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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.

Anti-heterogeneous nuclear ribonucleoprotein (RNP) autoantibodies (AAbs) are encountered in many autoimmune rheumatic diseases (ARDs). The potential diagnostic value of the RA33 AAb complex consisting of RNP A2 and alternative domains of the splicing proteins RNP B1 and RNP B2 is now of interest to rheumatologists. Subjects and methods. The authors studied the frequency of anti-RNP B1 AAbs in 300 patients with systemic ARDs, including those with rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjo gren's syndrome (SS) and in 53 people without ARDs, who constituted a control group. Serum anti-RNP B1 AAbs were assessed by enzyme immunoassay. Results and discussion. The frequency of anti-RNP B1 AAbs in patients with ARDs was much higher than that in the control group: 170/300 (56.6%) and 8/53 (13%) patients, respectively. Anti-RNP B1 AAbs were detected in 78.5% (113/144) of the patients with RA; 40.3% (23/57) of those with AS, in 67.5% (27/40) of those with SSc, in 36.4% (16/44) of those with SLE, and in 13.3% (2/15) of those with SS. The diagnostic sensitivity of the marker for RA was 78.5%, its diagnostic specificity was 84.9%; the likelihood ratio of positive and negative results was 5.24 and 0.24, respectively. In the patients with RA, the level of anti-RNP B1 AAbs significantly correlated with that of C-reactive protein and erythrocyte sedimentation rate, while in those with SSc the detection of anti-RNP B1 AAbs was related to the rigidity of the vascular wall and the presence of hypertension. The frequency of anti-RNP B1 AAbs among the RA patients seronegative for rheumatoid factor and anti-cyclic citrullinated peptide antibodies was 15.4%. Conclusion. Anti-RNP B1 AAs are a useful laboratory marker (with the upper limit of the normal range being 3.3 U/ml), but are of limited value in the diagnosis of RA. Anti-RNP B1 AAbs may be regarded as an additional diagnostic marker for RA.

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 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 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

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.

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.

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.

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 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

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.

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 SLE is a systemic autoimmune disease with distinct immunological characteristics including defective T cell functions, especially concerning IL2 production and proliferation. Furthermore, B-cell hyperactivity is observed leading to the formation of several characteristic autoantibodies (ab), among them ab to the heterogenous nuclear ribonucleoprotein A2/B1 (hnRNP/RA33). These antibodies are known to occur in over 20% of SLE patients. Objectives In order to elucidate the role of T cells and their influence in antibody production in SLE, we studied proliferation of PBMC to purified hnRNP-A2/B1 in SLE patients and healthy controls. Methods Stimulation assays with PBMC of 34 SLE patients and 21 healthy controls were performed. We then proceeded to draw RA33-specific T cell clones (TCC) by cultivation and limiting-dilution cloning of T cell lines. Results While the stimulation indices (SI) in the healthy control group ranged from 0.5 to 3.5 (mean SI: 1.5 ± 0.9), the proliferative response of PBMC of the patient group ranged from 0.7 to 17 with a mean SI of 4.8 ± 4.0 (only 6 of 34 patients had an SI Conclusion Our data reveal that more than 80% of SLE-patients have a significant T cell reactivity (SI >2) to the nuclear protein hnRNP-A2/B1 indicating that the antibody response might be T cell driven. Furthermore, almost 60% of TCC derived from SLE patients were CD8+, which supports the importance of these T cells in SLE. Further studies will have to elucidate the pathogenetic implications of these findings.

SLE is a systemic autoimmune disease with distinct immunological characteristics including defective T cell functions, especially concerning IL2 production and proliferation. Furthermore, B-cell hyperactivity is observed leading to the formation of several characteristic autoantibodies (ab), among them ab to the heterogenous nuclear ribonucleoprotein A2/B1 (hnRNP/RA33). These antibodies are known to occur in over 20% of SLE patients. In order to elucidate the role of T cells and their influence in antibody production in SLE, we studied proliferation of PBMC to purified hnRNP-A2/B1 in 34 SLE patients and 21 healthy controls. While the stimulation indices (SI) in the healthy control group ranged from 0.5 to 3.5 (mean SI: 1.5 ± 0.9), the proliferative response of PBMC of the patient group ranged from 0.7 to 17 with a mean SI of 4.8 ± 4.0 (only 6 of 34 patients had an SI

Rheumatoid arthritis (RA) is characterized by the occurence of autoreactive antibodies and T cells. RA is heterogneous disease also with respect to these autoreactivities, since none of them is present in every RA patient and they are additionally also present - although to a considerably lesser extent - in other autoimmune diseases and even in healthy individuals. It has now been analyzed if there are clusters of autoreactivites that are absolutely specific for RA. Therefore, the RA-associated autoantigens RA33 (hnRNP A2), cit-rulline, rheumatoid factor (RF), the stress protein BiP (heavy chain binding protein), calpastatin (Calp) and calreticulin (Calr) have either been biochemically purified or used as a kit of of recombinant antigen or chemically synthesized peptides. These antigens have been applied to screen sera and PBMCs from RA and control patients for reactivity and the data have subsequently been subjected to cluster analyses. Analyzing the reactivities of 100 RA and 100 control patients, the following patterns of the three combined autoreactivities were determined to be absolutly specific for RA: RF+Cit+BiP+, RF-Cit+BiP+. RA-specific patterns composed of four autoreactivities are RA33+RF+Cit+BiP+, RA33-RF+Cit+BiP+, RA33+RF+Cit+ BiP-, RA33+RF-Cit+BiP+, RA33+RF+Cit-BiP+, RA33-RF+Cit-BiP+. RA-specific patterns composed of five autoreactivites are RA33+RF+Cit+BiP+Calp+, RA33-RF+Cit+BiP+Calp+, RA33+ RF+Cit+BiP-Calp+, RA33+RF-Cit+BiP+Calp+, RA33+RF+ Cit+BiP+Calp-, RA33+RF+Cit+BiP-Calp-. RA-specific autoreactiv-ities composed of six autoreactivities are RA33+RF+ Cit+BiP+Calp+Calr+, RA33-RF+Cit+BiP+Calp+Calr+, RA33+ RF+Cit+BiP-Calp-Calr+, RA33-RF+Cit+BiP-Calp+Calr+, RA33-RF+Cit-BiP-Calp+Calr-. Other patterns also occured exclusively in RA patients, but the differences did not reach statistical significance. Patterns including p205-reactivity have yet to be analyzed and will be presented. Summing up the RA-specific patterns that are complementary to each other, the sensitivity of the analysis for RA is 54%. It appears evident that analyzing other known and unknown RA-associated autoantigens will further increase the sensitivity of the autoreactivity cluster analysis. The diagnostic confidence will markedly improve by testing autoreactivity patterns that clearly distinguish RA from other rheumatic diseases. The composition of the autoreactivity patterns will also improve our understanding of the pathogenesis.

