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ObjectivePatients with inflammatory arthritis (IA) associated with immune checkpoint inhibitor (ICI) treatment for cancer are typically seronegative for anti-cyclic citrullinated peptide (CCP) antibodies and rheumatoid factor, but little is known about the presence of other autoantibodies in this patient population. We investigated the prevalence and characteristics of anti-RA33 antibodies in patients with ICI-induced IA.MethodsAnti-RA33 ELISAs were performed on sera from four groups of patients: 79 with ICI-induced IA, 52 with rheumatoid arthritis (RA), 35 treated with ICIs without IA during follow-up and 50 healthy controls. Anti-RA33 positivity and level, clinical and demographic data were compared across groups.ResultsAnti-RA33 antibodies were found in 9/79 (11.4%) patients with ICI-induced IA but in 0/35 patients treated with ICIs who did not develop IA (0%; p=0.04). Of the patients positive for anti-RA33, two had sera available from before ICI treatment; anti-RA33 antibodies were present in both pre-ICI treatments. In patients with RA, 7.7% were positive for anti-RA33 antibodies as were 2% of healthy controls. In ICI-induced IA, anti-RA33 antibodies were associated with anti-CCP antibodies (p=0.001). We found no statistically significant differences in other clinical characteristics in those with and without anti-RA33 antibodies.ConclusionsAnti-RA33 antibodies are present in a subset of patients with ICI-induced IA, absent in other ICI-treated patients and may be a biomarker for developing IA. Additional studies evaluating serial samples before and after ICI treatment will further establish the temporal relationship of these antibodies to IA development.

The aim of the study was to evaluate the frequency of anti-mutated citrullinated vimentin antibodies (a-Sa), anti-citrullinated α-enolase peptide 1 antibodies (a-CEP-1), anti-filaggrin antibodies (AFAs), heterogeneous nuclear ribonucleoprotein compies/anti-RA33-antibodies (a-hnRNP/RA33), anti-carbamylated protein antibodies (a-CarP), and metalloproteinase (MMPs) activity in patients with early inflammatory arthritis (EIA). Seventy-four patients with EIA: 51 diagnosed with RA (rheumatoid arthritis) and 23 with UA (undifferentiated arthritis), and 20 healthy volunteers were enrolled to the study. Inflammatory markers, rheumatoid factor (RF), and antibodies mentioned above were assessed in all patients. In the EIA group, we observed significantly higher concentration of a-CEP-1 (65.8 ± 111.6 RU/mL) than in controls (2.0 ± 0.0 RU/mL). In RF(+) RA patients, we observed higher concentration of a-Sa and a-CEP-1 than in other groups. A-Sa were positive in 69% of RF(+) RA, 37% of RF(−) RA, 26% of UA patients and in 10% of controls. A–CEP-1 were positive in 77% of RF(+) RA patients, in 56% of RF(−) RA patients, in 8.7% of UA patients, but they were negative in controls. In patients with RF(+) RA, positive a-CarP were present statistically significantly more often than in RF (−) RA patients. No statistically significant difference in frequency of a-hnRNP/RA33 and AFA between RF(+) RA, RF(−) RA, and UA was observed. Our results suggest that a-CEP-1 may help in differentiation between RF(−) RA and UA. a-CEP-1 and a-Sa may be useful while diagnosing EIA. a-CarP may be used in differentiation of RA RF(−) and UA. However, a follow-up study is needed to evaluate the prognostic value of analyzed antibodies.

Background:The mechanisms affecting anti-RA33 antibody’s involvement in RA pathogenesis is still unclear. Refining our understanding of anti-RA33’s role in RA in relation to known RA-associated genes and serological elements is needed.Objectives:We investigated the relationship between RA-associated HLA-DRB1 epitope (SE) allele and presence of anti-RA33 antibodies in different serological subsets of rheumatoid arthritis in a Malaysian population.Methods:Serum samples from 550 RA cases comprising seronegative (negative for anti-CCP2, IgG and IgM, n=250), seropositive (triple-autoantibody positive, n=150), singular anti-CCP2 positive (n=100), and double RF positive RA (n=50) were chosen from the Malaysian Epidemiological Investigations of RA (MyEIRA) case-control study. Three hundred MyEIRA population controls were used for comparison. All serum samples were assayed using a commercial anti-RA33 ELISA kit. All genetic samples were genotyped for four-digit HLA-DRB1 alleles using the PCR-SSO method on Luminex platform.Results:The proportions of anti-RA33 positive was 20.9% in all RA cases (i.e. 34% in RF only positive RA; 25% in seropositive RA; 18% in seronegative RA and 18% in anti-CCP2 only positive RA). The HLA-DRB1 shared epitope alleles were significantly associated with anti-RA33 positive in the seropositive RA subgroup (OR=6.9, 95% CI 1.4-34.8; p=0.02). We observed significant association between anti-RA33 negative and HLA-DRB1 SE alleles among the seropositive RA patients (OR=4.5, 95% CI 2.8-7.2; pConclusion:The HLA-DRB1 SE alleles increased the risk of seropositive and CCP only positive RA independent of anti-RA33 positivity.References:[1]Boeters, Debbie M et al. “The 2010 ACR/EULAR criteria are not sufficiently accurate in the early identification of autoantibody-negative rheumatoid arthritis: Results from the Leiden-EAC and ESPOIR cohorts.” Seminars in arthritis and rheumatism vol. 47,2 (2017): 170-174.[2]de Brito Rocha, Sara et al. “Clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis.” Advances in rheumatology (London, England) vol. 59,1 2. 17 Jan. 2019.Acknowledgements:The authors would like to thank the Director General of Health, Ministry of Health Malaysia for supporting this study. The authors are also indebted to participants for their kind participation. This study was financially supported by the Ministry of Health, Malaysia (JPP-IMR 08-012).Disclosure of Interests:None declared

BackgroundAnti-citrullinated protein antibodies (ACPAs) are the hallmark of rheumatoid arthritis (RA). Protein citrullination is believed to drive autoantigen selection in RA. Nonetheless, several autoantigens in RA are targeted as native (unmodified) proteins. Here, the study of hnRNP A2/B1 (RA33) provides a framework to understand the humoral response to native and citrullinated autoantigens in RA.MethodsRA synovial fluid (SF) cells were analysed by immunoblotting and mass spectrometry. RA33 was cloned from RASF cells and splice variants expressed as recombinant proteins. Antibodies against native and citrullinated RA33 were characterised by ELISA, immunoblotting and immunoprecipitation.ResultsRA33 is citrullinated in the rheumatoid joint and targeted either as a citrullinated or native protein in distinct patient subsets with RA. A novel splice variant (hnRNP B1b) previously associated with disease initiation in experimental arthritis was identified in the RA joint and acts as the major target of the anti-RA33 response. Antibodies exclusively targeting citrullinated RA33 were positively associated with disease duration and erosive disease. In contrast, anti-(native) RA33 antibodies were detected almost exclusively in early RA and identified patients with low radiographic erosion scores. Finally, a unique subset of double-reactive patients demonstrated intermediate severity, but rapid disease progression, suggesting a transitional disease phase in the evolution of an anti-native protein antibody to ACPA response in RA.ConclusionsThese data suggest that native and citrullinated proteins targeted by autoantibodies in RA may be part of a single antibody system and challenge the paradigm of citrullination as the unifying principle underlying loss of tolerance in RA.

Background:In rheumatoid arthritis (RA), the endocardium is involved in the inflammatory process, which is caused by immunopathogenetic mechanisms involving CD4 +, T-cells and proinflammatory macrophages. Minor changes in the cardiovascular system can be successfully detected by ultrasound in patients with RA.Objectives:to improve the quality of non-invasive diagnostics of heart disease in patients with RA.Methods:57 patients with RA were under observation: 7 men and 50 women aged from 26 to 70 years; mean age 50.45 ± 10.12 years; activity (according to DAS28) was low for 3.5%, medium for 86%, and high for 10.5%. Immunological examination included determination of serum IgM-RF, CRP, antibodies to cyclic citrulline peptide (anti-CCP), antibodies to modified vimentin (anti-MCV), antibodies to antigen RA33, levels of angiopoietin-like proteins 3 (ANGPTL3) and 4 (ANGPTL4) types (classical ELISA test), as well as the detection of IgG antibodies to 5’-nucleotidase (5’-NT) and xanthine oxidase (XO) (modified ELISA test). Data from ultrasound, magnetic resonance (MRT) and computer (CT) tomography were used in assessing the state of the heart structures.Results:The pathology of cardiovascular system was diagnosed in 28 (49.1%) patients with RA. Signs of the heart damage were noted in 33.3% of cases (pericarditis and valvular heart disease were most often detected). In groups of patients with RA with elevated levels of antibodies to 5’-NT and XO (compared with RA patients with normal parameters), there was a significantly more frequent heart damage (for antibodies to 5’-NT: χ2= 3.8, p = 0.047; for antibodies to XO: χ2= 3.92, p = 0.041). It was discovered that in all patients with an increased level of antibodies to XO, one of the lesions of the heart valvular apparatus of varying severity was noted. According to ultrasound data (usually confirmed by CT and / or MRT), signs of valvular dysfunction were found in 21 (36.8%) patients with RA. The high frequency of mitral prolapse (28.6%) may be associated with the presence of a chronic inflammatory process that is able to accelerate the development of atherosclerosis and heart disease in RA patients. A tendency to an increase in the prevalence of mitral prolapse in patients with a longer duration of the disease (p = 0.062) and with high levels of serum ANGPTL4 (p = 0.058) was found.One of the main factors leading to the development of vascular pathology in RA is the accumulation of reactive oxygen intermediate. Antibodies to XO can affect the biochemical activity of the enzyme in the serum of patients with RA, stimulating the increased formation of O-form XO, which has a pronounced prooxidant effect (especially to lipids). ANGPTL4, acting as a potent inhibitor of endothelial lipoprotein lipase, can suppress the release of non-esterified fatty acids and their transfer to the heart muscle.Conclusion:To identify subclinical signs of involvement of cardiovascular system in the pathological process in the early asymptomatic stages of RA, it is advisable to use imaging techniques in combination with immunological markers of heart damage, which can be especially useful for screening, diagnostic evaluation and determining cardiovascular risk.Disclosure of Interests: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 The high frequency of detection of antinuclear antibodies in rheumatoid arthritis (RA), although predominantly in low titres, allows us to consider the possibility of using varieties of these antibodies (primarily antibodies to RA-33) markers of RA especially in the early stages of the disease. Objectives To study the frequency of occurrence and determine the prognostic significance of antibodies to RA-33 in RA patients. Methods 57 RA patients were examined (mean age 50.5±10.1 years). The patients with the developed stage of the disease (47.4%) prevailed, the average activity (DAS28=3.2–5.1) of the pathological process (86%); with the second radiographic stage (42%) and functional class 2 (77%). The antinuclear factor was determined in the indirect immunofluorescence reaction on the HEp-2 cell line (norm Results ANF was detected in 18 patients with RA (32%), and in 94.4% of cases (17 patients) a diagnostic titer of 1:80 and a diffuse type of glow of the nucleus were noted. The ANF titres did not correlate either with the activity of the disease or with extra-articular manifestations of RA (p>0.1). Anti-RA33 was detected in 20 (35%) RA patients: 18 positive people (90%) had low positive anti-RA33 values (25 to 75 U/ml). In 35 (61.4%) of RA patients, anti-CCP was detected: 19 (33.3%) had low positive values (20 to 60 U/ml), 16 (28%) had highly positive values (more 60 U/ml). IgM-RF was detected in 26 (45.6%) patients in the values>48 IU/ml, as well as in 17 (30%) patients with RA in the values from 24 to 48 IU/ml. 14 people (54%) of patients with high IgM-RF levels had systemic manifestations of RA. It should be noted that anti-RA33 was detected in 9 patients with seropositive for anti-CCP and in 11 patients with seronegative both for anti-CCP and IgM-RF. Thus, when the results of standard serological tests are negative, an additional study of anti-RA33 is recommended to diagnose seronegative RA. When assessing the prognostic significance of the available clinical and laboratory and instrumental data we have analysed the results of the study of anti-CCP, anti-RA33, as well as all data on the presence of X-ray erosions in patients with RA with magnetic resonance imaging or ultrasound examination of affected joints. The presence of erosion was noted in 23 (40.4%) RA patients. The frequency of detection of anti-CCP in RA patients was significantly higher in the presence of erosive lesions of joints (19 of 35 patients were positive by anti-CCP, compared with 4 of 22 patients, negative for anti-CCP, χ2=5.89, p=0.015). In the RA group of patients positive for a wide range of antibodies (IgM-RF, anti-CCP, anti-RA33), the signs of joint erosion were identified in 22 of 46 patients, and in isolated increase only anti-RA33 – in 1 patient out of 11 people in this group χ2=4.04, p=0.044). Conclusions In the presence of highly positive anti-CCP values, RA patients have a more unfavourable prognosis, while an isolated increase in anti-RA33 is associated with a ”milder progression” of the disease and inversely proportional to erosive processes in the joints. Disclosure of Interest None declared

