used to identify MPO-specific B cells by flow cytometry. An antigen-specific staining protocol was developed and validated using MPO- and PR3-specific hybridoma cells. MPO-specific B cells were phenotypically characterized and isolated from the peripheral blood of AAV patients by fluorescence activated cell sorting (FACS) and cultured as single cells. MPO-specificity was confirmed by ELISA on culture supernatants. B cell receptor (BCR) sequences were obtained from MPO-positive clones by full length ARTISAN-PCR and Sanger sequencing. MPO-specific IgG and IgM monoclonal antibodies (mAb) were produced to validate specificity and to examine their ability to activate complement. Finally, MPO-positive AAV patient plasma and plasma depleted of IgG or IgM was tested in in vitro complement assays.ResultsThe newly developed, differential antigen labelling approach successfully identified MPO-specific but not PR3-specific hybridoma cells. Subsequently, we detected MPO-specific B cells in the circulation of MPO-positive AAV patients at a frequency of up to 1:1000 B cells. FACS sorting and single cell culture yielded an enrichment of MPO-specificity of ~80%. Notably, the majority of isolated, MPO-specific B cells (60-95%) displayed an IgM memory phenotype, which corresponded to the presence of anti-MPO IgM in plasma. The remainder of the MPO-specific cells were mainly IgG memory B cells and few naive cells. BCR sequencing revealed a polyclonal IgM response with diverse V-gene usage, consisting of both germline and highly mutated clones. Generation of mAb (n=5) confirmed MPO specificity by inhibition ELISA for both germline and somatically mutated clones. Interestingly, anti-MPO IgM mAb showed a high capacity for complement factor deposition upon MPO binding. MPO-specific complement assays with IgG- and IgM-depleted patient plasma showed that anti-MPO IgM activated complement much more efficiently than anti-MPO IgG.ConclusionWe demonstrate the direct ex-vivo identification, isolation and characterization of MPO-specific B cells in human AAV. Intriguingly, we observed a remarkable expansion of MPO-specific, IgM-expressing memory B cells in patients. This so far unrecognized, active IgM-compartment may be clinically relevant, as both mAb and plasma-derived polyclonal MPO-specific IgM strongly activated complement, a pathway thought to play a central role in AAV. Next to these novel insights into autoreactive B cell biology in AAV, our findings now provide new opportunities for studying auto-reactive B cell responses in different clinical phases of AAV, amongst which active disease, remission and (imminent) flares.Disclosure of InterestsNone declared.

Anti-Carbamylated Protein Antibodies Precede the Onset of Symptoms of Rheumatoid Arthritis in A Swedish Biobank Cohort