Autoantibodies have the potential of pathogenicity in several diseases. In rheumatoid arthritis (RA), however, this has not been ultimately proven. RA is characterized by a variety of autoantibodies. Newer insights into characteristics of rheumatoid factors indirectly suggest their pathogenetic involvement. In contrast, antibodies to collagen, despite the availability of an experimental model, do not appear to be pathogenetic in man. Anti-hnRNP antibodies, particularly anti-A2/RA33, are present in RA and experimental models of RA, and therefore, aside from their diagnostic value in established and early RA, could also be involved in the disease process. The nature of Sa, another target antigen in RA, has not yet been elucidated. Filaggrin is the antigen recognized by antikeratin antibodies and antiperinuclear factor; however, citrullin is the target amino acid in filaggrin, and anticitrullin antibodies have a high predictive value. Among a series of cartilage proteins, most have not yet been characterized sufficiently; one, gp39, appears to be of particular interest. Whether or not these antibodies are involved in RA pathogenesis is not yet known. It can be speculated that autoimmunity to some, if not all, of these autoantigens mirrors events important in the development of RA, but further studies on T-cell reactivities and in experimental models are needed to fully understand the involvement.

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.

Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis of rheumatoid arthritis (RA). The only accepted serologic parameter (rheumatoid factor [RF]) is not disease specific, nor are any of several novel RA autoantibodies. We aimed at identifying profiles instead of individual autoreactivities allowing for unambiguous prediction of RA. Selected RA autoantigens were tested by ELISA (RF and anti-cyclic citrullinated peptide [anti-CCP]) or Western blot (heavy-chain-binding protein [BiP], heterogeneous ribonucleoprotein particle A2 [RA33/hnRNP A2], calpastatin and calreticulin). Antibody reactivities were assayed from serum samples of 149 RA patients and 132 patients with other rheumatic diseases and from synovial fluids (SF) (58 RA, 65 non-RA). No single autoreactivity was sufficient for unambiguous prediction of RA. Frequencies of multiparameter profiles consisting of 3, 4, 5 and 6 autoreactivites were determined. Fifteen six-parameter serum profiles were exclusively expressed in RA patients, representing a cumulative sensitivity of 59%. Twelve SF profiles were exclusively expressed in 64% of RA patients. The self-learning classification algorithm CLASSIF1 was capable of accurately predicting RA when these profiles were present. Data profile analysis of RF/CCP/BiP/calpastatin/calreticulin/RA33 provided specific discrimination of 64% of RA. Most importantly, RA specific profiles were observed in 64% of patients with early disease (

The autoantigen hnRNP-A2 (RA33) is targeted by autoantibodies (autoAbs) of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). To define the humoral autoimmune response in more detail, a series of overlapping peptides covering the N-terminal part hnRNP-A2 known to harbour the major epitopes was studied by ELISA in sera from patients with SLE (n = 40), RA (n = 50), MCTD (n = 11) and other rheumatic diseases (n = 86) and from healthy subjects (n = 29). Anti-peptide reactivities were detected in 25% of SLE sera but only rarely in patients with RA or MCTD. Since most of the SLE patients investigated had inactive disease, we next studied sequential sera of 15 individual patients. Of those, only three patients were completely negative for hnRNP-A2 antibodies, while all the other patients showed at least one positive reaction during the observation period. These were directed to the full-length protein and/or to 4 of the 13 peptides used: p35–55, p50–70, p90–116 and p155–175. Interestingly, autoreactivities to the first three peptides were significantly associated with each other but not with reactivity to p155–175 or to the complete protein. This cluster of reactivity was also not linked to any clinical marker of disease. In contrast, p155–175 was strongly correlated with reactivity to the complete protein (P < 0.001) and both reactivities were associated with autoAb to dsDNA and correlated significantly with clinical disease activity, skin involvement and proteinuria (P < 0.01). Remarkably, immunohistochemical analysis revealed overexpression of hnRNP-A2 in affected skin of SLE patients. These data, together with previously published findings in murine SLE models, are suggestive of an involvement of hnRNP-A2 autoimmunity in the pathogenesis of SLE.

In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. Stimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. Proliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. Taken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.

Using immunoblot analysis with soluble nuclear extracts from HeLa cells, we identified autoantibodies to an antigen with a molecular weight of approximately 33,000 in 36% of 95 sera from rheumatoid arthritis patients, but in only 1 of 170 controls. The antigen, termed RA33, was resistant to DNase and RNase digestion but sensitive to proteinase K treatment. There was no discernible relation to other autoantibodies. Thus, this newly described autoantibody appears to be highly specific for rheumatoid arthritis.

The recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.