Background Recently heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as antigenic targets for autoantibodies in RA. Autoantibodies to RNP B1, B2 and heterogeneous complex A2/b are the most studied and are referred to as ”antigen RA33”. Clinical value of anti-RA33 in RA continues to be studied, so the question is about the diagnostic and pathogenic significance of these autoantibodies in patients with RA remains open. Objectives The aim of this study was to investigate the frequency of occurrence of anti – RA33y patients with RA and compare them with clinical manifestations and immunological parameters of disease. Methods The study included 139 patients with RA. The diagnosis was verified according to the classification criteria of ACR/EULAR 2010 Autoantibodies to RNP B1 IgG was assessed in samples of blood serum by enzyme immunoassay (ELISA; Medipan AG, Germany). A recombinant human RNP antigen B1 was used as the antigen, which was synthesised in the E. coli. system of the protein production. Anti-CCP, RF and antibodies to Sa-antigen (anti-Sa) was evaluated by ELISA according to the manufacturer’s instructions (Euroimmun AG company, Germany). The results were expressed in relative units (U/ml). Statistical data processing was carried out using the software Statistica 7.0 (StatSoft, USA) and Medcalc 12.5.0.0 (USA), including standard methods of parametric and nonparametric analysis. Differences were considered significant at p Results Levels and frequency of occurrence of anti-RA33, anti-CCP, RF IgM and anti-Sa were analysed for the examined patients depending on disease duration. Patients were divided into two groups: patients with early RA (disease duration less than 12 months) and patients with established RA (disease duration over 12 months). In the analysis it was found that the incidence of anti-Sa in established RA are much higher in comparison with the early RA (77.98% vs. 52.00% respectively, p Conclusions Thus, anti-RA33 is a perspective independent biomarker of RA, which has its own potential. Further study of the anti-RA33 as a diagnostic biomarker of early stage RA is promising. Based on preliminary studies the anti-RA33 could have diagnostic and prognostic value in diagnosis and evaluation of patients with early RA, and its differentiation from other small joint disorders, particularly when the other serologic tests are negative. To clarify the diagnostic and pathogenic value of anti-RA33 further research with a large sample size, comparison of immunological data with genetic factors, the results of other laboratory and instrumental studies, clinical manifestations of the disease are required. The obtained data will allow us to more thoroughly study the pathogenesis of RA and contribute to the search for new therapeutic options. Disclosure of Interest None declared

Background As it is very important to identify patients with a high risk of developing rheumatoid arthritis (RA), new, diagnostic methods, evaluating the possibility of progression from undifferentiated arthritis (UA) to RA are needed. Objectives The aim of this work was the evaluation of the frequency of rheumatoid factor, anti-cyclic citrullinated peptide (anti-CCP) antibodies, mutated citrullinated vimentin antibodies (a-Sa), anti–CEP-1 antibodies, anti-filaggrin antibodies (AFA), heterogeneous nuclear ribonucleoprotein compies/anti-RA33-antibody (HnRNP/RA33), anti-CarP antibodies (a-Carp) in patients with early arthritis. Methods 74 patients with early arthritis and 20 healthy volunteers were enrolled to the study. 51 patients were diagnosed with RA, 23 with UA. Exclusion criteria were the application of disease-modifying antirheumatic drugs or glucocorticosteroids. In all patients the following laboratory tests were performed: inflammatory markers, rheumatoid factor (RF) and antibodies mentioned above, together with necessary diagnostic that enables diagnosis. Results In patients with early arthritis the sensitivity and specificity of the presence of RF was 69% and 95%, respectively, and of anti-CCP was 67% and 97%. In patients with early arthritis we observed significantly higher concentration of CEP-1 (65,8±111,6) than in the healthy group (2,0±0,0). In RF(+) RA patients we observed higher concentration of antibodies a-Sa and CEP-1 than in other groups. Antibodies a-Sa were positive in 69% of RF(+) RA patients, in 37% of RF(-) RA patients, in 26% of UA patients and in 10% of healthy people. In 8 aCCP (-) and RF(-) patients we observed the presence of a-Sa; 3 of them were diagnosed with RF(-) RA with and 5 with UA. Anti–CEP-1 antibodies were positive in 77% of RF(+) RA patients, in 56% of RF(-) RA patients, in 4,5% of UA patients, but their presence were not observed in the healthy people. In 8 aCPP (-) and RF (-) patients we observed positive anti–CEP-1 antibodies; 6 of them were diagnosed with RA, 2 of them with UA. Anti–CEP-1 antibodies were positive in 50% of RF(-) RA patients, in whom there was no aCCP nor RF, and only in 4,5% of UA patients. In case of marking a-Car-P, positive values were present in: the group of RF(+) RA in 40% of patients, in patients diagnosed with RF(-) RA in 6%, in case of UA in 22% of patients. In patients with RF(+) RA, positive anti-CarP antibodies are present statistically significantly more often than in the group of RF (-) RA patients (p In case of marking hnRNP/RA33 and AFA no statistically significant differences between RF(+) RA, RF(-) RA and UA in their occurrence were observed. In patients with arthritis no correlation between smoking and analysed autoantibodies was observed. In smokers higher CRP concentration and ESR values was observed. Conclusions Our results suggest that a-Sa and CEP-1 parameters allow to differentiate RF(+) RA, RF(-) RA and UA, but do not differentiate UA from RF(-) RA. Marking CEP-1 in patients with early arthritis may help in differentiation between RF(-) RA and UA. Marking CEP-1 and a-Sa may be useful in diagnosing early arthritis patients. The presence of Anti-CarP antibodies in UA patients is probably considerable risk factor in RA development. Smoking cessation may contribute to decrease RA activity. Disclosure of Interest None declared

Background The anti-heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) antibody is specific for rheumatoid arthritis (RA). But, it is also reported that there is no significant difference between RA and systemic lupus erythematosus (SLE) in terms of the antibody. Objectives To investigate the distribution and clinical significance of anti-hnRNP-A2 antibody in connective tissue diseases. Methods Serum anti-hnRNP A2 antibody level was measured by solid-phase enzyme linked immunosorbent assay (ELISA) in 1464 patients with RA, 209 patients with SLE, 204 patients with ankylosing spondylitis (AS), 63 patients with mixed connective tissue disease (MCTD), 133 patients with undifferentiated connective tissue disease (UCTD), 60 patients with Sjogren syndrome (SS), 47 patients with polymyositis/dermatomyositis (PM/DM), and 45 patients with systemic sclerosis (SSc). The positivity rate of anti-hnRNP-A2 antibody was compared among various patient groups, and its correlation to clinical and laboratory parameters and its diagnostic significance were analysed. Results The positivity rate of anti-hnRNP-A2 antibody was 38.0% (556/1464), 36.8% (77/209), 3.9% (8/204), 52.4% (33/63), 17.3% (23/133), 5.0% (3/60), 4.3% (2/47), and 8.9% (4/45) in RA, SLE, AS, MCTD, UCTD, SS, PM/DM and SSc, respectively. The rate differed insignificantly between the RA, SLE and MCTD groups (p>0.05), but was significantly higher than in other disease groups (p 0.05). In RA patients, anti-hnRNP-A2 antibody weakly correlated negatively to anti-Cyclic citrullinated peptide (CCP) antibody (r=-0.135, p 0.05). Conclusions Anti-hnRNP-A2 antibody can be found in various connective tissue diseases, and its positivity rate is relatively high in RA, SLE and MCTD. It is not a RA-specific antibody. In RA, anti-hnRNP-A2 antibody does not coincide with other RA-related serological indicators; hence, it may serve as an adjunctive indicator for RA diagnosis. References [1] Smolen JS, Aletaha D, McInnes IB: Rheumatoid arthritis [J]. Lancet2016;388(10055):2023–2038. [2] Gavrila BI, Ciofu C, Stoica V. Biomarkers in Rheumatoid Arthritis, what is new? [J]. J Med Life2016;9(2):144–8. [3] Hassfeld W, Steiner G, Hartmunth K, et al. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis [J]. Arthritis Rheum1989,32:1515–1520. [4] 2004;4(4):288–290. [5] Tomoum HY, Mostafa GA, EI-Shahat EM, Autoantibody to heterogeneous nuclear ribonucleoprotein-A2(RA33) in juvenile idiopathic arthritis: clinical sig. Acknowledgements This work was financially supported by the Science and Technology Innovation Plan of Southwest Hospital (No. SWH2016JCZD-06) and the National Natural Science Foundation of China (No. 30973827). Disclosure of Interest None declared

Background Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most specific diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the IgG (ACPA) or IgM (RF) isotype. Other subtypes of both antibodies – such as IgA – and other autoantibodies like RA33 have been repeatedly reported but their diagnostic value has still not been fully elucidated. Objectives To investigate the diagnostic value of IgA, IgG and IgM subtypes of RF, ACPA and RA33 antibodies in patients with RA and their potential predictive value regarding therapeutic response to methotrexate (MTX). Methods To determine the diagnostic specificity and sensitivity, sera from 290 RA patients (including 165 MTX starter), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG and IgM isotypes of RF, ACPA and RA33 by EliATM (Thermo Fisher Scientific). Cut-offs for prototype anti-RA33 (IgA, IgG and IgM) and the IgM-ACPA EliATM were calculated by Receiver Operating Characteristic (ROC) curve analysis against disease controls and healthy subjects. In addition, RF and ACPA had been routinely measured by nephelometry and the anti-CCP EliATM, respectively. Results The most specific antibodies were IgG and IgA-ACPA as well as IgG-RF, closely followed by IgG- and IgA-RA33 while IgM isotypes were found to be less specific. However, IgM-RF was the most sensitive isotype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less prevalent ranging from 35% (IgA-ACPA) to 6% (IgA-RA33). Concerning RA33 antibodies, 14 patients were positive for IgA- and 18 for IgG-RA33. Interestingly, the major RA33 subtype was IgM which was detected in 43 patients. However, in contrast to RF and ACPA the overlap between the RA33 isotypes was marginal. RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were found also in 22% of seronegative patients. Moreover, analysing IgM-RF by EliATM proved more sensitive than RF measured by nephelometry which further reduced the number of seronegative patients. Thus, additional antibodies were detected in 30% of the seronegative population and, importantly, most patients had several antibodies, in contrast to disease controls which generally showed only one antibody species. The majority of antibody positive RA patients was found to be triple positive for RF, ACPA and either IgA-RF or IgA-ACPA. Among the 64 RA33 positive patients 48 were also positive for IgA-RF and/or IgA-ACPA (figure 1). Interestingly, we found high levels of IgM-RF (>124 IU/ml) to be associated with achieving a SDAI50 response to MTX (17 of 24 cases). Furthermore, also the presence of RA33 antibodies was associated with a MTX response as 50% of RA33 positive patients (n=32) achieved a SDAI50 response compared to 34% in the RA33 negative population (p=0.034). Conclusions Thus, increasing the number of antibodies in serological routine testing would provide valuable additional information allowing to better distinguish between RA and other rheumatic disorders also in patients negative in current routine diagnostics and may provide valuable additional information regarding the prediction of treatment responses. Disclosure of Interest D. Sieghart: None declared, A. Platzer: None declared, P. Studenic: None declared, F. Alasti: None declared, M. Grundhuber Employee of: Thermo Fisher Scientific, Phadia GmbH, S. Swiniarski Employee of: Thermo Fisher Scientific, Phadia GmbH, S. Blueml: None declared, H. Haslacher: None declared, J. Smolen: None declared, G. Steiner: None declared

Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most commonly used diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the immunoglobulin (Ig) M (RF) or IgG (ACPA) isotype. Other subtypes of both antibodies—particularly IgA isotypes and other autoantibodies—such as RA33 antibodies—have been repeatedly reported but their diagnostic value has still not been fully elucidated. Here, we investigated the prevalence of IgA, IgG, and IgM subtypes of RF, ACPA, and RA33 antibodies in patients with RA. To determine the diagnostic specificity and sensitivity sera from 290 RA patients (165 early and 125 established disease), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA, and RA33 by EliA™ platform (Phadia AB, Uppsala, Sweden). The most specific antibodies were IgG-ACPA, IgA-ACPA, and IgG-RF showing specificities >98%, closely followed by IgG- and IgA-RA33 while IgM subtypes were somewhat less specific, ranging from 95.8% (RA33) to 90% (RF). On the other hand, IgM-RF was the most sensitive subtype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less sensitive ranging from 35 (IgA-ACPA) to 6% (IgA-RA33). RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were detected also in seronegative patients reducing their number from 109 to 85. Moreover, analyzing IgM-RF by EliA™ proved more sensitive than measuring RF by nephelometry and further reduced the number of seronegative patients to 76 individuals. Importantly, among antibody positive individuals, RA patients were found having significantly more antibodies (≥3) than disease controls which generally showed one or two antibody species. Thus, increasing the number of autoantibodies in serological routine testing provides valuable additional information allowing to better distinguish between RA and other rheumatic disorders, also in patients not showing antibodies in current routine diagnostics. In conclusion, testing for multiple autoantibody specificities increases the diagnostic power of autoimmune diagnostics and could further support physicians in clinical decision-making.

Introduction Besides the determination of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA), anti-RA33 antibodies (which are directed to the nuclear antigen hnRNP-A2) could be of additional diagnostic and/or prognostic value in patients with rheumatoid arthritis (RA) because they are also found in RF/ACPA negative patients.1 Objectives So far, published data on anti-RA33 antibodies refer only to the IgG isotype.2 It was therefore the aim of this study to measure the prevalence of anti-RA33 IgG, IgM and IgA antibodies in patients with RA and to determine their potential prognostic value regarding prediction of response to treatment. Methods To determine the diagnostic sensitivity and specificity of anti-RA33 subtypes sera from 255 RA patients, 258 disease controls and 100 healthy subjects were tested by a prototype anti-RA33 EliA (Thermo Fisher Scientific) for the presence of anti-RA33 IgG, IgA and IgM antibodies. ACPA and RF were routinely measured by EliA and nephelometry, respectively. All RA patients had initially been treated with conventional synthetic drugs (mostly methotrexate, MTX) and were subsequently treated with at least one TNF inhibitor (TNFi). Therapeutic responses to MTX and TNF blocking biologicals were analysed in an inception cohort (n=104) who had started their DMARD therapy at our clinic. To define therapeutic responses the simplified disease activity index (SDAI) and American College of Rheumatology (ACR) responses were calculated. Results Diagnostic specificity was >96% for all 3 anti-RA33 subtypes. Among the 255 RA patients, 11% tested positive for anti-RA33 IgG antibodies, 15% for IgM antibodies and 6% for IgA antibodies. Altogether, 62 patients (24%) had at least one type of anti-RA33 antibody: among these, 24 patients were RF-negative, 26 were ACPA-negative and 18 were RF/ACPA double negative. Thus, in 32 patients (13%), anti-RA33 was the only antibody specificity. Regarding responses to MTX therapy, the percentage of SDAI50 or ACR20 responders, respectively, was significantly higher (p=0.034 for SDAI50 and p=0.005 for ACR20) among anti-RA33 positive patients (with or without RF/ACPA) compared to anti-RA33 negative (but RF/ACPA positive) patients and RF/ACPA negative patients. Thus, 60% of the anti-RA33 positive patients as compared to 37% anti-RA33 negative patients showed a SDAI50 response; similar values were seen for ACR20 responses. Conclusions Apart from their added diagnostic value anti-RA33 antibodies may have also prognostic value for prediction of therapeutic responses to MTX treatment. Therefore anti-RA33 antibodies may be taken into consideration as additional markers that might become helpful tools in therapeutic decision making. References . Nell VPK, Machold KM, Stamm TA, Eberl G, Heinzl H, Uffmann M, Smolen JS, Steiner G. Autoantibody profiling as early diagnostic and prognostic tool for rheumatoid arthritis. Ann Rheum Dis2005;64:1731–6. . Yand X, Wang M, Zhang X, et al. Diagnostic accuracy of anti-RA33 antibody for rheumatoid arthritis: Systematic review and meta-analysis. Clin Exp Rheumatol2016;34(3):539–47. Disclosure of interest D. Sieghart: None declared, P. Studenic: None declared, M. Grundhuber Employee of: Thermo Fisher Scientific-Phadia GmbH, S. Swiniarski Employee of: Thermo Fisher Scientific-Phadia GmbH, A. Platzer: None declared, J. Smolen: None declared, G. Steiner: None declared

Background Joint involvement in SLE is very common, affecting 90% of patients at some stage in the course of their disease (1).Arthritis featuring prominent radiological erosion in SLE is less common; however, in a small subset of patients an erosive pattern similar to RA develops (2). Anti- heterogenous nuclear ribonucleoprotein A2 (anti-hnRNP-A2) occur in about one-third of patients with RA but rarely in other arthritides such as OA, PsA or reactive arthritis. Interestingly, in SLE patients anti-hnRNP-A2 autoantibodies were found to be significantly associated with erosive arthritis (3). Objectives To investigate joint involvement in SLE and its relationship with autoantibodies to the hnRNP Ab A1 and A2, rheumatoid factor (R.F), Antinuclear antibody (A.N.A) and Anti-double stranded DNA (Anti-DSDNA) and correlation with articular involvement by joint x-ray. Methods Case series study comparing diagnosis of arthritis by hand and wrist x-ray with anti-hnRNP A1 and A2 in Fourty SLE patients aged 17–60 years old with disease duration 1–17 years complaining of arthralgia or arthritis. A controlled group of 21 clinically normal persons, age and sex matched and blood samples are drawn and centrifuged. RF and auto-antibodies to nuclear antigens anti-nuclear antibodies (ANA) and anti-double stranded DNA were determined in all the patients. All patients underwent X-rays of the hands and wrists. Results Anti-hnRNP A1 showed highly significant difference between study and control. Anti-hnRNP A2 showed significant difference between study and control. Conclusions This study showed a high frequency of erosive arthropathy and autoantibody to both hnRNP antigens might become useful marker for joint involvement in SLE patients and identify SLE patients prone to develop joint damage References Pipili C, Sfritzeri A, Cholongitas E. Deforming arthropathy in systemic lupus erythematosus. Eur J Intern Med 2008;19(7):482–7. Pipili C, Sfritzeri A, Cholongitas E. Deforming arthropathy in SLE: review in the literature apropos of one case. Rheumatol Int 2009;29(10):1219–21. Hassfeld W, Steiner G, Graninger W, Witzmann G, Schweitzer H, Smolen JS. Autoantibody to the nuclear antigen RA33 – a marker for early rheumatoid arthritis.Br J Rheumatol 1993;86:19–203. Disclosure of Interest None declared

Objectives The aims of this study were to investigate the extent of MRI-determined joint disease (erosion and synovitis) in SLE and to link this to autoantibody profiles known to be relevant to SLE, including ACPA, RF and anti-RA33 antibodies. Methods Contrast-enhanced MRI of the hand and wrist was performed in 34 symptomatic SLE patients and in 15 RA patients with similar disease duration. Images were scored by two observers using the OMERACT rheumatoid arthritis MRI scoring (RAMRIS) system. Findings were correlated with clinical examination and autoantibody status. Results Erosions were present at the wrist in 93% of SLE patients and at the MCP joints in 61% of SLE patients. Despite the high prevalence of MRI-determined erosion, only 8.8% of SLE patients were ACPA positive, although these patients had a higher burden of erosive disease. There was no positive correlation with anti-RA33 titres and erosion scores in the SLE patients, but there was a negative correlation with anti-RA33 titres and total bone oedema scores in the SLE patients. Ninety-three per cent of SLE patients had at least grade 1 synovitis at one or more MCP joints, and wrist joint synovitis was present in all the SLE patients. Conclusion An MRI-determined joint erosive phenotype is common in SLE, even in ACPA-negative cases. The conventional radiographic observation that anti-RA33 is not positively associated with erosion in patients with RA was also found to be the case in SLE patients.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and damage of the joints affecting about 0.5% of the general population. Early treatment in RA is important as it can prevent disease progression and irreversible damage of the joints. Despite the high diagnostic value of anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF), there is a strong demand for novel serological biomarkers to further improve the diagnosis of this abundant disease. During the last decades, several autoantigens have been described in RA including Ra33 (hnRNP A2), fibrinogen, fibronectin, alpha-enolase, type II collagen, immunoglobulin binding protein (BiP), annexins and viral citrullinated peptide (VCP) derived from Epstein Barr Virus-encoded protein (EBNA-2). More recent discoveries include antibodies to carbamylated antigens (anti-CarP), to peptidyl arginine deiminase type 4 (PAD4), to BRAF (v raf murine sarcoma viral oncogene homologue B1) and to 14 autoantigens identified by phage display technology. This review provides a current overview of novel biomarkers for RA and discusses their future potential to improve the diagnosis of the disease.

This study evaluated the diagnostic accuracy of anti-Sa and anti-RA33 antibodies in rheumatoid arthritis (RA).PubMed, Embase, and Cochrane library databases were searched for relevant studies, and 2 meta-analyses were performed to determine the diagnostic accuracy of anti-Sa and anti-RA33 antibodies in patients with RA.The meta-analysis included 17 studies. Pooled sensitivity and specificity of anti-Sa antibody were 39.5 % (95 % confidence interval [CI] 36.5-42.4) and 96.8 % (95 % CI 95.9-97.4), respectively, and those of anti-RA33 antibody were 31.8 % (95 % CI 28.7-35.0) and 90.1 % (95 % CI 87.8-92.1), respectively. PLR, NLR, and DOR of anti-Sa antibody were 14.11 (95 % CI 7.076-28.13), 0.607 (95 % CI 0.558-0.703), and 22.76 (95 % C, 11.10-46.69), respectively, and those of anti-RA33 antibody were 3.429 (95 % CI 2.039-5.765), 0.761 (95 % CI 0.681-0.851), and 4.597 (95 % CI 2.602-8.121), respectively. AUC and QOur meta-analysis indicated that both anti-Sa and anti-RA33 antibodies were highly specific but not sensitive for diagnosing RA.

Rheumatoid arthritis (RA) is the most common connective tissue disease of autoimmune origin. The disease is characterized by chronic inflammation leading to bone erosions and organ involvement. RA is a progressive disease. It affects the quality of life, leading to disability and death mainly due to premature cardiovascular disease. Early diagnosis and appropriate treatment are essential for prognosis and quality of life improvement. In 2010 the American College of Rheumatology (ACR) and The European League Against Rheumatism (EULAR) established new RA classification criteria. Besides clinical symptoms it includes two immunologic criteria: rheumatoid factor (RF) and anti-citrullinated protein antibodies (anti-CCP antibodies). RF is the first well-known RA immunologic marker. It is observed in 80-85% of patients with RA. Elevated serum level of RF has been associated with increased disease activity, radiographic progression, and the presence of extraarticular manifestations. The sensitivity of RF is 50-90%, and specificity is 50-95%. Anti-CCP antibodies appear to be a more specific marker than RF. They are often present at the very beginning of the disease, or even years before the first symptoms. The prognostic value of anti-CCP antibodies is well established. High serum level of anti-CCP correlates with poor prognosis and early erosions of the joints. The sensitivity of anti-CCP2 is 48-80%, and specificity is 96-98%. New immunologic markers include anti-carbamylated protein antibodies (anti-CarP) and antibodies against heterogeneous nuclear ribonucleoproteins (anti-hnRNP A2/B1, RA33). Scientists aim to identify a highly sensitive and specific biomarker of the disease that not only has diagnostic and prognostic value but also may predict the response to treatment.

Contains fulltext : 153179.pdf (Publisher’s version ) (Closed access) OBJECTIVE: The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up-regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA. METHODS: Collagen-induced arthritis (CIA) and the K/BxN serum-transfer model were used as animal models of RA. Efficient silencing of hnRNP A2/B1 was achieved using a liposome-based carrier system for delivery of small interfering RNAs. Expression of hnRNP A2/B1 was analyzed by flow cytometry, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Cytokine levels and anticollagen antibody levels were measured by enzyme-linked immunosorbent assay. RESULTS: Efficient silencing of hnRNP A2/B1 was achieved in all lymphoid organs. In both experimental models, the incidence and severity of arthritis were largely reduced and bone erosion was not detectable as compared to the control groups. Down-modulation of hnRNP A2/B1 significantly interfered with the production of proinflammatory cytokines from monocyte/macrophages, but not from T cells. Consistent with these findings, production of T cell cytokines was not impaired when cells were restimulated in vitro with type II collagen. Furthermore, levels of anticollagen antibodies were not affected by hnRNP A2/B1 silencing. CONCLUSION: Our findings suggest that hnRNP A2/B1 has an important role in regulation of the innate immune system, especially at the level of monocyte/macrophage activation. Therefore, down-modulation of hnRNP A2/B1 seems to affect primarily the effector phase of autoimmune arthritis.

The primary objective of this study was to evaluate and compare the immunodiagnostic significance and utility of anti-RA33 with anti-CCP, RF, and CRP in Saudi patients with rheumatoid arthritis.Methods. This was a prospective controlled clinical study conducted at King Abdul Aziz University Tertiary Medical Centre. The sera of 41 RA patients, 31 non-RA patients, and 29 healthy controls were collected. Anti-RA33 and anti-CCP were measured using commercially available ELISA principle kits. RF and CRP were measured using nephelometry.Results. Anti-RA33 antibodies had the lowest positive and negative predictive values and showed a sensitivity of 7.32% with 95.12% specificity. Of the other three markers (including anti-CCP antibodies, CRP, and RF), only anti-CCP showed specificity of 90.46% with sensitivity of 63.41% compared to non-RA patients + healthy control. There was a significant correlation with rheumatoid factor positivity with anti-CCP. With respect to CRP, a notable correlation was seen only with anti-RA33.Conclusion. Compared to rheumatoid factor, anti-CCP antibodies, and C-reactive proteins, the anti-RA33 autoantibodies seem to be not representing as an important additional immunodiagnostic marker in Saudi patients with established RA. RA33 may have more interest in early RA or less severe RA and other systemic connective tissue disorders.

Human TNF-α transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-α leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-γ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-γ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

Aim: Accurate diagnosis and early treatment in rheumatoid arthritis (RA) can lead to a good outcome and a correct management of the disease. We aimed toinvestigate the prognostic value of anti-RA33 antibodies, by evaluating the relationship with ultrasonographic (US) findings in patients with early RA.Material and methods: We performed a prospective study which included 29 patients, diagnosed with early RA according to the ACR/EULAR 2010 criteria and 21 sex and age-matched control subjects. All patients underwent clinical and biological assessment, followed by US examination in grayscale (GS) and power Doppler (PD) at baseline and after 12 months [from the second to the fifth metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints and wrists (RCC), in dorsal aspect]. The second and fifth MCP joints were scanned also in lateral aspects.Results: The initial GS evaluation revealed the presence of synovitis in all 29 patients; PD found at least one joint with a PD grade higher than 1 in 23 patients, higher than 2 in 20 patients, and grade 3 in 6 patients; at 12 months, we revealed the presence of GSUS synovitis in 25 patients and PDUS examination found active synovitis in 12 subjects. In those patients, the anti-RA33 titre was significantly lower compared to those without PDUS active synovitis (p=0.031), with a moderately negative correlation (r=-0.519, p=0.0039).Conclusions: The current study shows that anti-RA33 antibodies might constitute an additional tool for diagnosing early RA patients and can help identify patients with mild disease and a low level of activesynovitis.

Background: Rheumatoid arthritis is a chronic inflammatory disease with uncertain etiology. It is characterized by symmetric polyarthritis in peripheral joints. Its diagnosis is based on clinical findings and serologic tests. However, its diagnosis is rarely conclusive in early course of the disease. So, its early diagnosis could be difficult. The present study was designed to evaluate the role of anti -RA33; an auto-antibody against RA33 in early diagnosis of the disease. Materials and Methods: forty three patients with RA who had been visited in a rheumatology clinic were randomly selected. Their disease has been diagnosed by a rheumatologist. They served as the case group. 55 persons were also chosen from healthy individuals who had attended in other clinic. They served as control. Their age and sex were matched with the case group. Anti-RA33 and RF titers were measured in their blood sample using standard methods. Findings: RF and anti-RA33 titers had significant correlation in the case group (p=0.015). Anti -RA33 test had 98% sensitivity, 20% specificity, 50% positive predictive value, and 90% negative predictive value. Conclusion: Anti -RA33 could have diagnostic and prognostic importance in diagnosis and evaluation of patients with RA, and its differentiation from other small joint disorders, particularly when the other serologic tests are negative.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are potent autoantigenic targets in systemic autoimmune rheumatic diseases (SARD). Loss of tolerance to the RA33 complex consisting of hnRNP A2 and its alternatively spliced variants B1 and B2 has been the interest of rheumatologists. A novel ELISA for the detection of anti-hnRNP B1 autoantibodies has been developed to investigate the prevalence thereof in 397 patients with SARD, including patients with rheumatoid arthritis (RA), spondyloarthropathy (SPA), juvenile chronic arthritis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren’s syndrome (SS), in comparison to 174 controls. Anti-hnRNP B1 autoantibodies were significantly more prevalent in patients with SARD than controls (47/397, 11.8% versus 2/174, 1.1%;P<0.001). In particular, anti-hnRNP B1 were found more frequently in the disease cohorts than in the controls and were present in 24/165 (14.5%) patients with RA, 6/58 (10.3%) SPA, 11/65 (16.9%) SSc, and 4/50 (8.0%) SLE. In RA patients, anti-hnRNP B1 autoantibodies correlated significantly with C-reactive protein levels and erythrocyte sedimentation rate, while in patients with SSc it was associated with features of arterial wall stiffness and presence of hypertension. Anti-hnRNP B1 autoantibodies occur in SARD and seem to be correlated with distinct clinical characteristics in patients with RA and SSc.

The clinical presentation of rheumatoid arthritis (RA) poses intriguing challenges to the elucidation of underlying pathogenesis. Unifying hypotheses must explain onset and perpetuation of inflammation, angiogenesis, and hyperplasia within the synovial membrane; articular destruction in cartilage and bone compartments; and the propensity in subsets of patients to manifest extra-articular involvement, variable prognosis, functional decline, and mortality. This broad clinical heterogeneity could alternatively instruct the investigator that several disease processes must coexist within the clinical cohort defined by the American College of Rheumatology classification criteria. 3 During the last decade, extraordinary advances have been made in exploring the pathogenetic events ongoing in RA patients, culminating in the development and clinical application of specific cytokine- and cell-targeting agents with variable success. In parallel, radical alterations have occurred in the use of "traditional" disease-modifying antirheumatic drugs such that most patients are now receiving combinations of drugs according to variable regimens. 50 Such combinations have not, however, always been derived from proven synergistic modes of action. This short review explores the hypothesis that translational clinical science involving clinicians and basic scientists with common interests in RA are essential to maintain further progress. A significant problem in the investigation of the pathogenesis of human autoimmune diseases has been the absence of reliable measures of immune functional activity, reflecting, by analogy, the absence of an "immunologic stethoscope." For many decades, RA has been associated with autoantibody production, particularly through association with rheumatoid factor expression. Autoimmune humoral responses may be identified through the detection of autoantibodies in serum; as such, they can be related to clinical cohorts with relative ease. Several novel autoantibody specificities, including citrullinated peptides, Sa, RA33, and (pro)filaggrin, have been identified in RA patients. Some antibodies (e.g., anticitrullinated peptides and anti-Sa) are present in early disease and carry high specificity for RA, perhaps identifying clinically distinct subgroups. 21,57 Sensitivities remain low, however, for many of these antibodies, and the presence of multiple specificities in individual patients is generally not the rule. Much less is known as yet of the precise cellular immune responses that contribute to RA pathogenesis. Investigation of cell-mediated immunity poses significant logistic difficulties. Peripheral blood–derived leukocytes have traditionally been employed but likely represent less than 2% of total immune active cells. Critical immune-regulatory activities almost certainly occur in lymph nodes and target tissues, which may be difficult to access. The fact that host tissues may also directly contribute to the development of immune responses, through activities of "nonprofessional immune" cells (e.g., activated fibroblasts, epithelial cells) has increasingly become evident and further emphasizes the importance of evaluating responses in situ. The absolute phenotype, tissue origin, and kinetics of developing autoimmune responses have proven elusive thus far. Formerly, RA studies relied almost exclusively on examination of synovial fluid or synovial tissues obtained at arthroplasty for end-stage disease or by closed-needle biopsies. The advent of needle arthroscopy/direct vision synovial biopsy is now contributing significant advances to our understanding of the architecture of the synovial membrane at the macro and micro levels through distinct disease phases. 71 A potential integrated pathway leading from basic to applied clinical science is shown in Figure 1. Well-characterized clinical cohorts are a prerequisite from which representative synovial and peripheral blood tissues can be obtained. The existence and functional significance of basic pathways of interest can then be tested in vitro and in vivo using animal model systems as appropriate. Recent advances at each stage of this pathway are reviewed.

Amaç: RA tanısının erken konulması, tedavi ile eklem dokusundaki hasarın önüne geçilebilmesi açısından çok önemlidir. Laboratuar testleri, RA aktivitesinin izlenmesinde ve tedaviye yanıtın değerlendirilmesinde oldukça önemlidir. RA şüphesi olan vakalarda bugüne kadar en yaygın kullanılan test romatoid faktördür, RA'ya duyarlı olmasına rağmen özgül olmayan bir parametredir. Şüpheli vakalarda ya da düşük RF sevyeli vakalarda otoantikor ölçümü tanı koymada yardımcı olabilir. Anti RA-33 otoantikor ölçümü RA'lı hastalarda kullanılan otoantikor testlerinden biridir. Bu çalışmanın amacı sağlıklı bireyler ile RA'lı bireylerin Anti-RA 33 seviyesini karşılaştırmak ve Anti-RA 33 seviyesinin RA tanısında önemini değerlendirmektir. Yöntem: American College of Rheumatology (ACR) RA klasifikasyon kriterlerine göre belirlenmiş hastalık süresi en az iki yıl olan 28 i bayan,12 si erkek toplam 40 RA'li hasta ile aynı sayıda kontrol grubu sağlıklı kişiler çalışmaya alındı. Bulgular: RA'li hasta grubunda AntiRA-33 düzeyi 12,47±49,76 U/ml, sağlıklı kontrol grubunda AntiRA-33 düzeyi 1,96±1.82 U/ml olup gruplar arası istatiksel olarak bir fark bulunmadı. Sonuç: RA'lı hastalarda AntiRA-33 düzeyi ile sağlıklı kontrol grubu arasında anlamlı bir fark bulunamadı., Objective: The early diagnosis of Rheumatoid Arthritis (RA) is of due significance for the prevention of possible damage to the joint tissue by means of treatment. Laboratory tests are very important for monitoring RA activity and for the evaluation of the patients' response to the treatment. Rheumatoid factor, so far the most commonly used test for the cases with suspected RA, is not a specific parameter despite its sensitivity to RA. The measurement of other autoantibodies might be helpful to the diagnosis particularly for the cases with low RF titer or with suspected diagnosis. The measurement of anti-RA33 autoantibodies is one of the tests used in the diagnosis of the RA cases. The present study was conducted to compare anti-RA33 levels of healthy individuals with long-term RA cases and to evaluate the value of anti-RA33 test for the RA diagnosis. Method: Total 40 patients (28 female and 12 male) identified with RA at least for the last five years according to the classification criteria of American College of Rheumatology (ACR) reviewed in 1987 and 40 healthy controls included in the present study. Results: AntiRA-33 level was determined as 12.47±4.97 U/mL for the patients with RA and as 1.96±1.82 U/mL for the healthy controls, and there is no statistically significant difference between the groups. Conclusions: As a result AntiRA-33 level was evaluated in RA patients and no difference was found compared to control group.

To assess the sensitivity and specificity of the recently described anti-Sa autoantibodies in order to determine their potential usefulness for the diagnosis of rheumatoid arthritis (RA).Sera from 67 patients with RA (including 31 patients with early RA of3 months duration), from 180 patients with other rheumatic diseases and from 30 healthy control subjects were investigated by immunoblotting employing partially purified Sa antigen. Additionally, all sera were tested for rheumatoid factor (RF), anti-A2/RA33, antinuclear antibodies (ANA) and ANA subsets.Twenty-one (31%) of the 67 patients with RA, but only four of the 180 control patients, were anti-Sa positive; therefore, anti-Sa was approximately 98% specific for RA. Anti-Sa was not associated with either RF or with anti-A2/RA33. The latter antibody was present in 21 RA sera, only eight of which also contained anti-Sa. Thus, 34 RA sera (51%) showed at least one of these two autoantibodies and, importantly, 18 of these sera were RF negative. Furthermore, of the 31 patients with early RA, 12 (40%) were anti-Sa and/or anti-A2/RA33 positive.Although the numbers studied remain small, taken together, these data suggest that anti-Sa may represent a promising novel serological marker with high specificity for RA.

Objective The characteristics of 3 patients with longstanding rheumatoid arthritis (RA) and consecutive evolution of limited cutaneous systemic sclerosis (lcSSc) were evaluated and compared with those of patients with lcSSc alone (n = 20) or with RA alone (n = 120). Methods Clinical features of the different patient populations were compared. Serologic analyses included tests for antinuclear antibodies (ANA) and ANA subsets, in particular anticentromere antibodies (ACA) and anti-heterogeneous nuclear RNP (hnRNP)-A2/RA33 (anti-A2/RA33). Results The 3 patients with RA developed lcSSc 11, 29, or 50 years after the onset of RA. Features of lcSSc were Raynaud's phenomenon, sclerodactyly, and telangiactasias in all 3 patients, and esophageal dysmotility in 1 patient. Rheumatoid factor (RF) and anti-A2/RA33 were each found in 2 patients, and 1 of these patients was seropositive for both RF and anti-A2/RA33. ACA titers were positive in all cases. However, similar to the development of RA prior to lcSSc, the occurrence of autoantibodies typical of RA preceded the occurrence of ACA, at least in 2 of the patients. Using affinity-purified antibodies, cross-reactivities between anti-centromere protein A (CENP-A) and anti-CENP-B antibodies with anti-A2/RA33 antigens were seen in the 2 anti-A2/RA33-positive patients. Such cross-reactivities were not found in lcSSc patients without concomitant RA. Epitope mapping revealed that both autoantibody specificities recognized the known major epitopes: anti-CENP-B reacted with the C-terminal region and anti-A2/RA33 with the second RNA binding domain in the N-terminal region of hnRNP-A2. Conclusion The RA-lcSSc overlap syndrome in these 3 patients with longstanding RA was characterized by an incomplete CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome. The study demonstrated the presence of autoantibodies typical of both diseases and cross-reactivity of ACA with hnRNP-A2/RA33 in the sera of these patients.

Cationic germ line gene-encoded anti-DNA antibodies appear to cause inflammatory lesions via deposition and in situ formation of immune complexes, but also perhaps through apoptosis of glomerular mesangial cells. Somatic mutation is not critical for the expression of the electrical net charge of anti-DNA. In one transgenic mouse model, the nephrogenicity of anti-DNA was dependent on the expression of interleukin alpha by epidermal cells. Anti-P autoantibodies are present in the serum of healthy children but are masked by IgG antibodies. Some anti-P antibodies appear to be a subset of antilymphocyte autoantibodies. It was confirmed that anti-Ro/SS-A antibodies react with structurally unrelated conformational epitopes. The combined results of immunofluorescence and enzyme-linked immunosorbent assay in the detection of antineutrophil cytoplasmic antibody has a 99.5% specificity for vasculitis among patients with connective tissue diseases. Antinucleosome antibodies are highly prevalent in patients with systemic lupus erythematosus and show an inverse correlation with nucleosome plasma levels. Transfected T-cell receptor alpha chains specific for nucleosomal autoepitopes from a Th clone that accelerates lupus nephritis, recognized the nucleosomal epitopes presented by antigen presenting cell-bearing 1-A molecules, as well as human DR molecules, via the classical major histocompatibility complex class II groove. IgG antibodies against (beta 2 -glycoprotein (GP)-1 are significantly associated with thrombosis in patients with antiphospholipid syndrome, with a specificity and sensitivity that ranges from 85% to 98% and 32% to 54%, respectively. The lupus anticoagulant activity, or the lack of it, of anti-beta 2-glycoprotein-1 appears to reside on the epitope specificity. Beta 2-glycoprotein-1 binds to apoptotic cells; in turn, anti-beta 2-glycoprotein-1 facilitate apoptotic cell clearance by macrophages. The recently described anti-A2/RA33 autoantibody appears to recognize an epitope capable of distinguishing patients with lupus or with rheumatoid arthritis from those with mixed connective tissue disease.

Despite commonly applied clinical criteria, the early diagnosis of rheumatoid arthritis (RA) often remains difficult, thus delaying on suitable early treatment. In search for a test furthering the early and reliable diagnosis of RA, we have screened for novel disease specific autoantibodies. To this end proteins were isolated from synovial membranes and other tissues following a special protein purification protocol, and these were separated electrophoretically. Western blots were then used to screen sera of RA patients and of individuals suffering from other rheumatic diseases for antibodies to any of these proteins. The most prominent RA specific immunoreaction was with a 68k antigen, occurring in 110 of 167 RA patients (sensitivity is 66%). The antibody could also be identified in seronegative RA patients but not in healthy individuals (55 tested), in only 1 SLE patient of a group of 98 patients with other rheumatic diseases and in 1 out of 22 HIV patients, resulting in a specificity of 99%. Moreover, the anti-68k antibody could be correlated with a more severe course of RA. 13 out of 20 anti-68k positive RA patients (58%) had subcutaneous nodules, while only 2 out of 11 anti-68k negative (20%) did. The mean sedimentation rate of these antibody positive patients was 51 mm/h and 26 mm/h for the negative respectively. The 68k antigen was shown to be present in all human tissues investigated and is probably ubiquitously expressed. It is either located in the endoplasmatic reticulum or cytoplasm or both. Its isoelectric point is 5.1. It proved to be O-glycosylated and contains only one or a few sugar residues as the untreated and the deglycosylated antigen identical electrophoretical mobilities. The patient derived anti-68k antibodies were directed against the sugar residue: deglycosylation of the antigen completely abolished its immunoreactivity. N-acetylglucosamine competes with the antibody for binding the 68k antigen. The antigen physicochemical data of the 68k antigen argue against identity with one of the autoantigens in this molecular mass range already known to be associated with RA or other autoimmune diseases. It is neither identical to the 62k human antigen (EBNA-1) nor to RA33 (A2hnRNP), the 50k Sa antigen or the Hsp70 class of heats-hock proteins. It is argued that the particular method of protein purification applied in combination with separation via SDS-PAGE in the presence of urea, made it possible to detect a hitherto unidentified antigen. Considering the striking disease specificity of the anti-68k antibody it is now worthwhile to look for corresponding autoreactive T cells in order to analyse its role in the pathogenesis of RA.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder, characterized by progressive joint destruction and disability. Classical autoantibodies of RA are rheumatoid factors and citrulline antibodies. Patients positive for these autoantibodies are usually associated with a progressive disease course. A subgroup of RA patients does not express citrulline antibodies, instead are approximately 35% of these anti-citrulline-negative patients reported to express autoantibodies to the heterogeneous nucleoriboprotein A2, a ribonucleoprotein involved in RNA transport and processing also referred to as RA33. In the absence of citrulline antibodies, RA33 antibodies have been suggested to be associated with a milder disease course. In this study we screened the reactivity of a monoclonal antibody to RA33-derived peptides by modified enzyme-linked immunosorbent assays (ELISA). Terminally truncated resin-bound peptides were applied for determination of the functional epitope necessary for antibody recognition. In addition, screening of substituted peptides by modified ELISA identified amino acids necessary for antibody reactivity. A potential epitope was identified in the region 71-79 (PHSIDGRVV), where the amino acids Ser, Ile and Asp were found to be essential for antibody reactivity. These amino acids were found to contribute to the antibody-antigen interface through side-chain interactions, possibly in combination with a positively charged amino acid in position 77. Moreover, the amino acids in the N-terminal end (Pro and His) were found to contribute to the interface through backbone contributions. No notable reactivity was found with RA-positive patient sera, thus screening of RA33 antibodies does not seem to be a supplementary for the diagnosis of RA.

Objective. To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. Methods. Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of crossreactivities, autoantibodies were affinity purified by blot elution. Results. Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 doublepositive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. Conclusion. Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.

Background Rheumatoid Arthritis (RA) is an inflammatory autoimmune disease characterized by joint involvement, pain and inflammation with or without extra-articular joint commitment. Serological markers can guide early diagnosis before that clinical activity takes place, optimizing early diagnosis, less activity and functional disability. RA 33 is considered a diagnosis-marker in RA patients related to prognosis and therapeutic response compared with conventional markers. RA 33 is common in patients with negative conventional markers and with few clinical symptoms. Objectives Evaluated the frecuency of RA33 in individuals with first-degree relatives of patients with RA and patients diagnosed with early rheumatoid arthritis as a good prognosis marker Methods A transversal study was conducted evaluated rheumatologic and periodontal condition of 112 individuals with first-degree relatives of patients with RA and 46 patients diagnosed with early rheumatoid arthritis, according to classification criteria for RA (ACR-EULAR 2010). In addition, 47 healthy individuals were included. RA patients and control group were matched for age and gender. Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor RF, ACPAs, and RA 33 were performed. VAS Pain Scale, SDAI Index, HAQ and DAS28 were evaluated. Porphyromona gingivalis (Pg) by CRP, IgG1, and IgG2 were determined. The data included in the study were analyzed using STATA 11.1. Associations were determined by the statistical test Chi 2, and Comparisons between groups of rheumatologic diagnosis and median values for continuous variables was performed using Mann-Whitney Test. Approved by the Ethics Committee of the Institution. Results 112 healthy individuals with first-degree relatives of patients with RA were assessed. Of them 78.3% has periodontal disease, presence of painful joints (30.63%). 36.94% patients had Pg in subgingival plaque samples. In the control group the presence of periodontal disease was 71.74%, tender joints (19.57%), swollen joints (6.52%). Disease activity measures were not representative. Only a woman in the group of family related RA patients was positive for RA 33 (49 years old, without treatment, painful and swollen joints and the presence of periodontal disease and Pg , ESR and CRP positives but RF and ACPAs negative). No patient with RA of less than two years of diagnosis was positive for RA 33. Conclusions The diagnostic value of anti RA33 was low in this population, with regard of worst prognosis markers and higher disease severity. Ra33 should be more follow-up clinical and paraclinical to subjects with risk factors who could have positive markers, without symptoms. References Fritsch R, Eselbock D.Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis. The Journal of Immunology.2002; 169 (2):1068–1076. Massardo L.Artritis reumatoide temprana early rheumatoid arthritis.Rev med Chile. 2008; 136: 1468–1475 Disclosure of Interest None declared

Background Anti-Sa antibody was identified in 1994 and belongs to a class of antibodies that target citrullinated antigens. Anti-Sa antibody is highly specific for diagnosing rheumatoid arthritis (RA, but it is associated with low prevalence and sensitivity. Anti-RA33 antibody was identified in 1989 and targets RA33 complex or heterogeneous nuclear ribonucleic protein. In contrast to anti-Sa antibody, anti-RA33 antibody does not target citrullinated antigens. Both anti-Sa and anti-RA33 antibodies are considered as specific serological markers for RA. However, studies assessing the diagnostic value of these antibodies in RA have provided inconsistent results. Objectives The aim of this study is to evaluate the diagnostic performance of anti-Sa and anti-RA33 antibodies in RA. Methods PubMed, Embase, and Cochrane library databases were searched for relevant studies and 2 meta-analyses were performed to determine the diagnostic accuracy of anti-Sa and anti-RA33 antibodies in patients with RA. Results The meta-analysis included 17 studies. Of these, 9 studies, which included 1,095 patients and 1,667 controls, were on anti-Sa antibody and 8 studies, which included 905 patients and 835 controls, were on anti-RA33 antibody. Pooled sensitivity and specificity of anti-Sa antibody were 39.5% (95% confidence interval [CI], 36.5–42.4) and 96.8% (95% CI, 95.9–97.4), respectively, and those of anti-RA33 antibody were 31.8% (95% CI, 28.7–35.0) and 90.1% (95% CI, 87.8–92.1), respectively. PLR, NLR, and DOR of anti-Sa antibody were 14.11 (95% CI, 7.076–28.13), 0.607 (95% CI, 0.558–0.703, and 22.76 (95% CI, 11.10–46.69), respectively, and those of anti-RA33 antibody were 3.429 (95% CI, 2.039–5.765), 0.761 (95% CI, 0.681–0.851), and 4.597 (95% CI, 2.602–8.121), respectively. AUC and Q * index for anti-Sa antibody were 0.558 and 0.543, respectively, while those for anti-RA33 antibody were 0.501 and 0.500, respectively. Conclusions Our meta-analysis indicated that both anti-Sa and anti-RA33 antibodies were highly specific and the PLR of anti-Sa antibody was very useful for diagnosing RA, although they were not sensitive for diagnosing RA. Disclosure of Interest None declared

Background Accurate and early diagnosis of rheumatoid arthritis (RA) is vital for the timely implementation of a comprehensive treatment aimed at halting or slowing its progression (1). The sensitivity of existing biomarkers, rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP) is low (2). It requires a new diagnostic biomarker with better performance in RA. Objectives To evaluate the diagnostic value of anti-RA33 (3) test in a simple of RA patientes and controls of Bogota, Colombia, by measuring its sensitivity, specificity, positive and negative likehood ratio compared to RF and anti-CCP. Additionally, to asses the association between clinical and serological variables with the functional impairment degree in patients with RA. Methods A cross-sectional study in a sample of 80 RA patients and 20 controls without RA who attended to outpatient service in two hospitals of Bogota from 2014 until 2015 was conducted by measuring their sociodemographic characteristics, arthritis activity degree by DAS-28 index, functional ability by HAQ-DI score and serological status for anti-RA33, RF and anti-CCP. A statistic analysis with proportions, summary and dispersion measures, contingency tables for measure of sensitivity, specificity, likehood ratios, ROC curves and multiple linear regression analysis was performed. Results Sensitivity score for anti-RA33, RF and anti-CCP was 15%, 85% and 93% respectively; while specificity was 100%, 90% and 85% respectively. The positive likelihood ratio was 8.5 for RF and 6.2 for anti-CCP. Finally, negative likelihood ratio was 0.87 for anti-RA33, 0.16 for RF and 0.08 for anti-CCP. The anti-RA33 seropositive patients were greater arthritis activity and functional disability as well as higher titers of RF and anti-CCP although these differences were not statistically significant. Finally, a moderately strong positive correlation was found between DAS-28 and HAQ-DI scores (R 2 =0.51). In the multiple linear regression model, DAS-28 index was the only predictor variable that explained the changes in the HAQ-DI functional ability score. Conclusions Findings suggest that anti-RA33 test does not seem to have any clinical utility for the diagnosis of RA in Colombian population. The importance of DAS-28 activity index as a score of association with functional disability in patients with RA was confirmed. References Torpy JM, Perazza GD, Golub RM. Rheumatoid Arthritis. JAMA 2011;305(17):1824. Chang PY, Yang CT, Cheng CH, Yu KH. Diagnostic performance of anti-cyclic citrullinated peptide and rheumatoid factor in patients with rheumatoid arthritis. Int J Rheum Dis. 2015 May 4. Hassfeld W, Steiner G, Hartmuth K, Kolarz G, Scherak O, Graninger W, Thumb N, Smolen JS. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis Rheum. 1989 Dec;32(12):1515–20 Disclosure of Interest None declared

Sera from 47 patients with early (3 months) arthritis of any type were investigated for anti-RA33, a new anti-nuclear autoantibody characteristic of RA, and the diagnoses determined within the following 8-14 months. In addition, seven patients with unclassified arthritis of4 months duration, who were all anti-RA33 positive, were followed for up to 2 years to establish their final rheumatologic diagnoses. Four of 47 early arthritis patients' sera were anti-RA33 positive at the initial evaluation; 14 of these 47 patients (30%) could be classified as RA (according to established criteria) at the final evaluation. All four anti-RA33 positive patients belonged to the RA group (27% of RA patients); of the 33 non-RA patients none had anti-RA33 (P = 0.005). Rheumatoid factor was found in four RA (none of whom had anti-RA33), but also in two non-RA patients (P = 0.05). Finally, the study involved seven additional patients with longer standing, initially unclassified, anti-RA33 positive arthritis: in all of them a diagnosis of RA could be established within 3 years of disease onset. These results suggest that anti-RA33 helps to discriminate early RA from other forms of early arthritis and, in the absence of an established diagnoses, it is predictive of RA. Its discriminative capacity appears to be better than and complementary to that of RF.

Josef S. Smolen and Gu¨nter SteinerAutoimmunity, characterized by autoantibodyexpression, is one of the hallmarks of rheumatoid arthri-tis (RA). This feature distinguishes RA from otherinflammatory and degenerative joint diseases, such aspsoriatic arthritis, reactive arthritis, or osteoarthritis(OA), where autoantibody responses are absent. Theprominence of serologic manifestations of RA suggeststhat, despite the occurrence of joint inflammation anddestruction in the various forms of arthritis, these dis-eases display significant differences in etiopathogenesis.The first autoantibody described in RA morethan 60 years ago was rheumatoid factor (RF) (1). Sincethen, an increasing number of autoimmune responsesfor RA have been detected, some of which appear to bemuch more specific for RA than RF. These autoanti-bodies include the antiperinuclear factor (APF) andantikeratin antibodies (AKA) (2), anticollagen antibod-ies (3), antibodies to nuclear antigens such as Epstein-Barr nuclear antigen and RA33 (4,5), anti-Sa (6), andanti-p68 (7). Many of the autoantigens targeted by theseantibodies have been characterized: RF is directedagainst IgG (8); APF and AKA are directed againstcitrullinated filaggrin and other proteins containing cit-rulline, a posttranslationally modified arginine residue(9); RA33 is identical to the heterogeneous nuclearribonucleoprotein A2 (hnRNP-A2) (10); and p68 hasrecently been identified as the heavy chain bindingprotein BiP (11).Neither the histologic features of the RA synovialmembrane nor the cytokine profile are pathognomonic forthe disease: synovial lining cell hyperplasia and cellularinfiltrates can be found in many disorders (12), and theproinflammatory cytokines tumor necrosis factor (TNF ) and interleukin-1, which are considered hallmarksof RA, can also be found in other disorders, such aspsoriatic arthritis (13), in which TNF blockade can be usedas successfully as in RA (14). Despite these similarities, thetypical bone and cartilage destruction distinguishes RAfrom most other joint disorders. The only other distinguish-ing characteristic of RA is autoimmunity.Is there a link between autoimmunity and RAjoint destruction? Although the role of various auto-immune responses in the pathogenesis of RA is cur-rently unknown, RF, anticollagen, anti-RA33, and anti-citrulline antibodies are all present in synovial fluid. Thisfinding suggests local production (15–18). Therefore, itis tempting to hypothesize that autoimmune responsesin the joint might fuel the inflammatory process andcontribute to destruction by virtue of immune complexformation and cell-mediated reactivities.* Indeed, RAsynovial fluid shows low complement levels (20), andimmune complexes have been found in the fluid as wellas trapped in cartilage of RA patients (21,22). Cellularreactivity against immunoglobulins, collagen, and RA33/hnRNP-A2 has been observed among T cells from RA(and less so from non-RA) patients (23–25). Such auto-reactive T cells appear to be almost exclusively of theTh1 type (25), like those found in the RA synovialmembrane (26–28).One piece of evidence for the pathogenicity inRA of autoimmune responses in general and autoanti-bodies in particular is the passive transfer of disease byautoantibodies or autoreactive cells and/or the inductionof autoimmunity and autoimmune disease by the puta-tive autoantigen (29). Although only collagen amongknown antigens targeted in RA (30) can elicit erosivearthritis in experimental animal models, RF can beobserved in the destructive arthritis of MRL/lpr mice(31) and, interestingly, anti-RA33 has been found in seraof both MRL/

As the potential of epitope chips for routine application in diagnostics relies on the careful selection of peptides, reliable epitope mapping results are of utmost interest to the medical community. Mass spectrometric epitope mapping, in combination with peptide chip analysis, showed that autoantibodies from patients who suffered from rheumatoid arthritis (RA) were directed against distinct surface structures on the full-length human autoantigen RA33 as well as against partial sequences. Using the combined mass spectrometric epitope extraction and peptide chip analysis approach, four sequence motifs on RA33 emerged as immuno-positive, showing that epitopes were not randomly distributed on the entire RA33 amino acid sequence. A sequential epitope motif (245GYGGG249) was determined on the C-terminal part of RA33 which matched with the Western blot patient screening results using the full-length protein and, thus, was regarded as a disease-associated epitope. Other epitope motifs were found on N-terminal partial sequences (59RSRGFGF65, 111KKLFVG116) and again on the C-terminal part (266NQQPSNYG273) of RA33. As recognition of these latter three motifs was also recorded by peptide chip analysis using control samples which were negative in the Western blot screening, these latter motifs were regarded as “cryptic epitopes”. Knowledge of disease-associated epitopes is crucial for improving the design of a customized epitope peptide chip for RA and mass spectrometric epitope mapping pivotally assisted with selecting the most informative peptide(s) to be used for future diagnostic purposes.

Summary A large number of laboratory tests have been developed within the past decade to measure factors involved in the immune inflammation of RA. These can be divided into genetic markers, general measures of inflammation, autoantibodies and tissue-specific markers. In general, it is simpler to prove the power of a certain test to measure the disease process than to predict outcome. Apart from RF positivity and CRP/ESR, few, if any, tests have proven to be of importance in independent studies from different centres. Among the promising candidates for future work are detailed analysis of the HLA-D region genes, sulphoxidation status, the autoantibody against RA33 nuclear antigen, soluble IL-2 receptor measuring lymphocyte activity, hyaluronate/hyaluronan or PIIINP from synovial tissue, the combined use of COMP and proteoglycan epitope tests for cartilage matrix, and pyrodinoline cross-links for collagen from bone and cartilage. The ideal setting for testing such markers are prospective cohort studies starting early in the disease, and since many such studies have been initiated recently, one can expect much new information in coming years. Attention needs to be devoted to the kinetics of marker metabolism, since many are degraded or removed at very fast rates from the circulation, making serum assays less informative.

Background: Objective biomarkers are needed for early diagnosis of juvenile idiopathic arthritis (JIA). Anti-A33 antibodies are considered good markers for adult rheumatoid arthritis (RA), but little information is available on their occurrence in JIA. The aim of the present study was therefore to investigate the value of anti-RA33 for diagnosis of JIA (both early and established disease), and its relation to markers of disease activity, and bone resorption. Subjects: This case–control study was conducted on 34 children with JIA. Ten patients with arthritis of short duration (

Spliceosomal small nuclear ribonucleoproteins (U-snRNPs) are frequent and specific targets of autoantibodies in systemic rheumatic diseases. The abundant, functionally related heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) have later defined as a new target of autoantibodies, of which their immunochemical/immunogenic and pathogenic properties are still under investigation. Among hnRNP proteins, those belonging to the A/B type are considered as the major autoantigens targeted by antibodies in sera of patients suffering with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). By performing an extensive screening using rat liver 40S hnRNP antigenic material, we document here the existence of multiple specificities of anti-hnRNP A/B autoantibodies in sera of Greek patients suffering with a spectrum of systemic rheumatic diseases. This included patients with SLE, Sjogren's syndrome (SS), Scleroderma (SSc) and a specific group of patients mostly with undifferentiated disease (UD patients). In total, four distinct types of anti-hnRNP A/B autoantibodies have been recognized. The first two referred to the known anti-hnRNPA2(RA33) and anti-hnRNP A1; the latter appearing very rarely. The third was of the new type selectively reacting with hnRNP B2 and an hnRNP A3 variant, while the fourth was a rare case of anti-hnRNP B2 alone. In addition, a novel specificity of autoantibodies against hnRNP L protein was identified in association with anti-hnRNP A/B antibodies. The co-existence within a serum of autoantibodies having variable specificity for hnRNP A/B and L autoantigens was shown. Specific immunochemical features of the identified autoantibodies are presented and a possible mechanism of autoepitope spreading within protein components of hnRNP complexes is discussed.

A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) is driven by autoreactive T cells but no autoantigen has been identified to date. We therefore analyzed B and T cell responses to autoantigens potentially involved in the pathogenesis of RA, including IgG, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (RA33). IgG and IgM autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed DA.1F rats already 1 wk before disease onset, reached maximum levels during the acute phase, and correlated with arthritis severity. Apart from rheumatoid factor, autoantibodies to other Ags were not observed. CD4+ lymph node cells isolated 10 days after pristane injection produced IFN-γ but not IL-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate Ags elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated lymph node cells of naive animals to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in the joints of rats injected with pristane. Overexpression coincided with the appearance of anti-RA33 Abs and preceded the onset of clinical symptoms of PIA by several days. Taken together, these data suggest hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this Ag might play a pivotal role in the pathogenesis of PIA and possibly also human RA.

Anti-RA33 antibodies (Ab) are directed to the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, an abundant nuclear protein that is involved in processing, transport and translation of mRNA. Although hnRNP-A2 appears to be constitutively expressed in most organs and tissues, aberrant overexpression has been observed in certain cancers and in inflamed synovial tissue of patients with rheumatoid arthritis (RA). Anti-RA33 Ab occur in approximately one-third of patients with RA but are rarely detected in other rheumatic diseases, with the exception of systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) where they are as prevalent as in RA. Since anti-RA33 Ab are already detectable in the earliest stages of RA they are useful diagnostic markers showing a positive predictive value (PPV) for RA of approximately 74%. In the absence of rheumatoid factor (RF) and anti-citrullinated protein Ab (ACPA), anti-RA33 Ab appear to be associated with a relatively favourable prognosis. Similar to RF and ACPA, their role in the pathogenesis of RA is unclear. However, autoreactive T cells directed to hnRNP-A2 appear to be commonly present in blood and synovial fluid of RA patients. Their Th1-like phenotype is suggestive of pathogenic involvement of anti-RA33 autoimmunity.

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial joints, often with a progressive and destructive course, associated with the production of wide range of autoantibodies, and their detection has diagnostic and prognostic implication. Objectives This study was to estimate the diagnostic value of antibodies against cyclic citrullinated peptide (ACCP) and nuclear antigen RA33 of the IgA rheumatoid factor (RF) versus the value of routinely used profile of autoantibodies in diagnostic work-up of RA. Methods We identified 230 patients with RA prehistory of varying duration. The control group was comprised of 96 patients, including patients with seronegative spondyloarthropathies (SpA), Psoriatic Arthritis (PsA) and diffuse connective tissue diseases, as well as sex and age matched healthy controls. Serum levels of IgM and IgA RF, antibodies against cyclic citrullinated peptide (ACCP), antibodies against RA33 antigen (ARA33) and antinuclear factor (ANF) were measured in all patients and controls. Results Diagnostic sensitivity of ACCP equaled 81%, IgM RF - 76%, IgA RF - 46%, ARA33 - 36% and ANF - 29%. ACCP was significantly more commonly associated with IgM RF. Among RF seronegative patients ACCP were found in 37% cases with 26 IU/Ml detection threshold, and in 26% - with 32 IU/Ml, allowing to increase diagnostic specificity of the test up to 93% with the increment of diagnostic threshold. Incidence of ARA33 was not significantly different among the RF and ACCP positive or negative subgroups, thus making ARA33 an independent RA marker. Specificity of this marker was 83,4%, thus making it inferior to RF and ACCP by a composite of diagnostic characteristics. Conclusions The measurement of RA33 is a rational approach at the next second stage of serologic testing work-up in suspected cases of RA onset, when initial RF and ACCP tests were negative. References Emery P., Breedveld F.C., Dougados M. et al. Early referral recommendation for newly diagnosed rheumatoid arthritis: evidence based development of a clinical guide. Ann Rheum Dis 2002;61(4):290-7. Malakoutikhah M., Gomara M.J., Gomez-Puerta J.A. et al. The use of chimeric vimentin citrullinated peptides for the diagnosis of rheumatoid arthritis. J Med Chem 2011;54(21):7486-92. Schneeberger E., Citera G., Heredia M. et al. Clinical significance of anti-Ro antibodies in rheumatoid arthritis. Clin Rheumatol 2008;27(4):517-9. Pincus T., Sokka T. Laboratory tests to assess patients with rheumatoid arthritis: advantages andlimitations.Rheum Dis Clin North Am 2009;35(4):731-4. Aletaha D., Neogi T., Silman A.J. et al. Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010;69(9):1580-8. Metz C.E. Basic principles of ROC analysis. Semin Nucl Med 1978;8(4):283-98. Nell V.P., Machold K.P., Stamm T.A. et al. Autoantibody profiling as early diagnostic and prognostic tool forrheumatoid arthritis. Ann Rheum Dis 2005;64(12):1731-6. Nell-DuxneunerV., Machold K., Stamm T. et al. Autoantibody profiling in patients with very early rheumatoid arthritis: a follow-up study. Ann Rheum Dis 2010;69(1):169-74. Acknowledgements All employees of Department of Rheumatology National Center of Cardiology and Internal Medicine, Bishkek, Kyrgyz Republic Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by the occurrence of a wide range of autoantibodies (Aab) which can predict distinct clinical features of SSc. Aab to hnRNP A2 and its alternatively spliced variants B1 (anti-hnRNP B1) and B2 are generally referred to as anti-RA33 and have been extensively studied in rheumatoid arthritis (RA) [1]. However, the prevalence of anti-RA33 Aab in other autoimmune diseases, such as scleroderma, mixed connective tissue disease and systemic vasculitides, is far from determined [2–4]. Objectives To compare the frequency of anti-hnRNP B1 with other SSc-specific Aab and to determine an association thereof with the clinical phenotype in patients with SSc Methods We studied Aab prevalence in 64 patients with SSc, 29 with diffuse cutaneous SSc and 26 with limited cutaneous disease as well as 9 patients with overlap syndrome. Diagnosis of SSc in all patients was based on 1980 ACR (ARA) criteria and the disease phenotype was analyzed according to LeRoy9s classification criteria [7]. Serum samples from clinically healthy blood donors (n=174) were used as a control group. Skin involvement and disease severity was measured with Rodnan9s skin score and Valentini activity index. Vascular involvement was accessed by measurement of pulse wave velocity (PWV) and augmentation index (AI) with applanation tonometry (SphygmoCor system, AtCor Medical Pty Ltd., Sydney, Australia). Anti-hnRNP B1 IgG was assessed by an ELISA employing recombinant human hnRNP B1 expressed in E.coli (in.vent DIAGNOSTICA GmbH, Germany). SSc-specific Aab were measured with line immunoassay (Euroimmun AG, Germany) according to the manufacturer9s instructions. Results Anti-hnRNP B1 were found in 18.5% (12/64) of SSc patients and in 1.1% (2/174) of controls (p Conclusions Anti-hnRNP B1 (RA33) is an independent serological marker in SSc and is associated with vascular involvement. References Nell-Duxneuner Vet al. Ann Rheum Dis. 2010; 69(1):169-74. Tomoum HY et al. Pediatr Int. 2009;51(2):188-92. Siapka Set al. Autoimmunity. 2007;40(3):223-33. Cho SBetal.J Invest Dermatol. 2012;132:601-8. Hoffmann MH et al. J Immunol. 2007;179(11):7568-76. Steiner Get al. ClinExpRheumatol. 2002 Jul-Aug;20(4):517-24. LeRoy EC, et al. J Rheumatol 1988; 15:202-205. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2222